Purification and characterization of a methanol dehydrogenase derived fromMethylomicrobium sp. HG-1 cultivated using a compulsory circulation diffusion system

Methanotrophs are microorganisms that possess the unique ability to utilize methane as their sole source of carbon and energy. A novel culture system, known as the compulsory circulation diffusion system, was developed for rapid growth of methanotrophic bacteria. Methanol dehydrogenase (MDH, EC 1.1.99.8) fromMethylomicrobium sp. HG-1, which belongs to the type 1 group of methanotrophic bacteria, can catalyze the oxidation of methanol directly into formaldehyde. This enzyme was purified 8-fold to electrophoretic homogeneity by means of a 4 step procedure and was found in the soluble fraction. The relative molecular weight of the native enzyme was estimated by gel filtration to be 120 kDa. The enzyme consisted of two identical dimers which, in turn, consisted of large and small subunits in anα ₂ β ₂ conformation. The isoelectric point was 5.4. The enzymatic activity of purified MDH was optimum at pH 9.0 and 60°C, and remained stable at that temperature for 20 min. MDH was able to oxidize primary alcohols from methanol to octanol and formaldehyde.     

Co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of recombinant Klebsiella pneumoniae J2B strain overexpressing aldehyde dehydrogenase

The co-production of 3-hydroxypropionic acid (3HP) and 1,3-propanediol (PDO) from glycerol was studied using the resting cells of a recombinant Klebsiella pneumoniae J2B strain that overexpresses an aldehyde dehydrogenase (KGSADH). Active biomass was produced in a mineral salt medium containing yeast extract and glycerol under a range of aeration conditions, and shifted to potassium phosphate buffer containing glycerol for bioconversion. The microaerobic or anaerobic conditions were favorable for both the production of active biomass and subsequent bioconversion. At the flask level, the recombinant strain (2.0?g?CDW/L) grown under microaerobic conditions produced 43.2?mM 3HP and 59.0?mM PDO from glycerol (117?mM) in 30?min with a cumulative yield of 0.87?(mol/mol). The fed-batch bioconversion, which was performed in a 1.5-L bioreactor with 1.0?g?CDW/L at a constant pH?7.0 under anaerobic conditions, resulted in 125.6?mM 3HP and 209.5?mM PDO in 12?h with a cumulative overall productivity, yield, and maximum specific production rate of 27.9?mmol/L/h, 0.71 (mol/mol), and 128.5?mmol/g CDW/h, respectively. Lactate, succinate and 2,3-butanediol were the major by-products, whereas the production of acetate and ethanol was marginal. This is the first report of the simultaneous production of 3HP and PDO from glycerol using a resting cell system.     

Immobilization of <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b for methanol production

Due to the natural gas boom in North America, there is renewed interest in the production of other chemical products from methane. We investigated the feasibility of immobilizing the obligate methanotrophic bacterium Methylosinus trichosporium OB3b in alginate beads, and selectively inactivating methanol dehydrogenase (MDH) with cyclopropane to produce methanol. In batch cultures and in semi-continuous flow columns, the exposure of alginate-immobilized cells to cyclopropane or cyclopropanol resulted in the loss of the majority of MDH activity (> 80%), allowing methanol to accumulate to significant concentrations while retaining all of M. trichosporium OB3b’s methane monooxygenase capacity. Thereafter, the efficiency of methanol production fell due to recovery of most of the MDH activity; however, subsequent inhibition periods resulted in renewed methanol production efficiency, and immobilized cells retained methane-oxidizing activity for at least 14 days.     

Selective bio-oxidation of propane to acetone using methane-oxidizing <Emphasis Type="Italic">Methylomonas</Emphasis> sp. DH-1

Propane is the major component of liquefied petroleum gas (LPG). Nowadays, the use of LPG is decreasing, and thus utilization of propane as a chemical feedstock is in need of development. An efficient biological conversion of propane to acetone using a methanotrophic whole cell as the biocatalyst was proposed and investigated. A bio-oxidation pathway of propane to acetone in Methylomonas sp. DH-1 was analyzed by gene expression profiling via RNA sequencing. Propane was oxidized to 2-propanol by particulate methane monooxygenase and subsequently to acetone by methanol dehydrogenases. Methylomonas sp. DH-1 was deficient in acetone-converting enzymes and thus accumulated acetone in the absence of any enzyme inhibition. The maximum accumulation, average productivity and specific productivity of acetone were 16.62 mM, 0.678 mM/h and 0.141 mmol/g cell/h, respectively, under the optimized conditions. Our study demonstrates a novel method for the bioconversion of propane to acetone using methanotrophs under mild reaction condition.     

Optimization of lab scale methanol production by <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

Methylosinus trichosporium OB3b is a methanotrophic bacterium containing particulate methane monooxygenase (MMO), which catalyzes the hydroxylation of methane to methanol. The methanol is further oxidized to formaldehyde by methanol dehydrogenase (MDH). We developed a novel compulsory circulation diffusion system for cell cultivation. A methane/air mixture (1:1, v/v) was prepared in a tightly sealed gas reservoir and pumped into a nitrate mineral salt culture medium under optimal conditions (5 μM CuSO₄, pH 7.0, 30°C). Cells were harvested, washed, and resuspended (0.6 mg dry cells/mL) in a 500 mL flask in 100 mL of 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA as MDH inhibitors, and 20 mM sodium formate. A single 12 h batch reaction at 25°C yielded a final concentration of 13.2 mM methanol. The use of a repeated batch mode, in which the accumulated methanol was removed after each of three 8 h cycles over a 24 h period, showed a productivity of 2.17 μmol methanol/h/mg dry cell wt. Finally, a lab-scale reaction performed using a 3 L cylindrical reactor with a working volume of 1 L produced 13.7 mM methanol after 16 h. Our results identify a simple process for improving the productivity of biologically derived methanol and, therefore the utility of methane as an energy source.     

Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

Acetogenic bacteria use CO and/or CO₂ plus H₂ as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h⁻¹ optical density unit⁻¹), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production.     

Potential and limitations of Klebsiella pneumoniae as a microbial cell factory utilizing glycerol as the carbon source

Klebsiella pneumoniae is a Gram-negative facultative anaerobe that metabolizes glycerol efficiently under both aerobic and anaerobic conditions. This microbe is considered an outstanding biocatalyst for transforming glycerol into a variety of value-added products. Crude glycerol is a cheap carbon source and can be converted by K. pneumoniae into useful compounds such as lactic acid, 3-hydroxypropionic acid, ethanol, 1,3-propanediol, 2,3-butanediol, and succinic acid. This review summarizes glycerol metabolism in K. pneumoniae and its potential as a microbial cell factory for the production of commercially important acids and alcohols. Although many challenges remain, K. pneumoniae is a promising workhorse when glycerol is used as the carbon source.     

Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria,using α-humulene as a model compound

Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden–Meyerhof–Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria.     

甲醇的生物利用与转化*

甲醇作为一种来源广泛、价格低廉、还原度高的非粮原料有望成为下一代生物制造的关键原料。利用合成生物学技术构建能够高效利用甲醇的重组微生物以实现从甲醇到高值化学品的生物转化已成国内外研究热点,但由于甲醇代谢过程的特殊性及复杂性,目前人工设计的甲基营养菌还难以实现以甲醇为唯一碳源进行生长及产物合成。基于对天然甲基营养菌甲醇代谢过程的分析,从甲醇脱氢酶的筛选与改造、甲醛同化途径的重构与优化、甲醇到化学品的生物转化几个方面对合成型甲基营养菌的构建策略及面临的挑战进行总结与分析,以期为今后合成型甲基营养菌的人工设计和利用提供一定的借鉴。     

一碳代谢关键酶——甲醇脱氢酶的研究进展与展望

甲醇和甲烷等一碳原料来源广泛,价格低廉,是生物制造的理想原料。甲醇脱氢酶(Methanol dehydrogenase,MDH)催化甲醇生成甲醛是一碳代谢的关键反应。目前已从天然甲基营养菌中发现了多种利用不同辅因子,具有不同酶学性质的MDH。其中,烟酰胺腺嘌呤双核苷酸(NAD)依赖型MDH被广泛应用于构建人工甲基营养菌。但是,NAD依赖型MDH的甲醇氧化活性较低,对甲醇的亲和力较差,导致甲醇氧化成为人工甲基营养菌代谢甲醇的限速步骤。为了提高甲醇氧化速率,进而提高人工甲基营养菌的甲醇利用效率,近年来大量研究集中于MDH的挖掘与改造研究。文中系统综述了不同类型MDH的发现、表征、改造以及在人工甲基营养菌中的应用进展,详细阐述了MDH的定向进化和多酶复合体的构建,并展望了通过细胞生长偶联的蛋白质进化和蛋白质理性设计获得高活性MDH的潜在策略。     

Chloromethane stimulates growth of Methylomicrobium album BG8 on methanol

Studies were performed to determine if the growth of Methylomicrobium album BG8 on methanol could be enhanced through the provision of chloromethane. M. album BG8 was found to be able to oxidize chloromethane via the particulate methane monooxygenase with an apparent K(s) of 11+/-3 microM and V(max) of 15+/-0.6 nmol (min mg protein)(-1). When up to 2.6 mM chloromethane was provided with 5 mM methanol, methanotrophic growth was significantly enhanced in comparison to the absence of chloromethane, indicating that methanotrophs can utilize chloromethane to support growth, although it could not serve as a sole growth substrate. [(14)C]chloromethane was found to be oxidized to [(14)C]CO(2) as well as incorporated into biomass. These results indicate that reactions previously thought to be cometabolic may actually provide some benefit to methanotrophs and that these cells can use multiple compounds to enhance growth.     

Production of 2,3-Butanediol from Sucrose by a <Emphasis Type="Italic">Klebsiella</Emphasis> Species

Chemical 2,3-butanediol is an important platform compound possessing diverse industrial applications. So far, it is mainly produced by using petrochemical feedstock which is associated with high cost and adverse environmental impacts. Hence, finding alternative routes (e.g., via fermentation using renewable carbon sources) to produce 2,3-butanediol are urgently needed. In this study, we report a wild-type Klebsiella sp. strain XRM21, which is capable of producing 2,3-butanediol from a wide variety of carbon sources including glucose, sucrose, xylose, and glycerol. Among them, fermentation of sucrose leads to the highest production of 2,3-butanediol. To maximize the production of 2,3-butanediol, fermentation conditions were first optimized for strain XMR21 by using response surface methodology (RSM) in batch reactors. Subsequently, a fed-batch fermentation strategy was designed based on the optimized parameters, where 91.2 g/L of 2,3-butanediol could be produced from substrate sucrose dosing in 100 g/L for three times. Moreover, random mutagenesis of stain XMR21 resulted in a highly productive mutant strain, capable of producing 119.4 and 22.5 g/L of 2,3-butanediol and ethanol under optimized fed-batch fermentation process within 65 h with a total productivity of 2.18 g/L/h, which is comparable to the reported highest 2,3-butanediol concentration produced by previous strains. This study provides a potential strategy to produce industrially important 2,3-butanediol from low-cost sucrose.     

Recent advances toward the bioconversion of methane and methanol in synthetic methylotrophs

Abundant natural gas reserves, along with increased biogas production, have prompted recent interest in harnessing methane as an industrial feedstock for the production of liquid fuels and chemicals. Methane can either be used directly for fermentation or first oxidized to methanol via biological or chemical means. Methanol is advantageous due to its liquid state under normal conditions. Methylotrophy, defined as the ability of microorganisms to utilize reduced one-carbon compounds like methane and methanol as sole carbon and energy sources for growth, is widespread in bacterial communities. However, native methylotrophs lack the extensive and well-characterized synthetic biology toolbox of platform microorganisms like Escherichia coli, which results in slow and inefficient design-build-test cycles. If a heterologous production pathway can be engineered, the slow growth and uptake rates of native methylotrophs generally limit their industrial potential. Therefore, much focus has been placed on engineering synthetic methylotrophs, or non-methylotrophic platform microorganisms, like E. coli, that have been engineered with synthetic methanol utilization pathways. These platform hosts allow for rapid design-build-test cycles and are well-suited for industrial application at the current time. In this review, recent progress made toward synthetic methylotrophy (including methanotrophy) is discussed. Specifically, the importance of amino acid metabolism and alternative one-carbon assimilation pathways are detailed. A recent study that has achieved methane bioconversion to liquid chemicals in a synthetic E. coli methanotroph is also briefly discussed. We also discuss strategies for the way forward in order to realize the industrial potential of synthetic methanotrophs and methylotrophs.     

Growth characteristics and corrinoid production of Methanosarcina barkeri on methanol-acetate medium

Methanosarcina barkeri strain Fusaro was grown on a mixed substrate medium of methanol and acetate. When 50 mM of acetate was added to the methanol basal medium (250 mM), the rates of methane production, methanol consumption, cell growth and corrinoid production were stimulated 3.2, 2.7, 3.5, and 2.4 times, respectively compared with those in methanol alone. Addition of acetate also has significant influence on corrinoid distribution decreasing the intracellular corrinoid content from 6.8 to 3.0 mg/g dry cell and increasing the extracellular corrinoid concentration from 4.0 to 5.4 mg/l. The carbon balance analysis for methanogenesis and cellular growth with or without acetate addition revealed that about 50% of the utilized acetate carbon might be incorporated in the cellular materials and the remaining might be oxidized to generate the electrons which stimulate the methanol reduction to methane, accelerating the metabolic activities of the methanogenesis from methanol consequently enhancing the rates of methane and corrinoid production, and cell growth.     

Expression of recombinant green fluorescent protein in Bacillus methanolicus

Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein.     

Syntrophic co-culture of a methanotroph and heterotroph for the efficient conversion of methane to mevalonate

As the bioconversion of methane becomes increasingly important for bio-industrial and environmental applications, methanotrophs have received much attention for their ability to convert methane under ambient conditions. This includes the extensive reporting of methanotroph engineering for the conversion of methane to biochemicals. To further increase methane usability, we demonstrated a highly flexible and efficient modular approach based on a synthetic consortium of methanotrophs and heterotrophs mimicking the natural methane ecosystem to produce mevalonate (MVA) from methane. In the methane-conversion module, we used Methylococcus capsulatus Bath as a highly efficient methane biocatalyst and optimized the culture conditions for the production of high amounts of organic acids. In the MVA-synthesis module, we used Escherichia coli SBA01, an evolved strain with high organic acid tolerance and utilization ability, to convert organic acids to MVA. Using recombinant E. coli SBA01 possessing genes for the MVA pathway, 61 mg/L (0.4 mM) of MVA was successfully produced in 48 h without any addition of nutrients except methane. Our platform exhibited high stability and reproducibility with regard to cell growth and MVA production. We believe that this versatile system can be easily extended to many other value-added processes and has a variety of potential applications.     

Detection of Metabolic Key Enzymes of Methane Turnover Processes in Cold Seep Microbial Biofilms

In anoxic environments, methane oxidation is conducted in a syntrophic process between methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Microbial mats consisting of ANME, SRB and other microorganisms form methane seep-related carbonate buildups in the anoxic bottom waters of the Black Sea Crimean shelf. To shed light on the localization of the biochemical processes at the level of single cells in the Black Sea microbial mats, we applied antibody-based markers for key enzymes of the relevant metabolic pathways. The dissimilatory adenosine-5′-phosphosulfate (APS) reductase, methyl-coenzyme M reductase (MCR) and methanol dehydrogenase (MDH) were selected to localize sulfate respiration, reverse methanogenesis and aerobic methane oxidation, respectively. The key enzymes could be localized by double immunofluorescence and immunocytochemistry at light- and electron microscopic levels. In this study we show that sulfate reduction is conducted synchronized and in direct proximity to reverse methanogenesis of ANME archaea. Microcolonies in interspaces between ANME/SRB express methanol dehydrogenase, which is indicative for oxidation of C₁ compounds by methylotrophic or methanotrophic bacteria. Thus, in addition to syntrophic AOM, oxygen-dependent processes are also conducted by a small proportion of the microbial population.     

Production of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolated Klebsiella oxytoca strain growing on biodiesel-derived glycerol based media

The production of 1,3-propanediol, 2,3-butanediol and ethanol was studied, during cultivations of strain Klebsiella oxytoca FMCC-197 on biodiesel-derived glycerol based media. Different kinds of glycerol feedstocks and experimental conditions had an important impact upon the distribution of metabolic products; production of 1,3-propanediol was positively influenced by stable pH conditions and by the absence of N₂ gas infusions throughout the fermentation. Thus, during batch bioreactor fermentations conducted at increasing glycerol concentrations, 1,3-propanediol at 41.3 g/L and yield ～47% (w/w) was achieved at initial glycerol concentration ～120 g/L. At even higher initial glycerol media (150 and 170 g/L), growth was not ceased, but 1,3-propanediol production declined. During fed-batch fermentation under optimal experimental conditions, 126 g/L of glycerol were converted into 50.1 g/L of 1,3-propanediol. In this experiment, also 25.2 g/L of ethanol (conversion yield ～20%, w/w) were formed. A batch-bioreactor culture was performed under non-sterilized conditions and the 1,3-propanediol production was almost equivalent to the sterilized process. Concerning 2,3-butanediol formation, the most detrimental parameter was the absence of N₂ sparging and as a result, no 2,3-butanediol was produced. The presence of glucose as co-substrate seriously enhanced 2,3-butanediol production; when commercial glucose was employed as sole substrate, 32.1 g/L of 2,3-butanediol were formed.     

Harnessing a methane‐fueled,sediment‐free mixed microbial community for utilization of distributed sources of natural gas

Harnessing the metabolic potential of uncultured microbial communities is a compelling opportunity for the biotechnology industry, an approach that would vastly expand the portfolio of usable feedstocks. Methane is particularly promising because it is abundant and energy‐rich, yet the most efficient methane‐activating metabolic pathways involve mixed communities of anaerobic methanotrophic archaea and sulfate reducing bacteria. These communities oxidize methane at high catabolic efficiency and produce chemically reduced by‐products at a comparable rate and in near‐stoichiometric proportion to methane consumption. These reduced compounds can be used for feedstock and downstream chemical production, and at the production rates observed in situ they are an appealing, cost‐effective prospect. Notably, the microbial constituents responsible for this bioconversion are most prominent in select deep‐sea sediments, and while they can be kept active at surface pressures, they have not yet been cultured in the lab. In an industrial capacity, deep‐sea sediments could be periodically recovered and replenished, but the associated technical challenges and substantial costs make this an untenable approach for full‐scale operations. In this study, we present a novel method for incorporating methanotrophic communities into bioindustrial processes through abstraction onto low mass, easily transportable carbon cloth artificial substrates. Using Gulf of Mexico methane seep sediment as inoculum, optimal physicochemical parameters were established for methane‐oxidizing, sulfide‐generating mesocosm incubations. Metabolic activity required >～40% seawater salinity, peaking at 100% salinity and 35 °C. Microbial communities were successfully transferred to a carbon cloth substrate, and rates of methane‐dependent sulfide production increased more than threefold per unit volume. Phylogenetic analyses indicated that carbon cloth‐based communities were substantially streamlined and were dominated by Desulfotomaculum geothermicum. Fluorescence in situ hybridization microscopy with carbon cloth fibers revealed a novel spatial arrangement of anaerobic methanotrophs and sulfate reducing bacteria suggestive of an electronic coupling enabled by the artificial substrate. This system: 1) enables a more targeted manipulation of methane‐activating microbial communities using a low‐mass and sediment‐free substrate; 2) holds promise for the simultaneous consumption of a strong greenhouse gas and the generation of usable downstream products; and 3) furthers the broader adoption of uncultured, mixed microbial communities for biotechnological use.     

pH对克雷伯氏菌1,3-丙二醇合成关键酶酶活及发酵特性的影响

在5 L发酵罐进行甘油脉冲流加发酵,分析了不同pH值对克雷伯氏肺炎杆菌发酵特性的影响,pH 6.5为菌体最佳生长条件,克雷伯氏肺炎杆菌合成1,3-丙二醇的产量最高。在1,3-丙二醇合成速率较大的对数中前期,进行甘油脉冲流加发酵,提高甘油浓度促进甘油脱水酶、1,3-丙二醇氧化还原酶和甘油脱氢酶活性。不同pH值的脉冲试验表明,甘油脱水酶,2,3-丁二醇脱氢酶比酶活随着pH值的升高而升高,1,3-丙二醇氧化还原酶,乳酸脱氢酶比酶活在pH6.5最高,因此偏酸性的发酵条件和对数期维持一定的甘油浓度能够促进1,3-丙二醇的合成。