发酵过程中溶氧浓度对D-核糖发酵的影响

通过固定不同溶氧浓度(DOT)对短小芽孢杆菌(Bacillus pumlus)进行分批发酵的过程参数变化的比较,发现发酵前期与后期对氧的需求不尽相同,探讨了氧代谢途径及溶氧浓度对核糖发酵的影响机理,并提出分阶段供氧模式。结果表明,发酵时间44h后,整个发酵过程保持了较高的核糖产率和葡萄糖消耗率,最终核糖产量和细胞生成量分别提高了5.0％和18.8％。     

透明颤菌血红蛋白基因在产PHB重组大肠杆菌中的引入

将透明颤菌血红蛋白基因 (vgb)采用插入染色体的方式引入产聚 β 羟基丁酸酯(PHB)重组大肠杆菌VG1 (pTU1 4)中 ,以从分子水平上提高克隆菌对氧的利用率 ,解决PHB发酵生产过程中的供氧矛盾 ,透明颤菌血红蛋白的一氧化碳差光谱分析明表 ,vgb基因可以在VG1 (pTU1 4)中成功表达 ,且其表达量受溶氧水平的调控。Vgb基因的引入可以同时促进菌体生长和PHB产品的积累 ,且溶氧水平越低 ,VHB表达量越高 ,这种促进作用就越明显     

真养产碱杆菌积聚-β-羟基丁酸发酵条件的研究

igenes eutrophus培养过程的研究表明,氮源的限制或缺乏可刺激细胞大量积累聚-β-羟基丁酸(PHB),但PHB合成期氮源的完全缺乏,会导致细胞的PHB合成速率迅速下降;氧的限制也可刺激A.eutrophus合成PHB,但胞内PHB的积累量远小于氮源控制下的情况。在细胞的不同生长期限制氮源的供应会明显影响PHB的发酵过程,当残留菌体浓度达到20g/L至30g/L时停止流加氨水,可以得到较好的发酵水平,细胞干重,PHB含量和PHB浓度可分别达到61.9g/L、80.5%和49.0g/L。     

真养产碱菌利用高果糖浆积累聚β-羟基丁酸的研究

真养产碱菌(Alcaligenes eutrophus)H16能以果糖为碳源在无机合成培养基上积累聚p-羟基丁酸(PHB)。将该菌用富含果糖的高果糖浆(HFS)培养,PHB的积累可达到果糖发酵水平,但发现高浓度的果糖和葡萄糖对菌体生长及PHB积累有抑制作用。采用补料分批培养技术可降低果糖和葡萄糖的抑制。并可大幅度提高产量,菌体干重达16—20g/L,PHB产量7.0—7.6g/L,PHB的产率达0.24g/g果糖。     

氮源流加对Alcaligenes eutrophus积累聚β-羟基丁酸的影响

以真养产碱杆菌(Alcaligenes eutrophus)为 聚β羟基丁酸(PHB)的生产菌株,在分析了PHB发酵过程参数变化的基础上,进一步探讨了PHB合成期不同的硫酸铵流加速率对PHB合成的影响。研究结果表明,在PHB合成阶段,培养基中氮源的完全缺乏,导致细胞合成PHB能力的下降;在PHB合成期,不同的氮源流加速率对PHB合成过程存在着显著的影响,当流加速率较小时,尽管最终胞内PHB含量很高,但细胞干重、PHB浓度和PHB生产强度都较低。当氮源流加速率过大时,会导致最终胞内PHB含量显著下降,使PHB浓度和PHB生产强度降低。当硫酸铵流加速率在05g/h左右时,可以得到较好的发酵效果。     

爱德华氏菌产乳酸氧化酶的条件研究

在以前工作的基础上,对已获得的产乳酸氧化酶的5株菌进行复筛,对产酶量大的一株菌进行了分类鉴定,确定该菌株属迟钝爱德华氏菌生物群I(Edwardsiella tarda Biogroup I)。这与曾报道的产乳酸氧化酶分枝杆菌(Mycobacterium)和片球菌(Pediococcus)是不同的菌。分别研究了培养基中的培养初始pH、核黄素、乳酸钠以及硫酸铵对发酵产乳酸氧化酶的影响。这一酶源在酶法生产丙酮酸及医疗诊断和酶电极应用上有意义。     

乙酸菌糖磷酸化作用的研究

对7种乙酸菌(Acetitomaculum ruminis, Acetobacterium woodii, Eubacterium limosum和分离株A2、A4、A10、H3HH)的葡萄糖和2脱氧葡萄糖磷酸化作用进行了研究。尽管所有机体都存在磷酸化反应,但它们在依赖PEP和ATP的比例上有实质性区别。分离菌株A10具有最高的依赖PEP的葡萄糖磷酸化活力(1162nmol·L-1·mg-1·min-1),A10、H3HH和E.limosum都具有葡萄糖磷酸转移酶系统(phosphotransferase systemPTS)。相反,Ａ.ruminis、A.woodii、A2和A4则不具有PTS活力。这七株菌的葡萄糖依赖ATP的磷酸化活力都高于依赖PEP的磷酸化活力,但其程度有所不同。A10和H3HH的葡萄糖PTS可通过胞外葡萄糖诱导,并且其比活在对数期随培养时间延长而增加。此外,还检测到A10和H3HH对麦芽糖和果糖的依赖ATP和PEP的磷酸化活力。     

滇重楼寄生菌的研究

从滇重楼(Paris polyphylla var.yunnanensis)地下茎中分离和鉴定出两种细菌——蜡状芽孢杆菌(Bacillus cereus)和产碱假单胞菌(Pseudomonas alcaligenes),以及三种真菌——黑团孢霉(Periconia sp.)、白色厚顶孢霉(Pachnocybe albida)和重楼索霉(Hormomyces paridiphilus)。对蜡状芽孢杆菌、产碱假单胞菌和重楼索霉进行了液体培养并测定了胞外多糖含量,结果表明重楼索霉可分泌大量胞外多糖,这可能是导致滇重楼地下茎胶质化和多糖含量增加的原因。     

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatum Z7产氢效率的因素,采用生理学方法对其进行了研究。结果表明：乙醇型发酵菌A. elongatum Z7的最适产氢温度为37℃, 最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度> 60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。     

从代谢流量分析角度探讨培养条件改变下对放射型根瘤菌WSH2601合成辅酶Q10的影响

分析了放射型根瘤菌(R. radiobacter) WSH2601生物合成辅酶Q10的代谢途径网络,并在溶氧条件改变和培养基中添加玉米浆条件下对辅酶Q10发酵细胞内代谢途径流量变化作定量的分析,结果表明：提高溶氧浓度（20%）5_磷酸核酮糖（Ru5P）物流（r7）增加26.6,即糖酵解途径（EMP）途径向磷酸戊糖途径（HMP）转移;添加1%玉米浆r7增加17.2,EMP与HMP途径物流比值与三羧酸循环（TCA）途径物流都下降,而癸异戊烯基焦磷酸（DPP）生成物流通量（绝对值）变化都较小,即辅酶Q10的生物合成更大程度地取决于辅酶Q10生物合成途径中催化DPP的合成和4_羟基苯甲酸（PHB）与DPP的缩合反应的两种关键酶活性。6_磷酸葡萄糖（G6P）节点是辅酶Q10生物合成代谢途径的柔性节点,而丙酮酸节点是半柔性节点。细胞生物量的提高与HMP途径物流增加有关。     

Modeling of the mixed culture and periodic control for PHB production

The unstructured mathematical model was developed in the present investigation for the mixed culture, where the metabolites produced by one microorganism is assimilated by the other microorganism. For this, we specifically employed such model system in which sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted in turn to poly-β-hydroxybutyrate (PHB) by Ralstonia eutropha in one fermentor. Several batch and fed-batch culture experiments were conducted using each microorganism at different dissolved oxygen (DO) concentrations. Those experimental data were then fitted to the mathematical model, which can describe the dynamics of a mixed culture. Some of the model parameters were expressed as functions of DO concentrations, and some of the other model parameters were tuned based on the mixed culture experiments. The model developed describes the effects of such concentrations of glucose, lactate, DO, and NH₃ on the dynamic behavior of such concentrations as both microorganisms, glucose, lactate, and PHB. Optimal operating condition was then investigated using the model developed. It was found that the periodic change in DO concentration improved such performance as PHB yield, and it was verified by experiments. The optimal NH₃ concentration profile was also obtained for the efficient PHB production by the application of the maximum principle.     

Metabolic and kinetic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli

A quantitatively repeatable protocol was developed for poly(3-hydroxybutyrate) (PHB) production by Escherichia coli XL1-Blue (pSYL107). Two constant-glucose fed-batch fermentations of duration 25 h were carried out in a 5-L bioreactor, with the measured oxygen volumetric mass-transfer coefficient (k(L)a) held constant at 1.1 min(-1). All major consumption and production rates were quantified. The intracellular concentration profiles of acetyl-CoA (300 to 600 microg x g RCM(-1)) and 3-hydroxybutyryl-CoA (20 to 40 microg x g RCM(-1)) were measured, which is the first time this has been performed for E. coli during PHB production. The kinetics of PHB production were examined and likely ranges were established for polyhydroxyalkanoate (PHA) enzyme activity and the concentration of pathway metabolites. These measured and estimated values are quite similar to the available literature estimates for the native PHB producer Ralstonia eutropha. Metabolic control analysis performed on the PHB metabolic pathway showed that the PHB flux was highly sensitive to acetyl-CoA/CoA ratio (response coefficient 0.8), total acetyl-CoA + CoA concentration (response coefficient 0.7), and pH (response coefficient -1.25). It was less sensitive (response coefficient 0.25) to NADPH/NADP ratio. NADP(H) concentration (NADPH + NADP) had a negligible effect. No single enzyme had a dominant flux control coefficient under the experimental conditions examined (0.6, 0.25, and 0.15 for 3-ketoacyl-CoA reductase, PHA synthase, and 3-ketothiolase, respectively). In conjunction with metabolic flux analysis, kinetic analysis was used to provide a metabolic explanation for the observed fermentation profile. In particular, the rapid onset of PHB production was shown to be caused by oxygen limitation, which initiated a cascade of secondary metabolic events, including cessation of TCA cycle flux and an increase in acetyl-CoA/CoA ratio.     

Metabolic engineering of pentose phosphate pathway in Ralstoniaeutropha for enhanced biosynthesis of poly-beta-hydroxybutyrate

Poly-beta-hydroxybutyrate (PHB) biosynthesis in Ralstonia eutropha from gluconate as a carbon source is carried out through the Entner-Doudoroff (ED) pathway and the pentose-phosphate (PP) pathway generating NADPH and glyceraldehyde-3-phosphate that flows to acetyl-CoA, actively in the unbalanced PHB accumulation phase. The gnd gene encoding 6-phosphogluconate dehydrogenase (6PGDH) and the tktA gene encoding the transketolase (TK) in PP pathway of E. coli were transformed into R. eutropha H16 to modify the metabolic flux of gluconate to the PHB biosynthesis. Over-generated NADPH by the amplified gnd gene tended to depress the cell growth and PHB concentration. Meanwhile, the amplified tktA gene significantly increased both PHB biosynthesis and cell growth as a result of the effective flow of glyceraldehyde-3-phosphate into acetyl-CoA along with the concomitant supplementation of NADPH. The amplified tktA gene also activated the enzyme activities directly associated with PHB biosynthesis. The transformant R. eutropha harboring tktA gene was cultivated using pH-stat-fed-batch to achieve the overproduction of PHB.     

The effect of dissolved oxygen on PHB accumulation in activated sludge cultures

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields. The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth.     

Effects of dissolved oxygen on hybridoma cell growth, metabolism, and antibody production kinetics in continuous culture.

The effects of dissolved oxygen concentration (DO) on hybridoma cell physiology were examined in a continuous stirred tank bioreactor with a murine hybridoma cell line (167.4G5.3). Dissolved oxygen concentration was varied between 0% and 100% air saturation. Cell growth and viability, carbohydrate, amino acid, and energy metabolism, oxygen uptake, and antibody production rates were investigated. Cell growth was inhibited at both high and low DO. Cells could grow at 0% DO and maintain viability under a nitrogen atmosphere. Cell viability was higher at low DO. Glucose, glutamine, and oxygen consumption rates changed little at DO above 1% air saturation. However, the metabolic uptake rates changed below 1% DO, where growth became oxygen limited, and a Km value of 0.6% DO was obtained for the specific oxygen uptake rate. The metabolic rates of glucose, glutamine, lactate, and ammonia increased 2-3-fold as the DO dropped from 1% to 0%. Amino acid metabolism followed the same general pattern as that of glutamine and glucose. Alanine was the only amino acid produced. The consumption rates of amino acids changed little above 1% DO, but under anaerobic conditions the consumption rates of all amino acids increased severalfold. Cells obtained most of their metabolic energy from glutamine oxidation except under oxygen limitation, when glucose provided most of the energy. The calculated ATP production rate was only slightly influenced by DO and rose at 0% DO. Antibody concentration was highest at 35% DO, while the specific antibody production rate was insensitive to DO.     

Periodic change in DO concentration for efficient poly-β-hydroxy-butyrate production using temperature-inducible recombinant<Emphasis Type="Italic">Escherichia coli</Emphasis> with proteome analysis

RecombinantEscherichia coli strain harboring the λp _R-p _L promotor and heterologus poly-β-hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression ofphb genes was induced by a temperature upshift from 33°C to 38°C. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lactate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.     

Dynamics and modeling on fermentative production of poly (β-hydroxybutyric acid) from sugars via lactate by a mixed culture of Lactobacillus delbrueckii and Alcaligenes eutrophus

The mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly β-hydroxybutyrate (PHB) by Alcaligenes eutrophus in one fermentor. For the modeling of the effect of NH₃ concentration on the cell growth of A. eutrophus and PHB production rates, metabolic flux distributions were computed at two culture phases of cell growth and PHB production periods. It was found that the NADPH, generated through isocitrate dehydrogenate in TCA cycle, was predominantly utilized for the reaction from α-ketoglutalate to glutamate when NH₃ was abundant, while it tended to be utilized for the PHB production through acetoacetyl CoA reductase as NH₃ concentration decreased. This phenomenon was reflected in the development of mathematical model. In the mixed culture experiments, the two phases were observed, namely the lactate production phase due to L. delbrueckii and the lactate consumption phase due to A. eutrophus. The lactate concentration could be estimated on-line by the amount of NaOH solution and HCl solution supplied to keep the culture pH at constant level. Several mixed culture experiments were conducted to see the dynamics of the system. Finally, a mathematical model which can describe the dynamic behavior of the present mixed culture was developed and the model parameters were tuned for fitting the experimental data. The model may be used for several purposes such as control, optimization, and understanding process dynamics etc.     

Metabolic flux modeling of detoxification of acetic acid by Ralstonia eutropha at slightly alkaline pH levels

Ralstonia eutropha grows on and produces polyhydroxyalkanoates (PHAs) from fermentation acids. Acetic acid, one major organic acid from acidogenesis of organic wastes, has an inhibitory effect on the bacterium at slightly alkaline pH (6 g HAc/L at pH 8). The tolerance of R. eutropha to acetate, however, was increased significantly up to 15 g/L at the slightly alkaline pH level with high cell mass concentration. A metabolic cell model with five fluxes is proposed to depict the detoxification mechanism including mass transfer and acetyl-CoA formation of acetic acid and the formation of three final metabolic products, polyhydroxybutyrate (PHB), active biomass, and CO(2). The fluxes were measured under different conditions such as cell mass concentration, acetic acid concentration, and medium composition. The experimental results indicate that the acetate detoxification by high cell mass concentration is attributed to the increased fluxes at high extracellular acetate concentrations. The fluxes could be doubled to reduce and hence detoxify the accumulated intracellular acetate anions.     

Cloning of an intracellular Poly[D(-)-3-Hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product

An intracellular poly[D(-)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(-)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.     

Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha

Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.