Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Improving intracellular production of recombinant protein in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using an optimized preinduction glycerol-feeding scheme

High-cell-density production of recombinant growth hormone of Lateolabrax japonicus (rljGH) expressed intracellularly in Pichia pastoris was investigated. In the regular strategy of induction at a cell density of 160 g l⁻¹, short duration of intracellular rljGH accumulation (17 h) resulted in a low final cell density of 226 g l⁻¹. Thus, a novel strategy of induction at a cell density of 320 g l⁻¹ was investigated. In this strategy, the preinduction glycerol-feeding scheme had a significant effect on the post-induction production. Constant glycerol feeding led to a decrease of the specific rljGH production and specific production rate because of low preinduction specific growth rate. This decrease was avoided by exponential glycerol feeding to maintain a preinduction specific growth rate of 0.16 h⁻¹. The results from exponential glycerol feeding indicated that the rljGH production depended on the preinduction specific growth rate. Moreover, mixed feeding of methanol and glycerol during induction improved the specific production rate to 0.07 mg g⁻¹ h⁻¹ from 0.043 mg g⁻¹ h⁻¹. Consequently, both high cell density (428 g l⁻¹) and high rljGH production could be achieved by the novel strategy: growing the cells at the specific growth rate of 0.16 h⁻¹ to the cell density of 320 g l⁻¹ and inducing the expression by mixed feeding.     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Heterotrophic production of eicosapentaenoid acid by the diatom Nitzschia laevis: effects of silicate and glucose

The effects of silicate and glucose on growth and eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis were studied. By alternately altering the concentrations of silicate (2.7–64 mg l⁻¹) and glucose (1–40 g l⁻¹) in the medium, the highest cell dry weight (ca. 5.5 g l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest specific growth rate (ca. 0.65 day⁻¹) was obtained at a relatively low glucose concentration (5 g l⁻¹) and high silicate concentrations (32–64 mg l⁻¹). At glucose levels of 5 and 20 g l⁻¹, EPA content was higher with lower silicate concentrations (2.7 and 16 mg l⁻¹ silicate, respectively), while at a silicate level of 16 mg l⁻¹, higher glucose concentrations (20–40 g l⁻¹) facilitated EPA formation. The highest EPA yield (131 mg l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest EPA productivity (15.1 mg l⁻¹ day⁻¹) was obtained at 20 g l⁻¹ glucose and 64 mg l⁻¹ silicate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 218–224. Received 08 May 2000/ Accepted in revised form 21 July 2000     

Improvement in Production and Quality of Gellan Gum by Sphingomonas paucimobilis Under High Dissolved Oxygen Tension Levels

The effect of agitation rate and dissolved oxygen tension (DOT) on growth and gellan production by Sphingomonas paucimobilis was studied. Higher cell growth of 5.4 g l⁻¹ was␣obtained at 700 rpm but maximum gellan (15 g l⁻¹) was produced at 500 rpm. DOT levels above 20% had no effect on cell growth but gellan yield was increased to 23 g l⁻¹ with increase in DOT level to 100%. Higher DOT levels improved the viscosity and molecular weight of the polymer with change in acetate and glycerate content of the polymer.     

Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101

Spray-dried soy molasses (SDSM) contains the sugars dextrose, sucrose, fructose, pinitol, raffinose, verbascose, melibiose, and stachyose. Of the 746 g kg⁻¹ total sugars in SDSM, 434 g kg⁻¹ is fermentable using Clostridium beijerinckii BA101. SDSM was used to produce acetone, butanol, and ethanol (ABE) by C. beijerinckii BA101 in batch cultures. Using 80 g l⁻¹ SDSM, 10.7 g l⁻¹ ABE was produced in P2 medium. Higher concentrations of SDSM resulted in poor solvent production due to the presence of excessive salt and inhibitory components. C. beijerinckii BA101 in SDSM at 80 g l⁻¹ concentration produced 22.8 g l⁻¹ ABE when supplemented with 25.3 g l⁻¹ glucose. SDSM contains 57.4 g kg⁻¹ mineral ash and 2% tri-calcium phosphate. Tri-calcium phosphate up to 43.1 g l⁻¹ was not inhibitory and at a tri-calcium phosphate concentration of 28.8 g l⁻¹, the culture produced more solvents (30.1 g l⁻¹) than the control experiment (23.8 g l⁻¹). In contrast, sodium chloride was a strong inhibitor of C. beijerinckii BA101 cell growth. At a concentration of 10 g l⁻¹ sodium chloride, a maximum cell concentration of 0.6 g l⁻¹ was achieved compared to 1.7 g l⁻¹ in the control experiment. The effects of two salts on specific growth rate constant (μ) and specific rate of ABE production (ν) for C. beijerinckii BA101 were examined. Journal of Industrial Microbiology & Biotechnology (2001) 26, 290–295. Received 20 September 2000/ Accepted in revised form 16 February 2001     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Enhancement of growth and polysaccharide production in suspension cultures of protocorm-like bodies from Dendrobium huoshanense by the addition of putrescine

Putrescine at 0.6 mM stimulated protocorm-like body growth and polysaccharide synthesis in suspension cultures of Dendrobium huoshanense. The specific growth rate of protocorm-like body increased from 0.047 to 0.056 day⁻¹, and the maximum dry weight and polysaccharide production reached 33.2 and 2.94 g l⁻¹, respectively, while they were 24.6 and 2.12 g l⁻¹, respectively, in the control. The administration of polyamine inhibitor, α-dl-difluoromethylarginine, at 1 mM, decreased protocorm-like body growth and polysaccharide production to 21.4 and 1.76 g l⁻¹, respectively.     

Reduction of phenolics content and COD in olive oil mill wastewaters by indigenous yeasts and fungi

Olive oil mill wastewaters (OOMW) cause a recurrent environmental pollution problem. The large concentration of phenolic compounds in the organic fraction of OOMW is principally responsible for the phytotoxicity and microbial growth inhibitory effects of the effluent. Candida boidinii, Geotrichum candidum, a Penicillium sp. and Aspergillus niger HA37 were isolated from OOMW. When cultivated directly on an undiluted OOMW-based medium containing 82 g l⁻¹ COD, these strains removed only 4–8% of chemical oxygen demand (COD) and phenolics. In contrast, reduction values attaining respectively 40–73% for phenolics and 45–78% for COD removal in the undiluted OOMW-based medium were obtained when using the strains gradually acclimated to high concentration of OOMW by successive stepwise transfer from media containing COD of 20.5 up to 82 g l⁻¹. Possibly, a sufficient production level of degradation and/or detoxification enzymes has to be attained to overcome the toxic effects of the phenolic fraction of concentrated OOMW. The present investigation calls attention to the necessity of acclimation for certain fungal and yeasts strains potentially useful for treating highly polluted effluents.     

Improvement of the multiple-stress tolerance of an ethanologenic <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain by freeze-thaw treatment

An effective, simple, and convenient method to improve yeast’s multiple-stress tolerance, and ethanol production was developed. After an ethanologenic Saccharomyces cerevisiae strain SC521 was treated by nine cycles of freeze-thaw, a mutant FT9-11 strain with higher multiple-stress tolerance was isolated, whose viabilities under acetic acid, ethanol, freeze-thaw, H₂O₂, and heat-shock stresses were, respectively, 23-, 26-, 10- and 7-fold more than the parent strain at an initial value 2 × 10⁷ c.f.u. per ml. Ethanol production of FT9-11 was similar (91.5 g ethanol l⁻¹) to SC521 at 30°C with 200 g glucose l⁻¹, and was better than the parent strain at 37°C (72.5 g ethanol l⁻¹), with 300 (111 g ethanol l⁻¹) or with 400 (85 g ethanol l⁻¹) g glucose l⁻¹.     

Optimization of culture conditions and scale-up to pilot and plant scales for coenzyme Q<Subscript>10</Subscript> production by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

This report describes the optimization of culture conditions for coenzyme Q₁₀ (CoQ₁₀) production by Agrobacterium tumefaciens KCCM 10413, an identified high-CoQ₁₀-producing strain (Kim et al., Korean patent. 10-0458818, 2002b). Among the conditions tested, the pH and the dissolved oxygen (DO) levels were the key factors affecting CoQ₁₀ production. When the pH and DO levels were controlled at 7.0 and 0–10%, respectively, a dry cell weight (DCW) of 48.4 g l⁻¹ and a CoQ₁₀ production of 320 mg l⁻¹ were obtained after 96 h of batch culture, corresponding to a specific CoQ₁₀ content of 6.61 mg g-DCW⁻¹. In a fed-batch culture of sucrose, the DCW, specific CoQ₁₀ content, and CoQ₁₀ production increased to 53.6 g l⁻¹, 8.54 mg g-DCW⁻¹, and 458 mg l⁻¹, respectively. CoQ₁₀ production was scaled up from a laboratory scale (5-l fermentor) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity (V _tip) as a scale-up parameter. CoQ₁₀ production at the laboratory scale was similar to those at the pilot and plant scales. This is the first report of pilot- and plant-scale productions of CoQ₁₀ in A. tumefaciens.     

Improvement of artemisinin production by chitosan in hairy root cultures of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg⁻¹ dry wt over 6 days by adding 150 mg chitosan l⁻¹. The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml⁻¹) increased artemisinin production to 1.5 and 0.9 μg mg⁻¹ dry wt, respectively.     

Enhancement of xylitol productivity and yield using a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis under fully aerobic conditions

The effects of glycerol and the oxygen transfer rate on the xylitol production rate by a xylitol dehydrogenase gene (XYL2)-disrupted mutant of Candida tropicalis were investigated. The mutant produced xylitol near the almost yield of 100% from d-xylose using glycerol as a co-substrate for cell growth and NADPH regeneration: 50 g d-xylose l⁻¹ was completely converted into xylitol when at least 20 g glycerol l⁻¹ was used as a co-substrate. The xylitol production rate increased with the O₂ transfer rate until saturation and it was not necessary to control the dissolved O₂ tension precisely. Under the optimum conditions, the volumetric productivity and xylitol yield were 3.2 g l⁻¹ h⁻¹ and 97% (w/w), respectively.     

New insights on toluene biodegradation by Pseudomonas putida F1: influence of pollutant concentration and excreted metabolites

The influence of toluene concentration on the specific growth rate, cellular yield, specific CO₂, and metabolite production by Pseudomonas putida F1 (PpF1) was investigated. Both cellular yield and specific CO₂ production remained constant at 1.0 ± 0.1 g biomass dry weight (DW) g⁻¹ toluene and 1.91 ± 0.31 g CO₂ g⁻¹ biomass, respectively, under the tested range of concentrations (2–250 mg toluene l⁻¹). The specific growth rate increased up to 70 mg toluene l⁻¹. Further increases in toluene concentration inhibited PpF1 growth, although inhibitory concentrations were far from the application range of biological treatment processes. The specific ATP content increased with toluene concentration up to toluene concentrations of 170 mg l⁻¹. 3-Methyl catechol (3-MC) was never detected in the cultivation medium despite being an intermediary in the TOD pathway. This suggested that the transformation from toluene to 3-MC was the limiting step in the biodegradation process. On the other hand, benzyl alcohol (BA) was produced from toluene in a side chain reaction. This is, to the best of our knowledge, the first reported case of methyl monoxygenation of toluene by PpF1 not harboring the pWW0 TOL plasmid. In addition, the influence of 3-MC, BA, and o-cresol on toluene degradation was investigated respirometrically, showing that toluene-associated respiration was not significantly inhibited in the presence of 10–100 mg l⁻¹ of the above-mentioned compounds.     

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l⁻¹) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l⁻¹ to 180 mg l⁻¹, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)⁻¹ h⁻¹. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.     

Bacterial and phytoplankton dynamics in a sub-tropical estuary

Heterotrophic bacterial and phytoplankton biomass, production, specific growth rates and growth efficiencies were studied in July 2001 and January 2002 during both spring and neap tides, along a tidal cycle, at three sites in a subtropical estuary. Major freshwater inputs located in the Northern region led to differences in both phytoplankton and bacterioplankton biomass and activity along the estuary. While in the Northern region phytoplankton is light-limited, with mean phytoplankton production (PP) between 1.1 and 1.9 μg C l⁻¹ h⁻¹ and mean specific growth rates (PSG) between 0.14 and 0.16 d⁻¹, the Southern region registered values as high as 24.7 μg C l⁻¹ h⁻¹ for PP and 2.45 d⁻¹ (mean PP between 3.4 and 7.3 μg C l⁻¹ h⁻¹; mean PSG between 0.28 and 0.57 d⁻¹). On the other hand, maximum bacterial production (BP: 63.8 μg C l⁻¹ h⁻¹) and specific growth rate (BSG: 32.26 d⁻¹) were observed in the Northern region (mean BP between 3.4 and 12.8 μg C l⁻¹ h⁻¹; mean BSG between 1.98 and 6.67 day⁻¹). These bacterial activity rates are among the highest recorded rates in estuarine and coastal waters, indicating that this system can be highly heterotrophic, due to high loads of allochthonous carbon (mainly derived from mangrove forest). Our results also showed that, despite that BP rates usually exceeded PP, in the Southern region BP may be partially supported (∼45%) by PP, since a significant regression was observed between BP and PP (r = 0.455, P < 0.001). Handling editor: P. Viaroli     

Improvement of echinacoside and acteoside production by two-stage elicitation in cell suspension culture of <Emphasis Type="Italic">Cistanche deserticola</Emphasis>

At appropriate concentrations, polyamines promoted the callus growth and echinacoside content of Cistanche deserticola while Ag⁺ increased the content of echinacoside and acteoside. In a 20-day culture period, when putrescine (25 μM) and Ag⁺ (10 μM) were added on day 8 and day 16, respectively, the echinacoside production (1.7 g l⁻¹) and acteoside production (0.4 g l⁻¹) reached the maximum, which were 1.4-fold and 1.5-fold of those in single putrescine treatment, 1.6-fold and 1.4-fold of those in single Ag⁺ treatment, respectively. Exogenous putrescine enhanced cell viability and antioxidant enzyme activity markedly, so increased the final biomass. Ag⁺ addition increased H₂O₂ content and phenylalanine ammonia-lyase activity significantly which led to higher echinacoside and acteoside contents.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study

The effect of individual environmental conditions (pH, pO₂, temperature, salinity, concentration of ethanol, propanol, tryptone and yeast extract) on the specific growth rate as well as ethanol and glycerol production rate of Saccharomyces cerevisiae S288C was mapped during the fermentative growth in aerobic auxo-accelerostat cultures. The obtained steady-state values of the glycerol to ethanol formation ratio (0.1 mol mol⁻¹) corresponding to those predicted from the stoichiometric model of fermentative yeast growth showed that the complete repression of respiration was obtained in auxostat culture and that the model is suitable for calculation of Y _ATP and Q _ATP values for the aerobic fermentative growth. Smooth decrease in the culture pH and dissolved oxygen concentration (pO₂) down to the critical values of 2.3 and 0.8%, respectively, resulted in decrease in growth yield (Y _ATP) and specific growth rate, however the specific ATP production rate (Q _ATP) stayed almost constant. Increase in the concentration of biomass (>0.8 g dwt l⁻¹), propanol (>2 g l⁻¹) or NaCl (>15 g l⁻¹) lead at first to the decrease in the specific growth rate and Q _ATP, while Y _ATP was affected only at higher concentrations. The observed decrease in Q _ATP was caused by indirect rather than direct inhibition of glycolysis. The increase in tryptone concentration resulted in an increase in the specific growth rate from 0.44 to 0.62 h⁻¹ and Y _ATP from 12.5 to 18.5 mol ATP g dwt⁻¹. This study demonstrates that the auxo-accelerostat method, besides being an efficient tool for obtaining the culture characteristics, provides also decent conditions for the experiments elucidating the control mechanisms of cell growth.