Eliciting expert judgements to underpin our understanding of faecal indicator organism loss from septic tank systems.

Septic tank systems (STS) in rural catchments represent a potential source of microbial pollution to watercourses; however, data concerning the risk of faecal indicator organism (FIO) export from STS to surface waters are scarce. In the absence of empirical data, elicitation of expert judgements can provide an alternative approach to aid understanding of FIO pollution risk from STS. Our study employed a structured elicitation process using the Sheffield Elicitation Framework to obtain expert judgements on the proportion of FIOs likely to be delivered from STS to watercourses, based on 36 scenarios combining: (i) septic tank effluent movement risk, driven by soil hydro-morphological characteristics; (ii) distance of septic tank to watercourse; and (iii) degree of slope. Experts used the tertile method to elicit a range of values representing their beliefs of the proportion of FIOs likely to be delivered to a watercourse for each scenario. The experts judged that 93 % of FIOs would likely be delivered from an STS to a watercourse under the highest risk scenario that combined (i) very high STS effluent movement risk, (ii) STS distance to watercourse <10 m, and (iii) a location on a steep slope with gradient >25 %. Under the lowest risk scenario, the proportion of FIOs reaching a watercourse would likely reduce to 5 %. Expert confidence was high for scenarios that represented extremes of risk, while uncertainty increased for scenarios depicting intermediate risk conditions. The behavioural aggregation process employed to obtain a consensus among the experts proved to be useful for highlighting both areas of strong consensus and high uncertainty. The latter therefore represent priorities for future empirical research to further improve our understanding of potential pollution risk from septic tanks and in turn enable better assessments of potential threats to water quality in rural catchments throughout the world where decentralised wastewater systems are common.

Rapid extraction of high- and low-density microplastics from soil using high-gradient magnetic separation.

Microplastics (MPs) are present in all environments, and concerns over their possible detrimental effects on flora and fauna have arisen. Density separation (DS) is commonly used to separate MPs from soils to allow MP quantification; however, it frequently fails to extract high-density MPs sufficiently, resulting in under-estimation of MP abundances. In this proof-of-concept study, a novel three-stage extraction method was developed, involving high-gradient magnetic separation and removal of magnetic soil (Stage 1), magnetic tagging of MPs using surface modified iron nanoparticles (Stage 2), and high-gradient magnetic recovery of surface-modified MPs (Stage 3). The method was optimised for four different soil types (loam, high­carbon loamy sand, sandy loam and high-clay sandy loam) spiked with different MP types (polyethylene, polyethylene terephthalate, and polytetrafluoroethylene) of different particle sizes (63 µm to 2 mm) as well as polyethylene fibres (2-4 mm). The optimised method achieved average recoveries of 96% for fibres and 92% for particles in loam, 91% for fibres and 87% for particles in high­carbon loamy sand, 96% for fibres and 89% for particles in sandy loam, and 97% for fibres and 94% for particles in high-clay sandy loam. These were significantly higher than recoveries achieved by DS, particularly for fibres and high-density MPs (p < 0.05). To demonstrate the practical application of the HGMS method, it was applied to a farm soil sample, and high-density MP particles were only recovered by HGMS. Furthermore, this study showed that HGMS can recover fibre-aggregate complexes. This improved extraction method will provide better estimates of MP quantities in future studies focused on monitoring the prevalence of MPs in soils.

Water safety plan enhancements with improved drinking water quality detection techniques.

Drinking water quality has been regulated in most European countries for nearly two decades by the drinking water directive 98/83/EC. The directive is now under revision with the goal of meeting stricter demands for safe water for all citizens, as safe water has been recognized as a human right by the United Nations. An important change to the directive is the implementation of a risk-based approach in all regulated water supplies. The European Union Framework Seventh Programme Aquavalens project has developed several new detection technologies for pathogens and indicators and tested them in water supplies in seven European countries. One of the tasks of the project was to evaluate the impact of these new techniques on water safety and on water safety management. Data were collected on risk factors to water safety for five large supplies in Denmark, Germany, Spain and the UK, and for fifteen small water supplies in Scotland, Portugal and Serbia, via a questionnaire aiming to ascertain risk factors and the stage of implementation of Water Safety Plans, and via site-specific surveys known as Sanitary Site Inspection. Samples were collected from the water supplies from all stages of water production to delivery. Pathogens were detected in around 23% of the 470 samples tested. Fecal contamination was high in raw water and even in treated water at the small supplies. Old infrastructure was considered a challenge at all the water supplies. The results showed that some of the technique, if implemented as part of the water safety management, can detect rapidly the most common waterborne pathogens and fecal pollution indicators and therefore have a great early warning potential; can improve water safety for the consumer; can validate whether mitigation methods are working as intended; and can confirm the quality of the water at source and at the tap.

Diversity-Function Relationships in Natural, Applied, and Engineered Microbial Ecosystems.

The connection between ecosystem function and taxonomic diversity has been of interest and relevance to macroecologists for decades. After many years of lagging behind due to the difficulty of assigning both taxonomy and function to poorly distinguishable microscopic cells, microbial ecology now has access to a suite of powerful molecular tools which allow its practitioners to generate data relating to diversity and function of a microbial community on an unprecedented scale. Instead, the problem facing today's microbial ecologists is coupling the ease of generation of these datasets with the formulation and testing of workable hypotheses relating the diversity and function of environmental, host-associated, and engineered microbial communities. Here, we review the current state of knowledge regarding the links between taxonomic alpha- and beta-diversity and ecosystem function, comparing our knowledge in this area to that obtained by macroecologists who use more traditional techniques. We consider the methodologies that can be applied to study these properties and how successful they are at linking function to diversity, using examples from the study of model microbial ecosystems, methanogenic bioreactors (anaerobic digesters), and host-associated microbiota. Finally, we assess ways in which our newly acquired understanding might be used to manipulate diversity in ecosystems of interest in order to improve function for the benefit of us or the environment in general through the provision of ecosystem services.

Assembly of microbial communities in replicate nutrient-cycling model ecosystems follows divergent trajectories, leading to alternate stable states.

We studied in detail the reproducibility of community development in replicate nutrient-cycling microbial microcosms that were set up identically and allowed to develop under the same environmental conditions. Multiple replicate closed microcosms were constructed using pond sediment and water, enriched with cellulose and sulphate, and allowed to develop over several months under constant environmental conditions, after which their microbial communities were characterized using 16S rRNA gene sequencing. Our results show that initially similar microbial communities can follow alternative - yet stable - trajectories, diverging in time in a system size-dependent manner. The divergence between replicate communities increased in time and decreased with larger system size. In particular, notable differences emerged in the heterotrophic degrader communities in our microcosms; one group of steady state communities was enriched with Firmicutes, while the other was enriched with Bacteroidetes. The communities dominated by these two phyla also contained distinct populations of sulphate-reducing bacteria. This biomodality in community composition appeared to arise during recovery from a low-diversity state that followed initial cellulose degradation and sulphate reduction.

Phylogenetic diversity of ceftriaxone resistance and the presence of extended-spectrum ß-lactamase genes in the culturable soil resistome.

The aim of this study was to determine the phylogenetic diversity of ceftriaxone resistance and the presence of known extended-spectrum ß-lactamase (ESBL) genes in culturable soil resistomes. Libraries of soil bacterial isolates resistant to ceftriaxone were established from six physicochemically diverse soils collected in Hawaii (USA) and Israel. The phylogenetic affiliation, ceftriaxone and multidrug resistance levels, and presence of known ESBL genes of the isolates were determined. The soil bacterial isolates were phylogenetically grouped with the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Ceftriaxone minimum inhibitory concentrations (MICs) largely followed the phylogeny structure and higher levels of ceftriaxone resistance corresponded to higher multidrug resistance. Three distinct blaTEM variants were detected in soil bacterial isolates belonging to nine different genera. In conclusion, the culturable soil resistomes for ceftriaxone exhibited high phylogenetic diversity and multidrug resistance. blaTEM was the only known ESBL detected in the soil resistomes, and its distribution in different phylogenetic groups suggests its ubiquitous presence and/or possible horizontal gene transfer within the soil microbiomes.

Resistance of Undisturbed Soil Microbiomes to Ceftriaxone Indicates Extended Spectrum ß-Lactamase Activity.

Emergence and spread of antibiotic resistance, and specifically resistance to third generation cephalosporins associated with extended spectrum ß-lactamase (ESBL) activity, is one of the greatest epidemiological challenges of our time. In this study we addressed the impact of the third generation cephalosporin ceftriaxone on microbial activity and bacterial community composition of two physically and chemically distinct undisturbed soils in highly regulated microcosm experiments. Surprisingly, periodical irrigation of the soils with clinical doses of ceftriaxone did not affect their microbial activity; and only moderately impacted the microbial diversity (α and ß) of the two soils. Corresponding slurry experiments demonstrated that the antibiotic capacity of ceftriaxone rapidly diminished in the presence of soil, and ∼70% of this inactivation could be explained by biological activity. The biological nature of ceftriaxone degradation in soil was supported by microcosm experiments that amended model Escherichia coli strains to sterile and non-sterile soils in the presence and absence of ceftriaxone and by the ubiquitous presence of ESBL genes (blaTEM, blaCTX-M, and blaOXA) in soil DNA extracts. Collectively, these results suggest that the resistance of soil microbiomes to ceftriaxone stems from biological activity and even more, from broad-spectrum ß-lactamase activity; raising questions regarding the scope and clinical implications of ESBLs in soil microbiomes.

Estimating the prevalence of potential enteropathogenic Escherichia coli and intimin gene diversity in a human community by monitoring sanitary sewage.

Presently, the understanding of bacterial enteric diseases in the community and their virulence factors relies almost exclusively on clinical disease reporting and examination of clinical pathogen isolates. This study aimed to investigate the feasibility of an alternative approach that monitors potential enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) prevalence and intimin gene (eae) diversity in a community by directly quantifying and characterizing target virulence genes in the sanitary sewage. The quantitative PCR (qPCR) quantification of the eae, stx1, and stx2 genes in sanitary sewage samples collected over a 13-month period detected eae in all 13 monthly sewage samples at significantly higher abundance (93 to 7,240 calibrator cell equivalents [CCE]/100 ml) than stx1 and stx2, which were detected sporadically. The prevalence level of potential EPEC in the sanitary sewage was estimated by calculating the ratio of eae to uidA, which averaged 1.0% (σ = 0.4%) over the 13-month period. Cloning and sequencing of the eae gene directly from the sewage samples covered the majority of the eae diversity in the sewage and detected 17 unique eae alleles belonging to 14 subtypes. Among them, eae-ß2 was identified to be the most prevalent subtype in the sewage, with the highest detection frequency in the clone libraries (41.2%) and within the different sampling months (85.7%). Additionally, sewage and environmental E. coli isolates were also obtained and used to determine the detection frequencies of the virulence genes as well as eae genetic diversity for comparison.

Impact of substratum surface on microbial community structure and treatment performance in biological aerated filters.

The impact of substratum surface property change on biofilm community structure was investigated using laboratory biological aerated filter (BAF) reactors and molecular microbial community analysis. Two substratum surfaces that differed in surface properties were created via surface coating and used to develop biofilms in test (modified surface) and control (original surface) BAF reactors. Microbial community analysis by 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis (DGGE) showed that the surface property change consistently resulted in distinct profiles of microbial populations during replicate reactor start-ups. Pyrosequencing of the bar-coded 16S rRNA gene amplicons surveyed more than 90% of the microbial diversity in the microbial communities and identified 72 unique bacterial species within 19 bacterial orders. Among the 19 orders of bacteria detected, Burkholderiales and Rhodocyclales of the Betaproteobacteria class were numerically dominant and accounted for 90.5 to 97.4% of the sequence reads, and their relative abundances in the test and control BAF reactors were different in consistent patterns during the two reactor start-ups. Three of the five dominant bacterial species also showed consistent relative abundance changes between the test and control BAF reactors. The different biofilm microbial communities led to different treatment efficiencies, with consistently higher total organic carbon (TOC) removal in the test reactor than in the control reactor. Further understanding of how surface properties affect biofilm microbial communities and functional performance would enable the rational design of new generations of substrata for the improvement of biofilm-based biological treatment processes.

Community history affects the predictability of microbial ecosystem development.

Microbial communities mediate crucial biogeochemical, biomedical and biotechnological processes, yet our understanding of their assembly, and our ability to control its outcome, remain poor. Existing evidence presents conflicting views on whether microbial ecosystem assembly is predictable, or inherently unpredictable. We address this issue using a well-controlled laboratory model system, in which source microbial communities colonize a pristine environment to form complex, nutrient-cycling ecosystems. When the source communities colonize a novel environment, final community composition and function (as measured by redox potential) are unpredictable, although a signature of the community's previous history is maintained. However, when the source communities are pre-conditioned to their new habitat, community development is more reproducible. This situation contrasts with some studies of communities of macro-organisms, where strong selection under novel environmental conditions leads to reproducible community structure, whereas communities under weaker selection show more variability. Our results suggest that the microbial rare biosphere may have an important role in the predictability of microbial community development, and that pre-conditioning may help to reduce unpredictability in the design of microbial communities for biotechnological applications.

Towards a hybrid anaerobic digester-microbial fuel cell integrated energy recovery system: an overview of the development of an electrogenic biofilm.

An electrogenic biofilm was developed on a macroporous chitosan-carbon nanotube (CHIT-CNT) electrode under constant poised potential (-0.25V versus Ag/AgCl reference electrode) and flow through conditions utilizing the effluent of an anaerobic digester as both the inoculant and substrate for the electrogenic biofilm. After 125 days of inoculation the bioelectrode demonstrated an open circuit potential of -0.62V and a current density of 9.43µAcm(-3) (at -0.25V). Scanning electron microscopy images indicate thorough surface coverage of the biofilm with a high density of bacterial nanowires physically connecting bacteria to bacteria and bacteria to carbon nanotube (electrode surface) suggesting the nanowires are electrically conductive. DGGE was used to identify the major bacterial and archaeal populations.

Spatial and temporal variation in enterococcal abundance and its relationship to the microbial community in Hawaii beach sand and water.

Recent studies have reported high levels of fecal indicator enterococci in marine beach sand. This study aimed to determine the spatial and temporal variation of enterococcal abundance and to evaluate its relationships with microbial community parameters in Hawaii beach sand and water. Sampling at 23 beaches on the Island of Oahu detected higher levels of enterococci in beach foreshore sand than in beach water on a mass unit basis. Subsequent 8-week consecutive samplings at two selected beaches (Waialae and Kualoa) consistently detected significantly higher levels of enterococci in backshore sand than in foreshore/nearshore sand and beach water. Comparison between the abundance of enterococci and the microbial communities showed that enterococci correlated significantly with total Vibrio in all beach zones but less significantly with total bacterial density and Escherichia coli. Samples from the different zones of Waialae beach were sequenced by 16S rRNA gene pyrosequencing to determine the microbial community structure and diversity. The backshore sand had a significantly more diverse community and contained different major bacterial populations than the other beach zones, which corresponded to the spatial distribution pattern of enterococcal abundance. Taken together, multiple lines of evidence support the possibility of enterococci as autochthonous members of the microbial community in Hawaii beach sand.

Bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong, China.

We investigated the bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong in the Yunnan Province, China, using direct molecular analyses. The Langpu (LP) laminated mat was found by the side of a boiling pool with temperature of 60-65 °C and a pH of 8.5, while the Tengchong (TC) streamer mat consisted of white streamers in a slightly acidic (pH 6.5) hot pool outflow with a temperature of 72 °C. Four 16S rRNA gene clone libraries were constructed and restriction enzyme analysis of the inserts was used to identify unique sequences and clone frequencies. From almost 200 clones screened, 55 unique sequences were retrieved. Phylogenetic analysis showed that the LP mat consisted of a diverse bacterial population [Cyanobacteria, Chloroflexi, Chlorobia, Nitrospirae, 'Deinococcus-Thermus', Proteobacteria (alpha, beta and delta subdivisions), Firmicutes, Bacteroidetes and Actinobacteria], while the archaeal population was dominated by methanogenic Euryarchaeota and Crenarchaeota. In contrast, the TC streamer mat consisted of a bacterial population dominated by Aquificae, while the archaeal population also contained Korarchaeota as well as Crenarchaeota and methanogenic Euryarchaeota. These mats harboured clone sequences affiliated to unidentified lineages, suggesting that they are a potential source for discovering novel bacteria and archaea.

Novel electrochemically active bacterium phylogenetically related to Arcobacter butzleri, isolated from a microbial fuel cell.

Exoelectrogenic bacteria are organisms that can transfer electrons to extracellular insoluble electron acceptors and have the potential to be used in devices such as microbial fuel cells (MFCs). Currently, exoelectrogens have been identified in the Alpha-, Beta-, Gamma- and Deltaproteobacteria, as well as in the Firmicutes and Acidobacteria. Here, we describe use of culture-independent methods to identify two members of the genus Arcobacter in the Epsilonproteobacteria that are selectively enriched in an acetate-fed MFC. One of these organisms, Arcobacter butzleri strain ED-1, associates with the electrode and rapidly generates a strong electronegative potential as a pure culture when it is supplied with acetate. A mixed-community MFC in which approximately 90% of the population is comprised of the two Arcobacter species generates a maximal power density of 296 mW/liter. This demonstration of exoelectrogenesis by strain ED-1 is the first time that this property has been shown for members of this genus.

Microbial biogeography of six salt lakes in Inner Mongolia, China, and a salt lake in Argentina.

We used cultivation-independent methods to investigate the prokaryotic biogeography of the water column in six salt lakes in Inner Mongolia, China, and a salt lake in Argentina. These lakes had different salt compositions and pH values and were at variable geographic distances, on both local and intercontinental scales, which allowed us to explore the microbial community composition within the context of both contemporary environmental conditions and geographic distance. Fourteen 16S rRNA gene clone libraries were constructed, and over 200 16S rRNA gene sequences were obtained. These sequences were used to construct biotic similarity matrices, which were used in combination with environmental similarity matrices and a distance matrix in the Mantel test to discover which factors significantly influenced biotic similarity. We showed that archaeal biogeography was influenced by contemporary environmental factors alone (Na+, CO3(2-), and HCO3(-) ion concentrations; pH; and temperature). Bacterial biogeography was influenced both by contemporary environmental factors (Na+, Mg2+, and HCO3(-) ion concentrations and pH) and by geographic distance.

Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China.

BACKGROUND: We are profoundly ignorant about the diversity of viruses that infect the domain Archaea. Less than 100 have been identified and described and very few of these have had their genomic sequences determined. Here we report the genomic sequence of a previously undescribed archaeal virus. RESULTS: Haloarchaeal strains with 16S rRNA gene sequences 98% identical to Halorubrum saccharovorum were isolated from a hypersaline lake in Inner Mongolia. Two lytic viruses infecting these were isolated from the lake water. The BJ1 virus is described in this paper. It has an icosahedral head and tail morphology and most likely a linear double stranded DNA genome exhibiting terminal redundancy. Its genome sequence has 42,271 base pairs with a GC content ofapproximately 65 mol%. The genome of BJ1 is predicted to encode 70 ORFs, including one for a tRNA. Fifty of the seventy ORFs had no identity to data base entries; twenty showed sequence identity matches to archaeal viruses and to haloarchaea. ORFs possibly coding for an origin of replication complex, integrase, helicase and structural capsid proteins were identified. Evidence for viral integration was obtained. CONCLUSION: The virus described here has a very low sequence identity to any previously described virus. Fifty of the seventy ORFs could not be annotated in any way based on amino acid identities with sequences already present in the databases. Determining functions for ORFs such as these is probably easier using a simple virus as a model system.