Optimization of γ-polyglutamic acid production byBacillus subtilis ZJU-7 using a surface-response methodology

The components of the media used to elicit the biosynthesis of poly-γ-glutamic acid (γ-PGA) byBacillus subtilis ZJU-7 were investigated, particularly the carbon and nitrogen sources. Of the 7 carbon sources investigated, sucrose induced the highest rate of γ-PGA productivity; among the nitrogen sources, tryptone had the best effect for γ-PGA production. A 2⁶⁻² fractional factorial design was used to screen factors that influence γ-PGA production significantly, and a central composite design was finally adopted to formulate the optimal medium. γ-PGA productivity improved approximately 2-fold when the optimal medium was used compared with the original nonoptimized medium, and volumetric productivity reached a maximum of 58.2 g/L after a 24-h cultivation period.     

Cloning,production and secretion of β-galactosidase inAcetobacter pasteurianus

The gene coding for β-galactosidase fromEscherichia coli was cloned into plasmid pACT71 containing the replicon from plasmid pAC1 fromAcetobacter pasteurianus. E. coli MC4100,E. coli JM105,E. coli LE392.23 andA. pasteurianus 3614 were transformed with the recombinant plasmid pACB815. Cells were cultivated in LB, YPG and M media supplemented with glucose, glycerol, lactose or ethanol and β-galactosidase activity was detected in the cells and in the cultivation medium. The best substrate for production of β-galactosidase was lactose. To release β-galactosidase fromA. pasteurianus cells amino acids were added to the cultivation medium. The highest secretory activity was achieved using 1.5% glycine after 36 h of cultivation in the M medium.     

Simocyclinones: diversity of metabolites is dependent on fermentation conditions3

Simocyclinones, a novel group of angucyclinone antibiotics, are produced by Streptomyces antibioticus Tü 6040. The compounds show antibacterial and antitumor properties. In submerged cultivation, the production of simocyclinones is strongly dependent on the carbon and nitrogen sources used in a chemically defined medium. Productivity of distinct components and diversity of simocyclinone compounds are influenced by the medium composition. Four series of simocyclinone compounds were detected by high-performance liquid chromatography (HPLC) diode array detector (DAD) and HPLC electrospray ionization (ESI) mass spectrometry (MS) analysis, isolated and the structures determined by nuclear magnetic resonance (NMR) techniques. Under optimized conditions, simocyclinone D8 was produced in an amount of 300 mg l⁻¹ and simocyclinone C4 in a concentration up to 50 mg l⁻¹. Journal of Industrial Microbiology & Biotechnology (2001) 27, 144–148. Received 21 September 1999/ Accepted in revised form 20 January 2000     

α-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans

The effect of biomass concentration on the formation of Aspergillus oryzaeα-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of α-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2–12 g dry weight kg⁻¹. When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzaeα-amylase gene (creA) was deleted, no significant decrease in the specific rate of α-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific α-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium. Received: 13 January 2000 / Received revision: 30 June 2000 / Accepted: 1 July 2000     

Efficient production of menaquinone (vitamin K2) by a menadione-resistant mutant of Bacillus subtilis

Efficient production of menaquinone (MK) by Bacillus subtilis was achieved. An edible strain of B. subtilis, isolated from the traditional Japanese food natto, was mutated to improve MK productivity. A menadione-resistant mutant producing 30% more MK than its parent strain was obtained. Soybean extract and glycerol were the best nitrogen and carbon sources, respectively, among the sources tested. Addition of yeast extract also increased MK productivity. The maximum concentration of MK reached about 35.0 mg/l after 4 days of culture in a jar fermenter. The pH of the medium decreased to 5.5 after the start of cultivation, then spontaneously increased to 7.7–8.0. This pH change might be important in the production of MK because only small amounts of MK were obtained when pH was controlled at 5.7, 6.0, 7.0, 7.5 or 8.0. Journal of Industrial Microbiology & Biotechnology (2001) 26, 115–120. Received 24 April 2000/ Accepted in revised form 14 August 2000     

beta-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant

Several wild strains and mutants of Rhodotorula spp. were screened for growth, carotenoid production and the proportion of -carotene produced in sugarcane molasses. A better producer, Rhodotorula glutinis mutant 32, was optimized for carotenoid production with respect to total reducing sugar (TRS) concentration and pH. In shake flasks, when molasses was used as the sole nutrient medium with 40 g l⁻¹ TRS, at pH 6, the carotenoid yield was 14 mg l⁻¹ and -carotene accounted for 70% of the total carotenoids. In a 14-l stirred tank fermenter, a 20% increase in torulene content was observed in plain molasses medium. However, by addition of yeast extract, this effect was reversed and a 31% increase in -carotene content was observed. Dissolved oxygen (DO) stat fed-batch cultivation of mutant 32 in plain molasses medium yielded 71 and 185 mg l⁻¹ total carotenoids in double- and triple-strength medium, respectively. When supplemented with yeast extract, the yields were 97 and 183 mg l⁻¹ total carotenoid with a 30% increase in -carotene and a simultaneous 40% decrease in torulene proportion. Higher cell mass was also achieved by double- and triple-strength fed-batch fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 327–332. Received 18 September 2000/ Accepted in revised form 02 March 2001     

α-mannosidase and mannanase of some wood-rotting fungi

Cultivation media from 11 wood-rotting fungi contained α-mannosidase and mannanase activity. α-Mannosidase was studied in more detail inPhellinus abietis and mannanase was studied more intimately in basidiomycetesPhellinus abietis, Trametes sanguinea andPholiota aurivella. Suitable cultivation conditions and optimum conditions for the production of α-mannosidase and mannanase were determined. Both enzymes are constitutive; mannanase is extracellular, α-mannosidase was found in both mycelium and cultivation medium.     

Kinetic analysis of oil biosynthesis by an arachidonic acid-producing fungus, Mortierella alpina 1S-4

 Kinetic analysis of arachidonic acid (AA)-oil biosynthesis by Mortierella alpina 1S-4 growing under lipid-accumulating (LN medium) and non-lipid-accumulating (HN medium) conditions was investigated and compared with industrial AA fermentation. Various kinetic parameters of these cultivation processes demonstrate a characteristic pattern of the lipogenesis in this fungus, where growth phase, phase of oil accumulation and phase of AA synthesis are distinct from each other. The fungus utilizing LN medium synthesized 32.3 g fatty acid 100 g⁻¹ glucose on the 4th day of cultivation and reached the maximum daily fatty acid accumulation (expressed as differential specific rate q _D(FA/B)) of 9.5%. Our results also indicate that a q _D(FA/B) value of about 2.5% might be critical for lipid overproduction in M. alpina. AA was rapidly incorporated into triacylglycerols (90% of total AA) at the later cultivation phase and overall AA yield was directly related to the total yield of fatty acid. Received: 10 December 1999 / Accepted: 13 February 2000     

Interaction between the diazo dye, “Vermelho Reanil” P8B,and Neurospora crassa strain 74A

Summary The cultivation of Neurospora crassa, strain 74A, in Neurospora complete medium containing a azo dye, “Vermelho Reanil P8B” from Imperial Chemical Industries showed that after 24 h of contact, most of the dye was removed from the medium. The greatest extent of dye removal (91.3 to 89.1%) was achieved when the concentration of the dye was between 16.0 and 32.0 μg ml⁻¹ which is the range found in industrial wastes. The biomass production was unaffected in the presence of the dye at all the concentrations analysed.     

Polymer production by two newly isolated extremely halophilic archaea: application of a novel corrosion-resistant bioreactor

A novel corrosion-resistant bioreactor composed of polyetherether ketone (PEEK), tech glass and silicium nitrite ceramics was constructed and applied for the cultivation of two newly isolated, extremely halophilic archaea producing poly(γ-glutamic acid) (PGA), or poly(β-hydroxy butyric acid) (PHB), respectively. These bacteria were isolated from hypersaline soil close to Aswan (Egypt). The isolate strain 40, which is related to the genus Natrialba, produced large amounts of PGA when cultivated on solid medium. Culture conditions were optimised applying the corrosion-resistant bioreactor. PGA production was dependent on NaCl concentration and occurred about at 20% (w/v) NaCl in the medium. A maximum cell density of about 1.6 g cell dry matter/l was obtained when the bioreactor was stirred and aerated in a batch fermentation process using proteose-peptone medium. The supernatant was monitored with respect to PGA formation, and after 90 h a maximum of 470 mg/l culture volume was detected by HPLC analysis. Culture conditions were optimized for the isolate 56, which accumulated PHB as intracellular granules. Batch fermentations in the stirred and aerated bioreactor applying acetate and n-butyric acid as carbon sources led to cell density of 2.28 g cell dry matter/l and a maximum PHB accumulation contributing to about 53% of cellular dry weight. About 4.6 g PHB were isolated from 10.6 g dried cells of strain 56, which exhibited a weight average molar mass of 2.3 × 10⁵ g mol⁻¹ and a polydispersity of about 1.4. Received: 3 December 1999 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

A genetically modified solvent-tolerant bacterium for optimized production of a toxic fine chemical

The aim of the study was to investigate whether toxic fine chemical production can be improved using the solvent-tolerant Pseudomonas putida S12 in a two-liquid-phase system consisting of aqueous media and a water-immiscible octanol phase with production of 3-methylcatechol from toluene as the model conversion. For this purpose the genes involved in this conversion, todC1C2BAD from P. putida F1, were introduced into P. putida S12 with high stable expression. Production of 3-methylcatechol was monitored in batch incubations with different media using a single medium and a two-liquid medium–octanol system. The maximum concentration of 3-methylcatechol increased two-fold using the two-liquid medium–octanol system, irrespective of the selected medium. Received: 29 December 1999 / Received revision: 29 February 2000 / Accepted: 6 March 2000     

Optimization of Nutritional Factors for Exopolysaccharide Production by Submerged Cultivation of the Medicinal Mushroom Oudemansiella radicata

Summary Effects of nutritional factors on exopolysaccharide production by submerged cultivation of the medicinal mushroom Oudemansiella radicata were investigated in shake flasks. Sucrose and peptone were optimal carbon and nitrogen sources for cell growth and exopolysaccharide production. The exopolysaccharide production was increased with an increase in initial sucrose concentration within the range of 10–40 g l⁻¹ and initial peptone concentration within the range of 1–3 g l⁻¹. To enhance further exopolysaccharide production, the effect of carbon/nitrogen ratios was studied using central composite design (CCD) and response surface analysis. The maximum exopolysaccharide production of 2.67 ± 0.15 g l⁻¹ was achieved in medium with optimized carbon and nitrogen sources, i.e. 39.3 g sucrose l⁻¹ and 3.16 g peptone l⁻¹ in the same cultivation conditions. The information obtained is helpful for the hyperproduction of exopolysaccharide by submerged cultivation of O. radicata on a large scale.     

Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus

A chemically defined medium has been developed for anaerobic cultivation of hyaluronic-acid(HA)-producing Streptococcus zooepidemicus, which contains 11 amino acids essential to growth, and glutamine as a principal nitrogen source. The final HA concentration, the specific rate of HA production and HA-to-glucose yields were similar for growth in the chemically defined medium relative to growth in complex medium. Consequently cells cultivated on chemically defined medium can be expected to have similar activity regarding HA synthesis as compared to cells grown on complex media. However, the specific growth rate and volumetric HA production rate were found to be less favourable in the chemically defined media. Received: 4 October 1996 / Accepted: 25 October 1996     

Statistical medium optimization for enhanced azadirachtin production in hairy root culture of Azadirachta indica

Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg’s medium vitamins, and 30 g l⁻¹ sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g⁻¹) and azadirachtin production (∼44 mg l⁻¹) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l⁻¹ dry weight. To enhance the production of azadirachtin, a Plackett–Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l⁻¹ sucrose, 0.19 g l⁻¹ potassium dihydrogen phosphate, 3.1 g l⁻¹ potassium nitrate, and 0.41 g l⁻¹ magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l⁻¹ and azadirachtin accumulation of 5.2 mg g⁻¹, which was equivalent to an overall azadirachtin production of 73.84 mg l⁻¹, 68% more than that obtained under non-optimized conditions.     

Use of whey for production of exocellular polysaccharide by a mutant strain ofXanthomonas campestris

Growth and kinetics of the production of exocellular polysaccharide was studied in a mutant strain ofXanthomonas campestris lac ⁺ during cultivation in a submerged culture in a medium containing whey. The maximum production of the polymer was observed at the initial stage of the stationary growth phase of the culture. The mean production yield was about 1.4%. The results were comparable with those obtained during cultivation on a lactose medium. Translated by Č. Novotny     

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca⁺² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 g l⁻¹) in the fermentation medium further increased naringinase production (8.9 IU ml⁻¹) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch cultures of Pseudomonas sp EL-2

Pseudomonas sp EL-2 was cultivated to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from a structurally unrelated carbon source, glucose, by a fed-batch culture technique. Variation of the carbon to nitrogen (C/N) ratio of the medium produced optimal P(3HB-co-3HV) production at a C/N ratio of 95. Production of P(3HB-co-3HV) was favored by a dissolved oxygen tension of 40%. A maximum biomass concentration of 38 g L⁻¹ containing 53% P(3HB-co-3HV) was achieved after 45 h of cultivation. This corresponds to a volumetric productivity of 0.84 g L⁻¹ h⁻¹. The copolymer contained 7.5 mol% 3-hydroxyvalerate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 36–40. Received 28 January 1999/ Accepted in revised form 11 September 1999     

Production of Cold-Adapted Amylase by Marine Bacterium <Emphasis Type="Italic">Wangia</Emphasis> sp. C52: Optimization,Modeling, and Partial Characterization

The aim of this work was to optimize the fermentation parameters in the shake-flask culture of marine bacterium Wangia sp. C52 to increase cold-adapted amylase production using two statistical experimental methods including Plackett–Burman design, which was applied to find the key ingredients for the best medium composition, and response surface methodology, which was used to determine the optimal concentrations of these components. The results showed starch, tryptone, and initial pH had significant effects on the cold-adapted amylase production. A central composite design was then employed to further optimize these three factors. The experimental results indicated that the optimized composition of medium was 6.38 g L⁻¹ starch, 33.84 g L⁻¹ tryptone, 3.00 g L⁻¹ yeast extract, 30 g L⁻¹ NaCl, 0.60 g L⁻¹ MgSO₄ and 0.56 g L⁻¹ CaCl₂. The optimized cultivation conditions for amylase production were pH 7.18, a temperature of 20°C, and a shaking speed of 180 rpm. Under the proposed optimized conditions, the amylase experimental yield (676.63 U mL⁻¹) closely matched the yield (685.60 U mL⁻¹) predicted by the statistical model. The optimization of the medium contributed to tenfold higher amylase production than that of the control in shake-flask experiments.