Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 I: production of xylitol and ethanol

An endophytic yeast, Rhodotorula mucilaginosa strain PTD3, that was isolated from stems of hybrid poplar was found to be capable of production of xylitol from xylose, of ethanol from glucose, galactose, and mannose, and of arabitol from arabinose. The utilization of 30 g/L of each of the five sugars during fermentation by PTD3 was studied in liquid batch cultures. Glucose-acclimated PTD3 produced enhanced yields of xylitol (67% of theoretical yield) from xylose and of ethanol (84, 86, and 94% of theoretical yield, respectively) from glucose, galactose, and mannose. Additionally, this yeast was capable of metabolizing high concentrations of mixed sugars (150 g/L), with high yields of xylitol (61% of theoretical yield) and ethanol (83% of theoretical yield). A 1:1 glucose:xylose ratio with 30 g/L of each during double sugar fermentation did not affect PTD3's ability to produce high yields of xylitol (65% of theoretical yield) and ethanol (92% of theoretical yield). Surprisingly, the highest yields of xylitol (76% of theoretical yield) and ethanol (100% of theoretical yield) were observed during fermentation of sugars present in the lignocellulosic hydrolysate obtained after steam pretreatment of a mixture of hybrid poplar and Douglas fir. PTD3 demonstrated an exceptional ability to ferment the hydrolysate, overcome hexose repression of xylose utilization with a short lag period of 10 h, and tolerate sugar degradation products. In direct comparison, PTD3 had higher xylitol yields from the mixed sugar hydrolysate compared with the widely studied and used xylitol producer Candida guilliermondii.     

Biotechnological production of xylitol in a three-phase fluidized bed bioreactor with immobilized yeast cells in Ca-alginate beads

Cells of Candida guilliermondii entrapped in Ca-alginate beads were used for xylitol production, from concentrated hemicellulose hydrolyzate of sugarcane bagasse, in a fluidized bed bioreactor (FBR). The maximum xylitol concentration 28.9 g xylitol/L was obtained at a high aeration rate of 600 mL/min after 70 h of fermentation, indicating that the use of high aeration rate in this system is favored for better oxygen transfer into the immobilized cells. The specific xylitol productivity and the xylitol yield were of 0.4 g xylitol/L.h and 0.58 g xylitol/g xylose respectively. The immobilization efficiency at the end of the fermentation was of 65 %. After 90 h of fermentation xylitol productivity and yield decreased to 0.25 g xylitol/L.h and 0.47 g xylitol/g xylose respectively, indicating the beginning of xylitol consumption by the yeast. The use of FBR system with immobilized cells presented high xylitol yield and productivity.     

Adaptation yields a highly efficient xylose-fermenting Zymomonas mobilis strain

Zymomonas mobilis is a superb ethanol producer with productivity exceeding yeast strains by several fold. Although metabolic engineering was successfully applied to expand its substrate range to include xylose, xylose fermentation lagged far behind glucose. In addition, xylose fermentation was often incomplete when its initial concentration was higher than 5%. Improvement of xylose fermentation is therefore necessary. In this work, we applied adaptation to improve xylose fermentation in metabolically engineered strains. As a result of adaptation over 80 days and 30 serial transfers in a medium containing high concentration of xylose, a strain, referred as A3, with markedly improved xylose metabolism was obtained. The strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, a total of 9% (w/v) ethanol was obtained from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Further investigation reveals evidence for an altered xylitol metabolism in A3 with reduced xylitol formation. Additionally xylitol tolerance in A3 was increased. Furthermore, xylose isomerase activity was increased by several times in A3, allowing cells to channel more xylose to ethanol than to xylitol. Taken together, these results strongly suggest that altered xylitol metabolism is key to improved xylose metabolism in adapted A3 strain. This work further demonstrates that adaptation and metabolic engineering can be used synergistically for strain improvement.     

Improvement of xylitol production by Candida guilliermondii FTI 20037 previously adapted to rice straw hemicellulosic hydrolysate

AIMS: To evaluate a simple and economical technique to improve xylitol production using concentrated xylose solutions prepared from rice straw hemicellulosic hydrolysate. METHODS AND RESULTS: Experiments were carried out with rice straw hemicellulosic hydrolysate containing 90 g l-1 xylose, with and without the addition of nutrients, using the yeast Candida guilliermondii previously grown on the hydrolysate (adapted cells) or on semi-defined medium (unadapted cells). By this method, the yield of xylitol increased from 17 g l-1 to 50 g l-1, and xylose consumption increased from 52% to 83%, after 120 h of fermentation. The xylitol production rates were very close to that (0.42 g l-1 h-1) attained in a medium simulating hydrolysate sugars. CONCLUSION: Yeast strain adaptation to the hydrolysate showed to be a suitable method to alleviate the inhibitory effects of the toxic compounds. Adapted cells of Candida guilliermondii can efficiently produce xylitol from hydrolysate with high xylose concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeast adaptation helps the bioconversion process in hydrolysate made from lignocellulosic materials. This low-cost technique provides an alternative to the detoxification methods used for removal of inhibitory compounds. In addition, the use of adapted inocula makes it possible to schedule a series of batch cultures so that the whole plant can be operated almost continuously with a concomitant reduction in the overall operation time.     

Controlled transient changes reveal differences in metabolite production in two<Emphasis Type="Italic"> Candida</Emphasis> yeasts

Physiological responses during growth on xylose and the xylose-degrading pathway of Candida tropicalis and Candida guilliermondii yeasts were investigated. The responses to a linearly decreasing oxygen transfer rate and a simultaneously increasing dilution rate were compared. C. guilliermondii produced acetate but no ethanol, and C. tropicalis ethanol but no acetate under oxygen limitation. Both strains produced glycerol. The D-xylose reductase of C. guilliermondii is exclusively NADPH-dependent. and acetate production regenerated NADPH. The xylose'reductase of C. tropicalis has a dual dependency for both NADH and NADPH. It regenerated NAD by producing ethanol. Both strains regenerated NAD by producing glycerol. The effect of intracellular NADH accumulation to xylose uptake and metabolite production was studied by using formate as a cosubstrate. Formate feeding in C. tropicalis triggered the accumulation of glycerol, ethanol and xylitol. Consequently, the specific xylose consumption increased 28% during formate feeding, from 477 to 609 C-mmol/C-mol cell dry-weight (CDW)/h. In C. guilliermondii cultures. formate feeding resulted only in glycerol accumulation. The specific xylose consumption increased 6%, from 301 to 319 C-mmol/C-mol CDW/h, until glycerol started to accumulate.     

粗糙脉孢菌(Neurospora crassa)木糖发酵的研究

研究了不同通氧条件和培养基初始pH等对粗糙脉孢菌(Neurospora crassa)AS3．1602木糖发酵的影响。结果表明,粗糙脉孢菌具有较强的发酵木糖产生乙醇及木糖醇的能力。通气量对木糖发酵有较大的影响。乙醇发酵适合在半好氧条件下进行,此时乙醇的转化率达到63．2％。木糖醇发酵适合在微好氧的条件下进行,转化率达到31．8％。木糖醇是在培养基中乙醇达到一定浓度后才开始积累。培养基的初始pH对木糖发酵产物有较大的影响,乙醇产生最适pH5．0,木糖醇产生最适pH4．0。在培养基pH为碱性条件时,木糖发酵受到很大的抑制。初始木糖浓度对产物乙醇及木糖醇的产率有很大的影响。葡萄糖的存在会抑制木糖的利用,对乙醇和木糖醇的产生也有很大的影响。     

Formation of xylitol in Candida guilliermondii 2581 culture

The yeast strain Candida guilliermondii 2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.     

Comparative study on a series of recombinant flocculent Saccharomyces cerevisiae strains with different expression levels of xylose reductase and xylulokinase

Ethanol production from xylose is important for the utilization of lignocellulosic biomass as raw materials. Recently, we reported the development of an industrial xylose-fermenting Saccharomyces cerevisiae strain, MA-R4, which was engineered by chromosomal integration to express the genes encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis along with S. cerevisiae xylulokinase gene constitutively using the alcohol-fermenting flocculent yeast strain, IR-2. IR-2 has the highest xylulose-fermenting ability of the industrial diploid strains, making it a useful host strain for genetically engineering xylose-utilizing S. cerevisiae. To optimize the activities of xylose metabolizing enzymes in the metabolic engineering of IR-2 for further improvement of ethanol production from xylose, we constructed a set of recombinant isogenic strains harboring different combinations of genetic modifications present in MA-R4, and investigated the effect of constitutive expression of xylulokinase and of different levels of xylulokinase and xylose reductase activity on xylose fermentation. This strain comparison showed that constitutive expression of xylulokinase increased ethanol production from xylose at the expense of xylitol excretion, and that high activity of xylose reductase resulted in an increased rate of xylose consumption and an increased glycerol yield. Moreover, strain MA-R6, which has moderate xylulokinase activity, grew slightly better but accumulated more xylitol than strain MA-R4. These results suggest that fine-tuning of introduced enzyme activity in S. cerevisiae is important for improving xylose fermentation to ethanol.     

Fed-batch fermentation of xylose by a fast-growing mutant of xylose-assimilating recombinant Saccharomyces cerevisiae

Mutants of xylose-assimilating recombinant Saccharomyces cerevisiae carrying the xylose reductase and xylitol dehydrogenase genes on plasmid pEXGD8 were selected, after ethyl methanesulfonate treatment, for their rapid growth on xylose medium. The fastest growing strain (strain IM2) showed a lower activity of xylose reductase but a higher ratio of xylitol dehydrogenase to xylose reductase activities than the parent strain, as well as high xylulokinase activity. Southern hybridization of the chromosomal DNA indicated that plasmid pEXGD8 was integrated into the chromosome of mutant IM2, resulting in an increase in the stability of the cloned genes. In batch fermentation under O₂ limitation, the yield and production rate of ethanol were improved 1.6 and 2.7 times, respectively, compared to the parent strain. In fed-batch culture with slow feeding of xylose and appropriate O₂ supply at a low level, xylitol excreted from the cells was limited and the ethanol yield increased 1.5 times over that in the batch culture, with a high initial concentration of xylose, although the production rate was reduced. The results suggested that slow conversion of xylose to xylitol led to a lower level of intracellular xylitol, resulting in less excretion of xylitol, and an increase in the ethanol yield. It was also observed that the oxidation of xylitol was strongly affected by the O₂ supply.Correspondence to: T. Yoshida     

混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究

【目的】构建可用于纤维素乙醇高效生产的混合糖发酵重组酿酒酵母菌株,并利用菊芋秸秆为原料进行乙醇发酵。【方法】筛选在木糖中生长较好的酿酒酵母YB-2625作为宿主菌,构建木糖共代谢菌株YB-2625 CCX。进一步通过r DNA位点多拷贝整合的方式,以YB-2625 CCX为出发菌株构建木糖脱氢酶过表达菌株,并筛选得到优势菌株YB-73。采用同步糖化发酵策略研究YB-73的菊芋秸秆发酵性能。【结果】YB-73菌株以90 g/L葡萄糖和30 g/L木糖为碳源进行混合糖发酵,乙醇产量比出发菌株YB-2625 CCX提高了13.9%,副产物木糖醇产率由0.89 g/g降低至0.31 g/g,下降了64.6%。利用重组菌YB-73对菊芋秸秆进行同步糖化发酵,48 h最高乙醇浓度达到6.10%(体积比)。【结论】通过转入木糖代谢途径以及r DNA位点多拷贝整合过表达木糖脱氢酶基因可有效提高菌株木糖发酵性能,并用于菊芋秸秆的纤维素乙醇生产。这是首次报道利用重组酿酒酵母进行菊芋秸秆原料的纤维素乙醇发酵。     

Deletion of FPS1, Encoding Aquaglyceroporin Fps1p,Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD⁺/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.     

Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate

Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae.     

Fed-batch xylitol production with recombinantXYL-1-expressingSaccharomyees cerevisiae using ethanol as a co-substrate

The bioconversion of xylose into xylitol in fed-batch fermentation with a recombinantSaccharomyces cerevisiae strain, transformed with the xylose-reductase gene ofPichia stipitis, was studied. When only xylose was fed into the fermentor, the production of xylitol continued until the ethanol that had been produced during an initial growth phase on glucose, was depleted. It was concluded that ethanol acted as a redox-balance-retaining co-substrate. The conversion of high amounts of xylose into xylitol required the addition of ethanol to the feed solution. Under O₂-limited conditions, acetic acid accumulated in the fermentation broth, causing poisoning of the yeast at low extracellular pH. Acetic acid toxicity could be avoided by either increasing the pH from 4.5 to 6.5 or by more effective aeration, leading to the further metabolism of acetic acid into cell mass. The best xylitol/ethanol yield, 2.4 gg^–1 was achieved under O₂-limited conditions. Under anaerobic conditions ethanol could not be used as a co-substrate, because the cell cannot produce ATP for maintenance requirements from ethanol anaerobically. The specific rate of xylitol production decreased with increasing aeration. The initial volumetric productivity increased when xylose was added in portions rather than by continuous feeding, due to a more complete saturation of the transport system and the xylose reductase enzyme.     

Screening and characterization of yeasts for xylitol production

AIMS: To discover novel naturally occurring xylitol producing yeast species with potential for industrial applications. METHODS AND RESULTS: Exactly 274 strains were cultivated on both solid and liquid screening medium with xylose as the sole carbon resource. Five strains were selected on the basis of significant growth and high degree of xylose assimilation. Their phylogenetic position was confirmed by the PCR-RFLP and sequence analysis of the D1/D2 domain of the 5' end of the large subunit rDNA gene (5'-LSU rDNA). Enzymatic analysis was conducted to compare xylose metabolism in each strain. Candida guilliermondii Xu280 and Candida maltosa Xu316 were found to have high xylose consumption rates and xylitol yields in the batch fermentation under micro-aerobic condition. The effect of the different media with high initial xylose concentration on biosynthesis of xylitol by both strains was investigated. CONCLUSIONS: We have identified Candida spp. strains, which exhibit high levels of xylitol production from xylose suggesting that these may have potential for industrial applications. SIGNIFICANCE AND IMPACTS OF THE STUDY: Microbial species are of importance for xylitol production. Xylitol production involves complicated metabolic regulation including xylose transport, production of key enzymes and cofactor regeneration. Thus, screening of naturally occurring xylose-utilizing micro-organisms is a viable and effective mean to obtain xylitol producing organisms with industrial application. Moreover, the research on selected strains will contribute to a better understanding of regulatory properties of xylose metabolism in different yeasts.     

Xylitol production from high xylose concentration: evaluation of the fermentation in bioreactor under different stirring rates

AIMS: To investigate the production of xylitol by the yeast Candida guilliermondii FTI 20037, in a bioreactor, from rice straw hemicellulosic hydrolysate with a high xylose concentration. METHODS AND RESULTS: Batch fermentation was carried out with rice straw hemicellulosic hydrolysate containing about 85 g xylose l(-1), in a stirred-tank bioreactor at 30 degrees C, under aeration of 1.3 vvm (volume of air per volume of medium per min) and different stirring rates (200, 300 and 500 rev min(-1)). The bioconversion of xylose into xylitol by the yeast depended on the stirring rate, the maximum xylitol yield (YP/S = 0.84 g g(-1)) being achieved at 300 rev min-1, with no need to pretreat the hydrolysate for purification. CONCLUSIONS: To determine the most adequate oxygen transfer rate is fundamental to improving the xylose-to-xylitol bioconversion by C. guilliermondii. SIGNIFICANCE AND IMPACT OF THE STUDY: For the microbial production of xylitol to be economically viable, the initial concentration of xylose in the lignocellulosic hydrolysate should be as high as possible, as with high substrate concentrations it is possible to increase the final product concentration. Nevertheless, there are few reports on the use of high xylose concentrations. Considering a process in bioreactor, from rice straw hemicellulosic hydrolysate, this is an innovator work.     

Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD(+). In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g(-1)) and the lowest xylitol yield (0.05 g g(-1)) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Expression of protein engineered NADP<Superscript>+</Superscript>-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD⁺-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP⁺. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP⁺-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP⁺-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.     

Xylitol production from sugarcane bagasse hydrolyzate in fluidized bed reactor. Effect of air flowrate

Cells of Candida guilliermondii immobilized onto porous glass spheres were cultured batchwise in a fluidized bed bioreactor for xylitol production from sugarcane bagasse hemicellulose hydrolyzate. An aeration rate of only 25 mL/min ensured minimum yields of xylose consumption (0.60) and biomass production (0.14 g(DM)/g(Xyl)), as well as maximum xylitol yield (0.54 g(Xyt)/g(Xyl)) and ratio of immobilized to total cells (0.83). These results suggest that cell metabolism, although slow because of oxygen limitation, was mainly addressed to xylitol production. A progressive increase in the aeration rate up to 140 mL/min accelerated both xylose consumption (from 0.36 to 0.78 g(Xyl)/L.h) and xylitol formation (from 0.19 to 0.28 g(Xyt)/L.h) but caused the fraction of immobilized to total cells and the xylitol yield to decrease up to 0.22 and 0.36 g(Xyt)/g(Xyl), respectively. The highest xylitol concentration (17.0 g(Xyt)/L) was obtained at 70 mL/min, but the specific xylitol productivity and the xylitol yield were 43% and 22% lower than the corresponding values obtained at the lowest air flowrate, respectively. The concentrations of consumed substrates and formed products were used in material balances to evaluate the xylose fractions consumed by C. guilliermondii for xylitol production, complete oxidation through the hexose monophosphate shunt, and cell growth. The experimental data collected at variable oxygen level allowed estimating a P/O ratio of 1.35 mol(ATP)/mol(O) and overall ATP requirements for biomass growth and maintenance of 3.4 mol(ATP)/C-mol(DM).     

Kinetic behavior of Candida guilliermondii yeast during xylitol production from Brewer's spent grain hemicellulosic hydrolysate

Brewer's spent grain, the main byproduct of breweries, was hydrolyzed with dilute sulfuric acid to produce a hemicellulosic hydrolysate (containing xylose as the main sugar). The obtained hydrolysate was used as cultivation medium by Candidaguilliermondii yeast in the raw form (containing 20 g/L xylose) and after concentration (85 g/L xylose), and the kinetic behavior of the yeast during xylitol production was evaluated in both media. Assays in semisynthetic media were also performed to compare the yeast performance in media without toxic compounds. According to the results, the kinetic behavior of the yeast cultivated in raw hydrolysate was as effective as in semisynthetic medium containing 20 g/L xylose. However, in concentrated hydrolysate medium, the xylitol production efficiency was 30.6% and 42.6% lower than in raw hydrolysate and semisynthetic medium containing 85 g/L xylose, respectively. In other words, the xylose-to-xylitol bioconversion from hydrolysate medium was strongly affected when the initial xylose concentration was increased; however, similar behavior did not occur from semisynthetic media. The lowest efficiency of xylitol production from concentrated hydrolysate can be attributed to the high concentration of toxic compounds present in this medium, resulting from the hydrolysate concentration process.     

Adaptation of the xylose fermenting yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> F12 for improving ethanol production in different fed-batch SSF processes

An efficient fermenting microorganism for bioethanol production from lignocellulose is highly tolerant to the inhibitors released during pretreatment and is able to ferment efficiently both glucose and xylose. In this study, directed evolution was employed to improve the xylose fermenting Saccharomyces cerevisiae F12 strain for bioethanol production at high substrate loading. Adapted and parental strains were compared with respect to xylose consumption and ethanol production. Adaptation led to an evolved strain more tolerant to the toxic compounds present in the medium. When using concentrated prehydrolysate from steam-pretreated wheat straw with high inhibitor concentration, an improvement of 65 and 20% in xylose consumption and final ethanol concentration, respectively, were achieved using the adapted strain. To address the need of high substrate loadings, fed-batch SSF experiments were performed and an ethanol concentration as high as 27.4 g/l (61% of the theoretical) was obtained with 11.25% (w/w) of water insoluble solids (WIS).