Removal of headspace CO2 increases biological hydrogen production

For biological hydrogen production by fermentation to be a useful method of hydrogen generation, molar yields of hydrogen must be increased. While heat treatment of a soil inoculum increases hydrogen yields by preventing loss of hydrogen to methanogenesis, hydrogen is still lost to acetic acid generation from hydrogen and CO2. To reduce hydrogen losses via acetogenesis, CO2 concentrations in the headspace were substantially reduced during hydrogen production using a chemical scavenger (KOH). CO2 in the headspace was decreased from 24.5% (control) to a maximum of 5.2% during the highest gas production phase, resulting in a hydrogen partial pressure of 87.4%. This reduction in CO2 increased the hydrogen yield by 43% (from 1.4 to 2.0 mol of H2/mol of glucose). The soluble byproducts in all tests consisted primarily of acetate and ethanol. Higher concentrations of ethanol (10.9 mM) remained in solution from bottles with CO2 removal than in the control (1.2 mM), likely as a result of hydrogen inhibition of biological ethanol conversion to acetic acid. These results show that hydrogen production can be increased by removing CO2 in the reactor vessel, likely as a result of suppression of acetogenesis.     

Regulation of lactate production in Streptococcus bovis: A spiraling effect that contributes to rumen acidosis

When Streptococcus bovis was grown in batch culture with 6 g/L glucose at pH 6.7, maximum specific growth rate was 1.47 h(-1), and lactate was the primary fermentation product. In continuous culture at pH 6.7 and growth rate equal to .10 h(-1), little lactate was formed, and formate, acetate, and ethanol accounted for most of the product. When extra-cellular pH decreased to 4.7, intra-cellular pH declined to 5.4, and organisms switched back to lactate production. Intracellular concentration of fructose 1,6-diphosphate of batch culture cells was greater than 12 mM, a concentration that allowed maximal lactate dehydrogenase activity. When Streptococcus bovis was grown in continuous culture at pH 6.7, intracellular fructose-l,6-diphosphate declined to .4 mM, a concentration which gave little lactate dehydrogenase activity at pH 6.5 or greater. Decreasing pH of continuous culture to 4.7 increased intracellular fructose-1,6-diphosphate concentration to .8 mM. This concentration was still limiting if lactate dehydrogenase was assayed at pH 6.5, but nearly maximal activity was obtained when enzyme was assayed at pH 5.5. The small increase in fructose-l,6-diphosphate and decreased requirement of lactate dehydrogenase for fructose-l,6-diphosphate under acidic assay conditions, accounted for increased lactate production during low pH (4.7) continuous culture. These and other aspects of lactate regulation by Streptococcus bovis are discussed as factors leading to rumen acidosis. This pattern of regulation also helps to explain why rumen acidosis is difficult to reverse.     

The relative effectiveness of pH control and heat treatment for enhancing biohydrogen gas production

Hydrogen gas can be recovered from the microbial fermentation of organic substrates at high concentrations when interspecies hydrogen transfer to methanogens is prevented. Two techniques that have been used to limit methanogenesis in mixed cultures are heat treatment, to remove nonsporeforming methanogens from an inoculum, and low pH during culture growth. We found that high hydrogen gas concentrations (57-72%) were produced in all tests and that heat treatment (HT) of the inoculum (pH 6.2 or 7.5) produced greater hydrogen yields than low pH (6.2) conditions with a nonheat-treated inoculum (NHT). Conversion efficiencies of glucose to hydrogen (based on a theoretical yield of 4 mol-H2/mol-glucose) were as follows: 24.2% (HT, pH = 6.2), 18.5% (HT, pH = 7.5), 14.9% (NHT, pH = 6.2), and 12.1% (NHT, pH = 7.5). The main products of glucose (3 g-COD/L) utilization (> or = 99%) in batch tests were acetate (3.4-24.1%), butyrate (6.4-29.4%), propionate (0.3-12.8%), ethanol (15.4-28.8%), and hydrogen (4.0-8.1%), with lesser amounts of acetone, propanol, and butanol (COD basis). Hydrogen gas phase concentrations in all batch cultures reached a maximum of 57-72% after 30 h but thereafter rapidly declined to nondetectable levels within 80 h. Separate experiments showed substantial hydrogen losses could occur via acetogenesis and that heat treatment did not prevent acetogenesis. Heat treatment consistently eliminated the production of measurable concentrations of methane. The disappearance of ethanol produced during hydrogen production was likely due to acetic acid production as thermodynamic calculations show that this reaction is spontaneous once hydrogen is depleted. Overall, these results show that low pH was, without heat treatment, sufficient to control hydrogen losses to methanogens in mixed batch cultures and suggest that methods will need to be found to limit acetogenesis in order to increase hydrogen gas yields by batch cultures.     

Inhibitory effect of acetic acid on growth of hyperthermophilic archaeon Pyrococcus furiosus

The growth inhibition of Pyrococcus furiosus by acetic acid was stronger than that by hydrogen and could be described by a non-competitive inhibition model in which the inhibition constants of undissociated acetic acid, K(p) and n, were estimated to be 0.69 mM (25 mM total acetic acid at pH 6.5; pKa=4.96; 98 degrees C) and 1.0, respectively. In order to reduce the acetic acid inhibition, repeated-batch culturing was performed using a filtration module. This yielded 0.49 g of dry cells l(-1) after growth for 12 h after inoculation. It became impossible, however, to continue repeated-batch culturing manually because the time intervals for medium replacement became too short. In order to automatically maintain a low concentration of acetic acid, a perfusion culture was carried out in which medium feeding coupled to a pH-auxostat was performed. In this perfusion culture, it was possible to maintain the acetic acid concentration below 7.6 mM during exponential growth of P. furiosus, resulting in 1.8 g of dry cells l(-1) at 15 h after inoculation.     

Inhibition of biohydrogen production by undissociated acetic and butyric acids

Glucose fermentation to hydrogen results in the production of acetic and butyric acids. The inhibitory effect of these acids on hydrogen yield was examined by either adding these acids into the feed of continuous flow reactors (external acids), or by increasing glucose concentrations to increase the concentrations of acids produced by the bacteria (self-produced). Acids added to the feed at a concentration of 25 mM decreased H2 yields by 13% (acetic) and 22% (butyric), and 60 mM (pH 5.0) of either acid decreased H2 production by >93% (undissociated acid concentrations). H2 yields were constant at 2.0 +/- 0.2 mol H2/mol glucose for an influent glucose concentration of 10-30 g/L. At 40 g glucose/L, H2 yields decreased to 1.6 +/- 0.1 mol H2/mol glucose, and a switch to solventogenesis occurred. A total undissociated acid concentration of 19 mM (self-produced acids) was found to be a threshold concentration for significantly decreasing H2 yields and initiating solventogenesis. Hydrogen yields were inhibited more by self-produced acids (produced at high glucose feed concentrations) than by similar concentrations of externally added acids (lower glucose feed concentrations). These results show the reason hydrogen yields can be maximized by using lower glucose feed concentrations is that the concentrations of self-produced volatile acids (particularly butyric acid) are minimized.     

“酒精沼气双发酵耦联工艺”中硫化物对酒精发酵的影响

"酒精沼气双发酵耦联工艺"中硫化物会对酒精发酵产生抑制,不利于工艺的稳定运行。与对照相比,pH 4.0,24 mmol/L的硫化物使酒精发酵时间从48 h延长至318 h,酒精产量由90.73 g/L下降至82.28 g/L,酵母数量由3.78×108/mL下降至2.20×108/mL,甘油产量由6.99 g/L上升至11.02 g/L。甘油产量上升是硫化物存在时酒精产量下降的原因之一。硫化物对酒精发酵的抑制性随添加时间的推迟而减弱,这是因为在酒精发酵过程中,硫化物会以H2S的形式随着CO2一起从发酵液逸出。pH 4.0时,硫化物对酒精发酵的临界抑制浓度为1.2 mmol/L。     

Batch growth of Salmonella typhimurium LT2: stoichiometry and factors leading to cessation of growth

Salmonella typhimurium LT2 was grown in batch culture (trypticase soy broth, with 0.3%(w/v) yeast extract, 1% (w/v) glucose and 0.5% (w/v/) NaCl, 20 degrees C) at a range of initial pH (4.4, 4.8, 5.0 and 7.0). The consumption of oxygen and glucose was found to be independent of initial pH, and stoichiometric with growth. Mean yield coefficients of 6.9 x 10(-15) and 15.5 x 10(-15) mol oxygen/cell were estimated. Calculation of the instantaneous state of carbon during the cultivation showed stoichiometric conversion of glucose into biomass, carbon dioxide and organic acids. The concentration of the undissociated form of the primary acidic product (acetic acid) was shown to be the factor limiting growth.     

Lactic acid tolerance determined by measurement of intracellular pH of single cells of Candida krusei and Saccharomyces cerevisiae isolated from fermented maize dough

Strains of Candida krusei and Saccharomyces cerevisiae were grown together at 30 degrees C in MYGP broth, pH 2.5, in the presence of 106.4 mM undissociated lactic acid. The two C. krusei strains investigated grew within 48 h from initial counts of 2 x 10(4) to approximately 10(7) cells/ml whereas the two S. cerevisiae strains investigated survived but did not grow in the presence of 106.4 mM undissociated lactic acid at pH 2.5. To explain the differences in lactic acid tolerance of the two yeast species, we used fluorescence-ratio-imaging microscopy and a perfusion system to determine the short-term intracellular pH (pH(i)) changes in single cells of C. krusei and S. cerevisiae. The changes were investigated both in the presence of low (20.7 mM) and high (106.4 mM) concentrations of undissociated lactic acid. For both the investigated species 20.7 mM undissociated lactic acid did not seem to influence the initial pH(i) which for C. krusei was found to be approximately 8.0 and for S. cerevisiae 6.9-7.5. For both C. krusei strains, perfusion with 106.4 mM undissociated lactic acid induced only weak short-term pH(i) responses with a decrease in pH(i) of less than one pH unit. Contrary, for both strains of S. cerevisiae perfusion with 106.4 mM undissociated lactic acid resulted in a significant decrease in pH(i) from initially 6.9-7.5 to 6.2-6.4 after 1 min and further to a pH(i) of < or = 5.5 after 3 min after which it remained constant. The results obtained show that C. krusei is more resistant to short-term pH(i) changes caused by lactic acid than S. cerevisiae, and this, in turn, may be part of the explanation why C. krusei is more tolerant to lactic acid than S. cerevisiae.     

Utilization and transport of acetic acid in Dekkera anomala and their implications on the survival of the yeast in acidic environments

The yeast Dekkera anomala IGC 5153 exhibited a restricted ability to use weak acids as the only carbon and energy sources. Of the monocarboxylic, dicarboxylic, and tricarboxylic acids tested, only acetic acid was used in such a way. The cells were able to grow at acetic acid concentrations of 0.1 to 3% (vol/vol) over a pH range of 3.5 to 5.5, and the specific growth rates decreased exponentially with the increase of the undissociated acetic acid concentration in the culture medium. Transport assays carried out in cells that exhibited higher specific growth rates showed the presence of an acetate-proton symport associated with a simple diffusion component of the undissociated acetic acid, the weight of the latter increasing with the undissociated acid concentration in the culture media. The acetate carrier was shared by propionic, formic, and sorbic acids and was inducible and repressed by glucose and concentrations of undissociated acetic acid in the culture medium above 0.3% (vol/vol). In undissociated acetic acid repression conditions, the lowest values for the yeast specific growth rates were obtained, and the simple diffusion of the undissociated acid was the only mechanism involved in the acetic acid uptake by the cells. The results will be discussed in terms of the high tolerance of D. anomala to the acidic stress conditions present in wine.     

Identification and biochemical characteristics of yeast microflora of Rokpol cheese

The inhibiting characteristics of lactic acid bacteria on Shiga toxin-producing Escherichia coli (STEC) O157:H7 (three strains, clinically isolated) was investigated by using a batch fermentation system. The species such as Lactobacillus casei strain Shirota or L. acidophilus YIT 0070 exert growth inhibitory and bactericidal activities on STEC. The pH value and undissociated lactic acid (U-LA) concentration of the culture medium of STEC cocultured with L. casei or L. acidophilus dramatically lowered or increased, respectively [corrected], when compared with those of the control culture. The cytotoxic properties of U-LA on STEC strain 89020087 analyzed in vitro was divided into two phases, i.e., the bacteriostatic phase (between 3.2 to 62 mM) and the bactericidal phase (over 62 mM). These data suggest that the bactericidal effect of Lactobacillus on STEC depends on its lactic acid production and pH reductive effect.     

Enhanced 2,3-butanediol production by addition of acetic acid in Paenibacillus polymyxa

By the addition of 150 mM acetate into a batch culture at an initial pH of 6.8, the production of 2,3-butanediol (BDL) by Paenibacillus polymyxa reached 248 mM, yielding 0.87 mol.mol(-1) glucose, where the ratio of acetate consumed to glucose consumed (A/C ratio) was calculated as 0.35 mol acetate mol(-1) glucose. Therefore, a fed-batch culture was carried out by feeding glucose and acetate at a ratio of 0.35 mol acetate mol(-1) glucose. In the fed-batch culture performed at pH 6.8, BDL production reached 637 mM, yielding 0.81 mol.mol(-1) glucose, although the A/C ratio was only 0.18 mol acetate mol(-1) glucose. By decreasing pH to 6.3 in the fed-batch culture, BDL production reached 566 mM, yielding 0.88 mol.mol(-1) glucose and the A/C ratio was 0.32 mol acetate mol(-1) glucose. The optical purity of BDL, which was expressed as enantiomeric excess, was retained at more than 98% of the (R, R)-stereoisomer at the end of culture, which was comparable to that without acetate addition.     

Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process

H2 and ethanol production from glycerol-containing wastes discharged after a manufacturing process for biodiesel fuel (biodiesel wastes) using Enterobacter aerogenes HU-101 was evaluated. The biodiesel wastes should be diluted with a synthetic medium to increase the rate of glycerol utilization and the addition of yeast extract and tryptone to the synthetic medium accelerated the production of H2 and ethanol. The yields of H2 and ethanol decreased with an increase in the concentrations of biodiesel wastes and commercially available glycerol (pure glycerol). Furthermore, the rates of H2 and ethanol production from biodiesel wastes were much lower than those at the same concentration of pure glycerol, partially due to a high salt content in the wastes. In continuous culture with a packed-bed reactor using self-immobilized cells, the maximum rate of H2 production from pure glycerol was 80 mmol/l/h yielding ethanol at 0.8 mol/mol-glycerol, while that from biodiesel wastes was only 30 mmol/l/h. However, using porous ceramics as a support material to fix cells in the reactor, the maximum H2 production rate from biodiesel wastes reached 63 mmol/l/h obtaining an ethanol yield of 0.85 mol/mol-glycerol.     

Efficient production of cellulolytic and xylanolytic enzymes by the rumen anaerobic fungus, Neocallimastix frontalis, in a repeated batch culture

In a batch culture of Neocallimastix frontalis in a medium (pH 6.8) containing 8 g.l(-1) cellulose, the concentrations of the fermentation products and the cellulolytic and xylanolytic enzymes did not increase in comparison with those of cultures in a medium containing 4 g.l(-1) cellulose. Therefore, kinetic studies were performed to determine the effect that products such as acetate, formate, lactate and ethanol have in inhibiting the growth. The reduction of the specific growth rate by the fermentation products could be expressed by using a noncompetitive inhibition model, and it was found that at low concentrations, acetate was the strongest inhibitor among the inhibitory products studied. In order to reduce the inhibition by the fermentation products, a repeated batch culture was carried out whereby none of the fatty acids exceeded 50 mM. In this repeated batch culture, xylanase, endoglucanase, beta-glucosidase, and avicelase were continuously produced in the medium during cultivation, and 18200, 4550, 3790 and 129 IU g.l(-1) of these enzymes, respectively, were produced up to 20 d of culture.     

Biohydrogen production from cattle wastewater by enriched anaerobic mixed consortia: influence of fermentation temperature and pH

Experiments were conducted to select a natural mixed microflora seed source and investigate the effect of temperature and pH on fermentative hydrogen (H2) production from cattle wastewater by sewage sludge. Sewage sludge was shown to have higher cumulative H2 production than other inoculum collected from cow dung compost, chicken manure compost, and river sludge. Experimental results show that H2 production from cattle wastewater was significantly affected by both pH and temperature of the culture. The maximum H2 yield was obtained at pH 5.5. H2 yield and the ratio of butyrate/acetate (Bu/Ac) followed a similar production trend, suggesting that butyrate formation might favor H2 production. The optimal temperature for H2 production from cattle wastewater was 45 degrees C with peak values of H2 production (368 ml), hydrogen yield of 319 ml H2/g chemical oxygen demand (COD) consumed, and butyrate/acetate ratio of 1.43. Presence of ethanol and propionic acid indicated decreased hydrogen production; their concentrations were also affected by pH and temperature. A modified Gompertz model adequately described H2 production and bacterial growth.     

Factors affecting the extreme acid resistance of Escherichia coli O157:H7

When stationary phase Escherichia coli O157:H7 cells were subjected to extreme acid shock (pH 2·0, 6 h, 37°C) cell survival was as great as 10%, but culture conditions greatly affected the acid resistance. Anaerobic cultures were more resistant to extreme acid shock if the glucose concentration of the growth medium was high, acids accumulated, and pH declined. By varying pH and acetate concentration, it was possible to demonstrate a high correlation (R²=0·86) between undissociated acetate and extreme acid resistance. Because dissociated acetate and extreme acid resistance were poorly correlated (R²<0·01), it appeared that the pH effects were being mediated via acetate dissociation. Propionate and butyrate were as effective as acetate, but formate, lactate, benzoate and the uncoupler, carbonylcyanide m -chlorophenylhydrazone (CCCP), were much less effective in promoting extreme acid-resistance. Acetate, propionate, butyrate, benzoate and CCCP all decreased the intracellular pH of E. coli O157:H7, but the correlation between intracellular pH and extreme acid resistance was low (R²<0·01). Cultures grown aerobically only needed half as much acetate to induce extreme acid resistance as those grown anaerobically, and the addition of the reducing agent, cysteine, to anaerobic media made the stationary phase cells less responsive to acetate. An rpoS mutant of E. coli O157:H7 was at least 100-fold more sensitive to acid shock than the wild-type, and large amounts of acetate were needed to promote even a small increase in viability.     

Effective onion vinegar production by a two-step fermentation system

A two-step fermentation system combining a repeated batch process using a flocculating yeast with a charcoal pellet bioreactor was developed for onion vinegar production. Juice from the red onion R-3, which contained 67.3 g/l total sugar, was smoothly converted to onion alcohol containing 30.6 g/l ethanol by repeated batch operation using the flocculating yeast Saccharomyces cerevisiae strain IR-2. Stable operation was possible and the maximum productivity was about 8.0 g/l/h. A packed bed bioreactor containing charcoal pellets produced from waste mushroom medium was then applied to continuous onion vinegar production from the onion alcohol. Onion vinegar was successfully produced, with a maximum productivity and acetic acid concentration of about 3.3 g/l/h and 37.9 g/l, respectively. The total acetic acid yield calculated from the amount of sugar consumed was 0.86. The two-step system was operated for 50 d and proved to be competitive with other systems in terms of its high productivity, high acetic acid yield, operational stability and low production costs.     

Effects of acetic acid and its assimilation in fed-batch cultures of recombinant Escherichia coli containing human-like collagen cDNA

The primary processing problem in recombinant Escherichia coli fermentation is the production of acetic acid, which can inhibit both cell growth and recombinant protein production. The ability of E. coli to assimilate acetate permits it to solve this problem in a rather creative manner. In this study, the effects of acetic acid assimilation through a glucose starvation period at different cell growth phases were investigated in fed-batch cultures of recombinant E. coli. Experimental results showed that the human-like collagen (HLC) production could be improved by introducing glucose starvation at the end of batch culture and pre-induction phase, while the glucose starvation at the induction phase resulted in a poor HLC productivity. The acetic acid assimilation was observed during all the glucose starvation periods. In addition, a systematic study for evaluating the effects of acetic acid was carried out by adding acetate into culture media at different cell growth phases and then employing a glucose starvation after several hours. It was found that obvious acetate inhibition on cell growth occurred in the batch culture phases while its inhibitory effect on HLC expression occurred only in the post-induction phase. The longer the elevated acetic acid concentration maintained, the stronger the inhibitory effects were. These results are of significance for optimizing and scaling-up fermentation processes.     

Culture pH interacts with corn oil concentration to affect biohydrogenation of unsaturated fatty acids and disappearance of neutral detergent fiber in batch culture

Effects of culture pH and corn oil (CO) concentration on biohydrogenation (BH) of unsaturated fatty acids and disappearance of neutral detergent fiber (NDF) in batch culture were evaluated in a 2 × 3 factorial design experiment. Culture vessels (100 mL; 4 replicates/treatment per time point) included ground alfalfa hay plus CO at 0, 1, or 2% dry matter inclusion rate and were incubated at pH 5.8 (low pH) or 6.2 (high pH) for 0, 6, 12, 18, or 24 h. Effects of culture pH, CO, time, and their interactions were determined. Adding CO increased total fatty acid concentration in substrates to 1.01, 2.31, and 3.58% dry matter for 0, 1, and 2% CO, respectively. Corn oil concentration interacted with culture pH and resulted in different effects on BH of cis-9,cis-12 18:2 at low or high culture pH. After 24 h of incubation, low pH, compared with high pH, reduced disappearance of NDF by 35% and BH extent of cis-9,cis-12 18:2 by 31%. Increasing CO increased disappearance of NDF across pH treatments and decreased BH extent of cis-9,cis-12 18:2 at low pH and increased it at high pH over 24 h. Compared with high pH, low pH reduced concentrations of 18:0 by 31% and increased concentrations of trans-10,cis-12 18:2 and trans-10 18:1 by 110 and 79% after 24 h, respectively. Adding CO at low pH had greater effect on BH intermediates of cis-9,cis-12 18:2 compared with adding oil at high pH. In particular, increasing CO to 1 and 2% DM at low pH, compared with at high pH, resulted in a 36 and 46% reduction in the concentration of 18:0, an 84 and 131% increase in the concentration of trans-10,cis-12 18:2, and an 81 and 129% increase in the concentration of trans-10 18:1, respectively. Despite the interactions between culture pH and CO concentration, main effects across time were also significant for the response variables of interest. In conclusion, culture pH interacted with CO concentration to affect BH of UFA and disappearance of NDF in batch culture, as the effects were greater at low culture pH than at high culture pH.     

双氧水对地衣芽胞杆菌合成杆菌肽的影响

在研究地衣芽胞杆菌浊液发酵生产杆菌肽过程中发现存在发酵前期溶氧为零、发酵中期菌体自溶以及二次生长现象,而H2O2是一种携氧剂,可释放O2以及不影响产物分离,因此在10L罐上,考察了流加H2O2对地衣芽胞杆菌浊液发酵合成杆菌肽的影响.在发酵24h时以速率为3mmol/(L·h)恒速流加H2O2至34h,菌体生物量在34h达到最大值为73.5× 109cfu/mL比对照最大值高6.5％(对照最高值为69× 109cfu/mL),自溶时间延后了8h,而且细胞自溶后的菌数最小值为42h的62.1×109cfu/mL,比对照自溶后最低值高了77％.24h～34h的挥发性脂肪酸(VFA)最高值为12.8g/L,比对照减少了11％,糖耗速率是0.36g/(L· h),比对照提高了3％.杆菌肽在24h～34h的合成速率为44.9U/(mL·h)比对照高12％(对照为40.1U/(mL·h)),杆菌肽最终效价为1021U/mL,比对照提高9.9％,说明适量流加H2O2不仅改善了细胞的生长环境,还能促进杆菌肽的生物合成效率.