Biosynthesis and characterization of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Alcaligenes eutrophus

Copolyesters of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) were produced by Alcaligenes eutrophus at 30 degrees C in nitrogen-free culture solutions containing gamma-butyrolactone alone or with fructose or butyric acid as the carbon sources. When gamma-butyrolactone was used as the sole carbon source, the 4HB fraction in copolyester increased from 9 to 21 mol% as the concentration of gamma-butyrolactone in the culture solution increased from 10 to 25 g/l. The addition of fructose to the culture solution of gamma-butyrolactone resulted in a decrease in the 4HB fraction in copolyester. The copolyesters produced from gamma-butyrolactone and fructose by A. eutrophus were shown to have random sequence distribution of 3HB and 4HB units by analysis of the 125 MHz 13C n.m.r. spectra. In contrast, a mixture of random copolyesters with two different 4HB fractions was produced by A. eutrophus when gamma-butyrolactone and butyric acid were used as the carbon sources. These results are discussed on the basis of a proposed biosynthetic pathway of P(3HB-co-4HB). The copolyester films became soft with an increase in the 4HB fraction, and the elongation to break at 23 degrees C increased from 5 to 444% as the 4HB fraction increased from 0 to 16 mol%. The P(3HB-co-10% 4HB) film was shown to be biodegradable in an activated sludge.     

Influence of dilution rate on NAD(P) and NAD(P)H concentrations and ratios in a Pseudomonas sp. grown in continuous culture.

A freshwater Pseudomonas sp. was grown in continuous culture under steady-state conditions in L-lactate-, succinate-, glucose- or ammonium-limited media. Under carbon limitation, the NAD(H) (i.e. NAD + NADH) concentration of the organisms increased exponentially from approximately 2 to 7 mumol/g dry wt as the culture dilution rate (D) was decreased from 0.5 to 0.02 h-1. Organisms grown at a given D in any of the carbon-limited media possessed very similar levels of NAD(H). Therefore, under these conditions, cellular NAD(H) was only a function of the culture O and was independent of the nature of the culture carbon source. D had no influence on the NAD(H) content of cells grown under ammonium limitation. In contrast, cellular NADH concentration was not influenced by D in carbon- or ammonium-limited media. In L-lactate-limited medium, bacteria possessed 0.14 mumol NADH/g dry wt; very similar levels were found in organisms grown in the other media. The results are consistent with those of Wimpenny & Firth (1972) that bacteria rigidly maintain a constant NADH level rather than a constant constant NADH: NAD ratio. NADP(H) (i.e. NADP + NADPH) and NADPH levels were also not influenced by changes in the culture carbon source or in D; in L-lactate-limited medium these concentrations were 0.97 and 0.53 mumol/g cell dry wt, respectively. The NADPH:NADP(H) ratio was much higher than the NADH:NAD(H) ratio, averaging 55% in carbon-limited cells.     

Regulation of the O-acetyl-L-serine(thiol)lyase activity in Monoraphidium braunii

Total level of O-acetyl-L-serine(thiol)lyase (OASTL) activity observed in Monoraphidium braunii fed-repleted cells decreases up to 40% after 24 h the carbon source was removed from the culture; however, no significant change in the activity is observed in N-starved cells. On the other hand, sulfur starvation induces OASTL activity in M. braunii, which may increase 2.5-fold after 36 h. Normal intracellular level of the activity is restored when a sulfur source, such as sulfate, sulfite, L-cysteine, L-methionine or glutathione is added to the culture. The induction of the OASTL activity requires de novo synthesis of protein, and thus the presence in the culture of adequate carbon and nitrogen sources. The OASTL isoenzymes from M. braunii cells are differently affected by S-starvation.     

Formation of Short-Chain Fatty Acids from H(2) and CO(2) by a Mixed Culture of Bacteria

The biological utilization of CO(2) and H(2) for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37 degrees C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Production of polyhydroxyalkanoates (PHAs) with canola oil as carbon source

Wautersia eutropha was able to synthesize medium chain length polyhydroxyalkanoates (PHAs) when canola oil was used as carbon source. W. eutropha was cultivated using fructose and ammonium sulphate as carbon and nitrogen sources, respectively, for growth and inoculum development. The experiments were done in a laboratory scale bioreactor in three stages. Initially, the biomass was adapted in a batch culture. Secondly, a fed-batch was used to increase the cell dry weight and PHA concentration to 4.36 g L(-1) and 0.36 g L(-1), respectively. Finally, after the addition of canola oil as carbon source a final concentration of 18.27 g L(-1) PHA was obtained after 40 h of fermentation. With canola oil as carbon source, the polymer content of the cell dry matter was 90%. The polymer was purified from dried cells and analyzed by FTIR, NMR and DSC using PHB as reference. The polymer produced by W. eutropha from canola oil had four carbon monomers in the structure of the PHA and identified by 1H and 13C NMR analysis as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxyoctanoate (3HO), and 3-hydroxydodecanoate (3HDD).     

Polysaccharide-hydrolyzing enzymes ofFrankia (Actinomycetales)

Studies were made of the polysaccharide-hydrolyzing activity inFrankia (Actinomycetales) grown in synthetic media using modifications of three standard assay procedures. In screening five different strains ofFrankia for cellulase activity, based on the method of utilization of cellulose in liquid culture, only one strain, CcI3, degraded filter paper cellulose to complete disintegration and only under very specific conditions of pH and primary carbon source. When carboxymethylcellulose (CMC) at 1% was used as substrate, all five strains showed the capacity to produce reducing sugars as hydrolytic products. Microcystalline cellulose, xylans and gum arabic were hydrolyzed to a lesser extent. Optimum activity depended upon pH and primary carbon source with pH 5.0 and pyruvate or propionate producing highest activities. In fractionation studies of culturedFrankia, assays for hydrolysis of 1% CMC in liquid medium showed that highest activity was in the enzyme preparation supernatant with lesser activity in the cell-free extract and cell wall fractions.Frankia strain CpI1 showed the greatest total hydrolytic activity against CMC after 2 weeks of culture. Strains ArI3 and CcI3 also showed good activity. The agar plate method for direct dye-polysaccharide interaction proved to be the least sensitive assay method with only ArI3 showing significant activity using CMC as substrate. It appears that theFranka strains grown in synthetic media all showed hydrolytic activity but the degree of hydrolysis of polysaccharides to reducing sugars depends upon strain of bacteria and very specific cultural conditions.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

重组嗜水气单胞菌Aeromonas hydrophila 4AK4中3-羟基丁酸3-羟基己酸共聚酯PHBHHx的发酵生产

3-羟基丁酸和3-羟基己酸共聚酯(PHBHHx)是一种性能优良的新型生物可降解材料,其机械和加工性能与3-羟基己酸(3HHx)在共聚物中的含量密切相关。在嗜水气单孢菌Aeromonas hydrophila 4AK4中引入了编码β-酮基硫解酶(β-ketothiolase)的phbA基因和编码乙酰乙酰辅酶A还原酶(Acetoacetyl-CoA reductase)的phbB基因,使重组菌增加了一条利用乙酰辅酶A合成3-羟基丁酸-CoA的代谢途径,这使得利用非相关性碳源调控PHBHHx的单体组成比例成为可能。利用葡萄糖酸钠和月桂酸作为碳源,对重组Aeromonas hydrophila 4AK4进行了摇瓶培养及5L发酵罐培养的研究。在摇瓶实验中,通过改变碳源中两种组分的比例,可以使A,hydrophila 4AK4合成的PHBHHx中的3HHx摩尔含量由原来的15％左右降低到3％～12％,成功地实现了对PHBHHx单体组成的调控;当以月桂酸为唯一碳源时,在5L发酵罐中,经过56h的培养,获得了51．5g／L的细胞干重(CDW),其中62％为PHBHHx,3HHx在PHBHHx中的摩尔含量为9．7％;当以1：1的葡萄糖酸钠和月桂酸为碳源时,48h的5L发酵罐培养获得了32．8g／L的CDW和52％的PHBHHx含量,其中3HHx在PHBHHx中的摩尔含量为6．7％。结果证明了该重组菌在大规模生产单体组成可控PHBHHx方面具有很大的应用潜力。     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) byPseudomonas acidovorans

Summary Terpolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvarelate (3HV) were produced byPseudomonas acidovorans in nitrogen-free culture solutions of 1,4-butanediol and pentanol. When 1,4-butanediol was used as the sole carbon source, a polyester with an unusually high 4HB fraction of 99 mol% was produced.     

Degradation of Extracellular beta-(1,3)(1,6)-d-Glucan by Botrytis cinerea

During growth on glucose, Botrytis cinerea produced extracellular beta-(1,3)(1,6)-d-glucan (cinerean), which formed an adhering capsule and slime. After glucose was exhausted from the medium, cinereanase activity increased from <0.4 to 30 U/liter, effecting a striking loss in the viscosity of the culture. Cinerean was cleaved into glucose and gentiobiose. Gentiobiose was then hydrolyzed to glucose. While cinereanase activity was strongest in the culture supernatant, gentiobiase activity was located mainly in the cell wall fraction. The addition of extra glucose or cycloheximide prevented the cinerean degradation caused by an effect on cinereanase formation. Cinerean degradation was accompanied by microconidiation and sclerotium formation. B. cinerea was found to grow on cinerean with the latter as its single carbon and energy source. In this case, cinerean degradation occurred during hyphal growth, and no microconidiation or sclerotium formation was observed. Growth experiments with various carbon sources indicated that cinerean had a positive effect on the formation of cinerean-degrading enzymes.     

Kinetics of growth of the hydrogen-oxidizing bacterium Alcaligenes eutrophus (ATCC 17707) in chemostat culture

The hydrogen-oxidizing bacterium, Alcaligenes eutrophus (ATCC 17707), was grown in chemostat culture with gas-phase (hydrogen, oxygen, and carbon dioxide) and liquid-phase (mineral nutrients) feedstreams; data were used to generate an analytical form for the specific growth rate equation. Model parameters obtained include Monod rate parameters for dissolved hydrogen and oxygen gases, yield coefficients, and specific maintenance rates under conditions of hydrogen or oxygen limitations. These values are similar to some obtained previously by Ohi et al. for another hydrogen bacterium. The observed increase in specific maintenance rates under hydrogen-versus-oxygen-limited culture may be associated with hydrogenase deactivation by oxygen.     

Modulation of glycogen and trehalose levels in Micromonospora echinospora (ATCC 15837)

The growth of Micromonospora echinospora was studied in high and low C/N ratio medium using both batch and continuous culture. Asparagine was consumed rapidly in batch cultures where it served as both a nitrogen and carbon source. Glucose consumption was low suggesting that asparagine functions as the major carbon source under these conditions. The effect of nutrient limitation on the accumulation of storage carbohydrate in batch culture revealed an intimate association between nitrogen limitation and the accumulation of carbonaceous reserves. This study revealed that glycogen constituted the major carbohydrate reserve associated with the onset of sporulation. Intracellular trehalose levels were found to be relatively low and may have been affected by the availability of carbon. Continuous culture studies revealed a correlation between glycogen accumulation and increasing growth rate. It was also found that elevated cellular ATP levels correlated with the increase in glycogen, and reduced glycolytic activity. At the higher growth rates cellular ATP levels were elevated and coincided with reduced activity of the key glycolytic enzyme, phosphofructokinase, suggesting that glycogen can act as a convenient energy reservoir when excess carbon flux dictates.     

Effect of simple and complex carbon sources,low temperature culture and complex carbon feeding policies on poly-3-hydroxybutyric acid (PHB) content and molecular weight (Mw) from Azotobacter chroococcum 6B

The molecular weight (M _w) of poly-3-hydroxybutyrate (PHB), produced by shake-flask culture of Azotobacter chroococcum showed little variation with increasing glucose concentration as carbon source (being in the range of 400–500 kDa), while M _w increased from 300–400 to 640 kDa when grown with increasing concentration of sugar cane molasses. Molecular weight increased nearly 30% from 48 to 72 h culture time when 5% molasses as carbon source was used, while with glucose the highest M _w was reached at 48 h. Under fermentor cultivation A. chroococcum produced PHB with a relatively high M _w of 1590 kDa at 53 h culture time when grown in modified Burk's medium with glucose as carbon source at an initial C/N ratio (molar basis) of 69 under fermentor cultivation. A batch glucose-grown ammonium-limited fermentor culture was repeatedly fed with sugar cane molasses (initial C/N ratio 69) and it was observed that PHB content curve decreased at a slower rate than in the fed-batch culture in which glucose and sucrose were not consumed in the culture medium after the feed.     

影响木薯次生胚状体发生及植株再生因素的研究

研究了在外植体的不同发育阶段中,碳源以及不同的生长激素配比对木薯次生胚状体诱导及植株再生的影响。结果表明：以固体成熟培养基上生长１５ｄ的胚状体子叶为外植体,次生胚状体的产量最高,达２９．３个成熟胚状体／１个外植体。在次生胚状体的诱导阶段,以麦芽糖（４０ｇ／Ｌ）代替蔗糖作碳源,能同时提高次生胚状体的产量（３２．５个胚次体／１个外植体）及植株再生频率（７４．３％）。２,４－Ｄ与ＰＰ３３３;（０．１ｍｇ／Ｌ）配合能提高植株再生频率到７７．６％。２,４－Ｄ与ＢＡＰ（２ｍｇ／Ｌ）或激动素（２．０ｍｇ／Ｌ）配合则大大降低了胚状体诱导及植株再生频率。     

Metabolism of 2,4,6-trinitrotoluene (TNT) by Desulfovibrio sp. (B strain)

A sulfate-reducing bacterium, Desulfovibrio sp. (B strain) isolated from an anaerobic reactor treating furfural-containing waste-water was studied for its ability to metabolize trinitrotoluene (TNT). The result showed that this isolate could transform 100 ppm TNT within 7 to 10 days of incubation at 37°C, when grown with 30 mm pyruvate as the primary carbon source and 20 mm sulfate as electron acceptor. Under these conditions, the main intermediate produced was 2,4-diamino-6-nitrotoluene. Under culture conditions where TNT served as the sole source of nitrogen for growth with pyruvate as electron donor and sulfate as electron acceptor, TNT was first converted to 2,4-diamino-6-nitrotoluene within 10 days of incubation. This intermediate was further converted to toluene by a reductive deamination process via triaminotoluene. Apart from pyruvate, various other carbon sources such as ethanol, lactate, formate and H₂ + CO₂ were also studied as potential electron donors for TNT metabolism. The rate of TNT biotransformation by Desulfovibrio sp. (B strain) was compared with other sulfate-reducing bacteria and the results were evaluated. This new strain may be useful in decontaminating TNT-contaminated soil and water under anaerobic conditions in conjunction with toluene-degrading denitrifiers (Pseudomonas spp.) or toluene-degrading sulfate reducers in a mixed culture system. Correspondence to: R. Boopathy     

Enhancement of poly(arginyl-histidine) production by Verticillium kibiense E18

An ergot fungus Verticillium kibiense E18 produced two cationic peptides, ɛ-poly-l-lysine (ePL) and poly(l-arginyl-d-histidine) (PRH). The ePL was used as a food preservative, and it was expected that PRH would be used as a novel material, such as cationic and antimicrobial peptide. To enhance PRH production of strain E18, various culture conditions were investigated. Glucose was a suitable carbon source for PRH production, although glycerol was a suitable carbon source for growth. The cultivation temperature significantly influenced both cell growth and PRH production. The optimal temperatures for cell growth and PRH production were 28 and 30 °C, respectively. Moreover, strain E18 produced more PRH when an additional 5.0 μg/L FeSO₄·7H₂O was added to the production medium. Under optimal conditions, strain E18 enhanced PRH production, while suppressing ePL production. The maximum PRH production was 183.9 mg/L, which is approximately 60-fold higher than that of the initial culture condition.     

Further Studies on Production of Chloramphenicol Analogs (Corynecins) from n-Alkanes

Further studies on culture condition of Corynebacterium hydrocarboclastus were carried out for more efficient production of corynecins (chloramphenicol analogs). The productivity was affected greatly by the concentration of phosphate and stimulated effectively by organic nutrients, particularly by yeast extract. The most effective production was obtained in the presence of 8 g of yeast extract and 0.2 g of K₂HPO₄ per one liter of chemically defined medium. The maximum amount was 1.42 g equivalent of l-base (free base of chloramphenicol) per liter of the broth culture, which corresponded to 4-fold of that reported in the previous paper.

Among fractions of n-alkanes, n-heptadecane was the best carbon source for the production. The proportion among corynecins homologs varied depending on carbon number of n-alkane used; odd number alkanes gave larger amounts of corynecin II (propionyl derivative), suggesting that carbon flow in the bacterial cells functioned significantly in changing the ratios of corynecin homologs.     

Effects of Mannose on the Growth of N(2)-Fixing Azotobacter vinelandii

Mannose is not a suitable substrate for N(2)-fixing Azotobacter vinelandii. However, when H(2) gas is provided, A. vinelandii can grow mixotrophically with H(2) as the energy source and mannose as the carbon source (T.-Y. Wong and R. J. Maier, J. Bacteriol. 163:528-533, 1985). In this report, seven sugars were used to determine whether A. vinelandii could derive energy from these sugars for mannose utilization. Supplementation of fructose- or galactose-limited medium with mannose did not influence the biomass produced by N(2)-fixing A. vinelandii. The presence of mannose in glucose- or maltose-limited cultures increased cell yield slightly. The addition of mannose decreased the total biomass in the melibiose-limited culture slightly. Mannose was a potent inhibitor of growth when sucrose or turanose was used as the primary sugar. The inhibitory effect of mannose on utilization of sucrose and turanose seems to be related to the energy requirement of the N(2)-fixing processes.     

Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Burkholderia cepacia D1.

Copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced by Burkholderia cepacia D1 at 30°C in nitrogen-free culture solutions containing n-butyric acid and/or n-valeric acid. When n-valeric acid was used as the sole carbon source, the 3HV fraction in copolyester increased from 36 to 90 mol% as the concentration of n-valeric acid in the culture solution increased from 1 to 20 g/l. The addition of n-butyric acid to the culture solution resulted in a decrease in the 3HV fraction in copolyester. The copolymers biosynthesized by this method were mixtures of random copolymers having a wide variety of composition of the 3HV component. The melting points of the fractionated copolymers show a concave curve with the minimum at the 3HV content of ≈40 mol%. The a-parameter of lattice indices of the P(3HB) crystal for the fractionated copolymers largely increased as the 3HV composition increased. Biodegradability of the copolymer increased with the lower content of 3HV composition and/or the lower crystallinity.     

Hydrophobic metabolites of 2,4-dichlorophenoxyacetic acid (2,4-D) in cultured coconut tissue

Cultures of inflorescence and plumular tissues of coconut palm (Cocos nucifera L.) were maintained in the presence of the auxin, [14C]2,4-dichlorophenoxyacetic acid (2,4-D), so that its metabolic fate could be studied. Thin layer chromatography of methanol extracts of the plumular tissue showed that four classes of metabolites, as well as the unchanged acid, were recovered in the extract. In inflorescence tissue, only the unchanged acid and the most polar class of metabolites (metabolite I) were recovered. Metabolite I was shown to consist mostly of a mixture of sugar conjugates and metabolite II (the next most polar) was an unidentified basic metabolite. Metabolites III and IV were both novel triacylglycerol analogues in which one of the natural fatty acids was replaced with a chain-elongated form of 2,4-D. Reversed-phase thin layer chromatography was used to identify the 2,4-D-derived acids and it was found that metabolite III contained the 2,4-dichlorophenoxy-moiety attached to a chain-length of between 2 and 12 carbons, whereas metabolite IV contained 12, 14 and 16 carbon chain lengths. In inflorescence tissue, and in plumular tissue at low sucrose or 2,4-D concentrations and after short periods in culture, metabolite I predominated. The other metabolites increased as a percentage when plumular culture was prolonged or when sucrose or 2,4-D concentrations were raised. These changes correlated with better development of the explant.