Characterization of a Recombinant Flocculent Saccharomyces cerevisiae Strain that Co-ferments Glucose and Xylose: I. Influence of the Ratio of Glucose/Xylose on Ethanol Production

Glucose/xylose mixtures (90 g/L total sugar) were evaluated for their effect on ethanol fermentation by a recombinant flocculent Saccharomyces cerevisiae, MA-R4. Glucose was utilized faster than xylose at any ratio of glucose/xylose, although MA-R4 can simultaneously co-ferment both sugars. A high percentage of glucose can increase cell biomass production and therefore increase the rate of glucose utilization (1.224 g glucose/g biomass/h maximum) and ethanol formation (0.493 g ethanol/g biomass/h maximum). However, the best ratio of glucose/xylose for the highest xylose consumption rate (0.209 g xylose/g biomass/h) was 2:3. Ethanol concentration and yield increased and by-product (xylitol, glycerol, and acetic acid) concentration decreased as the proportion of glucose increased. The maximum ethanol concentration was 41.6 and 21.9 g/L after 72 h of fermentation with 90 g/L glucose and 90 g/L xylose, respectively, while the ethanol yield was 0.454 and 0.335 g/g in 90 g/L glucose and 90 g/L xylose media, respectively. High ethanol yield when a high percentage of glucose is available is likely due to decreased production of by-products, such as glycerol and acetic acid. These results suggest that ethanol selectivity is increased when a higher proportion of glucose is available and reduced when a higher proportion of xylose is available.     

Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures

The fermentation characteristics of a recombinant strain of Zymomonas mobilis ZM4(pZB5) capable of converting both glucose and xylose to ethanol have been further investigated. Previous studies have shown that the strain ZM4(pZB5) was capable of converting a mixture o 65 g/L of glucose and 65 g/L of xylose to 62 g/L of ethanol in 48 h with an overall yield of 0.46 g/g. Higher sugar concentrations (e.g., 75/75 g/L) resulted in incomplete xylose utilization (80 h). In the present study, further kinetic evaluations at high sugar levels are reported. Acetate inhibition studies and evaluation of temperature and pH effects indicated increased maximum specific uptake rates of glucose and xylose under stressed conditions with increased metabolic uncoupling. A high-productivity system was developed that involved a membrane bioreactor with cell recycling. At sugar concentrations of approx 50/50 g/L of glucose/xylose, an ethanol concentration of 50 g/L, an ethanol productivity of approx 5 g/(L·h), and a yield (Y _p/s) of 0.50 g/g were achieved. Decreases in cell viability were found in this system after attainment of an initial steady state (40–60 h); a slow bleed of concentrated cells may be required to overcome this problem.     

Model compound studies

The influence of other hemicellulosic sugars (arabinose, galactose, mannose, and glucose), oxygen limitation, and initial xylose concentration on the fermentation of xylose to xylitol was in vestigated using experimental design methodology. Oxygen limitation and initial xylose concentration had strong influences on xylitol production by Candida tropicalis ATCC 96745. Under semiaerobic conditions, xylitol yield was highest (0.62 g/g), whereas under aerobic conditions volumetric productivity was highest (0.90g/[L·h]). In the presence of glucose, xylose utilization was strongly repressed and sequential sugar utilization was observed. Ethanol produced from the glucose caused a 50% reduction in xylitol yield when the ethanol con centration exceeded 30 g/L. When complex synthetic hemicellulosic sugars were fermented, glucose was initially consumed followed by a simultaneous uptake of the other sugars. The highest xylitol yield (0.84 g/g) and volumetric productivity (0.49 g/[L·h]) were obtained for substrates containing high arabinose and low glucose and mannose contents.     

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200–250°C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4c/gal of ethanol. This work examined the growth and fermentation performance of xyloseutilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated asthe “adapted” strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30h, followed by CP4:pZB5 at 48h, with corresponding volumetric productivities of 1.4 and 0.89 g/(L·h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to fermentarabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.     

Alcoholic Fermentation with Flocculant Saccharomyces cerevisiae in Fed-Batch Process

Studies have been conducted on selecting yeast strains for use in fermentation for ethanol production to improve the performance of industrial plants and decrease production costs. In this paper, we study alcoholic fermentation in a fed-batch process using a Saccharomyces cerevisiae yeast strain with flocculant characteristics. Central composite design (CCD) was used to determine the optimal combination of the variables involved, with the sucrose concentration of 170 g/L, a cellular concentration in the inoculum of 40 % (v/v), and a filling time of 6 h, which resulted in a 92.20 % yield relative to the theoretical maximum yield, a productivity of 6.01 g/L h and a residual sucrose concentration of 44.33 g/L. With some changes in the process such as recirculation of medium during the fermentation process and increase in cellular concentration in the inoculum after use of the CCD was possible to reduce the residual sucrose concentration to 2.8 g/L in 9 h of fermentation and increase yield and productivity for 92.75 % and 9.26 g/L h, respectively. A model was developed to describe the inhibition of alcoholic fermentation kinetics by the substrate and the product. The maximum specific growth rate was 0.103 h^?1, with K _I and K _s values of 109.86 and 30.24 g/L, respectively. The experimental results from the fed-batch reactor show a good fit with the proposed model, resulting in a maximum growth rate of 0.080 h^?1.     

Continuous culture studies of xylose-fermentingZymomonas mobilis

The continuous cofermentation performance of xylose-fermentingZymomonas mobilis at 30°C and pH 5.5 was characterized using a pure-sugar feed solution that contained 8 g/L glucose and 40 g/L xylose. Successful chemostat start up resulted in complete utilization of glucose and greater than 85% utilization of xylose, but was only reproducibly achieved using initial dilution rates at or less than 0.04/h; once initiated, cofermentation could be maintained at dilution rates of 0.04 to 0.10/h. Whereas xylose and cell-mass concentrations increased gradually with increasing dilution rate, ethanol concentrations and ethanol yields on available sugars remained approximately constant at 20–22 g/L and 80–90% of theoretical, respectively. Volumetric and specific ethanol productivities increased linearly with increasing dilution rate, rising from approx 1.0 each (g/L/h or g/g/h) at a dilution rate of 0.04/h to approx 2.0 each (g/L/h or g/g/h) at a dilution rate of 0.10/h. Similarly, specific sugar-utilization rates increased from approx 2.0 g/g/h at dilution rate 0.04/h to approx 3.5 g/g/h at dilution rate of 0.10/h. The estimated values of 0.042 g/g for the maximum Z.mobilis cell-mass yield on substrate and 1.13 g/g/h for the minimum specific substrate utilization rate required for cellular maintenance energy are within the range of values reported in the literature. Results are also presented which suggest that long-term adaptation in continuous culture is a powerful technique for developing strains with higher tolerance to inhibitory hemicellulose hydrolyzates.     

A Novel Process for Direct Production of Acetone–Butanol–Ethanol from Native Starches Using Granular Starch Hydrolyzing Enzyme by Clostridium saccharoperbutylacetonicum N1-4

In this work, a new approach for acetone–butanol–ethanol (ABE) production has been proposed. Direct fermentation of native starches (uncooked process) was investigated by using granular starch hydrolyzing enzyme (GSHE) and Clostridium saccharoperbutylacetonicum N1-4. Even the process was carried out under suboptimal condition for activity of GSHE, the production of ABE was similar with that observed in conventional process or cooked process in terms of final solvent concentration (21.3?±?0.4 to 22.4?±?0.4 g/L), butanol concentration (17.5?±?0.4 to 17.8?±?0.3 g/L) and butanol yield (0.33 to 0.37 g/g). The production of solvents was significantly dependent on the source of starches. Among investigated starches, corn starch was more susceptible to GSHE while cassava starch was the most resistant to this enzyme. Fermentation using native corn starch resulted in the solvent productivity of 0.47 g/L h, which was about 15 % higher than that achieved in cooked process. On the contrary, uncooked process using cassava and wheat starch resulted in the solvent productivity of 0.30 and 0.37 g/L h, which were respectively about 30 % lower than those obtained in cooked process. No contamination was observed during all trials even fermentation media were prepared without sterilization. During the fermentation using native starches, no formation of foam is observed. This uncooked process does not require cooking starchy material; therefore, the thermal energy consumption for solvent production would remarkably be reduced in comparison with cooked process.     

Selection of a Thermotolerant Kluyveromyces marxianus Strain with Potential Application for Cellulosic Ethanol Production by Simultaneous Saccharification and Fermentation

The development of technologies for cellulosic ethanol production by simultaneous saccharification and fermentation (SSF) depends on the use of microorganisms with high fermentative rates and thermotolerance. In this study, the ability of five Kluyveromyces marxianus strains to produce ethanol from glucose at 45 °C was investigated. The highest fermentative parameters were observed with K. marxianus NRRL Y-6860, which was then further studied. An initial evaluation of the oxygen supply on ethanol production by the selected yeast and a comparison of SSF process from acid pretreated rice straw between K. marxianus NRRL Y-6860 and Saccharomyces cerevisiae at 30 and 45 °C were carried out. Under the lowest evaluated conditions of aeration and agitation, K. marxianus NRRL Y-6860 produced 21.5 g/L ethanol from 51.3 g/L glucose corresponding to Y_P/S of 0.44 g/g and Q_P of 3.63 g/L h. In the SSF experiments, K. marxianus NRRL Y-6860 was more efficient than S. cerevisiae at both evaluated temperatures (30 and 45 °C), attained at the highest temperature an ethanol yield of 0.24 g/g and productivity of 1.44 g/L h.     

Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400(pLNH33)

Fermentation kinetics of ethanol production from glucose, xylose, and their mixtures using a recombinant Saccharomyces 1400 (pLNH33) are reported. Single-substrate kinetics indicate that the specific growth rate of the yeast and the specific ethanol productivity on glucose as the substrate was greater than on xylose as a substrate. Ethanol yields from glucose and xylose fermentation were typically 95 and 80% of the theoretical yield, respectively. The effect of ethanol inhibition is more pronounced for xylose fermentation than for glucose fermentation. Studies on glucose-xylose mixtures indicate that the recombinant yeast co-ferments glucose and xylose. Fermentation of a 52.8 g/L glucose and 56.3 g/L xylose mixture gave an ethanol concentration of 47.9 g/L after 36 h. Based on a theoretical yield of 0.51 g ethanol/g sugars, the ethanol yield from this experiment (for data up to 24 h) was calculated to be 0.46 g ethanol/g sugar or 90% of the theoretical yield. The specific growth rate of the yeast on glucose-xylose mixtures was found to lie between the specific growth rate on glucose and the specific growth rate on xylose. Kinetic studies were used to develop a fermentation model incorporating the effects of substrate inhibition, product inhibition, and inoculum size. Good agreements were obtained between model predictions and experimental data from batch fermentation of glucose, xylose, and their mixtures.     

Enhanced Reducing Equivalent Generation for 1,3-Propanediol Production Through Cofermentation of Glycerol and Xylose by Klebsiella pneumoniae

1,3-Propanediol (1,3-PD) biosynthesis plays a key role in NADH consumption to regulate the intracellular reducing equivalent balance of Klebsiella pneumoniae. This study aimed to increase reducing equivalent for enhancing 1,3-PD production through cofermentation of glycerol and xylose. Adding xylose as cosubstrate resulted in more reducing equivalent generation and higher cell growth. In batch fermentation under microaerobic condition, the 1,3-PD concentration, conversion from glycerol, and biomass (OD(600)) relative to cofermentation were increased significantly by 9.1%, 20%, and 15.8%, respectively. The reducing equivalent (NADH) was increased by 1-3 mg/g (cell dry weight) compared with that from glycerol alone. Furthermore, 2,3-butannediol was also doubly produced as major byproduct. In fed-batch fermentation with xylose as cosubstrate, the final 1,3-PD concentration, conversion from glycerol, and productivity were improved evidently from 60.78 to 67.21 g/l, 0.52 to 0.63 mol/mol, and 1.64 to 1.82 g/l/h, respectively.     

Improvement of Solvent Production from Xylose Mother Liquor by Engineering the Xylose Metabolic Pathway in Clostridium acetobutylicum EA 2018

Xylose mother liquor (XML) is a by-product of xylose production through acid hydrolysis from corncobs, which can be used potentially for alternative fermentation feedstock. Sixteen Clostridia including 13 wild-type, 1 industrial strain, and 2 genetically engineered strains were screened in XML, among which the industrial strain Clostridium acetobutylicum EA 2018 showed the highest titer of solvents (12.7 g/L) among non-genetic populations, whereas only 40 % of the xylose was consumed. An engineered strain (2018glcG-TBA) obtained by combination of glcG disruption and expression of the d-xylose proton-symporter, d-xylose isomerase, and xylulokinase was able to completely utilize glucose and l-arabinose, and 88 % xylose in XML. The 2018glcG-TBA produced total solvents up to 21 g/L with a 50 % enhancement of total solvent yield (0.33 g/g sugar) compared to that of EA 2018 (0.21 g/g sugar) in XML. This XML-based acetone–butanol–ethanol fermentation using recombinant 2018glcG-TBA was estimated to be economically promising for future production of solvents.     

Ethanol production from glucose and xylose by immobilized Zymomonas mobilis CP4 (pZB5)

Fermentation of glucose-xylose mixtures to ethanol was investigated in batch and continuous experiments using immobilized recombinant Zymomonas mobilis CP4(pZB5). This microorganism was immobilized by entrapment in κ-carrageenan beads having a diameter of 1.5–2.5 mm. Batch experiments showed that the immobilized cells cofermented glucose and xylose to ethanol and that the presence of glucose improved the xylose utilization rate. Batch fermentation of rice straw hydrolysate containing 76 g/L of glucose and 33.8 g/L of xylose gave an ethanol concentration of 44.3 g/L after 24 h, corresponding to a yield of 0.46 g of ethanol/g of sugars. Comparable results were achieved with a synthetic sugar control. Continuous fermentation experiments were performed in a laboratory-scale fluidized-bed bioreactor (FBR). Glucose-xylose feed mixtures were pumped through the FBR at residence times of 2–4 h. Glucose conversion to ethanol was maintained above 98% in all experiments. Xylose conversion to ethanol was highest at 91.5% for a feed containing 50 g/L of glucose and 13 g/L of xylose at a dilution rate of 0.24/h. The xylose conversion to ethanol decreased with increasing feed xylose concentration, dilution rate, and age of the immobilized cells. Volumetric ethanol productivities in the range of 6.5–15.3 g/L·h were obtained. The improved productivities achieved in the FBR compared to other bioreactor systems can help in reducing the production costs of fuel ethanol from lignocellulosic sugars. This article has been authored by a contractor of the US go vernment under contract DE-AC05-96OR22464. Accordingly, the US government retains a nonexclusive, royaltyfree license to publish or reproduce the published form of the contribution, or allow others to do so, for US government purposes.     

Studies on nutrient requirements and cost-effective supplements for ethanol production by recombinantE. coli

This article describes a systematic study of the nutritional requirements of a patented recombinant ethanologenicEscherichia coli (11303:pLOI297) and provides cost-effective formulations that are compatible with the production of fuel ethanol in fermentations of lignocellulosic prehydrolysate characterized by high xylose conversion efficiency. A complex and nutrient-rich laboratory medium, Luria broth (LB), provided the benchmark with respect to fermentation performance standard. Xylose fermentation performance was assessed in terms of the target values for operational process parameters established by the US National Renewable Energy Laboratory (NREL)—final ethanol concentration (25 g/L), xylose-to-ethanol conversion efficiency (90%), and volumetric productivity (0.52 g/L·h). Biomass prehydrolysates that are rich in xylose also contain acetic acid, and in anticipation of a need to reduce acetic acid toxicity, the fermentors were operated with a pH control set-point of 7.0 Growth and fermentation in the minimal defined salts (DS) medium was only about 15% compared to the reference medium. Amendment of the minimal medium containing 6 wt% xylose with both vitamins and amino acids resulted in improved growth, but the volume productivity (0.59 g/L·h) was still only about 54% of that with LB (1.1 g/L·h). Formulations directed at cost reduction through the use of less expensive commercial complex nutritional supplements were within 90% of the NREL process target with respect to yield and provided a productivity at about 80% of the LB medium, but were not economical. Corn steep liquor (CSL) at about 7–8 g/L was shown to be a complete source of nutritional requirements and supported a fermentation performance approaching that of LB. At a cost of CSL of $50/t (dry wt), the economic impact of using this amount CSL as the sole nutritional supplement in a cellulosic ethanol plant was estimated to be about 4¢/gal of ethanol.

     

Effect of starting xylose concentration on the microaerobic metabolism of Debaryomyces hansenii: the use of carbon material balances

Xylitol production by Debaryomyces hansenii NRRL Y-7426 was performed on synthetic medium varying the initial xylose concentration between 50 and 300 g/L. The experimental results of these tests were used to investigate the effect of substrate level on xylose consumption by this yeast. Satisfactory values of product yield on substrate (0.74–0.83 g/g) as well as volumetric productivity (0.481–0.694 g/L·h) were obtained over a wide range of xylose levels (90–200 g/L), while a worsening of kinetic parameters took place at higher concentration, likely due to a substrate inhibition phenomenon. The metabolic behavior of D. hansenii was studied, under these conditions, through a carbon material balance to estimate the fractions of xylose consumed by the cell for different activities (xylitol production, biomass growth, and respiration) during the lag, exponential, and stationary phases.     

Cofermentation of glucose,xylose, and arabinose by mixed cultures of two genetically engineeredZymomonas mobilis strains

Cofermentation of xylose and arabinose, in addition to glucose, is critical for complete bioconversion of lignocellulosic biomass, such as agricultural residues and herbaceous energy crops, to ethanol. A factorial design experiment was used to evaluate the cofermentation of glucose, xylose, and arabinose with mixed cultures of two genetically engineeredZymomonas mobilis strains (one ferments xylose and the other arabinose). The pH range studied was 5.0-6.0, and the temperature range was 30-37°C The individual sugar concentrations used were 30 g/L glucose, 30 g/L xylose, and 20 g/L arabinose. The optimal cofermentation conditions obtained by data analysis, using Design Expert software, were pH 5.85 and temperature 31.5°C. The cofermentation process yield at optimal conditions was 72.5% of theoritical maximum. The results showed that neither the arabinose strain nor arabinose affected the performance of the xylose strain; however, both xylose strain and xylose had a significant effect on the performance of the arabinose strain. Although cofermentation of all three sugars is achieved by the mixed cultures, there is a preferential order of sugar utilization. Glucose is used rapidly, then xylose, followed by arabinose.     

Efficient Non-sterilized Fermentation of Biomass-Derived Xylose to Lactic Acid by a Thermotolerant Bacillus coagulans NL01

Xylose is the major pentose and the second most abundant sugar in lignocellulosic feedstock. Its efficient utilization is regarded as a technical barrier to the commercial production of bulk chemicals from lignocellulosic biomass. This work aimed at evaluating the lactic acid production from the biomass-derived xylose using non-sterilized fermentation by Bacillus coagulans NL01. A maximum lactic acid concentration of about 75 g/L was achieved from xylose of 100 g/L after 72 h batch fermentation. Acetic acid and levulinic acid were identified as important inhibitors in xylose fermentation, which markedly reduced lactic acid productivity at 15 and 1.0 g/L, respectively. But low concentrations of formic acid (<2 g/L) exerted a stimulating effect on the lactic acid production. When prehydrolysate containing total 25.45 g/L monosaccharide was fermented with B. coagulans NL01, the same preference for glucose, xylose, and arabinose was observed and18.2 g/L lactic acid was obtained after 48 h fermentation. These results proved that B. coagulans NL01 was potentially well-suited for producing lactic acid from underutilized xylose-rich prehydrolysates.     

Application of factorial design to the study of xylitol production from eucalyptus hemicellulosic hydrolysate

This study deals with the bioconversion of xylose into xylitol by Candida guilliermondii FTI 20037 using eucalyptus hemicellulosic hydrolysate obtained by acid hydrolysis. The influence of various parameters (ammonium sulfate, rice bran, pH, and xylose concentration) on the production of xylitol was evaluated. The experiments were based on multivariate statistical concepts, with the application of factorial design techniques to identify the most important variables in the process. The levels of these variables were quantified by the response surface methodology, which permitted the establishment of a significant mathematical model with a coefficient determination of R ²=0.92. The best results (xylitol=10.0 g/L, yield factor=0.2 g/g, and productivity=0.1 g/[L·h]) were attained with hydrolysate containing ammonium sulfate (1.1 g/L), rice bran (5.0 g/L), and xylose (initial concentration of 60.0 g/L), after 72 h of fermentation. The pH of fermentation was adjusted to 8.0 and the inoculum level utilized was 3 g/L.     

Kinetics of ethanol production from carob pods extract by immobilizedSaccharomyces cerevisiae cells

Kinetics of ethanol production from carob pods extract by immobilizedS. cerevisiae cells in static and shake flask fermentation have been investigated. Shake flask fermentation proved to be a better fermentation system for the production of ethanol than static fermentation. The optimum values of ethanol concentration, ethanol productivity, ethanol yield, and fermentation efficiency were obtained at pH range 3.5–6.5 and temperature between 30–35°C. A maximum ethanol concentration (65 g/L), ethanol productivity (8.3 g/Lh), ethanol yield (0.44 g/g), and fermentation efficiency (95%) was achieved at an initial sugar concentration of 200, 150, 100, and 200 g/L, respectively. The highest values of specific ethanol production rate and specific sugar uptake rate were obtained at pH 6.5, temperature 40°C, and initial sugar concentration of 100 g/L. Other kinetic parameters, biomass concentration, biomass yield, and specific biomass production rate were maximum at pH 5.5, temperature 30°C, and initial sugar concentration 150 g/L. Under the same fermentation conditions non-sterilized carob pod extract gave higher ethanol concentration than sterilized medium. In repeated batch fermentations, the immobilizedS. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 5 d.     

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose–xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h^?1. With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.     

The Influence of Initial Xylose Concentration, Agitation, and Aeration on Ethanol Production by Pichia stipitis from Rice Straw Hemicellulosic Hydrolysate

Rice straw hemicellulosic hydrolysate was used as fermentation medium for ethanol production by Pichia stipitis NRRL Y-7124. Shaking bath experiments were initially performed aiming to establish the best initial xylose concentration to be used in this bioconversion process. In the sequence, assays were carried out under different agitation (100 to 200 rpm) and aeration (V _flask/V _medium ratio varying from 2.5 to 5.0) conditions, and the influence of these variables on the fermentative parameters values (ethanol yield factor, Y _P/S; cell yield factor, Y _X/S; and ethanol volumetric productivity, Q _P) was investigated through a 2² full-factorial design. Initial xylose concentration of about 50 g/l was the most suitable for the development of this process, since the yeast was able to convert substrate in product with high efficiency. The factorial design assays showed a strong influence of both process variables in all the evaluated responses. The agitation and aeration increase caused a deviation in the yeast metabolism from ethanol to biomass production. The best results (Y _P/S?= 0.37 g/g and Q _P?=?0.39 g/l.h) were found when the lowest aeration (2.5 V _flask/V _medium ratio) and highest agitation (200 rpm) levels were employed. Under this condition, a process efficiency of 72.5% was achieved. These results demonstrated that the establishment of adequate conditions of aeration is of great relevance to improve the ethanol production from xylose by Pichia stipitis, using rice straw hemicellulosic hydrolysate as fermentation medium.