Production of nisin Z using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1 from hydrolyzed sago starch

A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity of nisin Z, as 50,000 IU l⁻¹ h⁻¹ using a cell concentration of 15 g l⁻¹, 30^°C, pH 5.5 and a dilution rate of 1.24 h⁻¹. Adding 10 g l⁻¹ YE and 2 g l⁻¹ polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase mode with high metabolic activity.     

Influence of growth conditions on the nisin production of bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production of three bioengineered strains, (LAC338, LAC339 and LAC340) with immunity (nisFEG) and/or regulation (nisRK) genes of nisin biosynthesis on plasmids in the Lactococcus lactis LL27 nisin producer, was evaluated under pH-controlled and pH-uncontrolled batch fermentations. Optimization studies showed that fructose and yeast extract yielded the highest nisin activity. The strains LAC338, LAC339, and LAC340 produced 24, 45, and 44% more nisin, respectively, than wild-type L. lactis LL27 after 12-h incubation. However, sharp decreases in the yield of nisin were observed at the late phase of fermentation with LAC339 and LL27 in contrast to LAC340 and LAC338 strains for which the high level of nisin could be maintained longer. Obviously, increasing the copy number of the regulation genes together with immunity genes in the nisin producers retarded the loss of nisin in the late phase of the fermentation.     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

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.     

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.     

Isolation of sensitive nisin-sensing GFP<Subscript>uv</Subscript> bioassay <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains using FACS

Fluorescence-activated cell sorting (FACS) was used to isolate mutants of Lactococcus lactis LAC275, an indicator strain in GFP_uv nisin bioassay. It harbors the GFP_uv encoding gene under the nisA promoter and the nisin signal transduction nisRK genes whereby nisin concentration can be correlated to GFP_uv fluorescence. The sorted L. lactis cells, which showed higher fluorescence intensities at low inducer concentration, were analysed for higher responsiveness to low concentration of nisin. Two strains showed lower detection limits (0.2 pg ml⁻¹) for nisin than the parent strain (10 pg ml⁻¹). This showed that mutants of LAC275 could successfully be isolated using FACS.     

Continuous acetone–butanol–ethanol production by corn stalk immobilized cells

Corn stalk was used as a support to immobilize Clostridia beijerinckii ATCC 55025 in the fermentation process of acetone, butanol, and ethanol production. The effect of the dilution rate on solvent production was examined in a steady-state 20-day continuous flow operation. The maximum total solvent concentration of 8.99 g l⁻¹ was obtained at a dilution rate of 0.2 h⁻¹. Increasing the dilution rate between 0.2 and 1.0 h⁻¹ resulted in an increased solvent productivity, and the highest solvent productivity was obtained at 5.06 g l⁻¹ h⁻¹ with a dilution rate of 1 h⁻¹. The maximum solvent yield from glucose of 0.32 g g⁻¹ was observed at 0.25 h⁻¹. The cell adsorption and morphology change during the growth on corn stalk support were examined by the SEM.     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

High-yield production of erythritol from raw glycerol in fed-batch cultures of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

An acetate-negative mutant of Yarrowia lipolytica Wratislavia K1 was selected that, when grown with 300 g raw glycerol l⁻¹ at pH 3, produced 170 g erythritol l⁻¹ after 7 days, corresponding to a 56% yield and a productivity of 1 g l⁻¹ h⁻¹. The Wratislavia K1 strain did not produce citric acid.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

Continuous ethanol production from wheat straw hydrolysate by recombinant ethanologenic <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> strain FBR5

Continuous production of ethanol from alkaline peroxide pretreated and enzymatically saccharified wheat straw hydrolysate by ethanologenic recombinant Escherichia coli strain FBR5 was investigated under various conditions at controlled pH 6.5 and 35°C. The strain FBR5 was chosen because of its ability to ferment both hexose and pentose sugars under semi-anaerobic conditions without using antibiotics. The average ethanol produced from the available sugars (21.9–47.8 g/L) ranged from 8.8 to 17.3 g/L (0.28–0.45 g/g available sugars, 0.31–0.48 g/g sugar consumed) with ethanol productivity of 0.27–0.78 g l⁻¹ h⁻¹ in a set of 14 continuous culture (CC) runs (16–105 days). During these CC runs, no loss of ethanol productivity was observed. This is the first report on the continuous production of ethanol by the recombinant bacterium from a lignocellulosic hydrolysate.     

Production of 1,3-propanediol by Clostridium butyricum in continuous culture with cell recycling

The continuous fermentation of 1,3-propanediol from glycerol by Clostridium butyricum was subjected to cell recycling by filtration using hollow-fibre modules made from polysulphone. The performance of the culture system was checked at a retention ratio (dilution rate/bleed rate) of 5, dilution rates between 0.2 h⁻¹ and 1.0 h⁻¹ and glycerol input concentrations of 32 g l⁻¹ and 56 g l⁻¹. The near-to-optimum propanediol concentration of 26.5 g l⁻¹ (for 56 g l⁻¹ glycerol) was maintained up to a dilution rate of 0.5 h⁻¹ and then decreased while the propanediol productivity was highest at 0.7 h⁻¹. The productivity could be increased by a factor of four in comparison to the continuous culture without cell recycling. By application of the model of Zeng and Deckwer [(1995) Biotechnol Prog 11: 71–79] for cultures under substrate excess, it was shown that the limitations resulted exclusively from product inhibition and detrimental influences from the cell recycling system, such as shear stress, were not involved. Received: 20 October 1997 / Received revision: 12 December 1997 / Accepted: 14 December 1997     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.     

Production of fructose from inulin using mixed inulinases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

Two new effective microbial producers of inulinases were isolated from Jerusalem artichoke tubers grown in Thailand and identified as Aspergillus niger TISTR 3570 and Candida guilliermondii TISTR 5844. The inulinases produced by both these microorganisms were appropriate for hydrolysing inulin to fructose as the principal product. An initial inulin concentration of ∼100 g l⁻¹ and the enzyme concentration of 0.2 U g⁻¹ of substrate, yielded 37.5 g l⁻¹ of fructose in 20 h at 40°C when A. niger TISTR 3570 inulinase was the biocatalyst. The yield of fructose on inulin was 0.39 g g⁻¹. Under identical conditions, the yeast inulinase afforded 35.3 g l⁻¹ of fructose in 25 h. The fructose yield was 0.35 g g⁻¹ of substrate. The fructose productivities were 1.9 g l⁻¹ h⁻¹ and 1.4 g l⁻¹ h⁻¹ for the mold and yeast enzymes, respectively. After 20 h of reaction, the mold enzyme hydrolysate contained 53% fructose and more than 41% of initial inulin had been hydrolysed. Using the yeast enzymes, the hydrolysate contained nearly 38% fructose at 25 h and nearly 36% of initial inulin had been hydrolysed. The A. niger TISTR 3570 inulinases exhibited both endo-inulinase and exo-inulinase activities. In contrast, the yeast inulinases displayed mainly exo-inulinase activity. The mold and yeast crude inulinases mixed in the activity ratio of 5:1 proved superior to individual crude inulinases in hydrolysing inulin to fructose. The enzyme mixture provided a better combination of endo- and exo-inulinase activities than did the crude extracts of either the mold or the yeast individually.     

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.     

Factors affecting the production of <Emphasis Type="SmallCaps">l</Emphasis>-xylulose by resting cells of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors affecting the production of the rare sugar l-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol to l-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial xylitol concentration on l-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production rates were found at high shaking speed and at high temperature (over 44°C). The optimal pH for both productivity and conversion was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250–350 g l⁻¹. A specific productivity of 1.09 ± 0.10 g g⁻¹ h⁻¹ was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the operation of the process in a bioreactor with reasonable accuracy.     

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.     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.