Fermentative H2 Research Articles

Changes in the Physico-Chemical Properties of Maize During Natural Fermentation of Mawè

Abstract The physical and chemical changes that occurred during a 72-h fermentation period were studied in two differently processed maize doughs from Benin, referred to as home-produced and commercial mawe. The pH decreased from 6·1 to 3·5 in the commercial process and from 6·2 to 3·6 in the home-style process, whereas the titratable acidity increased from 0·2 to 1·7% (w/w, lactic acid) and from 0·3 to 2·3%, respectively. Home-produced mawe had significantly higher levels of crude fat, crude fibre and ash compared with the commercial mawe, as a consequence of the difference in the processing methods. No marked changes in proximate composition occurred during subsequent fermentation. Commercial mawe was whiter than home-produced mawe, and this whiteness increased with increasing fermentation time. Fermentation significantly increased the swelling and thickening capabilities of mawe, which were more pronounced in the commercial than in home-produced samples. Overall, the commercial mawe process appeared to be a technologically more advanced and effective method of mawe manufacture than the home process.

In Vitro Effects of the Thiopeptide A10255 on Ruminal Fermentation and Microbial Populations

Experiments used unadapted mixed cultures of ruminal microorganisms in batch or continuous culture fermentation to investigate the effect of a thiopeptide, A10255, on ruminal fermentation and microbial populations. After 24h of fermentation in batch culture, addition of A10255 (.5 to 20ppm of the culture fluid) to 0, 45, 60, and 75% concentrate diets had no effect on total VFA but increased molar proportion of propionate and decreased butyrate. The molar proportion of acetate was decreased by treatment only in the 0 and 75% concentrate diets. The increase in molar proportion of propionate by 20ppm of A10255 was less than the increase caused by a similar concentration of monensin. The same concentration of A10255 (20ppm) decreased ADF digestion less than 20ppm of monensin. In continuous culture, A10255 (33 mg/kg of dietary DM) did not affect total VFA concentration, culture pH, OM digestion, or ADF digestion. Ruminal bacterial populations of total anaerobes and lactate-producing, lactate-utilizing, cellulolytic and amylolytic bacteria were unaffected by treatment. However, molar proportions of acetate, butyrate, and isovalerate were decreased, and propionate was increased, by addition of A10255.

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

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.

Effect of Nutrient Media and Bile Salts on Growth and Antimicrobial Activity of Lactobacillus acidophilus

The Lactobacillus acidophilus strains produced variable quantities of antimicrobial substances in three different media: Elliker's, MRS (deMan, Rogosa, and Sharpe), and LBS (Lactobacillus selection) broths. The interaction between L. acidophilus strain and nutrient medium was statistically significant, indicating an influence of the nutrient medium on the level of antimicrobial activity produced. Also statistically significant different were levels of the antimicrobial activity of different strains and the pH of the medium at the end of the fermentation period. Bile salts were added to MRS broth to simulate concentrations in gastrointestinal tract of humans. Antimicrobial activity as well as growth decreased in the presence of high concentrations of bile salts. Sodium glycocholate was more inhibitory than sodium taurocholate at equimolar levels. Calcium hydroxide was added to MRS broth at hourly intervals for 8h to adjust the pH of the fermentation medium to pH of 6.5, which resulted in a doubling of cell numbers. However, there were no differences in the levels of antimicrobial activity at the end of 24h. Although there were no differences in pH of the fermentation medium at the end of 24 and 48h, the antimicrobial activity increased after 24h of fermentation.

Conversion of Glucose to 2-Keto- l -Gulonate, an Intermediate in l -Ascorbate Synthesis, by a Recombinant Strain of Erwinia citreus

A gene for 2,5-diketo-d-gluconate (25DKG) reductase, which encodes an enzyme composed of 277 amino acid residues catalyzing the reduction of 25DKG to 2-keto-l-gulonate (2KLG), was cloned from Corynebacterium sp. strain SHS752001 and expressed in Erwinia citreus SHS2003, a strain which oxidizes glucose to 25DKG. The recombinant microorganism converted glucose to 2KLG, a compound which can be readily converted to l-ascorbate (vitamin C). Improvements in the yield of 2KLG were obtained by changing fermentation conditions, using the p(l) promoter of bacteriophage lambda to express the reductase, and selecting a mutant of E. citreus which could use neither 25DKG nor 2KLG as a sole carbon source for growth. When a culture of the recombinant strain was fed with glucose to a total of 40 g/liter, 49.4% of the glucose was converted to 2KLG during a 72-h fermentation.

Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum.

The metabolic and enzymatic bases for growth tolerance to ethanol (4%) and H2 (2 atm [1 atm = 101.29 kPa]) fermentation products in Clostridium thermohydrosulfuricum were compared in a sensitive wild-type strain and an insensitive alcohol-adapted strain. In the wild-type strain, ethanol (4%) and H2 (2 atm) inhibited glucose but not pyruvate fermentation parameters (growth and end product formation). Inhibition of glucose fermentation by ethanol (4%) in the wild-type strain was reversed by addition of acetone (1%), which lowered H2 and ethanol production while increasing isopropanol and acetate production. Pulsing cells grown in continuous culture on glucose with 5% ethanol or 1 atm of H2 significantly raised the NADH/NAD ratio in the wild-type strain but not in the alcohol-adapted strain. Analysis of key oxidoreductases demonstrated that the alcohol-adapted strain lacked detectable levels of reduced ferredoxin-linked NAD reductase and NAD-linked alcohol dehydrogenase activities which were present in the wild-type strain. Differences in the glucose fermentation product ratios of the two strains were related to differences in lactate dehydrogenase and hydrogenase levels and sensitivity of glyceraldehyde 3-phosphate dehydrogenase activity to NADH inhibition. A biochemical model is proposed which describes a common enzymatic mechanism for growth tolerance of thermoanaerobes to moderate concentrations of both ethanol and hydrogen.

Effects of Stirring and Hydrogen on Fermentation Products of Clostridium thermocellum

Clostridium thermocellum produces ethanol, acetate, H(2), and CO(2) as major fermentation products from cellulose and cellobiose. The performance of three strains of this microorganism was studied to assess the potential use in producing ethanol directly from cellulosic fiber. Depending on the bacterial strain, an ethanol/acetate product ratio from 1 to as high as 3 was observed in unstirred cultures. Vigorous stirring during growth resulted in a threefold decrease in the ethanol/acetate ratio. The H(2) content in the unstirred culture broth was three times greater than that in the stirred one. Addition of exogenous H(2) to the gas phase during growth increased the ethanol/acetate ratio much more in the stirred than in the unstirred fermentations. The addition of sufficient H(2) to the gas phase almost relieved the effect of stirring, and the ethanol/acetate ratio approached that in the unstirred condition. Addition of tritium to the gas phase of the culture resulted in the formation of tritiated water (H(2)O), which indicates that C. thermocellum possesses hydrogenase(s) that catalyzes the reverse reaction. The rate of H(2)O formation was about three times higher in the stirred culture than in the unstirred culture. These results demonstrate that the H(2) concentration in the broth plays an important role in the product formation. The H(2) supersaturation present in the unstirred cultures is responsible for the observed effect of stirring. A hydrogen feedback control mechanism regulating the relative concentrations of reduced and oxidized electron carriers is proposed to account for the effect of hydrogen on the metabolite distribution.

The capacity of hydrogenotrophic anaerobic bacteria to compete for traces of hydrogen depends on the redox potential of the terminal electron acceptor

The effect of different electron acceptors on substrate degradation was studied in pure and mixed cultures of various hydrogenotrophic homoacetogenic, methanogenic, sulfate-reducing, fumarate-reducing and nitrate-ammonifying bacteria. Two different species of these bacteria which during organic substrate degradation produce and consume hydrogen, were cocultured on a substrate which was utilized only by one of them. Hydrogen, which was excreted as intermediate by the first strain (and reoxidized in pure culture), could, depending on the hydrogen acceptor present, also be used by the second organism, resulting in interspecies hydrogen transfer. The efficiency of H2 transfer was similar when methanol, lactate or fructose were used as organic substrate, although the free energy changes of fermentative H2 formation of these substrates are considerably different. In coculture experiments nitrate or fumarate>sulfate> CO2/CH4>sulfur or CO2/acetate were the preferred electron acceptors, and an increasing percentage of H2 was transferred to that bacterium which was able to utilize the preferred electron acceptor. In pure culture the threshold values for hydrogen oxidation decreased in the same order from ≤1,100 ppm for homoacetogenic bacteria to about 0.03 ppm for nitrate or fumarate reducing bacteria. The determined H2-threshold values as well as the percentage of H2 transfer in cocultures were related to the Gibbs free energy change of the respective hydrogen oxidizing reaction.

Hydrogen metabolism and sulfate-dependent inhibition of methanogenesis in a eutrophic lake sediment (Lake Mendota)

Hydrogen metabolism was studied in anoxic sediments of the stratified Lake Mendota; using a method which allowed the measurement of in situ H2 concentrations and the headspace-free analysis of turnover of dissolved H2. Addition of sulfate resulted in partial but immediate inhibition of H2-dependent methanogenesis. Sulfate addition did not result in an immediate decrease in the steady state concentration of dissolved H2, nor did it significantly stimulate the rate constant of H2 turnover. Sulfate-induced decrease in dissolved H2 was only observed after prolonged incubation or when endogenous H2 production was stimulated by added glucose. The turnover of the in situ H2 accounted for only 14% of the H2-dependent methanogenesis from bicarbonate. While rates of methanogenesis increased during the season, rates of H2 turnover decreased, accounting for only 2% of the H2-dependent methanogenesis at the end of summer stratification. These observations indicate that increasing proportions of CH4 were formed from H2 being directly transferred in syntrophic methanogenic associations. The rapid inhibition of H2-dependent methanogenesis by exogenous sulfate may be explained at least partially by assuming methanogenic associations in which syntrophic sulfate reducers change their metabolism from fermentative H2 production to sulfate reduction.

The predominant anaerobe from the spiral intestine of hatchery-raised sturgeon (Acipenser transmontanus), a new Bacteroides species

The anaerobic and aerobic bacterial flora from the spiral intestine of hatchery-raised sturgeon were enumerated. Among the obligate anaerobes, a new bacteroidelike organism was isolated and found to be the predominant strict anaerobe. The organism is Gram-negative, produces the fermentation products acetate, succinate, propionate and H2. The mol % G+C of DNA from various strains ranged from 33.17–33.65. The optimum sodium concentration for this organism was found to be 137 mM; the sodium level in the sturgeon spiral intestine ranged from 94.5–110.0mM. Based on these and other physiological characteristics, the organism appears to be a new species of the genus Bacteroides.

Methanogenesis from Sucrose by Defined Immobilized Consortia

A bacterial consortium capable of sucrose degradation primarily to CH(4) and CO(2) was constructed, with acetate as the key methanogenic precursor. In addition, the effect of agar immobilization on the activity of the consortium was determined. The primary fermentative organism, Escherichia coli, produced acetate, formate, H(2), and CO(2) (known substrates for methanogens), as well as ethanol and lactate, compounds that are not substrates for methanogens. Oxidation of the nonmethanogenic substrates, lactate and ethanol, to acetate was mediated by the addition of Acetobacterium woodii and Desulfovibrio vulgaris. The methanogenic stage was accomplished by the addition of the acetophilic methanogen Methanosarcina barkeri and the hydrogenophilic methanogen Methanobacterium formicicum. Results of studies with low substrate concentrations (0.05 to 0.2% [wt/vol]), a growth-limiting medium, and the five-component consortium indicated efficient conversion (40%) of sucrose carbon to CH(4). Significant decreases in yields of CH(4) and rates of CH(4) production were observed if any component of the consortium was omitted. Approximately 70% of the CH(4) generated occurred via acetate. Agar-immobilized cells of the consortium exhibited yields of CH(4) and rates of CH(4) production from sucrose similar to those of nonimmobilized cells. The rate of CH(4) production decreased by 25% when cysteine was omitted from reaction conditions and by 40% when the immobilized consortium was stored for 1 week at 4 degrees C.