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     

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     

Laccase and Manganese Peroxidase Activities of Phellinus Robustus and Ganoderma Adspersum Grown on Food Industry Wastes in Submerged Fermentation

Phellinus robustus produced both laccase (700–4,000 U l⁻¹) and manganese peroxidase (MnP) (1,000–11,300 U l⁻¹) in fermentation of nine food wastes, whereas Ganoderma adspersum produced only laccase (600–34,000 U l⁻¹). Glucose provided high laccase and MnP activity of P. robustus but repressed enzyme production by G. adspersum. Ammonium sulphate and ammonium tartrate increased the P. robustus laccase yield (3-fold), whereas the accumulation of MnP was not enhanced by additional nitrogen.     

Production of curdlan using sucrose or sugar cane molasses by two-step fed-batch cultivation of Agrobacterium species

Maltose and sucrose were efficient carbon sources for the production of curdlan by a strain of Agrobacterium sp. A two-step, fed-batch operation was designed in which biomass was first produced, followed by curdlan production which was stimulated by nitrogen limitation. There exists an optimal timing for nitrogen limitation for curdlan production in the two-step, fed-batch operation. Maximum curdlan production (60 g L⁻¹) was obtained from sucrose with a productivity of 0.2 g L⁻¹ h⁻¹ when nitrogen was limited at a cell concentration of 16.0 g L⁻¹. It was also noted that the curdlan yield from sucrose was as high as 0.45 g curdlan g⁻¹ sucrose, and the highest specific production rate was 1.0 g curdlan g⁻¹ cells h⁻¹ right after nitrogen limitation. Of particular importance was the use of molasses as a cheap carbon source to produce curdlan in the two-step, fed-batch cultivation. As high as 42 g L⁻¹ of curdlan with a yield of 0.35 g curdlan g⁻¹ total sugar was obtained after 120 h of fed-batch cultivation. Received 20 August 1996/ Accepted in revised form 26 November 1996     

Kinetic models for phycocyanin production by high cell density mixotrophic culture of the microalga Spirulina platensis

Phycocyanin production by high cell density cultivation of Spirulina platensis in batch and fed-batch modes in 3.7-L bioreactors with a programmed stepwise increase in light intensity program was investigated. The results showed that the cell density in fed-batch culture (10.2 g L⁻¹) was 4.29-fold that in batch culture (2.38 g L⁻¹), and the total phycocyanin production in the fed-batch culture (0.795 g L⁻¹) was 3.05-fold that in the batch culture (0.261 g L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, phycocyanin formation, as well as glucose consumption was proposed. The data fitted the models well (r ² > 0.99). Furthermore, based on the kinetic models, the potential effects of light limitation and photoinhibition on cell growth and phycocyanin formation can be examined in depth. The models demonstrated that the optimal light intensity for mixotrophic growth of Spirulina platensis in batch or fed-batch cultures using a 3.7-L bioreactor was 80160 μE m⁻² s⁻¹, and the stepwise increase in light intensity can be replaced by a constant light intensity mode. Received 28 July 1998/ Accepted in revised form 8 October 1998     

A simple and rapid method to gain high amounts of manganese peroxidase with immobilized mycelium of the agaric white-rot fungus Clitocybula dusenii

The agaric basidiomycete Clitocybula dusenii was used for the production of the extracellular ligninolytic enzyme, manganese (Mn) peroxidase. An immobilization technique is described using cellulose and polypropylene as carrier for the fungal mycelium. High amounts of Mn peroxidase were obtained with agitated cultures of immobilized fungus (up to 3,000 U l⁻¹) while the biomass was recovered and used for further production cycles. Purification of Mn peroxidase revealed the existence of two forms: MnP1 (molecular mass 43 kDa, pI 4.5) and MnP2 (42 kDa, pI 3.8). Received: 30 July 1999 / Received revision: 1 December 1999 / Accepted: 3 December 1999     

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.     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999     

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     

Characterisation of a novel thermotolerant yeast, Kluyveromyces marxianus var marxianus: development of an ethanol fermentation process

The fermentation characteristics of the novel, thermotolerant, isolate Kluyveromyces marxianus var marxianus were determined to evaluate its aptitude for use in an ethanol production process. Sustainable growth was not observed under anaerobic conditions, even in the presence of unsaturated fatty acid and sterol. A maximum ethanol concentration of 40 g L⁻¹ was produced at 45°C, with an initial specific ethanol production rate of 1.7 g g⁻¹ h⁻¹. This was observed at ethanol concentrations below 8 g L⁻¹ and under oxygen-limited conditions. The low ethanol tolerance and low growth under oxygen-limited conditions required for ethanol production implied that a simple continuous process was not feasible with this yeast strain. Improved productivity was achieved through recycling biomass into the fermenter, indicating that utilising an effective cell retention method such as cell recycle or immobilisation, could lead to the development of a viable industrial process using this novel yeast strain. Received 14 February 1998/ Accepted in revised form 19 May 1998     

Evaluation of sugar cane hemicellulose hydrolyzate for cultivation of yeasts and filamentous fungi

Sugar cane bagasse hemicellulosic fraction submitted to hydrolytic treatment with 100 mg of sulfuric acid per gram of dry mass, at 140°C for 20 min, was employed as a substrate for microbial protein production. Among the 22 species of microorganisms evaluated, Candida tropicalis IZ 1824 showed TRS consumption rate of 89.8%, net cell mass of 11.8 g L⁻¹ and yield coefficient (Y_x/s) of 0.50 g g⁻¹. The hydrolyzate supplemented with rice bran (20.0 g L⁻¹), P₂O₅ (2.0 g L⁻¹) and urea (2.0 g L⁻¹) provided a TRS consumption rate of 86.3% and a cell mass of 8.4 g L⁻¹. At pH 4.0 cellular metabolism was inhibited, whereas at pH 6.0 the highest yield was obtained. The presence of furfural (2.0 g L⁻¹) hydroxymethylfurfural (0.08 g L⁻¹) and acetic acid (3.7 g L⁻¹) in the hydrolyzate did not interfere with cultivation at pH 6.0. Received 25 October 1996/ Accepted in revised form 10 March 1997     

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.     

Comparison of exopolysaccharide production by strains of Lactobacillus rhamnosus and Lactobacillus paracasei grown in chemically defined medium and milk

Exopolysaccharide (EPS) production was compared among three strains of lactobacilli. Lactobacillus rhamnosus strain 9595M can be classified among the highest EPS-producing strains of lactic acid bacteria reported to date with a maximum EPS production of 1275 mg L⁻¹. Under controlled pH, no significant differences in the quantity of EPS produced could be detected between carbon source (glucose or lactose) or fermentation temperature (32 or 37°C). In milk, strains ATCC 9595M and R produced more than 280 mg L⁻¹ EPS whereas strain Type V produced less than 80 mg L⁻¹ EPS. Journal of Industrial Microbiology & Biotechnology (2000) 24, 251–255. Received 10 September 1999/ Accepted in revised form 22 December 1999     

Docosapentaenoic acid (C22:5, ω-3) production by Pythium acanthicum

The physiological roles of omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid have been investigated in detail and microbial strains producing these polyunsaturated fatty acids have been characterised. It has recently been suggested that docosapentaenoic acid may have an important role, especially in infant nutrition, and that its positive health effects have been overlooked. Docosapentaenoic acid (C22:5, ω-3) production by a strain of Pythium acanthicum ATCC 18660 was thus investigated. Optimum conditions for growth of P. acanthicum ATCC 18660 and docosapentaenoic acid production were: pH 6.0, temperature 20°C and incubation time, 10 days. Among different saccharides and complex nitrogen sources tested, glucose and sodium glutamate were preferred carbon and nitrogen sources, respectively. Maximum biomass content (10.4 g L⁻¹) and docosapentaenoic acid yield (49.9 mg L⁻¹) were obtained in 10 days. An increase in docosapentaenoic acid volumetric yields to 108–110 mg L⁻¹ was obtained when linseed oil was used to supplement glucose or soy flour-containing medium. Batch feeding of additional glucose or linseed oil further enhanced the docosapentaenoic acid volumetric yield to 132 mg L⁻¹ and 125 mg L⁻¹, respectively, in 14 days. The specific production of docosapentaenoic acid in preliminary experiments ranged from 1.0–5.0 mg g⁻¹ biomass. As conditions were optimised, docosapentaenoic acid specific production titers were generally in the range of 4.0–5.5 mg g⁻¹ and increases in docosapentaenoic acid volumetric production could be attributed to increased biomass production. The limited improvement obtained by modifying culture conditions indicates that increasing volumetric yields of docosapentaenoic acid by modifying culture conditions appears to represent a significant barrier to commercialisation of such a process and suggests a more fundamental manipulation of metabolism and physiology is required. Received 06 November 1997/ Accepted in revised form 10 January 1998     

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 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.     

Use of Acadian marine plant extract powder from Ascophyllum nodosum in tissue culture of Kappaphycus varieties

Three varieties of Kappaphycus alvarezii (Kapilaran, KAP), Tambalang purple (PUR), Adik-adik (AA), and one variety of Kappaphycus striatum var. sacol (green sacol (GS) were used to determine the efficiency of Acadian marine plant extract powder (AMPEP) as a culture medium at different concentrations, for the regeneration of young plants of Kappaphycus varieties, using tissue culture techniques for the production of seed stock for nursery and outplanting purposes for the commercial cultivation of carrageenophytes. A shorter duration for shoot formation was observed when the explant was treated with AMPEP + Plant Growth Regulator (PGR = PAA + zeatin at 1 mg L⁻¹) compared to AMPEP when used singly. However, four explants responded differently to the number of days required for shoot formation. The KAP variety took 46 days to form shoots at 3–4 mg L⁻¹ AMPEP + PGR; while PUR required 21 days at 3–5 mg L⁻¹ AMPEP and 3–4 mg L⁻¹ AMPEP + PGR. AA required 17 days at 3–5 mg L⁻¹ AMPEP and AMPEP + PGR; and GS 25 days at 1 mg L⁻¹ AMPEP + PGR. It was observed that among the four explants used, PUR and AA initiated shoot formation with the use of AMPEP only at higher concentrations (3–5 mg L⁻¹) after a shorter period. Only PUR responded positively to ESS/2 for shoot initiation. The use of AMPEP alone and/or in combination with PGR as a culture medium in the propagation of microplantlets using tissue culture technique is highly encouraging.     

Growth and bacteriocin production by Enterococcus faecium DPC1146 in batch and continuous culture

Production of the bacteriocin enterocin 1146 (E1146) by Enterococcus faecium DPC1146 was studied in batch and continuous fermentation. Growth was strongly inhibited by lactic acid. In batch fermentations maximum E1146 activity (2.8 MBU L⁻¹) was obtained in 9 h with 20 g L⁻¹ glucose. Increase in initial glucose concentration did not lead to a proportional increase in E1146 activity. A simple linear model was found to be adequate to explain the relationship between specific bacteriocin production rate and specific growth rate in batch fermentations with initial glucose concentration higher than 20 g L⁻¹. Maximum bacteriocin activity (2.9–3.2 MBU L⁻¹) was obtained in continuous fermentations at dilution rates between 0.12 and 0.17 h⁻¹ and specific bacteriocin production rate increased linearly with dilution rate. Received 31 July 1996/ Accepted in revised form 01 November 1996     

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.     

Effect of soya lecithin on the enzymatic system of the white-rot fungi Anthracophyllum discolor

The present work optimized the initial pH of the medium and the incubation temperature for ligninolytic enzymes produced by the white-rot fungus Anthracophyllum discolor. Additionally, the effect of soya lecithin on mycelial growth and the production of ligninolytic enzymes in static batch cultures were evaluated. The critical micelle concentration of soya lecithin was also studied by conductivity. The effects of the initial pH (3, 4, and 5) and incubation temperature (20, 25, and 30°C) on different enzymatic activities revealed that the optimum conditions to maximize ligninolytic activity were 26°C and pH 5.5 for laccase and manganese peroxidase (MnP) and 30°C and pH 5.5 for manganese-independent peroxidase (MiP). Under these culture conditions, the maximum enzyme production was 10.16, 484.46, and 112.50 U L⁻¹ for laccase, MnP, and manganese-independent peroxidase MiP, respectively. During the study of the effect of soya lecithin on A. discolor, we found that the increase in soya lecithin concentration from 0 to 10 g L⁻¹ caused an increase in mycelial growth. On the other hand, in the presence of soya lecithin, A. discolor produced mainly MnP, which reached a maximum concentration of 30.64 ± 4.61 U L⁻¹ after 25 days of incubation with 1 g L⁻¹ of the surfactant. The other enzymes were produced but to a lesser extent. The enzymatic activity of A. discolor was decreased when Tween 80 was used as a surfactant. The critical micelle concentration of soya lecithin calculated in our study was 0.61 g L⁻¹.