Application of mineral support on cephamycin C production in culture using soybean oil as the sole carbon source

A mineral support was used for cephamycin C production in a culture using soybean oil as the sole carbon source. When the support was added into an oil-water system, the soybean oil was emulsified as fine oil droplets, which was observed by a photomicroscope. Mycelia were also twined around the support, which was observed by a scanning electron microscope. That caused the formation of an oil-mycelia complex on or around the support, which provided a larger specific surface area of oil. When 15 g/L of the support was used in a batch culture of Streptomyces sp. P6621 with 50 g/L of soybean oil, the maximum cephamycin C concentration was 2.8 g/L, which was 2.2 times higher than that without the support. This indicates that the mineral support is useful for the culture system using vegetable oil as a carbon source. (c) 1997 John Wiley & Sons, Inc.     

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

Summary The effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h^–1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)^–1·h^–1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as -amylase, indicating that the protease production was selectively stimulated by the oils. Offprint requests to: Y. Fukushima     

Utilization of vegetable oil in the production of clavulanic acid by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>ATCC 27064

Production of clavulanic acid (CA) by Streptomyces clavuligerus ATCC 27064 in shake-flask culture (28 °C, 250 rev min^–1) was evaluated, with media containing different types and concentrations of edible vegetable oil. Firstly, four media based on those reported in the literature were examined. The medium containing soybean oil and starch as carbon and energy source gave the best production results. This medium, with the starch replaced by glycerol, and with various soybean oil concentrations (16, 23 and 30 g l^–1) was utilized to further investigate CA production. Medium containing 23 g l^–1 led to the highest CA productivity (722 mg l^–1 in 120 h) and that one containing 30 g l^–1 gave the highest CA titre (753 mg l^–1 in 130 h). Also, substitution of corn and sunflower edible oils furnished similarly good results in terms of CA titre and productivity. It can be concluded that easily available vegetable oil is a very promising substrate for CA production, since it is converted slowly to glycerol and fatty acids, which are the main carbon and energy source for the microorganism.     

Efficient lipid production with Trichosporon fermentans and its use for biodiesel preparation

Effects of medium components and culture conditions on biomass and lipid production of Trichosporon fermentans were studied. The optimal nitrogen source, carbon source and C/N molar ratio were peptone, glucose and 163, respectively. The favorable initial pH of the medium and temperature were 6.5 and 25 degrees C. Under the optimized conditions, a biomass of 28.1 g/l and a lipid content of 62.4% could be achieved after culture for 7 days, which were much higher than the original values (19.4 g/l and 50.8%) and the results reported by other groups. T. fermentans could grow well in pretreated waste molasses and a lipid yield of 12.8 g/l could be achieved with waste molasses of 15% total sugar concentration (w/v) at pH 6.0, representing the best result with oleaginous microorganisms on agro-industrial residues. Addition of various sugars to the pretreated molasses could efficiently enhance the accumulation of lipid and the lipid content reached as high as above 50%. Similar to vegetable oils, the lipid mainly contains palmitic acid, stearic acid, oleic acid and linoleic acid and the unsaturated fatty acids amount to about 64% of the total fatty acids. The microbial oil with an acid value of 5.6 mg KOH/g was transesterified to biodiesel by base catalysis after removal of free fatty acids and a high methyl ester yield of 92% was obtained.     

The fermentative production of acetone-butanol by Clostridium acetobutylicum.

Fourteen different media were used in the fermentative production of acetone-butanol. The highest total yields were achieved in medium I. Potato starch and soluble starch were suitable as carbon sources. The best concentrations of potato starch and soluble starch were 500.0 and 10.0 g/l, respectively. Peptone was the most favourable nitrogen source. The best concentration of peptone was 4.0 g/l. Calcium carbonate in 3.6 g/l acted as buffering agent in the fermentation process. The best initial pH value of the fermentation medium was 6.0. The optimum temperature was 32--33degreesC. The fermentation process required 120 h to obtain maximum yields of acetone-butanol.     

Tylosin production by Streptomyces fradiae using raw cornmeal in airlift bioreactor

Using a 50-l airlift bioreactor, for the effective production of tylosin from Streptomyces fradiae TM-224 using raw cornmeal as the energy source, various environmental factors were studied in flask cultures. The maximum tylosin concentration was obtained at 32 degrees C and pH between 7.0 and 7.5. When seed was inoculated after 24 h of culture, the maximum tylosin concentration, 5.7 g/l, was obtained after 4 days of culture. Various concentrations of raw cornmeal were tested to investigate the optimum initial concentration for the tylosin production. An initial raw cornmeal concentration of 80 g/l gave the highest tylosin concentration, 5.8 g/l, after 5 days of culture. Of the various nitrogen sources, soybean meal and fish meal were found to be the most effective for the production of tylosin. In particular, with the optimal mixing ratio, 12 g/l of soybean meal to 14 g/l of fish meal, 7.2 g/l of tylosin was obtained after 5 days of culture. To compare raw cornmeal and glucose for the production oftylosin in the 50-1 airlift bioreactor for 10 days, fed-batch cultures were carried out under the optimum culture conditions. When raw corn meal was used as the energy source, the tylosin production increased with increasing culture time. The maximum tylosin concentration after 10 days of culture was 13.5 g/l, with a product yield from raw cornmeal of 0.123 g/g of consumed carbon source, which was about 7.2 times higher than that obtained when glucose was used as the carbon source.     

Lipase activity and tacrolimus production in Streptomyces clavuligerus CKD 1119 mutant strains

The effect of carbon sources on tacrolimus production by a mutant strain of Streptomyces clavuligerus CKD 1119, an isolate from soil, was examined. Among the carbohydrates and oils tested in this work, a mixed carbon source of soluble starch and corn oil was the best. An analysis of the culture kinetics also showed that, in contrast to the carbohydrates, the corn oil was consumed later in the antibiotic production phase, implying that the oil substrate was the principal carbon source for the biosynthesis of tacrolimus, and this was directly proven by experiments using 14C-glucose and 14C-oleate substrates. Furthermore, corn oil induced the formation of lipase by the mutant strain, whereas the addition of glucose significantly repressed lipase activity. The lipase activity exhibited by the FK-506-overproducing mutants was also observed to be directly proportional to their tacrolimus yield, indicating that a high lipase activity is itself a crucial factor for tacrolimus production. A feasibility study with a 200-l pilot-scale fermentor and the best strain (Tc-XII- 15322) identified in this work revealed a high volumetric and specific productivity of about 495 mg/l and 0.34 mg/mg dry mycelium, respectively.     

Sophorolipid production by <Emphasis Type="Italic">Candida bombicola</Emphasis> on oils with a special fatty acid composition and their consequences on cell viability

Sophorolipids production by the yeast Candia bombicola is most favourable when glucose is used as a carbon source in combination with a hydrophobic carbon source such as a common vegetable oil. Most vegetable oils are comprised of C16–C18 fatty acids, an ideal range for sophorolipid production. The use of oils with either shorter or longer fatty acids, such has coconut oil or meadowfoam oil, respectively, was evaluated. Such oils did not contribute to enhanced sophorolipid production when compared to cultures run on glucose as the sole carbon source. Moreover, a toxic effect of medium-chain fatty acids towards stationary C. bombicola cells was demonstrated.     

Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.     

An inexpensive medium for production of arachidonic acid by <Emphasis Type="Italic">Mortierella alpina</Emphasis>

The production of arachidonic acid was studied in the fungus Mortierella alpina using an inexpensive medium. Glucose derived from maize starch hydrolysate was the sole carbon source and defatted soybean meal and sodium nitrate were the nitrogen sources. Optimal arachidonic acid yield (1.47 g l^-1) was observed at a glucose concentration of 100 g l^-1. Various treatments of defatted soybean meal to extract soluble nitrogen nutrients were evaluated. Alkali extract was the most effective for arachidonic acid production. A mixture of soybean alkali-extract protein and sodium nitrate was an excellent nitrogen source for fungal growth, lipid accumulation, and arachidonic acid production. A maximum yield of 1.87 g arachidonic acid l^-1 was obtained with a soybean protein concentration of 4.6 g l^-1 and a sodium nitrate concentration of 2.3 g l^-1. Electronic Publication     

Enhancing of erythromycin production by <Emphasis Type="Italic">Saccharopolyspora erythraea</Emphasis> with common and uncommon oils

The enhancing effect of various concentrations of 18 oils and a silicon antifoam agent on erythromycin production by Saccharopolyspora erythraea was evaluated in a complex medium containing soybean flour and dextrin as the main substrates. The oils used consisted of sunflower, pistachio, cottonseed, melon seed, water melon seed, lard, corn, olive, soybean, hazelnut, rapeseed, sesame, shark, safflower, coconut, walnut, black cherry kernel and grape seed oils. The biomass, erythromycin, dextrin and oil concentrations and the pH value were measured. Also, the kinds and frequencies of fatty acids in the oils were determined. The productivity of erythromycin in the oil-containing media was higher than that of the control medium. However, oil was not suitable as a main carbon source for erythromycin production by S. erythraea. The highest titer of erythromycin was produced in medium containing 55 g/l black cherry kernel oil (4.5 g/l). The titers of erythromycin in the other media were also recorded, with this result: black cherry kernel > water melon seed > melon seed > walnut > rapeseed > soybean > (corn = sesame) > (olive = pistachio = lard = sunflower) > (hazelnut = cotton seed) > grape seed > (shark = safflower = coconut). In media containing various oils, the hyphae of S. erythraea were longer and remained in a vegetative form after 8 days, while in the control medium, spores were formed and hyphae were lysed.     

密环菌胞外多糖的发酵条件研究

密环菌是天麻的共生菌,有研究证明,密环菌的菌丝及发酵液有与天麻相类似的药理作用,为此,我们进行密环菌深层发酵产胞外多糖条件的研究。研究结果表明：菌丝生长和多糖产生的最适初始pH为5．9左右;消泡剂用量在一定内增加,有利于菌丝的生长,但增加消泡剂用量会不利于多糖的产生;接种量大,菌丝得率高,但接种量为10％时的多糖得率最高;装液量试验表明,通气量大,有利于菌丝的生长和多糖的产生;机械搅拌对菌丝的生长和多糖的产生都不利;生长动态实验证明,密环菌可在6d内完成液体发酵;以豆粕粉作氮源时,菌丝得率最高,以麸皮作氮源时,多糖产量最高;红薯粉为密环菌菌丝生长和多糖产生的最适碳源。     

Influence of nutrients on enhancing laccase production by Botryosphaeria rhodina MAMB-05.

The physiological requirements needed to enhance the production of laccases by the ascomycete Botryosphaeria rhodina MAMB-05 in submerged cultivation were examined under non-induced and induced (veratryl alcohol, VA) conditions. Under non-induced conditions (-VA), the initial pH, C:N ratio, and inorganic N source did not influence laccase production, in contrast to Tween 80, soybean oil, and copper, which significantly increased laccase production, and proline and urea, which suppressed laccase formation. In addition, Tween 60 could serve as the sole carbon source for the production of these enzymes. Under VA-induced conditions of fungal growth, factors such as inoculum type, time-point of addition of inducer, initial pH, C:N ratio, and type of N source, influenced the production of laccases; however, unlike the non-induced conditions, proline and urea did not act as suppressors. Each of these physiological conditions exerted different effects on biomass production. The nutritional conditions examined for B. rhodina MAMB-05 are discussed in relation to their influence on fungal growth and laccase production.     

Extracellular lipase production by a sapwood-staining fungus,Ophiostoma piceae

The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH₄)₂SO₄ gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH₄)₂SO₄and peptone as nitrogen source.The authors are with Forest Products Biotechnology, Department of Wood Science, Facully of Forestry, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada     

Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source

Biosurfactant production by Pseudomonas aeruginosa A41, a strain isolated from seawater in the gulf of Thailand, was examined when grown in defined medium containing 2% vegetable oil or fatty acid as a carbon source in the presence of vitamins, trace elements and 0.4% NH(4)NO(3), at pH 7 and 30 degrees C with 200 rpm-shaking for 7 days. The yield of biosurfactant steadily increased even after a stationary phase. Under such conditions the surface tension of the medium was lowered from 55-70 mN/m to 27.8-30 mN/m with every carbon source tested. However, types of carbon sources were found to affect biosurfactant yield. The yields of rhamnolipid biosurfactant were 6.58 g/L, 2.91 g/L and 2.93 g/L determined as rhamnose content when olive oil, palm oil and coconut oil, respectively, were used as a carbon source. Among them, biosurfactant obtained from palm oil was the best in lowering surface tension of the medium. Increase in biosurfactant activities in terms of oil displacement test and rhamnose content were observed to be higher with shorter chain fatty acids than that of the longer chains (C12>C14>C16). In addition, we found that C18:2, highly unsaturated fatty acid, showed higher oil displacement activity and rhamnose content than that of C18:1. The optimal oil displacement activity was found at pH 7-9 and in the presence of 0.5-3% NaCl. The oil displacement activity was stable to temperatures up to 100 degrees C for 15 h. Surface tension reduction activity was relatively stable at pH 2-12 and 0-5% of NaCl. Emusification activity tested with various types of hydrocarbons and vegetable oils showed similarity of up to 60% stability. The partially purified biosurfactant via TLC and silica gel column chromatography gave three main peaks on HPLC with mass spectra of 527, 272, and 661 m/z respectively, corresponding to sodium-monorhamnodecanoate, hydroxyhexadecanoic acid and an unknown compound, respectively.     

Production of long-chain polyunsaturated fatty acids by monoxenic growth of labyrinthulids on oil-dispersed agar medium

A novel method is proposed for the production of long-chain polyunsaturated fatty acids (LCPUFA) by labyrinthulids. The method comprises a monoxenic culture with Psychlobacter phenylpyruvicus, using agar medium in which oil was dispersed. Soybean oil (SBO) was selected as the optimum material for an oil-dispersed agar medium. The labyrinthulids showed three-dimensional growth and an anastomosing ectoplasmic network in the SBO-dispersed agar medium. The oil plate changed from an opaque culture to a more transparent culture, due to growth of the labyrinthulids. The optimum culture conditions were 25-30 degrees C, an initial pH of 6-10 and artificial seawater with a salt concentration of 50-100%. These conditions are close to those where these strains were isolated. The maximum LCPUFA production (0.59 g/l) and dry cell weight (4.93 g/l) was obtained using strain S3-2 (isolated from Ishigaki Island) with 1.5% SBO at 14 days. This value was about 30 times more than that using glucose instead of SBO. The method proposed is promising in terms of the production of LCPUFA from reproducible oils.     

Kinetics of kojic acid fermentation byAspergillus flavus link S44-1 using sucrose as a carbon source under different pH conditions

Kojic acid production byAspergillus flavus strain S44-1 using sucrose as a carbon source was carried out in a 250-mL shake flask and a 2-L stirred tank fermenter. For comparison, production of kojic acid using glucose, fructose and its mixture was also carried out. Kojic acid production in shake flask fermentation was 25.8 g/L using glucose as the sole carbon source, 23.6 g/L with sucrose, and 6.4 g/L from fructose. Reduced kojic acid production (13.5 g/L) was observed when a combination of glucose and fructose was used as a carbon source. The highest production of kojic acid (40.2 g/L) was obtained from 150 g/L sucrose in a 2 L fermenter, while the lowest kojic acid production (10.3 g/L) was seen in fermentation using fructose as the sole carbon source. The experimental data from batch fermentation and resuspended cell system was analysed in order to form the basis for a kinetic model of the process. An unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe the growth, substrate consumption, and efficiency of kojic acid production byA. flavus in batch fermentation using sucrose. From this model, it was found that kojic acid production byA. flavus was not a growth-associated process. Fermentation without pH control (from an initial culture pH of 3.0) showed higher kojic acid production than single-phase pH-controlled fermentation (pH 2.5, 2.75, and 3.0).     

提高蛋白激发子产量的培养基及发酵条件的优化

为了进一步提高极细链格孢菌产蛋白激发子的产量,通过单因子和多因子试验与分析,筛选优化了适于极细链格孢菌产生蛋白激发子的培养基和培养条件,并检测了发酵过程中pH、还原糖、氨基氮和菌丝量变化以及与蛋白激发子产量的关系。结果表明,土豆淀粉和黄豆粉对蛋白激发子产量影响最大,其次是蛋白胨和无机盐。优化的发酵培养基主要成分（g／L）：碳源I 15、葡萄糖5、玉米淀粉5、土豆淀粉20、谷氨酸10、氮源I5、黄豆粉10、硫酸铵5。确定了优化的培养条件,调整培养基起始pH为7．0～7．5,将18h菌龄的种子培养液按10％接种量接种到装液量为75mL的500mL摇瓶中,在温度（28±1）℃、摇床转速180r／min下培养可获得理想的蛋白产量。在优化的培养基和培养条件下,发酵12～48h该菌进入对数生长期,48h进入稳定生长期,60h菌丝扣蛋白激发子产量达最高。蛋白产量与菌体生物量呈正相关,当还原糖、总糖量消耗到最低水平时,菌丝产量和蛋白激发子产量达最高。优化的培养基菌丝干重收率迭3．9g／100mL,蛋白激发子产量达到5．17g／L,比普通的土豆液体培养基提高近4倍。     

Candida albicans cell wall lytic enzyme produced by Streptomyces thermodiastaticus

The production of lytic enzyme by Streptomyces thermodiastaticus was found to be affected by some growth conditions and nutritional factors. The highest enzyme production was obtained after 18 h of incubation at pH 5.5 and at 50 degrees C. The carbon source influenced the lytic enzyme production. A higher enzyme yield was obtained when Candida albicans cell wall (1 g/100 ml) was used as the sole carbon source. NaNO3 at 0.1 g/100 ml was the best nitrogen source for enzyme production. From all phosphorous sources, microelements, and growth factors tested, KH2PO4 (1 g/l), ZnSO4 (1 mg/I) and Tween 80 (0.1%), respectively, were found to favour the highest production of lytic enzymes by S. thermodiastaticus. The lytic enzymes mainly produced chitinolytic and proteolytic activities.