Gavin Collins

Aerobic denitrification in permeable Wadden Sea sediments

Permeable or sandy sediments cover the majority of the seafloor on continental shelves worldwide, but little is known about their role in the coastal nitrogen cycle. We investigated the rates and controls of nitrogen loss at a sand flat (Janssand) in the central German Wadden Sea using multiple experimental approaches, including the nitrogen isotope pairing technique in intact core incubations, slurry incubations, a flow-through stirred retention reactor and microsensor measurements. Results indicate that permeable Janssand sediments are characterized by some of the highest potential denitrification rates (⩾0.19 mmol N m−2 h−1) in the marine environment. Moreover, several lines of evidence showed that denitrification occurred under oxic conditions. In intact cores, microsensor measurements showed that the zones of nitrate/nitrite and O2 consumption overlapped. In slurry incubations conducted with 15NO3− enrichment in gas-impermeable bags, denitrification assays revealed that N2 production occurred at initial O2 concentrations of up to ∼90 μM. Initial denitrification rates were not substantially affected by O2 in surficial (0–4 cm) sediments, whereas rates increased by twofold with O2 depletion in the at 4–6 cm depth interval. In a well mixed, flow-through stirred retention reactor (FTSRR), 29N2 and 30N2 were produced and O2 was consumed simultaneously, as measured online using membrane inlet mass spectrometry. We hypothesize that the observed high denitrification rates in the presence of O2 may result from the adaptation of denitrifying bacteria to recurrent tidally induced redox oscillations in permeable sediments at Janssand.

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.

Reactor performance and microbial community dynamics during anaerobic biological treatment of wastewaters at 16–37 °C

The anaerobic biological treatment of volatile fatty acid (VFA) - and sucrose - based wastewaters was investigated in two anaerobic bioreactors, R1 and R2, over a 300-day trial period. During the trial, the operating temperature of both reactors was lowered, in a stepwise fashion, from 37 to 16 degrees C. The VFA-fed reactor maintained an excellent level of performance, regardless of operating temperature, reaching COD removal efficiencies of 95% at 18 degrees C, and a biogas methane content in excess of 70% at 16 degrees C, at an imposed OLR of 20 kg COD m(-3) d(-1). However, an increase in the applied liquid upflow velocity to the bottom chamber of the reactor from 5 to 7.5 m h(-1)on day 236 resulted in a considerable decline in reactor performance. COD removal efficiencies in excess of 80% were achieved by the sucrose-fed reactor at 18 degrees C, at an imposed OLR of 20 kg COD m(-3) d(-1). An increase in the liquid upflow velocity applied to the sucrose-fed reactor resulted in enhanced reactor performance and stability, with respect to decreasing temperature. The different responses of both reactors to increased upflow velocity was associated with variations in the microbial population structure of the sludges, as determined by culture-independant molecular approaches, specifically the presence of high levels of delta-Proteobacteria and hydrogenotrophic methanogens in the VFA-fed biomass. High levels of Methanomicrobiales sp., in particular Methanocorpusculum parvum sp., were observed in both R1 and R2 during the trial. There was a distinct shift from acetoclastic methanogenic dominance to hydrogenotrophic dominance in both reactors in response to a decrease in the operating temperature.

Microbial community structure and methanogenic activity during start-up of psychrophilic anaerobic digesters treating synthetic industrial wastewaters

Culture-independent, molecular techniques were applied to the characterization of microbial communities of an anaerobic granular sludge obtained from a full-scale digester. Procedures were optimised for total DNA recovery and polymerase chain reaction (PCR) amplification of 16S rDNA using archaea- and eubacteria-specific oligonucleotide primers. Cloned PCR products were subsequently screened by amplified rDNA restriction analysis to identify operational taxonomic units (OTUs). Inserts from clones representing each OTU were sequenced and phylogenetic trees were prepared. In addition, the microbial communities were characterised using terminal restriction fragment length polymorphism (T-RFLP). The specific methanogenic activity of the biomass, against various substrates, was also ascertained. Two anaerobic bioreactors were seeded with granular and non-granular (i.e. crushed) aliquots of the characterised sludge, respectively, and used to investigate the treatment of a volatile fatty acid (VFA)-based synthetic wastewater, at a loading rate of 5 kg COD m(-3) day(-1) at low ambient temperatures (18 degrees C). DNA was isolated from sludge samples during the test period and shifts in archaeal and eubacterial population structures were elucidated. The start-up period was successful with methane yields and COD removal efficiencies of 60-75% and 65-85%, respectively. Specific methanogenic activities of reactor biomass, obtained at the conclusion of the trial, indicated the development of psychrotolerant biomass during the 90-day experiment. Furthermore, the efficacy of T-RFLP as a molecular tool for use in the surveyance of engineered ecosystems was confirmed.

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.

Quantitative and qualitative analysis of methanogenic communities in mesophilically and psychrophilically cultivated anaerobic granular biofilims

Anaerobic granulation describes the self-immobilisation of methanogenic consortia into dense, particulate biofilms. This procedure underpins the operation of several categories of high-rate anaerobic wastewater treatment system. Full-scale anaerobic granular sludge plants have been generally operated in the mesophilic (20-45 degrees C) or thermophilic (45-65 degrees C) temperature range. On the other hand, recent studies highlighted the economic advantages of treating wastewaters at their discharge temperatures (mostly under 18 degrees C), removing a costly heating process and increasing net biogas yield. However, as yet, relatively little information is available about the microbial behaviour and interactions in anaerobic granular sludge formed under psychrophilic conditions. To this end, and in order to provide a microbial insight into low-temperature anaerobic granulation, we monitored the changes in methanogenic community structure, associated with the changes in process performance. Three, laboratory-scale, expanded granular sludge bed (EGSB) bioreactors treating a synthetic glucose wastewater were tested at two temperatures of 37+/-1 degrees C (R1) and 15+/-1 degrees C (R2 and 3). Quantitative real-time PCR and specific methanogenic activity assays highlighted a community shift towards hydrogenotrophic methanogens, particularly the order Methanomicrobiales in the low-temperature bioreactors. Corresponding to this, denaturing gradient gel electrophoresis (DGGE) analysis identified the emergence and maintenance of a Methanocorpusculum-like organism. Our results indicate that hydrogenotrophic methanogens, particularly the Methanomicrobiales-related populations, are likely to play important roles in low-temperature anaerobic granular sludge systems. This suggests that the process efficiency could be improved by facilitating the growth and retention of this group.

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.

Long-term (1243 days), low-temperature (4–15 °C), anaerobic biotreatment of acidified wastewaters: Bioprocess performance and physiological characteristics

The feasibility of long-term (>3 years), low-temperature (4-15 degrees C) and anaerobic bioreactor operation, for the treatment of acidified wastewater, was investigated. A hybrid, expanded granular sludge bed-anaerobic filter bioreactor was seeded with a mesophilic inoculum and employed for the mineralization of moderate-strength (3.75-10 kg chemical oxygen demand (COD)m(-3)) volatile fatty acid-based wastewaters at 4-15 degrees C. Bioprocess performance was assessed in terms of COD removal efficiency (CODRE), methane biogas concentration, and yield, and biomass retention. Batch specific methanogenic activity assays were performed to physiologically characterise reactor biomass. Despite transient disimprovements, CODRE and methane biogas concentrations exceeded 80% and 65%, respectively, at an applied organic loading rate (OLR) of 10 kgCODm(-3)d(-1) between 9.5 and 15 degrees C (sludge loading rate (SLR), 0.6 kgCOD kg[VSS](-1)d(-1)). Over 50% of the granular sludge bed was lost to disintegration during operation at 9.5 degrees C, warranting a reduction in the applied OLR to 3.75-5 kgCODm(-3)d(-1) (SLR, c. 0.4-0.5kgCOD kg[VSS](-1)d(-1)). From that point forward, remarkably stable and efficient performance was observed during operation at 4-10 degrees C, with respect to CODRE (>or=82%), methane biogas concentration (>70%) and methane yields (>4l(Methane)d(-1)), suggesting the adaptation of our mesophilic inoculum to psychrophilic operating conditions. Physiological activity assays indicated the development of psychroactive syntrophic and methanogenic populations, including the emergence of putatively psychrophilic propionate-oxidising and hydrogenotrophic methanogenic activity. The data suggest that mesophilic inocula can physiologically adapt to sub-optimal operational temperatures: treatment efficiencies and sludge loading rates at 4 degrees C (day, 1243) were comparable to those achieved at 15 degrees C (day 0). Furthermore, long-term, low-temperature bioreactor operation may act as a selective enrichment for psychrophilic methanogenic activity from mesophilic inocula. The observed efficient and stable bioprocess performance highlights the potential for long-term, low-temperature bioreactor operation.

Distribution, Localization, and Phylogeny of Abundant Populations of Crenarchaeota in Anaerobic Granular Sludge

ABSTRACT Eight anaerobic granular sludges were surveyed for Crenarchaeota using rRNA gene cloning. Microbial arrangement and substrate uptake patterns were elucidated by fluorescent in situ hybridization and beta imaging. Group 1.3 Crenarchaeota represented up to 50% of Archaea and 25% of the total microbiota in five sludges. Crenarchaeota were localized in close association with methanogenic Archaea.

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.