Occurrence of sulfate-reducing bacteria in well water: identification of anaerobic sulfidogenic bacterial enrichment cultures

While approximately 97% of the fresh water on earth consists of groundwater, the remainder is composed of lakes, rivers, wetlands, and soil moisture (Quevauviller 2007). Among these freshwater sources, a tiny fraction (1%) is readily accessible to people (Vörösmarty et al. 2005). For this reason, different types of technologies have been developed to increase water availability such as digging wells for groundwater harvesting.

The microbial content of groundwater ecosystems including viruses, bacteria, archaea, protozoa, and fungi is relatively diverse (Griebler & Lueders 2009). The presence and activities of some microbial groups could be problematic with respect to water quality. Many countries consider the World Health Organization (WHO) Guidelines for setting national drinking water quality regulations and standards (WHO 2017). Groundwater management approaches focus on the entry of microorganisms into the groundwater sources. In this context, contamination of groundwater by pathogenic microorganisms is of particular concern because they are potentially harmful to human health, leading to outbreaks of waterborne diseases not only worldwide but also in Türkiye. A total of 14 waterborne outbreaks were reported in Türkiye between 2010 and 2020 (Akgül et al. 2023). These outbreaks include a gastroenteritis outbreak, affecting more than 1,000 people, which occurred due to bacterial (Shigella) and viral contamination of groundwater (Sezen et al. 2015). Unfortunately, very limited studies dealt with such microbial quality of groundwater in Türkiye.

Common bacterial pathogens distributed by the fecal–oral route include species of Vibrio, Shigella, and Salmonella. In this respect, fecal coliform bacteria (mainly Escherichia coli and enterococci) are used as water quality indicator organisms for fecal contamination and the possible presence of other pathogens in groundwater (Krauss & Griebler 2011). The microbiological quality of groundwater in Türkiye has also been assessed by this approach. The detected bacteria in groundwater, located in different geographical regions of Türkiye, were reported as E. coli, Enterococcus spp., Fecal streptococci, Salmonella sp., Staphylococcus spp., and Pseudomonas aeruginosa (Özler & Aydın 2008; Yolcubal et al. 2016; Gunes 2023). However, the absence of coliform bacteria in the water may not always indicate a safe water supply for humans. For instance, E. coli, proposed as the best indicator, becomes inactivated in chlorinated water, while the most resistant pathogens may survive for several hours. At this point, obligately anaerobic Clostridium perfringens is used as a water quality indicator, because C. perfringens spores are less affected by the chlorine (Cabral 2010). In addition to being resistant to water treatment, the longevity of spores makes C. perfringens a useful indicator for remote fecal contamination (Stelma 2018). In contrast to traditional aerobic indicators (E. coli and enterococci), apart from C. perfringens, different fecal anaerobes in the healthy gut microbiome may also be more reliable candidates for alternative indicators in groundwater because their survival is directly linked to the anaerobic environment in the gut (McLellan & Eren 2014).

Anaerobic bacteria are the residents of the groundwater environment, such as deep aquifers (Griebler & Lueders 2009). Due to the low redox potential of the water and the low presence or absence of oxygen in the water, anaerobiosis is highly favored over aerobiosis in an anaerobic zone of the water well. For instance, anaerobic Desulfovibrio africanus, a sulfate-reducing bacterium, was reported to be isolated from well water (Campbell et al. 1966). Anoxic subsurface of aquatic environments such as groundwater are typical habitats of sulfate-reducing bacteria (SRB) (Miao et al. 2012). However, the presence and activity of SRB may also influence well water quality and safety negatively, potentially leading to serious problems.

In anoxic well water environments, SRB use sulfate as an electron acceptor and generate hydrogen sulfide (H₂S), an acidic and toxic product. Released H₂S cause esthetic problems such as ‘rotten egg’ taste and odor in well water (Cullimore 1999). Exposure to high concentrations of H₂S gas or prolonged exposure at low concentrations may pose a great danger in terms of the health and safety of the people who use the well water for domestic purposes (Chou 2003). In addition, this gas can react with iron to generate iron sulfide (FeS) deposits in well water systems that causes colored water problems. The formation of SRB biofilms on the surface of metals may lead to clogging of the well and also corrosion of ferrous pipes or other materials in the well water system (Cullimore 1999). The occurrence of SRB in the human intestine has been recognized for a long time (Macfarlane et al. 2007). Although intestinal SRB are not considered as direct pathogens, recent studies suggest that SRB are associated with health problems, such as sepsis, liver abscess, and inflammatory bowel diseases (IBD) (e.g., ulcerative colitis) (Goldstein et al. 2003; Koyano et al. 2015; Kushkevych et al. 2020). Therefore, SRB should be evaluated as possible agents, and moreover, SRB detection in well water may be carried out routinely. For this reason, this study was first aimed to detect and identify SRB in well water. In the literature, the studies about the detection of SRB in groundwater were performed by only culture-independent molecular techniques or cultivation methods (Wargin et al. 2007; Keesari et al. 2015; Yang et al. 2015; An et al. 2016). However, in this study, the composition of the anaerobic bacterial community composed of SRB in sulfate-reducing enrichments obtained from well water samples was investigated using culture-dependent molecular analysis.

Apart from commonly used anaerobes, anaerobic partners of SRB can also be proposed as an alternative fecal contamination anaerobic indicator in water. In this respect, secondly, this study focused on the anaerobic bacteria that coexist with SRB in well water. Thus, the knowledge of anaerobic bacteria coexisting with SRB may be used for SRB indicators in well water. To the best of our knowledge, in this study, SRB and anaerobic bacteria coexisting with SRB in the enrichment culture obtained from a well water sample were identified for the first time.

Four mL of water samples were inoculated into 36 mL of Postgate's B (PB) medium under anaerobic conditions for obtaining SRB enrichment (Postgate 1984). PB medium of the following composition was prepared (per liter deionized water): KH₂PO₄ (0.5 g), NH₄Cl (1.0 g), CaSO₄ (1.0 g), MgSO₄ × 7 H₂O (2.0 g), yeast extract (1.0 g), FeSO₄ × 7 H₂O (0.5 g), sodium lactate (3.5 g), sodium acetate (2.46 g), sodium ascorbate, (0.1 g), sodium thioglycolate (0.1 g) and resazurin (0.001 g). The pH was adjusted to 7.2. The medium was heated to boiling point and purged with high-purity N₂ for 15 min, and autoclave-sterilized at 120 °C for 20 min. The sterile medium was cooled under a stream of N₂ gas. Ten mL of a vitamin solution containing biotin (2.0 mg), folic acid (2.0 mg), pyridoxine HCl (10.0 mg), thiamin (5.0 mg), riboflavin (5.0 mg), nicotinic acid (5.0 mg), calcium D-(þ)-pantothenate (5.0 mg), vitamin B12 (0.1 mg), p-aminobenzoic acid (5.0 mg), and lipoic acid (5.0 mg) was added to the sterile medium. The water samples were inoculated into serum bottles (capped with rubber stoppers and crimped with aluminum seals) containing PB medium. All manipulations were done in an anaerobic chamber (System One Glovebox, Innovative Technology, Amesbury, MA, USA) under a strict and controlled oxygen-free environment. The cultures were incubated for 2 months in the dark at 30 °C. SRB growth was monitored by observing the formation of a black FeS precipitate. SRB counts were determined by the most probable number (MPN) technique using PB medium. Standard MPN evaluation tables and 95% confidence intervals were used. MPN tubes were incubated in the dark at 30 °C for 2 months (The Institute of Petroleum 1995). In each inoculated tube, the growth of sulfate reducers was indicated by the formation of a black FeS precipitate and by turbidity.

Prior to DNA extraction, the internal method was performed. Three mL of culture sample was placed in a centrifuge tube and centrifuged at 10,000 rpm for 10 min, and then the supernatant was discarded. The pellet was used for DNA extraction, performed by Powersoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA), according to the manufacturer's instructions. The final volume of DNA was ∼ 100 μL. The yield and quality of the extracted DNA were evaluated using gel electrophoresis on 1% (w/v) agarose gel stained with GelRed (Biotium, Hayward, CA, USA), visualized on a UV transilluminator and photographed.

Bacterial 16S rDNA fragments were amplified by a two-step nested polymerase chain reaction (PCR) using primer sets, 27F/1495R and 341FGC/907R. The sequences of primers are listed in Table 1.

For the first step, PCR universal bacterial primers, 27F and 1495R, were used to amplify a fragment of about 1,400 bp in length. Each PCR reaction mix, with a final volume 25 μL, containe 1 μL genomic DNA (undiluted and diluted), 1.0 μL of each primer, 15.75 μL of Molecular Biology Grade Water (HiMedia, India), 5.0 μL of 5× PCR Dye Master Mix II (GeneMark, Taichung, Taiwan), and 1.25 μL dimethyl sulfoxide (DMSO) (Biomatik, Canada). Amplification was performed in a T100 thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) under the following conditions: initial denaturation for 5 min at 95%, followed by 25 cycles: denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 2 min, followed by a final extension at 72 °C for 5 min. After the first step, PCR products were analyzed by electrophoresis on a 1.5% agarose gel.

For the second-step PCR, 341F-GC and 907R primers were used to amplify a fragment of about 500 bp in length. The first PCR product was used as the template for the second PCR. Each PCR reaction mix, with a final volume of 25 μL, contained 1 μL DNA template (undiluted and diluted), 0.76 μL of each primer, 16.23 μL of Molecular Biology Grade Water (HiMedia, India), 5.0 μL of 5X PCR Dye Master Mix II (GeneMark, Taichung, Taiwan), and 1.25 μL DMSO (Biomatik, Canada). PCR amplification was performed on the same thermal cycler as mentioned earlier. This PCR was carried out under a touchdown protocol consisting of 5 min at 94 °C, followed by 10 cycles of 1 min at 94 °C, 1 min at 65 °C to 55 °C with a touchdown decrease of −1.0 °C cycle⁻¹, and 3 min at 72 °C, followed by 20 cycles of 1 min at 94 °C, 1 min at 55 °C, and 3 min at 72 °C, and was concluded with a final extension of 5 min at 72 °C. After the second step, PCR products were analyzed by electrophoresis on a 1.5% agarose gel.

Denaturing gradient gel electrophoresis (DGGE) analysis was performed using the Dcode Universal Mutation System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The PCR product of the second step was applied directly onto 1-mm-thick, 6% polyacrylamide (37.5: 1 acrylamide/bis-acrylamide) gel with a denaturing gradient ranging from 20 to 80% (w/v) (100% (w/v) denaturing solution containing 7 M urea and 40% (v/v) deionized formamide. Electrophoresis was run for 14 h at 80 V and 60 °C in 1X TAE (Tris-Acetate-EDTA) buffer. After electrophoresis, the gel was stained with SYBR Gold (Invitrogen, Carlsbad, CA, USA) for 20 min and photographed under UV transilluminator.

The DNA bands of interest were excised from the DGGE gel under UV transilluminator using sterile razor blades. The pieces of gel were placed in 40 μL of 1× Tris buffer (pH 8) and stored for 2 days at 4 °C to allow DNA diffusion. Re-amplification of the eluted DNA was performed in a 25-μL reaction volume containing 1.0 μL of template, 0.76 μL of primer 341F, 0.76 μL of primer 907R, 5.0 μL of 5× PCR Dye Master Mix II (GeneMark, Taichung, Taiwan) 16.23 μL of Molecular Biology Grade Water (HiMedia, India), and 1.25 μL DMSO (Biomatik, Canada). The PCR was run with an initial 2 min denaturation at 94 °C, 30 cycles of 60 s at 94 °C, 120 s at 55 °C, 120 s at 72 °C, and a final 5 min extension at 72 °C. After re-amplification, PCR products were verified by electrophoresis on a 1.5% agarose gel.

The resulting PCR products were sent to a commercial company (Eurofins Genomics, Constance, Germany) for purification and sequencing.

A consensus sequence was compiled for each DNA fragment obtained from both strands and consensus sequences were compared to published sequences deposited in GenBank using NCBI Nucleotide BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Benson et al. 2005). Multiple sequence alignments were performed using ClustalX (Thompson et al. 1997). Sequence data were processed with the GeneDoc sequence editor (Nicholas et al. 1997).

The sequences from DGGE bands 6 and 10 were identified (98.63 and 98.80% similarities, respectively) as Desulfolutivibrio sulfodismutans strain DSM 3696 (MN596860), a mesophilic sulfate-reducing bacterium isolated from freshwater mud (Thiel et al. 2020). D. sulfodismutans was known as Desulfovibrio sulfodismutans until Thiel et al. (2020) proposed the reclassification of Desulfovibrio sulfodismutans to Desulfolutivibrio sulfodismutans. The sequence of Desulfolutivibrio sulfodismutans was deposited in the GenBank database under the accession number OR647565.

The type strain of D. sulfodismutans DSM 3696 has been known to carry out a unique metabolic pathway enabling disproportionation of sulfite or thiosulfate to sulfide and sulfate. D. sulfodismutans could also carry out dissimilatory sulfate reduction by using lactate, ethanol, propanol, and butanol like typical sulfate reducers. However, this species is unable to utilize pyruvate as an electron donor and it grows very slowly when hydrogen is the only source (Bak & Pfennig 1987). Furthermore, although sulfate reducers are defined as obligate anaerobes, it was demonstrated that D. sulfodismutans is capable of microaerobic respiration (Dilling & Cypionka 1990). This type of respiration enables the sulfate-reducing bacterial species to grow at the oxic–anoxic interfaces of aquatic ecosystems such as marine sediments and oligotrophic freshwater lakes (Cypionka 2000). In this context, the detection of D. sulfodismutans in water samples flowing from groundwater to a pumping well in the present study proves that this species maintains its metabolic capacity throughout the well water system consisting of anoxic and oxic zones. It was also reported that D. sulfodismutans was able to reduce iron (III) and uranium (VI) (Lovley et al. 1993). Due to this metabolic capability, D. sulfodismutans is a potential candidate to be used for the remediation of heavy metal pollution such as the destruction of the ecosystem by mine wastes (Ayangbenro et al. 2018). On the other hand, the presence of D. sulfodismutans in well water may lead to severe issues such as corrosion in the well water system. As a matter of fact, it is known that hydrogen sulfide production and FeS formation due to SRB activity lead to the biocorrosion of the metal (Enning & Garrelfs 2014). Indeed, due to metal biocorrosion caused by SRB, a significant reduction in the operation life of the water well installations was reported previously (Calbo et al. 2018). However, the presence of D. sulfodismutans in well water has not been reported in the literature so far.

The presence of SRB, predominantly Desulfovibrio spp., in the large intestines of humans, has long been reported (Kushkevych et al. 2021). Despite being commonly known as nonpathogenic, different members of Desulfovibrio genus were reported to be associated with bacteremia (D. desulfuricans & D. fairfieldensis), incidents of an abdominal abscess (D. vulgaris), brain abscess and liver abscess (D. desulfuricans), and infections such as periodontitis (Desulfovibrio sp.), and IBD including ulcerative colitis and Crohn's disease (Desulfovibrio sp.) (Lozniewski et al. 1999; Langendijk et al. 2000; Goldstein et al. 2003; Koyano et al. 2015; Kushkevych et al. 2019; Kushkevych et al. 2020). Moreover, many studies have also reported a positive relationship between Desulfovibrio spp. and various human diseases such as Parkinson's disease, autism, obesity, and cancer (Singh et al. 2023). However, there is no reported information related to the correlation between the presence of D. sulfodismutans and human health.

The sequences of bands 1, 4, and 5 were related (with 99.15, 99.49, and 99.49 sequence similarities, respectively) to that of Anaerosinus sp. Jh2 (KX388181), an iron (III)-reducing strain that was detected in coastal riverine sediment (Zheng et al. 2017). The sequence of Anaerosinus sp. was deposited in the GenBank database under the accession number OR647564.

The genus Anaerosinus consists of obligately anaerobic, mesophilic, and chemo-organotrophic bacteria (Strömpl & Hippe 2015). Until now, only one species, Anaerosinus glycerini, was classified in this genus. A. glycerini was detected by DGGE analysis along with sulfidogenic bacteria, Clostridium genus, in the enrichment of groundwater contaminated by chlorinated ethene, a widely used industrial solvent. In that study, it was also reported that this enrichment showed dechlorinating activity and members of Clostridium genus were responsible for degrading chlorinated ethene (Arpita et al. 2013). On the contrary, the extracellular electron transfer capability of Anaerosinus sp. Jh2 in iron cycling was previously reported which makes this isolate a potential candidate for heavy metal bioremediation by reducing the toxicity of heavy metals in the environment (Zheng et al. 2017). In addition, the isolation of bacteria belonging to the genus Anaerosinus from the sub-surface horizons of a uranium deposit is important in terms of indicating the bioremediation potential of this genus (Babich et al. 2021). On the other hand, members of the Anaerosinus genus are found also in the human gut microbiome (Lin et al. 2018; Oluwagbemigun et al. 2019) and skin microbiome (Procopio et al. 2021). In this context, it is not surprising that Anaerosinus was detected in influent from the municipal wastewater treatment plant where domestic sewage is treated to control pathogenic risks. Although Anaerosinus genus are not defined as pathogens, their occurrence in the human sewage microbiome may indicate the presence of potential pathogens that may be co-existing (Cai et al. 2014). In other words, the presence of Anaerosinus sp. may be used as a potential indicator for pathogens. For example, it has been shown that the gut microbiome of children infected with rotavirus included higher levels of A. glycerini than those of healthy children (Sohail et al. 2021). It has also been reported that Anaerosinus, which is abundant in the gut microbiome, may affect the development of ulcerative colitis (Sahu et al. 2021), and colorectal cancer (Qingbo et al. 2024).

The genus Anaerosinus and Desulfovibrio survive in the same natural environments as well. For instance, they were stated as members of the microbial community of sub-surface horizons of a uranium deposit (Babich et al. 2021). In that study, while Anaerosinus was detected in the culture of aerobic organotrophic bacteria, Desulfovibrio was found in the medium for SRB. The coexistence of Anaerosinus and Desulfovibrio genera in the laboratory-scale environment has also been previously reported. They were both detected in the sulfidogenic upstream anaerobic sludge blanket reactor inoculated with methanogenic granular sludge after 110 days of operation (Mora et al. 2020).

In the literature, there are limited studies about the investigation of SRB in groundwater and groundwater associated environments, and in these studies, analyses were performed either by using culture-independent molecular technique or the cultivation method (Wargin et al. 2007; Yang et al. 2015; An et al. 2016). However, no study was found in which SRB was investigated in these environments by culture-dependent molecular methodology. In the present study, the presence of SRB in well water was investigated for the first time by cultivation based molecular fingerprinting technique in Türkiye. In this context, the bacterial community of the SRB enrichment was analyzed by a two-step nested PCR-DGGE because this approach makes it possible to detect even low numbers of SRB in complex microbial communities from natural environments (Dar et al. 2005).

The following conclusions can be drawn from the obtained data in this study:

• Even in low numbers, SRB can be found in a well water environment.

• Anaerobic sulfate-reducing D. sulfodismutans and anaerobic Anaerosinus sp. were isolated from the well water for the first time.

• Anaerosinus sp. may coexist with SRB.

• The presence of both SRB and Anaerosinus sp. in well water may be used as an indicator of water quality and may also be considered as potential microbial risk factors for public health.

• Anaerosinus genus, a member of the human gut microbiota, may be used as an alternative anaerobic indicator for fecal contamination of water quality. Further research is needed to confirm this suggestion.

• It is noteworthy that the detection of bacteria that can live in the same environmental conditions as the bacteria accepted as indicator microorganisms, using molecular analyzes following enrichment culture techniques, can bring new perspectives to evaluate microbial contamination in aquatic environments and to determine the possible origin and presence of alternative microbial indicators.

This study was funded by the Scientific Research Projects Coordination Unit of Istanbul University. Project number: 35889.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.