Vincent O'Flaherty

Effect of pH on growth kinetics and sulphide toxicity thresholds of a range of methanogenic, syntrophic and sulphate-reducing bacteria

Abstract The effect of pH on growth rates and sulphide toxicity thresholds of a variety of key bacterial groups involved in anaerobic digestion is described. At pH 7·0–7·5, the growth rates of methane-producing bacteria (MPB) and sulphate-reducing bacteria (SRB) were similar. Above and below this pH range, MPB and SRB, respectively, have more favourable growth properties. Inhibition of all the bacterial groups studied was related to the total sulphide concentration in the pH range 7·2–8·5. Propionate-utilising SRB were the most sensitive of the bacterial groups, both in pure culture and in sludge samples, to high concentrations of total sulphide. At lower pH levels (6·8–7·2), the levels of sulphide which resulted in 50% inhibition of the growth of the bacterial groups were similar, although values obtained were higher for sludge samples than for pure cultures.

Methanogenic population structure in a variety of anaerobic bioreactors

The methanogenic community structures of six anaerobic sludges were examined using culture-independent techniques. The sludges were obtained from full-scale and laboratory-scale bioreactors, treating a variety of low- and high-strength, simple and complex wastewaters at psychrophilic (10-14 degrees C), mesophilic (37 degrees C) and thermophilic (55 degrees C) temperatures. Amplified rDNA restriction analysis identified 18 methanogenic operational taxonomic units in the six samples. 16S rRNA gene sequencing and phylogenetic reconstruction demonstrated that five separate groups of methanogens were represented with Methanosaeta-like species dominant in all sludges, but particularly in samples from a psychrophilic bioreactor treating low-strength synthetic sewage (75% of all clones detected).

Quantitative analysis of methanogenic community dynamics in three anaerobic batch digesters treating different wastewaters.

Quantitative changes in methanogenic community structures, associated with performance data, were investigated in three anaerobic batch digesters treating synthetic glucose medium, whey permeate, and liquefied sewage sludge. All digesters were initially seeded with anaerobic sludge obtained from a local municipal wastewater treatment plant. Dynamics of methanogenic populations were monitored, at order and family levels, using real-time PCR based on the 16S rRNA gene. The molecular monitoring revealed that, in each digester, the quantitative structure of methanogenic community varied continuously over treatment time and the variation corresponded well to the changes in chemical profiles. Biphasic production of methane, associated with successive increases in aceticlastic (mainly Methanosarcinaceae) and hydrogenotrophic (mainly Methanomicrobiales) methanogenic groups, was observed in each digester. This corresponded to the diauxic utilization of acetate and longer-chain volatile fatty acids (C(3)-C(6)), mainly propionate. Additionally, the non-metric multidimensional scaling (NMDS) analysis of the quantification results demonstrated that the community shift patterns in three digesters were totally different from each other. Considering that the operating conditions in all trials were identical except substrates, the differences in quantitative shift profiles were suggested to be due to the different substrate compositions. This implied that the composition of wastewater could affect the evolution of quantitative methanogenic community structure in an anaerobic process. Overall, our results suggested that more attention to quantitative as well as qualitative approaches on microbial communities is needed for fundamental understanding of anaerobic processes, particularly under dynamic or transitional conditions.

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.

Anaerobic Granular Sludge Bioreactor Technology

Anaerobic digestion is a mature wastewater treatment technology, with worldwide application. The predominantly applied bioreactor designs, such as the upflow anaerobic sludge blanket and expanded granular sludge bed, are based on the spontaneous formation of granular sludge. Despite the exploitation of granular reactors at full-scale for more than two decades, the mechanisms of granulation are not completely understood and numerous theories have been put forward to describe the process from a biological, ecological and engineering point of view. New technological opportunities are emerging for anaerobic digestion, aided by an improved understanding of microbiological and environmental factors affecting the formation and activity of anaerobic granular sludge.

Long-Term Persistence and Leaching of Escherichia coli in Temperate Maritime Soils

ABSTRACT Enteropathogen contamination of groundwater, including potable water sources, is a global concern. The spreading on land of animal slurries and manures, which can contain a broad range of pathogenic microorganisms, is considered a major contributor to this contamination. Some of the pathogenic microorganisms applied to soil have been observed to leach through the soil into groundwater, which poses a risk to public health. There is a critical need, therefore, for characterization of pathogen movement through the vadose zone for assessment of the risk to groundwater quality due to agricultural activities. A lysimeter experiment was performed to investigate the effect of soil type and condition on the fate and transport of potential bacterial pathogens, using Escherichia coli as a marker, in four Irish soils (n = 9). Cattle slurry (34 tonnes per ha) was spread on intact soil monoliths (depth, 1 m; diameter, 0.6 m) in the spring and summer. No effect of treatment or the initial soil moisture on the E. coli that leached from the soil was observed. Leaching of E. coli was observed predominantly from one soil type (average, 1.11 ± 0.77 CFU ml−1), a poorly drained Luvic Stagnosol, under natural rainfall conditions, and preferential flow was an important transport mechanism. E. coli was found to have persisted in control soils for more than 9 years, indicating that autochthonous E. coli populations are capable of becoming naturalized in the low-temperature environments of temperate maritime soils and that they can move through soil. This may compromise the use of E. coli as an indicator of fecal pollution of waters in these regions.

Concentration of norovirus during wastewater treatment and its impact on oyster contamination

ABSTRACT The concentrations of Escherichia coli, F-specific RNA bacteriophage (FRNA bacteriophage), and norovirus genogroup I (NoV GI) and norovirus genogroup II (NoV GII) in wastewater were monitored weekly over a 1-year period at a wastewater treatment plant (WWTP) providing secondary wastewater treatment. A total of 49 samples of influent wastewater and wastewater that had been treated by primary and secondary wastewater treatment processes (primary and secondary treated wastewater) were analyzed. Using a real-time reverse transcription-quantitative PCR (RT-qPCR), the mean NoV GI and NoV GII concentrations detected in effluent wastewater were 2.53 and 2.63 log10 virus genome copies 100 ml−1, respectively. The mean NoV concentrations in wastewater during the winter period (January to March) (n = 12) were 0.82 (NoV GI) and 1.41 (NoV GII) log units greater than the mean concentrations for the rest of the year (n = 37). The mean reductions of NoV GI and GII during treatment were 0.80 and 0.92 log units, respectively, with no significant difference detected in the extent of NoV reductions due to season. No seasonal trend was detected in the concentrations of E. coli or FRNA bacteriophage in wastewater influent and showed mean reductions of 1.49 and 2.13 log units, respectively. Mean concentrations of 3.56 and 3.72 log10 virus genome copies 100 ml−1 for NoV GI and GII, respectively, were detected in oysters sampled adjacent to the WWTP discharge. A strong seasonal trend was observed, and the concentrations of NoV GI and GII detected in oysters were correlated with concentrations detected in the wastewater effluent. No seasonal difference was detected in concentrations of E. coli or FRNA bacteriophage detected in oysters.

Effect of sulphate addition on volatile fatty acid and ethanol degradation in an anaerobic hybrid reactor. I: process disturbance and remediation

Abstract The addition of sulphate (4 g/l) to the influent of a mesophilic (35 ± 2°C) laboratory-scale anaerobic hybrid reactor (volumetric loading rate 6 kg/m3/day) treating a propionate-, butyrate- and ethanol-containing (1:1:1 on a COD basis; total COD of 12 g/l) wastewater resulted in severe process disturbance, with a complete inhibition of the propionate-degrading ability of the sludge. Severe inhibition of acetate removal was also observed, with concentrations of propionate and acetate in the reactor effluent of 4000 and 1000 mg/l, respectively. An oscillating pattern of sulphate and sulphide concentrations was observed in the reactor effluent. A control reactor, which had no influent sulphate, maintained a COD removal efficiency of > 95% throughout the trial period. A number of remediation strategies were undertaken with the sulphate-fed reactor. Initially, the levels of total sulphide in the digester were reduced to 1000 mg/l by inclusion of a nitrogen gas sparging system at 4.12 l/l/day (5.36 l/m2/day). Although the levels of total sulphide were reduced in the reactor, little improvement in COD removal was observed. Re-inoculation of 21 of non-sulphate-adapted seed sludge (25 g VSS/l;6.0% inoculum) into the digester did not improve the reactor performance either, suggesting that no improvement could take place in the absence of appropriate sulphate-reducing bacteria or sulphate-adapted syntrophic and methanogenic bacteria. Successful bioaugmentation of the reactor was achieved by the inoculation of 21 sulphate-adapted sludge (25 g VSS/l;6.0% inoculum) from a full-scale digester. Improvement in reactor performance was observed after one retention time (48 h) and the COD removal efficiency exceeded 95% by the conclusion of the trial.

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.