Ethanol production from H2 and CO2 by a newly isolated thermophilic bacterium, Moorella sp. HUC22-1.

The thermophilic bacterium, Moorella sp. HUC22-1, newly isolated from a mud sample, produced ethanol from H(2) and CO(2) during growth at 55 degrees C. In batch cultures in serum bottles, 1.5 mM ethanol was produced from 270 mM H(2) and 130 mM CO(2) after 156 h, whereas less than 1 mM ethanol was produced from 23 mM fructose after 33 h. Alcohol dehydrogenase and acetaldehyde dehydrogenase activities were higher in cells grown with H(2) and CO(2) than those grown with fructose. The NADH/NAD(+) and NADPH/NADP(+) ratios in cells grown with H(2) and CO(2) were also higher than those in cells grown with fructose. When the culture pH was controlled at 5 with H(2) and CO(2) in a fermenter, ethanol production was 3.7-fold higher than that in a pH-uncontrolled culture after 220 h.

Metabolic analysis of acetate accumulation during xylose consumption by Paenibacillus polymyxa.

Paenibacillus polymyxa ATCC 12321 produced more acetic acid and less butanediol from xylose than from glucose. The product yields from xylose were ethanol (0.72 mol/mol sugar), (R,R)-2,3-butanediol (0.31 mol/mol sugar), and acetate (0.38 mol/mol sugar) while those from glucose were ethanol (0.74 mol/mol sugar), (R,R)-2,3-butanediol (0.46 mol/mol sugar), and acetate (0.05 mol/mol sugar). Higher acetate kinase activity and lower acetate uptake ability were found in xylose-grown cells than in glucose-grown cells. Furthermore, phosphoketolase activity was higher in xylose-grown cells than in glucose-grown cells. In fed-batch culture on xylose, glucose feeding raised the butanediol yield to 0.56 mol/mol sugar and reduced acetate accumulation to 0.04 mol/mol sugar.

Biological and chemical treatment of solid waste from soy sauce manufacture.

To reduce the solid waste in soy sauce refuse (SSR) that is a by-product of soy sauce fermentation, both biological and chemical treatment were examined. When anaerobic digestion of SSR was carried out at the amounts of 25 g/l under 30% (v/v) inocula of thermophilic methanogenic sludge at 50 degrees C, 120 mM of CH4 was produced and 50% (w/v) of mixed-liquor suspended solids in SSR was decreased after 35 days. Although in this culture 30 days' cultivation was required to get 100 mM of CH4, when the same amounts of SSR (25 g/l) were repeatedly added to the culture, the time requirement to get 100 mM of CH4 could be reduced to 20 days and 15 days at second and third batch cultures, respectively. When SSR was treated with 5% (w/v) NaOH for 24 h, the supernatant contained 98% (w/v) of protein that were alkaline-solubilized and insoluble in the intact refuse, and the residual pellet contained insoluble fiber.

Methane fermentation of coastal mud sediment by a two-stage upflow anaerobic sludge blanket (UASB) reactor system.

The removal of organic matter from a coastal mud sediment was carried out by a methane fermentation process under anaerobic conditions. In a batch acidogenic fermentation, the addition of vitamins containing thiamine, nicotinic acid and biotin dramatically enhanced acetate production from the mud sediment (200 g wet wt l(-1) artificial sea water), yielding 77 mM acetate after 6 days, which corresponded to 77% of the organic matter in the mud sediment, measured on the basis of chemical oxygen demand. Thereafter, the two-fold diluted, post-acidogenic fermentation liquor (PAF liquor) was continuously treated at 2.4x original dilution rate day(-1) for 30 days, using an upflow anaerobic sludge blanket methanogenic reactor containing the acclimated methanogenic sludge from the mud sediment. Acetate, 42 mM in the PAF liquor, was converted to methane at a maximum methane production rate of 96 mmol l(-1) day(-1); and 87.5% of the acetate and 88.7% of the total organic carbon in the PAF liquor were removed. Moreover, an efficient treatment of the mud sediment was carried out by a semi-continuous, two-stage reactor system, where the culture broth was circulated between acidogenic and methanogenic reactors. This two-stage reactor system gave a stable operation at 4-day intervals for one treatment period, yielding 112 mmol methane from the wet mud in the PAF liquor (278 g l(-1)).

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.

Asymmetric reduction of ethyl acetoacetate to ethyl (R)-3-hydroxybutyrate coupled with nitrate reduction by Paracoccus denitrificans.

We found that the asymmetric reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate (EHB) by Paracoccus denitrificans could be induced by nitrate addition under anaerobic conditions. However, the addition of electron donors such as glucose, ethanol, and methanol together with nitrate did not stimulate EHB production from EAA. When the reaction was carried out under optimum conditions (cell concentration, 10 g-d.w.l(-1); EAA, 150 mM; NO3-, 100 mM), after 8 h of reaction 49 mM of EHB with an optical purity of 98.9% e.e. was produced. Furthermore, a fed-batch reaction was carried out with an intermittent addition of nitrate and EAA to reduce substrate inhibition. The linear reduction of EAA to EHB proceeded for 40 h after initiation of the reaction, and finally 124 mM of EHB with an optical purity of 88.7% e.e. was produced after 104 h.

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.

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.

Flocculating property of extracellular polymeric substance derived from a marine photosynthetic bacterium, Rhodovulum sp.

The effect of the extracellular polymeric substance (EPS) derived from a marine photosynthetic bacterium, Rhodovulum sp., on the flocculation of kaolin is described. The amount of EPS obtained and its flocculating activity were highly dependent on the extraction temperature and duration: high flocculating activity was obtained at a relatively low temperature (30 degrees C) and long extraction time (60 min). The EPS was classified as a weak-anionic high-polymer group based on its pH profile, and its promoting effect on flocculation was dependent on the concentration of cationic ions and their valence. The relative influence of cations on the critical flocculating concentration (Cs,cr) followed the Schulze-Hardy rule for colloidal flocculation. This suggests that the promotion of flocculation by the additions of EPS and cations occurred as a result of the synergistic effect of the release of electrical repulsion by the cations and the formation of bridges between the EPS and the cations.

Influence of Oxygen and Glucose on Primary Metabolism and Astaxanthin Production by Phaffia rhodozyma in Batch and Fed-Batch Cultures: Kinetic and Stoichiometric Analysis.

The influence of the oxygen and glucose supply on primary metabolism (fermentation, respiration, and anabolism) and astaxanthin production in the yeast Phaffia rhodozyma was investigated. When P. rhodozyma grew under fermentative conditions with limited oxygen or high concentrations of glucose, the astaxanthin production rate decreased remarkably. On the other hand, when the yeast grew under aerobic conditions, the astaxanthin production rate increased with increasing oxygen uptake. A kinetic analysis showed that the respiration rate correlated positively with the astaxanthin production rate, whereas there was a negative correlation with the ethanol production rate. The influence of glucose concentration at a fixed nitrogen concentration with a high level of oxygen was then investigated. The results showed that astaxanthin production was enhanced by an initial high carbon/nitrogen ratio (C/N ratio) present in the medium, but cell growth was inhibited by a high glucose concentration. A stoichiometric analysis suggested that astaxanthin production was enhanced by decreasing the amount of NADPH required for anabolism, which could be achieved by the repression of protein biosynthesis with a high C/N ratio. Based on these results, we performed a two-stage fed-batch culture, in which cell growth was enhanced by a low C/N ratio in the first stage and astaxanthin production was enhanced by a high C/N ratio in the second stage. In this culture system, the highest astaxanthin production, 16.0 mg per liter, was obtained.