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.     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Starvation response of Saccharomyces cerevisiae grown in anaerobic nitrogen- or carbon-limited chemostat cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h(-1) at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Role of anaerobic spore-forming bacteria in the acidogenesis of glucose: Changes induced by discontinuous or low-rate feed supply

A mineral salts medium containing 1% (w/v) glucose providing carbon-limited growth conditions was subjected to anaerobic acidogenesis by mixed populations of bacteria in chemostat cultures. The formation of butyrate was shown to be dependent on the presence of saccharolytic anaerobic sporeformers in the acid-forming population. By the use of pasteurized activated sludge as an inoculum a culture was obtained consisting solely of anaerobic sporeformers that gave rise to the formation of butyrate, acetate, hydrogen and carbon dioxide as the main fermentation products. No formation of propionate could be detected. In this culture, the role of sporulation was investigated by applying periods of starvation and a single-step lowering of dilution rate (shift-down). In an experiment using a mineral salts medium supplemented with 1% (w/v) glucose and 0.5% (w/v) casein hydrolysate formation of refractile forespores as well as cell lysis could be demonstrated after 6 h starvation.In mixed cultures, initially inoculated with non-pasteurized activated sludge, a regular interruption of feed supply for 1 h per day resulted in selection of non-sporulatiog anaerobes. The fermentation pattern changed to a production of propionate and acetate, with a concomitant reduction of gas production. Similar results were obtained with shift-down in dilution rate.     

Hydrolysis and acidogenic fermentation of a protein,gelatin, in an anaerobic continuous culture

Summary Model studies of anaerobic protein digestion were performed using gelatin dissolved in a mineral medium, which was fed to a mixed population of bacteria in a carbon-substrate limited chemostat culture. The dilution rate and culture pH value were varied progressively in order to determine the optimal conditions for hydrolysis and acidification (i.e., fatty acids formation). The optimum pH value appeared to be in the neutral region (pH>6.3), and the maximal dilution rate allowing steady state growth was 0.23 h^-1. At this dilution rate and at pH 7 hydrolysis of gelatin was 78% complete, and 79% of the protein hydrolysed was fermented to identifiable products. At submaximal dilution rates both these values were higher. The main fermentation products were acetate, propionate, and valerate, and minor amounts of other volatile fatty acids. The product composition was relatively independent of the dilution rate, but varied substantially with the pH value.     

Microcalorimetry: A tool for the study of the biodegradability of straw by mixed bacterial cultures

Summary The biodegradability of straw by a mixed bacterial culture obtained from a pile of weeds was studied by microcalorimetry. All the cultures were grown at 30°C under anaerobic conditions in microcalorimetric vessels. The fermentation thermograms, obtained using well defined conditions, were very reproducible. The quantities of heat produced during straw degradation were found to be proportional to the quantity of straw introduced at the beginning of the fermentation.The recovered carbon was also found to be proportional to the initial quantity of straw. From both microcalorimetric and chemical analysis it was concluded that the limiting factor of the straw degradation was the cellulolytic activity of the mixed culture. This is supported by the fact that commercially available cellulase added to the growth medium increases the amount of straw degradation by about four times. The heat associated with fermentation of each cellulose monomer (C₆H₁₀O₅) was found to be 120 kJ, a value which is close to the heat associated with hexose fermentation by pure cultures. In conclusion, we propose that microcalorimetry can be used as a powerful tool for the analysis of the biodegradability of complex heterogeneous substrate by pure or mixed cultures.     

Significance of partial pre-acidification of glucose for methanogenesis in an anaerobic digestion process

Summary The effect of partial pre-acidification of carbohydrate containing wastewaters on anaerobic digester performance was investigated. The influent was a 1% (w/v) glucose solution in a mineral salts medium imposing carbon-limited growth conditions. Up to 13% of the Chemical Oxygen Demand (COD) was added as volatile fatty acids (VFA).In all cases, addition of VFA to the glucose medium resulted in significant increases in the maximum specific COD-conversion rates of the sludge (both with respect to continuous feeding and following a shock loading), as compared with values found on digestion of glucose media alone.     

Influence of anaerobic culture acclimation on the degradation kinetics of various substrates

The adaptation of an anaerobic culture (anaerobic sludge) to a specific substrate brings significant changes to its microbial population. These changes can be described by the sludge's ability to treat various substrates such as carbohydrates or proteins or "intermediate" products of anaerobic metabolism such as L-lactic, propionic, and acetic acids. The activity of the sludge with respect to a specific substrate is a critical parameter, because the anaerobic degradability of wastewaters depends strongly on it. This work examines and quantifies the differentiation of two anaerobic sludges of the same origin, following an adaptation period of about 18 months to lactose and gelatin, respectively. The acclimation has a significant effect on the maximum specific utilization rates of various compounds and on their apparent consumption kinetics. It is noticeable, however, that even if the anaerobic cultures were not exposed to a specific substrate for a prolonged period of time (more than a year), they still kept the ability of hydrolyzing or degrading it. In addition, the acclimation has an unquestionable effect on the stoichiometry of the production of volatile fatty acids and L-lactate. Finally, from codigestion experiments it is shown that codigestion of lactose and gelatin appears to have no effect on their hydrolysis kinetics in any of the lactose or gelatin acclimated cultures; specifically, the hydrolysis kinetics remained the same as calculated when lactose or gelatin were the only fed substrates. Similarly, the kinetics of L-lactate and D-glucose biodegradation seemed to be unchanged. On the other hand, codigestion has a significant effect on the production of L-lactic, propionic, and acetic acids, which can be attributed to the increased hydrogen production accompanying gelatin biodegradation.     

Pulsed feeding during fed-batch Aspergillus oryzae fermentation leads to improved oxygen mass transfer

Productivity in many fungal fermentations is detrimentally affected by high broth viscosity and consequent reduced oxygen mass transfer capacity. The goal here was to determine whether pulsed feeding of limiting carbon in a fungal fermentation could lead to reduced viscosity and improved oxygen mass transfer. As a model, an industrially relevant recombinant strain of Aspergillus oryzae was grown in carbon-limited, fed-batch mode. Maltodextrin was used as a carbon source and was added either continuously or in 1.5-min pulses, 3.5 min apart. In both feeding modes the same total amount of carbon was added, and carbon feed rate was at sufficiently low levels to ensure cultures were always carbon-limited. Compared to continuous feeding, pulsed addition of substrate led to smaller fungal elements, which resulted in a significant reduction in broth viscosity. This in turn led to higher dissolved oxygen concentrations and increased oxygen uptake rates during pulsed feeding.     

Simultaneous assay of dihydroxyacetone synthase and transketolase in a methylotrophic yeast grown in continuous culture. A cautionary note

The methylotrophic yeast Candida boidinii CBS 5777 was grown in continuous culture under carbon limitation on glucose, glucose plus methanol, and methanol as carbon and energy sources. During adaptation from glucose to methanol there was a rapid rise in the specific activities of triokinase, fructose-1,6-bisphosphatase and dihydroxyacetone synthase, which are key enzymes of the xylulose phosphate cycle of formaldehyde fixation. The specific activity of classical transketolase fell during this adaptation. Extracts from carbon-limited C. boidinii contained an enzyme which catalysed oxidation of NADH when some preparations or ribose 5-phosphate were added, which was not a transketolase. This enzyme activity was dependent on an impurity in such ribose 5-phosphate preparations and can be confused with transketolase activity.     

Aerobic and Anaerobic Starvation Metabolism in Methanotrophic Bacteria

The capacity for anaerobic metabolism of endogenous and selected exogenous substrates in carbon- and energy-starved methanotrophic bacteria was examined. The methanotrophic isolate strain WP 12 survived extended starvation under anoxic conditions while metabolizing 10-fold less endogenous substrate than did parallel cultures starved under oxic conditions. During aerobic starvation, the cell biomass decreased by 25% and protein and lipids were the preferred endogenous substrates. Aerobic protein degradation (24% of total protein) took place almost exclusively during the initial 24 h of starvation. Metabolized carbon was recovered mainly as CO(inf2) during aerobic starvation. In contrast, cell biomass decreased by only 2.4% during anaerobic starvation, and metabolized carbon was recovered mainly as organic solutes in the starvation medium. During anaerobic starvation, only the concentration of intracellular low-molecular-weight compounds decreased, whereas no significant changes were measured for cellular protein, lipids, polysaccharides, and nucleic acids. Strain WP 12 was also capable of a limited anaerobic glucose metabolism in the absence of added electron acceptors. Small amounts of CO(inf2) and organic acids, including acetate, were produced from exogenous glucose under anoxic conditions. Addition of potential anaerobic electron acceptors (fumarate, nitrate, nitrite, or sulfate) to starved cultures of the methanotrophs Methylobacter albus BG8, Methylosinus trichosporium OB3b, and strain WP 12 did not stimulate anaerobic survival. However, anaerobic starvation of these bacteria generally resulted in better survival than did aerobic starvation. The results suggest that methanotrophic bacteria can enter a state of anaerobic dormancy accompanied by a severe attenuation of endogenous metabolism. In this state, maintenance requirements are presumably provided for by fermentation of certain endogenous substrates. In addition, low-level catabolism of exogenous substrates may support long-term anaerobic survival of some methanotrophic bacteria.     

碳源对重组大肠杆菌两阶段发酵产丁二酸的影响

研究了在好氧培养基中分别添加不同碳源对两阶段发酵菌体生长、酶活及代谢产物分布的影响,结果表明添加4mmol/L葡萄糖和12,54,80mmol/L乙酸钠均可以提高好氧阶段的菌体密度和相关酶活。将不同条件下培养的菌体转接厌氧发酵后,厌氧阶段的酶活和代谢产物分布也发生改变。进一步对酶活及代谢产物分析表明：Escherichia coli NZN111(sfcA)厌氧发酵过程中,磷酸烯醇式丙酮酸羧化激酶(PCK)是产丁二酸的关键酶,丙酮酸激酶(PYK)主要和副产物丙酮酸的积累有关,异柠檬酸裂解酶(ICL)对丁二酸产量也有一定影响。好氧培养基中添加80mmol/L乙酸钠,厌氧发酵结束时丁二酸的质量收率可达89.0%,相比对照提高了16.6%。     

树干毕赤酵母酒精发酵碳元素代谢流向分析

实验对树干毕赤酵母（Pichia stipitis）进行了4个阶段共400 h连续恒化培养,在不同阶段以30.0 g/L葡萄糖作为基本碳源,添加30.0或15.0 g/L的木糖,通过控制温度（35±1）℃,进气量100～150mL/min,搅拌转速250～300 r/min。4个阶段共建立4个连续培养的"稳态"。对碳元素进行物料衡算发现,四个阶段碳元素回收率分别为118.0 %、105.6 %、113.5 %和94.7 %。对4个近似"稳态"的碳元素的代谢流向进行分析发现：将近50.0 %左右碳元素流向产物酒精,其次是CO2和酵母细胞;木糖醇浓度与流入底物中木糖浓度有直接关系,在相同发酵条件下流入的木糖浓度越大代谢生成木糖醇浓度也越高;实验所采用的通气条件更适合底物为30.0 g/L葡萄糖和30.0 g/L木糖混合液的连续发酵。     

Enhanced ethanol production through selective adsorption in bacterial fermentation

To alleviate the ethanol inhibition of Escherichia coli KO11 (ATCC 55124), during fermentation, online ethanol sequestration was achieved using F-600 activated carbon. Two separate schemes were tested, one involving direct addition of activated carbon to the fermentation flask for the purpose of in-situ adsorption and a second involving an externally located activated carbon packed bed. For the in-situ ethanol adsorption experiments, varying amounts of adsorbent were added to the medium at the start of the fermentation. The addition of the activated carbon in the fermentation broth resulted in increased glucose utilization and ethanol production for all flasks containing activated carbon. For the control flasks, approximately 75% of the available substrate was utilized before the fermentation was inhibited. The entire glucose supply of flasks containing activated carbon was depleted. Ethanol production was also increased from 28 g/L for the control containing no activated carbon to nearly 45 g/L (including the ethanol in the adsorbed phase) for the flasks containing activated carbon. The implementation of an externally located packed bed adsorber for the purpose of on-line ethanol removal was tested over a number of adsorption cycles to evaluate the performance of the adsorption bed and the ethanol productivity. Results indicate that maintaining ethanol fermentation medium concentrations below 20 ∼ 30 g/L extends and enhances ethanol productivity. After 3 cycles over a period of 180 h, an additional 80% ethanol was produced when compared to the control experiments, despite the suboptimal acidic pH of the medium.     

Anaerobic homolactate fermentation with Saccharomyces cerevisiae results in depletion of ATP and impaired metabolic activity

Conversion of glucose to lactic acid is stoichiometrically equivalent to ethanol formation with respect to ATP formation from substrate-level phosphorylation, redox equivalents and product yield. However, anaerobic growth cannot be sustained in homolactate fermenting Saccharomyces cerevisiae . ATP-dependent export of the lactate anion and/or proton, resulting in net zero ATP formation, is suspected as the underlying cause. In an effort to understand the mechanisms behind the decreased lactic acid production rate in anaerobic homolactate cultures of S. cerevisiae , aerobic carbon-limited chemostats were performed and subjected to anaerobic perturbations in the presence of high glucose concentrations. Intracellular measurements of adenosine phosphates confirmed ATP depletion and decreased energy charge immediately upon anaerobicity. Unexpectedly, readily available sources of carbon and energy, trehalose and glycogen, were not activated in homolactate strains as they were in reference strains that produce ethanol. Finally, the anticipated increase in maximal velocity ( V _max) of glycolytic enzymes was not observed in homolactate fermentation suggesting the absence of protein synthesis that may be attributed to decreased energy availability. Essentially, anaerobic homolactate fermentation results in energy depletion, which, in turn, hinders protein synthesis, central carbon metabolism and subsequent energy generation.     

产紫青霉中β-葡萄糖醛酸苷酶的诱导表达

针对产紫青霉(Penicillium purpurogenum)Li-3发酵生产β-葡萄糖醛酸苷酶存在的碳代谢阻遏现象,研究β-葡萄糖醛酸苷酶的高效诱导表达策略。在发酵条件优化的基础上,建立了新的产酶诱导工艺:葡萄糖的初始质量浓度5 g/L,在葡萄糖耗尽时加入20%诱导剂(10 g/L GL+1.2%Tween80)进行诱导,每24 h添加1次诱导剂,诱导72 h后立即转到40℃摇床发酵48 h。采用该工艺进行发酵,菌体出现了"二次生长"现象,比酶活从647.99 U/mL提高至2 356 U/mL,提高了近3倍。     

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.     

A method for the selective enumeration and isolation of ruminal Lactobacillus and Streptococcus

Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.     

Effects of glucose on phenol biodegradation by heterogeneous populations

The effect of the presence of more easily degradable alternative carbon sources on the biodegradation of toxic waste components is of great practical importance. In this work, a mixed phenol/glucose waste was fed to two heterogeneous populations acclimated to different conditions: one was acclimated to phenol as a sole source of carbon and one to a mixed phenol/glucose substrate. Batch substrate utilization experiments were performed under both growth and nonproliferating (no medium nitrogen source) conditions in order to assess substrate removal patterns at the levels of enzyme production and enzyme function. The results indicated that the substrate removal pattern exhibited by the cells was significantly influenced by the acclimation characteristics of the culture. The phenol acclimated cells showed an initial preference for phenol, but the presence of glucose hindered phenol removal rate under both growth and nonproliferating conditions. The cells acclimated to the mixed phenol/glucose waste demonstrated rapid initial glucose removal with a slower concomitant utilization of phenol; acclimation to the mixed waste evidently had a significant impact on the substrate removal pattern for this mixed substrate system.     

Multiple-carbon-source-limited growth kinetics of a marine coryneform bacterium.

The steady-state growth rate of a marine isolate was related to the concentrations of several carbon and energy source substrates when these substrates limited growth simultaneously in continuous culture. Glucose limitation was characterized by a threshold of 0.21 mg/liter for growth, a half-maximal growth rate at 0.48 mg/liter, U-shaped curves in extractable pool concentration-versus-growth velocity plots, and slow maximal growth rates. Arginine addition reduced the glucose threshold to 0.008 mg/liter, more than doubled the maximal growth rate, and stabilized pool concentrations at low growth rates. Addition of a third substrate, glutamate, caused further reduction of the glucose concentration a steady state. Maximal reduction of the glucose concentration was effected by adding a mixture of 20 amino acids. Steady-state limiting nutrient concentration was dependent on the specific identity of the auxiliary nutrients and on the concentration ratio at which they were supplied. When glucose was supplemented with an equal quantity of an amino acid mixture, the external steady-state glucose remained below 10 mug/liter. When 1 mug of glucose was added to a 2.5-mg/liter amino acid mixture, at least 70% of it was consumed at steady state in spite of the threshold observed. Lack of crossover between metabolic pathways, suggested by the absence of glucose carbon in pool glutamate of arginine-glucose-grown cells, may have been partly responsible for the mixed carbon source stimulation of nutrient accumulation observed. The affinity observed is sufficient to account for normal growth at a total organic substrate concentration of only 0.11 mg/liter when supplied from a suitable mixture.