Relationship between aquifer biofilms and unattached microbial indicators of urban groundwater contamination

Aquifers, springs and other groundwater‐dependent ecosystems are threatened by urban land use, which causes water quality deterioration through nutrient loading, sewage infiltration, groundwater extraction and, along coasts, seawater intrusion. The presence of certain microbes in groundwater can indicate that an aquifer is anthropogenically contaminated. Interpretations made from observations of indicator microbes in groundwater are limited because the relationship between the presumably allochthonous indicator microbes and relevant autochthonous microbial communities has not been characterized. This study addressed whether autochthonous aquifer biofilms can influence the presence of presumed microbial indicators in groundwater, and simultaneously used microbial indicators to trace sources of urban contamination at a karst spring of conservation concern. These questions were approached using a 17‐month time series analysis of attached biofilm and adjacent unattached bacteria in the submerged karst aquifer conduit associated with this spring. Environmental 16S rRNA gene sequencing was performed to characterize these communities, and community structure data were contextualized with groundwater geochemical and hydrogeological measurements. Linear regression models were developed to explain the relative abundance patterns of indicator microbes and other unattached microbes at this site. The results of this study suggest that dominant aquifer biofilms do not influence the presence of unattached microbial taxa that are presumed to be indicators of groundwater contamination, and generated new information about the origin of coliform bacteria at the study site. These results build confidence in the use of microbial indicators in groundwater‐dependent ecosystem conservation strategies and inform future management plans for urban aquifers and springs worldwide.     

Assessment of microbial aerosol emissions in an urban wastewater treatment plant operated with activated sludge process

Wastewater that enters wastewater treatment plants contains lots of pathogenic and nonpathogenic microorganisms which can become bioaerosols during treatment processes and pose health hazard to workers and nearby residents. The emission of the bioaerosols from an urban wastewater treatment plant in spring and summer in different locations and downwind of the plant adopting an extended mechanical aeration system was investigated. Samples of bacteria and fungi were collected within 6 months at 10 selected points by an Anderson one-stage impactor. The highest concentration of bacteria (mean 1373 CFU/m³, 741–2817 CFU/m³) and fungi (mean 1384 CFU/m³, 212–1610 CFU/m³) was found in downwind of the aeration basins. Statistical analysis showed a significant relationship between concentration of bacterial bioaerosols at downwind side of the aeration basins and wind speed (p value <0.05) and temperature (p value <0.05). Also, in the spring and summer, between the number of bacteria and fungi inside the plant and outside the plant (downwind) a significant correlation was observed (p value ≤0.05). The concentrations of bacteria at a distance of 500 m downwind were much higher than those at the background (upwind) point in spring and summer. The processes of wastewater treatment especially using mechanical equipment to create turbulence can be considered as a major source of spreading airborne microorganisms to ambient air of wastewater treatment plants, and the bioaerosols can be dispersed to downwind distances affecting the nearby neighboring. Therefore, in order to decrease the bioaerosols emission, doing some course of actions such as covering the surface of aeration basins, changing the aeration methods and aeration equipment (e. g using diffuser aerator) may be effective.     

Lachnospiraceae and Bacteroidales alternative fecal indicators reveal chronic human sewage contamination in an urban harbor

The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the Lachnospiraceae family, which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci and the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.     

Cyanobacteria as indicators of organic contamination

In two Costa Rican rivers used as receptors for domestic sewage, treated by primary stabilization ponds, were taken a total of 28 samplings located at the pond exit and at three different sites in each river: 100 m before the ponds discharge, at the discharge and 100 m after the discharge. These sampling were done for a five and a half years including dry and rainy seasons. In each sampling site, samples were collected of five different substrates: stones, submerge and semi submerge vegetation, tree trunks or sticks, water and artificial substrates. For each sample were used two types of artificial cultures, WC and BG110. A total of 55 cyanobacteria species isolations were obtained, belonging to a 26 genera, between these the most common were Phormidium with nine species, Microcystis with five species, Leptolyngbya and Pseudanabaena with four species each and Oscillatoria with three species. More cyanobacteria species were isolated in water substrate and less isolations in tree trunks and submerge vegetation. Konvophoron, Cyanarcus and Pilgeria only were isolate from water samples inoculated in culture media WC and in few opportunities, while three Leptolyngbya species and four Phormidium species were isolated very often. At the stabilization ponds Phormidium sp4 was dominant in 25 of 28 sampling while in the last others were the chlorophycea I. In this study were observed an increase in the frequency of cyanobacteria at the higher contamination places, and a species substitution between different sampling points. There were no biomass studies, therefore is not possible to relate between different cyanobacteria species and some specific types of water quality.     

Bovine enteroviruses as indicators of fecal contamination

Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.     

Assessment of microbial carcass contamination of hunted wild boars

To investigate the microbiological conditions of hunted wild boar carcasses and factors that contribute to the microbial carcass contamination, skin and carcass meat swab samples from 210 hunted wild boars were collected from freshly shot animals. The mean aerobic colony counts (ACCs) and Enterobacteriaceae counts on the skin were 5.2 and 3.6 log₁₀ CFU/cm², with 1.4% of animals’ skin tested positive for Salmonella spp. Slightly higher mean ACC and Enterobacteriaceae counts of 5.4 and 3.8 log₁₀ CFU/cm² were obtained from carcass meat with Salmonella spp. prevalence of 1.9%. Inadequate hygiene practices in handling and dressing wild boar carcasses, such as evisceration in the laying position on the ground and practice of skin and interior carcass surface washing after evisceration, were found to have the most significant influence on the microbiological conditions of final carcasses. Therefore, these findings indicate the need for the implementation and strict adherence to good hygiene practice in hunting estates and game handling establishments.     

Metabolites and biodegradation pathways of fatty alcohol ethoxylates in microbial biocenoses of sewage treatment plants.

The biodegradation of fatty alcohol polyglycol ethers was studied by analyzing the 14C-labeled intermediates isolated from the effluent of a model continuous-flow sewage treatment plant after dosage of either alkyl- or heptaglycol-labeled stearyl alcohol ethoxylate (SA-7EO). In each case, uncharged and carboxylated (mainly dicarboxylated) polyethylene glycols constituted the most prominent metabolites. The results indicate that there is a faster degradation of the alkyl than the polyethylene glycol moiety and that there are two distinct primary degradation mechanisms acting simultaneously in microbial biocenoses: intramolecular scission of the surfactant as well as omega- and beta-oxidation of the alkyl chain. Characterization of the bulk of 14C-labeled metabolites as a homologous series of neutral and acidic polyglycol units and identification of several C2-fragments accounted for the depolymerization of the hydrophilic part of the surfactant by stepwise cleavage of ether-bound EO units; from additional degradation studies employing either neutral or carboxylated 14C-labeled polyethylene glycols as model metabolites, it was concluded that hydrolytic as well as oxidative cleavage of C2-units is involved. Most of the identified low-molecular-weight 14C-labeled acids suggest an ultimate degradation of EO monomers by the oxidative dicarbonic acid cycle or the glycerate pathway or both. In addition, the finding of considerable amounts of oxalic and formic acids allow consideration of an additional mineralization route via glyoxylic, oxalic, and formic acids. The simultaneous action of different degradation mechanisms indicates the involvement of several distinct bacterial groups in the biodegradation of fatty alcohol ethoxylates under environmental conditions.     

Assessment of viral community functional potential from viral metagenomes may be hampered by contamination with cellular sequences

Although the importance of viruses in natural ecosystems is widely acknowledged, the functional potential of viral communities is yet to be determined. Viral genomes are traditionally believed to carry only those genes that are directly pertinent to the viral life cycle, though this view was challenged by the discovery of metabolism genes in several phage genomes. Metagenomic approaches extended these analyses to a community scale, and several studies concluded that microbial and viral communities encompass similar functional potentials. However, these conclusions could originate from the presence of cellular DNA within viral metagenomes. We developed a computational method to estimate the proportion and origin of cellular sequences in a set of 67 published viromes. A quarter of the datasets were found to contain a substantial amount of sequences originating from cellular genomes. When considering only viromes with no cellular DNA detected, the functional potential of viral and microbial communities was found to be fundamentally different—a conclusion more consistent with the actual picture drawn from known viruses. Yet a significant number of cellular metabolism genes was still retrieved in these viromes, suggesting that the presence of auxiliary genes involved in various metabolic pathways within viral genomes is a general trend in the virosphere.     

Plasmid profiles as indicators of the source of contamination of Staphylococcus aureus endemic within poultry processing plants

A total of 530 strains of Staphylococcus aureus were isolated from the defeathering machinery of a chicken processing plant and from neck skin samples of carcasses at different stages of processing in two visits 4 weeks apart. Eleven different plasmid profiles were detected in the isolates, eight being common to both visits. The plasmid profiles of the strains forming the majority of the population on the freshly slaughtered birds were rarely present in the strains isolated from the pluckers (except at the entry to the first plucker) and were present in only a small proportion of the strains isolated from carcasses after plucking. However, the profiles from the strains isolated from the pluckers on both visits were different from those forming the majority of the population on the incoming birds but formed the major part of the carcass flora after plucking, suggesting that such strains were endemic. These strains were found as a small proportion of the isolates made from the incoming birds, suggesting that this was the route by which the endemic strains were introduced into the plant. Such endemic strains exhibited a clumping growth, even in liquid shake culture, which may have made it easier for them to become established on the pluckers and to resist cleaning and disinfection. This clumping phenotype was correlated with the presence of a 7.5-megadalton plasmid.     

Some properties of methylotrophic bacteria isolated from sewage sludges derived from mechanical and biological sewage treatment plants.

The presented studies were carried out to get more information about physiological properties of methylotrophic bacteria selected from sewage sludges derived from mechanical and biological sewage treatment plants. All the isolated bacterial strains belonged to facultative methylotrophs. The majority of them utilized glucose, starch and lipids. Moreover, most of them were also found to possess proteolytic properties and were able to hydrolyze urea but they were incapable of asparagine ammonification.     

Growth,reproduction and longevity in nematodes from sewage treatment plants

The growth, reproduction and longevity of Diplogasteritus nudicapitatus, Paroigolaimella bernensis and Rhabditis curvicaudata were investigated under conditions of excess food within the temperature range 5°C–20°C. In all three species growth rate increased with temperature, and in D. nudicapitatus and R. curvicaudata the adult size attained varied significantly with temperature. P. bernensis did not reproduce at 5°C, but showed a progressive increase in reproductive output at higher temperatures. D. nudicaitatus showed increased egg production as temperature increased while R. curvicaudata had maximum egg output at 10°C. Longevity is temperature dependent, decreasing with higher temperatures. Virgin females survived for longer than reproducing females. The data indicate that while D. nudicapitatus and P. bernensis are thermophilic species, R. curvicaudata is adapted to lower temperatures.     

Plasmid profiles as indicators of the source of contamination of Staphylococcus aureus endemic within poultry processing plants.

A total of 530 strains of Staphylococcus aureus were isolated from the defeathering machinery of a chicken processing plant and from neck skin samples of carcasses at different stages of processing in two visits 4 weeks apart. Eleven different plasmid profiles were detected in the isolates, eight being common to both visits. The plasmid profiles of the strains forming the majority of the population on the freshly slaughtered birds were rarely present in the strains isolated from the pluckers (except at the entry to the first plucker) and were present in only a small proportion of the strains isolated from carcasses after plucking. However, the profiles from the strains isolated from the pluckers on both visits were different from those forming the majority of the population on the incoming birds but formed the major part of the carcass flora after plucking, suggesting that such strains were endemic. These strains were found as a small proportion of the isolates made from the incoming birds, suggesting that this was the route by which the endemic strains were introduced into the plant. Such endemic strains exhibited a clumping growth, even in liquid shake culture, which may have made it easier for them to become established on the pluckers and to resist cleaning and disinfection. This clumping phenotype was correlated with the presence of a 7.5-megadalton plasmid.     

Physicochemical and microbiological indicators of surface water body contamination with different sources of digestate from biogas plants

Transition from fossil energy sources to biogas production has resulted in a strong increase of leakage accidents from fermenters, but knowledge on the effects of fermentation product runoff into freshwater systems is currently restricted to direct toxicity due to oxygen depletion. This study provides first information about the influence of digestate runoff on the physicochemical habitat properties and the bacterial community composition of the hyporheic interstitial which is important in determining ecosystem functioning. We exposed natural stream beds to different concentrations of two different digestates from fermenters (corn and manure feedstock), hypothesizing that the digestate addition causes acute changes of the physicochemical parameters and has distinct effects on microbial community composition of the hyporheic interstitial depending on concentration and type of digestate. In line with the hypotheses, pH value, conductivity, redox potential and ammonium differed significantly from controls and among treatments after digestate addition, but only for a maximum of two days. pH values (controls: 7.8; corn: 7.9; manure: 7.9) and conductivity (controls: 813 μS/cm; corn: 969 μS/cm; manure: 1097 μS/cm) increased, the redox potential (controls: 153 mV; corn: 145 mV; manure: 144 mV) decreased the first two days. A high peak of ammonium-N was detected in the corn and manure treatments (controls: 5 mg/l, corn: 80 mg/l; manure: 60 mg/l) at day 1. In contrast, changes in bacterial community composition were detectable for longer periods of time (>5 days). Seventeen unique T-RF fingerprints of bacterial community response to each of the different digestate treatments (11 unique T-RFs in manure and 6 unique T-RFs in corn treatments) were found, suggesting that this approach provides a suitable ecological indicator for source tracking, e.g. in case of a biogas power plant leakage accident.     

某污灌区重金属与两种持久性有机污染物(POPs)污染趋势评价

对某污灌区的土壤剖面进行采样分析,利用不同的评价方法进行评价,结果表明,土壤中金属元素Cr、As、HG、Cd、Cu、Pb、Ni、Zn和持久性有机污染物BHC、DDT含量有上升趋势,80%以上的表层土壤样品污染物含量超过土壤底层,85%以上的土壤样品污染物含量明显超过当地土壤的自然背景值.通过污染物在土壤中的残留模型预测,两种有机污染物和元素Cr、As、Hg、Cd、Cu会在土壤中累积,持续的污水灌溉可能使在土壤中累积的这些污染物进入地下水或食物链,对环境健康造成危害.     

Release of microbial contamination from fractured solids

Estimation of the probability of release of microbial contamination from the interior of solids upon fracture due to impact is essential to the formulation of planetary quarantine and spacecraft sterilization requirements. A model system was designed in which known concentrations of bacterial spores were incorporated in methyl methacrylate plastic. Pieces of plastic were fractured in a uniform manner exposing interior surface areas of consistent and measurable size. Known surface areas were incubated in sets of 20 culture tubes containing liquid growth medium. The subsequent occurrence of visible growth expressed as percent of tubes positive was interpreted as an estimate of the probability of release of at least one viable micro-organism.From these experiments probability of release as a function of microbial concentration in plastic was estimated for exposed interior surface areas of 30.6, 61.2, 91.8 or 122.4 mm². Good agreement of the empirical results with a theoretical mathematical model of the probability of release of contamination from solids was demonstrated. Analysis of the data using the maximum likelihood procedure provided a means of calculating a proportionality constant representing the effective thickness of the exposed area and the characteristics of the recovery procedure.     

Microbiological parameters as indicators of compost maturity

AIMS: The objectives of this study were to determine the changes of microbial properties of pig manure collected from pens with different management strategies and composted using different turning and moisture regimes; relate their association with humification parameters and compost temperature; and identify the most suitable microbial indicators of compost maturity. METHODS AND RESULTS: Six different microbial parameters, including total bacterial count, oxygen consumption rate, ATP content, dehydrogenase activity, and microbial biomass C and N, along with humification parameters [humic acid (HA), fulvic acid (FA) and HA : FA ratio] and compost temperature were monitored during composting. Significant positive correlations were found between temperature and microbial properties, including O2 consumption rate, ATP content, dehydrogenase activity, and microbial biomass N. The humification parameters also showed significant correlations with microbial properties of the manure compost. For instance, HA contents of pig manures was positively correlated with total aerobic heterotrophs, and microbial biomass N and C; and negatively correlated with O2 consumption rate, ATP content, and dehydrogenase activity. Among the six microbial parameters examined, dehydrogenase activity was the most important factor affecting compost temperature and humification parameters. Composting strategies employed in this study affected the speed of composting and time of maturation. If the moisture content is maintained weekly at 60% with a 4-day turning frequency, the pig manure will reach maturity in 56 days. CONCLUSIONS: The composting process went through predictable changes in temperature, microbial properties and chemical components despite differences in the initial pig manure and composting strategies used. Among the six microbial parameters used, dehydrogenase activity is the most suitable indicator of compost maturity. Compared with respiration rate, ATP content and microbial biomass procedures, dehydrogenase activity is the simplest, quickest, and cheapest method that can be used to monitor the stability and maturity of composts. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here show that microbial parameters can be used in revealing differences between composts and compost maturity. The statistical relationship established between humification parameters and microbial parameters, particularly dehydrogenase activity, demonstrates that it is possible to monitor the composting process more easily and rapidly by avoiding longer and more expensive analytical procedures.     

454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants

Activated sludge (AS) contains highly complex microbial communities. In this study, PCR-based 454 pyrosequencing was applied to investigate the bacterial communities of AS samples from 14 sewage treatment plants of Asia (mainland China, Hong Kong, and Singapore), and North America (Canada and the United States). A total of 259 K effective sequences of 16S rRNA gene V4 region were obtained from these AS samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in AS, that is, 1183–3567 OTUs in a sludge sample, at 3% cutoff level and sequencing depth of 16 489 sequences. Clear geographical differences among the AS samples from Asia and North America were revealed by (1) cluster analyses based on abundances of OTUs or the genus/family/order assigned by Ribosomal Database Project (RDP) and (2) the principal coordinate analyses based on OTUs abundances, RDP taxa abundances and UniFrac of OTUs and their distances. In addition to certain unique bacterial populations in each AS sample, some genera were dominant, and core populations shared by multiple samples, including two commonly reported genera of Zoogloea and Dechloromonas, three genera not frequently reported (i.e., Prosthecobacter, Caldilinea and Tricoccus) and three genera not well described so far (i.e., Gp4 and Gp6 in Acidobacteria and Subdivision3 genera incertae sedis of Verrucomicrobia). Pyrosequencing analyses of multiple AS samples in this study also revealed the minority populations that are hard to be explored by traditional molecular methods and showed that a large proportion of sequences could not be assigned to taxonomic affiliations even at the phylum/class levels.