Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

Improvement of kojic acid production in Aspergillus oryzae B008 mutant strain and its uses in fermentation of concentrated corn stalk hydrolysate

A strain designated M866, producing kojic acid with a high yield, was obtained by combining induced mutation using ion beam implantation and ethyl methane sulfonate treatment of a wild type strain of Aspergillus oryzae B008. The amount of kojic acid produced by the strain M866 in a shaking flask was 40.2 g/L from 100 g/L of glucose, which was 1.7 times higher than that produced by wild strain (23.58 g/L). When the mixture of glucose and xylose was used as carbon source, the resulting kojic acid production was raised with the increasing of glucose ratios in the mixture. With concentrations of glucose at 75 g/L and xylose at 25 g/L mixed in the medium, the production of kojic acid reached 90.8 %, which was slightly lower than with glucose as the sole source of carbon. In addition, the kojic acid fermentation of the concentrated hydrolysate from corn stalk was also investigated in this study, the maximum concentration of kojic acid accumulated at the end of the fermentation was 33.1 g/L and this represents the yield based on reducing sugar consumed and the overall productivity of 0.36 g/g and 0.17 g/L/h, respectively.     

Kinetics of kojic acid fermentation by Aspergillus flavus using different types and concentrations of carbon and nitrogen sources

Kinetics of kojic acid fermentation by Aspergillus flavus Link 44-1 using various sources of carbon [glucose, xylose, sucrose, starch, maltose, lactose or fructose] and nitrogen [NH₄Cl, (NH₄)₂S₂O₈, (NH₄)₂NO₃, yeast extract or peptone] were analyzed using models based on logistic and Luedeking–Piret equations. The highest kojic acid production (39.90 g l⁻¹) in submerged batch fermentation was obtained when 100 g l⁻¹ glucose was used as a carbon source. Organic nitrogen sources such as peptone and yeast extract were favorable for kojic acid production as compared to inorganic nitrogen sources. Yeast extract at 5 g l⁻¹ was optimal. The optimal carbon to nitrogen (C/N) ratio for kojic acid fermentation was 93.3. In a resuspended cell system, the rate of glucose conversion to kojic acid by cell-bound enzymes increased with increasing glucose concentration up to 70 g l⁻¹, suggesting that the reaction followed the Michaelis–Menten enzyme kinetic model. The value of K _m and V _max for the reaction was 18.47 g l⁻¹ glucose and 0.154 g l⁻¹ h⁻¹, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 20–24. Received 13 October 1999/ Accepted in revised form 02 April 2000     

Fermentation of Detoxified Acid-Hydrolyzed Pyrolytic Anhydrosugars into Bioethanol with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 2.399

Pyrolysate obtained from the pyrolysis of waste cotton is a source of fermentable sugars that could be fermented into bioethanol fuel and other chemicals via microbial fermentation. However, pyrolysate is a complex mixture of fermentable and non-fermentable substrates causing inhibition of the microbial growth. The aim of this study was to detoxify the hydrolysate and then ferment it into bio-ethanol fuel in shake flasks and fermenter applying yeast strain Saccharomyces cerevisiae 2.399. Pyrolysate was hydrolyzed to glucose with 0.2 M sulfuric acid, neutralized with Ba(OH)₂ followed by treatment with ethyl acetate and activated carbon to remove fermentation inhibitors. The effect of various fermentation parameters such as inoculum concentration, pH and hydrolysate glucose was evaluated in shake flasks for optimum ethanol fermentation. With respect to inoculum concentration, 20% v/v inoculum i.e. 8.0 × 10⁸–1.2 × 10⁹ cells/mL was the optimum level for producing 8.62 ± 0.33 g/L ethanol at 9 h of fermentation with a maximum yield of 0.46 g ethanol/g glucose. The optimum pH for hydrolysate glucose fermentation was found to be 6.0 that produced 8.57 ± 0.66 g/L ethanol. Maximum ethanol concentration, 14.78 g/L was obtained for 4% hydrolysate glucose concentration after 16 h of fermentation. Scale-up studies in stirred fermenter produced much higher productivity (1.32 g/L/h^–1) compared to shake flask fermentation (0.92 g/L/h^–1). The yield of ethanol reached a maximum of 91% and 89% of the theoretical yield of ethanol in shake flasks and fermenter, respectively. The complex of integrated models of development was applied, that has been successfully tested previously for the mathematical analysis of the fermentation processes.     

枯草芽孢杆菌基因修饰生产核黄素

【目的】研究枯草芽孢杆菌核黄素合成途径、木糖代谢相关基因修饰对核黄素合成的影响。【方法】单独过表达或共同过表达核黄素操纵子中的基因、过表达木糖代谢相关基因构建相应的重组菌株。通过测定和比较重组菌株摇瓶发酵的核黄素产量和生物量,表征各个基因修饰的效应。采用摇瓶和5 L罐发酵,考察木糖作为主要碳源以及木糖与蔗糖共代谢对核黄素发酵的影响。【结果】ribA基因单独过表达,使核黄素产量提高99%,但生物量降低30%,出现细胞自溶现象。ribA-ribH基因共表达,使核黄素产量提高280%,并且无细胞自溶和生物量下降现象。1.5%蔗糖与6.5%木糖作为碳源,5 L发酵罐发酵70 h,核黄素产量达到3.6 g/L,与8%蔗糖为碳源的发酵相比,核黄素产量提高80%。木糖代谢相关基因过表达,均明显降低核黄素产量。【结论】与ribA基因单独过表达相比,ribA-ribH基因共表达可有效避免细胞自溶现象,并能进一步提高核黄素产量。蔗糖与木糖共代谢,能够改善前体物供给,有利于提高核黄素产量。     

Kinetics and modelling of kojic acid production by Aspergillus flavus Link in batch fermentation and resuspended mycelial system

An unstructured model based on logistic and Luedeking-Piret equations was proposed to describe growth, substrate consumption and kojic acid production by Aspergillus flavus Link strain 44-1 in batch fermentation and also in a resuspended cell system. The model showed that kojic acid production was non-growth associated. The maximum kojic acid and cell concentrations obtained in batch fermentations using the fermenter with optimized dissolved oxygen control (32.5 g/l and 11.8 g/l, respectively) and using a shake-flask (36.5 and 12.3 g/l, respectively) were not significantly different. However, the maximum specific growth rate and a non-growth-associated rate constant for kojic acid formation (n) for batch fermentation using the fermenter (0.085/h and 0.0125 g kojic acid/g cell.h, respectively) were approximately three and two times higher than the values obtained for fermentation using a shake-flask, respectively. Efficient conversion of glucose to kojic acid was achieved in a resuspended pellet or mycelial system, in a solution containing only glucose with citrate buffer at pH 3.5 and at a temperature of 30 °C. The resuspended cell material in the glucose solution was still active in synthesizing kojic acid after prolonged incubation (up to about 600 h). The rate constant of kojic acid production (n) in a resuspended cell system using 100 g glucose/l was almost constant at an average value of 0.011 g kojic acid/g cell.h up to a cell concentration of 19.2 g/l, above which it decreased. A drastic reduction of n was observed at a cell concentration of 26.1 g/l. However, the yield based on glucose consumed (0.45 g/g) was similar for all cell concentrations investigated.     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

Cultivation characteristics of immobilized Aspergillus oryzae for kojic acid production

Aspergillus oryzae in situ grown from spores entrapped in calcium alginate gel beads was used for the production of kojic acid. The immobilized cells in flask cultures produced kojic acid in a linear proportion while maintaining the stable metabolic activity for a prolonged production period. Kojic acid was accumulated up to a high concentration of 83 g/L, at which the kojic acid began to crystallize, and, thus, the culture had to be replaced with fresh media for the next batch culture. The overall productivities of two consecutive cultivations were higher than that of free mycelial fermentation. However, the production rate of kojic acid by the immobilized cells was suddenly decreased with the appearance of central cavernae inside the immobilized gel beads after 12 days of the third batch cultivation.     

Homofermentative production of optically pure <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from sucrose and mixed sugars by batch fermentation of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> RKY1

In the present study, lactic acid fermentation was carried out by batch culture of Enterococcus faecalis RKY1 using sucrose and mixed sugars as the major substrate. Maximum lactic acid productivity (5.2 g/L/h) was recorded when 50 and 100 g/L of sucrose were used as a carbon source. Sucrose concentration higher than 150 g/L resulted in the decrease of lactic acid productivity due to inhibition by high substrate concentration, but lactic acid productivity was remained > 3.0 g/L/h until the sucrose used for lactic acid fermentation increased up to 150 g/L. L-Lactic acid content of the total lactic acid produced from sucrose and mixed sugars was higher than 98%. When the fermentation media contained sucrose, the kinetic parameters showing specific rates such as μ, qS, and qP were relatively lower than those of fermentation using glucose as a sole carbon source, which might be due to additional time requirement to induce invertase enzyme for utilization of sucrose. There was no carbon catabolite repression observed when the sugar mixtures containing sucrose, glucose, and/ or fructose were used as a carbon source for lactic acid fermentation by E. faecalis RKY1.     

粘质沙雷氏菌发酵生产D-乳酸的研究

本文对粘质沙雷氏菌发酵生产D-乳酸进行了研究。以粘质沙雷氏菌G1(Serratia marcescens G1)为出发菌种,摇瓶试验确定了发酵培养方式:前12 h为菌体生长阶段,有氧培养,温度28℃,pH值7.0;后36 h为D-乳酸合成积累阶段,无氧培养,温度44℃,pH值6.0。且发现使用葡萄糖为碳源时更有利于D-乳酸的合成积累。采用缺失2,3-丁二醇合成能力的基因工程菌株R1为出发株,经筛选后得到耐受较高浓度乳酸盐的菌株R150,以R150为发酵菌种,在3.7 L发酵罐上采用两阶段发酵法,并通过增加起始菌体浓度的方法,发酵生成的D-乳酸浓度达到83.5 g/L,光学纯度达到98.9%。本研究成果为使用粘质沙雷氏菌发酵生产D-乳酸的深入研究打下了基础。     

Production of extracellular water insoluble β-1,3-glucan (curdlan) fromBacillus sp. SNC07

β-1,3-Glucan (curdlan) is a water-insoluble polysaccharide composed exclusively of β-1,3 linked glucose residues. Extracellular curdlan was mostly synthesized byAgrobacterium species andAlcaligenes faecalis under nitrogen-limiting conditions. In this study, we screened the microorganisms capable of producing extracellular curdlan from soil samples. For the first time, we reported Gram-positive bacteriumBacillus sp. SNC 107 capable of producing extracellular curdlan in appreciable amounts. The effect of different carbon sources on curdlan production was studied and found that the yield of curdlan was more when glucose was used as carbon source. It was also found that maximum production was achieved when the initial concentration of ammonium and phosphate in the medium was 0.5 and 1.9 g/L respectively. In this study the curdlan production was increased from 3 to 7 g/L in shake flask cultures.     

Strain improvement and up scaling of phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 563 under submerged fermentation conditions

Combination of physical and chemical mutagenesis was used to isolate hyper secretory strains of Aspergillus niger NCIM 563 for phytase production. Phytase activity of mutant N-1 and N-79 was about 17 and 47% higher than the parent strain. In shake flask the productivity of phytase in parent, mutant N-1 and N-79 was 6,181, 7,619 and 9,523 IU/L per day, respectively. Up scaling of the fermentation from shake flask to 3 and 14 L New Brunswick fermenter was studied. After optimizing various fermentation parameters like aeration, agitation and carbon source in fermentation medium the fermentation time to achieve highest phytase activity was reduced considerably from 14 days in shake flask to 8 days in 14 L fermenter. Highest phytase activity of 80 IU/ml was obtained in 1% rice bran–3.5% glucose containing medium with aeration 0.2 vvm and agitation 550 rpm at room temperature on 8th day of fermentation. Addition of either bavistin (0.1%), penicillin (0.1%), formalin (0.2%) and sodium chloride (10%) in fermented broth were effective in retaining 100% phytase activity for 8 days at room temperature while these reagents along with methanol (50%) and ethanol (50%) confer 100% stability of phytase activity at 4°C till 20 days. Among various carriers used for application of phytase in feed, wheat bran and rice bran were superior to silica and calcium carbonate. Thermo stabilization studies indicate 100% protection of phytase activity in presence of 12% skim milk at 70°C, which will be useful for its spray drying.     

Lycopene production from synthetic medium by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> NRRL 2895 (+) and 2896 (−) in a stirred-tank fermenter

The dissolved oxygen tension of 20% of air saturation, pH-shift from 4.0 to 5.5 on day 3, and a moderate shear stress (calculated as an impeller tip speed, V_\texttip = 0. 9 2 6- 2. 1 6 1  \textm/\texts V_{\text{tip}} = 0. 9 2 6- 2. 1 6 1 \, {\text{m}}/{\text{s}} ) were identified to be the key factors in scaling-up the mated fermentation of Blakeslea trispora NRRL 2895 (+) and 2896 (−) for lycopene production from a shake flask to a stirred-tank fermenter. The maximal lycopene production of 183.3 mg/L was obtained in 7.5-L stirred-tank fermenter, and then the mated fermentation process was successfully step-wise scaled-up from 7.5- to 200-L stirred-tank fermenter. The comparability of the fermentation process was well controlled and the lycopene production was maintained during the process scale-up. Furthermore, with the integrated addition of 150 μmol/L abscisic acid on day 3, 0.5 g/L leucine and 0.1 g/L penicillin on day 4, the highest lycopene production of 270.3 mg/L was achieved in the mated fermentation of B. trispora in stirred-tank fermenter.     

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS-22.

Production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae pneumoniae AS-22 was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, like carbon, nitrogen, phosphorus, and mineral sources as well as initial pH and temperature, were tested on enzyme production. The optimum concentrations of the selected media components were determined using statistical experimental designs. Two level fractional factorial designs in five variables, namely, dextrin, peptone, yeast extract, ammonium dihydrogen orthophosphate, and magnesium sulphate concentrations were constructed. The optimum medium composition thus found consisted of 49.3 g/L dextrin, 20.6 g/L peptone, 18.3 g/L yeast extract, 6.7 g/L ammonium dihydrogen orthophosphate, and 0.5 g/L magnesium sulphate. The maximum CGTase activity obtained was 21.4 U/mL in 28 h of incubation. The cell growth and CGTase production profiles were studied with the optimized medium in shake flasks and in 1-L fermenters. It was observed that the enzyme production was growth associated both in shake flask and in fermenter, although it was slower in shake flask. The maximum CGTase activity obtained in the fermenter was 32.5 U/mL in 16 h. The optimized medium resulted in about 9-fold increase in the enzyme activity as compared to that obtained in the basal medium in shake flask as well as in fermenter.     

Production of lactic acid and fructose from media with cane sugar using mutant of Lactobacillus delbrueckii NCIM 2365

AIMS: To examine the potential of Lactobacillus delbrueckii mutant, Uc-3 to produce lactic acid and fructose from sucrose-based media. METHODS AND RESULTS: The mutant of L. delbrueckii NCIM 2365 was cultivated in shake flask containing hydrolysed cane sugar (sucrose)-based medium. The lactic acid yield and volumetric productivity with hydrolysed cane concentration up to 200 g l(-1) were in the range of 92-97% of the theoretical value and between 2.7 and 3.8 g l(-1) h(-1), respectively. The fructose fraction of the syrup produced was more than 95% when the total initial sugar concentration in the medium was higher (150-200 g l(-1)). There are no unwanted byproducts detected in the fermentation broth. CONCLUSIONS: We demonstrated that L. delbrueckii mutant Uc-3 was able to utilize glucose preferentially to produce lactic acid and fructose from hydrolysed cane sugar in batch fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will be useful in the production of lactic acid and high fructose syrups using media with high concentrations of sucrose-based raw materials. This approach can lead to modification of the traditional fermentation processes to obtain value-added byproducts, attaining better process economics.     

Optimization of submerged culture conditions for the mycelial growth and exo-biopolymer production by Cordyceps militaris

AIMS: The objective of the present study was to determine the optimal culture conditions for exo-biopolymer production by Cordyceps militaris in shake flask culture. METHODS AND RESULTS: The optimal temperature and initial pH for both mycelial growth and exo-biopolymer production by Cordyceps militaris in shake flask culture were found to be 20 degrees C and 6.0, respectively. Sucrose (40 g x l(-1)) and corn steep powder (10 g x l(-1)) were the most suitable carbon and nitrogen source for both mycelial growth and exo-biopolymer production. CONCLUSION: Under optimal culture conditions, the maximum exo-biopolymer concentration in a 5-l jar fermenter indicated 10.3 g x l(-1), which was approximately three times higher than that in shake flask culture. SIGNIFICANCE AND IMPACT OF THE STUDY: This process can have a significant impact on the industrial scale when sucrose and corn steep powder were used as carbon and nitrogen source.     

The kinetics of simultaneous glucose and fructose uptake and product formation by Aspergillus niger in citric acid fermentation

The kinetics of substrate uptake and product formation in the process of citric acid accumulation by Aspergillus niger on sucrose as a sole carbon source are presented. The experiments are aimed at studying if glucose and fructose obtained from the hydrolysis of sucrose are equivalent carbon sources for A. niger and how the presence of the two different carbon substrates might influence the citric acid formation process. Beet sugar was used as a sole carbon source in the first series of experiments conducted in two types of bioreactors: stirred tank and air-lift. The fructose uptake rate was significantly lower than the glucose uptake rate in the late idiophase. A substrate utilisation breakpoint occurred when a large amount of citric acid was accumulated in the fermentation broth. A similar phenomenon was also detected in repeated fed-batch fermentation. This phenomenon was confirmed by the second series of parallel shake culture runs, in which fungal growth and citric acid accumulation by A. niger was simultaneously tested on the media containing the following carbon sources: sucrose, glucose and fructose, with and without addition of concentrated citric acid solution. Finally, it was shown that high concentration of citric acid strongly depleted fructose uptake rate.     

红酵母NZ-01发酵条件的优化

以红酵母菌株NZ-01为试验菌株,研究其发酵工艺与中试生产。采用摇瓶发酵优化的方式,研究培养基组分与发酵工艺条件对该菌发酵的影响,并进行中试放大生产。结果显示,该菌最适生长培养基组分为葡萄糖10g/L,蔗糖10g/L,酵母膏10g/L,牛肉膏2.5g/L;色素合成最适培养基组分为葡萄糖15g/L,蔗糖10g/L,酵母膏2.5g/L,牛肉膏5g/L。最适生长起始pH值为6.0,最适接种量为8%,生长周期为44h;最适色素合成起始pH值为7.0,最适色素合成接种量为8%,色素合成周期为48h。发酵优化后的色素产量3.88μg/mL较优化前1.71μg/mL提高了127%。中试产量达3.05μg/mL。红酵母菌NZ-01优化后的发酵条件可以应用于中试生产虾青素,有规模化生产应用潜力。     

被孢霉生物合成花生四烯酸的初步研究

花生四烯酸不仅是细胞膜的重要成分,在维持细胞膜的结构与功能上发挥重要作用,而且是人体前列腺素合成的前体物质,对人体生理功能具有重要的调节作用。近年来研究发现,花生四烯酸在保护皮肤、降低胆固醇、抑制血小板聚集、提高免疫能力、促进胎儿发育等方面具有独特的...     

5-酮基-D-葡萄糖酸发酵生产的工艺条件优化

葡萄糖酸氧化杆菌可将葡萄糖转化为5-酮基-D-葡萄糖酸(5-KGA),而5-KGA是重要食品添加剂L(+)-酒石酸的合成前体。为提高5-KGA产量及其对葡萄糖的转化率,对5-KGA发酵生产的工艺条件进行优化。在摇瓶水平最适的培养基和培养条件下,5-KGA最高产量为19.7 g/L,较优化前提高43.8%。在5 L发酵罐上控制恒定pH值5.5、溶氧浓度15%条件下,5-KGA产量达到46.0 g/L,较摇瓶最高产量提高1.3倍,应用葡萄糖流加工艺,5-KGA最高产量达到75.5 g/L,转化率超过70%,与已见报道的最高水平相比提高了32.0%,为实现微生物发酵生产5-KGA、进而合成L(+)-酒石酸的工业化提供了切实有效的途径。