Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 31100
Isolation and Probiotic Characterization of Arsenic-Resistant Lactic Acid Bacteria for Uptaking Arsenic

Authors: Jatindra N. Bhakta, Kouhei Ohnishi, Yukihiro Munekage, Kozo Iwasaki


The growing health hazardous impact of arsenic (As) contamination in environment is the impetus of the present investigation. Application of lactic acid bacteria (LAB) for the removal of toxic and heavy metals from water has been reported. This study was performed in order to isolate and characterize the Asresistant LAB from mud and sludge samples for using as efficient As uptaking probiotic. Isolation of As-resistant LAB colonies was performed by spread plate technique using bromocresol purple impregnated-MRS (BP-MRS) agar media provided with As @ 50 μg/ml. Isolated LAB were employed for probiotic characterization process, acid and bile tolerance, lactic acid production, antibacterial activity and antibiotic tolerance assays. After As-resistant and removal characterizations, the LAB were identified using 16S rDNA sequencing. A total of 103 isolates were identified as As-resistant strains of LAB. The survival of 6 strains (As99-1, As100-2, As101-3, As102-4, As105-7, and As112-9) was found after passing through the sequential probiotic characterizations. Resistant pattern pronounced hollow zones at As concentration >2000 μg/ml in As99-1, As100-2, and As101-3 LAB strains, whereas it was found at ~1000 μg/ml in rest 3 strains. Among 6 strains, the As uptake efficiency of As102-4 (0.006 μg/h/mg wet weight of cell) was higher (17 – 209%) compared to remaining LAB. 16S rDNA sequencing data of 3 (As99- 1, As100-2, and As101-3) and 3 (As102-4, As105-7, and As112-9) LAB strains clearly showed 97 to 99% (340 bp) homology to Pediococcus dextrinicus and Pediococcus acidilactici, respectively. Though, there was no correlation between the metal resistant and removal efficiency of LAB examined but identified elevated As removing LAB would probably be a potential As uptaking probiotic agent. Since present experiment concerned with only As removal from pure water, As removal and removal mechanism in natural condition of intestinal milieu should be assessed in future studies.

Keywords: Characterization, Lactic Acid Bacteria, As-resistant, Pediococcus sp, As removal probiotic

Digital Object Identifier (DOI):

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 2407


[1] H. M. Hemond, and H. F. Solo-Gabriele, "Children-s exposure to arsenic from CCA-treated wooden decks and playground structures," Risk Analysis, vol. 24, pp. 51-64, 2004.
[2] B. L. Murphy, A. P. Toole, and P. D. Bergstrom, "Health risk assessment for arsenic contaminated soil," Env. Geochem. Health, vol. 11, pp. 163-169, 1989.
[3] National Academy of Sciences, Arsenic-Medical and Biological Effects of Environmental Pollutants, U.S. Government Printing Office, DC: Washington, 1977.
[4] National Research Council, Arsenic in Drinking Water, National Academy Press, DC: Washington, 1999.
[5] F. N. Robertson, "Arsenic in ground-water under oxidizing conditions, south-west United States," Env. Geochem. Health, vol. 11, pp. 171-185, 1989.
[6] S. K. Gupta, and K. Y. Chen, "Arsenic removal by adsorption," J. of Water Poll. Cont. Federation, vol. 50(3), pp. 493-506, 1978.
[7] H. Marcussen, P. E. Holm1, L. T. Ha, and A. Dalsgaard, "Food safety aspects of toxic element accumulation in fish from wastewater-fed ponds in Hanoi, Vietnam," Trop. Med. Inter. Heal., vol. 12, no. 2, pp. 34-39, 2007.
[8] P. A. Amundsen, F. J. Staldvik, A. A. Lukin, N. A. Kashulin, O. A. Popova, and Y. S. Reshetnikov, "Heavy metal contamination in freshwater fish from the border region between Norway and Russia," Sc. Total Env., vol. 201, no. 3, pp. 211-24, 1997.
[9] L. Hollis, C. Hogstrand, and C. M. Wood, "Tissue-specific cadmium accumulation, metallothionein induction, and tissue zinc and copper levels during chronic sublethal cadmium exposure in juvenile rainbow trout," Arch. Env. Contamin. Toxicol., vol. 41, pp. 468-474, 2001.
[10] U.S.E.P.A., EPA Implements Standard of 10 ppb,, 2001.
[11] M. J. Brown, and J. N. Lester, "Role of bacterial extracellular polymers in metal uptake in pure bacterial culture and activated sludge-1 Effect of metal concentration," Wat. Res., vol. 16, p. pp. 1539, 1982.
[12] H. Min-sheng, P. Jing, and Z. Le-ping, "Removal of heavy metals from aqueous solutions using bacteria," J. Shanghai Univ., vol. 5 no. 3, pp. 253-259, 2001.
[13] T. Fenchel, and L. H. Kofoes, "Evidence for exploitative interspecific competition in mud snails," Oikos, vol. 27, pp. 367-376, 1976.
[14] J. Yingst, "The utilization of organic matter in shallow marine sediments by an epibenthic deposit feeding holothurian," J. Experim. Mar. Biol. Ecol., vol. 21, pp. 53-59, 1976.
[15] H. Sugita, T. Ishigaki, D. Iwai, Y. Suzuki, R. Okano, S. Matsuura, M. Asfie, E. Aono, and Y. Deguchi, "Antibacterial abilities of intestinal bacteria from three coastal fishes," Suisanzoshoku, vol. 46, pp. 563-568, 1998.
[16] H. Sugita, N. Matsuo, Y. Hirose, M. Iwato, and Y. Deguchi, "Vibrio sp. strain NM 10 with an inhibitory effect against Pasteurella piscicida from the intestine of Japanese coastal fish," Appl. Env. Microbio., vol. 63, pp. 4986-4989, 1997.
[17] H. Sugita, N. Matsuo, K. Shibuya, and Y. "Deguchi, Production of antibacterial substances by intestinal bacteria isolated from coastal crab and fish species," J. Mar. Biotech., vol. 4, pp. 220-223, 1996.
[18] H. Sugita, R. Okano, Y. Suzuki, D. Iwai, M. Mizukami, N. Akiyama, and S. Matsuura, "Antibacterial abilities of intestinal bacteria from larval and juvenile Japanese flounder against fish pathogens," Fish. Sc., vol. 68, pp. 1004-1011, 2002.
[19] T. Halttunen, P. Kankaanpää, R. Tahvonen, S. Salminen, and A.C. Ouwehand, "Cadmium removal by lactic acid bacteria," Bioscience Microflora, vol. 22, pp. 93-97, 2003.
[20] T. Halttunen, S. Salminen, and R. Tahvonen, "Rapid removal of lead and cadmium from water by speci.c lactic acid bacteria," Int. J. of Food Microbiol., vol. 114, pp. 30-35, 2007.
[21] T. Halttunen, S. Salminen, J. Meriluoto, R. Tahvonen, and K. Lertola, "Reversible surface binding of cadmium and lead by lactic acid and bifidobacteria," Inte. J. of Food Microbiol., vol. 125, pp. 170-175, 2008.
[22] M. Pierides, H. El-Nezami, K. Peltonen, S. Salminen, and J. Ahokas, "Ability of dairy strains of lactic acid bacteria to bind aflatoxin M1 in a food model," J. of Food Protection, vol. 63, pp. 645-650, 2000.
[23] J. Meriluoto, M. Gueimonde, C.A. Haskard, L. Spoof, O. Sjovall, and S. Salminen, "Removal of the cyanobacterial toxin microcystin-LR by human probiotics," Toxicon, vol. 46, pp. 111-114, 2005.
[24] S. M. K. Nybom, S. L. Salminen, and J. A. O. "Meriluoto, Removal of microcystin-LR by metabolically active probiotic bacteria," FEMS Microbiol. Lett., vol. 270, pp. 27-33, 2007.
[25] H. S. El-Nezami, N. N. Polychronaki, J. Ma, H. Zhu, W. Ling, E.K. Salminen, R. O. Juvonen, S. J. Salminen, T. Poussa, and H. M. Mykkänen, "Probiotic supplementation reduces a biomarker for increased risk of liver cancer in young men from Southern China," Am. J. of Clini. Nutr., vol. 83, pp. 1199-1203, 2006.
[26] K. Giller, E. Witter, and S. P. McGrath, "Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: a review," Soil B. Biochem., vol. 30, pp. 1389-1414, 1998.
[27] G. M. Gadd, "Heavy metal accumulation by bacteria and other microorganisms," Experientia, vol. 46, pp. 834-840, 1990.
[28] R. Idris, R. Trifonova, and M. Puschenreiter, "Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thaspi goesingense," Appl. Env. Microbiol., vol. 70, pp. 2667-2677, 2004.
[29] S. Erkkila, and E. Petaja, "Screening of commercial meat starter cultures at low pH in the presence of bile salts for potential probiotic use," J. Meat Sci., vol. 55, pp. 297-300, 2000.
[30] K. Arihara, H. Ota, M. Itoh, Y. Kondo, T. Sameshima, H. Yamanaka, M. Akimoto, S. Kanai, and T. Miki, "Lactobacillus acidophilus group lactic acid bacteria applied to meat fermentation," J. Food Sci., vol. 63, no. 3, pp. 544-547, 1998.
[31] J. L. Balcázar, D. Vendrell, I. de Blas, I. R.-Zarzuela, J. L. Muzquiz, and O. Girones, "Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish," Aquaculture, vol. 278, pp. 188-191, 2008.
[32] M. Pazirandeh, B. M. Wells, and R. L. Ryan, "Development of bacterium-based heavy metal biosorbents: Enhanced uptake of cadmium and mercury by Escherichia coli expressing a metal binding motif," Appl. Env. Microbio., vol. 64, no. 10, pp. 4068-4072, 1998.
[33] J. L. Ruiz-Barba, A. Maldonado, and R. Jiménez-D├¡az, "Small-scale total DNA extraction from bacteria and yeast for PCR applications," Anal. Biochem., vol. 347, pp. 333-335, 2005.
[34] B. Hyronimus, C. Le Marrec, A. Hadi Sassi, and A. Deschamps, "Acid and bile tolerance of spore-forming lactic acid bacteria," Int. J. Food Microbiol., vol. 61, pp. 193-197, 2000.
[35] C. Pennacchia, D. Ercolini, G. Blaiotta, O. Pepe, F. Mauriello, and F. Villani, "Selection of Lactobacillus strains from fermented sausages for their potential use as probiotics," J. Meat Sci., vol. 67, pp. 309-317, 2004.
[36] M. Succi, P. Tremonte, A. Reale, E. Sorrentino, L. Grazia, S. Pacifico, and R. Coppola, "Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese," FEMS Microbiol., vol. 244, pp. 129-137, 2005.
[37] J. Prasad, H. Gill, J. Smart, and P. K. Gopal, "Selection and characterization of Lactobacillus and Bifidobacterium strains for use as probiotics," Int. Dairy J., vol. 8, pp. 993-1002, 1998.
[38] V. Strompfova' and A. Laukova', "In vitro study on bacteriocin production of Enterococci associated with chickens," Anaerobe, vol. 13, pp. 228-237, 2007.
[39] S. E. Gilliland, T. E. Staley, and L. J. Bush, "Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct," J. of Dairy Sci., vol. 67, pp. 3045-3051, 1984.
[40] B. R. Goldin, S. L. Gorbach, M. Saxelin, S. Barakat, L. Gualtieri, and S. Salminen, "Survival of Lactobacillus species (strain GG) in human gastrointestinal tract," Digest. Disea. and Sci., vol. 37, pp. 121-128, 1992.
[41] S. Rengpipat, T. Rueangruklikhit, and S. Piyatiratitivorakul, "Evaluations of lactic acid bacteria as probiotics for juvenile seabass Lates calcarifer," Aqu. Res., vol. 39, pp. 134-143, 2008.
[42] S. E. Lindgren, and W.J. Dobrogosz, "Antagonistic activities of lactic acid bacteria in food and feed fermentations," FEMS Microbiol. Rev., vol. 87, pp. 149-163, 1990.
[43] N. Hwanhlem, N. Watthanasakphuban, S. Riebroy, S. Benjakul, A. HKittikun1, and S. Maneerat, "Probiotic lactic acid bacteria from Kung- Som: isolation, screening, inhibition of pathogenic bacteria," Int. J. of Food Sci. and Technol. vol. 45, pp. 594-601, 2010.
[44] C. Alvarado, A. B. E. Garcia, S. E. Martin, and C. Regalado, "Foodassociated lactic acid bacteria with antimicrobial potential from traditional Mexican foods," Revista Latinoamericana de Microbiologia, vol. 48, pp. 260-268, 2006.
[45] K. M. Sorrells, and M. L. Speck, "Inhibition of Salmonella gallinarum by culture filtrates of Leuconostoc citrovorum," J. of Dairy Sci., vol. 53, pp. 239-241, 1970.
[46] L. Gonza'lez, H. Sandoval, N. Sacrista'n, J. M. Castro, J. M. Fresno, and M. E. Tornadijo, "Identification of lactic acid bacteria isolated from Genestoso cheese throughout ripening and study of their antimicrobial activity," Food Contr., vol. 18, pp. 716-722, 2007.
[47] H. Qing, D. Min-Na, Q. Hong-Yan, X. Xiang-Ming, Z. Guo-Qiang, and Y. Min, "Detection, isolation, and identification of cadmium-resistant bacteria based on PCR-DGGE," J. Env. Sc., vol. 19, pp. 1114-1119, 2007.
[48] T. J. Foster, "Plasmid determined resistance to anti-microbial drugs and toxic metal ions in bacteria," Microbiol., vol. 47 pp. 361-409, 1983.
[49] P. W. Ramteke, "Plasmid mediated co-transfer of antibiotic resistance and heavy metal tolerance in coliforms," Ind. J. Microbiol., vol. 37, pp. 77-181, 1997.
[50] A. Anado'n M. R. MartÛ'nes-Larran˜aga, and M. A. MartÛ'nes, "Probiotic for animal nutrition in the European Union, Regulation and safety assessment," Regul. Toxicol. Pharmacol., vol. 45, pp. 91-95, 2006.