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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.

Electricity generation in microbial fuel cells (MFCs) has been a subject of significant research efforts. MFCs employ the ability of electricigenic bacteria to oxidize organic substrates using an electrode as an electron acceptor. While MFC application for electricity production from a variety of organic sources has been demonstrated, very little research on electricity production from carbon monoxide and synthesis gas (syngas) in an MFC has been reported. Although most of the syngas today is produced from non-renewable sources, syngas production from renewable biomass or poorly degradable organic matter makes energy generation from syngas a sustainable process, which combines energy production with the reprocessing of solid wastes. An MFC-based process of syngas conversion to electricity might offer a number of advantages such as high Coulombic efficiency and biocatalytic activity in the presence of carbon monoxide and sulfur components. This paper presents a discussion on microorganisms and reactor designs that can be used for operating an MFC on syngas.

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.

A special microbial consortium adapted to degrade petroleum hydrocarbons at limited availability of oxygen, transformed benzene, a highly toxic and carcinogenic contaminant of groundwater and soil, at low initial dissolved oxygen (DO) concentrations of 0.05-2 mg/L. The employed initial concentrations of dissolved oxygen were considerably lower than the previously reported values. Under these conditions, the overall transformation of benzene ranged from 34% � 1.7% to 100%, considerably higher than the theoretical predictions for complete mineralization of benzene based on the requirement of 3.08 mg oxygen/mg benzene. Unlike biotransformation that proceeded at the lowest examined DO concentration of 0.05 mg/L, the mineralization of benzene, defined by its conversion to CO2 and water, required a minimum DO concentration of 0.2 mg/L. The mineralization of benzene under microaerophilic conditions (DO < 2 mg/L), ranged from 0.83% � 0.06% to 89% � 1.3%, which was less than the theoretical predictions at any given initial DO concentration. The regulatory effects of dissolved oxygen concentration or its partial pressure on the activities of enzymes catalyzing the biotransformation of aromatic hydrocarbons was postulated to account for the reduced mineralization of benzene. The ratio between the transformed benzene and the consumed oxygen increased with the decrease of initial DO concentration, reaching a value of 2.8, considerably higher than the theoretical value of 0.33 obtained for a complete aerobic oxidation of benzene. Phenol was the major and the most stable intermediate metabolite during the biotransformation of benzene at low concentrations of DO. While benzene transformation stopped after the depletion of oxygen in the experimental system, phenol continued to accumulate under strictly anaerobic conditions, indicating its formation from an alternative carbon source, possibly biomass. � 2002 Government of Canada. Exclusive worldwide publication rights in this article have been transferred to Wiley Periodicals, Inc. Biotechnol Bioeng 79: 347-355, 2002.

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.

The impact of influent dissolved O(2) on the characteristics of anaerobic granular sludge was investigated at various dissolved O(2) concentrations (0.5-8.1 ppm) in 1- and 5-L laboratory-scale upflow anaerobic sludge bed (UASB)-like anaerobic/aerobic coupled reactors with a synthetic wastewater (carbon sources containing 75% sucrose and 25% acetate). The rate of dissolved O(2) supplied to the coupled reactor was as high as 0.40 g O(2)/L(rx).d, and the anaerobic/aerobic coupled reactors maintained excellent methanogenic performances at a COD loading rate of 3 g COD/L(rx).d even after the reactors had been operated with dissolved O(2) for 3 months. The activities of granular sludge on various substrates (glucose, propionate, and hydrogen) were not impaired, and acetate activity was even improved over a short term. However, after 3 months of operation, slight declines on the acetoclastic activities of granules were observed in the coupled reactor receiving the recirculated fluid containing 8.1 ppm dissolved O(2).Methane yield in the anaerobic control reactor and anaerobic/aerobic coupled reactors revealed that a significant aerobic elimination (up to 30%) of substrate occurred in the coupled reactors, as expected. The presence of dissolved O(2) in the recirculated fluid resulted in the development of fluffy biolayers on the granule surface, which imposed a negative impact on the settleability of granular sludge and caused a slightly higher sludge washout. This research shows that the anaerobic/aerobic coupled reactor can be successfully operated under O(2)-limited conditions and is an ideal engineered ecosystem integrating oxic and anaerobic niches. (c) 1996 John Wiley & Sons, Inc.

Aerobic biodegradation of gasoline and its constituents, benzene, toluene and ethylbenzene were studied by an enrichment from soil indigenous microbial population. The enrichment culture completely degraded 16.1-660 mg/l gasoline in 2.5-16 days respectively, without accumulation of any by-products. The kinetics of gasoline as well as benzene, toluene and ethylbenzene biodegradation was investigated with initial gasoline concentrations of 16.1-62.6 mg/l. The maximum specific rates of biodegradation of benzene, toluene and ethylbenzene were 0.12, 0.38 and 0.19 mg mg biomass-1 day-1 respectively. When benzene and toluene were used as sole substrate, the maximum specific rates of their biodegradation were 62.9 and 16.4 times greater than the corresponding values for a mixture (gasoline). The microbial culture was able to mineralize up to 200 mg/l pure toluene and benzene. Maximum mineralization efficiencies of benzene and toluene were 76.7 +/- 5.1% and 76.8 +/- 1.3% respectively. Self-inhibition and competitive inhibition patterns were observed during the biodegradation of benzene and toluene alone and in the mixture respectively. The observed kinetics was modeled according to Andrews' inhibition model.

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.

A model that portrays substrate profiles in a steady-state multispecies granular biofilm is developed and coupled with a biofilm detachment model. The model accounts for glucose, propionate, hydrogen, and acetate transformations performed by three bacterial trophic groups: acidogens, syntrophic bacterial consortia, and methanogens. This model adequately describes the phenomenon of propionate degradation under thermodynamically unfavorable bulk hydrogen concentrations. Also suggested is the superiority of the layered biofilm structure over homogeneous distribution of the trophic groups for anaerobic degradation of organic compounds. Furthermore, model analysis suggests that with increasing bulk glucose concentration biofilm thickness reaches a maximum that is then followed by biofilm disintegration. These results may have an important impact on the design and control of upflow anaerobic sludge bed reactors.

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.

Efficient anaerobic degradation may be completed only under low levels of dissolved hydrogen in the liquid surrounding the microorganisms. This restraint can be intensified by the limitations of liquid-to-gas H2 mass transfer, which results in H2 accumulation in the bulk liquid of the reactor. Dissolved hydrogen proved to be an interesting parameter for reactor monitoring by showing a good correlation with short-chain volatile fatty acid concentration, namely propionate, which was not the case for the H2 partial pressure. Biogas recycle was performed in a upflow anaerobic sludge bed and filter reactor. The effects of varying the ratio of recycled-to-produced gas from 2:1 (9 l/l reactor per day) to 8:1 (85 l/l reactor per day) were studied. By increasing the liquid—gas interface with biogas recycling, the dissolved hydrogen concentration could be lowered from 1.1 to 0.4 μ m . Accordingly, the H2 sursaturation factor was also reduced, leading to an important improvement of the H2 mass transfer rate, which reached 20.86 h−1 (±9.79) at a 8:1 gas recycling ratio, compared to 0.72 h−1 (±0.24) for the control experiment. Gas recycling also lowered the propionate concentration from 655 to 288 mg l−1 and improved the soluble chemical oxygen demand removal by 10–15%. The main problem encountered was the shorter solid retention time, which could lead to undesirable biomass washout at high gas recycling ratio. This could be circumvented by improving the reactor design to reduce the turbulence within the biomass bed.

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 (

A 450-m(3) multiplate anaerobic reactor (MPAR) has been started-up in April 1992 for treating wastewater (whey permeate and domestic wastewater) at the Nutrinor (Lactel) cheese factory in Chambord (Québec, Canada). The MPAR consists of four superimposed sections. The liquid flows upwards from one section to the next, while the gas is collected below each plate and evacuated through side-outlets. The wastewater is concurrently distributed at the bottom of the first, second, and third sections, as 50%, 33%, and 17% of the total influent stream, respectively. Granular anaerobic sludge at an initial concentration of 30 kg of volatile suspended solids (VSS) per cubic meter of reactor liquid volume was used to inoculate the reactor. Under normal operation of the factory, the chemical oxygen demand (COD) concentration of the influent ranged from 20 to 37 kg COD m(-3). The reactor organic loading rate (OLR) fluctuated between 9 and 14.7 kg COD m(-3) d(-1) for hydraulic retention times (HRT) maintained between 55 and 68 h. At the highest OLR, the MPAR showed an efficiency of 98% and 92% for soluble and total COD removal, respectively, and a methane production rate averaging around 4 m(3) m(-3) d(-1).Biomass-specific activities ranged between 7 and 51, 1.3 and 8.5, 5.3 and 12.2, 60 and 119, and 119 and 211 mmol g(-1) VSS d(-1) for glucose, propionate, acetate, formate, and hydrogen, respectively. Average equivalent-diameter of the granules was around 0.65 mm. The MPAR reactor generally showed a large capacity for solid retention with a biomass content between 32 and 37 kg VSS m(-3).

A 19.2 l multiplate anaerobic reactor (MPAR) was used to assess the impact of lime (Ca(OH)2) on the anaerobic treatment of whey permeate effluents. The amount of Ca(OH)2 required to maintain the pH of the whey permeate around 5 ranged between 3.0 and 4.5 kg/m3, which corresponded to concentration varying between 1.62 and 2.43 kg/m3. Soluble chemical oxygen demand (COD) removal efficiency exceeded 92% with a methane production rate of 6.7 m3/m3.d. at an organic loading rate (OLR) as high as 20 kg COD/m3.d. Extended operation of the MPAR resulted in the accumulation of significant amounts of calcium precipitates in the sludge bed which reached after three months of operation 0.19, 0.25 and 0.33 kg Ca2+ per kg of suspended solid (SS) in the lower, the middle and the upper compartment of the MPAR, respectively. The volatile suspended solids to suspended solids ratio (VSS/SS) decreased from 0.83 in inoculum to 0.37, 0.22 and 0.08 in the lower, the middle and the upper compartment of the MPAR, respectively. As a result, the soluble COD reduction and the methane production rate decreased to 31% and to 2.3 m3/m3.d. respectively, at OLR of 20 kg COD/m3.d.

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.

Electricity production from carbon monoxide (CO) is demonstrated in a single chamber microbial fuel cell (MFC) with a CoTMPP-based air cathode. The MFC was inoculated with anaerobic sludge and continuously sparged with CO as a sole carbon source. Volumetric power output was maximized at a CO flow rate of 4.8LLR(-1)d(-1) reaching 6.4mWLR(-1). Several soluble and gaseous degradation products including hydrogen, methane, and acetate were detected, resulting in a relatively low apparent Coulombic efficiency of 8.7%. Tests also demonstrated electricity production from hydrogen and acetate with the highest and fastest increase in voltage exhibited after acetate injection. It is hypothesized that electricity generation in a CO-fed MFC is accomplished by a consortium of carboxydotrophic and carbon monoxide - tolerant anodophilic microorganisms.

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.

Encapsulated cell bioaugmentation is a novel alternative solution to in situ bioremediation of contaminated aquifers. This study was conducted to evaluate the feasibility of such a remediation strategy based on the performance of encapsulated cells in the biodegradation of gasoline, a major groundwater contaminant. An enriched bacterial consortium, isolated from a gasoline-polluted site, was encapsulated in gellan gum microbeads (16–53 μm diameter). The capacity of the encapsulated cells to degrade gasoline under aerobic conditions was evaluated in comparison with free (non-encapsulated) cells. Encapsulated cells (2.6 mgcells g−1 bead) degraded over 90% gasoline hydrocarbons (initial concentration 50–600 mg L−1) within 5–10 days at 10°C. Equivalent levels of free cells removed comparable amounts of gasoline (initial concentration 50–400 mg L−1) within the same period but required up to 30 days to degrade the highest level of gasoline tested (600 mg L−1). Free cells exhibited a lag phase in biodegradation, which increased from 1 to 5 days with an increase in gasoline concentration (200–600 mg L−1). Encapsulation provided cells with a protective barrier against toxic hydrocarbons, eliminating the adaptation period required by free cells. The reduction of encapsulated cell mass loading from 2.6 to 1.0 mgcells g−1 bead caused a substantial decrease in the extent of biodegradation within a 30-day incubation period. Encapsulated cells dispersed within the porous soil matrix of saturated soil microcosms demonstrated a reduced performance in the removal of gasoline (initial concentrations of 400 and 600 mg L−1), removing 30–50% gasoline hydrocarbons compared to 40–60% by free cells within 21 days of incubation. The results of this study suggest that gellan gum-encapsulated bacterial cells have the potential to be used for biodegradation of gasoline hydrocarbons in aqueous systems. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 175–184, 2002.

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.

A new method for immobilization of anaerobic sludge in chitosan is described. It is based on the reaction of basic NH2 groups of chitosan with the acidic sulfonic-groups of lignosulfonate to form sulfonilamide linkages. The new procedure features simplicity, low-cost and mild immobilization conditions. Batch tests of acetate consumption along with a continuous reactor operation confirmed the effectiveness of the immobilization technique for maintenance of long-term stability of the polymer.

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 biohydrogen production from anaerobic fermentation was studied with thermophilic bacterium Carboxydothermus hydrogenoformans using carbon monoxide (CO) dissolved in fermentation broth as the substrate. Bacterium growth model, decay efficient and the maximum specific growth rate were observed based on the experimental study of biomass growing, flocculation and microbial characteristics. The results indicated the feasibility of continuous anaerobic fermentation for hydrogen production from CO with C. hydrogenoformans due to its good hydrogen yield and flocculation ability. Furthermore, by investigating the effect of procedure parameters during CO fermentation, the optimal feed/microorganism was proposed and the maximum CO concentration was observed to avoid CO inhibition which was the key factor to consider in the study of continuous biohydrogen production from CO fermentation.

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.

The Gibbs free energy changes in methanogenic granular biomass from sludge-bed reactors were evaluated using the in situ concentrations and partial pressures of metabolites during the metabolism of acetate, hydrogen, formate and propionate. Based on mass balance calculations it appeared that the degradation of propionate into acetate, hydrogen and bicarbonate was endergonic, even if propionate was effectively degraded. On the other hand, the methane-producing reactions, both from acetate and from hydrogen plus bicarbonate, were found to be exergonic and the free energy change was sufficient for the formation of ATP. Formate was detected in only one of the two reactors. When formate, instead of hydrogen, was considered as the electron carrier between propionate-degrading and methanogenic bacteria, similar thermodynamic results were obtained. The existence of trophic microniches in the granular biomass is suggested to explain propionate degradation even though the Gibbs free energy change in the liquid surrounding the granules was positive. Hence, to make propionate degradation exergonic the dissolved hydrogen concentration surrounding the propionate-degrading bacteria would have to be about 30 times lower than in the free liquid.

A miniature fuel cell, using a hydrophobic Teflon(R) membrane, designed to continuously measure dissolved H(2) in nonbiological media, was tested for use in anaerobic digestion conditions. In water, this detector responds quickly and efficiently to variation of hydrogen concentration in the range from 80 to 770 nM The media used, and the metabolites or products found in anaerobic digestion media, i. e. inorganic carbon and phosphate buffers, formate, acetate, and dissolved methane, did not interfere with the signal of the detector cell. Dissolved hydrogen sulfide did not poison the cell but was detected. In spite of the detector's high sensitivity to hydrogen (about 21,000 times higher for hydrogen than for hydrogen sulfide), interferences can occur in media containing high sulfide levels.In a methanogenic reactor, the detector cell response to dissolved hydrogen was fast and reliable with time. The observed values ranged values ranged from 2 to 3.5 microM. Dissolved hydrogen concentrations were 40 to 70 times higher than values calculated from measured hydrogen partial pressures and Henry's coefficient, suggesting a limitation of the process in the hydrogen transfer from the liquid to the gaseous phase.

A new hybrid reactor, the upflow blanket filter (UBF), which combines an open volume in the bottom two-thirds of the reactor for a sludge blanket and submerged plastic rings (Flexiring, Koch Inc., 235 squared m/cubic m) in the upper one-third of the reactor volume, was studied. This UBF reactor was operated at 27 degrees C at loading rates varying from 5 to 51 g chemical oxygen demand (COD)/l day with soluble sugar wastewater (2500 mg COD/l). Maximum removal rates of 34 g COD/l day and CH/sub 4/ production rates of 7 vol/vol day (standard temperature and pressure (STP)) were obtained. The biomass activity was about 1.2 g COD/g volatile suspended solids per day. Conversion (based on effluent soluble COD) was over 93% with loading rates up to 26 g COD/l day. At higher loading rates conversion decreased rapidly. The packing was very efficient in retaining biomass. 11 references.

The performance and biomass retention of a 4.2 L new hybrid reactor (upflow blanket filter, UBF) at 27 °C were determined at loading rates of 5 to 51 g Chemical Oxygen Demand (C0D)/L.d with sugar wastes of 2500 mg C0D/L strength. Maximum removal rates of 34 g C0D/L.d and C0D removal efficiency of over 93% were reached. The packing was very efficient in retaining biomass.

This article reports the steady-state performance of two hybrid anaerobic digesters treating soluble synthetic sugar wastes of 1 and 0.5% strength and the assessment of the associated macroenergetic parameters (growth yield, so-called maintenance coefficient). A theoretical development shows a “nongrowth” parameter concept to be more appropriate than maintenance or decay. Combined energy and mass balances are used to develop a model for growth rate which compares well with experimental data. The COD removal efficiency had no significant effect on growth yield and the maintenance parameter, although a dual combined balance indicated the possibility of such an effect. Macroenergetic parameters did not vary significantly with the specific feeding rate of the system. We thus conclude that a single model may be used over a broad range of feeding and performance conditions.

To calculate the transient accumulation of soluble organic matter in activated sludge, an equation based on COD and respirometry is described. In this approach the difference between the actual utilized organic matter and the metabolized matter is expressed as substrate transient accumulation. The amount of substrate generated by the lysis of dead microorganisms was taken into account, and the metabolized organic matter was expressed in two main endergonic functions of O(2) consumption, that is, assimilation and maintenance. The equation thus derived was simplified to contain parameters such as COD and O(2) consumption and tested on experimental results. The results show that the values obtained using this equation compared well to substrate removal in the absence of O(2) in short-term batch cultures.

Liquid-to-gas mass transfer in anaerobic processes was investigated theoretically and experimentally. By using the classical definition of k L a , the global volumetric mass transfer coefficient, theoretical development of mass balances in such processes demonstrates that the mass transfer of highly soluble gases is not limited in the usual conditions occurring in anaerobic fermentors (low-intensity mixing). Conversely, the limitation is important for poorly soluble gases, such as methane and hydrogen. The latter could be overconcentrated to as much as 80 times the value at thermodynamic equilibrium. Such overconcentrations bring into question the biological interpretations that have been deduced solely from gaseous measurements. Experimental results obtained in three different methanogenic reactors for a wide range of conditions of mixing and gas production confirmed the general existence of low mass transfer coefficients and consequently of large overconcentrations of dissolved methane and hydrogen (up to 12 and 70 times the equilibrium values, respectively). Hydrogen mass transfer coefficients were obtained from the direct measurements of dissolved and gaseous concentrations, while carbon dioxide coefficients were calculated from gas phase composition and calculation of related dissolved concentration. Methane transfer coefficients were based on calculations from the carbon dioxide coefficients. From mass balances performed on a gas bubble during its simulated growth and ascent to the surface of the liquid, the methane and carbon dioxide contents in the gas bubble appeared to be controlled by the bubble growth process, while the bubble ascent was largely responsible for a slight enrichment in hydrogen.

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.

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.