Assessment of Acrylamide Degradation Potential

of Pseudomonas aeruginosa BAC-6 Isolated

from Industrial Effluent
Vijayashree Chandrashekar & Chandrika Chandrashekar &
Rajath Shivakumar & Sourav Bhattacharya & Arijit Das &
Bhaskar Gouda & Subbaramiah Sundara Rajan
Received: 6 February 2014 / Accepted: 14 April 2014
#
Springer Science+Business Media New York 2014
Abstract Acrylamide finds diverse industrial applications but is considered an environmental
threat because of its neurotoxic, carcinogenic, and teratogenic effects. Certain bacteria enzymatically
degrade acrylamide to acrylic acid and ammonia. The present investigation was carried out to isolate
and identify an acrylamide-degrading bacteriumfromindustrial effluent. Bacterial growth and extent
of acrylamide degradation in the presence of different acrylamide concentrations, nutrients, varied
range of pH, and temperature were analyzed. Among the eight acrylamide-degrading isolates, isolate
BAC-6 demonstrated the highest degradation, and based upon the partial 16S rDNA sequencing, it
was identified as Pseudomonas aeruginosa. P. aeruginosa BAC-6 grew over a wide range of
acrylamide concentrations, but the highest degradation was recorded at 500 mg/Lconcentration with
concomitant cell growth. Among the carbon supplements, mannitol supported the highest growth
and degradation. Maximum degradation was reported at neutral pH. A mesophilic temperature
range (2540 C) facilitated conducive bacterial growth followed by degradation. The highest
degradation and bacterial growth were observed at 30 and 35 C, respectively. Thus, it could be
Appl Biochem Biotechnol
DOI 10.1007/s12010-014-0923-1
V. Chandrashekar
Department of Molecular Biology, Centre for Advanced Studies in Biosciences, Jain University,
Bangalore 560019 Karnataka, India
C. Chandrashekar
Department of Biochemistry, Centre for Advanced Studies in Biosciences, Jain University,
Bangalore 560019 Karnataka, India
R. Shivakumar
Department of Plant Biotechnology, Centre for Advanced Studies in Biosciences, Jain University,
Bangalore 560019 Karnataka, India
S. Bhattacharya (*)
:
A. Das
:
S. S. Rajan
Department of Microbiology, Centre for Advanced Studies in Biosciences, Jain University,
Bangalore 560019 Karnataka, India
e-mail: bhattsourav3011@gmail.com
B. Gouda
Department of Lipid Science and Traditional Foods, Central Food Technological Research Institute,
Mysore 570020 Karnataka, India
inferred from the present investigation that cultural conditions strongly affected the degradation
potential of P. aeruginosa BAC-6 and advocated the utilization of the isolate in bioremediation of
sites polluted with acrylamide.
Keywords Acrylamide
.
Pseudomonas aeruginosa
.
Degradation
.
High-performance liquid
chromatography
Introduction
Acrylamide (C
3
H
5
NO) is an important monomer used as a conjugated reactive molecule in
polyacrylamide or copolymer production [1]. Acrylamide often finds its use as a thickener or
binder in paints, coatings, and foundry sand, in toiletries, and in cosmetics as moisture-
retaining additive. It plays various roles in textile processing and in the production of
adhesives, tapes, and gels used for electrophoresis [2].
Acrylamide is a well-known neurotoxin, carcinogen, and teratogen. Neurotoxic effects
(severe symptoms in the central nervous system and peripheral polyneuropathy) in humans
have been observed towards high levels of exposure [3]. It is an irritant to the skin and
respiratory tract and is readily absorbed by all routes causing acute and subchronic/chronic
toxicity. Genotoxic effects include chromosomal aberrations, dominant lethality, sister chro-
matid exchanges, and unscheduled DNA synthesis in various in vitro and in vivo systems.
High concentrations of acrylamide in the human body can also contribute to cancer risk [4].
The widespread use and indiscriminate discharge of acrylamide and polyacrylamide has led to
contamination of soil and aquatic environments [5]. Since the presence of acrylamide in the
environment is hazardous, its degradation is desirable [6].
In spite of acrylamide being toxic in its monomeric form, some microorganisms are able to
utilize acrylamide [2]. Acrylamide-degrading Pseudomonas, Bacillus, Rhodococcus, and
Enterobacter species were isolated from the environment [4]. Microbial utilization of acryl-
amide and other amides is mediated by amidases that catalyze the hydrolysis of carboxylic
amides to free carboxylic acids and free ammonia [7].
Successful bioremediation depends on the introduction of specific microorganisms capable
of degrading toxic pollutants. Hence, the present study aimed at isolation and identification of
a bacterial strain capable of degrading acrylamide under different cultural conditions.
Materials and Methods
Chemicals
All the chemicals and media components used in the experiments were purchased from
HiMedia, Mumbai, India, and were of analytical grade.
Sample Collection and Processing
Effluent sample was collected from Jigani Industrial Area (12.46N, 77.38E), Bangalore, in
sterilized plastic containers and transported to the laboratory. To obtain pure bacterial cultures,
the samples were serially diluted and plated on nutrient agar and incubated at 37 C for 24 h.
Following the appearance of bacterial colonies, isolates were periodically subcultured on
nutrient agar slants.
Appl Biochem Biotechnol
Selection of the Best Acrylamide-Degrading Bacteria
Acrylamide (at a concentration of 500 mg/L) was added as sole nitrogen source to the modified
mineral salt medium of Syed et al. [4] containing grams per liter of glucose, 10; NaH
2
PO
4
, 6.8;
MgSO
4
.7H
2
O, 0.5; yeast extract, 0.01; and 10 mL of the following trace elements: ZnCl
2
, 0.03;
CoCl
2
.6H
2
O, 0.003; FeCl
3
, 0.002; CuCl
2
, 0.01; and H
3
BO
3
, 0.05; pH 7.0.
Suspension of the respective bacterial isolate (0.25 mL) was inoculated into mineral salt
medium (MSM), and uninoculated MSM served as blank. The inoculated medium was
incubated for 48 h at 37 C. Following incubation, selection of the potential acrylamide-
degrading bacteria was done based on the isolate showing the highest growth and extent of
acrylamide degradation based on high-performance liquid chromatography (HPLC) analysis.
Molecular Characterization of Selected Bacterial Isolate
The selected bacterial isolate was cultured in Luria Bertani broth and incubated at 37 C for
24 h in an orbital shaker at 150 rpm. Genomic DNA was extracted using Bacterial Genomic
DNA Isolation Kit (Chromous Biotech Pvt. Ltd., Bangalore, India) according to the manu-
facturer instructions and visualized using 0.8 % (w/v) agarose gel electrophoresis.
PCR Amplification
The PCR amplification reactions were performed in a total volume of 100 L. Each reaction
mixture contained the following solutions: 1 L genomic DNA; 400 ng of universal forward
16S ribosomal DNA (rDNA) primer (5-AGAGTTTGATCCTGGCTCAG-3); 400 ng of
universal reverse 16S rDNA primer (5-GGTTACCTTGTTACGACTT-3); 4 L of dNTPs
(2.5 mM each); 10 L of Taq DNA polymerase assay buffer; and 1 LTaq DNA polymerase
(3 U/L) (Chromous Biotech Pvt. Ltd., Bangalore, India), and the volume was made up to
100 L using sterile distilled water. The ABI 2720 thermal cycler (Applied Biosystems, USA)
was programmed as follows: 5-min initial denaturation at 94 C, followed by 35 cycles that
consisted of denaturation for 30 s at 94 C, annealing for 30 s at 55 C, and extension at 72 C
for 1 min and a final extension of 5 min at 72 C. The PCR-amplified product was eluted from
the gel using Gel Extraction Spin-50 kit (Chromous Biotech Pvt. Ltd., Bangalore, India)
according to the manufacturer instructions and detected by 1.2 % agarose gel (with ethidium
bromide) electrophoresis.
Partial 16S rDNA Sequencing and Analysis of Sequenced Data
The partial 16S rDNA sequencing of the PCR-amplified product was performed at Chromous
Biotech Pvt. Ltd., Bangalore, India, using Big Dye Terminator Version 3.1 cycles sequencing
kit and ABI 3500 XL Genetic Analyzer (Applied Biosystems, USA). Ten microliters of the
sequencing reaction mixture contained 4 L of Big Dye Terminator Ready Reaction Mix,
1 L of rDNA amplification product (100 ng/L), 2 L primer (10 pmol/), and 3 L Milli Q
water. The ABI 2720 Thermal Cycler (Applied Biosystems, USA) was programmed to
perform initial denaturation at 96 C for 1 min, followed by 25 cycles that consisted of
denaturation at 96 C for 10 s, hybridization at 50 C for 5 s, and elongation at 60 C for 4 min.
The resultant nucleotide sequence was analyzed using the software Seq Scape version 5.2. The
16S rDNA sequence was aligned manually with the available nucleotide sequences retrieved
from the NCBI database using BLASTn [8]. The nucleotide sequence was submitted to
GenBank database (NCBI, USA) and was provided an accession number.
Appl Biochem Biotechnol
Optimization of Cultural Conditions
Effect of acrylamide concentration (50, 100, 250, 500, 750, and 1,000 mg/L), carbon supple-
ments (glucose, fructose, maltose, sucrose, mannitol, and soluble starch), initial pH of the
medium (4, 5, 6, 7, 8, and 9), incubation temperature (25, 30, 35, 40, and 45 C) on bacterial
growth, and acrylamide degradation were optimized. Optimization of growth conditions was
carried out by changing one parameter at a time, while all other parameters remained constant.
Analytical Methods
Following incubation, 5 mL of aliquots from the acrylamide-degrading culture medium was
removed and divided into two portions. Serial dilution of one portion was carried out for
bacterial plate count and expressed in colony-forming units (CFU/mL). For the other portion,
the bacterial broth was centrifuged at 5,000 rpm for 20 min at 4 C to obtain the cell-free
supernatant. Cell-free supernatant of the broth culture was used for determining the extent of
acrylamide degradation. Residual acrylamide levels were measured by HPLC analysis.
HPLC Analysis
The cell-free supernatant was subjected to filtration using 0.25 nitrocellulose membrane
filter. The working standard solution of acrylamide (concentration of 500 mg/L) was prepared
using methanol. Ten microliters of the sample was injected into the HPLC system (Merck,
Germany). A C-18 column (1504.6 mm) was used. The mobile phase (100 % methanol) was
maintained at a flow rate of 1 mL/min. The concentration of the acrylamide standard solution
was determined at 196 nm. Area under the absorbance peak was used to estimate the
percentage of degradation using the formula: [(C
i
C
f
)/C
i
]100, where C
i
is the initial
concentration of acrylamide, and C
f
is the final concentration of acrylamide.
Statistical Analysis
Effect of each parameter was studied in triplicate, and the data are graphically presented as
mean standard deviation of triplicates (n=3). All the graphs have been prepared using
Microsoft Excel 2007.
Results and Discussion
Selection of the Best Acrylamide-Degrading Bacterial Isolate
From the effluent sample collected, 24 bacterial forms were isolated on nutrient agar medium.
When allowed to grow in MSM spiked with 500 mg/L of acrylamide, eight isolates (BAC-3, 6,
10, 11, 17, 19, 20 and 22) showed favorable growth and acrylamide degradation. Among these
eight isolates, BAC-6 demonstrated the highest growth and acrylamide degradation and was
selected for further study.
Molecular Identification of the Selected Isolate
16S rDNA sequencing is a powerful tool for rapid identification and phylogenetic analysis of
bacterial species. The apparent size of the PCR amplicon was ~1.5 kb. The obtained 633 bp
Appl Biochem Biotechnol
16S rDNA nucleotide sequence was compared with available 16S ribosomal sequences in the
NCBI database using BLASTn. The BAC-6 isolate has been enrolled into a cluster containing
Pseudomonas sp. and was found to be closely related to Pseudomonas aeruginosa strain
PAO1 with 99 % sequence similarity. Hence, it was designated as P. aeruginosa BAC-6. The
submitted nucleotide sequence was provided a GenBank accession number KJ381050.
Most reports on acrylamide or aliphatic amide degradation by bacteria had shown the
involvement of the genus Pseudomonas which includes P. aeruginosa [3, 9], Pseudomonas sp.
DRYJ7 [10], Pseudomonas putida [11], Pseudomonas azotoformans IAM 1603 [12], and
Pseudomonas chlororaphis B23 [13].
Effect of Acrylamide Concentration on its Degradation
Acrylamide concentration influenced the growth and degradation potential of P. aeruginosa
BAC-6. Maximum growth and degradation (1910
7
CFU/mL and 46.03 %) was recorded for
500 mg/L acrylamide concentration (Fig. 1). Growth was suppressed at higher concentrations
probably due to the inhibitory effect of acrylamide on thiol groups of proteins [14]. Earlier
reports have also shown the optimum concentration for acrylamide-degrading strain Pseudo-
monas sp. DRYJ7 to be 500 mg/L [10] which coincides with the present findings. Optimum
concentration for acrylamide degradation was found to be 440 mg/L for Pseudomonas stutzeri
[15]. P. aeruginosa could degrade 1,0002,000 mg/L of acrylamide [3].
Effect of Carbon Supplements on Acrylamide Degradation
Most Pseudomonas species have a remarkable nutritional versatility and can metabolize a wide
range of compounds by convergent catabolic pathways [16]. The synthesis of catabolic
enzymes is controlled by sequential induction of groups of enzymes by substrates or interme-
diates of the pathway and repression by later products [17].
Although numerous microorganisms catabolize aliphatic amides, acrylamide, because of its
inhibitory effect on sulfhydryl proteins, inhibits their growth [18]. However, this toxicity may
be reduced by the usage of carbon supplements. In the present study, extent of acrylamide
degradation was dependent on the composition of the medium. As compared to the control,
concomitant supplementation of carbon supplements resulted in improved growth of
Fig. 1 Effect of different concentrations of acrylamide on its degradation and P. aeruginosa BAC-6 biomass
formation. Data represent mean SD (n=3)
Appl Biochem Biotechnol
P. aeruginosa BAC-6 and acrylamide degradation (Fig. 2). Among the carbon supplements,
mannitol supported the highest bacterial growth and acrylamide degradation (19.710
7
CFU/
mL and 52.91 %). The possible reason for mannitol supporting the highest degradation and
bacterial growth may be due to the enhanced production of acrylamidase enzyme or increased
enzyme-specific activity resulting from reduced catabolite repression.
Production of microbial amidases responsible for the hydrolysis of acrylamide and similar
amides is influenced by the availability of nutrients. When amides were used as sole source of
carbon and nitrogen, growth of the organism was observed to be less, and therefore, the
production medium was supplemented with various sources of carbon and nitrogen to enhance
both growth and enzyme production [19]. Earlier, amidase-specific activity was low when
P. aeruginosa cells grew in minimal medium containing succinate (a preferred substrate
causing strong catabolite repression). Amidase levels were high with mannitol (a substrate
not causing catabolite repression) and intermediate with glucose (a substrate causing mild
catabolite repression) [20]. Previous studies on acrylamide degradation by P. chlororaphis B23
have identified glucose as the best carbon source when acrylamide was used as a nitrogen
source [13]. Besides glucose, starch had also been used as a supplement for acrylamide
degradation by Pseudomonas thermophila [21].
Effect of Initial pH of Medium
Every microorganism has a minimal, maximal, and optimal pH for growth and metabolism.
Microbial cells are significantly affected by the pH of their immediate environment because
they apparently have no mechanism for adjusting their internal pH [22]. Thus, studying the
effect of the media pH on the bacterial growth and acrylamide degradation was an important
criterion of this paper.
The external pH partly determines the cytoplasmic or intracellular pH, which affects
enzyme activity, reaction rates, protein stability, structure of nucleic acids and many other
biological molecules. Significant growth and degradation was observed between pH 6.08.0,
with the maximum (20.410
7
CFU/mL and 60.1 %) recorded at pH 7.0. Retardation in growth
and degradation was observed under acidic and alkaline conditions (Fig. 3).
Under acidic conditions, accumulation of organic acids in the cytoplasm of the isolate
might have resulted in partial or complete failure of pH homeostasis, which in turn retarded
Fig. 2 Effect of different carbon supplements on acrylamide degradation and P. aeruginosa BAC-6 biomass
formation. Data represent mean SD (n=3)
Appl Biochem Biotechnol
growth and metabolism [23]. Similarly, under alkaline conditions (in which the cytoplasmic
pH was lower than the external pH), membrane-permeable bases such as polyamines might
have accumulated in the cytoplasm. Inside the cytoplasm, these uncharged bases could have
been protonated, and the consumption of cytoplasmic protons could have impaired pH
homeostasis [24].
Several reports on acrylamide-degrading bacteria had shown that optimum conditions for
acrylamide biodegradation are achieved if pH and temperature are in the range of pH 68 and
mesophilic temperature [10, 25]. pH 7.0 was reported as optimum pH for acrylamide degra-
dation by Pseudomonas sp. MCI3434 [26]. Pseudomonas sp. DRY J7 grew over a relatively
wide pH range from 6.0 to 8.5 [27].
Effect of Incubation Temperature
Growth temperature is known to affect several outer membrane components such as outer
membrane proteins and lipopolysaccharide in Pseudomonas species such as P. aeruginosa
[28]. It has been suggested that the major outer membrane protein, the porin, may be
responsible for some nonspecific outer membrane permeability properties in P. aeruginosa
Fig. 3 Effect of initial pH of medium on acrylamide degradation and P. aeruginosa BAC-6 biomass formation.
Data represent mean SD (n=3)
Fig. 4 Effect of incubation temperature on acrylamide degradation and P. aeruginosa BAC-6 biomass formation.
Data represent mean SD (n=3)
Appl Biochem Biotechnol
[29]. Porins are important components of the interface between the bacterium and its sur-
rounding environment and are able to modulate its channel size as a function of the growth
temperature [30]. Behavior of porins in planar lipid bilayers suggests that they are temperature-
responsive proteins, creating large pores in vivo at physiological temperature, mediating the
transfer of hydrophilic molecules [31].
Information on temperature optimum for microbial growth would be useful for bioremedi-
ation purposes [32]. Most of the formerly studied acrylamide/aliphatic amides-degrading
bacteria are mesophiles with optimum temperature for growth between 25 and 40 C [3].
After 48 h of incubation, significant growth was observed at temperatures between 25 and
35 C. The optimum temperatures for maximum growth (21.710
7
CFU/mL) and degradation
(74.06 %) were 35 and 30 C, respectively, beyond which both growth and degradation
declined (Fig. 4). At 30 C, the porins in the bacterial membrane might have been fully
functional, stabilizing the membrane transport machinery. Both high and low temperatures
Fig. 5 HPLC chromatogram for acrylamide degradation a under unoptimized conditions and b under optimized
conditions
Appl Biochem Biotechnol
acted as important physical factors affecting the cell in numerous physical and biochemical
aspects, inducing a decrease in the bacterial growth rate.
Previously, 26 and 28 C were reported as optimum temperatures for the growth of
P. chlororaphis and P. aeruginosa, respectively [3, 14]. It was reported that 30 C was the
optimum temperature for the utilization of acrylamide by P. stutzeri [15].
HPLC chromatograms for acrylamide biodegradation by P. aeruginosa BAC-6 under
unoptimized and optimized media and growth conditions are shown in Fig. 5a, b. Extent of
acrylamide degradation under unoptimized and optimized condition was 44.52 and 78.24 %,
respectively. Following HPLC analysis, acrylamide peak was detected at retention time of
approximately 2.1 min. The peak of the degradation product, which could be of acrylic acid,
was detected at retention time between 12 min. Bacterial enzymes such as amidohydrolase
(amidase) and other synthesis proteins deaminate acrylamide to form acrylic acid and ammo-
nia as main degradation products [1, 5]. Previous studies reported detection of acrylic acid as
an intermediate during acrylamide degradation by Pseudomonas sp. strain DRYJ7 and
P. stutzeri [10, 15].
Conclusion
The present investigation reports that P. aeruginosa BAC-6 could tolerate high concentrations
of acrylamide. The isolate grew well at 500 mg/L concentration of acrylamide. Addition of
carbon supplements in the form of mannitol improved the extent of acrylamide degradation.
pH 7 and 30 C proved to be the best physical conditions supporting the highest degradation.
The results emphasize that P. aeruginosa BAC-6 may be used as a potent biological agent to
degrade acrylamide at polluted sites.
Acknowledgments We are thankful to Dr. R. Chenraj Jain, Chairman of Jain Group of Institutions, Dr. N.
Sundararajan, Vice Chancellor of Jain University, and Dr. S. Sundara Rajan, Director of Centre for Advanced
Studies in Biosciences, Jain University, Bangalore, for providing the laboratory facilities required for this
research work.
Conflict of Interest The authors declare that they have no conflict of interest.
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