Combined strategies for improving the heterologous expression of an alkaline lipase from Acinetobacter radioresistens CMC-1 in Pichia pastoris

In this study, a series of strategies was developed to enhance the expression of an alkaline lipase from Acinetobacter radioresistens (ARL) in Pichia pastoris. Activity of the lipase from recombinant strain carrying a single copy of codon-optimized ARL gene was 65 U/mL in shake flask culture with p-nitrophenyl caprylate as the substrate. The lipase yield was increased to 104 U/mL by introducing a short N-extension spacer peptide coding for the 10 amino acids (EEAEAEAEPK) between α-factor signal peptide and ARL. The N-terminal extension spacer did not affect the pH or temperature properties of the recombinant ARL. After the multi-copy constructs were identified by Q-PCR assay, a higher lipase activity of 180 U/mL was obtained. Further introduction of the spliced HAC1 gene into multi-copy integrants (>6 copies) extensively enhanced the ARL yield by 30–40%. As a result, the ARL yield reached 1.06 × 10⁴ U/mL in a 10-L scaled-up fed-batch fermenter as well as the lipase showed some better properties compared to that wild one from A. radioresistens.     

Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture

The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L).     

Bioprocess development of the production of the mutant P-219-L human d-amino acid oxidase for high soluble fraction expression in recombinant Escherichia coli

In order to examine the structure–activity relationship and the substrate specificity of human d-amino acid oxidase (h.DAO), a single amino acid mutation had been established as proline-219-luecine (P-219-L). The gene encoding mutant h.DAO has been cloned and expressed in Escherichia coli BL21 (DE3). It was observed that the host cell was negatively affected by the expressed mutant h.DAO, resulting in a remarkable decrease in the cell growth and consequently the amount of the produced enzyme. To overcome this problem, we investigated several factors that may affect the cell growth rate and the mutant h.DAO production such as optimization of the glucose concentration as a main carbon source and the yeast extract concentration as a main nitrogen source, optimization of dissolved oxygen (DO%) concentration and the addition of benzyl alcohol (BA, which can artificially induce a strong heat shock response at low temperature), to enhance the production of natively folded soluble fraction of the recombinant protein. These parameters were tested on both shake flask level and fed-batch bioreactor level. The Western blot analysis and the enzyme activity assay indicated the higher level of the mutant expression towards enhancement of the conditions by using our designed approach.The specific activity (which was used as an indicator for the level of the desired protein produced = U/mg protein) and the OD_600 nm of the host cells (which was used as an indicator for the cell growth), reached to be 0.061 U/mg protein and 3.44, respectively upon using fed-batch culture system containing the optimized medium composition (15 g/l glucose and 5 g/l yeast extract). While upon using the shake flask level, these values were 0.032 and 1.1, respectively. Enhancement of the cell growth and the enzyme production was noticed after DO% optimization upon using 500 rpm agitation speed and 1.8 v.v.m. (volume volume minute) aeration. The specific activity for the mutant enzyme and the OD_600 nm of the host cells reached to be 0.14 U/mg protein and 7.1, respectively. Finally upon using the optimized culture composition (15 g/l glucose and 5 g/l yeast extract), optimized DO% (using 500 rpm agitation speed and 1.8 v.v.m.) and 0.1 mM BA at the fed-batch bioreactor level, the specific activity and the OD_600 nm of the host cells increased significantly to be 0.21 U/mg protein and 11.3, respectively at 24 h culture. These results indicate the importance of our approaches to overproducing mutant h.DAO in soluble form in E. coli.     

Use of sea water as salts source in starch- and soya bean-based media,for the production of Bacillus thuringiensis bioinsecticides

In order to reduce the cost of Bacillus thuringiensis bioinsecticide-process production, a new medium only composed of starch, soya bean and diluted sea water, providing particularly 7.5 g/l NaCl, was optimized. The results obtained with cultures carried out into 1000 ml shake flasks showed 7% improvement of delta-endotoxin production in such cheap medium compared to that containing a mixture of pure minerals instead of sea water. Interestingly, this new formulated medium was shown to be efficient for delta-endotoxin production by several B. thuringiensis strains exhibiting various larvicidal activities towards either Diptera or Lepidoptera. Moreover, the use of such medium for large scale production of bioinsecticides was also evidenced in an automated full controlled 3 l fermenter. So that it was shown that sea water did not affect such requirements and have positive effect on growth, sporulation and delta-endotoxin synthesis. This should contribute to a significant reduction of the cost of B. thuringiensis bioinsecticide production and utilisation.     

Production of the postharvest biocontrol agent Bacillus subtilis CPA-8 using low cost commercial products and by-products

The aim of this research was to identify a low cost medium based on commercial products and by-products that provided maximum Bacillus subtilis CPA-8 growth and maintained biocontrol efficacy. Low cost media combining economical nitrogen and carbon sources such as yeast extract, peptone, soy products, sucrose, maltose and molasses were tested. Tests were carried out in 250-ml flasks containing 50 ml of each tested medium. Maximum cell growth (>3 × 10⁹ CFU ml^?1) was obtained in defatted soy flour 44% combined with sucrose or molasses media. Second, CPA-8 production was scaled up in a 5-l fermenter and CPA-8 population dynamics, pH and oxygen consumption in the optimized medium (defatted soy flour 44% – molasses) was recorded. In these tests, there was a 5-h lag phase before growth, after which exponential growth occurred and maximum production was 3 × 10⁹ CFU ml^?1 after 20 h. Fruit trials with cells and cell free supernatants from CPA-8 grown in optimized medium maintained biocontrol efficacy against Monilinia fructicola on peaches, resulting in disease reductions up to 95%. CPA-8 populations survived in wounds on inoculated peaches, regardless of the culture media used. The results show that B. subtilis CPA-8 can be produced in a low cost medium combining inexpensive nitrogen and carbon sources (40 g l^?1 defatted soy flour 44%, 5 g l^?1 molasses plus mineral trace supplements) in shake flasks and a laboratory fermenter (5 l). The results could be used to provide a reliable basis for scaling up the fermentation process to an industrial level.     

Overexpression of Candida rugosa lipase Lip1 via combined strategies in Pichia pastoris

In this study, combined strategies were employed to heterologously overexpress Candida rugosa lipase Lip1 (CRL1) in a Pichia pastoris system. The LIP1 gene was systematically codon-optimized and synthesized in vitro. The Lip1 activity of a recombinant strain harboring three copies of the codon-optimized LIP1 gene reached 1200 U/mL in a shake flask culture. Higher lipase activity, 1450 U/mL, was obtained using a five copy number construct. Co-expressing one copy of the ERO1p and BiP chaperones with Lip1p, the CRL1 lipase yield further reached 1758 U/mL, which was significantly higher than that achieved by expressing Lip1p alone or only co-expressing one molecular chaperone. When cultivated in a 3 L fermenter under optimal conditions, the recombinant strain GS115/87-ZA-ERO1p-BiP #7, expressing the molecular chaperones Ero1p and BiP, produced 13,490 U/mL of lipase activity at 130 h, which was greater than the 11,400 U/mL of activity for the recombinant strain GS115/pAO815-α-mCRL1 #87, which did not express a molecular chaperone. This study indicates that a strategy of combining codon optimization with co-expression of molecular chaperones has great potential for the industrial-scale production of pure CRL1.     

The role of volumetric power input in the growth,morphology, and production of a recombinant glycoprotein by Streptomyces lividans in shake flasks

The impact of flask geometry on Streptomyces lividans growth and morphology, production and O-mannosylation of a recombinant O-glycoprotein (APA from Mycobacterium tuberculosis) was described and associated to the evolution of the volumetric power input (P/V) in three shake flask geometries. During the exponential growth, the highest P/V was found in baffled flasks (BF) with 0.51 kW/m³, followed by coiled flasks (CF) with 0.44 kW/m³ and normal Erlenmeyer flasks (NF) with 0.20 kW/m³ (flasks volume of 250 mL, filling with 50 mL and agitated at 150 rpm). During the stationary phase, P/V decreased 20% in BF and CF, but increased two times in NF, surely due to changes in mycelial morphology and its effects on rheology. Also, NF cultures were carried out at a filling volume and agitation of 15 mL, 150 rpm (15 mL-NF), and 25 mL, 168 rpm (25 mL-NF), in order to raise P/V closely to the values obtained in CF. However, different growth, morphology and recombinant protein productivity were obtained. These data indicate that P/V is not a definitive parameter that can determine bacteria growth and morphology, not even glycoprotein production. But it can be proposed that the oxygen transfer in the center of the pellets and hydromechanical stress might be the more relevant parameters than P/V.     

Process development of eicosapentaenoic acid production

Eicosapentaenoic acid (EPA), a well-known member of omega-3 fatty acids, is considered to have a significant health promoting role in the human body. It is an essential fatty acid as the human body lacks the ability to produce it in vivo and must be supplemented through diet. Microbial EPA represents a potential commercial source. GC/MS analyses confirmed that bacterial isolate 717, similar to Shewanella pacifica on the basis of 16S rRNA sequencing, is a potential high EPA producer. Two types of bioreactors, a Stirred Tank Reactor (STR) and an Oscillatory Baffled Reactor (OBR), were investigated in order to choose the optimum system for EPA production. The EPA production media was optimised through the selection of media components in a Plackett–Burman (PB) design of experiment followed by a Central Composite Design (CCD) to optimise the concentration of medium components identified as significant in the Plackett–Burman experiment. The growth conditions for the bioreactor, using artificial sea water (ASW) medium, were optimised by applying Response Surface Methodology (RSM). This optimisation strategy resulted in an increase in EPA from 33 mg/l (10 mg/g biomass), representing 8% of the total fatty acids at shake flask level, to 350 mg/l (46 mg/g biomass) representing 25% of the total fatty acids at bioreactor level. During this study the main effects and the interactions between the bioreactor growth conditions were revealed and a polynomial model of EPA production was generated. Chemostat experiments were performed to test the effect of growth rate and temperature on EPA production.     

Characterization of the extracellular biodemulsifier of Bacillus mojavensis XH1 and the enhancement of demulsifying efficiency by optimization of the production medium composition

The demulsifying bacterium XH1 was identified as a Bacillus mojavensis by the 16S rDNA gene. The extracellular biodemulsifier produced by this species was purified by ethanol extraction and column chromatography through a sephadex and silicon gel column. Preliminary investigation using UV–vis and TLC indicated that the biodemulsifier had two components a protein and a lipopeptide. All major components of the medium, including the sources of soluble and insoluble carbon, nitrogen, phosphate, and metal ions were investigated to improve the biosynthesis and efficiency of the biodemulsifier. The optimal carbon sources were glucose and liquid paraffin. Glucose participated in the biosynthesis of the demulsifier, while liquid paraffin promoted the lipophilicity and secretion of biosurfactants. The absence of yeast extract, ammonium chloride or phosphate (K₂HPO₄/KH₂PO₄) had a negative effect on the production of the biodemulsifier and significantly inhibited its activity. To further enhance the biodemulsifier efficiency, the optimal medium composition was determined using the response surface methodology (RSM) based on the central composite rotation design (CCRD). Using the optimized biodemulsifier production medium: 8.5 g/l glucose; 3% (v/v) liquid paraffin; 1.5 g/l yeast extract; 3.36 g/l NH₄Cl and15 g/l phosphate, the demulsifying ratio increased 35.5% and biodemulsifier yield increased to 2.07 g/l.     

Bio-reduction of hexachloroplatinic acid to platinum nanoparticles employing Acinetobacter calcoaceticus

Acinetobacter calcoaceticus PUCM 1011 efficiently synthesized platinum nanoparticles (PtNP) of size 2–3 nm intracellularly when challenged with hexachloroplatinic acid. Salt concentration (1 mM), temperature (30 °C), pH (7) and incubation period (72 h) influenced the efficiency of monodisperse cuboidal PtNP synthesis. Resolution of ordered lattice fringes with “d” value of 0.23 nm corresponding to (1 1 1) plane and EDAX confirmed presence of metallic platinum. AFM, TEM and HR-TEM confirmed synthesis of PtNP and its effect on cell viability. Total cell protein profile for 120 h with an interval of 24 h after PtNP synthesis revealed prominent four protein bands (97, 66, 43 and 29 kDa) when compared to control. Combinations of three proteins initiated PtNP synthesis within 4 h in range of 1–4 nm and few in picometers under HR-TEM. This is the first report of PtNP synthesis employing whole cell and total cell protein of A. calcoaceticus.     

Significance of seed culture methods on mycelial morphology and production of a novel anti-cancer anthraquinone by marine mangrove endophytic fungus Halorosellinia sp. (No. 1403)

The effects of seed culture methods on the mycelial morphology and production of a novel promising anti-cancer anthraquinone 1403C by marine mangrove saprophytic fungus Halorosellinia sp. (No. 1403) was investigated. Inoculums were prepared using different seed culture methods, i.e., mycelia obtained by grinding biomass that was harvested from baffled flask culture (M1); biomass harvested from baffled flask culture (M2); biomass obtained from unbaffled flask culture with glass beads (M3); biomass attained from unbaffled flask culture (Control). The corresponding fermentations using M1, M2 and M3 enhanced 1403C production by 243.5%, 194.8% and 70.2%, respectively, as compared to that using Control (0.33 ± 0.03 g/l). Interestingly, 1403C production increased with the increase of ratio of number of clumps to pellets. Maximum 1403C production from baffled flask cultures was 4.8-fold of that from unbaffled flask culture. Increasing shaking speed from 170 rpm to 260 rpm could highly improve 1403C production by 151.8%.     

Spatial modulation of key pathway enzymes by DNA-guided scaffold system and respiration chain engineering for improved N-acetylglucosamine production by Bacillus subtilis

Previously we constructed a Bacillus subtilis strain for efficient production of N-acetylglucosamine (GlcNAc) by engineering of GlcNAc synthetic and catabolic pathways. However, the further improvement of GlcNAc titer is limited by the intrinsic inefficiency of GlcNAc synthetic pathway and undesirable cellular properties including sporulation and high maintenance metabolism. In this work, we further improved GlcNAc titer through spatial modulation of key pathway enzymes and by blocking sporulation and decreasing maintenance metabolism. Specifically, a DNA-guided scaffold system was firstly used to modulate the activities of glucosamine-6-phosphate synthase and GlcNAc-6-phosphate N-acetyltransferase, increasing the GlcNAc titer from 1.83 g/L to 4.55 g/L in a shake flask. Next, sporulation was blocked by respectively deleting spo0A (gene encoding the initiation regulon of sporulation) and sigE (gene encoding RNA polymerase sporulation-specific sigma factor). Deletion of sigE more effectively blocked sporulation without altering cell growth or GlcNAc production. The respiration chain was then engineered to decrease the maintenance metabolism of recombinant B. subtilis by deleting cydB and cydC, genes encoding cytochrome bd ubiquinol oxidase (subunit II) and ATP-binding protein for the expression of cytochrome bd, respectively. The respiration-engineered B. subtilis produced 6.15 g/L GlcNAc in a shake flask and 20.58 g/L GlcNAc in a 3-L fed-batch bioreactor. To the best of our knowledge, this report is the first to describe the modulation of pathway enzymes via a DNA-guided scaffold system in B. subtilis. The combination of spatial modulation of key pathway enzymes and optimization of cellular properties may be used to develop B. subtilis as a well-organized cell factory for the production of the other industrially useful chemicals.     

Novel biotransformation process of podophyllotoxin to produce podophyllic acid and picropodophyllotoxin by Pseudomonas aeruginosa CCTCC AB93066, Part II: Process optimization

This work optimized the novel biotransformation process of podophyllotoxin to produce podophyllic acid by Pseudomonas aeruginosa CCTCC AB93066. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/l of yeast extract and 5 g/l of peptone were favorable for podophyllic acid production (i.e. 25.3 ± 3.7 mg/l), while not beneficial for the cell growth of P. aeruginosa. This indicated that the accumulation of podophyllic acid was not corresponded well to the cell growth of P. aeruginosa. 0 g/l of sucrose was beneficial for podophyllic acid production (i.e. 34.3 ± 3.9 mg/l), which led to high podophyllotoxin conversion (i.e. 98.2 ± 0.1%). 1 g/l of NaCl was the best for podophyllic acid production (i.e. 47.6 ± 4.0 mg/l). Secondly, the production of podophyllic acid was significantly enhanced by fed-batch biotransformation. When each 100 mg/l of podophyllotoxin was added to the biotransformation system after 4, 10 and 25 h of culture, respectively, podophyllic acid concentration reached 99.9 ± 12.3 mg/l, enhanced by 284% comparing to one-time addition (i.e. 26.0 ± 2.1 mg/l). The fundamental information obtained in this study provides a simple and efficient way to produce podophyllic acid.     

The production of a cold-induced extracellular biopolymer by Pseudomonas fluorescens BM07 under various growth conditions and its role in heavy metals absorption

The psychrotrophic bacterium Pseudomonas fluorescens BM07 was induced to excrete an extracellular biopolymer when cells were grown aerobically at 10 °C and its secretion was inhibited at 30 °C. The biopolymer was easily torn apart from the cells by using a shear force under centrifugation (8700 × g, 30 min) and collected as a well-separated mucoid layer in centrifuge tube. The production of the biopolymer was affected by factors such as the types of carbon and nitrogen sources, temperature, and pH. The best production of 2.5 g/l was obtained when the cells were grown on M1 medium containing 70 mM sucrose and 0.2% (w/v) Casamino Acids. In Kings B enriched medium a maximum biopolymer production of up to 3.4 g/l and growth rate of 2.1 g/l, were achieved using 1:1 ratio of C/N. Addition of NaCl and ethanol to the medium led to a decrease in biopolymer production and growth rate of BM07 strain. FT-IR spectroscopy demonstrated the presence of carboxyl, amine, hydroxyl and methoxyl functional groups in the biopolymer. BM07 biopolymer showed high ion binding capacity with particular preference to uptake cadmium and mercury (∼45 and 70%, respectively). The percentage removal of cobalt, zinc, nickel and copper cations were between 20 and 30%. Overall ion uptake by BM07 biopolymer showed a definite preference for larger over smaller cations (Hg > Cd > Ni > Zn > Cu > Co).     

Biosynthesis of selenium-containing polysaccharides with antioxidant activity in liquid culture of Hericium erinaceum

The effects of inorganic selenium (sodium selenite) and Selol containing selenitetriglycerides synthesized from sunflower oil on mycelial growth and selenium-containing extracellular (EPS) and intracellular (IPS) polysaccharides production were examined in shake flask cultures of Hericium erinaceum.Unlike sodium selenite which inhibited mycelial growth, Selol increased in biomass production in a dose-dependent manner. Selol also dramatically enhanced EPS formation to 2.25 g/L which is 2.5–fold higher than in the control. Selenium content in EPS and IPS obtained from Selol-enriched medium reached a maximum of 4.89 and 4.69 mg/g, respectively.The in vitro antioxidant activities of polysaccharides were evaluated by reducing power, inhibition of lipid peroxidation, and 1,1-diphenyl-dipicrylhydrazyl radicals scavenging assays. The selenium-containing EPS showed an excellent antioxidant activity correlated well with increasing concentrations.The results suggested that selenium-containing EPS from H. erinaceum submerged culture should be explored as a novel selenium source in dietary supplements, with potent antioxidant properties.     

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Guanosine 5′-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.     

Macronutrients requirements of the dinoflagellate Protoceratium reticulatum

The basal L1 medium was found to be unsatisfactory for culturing the red tide dinoflagellate Protoceratium reticulatum at a high growth rate and biomass yield. The L1 medium enhanced with phosphate to a total concentration of 217 μM supported the highest attainable growth rate and biomass yield. Once the phosphate concentration exceeded 6× L1, phosphate inhibited the dinoflagellate growth and negatively affected cell viability. At the optimal phosphate concentration of 217 μM, an increase in nitrate concentration over the range of 882–8824 μM, did not affect cell growth and yield. Nitrate did not inhibit growth at any of the concentrations used. Clearly, the basal nitrate level in L1 is sufficient for effectively culturing P. reticulatum. At the ranges of phosphate and nitrate concentrations tested, cell volume was not sensitive to the concentration of nutrients but the concentration of phosphate affected both the specific cell number and cell volume growth rates. Elevated levels of nutrients supported their intracellular accumulation. Cell-specific production of yessotoxin was not influenced by concentration of phosphate in the culture medium, but elevated (>1764 μM) nitrate concentration did enhance the yessotoxin level. Phosphate concentration that maximized biomass yield also maximized volumetric production of yessotoxin in the culture broth.     

Enhanced phenolic antioxidants production in submerged cultures of Inonotus obliquus in a ground corn stover medium

The medicinal mushroom Inonotus obliquus has been a folk remedy for a long time in East-European and Asian countries. It is currently ascribed to a number of phenolic compounds as well as triterpenoids and polysaccharides responsible for significant biological and pharmacological properties. A study was conducted to determine the effects of inclusion of lignocellulosic material, in this case corn stover on production and antioxidant activity of extracellular (EPC) and intracellular phenolic compounds (IPC) by Inonotus obliquus in submerged fermentation. The corn stover medium contained 3% ground corn stover and 3.5% corn flour but the control medium contained 5% corn flour without corn stover. All of the other components were same in the two media. Decomposition rates of cellulose, hemicellulose, and lignin in the corn stover substrate were 20.9%, 17.9%, and 19.8% through 288 h of submerged cultivation. Lignocellulose decomposition in the corn stover-containing medium yielded significantly higher EPC (118.9/135.7 mg GAE (gallic acid equivalents)) and IPC (21.2/23.7 mg GAE) than in the control medium (34.7/42.5 mg GAE of EPC and 12.5/13.5 mg GAE of IPC) per liter of culture broth (EPC) and per gram of mycelia (IPC) in shake flask cultures/10 L fermenter runs. Both EPC and IPC from the corn stover medium showed a higher scavenging activity against hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals than those from the control medium during the later fermentation period. In dose-dependent experiments, EPC from the corn stover medium at 216 h demonstrated a significantly stronger free radical scavenger activity against DPPH and hydroxyl radicals, shown as much lower IC50 values, than that from the control medium and IPC from the two media.     

Improved 2,3-butanediol production from corncob acid hydrolysate by fed-batch fermentation using Klebsiella oxytoca

Corncob acid hydrolysate, detoxed by sequently boiling, overliming and activated charcoal adsorption, was used for 2,3-butanediol production by Klebsiella oxytoca ACCC 10370. The effects of acetate in hydrolysate and pH on 2,3-butanediol production were investigated. It was found that acetic acid in hydrolysate inhibited the growth of K. oxytoca while benefited the 2,3-butanediol yield. With the increase in acetic acid concentration in medium from 0 to 4 g/l, the lag phase was prolonged and the specific growth rate decreased. The acetic acid inhibition on cell growth can be alleviated by adjusting pH to 6.3 prior to fermentation and a substrate fed-batch strategy with a low initial acetic acid concentration. Under the optimum condition, a maximal 2,3-butanediol concentration of 35.7 g/l was obtained after 60 h of fed-batch fermentation, giving a yield of 0.5 g/g reducing sugar and a productivity of 0.59 g/h l.