394 536 JMTR Oct2020

ISSN 2056-5135
Johnson Matthey’s international journal of research

exploring science and technology in industrial
applications
Volume 64, Issue 4, October 2020

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Johnson Matthey’s international journal of research exploring science and

technology in industrial applications
Contents Volume 64, Issue 4, October 2020
394 Guest Editorial: Breaking Down Barriers and Borrowing from Biology
By Tom Sturgeon
396 Preparation and Evaluation of a Composite Filler Micro-Embedded with
Pseudomonas putida for the Biodegradation of Toluene
By Yuxi Yan, Rencheng Zhu and Shunyi Li
407 Unlocking the Full Evolutionary Potential of Artificial Metalloenzymes Through
Direct Metal-Protein Coordination
By George S. Biggs, Oskar James Klein, Sally R. Boss and Paul D. Barker
419 Reduction of Biofilm Formation on Cooling Tower Heat Exchangers using
Nano-silica Coating
By Irfan Turetgen
425 A Mini-Review of Shape-Memory Polymer-Based Materials
By Mathew J. Haskew and John G. Hardy
443 Application of Chitosan-Encapsulated Orange Oil onto Footwear Insock
Leathers
By Buket Yılmaz and Hüseyin Ata Karavana
452 Bacterial Community Composition in Produced Water of Diyarbakır Oil Fields in
Turkey
By Tuğçe Tüccar, Esra Ilhan-Sungur and Gerard Muyzer
466 The Biotechnological Potentials of Bacteria Isolated from Parsık Cave, Turkey
By Begüm Çandiroğlu and Nihal Doğruöz Güngör
480 Antibacterial Potential of Six Lichen Species against Enterococcus durans from
Leather Industry
By Didem Berber, İpek Türkmenoğlu and Nüzhet Cenk Sesal
489 The Destructive Effects of Extremely Halophilic Archaeal Strains on
Sheepskins, and Proposals for Remedial Curing Processes
By Meral Birbir, Pinar Caglayan and Yasar Birbir
504 Johnson Matthey Highlights
507 Antibiotic and Heavy Metal Resistant Bacteria Isolated from Aegean Sea Water
and Sediment in Güllük Bay, Turkey
By Gülşen Altuğ, Mine Çardak, Pelin Saliha Çiftçi Türetken, Samet Kalkan and
Sevan Gürün
526 “Nanomaterials and Environmental Biotechnology”
A book review by Martin Hayes
529 Biocatalytic Reduction of Activated Cinnamic Acid Derivatives
By Samantha Staniland, Tommaso Angelini, Ahir Pushpanath, Amin Bornadel,
Elina Siirola, Serena Bisagni, Antonio Zanotti-Gerosa and Beatriz Domínguez
https://doi.org/10.1595/205651320X15954136194837 Johnson Matthey Technol. Rev., 2020, 64, (4), 394–395
Guest Editorial
Breaking Down Barriers and Borrowing

from Biology
Introduction others, such as hides and textiles, show that as

boundaries within science are removed, previously
As humans, we seem to desire structure, distant industries will have much to learn from one
relationships and laws to understand the another.
universe. Through increased understanding, we
can solve the problems and challenges that we
Themes on Interdisciplinary Science
perceive. This method and the output are given
the label of science. At its best, science provides I have reflected on three themes as this issue has
exquisite understanding, life-changing solutions or come together. There are numerous examples on
sometimes both. each theme and I would challenge the reader to
The downside of the structures and rules we think “what next?” for each:
impose is that they can create inertia. Because 1. Interdisciplinary understanding coming into
the structure or rule served a purpose in the past, biology; for example, computational methods
we can be more willing to stand by it blindly than and coding which go hand-in-hand with the
openly seek the understanding or solutions we truly biological understanding required for directed
desire; a dynamic seen in the natural and social evolution of proteins
sciences alike and revealing more about human 2. Interdisciplinary understanding coming from
nature than the universe. One such structure is biology; for example, improved understanding
that of the disciplines within science. We should of biochemical pathways and the relevant
challenge ourselves to be very clear on the purpose biological structures being coupled with
of any structures we adhere to and be ready to synthetic chemistry understanding to
remove barriers that get in the way of progress. allow much more targeted small molecule
One such example is uncovering the fertile ground therapeutics to be designed
of interdisciplinary research. In recent years 3. Platform technologies; for example, clustered
interdisciplinary research has been of increasing regularly interspaced short palindromic
importance across the sciences. Volume 64 of the repeats and CRISPR-associated protein 9
Johnson Matthey Technology Review started a (CRISPR‑Cas9) genome editing where you
celebration of interdisciplinary science by looking at can custom design the edit while following
when chemistry collaborates with physics (1) and standardised procedures.
in this issue, we will celebrate the cross-disciplinary This third theme is perhaps the most important as
contributions of biology with other fields. it turns niche expertise into something accessible
This wide-ranging issue explores topics such as: to scientists across fields. Understanding the
what we can continue to learn from organisms in technology may be beneficial but is not a prerequisite
unusual environments; how we might leverage to accessing it. Biology follows favourably in the
biology in artificial situations; and even how we footsteps of computing in producing such platform
manage the interface between human-made, technologies and it is an attribute that perhaps we
controlled systems and the outside world. In should value and prioritise more in other fields. To
particular, the diversity of industrial applications is expand on this theme, it is exciting to look both
striking. Some are familiar to Johnson Matthey and backwards and forwards to the contributions made
this journal such as fine chemical synthesis, while possible by platform technologies from the field of
394 © 2020 Johnson Matthey

https://doi.org/10.1595/205651320X15954136194837 Johnson Matthey Technol. Rev., 2020, 64, (4)
biology. Often these point back to unlocking our Conclusions

understanding of the structure and function of DNA
at a molecular level and have resulted in some of As you read through this issue, I hope you enjoy
the most impactful scientific contributions of the reading something outside of your current field. I
last 50 years or so. Our health has been a significant would take you back to my earlier challenge and see
beneficiary of these advances with cancer drugs if you can gain any greater insights by not seeing
providing an illustrative case study. Looking back, the separation between your field and those of the
we can see recent classes of therapeutics that were authors. Rather, question what you can leverage,
significantly enabled by this flow of understanding what you can learn and what next?
and platform technologies such as tyrosine kinase
inhibitors and antibody-based therapies (2). Most TOM STURGEON
importantly, patient outcomes have improved Immaterial Ltd, 25 Cambridge Science Park,
substantially in part, thanks to these therapies (3). Milton Road, Cambridge, CB4 0FW, UK
Looking to the future, gene and cell therapies Email: t.sturgeon@immaterial.com
appear to be following a similar pattern and will
hopefully deliver similar patient benefits. Outside
References
of cancer treatments and healthcare, we can see
many industries set to benefit from being able to 1. A. Smith, Johnson Matthey Technol. Rev., 2020,
access biological understanding and technologies. 64, (2), 101
This is particularly as we seek to learn from biology 2. T. A. Baudino, Curr. Drug Discov. Technol., 2015,
and reduce our impact on the planet by using 12, (1), 3
materials and energy in keeping with what Earth 3. M. Quaresma, M. P. Coleman and B. Rachet, The
can sustain. Lancet, 2015, 385, (9974), 1206

Preparation and Evaluation of a Composite

Filler Micro-Embedded with Pseudomonas
putida for the Biodegradation of Toluene
Preparation of composite filler with high toluene removal efficiency
Yuxi Yan on the environment (1). Toluene is a common

School of Ecology and Environment, Zhengzhou pollutant in VOCs and is produced in a large number
University, Zhengzhou 450001, China; College of industrial activities, such as chemical refining
of Chemical Engineering, Zhengzhou University, and dye processing. Toluene stimulates skin
Zhengzhou 450001, China and mucosa, and when it reaches a certain high
concentration, it also causes paralysis of the human
Rencheng Zhu, Shunyi Li* nervous system. Compared with photocatalysis
School of Ecology and Environment, Zhengzhou and chemical oxidation, using a biofilter to remove
University, Zhengzhou 450001, China VOCs is more economical and environmentally
friendly (2). More important is that it does not
*Email: lsy76@zzu.edu.cn produce secondary pollution. The key element to
ensure the removal capacity of the biofilter is the
preparation of the filler. As a carrier for the transfer
The main objective of this study was to evaluate of pollutants, the filler can provide a suitable
the performance of a self-developed filler micro- growth environment for microorganisms (3).
embedded with Pseudomonas putida (P. putida) Micro-embedding technology is a method which
for toluene removal in a biofilter under various uses physical or chemical methods to keep
loading rates. The results show that the biofilter microorganisms in a defined space, ensuring
could reach 85% removal efficiency (RE) on the microorganisms with high activity. The principle
eighth day and remain above 90% RE when the of using micro-embedding technology to degrade
empty bed residence time (EBRT) was 18 s and the VOCs is to use a hollow porous membrane to
inlet loading was not higher than 41.4 g m–3 h–1. intercept microorganisms inside the filler. The pore
Moreover, the biofilter could tolerate substantial size of the hollow porous membrane is smaller than
transient shock loadings. After two shut-down that of microbial cells, so that microorganisms can
experiments, the removal efficiency could be be embedded. The VOCs can enter the interior
restored to above 80% after a recovery period of of the embedded carrier freely due to the small
three days and six days, respectively. Sequence particle size, and the degradation products can
analysis of the 16S rRNA gene of fillers in four flow out of the carrier through the pore size (4, 5).
operating periods revealed that the highly efficient A large number of studies have been carried out
bacterial colonies in fillers mainly included on different types of fillers. Chen et al. (6) used
Firmicutes, Actinobacteria and Proteobacteria and a two-layer biofilter filled with new mixed packing
that the abundance of Bacteroidetes increased materials to remove hydrogen sulfide gas. Dumont
significantly during the re-start period. et al. (7) prepared a nutritional slow-release filler
(UP20) to biodegrade H2S. In the above studies,
the fillers were not embedded with microorganisms.
1. Introduction
The concentration of microorganisms in the filler
The massive discharge of volatile organic was small, and the removal efficiency of the biofilter
compounds (VOCs) has a great negative impact was low in the start-up phase, resulting in a longer

start-up period. Zhu et al. (8) used a composite cross-linked in boric acid-calcium chloride solution
packing material with functional microorganisms to and dried at room temperature for 24 h. Taking the
remove H2S. However, toluene does not biodegrade mechanical strength as a single variable factor, the
easily due to the presence of a benzene ring. proportions of polyvinyl alcohol, sodium alginate
Zuo et al. (9) found that engineered P. putida and polypropylene fibre were adjusted to obtain
could simultaneously degrade organophosphates, the optimal ratio. After many tests and modifying
pyrethroids and carbamates. Muñoz et al. (10) the design, the optimum proportions of each
studied the long-term performance and stability of component of the filler were determined as follows:
P. putida in a toluene removal bioreactor. The above polyvinyl alcohol accounted for 30%~36%, sodium
studies have found that P. putida is highly effective alginate accounted for 12%~18%, polypropylene
in degrading organics containing benzene rings. fibre accounted for 4%~8%, decomposed plants
However, there is a lack of studies on filler micro- accounted for 15%~25%, calcium carbonate
embedded P. putida for toluene biodegradation. accounted for 15%~25%, activated carbon
Existing problems with biofilters packed with fillers accounted for 4%~10% and P. putida accounted
include bed clogging, low biomass concentration for 0.5%~1.5% (13). The schematic pictures of
and pressure drops. These problems become more the size and the composition of the composite
prominent when the biofilter is operated under high filler can be seen in Figure S1 and Figure S2 in the
VOC loading rates or long-term operation (11). For Supplementary Information.
example, Ryu et al. (12) found that the benzene
removal efficiency of a well-designed biofilter
2.2 Experimental Setup
decreased from greater than 90% to approximately
75% after 27 days of operation due to clogging The experimental system used in this experiment
caused by the excess growth of biomass. is shown in Figure 1. Three biofilters were
The main objective of this study was to evaluate constructed with transparent organic glass pipes.
the performance of a self-developed filler micro- Each biofilter consisted of three modules (each
embedded with P. putida for toluene removal under module is 105 mm in inner diameter and 500 mm
various inlet loading rates. The variations in start‑up in height), and all of them were filled with 300 mm
period, pressure drop, biomass concentration and composite fillers. A sampling port was set in the
tolerance to transient shock loading were monitored top of each module. Toluene gas was prepared by
throughout the experiments. Special attention was mixing fresh air with pure toluene in a mix chamber,
paid to the analysis of the microbial community and then introduced into the bottom of each
attached to these fillers and to monitoring the biofilter through the three models in sequence.
evolution of the microbial community in various Three biofilters, namely biofilter 1 (BF1), biofilter
periods. 2 (BF2) and biofilter 3 (BF3), were used in this
experiment to evaluate the start-up performance.
BF1 was packed with the composite filler micro-
2. Material and Methods
embedded with P. putida, and both BF2 and BF3
2.1 Preparation of Filler were packed with the sterilised fillers without any
microorganisms. However, the nutrient solution
The composite filler was mainly composed of used for BF2 at the start-up period was mixed
polyvinyl alcohol, sodium alginate, polypropylene with the P. putida suspension and the microbial
fibre, decomposed plants, calcium carbonate and concentration of the suspension was the same
activated carbon. First, polyvinyl alcohol and sodium as that of the P. putida suspension added in the
alginate, as the embedding and protective agents, preparation of the composite filler in BF1. Specially,
were heated, dissolved and cooled to 35°C. Then nutrient solution (0.11 K2HPO4, 0.04 KH2PO4,
polypropylene fibre as the skeleton, decomposed 0.025 NH4Cl, 0.067 MgSO4, 0.036 CaCl2, 0.25 FeCl3,
plants as nutrients and calcium carbonate as 0.03 MnSO4, 0.04 ZnSO4, 0.03 (NH4)2Mo7O4·4H2O;
the pH buffer were added into the liquid agent, unit: g l–1; adjusted to pH = 7.0 with NaOH) for
respectively. Additionally, activated carbon and microorganism growth was sprayed into the filler
P. putida BRJC1032 (screening from the activated bed from the top of three biofilters throughout the
sludge) were mixed with above agents to increase experiment. The nutrient solution was intermittently
the physical adsorption capacity and biodegradation sprayed onto the top of the three biofilters with a
capacity of toluene. After that, the mixtures were spray intensity of 1.5 l h–1 by a peristaltic pump for
stirred in a container for 15 min and extruded to one hour out of every three hours and the nutrient
spherical particles. Finally, these particles were solution was changed every seven days.

Gas outlet
Toluene
Sampling
points
Flowmeter
Mixing
chamber
Gas inlet Drainage
Air pump Peristaltic pump
Valve
Gas pump Circulation cistern
Fig. 1. Schematic diagram of the experimental set-up
2.3 Toluene Concentration Analysis volume of the whole biofilter (l) and Q is the gas
flow rate (l min–1).
The determination of toluene concentration was
carried out by adsorption of activated carbon and
2.4 Physical and Chemical Property
desorption of carbon disulfide, and then the toluene
Analysis
gas was injected into a gas chromatograph (GC-
2014, Shimadzu, Japan) equipped with a packed The specific surface area and the porosity of the
column (free fatty acid phase (FFAP) capillary filler were measured by a surface area analyser
column, 30 m × 0.25 mm × 0.25 μm) and a flame (Gemini® VII 2390, Micromeritics®, USA). Solid
ionisation detector (FID). The gas chromatography samples were filtered and the pH value of the filtrate
nitrogen was used as the carrier gas with a flow was detected using a Bioblock 90431 electrode
rate of 1 ml min–1. Temperatures of the injection connected to a C-835 Bioblock multiparameter
port, column and detection port were set to 150°C, analyser (Fisher Scientific, France).
65°C and 150°C, respectively. Gas samples were The mechanical strength of the composite filler was
collected from the inlet and outlet of the biofilter measured by using a compressive strength-testing
with a gas-tight syringe and injected into the GC instrument (YHKC-2A, Taizhou Yinhe Instrument
daily (14). Data were obtained from the workstation Plant, China). The pressure drop of the packed bed
by automatic comparison of the peak area of was measured using a digital pressure gauge (testo
the inlet and outlet samples with the baseline 510, Testo SE & Co KGaA, Germany) connecting two
of toluene. The performance of the biofilter was ends from the inlet and outlet. The pressure gauge
evaluated in terms of (%) RE and the elimination had a measuring range of 0–100 kPa, a resolution of
capacity (EC) as a function of toluene loading. The 1 Pa and an accuracy of ±0.3 Pa.
RE and EC were calculated as in Equations (i)–(iii): The saturated moisture content: some packing
fillers were chosen randomly and immersed into
Removal efficiency =
(Cin − Cout ) × 100% (i) distilled water for 2 h to adsorb as much water as
Cout possible. Then the packing fillers were removed
and placed in a vacuum oven (DZF6050, Yiheng
Q × Cin (ii) Scientific Instrument Co Ltd, China) at 105°C for at
Inlet loading =
V least 12 h until its weight remained stable.
Q × (Cin − Cout ) The concentration of microorganisms in the filler
Elimination capability = (iii) was determined by plate counting. Approximately
V
10 g fillers were taken out homogeneously from
where the Cin and Cout are the inlet and outlet the three modules of the running biofilter, and
toluene concentration (mg m–3), the V is the then put into a conical flask with 90 ml distilled

water. After that, the mixture was shaken in a of 30 s at 95°C, 30 s of annealing at 55°C, 45 s of
thermostatic shaker bath for 2 h at 25°C to obtain elongation at 72°C and a final extension at 72°C
the liquid containing microorganisms. Next, a series for 10 min. PCR was performed in triplicate in 20 μl
of solutions were prepared by different dilution mixtures containing 4 μl of 5 × FastPfu Buffer,
factors (1, 10, 102, 103, 104 and 105 times). Each 2 μl of 2.5 mM deoxyribonucleotide triphosphates
0.1 ml solution was taken and inoculated into three (dNTPs), 0.8 μl of each primer (5 μM), 0.4 μl of
types of plate cultures (beef-protein, Rose Bengal FastPfu Polymerase and 10 ng of template DNA.
medium and Gause’s No.1 medium) for bacteria, The resulting PCR products were extracted from a
fungi and actinomycetes, respectively. The plates 2% agarose gel, further purified using the Axygen®
were placed in a biochemical incubator (CLIN-250, AxyPrep DNA Gel Extraction Kit (Corning Inc, USA)
Tianjin Huabei Experimental Instrument Co Ltd, and quantified using QuantiFluor®-ST fluorometer
China) for 2–7 days at 28°C. Finally, the number (Promega, UK) according to the manufacturer’s
of microorganism colonies in each plate was protocol (16).
counted. Moreover, all the glass vessels used in Purified amplicons were pooled in equimolar
this experiment were sterilised by using a seating fashion and paired-end sequenced on a MiSeq
automatic electro-thermal pressure steam steriliser platform (Illumina Inc, USA) according to the
(Model ZDX-35B, Shanghai Medical Instrument standard protocols established by Shanghai Majorbio
Manufactory, China) (15, 16). Bio-Pharm Technology Co Ltd (Shanghai, China).
The acquired sequences were compared with 16S
rRNA gene sequences in the National Center for
2.5 DNA Extraction and Sequencing
Biotechnology Information (NCBI) database.
Approximately 10 g fillers were randomly sampled
from the lowest module of BF1 system at the 25th
3. Results and Discussion
day, 65th day, 95th day and 145th day. Then the
samples were sealed with aluminium foil and frozen 3.1 Physicochemical Properties of
at –4°C in a fridge. the Filler
Microbial DNA was extracted from the above four
samples using the E.Z.N.A.® soil DNA Kit (Omega Physicochemical properties of the experimental
Bio-tek Inc, USA) according to the manufacturer’s filler used in this study and some other materials
protocols. The final DNA concentration and from the references are listed in Table I (13). As
purification were determined by a NanoDropTM shown in Table I, the experimental filler is spherical
2000 UV-vis spectrophotometer (Thermo with a diameter of approximately 10 mm. The bulk
ScientificTM, USA), and DNA quality was checked by density of the experimental filler is approximately
1% agarose gel electrophoresis. Polymerase chain 271 kg m–3, similar to that of pine bark, and lighter
reaction (PCR) was conducted according to the than most of the reference fillers. The mechanical
following: 3 min of denaturation at 95°C, 27 cycles strength is greater than that of pine bark but
Table I Physicochemical Properties of the Fillers

Bulk Saturated Specific Organic
Size, Mechanical Porosity
Filler density, pH moisture surface matter
mm strength, N rate, %
kg m–3 content, % area, m2 g–1 rate, %
Experimental
10 ± 2 271 ± 17 153 ± 5 7.0 55.3 ± 3 13 ± 2 1.32 53 ± 4
filler
Pine
— 244 — 5.7 56.3 59.9 18.39 98.2
barka (17)
Lava
— 591 — 5.9 28.9 65.4 2.77 0.6
rocka (17)
UP20 (7) 7 920 — 6.9 47 — — —
Composite
12 471 427 10.5 49 38 3.91 —
filler (8)
Slow-release
50 164 — 7.9 46.7 88 — —
filler (16)
a
Due to the irregular shape of pine bark and lava rock, there is no corresponding size data. Other “—” data show that the author did
not determine its physicochemical property data in the corresponding literature

smaller than that of volcanic stone (>500 N) (17). efficiency of BF1 increased from the initial 40% to
The porosity rate is approximately 13%, which is 80%, and stabilised between 82% and 85% after
significantly smaller than other fillers and helps the eighth day (21, 22). The removal efficiency
toluene to better contact microorganisms in the of BF2 showed a downward trend in the first few
filler when entering the biofilter (18, 19). The initial days and then rose to approximately 85% at the
pH of the filler is 7.0 ± 0.2. The specific surface 14th day. The removal efficiency of BF3 gradually
area is approximately 1.3 ± 0.1 m2 g–1, which is declined from the beginning, and it decreased
similar to that of lava rock and composite filler. to almost zero on the 16th–18th days (14, 23).
Compared with lava rock, UP20 and slow-release The results showed that fillers embedded with
filler (7, 8, 16), the saturated moisture content and activated carbon and polypropylene fibres have a
organic matter rate are higher, which can provide certain adsorption capacity. However, the removal
water and nutrients for microorganisms in fillers. efficiency was gradually reduced when the filler
In addition, the decomposed plant fibre contained reached adsorption saturation, as shown in the
within the filler can provide nutrients for microbial BF3 trend line in Figure 2. For the same reason,
growth during experimental operation (20). The the BF2 line also showed a downward trend at
selected microbial source added to the filler was the beginning. Due to the substantial growth of
P. putida, and the activated carbon was contained microorganisms, the subsequent removal efficiency
in fillers, which can adsorb toluene quickly, gradually increased as shown in the BF2 trend line.
promoting toluene to enter the biofilter. The filler Compared with BF2, the fillers in BF1 embedded
in the biofilter did not appear to have deformation, with P. putida showed unique degradation of
accumulation or other phenomena after operating toluene at the beginning. The filler‑embedded
approximately 150 days. The results indicated that microorganisms entered the working state faster
the fillers had favourable properties as biofilter than those cultured with the bacterial solution.
media, and maintained characteristics under These results indicated that the biofilter packed
long‑term operation. with the composite fillers prepared by micro-
embedding could be quickly started up and the
microorganisms in the biofilter could well utilise
3.2 Start-up Performance
toluene as the carbon source (22).
The removal efficiency of the three biofilters
during the start-up period is presented in
3.3 Continuous Biodegradation
Figure 2. Three biofilters, operated at low toluene
Performance
concentrations (100–120 mg m–3) and an EBRT of
35 s, demonstrated different removal performance Toluene continuous removal experiments were
for toluene at the start-up period. The removal performed in three phases based on controlling the
100
Fig. 2. Removal performances
of BF1 (packed with the fillers
micro-embedded with P. putida),
80 BF2 and BF3 during the start-up
period
Removal efficiency, %
60
40 BF3
BF1
BF2
20
0 2 4 6 8 10 12 14 16 18
Time, d

EBRT of BF1 to 35 s (Phase 1, day 10 to day 49), 18 s biofilter. At a higher flow rate, the contact time
(Phase 2, day 50 to day 80) and 12 s (Phase 3, day between the toluene and the microorganisms
81 to day 110). The results of these experimental in the fillers was shortened and that resulted in
stages (Figure 3) are described below. Initially, deterioration of the biodegradation ability of the
the biofilter was operated at a low loading rate of filter bed, leading to lower removal efficiency (24).
toluene (10.5 g m–3 h–1) corresponding to a low Similarly, in Phase 3, the toluene inlet concentration
inlet concentration (100–120 mg m–3) and high increased from 100 mg m–3 to 400 mg m–3, and
EBRT (35 s) to facilitate proper microbial growth and the intake load increased to 123.3 g m–3 h–1 with a
establish steady-state conditions (8, 23). Steady corresponding EBRT of 12 s. During this phase, the
state was achieved on the 10th day of operation, removal efficiency of toluene gradually decreased
which was evident from the constant value of the to 80%, and no significant improvement in removal
removal efficiency (83%). On the 18th day, the inlet efficiency was observed (17, 22).
concentration increased to 200 mg m–3, the removal Elimination capacity, another important indicator of
efficiency was almost stable at 88% after a slight the biofilter, was also used to assess the ability of
decrease. On the 28th day, the inlet concentration the biofilter in terms of toluene removal. Figure 4
increased to 400 mg m–3, and the removal efficiency demonstrates the relationship of elimination capacity
dropped rapidly to 72% and finally stabilised at 90% upon the inlet loading. It could be seen from
after five days of continuous operation. However, Figure 4 that the elimination capacity presented a
when the inlet concentration was controlled at slow increase with the increase of inlet loading rates.
800 mg m–3, the removal efficiency did not reach The maximum elimination capacity of the biofilter
a correspondingly high state (less than 80%). In was 101 g m–3 h–1, which is better than other typical
Phase 1, the initial rapid increase within 90% of RE biofilters. For example, Zhu et al. (10) used composite
may be due to some extent to competition among packing materials to remove H2S and observed a
microorganisms in the filter unit (14, 21, 23). maximum elimination capacity of 65 g m–3 h–1. Liu
Again, in Phase 2, the inlet loading rate was et al. (18) reported compost-based biofilter with a
increased and maintained at 81.2 g m–3 h–1 with maximum elimination capacity of 50 g m–3 h–1 for
a corresponding EBRT of 18 s, and the toluene toluene.
inlet concentration varied between 100 mg m–3 The concentration of toluene in the nutrient
and 400 mg m–3. The removal efficiency reached solution was 0.3 ± 0.1 g l–1 (the saturated solubility
a maximum when the inlet loading rate was less of toluene in water was 0.5 ± 0.1 g l–1). This may
than 41.4 g m–3 h–1 and was stable above 90%. be due to the short contact time between toluene
However, the removal efficiency was only slightly and the nutrient solution. In addition, part of the
decreased and then stabilised close to 86% at the toluene dissolved in the nutrient solution was
end of this phase. This result might be attributed utilised by the filler with circulation of the nutrient
to the decrease in residence time of toluene in the solution.
EBRT = 35s EBRT = 18s EBRT = 12s Fig. 3. Time course of the inlet
1000 100
and outlet concentration and
the removal efficiency of BF1
Toluene concentration, mg m–3
800 80
Inlet concentration
600 Outlet concentration 60
Removal efficiency
400 40
200 20
0
0 10 20 30 40 50 60 70 80 90 100 110
Time, d

The above results showed that a sudden increase then biodegraded by microorganisms in the filler.
in the inlet loading will cause the removal rate to A certain amount of toluene will be dissolved in
decrease within a certain period of time. As the the nutrient solution, but with the circulation of
experiment proceeds, the system will gradually the nutrient solution, part of the toluene will be
return to a higher removal rate. When the degraded by the microorganisms in the filler again.
microorganisms grew under suitable conditions,
the recovery ability of the system also increased.
3.4 Tolerance for Transient Shock
However, when the inlet loading rate was too high,
Loading
the degradation ability of the microorganisms was
exceeded, resulting in a relatively low removal rate. To test the ability of the biofilter to resist sharp load
After entering the biofilter, toluene is first adsorbed change, two interference-shutdown experiments
by activated carbon and biofilms in the filler, and were operated after running for 114 days.
140 Fig. 4. Toluene elimination

capacity of BF1 versus the
120 inlet loading
Elimination capability, g m–3 h–1
100
80
60
40 y = 07989x + 2.9602
R2 = 0.995
20
0 20 40 60 80 100 120 140

Inlet loading, g m–3 h–1
3 days 7 days Fig. 5. Performance evaluation

1000 100
Inlet concentration during shutdown and restart
Outlet concentration periods of BF1 under transient
shock loading
Toluene concentration, mg m–3
Removal efficiency
800 80
600 60
400 40
200 20
0 0
110 115 120 125 130 135 140 145
Time, d

Figure 5 shows the performance evaluation forming units (CFU) g–1 (the filler was placed in
during shutdown and restart periods of BF1 under the refrigerator for 1 month, and the biomass
transient shock loading. When the inlet toluene concentration was reduced to 5 × 104 CFU g–1)
concentration decreased from 400 mg m–3 to to 4 × 108 CFU g–1 on the 60th day, which was
200 mg m–3, the removal efficiency increased consistent with the trend in the pressure drop (24,
to 90%. Then, the biofilter was subjected to 26). The above result indicates that the increase
a three-day shutdown experiment and the in system pressure drop was mainly due to the
removal efficiency was restored to 81.2% after rapid growth in microbial biofilm formation and
running three days. Compared with the shutdown inlet loading rates. The efficient growth and
experiments of Singh and Wang (22, 23), the reproduction of microbial biomass played an
interrupt experiment in this study better reflects important role in the efficient operation of the
the change of flow in actual operation. In the second system and the growth of the microorganisms
experiment, when the inlet toluene concentration affected the pressure drop across the packed
increased from 400 mg m–3 to 800 mg m–3, bed and the ease with which the packed bed
the removal efficiency decreased drastically to was clogged. Low biomass reduces the removal
62%, and time for the RE to reach at 80.9% efficiency. In contrast, excess biomass reduces the
was only six days after seven days of shutdown space required for gas and liquid to pass through
operation. This result clearly indicates that a the biofilter, which leads to an increase in the
certain amount of toluene absorbed in activated system pressure drop (27). Although the biomass
carbon was supplied to the microorganisms during concentrations in the biofilter increased and the
the shutdown operation of the system, and the porosity of the system was reduced, this process
microbial activity was maintained; in addition, the did not cause blockage of the system and had no
decomposed plant fibres also provided a carbon significant effect on the removal performance.
source for the microorganisms, as found by Jorge
and Livington (25).
4. Bacterial Community Analysis
To explore the bacterial communities in the
3.5 Biomass Concentration and
biomass attached to BF1, genetic sequencing
Pressure Drop in the Biofilter
analyses were carried out. Sequencing of 16S
The attached growth biomass concentration and rRNA genes amplified from the active bacterial
pressure inside the device were measured during communities during the operational stages revealed
1–60 days in the biofilter, as shown in Figure 6. 21 phyla, 41 classes, 96 orders, 184 families and
The pressure drop increased more obviously from 347 genera (28, 29). The community analysis at
56 Pa to 373 Pa. The biomass concentration in the phylum level of the fillers is shown in Figure 7.
biofilter gradually increased from 5 × 104 colony The four operational stages were sampled at the
500 1010 Fig. 6. Biomass concentration

and pressure drop changes in
BF1 during the first 60 days
Biomass concentration, CFU g–1
109
400
108
Pressure, Pa
300
107
200 106
Pressure
Biomass concentration 105
100
104
0 10 20 30 40 50 60
Time, d

Community barplot analysis

Firmicutes
Actinobacteria
Proteobacteria
Day_25 Bacteroidetes
Chloroflexi
Gemmatimonadetes
Others
Day_65
Samples
Day_95
Day_145
0 0.2 0.4 0.6 0.8 1

Percent of community abundance on Phylum level
Fig. 7. Bacterial community analysis of the fillers sampled at the 25th day, the 65th day, the 95th day and
the 145th day in BF1
25th day, the 65th day, the 95th day and the of Pseudomonas genus increased from 4.7 × 10–4
145th day, where the 25th day, the 65th day and to 1.9 × 10–3. After two intervention-shutdown
the 95th day had a different EBRT and the same experiments, the abundance of Pseudomonas genus
inlet toluene concentration, and the 145th day was decreased to 8.5 × 10–5, which indicates that the
after two interference-shutdown experiments. The biofilter was not in a sterile environment and that
dominant phyla were Firmicutes (63.4 ± 8.7%), there are other microorganisms competing with the
followed by Actinobacteria (14.6 ± 3.9%) and P. putida added to the filler. When the environmental
Proteobacteria (10.1 ± 4.2%). With decreased conditions and the nutrients in the biofilter became
EBRT, the abundance of Firmicutes remained unsuitable for the added microorganisms and
high, but the abundance of Actinobacteria were suitable for other microorganisms, the other
decreased, and the abundance of Proteobacteria microorganisms were activated and enriched (32).
increased. This is mainly due to a reduction However, in the start-up phase, the biofilter
in residence time leading to the inability of embedded with P. putida started quickly, and the
microorganisms to fully utilise toluene, and removal efficiency of toluene remained high, which
a reduction in the carbon source leading to a indicated that the added P. putida contributed to
change in the proportion of microorganisms (30). the efficient operation of the biofilter (33). These
After two interference-shutdown experiments, results indicated that the biomass could maintain
the abundance of Bacteroidetes increased and itself by microbial community changes, and the
the normal microecological balance was broken, rapid re-adaptation of the biofilter could contribute
which indicated that Bacteroidetes is a sensitive to the activity retention of its biomass during the
biological indicator, similar to the results found by starvation period.
Wolińska (31). Using this indicator (the increase
in Bacteroidetes), it can be judged whether the
5. Conclusions
biofilter is in an unstable state, which would
provide some guidance for practical engineering A composite filler micro-embedded with P. putida
applications. was prepared and evaluated for the biodegradation
In the four operational periods, few Pseudomonas of toluene. The biofilter packed with the fillers could
(abundance less than 1%, as shown in Figure S3) start up quickly with 85% RE on the eighth day,
were found in the sampling of the above four periods. and tolerate substantial transient shock loadings.
As the inlet loading rate increased, the abundance The RE of the biofilter remained above 90% when

the EBRT was 18 s and the intake load was not 10. R. Muñoz, M. Hernández, A. Segura, J. Gouveia,
higher than 41.4 g m–3 h–1. In the experimental A. Rojas, J. L. Ramos and S. Villaverde, Appl.
period of 145 days, no filter plugging phenomenon Microbiol. Biotechnol., 2009, 83, (1), 189
was observed. Moreover, the high removal 11. J. V. Littlejohns, K. B. McAuley and A. J. Daugulis,
efficiency and elimination capacity contributed J. Hazard. Mater., 2010, 175, (1–3), 872
to rich bacterial communities for the efficient 12. H. W. Ryu, K.-S. Cho and D. J. Chung, Bioresour.
biodegradation of toluene. The communities Technol., 2010, 101, (6), 1745
mainly included Firmicutes, Actinobacteria and 13. Y. Nie, R. Zhu, S. Li, S. Li, M. Wang and Y. Yan,
Proteobacteria, and the abundance of Bacteroidetes Chinese J. Environ. Eng., 2019, 13, (3), 678
increased significantly during the recovery 14. X. Chen, W. Qian, L. Kong, Y. Xiong and S. Tian,
period. The composite filler exhibited favourable Biochem. Eng. J., 2015, 98, 56
physicochemical properties in this experiment and 15. W.-F. Yang, H.-J. Hsing, Y.-C. Yang and J.-Y. Shyng,
its practicability in industrial engineering should be J. Hazard. Mater., 2007, 148, (3), 653
further investigated. 16. R. Zhu, S. Li, Z. Wu and É. Dumont, Environ.
Technol., 2017, 38, (8), 945
Acknowledgments 17. Y. Luo, S. Li, H. Ma and Y. Wang, Trans. Chinese
Soc. Agric. Eng., 2017, 33, (12), 218 (in Chinese)
The authors would like to acknowledge the support 18. Y. Liu, X. Quan, Y. Sun, J. Chen, D. Xue and
of the National Natural Science Foundation of China J. S. Chung, J. Hazard. Mater., 2002, 95, (1–2),
(No. U1304216), the Science and Technology Plan 199
of He’nan Province, China (No. 122102310366), 19. R. Logares, S. Sunagawa, G. Salazar,
the University Key Research Project of He’nan F. M. Cornejo-Castillo, I. Ferrera, H. Sarmento,
Province, China (No. 19A610002 and 19A150010), P. Hingamp, H. Ogata, C. de Vargas, G. Lima-Mendez,
and the China Postdoctoral Science Foundation J. Raes, J. Poulain, O. Jaillon, P. Wincker,
(No. 2018M632794). S. Kandels-Lewis, E. Karsenti, P. Bork and
S. G. Acinas, Environ. Microbiol., 2014, 16, (9),
2659
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128 2019, 285, 121317
The Authors
Yuxi Yan received a bachelor’s degree from Zhengzhou University, China, in 2018 and is
currently studying for a master’s degree at Zhengzhou University. His research interests
include the biodegradation of VOCs.
Rencheng Zhu received his PhD from Nanjing University of Aeronautics and Astronautics,
China, in 2017 and currently serves as an associate professor at Zhengzhou University. His
research interests include the governance of VOCs and the characteristics of automobile
exhaust emissions.
Shunyi Li received his PhD from Sun Yat-sen University, China, in 2005. He is currently
an executive director of the Henan Environmental Protection Federation, China, and a
professor at Zhengzhou University. His research interests include the management of
VOCs and the management of odorous gases.

Unlocking the Full Evolutionary Potential of

Artificial Metalloenzymes Through Direct
Metal-Protein Coordination
A review of recent advances for catalyst development
George S. Biggs, Oskar James Klein, stabilise transition states. This highlights the
Sally R. Boss*, Paul D. Barker** often neglected but crucial element of natural
Department of Chemistry, University of systems that is the energetic contribution
Cambridge, Lensfield Road, Cambridge, towards activating metal centres through protein
CB2 1EW, UK fold energy. Finally, general principles needed for
a different approach to the formation of ArMs are
Email: *srb39@cam.ac.uk; set out, utilising direct coordination inspired by
**pdb30@cam.ac.uk the activation of an organometallic cofactor upon
protein binding. This methodology, observed in
nature, delivers true interdependence between
Generation of artificial metalloenzymes (ArMs) metal and protein. When combined with the ability
has gained much inspiration from the general to efficiently evolve enzymes, new problems in
understanding of natural metalloenzymes. catalysis could be addressed in a faster and more
Over the last decade, a multitude of methods specific manner than with simpler small molecule
generating transition metal-protein hybrids have catalysts.
been developed and many of these new-to-
nature constructs catalyse reactions previously
1. Introduction
reserved for the realm of synthetic chemistry.
This perspective will focus on ArMs incorporating Metalloenzymes have been prominent in the
4d and 5d transition metals. It aims to summarise field of enzyme engineering since its emergence
the significant advances made to date and asks some 40 years ago, at the birth of protein
whether there are chemical strategies, used in and enzyme engineering (1, 2). Metal ions or
nature to optimise metal catalysts, that have yet cofactors in solution have an intrinsic chemistry
to be fully recognised in the synthetic enzyme that can be catalytic and these are accessible
world, particularly whether artificial enzymes to detailed mechanistic study. These properties
produced to date fully take advantage of the mean that co-localisation of substrate and metal
structural and energetic context provided by the within a peptidic scaffold can be sufficient in
protein. Further, the argument is put forward forming an ArM, without further influence from
that, based on precedence, in the majority of the protein on the catalytic mechanism. With
naturally evolved metalloenzymes the direct the advent of modern protein engineering and
coordination bonding between the metal and design technologies, ArMs were developed by
the protein scaffold is integral to catalysis. incorporating metal binding sites in or adjacent
Therefore, the protein can attenuate metal to hydrophobic pockets. While the resulting ArMs
activity by positioning ligand atoms in the form were active, they often displayed low efficiency
of amino acids, as well as making non-covalent and specificity. Therefore, directed evolution (i.e.
contributions to catalysis, through intermolecular iterative rounds of mutagenesis and selection
interactions that pre-organise substrates and for activity, Figure 1) has become a key step in

creating enzymes with new and useful properties. protein scaffold as well as the type of catalytic
The choice of starting point for such a forced centre and reactions involved. Advances in ArMs
evolution campaign, in this case the metal-protein have recently been reviewed and the reader
complex formed initially, is of great importance. is referred to these for further details of the
Since any particular enzyme follows a unique strategies used to find new systems (3–5). This
evolutionary trajectory as new mutations move it article aims to provide an overview of the strengths
along the fitness landscape towards (potentially and weakness of these different approaches and
local) maxima, choice of the starting point may to provide a perspective of some challenges that
directly predetermine the result. By nature of remain.
the selection process, it is further possible, that
trajectories leading to the global maximum fitness
2. Why Do We Want New Artificial
fall beneath the cut-off limit for further evolution,
Metalloenzymes?
becoming inaccessible. For instance, a mutation
introduced in the first round of mutagenesis One particular area that will greatly impact
may lead to a destabilisation of the protein at chemical production on this planet is synthetic
assay conditions, causing that initial variant biology. Replacing synthetic catalysts, acting on
to be discarded through selection. However, petrochemical feedstocks in non-aqueous solvents,
a compensating mutation to that variant in a with biocatalytic systems working in water with
subsequent round of mutagenesis could result simple carbon neutral feedstocks (carbon dioxide
in an enzyme which is stable, active and closer even?) is clearly highly desirable. But why engineer
to a global fitness maximum. Finally, not every new enzymes, particularly using expensive and
method of generating ArMs may be compatible relatively scarce transition metals, when the ability
with current methods for directed evolution and to find new catalysts amongst gene products from
therefore limit the extent of evolution that can all corners of the biological world has developed
be achieved. at staggering pace (6–8)? As a consequence of
In this perspective, different routes towards the latter, any target chemical can conceivably be
ArMs are considered in the context of the starting obtained by recombining pre-existing metabolic
Gene library
Mutagenesis Expression
Selection
and DNA Metal
recovery modification
Activity
assay
Fig. 1. The general overview of a directed evolution campaign for ArMs. The Darwinian algorithm can be
reproduced in the laboratory, greatly increasing the speed of evolution. Mutagenesis methods introduce
mutations with various levels of randomness, depending on the method used, to the starting point gene,
forming a gene library. This library can then be expressed in a manner that couples expression products
and genetic sequence information to yield the different proteins. Upon addition of the metal cofactor, the
ArMs are formed and can be selected for improved variants in regard to desired parameters (reaction rates,
yield, stereoselectivity, stability). The metal modification step must itself clearly be efficient and high yielding
to avoid limiting the library size at that stage. The sequence information of the improved candidates is
recovered and can be subjected to further rounds of directed evolution

pathways (9). What will new and unnatural metal-substrate proximity may be enough to
metalloenzymes provide? confer reactivity, directional metal-substrate orbital
One clear feature is orthogonality: the objective overlap also plays a crucial role in activating the
of introducing functionality into a cell that has no substrate to react. Indeed, it is via the formation
counterpart in the natural world could provide of metal ligand, including metal substrate,
chemistry that biology cannot currently catalyse, molecular orbitals that the substrate chemistry is
alkene metathesis for example. As there is a limit to attenuated by the presence of the metal and that
the number of additional transformations a viable catalytic reactivity can be achieved. Significant
cell will perform, these orthogonal reactions may computational advancements have been made
allow access to much shorter, and therefore more in the in silico design of catalytic metal binding
efficient, pathways. If not for a synthetic purpose, sites (10, 11) and the mechanistic understanding
one could also imagine orthogonal catalytic of reported ArMs (12–15). However, given the lack
chemistry providing a diagnostic or reporter output of reliable parameters for defining transition metal
without interference from the host endogenous bonding, and the immense complexity of the many
processes. For it to be truly orthogonal, it is low energy interactions that determine the coupling
difficult to imagine evolving a new enzyme based of protein folding to the binding of small molecules,
around metals already abundant in nature and it is beyond current computational capabilities to
already used as catalysts in biology. The transition predict what primary sequence and cofactors are
metals used by nature are very carefully controlled necessary to achieve the optimal arrangement for
by acquisition and regulatory networks that ensure metal catalysis. It therefore becomes important to
catalytic metal ions are not free to operate outside have a malleable, promiscuous starting system that
the endogenous metabolism. Therefore, there is can be used to sample a large space of different
significant advantage in trying to introduce metals structures (16). Hence, while choosing proteins
that biology currently has no evolved means of with well-defined properties and unique structures
metabolising. This work therefore focuses primarily has some advantages from a design point of view,
on non-biological transition metal cofactors as a starting points that do not fold into one specific
route to introducing novel orthogonal activity into structure may be desirable, since they are not as
a biologically viable system. closely constrained by any one particular energy
well. For similar reasons, in choosing a particular
chemical strategy for introducing a metal cofactor
3. Evolutionary Routes to Optimised
into the protein, it becomes essential to use a
Artificial Metalloenzymes
method that allows for high throughput selection
Natural evolution has provided numerous or screening (17).
examples of metal ions used by enzymes for a
plethora of different catalytic purposes. Rigorous
3.2 Considerations on Metal
mechanistic and structural biochemistry has
Chemistry in Proteins
advanced understanding of the mechanistic detail
of metalloenzyme activity significantly, to the point In addition to sampling sequence space to optimise
that a few underpinning principles can be identified, the geometrical factor, protein evolution offers the
linking protein structure and thermodynamics to unique possibility of sampling transition metal
catalytic activity of metal centres. Together with chemistry by poising the metal in energised states.
the knowledge garnered from extensive research In small molecule transition metal catalysis,
on transition metal catalysts, it is possible to ligands will arrange around the metal centre
establish key properties desirable for novel ArMs. to maximise bonding interactions and reach a
thermodynamic minimum. In order to maintain
the ligand exchange necessary for catalysis,
3.1 Considerations on
some ligands tend to be weakly bonding, with the
Protein-Substrate Interactions
presence of strongly bonding ligands (for instance
As mentioned above, the ability of enzymes to water or hydroxide) being a major factor in catalyst
organise reactants cooperatively can in itself give poisoning. In enzymes however, the intramolecular
rise to enhanced activity over background rates in bonds generated within the whole protein scaffold
solution and in highly evolved systems this may can be used to place and maintain coordinating
even be the greatest factor driving increased atoms from amino acids. These interactions can be
reaction rates. It is important to realise that while seen as the second coordination sphere, shaping

the metal complex and potentially leaving the first decorated with a linker moiety. These cannot
sphere ligand atoms in a suboptimal configuration make use of the protein fold energy to optimise
around the metal centre so that the energy of the chemical process of catalysis, a potential
the resulting complex is not at a minimum on the factor in why directed evolution campaigns of
coordination energy landscape. The stabilisation of ArMs have been of limited success. Whereas
this complex is made possible by the favourable improvements in enantioselectivity and turnover
intramolecular peptidic interactions (i.e. protein number have been reported, which can be traced
fold energy) offsetting the steric and electronic to substrate binding and the hydrophobic micro-
distortion of the optimum geometry (18). These environment respectively (23–25), significant
energised, or entatic, states have a reactivity that is increases in the chemical turnover rate (in many
not easily realised in conventional, synthetic metal systems characterised by the initial kcat) from the
catalysts, if it is possible at all (19). This effect is free cofactor to the formed ArM have so far been
most easily visualised by considering the common limited. Small changes in kcat can be explained
biological process of activation of inert cofactors by organisational effects and indirect interactions
by alteration of coordination upon binding to their with the substrate orbitals, such as charge
respective apoenzymes. For instance, on their own compensation. As demonstrated by Hilvert et al.,
the cobalt metallo-organic cofactor, vitamin B12 significant increases in kcat have been shown to be
and methionine synthase are catalytically inert; possible by fine tuning the actual centre of reaction,
upon protein-cofactor binding and coordination which is the first coordination sphere of the metal
of the cobalt centre to a specific histidine, methyl complex (26). From the perspective of the protein
transfer activity is unmasked with great control scaffold, the formation of an entatic state requires
and substrate specificity (20–22). Applying this the peptide to be at least partially folded before
principle, it can be envisioned that even with the binding the metal. The more defined the fold,
limited donor atoms available to proteins, a vast the greater the ability of the fold to energise the
number of different complexes with different metal complex. This is in contrast to the desirable
chemistries can be accessed, because the exact dynamic system for the evolutionary process.
positioning as well as characteristics of the ligands A potential compromise can be struck by using
dictate metal properties such as electron density, a starting scaffold that is partially folded as the
redox potential, Lewis acidity and ligand exchange apoprotein and upon cofactor binding rigidifies to
rates. Further, the metal cofactor does not need to a completely folded form. The initial folding energy
be a bare metal ion but could be incorporated with can be used to poise the metal in an activated
other ligands already attached. Interaction between state, while the folding process occurring during
these ligands (for instance π–π stacking with an cofactor binding allows for the system to adapt
arene ligand) and the protein can be relayed to during directed evolution. Once the ArM becomes
the metal centre and allow for an even finer tuning more specialised after rounds of evolution, the
of the metal centre. Again, current possibilities apoprotein will probably approach a more fully
for design are insufficient to predict these effects folded form, yielding an ArM after cofactor addition
which can be very subtle, highlighting the need for that is less promiscuous but contains a more
biochemical high throughput screening methods. energised and active metal centre.
To summarise, the number of different complex
chemical factors required of ArMs demand the use
3.3 The Optimal Method of ArM
of directed evolution in order to form enzymes
Formation
with industrially and medically relevant properties.
The above considerations define a range of In order to ensure a high level of engineerability,
requirements for potential methods of forming an optimal methodology for combining 4d and
ArMs. Primarily, there needs to be a direct 5d metals starts with a highly promiscuous and
connection between the protein scaffold and the malleable holoprotein that further has dative
metal ion in the form of at least one coordination bonds between the metal ion and the peptidic
bond, not only for localisation but also for poising moieties. A further point considering the cofactor
the metal reactivity. As will be detailed below, attachment point is that the cofactor should be
most of the successful methods of generating in a deep cleft within the protein topology rather
ArMs published to date are efficient but rely on than at the surface. This is to allow the protein
fully saturated, catalytically active cofactors to maximise substrate binding and secondary
such as commercial transition metal catalysts transition state stabilising effects, as well as

second sphere interactions influencing the metal been explored, with catalytic hydrogenation (31)
complex. and hydroformylation (32) demonstrated.
However, these rhodium metalloenzymes have
a much slower activity than commercial small
4. Strategies for Generating Artificial
molecule rhodium catalysts alone. Although in
Metalloenzymes
these examples it is demonstrated that unnatural
ArMs are generated either from the combination metal complexes can coordinate to the natural
of an unnatural transition metal cofactor being Zn(II) binding site, relatively low catalytic activity
introduced into a protein scaffold or a natural is observed. The highly evolved zinc binding site
metalloprotein being evolved in a laboratory to contains a complex secondary sphere architecture,
enhance or alter its natural catalytic reactivity. A in order to modulate the Lewis acidity of zinc.
detailed review of the field of the directed evolution The chemically different demands for rhodium
of natural metalloproteins is out of the scope of catalysed hydrogenation and hydroformylation
this perspective. However, the engineering and reactions will therefore not be met in this system.
evolutionary approaches developed by Frances Further, evolution of such a specialised system
Arnold and applied to haem metalloproteins (for may be difficult.
example, cytochrome P450) are particularly Hartwig et al. reported taking the metal-
noteworthy and applicable when evolving organic cofactor haem and substituting iron for
unnatural metal-protein hybrid catalysts (27–29). a range of different 4d and 5d metals (including
Four successful strategies have been employed rhodium, ruthenium, iridium and silver) (33).
to localise an unnatural metal to a well-defined In one particularly comprehensive example,
location within a protein matrix. an Ir(Me) porphyrin was incorporated into the
cytochrome P450 enzyme CYP119 and catalytic
functionalisation of C–H bonds to C–C bonds by
4.1 Metal Ion Substitution in Natural
carbene insertion was demonstrated, capable of
Enzymes
high stereospecificity (25). Evolutionary campaigns
Natural metal cofactors can be found in proteins on this artificial iridium metalloenzyme generated
encapsulated by ligands supplied by the protein variants with an impressive 4000-fold increase in
or with non-protein ligands also coordinated. This catalytic efficiency (defined by the kcat/KM), with
enables two different methods of metal substitution: kinetic parameters and selectivities matching those
(a) substituting the metal ion in a protein defined of native enzymes. These parameters highlight
coordination site; or (b) substituting the metal ion the potential of this attachment method, and in
in a natural metal-organic cofactor (such as haem) particular the advantages of introducing exogenous
(Figure 2). metal cofactors with non-protein ligands remaining
Many ArMs have been generated by substituting coordinated upon ArM formation.
the catalytic Zn(II) ion located in a His3 binding site In this case, the mutations made to this iridium
of carbonic anhydrase with different metals, for CYP119 metalloenzyme have greatly optimised the
example, Coleman et al. reported esterase activity binding and pre-organisation of the substrate for
of a Co(II) substituted carbonic anhydrase (30). catalysis, lowering the value for KM, (Figure 3).
Replacement with different Rh(I) species has also In this system there is no direct iridium-protein
coordination; the iridium metal is coordinatively
saturated by four haem nitrogens, one methyl
Remove ligand and coordination to the substrate. Therefore,
the moderate increase in kcat cannot have come
M2
M1
M1
Add through an electronic (through bond) contribution

M2 to catalysis from amino acid side chain ligands and
protein fold energy but must arise from other minor
contributions as discussed in the previous section.
Fig. 2. Schematic representation of metal ion Another limitation of such a system is that it does
substitution in natural enzymes. The natural not allow for the metal to interact with more than
cofactor (red) can be substituted with a suitable one substrate at a time, an essential feature of
unnatural cofactor (blue). This may include the
many interesting organometallic transformations
bare metal ion or larger cofactors such as haem
such as metathesis.

20-fold increase in kcat

M
Bare metal
cofactor
M
WT ArM system Evolved ArM
kcat = n/a kcat = 45.8 min–1

KM ≥ 5 mM KM = 0.17 mM
4000-fold increase in kcat/KM

Protein
Fig. 3. Comparisons between the activities of a bare cofactor and ArM before and after directed evolution.
The data in this figure are taken from the work of Hartwig et al. (25). This elegant study is a good example
of the issues encountered when using fully substituted artificial cofactors, even in highly optimised systems.
Whereas directed evolution was able to achieve an impressive 4000-fold increase in kcat/KM, the actual
chemical kcat was only moderately enhanced when compared to the cofactor in solution. This can be
explained by the enzyme evolving to more strongly bind the substrate and optimise the orientation of the
substrate-metal complex. However, as there is no direct metal-protein interface, directed evolution cannot
influence the metal chemistry, capping the chemical potential at that observed for the free cofactor in
solution
4.2 Supramolecular, Non-Covalent

Binding of Tagged Complexes
There are many specific complexes between M
proteins and small molecules which are well M
understood and have very high affinity. ArMs have

therefore been generated where a catalytic metal
complex has been attached to a small molecule
Fig. 4. Schematic representation of supramolecular,
with high affinity for a protein target (Figure 4).
non-covalent binding of tagged complexes. The
This means of localising the new cofactor into a metal cofactor (red) is localised by non-covalent
protein scaffold has been widely explored. Building interaction between a ligand bound recognition
on the work of Wilson and Whitesides in the group (blue) and the protein
1970s (34), Ward and coworkers have assembled
ArMs based on the high supramolecular affinity of
small molecule biotinylated metal catalysts for the metathesis catalyst was localised to a hydrophobic
protein streptavidin. As many as 12 different binding site in human serum albumin. The
catalytic transformations have been performed metalloenzyme was directed to cancerous tissue
by these metal-streptavidin hybrids, including (through specific glycosylation) and a pro-drug
ruthenium-catalysed olefin metathesis (17), was administered which upon metathesis induced
ruthenium-catalysed deallylation (35), iridium- cellular death (37).
catalysed transfer hydrogenation (24) and One key benefit of supramolecular assembly
dirhodium-catalysed cyclopropanation (36), all is apparent in the examples described above,
in vivo. and that is that the conjugation between metal
This strategy has also been employed in ArMs and protein is robust enough to be performed
that were reported by Tanaka et al. for potential in complex cellular environments. Furthermore,
therapeutic application. In this example, a unlike covalent attachment, supramolecular
coumarin derivative tagged with a ruthenium assembly can be a reversible process, which allows

for component recycling. In a recent report of scaffolds (44). Since then, the most successful
Duhme-Klair et al. catalytic transfer hydrogenation generation of ArMs involving a covalent linkage
is demonstrated from a siderophore-protein to an UAA were reported by Lewis et al. and
combination that enables strong but redox- involve a reaction between an alkyne-substituted
reversible catalyst anchoring (38). All current dirhodium catalyst and a genetically encoded
examples of ArMs generated by supramolecular L-4‑azidophenylalanine residue through strain-
assembly do, however, rely on the assembly of promoted azide-alkyne cycloaddition (SPAAC)
proteins with known, highly catalytically active (45–47). Hypothetically, UAAs could be encoded
metal complexes. As discussed previously, using into a specific residue of most proteins; here, the
complexes which maintain their ligand set during protein scaffold selected was a β-barrel prolyl
ArM formation does not allow the metal complex to oligopeptidase and the resulting metalloenzymes
be subjected to evolutionary pressures limiting the generated catalysed olefin cyclopropanation.
evolutionary potential. The effectiveness of introducing UAA via stop
codon methodology is that theoretically the
same conjugation technology is applicable to
4.3 Covalent Anchoring Through
many different proteins to generate diverse ArMs
Metal Ligands
through a specific, fast and irreversible covalent
Covalent anchoring relies on using a chemical conjugation. Beside commonly relying on pre-
reaction to covalently link a protein side chain to formed metal complexes, an overarching issue of
a strong ligand for a metal (Figure 5). Covalent covalent attachment and supramolecular assembly
anchoring methods can be split into two broad is that the protein scaffold is used predominantly
categories: (a) modification of a natural amino as an auxiliary providing a chiral and hydrophobic
acid side chain (for example cysteine, lysine or micro-environment. Further, many reported
tyrosine), via a nucleophilic–electrophilic reaction methods utilise a long flexible linker between the
and (b) coupling through a genetically encoded point of attachment and the metal complex which
unnatural amino acid (UAA). could remove the catalytic centre from the very
There is a resurgence in research for developing interactions needed for the protein to exert an
novel bioconjugation and protein modification influence on transition states.
techniques of natural amino acids (such as cysteine,
lysine or tyrosine) (39, 40). Generating ArMs
4.4 Direct Activation by Metal
through cysteine modification is attractive due to
Coordination to Protein Side Chains
the high nucleophilicity and rarity of free cysteines
allowing for greater control of reactivity. Salmain Dative ArMs have one or more coordination bonds
and coworkers have modified the free Cys25 in directly from the metal to a Lewis basic amino acid
the cysteine protease papain, using a variety residue (His, Cys, Ser, Glu, Asp) on the protein
of ruthenium, rhenium and rhodium complexes scaffold (Figure 6). The protein therefore has
all functionalised with either a maleimide or a direct electronic influence on the reactivity at
chloroacetamide group (41–43). the metal centre. The active hybrid molecule is
The pioneering work of the Schultz laboratory formed by substitution reactions from a precursor
enabled incorporation of UAAs into protein metal species and the apoprotein. This allows
M M
M
E M
Nu Nu LB LB LB LB
E
Fig. 6. Schematic representation of cofactor

Fig. 5. Schematic representation of covalent attachment via direct activation by metal
anchoring through metal ligands. The metal coordination to protein side chains. The free metal
cofactor (red) is attached to the protein by a cofactor (red) attaches to Lewis basic residues on
reaction forming a covalent bond, for instance the protein (LB) via ligand substitution reactions,
nucleophile (Nu) attacking an electrophile (E) forming a new metal-protein complex (blue)

for potentially very clean reaction conditions for cyclopentadiene with medium selectivity, however,
assembly of the metal-protein complex. Although to our knowledge no subsequent directed evolution
advances have been made, the complexity of experiments have been reported.
these metal-protein binding processes remain In contrast to these examples of forming the
a major challenge for the design of competently complete coordination sphere by binding a bare
folded and catalytically active metalloproteins from metal to the apoprotein state of the ArM, to the best
scratch. It is important to distinguish between of our knowledge there are only very few examples
metalloenzymes where coordination to the of adding exogenous metal complexes (particularly
metal is provided only by amino acid sidechains, 4d and 5d metal complexes) as precursor cofactors
substrates and solvents, and those in which the which then show catalytic activity upon direct
metal brings its own specific ligands with it. The coordination to a protein (57). This is a particularly
latter, metal cofactors would be artificial versions attractive methodology as the challenges of taking
of commonly encountered natural examples such unnatural ligands such as arenes, carbenes and
as haem, vitamin B12 and molybdopterin which phosphanes into biology become opportunities for
are (bio)synthesised separately and bind to the expanding the repertoire of chemistries available
protein through both non-covalent interactions and for catalysis. Controlling the ligand exchange
coordination. As pointed out above, their activity is behaviour of 4d and 5d metal complexes with
defined by the other ligands they carry to an active protein side chain ligands is challenging, not least
site as well as the coordination by the protein. because coordination bonds between ligands and
Degrado and coworkers have pioneered the heavier metals are often stronger than their 3d
design of a number of synthetic proteins which counterparts and hence exchange rates are slower.
directly coordinate bare metal atoms or metal This, however, remains an exciting area of research
cofactors (10, 48). For example, in some of the due to the catalytic diversity demonstrated by
earliest work, the His3-Zn(II) binding motif found many 4d and 5d metal complexes. In this specific
in carbonic anhydrase was introduced into a area our own work has focused upon ruthenium
designed four helical bundle protein, and hydrolytic complexes and their ligand exchange behaviour
activity was observed (49). More recently, with biological systems, laying the foundation for
de novo design has been coupled with directed future work into ArMs with direct metal-protein
evolutionary approaches to generate an artificial coordination (58, 59).
zinc metalloenzyme capable of accelerating ester
cleavage with un-paralleled catalytic efficiency
5. Summary and Outlook
(kcat/KM of 106 M–1 s–1) (26).
In a range of studies (13, 50–52), Roelfes and Significant advances in the incorporation of
coworkers use amber stop codon technology to organometallic complexes into proteins in order
introduce the UAA (2,2′-bipyridin-5yl)alanine into to generate ArMs have been made. The studies
a range of protein scaffolds. Upon addition of highlighted above reliably create hybrid molecules
different bare metal ions, they were able to obtain where the stability and turnover number of the
ArMs catalysing the Friedel-Crafts alkylation of metal centre is higher than the comparable small
indoles, enantioselective metallohydration and the molecule organometallic complex in aqueous
stabilisation of a semiquinone radical. By the use solution. Maybe unsurprisingly, the propensity
of sophisticated computational design, the group for side reactions and catalyst decomposition
was able to introduce beneficial point mutations in is lowered once the complex is in a hydrophobic
many of the novel hybrid molecules, improving both protein environment, already showcasing the
enantioselectivity and yield. The advances in stop usefulness of these hybrid systems. However,
codon technology to introduce UAAs, especially in the question remains, as to whether or not these
the context of directed evolution, make their use a strategies make full use of the protein component.
promising option and provides an enticing method The unique and numerous demands of ArMs call for
for expanding the ligand set available to the a highly integrated approach. To date, most of the
protein scaffold (53–55). In another study, Reetz work described in the literature attempts to exploit
and coworkers computationally designed a Cu(II) the chemistry of metal ions and their complexes
ion binding site into the thermostable protein in a protein scaffold but with limited influence
imidazole glycerol phosphate synthase (56). from the protein on any catalytic activity because
The resulting ArM was able to catalyse the metal-protein coordination is largely indirect and
Diels-Alder cycloaddition of an azachalcone and so cooperativity is limited.

The potential for synthetic organometallic in that reactive promiscuity is reduced. As pointed
chemistry to deliver cofactors which utilise ligand out above, such complexes would be designed to
chemistry not available to naturally evolved have a latent catalytic activity which emerges once
systems can vastly expand the orthogonal catalysis the metal complex is bound to a protein. The design
available in synthetic biological applications. Using challenges raised by this approach are not just as a
such molecules to embed novel metal-peptide result of a need to control the electronic and three-
hybrid complexes in protein scaffolds allows for dimensional steric coordination sphere of the metal
three-dimensional and electronic control around ion, but also to limit ligand exchange processes,
the metal centre that reduces the need for intricate restricting lability of a precursor complex (in the
synthetic catalyst generation. Instead, control of cellular milieu) until it reaches a specific protein
the steric and electronic environment around target. Since the metal-ligand exchange processes
the metal ion can be delivered via the protein for 4d and 5d metal complexes are typically slow,
coordination sphere, particularly where a direct they are particularly attractive from this point of
coordination bond is used to anchor the metal ion view but are hard to predict ab initio.
to the protein. When combined with the ability
to efficiently evolve enzymes, a sophisticated
6. Conclusion
organometallic precursor complex together with
a suitable apoprotein could potentially give rise to In conclusion, in order to optimise the chemistry
a number of diverse reactivities. Therefore, new and biochemistry of ArMs, directed evolutionary
problems in catalysis could be addressed in a faster campaigns coupled with high throughput screening
and more specific manner than with small molecule methods rather than individually-designed synthetic
catalysts. Together with non-covalent contributions strategies are much more likely to generate
to catalysis and the intermolecular interactions that optimised orthogonal catalysts for new and efficient
pre-organise substrates and stabilise transition metabolic processes. Direct coordination between
states, such a system contains many readily metal ions and enzymes is essential in order to
evolvable components. deliver truly interdependent systems, ideally where
The majority of protein scaffolds selected for entatic states deliver enhanced reactivity, efficiency
ArM construction have been chosen because of and selectivity that cannot easily be replicated
their apparent engineerability. However, in most in conventional, synthetic metal catalysis. Going
cases the focus seems to lie solely on the peptidic forward, methods of generating ArMs should be
component with little consideration for evolution of evaluated and developed for both their ability to
the metal complex. Although methods of selection be used in directed evolution procedures and the
and directed evolution have been applied, these extent to which the protein scaffold participates in
are often operating on already well-defined protein the activity of the metal complex.
scaffolds that carry an abiotic cofactor but not a
direct protein-metal complex, which inevitably
Acknowledgements
limits the scope for evolution. Arguably it is
desirable, therefore, to select for a promiscuous George Biggs is supported by the Engineering
and versatile protein starting point which is not and Physical Sciences Research Council (EPSRC)
constrained by one energy minima but instead can (EP/N509620/1) and Peterhouse, University of
potentially offer numerous distinct metal-binding Cambridge, UK. Oskar James Klein is supported
environments, both in terms of direct coordination by the EPSRC (EP/R513180/1). Sally Boss and
and through secondary, intramolecular spheres Paul Barker thank the Department of Chemistry,
of influence, ultimately generating differential University of Cambridge. We thank Florian
catalytic ArM activity. Hollfelder for deep discussions.
Performing catalysis with exogenous metal
complexes within cellular environments has
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The Authors
George Biggs completed an MChem in Chemistry at the University of Bath, UK, in 2016.
He is now a PhD student in the Department of Chemistry at the University of Cambridge.
Supervised by Paul Barker and Sally Boss, his project is focused on understanding the
reactivity of Ru(II) arene complexes with proteins for the development of novel ArMs.
Oskar James Klein obtained an MSc in Chemistry from the University of Cambridge in
2019, where he remains as a PhD student in the Department of Chemistry. Supervised by
Paul Barker and Sally Boss and in collaboration with Professor Florian Hollfelder his project
tries to develop a high throughout methodology for the formation and evolution of novel
ArMs.

Sally Boss studied for an MSci in Chemistry at the University of Bristol, UK, and Heidelberg
University, Germany, before moving to the University of Cambridge to begin a PhD on the
synthesis and reactivity of Lewis acidic, heterobimetallic main group compounds. Shortly
after obtaining her PhD in 2005, she was appointed to a joint College Lectureship in the
Department of Chemistry and at Churchill College, University of Cambridge. Her time is
split between teaching and research and her specific interest is in improving the utility
of heavy metals in biology by careful design of complexes, targeted direction of metal-
cofactors to protein targets and using spectroscopy to understand how they behave in situ.
Paul Barker is Senior Lecturer at the University of Cambridge, Department of Chemistry,

and a Fellow of Downing College, University of Cambridge. His research has always been
at the interface between inorganic chemistry and biology. It started in the field of electron
transfer proteins studied by biophysical methods and mutagenesis, in the early days of
protein engineering. After two, independent Medical Research Council (MRC), UK, and
Biotechnology and Biological Sciences Research Council (BBSRC), UK, fellowships in
Cambridge he joined the Chemistry faculty and has been combining protein engineering
with synthesis and self-assembly for the purposes of generating novel protein based
electronic and catalytic systems. His current interests span protein design and evolution,
self-assembling materials and synthesis of organometallic complexes.

Reduction of Biofilm Formation on Cooling

Tower Heat Exchangers using Nano-silica
Coating
Environmentally sustainable antifouling coating demonstrated on stainless steel
heat exchanger tubes
Irfan Turetgen towers provide cooling by spraying the heated

Basic and Industrial Microbiology Section, water coming from the system onto a fill material
Department of Biology, Faculty of Science, and rejecting the heat to the open atmosphere (1).
Istanbul University, 34134 Vezneciler, Istanbul, The cooled water returns to a basin to recirculate
Turkey again through the system. Common uses of wet
cooling towers include air conditioner systems,
Email: turetgen@istanbul.edu.tr manufacturing facilities, telecommunication
devices and power plants. Such man-made
installations provide an ideal environment for
Cooling towers are industrial cooling units bacterial growth similar to an incubator, supported
operating to dissipate heat. As with any surface in by water temperatures ranging between 24°C
contact with aqueous systems, biofilm formation and 38°C (2–4). The heated water comes from
appears on the surface of heat exchangers. the source to the heat exchanger that allows the
Although biofilm formation on plastic tower fill in exchange of heat between two liquids at different
wet cooling towers has been studied widely, no temperatures by indirect contact inside water
studies were found regarding biofilm formation jacketed tubes (5).
on steel heat exchangers in closed-loop systems. Wet cooling towers providing cool water
In this study, heat exchangers were coated with for heating, ventilating and air conditioning
nano‑silica, which is known to reduce the formation (HVAC) systems are known to be subject to
of biofilm. Natural biofilm formation was monitored contamination. Organic and inorganic substances
for six months. Biofouling was examined monthly in bulk water are deposited on the water contact
using epifluorescence microscopy by assessing the surfaces, reducing the heat transfer significantly
numbers of live and dead bacteria. It was observed and threatening the operating stability of
that the biofilm layer formed on the nano-silica the whole system. Established biofilms offer
coated heat exchanger surfaces was significantly cleaning challenges because they are resistant
lower than on the control surfaces. 3 log microbial to most chemical and physical cleaning
reduction was recorded on coated surfaces in the protocols and they also reduce the heat transfer
first month. After six months, total biomass on efficiency (6, 7). HVAC systems are responsible
control surfaces reached 1.28 × 1012 cell cm–2, for about half of the energy consumed in modern
while the biomass on nano-silica coated surfaces buildings and industrial facilities. Therefore,
was 6.3 × 104 cell cm–2. biofouling is always a significant issue for heat
exchangers and should be taken into account
during heat exchanger design and production. As
1. Introduction
a solution, altering the surface properties could
A cooling tower is a heat dissipation unit which be an effective approach to reduce biofouling in
cools bulk water in industrial systems. Cooling such hard-to-reach articulated systems (2, 8, 9).

A biofilm layer is a community formed by bacterial Materials and Method

cells living in a polymeric matrix that they produce;
a functional partnership adhered to a living or A brand new fabricated closed-loop cooling tower
inanimate surface organised by microorganisms was monitored for six months. A real size, fully
in a dense exopolymer matrix. The capability of working closed-loop cooling tower system was
microbes to stick to a substratum and to produce kept in operation by the manufacturer during the
a biofilm layer has great significance in a diversity experimental period at the factory test laboratory.
of cooling towers, where fouling can act as a The system was filled with distributed network
perpetual source of contamination. Biofilm layer water. Regular blowdown was implemented to
must be kept to a minimum in order to prolong limit the concentration of dissolved solids. In a
the operating life of man-made water systems and circulation rig, hot process water was kept separate
facilitate control of pathogens. Disinfectants may from the cooling water in a closed-loop system
be used for this purpose (4, 9). (Figure 1). For the experiments, a half portion of
Industrial cooling towers can be manufactured the stainless steel (316 SS) heat exchanger tubes
from different materials. Generally, towers were coated with nano-silica and the other part was
are made of reinforced concrete or fibreglass, left without coating. Coating was done by coaxial
stainless steel, wood or reinforced plastic electrospraying before assembly and left to cure in
sheets. The fill material is generally made of air for 24 hours. Coaxial electrospraying has several
plastic sheets (polypropylene, polyethylene or implicit advantages such as high encapsulation
polyvinylchloride) where heat dissipation occurs. efficiency and uniform particle distribution. The
For corrosion resistance, towers are specially coating thickness was between 4–6 µm. Before
treated, painted and covered with a protective coating, the heat exchanger tubes were sprayed
film layer (7). In the case of corrosive water or with 96% ethanol to remove any dirt, oil or grime.
atmospheric conditions, the use of plastic towers This application made the bonding of the coating
is recommended. But heat exchanger units are stronger.
made of stainless steel or copper for better Silica in powder form is hydrophilic. To produce
thermal conduction (5). The critical issue that hydrophobic nano-silica, the silica particles were
affects cooling is the aggregation of deposits transformed by fluorination to confer hydrophobicity.
over the heat exchanger surfaces which includes The final particle size was about 40 nm. The
biofouling. Conventional steel heat exchangers aqueous form of the nano-silica coating contains
may have corrosion or deposits may have formed ethanol as solvent to keep it in liquid form before
on the heat exchanger tubes. Both of these
factors reduce the heat transfer rate (10). To
solve this problem, novel anti-fouling coatings
are considered. Nano-silica can be used in the
form of liquid composites in many matrices as
coating materials. Nano-silica is used in the
textile and automotive industries because of its Fan
self-cleaning, abrasion resistant, hydrophobic Heat
and oleophobic features. It is known that exchanger
nano-silica is able to create low-cost, hard and Hot
tough coatings which are resistant to wear and water
weathering (11). inlet
Although biofilm formation on plastic fill surfaces

in wet cooling towers has been studied widely, no
studies were found on biofilm formation on steel
Cooled
heat exchangers in cooling towers. As coating of
water
heat exchangers is not common, the aim of the outlet
current work was to limit tenacious biofouling
on heat exchangers using a nano-silica coating,
which will lead to longer material life, better
Fig. 1. Schematic view of the cooling tower and
cooling of water and less clogging in closed-loop
heat exchanger
systems.

use. The final nano-silica product was supplied by carried out using special software (NIS-Elements,
a local company. After curing, the coating was solid Nikon Instruments Inc, Japan). Signals obtained
on the surfaces, and no colour change, shedding from 20 randomly selected regions were recorded.
or weight loss were observed on any of the coated Images were saved for later analysis.
test surfaces after the experimental period. The The LIVE/DEAD® kit stains dead cells red and
stability of the coating was tested in a different live cells green in colour. The LIVE/DEAD® test
study by the present author (9) and the mean kit contains two DNA-binding dyes, propidium
overall adhesion capability of the coating was iodide and SYTO® 9. These dyes differ in their
recorded as 1.6 using a pull-off adhesion tester, spectral properties and their ability to enter the
which matches very well with the general rating of living bacterial cell. The first dye in the kit is
adhesion. Water was circulated over the stainless SYTO® 9, which can pass through the membrane
steel (316 SS) heat exchanger tubes, where natural of all bacteria and stain the cells green. Propidium
biofilm formation was allowed to occur. Sampling of iodide only enters into cells with a damaged cell
the biofilm required dismantling the outer shell of membrane, allowing them to appear red under
the heat exchanger unit every month. The system fluorescent light. The number of viable and dead
temperature water was kept constant at 37°C using bacteria on surfaces can be determined in a single
an electrical heating unit to eliminate temperature step using a dual emission filter cube (Chroma
fluctuation which might influence biofilm formation Technology GmbH, Germany).
over time. For both parameters over the six-month duration
Pipe segments were cut monthly from the heat of the experiment, the difference between the
exchanger using an angle grinder, kept in a average bacterial numbers were compared
container filled with system water and brought by two-way analysis of variance. A follow-up
quickly to the laboratory for analysis. LIVE/DEAD® post‑hoc analysis was done in order to determine
BacLightTM Bacterial Viability Kit (InvitrogenTM, differences. The difference was considered
Thermo Fisher Scientific, USA) dye was added significant when p < 0.05. SPSS® Version 18.0
immediately to cover the surfaces completely to software (IBM Corp, USA) was used for the
stain the actively respiring and dead bacteria. statistical analyses.
After 15 min, the surfaces were rinsed with sterile
bi-distilled water to remove unattached cells, air
Results and Discussion
dried, covered with immersion oil and cover slip,
then examined in the dark. This was repeated The bacterial numbers from the LIVE/DEAD® test
every month until the study finished at the sixth kit were analysed in situ on the surfaces using the
month. An epifluorescence microscope (Eclipse manufacturer’s software during the experimental
80i, Nikon Instruments Inc, Japan) was used period for six months. The results are given in
to visualise the biofilm cells in situ. The camera Table I. The number of signals per cm2 were
enables counting and taking images of bacteria on calculated using the magnification factor. Since
solid surfaces, with the signals displayed on the the raw data were too scattered, the values are
computer monitor. Counting and recording were given in the logarithmic (log10) base for better
Table I Numbers with Standard Deviation of Live-Dead Bacteria Counted on Heat Exchanger
Surfaces
Nano-silica coated test surfaces, cell cm–2 Uncoated control surfaces, cell cm–2
Months Dead (log10) Live (log10) Total (log10) Dead (log10) Live (log10) Total (log10)
1 3.6 ± 0.07 3.6 ± 0.05 4.6 ± 0.09 6.8 ± 0.11 6.0 ± 0.08 8.1 ± 0.14
2 3.3 ± 0.09 3.6 ± 0.10 4.3 ± 0.12 7.7 ± 0.13 7.5 ± 0.11 9.7 ± 0.16
3 3.3 ± 0.04 3.7 ± 0.07 4.2 ± 0.08 6.9 ± 0.11 8.0 ± 0.14 10.1 ± 0.09
4 3.0 ± 0.02 3.8 ± 0.04 4.5 ± 0.10 7.0 ± 0.13 8.2 ± 0.12 11.5 ± 0.17
5 3.1 ± 0.09 3.8 ± 0.10 4.7 ± 0.11 7.5 ± 0.15 8.1 ± 0.15 11.9 ± 0.17
6 3.2 ± 0.08 3.9 ± 0.06 4.8 ± 0.12 7.8 ± 0.17 9.1 ± 0.18 12.1 ± 0.21

comparison. The logarithmic reduction was clearly properties of the material and supports less biofilm
significant starting from the first sampling. formation (16–18). Hydrophobic coatings limit the
The total bacterial numbers on coated tubes wettability of the surface, making it difficult for
were recorded as 49,090 cell cm–2 after the initial organic and inorganic matter or microorganisms to
month, and 13,016,957 cell cm–2 on uncoated adhere; and even if they do, they can easily be
surfaces after the first month. The results distinctly detached from the surface by physical factors such
showed that this type of coating reduces biofouling as laminar or turbulent water shear stress (19).
formation on heat exchanger surfaces from the The issue of antimicrobial coatings has been
start of the experimental set-up. The numbers extensively studied (20–24). The problem with these
of surface associated bacteria on uncoated products is development of bacterial resistance
control tubes gradually increased and reached against the agent (11, 25). Even antibiotic-
1.28 × 1012 cell cm–2 after the sixth month, at which containing coatings have been reported to promote
time the biomass on nano-silica coated tubes was biofilm formation (26). Silver compounds combined
6.3 × 104 cell cm–2. No significant rise (p < 0.05) of with silica, silane and titanium coatings in particular
bacterial numbers on nano-coated heat exchanger gave antimicrobial properties but the problem of
tubes was recorded during the six‑month period toxicity in medical devices was mentioned (27). In
in terms of total biofilm counts. This outcome industrial use, the resistance of microorganisms is
demonstrates that a nano-silica coating can clearly at the top of the list as a disadvantage (28). In
reduce the bacterial biofilm layers on coated heat addition, silver compounds in water systems will
exchanger surfaces. reach the aquatic environment and appear as a
As expected, nano-silica coating slowed down separate environmental problem.
the adhesion and colonisation of bacteria on It is also emphasised that anti-biofilm coatings
the substrata thanks to its strong hydrophobic are very important for preventing the formation of
properties. The pH, dissolved oxygen, total a biofilm layer at an early stage (29, 30). However,
dissolved matter and temperature values of the studies conducted to date are mostly aimed at
water in the system during the six-month test solving clinical problems and have been done
period were recorded and are given in Table II. in vitro with pure cultures (15, 17, 18, 31–33).
The values in Table II were important to monitor Using monospecies biofilms is a sterile approach
circulating water due to the blowdown regime. and cannot represent mixed cultures in the natural
It is known that even with conventional cleaning environment and their interaction with each other.
and disinfection regimens, there is a problem Sol-gel products and superhydrophobic coatings
fighting against biofilm formation and development which are more strongly water repellent (31, 34)
of microbial resistance (12). Based on previous have also been tried. It was observed that the life
studies conducted in this field (13, 14), it is of these coatings was not as long as hydrophobic
impossible to eliminate the formation of biofilm coatings. On the other hand, the high cost of
layers on surfaces, but biofilm formation can be superhydrophobic coatings was a drawback.
reduced (9, 15, 16). For this purpose, it is possible Contrary to hydrophobic coatings, some hydrophilic
to modify surfaces with different coatings. The coatings were also found to be effective against
nano-hydrophobic coating changes the surface biofouling. Holberg et al. (8) reported that
Table II pH, Dissolved Oxygen, Total Dissolved Solids and Temperature Values of Circulating
Water in the Systema
Months pH Dissolved oxygen, mg l–1 Total dissolved solids, ppm Temperature, °C
1 7.33 7.40 110 37
2 7.48 7.54 113 37
3 7.28 7.34 109 37
4 7.24 7.24 108 37
5 7.30 7.55 107 37
6 7.36 7.39 110 37

a
The numerical data were the average of three consecutive measurements

biocide-free silicone coatings showed promising 2. S. K. R. Namasivayam, A. L. Francis,
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This is the first report of a nano-silica coating on Technol., 2010, 43, (4), 573
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The study showed that the nano-silica coating
14. C. Gómez-Suárez, J. Pasma, A. J. van der Borden,
significantly reduced bacterial fouling on surfaces.
J. Wingender, H.-C. Flemming, H. J. Busscher and
There are many similar surfaces with biofouling H. C. van der Mei, Microbiology, 2002, 148, (4),
problems which have contact with water and 1161
require a solution. Nano-silica has proven to be 15. N. M. Dat, L. D. Manh, D. Hamanaka, D. V. Hung,
effective at reducing the formation of biofilms on F. Tanaka and T. Uchino, Food Control, 2014, 42,
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of system maintenance, different ways to limit
18. J. Azeredo and R. Oliveira, ‘Biofilm Characteristics:
biofilms in man-made water systems hold much Biofilm Formation: The Role of Hydrophobicity
expectation. and Exopolymers in Initial Adhesion and Biofilm
Formation’, in “Biofilms in Medicine, Industry
and Environmental Biotechnology”, eds.
Acknowledgments P. Lens, A. P. Moran, T. Mahony, P. Stoodley and
This study was supported by ‘Research Fund of the V. O’Flaherty, Pt. 1, Section 1, IWA Publishing,
Istanbul University’. Project number: 29220. London, UK, 2003, pp 16–32
19. B. Arkles, ‘Hydrophobicity, Hydrophilicity and
Silanes’, Paint and Coatings Industry Magazine,
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The Author
Irfan Turetgen is a Full Professor at the Department of Biology, Faculty of Science, Istanbul
University, Turkey. He received his PhD degree in Environmental Microbiology from Istanbul
University. His major area of research is disinfection of heterotrophic biofilms in man‑made
water systems, Legionella ecology in cooling towers and anti-biofilm coatings. He has
authored research papers published in reputed journals and conference proceedings.
Recently he is supervising thesis projects as a mentor. His current interest is modelling
man-made water systems to mimic their function at laboratory scale and test disinfectants
under conditions as close as possible to real life.

A Mini-Review of Shape-Memory
Polymer-Based Materials
Stimuli-responsive shape-memory polymers
Mathew J. Haskew, John G. Hardy* The reversible transformation of SMPs functions

Department of Chemistry and Materials by primary crosslinking net points (hard segments)
Science Institute, Faraday Building, Lancaster memorising and determining the permanent shape,
University, Lancaster, LA1 4YB, UK and secondary switching segments (soft segments)
with a transition (Ttrans) to reduce strain stress and
*Email: j.g.hardy@lancaster.ac.uk hold the temporary shape. Below the Ttrans, the
material will be in its permanent shape and be
stiffer than when Ttrans is reached and the SMPs are
Shape-memory polymers (SMPs) enable the more malleable and can be deformed into a desired
production of stimuli-responsive polymer-based shape (usually through application of an external
materials with the ability to undergo a large force). The deformed state is maintained once the
recoverable deformation upon the application of an external force has been removed and the system
external stimulus. Academic and industrial research is no longer at or above Ttrans. SMPs revert to their
interest in the shape-memory effects (SMEs) of these original state once the Ttrans conditions are met.
SMP-based materials is growing for task-specific This process describes the SME pathway of SMP-
applications. This mini-review covers interesting based materials that are thermally-induced (albeit
aspects of SMP-based materials, their properties, how not for some light or chemical-induced systems).
they may be investigated and highlights examples of While most SMP-based materials hold a single
the potential applications of these materials. permanent shape and a single temporary shape,
recent advances in SMP technology have allowed
the generation of multiple-shaped-memory
Introduction
materials with different stimuli responses (light or
SMEs refers to the ability of the material to chemical) (16, 20, 21). An interesting example of
memorise a shape and materials that possess these this is a triple shaped-memory material generated
properties have a multitude of exciting technical by combining two dual SMPs with different glass
and medical applications (1–14). For materials such transition temperatures (Tg) (22, 23), where the
as alloys this is commonly in a one-way SME (7, SMPs switch from one temporary shape to another at
15), however, there are a variety of materials the first Ttrans, and then back to the permanent shape
that are capable of reverting to their permanent at another, higher activation temperature (22).
shape or original state upon exposure to a stimulus SMPs have a large range of properties from stable
(such as a temperature change) or indeed multiple to biodegradable and transient, elastic to rigid or
stimuli (16). SMP-based materials have been soft to hard, depending on the structural units that
widely investigated since the 1980s because of the constitute the SMP. Consequently, SMPs not only
abundance of potential applications imparted by respond to temperature (24) and magnetism (25)
their interesting properties (for instance, stimuli- like shape-memory alloys (SMAs) (26), but also
responsiveness and ability to change shape), to moisture (27), electricity (28), light (29) and
which can lead to technological innovation and the chemical stimuli (such as a pH change) (30).
generation of new high value products for technical Moreover, there are other principles of SME; for
and medical applications (1, 17–19). instance, a thermal-responsive SMP can proceed

via a Diels-Alder reaction (chemical crosslinking/ hard segments form the net points that link the
reversible covalent bonds) (31). SMPs tend to have soft segments (acting as a fixed phase), whereas
much milder processing conditions than SMAs the soft segments work as the molecular switches
(<200°C, low pressure), have a greater extent (acting as a reversible phase). The fixed phase
of deformation (strain more than 200% for most prevents free flow of the surrounding polymer
materials) and tend to be based on cheap starting chains upon the application of stress. The reversible
materials with simple synthetic procedures (12, phase, on the other hand, undergoes deformation
32). After the term ‘shape-memory’ was first in a shape-memory cycle and is responsible for
proposed by Vernon in 1941 (32), the significance elasticity. For example, if the Ttrans is Tg, the micro-
of SMPs was not fully realised until the 1960s, Brownian motion of the network chains is fixed at
when crosslinked polyethylene (PE) was used low temperature (below Tg) and will be switched
to make heat-shrinkable tubes and films (33). back on at high temperature (above Tg), recovering
Significant investment in the development of its original state. When Ttrans is the crystal melting
SMPs began in the 1980s (34) with rapid progress temperature (Tm), the switching segments
realised in the last decade, particularly with a view crystallise at low temperature (below Tm), and then
to the generation of shape-memory materials with recover their original state at high temperature
exciting and versatile features. (above Tm). In addition, Tg normally extends over
a broader temperature range compared to Tm,
which tends to have relatively sharper transitions
Shape-Memory Polymer Function
in most cases (26). Moreover, after the exposure
Two important quantities used to describe SMEs to a specific stimulus and the Ttrans is achieved, the
are the strain recovery rate (Rr) and the strain strain energy in the deformed state is released,
fixity rate (Rf ). Rr describes the ability of a material resulting in the shape recovery phenomenon. The
to memorise its permanent shape, while Rf general process of this SME for SMPs is depicted in
describes the ability of switching segments to fix Figure 1, wherein the polymer network structure is
the mechanical deformation. Rr is calculated using either chemically or physically crosslinked and the
Equation (i): switching units are made from a semi-crystalline or
amorphous phase.
εm(N) – εp(N)
Rr(N) = × 100% (i) Shape-memory behaviour can be demonstrated
εm(N) – εp(N–1)
in various polymer systems that are significantly
different in molecular structure and morphology.
where N is the cycle number, εm is the maximum SME mechanisms differ according to the specific
strain imposed on the material and εp is the strain SMP(s); for instance, the SME mechanism of the
of the sample after recovery. Rf is calculated using chemically crosslinked semi-crystalline PE SMP.
Equation (ii): The crystalline phase, with a Ttrans being Tm, is
used as the molecular switching unit providing
εu(N)
Rf(N) = × 100% (ii) shape fixity. The chemically crosslinked PE network
εm(N)
memorises the permanent shape after deformation
upon heating (12, 36, 37), and the mechanism of
where εu is the strain in the fixed temporary shape. the thermally-induced shape-memory PE (SMPE) is
SMPs respond to specific stimuli through changes depicted in Figure 2.
in their macroscopic properties (for example, The associated modulus of elasticity is dictated
shape) (26). The polymer network underlying by configurational entropy reduction that occurs
active movement involves a dual system, one that with deformation of the constituent chains and
is highly elastic and another that can reduce the is therefore often termed entropy elasticity. For
stiffness upon application of a certain stimulus. T>Ttrans (Tg, Tm or other), polymer networks
The latter system incorporates either molecular exhibit super-elasticity wherein the polymer chain
switches or stimulus sensitive domains (35). segments between crosslink points can deform
Their shape-memory feature is a result of the quite freely and are prone to being twisted
combination of the polymer’s architecture, and a randomly via rotations about backbone bonds,
programming procedure that enables the formation maintaining a maximum entropy and minimum
of a temporary shape. Net points consist of covalent internal energy as macroscopic deformation
bonds or intermolecular interactions and the SMP’s occurs (12). The classic prediction from rubber

(a)
+ ∆conditions
SMP SMP SMP
Permanent Deformation stage, may Deformed

shape require an external force temporary shape
T<Ttrans e.g. (elongation under T>Ttrans
tensile stress T>Ttrans)
– ∆conditions
+ ∆conditions
SMP SMP
Permanent Deformed temporary

shape, shape shape, if force applied
recovery then the external force
T>Ttrans is removed
T<Ttrans
(b)
External applied force, Strain energy Strain energy released,
bending of polymer stored polymers shape recovery
Heating, a hot Cooling, taken off Heating, a hot

plate hot plate plate
Original state T>Tg/Tm Deformed state T>Tg/Tm Recovered shape,

T<Tg/Tm T<Tg/Tm original state
Fig. 1. (a) The general SME mechanism of SMPs; (b) thermally-responsive SMP
elastic theory is that the resulting elastic shear low temperature, and the ability to trigger shape
modulus (G) is proportional to both crosslink recovery at high temperature; super-elasticity
density and temperature (Equation (iii)): above Ttrans that leads to the eventual shape
pRT recovery and avoids residual strain (permanent
G = vKBT = (iii) deformation); and complete and rapid fixing of the
MC
temporary shape by immobilising the polymeric
chains without creep thereafter (12, 37). Thus
where ν is the number density of network chains, p far, the SME models describing how SMPs recover
the mass density, R the universal gas constant and their original state prominently involve thermo-
MC the molecular weight between crosslinks. From responsive SMPs. However, careful design of the
a macroscopic viewpoint, the SME in SMPs can be polymers allows the opportunity for SMPs to possess
graphically represented in three-dimensions (3D). different stimuli responses and applications.
Tensile strain vs. temperature and tensile stress
(for example, elongation) is depicted in Figure 3.
Shape-Memory Polymer Triggers
Using the shape-memory strain-temperature-
stress relationship description in Figure 3, the A multitude of different triggers for SMEs and SMPs
features of SMPs that allow for good shape- exist. However, an in-depth review is outside the
memory behaviour include: a sharp transition that scope of this mini-review, and therefore a few
can be used to quickly fix the temporary shape at examples are highlighted below.

Force of
T>Tm stretching
PE cross-linked T>Tm Heating at or

by radiation above Tm
T<Tm
Stretched at or
above Tm
Cooled at stretched
shape
Fig. 2. Molecular model of the thermally-induced SME mechanism of crosslinked SMPE
SMPs, with a schematic of the SME mechanism for

Shape fixity thermally-induced SMPs with Tg (amorphous cases)
Strain removal
εm and Tm (crystalline cases). Figure 2 presents a
200 specific example of the SME mechanism for SMPE
εu
with the Ttrans being Tm. In addition, advanced
150 thermomechanical constitutive models have
Strain (ε),%
been used to study the materials’ behaviour (for

100 example strain-temperature-stress development
Recovery with time) in a very accurate way (41). By applying
Deformation 3.0
50 these models to SME mechanistic studies and the
2.5
detailed characterisation of the SMPs (crosslinks,
2.0
0 Pa intermolecular and intramolecular interactions
20 1.5 M
),
40 εp 1.0 (σ involving the SMPs) (12), a deeper understanding
Tem 60 s
per 80 * 0.5 r es of the SME of SMPs can be achieved, which has
atu 100 St
re
(T) 120 0 proven beneficial for the development of new
, ˚C
SMPs and their proposed applications (31). For
Fig. 3. A general 3D plot of an SMP during a example, poly(ε-caprolactone) (PCL), typically
thermomechanical shape-memory cycle a biodegradable polymer, has been reported to
possess high shape fixity and recovery. This was
achieved by integrating reversible bonds within the
Thermally-Induced Shape-Memory PCL polymer network via the Diels-Alder addition of
Polymer 1,2,4-triazoline-3,5-dione (TAD)-anthracene and
Alder-ene addition of TAD-indole (42). These PCL
It is possible to generate thermally-induced SMEs SMPs were reported to attain recovery ratios greater
in a variety of materials (18–20, 38–40), however than 99% (43). Furthermore, a dual-functional
a comprehensive overview is outside the scope (self-healing and shape-memory) polymer network
of this mini-review. As previously discussed, the was achieved by crosslinking a polydimethylsiloxane
SME of SMPs can be thermally-induced, and these (PDMS) polymer containing dense carboxylate
SMPs are the most common (26). Figure 1 depicts groups (100% mol) (PDMS-COOH) with small
a general overview of the SME mechanism of amount of poly(ethylene glycol) diglycidyl ether

(PEGDGE) (44). This SMP (PDMS‑COO-E) actuates Light-Induced Shape-Memory

at body temperature (37°C) with possible strain Polymer
ca. 200% and shape recovery ratios at 98.06%.
In addition, a 25 mm × 4 mm × 1 mm sample It is possible to generate light-induced SMEs in a
cut into two separate pieces healed (the variety of materials (18–20, 38, 40, 45), however a
two pieces become one whole piece with no comprehensive overview is outside the scope of this
evidence of a cut) when the two cut surfaces mini-review. Light-activated SMPs (LASMPs) (46)
were brought into contact after 6 h at 25°C. typically use photothermal or photochemical
Thus, the unique material, PDMS‑COO-E, (photocrosslinking or photocleavage) triggers for
may have a wide range of applications in SMEs. For instance, photothermal LASMPs typically
many fields, including wearable electronics, employ photo-absorber molecules and particles
biomedical devices and four‑dimensional (4D) that convert light to heat, thereby increasing
printing (1, 19). Interestingly, the material was the temperature at the desired region within
also reported to possess a greater than 85% the LASMP. Photochemical LASMPs incorporate
light transmittance (425 nm to 700 nm) (44), photosensitive molecules to create or cleave bonds
therefore PDMS-COO-E has potential applications during irradiation with light, imparting potentially
in transparent electronic devices. Figure 4 very swift SMEs (47, 48). It is possible to improve
illustrates the possible SME mechanism of the response time of SMPs by increasing the
PDMS-COO-E. The short PDMS linear chains are thermal conductivity with various conductive
crosslinked by chemical covalent interactions and additives (49). However, the heating and cooling
abundant hydrogen bonds into a 3D network. The of materials with substantial thickness takes time,
covalent crosslinked networks of PDMS-COO-E which can be minimised by using light to trigger
maintain the permanent shape and resilience, transitions in LASMPs (46). It is also possible to
whereas, at ca. 37°C the weak hydrogen bonds generate multistimuli-responsive materials using
are broken, and the dynamics of polymer chains components of the materials that respond to
increase, resulting in recovering the permanent different wavelengths of light (for example, one
shape. Meanwhile, a large number of hydrogen wavelength of light to induce photocrosslinking,
bonds enable the samples to heal at temperature while a second wavelength of light cleaves bonds).
without external stimulus (44). It is possible to produce materials that can be
reversibly switched between an elastomer and
a rigid polymer employing polymers containing
cinnamic groups (48) that can be fixed into
pre‑determined shapes utilising ultraviolet (UV)
light illumination (>260 nm), and then recovered
their original state when exposed to UV light at a
different wavelength (<260 nm) (49). Figure 5
depicts one example of the process of LASMPs
shape recoverability.
Cooling Heating Electrically-Induced Shape-Memory

reshape recovery Polymer
It is possible to generate electrically-induced SMEs
in a variety of materials (18, 20, 50–55), however
a comprehensive overview is outside the scope of
this mini-review. A variety of electrically conductive
Stretched materials including organic electronic materials
(including conductive polymers such as polypyrrole
= PDMS = PEGGE
O H O
(PPy) (28, 56–58) and carbon nanotubes
= –COOH = (CNTs) (59, 60)) and inorganic electronic materials
O H O
(such as alloys, metals (61) and silver nanowires

Fig. 4. The possible mechanism about shape (NWs)), have been incorporated in materials
memory effect of PDMS-COO-E polymer. Reprinted
displaying SMEs to impart swift triggers to the
with permission from MDPI (44)
SMEs, enabling a variety of interesting applications.

A
hv2
Shape External force applied

recovery (e.g. elongation under
tensile stress)
Permanent shape,
D elastomer B
hv1
Deformed temporary
shape with external = elastomer network cross-link
force removed,
= photo-cross-link site
rigid polymer
= polymer chain backbone
Fig. 5. Schematic of an example of the SME function of LASMPs
Highlighting some of the potential of electrically- of SMP designs driving technological innovation. A
induced SMEs, electrically-induced SMP composites schematic of the composite is shown in Figure 6.
incorporating shape-memory polyurethane Polymeric blend SMPs can be constructed from
(SMPU) and Ag NWs in a bilayer structure exhibits two immiscible polymeric matrices. The shape-
flexibility and electrical conductivity (62–64), recovery of these systems can be controlled with
which may find applications as capacitive sensors, relative ease by varying the ratio of the polymer
healable transparent conductors and wearable blends (68). However, this process may have
electronics (65). In such materials the Ag NWs adverse effects on shape-memory characteristics
are randomly distributed on the surface layer of and diminish the material’s performance, thereby
the composite to form a conductive percolating limiting potential applications. On the other hand,
network that retains conductivity (200 Ω sq–1) after SMP functionality may also be enhanced with other
a 12% elongation. However, continual increase capabilities. For instance, it was recently reported
in elongation causes a dramatic increase to the that a new hybrid SMP was developed by combining
composites’ resistance value and the eventual loss single-walled CNTs (SWCNT) into a poly(lactic acid)
of electrical conductivity (66). When the material (PLA) and thermoplastic polyurethane (TPU) SMP
(deformed or in its original state), is connected to system, containing poly(ethylene glycol) (PEG)
a typical circuit, a low voltage of 1.5 V was enough plasticiser (68). By incorporating PEG, the hybrid
to activate a light-emitting diode (LED) (65). The SMP composite achieved a lower temperature Tg (for
composites possessing a higher Ag NW content example, 10 wt% of PEG lowered Tg of the PLA/TPU
exhibited a higher recovery ratio and reached the sample from 60°C to 40°C), meanwhile enhancing
maximum recovery speed quicker (66). It was the dispersion of SWCNT (for instance, even at
assumed that all the heat from electrical (Joule) 4 wt% of SWCNT loading, 100% SMP tensile strain
heating was absorbed by the sample, i.e. no was possible, much greater than previously reported
convective loss (67). Therefore, the composites with electrically-induced SMP studies, i.e. 12% discussed
higher Ag NW content had a lower resistance value previously). In addition, the presence of the SWCNT
and the heating effectiveness was promoted. Heat can stabilise the SMP system and enhance its shape-
initiates the thermal Ttrans of the SMPU leading to an fixity after deformations at room temperature
improved shape recovery, and voltages as low as conditions (68). Furthermore, the material was
5 V reverted bent composites to their original state capable of a conductivity above 10–7 S cm–1, which
within 3 s (66). This represents a good example of a can be considered conductive, as documented (68).
multifunctional SMP and demonstrates the potential The PLA/TPU SMP composite (2 wt% SWCNT and

(a) (b)
20 um
412.7 nm
500 nm
15
6.7
10
20
5
15
10
20 um
15
5
10
5
(c) AgNWs
(d)
Power
Spin coating
AgNWs LED
Composite
electrode
Release substrate Release substrate LED
Coating SMP Conductive

layer composite
SMP layer
Peel off
AgNW layer AgNW layer
SMP layer Release substrate
Fig. 6. (a) transmission electron microscopy (TEM) image of Ag NWs; (b) atomic force microscopy (AFM)
image of Ag NWs; (c) schematic illustration of composites fabrication process; (d) the LED turned on as the
composite was applied with voltages (the inset shows the circuit connecting with the composites). Reprinted
with permission. Copyright 2014 Elsevier (66)
10 wt% PEG) also achieved shape-recovery, via however a comprehensive overview is outside the
Joule heating derived from electricity, in 80 s when scope of this mini-review. Water is an important
currents of 125 mA were applied. The high stiffness stimulus due to the fact it is abundant in a multitude
of SWCNT filler results in decreasing shape-recovery of different environments, non-toxic and safe for a
performance because of the hindrance on the variety of applications.
polymer chain movements (68). As a result, under An interesting example highlighting the
room temperature stretching, the Rf and Rr values potential of such materials is based on strong
obtained were ca. 80% and 65%, respectively. and flexible composite films (73) utilising the
Therefore, when its shape-recoverability is compared combination of a flexible interpenetrating polyol-
to other SMPs (shape-recovery ratios being upwards borate network (74) and electroactive PPy (75,
of 98%), the material is lacking. However, the hybrid 76) that exchange water with the environment
SMP composite does possess electroactive ability, resulting in film expansion or contraction. The
thus a trade-off relationship between shape-memory/ free-standing multi-functional SMP films were
recovery and electroactive ability needs to be carefully prepared by electropolymerisation of pyrrole
considered when designing similar materials. in the presence of the polyol-borate complex
(composed of pentaerythritol ethoxylate (PEE)
coordinated to boron(III)) (74), wherein the
Water-Induced Shape-Memory
interpenetrating network enables water-gradient-
Polymer
induced displacement, converting chemical
It is possible to generate water-induced SMEs in potential energy in water gradients to mechanical
a variety of materials (18, 20, 38, 39, 69–72), work (73), and results in adaptation of the

architecture in response to an environmental pH-Induced Shape-Memory Polymer

condition change (i.e. sorption and desorption of
water which drives the SME process, as depicted It is possible to generate pH-induced SMEs in a
in Figure 7). The design of the water-responsive variety of materials (18, 20, 38, 77–80), however
PPy-PEE composites was creatively applied to a comprehensive overview is outside the scope of
prepare actuators and generators driven by this mini-review. An example of the interesting
water gradients. The film actuator can generate properties of such pH-responsive SMPs and their
contractile stress up to 27 MPa, lift objects composites is produced by blending poly(ethylene
380 times heavier than itself and transport cargo glycol)-poly(ε-caprolactone)-based polyurethane
10 times heavier than itself (73). An assembled (PECU) with functionalised cellulose nanocrystals
generator associating the actuator with a (CNCs) displaying pH responsive pyridine moieties
piezoelectric element driven by water gradients, (CNC-C6H4NO2) (81, 82). At high pH values the
outputs alternating electricity at ca. 0.3 Hz, with pyridine is deprotonated, facilitating hydrogen
a peak voltage of ca. 1 V (73). The electrical bonding interactions between the pyridine
energy can be stored in capacitors that could groups and hydroxyl moieties on the cellulose,
power micro and nanoelectronic devices (73). whereas at low pH values, the protonation of the
The SME mechanism for this SMP differs to that pyridine moieties diminishes these interactions.
of Figure 1 and Figure 2, utilising water as the By comparison, carboxylic acid functionalised
shape-memory trigger for Ttrans, and the original cellulose nanocrystals (CNC-CO2H) responded to
and deformed state interchange automatically via pH variation in the opposite manner (83–85). When
water sorption and desorption states. However, the functionalised CNCs were combined with PECU
the shape-memory phenomenon remains the polymer matrix to form a nanocomposite network,
same, further demonstrating the potential of SMP the mechanical properties of PECU were improved
designs driving technological innovation. along with the pH-responsiveness of CNCs (85).
(a) (b) (c)

Metal
electrode
Insulating
layer Vcap
Voutput RL G
PVDF
Wire Vrec G VG
PEE-PPy
(d) (e)
1.5 0.7
1.0 0.6
0.5
0.5
Voltage, V
Voltage, V
0.4
0.0
0.3 0.55
Voltage, V
–0.5
0.2
–1.0 0.1 0.50
290 310
Time, s
–1.5
0 20 40 60 80 100 120 0 100 200 300 400
Time, s Time, s
Fig. 7. Design and performance of a water-gradient–driven generator: (a) the assembly of a piezoelectric
polyvinylidene fluoride (PVDF) element with a PEE-PPy actuator to form the generator; (b) the connection of
the generator with a 10 MW resistor as load; (c) the configuration of the rectifying circuit and charge storage
capacitor; (d) the generator’s output voltage onto the 10 MW resistor; (e) voltage across a capacitor when
being charged by the generator. The inset shows a stepwise increase in the capacitor voltage accompanying
each cycle of the energy conversion process. Reprinted with permission. Copyright 2013 The American
Association for the Advancement of Science (73)

The percolated network of pH-sensitive CNC in the triggered by transition along the digestive
polymer matrix served as the switching units for tract) (83).
the shape-memory composite, the SME process
of this material is depicted in Figure 8 (81, 82).
Magnetically-Induced Shape-Memory
The CNC serves as the switching unit of the SMP
Polymer
composite within the matrix of PECU which is
physically crosslinked and microphase separated It is possible to generate magnetically-induced
to yield the net points. Such pH-responsive shape- SMEs in a variety of materials (18, 20, 38, 86– 88),
memory nanocomposites have promise in the however a comprehensive overview is outside
design of biomaterials for biomedical applications the scope of this mini-review. The SMP devices
(for example, SMP-based drug delivery systems discussed thus far are being researched with
potential application into wearable electronics,
nanoelectronics (such as actuators), biomaterials
and biomedical devices (1, 18, 19). However, in
some instances (such as medical devices) a key
challenge is the design and implementation of a
safe and effective method of actuating a variety of
device geometries in vivo. As previously discussed,
a pH‑triggered SMP design can be potentially
effective when utilised as drug delivery devices,
when the target environment has a substantial pH
difference (for instance, the digestive system) (83).
Force
pH = 8 However, the development of electrically and
Deformation pH = 4
thermally-triggered devices that safely operate
in vivo is difficult due to the (generally) high
temperatures these SMPs can reach (relative to
Fixity biological systems). For instance, the electroactive
PLA-TPU SMP composite (2 wt% SWCNT and
pH = 4 pH = 8 10 wt% PEG) reaches temperatures greater than
70°C in 80 s as shape-recovery is achieved (68).
Recovery An alternative method of achieving actuation is
inductive heating by loading ferromagnetic particles
into an SMP system and exposing the doped
device to an alternating electromagnetic field (89),
benefiting from the innate thermoregulation offered
by a ferromagnetic material’s Curie temperature
(Tc, at which a ferromagnetic material becomes
paramagnetic, losing its ability to generate heat
via a hysteresis loss mechanism) (90). By using
particle sizes and materials that will heat mainly
via a magnetic hysteresis loss mechanism over
an eddy current mechanism, it is possible to have
PECU matrix in solution Hydrogen bonding an innate thermoregulation mechanism that limits
O
CNC–C6H4NO2 CNC
the maximum achievable temperature to Tc (89).
+ C NH+
Therefore, by selecting ferromagnetic particle
materials with a Tc within safe medical limits,
Fig. 8. Schematic representation of the pH-
responsive shape-memory materials, which rely Curie thermoregulation eliminates the danger of
on hydrogen bonding switching mechanism in the overheating and the need for a feedback system
interactions between cellulose nanocrystals (CNC– to monitor implanted device temperatures (89).
C6H4NO2) within polymer matrix upon immersion However, this technology is not only useful when
in hydrochloric acid solution (pH = 4) or sodium applied to medical devices. Other useful applications
hydroxide solution (pH = 8). Reprinted with
include remote activation in which wires or
permission. Copyright 2015 American Chemical
Society (81) connections to SMP devices could be eliminated,
simplifying the design and reducing possible points

of failure. An example of this method of actuation composites did not exceed temperatures above
involves the incorporation of 10% by volume the respective Ni Zn particle Tc values, signifying
nickel zinc ferrites (for example C2050 (Ceramic a thermoregulation characteristic. In addition, it
Magnetics Inc, USA) and CMD5005 (Ceramic was stated that the 10% volume of Ni Zn particles
Magnetics Inc), particle sizes ca. 50 µm with did not impact the SMPs shape-memory properties
spherical shapes) with an ester-based thermoset significantly (89). The Tg increased from 55°C to
polyurethane (PU) SMP, MP5510 (SMP Technologies 61.4°C and the shape-recovery of a flower and
Inc, Japan) (Tg of 55°C) (89). The magnetic field foam-based device was achieved within 15 s to
utilised to achieve shape-recovery was a copper- 25 s, at a temperature range of 23°C to 78.6°C.
wound solenoid coil with a 2.54 cm diameter, The potential applications for this device are
7.62 cm length and with a total of 7.5 turns. The illustrated in Figure 9. Optimisation of this device/
unit possessed an adjustable power setting capable design is still required before it can be considered
of outputting 27 W to 1500 W at between 10 MHz clinically viable, however, this SMP composite
and 15 MHz frequency (note: this high frequency highlights very interesting characteristics, remote
may induce eddy currents in the tissue, causing activation (via magnetic fields inducing thermally-
undesirable direct heating of the human body in triggered actuation) and thermoregulation (via Tc
medical applications) (91). However, an alternating temperature of the material being employed).
magnetic field of 12.2 MHz and approx. 400 A m–1
(centre of the inductive coil) at room temperature
Shape-Memory Polymer
was used for actuation to demonstrate proof of
Classification
concept for the device. It was also reported that
clinically useable frequencies (50 kHz to 100 kHz) As highlighted above, SMP materials are diverse
(92) should still be effective (89), albeit this and respond to many different external stimuli
could result at a different quantitative level (i.e. (including temperature, light, electricity, water,
shape-recovery and memory performance may pH and electromagnetic fields) by a variety of
be reduced). Furthermore, C2050 and CMD5005 mechanisms. Although SMPs can be classified
possess a Tc of 340°C and 130°C, respectively. based on their composition and structure, stimulus
These temperatures exceed physiological limits and shape-memory function, their classification
and are therefore not practical for medical can be difficult, as organising these polymeric
devices currently, however, these doped SMP smart materials into one or two simple categories
(a)
Fig. 9. SMP devices used to evaluate
feasibility of actuation by inductive
heating: (a) flower shaped device
shown in collapsed and actuated
form; (b) SMP foam device shown
in collapsed and actuated form.
Reproduced with permission.
1.5 mm Copyright 2006 IEEE Transactions on
Biomedical Engineering (89)
(b)
5 mm

is an over-simplification of their abilities and polyesterurethanes (PEU), oligourethane segments

characteristics (93). are the hard-elastic segments, while polyester
SMPs are considered to consist of net points and serves as the switching segment (99).
molecular switches or stimuli sensitive domains.
These net points can be achieved by covalent
Thermoset Shape-Memory Polymers
bonds (chemically crosslinked) or intermolecular
interactions (physically crosslinked). Chemically For chemically crosslinked SMPs, two methods
crosslinked SMPs involve suitable crosslinking are commonly used to synthesise covalently
chemistry and are referred to as thermosets (94, crosslinked networks (36, 41). The first method
95). Physically crosslinked SMPs involve a polymer relies on addition of a multi-functional crosslinker
morphology consisting of at least two segregated during polymerisation (41), whereas the second
domains and are referred to as thermoplastics (96). method relies on the subsequent crosslinking of
The network chains of the SMP can be either a linear or branched polymer (36). The networks
amorphous or crystalline and therefore, the Ttrans are formed based on many different polymer
is either a Tg or Tm. The network architectures are backbones. Covalently crosslinked SMPs possess
thought to be constructed through crosslinking net chemically interconnected structures determining
points, with polymer segments connecting adjacent the original macroscopic shape. The switching
net points. The strongly crosslinked architectures segments of these materials are generally the
ensure the polymer can maintain a stable shape network chains between net points, and a Ttrans
on the macroscopic level (93). Thermoplastic of the polymer segments is used as the shape-
polymers exhibit a more reversible nature (97), memory switch. The chemical, thermal, mechanical
meaning the physical crosslinked net points can and shape-memory properties are determined
be disrupted and reformed with relative ease. The by the reaction conditions, curing times, the
interconnection of the individual polymer chains type and length of the network chains and the
in a physically crosslinked network is achieved by crosslinking density (35). Comparing physically
the formation of crystalline or glassy phases. For crosslinked SMPs with chemically crosslinked
thermoset polymers, the individual polymer chains SMPs, the chemically crosslinked SMPs often show
are connected by covalent bonds and are therefore less creep, thus, any irreversible deformation of
more stable than physically crosslinking networks the polymer during shape recovery is less. This is
and show an irreversible nature (98–100). because covalent crosslinked networks are more
Regarding thermo-responsive SMPs, they can stable than physical crosslinked networks. As a
be classified according to the nature of their result, chemically crosslinked SMPs usually show
permanent net points and the Ttrans related to the better chemical, thermal, mechanical and shape-
switching domains into four different categories: (a) memory properties than physically crosslinked
physically crosslinked thermoplastics, Ttrans = Tg; SMPs (96). For example, the shape recovery ratio
(b) physically crosslinked thermoplastics, of thermoplastic SMPU is usually in the range of
Ttrans = Tm; (c) chemically crosslinked amorphous 90% to 95% within 20 shape recovery cycles,
polymers, Ttrans = Tg; (d) chemically crosslinked and the elastic modulus is between 0.5 GPa and
semi-crystalline polymer networks Ttrans = Tm (93). 2.5 GPa at room temperature (26). Additionally,
when exposed to air, it is sensitive to moisture
and therefore possesses unstable mechanical
Thermoplastic Shape-Memory
properties. In contrast, an epoxy SMP shows
Polymers
better overall performance as a shape-memory
For the physically crosslinked SMPs, the formation material. The shape recovery ratio typically
of a phase-segregated morphology is the reaches 98–100%, the elastic modulus between
fundamental mechanism behind the thermally- 2 GPa and 4.5 GPa, and it is generally stable in
induced SME of these materials (93, 99). One the presence of moisture (26). Thermoplastic SMPs
phase provides the physical crosslinks while the (such as SMPU) are mostly researched and used
other acts as a molecular switch. They can be as functional materials at a small scale, such as
further classified into linear polymers, branched for biomaterials (30, 97). However, thermosetting
polymers or a polymer complex. Linear SMPs may SMPs (for example styrene-based SMP (SSMP)
consist of block copolymers and high molecular and epoxy SMPs) are generally used for structural
weight polymers, the typical physically crosslinked materials, such as space deployable structures and
SMP is linear block copolymers, such as PU. In automobile actuators (97, 98).

Shape-Memory Functionality Figure 10, and an integrated insight into the

classification of SMPs is shown in Figure 11.
The approaches to designing different shape- An example of a selective triple shape
memory functions become more abundant as multicomposite SMP was documented to incorporate a
scientists and engineers better understand the SME neat SSMP (112) and two SSMP composites (113).
mechanism of SMPs. For instance, discussed thus One incorporated iron(II, III) oxide nanoparticles
far are examples of SMPs with polymeric blends, while the other CNT nanoparticles. This unique
addition of crosslinking species, incorporation of SMP composite successfully possessed three
electroactive and ferromagnetic substances. All different regions within the sample: neat SSMP,
of which enhances an SMP device functionality, SSMP-Fe3O4 and SSMP-CNT. Because of this, the
enabling unique and interesting characteristics material also possessed distinct shape-memory
which can be tailored to a plethora of applications capabilities with different triggers. For instance,
(for example, self-healing and wearable the material was documented undergoing a three-
electronics, drug delivery and implantable medical step shape-memory recovery process, subjected
devices) (101–110). Further still, one-way SMEs, to an alternating magnetic field of 30 kHz, a radio
two-way SMEs (such as dual shape PPy-PEE, frequency (RF) field of 13.56 MHz and direct oven
discussed previously), triple SMEs, multiple SMEs heating at 130°C (113). Furthermore, the Rf and Rr
and even temperature-memory effects (TMEs) for the original shape to the first temporary shape
have been widely investigated in SMPs (34). As the (and back to the original shape) was reported at
types of SMP materials increasingly diversify, two 93% and 93%, respectively. Meanwhile, the Rf
and even three different types of shape-memory and Rr for the first temporary shape to the second
functions can be achieved simultaneously in the temporary shape (and back to the first temporary
same SMP material (34, 111). These types of shape) was at 95% and 99%, respectively (113).
materials can usually be achieved when combining The SME mechanism for this multicomposite is
different SMPs possessing different properties. represented in Figure 12 and it was concluded that
A schematic of one-way, two-way, dual shape this unique material has promising characteristics
and triple shape functionality SMPs is shown in to be used in biomimetic materials. Examples of
One-way SME Two-way SME
>Ttrans >Ttrans
Dual shape >Ttrans <Ttrans

Original Original
shape shape
Deformed Deformed
shape shape
maintained
>Ttrans1 >Ttrans2 >Ttrans1 >Ttrans2
Triple shape >Ttrans1 >Ttrans2 <Ttrans1 <Ttrans2
Original Deformed Original Deformed

shape shape1 shape shape1 Deformed
Deformed
maintained shape2
shape2
maintained
Fig. 10. The varying shape-memory functionality of SMPs

Composition and structure Stimulus Shape-memory function type
Block copolymer
TEMPERATURE
ONE-WAY SME
ELECTRICITY Thermo-
Supramolecular polymer sensitive
TWO-WAY SME
MAGNETIC
Polymer blend/composites
Water- TRIPLE SHAPE SME

WATER SENSITIVE
sensitive
e.g. PPy-PEE
discussed in
Figure 7
Cross-linked homopolymer
OXIDATION- Redox-
MULTI-SHAPE SME
REDUCTION sensitive
Polymer IPN/semi-IPN
Light-
LIGHT/RADIATION sensitive MULTI-FUNCTIONALITY
Fig. 11. The classification of SMPs based on composition and structure, stimulus triggers and the possible
type of shape-memory functions
applications of SMP-based materials and their SMPs by imparting new functional characteristics,
composites are highlighted in Table I. broadening the potential applications of these
materials and enabling a multipurpose material.
SMPs and their composites are capable of
Conclusion
industrially important applications (examples of
As the understanding of SMPs continually develops which include: self-healing (101–104), generators
among the academic and industrial communities, driven by water gradients (73), sensors (72), task-
the generation of new and potentially innovative specific medical devices (18, 105) and wearable
SMPs will be more rapid while we realise the full electronics (106–110), a few examples of which
potential of these materials. SMPs are one of the are highlighted in Table I. The literature published
most interesting of polymer classes within the field to date de-risks investment from governments and
of functional polymers. In addition, SMP composites industry to raise the technology readiness levels
can enhance the already impressive capabilities of towards products on the market.

a1 b1 c1 a2 b2 c2 Deformed a1 b1 c1 a2 b2 c2
at 130C
Fixed at
20C Temporary shape A
Original shape
Alternating magnetic
field heating of 30 kHz
a1
c1 a2 a1
c2 a2
b1 RF field heating
b2 b1 c1 b2 c2
13.56 MHz
Temporary shape C
Temporary shape B
Oven heating
130C
a b
SSMP-Fe3O4 Neat SSMP

a1 b1 c1 a2 c2
b2
c
SSMP-CNT an, bn, cn
Recovered shape
Fig. 12. Schematic of the selective shape recoveries of the multicomposite SSMP induced by alternating
magnetic field heating, RF field heating and oven heating, respectively (an, bn and cn stand for the n sections
of SSMP–Fe3O4, neat SSMP and SSMP–CNT, respectively). Reproduced by permission of The Royal Society of
Chemistry. Copyright 2015 The Royal Society of Chemistry (113)
Table I Examples of Applications of SMP-Based Materials and Their Composites

Application References
Actuators (for example, for generators) (73)
Biomedical devices (such as drug delivery systems, expanding foam and endovascular
(44, 83, 89)
thrombectomy device)
Multipurpose/multifunctionality (for example, self-healing, biocompatible, body
(44, 113)
temperature actuation and selective triple shape-memory)
Thermoregulators (89, 90)
Wearable electronics (65, 68)
Acknowledgements research aligned with some of the topics discussed

in this review, specifically the Glycoscience Tools
We acknowledge the Faculty of Science and for Biotechnology and Bioenergy (IBCarb) Network
Technology, Lancaster University, UK, for an in Industrial Biotechnology and Bioenergy (NIBB,
Early Career Internal Grant and The Royal BB/L013762/1); the BBSRC FoodWasteNet NIBB
Society, UK, for a Research Grant (RG160449). (BB/L0137971/1), the BBSRC From Plants to
We acknowledge the Engineering and Physical Products (P2P) NIBB (BB/L013819/1) and the
Sciences Research Council (EPSRC), UK, for Lignocellulosic Biorefinery Network (LBNet) NIBB
an EPSRC First Grant (EP/R003823/1) and (BB/L013738/1).
a Pathfinder Grant from the EPSRC Centre
for Innovative Manufacturing in Large Area
Electronics (EP/K03099X/1). We also thank the
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The Authors
Mathew John Haskew received his BSc in Chemistry from Lancaster University, UK, and
subsequently undertook an MSc by research on SMP-based materials (with John Hardy at
Lancaster University). He is currently undertaking a PhD in Engineering with Samuel Murphy
and John Hardy at Lancaster University. His PhD involves the development of biodegradable
biomaterials, and a combination of computational modelling and experimental validation
of their efficacy.
John George Hardy received his MSci and PhD in Chemistry from the University of Bristol, UK,
and the University of York, UK, respectively. Thereafter he undertook postdoctoral research
in Biochemistry, Biomedical Engineering, Materials Science and Pharmacy (in France,
Germany, Northern Ireland and the USA) before returning to the UK to lead a research
group developing stimuli-responsive materials for technical and medical applications.

Application of Chitosan-Encapsulated Orange

Oil onto Footwear Insock Leathers
Spray drying technique for an environmentally sustainable antibacterial
formulation
Buket Yılmaz leathers acquiring antibacterial properties. The

Department of Materials Science and products and process are biodegradable, nontoxic
Engineering, Graduate School of Natural and biocompatible.
and Applied Science, Ege University, 35100
Bornova-Izmir, Turkey
1. Introduction
Hüseyin Ata Karavana* Footwear is the most commonly worn apparel in
Department of Leather Engineering, Faculty of daily life, and its design features must prioritise
Engineering, Ege University, 35100 Bornova- anatomy, comfort and hygiene. For this reason, it
Izmir, Turkey; Department of Materials Science is important to develop sustainable improvements
and Engineering, Graduate School of Natural to footwear’s functional properties.
and Applied Science, Ege University, 35100 Footwear that carries the body’s weight during
Bornova-Izmir, Turkey the day can affect foot health physically, chemically
and microbiologically. Continuous contact with
*Email: huseyin.ata.karavana@ege.edu.tr; the external environment exposes footwear to
atakaravana@gmail.com microorganisms during normal use. All sorts of
footwear play a role in the transport, spread and
contamination of pathogenic or non-pathogenic
The purpose of this study was to devise an antibacterial microorganisms (1).
treatment for footwear insock leathers. Orange oil- There are different microorganisms in every part
loaded chitosan microparticles were utilised for of the human body. Sweat is regularly secreted
this purpose. Emulsion formulations with different from the body under normal conditions. It contains
ratios were prepared, and from these formulations 98% water and urea, uric acid, fatty acid, lactic acid
microparticles were manufactured using a spray and sulfates (2). The feet have more sweat glands
drying technique. Microparticles obtained in this way than other parts of the body. Sweat secreted from
were applied to the insock leathers using a finishing feet during the usage of footwear is decomposed
process. Successful encapsulation was confirmed by means of foot microbiota; as a result, bad
by ultraviolet-visible (UV-vis) spectrophotometry, odours emerge in footwear. Brevibacterium linens,
Fourier transform infrared (FTIR) spectroscopy and Staphylococcus epidermidis, Staphylococcus aureus
scanning electron microscopy (SEM) techniques. The and Escherichia coli are some microorganisms
microparticles exhibited highly spheroid shape with that make footwear unhygienic. As a result of the
a size range of 3–5 µm. Microparticle encapsulation breakdown of amino acids in sweat and skin by
efficiencies ranged from 79.41% ± 3.36% to 86.60% these microorganisms, bad odour arises in feet,
± 1.13%. After performing microbiological tests socks and footwear (3, 4).
and in vitro release studies on the insock leathers, Nowadays, in addition to individual foot care
it was determined that the prepared microparticles and hygiene for odour prevention in shoes,
are able to perform core material delivery. Also, commercial materials with various deodorising
successful microparticle application resulted in these and antimicrobial effects are also employed (5).

Footwear insock is a thin layer of materials put into Analytical grade chemicals were used in the
the shoe after manufacture to cover the insole. It analyses. Insock leathers, without dye and ready
directly contacts the sole of the wearer’s foot and for experimental application, were donated from
can provide a more sanitary environment when Ata Dilek Leather (Izmir, Turkey).
specially treated for antimicrobial purposes (6). For the microparticle production, chitosan (shell
Spray drying is an advantageous way to encapsulate material) was added to 1% w/w aqueous acetic
active substances and essential oils. Spray drying acid for preparing the chitosan solution. This
is a common and accepted encapsulation method solution was stirred at 45°C by using a magnetic
for industrial applications. With this method, it is stirrer until wholly dissolved. During the pre-
possible to mass produce capsules. The distribution emulsion preparation, orange oil (core material)
of particles is uniform (7–10). was gradually mixed into the chitosan solution and
Microencapsulation technology has been used for stirred for 1 min at 10,000 rpm. The surfactants
the application of orange oil to textiles and leathers, as compound emulsifiers used for pre-emulsion
being an economically viable, fast and efficient preparations were Tween 40 with Span 20 at
method by combining core and shell materials, the ratio of 8:2 w/w. Then, the microparticles
desirable perceptual and functional characteristics, were prepared by using an SD Basic spray dryer
and also allowing functional substances to be (Labplant, UK) with nozzle diameter of 0.5 mm.
released in a controlled manner. This technique The orange oil to chitosan ratios in the four
has also been used to microencapsulate a wide encapsulating compounds came to 1:1, 1:1.33,
range of active, functional, sensitive or volatile 1:1.67 and 1:2 w/w. The ingredients of the
substances (11–14). Tea tree oil containing formulations in the spray-drying process are shown
melamine formaldehyde microcapsules, essential in Table I. Homogeneous emulsions were fed to
oils (eucalyptus, lavender or oregano), polyurethane the spray dryer under the following conditions:
dispersions containing photoactive antimicrobial pump speed 12 ml min–1, outlet air temperature
agents, zinc oxide and silver nanoparticles are 114°C and inlet air temperature 175°C.
some substances that protect upper leathers from The microparticles’ morphology was examined by
the harmful propagation of microorganisms (3, 15). a Quanta 250 FEG scanning electron microscope
In addition, aromas confined to microcapsules are (FEI, USA) at 2 kV accelerating voltage. Before
also used to prevent bad odours in footwear (16, coating in an argon atmosphere with gold-palladium
17). Application of antibacterial and aromatic by a K550X sputter coating machine (Quorum
materials onto footwear insocks to control bad Emitech, UK), the samples were mounted onto an
odours is good for foot hygiene and desired shoe aluminium stub. The grain side of leathers coated
comfort. with microparticles was examined by a TM1000
The use of orange oil presents as an ecological tabletop scanning electron microscope (Hitachi,
alternative to synthetic chemicals, attracting Japan) after coating with gold-palladium.
the attention of the scientific community to the The FTIR spectra of the spray-dried microparticles
development of eco-friendly antimicrobials. In this and leathers with microparticles were procured by
study, microparticles were produced by a spray a Spectrum 100 FTIR attenuated total reflectance
drying method after the emulsions with orange (ATR) spectrometer (PerkinElmer, USA). The
oil and chitosan were prepared in different ratios. measurements were made using four scans with a
Microparticles manufactured in this way were then resolution of 4 cm–1 between 4000 cm–1 to 650 cm–1
transferred to the surface of the footwear insock wavenumber ranges at room temperature.
leathers using a finishing process. Afterward, some Encapsulation efficiencies of microparticles were
tests and analyses were performed on microparticle calculated as the amount of orange oil (core
coated footwear insock leather samples to material) encapsulated in the microparticles. The
evaluate the effectiveness of the microparticles,
their presence on the leather surface and their Table I Composition of the Formulations
antimicrobial properties. Formulation code Orange oil:chitosan,
w/w
2. Experiment T3 1:1
T4 1:1.33
Pharmaceutical grade cold pressed orange oil
T5 1:1.67
was donated from Ephesus, Turkey. Chitosan
T6 1:2
was purchased from Acros OrganicsTM (Belgium).

encapsulation efficiency was calculated using Table II Basic Finishing Recipe Applied to
Equation (i) (18). Insock Leathers
Materials Amount, part Practice
B (total amount of oil
content – the amount of Water 100 3 × Spray
surface oil content) Anionic wax 50
%EE = × 100 (i)
A (total amount of oil Non-ionic aliphatic
content) polyurethane 25
binder
A solvent extraction method was used to determine Orange oil loaded
12
total oil content. A 0.1 g measurement of orange oil microparticles
loaded microparticles was dissolved in 10 ml of 1%
acetic acid solution at room temperature for 45 min. were placed into an incubator for incubation at
Released orange oil which was obtained from the 37°C for 18 h in the Mueller Hinton broth (MHB)
completely dissolved microparticles was placed in medium. Then, microorganisms were inoculated
a beaker containing 50 ml n-hexane for extraction in petri dishes containing 105 colony forming unit
45 min. So as to determine the total amount of (CFU) ml–1 of Mueller Hinton agar (MHA) medium.
orange oil in the microparticles, this extract was Next, microparticle coated insock leather samples
filtered through a syringe filter (0.22 μm). Orange with 12.7 mm diameter were placed into the petri
oil content in the filtrate was measured using a UV- dishes (20, 25). All petri dishes were placed into
1800 UV-vis spectrophotometer (Shimadzu, Japan) an incubator for incubation at 37°C for 24 h, and
at 202 nm in triplicate. Surface oil content was also inhibition zones were measured to determine
determined by the same solvent extraction method antibacterial activity.
described above, except for a dissolving process in
1% acetic acid solution (11).
In vitro release studies of microparticles and
microparticle loaded leathers were carried out at In this study, orange oil microparticles were
a speed of 100 rpm in phosphate-buffered saline successfully prepared by spray drying method.
(PBS) and methanol at 37°C. 1 mg orange oil This method is a simple, viable method to obtain
loaded microparticles was suspended in beakers microparticles, suitable to prevent active substance
containing 4 ml methanol and 16 ml of PBS. Insock biological activity loss, avoiding exposure to
leathers with 2.5 cm2 area were placed in beakers elevated heating and to organic solvents.
containing 16 ml methanol and 16 ml of PBS for
in vitro release studies of microparticle loaded
3.1 Surface Appearance of
leathers. At suitable time intervals, the medium
Microparticles and Microparticle
in the beakers was filtered through a 0.22 μm
Coated Insock Leathers
syringe filter. Sink conditions were maintained in
the receptor compartment during in vitro release A scanning electron microscope was used to
studies. The released amount of orange oil was examine the morphology of the spray-dried
analysed by UV method, as previously described, microparticles. SEM micrographs revealed that all
for 5 h. Experiments were performed five times. microparticle formulations have a highly spheroid
A spraying pistol with nozzle diameter of 0.5 mm shape with a morphology approximating an orange
was used to apply microparticles to the insock peel effect. Microparticles of non-uniform size
leathers during the finishing process. Spray-dried were observed with clear distinction between shell
microparticles were added to the finishing recipe and core materials. These shape features indicate
as 20 g m–2 (19). The basic finishing recipe for the that orange oil is spread on the surface of the
insock leathers is given in Table II (20). microparticles. The morphology of spray-dried
The efficacy of microparticle coated insock leathers microparticle formulations is shown in Figure 1.
against test microorganisms Staphylococcus Particle morphology (surface, size and distribution)
aureus ATCC® 6538TM, Escherichia coli ATCC® was not affected by the polymer ratio or core:shell
25922TM, Candida albicans ATCC® 10231TM, ratio. It is observed that there was formation of
Klebsiella pneumoniae ATCC® 4352TM and Bacillus microcapsules, but they have stuck one to another
subtilis ATCC® 6633TM was examined by agar disc and an agglomerate of microcapsules occurred.
diffusion method (21–24). Test microorganisms Microparticles with similar morphology were also

3.2 Fourier Transform Infrared

(a) (b) Spectroscopy Studies
3.780 μm
Interactivity between the core material and shell
material usually leads to characteristic alterations in
2 μm 10 μm
the FTIR spectra. FTIR spectra of chitosan, orange
oil, microparticles and insock leather samples are
shown in Figure 3 and Figure 4. Characteristic
(c) (d) peaks at 1029 cm–1, 1149 cm–1, 1373 cm–1,
1419 cm–1, 1585 cm–1, 2867 cm–1 and 3362 cm–1
3.173 μm
were demonstrated in the FTIR spectrum of chitosan

(Figure 3). The peak at 3362 cm–1 (OH and NH2
2 μm 10 μm stretching) was attributed to the amino group of
chitosan. An intense absorption peak was seen at
2867–2922 cm–1 owing to C–H stretching in all
(e) (f)
spectra. The peak at 1585 cm–1 was attributed to
N–H bending of the NH3+ functional group present
4.523 μm
in the chitosan (28, 29). The peak at 1373 cm–1

confirmed the presence of an amide III band in the
2 μm 10 μm chitosan. The C–O–C stretching resulted from the
spectra at 1149 cm–1 and 1029 cm–1. The spectrum
(g) (h) at 660 cm–1 was attributed to stretching vibration
of pyranoside ring (30–34).
5.330 μm
The FTIR spectrum of the orange oil showed

the distinctive bands of D-limonene, which is the
primary constituent in orange oil (Figure 3).
2 μm 10 μm
Especially, the bands between 2919–2834 cm–1
were attributed to the C–H stretching vibrations in
Fig. 1. SEM micrographs of microparticle formulations:
(a) T3 formulation, 50,000 × magnification; –CH–, –CH2– and –CH3. The spectrum at 2965 cm–1
(b) T3 formulation, 10,000 × magnification; (c) was attributed to the stretching vibrations of =C–H.
T4 formulation, 50,000 × magnification; (d) The band 1644 cm–1 was attributed to the stretching
T4 formulation, 10,000 × magnification; (e) vibrations of C=C. The band seen at 1435 cm–1
T5 formulation, 50,000 × magnification; (f) T5 was attributed to the C–H bending vibrations in
formulation, 10,000 × magnification; (g) T6
–CH–, –CH2– and –CH3. The peaks at 885 cm–1 and
formulation, 50,000 × magnification; (h) T6
formulation, 10,000 × magnification 797 cm–1 were attributed to the bending vibrations
(out of plane) in =CH2 and =C(R)–H, respectively.
The band at 1376 cm–1 was also attributed to the
C–H bending vibrations in –CH3 (mostly used to
obtained in other spray drying experiments carried describe the existence of methyl) (35, 36).
out using natural polymeric mixtures (11, 26, 27). As seen in Figure 3, most bands in the FTIR spectra
We also examined the surface appearance of of the microparticles belonged to chitosan, which
microparticle-free and microparticle-coated insock indicated that orange oil droplets were trapped in
leather samples. The different microparticle chitosan (shell material) and that distinctive band
formulations were clearly observed on insock of orange oil vanished or declined. Evidently, the
leather surfaces after successful application of the free vibrations of orange oil molecules were blocked
finishing process. Micrographs of insock leather by the chitosan because of physical interactions
surfaces are shown in Figure 2. such as van der Waals or electrostatic interaction.
After the finishing process, the presence of Furthermore, the intensity of microparticle peaks
microparticles on the insock leather can be seen on the FTIR spectrum was lower than that of
very clearly for all formulations. The images indicate chitosan because of the interaction between orange
that the fixation was successfully achieved. Hence, oil and chitosan. The FTIR spectra of microparticles
the leather samples preserve the capsule content demonstrated the C–H bending vibrations of
even after the finishing process. –CH3 at 1376 cm–1, except the =C(R)–H bending

(a) (b) (c)
30 μm 100 μm 30 μm
(d) (e) Fig. 2. SEM micrographs

of the insock leather
after finishing process:
(a) microparticle free;
(b) T3 formulation;
(c) T4 formulation; (d)
100 μm 100 μm T5 formulation; (e) T6
formulation
Chitosan 1029.40 Fig. 3. The FTIR

3362.83 2867.87 1585.84 1419.87
1373.85 1149.73
spectrum of the chitosan,
892.61 660.57 orange oil and four
Orange oil different microparticle
1147.91 1016.92 758.92
2965.87 2834.88 formulations (T3, T4, T5
1644.85 1376.86 1051.92 914.86
2919.81 1435.80 956.93 797.80 and T6)
T3
885.61
3295.79 2922.80 1556.77
1511.79 1151.73 888.72
T, %
1407.75
T4 1067.53
1028.53
1376.76
3283.79 2874.81 1557.79
1510.80 1151.72 887.69 752.71
T5
1028.49
3288.76 2876.79 1557.76
1510.79 1151.70
1408.74 888.66
T6 1063.46 888.69
1027.45
2875.81 1593.82 1376.79
3351.78 1509.83 1151.72
4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650
cm–1
vibration at 797 cm–1, which was presumably due formulations were determined as 79.41% ± 3.36%,
to the fact of the D-limonene ring being covered 81.28% ± 1.69%, 83.56% ± 0.66% and
with chitosan (14, 36). 86.60% ± 1.13%, respectively. A great deal of
Figure 4 show that bands between encapsulated orange oil is preferred. These results
–1
1535–1547 cm attributed to the NH band of showed that the microparticles’ encapsulation
chitosan, did not appear in the blank leather efficiency is affected by the core:shell ratio. Increasing
sample (34). Similarly, it was determined that IR the chitosan weight resulted in more encapsulated
band vibration at 1095 cm–1 was observed in the orange oil, i.e. high encapsulation efficiency. This is
microparticle loaded leathers but absent from the an effect similar to the oil:polymer ratio given by Li
blank leather. That was evidence of the presence of and associates in their 2013 study (11).
terpenoid, a component in orange oil (37).
3.4 In Vitro Release Studies of

3.3 Encapsulation Efficiency Microparticles and Microparticle
Coated Insock Leathers
Orange oil loaded microparticles were produced
with a high orange oil encapsulation efficiency. Figure 5 shows the in vitro release behaviours
The encapsulation efficiency of T3, T4, T5 and T6 of orange oil released from microparticles in four

Blank leather Fig. 4. The FTIR

3309.92
1643.83 1379.80
852.85 spectrum of footwear
720.79
2849.72 1728.46 1552.83
1464.76 1237.63
1024.72 insock leather without
2917.62
1159.51 microparticles (blank
T3
leather) and with four
3285.90
2850.79 1644.83
1541.81 1379.80 1241.78
different microparticles
2916.72 1470.81
1155.66
formulations (T3, T4, T5
1730.66
T4 1024.55
and T6)
3309.93
T, %
1644.80 1380.81 851.84

2850.76 1728.54 1547.81 717.78
1464.78 1095.68
2916.66 1237.66 1024.67
T5 1159.59
3307.93
1644.81 1380.79 851.83
2850.77 1728.49 1544.80 718.78
2916.68 1464.78 1095.66
1237.64 1024.65
T6 1158.55
1023.64
3309.94 1236.61 1095.65
2850.82 1535.83 1157.52 851.83
2917.74 1463.79 732.78
1380.80
4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650
cm–1
different formulations. The quantity of released 90

orange oil was measured at 202 nm in PBS at 80
different times. Previous experiments used PBS 70
as an in vitro release and diffusion medium for 60

50 T3
topical applications (38, 39). Oil release from
C, %
40 T4
microparticulate systems occurs via different
30 T5
mechanisms including diffusion, desorption, 20 T6
disintegration and surface erosion (40). 10
The typical release pattern of the spray dried
0 30 60 90 120 150 180 210 240 270 300
microparticles is characterised by a small initial
Time, min
burst release and a sustained release rate following
Fig. 5. In vitro release of orange oil loaded
that. It can be seen that orange oil release from
microparticles
microparticles gradually increased over time with
exposure to PBS, which indicates that the orange
oil disintegrated swiftly in PBS. This circumstance 60
is presumably owed to the fact that PBS is slightly
50
alkaline; chitosan is inclined to dissolve in slightly
alkaline solution. Nonetheless, it can be seen that 40
the release rate was not affected by chitosan T3

C, %
30
T4
concentration in the formulations. Figure 5
20 T5
graphs release behaviour as a function of orange
T6
oil concentration, which was independent from 10
chitosan concentration.
0 500 1000 1500
The in vitro release results of the leathers
Time, min
impregnated with orange oil loaded microparticles
in pH 7.4 PBS at 37°C are presented in Figure 6. Fig. 6. In vitro release of microparticle-coated
This line graph shows controlled release behaviour insock leathers
from leather treated with all formulations.
Orange oil trapped inside the microparticles
caused sustained release up to 24 h. When the 3.5 Microbiologic Studies on
formulations are compared to each other, we Microparticle Coated Insock
see the oil release ratio of insock leathers was Leathers
affected by polymer concentration. High polymer
concentration caused a slow release ratio of Table III shows microbiologic test results of
orange oil. insock leathers treated with four microparticle

Table III Microbiologic Test Results of the Microparticle-Loaded Insock Leathers

Candida Escherichia Klebsiella Staphylococcus
Formulation Bacillus subtilis
albicans ATCC® coli ATCC® pneumoniae aureus ATCC®
codes ATCC® 6633TM
10231TM 25922TM ATCC® 4352TM 6538TM
T3
T4
T5
T6
formulations. An important revelation is that the 4. Conclusion

test microorganisms did not grow on these leather
samples. However, in some test groups, a meagre During the usage of footwear, perspiration and
antimicrobial inhibition zone around the insock bacterial activity negatively impact foot health and
leather samples meant that orange oil diffusion generate bad odours from both the feet and footwear.
did not occur. There is a visible zone on Candida Shoe production using natural and non-toxic materials
albicans in all formulations. T3 and T4 formulations, that prevent or inhibit bad odours and bacterial activity
whose orange oil releases are higher in 24 h, is one solution to this hygienic problem. Likewise, the
look more effective against Escherichia coli. The successful application of microparticles that release for
antimicrobial activity is dependent on chitosan’s a long time on footwear insock leather is an important
inherent behaviour and orange oil present on alternative to existing toxic products.
leather samples. When the inhibition zones in the Our research found that emulsions with orange
T6 formulation are examined, it can be seen that oil and chitosan have natural antibacterial activity.
orange oil found in the insock leather samples These emulsions, when successfully converted into
is more effective than the natural behaviour of encapsulated powders by a spray drying method,
chitosan on antimicrobial activity. The antimicrobial produce a core-shell material. SEM images showed
effect can be considered as proliferation or non- how an effective finishing process was used to
proliferation in the area under the insock leather apply laboratory produced microparticles to the
samples. This effect is also expressed as contact surface of footwear insock leather. Microbiological
inhibition. No proliferation was observed on the tests performed on microparticle coated leathers
contact surface of the insock leathers, that is, proved that footwear insock leathers were fortified
on the surface where it touches the medium and with antibacterial properties.
microorganism. Also, any proliferation on the These findings demonstrate that application of
surfaces or edges of insock leather samples was orange oil-chitosan microparticles onto footwear
not observed. There was no difference between insock leather surfaces is an alternative natural
leather formulations on the antimicrobial test. method to control hygiene and eliminate bad

odours. Non-toxic, functional, leather shoes J. Scapinello, C. N. Nesi, J. Dal Magro and
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The Authors
Buket Yılmaz graduated from Chemical Engineering, Faculty of Engineering, Anadolu
University, Turkey, in 2015. For a period during her undergraduate education, she benefited
from the FARABİ exchange programme for further chemical engineering studies at Ege
University. Yılmaz won and completed a competitive internship at Turkey’s two leading
companies involved in polymers and food production. In 2016, she started her master’s
degree at Ege University’s Institute of Science, Materials Science and Engineering. Her
scientific expertise has also been employed by the private sector in sales and in the quality
control unit of a food production enterprise.
Hüseyin Ata Karavana graduated from the Leather Technology Department, Faculty of
Agriculture, Ege University, Turkey. He earned his MSc degree in Leather Technology in
2001 from that institution’s Graduate School of Natural and Applied Science. From 2006
to 2007 he continued his studies as an Erasmus student in the Department of Footwear
Engineering and Hygiene at the Tomas Bata University’s Faculty of Technology (Zlin,
Czech Republic). Karavana completed his PhD degree in Leather Engineering at Ege
University in 2008. Karavana currently serves as Associate Professor in the Department
of Leather Engineering at Ege University’s Faculty of Engineering. His research interests
are in all manner of leather and footwear engineering including plastic composites,
microencapsulation, leather quality and control, footwear quality and control.

Bacterial Community Composition in Produced

Water of Diyarbakır Oil Fields in Turkey
Bacterial communities in produced waters of south-eastern Turkey reported in
detail for the first time
Tuğçe Tüccar* Among the classified bacteria, Proteobacteria

Department of Biology, Institute of Graduate (29.2%), Firmicutes (8.3%), Bacteroidetes (8.3%)
Studies in Sciences, Istanbul University, 34134, and Actinobacteria (4.2%) groups were identified.
Vezneciler, Istanbul, Turkey Pseudomonas was the dominant genus detected
in the produced water samples. The results of
Esra Ilhan-Sungur this research provide, for the first time, insight
Department of Biology, Faculty of Science, into the complexity of microbial communities in
Istanbul University, 34134, Vezneciler, Istanbul, the Diyarbakır oil reservoirs and their dominant
Turkey constituents.
Gerard Muyzer 1. Introduction

Department of Biotechnology, Delft University
of Technology, van der Maasweg 9, 2629 HZ Although much progress has been made in the use
Delft, The Netherlands; Microbial Systems of renewable energy in recent years, fossil fuels
Ecology, Department of Freshwater and Marine (especially oil and gas) still meet most of the global
Microbiology, Institute for Biodiversity and energy demand, and they will continue to be the
Ecosystem Dynamics, University of Amsterdam, dominant source of energy worldwide over the
PO Box 94240, 1090 GE, Amsterdam, The next few decades (1).
Netherlands Petroleum is a naturally occurring material found
in various geological formations (reservoirs)
*Email: tugce.tuccar1@gmail.com worldwide. Crude oil, the liquid part of petroleum, is
primarily composed of hydrocarbons (2). However,
it may also include compounds of nitrogen, sulfur,
Oil fields harbour a wide variety of microorganisms oxygen and metals (3). Because crude oil in
with different metabolic capabilities. To examine reservoirs is found as a mixture containing varying
the microbial ecology of petroleum reservoirs, a constituents and proportions, each crude oil has
molecular-based approach was used to assess the its own unique properties. The most important
composition of bacterial communities in produced specified properties are density and sulfur
water of Diyarbakır oil fields in Turkey. Denaturing content (4). The density of crude oil is reported
gradient gel electrophoresis (DGGE) of polymerase in terms of American Petroleum Institute (API)
chain reaction (PCR)-amplified 16S rRNA gene gravity (specific gravity). Based on the API gravity,
fragments was performed to characterise the crude oils can be classified into light, medium,
bacterial community structure of produced water heavy and extra heavy oils (3). Depending on the
samples and to identify predominant community amount of sulfur content (elemental sulfur or sulfur
members after sequencing of separated DGGE compounds such as hydrogen sulfide), the crude
bands. The majority of bacterial sequences oil is categorised as ‘sweet’ or ‘sour’. In addition
retrieved from DGGE analysis of produced water to chemical composition and physical properties,
samples belonged to unclassified bacteria (50%). crude oil typically is also identified by underground

reservoir (4). Reservoir characteristics (depth, reservoir souring and microbial corrosion. Reservoir
temperature, pressure and other factors) vary souring, which is characterised by an increase
significantly from one location to another, even in in production of H2S in the reservoir fluids, most
the same geologic formation (5, 6). The fact that commonly occurs when sulfidogenic microorganisms
microbial community composition and reservoir reduce sulfate to sulfide, a toxic and corrosive
conditions vary dramatically not only between product (20). Undesirable accumulation of sulfide
the different geographical areas, but also among minerals in reservoirs is one of the major challenging
different oil fields in the same region, makes each problems in oil production because it causes plugging
oil reservoir ecosystem unique. of reservoirs, decreasing the oil quality and value and
Despite the extreme environmental conditions increasing the refining costs. Moreover, exposure to
in the oil-bearing formations (i.e. anoxic, high H2S can be dangerous in terms of worker health
temperature, high salinity), many microorganisms and safety due to its high toxicity. Additionally, the
are capable of surviving in the oil and water produced H2S promotes corrosion of the metallic
phases of the oil wells (7, 8). Oil fields harbour equipment and structures used for oil production and
mainly facultative aerobic and strictly anaerobic processing (21). Another destructive phenomenon
microorganisms due to the low redox potential is biocorrosion, which is defined as microbial attack
in the reservoirs (8). These ecosystems contain on the surface of the metal infrastructure leading
different types of microbial communities (such as to disruption of the material (22). In addition to
mesophiles, thermophiles and halophiles) which sulfate-reducing bacteria, which play a major role
adapt to the reservoir conditions (9). Bacterial in biocorrosion, other corrosive microbes, such as
and archaeal groups identified in oil fields include acetogenic bacteria and methanogenic archaea,
sulfate-reducing bacteria (10), sulfur-oxidising are also associated with corrosion failures (23).
bacteria (11), methanogens (12), fermentative Biocorrosion is a great concern because it leads to loss
microorganisms (13), acetogens (14), nitrate of material, large economic losses and safety issues
reducers (15), manganese and iron reducers (16) in the oil industry (24). In contrast, hydrocarbon-
and hydrocarbon degraders (17). Among these degrading bacteria may be used for environmental
microbes, sulfate-reducing bacteria have attracted clean-up processes (6). Bacterial degradation of
much attention due to their detrimental effects hydrocarbons was carried out by both aerobic
such as reservoir souring and biocorrosion (7). In (for example, Rhodococcus sp., Sphingomonas
addition, different members of the oil microbial sp., Pseudomonas putida, Pseudomonas stutzeri,
community are involved in syntrophic interactions. Acinetobacter sp.) and anaerobic bacteria (such
Fermenting bacteria and methanogenic archaea as Fe(III)-reducing bacteria, sulfate-reducing
are involved in methanogenic hydrocarbon bacteria) (6, 17). Furthermore, microbial products
biodegradation through their close syntrophic such as biopolymers and biosurfactants can be
associations (18). This microbial process is used for facilitating oil movement in a widely used
undesirable in oil reservoirs because it causes a technology, known as microbial enhanced oil recovery
decrease in oil quality and value (19). Syntrophic (MEOR) (1). Compared with other conventional oil
microorganisms in oil reservoirs also play important recovery techniques, MEOR has advantages such as
roles in the global biogeochemical cycling of sulfur, low cost, wide application, high efficiency and low
carbon and nitrogen. For instance, sulfate-reducing environmental pollution (25). Therefore, diversity,
bacteria and sulfur-oxidising bacteria, the key metabolic processes and habitat conditions of
drivers in sulfur transformations, are involved microbial communities in oil reservoirs should be
in the sulfur cycle (11). Thus, knowledge of the investigated, so that their negative effects can be
microbial groups and microbial dynamics in oil decreased and their positive effects can be exploited.
fields enable us to obtain detailed insights into the This study aimed to determine the bacterial
microbial ecology of oil associated environments. community composition and to identify the
Understanding the microbial ecology of oil reservoirs predominant community members in produced
is crucial to the petroleum industry because the water from oil fields located in the Diyarbakır
success of oilfield operations is strongly influenced region in Turkey. To this end, we used PCR-DGGE
by the activity of microorganisms. Oil microbes to analyse 20 produced water samples from the
with different metabolic capabilities have significant Diyarbakır region. There are limited studies on
negative and positive impacts on the petroleum produced water from the Diyarbakır region and this
resources and the extraction processes (7). Microbial paper represents the only in situ study available.
activity may lead to severe problems such as The results of this study provide not only new

data about the microbial ecology of the Diyarbakır 2.2 DNA Extraction
oil fields, but also information on the bacterial
populations which may have potential roles in Bacteria in the produced water samples were
terms of increasing or decreasing the efficiency of collected by filtration over 0.20 μm pore size
industrial applications. polyamide filters (Sartolon®, Sartorius AG,
Germany). Genomic DNA was extracted with
the UltraClean® Microbial DNA isolation kit (MO
2. Materials and Methods
BIO Laboratories Inc, USA) according to the
2.1 Sampling Procedure manufacturer’s protocol.
The sampling site, the Diyarbakır region, is located

2.3 Polymerase Chain Reaction
at the boundary of the Anatolian plate and the
Amplification
Middle Eastern oil region in south-eastern Turkey.
A total of 20 crude oil samples (B1, B6, B8, B14, Extracted DNA was used as the template for PCR
B23, B32, B56, GK8, GS6, GS15, M3, K2, K3, amplification of partial 16S rRNA fragments. Primer
K32, K35, K44, S4, S15, Y18 and Y30) consisting pair consisting of 341F with a GC clamp and 907R
of an oil/water mixture were collected from the was used for DGGE analysis (26). A 40-base GC
production wells of Diyarbakır oil fields (Figure 1). clamp was used to prevent complete denaturation
These wells produced oils withdrawn from the of the fragment during DGGE (27).
oil sandstone deposits (depths from 1600 m to Due to the low DNA yield, a two-step PCR strategy
2620 m, API gravity from 24.3° to 42.3°, water was used. At the first step, a real-time PCR
content around 94%, an average pH of 7.0 and (quantitative PCR, qPCR) approach was applied to
salinity from 2966 mg l−1 to 26,961 mg l−1). The the produced water samples. The reaction mixture
samples were aseptically taken at the wellhead and in a final volume of 22.5 µl contained 0.2 µl of
put into sterile 500 ml serum bottles sealed with each primer, 12.5 µl iQTM SYBR® Green Supermix
rubber stoppers and aluminium caps. The samples (Bio-Rad Laboratories Inc, USA), 9.6 µl RNase-
were shipped at ambient temperature. Upon arrival Free Water (Qiagen, Germany) and 0.5 µl DNA
at the laboratory, the samples were immediately template. qPCR was performed in iCycler iQTM Real-
analysed. All samples were treated within 48 h Time PCR Detection System (Bio-Rad Laboratories
after collection. Decantation was used to separate Inc, USA) using the following conditions: 5 min at
produced water from the oil/water mixture. 95°C; 40 cycles of 95°C for 30 s, 57°C for 40 s,
37º00' 40º20' 43º40'
Black sea
Yerevan
39º40'
B1
36º20'
M3
Y18
S4 GS15
Beirut K44 Y30
K35
Damascus GS6
B32 K2
37º00' 40º20' 43º40'
B32
K3 GK8 N
B23 S15
B14
B56 K32 B8 B6
0 10 20
km
Fig. 1. Sampling locations in Diyarbakır region. Produced water samples were collected from 20 different oil
wells © Maphill / Creative Commons Attribution-NoDerivatives (CC BY-ND)

72°C for 40 s and 80°C for 25 s; and a final 72°C KF720820, KF720823, KF720825 - KF720826,
for 10 min. In the qPCR method, after each cycle, KF720828, KF720830 - KF720832, KF720839,
a signal was formed. By observing the signals for KF720844, KF720852, KF720855, KF720858,
each sample, PCR products could be detected. The KF720872, KF720877, KF720882 - KF720884,
reaction was terminated when the desired amount KF720886 - KF720889, KF720891, KF720893 -
of product was reached. At the second step, a KF720894, KF720896 and KF720903.
conventional PCR approach was applied to the qPCR
products. Reaction mixture in a final volume of 25 µl
3. Results
contained 0.2 µl of each primer, 12.5 µl Taq PCR
Master Mix (Qiagen, Germany), 9.6 µl RNase-Free 3.1 Molecular Analysis of Bacterial
Water (Qiagen, Germany) and 0.5 µl DNA template. Communities
The PCR was performed in TGradient thermocycler
(Biometra, Germany) using the following conditions: Bacterial DNA isolation could only be achieved for 16
5 min at 95°C; 12 cycles of 95°C for 30 s, 57°C for (B1, B8, B6, B14, B23, B32, B56, GS6, GK8, K35,
40 s and 72°C for 40 s; and a final 74°C for 30 min. K44, M3, S4, S15, Y18, Y30) of the 20 produced
water samples. Because the water phase could not
be separated from the oil phase for the other four
2.4 Denaturing Gradient Gel
produced water samples, DNA could not be extracted
Electrophoresis
from these samples. The extracted DNA was used
The DCodeTM system (Bio-Rad Laboratories, USA) as template DNA for the amplification of 16S rRNA
was used for DGGE analysis. 25 µl of each PCR gene fragment. Unfortunately, direct PCR with
product (200–300 ng) were loaded onto 6% bacterial primers did not yield a product from any
polyacrylamide gels (w/v) containing gradients of the produced water samples. For this reason, a
of 20% to 70% denaturants (urea/formamide). two-step PCR was applied: the first step was a qPCR
The gels were run for 16 h at 100 V and 60°C in to increase the concentration of genetic material to
1× Tris-acetate-EDTA buffer. After completion of measurable amounts (30), while the second step was
electrophoresis, the gels were stained with SYBR® a normal PCR to obtain enough material for DGGE
Gold Nucleic Acid Gel Stain (InvitrogenTM, Thermo analysis. For produced water samples, a total of 113
Fisher Scientific, USA) for 20 min, visualised and DGGE gel bands were analysed, but only 69 bands
photographed. Selected predominant DGGE bands yielded sequences of satisfactory quality (Figure 2).
were excised, eluted in 40 µl of 1× Tris buffer
(pH 8) for 2 d at 4°C and re-amplified with 25
cycles as described above. Reaction mixture in a B32 B6 B14 B23 S4 GK8 GS6 M3 B56 B1 B8 K35 Y30 Y18 S15
final volume of 25 µl contained 0.125 µl of primer 1 2 3 4 5 6 7 8 9 10
11 12 13
14 15
341F, 0.125 µl of primer 907R, 12.5 µl of Taq PCR (a) (b) (c)
Master Mix, 9.75 µl of ultra-pure water and 0.5 µl

of template. The PCR products were quantified on
a 1.5% (w/v) agarose gel and then sequenced by
Macrogen Inc (Seoul, South Korea).
2.5 Comparative Sequence Analysis

The resulting sequences were first aligned
and edited using CodonCode Aligner software
(CodonCode Corp, USA). Then they were compared
to sequences stored in the database GenBank®
using the National Center for Biotechnology
Information (NCBI) Basic Local Alignment Search
Tool (BLAST®) (28, 29). All obtained partial 16S Fig. 2. DGGE profiles of 16S rRNA gene fragments
rRNA gene sequences were deposited in GenBank® amplified from produced water samples. See
database under the following accession numbers: legend to Figure 1. (a) 1, B32; 2, B6; 3, B14; 4,
KF720792 - KF720796, KF720798, KF720801 - B23; 5, S4, 6, GK8; (b) 7, GS6; 8, M3; (c) 9, B56;
10, B1; 11, B8; 12, K35; 13, Y30; 14, Y14; 15,
KF720802, KF720804, KF720806 - KF720808,
S15
KF720810 - KF720811, KF720814, KF720818,

Comparative sequence analysis of the DGGE Acinetobacter comprises important soil organisms
bands indicated that 50% of the bacterial where they contribute to the mineralisation of
sequences belonged to ‘unclassified bacteria’. aromatic compounds and they are suited to
Among the classified bacteria, members of exploitation for biotechnological purposes, such as
the phyla Proteobacteria, Bacteroidetes, biodegradation (33). B8_30 was related (96%) to
Firmicutes and Actinobacteria, and the classes Marinobacter sp. Trimyema-2, a thermophilic strain
Alphaproteobacteria, Betaproteobacteria, that was isolated from the hydrothermally heated
Gammaproteobacteria, Sphingobacteriia, Bacilli sea floor at Vulcano Island, Italy (34). Members
and Actinobacteria were identified (Figure 3). of the genus Marinobacter were also identified in
the production water retrieved from a Dutch oil
field (35). The sequence from B32_2 was distantly
3.1.1 Proteobacteria
related (93%) to Thermithiobacillus sp. ParkerM
Proteobacteria was the dominant phylum, (HM173631) that is moderately thermophilic
comprising 29.2% of the total sequences and obligately chemolithoautotrophic on reduced
retrieved from the produced water samples inorganic sulfur compounds (36). Another member
(Figure 3). The sequences B6_19 and B14_37 of the class Gammaproteobacteria was close to
shared 100% and 99% identity with uncultured the sequence of uncultured hydrocarbon seep
bacteria (EU044497 and JF421153, respectively) bacterium (91% similarity) (AF154088) (Table I).
(Table I). The sequence B32_3 was distantly
(94%) related to a moderately thermophilic
3.1.2 Bacteroidetes
bacterium Phenylobacterium lituiforme, a
member of Alphaproteobacteria (31). Within 8.3% of the sequences detected among the
Betaproteobacteria, the sequence represented produced water samples fell into Bacteroidetes
by B32_55 was identified (98%) as Aquincola sp. (Figure 3). The sequence of band B6_14 was
THE-49 (JN128637), isolated from water reservoir affiliated to unclassified Chitinophagaceae. It
(published only in GenBank®). The produced shared 99% identity with Chitinophagaceae
water contained different members of the class bacterium F1 (AB535716), isolated from compost
Gammaproteobacteria. DGGE bands B32_4, (Table I). DGGE bands B6_16, S4_67 and B8_27
B14_35, S4_70, GS6_2 and GK8_79 were affiliated were identified (92% to 99% sequence identity) as
(100%, 94%, 99%, 93%, 99%, respectively) to uncultured Bacteroidetes bacteria (Table I). The
Pseudomonas stutzeri (Table I), a non-fluorescent sequences from S4_67 and B8_27 were related to
denitrifying bacterium (32). The sequence uncultured bacteria that were taught as members
from band B1_20 showed a 100% similarity to of biocorroding microbiota colonising on steel
Acinetobacter sp. VKPM 2838 (Table I). The genus surfaces immerged in coastal seawater (37).
(a) (b) Actinobacteria

4.2%
Firmicutes Actinobacteria Gammaproteobacteria
8.3% 15% 38%
Bacteroidetes
8.3% Sphingobacteriia
8%
Unclassified
bacteria
Bacilli
50%
8%
Betaproteobacteria
Proteobacteria 8%
29.2%
Alphaproteobacteria
23%
Fig. 3. Phylogenetic distribution of the 16S rRNA sequences of produced water samples from the Diyarbakır
oil wells at: (a) the phylum level; and (b) the class level

Table I Phylogenetic Affiliations of Bacterial Sequences Retrieved from Produced Water Samples Based on 16S rRNA Analysis and
457
GenBank® Accession Numbers Assigned to these Sequences
BLAST®
Well DGGE Accession Similarity,
Closest BLAST® match accession Phylum Class Isolation source
no. band number %
number
Gamma
B1_20 KF720883 Acinetobacter sp. VKPM 2838 JF891390 100 Proteobacteria —
proteobacteria
Aeribacillus pallidus strain MCM
B1 B1_23 KF720891 JN701188 89 Firmicutes Bacilli Petroleum reservoir
B-886
B1_24 KF720893 Uncultured Firmicutes bacterium HM041942 98 Firmicutes — Produced fluid
B6_14 KF720818 Chitinophagaceae bacterium F1 AB535716 99 Bacteroidetes Sphingobacteriia Compost
Uncultured Bacteroidetes Total copepod

B6_16 KF720830 FR871413 92 Bacteroidetes —
https://doi.org/10.1595/205651320X15911723486216
bacterium extracts
B6_17 KF720793 Uncultured bacterium GQ259593 98 — — Bioreactor

B6
Alpha
B6_19 KF720802 Uncultured Sphingomonas sp. EU044497 100 Proteobacteria Soil
proteobacteria
Charleston Harbor
B6_20 KF720808 Uncultured Firmicutes bacterium EU194836 96 Firmicutes —
sediment
Coriobacteriaceae bacterium Crude oil

B6_26 KF720798 HQ133029 100 Actinobacteria Actinobacteria
enrichment culture clone B3113 contaminated soil
Steel surfaces
Uncultured Bacteroidetes
B8_27 KF720882 EF491430 99 Bacteroidetes — immerged in marine
bacterium
water
Brackish water from

B8 anoxic fjord Nitinat
B8_29 KF720887 Uncultured bacterium FJ628289 96 — —
Lake at depth of
50 m
Gamma The hydrothermally
B8_30 KF720888 Marinobacter sp. Trimyema-2 AJ292528 96 Proteobacteria
proteobacteria heated sea floor
(Continued)
Johnson Matthey Technol. Rev., 2020, 64, (4)
© 2020 Johnson Matthey

BLAST®
458
Well DGGE Accession Similarity,
Closest BLAST® match accession Phylum Class Isolation source
no. band number %
number
Water/soil mix pile
Gamma
B14_35 KF720804 Pseudomonas stutzeri HQ189755 94 Proteobacteria of samples from oil
proteobacteria
wells
Petroleum-
Uncultured Caenispirillum sp. Alpha contaminated
B14_37 KF720814 JF421153 99 Proteobacteria
clone Ppss_Ma27 proteobacteria saline-alkali soil with
phytoremediation
B14 Wastewater of oil
B14_38 KF720820 Uncultured bacterium FN429535 98 — — refinery treatment
plant
Activated sludge
from industrial
B14_39 KF720825 Georgenia daeguensis HQ246163 100 Actinobacteria Actinobacteria
wastewater
https://doi.org/10.1595/205651320X15911723486216
treatment
B14_41 KF720794 Uncultured bacterium GQ457025 96 — — Rhizosphere
Domestic toilet
B23_52 KF720810 Uncultured bacterium FN401244 99 — —
biofilm
B23_55 KF720826 Aquincola sp. THE-49 JN128637 98 Proteobacteria Beta proteobacteria Water reservoir
B23
Groundwater from
B23_56 KF720831 Uncultured bacterium HM921144 99 — — drinking water
treatment plant
Metal and
B32_1 KF720792 Uncultured soil bacterium AY221598 99 — — hydrocarbon
contaminated soil
Gamma
B32_2 KF720796 Thermithiobacillus sp. ParkerM HM173631 93 Proteobacteria —
proteobacteria
B32
Alpha
B32_3 KF720801 Phenylobacterium lituiforme AY534887 94 Proteobacteria Subsurface aquifer
proteobacteria
Area contaminated
Gamma
B32_4 KF720807 Pseudomonas stutzeri FJ345693 100 Proteobacteria by crude oil and
proteobacteria
chemicals
B56 B56_17 KF720903 Uncultured Firmicutes bacterium HM041942 97 Firmicutes — Produced fluid
(Continued)

BLAST®
459
Well DGGE Accession ® Similarity,
Closest BLAST match accession Phylum Class Isolation source
no. band number %
number
Petroleum-
Gamma
GK8 GK8_79 KF720828 Pseudomonas stutzeri JF727663 99 Proteobacteria contaminated
proteobacteria
saline-alkali soils
Fissure water
GS6_1 KF720832 Uncultured bacterium JN030519 99 — — collected from a
borehole
Gamma
GS6_2 KF720839 Pseudomonas stutzeri JN228329 93 Proteobacteria —
GS6 proteobacteria
GS6_4 KF720852 Uncultured bacterium JF497820 90 — — Activated sludge
Microcosm
GS6_5 KF720858 Uncultured marine bacterium FM211087 90 — —
experiment
https://doi.org/10.1595/205651320X15911723486216
Uncultured bacterium PHOS-

M3_28 KF720855 AF314430 99 — — Batch reactor
HE31
Groundwater from
M3_31 KF720872 Uncultured bacterium HM921144 98 — — drinking water
treatment plant
M3
Bulking activated
M3_32 KF720877 Uncultured bacterium HQ538639 99 — —
sludge
Enhanced biological
M3_34 KF720844 Uncultured bacterium AB231448 99 — — phosphorus removal
(EBPR) sludge
Uncultured hydrocarbon seep Gamma Hydrocarbon seep
K35_44 KF720884 AF154088 91 Proteobacteria
bacterium BPC028 proteobacteria sediment
Groundwater from
K35_46 KF720889 Uncultured bacterium HM921144 98 — — drinking water
K35 treatment plant
K35_48 KF720894 Uncultured bacterium FJ623379 97 — — Batch reactor
K35_49 KF720896 Uncultured bacterium AB231448 99 — — EBPR sludge
(Continued)

3.1.3 Firmicutes
Brackish water from

immersed in marine
Isolation source
Groundwater from
Groundwater from
Hydrocarbon seep
treatment plant
treatment plant
Sequences belonging to members
drinking water
drinking water
Steel surfaces
Batch reactor
EBPR sludge
of Firmicutes accounted for 8.3% of
anoxic fjord
sediment
the bacteria in the produced water
(Figure 3). DGGE band B1_23 was
water
Sea
distantly related (89%) to Aeribacillus
—
pallidus strain MCM B-886 (JN701188),
isolated from petroleum reservoir
(published only in GenBank®)
proteobacteria
proteobacteria
(Table I). In addition, different strains
of Aeribacillus pallidus (with sequence
Gamma
Gamma
similarity values from 98% to 99.6%)
Class
were isolated previously from various

—
—
geothermal sites of Turkey (38). DGGE
band B6_20 was distantly related (96%)
Proteobacteria
Proteobacteria
Bacteroidetes
to an uncultured Firmicutes bacterium,

isolated from marine sediment in
Phylum
Charleston, South Carolina, USA (39).

B56_17 and B1_24 were affiliated (97%
—
—
and 98%, respectively) to an uncultured
Firmicutes bacterium (Table I), detected
Similarity,
in produced fluid from non-water-flooded

high-temperature reservoir of the Niibori
100
oilfield, Japan (40).
%
92
96
99
91
98
98
97
99
3.1.4 Actinobacteria
accession
HM921144
HM921144
AB231448
AF154088
EF491430
JN228329
FJ628289
JF514265
FJ623379
number
BLAST®
The phylum Actinobacteria comprised 4.2%

of the bacterial community recovered from
the produced water (Figure 3). DGGE band
B6_26 displayed 100% sequence similarity
to Coriobacteriaceae bacterium enrichment
Uncultured hydrocarbon seep
culture clone B3113 (HQ133029) isolated

Closest BLAST® match
Uncultured Bacteroidetes
from crude oil contaminated soil of Shengli

Pseudomonas stutzeri
Uncultured bacterium
oil fields, China (41). The sequence

bacterium BPC028
B14_39 was closely related (100%

similarity) to an aerobic bacterial strain
Georgenia daeguensis 2C6-43, isolated
bacterium
from an activated sludge sample collected

from an industrial wastewater treatment
plant in Daegu, South Korea (42).
Although little is known about the
Accession
presence of G. daeguensis in oil associated

KF720806
KF720811
KF720823
KF720795
KF720886
KF720889
KF720889
KF720894
KF720896
number
environments, it was reported that

different strains of G. daeguensis were
isolated from hydrocarbon contaminated
soil of an industrial zone and oil-saturated
S15_60
Y18_70
Y30_66
Y30_68
Y30_69
soil under laboratory conditions (43–45).

DGGE
S4_67
S4_68
S4_70
S4_73
band
4. Discussion
Well
In order to increase our knowledge about

Y18
Y30
S15
no.
S4
microbial diversity, culture-dependent

and molecular-based approaches are used for Ppss_Ma27 were detected in our study. This
describing the diversity of microbes. Molecular- result is consistent with the fact that the vast
based approaches such as PCR-DGGE methodology, majority of microorganisms are uncultured and
which is a useful tool for monitoring the genetic do not grow under laboratory conditions as stated
diversity of complex microbial populations (26), by Lewis et al. (52). In order to isolate more
provide valuable information about the microbial microbes, an appropriate identification laboratory
community structure and dynamics in nature. For protocol should be followed. At this point, different
these reasons, PCR-DGGE fingerprinting analysis strategies such as mimicking natural conditions via
of environmental samples was used in this study. decreased nutrient, extended incubation times,
The choice of appropriate primers for PCR the modification of isolating media formulations
amplification is a crucial step to accurately and different incubation parameters (for example,
characterise the microbial communities. In this temperature) were suggested for the cultivation
study, primer pair (341F-GC/907R), targeting the of microorganisms (53). For instance, pollutant
V3-V5 region of the 16S rRNA gene fragment, degrader Sphingomonas, which seemed to be
was selected due to its suitability for DGGE previously uncultured by nutrient-rich methods,
analysis of bacterial populations in environmental could be isolated from crude oil contaminated
samples (26). This primer pair designed by Muyzer soil by using an in situ method that mimics the
et al. (27, 28) has been used predominantly for original environment (54). In addition, culture-
microbial community analysis (26). dependent investigation should also be supported
The DNA yield obtained from produced water by molecular techniques.
samples was very low. It is known that crude oil Based on the sequences, organisms related to
samples contain low amounts of biomass which known mesophilic bacteria were predominant
makes DNA isolation difficult to achieve (46). In in the produced water samples. In addition,
this study, the permit included taking up to 500 ml some organisms related to thermophilic bacteria
of oil/water mixture from each sampling point so (Aeribacillus pallidus, Marinobacter sp. Trimyema-2,
that only ca. 25 ml of each produced water sample Phenylobacterium lituiforme and Thermithiobacillus
could be obtained. In this scope, the low sample sp.) were also identified. Bacteria having different
volumes of produced water separated from the oil/ metabolic capabilities (denitrifying, biodegrading
water mixture may be a reason for the low amount and sulfur removing bacteria) were also detected.
of DNA. It was reported in other studies that higher In addition, bacteria which may cause biocorrosion
sample volumes (100–4000 ml) of produced water on steel surfaces were detected.
were used for DNA isolation (35, 47–50). The The dominant bacterial phylum was the
low DNA yield affected the efficiency of the PCR Proteobacteria. The members of this phylum were
technique and for this reason, a two-step PCR was also frequently found in many other studies on
applied to the produced water samples. Thus, a microbial diversity of oil field produced waters
sufficient amount of PCR product for DGGE for the (55–58). Moreover, it was stated that Proteobacteria
produced water samples could be obtained. are ubiquitous in oil reservoirs over all temperature
Bacterial communities associated with the ranges (59).
produced waters was analysed by the PCR-DGGE In this study, among the detected genera in
approach. Although numerous bands were visible produced water samples that potentially contain
on the DGGE gel, only dominant bands could be hydrocarbon degrading bacteria were Aeribacillus,
excised and sequenced. Most of the sequences Acinetobacter, Sphingomonas, Marinobacter and
retrieved from produced water samples were Phenylobacterium. It has been known for years
related to unclassified bacteria. Different studies that the species belonging to these genera are
on oil reservoir microbiota have also shown that oil capable of degrading hydrocarbons (6, 17, 60, 61).
fields harbour new and still unidentified microbial In addition, G. daeguensis, a hydrocarbonoclastic
species. For example, Lenchi et al. described bacterium, was detected in produced water sample
microbial communities in production and injection with a 100% sequence similarity. G. daeguensis
waters from the Algerian oil fields. In their study, has also been demonstrated as a potential
they detected that a large number of unclassified microbe for bioremediation due to its hydrocarbon
bacterial and archaeal sequences were found in degradation ability (44). Further investigations
the water samples (51). Furthermore, uncultured are needed because our current knowledge of the
bacteria such as uncultured Sphingomonas metabolic capability of G. daeguensis is limited.
sp. and uncultured Caenispirillum sp. clone Moreover, sulfur-oxidising Thermithiobacillus sp.

was also identified in produced water sample. oil or rock surfaces (59). In addition, biofilm may
Sulfur-oxidising bacteria, which oxidise the sulfur form on the metal surfaces of the pipes in the oil-
compounds produced by the activity of sulfate- producing wells (70). Oil microbiome studies focus
reducing bacteria in oil reservoirs, may play a key mainly on the analysis of the water phase due to
role in the oil industry because they can be utilised its easy sampling. However, it should be noted that
to resolve processing problems such as reservoir the water phase itself contains only a minor portion
souring (11). of the microbes found in the oil reservoir (59). On
Pseudomonas was the dominant genus the other hand, the sampling of sessile microbes is
detected among the produced water samples. likely to be more challenging (70).
Pseudomonas stutzeri was the species identified
in five produced water samples. P. stutzeri was
5. Conclusion
previously isolated not only from formation
water, produced from the petroleum wells in This study reported for the first time the bacterial
Adıyaman (62), but also oil-contaminated soils in community composition of produced water
Batman petroleum refinery, Turkey (63). These from Diyarbakır oil reservoirs as obtained by
two areas are close to the Diyarbakır region from DGGE analysis of PCR-amplified 16S rRNA gene
where the samples in this study were collected and fragments. DGGE analysis of produced water
these findings show that P. stutzeri is distributed samples demonstrated that the majority of the
widely in south-eastern Turkey. In other different bacterial sequences belonged to unclassified
geographical areas, this species was also isolated bacteria, indicating that oil reservoirs harbour
from oil-associated environments, such as oil still undescribed microbial species. Among the
field production water (64), oil sludge (65) and classified bacteria, the members of Proteobacteria
oil contaminated soil (66). However, although were more abundant. Pseudomonas was the
P. stutzeri is often isolated from oil reservoirs, dominant genus detected in the produced water.
the origin of P. stutzeri in oil reservoirs is a Although the members of Pseudomonas were
debatable issue. Because oil reservoirs have known as exogenous organisms inoculated into oil
low redox potentials and contain little oxygen, reservoirs, Pseudomonas stutzeri was found in five
anaerobic microorganisms are considered as truly produced water samples. Bacteria having different
indigenous to oil reservoirs (67). In this regard, metabolic capabilities (denitrifying, biodegrading
it is believed that P. stutzeri, most of whose and sulfur removing bacteria) were also detected.
strains are aerobes, is an exogenous organism It can be stated that the metabolic capacities of
inoculated into oil reservoirs during the oil these bacteria make them potential candidates
production processes. Even if strains of P. stutzeri for utilising in biodegradation, bioremediation,
are introduced into oil reservoirs with injected the improvement of oil quality and oil recovery
fluids, they should adapt to the physicochemical processes. The knowledge of the bacterial
characteristics of the reservoir to survive. At this community composition in oil reservoirs of the
point, it has been proposed that extreme reservoir Diyarbakır region obtained in this study will be
conditions may act as special factors for the of great interest for both scientific research and
evolution of P. stutzeri, thereby forming mutant applications in the oil industry. To build on the data
strains (68). Furthermore, P. stutzeri, being found presented in this study, metagenomic analyses
in a wide variety of habitats, is known for its should be performed to explore the undescribed
diverse metabolism. Some strains of P. stutzeri microbes.
are capable of denitrification, degradation of
aromatic compounds and nitrogen fixation (32).
Acknowledgements
These metabolic features make P. stutzeri highly
attractive for biotechnological processes, such as This work was supported by ‘Research Fund of
reservoir souring control (69), microbial enhanced Istanbul University’ (Project number: 28699).
oil recovery (64) and bioremediation of oil-polluted Tuğçe Tüccar was awarded an Erasmus LLP
environments (65). Scholarship. Esra Ilhan-Sungur was awarded a
In undisturbed oil reservoirs, microorganisms Post-doctoral Research Scholarship by the Scientific
are found in different phases such as reservoir and Technological Research Council of Turkey
fluid containing crude oil and formation water, and (TUBITAK-BIDEB). We thank the Turkish Petroleum
rock surfaces. While planktonic microbes thrive in Corporation for permission to collect samples, and
the water phase, sessile microbes may attach to Ender Taptık and Hasan Kaya for their assistance

with the sample collection. We thank Ben Abbas Y. Kamagata and H. Tamaki, Int. J. Syst. Evol.
for his technical assistance. We acknowledge Microbiol., 2017, 67, (10), 3982
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The Authors
Tuğçe Tüccar is a PhD candidate in Fundamental and Industrial Microbiology at Istanbul
University, Turkey. She received her Bachelor’s degree in Biology from Middle East
Technical University, Turkey, in 2009. She obtained her Master’s degree in Fundamental
and Industrial Microbiology from Istanbul University, Turkey, in 2011. Her dissertation
was on investigation of sulfate-reducing bacteria in petroleum samples. She was awarded
an Erasmus LLP Scholarship and conducted her research work at Delft University of
Technology, The Netherlands. Areas of interest are microbial ecology, microbial genetics,
petroleum microbiology and microbial corrosion.
Esra Ilhan-Sungur is professor in the Biology Department at Istanbul University, Turkey,

since 2018. A key focus of her research is microbiologically induced corrosion and its
prevention. Further research interests lie in the area of anaerobic bacteria (especially
sulfate-reducing bacteria), petroleum microbiology, microbial diversity and ecology,
microbial genetics and biofilm. She was awarded a postdoctoral research scholarship by
the Scientific and Technological Research Council of Turkey (TUBITAK-BIDEB) and worked
as a guest researcher at Delft University of Technology.
Gerard Muyzer is Professor in Microbial Systems Ecology at the University of Amsterdam, The
Netherlands. He is studying the structure, function and dynamics of microbial communities,
their role in biogeochemical cycles and their application in biotechnological processes. For
this he is using a systems biology approach in which he combines experimental work,
the use of state-of-the-art omics techniques, and mathematical modelling. He is mainly
focusing on the microbial sulfur cycle, and in particular on sulfur bacteria that are present
in natural ecosystems (such as soda lakes, stratified lakes, rhizosphere of seagrasses)
as well as man-made ecosystems, such as full-scale bioreactors removing toxic sulfur
compounds from wastewater.

The Biotechnological Potentials of Bacteria

Isolated from Parsık Cave, Turkey
Measuring the enzyme profiles, antibiotic resistance and antimicrobial activity in
bacteria
Begüm Çandiroğlu** 1. Introduction

Institute of Graduate Studies in Sciences,
Istanbul University, Balabanaga Mah. Caves are dark environments with high humidity,
Sehzadebasi Cd., 34134 Vezneciler, low nutrients, stable temperature and high
Fatih-Istanbul, Turkey mineral diversity. They are natural geological
formations constituting ecological niches for
Nihal Doğruöz Güngör* microorganisms (1). Each cave is singular in
Department of Biology, Faculty of Science, its physical, chemical, biological and ecological
Istanbul University, Balabanaga Mah. factors. These conditions contribute to the
Sehzadebasi Cd., 34134 Vezneciler, formation of unique microbial communities in
Fatih-Istanbul, Turkey every cave. Moreover, caves contain some unique
microorganisms which lead to rock weathering
Email: *ndogruoz@istanbul.edu.tr; process and biomineralisation by carrying out
**begumcandiroglu@gmail.com various enzymatic reactions as a result of their
metabolism. These microorganisms play an
important and major role in the formation of cave
Since cave ecosystems have extraordinary structures such as stalactites, stalagmites, cave
environmental conditions, these ecosystems offer pearls and curtains (2–5). Studies have shown that
opportunities for microbiological studies. In this cave isolates have biotechnological and industrial
study, cultivable bacteria isolated from Parsık cave, applications such as microplastic degradation (6),
Turkey, were investigated regarding enzyme profiles, biological treatment of metal contaminated soil
antibiotic resistance and potential for production of and groundwater (7) and use in self-healing
antimicrobial agents. The metabolic properties of concrete (8).
321 bacterial isolates were determined. The most The insufficient nutrient levels in caves stimulate
produced enzyme by the isolates was found to be competition among microorganisms by forcing them
tyrosine arylamidase. The enzymatic reactions of to develop survival strategies such as producing
the bacteria showed that Parsık cave isolates have high amounts of exopolymeric substances, enzymes
high aminopeptidase activity. The highest antibiotic and antimicrobial metabolites. Hence, caves could
resistance frequency of the isolates was 38.6% be considered as incomparable environments for
against ampicillin. While the isolates displayed the discovery of new antibiotics and production of
variable inhibition rates against tested pathogenic novel enzymes (9–11).
microorganisms, they showed the highest inhibition Since microorganisms have the capacity to
against Candida albicans. The results show that the produce a high quantity of stable enzymes in a
bacteria isolated from Parsık cave have potential short period of time, they become the preferred
for further studies related to biotechnological source of industrial enzymes. Microbial enzymes are
applications. The study findings contribute increased used in the clinical field for diagnosis, treatment,
knowledge on metabolic peculiarities of bacteria biochemical tests and monitoring of various
isolated from cave ecosystems. diseases. Furthermore, cave microbial enzymes are

used in biotechnological and industrial fields such as which they can produce neutralising or detoxifying
biodegradation, recycling of waste (12), purification products which act against microorganisms in the
and dirt or waste-dissolving products. It is reported same environment. This explains the imperative
that enzymes from microorganisms isolated from production of antibiotics in these bacteria. Since the
cold cave or ocean environments offer economic resistance and antimicrobial biosynthesis genes are
benefits and contribute to energy conservation due often linked and coregulated, antibiotic resistance
to their activation at low temperatures (13, 14). in environmental bacteria remains a major indicator
Apart from the importance of enzymes isolated of antibiotic production, as is the case of bacteria
from cave microorganisms, it is interesting isolated from soil (19, 20). Therefore, it is important
to investigate the potential of producing new to establish antibiotic resistance profiles as well as
antimicrobial agents. Since the World Health the antibacterial properties of bacteria.
Organization pointed out the need for new antibiotics This study has two main goals:
because of increasing microbial resistance (15), • Detection of enzyme profiles of the isolates and
studies in this field are multiplying and many cave determination of isolates that have potential
isolates producing antimicrobial substances have uses in biotechnology
been discovered. Cervimycin A, B, C and D from • Investigation of antimicrobial agents and
Streptomyces tendae strain HKI 0179 isolated from antibiotic resistance of cave bacteria.
Grotta dei Cervi in Italy (16), Xiakemycin A from
Streptomyces sp. CC8-201 isolated from Chongqing
2. Experimental
City karst soil in China (17), and Hypogeamicin A,
B, C and D from Nonomuraea specus isolated from 2.1 Studying Area and Sampling
Hardin’s cave system in Tennessee, USA (18) were
the first produced and purified bioactive substances Parsık cave is located in Izmit-Aksığın village
from microorganisms of caves situated in different (Global Positioning System (GPS) coordinates 40°
geographical regions. 37’ 50.1060”N, 29° 57’ 56.5056”E), in the north-
Bacteria in environments far away from human west of Turkey. It is a horizontal cave with a length
influence are not expected to have antibiotic of 778 m and a depth of 166 m. There is an intense
resistance. However, studies have shown that bacteria water inlet in Parsık cave throughout four seasons.
isolated from such environments do have antibiotic Samples were taken from water, soil and surface
resistance. Some bacteria have resistance genes by formations (‘moonmilk’) (Figure 1). The selected
Plan
North
MANYETIK
KUZEY
0m 6m 12 m
0 cm 5 cm 10 cm
B C
Fig. 1. Map of Parsık cave (red dots show the sampling areas) from the Anatolian Speleology Association,
Turkey

sampling zones are the sole area away from the isolation from soil samples was on SCA, ISP4,
entrance area, trip and running water pathway. AIA-G, SEA and 1/2 tryptic soy agar (TSA) media,
Although Parsık cave is not a show cave, it is open and that from water samples was on 1/2 TSA only.
to cavers and researchers. Plates were incubated aerobically for a period of
Surface formation samples were collected 5–30 days at 20°C (21). At the end of incubation,
by sterile swabs under aseptic conditions and plates which contained between 30 and 300
cultivated on starch casein agar (SCA), inorganic colonies were considered for both soil and water
salt-starch agar (ISP4), soil extract agar (SEA) and samples. Colonies which appeared different were
Actinomycetes isolation agar (AIA-G) in duplicate selected for identification, then stored at –86°C for
for each region. Once the plates reached the subsequent uses.
laboratory, they were incubated aerobically for a
period of 5–30 days at 20°C (21). All water and soil
2.5 Identification of Cave Isolates
samples were taken in sterile sample containers.
and Their Enzymatic Reactions
Cave isolates were identified through biochemical
2.2 Physicochemical Measurements
tests performed in the VITEK® 2 system (bioMérieux
of Sampling Areas
SA, France). One of the three formats of this system
Humidity and temperature values of the sampling is the VITEK® 2 Compact 30 which focuses mainly
areas were measured by a portable temperature/ on the industrial microbiology-testing environment.
humidity meter. In addition, the temperature, Based on this industrial software, three reagent
conductivity, amount of dissolved substances and cards of VITEK® 2 Compact 30, named Gram-
pH values of the sampled water sources were negative fermenting and non-fermenting bacilli
measured during sampling and recorded by a (GN), Gram-positive cocci and non-spore-forming
HQ40D digital two channel multimeter (Hach Lange bacilli (GP) and Gram-positive spore-forming bacilli
GmbH, Germany). (BCL), were used to characterise the isolated
bacteria following the procedure and data given
by the system manufacturers. Reagent cards
2.3 Total (Live/Dead) Bacteria
are based on established biochemical methods
Number
and developed substrates (23). The results of
The redox dye 5-cyano-2,3-ditolyl-tetrazolium biochemical reactions were interpreted to establish
chloride (CTC) was used together with the enzymatic profiles of isolates.
DNA-binding fluorescent dye 4’,6-diamidino-2-
phenylindole (DAPI) to determine the total number
2.6 Ability of Cave Bacteria to
of bacterial cells and the viable count of bacteria
Produce Antimicrobial Materials
which actively respire. The concept is to distinguish
between the metabolically active cells and the dead The ability of Bacilli or Actinobacteria to produce
cells present in each of the water and soil samples. antimicrobial agents was tested on standard
The experimentation procedure is the same as strains of fungi species of Candida albicans (ATCC®
previously described by Güngör and Yurudu (22). 10231TM) and bacterial species of Escherichia coli
(ATCC® 8739TM), Pseudomonas aeruginosa (ATCC®
9027TM), Staphylococcus aureus (ATCC® 6538TM),
2.4 Enumeration and Isolation of
Bacillus subtilis (ATCC® 6633TM), Staphylococcus
Culturable Aerobic Heterotrophic
epidermidis (ATCC® 12228TM), Klebsiella
Bacteria ®
pneumoniae (ATCC 4352TM), Enterococcus
1 l of water samples were condensed by using hirae (ATCC® 10541TM), vancomycin-resistant
polyamide filters of 0.22 µm pore size. Filters were Enterococcus faecalis (VRE) (ATCC® 51299TM)
re-suspended in 20 ml of sterile physiological saline and methicillin-resistant Staphylococcus aureus
water. 1 g of the soil samples was homogenised (MRSA) (ATCC® 33591TM).
in 9 ml of sterile physiological saline water. All Bacterial suspensions containing 3 × 108 cells ml–1
samples were cultivated using the 10-fold serial of the selected isolates were prepared. 2.5 μl of each
dilution method. Diluted samples were cultured suspension were incubated on Mueller Hinton Agar
on tap water agar (TWA) and Reasoner’s 2A agar (MHA) plates at 20°C for 24 h. After incubation, all
(R2A) for enumeration and isolation of bacteria media in which bacterial colonies were observed,
from water and soil samples. In addition, bacterial were exposed to ultraviolet (UV) radiation in an

open laminar flow cabinet. Therefore, the vitality These details highlight the differences in chemical
of the bacteria was destroyed. 1.5 × 108 cells ml–1 environment that may exist within the cave areas.
of 24 h fresh cultures of the standard strains were
prepared. 100 μl of each suspension was mixed
3.2 Number of Determined Total
with TSA medium at 45°C. Subsequently, it was
(Live/Dead) Bacteria
poured into the previously UV exposed plates, then
incubated for 24 hours at 37°C after solidification. The highest vitality percentage of bacteria isolated in
At the end of the incubation period, the growth of soil samples was found in samples from point B with
the standard bacteria in the TSA was investigated 38.7%, whereas the highest vitality percentage in
and the zone diameters were measured (24). the water samples was found in samples from point
C with 26.3% (Table II). In cave environments, it
is observed that bacteria can survive metabolically
2.7 Susceptibility to Antibiotics
but cannot be cultured. This is because bacteria
The sensitivity of 101 selected isolates to antibiotics enter a viable but nonculturable cell form under
was examined by using the disc diffusion method extreme environmental conditions such as low or
of Kirby-Bauer (21) in which 10 antibiotics were high temperature, nutrient deficiency, osmolarity
used: piperacillin (100 µg), erythromycin (15 µg), and light. In addition, cave microorganisms obtain
vancomycin (30 µg), ampicillin (10 µg), their energy from the cave atmosphere or the cave
neomycin (10 µg), gentamycin (10 µg), surfaces to which they are attached (28, 29).
chloramphenicol (30 µg), tetracycline (10 µg),
rifampicin (30 µg) and ofloxacin (10 µg). The
3.3 Number and Classification of
incubation conditions were 24 h at 20°C.
Culturable Aerobic Heterotrophic
Escherichia coli (ATCC® 8739TM), Pseudomonas
Bacteria
aeruginosa (ATCC® 9027TM) and Staphylococcus
aureus (ATCC® 6538TM) were tested against the SCA, ISP4, SEA and AIA-G have been used
same antibiotics as control microorganisms (25). especially in surface and soil samples to increase
the probability of isolating bacteria belonging to
phylum Actinobacteria, which have an extremely
high potentials in terms of antimicrobial
3.1 Physicochemical Measurements production (30). TWA and R2A medium were used
of Sampling Areas for both isolation and counting of other bacterial
groups. Apart from these media, 1/2 TSA was
Temperature, pH, conductivity and hardness values used for isolation of other bacterial groups from
of water samples are shown in Table I. The air all samples. The cave environment in general is
temperature of the sample areas A, B, C (Figure 1) oligotrophic and these media provide a similar
was determined. The temperature of area A was environment to the culturable cave bacteria. The
9.8°C and that of B and C were determined as number of culturable aerobic heterotrophic bacteria
9.4°C. The moisture value was evaluated as 93% from water and soil samples obtained from R2A
in all these areas. The Parsık cave resembles most and TWA media is given in Figure 2.
cave systems with its high level of humidity and When the bacterial counts of water and soil
stable air temperature (26, 27). It was determined samples in R2A and TWA media were examined,
that the pH and hardness values of the waters at the highest bacterial numbers were found in R2A
points B and C were higher than those at point A. medium. These results were evaluated statistically
using the Kruskal-Wallis test. The p value was
found to be 0.09 and no significant difference was
Table I Physicochemical Measurements of found between the numbers of bacteria grown on
Water Samples the R2A and TWA media. In a study conducted in
Measured values Water sample areas 2014 (31), the efficiency of various media (SEA,
TWA, SCA, TSA) was compared to their suitability
A point B point C point
for bacterial counting. Efficient results for both
pH 8.2 9.8 9.8
isolation and counting were obtained in TWA.
Hardness, ppt 0.107 0.145 0.145 A total of 372 bacteria were isolated from all
Conductivity, mS 0.22 0.30 0.30 samples. VITEK® analysis was applied to only
Temperature, °C 10 9.2 9.2 321 bacteria which had different characteristics in

Table II Number of Bacteria in Water and Soil Samples According to DAPI/CTC Method
Samples Total number of signals, cm2 Vitality, %
CTC Total
SA 406,505,880 2,947,167,630 13.8
SB 135,501,960 643,634,310 21
SC 508,132,350 1,930,902,930 26.3
TA 1,084,015,680 5,318,451,930 20.3
TB 8,130,117,600 21,002,803,800 38.7
TC 6,097,588,200 20,664,048,900 29.5
and water isolates was Actinobacteria with 36%

7 and 35% respectively. Considering all the samples,
6 R2A at the class level, Actinobacteria was the most
log(10)
TWA
10
5 dominant with 33%, while Bacilli with 23% was

CFU ml–1 log,
4
detected as the second dominant class. It was
demonstrated through previous studies that
3
Actinobacteria existed mainly in cave walls, soil,
2 sediment and on speleothem surfaces, which might
1 have considerably contributed to the formation of
0 cave structures and the biomineralisation in the
TA TB TC SA SB SC cave ecosystems (4–37). Actinobacteria as well
Points of samples as Firmicutes are frequent among the microbial
Fig. 2. Number of aerobic heterotrophic bacteria population inhabiting the caves. Compared to
that can be cultured from water and soil samples the Proteobacteria group, Firmicutes are more
(TA = soil sample A; TB = soil sample B; TC = resistant to stress caused by dehydration as
soil sample C; SA = water sample A; SB = water well as restriction of nutrients (37). Contrary
sample B; SC = water sample C)
to our findings for Parsık cave, Proteobacteria
are a dominant group in heterotrophic bacterial
culture-based analyses. The results of the systematic communities in many caves (33, 34, 38–40). In
classification of the bacteria were compiled by the current study, Proteobacteria were determined
biochemical analysis using the VITEK® 2 Compact respectively as 10%, 21% and 17% in the surface,
30 automated system. Actinobacteria (33%) was water and soil samples. The dominant classes of this
determined to be the dominant phylum in this phylum were found to be Gammaproteobacteria
study while the other determined phyla were and Alphaproteobacteria with 9.2% and 6.4%,
Firmicutes (25%) and Proteobacteria (16%). In respectively. In our previous study in Kadıini
our previous work in Kadıini cave in Turkey, the cave, Alphaproteobacteria were detected at 2%,
dominant phylum was Firmicutes (86%), followed while Gammaproteobacteria were at 9% (32).
by Proteobacteria (12%) and Actinobacteria (2%) The phylum Proteobacteria, having a key role in
respectively (32). In addition, in the study done biogeochemical cycles, and being abundant in
by Tomova et al. (33), Proteobacteria (51.45%) samples from cave sediment, soil, dripping water
were found to be the dominant phylum in the and cave surface, is a cosmopolitan bacterial
samples taken from the Magura cave, Bulgaria, group (37).
followed by Actinobacteria (43.68%) and
Bacteroidetes (3.88%). Although the bacterial
3.4 Enzymatic Reactions of Parsık
habitat of each cave is specific, Proteobacteria,
Cave Bacteria
Actinobacteria, Bacteroidetes and Firmicutes
are the most identified groups in culture-based Enzymatic reactions of microorganisms give
microbiological studies in caves (34–36). us ideas of their metabolic activities which are
In our study, Firmicutes was the most common related to their environment. The biochemical
phylum in soil samples with a rate of 33%, while tests of our isolates in the VITEK® system were
the most common phylum determined in surface not only useful for bacterial identification but

also to provide more information about nutrients positive and Gram-positive spore forming bacilli
in Parsık cave. In addition, results of these tests revealed tyrosine-arylamidase activity. Tyrosine is
were used to evaluate the potentials of the isolates a non-essential amino acid which is synthesised
for biotechnological uses in terms of their enzyme through phenylalanine hydrolysis. It plays a
production. 76 Gram-negative bacteria, 194 Gram- major role in most enzyme synthesis as reported
positive and 51 Gram-positive spore forming by Kalkan and Altuğ (42), since it is the phosphate
bacteria have been tested using the GN, GP and and sulfate receptor of protein kinase during protein
BCL cards respectively in the VITEK® 2 compact synthesis. It is also used to reinforce the activity
device, and results are given in Figure 3, Figure 4 of proteins as demonstrated in a study conducted
and Figure 5 respectively. Most of the isolates in thrombin inhibitors showing that tyrosine
displayed peptidase (arylamidase) while only sulfation could open a way for the development
Gram-negative bacteria (less than 10%) showed of an anti-thrombotic drug (43). Hence, tyrosine
lipolytic activity. In the study conducted in Gumki arylamidase has a valuable role in biotechnology
cave, India, 75.5% of bacteria produced lipase, since it contributes to the liberation of the amino
47% were amylase producers and 24% produced acid tyrosine.
protease (41). Another study screening cave Enzymes like leucine arylamidase have been
bacteria for enzyme production found 40% lipase reported to be important in food processing
and 87% protease producers (33). This variation in industries and the treatment of waste products (44,
enzymatic profiles in cave bacteria reinforces the 45). The degradation of leucine and other amino
idea that every cave is unique. acids results in volatile molecules responsible for
The high activity of amino acids arylamidase the flavours of some foods like meat products as
determined in our tested isolates indicates their reported by Papamanoli et al. (46) and Lee et
potential for protein catabolism (42). The phyla al. (44). In addition, a study showed the roles
Firmicutes (31%) and Actinobacteria (30.7%) of bacteria in conversion of paper mill sludge
produced the highest amounts of arylamidases demonstrating the important contribution of amino
identified among the tested isolates. 85.52%, acid peptidase with leucine arylamidase (45). In
65.97% and 82.35% of Gram-negative, Gram- our study, 81.95% and 88.23% of Gram-positive
Gr– bacteria
70
60
50
40
30
20
10
0
APPA
GlvA
ODC
LDC
IHISa
ADO
PyrA
IARL
AGLTp
dGLU
OFF
URE
SAC
dTAG
dTRE
CIT
MNT
5KG
ILATk
AGLU
SUCT
NAGA
AGAL
PHOS
CMT
GGAA
IMLTa
ELLM
dCEL
GGT
dMAL
dMAN
dMNE
BAlap
ProA
LIP
PLE
TyrA
BGAL
BNAG
BGLU
BXYL
dSOR
0129R
O
Fig. 3. Biochemical properties of Gram-negative bacteria. Tests for Gram-negative bacteria by VITEK® 2
Compact 30 micro device. See Glossary in main text for explanation of terms

Gr+ bacteria
180
160
140
120
100
80
60
40
20
POLYB_R
ADH1
TyrA
AMY
PIPLC
AGLU
APPA
LeuA
URE
POLYB
dGAL
dRIB
ILATk
NAG
NOVO
NC6.5
dXYL
CDEX
AspA
AMAN
PHOS
ProA
PyrA
AGAL
AlaA
dMAL
dMAN
dMNE
MBdG
dTRE
ADH2s
OPTO
PUL
dRAF
BGAL
BGURr
dSOR
BACI
0129R
SAL
SAC
BGAR
BGUR
LAC
O
Fig. 4. Biochemical properties of Gram-positive bacteria. Tests for Gram-positive bacteria by VITEK® 2
Compact 30 micro device. See Glossary in main text for explanation of terms
Gr(+) bacilli bacteria

60
50
40
30
20
10
0
POLYB_R
NC6.5
LysA
AspA
LeuA
PheA
ProA
PyrA
TyrA
APPA
AGLU
INU
AGAL
AlaA
CDEX
GlyA
dMAN
IRHA
dGAL
GLYG
INO
ELLM
AMAN
MTE
dMNE
dMIZ
NAG
PLE
BGLU
PHC
PVATE
dTAG
dGLU
dRIB
NC6.5%
KAN
OLD
ESC
TTZ
BXYL
BGAL
BNAG
POLYB R
Fig. 5. Biochemical properties of Gram-positive Bacilli bacteria. Tests for Gram-positive Bacilli bacteria by
VITEK® 2 Compact 30 micro device. See Glossary in main text for explanation of terms
bacteria and Gram-positive bacilli showed leucine produce this enzyme could be used directly or
arylamidase activity. This enzyme was the indirectly by using their enzymes in both composting
second most produced enzyme, after the tyrosine of sludge and fermentation of food products such
arylamidase, by our isolates. Bacteria which can as meat and dairy products.

VITEK® results have showed that some Parsık cave the highest inhibition rate was observed against
isolates exhibit beta-galactosidase activity which is Candida albicans. Some of our cave isolates have
the more expressed carbohydrate hydrolase in this been found to have inhibitory effects against S.
study. Considering the whole of the tested isolates, aureus, S. epidermidis, VRE and P. aeruginosa. The
most of the bacteria producing beta-galactosidase zone diameters of cave bacteria with antimicrobial
belong to the Firmicutes phylum with 40.6%, while properties against tested microorganisms are
only 10.9% of beta-galactosidase producers were shown in Table III.
classified under the phylum Proteobacteria. In our study, the isolate which affected S.
The main role of the beta-galactosidase enzyme is epidermidis belongs to the Bacilli class and those
to convert lactose into monosaccharides. Glucose which inhibit VRE and S. aureus belong to the
and galactose resulting from this reaction not only Actinobacteria class. Some studies have shown
contribute to the development of the cell but can that bacteria with antimicrobial activity inhabiting
also be used in dairy product processing (44, 47). karst caves are often from the Actinobacteria
This enzyme is important since it solves the problem class (30, 31). However, cave bacteria belonging
of human lactose intolerance. The hydrolysation of to phyla Proteobacteria, Firmicutes (especially
lactose by this enzyme results in molecules like Bacilli class) and Bacteroides were determined to
galactooligosaccharides which have health benefits have antimicrobial and bioactive substances. Thus,
as prebiotics (47). Moreover, breakdown of some approximately 50% of the bacteria isolated from
sugars like D-mannose, D-mannitol and D-glucose the Magura cave, Bulgaria were detected to inhibit
by fermentation was reported, especially in Gram- the increase of P. aeruginosa (33). Cave bacteria
negative bacteria. inhibiting MRSA and VRE clinical strains were
Lipolytic activity was also observed in some of our determined in a study on Actinomycetes isolated
isolated Gram-negative bacteria (less than 10%). from 19 different caves in Turkey (30). Certainly the
Even if it was produced by a minimum number of bacteria belonging to the class Actinobacteria are
isolates, the activity of lipase was fully expressed the best known in terms of antimicrobial material
by bacteria belonging to the phylum Proteobacteria. synthesis, but the isolation of bacteria belonging
This class of enzymes which is used in hydrolysation to the other classes is very important especially in
of lipids could be important in bioremediation since karst environments.
it could participate in oil degradation. Sharma et
al. reported that microbial lipases are best for
3.6 Determination of Antibiotic
biodiesel production (48). Since they can use all
Resistance Profiles of Isolated
types of free fatty acids and glycerides, they exhibit
Bacteria
a high activity, thermostability, alcohol resistance,
less reaction time as well as less production Antibiotic resistant bacteria are widespread
inhibition (48). Other enzymes were produced in several environments. In this study, resistance
by some of the bacteria in Parsık caves. Further to 10 different antibiotics of 101 bacteria (76%
studies should be carried out to clarify them and Gram-positive; 25% Gram-negative) selected from
assess their biotechnological uses. the cave isolates was investigated. Isolates with a
metabolic reaction rate of at least 95% similarities
to the data in the VITEK® database were selected.
3.5 Antimicrobial Agent Production
When the antibiotic resistance profiles of the
Capability
isolates were examined, 7% of the bacteria
Microorganisms with broad-spectrum bioactive belonging to the cave were resistant to all antibiotics.
components, antifungal and antibacterial agents in The highest number of bacteria showed resistance
cave-specific habitats are common in these extreme against ampicillin with a rate of 38.6%. In addition,
environments (17). In our study, a total of 129 35.6% of the isolates showed resistance against
cave bacteria were tested for their antimicrobial two or more antibiotics.
effect against nine different standard bacterial Antibiotic resistance profiles of Gram-positive and
strains and one fungal strain. Experiments have Gram-negative cave isolates are shown in Figure 6.
shown that 10 of the selected bacteria (six from The lowest resistance was observed to rifampicin (9%
Actinobacteria class, four from Bacilli class) have for Gram-positive and 8% for Gram-negative).
antimicrobial effects against the standard strains. In parallel with our study, it was determined that
Parsık cave isolates displayed variable inhibition all the Pajsarjeva jama, Slovenia, isolates were
rates against the tested microorganisms and sensitive to rifampicin (49). Likewise, low levels

Table III Antimicrobial Agent Production Ability
474
Isolates/classes of Resulting zone diameters, mm
bacteria
E. coli E. faecalis B. subtilis S. aureus S. epidermidis MRSA VRE P. aeruginosa K. pneumoniae Candida
albicans
SA22/ Actinobacteria – – – – – – 9 – – –
TA44/ Actinobacteria – – – – – – – 16 – –
TA12/ Actinobacteria – – – – – – – 13.5 – –
SA56/ Actinomycetes – – – 13 – – – – – –
TB48/ Bacilli – – – – – – – – – 13
SB1/ Bacilli – – – – – – – – – 30
https://doi.org/10.1595/205651320X15923194903811
TA62/ Actinomycetes – – – – – – – – – 24
TB27/ Bacilli – – – – 15 – – – – –
SC3/ Bacilli – – – – – – – – – 30
TB64/ Actinobacteria – – – – – – – – – 13
Antibiotics
Piperacillin 11 24 19 13 20 9 31 16 22 ND
Vancomycin – 16 13 8 10 21 18 – – ND
Gentamicin 11 6.5 16 8 13 20 – 11 22 ND
Tetracycline 10 – 16 8 14 – 14 5 19 ND
Rifampicin 7 16 17 10 18 35 28 8 15 ND
Ofloxacin 15 18 21 13 15 31 30 23 35 ND
ND = not determined

(a) Gram-positive isolates Cell wall synthesis

35 inhibitors
30S ribosome synthesis
30 inhibitors
Percentage of bacteria
showing resistance
25 50S ribosome synthesis

inhibitors
20 DNA/RNA synthesis
inhibitors
15
10
0
in
in
e
n
in
ol
n
lin
i
lli
lli
ci
ci
ic
yc
yc
yc
yc
pi
xa
ci
ci
en
yc
om
om
ra
pi
flo
ac
ph
eo
ta
Am
pe
fa
nc
hr
O
en
tr
m
N
Ri
Pi
yt
Va
Te
ra
G
Er
lo
Ch
Antibiotics
(b) Gram-negative isolates

70
60
Percentage of bacteria
showing resistance
50
40
30
20
10
0
in
in
in
ol
n
n
in
lin
ci
ci
lli
lli
ic
yc
yc
yc
yc
pi
xa
ci
ci
en
yc
om
om
ra
pi
flo
ac
ph
eo
ta
Am
pe
fa
nc
hr
O
en
tr
m
N
Ri
Pi
yt
Va
Te
ra
G
Er
lo
Ch
Antibiotics
Fig. 6. Resistance levels of Parsık cave bacteria against various antibiotics which are grouped according to
their mode of action: (a) Gram-positive isolates; (b) Gram-negative isolates
of resistance to ofloxacin, which is a DNA/RNA and Gram-negative bacteria. Contrary to our study,
synthesis inhibitor like rifampicin, were observed in Lavoie et al. (50) showed that cave isolates were
Parsık cave isolates (11% in Gram-positive and 12% highly resistant to gentamycin, neomycin and
in Gram-negative). The resistance rate of Pajsarjeva chloramphenicol antibiotics (33–66% for Gram-
jama isolates to erythromycin was 73.6% for Gram- negative bacteria and 61–83% for Gram-positive
negative and 39% for Gram-positive bacteria. The bacteria).
resistance of Parsik cave isolates to erythromycin was Furthermore, the lowest resistance to cell wall
determined at lower levels of 20% and 21% for Gram- synthesis inhibitors was observed in piperacillin
negative and Gram-positive bacteria respectively. for both Gram-positive (12%) and Gram-
The levels of resistance to protein synthesis inhibitors negative (16%) bacteria. In the study conducted
other than erythromycin (gentamycin, neomycin, by Lavoie et al. (50), the resistance to piperacillin,
tetracycline and chloramphenicol) were determined compared to other antibiotic resistance, was found
to range from 12% to 20% for both Gram-positive to be lower (average 37.5%).

In our study, considering the cell wall synthesis produced by these isolates. Apart from the potential
inhibitors (vancomycin and ampicillin), Gram- of bacteria isolated from Parsık cave to produce
negative bacteria were found to be more resistant enzymes and antimicrobial agents, it is planned
than the Gram-positive ones. Similar to our study, to determine their potential use in biodegradation,
Avguštin et al. (49) revealed that cave Gram- self-healing concrete and production of
negative isolates showed higher resistance to antimicrobial drugs against phytopathogens and
ampicillin. entomopathogens.
According to VITEK® results, except ampicillin
and vancomycin, Actinobacteria were determined
5. Conclusion
to be the most resistant (47–70%) phylum to
all tested antibiotics. The highest resistance The microorganisms attached to the specific
to ampicillin and vancomycin was observed in environmental conditions of caves are important
the phylum Proteobacteria. Like the microbial in terms of exploring their uses and specific
diversity of caves, antibiotic resistance is also features. This study was the first microbiological
variable. While the antibiotic resistance rates study in Parsık cave. It has been demonstrated
were high, no isolate producing antimicrobial that enzymes such as arylamidases, carbohydrate
agent was detected in the study conducted by hydrolases and lipases found in bacteria isolated
Lavoie et al. (50). However, one of the antibiotic from Parsık cave can be important in industrial
resistance hypotheses in caves is that there is a as well as clinical fields. In addition, some of our
high rate of antibiotic resistance in the presence of isolates have shown antimicrobial activity and can
microorganisms producing antimicrobial agents. contribute to the development of new antibiotics.
Studies have shown that bacteria having antibiotic Antibiotic resistance profiles of Parsık cave isolates
genes can also produce antimicrobial agents (51, should be clarified in further studies through
52). In our study, it was found that 50% of the studies of their genes.
isolates producing antimicrobial agents were
resistant to at least two antibiotics. Therefore,
Acknowledgements
study of bacterial antibiotic resistance may
contribute to the development of new antibiotics. The author would like to thank the Anatolian
To clarify this issue, studies in this issue should be Speleology Association, Turkey, and Nahdhoit
continued. Ahamada Rachid, Department of Fundamental and
Industrial Microbiology of Istanbul University, for
their contributions. The author also thanks Istanbul
4. Further Work
University Scientific Project Unit, Turkey, (BAP
In our future studies, we are planning to purify Project No FYL-2016-20759 and FHZ-2017‑26457)
and use the enzymes and antimicrobial substances for financial support.
Glossary
Term Definition AspA L-aspartate arylamidase

5KG 5-keto-D-gluconate BACI bacitracin resistance
ADH1 arginine dihydrolase 1 BAlap beta-alanine arylamidase pNA
ADH2s arginine dihydrolase 2 BCL Gram-positive spore-forming bacilli
ADO Adonitol BGAL beta-galactosidase
AGAL alpha-galactosidase BGAR beta-galactopyranosidase
AGLTp glutamyl arylamidase pNA BGLU beta-glucosidase
AGLU alpha-glucosidase BGUR/
beta-glucuronidase
BGURr
AIA-G Actinomycetes isolation agar
BNAG beta-N-acetyl-glucosaminidase
AlaA alanine arylamidase
BXYL beta-xylosidase
AMAN alpha-mannosidase
CDEX cyclodextrin
AMY D-amygdalin
CIT citrate (sodium)
APPA Ala-Phe-Pro-arylamidase

CMT coumarate LIP lipase

5-cyano-2,3-ditolyl-tetrazolium LysA L-lysine-arylamidase
CTC
chloride MBdG methyl-beta-D-glucopyranoside
DAPI 4’,6-diamidino-2-phenylindole MHA Mueller Hinton Agar
dCEL D-cellobiose MNT malonate
dGAL D-galactose methicillin-resistant Staphylococcus
MRSA
dGLU D-glucose aureus
dMAL D-maltose MTE maltotriose
dMAN D-mannitol NAG N-acetyl-D-glucosamine

NAGA beta-N-acetyl-galactosaminidase
dMLZ D-melezitose
NC6.5 growth in 6.5% NaCl
dMNE D-mannose
NOVO novobiocin resistance
dRAF D-raffinose
O129R O/129 resistance (comp. vibrio.)
dRIB D-ribose
ODC ornithine decarboxylase
dSOR D-sorbitol
OFF fermentation/glucose
dTAG D-tagatose
OLD oleandomycin resistance
dTRE D-trehalose
OPTO optochin resistance
dXYL D-xylose
PHC phosphoryl choline
ELLM Ellman
PheA phenylalanine arylamidase
ESC esculin hydrolysis
PHOS phosphatase
GGAA Glu-Gly-Arg-arylamidase
PIPLC phosphatidylinositol phospholipase C
GGT gamma-glutamyl-transferase
PLE PalatinoseTM
GlyA glycine arylamidase POLYB_R polymyxin B resistance
GLYG glycogen ProA L-proline arylamidase
Gram-negative fermenting and non- PUL pullulan
GN
fermenting bacilli
PVATE pyruvate
Gram-positive cocci and non-spore-
GP PyrA L-pyrrolidonyl-arylamidase
forming bacilli
GPS Global Positioning System R2A Reasoner’s 2A agar
IARL L-arabitol SAC saccharose/sucrose

SAL salicin
IHISa L-histidine assimilation
SCA starch casein agar
ILATk L-lactate alkalinisation
SEA soil extract agar
IMLTa L-malate assimilation
SUCT succinate alkalinisation
INO myo-inositol
TSA 1/2 tryptic soy agar
INU inulin
TTZ tetrazolium red
IRHA L-rhamnose
TWA tap water agar
ISP4 inorganic salt-starch agar
TyrA tyrosine arylamidase
KAN kanamycin resistance
URE urease
LAC lactose
UV ultraviolet
LDC lysine decarboxylase
vancomycin-resistant Enterococcus
VRE
LeuA leucine arylamidase faecalis
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The Authors
Nihal Doğruöz Güngör is an Associate Professor in the Department of Fundamental and
Industrial Microbiology at Istanbul University, Turkey. She obtained her doctorate at
Istanbul University in 2008, focusing on microbiological corrosion of copper. Her research
interests include cave microbiology, antimicrobial activities of bacteria, microbial corrosion
and biotechnology.
Begüm Çandıroğlu is a biologist from Istanbul University. In 2014 she obtained her MSc in
microbiology in the Department of Fundamental and Industrial Microbiology at the same
university. She studied cave microbiology and worked with cave bacteria that live in soil,
water and cave walls. She is interested in biotechnology, enzymatic reactions of bacteria
and antibiotic resistance.

Antibacterial Potential of Six Lichen Species

against Enterococcus durans from Leather
Industry
Evaluation of acetone extracts obtained from several lichen species as
alternative natural antibacterial agents
Didem Berber* effect. The most successful lichen extract was

Department of Biology, Faculty of Arts and determined to be Usnea sp. at the concentrations
Sciences, Marmara University, Istanbul, Turkey; of 240 µg ml–1, 120 µg ml–1, 60 µg ml–1, 30 µg ml–1
Gastronomy and Culinary Arts Department, and 15 µg ml–1. In conclusion, lichen extracts seem
Faculty of Fine Arts, Maltepe University, to have potential antibacterial efficacies against
Marmara Eğitim Köyü, Istanbul, Turkey E. durans.
İpek Türkmenoğlu 1. Introduction

Biology Department, Institute of Pure and
Applied Sciences, Marmara University, Istanbul, The leather industry produces and exports high-
Turkey quality products with high added value to the world
market. However, several bacterial problems during
Nüzhet Cenk Sesal leather-making processes are reflected in finished
Department of Biology, Faculty of Arts and products and lead to economic losses. After the
Sciences, Marmara University, Istanbul, Turkey flaying process in slaughterhouses, microflora
on hide or skin surfaces change due to bacterial
*Email: yazi47@hotmail.com contamination originating from faeces, air, dust
or the animal skin itself and some bacteria easily
colonise (1–4).
Antibacterial resistant bacteria are a significant The soaking process is the first tannery operation
problem in the hide or skin soaking process due that recovers water loss during raw hide or skin
to their destructive properties on finished leather. curing applications. There are some criteria to be
Lichens may be a solution to overcome this taken into consideration during the soaking process
resistance problem. Enterococcus durans (99.86%) of raw hides or skins. Especially prolonged soaking
was isolated from soak liquor samples. For screening provides a convenient milieu for bacterial activity and
of possible antibacterial effects of lichen acetone damage to hides or skins may occur. Due to reduced
extracts, six lichen species (Hypogymnia tubulosa, salt content and high protein and lipid constituents,
H. physodes, Evernia divaricata, Pseudevernia hides or skins become defenceless against bacterial
furfuracea, Parmelia sulcata and Usnea sp.) were attacks in the soaking process (5–8). It has been
examined by nine-fold dilution against E. durans. reported that the number of bacterial populations
H. tubulosa, H. physodes and E. divaricata extracts in soak liquors may be up to 105 colony forming
showed antibacterial effects at the concentrations unit (CFU) ml–1 (5). But in a previous study, it was
of 240 µg ml–1, 120 µg ml–1 and 60 µg ml–1 demonstrated that total bacterial numbers were
whereas the extracts of P. furfuracea had an considerably higher than 105 CFU ml–1 in soak
antibacterial effect at 240 µg ml–1 and 120 µg ml–1. liquor samples (9). The adverse effects of the
On the other hand, P. sulcata had no antibacterial soaking process on the hide quality originate from

degradative enzymatic properties of bacteria such from numerous lichen extracts have been reported
as protease and lipase activities. These enzymatic to have biological activities such as antibacterial
activities can irreversibly affect the structure of activity against Gram-positive and Gram-negative
hide or skin substances that cannot be fixed at bacteria (24–27). It has been reported that
the subsequent stages of hide processing (10). approximately 2000 of the 20,000 lichen species
High numbers of bacteria with protease and lipase in the world are in Turkish lichen mycota. There
activities cause unwanted defects such as hair-slip, are many studies evaluating the bioactivities of
putrefaction, grain peeling, loose grain, holes on lichen species in Turkey against different bacterial
the hides or skins and light stains on the suede species (25–27). In the previous study, the acetone
surface (1, 3, 11–15). extracts of H. physodes, E. divaricata, P. furfuracea
Antibiotics are used in various industries as and Usnea sp. at different concentrations were
well as in the treatment of diseases. The World tested on some Bacillus species which were isolated
Health Organization declared that antimicrobial from soak liquor samples. These extracts were
resistance in most countries and industrial detected to have potential antibacterial effects (28).
sectors has increased dramatically (16, 17). From this point, lichen species may have potential
The emergence of antibiotic-resistant bacteria antibacterial efficacies against various antibacterial-
due to improperly used antibiotics in humans, resistant bacterial strains in the soaking process
animals and agriculture has been reported in the which cannot be exterminated by antimicrobial
literature (17). In the leather industry, to control agents. Therefore, the antibacterial effects of
bacterial numbers and their degradative properties acetone extracts of lichen species H. tubulosa,
on hides or skins, various antibacterial agents are H. physodes, E. divaricata, P. furfuracea, P. sulcata
utilised during the soaking process of beam house and Usnea sp. against Isolate 1 (E. durans), which
operations. The normal microflora in animals has protease and lipase acitivities, was evaluated
comprises many harmless bacteria but any of in the present study.
them may become resistant to commonly utilised
antibacterial agents due to intrinsic or acquired
resistance (17, 18). The resistant bacteria may
survive despite bactericides and may transfer their 2.1 Sample Collection
resistance properties to others through horizontal
gene transfer (5, 9, 18). Bactericides may remain Three soak liquor samples were collected from
ineffective against proteolytic and lipolytic bacteria Istanbul Leather Organized Industrial Zone, Tuzla,
in soak liquors because of high organic content Istanbul, Turkey. These samples were immediately
in soak liquors (9, 19). The existence of many placed into sterile sample bags and carried on ice
non-halophilic bacteria was demonstrated in the during transportation. Direct and serial dilutions
presence of an antimicrobial agent at twofold were spread onto nutrient agar plates. The
increased concentration (0.8 g l–1) (19). This morphologically different colony was picked up
finding emphasises the antibacterial resistance to obtain the pure culture of the isolate and was
of bacteria in the soaking process. More recently, numbered as Isolate 1.
it was reported that antimicrobial agents used in
the soaking process could not control multidrug-
2.2 Biochemical and Molecular
resistant Enterobacteriaceae from soaked
Analyses
sheepskins and cattle hides treated with an
antibacterial agent (20). Gram staining, catalase, oxidase, lipase and
Over the past decades, it has been suggested protease activities were examined. Protease
that alternative compounds from natural resources activity of Isolate 1 was examined on gelatin agar
may overcome the antimicrobial resistance of many medium containing 2% gelatin (w/v). The agar
bacteria. Previously, the potential of lichen derived plates were flooded with Frazier solution following
extracts from P. furfuracea (L.) Zopf was reported 24 h incubation. Clear zones around the colonies
in the leather industry (21). Lichens are symbiotic were evaluated as positive for protease activity.
organisms between a fungus and one or more Lipase activity was tested on Tween® 80 agar
algae or cyanobacteria. They synthesise unique medium containing 1% (w/v) Tween® 80. After
secondary metabolites that cannot be synthesised incubation, opaque zones around the colonies were
by higher plants (22, 23). Secondary metabolites accepted as evidence of lipase activity (29, 30).

Genomic DNA of Isolate 1 which was determined 2.5 Determination of Antibacterial

to have protease and lipase activities were Efficacies of Lichen Samples
extracted by phenol/chloroform extraction
and ethanol precipitation. DNA isolation was The test isolate was grown on Tryptic soy agar
confirmed by agarose gel electrophoresis. DNA media at 37°C for 24 h. The tests were performed
samples were stored at −20°C until use. The in 96-well CELLSTAR®, F-bottom microplates with
16S rRNA gene was amplified by polymerase lid (Greiner Bio-One GmbH, Austria). Tryptic soy
chain reaction (PCR) with the universal bacterial broth was added to each well and nine-fold serial
primers 27F (5-AGAGTTTGATCMTGGCTCAG) dilutions of the acetone extracts of H. tubulosa,
and 1492R (5-TACCTTGTTACGACTT). Negative H. physodes, E. divaricata, P. furfuracea, P. sulcata
control was included in PCR amplifications. and Usnea sp. were made. Final concentrations of
PCR amplification was carried out by an initial all lichen extracts were 240 µg ml–1, 120 µg ml– 1,
denaturation at 95°C for 4 min, followed by 30 60 µg ml–1, 30 µg ml–1, 15 µg ml–1, 7.5 µg ml– 1,
cycles at 95°C for 1 min, 57°C for 1 min and 73°C 3.75 µg ml–1, 1.9 µg ml–1 and 0.9 µg ml–1.
for 1 min. The reactions were finished by a final Overnight culture of the isolate was added to
extension at 73°C for 7 min. The PCR products were obtain a total volume of 100 µl with an optical
also monitored by agarose gel electrophoresis. density (OD) 600 nm of 0.01. The experiments
These products were purified by GeneJETTM Gel included untreated and blank controls. The tests
Extraction Kit (Thermo ScientificTM, Thermo were performed in three replicates. Bacterial
Fisher Scientific, USA). These purified samples growth ratios at an OD 600 nm were measured
were analysed by Medsantek Ltd Co, Istanbul, using CytationTM 3 Multi-Mode microplate reader
Turkey. The 16S rRNA sequence contigs were (BioTek Instruments Inc, USA).
generated by the software ChromasPro version
2.1.8 (Technelysium Pty Ltd, Australia). Then,
consensus sequences were exported in FASTA
format for each sample for data analysis. These In the present study, Isolate 1, which was obtained
sequences were compared with sequences in the from soak liquor samples collected from different
National Center for Biotechnology Information tanneries in Istanbul Leather Organized Industrial
(NCBI) using the Basic Local Alignment Search Zone, Turkey, was identified by biochemical and
Tool (BLAST®) search program. molecular techniques. To our knowledge, there is
no study on the antibacterial efficacies of lichen
extracts against E. durans from soak liquor samples.
2.3 Lichen Samples
For the first time, H. tubulosa, H. physodes,
The lichen samples belonging to H. tubulosa, E. divaricata, P. furfuracea, P. sulcata and Usnea
H. physodes, E. divaricata, P. furfuracea, P. sulcata sp. acetone extracts were examined against
and Usnea sp. were collected from fir trees of E. durans isolated from soak liquor samples.
Kastamonu province in the north-west of Turkey. Isolate 1 was Gram-positive, oxidase and
They were identified through classical taxonomical catalase-negative, protease and lipase positive.
methods by microscopic examination. The degradative protease and lipase activities of
H. tubulosa, H. physodes, E. divaricata, bacteria have an important role in the production
P. furfuracea, P. sulcata and Usnea sp: Turkey, of high-quality leather. There are many studies
Kastamonu province, Kapaklı Village, 41.24492, focused on protease and lipase activities of
34.18330, G. Çobanoğlu. halophilic, extremely halophilic and non-halophilic
bacteria on hides or skins in the literature.
McLaughlin and Highberger reported that bacterial
2.4 Extraction of Lichen Samples
strains with proteolytic activity were present in
The experiment steps included washing, drying in high percentages on salt-cured goat skins (31). The
air, weighing, pulverising by liquid nitrogen, adding proteolytic and lipolytic activities of halophilic and
acetone (ACS, ISO, Reag. Ph. Eur.), keeping in a extremely halophilic bacteria were also reported
dark place for 24 h followed by filtration through in previous studies. Birbir reported that 91% of
filter paper. Then, the evaporation of acetone in a 35 salt-cured skins had halophilic bacteria and
rotary evaporator was performed and crude lichen 67% of 85 extremely halophilic bacterial strains
acetone extracts were obtained (27). had proteolytic activities (32). Bailey and Birbir

detected that 98% of 131 brine-cured skin samples several unwanted defects on finished products
had extremely halophilic microorganisms and due to its enzymatic activities.
94% of 332 isolates from these samples showed Antibacterial agents that are commonly used in
proteolytic activity (12). Bitlisli et al. demonstrated the soaking process seem to be ineffective due
that 53–74% of halophilic bacteria from salt- to random or insufficient application and lead to
cured sheepskins had proteolytic activity and antimicrobial-resistant bacteria in soak liquors (12,
47–62% of them had lipolytic activity (33). There 19). From this point, we can suggest that E. durans
are also several studies revealing the proteolytic from salted hides or skins could not be exterminated
and lipolytic activities of non-halophilic bacteria by curing methods and also in the soaking process
from soak liquor samples. Veyselova et al. showed despite the use of antibacterial agents. There are
proteolytic activity of some bacteria belonging to the several studies focused on the determination of
genera Enterobacter, Pseudomonas, Enterococcus, effective concentrations of several antimicrobial
Lactococcus, Aerococcus, Vibrio, Kocuria, agents against various species of bacteria. Both
Staphylococcus and Micrococcus and lipolytic the ineffectiveness of antibacterial agents in some
activity of B. licheniformis, B. pumilus, P. luteola cases and possible harmful and toxic effects for the
and E. cloacae from soak liquor samples (10). environment and human health of some synthetic
In molecular analyses, the tested isolate was antimicrobial agents were emphasised in the
identified by comparative partial 16S rRNA gene literature (19, 21). In this respect, the need for
sequence analysis with the sequences deposited in safer, more ecological and effective materials has
the GenBank® database via the BLAST® program. come into prominence for the leather industry. In
The Isolate 1 had similarities with E. durans the previous study, the potential antibacterial effects
CMGB-120 (99.86%, GenBank® accession number of acetone extracts of H. physodes, E. divaricata,
MF348232.1). The existence of Enterococcus P. furfuracea and Usnea sp. at the concentrations of
species was previously reported from hides or skins 240 µg ml–1, 120 µg ml–1, 60 µg ml–1 and 30 µg ml–1
in the leather industry (6, 34). It is well known were demonstrated against Bacillus toyonensis,
that Enterococcus species are common in surface B. mojavensis, B. subtilis, B. amyloliquefaciens, B.
water, soil, vegetables and animal products and velezensis, B. cereus and B. licheniformis which were
they are naturally commensal members of gut isolated from soak liquor samples (28). In respect
microflora of human and warm-blooded animals. to these findings, we suggested that H. tubulosa,
Enterococcus avium, E. casseliflavus, E. durans, H. physodes, E. divaricata, P. furfuracea, P. sulcata
E. faecalis, E. faecium and E. gallinarum have and Usnea sp. acetone extracts may have
been isolated from salted hide samples (34). antibacterial potential against E. durans which has
Furthermore, despite increasing the concentration protease and lipase activities.
of antimicrobial agents containing didecyl According to our results, the acetone extracts of
dimethyl ammonium chloride from 0.4 g l–1 to P. sulcata had no antibacterial effect at all tested
0.8 g l–1, several bacteria including E. avium concentrations against E. durans (Figure 1).
and E. faecium were reported from soak liquor On the other hand, we observed a considerable
samples (19). These results suggest that some antibacterial effect for the acetone extracts of
Enterococcus species may come from salted hides H. tubulosa and H. physodes against E. durans.
and can survive in soak liquor samples even in the High inhibitory effects of these tested extracts
presence of antibacterial agents. Fluckey et al. for the growth of E. durans (above 50%
isolated 279 Enterococcus isolates from faecal inhibition) were detected at the concentrations
and hide samples. Among them, 169 isolates were of 240 µg ml–1, 120 µg ml–1 and 60 µg ml–1 with
detected to be E. durans by biochemical tests (35). inhibition ratios of 82.54%, 79.53% and 79.98%
E. durans is mostly found in pre-ruminant calves for H. tubulosa, and 86.8%, 78.2%, 77.75% for
and young chickens and can survive in moderately H. physodes, respectively (Figures 2 and 3).
harsh conditions such as various temperature The acetone extracts of P. furfuracea also had
ranges, pH degrees and salt concentrations as well antibacterial effect against E. durans at the
as detergents (36–38). Similarly to our results, concentrations of 240 µg ml–1 and 120 µg ml–1
the proteolytic and lipolytic activities of E. durans by the inhibition percentages of 80.63% and
were also demonstrated in previous studies. Aslan 85.2%. The other tested concentrations had
and Birbir detected that six E. durans isolates had also inhibitory effects on the tested bacteria but
proteolytic and lipolytic activities (34). In this the inhibition ratios recorded were below 50%
regard, Isolate 1 may have the potential to cause (Figure 4).

2.5
2.0
Optical density, OD
1.5
1.0
0.5
0
ed
60
30
15
75
9
24
0.
12
7.
1.
at
3.
re
nt
U
Concentration, µg ml–1
Fig. 1. Antibacterial effect of acetone extracts of P. sulcata against E. durans from soak liquor samples
2.0
Optical density, OD
1.5
1.0
0.5
0
ed
9
0
60
30
15
75
9
24
0.
12
7.
1.
at
3.
re
nt
U
Fig. 2. Antibacterial effect of acetone extracts of H. tubulosa against E. durans from soak liquor samples
Potential antibacterial efficacy was also detected and 89.5% respectively. Furthermore, a 58.1%
for the acetone extracts of E. divaricata against inhibition ratio was noted for the concentration of
E. durans. At the concentration of 240 µg ml–1, we 7.5 µg ml–1 (Figure 6).
detected 91% inhibition on the bacterial growth. All data showed that the acetone extracts of
Antibacterial effects were observed at the H. tubulosa, H. physodes, P. furfuracea, E. divaricata
concentrations of 120 µg ml–1 and 60 µg ml–1 with and Usnea sp. had potential antibacterial efficacies
inhibition ratios of 81% and 79% (Figure 5). at varying concentrations against E. durans.
Usnea sp. acetone extract was determined to be the Usnea sp. acetone extracts were found to have a
most successful among the tested lichen extracts. stronger inhibitory effect on the bacterial growth
240 µg ml–1, 120 µg ml–1, 60 µg ml–1, 30 µg ml–1 of E. durans, even at a low concentration of
and 15 µg ml–1 of the extracts belonging to Usnea 15 µg ml–1 (89.5% inhibition) compared to other
sp. had an antibacterial effect above 80% inhibition. extracts. These results emphasise the potential of
The inhibition ratios at these concentrations were lichens to be utilised as an antibacterial agent in
similar and recorded as 88.7%, 84.2%, 92%, 87.8% the leather industry. Further studies are needed

2.0
1.5
Optical density, OD
1.0
0.5
0
ed
60
30
15
75
9
24
0.
12
7.
1.
at
3.
re
nt
U
Fig. 3. Antibacterial effect of acetone extracts of H. physodes against E. durans from soak liquor samples
2.0
1.5
Optical density, OD
1.0
0.5
0
ed
60
30
15
75
9
24
12
7.
1.
0.
at
3.
re
nt
U
Fig. 4. Antibacterial effect of acetone extracts of P. furfuracea against E. durans from soak liquor samples
to detect potential compounds of these lichen P. furfuracea, E. divaricata and Usnea sp.) have
species and then these compounds may be used in antibacterial effects against E. durans with protease
formulations in the industry. and lipase properties. Whereas P. sulcata did not
have any antibacterial efficacy against E. durans,
other tested extracts were successful depending
4. Conclusions
on the lichen species and concentrations applied.
In the leather industry, bacteria with proteolytic and The acetone extracts of Usnea sp. had the highest
lipolytic activities are important in terms of finished antibacterial efficacy. The potential antibacterial
product quality. In this study, we tried to answer the efficacies of several lichen species suggest that
question of whether acetone extracts of six lichen compound(s) extracted from lichens as natural
species (H. tubulosa, H. physodes, P. sulcata, resources may be used in the leather industry. We

2.0
1.5
Optical density, OD
1.0
0.5
0
ed
9
0
60
30
15
75
9
24
0.
12
7.
1.
at
3.
re
nt
U
Fig. 5. Antibacterial effect of acetone extracts of E. divaricata against E. durans from soak liquor samples
2.0
1.5
Optical density, OD
1.0
0.5
0
ed
60
30
15
9
24
0.
12
7.
1.
at
3.
re
nt
U
Fig. 6. Antibacterial effect of acetone extracts of Usnea sp. against E. durans from soak liquor samples
believe that more comprehensive studies about (Marmara University) for sharing their experiences
their unique chemical compounds will provide new about the experiments.
insight to utilise them in this sector.
References
Acknowledgement
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The authors are grateful to Gülşah Çobanoğlu 103
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species tested. The authors would like to thank J. Am. Leather Chem. Assoc., 2016, 111, (10),
Arhun Ali Balkan, Ayla Yıldız and Barış Gökalsın 358

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The Authors
Didem Berber received her MSc degree from the Pediatric Allergy-Immunology Department,
School of Medicine, Marmara University, Turkey, in 2003 and PhD from the Department of
Biology, Faculty of Arts and Sciences, Marmara University in 2010. She has been studying
as postdoctoral researcher in the same department from 2016 up to date. She contributed
to projects (European Cooperation in Science and Technology (COST) and other bilateral
collaboration projects) on bacterial quorum sensing and biofilm inhibition. Her research
topics are hide microbiology, environmental microbiology, antimicrobial agents, fungi,
quorum sensing and biofilm formation.

İpek Türkmenoğlu graduated from the Biology Department, Atatürk Faculty of Education,
Marmara University in 2012. She is continuing to the master’s programme and she is
studying as a scholarship researcher with the support of Scientific and Technological
Research Council of Turkey (TÜBİTAK) on the determination and utilisation of species-
specific allosteric inhibition zones in glycolytic enzymes in pharmaceutical design. Her
research topics are hide microbiology, environmental microbiology, antimicrobial agents,
quorum sensing and biofilm formation.
Nüzhet Cenk Sesal graduated from the Biology Department, Atatürk Faculty of Education,
Marmara University. He has been working at the Department of Biology, Faculty of Arts
and Sciences, Marmara University since 2001. His research area is molecular microbiology.
He has been working as a principal investigator, researcher, and consultant in national and
international projects, especially about molecular diversity, environmental microbiology,
antimicrobial agents, quorum sensing and biofilm formation.

The Destructive Effects of Extremely Halophilic

Archaeal Strains on Sheepskins, and Proposals
for Remedial Curing Processes
Use of sterile brine or direct electric current to prevent red heat damage on
salted sheepskins
Meral Birbir*, Pinar Caglayan brine using DC to prevent archaeal damage on

Division of Plant Diseases and Microbiology, cured hides and skins in the leather industry.
Department of Biology, Faculty of Arts and
Sciences, Marmara University, Göztepe Campus,
1. Introduction
34722 Kadıköy, Istanbul, Turkey
Extremely halophilic archaea have been found in
Yasar Birbir hypersaline salt lakes, salterns, salt mines, salted
Department of Electrical and Electronics foods and salted hides. There have been numerous
Engineering, Faculty of Technology, Marmara studies on the presence of extremely halophilic archaea
University, Göztepe Campus, 34722 Kadıköy, in these hypersaline environments (1–12). Due to
Istanbul, Turkey the high salt requirements of extreme halophiles
(15–30% NaCl), these microorganisms have been
*Email: m.birbir64@hotmail.com denominated as extremely halophilic archaea (13,
14). Cells of Haloarchaea staining Gram-negatively
are irregular rods, cocci, pleomorphic rods, cups,
Proteolytic and lipolytic extremely halophilic irregular disks, flattened disks, irregular triangles,
archaea found in curing salt may contaminate skins rectangles and squares (2, 5, 15). Chemoorganotroph
during the brine curing process and damage skin extremely halophilic archaea, which can be motile or
structure. In the present study, three proteolytic non-motile, grow aerobically and use different amino
and lipolytic extremely halophilic archaea were acids. Colonies of these microorganisms are pink, red
isolated from deteriorated salted sheepskins and and orange due to C50‑carotenoid pigments called
characterised using conventional and molecular bacterioruberins (15, 16).
methods. Each test strain (Haloarcula salaria Observation of red or violet discolorations on
AT1, Halobacterium salinarum 22T6, Haloarcula the flesh side of salted hides and skins is the key
tradensis 7T3), a mixed culture of these strains and for detecting extremely halophilic archaea in the
the mixed culture treated with 1.5 A direct current leather industry. These discolorations are a sign of
(DC) were used for brine curing processes of fresh bacterial deterioration of hides and skins (17, 18).
sheepskins and examined during 47 days of storage Previous experiments reported that microorganisms
to evaluate the degree of destruction wreaked by in curing salts and raceway brines contaminated
these microorganisms. Both organoleptic properties hides and skins and caused red heat (10). The
and scanning electron microscopy (SEM) images of brine cured hides and skins were often stored in
sheepskins proved that each separate test strain hot warehouses, trucks or ships, and these high
and the mixed culture caused serious damage. temperature conditions, combined with moisture,
However, the mixed culture of strains treated with offer an ideal medium for proteolytic extremely
electric current did not damage sheepskin structure. halophilic archaea to grow and potentially digest
Therefore, we highly recommend sterilisation of collagen fibres in the hides and skins (10).

Extremely halophilic archaea (102–105 colony The adverse effects of extremely halophilic

forming units (CFU) g–1), proteolytic (102–104 archaeal hide isolates and ATCC strains of extremely
CFU g–1) and lipolytic (102–104 CFU g–1) extremely halophilic archaea on brine cured hides have been
halophilic archaea were detected in 40 curing reported in these studies, respectively (25, 26).
salt samples collected from different tanneries However, the destructive effects of salted sheepskin
in Turkey (19). Almost all salted hides and skins strains of extremely halophilic Haloarcula salaria,
contained extremely halophilic archaea, proteolytic Halobacterium salinarum and Haloarcula tradensis
and lipolytic extremely halophilic archaea originating on brine cured sheepskins have not been examined
in the curing salt. Extremely halophilic archaea were yet. Therefore, the aim of this study was to
also detected on 94% of 131 brine-cured cattle examine adverse effects of proteolytic and lipolytic
hides collected from USA, 91% of 35 salted hides archaeal sheepskin strains (Haloarcula salaria
cured in France and Russia and all salted hides cured AT1, Halobacterium salinarum 22T6, Haloarcula
in Turkey, Greece, the UK, USA, Serbia, Bulgaria, tradensis 7T3) and the mixed culture of these
Russia, South Africa and Australia (20–22). Five strains on sheepskins during a 47-day storage
extremely halophilic archaeal species, Halorubrum period at 33°C. We also investigated effective
saccharovorum, Halorubrum tebenquichense, curing methods to prevent the destructive
Halorubrum lacusprofundi, Natrinema pallidum effects of these microorganisms on sheepskins.
and Natrinema gari were isolated from five salted Additionally, we evaluated pH values, ash contents,
hides originating in England and Australia (22). moisture contents, salt saturations, total counts
Also, 101 extremely halophilic archaeal strains of extremely halophilic archaea and organoleptic
(Halorubrum tebenquichense, Halorubrum properties of the brine cured sheepskin samples
saccharovorum, Halorubrum kocurii, Halorubrum during different storage periods to determine the
terrestre, Halorubrum lipolyticum, Halococcus brine curing procedure’s efficiency and the test
dombrowskii, Halococcus qingdaonensis, microorganisms’ adverse effects of on sheepskins.
Halococcus morrhuae, Natrinema pellirubrum,
Natrinema versiforme, Halostagnicola larsenii
and Haloterrigena saccharevitans) were isolated
from four salted sheepskin samples (Spain) 2.1 Isolation of Extremely Halophilic
exhibiting bad odour, a slimy layer, hair slip, red Archaeal Strains from Deteriorated
and yellow discolorations (23). Moreover, 28 Salted Sheepskins
extremely halophilic archaeal strains (Natrialba
aegyptia, Halovivax asiaticus, Halococcus Two deteriorated salted sheepskins containing red
morrhuae, Halococcus thailandensis, Natrinema discolorations were collected from two tanneries
pallidum, Halococcus dombrowskii, Halomicrobium in the Istanbul Leather Organized Industrial
zhouii, Natronococcus jeotgali, Haloterrigena Zone (40°52′39.7″N,29°20′25.3″E) in Tuzla,
thermotolerans, Natrinema versiforme and Turkey. The samples were immediately placed
Halobacterium noricense) were isolated from eight into sterile sample bags and transported on ice to
salted hide and skin samples from Turkey, Iraq, the laboratory. Then, 20 g of the salt-pack cured
Turkmenistan and Kazakhstan (24). sheepskin samples were weighed and separately
While there are many reports that detect the soaked in flasks containing 180 ml 30% NaCl
presence of extremely halophilic archaea on salted (Merck KGaA, Germany) solution. The flasks were
hides and skins (10, 17, 20–25), the destructive placed into a shaking incubator at 90 rpm, 24°C
effects of these microorganisms on salted hides for 3 h. The suspension of the skin was diluted
have been studied much less (25, 26). In our with sterile physiological saline water (30% NaCl).
previous investigation, we found that extremely An aliquot of 100 µl each of direct and serial skin
halophilic archaeal strains, isolated from hides suspension dilutions was spread onto the surface
brine cured in the USA, damaged grain the surface of modified Brown agar media containing (per
of hides at 41°C after 49 days (25). An experiment litre): 1 g CaCl2·H2O, 2 g KCl, 20 g MgSO4·7H2O,
with extremely halophilic Haloferax gibbonsii 3 g trisodium citrate, 250 g NaCl, 5 g yeast extract,
(ATCC 33959TM) and Haloarcula hispanica (ATCC 20 g agar, pH 7 (5, 27). The plates were incubated
33960TM) obtained from American Type Culture at 39°C for 10 days. Following incubation, red
Collection (ATCC), USA, demonstrated that pigmented colonies on the agar media were
Haloferax gibbonsii caused hair slip, loss of hide selected and restreaked several times to obtain
substance and deterioration of brine cured hide pure cultures. A total of 22 isolates were obtained
after 45 days at 40°C (26). from the sheepskins and then, these strains were

examined the proteolytic and lipolytic activities. and oxidase activities, indole production, methyl
Proteolytic activity of each strain was detected red test, H2S and NH3 productions of each strain
on gelatin agar medium containing 2% gelatin. were investigated according to the procedures
After incubation, clear zones around the colonies described previously (4, 28, 31). Furthermore,
on the gelatin agar medium indicated protease each strain’s caseinase activity was determined on
production (5, 10). Lipolytic activity of each the agar medium containing 2% skim milk. After
strain was screened on Tween® 80 agar medium incubation, clear zones around the colonies were
containing 1% Tween® 80. After growth was evidence of positive caseinase activity (4). Urease
obtained, opaque zones around the colonies were production was investigated on Christensen urea
interpreted as positive lipase activity (5). In the agar medium. The tubes were examined for pink
present study three red pigmented proteolytic and or red colour change in the medium after seven
lipolytic strains (AT1, 22T6 and 7T3) were obtained days of incubation (28, 31). β-galactosidase
from two salted sheepskins and these strains were activity was screened in test tubes containing
used in the present study. ortho-nitrophenyl-β-galactoside (ONPG) discs and
1 ml of sterile saline water (30% NaCl). The yellow
colour formation in the test tube was accepted as
2.2 Phenotypic Characteristics of
positive β-galactosidase activity (5, 31). Amino
Test Strains
acid utilisation of each strain was examined in
Exponentially growing pure cultures of three strains the test medium containing 1% amino acid, 0.5%
designated as AT1, 22T6 and 7T3 were used in all beef extract, 0.5% peptone, 0.05% dextrose,
experiments. First, the strains’ salt requirement and 0.0005% cresol red, 0.001% bromocresol purple,
salt tolerance were examined on Brown agar plates 0.0005% pyridoxal and saline water (30% NaCl).
containing different salt concentrations (0%, 0.5%, Purple colour formation in the test tube containing
3%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25% archaeal culture was accepted as a positive test
and 30%) (27). After detection of optimum salt after 10 days incubation period at 39°C (31).
concentration for each strain, pH and temperature
ranges for growth of each strain (AT1, 22T6, 7T3)
2.3 Amplification and Sequencing of
were respectively examined at Brown agar plates
16S rRNA Genes of Test Strains
with different pH values (pH 4, pH 5, pH 6, pH 7,
pH 7.5, pH 8, pH 9, pH 10, pH 11 and pH 12) and Chromosomal DNA was isolated by QIAamp DNA
different temperatures (4°C, 10°C, 15°C, 24°C, Mini Kit (Qiagen, Germany) and purified by QIAquick
28°C, 35°C, 37°C, 39°C, 45°C, 50°C, 55°C, 60°C) PCR Purification Kit (Qiagen, Germany) according
according to the methods described in Proposed to the manufacturer’s directions. The 16S rRNA
Minimal Standards for Description of New Taxa in genes of the strains were amplified by polymerase
the Order Halobacteriales (28). Based on the pH, chain reaction (PCR) using forward primer 21F and
and temperature range of each test strain, the reverse primer 1492R (32). The 16S rRNA gene
optimal pH and growth temperature of each test sequences of three strains (AT1, 22T6 and 7T3)
strain were determined. were determined by IONTEK Laboratory (Turkey).
Pigmentation, size, margin, elevation and The sequences of these strains were analysed
opacity of colonies of the strains grown on using ChromasPro v.2.1.8 software (Technelysium,
Brown agar media were examined under optimal Australia) and then compared with the sequence
growth conditions (28). Cell morphology, cell on the EZBioCloud Database (ChunLab, South
length, cell width and motility of each strain Korea) (33).
were examined using both light microscopy and
electron microscopy. Microscopic observation of
2.4 Preparation of Test Strains and
each strain was made by using freshly prepared
Sheepskin Samples for Brine Curing
wet mount (28). For SEM observations, 20 ml of
Treatments
each test strain were separately passed through
0.2 μm pore size cellulose nitrate membrane filter 2.4.1 Preparation of Strains and
placed in the stainless steel funnel via vacuum Cultures Used in Brine Curing
pump (Sartorius AG, Germany). The archaeal Processes
cells of each strain trapped on the membrane
filters were observed under SEM (QuantaTM 450 Pure cultures of each test strain (AT1, 22T6,
FEG (FEI, USA)). Gram staining was performed 7T3) were separately grown in liquid Brown test
with acetic acid-fixed slides (28–30). Catalase medium containing 30% NaCl for 10 days at 39°C.

Each archaeal cell suspension’s turbidity was culture in the electrolysis cell before the electric
adjusted to 0.5 McFarland standard (108 CFU ml–1) current application, 100 µl of the test medium was
using densitometer (DEN-1, BIOSAN, Latvia). removed from the electrolysis cell and diluted to
Each cell suspension was diluted in sterile saline 10–2–10–4 using sterile 30% NaCl solution. The
solution (30% NaCl) to adjust the cell suspension diluted archaeal suspensions were spread over the
to 107 CFU ml–1. In addition, mixed cultures of Brown agar media. Then, 1.5 A DC was applied
these strains (107 CFU ml–1) were prepared. Then, to the electrolysis cell for 22 min (Figure 1). A
20 ml of each test strain, 20 ml of the mixed culture 100 µl quantity of test medium was removed from
were used in the brine curing solutions of T1–T4 the cell at intervals of 1 min, 4 min, 7 min, 10 min,
(Table I). 13 min, 16 min, 19 min and 22 min of electric
To prepare brine curing solution containing current application. Direct and diluted suspensions
electrically inactivated mixed culture (T5), 20 ml of electrically inactivated the mixed culture were
of the mixed culture containing AT1, 22T6, spread over Brown agar media. All inoculated
7T3 strains (107 CFU ml–1) were placed into the Brown media were incubated for 10 days at 39°C,
electrolysis cell consisting of a glass beaker having and colonies on the agar plates were counted.
two internally attached platinum wire electrodes This test medium was used for curing process of
and 180 ml of sterile brine solution (30% NaCl) (34, the sheepskin (T5) after 22 min of electric current
35). To detect the archaeal numbers of the mixed application on the mixed culture (Table I).
Table I Protocol for Brine Curing Treatments of Sheepskins Brine Curing Compositions
Control
59.5 g sheepskin sample + 200 ml sterile brine solution
Treatments
T1 59.5 g sheepskin sample + 180 ml sterile brine solution + 20 ml strain AT1 (107 CFU ml–1)
T2 59.5 g sheepskin sample + 180 ml sterile brine solution + 20 ml strain 22T6 (107 CFU ml–1)
T3 59.5 g sheepskin sample + 180 ml sterile brine solution + 20 ml strain 7T3 (107 CFU ml–1)
T4 59.5 g sheepskin sample + 180 ml sterile brine solution + 20 ml mixed culture (107 CFU ml–1)
59.5 g sheepskin sample + 180 ml sterile brine solution containing 20 ml electrically inactivated
T5
mixed culture
DC ammeter AC ammeter
Voltmeter DC 1 0 2 AC
A A
AC-DC Platinum electrodes

Main switch
Anode Cathode
Variac
100 0
220 V Fuse
50 Hz AC Test
0–220 V
DC output AC output medium
Input
+ –
R Mp
Insulated layer
Inverter switch Electrolysis cell
Fig. 1. Electrolysis cell system used 1.5 A DC treatment in this study (R: phase, Mp: ground)

2.4.2 Preparation of Sheepskin 2.6 Determination of pH, Moisture

Samples for Brine Curing Treatments Content, Ash Content and Salt
Saturation of Cured Sheepskin
One freshly slaughtered, de-fleshed whole sheepskin Samples
sample was obtained from a slaughterhouse in
Istanbul, Turkey. Then, the sheepskin sample was After curing processes, 5 g of the sheepskins were
immediately placed into sterile sample bag and cut and placed into flasks containing 100 ml of
transported on ice to the laboratory. The sheepskin sterile distilled water. The flasks were placed in a
was cut into six pieces perpendicular to backbone, shaking incubator for 1 h at 100 rpm and then pH
from backbone to belly. Next, we carried out the was measured with a pH meter. Hairs and dirt on
following six treatments for brine curing of the the samples were removed to properly determine
sheepskin samples. In each treatment, sterile the samples’ moisture content. 3 g of the samples
30% NaCl (Merck KGaA) solution was used. In all were placed into an oven at 102°C for 6 h. The
treatments, a 400% float of the brine solution (238 g dried samples were weighed, returned to the
of the brines without test strain, with each test oven for 1 h, and then were weighed again. The
strain or mixed culture/59.5 g of sheepskin) was drying procedure was repeated until the first dry
used (25). Sterile 30% NaCl solution containing weight was equal to the second dry weight. The
the sheepskin sample was used as Control. The samples were put into a desiccator for 30 min
sheepskin samples (T1–T4) were separately placed to cool. Next, we calculated the skins’ moisture
in a glass beaker containing the brine solution, each contents (20, 21). The dry sheepskins samples
test strain or mixed culture (T1–T4, Table I). In were placed in ceramic crucibles and ashed in a
the Treatment 5, the sheepskin sample was placed muffle furnace at 600°C for 8 h. After cooling, the
in a glass beaker containing the brine solution with samples were weighed to determine ash content.
electrically inactivated mixed culture (T5, Table I). Moisture content, ash content and salt saturations
The curing processes of all sheepskins were of skin samples were calculated according to the
carried out the protocol described in Table I. The aforementioned methods (30, 36). The pH value,
sheepskin samples were separately cured in the ash content, moisture content and salt saturation
brine solutions at 90 rpm for 18 h at 24°C. After of all cured sheepskin samples were examined at
the curing processes, all sheepskins were taken different storage periods.
from the brine solutions and stored for 47 days at
33°C.
2.7 Organoleptic Examination of
Brine Cured Sheepskin Samples
2.5 Determination of Extremely During Storage Periods
Halophilic Archaeal Counts in Curing
All cured sheepskin samples were examined
Solutions and Cured Sheepskin
organoleptically (hair slip, deterioration of skins,
Samples bad odour, sticky appearance, red heat, hole
To determine total counts of extremely halophilic formation) during different storage periods.
archaea in the curing solutions before the curing
processes, 100 µl of the test medium was removed
2.8 Preparation of Sheepskin
from the each curing solution and diluted to
Samples for Scanning Electron
10–2–10–4 using sterile 30% NaCl solution. The
Microscopy Observation
diluted archaeal suspensions were spread over
the Brown agar media. In addition, subsequent After a 47-day storage period, the sheepskin
to each brine curing process detailed above samples were prepared for SEM observation. The
(T1–T5), the suspensions of cured sheepskin samples were fixed in 4% glutaraldehyde solution
samples were prepared at intervals of 5 days, prepared in 0.1 M phosphate buffer (pH 7.2) for
16 days, 28 days and 47 days of storage. 2 g of 30 min. The samples were washed three times
each skin sample were put into a flask containing with 0.1 M phosphate buffer for 10 min and
18 ml sterile 30% NaCl solution and incubated were treated with 1% OsO4 prepared in 0.1 M
for 1 h at 24°C and 100 rpm. Direct and serial phosphate buffer at room temperature for 1 h.
dilutions of the suspensions were spread onto the The samples were washed two times in sterile
surface of Brown agar media. All inoculated Brown distilled water for 10 min. Then, the water in the
media were incubated at 39°C for 10 days and the sheepskins was gradually removed by 35%, 50%,
colonies grown on the test media were counted. 75%, 95% and absolute ethanol. The mixtures

of ethanol-hexamethyldisilazane (ethanol‑HMDS) lipase enzymes were selected and used as test

[1:1 (v/v)] (1 × 30 min), ethanol-HMDS strains (AT1, 22T6 and 7T3) in the present study.
[1:2 (v/v)] (1 × 30 min) and HMDS (2 × 30 min)

were used for air drying process. After drying,

3.2 Phenotypic Characteristics of
HMDS was poured from petri dishes and the
Test Strains
samples were placed in a desiccator for 12 h.
Later, the sheepskin samples were examined under Strains AT1, 22T6 and 7T3 grew at 15–30% NaCl,
SEM (QuantaTM 450 FEG) using sample stub with 15–30% NaCl, 20–30% NaCl concentrations,
double-sided sticky tape (37). respectively. Optimum salt concentrations of strains
AT1, 22T6 and 7T3 were determined as 25% NaCl.
Hence, these strains were accepted as extremely
halophilic archaea. The pH and temperature
3.1 Isolation and Selection of Test ranges for growth of strains AT1, 22T6 and 7T3
Strains from Sheepskins were respectively found as pH 6–11 and 20–50°C,
pH 6–11 and 15–55°C, pH 5–11 and 15–55°C. All
A total of 22 red coloured strains were isolated extremely halophilic archaeal strains optimally grew
from two deteriorated salted sheepskin samples at 39°C and pH 7. The colony pigmentation, size,
obtained from two tanneries in the Istanbul Leather margin, elevation and opacity of strains AT1, 22T6,
Organized Industrial Zone in Tuzla, Turkey. While 7T3 were respectively observed as: red, 0.6– 2 mm,
nine, seven and three strains respectively produced entire, convex, translucent; red, 1–2 mm, entire,
protease, lipase, both protease and lipase, three convex, translucent; red, 0.8–1.9 mm, entire,
strains did not produce either lipase or protease convex, translucent. The cells of strains AT1
enzymes. The red coloured three extremely (Figure 2(a)) and 7T3 (Figure 2(c)) were non-
halophilic strains producing both protease and motile, extremely pleomorphic (triangle, square,
(a) (b) Fig. 2. SEM micrographs

of pleomorphic test
strains of (a) Haloarcula
salaria (AT1) cells;
(b) Halobacterium
salinarum (22T6)
cells; (c) Haloarcula
tradensis (7T3)
cells trapped on the
membrane filter
5 μm 10 μm
(c)
5 μm

irregular disk, short rod). The cells of strains AT1 and of sheepskin causing loss of skin substance. When
7T3 were approximately 0.4–1.3 µm × 0.4–2.0 µm and the protein structure of salted skins is broken down
0.3–0.7 µm × 0.3–4 µm, respectively. The cells of by proteolytic extremely halophilic archaea, these
strain 22T6 (Figure 2(b)) were motile, pleomorphic microorganisms can utilise some amino acids as a
rods, approximately 0.5–1.2 µm × 3.2–6.6 µm. source of carbon, nitrogen and energy. Haloarcula
All strains were Gram-negative (Table II). While salaria AT1 and Halobacterium salinarum 22T6
all strains showed positive catalase, oxidase, utilised most of the amino acids examined. While
protease, lipase activities, indole production, the Haloarcula salaria AT1, Halobacterium salinarum
methyl red, caseinase, urease and β-galactosidase 22T6 utilised 17 amino acids, Haloarcula tradensis
reactions of all strains were negative. The strains 7T3 used only three amino acids (Table III). In
did not produce H2S and NH3 (Table II). another study, the liquid test media containing
Our experimental results showed that Haloarcula calfskin samples, 30% NaCl and proteolytic red and
salaria (AT1), Halobacterium salinarum (22T6), pink strains of the extremely halophilic archaea
Haloarcula tradensis (7T3) strains have protease were separately prepared to show disintegration
activities which can breakdown proteins in corium of the skin proteins. After an incubation period,
Table II Phenotypic Characteristics of Haloarcula salaria, Halobacterium salinarum,

Haloarcula tradensis
Characteristics Haloarcula salaria Halobacterium salinarum Haloarcula tradensis
Strain code AT1 22T6 7T3
Motility Non-motile Motile Non-motile
Extremely
Cell morphology Pleomorphic rods Extremely pleomorphic
pleomorphic
Cell width, µm 0.4–1.3 0.5–1.2 0.3–0.7
Cell length, µm 0.4–2 3.2–6.6 0.3–4
Gram staining Negative Negative Negative
Pigmentation Red Red Red
Colony size, mm 0.6–2 1–2 0.8–1.9
Colony margin Entire Entire Entire
Colony elevation Convex Convex Convex
Colony opacity Translucent Translucent Translucent
NaCl concentration, % 15–30 15–30 20–30
pH range 6–11 6–11 5–11
Temperature range, °C 20–50 15–55 15–55
Optimum NaCl 25 25 25
Optimum Temperature,
39 39 39
°C
Optimum pH range 7 7 7
Catalase activity + + +
Oxidase activity + + +
Methyl red reaction – – –
Caseinase activity – – –
Urease activity – – –
β-galactosidase activity – – –
Indole production – – –
H2S production – – –
NH3 production – – –
Protease activity + + +a
Lipase activity + + +
a
Haloarcula tradensis (7T3) showed weak protease activity

Table III Utilisation of Amino Acids by Strains

Halobacterium Haloarcula
Amino acids Haloarcula salaria (AT1)
salinarum (22T6) tradensis (7T3)
L-arginine + + +
L-cysteine – – –
L-glycine + + –
L-alanine + + –
L-tyrosine + + –
L-proline + + –
L-hydroxyproline + + –
L-glutamic acid – – –
L-methionine + + –
L-serine + + –
L-isoleucine + + –
myo-inositol + + –
L-lysine + + +
L-phenylalanine + + –
L-leucine + – –
L-valine + + –
L-threonine + + –
L-ornithine + + –
L-histidine + + +
L-aspartic acid – – –
L-cystine – + –
decomposition of the skin samples in the media Center for Biotechnology Information, USA) under
was detected by visual observation. While contents accession numbers as MN585896, MN585803,
of asparagine, threonine, serine, glutamine, MN585804.
proline, glycine, alanine, valine, isoleucine, In our previous study, extremely halophilic
leucine, phenylalanine, lysine and arginine in the archaeal strains were isolated from Tuz Lake and
test tubes were detected at high levels, contents of its salterns (5). In Turkish leather industry, curing
methionine, tyrosine and histidine were low (10). salt is mostly obtained from Tuz Lake and its
Phenotypic features of extremely halophilic AT1, salterns. Hence, we suspect that contaminations of
7T3 and 22T6 strains detected in this study were our sheepskin samples with Haloarcula salaria AT1,
fairly similar to phenotypic features of Haloarcula Halobacterium salinarum 22T6 and Haloarcula
salaria, Haloarcula tradensis and Halobacterium tradensis 7T3 were due to the curing salt obtained
salinarum isolated by other researchers (15, 38, 39). from Tuz Lake and its salterns.
3.3 16S rRNA Gene Sequences of 3.4 Extremely Halophilic Archaeal

Test Strains Counts in Curing Solutions Before
Curing
The phylogenetic analysis revealed that three
strains shared highly similar identities with their In the study carried out with 25 salted sheepskin
closest phylogenetic relatives. Strains AT1, 22T6, samples (Australia, Bulgaria, Dubai, Greece,
7T3 were respectively assigned to Haloarcula Israel, Kuwait, South Africa, Turkey, USA) and
salaria (98.36%-1344 base pairs), Halobacterium 25 salted goat skin samples (Australia, Turkey,
salinarum (99.78%-1345 base pairs), Haloarcula Bulgaria, Israel, South Africa, Russia, China,
tradensis (98.37%-1355 base pairs). The gene France), proteolytic extremely halophilic archaea
sequence data of the strains AT1, 22T6, 7T3 were and lipolytic extremely halophilic archaea were
respectively deposited in GenBank® (National detected as 102–105 CFU g–1; 102–106 CFU g–1

and 102–106 CFU g–1; 102–106 CFU g–1 on salted from 2.1 × 106 CFU ml–1 to 3.2 × 105 CFU ml–1
sheepskins and goat skins, respectively (40). after 1 min of DC treatment, the cell numbers of
The highest number of proteolytic and lipolytic 1.24 × 102 CFU ml–1 was detected after 4 min
extremely halophilic archaea on the salted skins of DC treatment. All archaeal cells in the mixed
was found as 106 CFU g–1 (40). Therefore, the culture were completely killed in 7 min of DC
archaeal cell numbers of test strains in the brine treatment. In the present study, log10 value of the
curing solutions were adjusted to 106 CFU ml –1. mixed culture of extremely halophilic archaea in
Before the curing processes of sheepskins, while the brine solution before the DC treatment was
the archaeal cell numbers in the brine solutions 6.32. After 1 min, 4 min and 7 min of 1.5 A DC
of Treatments 1, 3 and 4 were detected as treatment; 0.82, 4.23 and 6.32 log10 reduction
2.1 × 106 CFU ml–1, the archaeal cell numbers in values (CFU ml–1) of the mixed culture in the brine
the brine solution of Treatment 2 was detected were detected, respectively.
as 2.2 × 106 CFU ml–1. Temperature and pH of the electrolysis cell were
The archaeal cell numbers in the mixed culture was respectively measured as 31°C and pH 6 prior to the
detected as 2.1 × 106 CFU ml–1 in the electrolysis electric current treatment. After treating the brine
cell before 1.5 A DC application. While the archaeal solution with the electric current, the temperature
cell numbers in the mixed culture were reduced of the brine was adjusted to 24°C for using in
Table IV pH, Ash Content, Moisture Content and Salt Saturation Values, Total Extremely
Halophilic Archaeal Counts of the Sheepskin Samples After Different Storage Periods
Ash Moisture Salt Total count of extremely
Experiment pH
content, % content, % saturation, % halophilic archaea
After 5 days
Control 7.55 20 55 >100 0
T1 6.72 24 50 >100 2.0 × 107
T2 6.59 23 50 >100 3.4 × 107
T3 6.65 21 57 >100 2.2 × 107
T4 6.53 26 52 >100 3.8 × 107
T5 7.80 21 57 >100 0
After 16 days
Control 7.43 25 50 >100 0
T1 6.52 30 47 >100 3.0 × 107
T2 6.70 27 51 >100 6.0 × 107
T3 6.65 22 50 >100 3.4 × 107
T4 6.85 32 46 >100 8.4 × 107
T5 7.32 23 55 >100 0
After 28 days
Control 7.40 28 45 >100 0
T1 7.70 29 40 >100 1.2 × 107
T2 7.52 29 43 >100 2.0 × 107
T3 7.36 33 44 >100 2.0 × 107
T4 7.51 32 39 >100 3.4 × 107
T5 7.81 29 46 >100 0
After 47 days
Control 7.26 41 30 >100 0
T1 7.58 34 26 >100 1.0 × 107
T2 7.47 34 35 >100 1.8 × 107
T3 7.31 44 24 >100 1.7 × 107
T4 7.60 37 38 >100 2.0 × 107
T5 7.64 33 33 >100 0

curing process of sheepskin in the Treatment 5. 3.6 pH, Moisture Content, Ash
While the temperature and pH of the test medium Content and Salt Saturation Values
respectively increased from 31°C to 41°C and of Cured Sheepskin Samples
from pH 6 to pH 8.5 during the electric current
treatment, voltage values slightly decreased from After the curing processes of skins, pH values of the
4.7 V to 4.3 V. sheepskin samples were measured as pH 7.35 for
We also demonstrated the inactivation Control; pH 6.89 for T1; pH 7.09 for T2; pH 7.05
of extremely halophilic strains via DC and for T3; pH 7.16 for T4; pH 8.05 for T5. While salt
alternating electric current (AC) in our previous saturation values of all cured sheepskins were
studies (35, 41, 42). A 0.5 A DC was applied for higher than 100% during all storage periods, pH,
30 min to several strains of extremely halophilic ash content and moisture content values changed
archaea (107 CFU ml–1) isolated from Tuz Lake, during different storage periods. pH, ash content
Kaldırım and Kayacık salterns (35). While the and moisture content values of the cured skins
mixed culture of extremely halophilic archaea were detected between pH 6.52–7.81, 20–44%,
was exterminated in 10 min, protease producing 24–57%, respectively (Table IV).
extremely halophilic archaea were killed in 5 min. Moisture, minimum and maximum ash contents,
However, lipase or lipase and protease producing salt saturation values of adequately cured salted
extremely halophilic archaea were exterminated hides were suggested as 40–48%, 14–48%, higher
in 20 min (35). In another experiment, lipase than 85%, respectively (36). Due to detection of
and protease producing extremely halophilic high moisture content in all samples (between 50–
strains (105–106 CFU ml–1), separately grown 57%) after five days storage, sterile salt was added
in liquid Brown media, were inactivated by a to all sheepskins to reduce their moisture contents
10 min treatment with 0.5 A DC (41). It was according to curing procedure described in the
also detected that 1 min of 2 A AC treatment previous study (43). While all skin samples reached
was enough to kill extremely halophilic archaea the suggested moisture content values (39–46%)
found in brine solution (102–104 CFU ml–1). When after 28 days, the suggested saturation values
2 A AC was applied to lipolytic extremely halophilic were detected after five days. The samples’
archaea, proteolytic extremely halophilic lowest moisture content values were detected
archaea, both proteolytic and lipolytic extremely after 47 days. Ash contents of all skins (20–44%)
halophilic archaea, and a mixed culture of these were close to suggested values (36). While the
strains (106 CFU ml–1), all test microorganisms skins’ pH values changed during storage periods,
found in 25% NaCl solution were exterminated all values were found sufficient to support the
in 5 min (42). growth of extremely halophilic strains (Table IV).
The pH, moisture content, ash content and salt
saturation values detected in this study were also
3.5 Extremely Halophilic Archaeal
consistent with pH range (pH 6.53–8.01), moisture
Counts on Cured Sheepskin Samples
content (32–68%), ash content (12–30%) and
During Storage
salt saturation (58–100%) values of 25 salted
After the curing processes of sheepskins, we did sheepskin samples determined in the previous
not detect any extremely halophilic archaea on experiment (40).
the sheepskin sample cured with the sterile brine
solution (Control) and the sheepskin sample cured
3.7 Organoleptic Characteristics
with the brine solution containing electrically
of Brine Cured Sheepskin Samples
inactivated mixed culture (T5) during the all
During Storage
storage periods.
While extremely halophilic archaeal numbers on While hair slip and bad odour were detected on
both skin samples cured with each strain and the the sheepskin samples cured with each strain and
skin sample cured with mixed cultures of the strains the mixed culture after five days at 33°C, sticky
slowly increased from 106 CFU ml–1 to 107 CFU appearance and red heat were observed on the cured
during five days and 16 days storage periods, the sheepskin samples after 16 days (T1–T4, Figure
numbers of these strains slowly decreased 28 days 3). In addition to the aforementioned organoleptic
and 47 days storage periods due to attachment of properties, hole formations were observed on
these cells to sheepskins (Table IV). these sheepskin samples after 28 days. However,

we did not detect any organoleptic properties on significantly debilitated structural integrity of the
sheepskin samples cured with sterile brine and cells in the mixed culture trapped on the filter
the brine treated with 1.5 A DC (Control and T5, (Figure 5). The SEM images clearly showed that
Figure 3). electric current application damaged cell structures
In another study, the commercially cured of each strain in the mixed culture (Figure 5). As
hides stored one year in the USA were also seen in Figure 6, the sterile brine curing process
examined for proteolytic activity of extremely protected the sheepskin against microbial damage
halophilic archaea. Experimental results of during 47 days of storage.
that study showed that the flesh side of hides Attachment of Haloarcula salaria AT1,
containing extremely halophilic archaea had pink Halobacterium salinarum 22T6 and Haloarcula
discolorations called red heat. When these hides tradensis 7T3 to corium fibres and the consequent
were incubated at 35°C–40°C, bad odour, hair destructive effects on sheepskins are seen
slip and severe grain damage were detected. in Figures 7–10. Haloarcula salaria AT1,
Damaged grain surfaces were observed on Halobacterium salinarum 22T6 and the mixed
leather made from these hides (10). In another culture of the strains caused fibres in the corium to
experiment researchers emphasised that split and weaken (Figures 7, 8 and 10). In contrast
temperatures of the brines and hides should be with the skin samples treated with Haloarcula
maintained below 20°C to prevent growth of salaria AT1, Halobacterium salinarum 22T6, skin
extremely halophilic archaea (44). sample treated with Haloarcula tradensis 7T3 had
compact appearance, although the shredding of
the fibres was still present in corium (Figure 9).
3.8 Scanning Electron Microscopy
That damage was due to the proteolytic activities
Observation of Mixed Culture and
of these microorganisms.
Treated Sheepskin Samples
Figure 11 clearly shows that the curing process of
Figure 4 shows extremely halophilic archaeal cells sheepskin with the brine containing mixed culture
of the mixed culture on 0.2 µm pore-size cellulose treated with DC prevented extremely halophilic
nitrate membrane filter in pleomorphic shapes archaea from contaminating the sheepskin and
such as triangle, square, irregular disk and rod. As furthermore protected the skin very well against
seen in the SEM micrograph, 1.5 A DC treatment microbial damage during a long storage period.
(a) (b) (c) Fig. 3. Organoleptic

characteristics of brine
cured sheepskin samples
after 16 days storage
period: (a) Control,
sheepskin sample cured
with sterile brine (30%
NaCl); (b) T1, sheepskin
sample cured with brine
containing Haloarcula
salaria AT1; (c) T2,
(d) (e) (f) with brine containing
Halobacterium salinarum
22T6; (d) T3, sheepskin
sample cured with brine
containing Haloarcula
tradensis 7T3; (e) T4,
with brine containing
mixed culture; (f) T5,
with brine containing
electrically inactivated
mixed culture

20 μm 30 μm
Fig. 4. SEM micrograph of mixed culture of Fig. 7. SEM micrograph of the longitudinal section
undamaged pleomorphic cells of Haloarcula of damaged corium layer of sheepskin treated with
salaria (AT1), Halobacterium salinarum (22T6) Haloarcula salaria (AT1) stored for 47 days at 33°C
and Haloarcula tradensis (7T3) trapped on the
membrane filter
10 μm 40 μm
Fig. 5. SEM micrograph of mixed culture of Fig. 8. SEM micrograph of the longitudinal section
damaged Haloarcula salaria (AT1), Halobacterium of damaged corium layer of sheepskin treated with
salinarum (22T6) and Haloarcula tradensis (7T3) Halobacterium salinarum (22T6) stored for 47 days
cells treated with 1.5 A DC trapped on the at 33°C
membrane filter
50 μm
50 μm
Fig. 9. SEM micrograph of the longitudinal section
Fig. 6. SEM micrograph of the longitudinal section of damaged corium layer of sheepskin treated with
of undamaged sheepskin structure treated with Haloarcula tradensis (7T3) stored for 47 days at
sterile brine (Control) stored for 47 days at 33°C 33°C
The present study proved that organoleptic Electron micrographs also showed that each test
changes detected in the sheepskins were closely isolate and a mixed culture of extremely halophilic
related to proteolytic and lipolytic activities of strains destroyed the skins’ collagen fibres. We
extremely halophilic archaeal strains on the skin. did not detect any difference when assessing the

50 μm 100 μm
Fig. 10. SEM micrograph of the longitudinal section Fig. 11. SEM micrograph of the longitudinal section
of damaged corium layer of sheepskin treated of undamaged sheepskin structure treated with
with mixed culture of Haloarcula salaria (AT1), electrically inactivated mixed culture stored for 47
Halobacterium salinarum (22T6), Haloarcula days at 33°C
tradensis (7T3) stored for 47 days at 33°C
efficacy of sterile brine and electrically treated brine application of 1.5 A DC treatment in 7 min. Our
curing processes of sheepskin samples throughout experimental results proved that a curing process
47 days. We did not observe any damage to the using sterile brine or brine treated with electric
compactness of sheepskin structure cured with current prevented red heat and deterioration of
both the sterile brine and electrically treated brine sheepskins during long storage periods. Therefore,
containing the mixed culture. Both methods were an environmentally friendly, easy, cheap, very
found very effective for preventing archaeal growth simple electrolysis system is a very attractive
and damage on the brine cured sheepskins. alternative for brine disinfection: (a) it kills different
Our results were consistent with those of other species of microorganisms including proteolytic
experimental studies on the extremely halophilic and lipolytic extremely halophilic archaea; (b) it
strains and culture collection strains of extremely prevents development of resistant strains in leather
halophilic archaea (25, 26). In our previous factories; (c) it kills very effectively the aggregated
experiment, SEM images showed that hides cured microorganisms found in the brine containing high
with proteolytic extremely halophilic archaeal organic substances; (d) it can achieve a reduction
strains had red heat and severe grain damage after factor of more than 6 log10 for proteolytic and
49 days of storage at 41°C (25). In another study, lipolytic extremely halophilic archaea; and (e) it
the cured hides with extremely halophilic Haloferax has irreversibly lethal action. Hence, we suggest
gibbonsii (ATCC 33959TM) exhibited hair loss, using this effective brine disinfection system in
thinner and flaccid structure; these consequences of the leather industry after sufficient insulation and
deterioration and loss of hide substance. The open grounding are provided by an electrical engineer.
fibre structure was also detected in the corium of
the hide inoculated with Haloferax gibbonsii (27).
Acknowledgements
The SEM images showed that the fibre structures
of hide were broken down into the smaller fibres This study is dedicated to our wonderful teachers
after 43 days (27). who have contributed so much to science and
taught us to love this discipline. We thank Yeşim
Müge Şahin, Demet Sezgin Mansuroğlu (Istanbul
4. Conclusion
Arel University, Turkey) and Aslıhan Çetinbaş Genç
This is the first study that detects adverse effects (Marmara University) for the SEM micrographs. We
of characterised extremely halophilic archaeal are also grateful to Martin Louis Duncan (Virginia
strains on brine cured sheepskins with SEM. Union University, USA) for his critical reading of the
SEM images proved that proteolytic and lipolytic manuscript.
Haloarcula salaria AT1, Halobacterium salinarum
22T6, Haloarcula tradensis 7T3 caused corium
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The Authors
Meral Birbir graduated from the Biology Department, Ataturk Faculty of Education,
Marmara University, Turkey. She received her MSc and PhD Degrees in Biology (especially
microbiology) from the Institute of Pure and Applied Sciences, Marmara University. Professor
Birbir has been working in the Biology Department of Marmara University since 1985. She
was a research scientist at the Department of Pathology and Microbiology, Veterinary
Medical School, Purdue University, USA (1990) and Hides and Leather Department of
the United States Department of Agriculture (1992–1993). Her research interests are
halophilic microorganisms, hide/skin microbiology, antimicrobial agents, electric current
applications on microorganisms and microbial communities in hypersaline environments.
Pinar Caglayan graduated from the Biology Department, Ataturk Faculty of Education,
Marmara University. She received her MSc and PhD Degrees in Biology from the Institute
of Pure and Applied Sciences, Marmara University. She was an Erasmus student in the
Department of Microbiology and Parasitology, Faculty of Pharmacy, Sevilla University,
Spain (2008–2009). She has been working as a research and teaching assistant at the
Division of Plant Diseases and Microbiology, Marmara University since 2011. Her research
interests are moderately halophilic bacteria, extremely halophilic archaea, antimicrobial
agents, hide microbiology and electric current applications on microorganisms.
Yasar Birbir received his BSc Degree from Gazi University, Turkey, and MSc and PhD
Degrees in Electrical Education from Marmara University. He has been working at Marmara
University since 1983. He attended the World Bank Industrial Training Project at Indiana
and Purdue Universities, USA (1989–1990). He worked as a visiting research scientist at the
Electrical and Computer Engineering Department of Drexel University, USA (1992–1993).
He has been working as a Professor at the Technology Faculty, Department of Electrical
Engineering, Marmara University. His current interests are power electronic converters and
drivers, electromagnetic filtering processes in industry and applications of electric currents
for inactivation of different microorganisms.

Johnson Matthey Highlights

A selection of recent publications by Johnson Matthey R&D staff and
collaborators
A Stochastic Approach to Model Chemical Looping propylene selectivity had a decrease in response
Combustion to the metals.
M. A. Schnellmann, G. Williams and J. S. Dennis,
Ga2.52V2.48O7.33(OH)0.67, A Synthetic Member of the
Powder Technol., 2020, 365, 39
Nolanite/Akdalaite-Type Family of Oxyhydroxides
Using two coupled fluidised-bed reactors, a Containing Trivalent Vanadium
stochastic model for reactor-regenerator systems D. S. Cook, M. R. Lees, J. M. Fisher, D. Thompsett
was established. As a result the stochastic model and R. I. Walton, J. Solid State Chem., 2020, 288,
was able to achieve the simulation of the circulating 121396
fluidised bed with acceptable precision. This
Powder neutron diffraction shows oxyhydroxide
model had been used before to comprehend how
Ga2.52V2.48O7.33(OH)0.67 prepared by reaction
sensitive a chemical looping combustion (CLC)
between gallium metal and Na3VO4 in a 1:1
process is when other factors, for example the
monoethanolamine:water mixture at 240ºC
nature of gas-solid reactions, are included. To
demonstrates the material is isostructural with
show the stochastic model has value for simulation
nolanite and akdalaite (Figure 1). Rietveld
and optimisation formations of CLC it is applied
refinement was undertaken against the data
here with methane fuel gas in a laboratory-scale
showing all vanadium is octahedrally coordinated.
circulating fluidised bed.
Vanadium’s oxidation state is close to V3+ when
ZSM-5 Additive Deactivation with Nickel and vanadium K-edge XANES spectroscopy is used.
Vanadium Metals in the Fluid Catalytic Cracking There is dehydration around 300ºC (oxide
(FCC) Process Ga2.52V2.48O8 is produced and has a larger amount
A. A. Gusev, A. C. Psarras, K. S. Triantafyllidis, A. of V4+) followed by decomposition at 500ºC. While
A. Lappas, P. A. Diddams and I. A. Vasalos, Ind. both materials seem to follow the Curie-Weiss
Eng. Chem. Res., 2020, 59, (6), 2631 law at high temperatures, this is not so at low
temperature. No reducing gas atmospheres are
This article explores properties of ZSM-5 additives required in the preparation of V(III) oxides.
and the role of nickel and vanadium in fluid
catalytic cracking (FCC). Loadings of 4000 ppm
and 12,000 ppm nickel and vanadium were found.
There was deactivation of ZSM-5 in a cyclic
deactivation unit and vacuum gas oil (VGO) reacted
with nickel and vanadium naphthenates when
cracking-regeneration reactions were undertaken.
There was an even distribution of nickel across
a particle where characterisation of deactivated
ZSM-5 additives by nitrogen physiosorption, SEM
and pyridine FTIR techniques were used. The 1 µm
disparity was small in the Brønsted acidity, but Fig. 1. Reprinted from D. S. Cook et al., J. Solid
when nickel and vanadium were added there was State Chem., 2020, 288, 121396, Copyright
a rise in Lewis acidity. Further tests were carried (2020), with permission from Elsevier
out with VGO and where butylene increased,

Hierarchical ZSM-5 Catalysts: The Effect of Different with an impregnation of metal precursors onto
Intracrystalline Pore Dimensions on Catalyst activated carbon from a low boiling point solvent.
Deactivation Behaviour in the MTO Reaction Single-site gold, palladium, ruthenium and platinum
T. Weissenberger, A. G. F. Machoke, J. Bauer, R. catalysts supported on carbon were prepared in
Dotzel, J. L. Casci, M. Hartmann and W. Schwieger, a facile method. In addition, it is shown that a
ChemCatChem, 2020, 12, (9), 2461 single-site gold on carbon catalyst for acetylene
hydrochlorination can be produced by this method.
ZSM-5 zeolites used as methanol-to-olefins
(MTO) catalysts were studied to determine Efficient and Selective Solvent-Free Homogeneous
the effect of intracrystalline pore systems in Hydrogenation of Aldehydes Under Mild Reaction
different combinations. Intracrystalline mesopores, Conditions Using [RuCl2(dppb)(ampy)]
intracrystalline macropores and a novel ZSM-5 A. Zanotti-Gerosa, T. Angelini and S. Roseblade,
type zeolite with both intracrystalline meso and Tetrahedron Lett., 2020, 61, (13), 151677
macropores were used. There was a prolonged
catalyst lifetime with the hierarchical catalysts Using commercial grade aldehydes, effective,
unlike microporous only ZSM-5 catalyst. Using solvent-free homogeneous hydrogenation of
the intracrystalline mesopores and intracrystalline aldehydes was undertaken with catalysts
macropores as catalysts resulted in the ZSM-5 [RuCl2(dppb)(ampy)] and [RuCl2(dppf)(ampy)].
catalyst lasting up to three times longer. There were This gave high conversion to the related alcohols
a number of important outcomes which were also using molar catalyst loadings of 10,000/1– 50,000/1.
noted including how mesopores and macropores Aldehydes can be reduced preventing byproducts
effect catalyst deactivation. Overall, the study being formed with the minimum of waste which has
demonstrates intracrystalline macropores (alone led to a solvent-free protocol being established. This
or in combination with mesopores) significantly gives a straightforward hydrogenation technique for
enhance the ZSM-5 catalytic performance in the reduction of aldehydes to alcohols and commercial
MTO reaction. grade aldehydes require no further purification.
Accelerating Pharmaceutical Development via Innovation in Fischer–Tropsch: Developing

Metal-Mediated Bond Formation Fundamental Understanding to Support Commercial
Opportunities
C. J. Borths and S. D. Walker, Israel J. Chem., 2020,
60, (3–4), 340 M. Peacock, J. Paterson, L. Reed, S. Davies,
S. Carter, A. Coe and J. Clarkson, Top. Catal., 2020,
This article presents work being undertaken related 63, (3–4), 328
to progress with metal-mediated carbon–carbon
and carbon–heteroatom bond forming processes. The BP-Johnson Matthey proprietary Fischer‑Tropsch
The viewpoint of a current drug portfolio is used. technology and advanced CANSTM reactor and
Several case studies are discussed from the authors’ catalyst system are detailed. It provides improved
laboratories looking at synthetic challenges plus heat transfer, reduced pressure drop and higher
prospects offered by pharmaceutically pertinent productivity and subsequently less financial
platforms. In several instances, available synthetic expenditure. A clear understanding of how catalysts
methods are challenged by target structures. This behave is crucial to finding a catalyst stable during
drives development permitting the acceleration of its use and life. This report presents a study on
technology for drug development. There is also catalyst activation on different catalyst supports
a discussion about metal-mediated processes at and combines in situ techniques and reactor testing.
large scale. Logical and systematic catalyst programmes are
crucial for their development and are discussed
Facile Synthesis of Precious-Metal Single-Site in the results. Also, catalyst understanding,
Catalysts Using Organic Solvents optimisation and development in combination with
X. Sun, S. R. Dawson, T. E. Parmentier, G. Malta, the novel CANSTM reactor design can maximise
T. E. Davies, Q. He, L. Lu, D. J. Morgan, N. Carthey, potential.
P. Johnston, S. A. Kondrat, S. J. Freakley, C. J. Kiely
and G. J. Hutchings, Nature Chem., 2020, 12, (6), Cu/M:ZnO (M = Mg, Al, Cu) Colloidal Nanocatalysts
560 for the Solution Hydrogenation of Carbon Dioxide
to Methanol
In many catalytic reactions, high activity and
A. H. M. Leung, A. García-Trenco, A. Phanopoulos, A.
selectivity can be demonstrated by single-site
Regoutz, M. E. Schuster, S. D. Pike, M. S. P. Shaffer
catalysts. Creation of low metal loadings or a
and C. K. Williams, J. Mater. Chem. A, 2020, 8,
variety of metal species can be achieved by
(22), 11282
impregnation, for example, from strongly oxidising
aqueous solutions. This study shows an atomic A synthesis is undertaken using controlled
distribution of cationic metal species is achieved hydrolysis of a mixture of organometallic precursors

for doped-ZnO NPs capped with dioctylphosphinate Chem. Sci., 2020, 11, (27), 7040
ligands. Following substitutional doping and after
The environmental impact of acetylene
hydrolysis, colloidal nanoparticles (2–3 nm) were
hydrochlorination was substantially reduced by
characterised. Doped-ZnO nanoparticles and
replacing HgCl2/C with Au/C as a catalyst. Atomically
colloidal Cu(0) nanoparticles in solution were
dispersed cationic gold species are the catalytically
applied for hydrogenation catalysis of CO2 to
active site. There have been limited studies which
methanol in a liquid-phase continuous flow stirred
look at the ligand environment around the metal
tank reactor under the following conditions: 210ºC,
centre. This study uses K-edge soft XAS. Three
50 bar, CO2:H2 = 1:3, 150 ml min−1, mesitylene,
separate chlorine species are identified and how
20 h. Higher rates are displayed for all catalyst
they evolve in the reaction is demonstrated. Au–S
systems with respect to methanol production
interactions are established in catalysts prepared
compared to a benchmark catalyst. There is better
using thiosulfate precursors. The catalysts display
stability. There was around double the activity
evidence of high stability towards reduction to
for Al(III)‑doped nanocatalyst. Mg(II) doping
inactive metal NPs. Gas switching experiments
outperforms the benchmark catalyst but was worse
made clear this stability. C2H2 on its own did not
compared to undoped ZnO. There is an implication
particularly change the gold electronic structure
that Al(III) migrates to the catalyst surface, and
and the thiosulfate catalyst was not deactivated.
is proposed to enable stabilisation of the catalytic
ZnO/Cu interfaces. Optimization of Biomass Pyrolysis Vapor Upgrading
N-Functionalised Imidazoles as Stabilisers for Metal Using a Laminar Entrained-Flow Reactor System
Nanoparticles in Catalysis and Anion Binding B. Peterson, C. Engtrakul, T. J. Evans, K. Iisa,
C. J. Serpell, J. Cookson and P. D. Beer, M. J. Watson, M. W. Jarvis, D. J. Robichaud,
ChemistryOpen, 2020, 9, (6), 683 C. Mukarakate and M. R. Nimlos, Energy Fuels,
2020, 34, (5), 6030
The physicochemical properties of metal NPs are
discrete from bulk and molecular metal species. To obtain understanding of commercial scale
Consequently, this delivers opportunities in areas ex situ catalytic fast-pyrolysis (CFP) a customised
like catalysis and sensing, for example. The surface bench‑scale continuous-flow catalytic fast-
of the NPs usually need to be protected to hamper pyrolysis CFP reactor system was built. The study
aggregation. However, access to the surface can successfully carried out CFP of pine over two
also be blocked by these coatings preventing the commercial zeolite catalysts. The transmission of
ability to benefit from their uncommon properties. pyrolysis vapours to the vapour-phase upgrader
The article shows that palladium, platinum, gold was optimised to limit secondary thermal cracking
and silver NPs can be stabilised by alkyl imidazoles. and preserve carbon in the ex situ CFP process.
It also outlines the limits of their synthesis. Products attained were comparable to those from
Proof‑of-principle in catalysis and anion binding is fixed bed and fluidised bed reactor systems and
established showing that the ligands deliver a level entrained-flow riser reactor systems. Experiments
of surface protection. that duplicated the process provided a good
average mass balance closure and comparable
In Situ K-edge X-ray Absorption Spectroscopy of the trends in deactivation of catalyst were seen in the
Ligand Environment of Single-Site Au/C Catalysts laminar entrained-flow reactor system. There was
During Acetylene Hydrochlorination a decreasing catalyst‑to-biomass ratio. Optimised
G. Malta, S. A. Kondrat, S. J. Freakley, D. J. Morgan, conditions suggest a feasible option for CFP of
E. K. Gibson, P. P. Wells, M. Aramini, D. Gianolio, pine. For the two catalysts tested, minor variances
P. B. J. Thompson, P. Johnston and G. J. Hutchings, were detected.

Antibiotic and Heavy Metal Resistant Bacteria

Isolated from Aegean Sea Water and Sediment
in Güllük Bay, Turkey
Quantifying the resistance of identified bacteria species with potential for
environmental remediation applications
Gülşen Altuğ* in sediment and seawater samples taken from

Department of Marine Biology, Faculty the Aegean Sea, Turkey, between 2011 and 2013.
of Aquatic Sciences, Istanbul University, Bioindicator bacteria in seawater samples were
Balabanağa Mahallesi Ordu Caddesi No 8, Laleli, tested using the membrane filtration technique. The
Fatih, Istanbul, 34134, Turkey spread plate technique and VITEK® 2 Compact 30
micro identification system were used for
Mine Çardak heterotrophic aerobic bacteria in the samples. The
Department of Fisheries Technology, Faculty of minimum inhibition concentration method was
Çanakkale Applied Sciences, Çanakkale Onsekiz used for heavy metal-resistant bacteria. Antibiotic-
Mart University, Terzioğlu Campus, Çanakkale, resistant bacteria were tested using the disk diffusion
17020, Turkey method. All bacteria isolated from sediment samples
showed 100% resistance to rifampicin, sulfonamide,
Pelin Saliha Çiftçi Türetken tetracycline and ampicillin. 98% of isolates were
Department of Marine Biology, Faculty resistant against nitrofurantoin and oxytetracycline.
of Aquatic Sciences, Istanbul University, Higher antibiotic and heavy metal resistance was
Balabanağa Mahallesi Ordu Caddesi No 8, Laleli, recorded in bacteria isolated from sediment than
Fatih, Istanbul, 34134, Turkey seawater samples. The highest levels of bacterial
metal resistance were recorded against copper
Samet Kalkan (58.3%), zinc (33.8%), lead (32.1%), chromium
Department of Marine Biology, Faculty of (31%) and iron (25.2%). The results show that
Fisheries and Aquatic Sciences, Recep Tayyip antibiotic and heavy metal resistance in bacteria
Erdoğan University, Zihni Derin Campus, Rize, from sediment and seawater can be observed as
53100, Turkey responses to environmental influences including
pollution in marine areas.
Sevan Gürün
Department of Marine Biology, Faculty
1. Introduction
of Aquatic Sciences, Istanbul University,
Balabanağa Mahallesi Ordu Caddesi No 8, Laleli, In the era of Industry 4.0, with global climate change,
Fatih, Istanbul, 34134, Turkey increasing population and developing technology,
the spread of heavy metal pollutants in aquatic
*Email: galtug@istanbul.edu.tr areas is increasing. Bacterial resistance and metal
accumulation capability are common phenomena
that can be exploited for the bioremediation of
Heavy metal and antibiotic-resistant bacteria the environment, hence these resistant bacteria
have potential for environmental bioremediation may be potential candidates for biotechnological
applications. Resistant bacteria were investigated applications. Despite the risks caused by antibiotic-

resistant bacteria, heavy metal-resistant bacteria persistence of antibiotic resistance in bacterial

can be used in detoxification processes to convert pathogens is a threat and a source of considerable
a toxic form to a harmless form of a substance concern to public health (8–15). It is known that
by developing biotransformation mechanisms. environmental factors such as overpopulation,
Bioremediation studies have been carried out to livestock farming, insufficient drainage and
identify candidate species (1–4). sanitation infrastructure may provide hotspots
In recent years, the increase in pollution by toxic for environmental antibiotic-resistant bacteria
compounds and heavy metals in marine areas makes transmission (16).
it increasingly important to study the relationships In aquatic environments, antibiotic-resistant
between bacteria and toxic compounds. Studies bacteria can be accompanied by heavy metal-
related to the transformation of compounds into resistant bacteria that are often induced by the
different forms via bacterial metabolic processes presence of metal caused by anthropogenic activities
for the removal of toxic substances from the and environmental factors (16, 17). Heavy metals
environment have gained importance. Detection are introduced into the marine environment in
of bacteria that are resistant to heavy metals in different ways. Accumulation in sediment can affect
natural environments constitutes the first step to aquatic life negatively for a long time. Bacteria
provide data for remediation studies. that will take part in the transformation of heavy
Bacteria are some of the most important metal salts into harmless forms must be resistant
components in marine ecosystems. Since bacteria to the heavy metals. Bacteria that cannot adapt to
adapt to new conditions created by environmental the changes metabolically will be eliminated and
variables around them, knowledge of bacteria therefore various pollution inputs accumulated
provides data in terms of defining environmental in the sediment will affect the composition of
factors, public health status and ecosystem microbial diversity. Sediments containing harmful,
function. Marine areas are exposed to domestic and inorganic or organic particles are relatively
industrial wastes depending on local technology heterogeneous in terms of physical, chemical and
levels and population. Many xenobiotic micro biological properties and are an important source
pollutants, antibiotic derivatives and metabolites of heavy metal contamination (11). It has been
reach the sea from human activity. This concerning reported that microplastics mediate the spread of
issue is considered an important factor for global metal- and antibiotic-resistant pathogens due to
health with respect to the evolution and detection their ability to adsorb various pollutants (18, 19).
of antibiotic resistance in bacterial pathogens (5). Bacteria resistant to heavy metals in marine areas
Since the spread of antimicrobial resistance is not have developed various resistance mechanisms
restricted by phylogenetic, geographic or ecological to counteract heavy metal stress. Only bacteria
borders, studies describing regional status of that can withstand the current heavy metal
bacterial resistance in natural areas are important. concentration can survive in these areas.
Antibiotic resistance can spread rapidly among Heavy metals accumulate in biota via food chains
bacterial species (6). It is known that the and are transferred between organisms in marine
occurrence of antibiotic-resistant bacteria in environments. This cumulative process, named
natural environments reduces the effectiveness of biomagnification, is higher in the sea than in
antibiotics in the treatment of infectious diseases. terrestrial environments (15, 20) and this implies
Due to the increasing global resistance of bacteria significant effects of heavy metal pollution in
against antibiotics, humanity is constantly being marine areas. On the other hand, heavy metal-
forced to develop new antibiotic derivatives. resistant bacteria can play a role in detoxification by
This vicious circle is one of the most important converting a toxic form into a harmless form through
problems of our age and poses a threat for the biotransformation mechanisms that develop in
future. Thus, it is important to know the resistance natural environments. These mechanisms include
levels of bacteria and to produce regional antibiotic the formation and sequestration of heavy metals
resistance profiles in natural areas. Aquatic in complexes and the reduction of a metal to a less
environments constitute a way to disseminate toxic species (21, 22).
not only antibiotic-resistant bacteria but also the Metal-resistant bacteria have developed very
resistant genes in natural bacterial habitats (7). efficient and varying mechanisms for tolerating
It has been well documented that the aquatic high levels of toxic metals and thus they carry an
environment is a potential reservoir of antibiotic- important potential for controlling heavy metal
resistant bacteria, furthermore the prevalence and pollution (23). In many prokaryotes, it has been

shown that the mechanism for resisting heavy 2. Material and Methods
metals develops over time. This process has
2.1 Sampling Area
been studied in species such as Escherichia coli
and Staphylococcus aureus. It is reported that Güllük Bay is an important location due to its
many different species of Pseudomonas, Bacillus, natural resources. The region is open to different
Enterobacter, Providencia and Chryseobacterium environmental influences and inputs due to
are efficient for reducing heavy metals (1–4). tourism, port activities, marine transportation,
It is known that the occurrence of bacteria domestic and industrial wastes and fish farms.
resistant to antibiotics and heavy metal salts in The bay is also affected by the presence of Sarıçay
the sea is related to the pollutants present in the Creek, Kazıklı Port, Güllük Port and Akbük Port
environment. For the reasons highlighted above, it (24–26). Fish farms were operated in Güllük Bay
is important to determine the profile of antibiotic until 2008. Although they have been relocated
and heavy metal-resistant bacteria in marine away from the coastal regions to an offshore area,
environments. Marine areas which have different the indirect effects of this long-time pollution may
environmental inputs present novel media for have contributed to the sediment.
bacterial studies. The export of feldspar and bauxite from the region
For the present study, the Güllük Bay of the has been conducted from ports within the borders
Aegean Sea, Turkey, was chosen since it is a of Güllük Bay. The port is mainly used by dry cargo
dynamic area due to marine transportation, and other cargo-type ships. It is reported that an
seasonal population growth depending on tourism, annual average of 800,000 tonnes of ballast water
aquaculture, recreational and agricultural activities is transported to the bay from 157 different ports.
and terrestrial pollution inputs transported from The amount of ballast water carried is reported
rivers. as: 68% from the Mediterranean, 21% from the
Probable faecal source analysis conducted in Aegean Sea, 7% from the Sea of Marmara, 2%
Güllük Bay showed that the primary source of the from the Atlantic Ocean and 1% from the Black
detected bacteriological pollution is anthropogenic Sea and Red Sea, respectively (37). The operation
(24). A significant part of domestic wastewater of many tourism-oriented boats in Güllük Bay is
in the region collects in sealed septic tanks. It also among the possible polluters of the bay due
is possible for the wastewater to reach the sea to bilge water and wastewater. More than half of
by mixing the sedimentary septic tanks with Turkey’s sea bream and sea bass production was
groundwater. Chemical and biological studies in farms operating in the coastal areas of the
(24– 33) confirm that regional pollutants have Güllük Bay for many years. These farms have
reached Güllük Bay. been operating in the offshore areas of the region
It is well known that sewage transported via for the past 10 years. The domestic wastewater
domestic wastewater carries antibiotics to marine of the human population, reaching approximately
environments. This has an effect on metabolic 50,000 around the region in the summer months,
capabilities of bacteria in marine environments. and the wastes of small industrial establishments
For example, β-lactam antibiotic derivatives used such as yogurt, yeast and olive oil producers that
for human infection treatment may enter marine directly reach streams are the other main sources
environments via domestic wastewater. Bacteria of pollution in Güllük Bay. The population of the
may obtain resistance via intercellular contact Bodrum peninsula, which is 25,000 in winter, can
mostly using a conjugation mechanism (34). reach 1,500,000 in summer (27). The change in
The existence of antibiotic-resistant bacteria is the population between the seasons was among
an indicator of domestic pollution. Furthermore, the biggest pollution sources according to the
antibiotic-resistant bacteria may cause a vicious terrestrial bioindicator bacteria distribution in
cycle. This problem has grown in recent years due coastal areas in the region (24, 26).
to systematic use of antibiotics in animal husbandry Sampling stations were selected to represent
and overuse of antibiotics (35, 36). tourist areas (G1, G5, G7, G8); harbours (G4, G6);
The frequencies of heavy metal-resistant bacteria fresh water entry-exit points of the Sarıçay Creek
and antibiotic-resistant bacteria were investigated (G9); fish farms (G11, G12, G13); and the deepest
in seawater and sediment samples collected from point in the bay as a reference station (G14).
Güllük Bay in the period between May 2011 and Figure 1 shows the location of Güllük Bay and the
February 2013. sampling stations.

Code Mid point, m Max. depth, m
G1 25 50
G1 G4 25 50
G5 10 20
G4
G6 11 22
G7 14 28
G14 G6
G5 G8
G8 8 16
G7 G9 G9 4 8
G10 18 37
G10
Black Sea G11 5 10
G13
Marmara
Sea G12 25 50
G11
Aegean TURKEY G12
Sea G13 12 25
G14 33 66
Mediterranean Sea
Fig. 1. Location of Güllük Bay and seawater (0–30 cm surface, mid-point and bottom-point) and sediment
sampling stations
2.2 Sampling 2.3 Bacterial Isolation and

Identification
Seawater and sediment samples were collected
from 12 different sampling stations in Güllük Bay Bacterial heavy metal and antibiotic resistance
between May 2011 and February 2013. Three were tested in heterotrophic aerobic bacteria
units of seawater samples were taken from each isolated from seawater and sediment samples.
station at surface (0–30 cm), mid-point and Heavy metal and antibiotic resistance of indicator
bottom-point water (Figure 1). In each sampling bacteria (faecal coliform, coliform and faecal
process covering 12 stations, 36 seawater Streptococcus) isolated from the seawater samples
samples were collected. In the spring and summer were also tested.
months monthly, at other times seasonally, a total
of 432 seawater samples were collected in the
2.3.1 Seawater Samples
period between May 2011 and February 2013.
The seawater samples were collected using a Indicator bacteria and heterotrophic aerobic bacteria
Nansen bottle that was cleaned with acid (10% analyses were performed on the seawater samples.
HCl in distilled water), sterilised with alcohol The membrane filtration technique was used to
(50:50, v/v) and rinsed with sterile water. The detect indicator bacteria. A sample containing
seawater samples were then transferred into 300 ml seawater was diluted serially (10–5 dilution)
brown sterile glass bottles and transported to the and filtered through membrane filters (0.45 µm,
laboratory as a cold chain. Sartorius AG, Göttingen, Germany). The filters
Surface sediment samples were collected using were placed on m-Endo, m-FC and m-Azide media
Ekman grab (HYDRO-BIOS Apparatebau GmbH, (Sartorius AG). The plates were incubated for 24 h
Germany, 15 × 15) from the sampling stations (at 37 ± 0.1°C; at 44 ± 0.1°C for m-FC). Brown‑red
which have various depths from 8 m to 66 m colonies growing on the azide medium were
(Figure 1). A total of 144 units of sediment considered as suspicious faecal Streptococcus, blue
samples were collected during the two-year study colonies growing on the m-FC medium as suspicious
from 12 stations (one from each station). The faecal coliform and yellow-green colonies with yellow-
sediment samples were transferred into sterile zip metallic gloss on the m-Endo medium as suspicious
seal bags from Ekman grab and transferred in the coliform. Cytochrome oxidase test (API® 20 Strep,
cold chain to the laboratory. bioMérieux, France) was performed on suspicious

coliform colonies and oxidase negative colonies were Further processes related to heterotrophic bacteria
evaluated numerically. Cytochrome oxidase (API® 20 identification were continued by using VITEK® 2
Strep, bioMérieux) and indole tests were performed Compact 30 similarly to the seawater samples
on the suspicious colonies of faecal coliform. described above.
Colonies with oxidase negative and indole positive
results were evaluated as faecal coliform. Suspicious
2.4 Bacterial Resistance Against
Streptococcus colonies, to which the catalase test
Antibiotics
was applied (1 ml, 3% H2O2), were incubated on
Bile Esculin Agar (BEA) for 18 h at 37°C for esculin The antibiotic resistance of the isolates was
hydrolysis and 40% bile resistance control. Blackening examined by the Kirby–Bauer method with slight
in the medium and the formation of black shadow modifications. Two or three colonies of each isolate
around the colony, positive of esculin hydrolysis, were suspended with 5 ml of DifcoTM Marine Broth
and the number of colonies showing growth in the 2216 and diluted with sterile water against the
medium were evaluated as 40% bile resistant, and 0.5 McFarland turbidity standard to approximately
catalase negative and breeding colonies in BEA 106 cells ml–1 and swabbed as 2 ml on DifcoTM
were evaluated as faecal Streptococcus. Counted Marine Agar 2216. Antibiotic discs (Oxoid, UK)
colonies were multiplied by the 10–5 dilution factor to containing ampicillin (10 µg), nitrofurantoin
determine the number of colony forming units (CFU) (300 µg), oxytetracycline (30 µg), sulfonamide
100 ml–1 in the original sample (38). (300 µg), rifampicin (2 µg), tetracycline (10 µg)
The spread plate technique was used for and tetracycline (30 µg) were incubated for two to
heterotrophic aerobic bacteria analyses in seawater. three days at 37°C. The results were interpreted
Seawater samples 0.1 ml with 10–5 dilution were according to the guidelines of the Clinical Laboratory
used for duplicate spreading on the DifcoTM Marine Standard Institute (CLSI) (41). All isolates that
Agar 2216 (Becton, Dickinson and Company, USA) showed resistance were classified as ‘resistant’.
and the plates were incubated for five days at Other isolates that did not show resistance were
22 ± 0.1°C. At the end of the incubating period, classified as ‘sensitive’ or ‘susceptible’.
counted colonies were multiplied by the 10–5
dilution factor to determine the number of CFU ml–1
2.4.1 Multiple Antibiotic Resistance
in the original sample. An average of 10 different
colonies were picked and restreaked several times The multiple antibiotic resistance (MAR) index of
to obtain pure cultures. The pure isolates were a given sample was calculated by the equation:
Gram-stained. For identification of spore-forming a/ (bc), where a represents the aggregate antibiotic
bacilli, the isolates were stained with Indian ink resistance score of all isolates from a sample; b is
according to the negative staining technique the total number of isolates; and c is the number of
and were evaluated using a light microscope isolates from a sample (42). Bacterial isolates that
(Nikon E110, Nikon, Japan). The isolates were displayed resistance to three or more antibiotic
then tested using Gram‑negative fermenting agents were designated as multiple antibiotic
and non‑fermenting bacilli (GN), Gram-positive resistant (ranging from two to 10).
cocci and non-spore-forming bacilli (GP) and
Gram‑positive spore-forming bacilli (BCL) cards in
the automated micro identification system VITEK®
Heavy Metal Salts
2 Compact 30 (bioMérieux) (39).
Different concentrations (50 μg ml–1, 100 μg ml–1,
150 μg ml–1, 200 μg ml–1 and 250 μg ml–1) of heavy
2.3.2 Sediment Samples
metal salts (FeSO4, ZnSO4, CuSO4, Cr2(SO4)3 and
The spread plate technique was used for Pb(NO3)2) were used to test the bacteria resistivity
heterotrophic aerobic bacteria analyses in sediment against iron, zinc, copper, chromium and lead.
samples. Each sediment sample was mixed and The microdilution method was followed with minor
homogenised. Then 1 g sample was taken from modifications to determine the resistance of isolates
each and serially diluted with sterile commercial to heavy metals (43). Stock solutions of metal
seawater. 0.1 ml samples of 10–5 dilutions were salts prepared in distilled water were sterilised by
taken and spread on DifcoTM Marine Agar 2216. The filtration (0.20 μm). In U-well microtiter plates,
plates were incubated for five days at 22 ± 0.1°C. serial dilutions of heavy metals were prepared
Growing colonies were evaluated as CFU g–1 (40). and then each well was inoculated with bacteria

inoculation. The OxoidTM Turbidometer (Thermo Shewanella algae and Vibrio parahaemolyticus
Fisher Scientific Inc, USA) provides the inoculum isolates from the sediment samples showed
density standardisation for 0.5 McFarland which is resistance to all antibiotics (Table I).
necessary to ensure accurate reproducible results. The highest number of antibiotic-resistant
Before the addition of bacterial inoculation, no bacteria were detected from the sediment
precipitation was seen. The plates were incubated samples. The frequency of resistant bacteria (%)
at 37°C for 24 h and then examined for visual to oxytetracycline (30 µg), nitrofurantoin (300 µg),
turbidity. The lowest concentration of the metal rifampicin (2 µg), tetracycline (10 µg), tetracycline
salt, at which growth was inhibited (indicated (30 µg), sulfonamide (300 µg) and ampicillin
by lack of turbidity), was taken as the minimum (10 µg) from the seawater and sediment samples
inhibitory concentration (MIC) (44) Samples of are shown in Figure 2. The frequencies of antibiotic
10 μl were drawn from each well without turbidity resistance in bacteria species from seawater and
and were subcultured on agar plates to determine sediment samples are shown in Figure 3.
bactericidal concentration. A total of 258 and 158 isolates were tested
Reference strains of Escherichia coli (ATCC® against antibiotics from seawater and sediment
25922TM), Salmonella enterica (ATCC® 2577TM) samples, respectively. The frequencies of
and Staphylococcus epidermidis (ATCC® 12228TM) resistance against seven antibiotics in bacteria
which are susceptible to Cu2+, Zn2+, Pb2+, Cr2+ and species isolated from the seawater samples
Fe3+ and metal-free plates were used in the control were recorded as 49% in Gammaproteobacteria,
tests to evaluate the viability of the strains and 22% in Αlphaproteobacteria, 3% in
culture media. All of the experiments were carried Betaproteobacteria, 14% in Bacilli, 8% in
out in triplicate. Flavobacteriia and 4% in Actinomycetales. The
resistance frequencies against seven antibiotics
in bacteria isolated from the sediment samples
3. Results
were recorded as 43% in Gammaproteobacteria,
3.1 Bacterial Resistance Against 34% in Bacilli, 7% in Αlphaproteobacteria, 7%
Antibiotics in Betaproteobacteria, 7% in Flavobacteriia and
2% in Actinomycetales.
Table I shows the antibiotic-resistant, intermediate
or susceptible bacteria species isolated from the
3.2 Multiple Antibiotic Resistance
seawater and sediment samples in this study.
Indexes
Bacterial species isolated from the seawater samples
showed considerable resistance to rifampicin (98%), The MAR index was calculated for each of the
sulfonamide (98%) and ampicillin (76%) and antibiotic-resistant bacteria. If the MAR index
considerable sensitivity to tetracycline-30 µg (52%), is lower than 0.2, it shows a non-point based
tetracycline-10 µg (39%) and oxytetracycline source of pollution and if it is higher than 0.2 it
(33%). Almost all the bacterial species isolated from shows point‑based pollution and a high risk of
sediment samples showed resistance to rifampicin contamination by excessive antibiotic presence
(100%), sulfonamide (100%), ampicillin (100%), (23). Table II shows the MAR indexes.
nitrofurantoin (98%), tetracycline-30 µg (100%), The MAR indexes of the study showed possible
tetracycline-10 µg (100%) and oxytetracycline exposure of these bacterial isolates to the tested
(98%) while they showed almost no sensitivity antibiotics. The MAR index of bacteria isolated
to antibiotics except nitrofurantoin (2%) and from all stations around fish farm areas (0.0576)
oxytetracycline (2%). Pseudomonas aeruginosa was 2.6 times greater than the MAR index for the
(24%) and Sphingomonas paucimobilis (20%), combined non-fish farm areas (0.022).
isolated from seawater samples, showed higher
resistance to antibiotics than did Raoultella
oxytica, Staphylococcus xylosus, Kocuria kristinae,
Heavy Metals
Aeromonas salmonicida and Proteus vulgaris
strains. On the contrary, Aeromonas caviae, The frequencies of heavy metal resistance in the
Alicyclobacillus acidoterrestris, Brevundimonas bacteria species isolated from the seawater samples
diminuta, Chryseobacterium indologenes, were recorded as 76.72% in Gammaproteobacteria,
Lactococcus garvieae, Neisseria animaloris, 71.82% in Αlphaproteobacteria, 80.01% in
Pseudomonas aeruginosa, Serratia marcescens, Bacilli, 56.92% in Flavobacteriia and 75% in

Table I Antibiotic Resistant, Intermediate or Susceptible Bacteria Species Isolated from Seawater and Sediment
513
Antibioticsa
Order/class Bacterial isolates
tested (%) tested (n) AM TE S TE RD F/M OT
(10 µg) (30 µg) (300 µg) (10 µg) (2 µg) (300 µg) (30 µg)
Sample
R: 66.7% R: 33.3% R: 100% R: 33.3% R: 100% R: 66.7% R: 66.7%
Brevundimonas
I: 33.3% I: 0.0% I: 0.0% I: 33.3% I: 0.0% I: 0.0% I: 33.3%
diminuta (3)
S: 0.0% S: 66.7% S: 0.0% S: 33.3% S: 0.0% S: 33.3% S: 0.0%
R: 75% R: 50% R: 100% R: 50% R: 100% R: 50% R: 25%
Proteobacteria/ Brevundimonas
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
Alpha vesicularis (4)
S: 25% S: 50% S: 0.0% S: 50% S: 0.0% S: 50% S: 75%
proteobacteria
(27%) R: 71.05% R: 31.57% R: 97.38% R: 42.10% R: 97.36% R: 60.52% R: 26.31%
Sphingomonas
I: 2.63% I: 5.26% I: 0.0% I: 7.89% I: 0.0% I: 0.0% I: 34.21%
paucimobilis (38)
S: 26.31% S: 63.15% S: 2.63% S: 50% S: 2.63% S: 39.47% S: 39.47%
R: 50% R: 0.0% R: 100% R: 0% R: 100% R: 75% R: 25%
Sphingomonas
I: 0.0% I: 0.0% I: 0.0% I: 25% I: 0.0% I: 0% I: 25%
thalpophilum (4)
https://doi.org/10.1595/205651320X15953337767424
S: 50% S: 100% S: 0.0% S: 75% S: 0.0% S: 25% S: 50%

R: 100% R: 0.0% R: 100% R: 0.0% R: 100% R: 0.0% R: 0.0%
Burkholderia cepacia
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(3)
S: 0.0% S: 100% S: 0.0% S: 100% S: 0.0% S: 100% S: 100%
Proteobacteria/
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Beta proteobacteria Burkholderia mallei
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(%) (3)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Neisseria animaloris
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
Seawater
(3)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 0.0% R: 100% R: 0.0% R: 100% R: 100% R: 0.0%
Acinetobacter lwoffii
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 100%
(3)
S: 0.0% S: 100% S: 0.0% S: 100% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 50% R: 100% R: 50% R: 50% R: 50% R: 0.0%
Aeromonas hydrophila
I: 0.0% I: 0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 50%
(4)
S: 0.0% S: 50% S: 0.0% S: 50% S: 50% S: 50% S: 50%
Proteobacteria/
R: 50% R: 50% R: 100% R: 50% R: 100% R: 50% R: 50%
Gamma Aeromonas
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
proteobacteria salmonicida (4)
S: 50% S: 50% S: 0.0% S: 50% S: 0.0% S: 50% S: 50%
(53%)
R: 100% R: 0.0% R: 100% R: 0.0% R: 100% R: 100% R: 0.0%
Aeromonas sobria (3) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 100%
S: 0.0% S: 100% S: 0.0% S: 100% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 0.0% R: 100% R: 0.0% R: 100% R: 100% R: 0.0%

Aeromonas veronii (3) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 100% S: 0.0% S: 100% S: 0.0% S: 0.0% S: 100%

Antibiotics
514
Bacterial isolates
Order/class
tested (n) TE S TE RD F/M OT
tested (%)
AM (10 µg)
Sample
(30 µg) (300 µg) (10 µg) (2 µg) (300 µg) (30 µg)
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Citrobacter sedlakii
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(3)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
Cronobacter R: 0.0% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
dublinensis subsp. I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
lausannensis (3) S: 100% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 33.3% R: 33.3% R: 66.6% R: 66.6% R: 100% R: 33.3% R: 33.3%
Enterobacter
I: 33.3% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 66.6%
aerogenes (6)
S: 33.3% S: 66.6% S: 33.3% S: 33.3% S: 0.0% S: 66.6% S: 0.0%
R: 100% R: 0.0% R: 100% R: 100% R: 100% R: 100% R: 0.0%
Enterobacter cloacae
I: 0.0% I: 100% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
subsp. dissolvens (4)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 100%
R: 0.0% R: 0.0% R: 0.0% R: 100% R: 100% R: 100% R: 0.0%
https://doi.org/10.1595/205651320X15953337767424
I: 50% I: 0.0% I: 100% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(4)
S: 50% S: 100% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 100%
R: 100% R: 50% R: 100% R: 100% R: 100% R: 100% R: 50%
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 50%
complex (4)
S: 0.0% S: 50% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0%
R: 78.5% R: 71.4% R: 92.9% R: 89.3% R: 100.0% R: 100.0% R: 75%
Escherichia coli (30) I: 10.7% I: 10.7% I: 3.5% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 10.7% S: 17.8% S: 3.5% S: 10.7% S: 0.0% S: 0.0% S: 25%
Seawater
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Klebsiella pneumoniae
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
subsp. ozaenae (3)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 0.0% R: 100% R: 0.0% R: 100% R: 0.0% R: 0.0%
Pasteurella canis (3) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 100% S: 0.0% S: 100% S: 0.0% S: 100% S: 100%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Proteus vulgaris group
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
Proteus penneri (3)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 90.91% R: 100% R: 90.91% R: 100% R: 90.91% R: 90.91%
Pseudomonas
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
aeruginosa (13)
S: 0.0% S: 9.09% S: 0.0% S: 9.09% S: 0.0% S: 9.09% S: 9.09%
R: 100% R: 0.0% R: 100% R: 0.0% R: 100% R: 0.0% R: 0.0%
Raoultella
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
ornithinolytica (3)
S: 0.0% S: 100% S: 0.0% S: 100% S: 0.0% S: 100% S: 100%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Raoultella ytica (3) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%

Antibiotics
515
(10 µg) (30 µg) (300 µg) (10 µg) (2 µg) (300 µg) (30 µg)
Sample
R: 100% R: 66.6% R: 100% R: 66.6% R: 100% R: 100% R: 66.6%
Serratia marcescens
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(5)
S: 0.0% S: 33.4% S: 0.0% S: 33.4% S: 0.0% S: 0.0% S: 33.4%
R: 81.81% R: 45.45% R: 100% R: 72.72% R: 100% R: 63.63% R: 54.54%
Shewanella
I: 0.0% I: 18.18% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 36.36%
putrefaciens (13)
S: 18.18% S: 36.36% S: 0.0% S: 27.27% S: 0.0% S: 36.36% S: 9.06%
R: 80% R: 40% R: 100% R: 20% R: 100% R: 100% R: 20%
Stenotrophomonas
I: 0.0% I: 0.0% I: 0.0% I: 50% I: 0.0% I: 0.0% I: 0.0%
maltophilia (7)
S: 20% S: 60% S: 0.0% S: 50% S: 0.0% S: 0.0% S: 80%
R: 50% R: 0.0% R: 100% R: 0.0% R: 100% R: 0.0% R: 0.0%
Vibrio vulnificus (4) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 50%
S: 50% S: 100% S: 0.0% S: 100% S: 0.0% S: 100% S: 50%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
https://doi.org/10.1595/205651320X15953337767424
Enterococcus faecium
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(3)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 0.0% R: 0.0% R: 100% R: 100% R: 100% R: 100% R: 0.0%
Alicyclobacillus
I: 100% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 100%
acidocaldarius (3)
S: 0.0% S: 100% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 60% R: 100% R: 80% R: 100% R: 100% R: 60%
Bacillus cereus (7) I: 0.0% I: 0.0% I: 0.0% I: 20% I: 0.0% I: 0.0% I: 20%
S: 0.0% S: 40% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 20%
Seawater
R: 66.7% R: 66.7% R: 100% R: 100% R: 100% R: 66.7% R: 66.7%
Bacillus pumilus (5) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
Firmicutes/ S: 33.3% S: 33.3% S: 0.0% S: 0.0% S: 0.0% S: 33.3% S: 33.3%
Bacilli
(9%) R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Staphylococcus
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
xylosus (3)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 0.0% R: 0.0% R: 0.0% R: 100% R: 100% R: 100% R: 0.0%
Staphylococcus
I: 0.0% I: 0.0% I: 100% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
aureus (3)
S: 100% S: 100% S: 100% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 33.3% R: 33.4% R: 66.7% R: 66.7% R: 33.3% R: 100% R: 33.3%
Staphylococcus
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 66.7% I: 0.0% I: 0.0%
warneri (5)
S: 66.7% S: 66.7% S: 33.3% S: 33.3% S: 0.0% S: 0.0% S: 66.7%
R: 54.54% R: 36.36% R: 100% R: 45.45% R: 100% R: 54.54% R: 45.45%
Chryseobacterium
I: 18.18% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 18.18%
indologenes (13)
Bacteroidetes/ S: 27.27% S: 63.63% S: 0.0% S: 54.54% S: 0.0% S: 45.45% S: 36.36%
Flavobacteriia (8%) R: 100% R: 66.6% R: 100% R: 66.6% R: 100% R: 66.6% R: 66.6%
Myroides spp. (5) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 33.3% S: 0.0% S: 33.3% S: 0.0% S: 33.3% S: 33.3%

Antibiotics
516
(10 µg) (30 µg) (300 µg) (10 µg) (2 µg) (300 µg) (30 µg)
Sample
R: 0.0% R: 0.0% R: 100% R: 0.0% R: 100% R: 0.0% R: 0.0%
Dermacoccus
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
nishinomiyaensis (3)
S: 100% S: 100% S: 0.0% S: 100% S: 0.0% S: 100% S: 100%
R: 100% R: 100% R: 100% R: 50% R: 100% R: 100% R: 50%
Actinobacteria/ Kocuria kristinae (4) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
Actinomycetales S: 0.0% S: 0.0% S: 0.0% S: 50% S: 0.0% S: 0.0% S: 50%
(3%) R: 0.0% R: 0.0% R: 100% R: 100% R: 100% R: 0.0% R: 0.0%
Kocuria varians (3) I: 100% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 1000%
Seawater
S: 0.0% S: 100% S: 0.0% S: 0.0% S: 0.0% S: 100% S: 0.0%
R: 50% R: 50% R: 100% R: 50% R: 100% R: 50% R: 0.0%
Micrococcus luteus (4) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 100%
S: 50% S: 50% S: 0.0% S: 50% S: 0.0% S: 50% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
https://doi.org/10.1595/205651320X15953337767424
Brevundimonas
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
diminuta (1)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
Proteobacteria/
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Alpha Sphingomonas
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
proteobacteria paucimobilis (1)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
(7%)
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Sphingomonas
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
thalpophilum (1)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Neisseria animaloris
Proteobacteria/ I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(3)
Beta proteobacteria S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
(7%) R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Chromobacterium
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
violaceum (1)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
Sediment
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Aeromonas caviae (1) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Proteobacteria/ Aeromonas sobria (1) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
Gamma S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
proteobacteria R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
(43%) Pseudomonas
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
aeruginosa (1)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Serratia marcescens
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(5)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%

Antibiotics
517
tested (%) tested (n) AM (10 µg) TE (30 µg) S (300 µg) TE (10 µg) RD (2 µg) F/M (300 µg) OT (30 µg)
Sample
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Shewanella algae (15) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Shewanella
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
putrefaciens (11)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 66.7% R: 66.7%
Vibrio alginolyticus
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(14)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 33.3% S: 33.3%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Vibrio fluvialis (11) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
Vibrio R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
https://doi.org/10.1595/205651320X15953337767424
parahaemolyticus I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%

(13) S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Vibrio vulnificus (11) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Alicyclobacillus
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
acidoterrestris (11)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
Sediment
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Bacillus cereus (23) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
Firmicutes/ Bacilli S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
(34%)
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Bacillus pumilus (11) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Lactococcus garvieae
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(13)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Chryseobacterium
I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
Bacteroidetes/ indologenes (12)
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
Flavobacteriia
(7%) R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Myroides spp. (12) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
Actinobacteria/ R: 100% R: 100% R: 100% R: 100% R: 100% R: 100% R: 100%
Actinomycetales Micrococcus lylae (11) I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0% I: 0.0%
(2%) S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0% S: 0.0%
a
Ampicillin (AM, 10 µg), nitrofurantoin (F/M, 300 µg), oxytetracycline (OT, 30 µg), sulfonamide (S, 300 µg), rifampicin (RD, 2 µg), tetracycline (TE, 10 µg) and tetracycline (TE, 30 µg). Resistant
(R), intermediate (I) or susceptible (S)

Sediment Seawater Fig. 2. The frequency

of bacteria resistant to
Ampicillin, 10 µg
specific antibiotics (%)
Sulfonamide, 300 µg in the seawater and
sediment samples
Oxytetracycline, 30 µg
Tetracycline, 30 µg
Tetracycline, 10 µg
Rifampicin, 2 µg
Nitrofurantoin, 300 µg
0 20 40 60 80 100
Resistance, %
(a) (b)
Betaproteobacteria 3% Actinomycetales 2%
Actinomycetales 4% Flavobacteriia 7%
Flavobacteriia 8% Betaproteobacteria 7%
Bacilli 14% Alphaproteobacteria 7%
Alphaproteobacteria 22% Bacilli 34%
Gammaproteobacteria 49% Gammaproteobacteria 43%
0 20 40 60 0 10 20 30 40 50
Resistance, % Resistance, %
Fig 3. The frequencies of antibiotic resistance in bacteria species from (a) seawater samples and (b)
sediment samples
Table II Multiple Antibiotic Resistance Indexes and Resistance Ratios

Number of antibiotics to which bacteria show
MAR Index Resistance, % p-value
resistance
1 0.0052 0.75187 0.3635
2 0.0104 18.0451 0.6513

3 0.0022 12.7819 0.1323
4 0.0294 12.7819 0.1325
5 0.048 9.7744 0.3635
6 0.0576 14.2857 0.4084
7 0.0208 31.57894 0.1234
Actinomycetales. The frequencies of resistance to and Fe2+ were detected as an average of 33.3%,
Cu2+, Zn2+, Pb2+, Cr2+ and Fe2+ were detected as 30.3%, 25.5%, 35.3% and 28.4% respectively in
an average of 58.3%, 33.8%, 32.1%, 31.0% and 158 strains isolated from the sediment samples.
25.2% respectively in 258 bacterial strains isolated The frequencies of heavy metal-resistant bacteria
from seawater samples. isolated from sediment samples were higher than
The frequencies of heavy metal resistance in the frequencies of heavy metal-resistant bacteria
bacteria species isolated from the sediment samples isolated from the seawater samples. Table III
were recorded as 100% in Αlphaproteobacteria, shows the heavy metal resistance in bacteria
100% in Betaproteobacteria, 97.5% in isolates from seawater and sediment in Güllük Bay.
Flavobacteriia, 95% in Gammaproteobacteria, The MICs of the isolates ranged from 0.004 mM
72.5% in Bacilli and 66.6% in Actinomycetales. The to 2.5 mM. The isolates from sediment samples
frequencies of resistance to Cu2+, Zn2+, Pb2+, Cr2+ obtained from stations close to fish farms showed

Table III Heavy Metal Resistance in Bacteria Species from Seawater and Sediment in Güllük
Bay, Turkey
–1 Resistant
Heavy Sampling Metal concentrations, µg ml Isolates
isolates
metals sides
0.8 1.6 3.1 6.5 12.5 25 50 100 200 >200 n n %
Seawater 3 3 3 7 8 9 10 11 6 – 258 149 58.3
Cu2+
Sediment 7 4 9 3 6 17 17 29 11 – 158 53 33.3
Seawater 3 6 4 9 8 7 11 8 2 – 258 86 33.8
Zn2+
Sediment 4 2 2 8 4 19 36 22 6 – 158 48 30.3
Seawater 1 8 2 7 7 10 11 12 2 – 258 82 32.1
Pb2+
Sediment 1 9 4 4 3 18 13 23 17 – 158 40 25.5
Seawater 3 2 7 4 6 7 12 6 4 16 258 79 31.0
Cr2+
Sediment 3 4 5 8 4 9 11 10 27 32 158 56 35.3
Seawater 1 2 – – 9 24 15 6 – – 258 67 25.2
Fe2+
Sediment 3 13 3 5 9 15 24 29 – – 158 45 28.4
Total number of tested isolates 416
higher frequency of resistance against chromium, in another study (48). For example, there were
copper and zinc than other stations. The highest significant increases in numbers of bacteria resistant
resistance (MIC value: 2.5 mM) was displayed to oxytetracycline, oxolinic acid and florfenicol
against Cr+ by all isolates. Bacillus isolates showed a in sediments from an aquaculture site compared
higher resistance to chromium, lead and copper than with those from a non-aquaculture control site.
Pseudomonas isolates, and Vibrio isolates showed Interestingly, in another study a similar number
higher resistance to zinc, copper and chromium than of antibiotic-resistant bacteria were isolated from
Escherichia coli. Tolerance to the maximum MIC aquaculture and non-aquaculture sites (49). Gram-
(>2.5 mM) for chromium was 10.1% for Bacillus negative bacteria (predominantly Plesiomonas
and 0.8% for Pseudomonas isolates. Bacillus isolates shigelloides and Aeromonas hydrophila) were
from sediment samples showed higher resistance to isolated from aquaculture ponds in the south-
chromium, lead, iron and copper than Klebsiella spp. eastern USA and it was reported that antibiotic
and Escherichia coli strains from seawater samples. resistance to tetracycline, oxytetracycline,
Similarly, Shewanella spp. and Serratia spp. strains chloramphenicol, ampicillin and nitrofurantoin
from the sediment samples also showed higher were higher in antibiotic-treated ponds compared
resistance than the species mentioned above. to non-treated rivers (50). It was determined that
bacteria isolated from Sopot Beach, Poland, were
resistant to ampicillin (51). A high percentage of
4. Discussion
bacteria were reported as resistant to streptomycin
Indicator bacteria levels reported in Güllük Bay (100%), cefazolin (89.8%), ampicillin (83.7%) and
and the presence of pathogenic bacteria (25, 26) trimethoprim-sulfamethoxazole (69.4%), whereas
support the relationship between the resistance data a low percentage of bacteria were resistant to
detected in the current study with bacteriological cefepime (12.3%) and meropenem (14.3%) in the
pollution levels. In the present study, bioindicator aquaculture region of İskenderun Bay, Turkey (52).
bacteria showing human-induced pollution input In the current study, higher numbers of sulfonamide,
isolated from seawater had the highest frequency rifampicin and ampicillin-resistant bacteria were
of resistance against nitrofurantoin (100%) and recorded in the stations around aquaculture areas
sulfonamide (95%). Sulfonamides were the first than other stations. Sphingomonas paucimobilis,
antibiotics developed for clinical use. Sulfonamides Escherichia coli and Enterobacter cloacae isolated
have been widely used to treat bacterial and from both seawater and sediment at the stations
protozoan infections in humans, domestic animals around aquaculture areas had the highest levels
and fish since their introduction to clinical practice of antibiotic resistance. The development of
in 1935 (45–47). The results of higher resistance resistant pathogens in aquaculture environments
against sulfonamide in the present study were is well documented (53, 54) and evidence of
similar to the findings of sulfonamide resistance transfer of resistance encoding plasmids between

aquaculture environments and humans has been was found to be 2.6 times greater in the stations
presented recently (55). It has been reported that around fish farm areas (0.057) than the other
antibiotic‑resistant bacteria are present in a seafood stations (0.022).
ecosystem where antibiotics have never been used Marine sediments offer more informative results
(56). This is interesting in terms of showing that than seawater about environmental pollution
aquaculture areas may be adversely affected by due to the accumulation of various pollutants
the presence of environmental antibiotic-resistant at the bottom of the sea, therefore analysis of
bacteria. sediments is widely used in tests. The association
In the present study, a high percentage of the of microorganisms with sediment particles is one
bacteria Sphingomonas paucimobilis were isolated, of the primary factors in assessing microbial fate
which was especially prevalent in Güllük Bay. The in aquatic systems. In this study, the bacteria
natural habitat of Sphingomonas has not been isolated from sediment in all samples showed a
defined, but it is widely distributed in the natural higher resistance rate than bacteria isolated from
environment especially in water and soil (57). The seawater. Detection of higher antibiotic resistance
second most prevalent species were Escherichia in sediment bacteria than bacteria isolated from
coli and Enterobacter cloacae. Escherichia coli is seawater showed that sediment bacteria were
an indicator of faecal contamination in aquatic exposed to more antibiotics. Natural ecosystems
environments. Enterobacter cloacae is the most containing high concentrations of heavy metals
frequent species associated with nosocomial are also frequent. Heavy metal resistance
infections along with Klebsiella pneumoniae that genes are commonly found in environmental
is a growing problem in human healthcare. The bacteria (71). The resistance to seven
highest number of Bacillus cereus was isolated heavy metals has been reported in the order
from the sediment underneath fish farms. A few Cu > Mn > Ni > Zn > Pb > Cd > Fe for seawater
Bacilli of marine origin have been reported to bacteria isolated from the Golden Horn, Istanbul,
produce unusual metabolites different from those Turkey (17). Heavy metal resistance in bacteria
isolated from terrestrial bacteria (58). Due to the found in seawater from the Mediterranean has
ubiquity and ability of the Bacillus species to survive been reported as Cd > Cu > Cr = Pb > Mn; in
under difficult circumstances, Bacillus strains Karataş, Turkey Cd > Cu > Cr = Mn > Pb; and
are considered to be species of certain habitats İskenderun Bay, Cu > Cd > Mn > Cr > Pb (72).
(59, 60). In the current study, Bacillus pumilus, In the present study, resistance to five different
B. thuringiensis, B. mycoides and B. cereus were heavy metals (Zn2+, Pb2+, Cu2+, Cr3+ and
isolated from the sediment samples of the stations Fe3+) were investigated for all isolates. Trends
around fish farms. in heavy metal resistance vary depending on
The high frequency of resistance among bacterial the sample sites: Güllük Bay, fish farm water
isolates in the present study confirms the earlier column: Cu > Zn > Pb > Cr > Fe; sediment:
reports regarding the role of antimicrobial use that Cr > Cu> Zn > Fe > Pb. Frequency of bacteria
plays a role in selecting antibiotic-resistant bacteria resistance to heavy metals shows the direct
in water and aquatic sediments (46–52). Many effects of metal pollution. Neisseria animaloris,
previous studies have shown that the increases in Aeromonas caviae and Bacillus cereus isolated
antibiotic resistance in human medicine, agriculture from sediment samples were the most tolerant
and aquaculture are directly related to the amounts of all the heavy metal salts. Chryseobacterium
of antimicrobials used (61–65). indologenes displayed the highest degree of
Infections caused by antibiotic-resistant bacteria sensitivity to all metal salts while Lactococcus
are one of the most important public health garvieae showed the highest degree of sensitivity
concerns worldwide. Currently, MARs have been to Zn2+, Pb2+, Cu2+ and Fe3+. Kocuria kristinae,
reported in a wide range of human pathogenic Escherichia coli and Acinetobacter lwoffii, which
or opportunistic bacteria such as Vibrio sp. (66), were isolated from the seawater underneath the
Klebsiella pneumoniae (67), Salmonella sp. (68), fish farm, displayed similar sensitivities to all
Pseudomonas aeruginosa and also in pathogens tested heavy metal salts. Resistances to heavy
(69, 70). Reservoirs of antibiotic resistance can metals for Aeromonas and Pseudomonas isolates
interact between different ecological systems and were similar to those from İskenderun Bay, with
potential transfer of resistant bacteria or resistant cadmium, 35.0% and 56.5%; copper, 98.3% and
genes from animals to humans may occur through 75.4%; chromium, 38.3% and 31.9%; lead, 1.7%
the food chain (70). In the current study, the and 7.2%; manganese, 43.3% and 44.9%; and
MAR index of multiple antibiotic-resistant bacteria zinc 35.0% and 41.3%, respectively (72).

Both Gram-positive and Gram-negative bacteria pesticides and heavy metals might encourage
can resist heavy metals (73). Resistance to selection and result in antibiotic and heavy metal
toxic metals in bacteria probably reflects the resistance. Marine environmental conditions are
level of environmental contamination with extremely dynamic compared to the terrestrial
these substances and it may be related to the environment, allowing bacteria to bring resistance
concentration of bacteria (74). The present project mechanisms they have developed together while
found heavy metal pollution in Güllük Bay sediment being adapted to the varying conditions. This
samples at all stations. In the sediment samples, makes the isolation of various bacteria useful to
the heavy metal contents were reported at varying assess environmental pollution and provides a
rates: between 1 μg g–1 and 209 μg g–1 for lead; pathway to possible solutions to remove pollution
10 μg g–1 and 259 μg g–1 for zinc; 1 μg g–1 and from marine environments. For bacteria to take
59 μg g–1 for copper; 0.1 μg g–1 and 46 μg g–1 part in the transformation of any heavy metal salt
for chromium; <0.01 μg g–1 and 2.8 μg g–1 for into a harmless form, those bacteria must firstly
cadmium; <0.01 μg g–1 and 0.4 μg g–1 for arsenic; be resistant to the heavy metal; thus the data
and 0.6% and 5.9% for aluminium, respectively. related to frequency of metal resistant bacteria can
The region was defined according to cadmium, lead provide knowledge on the continual accumulation
and zinc levels as moderately polluted. Recorded or transformation of heavy metals in the marine
high metal values were evaluated as an indicator of environment.
domestic and industrial inputs, carried via Sarıçay The findings of the current study provide
Creek, port operations and tourism activities data regarding the distribution of heavy metal-
within Güllük Bay (75). In the current study, the and antibiotic-resistant bacteria in seawater
high frequencies of heavy metal-resistant bacteria and sediment samples of Güllük Bay, Aegean
detected in the sediment samples support this Sea, Turkey. As a result, preliminary data on
data. Bacterial heavy metal resistance detected candidate bacteria will offer opportunities for
in the study may depend on many factors. A further studies on the elimination of heavy metal
possible explanation for differences in heavy metal contamination by the detection of heavy metal-
resistance is the proximity of Güllük Bay to iron-steel resistant bacteria.
factories. Additionally, Güllük Harbour is a serious
pollution source. It was reported that 2862‑unit
5. Conclusions
ships carried 4.8 million tonnes of ballast water to
Güllük Harbour during 2007–2012 (37). Another Analyses of the presence of antibiotic resistance
potential source of increased resistance may be the in bacteria provide knowledge on pollution sources
discharge of thermal power plants located 107 km, such as septic systems on regional ecosystems.
46 km and 39 km away from Güllük Bay. The Since antibiotic-resistant bacteria can affect
effects of thermal power plant discharge on the pathogen virulence, these pollution sources can
accumulation of heavy metals have been reported induce pathogens and can create health risks
in other studies (29, 75). for both humans and the ecosystem. In the
The association between antibiotic resistance present study, bacteria resistant to antibiotics
and resistance to heavy metals is quite common and heavy metals in seawater and sediment were
in the same organism. The increasing numbers investigated. The bacterial information obtained
of antibiotic and heavy metal-resistant bacteria provides essential data for identifying the regional
could be a result of gene transfer activities distribution of resistant bacteria. Levels of
demonstrating that industrial pollution most resistance against heavy metals and antibiotics in
likely selects for antibiotic resistance and vice bacteria isolated from seawater and sediments of
versa (58). In this study, similarly, the most the Aegean Sea were quantified. Bacteria isolated
antibiotic-resistant bacteria such as Sphingomonas from Güllük Bay sediment were resistant to all
paucimobilis, Escherichia coli and Enterobacter antibiotics tested and exhibited higher resistance
cloacae were also resistant to heavy metals. than those isolated from seawater. The frequency
Metal‑resistant isolates from Güllük Bay also of antibiotic-resistant bacteria was higher around
showed high resistance to sulfonamide, rifampicin fish farms and near the exit of Sarıçay Creek. The
and ampicillin. Bacteria from different sources widespread resistances of indicator bacteria to
such as humans, animals and soil can transfer or antibiotics suggest the presence of anthropogenic
exchange their resistance genes. At the same time, influences due to domestic waste and maritime
water contaminated with antibiotics, disinfectants, transport.

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228, (2), 175 2020, 63, (1), 117
The Authors
Gülşen Altuğ is a professor in the Department of Marine Biology of the Faculty of Aquatic
Science at Istanbul University, Turkey. Her research focuses on marine bacteriology,
including bacterial diversity and micro-geographical variations, clinical, industrial and
ecological uses of marine isolates, bacterial pollution, epibiotic bacterial communities
and anti-bacterial characteristics, bacterial remediation (oil degrading capacity of marine
isolates) and resistant bacterial isolates against heavy metals and antibiotics. She is also
the inventing founder of the biotechnology company named BIYOTEK15 R&D Training and
Consulting Industry and Trade Ltd Company in Entertech of Istanbul University Technocity.
Mine Çardak is an associate professor at Çanakkale Onsekiz Mart University, School of

Çanakkale Applied Sciences, Department of Fisheries Technology, Turkey. Her researches
focus on marine bacteriology, bacterial resistance against heavy metals and antibiotics,
bacterial pollution and biotechnology. She has worked as a scientist since 2000.
Pelin Saliha Çiftçi Türetken is a researcher at İstanbul University, Faculty of Aquatic

Sciences, Department of Marine Biology. Her research focus on marine bacteriology,
bacterial remediation, bacterial resistance and biotechnology. She has a PhD degree in
marine biology. She has worked as an academic at university since 2005.

Samet Kalkan has a PhD degree from Istanbul University, Institute of Graduate Studies in
Science and Engineering, Department of Marine Biology. He currently works as a doctor
scientist at Recep Tayyip Erdogan University- Faculty of Fisheries, Department of Marine
Biology, Turkey. He has worked as academic at university since 2010. His main researches
focus on marine bacteria, bacterial diversity, bacterial pollution, resistant bacteria against
heavy metals-antibiotics, also marine biotechnology. He has scientific abroad experiences
in Italy and Portugal.
Sevan Gürün graduated with a degree in Biology from Istanbul University. He has a PhD
degree from Istanbul University, Institute of Graduate Studies in Science and Engineering,
Department of Marine Biology. He worked as a researcher in various scientific projects.
He has been working as a researcher in a private company since 2016. His expertise
focuses on bacterial diversity, marine bacteria, bacterial pollution, bacterial biotechnology,
resistant bacteria against heavy metals and antibiotics.

“Nanomaterials and Environmental

Biotechnology”
Edited by Indu Bhushan (Shri Mata Vaishno Devi University, India), Vivek Kumar
Singh (Shri Mata Vaishno Devi University, India), Durgesh Kumar Tripathi (Amity
University, India), Nanotechnology in the Life Sciences Series, Springer Nature
Switzerland AG, Cham, Switzerland, 2020, 434 pp, ISBN: 978-3-030-34543-3,
£129.99, €155.99, US$179.99
Reviewed by Martin Hayes on aluminium oxide and zinc oxide nanoparticles

Johnson Matthey, 28 Cambridge Science Park, hindering root growth rate.
Milton Road, Cambridge, CB4 0FP, UK Chapter 2 considers an entirely separate but
equally important area of use of nanomaterials in
Email: martin.hayes@matthey.com food packaging. It concludes that “it is essential to
perform safety assessment of nanomaterials before
their application in food packaging or processing”
Introduction and provide a citation on how to do this using a
“decision tree”.
This book is a fascinating account of how
nanoparticles and nanotechnology are increasingly
Biosensors from Nanobiotechnology
employed in a diverse array of applications ranging
from plant growth to food packaging, biosensing, Chapter 7 introduces how biosensors derived
enzyme immobilisation and more. The book is from nanobiotechnology can be used to monitor
divided into 20 chapters, each dealing with a the environment and gain information relating
specific application of nanotechnology and written to its health and the detrimental effects that
by a different group of eminent academics from modernisation and industrialisation have had
Indian universities and research institutes. on the planet. Biosensors need to be specific,
Each chapter is a review of its own topic area and the rapid, sensitive and cost-effective. The advent
literature citations at the end of each chapter make of nanotechnology and biosensors has made this
it easy for the reader to use this book as a reference possible and the authors of this chapter (Gupta and
volume from which further in-depth reading can be Kakkar) explore the different types of biosensors
pursued by following the cited literature. that have been developed over recent years. The
authors give a brief explanation of how different
types of sensors work using a combination of bio-
Safety of Nanoparticles in Plants and
recognition components and different transduction
Packaging
principles. Types include: (a) immunosensors;
Chapter 1 deals with the phytotoxicity of (b) enzymatic biosensors; (c) whole-cell based
nanoparticles in plants which can lead to both sensors; (d) biosensors; (e) genosensors;
positive and negative outcomes. For example, (f) aptasensors and (g) biomimetic biosensors. The
improvement in germination rate and growth role of the transducer is to convert the biochemical
have been reported in seeds of rice exposed to response into an analysable and measurable
carbon nanotubes; on the other hand, toxicity signal. The outputs can be electrochemical, optical,
has also been widely reported, including studies piezoelectric, thermometric or magnetic.

Enzyme Immobilisation to traditional drug delivery systems, offering

multiple advantages in terms of drug delivery
Chapter 10 tackles the interesting area of enzyme and bioavailability, as well as being economically
immobilisation and the use of chitosan nanoparticles efficient and easy to produce on scale.
therein. The biopolymer’s distinct physicochemical
properties have been described to offer an excellent
FDA-Approved Nanomedicines
microenvironment for enzyme immobilisation
through adsorption, covalent binding or cross- An extensive summary of FDA-approved
linking, to achieve desirable enzymatic activity nanomedicines is included in Table 19.1
and stability. On the other hand, nanoparticles as (Chapter 19), which also summarises the
materials of enhanced properties, owing to their advantages of these specific formulations. The
high surface to volume ratio, have been introduced main types of nanocarriers described in Chapter 19
as attractive candidates for enzyme immobilisation. are vesicular carriers (liposomes and niosomes),
The chapter briefly discussed various methods polymeric nanoparticles and inorganic carriers
for the preparation of chitosan nanoparticles for (silica, gold and calcium nanoparticles). Liposomes
enzyme immobilisation including reverse micelle, and solid lipid nanoparticles (see also Chapter 13)
coprecipitation, ionotropic gelation and ionic or are suitable for the delivery of drugs by any
emulsion cross-linking methods. Different methods route, either oral or parenteral and can be used
for enzyme immobilisation such as support binding, with both hydrophilic and lipophilic drugs. Their
cross-linking and entrapment, as well as different main advantages reside in protecting labile drugs,
materials used as supports have been explained limited toxicity and a sustainable targeted release
too. This section is then closed by presenting some of the drug.
examples for immobilisation of different enzyme Inorganic nanocarriers exhibit higher stability and
families (for example, α-amylase, β-galactosidase, resistance to microbial growth, while having a low
cellulase, laccase, lipase or protease) through toxicity and allowing facile surface modifications.
applying chitosan nanoparticles. Mesoporous silica nanoparticles allow encapsulation
of the therapeutic agent and targeted delivery to
tumour cells in cancer therapy. Gold nanoparticles
Solid Lipid Nanoparticles
are biocompatible and bio-inert and have been
Chapter 13 offers an overview of solid lipid successfully used in covalent conjugation with
nanoparticles (SLN) as pharmaceuticals delivery protein antigens in developing vaccines for cancer
systems whereas Chapter 19 gives a review of the immunotherapy. Calcium phosphate nanoparticles
most commonly used nanocarriers for drug delivery are excellent candidates for developing ceramic-
systems, with a focus on vesicular, polymeric and based carriers for peptide drugs prone to
inorganic carriers. degradation, such as insulin.
SLN are lipid-based formulations, containing
typically non-toxic biodegradable polymers
Summary
forming a solid hydrophobic core suspended in
an aqueous phase, the whole structure being In conclusion, I consider this book to be a positive
stabilised by surfactants. The therapeutic agent is contribution to the biotechnology literature,
dissolved or dispersed in the solid lipid core, the although I do not recommend reading this book
SLN being suitable for incorporation and delivery sequentially from Page 1 as the variety of topics
of both hydrophilic and hydrophobic drugs. SLN introduced is too great and each individual topic
present significant advantages over conventional is not explored in depth. It is best used (and
drug delivery systems, including but not limited deserves recommendation) as a reference source
to biocompatibility and bioavailability, reduced from which each chapter can be used as the
drug leakage and increased physical stability starting point to a more in-depth study or review
of the drugs. In addition, they have been used of a particular topic. There are some negative
successfully in various drug delivery techniques. aspects of the presentation of this work which do,
Novel applications of SLN as drug carriers are unfortunately, detract from its enjoyment. These
described in the field of gene therapy, peptide drug are exemplified in the poor quality of the diagrams,
delivery and vaccines. SLN production methods the grammatical errors and the somewhat odd
use low mechanical force, allowing successful references of Chapter 7.
incorporation and delivery of nucleic acids in gene Overall, though, this book is a positive addition to
therapy. Overall, SLN are promising alternatives the biotechnology reference bookshelf.

"Nanomaterials and Environmental Biotechnology"
The Reviewer
Martin Hayes is Biotechnology Lead at Johnson Matthey based in Cambridge, UK.
He has worked with Johnson Matthey since 1997 and has held multiple research,
development and customer-facing technical roles across the company. He holds a
PhD in heterogeneous catalysis and is interested in the application of biology as
a technology to realise circular chemical processing and accelerate the transition
to “Net Zero”.

Biocatalytic Reduction of Activated Cinnamic

Acid Derivatives
Asymmetric reduction of C=C double bonds using Johnson Matthey enzymes
Samantha Staniland, Tommaso hydrogenation. Traditionally, whole cell

Angelini, Ahir Pushpanath, Amin microorganisms were used for this purpose but
Bornadel, Elina Siirola, Serena a recent increase in the number of isolated and
Bisagni, Antonio Zanotti-Gerosa, characterised ENEs means that recombinantly-
Beatriz Domínguez* expressed enzyme preparations are now generally
Johnson Matthey, 260 Cambridge Science Park, favoured over whole cells, as a number of recent
Milton Road, Cambridge, CB4 0WE, UK publications demonstrate (1–10).
Double bond ‘activation’ to facilitate ENEs mediated
*Email: beatriz.dominguez@matthey.com reduction can be achieved in many cases by alpha
substituted functional groups including aldehydes,
ketones or nitro moieties. Carboxylate derivatives
The asymmetric reduction of C=C double bonds is (such as esters, lactones and anhydrides) can
a sought-after chemical transformation to obtain also act as activating groups but their ability to
chiral molecules used in the synthesis of fine sufficiently activate the C=C bond in the absence of
chemicals. Biocatalytic C=C double bond reduction other groups is less evident (11, 12). The traditional
is a particularly interesting transformation approach in these cases is to turn to chemocatalytic
complementary to more established chemocatalytic hydrogenation (see (13–15) for reviews focused on
methods. The enzymes capable of catalysing this industrial applications). Herein we describe a new
reaction are called ene-reductases (ENEs). For approach to activate α,β-unsaturated carboxylic
the reaction to take place, ENEs need an electron acids for the reduction with ENEs using a substrate
withdrawing group (EWG) in conjugation with engineering approach.
the double bond. Especially favourable EWGs
are carbonyls and nitro groups; other EWGs,
2. Experimental
such as carboxylic acids, esters or nitriles, often
give poor results. In this work, a substrate 2.1 General
engineering strategy is proposed whereby a
simple transformation of the carboxylic acid into a All reagents and solvents were purchased from
fluorinated ester or a cyclic imide allows to increase Sigma-Aldrich and Alfa Aesar, Thermo Fisher
the ability of ENEs to reduce the conjugated double Scientific. They were of the highest available purity
bond. Up to complete conversion of the substrates and were used without further purification. 1H
tested was observed with enzymes ENE-105 and nuclear magnetic resonance (NMR) spectra were
*ENE-69. recorded using a Bruker 400 MHz Avance III HD
equipped with SMART probe (Bruker Corporation,
USA) where spectra are referenced to deuterated
1. Introduction
chloroform (CDCl3) 7.26 ppm, shifts are recorded
The use of enzymes for the asymmetric reduction in parts per million and J values in hertz. The
of activated C=C double bonds can be a viable NMR results can be found in the Supplementary
and straightforward alternative to asymmetric Information.

2.2 Enzyme Preparations was then quenched by addition of saturated

aqueous NaHCO3 (20 ml) and extracted with
Genes coding for Johnson Matthey, ENEs (ENE-101, dichloromethane (2 × 20 ml), dried over MgSO4,
ENE-102, ENE-103, ENE-104, ENE-105, *ENE-69 filtered and concentrated under reduced pressure
and GDH-101) were ordered codon-optimised from to afford the corresponding fluorinated esters 6a
GeneArt (Thermo Fisher Scientific) and cloned and 7a in 95% to 99% yield.
into T5 vector pJEx401 (ATUM). Enzymes were
expressed recombinantly in Escherichia coli BL21
2.5 1-Cinnamoylpyrrolidin-2-one
in both shake flasks and fed batch fermentations,
(9a)
whereby induction was carried out with isopropyl
β-D thiogalactopyranoside (IPTG) at 30°C. Cinnamoyl chloride (5 g, 30.01 mmol), pyrrolidinone
Harvested biomass was resuspended in 100 mM (2.3 ml, 36.01 mmol) and triethylamine (13 ml,
potassium phosphate buffer (pH 7) and cells were 90.03 mmol) in dichloromethane (50 ml) were
broken up either by sonication or homogenisation. stirred at room temperature overnight. The reaction
The so-obtained cell lysate was clarified by was quenched by addition of water (20 ml), the
centrifugation and filtrated prior to lyophilisation. organic layer was separated and washed with
Protein expression was assessed by sodium saturated aqueous NaCl (20 ml), dried over MgSO4,
dodecyl sulfate polyacrylamide gel electrophoresis filtered and concentrated under reduced pressure
(SDS-PAGE) and chromatographic activity assays. to afford 9a in 81% yield.
Enzymes ERED-103, ERED-110, ERED-112,
ERED-207, ERED-P1-A04, ERED-P1-E04 and ERED-
2.6 3-Cinnamoyloxazolidin-2-one
P1-H09 were purchased from Codexis.
(8a)
Cinnamic acid 1a (5 g, 33.56 mmol) and oxalyl
2.3 2,2,2-Trifluoroethyl Cinnamate
chloride (2.85 ml, 33.56 mmol) in dichloromethane
(3a) and 3-Phenyl-Acrylic Acid
(5 ml) were stirred at room temperature overnight
2,2,2-Trifluoro-1-Trifluoromethyl-
before removing the solvent under reduced
Ethyl Ester (5a) pressure. The reaction crude was dissolved
Cinnamic acid 1a (5 g, 33.75 mmol) and oxalyl in anhydrous tetrahydrofuran (THF) (20 ml)
chloride (2.85 ml, 33.75 mmol) in dichloromethane and n-butyllithium (1.6 M in hexane, 21 ml,
(5 ml) were stirred at 25°C for 2 h before adding the 33.56 mmol, one equivalent) was added dropwise
fluorinated alcohol-trifluoro ethanol for 3a (2.47 ml, over 30 min. The cinnamoyl chloride solution was
33.75 mmol) and 1,1,1,3,3,3-hexafluoropropan- then added dropwise to a solution of oxazolidinone
2-ol for 5a (3.50 ml, 33.75 mmol). The reaction (2.92 g, 33.56 mmol) in anhydrous THF (100 ml) at
was then stirred at room temperature overnight 0°C before stirring at room temperature overnight.
before being quenched by addition of saturated The reaction was quenched with water (50 ml),
aqueous NaHCO3 (20 ml) and extracted with extracted with ethyl acetate (EtOAc) (2 × 100 ml),
dichloromethane (2 × 20 ml), dried over MgSO4, washed with saturated aqueous NaHCO3 (20
filtered and concentrated under reduced pressure ml) and saturated aqueous NaCl (20 ml). The
to afford the corresponding fluorinated esters 3a solvent was removed under reduced pressure and
and 5a in quantitative yield. the solid was recrystallised from a 1:1 mixture
EtOAc:heptane (20 ml). The solid was filtered and
washed with hexane (10 ml) to give crystals of 8a
2.4 3-Phenyl-Acrylic Acid
in 80% yield.
2,2,3,3,4,4,4-Heptafluoro-Butyl Ester
(6a) and (Perfluorophenyl)Methyl
Cinnamate (7a) 2.7 (E)-1-(2-Methyl-3-
Phenylacryloyl)Pyrrolidin-
Cinnamoyl chloride (0.75 g, 4.50 mmol)
2-one (10a) and (E)-1-(2,3-
and the corresponding fluorinated alcohols
– 2,2,3,3,4,4,4-heptafluorobutan-1-ol for
Diphenylacryloyl)Pyrrolidin-2-one
6a (0.98 g, 4.50 mmol) and pentafluroro (11a)
benzyl alcohol for 7a (0.89 g, 4.50 mmol) –
in dichloromethane (2.5 ml) were stirred at (E)-2-methyl-3-phenylacrylic acid (5 g,
room temperature overnight. The reaction 30.86 mmol) was converted to the corresponding

acid chloride by addition of oxalyl chloride (1.4 ml, C18 SunFire Column (Waters Corporation, USA,
30.86 mmol) in dichloromethane (5 ml). The 150 × 4.6 mm, 3.5 µm) with an isocratic method
reaction was stirred at room temperature for (MeCN:Water, 30:70 + 0.1% trifluoroacetic acid)
3 h. Pyrrolidinone (2.82 ml, 37.03 mmol) and and a flow rate of 1 ml min–1.
triethylamine (13 ml, 92.58 mmol) were added Chiral HPLC analysis was performed on a Varian
before stirring the reaction overnight. The reaction ProStar series (Agilent) with a CHIRALCEL® OD-H
was quenched by addition of water (20 ml) and column (Chiral Technologies, USA, 250 × 4.6 mm,
saturated aqueous NaCl (20 ml). The solvent 5 µm) with an isocratic method A (heptane:isopropyl
was removed under reduced pressure and the alcohol (IPA), 88:12) and a flow rate of 1 ml min–1
solid was dissolved in EtOAc and treated with or isocratic method B (heptane:IPA, 98:2).
activated charcoal (1 g), filtered through Celite® GC analysis of conversion was performed on
and concentrated. The solid was recrystallised from a Varian CP-3800 (Agilent) using γ-DEX™ 225
heptane (10 ml) to give 10a in 55% yield. capillary column (Sigma-Aldrich, 30 m × 0.25 mm
Following an identical procedure, 11a was × 0.25 μm) and using helium as carrier gas.
synthesised in 53% yield from (E)-2,3- Percentage conversion was measured by integration
diphenylacrylic acid (10 g, 44.64 mmol). of the product peak in the GC (uncorrected area
under curve (AUC)), values below 100% indicate
that unreacted starting material was detected. No
2.8 Small Scale Screening Reactions
side products were detected in any of the reported
Substrates 1a–9a (0.025 mmol) and enzymes reactions. GC program parameters: injector 250°C,
ENE-101, ENE-102, ENE-103, ENE-104, ENE-105 flame ionization detector (FID) 250°C, 80°C for
or *ENE-69 (2.5 mg), were added to reaction 3 min then 5°C min–1 up to 160°C, hold 1 min
vials containing 500 µl of aqueous media at pH 7 (total time 20 min), constant flow 5 ml min–1.
(250 mM potassium phosphate buffer pH 7, 1.1 mM
NAD(P)+, 100 mM D-glucose, 10 U ml–1 GDH-101)
to give a final concentration of substrate of 50 mM.
The vials were shaken at 400 rpm, 30°C for 18 h. It has been found that a particular ENE in Johnson
For high-performance liquid chromatography Matthey’s collection, a homologue from the tobacco
(HPLC) analysis, the reactions were quenched with ENE reductase fold (16), ENE-105, was capable
acetonitrile (MeCN) (1 ml), vortexed, centrifuged of reducing methyl ester 2a (Figure 1), albeit
and aliquoted. For gas chromatography (GC) in a very low yield of 3% (Entry 2, Table I). By
analysis, samples were extracted with EtOAc comparison, cinnamic acid 1a was a poor substrate
(2 × 0.5 ml), dried over MgSO4 and analysed and showed no conversion to the reduced product
directly. For NMR analysis, the reactions were 1b at pH 7.0 (Entry 1, Table I). The pKa of
extracted with CDCl3 and analysed directly. cinnamic acid 1a is 4.4 and therefore, at pH 7.0,
the carboxylic acid should be deprotonated
affecting its ability to bind to the enzyme active
2.9 Preparative Scale Screening
site. This observation is in line with other literature
Reactions
examples where carboxylates were found to be
Reactions were scaled up using three-neck round poor activating groups (17). Encouraged by this
bottom flask equipped with stir bar and pH initial result, we turned our efforts towards the use
titrator (10 M NaOH). To the flask was weighed of more activated esters. It was envisaged that
100–500 mg substrate (40–100 mM final converting the alkyl chain in the ester moiety to a
concentration) and 5 mg ml–1 enzyme which was more EWG could lead to an increase in double bond
suspended in aqueous media at pH 7 (250 mM activation. A similar approach has been reported
potassium phosphate buffer pH 7, 1.1 mM previously by BASF SE for the lipase-catalysed
NAD(P)+, 100–200 mM D-glucose (two equivalent), kinetic resolution of racemic amines and alcohols,
10 U ml–1 GDH-101) the reactions were stirred at where the choice of acylating agent proved critical
30°C, 400 rpm for 18 h. (18). We chose trifluoroethyl ester 3a as a starting
point which was reduced by ENE-105 and *ENE-69
in 6% and 12% conversion respectively (Entry 3,
2.10 Analytical Methods
Table I) suggesting that the addition of an EWG
HPLC analysis of conversion was conducted on an had a positive activating-effect on the reduction. To
1260 Infinity II LC system (Agilent, USA) using a consolidate this theory, ethyl ester 4a was tested

O Fig. 1. Reduction of
O
cinnamic acid and
R [a]
R cinnamoyl esters.
[a] = 1–7a (50 mM
1a R = OH 1b R = OH concentration), ENE-
2a R = OCH3 2b R = OCH3 105 or ENE-69 (5 mg
3a R = OCH2CF3 3b R = OCH2CF3 ml–1), 500 µl buffer
4a R = OCH2CH3 4b R = OCH2CH3 (250 mM KPi, pH 7, 1.1
5a R = OCH(CF3)2 5b R = OCH(CF3)2 mM NAD(P)+, 100 mM
6a R = OCH2CF2CF2CF3 6b R = OCH2CF2CF2CF3 D-glucose, 10 U ml–1
7a R = OCH2C6F5 7b R = OCH2C6F5 GDH-101), 400 rpm,
30°C, 18 h
Table I Reduction of Cinnamoyl Esters at Table II Reduction of Cinnamoyl Cyclic

50 mM Substrate Concentration, Imide Derivatives at 50 mM
pH 7, 30°C, 18 h Substrate Concentration, pH 7,
Conversion, %a 30°C, 18 h
Entry Substrate ENE-105 *ENE-69 Conversion, %
1b 1a 0 0 Entry Substrate ENE-105 *ENE-69
a
2 2a 3 1 1 8a 51 39
3 3a 6 12 2b 9a >95 >95
a
Integration of the product peak in the HPLC (chiral method,
4 4a <0.5 <0.5
uncorrected AUC), values below 100% indicate that unreacted
c
5 5a 0 0 starting material was detected; no side products were
detected for these reactions
d
6 6a <0.5 <0.5 b
Conversion calculated by 1H NMR
7d 7a 0 0
a
Integration of the product peak in the GC (uncorrected AUC),
values below 100% indicate that unreacted starting material cyclic imides since activated substrates 8a and
was detected; no side products were detected for these
reactions
9a have been shown to be highly activated
b
Integration of the product peak in the HPLC (achiral method, towards Michael addition reactions (19, 20, 21)
uncorrected AUC), values below 100% indicate that unreacted (Figure 2). Compounds 8a and 9a were
starting material was detected; no side products were
detected for these reactions synthesised and tested with enzymes ENE-105
c
10% cinnamic acid observed and *ENE-69. Pleasingly, oxazolidinone 8a was
d
Conversion calculated by 1H NMR successfully reduced by both ENEs (51% and
39% conversion to 8b, Entry 1, Table II) and
with the novel ENEs; only a trace of reduction was pyrrolidinone 9a was reduced to 9b in >95%
observed <0.5% (Entry 4, Table I). conversion (Entry 2, Table II), proving to be
Other EWGs such as hexafluoroethyl in an excellent activating group. The 1H NMR shift
compound 5a, heptafluorobutyl in 6a and of the alkene proton alpha to the carbonyl for
pentafluorobenzyl in 7a could also activate the pyrrolidinone 9a is shifted down field (7.92 ppm)
double bond in the same way, so 5a, 6a and 7a compared to cinnamic acid 1a (6.46 ppm),
were prepared by reacting cinnamoyl chloride therefore supporting the electron-withdrawing
with the corresponding fluorinated alcohols nature of the activating group.
and these substrates were subsequently tested The enzymes were then tested for their ability to
with the ENEs. Hexafluoro 5a was not reduced reduce α-substituted cinnamic acid derivatives such
by ENE-105 or *ENE-69 (Entry 5, Table I), as α-methyl 10a and α-phenyl 11a (Figure 3).
instead, a significant amount of hydrolysis Encouragingly, the tri-substituted double bond in
product (cinnamic acid 1a, 10%) was observed. 10a was reduced to 10b in >95% conversion by
1
Heptafluorobutyl 6a and pentafluoro 7a were H NMR analysis (Entry 2, Table III). However,
poor activating groups with 6a showing only a bulkier substrate 11a, was not tolerated so well on
trace amount of product 6b (Entry 6, Table I) an analytical scale due to solubility issues causing
and 7a giving no conversion (Entry 7, Table I). mass-transfer limitations (Entry 3, Table III).
With only limited success with the fluorinated The reaction was repeated on a larger scale with
activating groups, our efforts turned towards stirring (Entry 4, Table III) and >95% conversion

O O Fig. 2. Cinnamoyl cyclic

imide derivatives.
[a]
R R [a] = 8a–9a (50 mM
concentration), ENE-
105 or ENE-69 (5 mg
O O O O
ml–1), 500 µl buffer
(250 mM KPi, pH 7, 1.1
N O N N O N mM NAD(P)+, 100 mM
R= R= D-glucose, 10 U ml–1
8a 9a 8b 9b
GDH-101), 400 rpm,
30°C, 18 h
O O O O Fig. 3. Reduction
[a] of α-substituted
N N
cinnamoyl
R R pyrrolidinones. [a] =
9a R = H 9b R = H 9a–11a (40–100
10a R = Me 10b R = Me mM concentration),
11a R = Ph 11b R = Ph ENE-105 or ENE-69
(5 mg ml–1), buffer
(250 mM KPi, pH 7,
1.1 mM NAD(P)+, two
equivalent D-glucose,
10 U ml–1 GDH-101),
400 rpm, 30°C, 18 h
Table III Reduction of α-Substituted carboxylic acids, other commercially available

Cinnamoyl Pyrrolidinones at enzymes were tested as a comparison on the
50 mM Substrate Concentration, reduction of 10a (Table V). Six enzymes
pH 7, 30°C, 18 h
from Johnson Matthey collection (Entries 3
Conversion, %a to 6, Table V) and seven enzymes purchased
Entry Substrate ENE-105 *ENE-69 from Codexis (Entries 7 to 13, Table V) were
1 9a >95 >95 compared with ENE-105 and ENE-69* (Entries 1
2 10a >95 >95 and 2, Table V). It was found that, despite the
3 11a 24 2 extra activation of the C=C double bond, none of
b the tested enzymes could reduce cinnamic acid
9 11a >95 –
a
derivative 10a, highlighting the unique ability of
b
100 mg scale with stirring
ENE-105 and *ENE-69 within the focused library
(13 enzymes) screened.
In summary, we have shown that cinnamic acid
was achieved. 10b and 11b were obtained as derivatives activated as fluorinated esters or
racemic mixtures. as cyclic imides can be reduced using Johnson
With a successful activating group found, the Matthey enzymes ENE-105 or *ENE-69. The
reaction was repeated on a preparative scale to concept of ‘substrate engineering’ as opposed
test reproducibility and scalability (Table IV). to ‘enzyme engineering’, offers a complimentary
Pyrrolidinone 9a was successfully reduced using and faster approach to developing a bioprocess,
enzyme ENE-105 at 130 mg scale with the desired making difficult transformations possible. The
product 9b being obtained in 95% conversion by reduced products can be subsequently converted
1
H NMR (Entry 1, Table IV). 72% conversion to to the parent carboxylic acids by LiOH hydrolysis
10b was achieved after 20 h (Entry 3, Table IV) (22, 23) and the potential re-use of these
on the reduction of pyrrolidinone 10a at 500 mg activating groups will be investigated in the
scale. future. It is envisaged that the work will lead
Having found enzymes in Johnson Matthey’s to further examples of activated acids or esters
collection that could successfully reduce masked being reduced by ENEs.

Table IV Reduction of Cinnamoyl Pyrrolidinones by ENE-105 at pH 7b and 30°C

Entry Substrate Scale, mg Concentration, mM Time, h Conversion, %a
1 9a 130 40 16 >95
2 10a 500 100 4 33
3 10a 500 100 20 72
a
b
pH controlled with NaOH titration (10 M)
Table V Reduction of Cinnamoyl procedure, and in combination with the right

Pyrrolidinone 10a at 50 mM enzyme, it was possible to biocatalytically reduce
Substrate Concentration, pH 7, the conjugated double bond of cinnamic acid and
30°C, 18 h substituted derivatives.
Entry Enzyme Conversion, %a
1 ENE-105 >95 References
2 *ENE-69 >95
1. B. Dominguez, U. Schell, S. Bisagni and T. Kalthoff,
3 ENE-101 <0.5
Johnson Matthey Technol. Rev., 2016, 60, (4),
4 ENE-102 1 243
5 ENE-103 0
2. M. Hall, C. Stueckler, H. Ehammer, E. Pointner, G.
6 ENE-104 0 Oberdorfer, K. Gruber, B. Hauer, R. Stuermer, W.
7 ERED-103 0 Kroutil, P. Macheroux and K. Faber, Adv. Synth.
8 ERED-110 0.5 Catal., 2008, 350, (3), 411
9 ERED-112 0 3. M. Hall, C. Stueckler, W. Kroutil, P. Macheroux and

K. Faber, Angew. Chem., Int. Ed., 2007, 46, (21),
10 ERED-207 <0.5
3934
11 ERED-P1-A04 1
4. J. F. Chaparro-Riggers, T. A. Rogers, E. Vazquez-
12 ERED-P1-E04 0
Figueroa, K. M. Polizzi and A. S. Bommarius, Adv.
13 ERED-P1-H09 0 Synth. Catal., 2007, 349, (8–9), 1521
a
5. A. Müller, B. Hauer and B. Rosche, Biotechnol.
Bioeng., 2007, 98, (1), 22
6. M. A. Swiderska and J. D. Stewart, J. Mol. Catal.
4. Conclusions B: Enzym., 2006, 42, (1–2), 52
7. D. Dobrijevic, L. Benhamou, A. E. Aliev, D. Méndez-
The biocatalysed reduction of the double bond of Sánchez, N. Dawson, D. Baud, N. Tappertzhofen,
cinnamic acid derivatives is strongly influenced by T. S. Moody, C. A. Orengo, H. C. Hailes and J. M.
the nature of the EWG. While no conversion was Ward, RSC Adv., 2019, 9, (63), 36608
observed on the biocatalysed reduction of cinnamic 8. H. S. Toogood and N. S. Scrutton, ACS Catal.,
acid 1a, an enzyme in Johnson Matthey’s collection, 2018, 8, (4), 3532
ENE-105, was capable of reducing methyl ester 9. G. Brown, T. S. Moody, M. Smyth, S. J. C.
derivative 2a in low conversion. By replacing the Taylor, ‘Almac: An Industrial Perspective of Ene
alkyl chain in the ester moiety by a more EWG, Reductase (ERED) Biocatalysis’, in “Biocatalysis:
such as fluorinated alkanes, and in the presence An Industrial Perspective”, eds. G. de Gonzalo and
of enzymes ENE-105 and *ENE-69, we were able P. Domínguez de María, ch. 8, Royal Society of
to significantly increase conversion to the reduced Chemistry, London, UK, 2018, pp. 229–256
product. Furthermore, other electronegative 10. D. Mangan, I. Miskelly and T. S. Moody, Adv.
derivatives such as cyclic imides proved to be even Synth. Catal., 2012, 354, (11–12), 2185
better activating groups, allowing the reduction 11. R. Stuermer, B. Hauer, M. Hall and K. Faber, Curr.
of challenging substituted double bonds such as Opin. Chem. Biol., 2007, 11, (2), 203
substrates 10a and 11a. 12. Y. Kawai, M. Hayashi, Y. Inaba, K. Saitou and A.
In summary, by ‘masking’ the carboxylic acid Ohno, Tetrahedron Lett., 1998, 39, (29), 5225
moiety into a fluorinated alkyl ester or a cyclic 13. H. U. Blaser, B. Pugin and F. Spindler, ‘Asymmetric
imide, following a straightforward synthetic Hydrogenation’, in “Topics in Organometallic

Chemistry: Organometallics as Catalysts in the 18. M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M.

Fine Chemical Industry”, eds., M. Beller, H. U. Keßeler, R. Stürmer and T. Zelinski, Angew.
Blaser, Vol. 42, Springer-Verlag, Berlin, Germany, Chem., Int. Ed., 2004, 43, (7), 788
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The Authors
Samantha Staniland graduated from The University of Manchester, UK, in 2011 with an
MChem in Chemistry with Industrial Experience, while carrying out her industrial placement
at Pfizer, UK, in Medicinal Chemistry. In 2011–2015, Sam did a PhD in the groups of
Professor Jonathan Clayden and Professor Nicholas Turner on the biocatalytic asymmetric
synthesis of atropisomers. Sam joined Johnson Matthey in 2015 as a research chemist in
catalysis.
Tommaso Angelini completed his PhD in Chemical Science in 2010 from University of
Perugia, Italy, working on the development of environmentally friendly synthetic protocols.
During his postdoctoral studies, he finalised his work designing new continuous flow
devices for the use of solid supported catalyst in low E-Factor transformations. Later, he
gained experience in developing active pharmaceutical ingredient (API) production process
at Procos (Italy). In 2015, he joined Johnson Matthey as Research Chemist, designing
new enantioselective synthetic process for the preparation of APIs. He is now a Research
Expert at Evotec Verona (Italy), working on the production of preclinical and Phase 1 API
candidates.
Ahir Pushpanath obtained his PhD in Birkbeck College (University of London, UK) working
on the engineering of enzymes for industrial biofuel production. With a biochemistry
background, he specialises in the use of bioinformatics and computational biology in the
rational design of new enzyme variants. Ahir joined Johnson Matthey in 2013 as a Senior
Biologist and was instrumental in demonstrating the utility of computational techniques
for rapid enzyme discovery through genome mining, in silico design and targeted enzyme
engineering. He currently leads the enzyme development arm of biocatalysis, continuing
to develop faster, more effective methods for ‘predictive biocatalysis’.

Amin Bornadel studied chemical engineering and received a PhD in biotechnology from
Lund University in Sweden. For postdoctoral work, Amin went to Germany, where he
carried out research within biocatalysis at University of Dresden and Technical University
of Hamburg. In 2016, Amin joined Johnson Matthey to work as a biocatalysis researcher.
He is currently a senior scientist working in the Biotech team.
Elina Siirola completed her PhD in 2012 from the University of Graz, Austria, where she
worked on biocatalytic C=C bond hydrolysis. After a postdoctoral position in enzyme
engineering at the Max Planck Institute for Coal Research, Germany, she joined Johnson
Matthey in 2013, where she worked on biocatalysis research and development (R&D).
Since 2017 she is a Principal Scientist in the Bioreactions group at Novartis Pharma in
Basel, Switzerland.
Serena Bisagni completed her MSc in Industrial Biotechnology from the University of Pavia,
Italy, in 2010 and then moved to Lund University, Sweden, for her postgraduate studies.
In 2014 she obtained her PhD in Biotechnology in which she focused on the identification
of new Baeyer-Villiger monooxygenases for fine chemicals synthesis within the Marie Curie
Innovative Training Networks (ITN) ‘Biotrains’. In 2015 Serena joined Johnson Matthey.
Her main interests are enzyme screening for synthesis of active pharmaceutical ingredients
and fine chemicals and identification of novel biocatalysts.
Antonio Zanotti-Gerosa studied in Milano, Italy, completing his PhD in 1994 (organometallic
chemistry). His academic experience include secondments to Imperial College, UK
(Professor S. V. Ley), Nagoya University, Japan (Professor R. Noyori) and postdoctoral
research at the University of Lausanne, Switzerland (Professor C. Floriani). Since 1997 he
has been working on industrial applications of homogeneous catalysis. In 2003 he joined
Johnson Matthey and, as R&D Director, he is leading the chemocatalysis group in the
Cambridge laboratories.
Beatriz Domínguez gained her PhD in Synthetic Organic Chemistry from the University of
Vigo, Spain, and then moved to the UK where she worked with Professor Tom Brown at
the University of Southampton, UK, and with Professor Guy Lloyd-Jones at the University
of Bristol, UK. In 2002 she joined Synetix, soon to become Johnson Matthey Catalysts and
Chiral Technologies and has worked at Johnson Matthey’s facilities in Cambridge since.
Beatriz has gained broad experience in the application of metal catalysis and biocatalysis,
working closely with fine chemicals companies to deliver optimal catalysts for chemical
processes.

Johnson Matthey Technology Review is Johnson Matthey’s international journal of research exploring science
and technology in industrial applications
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ISSN 2056-5135

Johnson Matthey’s international journal of research

Volume 64, Issue 4, October 2020

Johnson Matthey Technology Review is published by Johnson Matthey Plc.

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International

Johnson Matthey’s international journal of research exploring science and

Contents Volume 64, Issue 4, October 2020

Breaking Down Barriers and Borrowing

Introduction others, such as hides and textiles, show that as

394 © 2020 Johnson Matthey

biology. Often these point back to unlocking our Conclusions

395 © 2020 Johnson Matthey

Preparation and Evaluation of a Composite

Yuxi Yan on the environment (1). Toluene is a common

396 © 2020 Johnson Matthey

397 © 2020 Johnson Matthey

Air pump Peristaltic pump

Fig. 1. Schematic diagram of the experimental set-up

398 © 2020 Johnson Matthey

Table I Physicochemical Properties of the Fillers

399 © 2020 Johnson Matthey

400 © 2020 Johnson Matthey

401 © 2020 Johnson Matthey

140 Fig. 4. Toluene elimination

0 20 40 60 80 100 120 140

3 days 7 days Fig. 5. Performance evaluation

402 © 2020 Johnson Matthey

500 1010 Fig. 6. Biomass concentration

403 © 2020 Johnson Matthey

Community barplot analysis

0 0.2 0.4 0.6 0.8 1

404 © 2020 Johnson Matthey

405 © 2020 Johnson Matthey

406 © 2020 Johnson Matthey

Unlocking the Full Evolutionary Potential of

407 © 2020 Johnson Matthey

408 © 2020 Johnson Matthey

409 © 2020 Johnson Matthey

410 © 2020 Johnson Matthey

Add through an electronic (through bond) contribution

411 © 2020 Johnson Matthey

20-fold increase in kcat

kcat = n/a kcat = 45.8 min–1

4000-fold increase in kcat/KM

4.2 Supramolecular, Non-Covalent

proteins and small molecules which are well M

understood and have very high affinity. ArMs have

412 © 2020 Johnson Matthey

Fig. 6. Schematic representation of cofactor

413 © 2020 Johnson Matthey

414 © 2020 Johnson Matthey

415 © 2020 Johnson Matthey

416 © 2020 Johnson Matthey

417 © 2020 Johnson Matthey

Paul Barker is Senior Lecturer at the University of Cambridge, Department of Chemistry,

418 © 2020 Johnson Matthey

Reduction of Biofilm Formation on Cooling

Irfan Turetgen towers provide cooling by spraying the heated

419 © 2020 Johnson Matthey

A biofilm layer is a community formed by bacterial Materials and Method

Although biofilm formation on plastic fill surfaces

420 © 2020 Johnson Matthey

421 © 2020 Johnson Matthey