Development of a cost-effective glucose-corn steep medium for production of butanol by Clostridium beijerinckii

Corn steep water (CSW) medium (1.6% solids plus 6% glucose) was evaluated for growth and butanol production by Clostridium beijerinckii NCIMB 8052 wild-type and hyper-amylolytic, hyper-butanol-producing mutant strain BA101. CSW alone was not a suitable substrate, whereas addition of glucose supported growth and butanol production by both strains. In a batch-scale fermentation using an optimized 6% glucose-1.6% solids CSW medium, C. beijerinckii NCIMB 8052 and strain BA101 produced 10.7 g L⁻¹ and 14.5 g L⁻¹ of butanol, respectively. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and strain BA101 were 14 g L⁻¹ and 20 g L⁻¹, respectively. Initial fermentation in small-scale flasks containing 6% maltodextrin-1.6% solids concentration CSW medium resulted in 6 g L⁻¹ and 12.6 g L⁻¹ of butanol production by C. beijerinckii NCIMB 8052 and strain BA101, respectively. CSW can serve as an economic source of nitrogen, vitamins, amino acids, minerals, and other nutrients. Thus, it is feasible to use 6% glucose-1.6% solids CSW medium in place of semi-defined P2 medium. Received 9 February 1998/ Accepted in revised form 1 September 1998     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Production of Cold-Adapted Amylase by Marine Bacterium <Emphasis Type="Italic">Wangia</Emphasis> sp. C52: Optimization,Modeling, and Partial Characterization

The aim of this work was to optimize the fermentation parameters in the shake-flask culture of marine bacterium Wangia sp. C52 to increase cold-adapted amylase production using two statistical experimental methods including Plackett–Burman design, which was applied to find the key ingredients for the best medium composition, and response surface methodology, which was used to determine the optimal concentrations of these components. The results showed starch, tryptone, and initial pH had significant effects on the cold-adapted amylase production. A central composite design was then employed to further optimize these three factors. The experimental results indicated that the optimized composition of medium was 6.38 g L⁻¹ starch, 33.84 g L⁻¹ tryptone, 3.00 g L⁻¹ yeast extract, 30 g L⁻¹ NaCl, 0.60 g L⁻¹ MgSO₄ and 0.56 g L⁻¹ CaCl₂. The optimized cultivation conditions for amylase production were pH 7.18, a temperature of 20°C, and a shaking speed of 180 rpm. Under the proposed optimized conditions, the amylase experimental yield (676.63 U mL⁻¹) closely matched the yield (685.60 U mL⁻¹) predicted by the statistical model. The optimization of the medium contributed to tenfold higher amylase production than that of the control in shake-flask experiments.     

Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis>

Butanol, a four-carbon primary alcohol (C₄H₁₀O), is an important industrial chemical and has a good potential to be used as a superior biofuel. Bio-based production of butanol from renewable feedstock is a promising and sustainable alternative to substitute petroleum-based fuels. Here, we report the development of a process for butanol production from glycerol, which is abundantly available as a byproduct of biodiesel production. First, a hyper butanol producing strain of Clostridium pasteurianum was isolated by chemical mutagenesis. The best mutant strain, C. pasteurianum MBEL_GLY2, was able to produce 10.8 g l⁻¹ butanol from 80 g l⁻¹ glycerol as compared to 7.6 g l⁻¹ butanol produced by the parent strain. Next, the process parameters were optimized to maximize butanol production from glycerol. Under the optimized batch condition, the butanol concentration, yield, and productivity of 17.8 g l⁻¹, 0.30 g g⁻¹, and 0.43 g l⁻¹ h⁻¹ could be achieved. Finally, continuous fermentation of C. pasteurianum MBEL_GLY2 with cell recycling was carried out using glycerol as a major carbon source at several different dilution rates. The continuous fermentation was run for 710 h without strain degeneration. The acetone–butanol–ethanol productivity and the butanol productivity of 8.3 and 7.8 g l⁻¹ h⁻¹, respectively, could be achieved at the dilution rate of 0.9 h⁻¹. This study reports continuous production of butanol with reduced byproducts formation from glycerol using C. pasteurianum, and thus could help design a bioprocess for the improved production of butanol.     

Continuous production of isopropanol and butanol using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> DSM 6423

Clostridium beijerinckii DSM 6423 was studied using different continuous production methods to give maximum and stable production of isopropanol and n-butanol. In a single-stage continuous culture, when wood pulp was added as a cell holding material, we could increase the solvent productivity from 0.47 to 5.52 g L⁻¹ h⁻¹ with the yield of 54% from glucose. The overall solvent concentration of 7.51 g L⁻¹ (39.4% isopropanol and 60.6% n-butanol) with the maximum solvent productivity of 0.84 g L⁻¹ h⁻¹ was obtained with two-stage continuous culture. We were able to run the process for more than 48 overall retention times without losing the ability to produce solvents.     

Bioreduction of Cu(II) by Cell-Free Copper Reductase from a Copper Resistant <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NA

Environmental copper contamination is a serious human health problem. Copper reductase is produced by microorganisms to facilitate copper uptake by ATPases into the cells increasing copper biosorption. This study assessed the reduction of Cu(II) by cell-free extracts of a highly copper-resistant bacterium, Pseudomonas sp. strain NA, isolated from vineyard soil contaminated with copper. Both intact cells and cell-free extract of Pseudomonas sp. strain NA displayed substantial reduction of Cu(II). Intact cells reduced more then 80 mg L⁻¹ of Cu(II) from medium amended with 200 mg L⁻¹ of copper after 24 h of incubation. Cell-free extract of the isolate reduced more than 65% of the Cu(II) at initial copper concentration of 200 mg L⁻¹ after 24 h. Soluble protein production was high at 72 h of incubation at 100 mg L⁻¹ of copper, with more then 60 μg L⁻¹ of total soluble protein in cell-free extract recorded. Cu(II) reduction by isolate NA was increased when copper concentration increased for both intact cells and cell-free extract. Results indicate that Pseudomonas sp. strain NA produces copper reductase enzyme as the key mechanism of copper biotransformation.     

Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris

We tested 10 different Chlorella and Parachlorella strains under lipid induction growth conditions in autotrophic laboratory cultures. Between tested strains, substantial differences in both biomass and lipid productivity as well as in the final content of lipids were found. The most productive strain (Chlorella vulgaris CCALA 256) was subsequently studied in detail. The availability of nitrates and/or phosphates strongly influenced growth and accumulation of lipids in cells by affecting cell division. Nutrient limitation substantially enhanced lipid productivity up to a maximal value of 1.5 g l⁻¹ day⁻¹. We also demonstrated the production of lipids through large-scale cultivation of C. vulgaris in a thin layer photobioreactor, even under suboptimal conditions. After 8 days of cultivation, maximal lipid productivity was 0.33 g l⁻¹ day⁻¹, biomass density was 5.7 g l⁻¹ dry weight and total lipid content was more than 30% dry weight. C. vulgaris lipids comprise fatty acids with a relatively high degree of saturation compared with canola oil offering a possible alternative to the use of higher plant oils.     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Reaction engineering studies for the production of 2-hydroxyisobutyric acid with recombinant Cupriavidus necator H 16

Recombinant Cupriavidus necator H 16 with a novel metabolic pathway using a cobalamin-dependent mutase was exploited to produce 2-hydroxyisobutyric acid (2-HIBA) from renewable resources through microbial fermentation. 2-HIBA production capacities of different strains of C. necator H 16 deficient in the PHB synthase gene and genetically engineered to enable the production of 2-HIBA from the intracellular PHB precursor (R)-3-hydroxybutyryl-CoA were evaluated in 48 parallel milliliter-scale stirred tank bioreactors (V = 11 mL). The effects of media composition, limitations, pH, and feed rate were studied with respect to the overall process performances of the different recombinant strains. 2-HIBA production was at a maximum at nitrogen limiting conditions and if the pH was controlled between 6.8 and 7.2 under fed-batch operating conditions (intermittent fructose addition). The final concentration of 2-HIBA was 7.4 g L⁻¹ on a milliliter scale. Best reaction conditions identified on the milliliter scale were transferred to a laboratory-scale fed-batch process in a stirred tank bioreactor (V = 2 L). Two different process modes for the production of 2-HIBA, a single-phase and a dual-phase fermentation procedure, were evaluated and compared on a liter scale. The final concentration of 2-HIBA was 6.4 g L⁻¹ on a liter scale after 2 days of cultivation.     

Ethanol adaptation in a thermotolerant yeast strain Kluyveromyces marxianus IMB3

The maximum ethanol concentration produced from glucose in defined media at 45°C by the thermotolerant yeast Kluyveromyces marxianus IMB3 was 44 g L⁻¹. Acclimatisation of the strain through continuous culture at ethanol concentrations up to 80 g L⁻¹, shifted the maximum ethanol concentration at which growth was observed from 40 g L⁻¹ to 70 g L⁻¹. Four isolates were selected from the continuous culture, only one of which produced a significant increase in final ethanol concentration (50 ± 0.4 g L⁻¹), however in subsequent fermentations, following storage on nutrient agar plates, the maximum ethanol concentration was comparable with the original isolate. The maximum specific ethanol production rates (approximately 1.5 g (gh)⁻¹) were also comparable with the original strain except for one isolate (0.7 g (gh)⁻¹). The specific ethanol productivity decreased with ethanol concentration; this decrease correlated linearly (rval 0.92) with cell viability. Due to the transience of induced ethanol tolerance in the strain it was concluded that this was not a valid method for improving final ethanol concentrations or production rates. Received 18 July 1997/ Accepted in revised form 19 February 1998     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

Continuous bio-catalytic conversion of sugar mixture to acetone–butanol–ethanol by immobilized <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> DSM 792

Continuous production of acetone, n-butanol, and ethanol (ABE) was carried out using immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and sugar mixture as a substrate. Among various lignocellulosic materials screened as a support matrix, coconut fibers and wood pulp fibers were found to be promising in batch experiments. With a motive of promoting wood-based bio-refinery concept, wood pulp was used as a cell holding material. Glucose and sugar mixture (glucose, mannose, galactose, arabinose, and xylose) comparable to lignocellulose hydrolysate was used as a substrate for continuous production of ABE. We report the best solvent productivity among wild-type strains using column reactor. The maximum total solvent concentration of 14.32 g L⁻¹ was obtained at a dilution rate of 0.22 h⁻¹ with glucose as a substrate compared to 12.64 g L⁻¹ at 0.5 h⁻¹ dilution rate with sugar mixture. The maximum solvent productivity (13.66 g L⁻¹ h⁻¹) was obtained at a dilution rate of 1.9 h⁻¹ with glucose as a substrate whereas solvent productivity (12.14 g L⁻¹ h⁻¹) was obtained at a dilution rate of 1.5 h⁻¹ with sugar mixture. The immobilized column reactor with wood pulp can become an efficient technology to be integrated with existing pulp mills to convert them into wood-based bio-refineries.     

Production of pig liver esterase in batch fermentation of E. coli Origami

The establishment of a fermentation process for the production of pig liver esterase (PLE) in high yields is necessary for industrial applications. In our previous studies, we reported the recombinant expression of PLE in Escherichia coli Origami™ (DE3) in shake flask. Only a coexpression with chaperones GroEL/ES allowed the production of soluble and active enzyme. The optimization of the cultivation conditions, such as temperature, inducer concentrations, or media compositions to increase enzyme yield in a fermentation process is described here. Using fed-batch fermentation cell densities up to OD = 50 were obtained, but almost no active enzyme was expressed. Only batch fermentation was found suitable for production of active pig liver esterase and cell densities between OD = 7–13 and activities of 300–400 U L⁻¹ for isoenzyme PLE-1 (γPLE) and 1,400 U L⁻¹ for PLE-5 were obtained after 22 h total cultivation time or 18 h after induction of PLE expression, respectively.     

Continuous cultures of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> mt-2 overcome catabolic function loss under real case operating conditions

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h⁻¹ under toluene loading rates of 259 ± 23 and 801 ± 78 g m⁻³ h⁻¹ (inlet toluene concentrations of 3.5 and 10.9 g m⁻³, respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol⁻ population at day 4 to approx. 100% Tol⁺ cells from day 13 onward, sustaining elimination capacities of 232 ± 10 g m⁻³ h⁻¹ at 3.5 g Tol m⁻³ and 377 ± 13 g m⁻³ h⁻¹ at 10.9 g Tol m⁻³, which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol⁻ variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique

An efficient procedure has been developed for inducing somatic embryogenesis and regeneration of plants from tissue cultures of oil palm (Elaeis guineensis Jacq.). Thin transverse sections (thin cell layer explants) of different position in the shoot apex and leaf sheath of oil palm were cultivated in Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented with 0–450 μM picloram and 2,4-D with 3.0% sucrose, 500 mg L⁻¹ glutamine, and 0.3 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel. Embryogenic calluses were evaluated 12 wk after inoculation. Picloram (450 μM) was effective in inducing embryogenic calluses in 41.5% of the basal explants. Embryogenic calluses were maintained on a maturation medium composed of basal media, plus 0.6 μM NAA and 12.30 μM 2iP, 0.3 g L⁻¹ activated charcoal, and 500 mg L⁻¹ glutamine, with subcultures at 4-wk intervals. Somatic embryos were converted to plants on MS medium with macro- and micronutrients at half-strength, 2% sucrose, and 1.0 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel.     

Production of xylitol by Candida peltata

Candida peltata NRRL Y-6888 to ferment xylose to xylitol was evaluated under different fermentation conditions such as pH, temperature, aeration, substrate concentration and in the presence of glucose, arabinose, ethanol, methanol and organic acids. Maximum xylitol yield of 0.56 g g⁻¹ xylose was obtained when the yeast was cultivated at pH 6.0, 28°C and 200 rpm on 50 g L⁻¹ xylose. The yeast produced ethanol (0.41 g g⁻¹ in 40 h) from glucose (50 g L⁻¹) and arabitol (0.55 g g⁻¹ in 87 h) from arabinose (50 g L⁻¹). It preferentially utilized glucose > xylose > arabinose from mixed substrates. Glucose (10 g L⁻¹), ethanol (7.5 g L⁻¹) and acetate (5 g L⁻¹) inhibited xylitol production by 61, 84 and 68%, respectively. Arabinose (10 g L⁻¹) had no inhibitory effect on xylitol production. Received 24 December 1998/ Accepted in revised form 18 March 1999     

Application of oscillation for efficiency improvement of continuous ethanol fermentation with Saccharomyces cerevisiae under very-high-gravity conditions

Compared with steady state, oscillation in continuous very-high-gravity ethanol fermentation with Saccharomyces cerevisiae improved process productivity, which was thus introduced for the fermentation system composed of a tank fermentor followed by four-stage packed tubular bioreactors. When the very-high-gravity medium containing 280 g l⁻¹ glucose was fed at the dilution rate of 0.04 h⁻¹, the average ethanol of 15.8% (v/v) and residual glucose of 1.5 g l⁻¹ were achieved under the oscillatory state, with an average ethanol productivity of 2.14 g h⁻¹ l⁻¹. By contrast, only 14.8% (v/v) ethanol was achieved under the steady state at the same dilution rate, and the residual glucose was as high as 17.1 g l⁻¹, with an ethanol productivity of 2.00 g h⁻¹ l⁻¹, indicating a 7% improvement under the oscillatory state. When the fermentation system was operated under the steady state at the dilution rate of 0.027 h⁻¹ to extend the average fermentation time to 88 h from 59 h, the ethanol concentration increased slightly to 15.4% (v/v) and residual glucose decreased to 7.3 g l⁻¹, correspondingly, but the ethanol productivity was decreased drastically to 1.43 g h⁻¹ l⁻¹, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h⁻¹.     

The viability to a wall shear stress and propagation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> in the intensive membrane bioreactor

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.     

The effect of complex carbon and nitrogen, salt, Tween-80 and acetate on delta-endotoxin production by a Bacillus thuringiensis subsp kurstaki

Delta-endotoxin production by a strain of Bacillus thuringiensis subsp kurstakion complex media based on crude gruel and fish meal was investigated. High proteolytic activities were concomitantly produced with the bioinsecticide. In such complex media, the repressive regulation due to readily consumed carbon sources was partially overcome. In order to improve substrate assimilation, 0.5 g L⁻¹ sodium chloride and 0.1% Tween-80 were supplemented to the production medium, increasing delta-endotoxin yields when using gruel concentrations below 59 g L⁻¹. At and beyond 75 g L⁻¹ gruel, delta-endotoxin yields were not affected in the presence of 0.5 g L⁻¹ NaCl and 0.1% Tween-80, but proteolytic activity yields were remarkably reduced. Thus, the use of sodium chloride and Tween-80 allowed reduction of the initial gruel concentration to 42 g L⁻¹ for the production of 3350 mg L⁻¹ delta-endotoxin, while it was only 3800 mg L⁻¹ with 92 g L⁻¹ gruel. Moreover, similar to 0.5 g L⁻¹ NaCl and 0.1% Tween-80, the use of 10 g L⁻¹ sodium acetate significantly improved delta-endotoxin production and also reduced the proteolytic activity to 250 U ml⁻¹. Received 05 November 1998/ Accepted in revised form 19 August 1999