Conditions for Ethanol Production during Bioconversion of Cellulose-Containing Raw Material

Several natural associations composed of thermophilic anaerobic bacteria capable of utilizing various cellulose materials at 60 ± 2°C and pH 6.0–7.0 were isolated from the sludge of Kamchatka geothermal springs. The rate of ethanol production (up to 1.7 g/l per day) and the concentration of ethanol in the medium (up to 1.2%), as well as the fermentation period (10–15 days), were determined under anaerobic conditions in the presence of cellulose, coniferous sawdust, newsprint, or paper pulp as a carbon source. Microorganisms were found that inhibited the production of ethanol. The initial pH value was found to influence both the ethanol production rate and ethanol/acetate ratio. A pH decrease from 7.0 to 5.0 led to a 6.7-fold increase in ethanol production and caused a 23.8-fold increase in the ethanol/acetate ratio.     

Production of patulin by Micromycetes

In the context of the screening for antifungal and antibacterial molecules among the Micromycetes, we investigated the ability of 850 strains grown in liquid medium to produce patulin. The compound was produced by 58 fungi, most of which had not been mentioned in the literature, especially Mucorales and Fungi Imperfecti. Biological tests showed that dermatophytes are the most sensitive to this molecule. It also appears that the origin of the producing strains influences their toxin producing activity.     

Bioconversion of compactin into pravastatin by Streptomyces sp.

Streptomyces sp. Y-110, isolated from soil, modified compactin to pravastatin, a therapeutic agent for hypercholesterolemia. In a batch culture, the highest production of pravastatin was 340 mg l^–1 from 750 mg compactin l^–1 in 24 h. By intermittent feeding of compactin into the culture medium, both the compactin concentration and its conversion increased to 2000 mg l^–1 and 1000 mg pravastatin l^–1, respectively, with the conversion rate of 10 mg l^–1 h^–1. Continuous feeding of compactin increased production of pravastatin to 15 mg l^–1 h^–1.     

Bioconversion of cellulose into ethanol by Clostridium thermocellum--product inhibition

Direct anaerobic bioconversion of cellulosic substances into ethanol by Clostridium thermocellum ATCC 27405 has been carried out at 60 degrees C and pH 7.0 (initial for 100 L) under continuous sparging of oxygen free nitrogen in a culture vessel. Raw bagasse, mild alkali-treated bagasse, and solka floc were used as substrates. The extent of conversion of raw bagasse (cellulose, 50%; hemicellulose, 25%; lignin, 19%) was observed as 52% (w/w) and 79% (w/w) in the case of mild alkali and steam-treated bagasse (cellulose, 72%; hemicellulose, 11%; lignin, 12%), respectively. Use of bagasse concentration above 10 g/L showed a decreased rate in ethanol production. An inoculum age between 28-30 h and cell mass content of 0.027-0.036 g/L (dry basis) were used. The results obtained with raw and pretreated bagasse have been compared with those of highly pure Solka Floc (hemicellulose, 10%). Studies on the product inhibition indicated a linear fall of the percent of survivors with time. An Arrhenius type correlation between the cell decay rate constant and the product concentration was predicted. Even at low levels, the inhibitory effects of products on cell viability, the specific growth rate, and extracellular cellulase enzyme were observed.     

Metabolism of vanillic acid by Micromycetes

The ability of 953 strains of Micromycetes to grow with vanillic acid (0.5 g/l) was investigated. Toxicity assays were performed on malt extract/agar medium, while consumption was estimated by growing fungi on solid synthetic medium with vanillic acid as sole carbon source. More than half of the tested strains grew in both conditions. After cultivation on solid media, 296 strains were selected and cultivated in liquid synthetic medium. These experiments allowed division of the Micromycetes into different groups according to their consumption of the phenolic compound and the appearance of new metabolites. Results were related to the taxonomic position of the strains.     

Bioconversion of agro-wastes into acetone butanol

Summary Agricultural residues such as bagasse and rice straw have been hydrolyzed by mixed culture filtrates of T. reesei and A. wentii to obtain fermentable sugars. After the hydrolyzate had been treated to remove undesired impurities, Cl. saccharoper butylacetonicum was used to produce butanol to the extent of 16 g/l.     

Bioconversion of Pinoresinol into Matairesinol by Use of Recombinant Escherichia coli

Lignans, a class of dimeric phenylpropanoid derivative found in plants, such as whole grains and sesame and flax seeds, have anticancer activity and can act as phytoestrogens. The lignans secoisolariciresinol and matairesinol can be converted in the mammalian proximal colon into enterolactone and enterodiol, respectively, which reduce the risk of breast and colon cancer. To establish an efficient bioconversion system to generate matairesinol from pinoresinol, the genes encoding pinoresinol-lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase (SDH) were cloned from Podophyllum pleianthum Hance, an endangered herb in Taiwan, and the recombinant proteins, rPLR and rSDH, were expressed in Escherichia coli and purified. The two genes, termed plr-PpH and sdh-PpH, were also linked to form two bifunctional fusion genes, plr-sdh and sdh-plr, which were also expressed in E. coli and purified. Bioconversion in vitro at 22°C for 60 min showed that the conversion efficiency of fusion protein PLR-SDH was higher than that of the mixture of rPLR and rSDH. The percent conversion of (+)-pinoresinol to matairesinol was 49.8% using PLR-SDH and only 17.7% using a mixture of rPLR and rSDH. However, conversion of (+)-pinoresinol by fusion protein SDH-PLR stopped at the intermediate product, secoisolariciresinol. In vivo, (+)-pinoresinol was completely converted to matairesinol by living recombinant E. coli expressing PLR-SDH without addition of cofactors.     

Bioconversion of kinnow-mandarin waste into single-cell protein

Summary Kinnow-mandarin waste (peel, pulp and seeds) was assessed for single-cell protein (SCP) production byChaetomium globosum andSporotrichum pulverulentum in shake-flask culture. The maximum protein enrichment (32% and 34%) of the substrate was achieved after 5 and 7 days of incubation by the two organisms, respectively. Of various nitrogen sources, NaNO₃ and NH₄Cl gave maximum protein enrichment of the substrate byC. globosum andS. pulverulentum, respectively.

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Bioconversion des résidus de la mandarine-kinnow en protéine uni-cellulaire

Résumé On a examiné la possibilité de produire des protéines uni-cellulaires (POU) à partir de résidus de la mandarine-kinnow (pelures, pulpe, et pépins) par la culture deChaetomium globosum et deSporotrichum pulverulentum en flacons agités. L'enrichissement maximum en protéines du substrat, soit 32 et 34% est obtenu après 5 et 7 jours d'incubation respectivement par les deux organismes. Parmi les diverses sources d'azote, NaNO₃ et NH₄Cl ont permis respectivement l'enrichissement maximum en protéines du substrat parC. globosum etS. pulverulentum.

     

Bioconversion of avermectin into 27-OH avermectin

Summary The bioconversion of avermectin to its 27-hydroxy derivative is achieved withNocardia autotrophica subsp.canberrica. The approach of increasing bioconversion productivity rather than efficiency was adopted in these studies. Process improvement studies focused on the physico-chemical conditions of the fermentation, examined initially at the shake-flask scale. Bioconversion yields were affected by pH, substrate concentration, time of substrate addition, substrate solubilization, carbon to nitrogen ratio, and medium strength. Optimization of these parameters resulted in a 8-fold process improvement. During pre scale-up studies, the sensitivity of this bioconversion to the antifoam employed was demonstrated and lard oil was selected as giving the best results. Additional process changes were required during scale-up efforts in larger vessels, including replacement of the original substrate solvent with dimethylsulfoxide.     

Bioconversion of linoleic acid into conjugated linoleic acid by immobilized Lactobacillus reuteri

Lactobacillus reuteri was immobilized on silica gel to evaluate the bioconversion of linoleic acid (LA) into conjugated linoleic acid (CLA), consisting of cis-9,trans-11 and trans-10,cis-12 isomers. The amount of cell to carrier, the reaction time, and the substrate concentration, pH, and temperature for CLA production were optimized at 10 mg of cells/(g of carrier), 1 h, 500 mg/L LA, 10.5, and 55 degrees C, respectively. In the presence of 1.0 mM Cu(2+), CLA production increased by 110%. Under the optimal conditions, the immobilized cells produced 175 mg/L CLA from 500 mg/L LA for 1 h with a productivity of 175 mg/(L.h) and accumulated 5.5 times more CLA than that obtained from bioconversion by free washed cells. The CLA-producing ability of reused cells was investigated over five reuse reactions and was maximal at pH 7.5, 25 degrees C, and 1.0 mM Cu(2+). The total amount of CLA by the combined five reuse reactions was 344 mg of CLA/L reaction volume. This was 8.6 times higher than the amount obtained from reuse reactions by free washed cells.     

啤酒酵母菌对无机硒的有机转化

利用啤酒酵母菌对无机硒(亚硒酸钠)进行有机转化。通过在培养基中加入不同浓度的无机硒溶液和不同时间加入无机硒溶液,于28℃、220 r/min摇床条件下培养5 d,离心得菌细胞,测定前样品预处理:破碎菌细胞,显微镜下计数,计算破碎率,破碎后的菌体装入透析袋于蒸馏水中透析除去无机硒。准确测定无机硒,用浓硫酸-高氯酸的消化体系消化样品后,紫外分光光度法于335 nm处测量吸光度,在标准曲线上查出硒含量,计算无机硒的转化率。啤酒酵母菌的最佳加硒时间为24 h,亚硒酸钠浓度大于12μg/mL对啤酒酵母菌转化无机硒有明显抑制作用,啤酒酵母菌对无机硒的摄入率约为62%,转化率约为53%;超生波细胞粉碎仪破碎细胞的破碎率为55%左右。结果表明,啤酒酵母菌可以转化无机硒。     

Secretion of Extracellular Proteinases Active against Fibrillar Proteins by Micromycetes

It has been shown that micromycetes Aspergillus ustus 1 and Tolypocladium inflatum k1 secrete proteolytic enzymes that possess high collagenolytic, fibrinolytic, and elastolytic activity. The activity of proteinases hydrolyzing fibrillar proteins, which was determined by the cleavage of azo-collagen, was 122.6 × 10^–3E_Azc/mL in A. ustus 1 and 69.7 × 10^–3E_Azc/mL in T. inflatum k1 (E_Azc is the amount of azocollagen cleaved in 1 min (μg). The maximum values of activity were observed during submerged cultivation of A. ustus 1 for 4 days and of T. inflatum k1 for 5 days. It has been shown that the maximum of collagenolytic and general proteolytic activity during the cultivation of A. ustus 1 are time-separated, unlike T. inflatum k1, which, presumably, can simplify the procedure for obtaining proteinases active against fibrillar proteins.     

Bioconversion of Pinoresinol Diglucoside and Pinoresinol from Substrates in the Phenylpropanoid Pathway by Resting Cells of Phomopsis sp.XP-8

Pinoresinol diglucoside (PDG) and pinoresinol (Pin) are normally produced by plant cells via the phenylpropanoid pathway. This study reveals the existence of a related pathway in Phomopsis sp. XP-8, a PDG-producing fungal strain isolated from the bark of the Tu-chung tree (Eucommiaulmoides Oliv.). After addition of 0.15 g/L glucose to Phomopsis sp. XP-8, PDG and Pin formed when phenylalanine, tyrosine, leucine, cinnamic acid, and p-coumaric acid were used as the substrates respectively. No PDG formed in the absence of glucose, but Pin was detected after addition of all these substrates except leucine. In all systems in the presence of glucose, production of PDG and/or Pin and the accumulation of phenylalanine, cinnamic acid, or p-coumaric acid correlated directly with added substrate in a time- and substrate concentration- dependent manner. After analysis of products produced after addition of each substrate, the mass flow sequence for PDG and Pin biosynthesis was defined as: glucose to phenylalanine, phenylalanine to cinnamic acid, then to p-coumaric acid, and finally to Pin or PDG. During the bioconversion, the activities of four key enzymes in the phenylpropanoid pathway were also determined and correlated with accumulation of their corresponding products. PDG production by Phomopsis sp. exhibits greater efficiency and cost effectiveness than the currently-used plant-based system and will pave the way for large scale production of PDG and/or Pin for medical applications.     

Bioconversion of citronellol by Botrytis cinerea

Summary Bioconversion of citronellol 1 was studied with four strains of Botrytis cinerea. Using grape must predominant transformation of 1 to 2,6-dimethyl-1,8-octandiol 2 and (E)-2,6-dimethyl-2-octen-1,8-diol 3 was observed. In minor amounts 2,6-dimethyl-2,8-octandiol 4, two p-menthan-3,8-diol isomers 5a, 5b, (Z)-2,6-dimethyl-2-octen-1,8-diol 6, isopulegol 7, 2-methyl-2-hepten-6-one-1-ol 8 and 2-methyl--butyrolactone 9 were found. Using a small amount of grape must in a synthetic medium (1:700) the bioconversion products 2, 4, 5a and 5b were absent, but additionally 2-methyl-2-hepten-6-one 10, 2-methyl-2-hepten-6-ol 11 and citronellic acid 12 were detected. The results obtained were strongly dependent on the strains used; one strain did not show any metabolic activity against 1. The bioconversion products were identified by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e. on-line — mass spectrometry (HRGC-MS) and — Fourier transform infrared spectroscopy (HRGC-FTIR).     

Bioconversion of alpha-Damascone by Botrytis cinerea

Bioconversion of alpha-damascone (compound 1) was studied with four strains of Botrytis cinerea in grape must (pH 3.2). As biotransformation products of compound 1, 3-oxo-alpha-damascone, cis- and trans-3-hydroxy-alpha-damascone, gamma-damascenone, 3-oxo-8, 9-dihydro-alpha-damascone, and cis- and trans-3-hydroxy-8,9-dihydro-alpha-damascone were identified. In addition, acid-catalyzed chemical transformation of compound 1 to the diastereomers of 9-hydroxy-8,9-dihydro-alpha-damascone was observed. Identifications were performed by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e., on-line HRGC-mass spectrometry and HRGC-Fourier transform infrared spectroscopy, after extractive sample preparation.     

Bioconversion of para-substituted monophenolic compounds into corresponding catechols by alginate entrapped cells ofMucuna pruriens

Alginate-entrapped cells ofM. pruriens were able to convert a number of parasubstituted monophenolic compounds into the corresponding catechols. All catechols produced were released into the medium, which offered the opportunity to isolate these products via a relatively simple procedure. Prepurification was performed on a Sephadex G10 gel and catechols were concentrated on Affigel 601. The identity of all products was confirmed with combined liquid chromatography/mass spectrometry (LC/MS) or MS using the desorption chemical ionization technique, depending on the catechol. For the entrapped cells and for a cell homogenate prepared of the same cell line ofM. pruriens the substrate specificities were qualitatively identical when judged on initial rates of synthesis calculated on protein basis.     

Bioconversion of grape must into modulated gluconic acid production by Aspergillus niger ORS-4.410

AIMS: Analysis of regulators for modulated gluconic acid production under surface fermentation (SF) condition using grape must as the cheap carbohydrate source, by mutant Aspergillus niger ORS-4.410. Replacement of conventional fermentation condition by solid-state surface fermentation (SSF) for semi-continuous production of gluconic acid by pseudo-immobilization of A. niger ORS-4.410. METHODS AND RESULTS: Grape must after rectification was utilized for gluconic acid production in batch fermentation in SF and SSF processes using mutant strain of A. niger ORS-4.410. Use of rectified grape must led to the improved levels of gluconic acid production (80-85 g l(-1)) in the fermentation medium containing 0.075% (NH4)2HPO4; 0.1% KH2PO4 and 0.015% MgSO4.7H2O at an initial pH 6.6 (+/-0.1) under surface fermentation. Gluconic acid production was modulated by incorporating the 2% soybean oil, 2% starch and 1% H2O2 in fermentation medium at continuously high aeration rate (2.0 l min(-1)). Interestingly, 95.8% yield of gluconic acid was obtained when A. niger ORS-4.410 was pseudo-immobilized on cellulose fibres (bagasse) under SSF. Four consecutive fermentation cycles were achieved with a conversion rate of 0.752-0.804 g g(-1) of substrate into gluconic acid under SSF. CONCLUSIONS: Use of additives modulated the gluconic acid production under SF condition. Semi-continuous production of gluconic acid was achieved with pseudo-immobilized mycelia of A. niger ORS-4.410 having a promising yield (95.8%) under SSF condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioconversion of grape must into modulated gluconic acid production under SSF conditions can further be employed in fermentation industries by replacing the conventional carbohydrate sources and expensive, energy consuming fermentation processes.     

Bioconversion of Dethiobiotin into Biotin by Resting Cells and Protoplasts of Bacillus sphaericus bioB Transformant

The reaction system for the bioconversion of dethiobiotin into biotin by resting cells and protoplasts of a Bacillus sphaericus bioB transformant was established. The reaction mixtures consisted of completely synthetic components, such as amino acids and metal salts. Among the sulfur compounds tested, L-CyS and L-cystine were effective in the biosynthesis of biotin from dethiobiotin both by resting cells and by protoplasts. The optimum concentrations of L-Cys were 2 to 3 mM and more than 0.25 mM for resting cell and protoplast systems, respectively. Vigorous shaking enhanced the biotin biosynthesis by protoplasts. The addition of yeast extract to the reaction mixture without a mixture of amino acids brought about a three-fold increase in the, amount of biotin synthesized by protoplasts when compard to the case with the reaction mixture containing the amino acid mixture. The amount of biotin synthesized by protoplasts increased with the incubation time up to 6 h and reached about 2 μg/ml. There was a clear correlation between the number of remaining protopiasts and their biotin-biosynthesizing activity during the incubation.