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Gas diffusion cathodes with electrodeposited nickel (Ni) particles have been developed and tested for hydrogen production in a continuous flow microbial electrolysis cell (MEC). A high catalytic activity of electrodeposited Ni particles in such a MEC was obtained without a proton exchange membrane, i.e. under direct cathode exposure to anodic liquid. Co-electrodeposition of Pt and Ni particles did not improve any further hydrogen production. The maximum hydrogen production rate was 5.4 L/LR/day, corresponding to Ni loads between 0.2 and 0.4 mg cm−2. Continuous MEC operation demonstrated stable hydrogen production for over one month. Owing to the fast hydrogen transport through the cathodic gas diffusion layer, the loss of hydrogen production to methanogenic activity was minimal, generally with less than 5% methane in the off-gas. Overall, gas diffusion cathodes with electrodeposited Ni particles demonstrated excellent stability for hydrogen production compared to expensive Pt cathodes.

Gas diffusion cathodes with Ni alloy and Ni catalysts manufactured by chemical deposition were tested for H2 production in a microbial electrolysis cell (MEC). In a continuous flow MEC, multi-component cathodes containing Ni, Mo, Cr, and Fe, at a total catalyst load of 1 mg cm−2 on carbon support demonstrated stable H2 production at rates of 2.8 – 3.7 L L R − 1 d − 1 with only 5% methane in the gas stream. Furthermore, a Ni-only gas diffusion cathode, with a Ni load of 0.6 mg cm−2, demonstrated a H2 production rate of 4.1 L L R − 1 d − 1 . Overall, H2 production was found to be proportional to the Ni load implying that inexpensive gas diffusion cathodes prepared by chemical deposition of Ni can be successfully used for continuous production of H2 in a MEC.

Anaerobic digestion of manures induces deodorization and conserves nitrogen. Livestock producers could transform their long-term (250–300 day) manure storages into psychrophilic anaerobic digesters, thus greatly reducing the investment, operational and technical cost associated with the treatment. But, sufficient inoculation is required and the feeding regime differs from that of a fixed volume digester, as the fed manure volume to digester content ratio is ever decreasing over time and the retention time is as long as the storage period. The project was designed to establish, in the laboratory, the inoculation conditions and feeding regime leading to the efficient establishment and operation of anaerobic digestion in conventional storages. In the laboratory using the biochemical methane potential test (BMP), methane production was monitored from swine manure inoculated with one of the three inocula (cattle manure—B; municipal wastewater anaerobic sludge—R, and; food processing wastewater anaerobic sludge—C) at four ratios (0, 10, 30 and 50%). Also, six prototype digesters were used to simulate VSS digestion in conventional storages, under two feeding regimes (once every week and every 2 weeks) and using three inocula. With the BMP and for all three inocula, a ratio of 10% provided the fastest initiation and the highest methane production. Both R and C initiated methane production as of the 1st day because of their active bacterial population, while B, obtained from a facultative system, required 24 days. After 100 days, all three inocula had produced the same amount of methane, for the same inoculation ratio. In the prototype digesters, both R and C inocula produced methane as of the first day, while the B inoculum produced methane after day 45 and 78 days, for the 1/week and 1/2week feeding regime, respectively. For both feeding regimes and after 148 days, all three inocula produced the same cumulative amount of methane. Thus, 10% B can initiate anaerobic conditions in swine manure during storage, as long as the storage period exceeds 140 days and a 45 day initiation period at a temperature is 35 °C.

Hydrogen production from glycerol was studied in a microbial electrolysis cell (MEC) with a 250 mL anodic chamber and a gas-phase cathode. A membraneless MEC design was employed, where a graphite felt anode and gas diffusion cathode were only separated by a 0.7 mm thick highly porous synthetic fabric (J-cloth). Glycerol (fuel) was continuously fed to the anodic chamber at loads of 0.3-5.3 g La⁻¹ d⁻¹. Fast conversion of glycerol to fermentation products, mainly 1,3-propanediol, propionate, and acetate was observed, that is, the fermentation products rather than glycerol were the most likely source of electrons for the anodophilic microorganisms. Hydrogen formation at the cathode required additional input of energy, which was provided by a controllable power supply. Hydrogen formation was observed starting from an applied voltage of 0.5 V. The highest volumetric rate of hydrogen production was 0.6 L La⁻¹ d⁻¹, which was obtained at a glycerol load of 2.7 gLa⁻¹ d⁻¹ and an applied voltage of 1.0 V. Hydrogen yield reached 5.4 mol per mol glycerol consumed, which corresponded to 77% of the theoretical value.

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 hybrid upflow sludge bed-filter (UBF) anaerobic reactor was successfully used for treatment of synthetic soluble 1% sugar waste. An hysteresis phenomenon was observed and discussed with respect to changes in acidogenic/acetoclastic specific activities ratio. The Haldane equation was adapted for predicting performance inhibition by undissociated acids as a function of substrate-COD and pH. The Monod and adapted Haldane models were solved simultaneously with substrate mass balance for a UBF reactor. The soluble COD removal efficiency and methane productivity were predicted as a function of the dilution rate. Critical values of solid and hydraulic residence times were also estimated and discussed in detail.

This study demonstrates hydrogen production in a membrane-less continuous flow microbial electrolysis cell (MEC) with a gas-phase cathode. The MEC used a carbon felt anode and a gas diffusion cathode with a Pt loading of 0.5 mg cm−2. No proton exchange membrane (PEM) was used in the setup. Instead, the electrodes were separated by a J-cloth. The absence of a PEM as well as a short distance maintained between the electrodes (0.3 mm) resulted in a low internal resistance of 19 Ω. Due to an improved design, the volumetric hydrogen production rate reached 6.3 LSTP L A − 1 d−1. In spite of the PEM absence, methane concentration in the gas collection chamber was below 2.1% and the presence of hydrogen in the anodic chamber was never observed.

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.

This study presents a temperature-based control strategy for the stabilization of an anaerobic reactor during organic overloads. To prove feasibility of the proposed approach the rate of methane production was followed in batch activity tests and reactor runs during mesophilic-thermophilic transitions. Within the first 0.25-6 h of temperature augmentation, an increase in the rate of methane production was observed with higher rates measured under thermophilic (above 40 degrees C) conditions. However, 24 h after startup both in batch tests and reactor runs, the rate of methane production under thermophilic conditions was inferior to that under optimal mesophilic conditions (35 degrees C). Following these results, a control strategy based on short-term augmentation of the reactor temperature was proposed and tested in a 10 L UASB reactor. The control strategy employed a multi-model observer-based estimator to stabilize the effluent COD concentration during organic overloads. The temperature-based control resulted in an increased methanization rate and improved reactor stability overall.

A proof of concept was performed in order to verify if the coupling of anaerobic and aerobic conditions inside the same digester could efficiently treat a reconstituted whey wastewater at 21 degrees C. The sequencing batch reactor (SBR) cycles combined initial anaerobic phase and final aerobic phase with reduced aeration. A series of 24 h cycles in 0.5 L digesters, with four different levels of oxygenation (none, 54, 108 and 182 mgO2 per gram of chemical oxygen demand (COD)), showed residual soluble chemical oxygen demand (sCOD) of 683 +/- 46, 720 +/- 33, 581 +/- 45, 1239 +/- 15 mg L(-1), respectively. Acetate and hydrogen specific activities were maintained for the anaerobic digester, but decreased by 10-25% for the acetate and by 20-50% for the hydrogen, in the coupled digesters. The experiment was repeated using 48 h cycles with limited aeration during 6 or 16 hours at 54 and 108 mgO2gCODinitial(-1), displaying residual sCOD of 177 +/- 43, 137 +/- 38, 104 +/- 22 and 112 +/- 9 mgL(-1) for the anaerobic and the coupled digesters, respectively. The coupled digesters recovered after a pH shock with residual sCOD as low as 132 mg L(-1) compared to 636 mg L(-1) for the anaerobic digester. With regard to the obtained results, the feasibility of the anaerobic-aerobic coupling in SBR digesters for the treatment of whey wastewater was demonstrated.

Anaerobic digestion model no.1 (ADM1) was used for tuning and performance analysis of the multi-model observer based estimator (mmOBE). The mmOBE was based on the variable structure model (VSM) of the anaerobic digestion model, which consists of several local submodels, each of which describes a typical process state. Depending on the hydraulic retention time, ADM1 simulated the methanogenic, organic overload, and acidogenic states of the process. These simulations allowed for optimising tunable parameters of the mmOBE. Owing to relatively slow process dynamics, a data acquisition interval as large as one day was sufficient to obtain acceptable accuracy. The simulations of mmOBE performance showed excellent rate of mmOBE convergence to ADM1 outputs. Moreover, mmOBE successfully estimated key kinetic parameters, such as maximal transformation rates of CODs, VFAs, and methane. These estimations can be used in the development of the advanced knowledge-based process system, which uses both available measurements and estimations of key kinetic parameters for extended diagnosis of failures and process trend analysis.

As opposed to mesophilic, thermophilic anaerobic digestion of food waste can increase the biogas output of reactors. To facilitate the transition of anaerobic digesters, this paper investigated the impact of adapting mesophilic sludge to thermophilic conditions. A 5L bench scale reactor was seeded with mesophilic granular sludge obtained from an up-flow anaerobic sludge blanket digester. After 13 days of operation at 35 degrees C, the reactor temperature was instantaneously increased to 55 degrees C and operated at this temperature until day 21. The biomass was then fed food waste on days 21, 42 and 63, each time with an F/M (Food/Microorganism) ratio increasing from 0.12 to 4.43 gVS/gVSS. Sludge samples were collected on days 0, 21, 42 and 63 to conduct substrate activity tests, and reactor biogas production was monitored during the full experimental period. The sludge collected on day 21 demonstrated that the abrupt temperature change had no pasteurization effect, but rather lead to a biomass with a fermentative activity of 3.58 g Glucose/gVSS/d and a methanogenic activity of 0.47 and 0.26 g Substrate/gVSS/d, related respectively, to acetoclastic and hydrogenophilic microorganisms. At 55 degrees C, an ultimate gas production (Go) and a biodegradation potential (Bo) of 0.2-1.4 L(STP)/gVS(fed) and of 0.1-0.84 L(STP) CH(4)/gVS(fed) were obtained, respectively. For the treatment of food waste, a fully adapted inoculum was developed by eliminating the initial time-consuming acclimatization stage from mesophilic to thermophilic conditions. The feeding stage was initiated within 20 days, but to increase the population of thermophilic methanogenic microorganisms, a substrate supply program must be carefully observed.

Observer-based estimators (OBE) were used for estimation of state variables and kinetic parameters in an anaerobic digestion (AD) process. A simplified first-order model with time-varying kinetic parameters was used to design an OBE for kinetic parameter estimation. This approach was validated on a laboratory-scale anaerobic reactor equipped with a multiwavelength fluorometer for on-line measurements of COD and VFA concentrations in the reactor effluent. The proposed estimators provide continuous adjustment of kinetic parameters and can be used for predictions of state variables between samples acquisition and during sensor failure.

In this study, a two-compartment continuous flow microbial fuel cell (MFC) reactor was used to compare the efficiencies of cathode oxygenation by air and by hydrogen peroxide. The MFC reactor had neither a proton-selective membrane nor an electron transfer mediator. At startup, the cathodic compartment was continuously aerated and the anodic compartment was fed with a glucose solution. An increase of electrical power generation from 0.008 to 7.2 mW m(-2) of anode surface with a steady-state potential of 215-225 mV was observed within a period of 12 days. The performance of the air-oxygenated MFC reactor progressively declined over time because of biofilm proliferation in the cathodic compartment. Oxygenation of the cathodic compartment using 300 mL d(-1) of 0.3% hydrogen peroxide solution resulted in a power density of up to 22 mW m(-2) (68.2 mA m(-2)) of anode surface at a potential of 340-350 mV. The use of H2O2 for oxygenation was found to improve the long-term stability of the MFC reactor.

In this work, the axial distribution of a conservative tracer in the upflow anaerobic sludge bed (UASB) reactor was modelled by a two-compartment model describing sludge bed and liquid zones. The orthogonal collocation algorithm was used for numerical solution of the model. The model parameters were estimated using on-line fluorescence-based measurements of an inert tracer rhodamine. Rhodamine concentrations were simultaneously measured at the bottom, middle and top of the sludge bed, as well as in the reactor effluent. The model used either a constant or variable axial dispersion coefficient in the sludge bed zone. A significantly better agreement between model outputs and observed tracer distribution was obtained when the dispersion coefficient was proportional to a constant raised to the power of the reactor height. In addition, a near linear dependence of the dispersion coefficient on the liquid upflow velocity was found. These findings, along with high efficiency of the orthogonal collocation algorithm used for model solution, enable future development of a comprehensive model, which accounts for both hydraulics and biokinetics of the anaerobic digestion process.

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.

This study presents a comparison of denitrification rates and denitrification stoichiometry when using different sources of carbon. Denitrification tests were carried out in test bottles containing water and soil samples acquired at a nitrate-contaminated site and supplemented with either sucrose, acetate or ethanol. The tests demonstrated nitrate removal in all carbon source supplemented bottles. The rate of denitrification and the required amount of a carbon source, however, depended on the choice of substrate. Ethanol and acetate were found to provide the highest denitrification rate, that of 1.4 - 1.6 mg-N l(-1) d(-1). Sucrose-supplemented bottles demonstrated a significantly slower denitrification rate, that of 0.6 - 0.9 mg-N l(-1) d(-1). In addition to slow denitrification rates, sucrose-supported denitrification required more carbon source.

A two-phase dispersion technique, termed emulsification-internal gelation, is proposed for encapsulation of activated sludge in gellan gum microbeads. The influence of emulsion parameters on size distribution of microbeads was investigated. Mean diameter of microbeads varied within a range of 34-265 microm as a descending function of emulsion stirring rate (1,000-5,000 rpm), emulsification time (10-40 min), and emulsifier concentration (0-0.1% w/w), and as an ascending function of disperse phase volume fraction (0.08-0.25). Encapsulated sludge expressed a high biodegradation activity compared with non-encapsulated sludge cultures even at 4.4 times lower level of overall biomass loading. Over 90% of gasoline at an initial concentration of 35 and 70 mg l(-1) was removed by both encapsulated and non-encapsulated sludge cultures in sealed serum bottles within 7 days. Encapsulation of activated sludge in gellan gum microbeads enhanced the biological activity of microbial populations in the removal of gasoline hydrocarbons. The results of this study demonstrated the feasibility of the production of size-controlled gellan gum-encapsulated sludge microbeads and their use in the biodegradation of gasoline.

The influence of ultraviolet photolysis as a pretreatment to the aerobic and anaerobic biological mineralization of a 14C-polyacrylamide was assessed using a series of radiorespirometry bioassays. The polyacrylamide studied was non-ionic with molecular weights ranging between 100,000 and 1 million. Aerobic and anaerobic biomineralization of the unphotolysed (raw) polyacrylamide was found to be only 0.60% and 0.70%, respectively, after 6 weeks of incubation, and hence indicative of the natural recalcitrance of polyacrylamide to microbial degradation. The effectiveness of UV irradiation in the physical breakdown of the polyacrylamide chain into oligomers was demonstrated by the shift in the molecular weight distribution and the positive correlation between the time of irradiation and the degree of its biological mineralization. The molecular weight fraction below 3 kD, which represents only 2% of the raw polyacrylamide, was increased to 41, 60 and 80% after 12, 24 and 48 hours of photolysis, respectively. This in turn, yielded, after 6 weeks of incubation, an aerobic mineralization of 5, 17 and 29% of 150 mg/L polyacrylamide, respectively, and an anaerobic mineralization of 3, 5 and 17%, respectively. Biomass acclimation substantially improved the specific initial rate of biomineralization of the photolysed polyacrylamides, but not the overall percentage of polyacrylamides mineralized.

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.

In this work, a variable structure model (VSM) of an anaerobic digestion process was developed. The anaerobic biodegradation process was described by four nonlinear submodels representing methanogenic, chemical oxygen demand overload, acidogenic, and hydrogen-inhibited states of the anaerobic process. At any instant, process dynamics was modeled only by one of the submodels, while the others were considered trailing. The choice of a leading submodel was handled by a knowledge-based system, which analyzed available process variables, such as off-gas composition and reactor pH. The feasibility of the proposed method was demonstrated both by using the VSM to predict the outputs of a comprehensive process model, and the experimental results obtained in a pilot scale anaerobic fixed-bed bioreactor.

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.

A laboratory-scale permeable biobarrier exhibited high removal efficiencies of benzene at inlet concentrations of 0.4 to 35.1 mg/L and with a limited supply of dissolved oxygen. The supplied oxygen was less than the demand for a complete aerobic oxidation of benzene. Stainless steel pieces or granulated peat moss were used as packing material for microbial support in the biobarrier. Removal efficiencies ranged from 63.9% to 99.9% in the stainless steel-packed biobarrier and from 70.4% to 97.2% in the peat moss-packed biobarrier, while benzene elimination rate changed from 0.2 to 10.4 mg/L-d and from 0.1 to 3.7 mg/L-d in the two biobarriers, respectively. The consumption of sulfate and the presence of sulfate-reducing bacteria suggested the contribution of anaerobic metabolism in the biodegradation of benzene. The biodegradation of benzene under microaerophilic conditions (defined as dissolved oxygen concentrations

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.

Disappearance of Aroclor 1242 was observed in a continuously operated upflow anaerobic sludge bed (UASB) reactor inoculated with granular anaerobic sludge. To distinguish between sorption and dechlorination processes, extraction of Aroclor from the sludge was performed along with the measurements of chloride and Aroclor in the reactor effluent. A release of inorganic chloride combined with the enrichment in lightly chlorinated congeners was observed. Based on the chloride balance, the dechlorination process was found to be Aroclor concentration-limited at an Aroclor load of 0.4 mg/(g VSS day) due to a low solubility of Aroclor in water. While an addition of Tween 80 to the influent stream improved solubility of Aroclor, it led to self-inhibition of the dechlorination process at a load of 1.3 mg/(g VSS day). The maximal dechlorination rate observed in this study was 0.6 mg Aroclor/(g VSS day).

Native soil microbial populations and unadapted municipal anaerobic sludges were compared for nitramine explosive degradation in microcosm assays under various conditions. Microbial populations from an explosive-contaminated soil were only able to mineralize 12% hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (at a concentration of 800 mg/kg slurry) or 4% octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (at a concentration of 267 mg/kg slurry). In contrast, municipal anaerobic sludges were able to mineralize them to carbon dioxide, with efficiencies of up to 65%. Reduction of RDX and HMX into their corresponding nitroso-derivatives was notably faster than their mineralization. The biodegradation of HMX was typically delayed by the presence of RDX in the microcosm, confirming RDX is used as an electron acceptor preferentially to HMX. The laboratory-scale bioslurry reactor reproduced the results of the microcosm assays, yet with much higher RDX and HMX degradation rates. A radiolabel-based mass balance in the soil slurry indicated that, besides a significant mineralization to carbon dioxide, 25% and 31% of RDX and HMX, respectively, appeared as acetonitrile-extractable metabolites, while the remaining part was incorporated into biomass and irreversibly bound to the soil matrix. About 10% of the HMX derivatives were estimated to be chemically bound to the soil matrix, while for RDX the estimation was nil.

Four techniques (microscope sizing, calculation from settling velocities, image and laser analysis) are available nowadays for determining the particle size distribution of upflow anaerobic sludge blanket (UASB) reactor sludge. These techniques present however the disadvantage of being either tedious, imprecise or expensive and hardly applicable in full scale treatment plants. There was then the need for a simple and low cost technique. In this study, a granulometry procedure based on manual humid sieving was evaluated. It was shown that no solid loss occured during the screening and that the particle size profiles were reproducible when performed with sludge samples of 5, 10, 25 and 150 ml, but not 1 ml. Only the results between 10 and 25 ml were however fully identical. It was shown also that the sieving could be performed on sludge samples stored for as long as 50 days at refrigerator temperature and that tap water could be use for the wash and backwash operations without any impact on the particle size profile. The granulometry obtained by image analysis was not comparable to that given by sieving. Nevertheless, no evidence of granule erosion could be found. In any case, the technique allowed us to follow the evolution of sludge granulometry perfectly over time. As a consequence, the manual humid sieving appears to be an adequate technique for determining the granule size distribution of UASB sludges.

The ecotoxicological effects of four bioslurry reactors treating 2,4,6-trinitotoluene (TNT)- and 1,3,5-trinitro-1,3,5- triazacyclohexane (RDX)-spiked soil were evaluated. A control bioslurry reactor was used to assess the endogenous toxicity of the bioslurry operation conditions. A battery of ecotoxicity tests was used: Microtox, green algae growth inhibition, bacterial genotoxicity and mutagenicity, and earthworm mortality and growth inhibition. Bioslurry soluble and solid phases were separated by centrifugation in order to identify toxicity and possible toxicants associated with each phase. Microtox toxicity values were initially very high in both bioslurry reactors spiked with TNT, in relation with TNT concentration. Initial toxicity was also detected by algal growth inhibition, earthworm lethality, genotoxicity and mutagenicity tests. An endogenous toxicity was detected in the control bioreactor using the Microtox and the SOS Chromotest. The soluble phase of the control bioslurry was genotoxic, suggesting that some potentially genotoxic agents were induced in the bioslurry samples. At the end of the bioremediation treatment, data showed that toxicity was reduced using all of the bioassays, except for earthworm lethality and growth inhibition tests in both RDX-spiked bioslurries. This study demonstrates the usefulness of a battery of toxicity tests to monitor bioremediation processes

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.

The influence of synthetic Percol 763 and natural chitosan polymers on the granulation rate of suspended anaerobic sludge was studied in laboratory-scale upflow anaerobic sludge bed (UASB)-like reactors. The results showed that supplementation of the reactors with polymers, either natural or synthetic, enhanced the granulation in comparison to control reactors (without the addition of polymers). A greater granulation was, however, obtained with chitosan compared to that with Percol 763. Chitosan yielded a granulation rate as high as 56 m/d compared to 35 m/d with Percol 763. The superior granulation performance of chitosan was probably related to its polysaccharidic structure, hence acting similarly to the extracellular polymeric substances (EPS) in aggregating anaerobic sludge. The granules specific activities were enhanced in the reactors supplemented with both polymers compared to those in control reactors. Overall, the results showed that polymers play a more critical role than microbial trophic groups in enhancing anaerobic granulation in UASB-like reactors.

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.

Dehalogenation of highly substituted chloro-organics is more effective under anaerobic conditions compared to aerobic conditions. The less chlorinated homologs produced under anaerobic conditions are then mineralized more easily in a subsequent aerated unit. However an anaerobic/aerobic coupled biofilm single unit could be more effective due to the synergism created between the oxidative and reductive catabolisms. The bacterial granules of upflow anaerobic sludge bed (UASB)-like reactors showed an excellent resistance to the extensive presence of O 2 in reactor. It is believed that the granular biofilm's outermost layer whose members include facultative anaerobes was limiting the O 2 penetration into the biofilm and shielding the innermost methanogens against toxic levels of O 2 . These results suggested that is could be interesting to develop granular biofilms intergrating both aerobic and anaerobic populations and to apply such biosystems for complete mineralization of recalcitrant compounds. Treatment of resin acids-, pentachlorophenol (PCP)- and tetrachloroethylene (PCE)-containing effluents have been tested within both anaerobic and coupled anaerobic/aerobic systems (anaerobic granule beds supplied with pure O 2 ). As oxygenation rate was controlled in the coupled anaerobic/aerobic systems, most of the co-substrate carbon was still anaerobically converted. At the highest PCP and PCE loading, the coupled anaerobic/aerobic reactor showed a specific complete dehalogenation higher than that of the control UASB (10 against 7 mg PCP g −1 volatile suspended solid (VSS) d −1 and 3.1 against 1.2 mg PCE g −1 VSS d −1 , respectively). The coupled reactor also showed better overall performance in resin acid removal by having lower concentrations in their effluents and lower amounts adsorbed onto the biomass (8–12 mg resin acids/g VSS in the coupled reactor against 67, in the control UASB). It is thought that the oxygenated granules were colonized by aerobic or microaerobic bacteria which can complete the degradation process in mineralizing the less chlorinated intermediates, or in degrading the resin acids which otherwise accumulate in the reactor. The concept might thus be promising, namely because of its immediate application to large-scale processes.

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.

As indicated in most literature reports, the solubility of pentachlorophenol (PCP) in water ranges between 10 and 20 mg l−1. Since PCP is a weak acid (pKa 4.35), its solubility increases drastically with increasing pH. When PCP dissolves in water two forms are normally present: undissociated PCP (PCP°) and a dissociated anionic form, i.e. pentachlorophenolate (PCP−). Both forms differ in their physico-chemical properties and in their microbial response and toxicity. It is therefore important to know the solubility behaviour of PCP at specific pHs when working with site remediation and toxicity assessment. While anionic PCP is very soluble, experimental data have shown that undissociated PCP has a solubility limited to 10 μM (3 mg l−1). Theoretical calculations have confirmed this value to be independent of the solution's pH. A non-empirical model was developed for estimating the “total” PCP aqueous solubility at different pHs using the standard solubility value and PCP's dissociation constant. The method may be extended to other chlorophenols and to most wastewaters.

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 present study evaluates the capability of sulphonated lignin (S-lignin) and calcium to promote anaerobic sludge granulation, which is of prime importance in the operation of upflow anaerobic sludge bed (UASB)-like reactors. One of two reactors fed with a synthetic sugar waste was stepwise supplemented with a spent liquor rich in S-lignin from a neutral sulphite semi-chemical pulping plant to reach an addition rate of 0.17 g S-lignin per gram volatile suspended solid (VSS) per day. This resulted in biomass pre-granulation (50-60 % of the biomass aggregates had a size > 0.3 mm). No change was observed in the surface properties (hydrophobicity and zeta potential) of the bacterial aggregates. The S- lignin-fed sludge aggregated in an ordered and cluster-like manner and exhibited a more compact ultrastructure than the control sludge. Moreover, the addition of S-lignin slightly enhanced the metabolic activities of the biomass. Calcium ion addition following the S-lignin treatment improved the size and the settleability of the aggregates and increased the solid retention time. Key words

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.

The upflow anaerobic sludge bed (UASB) concept, first proposed by Hemens et al. (1962), has been developed and extensively promoted by the Lettinga school, and applied to a large variety of wastewaters (Lettinga et al. 1980, 1983). The UBF reactor used in this study hybridized the UASB reactor with a packing filter (Guiot and van den Berg 1984). These reactors are characterized by the ability to accumulate a large amount of biomass due to adhesion of bacterial cells to each other. In the UASB and UBF reactors, the upflow velocity will select for organisms which can adhere to each other to form well settling granules which can be several millimeters in diameter. The granules accumulate in the reactor and then are exposed to the continuous feeding which is injected into the bottom of the reactor.

Anaerobic treatment of dilute synthetic wastewater (300-1,000 mg chemical oxygen demand/L using laboratory upflow sludge blanket filter reactors with and without effluent recycle is described. Treatment of dilute synthetic wastewater at hydraulic retention times less than 1 and 2 h in reactors without and with recycle, respectively, resulted in biomass washout as the solids retention time decreased to less than 12 d. Reseeding would be required to operate at these critical hydraulic retention times for extended periods. Treatment of dilute synthetic wastewater at hydraulic retention times between 3-12 h resulted in soluble COD removal efficiencies between 84-95% treating 300 mg COD/L. At a 3 h hydraulic retention time, solids retention time of 80 d and stable reactor biomass concentrations of 25 g volatile suspended solids/L were maintained.

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.