A high‐throughput and quantitative hierarchical oligonucleotide primer extension (HOPE)‐based approach to identify sources of faecal contamination in water bodies

As faecal contamination of recreational and drinking water impairs the water quality and threatens public health, water bodies are routinely monitored for faecal coliforms to detect contamination. However, faecal coliforms are facultative anaerobes that survive and reproduce in ambient waters, and their presence does not depict the origin of contamination. Therefore, the use of Bacteroides‐Prevotella 16S rRNA gene to perform faecal source tracking has been proposed and applied. Here, we demonstrate the use of a new molecular method termed hierarchical oligonucleotide primer extension (HOPE) to simultaneously detect human‐associated Bacteroides spp. and three clusters of cow‐, pig‐ and dog‐specific uncultivated Bacteroidales. The method correctly identifies the origin of faecal contamination when tested against human, cow, pig and dog faeces (n = 17, 17, 16 and 13 respectively), and in waters contaminated with faeces of known origins. Subsequent tests with a total of 21 blind samples show that HOPE is able to accurately indicate single or multiple sources of faecal contamination originating from pigs, cows and humans in 81% of the blind samples. HOPE can further correctly detect and identify faecal contamination in five sampling sites located along a canal in southern Taiwan, and the results are validated against conventional faecal coliform tests and quantitative PCR. Overall, this study demonstrates HOPE as a quantitative and high‐throughput method that can identify sources of faecal contamination.     

Microbial Load of Drinking Water Reservoir Tributaries during Extreme Rainfall and Runoff

Hygienic and microbiological examinations of watercourses are usually not carried out during heavy rainfall and runoff events. After rainfall or snowmelt, there are often massive increases in turbidity in flooding creeks in mountain ranges, which are frequently interpreted as an indication of microbial contamination. The aim of this study was to quantify the microbial loads of watercourses during such runoff events and to compare these loads with loads occurring during regular conditions. In a 14-month monitoring period we investigated the microbial loads of three tributaries of different drinking water reservoirs. A total of 99 water samples were taken under different runoff conditions and analyzed to determine physical, chemical, bacterial, and parasitic parameters. Thirty-two water samples were considered event samples during nine measuring series. The criteria for events, based on duration and intensity of precipitation, water depth gauge measurements, and dynamics, had been fixed before the investigation for each creek individually. Of the physical and chemical parameters examined, only the turbidity, pH, and nitrate values differed clearly from the values obtained for regular samples. Most of the bacteriological parameters investigated (colony, Escherichia coli, coliform, fecal streptococcal, and Clostridium perfringens counts) increased considerably during extreme runoff events. If relevant sources of parasitic contamination occurred in catchment areas, the concentrations of Giardia and Cryptosporidium rose significantly during events. The results show that substantial shares of the total microbial loads in watercourses and in drinking water reservoirs result from rainfall and extreme runoff events. Consequently, regular samples are considered inadequate for representing the microbial contamination of watercourse systems. The procedures for raw water surveillance in the context of multiple-barrier protection and risk assessment ought to include sampling during extreme runoff situations.     

三峡库区水体中固氮微生物多样性及其影响因素

【目的】研究分析不同时空条件下三峡库区水体固氮微生物多样性,并探讨其与地球化学参数的相关性。【方法】采集三峡库区不同时间(三月份和六月份)和空间(干流与支流)的水体样品,对其进行地球化学参数分析,并通过构建克隆文库分析样品中固氮功能基因(nifH)的多样性进而探讨其与水体地化参数的相关关系。【结果】统计分析显示三峡库区水体固氮微生物α-多样性和群落组成具有时空差异。支流水体样品的固氮微生物α-多样性高于干流水体样品;六月水体样品的固氮微生物α-多样性高于三月水体样品。三峡库区三月水体样品中的固氮微生物群落以Proteobacteria (50.3%)和Firmicutes (40.0%)为主;六月水体样品的固氮微生物群落以Proteobacteria(48.4%)、Firmicutes(25.4%)和Cyanobacteria(19.0%)为主。Mantel检验结果显示:固氮微生物群落结构的差异与温度、pH和DIC等地球化学参数具有显著(P0.05)相关性,其中温度和pH的相关性系数最大。【结论】三峡库区固氮微生物的种群结构和多样性具有时空差异,影响三峡水库水体中固氮微生物群落结构与多样性的主要环境因素为温度和pH,同时浊度、DIC、氨氮也对库区水体固氮微生物群落结构和多样性有一定的影响。     

High Diversity of Fungi may Mitigate the Impact of Pollution on Plant Litter Decomposition in Streams

We investigated how a community of microbial decomposers adapted to a reference site responds to a sudden decrease in the water quality. For that, we assessed the activity and diversity of fungi and bacteria on decomposing leaves that were transplanted from a reference (E1) to a polluted site (E2), and results were compared to those from decomposing leaves either at E1 or E2. The two sites had contrasting concentrations of organic and inorganic nutrients and heavy metals in the stream water. At E2, leaf decomposition rates, fungal biomass, and sporulation were reduced, while bacterial biomass was stimulated. Fungal diversity was four times lower at the polluted site. The structure of fungal community on leaves decomposing at E2 significantly differed from that decomposing at E1, as indicated by the principal response curves analysis. Articulospora tetracladia, Anguillospora filiformis, and Lunulospora curvula were dominant species on leaves decomposing at E1 and were the most negatively affected by the transfer to the polluted site. The transfer of leaves colonized at the reference site to the polluted site reduced fungal diversity and sporulation but not fungal biomass and leaf decomposition. Overall, results suggest that the high diversity on leaves from the upstream site might have mitigated the impact of anthropogenic stress on microbial decomposition of leaves transplanted to the polluted site.     

Microbial load of drinking water reservoir tributaries during extreme rainfall and runoff

Hygienic and microbiological examinations of watercourses are usually not carried out during heavy rainfall and runoff events. After rainfall or snowmelt, there are often massive increases in turbidity in flooding creeks in mountain ranges, which are frequently interpreted as an indication of microbial contamination. The aim of this study was to quantify the microbial loads of watercourses during such runoff events and to compare these loads with loads occurring during regular conditions. In a 14-month monitoring period we investigated the microbial loads of three tributaries of different drinking water reservoirs. A total of 99 water samples were taken under different runoff conditions and analyzed to determine physical, chemical, bacterial, and parasitic parameters. Thirty-two water samples were considered event samples during nine measuring series. The criteria for events, based on duration and intensity of precipitation, water depth gauge measurements, and dynamics, had been fixed before the investigation for each creek individually. Of the physical and chemical parameters examined, only the turbidity, pH, and nitrate values differed clearly from the values obtained for regular samples. Most of the bacteriological parameters investigated (colony, Escherichia coli, coliform, fecal streptococcal, and Clostridium perfringens counts) increased considerably during extreme runoff events. If relevant sources of parasitic contamination occurred in catchment areas, the concentrations of Giardia and Cryptosporidium rose significantly during events. The results show that substantial shares of the total microbial loads in watercourses and in drinking water reservoirs result from rainfall and extreme runoff events. Consequently, regular samples are considered inadequate for representing the microbial contamination of watercourse systems. The procedures for raw water surveillance in the context of multiple-barrier protection and risk assessment ought to include sampling during extreme runoff situations.     

Detection of Helicobacter pylori DNA in human faeces and water with different levels of faecal pollution in the north-east of Spain

AIMS: To assess the role of water in the faecal transmission of Helicobacter pylori by detecting the DNA of this pathogen in human faecal samples and environmental water samples with a range of faecal pollution from the north-east of Spain. METHODS AND RESULTS: Semi-nested PCR was used to detect H. pylori in stools and water, both matrices with a complex biota. DNA was detected using highly specific primers of an ureA gene fragment. In addition, antigens were used to detect the bacteria in stools. Helicobacter pylori was detected in 33% of 36 human faecal samples and in 66% of wastewater samples, and 11% of river samples, but in none of the spring waters samples. Faecal pollution of the aquatic environment was tested analysing the presence of microbial indicators. CONCLUSIONS: We report the presence of H. pylori DNA in stools and in aquatic environments with different levels of faecal pollution, from the north-east of Spain. In this study a higher number of positive results were obtained in the more faecally polluted waters. SIGNIFICANCE AND IMPACT OF THE STUDY: These data indicate that water may be a vector of H. pylori in its faecal-oral route.     

Microbial community analyses of produced waters from high‐temperature oil reservoirs reveal unexpected similarity between geographically distant oil reservoirs

As a preliminary investigation for the development of microbial‐enhanced oil recovery strategies for high‐temperature oil reservoirs (~70 to 90°C), we have investigated the indigenous microbial community compositions of produced waters from five different high‐temperature oil reservoirs near Segno, Texas, U.S. (~80 to 85°C) and Crossfield, Alberta, Canada (~75°C). The DNA extracted from these low‐biomass‐produced water samples were analysed with MiSeq amplicon sequencing of partial 16S rRNA genes. These sequences were analysed along with additional sequence data sets available from existing databases. Despite the geographical distance and difference in the physicochemical properties, the microbial compositions of the Segno and Crossfield produced waters exhibited unexpectedly high similarity, as indicated by the results of beta diversity analyses. The major operational taxonomic units included acetoclastic and hydrogenotrophic methanogens (Methanosaetaceae, Methanobacterium and Methanoculleus), as well as bacteria belonging to the families Clostridiaceae and Thermotogaceae, which have been recognized to include thermophilic, thermotolerant, and/or spore‐forming subtaxa. The sequence data retrieved from the databases exhibited different clustering patterns, as the communities from close geographical locations invariably had low beta diversity and the physicochemical properties and conditions of the reservoirs apparently did not have a substantial role in shaping of microbial communities.     

Nitrate-reducing community in production water of three oil reservoirs and their responses to different carbon sources revealed by nitrate-reductase encoding gene (napA)

The nitrate-reducing microbial community in oil reservoirs was examined by PCR using primers to amplify a segment of napA gene encoding for a subunit of the nitrate reductase, and the effects of different organic carbon additions on the nitrate-reducing community were also evaluated. The orders Rhodocyclales and Burkholderiales within Betaproteobacteria and Pseudomonadales within Gammaproteobacteria were recovered in production water of all three oil reservoirs. Amendment of organic acids promoted Enterobacteriales within Gammaproteobacteria and orders of Rhodocyclales, Pseudomonadales, and Burkholderiales, while alkanes favored the Rhizobiaceae family within Alphaproteobacteria and orders of Rhodocyclales and Pseudomonadales. Results indicated that the functional gene napA can be used as a valuable biomarker in analyzing the diversity of nitrate reducers in oil reservoirs. Nitrate-reducing microbial community shifts following the available carbon sources. Information about napA gene in oil reservoirs environment is scarce. This is the first study that combines molecular and culture-dependent approaches to reveal the diversity of the nitrate-reducing microbial community in production water of oil reservoirs by using the napA gene.     

Assessment of norovirus contamination in environmental samples from Florianópolis City,Southern Brazil

Aims: To assess norovirus (NoV) contamination in aquatic ecosystems in the city of Florianópolis, in Southern Brazil, to provide epidemiological data that can support actions for environmental contamination control. Methods and Results: An adsorption–elution method, followed by ultrafiltration, was performed to concentrate the viruses. NoV were detected using semi‐nested PCR and quantified by real‐time PCR. From June 2007 to May 2008, NoV were detected in 23% (22/94) of the samples analysed, including seawater, drinking water, superficial water (creek and brackish lagoon) and treated sewage. The mean viral loads for genogroups (G)I and GII in treated sewage samples were 297 and 440 genomic copies (gc) l^?1, respectively, whereas creek water samples contained 2603 and 1361 gc l^?1, respectively. Six samples were sequenced: two samples were GII.4, two were GII.2 and two were GI.3. Conclusions: NoV were detected in all water types analysed, demonstrating the widespread contamination of this geographical area with several cocirculating strains belonging to GI and GII. Significance and Impact of the Study: This study demonstrates the environmental spread of NoV in environmental waters and highlights the potential hazard for human health following the consumption of or contact with these waters, which could result in waterborne or foodborne acute gastroenteritis.     

Diversity and population structure of sewage‐derived microorganisms in wastewater treatment plant influent

The release of untreated sewage introduces non‐indigenous microbial populations of uncertain composition into surface waters. We used massively parallel 454 pyrosequencing of hypervariable regions in rRNA genes to profile microbial communities from eight untreated sewage influent samples of two wastewater treatment plants (WWTPs) in metropolitan Milwaukee. The sewage profiles included a discernible human faecal signature made up of several taxonomic groups including multiple Bifidobacteriaceae, Coriobacteriaceae, Bacteroidaceae, Lachnospiraceae and Ruminococcaceae genera. The faecal signature made up a small fraction of the taxa present in sewage but the relative abundance of these sequence tags mirrored the population structures of human faecal samples. These genera were much more prevalent in the sewage influent than standard indicators species. High‐abundance sequences from taxonomic groups within the Beta‐ and Gammaproteobacteria dominated the sewage samples but occurred at very low levels in faecal and surface water samples, suggesting that these organisms proliferate within the sewer system. Samples from Jones Island (JI – servicing residential plus a combined sewer system) and South Shore (SS – servicing a residential area) WWTPs had very consistent community profiles, with greater similarity between WWTPs on a given collection day than the same plant collected on different days. Rainfall increased influent flows at SS and JI WWTPs, and this corresponded to greater diversity in the community at both plants. Overall, the sewer system appears to be a defined environment with both infiltration of rainwater and stormwater inputs modulating community composition. Microbial sewage communities represent a combination of inputs from human faecal microbes and enrichment of specific microbes from the environment to form a unique population structure.     

Effect of biofilm in irrigation pipes on microbial quality of irrigation water

Aims: The focus of this work was to investigate the contribution of native Escherichia coli to the microbial quality of irrigation water and to determine the potential for contamination by E. coli associated with heterotrophic biofilms in pipe‐based irrigation water delivery systems. Methods and Results: The aluminium pipes in the sprinkler irrigation system were outfitted with coupons that were extracted before each of the 2‐h long irrigations carried out with weekly intervals. Water from the creek water and sprinklers, residual water from the previous irrigation and biofilms on the coupons were analysed for E. coli. High E. coli concentrations in water remaining in irrigation pipes between irrigation events were indicative of E. coli growth. In two of the four irrigations, the probability of the sample source, (creek vs sprinkler), being a noninfluential factor, was only 0·14, that is, source was an important factor. The population of bacteria associated with the biofilm on pipe walls was estimated to be larger than that in water in pipes in the first three irrigation events and comparable to one in the fourth event. Conclusion: Biofilm‐associated E. coli can affect microbial quality of irrigation water and, therefore, should not be neglected when estimating bacterial mass balances for irrigation systems. Significance and Impact of the Study: This work is the first peer‐reviewed report on the impact of biofilms on microbial quality of irrigation waters. Flushing of the irrigation system may be a useful management practice to decrease the risk of microbial contamination of produce. Because microbial water quality can be substantially modified while water is transported in an irrigation system, it becomes imperative to monitor water quality at fields, rather than just at the intake.     

Detection of the nifH gene of Methanobrevibacter smithii: a potential tool to identify sewage pollution in recreational waters

AIMS: The goal of this study was to develop and test the efficacy of a PCR assay for the environmental detection of the nifH gene of Methanobrevibacter smithii, a methanogen found in human faeces and sewage. METHODS AND RESULTS: PCR primers for the nifH gene of M. smithii were designed, tested and used to detect the presence or absence of this organism in faecal and environmental samples. Specificity analysis showed that the Mnif primers amplified products only in M. smithii pure culture strains (100%), human faeces (29%), human sewage samples (93%) and sewage-contaminated water samples (100%). No amplification was observed when primers were tested against 43 bacterial stock cultures, 204 animal faecal samples, 548 environmental bacterial isolates and water samples from a bovine waste lagoon and adjacent polluted creek. Sequencing of PCR products from sewers demonstrated that a 222-bp product was the nifH gene of M. smithii. The minimal amount of total DNA required for the detection of M. smithii was 10 ng for human faeces, 10 ng for faecally contaminated water and 5 ng for sewage. Recreational water seeded with M. smithii established a lower detection limit of 13 cells ml(-1). CONCLUSIONS: The Mnif assay developed during this investigation showed successful detection of M. smithii in individual human faecal samples, sewage and sewage-contaminated water but not in uncontaminated marine water or bovine-contaminated waters. The Mnif assay appears to be a potentially useful method to detect sewage-polluted coastal waters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to utilize methanogens as an indicator of sewage pollution. Mnif PCR detection of M. smithii was shown to be a rapid, inexpensive and reliable test for determining the presence or absence of sewage pollution in coastal recreational waters.     

Changes in the zooperiphyton structure in a small river along the spatial gradient

A total of 165 taxa have been recorded in the zooperiphyton of the Sestra River, among which larvae of chironomids (40), nematodes (24), and oligochaetes (21) have the highest species richness. The increase in the taxonomic richness of zoocenoses from the river head to its mouth is weakly expressed. The highest diversity of zoocenoses is characteristic for the contact zones of the river water with its tributaries and receiving waters. The discreteness of spatial characteristic of the taxonomic composition of zooperiphyton reflects the heterogeneity and diversity of habitat conditions of invertebrates. In regards to its effect on zoocenoses, anthropogenic pollution can exceed the impact of many environmental factors (including the current velocities) which affect the formation of rheophilic communities in river ecosystems. The dominant complexes of zooperiphyton include all trophic groups of invertebrates, among which the group of detritophages-collectors has the highest taxonomic diversity. The group of phytodetritophages-filterers + collectors mainly includes chironomid larvae of the genus Chironomus, which are associated with strongly polluted zones. Invertebrate filterers, sponges, and bryozoans prevail mainly in zooperiphyton in the lower reaches of the river, which are not subjected to the pollution.     

Phylogenetic diversity of microbial communities in real drinking water distribution systems

Microbial regrowth in drinking water distribution systems (DWDS) is a major concern in the water supply industry. Detailed knowledge of the microbial community in DWDS will be of great importance for assessing the microbiological risks of drinking water. The spatial heterogeneity of microbial community structures in the bulk waters of a large real DWDS was investigated using 16S rRNA clone library analysis. The results indicate that high residual chlorine in drinking water could not control microbial regrowth in DWDS. The bacterial communities in the bulk waters were spatially heterogenic, mainly composed of Alphaproteobacteria and Betaproteobacteria (or Cyanobacteria). Microorganisms from the genera Acinetobacter, Sphingomonas and Gemella were detected, implying there is microbiological risk from drinking water. This work provides new insight into microbial ecology in DWDS.     

Multi‐scale effects of impoundments on genetic structure of creek chub (Semotilus atromaculatus) in the Kansas River basin

1. Habitat fragmentation has been implicated as a primary cause for the ongoing erosion of global biodiversity, yet our understanding of the consequences in lotic systems is limited for many species and regions. Because of harsh environmental conditions that select for high colonisation rates, prairie stream fishes may be particularly vulnerable to the effects of fragmentation. Hence, there is urgent need for broader understanding of fragmentation in prairie streams such that meaningful conservation strategies can be developed. Further, examination at large spatial scales, including multiple impoundments and un‐impounded catchments, will help identify the spatial extent of species movement through the landscape. 2. Our study used data from 10 microsatellite loci to describe the genetic structure of creek chub (Semotilus atromaculatus) populations across four catchments (three impounded and one un‐impounded) in the Kansas River basin. We investigated whether genetic diversity was eroded in response to habitat fragmentation imposed by reservoirs and whether intervening lentic habitat increased resistance to dispersal among sites within a catchment. 3. Our analyses revealed that genetic diversity estimates were consistent with large populations regardless of the location of the sampled tributaries, and there was little evidence of recent population reductions. Nevertheless, we found a high degree of spatial genetic structure, suggesting that catchments comprise a set of isolated genetic units and that sample sites within catchments are subdivided into groups largely defined by intervening habitat type. Our data therefore suggest that lentic habitat is a barrier to dispersal among tributaries, thus reducing the opportunity for genetic rescue of populations in tributaries draining into reservoirs. Isolation by a reservoir, however, may not be immediately deleterious if the isolated tributary basin supports a large population.     

Spatiotemporal variation of bacterial and archaeal communities in sediments of a drinking reservoir,Beijing, China

Bacterial and archaeal assemblages are one of the most important contributors to the recycling of nutrients and the decomposition of organic matter in aquatic sediments. However, their spatiotemporal variation and its driving factors remain unclear, especially for drinking reservoirs, which are strongly affected by human consumption. Using quantitative PCR and Illumina MiSeq sequencing, we investigated the bacterial and archaeal communities in the sediments of a drinking reservoir, the Miyun Reservoir, one of the most important drinking sources for Beijing City. The abundance of bacteria and archaea presented no spatiotemporal variation. With respect to community diversity, visible spatial and temporal differences were observed in archaea, whereas the bacterial community showed minor variation. The bacterial communities in the reservoir sediment mainly included Proteobacteria, Bacteroidetes, Nitrospirae, Acidobacteria, and Verrucomicrobia. The bacterial community structure showed obvious spatial variation. The composition of the bacterial operational taxonomic units (OTUs) and main phyla were dam-specific; the composition of samples in front of the dam were significantly different from the composition of the other samples. The archaeal communities were mainly represented by Woesearchaeota and Euryarchaeota. Distinctly spatial and seasonal variation was observed in the archaeal community structure. The sediment NH₄ ⁺–N, pH, and water depth were identified as the key driving factors of changes in the composition of the bacterial and archaeal communities. Water depth might have the greatest influence on the microbial community structure. The dam-specific community structure may be related to the greater water depth in front of the dam. This finding indicates that water depth might be the greatest contributor to the microbial community structure in the Miyun Reservoir.

     

Decoding the DNA and RNA viromes of a tropical urban lagoon

Human and livestock sewage is one of the major causes of excess nutrients, leading to the eutrophication of aquatic ecosystems and potentially to the emergence or spread of pathogenic viruses. This study aimed to investigate the composition and diversity of aquatic viromes in a highly anthropized lagoon, to identify the presence of pathogenic taxa and to explore their use as possible viral indicators of faecal contamination. For this, water and sediment samples were collected in the Ebrié Lagoon (Ivory Coast) at seven stations with contrasting levels of eutrophication. The DNA viromes of the planktonic and the benthic compartments were highly divergent, but were not influenced by the level of eutrophication. Conversely, the RNA viromes in the water column were comparable to those found in sediment, but showed significant differences between the stations. We detected the presence of viral DNA and RNA sequences we had assigned as indicators of faecal contamination (smacovirus, pecovirus and pepper mild mottle virus) as well as human pathogens (human cyclovirus, coxsackie B virus and picobirnavirus), which were all enriched in the most eutrophicated sites. These findings suggest that the examination of viromes represents a promising tool for assessing the state of human-induced contamination of aquatic ecosystems.     

鄱阳湖湖泊细菌群落组成及结构——以松门山为例

于2011年5月在鄱阳湖——松门山湖区采集底泥与表层水样,分别提取了表层水体浮游和底泥微生物基因组DNA,利用454高通量测序技术对细菌的16S rRNA基因进行了序列测定,分析了湖泊底泥细菌、水体浮游细菌群落结构特征。结果显示:底泥细菌OTUs(Operational Taxonomic Units)为1454,表层水体浮游细菌OTUs为269;底泥细菌群落比表层水体更加多样化,底泥细菌的物种数大大多于表层水体。同时,底泥细菌群落与表层浮游细菌群落结构存在显著差异。物种分类显示鄱阳湖底泥细菌种类隶属于20门,228属,其中优势种群为δ-变形菌纲(Deltaproteobacteria)、β-变形菌纲(Betaproteobacteria)和疣微菌门(Verrucomicrobia);表层水体浮游细菌隶属于13门116属,优势种群为β-变形菌纲、拟杆菌门(Bacteroidetes)和放线菌门(Actinobacteria)。结果进一步揭示,无论是浮游细菌群落还是沉积物细菌群落,优势细菌种群的基因型多样性更高。     

Archaeal and bacterial communities of heavy metal contaminated acidic waters from zinc mine residues in Sepetiba Bay

Mining of metallic sulfide ore produces acidic water with high metal concentrations that have harmful consequences for aquatic life. To understand the composition and structure of microbial communities in acid mine drainage (AMD) waters associated with Zn mine tailings, molecular diversity of 16S genes was examined using a PCR, cloning, and sequencing approach. A total of 78 operational taxonomic units (OTUs) were obtained from samples collected at five different sites in and around mining residues in Sepetiba Bay, Brazil. We analyzed metal concentration, physical, chemical, and microbiological parameters related to prokaryotic diversity in low metal impacted compared to highly polluted environments with Zn at level of gram per liter and Cd–Pb at level of microgram per liter. Application of molecular methods for community structure analyses showed that Archaea and Bacteria groups present a phylogenetic relationship with uncultured environmental organisms. Phylogenetic analysis revealed that bacteria present at the five sites fell into seven known divisions, α-Proteobacteria (13.4%), β-Proteobacteria (16.3%), γ-Proteobacteria (4.3%), Sphingobacteriales (4.3%), Actinobacteria (3.2%) Acidobacteria (2.1%), Cyanobacteria (11.9%), and unclassified bacteria (44.5%). Almost all archaeal clones were related to uncultivated Crenarchaeota species, which were shared between high impacted and low impacted waters. Rarefaction curves showed that bacterial groups are more diverse than archaeal groups while the overall prokaryotic biodiversity is lower in high metal impacted environments than in less polluted habitats. Knowledge of this microbial community structure will help in understanding prokaryotic diversity, biogeography, and the role of microorganisms in zinc smelting AMD generation and perhaps it may be exploited for environmental remediation procedures in this area.