A media design program for lactic acid production coupled with extraction by electrodialysis

The aim of this study was to investigate industrial media for lactic acid fermentation to reduce the cost of nitrogen sources. Corn steep liquor (CSL) was successfully used at 5% (v/v) in batch fermentations. Use of soluble CSL improved the productivity approximately 20% with an advantage of clearer fermentation broth. Yeast extract (YE)-complemented CSL media further increased the productivity. It was found that 3.1 g L(-1) yeast extract and 5% CSL could be an effective substitute for 15 g L(-1) yeast extract in 10% glucose medium. Spent brewery yeast was also used as a sole nitrogen source equivalent to 5% CSL. Lactic acid was recovered by electrodialysis from the cell free broth. Depleted cell free broth supplemented with 5 g L(-1) of yeast extract performed reasonably in batch cultures. Reuse of the fermentation broth may reduce the cost of raw materials as well as minimize the fermentation wastes.     

Pilot-scale production of intracellular and extracellular enzymes

Optimization of pilot-scale enzyme production is described for the case of an extracellular protease and an intracellular esterase. Media optimization was conducted to reduce medium costs and to determine the effect of various defined ingredients as well as complex nitrogen sources on enzyme production. Fermentation conditions such as inoculum transfer timing, agitation rate, and cultivation temperature were evaluated for their effect on enzyme production. Broths were harvested via microfiltration, diafiltered, and in the case of the extracellular enzyme, lysed via homogenization. An improvement in enzyme titer and reduction in medium costs for the extracellular protease was realized through replacement of Sabouraud dextrose broth medium with more reasonably priced complex nitrogen sources such as N-Z-amine A. An improvement in enzyme titer and reduction in medium costs for the intracellular esterase was realized by decreasing the amount of malt extract and omitting glycerol from the medium. An improvement in the harvest conditions for both enzymes was realized by using large-lumen-diameter hollow-fiber membranes (2.7 mm) which seemed wide enough to pass clumps of fungal and filamentous bacterial fermentation broth without clogging.     

Influence of Environmental Factors on Bacteriocin Production by Human Isolates of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> MM19 and <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> MM33

The influence of temperature, initial pH, and carbon and nitrogen sources on bacteriocin secreted by Lactococcus lactis MM19 (MM19) and Pediococcus acidilactici MM33 (MM33) was evaluated. It was found that 30 and 45 °C were the growth temperatures for higher nisin and pediocin production by MM19 and MM33, respectively. The initial pH values for higher production of nisin and pediocin were 9 and 6, respectively. Glucose and wheat peptone E430 were found as suitable carbon and nitrogen sources, respectively, for highest nisin production by MM19 at 30 °C and initial pH of 9. In these conditions, nisin production could be increased by 6.7 times as compared to the control medium (de Man, Rogosa, and Sharpe—MRS broth). Similarly, fructose and pea peptone were suitable carbon and nitrogen sources, respectively, for highest production of pediocin by MM33 at 45 °C and initial pH of 6. In these conditions, pediocin production by MM33 was increased by three times as compared to the control medium (tryptone-glucose-yeast extract—TGE broth).     

长双歧杆菌发酵培养基的优化

目的对长双歧杆菌液态发酵培养基进行优化。方法以长双歧杆菌（Bifidobacteriumlongum）为发酵菌株,以MRS培养基为基础培养基,以发酵液活菌数为指标,通过单因素添加实验考察发酵培养基的碳源和氮源的种类,并验证优化后培养基的效果。结果优化后培养基的最适碳源为葡萄糖,最适氮源为酪蛋白胨、牛肉蛋白胨、水解乳蛋白,发酵液活菌数达到2．09×10^9CFU／mL,比原MRS培养基（1．22×10^9CFU／mL）提高了71．30％。结论优化后培养基优于原MRS基础培养基,可应用于长双歧杆菌的液态发酵。     

Optimal protease production condition for Prevotella ruminicola 23 and characterization of its extracellular crude protease

In this study, Prevotella ruminicola 23 (ATCC 19189), a ruminal proteolytic bacterium, was used as protease producer to examine the optimal condition for protease production. The best carbon and nitrogen sources for the maximum growth were glucose with peptone. Both sucrose and glucose could stimulate high protease production. Casein and peptone are better nitrogen sources for protease production than other choice in this study. The best enzyme production condition was 18-20 h incubation which was at late log phase in the broth of 5% glucose or sucrose as carbon source with 0.1% ammonium chloride and 0.2% peptone as nitrogen sources. Most of the protease activity was secreted into broth (65%) and on cell surface (18%). The optimal temperature and pH for protease reaction were 40 degrees C and pH 6.8, respectively. After incubation for 6h, the crude extract maintained 50% of original protease activity at 30 and 50 degrees C, and protease activity was stable between pH 6 and 8. The protease inhibitor test showed that serine, aspartic acid and metallo-protease inhibitors could cause inhibition of proteolysis. Protein feedstuff degradation experiments suggested that protease in crude extract had higher degradation ability on fish meal, whey, and feather meal (2.39, 2.60 and 1.76 micromol aminoacid/mg enzyme/h) in comparison to soybean meal and blood meal (1.11 and 1.09 micromol aminoacid/mg enzyme/h). The protease in the crude extract should have application potential in term of improving utilization of fish meal and feather meal for monogastric animals.     

Impact of nitrogen assimilation on regulation of antibiotic production in Streptomyces hygroscopicus 155

The enzymes of the assimilation pathways in cultures of S. hygroscopicus grown in the presence of various nitrogen sources were investigated. No assimilation activity of glutamate dehydrogenase (GDH) was observed. Activities of alanine dehydrogenase (ADH), GDH, glutamine: 2-oxoglutarate aminotransferase (GOGAT) and glutamate synthetase (GS) were studied. High concentrations of ammonium and alanine induced ADH formation. The levels of GS remained low in media with NH4Cl. Various nitrogen sources had no impact on the activity of GOGAT which suggested the involvement of constitutive synthesis. ADH was likely to play an alternative role. Determination of the quantitative and qualitative composition of the free amino acids confirmed the involvement of the GS-GOGAT pathway in nitrogen assimilation. The concentration of ammonium ions in the media with one amino acid or in the presence of several amino acids lowered the antibiotic activity while in the media with alanine and the other nitrogen compounds it increased the antibiotic activity.     

Development of a fructose based medium for biosynthesis of cyclosporin-A byBeauveria nivea

Summary A new fructose based medium was developed to growBeauveria nivea ATCC 34921 for the production of Cyclosporin A (CyA). Two different sugars and three different nitrogen sources were used for CyA production. Different pH levels in the broth revealed that the best pH for CyA production was 5.5. Maximum concentration of 170 mg CyA/L of broth was obtained with the new medium in 8 days corresponding to yields of 14.8 mg CyA/g dry weight biomass and 5.67 mg CyA/g fructose supplied.     

Production of Aflatoxins in Submerged Culture

Aflatoxins can be produced on a synthetic medium in submerged culture. Glucose, sucrose, or fructose are the preferred carbon sources, and Casamino Acids are the preferred nitrogen source. Ammonia is almost as good a nitrogen source. Zinc is required at levels of at least 0.4 mg per liter. Concentrations of aflatoxin of 60 to 80 mg per liter (as determined by optical-density measurements of a chloroform extract of the unfiltered broth) can readily be obtained in indented shake flasks; somewhat lower yields were obtained in 5-liter fermentors.     

Influence of medium composition on the growth and antigen expression of Helicobacter pylori

An evaluation of the ability of various solid and liquid media to support both growth and antigen expression, particularly lipopolysaccharide (LPS) expression, by Helicobacter pylori culture collection strains and clinical isolates was performed. Liquid-based basal media (brain heart infusion, Brucella broth, Mueller–Hinton broth and tryptone soya broth) supported the growth of strains, whereas solid basal media of the same formulation did not support growth. Optimal growth of all strains was obtained on solid and in liquid media containing blood. Supplemented solid media containing supplements other than blood supported growth but only to a small extent. In liquid media excluding blood, serum supplements enhanced growth and horse serum was found to be superior to fetal calf serum. In general, β-cyclodextrin did not increase growth. Mueller–Hinton broth or tryptone soya broth containing horse serum and a nitrogen source such as yeast extract or proteose peptone no. 3 were found to give optimal growth of H. pylori in a blood-free environment. Strains after cultivation in liquid media, irrespective of composition, maintained production of high-molecular weight (mol. wt) LPS with an O side chain independent of medium composition, whereas subculturing on solid media resulted in production of low-mol. wt LPS. Expression of proteins differed in liquid and on solid media, particularly proteins of 57 and 60 kDa, but qualitatively no differences were observed upon supplementation of basal media.     

Effect of Mycelial Forms on Citric Acid Fermentation in Submerged Mold Culture

A number of reports have been published on the production of citric acid by submerged mold culture. Most of them, however, have laid stresses on the effects of chemical factors, such as metal ions, nitrogen sources, potassium ferrocyanide and methanol, and very little has been reported on the effect of other factors.

The form of mycelia of mold changes depending on the physical characters cf broth, and it was found that Aspergillus niger 93A, which showed a constant activity of citric acid production, could increase its acid production activity when mycelial forms were controlled to filamentous by adding suitable non-ionic surface active agents to the broth.     

Molecular composition of diphtheria toxoid produced using semi-synthetic and meat extract-based broths

The use of semi-synthetic broths for cultivation of Corynebacterium diphtheriae instead of a meat extract-based broth avoids the presence of highly undesirable bovine meat antigens in the diphtheria toxoid. As information on the composition of casein digest-based broths used for the production of diphtheria toxoid is scarce, we have now developed one. The composition of a casein-based medium that supports vigorous bacterial growth as well as high toxin production is described below. The comparative analysis of the toxoids, produced using the meat-based Pope–Lingood and the casein digest-based broths, showed considerable differences in their molecular composition. The variance of weight distribution of toxoid-containing molecular complexes was smaller when the semi-synthetic broth was used. Normal human therapeutic IgG recognizes some of the proteins in the meat-based medium but does not react with any components of the semi-synthetic medium. While precipitation at the isoelectric point of the diphtheria toxoid produced by culturing the C. diphtheriae strain in the semi-synthetic medium resulted in a preparation meeting the requirement for purity (more than 1500 limit floculation Lf/mg protein nitrogen PN), the toxoid produced in the Pope–Lingood broth failed to meet this requirement in some cases, even after a second purification step using ultrafiltration.     

双碳源单细胞蛋白高密度培养

单细胞蛋白(SCP)培养具有增殖速度快、原料来源广、蛋白质含量高等优点,正在成为重要的蛋白质来源之一。生产SCP的原料包括烷烃、低碳醇类及碳水化合物。由碳水化合物,尤其是由各种农副产品加工的废料、造纸工业废水及木质纤维素水解产物等,生产SCP属于废弃资源的综合利用,具有重要的经济和社会意义。这类原料中常含有多种碳源,例如,在木质纤维水解液中,有以木糖为主的五碳糖,也有以葡萄糖为主的六碳糖,木糖与葡萄糖之比约为1:2。这样,木糖的利用就成了一个关键问题。葡萄糖对木糖代谢的抑制作用随葡萄糖比例的降低而减轻;Slinger和Bothast研究了用混合糖培养Candida shehetane的过程,当木糖与葡萄糖的比例为3:1及1:1时,葡萄糖的利用速度分别比木糖高1.6倍及3.4倍;在低还原势时,酵母产酒精速率降低而细胞增长速率加快,可以达到较高细胞浓度。     

费氏链霉菌中子辐射诱变菌株对新霉素的生物转化

对新霉素产生菌费氏链霉菌进行中子辐射诱变和突变株筛选, 获得不产新霉素的突变株。以突变株为转化菌种, 以新霉素为底物, 对转化发酵液进行高效液相色谱分析, 研究了不同转化条件对新霉素转化的影响。结果表明, 底物浓度、底物添加时间、底物添加方式、接种量、培养基装量、转化时间、碳源、氮源、pH、温度对新霉素转化具有不同程度的影响。以转化条件优化参数进行转化大量培养, 转化液经4步离子交换层析进行分离纯化, 薄层层析检测纯化样品为单一斑点。采用薄层生物自显影对获得的4个转化产物分离样品做生物活性检测, 发现4个样品对金黄色葡萄球菌和姜青枯假单孢杆菌都具有抑制活性, 1个样品对大白菜软腐样品具有明显的抑制活性。     

Effects of food quality on growth and body composition of the collembolan Orchesella cincta

Abstract. The effects of food quality on growth and body composition of the collembolan Orchesella cincta (L.) (Entomobryidae) were investigated to determine optimal values for dietary nitrogen.Hyphae of Cladosporium cladosporioides containing 2.2%, 2.7%, 4.0% and 6.2% nitrogen were used as food sources.These different nitrogen contents were obtained by adjusting the medium on which the fungi were grown.Collembola were fed on one of these four food sources for 8 weeks.
O. cincta grew faster and reached a higher asymptotic body mass when fed on fungi with 4% or 6.2% nitrogen content: this effect decreased with higher nitrogen content.The lipid concentration in the animals was negatively correlated with dietary nitrogen content, whereas protein concentration was positively correlated.Glycogen concentration was not found to correlate with the composition of the food.
A mechanism is proposed by which O. cincta compensates for low dietary nitrogen by consuming more to maintain a relatively constant amount of protein in its body and, as a result, accumulates more fat than animals fed on fungi with a higher nitrogen content.     

Developing an understanding of sophorolipid synthesis through application of a central composite design model

A key barrier to market penetration for sophorolipid biosurfactants is the ability to improve productivity and utilize alternative feedstocks to reduce the cost of production. To do this, a suitable screening tool is required that is able to model the interactions between media components and alter conditions to maximize productivity. In the following work, a central composite design is applied to analyse the effects of altering glucose, rapeseed oil, corn steep liquor and ammonium sulphate concentrations on sophorolipid production with Starmerella bombicola ATCC 222144 after 168 h. Sophorolipid production was analysed using standard least squares regression and the findings related to the growth (OD₆₀₀) and broth conditions (glucose, glycerol and oil concentration). An optimum media composition was found that was capable of producing 39.5 g l^–1 sophorolipid. Nitrogen and rapeseed oil sources were found to be significant, linked to their role in growth and substrate supply respectively. Glucose did not demonstrate a significant effect on production despite its importance to biosynthesis and its depletion in the broth within 96 h, instead being replaced by glycerol (via triglyceride breakdown) as the hydrophilic carbon source at the point of glucose depletion. A large dataset was obtained, and a regression model with applications towards substrate screening and process optimisation developed.     

盾壳霉产生几丁质酶的条件研究

本实验采用摇瓶培养研究了盾壳霉(Coniothyrium minitans)产生几丁质酶的条件。改良的天然马铃薯葡萄糖培养基(mPDB)较合成培养基(SMCS)更适宜作为盾壳霉产生几丁质酶的基础培养基。添加9种不同碳源试验表明葡萄糖较适宜于盾壳霉产几丁质酶;氮源试验表明硝酸钾是产酶的较适宜氮源。盾壳霉形成几丁质酶的培养时间以15 d为佳,培养的适宜pH值为6.5。     

Effects of nitrogen sources on production and composition of sophorolipids by <Emphasis Type="Italic">Wickerhamiella domercqiae</Emphasis> var. <Emphasis Type="Italic">sophorolipid</Emphasis> CGMCC 1576

The effects of nitrogen sources on growth of sophorolipid-producing yeast, Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 and on production and composition of sophorolipids were studied. Organic nitrogen sources are more favorable for accumulation of biomass than inorganic ones. Presence of ammonium ion from different inorganic nitrogen sources (except NH₄HCO₃) greatly inhibited the production of lactonic sophorolipids. However, when organic nitrogen sources were used, lactonic sophorolipid production was strongly increased. Production of crystalline lactonic sophorolipids from organic/inorganic nitrogen sources was enhanced with the increase of pH value adjusted by sodium hydroxide or sodium citrate solution. Fourier-transform infrared (FT-IR), gas chromatography mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and mass spectra (MS) were employed to compare the composition of sophorolipid mixture obtained from different nitrogen sources. More than 15 acidic sophorolipid molecules and only 4 lactonic sophorolipid molecules were produced by using 1.27 g/l ammonium sulfate as nitrogen source; they were separated by preparative HPLC and their structures were elucidated by MS. These results suggest extraordinary regulatory effects of nitrogen source on growth and sophorolipid synthesis of W. domercqiae var. sophorolipid.     

产微生物絮凝剂的菌株筛选、鉴定及发酵条件优化

微生物絮凝剂是一类新型的水处理制剂,因其具有高效、无毒、易降解等特点,已成为水处理剂开发的研究热点。为了扩大微生物絮凝剂的种类,本研究从活性污泥和土壤样品中筛选絮凝剂产生菌,最终获得一株具有絮凝活性的菌株,通过16S rDNA鉴定,该菌株为蒙氏假单胞菌(Pseudomonas monteilii),因此命名为P. monteilii YR-1。利用单因素实验和正交试验对该菌株的发酵条件进行了优化,其最佳碳、氮源分别为25 g/L葡萄糖和2 g/L复合氮源(酵母浸粉:尿素:硫酸铵=5:5:2),培养基初始pH值为10,培养温度为28℃,发酵时间为72 h。在最佳培养条件下,其发酵液的絮凝率从37.68%提高到63.52%。这是关于蒙氏假单胞菌产絮凝剂的首次报道。     

Evaluation of novel assays to assess the influence of different iron sources on the growth of Clostridium difficile

The ability of four Clostridium difficile strains to utilize various exogenous organic and inorganic iron sources for growth under iron-depleted (250 μM DPP) and iron-limited (75 μM DPP) conditions was analyzed in liquid broth cultures grown in tubes and in microtiter plates, and data compared with results from a bioassay developed on solid media. The growth profile of C. difficile varied depending on the iron source and availability. Addition of FeSO(4), FeCl(3), Fe citrate and ferritin allowed growth in an iron-depleted environment whereas glycoproteins (iron-saturated and low-iron lactoferrin, apo- and holo-transferrin) and heme proteins (hemoglobin, hematin and hemin) did not. All iron sources, except lactoferrin, were able to restore bacterial growth under iron-limited conditions to varying extents. The results demonstrated that the broth microtiter assay developed here was reproducible, reliable and convenient for high-throughput analysis of the growth of C. difficile compared to alternative traditional methods.