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.     

Biodegradation of octylphenol polyethoxylate surfactant Triton X-100 by selected microorganisms

Octylphenol polyethoxylate (OPEO(n)) surfactants are used in numerous commercial and industrial products. Large amounts of such surfactants and their various residual biodegradation by-products are ultimately released into the environment. OPEO(n) biodegradation was performed in this study using pure cultures of Pseudomonas species and strains under different environmental conditions. Environmental factors including the pH, nitrogen sources, and growth kinetics of the cells were investigated. The intermediates of Triton X-100 biotransformation were detected by high performance liquid chromatography-mass spectrophotograph (HPLC-MS). We found the highest specific growth rate (mu) was 0.56 h(-1) and this was achieved by strain E with an initial concentration of Triton X-100 of 5000 mg L(-1). A pH level of 7 was most favorable for cell growth for all five strains. The highest specific growth rate was achieved using (NH(4))(2)SO(4) as the sole nitrogen source for strain E. Strain A showed an enhancement of growth when between 0.2 and 1.4 mg L(-1) of H(2)O(2) was added. Detection of intermediates was possible after four days of transformation and the octylphenol triethoxylate (OPEO(3)) peak was predominant, while the high molecular weight peaks had all disappeared. The kinetic analysis demonstrated that the greatest maximum specific growth rate (mu(max)) and the greatest saturation constant (K(s)) of 0.83 h(-1) and 5.24 mg L(-1), respectively, were obtained for strain E in 5000 mg L(-1) Triton X-100. The higher K(i) revealed that strain A was resistant to higher Triton X-100 concentrations.     

Cost-effective optimization of gellan gum production by Sphingomonas paucimobilis using corn steep liquor

Abstract

Gellan gum, produced by Sphingomonas paucimobilis, is increasingly used in food and pharmaceutical industries as stabilizing, emulsifying, texturing and gelling agents. However, its high production costs may limit its full commercial potential. Therefore, in this study, we investigated ways to reduce gellan gum production costs and improve yields. We first revealed corn steep liquor (CSL) as a cost-effective nutrient source that can improve gellan gum yields. We then systematically optimized culture conditions even further, and revealed that the addition of Triton X-100 surfactant and selected inorganic nitrogen sources improved gellan gum production. Under our optimized conditions (glucose 33.75?g/L, CSL 10?g/L, urea 2.5?g/L, MgSO₄ 1.08?g/L, KH₂PO₄ 3.24?g/L, K₂SO₄ 1?g/L and Triton X-100 0.75?g/L), we yielded a maximum concentration of 14.41?g/L, which was about 1.5-fold higher than non-optimized CSL-based medium. Our findings highlight the use of CSL as a cost effective and promising nutrient source for industrial production of gellan gum.     

Growth and amino acid contents of<Emphasis Type="Italic">Spirulina platensis</Emphasis> with different nitrogen sources

The growth and amino acid contents of the cyanobacterium,Spirulina platensis strain NIES 46, were investigated using ammonium, nitrate, nitrite, or urea as the sole nitrogen source in a batch culture. Chlorophylla concentration was highest at 2,096 μg/L in the nitrate group after 10 days of cultivation, while the dry weight ofS. platensis was highest at 4.5 g/L in the ammonium group after 30 days of cultivation. The total amino acid content was highest at 174 mg/g dry weight ofS. platensis in the urea group at the end of the cultivation period, yet the amino acid patterns forS. platensis were similar for all the experimental groups. Therefore, it seemed that the growth and amino acid composition ofS. platensis varied depending on the type of nitrogen sources, while the amino acid patterns were not changed. Also, the most efficient harvesting time forS. platensis seemed to be approximately 10 days after cultivation.     

吸水链霉菌ATCC 29253产Hygrocin A发酵条件的优化

【背景】Hygrocins是一种萘安莎抗生素,具有良好的新药开发潜能。但在常见培养基及发酵条件下菌体内Hygrocin A含量一般很低,甚至难以直接进行准确检测。【目的】提高吸水链霉菌ATCC 29253发酵物中Hygrocin A的产量。【方法】采用单因素与正交试验设计优化相结合的方法系统考察碳源、氮源、磷酸盐、MgCl_2浓度、NaCl浓度、种子菌龄等因素对吸水链霉菌ATCC 29253产Hygrocin A能力的影响。【结果】最佳发酵条件为(g/L):葡萄糖4.0,黄豆饼粉8.0,麦芽提取物10.0,K_2HPO_4 1.5,KH_2PO_4 1.5,NaCl 1.5,Mg Cl2 1.0;种子最佳活化时间为48 h;培养参数:摇床转速200 r/min,初始pH为6.8-7.0,瓶装量50 m L/250 m L,接种量5%,30°C培养10 d。在优化条件下,Hygrocin A产量与其原始培养基M10相比提高了500%,Rapamycin产量同时下降了95%。【结论】通过培养基优化,可显著提高吸水链霉菌ATCC 29253中Hygrocin A产量,为Hygrocin A合成应用研究奠定基础,同时可使Rapamycin产量明显下降。这说明可通过选择培养条件有目的地调节两种抗生素的代谢通量,进而开展多种抗生素同时表达的代谢调控研究。     

碳源和氮源对5-酮基-葡萄糖酸生成的影响

氧化葡萄糖杆菌Gluconobacter oxydans可以将葡萄糖氧化成葡萄糖酸,并进一步氧化成2-酮基-葡萄糖酸(2KGA)和5-酮基-葡萄糖酸(5KGA),其中5KGA在催化剂的作用下能够转化为L(+)-酒石酸。为了提高5-酮基-葡萄糖酸产量,以仅生成5KGA的氧化葡萄糖杆菌Gluconobacter oxydans HGI-1为出发菌株,研究不同碳源(蔗糖、乳糖、麦芽糖、淀粉、葡萄糖)和有机氮源(酵母浸粉、鱼粉、玉米浆、黄豆饼粉、棉籽饼粉)对5KGA产量的影响。500 mL摇瓶试验结果表明,当葡萄糖浓度为100 g/L时,5KGA产量最高为98.20 g/L;当有机氮源为酵母浸粉、鱼粉和玉米浆,其添加量的蛋白含量为1.60%时,5KGA产量分别为100.20 g/L、109.10 g/L和99.83 g/L,其中,使用鱼粉的5KGA产量最高,使用玉米浆的5KGA产量比酵母浸粉略低。出于经济考虑,文中选择玉米浆作有机氮源,并在5 L发酵罐中进行分批发酵放大试验,5KGA的产量为93.80 g/L,最大生成速率为3.48 g/(L·h),平均生成速率为1.56 g/(L·h)。结果表明,葡萄糖和玉米浆分别为Gluconobacter oxydans HGI-1规模化生产5KGA的最适碳源和氮源,可利用葡萄糖几乎全部(85.93%)转化为5KGA。     

Beta vulgaris L. suspension cultures permeabilized with triton X-100 retain cell viability and betacyanines production ability: a digital image analysis study

The influence of Triton X-100 on Beta vulgaris L. permeabilized cell culture viability, regrowth, and ability to produce betacyanines was evaluated in this study. A non-destructive method based on the analysis of images in the RGB (red, green, blue) system was developed to estimate betacyanines content. A treatment for 15 min with 0.7 mM Triton X-100 induced the release of 30% of betacyanines without loss of cell viability (>or=70%). After this permeabilization treatment, B. vulgaris cultures regrew normally, reaching a maximum biomass concentration of 48% higher than non-permeabilized cultures after 14 days of culture. Also, maximum betacyanines concentration was only 25% lower than that of non-permeabilized cultures.     

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     

富营养废水中碳氮磷浓度对布朗葡萄藻总脂和总烃的影响

以经过二次过滤的富营养化鱼塘养殖污水为培养液,添加外源的碳、氮、磷元索,研究了污水中不同的外源无机碳、总氮和总磷浓度对布朗葡萄藻(Botryococcus braunii)生物量、总脂和总烃含量的影响.结果表明:(1)以NaHCO3作为碳源,布朗葡萄藻的生物量和总脂含量在外源无机碳浓度为5～10 mg/L时最高,总烃含量在外源无机碳浓度为15mg/L时最高.(2)以KNO3作为氮源,布朗葡萄藻的生物量在总氮浓度为15mg/L时最高,总脂含量在总氮浓度为2mg/L时最高,总烃含量在总氮浓度为20mg/L时最高.(3)以KH2 PO4作为磷源,布朗葡萄藻生物量在总磷浓度为2mg/L时最高,总脂含量和总烃含量在总磷浓度为1.5 mng/L时最高.     

林可霉素生物合成培养基的优化

以花生粉和棉籽蛋白粉取代了原培养基中的黄豆饼粉,采用响应面法对林可霉素产生菌的发酵培养基进行了优化.首先通过单因素试验及正交实验确定替代氮源及其浓度,采用Plackett-Burman实验分析各因素的主效应,选出对响应值影响较大的3个因素,即花生粉、K2HPO4和玉米浆.对这些因素做爬坡实验,确定三个重要因素的中心点浓...     

Kinetic characterization and Mg2+ enhancement of Streptomyces griseocarneus sphingomyelinase C produced by recombinant Streptomyces lividans

Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550-950 U/mg) and was obtained in high yields (approximately 120 mg/L of culture). SMC activity was enhanced by MgCl(2), and the maximum activity (542±25 U/mg) was observed in the presence of 1.5 mol/L (M) MgCl(2). Dynamic light scattering analysis proved that the highest specific SMC activity was obtained with the smallest mixed micelles of sphingomyelin (SM) and Triton X-100. The turnover rate (k(cat)), K(m) and k(cat)/K(m) values for SM were 346 s(-1), 0.458 mM, and 756 mM(-1)s(-1), respectively, in the presence of 1M MgCl(2). The k(cat) was strongly influenced by the MgCl(2) concentration. By contrast, the K(m) value was independent of the MgCl(2) concentration and was almost constant. Circular dichroism spectroscopy indicated that MgCl(2) did not cause local structural changes in SMC. From these results, we concluded that the SMC activity enhancement by MgCl(2) was caused by the increased specific surface area of the mixed micelles composed of substrate, SM, and Triton X-100.     

Biological removal of pyridine in heavy oil byRhodococcus sp. KCTC 3218

The removal of organic nitrogen compounds present in crude perroleum and shale oils poses a challenging problem in petroleum industries. The deleterious effect of nitrogen compounds on cracking catalysts and the indication that they contribute to gum formation in gasolines are some of these aspects. Pyridine, a representative nitrogen compound in gaavy oil—was degraded byRhodoccus sp. KCTC 3218 in a water-heavy oil two-phase system. The pyridine degradation rate was affected by the presence of hydrocarbons such as n-hexadecane. This microorganism formed flocs which could be a barrier to mass transfer between the cells in flocs and the pyridine dissolved in water. This problem could be overcome by the addition of a surfactant such as Triton X-100. The ratio of water to heavy oil was important to separate the heavy oil phase from the water phase after treating the heavy oil. The culture medium was emulsified by a sort of biosurfactant secreted by this microorganism. The emulsified oil phase returned to its natural state when the ratio of water to heavy oil was 1.5. Above this ratio, the emulsified oil phase remained an emulsion after decantation. Pyridine in heavy oil was completely degraded in 15 hr at this water to heavy oil ratio of 1.5 when the concentration of pyridine in heavy oil was 700 ppm and the cell concentration was 0.32 g DCW/L.     

Toxicity of phenanthrene dissolved in nonionic surfactant solutions to Pseudomonas putida P2

The fraction in which direct contact occurs between micellar-phase phenanthrene and the bacterial cell surface was estimated by measuring the toxicity of nonionic surfactant (Tween 80 and Triton X-100) solutions to the phenanthrene-degrading bacterium, Pseudomonas putida P2. Cell viability of completely dissolved phenanthrene decreased by 30% at concentrations greater than 0.3 mg L(-1), which is equal to approximately one third of its solubility. Both nonionic surfactants had no effect on cell viability up to 5 g L(-1). Cell viability increased with increasing surfactant concentration at a fixed phenanthrene concentration, due to the decreased concentration of aqueous-pseudophase phenanthrene and the reduced fraction of direct contact. The fraction of direct contact was c. 20% or more below 3 g L(-1) of Triton X-100. The fraction of direct contact for Tween 80 was estimated to be lower than Triton X-100.     

Immobilization in the presence of Triton X-100: modifications in activity and thermostability of <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> CCR11 lipase

A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100. Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration (co. 500,000 Da). Immobilization conditions were established at 25 °C, pH 6, and a protein concentration of 0.9 mg/mL in the presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 °C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions.     

Highly efficient production of <Emphasis Type="SmallCaps">d</Emphasis>-lactate by <Emphasis Type="Italic">Sporolactobacillus</Emphasis> sp. CASD with simultaneous enzymatic hydrolysis of peanut meal

Highly efficient d-lactate production by Sporolactobacillus sp. strain CASD was demonstrated in this study. Peanut meal was found to be a better nutrient than yeast extract, soybean meal, soybean peptone, corn steep, liquor beef extract, and ammonium sulfate in the production of d-lactate. To improve the utilization of peanut meal, the material was enzymatically hydrolyzed and simultaneously utilized as the nitrogen source in d-lactate fermentation. Very high d-lactate production (207 g/L) was obtained using 40 g/L of peanut meal in 30-L fed-batch fermentation, with the average productivity of 3.8 g/(L·h) and optical purity of 99.3%. The production of such a high concentration of optically pure d-lactate by strain CASD, with the simultaneous enzymatic hydrolysis of peanut meal and fermentation, represents a new cost-efficient and integrated method for d-lactate production using agricultural by-products.     

Effect of nonionic surfactants on Rhizopus homothallicus lipase activity: a comparative kinetic study

Based on amino-terminal sequencing and mass spectrometry data on the Rhizopus homothallicus lipase extracted using solid (SSF) and submerged state fermentation (SmF) methods, we previously established that the two enzymes were identical. Differences were observed, however, in terms of the specific activity of these lipases and their inhibition by diethyl p-nitrophenyl phosphate (E600). The specific activity of the SSF lipase (10,700 μmol/min/mg) was found to be 1.2-fold that of SmF lipase (8600 μmol/min/mg). These differences might be the result of residual Triton X-100 molecules interacting with the SSF lipase. To check this hypothesis, the SmF lipase was incubated with submicellar concentrations of Triton X-100. The specific activity of the lipase increased after this treatment, reaching similar values to those measured with the SSF lipase. Preincubating SSF and SmF lipases with E600 at a molar excess of 100 for 1 h resulted in 80% and 60% enzyme inhibition levels, respectively. When the SmF lipase was preincubated with Triton X-100 for 1 h at a concentration 100 times lower than the Trition X-100 critical micellar concentration, the inhibition of the lipase by E600 increased from 60% to 80%. These results suggest that residual detergent monomers interacting with the enzyme may after the kinetic properties of the Rh. homothallicus lipase.     

Dependence of apparent viscosity on mycelial morphology of Streptomyces fradiae culture in various nitrogen sources

To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa.s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration, which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa.s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 x 10(4) microm(2). In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 x 10(4) microm(2) comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology.     

Optimization of growth and hydrolytic enzymes production by Fusarium culmorum using response surface method

Production of hydrolytic enzymes by a phytopathogenic fungus Fusarium culmorum was investigated. The proteolytic activity was observed when the fungus was grown in the medium containing starch or soybean meal as a carbon source. The amylolytic and lipolytic activities were not found. Response surface modeling was applied to shake-flask culture of the fungus to determine the optimum concentration of carbon source and optimal culture time for growth and protease production. The results indicated that the maximum yield of protease production corresponded to the concentration of soybean meal of 1.4?g/ml and culture time of 4.5?days. The fungus growth depends on the concentration of carbon source in the medium whereas the enzyme production was also influenced by the culture time and interaction between these two variables.     

Examination of Le and lele Genotypes of Glycine max (L.) Merr. for Membrane-Bound and Buffer-Soluble Soybean Lectin

Membrane fractions from seedlings of four soybean [Glycine max (L.) Merr.] lines were examined by radioimmunoassay and hemagglutination assay for the 120,000 dalton soybean lectin. Two of the lines (Sooty and T-102) are genotypically lele and lack buffer-soluble soybean lectin; the remaining two lines (Beeson and Harosoy 63) are Le and produce seeds that contain the lectin (Su et al. 1980 Biochim. Biophys. Acta 629: 292-304). Both Triton X-100 (0.5% v/v) and nonidet P-40 (0.05% v/v) solubilized soybean lectin from membrane fractions of Le cotyledons. Triton X-100 interfered substantially with the assay of protein and hemagglutinating activity and was unacceptable for use in quantitative measurements. The nonidet P-40-solubilized soybean lectin from Le cotyledons was consistently present both in washed 13,000g and 82,500g membrane fractions, but it accounted for less than 1.5% of the total (buffer-soluble plus membrane-bound) soybean lectin. The membrane lectin was purified by the affinity chromatography procedure devised for soluble soybean lectin, and it was immunologically indistinguishable from authentic soybean lectin. Membrane fractions from Le cotyledons contained insignificant amounts of radioisotope-labeled soybean lectin that had been added during homogenization, and purified membrane fractions did not bind the lectin in the presence of the hapten, d-galactose. These controls make it unlikely that the membrane soybean lectin was of cytoplasmic origin. Soybean lectin and other hemagglutinins were not present in buffer-soluble or membrane fractions from lele cotyledons or from roots and hypocotyls of any of the lines.     

Optimization of conditions for transient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated gene expression assays in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.