Long-term surveillance of sulfate-reducing bacteria in highly saline industrial wastewater evaporation ponds
© Ben-Dov et al; licensee BioMed Central Ltd. 2009
Received: 11 December 2008
Accepted: 18 February 2009
Published: 18 February 2009
Abundance and seasonal dynamics of sulfate-reducing bacteria (SRB), in general, and of extreme halophilic SRB (belonging to Desulfocella halophila) in particular, were examined in highly saline industrial wastewater evaporation ponds over a forty one month period. Industrial wastewater was sampled and the presence of SRB was determined by quantitative real-time PCR (qPCR) with a set of primers designed to amplify the dissimilatory sulfite reductase (dsrA) gene. SRB displayed higher abundance during the summer (106–108 targets ml-1) and lower abundance from the autumn-spring (103–105 targets ml-1). However, addition of concentrated dissolved organic matter into the evaporation ponds during winter immediately resulted in a proliferation of SRB, despite the lower wastewater temperature (12–14°C). These results indicate that the qPCR approach can be used for rapid measurement of SRB to provide valuable information about the abundance of SRB in harsh environments, such as highly saline industrial wastewaters. Low level of H2S has been maintained over five years, which indicates a possible inhibition of SRB activity, following artificial salination (≈16% w/v of NaCl) of wastewater evaporation ponds, despite SRB reproduction being detected by qPCR.
Sulfate-reducing bacteria (SRB) are anaerobic microorganisms that use sulfate as an electron acceptor. They are known to grow both heterotrophically, relying on small organic molecules, and autotrophically, using H2 as the electron donor and CO2 as the carbon source . However, several studies have demonstrated that certain species of SRB are not only able to tolerate high concentrations of oxygen but can also utilize oxygen as a terminal electron acceptor . SRBs are known to be present in the complex consortia of microorganisms involved in the anaerobic digestion processes used in municipal and industrial wastewater treatment. Furthermore, sulfate reduction may account for up to 50% of the mineralization of organic matter in aerobic wastewater treatment systems . A major drawback of sulfate reduction in wastewater treatment is the production of the toxic odorant H2S, which in addition, is an agent that significantly enhances microbially-mediated corrosion of treatment facilities . This is especially true in the oil industry, where sulfate reduction causes severe problems, including souring of oil and gas deposits .
Dissimilatory sulfate reduction occurs up to quite high salt concentrations. Black sediments are often found on the bottom of salt lakes and saltern ponds approaching NaCl saturation [5, 6]. Some culturable halophilic sulfate reducers, such as Desulfovibrio halophilus, Desulfocella halophila, Desulfovibrio oxyclinae and Desulfohalobium retbaense, may grow from 18 up to 24% NaCl concentration at the upper limit [7–10]. The dsrAB genes which encode dissimilatory sulfite reductase, the key enzyme in dissimilatory sulfate reduction, can be used as a phylogenetic marker for identification of SRB . These genes are found in all known sulfate-reducing prokaryotes . Thus, dsrAB from all sulfate-reducing lineages can be targeted by a single set of conserved primers or using specific primers from variable regions of dsrAB.
In this study, a pair of universal PCR primers for the functional gene dsrA  and two specific sets of primers for dsrA of halophilic SRB, were designed and used for long-term surveillance of SRB populations within five highly saline industrial wastewater evaporation ponds by quantitative real-time PCR (qPCR). These ponds are the final treatment stage of a combined wastewater stream contributed to by several chemical plants (manufacturing various pesticides, pharmaceuticals, aliphatic and aromatic halogens) at the Ramat-Hovav industrial park in the Negev desert, Israel . Organic matter concentration in the wastewater stream is 2–2.5 gC/l (on the basis of total organic carbon measure), of which over 30% reaches the evaporation ponds. Receiving a mixture of saline, high strength industrial wastewater, these ponds offer a unique habitat for various microorganisms . In order to reduce the foul odors emitted by the ponds and, in particular, the H2S that results from SRB activity, salinity of the evaporation ponds was artificially raised (August–October 2003) from an initial 3–7% to a final concentration of about 12% (w/v) by addition of NaCl .
Total genomic DNA of wastewater samples was extracted  from obtained pellets (derived from 30 ml samples) using the MoBio Power Soil DNA isolation kit (MoBio Laboratories, Solana Beach, CA). The pair of PCR primers (DSR1F and RH3-dsr-R) that specifically detect and quantify SRB was used as previously described . The measured values were transformed to targets per milliliter of wastewater.
Oligonucleotides used for real-time PCR to amplify halophilic dsrA genes closely related to Desulfocella halophila.
Primer binding sitec
Product size (bp)
quantitative real-time PCR
- dsrA :
dissimilatory sulfIte reductase
weight per volume.
This project was supported by a grant from the Ramat-Hovav Industrial Council, Israel and BMBF-MOST Cooperation in Water Technologies Grant WT-501. Special thanks are conveyed to the management and staff of the Ramat-Hovav Council for their cooperation. The authors thank Larissa Shemtov, Lisa Arkhangelsky, Ronnen Veitsman, Nachshon Siboni and Orr Shapiro, for technical support and useful comments on the manuscript.
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