Batch and membrane-assisted cell recycling in ethanol production byCandida shehatae

Summary During xylose fermentation byCandida shehatae ATCC 22984 with batch cell recycling, the volumetric ethanol fermentation rate increased two-fold, and the xylitol production rate increased three-fold as the cell density increased to ten-fold. In continuous fermentation with membrane-assisted cell recycle, the fermentation rates increased almost linearly with increasing agitation rates up to 300 rpm. The maximum continuous ethanol production rates obtained with 90 and 200 g L^–1 xylose were respectively 2.4 and 4.4 g L^–1h^–1. The cell density was 65–70 g (dry wt) L^–1. Ethanol yields ranged from 0.26 to 0.41 g g^–1.     

Hydrolysate detoxification with activated charcoal for xylitol production by Candida guilliermondii

A detoxification method using activated charcoal with concentrated rice straw hemicellulosic hydrolysate improved the conversion of xylose to xylitol by the yeast Candida guilliermondii by 22%. This was achieved when the hydrolysate:charcoal ratio was 40 g g^–1, resulting in removal of 27% of phenolic compounds. Under this condition, the xylitol yield factor (0.72 g g^–1) and volumetric productivity (0.61 g l^–1 h^–1) were close to those attained in a semi-defined medium simulating hydrolysate sugars.     

Comparison of different pretreatments in ethanol fermentation using corn cob hemicellulosic hydrolysate with Pichia stipitis and Candida shehatae

A hemicellulosic hydrolysate was prepared with 0.3 M H₂SO₄ at 98 °C for 1 h. The total initial reducing sugar was maintained at 45 g l^–1 by synthetic xylose supplementation. The seven detoxification methods were employed including either the single addition of solid CaO (to pH 10 or 6) or its combinations with zeolite shaking. Over-liming gave the hydrolysate that was most completely fermented by Pichia stipitis and Candida shehatae at 30 °C, pH 6, among the tested methods.     

The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis

Summary The fermentation by Candida shehatae and Pichia stipitis of xylitol and the various sugars which are liberated upon hydrolysis of lignocellulosic biomass was investigated. Both yeasts produced ethanol from d-glucose, d-mannose, d-galactose and d-xylose. Only P. stipitis fermented d-cellobiose, producing 6.5 g·l^-1 ethanol from 20 g·l^-1 cellobiose within 48 h. No ethanol was produced from l-arabinose, l-rhamnose or xylitol. Diauxie was evident during the fermentation of a sugar mixture. Following the depletion of glucose, P. stipitis fermented galactose, mannose, xylose and cellobiose simultaneously with no noticeable preceding lag period. A similar fermentation pattern was observed with C. shehatae, except that it failed to utilize cellobiose even though it grew on cellobiose when supplied as the sole sugar. P. stipitis produced considerably more ethanol from the sugar mixture than C. shehatae, primarily due to its ability to ferment cellobiose. In general P. stipitis exhibited a higher volumetric rate and yield of ethanol production. This yeast fermented glucose 30–50% more rapidly than xylose, whereas the rates of ethanol production from these two sugars by C. shehatae were similar. P. stipitis had no absolute vitamin requirement for xylose fermentation, but biotin and thiamine enhanced the rate and yield of ethanol production significantly.Nomenclature _max Maximum specific growth rate, h^-1 - Q _p Maximum volumetric rate of ethanol production, calculated from the slope of the ethanol vs. time curve, g·(l·h)^-1 - q _p Maximum specific rate of ethanol production, g·(g cells·h) - Y _p/s Ethanol yield coefficient, g ethanol·(g substrate utilized)^-1 - Y _x/s Cell yield coefficient, g biomass·(g substrate utilized)^-1 - E Efficiency of substrate utilization, g substrate consumed·(g initial substrate)^-1·100     

Effect of inoculum level on xylitol production from rice straw hemicellulose hydrolysate byCandida guilliermondii

The effect of inoculum level on xylitol production byCandida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate. High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L^–1 to 2.41 g L^–1 decreased both the rate of xylose utilization and xylitol accumulation. The maximum xylitol yield (0.71 g g^–1) and volumetric productivity (0.56 g L^–1 h^–1) were reached with an inoculum level of 0.9 g L^–1. These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeastC. guilliermondii with efficiency values as high as 77% of the theoretical maximum.     

Influence of the oxygenation level on d-xylose fermentation by free and agar-entrapped cultures of Candida shehatae

This paper investigates the effects of the oxygenation level on the performance of d-xylose alcoholic fermentation by free- and immobilized-cell batch cultures of Candida shehatae (ATCC 22984). Yeast cells were immobilized in composite agar layer/microporous membrane structures. Fermentations were performed under varying oxygenation levels corresponding to different O₂ flow rates (OFRs). Low OFRs enhanced the fermentation performance of free and immobilized yeasts. The best ethanol yield coefficient, obtained at an OFR of 5 mmol O₂ h^–1 dm^–3 for both culture modes, was slightly higher (0.425 g g^–1) for immobilized cultures than for their free counterparts (0.39 g g^–1). More sustained aeration inhibited ethanol production by free and immobilized organisms. However, this inhibition was more pronounced for agar-entrapped cultures. Xylitol production of free cultures normally decreased as the OFR increased. At high OFR, however, immobilized organisms surprisingly produced more xylitol than at lower OFR or in anaerobiosis. This effect is discussed by referring to the mass transfer limitations that occur inside the immobilized-cell structures. Gel-entrapped cultures displayed higher specific and volumetric production rates of ethanol and xylitol than free-cell cultures.     

Growth kinetics ofSaccharomyces cerevisiae in batch and fedbatch cultivation using sugarcane molasses and glucose syrup from cassava starch

Growth kinetics ofSaccharomyces cerevisiae in glucose syrup from cassava starch and sugarcane molasses were studied using batch and fed-batch cultivation. The optimum temperature and pH required for growth were 30°C and pH 5.5, respectively. In batch culture the productivity and overall cell yield were 0.31 g L^–1 h^–1 and 0.23 g cells g^–1 sugar, respectively, on glucose syrup and 0.22 g L^–1 h^–1 and 0.18 g cells g^–1 sugar, respectively, on molasses. In fed-batch cultivation, a productivity of 3.12 g L^–1 h^–1 and an overall cell yield of 0.52 g cells g^–1 sugar in glucose syrup cultivation and a productivity of 2.33 g L^–1 h^–1 and an overall cell yield of 0.46 g cells g^–1 sugar were achieved in molasses cultivation by controlling the reducing sugar concentration at its optimum level obtained from the fermentation model. By using an on-line ethanol sensor combined with a porous Teflon^® tubing method in automating the feeding of substrate in the fed-batch culture, a productivity of 2.15 g L^–1 h^–1 with a yield of 0.47 g cells g^–1 sugar was achieved using glucose syrup as substrate when ethanol concentration was kept at a constant level by automatic control.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Alcoholic fermentation of glucose and xylose by Pichia stipitis,Candida shehatae,Saccharomyces cerevisiae and Zymomonas mobilis: oxygen requirement as a key factor

Summary To investigate simultaneous alcoholic fermentation of glucose and xylose derived from lignocellulosic material by separate or co-culture processes, the effect of oxygen transfer rate (OTR) on the fermentation of 50 g/l xylose by Pichia stipitis NRRL Y 7124 and Candida shehatae ATCC 22984, and the fermentation of 50 g/l glucose by Saccharomyces cerevisiae CBS 1200 and Zymomonas mobilis ATCC 10988 was carried out in batch cultures. The kinetic parameters of the xylose-fermenting yeasts were greatly dependent on the OTR. The optimum OTR values were found to be 3.9 and 1.75 mmol·1^–1·h^–1 for C. shehatae and P. stipitis, respectively. By contrast the fermentative parameters of S. cerevisiae were poorly affected by the OTR range tested (0.0–3.5 mmol·l^–1·h^–1) Under these conditions the ethanol yields ranged from 0.41 g·g^–1 to 0.45 g·g^–1 and the specific ethanol productivity was around 0.70 g·g^–1·h^–1. Z. mobilis gave the highest fermentative performance under strictly anaerobic conditions (medium continually flushed with nitrogen): under these conditions, the ethanol yield was 0.43 g·g^–1 and the average specific ethanol productivity was 2.3 g·g^–1·h^–1. Process considerations in relation to the effect of OTR on the fermentative performance of the tested strains are discussed. Offprint requests to: J. P. Delgenes     

Xylose chemostat isolates of Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose, glucose, and ethanol metabolism of the original strain

The efficient conversion of xylose-containing biomass hydrolysate by the ethanologenic yeast Saccharomyces cerevisiae to useful chemicals such as ethanol still remains elusive, despite significant efforts in both strain and process development. This study focused on the recovery and characterization of xylose chemostat isolates of a S. cerevisiae strain that overexpresses xylose reductase- and xylitol dehydrogenase-encoding genes from Pichia stipitis and the gene encoding the endogenous xylulokinase. The isolates were recovered from aerobic chemostat cultivations on xylose as the sole or main carbon source. Under aerobic conditions, on minimal medium with 30 g l^–1 xylose, the growth rate of the chemostat isolates was 3-fold higher than that of the original strain (0.15 h^–1 vs 0.05 h^–1). In a detailed characterization comparing the metabolism of the isolates with the metabolism of xylose, glucose, and ethanol in the original strain, the isolates showed improved properties in the assumed bottlenecks of xylose metabolism. The xylose uptake rate was increased almost 2-fold. Activities of the key enzymes in the pentose phosphate pathway (transketolase, transaldolase) increased 2-fold while the concentrations of their substrates (pentose 5-phosphates, sedoheptulose 7-phosphate) decreased correspondingly. Under anaerobic conditions, on minimal medium with 45 g l^–1 xylose, the ethanol productivity (in terms of cell dry weight; CDW) of one of the isolates increased from 0.012 g g^–1 CDW h^–1 to 0.017 g g^–1 CDW h^–1 and the yield from 0.09 g g^–1 xylose to 0.14 g g^–1 xylose, respectively.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose,glucose and sugarcane bagasse hydrolysate

Fifty-five bacterial strains isolated from soil were screened for efficient poly-3-hydroxybutyrate (P3HB) biosynthesis from xylose. Three strains were also evaluated for the utilization of bagasse hydrolysate after different detoxification steps. The results showed that activated charcoal treatment is pivotal to the production of a hydrolysate easy to assimilate. Burkholderia cepacia IPT 048 and B. sacchari IPT 101 were selected for bioreactor studies, in which higher polymer contents and yields from the carbon source were observed with bagasse hydrolysate, compared with the use of analytical grade carbon sources. Polymer contents and yields, respectively, reached 62% and 0.39 g g^–1 with strain IPT 101 and 53% and 0.29 g g^–1 with strain IPT 048. A higher polymer content and yield from the carbon source was observed under P limitation, compared with N limitation, for strain IPT 101. IPT 048 showed similar performances in the presence of either growth-limiting nutrient. In high-cell-density cultures using xylose plus glucose under P limitation, both strains reached about 60 g l^–1 dry biomass, containing 60% P3HB. Polymer productivity and yield from this carbon source reached 0.47 g l^–1 h^–1 and 0.22 g g^–1, respectively.     

Glucose repression of xylitol production in Candida tropicalis mixed-sugar fermentations

Glucose repressed xylose utilization inCandida tropicalis pre-grown on xylose until glucose reached approximately 0–5 g l^–1. In fermentations consisting of xylose (93 g l^–1) and glucose (47 g l^–1), xylitol was produced with a yield of 0.65 g g^–1 and a specific rate of 0.09 g g^–1 h^–1, and high concentrations of ethanol were also produced (25 g l^–1). If the initial glucose was decreased to 8 g l^–1, the xylitol yield (0.79 g g^–1) and specific rate (0.24 g g^–1 h^–1) increased with little ethanol formation (<5 g l^–1). To minimize glucose repression, batch fermentations were performed using an aerobic, glucose growth phase followed by xylitol production. Xylitol was produced under O₂ limited and anaerobic conditions, but the specific production rate was higher under O₂ limited conditions (0.1–0.4 vs. 0.03 g g^–1 h^–1). On-line analysis of the respiratory quotient defined the time of xylose reductase induction.     

Xylitol production from aspenwood hemicellulose hydrolysate by Candida guilliermondii

The production of xylitol by the yeast Candida guilliermondii was investigated in batch fermentations with aspenwood hemicellulose hydrolysate and compared with results obtained in semi-defined media with a mixture of glucose and xylose. The hemicellulose hydrolysate had to be supplemented by yeast extract and the maximum xylitol yield (0.8 g g^–1) and productivity (0.6 g l^–1 h^–1) were reached by controlling oxygen input.     

Fermentation of hexoses and pentoses from sugarcane bagasse hydrolysates into ethanol by Spathaspora hagerdaliae

The present study evaluated 13 strains of yeast for ethanol and xylitol production from xylose. Among them, Spathaspora hagerdaliae UFMG-CM-Y303 produced ethanol yields (Y_P/S) of 0.25 g g^− 1 and 0.39 g g^− 1 under aerobic and microaerophilic conditions, respectively, from a mixture of glucose and xylose in flasks. A pH of 5.0 and an inoculum of 3.0 × 10⁸ cells mL^− 1r resulted in the highest ethanol yields. These conditions were tested in a bioreactor for fermenting a medium containing an enzymatic hydrolysate of sugarcane bagasse with 15.5 g L^− 1 of glucose and 3 g L^− 1 of xylose, and achieved a Y_P/S of 0.47 g g^− 1, in relation to total available sugar. These results suggest that S. hagerdaliae UFMG-CM-Y303 has potential for use in second-generation ethanol studies.

     

Fed-batch production of baker's yeast using millet (Pennisetum typhoides) flour hydrolysate as the carbon source

A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of baker's yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4--d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca²⁺, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4--d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h^–1, biomass yield coefficient of 0.5 g g^–1 and feed substrate concentration of 200 g L^–1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L^–1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g^–1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.Nomenclature C _ox Dissolved oxygen concentration (mg L^–1) - CPR Carbon dioxide production rate (mmol h^–1) - C _s0 Glucose concentration in the feed (g L^–1) - C _s Substrate concentration in the fermenter (g L^–1) - C _s.crit Critical substrate concentration (g L^–1) - E Ethanol concentration (g L^–1) - F _s Substrate flow rate (g h^–1) - i Sample number (–) - K _e Constant in Equation 6 (g L^–1) - K _o Constant in Equation 7 (mg L^–1) - K _s Constant in Equation 5 (g L^–1) - m Specific maintenance term (h^–1) - OUR Oxygen up-take rate (mmol h^–1) - q _ox Specific oxygen up-take rate (h^–1) - q _ox.max Maximum specific oxygen up-take rate (h^–1) - q _p Specific product formation rate (h^–1) - q _s Specific substrate up-take rate (g g^–1 h^–1) - q _s.max Maximum specific substrate up-take rate (g g^–1 h^–1) - RQ Respiratory quotient (–) - S Total substrate in the fermenter at timet (g) - S ₀ Substrate mass fraction in the feed (g g^–1) - t Fermentation time (h) - V Instantaneous volume of the broth in the fermenter (L) - V ₀ Starting volume in the fermenter (L) - V _si Volume of samplei (L) - x Biomass concentration in the fermenter (g L^–1) - X ₀ Total amount of initial biomass (g) - X _t Total amount of biomass at timet (g) - Y _p/s Product yield coefficient on substrate (–) - Y _x/e Biomass yield coefficient on ethanol (–) - Y _x/s Biomass yield coefficient on substrate (–) Greek letters Moles of carbon per mole of yeast (–) - Moles of hydrogen atom per mole of yeast (–) - Moles of oxygen atom per mole of yeast (–) - Moles of nitrogen atom per mole of yeast (–) - Specific growth rate (h^–1) - _crit Critical specific growth rate (h^–1) - _E Specific ethanol up-take rate (h^–1) - _max.E Maximum specific ethanol up-take rate (h^–1)     

Grape skins as a natural support for yeast immobilization

Grape skins were used to immobilize Saccharomyces cerevisiae. In repeated batch fermentations of grape by immobilized and free cells, the maximum specific rate of alcohol production on glucose decreased from 7.98 h^–1 at 25 °C to 0.7 h^–1 at 5 °C. The rate was approximately twice as high as that on fructose. The rates for free cells were very low. The maximum alcohol yield (0.45 g g^–1) was obtained at 5 °C when the immobilized biocatalyst was used.     

Batch xylitol production from wheat straw hemicellulosic hydrolysate using Candida guilliermondii in a stirred tank reactor

Batch production of xylitol from the hydrolysate of wheat straw hemicellulose using Candida guilliermondii was carried out in a stirred tank reactor (agitation speed of 300 rpm, aeration rate of 0.6 vvm and initial cell concentration of 0.5 g l^–1). After 54 h, xylitol production from 30.5 g xylose l^–1 reached 27.5 g l^–1, resulting in a xylose-to-xylitol bioconversion yield of 0.9 g g^–1 and a productivity of 0.5 g l^–1 h^–1.     

Evaluation of xylitol production from corn cob hemicellulose hydrolysate by Candida parapsilosis

Candida parapsilosis was grown for 59 h in a medium containing corn cob hydrolysate consisting of 50 g xylose l^–1, 3.0 g glucose l^–1, 2.0 g arabinose l^–1, and 0.9 g acetic acid l^–1. A biomass of 9.1 g l^–1 was produced with 36 g xylitol l^–1 and 2.5 g ethanol l^–1. In a medium containing 50 g xylose l^–1 instead of corn cob hydrolysate, the concentrations of cells, xylitol, and ethanol were 8.6 g l^–1, 33 g l^–1, and 0.2 g l^–1, respectively. The differences between two cultures were due to the glucose and arabinose in the corn cob hydrolysate stimulating growth and the low concentration of acetic acid stimulating xylitol production.     

Cultivation ofCandida blankii in simulated bagasse hemicellulose hydrolysate

Summary All fourCandida blankii isolates evaluated for growth in simulated bagasse hemicellulose hydrolysate utilized the sugars and acetic acid completely. The utilization ofd-xylose,l-arabinose and acetic acid were delayed by the presence ofd-glucose, but after glucose depletion the other carbon sources were utilized simultaneously. The maximum specific growth rate of 0.36 h^–1 and cell yield of 0.47 g cells/g carbon source assimilate compared with published results obtained withC. utilis. C. blankii appeared superior toC. utilis for biomass production from hemicellulose hydrolysate in that it utilizedl-arabinose and was capable of growth at higher temperatures.