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.     

Protein degradation in anaerobic digestion: influence of volatile fatty acids and carbohydrates on hydrolysis and acidogenic fermentation of gelatin

Summary The hydrolysis and fermentation of gelatin in the presence of a carbohydrate by gelatin-adapted mixed anaerobic bacterial populations in putatively carbon-limited chemostat cultures is investigated. It was shown that the degradation of the protein is progressively retarded with increasing dilution rates, as well as with increased concentrations of carbohydrates present in the feed as a second substrate. That this is not due to high concentrations of fermentation products in the reactor was established. Moreover, the carbohydrate is totally fermented at all dilution rates. It is concluded that for optimal performance of an anaerobic digestion system purifying waste waters containing carbohydrate/protein mixtures, fermentation of carbohydrates should be spatially separated from hydrolysis and fermentation of the protein.     

Characterization of anaerobic microbial culture with high acidogenic activity

The mixed cultures which were used were isolated from municipal sludge digesters, and the production of organic acids (acetic, propionic, butyric, etc.) from carbohydrates was tested. The behavior of the reference population (culture R) obtained directly from the sewage treatment plant, is compared to that obtained after three months in a plug-flow reactor (Gradostat fermentor) without pH control (culture A) and after six months with pH control (culture B). For culture B, the specific rate of acid production is related to the cell growth rate by (1/X)r_p= 17 µ + 1.6 with a maximal acid concentration of 40 g/liter. The batch culture yields are improved from 0.36g/g for the initial culture (R) to 0.72 g/g for culture B after six months in continuous culture, and 0.8 g/g in plug-flow continuous culture. The productivity of organic acids reaches 1.7 g/liter·hr. It is suggested that the acidogenic fermentation, the first step of methanogenesis, is a potential process to produce acetic, propionic, and butyric acids.     

污泥厌氧消化中同型产乙酸菌富集研究

在市政污泥厌氧消化过程中,采用三阶段选择性富集同型产乙酸菌,通过监测培养过程中pH值、挥发性脂肪酸、关键酶的活性以及不同碳源利用率等,研究同型产乙酸菌在系统中的累积情况.实验数据表明:经过40 d的富集培养,pH值稳定在8.0,乙酸含量逐渐趋于稳定,占COD的比例为46.4％,同型产乙酸菌对底物的利用率达到了87％,乙酸激酶和乙酰磷酸转移酶呈现明显上升的趋势.结果显示,通过本方法可在市政污泥中有效地富集同型产乙酸菌.     

Characterization of aerobic,facultative anaerobic,and anaerobic bacteria in an acidogenic phase reactor and their metabolite formation

Fifty-two aerobic and facultative anaerobic and 57 anaerobic bacterial isolates were obtained from an acidogenic phase digestion system. These isolates were characterized and the similarities between the different strains were calculated using Sokal and Michener's similarity coefficient. The aerobic and facultative anaerobic strains clustered in two major groups with the strains of the first main group being gram-negative fermentative rods, representing the generaKlebsiella, Enterobacter, Escherichia andAeromonas. Isolates of the second group were gram-positive streptococci similar toStreptococcus lactis. The strict anaerobic isolates also clustered into two main groups with strains of cluster A being identified as members of the genusFusobacterium while strains in cluster B were members of the genusBacteroides. Hypothetical mean organisms were calculated for each cluster and used in further culture studies. The major products of the continuously fed acidogenic phase reactor were ethanol and acetic, propionic, and butyric acids. In batch cultures, ethanol, acetic acid, diacetyl, and 2,3-butanediol were formed by the strains as major products both under aerobic and anaerobic conditions. The ability of the aerobic and facultative anaerobic strains to be metabolically active under anaerobic conditions indicates a prominent role in acidogenic reactors.     

Improving acidogenic performance in anaerobic degradation of solid organic waste using a rotational drum fermentation system

The effects of leachate from a methanogenic process on acidogenic performance of a solid recycle (SR) process by a rotational drum fermentation (RDF) system were evaluated under mesophilic condition and a hydraulic retention time (HRT) of 20 days. Two SR process configurations, SR1 and SR2, were evaluated, using fresh soybean meal or Okara as substrates. An apparent first-order hydrolysis rate constant of 5.0 x 10(-3)/d for SR1 at pH values of 4.4 and 14.4 x 10(-3)/d for SR2 at pH of 5.0, were obtained. The apparent volatile solids (VS) degradation ratio ranged from 9.6% to 19.4% and total volatile acid (as acetic acid) from 10.8 to 14.9 g/L. Occupying ratios for ionized volatile acid (VA) increased from 30.6% to 63.4% after recycling the leachate to process. However, occupying ratios of acetic acid decreased from 93.3% to 42.0% whereas propionic acid and butyric acid ratios increased in SR2. Integrating the VA production with the hydrolysis rate constants, it is clear that the recirculation of leachate considerably enhanced acidogenic performance of solid recycle process.     

Methanogenic and acidogenic activity test in an anaerobic thermophilic reactor

Summary The experimental conditions selected (addition to assay medium of micronutrients, macronutrients and pH control), the experimental design and the procedure for maximum acidogenic and methanogenic activity determination tests in thermophilic biomass, are presented. The proposed tests are highly reproducible and can be carried out in a short space of time. Specific tests are applied to measure the maximum acidogenic and methanogenic activities in lab-scale anaerobic reactors treating wine-distillery wastes in the thermophilic range (55°C).     

Effect of different inoculum levels of heterogeneous mixed culture in acidogenic fermentation

Summary Acidogenesis during fermentation by a heterogeneous mixed population inoculated at different initial biomass levels was studied. Butyric and lactic acid production and biomass formation were measured. Acidogenesis appeared to be a two step process, lactic acid being produced first, followed by butyric. Fermentation started more rapidly with higher levels of initial biomass, after which the fermentation rate remained unaffected indicating a change in the fermentation profile     

Study of the acidogenic phase of the anaerobic fermentation of stillage from ethanol distilleries

Summary A bench scale continuously stirred reactor was used to study the acidogenic phase of the anaerobic fermentation of stillage. The residence time of the effluent in the reactor ranged from 15.7 to 8.2 hours, pH was around 5.0 and temperature was maintained at 35°C. The results indicate that the residence time had no appreciable effect on the production or composition of the organic acids. The main acid products found in the reactor effluent were acetic, propionic and butiric acids.     

Trophic link between syntrophic acetogens and homoacetogens during the anaerobic acidogenic fermentation of sewage sludge

Acetate production during anaerobic sludge treatment has significant economic and environmental benefits. In this study, trophic links between syntrophic acetogens and homoacetogens in the anaerobic acidogenic fermentation of sewage sludge were investigated using methanogenic inhibitor 2-bromoethanesulfonate (BES) to block the methanogenesis pathway and butyrate to enhance syntrophic acetogenesis. The Gibbs free energies (ΔG) of the butyrate-degrading and homoacetogenic processes were close to the thermodynamic threshold of the reaction activity (−15 kJ/mol). In addition, microbial quantification analysis revealed that the growth of syntrophic acetogenic bacteria and homoacetogens in the treatment incubations was higher than that of the control. The results indicated that hydrogen-producing butyrate degraders are stimulated with homoacetogens when methanogenesis was specifically inhibited.     

Influence of whey protein on continuous acidogenic degradation of lactose

In previous studies on the acidogenic phase of anaerobic fermentation of lactose, a pathway for the reaction and a rate equation have been proposed. The question then remained as to the effect of the protein in whole whey on the mechanism and on the overall organic substrate conversion. In this study, it was found that as much as 70% of the protein was broken down in the acidogenic reactor. Radiotracer tests showed that the inclusion of protein had no effect on the reaction pathway for lactose degradation. Thus, the whole sweet cheese whey can be fermented as efficiently as whey from which the protein has been removed.     

Inhibition of the acidogenic dissimilation of glucose in anaerobic continuous cultures by free butyric acid

Summary In anaerobic wastewater treatment the separation of fermentative and methanogenic bacteria is aimed at an increased performance of the total digestion process. It is known that the attainable growth rate of the acidogenic population in continuous culture decreases at increasing influent concentrations of glucose. To account for this phenomenon, a new kinetic model was developed that combines substrate and product inhibition. In the present research product inhibition was investigated quantitatively in a continuous culture fermenting 50 mmol/l glucose. Extra acetate and butyrate were added up to 200 mmol/l at different pH values, and it turned out that only free butyric acid inhibited growth. The lower attainable growth rates of cultures producing comparable amounts of butyrate when fed with concentrated influents, strongly indicated substrate inhibition. Evidence is presented that transitions to low-conversion steady states predicted by the kinetic model, play a role and decrease the stability of the culture.Nomenclature D dilution rate, h^-1 - D_att highest D using certain experimental procedure h^-1 - K_i substrate inhibition constant, mol·m^-3 - K_p product inhibition constant mol·m^-3 - K_s substrate saturation constant, mol·m^-3 - P concentration inhibitory product mol·m^-3 - S substrate concentration, mol·m^-3 - S_o influent substrate concentration, mol·m^-3 - S _max ^c substrate concentration at _max ^c , mol·m^-3 - S _max ^h substrate concentration at _max ^h , mol·m^-3 - specific growth rate, h^-1 - experimental realization of at D_att, h^-1 - _max maximum specific growth rate, h^-1 - _max ^c maximum attainable specific growth rate according to combined substrate/product inhibition model, h^-1 - _h ⁰ specific growth rate at S₀ according to Haldane kinetics, h^-1 - _max ^c maximum attainable specific growth rate according to Haldane kinetics, h^-1 - Y_p yield inhibitory product, mol·mol^-1 - Y_x yield biomass, kg dry weight·kg^-1 - bio biomass - EtOH ethanol - gluc glucose - HAc acetate - HBt butyrate - HCap caproate - HFo formate - HPr propionate - HVal valerate - prod produced - lact lactate     

Heterolactic fermentation by a homofermentative Lactobacillus sp. during glucose limitation in anaerobic continuous culture with complete cell recycle

E. BORCH, H. BERG AND O. HOLST. 1991. The homofermentative Lactobacillus sp. 93 SMRICC 235 was grown anaerobically in batch culture and subsequent continuous culture, with complete cell recycle at pH 6.0 and 25^.C, on a semi-defined medium. During cell recycle culture the biomass was concentrated in the fermenter to a final dry weight of 37 g/1 and a viable count of 10.6 log cfu/ml. The corresponding final values in batch culture were 2.4 g/1 and 9.3 log cfu/ml. A switch from homo- to heterolactic fermentation was observed during starvation, due to glucose depletion in the cell recycle culture. High levels of acetate and formate were produced in addition to ethanol. Amino acid profiles of hydrolysed samples showed extensive utilization of amino acids, particularly in cell recycle culture. Sulphide was produced during cell recycle culture. The change from homo- to heterolactic fermentation observed during semi-starvation is likely to affect the properties of lactobacilli as spoilage bacteria of meat and meat products, as well as starter cultures.     

Growth and genetic responses of Salmonella Typhimurium to pH-shifts in an anaerobic continuous culture

Salmonella infection of chickens that leads to potential human foodborne salmonellosis continues to be a concern. Changes in the pH of poultry gastrointestinal tract could influence Salmonella growth and virulence response. In the current study, growth responses of a chicken isolate Salmonella enterica serovar Typhimurium (ST) to three incremental pH-shifts (6.17-7.35) in continuous cultures (CC) were evaluated. The expression of rpoS and hilA was determined by real time-polymerase chain reaction (RT-PCR) as well. Increases in pH resulted in higher cell protein concentrations, glucose disappearance, and glucose and ATP yields. Although with some inconsistency between the two trials, the data indicated that the ammonia release into media was favored by low pH. The pH shifts did not significantly affect acetate biosynthesis. No consistent trends of pH influence on propionate and butyrate production could be detected. In all three pH shifts, relative expression of hilA was dominant at 0h which represented CC steady state. In pH shift 7.35-6.86 (Trial 1), the relative expression of rpoS at time 0 and 1h were over five-fold higher than after 3 and 6h of growth. Overall, the results suggest that ST physiology is altered by changes in pH, which could be determinant factors for ST survival in the poultry gastrointestinal ecosystems.     

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.     

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.     

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.