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Nanaerobes are a newly described class of microorganisms that use a unique cytochrome bd oxidase to achieve nanaerobic respiration at <2 μM dissolved oxygen (∼1% of atmospheric oxygen) but are not viable above this value due to the lack of other terminal oxidases. Although sharing an overlapping ecological niche with methanogenic archaea, the role of nanaerobes in methanogenic systems has not been studied so far. To explore their occurrence and significance, we re-analyzed published meta-omic datasets from animal rumina and waste-to-energy digesters, including conventional anaerobic digesters and anaerobic digesters with ultra-low oxygenation. Results show that animal rumina share broad similarities in the microbial community and system performance with oxygenated digesters, rather than with conventional anaerobic digesters, implying that trace levels of oxygen drive the efficient digestion in ruminants. The rumen system serves as an ideal model for the newly named nanaerobic digestion, as it relies on the synergistic co-occurrence of nanaerobes and methanogens for methane yield enhancement. The most abundant ruminal bacterial family Prevotellacea e contains many nanaerobes, which perform not only anaerobic fermentation but also nanaerobic respiration using cytochrome bd oxidase. These nanaerobes generally accompany hydrogenotrophic methanogens to constitute a thermodynamically and physiologically consistent framework for efficient methane generation. Our findings provide new insights into ruminal methane emissions and strategies to enhance methane generation from biomass.

Anaerobic digestion is a long-established technology for the valorization of diverse organic wastes with concomitant generation of valuable resources. However, mono-digestion (i.e., anaerobic digestion using one feedstock) suffers from challenges associated with feedstock characteristics. Co-digestion using multiple feedstocks provides the potential to overcome these limitations. Significant research and development efforts have highlighted several inherent merits of co-digestion, including enhanced digestibility due to synergistic effects of co-substrates, better process stability, and higher nutrient value of the produced co-digestate. However, studies focused on the underlying effects of diverse co-feedstocks on digester performance and stability have not been synthesized so far. This review fills this gap by highlighting the limitations of mono-digestion and critically examining the benefits of co-digestion. Furthermore, this review discusses synergistic effect of co-substrates, characterization of microbial communities, the prediction of biogas production via different kinetic models, and highlights future research directions for the development of a sustainable biorefinery., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This study developed an intermittent oxidation-reduction potential (ORP)-controlled micro-aeration system for high solids anaerobic digestion (AD) of lignocellulosic biomass without volatile fatty acids (VFA) accumulation at high organic loading rate (OLR). Traditional AD of Napier grass, a model lignocellulosic biomass, at an OLR of 5 g volatile solids (VS)/L/day resulted in an accumulation of total VFA concentration up to 9.2 g/L as acetic acid (HAc) equivalent, causing rapid drops in pH and methane yield, and driving the digester to the verge of failure. Once intermittent (every 24 h) ORP-controlled micro-aeration (at ORP of -470 mV) was initiated, the total VFA concentration rapidly decreased to 3.0 g HAc/L and the methane yield improved, resulting in stable digester performance without the need for alkalinity supplementation or OLR reduction. By combining reactor performance results, mass balance analyses, microbial community characterization data, and a bioenergetic evaluation, this study suggested that rapid VFA conversion and CH 4 production were carried out by facultative anaerobes and hydrogenotrophic methanogens under micro-aerobic conditions. This novel operating approach can be applied as an effective control strategy for high OLR AD processes especially in the event of VFA accumulation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The objective of this study was to evaluate emerging anaerobic membrane bioreactor (AnMBR) technology in comparison with conventional wastewater energy recovery technologies. Wastewater treatment process modeling and systems analyses were combined to evaluate the conditions under which AnMBR may produce more net energy and have lower life cycle environmental emissions than high rate activated sludge with anaerobic digestion (HRAS+AD), conventional activated sludge with anaerobic digestion (CAS+AD), and an aerobic membrane bioreactor with anaerobic digestion (AeMBR+AD). For medium strength domestic wastewater treatment under baseline assumptions at 15 °C, AnMBR recovered 49% more energy as biogas than HRAS+AD, the most energy positive conventional technology considered, but had significantly higher energy demands and environmental emissions. Global warming impacts associated with AnMBR were largely due to emissions of effluent dissolved methane. For high strength domestic wastewater treatment, AnMBR recovered 15% more net energy than HRAS+AD, and the environmental emissions gap between the two systems was reduced. Future developments of AnMBR technology in low energy fouling control, increased flux, and management of effluent methane emissions would make AnMBR competitive with HRAS+AD. Rapid advancements in AnMBR technology must continue to achieve its full economic and environmental potential as an energy recovery strategy for domestic wastewater.

A bench-scale anaerobic membrane bioreactor (AnMBR) equipped with submerged flat-sheet microfiltration membranes was operated at psychrophilic temperature (15 °C) treating simulated and actual domestic wastewater (DWW). Chemical oxygen demand (COD) removal during simulated DWW operation averaged 92 ± 5% corresponding to an average permeate COD of 36 ± 21 mg/L. Dissolved methane in the permeate stream represented a substantial fraction (40-50%) of the total methane generated by the system due to methane solubility at psychrophilic temperatures and oversaturation relative to Henry's law. During actual DWW operation, COD removal averaged 69 ± 10%. The permeate COD and 5-day biochemical oxygen demand (BOD(5)) averaged 76 ± 10 mg/L and 24 ± 3 mg/L, respectively, indicating compliance with the U.S. EPA's standard for secondary effluent (30 mg/L BOD(5)). Membrane fouling was managed using biogas sparging and permeate backflushing and a flux greater than 7 LMH was maintained for 30 days. Comparative fouling experiments suggested that the combination of the two fouling control measures was more effective than either fouling prevention method alone. A UniFrac based comparison of bacterial and archaeal microbial communities in the AnMBR and three different inocula using pyrosequencing targeting 16S rRNA genes suggested that mesophilic inocula are suitable for seeding psychrophilic AnMBRs treating low strength wastewater. Overall, the research described relatively stable COD removal, acceptable flux, and the ability to seed a psychrophilic AnMBR with mesophilic inocula, indicating future potential for the technology in practice, particularly in cold and temperate climates where DWW temperatures are low during part of the year., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Terminal electron accepting process (TEAP) zones developed when a simulated groundwater containing dissolved oxygen (DO), nitrate, arsenate, and sulfate was treated in a fixed-bed bioreactor system consisting of two reactors (reactors A and B) in series. When the reactors were operated with an empty bed contact time (EBCT) of 20 min each, DO-, nitrate-, sulfate-, and arsenate-reducing TEAP zones were located within reactor A. As a consequence, sulfate reduction and subsequent arsenic removal through arsenic sulfide precipitation and/or arsenic adsorption on or coprecipitation with iron sulfides occurred in reactor A. This resulted in the removal of arsenic-laden solids during backwashing of reactor A. To minimize this by shifting the sulfate-reducing zone to reactor B, the EBCT of reactor A was sequentially lowered from 20 min to 15, 10, and 7 min. While 50 mg/L (0.81 mM) nitrate was completely removed at all EBCTs, more than 90% of 300 μg/L (4 μM) arsenic was removed with the total EBCT as low as 27 min. Sulfate- and arsenate-reducing bacteria were identified throughout the system through clone libraries and quantitative PCR targeting the 16S rRNA, dissimilatory (bi)sulfite reductase (dsrAB), and dissimilatory arsenate reductase (arrA) genes. Results of reverse transcriptase (RT) qPCR of partial dsrAB (i.e., dsrA) and arrA transcripts corresponded with system performance. The RT qPCR results indicated colocation of sulfate- and arsenate-reducing activities, in the presence of iron(II), suggesting their importance in arsenic removal.

Linkages among bioreactor operation and performance and microbial community structure were investigated for a fixed-bed biofilm system designed to remove perchlorate from drinking water. Perchlorate removal was monitored to evaluate reactor performance during and after the frequency and intensity of the backwash procedure were changed, while the microbial community structure was studied using clone libraries and quantitative PCR targeting the 16S rRNA gene. When backwash frequency was increased from once per month to once per day, perchlorate removal initially deteriorated and then recovered, and the relative abundance of perchlorate-reducing bacteria (PRB) initially increased and then decreased. This apparent discrepancy suggested that bacterial populations other than PRB played an indirect role in perchlorate removal, likely by consuming dissolved oxygen, a competing electron acceptor. When backwash intensity was increased, the reactor gradually lost its ability to remove perchlorate, and concurrently the relative abundance of PRB decreased. The results indicated that changes in reactor operation had a profound impact on reactor performance through altering the microbial community structure. Backwashing is an important yet poorly characterized procedure when operating fixed-bed biofilm reactors. Compared to backwash intensity, changes in backwash frequency exerted less disturbance on the microbial community in the current study. If this finding can be confirmed in future work, backwash frequency may serve as the primary parameter when optimizing backwash procedures.

Interest in increasing the sustainability of water management is leading to a reevaluation of domestic wastewater (DWW) treatment practices. A central goal is to reduce energy demands and environmental impacts while recovering resources. Anaerobic membrane bioreactors (AnMBRs) have the ability to produce a similar quality effluent to aerobic treatment, while generating useful energy and producing substantially less residuals. This review focuses on operational considerations that require further research to allow implementation of AnMBR DWW treatment. Specific topics include membrane fouling, the lower limits of hydraulic retention time and temperature allowing for adequate treatment, complications with methane recovery, and nutrient removal options. Based on the current literature, future research efforts should focus on increasing the likelihood of net energy recovery through advancements in fouling control and development of efficient methods for dissolved methane recovery. Furthermore, assessing the sustainability of AnMBR treatment requires establishment of a quantitative environmental and economic evaluation framework., (Copyright © 2012. Published by Elsevier Ltd.)

Contaminant removal from drinking water sources under reducing conditions conducive for the growth of denitrifying, arsenate reducing, and sulfate reducing microbes using a fixed-bed bioreactor may require oxygen-free gas (e.g., N2 gas) during backwashing. However, the use of air-assisted backwashing has practical advantages, including simpler operation, improved safety, and lower cost. A study was conducted to evaluate whether replacing N2 gas with air during backwashing would impact performance in a nitrate and arsenic removing anaerobic bioreactor system that consisted of two biologically active carbon reactors in series. Gas-assisted backwashing, comprised of 2 min of gas injection to fluidize the bed and dislodge biomass and solid phase products, was performed in the first reactor (reactor A) every two days. The second reactor (reactor B) was subjected to N2 gas-assisted backwashing every 3-4 months. Complete removal of 50 mg/L NO3- was achieved in reactor A before and after the switch from N2-assisted backwashing (NAB) to air-assisted backwashing (AAB). Substantial sulfate removal was achieved with both backwashing strategies. Prolonged practice of AAB (more than two months), however, diminished sulfate reduction in reactor B somewhat. Arsenic removal in reactor A was impacted slightly by long-term use of AAB, but arsenic removals achieved by the entire system during NAB and AAB periods were not significantly different (p>0.05) and arsenic concentrations were reduced from approximately 200 μg/L to below 20 μg/L. These results indicate that AAB can be implemented in anaerobic nitrate and arsenic removal systems., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aims: Optimization of full-scale, biological perchlorate treatment processes for drinking water would benefit from knowledge of the location and quantity of perchlorate-reducing bacteria (PRB) and expression of perchlorate-related genes in bioreactors. The aim of this study was to quantify perchlorate removal and perchlorate-related genes (pcrA and cld) and their transcripts in bioreactors and to determine whether these genes or transcripts could serve as useful biomarkers for perchlorate treatment processes., Methods and Results: Quantitative PCR (qPCR) assays targeting pcrA and cld were applied to two pilot-scale, fixed-bed bioreactors treating perchlorate-contaminated groundwater. pcrA and cld genes per microgram of DNA were two- to threefold higher and three- to fourfold higher, respectively, in the bioreactor showing superior perchlorate-removal performance. In a laboratory-scale bioreactor, quantities of pcrA and cld genes and transcripts were compared under two distinct performance conditions (c.60 and 20% perchlorate removal) for a 5-min empty bed contact time. cld genes per microgram of DNA were approximately threefold higher and cld transcripts per microgram of RNA were approximately sixfold higher under the higher perchlorate-removal condition. No differences in pcrA genes or transcripts per microgram of DNA or RNA, respectively, were detected between the c.60 and 20% perchlorate-removal conditions, possibly because these assays did not accurately quantify pcrA genes and transcripts in the mixed culture present., Conclusions: Quantities of cld genes and transcripts per microgram of DNA and RNA, respectively, were found to be higher when perchlorate removal was higher. However, quantities of pcrA and cld genes or transcripts were not found to directly correlate with perchlorate-removal rates., Significance and Impact of the Study: To our knowledge, this study represents the first application of qPCR assays to quantify perchlorate-related genes and transcripts in continuous-flow bioreactors. The results indicate that cld gene and transcript quantities can provide insights regarding the quantity, location and gene expression of PRB in bioreactors., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

The effects of the antimicrobial tylosin on a methanogenic microbial community were studied in a glucose-fed laboratory-scale anaerobic sequencing batch reactor (ASBR) exposed to stepwise increases of tylosin (0, 1.67, and 167 mg/L). The microbial community structure was determined using quantitative fluorescence in situ hybridization (FISH) and phylogenetic analyses of bacterial 16S ribosomal RNA (rRNA) gene clone libraries of biomass samples. During the periods without tylosin addition and with an influent tylosin concentration of 1.67 mg/L, 16S rRNA gene sequences related to Syntrophobacter were detected and the relative abundance of Methanosaeta species was high. During the highest tylosin dose of 167 mg/L, 16S rRNA gene sequences related to Syntrophobacter species were not detected and the relative abundance of Methanosaeta decreased considerably. Throughout the experimental period, Propionibacteriaceae and high GC Gram-positive bacteria were present, based on 16S rRNA gene sequences and FISH analyses, respectively. The accumulation of propionate and subsequent reactor failure after long-term exposure to tylosin are attributed to the direct inhibition of propionate-oxidizing syntrophic bacteria closely related to Syntrophobacter and the indirect inhibition of Methanosaeta by high propionate concentrations and low pH., (© 2010 Wiley Periodicals, Inc.)

The microbial processes involved in two-phase anaerobic digestion were investigated by operating a laboratory-scale acid-phase (AP) reactor and analyzing two full-scale, two-phase anaerobic digesters operated under mesophilic (35 °C) conditions. The digesters received a blend of primary sludge and waste activated sludge (WAS). Methane levels of 20% in the laboratory-scale reactor indicated the presence of methanogenic activity in the AP. A phylogenetic analysis of an archaeal 16S rRNA gene clone library of one of the full-scale AP digesters showed that 82% and 5% of the clones were affiliated with the orders Methanobacteriales and Methanosarcinales, respectively. These results indicate that substantial levels of aceticlastic methanogens (order Methanosarcinales) were not maintained at the low solids retention times and acidic conditions (pH 5.2-5.5) of the AP, and that methanogenesis was carried out by hydrogen-utilizing methanogens of the order Methanobacteriales. Approximately 43, 31, and 9% of the archaeal clones from the methanogenic phase (MP) digester were affiliated with the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales, respectively. A phylogenetic analysis of a bacterial 16S rRNA gene clone library suggested the presence of acetate-oxidizing bacteria (close relatives of Thermacetogenium phaeum, 'Syntrophaceticus schinkii,' and Clostridium ultunense). The high abundance of hydrogen consuming methanogens and the presence of known acetate-oxidizing bacteria suggest that acetate utilization by acetate oxidizing bacteria in syntrophic interaction with hydrogen-utilizing methanogens was an important pathway in the second-stage of the two-phase digestion, which was operated at high ammonium-N concentrations (1.0 and 1.4 g/L). A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for syntrophic acetate oxidation and weak-acid inhibition adequately described the dynamic profiles of volatile acid production/degradation and methane generation observed in the laboratory-scale AP reactor. The model was validated with historical data from the full-scale digesters.

Phosphorus was added as a nutrient to bench-scale and pilot-scale biologically active carbon (BAC) reactors operated for perchlorate and nitrate removal from contaminated groundwater. The two bioreactors responded similarly to phosphorus addition in terms of microbial community function (i.e., reactor performance), while drastically different responses in microbial community structure were detected. Improvement in reactor performance with respect to perchlorate and nitrate removal started within a few days after phosphorus addition for both reactors. Microbial community structures were evaluated using molecular techniques targeting 16S rRNA genes. Clone library results showed that the relative abundance of perchlorate-reducing bacteria (PRB) Dechloromonas and Azospira in the bench-scale reactor increased from 15.2% and 0.6% to 54.2% and 11.7% after phosphorus addition, respectively. Real-time quantitative PCR (qPCR) experiments revealed that these increases started within a few days after phosphorus addition. In contrast, after phosphorus addition, the relative abundance of Dechloromonas in the pilot-scale reactor decreased from 7.1 to 0.6%, while Zoogloea increased from 17.9 to 52.0%. The results of this study demonstrated that similar operating conditions for bench-scale and pilot-scale reactors resulted in similar contaminant removal performances, despite dramatically different responses from microbial communities. These findings suggest that it is important to evaluate the microbial community compositions inside bioreactors used for drinking water treatment, as they determine the microbial composition in the effluent and impact downstream treatment requirements for drinking water production. This information could be particularly relevant to drinking water safety, if pathogens or disinfectant-resistant bacteria are detected in the bioreactors.

A novel bioreactor system, consisting of two biologically active carbon (BAC) reactors in series, was developed for the simultaneous removal of nitrate and arsenic from a synthetic groundwater supplemented with acetic acid. A mixed biofilm microbial community that developed on the BAC was capable of utilizing dissolved oxygen, nitrate, arsenate, and sulfate as the electron acceptors. Nitrate was removed from a concentration of approximately 50 mg/L in the influent to below the detection limit of 0.2 mg/L. Biologically generated sulfides resulted in the precipitation of the iron sulfides mackinawite and greigite, which concomitantly removed arsenic from an influent concentration of approximately 200 ug/L to below 20 ug/L through arsenic sulfide precipitation and surface precipitation on iron sulfides. This study showed for the first time that arsenic and nitrate can be simultaneously removed from drinking water sources utilizing a bioreactor system., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A multi-compartment anaerobic bioreactor, designated the anaerobic migrating blanket reactor (AMBR), did not perform well in terms of chemical oxygen demand (COD) removal after an increase in sulfate load, compared to a conventional upflow anaerobic sludge blanket (UASB) reactor. The trophic structures of the bioreactors were characterized by analyzing the electron flows, formation and consumption of fermentation intermediates and terminal product (methane and hydrogen sulfide) formation. Critical performance parameters were linked to operational perturbations such as increase in sulfate load and changes in flow reversal schemes in the AMBR. Both of these manipulations affected the microbial communities, which were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the bacterial and archaeal domains. The less stable AMBR did not produce granular biomass, and in response to increased sulfate concentrations, experienced a reversal in the distribution of hydrogenotrophic methanogens that correlated with a shift in electron flow from butyrate to propionate. As this shift occurred, bacterial populations such as butyrate-producing clostridia, became predominant, thus leading to reactor imbalance. The stable UASB reactor developed and retained granules and maintained a relatively stable archaeal community. Sulfate perturbation led to the selection of a novel bacterial group (Thermotogaceae), which was most likely well adapted to the increasingly sulfidogenic conditions in the bioreactor.

A laboratory-scale anaerobic sequencing batch reactor (ASBR) was operated using a glucose-based synthetic wastewater to study the effects of tylosin, a macrolide antimicrobial commonly used in swine production, on treatment performance. The experimental period was divided into three consecutive phases with different influent tylosin concentrations (0, 1.67, and 167 mg/L). The addition of 1.67 mg/L tylosin to the reactor had negligible effects on the overall treatment performance, that is, total methane production and effluent chemical oxygen demand did not change significantly (P < 0.05), yet analyses of individual ASBR cycles revealed a decrease in the rates of both methane production and propionate uptake after tylosin was added. The addition of 167 mg/L tylosin to the reactor resulted in a gradual decrease in methane production and the accumulation of propionate and acetate. Subsequent inhibition of methanogenesis was attributed to a decrease in the pH of the reactor. After the addition of 167 mg/L tylosin to the reactor, an initial decrease in the rate of glucose uptake during the ASBR cycle followed by a gradual recovery was observed. In batch tests, the specific biogas production with the substrate butyrate was completely inhibited in the presence of tylosin. This study indicated that tylosin inhibited propionate- and butyrate-oxidizing syntrophic bacteria and fermenting bacteria resulting in unfavorable effects on methanogenesis.

A laboratory-scale anaerobic sequencing batch reactor (ASBR) was fed a synthetic wastewater containing glucose to study the effects of the antimicrobial tylosin on treatment performance. Measurements of methane, volatile fatty acids, and COD concentrations suggested that the addition of 1.67 mg/L and 167 mg/l of tylosin to the synthetic wastewater inhibited propionate oxidizing syntrophic bacteria and aceticlastic methanogens. The latter is presumed to be an indirect effect. A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for microbial storage and hydrolysis of reserve carbohydrates, and tylosin liquid-solid mass transfer and inhibition adequately described the dynamic profiles observed in the ASBR.

This study demonstrates the accumulation and degradation of trehalose as a storage compound in a glucose-fed anaerobic sequencing batch reactor (ASBR). One hour after substrate addition, only 40% of the added organic matter (as chemical oxygen demand, COD) was accounted for by the cumulative methane production and soluble COD remaining in the reactor. All influent COD was accounted for by methane and biomass production by the end of the 24-h ASBR cycle. These dynamics can be explained by the production of an intracellular storage product. Total carbohydrate analysis showed that 26% of the glucose added to the reactor transiently accumulated within the biomass. Based on (13)C-nuclear magnetic resonance (NMR) analysis, trehalose (alpha-D-glucopyranosyl-(D-glucopyranoside)) was identified as the main carbohydrate produced. Mathematical modeling was performed and the IWA Anaerobic Digestion Model No. 1 (ADM1) was modified to include microbial storage. The modified model adequately described the ASBR dynamics during a 24-h cycle.

Biological hydrogen production by anaerobic mixed communities was studied in laboratory-scale bioreactors using sucrose as the substrate. A bioreactor in which a fraction of the return sludge was exposed to repeated heat treatments performed better than a control bioreactor without repeated heat treatment of return sludge and produced a yield of 2.15 moles of hydrogen per mole of sucrose, with 50% hydrogen in the biogas. Terminal restriction fragment length polymorphism analysis showed that two different Clostridium groups (comprised of one or more species) were dominant during hydrogen production. The relative abundance of two other non-Clostridium groups increased during periods of decreased hydrogen production. The first group consisted of Bifidobacterium thermophilum, and the second group included one or more of Bacillus, Melissococcus, Spirochaeta, and Spiroplasma spp.

The influence of backwashing on biological perchlorate reduction was evaluated in two laboratory scale fixed bed biofilm reactors using 1- or 3-mm glass beads as support media. Influent perchlorate concentrations were 50 microg/L and acetate was added as the electron donor at a concentration of 2 mg C/L. Perchlorate removal was evaluated at various influent dissolved oxygen (DO) concentrations. Complete perchlorate removal was achieved with an influent DO concentration of 1mg/L resulting in bulk phase DO concentrations below the detection limit of 0.01 mg/L. The influence of increasing influent DO concentrations for 12 h periods was evaluated before and after individual backwash events. Partial perchlorate removal was achieved with an influent DO concentration of 3.5 mg/L before a strong backwash (bulk phase DO concentrations of approximately 0.2mg/L), while no perchlorate removal was observed after the strong backwash at the same influent DO level (bulk phase DO concentrations of approximately 0.8 mg/L). The immediate effect of backwashing depended on influent DO concentrations. With influent DO concentrations of 1 mg/L, strong backwashing resulted in a brief (<12 h) increase of effluent perchlorate concentrations up to 20 microg/L; more pronounced effects were observed with influent DO concentrations of 3mg/L. Daily weak backwashing had a small and, over time, decreasing negative influence on perchlorate reduction, while daily strong backwashing ultimately resulted in the breakdown of perchlorate removal with influent DO concentrations of 3 mg/L.

A 6-L, completely mixed anaerobic bioreactor with an external ultrafiltration membrane module was operated for 300 days to evaluate the startup and performance of an anaerobic membrane bioreactor (AnMBR) treating swine manure. The reactor had a successful startup at the initial loading rate of 1g volatile solids (VS)/L/day. After a two-fold increase in loading rate followed by a sudden, two-fold increase in flow velocity through the membrane module on day 75, the performance of the AnMBR deteriorated as measured by volatile fatty acid (VFA) accumulation, decrease in pH, and decrease in biogas production. The methanogenic population dynamics in the reactor were monitored with terminal restriction fragment length polymorphism (T-RFLP). Changes in the relative levels of Methanosarcinaceae and Methanosaetaceae were consistent with changes in VFA concentrations, i.e., high and low levels of acetate corresponded to a high abundance of Methanosarcinaceae and Methanosaetaceae, respectively. The levels of hydrogenotrophic methanogens of the order of Methanomicrobiales increased during decreased reactor performance suggesting that syntrophic interactions involving hydrogenotrophic methanogens remained intact regardless of the degree of shear in the AnMBR.

We investigated bacterial and archaeal community structures and population dynamics in two anaerobic bioreactors processing a carbohydrate- and sulfate-rich synthetic wastewater. A five-compartment anaerobic migrating blanket reactor (AMBR) was designed to promote biomass and substrate staging, which partially separates the processes of methanogenesis and sulfidogenesis in the middle and outer compartment(s) respectively. The second reactor was a conventional, single-compartment upflow anaerobic sludge blanket (UASB) reactor. Both reactors, which were seeded with the same inoculum, performed well when the influent chemical oxygen demand (COD)/SO(4) (2-) mass ratio was 24.4. The AMBR performed worse than the UASB reactor when the influent COD/SO(4) (2-) mass ratio was decreased to 5.0 by raising the sulfate load. Terminal restriction fragment length polymorphism analyses of bacterial 16S rRNA genes showed that the increase in sulfate load had a greater impact on bacterial diversity and community structure for the five AMBR compartments than for the UASB reactor. Moreover, bacterial community profiles across AMBR compartments became more similar through time, indicating a converging, rather than a staged community. While similar populations were abundant in both reactors at the beginning of the experiment, fermenting bacteria (clostridia, streptococci), and sulfate-reducing bacteria became more abundant in the AMBR, after shifting to a higher sulfate load, while a novel Thermotogales-like population eventually became predominant in the UASB reactor. A similar shift in the community structure of the hydrogenotrophic methanogens in the AMBR occurred: representatives of the Methanobacteriaceae out-competed the Methanospirillaceae after increasing the sulfate load in the AMBR, while the archaeal community structure was maintained in the UASB.

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate., (2006 Wiley Periodicals, Inc.)

A laboratory-scale anaerobic sequencing batch reactor was used to treat a model substrate mixture representing pharmaceutical wastewater at an organic loading rate of 2.9 g COD/(L d). After reaching stable operation the reactor was first exposed to low (1 mg/L) and, subsequently, to high (200 mg/L) concentrations of the antibiotic erythromycin. The addition of low levels of erythromycin resulted in a significant but limited reduction of biogas production by 5% and the higher level of erythromycin did not impact biogas production further, suggesting that a substantial fraction of the microbial populations in the ASBR were resistant to the antibiotic. Effluent soluble COD could not be accounted for in measured volatile fatty acids, perhaps suggesting the production of soluble microbial products. In batch tests evaluating the specific methanogenic activity, conversion of the model substrate mixture was only slightly affected by the presence of erythromycin. However, the conversion of butyric acid was inhibited when erythromycin was present. After 47 days of exposure to erythromycin, the conversion of butyric acid was inhibited to a lesser extent, suggesting the development of antibiotic resistance in the biomass. Exposure to antibiotics can affect specific substrate degradation pathways, leading to the accumulation of volatile fatty acids, soluble microbial products, and potentially to overall system instabilities.

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-beta-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage.

The effect of acclimating anaerobic granules from commercial bioreactors with different carbon/electron sources on their ability to reductively dechlorinate a tri-(2,3,4-CB) and heptachlorobiphenyl (2,2',3,3',4,5,6-CB) was studied. The anaerobic granules were first grown in upflow anaerobic sludge blanket (UASB) reactors fed with two different mixtures of carbon/electron sources, i.e., propionate/butyrate/methanol and formate/methanol. Differences in dechlorination patterns for 2,2',3,3',4,5,6-CB were observed in batch experiments inoculated with granules from these two sets of UASB reactors. Variation of the carbon/electron source, during the dechlorination process, had no effect on the dechlorination pathway, but the extents and rates of dechlorination were highest for ethanol and formate and lowest for pyruvate fed batches. Pre-acclimation of different anaerobic sludges to polychlorinated biphenyls (PCBs) shortened the lag period, but did not influence the PCB dechlorination pathway. This is the first time that similar acclimation conditions for several anaerobic microbial communities prior to inoculation were reported to yield similar substrate specificities for the reductive dechlorination of specific PCB congeners. This research demonstrates a successful strategy for the development of biocatalysts to serve as the inoculum of partially decontaminated sites in order to provide microorganisms with specificities complementary to those of naturally occurring dechlorinators.

Microbial population dynamics were investigated during start-up and during periods of overload conditions in anaerobic co-digesters treating municipal solid waste and sewage sludge. Changes in community structure were monitored using ribosomal RNA-based oligonucleotide probe hybridization to measure the abundance of syntrophic propionate-oxidizing bacteria (SPOB), saturated fatty acid-beta-oxidizing syntrophs (SFAS), and methanogens. These changes were linked to traditional performance parameters such as biogas production and volatile fatty acid (VFA) concentrations. Digesters with high levels of Archaea started up successfully. Methanosaeta concilii was the dominant aceticlastic methanogen in these systems. In contrast, digesters that experienced a difficult start-up period had lower levels of Archaea with proportionally more abundant Methanosarcina spp. Syntrophic propionate-oxidizing bacteria and saturated fatty acid-beta-oxidizing syntrophs were present at low levels in all digesters, and SPOB appeared to play a role in stabilizing propionate levels during start-up of one digester. Digesters with a history of poor performance tolerated a severe organic overload event better than digesters that had previously performed well. It is hypothesized that higher levels of SPOB and SFAS and their methanogenic partners in previously unstable digesters are responsible for this behavior., (Copyright 2004 Wiley Periodicals, Inc.)

An integrated chemical-physical-biological treatment concept for the low-cost treatment of domestic wastewater is proposed. Domestic wastewater was subjected to a chemically enhanced primary treatment (CEPT), followed by treatment in an upflow anaerobic sludge blanket (UASB) reactor. In addition, a regenerable zeolite was used to remove NH4+, either after CEPT pretreatment or after biological treatment in the UASB reactor. The CEPT pretreatment consisted of the addition of a coagulant (FeCl3) and an anionic organic flocculant and removed on average 73% of the total chemical oxygen demand (COD(t)), 85% of the total suspended solids, and 80% of PO4(3-) present in the wastewater. The UASB system, which consequently received a low COD(t) input of approximately 140 mg/L, was operated using a volumetric loading rate of 0.4 g COD(t)/L. d (hydraulic retention time [HRT]=10 h) and 0.7 g COD(t)/L. d (HRT=5 h). For these conditions, the system removed about 55% of the COD(t) in its influent, thus producing an effluent with a low COD(t) of approximately 50 mg/L. The zeolite, when applied in batch mode before the UASB reactor, removed approximately 45% of the NH4+, whereas its application as a post-treatment cartridge resulted in almost 100% NH4+ removal. The simple design and relatively low operating costs, due to low costs of added chemicals and low energy input (estimated at Euro 0.07-0.1 per m3 wastewater treated), combined with excellent treatment performance, means that this system can be used as a novel domestic wastewater treatment system for developing countries. Therefore, the system is called a Low Investment Sewage Treatment (LIST) system.

It has generally been accepted that the formation of granules in anaerobic wastewater treatment systems requires a hydraulic upflow pattern. To evaluate this hypothesis, we operated an anaerobic migrating blanket reactor (AMBR) without a hydraulic upflow pattern, using a synthetic wastewater containing acetate, propionate, butyrate and sucrose. We provided conditions amenable to the formation of granules by operating the system with a moderate hydraulic selection pressure, which in this system was not the result of a hydraulic upflow pattern, but was provided by migration of biomass and intermittent mechanical mixing. Granules were first noticed after 2 months of operation, and it took another 2 months for a mature granular blanket to develop. Besides granules, approximately 1-cm-long Methanosaeta fibres developed and, after 6 months of operation, 30% of biomass consisted of these fibres. Quantitative membrane hybridization showed that almost all the total 16S rRNA extracted from fibres consisted of 16S rRNA from Methanosaeta concilii. This finding indicates that it was possible to develop pockets consisting almost entirely of an organism with a very limited substrate utilization spectrum (only acetate) in a system that was fed a synthetic wastewater containing acetate, propionate, butyrate and sucrose and that is known for its ability to develop biomass with a complex microbial community structure.

The availability of molecular biological tools for studying microbial communities in bioreactors and other engineered systems has resulted in remarkable insights linking diversity and dynamics to process stability. As engineered systems are often more manageable than large-scale ecosystems, and because parallels between engineered environments and other ecosystems exist, the former can be used to elucidate some unresolved ecological issues. For example, the process stability of methanogenic bioreactors containing well-defined trophic groups appears to depend on the diversity of the functional groups within each trophic level as well as on how these functional groups complement each other. In addition to using engineered systems to study general ecological questions, microbial ecologists and environmental engineers need to investigate conditions, processes, and interactions in engineered environments in order to make the ecological engineering of bioreactor design and operation more practicable.

Anaerobic reactor systems are essential for the treatment of solid and liquid wastes and constitute a core facility in many waste treatment plants. Although much is known about the basic metabolism in different types of anaerobic reactors, little is known about the microbes responsible for these processes. Only a few percent of Bacteria and Archaea have so far been isolated, and almost nothing is known about the dynamics and interactions between these and other microorganisms. This lack of knowledge is most clearly exemplified by the sometimes unpredictable and unexplainable failures and malfunctions of anaerobic digesters occasionally experienced, leading to sub-optimal methane production and wastewater treatment. Using a variety of molecular techniques, we are able to determine which microorganisms are active, where they are active, and when they are active, but we still need to determine why and what they are doing. As genetic manipulations of anaerobes have been shown in only a few species permitting in-situ gene expression studies, the only way to elucidate the function of different microbes is to correlate the metabolic capabilities of isolated microbes in pure culture to the abundance of each microbe in anaerobic reactor systems by rRNA probing. This chapter focuses on various molecular techniques employed and problems encountered when elucidating the microbial ecology of anaerobic reactor systems. Methods such as quantitative dot blot/fluorescence in-situ probing using various specific nucleic acid probes are discussed and exemplified by studies of anaerobic granular sludge, biofilm and digester systems.

Flow rate, electron donor addition, and biomass control were evaluated in order to optimize perchlorate (ClO4-) removal from drinking water using biologically active carbon (BAC) filtration. Influent dissolved oxygen (DO) was lowered from ambient conditions to approximately 2.5 mg/L for all experiments using a nitrogen sparge. When influent nitrate concentration was 0-2.0 mg/L, 1.6-2.8 mg/L as carbon of acetate or ethanol was required to achieve and sustain the complete removal of 50 microg/L perchlorate in a BAC filter. Most or all of the exogenous acetate and ethanol was removed during biofiltration. When a 72-h electron donor feed failure was simulated, a maximum perchlorate breakthrough of 18 microg/L was observed and, once electron donor was reapplied, 9 days were required to reestablish complete perchlorate removal. During a 24-h electron donor feed failure simulation, the maximum effluent perchlorate concentration detected was 6.7 microg/L. Within 24 h of reactivating the electron donor, the filter regained its capacity to consistently remove 50 microg/L perchlorate to below detection. Although biomass growth diminished the filter's ability to consistently remove perchlorate, a cleaning procedure immediately restored stable, complete perchlorate removal. This cleaning procedure was required approximately every 50 days (4800 bed volumes) when influent DO concentration was 2.5 mg/L. Empty-bed contact time (EBCT) experiments showed that 80% perchlorate removal was achieved using a 5-min EBCT, and complete perchlorate removal was observed for an EBCT of 9 min. It was also demonstrated that BAC filtration consistently removed perchlorate to below detection for influent perchlorate concentrations ranging from 10 to 300 microg/L, influent sulfate concentrations between 0 and 220 mg/L, influent pH values of 6.5-9.0, and operating temperatures of 5-22 degrees C.

Changes in methanogenic population levels were followed during startup of a full-scale, farm-based anaerobic sequencing batch reactor (ASBR) and these changes were linked to operational and performance data. The ASBR was inoculated with anaerobic digester sludge from a municipal wastewater treatment facility. During an acclimation period of approximately 3 months, the ASBR content was diluted to maintain a total ammonia-N level of approximately 2000mg l(-1). After this acclimation period, the volatile solids loading rate was increased to its design value of 1.7g l(-1) day(-1) with a 15-day hydraulic retention time, which increased the total ammonia-N level in the ASBR to approximately 3,600 mg l(-1). The 16S ribosomal RNA (rRNA) levels of the acetate-utilizing methanogens of the genus Methanosarcina decreased from 3.8% to 1.2% (expressed as a percentage of the total 16S rRNA levels) during this period, while the 16S rRNA levels of Methanosaeta concilii remained low (below 2.2%). Methane production and reactor performance were not affected as the 16S rRNA levels of the hydrogen-utilizing methanogens of the order Methanomicrobiales increased from 2.3% to 7.0%. Hence, it is likely that during operation with high ammonia levels, the major route of methane production is through a syntrophic relationship between acetate-oxidizing bacteria and hydrogen-utilizing methanogens. Anaerobic digestion at total ammonia-N levels exceeding 3500mg l(-1) was sustainable apparently due to the acclimation of hydrogen-utilizing methanogens to high ammonia levels.

The objective of this study was to evaluate staging and its effects on reactor performance in a compartmentalized bioreactor, designated the anaerobic migrating blanket reactor (AMBR). The AMBR was operated by reversing the flow several times per day, which allowed for substantial biomass migration without biomass accumulation in the final compartment. During reactor startup, the structures of the microbial communities in the five compartments were quite similar despite substantial differences in substrate types and concentrations in the different compartments. During the rest of the operational period, biomass migration was reduced by changing operating conditions and, as a result, a larger difference in the structures of the microbial communities developed for the different compartments (biomass staging). For example, after changing operating conditions, rRNA levels for the acetate-utilizing methanogen Methanosaeta concilii were approximately 35 and 10% of the total rRNA in the middle and outside compartments, respectively (before changing operating conditions these levels were approximately 20 and 12% of the total rRNA, respectively). Promoting larger differences in the structures of the microbial communities for the different compartments did not improve reactor performance as lower levels of M. concilii in the outside compartments hindered acetate removal and compromised effluent quality.

The relationship between the levels of mycolic acid-containing actinomycetes (mycolata), Gordonia spp. and Gordonia amarae, and foam initiation and stability was characterized using: (1) batch tests involving addition of G. amarace cells to activated sludge, (2) analysis of a full-scale activated sludge plant that experienced seasonal foaming, and (3) a study of lab-scale activated sludge reactors augmented with G. amarae. Using batch tests, threshold Gordonia levels for foam formation and foam stability were determined to be approximately 2 x 10(8) microm ml(-1) and 1 x 10(9) microm ml(-1), respectively. In the full-scale plant, the levels of Gordonia spp. and G. amarae increased during the course of foaming, and the foam formation threshold of 2 x 10 microm ml(-1) corresponded to the onset of foaming. This value was also verified in lab-scale reactor washout experiments, where decreasing mycolata levels were observed during the course of foam dissipation. The foam stability threshold of 1 x 10(9) micorm ml(-1) was verified in lab-scale reactor studies. The increase in the levels of Gordonia spp. and G. amarae in the full-scale plant corresponded to an increase in temperature, suggesting that the growth of Gordonia spp. was favored during warmer periods.

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050-4057, 2000).

Biological hydrogen production by anaerobic mixed communities was studied in laboratory-scale bioreactors using sucrose as the substrate. A bioreactor in which a fraction of the return sludge was exposed to repeated heat treatments performed better than a control bioreactor without repeated heat treatment of return sludge and produced a yield of 2.15 moles of hydrogen per mole of sucrose, with 50% hydrogen in the biogas. Terminal restriction fragment length polymorphism analysis showed that two different Clostridium groups (comprised of one or more species) were dominant during hydrogen production. The relative abundance of two other non-Clostridium groups increased during periods of decreased hydrogen production. The first group consisted of Bifidobacterium thermophilum, and the second group included one or more of Bacillus, Melissococcus, Spirochaeta, and Spiroplasma spp. [ABSTRACT FROM AUTHOR]

rid=""id=""Correspondence to: L. Raskin; Fax (217) 333-6968; E-mail: lraskin@uiuc.eduAbstract To evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized. The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed probes together with previously designed probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA. [ABSTRACT FROM AUTHOR]

Abstract: Membrane fouling was evaluated in a side stream anaerobic membrane bioreactor (AnMBR) operated for the treatment of swine manure. The AnMBR consisted of an external tubular polyethersulphone ultrafiltration membrane module (diameter = 12 mm) connected to a six-liter mixed bioreactor. The system was operated for 135 days without chemical membrane cleaning resulting in a membrane flux of 5–10 L/m2 h. Membrane fouling was dominated by a loosely attached fouling layer, which could be removed by flushing the tubular membrane. Intensive chemical cleaning after the 135 days of continuous operation resulted in an irreversible resistance of 3×1012 1/m, equivalent to 1.3 times the resistance of the new membrane. More frequent chemical membrane cleaning using HNO3 could not prevent the development of irreversible fouling. Equilibrium calculations and scanning electron microscopy with energy dispersive spectroscopy demonstrated that inorganic precipitation contributed to fouling of the membrane surface and in the membrane pores. [Copyright &y& Elsevier]