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1AbstractThis work evaluates four different physicochemical pretreatments (acidic, thermal, acidic-thermal and thermal-acidic) on an anaerobic inoculum used for alcohol production from acetate and butyrate. All experiments were conducted in single batches using acetate and butyrate as substrates at 30 °C and with a pressurized headspace of pure H2 at 2.15 atm (218.2 MPa). Thermal and acidic-thermal pretreatments lead to higher production of both ethanol and butanol. Mathematical modelling shows that the highest attainable concentrations of ethanol and butanol produced were 122 mg L-1 and 97 mg L-1 for the thermal pretreatment (after 17.5 days) and 87 mg L-1 and 143 mg L-1 for the acidic-thermal pretreatment (after 18.9 days). Acetate was produced in all assays. Thermodynamic data indicated that a high H2 partial pressure favoured solventogenic metabolic pathways. Finally, sequencing data showed that both thermal and acidic-thermal pretreatments selected mainly the bacterial genera Pseudomonas, Brevundimonas and Clostridium. The acidic-thermal pretreatment selected a bacterial community more adapted to the conversion of acetate and butyrate into ethanol and butanol, respectively. Thermal-acidic pretreatment was unstable, showing significant variability between replicates. Acidic pretreatment showed the lowest alcohol production.

Extracellular polymeric substances (EPS) play major roles in the efficacy of biofilms such as anaerobic granules, ranging from structural stability to more specific functions. The EPS of three granular anaerobic sludges of different origins were studied and compared. Particularly, the peptides from the protein fraction were identified by mass spectrometry. Desulfoglaeba and Treponema bacterial genera and Methanosaeta and Methanobacterium archaeal genera were prominent in all three sludges. Methanosaeta concilii proteins were the most represented in EPS of all three sludges studied. Principally, four proteins found in the three sludges, the S-layer protein, the CO-methylating acetyl-CoA synthase, an ABC transporter substrate-binding protein and the methyl-coenzyme M reductase, were expressed by Methanosaeta concilii. Mainly catabolic enzymes were found from the 45 proteins identified in the protein fraction of EPS. This suggests that EPS may have a role in allowing extracellular catabolic reactions. Electronic supplementary material The online version of this article (10.1186/s13568-019-0746-0) contains supplementary material, which is available to authorized users.

Direct interspecies electron transfer (DIET) has been typically proposed as mechanism of electron transfer among methanogenic populations in granules during anaerobic digestion where Geobacter species play a key role. Using anaerobic granules where Geobacteraceae members were not prevalent − representing only 0.3% of total bacteria −, tests incubated with two co-substrates showed that the rate of methanogenesis from formate and hydrogen diminished in the presence of a non-methanogenic co-substrate such as ethanol. This could indicate that biological DIET occurs and competes with hydrogen and formate during methanogenesis. Moreover, the addition of conductive microparticles, such as stainless steel and granular activated carbon, was found to increase methanogenic activity in disintegrated granules by 190 ± 18% and 175 ± 22% respectively as compared to disintegrated granules devoid of microparticles. The addition of non-conductive microparticles such as porcelain however decreased methanogenic activity by 65 ± 3% of the disrupted granules without microparticle activity. These results indicate that syntrophic bacteria from anaerobic sludge excluding Geobacter species can also carry out conductive mineral mediated DIET.

Methane production from the microalga Scenedesmus sp. AMDD digested in a continuously operated anaerobic reactor was studied under a range of conditions. Preliminary anaerobic toxicity assays revealed that methane yields and glucose consumption were inhibited by algae/sludge ratios in excess of 0.1 (g/g). Continuous digestions of microalgae at two hydraulic retention times (16 and 58 days) and two influent concentrations of algal feed (11 and 20 gTVS L− 1) were evaluated. Based on the amount of degraded algal biomass, a methane yield of 0.4 LCH4 gTVS‐ 1 was estimated. However, at a hydraulic retention time of 16 days only 52–53% of the algal biomass was degraded, primarily due to the limited hydrolysis of the algal cellular material. An increase of retention time to 58 days did not improve the degradation efficiency. Anaerobic decomposition of microalgae biomass appeared to affect composition of the microbial consortium in the digester, promoting the growth of sulfate-reducing bacteria leading to high levels of H2S in the biogas.

Hydrogen conversion of CO by a pure culture of Carboxydothermus hydrogenoformans was investigated and optimized in a lab-scale hollow fiber membrane bioreactor (HFMBR). The reactor was operated under strict anaerobic, extremely thermophilic (70 °C) conditions with a continuous supply of gas, for four months. Reactor performance was evaluated under various operational conditions, such as liquid velocity (vliq) (13, 65 and 130 m h−1), temperature (70, 65, and 60 °C), CO pressure (from 1 to 2.5 atm) and CO loading rate (from 1.3 to 16 . 5 mol L rxr − 1 d − 1 ). Overall, results indicated a relatively constant H2 yield of 92 ± 4% (mol mol−1) regardless of the operational condition tested. Permeation across the colonized membrane was improved by three orders of magnitude as compared to the abiotic membrane, because of dissolved CO concentration was constantly maintained low in the liquid on the shell side of the membrane as continually depleted by the microorganisms. Once the biofilm was sufficiently developed, a maximum CO conversion activity of 0.44 mol CO g−1 volatile suspended solid (VSS) d−1 was achieved at a pCO of 2 atm or above and a vliq of 65 m h−1. However, this highest activity represented only 15% of the maximal activity potential of the strain under non-limiting conditions, attributed to the low concentration of dissolved CO (0.01–0.07 mM) present in the HFMBR liquid. Higher vliq (130 m h−1) produced shearing stress, which detached a significant portion of the biofilm from the membrane, and/or prevented less sessile growth (57% total biomass as biofilm, as opposed to 84–86% at lower vliq). One may deduce from this work that the volumetric CO conversion performance of such a membrane bioreactor would be at the most in the range of 5 mol CO L rxr − 1 d − 1 . Overall, the CO conversion performance in the HFMBR was biokinetically limited, when not limited by gas–liquid mass transfer. Additionally, over time, membrane fouling and aging decreased membrane permeability such that the CO transfer rate would be the most limiting factor in the long run.

This study compared the acidogenic fermentation of Scenedesmus sp.-AMDD at laboratory-scale, under mesophilic (35 °C) and thermophilic conditions (55 °C). Preliminary batch tests were performed to evaluate best conditions for volatile fatty acid (VFA) production from microalgal biomass, with respect to the inoculum, pH and nutrients. The use of bovine manure as inoculum, the operating pH of 4.5 and the addition of a nutrient mix, resulted in a high VFA production of up to 222 mg g-1 total volatile solid (TVS), with a butyrate share of 27%. Both digesters displayed similar hydrolytic activity with 0.38±0.02 and 0.42±0.03 g soluble chemical oxygen demand (COD) g-1 TVS for the digesters operated at 35 and 55 °C, respectively. Mesophilic conditions were more favourable for VFA production, which reached 171±5, compared to 88±12 mg soluble COD g-1 TVS added under thermophilic conditions (94% more). It was shown that in both digesters, butyrate was the predominant VFA.

In-storage psychrophilic anaerobic digestion develops by microbial acclimation in covered swine-manure storage tanks, producing CH4 and stabilizing organic matter. To optimize the system's performance, the process kinetics must be understood. The objective of this study was to evaluate kinetic parameters describing the major stages in the digestion process, and to investigate the effect of temperature acclimation on these parameters. Specific activity tests were performed using manure inocula and five substrates at three incubation temperatures. Extant substrate activities were determined analytically for each case, and intrinsic kinetic parameters for glucose uptake were estimated by grid search fitting to the Monod model. The results demonstrate that this acclimated microbial community exhibits different kinetic parameters to those of the mesophilic communities currently modelled in the literature, with increased activity at low temperatures, varying with substrate and temperature. For glucose, the higher uptake is accompanied by lower microbial yield and half-saturation constant. Decomposing these values suggests that active psychrophilic and mesophilic microbial populations co-exist within the community. This work also confirms that a new method of assessing microbial substrate kinetics must be developed for manure microbial communities, separating microbial mass from other suspended organics.

The conversion of cellulosic crops into biofuels, including methane, is receiving a lot of attention lately. Panicum vergatum , or switchgrass, is a warm season perennial grass well adapted to grow in North America. Different pre-treatments were tested in 0.5 l batch reactors, at 35 °C, in order to enhance the methane production from switchgrass, including temperature, sonication, alkalinization and autoclaving. The methane production on the basis of volatile solids (VS) added to the fermentation were 112.4 ± 8.4, 132.5 ± 9.7 and 139.8 ml g −1 after 38 days of incubation for winter harvested switchgrass (WHS) after grinding, grinding with alkalinization, and grinding with alkalinization and autoclaving, respectively. The methane production was higher for fresh summer harvested switchgrass (SHS), with a production of 256.6 ± 8.2 ml g −1 VS after mulching, alkalinization and autoclaving. The methane production from SHS was improved by 29 and 42% when applying lignin (LiP) or manganese peroxidase (MnP), at 202.1 ± 9.8 and 222.9 ± 22.5 ml g −1 VS, respectively. The combination of an alkali pre-treatment with the MnP increased the methane production furthermore at 297.7 ml g −1 VS. The use of pectinases without chemical pre-treatment showed promising yields at 287.4 and 239.5 ml g −1 VS for pectate-lyase and poly-galacturonase, respectively. An estimation of the methane yield per hectare of crop harvested resulted in net energy production of 29.8, 49.7 and 78.1 GJ for winter harvested switchgrass, mulched and pretreated summer harvested switchgrass, respectively. Switchgrass represents an interesting candidate as a lignocellulosic crop for methane production.

Covering a concrete manure storage tank with an air-tight floating membrane should induce anaerobic digestion of the stored manure. If the microbial community in the manure can acclimate to the ambient conditions, then In-Storage Psychrophilic Anaerobic Digestion (ISPAD) could be used by Canadian livestock producers to produce methane and stabilize manure. The objective of this study was to determine whether the microbial community in swine manure can successfully acclimate to the psychrophilic operating conditions in ISPAD and develop robust anaerobic digestion. This was done in the laboratory by analyzing manure from a three-year old full-scale pilot ISPAD facility located in St. Francois Xavier, Quebec, Canada, along with fresh manure and manure from an uncovered storage tank. Biochemical methane production assays performed at the three temperatures were used to quantify the performance of the microbial community and its temperature dependence. The ISPAD microbial community produced methane, in terms of VS added, at rates of 44.6, 9.8 and 8.5 dm 3 kg −1 d −1 , at 35, 18 and 8 °C, respectively. The ISPAD process reduced the organic matter content of the manure by 24% while releasing 63% of the potential methane in the manure, as opposed to the open storage tank where no measurable reduction in solids occurred, and only 15% of the potential methane was released. These results indicate that a robust, acclimated microbial community actively digests manure in the pilot ISPAD installation.

Mycobacterium austroafricanum IFP 2012 is able to slowly grow on methyl tert-butyl ether (MTBE), a fuel oxygenate widely used as a gasoline additive. The potential of M. austroafricanum IFP 2012 for aerobic MTBE degradation was investigated in the presence of a secondary carbon source, isopropanol. The strain was then tested for MTBE biodegradation at the laboratory-scale in a fixed-bed reactor using perlite as the matrix, and isopropanol was injected once a week to maintain M. austroafricanum IFP 2012 biomass inside the perlite bed. The biofilter was operated for 85 days at an influent flow rate of 20 ml/h by varying the MTBE concentration from 10 to 20 mg/l. The hydraulic retention time was fixed at 5 days. The removal of MTBE depended on the inlet MTBE concentration and a MTBE removal efficiency higher than 99% was obtained for MTBE concentrations up to 15 mg/l. A set of 16S rRNA gene primers specific for M. austroafricanum species was used to analyze the DNA extracted from the biofilter effluent in order to detect the presence of M. austroafricanum IFP 2012 and to estimate the effect of periodic injections of isopropanol on the release of the strain from the perlite bed. The results demonstrated that the injection of isopropanol served to maintain an active MTBE degrading biomass in the biofilter and that this system could be used to effectively treat MTBE contaminated groundwater.

Interest in the use of microalgae for the production of biofuels has grown in recent years. Biomethane is a biofuel that can be obtained with high efficiency from anaerobic digestion of various organic feedstocks. In this study, a selection of freshwater (n = 15) and marine (n = 5) microalgae were tested in order to identify a microalgal strain that could be used as a model for large scale production of methane. Analysis of pH, volatile suspended solids and ammonium at the end of the assay ranged between 6.98–7.66, 16.0–25.9 g/L and 495–1622 mg/L respectively. No significant differences in these values were detected between freshwater and marine strains. There was no significant difference in the methane yield from freshwater microalgae (329 ± 43 mL CH4/g TVS) and marine microalgae (298 ± 83 mL CH4/g TVS) although it varied greatly within the tested strains. A statistical analysis of the microalgae grown under two different culture media showed that the type of medium was more determinant than the type of microalgae (freshwater or marine) for the methane yield, with 310 ± 35, 365 ± 25 and 303 ± 77 mL CH4/g TVS for the freshwater microalgae grown in Bold’s-3NV, f/2 and marine microalgae grown in f/2 media, respectively. The strains Scenedesmus sp.-AMDD, Isochrysis sp. and Scenedesmus dimorphus displayed the best methane yield with 410 ± 6, 408 ± 4 and 397 ± 10 mL CH4/g TVS, respectively. The strain Scenedesmus sp.-AMDD was chosen as a model strain for future work development with continuously fed digesters.

Direct interspecies electrons transfer (DIET) is a syntrophic metabolism in which free electrons flow from one cell to another without being shuttled by reduced molecules such as molecular hydrogen or formate. As more and more microorganisms show a capacity for electron exchange, either to export or import them, it becomes obvious that DIET is a syntrophic metabolism that is much more present in nature than previously thought. This article reviews literature related to DIET, specifically in reference to anaerobic digestion. Anaerobic granular sludge, a biofilm, is a specialized microenvironment where syntrophic bacterial and archaeal organisms grow together in close proximity. Exoelectrogenic bacteria degrading organic substrates or intermediates need an electron sink and electrotrophic methanogens represent perfect partners to assimilate those electrons and produce methane. The granule extracellular polymeric substances by making the biofilm matrix more conductive, play a role as electrons carrier in DIET., Series: Advances in Biochemical Engineering/Biotechnology; no. 151

Biomethane production from the microalga Scenedesmus sp. AMDD was demonstrated in an upflow anaerobic sludge bed (UASB) reactor. A full factorial design experiment was used to identify the effect of organic loading rate (OLR) of algal biomass and hydraulic retention time (HRT) on the volumetric rate of methane production. At an HRT of 4 days and an OLR of 3.23 gTVS LR− 1 d− 1 corresponding to an influent microalgae concentration of 12 g total volatile solids (TVS) L− 1, the volumetric rate of CH4 production reached 0.6 LSTP L− 1R d− 1. A methane yield of 0.18–0.2 L per gTVS of fed microalgae was estimated. A stable performance was observed throughout 3 months of UASB reactor operation. Due to the short HRT and the good performance of UASB reactor, operated at influent microalgae concentrations in a range of 4–12 gTVS L− 1, this reactor type is suitable for coupling with photobioreactors equipped with gravitational settlers.

The literature is showing an increasing trend in the use of microalgal biomass for the production of biofuel, with a recent keen interest for the conversion of microalgae into methane. The methane potential from Phaeodactylum tricornutum was similar for the whole algae, 362. ±. 5. L/kg total volatile solid (TVS), compared with a sample from which the lipids were extracted, at 358. ±. 6. L/kg TVS. The anaerobic digestion of long chain fatty acids (LCFAs) representative of the dominant acids found in P. tricornutum during 22. weeks of incubation demonstrated that the usual incubation time of typical anaerobic digesters (3-6. weeks) is insufficient to hydrolyze the lipids and maximize the energy yield contained in the microalgae. The results from methane potential assays targeting the LCFAs that were found in significant concentrations in P. tricornutum, namely palmitic, palmitoleic and eicosapentaenoic (EPA) acids), at concentration of 2.5 and 1.25. g/L, showed a slow but high yield with palmitic acid (802. ±. 16. L/kg), inhibition from palmitoleic acid and fair degradation of EPA (442. ±. 67. L/kg). There was a 27% increase in the methane yield from a reconstituted P. tricornutum containing 25% of LCFA compared to 10%, at 456. ±. 1 vs 363. ±. 5. mL. STP/g TVS. Increasing the free fatty acid fraction to 50% in the microalgal biomass resulted in the inhibition of the digestion after production of methane during the first 11. days of incubation.

Electricity generation in a microbial fuel cell (MFC) fed with carbon monoxide (CO) has been recently demonstrated; however, the microbial ecology of this system has not yet been described. In this work the diversity of the microbial community present at the anode of CO-fed MFCs was studied by performing denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing (HTS) analyses. HTS indicated a significant increase of the archaeal genus Methanobacterium and of the bacterial order Clostridiales, notably including Clostridium species, while in both MFCs DGGE identified members of the bacterial genera Geobacter, Desulfovibrio, and Clostridium, and of the archaeal genera Methanobacterium, Methanofollis, and Methanosaeta. In particular, the presence of Geobacter sulfurreducens was identified. Tolerance of G. sulfurreducens to CO was confirmed by growing G. sulfurreducens with acetate under a 100 % CO atmosphere. This observation, along with the identification of acetogens, supports the hypothesis of the two-step process in which CO is converted to acetate by the carboxidotrophic Bacteria and acetate is then oxidized by CO-tolerant electricigenic Bacteria to produce electricity. © 2013 Her Majesty the Queen in Right of Canada.

Two previously unknown modes of biomineralization observed in the presence of Carboxydothermus hydrogenoformans are presented. Following the addition of NaHCO3 and the formation of an amorphous calcium phosphate precipitate in a DSMZ medium inoculated with C. hydrogenoformans , two distinct crystalline solids were recovered after 15 and 30 days of incubation. The first of these solids occurred as micrometric clusters of blocky, angular crystals, which were associated with bacterial biofilm. The second solid occurred as 30-50 nm nanorods that were found scattered among the organic products of bacterial lysis. The biphasic mixture of solids was clearly dominated by the first phase. The X-ray diffractometry (XRD) peaks and Fourier transform infrared spectroscopy (FTIR) spectrum of this biphasic material consistently showed features characteristic of Mg-whitlockite. No organic content or protein could be identified by dissolving the solids. In both cases, the mode of biomineralization appears to be biologically induced rather than biologically controlled. Since Mg is known to be a strong inhibitor of the nucleation and growth of CaP, C. hydrogenoformans may act by providing sites that chelate Mg or form complexes with it, thus decreasing its activity as nucleation and crystal growth inhibitor. The synthesis of whitlockite and nano-HAP-like material by C. hydrogenoformans demonstrates the versatility of this organism also known for its ability to perform the water-gas shift reaction, and may have applications in bacterially mediated synthesis of CaP materials, as an environmentally friendly alternative process. © 2014 Haddad et al.

The conversion of carbon monoxide (CO) into hydrogen (H2) by a Carboxydothermus hydrogenoformans pure culture was investigated and optimized in a 35 L gas-lift reactor. The reactor was operated with a continuous supply of gas for 3 months. Reactor performance was evaluated under various operational conditions, such as gas recirculation rates (0.3 and 1.5 L min−1), CO feeding rates (from 0.05 to 0.46 mol L−1reactor day−1) and bacto-peptone addition to the medium. Overall, the H2 yields were constant at 95 ± 1% and 82 ± 1% ( mol H 2 ⋅ mol CO − 1 ) with growth supported by peptone and unsupported respectively, regardless of the operational conditions tested. At the highest biomass density, a maximum CO conversion activity of 0.17 molCO L−1reactor day−1 or 3.79 LCO L−1reactor day−1 was achieved. The ratio of gas recirculation over CO feed flow rates (QR:Qin) was the major parameter that impacted both biological activity and volumetric gas–liquid mass transfer. The CO conversion performance of the gas-lift reactor was kinetically limited over a QR:Qin ratio of 40, and mass transfer limited below that ratio, resulting in a maximum conversion efficiency of 90.4 ± 0.3% and a biological activity of 2.7 ± 0.4 molCO g−1VSS day−1. Overall, the CO conversion performance in the gas-lift reactor was limited by a low cell density, typical of C. hydrogenoformans planktonic growth. This limitation was found to be the most restrictive factor for higher CO loading rates.

The effects of aging trichloroethylene (TCE) with a surrogate soil organic matter (SSOM) on its anaerobic biodegradation and desorption characteristics were studied. Surrogate soil organic matter comprised of composted sphagnum moss was used as a model sorbent, and Desulfomonile tiedjei was used as a model member of an anaerobic consortium capable of reductive dechlorination. After TCE was aged with the sterile SSOM for periods of 2, 14, and 30 d, the completely mixed batch reactors were inoculated with D. tiedjei cells and incubated for 5 d. The mass of TCE dechlorinated to cis-1,2-dichloroethylene (DCE) by D. tiedjei in this period decreased by 72% from 2 to 30 d of aging. Although there was an increase in the equilibrium desorption coefficient (Kd) with aging, sorption was completely reversible. Hence, the reduced availability of TCE for degradation was not coupled with irreversible sorption. To examine the longer-term fate of TCE, the unaged TCE-SSOM slurries were inoculated with D. tiedjei and then incubated for longer times (15, 24, and 29 d vs. 5 d). The conversion of TCE to cis-1,2-DCE was up to eight times greater than what was observed in control experiments without SSOM. Therefore, although aging resulted in reduced TCE biodegradation for short-term incubation (5 d), longer-term incubation (29 d) with the unaged SSOM resulted in extensive TCE dechlorination (up to 40%) to cis-1,2-DCE.

Systems of co-immobilized microorganisms are highly effective for conducting two or more consecutive bioprocesses within close proximity of the source and the sink of substrates. In this work, a general model of a co-immobilized aerobic/anaerobic bacterial system was developed and analyzed. The model was used to optimize pellet design and to study the effect of slow cell growth on process efficiency. The analysis demonstrated that co-immobilization of bacterial species accelerates the rate of biotransformation and provides complete transformation of toxic intermediates.

A commensal system of the aerobic methanotroph Methylosinus sporium with anaerobic methanogens was obtained using cell coimmobilization in a chitosan-lignosulfonate matrix and applied for the dechlorination of tetrachloroethylene (PCE) under coupled anaerobic/aerobic conditions. Due to the oxygen gradient in a bead of immobilized cells, anaerobic transformation of PCE into lower chlorinated compounds was coupled with aerobic dechlorination of these intermediates. At a PCE loading rate of 42.9 mg PCE L r −1day−1 and an oxygenation rate of 4.9 l O2/L r −1day−1, the mineralization efficiency of the commensal system was 88% as opposed to 38% in the control system which was not inoculated with the methanotrophs. The dechlorination rates were 6.7 and 3.0 mg PCE g−1 VSS day−1 for the commensal and control systems, respectively.

The influence of four different granulation precursors, syntroph-enriched methanogenic consortia, Methanosaeta-enriched, Methanosarcina-enriched nuclei and acidogenic flocs, on the time course of complex granule development and the lag time for start-up was investigated in four upflow anaerobic sludge-bed and filter reactors. Although the operational conditions allowed the maintenance of the same specific growth rate of biomass in the four reactors, granulation proceeded rapidly with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei. However, granulation was significantly retarded when acidogenic flocs were used as precursors. The granule mean Sauter diameter increased rapidly in the reactor inoculated with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei and reached, at the end of the experiment, 3.1, 2.7 and 2.4 mm compared to 1.1 mm in that inoculated with acidogenic flocs. This corresponded to a rate of granule size increase of 31, 21, 18 μm/day in syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei, respectively, compared to 7 μm/day in acidogenic flocs. Biomass specific activities (i.e. acidogenic, syntrophic and methanogenic activities) increased stepwise in all reactors with time, especially in those inoculated with syntroph/methanogenic consortia and Methanosaeta nuclei. From these results it appears that syntrophs and Methanosaeta spp. play an important role in the anaerobic granulation process.

Carboxydothermus hydrogenoformans is a thermophilic anaerobic strain most widely known for its ability to produce hydrogen (H2) when grown on carbon monoxide (CO). Although relatively well studied, growth characterization on pyruvate has never been assessed. The present work fully characterizes growth of the bacterium on pyruvate as a sole carbon source. C. hydrogenoformans demonstrated a growth rate of 0.03 h-1, with pyruvate consumption ranging between 0.21 and 0.48 mol · g-1 volatile suspended solid · d-1. A lag phase was also observed when switching from pyruvate to CO. When grown simultaneously on pyruvate and CO, pyruvate consumption was initiated upon CO depletion. This was attributed to pyruvate oxidation inhibition by CO, and not to a diauxic phenomenom. The strain also showed homoacetogenic activity.

The co-culture between Methylosinus sporium, a strictly aerobic methanotroph, and strictly anaerobic methanogens was studied in 5 L aerobic/anaerobic coupled granular sludge reactors under O2-limited conditions. The methanogenic bacteria maintained very good metabolic activities and were able to produce sufficient methane which serviced as substrate for methanotrophic growth. Although other strictly aerobic population proliferated by two orders of magnitude after the granular sludge had been operated under O2-limited conditions for one month, only a limited amount of the added methanotroph remained in the sludge. This result may indicate that M. sporium lacks sufficient O2 affinity to compete with facultative bacteria for the dissolved O2 for their growth.

Anaerobic granule nuclei enriched in either acidogens, syntrophic consortia, Methanosaeta spp. or Methanosarcina spp. were developed in four upflow bed filter reactors fed with sucrose, an ethanol/acetate mixture, acetate and methanol, respectively. The enrichment process was evaluated by scanning electron microscopy and by the change in trophic specific activities. The four developed granule nuclei presented different settling velocities: 3.2, 8.7, 10.5 and 11.3 m h−1 for the acidogenic floes, the Methanosarcina-, the syntrophic consortia and the Methanosaeta-enriched nuclei, respectively. These velocities were related to the size and the ash content of the various aggregates. Acidogenic floes were relatively small in size (

Municipal wastewater is a major source of nutrients and pollutants to freshwater and marine ecosystems and current treatment technologies are either expensive or only partially effective at removing them. We cultivated the alga Scenedesmus sp. AMDD at different pH in chemostats and batch photobioreactors in wastewater obtained from a local treatment plant. In batch mode, biomass productivities averaged 130 mg dry weight L − 1 d − 1 , with no significant effect of growth pH detected. Maximum nitrogen and phosphorus removal rates were equivalent to 7% and 0.7% of the biomass productivity rates. Average hydraulic retention times for 90% N and P removal ranged from 6.55 to 6.65 days and 6.50–6.56 days, respectively. Recovered biomass yields ranged from 0.23 to 0.65 kg m − 3 wastewater, equivalent to approximately 5–15 MJ bioenergy m − 3 wastewater based on an average calorific value of 23 MJ kg − 1 d.w. algal biomass. Approximately 65% of energy equivalent could potentially be recovered from the biomass through anaerobic digestion to methane. Cellular N and P content varied, with cells held longer in stationary phase showing higher C:N and C:P ratios indicative of N- and P-limitation, respectively. Analysis of trace metals in the algal biomass indicated near total depletion of Fe, Zn and Cd from the wastewater, and lower, but substantial, uptake and/or adsorption of seven other elements. Cultivation in 2 L continuous chemostats containing wastewater was also conducted. Biomass productivities in chemostats were almost 2-fold greater than the maximum rates in batch cultures. Dissolved N and P in chemostats were both either undetectable or > 99% reduced compared to the wastewater. Production of bioenergy from the chemostats was estimated to be roughly 5.3–6.1 MJ m − 3 d − 1 , significantly higher than in batch culture.

The objective of the present study was to investigate the optimal nutritional requirements for hydrogen production from carbon monoxide by biological water-gas shift (WGS) reaction with Carboxydothermus hydrogenoformans using orthogonal layout methods. Cultures of C. hydrogenoformans on the medium as formulated by the strain supplier (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) unexpectedly showed a large content in inorganic material. This was confirmed by the rather elevated levels of calcium and phosphorus contained in the grown biomass. As an excessive production of those minerals may interfere with the growth and catabolism rates as well as clog up biofilm-based reactors, it was desirable to minimize the mineral accumulation during growth, while keeping at maximal levels both the H2 yield and the specific H2 production rate (SHPR). PO 4 3 − , HCO 3 − , Ca2+ and Mg2+ were considered as potentially the major factors of the mineral accumulation. The experiments were designed according to the Taguchi’s orthogonal method, using the above factors at three levels, and considering the culture mineral content, the H2 yield and the SHPR as optimization criteria. Optimal concentrations of PO 4 3 − , HCO 3 − , Ca2+ and Mg2+ were determined as (mM): 1.0, 5.0, 0.1 and 0.5, respectively. Under those culture conditions, Ca + P content decreased from 55.6 ± 1.8 to 9.5 ± 3% while the highest H2 yield at 90.9 ± 1.2% and SHPR at 0.85 ± 0.06 mol H2 g−1 VSS d−1 were achieved in bottle batch tests at 100% CO headspace atmosphere, neutral pH, a temperature of 70 °C, and an agitation of 100 rpm.

The objective of this study was to improve the biological water-gas shift reaction for producing hydrogen (H2) by conversion of carbon monoxide (CO) using an anaerobic thermophilic pure strain, Carboxydothermus hydrogenoformans. Specific hydrogen production rates and yields were investigated at initial biomass densities varying from 5 to 20 mg volatile suspended solid (VSS) L-1. Results showed that the gas-liquid mass transfer limits the CO conversion rate at high biomass concentrations. At 100-rpm agitation and at CO partial pressure of 1 atm, the optimal substrate/biomass ratio must exceed 5 mol CO g-1 biomass VSS in order to avoid gas-liquid substrate transfer limitation. An average H2 yield of 94±3% and a specific hydrogen production rate of ca. 3 mol g-1 VSS day-1 were obtained at initial biomass densities between 5 and 8 mg VSS-1. In addition, CO bioconversion kinetics was assessed at CO partial pressure from 0.16 to 2 atm, corresponding to a dissolved CO concentration at 70°C from 0.09 to 1.1 mM. Specific bioactivity was maximal at 3.5 mol CO g-1 VSS day-1 for a dissolved CO concentration of 0.55 mM in the culture. This optimal concentration is higher than with most other hydrogenogenic carboxydotrophic species. © 2011 Her Majesty the Queen in Right of Canada.

Herbivorous mammals and wood-eating insects are fairly effective in the digestion of plant polymers, such as lignocellulosics. In order to improve methane production from the lignocellulosic biomass, several kinds of anaerobic digestion processes derived from animal models have been devised. However, the rates of biodegradation occurring in the anaerobic bioreactors still remain lower than in animal guts. The effectiveness of the digestive systems of those animals results from the concerted action of the various enzymes (e.g. cellulases, xylanases, esterases, ligninases) produced in their guts as well as their integration with mechanical and chemical actions. Powerful pretreatment (prefermentation) operations are integrated to and support efficiently the microbial fermentation system, e.g. the rumination (i.e. mechanical) in ruminants and the secretion of endogenous cellulases (i.e. enzymatic) or the alkaline treatment (chemical) at mid-way in xylophagous insects. The oxygen gradients along the gastrointestinal tract may also stimulate the hydrolytic activities of some microbial populations. In addition, the solid retention time, the digesta flow and the removal of the end-products are well ordered to enable animals to thrive on a complex polymer such as lignocellulose. At the same time, technologies were developed to degrade lignocellulosic biomass, such as the rumen derived anaerobic digestion (RUDAD) process and the rumen simulating technique (RUSITEC), more elaborated and using rumen microbial consortia. Overall, they showed that the fermentation taking place in the rumen fermentation and even in the hindgut are biological processes that go beyond the limited environmental conditions generally found in anaerobic digesters. Hence, knowledge on herbivores' digestion mechanisms might be better exploited in the design and operation of anaerobic digesters. This literature review is a cross-analysis of the relevant information about the digestive strategies of herbivorous and wood-eating animals and the bioengineering techniques in lignocelluloses degradation. The aim is to highlight strategies of animals' digestion simulation for more effective anaerobic digestion of lignocellulosic compounds and other solid residues. © 2010 Her Majesty.

Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7(T) possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO(2) fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7(T) chromosome. The functionality of these pathways was also confirmed by growth of P7(T) on CO and production of CO(2) as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7(T) was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7(T) genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans's natural capacity to produce potential biofuels from syngas.

Anaerobic granule nuclei enriched in either acidogens (AF), syntrophic consortia (SN), Methanosaeta spp. (MT) and Methanosarcina spp. (MN) were developed in four upflow bed filter reactors fed with sucrose, an ethanol/acetate mixture, acetate and methanol, respectively. The four developed granule nuclei presented different settling velocities: 3.2, 8.7, 10.5 and 11.3 m/h for the AF flocs, the MS-, the SN- and the MT-nuclei, respectively. The ash content represented 60%, 40%, 30% and 16% of dry weight for the MT-, MS-, SN-enriched nuclei, and AF flocs, respectively. Acidogenic flocs contained high amount of extracellular polymeric substances. The influence of these four different nuclei on the time course of complex granule development was investigated by shifting the feed carbon of all of the four reactors to sucrose. Granulation proceeded rapidly both on syntrophic and Methanosaeta nuclei. The largest granules (Sauter diameter of 2.36 mm), however, were obtained within the shortest period in the reactor started with syntrophic nuclei. These nuclei presented also the best colonization by fermentative bacteria as shown by the evolution of their glucotrophic activities. Less satisfying granulation was obtained on Methanosarcina nuclei. In contrast, granulation was significantly retarded when acidogens were used as precursors. From these results it appears that syntrophs and Methanosaeta spp. play the principal role in anaerobic granulation process.

A strain of Mycobacterium austroafricanum capable of mineralizing methyl tert-butyl ether (MtBE) as its sole carbon and energy source had previously been isolated from activated sludge. The use of this bacteria in a biobarrier system for the in situ treatment of contaminated groundwater was the ultimate goal of this work. This paper reports the results of initial studies to find an appropriate immobilizing or support material. Several materials (peat, perlite, pozzolan, pumice and granulated activated carbon) were considered. Selection was based on the measurement and comparison of their abilities to immobilize or retain the bacteria, and to adsorb MtBE. The immobilization test was based on monitoring the respiration of bacteria remaining on the material after initial contact times of either 24 or 72 h. O2 consumption and CO2 production were measured over a period of time. The study showed that perlite was the best material. Adsorption isotherms for MtBE were constructed for each support. The strength of adsorption coefficient, Kf, in the Freundlich equation was calculated and used as the basis for comparison of their adsorptive capacities. The strength of adsorption was highest for the granulated activated carbon, and the Kf values for the other materials were relatively low and fell within the same range. However, since GAC performed poorly in the immobilization test, perlite was selected as the best support material.

O. TRESSE, F. MOUNIEN, M.-J. LEVESQUE AND S. GUIOT. 2004. Aims: To understand the microbial ecology underlying trichloethene (TCE) degradation in a coupled anaerobic/ aerobic single stage (CANOXIS) reactor oxygenated with hydrogen peroxide (H2O2) and in an upflow anaerobic sludge bed (UASB) reactor. Methods and Results: The molecular study of the microbial population dynamics and a phylogenetic characterization were conducted using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR- DGGE). In both reactors, TCE had a toxic effect on two uncultured bacterial populations whereas oxygen favoured the growth of aerobic species belonging to Rhizobiaceae and Dechloromonas. No methanotrophic bacteria were detected when targeting 16S rRNA gene with universal primers. Alternatively, pmo gene encoding the particulate methane monooxygenase of Methylomonas sp. LW21 could be detected in the coupled reactor when H2O2 was supplied at 0AE 7gO 2 l � 1 R day )1 . Conclusions: Methylomonas sp. LW21 that could be responsible for the aerobic degradation of the TCE by- products is not among the predominant bacterial populations in the coupled reactor. It seems to have been outcompeted by heterotrophic bacteria (Rhizobiaceae and Dechloromonas sp.) for oxygen. Significance and Impact of the Study: The results obtained show the limitations of the coupled reactor examined in this study. Further investigations should focus on the operating conditions of this reactor in order to favour the growth of the methanotrophs.

Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with Desulfitobacterium frappieri PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for Eubacteria and Archaea probes showed that Eubacteria predominantly colonized the outer layer, while Archaea were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day −1 and a low bacterial diffusion of 10 −7 dm 2 day −1 . Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.

The effects of four aeration and four organic loading (OLR) rates on trichloroethylene (TCE) degradation in methanogenic-methanotrophic coupled reactors were studied using ethanol as the carbon source for the methanogens. Microcosm and PCR studies demonstrated that methanotrophs capable of mineralizing TCE and methanogens were present in the biomass throughout the study. The gene for the particulate form of methane monooxygenase (pMMO) was detected by PCR, but not that for the soluble form (sMMO). TCE mineralization by methanotrophs was therefore due primarily to pMMO activity. Low TCE concentrations were measured in effluent and off-gas samples in all cases. Volatilization losses were 0–5%. Dichloroethylene (DCE) was also observed, but vinyl chloride and ethylene were never detected. Changes in the aeration rate had no effect on TCE removal, but did influence DCE degradation. Reductive dechlorination of TCE to DCE was favored at low and no-aeration conditions, and DCE accumulation occurred due to slow DCE degradation. Low DCE levels were observed at the higher aeration rates, which indicated that conditions in these reactors were amenable to the aerobic co-metabolism of TCE and DCE. The OLR did have an effect on TCE removal. TCE and DCE removal were negatively affected when the OLR was increased. An OLR of 0.3 g COD lrx −1day−1 or lower with an aeration rate of 3 lO2 lrx −1day−1 and higher is the recommended operating condition of a coupled reactor for removal of TCE.

Controlling the mean diameter of polymeric carriers is crucial to the successful application of encapsulated cells for in situ bioaugmentation of contaminated aquifers. The cell carriers should be small enough to be transported through a granular soil matrix, thus an emulsification-internal gelation technique for production of cell-encapsulating gellan gum microbeads is proposed. Mean diameter and size distribution of microbeads were investigated as a function of the water-in-oil emulsion parameters. The mean diameter of the microbeads ranged from 12 to 135 μm, varying as a descending function of the stirring rate (1000–5500 rpm) and emulsifier concentration (0–0.20% w/w), and as an ascending function of the disperse phase volume fraction (0.08–0.25). A bacterial consortium encapsulated within the microbeads (23 μm mean diameter) showed improved biodegradation activity in the removal of gasoline (400 mg L −1 ), as compared to free cells. A high degree of repeatability in the microbead formation process and particle size measurements was demonstrated. The results of this study suggest that the emulsification process can potentially be used for the large-scale production of controlled-diameter gellan gum-encapsulated cell microbeads for subsurface bioremediation applications.

Degradation of Aroclor 1242 was studied in granular biofilm reactors with limited aeration. An aerobic biphenyl degrader, Rhodococcus sp. M5, was used to supplement a natural bacterial population present in a "bioaugmented" reactor, while the "non-bioaugmented" reactor only contained natural granular sludge. The bioaugmentation, however appeared to have no effect on the reactor performance. Aroclor measurements showed its disappearance in both reactors with only 16-19% of Aroclor recovered from the reactor biomass and effluent. Simultaneously, a chlorine balance indicated that dechlorination occurred at a specific rate of 1.43 mg PCB (g volatile suspended solids)(-1) d(-1), which was comparable to the observed rate of Aroclor disappearance. Intermediates detected in both reactors were biphenyl, benzoic acid, and mono-hydroxybiphenyls. This suggests that a near-complete mineralization of Aroclor can be achieved in a single-stage anaerobic/aerobic system due to a combination of reductive and oxidative degradation mechanisms.

A rapid enrichment approach based on a pen- tachlorophenol (PCP) feeding strategy which linked the PCP loading rate to methane production was applied to an upflow anaerobic sludge bed reactor inoculated with anaerobic sludge. Due to this strategy, over a 140-day experimental period the PCP volumetric load increased from 2t o 65 mg L R day ˛1 with a near zero effluent con- centration of PCP. Dechlorination dynamics featured sequential appearance of 3,4,5-chlorophenol, 3,5-chloro- phenol, and 3-chlorophenol in the reactor effluent. Pro- filing of the reactor population by denaturing gradient gel electrophoresis (DGGE) revealed a correlation be- tween the appearance of dechlorination intermediates and bands on the DGGE profile. Nucleotide sequencing of newly detected 16S rDNA fragments suggested the proliferation of Clostridium and Syntrophobacter/ Syntrophomonas spp. in the reactor during PCP degra- dation. Published by John Wiley & Sons, Inc. Biotechnol Bio- eng 73: 476-483, 2001. indigenous bacterial populations often demonstrate biodeg- radation rates which are comparable or even superior to those of the bioaugmented systems. In this study, a rapid enrichment approach which relies on the proliferation of indigenous bacteria in response to stress- ing environmental conditions was used to develop an effi- cient consortium of anaerobic PCP degrading bacteria. This approach attempted to continuously increase the volumetric load of PCP while avoiding PCP toxicity through on-line monitoring of methane production. Methanogens (archeo- bacteria) are not directly implicated in PCP dechlorination. However, methanogens which are obligate members of the consortium are highly sensitive to chlorophenols (Stuart and Woods, 1998; Wu et al., 1993). It is thus assumed that the chlorophenol threshold concentration in the reactor bulk liq- uid for the chlorophenol-dechlorinating microorganisms is higher than for the methanogenic populations. Such a se- lective stress strategy is only possible through the computer- based on-line feedback control of a continuously fed reac- tor, using the volumetric PCP loading rate as the control parameter and the methane production rate as the process output. Thus, severe inhibition of the entire anaerobic con- sortium is avoided. Although monitoring of the population dynamics was of primary importance in this study, species identification in a mixed anaerobic culture by conventional microbiological methods is a particularly difficult process. Consequently, denaturing gradient gel electrophoresis (DGGE) was chosen to follow the bacterial diversity of the sludge population. DGGE analysis was accompanied by a thorough chemical analysis of the reactor effluent, which allowed for associa- tions to be drawn between degradation intermediates and growth dynamics of different bacterial species.

The effect of a nonionic surfactant (Tween 80) on 2,4,6-trinitrotoluene (TNT) mineralization by the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767, was investigated in a liquid culture at 20, 50, and 100 mg TNT·L-1. The presence of 1% (w/v) Tween 80, at 20 mg·L-1 TNT, added to a 4-d-old culture, allowed the highest TNT mineralization level, that is 29.3% after 24 d, which is two times more than the control culture, without Tween 80 (13.9%). The mineralization of TNT resumed upon additional Tween 80 supplementation, consequently, 39.0% of the TNT was respired on day 68. Orbital agitation of the fungal culture was found detrimental to TNT mineralization, with or without Tween 80 in the culture medium. The surfactant also stimulated the growth of P. chrysosporium without any notable effect on either the glycerol consumption rate or the extracellular LiP and MnP activity levels. Respirometric assays highlighted some differences between the oxygen uptake rate of the fungal culture supplemented with or without Tween 80. Key words: 2,4,6-trinitrotoluene, TNT, surfactant, white-rot fungus, Phanerochaete chrysosporium, lignin peroxidase, manganese peroxidase.

International audience; The white-rot basidiomycete Phanerochaete chrysosporium BKM-F-1767 was tested for its capacity to degrade dehydroabietic acid (DHA). In anaerobic treatment, this molecule is the most recalcitrant member of the resin acid group, which is known to cause operational problems to anaerobic reactors treating pulp and paper industry wastewaters. In this study the effect of DHA on different parameters, such as growth, ligninolytic enzyme activity, extracellular protein production as well as both glycerol and ammonium consumption by the fungus, was determined. Although the above parameters were affected by the addition of DHA, the results show that the fungus could still produce significant titres of ligninolytic enzymes. The fungus removed 47% of the DHA initially present in the static culture, after 10 days of incubation. Anaerobic toxicity assays showed that the treatment of DHA with P. chrysosporium reduced the methanogenesis and acetogenesis inhibition caused by DHA and allowed improved methane production by the anaerobic bacteria.

Scanning electron microscopy revealed that collapsed extracellular polymeric substances (EPS) surrounded bacteria present in granular sludge. Treatment of granular sludge with whole-cell antiserum and staining with polycationic ferritin demonstrated that bacteria were enveloped by extensive EPS. Antibody stabilization permitted a visualization of the EPS which more closely resembled its natural hydrated state. The EPS was seen to completely fill the intercellular spaces in the microcolonies. Both pure and mixed microcolonies were observed to be enclosed by EPS. The presence of these large amounts of EPS indicates that this extracellular layer is important in maintaining the structural integrity of granular sludge.

The ultrastructure of bacterial granules that were maintained in an upflow anaerobic sludge bed and filter reactor was examined. The reactor was fed a sucrose medium, and it was operated at 35 degrees C. Scanning and transmission electron microscopy revealed that the granular aggregates were three-layered structures. The exterior layer of the granule contained a very heterogeneous population that included rods, cocci, and filaments of various sizes. The middle layer consisted of a slightly less heterogeneous population than the exterior layer. A more ordered arrangement, made up predominantly of bacterial rods, was evident in this second layer. The third layer formed the internal core of the granules. It consisted of large numbers of Methanothrix-like cells. Large cavities, indicative of vigorous gas production, were evident in the third layer. On the basis of these ultrastructural results, a model that presents a possible explanation of granule development is offered.

Significant downward gradient of glucotrophic (acidogenic) activity was experimentally observed as anaerobic granules were stripped off their surface bacteria. This indicated a clear predominance of fermentative bacteria in the external layer of the granules. This is consistent with the multilayer microstructural model evidenced by scanning electron microscopy (SEM) of cleaved granules. In contrast, acetoclastic activities were evenly distributed along the granule depth even though SEM had shown the core of granules of various sources to be almost exclusively composed of Methanosaeta-like bacteria. A detailed distributed diffusion-reaction mathematical model was developed to predict local substance and biomass concentrations at any depth within the granular biofilm. This intragranular model combines the kinetics of ten substances and eight trophic groups of microorganisms as well as physico-chemical constraints such as ionic dissociation and gas-liquid equilibrium, internal and bulk-surface interfacial diffusion mechanisms. Predicted results of this intragranular kinetic model have shown a drastic downward glucose gradient towards the centre, as a concentration of 10 μM in the bulk liquid boundary layer dropped to almost 0 at a distance less than 100 μm below the surface. In contrast, the model predicted very low to no gradients of acetate. However, the model predicted pH values 1 mm inside the granule to be over one pH unit more alkaline than in the bulk liquid. Hence this significant pH gradient could be the major factor which gives a competitive advantage for Methanosaeta-like population to proliferate in the granule core.

We attempted to mimic in small upflow anaerobic sludge bed (UASB) bioreactors the metabolic association found in nature between methanogens and methanotrophs. UASB bioreactors were inoculated with pure cultures of methanotrophs, and the bioreactors were operated by using continuous low-level oxygenation in order to favor growth and/or survival of methanotrophs. Unlike the reactors in other similar studies, the hybrid anaerobic-aerobic bioreactors which we used were operated synchronously, not sequentially. Here, emphasis was placed on monitoring various methanotrophic populations by using classical methods and also a PCR amplification assay based on the mmoX gene fragment of the soluble methane monooxygenase (sMMO). The following results were obtained: (i) under the conditions used, Methylosinus sporium appeared to survive better than Methylosinus trichosporium ; (ii) the PCR method which we used could detect as few as about 2,000 sMMO gene-containing methanotrophs per g (wet weight) of granular sludge; (iii) inoculation of the bioreactors with pure cultures of methanotrophs contributed greatly to increases in the sMMO-containing population (although the sMMO-containing population decreased gradually with time, at the end of an experiment it was always at least 2 logs larger than the initial population before inoculation); (iv) in general, there was a good correlation between populations with the sMMO gene and populations that exhibited sMMO activity; and (v) inoculation with sMMO-positive cultures helped increase significantly the proportion of sMMO-positive methanotrophs in reactors, even after several weeks of operation under various regimes. At some point, anaerobic-aerobic bioreactors like those described here might be used for biodegradation of various chlorinated pollutants.

When TNT (N-source) was treated with anaerobic sludge it biotransformed into triaminotoluene (TAT) in high yield (80%). Results of experiments using ¹³C-labeling indicate that denitrated or deaminated products such as p-cresol and toluene were not formed. Whereas ¹⁴C-labeling showed negligible mineralization (, International Conference on Waste Minimisation and End of Pipe Treatment in Chemical and Petrochemical Industries (IAWQ 1999), Nov. 14-18, 1999, Yucatan, Mexico

International audience; This paper presents a synthetic review of studies conducted recently and consolidated by other literature insights that substantiate a multilayer structured model of the population organization in the anaerobic granule. Syntrophic bacteria associations would be located between an external, predominantly acidogenic, layer including also H2-consuming bacteria, and an aceticlastic core. This was evidenced conjointly by electron microscopic observations of cross-cleaved granules, by the specific activity analysis of stepwise abraded granules and by the changes in the specific metabolic activities with granule size distribution and enlargement. Such a structure was deduced from observations of propionate degradation in spite of endergonic conditions in the bulk liquid, of the two-step transfer of CH4 and H2 from liquid to the gas bubbles and from a substrate and product diffusion model which differentiates active zones for trophic groups along the granule radius. Such a structured aggregate is a stable metabolic arrangement that creates optimal nutritional conditions for all its members.

Degradation of phenol, ortho- and para-cresol was investigated in upflow anaerobic sludge bed (UASB) reactors bioaugmented with a methanogenic enrichment consortium able to degrade a mixture of phenolic compounds, in comparison to a reactor, which was inoculated only with anaerobic granules: 1) natural attachment of free cells to the granules, and 2) encapsulation within alginate beads. The increase of the percentage of enrichment from 2 to 5% improved considerably the startup of the reactors. Going from 5 to 10% had no effect on the removal of the phenolic compounds. Following a period of continuous operation at a hydraulic retention time of 3 days, the bioaugmented reactors showed specific activities on phenol, ortho- and para-cresol, at least twofold higher than those of the control reactor. This increase was attributed to the immobilization of phenol, ortho- and para-cresol-degrading bacteria on the granules.

In this work, a strain of anaerobic pentachlorophenol (PCP) degrader, Desulfitobacterium frappieri PCP-1, was used to augment a mixed bacterial community of an anaerobic upflow sludge bed reactor degrading PCP. To estimate the efficiency of augmentation, the population of PCP-1 in the reactor was enumerated by a competitive PCR technique. The PCP-1 strain appeared to compete well with other microorganisms of the mixed bacterial community, with its population increasing from 10 6 to 10 10 cells/g of volatile suspended solids within a period of 70 days. Proliferation of strain PCP-1 allowed for a substantial increase of the volumetric PCP load from 5 to 80 mg/liter of reaction volume/day. A PCP removal efficiency of 99% and a dechlorination efficiency of not less than 90.5% were observed throughout the experiment, with 3-Cl-phenol and phenol being observable dechlorination intermediates.