Assessment of perchlorate-reducing bacteria in a highly polluted river

Assessment of perchlorate-reducing bacteria in a highly polluted river

International Journal of Hygiene and Environmental Health 213 (2010) 437–443 Contents lists available at ScienceDirect International Journal of Hygi...

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International Journal of Hygiene and Environmental Health 213 (2010) 437–443

Contents lists available at ScienceDirect

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Assessment of perchlorate-reducing bacteria in a highly polluted river Giovanni Vigliotta a , Oriana Motta b,∗ , Francesco Guarino a , Patrizia Iannece a , Antonio Proto a a b

Department of Chemistry, University of Salerno, via Ponte don Melillo, 84084 Fisciano, SA, Italy Department of Educational Science, University of Salerno, via Ponte don Melillo, 84084 Fisciano, SA, Italy

a r t i c l e

i n f o

Article history: Received 10 February 2010 Received in revised form 29 July 2010 Accepted 3 August 2010 Keywords: Sarno River Perchlorate reduction Dechlorospirillum Dechlorosoma PCR

a b s t r a c t A 1-year monitoring experiment of the Sarno River basin was conducted during 2008 to evaluate the overall quality of the water over time and to compare the results with those obtained previously. The physico-chemical and microbiological characteristics of the water course had not changed appreciably with respect to previous determinations, thus emphasizing the major contribution of untreated urban wastewater to the overall pollution of the river. Moreover, attention was paid to the perchlorate ion, one of the so-called emerging contaminants, which is widespread in natural environments and is known to have adverse effects on the human thyroid gland. Over the entire monitoring program, we did not find appreciable levels of perchlorate, although the particular environmental condition could support its development. Thus, a dedicated study was designed to assess the presence of bacteria that can reasonably reduce perchlorate levels. By enrichment and molecular procedures, we identified ␣- and ␤-Proteobacteria strains, classified by 16S rDNA sequences as Dechlorospirillum sp. and Dechlorosoma sp., respectively. Further physiologic characterization and the presence of the alpha subunit gene (pcrA) of the perchlorate reductase in both strains confirmed the presence in the river of viable and active perchlorate dissimilatory bacteria. © 2010 Elsevier GmbH. All rights reserved.

Introduction In a previous work, we reported the results of a 1-year monitoring study of Sarno River water quality. We identified high anthropogenic activity as the predominant source of microbial contamination, whereas chemical contamination was mainly related to uncontrolled release of polluting materials as well as to byproducts of the wastewater treatment plant (WWTP) located along one of the tributaries (Motta et al., 2008). The river and its tributaries are used extensively as sources of irrigation water for the intensive agricultural activity in the basin. Therefore, contamination of the river, for which the indiscriminate increase in pollution factors has made natural purification capacities insufficient, is of particular concern to public health. Many years after the Ministry of the Environment declared the area to be at high environmental risk (Ministry Council, 1994), it was found that adequate chemical and bacteriological data to support this claim were almost completely absent. A further 1-year monitoring study of the Sarno River basin was conducted during 2008 to evaluate the overall water quality over time. Moreover, attention was paid to the perchlorate ion, one of the so-called emerging contaminants because the characteristic pollu-

∗ Corresponding author. Tel.: +39 089 963083; fax: +39 089 963083. E-mail address: [email protected] (O. Motta). 1438-4639/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2010.08.001

tion and the particularly anoxic condition of the river suggested it could be present. The importance of perchlorate in the environment was first noted in the United States because of the high concentration of military and spatial activities, especially in areas of Southwestern states such as Nevada, Utah and California where contamination levels in many rivers and water wells used for human consumption and rural uses reached values associated with adverse health outcomes (Kirk, 2006; NRC, 2005; Renner, 1998). Health concerns mainly arise from the ability of perchlorate to disrupt thyroid gland utilization of iodine in metabolic hormones, which could affect normal metabolism, growth and development (Greer et al., 2002; Wolterink et al., 1998; Xu et al., 2003). Perchlorate is a ubiquitous salt that is released into the environment by both anthropogenic and natural sources. Anthropogenic perchlorate sources include military and commercial application of perchlorate salts as oxidizers in propellants, flares, munitions, matches, fireworks, blasting agents and other materials. Although the natural occurrence of perchlorate is poorly understood and the exact role of atmospheric processes in the formation of perchlorate remains uncertain, recent studies have demonstrated that significant quantities of perchlorate can form naturally in the atmosphere, especially during thunderstorms (Dasgupta et al., 2005; Parker et al., 2008). However, it has also been demonstrated that the perchlorate concentration ultimately occurring in groundwater is mitigated by factors such as hydrologic balance and microbial reduction (Plummer et al., 2006).


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Fig. 1. Locations of sampling stations along the Sarno River basin. Sites S1–S6 were along the Sarno River, Sol1–Sol2 were along the Solofrana tributary, Cav1–Cav2 were along the Cavaiola tributary, and AC was in the confluence between Solofrana and Cavaiola tributaries.

Currently, it is known that perchlorate contamination not only affects U.S. waters, but also, massive doses of perchlorate have been found in cow’s milk and in a variety of foods of vegetable origin from different areas worldwide (Dyke et al., 2007; Takatsuki et al., 2009; Wang et al., 2009). We were particularly interested in the presence of perchlorate in the Sarno River basin, which is located in an important economic area southeast of the volcano Vesuvius (South Italy). This area, with an economy largely based on agriculture, has undergone rapid and uncontrolled urban expansion and is now populated by about one million people, who are mostly concentrated in small towns that are important sites of firework displays. The relationship between fireworks displays and the occurrence of perchlorate in adjacent surface waters has been documented in a work by Wilkin et al. (2007). In our research, we did not find appreciable levels of perchlorate in the watercourse, but it was detected in the headwater. This suggested the need to design a dedicated study to determine whether there were microbial communities capable of degrading perchlorate. In the paper, new viable and active perchlorate-reducing bacteria are reported. Experimental Sampling Sampling was done every fortnight during the summer and winter of 2008. Water samples were collected from six sampling stations on the Sarno River (S1–S6), two sampling stations each on the Cavaiola and Solofrana tributaries (Cav1, Cav2, Sol1, Sol2) and one sampling station at the confluence between the Cavaiola and Solofrana tributaries (AC) (Fig. 1).

surements and some physical properties, such as pH, electrical conductivity and water temperature, were measured at the time of collection by means of a Hanna Instruments mod. HI 9625. Chemical oxygen demand (COD) was determined with K2 Cr2 O7 and H2 SO4 in a 1:1 ratio by the open reflux method with AgSO4 as a catalyst and HgSO4 to remove Cl− interference. Excess dichromate was titrated with Fe2+ using phenanthroline as an indicator. Inorganic anions (bromides, chlorides, fluorides, sulfates, nitrites, nitrates and phosphates) were determined using a Dionex DX 120 ion chromatograph (Dionex, Sunnyvale, CA, USA), equipped with an Ion Pac AS14 column (4 mm × 250 mm). The eluent was 1.8 mM Na2 CO3 :1.6 mM NaHCO3 at a flow rate of 2 mL min−1 at a pressure of 970 psi. Perchlorates were determined using a Dionex DX 120 ion chromatograph equipped with an Ion Pac AS20 (4 mm × 250 mm) analytical column and an Ion Pac AG20 (4 mm × 50 mm) guard column and detected by suppressed conductivity. The eluent was 10 mM NaOH at a flow rate of 0.25 mL/min. The quality control was ensured by analyzing blank samples and international standards. To detect trace amounts of perchlorate ions, EPA Method 314.1 was used (EPA, 2005). The concentration was determined with a 4 mm × 35 mm cryptand concentrator column in the sample loop position. The cryptand concentrator column was combined with a primary AS16 analytical column and a confirmation AS20 analytical column. Metallic cations were determined with a Perkin Elmer model Analyst 100 atomic absorption spectrometer equipped with deuterium-arc background correction in acidic samples (pH below 2.0). All reagents used for the analytical determinations were analytical grade. Microbial analyses

Physical and chemical analyses Collected water samples were maintained at 4 ◦ C, either in lowdensity polyethylene or Pyrex-glass containers and were delivered to the laboratory within 4 h. Standard water-chemistry mea-

Samples were collected in pre-sterilized Teflon bottles and shipped on ice to the laboratory for analysis. Water samples were analyzed for heterotrophic plate count (HPC) bacteria by placing 1 mL of raw sample on an agar plate and for total and fecal coliform

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Table 1 Oligonucleotide primers used in this study. Primer name PR-v1 PR-v2 F8 1525R pcrA-v1 pcrA-v2 narG-v1 narG-v3 narG-v4 narG-v6 a b

Target gene 16S rRNA 16S rRNA 16S rRNA 16s rRNA Perchlorate reductase alpha subunit (pcrA) Perchlorate reductase alpha subunit (pcrA) Nitrate reductase alpha subunit (narG) Nitrate reductase alpha subunit (narG) Nitrate reductase alpha subunit (narG) Nitrate reductase alpha subunit (narG)


Annealing position 



578–601 1013–1037a

325–349a 941–961a 1741–1759b 2360–2380b 3007–3023b 3482–3500b

Reference This study This study Bruce et al. (1999) Bruce et al. (1999) This study This study This study This study This study This study

Numbering is relative to the Dechloromonas agitata 16S rRNA and pcrA genes. Numbering is relative to the Escherichia coli strain K-12 narG gene.

(TC, FC), fecal streptococci (FS) and E. coli by employing the filtration membrane method (0.45-␮m pore-size filters, Millipore). For samples analyzed by the filtration membrane method, 100-mL portions of the water sample were filtered, and the filters were then placed on separate RODAC plates containing selective broth. Plates were incubated at 36 and 22 ◦ C for HPC and examined after 24 and 72 h and at 36 and 44 ◦ C for TC-FS and FC, respectively, and examined after 24 h. Source, media and enrichment procedure for perchlorate-reducing bacteria Microorganisms were isolated from samples collected from one point located between S2 and S3, where the chemical and physical conditions, such as the low concentration of oxygen and chloride levels, were consistent with those of a possible development site for these bacteria. Enrichment occurred in chemically defined VG medium (Logan, 2001; Rikken et al., 1996; Xu and Logan, 2003) modified for growth of more exigent microorganisms by the addition of a mixture of B vitamins (2 mg/L nicotinamide, 0.9 mg/L pantothenic acid, 0.3 mg/L pyridoxine, 0.16 mg/L riboflavin, 0.14 mg/L thiamine, 0.2 ng/L folic acid, 0.16 mg/L Vitamin B12 ), at pH 7.00 (adjusted with NaOH), with 15 mM CH3 COONa as the single carbon source and 10 mM sodium perchlorate (NaClO4 ) as the electron acceptor. Medium was prepared in an anaerobic condition by boiling to remove O2 and was dispensed in serum bottles that were capped with thick butyl rubber stoppers and autoclaved (20 min, 121 ◦ C) for sterilization. All of the anaerobic manipulations were performed using an anaerobic Glove box under a N2 /CO2 (80:20, v/v) atmosphere. The enrichment was conducted in the dark at 28 ◦ C under constant shaking (150–200 rpm), as described by Logan (2001). Antibiotic selection was performed with 50 ␮g/mL ampicillin and 2.5 ␮g/mL vancomycin. To follow population evolution, all stages were monitored by polymerase chain reaction (PCR) analysis, with specific primers for perchlorate reductase alpha subunit (pcrA) and 16S rDNA of perchlorate-reducing bacteria, respectively PR-v1/PR-v2 and pcrA-v1/pcrA-v2, as indicated in the DNA procedures section. Kinetics of growth and perchlorate/nitrate utilization Kinetic analysis of the enriched population was conducted in VG modified medium in the presence of 15 mM sodium acetate and 10 mM sodium perchlorate or 7 mM nitrate. Experiments were conducted in triplicate. Pre-inoculated samples in anoxic conditions in the exponential phase of growth were diluted to optical density at 600 nm (OD600 ) of 0.005/mL in fresh VG modified medium, incubated in anaerobiotic conditions at 28 ◦ C in the dark with constant shaking (150–200 rpm) and monitored for 13 days (advanced stationary phase). At the indicated times, two aliquots of culture were collected, one for the OD600 measurement and the other for

perchlorate or nitrate concentration evaluation. Linear regression analysis of the natural logarithms of the OD600 values yielded estimates of growth rates (R2 ≥ 0.9).

DNA procedures To extract genomic DNAs from the enriched population, samples were inoculated in 20 mL of VG modified medium containing 15 mM acetate and 10 mM perchlorate until the exponential phase of growth. DNA was extracted as described by Vigliotta et al. (2005) and used in PCR experiments with specific primers for rDNA, pcrA and narG, as indicated in Table 1. Full-length (>1500 bp) 16S rRNA genes were produced by primers F8 and 1525R, as specified by Bruce et al. (1999). Partial regions of 16S rDNAs and pcrA genes were amplified by primer pairs PR-v1/PR-v2 and pcrA-v1/pcrA-v2, respectively. For nitrate reductase analysis, primer pairs narG-v1/narG-v6, narG-v3/narG-v4, narG-v3/narGv6 were used. For heterologous primers construction, databases of the National Center for Biotechnology Information (NCBI) ( and Ribosomal Database Project II (RDP) ( were utilized as sequence sources, and the CLUSTAL W program from the European Bioinformatics Institute ( was used for multiple sequence alignments. The specificity of primers for 16S rDNA was evaluated by the Probe Match tool of the RDP. All PCR reactions were performed in a 25-␮L reaction mixture containing approximately 100 ng of total genomic DNA and 0.5 U of Taq polymerase (AmpliTaq® DNA polymerase, Applied Biosystems), according to the manufacturer’s instructions, as follows: 5 min of initial denaturation at 94 ◦ C, followed by 35 cycles at 94 ◦ C for 1 min, 60 ◦ C for 1 min and 72 ◦ C for 1 min. The final extension was set at 72 ◦ C for 7 min. PCR products were separated by electrophoresis on 1% agarose gels and recovered using the Qiaex II DNA purification kit (QIAGEN) for cloning and sequence analysis. Libraries were constructed by cloning F8/1525R amplified 16S rDNAs and pcrA-v1/pcrA-v2 amplified pcrA genes into the pGEM® T Easy Vector (pGEM® -T Easy Vector System II, Promega) according to the manufacturer’s instructions and introduced in the JM109 strain of E. coli. Fifty clones from each library were collected, and plasmids were prepared by alkaline lysis (Sambrook and Russel, 2001) and sequenced using primers M13F and M13R on both strands by Eurofins MWG Operon custom DNA sequencing ( The determined sequences were deposited in the GenBank database with accession numbers GU294119, GU294120, and GU294121 for 16S rDNA sequences of Dechlorosoma sp. cl-6-Sarno River, Azospirillum sp. cl-19-Sarno River, and Dechlorospirillum sp. cl-31-Sarno River, respectively, GU320252 for the pcrA sequence of Dechlorosoma sp. cl-6-Sarno River and GU320253 for the pcrA sequence of Dechlorospirillum sp. cl-31-Sarno River.


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Fig. 2. Phylogenetic relationships of Sarno River perchlorate-reducing bacteria and the closest relative perchlorate- and non-perchlorate-reducing Proteobacteria. Brackets indicate subclasses of Proteobacteria. Bootstrap values (expressed as percentages of 1000 replications) >50 are reported at each node.

Phylogenetic analysis

Results and discussion

16S rRNA and pcrA gene sequences were analyzed with a similarity search with the BLAST function of GenBank on the NCBI electronic site and the Seq Match tool of the Ribosomal Database Project II. For phylogenetic analysis, only the 16S rDNA sequences >1100 bp deposited in both the NCBI and RDP databases were considered, and multiple sequence alignments were conducted using the CLUSTAL W program from the European Bioinformatics Institute. The phylogenetic tree was constructed using the MEGA 3 program (Kumar et al., 2004) with the Kimura two-parameter algorithm and the neighbor-joining method. The robustness of the inferred phylogenies was determined by bootstrap analysis based on 1000 resamplings of date.

In this study, the physico-chemical and microbiological characteristics of the Sarno River basin were explored to determine the overall quality of water over time and to compare the results with those obtained 2 years prior during the first analytical monitoring program (Motta et al., 2008). Two extensive sampling campaigns were conducted in the summer and winter of 2008. The results showed that the physico-chemical and microbiological characteristics of the water course had not changed appreciably with respect to previous determinations, thus emphasizing the major contribution of untreated urban wastewater to the overall pollution of the river, which accounted for the high level of bacterial contamination. Conversely, the overall level of chemical contamination was

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low to moderate and below the Italian State water quality thresholds in all cases (Italian Parliament, 2006). We did not see the value in reporting the new analytical data in this paper because they do not add any relevant information about the critical environmental condition of the river. However, we focused our attention on the characterization of perchlorate-degrading bacteria because in our research, we did not find appreciable levels of perchlorate in the watercourse, which was likely to develop due to the particularly polluted and anoxic conditions. Research conducted previously demonstrated that there is a diverse group of perchlorate-respiring bacteria (PRB) capable of degrading perchlorate and chlorate [(per)chlorate] under anoxic and microaerophilic conditions by using the molecule as a terminal electron acceptor (Sturchio et al., 2007). Biological degradation of perchlorate results in complete reduction of the chlorine atom through an initial two-step reduction of perchlorate to chlorate and then chlorite anions (ClO4 − , ClO3 − , ClO2 − ,), which is mediated by perchlorate reductase (Coates and Achenbach, 2004; Kengen et al., 1999; van Ginkel et al., 1996). Then the chlorite undergoes a disproportionation reaction catalyzed by chlorite dismutase to yield chloride and oxygen (Coates and Achenbach, 2004, Coates et al., 1999). Bacteria capable of (per)chlorate reduction are widely distributed in the environment (Coates et al., 1999; Wu et al., 2001), and phylogenetically, they are distributed in ␣-, ␤-, ␥and ␧-subclasses of the Proteobacteria phylum (Achenbach et al., 2001; Wallace et al., 1996). The majority of the species described thus far are ␤-Proteobacteria and are grouped in two genera of monophyletic origin Dechloromonas and Dechlorosoma (synonym of Azospira), respectively (Achenbach et al., 2001; Michaelidou et al., 2000; Wolterink et al., 2005). Over the entire period of analysis of Sarno River, we surprisingly found no evidence of perchlorate ions along the river course, although a detectable amount was found in the headwater (0.29 ␮g/L). Therefore, we next set to determine the occurrence of microbial communities capable of reducing perchlorate to find an explanation of its absence in an extremely polluted river. Using an enrichment strategy, alternating serial tube-to-plate transfer method and antibiotics selection, we obtained two perchloratereducing bacteria, as demonstrated by sequence analysis of libraries of amplified 16S rDNA and pcrA gene from the enriched population. Vancomycin and ampicillin were useful in further reducing Gram-positive and unwanted Gram-negative bacterial populations, as evidenced by the analysis of the 16S rDNA and microscopic evaluation. Sequence analysis of 16S rDNA libraries indicated that in the enriched sample, one of the two perchloratereducing microorganisms was a ␤-proteobacterium belonging to the Dechlorosoma genus, with more than 99.8% identity with perchlorate reducing Azospira oryzae GR-1 (Achenbach et al., 2001; Dudley et al., 2008; Michaelidou et al., 2000), Dechlorosoma suillum PST and Dechlorosoma spp. strains PCC, Iso1, Iso2 and PDX. The second was an ␣-proteobacterium, with more than 99.9% identity with 16S rDNA of Dechlorospirillum sp. DB, Dechlorospirillum sp. WD and Dechlorospirillum sp. VDY (Coates et al., 1999; NozawaInoue et al., 2008). These data were in accordance with pcrA gene analysis. In fact, in the pcrA gene library, we found only two different clones with 99.6% and 96% identity, respectively, with pcrA of Dechlorosoma sp. PCC and Dechlorospirillum sp. WD; furthermore, the latter was the only pcrA sequence present in databases for the Dechlorospirillum genus. The phylogenetic analysis based on 16S rDNA (Fig. 2) clearly indicated the relationship between the strains identified in Sarno River with the main described perchlorate-reducing ␣- and ␤Proteobacteria, placing the Dechlorosoma strain within the group of Azospira oryzae (Dechlorosoma suillum PST and Azospira oryzae GR-1) and the Dechlorospirillum strain near Dechlorospirillum spp.


Fig. 3. Kinetics of growth and perchlorate utilization of perchlorate-reducing strains from Sarno River in minimal medium and in the presence of 10 mM perchlorate.

strains VDY and WD. Based on 16S rDNA molecular taxonomy results and identity analysis of pcrA, we concluded that both bacteria identified in Sarno River represent two new strains of the genera Dechlorosoma and Dechlorospirillum, respectively, and that the first might be included in suillum (oryzae) species (16S rDNA identity of 99.93% with Dechlorosoma suillum PST ) and the second one in the same species of Dechlorospirillum sp. VDY (16S rDNA identity of 100%). Furthermore, we found a third ␣-proteobacterium 16S rDNA sequence that showed more than 99.3% identity with the 16S rRNA gene of different strains of the Azospirillum genus and was phylogenetically related to Azospirillum sp. NS01 and Azospirillum brasilense N8 (Fig. 2). In these taxa, strains with perchlorate reductase have been previously described (Coates et al., 1999; Nozawa-Inoue et al., 2008); therefore, we are presently assessing the presence of the pcrA gene of Azospirillum strains in Sarno River. To test the ability of our strains to use perchlorate as an electron acceptor, batch experiments were conducted to study growth kinetics. Enriched samples in the exponential phase of growth were inoculated in minimal VG medium without nitrate supplemented with 10 mM perchlorate and 10 mM acetate as the single carbon source. After 6 days, exponential growth ended, and the cultures entered the stationary phase, requiring more than 6 cycles of duplication; the growth rate calculated in the exponential phase was 0.87 generations per day (Fig. 3). The perchlorate concentration in the first 6 days was reduced by as much as 79% of the initial value, from 10 to 2.1 mM, and was decreased by 85% on the 12th day (advanced stationary phase). As expected, the major utilization rate of perchlorate occurred during exponential growth. In noninoculated samples, we could not detect changes in perchlorate concentrations, in accordance with the biological reduction of this compound in our conditions (data not shown). Thus, both molecular and kinetic data confirmed the presence of viable and active perchlorate-reducing bacteria in the river. Moreover, the ability to reduce nitrate has been described for some perchlorate-reducing bacteria within both Dechlorosoma and Dechloromonas spp. groups (Bruce et al., 1999; Coates et al., 1999; Horn et al., 2005; Xu et al., 2004). Thus, we assessed nitratereducing activity in our sample by measuring bacterial growth and nitrate utilization in minimal medium in the presence of nitrate as the only electron acceptor and acetate as the carbon source. As shown in Fig. 4, in anoxic conditions, nitrate supported the growth of microorganisms. More than 80% of the initial nitrate (7 mM) was utilized during the exponential phase, while the total number of generations and final biomass were slightly lower than their totals in perchlorate-supplemented medium (Figs. 3 and 4). PCR analysis with universal primers specific for the nitrate reductase alpha subunit (narG) gene of Gram-negative nitrate-respiring bacteria failed


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on environmental contamination from perchlorate and searched for new perchlorate-degrading bacteria. Acknowledgements The authors acknowledge the financial support from Italian Minister of Research MURST – FARB 2009. References

Fig. 4. Kinetics of growth and nitrate utilization of perchlorate-reducing strains from Sarno River in minimal medium and in the presence of 7 mM nitrate.

Fig. 5. PCR analysis of the alpha subunit (narG) of respiratory nitrate reductase. Several universal primers specific for narG of Gram-negative bacteria were tested. Escherichia coli and Pseudomonas fluorescens were used as positive controls. M: molecular marker; lane 1: amplification by primer pair narG3/4; lane 2: narG 3/6; lane 3: narG 1/6. Expected length of PCR fragments for E. coli: 664, 1141, 1760 bp, respectively, for primer pairs narG3/4, narG 3/6, and narG 1/6.

to detect the presence of the gene in our sample (Fig. 5), opening the possibility for a role of perchlorate reductase in this process as also suggested by other authors (Chaudhuri et al., 2002; Kengen et al., 1999). Therefore, future work has been planned to better define basis of this activity. Conclusion In this work, we have emphasized that in Sarno River, one of the most polluted rivers in Europe that crosses an area with high human activities and also has a high risk of perchlorate contamination, this compound is not quantifiable in most of its course, although it was present in the headwater. Because we found bacteria in the river that, in the presence of organic substances, were able to efficiently degrade perchlorate, we believe that its reduction by these organisms could be a possible cause of its absence from the river. This study further defined the group of bacteria dissimilators of perchlorate, describing two new strains of the genus Dechlorosoma spp. and Dechlorospirillum spp. Moreover, experiments conducted in the presence of nitrate as an electron acceptor provided evidence of nitrate reduction in the samples. Thus, work is underway to better define the basis of this activity. To our knowledge, based on the literature reported to date, we can state that this is the first study conducted in Europe that focused

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