Continuous nisin production with bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h^–1 dilution rate and 12.5 g l^–1 fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h⁻¹ compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h^–1. For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h^–1 dilution rates and 11.95, 12.01, 11.63, and 12.50 g l^–1 fructose concentrations, respectively. The highest nisin productivity, 496 IU ml^–1 h^–1, was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations.     

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.     

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.     

Bioethanol production in batch mode by a native strain of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Two wild strains of Zymomonas mobilis were isolated (named as ML1 and ML2) from sugar cane molasses obtained from different farms of Santander, Colombia. Initially, selection of the best ethanol-producer strains was carried out using ethanol production parameters obtained with a commercial strain Z. mobilis DSM 3580. Three isolated strains were cultivated in a culture medium containing yeast extract, peptone, glucose and salts, at pH 6 and 32°C with stirring rate of 65 rpm during 62 h. The best results of ethanol production were obtained with the native strain ML1, reaching a maximum ethanol concentration of 79.78 g l⁻¹. ML1 and ML2 strains were identified as Z. mobilis, according to the morphology, biochemical tests and molecular characterization by PCR of specific DNA sequences from Z. mobilis. Subsequently, the effect of different nitrogen sources on production of ethanol was evaluated. The best results were obtained using urea at a 0.73 g/l. In this case, maximum concentration of ethanol was 83.81 g l⁻¹, with kinetic parameters of yield of ethanol on biomass (Y_P/X) = 69.01(g g⁻¹), maximum volumetric productivity of ethanol (Qp_max) = 2.28 (g l⁻¹ h⁻¹), specific productivity of ethanol (q_P) = 3.54 (h⁻¹) and specific growth rate (μ) = 0.12 h⁻¹. Finally, we studied the effect of different culture conditions (pH, temperature, stirring, C/N ratio) with a Placket-Burman′s experimental design. This optimization indicated that the most significant variables were temperature and stirring. In the best culture conditions a significant increase in all variables of response was achieved, reaching a maximum ethanol concentration of 93.55 g l⁻¹.     

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.     

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.     

Increase of lycopene production by supplementing auxiliary carbon sources in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

In the fed-batch culture of glycerol using a metabolically engineered strain of Escherichia coli, supplementation with glucose as an auxiliary carbon source increased lycopene production due to a significant increase in cell mass, despite a reduction in specific lycopene content. l-Arabinose supplementation increased lycopene production due to increases in cell mass and specific lycopene content. Supplementation with both glucose and l-arabinose increased lycopene production significantly due to the synergistic effect of the two sugars. Cell growth by the consumption of carbon sources was related to endogenous metabolism in the host E. coli. Supplementation with l-arabinose stimulated only the mevalonate pathway for lycopene biosynthesis and supplementation with both glucose and l-arabinose stimulated synergistically only the mevalonate pathway. In the fed-batch culture of glycerol with 10 g l⁻¹ glucose and 7.5 g l⁻¹ l-arabinose, the cell mass, lycopene concentration, specific lycopene content, and lycopene productivity after 34 h were 42 g l⁻¹, 1,350 mg l⁻¹, 32 mg g cells⁻¹, and 40 mg l⁻¹ h⁻¹, respectively. These values were 3.9-, 7.1-, 1.9-, and 11.7-fold higher than those without the auxiliary carbon sources, respectively. This is the highest reported concentration and productivity of lycopene.     

Lipid production by <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> Y4 using different substrate feeding strategies

Microbial lipid is a potential alternative feedstock for the biodiesel industry. New culture strategies remain to be developed to improve the economics of microbial lipid technology. This work describes lipid production by the oleaginous yeast Rhodosporidium toruloides Y4 using a 15-l bioreactor with different substrate feeding strategies. Among these strategies, the intermittent feeding mode gave a lipid productivity of 0.36 g l⁻¹ h⁻¹, whereas the constant glucose concentration II (CC-II) mode gave the highest lipid productivity of 0.57 g l⁻¹ h⁻¹. The repeated fed-batch mode according to the CC-II mode was performed with a duration time of 358 h, and the overall lipid productivity was 0.55 g l⁻¹ h⁻¹. Our results suggested that substrate feeding modes had a great impact on lipid productivity and that the repeated fed-batch process was the most appealing method by which to enhance microbial lipid production.     

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.     

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.     

Fungal autolysate as a nutrient supplement for ethanol and chitosan production by <Emphasis Type="Italic">Mucor indicus</Emphasis>

Mucor indicus can be used to produce ethanol from a variety of sugars, including pentose’s. An extract of it, produced by autolysis, could replace yeast extract in culture medium with improved production of ethanol. At 10 g l⁻¹, the extract gave a higher ethanol yield (0.47 g g⁻¹) and productivity (0.71 g l⁻¹ h⁻¹) compared to medium containing yeast extract (yield 0.45 g g⁻¹; productivity 0.67 g l⁻¹ h⁻¹).     

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.     

Carbon source pulsed feeding to attain high yield and high productivity in poly(3-hydroxybutyrate) (PHB) production from soybean oil using <Emphasis Type="Italic">Cupriavidus necator</Emphasis>

Poly(3-hydroxybutyrate) (PHB) biosynthesis from soybean oil by Cupriavidus necator was studied using a bench scale bioreactor. The highest cell concentration (83 g l⁻¹) was achieved using soybean oil at 40 g l⁻¹ and a pulse of the same concentration. The PHB content was 81% (w/w), PHB productivity was 2.5 g l⁻¹ h⁻¹, and the calculated Y_p/s value was 0.85 g g⁻¹. Growth limitation and the onset of PHB biosynthesis took place due to exhaustion of P, and probably also Cu, Ca, and Fe.     

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.     

Succinic acid production by a newly isolated bacterium

A new bacterial strain producing succinic acid was enriched from bovine rumen content. It is facultatively anaerobic, belongs to the family Pasteurellaceae and has similarity to the genus Mannheimia. In batch cultivations with D-glucose or sucrose the strain produced up to 5.8 g succinic acid l⁻¹ with a productivity and a yield of up to 1.5 g l⁻¹ h⁻¹ and 0.6 g g⁻¹, respectively. With crude glycerol up to 8.4 g l⁻¹, 0.9 g l⁻¹ h⁻¹ and 1.2 g g⁻¹ were obtained. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Biomass and lipid productivities of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> under autotrophic,heterotrophic and mixotrophic growth conditions

Biomass and lipid productivities of Chlorella vulgaris under different growth conditions were investigated. While autotrophic growth did provide higher cellular lipid content (38%), the lipid productivity was much lower compared with those from heterotrophic growth with acetate, glucose, or glycerol. Optimal cell growth (2 g l⁻¹) and lipid productivity (54 mg l⁻¹ day⁻¹) were attained using glucose at 1% (w/v) whereas higher concentrations were inhibitory. Growth of C. vulgaris on glycerol had a similar dose effects as those from glucose. Overall, C. vulgaris is mixotrophic.     

1,3-Propanediol production in a two-step process fermentation from renewable feedstock

In this work, the production of 1,3-propanediol from glucose and molasses was studied in a two-step process using two recombinant microorganisms. The first step of the process is the conversion of glucose or other sugar into glycerol by the metabolic engineered Saccharomyces cerevisiae strain HC42 adapted to high (>200 g l⁻¹) glucose concentrations. The second step, carried out in the same bioreactor, was performed by the engineered strain Clostridium acetobutylicum DG1 (pSPD5) that converts glycerol to 1,3-propanediol. This two-step strategy led to a flexible process, resulting in a 1,3-propanediol production and yield that depended on the initial sugar concentration. Below 56.2 g l⁻¹ of sugar concentration, cultivation on molasses or glucose showed no significant differences. However, at higher molasses concentrations, glycerol initially produced by yeast could not be totally converted into 1,3-propanediol by C. acetobutylicum and a lower 1,3-propanediol overall yield was observed. In our hand, the best results were obtained with an initial glucose concentration of 103 g l⁻¹, leading to a final 1,3-propanediol concentration of 25.5 g l⁻¹, a productivity of 0.16 g l⁻¹ h⁻¹ and 1,3-propanediol yields of 0.56 g g⁻¹ glycerol and 0.24 g g⁻¹ sugar, which is the highest value reported for a two-step process. For an initial sugar concentration (from molasses) of 56.2 g l⁻¹, 27.4 g l⁻¹ of glycerol were produced, leading to 14.6 g l⁻¹ of 1.3-propanediol and similar values of productivity, 0.15 g l⁻¹ h⁻¹, and overall yield, 0.26 g g⁻¹ sugar.     

Citric acid production from glycerol-containing waste of biodiesel industry by Yarrowia lipolytica in batch, repeated batch, and cell recycle regimes

Yarrowia lipolytica A-101-1.22 produces high citric acid (112 g l⁻¹) with a yield of 0.6 g g⁻¹ and a productivity of 0.71 g l⁻¹ h⁻¹ during batch cultivation in the medium with glycerol-containing waste of biodiesel industry. However, it was observed that the specific citric acid production rate, which was maximal at the beginning of the biosynthesis, gradually decreases in the late production phase and it makes continuation of the process over 100 h pointless. The cell recycle and the repeated batch regimes were performed as ways for prolongation of citric acid synthesis by yeast. Using cell recycle, the active citric acid biosynthesis (96–107 g l⁻¹) with a yield of 0.64 g g⁻¹ and a productivity of 1.42 g l⁻¹ h⁻¹ was prolongated up to 300 h. Repeated batch culture remained stable for over 1000 h; the RB variant of 30% feed every 3 days showed the best results: 124.2 g l^-1 citric acid with a yield of 0.77 g g^-1 and a productivity of 0.85 g l^-1 h^-1.     

Efficient production of ectoine using ectoine-excreting strain

Halophilic bacteria strain Halomonas salina DSM 5928 was found to excrete ectoine, suggesting its potential in the development of a new method of ectoine production. We performed HPLC and LC–MS analyses that showed that Halomonas salina DSM 5928 excreted ectoine under constant extracellular osmolarity. Medium adopting monosodium glutamate as a sole source of carbon and nitrogen was beneficial for ectoine synthesis. The total concentration of ectoine was not affected by NaCl concentration in the range 0.5–2 mol l⁻¹. The total concentration of ectoine and productivity in a 10-l fermentor with 0.5 mol l⁻¹ NaCl were 6.9 g l⁻¹ and 7.9 g l⁻¹ d⁻¹, respectively. These findings show that Halomonas salina DSM 5928 efficiently produces ectoine at relatively low NaCl concentration. This research also indicates the potential application of free or immobilized cells for continuous culture to produce ectoine.