Isolation of hydrogen-producing bacteria from granular sludge of an upflow anaerobic sludge blanket reactor

H₂-producing bacteria were isolated from anaerobic granular sludge. Out of 72 colonies (36 grown under aerobic conditions and 36 under anaerobic conditions) arbitrarily chosen from the agar plate cultures of a suspended sludge, 34 colonies (15 under aerobic conditions and 19 under anaerobic conditions) produced H₂ under anaerobic conditions. Based on various biochemical tests and microscopic observations, they were classified into 13 groups and tentatively identified as follows: From aerobic isolates,Aeromonas spp. (7 strains),Pseudomonas spp. (3 strains), andVibrio spp. (5 strains); from anaerobic isolates,Actinomyces spp. (11 strains),Clostridium spp. (7 strains), andPorphyromonas sp. When glucose was used as the carbon substrate, all isolates showed a similar cell density and a H₂ production yield in the batch cultivations after 12h (2.24–2.74 OD at 600 nm and 1.02–1.22 mol H₂/mol glucose, respectively). The major fermentation by-products were ethanol and acetate for the aerobic isolates, and ethanol, acetate and propionate for the anaerobic isolates. This study demonstrated that several H₂ producers in an anaerobic granular sludge exist in large proportions and their performance in terms of H₂ production is quite similar.     

The effect of CO2 ventilation on kinetics and yields of cell-mass and ethanol in batch cultures ofZymomonas mobilis

Summary The effect of CO₂ removal by continuous sparging of N₂ in batch cultures ofZymomonas mobilis (ATCC10988) was examined. N₂ sparging considerably reduces lag times in batch cultures, possibly because of continuous removal of CO₂ from the culture media. Ventilation of CO₂ from culture media results in an increase of about 15% in the average specific growth rate and about 12% in the cell-mass yield with no noticeable trend in the average specific glucose uptake and ethanol production rates. The overall ethanol yield on glucose, however, decreases slightly by 5%. Analysis of ventilated experiments show that the CO₂ production is directly coupled with the ethanol formation but not necessarily with the cell-mass production, indicating a decoupling of growth from ethanol production. Further, comparison of ventilated and non-ventilated experiments rules out the possibility of CO₂ accumulation in the culture media as a factor responsible for increasing growth inhibition and decoupling of growth from ethanol fermentation at increasing initial glucose concentrations in batch cultures.     

Fed‐Batch Process for Pyruvate Production by Recombinant Escherichia coli YYC202 Strain

Fed‐batch fermentation was applied to the production of pyruvate by using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate into acetyl‐CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. By controlling acetate and glucose feed rate, a series of lab‐scale fed‐batch experiments were performed at pH 7 and 37 °C. CO₂ production rate (CTR) was used for on‐line regulation of the acetate feed rate. The correlation between CTR and acetate consumption rate (ACR) was determined experimentally. At optimal process conditions a final pyruvate concentration higher than 62 g/L, a space‐time yield of up to 42 g/L/d and pyruvate/glucose molar yield of 1.11 mol/mol were achieved. Experimental evidence was gathered that pyruvate export is active.     

Microcalorimetric investigations of the metabolism of yeasts VII. Flow-calorimetry of aerobic batch cultures

Summary The heat evolution of aerobic batch cultures of growing yeast (Saccharomyces cerevisiae) in glucose media was investigated by a combination of a flow-microcalorimeter with a fermentor vessel. The course of heat production, cell production and the rate of oxygen consumption were qualitatively the same for all glucose concentrations between 10 mM and 100 mM. Under optimal aerobic conditions a triphasic growth was observed due to the fermentation of glucose to ethanol, respiration of ethanol to CO₂ and acetate, and respiration of acetate to C0₂. Energy and carbon were found to be in balance for all glucose concentrations.     

An on-line technique for monitoring propionic acid fermentation

An on-line technique, based on measuring the increase in pressure due to CO₂ release in a closed air-tight reactor, was used to evaluate the fermentation of lactate by propionibacteria. The method was applied to batch cultures of Propionibacterium shermanii grown in yeast extract/sodium lactate medium containing lactate as a carbon source under micro-aerophilic conditions. Gas pressure evolution was compared both with substrate consumption and metabolites production and with acidification and growth. Linear relationships were found between gas pressure variation, lactate consumption and propionate and acetate production. The technique also enabled the evaluation of total CO₂ produced, by taking account of pressure, oxygen and pH measurements. These results tend to show that this simple and rapid method could be useful to monitor propionic acid bacteria growth.     

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature,oxygen, and pH on hydrogen production

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H₂) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H₂ production at various growth temperatures, (2) investigate the effect of oxygen (O₂) on H₂ production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85°C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H₂ production reached maximum levels in both the 77°C and 85°C experiments. The fermentation stoichiometry for T. neapolitana at 85°C was 3.8 mol H₂, 2 mol CO₂, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H₂ production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H₂ through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

High production of 1,3-propanediol from glycerol by Clostridium butyricum VPI 3266 in a simply controlled fed-batch system

Summary A simple fed-batch system which controls substrate feeding by measuring the CO₂ produced during the fermentation, was developped. This Fed-batch approach allowed high production of 1,3-propanediol from glycerol by Clostridium butyricum by avoiding substrate inhibition phenomena. 65 g/l of 1,3-propanediol was produced with a productivity of 1.21 g/l.h and a yield of 0.56. The concentration of 1,3-propanediol obtained and the productivity were significantly higher than those reached in batch culture.     

Whey Fermentation by Anaerobiospirillum succiniciproducens for Production of a Succinate-Based Animal Feed Additive

Anaerobic fermentation processes for the production of a succinate-rich animal feed supplement from raw whey were investigated with batch, continuous, and variable-volume fed-batch cultures with Anaerobiospirillum succiniciproducens. The highest succinate yield, 90%, was obtained in a variable-volume fed-batch process in comparison to 80% yield in a batch cultivation mode. In continuous culture, succinate productivity was 3 g/liter/h, and the yield was 60%. Under conditions of excess CO₂, more than 90% of the whey-lactose was consumed, with an end product ratio of 4 succinate to 1 acetate. Under conditions of limited CO₂, lactose was only partially consumed and lactate was the major end product, with lower levels of ethanol, succinate, and acetate. When the succinic acid in this fermentation product was added to rumen fluid, it was completely consumed by a mixed rumen population and was 90% decarboxylated to propionate on a molar basis. The whey fermentation product formed under excess CO₂, which contained mainly organic acids and cells, could potentially be used as an animal feed supplement.     

Enhancement of yeast ethanol tolerance by calcium and magnesium

Ethanol-induced changes of CO₂ production were compared in three strains ofSaccharomyces cerevisiœ. CaCl₂ and MgCl₂ exerted protective effects against the action of ethanol. Optimal concentrations ensuring maximum of CO₂ production at 10% (V/V) of ethanol under non-growing conditions were 3 mmol/L Ca²⁺ and 2 mmol/L Mg²⁺. Yeast growth with and without ethanol addition was stimulated by Mg²⁺ more than by Ca²⁺ during fermentation, whereas ethanol production was more efificient when both Ca²⁺ and Mg²⁺ were added.     

RESPONSES OF THE ACIDOPHILIC ALGA EUGLENA MUTABILIS (EUGLENOPHYCEAE) TO CARBON ENRICHMENT AT pH 31

A defined medium (MAM) simulating acid mine drainage waters was developed which supported reproducible growth rates of three axenic strains of Euglena mutabilis Schmitz. Growth responses to various pHs and carbon sources were examined under defined culture conditions. A lab strain and two 5eld isolates, tested over pH range 1.5-9.0, grew best under acidic conditions (pH < 5.5) with highest growth rates at pH 3-4. Photoauxotrophic growth rates of all strains at pH 3 were improved significantly over unstirred batch controls by bubbling with air and even more by enrichment with 5% CO₂ in air. These results confirmed inorganic carbon limitation in batch culture. Organic carbon substrates were tested as possible carbon supplements in batch culture at pH 3. None of the strains survived in the dark on any of the twenty organic sources added. In the light, the lab strain exhibited some photoheterotrophic growth potential on glucose, sucrose, ethanol, and amino acids but growth was inhibited by acetate. Field strains showed little or no growth improvement with any organic substrate addition. Under simultaneous enrichment with acetate and 5% CO₂ acetate continued to be inhibitory. Simultaneous enrichment with glucose and 5% CO₂ gave higher yields of the lab strain than with CO₂ alone but did not enhance growth of the field strain. We conclude that E. mutabilis is an acidophilic photoauxotroph which appears unable to use organic carbon supplements for growth even under conditions of carbon limitation.     

Characterization of Growth and Metabolism of Commercial Strains of Propionic Acid Bacteria by Pressure Measurement

Dairy propionibacteria are essential starters for Emmental cheese manufacture. The behavior of three commercial strains of Propionibacterium freudenreichii subsp. shermanii (P.f. 1, P.f. 2 and P.f 3) were studied in a liquid medium under air and N₂ atmosphere using an on‐line pressure measurement technique. Growth kinetics and metabolite production were characterized under conditions usually reported as “optimal conditions” (pH 6.5, NaCl 0 %, temperature 30 °C) and also evaluated under “stressful conditions” (pH 5.2, NaCl 2 %, temperature 20 °C) simulating the cheese ripening conditions. In both cases, the effects of oxygen on growth were strain‐dependent. Under “stressful conditions”, two of the three strains were inhibited by oxygen under conditions of air atmosphere, while all three strains grew under conditions of N₂ atmosphere. In the latter case, the duration of the lag phase and the maximum rate of pressure variation were significantly different, however, no significant differences were found between the strains with regard to the total fermentation time. Under “optimal conditions” metabolite production was strain‐dependent. In an air atmosphere, all strains produced more acetate and CO₂ and less propionate than in a nitrogen atmosphere.     

The influence of H2, CO2 and dilution rate on the continuous fermentation of acetone-butanol

Summary The effect of product gases, H₂ and CO₂, on solvent production was studied using a continuous culture of alginate-immobilized Clostridium acetobutylicum. Initially, in order to find the optimum dilution rate for aceton--butanol production in this system, fermentations were carried out at various dilution rates. With 10% H₂ and 10% CO₂ in the sparging gas, a dilution rate of 0.07 h^–1 was found to maximize volumetric productivity (0.58 g·l^–1·h^–1), while the maximum specific productivity of 0.27 g^–·h^–1 occured at 0.12 h^–1. Continuous cultures with vigorous sparging of N₂ produced only acids. It was concluded that in the case of continuous fermentation H₂ is essential for good solvent production, although good solvent production is possible in an H₂-absent environment in the case of batch fermentations. When the fermentation was carried out at atmospheric pressure under H₂-enriched conditions, the presence of CO₂ in the sparging gas did not slow down glucose metabolism; rather it changed the direction of the phosphoroclastic reaction and as a result increased the butanol/acetone ratio.     

Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large‐scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal‐derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal‐derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design‐of‐experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two‐fold increase in OD₆₀₀ compared to YEB medium during a 4‐L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal‐derived components, thus facilitating the GMP‐compliant large‐scale transient expression of recombinant proteins in plants.     

Carbon dioxide inhibition of yeast growth in biomass production

Saccharomyces cerevisiae was grown under aerobic and substrate-limiting conditions for efficient biomass production. Under these conditions, where the sugar substrate was fed incrementally, the growth pattern of the yeast cells was found to be uniform, as indicated by a constant respiratory quotient during the entire growing period. The effect of carbon dioxide was investigated by replacing portions of the nitrogen in the air stream with carbon dioxide, while maintaining the oxygen content at the normal 20% level, so that identical oxygen transfer rate and atmospheric pressure were maintained for all experiments with different partial pressures of carbon dioxide. Inhibition of yeast growth was negligible below 20% CO₂ in the aeration mixture. Slight inhibition was noted at the 40% CO₂ level and significant inhibition was noted above the 50% CO₂, level, corresponding to 1.6 × 10^?2M of dissolved CO₂ in the fermentor broth. High carbon dioxide content in the gas phase also inhibited the fermentation activity of baker's yeast.     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Influence of the curing of the killer phenotype inSaccharomyces cerevisiae wine strains on their fermentative behaviour

Fermentative behaviour and cell growth have been studied in grape juice inoculated either with two killerSaccharomyces cerevisiae wild strains or with their Acridine Orange-cured isogenic counterparts. The number of viable cells/ml at the beginning of the fermentation, as well as during exponential growth, were higher in grape juices inoculated with the cured strains. The CO₂ production, fermentative rate and ethanol and acetic acid production were also higher in the cured strains, particularly during the stage of active fermentation. These differences, however, were minimal at the end of the fermentations.     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...     

Enhanced fermentative capacity of yeasts engineered in storage carbohydrate metabolism

During yeast biomass production, cells are grown through several batch and fed‐batch cultures on molasses. This industrial process produces several types of stresses along the process, including thermic, osmotic, starvation, and oxidative stress. It has been shown that Saccharomyces cerevisiae strains with enhanced stress resistance present enhanced fermentative capacity of yeast biomass produced. On the other hand, storage carbohydrates have been related to several types of stress resistance in S. cerevisiae. Here we have engineered industrial strains in storage carbohydrate metabolism by overexpressing the GSY2 gene, that encodes the glycogen synthase enzyme, and deleting NTH1 gene, that encodes the neutral trehalase enzyme. Industrial biomass production process simulations were performed with control and modified strains to measure cellular carbohydrates and fermentation capacity of the produced biomass. These modifications increased glycogen and trehalose levels respectively during bench‐top trials of industrial biomass propagation. We finally show that these strains display an improved fermentative capacity than its parental strain after biomass production. Modification of storage carbohydrate content increases fermentation or metabolic capacity of yeast which can be an interesting application for the food industry. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:20–24, 2015     

蝉拟青霉液体发酵条件优化

采用液体发酵蝉拟青霉,对蝉拟青霉的发酵条件进行优化,以提高蝉拟青霉胞外多糖产量及生物量。摇瓶发酵条件下,在单因素基础上设计正交实验确定各因素的最佳组合。优化后得最佳发酵培养基:蔗糖8%,牛肉膏0.75%,酵母膏0.125%,MgSO_4·7H_2O 0.3%,KH_2PO_4 0.2%,麸皮0.5%。该条件下胞外多糖产量为5.96 g/L,生物量为42 g/L,较优化前提高了1倍。采用发酵罐进行扩大培养,对分批发酵时的初糖浓度进行了优化,并分析了补料分批发酵对发酵过程的影响。发酵罐培养时最适初糖浓度为5%,此时生物量最高为38 g/L,多糖含量最高为5.5 g/L;采用补料分批发酵时,多糖产量最高为5.89 g/L,生物量最高为40 g/L,效果优于分批发酵。