H2 production from starch by a mixed culture of Clostridium butyricum and Enterobacter aerogenes

A mixed continuous culture of Clostridium butyricum and Enterobacter aerogenes removed O2 in a reactor and produced H2 from starch with yield of more than 2 mol H2/mol glucose without any reducing agents in the medium. Co-immobilized cells of the bacteria on porous glass beads evolved H2 from starch at 1.3 l/l.h, with H2 yield of 2.6 mol H2/ mol glucose at dilution rate of 1.0 h–1 in a continuous culture.     

Thermophilic biohydrogen production from glucose with trickling biofilter

Thermophilic H2 production from glucose was studied at 55-64 degrees C for 234 days using a continuous trickling biofilter reactor (TBR) packed with a fibrous support matrix. Important parameters investigated included pH, temperature, hydraulic retention time (HRT), and glucose concentration in the feed. The optimal pH and temperature were 5.5 and 60 degrees C, respectively. With decreasing HRT or increasing inlet glucose concentration, volumetric H2 production rate increased but the H2 production yield to glucose decreased gradually. The biogas composition was almost constant at 53 +/- 4% (v/v) of H2 and 47 +/- 4% (v/v) of CO2. No appreciable CH4 was detected when the reactor was under a normal operation. The carbon mass balance showed that, in addition to cell mass, lactate, n-butyrate, CO2, and acetate were major products that comprised more than 85% of the carbon consumed. The maximal volumetric H2 production rate and H2 yield to glucose were 1,050 +/- 63 mmol H2/l.d and 1.11 +/- 0.12 mol H2/mol glucose, respectively. These results indicate that the thermophilic TBR is superior to most suspended or immobilized reactor systems reported thus far. This is the first report on continuous H2 production by a thermophilic TBR system.     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

Growth and butyric acid production by Clostridium populeti

The growth of Clostridium populeti in 2% (w/v) glucose medium containing 0.2% (w/v) yeast extract was optimal with 10 mM NH₄Cl as the nitrogen source. Although the maximum specific growth rate (=0.32 h^-1) with 5 mM NH₄Cl was similar, the biomass yield was about 30% lower than that at the optimum. Either sodium sulphide or cysteine-HCl at an optimum concentration of 0.33 mM and 5.0 mM respectively, could serve as the sole sulphur source for growth. The growth rate was unaffected by initial glucose concentrations of up to 10% (w/v), but in the presence of 15% glucose it declined by about 35%. The molar yield of butyric acid (mol/mol glucose) declined from 0.70 in 1% (w/v) initial glucose medium to 0.39 in 10% glucose medium. In 5.7% initial glucose medium, butyric acid levels of 6.3 g/l were obtained (0.56 mol butyrate/mol glucose) after 72 h of incubation in 2.5 l batch cultures. A decrease of about 50% in the maximum specific growth rate of C. populeti was observed in the presence of an initial concentration of either 1.2 g/l of butyric acid or 18.9 g/l of acetic acid.This paper is issued as NRCC No. 29032     

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatumZ7产氢效率的因素,采用生理学方法对其进行了研究。结果表明:乙醇型发酵菌A.elongatumZ7的最适产氢温度为37℃,最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度>60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。     

Galanin in the porcine pancreas.

Galanin, a 29 amino acid neuropeptide, was recently isolated from pig intestine. We studied the localization, nature and effect of galanin in pig pancreas. Galanin immunoreactive nerve fibers were regularly found in the pancreas. A peptide chromatographically similar to synthetic galanin was identified in pancreas extracts. The effect of galanin on the endocrine and exocrine secretion was studied in isolated pancreases, perfused with a synthetic medium containing 3.5, 5 or 8 mmol/l glucose and synthetic galanin (10(-10)-10(-8) mol/l). There was no effect on the basal exocrine secretion. The output of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) was measured in the effluent. There was no effect on PP secretion. At a perfusate glucose concentration of 5 mmol/l, galanin at 10(-9) mol/l increased insulin secretion by 55 +/- 14% (mean +/- S.E.M., n = 5) of basal secretion, and at 10(-8) mol/l by 58 +/- 27% (n = 6). At 8 mmol/l glucose, insulin secretion increased by 25 +/- 10% (n = 6) and 62 +/- 17% (n = 8). At 5 mmol/l glucose glucagon secretion was increased by 15 +/- 3% (n = 5) by galanin at 10(-9) mol/l and by 29 +/- 11% (n = 5) by galanin at 10(-8) mol/l, and at 8 mmol/l glucose by 66 +/- 27% and 41 +/- 25%. Somatostatin secretion was inhibited to 72 +/- 2% (n = 5) of basal secretion by galanin at 10(-9) mol/l and to 65 +/- 7% (n = 7) at galanin at 10(-8) mol/l, both at 5 mmol/l glucose. At 8 mmol/l the figures were 83 +/- 6% and 70 +/- 10%. Insulin secretion in response to square wave increases in glucose concentration from 3.5 to 11 mmol/l (n = 5) increased 2-fold during simultaneous perfusion with galanin (10(-8) mol/l).     

Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate

In this study, local sewage sludge was acclimated to establish H2-producing enrichment cultures, which were used to convert sucrose to H2 with continuously stirred anaerobic bioreactors. The steady-state behaviors of cell growth, substrate utilization, and product formation were closely monitored. Kinetic models were developed to describe and predict the experimental results from the H2-producing cultures. Operation at dilution rates (D) of 0.075-0.167 h(-1) was preferable for H2 production, resulting in a H2 concentration of nearly 0.02 mol/l. The optimal hydrogen production rate was 0.105 mol/h occurring at D=0.125 h(-1). The major volatile fatty acid produced was butyric acid (HBu), while acetic acid and propionic acid were also produced in lesser quantities. The major solvent product was ethanol, whose concentration was only 15% of that of HBu, indicating that the metabolic flow favors H2 production. The proposed model was able to interpret the trends of the experimental data. The maximum specific growth rate (mu(max)), Monod constant (Ks), and yield coefficient for cell growth (Y(x/s)) were estimated as 0.172 h(-1), 68 mg COD/l, and 0.1 g/g, respectively. The model study also suggests that product formation in the continuous hydrogen-producing cultures was essentially a linear function of biomass concentration.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

Acid-hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol/l sulfuric acid using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol after 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol after 18 h. The results showed that acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by it in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

The effect of temperature and effluent recycle rate on hydrogen production by undefined bacterial granules

Biohydrogen production in an anaerobic fluidized granular bed bioreactor was strongly dependent on temperature and effluent recycle rates. At 45 °C as the effluent recycle rate was increased from 1.3 to 3.5 L/min, the total H? output for the bioreactor increased from 10.6 to 43.2 L/h. Volumetric H(2) productivity also increased from 2.1 to 8.7 L H?/L/h. At 70°C as the effluent recycle was increased from 1.3 to 3.5 L/min, the total H? output for the bioreactor increased from 13.8 to 73.8L/h. At 70 °C volumetric H(2) productivities increased from 2.8 to 14.8L H?/L/h as the effluent recycle rate was increased from 1.3 to 3.5 L/min. At 45 °C % H? was 45% and reached 67% at 70 °C. Maximum hydrogen yields at 45 °C were 1.24 and 2.2 mol H?/mol glucose at 70 °C.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

The acid hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol sulfuric acid per liter pyrolysate using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol in 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol in 18 h. The results showed that the acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by S. cerevisiae (R) in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Polygalacturonase biosynthesis by Lactobacillus plantarum: effect of cultural conditions on enzyme production

Lactobacillus plantarum was found to produce extracellular polygalacturonase (EC 3.2.1.15.). Maximum enzyme production was obtained in a medium containing 0.5% glucose and 1.5% low methyl-pectin as inducer at 27°C at an initial pH of 6.8. Enzyme production was strongly inhibited by 5 μmol/l NiCl₂, 5 μmol/l CoCl₂, 5 μmol/l CuSO₄, and 10 μmol/l ZnCl₂. MnSO₄ and MgSO₄ at 200 μmol/l and 50 μmol/l respectively seemed to enhance enzyme biosynthesis. The optimal pH and temperature for enzyme activity were 4.5 and 30°C respectively. Enzyme production in batch culture accompanied growth.     

Fermentative production of hydrogen from a wheat flour industry co-product

The global flour industry produces 96 million ton/year of wheatfeed, which is mainly used for animal feed. This co-product is high in carbohydrates and potentially a significant substrate for biohydrogen production. A 10 l bioreactor, inoculated with sewage sludge, was operated on wheatfeed (10 g l(-1)) at pH 5.5 and 35 degrees C in batch and semi-continuous mode (15 h hydraulic retention time (HRT)). Wheatfeed hydrolysate was also investigated in continuous mode (15 h HRT). NaOH-H2O2 treatment of 25 g l(-1) wheatfeed resulted in hydrolysate containing on average 8.1 g l(-1) total sugar. Hydrogen yields of 64 and 56 m3 H(2) per ton dry weight were produced from wheatfeed in batch and 56 m3 H2 per ton dry weight of wheatfeed in semi-continuous mode. Hydrogen yields from hydrolysate were only 22 and 31 m3 H2 per ton dry weight, (or 0.9 mol H2 per mol hexose degraded, assuming all sugar is hexose). Fermentation of unhydrolysed wheatfeed is therefore recommended. It is calculated that approximately 264 m3/ton of CH4 can be produced from a subsequent anaerobic digestion stage. The biohydrogen produced (diesel equivalents) from the 1.2 million ton/year of wheatfeed in the UK would be more than twice that required for transportation by the UK flour industry.     

Effect of Maillard reaction products on the growth of selected food-poisoning micro-organisms

The activity of the Maillard reaction products (MRP) prepared by heating (15 h at 90°C) a solution of 1·71 mol/l glucose and 2·05 mol/l glycine at pH values 6·0 and 8·8, against food-poisoning micro-organisms, including Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Salmonella enteritidis and Aeromonas hydrophila , was investigated. High and low pH MRPs strongly inhibited A. hydrophila , whereas Staph. aureus and L. monocytogenes were slightly inhibited by the high pH MRPs only and Salmonella strains were resistant to both.     

Enhanced photo-H2 production of R. faecalis RLD-53 by separation of CO2 from reaction system

The effect of different gases, CO(2) concentration, and separation of CO(2) from reaction system on photo-fermentation H(2) production was investigated by batch culture in this study. Experimental results showed that different gases (Ar,N(2),CO(2), and air) as gas phase have obviously affected on photo-H(2) production and a high concentration of CO(2) can inhibit the growth and H(2) evolution of Rhodopseudomonas faecalis RLD-53. When CO(2) concentration at 5%, cell increased most rapidly the specific growth rate of 0.489 g/l/h and the specific growth rate fell to be 0.265 g/l/h when CO(2) concentration at 40%. However, the growth of RLD-53 at CO(2) concentration of 60-100% was almost completely inhibited. At CO(2) concentrations of 5% and 10%, the maximum H(2) yield was 2.54 and 2.59 mol-H(2)/mol acetate, respectively, and it was similar with the control (2.61 mol-H(2)/mol acetate). H(2) not produced when CO(2) concentration at 60-100%. In conclusion, separation of CO(2) from reaction system can stimulate H(2) production in the entire photo-H(2) production process and H(2) yield increased about 12.8-18.85% than the control.     

Metabolism of H2-CO2, methanol, and glucose by Butyribacterium methylotrophicum.

The fermentative metabolism of Butyribacterium methylotrophicum grown on either H2-CO2, methanol, glucose, or CO is described. The following reaction stoichiometries were obtained: 1.00 H2 + 0.52 CO2 leads to 0.22 acetate + 0.06 cell C; 1 methanol + 0.18 CO2 + 0.01 acetate leads to 0.24 butyrate + 0.29 cell C; and 1.00 glucose leads to 0.31 CO2 + 1.59 acetate + 0.21 butyrate + 0.13 H2 + 1.58 cell C. Cell yields of 1.7 g (dry weight) per mol of H2, 8.2 g (dry weight) per mol of methanol, 42.7 g (dry weight) per mol of glucose, and 3.0 g (dry weight) per mol of CO were obtained from linear plots of cell synthesis and substrate consumption. Doubling times of 9.0, 9.0, and 3 to 4 h were observed during batch growth on H2-CO2, methanol, and glucose, respectively. Indicative of a growth factor limitation, glucose fermentation in defined medium displayed a lower cell synthesis efficiency than when yeast extract (0.05%) was present. B. methylotrophicum fermentation displayed atypically high substrate/cell carbon synthesis conversion ratios for an anaerobe, as greater than 24% of the carbon was assimilated into cells during growth on methanol or glucose. The data indicate that B. methylotrophicum conserves carbon-bound electrons during growth on single-carbon or multicarbon substrates.     

H2 production from starch by a mixed culture of Clostridium butyricum and Rhodobacter sp. M[h]19

A photosynthetic bacterium having ability to produce H2 from acetic, butyric and lactic acids, Rhodobacter sp. M-19 was isolated. H2 was produced from starch in a batch culture by Clostridium butyricum and in a two-step batch culture by C. butyricum and Rhodobacter sp. M-19 in yields of 1.9 and 3.6 mol H2/mol glucose, respectively. A mixed culture of C. butyricum and Rhodobacter sp. M-19 produced H2 from starch with a yield of 6.6 mol H2/mol glucose in a fed-batch culture. © Rapid Science Ltd. 1998     

Metabolic engineering of Bacillus subtilis for enhanced production of acetoin

Acetoin is widely used in food and other industries. A bdhA and acoA double-knockout strain of Bacillus subtilis produced acetoin at 0.72?mol/mol, a 16.4?% increased compared to the wild type. Subsequent overexpression of the alsSD operon enhanced the acetolactate synthase activity by 52 and 66?% in growth and stationary phases, respectively. However, deletion of pta gene caused little increase of acetoin production. For acetoin production by the final engineered strain, BSUW06, acetoin productivity was improved from 0.087?g/l?h, using M9 medium plus 30?g glucose/l under micro-aerobic conditions, to 0.273?g/h?l using LB medium plus 50?g glucose/l under aerobic conditions. In fermentor culture, BSUW06 produced acetoin up to 20?g/l.     

Evaluation of metabolism using stoichiometry in fermentative biohydrogen

We first constructed full stoichiometry, including cell synthesis, for glucose mixed-acid fermentation at different initial substrate concentrations (0.8-6 g-glucose/L) and pH conditions (final pH 4.0-8.6), based on experimentally determined electron-equivalent balances. The fermentative bioH2 reactions had good electron closure (-9.8 to +12.7% for variations in glucose concentration and -3 to +2% for variations in pH), and C, H, and O errors were below 1%. From the stoichiometry, we computed the ATP yield based on known fermentation pathways. Glucose-variation tests (final pH 4.2-5.1) gave a consistent fermentation pattern of acetate + butyrate + large H2, while pH significantly shifted the catabolic pattern: acetate + butyrate + large H2 at final pH 4.0, acetate + ethanol + modest H2 at final pH 6.8, and acetate + lactate + trivial H2 at final pH 8.6. When lactate or propionate was a dominant soluble end product, the H2 yield was very low, which is in agreement with the theory that reduced ferredoxin (Fd(red)) formation is required for proton reduction to H2. Also consistent with this hypothesis is that high H2 production correlated with a high ratio of butyrate to acetate. Biomass was not a dominant sink for electron equivalents in H2 formation, but became significant (12%) for the lowest glucose concentration (i.e., the most oligotrophic condition). The fermenting bacteria conserved energy similarly at approximately 3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had approximately 3.5 mol ATP/mol glucose) over a wide range of H2 production. The observed biomass yield did not correlate with ATP conservation; low observed biomass yields probably were caused by accelerated rates of decay or production of soluble microbial products.     

热量限制对SH-SY5Y细胞氧化损伤的影响

目的观察热量限制培养条件下,SH-SY5Y细胞抗氧化应激损伤的能力。方法建立过氧化氢诱导的SH-SY5Y细胞损伤模型。体外培养SH-SY5Y细胞,分为对照组、损伤组（50、100、250、500、1 000μmol/L H2O2）、低糖组（2 g/L）、低糖＋损伤组,进行细胞形态观察、测定各组细胞的噻唑蓝（MTT）代谢率、乳酸脱氢酶（LDH）漏出率。结果与对照组比较,（50、100、250、500、1 000）μmol/L H2O2损伤1 h后MTT代谢率测定细胞活力,50μmol/L组与对照组比较差异无统计学意义（P〉0.05）;其他组与对照组比较,随着H2O2浓度的增加,细胞活力呈递减趋势,差异具有显著性（P〈0.01）;选定250μmol/L H2O2组为损伤应激源。用低糖预处理细胞24 h,给与250μmol/L H2O2损伤1 h后测定MTT代谢率显示,与对照组比较,损伤组活力明显下降,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（p〈0.01）;继续培养至7 h发现,与对照组比较,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（P〈0.01）。进一步检测LDH漏出率显示,损伤1 h后结果显示,与对照组比较,损伤组漏出率明显增加（P〈0.05）,低糖组漏出率稍有减少（P〉0.05）;与损伤组比较,低糖＋损伤组漏出率明显减少（P〈0.01）;继续培养7h显示,低糖7h组与低糖1 h组比较,漏出稍有增多（P〉0.05）,低糖＋损伤组7 h组与低糖＋损伤组1 h比较漏出率稍有增加（P〈0.05）;细胞形态学观察显示,未加损伤之前,低糖组的细胞形态,与对照组比较无明显改变。加入损伤药物1h后的细胞形态与对照组比较无明显改变。加入损伤药物7 h后的细胞形态,低糖组和对照组细胞突起伸展良好细长,损伤组可见细胞数目明显减少,死细胞多,突起回缩,细胞明显变圆,贴壁性不好,透光性差。结论热量限制能提高神经细胞的抗氧化应激能力,增加细胞生存率,降低死亡率。     

Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability

The promising advantages of Prussian Blue (PB) as catalyst and of the thick film screen printing technology have been combined to assemble sensors with improved characteristics for the amperometric determination of H(2)O(2). PB-modified screen printed electrodes were applied to detect H(2)O(2) at an applied potential of -0.05 V versus the internal screen printed Ag pseudoreference electrode, showing a detection limit of 10(-7) mol l(-1), a linearity range from 10(-7) to 5x10(-5) mol l(-1), a sensitivity of 234 microA mmol l(-1) cm(-2), and a high selectivity. Improved stability at alkaline pH values was also observed, which made possible their use with enzymes having an optimum basic pH. Then, the immobilisation of a single enzyme (glucose oxidase (GOD) or choline oxidase (ChOX)) or of two enzymes, acetylcholinesterase (AchE) coimmobilised with ChOX, has been performed on the surface of PB modified screen-printed electrodes (SPEs) using glutaraldehyde and Nafion. ChOX has been selected as an example of enzyme working at alkaline pH. The choline biosensors showed a detection limit of 5x10(-7) mol l(-1), a wide linearity range (5x10(-7)-10(-4) mol l(-1)), a high selectivity and a remarkable long term stability of 9 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical characteristics and stability were observed with the acetylcholine biosensors.