Continuous ethanol production by thermotolerant Kluyveromyces marxianus IMB3 immobilized on mineral Kissiris at 45°C

Thermotolerant Kluyveromyces marxianus var. marxianus IMB3 yeast strain was immobilized on Kissiris (mineral glass foam derived from lava) in column packed reactors, and used for ethanol production from glucose or molasses under continuous culture conditions at temperatures between 40 and 50°C. Both ethanol yield and fermentation efficiency were highest at 45°C and a dilution rate (D) of 0.15/h. Increasing sugar concentration led to an increase in ethanol yield of up to 68.6 and 55.9 g/l on approx. 200g glucose or molasses, respectively. Optimum fermentation efficiency (experimental yields over theoretical maximum yields) however was at about 15% sugar for both glucose and molasses. Slight aeration (25 ml of air/min) through the medium addition line was found advantageous due to its mixing effect and probable maintenance of activity.     

Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs

In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of citric acid in submerged culture. For screening of fermentation medium composition significantly influencing citric acid production, the two-level Plackett-Burman design was used. Under our experimental conditions, beet molasses and corn steep liquor were found to be the major factors of the acid production. A near optimum medium formulation was obtained using this method with increased citric acid yield by five-folds. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, the three-level Box-Behnken design was applied. A polynomial model was created to correlate the relationship between the three variables (beet molasses, corn steep liquor and inoculum concentration) and citric acid yield. Estimated optimum composition for the production of citric acid is as follows pretreated beet molasses, 240.1g/l; corn steep liquor, 10.5g/l; and spores concentration, 10(8)spores/ml. The optimum citric acid yield was 87.81% which is 14 times than the basal medium. The five level central composite design was used for outlining the optimum values of the fermentation factors initial pH, aeration rate and temperature on citric acid production. Estimated optimum values for the production of citric acid are as follows initial pH 4.0; aeration rate, 6500ml/min and fermentation temperature, 31.5 degrees C.     

Effect of the carbon source and aeration conditions on homoserine lysine biosynthesis in the threonine-dependent mutant Brevibacterium flavum 2T

The effect of two carbon sources (sucrose and acetate), aeration conditions and threonine concentration on the homoserine and lysine biosynthesis by the threonine-dependent mutant Brevibacterium flavum 2T was examined. It was demonstrated that acetate provided the predominant synthesis of homoserine to a far greater extent than sucrose (with the weight/weight ratio of homoserine : lysine being 2.5-5.0 and 0.8-1,2, respectively). The maximal level of homoserine and lysine was 18-21 and 3-7 g/l on the acetate containing medium and 18-22 and 12-16 g/l on the sucrose containing medium, respectively. On sucrose the total amount of amino acids and the total yield of products as related to the consumed substrate were greater than on acetate. Using the sucrose medium, the effect of aeration conditions and threonine concentration on the biosynthesis of both compounds was investigated. With an aeration increase from 1.3 to 4.6 g O2/l.hr the optimal concentration of threonine in the medium grow. The biosynthesis of homoserine was less sensitive to the inhibitory effect of excessive threonine than that of lysine. With an increase of the threonine concentration in the medium from 0.25 to 3.0 g/l the ratio homoserine : lysine grew from 1.03 to 5.20 (with the sulphite number being 4.6 g O2/l.hr). This effect was independent of the aeration conditions.     

The utilization of beet molasses as a novel carbon source for cephalosporin C production by Acremonium chrysogenum: Optimization of process parameters through statistical experimental designs

In this work, cephalosporin C (CPC) production on pilot scale fermenters of 600l capacity with 350l working volume by Acremonium chrysogenum EMCC 904 was performed. The effects of fermentation medium composition, inoculum concentration, initial pH and aeration rate on CPC production by A. chrysogenum strain was investigated by using response surface methodology (RSM). The Plackett-Burman design which involves two concentrations of each nutrient was effective in searching for the major medium components promoting CPC production. Under our experimental conditions; Soya oil, beet molasses and corn steep liquor were found to be the major factors contributing to the antibiotic production. Subsequently, a Box-Behnken design was used for outlining the concentration of the most effective medium constituents. Estimated optimum composition for the production of CPC was as follows: soya oil, 40g/l; beet molasses, 180g/l; and corn steep liquor, 330g/l. The central composite design was used for outlining the optimum values of the fermentation parameters. Estimated optimum values for the production of CPC are as follows: inoculum level, 10(5.5)spores/ml; initial pH, 4.3; and aeration rate, 9364ml/min.     

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).     

The production of auxins by the endophytic bacteria of winter rye

The ability of the actinomycetes and coryneform bacteria isolated from the root tissues of winter rye to produce auxin in a liquid culture was studied. The isolates of coryneform bacteria produced indolyl-3-acetic acid (IAA) into the medium in the amount of 9.0–95.0 μg/ml and the isolates of actinomycetes in the amount of 39.5–83.0 μg/ml. The maximal IAA accumulation in culture liquid of actinomycetes coincided, in general, with the beginning of the stationary growth phase. The dependences of IAA synthesis by actino-mycetes on the composition and pH of nutrient medium, tryptophan concentration, and aeration conditions were determined. Biological activity of the bacterial IAA was assessed. Treatment of winter rye seeds with coryneform auxin-producing bacteria increased the germination capacity and enhanced an intensive seedling growth in vitro.     

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.     

松茸液态发酵菌丝生长量因子的研究

研究了液态发酵条件下通气量、转速、pH、可溶性淀粉浓度对松茸菌丝生长量的影响,探讨了可溶性淀粉稳定pH的作用机理。试验结果表明：通气量≤0.4vvm时松茸菌丝产量随通气量的增大而增长,当通气量＞0.5vvm时,菌丝产量增加不明显;转速增大菌丝产量增加,转速＞80r/min,菌丝产量下降;松茸菌丝发酵的最适pH为5.5,初始pH不同,发酵罐中培养基最终的pH趋于6.0;可溶性淀粉是影响松茸菌丝量的重要因子,可溶性淀粉浓度≤4g/100mL时菌丝量随可溶性淀粉浓度的增加而增大,当其浓度为4g/100mL时,菌     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

Stirred tank culture of Bacillus thuringiensis H-14 for production of the mosquitocidal δ-endotoxin: mathematical modelling and scaling-up studies

The effect of culture conditions on -endotoxin production by strain S128 of Bacillus thuringiensis H-14 (locally isolated in Egypt) was investigated using a 10l working volume fermenter. Fermentation medium formulated from the inexpensive locally available soya (as a nitrogen source) and molasses (as a carbon source) was used. Aeration, agitation, pH and initial concentration of molasses were chosen as experimental factors and their influence on toxin yield was investigated using 2⁴ central composite experimental design. The mathematical model obtained revealed that the optimal batch cultivation conditions with respect to agitation, pH, and initial concentration of molasses were 325 rev min^–1, 7.1 and 2.1% (w/v) respectively. The mathematical model obtained indicated that by increasing the aeration rate over 0.89 v/v per minute the productivity could still be increased. A simulated scaling-up study, in which the Simplex method was applied, is also presented. The results of this investigation could be of great help for large-scale production of a cheap mosquito-larvicide in developing countries where mosquito-borne diseases are still a serious health and economic problem.     

A novel circulating loop bioreactor with cells immobilized in loofa (<Emphasis Type="Italic">Luffa cylindrica</Emphasis>) sponge for the bioconversion of raw cassava starch to ethanol

A circulating loop bioreactor (CLB) with cells immobilized in loofa sponge was constructed for simultaneous aerobic and anaerobic processes. The CLB consists of an aerated riser and a non-aerated downcomer column connected at the top and bottom by cylindrical pipes. Ethanol production from raw cassava starch was investigated in the CLB. Aspergillus awamori IAM 2389 and Saccharomyces cerevisiae IR2 immobilized on loofa sponge were placed, respectively, in the aerated riser column and non-aerated downcomer column. Both alpha-amylase and glucoamylase activities increased as the aeration rate was increased. Ethanol yield and productivity increased with an increase in the aeration rate up to 0.5 vvm, but decreased at higher aeration rates. The CLB was operated at an aeration rate of 0.5 vvm for more than 600 h, resulting in an average ethanol productivity and yield from raw cassava starch of 0.5 g-ethanol l(-1) x h(-1) and 0.45 g ethanol/g starch, respectively. In order to increase ethanol productivity, it was necessary to increase the dissolved oxygen (DO) concentration in the riser column and decrease the DO concentration in the downcomer column. However, increasing the aeration rate resulted in increases in the DO concentration in both the riser and the downcomer columns. At high aeration rate, there was no significant difference in the DO concentration in the riser and downcomer columns. The aeration rate was therefore uncoupled from the liquid circulation by attaching a time-controlled valve in the upper connecting pipe. By optimizing the time and frequency of valve opening, and operation at high aeration rate, it was possible to maintain a very high DO concentration in the riser column and a low DO concentration in the downcomer column. Under these conditions, ethanol productivity increased by more than 100%, to 1.17 g l(-1) x h(-1).     

Production of poly-b-hydroxybutyrate by Azotobacter vinelandii in a two-stage fermentation process

Poly-b-hydroxybutyrate (PHB) production in Azotobacter vinelandii UWD, a mutant that produces PHB constitutively, was suppressed by high aeration of beet molasses medium. Thus a two-stage process was designed using aeration to promote growth and suppress PHB production in the first phase, while lower aeration of raw sugar medium containing fish peptone was used to promote PHB formation in the second phase. A PHB yield of 36 g/l and productivity of > 1 g polymer l -1 .h was obtained by this approach.     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

羊肚菌液态发酵的研究*

通过摇瓶正交试验研究了圆锥羊肚菌液体发酵的条件,选出最佳培养基配方、接种量、培养温度、pH、通气量,培养时间等参数。在此基础上,应用VIRTIS公司生产的2L－自控发酵罐进行了分批发酵,摸索到了羊肚菌液态深层发酵的基本规律,然后分别以淀粉和玉米粉为碳源进行了补料分批发酵,获得其干细胞产量为25．4g／L和27．4g／L,生长速率为0．45g／（L·h）和0．51g／（L·h）,以碳源为基准的产量得率系数（yx／s）为0.72（g／g）和0.75（g／g）等数据。实验结果表明,羊肚菌的液态深层发酵已达到了真菌菌丝液态发酵的正常水平。     

Effect of carbon and nitrogen sources on growth and biological efficacy of Pseudomonas fluorescens and Bacillus subtilis against Rhizoctonia solani, the causal agent of bean damping-off

One of the most important environmental factors that regulate the growth and antagonistic efficacy of biocontrol agents is the medium. The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strains P-5 and P-6 (Pseudomonas fluorescens), B-3 and B-16 (Bacillus subtilis) and minimum cost of media, whilst maintaining biocontrol efficacy. All of the strains were grown in seven liquid media (pH=6.9) including: sucrose + yeast extract, molasses of sugar beet + yeast extract in 2:1 and 1:1 w/w ratios, molasses of sugar beet + urea, nutrient broth, molasses and malt extract, at an initial inoculation of 1 x 10(5) CFU ml(-1). Cells from over night cultures used to inoculate soil at 1 x 10(9) CFU cm(-3) soil. At the same time, fungal inoculum (infected millet seed with Rhizoctonia solani) was added to soil at the rate of 2 g kg(-1) soil. Results indicated that growth of P-6, B-3 and B-16 in molasses + yeast extract (1:1 w/w) medium was significantly higher than in the other media. Molasses + yeast extract (1:1 and 2:1 w/w) media supported rapid growth and high cell yields in P-5. In greenhouse condition, results indicated that the influence of the media on the biocontrol efficacy of P-5, P-6, B-3 and B-16 was the same and Pseudomonas fluorescens P-5 in molasses and malt extract media reduced the severity of disease up to 72.8 percent. On the other hand, there were observed significant differences on bean growth after one month in greenhouse. P-5 in molasses + yeast extract (1:1 w/w) medium had the most effects on bean growth promotion. In this study molasses media showed good yield efficacy in all of the strains. The high sucrose concentration in molasses justifies the high biomass in all of the strains. Also, the low cost of molasses allows its concentration to be increased in media. On the other hand, yeast extract was the best organic nitrogen source for antagonist bacteria but it is expensive for an industrial process. So it should be replaced by another industrial product instead of yeast extract, which confirm by an economic and technological study. The results obtained in this study could be used to provide a reliable basis to increase the population of biocontrol agents in fermentation process.     

Use of a sequential strategy of experimental design to optimize the inulinase production in a batch bioreactor

A potential application of inulinase in the food industry is the production of fructoligosaccharides (FOS) by the transfructosilation of sucrose. The FOSs present many interesting functional properties besides their ability to increase the shelf-life and flavor of many products. The use of an industrial medium represents a good alternative to producing inulinase at low cost, since the activity may improve, or at least remain the same, as that obtained using a synthetic medium. This work was an optimization study of the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using industrial pre-treated culture medium in a bioreactor employing a sequential strategy of experimental design. Initially, a Plackett–Burman (Screening Design) design was used, where the studied variables were molasses, corn steep liquor, yeast extract concentration, and agitation and aeration rates. After the analysis of the effects, a central composite rotational design (CCRD) was carried out. The optimized condition for the inulinase production was: 250 g/l of molasses, 80 g/l of corn steep liquor, 6 g/l of yeast extract, 300 rpm of agitation and 1.5 vvm aeration rate, which resulted in an enzymatic activity of 1,317 ± 65 U/ml.     

Production of bacterial cellulose by Acetobacter xylinum BPR2001 using molasses medium in a jar fermentor

Bacterial cellulose (BC) production by Acetobacter xylinum subsp. sucrofermentans BPR2001 using molasses medium was carried out in a jar fermentor. When molasses was subjected to H₂SO₄-heat treatment, the maximum BC concentration increased to 76% more than that achieved using untreated molasses, and the specific growth rate increased 2-fold. When the initial sugar concentrations in the H₂SO₄-heat treated molasses were varied from 23 g/l to 72 g/l, BC concentration, production rate, and yield were maximum at sugar concentrations of 23 g/l and 37 g/l, and production of by-products, such as polysaccharides and CO₂, was lower than at sugar concentrations of 48 g/l and 72 g/l, indicating that maintaining a lower molasses concentration is essential for efficient BC production in jar fermentors, this being due mainly to the complex nature of molasses. Molasses has a clear advantage over pure sugars as a carbon source from an economic viewpoint.     

荒漠微藻的碳氧转换与调控

为了提高微藻的生物燃料生产效率及其在密闭环境中的碳氧转换效率,以两株荒漠微藻BG18-3、BE6-2和一株淡水蓝藻7924为研究对象,对其进行逆境条件培养,发现荒漠微藻BG18-3在各种逆境中表现最佳。在静态培养中,荒漠微藻BG1-3也具有明显的优势,其生物量干重达到0.26 g/L,硝态氮和磷酸盐去除率分别为36%和99%。在荒漠微藻BG18-3的通气培养中,生物干重量最高（3% CO₂通气培养16天）达到2.63 g/L,生物量产率为164.0 mg/L·d,出口CO₂浓度最低降到0.04%,O₂净含量增加0.68%,这表明荒漠微藻BG18-3具有较高的碳氧转化效率,具有生产生物燃料的潜质。最后根据18s rDNA分析结果将荒漠微藻BG18-3鉴定为栅列藻Scenedesmus littoralis。     

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.     

Halogenated monoterpene production by microplantlets of the marine red alga Ochtodes secundiramea within an airlift photobioreactor under nutrient medium perfusion

Macrophytic marine red algae are a unique source of novel and bioactive terpenoids, including halogenated monoterpenes. Biomass and halogenated monoterpene production by regenerated microplantlet suspension cultures derived from the red alga Ochtodes secundiramea were studied within a perfusion airlift photobioreactor. Photobioreactor cultivations were carried out at 26 degrees C, 140 microE m(-2)s(-1) light intensity, 0.3 air L(-1) culture min(-1) aeration (3500 ppm CO(2)), and ESS/seawater medium perfusion rate of 0.2 L medium L(-1) culture d(-1). Macronutrient concentrations in the perfusion medium were adjusted to provide nitrate delivery rates of 0.0063, 0.077, and 0.74 mmol L(-1) d(-1) at a fixed N:P ratio of 19:1. Growth was maximized at the highest nutrient delivery rate, where 10 g dry biomass L(-1) culture was achieved after 30 days of cultivation. GC-MS analysis of dichloromethane extracts from cell biomass revealed that O. secundiramea microplantlets produced myrcene, three acyclic halogenated monoterpenes (10-bromomyrcene, 10-bromo-7-chloromyrcene, 3,10-dibromomyrcene), and one cyclic halogenated monoterpene (6-bromo-1,2,8-trichloro-3,4-ochtodene). 10E-bromomyrcene levels were much higher than those of its isomer 10Z-bromomyrcene, demonstrating stereoselective halogenation. Maximum yields of 10E-bromomyrcene and 6-bromo-1,2,8-trichloro-3,4-ochtodene were 15 and 13 micromol/g dry cell mass, respectively. Increasing the rate of nutrient delivery increased the accumulation of myrcene and 10-bromomyrcene during the first 14 days in culture. Furthermore, the yield selectivity toward higher halogenated monoterpenes increased as the rate of nutrient delivery decreased. From this data, a biogenic scheme was proposed where cyclic and acyclic halogenated monoterpenes are derived from sequential halogenation of myrcene, their common precursor.