This document summarizes the work of Sayan Sarkar on isolating and characterizing arsenic-tolerant bacteria from soil and water samples. Three bacterial strains, CRS2, CRS4, and CMW3, were isolated that could grow in media containing high concentrations of arsenic. Initial tests showed the strains were Gram negative, and CRS4 and CMW3 tested positive for catalase. A soil-water study found the strains could mobilize arsenic from soil into water. Further sequencing and analysis of the strains' arsenic transformation abilities is planned to understand their potential for bioremediating arsenic-contaminated environments.

1. Name: Sayan Sarkar
Exam roll no.: M4EBT1601
Under the supervision of
Dr. Ujjal Kumar Mukhopadhyay
Chief Scientist
West Bengal Pollution Control Board

2. Arsenic
 A metalloid having atomic number 33.
 Commonly used in copper and lead alloys, semiconductor materials, optoelectronic materials, pesticides and
wood preservatives.
 Highly toxic and disrupts the disulphide bonds in the enzymes.
 Primarily affects the skin, while, prolonged exposure leads to skin and lung cancer, neurological and obstetric problems.
[Chakraborti et. al., 2002; Chakraborti et. al., 2008; Rahman et. al., 2001; Mukherjee et. al., 2003; Chakraborti et. al., 2003]

3.  Arsenic occurs in four oxidation states – elemental (As0), As3+, As5+ and arsenide (As3-).
 As3+ and As5+ are the most common species found in aquatic environment. [Kudo et. al. 2013]
 Arsenate, containing arsenic in the form of As5+, gets easily adsorbed on the minerals. [O’Day et. al., 2004]
 As3+ is more soluble and gets easily mobilized into water.
Arsenic

4. Arsenic transformation by bacteria
Reduction
o Cytosolic reduction occurs by the expression of the ars operon.
o The core genes of the ars operon are – cytosolic As5+ reductase arsC, membrane bound As3+ efflux pump arsB and
transcriptional repressor arsR.
o Another five membered ars operon (arsRDABC) is found containing additional arsA, an ATPase for arsB, and arsD, a
chaperone to the arsAB pump.
o The ars operon occurs in chromosomes or plasmids of Gram negative α-, β- and γ-Proteobacteria and Gram positive
Firmicutes and Actinobacteria.
[Escudero et. al., 2013; Cavalca et. al., 2013; Lin et. al., 2007]

5. Arsenic transformation by bacteria
Reduction
 Respiratory or dissimilatory reduction occurs in various obligate or facultative anaerobes.
 Bacteria obtain metabolic energy by respiration with As5+ as the terminal electron acceptor.
 Respiratory As5+ reductase (Arr) is a heterodimer protein having a catalytic subunit, ArrA, and an electron transfer
subunit, ArrB.
 ArrA consists of a molybdopterin centre and a [3Fe-4S] cluster.
 ArrB subunit contains four [4Fe-4S] clusters.
[Saltikov and Newman, 2003; Malasarn et. al. 2004]

6. Arsenic transformation by bacteria
Oxidation
 Numerous bacteria can oxidize As3+ enzymatically.
 The energy and reducing power of As3+ oxidation is used in cellular growth and carbon dioxide fixation.
 The enzyme As3+ oxidase is heterodimeric and consists of –
1) a small subunit with a [2Fe-2S] Rieske cluster
2) a large subunit having a molybdopterin guanosine dinucleotide at the active site and an iron binding [3Fe-4S] cluster.
 The expression of the enzyme is regulated by As3+.
[Santini et. al., 2000; Oremland et. al., 2002; Ellis et. al., 2001; Lebrun et. al., 2003]

7. Arsenic transformation by bacteria
Methylation and Demethylation
 Methylation is considered to be a detoxification process.
 It is more common among the eukaryotes.
 As5+ is reduced followed by oxidative addition of a methyl group.
 For prokaryotes, the same mechanism follows except for formation of arsine.
 A methyl transferase, ArsM, confer arsenic resistance and generation of trimethylarsine.
 Several soil bacteria have been observed to demethylate mono- and dimethyl arsenic compounds.
[Bentley and Chesteen, 2002; Dopp et. al., 2010; Qin et. al., 2006; Yin et. al., 2011; Chen et. al., 2013]

8. Arsenic release by bacteria
o Two mechanisms for microbial release of arsenic in water are –
1) cytosolic reduction of As5+ into As3+ by certain heterotrophic and chemolithoautotrophic bacteria
2) direct respiration of As5+ present in minerals by dissimilatory As5+ reducing bacteria
[Drewniak et. al., 2008; Sultana et. al., 2012; Liao et. al., 2011; Mumford et. al., 2012]
o In case of the Bengal Delta Plain, analyses have revealed the role of metal-reducing bacteria in arsenic release.
[Sutton et. al., 2009; Sultana et. al., 2012; Akai et. al., 2004; Islam et. al., 2012]

9. Purpose of the work
 Characterization and identification of bacterial strains that are aerobic and can tolerate high amount of arsenic.
 To look for novel bacterial species.
 To determine the role of these strains in arsenic mobilization.
 To determine the potential of these strains in oxidation of arsenite.
 To determine whether these strains can be utilized for bioremediation purpose.

10. Plan of work
 Isolation of arsenic tolerant bacteria
 Determination of growth curves
 Gram’s staining
Catalase test
 Determination of minimal inhibitory concentration (MIC)
 Isolation of DNA
 Spectrophotometric analysis of the DNA
 Amplification of the 16S rRNA genes
 Agarose gel electrophoresis
 Soil water partitioning study

11. Isolation of arsenic tolerant bacteria
CRS2 CRS4 CMW3
Pure culture of the isolated strains in nutrient agar medium [5mM As(III)]

12. Growth curves of the isolated strains
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 1 2 3 4 5 6 7 8 9 10 11 12 13
OD
Time (hours)
CRS2
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25
OD
Time (hours)
CRS4
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 1 2 3 4 5 6 7 8 9 10 11
OD
Time (hours)
CMW3
Grown in nutrient broth containing 5 mM As(III)

13. Gram’s staining of the isolated strains
CRS2 CRS4
CMW3
Microscopic images of the Gram stained cells (at 100X)

14. Catalase Test
Strain Catalase test
CRS2 Negative
CRS4 Positive
CMW3 Positive
Result of the catalase test

15. Minimal Inhibitory Concentration
Concentrations
(in mM)
Strains
CRS2 CRS4 CMW3
0 +++ +++ +++
0.05 + + +
0.1 + + +
0.5 ++ + +
1.0 + + +
5.0 +++ - +
10.0 +++ ++ +
15.0 + - -
[+++ = good growth; ++ = moderate growth; + = very low growth; - = no growth]

16. Quantification of isolated DNA
Strains CRS2 CRS4 CMW3
DNA concentration
(in μg/ml)
22.96 560.91 25.732

17. Agarose gel electrophoresis of the amplified DNA samples
Image of the agarose gel containing the PCR products of the isolated strains viewed under UV light

18. Soil water partitioning study
Set no. Soil (60 g) Water (150 ml) Inoculum (20 ml) As (μg/l) Mean As (μg/l)
1 Sterilized Sterilized - 218.60
164.232 Sterilized Sterilized - 134.90
3 Sterilized Sterilized - 139.20
4 Sterilized Unsterilized - 196.20
190.605 Sterilized Unsterilized - 202.60
6 Sterilized Unsterilized - 173.00
7 Sterilized Sterilized CMW3 150.80 -
8 Sterilized Unsterilized CMW3 337.40 -
9 Sterilized Sterilized CRS4 160.20 -
10 Sterilized Unsterilized CRS4 505.00 -

19. Conclusion
• The isolated strains are moderately tolerant to high arsenic concentration.
• The strains can mobilize arsenic from soil to water.
• The arsenic mobilization by these strains are facilitated by the microbial consortia already present in the water.
• These strains are expected to oxidize arsenite into arsenate.
• The strains are indigenous, therefore, can be used in natural water or any other environment.

20. Scope of further research
• Sequencing of the amplified 16S rRNA genes to identify the bacterial species.
• Determination of arsenic transforming characteristics of these strains – both qualitative and quantitative.
• Isolation and identification of arsenic transforming genes present in these strains.
• Determination of potential of these strains in bioremediation of arsenic contaminated water and/or soil.

21. References
Akai J., Izumi K., Fukuhara H., Masuda H., Nakano S., Yoshimura T., Ohfuji H., Anawar H.M., Akai K.; 2004: Mineralogical and geomicrobiological
investigations on groundwater arsenic enrichment in Bangladesh. Appl. Geochem. 19, 215-230.
Cavalca L., Corsini A., Andreoni V., Muyzer G; 2013: Microbial transformations of arsenic: Perspective for biological removal of arsenic from water.
Future Microbiol. 8, 753-768.
Chakraborti D., Das B., Nayak B., Pal A., Sengupta M.K., Hossain M.A., Ahamed S., Sahu M., Saha K.C., Mukherjee S.C., Pati S., Dutta R.N.,
Quamruzzaman Q.,; 2008: Groundwater arsenic contamination in Ganga-Meghna-Brahmaputra plain, its health effects and approach for mitigation.
Environ. Earth Sci. 70(5), 1993-2008.
Chakraborti D., Mukherjee S.C., Pati S., Sengupta M.K., Rahman M.M., Chowdhury U.K., Lodh D., Chanda C.R., Chakraborty A.K., Basu G.K.; 2003:
Arsenic groundwater contamination in Middle Ganga Plain, Bihar, India: A future danger. Environ. Health Perspec. 111, 1194-1201.
Chakraborti D., Rahman M.M., Paul K., Chowdhury U.K., Sengupta M.K., Lodh D., Chanda C.R., Saha K.C., Mukherjee S.C.; 2002: Arsenic calamity in
the Indian subcontinent – what lessons have been learned? Talanta 58, 3-22.
Drewniak L., Styczek A., Lopatka M.M., Sklodowska A.; 2008: Bacteria, hypertolerant to arsenic in the rocks of an ancient gold mine, and their potential
role in dissemination of arsenic pollution. Environ. Pollut. 156, 1069-1074.

22. References
Ellis P.J., Conrads T., Hille R., Kuhn P.; 2001: Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 Å
and 2.03 Å. Structure 9, 125-132.
Escudero L.V., Casamayor E.O., Chong G., Pedro’s-Alio’ C., Demergasso; 2013: Distribution of microbial arsenic reduction, oxidation and extrusion
genes along a wide range of environmental arsenic contaminations. PLoS ONE 8, e78890.
Islam A.B.M.R., Maity J.P., Bundschuh J., Chen C.Y., Bhowmik B.K., Tazaki K.; 2012: Arsenic mineral dissolution and possible mobilization in mineral-
microbe-groundwater environment. J. Hazard. Mater. 262, 989-996.
Kudo K., Yamaguchi N., Makino T., Ohtsuka T., Kimura K., Dong D.T., Amachi S.; 2013: Arsenic release from soil by a novel dissimilatory arsenate-
reducing bacterium Anaeromyxobacter sp. PSR-1. Appl. Environ. Microbiol. 79, 4635-4642.
Lebrun E., Brugna M., Baymann F., Muller D., Lièvremont D., Lett M-C., Nitschke W.; 2003: Arsenite oxidase, an ancient bioenergetic enzyme. Mol.
Biol. Evol. 20, 686-693.
Liao VH-C., Chu Y.J., Su Y.C., Hsiao S.Y., Wei C.C., Liu C.W., Liao C.M., Shen W.C., Chang F.J.; 2011: Arsenite-oxidizing and arsenate-reducing
bacteria associated with arsenic-rich groundwater in Taiwan. J. Contam. Hydrol. 123, 20-29.

23. Lin Y.F., Yang J., Rosen B.P.; 2007: ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase. J. Bioener. Biomembr. 39, 453-458.
Malasarn D., Saltikov C.W., Campbell K.M., Santini J.M., Hering J.G., Newman D.K.; 2004: arrA is a reliable marker for As(V) respiration. Science 306,
455-468.
Mukherjee S.C., Rahman M.M., Chowdhury U.K., Sengupta M.K., Lodh D., Chanda C.R., Saha K.C., Chakraborti D.; 2003: Neuropathy in arsenic
toxicity from groundwater arsenic contamination in West Bengal-India. J. Environ. Sci. Health 38(1), 165-183.
Mumford A.C., Barringer J.L., Benzel W.M., Reilly P.A., Young L.Y.; 2012: Microbial transformations of arsenic: Mobilization from glauconitic
sediments to water. Water Res. 56, 2859-2868.
O’Day P.A., Vlassopoulos D., Root R., Rivera N.; 2004: The influence of sulphur and iron on dissolved arsenic concentrations in the shallow subsurface
under changing redox conditions. Proc. Nation. Acad. Sci. USA 101, 13703-13708.
Oremland R.S., Hoeft S.E., Santini J.M., Bano N., Hollibaugh R.A., Hollibaugh J.T.; 2002: Anaerobic oxidation of arsenite in Mono Lake water by a
facultative arsenite-oxidizing chemoautotroph, strain MLHE-1. Appl. Environ. Microbiol. 68, 4795-4802.
References

24. Rahman M.M., Chowdhury U.K., Mukherjee S.C., Mondal B.K., Paul K., Lodh D., Chanda C.R., Basu G.K., Saha K.C., Roy S., Das R., Palit S.K.,
Quamruzzaman Q., Chakraborti D.; 2001: Chronic arsenic toxicity in Bangladesh and West Bengal-India – A review and commentary. J. Toxic. 39(7),
683-700.
Saltikov C.W., Newman D.K.; 2003: Genetic identification of a respiratory arsenate reductase. Proc. Nation. Acad. Sci. USA 100, 10983-10988.
Santini J.M., Sly L.I., Schnagl R.D., Macy J.M.; 2000: A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine:
phylogenetic, physiological and preliminary biochemical studies. Appl. Environ. Microbiol. 66, 92-97.
Sultana M., Vogler S., Zargar K., Schmidt A-C., Saltikov C., Seifert J., Schlömann M.; 2012: New clusters of arsenite oxidase and unusual bacterial
groups in enrichments from arsenic-contaminated soil. Arch. Microbiol. 194, 623-635.
Sutton N.B., van der Kraan G.M., van Loosdrecht M.C., Muyzer G., Bruining J., Schotting R.J.; 2009: Characterization of geochemical constituents and
bacterial populations associated with As mobilization in deep and shallow tube wells in Bangladesh. Water Res. 43, 1720-1730.
References