Stepanov et al. BMC Microbiology 2014, 14:225 http://www.biomedcentral.com/1471-2180/14/225 RESEARCH ARTICLE Open Access The presence of nitrate dramatically changed the predominant microbial community in perchlorate degrading cultures under saline conditions Victor G Stepanov2†, Yeyuan Xiao1†, Quyen Tran2, Mark Rojas3, Richard C Willson2,4,5, Yuriy Fofanov3, George E Fox2,4 and Deborah J Roberts1* Abstract Background: Perchlorate contamination has been detected in both ground water and drinking water. An attractive treatment option is the use of ion-exchange to remove and concentrate perchlorate in brine. Biological treatment can subsequently remove the perchlorate from the brine. When nitrate is present, it will also be concentrated in the brine and must also be removed by biological treatment. The primary objective was to obtain an in-depth characterization of the microbial populations of two salt-tolerant cultures each of which is capable of metabolizing perchlorate. The cultures were derived from a single ancestral culture and have been maintained in the laboratory for more than 10 years. One culture was fed perchlorate only, while the other was fed both perchlorate and nitrate. Results: A metagenomic characterization was performed using Illumina DNA sequencing technology, and the 16S rDNA of several pure strains isolated from the mixed cultures were sequenced. In the absence of nitrate, members of the Rhodobacteraceae constituted the prevailing taxonomic group. Second in abundance were the Rhodocyclaceae. In the nitrate fed culture, the Rhodobacteraceae are essentially absent. They are replaced by a major expansion of the Rhodocyclaceae and the emergence of the Alteromonadaceae as a significant community member. Gene sequences exhibiting significant homology to known perchlorate and nitrate reduction enzymes were found in both cultures. Conclusions: The structure of the two microbial ecosystems of interest has been established and some representative strains obtained in pure culture. The results illustrate that under favorable conditions a group of organisms can readily dominate an ecosystem and yet be effectively eliminated when their advantage is lost. Almost all known perchlorate-reducing organisms can also effectively reduce nitrate. This is certainly not the case for the Rhodobacteraceae that were found to dominate in the absence of nitrate, but effectively disappeared in its presence. This study is significant in that it reveals the existence of a novel group of organisms that play a role in the reduction of perchlorate under saline conditions. These Rhodobacteraceae especially, as well as other organisms present in these communities may be a promising source of unique salt-tolerant enzymes for perchlorate reduction. Keywords: Perchlorate contamination, Nitrate, Metagenomic sequencing, Community analysis, Salt-tolerance * Correspondence: [email protected] †Equal contributors 1School of Engineering, University of British Columbia, EME 4261, 3333 University Way, Kelowna, BC, Canada Full list of author information is available at the end of the article © 2014 Stepanov et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stepanov et al. BMC Microbiology 2014, 14:225 Page 2 of 10 http://www.biomedcentral.com/1471-2180/14/225 Background rRNA genes of several strains that have been isolated The perchlorate ion has been found to contaminate from the cultures and successfully cultivated in the la- ground and surface water, as well as food, vitamins, and boratory. The combination of these two approaches pro- drinking water [1-5]. Perchlorate is known to inhibit the vides new insight into the major and minor components sodium-iodide symporter, which is responsible for the of the cultures and their contribution to perchlorate and supply of iodine to the thyroid and to milk in the mam- nitrate removal from contaminated water. mary glands [6]. The environmentally stable perchlorate ion can be re- Results moved from contaminated water using ion-exchange in Pure culture isolation combination with a microbial treatment processes. Dur- Four pure strains were obtained using anaerobic incuba- ing bacterial respiration perchlorate acts as an electron tions: NW (white colonies) and NY (yellow colonies), acceptor and requires the addition of an electron donor originated from NP30; PW (white colonies) and PY (yel- [7-9]. Perchlorate is metabolized through a series of re- low colonies), originated from P30. Another four pure ductions to chlorite and then a dismutation to chloride strains were obtained from aerobically incubated plates and molecular oxygen [10]. In many cases, perchlorate is and designated as NWO, NYO, PWO and PYO follow- present in very low concentrations and must be treated ing the naming scheme for anaerobic isolates. When to even lower levels, e.g., 6 ppb in California [11], and these new strains were transferred back into P medium, 2 ppb in Massachusetts [12]. This makes biological treat- none of them could reduce perchlorate after 120 days’ ment very difficult as a stand-alone process since other incubation, although growth was observed. A single add- electron acceptors such as nitrate and oxygen are often itional isolate that can reduce perchlorate was obtained preferred. from the NP30 culture and characterized in detail as re- Ion-exchange processes have been used to remove per- ported elsewhere [24]. This strain was identified as Marino- chlorate to meet the required treatment goals, but the bacter sp. strain P4B1 a member of the Alteromonadaceae. perchlorate and other co-contaminant anions such as ni- 16S rDNA sequences were obtained for all the pure strains trate are merely transferred to a concentrated brine except the NW isolate, which produced heterogeneous se- stream, which in turn must be treated [13,14]. The com- quencing patterns. Table 1 presents a summary of the re- bination of ion-exchange to remove and concentrate the sults obtained by comparing the sequences obtained for perchlorate from the contaminated water source and the pure cultures with those from the NCBI nt database. biological treatment to remove the perchlorate from the All belong to the phylum Proteobacteria. Two of the brine is an attractive option for drinking water treat- NP30 strains were identified as members of the Rhodobac- ment. Since nitrate is often present with perchlorate and teraceae closely related to Stappia indica.Theother will also be concentrated in the ion-exchange brine, the was an Azoarcus like isolate close to the members of a re- biological system should also remove nitrate. cently discovered and vaguely defined genus Denitromo- The cultures examined here were initially enriched nas (Rhodocyclaceae). Three of the five P30 isolates were from marine sediment in the year 2001. The culture des- identified as Hyphomonas jannaschiana. The remaining ignated as P30 has been maintained continuously in the P30 isolate was identified as a Cellulomonas strain that be- laboratory in a defined 3% NaCl saline medium with longs to the Cellulomonadaceae and is closely related to perchlorate and acetate as the sole electron acceptor and the C. hominis type strain. donor respectively in a 1.5 L batch reactor [15]. In 2003, half of the P30 culture was transferred to a new reactor Comparative metagenomic analysis and fed with both nitrate and perchlorate as the electron Illumina sequencing of the two cultures yielded approxi- acceptors and acetate as the sole electron donor. This mately 7.5 million reads for P30 and 6.3 million for culture was designated as NP30. The cultures have been NP30. Table 2 summarizes the assembly of the metage- used as the basis for many published studies over the nomic contigs. The data analysis strategy yielded conver- last 10 years [13,15-23]. ging estimates for the taxonomic composition of the P30 In order to better understand these cultures, an initial and NP30 microbial communities at the family level and characterization of the microbial populations was ob- above (Additional files 1, 2 and 3). As shown in Figure 1, tained by cloning 16S rDNA genes and characterizing both mixed cultures were found to be dominated by them by either DGGE or 16S rDNA sequencing [21]. In Alpha, Beta, and Gamma Proteobacteria, while the pro- the work reported here, we further our understanding of portion of other phyla is substantially lower. As shown the biota in these cultures by characterizing the total in Figure 2, Alphaproteobacteria from the family Rhodo- community DNA of both the P30 and NP30 cultures bacteraceae constitute a prevailing taxonomic group in using Illumina DNA sequencing technology. This meta- the P30 culture (Figure 2, Additional file 4). The second genome analysis is supplemented by sequencing the 16S most abundant fraction is formed by Betaproteobacteria Stepanov et al. BMC Microbiology 2014, 14:225 Page 3 of 10 http://www.biomedcentral.com/1471-2180/14/225 Table 1 Sequencing summary for 16S rDNA from pure cultures ID Origin Accession Length Closest BLAST match in NCBI nt database*) Identity Bit Taxonomic affiliation number (bp) (%) score (excluding uncultured/environmental