Vincent O’Flaherty

DNA extraction method affects microbial community profiles from soils and sediment

To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles generated. This was reflected both in the number of bands or ribotypes detected from each sample and in subsequent principle coordinate analysis and unweighted-pair group method using arithmetic average analyses. The methods also differed significantly in their robustness, i.e. reproducibility across multiple analyses. Two methods, both based on bead beating, were demonstrated to be suitable for comparative studies of a range of soil and sediment types.

Quantitative and qualitative analyses of methanogenic community development in high-rate anaerobic bioreactors.

Methanogenic community structure and population dynamics were investigated in two anaerobic reactors treating a dairy wastewater, an Inverted Fluidized Bed (IFB) and Expanded Granular Sludge Bed (EGSB). A combination of real-time PCR, denaturing gradient gel electrophoresis and statistical techniques was employed. Distinct methanogenic communities developed in the IFB and EGSB reactors reflecting step-wise reductions in the applied hydraulic retention time from 72 to 12 h during the 200-day trial. The aceticlastic family Methanosarcinaceae was only detected in the IFB and the order Methanomicrobiales was also much more abundant in this reactor, while the aceticlastic family Methanosaetaceae was more abundant in the EGSB. The hydrogenotrophic order, Methanobacteriales, predominated in both reactors under all applied operational conditions. Non-metric multidimensional scaling (NMS) and moving-window analyses, based on absolute and relative abundance quantification data, demonstrated that the methanogenic communities developed in a different manner in the IFB, compared to the EGSB reactor. In our study, relative abundance-based quantification by NMS and moving-window analysis appeared to be a valuable molecular approach that was more applicable to reflect the changes in the anaerobic digestion process than approaches based either on qualitative analysis, or solely on absolute quantification of the various methanogenic groups. The overall results and findings provided a comparative, quantitative and qualitative insight into anaerobic digestion processes, which could be helpful for better future reactor design and process control.

A metaproteomic approach gives functional insights into anaerobic digestion.

Aims:  The objective of this work was to provide functional evidence of key metabolic pathways important for anaerobic digestion processes through the identification of highly expressed proteins in a mixed anaerobic microbial consortium.

Quantitative and qualitative transitions of methanogen community structure during the batch anaerobic digestion of cheese-processing wastewater.

Qualitative and quantitative shifts in methanogen community structure, associated with process performance data, were investigated during the batch anaerobic digestion of a cheese-processing wastewater, whey permeate. Denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques were applied to obtain qualitative and quantitative microbial data sets, respectively, based on methanogen 16S rRNA genes. Throughout the operation, dynamic variations in both qualitative and quantitative community structures were observed, with repeated shifts in dominance between the aceticlastic Methanosarcinaceae (suggested mainly by the detection of a Methanosarcina-like population) and the hydrogenotrophic Methanomicrobiales (suggested mainly by the detection of a Methanofollis-like population). This trend corresponded well to the diauxic utilization of acetate and longer-chain fatty acids (C3–C6), mainly propionate. Joint-plot non-metric multidimensional scaling (NMS) analysis demonstrated that the qualitative and quantitative community shifts had significant correlations with the composition of residual organic acids and the methane production rate, respectively. This suggests the potential use of microbial community shift analysis as an indicative tool for diagnosing anaerobic digestion processes. The results suggest that more attention should be directed to quantitative, as well as qualitative, approaches for a better understanding of anaerobic digestion, particularly in terms of biogas production efficiency, under dynamic and transitional conditions.

Microbial Exposure Assessment of Waterborne Pathogens

ABSTRACT The large number of waterborne illnesses in Ireland and worldwide has highlighted the need to enhance strategies that minimize human exposure to pathogens in drinking water supplies. Waterborne pathogens of public concern together with relevant national and international legislation are reviewed in this study. Cryptosporidium species and pathogenic Escherichia coli are among pathogens of primary concern. The organisms originate from the gastrointestinal tract of animals and humans. They may be associated with persistent contamination of water sources, survive for long periods in the environment, and, in particular in the case of Cryptosporidium species, may survive in chlorinated water supplies. Prevention of waterborne infection should emphasize source protection in addition to water treatment. Risk assessment models can play an important role in protecting natural water systems from contamination with these pathogens. Qualitative approaches can provide an effective means of assessing risks with minimum resources and limited data; however, they lack the precision and predictive ability of fully quantitative approaches. Thirteen quantitative simulation models that could potentially be used for modeling bacterial pollutants in agricultural watersheds have been assessed in this study. No one model suits all modeling criteria. Pathogen predictions have proved variable and no model was capable of accounting for all geological and hydrological factors in addition to modeling the physical transport of bacteria in surface runoff. This assessment summarizes commonly used models and their capacity to model water pollution while also providing a good reference point for the microbial risk assessment of waterborne pathogens.

Low-temperature anaerobic digestion for wastewater treatment

Methanogenesis is an important biogeochemical process for the degradation of organic matter within cold environments, and is associated with the release of the potent greenhouse gas, methane. Cold methanogenesis has been harnessed, in engineered systems, as low-temperature anaerobic digestion (LTAD) for wastewater treatment and bioenergy generation. LTAD represents a nascent wastewater treatment biotechnology, which offers an attractive alternative to conventional aerobic and anaerobic processes. Successful, high-rate, LTAD of sewage and industrial wastewaters (e.g. from the brewery, food-processing and pharmaceutical sectors), with concomitant biogas generation, has been demonstrated at laboratory-scale and pilot-scale. A holistic, polyphasic approach, which integrates bioprocess, physiological and molecular biological datasets has been critical to the development of the LTAD concept.

Temperature dependent (37–15 °C) anaerobic digestion of a trichloroethylene-contaminated wastewater

The impact of a trichloroethylene (TCE) contaminated wastewater on the microbial community structure of an anaerobic granular biomass at 15°C compared to 37°C was investigated. Four expanded granular sludge bed (EGSB) bioreactors (R1-R4) were employed in pairs at 37 and 15°C. The influents of one of each pair were supplemented with increasing concentrations of TCE (max. 60 mgl(-1)). At 37°C, stable operation was maintained with 88% COD removal and >99% TCE removal at maximum influent TCE concentrations. R3 performance decreased at influent TCE concentration of 60 mgl(-1), although TCE removal rates of >97% were recorded. Archaeal community analysis via clone library and quantitative polymerase chain reaction (qPCR) analysis, and bacterial community analysis via denaturing gradient gel electrophoresis (DGGE), indicated that temperature resulted in a greater change in community structure than the presence of TCE, and clones related to cold adaptation of biomass were identified at 15°C.

Charge transport through Geobacter sulfurreducens biofilms grown on graphite rods

Biofilms of the electroactive bacterium Geobacter sulfurreducens were induced to grow on graphite-rod electrodes under a potential of 0 V (vs Ag/AgCl) in the presence of acetate as an electron donor. Increased anodic currents for bioelectrocatalytic oxidation of acetate were obtained when the electrodes were incubated for longer periods with periodic electron-donor feeding. The maximum current density for acetate oxidation increased 2.8-fold, and the biofilm thickness increased by 4.25-fold, over a time period of 83-147 h. Cyclic voltammetry in the presence of acetate supports a model of heterogeneous electron transfer, one electron at time, from biofilm to electrode through a dominant redox species centered at -0.41 V vs Ag/AgCl. Voltammetry performed under nonturnover conditions provided an estimate of the surface coverage of the redox species of 25 nmol/cm(2). This value was used to estimate a redox species concentration of 7.3 mM within the 34-μm-thick biofilm and a charge-transport diffusion coefficient of 3.6 × 10(-7) cm(2)/s. This value of diffusion coefficient is greater than that observed in traditional thin-film voltammetric studies with redox polymer films containing much higher surface concentrations of redox species and might be associated with proton transport to ensure electroneutrality within the biofilm upon electrolysis.

Effect of seed sludge and operation conditions on performance and archaeal community structure of low-temperature anaerobic solvent-degrading bioreactors.

Two laboratory-scale expanded granular sludge bed (EGSB) anaerobic bioreactors (R1 and R2) were inoculated with biomass from different mesophilic (37 degrees C) treatment plants, and used for the treatment of an organic solvent-based wastewater at 9-14 degrees C at applied organic loading rates (OLRs) of 1.2-3.6kg chemical oxygen demand (COD)m(-3)d(-1). Replicated treatment performance was observed at 10-14 degrees C, which suggested the feasibility of the process at pilot-scale. Stable and efficient COD removal, along with high methane productivity, was demonstrated at 9 degrees C at an applied OLR of 2.4kgCODm(-3)d(-1). Clonal libraries and fluorescence in situ hybridization (FISH) indicated that the seed sludges were dominated (>60%) by acetoclastic Methanosaeta-like organisms. Specific methanogenic activity (SMA) profiles indicated shifts in the physiological profiles of R1 and R2 biomass, including the development of psychrotolerant methanogenic activity. Acetoclastic methanogenesis represented the primary route of methane production in R1 and R2, which is in contrast with several previous reports from low-temperature bioreactor trials. A reduction in the abundance of Methanosaeta-like clones (R2), along with the detection of hydrogenotrophic methanogenic species, coincided with altered granule (sludge) morphology and the development of hydrogenotrophic SMA after prolonged operation at 9 degrees C.

Microbial community dynamics associated with biomass granulation in low-temperature (15°C) anaerobic wastewater treatment bioreactors

Granular biofilms underpin the operation of several categories of anaerobic wastewater treatment bioreactors. Recent studies have demonstrated the feasibility of treating both industrial and domestic wastewaters at their discharge temperatures (usually <18 degrees C), thereby avoiding the heating expenses of mesophilic (20-45 degrees C) or thermophilic (45-65 degrees C) treatments. Previous low-temperature trials used mesophilic inocula and little information is available on the viability of low-temperature anaerobic granulation. Six laboratory-scale, expanded granular sludge bed bioreactors (R1-6) were operated at 15 degrees C (R1-2 and R4-5) and 37 degrees C (R3 and R6). R1-3 were fed glucose-based wastewater and R4-6 were fed volatile fatty acid-based wastewater. Quantitative real-time PCR and qualitative denaturing gradient gel electrophoresis of 16S rRNA genes identified the dominance of Methanomicrobiales (mainly Methanocorpusculum-like organisms) during low-temperature granulation. Granulation only occurred in glucose-fed bioreactors. The results suggest that (i) granulation is feasible in low-temperature bioreactors; (ii) carbohydrate decomposition likely favoured granulation, (iii) Methanocorpusculum-like organisms play a critical role in low-temperature granulation.