Biokinetics in acidogenesis of highly suspended organic wastewater by adenosine 5' triphosphate analysis

In this paper, we pointed out the problems of using conventional volatile suspended solids (VSS) and chemical oxygen demand (COD) to evaluate biokinetic coefficients, especially for the treatment of highly suspended organic wastewater. We also introduced a novel approach to evaluate biokinetic coefficients by measurement of adenosine 5'-triphosphate (ATP) of microorganisms. The concept of using ATP analysis in biokinetic evaluations with highly suspended wastewater was shown to be effective. This study also showed that the conventional VSS and COD methods were strongly affected by incoming suspended organics in the wastewater and by biokinetics of microorganisms. A cheese-processing wastewater was used in evaluating the biokinetics of mesophilic acidogens. The concentration of COD and total suspended solids in the wastewater was 63.3 g/L and 12.4 g/L, respectively. The TSS was 23.6% of total solids concentration. A high ratio of VSS to total suspended solids of 96.7% indicated that most of the suspended particles were organic materials. Lactose and protein were the major organic components contributing COD in the wastewater, and a total of 94.2% of the COD in the wastewater was due to the presence of lactose and protein. Two different physiological conditions where the maximum rates of acetate and butyrate production occurred were tested. These were pH 7 (condition A for acetate production) and pH 7.3 (condition B for butyrate production) at 36.2C, respectively. Based on the molecular structures of the major organic substances and microbial ATP analysis, the residual substrate and microbial concentrations were stoichiometrically converted to substrate COD (SuCOD) and microbial VSS (MVSS), respectively, using correlation coefficients reported previously. These SuCOD and MVSS were simultaneously used to evaluate the biokinetic coefficients using Monod-based mathematical equations. The nonlinear least squares method with 95% confidence interval was used to evaluate biokinetic coefficients. The maximum microbial growth rate, mu(max) and half saturation coefficient, K(s), for conditions A and B were determined to be 9.9 +/- 0.3 and 9.3 +/- 1.0 day(-1) and 134.0 +/- 58.3 and 482.5 +/- 156.5 mg SuCOD/L, respectively. The microbial yield coefficient, Y, and microbial decay rate coefficient, k(d) for conditions A and B were determined to be 0.29 +/- 0.03 and 0.20 +/- 0.05 mg MVSS/mg SuCOD, and 0.14 +/- 0.05 and 0.25 +/- 0.05 day(-1), respectively. Specific substrate utilization rate at condition B was 43.8 +/- 20.6 mg SuCOD/mg MVSS/day, which was 31% higher than that at condition A.     

Effect of the concentration of suspended solids on the enzymatic activities and biodiversity of a submerged membrane bioreactor for aerobic treatment of domestic wastewater

A pilot-scale submerged membrane bioreactor was used for the treatment of domestic wastewater in order to study the influence of the variations in the concentration of volatile suspended solids (VSS) on the enzymatic activities (acid and alkaline phosphatases, glucosidase, protease, esterase, and dehydrogenase) and biodiversity of the bacterial community in the sludge. The influence of VSS concentration was evaluated in two separated experiments, which were carried out in two different seasons of the year (experiment 1 through spring–summer and experiment 2 through autumn–winter). Cluster analysis of the temperature gradient gel electrophoresis (TGGE) profiles demonstrated that the community composition was significantly different in both experiments. Within the same experiment, the bacterial community experienced sequential shifts as the biomass accumulated, as shown by the evolution of the population profiles through time as VSS concentration increased. All enzymatic activities studied were significantly lower during experiment 2, except for glucosidase. Concentrations of VSS over 8 g/l induced a strong descent of all enzymatic activities, which overlapped with a significant modification of the community composition. Sequences of the major TGGE bands were identified as representatives of the Alpha-proteobacteria, filamentous bacteria (Thiotrix), and nitrite oxidizers (Nitrospira). Some sequences which were poorly related to any validated bacterial taxon were obtained.     

Effect of stabilization on biomass activity

The study aimed to compare aerobic and aerobic/anoxic stabilization processes in terms of organic matter and the biomass removal efficiencies using a municipal sludge sample. The efficiency of stabilization process was assessed monitoring suspended solids (SS), volatile suspended solids (VSS), total and dissolved organic carbon (TOC, DOC), nitrate, nitrite, and phosphate parameters. The oxygen uptake rate (OUR) measurements were conducted to determine active biomass concentration. On the 30th day of the aerobic stabilization, the SS, VSS and TOC removal efficiencies were 22%, 28% and 55%, respectively. Under aerobic/anoxic conditions, removal efficiencies for SS, VSS and TOC were 25%, 27% and 67%. On the 17th day of the stabilization, SS and VSS removal rates were 60 mg SS/L day and 47 mg VSS/L day for aerobic and 102 mg SS/L day and 63 mg VSS/L day for aerobic/anoxic conditions, respectively. These findings reflected the higher stabilization performance of the aerobic/anoxic conditions. Based on respirometric results, the ratios of the active biomass were decreased to 30% and 24% for the 17th and 30th day of the aerobic stabilization, respectively. Such results have significant implications relative to the activity decrease quantification of the biomass as well as its further application potentials after aerobic or aerobic/anoxic sludge stabilization.     

Biological decolorization of xanthene dyes by anaerobic granular biomass

Biodegradation of a xanthene dyes was investigated for the first time using anaerobic granular sludge. On a first screening, biomass was able to decolorize, at different extents, six azo dye solutions: acid orange 7, direct black 19, direct blue 71, mordant yellow 10, reactive red 2 and reactive red 120 and two xanthene dyes--Erythrosine B and Eosin Y. Biomass concentration, type of electron donor, induction of biomass with dye and mediation with activated carbon (AC) were variables studied for Erythrosine B (Ery) as model dye. Maximum color removal efficiency was achieved with 4.71 g VSS L?1, while the process rates were independent of the biomass concentration above 1.89 g VSS L?1. No considerable effects were observed when different substrates were used as electron donors (VFA, glucose or lactose). Addition of Ery in the incubation period of biomass led to a fivefold increase of the decolorization rate. The rate of Ery decolorization almost duplicated in the presence of commercial AC (0.1 g L?1 AC?). Using different modified AC samples (from the treatment of AC?), a threefold higher rate was obtained with the most basic one, AC(H?), as compared with non-mediated reaction. Higher rates were obtained at pH 6.0. Chemical reduction using Na?S confirmed the recalcitrant nature of this dye. The results attest that decolorization of Ery is essentially due to enzymatic and adsorption phenomena.     

Kinetic study of anaerobic digestion of brewery wastewater

A study of the kinetics of the anaerobic digestion of brewery wastewater was carried out using a 1-litre, continuous-flow, completely-mixed, bioreactor operating at 35°C and containing a saponite-immobilized biomass at a concentration of 6·2 g volatile suspended solids (VSS)/litre. The bioreactor worked satisfactorily in a range of hydraulic retention times from 1·2 to 10 days and eliminated more than 95% of the initial chemical oxygen demand (COD) in all instances.

Guiot's kinetic model was used to determine the macroenergetic parameters of the system, and showed it to have a yield coefficient for the biomass (Y) of 0·080 g VSS/g COD and a specific rate of substrate uptake for cell maintenance (m) of 0·045 g COD/g VSS day.

The experimental results showed the rate of substrate uptake (R_s; g COD/g VSS day), correlated with the concentration of biodegradable substrate (S_b; g COD/litre), through an equation of the Michaelis-Menten type.     

Anaerobic treatment of low-strength municipal wastewater by a two-stage pilot plant under psychrophilic conditions

A two-stage anaerobic treatment pilot plant was tested for the treatment of raw domestic wastewater under temperatures ranging from 21 to 14 degrees C. The plant consisted of a hydrolytic upflow sludge bed (HUSB) digester (25.5m3) followed by an upflow anaerobic sludge blanket (UASB) digester (20.36m3). The hydraulic retention time (HRT) varied from 5.7 to 2.8h for the first stage (HUSB digester) and from 13.9 to 6.5h for the second stage (UASB digester). Total suspended solids (TSS), total chemical oxygen demand (TCOD), and biochemical oxygen demand (BOD) removals ranged from 76% to 89%, from 49% to 65%, and from 50% to 77%, respectively, for the overall system. The percentage of influent COD converted to methane was 36.1%, the hydrolysis of influent volatile suspended solids (VSS) reached 59.7% and excess biomass was 21.6% of the incoming VSS. Plant performance was influenced by the wastewater concentration and temperature, yet better results were obtained for influent COD higher than 250mg/l.     

Kinetic behaviour of waste tyre rubber as microorganism support in an anaerobic digester treating cane molasses distillery slops

The kinetics of anaerobic digestion of cane molasses distillery slops was investigated using a continuous-flow bioreactor which contained waste tyre rubber as support, to which the microorganisms became immobilized. Hydraulic retention times (HRT) ranging from 1 to 10 days were investigated at an average influent chemical oxygen demand (COD) concentration of 47.7?g/l. The maximum substrate utilization rate, k, and half saturation coefficient, K _L, were determined to be 1.82?kg COD_removed/kg VSS day and 0.33?kg COD/kg VSS day. The yield coefficient, Y, and sludge decay rate coefficient, K _d, were also determined to be 0.06?kg VSS/kg COD_removed and 0.05?day^-1, respectively. Methane production was maximum (6.75?l/l day) at a 2 day HRT corresponding to a biomass loading rate of 2.578?kg COD/kg VSS day. Biogas yield ranged between 0.51?l/g COD (HRT=2 days) and 0.25?l/g COD (HRT=1?day). In addition, the methane percentage in the biogas varied between 70.5% (HRT=10?days) and 47.5% (HRT=1?day). The close relationship between biomass loading rate and specific substrate utilization rate supported the use of Monod equations. Finally, the experimental values of effluent substrate concentration were reproduced with deviations equal to or less than 10% in every case.     

Kinetics of nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium

A mathematical model system was derived to describe the kinetics of ammonium nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium. The model incorporates diffusive mass transport and Monod kinetics. The model was solved using a combination of the orthogonal collocation method and Gear’s method. A batch test was conducted to observe the nitrification of ammonium-nitrogen ( \({\text{NH}}_{4}^{ + }\) -N) and the growth of nitrifying biomass. The compositions of nitrifying bacterial community in the batch kinetic test were analyzed using PCR–DGGE method. The experimental results show that the most staining intensity abundance of bands occurred on day 2.75 with the highest biomass concentration of 46.5 mg/L. Chemostat kinetic tests were performed independently to evaluate the biokinetic parameters used in the model prediction. In the column test, the removal efficiency of \({\text{NH}}_{4}^{ + }\) -N was approximately 96 % while the concentration of suspended nitrifying biomass was approximately 16 mg VSS/L and model-predicted biofilm thickness reached up to 0.21 cm in the steady state. The profiles of denaturing gradient gel electrophoresis (DGGE) of different microbial communities demonstrated that indigenous nitrifying bacteria (Nitrospira and Nitrobacter) existed and were the dominant species in the fixed biofilm process.     

Simple and rapid methods to evaluate methane potential and biomass yield for a range of mixed solid wastes

This paper describes rapid techniques to evaluate the methane potential and biomass yield of solid wastes. A number of solid wastes were mixed to provide a range of C:N ratios. Empirical formulae were calculated for each waste based on the results of chemical analysis and these formulae were used to estimate the COD equivalent and stoichiometric methane potential (SMP). The actual COD and biochemical methane potential (BMP) were determined experimentally for each waste and for both parameters there was a good agreement between the empirical and experimental values. The potential of adenosine triphosphate (ATP) to act as an indicator of biomass yield (mg VSS mg(-1) COD removed) was determined during the anaerobic digestion process. The biomass yield determined from ATP analysis was in the range 0.01-0.25mg VSS mg(-1) COD removed which corroborated well with previously reported studies. Empirical formula based SMP together with ATP measurement were shown to provide rapid methods to replace or augment the traditional BMP and VSS measurements and are useful for evaluating the bioenergy and biomass potential of solid wastes for anaerobic digestion.     

The dielectric permittivity at radio frequencies and the bruggeman probe: novel techniques for the on-line determination of biomass concentrations in plant cell cultures

A novel technique is described for the measurement of the volume fraction of biomass in a suspension by the simultaneous measurement of the conductivity of a suspension containing cells and of the medium in which the cells are suspended. The presence of non-conducting particulate matter in a suspension will cause the conductivity of a suspension to be decreased relative to that of the medium in which the particles are suspended. A simple equation (the Bruggeman equation) describes the relationship between the volume fraction of non-conducting particulate matter and the decrease in conductivity. The accuracy of this method for the determination of the biomass concentration of plant cells (Festuca arundinacea) in culture was shown. The method was successfully applied to the on-line determination of biomass concentrations during the growth of F. arundinacea cultures, and gave good agreement with biomass levels as determined from measurements of the radio-frequency dielectric permittivity of such cultures.     

Mathematical modelling of the aerobic degradation of two-phase olive mill effluents in a batch reactor

A laboratory-scale study was conducted on the aerobic degradation of two-phase olive mill effluents (TPOME) made up of the mixture of the washwaters derived from the initial cleansing of the olives and those obtained in the washing and purification of virgin olive oil. The process was carried out in a 1-l working volume stirred tank reactor operating in batch mode at room temperature (25 °C). The reactor was operated at influent substrate concentrations of 2.80 g COD/l (TPOME 25%), 5.45 g COD/l (TPOME 50%), 8.18 g COD/l (TPOME 75%) and 10.90 g COD/l (TPOME 100%). After five days of operation time, total and soluble COD removal efficiencies of 64.3% and 66.6% were achieved respectively for the most concentrated influent used (TPOME 100%). A simplified kinetic model for studying the hydrolysis of insoluble organic matter, oxidation of soluble substrate and biomass production was proposed on the basis of the experimental results obtained. The following kinetic constants with their standard deviations were obtained for the above stages in the case of the most concentrated influent used (TPOME 100%): k₁ (kinetic constant for hydrolysis of suspended organic matter): 0.11 ± 0.01 l/(g VSS day); k₂ (kinetic constant for total consumption of soluble substrate): 0.30 ± 0.02 l/(g VSS day); k₃ (endogenous metabolism constant): 0.07 ± 0.01 per day). Finally, the biomass yield coefficient was found to be 0.30 g VSS/g COD_removed. The values of non-biodegradable total and soluble CODs obtained from the model were found to be 3 and 2 g/l, respectively. The kinetic constants obtained and the proposed equations were used to simulate the aerobic degradation process of TPOME and to obtain the theoretical values of non-soluble and soluble CODs and biomass concentration. The small deviations obtained (equal or lower than 10%) between the theoretical and experimental values suggest that the parameters obtained represent and predict the activity of the microorganisms involved in the overall aerobic degradation process of this wastewater.     

Converting corn wet-milling effluent into high-value fungal biomass in a biofilm reactor

Rhizopus microsporus was grown in an attached growth system using corn wet-milling effluent as a growth medium. This strain was chosen due to its ability to effectively degrade organic matter in corn wet-milling effluent and for its properties to produce significant levels of protein, chitin and chitosan. Fungal growth and organic removal efficiency were examined under both aseptic and non-aseptic conditions with and without nutrient supplementation. Plastic composite support (PCS) tubes, composed of 50% (w/w) polypropylene (PP) and 50% (w/w) agricultural products were used as support media. Significantly higher organic removal measured as chemical oxygen demand (COD) and biomass yield were observed in the bioreactor with PCS tubes than in two control bioreactors; that is with PP tubes alone and suspended growth (without support media). This confirmed that the PCS support medium with agricultural components enhanced fungal growth and organic removal. The results showed that supplementation of nutrients (e.g., mineral salts) under aseptic conditions enhanced the COD removal from 50% to 55% and observed biomass yield from 0.11 to 0.16 g (dry-weight)/g COD(removed) (i.e., from 0.10 to 0.14 g volatile solids (VS)/g COD(removed) approximately). Non-aseptic operation without nutrient supplementation resulted in an observed biomass yield of 0.32 g volatile suspended solids (VSS)/g COD(removed) with no significant improvement in COD removal ( approximately 53%); whereas with nutrient supplementation, the observed biomass yield increased to 0.56 g VSS/g COD(removed) and COD removal improved to 85%. The fungal system was able to degrade the organic matter with concomitant production of high-value fungal biomass. This is the first study that examined the conversion of corn milling waste stream into high value fungal protein.     

Detachment of multi species biofilm in circulating fluidized bed bioreactor

In this study, the detachment rates of various microbial species from the aerobic and anoxic biofilms in a circulating fluidized bed bioreactor (CFBB) with two entirely separate aerobic and anoxic beds were investigated. Overall detachment rate coefficients for biomass, determined on the basis of volatile suspended solids (VSS), glucose and protein as well as for specific microbial groups, i.e., for nitrifiers, denitrifiers, and phosphorous accumulating organisms (PAOs), were established. Biomass detachment rates were found to increase with biomass attachment on carrier media in both beds. The detachment rate coefficients based on VSS were significantly affected by shear stress, whereas for protein, glucose and specific microbial groups, no significant effect of shear stress was observed. High detachment rates were observed for the more porous biofilm structure. The presence of nitrifiers in the anoxic biofilm and denitrifiers in the aerobic biofilm was established by the specific activity measurements. Detachment rates of PAOs in aerobic and anoxic biofilms were evaluated.     

Kinetics of CO conversion into H<Subscript>2</Subscript> by <Emphasis Type="Italic">Carboxydothermus hydrogenoformans</Emphasis>

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.     

新疆天山中段巴音布鲁克高山草地碳含量及其垂直分布

对新疆天山中段巴音布鲁克高山草地(高山草原和高山草甸)的生物量和土壤有机碳进行了测定。结果表明积分和分层两种估算方法得到的土壤有机碳含量没有显著差异,但积分算法的优势在于能推算不同深度的土壤有机碳含量,便于与以往的研究进行比较;高山草甸的生物量和土壤有机碳含量均大于高山草原;其地上生物量分别为71.4和94.9 g C·m^-2,地下生物量分别为1 033.5和1 285.2 g C·m^-2; 1 m深度的土壤有机碳含量分别为25.7和38.8 kg·m^-2;地上生物量呈现较为明显的垂直分布格局,即随着海拔的增加,地上生物量先呈增加趋势,但当海拔超过一定界限后生物量突然下降;土壤含水率是导致南坡(阳坡)土壤有机碳含量空间分异的重要因素,但北坡(阴坡) 土壤有机碳含量还可能与地形、土壤质地等其它因素有关;两种高山草地(高山草原和高山草甸)的根系集中分布在40 cm以内,0~20 cm根系分别占其总量的76%和80%;土壤有机碳集中分布在60 cm以内,0~20 cm土壤有机碳分别占其总量的55%和49%;高山草原根系分布比高山草甸深,但较低的地下/地上比使得其有机碳分布比高山草甸浅。     

Model-based analysis on growth of activated sludge in a sequencing batch reactor

A mathematical model is developed to describe the growth of multiple microbial species such as heterotrophs and autotrophs in activated sludge system. Performance of a lab-scale sequencing batch reactor involving storage process is used to evaluate the model. Results show that the model is appropriate for predicting the fate of major model components, i.e., chemical oxygen demand, storage polymers (X _STO), volatile suspended solid (VSS), ammonia, and oxygen uptake rate (OUR). The influence of sludge retention time (SRT) on reactor performance is analyzed by model simulation. The biomass components require different time periods from one to four times of SRT to reach steady state. At an SRT of 20 days, the active bacteria (autotrophs and heterotrophs) constitute about 57% of the VSS; the remaining biomass is not active. The model established demonstrates its capacity of simulating the reactor performance and getting insight in autotrophic and heterotrophic growth in complex activated sludge systems.     

Methane fermentation of brewery by-products

A laboratory-scale research program was undertaken to investigate the kinetics of the mesophilic (37°C) anaerobic digestion of brewery industry by-product. The purpose was to develop data for the design and operation of full-scale units which could be used to generate methane fuel gas from these materials. This is important because the brewery industry has been susceptible to shortages of natural gas in recent years. The minimum SRT is 2.3 days, although for design purposes as much as ten days is recommended. The biomass yield is 0.512 g volatile suspended solids (VSS)/g volatile solids (VS) or 0.421 g VSS/g chemical oxygen demand (COD). The maintenance requirement is 0.052 g VS/g VSS per day or 0.061 g COD/g VSS per day. The specific methane yield is 2.51 liter/g VSS, and the methane productivity is 0.32–0.41 liter/g dry substrate added or 0.69–0.91 liter/g destroyed. The maximum loading rate for which substrate inhibition is not observed is 6 g dry substrate added per liter per day. The results of the entire program indicate that processing brewery by-product in this manner is both technically feasible and economically attractive.     

Bacterial community dynamics involved in Reactive Orange M2R dye degradation using a real time quantitative PCR and scale up studies using sequence batch reactor

Bacterial consortium volatile suspended soil (VSS) (Vatva soil sample) with a capability of azo dye Reactive Orange M2R (ROM2R) decolorization and degradation (shown in our earlier studies using Fourier transform infrared spectroscopy (FTIR) and phytotoxicity studies) was isolated from industrial wastewaters by enrichment culture technique. The present study was carried out to study bacterial population dynamics in consortium Vatva soil sample (VSS) during azo dye ROM2R degradation and to identify the consortium members that were actively involved in the degradation process. To achieve this goal, a real-time Polymerase chain reaction (PCR) assay targeting species-specific region of 16S rDNA of each consortial bacteria was developed to provide quantitative information about the bacterial abundance during azo dye degradation. The real-time PCR assay indicated that Pseudomonas aeruginosa (VSS-6) dominated consortium bacterial community during the active continuous bioremediation process. Attempt has been made to scale up from 100 ml volume to 10 L operation volume with intermittent additions (batch fed loadings) in a Sequence batch reactor (SBR). The development of VSS consortium biomass (MLVSS), changes in COD and biochemical oxygen demand, and the dye degradation were studied under conditions simulating the operations of biological effluent treatment in an attempt to develop a commercially applicable dye effluent treatment process unit.     

Production of polyhydroxybutyrate by activated sludge performing enhanced biological phosphorus removal

In this study, polyhydroxybutyrate (PHB) – a biodegradable plastics material – was produced by activated sludge performing enhanced biological phosphorus removal (EBPR) in batch experiments under anaerobic, aerobic and anaerobic/aerobic conditions. Under anaerobic conditions, the maximum PHB content of the dry biomass was 28.8% by weight, while under aerobic or anaerobic/aerobic conditions, the maximum PHB content was about 50%. The PHB production rate with respect to the volatile suspended solids (VSS) was: (i) 70 mg/(g VSS) h under aerobic conditions that followed anaerobic conditions, (ii) 156 mg/(g VSS) h under anaerobic condition, and (iii) 200 mg/(g VSS) h under aerobic conditions with energy also supplied from polyphosphate. A side stream, with initially anaerobic conditions for PHB accumulation and phosphorus release, and then aerobic conditions for PHB accumulation, was proposed. In this side stream, biomass with a high PHB content and a high PHB production rate could be both achieved.     

Dynamic determination of anaerobic acetate kinetics using membrane mass spectrometry

A small, stirred, 14.4-mL tank reactor was designed to serve as a measurement cell for short-term investigation of microbial kinetics. A mass spectrometer membrane probe allowed the measurement of the dissolved gases of hydrogen, methane, oxygen, and carbon dioxide. pH was measured by an electrode and controlled by addition of acid or alkali. The highly sensitive measurement of gases with low solubility allowed rapid measurements at very low conversion. In kinetic experiments, a stepwise increase of substrate concentration (method A) and continuous feed of substrate (method B) were used, allowing quick estimation of substrate kinetics. Acetate conversion in mixed culture biofilms from a fluidized bed reactor was investigated. Substrate inhibition was found to be negligible in the concentration range studied. Experiments at various pH values showed that the undissociated acid form was the kinetic determinant. Kinetic parameters for Haldane kinetics of protons were KSH = 1.3 x 10(-5) mol m-3 and KIH = 8.1 x 10(-3) mol m-3. With free acid (HAc) as the rate determining species, the kinetic parameters for method A were KSHAc = 0.005 mol m-3 and KIHAc = 100 mol m-3 and for method B were KSHAc = 0.2 mol m-3 and KIHAc = 50 mol m-3. The maximum biomass activity occurred at around pH 6.5. Acetate was exclusively converted to methane and CO2 at pH > 6. Copyright 1998 John Wiley & Sons, Inc.