Continuous high-ethanol fermentation from cane molasses by a flocculating yeast

Repeated-batch fermentation by a flocculating fusant, Saccharomyces cerevisiae HA 2, was done in a molasses medium that contained 20% (w/v) total sugar, at 30°C in an automatically controlled fermentor, and the effects of ethanol concentration on the specific growth rate and the specific production rate of ethanol were studied. Both the specific growth rate and the specific production rate of ethanol fell with increase of ethanol concentration, and there was a linear correlation between each rate and the concentration of thanol. The maximum specific growth rate (μ_max) and the maximum specific production rate of ethanol (q_max) were 0.12 h⁻¹ and 0.1 g ethanol/10⁹ cells·h, respectively. The specific growth rate and the specific production rate of ethanol fell to zero at ethanol concentration of 89 g/l and 95 g/l, respectively. The number of viable cells, calculated from the linear inhibition equation, was 1.3 × 10⁹ cells/ml for production of 85 g/l ethanol at a dilution rate (D₁) of 0.2 h⁻¹. Based on this estimation, a laboratory-scale continuous fermentation, using two fermentors in series, was done. In the second fermentor, 85 g/l ethanol was produced at a dilution rate (D₁) of 0.2 h⁻¹ by the active feedig of the fermented mash from the first fermentor into the second fermentor by pumping (hereafter called active feeding). To maintain the number of viable cells above 10⁹ cells/ml in the second fermentor, a active feeding ratio of more than 23% was required. Under these conditions, 81 g/l ethanol was produced in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹, and the high ethanol productivity of 20.3 g/l·h could be achieved. A bench-scale continuous fermentation, using two fermentors in series, with a active feeding ratio of 25% was done. An ethanol concentration of 84 g/l in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹ was achieved, just as it was in the laboratory-scale fermentation test.     

Influence of the concentrations of d-xylose and yeast extract on ethanol production by Pachysolen tannophilus

To determine the most favorable conditions for the production of ethanol by Pachysolen tannophilus, this yeast was grown in batch cultures with various initial concentrations of two of the constituents of the culture medium: d-xylose (s_o), ranging from 1 g·l⁻¹ to 200 g·l⁻¹, and yeast extract (l_o), ranging from 0 g·l⁻¹ to 8 g·l⁻¹. The most favorable conditions proved to be initial concentrations of S_o=25 g·l⁻¹ and l_o=4 g·l⁻¹, which gave a maximum specific growth rate of 0.26 h⁻¹, biomass productivity of 0.023 g·l⁻¹·h⁻¹, overall biomass yield of 0.094 g·g⁻¹, specific xylose-uptake rate (q_s) of 0.3 g·g⁻¹·h⁻¹ (for t=50 h), specific ethanol-production rate (q_E) of 0.065 g·g⁻¹·h⁻¹ and overall ethanol yield of 0.34 g·g⁻¹; q_s values decreased after the exponential growth phase while q_E remained practically constant.     

Enhanced production of peroxidase in continuous culture of Arthromyces ramosus by selective bleeding of mycelium

A new method of continuous culture with selective bleeding of mycelia using 9-mesh screen was developed to improve the production rate of peroxidase (POD) by Arthromyces ramosus. At the dilution rate of 0.05 h⁻¹ with the mycelium leakage rate of 60%, a high production rate (average value was 1.67 U·ml⁻¹·h⁻¹) was maintained for over 100 h: the rate was 3.2 times that in a glucose-fed batch culture. At the same dilution rate, the volumetric and specific production rates of POD in the continuous culture without the screen were lower than those in the first continuous culture and decreased gradually in the later phase of the culture. In the continuous culture with low mycelium leakage rate of 1.6%, the POD production rate was not improved further, although the mycelial concentration (43 g·l⁻¹) increased 2.9 times. It is suggested that the high agitation rate required to meet the oxygen demand is unfavorable for the POD production.     

Production of l-Methionine-enriched cells of a mutant derived from a methylotrophic yeast,Candida boidinii

l-Methionine-enriched cells production of an ethionine-resistant mutant of Candida boidinii no. 2201 was greatly improved by the control of pH and by feeding of methanol and other medium components during cultivation in a jar fermentor. Under the optimal conditions, 38.5 g (as dry weight)_of cells abd 282 mg of pool methionine (intracellular pool of free l-methionine) per l of culture broth were obtained after 11 d of cultivation.The culture conditions for production of l-methionine-enriched cells in continuous culture were investigated. With limited methanol in continuous cultivation, pool methionine productivity reached a maximum value of 1.14 mg·l⁻¹·h⁻¹ at a dilution rate of 0.05·h⁻¹. During methanol-limited growth in continuous cultivation, the pool methionine content of the mutant was about 20–35% higher than that in batch cultivation.     

Influence of planting geometry on photosynthetically active radiation interception and dry matter production relationships in pearl millet

A field experiment was conducted at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Center, Patancheru, India to study photosynthetically active radiation (PAR) interception and dry matter production relationships in pearl millet (Pennisetum americanum (L.) Leeke). Two pearl millet genotypes, BJ 104 (G₁) and ICH 226 (G₂) were sown at three planting geometries obtained by using combinations of row and plant spacings (S₁: 37·5 cm × 26·6 cm; S₂: 75·0 cm × 13·3 cm; S₃: 150·0 cm × 6·6 cm) such that plant population was constant at 100 000 ha⁻¹ in all treatments. Cumulative intercepted PAR was maximum (330 MJ m⁻²) in G₂S₂ and minimum (268 MJ m⁻²) in G₁S₃. Conversion efficiency values ranged from 1·87 g MJ⁻¹ in G₁S₂ to 2·32 g MJ⁻¹ in G₂S₃. Final above-ground dry matter followed the pattern of cumulative intercepted PAR and maximum dry matter (7·22 Mg ha⁻¹) was produced by G₂S₂ while G₁S₃ produced minimum dry matter (4·97 Mg ha⁻¹).     

Effect and control of glucose feeding on bacitracin production by fed-batch culture of Bacillus licheniformis

The effect of glucose feeding on bacitracin production was investigated by fed-batch culture of Bacillus licheniformis. In batch culture, bacitracin secretion was induced after the glucose initially contained in the medium was completely consumed. The concentration of bacitracin, however, increased to no more than 340 units·ml⁻¹ in the batch cultivations. Therefore, additional glucose was supplied after exhaustion of the initial glucose. The effect of glucose feeding on bacitracin biosynthesss was investigated in two ways, the pH-stat modal feeding method and the CO₂-dependent feeding method. A kinetic study of bacitracin production found that some glucose was necessary, even during the bacitracin production phase. Excessive feeding of glucose, however, caused a reduction in bacitracin biosynthetic activity. When 50 g·l⁻¹ of defatted soy bean meal (SBM) was used, the bacitracin concentration reached 670 units·ml⁻¹ with the pH-stat modal feeding method and 610 units·ml⁻¹ with the CO₂-dependent feeding method, respectively. The yield of bacitracin from consumed glucose was better for the pH-stat method. Using this control strategy, the highest concentration of bacitracin (940 units·ml⁻¹) was obtained with 150 g·l⁻¹ of SBM.     

Effect of oxygen supply and dilution rate on the production of 2,3-butanediol in continuous bioreactor by Klebsiella pneumoniae

Kinetics of 2,3-butanediol production by Klebsiella pneumoniae (NRRL B199) from glucose have been studied in a continuous bioreactor. The effect of oxygen supply rate and dilution rate on the product output rate and yield of 2,3-butanediol were investigated. For a feed glucose concentration of 100 g l⁻¹, the optimum oxygen transfer rate is between 25.0–35.0 mmol l⁻¹ h⁻¹. Under these conditions, maximum product concentration obtained was 35 g l⁻¹ at a dilution rate of 0.1 h⁻¹ and the maximum product output rate obtained was 4.25 g l⁻¹ h⁻¹. The product yield based on the substrate utilized approached the theoretical value (50%) at low values of oxygen transfer rate but decreased with increasing oxygen transfer rate.     

Factors affecting l-arginine production in the continuous culture of an l-arginine producer of Corynebacterium acetoacidophilum

The effects of culture conditions on l-arginine production by continuous culture were studied using a stable l-arginine hyperproducing strain of Corynebacterium aceto-acidophilum, SC-190. Strain SC-190 demonstrated a volumetric productivity of 35 g l⁻¹·h⁻¹ at a dilution rate of 0.083h⁻¹ and feeding sugar concentration of 8%, and a product yield of 29.2% at a dilution rate 0.021h⁻¹ and feeding sugar concentration of 15%. The corresponding values for fed-batch culture are 0.85 g·l⁻¹·h⁻¹ and 26%. However, the product yield decreased with an increase in the volumetric productivity. To achieve stable l-arginine production, aeration and agitation conditions sufficient to maintain an optimal level of redox potential (>−100 mV) were necessary. The addition of phosphate to the feeding medium led to a decrease in l-arginine production. It was confirmed in the steady state that growth and l-arginine formation were inhibited by a high concentration of l-arginine.     

Disruption of glpF gene encoding the glycerol facilitator improves 1,3-propanediol production from glucose via glycerol in Escherichia coli

Engineered Escherichia coli has recently been applied to produce 1,3-propanediol (1,3-PDO) from glucose. A metabolic intermediate in the production pathway, glycerol, is partially secreted into the extracellular of E. coli through a glycerol facilitator encoded by glpF, and this secretion consequently decreases 1,3-PDO production. Therefore, we aimed to determine whether disrupting the glpF gene would improve 1,3-PDO production in E. coli. The intracellular glycerol concentration in a glpF-disruptant was 7·5 times higher than in a non-disruptant. The glpF-disrupted and non-disrupted E. coli strains produced 0·26 and 0·09 g l⁻¹ of 1,3-PDO, respectively, from 1% glucose after 72 h of cultivation. The specific growth rate (μ) and the 1,3-PDO yield from glucose (Y_P/S) in the disruptant were higher than those in the non-disruptant (ΔglpF, μ = 0·08 ± 0·00 h⁻¹, Y_P/S = 0·06 mol mol-glucose⁻¹; BW25113, μ = 0·06 ± 0·00 h⁻¹, Y_P/S = 0·02 mol mol-glucose⁻¹). Disruption of the glpF gene decreased the production of the by-product, acetic acid. These results indicated that disruption of glpF increased the intracellular concentration of glycerol and consequently increased 1,3-PDO production in E. coli.     

Continuous ethanol production by yeast immobilized on to channeled alumina beads

Vertical and near-horizontal (15° angle) packed-bed columns were compared for continuous ethanol fermentation using an alcohol- and glucose-tolerant Saccharomyces cerevisiae strain immobilized on to channeled alumina beads (5·0 × 10⁹ cells g⁻¹ beads). Spaces between beads (1·0–6·5 mm) and angle (15°) of near-horizontal reactor columns (with six ports in each) efficiently removed CO₂ and increased ethanol productivity. Malt-glucose-yeast-extract broth containing 16·7% glucose at 35°C fed at a dilution rate of 3· h⁻¹ to thw two horizontal columns (in series) yielded maximum ethanol productivity of 40·0 g liter⁻¹ h⁻¹. Feedstock flow rate and other factors (temperature, pH, nutrients, and glucose levels) affected productivities. The immobilized-cell system showed operational stability for >3 months without plugging, and could be stored for at least one year with no loss of bioreactor performance. Scanning electron micrographs of the beads revealed large numbers of yeast-cells attached on to internal and external surfaces of beads.     

Ethanol production from wheat flour by Zymomonas mobilis

Starch from wheat flour was enzymatically hydrolyzed and used for ethanol production by Zymmonas mobilis. The addition of a nitrogen source like ammonium sulfate was sufficient to obtain a complete fermentation of the hdyrolyzed strach. In batch culture a glucose concentration as high as 223 g/l could be fermented (conversion 99.5%) to 105 g/l of ethanol in 70 h with an ethanol yield of 0.47 g/g (92% of theoretical). In continuous culture the use of a flocculent strain and a fermentor with an internal settler resulted (D=1,4 h⁻¹) in a high ethanol productivity of 70.7 g/l·h with: ethanol concentration 49.5 g/l, ethanol yield 0.50 g/g (98% of theoretical and substrate conversion 99%.     

Influence of pH on methane and sulfide production from methanol

To investigate the influence of pH on methane and sulfide production, continuous cultures were done using a bio-reactor packed with pumice stone. Sulfate (1 g SO₄²⁻·l⁻¹) in a methanol defined medium (10 g·l⁻¹) was almost completely reduced to sulfide at pHs between 7.0 and 7.5 in methane fermentation, but at pHs between 6.2 and 6.8, sulfate reduction to sulfide was suppressed up to 40%. In addition, methane fermentation was not inhibited by 10 g sulfate·l⁻¹.     

Prodigiosin formation by Serratia marcescens in a chemostat

In a study of the control of metabolite formation, prodigiosin production by Serratia marcescens was used as a model. Specific production rates of prodigiosin formation were determined using batch culture technique. Sucrose as carbon source and NH₄NO₃ as nitrogen source resulted in a specific production rate of 0.476 mg prodigiosin (g cell dry weight)⁻¹ h⁻¹. Prodigiosin formation and productivity was inversely correlated to growth rate when the bacterium was grown under carbon limitation on a defined medium in a chemostat culture. The maximum specific growth rate (μ_max) was 0.54 h⁻¹ and prodigiosin was formed in amounts over 1 mg l⁻¹ up to a growth rate (μ) of 0.3 h⁻¹ at steady state conditions. At a dilution rate of 0.1 h⁻¹ growth at steady state with carbon and phosphate limitation supported prodigiosin formation giving a similar specific yield [1.17 mg prodigiosin (g cell dry weight)⁻¹ and 0.94 mg g⁻¹, respectively], however, cells grown with nitrogen limitation [(NH₄)₂SO₄] did not form prodigiosin. Productivity in batch culture was 1.33 mg l⁻¹ h⁻¹ as compared to 0.57 mg l⁻¹ h⁻¹ in the chemostat.     

Lipid and biomass production by the halotolerant microalga Nannochloropsis salina

The effect of environmental factors on cell-lipid content, on the growth rate and on the overall productivity of Nannochloropsis salina was tested in the laboratory and in outdoor cultures. Under optimum conditions in the laboratory, the maximum growth rate (μ_max) was 0·030 h⁻¹, which corresponds to a doubling time of 23 h. Cellular lipid content was affected by the phase of growth and the temperature, but not by nitrogen starvation, pH or the source of sea water. The most important factor affecting the output rate of biomass was the cell concentration. The maximum biomass productivity obtained in outdoor ponds was 24·5 g·m⁻²·day⁻¹, and the lipid production rate was 4·0 g m⁻²·day⁻¹.     

Limitations in substrate utilization efficiency by Zymomonas mobilis

Summary Optimal growth conditions for Zymomonas mobilis have been established using continuous cultivation methods. Optimal substrate utilization efficiency occurs with 2.5 g l^–1 yeast extract, 2.0 g l^–1 ammonium sulfate and 6.0 g l^–1 magnesium sulfate in the media. Catabolic activity is at its maximum with glucose uptake rates of 16–18 g l^–1 h^–1 and ethanol production rates of 8–9 g l^–1 h^–1, Q_g values of 22–26 and Q_p values between 11 and 13, which results in 40 g l^–1 h^–1 ethanol yields using a 100 g l^–1 substrate feed. Any increase in these parameters goes on cost of substrate utilization efficiency. Calcium pantothenate can not substitute yeast extract.Abbreviations G Glucose (%) - Pant Calcium pantothenate (mg l^–1) - D Dilution rate (h^–1) - NH₄ Ammonium sulfate (%) - M_g Magnesium sulfate (%) - S₁ Residual glucose in the fermenter (g l^–1) - S₀ Glucose feed (g l^–1) - Eth Ethanol concentration (g l^–1) - GUR Glucose uptake rate (g l^–1 h^–1) - Q_g Specific glucose uptake rate (g g^–1 h^–1) - Q_p Specific ethanol production rate (g g^–1 h^–1) - EPR Ethanol production rate (g l^–1 h^–1) - Y_g Yield coefficient for glucose (g g^–1) - Y_p Conversion efficiency (%) - C Biomass concentration (g l^–1) Present address: (Until June 1982) Institut für Mikrobiologie, TH Darmstadt, 6100 Darmstdt, Federal Republic of Germany     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Effects of dehydration and low temperatures on the oxidation of high-potential cytochrome c by photosynthetic reaction centers in Ectothiorhodospira shaposhnikovii

Effects of temperature and dehydration on the efficiency of electron transfer from membrane-bound high-potential cytochromes c_h to the reaction-center bacteriochlorophyll (P-890) in Ectothiorhodospira shaposhnikovii have been studied. A kinetic analysis of the cytochrome oxidation suggests that there are at least two conformational states of the c_h-P-890 complex, of which only one allows photoinduced electron transfer from cytochrome to P-890⁺. Lowering the temperature of dehydration leads to a change in the proportion of the populations in the two conformations. The observed 2-fold deceleration of cytochrome oxidation can be related only to the diminution of the amount of photoactive cytochromes per reaction center. The rate constant for the transfer of an electron from cytochrome c_h to bacteriochlorophyll is 2.8 · 10⁵ s⁻¹ and is independent of temperature and dehydration (as estimated within the accuracy of the experiments). The effects produced by low temperature and dehydration are completely reversible. The thermodynamic parameters of the transition of the cytochrome from the nontransfer to electron-transfer conformation were estimated. For room temperature (+ 20°C) in chromatophore preparations, ΔG = −5.4 kJ · M⁻¹, ΔH = 60 kJ · M⁻¹, ΔS = 0.22 kJ · M⁻¹ · K⁻¹. For Triton X-100 subchromatophore preparations, the absolute values of the above parameters are significantly lower: ΔG = −2.8 kJ · M⁻¹, ΔH = 18 kJ · M⁻¹, and ΔS = 0.075 kJ · M⁻¹ · K⁻¹. To a larger extent, the above parameters are diminished for chromatophore preparations in an 80% glycerol solution: ΔG = −1.7 kJ · M⁻¹, ΔH = 6 kJ · M⁻¹, ΔS = 0.025 kJ · M⁻¹ · K⁻¹. The data suggest the hydrophobic character of the forces that maintain the P-890-c_h complex in the electron-transfer conformation. The results obtained suggest that electron tunneling within the complex cannot occur until a specific conformational configuration of the complex is formed. The efficiency of cytochrome c_h oxidation is determined by the temperature, the degree of dehydration and the environmental conditions, whereas the transfer of an electron itself in the electron-transfer configuration is essentially independent of temperature and hydration.     

Influence of amino acid supplements on the metabolism of Clostridium acetobutylicum

The effects of organic nitrogen on the metabolism of Clostridium acetobutylicum were investigated in batch fermentations. For this study, amino acids were added to a chemically defined medium in groups from the same biosynthetic pathways. In all cases the addition of amino acids shifted the solvent ratio to higher butanol production at the expense of that of acetone (except for the glutamic acid group) and ethanol (except for histidine). Highest biomass production was obtained from media containing aromatic amino acids and histidine (4.57 g · l⁻¹ and 5.4 g · l⁻¹, respectively). However, the solvent production (ca. 20 g · l⁻¹) and the solvent yield (ca. 33%) in both cases, were similar to those obtained from the synthetic medium. Lower values were obtained from fermentations carried out with other families of amino acids. The strongest inhibition of cell growth (1.13 g · l⁻¹) which related to the lowest solvent production (3.15 g · l⁻¹) was observed on a medium complemented with amino acids of the pyruvic acid group. During the second phase of fermentation, amino acids-complemented media caused a less efficient remetabolization of acetic and butyric acids. Highest production of acids was obtained with the aspartic acid group (7.4 g · l⁻¹). These observations suggest that amino acids can be used as a competitive nitrogen source and also modify the level of enzyme activities involved in acid and solvent production.     

Production of biomass and lutein by Chlorella protothecoides at various glucose concentrations in heterotrophic cultures

The influence of initial glucose concentrations on the production of biomass and lutein by Chlorella protothecoides CS-41 was investigated in batch cultures using both shake flasks and fermentors. The maximum biomass concentration increased from 4·9 to 31·2 g litre⁻¹ dry cells with an increase in initial glucose concentration from 10 to 80 g litre⁻¹. An even higher initial glucose concentration (100 g litre⁻¹) resulted in a lower biomass concentration, a lower specific growth rate, a lower growth yield coefficient and a considerably longer lag phase, which might be due to substrate inhibition. The initial glucose level also had a significant effect on the production of lutein. In a 3·7-litre fermentor an increase in lutein production from 19·39 to 76·56 mg litre⁻¹ was obtained with an increase in initial glucose concentration from 10 to 40 g litre⁻¹, within which range, lutein yield coefficient was constant (Y_Itn=1·90±0·02 mg g⁻¹). A simple substrate inhibition model was developed, which fitted the experimental data better than the classical Haldane model. A group of time-dependent kinetic models for algal cultivation in fermentors were also constructed, which were in good agreement with the experimental results and could be employed to predict the production of biomass and lutein, and the consumption of glucose in fermentor cultures.