Effect of different heterogeneous inocula in acidogenic fermentation of whey permeate

Summary The effect of different heterogeneous mixed bacterial inocula has been investigated on the acidogenic fermentation of cheese whey permeate. After a few months of continuous cultivation at pH 6 and at a retention time of 40 h, all mixed bacterial populations produced large amounts of lactate and butyrate, whereas the production of ethanol and acetate was largely depending on the source of the inoculum.     

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.     

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.     

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.     

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.     

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.     

A non-passive mechanism of butyrate excretion operates during acidogenic fermentation of methanol by Eubacterium limosum

The inhibitory effects of organic acids produced as fermentation end-products during methylotrophic growth of the acidogenic anaerobe, Eubacterium limosum have been investigated. Precise quantification of the intracellular concentrations of acetate and butyrate, together with delta pH measurements indicate that butyrate efflux cannot be explained by a process of passive diffusion. Intracellular concentrations of butyrate were significantly lower than those of the culture broth. It is argued that growth inhibition by butyrate is due to energetic limitations resulting from the energy drain associated with this non-passive efflux mechanism.     

Short-term effect of acetate and ethanol on methane formation in biogas sludge

Biochemical processes in biogas plants are still not fully understood. Especially, the identification of possible bottlenecks in the complex fermentation processes during biogas production might provide potential to increase the performance of biogas plants. To shed light on the question which group of organism constitutes the limiting factor in the anaerobic breakdown of organic material, biogas sludge from different mesophilic biogas plants was examined under various conditions. Therefore, biogas sludge was incubated and analyzed in anaerobic serum flasks under an atmosphere of N₂/CO₂. The batch reactors mirrored the conditions and the performance of the full-scale biogas plants and were suitable test systems for a period of 24 h. Methane production rates were compared after supplementation with substrates for syntrophic bacteria, such as butyrate, propionate, or ethanol, as well as with acetate and H₂+CO₂ as substrates for methanogenic archaea. Methane formation rates increased significantly by 35 to 126 % when sludge from different biogas plants was supplemented with acetate or ethanol. The stability of important process parameters such as concentration of volatile fatty acids and pH indicate that ethanol and acetate increase biogas formation without affecting normally occurring fermentation processes. In contrast to ethanol or acetate, other fermentation products such as propionate, butyrate, or H₂ did not result in increased methane formation rates. These results provide evidence that aceticlastic methanogenesis and ethanol-oxidizing syntrophic bacteria are not the limiting factor during biogas formation, respectively, and that biogas plant optimization is possible with special focus on methanogenesis from acetate.     

Anaerobic bio-hydrogen production from ethanol fermentation: the role of pH

Hydrogen was produced by an ethanol-acetate fermentation at pH of 5.0 +/- 0.2 and HRT of 3 days. The yield of hydrogen was 100-200 ml g Glu(-1) with a hydrogen content of 25-40%. This fluctuation in the hydrogen yield was attributed to the formation of propionate and the activity of hydrogen utilizing methanogens. The change in the operational pH for the inhibition of this methanogenic activity induced a change in the main fermentation pathway. In this study, the main products were butyrate, ethanol and propionate, in the pH ranges 4.0-4.5, 4.5-5.0 and 5.0-6.0, respectively. However, the activity of all the microorganisms was inhibited below pH 4.0. Therefore, pH 4.0 was regarded as the operational limit for the anaerobic bio-hydrogen production process. These results indicate that the pH plays an important role in determining the type of anaerobic fermentation pathway in anaerobic bio-hydrogen processes.     

Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta-deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose.     

Thermodynamic analysis of product formation in mesophilic acidogenesis of lactose

Thermodynamic analysis on the acidogenesis of lactose was performed to evaluate the different acidogenic patterns and mechanisms by using Gibbs free energy calculation. Batch acidogenesis of lactose was investigated by using an enriched culture at 37 degrees C, pH 5.5 and varied substrate levels. In addition to usual acidogenic products, i-butyrate, valerate, i-valerate, caproate, and propanol were also produced at a significant level. Thermodynamic analysis shows that valerate might be formed through the reaction requiring hydrogen as electron donor and consuming of propionate and carbon dioxide. Caproate was most likely produced directly from butyrate, hydrogen, and carbon dioxide. The minimum amount of Gibbs free energies needed to sustain isomerization of butyrate and valerate were approximately 5.7-5.8 and 4.5-4.6 kJ/mol, respectively. Propanol was produced from acetate, hydrogen, and carbon dioxide with a minimum amount of Gibbs free energy of 41.8-42.0 kJ/mol. Formation of butanol was controlled more by substrate level or population dynamics than by thermodynamics.     

Influence of External pH and Fermentation Products on Clostridium acetobutylicum Intracellular pH and Cellular Distribution of Fermentation Products

Clostridium acetobutylicum ATCC 824 cells harvested from a phosphate-limited chemostat culture maintained at pH 4.5 had intracellular concentrations of acetate, butyrate, and butanol which were 13-, 7-, and 1.3-fold higher, respectively, than the corresponding extracellular concentrations. Cells from a culture grown at pH 6.5 had intracellular concentrations of acetate and butyrate which were only 2.2-fold higher than the respective external concentrations. The highest intracellular concentrations of these acids were attained at ca. pH 5.5. When cells were suspended in anaerobic citrate-phosphate buffer at pH 4.5, exogenous acetate and butyrate caused a concentration-dependent decrease in the intracellular pH, while butanol had relatively little effect until the external concentration reached 150 mM. Acetone had no effect at concentrations up to 200 mM. These data demonstrate that acetate and butyrate are concentrated within the cell under acidic conditions and thus tend to lower the intracellular pH. The high intracellular butyrate concentration presumably leads to induction of solvent production, thereby circumventing a decrease in the intracellular pH great enough to be deleterious to the cell.     

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.     

Acetone-butanol production by Clostridium acetobutylicum in an ammonium-limited chemostat at low pH values

Clostridium acetobutylicum was grown in continuous culture under ammonium limitation (15.15 mM NH₄ ⁺). At a pH of 6.0 and at various dilution rates only acetate, butyrate and ethanol were formed as non-gaseous products. A decrease of the pH to values between 5.2 and 4.3 resulted in a shift of the fermentation towards acetone-butanol formation.     

Response of a biohydrogen-producing reactor to the substrate shift from sucrose to lactose

The response of an upflow acidogenic granule-based reactor to the substrate shift from sucrose to lactose was investigated in this study. Experimental results show that it took 60h for the reactor to completely degrade the new substrate. Hydrogen production performance, in terms of H(2) partial pressure, H(2) production rate and H(2) yield, was affected. Acetate, propionate, butyrate, valerate, caporate, ethanol and propanol were present in the reactor effluent, and their distribution changed significantly after the substrate shift. As the substrate was changed, the caproate- and ethanol-type fermentation was weakened, while the propionate-type fermentation was strengthened. Throughout the experiment, the butyrate-type fermentation played an important role. The H(2) yield had a close correlation with both propionate and B/A (butyrate/acetate) in this substrate shift process.     

Formation and Fate of Fermentation Products in Hot Spring Cyanobacterial Mats

The fate of representative fermentation products (acetate, propionate, butyrate, lactate, and ethanol) in hot spring cyanobacterial mats was investigated. The major fate during incubations in the light was photoassimilation by filamentous bacteria resembling Chloroflexus aurantiacus. Some metabolism of all compounds occurred under dark aerobic conditions. Under dark anaerobic conditions, only lactate was oxidized extensively to carbon dioxide. Extended preincubation under dark anaerobic conditions did not enhance anaerobic catabolism of acetate, propionate, or ethanol. Acetogenesis of butyrate was suggested by the hydrogen sensitivity of butyrate conversion to acetate and by the enrichment of butyrate-degrading acetogenic bacteria. Accumulation of fermentation products which were not catabolized under dark anaerobic conditions revealed their importance. Acetate and propionate were the major fermentation products which accumulated in samples collected at temperatures ranging from 50 to 70°C. Other organic acids and alcohols accumulated to a much lesser extent. Fermentation occurred mainly in the top 4 mm of the mat. Exposure to light decreased the accumulation of acetate and presumably of other fermentation products. The importance of interspecies hydrogen transfer was investigated by comparing fermentation product accumulation at a 65°C site, with naturally high hydrogen levels, and a 55°C site, where active methanogenesis prevented significant hydrogen accumulation. There was a greater relative accumulation of reduced products, notably ethanol, in the 65°C mat.     

Hydrolysis and acidogenic fermentation of gelatin under anaerobic conditions in a laboratory scale upflow reactor

Summary Gelatin was dissolved in a mineral salts medium for growth under carbon limitation and fed to a mixed population of bacteria in a lab-scale upflow reactor for hydrolysis and acidogenic fermentation under anaerobic conditions, at pH=7 and 30°C.With the shortest applied liquid retention time (30 min), 84% of the protein was hydrolysed. The majority (85%) of the hydrolysed protein was fermented. The ammonia concentration in the reactor was about 1,400 mg N·l^-1.The fermentation products were mainly acetate, propionate, and valerate. Iso-butyrate, butyrate, and iso-valerate were formed to a limited extent. Gas production was very low and consisted of carbon dioxide and methane. The product composition was independent of the retention time applied. The sludge formed was slimy. No granules were formed, however a hold-up factor of 46 could be obtained.     

Production of ethanol from cassava whey

Investigations were conducted into the potential use of enzyme hydrolysed cassava whey for ethanol production by Saccharomyces cerevisiae Aspergillus niger grown on whct bran was used as crude enzyme source to saccharify the whey starch. The whey with an initial HCN concentration of 54.0μg/ml was fermented at pH 4.5 and 30°C in a one-step process to produce ethanol. A maximum ethanol concentration of 4.5% (v/v) was obtained in 120 h with a decrease in HCN level to 4.0 μg/ml. In a two-stage fermentation, in which the raw whey was pre-hydrolysed and under the same fermentation conditions, the unsterilized hydrolysate yielded alcohol content of 5.5% (v/v), while the sterilized hydrolysate gave higher alcohol yield, 7.5% (v/v), in 48 h. No HCN was detected in the fermented liquour at the end of the two-stage process.     

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.     

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。