Journal of Fermentation and Bioengineering最新文献

Previously, we had developed a simple gene transfection technique for animal cells using cationic lipid vesicles; a commercially available synthetic cationic surfactant, dimethyldioctadecyl ammonium bromide (DDAB) was used for making lipid vesicles. In the present study, the lipid vesicles for receptor mediated gene transfer were modified with a ligand such as insulin and galactose residues to realize enhanced transfection efficiency and/or cell-specific gene transfection. The insulin-modified lipid vesicle solution mixed with the plasmid DNA (pCMVβ) was added to COS-7, NIH3T3, Hela or HepG2 cells; the transfection efficiency was increased 3–4-fold in all the cell lines tested. Furthermore, a mixture of the galactose-modified lipid vesicles and plasmid pCMVβ was added to HepG2 or HuH-6 cells expressing asialoglycoprotein receptors, and the transfection efficiency was increased 3–4-fold in these cell lines.

A high cell density culture of Rhodococcus rhodochrous IGTS8 was investigated. Acetic acid was one of the most suitable carbon sources for cell growth and sulfate ion was more suitable than dibenzothiophene (DBT) as a sulfur source. Fed-batch culture was conducted in a 1-l jar fermentor with FB medium containing acetic acid and sulfate ion as carbon and sulfur sources. Cell growth was found to be inhibited when the concentrations of acetic acid and ammonium ion were above 3 g/l. To control the concentrations of the two components below 3 g/l, a mixture of acetic acid and ammonium acetate was supplied by means of pH-stat feeding. As a result, a cell concentration of 33 g dry cells/l was obtained after 28-h cultivation. When the cells obtained were incubated in a fresh medium containing DBT as a substrate, hydroxybiphenyl (HBP), which is the end-product of the DBT degradation pathway, was detected and its production rate gradually increased with incubation time. Incubation for 3 to 4 h was enough for the full induction of DBT-degrading enzymes, and the specific production rate of HBP was about 6.1 mmol/kg dry cells/h. A two-phase cultivation (cell growth phase and induction phase) is proposed in order to obtain a high cell density and full induction of DBT-degrading enzymes.

Xylitol, a five carbon sugar alcohol, occurs widely in nature but it is also a normal intermediate in human metabolism. As an alternative sweetener, it is recommended for diabetics and for the prevention of dental caries. Xylitol is currently produced chemically on a large scale. Microbial production is lately becoming more attractive since the downstream processing is expected to be cheaper. Among microorganisms, yeasts are the best xylitol producers, particularly those belonging to the genus Candida. The key enzymes for xylitol production in yeasts are d-xylose reductase which, using either NADH or NADPH, reduces d-xylose to xylitol, and predominantly, NAD-linked xylitol dehydrogenase which reoxidizes xylitol to d-xylulose. Xylitol accumulation in yeasts is sensitive to environmental conditions such as nutrition, temperature, pH, inoculum, substrate and aeration, with the last two being critical for yeast growth and fermentation. Hemicellulosic hydrolysates derived from hardwood and particularly from agricultural residues, such as sugar cane bagasse, corn cobs, wheat and rice straw, are used as feedstock for xylitol production. Due to the presence of inhibitory components, some of the hydrolysates have to be treated prior to microbial utilization. The most investigated types of processes have been batch ones, although fed-batch and immobilized systems have been characterized by the highest yields and productivities. Apart from the naturally occurring yeasts, recombinant strains of Saccharomyces cerevisiae in free and immobilized form were also investigated for xylitol production.

A strain of Agrobacterium tumefaciens isolated from soil was cultivated under various conditions in shake flasks to study exopolysaccharides (EPS) production. NMR analysis revealed that the EPS obtained was a succinoglycan-like polymer. Optimal yields of EPS were obtained using sucrose and lysine as the carbon and nitrogen sources, respectively. Supplementation of the medium with various chemicals resulted in a more or less marked effect on the polymer yield and properties: in particular, high phosphate levels and non-ionic surfactants led to the production of polymers of different molecular sizes in yields up to 13.7 g/l. Oxygen availability also affected the polymer yield and quality. The chemical structure was substantially unaffected by the various fermentation conditions tested. A fermentation carried out in a laboratory-scale fermentor yielded 9.6 g/l succinoglycan from 15 g/l sucrose in the basic medium without further supplements.

The effects of electric current on the fermentation characteristics of yeast were investigated. When 10 mA direct current (DC) or 100 mA alternating current (AC) was applied to the culture broth, significant increases in cell growth and alcohol production rates occurred. The contents of higher alcohols, esters and organic acids in the culture broths to which AC and DC were applied, were different from those in the control culture (no current application). Several compounds such as acetaldehyde and acetic acid, were formed from ethanol as a result of electrode reaction.

Sugar feeding based on the CO₂ production rate was investigated for the control of the cell growth rate during cephalosporin C fermentation in which sugar concentration was the growth limiting factor. The rates of cell growth and sugar consumption were apparently influenced by the sugar feed rate, as the sugar concentration in the broth was kept at approximately 1 g/l during the cultivation. The ratio of the CO₂ production rate to the sugar consumption rate was maintained almost constant after 40 h cultivation. Stepwise alteration in sugar feed rate after the culture time of 50 h caused a change in the CO₂ production rate within 15 min. These results indicated that the CO₂ production rate could be used as an effective parameter of sugar consumption and cell growth rates. Consequently, a control strategy was developed that involved the control of sugar feed rate so that a CO₂ production rate profile corresponded to a preset standard profile. For the half the usual amount of inoculum, the sugar feeding control system enabled the cell concentration to increase faster to that in the usual inoculum. The cell concentration deviation during exponential growth phase among several batches, decreased to half using this sugar feeding control system compared to the culture without such control. These results indicated that the sugar feeding control system developed in this report was found to be efficient for control of cell growth.

For the purpose of separating microorganisms from culture broth by magnetic force, magnetic particles were conjugated with a polymer to produce polymer-conjugated magnetite (polymer-mag). Among 4 preparation methods investigated—aminosilane coupling, glycidylsilane coupling, crosslinking, and co-precipitation—, polymer-mag prepared by co-precipitation showed the highest cell recovery and high dispersibility. When various cationic, anionic, and nonionic polymers were used to prepare polymer-mag and applied to the magnetic separation for Escherichia coli, magnetite conjugated with chitosan (chitosan-mag) gave the highest cell recovery. In addition, E. coli cells could be recovered as the precipitant only 1 min after chitosan-mag was added to a cell suspension, and a clarified supernatant was obtained. The amount of E. coli cells adsorbed to the chitosan-mag was about 1 g-dry cells/g-chitosan-mag, and cell recovery of over 90% was attained in wide pH range from 3.0 to 7.0. Of 12 microorganisms tested, 4 could be recovered with chitosan-mag at recovery levels above 90%, and the adsorbed amounts exceeded 0.5 g-dry cells/g-chitosan-mag. Differences in adsorbed amounts were considered to be mainly due to the different zeta potential of the microorganisms tested.

The interaction between homoacetogens and methanogens in lake sediments was investigated using hydrogen consumption as an indicator. Sediments samples were obtained from Lake Izunuma, Miyagi prefecture, Japan, a wintering place for migratory birds from Siberia. A batch experiment using $\text{H}{}_2\text{CO}{}_2$ as a substrate was conducted to determine the acetate generation and methane production potential of the sediments. Incubation for 4 d at 37°C gave the following stoichiometric equation: 88H₂ + 39HCO₃⁻ + 22H⁺ → 17CH₃COO⁻ + 5CH₄ + 83H₂O. The activities, ν_m, of hydrogen-utilizing homoacetogens and methanogens respectively ranged from 3.2 to 48 and from 1.8 to 3.2 mgCOD·gVSS⁻¹·h⁻¹. The population of hydrogen-utilizing homoacetogens was determined to be 2.6 × 10⁸ MPN·gVSS⁻¹, which was approximately two orders of magnitude higher than that of hydrogen-utilizing methanogens. The results suggest that homoacetogens in the sediments functioned not only as hydrogen consumers but also as major degraders of organic matter, forming acetate as the major reduction product.

Uridine 5′-monophosphate α-d-glucose (UMPG) was evaluated as a novel and potent inhibitor of the enzymatic reaction involved in sugar nucleotide metabolism. UMPG was synthesized by chemical coupling of 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide with uridine 5′-monophosphate (UMP) to give uridine 5′-monophosphate 2″,3″,4″,6″-tetra-O-acetyl-α-d-glucose (UMPTAG), followed by deacetylation of UMPTAG with sodium methoxide. In addition to UMPG, UMPTAG showed potent inhibitory activity toward yeast UDPG pyrophosphorylase (UDPG synthetase). UMPG and UMPTAG were competitive with UDPG in the pyrophosphorolytic reaction, with inhibition constants (K_i) of 4.8 and 20.7 μM, respectively, but non-competitive with inorganic pyrophosphate. UMPG and UMPTAG also inhibited the enzyme non-competitively in the reverse reaction to synthesize UDPG from UTP and glucose 1-phosphate (G1P). The acetyl group of UMPTAG was thought to enhance its hydrophobic interaction, possibly with an active site region of the enzyme functional for binding with UDPG.