Fermentative production of L(+)‐lactic acid from starch hydrolyzate and corn steep liquor as inexpensive nutrients by batch culture of Enterococcus faecalis RKY1

BACKGROUND: Attempts were made to determine the lactic acid production efficiency of novel isolate, Enterococcus faecalis RKY1 using four different starches (corn, tapioca, potato, and wheat starch) with different concentrations (50, 75, 100, and 125 g L^?1) and corn steep liquor as an inexpensive nitrogen source. RESULTS: The yield of lactic acid from each starch was higher than 95% based on initial starch concentrations. High lactic acid concentration (129.9 g L^?1) and yield (1.04 g‐lactic acid g^?1‐starch) were achieved faster (84 h) from 125 g L^?1 of corn starch. Among the starches used, tapioca starch fermentation usually completed in a shorter incubation period. The final dry cell weight was highest (7.0 g L^?1) for the medium containing 75 g L^?1 of corn starch, which resulted in maximum volumetric productivity of lactic acid (3.6 g L^?1 h^?1). The addition of 30 g L^?1 corn steep liquor supplemented with a minimal amount of yeast extract supported both cell growth and lactic acid fermentation. CONCLUSION: Enterococcus faecalis RKY1 was found to be capable of growing well on inexpensive nutrients and producing maximum lactic acid from starches and corn steep liquor as lower‐cost raw materials than conventionally‐used refined sugars such as glucose, and yeast extract as an organic nitrogen source in laboratory‐scale studies. These fermentation characteristics are prerequisites for the industrial scale production of lactic acid. Copyright © 2008 Society of Chemical Industry     

Production of Lipase from Geotrichum candidum Using Corn Steep Liquor in Different Bioreactors

The production of an extracellular lipase using corn steep liquor (CSL) as the nitrogen source in the cultivation of Geotrichum candidum NRRLY‐552 was evaluated. The optimized conditions in shake flasks were CSL, 8.0 % w/v, soybean oil, 0.6 % w/v, pH 7.0, 30 °C, 250 rpm, and 48 h, resulting in a maximum lipase productivity of 0.438 U mL^?1 h^?1(U = the amount of enzyme required to liberate 1 μmol of fatty acid per minute). Scale‐up was evaluated with airlift and stirred tank reactors; the best conditions, respectively, were 1 vvm(volume of gas per volume of medium per minute) of aeration which resulted in 0.535 U mL^?1 h^?1 (32 h) and 1 vvm and 300 rpm resulting in 0.563 U mL^?1 h^?1 (16 h). To facilitate downstream processes, lipase production was also evaluated using CSL previously clarified with activated charcoal resulting in 0.275 U mL^?1 h^?1 (24 h) using 12 % (w/v) of clarified CSL in shake flasks. The obtained results showed that CSL leads to similar productivity compared to peptone using the same microorganism under similar conditions. In addition the cost of fermentation medium using CSL is much lower because it is a very inexpensive by‐product from corn processing.     

Study of the bio‐production of carotenoids by Sporidiobolus salmonicolor (CBS 2636) using pre‐treated agro‐industrial substrates

BACKGROUND: The increasing industrial demand for carotenoids has aroused interest in their bio‐production, and the need to reduce production costs has encouraged the use of low cost industrial substrates, such as agro‐industrial residues. Thus the objective of this research was the bio‐production of carotenoids by Sporidiobolus salmonicolor using agro‐industrial substrates (corn steep liquor and sugarcane molasses), pre‐treated with acids (sulphuric and phosphoric). RESULTS: Bio‐production was carried out in an orbital shaker using a 10% (v/v) inoculum, incubation at 25 °C, and agitation at 180 rpm for 120 h in a non‐illuminated environment. The carotenoids were recovered using liquid N₂ combined with dimethylsulphoxide for cell rupture, and an acetone/methanol mixture (7:3 v/v) for extraction. CONCLUSION: The complete second‐order design allowed for optimisation of the carotenoid concentration obtained from industrial substrates pre‐treated with acids (sulphuric and phosphoric), obtaining a total carotenoid content of 541.5 µg L^?1 using 10 g L^?1 sugarcane molasses, 5 g L^?1 corn steep liquor and 5 g L^?1 yeast hydrolysate at 25 °C, with agitation at 180 rpm and an initial pH of 4.0. Copyright © 2008 Society of Chemical Industry     

Optimization of growth medium for the production of α‐amylase from Bacillus amyloliquefaciens using response surface methodology

The optimization of nutrient levels for the production of α‐amylase by Bacillus amyloliquefaciens was carried out using response surface methodology (RSM) based on the 2³ factorial central composite design (CCD). This procedure limited the number of actual experiments performed while allowing for possible interactions between three components. RSM was adopted to derive a statistical model for the effect of starch, peptone and yeast extract (YE) on α‐amylase production. The P‐value of the coefficient for linear effects of starch and YE concentration was <0.0001, suggesting that this was the principal experimental variable, having the greatest effect on the production of α‐amylase. The optimal combinations of media constituents for maximum α‐amylase production were determined as 12.61 g L^?1 starch, 2.83 g L^?1 peptone and 1.25 g L^?1 YE. The optimization of the medium resulted not only in a 34% higher enzyme activity than unoptimized medium but also in a reduced amount of the required medium constituents. Copyright © 2006 Society of Chemical Industry     

Optimization of inulinase production by solid‐state fermentation in a packed‐bed bioreactor

BACKGROUND: This work is focused on inulinase production by solid‐sate fermentation (SSF) using sugarcane bagasse, corn steep liquor (CSL), pre‐treated cane molasses, and soybean bran as substrates in a 3‐kg (dry basis) packed‐bed bioreactor. SSF was carried out by the yeast Kluyveromyces marxianus NRRL Y‐7571 and response surface methodology was used to optimize the temperature, air flow rate and initial mass of cells. RESULTS: The optimum inulinase activity (436.7 ± 36.3 U g^?1 dry substrate) was obtained at 24 h at an inlet air temperature of 30 °C, air flow rate 2.2 m³ h^?1 and 22 g of cells for fermentation. Inulinase productivity at these conditions was 18.2 U gds^?1 h^?1. Kinetic evaluation at the optimized conditions showed that the maximum inulinase production was verified at 24 h of fermentation. The carbon dioxide and the metabolic heat generation are directly associated with the consumption of total reducing sugars present in the medium. CONCLUSION: The high productivity achieved in this work shows the technical viability of inulinase production by SSF in a packed‐bed bioreactor. Copyright © 2009 Society of Chemical Industry     

Production of L‐lactic acid with very high gravity distillery wastewater from ethanol fermentation by a newly isolated Enterococcus hawaiiensis

BACKGROUND: A great amount of wastewater with high contents of chemical oxygen demand (COD) are produced by ethanol production. It would be useful to utilize distillery wastewater to produce L‐lactic acid, which could be a high additional value byproduct of ethanol production. The fermentation process of L‐lactic acid production by a newly isolated Enterococcus hawaiiensis CICIM‐CU B0114 is reported for the first time. RESULTS: The strain produced 56 g L^?1 of L‐lactic acid after cultivation for 48 h in optimized medium consisting of (g L^?1) 80 glucose, 10 peptone, 10 yeast extract, 1.5 Na₂HPO₄ and 0.2 MgSO₄. E. hawaiiensis CICIM‐CU B0114 was isolated and purified by subculture for growing and producing L‐lactic acid in distillery wastewater of very high gravity (VHG) from ethanol fermentation. L‐lactic acid fermentation was further studied with distillery wastewater substrate in 7 L and 15 L fermentors. The results showed that L‐lactic acid concentrations of 52 g L^?1 and 68 g L^?1 was achieved in 7 L and 15 L fermentors with the initial sugar concentrations of 67 g L^?1 and 87 g L^?1, respectively. CONCLUSION: The production of L‐lactic acid by the newly isolated E. hawaiiensis CICIM‐CU B0114 was carried out and the fermentation medium was optimized by orthogonal experimental design. This new strain holds the promise of L‐lactic acid production utilizing distillery wastewater from VHG ethanol fermentation. Copyright © 2010 Society of Chemical Industry     

玉米浆为有机氮源的L-乳酸发酵的研究

利用细菌厌氧发酵生产L-乳酸,以降低L-乳酸生产成本为主要目的,实验以玉米浆为有机氮源,以硫酸铵为主要无机氮源,研究了不同接种量对发酵过程的影响;并就以玉米浆替代酵母粉、豆粕水解液、生物素为有机氮源的L-乳酸发酵进行了对比研究实验,实验证明了玉米浆做为有机氮源用于L-乳酸发酵的可行性。在保证生产能力的同时,使用玉米浆为有机氮源对进一步降低L-乳酸生产成本,减少玉米浆对环境的污染进行了探索。     

Effect of acetic acid on lipid accumulation by glucose‐fed activated sludge cultures

BACKGROUND: The effect of acetic acid, a lignocellulose hydrolysis by‐product, on lipid accumulation by activated sludge cultures grown on glucose was investigated. This was done to assess the possible application of lignocellulose as low‐cost and renewable fermentation substrates for biofuel feedstock production. RESULTS: Biomass yield was reduced by around 54% at a 2 g L^?1 acetic acid dosage but was increased by around 18% at 10 g L^?1 acetic acid dosage relative to the control run. The final gravimetric lipid contents at 2 and 10 g L^?1 acetic acid levels were 12.5 ± 0.7% and 8.8 ± 3.2% w/w, respectively, which were lower than the control (17.8 ± 2.8% w/w). However, biodiesel yields from activated sludge grown with acetic acid (5.6 ± 0.6% w/w for 2 g L^?1 acetic acid and 4.2 ± 3.0% w/w for 10 g L^?1 acetic acid) were higher than in raw activated sludge (1–2% w/w). The fatty acid profiles of the accumulated lipids were similar with conventional plant oil biodiesel feedstocks. CONCLUSIONS: Acetic acid enhanced biomass production by activated sludge at high levels but reduced lipid production. Further studies are needed to enhance acetic acid utilization by activated sludge microorganisms for lipid biosynthesis. Copyright © 2011 Society of Chemical Industry     

D‐Lactic acid production from waste cardboard

The effects caused by alkaline treatment on the susceptibility of waste cardboard to enzymatic hydrolysis have been studied. Optimised conditions leading to extensive saccharification of both cellulose (870 g kg^?1 conversion) and hemicelluloses (845 g kg^?1 conversion) were identified. Samples treated under selected operational conditions were employed for producing D ‐lactic acid by simultaneous saccharification and fermentation (SSF) in media containing cellulases, β‐glucosidase and Lactobacillus coryniformis ssp torquens cells. SSF fed‐batch experiments led to D ‐lactic acid concentrations up to 23.4 g dm^?3 at a product yield of 514 g lactic acid kg^?1 of potential glucose and a volumetric productivity of 0.48 g dm^?3 h^?1. Copyright © 2004 Society of Chemical Industry     

Lactic acid production from dining‐hall food waste by Lactobacillus plantarum using response surface methodology

BACKGROUND: Food waste generally has a high starch content and is rich in nutritional compounds, including lipids and proteins. It therefore represents a potential renewable resource. In this study, dining‐hall food waste was used as a substrate for lactic acid production, and response surface methodology was employed to optimise the fermentation conditions. RESULTS: Lactic acid biosynthesis was significantly affected by the interaction of protease and temperature. Protease, temperature and CaCO₃ had significant linear effects on lactic acid production, while α‐amylase and yeast extract had insignificant effects. The optimal conditions were found to be an α‐amylase activity of 13.86 U g^?1 dried food waste, a protease activity of 2.12 U g^?1 dried food waste, a temperature of 29.31 °C and a CaCO₃ concentration of 62.67 g L^?1, which resulted in a maximum lactic acid concentration of 98.51 g L^?1 (88.75% yield). An increase in inoculum size would be appropriate for accelerating the depletion of initial soluble carbohydrate to enhance the efficiency of α‐amylase in dining‐hall food waste fermentation. CONCLUSION: A suitable regression model for lactic acid production was developed based on the experimental results. Dining‐hall food waste was found to be a good substrate for lactic acid fermentation with high product yield and without nutrient supplementation. Copyright © 2008 Society of Chemical Industry     

Kinetic modelling of lactic acid production from whey by Lactobacillus casei (NRRL B‐441)

The biomass growth, lactic acid production and lactose utilisation kinetics of lactic acid production from whey by Lactobacillus casei was studied. Batch fermentation experiments were performed at controlled pH and temperature with six different initial whey lactose concentrations (9‐77 g dm^?3) in a 3 dm³ working volume bioreactor. Biomass growth was well described by the logistic equation with a product inhibition term. In addition, biomass and product inhibition effects were defined with corresponding power terms, which enabled adjustment of the model for low‐ and high‐substrate conditions. The Luedeking‐Piret equation defined the product formation kinetics. Substrate consumption was explained by production rate and maintenance requirements. A maximum productivity of 2.5 g dm^?3 h^?1 was attained with an initial lactose concentration of 35.5 g dm^?3. Copyright © 2006 Society of Chemical Industry     

Inulinase bio‐production using agroindustrial residues: screening of microorganisms and process parameters optimization

BACKGROUND: The use of agroindustrial residues as substrate for inulinase bio‐production is a good choice to reduce production costs, since enzyme activity will be improved and the downstream step of the process will be viable technically and economically. In addition, the screening of microorganisms that are able to overproduce inulinase using these substrates is fundamental to guarantee successful medium substitution. Based on these considerations, the objective of this present work was to select different strains of yeasts of the genus Kluyveromyces to produce the inulinase. RESULTS: Initially, 10 strains were tested in synthetic medium and six of these selected to perform tests in agroindustrial medium. Screening showed that the strains NCYC 587 and NRRL Y‐7571 were able to produce the enzyme using agroindustrial residues as substrate. Optimization of inulinase activity as a function of pH, and concentrations of molasses, corn steep liquor (CSL) and yeast extract (YE) was carried out for the two strains. The maximum inulinase activity under optimized conditions was about 750 U mL^?1. CONCLUSION: These values are approximately seven times greater than the values obtained using synthetic medium, showing the technical viability of the use agroindustrial residues for the inulinase production. Copyright © 2009 Society of Chemical Industry     

Optimization of the production of β‐carotene from molasses by Blakeslea trispora: a statistical approach

The effect of pretreatment of molasses, nitrogen sources, natural oils, fatty acids, antioxidant, precursors, and mixtures of the above substances on β‐carotene production by Blakeslea trispora in shake flask culture was investigated. Also, a central composite design was employed to determine the maximum β‐carotene concentration at optimum values for the process variables (linoleic acid, kerosene, antioxidant). The highest concentration of the carotenoid pigment was obtained in molasses solution treated with invertase. Corn steep liquor and yeast extract at concentrations of 5.0% and 0.5% (w/v), respectively, increased slightly the concentration of β‐carotene, while the natural oils, fatty acids, and precursors (except kerosene) did not improve the production of pigment when they were added separately to the medium. On the other hand, the mixture of linoleic acid, kerosene and antioxidant increased significantly the concentration of β‐carotene. The fit of the model was found to be good. Linoleic acid, kerosene and antioxidant had a strong linear effect on β‐carotene concentration. The concentration of β‐carotene was significantly affected by linoleic acid–antioxidant and kerosene–antioxidant interactions as well as by the negative quadratic effects of these variables. The interaction between linoleic acid–kerosene had no significant linear effect. Maximum β‐carotene concentration (790.0 mg dm^?3) was obtained in culture grown in molasses solution supplemented with linoleic acid (30.74 g dm^?3), kerosene (27.79 g dm^?3) and antioxidant (10.22 g dm^?3). © 2002 Society of Chemical Industry     

L(+)‐Lactic acid production using poly(vinyl alcohol)‐cryogel‐entrapped Rhizopus oryzae fungal cells

A new immobilized biocatalyst based on Rhizopus oryzae fungal cells entrapped in poly(vinyl alcohol)‐cryogel was evaluated in both the batch and semi‐batch processes of L (+)‐lactic acid (LA) production, when glucose, acid hydrolysates of starch or gelatinized potato starch were used as the main substrates. Under the batch conditions, the immobilized biocatalyst developed produced LA with yields of 94% and 78% from glucose and acid starch hydrolysates, respectively. Semi‐batch conditions enabled product yields of 52% and 45% to be obtained with the corresponding substrates. The highest process productivity (up to 173 g L^?1) was reached under semi‐batch conditions. Potato starch (5–70 g L^?1) was also transformed into lactic acid by immobilized R. oryzae. It was shown that long‐term operation of the immobilized biocatalyst (for at least 480 h) produced a low decrease in metabolic activity. Copyright © 2006 Society of Chemical Industry     

Optimization of lactic acid production from whey by L casei NRRL B‐441 immobilized in chitosan stabilized Ca‐alginate beads

The production of lactic acid from whey by Lactobacillus casei NRRL B‐441 immobilized in chitosan‐stabilized Ca‐alginate beads was investigated. Higher lactic acid production and lower cell leakage were observed with alginate–chitosan beads compared with Ca‐alginate beads. The highest lactic acid concentration (131.2 g dm^?3) was obtained with cells entrapped in 1.3–1.7 mm alginate–chitosan beads prepared from 2% (w/v) Na‐alginate. The gel beads produced lactic acid for five consecutive batch fermentations without marked activity loss and deformation. Response surface methodology was used to investigate the effects of three fermentation parameters (initial sugar, yeast extract and calcium carbonate concentrations) on the concentration of lactic acid. Results of the statistical analysis showed that the fit of the model was good in all cases. Initial sugar, yeast extract and calcium carbonate concentrations had a strong linear effect on lactic acid production. The maximum lactic acid concentration of 136.3 g dm^?3 was obtained at the optimum concentrations of process variables (initial sugar 147.35 g dm^?3, yeast extract 28.81 g dm^?3, CaCO₃ 97.55 g dm^?3). These values were obtained by fitting of the experimental data to the model equation. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in lactic acid production using alginate–chitosan‐immobilized cells. Copyright © 2005 Society of Chemical Industry     

The effect of raw glycerol concentration on the production of 1,3‐propanediol by Clostridium butyricum

Raw glycerol, the main by‐product of the bio‐diesel production process, was converted to 1,3‐propanediol by Clostridium butyricum F2b. In batch cultures, 47.1 g dm^?3 of 1,3‐propanediol were produced. Continuous cultures were conducted at a constant dilution rate (= 0.04 h^?1) and various inlet glycerol concentrations with 1,3‐propanediol produced at levels up to 44.0 g dm^?3. At increasing glycerol concentrations in the inlet medium, biomass yield decreased. This decrease was attributed to the microbial metabolism being directed towards the biosynthesis of organic acids (and hence carbon losses as CO₂) instead of biochemical anabolic reactions. An autonomous analytical model was developed, and quantified the effect of inlet glycerol concentration on the production of biomass and metabolites. Indeed, high inlet substrate concentrations positively affected the biosynthesis, principally of butyric acid and to a lesser extent that of acetic acid. In contrast, at increased glycerol concentrations, the relative increase of 1,3‐propanediol production per unit of substrate consumed was lower as compared with that of acetic and, mainly, butyric acid. This could be explained by the fact that the butyric acid pathway represents an alternative and competitive one to that of 1,3‐propanediol for re‐generation of NADH₂ equivalents in the microbial cell. Copyright © 2004 Society of Chemical Industry     

Volatile fatty acid production during anaerobic mesophilic digestion of solid potato waste

The production of volatile fatty acids by anaerobic digestion of solid potato waste was investigated using a batch solid waste reactor with a working capacity of 2 dm^?3 at 37°C. Solid potato waste was packed into the digester and the organic content of the waste was released by microbial activity by circulating water over the bed, using batch loads of 500 g or 1000 g potato waste. The sequence of appearance of the volatile fatty acids was (acetic, propionic); (n‐butyric); (n‐valeric, iso‐valeric, caproic); (iso‐butyric). After 300 h digestion of potato waste on a small scale, the fermentation products were chiefly (mg g^?1 total VFAs): acetic acid (420), butyric acid (310), propionic acid (140) and caproic acid (90), with insignificant amounts of iso‐butyric acid, n‐valeric and iso‐valeric acids. When the load of potato solids was increased, the volatile fatty acid content was similar, but butyric acid constituted 110 mg g^?1 and lactic acid 400 mg g^?1 of the total volatile fatty acids. The maximum soluble chemical oxygen demand (COD) achieved under the experimental conditions used was 27 and 37 g COD dm^?3 at low and high loadings of potato solids, respectively. The total volatile fatty acids reached 19 g dm^?3 of leachate at both loads of potato solid waste. Gas production was negligible, indicating that methanogenic activity was effectively inhibited. Copyright © 2004 Society of Chemical Industry     

Strategies to improve the bioconversion of processed wood into lactic acid by simultaneous saccharification and fermentation

In the one‐step conversion of wood into lactic acid by Simultaneous Saccharification and Fermentation (SSF), inhibition effects caused by hydrolysis‐ and fermentation‐derived compounds on both enzymatic activity and fermentative ability of microorganisms appear when the operation is carried out under conditions leading to high productivities. The main effects inhibiting SSF have been assessed, and the results obtained in fed‐batch experiments allowed the definition of strategies for improving the overall bioconversion process. As cellobiose caused significant inhibition of cellulases, the supplementation of media with β‐glucosidase resulted in improved kinetics and yields. The inhibition of both enzymatic activity and microbial metabolism by lactic acid was confirmed. Intermittent removal of lactic acid by passing the fermentation media through an anion‐exchange resin column resulted in increased productivities and yields. Improved conversion of pretreated wood into lactic acid (67% conversion of cellulose into lactic acid, with maximum lactic acid concentration of 108 g dm^?3 and a productivity of 0.94 g dm^?3 h^?1) was achieved combining multiple substrate addition, supplementation with fresh nutrients and enzymes and removal of lactic acid. © 2001 Society of Chemical Industry     

Strain isolation and optimization of process parameters for bioconversion of glycerol to lactic acid

BACKGROUND: The crude glycerol from biodiesel production represents an abundant and inexpensive source which can be used as raw material for lactic acid production. The first aim of this investigation was to select a strain suitable for producing lactic acid from glycerol with a high concentration and productivity. The second aim was to obtain the optimum fermentation conditions, as a basis for large‐scale lactate production in the future. RESULTS: Eight bacterial strains, which could aerobically convert glycerol to lactic acid, were screened from soil samples. One of the strains, AC‐521, which synthesized lactic acid with a higher concentration, was identified based on its 16S rDNA sequences and physiological characteristics. These results indicated that this strain was a member of Escherichia coli. The optimal fermentation conditions for Escherichia coli AC‐521 were 42 °C, pH 6.5, 0.85 min^?1 (K_La). CONCLUSION: Escherichia coli AC‐521 suitable for producing lactic acid from glycerol with high concentration and productivity was identified. After 88 h of fed‐batch fermentation, both the lactic acid concentration and glycerol consumption reached maximum, giving 85.8 g L^?1 of lactic acid with a productivity of 0.97 g L^?1 h^?1 and a yield of 0.9 mol mol^?1 glycerol. Copyright © 2009 Society of Chemical Industry     

Effective biosynthesis of poly(3‐hydoxy‐ butyrate‐co‐4‐hydroxybutyrate) with high 4‐hydroxybutyrate fractions by Wautersia eutropha in the presence of α‐amino acids

BACKGROUND: Biopolymers produced by microbes are in demand as their biodegradable and biocompatible properties make them suitable for disposable products and for potential use as biomaterials for medical applications. The effective microbial production of copolyesters of 3‐hydroxybutyrate (3HB) and 4‐hydroxybutyrate(4HB) with high molar fractions of 4HB unit by a wild‐type Wautersia eutropha H16 was investigated in culture media containing 4‐hydroxybutyric acid (4HBA) and different carbon substrates in the presence of various α‐amino acids. RESULTS: The addition of carbon sources such as glucose, fructose and acetic acid to the culture medium containing 4HBA in the presence of α‐amino acids resulted in the production of random poly(3HB‐co‐4HB) with compositions of up to 77 mol% 4HB unit, but the yields of copolyesters with 60–77 mol% 4HB units were less than 15 wt% of dried cell weights. In contrast, when carbon sources such as propionic acid and butyric acid were used as the co‐substrates of 4HBA in the presence of α‐amino acids, poly(3HB‐co‐4HB) copolyesters with compositions of 72–86 mol% 4HB were produced at maximally 47.2 wt% of dried cell weight (11.3 g L^?1) and the molar conversion yield of 4HBA to 4HB fraction in copolyesters was as high as 31.4 mol%. Further, poly(3HB‐co‐4HB) copolyesters with compositions of 93–96 mol% 4HB were isolated at up to 35.2 wt% of dried cell weights by fractionation of the above copolymers with chloroform/n‐hexane. CONCLUSION: The productivity of copolyesters with over 80 mol% 4HB fractions was as high as 0.146 g L^?1 h^?1 (3.51 g L^?1 for 24 h) by flask batch cultivation. Copyright © 2007 Society of Chemical Industry