Extracellular lysine production from hydrocarbons byArthrobacter globiformis

A bacterium isolated from Burdwan (India) soil was found to accumulatel-lysine in the growth medium and was identified asArthrobacter globiformis. The strain grew and accumulated lysine in a purely synthetic medium. Supplementation of the synthetic medium stimulated growth but did not improve the yield. The entire fermentation period could be divided into a growth phase and a production phase, which could be prolonged by adjustment of pH to the neutral range. Among the different hydrocarbon and nitrogen sources tested SR gas oil at 4 % and ammonium sulphate at 0.4 %, respectively, were found most to be suitable. Different vitamins and antibiotics stimulated growth and lysine yield; inoculum of 7 % (V/V) of the medium was found to be optimal. The yield of lysine under optimal conditions was 3.4 g per litre medium. Lysine was isolated in crystalline form from the fermented broth by IEC and found to be a purel-isomer.     

Lysine production from hydrocarbon by micrococcus species

A bacterium isolated from Assam (India) soil was found to accumulate L-lysine in the mineral salt-hydrocarbon medium and identified to be a strain of Micrococcus luteus. The strain is able to grow and accumulate l-lysine in a purely synthetic medium, but supplementation of the synthetic medium with casamino acid or yeast extract or both, improves the yield. The entire fermentation period can be divided into a growth phase and a production phase, which can be prolonged by adjustment of the pH to the neutral range. Among the different hydrocarbon and nitrogen sources tested straight run gas oil (47percnt;) and ammonium sulphate (0.4%), respectively, were found most suitable. Erythromycin at 1 μg/ml level inhibited growth bu¸t stimulated lysine excretion. An inoculum level of 10% (v/v) of the medium was optimal for lysine production. The yield of lysine under optimal conditions was found to be 3.25 g per litre of the medium. Lysine has been isolated in crystalline form from the fermented broth by ion exchange resin chromatography and found to be pure sample of L-isomer.     

Growth and production characteristics of permeabilized Coleus blumei cells in immobilized fed-batch culture

Summary Permeabilized Coleus blumei cells were cultivated in an immobilized state to study the effect of dimethyl sulfoxide (DMSO) concentrations and growth regulators on cell growth and rosmarinic acid (RA) production characteristics. Luffa (the fibrous skeleton of mature fruit of Luffa cylindrica) was a good support matrix for cell immobilization because of its high void volume. Maximum cell loading capacity was 1.33 g dry cell weight (DCW)/g dry Luffa. The experiments were done in shake flasks with no free medium. The medium was supplied in a fed-batch mode to avoid the flotation of Luffa pieces. The sucrose in the medium was completely hydrolyzed to glucose and fructose without any sugar accumulation in the medium. The cell viability was slightly higher in the cells on top of the Luffa than those in the middle. Cell growth rate and rosmarinic acid (RA) production were approximately half that obtained in cell suspension cultures. Cell yield (g DCW/g glucose) was similar to that of cell suspension cultures. The absence of growth regulators did not promote an increase of RA production but did decrease the cell mass. The second step preconditioning with 0.5% DMSO did not improve the cell's adaptability to higher DMSO concentrations and the cell mass did not increase with 2.5% DMSO.     

Production of lysine by using immobilized livingCorynebacterium sp cells

Summary Lysine production by immobilizedCorynebacterium sp cells in alginate gel beads was investigated in flasks. ImmobilizedCorynebacterium sp cells exhibited a slightly greater lysine production than free cells and accumulated 60 g/l of L-lysine at maximum, when cultured for 120h in a medium containing 200g/l glucose as carbon source. Several factors, such as inoculum size, incubation time and alginate gel concentration were examined in order to improve lysine production by immobilized growing cells.     

The improvement of cephalosporin C production by fed-batch culture ofCephalosporium acremonium M25 using rice oil

The objective of this study is to improve cephalosporin C (CPC) production by optimization of medium and culture conditions. A statistical method was introduced to optimize the main culture medium. The main medium for CPC production was optimized using a statistical method. Glucose and corn steep liquor (CSL) were found to be the most effective factors for CPC production. Glucose and CSL were optimized to 2.84 and 6.68%, respectively. CPC production was improved 50% by feeding of 5% rice oil at day 3rd and 5th day during the shake flask culture ofC. acremonium M25. The effect of agitation speeds on CPC production in a 2.5-L bioreactor was also investigated with fed-batch mode. The maximum cell mass (54.5 g/L) was obtained at 600 rpm. However, the maximum CPC production (0.98 g/L) was obtained at 500 rpm. At this condition, the maximum CPC production was improved about 132% compared to the result with batch flask culture.     

Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalose purification

In this study, corn starch was used as the substrate for cell growth and trehalose accumulation by Saccharomycopsis fibuligera A11. Effect of different aeration rates, agitation speeds, and concentrations of corn starch on direct conversion of corn starch to trehalose by S. fibuligera A11 were examined using a Biostat B2 2-l fermentor. We found that the optimal conditions for direct conversion of corn starch to trehalose by this yeast strain were that agitation speed was 200 rpm, aeration rate was 4.0 l/min, concentration of corn starch was 2.0% (w/v), initial pH was 5.5, fermentation temperature was 30°C. Under these conditions, over 22.9 g of trehalose per 100 g of cell dry weight was accumulated in the yeast cells, cell mass was 15.2 g/l of the fermentation medium, 0.12% (w/v) of reducing sugar, and 0.21% (w/v) of total sugar were left in the fermented medium within 48 h of the fermentation. It was found that trehalose in the yeast cells could be efficiently extracted by the hot distilled water (80°C). After isolation and purification, the crystal trehalose was obtained from the extract of the cells.     

Sustainable, High-Yielding Outdoor Mass Cultures of Chaetoceros muelleri var. subsalsum and Isochrysis galbana in Vertical Plate Reactors

Continuous cultures of Chaetoceros muelleri and Isochrysis galbana were grown outdoors in flat plate-glass reactors in which light-path length (LPL) varied from 5 to 30 cm. High daily productivity (13 to 16 g cell mass per square meter of irradiated reactor surface) for long periods of time was obtained in reactors in which the optical path as well as cell density were optimized. 'Twenty centimeters was the optimal LPL, yielding the highest areal productivity of cell mass (g m^–2d^–1), eicosapentaenoic acid, and docosahexaenoic acid, which was identical with that previously found for polysaccharide production of Porphyridium and not far from the optimal LPL affecting maximal productivity in Nannochloropsis species. Relating the energy impinging on a given reactor surface area to the appropriate number of cells showed that the most efficient light dose per cell, obtained with the 20-cm LPL reactor, was approximately 2.5 times lower than the light dose available per cell in the 5-cm LPL reactor, in which a significant decline in areal cell density accompanied the lowest areal output of cell mass. The most effective harvesting regimen was in the range of 10% to 15% of culture volume harvested daily and replaced with fresh growth medium, resulting in a sustainable culture density of 24 × 10⁶ and 28 × 10⁶ cells/ml of C. muelleri and I. galbana, respectively.     

碳源对早花百子莲愈伤组织诱导及其增殖的生理特性影响

为揭示碳源对早花百子莲愈伤组织诱导与增殖的影响机理,该研究以早花百子莲的小花梗为外植体,比较分析30.0 g/L蔗糖、葡萄糖、麦芽糖在愈伤组织诱导、增殖中的效果,测定不同碳源种类处理下愈伤组织增殖相关生理特性,并根据细胞增殖效果、生理指标相关性进行优化验证。结果表明:(1)蔗糖、葡萄糖和麦芽糖碳源处理下,愈伤组织诱导率分别为86.00%、72.00%和59.67%,蔗糖碳源的愈伤组织诱导率比葡萄糖和麦芽糖分别显著提高19.44%和44.13%(P<0.05),蔗糖碳源较葡萄糖和麦芽糖碳源的愈伤组织大小分别显著增加22.44%和90.09%(P<0.05);愈伤组织增殖阶段,蔗糖碳源能够同时维持良好的细胞增殖效率及活性,而葡萄糖碳源的愈伤组织增殖快、状态差,麦芽糖处理增殖慢、状态佳;蔗糖转换葡萄糖碳源后愈伤组织细胞团大小、细胞活性明显下降;蔗糖转换蔗糖、蔗糖转换麦芽糖的效果较好。(2)培养基碳源显著调节愈伤组织增殖阶段的糖代谢、内源激素代谢和氧化胁迫平衡。(3)愈伤组织的主要糖组分为淀粉、葡萄糖;淀粉、麦芽糖含量与细胞团大小相关性高,以蔗糖为碳源的培养基中添加麦芽糖,愈伤组织...     

Establishment of <Emphasis Type="Italic">Cyperus aromaticus</Emphasis> cell suspension cultures for the production of Juvenile hormone III

Cell suspension cultures of Cyperus aromaticus were established from the yellow friable callus derived from the root explants of in vitro plantlets. Four callus cell lines were selected based on their growth index from two populations of callus cultures originated from the mother plants grown in two different locations. The selected four cell lines (Z1, Z6, P4, P9) showed uniform cell growth but produced different amounts of juvenile hormone III (JHIII). The Z1 cell line possessed fast-growing characteristics, produced a high JHIII content, and was chosen as the elite cell line for an optimization study of C. aromaticus cell suspension cultures. An inoculum cell mass of 0.3 g from 12-d cultures in 30 ml culture medium was found to be the optimum inoculum size and culture age for establishing the cell suspension culture of C. aromaticus. MS basal medium supplemented with 4.5 mg/l 2,4-D and 5.5 mg/l NAA was found to be the best medium for production of maximum cell biomass and JHIII. These results indicated that JHIII can be produced from suspension culture of C. aromaticus using a single-stage cell-culture system.     

Increased production of cellulase of Trichoderma sp. by pH cycling and temperature profiling

Cultivation of Trichoderma reesei QM 9414 on 3% (w/v) cellulose medium (C/N ratio = 8.5) produced 4.5 IU/ml celulase 180 hr at a cell growth of 8.0 g/liter (0.266 g cell/g cellulose). It corresponded to an average cellulase productivity 25.0 IU/liter/hr (3.5 IU/g cell/hr). In the same medium 9.5 g/liter cell mass (0.316 g cell/g cellulose), 6.2 IU/ml cellulase, and 38.75 IU/liter/hr (4.0 IU/g cell/hr) cellulase productivity could be obtained using pH cycling condition during cultivation. Cell mass, cellulase yield, and productivity were further increased to 10.0 g/liter, 7.2 IU/ml, and 44.0 IU/liter/hr (4.5 IU/g cell/hr), respectively, by simultaneous pH cycling and temperature profiling strategy. Results are described.     

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and its nutritive analysis

After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0°C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C_16:0 (19.0%), C_18:0 (46.3%), and C_18:1 (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production.     

Optimization of culture medium for lactosucrose ( G-beta-D-galactosylsucrose) Production by Sterigmatomyces elviae mutant using statistical analysis

In this study, the optimization of culture medium using a Sterigmatomyces elviae mutant was investigated using statistical analysis to increase the cell mass and lactosucrose ((4)G-beta-D-galactosylsucrose) production. In basal medium, the cell mass and lactosucrose production were 4.12 g/l and 140.91 g/l, respectively. However, because of the low cell mass and lactosucrose production, optimization of culture medium was carried out to increase the cell mass and lactosucrose production. Culture media were optimized by the S. elviae mutant using analysis of variance (ANOVA) and response surface methodology (RSM). Central composite designs using RSM were utilized in this investigation. Quadratic models were obtained for cell mass and lactosucrose production. In the case of cell mass, optimal components of the medium were as follows: sucrose 1.13%, yeast extract 0.99%, bactopeptone 2.96%, and ammonium sulfate 0.40%. The predicted maximum value of cell mass was about 5.20 g/l and its experimental value was 5.08 g/l. In the case of lactosucrose production, optimal components of the medium were as follows: sucrose 0.96%, yeast extract 1.2%, bactopeptone 3.0%, and ammonium sulfate 0.48%. Then, the predicted maximum value of lactosucrose production was about 194.12 g/l and the corresponding experimental value was about 183.78 g/l. Therefore, by culturing using predicted conditions, the real cell mass and lactosucrose production increased to 23.3% and 30.42%, respectively.     

Isolation and characterization of siderophore from cowpeaRhizobium (peanut isolate)

In an iron-depleted broth culture of cowpeaRhizobium (a peanut isolate), phenolate type of compounds were detected. Chemical characterization showed the presence of 2,3-dihydroxy benzoic acid (DHBA) and 3,4-DHBA in the siderophore extract. Lysine and alanine were identified as conjugated amino acids of the siderophore. Maximum concentration of the siderophore in the culture supernatant was found after 24 h of growth. The compounds in the extracted siderophore induced growth ofRhizobium in a medium containing EDTA. Addition of lysine and alanine in the growth medium (20 mM each) led to a fourfold increase in siderophore production.     

Growth and copper resistance of recombinantSaccharomyces cerevisiae containing a metallothionein gene

Summary Resistance to copper's toxicity inS. cerevisiae is mediated by a copper-binding protein, metallothionein (MT). Overexpression of MT in the recombinant yeast containing multiple copies of the MT gene on plasmid leads to a four-fold increase in the critical copper resistance level and an eight-fold enhancement of specific growth rate in a 2mM CuSO₄ medium compared with the parental host strain. The recombinant yeast grown in a 5mM CuSO₄ medium was found to accumulate 24.1mg of Cu per g of dry cell mass.     

<Emphasis Type="Italic">Trachydiscus minutus</Emphasis>, a new biotechnological source of eicosapentaenoic acid

The yellow-green alga Trachydiscus minutus (class Xanthophyta) was cultivated in a standard medium and in media without sulfur and nitrogen. Its yield after a 16-d cultivation reached 13 g dry mass per 1 L medium. The content of oligoenoic (‘polyenoic’) fatty acid (PUFA), i.e. eicosapentaenoic (EPA), was in excess of 35 % of total fatty acids; the productivity was thus 88 mg/L per d. This result makes the alga a very prospective organism that may serve as a new biotechnological source of single cell oil.     

Bioconversion of synthesis gas to hydrogen using a light-dependent photosynthetic bacterium,Rhodospirillum rubrum

Biological hydrogen production from synthesis gas was carried out in batch culture. The phototrophic anaerobic bacterium, Rhodospirillum rubrum was used to oxidize CO and water to CO₂ and hydrogen. The bacteria were grown under anaerobic conditions in liquid medium; also acetate was used as carbon source in presence of synthesis gas. Biological hydrogen production was catalysed by R. rubrum via the water–gas shift reaction. A light-dependent cell growth modelled with a desired rate of hydrogen production and CO uptake was determined. The effect of light intensity on microbial cell growth was also studied at 500, 1,000 and 1,500 m.cd. A complete conversion of CO to hydrogen and maximum light efficiency were obtained with an acetate concentration of 1 g/l and light intensity of 500 m.cd. Utilization of the carbon monoxide from the gas phase was often considered as a mass transfer limited process, which needed to diffuse through the gas–liquid interface and then further diffuse into liquid medium prior to reaction. The results from this study showed that maximum cell propagation and hydrogen production were achieved with a limited light intensity of 1,000 m.cd. It was also found that high-light intensity may interfere with cell metabolism. In low-light intensity and substrate concentration, no inhibition was observed, however at extreme conditions, non-competitive inhibition was identified. The adverse effect of high-light intensity was shown at 5,000 m.cd, where the CO conversion drastically dropped to as low as 21%. Maximum CO conversion of 98% and maximum yield of 86% with an acetate concentration of 1.5 g/l and a light intensity of 1,000 m.cd were achieved.     

Influence of growth conditions on production of capsular and extracellular polysaccharides byRhizobium leguminosarum

The influence of growth rate and medium composition on exopolymer production byRhizobium leguminosarum was studied. When grown in medium containing 10g/l mannitol and 1g/l glutamic acid,Rhizobium leguminosarum biovartrifolii TA-1 synthesized up to 2.0g/l of extracellular polysaccharide (EPS), and up to 1.6g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8g/l). Maximal CPS-yield was 2.9g CPS/l medium in a medium containing 20g/l mannitol and 2g/l glutamic acid. TheEPS-deficient strain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h^–1). The production rates were 50–100mg/g protein/h for EPS and 15–20mg/g protein/h for CPS. Only low amounts of cyclic -(1,2)-glucans were excreted (10–30 mg/l) over the entire range of growth rates.Abbreviations bv biovar - CPS capsular polysaccharide - EPS extracellular polysaccharide - HM_r high molecular mass - LM_r low molecular mass - YEMCR Yeast Extract-Mannitol-Congo Red agar     

Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar ‘Villafranca’) cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml^?1, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml^?1 and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin‐induced NO production was sensitive to sodium azide and N^G‐monomethyl‐l ‐arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.     

Growth of microorganisms on chemically synthesized carbohydrate ("formose") syrups

Formose syrup was studied as a carbon source for growth of a series of microorganisms obtained from various collections. Approximately 80 strains of bacteria, yeasts, and molds were inoculated into a medium containing formose syrup and mineral salts supplemented with small amounts of yeast extract and casein hydrolysate to supply accessory growth factors. Two preparations of formose syrup, produced by two different laboratories, were employed. Formose syrup I, characterized by a low sugar content, was poorly utilized; syrup II, containing a higher sugar concentration, was utilized to a greater extent. Two strains of Aerobacter acrogenes yielded 1.3 g dry cell mass from an initial charge of 10 g of formose II solids, whereas growth on 10 g of D -glucose amounted to 3.7 g. Klebsiella aerogenes MIT-B44, the best microbial strain isolated from soil by an enrichment technique, produced 1.3 g cells from 10 g fromose syrup II solids in supplemented medium; in direct comparisons, it produced 10–15% more cell 0.7–0.9 g cells per 10 g formose and grew with a doubling time of 55–70 min. Under such conditions, its macromolecular composition was 52% protein, 22% RNA, and 2% DNA. Although the apparent yield of cells from formose was only 8–11%, the actual yield based on formose utilized was 30%, the same as observed with glucose. A second strain was isolated from soil by enrichment with spent broth from K. aerogenes. This unidentified gram-negative, short rod-shaped bacterium grew in mixed culture with strain MIT-B44; in unsupplemented media they produced 1.55 g cells from 10 g formose II solids and 2.9 g cells from 10 g glucose.     

Protoplast isolation from cultured lichen <Emphasis Type="Italic">Usnea ghattensis</Emphasis>, their fusion with protoplasts of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>, fusant regeneration and production of usnic acid

Protoplasts isolated from the mycobiont of a cultured lichen Usnea ghattensis were fused with protoplasts of the fungus Aspergillus nidulans in order to increase the growth rate of the cultured lichen mycobiont in vitro. The maximum protoplast yield (102 × 10⁴/g fresh cell mass) was reached in citrate buffer with 50 mmol/L 2-sulfanylethanol (‘2-mercaptoethanol’) containing 0.1 % Novozym after 1.5 h at pH 5 and ≤25 °C. The increase in the concentration of the above effectors or the addition of others (e.g., MgSO₄) as well as increase in time, shaking frequency, etc. caused the lower yield of protoplasts. The fused protoplasts were regenerated after transfer to malt extract-yeast extract medium and produced, after a 45-d cultivation, a fresh cell mass of 0.232 g (from starting 0.3 g) along with the lichen substance usnic acid.