Production of red pigments byMonascus purpureus in submerged culture

For the purpose of mass producingMonascus red pigments optimum medium composition and environmental conditions were investigated in submerged flask cultures. The optimum carbon and nitrogen sources were determined to be 30 g/L of glucose and 1.5 g/L of monosodium glutamate (MSG). Of the three metals examined, Fe²⁺ showed the stronges stimulatory effect on pigment production and some stimulatory effect was also found in Mn²⁺. Optimum pH and agitation speed were determined to be 6.5 and 700 rpm, respectively. Under the optimum culture conditions batch fermentation showed that the maximum biomass yield and specific productivity of red pigments were 0.20 g DCW/g glucose and, 32.5 OD₅₀₀ g DCW⁻¹ h⁻¹, respectively.     

Effect of pH and nitrogen source on pigment production by Monascus purpureus

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions. Correspondence to: M. R. Johns     

Production of red pigments byMonascus purpureus in solid-state culture

To maximize and sustain the productivity ofMonascus pigments, various environmental and nutritional parameters, such as the initial moisture content, pH, inoculum size, sample size, and nutrient supplement, that influence pigment production were evaluated in solid-state cultures as follows: initial moisture content, 50%; pH, 6.0; inoculum size 1 x 10⁴ spore cells (grams of dry solid substrate)⁻¹; sample size, 300 g. All supplementary nutrients (carbon, nitrogen, and mineral sources) added has inhibitory effects on the cell growth and red pigment production. In open tray culture the maximum biomass yield and specific productivity of red pigments were 223 mg DCW (grams of initial dry substrate)⁻¹ and, 47.6 OD₅₀₀ (DCW grams)⁻¹ h⁻¹, respectively.     

Biological activity of Bifidobacterium longum in response to environmental pH

The influence of environmental pH on biological activity of Bifidobacterium longum CRL 849 grown in MRS-raffinose was evaluated. At pH 6.0, 5.5 and 5.0, raffinose was completely consumed by this microorganism, showing different consumption rates at each pH value (between 3.03 and 0.76 mmol l⁻¹ h⁻¹). At pH 4.5, the growth was lowest. The removal of raffinose was due to the α-galactosidase (α-gal) activity of this bifidobacteria, which was highest at pH 6.0–5.5 (1,280–1,223 mU ml⁻¹). The production of β-glucosidase (β-glu) showed a similar pattern to α-gal activity with major values. The yield of organic acids produced during raffinose consumption was also highest at pH 6.0–5.5. The results of this study will allow the selection of the optimum growth conditions of B. longum CRL 849, with elevated levels of α-gal to be used in the reduction of nondigestible α-oligosaccharide in soy products and β-glu activities involved in isoflavone conversion to bioactive forms when used as starter culture.     

Kinetic control of ethanol production by Zymomonas mobilis

Summary Zymomonas mobilis UQM 2716 was grown anaerobically in continuous culture (D = 0.1/h; 30° C) 3nder glucose or nitrogen limitation at pH 6.5 or 4.0. The rates of glucose consumption and ethanol production were lowest during glucose-limited growth at pH 6.5, but increased during growth at pH 4.0 or under nitrogen limitation, and were highest during nitrogen-limited growth at pH 4.0. The uncoupling agent CCCP substantially increased the rate of glucose consumption by glucose-limited cultures at pH 6.5, but had much less effect at pH 4.0. Washed cells also metabolised glucose rapidly, irrespective of the conditions under which the original cultures were grown, and the rates were variably increased by low pH and CCCP. Broken cells exhibited substantial ATPase activity, which was increased by growth at low pH. It was concluded that the fermentation rates of cultures growing under glucose or nitrogen limitation at pH 6.5, or under glucose limitation at pH 4.0, are determined by the rate at which energy is dissipated by various cellular activities (including growth, ATP-dependent proton extrusion for maintenance of the protonmotive force and the intracellular pH, and an essentially constitutive ATP-wasting reaction that only operates in the presence of excess glucose). During growth under nitrogen limitation at pH 4.0 the rate of energy dissipation is sufficiently high for the fermentation rate to be determined by the inherent catalytic activity of the catabolic pathway.Abbreviations CCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - qG rate of glucose consumption (g glucose/g dry wt cells/h) - qE rate of ethanol production (g ethanol/g dry wt cells/h) - Y growth yield (g dry wt cells/g glucose) - D dilution rate Offprint requests to: C. W. Jones     

Kinetic study on succinic acid and acetic acid formation during continuous cultures of Anaerobiospirillum succiniciproducens grown on glycerol

Succinic acid-producing Anaerobiospirillum succiniciproducens was anaerobically grown in a glycerol-fed continuous bioreactor in order to investigate the physiological responses of the cell to different pH values (5.9, 6.2, or 6.5) and various dilution rates, D. In these experiments, A. succiniciproducens showed a pH-dependent glycerol consumption behavior. When pH was maintained at 5.9 or 6.5, glycerol started to accumulate even at a very low D of 0.027 h⁻¹. Succinic acid yield was not significantly affected by the pH of the culture or the Ds. However, more acetic acid formation was observed when the growth rate of A. succiniciproducens was fast on glycerol at pH 6.2 (at D ≥ 0.15 h⁻¹). The highest obtainable succinic acid/acetic acid ratio was 40:1, which was 10 times higher than that obtained by batch cultures grown on glucose. The maximum obtainable productivity of succinic acid was 2.1 g L⁻¹ h⁻¹), which was 14 times higher than that obtained by batch culture.     

Evaluation of electrocoagulation,flocculation, and sedimentation harvesting methods on microalgae consortium grown in anaerobically digested abattoir effluent

Microalgae dewatering is a major bottleneck for biomass production in a large-scale microalgal production system which accounts for 20–60% of production cost. In this study, three dewatering systems of electrocoagulation, flocculation, and pH-induced flocculation were evaluated for microalgal consortium grown in anaerobically digested abattoir effluent at pH 6.5 and 9.5. At the shortest time (15 min) and the highest current density (0.08 A cm^?2), the highest microalgae recoveries of 78 and 84% were obtained with the corresponding power consumptions of 1.25 and 1.07 kWh kg^?1 for cultures at pH 6.5 and 9.5. For microalgae suspension at pH 6.5, the highest biomass recovery of 77% was obtained when 100 mg L^?1 of FeCl₃·6H₂O (after 15 min) or 100 mg L^?1 of Al₂(SO₄)₃·18H₂O (after 30 min) was added. However, microalgal recoveries significantly increased when FeCl₃·6H₂O or Al₂(SO₄)₃·18H₂O was used with the culture at pH 9.5. pH-Induced experiments showed that cultures adjusted at pH 10.5 had 36% higher biomass recovery compared to that in cultures at pH 8.5 after 2 h. The results of this study showed that cultures at higher pH (9.5) had a better microalgae recovery in all dewatering systems than cultures at lower pH (6.5).

     

Composition and accumulation of secondary carotenoids in Chlorococcum sp.

A locally isolated Chlorococcum sp. could accumulate astaxanthin and its esters as secondary carotenoids. The secondary carotenoids could reach a concentration of 5.2 mg g⁻¹ d. wt, and were located in the cytoplasm and chloroplast as globules. Cells grew best at pH 8.0 and 30 °C, at which the growth rate was about 0.066 h⁻¹. Acidic condition (pH 5.5 and 6.5) and slightly elevated temperature (35 °C) enhanced the cellular accumulation of astaxanthin. Outdoor studies indicated that Chlorococcum sp. grew well in a tubular photobioreactor. In medium containing 2 mM and 10 mM NH₄CI, the cellular contents of total secondary carotenoids and astaxanthin reached similar levels (5.0 mg g⁻¹ d. wt and 2.0 mg g⁻¹ d. wt, respectively) in the 15 days of cultivation, while the yield of total secondary carotenoids and astaxanthin in 10 mM NH₄CI were higher (45 mg L⁻¹ and 18 mg L⁻¹, respectively). The advantages of tolerance to high temperature and extreme pH values, relative fast growth rate and ease of cultivation in outdoor system suggest that Chlorococcum sp. could be a potential candidate for mass production of secondary carotenoids in particular astaxanthin. This revised version was published online in September 2006 with corrections to the Cover Date.     

Butanol production from wheat straw hydrolysate using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In these studies, butanol (acetone butanol ethanol or ABE) was produced from wheat straw hydrolysate (WSH) in batch cultures using Clostridium beijerinckii P260. In control fermentation 48.9 g L⁻¹ glucose (initial sugar 62.0 g L⁻¹) was used to produce 20.1 g L⁻¹ ABE with a productivity and yield of 0.28 g L⁻¹ h⁻¹ and 0.41, respectively. In a similar experiment where WSH (60.2 g L⁻¹ total sugars obtained from hydrolysis of 86 g L⁻¹ wheat straw) was used, the culture produced 25.0 g L⁻¹ ABE with a productivity and yield of 0.60 g L⁻¹ h⁻¹ and 0.42, respectively. These results are superior to the control experiment and productivity was improved by 214%. When WSH was supplemented with 35 g L⁻¹ glucose, a reactor productivity was improved to 0.63 g L⁻¹ h⁻¹ with a yield of 0.42. In this case, ABE concentration in the broth was 28.2 g L⁻¹. When WSH was supplemented with 60 g L⁻¹ glucose, the resultant medium containing 128.3 g L⁻¹ sugars was successfully fermented (due to product removal) to produce 47.6 g L⁻¹ ABE, and the culture utilized all the sugars (glucose, xylose, arabinose, galactose, and mannose). These results demonstrate that C. beijerinckii P260 has excellent capacity to convert biomass derived sugars to solvents and can produce over 28 g L⁻¹ (in one case 41.7 g L⁻¹ from glucose) ABE from WSH. Medium containing 250 g L⁻¹ glucose resulted in no growth and no ABE production. Mixtures containing WSH + 140 g L⁻¹ glucose (total sugar approximately 200 g L⁻¹) showed poor growth and poor ABE production. Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Heterotrophic high-cell-density fed-batch and continuous-flow cultures of <Emphasis Type="Italic">Galdieria sulphuraria</Emphasis> and production of phycocyanin

Production of biomass and phycocyanin (PC) were investigated in highly pigmented variants of the unicellular rhodophyte Galdieria sulphuraria, which maintained high specific pigment concentrations when grown heterotrophically in darkness. The parental culture, G. sulphuraria 074G was grown on solidified growth media, and intensely coloured colonies were isolated and grown in high-cell-density fed-batch and continuous-flow cultures. These cultures contained 80–110 g L⁻¹ biomass and 1.4–2.9 g L⁻¹ PC. The volumetric PC production rates were 0.5–0.9 g L⁻¹ day⁻¹. The PC production rates were 11–21 times higher than previously reported for heterotrophic G. sulphuraria 074G grown on glucose and 20–287 times higher than found in phototrophic cultures of Spirulina platensis, the organism presently used for commercial production of PC.     

High-Efficiency Conversion of Pyruvate to Acetoin by Lactobacillus plantarum during pH-Controlled and Fed-Batch Fermentations

The influence of pH on the type and concentration of metabolites produced from pyruvate by Lactobacillus plantarum ATCC 8014 was examined in pH-controlled fermentors at pH values of 4.5 to 6.5. Specific growth rates, cell dry weights, and diacetyl concentrations were highest at pH 5.5, with values of 0.78 h⁻¹, 190 mg/liter, and 1.2 mM, respectively. While the conversion efficiency (millimoles of acetoin formed per millimoles of pyruvate utilized) was highest (94.6%) at pH 4.5, acetoin levels were similar (20 mM) between pH 4.5 and 5.5. Feeding stationary-phase cells exogenous pyruvate increased acetoin levels to 78 mM.     

High yield mixotrophic cultures of the marine microalga Tetraselmis suecica (Kylin) Butcher (Prasinophyceae)

The effects of three organic compounds were tested on one of the most used marine micro-algae in the aquaculture of molluscs and crustaceans, Tetraselmis suecica. Studies were made in axenic conditions with yeast extract, peptone and glucose added to the culture medium, each alone, in combinations of two or all together. Medium without any organic compound was used for the control. Cultures containing yeast extract grew best, reaching maximum cell density of 3.79 × 10⁶ and 3.84 × 10⁶ cells ml⁻¹. The organic carbon source affected the biochemical composition. The components most affected were the carbohydrates, with values between 6.5 pg cell⁻¹ in control cultures and 48.5 pg cell⁻¹ in glucose cultures. Protein content ranged between 27.5 pg cell⁻¹ in control cultures and 88.6 pg cell⁻¹ in yeast + glucose + peptone cultures. The lipid content changed little. Maximum protein yields were reached in cultures with yeast + glucose and with yeast - glucose - peptone, with values of 24.6 and 28.2 mg 1⁻¹ d⁻¹, respectively. These values are 22 and 25 times those in control cultures. A maximum carbohydrate yield of 7.9 mg carbohydrate per litre per day was obtained in yeast + glucose + peptone cultures, 27 times that in the control cultures. The maximum lipid yield was obtained with yeast + glucose + peptone and yeast + glucose. Maximum energy values were 308 kcal 1⁻ in yeast extract - glucose - peptone cultures and 279 kcal 1⁻¹ in yeast extract + glucose cultures. Gross energy values in control cultures were 24.5 kcal 1⁻¹, but peptone cultures presented the minimum energy value, 22 kcal 1⁻¹. The yeast extract: glucose ratio in the culture medium was optimized. A ratio 2:1 produced the best yields in cells, protein, carbohydrate and gross energy.     

Growth conditions of clostridium perfringens type B for production of toxins used to obtain veterinary vaccines

The diseases caused for Clostridium perfringens are generically called enterotoxemias because toxins produced in the intestine may be absorbed into the general circulation. C. perfringens type B, grown in batch fermentation, produced toxins used to obtain veterinary vaccines. Glucose in concentrations of 1.4–111.1 mM was used to define the culture medium. The minimum concentration for a satisfactory production of vaccines against clostridial diseases was 55.6 mM. Best results were brought forth by meat and casein peptones, both in the concentration 5.0 g l^?1 in combination with glucose and a culture pH maintained at 6.5 throughout the fermentation process. The production of lactic, acetic and propionic organic acids was observed. Ethanol was the metabolite produced in the highest concentration when cultures maintained steady pH of 6.5 with exception of cultures with initial glucose concentration of 1.4 mM, where the highest production was of propionic acid. Maximal cell concentration and the highest toxin title concomitantly low yield coefficient to organic acids and ethanol were obtained using basal medium containing 111.1 mM glucose under a controlled pH culture (pH) 6.5 in batch fermentations of C. perfringens type B. These data contribute to improve process for industrial toxin production allowing better condition to produce a toxoid vaccine.     

Effect of pH on growth and ethanol production byZymomonas

Summary In a mineral salts medium containing yeast extract, NH₄Cl and glucose (50g/L), the pH range producing the fastest growth ofZ. mobilis was 5.5–6.5 with an apparent optimum at 6.5. At constant growth rate of 0.15hr^–1, the specific rates of glucose utilization (q_s) and ethanol production (q_p) were relatively unaffected by pH over the range 7.0–5.5 but increased sharply as the pH was further decreased below 5.5 to 4.0. Under these conditions the ethanol yield was unaffected by pH over the range 4.0–6.5 but decreased markedly at pH of 7.     

Effects of oxygen volumetric mass transfer coefficient and pH on lipase production by Staphylococcus warneri EX17

The principal objectives of this study were to evaluate the kinetics of lipase production by Staphylococcus warneri EX17 under different oxygen volumetric mass transfer coefficients (k_La) and pH conditions in submerged bioreactors, using glycerol (a biodiesel by-product) as a carbon source. Cultivations were conducted at different k_La (26, 38, 50, and 83 h⁻¹) and pH values (6.0, 7.0, and 8.0). The optimal k_La and pH were 38 h⁻¹ and 7.0, respectively. Under these conditions, the maximal cell production obtained was 8.0 g/L, and the volumetric and specific lipase production reached high levels of activity, approximately 800 U/L and 150 U/g cell, respectively, after 12 h of cultivation. This result was approximately five times higher than that obtained in the shake flask cultures. The relationship between cell growth and lipase production was found to be associated with growth by the Luedeking-Piret model.     

Effects of initial medium pH on growth and metabolism ofCatharanthus roseus hairy root cultures —A study with31P and13C NMR spectroscopy

Summary Hairy root cultures ofCatharanthus roseus were grown for 26 days in half-strength Gamborg's B5 liquid medium at different initial pH values of 4.2, 5.7, 6.5, and 7.3. Maximum growth was obtained for cultures with an initial medium pH 6.5. The lowest growth rate was found in cultures at initial pH values of 4.2 and 7.3. Roots in cultures at initial pH of 4.2 had a thickened and stunted morphology in contrast to the other cultures. Also, cultures at initial medium pH of 4.2 exhibited an increase in medium pH in the first few days instead of the characteristic acidification. All cultures maintained a cytoplasmic pH of 7.4 throughout the growth cycle. However, vacuolar pH was 5.1–5.2 in cultures of initial pH 4.2, as opposed to 5.4–5.5 for other cultures. Sucrose was hydrolyzed completely to glucose and fructose by day 26 except for cultures at initial pH of 7.3. Glucose was the preferred substrate throughout the growth cycle for cultures with initial pH values of 7.3 and 6.5, after day 20 for an initial pH of 5.7, and after day 26 for pH 4.2.     

Selection and Optimization of Culture Medium for Exopolysaccharide Production by Coriolus (Trametes) Versicolor

Summary Coriolus versicolor is a medicinal fungus producing exopolysaccharides (EPS). Five well-defined culture media were studied to select the medium that maximizes production of EPS by C. versicolor. Biomass, reducing sugars and EPS concentrations along with the rheological behaviour of the broth were followed during fermentations lasting 9 days. The yeast malt extract medium (YM) was shown to yield the highest production of EPS. Fermentation conditions with YM medium were further investigated to optimize EPS production by C. versicolor. An experimental design to do this was adopted, in which the effects of pH and initial substrate concentration were considered. The effects of initial glucose concentration (5, 15 and 25 g l⁻¹) and pH (4.0, 5.5 and 7.0) were evaluated. The initial glucose concentration was found to be the most important factor in EPS production and also cell growth.     

Cultivation ofLactobacillus crispatus KLB46 isolated from human vagina

Bacterial vaginosis can be treated by restoring the normal vaginal flora using lactobacilli.Lactobacillus crispatus KLB46 that was isolated from the human vagina has a strong antimicrobial activity and was grown in a batch and in a continuous fermentor. During batch cultivation, the maximum specific growth rate ofL. crispatus KLB 46 was 0.63 h⁻¹ and the highest viable cell count (1.9×10⁹ CFU/mL) was obtained at pH 5.5.L. crispatus KLB 46 did not grow well at either pH 3.5 or 7.5. During continuous cultivation, the highest viable cell count (1.53×10⁹ CFU/mL) was obtained at a dilution rate of 0.32 h⁻¹. However, the maximum productivity of viable cells was obtained at a dilution rate of 0.52 h⁻¹, and was 7.33×10¹¹ CFU L⁻¹ h⁻¹, that is approximately 5 times higher than that obtained from batch culture.     

The effect of pH on nitrogen supply, cell lysis, and solvent production in fermentations of Clostridium acetobutylicum

In batch fermentations of C. acetobutylicum, with 5 g/L yeast extract and 50mM glucose, the ratio of ammonium to glucose affected solvent production when the pH was left to vary uncontrolled from 4.5 to 3.65. High solvent production was observed for a low ratio. When the pH was controlled at 4.5, only acids were produced for all ratio values. At a low ammonium-to-glucose ratio, solvents were produced when the pH was controlled at 3.7. Acids only were produced for a low ratio value at pH 4.0 or for a high ratio value at pH 3.7. In continuous cultures, mostly acids were produced under glucose limitation, but solvents were produced under nitrogen limitation. It was concluded that the nitrogen availability controls solvent production and that the pH affects the availability of organic nitrogen. Biomass autolysis at the stationary phase of batch cultures was reversibly inhibited at pH values less than 3.8. In batch fermentations, the overall molar growth yields on ATP (Y(ATP)) varied from 5.5 to 9.0 and the transient yields from 5.5 to 15.5. In continuous cultures, the Y(ATP) values varied from 5.5 to 14.7 under glucose limitation, and from 6.1 to 9.3 under nitrogen limitation. The Y(ATP) depended on the ammonium to glucose ratio and the culture pH, but did not show the usual dependence on the specific growth rate in batch cultures. The experiments seem to confirm the hypothesis that solvent production is controlled by the demand and availability of ATP.     

Metabolic and energetic aspects of the growth of Bacillus stearothermophilus in glucose-limited and glucose-sufficient chemostat culture

With a glucose-limited chemostat culture of Bacillus stearothermophilus, increasing the incubation temperature progressively from 45°C to 63°C led to a progressive marked increase in the maintenance rates of glucose and oxygen consumption. Hence, at a fixed low dilution rate the yield values with respect to glucose and oxygen decreased substantially with increased temperature. However, the apparent Y _glucose ^max and values did not decrease but actually increased with temperature, being highest at 63°C (i.e., close to the maximum growth temperature). With glucose-sufficient cultures growing at a fixed low dilution rate (0.2 h^–1) and at their optimum temperature (55°C), glucose and oxygen consumption rates invariably were higher than that of a corresponding glucose-limited culture. Cation (K⁺ or Mg²⁺)-limited cultures expressed the highest metabolic rates and with the K⁺ limited culture this rate was found to be very markedly temperature dependent. As the temperature was increased from 45°C to 63°C the rate of glucose consumption increased 1.8-fold, and that of oxygen consumption by 3.7-fold. The culture pH value also exerted a noticeable effect on the metabolic rate of a glucose-limited culture, particularly at the extremes of pH tolerance (5.5 and 8.5, respectively). A K⁺-limited culture was less affected with respect to metabolic rate by the culture pH value though the steady state bacterial concentration, and thus the cellular K⁺ content, changed substantially. These results are discussed in relation to previous findings of the behaviour of this organism in batch culture, and to the behaviour of other thermophilic Bacillus species in chemostat culture.