Effect of oleic acid and Tween-80 on lysine synthesis by the culture Corynebacterium glutamicum]

The effect of oxidized and unoxidized oleic acid and Tween-80 on the growth and lysine synthesis by the producers C. glutamicum strains 95, 8, 28 was investigated. Surface active substances like oxidized and unoxidized oleic acid and Tween-80 during cultivation of the lysine producers on the glucose medium (the synthetic medium) and the medium with molasses and corn extract either inhibited the culture growth, thus reducing lysine yield, or accelerated the culture growth, thus increasing lysine yield. Oxidized and unoxidized oleic acid produced the greatest effect when added to the nutrient medium on the 48th cultivation hour. The increment of synthesized lysine was 120-150% of the control. Tween-80 proved to be very effective when added at early stages of fermentation (20 hours).     

Effect of tweens on the lipase activity of Oospora lactis

Oospora lactis was found to use Tweens 20, 40, 60 and 80 as a source of carbon in a nutrient medium for lipase biosynthesis. The highest production of lipase was registered in the medium with Tween-80. The lipase activity of the culture increased if even small quantities of Tweens 40, 60 and, particularly, 80 (0.05--1.0%) were added to the medium containing cottonseed oil. All the tested Tweens stimulating the biosynthesis of lipase were also, at a particular concentration, the inhibitors of this enzyme.     

Efficient clonal propagation method for Decalepis hamiltonii, an endangered shrub, under the influence of phloroglucinol

A highly efficient two stage protocol was developed for induction of multiple shoots from single node in vitro shoot tip explants of Decalepis hamiltonii. It was found that phloroglucinol (PG) had synergistic effect on shoot multiplication when added with N6-benzyladenine and gibberellic acid. This protocol uses PG for both multiple shoot induction from nodal explants, elongation of primary shoots and initiation of adventitious shoot formation from primary shoots, which was more in presence of triacontanol (TRIA). Maximum number of shoots per culture was observed on the medium containing N6-benzyladenine (1.1 microM; BA), GA3 (5.8 microM) and PG (800 microM). Sub-culturing of the shoots onto MS medium containing optimum concentration of BA (5.6 microM), PG (200 microM) and TRIA (0.011 microM) produced elongated shoots along with secondary shoot formation. The long shoots were rooted on alpha-naphthalene acetic acid (5.38 microM; NAA) and PG (400 microM) containing medium. The rooted plantlets were hardened and their field survival rate was 80-90%.     

In vitro micropropagation of Gymnema sylvestre – A multipurpose medicinal plant

The nature of the explant, seedling age, medium type, plant growth regulators, complex extracts (casein hydrolysate, coconut milk, malt extract and yeast extract) and antioxidants (activated charcoal, ascorbic acid, citric acid and polyvinylpyrrolidone) markedly influenced in vitro propagation of Gymnema sylvestre. A maximum number of shoots (57.2) were induced from 30 day old seedling axillary node explants on Murashige and Skoog (MS) medium containing 6-benzyladenine (1 mg l⁻¹), kinetin (0.5 mg l⁻¹), 1-napthalene acetic acid (0.1 mg l⁻¹), malt extract (100 mg l⁻¹) and citric acid (100 mg l⁻¹). High frequency of rooting was obtained in axillary node explant derived shoots (50%) on half strength MS medium supplemented with IBA (3 mg l⁻¹). The plantlets, thus developed, were hardened and successfully established in natural soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fluorene biodegradation and identification of transformation products by white-rot fungus Armillaria sp. F022

A diverse surfactant, including the nonionic Tween 80 and Brij 30, the anionic sodium dodecyl sulphate, the cationic surfactant Tetradecyltrimethylammonium bromide, and biosurfactant Rhamnolipid were investigated under fluorine-enriched medium by Armilaria sp. F022. The cultures were performed at 25 °C in malt extract medium containing 1 % of surfactant and 5 mg/L of fluorene. The results showed among the tested surfactants, Tween-80 harvested the highest cell density and obtained the maximum specific growth rate. This due Tween-80 provide a suitable carbon source for fungi. Fluorane was also successfully eliminated (>95 %) from the cultures within 30 days in all flasks. During the experiment, laccase production was the highest among other enzymes and Armillaria sp. F022-enriched culture containing Non-ionic Tween 80 showed a significant result for laccase activity (1,945 U/L). The increased enzyme activity was resulted by the increased biodegradation activity as results of the addition of suitable surfactants. The biotransformation of fluorene was accelerated by Tween 80 at the concentration level of 10 mg/L. Fluorene was initially oxidized at C-2,3 positions resulting 9-fluorenone. Through oxidative decarboxylation, 9-fluorenone subjected to meta-cleavage to form salicylic acid. One metabolite detected in the end of experiment, was identified as catechol. Armillaria sp. F022 evidently posses efficient, high effective degrader and potential for further application on the enhanced bioremediation technologies for treating fluorene-contaminated soil.     

Strain improvement of Serratia marcescens ECU1010 and medium cost reduction for economic production of lipase

Industrial strain improvement plays a central role in the commercial development of microbial fermentation processes. The strain of Serratia marcescens ECU1010, a wild-type lipase-producer capable of stereospecific synthesis of a Diltiazem precursor, was subjected to physical mutation involving treatment by UV-irradiation for 30 s. A mutant strain, no. UV-01, showed enhanced lipase production, but lost the capability of producing red pigment (prodigiosin). The variant strain UV-01 had a 2.3-fold higher activity than the wild type and was stable in its enzyme production for ten serial transfers. For reduction of the fermentation medium cost, dried powder of corn steep liquor was used as an inexpensive substitute for beef extract in the medium. Dextrin as an organic carbon source and Tween-80 as an important element were further optimized, respectively. The high primary biodegradation of the Tween-80 by S. marcescens ECU1010 and its variant demonstrated their potential ability of degrading alkyl polyethoxylates to remove harmful nonionic surfactants from polluted effluents and streams. The optimal cultivation time for lipase biosynthesis was 24 h. These optimized compositions resulted in an economic production of lipase by S. marcescens ECU1010 var. UV-01, with a dramatically reduced cost (1/8–1/7 of the initial one) which is more suitable for industrial application.     

响应面法优化重组巴斯德毕赤酵母合成ECH42的培养基组分及其重组酶降解几丁质的研究

采用响应面法在摇瓶水平对重组巴斯德毕赤酵母合成内切几丁质酶的培养基组分进行优化,并探讨重组内切几丁质酶降解几丁质的最佳反应条件。首先对培养基中显著影响内切几丁质酶活力的关键组分通过Plackett-Burman试验设计进行筛选;然后通过Box-Behnken试验设计和响应面法确定关键组分的最佳浓度。结果筛选出3个具有显著效应的关键组分为酵母膏、油酸和吐温-80,最佳浓度分别为：2.45%、0.17%和0.62%。优化后的最佳培养基组成为：2.45%酵母膏、2.00%蛋白胨、0.50%酵母氮碱（YNB）、0.50%甲醇、0.17%油酸、0.62%吐温-80和0.40% PTM₁。在该培养基中,重组巴斯德毕赤酵母在摇瓶水平（25mL/250mL）发酵生产内切几丁质酶的活力高达92.26U/mL。重组内切几丁质酶催化几丁质降解的最佳反应条件为：粉末几丁质浓度为4%,pH和温度分别为7.0和30℃,反应时间为10h。研究结果为后期在发酵罐中大规模生产内切几丁质酶和几丁寡糖提供了基础。     

Factors affecting viable cell counts of freeze-driedCryptococcus terricolus cells

Freeze-drying ofCryptococcus terricolus cells in distilled water resulted in a survival of only 0.1% of the cells. The viability could be increased to 16% by the use of a dextran-sucrose-sodium glutamate solution as suspending medium.For freeze-dried material with both low and high survival rates, and for five as well as for ten days old cultures, malt extract solution was the superior reconstitution medium. Less, but still distinct, protective action was found with a synthetic glucose-urea-salt solution.The viable cell counts of cells freeze-dried in dextran-sucrose-sodium glutamate solution were independent of the medium used for plating. When distilled water was used as a medium for freeze-drying, two to four times higher counts were obtained with malt extract agar than with synthetic glucose-urea-salt agar.Of the twenty different media tried for freeze-drying, sucrose solution gave the best protection. The viability was greatly influenced by the concentration used, maximum values being obtained when more than 10% of sucrose was added. The survival rate increased with the age of the cells until the fifth day, but was independent of the concentration of cells in the suspension. Under optimum conditions a survival rate of more than 80% was reached.     

Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production byPropionibacterium acidipropionici

Fed-batch propionic and acetic acid fermentations were performed in semi-defined laboratory medium and in corn steep liquor withPropionibacterium acidipropionici strain P9. On average, over four experiments, 34.5 g/l propionic acid and 12.8 g/l acetic acid were obtained in about 146 h in laboratory medium with 79 g/l glucose added over five feeding periods. The highest concentration of propionic acid, 45 g/l, was obtained when the glucose concentration was not allowed to drop to zero. In corn steep liquor 35 g/l propionic acid and 11 g/l acetic acid were produced in 108 h from 59.4 g/l total lactic acid provided as seven feedings of corn steep liquor. Extractive fed-batch fermentations were conducted in semi-defined medium using either flat-sheet-supported liquid membranes or hollow-fiber membrane extraction to remove organic acids from the culture medium. As operated during the course of the fermentation, these systems extracted 25% and 22% of the acetic acid and 36.5% and 44.5% of the propionic acid, respectively, produced in the fermentation. Total amounts of acids produced were about the same as in comparable nonextractive fermentations: 30–37 g/l propionic acid and 13 g/l acetic acid were produced in 150 h. Limitations on acid production can be attributed to limited substrate feed, not to failure of the extraction system.Journal paper J-16303 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 3122.     

Use of malt extract as a nitrogen source for continuous cultivation of Candida utilis]

The effect of an aqueous extract from malt sprouts on the growth and development of fodder yeast was investigated during their continuous cultivation. The extract can be used as a nitrogen source during continuous cultivation of fodder yeast. It is supposed that the extract contains compounds capable to inhibit yeast growth. The extract should be added to the nutrient medium in the amount of 4 g/l of dry weight.     

Organic Nutrients in the Media for Propagation of Cymbidium in vitro

Protocorms of Cymbidium (Orchidaceae) were grown on media containing different organic nutrients. Of the sugars tested sucrose was better than maltose, glucose and fructose, and sucrose had an optimum concentration of 3 to 4 %. D-Mannose was significantly less effective than the other sugars. The amino acid mixtures casamino acids (casein hydrolysate) and tryptone increased growth while yeast extract was inhibitory and malt extract without effect. Optimal concentrations were 2 to 3 g · l^-1 casamino acids and 3 to 4 g · l^-1 tryptone. It was to some extent possible to substitute the amino acid mixtures with a single amino acid (glutamine at 300 mg · l^-1). Arginine was inhibitory and asparagine was without any effect. Vitamins proved to be unnecessary although there was a tendency towards increased growth with nicotinic acid and meso-inositol. Purines and pyrimidines were added to the medium but with no effect. Liquid endosperm from coconuts (10 to 15%) increased growth while the liquid endosperm from Aesculus hippocastanum was inhibitory. On the basis of these results a revised medium is proposed for the in vitro propagation of Cymbidium.     

Incorporation and accumulation of docosahexaenoic acid from the medium by Pichia methanolica HA-32

Yeast species were screened for the incorporation and accumulation of docosahexaenoic acid (DHA) with a yeast-malt medium containing 0.5% free fatty acid prepared from fish oil (DHA, 28% of total fatty acids in fish oil). The most suitable strain was Pichia methanolica HA-32. The optimum cultivation conditions for the accumulation of lipids and incorporation of DHA were as follows: 5% glucose, 20% yeast extract, and 3% free fatty acid in the medium, at pH 6.0 and with incubated at 25 degrees C for 3 days. Under these conditions, about 200 mg of total lipids and 60 mg of DHA were recovered from 1 g of dry cells. The accumulation of DHA in cells increased in conjunction with the amount of yeast extract added to the medium. Vitamin B groups and minerals also had an effect on the accumulation of DHA. Choline and K2HPO4, which caused browning of the medium, promoted the accumulation of DHA in cells.     

重组巴斯德毕赤酵母发酵生产内切几丁质酶的培养条件优化

探讨重组巴斯德毕赤酵母发酵生产内切几丁质酶的最适培养条件,以期获得最佳的内切几丁质酶活力。以内切几丁质酶活力为指标,通过部分因子试验设计以及响应面法优化确定重组巴斯德毕赤酵母高产内切几丁质酶的最适培养条件。部分因子试验设计筛选的影响重组巴斯德毕赤酵母高产内切几丁质酶的3个关键因子为甲醇、油酸和吐温-80。响应面法优化的上述3个关键因子的最佳浓度分别为0.71%、0.086%和0.31%。重组巴斯德毕赤酵母发酵生产内切几丁质酶的最适培养条件为：酵母膏1%、酵母氮碱（YNB）1.34%、蛋白胨2%、甲醇0.71%、油酸0.086%、吐温-80 0.31%、PTM₁ 0.8%、pH 6.0。在上述培养条件下,重组巴斯德毕赤酵母产内切几丁质酶的活力高达30.92U/mL。与未优化前相比,酶活力提高了1.44倍。研究结果为内切几丁质酶的产业化生产和应用奠定了良好基础。     

Incorporation and Accumulation of Docosahexaenoic Acid from the Medium by Pichia methanolica HA-32

Yeast species were screened for the incorporation and accumulation of docosahexaenoic acid (DHA) with a yeast-malt medium containing 0.5% free fatty acid prepared from fish oil (DHA, 28% of total fatty acids in fish oil). The most suitable strain was Pichia methanolica HA-32. The optimum cultivation conditions for the accumulation of lipids and incorporation of DHA were as follows: 5% glucose, 20% yeast extract, and 3% free fatty acid in the medium, at pH 6.0 and with incubated at 25°C for 3 days. Under these conditions, about 200 mg of total lipids and 60 mg of DHA were recovered from 1 g of dry cells. The accumulation of DHA in cells increased in conjunction with the amount of yeast extract added to the medium. Vitamin B groups and minerals also had an effect on the accumulation of DHA. Choline and K₂HPO₄, which caused browning of the medium, promoted the accumulation of DHA in cells.     

Production of Luteoskyrin, a Hepatotoxic Pigment, by Penicillium islandicum Sopp

Various factors affecting the yields of luteoskyrin, a hepatotoxic mycotoxin, and related pigments in the liquid medium were studied. Maximal yields of luteoskyrin (0.13% by isolation) and of other pigments were attained in the late phase of the cultivation. The yield of the pigment was increased by supplying malt extract, malonic acid, glutamic acid, or asparagine. A useful material for preparation of (14)C-labeled luteoskyrin was 2-(14)C-malonate.     

Optimization of novel halophilic lipase production by Fusarium solani strain NFCCL 4084 using palm oil mill effluent

Among different sources of lipases, fungal lipases have continued to attract a wide range of applications. Further, halophilic lipases are highly desirable for biodiesel production due to the need to mitigate environmental pollution caused as result of extensive use of fossil fuels. However, currently, the high production cost limits the industrial application of lipases. In order to address this issue, we have attempted to optimize lipase production by Fusarium solani NFCCL 4084 and using palm oil mill effluent (POME) based medium. The production was optimized using a combinatory approach of Plackett-Burman (PB) design, one factor at a time (OFAT) design and face centred central composite design (FCCCD). The variables (malt extract, (NH₄)₂SO₄, CaCl₂, MgSO₄, olive oil, peptone, K₂HPO₄, NaNO₃, Tween-80, POME and pH) were analyzed using PB design and the variables with positive contrast coefficient were found to be K₂HPO₄, NaNO₃, Tween-80, POME and pH. The significant variables selected were further analyzed for possible optimum range by using OFAT approach and the findings revealed that K₂HPO₄, NaNO₃, and Tween-80 as the most significant medium components, and thus were further optimized by using FCCCD. The optimum medium yielded a lipase with an activity of 7.8 U/ml, a significant 3.2-fold increase compared to un-optimized medium. The present findings revealed that POME is an alternative and suitable substrate for halophilic lipase production at low cost. Also, it is clearly evident that the combinatory approach employed here proved to be very effective in producing high activity halophilic lipases, in general.     

吐温-80对野葛毛状根生长及异黄酮含量的影响

将不同浓度的吐温-80添加到野葛毛状根悬浮培养液中,研究在一定的作用时间内其对毛状根生长及次生代谢物合成与分泌的影响。结果表明,采用2％浓度处理较为适宜,不仅可以提高毛状根内葛根素的含量,而且有利于培养液中葛根素、大豆甙元及总异黄酮的积累,与对照相比,其含量可分别提高24．2％、50％和46．7％。在该浓度下连续处理毛状根72h后,发现毛状根仍生长旺盛,其生长量已是对照的1．5倍。但不同时间的连续处理对毛状根及培养液中几种异黄酮物质的积累与释放作用不同,其中以处理48h最有利于培养液中总异黄酮的累积,其含量是毛状根中的38倍。     

gamma-Linolenic acid production by solid-state fermentation of Mucorales strains on cereals

Oleaginous fungi of the genus Mucorales were screened for gamma-linolenic acid (GLA) production on solid substrates containing moistened cereals. Cunninghamella elegans CCF 1318 produced the highest yields of GLA when cultivated on barley. Substrate moisture and cultivation temperature proved critical for effective GLA production. Vegetable oil supplied to the cultures improved GLA production. Rotating bottles and plastic bags were used as cultivation vessels to reproduce the conditions found in rotating drums and tray bioreactors, respectively. After 11 days of cultivation at 21 degrees C, C. elegans produced 14.2 mg of GLA per gram of dry substrate, composed of a mixture of barley, spent malt grains (SMG) and peanut oil. GLA represented 15.6% of the total fatty acids in the lipid extract.     

The effect of acetic acid concentration on the growth and production of gamma-linolenic acid byMucor circinelloides CBS 203.28 in fed-batch culture

The effect of different initial acetic acid concentrations on the growth of and lipid and gamma-linolenic acid (GLA) production byMucor circinelloides CBS 203.28 was determined in a 14 litre stirred tank reactor operated in a fedbatch, pH-stat mode with acetic acid as carbon source and pH titrant. Increased acetic acid concentrations in the culture resulted in a significant increase in the crude oil content of the biomass. By contrast, all the other parameters such as the biomass concentration, GLA and oil yield on acetic acid, the GLA content of the biomass and oil, the growth rate and volumetric rate of GLA production decreased with an increase in acetic acid concentration. The best results were obtained with acetic acid at 2 g/1, which gave 39.8 mg GLA/g biomass and 15.6% GLA in the neutral lipid fraction, amounting to 340 mg GLA/1 culture. A decrease in the glyco- and phospho-lipid fractions during the cultivation coincided with an increase in the neutral lipid fraction. The GLA content of the biomass remained within rather narrow limits of 3.5% to 4% of the biomass, irrespective of the oil content of the biomass. The fatty acid profile was not greatly affected by the acetic acid concentration. The hyphae of the fungus were characterized by the accumulation of large intracellular oil droplets and some septa delimited the hyphae.     

Effect of various factors on the biosynthesis of piscarinines,secondary metabolites of the fungus <Emphasis Type="Italic">Penicillium piscarium</Emphasis> Westling

Piscarinines A and B were synthesized most actively during the surface cultivation of the fungus Penicillium piscarium in a complex medium (5.5 mg/l). Under conditions of submerged cultivation in a mineral medium, the yield of piscarinines was two times lower. An increase in the inoculum quantity of conidia treated with Tween-80 increased the culture productivity. The biosynthesis of the alkaloid was completely suppressed when mannitol was replaced with glucose or when zinc, iron, or copper ions were added to the culture medium. The metabolites were active against the prostate cancer cell line LNCAP (IC₅₀ were 2.195 and 1.914 μg/ml for piscarinines A and B, respectively).