Glycerol fermentation by (open) mixed cultures: a chemostat study

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.     

Evaluation of metabolism using stoichiometry in fermentative biohydrogen

We first constructed full stoichiometry, including cell synthesis, for glucose mixed-acid fermentation at different initial substrate concentrations (0.8-6 g-glucose/L) and pH conditions (final pH 4.0-8.6), based on experimentally determined electron-equivalent balances. The fermentative bioH2 reactions had good electron closure (-9.8 to +12.7% for variations in glucose concentration and -3 to +2% for variations in pH), and C, H, and O errors were below 1%. From the stoichiometry, we computed the ATP yield based on known fermentation pathways. Glucose-variation tests (final pH 4.2-5.1) gave a consistent fermentation pattern of acetate + butyrate + large H2, while pH significantly shifted the catabolic pattern: acetate + butyrate + large H2 at final pH 4.0, acetate + ethanol + modest H2 at final pH 6.8, and acetate + lactate + trivial H2 at final pH 8.6. When lactate or propionate was a dominant soluble end product, the H2 yield was very low, which is in agreement with the theory that reduced ferredoxin (Fd(red)) formation is required for proton reduction to H2. Also consistent with this hypothesis is that high H2 production correlated with a high ratio of butyrate to acetate. Biomass was not a dominant sink for electron equivalents in H2 formation, but became significant (12%) for the lowest glucose concentration (i.e., the most oligotrophic condition). The fermenting bacteria conserved energy similarly at approximately 3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had approximately 3.5 mol ATP/mol glucose) over a wide range of H2 production. The observed biomass yield did not correlate with ATP conservation; low observed biomass yields probably were caused by accelerated rates of decay or production of soluble microbial products.     

Changes in bacterial community during fermentative hydrogen and acid production from organic waste by thermophilic anaerobic microflora

AIMS: Changes in fermentation pattern during the treatment of organic wastes containing solid materials by thermophilic anaerobic microflora were investigated with respect to product formation and bacterial community structure during hydrogen production. METHODS AND RESULTS: Anaerobic microflora enriched from sludge compost was cultivated using artificial garbage slurry in a continuous flow-stirred tank reactor. Product formation varied depending on pH and hydraulic retention time (HRT) applied. Community analysis by terminal restriction fragment length polymorphism and clone library analysis of polymerase chain reaction-amplified bacterial 16S rDNA indicated that difference in the fermentative product distribution could be caused by different populations of micro-organisms in the microflora. CONCLUSION: Hydrogen fermentation with acetate/butyrate formation was optimized at <1.0 d HRT at pH 5.0 and 6.0. Thermoanaerobacterium thermosaccharolyticum was the dominant hydrogen-producing micro-organism. Conversely, unidentified organisms became dominant after 4.0 d HRT at pH 7.0 and 8.0, where relatively high-solubilization efficiency of solid materials was observed with no production of hydrogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing product formation in the fermentation of solid organic wastes by a mixed population of micro-organisms. Various fermentation patterns including hydrogen fermentation were characterized and evaluated from engineering and microbial aspects.     

Metabolic Energy-Based Modelling Explains Product Yielding in Anaerobic Mixed Culture Fermentations

The fermentation of glucose using microbial mixed cultures is of great interest given its potential to convert wastes into valuable products at low cost, however, the difficulties associated with the control of the process still pose important challenges for its industrial implementation. A deeper understanding of the fermentation process involving metabolic and biochemical principles is very necessary to overcome these difficulties. In this work a novel metabolic energy based model is presented that accurately predicts for the first time the experimentally observed changes in product spectrum with pH. The model predicts the observed shift towards formate production at high pH, accompanied with ethanol and acetate production. Acetate (accompanied with a more reduced product) and butyrate are predicted main products at low pH. The production of propionate between pH 6 and 8 is also predicted. These results are mechanistically explained for the first time considering the impact that variable proton motive potential and active transport energy costs have in terms of energy harvest over different products yielding. The model results, in line with numerous reported experiments, validate the mechanistic and bioenergetics hypotheses that fermentative mixed cultures products yielding appears to be controlled by the principle of maximum energy harvest and the necessity of balancing the redox equivalents in absence of external electron acceptors.     

Substrate and product inhibition of hydrogen production by the extreme thermophile,Caldicellulosiruptor saccharolyticus

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H(2) in the gas phase (identical with partial hydrogen pressure (pH(2)) of (1-2) x 10(4) Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H(2) (identical with pH(2) of 5.6 x 10(4) Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70 degrees C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins.     

Anaerobic fermentation: Microbes from ruminants

Fed-batch fermentation of biomass could provide a route for direct conversion of renewable resources to commercially significant chemicals. The ecosystem in the forestomach (rumen) of ruminants provides a highly reduced environment (oxidation-reduction potential of ?250 to ?450 mV) in which anaerobic bacteria directly utilize cellulose, hemicellulose, and other fermentable biomass constituents to produce acetate, butyrate, propionate, methane and carbon dioxide at pH 5.7 to 7.3. The cellulose fermentation in the rumen is impacted by the physically and chemically heterogeneous character of the insoluble substrate, as well as the properties of the mixed culture responsible for fibre hydrolysis and carbohydrate utilization. The rumen system provides an interesting case study in the context of possible process concepts for direct fermentation of biomass to commercially important chemicals such as acetate, propionate, succinate, lactate and ethanol. The role of the chemical and physical characteristics of the substrate, the microbes in the rumen system and the metabolic pathways of soluble carbohydrates are discussed in the context of cellulose and hemicellulose fermentation.     

Influence of the pH on (open) mixed culture fermentation of glucose: a chemostat study

Catabolic products from anaerobic fermentation processes are potentially of industrial interest. The volatile fatty acids and alcohols produced can be used as building blocks in chemical processes or applied directly as substrates in a mixed culture process to produce bioplastics. Development of such applications requires a predictable and controllable product spectrum of the fermentation process. The aim of the research described in this paper was (i) to investigate the product spectrum of an open mixed culture fermentation (MCF) process as a function of the pH, using glucose as substrate, and (ii) to relate the product spectrum obtained to generalized biochemical and thermodynamic considerations. A chemostat was operated under carbon and energy limitation in order to investigate the pH effect on the product spectrum in a MCF process. A transition from CO(2)/H(2) production at lower pH values to formate production at higher pH values was observed. The ratio of CO(2)/H(2) versus formate production was found to be related to the thermodynamics of formate dehydrogenation to CO(2)/H(2). This transition was associated with a shift in the catabolic products, from butyrate and acetate to ethanol and acetate, likely due to a decrease in the oxidation state of the electron carriers in the cell. The product spectrum of the MCF process as a function of the pH could largely be explained using general biochemical considerations.     

Kinetic modeling of batch hydrogen production process by mixed anaerobic cultures

The kinetics of batch anaerobic hydrogen production by mixed anaerobic cultures was systemically investigated in this study. Unstructured models were used to describe the substrate utilization, biomass growth and product formation in the hydrogen production process. The relationship between the substrate, biomass and products were also evaluated. Experimental results show that the Michaelis-Menten equation, Logistic model and modified Gompertz equation all could be adopted to respectively describe the kinetics of substrate utilization, biomass growth and product formation. Furthermore, the relationship between the acidogenic products and biomass was simulated by Luedeking-Piret model very well. The experimental results suggest that the formation of hydrogen and the main aqueous products, i.e., butyrate and acetate, was all growth-associated.     

Effects of Acetate and Butyrate During Glycerol Fermentation by Clostridium butyricum

The effects of acetate and butyrate during glycerol fermentation to 1,3-propanediol at pH 7.0 by Clostridium butyricum CNCM 1211 were studied. At pH 7.0, the calculated quantities of undissociated acetic and butyric acids were insufficient to inhibit bacterial growth. The initial addition of acetate or butyrate at concentrations of 2.5 to 15 gL⁻¹ had distinct effects on the metabolism and growth of Clostridium butyricum. Acetate increased the biomass and butyrate production, reducing the lag time and 1,3-propanediol production. In contrast, the addition of butyrate induced an increase in 1,3-propanediol production (yield: 0.75 mol/mol glycerol, versus 0.68 mol/mol in the butyrate-free culture), and reduced the biomass and butyrate production. It was calculated that reduction of butyrate production could provide sufficient NADH to increase 1,3-propanediol production. The effects of acetate and butyrate highlight the metabolic flexibility of Cl. butyricum CNCM 1211 during glycerol fermentation. Received: 2 January 2001 / Accepted: 6 February 2001     

Improvement of biohydrogen production by Enterobacter cloacae IIT-BT 08 under regulated pH

The present study investigates the effect of pH and intermediate products formation on biological hydrogen production using Enterobacter cloacae IIT-BT 08. Initial pH was found to have a profound effect on hydrogen production potential, while regulating the pH 6.5 throughout the fermentation was found to increase the cumulative hydrogen production rate and yield significantly. Modified Gompertz equation was used to fit the cumulative hydrogen production curves to obtain the hydrogen production potential P, the hydrogen production rate R and lag phase λ. At regulated pH 6.5, higher H(2) yield (3.1molH(2)mol(-1) glucose), specific hydrogen production potential (798.1mL/g) and specific rate of H(2) production (72.1mLL(-1)h(-1)g(-1)) were obtained. The volatile fatty acid profile showed butyrate, ethanol and acetate as the major end metabolites of fermentation under the operating pH conditions tested; however, their pattern of distribution was pH dependent. At the optimum pH of 6.5, the acetate to butyrate ratio (A/B ratio) was found to be higher than that at any other pH. The study also investigates the effect of sodium ions on biohydrogen production potential. It was also found that sodium ion concentration up to 250mM enhanced the hydrogen production potential; however, any further increase in the metal ion concentration had an inhibitory effect.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the ruminal ciliate Polyplastron multivesiculatum.

The effects of ruminal concentrations of CO2 and oxygen on the end products of endogenous metabolism and fermentation of D-glucose by the ruminal entodiniomorphid ciliate Polyplastron multivesiculatum were investigated. The principal metabolic products were butyric, acetic, and lactic acids, H2, and CO2. 13C nuclear magnetic resonance spectroscopy identified glycerol as a previously unknown major product of D-[1-13C]glucose fermentation by this protozoan. Metabolite formation rates were clearly influenced by the headspace gas composition. In the presence of 1 to 3 microM O2, acetate, H2, and CO2 formation was partially depressed. A gas headspace with a high CO2 content (66 kPa) was found to suppress hydrogenosomal pathways and to favor butyrate accumulation. Cytochromes were not detected (less than 2 pmol/mg of protein) in P. multivesiculatum; protozoal suspensions, however, consumed O2 for up to 3 h at 1 kPa of O2. Under gas phases of greater than 2.6 kPa of O2, the organisms rapidly became vacuolate and the cilia became inactive. The results suggest that fermentative pathways in P. multivesiculatum are influenced by the O2 and CO2 concentrations that prevail in situ in the rumen.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the ruminal ciliate Polyplastron multivesiculatum

The effects of ruminal concentrations of CO2 and oxygen on the end products of endogenous metabolism and fermentation of D-glucose by the ruminal entodiniomorphid ciliate Polyplastron multivesiculatum were investigated. The principal metabolic products were butyric, acetic, and lactic acids, H2, and CO2. 13C nuclear magnetic resonance spectroscopy identified glycerol as a previously unknown major product of D-[1-13C]glucose fermentation by this protozoan. Metabolite formation rates were clearly influenced by the headspace gas composition. In the presence of 1 to 3 microM O2, acetate, H2, and CO2 formation was partially depressed. A gas headspace with a high CO2 content (66 kPa) was found to suppress hydrogenosomal pathways and to favor butyrate accumulation. Cytochromes were not detected (less than 2 pmol/mg of protein) in P. multivesiculatum; protozoal suspensions, however, consumed O2 for up to 3 h at 1 kPa of O2. Under gas phases of greater than 2.6 kPa of O2, the organisms rapidly became vacuolate and the cilia became inactive. The results suggest that fermentative pathways in P. multivesiculatum are influenced by the O2 and CO2 concentrations that prevail in situ in the rumen.     

Influence of dilution rate on the acidogenic phase products distribution during two-phase lactose anaerobiosis

Acidogenic fermentation of lactose was carried out in a continuous stirred reactor with a mixed anaerobic culture. From the variation of the reactor products with pH and dilution rate two possible carbon flow schemes were proposed for the reaction. In both schemes the carbon flow from pyruvate to butyrate and lactate was assumed to occur in parallel. A change in gas composition and in product concentrations at dilution rates between 0.1 and 0.15 h(-1) for pH levels between 4.5 and 6.0 was ascribed to a shift in microbial population. To clarify the mechanism radiotracer tests were made using [U-(14)C]-butyrate, [2-(14)C]-propionate and [U-(14)C]-lactate to determine the path of carbon flow during acidogenesis of lactose using a mixed culture. At a dilution rate between 0.1 and 0.15 h(-1) and pH from 4.5 to 6.0 a rise in the lactate concentration in the product was shown to be due to a microbial population shift which disabled the conversion of lactate to other intermediary metabolites. It was also found that the flow of carbon from pyruvate to butyrate and lactate occurred by parallel pathways. Also, in the presence of hydrogen reducing methanogens, lactate was almost completely converted to acetate and not propionate. Butyrate was found to be converted to acetate at a slow rate as long as hydrogen reducing methanogens were present. The role played by propionibacteria in this lactose acidogenic eocosystem was minor. From the carbon flow model it can be concluded that lactate is the most suitable marker for optimizing an acidogenic reactor in a two-phase biomethanation process.     

Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum

The effect of pH (between 5.0 and 6.3) on butyric acid fermentation of xylose by Clostridium tyrobutyricum was studied. At pH 6.3, the fermentation gave a high butyrate production of 57.9 g l(-1) with a yield of 0.38-0.59 g g(-1) xylose and a reactor productivity up to 3.19 g l(-1)h(-1). However, at low pHs (<5.7), the fermentation produced more acetate and lactate as the main products, with only a small amount of butyric acid. The metabolic shift from butyrate formation to lactate and acetate formation in the fermentation was found to be associated with changes in the activities of several key enzymes. The activities of phosphotransbutyrylase (PTB), which is the key enzyme controlling butyrate formation, and NAD-independent lactate dehydrogenase (iLDH), which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3. In contrast, cells at pH 5.0 had higher activities of phosphotransacetylase (PTA), which is the key enzyme controlling acetate formation, and lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate. Also, PTA was very sensitive to the inhibition by butyric acid. Difference in the specific metabolic rate of xylose at different pHs suggests that the balance in NADH is a key in controlling the metabolic pathway used by the cells in the fermentation.     

A modified metabolic model for mixed culture fermentation with energy conserving electron bifurcation reaction and metabolite transport energy

A modified metabolic model for mixed culture fermentation (MCF) is proposed with the consideration of an energy conserving electron bifurcation reaction and the transport energy of metabolites. The production of H₂ related to NADH/NAD⁺ and Fdred/Fdox is proposed to be divided in three processes in view of energy conserving electron bifurcation reaction. This assumption could fine‐tune the intracellular redox balance and regulate the distribution of metabolites. With respect to metabolite transport energy, the proton motive force is considered to be constant, while the transport rate coefficient is proposed to be proportional to the octanol–water partition coefficient. The modeling results for a glucose fermentation in a continuous stirred tank reactor show that the metabolite distribution is consistent with the literature: (1) acetate, butyrate, and ethanol are main products at acidic pH, while the production shifts to acetate and propionate at neutral and alkali pH; (2) the main products acetate, ethanol, and butyrate shift to ethanol at higher glucose concentration; (3) the changes for acetate and butyrate are following an increasing hydrogen partial pressure. The findings demonstrate that our modified model is more realistic than previous proposed model concepts. It also indicates that inclusion of an energy conserving electron bifurcation reaction and metabolite transport energy for MCF is sound in the viewpoint of biochemistry and physiology. Biotechnol. Bioeng. 2013; 110: 1884–1894. © 2013 Wiley Periodicals, Inc.     

Isolation and Characterization of Clostridium butyricum DSM 5431 Mutants with Increased Resistance to 1,3-Propanediol and Altered Production of Acids

Clostridium butyricum mutants were isolated from the parent strain DSM 5431 after mutagenesis with N-methyl-N(prm1)-nitro-N-nitrosoguanidine and two selection procedures: osmotic pressure and the proton suicide method. Isolated mutants were more resistant to glycerol and to 1,3-propanediol (1,3-PD) than was the wild type, and they produced more biomass. In batch culture on 62 g of glycerol per liter, the wild type produced more acetic acid than butyrate, with an acetate/butyrate ratio of 5.0, whereas the mutants produced almost the same quantities of both acids or more butyrate than acetate with acetate/butyrate ratios from 0.6 to 1.1. The total acid formation was higher in the wild-type strain. Results of analysis of key metabolic enzymatic activities were in accordance with the pattern of fermentation product formation: either the butyrate kinase activity increased or the acetate kinase activity decreased in cell extracts of the mutants. A decreased level of the hydrogenase and NADH-ferredoxin activities concomitant with an increase in ferredoxin-NAD(sup+) reductase activities supports the conclusion that the maximum percentage of NADH available and used for the formation of 1,3-PD was higher for the mutants (97 to 100%) than for the wild type (70%). In fed-batch culture, at the end of the fermentation (72 h for the wild-type strain and 80 to 85 h for the mutants), 44% more glycerol was consumed and 50% more 1,3-PD was produced by the mutants than by the wild-type strain.     

Regulation of carbon and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H(+) ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.     

CO2-dependent fermentation of phenol to acetate, butyrate and benzoate by an anaerobic, pasteurised culture

Fermentative degradation of phenol was studied using a non-methanogenic, pasteurised enrichment culture containing two morphologically different bacteria. Phenol was fermented to benzoate, acetate and butyrate and their relative occurrence depended on the concentration of hydrogen. Proportionately more benzoate was formed with high initial levels of H2. The influence of PH2 on the fermentation pattern was studied both in dense cell suspensions and in growing cultures by addition of hydrogen. An increase in growth yield (OD578) was observed, compared to controls, as a consequence of phenol degradation; however, the increase was less in H2-amended treatments, in which most of the phenol ended up as benzoate. The degradation of phenol in the dense cell suspension experiments was dependent on CO2. Benzoate was not degraded when added as a substrate to the growing culture. This is, to our knowledge, the first report concerning the fermentative degradation of phenol to nonaromatic products.     

果蔬废弃物两相厌氧发酵产己酸的效能研究

利用厌氧菌群生物合成己酸被认为是一种非常有潜力的新型废弃物资源化技术,但是其合成效能的提高是目前亟待解决的关键问题。本研究以实际果蔬废弃物为原料,对两相厌氧发酵产己酸的效能进行了研究。首先优化接种比以提高酸化相的水解转化效率;在此基础上通过调控醇酸比和pH以强化产己酸相的发酵效能。结果显示,果蔬废弃物厌氧产酸的最佳接种比为2∶1,此时水解率和酸化率分别可达到98.1%和83.2%,乙酸和丁酸产量分别达到5.4 g/L和3.3 g/L。合理控制醇酸比和pH对提高产己酸相的发酵效能非常关键。当醇酸比和pH控制为4∶1和7.5时,己酸生成量可达14.9 g/L,约占液相总COD的80.84%;而低醇酸比和低pH易造成丁酸的累积,从而降低了己酸产量。己酸发酵过程属于非生长偶联型,己酸菌(Clostridium kluyveri)指数增长期伴随着丁酸的生成,而己酸合成主要发生在生长中后期。此外,己酸菌对于pH变化较为敏感,适当提高pH有助于减轻有机酸毒性,提高生物量;但是碱性环境会严重抑制己酸菌的生长繁殖。研究表明,通过分别对酸化相和产己酸相进行优化和调控,两相发酵策略更有利于提高己酸合成效能。     

Intrinsic fermentation kinetics of lactose in acidogenic biofilms

The intrinsic fermentation kinetics of lactose in acidogenic biofilms were investigated in situ in a continuous flow fermentor at 35 degrees C and pH 4.6. The external and internal mass transfer resistances to lactose molecules from bulk solution to inside the biofilms were experimentally minimized or eliminated in a thin biofilm and recycled medium. In a chemically defined culture medium, the immobilized acidogens converted lactose mainly to acetate and butyrate; the minor products included ethanol. propionate, lactate, and hydrogen. The utilization rate of lactose, as a function of lactose concentration in the fermentor, can be described by a Michaelis-Menten equation, as can the formation rates of acetate, butyrate, and ethanol. The production rates of propionate and lactate had a liner relationship with lactose concentration under the experimental conditions. The low pH (4.6) of culture medium could depress the formation of propionate, and intermediate which is most difficulty digested by acetogenic bacteria located in the second fermentor in a two-phase process. Production rate of acetate quickly reached a constant, and additional utilization of lactose produced more butyrate and other minor products. (c) 1993 John Wiley & Sons, Inc.