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Background Scheffersomyces stipitis is an important yeast species in the field of biorenewables due to its desired capacity for xylose utilization. It has been recognized that redox balance plays a critical role in S. stipitis due to the different cofactor preferences in xylose assimilation pathway. However, there has not been any systems level understanding on how the shift in redox balance contributes to the overall metabolic shift in S. stipitis to cope with reduced oxygen uptake. Genome-scale metabolic network models (GEMs) offer the opportunity to gain such systems level understanding; however, currently the two published GEMs for S. stipitis cannot be used for this purpose, as neither of them is able to capture the strain’s fermentative metabolism reasonably well due to their poor prediction of xylitol production, a key by-product under oxygen limited conditions. Results A system identification-based (SID-based) framework that we previously developed for GEM validation is expanded and applied to refine a published GEM for S. stipitis, iBB814. After the modified GEM, named iDH814, was validated using literature data, it is used to obtain genome-scale understanding on how redox cofactor shifts when cells respond to reduced oxygen supply. The SID-based framework for GEM analysis was applied to examine how the environmental perturbation (i.e., reduced oxygen supply) propagates through the metabolic network, and key reactions that contribute to the shifts of redox and metabolic state were identified. Finally, the findings obtained through GEM analysis were validated using transcriptomic data. Conclusions iDH814, the modified model, was shown to offer significantly improved performance in terms of matching available experimental results and better capturing available knowledge on the organism. More importantly, our analysis based on iDH814 provides the first genome-scale understanding on how redox balance in S. stipitis was shifted as a result of reduced oxygen supply. The systems level analysis identified the key contributors to the overall metabolic state shift, which were validated using transcriptomic data. The analysis confirmed that S. stipitis uses a concerted approach to cope with the stress associated with reduced oxygen supply, and the shift of reducing power from NADPH to NADH seems to be the center theme that directs the overall shift in metabolic states. Electronic supplementary material The online version of this article (10.1186/s12934-018-0983-y) contains supplementary material, which is available to authorized users.

Lipid production by oleaginous yeasts is optimal at high carbon-to-nitrogen ratios. In the current study, nitrogen and carbon consumption by Lipomyces starkeyi were directly measured in defined minimal media with nitrogen content and agitation rates as variables. Shake flask cultures with an initial C:N ratio of 72:1 cultivated at 200rpm resulted in a lipid output of 10g/L, content of 55%, yield of 0.170g/g, and productivity of 0.06g/L/h. All of these values decreased by ≈50-60% when the agitation rate was raised to 300rpm or when the C:N ratio was lowered to 24:1, demonstrating the importance of these parameters. Under all conditions, L. starkeyi cultures tolerated acidified media (pH≈2.6) without difficulty, and produced considerable amounts of alcohols; including ethanol, mannitol, arabitol, and 2,3-butanediol. L. starkeyi also produced lipids from a corn stover hydrolysate, showing its potential to produce biofuels from renewable agricultural feedstocks.

This study employed cell recycling, batch adaptation, cell mating and high-throughput screening to select adapted Spathaspora passalidarum strains with improved fermentative ability. The most promising candidate YK208-E11 (E11) showed a 3-fold increase in specific fermentation rate compared to the parental strain and an ethanol yield greater than 0.45 g/g substrate while co-utilizing cellobiose, glucose and xylose. Further characterization showed that strain E11 also makes 40% less biomass compared to the parental strain when cultivated in rich media under aerobic conditions. A tetrazolium agar overlay assay in the presence of respiration inhibitors, including rotenone, antimycin A, KCN and salicylhydroxamic acid elucidated the nature of the mutational events. Results indicated that E11 has a deficiency in its respiration system that could contribute to its low cell yield. Strain E11 was subjected to whole genome sequencing and an ∼11 kb deletion was identified; the open reading frames absent in strain E11 code for proteins with predicted functions in respiration, cell division and the actin cytoskeleton, and may contribute to the observed physiology of the adapted strain. Results of the tetrazolium overlay also suggest that cultivation on xylose affects the respiration capacity in the wild-type strain, which could account for its faster fermentation of xylose as compared to glucose. These results support our previous finding that S. passalidarum has highly unusual physiological responses to xylose under oxygen limitation.

Biomass pretreatment is essential to overcome recalcitrance of lignocellulose for ethanol production. In the present study we pretreated giant reed (Arundo donax L.), a perennial, rhizomatous lignocellulosic grass with dilute oxalic acid. The effects of temperature (170–190 °C), acid loading (2–10% w/w) and reaction time (15–40 min) were handled as a single parameter, combined severity. We explored the change in hemicellulose, cellulose and lignin composition following pretreatment and glucan conversion after enzymatic hydrolysis of the solid residue. Two different yeast strains, Scheffersomyces (Pichia) stipitis CBS 6054, which is a native xylose and cellobiose fermenter, and Saccharomyces carlsbergensis FPL-450, which does not ferment xylose or cellobiose, were used along with commercial cellulolytic enzymes in simultaneous saccharification and fermentation (SSF). S. carlsbergensis attained a maximum ethanol concentration of 15.9 g/l after 48 h at pH 5.0, while S. stipitis, at the same condition, took 96 h to reach a similar ethanol value; increasing the pH to 6.0 reduced the S. stipitis lag phase and attained 18.0 g/l of ethanol within 72 h.

Dicarboxylic organic acids have properties that differ from those of sulfuric acid during hydrolysis of lignocellulose. To investigate the effects of different acid catalysts on the hydrolysis and degradation of biomass compounds over a range of thermochemical pretreatments, maleic, oxalic and sulfuric acids were each used at the same combined severity factor (CSF) values during hydrolysis. Xylose and glucose concentrations in hydrolysates were highest with maleic acid. Oxalic acid gave the next highest followed by sulfuric acid. This ranking was particularly true at low CSF values. The concentrations of glucose and xylose increased with oxalic and sulfuric acid pretreatments as the CSF increased, but they never attained the levels observed with maleic acid. Among sulfuric, oxalic and maleic acid treatments, the amount of xylose released as xylooligosaccharide was highest with sulfuric acid. The fraction of xylooligosaccharide was lowest with the maleic acid and the oligosaccharide fraction with oxalic acid fell in between. Furfural and hydroxymethyl furfural levels were also highest with maleic acid. In subsequent fermentations with pretreated biomass, the ethanol concentration was maximal at 19.2g/l at CSF 1.9 when maleic acid was used as the pretreatment catalyst. This corresponded to an ethanol volumetric production rate of 0.27 g ethanol/l per h. This was the same condition showing the highest xylose production in following pretreatment with various acid catalysts. These findings suggest that maleic and oxalic dicarboxylic acids degrade hemicelluloses more efficiently than does sulfuric acid.

Estrogen has been shown to increase endothelial cell (EC) production of nitric oxide (NO) by the rapid activation of endothelial nitric oxide synthase (eNOS). To better understand the mechanisms by which estrogen acutely increases endothelial NO production, proteins of the mitogen activated protein kinase (MAPK) family, ERK, JNK, and p38 were examined to determine their potential role in this process. Bovine aortic endothelial cells (BAEC) were grown to confluence in phenol red-free DMEM F-12, supplemented with 10% fetal calf serum, on 35 cm2 dishes. After confluence, cells were starved for 12–24 hours and then exposed to 10 nM 17β-estradiol (E2) for 1–60 minutes. The cells were then lysed and proteins resolved by SDS-PAGE followed by transfer to nitrocellulose membranes. Membranes were then probed with anti-phosphospecific antibodies for ERK, JNK, and p38. Antibodies specific for total ERK and JNK were used to assure even protein loading. The level of ERK phosphorylation rapidly increased in biphasic pattern after E2 exposure: 1 min, 4.3 fold; 5 min, 1 fold; and 10 min, 7.3 fold (max). However, the level of JNK phosphorylation did not increase throughout the time course. The level of p38 phosphorylation increased rapidly to 3.6 fold at 10 minutes. We demonstrate that E2 rapidly activates ERK in a biphasic manner and p38 in a monophasic manner, but has no effect on the activity of JNK. Our data suggest that rapid eNOS activation by estrogen in vascular endothelial cells is predominantly mediated through ERK and p38 pathway. Rapid activation of ERK and p38 resulting in NO production may substantially contribute to the atheroprotective effect of estrogen.

This research examined cellulolytic effects of fungi and other microbes present in cured sausages on the strength and stability of regenerated cellulose casings (RCC) used in the sausage industry. Occasionally during the curing process, RCC would split or fail, thereby leading to loss of product. The fungus Penicillium sp. BT-F-1, which was isolated from fermented sausages, and other fungi, which were introduced to enable the curing process, produced small amounts of cellulases on RCC in both liquid and solid cultivations. During continued incubation for 15-60 days in solid substrate cultivation (SSC) on RCC support, the fungus Penicillium sp isolate BT-F-1 degraded the casings' dry weights by 15-50% and decreased their tensile strengths by ~75%. Similarly commercial cellulase(s) resulted in 20-50% degradation of RCC in 48 h. During incubation with Penicillium sp BT-F-1, the surface structure of RCC collapsed, resulting in loss of strength and stability of casings. The matrix of industrial RCC comprised 88-93% glucose polymer residues with 0.8-4% xylan impurities. Premature casing failure appeared to result from operating conditions in the manufacturing process that allowed xylan to build up in the extrusion bath. The sausage fungus Penicillium sp BT-F-1 produced xylanases to break down soft xylan pockets prior to slow cellulosic dissolution of RCC.

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono- and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a D-xylulokinase mutant of Pichia stipitis, FPL-YS30 (xyl3-∆1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic acids used for neutralization and urea or ammonium sulfate used as nitrogen sources. Phosphoric acid improved color and removal of phenolic compounds. D-Gluconic acid enhanced cell growth. Ammonium sulfate increased cell yield and maximum specific cell growth rate independently of the acid used for neutralization. The highest xylitol yield (0.61 g(xylitol)/g(xylose)) and volumetric productivity (0.18 g(xylitol)/g(xylose )l) were obtained in hydrolysate neutralized with phosphoric acid. However, when urea was the nitrogen source the cell yield was less than half of that obtained with ammonium sulfate.

이 연구에서는 백합나무(Liriodendron tulipifera)를 옥살산으로 전처리한 시료로부터 에탄올 생산 가능성을 조사하고, response surface methodology(RSM)를 도입하여 전처리 조건을 분석하고자 한다. 산농도, 전처리 시간, 반응 온도를 조절하여 2(3) factorial central composite experimental design을 바탕으로 각기 다른 l5가지의 전처리 조건에서 시험하였다(central point에서 2반복). 전처리 후 고체 시료는 발효 균주인 Pichia stipitis를 사용하여 동시 당화발효로 에탄올 생산에 이용되었으며, 각각의 시료에서의 72시간에서의 에탄올 생산량(y,g/l)이 최대값으로, 종속변수로써 RSM에 적용되었다. 180℃에서 40분간 0.013 g/ g의 옥살산으로 처리한 시료가 가장 많은 양의 에탄올(9.7 g/l)을 생산하였으며, response surface methodology 분석에 따르면, 전처리 조건에서 온도 인자가 ethanol에 가장 큰 영향을 미치는 것으로 제시되었으며, 결과는 수식화되어 나타내었다.

In this study, we evaluated optimal conditions for ethanol production of the spruce hydrolysate (SH) obtained from diethyl oxalate pretreatment. Fermentable sugar concentration in SH was 29.04 g/l except arabinose. Monosaccharides obtained from the oligomer degradation were mainly mannose (39.26 g/l) and galactose (12.83 g/l). Concentration of 5-HMF and furfural which are inhibitors on ethanol fermentation were 0.09 g/l and 0.04 g/ respectively. Concentration of acetic acid and total phenolic compounds in SH were 1.4 g/l and 2.83 g/. Ethanol production using hydrolysate was 11.7 g/l at optimal pH 6.0 after 48 h. Specific ethanol production was 0.15 (g/(l*h)) at pH 5.0 and 5.5. while that was 0.24 (g/(l*h)) at pH 6.0. Specific ethanol production has difference depend on initial pH for fermentation. Ethanol production was 14.3 g/l after 48 h when xylanase 20 IU was added in SH for degradation of oligomer during fermentation. It implied that ethanol production increased by 22.2% compare with control (without xylanase).

Aqueous dilute acid pretreatments of corncob were conducted using cylindrical pressure vessels in an oil bath. Pretreatments were conducted in a temperature range of 160-190 °C with acid-solution-to-solid-corncob ratio of 2. The acid concentration (v/v) in the pretreatment solution was varied from 0% to 0.7%, depending on temperature. This gives acid charge on ovendry-weight corncob of 0-2.58%. It was found that optimal pretreatment temperature is between 160 and 170 °C based on total xylose and glucose yields and thermal energy consumption in pretreatment. At 170 °C and acid charge of 2.2% on cob, total glucose yield and xylose recovery were 97% and 75%, respectively, which resulted in an overall monomeric sugar recovery of about 88%. Xylose concentration in the hydrolysate was about 12%, with xylose-to-acetic-acid ratio of 8 and to furan (furfural and hydroxymethylfurfural) of about 15.

Saccharum (Saccharum spontaneum L. ssp. aegyptiacum (Willd.) Hack.), is a rapidly growing, wide ranging high-yield perennial, suitable for second generation bioethanol production. This study evaluated oxalic acid as a pretreatment for bioconversion. Overall sugar yields, sugar degradation products, enzymatic glucan hydrolysis and ethanol production were studied as effects of temperature (150-190 degrees C), reaction time (10-40 min) and oxalic acid concentration 2-8% (w/w). Time and temperature were combined into a single parameter, Severity Factor (SF) [Log(R(0))], and related to oxalic acid using a response surface methodology. Maximum total sugar yield was attained at a SF of 2.93 and 6.79% (w/w) oxalic acid, while maximum formation of sugar degradation products was observed at the highest SF (4.05) and 5% (w/w) oxalic acid. These were also the conditions for maximum simultaneous saccharification and fermentation (SSF) of the residual solids. Commercial cellulases and Saccharomyces cerevisiae attained 89.9% glucan conversion and 17.8 g/l ethanol. Pichia stipitis CBS 6054 fermented hemicellulosic hydrolysates from less severe conditions to ethanol with a yield of 0.35 (g(e)/g(s)). Maximal product yields were 69% of theoretical value and 90% of the SSF conversion efficiency for hydrolysate fermentation and SSF, respectively.

Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 2 3 full factorial design with six axial points. Temperatures ranged from 132 to 180 C, time from 10 to 90 min and oxalic acid loadings from 0.01 to 0.038 g/g solids. Separate maxima were found for enzymatic saccharification and hemicellulose fermentation, respectively, with the condition for maximum saccharification being significantly more severe. Ethanol production was affected by reaction temperature more than by oxalic acid and reaction time over the ranges examined. The effect of reaction temperature was significant at a 95% confidence level in its effect on ethanol production. Oxalic acid and reaction time were statistically significant at the 90% level. The highest ethanol concentration (20 g/l) was obtained after 48 h with an ethanol volumetric production rate of 0.42 g ethanol l � 1 h � 1 . The ethanol yield after SSF with P. stipitis was significantly higher than predicted by sequential saccharification and fermentation of substrate pretreated under the same condition. This was attributed to the secretion of b-glucosidase by P. stipitis. During SSF, free extracellular b-glucosidase activity was 1.30 pNPG U/g with P. stipitis, while saccharification without the yeast was 0.66 pNPG U/g. Published by Elsevier Ltd.

Efficient fermentation of hemicellulosic sugars is critical for the bioconversion of lignocellulosics to ethanol. Efficient sugar uptake through the heterologous expression of yeast and fungal xylose/glucose transporters can improve fermentation if other metabolic steps are not rate limiting. Rectification of cofactor imbalances through heterologous expression of fungal xylose isomerase or modification of cofactor requirements in the yeast oxidoreductase pathway can reduce xylitol production while increasing ethanol yields, but these changes often occur at the expense of xylose utilization rates. Genetic engineering and evolutionary adaptation to increase glycolytic flux coupled with transcriptomic and proteomic studies have identified targets for further modification, as have genomic and metabolic engineering studies in native xylose fermenting yeasts.

Overexpression of D-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled bioreactors using engineered S. cerevisiae CEN.PK. Growth on glucose was not significantly affected in pho13Delta mutants, but acetate production increased by 75%. Cell growth, ethanol production, and xylose consumption all increased markedly in pho13Delta mutants. The specific growth rate and rate of specific xylose uptake were approximately 1.5 times higher in the deletion strain than in the parental strain when growing on glucose-xylose mixtures and up to 10-fold higher when growing on xylose alone. In addition to showing higher acetate levels, pho13Delta mutants also produced less glycerol on xylose, suggesting that deletion of Pho13p could improve growth by altering redox levels when cells are grown on xylose.

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3Delta) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h(-1)). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 g(xylose)/g(cel) h) and xylitol production (0.059 g(xylitol)/g(cel) h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. Selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentation mixture by shuffling the promoters for GND2 and HXK2 with the genes for transaldolase ( TAL1 ), transketolase ( TKL1 ), and pyruvate kinase ( PYK1 ) in the Saccharomyces cerevisiae strain FPL-YSX3. This host strain has integrated xylose-metabolizing genes, including xylose reductase, xylitol dehydrogenase, and xylulose kinase. The optimal expression levels for TAL1 , TKL1 , and PYK1 were identified by analysis of volumetric ethanol production by transformed cells. We found the optimal combination for ethanol production to be GND2 - TAL1 - HXK2 - TKL1 - HXK2 - PYK1 . The MGPS method could easily be adapted for other eukaryotic and prokaryotic organisms to optimize expression of genes for industrial fermentation.

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was produced when the initial cell concentrations were high, cell density had no effect on the final ethanol yield. A two-parameter mathematical model was used to predict the cell population dynamics at the different initial cell concentrations. The model parameters, a and b correlate with the initial cell concentrations used with an R(2) of 0.99.

Technologies for the production of alternative fuels are receiving increased attention owing to concerns over the rising cost of petrol and global warming. One such technology under development is the use of yeasts for the commercial fermentation of xylose to ethanol. Several approaches have been employed to engineer xylose metabolism. These involve modeling, flux analysis, and expression analysis followed by the targeted deletion or altered expression of key genes. Expression analysis is increasingly being used to target rate-limiting steps. Quantitative metabolic models have also proved extremely useful: they can be calculated from stoichiometric balances or inferred from the labeling of intermediate metabolites. The recent determination of the genome sequence for P. stipitis is important, as its genome characteristics and regulatory patterns could serve as guides for further development in this natural xylose-fermenting yeast or in engineered Saccharomyces cerevisiae. Lastly, strain selection through mutagenesis, adaptive evolution or from nature can also be employed to further improve activity.

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g(-1) sugar consumed, so commercialization seems feasible for some applications.

The lack of industrially suitable microorganisms for converting biomass into fuel ethanol has traditionally been cited as a major technical roadblock to developing a bioethanol industry. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. Lignocellulosic biomass contains complex carbohydrates that necessitate utilizing microorganisms capable of fermenting sugars not fermentable by brewers' yeast. The most significant of these is xylose. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars, and work has concentrated on engineering these strains to selectively produce ethanol. Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work on this organism has concentrated on introducing pathways for the fermentation of arabinose and xylose. The history of constructing these strains and current progress in refining them are detailed in this review.

d -Xylulokinase (XK) is essential for the metabolism of d -xylose in yeasts. However, overexpression of genes for XK, such as the Pichia stipitis XYL3 gene and the Saccharomyces cerevisiae XKS gene, can inhibit growth of S . cerevisiae on xylose. We varied the copy number and promoter strength of XYL3 or XKS1 to see how XK activity can affect xylose metabolism in S . cerevisiae . The S . cerevisiae genetic background included single integrated copies of P . stipitis XYL1 and XYL2 driven by the S . cerevisiae TDH1 promoter. Multicopy and single-copy constructs with either XYL3 or XKS1 , likewise under control of the TDH1 promoter, or with the native P . stipitis promoter were introduced into the recombinant S . cerevisiae . In vitro enzymatic activity of XK increased with copy number and promoter strength. Overexpression of XYL3 and XKS1 inhibited growth on xylose but did not affect growth on glucose even though XK activities were three times higher in glucose-grown cells. Growth inhibition increased and ethanol yields from xylose decreased with increasing XK activity. Uncontrolled XK expression in recombinant S . cerevisiae is inhibitory in a manner analogous to the substrate-accelerated cell death observed with an S . cerevisiae tps1 mutant during glucose metabolism. To bypass this effect, we transformed cells with a tunable expression vector containing XYL3 under the control of its native promoter into the FPL-YS1020 strain and screened the transformants for growth on, and ethanol production from, xylose. The selected transformant had approximately four copies of XYL3 per haploid genome and had moderate XK activity. It converted xylose into ethanol efficiently.

Pichia stipitis is a source of genes for engineering xylose metabolism in Saccharomyces cerevisiaea task undertaken in numerous laboratories around the world. Increasing the capacity of P. stipitis for rapid xylose fermentation can greatly improve its usefulness in commercial xylose fermentations. Genetic transformation with URA3 is more efficient than transformation with the modified Sh ble marker. Cultivation conditions can strongly affect expression of fermentative enzymes in P. stipitis. Unlike S. cerevisiae, which regulates fermentation by sensing the presence of glucose, P. stipitis induces fermentative activity in response to oxygen limitation. The coupling of Xyl1 and Xyl2 activities therefore tends to result in the consumption of NADPH and accumulation of NADH. Preliminary data based on expressed sequence tags indicate that transcripts for fatty acid synthase (FAS2) stearoyl-coenzyme A desaturase (OLE1) are induced under oxygen-limiting conditions. Xylanase production by P. stipitis has been recognized, and the organism has also been transformed with heterologous xylanases to increase xylanase activity. A genetic system has been developed that includes the auxotrophic markers URA3 and LEU3 along with modified forms of the phleomycin D1 resistance marker, Sh ble, and the Cre recombinase. Many genes in P. stipitis are found in functionally related clusters. Further metabolic engineering and strain selection are needed to increase the overall fermentation rate and ethanol tolerance of P. stipitis for the commercial bioconversion of hemicellulose hydrolysates.

This work describes ethanol production from alfalfa fiber using separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) with and without liquid hot water (LHW) pretreatment. Candida shehatae FPL-702 produced 5 and 6.4 g/l ethanol with a yield of 0.25 and 0.16 g ethanol/g sugar respectively by SHF and SSF from alfalfa fiber without pretreatment. With LHW pretreatment using SSF, C. shehatae FPL-702 produced 18.0 g/l ethanol, a yield of 0.45 g ethanol/g sugar from cellulosic solids or ‘raffinate’. Using SHF, it produced 9.6 g/l ethanol, a yield of 0.47 g ethanol/g sugar from raffinate. However, the soluble extract fraction containing hemicelluloses was poorly fermented in both SHF and SSF due to the presence of inhibitors. Addition of dilute acid during LHW pretreatment of alfalfa fiber resulted in fractions that were poorly saccharified and fermented. These results show that unpretreated alfalfa fiber produced a lower ethanol yield. Although LHW pretreatment can increase ethanol production from raffinate fiber fractions, it does not increase production from the hemicellulosic and pectin fractions.

A total of 43 Forest Products Laboratory (FPL) strains of Pichia stipitis and Candida shehatae were tested for their ability to ferment a 1:1 mixture of glucose and xylose to ethanol prior to fermentation of partially deacidified wood hydrolyzates. The starting sugar composition, pH, and concentrations of inhibitors such as acetic acid, furfural, and hydroxy methyl furfural varied from one batch to another. The delay observed in growth and fermentation depended on the amounts of inhibitors present and on the capacity of the strain to resist them. The ethanol production rates and yields obtained with C. shehatae were higher than those with P. stipitis . C. shehatae strain FPL-Y-049 produced up to 34 g/l ethanol from batch VII of wood hydrolyzates. All the glucose, mannose, galactose and xylose in the wood hydrolyzate were consumed during fermentation. Only arabinose was unused. Addition of 10 mg/l zinc to acid hydrolyzate did not affect peak ethanol production, but it did increase rates of sugar utilization and ethanol production. The fermentation of hydrolyzate with recycled cells of C. shehatae Y-049 reduced the fermentation lag and increased the final ethanol concentration. The ethanol production rate was optimum in the pH range of 5.5–6.0, and an ethanol yield of 0.41–0.46 g/g was obtained in these fermentations. Because of heterogeneity between wood hydrolyzate batches, ethanol production was found to be influenced by hydrolyzate composition, pH, acetate concentration, amount of cells, and recycling of cells.

Liquid hot water (LHW) at a high temperature has been advocated as a pretreatment for herbaceous and lignocellulosic materials prior to enzymic saccharification. The focus of our research was the suitability of LHW pretreatment of alfalfa (Medicago saliva) fibre in the presence and absence of mild acid for optimum saccharification using enzymes. Enzymic saccharification was optimised in terms of substrate and enzyme concentrations. The main components of the enzymic hydrolyzates were sucrose, glucose, xylose, arabinose, and low amounts of uronic acid. After LHW pretreatment, both the soluble hemicellulose-rich ‘extract’ fraction and the insoluble residue ‘raffinate’ fraction (which contains predominantly cellulose) of alfalfa were recovered; the yield was 48 and 41%, respectively. Enzymic saccharification released more reducing sugars from pretreated fibre than from untreated fibre. For untreated alfalfa, a commercial pectinase and cellulase mixture caused maximum release of reducing sugars; using 2 and 4% (w/v) enzyme, a maximum of 51 g/l reducing sugars was released from 100 g/l untreated alfalfa. The soluble extract, LHW-pretreated alfalfa, was clarified and saccharified with the same cellulase and pectinase mixture releasing 8.4 g/l reducing sugars from 15.5 g/l solids. Release of reducing sugars from the insoluble raffinate ranged from 59 to 65 g/l from 100 g/l substrate, using 2 and 4% (w/v) cellulase. Addition of 0.07% sulphuric acid to the LHW pretreatment facilitated hemicellulose solubilisation; 9.9 g/l reducing sugars were released from the alfalfa extract by enzymic saccharification. However, pretreatment with sulphuric acid reduced sugar release and decreased fibre degradation of the raffinate.

The xylose-utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non-cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild-type P. stipitis CBS 6054 by cross-hybridization to CYC1 from Saccharomyces cerevisiae. The 333 bp open reading frame of PsCYC1 showed 74% and 69% identity to ScCYC1 and ScCYC7, respectively, at the DNA level. Disruption of PsCYC1 resulted in a mutant that uses the salicylhydroxamic acid (SHAM)-sensitive respiratory pathway for aerobic energy production. Cytochrome spectra revealed that cytochromes c and a.a(3) both disappeared in the cyc1-Delta mutant, so no electron flow through the cytochrome c oxidase was possible. The cyc1-Delta mutant showed 50% lower growth rates than the parent when grown on fermentable sugars. The cyc1-Delta mutant was also found to be unable to grow on glycerol. Interestingly, the mutant produced 0.46 g/g ethanol from 8% xylose, which was 21% higher in yield than the parental strain (0.38 g/g). These results suggested that the alternative pathway might play an important role in supporting xylose conversion to ethanol under oxygen-limiting conditions.

The glucose analog 2-deoxyglucose (2-DOG) has been used to obtain mutants depressed for pentose metabolism. Some researchers have used 2-DOG alone whereas others have used it in the presence of a glucose-repressible carbon source. We examined both methods and screened mutant strains for improved use of xylose in the presence of glucose. Pichia stipitis mutants selected for growth on d-xylose in the presence of 2-DOG used xylose from a 1∶1 glucose:xylose mixture more rapidly than did their parents. One of these mutants, FPL-DX26, completely consumed xylose in the presence of glucose and produced 33g/L ethanol in 45h from 80 g/L of this sugar mixture. Mutants selected for growth on 2-DOG alone did not show significant improvement. Selection for growth on d-xylose in the presence of 2-DOG has been useful in developing parental strains for further genetic manipulation.

Sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) was applied to an empty fruit bunches (EFB) for ethanol production. SPORL facilitated delignification through lignin sulfonation and dissolution of xylan to result in a highly digestible substrate. The pretreated whole slurry was enzymatically saccharified at a solids loading of 18% using a relatively low cellulase loading of 15 FPU/g glucan and simultaneously fermented without detoxification using Saccharomyces cerevisiae of YRH400. An ethanol yield of 217 L/tonne EFB was achieved at titer of 32 g/L. Compared with literature studies, SPORL produced high ethanol yield and titer with much lower cellulase loading without detoxification.

Spathaspora passalidarum NN245 (NRRL-Y27907) is an ascomycetous yeast that displays a higher specific fermentation rate with xylose than with glucose. Previous studies have shown that its capacity for xylose fermentation increases while cell yield decreases with decreasing aeration. Aeration optimization plays a crucial role in maximizing bioethanol production from lignocellulosic hydrolysates. Here, we compared the kinetics of S. passalidarum NN245 and Scheffersomyces (Pichia) stipitis NRRL Y-7124 fermenting 15% glucose, 15% xylose, or 12% xylose plus 3% glucose under four different aeration conditions. The maximum specific fermentation rate for S. passalidarum was 0.153 g ethanol/g CDW · h with a yield of 0.448 g/g from 150 g/L xylose at an oxygen transfer rate of 2.47 mmol O2 /L h. Increasing the OTR to 4.27 mmol O2 /L h. decreased the ethanol yield from 0.46 to 0.42 g/g xylose while increasing volumetric ethanol productivity from 0.52 to 0.8 g/L h. Both yeasts had lower cells yields and higher ethanol yields when growing on xylose than when growing on glucose. Acetic acid accretions of both strains correlated positively with increasing aeration. S. passalidarum secreted lower amounts of polyols compared to S. stipitis under most circumstances. In addition, the composition of polyols differed: S. passalidarum accumulated mostly xylitol and R,R-2,3-butanediol (BD) whereas S. stipitis accumulated mostly xylitol and ribitol when cultivated in xylose or a mixture of 12% xylose and 3% glucose. R,R-2,3-BD accumulation by S. passalidarum during xylose fermentation can be as much as four times of that by S. stipitis, and R,R-2,3-BD is also the most abundant byproduct after xylitol. The ratios of polyols accumulated by the two species under different aeration conditions and the implications of these observations for strain and process engineering are discussed.

We report the development of an efficient genetic transformation system for Lipomyces starkeyi based on a modified lithium acetate transformation protocol. L. starkeyi is a highly lipogenic yeast that grows on a wide range of substrates. The initial transformation rate for this species was extremely low, and required very high concentrations of DNA. A systematic approach for optimizing the protocol resulted in an increase in the transformation efficiency by four orders of magnitude. Important parameters included cell density, the duration of incubation and recovery periods, the heat shock temperature, and the concentration of lithium acetate and carrier DNA within the transformation mixture. We have achieved efficiencies in excess of 8,000 transformants/µg DNA, which now make it possible to screen libraries in the metabolic engineering of this yeast. Metabolic engineering based on this transformation system could improve lipogenesis and enable formation of higher value products.

Production of xylitol by Candida boidinii NRRL Y-17213 occurs under conditions of an oxygen limitation. The extent to which substrate is converted to xylitol and its coproducts (ethanol, other polyols, acetic acid), and the relative flow rates of substrate to energetic and biosynthetic pathways is controlled by the degree of oxygen limitation. With decrease in oxygen concentration in the inlet gas, for a constant dilution rate of 0.05 1/h. the specific oxygen uptake rate decreased from 1.30 to 0.36 mmol/gh Xylitol was not produced at specific oxygen uptake rates above 0.91 mmol/gh. Upon shift to lower oxygen rates, specific xylitol production rate increased more rapidly than specific ethanol production rate

Giant reed was evaluated for enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) using a commercial cellulase/β-glucosidase and Scheffersomyces ( Pichia ) stipitis CBS 6054 for ethanol production following dilute-oxalic acid pretreatment. A response surface methodology with two input parameters – severity factor (SF) and oxalic acid concentration (OA) – was employed to optimize both enzymatic hydrolysis and SSF. Xylan content after dilute-OA pretreatment decreased with increasing SF and OA; almost complete hydrolysis was observed when the harsher pretreatment conditions were used. Glucan and lignin content showed an opposite trend with respect to xylan content after dilute-OA pretreatment. Accordingly, enzymatic hydrolysis and ethanol production reached 95% of glucan conversion and 18 g l −1 (75.3% of the maximum theoretical ethanol yield), respectively, with the pretreatment condition 4.05 SF and 5% OA w/w. Dilute-OA mediated pretreatment of giant reed followed by coupled saccharification and fermentation can be considered a promising methodology for second generation bioethanol production.

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (μmol xylose min−1 ml−1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.

Microbial endoxylanases reduce chemical demand when they are applied to pulps prior to bleaching sequences. However, their mechanism of action is not fully understood, and the criteria for determining which enzymes are most effective are not well known. We examined the effects of two types of well-characterized endoxylanases from Streptomyces sp. TUB B-12-2 on the bleachability of kraft softwood and hardwood pulps. xyl 1a is a low molecular mass, high pl xylanase that forms mainly xylotriose but no xylose from oat spelts xylan. xyl 3 is a high molecular mass, low pl xylanase that forms mainly xylose and xylobiose as end products. xyl 3 released more reducing sugars than did xyl 1a, but both enzymes released about the same amount of total sugars. High performance liquid chromatography analysis of product sugars from pulp showed that at pH 7.0, xyl 1a formed oligosaccharides with a high degree of polymerization, but at pH 9.0 the action patterns of the two enzymes were similar. xyl 3 released 12% more chromophores from hardwood and 30% more from softwood pulp than did xyl 1a. Both enzymes enhanced brightness while reducing chemical demand, but xyl 3 was more effective. Synergism was noted in the prebleaching of softwood but not hardwood pulp. Both enzymes were active at pH 9, and the presence of pulp increased thermal stability.

Candida boidinii NRRL Y-17213 produced more xylitol thanC. magnolia (NRRL Y-4226 and NRRL Y-7621),Debaryomyces hansenii (C-98 M-21, C-56 M-9 and NRRL Y-7425), orPichia (Hansenula) anomala (NRRL Y-366). WithC. boidinii, highest xylitol productivity was at pH 7 but highest yield was at pH 8, using 5 g urea and 5 g Casamino acids/I. Decreasing the aeration rate decreased xylose consumption and cell growth but increased the xylitol yield. When an initial cell density of 5.1 g/l was used instead of 1.3 g/l, xylitol yield and the specific xylitol production rate doubled. Substrate concentration had the greatest effect on xylitol production; increasing xylose concentration 7.5-fold (to 150 g/l) gave a 71-fold increase in xylitol production (53 g/l) and a 10-fold increase in xylitol/ethanol ratio. The highest xylitol yield (0.47 g/g), corresponding to 52% of the theoretical yield, was obtained with 150 g xylose/l after 14 days. Xylose at 200 g/l inhibited xylitol production.

Under oxygen transfer rates (OTR), from 10 to 30 mmol· l −1 h −1 , Candida boidinii NRRL Y-17213 exhibited both NADH and NADPH linked d -xylose reductase activities with the former being higher. Xylitol dehydrogenase was mainly NAD dependent. Maximum xylitol production was attained at OTR of 14 mmol· l −1 h −1 . Ethanol, glycerol and ribitol were also produced. A correlation between xylitol accumulation, oxygen availability and key enzyme activities was viewed.

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spathaspora passalidarum , which ferments xylose and cellobiose natively, can also coferment these two sugars in the presence of 30 g/liter glucose. S. passalidarum simultaneously assimilates glucose and xylose aerobically, it simultaneously coferments glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, an adapted strain of S. passalidarum produced 39 g/liter ethanol with a yield of 0.37 g/g sugars from a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the flux of glycolytic intermediates is significantly higher on xylose than on glucose. The high affinity of its xylose reductase activities for NADH and xylose combined with allosteric activation of glycolysis probably accounts in part for its unusual capacities. These features make S. passalidarum very attractive for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts.

Xylose utilization is essential for the efficient conversion of lignocellulose to ethanol. The objective of this review is to trace the development of xylose-fermenting yeast strains from their discovery in 1980. following initial reports, screens of known yeast identified five species of interest: Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis , and Pichia stipitis, Candida shehatae strains can be deivided into three varieties. Pachysolen tannophilus and Pichia stipilis have been studied most extensively and have the best-understood genetic systems. Improved mutants of P. tannophilis have been obtained by selecting for an inability to oxidize ethanol ( eth ) and for rapid growth on xylitol and nitrate,. Improved P. stipitis mutants have been obtained by selecting for flocculation decreased utilization of glucose,a nd growth on noninductive carbon sources. Bacterial xylose isomerase has been cloned and expressed in S. cerevisiae and Schizosaccharomyces pombe , but he heterologous enzyme is inactive. Xylose reductase and xylitol dehydrogenase have been cloned from P. stipitis and expressed in Saccharomyces cerevisiae , giveing rise to transformant S. cerevisiae that grow on oxylose but that ferment it poorly. A transformation and expression system based on the URA3 marker has recently been developed for P. stipilitis so that contemporary genetic methods may be brough to bear on this organism.

Pichia stipitis CBS 6054 ferments D-Xylose to ethanol in a medium containing corn steep liquor as the only source of nitrogen, amino acids, vitamins and other nutrients. The ethanol yield and fermentation rate compare favorably to those obtained with media containing more expensive sources of nitrogen, vitamins and amino acids. Corn steep liquor is a good source of nutrients that can support growth and fermentation activity of this xylose fermenting yeast.

Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S similar to cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S similar to cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S similar to cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling of this strain was employed to further elucidate the observed physiology confirms a strongly up-regulated glyoxylate pathway enabling respiratory metabolism. The resulting strain is a desirable platform for the industrial production of biomass-related products using xylose as a sole carbon source.

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis . To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae , demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.

Response surface methodology (RSM), based on a 2 2 full factorial design, evaluated the moisture effects in recovering xylose by diethyloxalate (DEO) hydrolysis. Experiments were carried out in laboratory reactors (10 mL glass ampoules) containing corn stover (0.5 g) properly ground. The ampoules were kept at 160 °C for 90 min. Both DEO concentration and corn stover moisture content were statistically significant at 99% confidence level. The maximum xylose recovery by the response surface methodology was achieved employing both DEO concentration and corn stover moisture at near their highest levels area. We amplified this area by using an overlay plot as a graphical optimization using a response of xylose recovery more than 80%. The mathematical statistical model was validated by testing a specific condition in the satisfied overlay plot area. Experimentally, a maximum xylose recovery (81.2%) was achieved by using initial corn stover moisture of 60% and a DEO concentration of 4% w/w. The mathematical statistical model showed that xylose recovery increases during DEO corn stover acid hydrolysis as the corn stover moisture level increases. This observation could be important during the harvesting of corn before it is fully dried in the field. The corn stover moisture was an important variable to improve xylose recovery by DEO acid hydrolysis.

Building on our laboratory-scale optimization, oxalic acid was used to pretreat corncobs on the pilot-scale. The hydrolysate obtained after washing the pretreated biomass contained 32.55g/l of xylose, 2.74g/l of glucose and low concentrations of inhibitors. Ethanol production, using Scheffersomyces stipitis, from this hydrolysate was 10.3g/l, which approached the predicted value of 11.9g/l. Diafiltration using a membrane system effectively reduced acetic acid in the hydrolysate, which increased the fermentation rate. The hemicellulose content of the recovered solids decreased from 27.86% before pretreatment to only 6.76% after pretreatment. Most of the cellulose remained in the pretreated biomass. The highest ethanol production after simultaneous saccharification and fermentation (SSF) of washed biomass with S. stipitis was 21.1g/l.

Nitrogen, carbon, and manganese are potent regulators of lignin degradation, but although nitrogen and carbon elicit a generalizated response when cells are starved, manganese is a relatively specific regulator of lignin and manganese peroxidase (LiP and MnP, respectively). At high manganese levels, MnP is induced, and LiP is repressed. At low Mn levels, MnP is repressed, and LiP is induced. Organic acid chelators are very important in attaining LiP repression with high Mn. Both mineralization and lignin depolymerization are regulated by manganese in the presence of organic acid chelators. As long as the chelators keep Mn(II) and Mn(III) in solution, repression is observed, but eventually, dismutation reactions cause the formation and precipitation of Mn (IV) as MnO2. Repression is immediately relieved, and depolymerization and mineralization proceed at a high rate.

In scale-up, the potential of ethanol production by dilute sulfuric acid pretreatment using corncob was investigated. Pretreatments were performed at 170 °C with various acid concentrations ranging from 0% to 1.656% based on oven dry weight. Following pretreatment, pretreated biomass yield ranged from 59% to 67%. More than 90% of xylan was removed at 0.828% of sulfuric acid. At same pretreatment condition, the highest glucose yield obtained from pretreated biomass by enzymatic hydrolysis was about 76%, based on a glucan content of 37/100 g. In hydrolysate obtained by pretreatment, glucose concentration was low, while xylose concentration was significantly increased above 0.368% of sulfuric acid. At 1.656% of sulfuric acid, xylose and glucose concentration was highest. In subsequent, fermentation with hydrolysate, maximal ethanol yield was attained after 24 h with 0.368% of sulfuric acid. The fermentation efficiency of hydrolysate obtained by enzymatic hydrolysis reached a maximum of 75% at an acid charge of 0.368%.

The primary goal of this study was to determine the optimal condition to obtain fermentable monosaccharides (xylose and glucose) from hydrolysates of yellow poplar ( Liriodendron tulipifera ) by oxalic acid pretreatment as a potential bio-ethanol source. Based on 2 3 factorial design, fifteen operations were performed by varying on acid loading, reaction time and temperature, and the components of the solid and liquid fractions were analyzed. The sugar concentration (g/L) in hydrolysates and xylose solubilization (%) were applied to response surface methodology. The optimal condition for producing sugars was 151 °C, 0.042 g/g (weight of oxalic acid/dry matter), 13 min with predicted yield of 37.4 g/L, and for the xylose solubilization was 158 °C, 0.037 g/g, 13 min yielding 72.0% of the predicted value. Severe conditions generated inhibitors. By measuring the concentrations, the possibility utilizing hydrolysates for fermentation were estimated.

High yields of hemicellulosic and cellulosic sugars are critical in obtaining economical conversion of agricultural residues to ethanol. To optimize pretreatment conditions, we evaluated oxalic acid loading rates, treatment temperatures and times in a 2(3) full factorial design. Response-surface analysis revealed an optimal oxalic acid pretreatment condition to release sugar from the cob of Zea mays L. ssp. and for Pichia stipitis CBS 6054. To ferment the residual cellulosic sugars to ethanol following enzymatic hydrolysis, highest saccharification and fermentation yields were obtained following pretreatment at 180 degrees C for 50 min with 0.024 g oxalic acid/g substrate. Under these conditions, only 7.5% hemicellulose remained in the pretreated substrate. The rate of cellulose degradation was significantly less than that of hemicellulose and its hydrolysis was not as extensive. Subsequent enzymatic saccharification of the residual cellulose was strongly affected by the pretreatment condition with cellulose hydrolysis ranging between 26.0% and 76.2%. The residual xylan/lignin ratio ranged from 0.31 to 1.85 depending on the pretreatment condition. Fermentable sugar and ethanol were maximal at the lowest ratio of xylan/lignin and at high glucan contents. The model predicts optimal condition of oxalic acid pretreatment at 168 degrees C, 74 min and 0.027 g/g of oxalic acid. From these findings, we surmised that low residual xylan was critical in obtaining maximal glucose yields from saccharification.

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus ( Chainia rosea ) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching β-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60°C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K m of 7.9 mg/ml and an apparent V max of 305 μmol · min -1 · mg of protein -1 . The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.

A universal, rapid DNA assembly method for efficient multigene plasmid construction is important for biological research and for optimizing gene expression in industrial microbes. Three different approaches to achieve this goal were evaluated. These included creating long complementary extensions using a uracil-DNA glycosylase technique, overlap extension polymerase chain reaction, and a SfiI-based ligation method. SfiI ligation was the only successful approach for assembling large DNA fragments that contained repeated homologous regions. In addition, the SfiI method has been improved over a similar, previous published technique so that it is more flexible and does not require polymerase chain reaction to incorporate adaptors. In the present study, Saccharomyces cerevisiae genes TAL1, TKL1, and PYK1 under control of the 6-phosphogluconate dehydrogenase promoter were successfully ligated together using multiple unique SfiI restriction sites. The desired construct was obtained 65% of the time during vector construction using four-piece ligations. The SfiI method consists of three steps: first a SfiI linker vector is constructed, whose multiple cloning site is flanked by two three-base linkers matching the neighboring SfiI linkers on SfiI digestion; second, the linkers are attached to the desired genes by cloning them into SfiI linker vectors; third, the genes flanked by the three-base linkers, are released by SfiI digestion. In the final step, genes of interest are joined together in a simple one-step ligation.