Yield of poly-D(-)-3-hydroxybutyrate from various carbon sources: a theoretical study

The theoretical yield of poly-D(-)-3-hydroxybutyrate (PHB) has been estimated from the biochemical pathway leading to PHB when a carbohydrate (glucose), a C(1) compound (methanol), a C(2) compound (acetic acid), or a C(4) compound (butyric acid) is used as a carbon source. In estimating the yield, recycling (or regeneration) of NADP(+)/ (NADPH + H(+)) and NAD(+) /(NADH + H(+)) have been taken into account. A special emphasis is made on te regeneration of NADPH, which is the coenzyme of acetoacetyl-CoA reductase, one of three key enzymes involved in the biosynthesis of PHB. As a NADPH-regenerating enzyme, glucose-6-phosphate dehydrogenase or isocitrate dehydrogenase is conceived. An equation which predicts the overall yield of PHB when non-PHB residual biomass is actually formed has been derived as a function of both the theoretical yield and PHB content of the dry cell mass. The ratio of the overall yield to the theoretical yield is roughly proportional to the PHB content. (c) 1993 John Wiley & Sons, Inc.     

Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019

This study demonstrated the improved polyhydroxybutyrate (PHB) production via high cell density cultivation of Bacillus megaterium BA-019 with balanced initial total sugar concentration and carbon to nitrogen (C/N) weight ratio. In the 10 L stirred fermentor operated at 30 °C, pH 7.0, 600 rpm, and 1.0 vvm air, with the initial total sugar concentration of 60 g/L and urea at the C/N weight ratio of 10:1, 32.48 g/L cell biomass with the corresponding PHB weight content of 26.94 % and volumetric productivity of 0.73 g/L h were obtained from batch cultivation. Continuing cultivation by intermittent feeding of the sugarcane molasses along with urea at the C/N weight ratio of 12.5:1 gave much improved biomass and PHB production (90.71 g/L biomass with 45.84 % PHB content and 1.73 g/L h PHB productivity). Similar biomass and PHB yields were obtained in the 90 L stirred fermentor when using the impeller tip speed as the scale-up criterion.     

NADP+-malate dehydrogenase in C3-plants: Regulation and role of a light-activated enzyme

The thioredoxin-dependent light/dark modulation system of the chloroplast is described as a prerequisite enabling the flexible control of fluxes through the various parts of the CO₂-fixation pathway. Both the rapid turnover of the reduced thiol-containing form of the respective target enzyme, and the metabolite effect upon the reductive enzyme modulation, allow rapid adjustment of the amount of active species to the actual requirements. The structural basis of the regulation of chloroplast NADP⁺-malate dehydrogenase (EC 1.1.1.82) is described in more detail. The modulable plastid enzyme is characterized by two sequence extensions not present in any other known NADP⁺- and/or NAD⁺-specific malate dehydrogenase. The NADP⁺-malate dehydrogenase of C₃-plants is part of the "malate valve", which catalyzes the export of reducing equivalents in the form of malate from the chloroplast only when the NADPH to NADP⁺ ratio is high, thus poising the NADPH to ATP ratio required for optimal carbon reduction in the light. The mode of regulation of other light/dark modulated enzymes is discussed.     

Utilization of oil extracted from spent coffee grounds for sustainable production of polyhydroxyalkanoates

Spent coffee grounds (SCG), an important waste product of the coffee industry, contain approximately 15 wt% of coffee oil. The aim of this work was to investigate the utilization of oil extracted from SCG as a substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16. When compared to other waste/inexpensive oils, the utilization of coffee oil resulted in the highest biomass as well as PHB yields. Since the correlation of PHB yields and the acid value of oil indicated a positive effect of the presence of free fatty acids in oil on PHB production (correlation coefficient R ²?=?0.9058), superior properties of coffee oil can be probably attributed to the high content of free fatty acids which can be simply utilized by the bacteria culture. Employing the fed-batch mode of cultivation, the PHB yields, the PHB content in biomass, the volumetric productivity, and the Y _P/S yield coefficient reached 49.4 g/l, 89.1 wt%, 1.33 g/(l h), and 0.82 g per g of oil, respectively. SCG are annually produced worldwide in extensive amounts and are disposed as solid waste. Hence, the utilization of coffee oil extracted from SCG is likely to improve significantly the economic aspects of PHB production. Moreover, since oil extraction decreased the calorific value of SCG by only about 9 % (from 19.61 to 17.86 MJ/kg), residual SCG after oil extraction can be used as fuel to at least partially cover heat and energy demands of fermentation, which should even improve the economic feasibility of the process.     

Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli

The gene for Escherichia coli glutamate dehydrogenase (EcGDH) has been overexpressed, and a simplified purification procedure afforded greatly increased yields of c. 40 mg pure EcGDH L⁻¹ culture. EcGDH was unstable at a low concentration in plastic tubes, but stabilization measures allowed a robust kinetic characterization. Contrary to past reports, EcGDH deviates from Michaelis–Menten kinetics, exhibiting apparent mild negative co-operativity with both l -glutamate and NADP⁺, with Hill coefficients of 0.90 and 0.92, respectively. NADPH yielded simple Michaelis–Menten kinetics but both 2-oxoglutarate and NH₄⁺ showed substrate inhibition. pH optima were 9 for oxidative deamination and 8 for reductive amination.     

NADP+-isocitrate dehydrogenase in germinating cucumber cotyledons: Purification and characterization of a cytosolic isoenzyme

The activity of NADP⁺-dependent isocitrate dehydrogenase (ICDH, EC 1.1.1.42) was investigated during the post-germinative growth of cucumber ( Cucumis sativus L. cv. Marketmore) seedlings. Isoelectric focusing showed the presence of several isoenzymes, two of which represented 70–80% of the total NADP⁺-ICDH activity in cotyledons of seedlings grown in the dark. They had pI values between 4.8 and 5.8. The isoenzyme with higher pI was purified to homogeneity by hydrophobic interaction, affinity, hydroxylapatite and anion exchange chromatography. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 43-kDa subunits. It is specific for NADP⁺, inhibited by ATP and by 2-oxoglutarate, whereas it is not inhibited by citrate, succinate, and glyoxylate. The data indicate that NADP⁺-ICDH from cucumber is structurally similar to ICDHs from other plants, but it shows some peculiar biochemical characteristics.     

Cultivation of Thermoanaerobium brockii in continuous culture with complete cell recycling

The growth behaviour of the thermophilic anaerobic bacterium Thermoanaerobium brockii for the production of its intracellular secondary alcohol dehydrogenase (sADH) has been studied in batch cultures as well as in continuous cultivation with complete cell recycling. In batch culture the maximum specific growth rate, μ_MAX, was 0·5 h⁻¹, resulting in a cell density of 1·2 g l⁻¹ and an sADH activity of 1·3 units ml⁻¹. Higher glucose concentrations resulted in a decrease in ep cf7 max rs, enzyme productivity as well as biomass yield although an increase in total biomass was achieved. To improve cell density and productivity, continuous culture with complete cell recycling was used, resulting in an increase in cell density by 5 times and in productivity of the sADH by 3 times in comparison to those obtained in batch culture.     

Production of poly-3-hydroxybutyrate from lactose and whey by Methylobacterium sp. ZP24

Methylobacterium sp. ZP24, isolated from a local pond, is able to grow in a medium containing 12 g l⁻¹ lactose as a sole source of carbon, giving 5·25 g l⁻¹ biomass yield and poly-3-hydroxybutyrate (PHB) up to 59% of its dry weight in 40 h. The isolate was also able to utilize cheese whey and produce 1·1 g l⁻¹ PHB. Addition of ammonium sulphate increased the production of PHB from whey 2·5-fold. The potential of Methylobacterium sp. ZP24 in PHB production from cheese whey is discussed.     

Prediction of volumetric productivity of an outdoor photobioreactor

Volumetric productivity of Monodus subterraneus cultivated in an outdoor pilot-plant bubble column was predicted with a mathematical model. Two border cases to model the photobioreactor were chosen. Firstly, a model with no light integration in which it is assumed that microalgae can adapt immediately to local light conditions. Secondly, full light integration implicating that microalga can convert all absorbed light with a photosynthetic yield based on average light intensity. Because temperature and light conditions in our photobioreactor changed during the day, photosynthetic yields at any combination of temperature and light intensity were needed. These were determined in repeated-batch lab-scale experiments with an experimental design. The model was evaluated in an outdoor bubble column at different natural light conditions and different temperatures. Volumetric productivities in the bubble column were predicted and compared with experimental volumetric productivities. The light integration model over-estimated productivity, while the model in which we assumed no light integration under-estimated productivity. Light integration occurred partly (47%) during the period investigated. The average observed biomass yield on light was 0.60 g.mol(-1). The model of partly light integration predicted an average biomass yield on light of 0.57 g.mol(-1) and predicted that productivity could have been increased by 19% if culture temperature would have been maintained at 24 degrees C.     

利用克雷伯肺炎杆菌限制性底物发酵生产1，3-丙二醇

研究了克雷伯肺炎杆菌（Klebsiella pneumoniae）批式流加发酵生产1,3-丙二醇的发酵工艺,根据1,3-丙二醇的生产和菌体生长相关的特点,采用营养基质限制性流加的发酵工艺,通过控制氮源氯化铵以保持细胞稳定生长。结果表明：过低的氮源浓度,细胞生长受到限制,影响产物1,3-PD的合成;过高的氮源浓度,细胞比生长速率增加,但1,3-PD关于消耗甘油的得率降低,用于生长和维持代谢所消耗的甘油量增加。以0.41 g/(L·h)的氮源流加速率,残余氯化铵浓度在0.1 g/L时,转化率和生产强度最高。发酵25 h～28 h后,1,3-丙二醇最终浓度达到52.03 g/L,生产强度为2.04 g/(L·h),相对于甘油的摩尔转化率为0.66,分别比氮源限制前提高了28.0 ％、35.1 ％及29.4 ％。通过限制性流加氯化铵,控制细胞的比生长速率,使底物甘油有效转变为发酵的目标产物1,3-PD,有效实现产物1,3-PD的高生产强度以及对甘油的高转化率。     

Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum

The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield Y_P/S = 0·24 g/g and process productivity 2·29 g/litre·h.     

Characteristics of the reverse reaction of NADP+-isocitrate dehydrogenase from castor bean seeds

The reductive carboxylation of α-ketoglutarate by purified NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) from maturing castor bean seeds ( Ricinus communis L. ) has been characterized. The optimum pH for the reaction was 6.5, whereas pH 8.5 was optimum for oxidation of isocitrate (forward reaction). The enzyme utilized NADH as well as NADPH as the reducing agent in the reverse reaction, but only NADP⁺ in the forward reaction. The Km values for NADPH and NADH were 0.044 and 2.8 m M respectively, and for α-ketoglutarate and HCO₃ 4.1 and 3.7 m M. The enzyme was activated by various cations including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺ and Co²⁺. Km values for Mg²⁺ Mn²⁺, Co²⁺ and Zn²⁺ were 12, 34, 37 and 49μ M respectively.     

Electron transfer reactions for the reduction of NADP+ in Methanosphaera stadtmanae

Abstract Methanosphaera stadtmanae , a member of the Methanobacteriales reduces methanol, but not CO₂ with H₂ or 2-propanol to produce methane. In cell-free extracts of M. stadtmanae the activities of several enzymes involved in electron transfer were measured. The activities of an F₄₂₀-nonreactive hydrogenase, NADP⁺: F₄₂₀ oxidoreductase, NADP⁺-dependent 2-propanol dehydrogenase, and a methyl viologen dependent F₄₂₀ dehydrogenase were observed. Based on the presence of these particular enzyme activities, their cofactor requirements and the absence of F₄₂₀-dependent hydrogenase activity, a model of the electron transport pathway through the coenzyme F₄₂₀ to provide electrons for biosynthesis, was formulated.     

Characterization of NADP+-isocitrate dehydrogenase from the host plant cytosol of lucerne (Medicago sativa) root nodules

NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) was purified more than 1500-fold from the host-plant cytosol of Medicago sativa L. cv. Saranac root nodules by ion exchange and affinity chromatography. The forward reaction was characterized. The enzyme exhibited an absolute requirement for a divalent cation (preferably Mn²⁺), had a broad activity optimum from pH 7.5 to 9.0, and was most stable at pH 7.5. The apparent Arrhenius energy of activation was 70.7 kJ mol⁻¹ (20 to 30°C) indicating that the reaction rate of the enzyme was relatively sensitive to temperature. The K_m for isocitrate was 20 μ M and for NADP⁺ 10.7 μ M . Initial velocity and end product inhibition studies of the forward reaction indicate a random bi ter mechanism. End product studies indicated that NADPH was a competitive inhibitor and α-ketoglutarate was a non-competitive inhibitor with respect to isocitrate and NADP⁺. Citrate was a competitive inhibitor with respect to isocitrate. Glutamine was identified as a positive effector when assays were conducted at non-saturating isocitrate concentrations. The potential significance of glutamine regulation of α-ketoglutarate production in a dinitrogen-fixing tissue is discussed.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Controlling substrate concentration in fed-batch candida magnoliae culture increases mannitol production

Candida magnoliae HH-01, a yeast strain that is currently used for the industrial production of mannitol, has the highest mannitol production ever reported for a mannitol-producing microorganism. However, when the fructose concentration exceeds 150 g/L, the volumetric mannitol production rate decreases because of a lag in mannitol production, and the yield decreases as a result of the formation of side products. In fed-batch culture, the volumetric production rate and mannitol yield from fructose vary substantially with the fructose concentration and are maximal at a controlled fructose concentration of 50 g/L. In continuous feeding experiments, the maximum mannitol yield was 85% (g/g) at a glucose/fructose feeding ratio of 1/20. A high glucose concentration in the production phase resulted in the formation of ethanol followed by a decrease in yield and productivity. NAD(P)H-dependent mannitol dehydrogenase was purified to homogeneity from C. magnoliae. In vitro, mannitol dehydrogenase was inhibited by increasing ethanol concentration. Mannitol product was also found to be inhibitory with a K(i) of 183 mM. Under optimum conditions, a final mannitol production of 213 g/L was obtained from 250 g fructose/L after 110 h.     

Study of metabolic network of Cupriavidus necator DSM 545 growing on glycerol by applying elementary flux modes and yield space analysis

A metabolic network consisting of 48 reactions was established to describe intracellular processes during growth and poly-3-hydroxybutyrate (PHB) production for Cupriavidus necator DSM 545. Glycerol acted as the sole carbon source during exponential, steady-state cultivation conditions. Elementary flux modes were obtained by the program Metatool and analyzed by using yield space analysis. Four sets of elementary modes were obtained, depending on whether the pair NAD/NADH or FAD/FADH₂ contributes to the reaction of glycerol-3-phosphate dehydrogenase (GLY-3-P DH), and whether 6-phosphogluconate dehydrogenase (6-PG DH) is present or not. Established metabolic network and the related system of equations provide multiple solutions for the simultaneous synthesis of PHB and biomass; this number of solutions can be further increased if NAD/NADH or FAD/FADH₂ were assumed to contribute in the reaction of GLY-3-P DH. As a major outcome, it was demonstrated that experimentally determined yields for biomass and PHB with respect to glycerol fit well to the values obtained in silico when the Entner–Doudoroff pathway (ED) dominates over the glycolytic pathway; this is also the case if the Embden–Meyerhof–Parnas pathway dominates over the ED.     

Growth of Methylosinus trichosporium OB3b on methane and poly-beta-hydroxybutyrate biosynthesis

Optimal conditions for batch cultivation of the obligate methanotroph Methylosinus trichosporium OB3b on methane without superatmospheric pressure were chosen. The yield of absolutely dry biomass after 120 h of growth reached 20 g/l. This biomass contained 30% poly-beta-hydroxybutyrate (PHB) with molecular weight 300 kDa. The growth process included the stages of biomass growth and PHB biosynthesis. The latter stage occurred under nitrogen-deficiency conditions. It was accompanied by an increase in the activity of PHB biosynthesis enzymes (beta-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase) and the main NAD(P)H producer, methylenetetrahydromethanopterin dehydrogenase. The activity of PHB depolymerase increased insignificantly.     

The physiology of lactate production by Lactobacillus delbreuckii in a chemostat with cell recycle

The physiology of lactate production by Lactobacillus delbreuckii NRRL B-445 in a continuous fermenter with partial cell recycle has been studied and compared with that observed in a conventional chemostat. Partial cell recycle was achieved using a hollow-fiber ultrafiltration cartridge. The biomass growth yield was reduced in the recycle fermenter while culture viability and the cellular content of polysaccharide, protein, carbon, and nitrogen remained constant, suggesting an enlarged specific rate of glucose consumption for nonanabolic (e.g., maintenance) functions. The volumetric productivity of lactate was enhanced in the recycle fermenter due to the complete utilization of glucose. The yield of lactate from biomass and the molar product ratio, lactate: ethanol plus acetate, decreased with increasing recycle ratio. Enhanced formation of ethanol and acetate occurred in the recycle fermenter although lactate remained the major product. The change in product profile was due to glucose limitation. The specific activity of lactate dehydrogenase remained constant during recycle fermentation. These physiological observations have implications for the future application of cell recycle to production processes.     

Growth of <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b on methane and poly-β-hydroxybutyrate biosynthesis

Optimal conditions for batch cultivation of the obligate methanotroph Methylosinus trichosporium OB3b on methane without superatmospheric pressure were chosen. The yield of absolutely dry biomass after 120 h of growth reached 20 g/l. This biomass contained 30% poly-β-hydroxybutyrate (PHB) with molecular weight 300 kDa. The growth process included the stages of biomass growth and PHB biosynthesis. The latter stage occurred under nitrogen-deficiency conditions. It was accompanied by an increase in the activity of PHB biosynthesis enzymes (β-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase) and the main NAD(P)H producer, methylenetetrahydromethanopterin dehydrogenase. The activity of PHB depolymerase increased insignificantly.