Cleaner Materials 6 (2022) 100149

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Cleaner Materials
journal homepage: www.journals.elsevier.com/cleaner-materials

Investigation of Ochrobacter pseudintermedium ASMCS06 for Cleaner

biodegradation and bioelectricity production
Mira chares Subash a, *, A.S. Maheshwari b
a
Department of Chemical Engineering, National Institute of Technology, Tiruchirappalli, Tamil Nadu, India
b
Department of Biotechnology, BIT-Campus, Anna University, Tiruchirappalli, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Keywords: One-fifth of the world’s water pollution has been generated by textile dyeing and fabric finishing process. Textile
Laccase dyes affect the photosynthetic process and reduce the oxygen content in water leading to the death of aquatic
O. pseudintermedium ASMCS06 plants. Industrial wastewater can be subjected to bioremediation by employing efficient ligninolytic laccase
Dye-degradation
enzymes. The laccases have gained popularity with efficient oxidation of recalcitrant environment pollutants.
Bioelectricity
Similarly, microbial fuel cells can also be concerned with bioremediation treatment with atmospheric carbon
dioxide reduction and production of potential energy sources. In this study, a new laccase-producing bacterium
was isolated and identified as Ochrobacter pseudintermedium ASMCS06 by 16sr-RNA sequencing. Rice bran
exhibited enhanced laccase activity compared to carbon and nitrogen sources. Laccase produced by
O. pseudintermedium ASMCS06 was found to be stable at 75 ◦ C and also at alkaline pH 8. Further, ASMCS06 was
capable of degrading synthetic azo dye. After 72 h, the decolourization and degradation efficiencies of azo dye
were 65 ± 2 % and 73 ± 1.8 %. Suggesting the possible degradation pathway, ASMSC06 was found to be po­
tential with higher laccase activity and is suitable for dye degradation and other bioremediation applications. In
addition, isolated bacteria also play a major role in bioelectricity production. The maximum voltage produced
was identified as 415 mV on day 7. The isolated bacterium O. pseudintermedium ASMCS06 was able to produce a
maximum current density of 930 (mA/cm2) and a maximum power density of 350 (mW/cm2).

Introduction
Increased ecological pollution due to modernization and industrial­
ization has been identified in industrial effluents, landfills, aquatic en­
vironments, and municipal sewage (Bilal et al., 2019). Annually, 0.7
million tons of synthetic dyes and pigments are produced all over the
world with industrial applications including textile, paint, food-based,
paper & printing, plastic manufacturing, cosmetics etc. Majorly, textile
and dyeing industries often disperse colorants in their effluents which
comprise 10–15 % residual azo dyes. Azo dyes are of a variety of colors
and functional groups which have adverse effects on both environment
and humans. They are very toxic, mutagenic and carcinogenic, so they
have to be eliminated from the effluents (Chauhan and Choudhury,
2021). Amid azo dyes, sulfonated azo dyes are more recalcitrant than
carboxylated groups. Physicochemical treatments such as adsorption,
chemical precipitation, photolysis, chemical oxidation and reduction,
and electrochemical treatment are conventionally used for removing
these pollutants. But these are not efficient enough as their costlier with
low efficiency and are sometimes not applicable to certain dyes in the
groups. Bioremediation is gaining attraction owing to its low cost and
eco-friendly approach with help of microorganisms. These azo dyes
could be easily degraded into aromatic amines by anaerobic bacteria
(Moreno et al., 2020). India is addressed to be one of the largest elec­
tricity consumers in the world. Despite, India being the fifth-largest
electricity consumer, nearly 43–45 % of rural and 20–22 % of urban
people are devoid of electricity (Thulasinathan et al., 2021). Microbial
or biological fuel cells have created a positive impact in people’s minds
as alternative energy technology (Lai et al., 2017). Microbial Fuel Cells
(MFCs) involves electricity production by the conversion of complex
organic or inorganic substance through microbial metabolism. The
conversion is possible owing to the possible role of organic compounds,
carbohydrates, derivatives of sugar molecules, alcohols and amino acids
as electron donors.
Belonging to a family of oxidoreductase enzymes, laccase is
renowned for the bioremediation of environmental pollutants (Zdarta
et al., 2019). The main role of laccase has been categorized as cross-

* Corresponding author.
E-mail addresses: charesmira@gmail.com (M. Subash), asmaheshwari@yahoo.com (A.S. Maheshwari).

https://doi.org/10.1016/j.clema.2022.100149
Received 25 March 2022; Received in revised form 2 September 2022; Accepted 12 September 2022
Available online 15 September 2022
2772-3976/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M. Subash and A.S. Maheshwari
Table 1
Composition of Modified Minimal Medium.
linking of monomers, polymer degradation, and ring cleavage of aro­
matic compounds (Dwivedi et al., 2011). The biocatalytic efficiency of
laccases reckons upon the potential of T1 for the oxidation–reduction
process in which oxidation of substrate occurs and the formation of a
cluster (T2/T3) for molecular reduction of the substrate by transferring
an electron from T1 to the trinuclear site (Morsi et al., 2020). The copper
oxidases act as the backbone of laccase by oxidizing phenolic and aro­
matic compounds with variant functional groups namely methoxy,
amino, diamino, hydroxyindols, [Mo(CN)8]4-, [Fe(CN)6]4- and [Os
(CN)6]4 (Senthivelan et al., 2016). Laccase from the Monocillium indi­
cum was the first laccase to be characterized by the ascomycetes family
which shows peroxidase activity (Shekher et al., 2011). The majority of
the laccases have been reported from white-rot fungi such as Pleurotus
ostreatus, Trametes Versicolor, Agaricus bisporus, Cerrena unicolour,
Coprinopsis cinerea, Coriolopsis gallica, Cryptococcus neoformans, Cyathus
bulleri, Fomes fomentarius, Ganoderma lucidum, Panus rudis, Phlebia
radiata, Polyporus brumalis, Pycnoporus cinnabarinus, Pycnoporus sangui­
neus, Rigidoporus microporous, Schizophyllum commune (Forootanfar,
2015). Even though fungal laccases are prevalent, they have a drawback
of the prolonged fermentation period, and deficient yield. Fungal Lac­
cases cannot meet the industrial demand as they fail at high temperature
and pH rate. In this aspect, bacterial laccases are recognized to be cost-
effective, broad substrate specificity, higher yield, short fermentation
period, compatible with industrial process and also desirable in cloning
with genetic manipulation. Based on the source such as plants, insects
and microorganisms, the characteristic feature of laccase enzyme varies
in pH and molecular weight (Benfield, 1964). The ecological demand of
microbial Laccase production can be met with commonly available
agricultural wastes residues, which serves as cost-effective excellent
sources (Singh and Gupta, 2020). With these advantageous of bacterial
laccases, microorganisms associated with these oxido-reductases could
be employed in removal of azo dye with focus of microbial degradation
and bioelectricity production.
In this present investigation, a novel laccase producing strain was
isolated from contaminated soil containing vegetables and plant resi­
dues. The isolation and phylogenetic identification of Ochrobacter
pseudintermedium ASMCS06 was performed through biochemical
2
Cleaner Materials 6 (2022) 100149
characterization and 16sr-RNA sequencing. Laccase production by this
strain was investigated with the effect of parameters such as nutrient
source, pH and temperature. The potentiality of ASMCS06 was
demonstrated with its ability in biodegradation of azo dye and
bioelectricity production. Also, practical difficulties involved in the
implementation of bacteria in the bioremediation of azo dye and
bioelectricity production were proposed in this study.
Materials and methods
Isolation of laccase-producing strain
The soil sample was collected from the residues of vegetable waste in
the Tiruchirappalli market (10◦ 48′ latitude and 78◦ 41′ 8′′ longitude),
Tamilnadu, India. 1 g of the isolated soil sample was added to the conical
flask containing 100 mL distilled water and 0.01 M of 100ul guaiacol
substrate incubated at 37 ◦ C for 24 h. From the overnight culture, 1 mL
was pipetted and serially diluted. Nearly 30 isolates were identified from
10− 6 to 10− 10 serial dilution and patched in modified media composi­
tion (Table 1 represents the minimal medium composition) to screen the
Laccase-producing isolates. The optimized screening minimal media
composition includes peptone 0.2 %, meat extract 0.2 %, agar 2 %,
dipotassium hydrogen phosphate 0.05 %, magnesium chloride 0.05 %
and guaiacol 2 mM. The isolates were analyzed for the appearance of
brown colour and halo zone formation concerning a change in medium
composition. The isolates that were distinguishable by halo zone for­
mation and colour change were subjected to guaiacol screening (Devasia
and Nair, 2016). The screened isolates were again patched in an opti­
mized medium employing guaiacol as the substrate. The positive isolate
was streaked for the isolation of a single colony in guaiacol. The growth
of the positive (isolate no 6) was analyzed periodically at two hours
intervals for a period of 24 h using a UV spectrophotometer at 600 nm.
The isolate was viewed using a light microscope under oil- immersion by
following the morphological characterization (gram-staining method).
The isolated bacterial strain was stored at − 20 ◦ C in glycerol (20 %) for
future use.
M. Subash and A.S. Maheshwari Cleaner Materials 6 (2022) 100149

Fig. 1. The 30 isolates patched in MM3 & MM4, *Isolate 6-Positive.

Fig. 2. a) Guaiacol screening in Modified minimal medium, b) Streak plate of Isolate 6 showing brown in presence of guaiacol.

Fig. 3. Phylogenetic analysis of Isolate 6.

Molecular characterization sequenced at Yazh Xenomics, Coimbatore, India. The nucleotide

The isolated bacteria is identified using the 16Sr-RNA gene
sequencing. The 5 μl of isolated DNA was taken in 25 μl of PCR reaction
solution which contains (1.5 μl of forward Primer and Reverse Primer, 5
μl of deionized water, and 12 μl of Taq Master Mix). The PCR reaction
was carried out with initial Denaturation (94˚C for 3 min), denaturation
(94˚C for 30 sec), Annealing (60˚C for 30 sec), Extension (72˚C for 1 min),
Final extension (72˚C for 10 min) and Hold at 4˚C for 30 cycles. The DNA
was extracted, purified and then amplified using a clean gene kit and
sequencing was done using the Basic Local Alignment Search Tool
(BLAST) algorithm and further compared with the public nucleotide
databases at the National Center for Biotechnology Information (NCBI)
(http://www.ncbi.nlm.nih.gov). The bacterial sequence was deposited
at the GenBank to obtain the accession numbers. Further, the alignments
of the isolated bacteria and the phylogenetic tree were performed using
the neighbour-joining method in Molecular Evolutionary Genetics
Analysis software (MEGA 5.0).

3
M. Subash and A.S. Maheshwari
Fig. 4. Effect of a) carbon, b) nitrogen and c) natural substrate on Laccase
production by O. pseudintermedium ASMCS06.
Laccase activity
The oxidation of guaiacol has been reported for laccase assay (Abd El
Monssef et al., 2016). The brown colour developed due to oxidation of
guaiacol by laccase is used to measure enzyme activity at 450 nm. The
reaction mixture was prepared by adding Guaiacol (2 mM) 1 mL, So­
dium acetate buffer (10 mM) 3 mL, and Enzyme source 1 mL (bacterial
4
Cleaner Materials 6 (2022) 100149
supernatant). Blank contains 1 mL of distilled water instead of the
enzyme. The mixture was incubated at 30 ◦ C for 15 min and the
absorbance was read at 450 nm using a UV spectrophotometer. The
laccase activity in U/mL corresponds to the amount of enzyme required
to oxidize 1 mol of guaiacol per min.
Effect of nutrient sources
The effect of carbon source in Laccase enzyme production by
Ochrobacter pseudintermedium ASMCS06 was analyzed by utilizing
variant (1 %W/V) carbon sources such as dextrose, Sucrose, Fructose,
starch, Maltose, and Lactose. Similarly, the nitrogen source in Laccase
enzyme production by the species was also experimented with by
employing Yeast extract, Ammonium chloride, Ammonium sulfate,
Peptone and Meat extract as evident nitrogen sources (Patel and Gupte,
2016). In addition to the above, the aftermath of a natural substrate was
also studied by availing Sawdust, Rice bran, and Coconut pith. For
screening the effective carbon, nitrogen and natural substrate, the
enzyme activity of Ochrobacter pseudintermedium ASMCS06 was evalu­
ated at 450 nm and for control, no exogenous sources were added.
Effect of operational parameters
The influence of temperature and pH in Laccase enzyme production
by Ochrobacter pseudintermedium ASMCS06 was analyzed at variant
temperatures (35◦ C to 95◦ C) and at different (pH 2–12). The enzyme
activity was measured at 450 nm to identify the stability of the species in
enzyme production. The effect of pH in Laccase enzyme production by
the isolated species Ochrobacter pseudintermedium ASMCS06 were
analyzed (Gupta et al., 2015).
Partial purification
The crude sample was allowed for partial purification by the
ammonium sulfate precipitation method. The maximum activity was
achieved with 70 % salt saturation. The precipitates obtained by the
ammonium sulphate precipitation method were further subjected to
Dialysis. Dialysis is the separation of particles in a liquid based on dif­
ferences in their ability to pass through a membrane. Dialysis was car­
ried out against the membrane of 10 kDa and 2.5 kDa.
SDS PAGE
The Casting frames were set on the casting stands. The appropriate
amount of separating gel was pipetted into the gap between the glass
plates and kept for 20 min. After complete gelation, the stacking gel was
pipetted into the glass plate along with the well-forming comb without
trapping air under the teeth. The comb was removed after the complete
gelation of the stacking gel. The running buffer (electrophoresis buffer)
was poured into the inner chamber until the buffer surface reaches the
required level in the outer chamber. The samples were mixed with the
loading buffer and subjected to heating for 5–10 min. The prepared
samples were loaded into the wells along with the protein marker in the
first lane. The appropriate voltage was set to run the electrophoresis.
Decolorization test
Decolourization tests were performed in 250 mL conical flasks with a
100 mL optimized medium in an orbital shaker. Methyl orange dye was
chosen among the sulfonated azo dyes for investigating the decolouri­
zation efficiency of O. pseudintermedium ASMCS06. Decolourization was
monitored using a UV–vis spectrophotometer and the peak detection of
Methyl orange dye was observed at 465 nm. At optimized operating
conditions (pH and temperature), 6 % cell suspension (volume) of the
bacterial strain was cultured in a 100 mL medium containing 10 mg L− 1
dye and incubated at 37C for 12–24 h. Subsequently, the effect of dye
M. Subash and A.S. Maheshwari
1
dosage such as 15 to 50 mg L− was investigated along with microbial
growth.
Decolorization ratio (%) = (A0/A1)/A0 *100,
where A0 and A1 represented the absorbance of the dye solution
before and after decolourization, respectively. The concentration of pure
strains was analyzed by spectrophotometry at the absorbance of 600 nm
(OD600). The supernatant after centrifugation (5000 rpm for 10 min)
was used as a reference solution to eliminate the influence of possible
residual absorbance on the analysis of OD600.
Microbial fuel cell
A single-chambered Microbial Fuel cell (MFC) was constructed using
polyacrylic sheets in the dimensions of 10 cm × 5 cm × 7 cm. The
graphite plate is employed as an electrode with a working volume of
250 mL. The proton exchange membrane is kept between the anode and
cathode compartments (Lai et al., 2017). For the substrate, the rice bran
was soaked in the distilled water for 24 h and filtered, and sterilized at
121 ◦ C for 15 min to kill the native microorganisms. The sterilized rice
bran water was used as a substrate in the anode chamber. 1 % inoculum
from the isolated bacterial culture was utilized as a biocatalyst in the
anode chamber. The cathode chamber was filled with (50 mM) phos­
phate buffer. The experiment was conducted for 10 days at 37 ◦ C. The
corresponding voltage (mV) was measured using a digital multimeter
(Fluke 107). The external resistance was calculated at 10,000 Ώ, 8000
Ώ, 1000 Ώ, 470 Ώ, 220 Ώ and 68 Ώ for 30 min time interval. The po­
larization curve was obtained with measurement of open circuit voltage
for the corresponding external resistance feed. The current density and
power density were calculated by the following equations (Thulasina­
than et al., 2021).
Ohm’s law: V = IR
5
Cleaner Materials 6 (2022) 100149
Power: P = V2/R
Current: I = V/R
Power density = IV/A
Current density = I/A
Where V is the voltage (mV), R is Resistance, A is the anode surface
area (m2 or cm2).
Flowchart of current research study
Results and discussion
Isolation and screening of Laccase producing bacteria
Laccases are versatile enzymes and ubiquitous in higher plants,
bacteria, fungi, and insects. Bacterial laccases have higher thermosta­
bility, and alkaline pH optimum and are halotolerant due to the wide­
spread existence and versatility of bacteria (Chandra and Chowdhary,
2015). Bacterial laccases have received much attention due to their
peculiar nature of oxidation ability in biotechnological processes (Dhi­
man and Shirkot, 2015). In this investigation, the status of indigenous
laccase-producing bacteria was explored from vegetable and fruit waste
dumped soil in the state of the Tiruchirappalli market. The samples were
collected from waste dumped soil because they were rich in lignocel­
lulose which may act as a substrate for Laccase. A literature survey
suggested the isolation of laccase like multicopper oxidase Streptomyces
sp. C1 from the agricultural waste compost (Lu et al., 2013). Similarly,
Trametes versicolor has been also isolated from the agricultural waste for
simultaneous production of Laccase and degradation of bisphenol A
M. Subash and A.S. Maheshwari
Fig. 5. Effect of Temperature and pH on Laccase production by O. pseudintermedium ASMCS06.
Fig. 6. Lane 1 – 4: 25, 50, 75 & 100 µl of Ammonium Sulphate precipitate of
supernatant dialysed.
(Zeng et al., 2017). The collected soil samples were serially diluted
which resulted in 30 isolates. The 30 isolates were patched in a Minimal
Modified medium containing guaiacol by varying the composition to
screen the positive laccase-producing isolate. Among the 30 isolates,
isolate 6 was only able to oxidize guaiacol and produce a brown colour
(Fig. 1 indicates the positive isolate (6) among the 30 isolates). Guaiacol
is chromogenic thereby serving as a successful platform for screening
laccase producers by synthesizing extracellular guaiacol oxidizing en­
zymes (Devasia and Nair, 2016).
The isolates that were distinguishable in colour change and halo zone
formation were further subjected to guaiacol screening (secondary
screening). Guaiacol screening was used for secondary screening
(Fig. 2a). Isolate 6 was able to oxidize guaiacol and produce brown
colour in Minimal modified medium 3 (Fig. 2b) (Dhiman and Shirkot,
2015). Therefore isolate 6 was recognized as positive laccase-producing
bacteria and subjected to the biochemical testing method. The gram
staining of isolate 6 in Minimal media was subjected to gram staining
and visualized under a microscope. The stained bacteria were observed
to be pink in colour and identified as gram-negative bacteria. The isolate
6 growth in Minimal media 3(MM3) composition was analyzed for
6
Cleaner Materials 6 (2022) 100149
growth for 24 h. The lag phase of isolate 6 was identified as between 14
and 18 h. The isolate 6 tested positive for catalase, nitrate reduction, and
oxidase and tested negative for spore formation, indole test and urease.
Further, isolate 6 was analyzed by 16sr-Rna gene sequencing.
16sr-RNA identification of selected strain
The most attractive potential uses of the 16Sr-RNA sequence are to
provide information about genus and species for the identification of
isolates that do not fit any recognized biochemical profiles (Gupta et al.,
2017). The 16Sr-RNA generates a “low likelihood” or “acceptable”
identification according to commercial systems, or for taxa that are
rarely associated with human infectious diseases (Al et al., 2017). Isolate
6 was subjected to strain identification by 16Sr-RNA analysis. The
neighbour-joining phylogenetic tree was constructed for the isolated
positive bacterial strain. The phylogenetic analysis infers that the iso­
lated strain has a sequential relationship with the organism orchobac­
trum pseudintermedium. The phylogenetic analysis of isolate 6 is
represented in Fig. 3. The blast search was carried out for the nucleic
acid sequence (query length-711). For our query sequence, we have got
nearly a hundred blast hits with 97 % similarity and 1214 bp. The
identified strain O. pseudintermedium ASMCS06 was submitted in Gen­
Bank with accession number MG280942. According to Stackebrandt and
Goebel (1994), strains belonging to the same genus with 97 % or lesser
16Sr-RNA gene sequence similarity can be regarded as members of
different species (Stackebrandt and Goebel, 1994).
Effect of Carbon, nitrogen and natural substrates
The carbon sources have a notable effect on enzyme production. To
interrupt the effect of carbon on Laccase production, dextrose, sucrose,
fructose, starch, maltose, and lactose were used (Gawande and Kamat,
2000). The enzyme activity of different carbon sources was determined
using a Guaiacol assay at 450 nm by UV spectrophotometer after 24 h of
incubation. Among the different carbon sources, dextrose and fructose
were identified as good carbon sources for Laccase production with 28
U/mL and 23 U/mL respectively (Fig. 4a). In agreement with the ob­
tained result, Afreen et al.,2018 reported that sucrose and fructose
showed much higher activity (Afreen et al., 2018) and also Willian
Daniel Hahn Schneider et al., 2018 also reported Glucose and casein as
the best sources for ligninolytic enzymes (Schneider et al., 2018). In
contradiction with the above result, Ashok Rao et al., 2018 highlighted
sodium potassium tartrate for laccase production as an efficient carbon
source (Rao et al., 2019).
Similarly, various nitrogen sources such as yeast extract, NH4Cl,
M. Subash and A.S. Maheshwari Cleaner Materials 6 (2022) 100149

Fig. 7. Growth rate and dye decolourization effect of Laccase at 600 nm and 450 nm at a)10 ppm b)50 ppm and c)100 ppm.

Fig. 8a. Voltage measurement in Microbial fuel cell b- Polarization curves of Fig. 8b. b – Polarization curves of voltage vs current density and power density
voltage vs current density and power density vs current density. vs current density.

(NH4)2SO4, peptone, and meat extract were opted for analyzing the activity (24 U/mL) (Fig. 4b). Following the obtained result Zhu et al.,
evident nitrogen source. The Laccase activity of variant nitrogen sources 2016 optimized Laccase production with yeast as a nitrogen source
was determined by the Guaiacol assay. Amid nitrogen sources, yeast using the white-rot fungus Pleurotus ostreaus (Zhu et al., 2016). Simi­
extract was identified as the best source for better growth and enzyme larly, Ahmed et al., 2015 also reported the importance of yeast extract in

7
M. Subash and A.S. Maheshwari
Laccase production using Pleurotus ostreaus (El-Batal et al., 2015).
Contrary to the above result, Rajesh Kumar et al., 2016 optimized
peptone as an efficient nitrogen source for Laccase production by
Aspergillus flavus (Kumar et al., 2016). Murugesan Rajeshwari et al.,
2016 isolated the Laccase-producing bacteria Bacillus sp. PK4 with yeast
extract as a nitrogen source (Murugesan and Vembu, 2016).
To examine the influence of the natural substrate on Laccase pro­
duction, sawdust, rice bran, and coconut pith were used. The enzyme
activity of different natural substrates was analyzed. Rice bran seems to
be an efficient natural substrate compared to carbon and nitrogen
sources. Rice bran exhibited maximum laccase activity of 38 U/mL
(Fig. 4c). Vaneesa Elisa Pinheiro et al., 2020 suggested Laccase pro­
duction using agricultural waste by Trametes versicolor (Pinheiro et al.,
2020). In agreement with the previous report, the production of extra­
cellular Laccase from bacteria was also reported using agricultural res­
idues as potential substrates (Muthukumarasamy et al., 2015). Rim
et al., 2002 confessed the influence of the natural substrate on the sta­
bility of Laccase production using the fungal species Trametes trogii
(Khlifi-slama et al., 2012).
Stability studies
The effect of temperature on Laccase production was analyzed by
varying the temperature from 30 ◦ C to 100 ◦ C. Among the temperature
variance, 75 ◦ C was found to be the most effective temperature. The
effect of pH in Laccase production was analyzed by varying the pH range
from 2 to 12. Among the pH variance, pH 8 was found to be the most
effective (Fig. 5). O. pseudintermedium ASMCS06 was thermostable with
maximum activity at 75 ◦ C and identified to be active under alkaline
conditions. The laccase activity was found to be higher with tempera­
tures 65–85 ◦ C. In agreement with the experimented result, few reports
highlight the thermal and alkaline stability Laccase producing bacteria.
Zhongyang Ding et al., 2012 reported that optimum pH and temperature
were 3.0 and 60 ◦ C respectively for laccase production using G. lucidum
with residual activity of 46 % (Ding et al., 2012). Similarly, the laccase
activity was optimal at 90 ◦ C and pH 9 using the thermophilic bacterium
Anoxybacillus sp. Strain UARK-01 (Al et al., 2017). The laccase exhibited
optimal activity at 70 ◦ C and pH9.0 for extracellular Laccase production
using Bacillus subtilis (Muthukumarasamy et al., 2015). Further, thermo
alkali stable laccase using Staphylococcus arlettae S1-20 with an optimum
temperature of 85 ◦ C and pH of 9.0 was successfully reported (Chauhan
et al., 2018). Contrary to the previous reports, Laccase production was
reported using Endophytic fungi from Euphorbia milii with optimal pH 5
and temperature of 28 ◦ C (Rao et al., 2019). Similarly, the fungal species
Aspergillus flavus was able to produce maximum Laccase activity at a
temperature of 35 ◦ C and pH 7 (Kumar et al., 2016).
Molecular weight determination
Ammonium sulphate precipitated and dialysed samples were loaded
into lanes 1, 2,3 and 4 as 24, 50 75 and 100ul respectively. SDS PAGE
analysis of O. pseudintermedium ASMCS06 suggests that the molecular
weight of the protein will be in the range of 60–90 kDa (Fig. 6). Most
laccases show mobilities corresponding to the molecular weight of
60–100 kDa, of which 10–50 % may be attributed to glycosylation.
Mannose is one of the major components of the carbohydrate attached to
laccase. The molecular masses of laccases in the 55–90 kDa range have
previously been reported (Quaratino et al. 2007).
Dye degradation
The medium in the presence of O. pseudintermedium ASMCS06 was
able to degrade dye at a concentration of 10,50 and 100 ppm. Dye
decolourization was analyzed for three days and was found to be nearly
65 %. O. pseudintermedium ASMCS06 was subjected to degraded dye at a
concentration of 50 ppm and 100 ppm further and the maximum
8
Cleaner Materials 6 (2022) 100149
decolourization obtained was found to be nearly 60 % and 63 %
respectively. Fig. 7 indicates the role of O. pseudintermedium ASMCS06 in
dye degradation.
Microbial fuel cell
O. pseudintermedium ASMCS06 was analyzed for voltage production
by inoculating them in the anode chamber and phosphate buffer was
added in the cathode chamber to maintain the pH. The performance of
the bacteria in electricity production was investigated. From the results,
it is evident that O. pseudintermedium ASMCS06 can be successfully
employed for bioelectricity production. The maximum voltage produced
was identified as 415 mV on day 7. After day 7, there is no further in­
crease in voltage, the voltage gets stabilized. Fig. 8a represents the
voltage measurement in a microbial fuel cell. The polarization curve
magnifies the voltage concerning current density (Fig. 8b). From the
result, it is inferred that current density increases with an increase in the
voltage of microbial fuel cells. The common regions of the polarization
curves are recognized as activation loss, ohmic loss and mass transfer
loss. The isolated bacterium O. pseudintermedium ASMCS06 was able to
produce a maximum current density of 930 (mA/cm2) and a maximum
power density of 350 (mW/cm2).
Conclusion
Bioremediation is an emerging technology with significance for
environmental pollution management. The bioremediation technology
is considered a safe, sustainable and suitable pathway for contaminant
removal using microorganisms. The isolated bacterial strain
O. pseudintermedium ASMCS06 was efficient in laccase production, a
sustainable enzyme for bioremediation. The current research studies
provoke the efficient application of laccase enzyme in the oxidation of
toxic substances, decolourization of environmental pollutants, detoxi­
fication of industrial effluents and variant biotechnological application.
The decolourization results provide positive feedback on enzyme prop­
erties for dye decolourization. Further, Laccase can be employed as a
biocatalyst on the cathodic surface of the microbial fuel cell. The iso­
lated bacterium O. pseudintermedium ASMCS06 was able to a maximum
current density of 930 (mA/cm2) and a maximum power density of 350
(mW/cm2). The investigation highlights the reader to implement the
laccase as bioremediation enzyme and the efficiency of laccase in dye
decolourization and bioelectricity production in an eco-friendly manner.
Funding
The above research was not supported by any funding agency.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
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