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.     

Stimulation of citric acid production in Aspergillus niger by addition of viscous substances in shake culture

When glucose (120mg/ml) was used as a carbon source, Aspergillus niger Yang no. 2. showed a markedly low citric acid productivity in shake culture (15.4 mg/ml) but a high productivity in semi-solid and surface cultures (72.3 mg/ml and 67.6 mg/ml, respectively). Since the viscosity of the medium was assumed to be one of the important factors for citric acid productivity in shake culture, the effects of the addition of viscous substances on citric acid productivity of strain Yang no. 2 were examined. The addition of 2.0–6.0 mg gelatin/ml as a viscous additive to the medium containing glucose as a carbon source increased slightly the medium viscosity but substantially increased the citric acid productivity in shake culture to levels of 52.0–53.3 mg/ml, about 3.4 times as much as that without gelatin. However, no influence of gelatin addition was observed in semi-solid and surface cultures, i.e. under static cultivation conditions. Different mycelial morphologies of the strain were observed when cultivations were done in shake culture with or without the addition of gelatin. Addition of 5.0 mg agar/ml, 5.0 mg carageenan/ml, 2.5 mg carboxymethylcellulose/ml and 2.5 mg polyethylene glycol 6000/ml, to the medium containing glucose as a carbon source also increased the citric acid productivity in shake culture to levels of 39.2–54.7 mg/ml. Since Yang no. 2 does not utilize these viscous substances, these results suggested that the viscous substances functioned as protectants for the mycelium from physiological stresses due to shaking and as a consequence resulted in a remarkably increased citric acid productivity in shake culture.     

Optimization of single cell protein production from cassava starch (Rhizopus oligosporus)

The fermentation pattern of cassava starch utilization was investigated at 37°C using Rhizopus oligosporus UQM 145 F and eight different media. Depending on the medium used, the addition of zinc or zinc plus iron to a combination of calcium plus manganese switches the fermentation from glucose accumulation to biomass (single cell protein) production. Complete starch hydrolyzation was obtained in both cases, with a complete glucose utilization resulting in 24 g biomass containing 30% true protein per 100 g cassava starch (= 7.45 g SCP/100 g substrate) in 24 hours. In the case of glucose accumulation, biomass was kept low and 15.5 g/l glucose representing 57.3% of starch supplied were obtained in 36 hours. R. oligosporus UQM 145 F grows well between 30° and 45°C. At 45°C and pH 5.0, 7.0 g SCP/100 g substrate were obtained, which rose to 8.6 g if cassava starch is replaced by ground cassava tuber.     

The production of ethanol from D-glucose and D-xylose by different Fusarium strains

Summary Production of ethanol from glucose and xylose by different Fusarium strains has been studied in shake flask cultures. The best strain was Fusarium oxysporum VTT-D-80134. The best ethanol yields were 50 % ethanol on both sugars. The fermentation time was 3 days on glucose and 6 days on xylose.     

Kinetics of kojic acid fermentation byAspergillus flavus link S44-1 using sucrose as a carbon source under different pH conditions

Kojic acid production byAspergillus flavus strain S44-1 using sucrose as a carbon source was carried out in a 250-mL shake flask and a 2-L stirred tank fermenter. For comparison, production of kojic acid using glucose, fructose and its mixture was also carried out. Kojic acid production in shake flask fermentation was 25.8 g/L using glucose as the sole carbon source, 23.6 g/L with sucrose, and 6.4 g/L from fructose. Reduced kojic acid production (13.5 g/L) was observed when a combination of glucose and fructose was used as a carbon source. The highest production of kojic acid (40.2 g/L) was obtained from 150 g/L sucrose in a 2 L fermenter, while the lowest kojic acid production (10.3 g/L) was seen in fermentation using fructose as the sole carbon source. The experimental data from batch fermentation and resuspended cell system was analysed in order to form the basis for a kinetic model of the process. An unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe the growth, substrate consumption, and efficiency of kojic acid production byA. flavus in batch fermentation using sucrose. From this model, it was found that kojic acid production byA. flavus was not a growth-associated process. Fermentation without pH control (from an initial culture pH of 3.0) showed higher kojic acid production than single-phase pH-controlled fermentation (pH 2.5, 2.75, and 3.0).     

Anaerobic fermentation of gelatinized sago starch-derived sugars to acetone—1-butanol—Ethanol solvent byClostridium acetobutylicum

A study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch byClostridium acetobutylicum showed that the use of 30 g/L gelatinized sago starch as the sole carbon source produced 11.2 g/L total solvent,i.e. 1.5–2 times more than with pure maltose or glucose used as carbon sources. Enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinized sago starch. The solvent yield of direct gelatinized sago starch fermentation depended on the activity and stability of amylolytic enzymes produced during the fermentation. The pH optima for α-amylase and glucoamylase were found to be at 5.3 and 4.0–4.4, respectively. α-Amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0–8.0 after a 1-d incubation at 37°C. SinceC. acetobutylicum α-amylase has a high activity and stability at low pH, this strain can potentially be employed in a one-step direct solvent-yielding fermentation of sago starch. However, theC. acetobutylicum glucoamylase was only stable at pH 4–5, maintaining more than 90% of its maximum activity after a 1-d incubation at 37°C.     

Direct fermentation of starch to L-(+)-lactic acid using Lactobacillus amylophilus

Summary Fermentation of L-(+)-lactic acid from soluble starch by Lactobacillus amylophilus was studied. The bacterium produced about 30 g of L-(+)-lactic acid from 50 g of soluble starch when the pH of the culture was ranging from pH 5 to pH 6.8 at 28°C. 53.4 g of L-(+)-lactic acid was produced when 100 g of starch was added in the medium. The fermentation procedures will reduce the cost of complete hydrolysis of starch to glucose prior to fermentation.     

Improvement in lactic acid production from starch using α-amylase-secreting <Emphasis Type="Italic">Lactococcus lactis</Emphasis> cells adapted to maltose or starch

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l⁻¹ h⁻¹ lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l⁻¹ h⁻¹ lactate). Maximum volumetric lactate productivity was further increased (1.57 g l⁻¹ h⁻¹ lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.     

Starch industry wastewater as a substrate for antagonist, Trichoderma viride production

Starch industry wastewater was investigated to assess and improve its potential as a raw material for the conidia production of biocontrol fungi, Trichoderma viride. The wastewater was tested with and without supplements of glucose, soluble starch, meat peptone and probable conidiation inducer chemicals in shake flask culture. Addition of complex carbon source (soluble starch, 1% and 2% w/v) produced maximum conidia ( approximately 3.02 and 4.2 x 10(10)CFU/mL, respectively). On the other hand, glucose addition as a simpler carbon source was either ineffective or, reduced conidia production (from 1.6 x 10(8) in control to 3.0 x 10(7)CFU/mL in 5% w/v glucose supplement). Supplement of nitrogen source showed a small increase of conidia concentration. Propionic, maleic and humic acids, EDTA, pyridine, glycerol and CaCO(3) were examined as probable conidiation inducers and showed effect only on initial rate of conidiation with no increase in final conidia concentration. Intra and extracellular ATP correlation with spore production showed dependence on growth media used and conidia concentration at the end of fermentation. Addition of carbon and nitrogen sources showed an increase in protease activity (from 0.4985 to 2.43 IU/mL) and entomotoxicity (from 10448 to 12335 spruce budworm unit (SBU)/microL). Entomotoxicity was improved by 11% in fermenter over shake flask when starch industry wastewater was supplemented with meat peptone.     

Protein entrainment during baker's yeast fermentation on a semi-solid substrate in an air-fluidized bed fermentor

Baker's yeast was grown on a semi-solid substrate (homogenized whole potatoes) in an air-fluidized bed fermentor, in which a rapid stream of air simultaneously supplied oxygen and mixed the semi-solid substrate. The potato starch was converted to reducing sugars by -amylase (from Aspergillus).During the course of the batch fermentation, some secreted yeast proteins were trapped by sparging the effluent air into a water chamber. Surprisingly, neither the -amylase nor the potato proteins were the most predominant proteins carried over to the overhead collector during the 24 h run, even though they were the most abundant proteins in the fermentation mash. Fractionation of the yeast-produced proteins during this carry-over process is described, based on gel electrophoresis analyses of both the carried-over proteins and the extracellular proteins in the fermentation bed. Effects of the operating variables on the extracellular protein levels in the fermentation bed and the proteins in the overhead collector are also discussed.     

THE STUDY OF THE INFLUENCE OF TEMPERATURE AND INITIAL GLUCOSE CONCENTRATION ON THE FERMENTATION PROCESS IN THE PRESENCE OF Saccharomyces cerevisiae YEAST STRAIN IMMOBILIZED ON STARCH GELS BY REVERSED-FLOW GAS CHROMATOGRAPHY

The technique of reversed-flow gas chromatography (RFGC) was employed for the determination of the alcoholic fermentation phases and of kinetic parameters for free and immobilized cell systems, at different initial glucose concentrations and temperature values. In addition to this, due to its considerable advantages over other techniques, RFGC was used for the characterization of a new biocatalyst, yeast cells immobilized on starch gel, and especially wheat starch gel. Immobilization of wine yeast Saccharomyces cerevisiae AXAZ-1 was accomplished on wheat and corn starch gels in order to prepare new biocatalysts with great interest for the fermentation industry. The RFGC led with great accuracy, resulting from a literature review, to the determination of reaction rate constants and activation energies at each phase of the fermentation processes. A maximum value of rate constants was observed at initial glucose concentration of 205 g/L, where a higher number of yeast cells was observed. The increase of glucose concentrations had a negative influence on the growth of AXAZ-1 cells and rate constants were decreased. The decrease of fermentation temperature caused a substantial reduction in the viability of immobilized cells as well as in rate constant values. Activation energies of corn starch gel presented lower values than those of wheat starch gel. However, the two supports showed higher catalytic efficiency than free cell systems, proving that starch gels may act as a promoter of the catalytic activity of the yeast cells involved in the fermentation process.     

Lactic acid production from enzyme-thinned corn starch usingLactobacillus amylovorus

Summary An alternative process for industrial lactic acid production was deveooped using a starch degrading lactic acid producing organism,Lactobacillus amylovorus B-4542. In this process, saccharification takes place during the fermentation, eliminating the need for complete hydrolysis of the starch to glucose prior to fermentation. The cost savings of this alternative are substantial since it eliminates the energy input, separate reactor tank, time, and enzyme associated with the typical pre-fermentation saccharification step. The only pre-treatment was gelatinization and enzyme-thinning of the starch to overcome viscosity problems associated with high starch concentrations and to make the starch more rapidly degradable. This fermentation process was optimized for temperature, substrate level, nitrogen source and level, mineral level, B-vitamins, volatile fatty acids, pH, and buffer source. The rate of the reaction and the final level of lactic acid obtained in the optimized liquefied starch process was similar to that obtained withL. delbrueckii B-445 using glucose as the substrate.     

Lipids-free waxy corn starch as a substrate for distillery yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Utilization of lipids-free waxy starch by distillery yeasts in fuel ethanol production can contribute to better management of renewable resources, like cereals, especially maize Zea mays L. But the efficient conversion of starch into glucose-rich fermentable substrate, and subsequently to ethanol, needs more research on hydrolysis and fermentation conditions. The aim of this study was to evaluate the lack of natural corn grain lipids on the process of simultaneous saccharification and fermentation using chemometric techniques of designed experiments, commercial enzymatic preparations and distillery yeasts Saccharomyces cerevisiae CCY-11-3. Based on the results and statistical software support we can conclude that extraction of lipids from corn grains did not lead to statistically significant increase or decrease of glucose concentration in starch hydrolysis. The ethanol concentration in fermentation mash according to analysis was not statistically significantly affected by lipids extraction. The separated lipids could serve as a source of very valuable corn oil.     

Citric acid production from cellobiose by 2-deoxyglucose-resistant mutant strains of Aspergillus niger in semi-solid culture

Citric acid production from cellobiose by Aspergillus niger was studied by a semi-solid culture method using bagasse as a carrier. From the parental strain Yang no. 2, mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glucose as a carbon source were induced. The representative mutant strain M155 was selected and subjected to further mutation. The new series of mutant strains showing resistance to DG on minimal medium containing cellobiose as a carbon source was induced, and among them the best mutant strain C192 showed higher citric acid productivity than Yang no. 2 in semi-solid culture when glucose was used as a carbon source. Moreover, in semi-solid culture, the strain C192 produced 49.6 g/l of citric acid, 1.6 times as much citric acid as Yang no. 2 produced, from 100 g cellobiose/l and showed enhanced -glucosidase production. In shake culture, the extracellular -glucosidase activity of C192 was higher than that of Yang no. 2 when not only cellobiose but also glucose and glycerol, catabolite repressors, were used as a carbon source. These results indicate that mutant strains such as C192 are insensitive to catabolite repression. Correspondence to: S. Usami     

Isolation of a kojic acid-producing fungus capable of using starch as a carbon source

A fungal strain (S33-2), able to grow on cooked starch and produce a substantially high level of kojic acid, was isolated from morning glory flower ( Bixa orellana ). The fungus was characterized and identified as Aspergillus flavus. The effect of different types of starch (sago, potato and corn starch) on growth of strain S33-2 and kojic acid production was examined using shake flasks. It was found that strain S33-2 grew well on all types of starch investigated. However, kojic acid production was highest when corn starch was used, with the maximum kojic acid obtained being comparable to fermentation using glucose. The highest kojic acid production (19·2 g l⁻¹) was obtained when 75 g l⁻¹ corn starch was used. This gave a yield, based on starch consumed, and an overall productivity of 0·256 g g⁻¹ and 0·04 g l⁻¹ h⁻¹, respectively.     

Streptomyces sp.FIM-16-06产他克莫司摇瓶发酵条件的优化

采用单因素试验和正交试验研究发酵培养基组成,优化Streptomyces sp. FIM-16-06产他克莫司的发酵条件,探讨摇瓶发酵的主要影响因子初始p H、装液量、转速等发酵参数的影响。确定了适宜的发酵培养基和发酵参数:6. 0%玉米淀粉、2. 0%黄豆粉、2. 0%葡萄糖和0. 5%玉米浆,初始pH 7. 5,装液量50 m L/500 m L三角瓶,种子菌龄48 h,接种量10%,摇床转速250 r/min,发酵温度27℃,发酵周期7 d。优化后的发酵水平较原生产工艺提高60%以上。他克莫司的优化发酵工艺为其工业化生产奠定了基础。     

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.     

Fermentation of carbohydrates and yield of microbial protein in mixed cultures of rabbitcaecal microorganisms

Fermentation pattern and yields of microbial protein were investigated in cultures of the rabbit caecal contents supplied with glucose, xylose, starch, pectin and xylan. Rabbits at the age of 4 weeks (before weaning) and 3 months were slaughtered, their caecal contents added at 1.1% to growth media and incubated anaerobically at 39°C for 18 h. Caecal microorganisms of 4‐week‐old rabbits produced no methane and caproate, less butyrate, but more propionate than microorganisms of 3‐month‐old rabbits. In both groups of rabbits, fermentation of xylose produced significantly more propionate and less butyrate than fermentation of glucose. More propionate and less acetate was formed from starch than from pectin. In caecal cultures from 4‐week‐old rabbits with pectin, the molar percentages of acetate was significantly higher and percentages of other short‐chain fatty acids (SCFA) lower than in cultures with starch or xylan. In cultures from 3‐month‐old rabbits, fermentation of pectin and xylan produced similar SCFA profiles, different from SCFA molar composition in cultures with starch. Average production of microbial protein was 129mg per lg of carbohydrate digested (range 110 to 141mg/g). Protein yields were the same on glucose and xylose, but nonsignificantly higher on starch than on pectin and xylan. It can be concluded that the characteristics of substrate affected fermentation pattern in mixed cultures of rabbit caecal microorganisms. Substrate effects on protein yields were not statistically significant, due to high variation.     

High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.