Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l-h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h^?1.     

Continuous alpha-amylase production using Bacillus amyloliquefaciens adsorbed on an ion exchange resin

Summary A method for the continuous production of extracellular alpha amylase by surface immobilized cells of Bacillus amyloliquefaciens NRC 2147 has been developed. A large-pore, macroreticular anionic exchange resin was capable of initially immobilizing an effective cell concentration of 17.5 g DW/1 (based on a total reactor volume of 160 ml). The reactor was operated continuously with a nutrient medium containing 15 g/l soluble starch, as well as yeast extract and salts. Aeration was achieved by sparging oxygen enriched air into the column inlet. Fermentor plugging by cells was avoided by periodically substituting the nutrient medium with medium lacking in both soluble starch and yeast extract. This fermentor was operated for over 200 h and obtained a steady state enzyme concentration of 18700 amylase activity units per litre (18.7 kU/l), and an enzyme volumetric productivity of 9700 amylase activity units per litre per hour (9.7 kU/l-h). Parallel fermentations were performed using a 2 l stirred vessel fermentor capable of operation in batch and continuous mode. All fermentation conditions employed were identical to those of the immobilized cell experiments in order to assess the performance of the immobilized cell reactor. Batch stirred tank operation yielded a maximum amylase activity of 150 kU/l and a volumetric productivity of 2.45 kU/l-h. The maximum cell concentration obtained was 5.85 g DW/l. Continuous stirred tank fermentation obtained a maximum effluent amylase activity of 6.9 kU/l and a maximum enzyme volumetric productivity of 2.73 kU/l-h. Both of these maximum values were observed at a dilution rate of 0.345 l/h. The immobilized cell reactor was observed to achieve larger volumetric productivities than either mode of stirred tank fermentation, but achieved an enzyme activity concentration lower than that of the batch stirred tank fermentor.     

Exopolysaccharide biosynthesis by a fast-producing strain of Aureobasidium pullulans

Summary The mutant strain Aureobasidium pullulans ICCF-68 was able to produce in batch fermentation on a glucose medium of 80 g/l, exopolysaccharide at high volumetric productivity and final concentration (1.05 g/l.h and 50.2 g/l, respectively). A specific pH pattern and very high oxygen requirement were shown.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

Continuous-flow fermentation of banana fruit pulp sugar into ethanol by carrageenan-immobilized yeast

Summary The extracted juice of ripe Cavendish banana fruit pulp, which contained about 126 g/l total sugars, was fermented into ethanol by yeast immobilized on kappa-carrageenan. The volumetric productivity and fermentation efficiency were about 15 g/l-h and 94%, respectively. The concentrations of alcohol and residual sugar in the beer were 54 g/l and 12.8–14.5 g/l, respectively.     

Performance,kinetics, and substrate utilization in a continuous yeast fermentation with cell recycle by ultrafiltration membranes

Summary A continuous single stage yeast fermentation with cell recycle by ultrafiltration membranes was operated at various recycle ratios. Cell concentration was increased 10.6 times, and ethanol concentration and fermentor productivity both 5.3 times with 97% recycle as compared to no recycle. Both specific growth rate and specific ethanol productivity followed the exponential ethanol inhibition form (specific productivity was constant up to 37.5 g/l of ethanol before decreasing), similar to that obtained without recycle, but with greater inhibition constants most likely due to toxins retained in the system at hight recycle ratios.By analyzing steady state data, the fractions of substrate used for cell growth, ethanol formation, and what which were wasted were accounted for. Yeast metabolism varied from mostly aerobic at low recycle ratios to mostly anaerobic at high recycle ratios at a constant dissolved oxygen concentration of 0.8 mg/kg. By increasing the cell recycle ratio, wasted substrate was reduced. When applied to ethanol fermentation, the familiar terminology of substrate used for Maintenance must be used with caution: it is not the same as the wasted substrate reported here.A general method for determining the best recycle ratio is presented; a balance among fermentor productivity, specific productivity, and wasted substrate needs to be made in recycle systems to approach an optimal design.Nomenclature B Bleed flow rate, l/h - C _T Concentration of toxins, arbitrary units - D Dilution rate, h^-1 - F Filtrate or permeate flow rate, removed from system, l/h - F _o Total feed flow rate to system, l/h - K _s Monod form constant, g/l - P Product (ethanol) concentration, g/l - P _o Ethanol concentration in feed, g/l - PP} Adjusted product concentration, g/l - PD Fermentor productivity, g/l-h - R Recycle ratio, F/F _o - S Substrate concentration in fermentor, g/l - S _o Substrate concentration in feed, g/l - V Working volume of fermentor, l - V _MB Viability based on methylene blue test - X Cell concentration, g dry cell/l - X _o Cell concentration in feed, g/l - Y _ATP Cellular yield from ATP, g cells/mol ATP - Y _ATPS Yield of ATP from substrate, mole ATP/mole glucose - Y _G True growth yield or maximum yield of cells from substrate, g cell/g glucose - Y _P Maximum theoretical yield of ethanol from glucose, 0.511 g ethanol/g glucose - Y _P/S Experimental yield of product from substrate, g ethanol/g glucose - Y _x/s Experimental yield of cells from substrate, g cell/g glucose - S _NP/X Non-product associated substrate utilization, g glucose/g cell - k ₁, k₂, k₃, k₄ Constants - k ₁ ^APP , k ₂ ^APP Apparent k ₁, k₃ - k ₁ ^TRUE True k ₁ - m Maintenance coefficient, g glucose/g cell-h - m ^* Coefficient of substrate not used for growth nor for ethanol formation, g glucose/g cell-h - Specific growth rate, g cells/g cells-h, reported as h^-1 - _m Maximum specific growth rate, h^-1 - v Specific productivity, g ethanol/g cell-h, reported as h^-1 - v _m Maximum specific productivity, h^-1     

Stability studies and effect of the initial oleic acid concentration on lipase production by Candida rugosa

The production of lipase by Candida rugosa in batch cultures was studied. The initial concentration of the carbon source employed, oleic acid, had an important effect on the final lipolytic activity levels. The maximum lipase/substrate yield and specific productivity obtained correspond to an initial oleic acid concentration of 2 g/l. At higher concentrations, up to 8 g/l oleic acid, specific productivity decreased. Lipase production was not observed below 1 g/l oleic acid. Lipase inactivation in culture broth due to surface forces and shear stress at the gas/liquid interface was not observed. There was no shear stress denaturation at stirring rates of 250, 500 and 750 rpm. No temperature inactivation was detected up to 50° C. Two different lipases with a similar molecular weight of 60kDa were purified from culture broth.     

Isolation and characterization of the exopolysaccharide produced by Daedalea quercina

The production of exopolysaccharide (EPS) by a strain of the basidiomycete Daedalea quercina was investigated. Of seven different carbon sources, glucose and dextrins gave the highest crude polysaccharide yield (4.7–5 g l^–1, 55–60% carbohydrate content) in shake-flask cultures, at 14 days of fermentation. Experiments carried out in a 10 l fermenter, at two different agitation speeds, gave the best results at 300 rpm, resulting in 12–14 g l^–1 of crude exopolysaccharide in 9–11 days. Fractionation of the EPS samples, carried out by tangential flow ultrafiltration, evidenced a single EPS fraction (MW >30 000 Da) in samples from glucose, while two fractions (MW > 30 000 Da and 30 000 > MW > 10 000 Da) were present in samples from dextrins. Fractions characterization by HPLC and proton NMR spectroscopy revealed diversity in composition and structure in the obtained EPS: from glucose mainly an -linked mannan, and from dextrins mainly an - and -linked glucan.     

Production of beta-fructofuranosidase with transfructosylating activity for fructooligosaccharides synthesis by Aspergillus japonicus NTU-1249.

Microbial beta-fructofuranosidases with transfructosylating activity can catalyze the transfructosylation of sucrose and synthesize fructooligosaccharides. Aspergillus japonicus NTU-1249 isolated from natural habitat was found to produce a significant amount of beta-fructofuranosidase with high transfructosylating activity and to have the potential for industrial production of fructooligosaccharides. In order to improve it's enzyme productivity, the medium composition and the cultivation conditions for A. japonicus NTU-1249 were studied. A. japonicus NTU-1249 can produce 83.5 units of transfructosylating activity per ml broth when cultivated in a shaking flask at 28 degrees C for 72 hours with a modified medium containing 80 g/l sucrose, 15 g/l soybean flour, 5 g/l yeast extract and 5 g/l NaCl at an initial pH of 6.0. The enzyme productivity was also optimized by submerged cultivation in a 5-litre jar fermentor with aeration at 1.5 vvm and agitation at 500 rpm. Under these operating conditions, the productivity of transfructosylating activity increased to 185.6 U/ml. Furthermore, the transfructosylating activity was improved to 256.1 U/ml in 1,000-litre pilot-scale fermentor. Enzymatic synthesis of fructooligosaccharides by beta-fructofuranosidase from A. japonicus NTU-1249 was performed in batch type by adding 5.6 units of transfructosylating activity per gram of sucrose to a 50% (w/v) sucrose solution at pH 5.0 and 50 degrees C. The yield of fructooligosaccharides was about 60% after reaction for 24 hours, and the syrup produced contained 29.8% (w/v) fructooligosaccharides, 15.2% (w/v) glucose and 5.0% (w/v) sucrose.     

Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy

The effect of agitation speeds on the performance of producing pyruvate by a multi-vitamin auxotrophic yeast, Torulopsis glabrata, was investigated in batch fermentation. High pyruvate yield on glucose (0.797 g g(-1)) was achieved under high agitation speed (700 rpm), but the glucose consumption rate was rather low (1.14 g l(-1) h(-1)). Glucose consumption was enhanced under low agitation speed (500 rpm), but the pyruvate yield on glucose decreased to 0.483 g g(-1). Glycerol production was observed under low agitation speed and decreased with increasing agitation speed. Based on process analysis and carbon flux distribution calculation, a two-stage oxygen supply control strategy was proposed, in which the agitation speed was controlled at 700 rpm in the first 16 h and then switched to 500 rpm. This was experimentally proven to be successful. Relatively high concentration of pyruvate (69.4 g l(-1)), high pyruvate yield on glucose (0.636 g g(-1)), and high glucose consumption rate (1.95 g l(-1)h(-1)) were achieved by applying this strategy. The productivity (1.24 g l(-1) h(-1)) was improved by 36%, 23% and 31%, respectively, compared with fermentations in which agitation speeds were kept constant at 700 rpm, 600 rpm, and 500 rpm. Experimental results indicate that the difference between the performances for producing pyruvate under a favorable state of oxygen supply (dissolved oxygen concentration >50%) was caused by the different regeneration pathways of NADH generated from glycolysis.     

Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus

Kluyveromyces marxianus DMKU 3-1042, isolated by an enrichment technique in a sugar cane juice medium supplemented with 4% (w/v) ethanol at 35 degrees C, produced high concentrations of ethanol at both 40 and 45 degrees C. Ethanol production by this strain in shaking flask cultivation in sugar cane juice media at 37 degrees C was highest in a medium containing 22% total sugars, 0.05% (NH(4))(2)SO(4), 0.05% KH(2)PO(4), and 0.15% MgSO(4).7H(2)O and having a pH of 5.0; the ethanol concentration reached 8.7% (w/v), productivity 1.45 g/l/h and yield 77.5% of theoretical yield. At 40 degrees C, a maximal ethanol concentration of 6.78% (w/v), a productivity of 1.13 and a yield 60.4% of theoretical yield were obtained from the same medium, except that the pH was adjusted to 5.5. In a study on ethanol production in a 5l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.2 vvm throughout the fermentation, K. marxianus DMKU 3-1042 yielded a final ethanol concentration of 6.43% (w/v), a productivity of 1.3g/l/h and a yield of 57.1% of theoretical yield.     

Direct bioconversion of rice residue from canteen waste into lipids by new amylolytic oleaginous yeast Sporidiobolus pararoseus KX709872

The new amylolytic oleaginous red yeast, Sporidiobolus pararoseus KX709872, produced both α-amylase (540?±?0.09?mU/mL) and amyloglucosidase (23?±?0.00?mU/mL) and showed good ability to directly convert rice residue from canteen waste to biomass and lipids. Effects of medium composition and cultivation conditions on growth and lipid accumulation for strain KX709872 were investigated under shaking flask and upscaling levels. At C?:?N ratio of 25?:?1, pH 5.45, 22.36°C, and 199.40?rpm for 7 days, volumetric production of biomass and lipids, lipid content, and lipid productivity reached 17.69?±?0.44, 8.35?±?0.19?g/L, 49.48?±?0.41% (w/w), and 1.67?±?0.11?g/L/day, respectively. Production of lipids was also implemented in 5.0-L stirred tank bioreactor with 2.5?L of optimized medium at 300?rpm and 3.0 vvm for 5 days. Volumetric production of biomass and lipids, lipid content, and lipid productivity were 16.33?±?0.49, 8.75?±?0.13?g/L, 56.61?±?0.04% (w/w), and 2.19?±?0.03?g/L/day, respectively. Meanwhile, the fatty acids of lipids from strain KX709872 had high oleic acid content (60?62%) which was similar to those of vegetable oils, indicating that these lipids are promising as an alternative biodiesel feedstock. Moreover, the biodiesel derived from lipids of strain KX709872 had properties satisfying the criteria of ASTM D6751 and EN 14214 standards.     

Fed-batch fermentation to produce oligonucleotides from Kluyveromyces marxianus grown on whey

Oligonucleotides (ON) extracted from yeasts are used as antiviral agents, immunostimulators, and flavour enhancers. Fed-batch fermentation of cheese whey by Kluyveromyces marxianus was carried out to produce high biomass yields to extract ON. K marxianus was grown for 20 h in medium containing 5% (w/v) dehydrated whey, at 30°C (pH 4.5), with agitation (350 rpm), and under aeration (1.0–2.0 vvm). After 20 h, media containing 10–15% (w/v) of dehydrated whey were added at different flow rates (180–230 ml/h). Samples were analyzed at 6–8 h intervals for cell count, lactose consumption, and ethanol production. Maximum production of biomass (28.13 g/l), yield (0.58 g/g), productivity (2.42 g/l per h), and specific growth rate (0.63 1/h) were obtained when medium containing 15% (w/v) of whey was added at 180 ml/h under 2 vvm aeration. Fed-batch fermentation converted 95% of whey lactose into biomass.     

Xylitol production by <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> in brewery spent grain dilute-acid hydrolysate: effect of supplementation

A brewery spent-grain hemicellulosic hydrolysate was used for xylitol production by Debaryomyces hansenii. Addition of 6 g yeast extract/l increased the xylitol yield to 0.57 g/g, and productivity to 0.51 g/l h that were, respectively, 1.4 -and 1.8-times higher than the values obtained with non-supplemented hydrolysate. When corn steep liquor was combined with 3 g yeast extract/l, the highest xylitol yield, 0.58 g/g, was obtained with a similar productivity.     

Exopolysaccharide production and mycelial growth in an air-lift bioreactor using Fomitopsis pinicola

For effective exopolysaccharide production and mycelial growth by a liquid culture of Fomitopsis pinicola in an air-lift bioreactor, the culture temperature, pH, carbon source, nitrogen source, and mineral source were initially investigated in a flask. The optimal temperature and pH for mycelial growth and exopolysaccharide production were 25degrees C and 6.0, respectively. Among the various carbon sources tested, glucose was found to be the most suitable carbon source. In particular, the maximum mycelial growth and exopolysaccharide production were achieved in 4% glucose. The best nitrogen sources were yeast extract and malt extract. The optimal concentrations of yeast extract and malt extract were 0.5 and 0.1%, respectively. K2HPO4 and MgSO4 x 7H2O were found to be the best mineral sources for mycelial growth and exopolysaccharide production. In order to investigate the effect of aeration on mycelial growth and exopolysaccharide production in an air-lift bioreactor, various aerations were tested for 8 days. The maximum mycelial growth and exopolysaccharide production were 7.9 g/l and 2.6 g/l, respectively, at 1.5 vvm of aeration. In addition, a batch culture in an air-lift bioreactor was carried out for 11 days under the optimal conditions. The maximum mycelial growth was 10.4 g/l, which was approximately 1.7-fold higher than that of basal medium. The exopolysaccharide production was increased with increased culture time. The maximum concentration of exopolysaccharide was 4.4 g/l, which was about 3.3-fold higher than that of basal medium. These results indicate that exopolysaccharide production increased in parallel with the growth of mycelium, and also show that product formation is associated with mycelial growth. The developed model in an air-lift bioreactor showed good agreement with experimental data and simulated results on mycelial growth and exopolysaccharide production in the culture of F pinicola.     

Influence of cultivation conditions on lipid production by Cryptococcus albidus

Summary Cryptococcus albidus var. albidus CBS 4517 was able to accumulate lipid under nitrogen-limited as well as excess-nitrogen conditions. The highest lipid-producting capacity was, however, observed in nitrogen-limited cultivations. In nitrogen-limited batch cultures, a lipid content of 34% (w/w) in biomass and a maximum specific lipid productivity of 37 mg lipid/g lipid-free biomass·h, was determined. The yield of lipid from glucose was about 0.15 g/g in nitrogen-limited and 0.11 g/g in excess-nitrogen cultures.In a nitrogen-limited fed-batch culture, 12.4 g/l lipid was produced at 90 h of cultivation and the cells contained 46.3% (w/w) lipid.Higher lipid yield and cellular lipid content were observed when inorganic nitrogen sources were used compared with organic. The choice of carbon source was seen to influence growth as well as lipid production and the highest yields of lipid were obtained when glucose, maltose or mannitol was used.A cultivation temperature of 20°C provided the highest lipid productivity compared to 25°C and 30°C. Addition of citrate to the growth medium was seen to have a stimulating effect on the specific lipid productivity.     

Ethanol production at 45°C by Kluyveromyces marxianus IMB3 immobilized in magnetically responsive alginate matrices

Summary The thermotolerant yeast, Kluyveromyces marxianus IMB3 produced 11g ethanol/l during growth at 45°C on media containing 4% (w/v) lactose when immobilized in alginate beads whereas the free cells produced 5g ethanol/l. A magnetically responsive biocatalyst, prepared by incorporating Fe₃O₄ into the alginate matrix increased ethanol production to 12g/l in batch-fed reactors. Ethanol concentrations were further increased to a maximum of 18g/l by immobilization of the endogenous K. marxianus -galactosidase to the Fe₃O₄ particles prior to inclusion into the alginate matrix. Maximum ethanol productivity by the system was 87% of the maximum theoretical yield.     

Production of poly(β-hydroxybutyric acid) and exopolysaccharide by Azotobacter beijerinckii WDN-01

The physico-chemical factors influencing the production of poly(-hydroxybutyric acid) [PHB] and exopolysaccharide (EPS) by a yellow pigmented Azotobacter beijerinckii strain WDN-01 were investigated. Under N-free condition with excess carbon, PHB accumulation attained its maximum at the late exponential phase followed by a sharp decline while EPS production was more or less parallel with growth. Polymer synthesis, however, was carbon-source-specific, the highest yield of PHB (2.73 g/l) and EPS (1.5 g/l) was obtained with 3% (w/v) glucose and mannitol respectively. Organic N-sources enhanced PHB production significantly, but inorganic nitrogenous compounds were inhibitory to both PHB and EPS synthesis. At optimum K₂HPO₄ concentration, the polymer yield was attributed to biomass yield. Oxygen-limiting conditions, irrespective of carbon sources favoured production of PHB and EPS.     

A novel two-step fermentation process for improved arachidonic acid production by Mortierella alpina

A novel two-step fermentation process was developed to enhance arachidonic acid (ARA) production by Mortierella alpina ME-1 in a 5 l fermentor. Agitation speed and aeration rate were adjusted from 180 to 40 rpm and from 0.6 to1 vvm, respectively, after 5 days cultivation, to decrease physical damage to the mycelia and to extend the stationary phase. Moreover, 3% (w/v) and 2% (w/v) ethanol were fed after 5 and 7 days cultivation, respectively, to enhance ARA content of total lipid. Eventually, an ARA yield of 19.8 g/l was achieved, which was 1.7 times higher than that of a one-step fed-batch cultivation.     

High scleroglucan production by Sclerotium rolfsii: Influence of medium composition

Scleroglucan production by Sclerotium rolfsii was markedly affected by the C-source concentration, showing a highest value with 150 g sucrose l–1. Production was also influenced by the N-source, being considerably higher in media containing NO3– than in those containing NH4, which had a clear inhibitory effect. Once defined the optimum culture medium composition, the highest exopolysaccharide production (ca. 26 g scleroglucan l–1) was achieved after 72 h of fermentation at shake flask scale. High values of yield (Yp/c = 0.49), productivity (Pr = 0.365 g l–1 h–1) and specific productivity (Pr/x = 0.031 g (g biomass)–1 h–1) were observed, and productivity was 1.5 times further increased by scalling-up to fermenter scale. Addition of L-threonine, sunflower oil and ascorbic acid diminished exopolysaccharide production. © Rapid Science Ltd. 1998