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Up-flow anaerobic bioreactors are widely applied for high-rate digestion of industrial wastewaters and rely on formation, and retention, of methanogenic granules, comprising of dense, fast-settling, microbial aggregates (approx. 0.5–4.0 mm in diameter). Granule formation (granulation) mechanisms have been reasonably well hypothesized and documented. However, this study used laboratory-scale bioreactors, inoculated with size-separated granular sludge to follow new granule formation, maturation, disintegration and re-formation. Temporal size profiles, volatile solids content, settling velocity, and ultrastructure of granules were determined from each of four bioreactors inoculated only with small granules, four with only large granules, and four with a full complement of naturally-size-distributed granules. Constrained granule size profiles shifted toward the natural distribution, which was associated with maximal bioreactor performance. Distinct morphological features characterized different granule sizes and biofilm development stages, including ‘young’, ‘juvenile’, ‘mature’ and ‘old’. The findings offer opportunities toward optimizing management of high-rate, anaerobic digesters by shedding light on the rates of granule growth, the role of flocculent sludge in granulation and how shifting size distributions should be considered when setting upflow velocities.

The feasibility of NO3- removal by the synergistic action of a prevailing denitrifying anoxic methane oxidising (DAMO), and nitrate-reducing and sulfide-oxidising bacterial (NR-SOB) consortium, using CH4 and H2S from biogas as electron donors in a biotrickling filter was investigated. The influence of NO3- concentration on N2O production during this process was also evaluated. The results showed that NO3- was removed at rates up to 2.8 g mreactor−3 h−1 using CH4 as electron donor. N2O production rates correlated with NO3- concentration in the liquid phase, with a 10-fold increase in N2O production as NO3- concentration increased from 50 to 200 g m−3. The use of H2S as co-electron donor resulted in a 13-fold increase in NO3- removal rates (∼18 gNO3- m−3 h−1) and complete denitrification under steady-state conditions, which was supported by higher abundances of narG, nirK and nosZ denitrifying genes. Although the relative abundance of the DAMO population in the consortium was reduced from 60 to 13% after H2S addition, CH4 removals were not compromised and H2S removal efficiencies of 100% were achieved. This study confirmed (i) the feasibility of co-oxidising CH4 and H2S with denitrification, as well as (ii) the critical need to control NO3- concentration to minimize N2O production by anoxic denitrifiers. This article is protected by copyright. All rights reserved

Exposure to endocrine-disrupting chemicals (EDCs), such as estrogens, is a growing issue for human and animal health as they have been shown to cause reproductive and developmental abnormalities in wildlife and plants and have been linked to male infertility disorders in humans. Intensive farming and weather events, such as storms, flash flooding, and landslides, contribute estrogen to waterways used to supply drinking water. This paper explores the impact of estrogen exposure on the performance of slow sand filters (SSFs) used for water treatment. The feasibility and efficacy of SSF bioaugmentation with estrogen-degrading bacteria was also investigated, to determine whether removal of natural estrogens (estrone, estradiol, and estriol) and overall SSF performance for drinking water treatment could be improved. Strains for SSF augmentation were isolated from full-scale, municipal SSFs so as to optimize survival in the laboratory-scale SSFs used. Concentrations of the natural estrogens, determined by gas chromatography coupled with mass spectrometry (GC-MS), revealed augmented SSFs reduced the overall estrogenic potency of the supplied water by 25% on average and removed significantly more estrone and estradiol than nonaugmented filters. A negative correlation was found between coliform removal and estrogen concentration in nonaugmented filters. This was due to the toxic inhibition of protozoa, indicating that high estrogen concentrations can have functional implications for SSFs (such as impairing coliform removal). Consequently, we suggest that high estrogen concentrations could impact significantly on water quality production and, in particular, on pathogen removal in biological water filters.

The literature is conflicted on the influence of ammonium on the kinetics and microbial ecology of methanotrophy. In this study, methanotrophic cultures were enriched, under ammonium concentrations ranging from 0 to 200 mM, from an inoculum comprising leachate and top-cover soil from a landfill. Specific CH4 biodegradation rates were highest (7.8 × 10−4 ± 6.0 × 10−5 gCH4 gX−1 h−1) in cultures enriched at 4 mM NH4+, which were mainly dominated by type II methanotrophs belonging to Methylocystis spp. Lower specific CH4 oxidation rates (average values of 1.8–3.6 × 10−4 gCH4 gX−1 h−1) were achieved by cultures enriched at higher NH4+ concentrations (20 and 80 mM), and had higher affinity for CH4 compared to 4 mM enrichments. These lower affinities were attributed to lower diversity dominated by type I methanotrophs, of the Methylosarcina, Methylobacter and Methylomicrobium genera, encountered with increasing concentrations of NH4+. The study indicates that CH4 oxidation biotechnologies applied at low NH4+ concentrations can support efficient abatement of CH4 and high diversity of methanotrophic consortia, whilst enriching type II methanotrophs.

A mesophilic (37 °C) and a thermophilic (55 °C) two-chamber microbial fuel cell (MFC) were studied and compared for their power production from xylose and the microbial communities involved. The anode-Attached, membrane-Attached, and planktonic microbial communities, and their respective active subpopulations, were determined by next generation sequencing (Illumina MiSeq), based on the presence and expression of the 16S rRNA gene. Geobacteraceae accounted for 65% of the anode-Attached active microbial community in the mesophilic MFC, and were associated to electricity generation likely through direct electron transfer, resulting in the highest power production of 1.1 W m-3. A lower maximum power was generated in the thermophilic MFC (0.2 W m-3), likely due to limited acetate oxidation and the competition for electrons by hydrogen oxidizing bacteria and hydrogenotrophic methanogenic archaea. Aerobic microorganisms, detected among the membrane-Attached active community in both the mesophilic and thermophilic MFC, likely acted as a barrier for oxygen flowing from the cathodic chamber through the membrane, favoring the strictly anaerobic exoelectrogenic microorganisms, but competing with them for xylose and its degradation products. This study provides novel information on the active microbial communities populating the anodic chamber of mesophilic and thermophilic xylose-fed MFCs, which may help in developing strategies to favor exoelectrogenic microorganisms at the expenses of competing microorganisms. publishedVersion

High-resolution environmental observations from satellite remote sensing are useful for gaining insight into climate variability over large regions of the earth. However, data products derived from infrared remote sensing can have large amounts of missing data due to periodic cloud cover obscuring the earth's surface. In addition, these products are commonly derived from instruments aboard polar-orbiting satellites, which make relatively infrequent passes over each region of the planet, leaving large blocks of data that are missing in time. The goal of this research is to overcome these issues by spatially and temporally infilling temperature data using satellite observations. To accomplish this, we propose novel statistical modeling techniques to account for dissonance between the areal spatial support of the data and the continuous temporal domain. In addition, we develop a novel multiresolution approach that accommodates the large spatial dimensionality of high-resolution satellite images. The result of our work is a computationally efficient methodology that provides the means to infill satellite data products both spatially and temporally. The methodology is demonstrated using land surface temperature fields from the Moderate Resolution Imaging Spectroradiometer (MODIS) instrument onboard NASA's Terra and Aqua satellites, over Harris County, Texas, USA.

Studies investigating the feasibility of new, or improved, biotechnologies, such as wastewater treatment digesters, inevitably start with laboratory-scale trials. However, it is rarely determined whether laboratory-scale results reflect full-scale performance or microbial ecology. The Expanded Granular Sludge Bed (EGSB) bioreactor, which is a high-rate anaerobic digester configuration, was used as a model to address that knowledge gap in this study. Two laboratory-scale idealizations of the EGSB-a one-dimensional and a three-dimensional scale-down of a full-scale design-were built and operated in triplicate under near-identical conditions to a full-scale EGSB. The laboratory-scale bioreactors were seeded using biomass obtained from the full-scale bioreactor, and, spent water from the distillation of whisky from maize was applied as substrate at both scales. Over 70 days, bioreactor performance, microbial ecology, and microbial community physiology were monitored at various depths in the sludge-beds using 16S rRNA gene sequencing (V4 region), specific methanogenic activity (SMA) assays, and a range of physical and chemicalmonitoringmethods. SMA assays indicated dominance of the hydrogenotrophic pathway at full-scale whilst a more balanced activity profile developed during the laboratory-scale trials. At each scale, Methanobacterium was the dominant methanogenic genus present. Bioreactor performance overall was better at laboratory-scale than full-scale. We observed that bioreactor design at laboratory-scale significantly influenced spatial distribution of microbial community physiology and taxonomy in the bioreactor sludge-bed, with 1-D bioreactor types promoting stratification of each. In the 1-D laboratory bioreactors, increased abundance of Firmicutes was associated with both granule position in the sludge bed and increased activity against acetate and ethanol as substrates. We further observed that stratification in the sludge-bed in 1-D laboratory-scale bioreactors was associated with increased richness in the underlying microbial community at species (OTU) level and improved overall performance. The authors thank Carole Jude and North British Distillery for providing support throughout the sampling of the full-scale bioreactor, and for making available performance data from full-scale. The study was funded by the Engineering and Physical Sciences Research Council, UK (EP/J00538X/1; EP/K038885/1). GC was supported by a European Research Council Starting Grant (3C-BIOTECH 261330). UI was funded by NERC IRF NE/L011956/1. CQ was funded by an MRC fellowship MR/M50161X/1 as part of the CLoud Infrastructure for Microbial Genomics (CLIMB) consortium MR/L015080/1. peer-reviewed

Previous laboratory-scale studies to characterise the functional microbial ecology of slow sand filters have suffered from methodological limitations that could compromise their relevance to full-scale systems. Therefore, to ascertain if laboratory-scale slow sand filters (L-SSFs) can replicate the microbial community and water quality production of industrially operated full-scale slow sand filters (I-SSFs), eight cylindrical L-SSFs were constructed and were used to treat water from the same source as the I-SSFs. Half of the L-SSFs sand beds were composed of sterilized sand (sterile) from the industrial filters and the other half with sand taken directly from the same industrial filter (non-sterile). All filters were operated for 10 weeks, with the microbial community and water quality parameters sampled and analysed weekly. To characterize the microbial community phyla-specific qPCR assays and 454 pyrosequencing of the 16S rRNA gene were used in conjunction with an array of statistical techniques. The results demonstrate that it is possible to mimic both the water quality production and the structure of the microbial community of full-scale filters in the laboratory - at all levels of taxonomic classification except OTU - thus allowing comparison of LSSF experiments with full-scale units. Further, it was found that the sand type composing the filter bed (non-sterile or sterile), the water quality produced, the age of the filters and the depth of sand samples were all significant factors in explaining observed differences in the structure of the microbial consortia. This study is the first to the authors' knowledge that demonstrates that scaled-down slow sand filters can accurately reproduce the water quality and microbial consortia of full-scale slow sand filters.

Methane (CH4) is a potent greenhouse gas often emitted in low concentrations from waste sector activities. Biological oxidation techniques have the potential to offer effective methods for the remediation of such emissions. In this paper, methods of improving the CH4 oxidation performance of a horizontal flow biofilm reactor (HFBR) technology, operated at low temperatures, are investigated.Three pilot scale HFBRs were operated over three studies (Study 1,2 & 3) lasting 310 days in total. The reactors were loaded with 13.2 g CH4/m(3)/h during each study and operated at an average temperature of 10 degrees C.In Study 1, nutrients were added to the biofilm via a liquid nutrient feed (LNF) comprising water and nutrient mineral salts. Average removals were 4.2, 3.1 and 2.3 g CH4/m(3)/h for HFBRs 1, 2 and 3 respectively.In Study 2 silicone oil was added to the LNF of all three HFBRs. Average removals increased, when compared to Study 1, by 31%, 79% and 78% for HFBRs 1, 2 and 3 respectively.In Study 3 a non ionic surfactant (Brij 35) was added to the LNF and silicone oil liquid phase of HFBRs 1 and 2. The operating conditions of HFBR 3 were not changed and it was used as a control. A concentration of 1.0 g Brij 35/L proved most effective in improving reactor performance; with removal rates increasing by 105% and 171% for HFBRs 1 and 2 respectively when compared to Study 1.These results indicate the potential of liquid phase optimisation for improving mass transfer rates and removal performances in biological reactors treating CH4. (C) 2014 Elsevier B.V. All rights reserved. The authors would like to gratefully acknowledge financial support from Science Foundation Ireland (SFI) and Enterprise Ireland. peer-reviewed

This study examined the relationship between the abundance of bacterial denitrifiers in groundwater at four sites, differing with respect to overlaying land management and peizometer depth. Groundwater was sourced from 36 multilevel piezometers, which were installed to target different groundwater zones: (1) subsoil, (2) subsoil to bedrock interface, and (3) bedrock. The gene copy concentrations (GCCs), as gene copies per liter, for bacterial 16S rRNA genes and the denitrifying functional genes, nirK, nirS, and nosZ, were determined using quantitative polymerase chain reaction assays. The results were related to gaseous nitrogen emissions and to the physicochemical properties of the four sites. Overall, nirK and nirS abundance appeared to show no significant correlation to N2O production (P = 0.9989; P = 0.3188); and no significant correlation was observed between nosZ and excess N2 concentrations (P = 0.0793). In the majority of piezometers investigated, the variation of nirK and nirS gene copy concentrations was considered significant (P 0.0001). Dissolved organic carbon (DOC) decreased with aquifer depth and ranged from 1.0-4.0 mg l(-1), 0.9-2.4 mg l(-1), and 0.8-2.4 mg l(-1) within piezometers located in the subsoil, subsoil/bedrock interface, and bedrock depths, respectively. The availability of increasing DOC and the depth of the water table were positively correlated with increasing nir and nosZ GCCs (P = 0.0012). A significant temporal correlation was noted between nirS and piezometer depth (P 0.001). Interestingly, the nirK, nirS, and nosZ GCCs varied between piezometer depths within specific sites, while GCCs remained relatively constant from site to site, thus indicating no direct impact of agricultural land management strategies investigated on denitrifier abundance.

BackgroundWe introduceDESMANfor De novo Extraction of Strains from MetAgeNomes. Metagenome sequencing generates short reads from throughout the genomes of a microbial community. Increasingly large, multi-sample metagenomes, stratified in space and time are being generated from communities with thousands of species. Repeats result in fragmentary co-assemblies with potentially millions of contigs. Contigs can be binned into metagenome assembled genomes (MAGs) but strain level variation will remain. DESMAN identifies variants on core genes, then uses co-occurrence across samples to link variants into strain sequences and abundance profiles. These strain profiles are then searched for on non-core genes to determine the accessory genes present in each strain.ResultsWe validatedDESMANon a synthetic twenty genome community with 64 samples. We could resolve the fiveE. colistrains present with 99.58% accuracy across core gene variable sites and their gene complement with 95.7% accuracy. Similarly, on real fecal metagenomes from the 2011E. coli(STEC) O104:H4 outbreak, the outbreak strain was reconstructed with 99.8% core sequence accuracy. Application to an anaerobic digester metagenome time series reveals that strain level variation is endemic with 16 out of 26 MAGs (61.5%) examined exhibiting two strains. In almost all cases the strain proportions were not statistically different between replicate reactors, suggesting intra-species niche partitioning. The only exception being when the two strains had almost identical gene complement and, hence, functional capability.ConclusionsDESMANwill provide a provide a powerful tool forde novoresolution of fine-scale variation in microbial communities. It is available as open source software fromhttps://github.com/chrisquince/DESMAN.

The retention of selective biofilms of Methanosarcina species within anaerobic digesters could reduce start-up times and enhance the efficiency of the process in treating high-strength domestic sewage. The objective of the study was to examine the effect of the surface characteristics of six common polymer support materials on the initial adhesion of the model methanogen, Methanosarcina barkeri, and to assess the potential of these support materials as selective biofilm carriers. Results from both the initial adhesion tests and extended DLVO (xDLVO) model correlated with each other, with PVC (12% surface coverage/mm(2)), PTFE (6% surface coverage/mm(2)), and PP (6% surface coverage/mm(2)), shown to be the better performing support materials for initial adhesion, as well as subsequent biofilm formation by M. barkeri after 72h. Experimental results of these three support materials showed that the type of material strongly influenced the extent of adhesion from M. barkeri (p

The feasibility of NO

Hydrogen Sulfide (H2S) is an odourous, highly toxic gas commonly encountered in various commercial and municipal sectors. Three novel, laboratory-scale, Horizontal-Flow Biofilm Reactors (HFBRs) were tested for the removal of H2S gas from air streams over a 178-day trial at 10 degrees C. Removal rates of up to 15.1 g [H2S] m(-3) h(-1) were achieved, demonstrating the HFBRs as a feasible technology for the treatment of H2S-contaminated airstreams at low temperatures. Bio-oxidation of H2S in the reactors led to the production of H+ and sulfate (SO42-) ions, resulting in the acidification of the liquid phase. Reduced removal efficiency was observed at loading rates of 15.1 g [H2S] m(-3) h(-1). NaHCO3 addition to the liquid nutrient feed (synthetic wastewater (SWW)) resulted in improved H2S removal. Bacterial diversity, which was investigated by sequencing and fingerprinting 16S rRNA genes, was low, likely due to the harsh conditions prevailing in the systems. The HFBRs were dominated by two species from the genus Acidithiobacillus and Thiobacillus. Nonetheless, there were significant differences in microbial community structure between distinct HFBR zones due to the influence of alkalinity, pH and SO4 concentrations. Despite the low temperature, this study indicates HFBRs have an excellent potential to biologically treat H2S-contaminated airstreams. This work was supported by Science Foundation Ireland [grant number 08/RFP/ENM1762] and a European Research Council Starting Grant (3C-BIOTECH; no. 261330). peer-reviewed

The nitrite reductase (nirS and nirK) and nitrous oxide reductase-encoding (nosZ) genes of denitrifying populations present in an agricultural grassland soil were quantified using real-time polymerase chain reaction (PCR) assays. Samples from three separate pedological depths at the chosen site were investigated: horizon A (0-10 cm), horizon B (45-55 cm), and horizon C (120-130 cm). The effect of carbon addition (treatment 1, control; treatment 2, glucose-C; treatment 3, dissolved organic carbon (DOC)) on denitrifier gene abundance and N2O and N2 fluxes was determined. In general, denitrifier abundance correlated well with flux measurements; nirS was positively correlated with N2O, and nosZ was positively correlated with N2 (P

Evidence from 16S rRNA gene sequences indicates that as yet uncultured microorganisms represent the vast majority of organisms in most environments on earth. However, since many species cannot be cultured within a laboratory setting, these communities are mostly unstudied and consequently there has been little insight into the genetics, physiology and biochemistry of their members. The new field of community metabolomics is about to change this scenario. In the same way as metagenomics indicates the analyses of all DNA from a given sample, community metabolomics looks at the entirety of the thousands of naturally occurring metabolites from the meta-population of a sample of a given environment such as soil or water, and perhaps even air. In this chapter we outline how this new field has recently been applied to generate new insights into these unexplored areas of the bacterial realm in the fields of environmental science and technology, within natural, laboratory and even industrial, settings. Potential future applications in this area are also discussed.

91 Páginas, 4 Tablas, 6 Figuras, Optimal supply of trace elements (TE) is a prerequisite for microbial growth and activity in anaerobic digestion (AD) bioprocesses. However, the required concentrations and ratios of essential TE for AD biotechnologies strongly depend on prevailing operating conditions as well as feedstock composition. Furthermore, TE in AD bioreactors undergo complex physico-chemical reactions and may be present as free ions, complex bound or as precipitates depending on pH, or on the presence of sulfur compounds or organic macromolecules. To overcome TE deficiency various commercial mineral products are typically applied to AD processes. The addition of heavy metals poses the risk of overdosing operating systems, which may be toxic to microbial consortia and ultimately the environment. Adequate supplementation, therefore, requires not only appropriate knowledge about the composition, but also on the speciation and bioavailability of TE. However, very little is yet fully understood on this specific issue. Evaluations of TE typically only include the measurement of total TE concentrations but do not consider the chemical forms in which TE exist. Thus detailed information on bioavailability and potential toxicity cannot be provided. This review provides an overview of the state-of-the-art in approaches to determine bioavailable TE in anaerobic bioprocesses, including sequential fractionation and speciation techniques. Critical aspects and considerations, including with respect to sampling and analytical procedures, as well as mathematical modelling, are examined. The approaches discussed in this review are based on our experiences and on previously published studies in the context of the ‘COST Action 1302: European Network on Ecological Roles of Trace Metals in Anaerobic Biotechnologies'., This article is based upon work from COST Action 1302 (‘European Network on Ecological Roles of Trace Metals in Anaerobic Biotechnologies’) supported by COST (European Cooperation in Science and Technology). EvH and GE acknowledge support from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 643071 (‘Advanced Biological Waste-to-Energy Technologies – ABWET’). GC acknowledges funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme grant agreement No. 261330. The authors thank the two anonymous reviewers for their comments, which significantly improved the quality of the manuscript.

For over 200 years, slow sand filtration (SSF) has been an effective means of treating water for the control of microbiological contaminants in both small and large community water supplies. However, such systems lost popularity to rapid sand filters mainly due to smaller land requirements and less sensitivity to water quality variations. SSF is still a particularly attractive process because its operation does not require chemicals or electricity. It can achieve a high level of treatment, which is mainly attributed to naturally-occurring, biochemical processes in the filter. Several microbiologically-mediated purification mechanisms (e.g. predation, scavenging, adsorption and bio-oxidation) have been hypothesised or assumed to occur in the biofilm that forms in the filter but these have not yet been comprehensively verified. Thus, SSFs are operated as ‘black boxes’ and knowledge gaps pertaining to the underlying ecology and ecophysiology limit the design and optimisation of the technology. The objective of this review is to outline the biological aspects of SSF in to the context of recent developments in molecular microbial ecology.

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. Methods and Results: The microbial communities from an anaerobic industrial-like wastewater treatment bioreactor were characterized using phylogenetic analyses and metaproteomics. Clone libraries indicated that the bacterial community in the bioreactor was diverse while the archaeal population was mainly composed of Methanocorpusculum-like (76%) micro-organisms. Three hundred and eighty-eight reproducible protein spots were obtained on 2-D gels, of which 70 were excised and 33 were identified. The putative functions of the proteins detected in the anaerobic bioreactor were related to cellular processes, including methanogenesis from CO2 and acetate, glycolysis and the pentose phosphate pathway. Metaproteomics also indicated, by protein assignment, the presence of specific micro-organisms in the bioreactor. However, only a limited overlap was observed between the phylogenetic and metaproteomic analyses. Conclusions: This study provides some direct evidence of the microbial activities taking place during anaerobic digestion. Significance and Impact of Study: This study demonstrates metaproteomics as a useful tool to uncover key biochemical pathways underpinning specific anaerobic bioprocesses.

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

The reproducibility and stability of low- temperature anaerobic wastewater treatment systems undergoing transient perturbations was investigated. Three identical anaerobic expanded granular sludge bed-based bioreactors were used to degrade a volatile fatty acid and glucose-based wastewater under sub-ambient (15 degrees C) conditions. The effect of a variety of environmental perturbations on bioreactor performance was assessed by chemical oxygen demand removal. Temporal microbial community development was monitored by denaturation gradient gel electrophoresis (DGGE) of 16S rRNA genes extracted from sludge granules. Methanogenic activity was monitored using specific methanogenic activity assays. Bioreactor performance and microbial population dynamics were each well replicated between both experimental bioreactors and the control bioreactor prior to, and after the implementation of most of the applied perturbations. Gene fingerprinting data indicated that Methanosaeta sp. were the persistent, keystone members of the archaeal community, and likely were pivotal for the physical stability and maintenance of the granular biofilms. Cluster analyses of DGGE data suggested that temporal shifts in microbial community structure were predominantly independent of the applied perturbations.

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.

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.

The effect of nickel deprivation from the influent of a mesophilic (30 degrees C) methanol fed upflow anaerobic sludge bed (UASB) reactor was investigated by coupling the reactor performance to the evolution of the Methanosarcina population of the bioreactor sludge. The reactor was operated at pH 7.0 and an organic loading rate (OLR) of 5-15 g COD l(-1) day(-1) for 191 days. A clear limitation of the specific methanogenic activity (SMA) on methanol due to the absence of nickel was observed after 129 days of bioreactor operation: the SMA of the sludge in medium with the complete trace metal solution except nickel amounted to 1.164 (+/- 0.167) g CH(4)-COD g VSS(-1) day(-1) compared to 2.027 (+/- 0.111) g CH(4)-COD g VSS(-1) day(-1) in a medium with the complete (including nickel) trace metal solution. The methanol removal efficiency during these 129 days was 99%, no volatile fatty acid (VFA) accumulation was observed and the size of the Methanosarcina population increased compared to the seed sludge. Continuation of the UASB reactor operation with the nickel limited sludge lead to incomplete methanol removal, and thus methanol accumulation in the reactor effluent from day 142 onwards. This methanol accumulation subsequently induced an increase of the acetogenic activity in the UASB reactor on day 160. On day 165, 77% of the methanol fed to the system was converted to acetate and the Methanosarcina population size had substantially decreased. Inclusion of 0.5 mu M Ni (dosed as NiCl(2)) to the influent from day 165 onwards lead to the recovery of the methanol removal efficiency to 99% without VFA accumulation within 2 days of bioreactor operation.

Radiotracer incubation experiments and beta microimaging, along with fluorescent in situ hybridisation (FISH), are proposed as a complementary approach to specific methanogenic activity testing and measurement of in vitro substrate utilisation rates to understand better the ecophysiology of anaerobic granular biofilms from wastewater treatment reactors.

This study investigated the efficiency of a pyrrhotite autotrophic denitrification biofilter (PADB) technology for simultaneous N and P removal from wastewater lacking organic matter. A PADB was constructed with natural pyrrhotite as the biofilter medium and inoculated with autotrophic denitrifies enriched from anaerobic sludge. Over an operating period of 247 days, PADB efficiently removed NO3(-) and PO4(3-) simultaneously from wastewater that lacked organic matter. The hydraulic retention time (HRT), and influent NO3(-) and PO4(3-) concentrations affected the removal of NO3(-) and PO4(3-). A longer HRT led to lower concentrations of NO3(-) and PO4(3-) in the effluent. The PO4(3-) removal was influenced by NO3(-) removed; the more NO3(-) removed, the more PO4(3-) removed. As the synthetic wastewater containing NO3(-)-N of 28 mg L(-1) and PO4(3-)-P of 6 mg L(-1) in the absence of organic matter was treated by PADB at HRT of 24 h, total oxidized nitrogen (TON; NO2(-)-N + NO3(-)-N) and PO4(3-)-P concentrations of effluent were as low as 1.13 and 0.28 mg L(-1), respectively. When treatment of municipal wastewater treatment plant (WWTP) secondary effluent with TON of 21.11 mg L(-1) and PO4(3-)-P of 2.62 mg L(-1) at HRT of 24 h, the effluent TON was 1.89 mg L(-1) and PO4(3-)-P was 0.34 mg L(-1). PO4(3-) was removed through the formation of secondary minerals with Fe and Ca. These secondary minerals contained elevated phosphorus, which presents a potential for P recovery from wastewater.

The objective of this study was to demonstrate the feasibility of novel, Horizontal Flow Biofilm Reactor (HFBR) technology for the treatment of ammonia (NH3)-contaminated airstreams. Three laboratory-scale HFBRs were used for remediation of an NH3-containing airstream at 10 °C during a 90-d trial to test the efficacy of low-temperature treatment. Average ammonia removal efficiencies of 99.7 % were achieved at maximum loading rates of 4.8 g NH3 m3 h−1. Biological nitrification of ammonia to nitrite (NO2−) and nitrate (NO3−) was mediated by nitrifying bacterial and archaeal biofilm populations. Ammonia-oxidising bacteria (AOB) were significantly more abundant than ammonia-oxidising archaea (AOA) vertically at each of seven sampling zones along the vertical HFBRs. Nitrosomonas and Nitrosospira, were the two most dominant bacterial genera detected in the HFBRs, while an uncultured archaeal clone dominated the AOA community. The bacterial community composition across the three HFBRs was highly conserved, although variations occurred between HFBR zones and were driven by physicochemical variables. The study demonstrates the feasibility of HFBRs for the treatment of ammonia-contaminated airstreams at low temperatures; identifies key nitrifying microorganisms driving the removal process; and provides insights for process optimisation and control. The findings are significant for industrial applications of gas oxidation technology in temperate climates.

© Springer International Publishing Switzerland 2015. A challenging, and largely uncharted, area of research in the field of anaerobic digestion science and technology is in understanding the roles of trace metals in enabling biogas production. This is a major knowledge gap and a multifaceted problem involving metal chemistry; physical interactions of metal and solids; microbiology; and technology optimization. Moreover, the fate of trace metals, and the chemical speciation and transport of trace metals in environments— often agricultural lands receiving discharge waters from anaerobic digestion processes— simultaneously represents challenges for environmental protection and opportunities to close process loops in anaerobic digestion., The authors acknowledge funding within the framework of the COST Action 1302 (‘European Network on Ecological Roles of Trace Metals in Anaerobic Biotechnologies’). GC is supported by a European Research Council Starting Grant (‘3C-BIOTECH; No. 261330).

The aims of this study were to demonstrate the (1) feasibility of psychrophilic, or low-temperature, anaerobic digestion (PAD) of phenolic wastewaters at 10–15 °C; (2) economic attractiveness of PAD for the treatment of phenol as measured by daily biogas yields and (3) impact on bioreactor performance of phenol loading rates (PLRs) in excess of those previously documented (1.2 kg phenol m −3 d −1 ). Two expanded granular sludge bed (EGSB)-based bioreactors, R1 and R2, were employed to mineralise a volatile fatty acid-based wastewater. R2 influent wastewater was supplemented with phenol at an initial concentration of 500 mg l −1 (PLR, 1 kg m −3 d −1 ). Reactor performance was measured by chemical oxygen demand (COD) removal efficiency, CH 4 composition of biogas and phenol removal (R2 only). Specific methanogenic activity, biodegradability and toxicity assays were employed to monitor the physiological capacity of reactor biomass samples. The applied PLR was increased to 2 kg m −3 d −1 on day 147 and phenol removal by day 415 was 99% efficient, with ⩽4 mg l −1 present in R2 effluent. The operational temperature of R1 (control) and R2 was reduced by stepwise decrements from 15 °C through to a final operating temperature of 9.5 °C. COD removal efficiencies of c. 90% were recorded in both bioreactors at the conclusion of the trial (day 673), when the phenol concentration in R2 effluent was below 30 mg l −1 . Daily biogas yields were determined during the final (9.5 °C) operating period, when typical daily R2 CH 4 yields of c. 3.3 l CH 4 g −1 COD removed d −1 were recorded. The rate of phenol depletion and methanation by R2 biomass by day 673 were 68 mg phenol g VSS −1 d −1 and 12–20 ml CH 4 g VSS −1 d −1 , respectively.

Thirteen anaerobic hybrid expanded granular sludge bed-anaerobic filter bioreactors were used for psychrophilic (15–18 °C) anaerobic digestion of a variety of synthetic and non-synthetic wastewaters, including: food-processing, dairy, aromatic- and aliphatic-containing and brewery discharges. Specific methanogenic activity assays were employed to assess temporal physiological activity dynamics. Terminal restriction fragment length polymorphism genetic fingerprinting and fluorescent in situ hybridization were used to monitor shifts in the structure of the microbial communities in the bioreactors in response to operating conditions. Treatment efficiencies obtained were comparable to previous mesophilic (37 °C) trials. Methanogenic activity developed under psychrophilic conditions and putative psychrophilic populations were detected within otherwise psychrotolerant mesophilic communities. Shifts in the population structure of archaeal (methanogenic) communities were more indicative of process disruption than bacterial communities. Biomolecular techniques were demonstrated as valuable tools for anaerobic wastewater treatment plant monitoring.

Two expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactors (3.38 l active volume) were used to directly compare psychrophilic (15 degrees C), anaerobic digestion (PAD) to mesophilic (37 degrees C) anaerobic digestion (MAD) for the treatment of a brewery wastewater (chemical oxygen demand (COD) concentration of 3,136+/-891 mg l(-1)). Bioreactor performance was evaluated by COD removal efficiency and biogas yields at a range of hydraulic and organic loading rates. Specific methanogenic activity (SMA) assays were also employed to investigate the activity of the biomass in the bioreactors. No significant difference in the COD removal efficiencies (which ranged from 85-93%) were recorded between PAD and MAD during the 194-d trial at maximum organic and hydraulic loading rates of 4.47 kg m(-3) day(-1) and 1.33 m(3) m(-3) day(-1), respectively. In addition, the methane content (%) of the biogas was very similar. The volumetric biogas yield from the PAD bioreactor was approximately 50% of that from the MAD bioreactor at an organic loading rate of 4.47 kg COD m(-3) day(-3) and an applied liquid up-flow velocity (V(up)) of 2.5 m h(-1). Increasing the V(up) in the PAD bioreactor to 5 m h(-1) resulted in a volumetric biogas production rate of approximately 4.1 l d(-1) and a methane yield of 0.28 l CH(4) g(-1) COD d(-1), which were very similar to the MAD bioreactor. Significant and negligible biomass washout was observed in the mesophilic and psychrophilic systems, respectively, thus increasing the sludge loading rate applied to the former and underlining the robustness of the latter, which appeared underloaded. A psychrotolerant mesophilic, but not truly psychrophilic, biomass developed in the PAD bioreactor biomass, with comparable maximum SMA values to the MAD bioreactor biomass. PAD, therefore, was shown to be favourably comparable to MAD for brewery wastewater treatment and biogas generation.

The reproducibility of low-temperature anaerobic biological wastewater treatment trials was evaluated. Two identical anaerobic expanded granular sludge bed bioreactors were used to treat synthetic volatile fatty acid-based industrial wastewater under ambient conditions (18–20°C) and to investigate the effect of various environmental perturbations on reactor performance and microbial community dynamics, which were assessed by chemical oxygen demand removal or effluent volatile fatty acid determination and terminal restriction fragment length polymorphism analysis, respectively. Methanogenic activity was monitored using specific methanogenic activity assays. Reactor performance and microbial community dynamics were each well replicated between Reactor 1 and Reactor 2. Archaeal dynamics, in particular, were associated with reactor operating parameters. Terminal restriction fragment length polymorphism data suggested dynamic acetoclastic and hydrogenophilic methanogenic populations and were in agreement with temporal specific methanogenic activity data. Putative psychrophilic populations were observed in anaerobic bioreactor sludge for the first time.

Anaerobic digestion in the psychrophilic (20 degrees C) or sub-mesophilic temperature range has recently been proven as an effective treatment option for the mineralization of a wide variety of problematic wastewaters. In this study, an expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactor was seeded with a full-scale, mesophilic sludge and employed to evaluate the long-term operational potential, and underlying microbial ecology, of this approach for the treatment of a medium-strength (5 g chemical oxygen demand [COD] l(-1)), synthetic, volatile fatty acid-based wastewater. Throughout the trial period of 625 days, extended intervals of consistently stable and efficient wastewater treatment were sustained. These results were highlighted by a short start-up period (21 d), low hydraulic retention times (4.88h), high organic (up to 24.64kg CODm(-3)d(-1)), and volumetric loading rates (up to 4.92 m3 m(-3) d(-1)). A stable, well-settling granular sludge bed was maintained in the bioreactor for the majority of the trial; however, reduced treatment efficiency and biomass washout were observed at an imposed OLR of 36.96 kg COD m(-3) d(-1). The microbial biomass in the bioreactor was investigated using maximum specific methanogenic activity assays and polymerase chain reaction-denaturing gradient gel electrophoresis. A temporal succession of both the bacterial and archaeal populations was noted during the trial, compared to the seed sludge, in response to bioreactor operation at lower temperatures, loading rate increases and to VFA accumulation in the bioreactor. During the trial, an increased contribution of hydrogenotrophic methanogenesis as a pathway of methane production was observed, along with the overall emergence of a highly active psychrotolerent-though still mesophilic biomass.

Anaerobic granular and fixed-film reactors have been successfully operated for wastewater treatment at full scale for over two decades and represent a sustainable, energy-producing approach, which is increasingly being directed towards treatment of domestic sewage. Research over the past two decades, and significant operational experience, has demonstrated that there are no fundamental microbiological barriers to the implementation of AD for domestic sewage treatment in regions with warm and temperate climates. Despite this, the underlying microbiology of methanogenesis is not fully understood and novel groups of microbes have been identified in sludge, with unknown functions. The methanogenic process has recently been subject to systematic investigation using newly developed analytical and microbiological approaches. A combination of process monitoring, physiological, molecular microbiological and microscopic methods are beginning to generate a comprehensive, integrated data set at micro-organism, granule and reactor level and the current state of knowledge is reviewed here. Information on the formation of granules, on the relationship between reactor operating conditions and microbial consortia and on the impact of process changes on the microorganisms in reactors will, in future, enable the link between the processes occurring at microorganism level (scale ca. 1 μm–1 mm) and the processes occurring within reactors (scale >1 m), which will enhance the efficiency and applicability of anaerobic sewage treatment.

The microbial composition, methanogenic activity and architecture of particulate and fixed biofilms within four anaerobic hybrid reactors, R1–R4, operating at psychrophilic temperatures were investigated. The reactors treated low-strength (1 g COD l−1; R1) and high-strength (10 g COD l−1; R2–R4) wastewaters from the food-processing sector (R1, R2 – whey; R3 – sucrose; R4 – volatile fatty acids). Successful start-up and long-term psychrophilic operation was observed for all four reactors, with COD removal efficiencies of 80–99% achieved at 12–20 °C at organic loading rates of 1.3–20 kg COD m−3 d−1. The formation and maintenance of a well-settling granular sludge bed and an attached biofilm were shown to occur under psychrophilic conditions, an important consideration for the successful implementation of low temperature biofilm reactor technology. Culture-independent molecular techniques (terminal restriction fragment length polymorphism, clone library analysis and 16S rRNA gene sequencing) revealed that microbial population structure could be a key factor in reactor performance, with changes in the community structure of the three high-strength reactors preceding granular instability and a subsequent decline in COD removal efficiency. Biomonitoring of microbial population structure and dynamics within anaerobic reactors may, therefore, allow for the early recognition of potential operational problems.

Low-temperature, or psychrophilic ( −3 d −1 and 0.4–1.2 kg phenol m −3 d −1 (400–1200 mg phenol [l wastewater] −1 ), respectively. There was no long-term accumulation of volatile fatty acids in the reactor systems. Methanogenic activity was developed under psychrophilic conditions but anaerobic methane-producing populations remained mesophilic throughout the trial of 415 days.

In this laboratory study, a novel wastewater treatment technology, the air suction flow-biofilm reactor (ASF-BR) - a sequencing batch biofilm reactor technology with a passive aeration mechanism - was investigated for its efficiency in removing organic carbon, nitrogen and phosphorus, from high-strength synthetic wastewaters. A laboratory-scale ASF-BR comprising 2 reactors, 350 mm in diameter and 450 mm in height, was investigated over 2 studies (Studies 1 and 2) for a total of 430 days. Study 1 lasted a total of 166 days and involved a 9-step sequence alternating between aeration, anoxic treatment and settlement. The cycle time was 12.1 h and the reactors were operated at a substrate loading rate of 3.60 g filtered chemical oxygen demand (CODf)/m(2) media/d, 0.28 g filtered total nitrogen (TNf)/m(2) media/d, 0.24 g ammonium-nitrogen (NH4-N)/m(2) media/d and 0.07 g ortho-phosphate (PO4-P)/m(2) media/d. The average removal rates achieved during Study 1 were 98% CODf, 88% TNf, 97% NH4-N and 35% PO4-P. During Study 2 (264 days), the unit was operated at a loading rate of 2.49 g CODf/m(2) media/d, 0.24 g TNf/m(2) media/d, 0.20 g NH4-N/m(2) media/d and 0.06 PO4-P/m(2) media/d. The energy requirement during this study was reduced by modifying the treatment cycle in include fewer pumping cycles. Removal rates in Study 2 averaged 97% CODf, 86% TNf, 99% NH4-N and 76% PO4-P. The excess sludge production of the system was evaluated and detailed analyses of the treatment cycles were carried out. Biomass yields were estimated at 0.09 g SS/g CODf, removed and 0.21 g SS/g CODf, removed for Studies 1 and 2, respectively. Gene analysis showed that the use of a partial vacuum did not affect the growth of ammonia-oxidizing bacteria. The results indicate that the ASF-BR and passive aeration technologies can offer efficient alternatives to existing technologies. The authors would like to acknowledge the financial support provided by Enterprise Ireland who funded this research project (Grant No CFTD-2008-315). The authors also thank M. Barrett and K. Kilroy for kindly providing bacterial and amoA plasmids for real-time PCR experiments. This publication has also emanated from research conducted with the financial support of the European Research Council (ERC Starting Grant #: 2613303 “C-BIOTECH”). peer-reviewed

Stable-isotope probing and metagenomics were applied to study samples taken from laboratory-scale slow sand filters 0.5, 1, 2, 3 and 4 h after challenging with (13)C-labelled Escherichia coli to determine the mechanisms and organisms responsible for coliform removal. Before spiking, the filters had been continuously operated for 7 weeks using water from the River Kelvin, Glasgow as their influent source. Direct counts and quantitative PCR assays revealed a clear predator-prey response between protozoa and E. coli. The importance of top-down trophic-interactions was confirmed by metagenomic analysis, identifying several protozoan and viral species connected to E. coli attrition, with protozoan grazing responsible for the majority of the removal. In addition to top-down mechanisms, indirect mechanisms, such as algal reactive oxygen species-induced lysis, and mutualistic interactions between algae and fungi, were also associated with coliform removal. The findings significantly further our understanding of the processes and trophic interactions underpinning E. coli removal. This study provides an example for similar studies, and the opportunity to better understand, manage and enhance E. coli removal by allowing the creation of more complex trophic interaction models.

The effect of sulfate addition on the stability of, and microbial community behavior in, low-temperature anaerobic expanded granular sludge bed-based bioreactors was investigated at 15 degrees C. Efficient bioreactor performance was observed, with chemical oxygen demand (COD) removal efficiencies of >90%, and a mean SO42- removal rate of 98.3%. In situ methanogensis appeared unaffected at a COD: SO42- influent ratio of 8:1, and subsequently of 3:1, and was impacted marginally only when the COD: SO42- ratio was 1:2. Specific methanogenic activity assays indicated a complex set of interactions between sulfate-reducing bacteria (SRB), methanogens and homoacetogenic bacteria. SO42- addition resulted in predominantly acetoclastic, rather than hydrogenotrophic, methanogenesis until >600 days of SO42--influenced bioreactor operation. Temporal microbial community development was monitored by denaturation gradient gel electrophoresis (DGGE) of 16S rRNA genes. Fluorescence in situ hybridizations (FISH), qPCR and microsensor analysis were combined to investigate the distribution of microbial groups, and particularly SRB and methanogens, along the structure of granular biofilms. qPCR data indicated that sulfidogenic genes were present in methanogenic and sulfidogenic biofilms, indicating the potential for sulfate reduction even in bioreactors not exposed to SO42-. Although the architecture of methanogenic and sulfidogenic granules was similar, indicating the presence of SRB even in methanogenic systems, FISH with rRNA targets found that the SRB were more abundant in the sulfidogenic biofilms. Methanosaeta species were the predominant, keystone members of the archaeal community, with the complete absence of the Methanosarcina species in the experimental bioreactor by trial conclusion. Microsensor data suggested the ordered distribution of sulfate reduction and sulfide accumulation, even in methanogenic granules. Pádhraig Madden was supported by a scholarship from the Irish Research Council. Gavin Collins is supported by a European Research Council (ERC) Starting Grant (‘3C-BIOTECH’; project no. 261330). Profs. Michael Böttcher and Tim Ferdelman, and Dr. Raeid Abed, are thanked for their insightful conversations. peer-reviewed

Three pilot-scale, horizontal-flow biofilm reactors (HFBRs 1-3) were used to treat methane (CH4)-contaminated air to assess the potential of this technology to manage emissions from agricultural activities, waste and wastewater treatment facilities, and landfills. The study was conducted over two phases (Phase 1, lasting 90 days and Phase 2, lasting 45 days). The reactors were operated at 10 degrees C (typical of ambient air and wastewater temperatures in northern Europe), and were simultaneously dosed with CH4-contaminated air and a synthetic wastewater (SWW). The influent loading rates to the reactors were 8.6 g CH4/m3/hr (4.3 g CH4/m2 TPSA/hr; where TPSA is top plan surface area). Despite the low operating temperatures, an overall average removal of 4.63 g CH4/m3/day was observed during Phase 2. The maximum removal efficiency (RE) for the trial was 88%. Potential (maximum) rates of methane oxidation were measured and indicated that biofilm samples taken from various regions in the HFBRs had mostly equal CH4 removal potential. In situ activity rates were dependent on which part of the reactor samples were obtained. The results indicate the potential of the HFBR, a simple and robust technology, to biologically treat CH4 emissions.The results of this study indicate that the HFBR technology could be effectively applied to the reduction of greenhouse gas emissions from wastewater treatment plants and agricultural facilities at lower temperatures common to northern Europe. This could reduce the carbon footprint of waste treatment and agricultural livestock facilities. Activity tests indicate that methanotrophic communities can be supported at these temperatures. Furthermore, these data can lead to improved reactor design and optimization by allowing conditions to be engineered to allow for improved removal rates, particularly at lower temperatures. The technology is simple to construct and operate, and with some optimization of the liquid phase to improve mass transfer, the HFBR represents a viable, cost-effective solution for these emissions.

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.

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.

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 (> or =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 O(2) consumption overlapped. In slurry incubations conducted with (15)NO(3)(-) enrichment in gas-impermeable bags, denitrification assays revealed that N(2) production occurred at initial O(2) concentrations of up to approximately 90 microM. Initial denitrification rates were not substantially affected by O(2) in surficial (0-4 cm) sediments, whereas rates increased by twofold with O(2) depletion in the at 4-6 cm depth interval. In a well mixed, flow-through stirred retention reactor (FTSRR), (29)N(2) and (30)N(2) were produced and O(2) was consumed simultaneously, as measured online using membrane inlet mass spectrometry. We hypothesize that the observed high denitrification rates in the presence of O(2) may result from the adaptation of denitrifying bacteria to recurrent tidally induced redox oscillations in permeable sediments at Janssand.