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.     

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.     

Fe(III) and S0 reduction by Pelobacter carbinolicus.

There is a close phylogenetic relationship between Pelobacter species and members of the genera Desulfuromonas and Geobacter, and yet there has been a perplexing lack of physiological similarities. Pelobacter species have been considered to have a fermentative metabolism. In contrast, Desulfuromonas and Geobacter species have a respiratory metabolism with Fe(III) serving as the common terminal electron acceptor in all species. However, the ability of Pelobacter species to reduce Fe(III) had not been previously evaluated. When a culture of Pelobacter carbinolicus that had grown by fermentation of 2,3-butanediol was inoculated into the same medium supplemented with Fe(III), the Fe(III) was reduced. There was less accumulation of ethanol and more production of acetate in the presence of Fe(III). P. carbinolicus grew with ethanol as the sole electron donor and Fe(III) as the sole electron acceptor. Ethanol was metabolized to acetate. Growth was also possible on Fe(III) with the oxidation of propanol to propionate or butanol to butyrate if acetate was provided as a carbon source. P. carbinolicus appears capable of conserving energy to support growth from Fe(III) respiration as it also grew with H2 or formate as the electron donor and Fe(III) as the electron acceptor. Once adapted to Fe(III) reduction, P. carbinolicus could also grow on ethanol or H2 with S0 as the electron acceptor. P. carbinolicus did not contain detectable concentrations of the c-type cytochromes that previous studies have suggested are involved in electron transport to Fe(III) in other organisms that conserve energy to support growth from Fe(III) reduction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energetics of syntrophic fatty acid oxidation

Abstract: Fatty acids are key intermediates in methanogenic degradation of organic matter in sediments as well as in anaerobic reactors. Conversion of butyrate or propionate to acetate, (CO₂), and hydrogen is endergonic under standard conditions, and becomes possible only at low hydrogen concentrations (10^-4-10^-5 bar). A model of energy sharing between fermenting and methanogenic bacteria attributes a maximum amount of about 20 kJ per mol reaction to each partner in this syntrophic cooperation system. This amount corresponds to synthesis of only a fraction (one-third) of an ATP to be synthesized per reaction. Recent studies on the biochemistry of syntrophic fatty acid-oxidizing bacteria have revealed that hydrogen release from butyrate by these bacteria is inhibited by a protonophore or the ATPase inhibitor DCCD ( N , N '-dicyclohexyl carbodiimide), indicating that a reversed electron transport step is involved in butyrate or propionate oxidation. Hydrogenase, butyryl-CoA dehydrogenase, and succinate dehydrogenase acitivities were found to be partially associated with the cytoplasmic membrane fraction. Also glycolic acid is degraded to methane and CO₂ by a defined syntrophic coculture. Here the most difficult step for hydrogen release is the glycolate dehydrogenase reaction ( E '₀=−92 mV). Glycolate dehydrogenase, hydrogenase, and ATPase were found to be membrane-bound enzymes. Membrane vesicles produced hydrogen from glycolate only in the presence of ATP; protonophores and DCCD inhibited this hydrogen release. This system provides a suitable model to study reversed electron transport in interspecies hydrogen transfer between fermenting and methanogenic bacteria in methanogenic biomass degradation.     

Kinetic modeling and sensitivity analysis of acetone-butanol-ethanol production

A kinetic simulation model of metabolic pathways that describes the dynamic behaviors of metabolites in acetone-butanol-ethanol (ABE) production by Clostridium saccharoperbutylacetonicum N1-4 was proposed using a novel simulator WinBEST-KIT. This model was validated by comparing with experimental time-course data of metabolites in batch cultures over a wide range of initial glucose concentrations (36.1-295 mM). By introducing substrate inhibition, product inhibition of butanol, activation of butyrate and considering the cessation of metabolic reactions in the case of insufficiency of energy after glucose exhaustion, the revised model showed 0.901 of squared correlation coefficient (r(2)) between experimental time-course of metabolites and calculated ones. Thus, the final revised model is assumed to be one of the best candidates for kinetic simulation describing dynamic behavior of metabolites in ABE production. Sensitivity analysis revealed that 5% increase in reaction of reverse pathway of butyrate production (R(17)) and 5% decrease in reaction of CoA transferase for butyrate (R(15)) highly contribute to high production of butanol. These system analyses should be effective in the elucidation which pathway is metabolic bottleneck for high production of butanol.     

Production of optically active 2,3-butanediol by Bacillus polymyxa

Bacillus polymyxa produces (R, R)-2,3-butanediol from a variety of carbohydrates. Other metabolites are also produced including acetoin, acetate, lactate, and ethanol. The excretion of each metabolite was found to depend on the relative availability of oxygen to the culture. When the relative oxygen uptake rate was high, enhanced yields of acetate and acetoin were noted. At an intermediate oxygen availability, the butanediol yield was maximal. When the availability of oxygen was more restricted, higher yields of lactate and ethanol occurred. The cells appeared to regulate themselves such that energy generation is optimal subject to the constraint that the cells do not produce more reducing equivalents than can be oxidized by the electron transport system. The dependence of each product yield on the relative oxygen availability was determined, and this knowledge was used to carry out a fed-batch fermentation that attained a final butanediol concentration of over 40 g/L in 50 h.     

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.     

己酸菌LⅡ己酸发酵过程中生理及代谢特点

在了解了产己酸细菌ＬＩＩ的发酵最适条件的基础上,进一步对己酸发酵过程中的生理及代谢特性作了较详细的分析。结果表明,在己酸发酵过程中除产生正常代谢产物己酸外,还产生丁酸、乙酸、戊酸等其它酸类和甲烷,氢气和ＣＯ_２等气体。用１—^１３Ｃ－乙酸钠和未标记的乙醇作为底物发酵,用色质联用仪（ＧＣ／ＭＳ）分析,结果表明：在己酸合成中碳的来源为乙醇和乙酸,中间代谢产物为丁酸。     

Metabolism of position-labelled glucose in anoxic methanogenic paddy soil and lake sediment

Abstract Turnover times of radioactive glucose were shorter in paddy soil (4–16 min) than in Lake Constance sediment (18–62 min). In the paddy soil, 65–75% of the radioactive glucose was converted to soluble metabolites. In the sediment, only about 25% of the radioactive glucose was converted to soluble metabolites, the rest to particulate material. In anoxic paddy soil, the degradation pattern of position-labelled glucose was largely consistent with glucose degradation via the Embden-Meyerhof-Parnas (EMP) pathway followed by methanogenic acetate cleavage: CO₂ mainly originated from C-3,4, whereas CH₄ mainly originated from C-1 and C-6 of glucose. Acetate-carbon originated from C-1, C-2 and C-6 rather than from C-3,4 of glucose. In both paddy soil and Lake Constance sediment acetate and CO₂ were the most important early metabolites of radioactive glucose. Other early products included propionate, ethanol/butyrate, succinate, and lactate, but accounted each for less than 1–8% of the glucose utilized. The labelling of propionate by [3,4-¹⁴C]glucose suggests that it was mainly produced from glucose or lactate rather than from ethanol. Isopropanol and caproate were also detectable in paddy soil, but were not produced from radioactive glucose. Chloroform inhibited methanogenesis, inhibited the further degradation of radioactive acetate and resulted in the accumulation of H₂, however, did not inhibit glucose degradation. Since acetate was the main soluble fermentation product of glucose and was produced at a relatively high molar acetate: CO₂ ratio (2.5:1), homoacetogenesis appeared to be the most important glucose fermentation pathway.     

Modeling product formation in anaerobic mixed culture fermentations

The anaerobic conversion of organic matter to fermentation products is an important biotechnological process. The prediction of the fermentation products is until now a complicated issue for mixed cultures. A modeling approach is presented here as an effort to develop a methodology for modeling fermentative mixed culture systems. To illustrate this methodology, a steady-state metabolic model was developed for prediction of product formation in mixed culture fermentations as a function of the environmental conditions. The model predicts product formation from glucose as a function of the hydrogen partial pressure (P(H2)), reactor pH, and substrate concentration. The model treats the mixed culture as a single virtual microorganism catalyzing the most common fermentative pathways, producing ethanol, acetate, propionate, butyrate, lactate, hydrogen, carbon dioxide, and biomass. The product spectrum is obtained by maximizing the biomass growth yield which is limited by catabolic energy production. The optimization is constrained by mass balances and thermodynamics of the bioreactions involved. Energetic implications of concentration gradients across the cytoplasmic membrane are considered and transport processes are associated with metabolic energy exchange to model the pH effect. Preliminary results confirmed qualitatively the anticipated behavior of the system at variable pH and P(H2) values. A shift from acetate to butyrate as main product when either P(H2) increases and/or pH decreases is predicted as well as ethanol formation at lower pH values. Future work aims at extension of the model and structural validation with experimental data.     

Effect of carbon sources differing in oxidation state and transport route on succinate production in metabolically engineered Escherichia coli

In mixed-acid fermentation, succinate synthesis requires one mole of phosphoenolpyruvate (PEP), one mole of CO₂, and two moles of NADH for every mole of succinate to be formed. Different carbon sources with different properties were used to address these requirements. Sorbitol generates one more mole of NADH than glucose. Fermentation of sorbitol was shown in this study (and by others) to produce significantly more succinate than fermentation of glucose, due to increased NADH availability. Xylose fermentation conserves the intracellular PEP pool, since its transport does not require the phosphotransferase system normally used for glucose transport. The extra PEP can then be assimilated in the succinate pathway to improve production. In this study, fermentation of xylose did yield higher succinate production than glucose fermentation. Subsequent inactivation of the acetate and lactate pathways was performed to study metabolite redistribution and the effect on succinate production. With the acetate pathway inactivated, significant carbon flux shifted toward lactate rather than succinate. When both acetate and lactate pathways were inactivated, succinate yield ultimately increased with a concomitant increase in ethanol yield.     

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.     

Clostridium thermobutyricum: growth studies and stimulation of butyrate formation by acetate supplementation

Clostridium thermobutyricum produces butyrate as the main fermentation product from glucose, and from yeast extract, which is required for substantial growth. After sequential transfer in the presence of increasing butyrate concentrations, strain JW 171 K grew in the presence of up to 350 mM butyrate either at pH 5.5 or at pH 8.0 and at 40 degrees C as well as at 60 degrees C. This result indicated that butyrate-dependent growth inhibition was independent from the concentration of undissociated butyric acid. Increased butyrate concentration decreased the level of tolerated glucose from above 15% to below 10%. At 0.05 and 2.0% (wt/vol) yeast extract, the Y(Glucose) was 30 and 55 g dry weight cells per mole glucose, respectively. Y(ATP) values between 18 and 21 g weight cells per mole ATP, obtained after growth in the presence of 2% yeast extract, indicate that the butyrate fermentation under thermophilic growth conditions is as energy efficient as it is under mesophilic conditions. Externally added acetate stimulated the production of butyrate. Supplemented 14C-acetate was converted to butyrate, resulting in the formation of 44% labeled butyrate (i.e. formed from 14C-acetate) and 56% unlabeled butyrate (formed from glucose and yeast extract). Continuous removal of H2 in batch cultures led to a shift in the fermentation products from more butyrate to the more oxidized and more energy yielding acetate.     

Acidogenic fermentation of lactose

Cheese whey is the main component of waste streams from cheese manufacturing plants. Whey is a high biochemical oxygen demand (BOD) effluent that must be reduced before the streams are sent to the sewer. It is proposed in this article that the production of methane by anaerobic fermentation would be the best use of this stream, especially for small plants. Single-stage fermentation of lactose, the main component of whey, results in a very low pH and a stalled process. Two-phase fermentation will eliminate this problem. The acidogenic stage of fermentation has been studied at pH of between 4 and 6.5. The nature of the main products of the reaction have been found to be pH dependent. Below a pH of 4.5 a gas (CO(2) and H(2)) is produced along with ethanol, acetate, and butyrate. Above a pH of 4.5 no gas was produced, and the liquid products included less ethanol and butyrate and more acetate. A separate study on the conditions for gas formation showed that if the pH dropped for a short time below 4.5 gases were formed at all subsequent pH. This would indicate a change in population distribution due to the period at a low pH. By assuming that the desired products from the acidogenic stage were butyrate, acetate, and no gases, the optimum pH range was found to be between 6.0 and 6.5.     

Physiological, biochemical, and mathematical studies of micro-aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. II: intracellular metabolite and enzyme assays at steady state chemostat cultures

Intracellular metabolite concentration and enzyme activity measurements were made to explain the new metabolic and growth phenomena seen in the micro-aerobic, continuous yeast cultures described in Part I. The results of these assays suggested mechanisms for the observed maximum in the specific ethanol productivity as a function of the oxygen feed rate, changing ATP yields, the effects of antifoam, and the sharp changes in the biomass concentration with small changes in the oxygenation. Measured were the intracellular concentrations of ATP, NADH, glucose 6-phosphate, pyruvate, glycerol, and ethanol, and the activities of hexokinase and alcohol dehydrogenase. Rate-limiting steps were identified by the accumulation of metabolites upstream and the depletion of metabolites downstream of the step.A potential mechanism for the stimulation of fermentation with decreasing oxygenation was an activation of glucose transport by an accumulating intracellular ATP concentration. The inhibition of fermentation at yet lower oxygenation rates may have been caused by the continued accumulation of ATP to the point that the glycolytic kineses were inhibited. A mechanism for the changing ATP yields and intracellular ATP concentration proposed the existence of ATPases or ATP waste reactions stimulated by both oxygen and ATP. Antifoam had the effect of decreasing the resistance for glycerol transport out of the cell. The resulting stimulation of glycerol production and inhibition of ethanol production decreased the intracellular ATP content. Finally, intracellular ethanol was found not to accumulate to levels of higher than the extracellular concentration.     

Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor

Changes in product formation during carbohydrate fermentation by anaerobic microflora in a continuous flow stirred tank reactor were investigated with respect to the dilution rate in the reactor. In the fermentation by methanogenic microflora, stable methane fermentation, producing methane and carbon dioxide, was observed at relatively low dilution rates (less than 0.33 d(-1) on glucose and 0.20 d(-1) on cellulose). Decomposition of cellulose in the medium was a rate-limiting step in the reaction, because glucose was easily consumed at all applied dilution rates (0.07-4.81 d(-1)). Intermediate metabolites of methane fermentation, such as lactate, ethanol, acetate, butyrate, formate, hydrogen, and carbon dioxide, were accumulated as dilution rate increased. Maximum yield of hydrogen was obtained at 4.81 d(-1) of dilution rate (0.1 mol/mol glucose on glucose or 0.7 mol/mol hexose on cellulose). Lactate was the major product on glucose (1.2 mol/mol glucose), whereas ethanol was predominant on cellulose (0.7 mol/mol hexose). An analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S rDNA of the microflora indicated that changes in the microbial community took place at various dilution rates, and these changes appeared to correspond to the changes in product distributions. Sequence analyses of the DGGE fragments revealed the probable major population of the microflora. A band closely related to the microorganisms of thermophilic anaerobic bacteria was detected with strong intensity on both glucose and cellulose. Differences in the production yield of hydrogen could have been caused by different populations of microorganisms in each microflora. In the case of cellulose, increasing the dilution rate brought about an accumulation of microorganisms related to Clostridia species that have cellulolytic activity, this being in accordance with the notion of cellulose decomposition being the rate-limiting reaction.     

Control of the selectivity of butyric acid production and improvement of fermentation performance withClostridium tyrobutyricum

Summary The parameters that control fermentation performance of butyrate production have been studied with a selected strain ofClostridium tyrobutyricum. Fed-batch supply of glucose increased productivity for butyrate. The ratio of butyrate to total acids was strongly influenced by the growth rate of the bacteria, acetate being produced along with butyrate at higher growth rates. In glucose-limited, fed-batch cultures, initially produced acetate was re-utilized, resulting in exclusive production of butyrate. In cultures with non-limiting glucose feeding, the butyrate concentration reached 42.5 g·1^–1 with a selectivity of 0.90, a productivity of 0.82 g·^–1 per hour and a yield of 0.36 g·g^–1 The effects of the mode of supply of glucose on the production of butyrate and acetate are discussed in relation with the energy requirements for cell growth.     

13C NMR studies of butyric fermentation in Clostridium kluyveri

The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR. The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate. If butyryl-CoA is involved in the condensation, caproate is the ultimate product. ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway. The rapid initial production of acetate indicates equally rapid production of ATP. Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate. The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium. The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds. 13C-13C coupling in the butyrate and caproate produced from [1-13C]ethanol and [2-13C]acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated. The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources.     

Acetoin production in Leuconostoc mesenteroides NCDO 518

Abstract Cell suspensions of Leuconostoc mesenteroides NCDO 518 converted pyruvate to acetoin and a small amount of lactate and acetate. Acetoin was not produced from mixtures of pyruvate and glucose unless the ratio of pyruvate to glucose was greater than 2:1. In the presence of glucose, external pyruvate was first used as an electron acceptor, being reduced to lactate, and was converted to acetoin only after the exhaustion of glucose. Use of added pyruvate as an electron acceptor suppressed ethanol formation and the products of glucose fermentation were then lactate and acetate; 2 mol of pyruvate per mol of glucose were required to completely suppress ethanol formation. It is suggested that acetoin is produced by heterofermentative organisms when available pyruvate is in excess of that required for reoxidation of all NADH produced during glucose fermentation.