Microbial community in packed bed bioreactor ... - Wiley Online Library

Environment Health Techniques 198

Madhusmita Mishra et al.

Short Communication Microbial community in packed bed bioreactor involved in nitrate remediation from low level radioactive waste Madhusmita Mishra1, Savita Jain2, Ashoke Ranjan Thakur3 and Shaon RayChaudhuri1 1

2

3

Department of Biotechnology, West Bengal University of Technology, BF-142, Sector-1, Saltlake, Calcutta, West Bengal, India Waste Management Division, Bhabha Atomic Research Centre, Anusakti Nagar, Trombay, Mumbai, Maharashtra, India Vice Chancellor’s Office, Techno India University, EM4/1, sector V, Salt Lake, Kolkata, West Bengal, India

Nitrate is the second largest contaminant of agriculture soil after pesticides. It also is a major pollutant from nuclear and metallurgical operations. Conventional methods for nitrate removal suffers from high cost and complexity leaving bioremediation as a viable alternative strategy. A pilot plant of 2.5 m3/day capacity has been functioning since 2005 based on microbial consortia treating actual effluent from nuclear power plant having pH of 7–8.5 (optimum) with N:C ratio of 1:1.7. The maximum biodegradable nitrate concentration of 3000 ppm could be reduced to below permissible limit (44.2 ppm) within 24 h in presence of sodium acetate as carbon source. Culture independent analysis (16S rDNA based) revealed clones having closest identity with uncultured bacterium, Pseudomonas stutzeri and Azoarcus sp. The existence of dissimilatory pathway of nitrate reduction in the community DNA is indicated by presence of nirS and nirK gene. Though the microbial mass was developed using municipal sewage, absence of Mycobacterium sp was confirmed using PCR. The understanding of the molecular identification of the consortium would help in developing the preservation strategy of the microbial mass for replication and perpetuation of the system. Keywords: Community analysis / Nitrate removal / Bioremediation / Culture independent approach Received: November 6, 2012; accepted: December 8, 2012 DOI 10.1002/jobm.201200676

Introduction Large amount of nitrate ions containing aqueous solution is generated from fertilizer, nuclear, and metallurgical [1– 5] industries due to use of nitric acid. The accumulation of nitrates in the environment results from the over application of nitrogenous fertilizers of which only a small fraction is utilized while the rest is washed in agricultural runoff. In nuclear industry, the plants handling uranium and plutonium generate radioactive aqueous waste in various volume and concentration. The waste generated from the nuclear industry may contain nitrate as a result of use of nitric acid in addition to radioCorrespondence: Shaon RayChaudhuri, Department of Biotechnology, West Bengal University of Technology, BF-142, Sector-1, Salt Lake, Calcutta 700064, West Bengal, India E-mail: [email protected] Phone: 00913323210731 Ext 108 Fax: 00913323341030 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

nuclides. To minimize the concentration of nitrates in the effluents, various processes are under development for conversion of nitrate to nitrogen. Nitrate pollution is reported from paddy as well as tea cultivation where the nitrate leaching causes acidification of soil and increases the concentration of nitrate level in ground water [6]. Nitrate mobility or leaching is responsible for contaminating water bodies. Mobility in soil is also dependent on type of soil (clay or sandy) and slope of cultivable land [7]. When it leaches into the marine environment, it triggers plankton bloom which in turn results in local increase of BOD, a process that decreases the available oxygen level and thus kills the preexisting flora and fauna in those regions [8]. Nitrate is also a potential human health threat, especially to infants, causing the condition known as methemoglobinemia, also called “blue baby syndrome”. Central Nervous System and Cardiovascular System may also be affected while it poses to be carcinogenic.

www.jbm-journal.com

J. Basic Microbiol. 2014, 54, 198–203

Culture independent analysis of nitrate reducing microbial community

According to Environmental Protection Agency, the maximum contaminant level in ground water has been demarcated as 10 mg/L for NO3N and 45 mg/L for NO3 concentration. Conventional methods like distillation, reverse osmosis and ion exchange are not suitable for nitrate removal because of their complexity and cost of operation. One of the more advanced technologies would be the bioremediation [9–10]. Nitrate reducing bacteria (NRB) are a group of microbes that use nitrate as a nutrient. NRB can either use it for the synthesis of amino acids (assimilatory) or as a terminal electron acceptor in their respiratory mechanism to generate molecular nitrogen (dissimilatory). The later is known as denitrifiers which are helping to regenerate nitrogen gas and thus is an integral part of the nitrogen cycle [11]. The diversity of denitrifiers is marked from all main phyla, represented by heterotrophs like Paracoccus dentrificans, Pseudomonads; autotrophic bacteria like Thiobacillus

199

denitrificans. Other than denitrification, another significant pathway is reported to be involved in dissimilatory nitrate reduction to ammonia (DNRA) [12]. Gene clusters like nasCA, narB, nasB, nirA are necessary for assimilatory nitrate reduction where as narGHI, nirS, nirK, norC, norB, nosZ are responsible for dissimilatory or denitrification pathway [11]. DNRA is governed by nrf genes. The wide diversity of the group implies application of the culture independent technique to explore the existing population. Aim of this study is to focus on identifying bacterial population thriving in the packed bed reactor since 2005. Culture independent approach was used for this purpose since only 1–4% of the microbes could be cultivated under standard cultivable condition [13]. By way of understanding the underlying mechanism, the molecular identifiers for nitrate reduction pathway was assessed. The occupational hazard, if any, was identified.

Table 1. Table depicting the molecular characterization of the nitrate reducing microbial community based on 16S rDNA analysis. Clone no.

GenBank accession no.

Maximum similarity with organism

% identity

1 2 3 4 5 6 7 8 9 10

GQ849493 GQ849494 GQ849495 GQ849496 GQ849497 GQ849498 GQ849499 GQ849500 GQ979941 GQ979942

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

GQ979943 GQ979944 GQ979945 GQ979946 GQ979947 GQ979948 GQ979949 GQ979950 GQ979951 GQ979952 GQ979953 GQ979954 GQ979955 GQ979956 GQ979957 GQ979958 GQ979959 GQ979960 GQ979961 GQ979962 GQ979963 GQ979964 GQ979965

Pseudomonas stutzeri 13636J Uncultured bacterium clone O7 Pseudomonas stutzeri A1501 Azoarcus sp. NSC3 Uncultured bacterium H2SRC229x Uncultured bacterium clone O7 Uncultured bacterium clone HB99 Uncultured Azoarcus sp. Pseudomonas stutzeri A1501 Uncultured bacterium clone O7Pseudomonas sp. 13636J Pseudomonas stutzeri A1501 Azoarcus sp. NSC3 Uncultured bacterium H2SRC229x Uncultured bacterium clone O7 Uncultured bacterium clone HB99 Azoarcus sp. NSC3 Pseudomonas stutzeri A1501 Pseudomonas stutzeri A1501 Uncultured bacterium clone O7 Uncultured bacterium clone O7 Azoarcus sp. NSC3 Azoarcus sp. NSC3 Uncultured bacterium clone O7 Uncultured bacterium clone O7 Azoarcus sp. NSC3 Azoarcus sp. NSC3 Uncultured bacterium H2SRC229x Uncultured bacterium H2SRC229x Uncultured bacterium HB99 Uncultured bacterium HB99 Azoarcus sp. NSC3 Azoarcus sp. NSC3 Azoarcus sp. NSC3

99 98 99 95 92 98 96 95 99 98 97 99 95 92 98 96 99 99 99 98 98 95 95 93 93 99 99 92 92 96 96 99 97 97

The molecular analysis was based on culture independent approach. The 33 novel sequences were subjected to BLAST N analysis and the identity was deciphered on the basis of the closest neighbor within the existing database showing maximum % of identity. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

www.jbm-journal.com


200


Materials and methods Source of microbes Municipal sewage was passed through the perforated corrugated sheets of a packed bed reactor at Waste Management Department (WMD) of Bhabha Atomic Research Centre, Mumbai, India for 7 days. The consortium sticking to the solid matrix was allowed to stabilize for 6 h before passing the solution containing sodium acetate as a carbon source. The denitrification reaction occurs as follows: 8NO3 þ 5CH3 COONa ! 4N2 þ 10CO2 þ H2 O þ 5NaOH þ 8OH The consortium so developed has been removing nitrate from radioactive waste since 2005. The stabilized microbial consortium was used for community analysis using culture independent technique in 2009.

Community analysis Community DNA was isolated from microbial biomass according to modified alkaline lysis method [14] followed by PCR amplification of 16S rDNA using degenerate universal primer. The PCR conditions include initial denaturation for 2 min at 92°C followed by 40 cycles of 1 min at 92°C, 1 min at 50°C, and 2 min at 72°C. Further steps in molecular characterization involved TA cloning (as per manufacturer’s protocol) and partial sequencing of 16S rDNA gene sequence. Novel sequences were submitted to GenBank after BLAST analysis. The phylogenetic analysis was done by Neighbor Joining method. The rarefaction curve was constructed as per earlier studies [14]. The richness and evenness of diversity was calculated as per standard methods [15] of Shannon diversity index and equitability index, respectively.

Figure 1. a: Phylogenetic analysis based on neighbor joining method depicting the molecular identity of one of the isolates of the bacterial population within the nitrate reducing microbial community. These sequences were obtained by culture independent approach where the community DNA was isolated from the microbial biomass on the corrugated sheet followed by amplification, cloning, and partial sequencing of 16Sr DNA gene sequence. The branch lengths were provided at the bottom of the tree. b: Rarefaction curve for the different bacterial species within the nitrate reducing community. The number of OTUs observed within the 16S rDNA gene sequence library indicated that there was saturation of species diversity in the study. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

www.jbm-journal.com



Detection of nirS and nirK gene Since most of the microbes detected during community analysis are novel, the associated pathway for nitrate removal was analyzed using molecular tools. To detect the presence of denitrification process operating within the consortium, two nitrate reductase genes, i.e., nirS and nirK were checked by using specific primers reported earlier [16] and amplification reactions were performed in a thermo cycler (Biorad MyCycler ThermoCycler). The nirS and nirK were amplified by using touchdown PCR. The protocol for amplification of nirS and nirk were as follows: annealing temperature starting at 54°C and decreasing 0.5°C each cycle until an annealing temperature of 49°C was reached, followed by 25 cycles at 52°C; annealing temperature starting at 56°C and decreasing 0.5°C each cycle until an annealing temperature of 51°C was reached, followed by 25 cycles at 54°C, respectively. The amplicons were checked on 2% agarose gel.

201

diversity value was calculated to be 1.03 which indicates existence of rich microbial diversity within the nitrate reducing consortium. The equitability index value was 0.92 which was close to 1. This explains that the different varieties observed were evenly distributed within the community. Thus, the above study reflects a rich microbial diversity associated with nitrate reduction from radioactive waste.

Detection of Mycobacterium sp. within the microbial community The consortium in the bioreactor was selectively enriched from municipal sewage. Its origin raises concern about the occupational hazard of workers handling the treatment plant. To check for the presence of Mycobacterium within the population, specific primers reported to target the IS61 10 gene of Mycobacterium genus were used as reported by Mustafa et al. [17] and the community DNA obtained from the microbial biomass was used as a template. Genomic DNA from a known Mycobacterium species was used as a positive control.

Results and discussion The 16S rDNA gene sequence analysis revealed the diversity of population within the biomass obtained from the corrugated sheet in the nitrate reducing bioreactor. Thirty-three novel sequences were obtained in this study. The BLAST analysis provided the identity of closest matching strains and the percentage of identity with them (Table 1). The closest neighbors like Pseudomonas stutzeri, Pseudomonas sp., and Azoarcus sp. were already known to be involved in nitrate reduction [18–21]. All the sequences were found to be novel and they were submitted to GenBank (Table 1). The relationship among the isolate and those of the type strains within the family is presented in the form of a phylogenetic tree (Fig. 1a). The number of clones was plotted against the number of operational taxonomic units (OTUs) to generate the rarefaction curve (Fig. 1b) to understand the saturation of screening of the microbial community. The Shannon ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

Figure 2. Two percent agarose gel photograph representing the following amplified products: (a) nirS (890 bp) and nirK gene (514 bp) and (b) Mycobacterium sp. specific gene (IS 16 10). M represents 500 bp DNA molecular weight marker for DNA (Fermentus) where as CN represents community DNA of microbial biomass obtained from corrugated sheet. MS represents the DNA from known Mycobacterium sp. and NS was the negative control where DNA template was absent. Distinct PCR product of 580 bp was observed in case of known Mycobacterium sp whereas no amplification was there in case of the nitrate reducing community.

www.jbm-journal.com


202


The agarose gel depicted in Fig. 2b shows the presence of nirS (890 bp) and nirK (514 bp) gene in the community DNA isolated from the microbial consortium indicating the existence of dissimilarity process of nitrate reduction. The same DNA failed to show amplification (Fig. 2a) of Mycobacterium specific gene whereas for the known Mycobacterium sp., distinct amplicon of 580 bp size was observed. This indicates that in spite of its origin from the municipal sewage, there is no risk of Mycobacterium infection from this bioreactor.

[3] Yong, L., Zhang, J., 1999. Agricultural diffuse pollution from fertilizers and pesticides in China. Water Sci. Technol., 39, 25–32. [4] Harrington, C.D., Ruchle, A.E., 1959. Uranium Production Technology, D. Van Nostrand Company, Princeton, NJ. [5] Benedict, M., Pigfond, T., Levi, H., 1981. Nuclear Chemical Engineering, 2nd edn., McGraw-Hill, New York. [6] Nakasone, H., Yamamoto, T., 2004. The impacts of the water quality of the inflow water from tea fields on irrigation reservoir ecosystems. Paddy Water Environ., 2, 45–50.

Conclusions The selective enrichment of the efficient nitrate reducing microbes from the biomass was achieved. The molecular characterization of the biomass showed 13 different varieties. The Shannon diversity and equitability index values were calculated to be 1.03 and 0.92 which indicates that microbial diversity is rich and evenly distributed within the nitrate reducing consortium. The presence of nirS and nirK genes reflects the presence of denitrifiers within the nitrate reducing consortium; presence of this kind of consortium is beneficial as it produce molecular nitrogen from nitrate instead of ammonium. The possibility of presence of pathogenic bacteria like Mycobacterium within the microbial community would be hazardous for human beings. Therefore, the presence of these groups was checked by molecular approach using specific primers which were reported to target the IS61 10 gene of Mycobacterium species. It was found from the PCR analysis that the current consortium is free from tuberculosis causing germs. On the basis of the above result it could be stated that the bioreactor consortium is non-hazardous from human health and ecological point of view.

Acknowledgments The authors would like to acknowledge the financial assistance of Department of Atomic Energy under the BRNS scheme. They are thankful to Dr. L.M. Gantayet of BARC for initiating the microbial analysis study and his constant support as well as intellectual input to the research group.

References [1] Burkart, M.R., Kolpin, D.W., James, D.E., 1999. Assessing groundwater vulnerability to agrichemical contamination in the Midwest US. Water Sci. Technol., 39, 103–112.

ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

[2] Giupponi, C., Rosato, P., 1999. Agricultural tural land use changes and water quality: a case study in the water shed of the lagoon of Venice. Water Sci. Technol., 39, 135– 148.

[7] Chong, K.P., Ho, T.Y., Jalloh, M.B., 2008. Soil nitrogen phosphorus and tea leaf growth in organic and conventional farming of selected fields at Sabah Tea Plantation Slope. J. Sust. Dev., 1, 117–122. [8] Thorburn, P.J., Biggs, J.S., Weier, K.L., Keating, B.A., 2003. Nitrate in groundwater of intensive agricultural areas in coastal Northeastern Australia. Agric. Ecosyst. Environ., 94, 49–58. [9] Pinar, G., Duque, E., Haidour, A., Oliva, J. et al., 1997. Removal of high concentrations of nitrate from industrial wastewaters by Bacteria. Appl. Environ. Microbiol., 63, 2071–2073. [10] Awadallah, R.M., Soltan, M.E., Shabeb, M.S.A., Moalla, S.M. N., 1998. Bacterial removal of nitrate, nitrite and sulphate in wastewater. Water Res., 32, 3080–3084. [11] Moreno-Vivian, C., Cabello, P., Martinez-Luque, M., Blasco, R., Castillo, F., 1999. Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases. J. Bacteriol., 181, 6573– 6584. [12] Shailaja, M.S., Navekar, P.V., Alagarsamy R., Naqvi, S.W.A., 2006. Nitrogen transformations as inferred from the activities of key enzymes in Arabian sea oxygen minimum zone. Deep-Sea Res., 53, 960–970. [13] Amann, R.I., Ludwig, W., Schleifer, KH., 1995. Phylogenetic identification and in situ detection individual microbial cells without cultivation. Microbiol. Rev., 59, 143– 169. [14] RayChaudhuri, S., Thakur, A.R., 2006. Microbial DNA extraction from sample of varied origin. Curr. Sci., 12, 1697–1700. [15] Nasipuri, P., Pandit, G.G., Thakur, A.R., RayChaudhuri, S., 2010. Comparative study of soluble sulfate reduction by acterial consortia from varied regions of India. Am. J. Environ. Sci., 6(2), 152–158. [16] Balbina, N., Kenneth, N.T., David, B.N., Osborn, A.M., 2002. Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription PCR amplification from mRNA. Appl. Environ. Microbiol., 68, 5017–5025. [17] Mustafa, A.S., Abal, A.T., Chugh, T.D., 1999. Detection of Mycobacterium tuberculosis complex and nontuberculous Mycobacteria by multiplex polymerase chain reactions. East. Mediterr. Health J., 5, 61–70.

www.jbm-journal.com



[18] Strohm, T.O., Griffin, B., Zumft, W.G., Schink, B., 2007. Growth yields in bacterial denitrification and nitrate ammonification. Appl. Environ. Microbiol., 73, 1420– 1424. [19] Li, A., Gai, Z., Cui, D., Ma, F. et al., 2012. Genome sequence of a highly efficient aerobic denitrifying bacterium, Pseudomonas stutzeri T13. J. Bacteriol., 194, 5720.

ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

203

[20] Jormakka, M., Richardson, D., Byrne, B., Iwata, S., 2004. Architecture of NarGH reveals a structural classification of Mo-bisMGD enzymes. Structure, 12, 95–104. [21] Krause, A., Ramakumar, A., Bartels, D., Battistoni, F. et al., 2006. Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72. Nat. Biotechnol., 24, 1385–1391.

www.jbm-journal.com
