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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #2)

Biorefinery: an Efficient Way to Sustainable Development of Chemical Industry—a Special Issue for International Conference on Biorefinery (ICB 07) and the 5th International Conference on Separation Science and Technology (ICSST2007) by Tianwei Tan; Jian-He Xu (pp. 301-302).

Purification, Properties, and Application of a Novel Acid Urease from Enterobacter sp. by Lu-qiang Yang; Song-hua Wang; Ya-ping Tian (pp. 303-313).

It has been demonstrated that acid urease is capable of decomposing urea in fermented beverage and foods. As urea is a precursor of ethylcarbamate, a potential carcinogenic compound, measures must be taken to control the level of urea. We herein describe the purification and characterization of a novel acid urease from Enterobacter sp. R-SYB082 and its application to the removal of urea in Chinese rice wine. The enzyme was purified to electrophoretic homogeneity using ethanol precipitation, Superdex 200 and Mono Q with a fold purification of 21.1 and a recovery of 49%. The molecular weight of the enzyme was 430,000 Da by gel filtration and 72,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that it was a hexamer. The activity of this purified enzyme was optimal at pH 4.5 and 35 °C. The temperature stability was under 55 °C, and the pH stability was 4.0~5.0. The enzyme exhibited an apparent K _m of 19.5 μmol/l and a V _max of 109 μmol urea/mg·min at 35 °C and pH 4.5. When incubating two different kinds of Chinese rice wine with the enzyme (0.08 U/ml) at 35 °C for 7 days, over 85% of urea was decomposed, and at 20 °C, above 78% was removed. The result showed that the enzyme is applicable to elimination of urea in Chinese rice wine.

Keywords: Enterobacter sp.; Acid urease; Ethylcarbamate; Urea; Chinese rice wine

Breeding of d(–)-Lactic Acid High Producing Strain by Low-energy Ion Implantation and Preliminary Analysis of Related Metabolism by Ting-Ting Xu; Zhong-Zhong Bai; Li-Juan Wang; Bing-Fang He (pp. 314-321).

The low-energy nitrogen ion beam implantation technique was used in the breeding of mutant d(–)-lactic-acid-producing strains. The wild strain Sporolactobacillus sp. DX12 was mutated by an N⁺ ion beam with energy of 10keV and doses ranging from 0.4 × 10¹⁵ to 6.60 × 10¹⁵ ions/cm². Combined with an efficient screening method, an efficient mutant Y2-8 was selected after two times N⁺ ion beam implantation. By using the mutant Y2-8, 121.6g/l of d-lactic acid was produced with the molar yields of 162.1% to the glucose. The yield of d-lactic acid by strain Y2-8 was 198.8% higher than the wild strain. Determination of anaerobic metabolism by Biolog MT2 was used to analyze the activities of the concerned enzymes in the lactic acid metabolic pathway. The results showed that the activities of the key enzymes responded on the substrates such as 6-phosphofructokinase, pyruvate kinase, and d-lactate dehydrogenase were considerably higher in the mutants than the wild strain. These might be affected by ion beam implantation.

Keywords: Nitrogen ion beam implantation; d(–)-Lactic-acid-producing strain; Mutation; Breeding; Metabolic influence

An Improved RNA Isolation Method for Filamentous Fungus Blakeslea trispora Rich in Polysaccharides by Ye Li; Wenya Wang; Xueling Du; Qipeng Yuan (pp. 322-327).

Isolation and purification of biologically active RNA from filamentous fungi is difficult because of the complex cell wall and the high level of polysaccharides which bind to or co-precipitate with RNA. Using benzyl chloride and guanidine thiocyanate, RNA was successfully isolated from Blakeslea trispora in which other RNA extraction methods and commercially available kits failed to deliver suitable results. The RNA isolated by this procedure appears to be relatively pure, as it has a ratio of absorbance at 260/280 nm of 1.8–1.9. The integrity of the RNA was further substantiated by RT-PCR and Northern hybridization, respectively. This procedure should be useful for isolating RNA from other filamentous fungi and, therefore, will serve as an important tool for the molecular analysis of these organisms.

Keywords: RNA isolation; Benzyl chloride; Filamentous fungus; Blakeslea trispora

Rational Design and Study on Recognition Property of Paracetamol-Imprinted Polymer by Ying Liu; Fang Wang; Tianwei Tan; Ming Lei (pp. 328-342).

The design and study on recognition of paracetamol-imprinted polymer for application in quantification of drugs was reported. Base on our previous work, the promising monomer, itaconic acid (IA), was computationally selected rapidly from the virtual library using the interaction energy (ΔE) between a paracetamol (PR) molecule and four monomer molecules as a measure of their interaction. The possible conformation of PR interacting with IA displayed the nature of the interaction between PR and IA; hydrogen bonds (hbs) mainly contribute to this interaction. UV spectra analysis confirmed the occurrence of the hbs interaction between PR and IA at the polymerization stage. The optimal solvents for porogen and eluant were determined by the strength of hbs interaction between PR and the solvents, which were calculated employing density functional theory. The corresponding molecularly imprinted polymers (MIPs) and non-imprinted polymers were prepared and evaluated. The experimental results were consistent with those calculated, which confirmed the validity of the above-related calculation believed to facilitate the selection of monomers and solvents for the synthesis of MIP at molecular level.

Keywords: Molecularly imprinted polymer (MIP); Design; Interaction energy; Solvent; Hydrogen bond (hb)

Kinetic Analysis and pH-Shift Control Strategy for Propionic Acid Production with Propionibacterium Freudenreichii CCTCC M207015 by Xiaohai Feng; Hong Xu; Jun Yao; Sha Li; Hongyang Zhu; Pingkai Ouyang (pp. 343-349).

The production of propionic acid by Propionibacterium freudenreichii CCTCC M207015 was investigated in a 7.5-l stirred-tank fermentor. Batch fermentations by P. freudenreichii CCTCC M207015 at various pH values ranging from 5.5 to 7.0 were studied. Based on the analysis of the time course of specific cell growth rate (μ _x) and specific propionic acid formation rate (μ _p), a two-stage pH-shift control strategy was proposed. At first 48 h, pH was controlled at 6.5 to obtain the maximal μ _x, subsequently pH 6.0 was used to maintain high μ _p to enhance the production of propionic acid. By applying this pH-shift control strategy in propionic acid fermentation, the maximal propionic acid and glucose conversion efficiency had a significant improvement and reached 19.21 g/l and 48.03%, respectively, compared with those of constant pH operation (14.58 g/l and 36.45%). Fed-batch fermentation with pH-shift control strategy was also applied to produce propionic acid; the maximal propionic acid yield and glucose conversion efficiency reached 25.23 g/l and 47.76%, respectively.

Keywords: Propionibacterium freudenreichii CCTCC M207015; Propionic acid; Kinetic analysis; pH-Shift Control Strategy; Fed-batch fermentation

Production of Butyric Acid from Glucose and Xylose with Immobilized Cells of Clostridium tyrobutyricum in a Fibrous-bed Bioreactor by Ling Jiang; Jufang Wang; Shizhong Liang; Xiaoning Wang; Peilin Cen; Zhinan Xu (pp. 350-359).

Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g⁻¹, respectively, in batch fermentation when 30 g L⁻¹ glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L⁻¹ and yield 0.40 g g⁻¹ of butyric acid were obtained with 25 g L⁻¹ xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L⁻¹ with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).

Keywords: Butyric acid; Clostridium tyrobutyricum ; Biomass resource; Fibrous-bed bioreactor; Immobilization

Influence of High Solid Concentration on Enzymatic Hydrolysis and Fermentation of Steam-Exploded Corn Stover Biomass by Yifeng Lu; Yonghong Wang; Guoqian Xu; Ju Chu; Yingping Zhuang; Siliang Zhang (pp. 360-369).

Steam-exploded corn stover biomass was used as the substrate for fed-batch separate enzymatic hydrolysis and fermentation (SHF) to investigate the solid concentration ranging from 10% to 30% (w/w) on the lignocellulose enzymatic hydrolysis and fermentation. The treatment of washing the steam-exploded material was also evaluated by experiments. The results showed that cellulose conversion changed little with increasing solid concentration, and fermentation by Saccharomyces cerevisiae revealed a nearly same ethanol yield with the water-washed steam-exploded corn stover. For the washed material at 30% substrate concentration, i.e., 30% water insoluble solids (WIS), enzymatic hydrolysis yielded 103.3 g/l glucose solution and a cellulose conversion of 72.5%, thus a high ethanol level up to 49.5 g/l. With the unwashed steam-exploded corn stover, though a cellulose conversion of 70.9% was obtained in hydrolysis at 30% solid concentration (27.9% WIS), its hydrolysate did not ferment at all, and the hydrolysate of 20% solid loading containing 3.3 g/l acetic acid and 145 mg/l furfural already exerted a strong inhibition on the fermentation and ethanol production.

Keywords: Bioethanol; Corn stover; High solid concentration; Enzymatic hydrolysis; Fermentation

Effects of Cultivation Conditions on the Production of γ-PGA with Bacillus subtilis ZJU-7 by Jie Chen; Feng Shi; Bin Zhang; Fan Zhu; Weifeng Cao; Zhinan Xu; Guohua Xu; Peilin Cen (pp. 370-377).

Poly-γ-glutamic acid (γ-PGA) is a kind of water-soluble and biodegradable polymer made from d- and l-glutamic acid units, which are linked by amide bonds formed between α-amino and γ-carboxylic acid groups. As a potential targeted biopolymer that can be refined from biomass directly, γ-PGA has been increasingly applied to food, cosmetic, and pharmaceutical industries. In this work, a suitable nitrogen source was screened out for the high and cost-effective production of γ-PGA in Bacillus subtilis ZJU-7. The effects of inoculation time and initial glucose concentration on the γ-PGA production were investigated systematically in both shake flasks and a bench-top 15-l fermentor. Under the optimized culture conditions, a high γ-PGA productivity (46.4 g/l) was obtained after 48 h cultivation at 37 °C. Finally, the large-scale fermentation of γ-PGA production was successfully scaled up to a 100-l fermentor, with the highest γ-PGA productivity for over 54.0 g/l.

Keywords: Poly-γ-glutamic acid; l-glutamic acid; Bacillus subtilis ; Cultivation conditions; Optimization; Scale up

The Ethanol Tolerance of Pachysolen tannophilus in Fermentation on Xylose by Lei Zhao; Jianliang Yu; Xu Zhang; Tianwei Tan (pp. 378-385).

The influence of ethanol on fermentation by Pachysolen tannophilus was studied. When xylose utilization rate was 80%, ethanol concentration began to decline. Fermentation of P. tannophilus was affected by ethanol addition in the beginning of fermentation; average xylose consumption rate was 0.065 g·l⁻¹·h⁻¹, and maximum specific growth rate was 0.07 h⁻¹ at 28 g·l⁻¹ ethanol, comparing with the average xylose consumption rate of 0.38 g·l⁻¹·h⁻¹ and maximum specific growth rate of 0.14 h⁻¹ in fermentation with no ethanol addition; P. tannophilus stopped growth at 40 g·l⁻¹ ethanol. When the initial ethanol concentration was 30 g·l⁻¹, the addition of glucose in xylose media made the growth of P. tannophilus better, and the most favorable glucose concentration was 15 g·l⁻¹ with the highest biomass of 1.51 g·l⁻¹ as compared with that of 0.95 g·l⁻¹ in pure xylose media.

Keywords: Ethanol; Glucose; Pachysolen tannophilus ; Xylose

Contribution of Glycerol on Production of Poly(γ-Glutamic Acid) in Bacillus subtilis NX-2 by Qun Wu; Hong Xu; Jinfeng Liang; Jun Yao (pp. 386-392).

Glycerol would stimulate the production of poly(γ-glutamic acid) (γ-PGA) and decrease its molecular weight in Bacillus subtilis NX-2. When 20 g/l glycerol was added in the medium, the yield of γ-PGA increased from 26.7 ± 1.0 to 31.7 ± 1.3 g/l, and molecular weight of γ-PGA decreased from 2.43 ± 0.07 × 10⁶ to 1.86 ± 0.06 × 10⁶ Da. In addition, it was found that the decrease of γ-PGA chain length by glycerol would lead to the decrease of broth viscosity during the fermentation and enhanced the uptake of substrates, which could not only improve cell growth but also stimulate γ-PGA production. Moreover, it was also found that glycerol could effectively regulate molecular weight between 2.43 ± 0.07 × 10⁶ and 1.42 ± 0.05 × 10⁶ Da with the concentration ranging from 0 to 60 g/l. This was the first time to discover such contribution of glycerol on γ-PGA production in Bacillus genus. And the effects of glycerol on molecular weight of γ-PGA would be developed to be an approach for the regulation of microbial γ-PGA chain length, which is of practical importance for future commercial development of this polymer.

Keywords: Bacillus subtilis NX-2; Fermentation; Glycerol; Molecular weight; Poly(γ-glutamic acid)

Gene Cloning, Overexpression, and Characterization of the Nitrilase from Rhodococcus rhodochrous tg1-A6 in E. coli by Hui Luo; Lu Fan; Yanhong Chang; Jinwei Ma; Huimin Yu; Zhongyao Shen (pp. 393-400).

A DNA fragment containing the entire coding sequence of nitrilase gene was amplified from Rhodococcus rhodochrous tg1-A6 with high nitrilase activity using PCR and sequenced. The open reading frame of the nitrilase gene contains 1,101 base pairs, which encodes a putative polypeptide of 366 amino acid residues. The nitrilase gene was cloned into an expression vector pET-28a and expressed in an Escherichia coli strain BL21(DE3). The enzymatic activity of nitrilase, which converts various nitriles to the corresponding carboxylic acids, was detected to reach 24.5 U/ml at 9 h in the recombinant bacteria.

Keywords: Nitrilase; Nitrile; Carboxylic acid; Acrylic acid; Recombinant E. coli ; Induction; Expression

Ethanol Production from H₂SO₃-Steam-Pretreated Fresh Sweet Sorghum Stem by Simultaneous Saccharification and Fermentation by Jianliang Yu; Jing Zhong; Xu Zhang; Tianwei Tan (pp. 401-409).

The present work presents an alternative approach to ethanol production from sweet sorghum: without detoxification, acid-impregnated fresh sweet sorghum stem which contains soluble (glucose and sucrose) and insoluble carbohydrates (cellulose and hemicellulose) was steam pretreated under mild temperature of 100 °C. Simultaneous saccharification and fermentation experiments were performed on the pretreated slurries using Saccharomyces cerevisiae. Experimentally, ground fresh sweet sorghum stem was combined with H₂SO₃ at dosages of 0.25, 0.50, and 0.75 g/g dry matter (DM) and steam pretreated by varying the residence time (60, 120, or 240 min). According to enzymatic hydrolysis results and ethanol yields, H₂SO₃ was a powerful and mild acid for improving enzymatic digestibility of sorghum stem. At a solid loading of 10% (w/v) and acid dosage of 0.25 g/g DM H₂SO₃ at 100 °C for 120 min, 44.5 g/L ethanol was obtained after 48 ± 4 h of simultaneous saccharification and fermentation. This corresponded to an overall ethanol yield of 110% of the theoretical one, based on the soluble carbohydrates in the fresh sweet sorghum stem. The concentrations of hydroxymethylfurfural and furfural of the sulfurous acid pretreated samples were below 0.4 g/L. Ethanol would not inhibit the cellulase activity, at least under the concentration of 34 g/L.

Keywords: Ethanol; Steam pretreatment; Simultaneous saccharification and fermentation; SSF; Sweet sorghum; Sulfurous acid (H₂SO₃)

Effects of Soya Fatty Acids on Cassava Ethanol Fermentation by Dongguang Xiao; Shuai Wu; Xudong Zhu; Yefu Chen; Xuewu Guo (pp. 410-420).

Ethanol tolerance is a key trait of microbes in bioethanol production. Previous studies have shown that soya flour contributed to the increase of ethanol tolerance of yeast cells. In this paper, the mechanism of this ethanol tolerance improvement was investigated in cassava ethanol fermentation supplemented with soya flour or defatted soya flour, respectively. Experiment results showed that ethanol tolerance of cells from soya flour supplemented medium increased by 4–6% (v/v) than the control with defatted soya flour. Microscopic observation found that soya flour can retain the cell shape while dramatic elongations of cells were observed with the defatted soya flour supplemented medium. Unsaturated fatty acids (UFAs) compositions of cell membrane were analyzed and the UFAs amounts increased significantly in all tested strains grown in soya flour supplemented medium. Growth study also showed that soya flour stimulated the cell growth rate by approximately tenfolds at 72-h fermentation. All these results suggested that soya fatty acids play an important role to protect yeast cells from ethanol stress during fermentation process.

Keywords: Soy fatty acids; Soya flour; Cassava; Ethanol; Tolerance

Investigation on α-Galactosidase Production by Streptomyces griseoloalbus in a Forcefully Aerated Packed-Bed Bioreactor Operating in Solid-State Fermentation Condition by G. S. Anisha; Rojan P. John; P. Prema; Ashok Pandey (pp. 421-427).

Solid-state fermentation (SSF) was carried out for the production of extracellular α-galactosidase by Streptomyces griseoloalbus. Soybean flour was the best solid substrate for α-galactosidase production. Packed-bed bioreactor performed well in enhancing the enzyme yield and resulted in a highest yield of 197.2 ± 0.02 U/gds with a forced aeration of 2 vvm, which was approximately twofold the yield obtained in flasks. The α-galactosidase production was growth-associated, and the highest enzyme yield was obtained after 96 h of incubation. It is significant that this is the first report on α-galactosidase production by a filamentous bacterium in SSF using packed-bed bioreactor.

Keywords: α-Galactosidase; Filamentous bacteria; Streptomyces griseoloalbus ; Solid-state fermentation; Packed-bed bioreactor

An Effective Device for Gas–Liquid Oxygen Removal in Enclosed Microalgae Culture by Zhenfeng Su; Ruijuan Kang; Shaoyuan Shi; Wei Cong; Zhaoling Cai (pp. 428-437).

A high-performance gas–liquid transmission device (HPTD) was described in this paper. To investigate the HPTD mass transfer characteristics, the overall volumetric mass transfer coefficients, $$K_{{ ext{La}},{ ext{CO}}_2 }^{ ext{A}} $$ for the absorption of gaseous CO₂ and $$K_{{ ext{La}},{ ext{O}}_2 }^{ ext{D}} $$ for the desorption of dissolved O₂ were determined, respectively, by titration and dissolved oxygen electrode. The mass transfer capability of carbon dioxide was compared with that of dissolved oxygen in the device, and the operating conditions were optimized to suit for the large-scale enclosed micro-algae cultivation. Based on the effectiveness evaluation of the HPTD applied in one enclosed flat plate Spirulina culture system, it was confirmed that the HPTD can satisfy the demand of the enclosed system for carbon supplement and excessive oxygen removal.

Keywords: Micro-algae; Enclosed photo-bioreactor; Volumetric transfer coefficient; Retention time; Lag time

pH Neutralization while Succinic Acid Adsorption onto Anion-Exchange Resins by Qiang Li; Wang-liang Li; Dan Wang; Bin-bin Liu; Huang Tang; Mao-hua Yang; Qing-fen Liu; Jian-min Xing; Zhi-guo Su (pp. 438-445).

Succinic acid is a useful chemical and its purification from fermentation broth by ion-exchange resins has widely drawn attention. In this study, pH neutralization in the process of adsorption of succinic acid from model solutions and fermentation broth by anion-exchange resin NERCB 04 has been tested. Adsorption capacity of NERCB 04 was about 0.41 g succinic acid/g resin at concentrations of succinic acid in the range of 10–50 g/L in packed column. In the process of succinic acid removal, pH of the system could also be neutralized. The neutralizing ability of the resin as a neutralizing agent has also been studied in the model cycle system and in the real fermentation cycle process. It was found that NERCB 04 showed stable adsorption capacity and pH neutralization ability after each regeneration. A certain amount of anion-exchange resin could neutralize the low pH values (pH 2–5) and maintain the system around pH 7.0. This means the anion-exchange resins have the function of neutralizing reagent in the process of adsorbing succinic acid.

Keywords: Succinic acid; Adsorption; pH neutralizing agent; Resin; Fermentation

Symbiosis between Microorganisms from Kombucha and Kefir: Potential Significance to the Enhancement of Kombucha Function by Zhiwei Yang; Feng Zhou; Baoping Ji; Bo Li; Yangchao Luo; Li Yang; Tao Li (pp. 446-455).

Gluconacetobacter sp. A4 (G. sp. A4), which had strong ability to produce d-saccharic acid 1, 4 lactone (DSL), was the key functional bacteria isolated from the kombucha preserved. This paper investigated the interaction between G. sp. A4 and ten different strains of lactic acid bacteria (LAB) obtained from kefir. The result suggested that the LAB promoted DSL production of G. sp. A4 to different extents, ranging from 4.86% to 86.70%. Symbiosis between G. sp. A4 and LAB was studied. LAB’s metabolites, xylitol, and acetic acid, were utilized by G. sp. A4, and it promoted the growth of G. sp. A4 and yield of DSL. Therefore, in developing starter cultures for kombucha fermentation process, a mixed flora of LAB and G. sp. A4 would be the optimal combination.

Keywords: Kombucha; Gluconacetobacter sp; Kefir; Lactic acid bacteria; Symbiosis; D-saccharic acid 1,4 lacton

Expression of a hemA Gene from Agrobacterium radiobacter in a Rare Codon Optimizing Escherichia coli for Improving 5-aminolevulinate Production by Weiqi Fu; Jianping Lin; Peilin Cen (pp. 456-466).

The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.

Keywords: 5-aminolevulinate; Agrobacterium radiobacter ; Escherichia coli Rosetta(DE3); hemA ; Rare codons

Synthesis and Properties of Surface Molecular Imprinting Adsorbent for Removal of Pb²⁺ by Wei Jiang; Haijia Su; Hongyan Huo; Tianwei Tan (pp. 467-476).

A new chitosan imprinting adsorbent using diatomite as core material was prepared by using the surface molecular imprinting technology with the Pb²⁺ as imprinted ion. The preparation process conditions of the surface molecular imprinting adsorbent were studied. The adsorbent was characterized by using Fourier transform infrared (FTIR) spectrum. FTIR spectrum indicated that it was cross-linked by epichlorohydrin. The new imprinting adsorbent could provide a higher adsorption capacity for Pb²⁺, which reached 139.6 mg/g increasing 32.3% compared with cross-linking chitosan adsorbent (the initial Pb²⁺ concentration of 600 mg/L). The adsorption velocity was quick and the equilibration time of the imprinting adsorbent for Pb²⁺ was 3 h that shortened about 40% compared with cross-linking chitosan adsorbent. It had a more wide pH range of 5–7 than that of cross-linking chitosan adsorbent. The new imprinting adsorbent can be reused for up to ten cycles without loss of adsorption capacity. In the kinetics and isotherm study, the pseudosecond order model and Langmuir model could represent the adsorption process.

Keywords: Surface molecular imprinting; Chitosan; Diatomite; Adsorption; Lead ion

Succinic Acid Production from Acid Hydrolysate of Corn Fiber by Actinobacillus succinogenes by Kequan Chen; Min Jiang; Ping Wei; Jiaming Yao; Hao Wu (pp. 477-485).

Dilute acid hydrolysate of corn fiber was used as carbon source for the production of succinic acid by Actinobacillus succinogenes NJ113. The optimized hydrolysis conditions were obtained by orthogonal experiments. When corn fiber particles were of 20 mesh in size and treated with 1.0% sulfuric acid at 121 °C for 2 h, the total sugar yield could reach 63.3%. It was found that CaCO₃ neutralization combined with activated carbon adsorption was an effective method to remove fermentation inhibitors especially furfural that presented in the acid hydrolysate of corn fiber. Only 5.2% of the total sugar was lost, while 91.9% of furfural was removed. The yield of succinic acid was higher than 72.0% with the detoxified corn fiber hydrolysate as the carbon source in anaerobic bottles or 7.5 L fermentor cultures. It was proved that the corn fiber hydrolysate could be an alternative to glucose for the production of succinic acid by A. succinogenes NJ113.

Keywords: Succinic acid; Corn fiber; Acid hydrolysate; Detoxification; Actinobacillus succinogenes NJ113

Pervaporation Separation of Thiophene–Heptane Mixtures with Polydimethylsiloxane (PDMS) Membrane for Desulfurization by Jian Chen; Jiding Li; Rongbin Qi; Hong Ye; Cuixian Chen (pp. 486-497).

Cross-linked polydimethylsiloxane (PDMS)–polyetherimide (PEI) composite membranes were prepared, in which asymmetric microporous PEI membrane prepared with phase inversion method was acted as the microporous supporting layer in the flat-plate composite membrane. Membrane characterization was conducted by Fourier transform infrared and scanning electronic microscopy analysis. The composite membranes were employed in pervaporation separation of n-heptane–thiophene mixtures. Effect of amount of PDMS, cross-linking temperature, amount of cross-linking agent, and cross-linking time on the separation efficiency of n-heptane–thiophene mixtures was investigated experimentally. Experiment results demonstrated that 80–100°°C of cross-linking temperature was more preferable for practical application, as the amount of cross-linking agent was up to 20 wt.%, and 25 wt.% of PDMS amount was more optimal as far as flux and sulfur enrichment factor were concerned. In addition, the swelling degree of and stableness of composite membrane during long-time operation were studied, which should be significant for practical application.

Keywords: Polydimethylsiloxane; Pervaporation; Thiophene–heptane mixtures; Desulfurization

A New Strategy for Lipid Production by Mix Cultivation of Spirulina platensis and Rhodotorula glutinis by Feiyan Xue; Jinxin Miao; Xu Zhang; Tianwei Tan (pp. 498-503).

Mix cultivation of microalgae (Spirulina platensis) and yeast (Rhodotorula glutinis) for lipid production was studied. Mixing cultivation of the two microorganisms significantly increased the accumulation of total biomass and total lipid yield. Dissolved oxygen and medium components in the mixed fermentation medium were analyzed. Mix cultivation in monosodium glutamate wastewater was further studied. Result indicated 1,600 mg/L of biomass was obtained and 73% of COD were removed.

Keywords: Spirulina platensis ; Rhodotorula glutinis ; Mix cultivation; Wastewater treatment; Lipid

Optimization of Lipase-Catalyzed Transesterification of Lard for Biodiesel Production Using Response Surface Methodology by Ying Huang; Hai Zheng; Yunjun Yan (pp. 504-515).

Biodiesel, an alternative diesel fuel made from renewable biological resources, has become more and more attractive recently. Combined use of two immobilized lipases with complementary position specificity instead of one lipase is a potential way to significantly reduce cost of lipase-catalyzed biodiesel production. In this study, the process of biodiesel production from lard catalyzed by the combined use of Novozym435 (non-specific) and Lipozyme TLIM (1,3-specific) was optimized by response surface methodology. The optimal reaction conditions were 0.04 of amount of lipase/oil (w/w), 0.49 of proportion of Novozym435/total lipases (w/w), 0.55 of quantity of tert-butanol/oil (v/v), 5.12 of quantity of methanol/oil (mol/mol), and 20 h of reaction time, by which 97.2% of methyl ester (ME) yield was attained, very close to the predicted value (97.6%). This optimal reaction condition could be true of other similar reactions with plant and animal oil resources; their ME yield could be higher than 95%. The lipases regenerated by washing with organic solvent after each reaction cycle could be continuously reused for 20 cycles without any loss of activity, exhibiting very high manipulation stability.

Keywords: Biodiesel; Lipase; Novozym435; Lipozyme TLIM; RSM

Liquid–Liquid Equilibria of the Ternary System Thiophene + Octane + Dimethyl Sulfoxide at Several Temperatures by Wenlin Zhang; Kaihu Hou; Guanjie Mi; Na Chen (pp. 516-522).

Liquid–liquid equilibria (LLE) data of the ternary system thiophene + octane + dimethyl sulfoxide at 40°C, 50°C, and 60°C under atmospheric pressure were determined using an equilibrium cell with the standard curve method. The distribution of thiophene between extract and raffinate was measured and a practical formula of equilibria data for industrial extraction was proposed. NRTL model and UNIQUAC model were used to correlate and calculate LLE data of the system, and model parameters were determined using the simplex optimization method and imitative Newton method with a minimized objective function of mole fraction deviation. The rule of thermodynamic equilibria was used to deal with multi-roots problem in correlating process. Agreement between predicted and experimental data was satisfactory. The average absolute deviations of the NRTL and UNIQUAC models of thiophene mass fraction were 0.0040 and 0.0078, respectively. Both NRTL and UNIQUAC models were suitable for the calculation of LLE data of the ternary system thiophene + octane + dimethyl sulfoxide. The correlation accuracy of NRTL model is inferior to that of UNIQUAC model.

Keywords: Liquid–liquid equilibria; Thiophene; Octane; Dimethyl sulfoxide; NRTL model; UNIQUAC model

Enhanced Production of Coenzyme Q10 by Overexpressing HMG-CoA Reductase and Induction with Arachidonic Acid in Schizosaccharomyces pombe by Bing Cheng; Qi-peng Yuan; Xin-xiao Sun; Wen-jin Li (pp. 523-531).

Coenzyme Q10 (CoQ10) is a vitamin-like substance which plays a crucial role in the respiratory chain ranging from bacteria to humans and in the radical scavenging in human body. In this study, the full-length hmgR gene (encoding 3-hydroxy-3-methyl-glutaryl-CoA reductase, HMG-CoA reductase) was cloned and overexpressed in Schizosaccharomyces pombe. Using the pREPG yeast depressed under the thiamine as the control, CoQ10 contents increased up to 2.68 and 3.09 times when recombinant cells were incubated without and with arachidonic acid, respectively. It demonstrated that arachidonic acid could upregulate the activity of HMG-CoA reductase and that hmgR gene played a significant role in CoQ10 biosynthesis. So, it has an importance to be utilized for fermentation.

Keywords: HMG-CoA Reductase; Schizosaccharomyces pombe (fission yeast); Coenzyme Q10; Arachidonic acid

Improved Ethanol Production by Mixed Immobilized Cells of Kluyveromyces marxianus and Saccharomyces cerevisiae from Cheese Whey Powder Solution Fermentation by Xuewu Guo; Jun Zhou; Dongguang Xiao (pp. 532-538).

Ethanol productions from cheese whey powder (CWP) solution were investigated by using free or immobilized cells of Kluyveromyces marxianus in monocultures or mixed cultures with free or immobilized cells of K. marxianus and Saccharomyces cerevisiae. K. marxianus free cells produced 3.8% v/v ethanol in monocultures, while S. cerevisiae immobilized cells produced 5.3% v/v ethanol in mixed cultures. The percentage of theoretical yield was found to be higher in mixed cultures than that in monocultures. The maximum ethanol fermentation efficiency was achieved (79.9% of the theoretical value) using mixed cultures of immobilized cells of K. marxianus and S. cerevisiae. The beads were relatively stable without significant reduction in activity for about eight batches of fermentation.

Keywords: Ethanol; Mixed cultures; Immobilization; Cheese whey powder

Pretreatment of Rice Straw by a Hot-Compressed Water Process for Enzymatic Hydrolysis by Guoce Yu; Shinichi Yano; Hiroyuki Inoue; Seiichi Inoue; Takashi Endo; Shigeki Sawayama (pp. 539-551).

Hot-compressed water (HCW) is among several cost-effective pretreatment processes of lignocellulosic biomass for enzymatic hydrolysis. The present work investigated the characteristics of HCW pretreatment of rice straw including sugar production and inhibitor formation in the liquid fraction and enzymatic hydrolysis of pretreated material. Pretreatment was carried out at a temperature ranging from 140 to 240 °C for 10 or 30 min. Soluble oligosaccharides were found to constitute almost all the components of total sugars in the liquid fraction. The maximal production of total glucose at 180 °C and below accounted for 4.4–4.9% of glucan in raw material. Total xylose production peaked at 180 °C, accounting for 43.3% of xylan in raw material for 10-min pretreatment and 29.8% for 30-min pretreatment. The production of acetic acid increased at higher temperatures and longer treatment time, indicating more significant disruption of lignocellulosic structure, and furfural production achieved the maximum (2.8 mg/ml) at 200 °C for both 10-min and 30-min processes. The glucose yield by enzymatic hydrolysis of pretreated rice straw was no less than 85% at 180 °C and above for 30-min pretreatment and at 200 °C and above for 10-min pretreatment. Considering sugar recovery, inhibitor formation, and process severity, it is recommended that a temperature of 180 °C for a time of 30 min can be the most efficient process for HCW pretreatment of rice straw.

Keywords: Rice straw; Pretreatment; Hot-compressed water; Sugar production; Inhibitor formation; Enzymatic hydrolysis; Fermentation

Accumulation of Exopolysaccharides in Liquid Suspension Culture of Nostoc flagelliforme Cells by Haifeng Yu; Shiru Jia; Yujie Dai (pp. 552-560).

The liquid suspension culture of dissociated Nostoc flagelliforme cells was investigated. It was found that the growth rate of N. flagelliforme cells and the accumulation of exopolysaccharides (EPS) increased prominently when NaNO₃ and KH₂PO₄ were added in the liquid BG-11culture medium though phosphate had little effect on EPS yield for specific mass of cells. N. flagelliforme cells grew well at 25 °C and neutral pH, however, a lower or higher temperature and weak alkaline can promote EPS accumulation. With the increase of the light intensity, the growth rate of N. flagelliforme cells and the EPS accumulation increase accordingly. When N. flagelliforme cells was cultured in BG-11 medium added with 2.5 g L⁻¹ of NaNO₃ and 0.956 g L⁻¹ of KH₂PO₄ at 25 °C with 60 μmol photon m⁻² s⁻¹ of light intensity, 1.05 g L⁻¹ cell density and 89.9 mg L⁻¹ EPS yield were achieved respectively. Adopting the optimal conditions established in flask culture, the liquid culture of N. flagelliforme cells in 20-L photobioreactor for 16 days was conducted and a maximum biomass of 1.32 g L⁻¹ was achieved, which was about 17.6-fold of that in the initial inoculation. The yield of EPS was 228.56 mg L⁻¹and about 2.23-fold of that in flask culture. Moreover, the polysaccharides’ material was released into the culture medium during cell growth. These released polysaccharides (RPSs), which can be easily recovered from the medium, are favorable for industrial applications.

Keywords: Nostoc flagelliforme cells; Liquid suspension culture; Exopolysaccharides; Culture conditions

Study of Phase Transformation of Guanosine 5′-Monophosphate in Drowning-Out Crystallization by Jeongki Kang; Nguyen Anh Tuan; Jong-Min Kim; Sang-Mok Chang; Woo-Sik Kim (pp. 561-573).

The present study used a mechanistic approach to control the phase transformation of guanosine 5′-monophosphate (GMP) via the operating conditions of agitation and feed concentration during drowning-out crystallization. First, Fourier transform infrared and UV/vis spectrophotometry were successfully applied to monitor the mass fraction of GMP polymorphs (amorphous and hydrate crystalline GMPs) and GMP supersaturation, respectively, during the crystallization. The phase transformation of amorphous GMP into hydrate crystals was significantly influenced by the agitation, which promoted the mass transfer of GMP dissolution and growth. Therefore, the phase transformation was quickly finished when increasing the agitation speed. However, a high agitation caused breakage of the hydrate crystals, resulting in a reduced crystal size with a bimodal distribution. The phase transformation was also influenced by the GMP feed concentration, as the crystal growth was promoted and the crystal size increased when increasing the feed concentration up to 61 g/l. However, a further increase in the feed concentration caused secondary nucleation due to the induction of a high supersaturation level during the phase transformation, leading to a small crystal size with a bimodal distribution. In addition, the rectangular-shaped hydrate GMP crystals exhibited a higher growth rate in the b direction rather than the a direction. Therefore, the crystal morphology shifted from a long rectangle to a square when increasing the feed concentration.

Keywords: Guanosine 5′-monophosphate; Phase transformation; Drowning-out crystallization; Polymorphs

gTME for Improved Xylose Fermentation of Saccharomyces cerevisiae by Hongmei Liu; Ming Yan; Cangang Lai; Lin Xu; Pingkai Ouyang (pp. 574-582).

Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. In our study, the application of gTME to Saccharomyces cerevisiae was to improve xylose utilization and tolerance, which is a key trait for many biofuel programs. Mutation of the transcription factor spt15 was introduced by error-prone polymerase chain reaction and then screened on media using xylose as the sole carbon source. The selected out strain spt15-25 showed modest growth rates in the media containing 50, 100, and 150 g/L of xylose or glucose. Under the following fermentation condition: 30 °C, rotating speed of 200 r/min, 500-mL Erlenmeyer flask containing 100-mL media, after 109 h, 93.5% of xylose was consumed in 50 g/L xylose medium. Meanwhile, 98.3% glucose can be metabolized in 50-g/L glucose medium. And the carbon source was 50 g/L glucose–xylose (w/w = 1); the utilization ratio of xylose and glucose was 90.8% and 97.3%, respectively. And all the xylitol concentration was below 2.48 g/L.

Keywords: gTME; Xylose uptake; Saccharomyces cerevisiae

An Enhanced Adsorption in Puerarin Retention on Oligo-β-Cyclodextrin Grafted Poly(Glycidyl Methacrylate) Media by Li Yang; Tianwei Tan (pp. 583-592).

The poly(glycidyl methacrylate) adsorbents P(GMA–EDMA) and P(GMA–DVB) were synthesized by the radical suspension–polymerization method and farther coupled by oligo-β-cyclodextrin (CDP) to obtain P(GMA–EDMA)–CDP and P(GMA–DVB)–CDP. The synthesized polymeric media were characterized by Fourier transform infrared (FTIR) spectrometer, scanning electron microscopy, and BET surface area. The adsorption of puerarin from aqueous solution onto the four media, i.e., P(GMA–EDMA), P(GMA–DVB), P(GMA–EDMA)–CDP, and P(GMA–DVB)–CDP, was studied. An enhanced adsorption of puerarin apparently presented on grafted media. The interaction between the polymeric media and the puerarin was researched by FTIR. The result shows that the adsorption efficiency on P(GMA–DVB)–CDP driven by multiple weak interactions is much higher than that on P(GMA–EDMA) driven by hydrogen bonding interaction only and on P(GMA–DVB) or P(GMA–EDMA)–CDP driven by two interactions.

Keywords: Polymeric media; Weak interactions; Enhanced adsorption; Synthesis; Oligo-β-cyclodextrin

Desulfurization of Gasoline using Molecularly Imprinted Chitosan as Selective Adsorbents by Yonghui Chang; Lei Zhang; Hanjie Ying; Zhenjiang Li; Hao Lv; Pingkai Ouyang (pp. 593-603).

For desulfurization of gasoline, novel chitosan-based molecularly imprinted polymer (MIP) was prepared by cross-linking chitosan with epichlorohydrin in the presence of dibenzothiophene (DBT) as the template. The influence of cross-linking ratio on the specific adsorption was evaluated. The effects of the types and the amounts of porogen on selectivity of the chitosan MIP were also examined. Results showed that MIP has a higher recognition property to DBT. The maximum rebinding capacities of the MIP reached 22.69 mg g⁻¹ in the model solution. The adsorption behaviors of the MIP including adsorption kinetics, isotherms, and thermodynamic parameters were investigated and the experimental data agreed well with the Langmuir model. The dynamical adsorption behaved in first-order kinetics. Negative values for the Gibbs free energy showed that the adsorptions were spontaneous processes. The MIP was further used to selectively adsorb organosulfur from gasoline.

Keywords: Molecularly imprinted polymer; Molecular imprinting technology; Dibenzothiophene; Desulfurization

Statistical Optimization of Recycled-Paper Enzymatic Hydrolysis for Simultaneous Saccharification and Fermentation Via Central Composite Design by Qing Liu; Ke-ke Cheng; Jian-an Zhang; Jin-ping Li; Ge-hua Wang (pp. 604-612).

A central composite design of the response surface methodology (RSM) was employed to study the effects of temperature, enzyme concentration, and stirring rate on recycled-paper enzymatic hydrolysis. Among the three variables, temperature and enzyme concentration significantly affected the conversion efficiency of substrate, whereas stirring rate was not effective. A quadratic polynomial equation was obtained for enzymatic hydrolysis by multiple regression analysis using RSM. The results of validation experiments were coincident with the predicted model. The optimum conditions for enzymatic hydrolysis were temperature, enzyme concentration, and stirring rate of 43.1 °C, 20 FPU g⁻¹ substrate, and 145 rpm, respectively. In the subsequent simultaneous saccharification and fermentation (SSF) experiment under the optimum conditions, the highest 28.7 g ethanol l⁻¹ was reached in the fed-batch SSF when 5% (w/v) substrate concentration was used initially, and another 5% added after 12 h fermentation. This ethanol output corresponded to 77.7% of the theoretical yield based on the glucose content in the raw material.

Keywords: Enzymatic hydrolysis; Cellulose; Response surface methodology (RSM); Central composite design (CCD)

Trehalose Biosynthesis Enhancement for Six Yeast Strains Under Pressurized Culture by Changsheng Qiao; Shiru Jia; Yujie Dai; Rui Wang; Aiyou Sun (pp. 613-620).

Six yeast strains of the commercial brewing yeasts CICC1391 and CICC1471, the commercial baker yeasts CICC1339 and CICC1447, and the commercial alcohol yeasts CICC1286 and CICC1291 have been cultured under 1.0 MPa of pressure with N₂ and CO₂ as pressure media. The concentration of intracellular trehalose and the activity of trehalose synthases complex have been measured. Also, the morphology changes of yeast cells have been observed by scanning electronic microscope. There was a positive correlation between the activity of trehalose synthase complex and the concentration of intracellular trehalose; and there was a negative correlation between the activity of trehalose synthase complex and the viability of yeast strains. Having been cultured for 3 h at high pressure of 1.0 MPa, the concentration of intracellular trehalose and the activity of trehalose synthases complex were improved by 50.1% to 116.4% and 45.2% to 219.1%, respectively, compared to those of atmospheric pressure culture. Under high pressure, many wrinkles appeared on the membrane surface of yeast cells. It has been found that yeasts are more sensitive to high pressure for having more and sharper wrinkles on their cell membranes.

Keywords: Pressurized culture; Yeasts; Trehalose; Trehalose synthase complex

Optimization of Effect Factors for Mycelial Growth and Exopolysaccharide Production by Schizophyllum commune by Li-min Hao; Xin-hui Xing; Zheng Li; Jian-chun Zhang; Jin-xu Sun; Shi-ru Jia; Chang-sheng Qiao; Tianyi Wu (pp. 621-631).

This paper is concerned with the optimization of effect factors for mycelial growth and exopolysaccharide production by Schizophyllum commune by one-factor-at-a-time and orthogonal methods. The one-factor-at-a-time method was adopted to investigate the effects of six different compounds (sodium carboxymethylcellulose, l-glutamic acid, V_B1, naphthalene acetic acid, oleic acid, and Tween 80) on mycelial growth and exopolysaccharide production. Among these factors, oleic acid, V_B1 and Tween 80 were identified to be the most important factors. Subsequently, the concentration of oleic acid, V_B1 and Tween 80 were optimized using the orthogonal matrix method. The effects of the factors on the mycelial growth of S. commune were in the order of oleic acid > V_B1 > Tween 80, and those on exopolysaccharide production were in the same order. The optimal concentration for mycelia and exopolysaccharide were determined as oleic acid 0.1% (v/v), V_B1 0.5 mg/L, and Tween 80 6 mg/L. The subsequent verification experiments confirmed the validity of the models. Under this optimized conditions in shake flask culture, the mycelial yield and exo-biopolymer production were 25.93 and 2.79 g/L, respectively, which were considerably higher than those obtained in the preliminary studies. The result was further confirmed in a 7-L fermentor experiments.

Keywords: Schizophyllum commune ; Exopolysaccharide; Mycelia; Optimization

Enhanced Pervaporation Performance of Multi-layer PDMS/PVDF Composite Membrane for Ethanol Recovery from Aqueous Solution by Xia Zhan; Jiding Li; Junqi Huang; Cuixian Chen (pp. 632-642).

Multi-layer PDMS/PVDF composite membrane with an alternative PDMS/PVDF/non-woven-fiber/PVDF/PDMS configuration was prepared in this paper. The porous PVDF substrate was obtained by casting PVDF solution on both sides of non-woven fiber with immersion precipitation phase inversion method. Polydimethylsiloxane (PDMS) was then cured by phenyltrimethoxylsilane (PTMOS) and coated onto the surface of porous PVDF substrate one layer by the other to obtain multi-layer PDMS/PVDF composite membrane. The multi-layer composite membrane was used for ethanol recovery from aqueous solution by pervaporation, and exhibited enhanced separation performance compared with one side PDMS/PVDF composite membranes, especially in the low ethanol concentration range. The maximum separation factor of multi-layer PDMS/PVDF composite membrane was obtained at 60 °C, and the total flux increased exponentially along with the increase of temperature. The composite membrane gave the best pervaporation performance with a separation factor of 15, permeation rate of 450 g/m²h with a 5 wt.% ethanol concentration at 60 °C.

Keywords: Multi-layer; PDMS/PVDF; Composite membrane; Pervaporation

Biogas Production from Anaerobic Co-digestion of Food Waste with Dairy Manure in a Two-Phase Digestion System by Rongping Li; Shulin Chen; Xiujiu Li (pp. 643-654).

Co-digestion of food waste and dairy manure in a two-phase digestion system was conducted in laboratory scale. Four influents of R0, R1, R2, and R3 were tested, which were made by mixing food waste with dairy manure at different ratios of 0:1, 1:1, 3:1, and 6:1, respectively. For each influent, three runs of experiments were performed with the same overall hydraulic retention time (HRT) of 13 days but different HRT for acidification (1, 2, and 3 days) and methanogenesis (12, 11, and 10 days) in two-phase digesters. The results showed that the gas production rate (GPR) of co-digestion of food waste with dairy manure was enhanced by 0.8–5.5 times as compared to the digestion with dairy manure alone. Appropriate HRT for acidification was mainly determined by the biodegradability of the substrate digested. Three-, 2-, and 1-day HRT for acidification were found to be optimal for the digestion of R0, R1, and R2/R3, respectively, when overall HRT of 13 days was used. The highest GPR of 3.97 L/L·day was achieved for R3(6:1) in Run 1 (1 + 12 days), therefore, the mixing ratio of 6:1 and HRT of 1 day for acidification were considered to be the optimal ones and thus recommended for co-digestion of food waste and dairy manure. There were close correlations between degradation of organic matters and GPR. The highest VS removal rate was achieved at the same HRT for acidification and mixing ratio of food waste and dairy manure as GPR in the co-digestion. The two-phase digestion system showed good stability, which was mainly attributed to the strong buffering capacity with two-phase system and the high alkalinity from dairy manure when co-digested with food waste.

Keywords: Anaerobic co-digestion; Biogas; Dairy manure; Food waste; Mixing ratio; Two-phase digestion system

Induction of an Oxalate decarboxylase in the Filamentous Fungus Trametes versicolor by Addition of Inorganic Acids by Cui Xia Zhu; Feng Hong (pp. 655-664).

In order to improve yields and to reduce the cost of oxalate decarboxylase (OxDC, EC 4.1.1.2), the induction of OxDC in the white-rot fungus Trametes versicolor was studied in this work. OxDC was induced by addition of inorganic acids including hydrochloric acid, sulfuric acid, and phosphoric acid to culture media. The results showed that all the acids could enhance OxDC expression. The activity of the acid-induced OxDC rose continuously. All of the OxDC volumetric activities induced by the inorganic acids were higher than 20.0 U/L and were two times higher than that obtained with oxalic acid. OxDC productivity was around 4.0 U•L⁻¹•day⁻¹. The highest specific activity against total protein was 3.2 U/mg protein at day 8 after induction of sulfuric acid, and the specific activity against mycelial dry weight was 10.6 U/g at day 9 after induction of hydrochloric acid. The growth of mycelia was inhibited slightly when the pH values in culture media was around 2.5–3.0, while the growth was inhibited heavily when the pH was lower than 2.5.

Keywords: Oxalate decarboxylase; Induction; Trametes versicolor ; Low pH environment; Inorganic acid

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #2)