Bioprocess and Biosystems Engineering (2019) 42:187–198

https://doi.org/10.1007/s00449-018-2024-y

RESEARCH PAPER

Role of zinc oxide nanoparticles for effluent treatment using

Pseudomonas putida and Pseudomonas aureofaciens
Dharmesh H. Sur1,2 · Mausumi Mukhopadhyay1

Received: 16 January 2018 / Accepted: 6 October 2018 / Published online: 25 October 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
The biological treatment of textile effluent is enhanced by the use of zinc oxide (ZnO) nanoparticles for the reduction in
chemical oxygen demand (COD) from its initial value of 1700 ppm. The present research investigated the effect of ZnO
nanoparticles when microbial cultures of Pseudomonas putida and Pseudomonas aureofaciens were used to treat textile
effluent in three-phase inverse fluidized bed bioreactor. The parameters like—size of ZnO nanoparticles, static bed height,
superficial gas velocities and solid media particle size—together affected the COD reduction and all of these were inves-
tigated in this paper. ZnO nanoparticles of 280 nm reduced the maximum COD to 47 ppm (97.24%) at low gas velocity of
0.0027 m/s at 10% inoculum size and at a static bed height of 2.43 cm.
Graphical abstract 

Keywords  Zinc oxide nanoparticles · Aerobic treatment · COD reduction · Three-phase inverse fluidized bed bioreactor ·
Textile effluent

Introduction
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s0044​9-018-2024-y) contains
supplementary material, which is available to authorized users. The existing wastewater treatment strategies are in need of
revival in context to improvement in technology, since envi-
* Mausumi Mukhopadhyay ronmental pollution control demands cheaper wastewater
mmu@ched.svnit.ac.in;
treatment technologies. The need of the time is to develop
mausumi_mukhopadhyay@yahoo.com
technologies that treat pollutants which are highly toxic,
Extended author information available on the last page of the article

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188
persistent, and difficult to treat. Various physical, chemical
and biological treatment processes are used for the efflu-
ent treatment. Recently, using biotechnological application
to treat the industrial effluent was a success [1]. Currently,
nanotechnology is extensively studied to conquer its poten-
tial advantages like low cost and high efficiency in removing
and recovering the pollutants [2, 3]. The benefits of nano-
technology might bring attenuation of the environmental
pollution through a convenient and ecofriendly means of
wastewater treatment [4]. Application of nanotechnology
through the breakdown of contaminants by nanoparticles is
one of the means of treating wastewater.
Different types of nanoparticles such as nanosized met-
als, metal oxides [5], catioinic exchangers [2, 6], and nano-
filtration membranes [3] have been proven to be effective
in detection, removal and/or destruction of contaminants.
The applications of nanotechnology in wastewater treatment
are intensively reviewed with their process concerns [7, 8].
Most of these are discussed for its use at laboratory scale or
pilot stage. Qu et al. [7] represented the summary of various
methods applied under nanotechnology-driven mechanisms
for wastewater treatment. It indicates the remarks on differ-
ent parameters which still require more focus indicating the
incompleteness of that particular method for its industrial
application. In this regard, there exists a good scope to select
the proper method of treatment for industrial effluents.
Selection and comparison of potential
nanomaterials for wastewater treatment
to remove COD
The nanoparticles like titanium dioxide (­ TiO2), zinc oxide
(ZnO) and cerium oxide (­ CeO2) have roles in chemical or
physical treatments [9, 10]. They possess high reactivity and
photolytic properties. They are good adsorbents for water
purification due to their surface area. Nano ­TiO2 is used
for electrocatalytic oxidation of organic compounds and
COD removal is observed to be high [11]. For cerium oxide,
researchers found no evidence of nanoparticle uptake by
cells, but observed that their toxic mode of action required
direct contact between nanoparticles and cells. Cell damage
most probably took place by cell wall and membrane dis-
ruption. Researchers further needed to find out whether the
oxidative activity of ceria could be responsible [12]. ­TiO2
and ZnO are stated as the benchmark catalysts [10]. ­TiO2
nanoparticles are used to successfully inactivate different
genera of bacteria including E. coli, Staphylococcus aureus,
Saccharomyces cerevisiae, and Aspergillus niger [13] as
well as Pseudomonas aeruginosa, Salmonella typhimurium
and Enterobacter cloacae [14]. ­TiO2 nanoparticles inacti-
vate the bacteria, fungi, protozoa, spores and cysts under
solar light irradiation. However, T­ iO2 is insoluble in water
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Bioprocess and Biosystems Engineering (2019) 42:187–198
and the nanoparticles can be present in the environmental
contaminants [10, 15].
A recent study showed the effect of ZnO nanoparticles
for biological wastewater treatment [16] and their removal
behaviour is also exercised [17]. ZnO emerged as an attrac-
tive material for water treatment, as it can be tailored to
absorb visible light with a high surface reactivity owing to
large number of active sites [10]. The ZnO nanoparticles
have shown more enhanced biocidal activity against vari-
ous pathogens when compared to chemical ZnO particles
[18]. ZnO is more biocompatible than T ­ iO2 [19]. It has an
edge over other metal oxides like ­TiO2 in water purifica-
tion and other environmental remediation processes, as it is
soluble in water [20]. Further, ZnO can be synthesized under
mild conditions and the asymmetry in the crystal structure
allows aniotropic growth that can provide large surface-to-
volume ratios [3]. The nanosized ZnO interacts with the
bacterial surface and with the bacterial core, wherefrom it
enters inside the cell, and subsequently exhibits distinct bac-
tericidal (killing bacteria) mechanisms. However, toxicity
differs from one study to another based on test conditions.
Additional research is required to investigate exact effects
(bactericidal/ bacteriostatic) to elucidate the sensitivity of
bacteria to ZnO nanoparticles [21]. It means that their con-
centration and size must be specified for the range where
they do not kill or deactivate microorganisms. And that is
worked out here in this paper.
Interaction between ZnO nanoparticles
and microorganisms
The interactions between ZnO nanoparticles and micro-
organisms have been studied by Sirelkhatim et al. [21].
ZnO exhibits photo oxidizing and photo catalytic impacts
on chemical and biological species. ZnO provides signifi-
cant antibacterial activity when particle size is reduced
to nanometer range, then nano-sized ZnO can interact
with bacterial surface and/or with bacterial core enter-
ing inside the cell and disturbs bactericidal mechanism
[22]. Particle size and concentration is one of the impor-
tant properties affecting the antibacterial activity of ZnO
nanoparticles [21]. Larger surface area and higher con-
centration are accountable for antibacterial activity of
ZnO nanoparticles [23, 24]. ZnO nanoparticles of smaller
size can easily penetrate into bacterial membranes due to
their large interfacial area. Thus, it improves antibacterial
efficiency. The superoxide and hydroxyl radicals cannot
penetrate into the membrane due to their negative charges
[25]. Thus, these species are found on the outer surface of
the bacteria, by contrast, ­H2O2 molecules are able to pass
through the bacterial cell wall, subsequently leading to
injuries and destroy, and finally triggering cell death [23].
Bioprocess and Biosystems Engineering (2019) 42:187–198 189
The larger the surface area and the higher the concentra-
tion of reactive oxygen species (ROS) on the surface of
nanoparticles, the easier is for smaller particles to obtain
higher antibacterial activity [26]. It was contradicting
other works which found no size-related effect [27]. Pad-
mavathy and Vijayaraghavan [26] used three different sizes
of ZnO nanoparticles (45 nm, 12 nm, 2 µm) to find out the
bactericidal efficiency. A smaller size was found to kill
the bacteria that were attributed to ROS release on ZnO
surface under both UV and visible light. The 12 nm size
particle of ZnO showed best efficiency compared to 45 nm
for bactericidal activity [26]. ZnO nanoparticles’ antibac-
terial activity is inversely proportion to particle size as
deduced by Raghupathi et al. [28]. ZnO (100–300 nm) has
bacteriostatic effects on two bacterial strains Lactobacillus
salivarius and Streptococcus sobrinus. However, few inhi-
bitions are observed on three other types (P. aeruginosa,
Streptococcus mutants, and S. aureus) according to test
conditions [29].
COD removal using ZnO nanoparticles
Recent research on effect of zinc oxide nanoparticles on bio-
logical wastewater treatment revealed that ZnO nanoparti-
cles caused poor settleability of activated sludge and a sig-
nificant decrease in the removal of nitrogen and phosphorous
over a time [16]. However, there was no discussion on COD
reductions using ZnO nanoparticles and that makes it as a
scope of this work. Long-term effects of ZnO nanoparticles
on the system performance of membrane bioreactor are also
investigated [17].
Another vital parameter is the selection of microorgan-
isms to degrade the wastewater. Inherently, it depends on
the type of industry wherefrom wastewater is generated.
Recently, COD reduction is achieved with specific microor-
ganisms for textile effluent, in an aerobic mode using TPIFB
[1]. The application of TPIFB is well-reviewed for waste-
water treatment [30]. The present work is aimed for textile
effluent treatment. This idea encouraged to investigate the
COD reduction combining nanotechnology with biotechnol-
ogy to treat textile effluent. Nevertheless, the size of nano-
particles and other operating variables of the system need
to be understood.
The present work investigates the potential of ZnO nano-
particles for the biological treatment of textile effluent to
reduce its COD. This may serve as an example showing
combined nano-bio treatment of textile effluent. So far, no
research addressed the effect of nanoparticles in a bioreactor.
That is presented in this paper demonstrating reduction in
the chemical oxygen demand (COD) of the textile effluent
using two different ZnO nanoparticles (90 nm, 280 nm).
Materials and methods
Collection and characterization of sample
The textile effluent (Table 1) was collected from the tex-
tile industry in Rajkot (Gujarat, India). The wastewater
used in the experiment was the wastewater collected after
mercerizing operation (mercerization is a process applied
to cotton to increase luster and done after weaving the cot-
ton) from the Textile plant. The effluent was stored at 4 °C
before use to prevent any metabolic activity of microorgan-
isms present in that. The chemical oxygen demand (COD)
was measured when the reactor was in steady state. The
pH was determined using pH meter (Eppendorf, USA).
The effluent samples were collected for analysis every 2 h
after the ZnO nanoparticles were dosed into the TPIFB.
Reactor set up and operation
The effluent was treated in a TPIFB which was fabricated
[1]. Liquid phase was a textile effluent that had an initial
COD of 1700 mg/L. The analysis of solid media particles
(made of polypropylene) via SEM confirmed the size of both
the particles. FTIR imparted the structural morphology. At
a time, the bed of solid media became steady with expan-
sion of bed and then remained constant. This was the condi-
tion after which COD removal was observed at a zero liquid
velocity and superficial gas velocities. The steady state for
the TPIFB was reached in 18 min. Having ensured inverse
condition of the fluidized bed while maintaining the constant
superficial gas flow, the experiments were carried out at all
possible combinations of parameters including gas veloci-
ties (0.006 m/s, 0.004068 m/s and 0.00343 m/s), bed heights
(0.3 m, 0.6 m and 0.9 m) and the H/D of 0.5 and 0.25. When
the bioreactor operation began, gas bubbles entered the bio-
reactor through gas sparger. These bubbles were moving in
different fashions developing flow patterns including inver-
sion of bed where particles moved downward.
Table 1  Characteristics of Parameters Value
textile effluents
pH 8.2
TSS (mg/L) 97.8
BOD (mg/L) 105
COD (mg/L) 1700
NO3− (mg/L) 7.6
NO2− (mg/L) 0.48
NH4+ (mg/L) 6.8
Chlorides 108.2
H2S 0.5
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190 Bioprocess and Biosystems Engineering (2019) 42:187–198

Microorganisms Results and discussions

The microbial cultures of P. putida (NCIM 2872), P. aureo- Characterization of samples

faciens (NCIM 2026), Pseudomonas fluorescens (NCIM
2174), Bacillus subtilis (NCIM 5433) and Escherichia coli Figure 1 illustrated the characteristics of 90 nm sized nan-
(NCIM 2805) were purchased from National Chemical Lab- oparticles through its SEM image and a similar image is
oratory, Pune, India. Their pH at various concentrations was captured for 280 nm (Online resources: ESM: Figure S1).
measured to observe the optimal growth at different concen- Since each element has a unique atomic structure allowing
trations (Table 2). The COD degradation capability of each a unique set of peaks on its electromagnetic emission spec-
of these cultures was determined in presence of ZnO nano- trum, energy-dispersive X-ray spectroscopy (EDS or EDX)
materials (COD reduction: 87% for Pseudomonas putida, was used for size and morphology of the samples (Online
98% for P. aureofaciens, 56% for Pseudomonas fluorescens, resources: ESM: Figure S2). EDX imparted the elemental
26% for Bacillus subtilis and 11% for Escherichia coli). composition of the specimen to be monitored. The structural
Finally, two microbial cultures of P. putida and P. aureo- morphology was further studied by FTIR of ZnO nanopar-
faciens were selected considering their capacity to reduce ticles (Fig. 2a, b).
the COD and continued with the same for further experi-
mentation. These inoculated cultures were used for the entire Reactor operation
bioreactor operation in a batch mode for inoculum sizes of
5%, 10% and 20% of the working volume of the reactor. Textile effluent was treated in TPIFB with 90  nm and
280 nm sized ZnO nanoparticles to reduce COD from its
Nanoparticles initial value of 1700 ppm using aerobic biological treat-
ment. The bed of solid media particles (polypropylene
The size of ZnO nanoparticles, at which it was not show-
ing bacteriostatic and bactericidal effect for the cultures of
P. putida and P. aureofaciens, were selected. Considering
that fact, ZnO nanoparticles were synthesized at 90 nm and
280 nm using the Solgel method. The suspension of ZnO
nanoparticles was prepared by adding 0.125 g of ZnO nan-
opowder to 1 L deionized water. These suspensions were
sonicated at 20 kHz for 25 min to break the aggregates. Only
after this, this suspension was added to the TPIFB. Each
concentration was set in such a way that the total working
volume in the reactor becomes 2.43 l approximately. These
nanoparticles were characterized by various tests such as
SEM (scanning electron microscopy), EDS or EDX (energy-
dispersive X-ray spectroscopy) and Fourier transform infra-
red spectroscopy (FTIR).

Fig. 1  Images of 90  nm ZnO nanoparticles by scanning electron

microscope

Table 2  pH values for various microbial strains for different concentrations of ZnO nanoparticles
Species NCIM reference Optimal pH at 60 mM pH at 100 mM pH at 140 mM pH at 180 mM pH at 280 mM
range of
pH

Pseudomonas fluorescens 2174 8.5–9.5 8.5 8.5 8.0 7.5 7.0

Pseudomonas aureofaciens 2026 7.5–8.5 7.0 7.5 7.5 7.5 7.5
Escherichia coli 2805 5.5–8.5 5.5 5.5 5.5 5.0 5.0
Pseudomonas putida 2872 5–6 6.5 6.0 6.0 5.5 5.0
Bacillus subtilis 5433 7-7.5 8.5 8.5 8.0 8.0 8.0

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Bioprocess and Biosystems Engineering (2019) 42:187–198 191

Fig. 2  a FTIR spectrum for 90 nm ZnO nanoparticles. b FTIR spectrum for 280 nm ZnO nanoparticles

13

192
particles) was stationary at no gas flow rate. As the reac-
tor was injected with gas flow, only the lower portion of
bed started fluidizing at 0.0027 m/s (much lower due to
induced liquid circulations promoted by gas bubbles) show-
ing inversion. As the gas velocity increased, the remaining
fixed bed also moved progressively until the full fluidization
was achieved (incipient fluidization). The media particles
were distributed along the total height of the reactor column
non-uniformly with the further increase in the gas velocity.
The concentration of media particles was still higher at the
top and progressively decreased downward. The pH values
contributed by microorganisms, at working range of ZnO
concentrations, ensured the bioreactor operation for COD
reduction (Table 2). The actual working pH values were
determined for each of the microbial cultures at different
concentrations of ZnO nanoparticles. These pH values were
investigated for its optimal range for each of the microorgan-
isms tested, initially. Finally, at all ZnO concentrations, the
microbial cultures of P. putida as well as P. aureofaciens
were found in optimal pH range and employed for the entire
experiments. ZnO particles were added after 1 h of each
batch of the bioreactor to ensure sufficient growth of mixed
cultures of P. putida and P. aureofaciens. The experimental
trials were carried out at 0.00343 m/s and 0.004068 m/s for
measurement of reduction in COD. The bed showed no more
inverse flow beyond 0.0068 m/s and exhibited slug formation
at or beyond 0.0068 m/s. The images from scanning electron
microscope further clarify the structural morphology of the
solid media particles (Online resources: ESM: Figure S3).
The structural emission spectrum is characterized by FTIR
for both the solid media particles (Fig. 3a, b). Most of the
time TPIFB exhibited partially fluidized bed with dispersed
bubble flow regime. The performance of the bioreactor was
investigated to check the effect of superficial gas velocities
(0.0027 m/s, 0.00342 m/s, 0.0068 m/s) and static bed height
(4.85 and 2.43 cm) using polypropylene particles (12 mm
hollow). This batch reactor was operated until maximum
COD reduced. Nanoparticles were added to the bioreactor in
a quantity of 0.75 g for 2.12 l of volume of bioreactor with
4.85 cm and 2.43 cm static bed heights.
Initial trails were conducted to find out the best set of
operating parameters leading to maximum reduction in
COD. Three bed heights were taken as 30 cm, 60 cm and
90 cm each at 5%, 10% and 20% inoculum size (percentage
is of the total working volume of the reactor) and three gas
velocities. At lower bed height of 30 cm, the gas holdup
formed faster but for a lesser period of time in comparison
with that of 60 cm. Both the heights—30 and 60 cm exhib-
ited the discrete bubble regime. The pattern of degradation
of COD was similar for all bed heights. However, the ini-
tial adaptation period was different for mixed culture of P.
putida and P. aureofaciens. Consistent gas holdup was also
visualized with both—30 cm and 60 cm. However, inversion
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Bioprocess and Biosystems Engineering (2019) 42:187–198
sustained for all retention time with 30 cm bed height. So,
it was only considered across the whole experimentation.
However, 90 cm bed height produced slugs at regular inter-
val and the tendency of the bioreactor at this height was to
form slugs at regular interval. This contributed poor expo-
sure of solid media particles to gas bubbles making micro-
organisms deficient in growth. The reason was that micro-
organisms were not being able to degrade the full oxygen
required for their growth. Therefore, slugs must be prevented
in exercising suitable bed height for better performance.
Selection of size of ZnO nanoparticles
From the trial experiments, the ZnO nanoparticles showed
slow to moderate growth for 90–280 nm size and no growth
inhibition was observed between 90 and 280 nm. The experi-
ments were performed to find out representative values of
ZnO nanoparticles size that can result in COD reduction.
These initial experiments were conducted to find out the
size of ZnO nanoparticles within which it does not kill P.
putida and P. aureofaciens. The antibacterial activities are
not desirable within some size of ZnO nanoparticles, but
the same was observed for higher concentration of ZnO
nanoparticles. This was due to its higher surface area corre-
sponding to that size. It was concluded that increase in ZnO
particle size will decrease the antibacterial activity. There-
fore, ZnO nanoparticles’ antibacterial activity was inversely
proportional to the particle size. This can be explained by
the fact that smaller size ZnO nanoparticles can penetrate
the bacterial cell easily due to its large interfacial area and
enhance antibacterial activity. The reaction mechanism is
illustrated in Fig. 4. The generation of reactive oxygen spe-
cies (ROS) is strongly dependent on the surface area avail-
able. The ROS species generated would consequently dam-
age the cell wall as bacterial cells come in contact with the
ZnO nanoparticles.
Effects of static bed height using nanoparticles
The ratio of static bed height to column diameter (H/D) was
an important parameter in evaluating bioreactor performance
apart from considerations of gas velocities [1, 30]. H/D was
taken as 0.5 and 0.25 but since the diameter was constant
here, H equivalent to H/D of 0.5 and 0.25 was calculated
to be equal to the height of 4.85 cm and 2.43 cm of solid
media particles in a bioreactor filled with the wastewater.
ZnO nanoparticles of the two different sizes 90 nm (Fig. 5a)
and 280 nm (Fig. 5b) reacted differently when its effects
were investigated with two static bed heights at 4.85 cm and
2.43 cm. The faster reduction was achieved between 5 and
10 h for 90 nm particles and between 3 and 10 h for 280 nm
particles. The use of ZnO nanoparticles definitely enhanced
the rate of reaction of the process. The 280 nm particles
Bioprocess and Biosystems Engineering (2019) 42:187–198 193

Fig. 3  a FTIR spectra for 4 mm polypropylene solid media particles. b FTIR spectra for 12 mm polypropylene solid media particles

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194 Bioprocess and Biosystems Engineering (2019) 42:187–198

reduced COD much faster than that by 90 nm particles. The

photocatalytic activity of ZnO with high surface reactivity
made it possible to have nano-bioreaction: biological growth
of P. putida and P. aureofaciens degrading organic pollutants
and treatment by ZnO nanopowder. Oxygen entered via air
through gas sparger destructed organics through photo reac-
tions on catalytic surfaces of ZnO. The higher sizes of ZnO
nanoparticles (> 280 nm) provided lower surface area. Less
surface area implied very low generation of reactive oxygen
species (ROS) like oxygen and hydrogen peroxides. Less
ROS generation meant that there would have been bacte-
riostatic effect causing inactivation of both P. putida and P.
aureofaciens but would not have killed them.
If the size was reduced in the range of 12–45 nm [26], the
same effect was bactericidal and that is not desirable here,
since the growth of P. putida and P. aureofaciens helped
reducing COD apart from same role by nanoparticles. The
Fig. 4  ZnO reaction mechanism
ZnO concentration than 45 nm killed the microbial cultures.
However, the range of nanoparticles between 90 nm and
280 nm did not kill the cultures of P. putida and P. aureofa-
ciens but allowed them to grow and reduce COD as exam-
ined. This was the reason why they were added after 2 h for
the entire experimentation and elucidated the sensitivity of
P. putida and P. aureofaciens to the ZnO nanoparticles. The
result confirmed the mechanism on the basis of morphologi-
cal features of nanoparticles, as described by other research-
ers [21, 26]. The highest COD reduction was achieved to be
47 ppm (97.24%) for 2.43 cm static bed and 280 nm sized
ZnO particles (Fig. 5b).

Effects of superficial gas velocities using

nanoparticles

The chief advantage of TPIFB was that very low superfi-

Fig. 5  a Effect of static bed heights at 0.0027  m/s gas velocity and
90 nm ZnO nanoparticles. b Effect of static bed heights at 0.0027 m/s
gas velocity and 280 nm ZnO nanoparticles
cial gas velocities were sufficient even in presence of ZnO
nanoparticles, to degrade textile effluent. Superficial gas
velocities were observed within all possible ranges, ensur-
ing inversion process of solid media particles onto which P.
putida and P. aureofaciens rest for growth to degrade COD.
As the gas velocity increased, the gas hold up increased
over a period of time and then remained steady. Addition
of ZnO was done at this juncture of process to enhance the
COD removal across all retention time. Gas holdup was, in
turn, dependent on the rate of consumption of oxygen by the
mixed cultures P. putida and P. aureofaciens. This actually
decided the extent of degradation of the pollutants present in
textile effluent. Figure 6a indicated consistent degradation in
COD for 0.0027 m/s and 0.00343 m/s of gas velocities. At a
velocity 0.00342 m/s, the growth of P. putida and P. aureo-
faciens increased between 5 and 8 h, since there was holdup
comprising bigger than normal size gas bubbles. These big-
ger bubbles reduced the surface area available for both P.
putida and P. aureofaciens to degrade COD from effluent

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Bioprocess and Biosystems Engineering (2019) 42:187–198 195
Fig. 6  a Effect of superficial gas velocity at 10% inoculum size, with
90 nm ZnO nanoparticles. b Effect of superficial gas velocity at 10%
inoculum size, with 280 nm ZnO nanoparticles
phase. Not only that but ZnO particles were also introduced
at 1 h and they contributed significantly to the 10 h reten-
tion period. At 5–15 h, ZnO reacted consistently with gas
(oxygen) making COD degradation faster. Then after, the
bubbles were constantly formed in an uneven manner until
16 h. The retention period from 6 to 15 h for 90 nm particles
was the one where formation of ROS was observed. This
ROS generation would have been as comparatively higher in
particles of 90 nm, leading to bactericidal effect as compared
to that with 280 nm (Fig. 6b).
Effects of size of solid media particles
Two different particles—4 mm and 12 mm diameters—were
used in the bioreactor operation [31]. They exhibited reduction
in COD (Fig. 7). Both the particles were made from polypro-
pylene. Once the packed bed was broken and bioreactor was
in inverse conditions, minimum fluidization velocity prevailed.
Fig. 7  Effect of solid media particle size: 0.0027 m/s superficial gas
velocity; 10% inoculum size; 280 nm ZnO nanoparticles
The gas flow rate was further reduced to a value where par-
ticles were reaggregated. Bed was in the expanded state and
ready for operation. All the experimentations were conducted
with 4 mm diameter particles since they had large surface area
and density lower than that of 12 mm diameter spheres of
polypropylene particles. Both these factors were important
in achieving COD reduction because the higher the surface
area of solid media particles, the better was the degradation
of organic load present in the electroplating effluent. P. putida
and P. aureofaciens used all surfaces present on particles to
grow over this entire surface and degrade the waste in pres-
ence of oxygen. The difference of COD reduced with both
these particles was 11.48% which was significant from point
of view of hydraulic retention time for a batch process. Both
the particles bring the COD down in the permissible limit of
250 ppm set for the textile effluent, but 12 mm particles it is
faster than 4 mm sized particles.
The optimal operating parameters were found this way
and from the present experiments, it is ensured that low gas
velocities and very low minimum fluidization velocity gave
much lower COD removal in batch mode in TPIFB. Table 3
illustrated the comparisons of other similar works [32–41] for
COD reduction. From all the above trials, three-phase inverse
fluidized bed bioreactor operation ensured optimal COD
reduction for the textile effluent at 10% inoculum size with low
gas velocity 0.0027 m/s and static bed height of 2.43 cm. The
maximum COD removal was achieved at 47 ppm (97.24%)
with the use of 280 nm ZnO nanoparticles exhibiting its effi-
ciency in oxidizing the pollutants. Therefore, it can also be
concluded that bio-nano treatment for industrial effluent was
accomplished using ZnO nanoparticles in an effective way.
Conclusions
In this study, experimentations carried out to: (1) deter-
mine the appropriate size of ZnO nanoparticles that do
not show either bacteriostatic or bactericidal effect (2)
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196 Bioprocess and Biosystems Engineering (2019) 42:187–198

Table 3  Comparisons of various research works for COD reduction

Sr. no. Effluent type Reactor system Maximum Time consideration Process characteristics References
(%) COD (HRT or operation time)
removal and mode of operation

1 Textile wastewater Three-phase inverse flu- 97.24% HRT = 12–16 h, batch Aerobic treatment (nano- Present work
idized bed bioreactor operation bio)
2 Low-strength synthetic Anaerobic fluidized bed 96% HRT = 0.27–1.33 h, Anaerobic treatment [32]
wastewater reactor (AFBR) 3–46 days of operation,
continuous mode
3 Textile Sequencing batch reactor 97% 24 h cycle, 12–97 days Anaerobic–aerobic [33]
of operation, semi-
continuous mode
4 Textile Submerged filtration 91.50% Reaction time = 50 h, Physical–chemical [34]
semi-continuous mode
5 Textile UASB reactor coupled 81.20% HRT = 5.3 h, 230 days of Physical–chemical [35]
with adsorption operation, continuous
mode
6 Textile Fluidized bed reactor 34–49% Single reaction Chemical [36]
time = 100 m, batch
mode
7 Textile Microbial degradation 97% 14 days of operation, Biodegradation [37]
batch mode
8 Cotton textile (dye-bath Fixed and fluidized bed 70–93% HRT = 19.8–30.8 h, Physical–chemical [38]
effluent) (optimal), semi-continuous mode
95.60% HRT = 40 h (max.),
(maxi- semi-continuous mode
mum)
9 Domestic wastewater Inverse fluidized bed 97.50% HRT = 6–40 h, batch Aerobic treatment [39]
bioreactor mode
10 Industrial wastewater Inverse fluidized bed 96% HRT = 65 h, continuous Aerobic treatment [40]
biofilm reactor mode
11 Refinery wastewater Inverse fluidized bed 96% HRT = 27.5–80 h, con- Aerobic treatment [41]
biological reactor tinuous mode

examine the effect of other parameters in TPIFB, respon-
sible for COD reduction (3) examine and investigate the
species of bacteria that follows reduction in COD with
possible concentrations of ZnO nanoparticles. The follow-
ing inferences are drawn:
1. 280  nm sized ZnO nanoparticles reduced COD to
47  ppm (97.24%) in 14  h and that was much below
its permissible limit (250  ppm) for industrial efflu-
ents. 90 nm sized ZnO nanoparticles reduced COD to
248 ppm (85.4%) in 16 h.
2. Size of nanoparticles, static bed height, superficial gas
velocities and solid media particle size affects the COD
reduction. The work concludes that three-phase inverse
fluidized bed bioreactor performed optimal for the tex-
tile effluent at 10% inoculum size with low gas velocity
0.0027 m/s and static bed height of 2.43 cm for 280 nm
sized ZnO nanoparticles.
3. The maximum COD removal is enhanced by nano-
particles of ZnO and achieved to be 47 ppm (97.24%)
by 280 nm ZnO nanoparticles, as compared to that of
248 ppm (85.4%) for 90 nm ZnO nanoparticles, for tex-
tile effluent using P. putida and P. aureofaciens.
Acknowledgements  Mr. Dharmesh H Sur acknowledges the help pro-
vided by Dr. Jaysukh Marakana and Mr. Chirag Savaliya of Department
of Nanotechnology, V V P Engineering College, Rajkot. The author
remains grateful for the support provided by the management of V V
P Engineering College, Rajkot, Gujarat (India).
Compliance with ethical standards 
Conflict of interest  This manuscript is the authors’ original work and
has not been published nor has it been submitted simultaneously else-
where. All authors have checked the manuscript and have agreed to
the submission.
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198 Bioprocess and Biosystems Engineering (2019) 42:187–198

Affiliations

Dharmesh H. Sur1,2 · Mausumi Mukhopadhyay1

1 2
Department of Chemical Engineering, Sardar Vallabhbhai Department of Biotechnology, V. V. P. Engineering College,
National Institute of Technology, Surat, Gujarat 395007, Rajkot, Gujarat 360005, India
India

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