A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Effect of eight growth media upon fermentation profiles of ten anaerobic bacteria

A study was made of the influence of eight growth media upon the fermentation end-product patterns obtained with ten species of anaerobes. Acidic and neutral end-products of fermentation were analysed by gas chromatography and the results were examined statistically by computer. Differences in end-product profile were at least as great between different media used to grow a single anaerobic species, as between different anaerobes grown in a single type of medium. When individual fermentation end-products produced by a single anaerobe species were examined, statistically significant differences were found between different media for individual end-products. It is concluded that standardisation of growth medium is essential in diagnostic laboratories concerned with identification of anaerobes with the aid of gas chromatography.     

The effect of surfactants on the phytase production and the reduction of the phytic acid content in canola meal byAspergillus carbonarius during a solid state fermentation process

Summary During the growth of A. carbonarius, the rates of biomass growth, phytase production and phytic acid content reduction in canola meal media during solid state fermentation were higher in the presence of Na-oleate or Tween-80 than in the control medium which was not supplemented with these surfactants. Addition of Triton X-100 had a negative effect on the studied processes.The optimum concentration of Na-oleate in solid state culture media was 1%.     

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

AIMS: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. METHODS AND RESULTS: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l(-1)) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. CONCLUSIONS: The yeast strain required a minimum of 267 mg N l(-1) to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality.     

Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation

Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P.pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P.pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P.pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10–100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N- labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment.     

Polygalacturonase production by Kluyveromyces marxianus: effect of medium composition

A strain of Kluyveromyces marxianus (CCT 3172), isolated from a cocoa fermentation in Brazil, secreted an endopolygalacturonase (PG) when grown under self-induced anaerobic conditions; neither polymethylesterase nor pectate lyase appeared in culture filtrates. Replacing glucose in the medium with sucrose had no effect on PG secretion or ethanol production. Growth in fructose-containing medium retarded secretion of PG and ethanol, but had no effect on growth. Growth and ethanol production in media containing galactose resembled those in fructose-containing medium, although PG secretion was lowered. Growth and PG secretion were considerably retarded in xylose-containing medium, and were similarly affected in media containing different concentrations of glucose. Varying the concentration of ammonium sulphate in media had no effect on growth or PG secretion.     

Evaluation of the fermentability of oat fractions obtained by debranning using lactic acid bacteria

Aims: The overall kinetics of the fermentation of four oat fractions obtained by debranning using three potentially probiotic lactic acid bacteria were investigated. The main objective was to study the suitability of these fractions as fermentation media for the growth and the metabolic production of bacteria isolated from human intestine. Methods and Results: The cell growth, lactic acid production and substrate uptakes of the three lactobacilli was monitored for 30 h. An unstructured mathematical model was used to describe and fit the experimental data. In the medium from fraction B (1–3% pearlings or β-glucan-rich fraction) all strains reached the highest cell populations, maximum growth rates and maximum lactic acid productions. This could be because of the high levels of total fibre and β-glucan of this fraction. Limited growth and lactic acid formation was found in medium A (0–1% pearlings or bran-rich fraction). Conclusions: Medium B (1–3% pearling fraction) is the most suitable for fermentation and produces considerably higher probiotic cell concentrations. Significance and Impact of the Study: Debranning technology could be used to separate fractions from cereal grains for the production of functional formulations with higher probiotic levels than the ones that were obtained with the whole grain.     

大肠杆菌O121侵袭性菌株的发现与研究

本文报告从一急性腹泻患儿的脓血便中分离的具有侵袭性的大肠杆菌。该菌株发酵葡萄糖,产酸产气,不发酵乳糖,β-半乳糖苷酶试验阳性,在醋酸钠培养基上生长,赖氨酸脱羧酶阴性,无动力,能引起豚鼠角膜结膜炎,侵入上皮细胞,具有140Md的质粒带,经血清学试验证实其血清型为O121:H-,是国内外首次报道的新的侵袭性大肠杆菌血清型。     

Relationship of low lysine and high arginine concentrations to efficient ethanolic fermentation of wheat mash.

Very high gravity wheat mashes containing 20 or more grams of carbohydrates per 100 mL were fermented completely by Saccharomyces cerevisiae, even though these mashes contained low amounts of assimilable nitrogen. Supplementation of wheat mashes with various amino acids or with yeast extract, urea, or ammonium sulfate reduced the fermentation time. However, lysine or glycine added as single supplements, inhibited yeast growth and fermentation. With lysine, yeast growth was severely inhibited, and a loss of cell viability as high as 80% was seen. Partial or complete reversal of lysine-induced inhibition was achieved by the addition of a number of nitrogen sources. All nitrogen sources that relieved lysine-induced inhibition of yeast growth also promoted uptake of lysine and restored cell viability to the level observed in the control. They also increased the rate of fermentation. Experiments with minimal media showed that for lysine to be inhibitory to yeast growth, assimilable nitrogen in the medium must be in growth-limiting concentrations or totally absent. In the presence of excess nitrogen, lysine stimulated yeast growth and fermentation. Results indicate that supplementing wheat mash with other nitrogen sources increases the rate of fermentation not only by providing extra nitrogen but also by reducing or eliminating the inhibitory effect of lysine on yeast growth.     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.     

莲子草假隔链格孢SF 193固体发酵培养基的筛选及其对空心莲子草的控制效果

【背景】空心莲子草是一种源于南美洲且正在我国不断传播的外来入侵植物。莲子草假隔链格孢是空心莲子草的强致病菌,能引起空心莲子草茎叶发病,抑制空心莲子草生长。【方法】选择以麦麸、大米、玉米芯粉、玉米粉、稻秸秆粉为基料,棉籽壳、稻秕壳和大豆粉为辅料,按照一定质量比配制固体培养基,筛选适合莲子草假隔链格孢SF-193大量生产的有效组分;同时研究培养基含水量和6种金属离子对SF-193菌落生长的影响。【结果】在以大米和大豆粉质量比为3:1和5:1的培养基上SF-193菌落面积最大,其次为大米和棉籽壳质量比为5:1的培养基。在含水量为20%和30%的培养基上SF-193菌落面积显著大于其他培养基。Mg2+和Mn2+对SF-193菌落生长影响较小,Zn2+对SF-193菌落生长具有显著的抑制作用,而SF-193菌落在含Cu2+和Fe3+的培养基上不能生长。田间控草试验表明,固体发酵生产的菌粉对空心莲子草的致病性显著高于液体菌,用量为50g.m-2时,10d后空心莲子草的病情指数达86.1。【结论与意义】利用固体培养基发酵生产的莲子草假隔链格孢对空心莲子草的防除效果显著优于液体发酵。因此,莲子草假隔链格孢SF-193固体发酵菌粉可能在生物防治空心莲子草方面具有重要作用。     

红树林植物促生菌SZ7-1 菌株的培养基优化

采用响应面分析法对红树林植物促生菌SZ7-1 菌株发酵培养基进行了优化。首先利用Plackett-Burman (PB)设计对影响SZ7-1 生长的11 个营养因素进行评价, 并筛选出显著影响因子:玉米糖浆、酵母膏和K₂HPO₄; 其次用最陡爬坡实验逼近以上三因素最优水平; 最后采用响应面法对该3 个显著因素的最佳水平范围进行研究, 得到的最佳浓度为: 玉米浆28 g/L、酵母膏14 g/L和K₂HPO₄ 2.2 g/L。在此条件下, 培养20 h 后SZ7-1 菌数达到2.6×10¹⁰ CFU/mL, 与优化前基础培养基、LB 和牛肉膏蛋白胨培养基中生长的菌数相比, 分别提高了12.4、2.4 和5.5 倍。     

The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations

AIM: To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. METHODS AND RESULTS: Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l(-1)) and biotin (0, 1 or 10 microg l(-1)) as variables. All media contained 240 g l(-1) glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 microg l(-1) had no effect. Nitrogen x biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H(2)S). Maximum H(2)S production occurred in the medium containing 60 mg l(-1) YAN and 1 microg l(-1) biotin. CONCLUSIONS: Nitrogen x biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.     

Effect of soy skim from soybean aqueous processing on the performance of corn ethanol fermentation

The feasibility of using soy skim, a co-product of the aqueous processing of soybeans, in ethanol production from corn was evaluated. Specific growth rates were compared when Saccharomyces cerevisiae was grown in soy skim and peptone-yeast extract media supplemented with glucose. Such soy skim was proved to be a good nitrogen source for yeast growth. Next, fermentation of dry-ground corn to ethanol using soy skim as the media was simulated on 1.5-L scale. Replacing water with soy skim increased the initial ethanol production rates by 4-32% while final ethanol yield was about 39 g/100 g dry corn, similar to the result when water was used. Solid and protein contents in the finished beer increased with the addition of soy skim. Thus, replacing water in corn-ethanol fermentation with soy skim is feasible, and may improve the economics of both aqueous soybean processing and corn ethanol fermentation.     

Using highly efficient nonlinear experimental design methods for optimization of Lactococcus lactis fermentation in chemically defined media

Optimization of fermentation media and processes is a difficult task due to the potential for high dimensionality and nonlinearity. Here we develop and evaluate variations on two novel and highly efficient methods for experimental fermentation optimization. The first approach is based on using a truncated genetic algorithm with a developing neural network model to choose the best experiments to run. The second approach uses information theory, along with Bayesian regularized neural network models, for experiment selection. To evaluate these methods experimentally, we used them to develop a new chemically defined medium for Lactococcus lactis IL1403, along with an optimal temperature and initial pH, to achieve maximum cell growth. The media consisted of 19 defined components or groups of components. The optimization results show that the maximum cell growth from the optimal process of each novel method is generally comparable to or higher than that achieved using a traditional statistical experimental design method, but these optima are reached in about half of the experiments (73–94 vs. 161, depending on the variants of methods). The optimal chemically defined media developed in this work are rich media that can support high cell density growth 3.5–4 times higher than the best reported synthetic medium and 72% higher than a commonly used complex medium (M17) at optimization scale. The best chemically defined medium found using the method was evaluated and compared with other defined or complex media at flask‐ and fermentor‐scales. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Growth and cyclosporin a production by an indigenously isolated strain ofTolypocladium inflatum

A fungal culture isolated from a local soil sample which showed antifungal activity and produced cyclosporin A, was identified asTolypocladium inflatum. The culture grew best in a medium containing 1% maltose (pH 5–6) when inoculated with a one-day-old inoculum at 2% (V/V) concentration. Under batch fermentation conditions, growth and cyclosporin A production were better in complex media (24.6 g biomass and 205 mg cyclosporin A per liter) in comparison with synthetic media (6.84 g biomass and 35 mg cyclosporin A per liter). While addition of peptone increased culture growth (high biomass yield), supplementation with casein acid hydrolyzate favored cyclosporin A production.     

Product inhibition and its removal on josamycin fermentation by Streptomyces narbonensis var. josamyceticus

Josamycin formation was shown to be closely associated with the degree of mycelial growth. Media using glycerol or glucose were slight in josamycin accumulation. However, the mycelial growth and product formation were interrupted in the course of the fermentation. The self-resistance level of the producing organism against josamycin was shown to be not higher than 1 mg per ml. In media using fatty oil, the organism grew smoothly and gave a concentration of josamycin at least 5 times higher than the self-resistance levelMost of the josamycin was distributed in the oily phase of the broth. The josamycin concentration of the water phase of the broth was less than the self-resistance level, except in the later period of the fermentation. Unsaturated fatty acids remaining in the broth were found to be the best extractors of josamycin. It was concluded that the enhanced josamycin accumulation of this fermentation resulted from the removal of the product inhibition.     

Effects of potassium chloride on ethanol production by an osmotolerant mutant of Zymomonas mobilis

The effect of increasing the KCl concentration in the culture medium of an alcoholic fermentation of glucose using the bacterium Zymomonas mobilis was investigated. Data obtained with the wild-type strain (ZM4, ATCC 31821) and with a newly isolated osmotolerant mutant (SBE15) were compared. It was observed that, at high salt concentration, inhibition of growth occured (specific growth rate and biomass yield) while ethanol production (specific ethanol productivity and ethanol yield) was unaffected. In contrast, the specific rate of in-vitro ethanol production, using either cell-free extract or washed cells, was strongly inhibited by increasing the KCl concentration in the incubation mixture. Therefore, it was concluded that the intracellular concentration of KCl was maintained below the inhibitory concentration by an active transport system. In addition, the fermentation performances of the osmotolerant mutant strain were higher than those of the parent strain at all the KCl concentrations tested, suggesting the utility of the former to run ethanolic fermentations in crude industrial media with a high salt content. Furthermore, the fermentation data on media containing added KCl agreed well with those obtained on molasses media, suggesting that the inhibition observed on these media was due to their high osmolality. Correspondence to: J. Baratti     

Lithotrophic growth and hydrogen metabolism by Clostridium magnum

Clostridium magnum, originally described as a non-autotrophic homoacetogenic bacterium, was found to be able to grow with H2/CO2, formate, or methanol with stoichiometric acetate formation, provided that the growth medium contained at least 0.025% (w/v) yeast extract. Hydrogen was also formed as a byproduct of glucose fermentation, and was consumed again after glucose consumption. Hydrogen formation from glucose was independent of growth conditions and reached similar maximal concentrations in mineral media with or without ammonia added as well as in non-growing cultures or in the presence of carbon monoxide.     

红缘拟层孔菌发酵工艺的研究及其升罐培养

为筛选出红缘拟层孔菌发酵的最佳条件,并用于升罐发酵,试验优选出5个基础培养基,再以发酵菌丝体的生物量为指标,确定红缘拟层孔菌发酵的最佳条件。进行升罐发酵,间隔取出少量发酵液进行显微观察并检测葡萄糖含量,描述出菌丝体的生长状态。结果表明:红缘拟层孔菌发酵的最佳条件为马铃薯200 g/L,葡萄糖20 g/L,磷酸二氢钾1 g/L,硫酸镁1.5 g/L,维生素B10.4 g/L。升罐发酵中,菌丝干质量在前60 h处于快速的增长状态,最大为6.890 1 g/L,而60 h之后菌丝的生长变缓慢,到84 h之后,菌丝自溶,菌丝的产量开始下降。菌丝的葡萄糖含量经血糖仪检测在60 h时最高,达到0.296 5 g/L,之后呈现下降趋势。红缘拟层孔菌升罐发酵的最佳发酵时间为56~64 h。