NRRL Y-7124 Research Articles

Cellulosic Ethanol Production Using a Dual Functional Novel Yeast.

Reducing the cost of cellulosic ethanol production, especially for cellulose hydrolytic enzymes, is vital to growing a sustainable and efficient cellulosic ethanol industry and bio-based economy. Using an ethanologenic yeast able to produce hydrolytic enzymes, such as Clavispora NRRL Y-50464, is one solution. NRRL Y-50464 is fast-growing and robust, and tolerates inhibitory compounds 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) associated with lignocellulose-to-fuel conversion. It produces three forms of β-glucosidase isozymes, BGL1, BGL2, and BGL3, and ferment cellobiose as the sole carbon source. These β-glucosidases exhibited desirable enzyme kinetic parameters and high levels of enzyme-specific activity toward cellobiose and many oligosaccharide substrates. They tolerate the product inhibition of glucose and ethanol, and are stable to temperature and pH conditions. These characteristics are desirable for more efficient cellulosic ethanol production by simultaneous saccharification and fermentation. NRRL Y-50464 provided the highest cellulosic ethanol titers and conversion rates at lower cellulase loadings, using either pure cellulose or agricultural residues, as so far reported in the literature. This review summarizes NRRL Y-50464 performance on cellulosic ethanol production from refined cellulose, rice straw, and corn stover processed in various ways, in the presence or absence of furfural and HMF. This dual functional yeast has potential to serve as a prototype for the development of next-generation biocatalysts. Perspectives on continued strain development and process engineering improvements for more efficient cellulosic ethanol production from lignocellulosic materials are also discussed.

Detoxification of sugarcane bagasse hydrolysate with different adsorbents to improve the fermentative process.

Second generation ethanol has the prospect of becoming an important bioenergy alternative. The development of this technology is associated with the lignocellulosic materials' use, with emphasis on agricultural and agroindustrial by-products from which fermentable sugar can be produced. The acid hydrolysis depolymerizes the hemicellulose releasing mainly xylose. Subsequently, the cellulose can be converted into glucose by enzymatic hydrolysis. However, the acid hydrolysis produces toxic compounds, such as furan derivatives, phenolics, and organic acids, which are harmful to fermentative microorganisms. This study investigated different acid concentrations in the sulfuric acid hydrolysis of sugarcane bagasse (1- 5% m/v) and the use of adsorbents with the prerogative to improve the acid hydrolysate (AH) quality for microbial ethanolic fermentation. Cell growth and fermentative yield of Saccharomyces cerevisiae (PE-2) and Scheffersomyces stipitis (NRRL Y-7124) were evaluated. AH was used as a source of pentoses (17.7gL-1) and molasses (ME) sugarcane as source of hexoses (47gL-1). The following adsorbents were used: activated charcoal, clay, hydrotalcite and active and inactive cells of PE-2 and NRRL Y-7124, at concentrations ranging (1 - 8% m/v). Results of cell growth and chemical characterization allowed to select the most effective adsorbents with emphasis for active cells that removed 66% furfural and 51% 5-(hydroxymethyl) furfural) (5-HMF) and alcoholic productivity of 23.5gL-1 in AH and ME substrates, in the presence of mixed culture. These results indicate the application of active yeast cells in the detoxification of acid hydrolysates of the sugarcane bagasse previously to the fermentation.

Improved cellulosic ethanol production from corn stover with a low cellulase input using a β-glucosidase-producing yeast following a dry biorefining process.

A low-cost and sustainable cellulosic ethanol production is vital for fermentation-based industrial applications. Reducing the expenses of cellulose-deconstruction enzymes is one of the significant challenges to economic cellulose-to-ethanol conversion. Here, we report the improved ethanol production from corn stover after dry biorefining using a natural β-glucosidase-producing strain Clavispora NRRL Y-50464 with a low cellulase dose of 5mg protein/g glucan under separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) conditions. Strain Clavispora NRRL Y-50464 exhibited a superior ethanol fermentation performance over Saccharomyces cerevisiae DQ1 under both conditions. It produced an ethanol titer of 38.1g/L within 96h at a conversion efficiency of 55.5% with 25% solids loading (w/w) via SSF without addition of extra β-glucosidase supplement. Improved performance of Y-50464 on a bioreactor with a helical stirring apparatus confirmed its advantage over the conventional bioreactors originally designed for liquid fermentations in cellulosic ethanol conversion by SSF. The results of this study suggested that the strain Clavispora NRRL Y-50464 has a potential as a candidate for lower-cost cellulosic ethanol production from lignocellulosic materials.

Real‐time monitoring of ethanol production during Pichia stipitis NRRL Y‐7124 alcoholic fermentation using transflection near infrared spectroscopy

The application of in situ near-infrared spectroscopy monitoring of xylose metabolizing yeast such as Pichia stipitis for ethanol production with semisynthetic media, applying chemometrics, was investigated. During the process in a bioreactor, biomass, glucose, xylose, ethanol, acetic acid, and glycerol determinations were performed by a transflection probe immersed in the culture broth and connected to a near-infrared process analyzer. Wavelength windows in near-infrared spectra recorded between 800 and 2200nm were pretreated using Savitzky-Golay smoothing, second derivative and multiplicative scattering correction in order to perform a partial least squares regression and generate the calibration models. These calibration models were tested by external validation (78 samples). Calibration and validation criteria were defined and evaluated in order to generate robust and reliable models for an alcoholic fermentation process matrix. Moreover, regressions coefficients (β) and variable influence in the projection plots were used to assess the results. A novelty is the use of β versus VIP dispersion plots to determine which vectors have more influence on the response in order to improve process comprehension and operability. Validated models were used in a real-time monitoring during P. stipitis NRRL Y7124 semisynthetic media fermentations.

Technical assessment of natural deep eutectic solvent (NADES) mediated biorefinery process: A case study

Comprehensive evaluation of an integrated process for cellulosic ethanol production from lactic acid+choline chloride+water (LA-CC-H2O) mixture based natural deep eutectic solvent (NADES) pretreated rice straw; solvent recovery and its reusability; extraction of value added products viz., lignin and xylan; enzymatic saccharification and fermentation were studied. Four separate processes viz. pretreatment at 5% (w/v) and 10% (w/v) solids loading and enzymatic hydrolysis at 10% (w/v) and 25% (w/v) solids loading were investigated. No significant difference was observed in holocellulose recovery and lignin solubility in all the tested processes. Hydrolysis at 10% (w/v) and 25% (w/v) solids loading produced maximum glucose yields of 38.6±1.2g/L and 90.3±2.3g/L, respectively. Additional concentration step of sugars is needed from 10% (w/v) hydrolysis for direct ethanol fermentation using Clavispora NRRL Y-50464, while this could be voided with 25% (w/v) hydrolysis sugars. Maximum ethanol yield of 36.7g/L was obtained with 9% (w/v) glucose with a theoretical conversion efficiency of 79.9% which estimated to produce ~129L of ethanol per ton of raw biomass. Alongside, NADES pretreatment resulted in solubilization of 72-75% (w/v) of biomass bound lignin from which 86–90% (w/v) of lignin was recovered. Additionally, >98% (v/v) of the treated NADES reagent was recovered and reused in three consecutive biomass pretreatment cycles with no loss in pretreatment efficiency. This study clearly demonstrated that pretreatment at 10% (w/v) solids loading followed by enzymatic hydrolysis at 25% (w/v) solids loading is the most effective integrated biorefinery process using NADES pretreated rice straw.

Application of Natural Deep Eutectic Solvents in Biomass Pretreatment, Enzymatic Saccharification and Cellulosic Ethanol Production

Currently a revolutionary transition is needed in use of greener solvents in chemical industries for a more sustainable world. Natural Deep Eutectic Solvents (NADES) are recently developed as one of these types of new solvents considered as bio-based Ionic liquids (ILs) or Deep Eutectic Solvents (DES). Foreseeing the potential applications of NADES in diverse areas of research, here we have evaluated the applicability of NADES reagents in cellulosic ethanol production from lignocellulosic biomass residue rice straw using Cellic Ctec2 and cellobiose-fermenting yeast strain Clavispora NRRL Y-50464. Enzymatic saccharification of NADES pretreated rice straw produced maximum reducing sugars of 389 g/L was achieved at 40 % solids loading and 12 FPU/g of cellulase enzyme in 48 h. This was marginally less when compared to mild-alkali pretreated rice straw where maximum reducing sugars of 414 g/L was achieved at similar solids and enzyme loading. Besides, increase in solids loading from 10 % to 40 % increased the rate of sugars production significantly in both batch and fed-batch saccharification. Subsequently with the yeast strain NRRL Y-50464 an ethanol production of 36.7 g/L was achieved within 36 h from the so-formed reducing sugars (at 8 % measured glucose concentration) with a conversion efficiency of 90.1%.

Biosurfactants produced by Scheffersomyces stipitis cultured in sugarcane bagasse hydrolysate as new green larvicides for the control of Aedes aegypti, a vector of neglected tropical diseases.

Biosurfactants are microbial metabolites with possible applications in various industrial sectors that are considered ecofriendly molecules. In recent years, some studies identified these compounds as alternatives for the elimination of vectors of tropical diseases, such as Aedes aegypti. The major bottlenecks of biosurfactant industrial production have been the use of conventional raw materials that increase production costs as well as opportunistic or pathogenic bacteria, which restrict the application of these biomolecules. The present study shows the potential of hemicellulosic sugarcane bagasse hydrolysate as a raw material for the production of a crystalline glycolipidic BS by Scheffersomyces stipitis NRRL Y-7124, which resulted in an emulsifying index (EI24) of 70 ± 3.4% and a superficial tension of 52 ± 2.9 mN.m-1. Additionally, a possible new application of these compounds as biolarvicides, mainly against A. aegypti, was evaluated. At a concentration of 800 mg.L-1, the produced biosurfactant caused destruction to the larval exoskeletons 12 h after application and presented an letal concentration (LC50) of 660 mg.L-1. Thus, a new alternative for biosurfactant production using vegetal biomass as raw material within the concept of biorefineries was proposed, and the potential of the crystalline glycolipidic biosurfactant in larvicidal formulations against neglected tropical disease vectors was demonstrated.

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Alkaline deacetylation as a strategy to improve sugars recovery and ethanol production from rice straw hemicellulose and cellulose

A mild alkaline pretreatment (deacetylation) prior to the dilute acid pretreatment was evaluated as a strategy to improve the sugars recovery and ethanol production from both hemicellulose and cellulose fractions of rice straw. This pretreatment was carried out using different conditions of temperature (50–70°C) and NaOH loading (20–80mg NaOH/g biomass), which were combined according to a 22 central composite design. In this step the removal of acetyl groups as well as the impact of this step on biomass composition were evaluated. In order to assess the impact of the deacetylation on hemicellulosic hydrolysate composition, the influence of the reaction time (30–90min) and sulfuric acid concentration (0.5–1.5% w/v) was also studied, using the alkaline-pretreated solid (deacetylated) and rice straw in natura. The best sequential pretreatment conditions were scaled-up to 50-L reactor, being obtained a cellulose-rich pretreated solid (cellulignin) and a hemicellulosic hydrolysate, which was concentrated to 70g/L xylose to be used as fermentation medium. A significant improvement on ethanol production from xylose by Scheffersomyces stipitis NRRL Y-7124 was observed when the biomass was submitted to deacetylation (about 4-fold). The enzymatic conversion of cellulose was also improved (from 73 to 89%) when the deacetylated cellulignin was used, resulting in an enhancement of the ethanol production (from 12.7 to 20.4g/L) during the simultaneous saccharification and fermentation with Kluyveromyces marxianus NRRL Y-6860. In brief, biomass deacetylation prior to dilute acid pretreatment was an efficient strategy for rice straw processing, substantially improving the ethanol production from both pentose and hexose sugars.

Cellulosic ethanol production from green solvent-pretreated rice straw

Cellulosic ethanol production from green solvent (GS) -pretreated rice straw and the effects of green solvents on cellulase enzyme cellic ctec2 and cellobiose-fermenting yeast strain Clavispora NRRL Y-50464 was comprehensively investigated. Using choline chloride/glycerol (CC-GLY) treated rice straw, maximum reducing sugars of 226.7 g/L was obtained with a saccharification efficiency of 87.1% at 20% solids loading and 12 FPU cellic ctec2. An ethanol production of 36.7 g/L was observed from 8% of glucose within 36 h with a conversion efficiency of 90.1%. Incubation of cellulase in green solvents (CC-GLY, choline chloride/1,2-propane diol (CC-PD), choline chloride/ethylene glycol (CC-EG)) at high concentrations (up to 30%, v/v) had no inhibitory effect on cellic ctec2. Moreover, CC-GLY and CC-PD at 10% (v/v) did not affect the growth rate, sugar consumption and ethanol production from Clavispora NRRL Y-50464, while 10% (v/v) CC-EG repressed and delayed the cell growth of the microbe. While, strain Y-50464 was highly sensitive in other green reagents that were evaluated in this study. The green solvent-pretreatment efficiently removed lignin from the ricestraw and improved fermentation efficiency significantly. Green solvents with pH<3.0 inhibited of cellic ctec2 enzyme activity and growth of Clavispora strain NRRL Y-50464. When pH of the green reagents was neutralized or adjusted to pH 5.0, normal enzyme activity and cell growth was observed. Moreover, with selective green reagents i.e. CC-GLY, CC-PD, and CC-EG, cellulosic ethanol production can be accomplished using cellic ctec2 and strain NRRL Y-50464.

Two New Native β-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast.

Yeast strain Clavispora NRRL Y-50464 is able to produce cellulosic ethanol from lignocellulosic materials without addition of external β-glucosidase by simultaneous saccharification and fermentation. A β-glucosidase BGL1 protein from this strain was recently reported supporting its cellobiose utilization capability. Here, we report two additional new β-glucosidase genes encoding enzymes designated as BGL2 and BGL3 from strain NRRL Y-50464. Quantitative gene expression was analyzed and the gene function of BGL2 and BGL3 was confirmed by heterologous expression using cellobiose as a sole carbon source. Each gene was cloned and partially purified protein obtained separately for direct enzyme assay using varied substrates. Both proteins showed the highest specific activity at pH 5 and relatively strong affinity with a Km of 0.08 and 0.18 mM for BGL2 and BGL3, respectively. The optimum temperature was found to be 50°C for BGL2 and 55°C for BGL3. Both proteins were able to hydrolyze 1,4 oligosaccharides evaluated in this study. They also showed a strong resistance to glucose product inhibition with a Ki of 61.97 and 38.33 mM for BGL2 and BGL3, respectively. While BGL3 was sensitive showing a significantly reduced activity to 4% ethanol, BGL2 demonstrated tolerance to ethanol. Its activity was enhanced in the presence of ethanol but reduced at concentrations greater than 16%. The presence of the fermentation inhibitors furfural and HMF did not affect the enzyme activity. Our results suggest that a β-glucosidase gene family exists in Clavispora NRRL Y-50464 with at least three members in this group that validate its cellobiose hydrolysis functions for lower-cost cellulosic ethanol production. Results of this study confirmed the cellobiose hydrolysis function of strain NRRL Y-50464, and further supported this dual functional yeast as a candidate for lower-cost cellulosic ethanol production and next-generation biocatalyst development in potential industrial applications.

Evaluation of oxygen availability on ethanol production from sugarcane bagasse hydrolysate in a batch bioreactor using two strains of xylose-fermenting yeast

Ethanol production from biomass-derived pentose sugar challenges the development of low cost technologies to produce 2G ethanol. Although some studies describe ethanol production from yeast, few reports describe its manufacturing from hemicelluloses sugars in stirred-tank fermenter under controlled conditions. Experimental assays were performed to evaluate the influence of aeration, agitation rate and initial pH on ethanol production using sugarcane bagasse hemicelluloses hydrolysate by Scheffersomyces stipitis NRRL Y-7124 and Scheffersomyces shehatae UFMG HM 52.2. Ethanol production from these two yeasts was favored by initial pH increase and agitation rate decrease. The maximum fermentative yield was attained by S. stipitis and S. shehatae yeasts at 100 RPM, initial pH 6.50 and under oxygen limited conditions (0.1 and 3.2 h−1). Yield, ethanol productivity and process efficiency in S. shehatae and S. stipitis were 0.42 and 0.16 g g−1, 0.25 and 0.1 g L−1 h−1 and 85 and 31%, respectively. It showed that S. shehatae presented the best ethanol production performance. The current study is the first report describing the influence of these variables on hemicelluloses hydrolysates under controlled conditions. It highlights these xylose-fermenting yeasts potential to produce ethanol from biomass.

Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading.

BackgroundLignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment.ResultsRepetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol-challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARS Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions.ConclusionsRobust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.

Effects of aeration on growth, ethanol and polyol accumulation by Spathaspora passalidarum NRRL Y-27907 and Scheffersomyces stipitis NRRL Y-7124.

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.

Technical Assessment of Cellulosic Ethanol Production Using β-Glucosidase Producing Yeast Clavispora NRRL Y-50464

Reducing the cost of cellulosic ethanol production, especially the use of expensive exogenous cellulose hydrolytic enzymes such as cellulase and β-glucosidase, is a critical challenge and vital for a sustainable advanced biofuel industry. Here, we report a novel ethanologenic yeast strain Clavispora NRRL Y-50464 that produces sufficient innate β-glucosidase enzyme activity for cellulosic ethanol production by simultaneous saccharification and fermentation (SSF). In a bottled SSF, strain Y-50464 produced 40.44 g/L ethanol from pure cellulose within 72 h at a conversion rate of 0.04 g/L/h, applying conventional cellulase without supplementary β-glucosidase. Ethanol conversion from delignified corn stover by Y-50464 showed significantly higher titers and rates at various solids loading levels than that from conventional pretreated corn stover with over 40 to 60 % improved efficiency in a bottled SSF. However, the bottled SSF was inefficient for mixing higher levels of cellulose feedstock and should be replaced by a more suitable experimental apparatus. In a 2-L bioreactor SSF using conventional dilute acid pretreated corn stover, strain Y-50464 produced 32 g/L ethanol from 20 % solids loading at 48 h applying cellulase alone without addition of β-glucosidase. This represented a conversion rate of 0.088 g/L/h, the highest rate so far for cellulosic ethanol production from lignocellulosic materials. Elimination of β-glucosidase in cellulose-to-ethanol fermentation would be expected to reduce cost of cellulose conversion. The robustness, fast growth rate, and the capability of producing both ethanol and β-glucosidase illustrated the potential of strain Y-50464 as a potential candidate biocatalyst for advanced biofuel production from lignocellulosic biomass.

Bioethanol production from hydrolyzed sugarcane bagasse supplemented with molasses “B” in a mixed yeast culture

Lignocellulosic biomass is an abundant renewable source of xylose and glucose, which can be converted into ethanol using microorganisms. The aim of this study was to use sugarcane bagasse hydrolyzate (HBC) supplemented with sugarcane molasses “B”, one of the liquors resulting from the crystallization step in cane sugar production process, in a mixed yeast culture (Saccharomyces cerevisiae ITV-01 and Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124) in order to increase ethanol production and the complete utilization of hydrolyzate sugars. The culture of mixed yeast in a medium with 50% HBC supplemented with 8% v/v intermediate molasses “B” after 42 h fermentation exhibited a significant increase in ethanol production (53.80 gL−1), yield (0.45 gg−1) and productivity (1.07 gL−1 h−1). The novel strategy of using 8% v/v sugar cane molasses “B”, promotes complete sugar consumption and a 74% increase in ethanol fermentation by the mixed culture of S. cerevisiae ITV-01 and Scheffersomyces stipitis NRRL Y-7124.

Evaluation of the tolerance of acetic acid and 2-furaldehyde on the growth of Pichia stipitis and its respiratory deficient

The use of lignocellulosic residues for ethanol production is limited by toxic compounds in fermenting yeasts present in diluted acid hydrolysates like acetic acid and 2-furaldehyde. The respiratory deficient phenotype gives the cell the ability to resist several toxic compounds. So the aim of this work was to evaluate the tolerance to toxic compounds present in lignocellulosic hydrolysates like acetic acid and 2-furaldehyde in Pichia stipitis and its respiratory deficient strains. The respiratory deficient phenotype was induced by exposure to chemical agents such as acriflavine, acrylamide and rhodamine; 23 strains were obtained. The selection criterion was based on increasing specific ethanol yield (gethanolg(-1)biomass) with acetic acid and furaldehyde tolerance. The screening showed that P. stipitis NRRL Y-7124 ACL 2-1RD (lacking cytochrome c), obtained using acrylamide, presented the highest specific ethanol production rate (1.82gg(-1)h(-1)). Meanwhile, the ACF8-3RD strain showed the highest acetic acid tolerance (7.80gL(-1)) and the RHO2-3RD strain was able to tolerate up to 1.5gL(-1) 2-furaldehyde with a growth and ethanol production inhibition of 23 and 22%, respectively. The use of respiratory deficient yeast phenotype is a strategy for ethanol production improvement in a medium with toxic compounds such as hydrolysed sugarcane bagasse amongst others.

Evaluation of cellulosic and hemicellulosic hydrolysate fermentability from sugarcane bagasse hybrids with different compositions

Seven sugarcane hybrids with varying initial level of cell wall components were evaluated as substrates to ethanol production from cellulosic and hemicellulosic fractions by Pichia stipitis NRRL Y-7124, following pretreatment and enzymatic hydrolysis. The bagasses were pretreated with diluted sulfuric acid to separate the hemicellulose hydrolysate, whereas the cellulignin was hydrolyzed by commercial cellulase to attain the cellulosic hydrolysate. The cellulose conversion varied from 35 to 58 %, and based on the cellulignin composition, it was possible to describe a correlation between the enzymatic conversion of cellulose (ECC, %) as a function of lignin/hemicellulose ratio. For all the cellulosic hydrolysates, the yeast P. stipitis was able to convert glucose into ethanol with yield and volumetric productivity varying from 0.29 to 0.34 g/g and from 0.55 to 0.88 g/L h, respectively. On the other hand, the hemicellulosic hydrolysates were poorly utilized by this yeast due to the high level of inhibitory compounds, such as acetic acid and furans. Under the pretreatment conditions used, it was possible to point out a clone (referred as number 8) with potential for fermentation of both cellulosic and hemicellulosic hydrolysates.

Detoxification of rice straw and olive tree pruning hemicellulosic hydrolysates employing Saccharomyces cerevisiae and its effect on the ethanol production by Pichia stipitis

The aim of this work was to study the ability of Saccharomyces cerevisiae (baker's yeast) to metabolize a variety of aromatic compounds found in rice straw (RSHH) and olive tree pruning (OTHH) hemicellulosic hydrolysates, obtained by acid hydrolysis at different sugar and toxic compound concentrations. Initially, the hydrolysates were inoculated with S. cerevisiae (10 g L(-1)) and incubated at 30 °C under agitation at 200 rpm for 6 h. The results showed that this yeast was able to utilize phenolic and furan compounds in both hemicellulose hydrolysates. Next, the treated hydrolysates were inoculated with Pichia stipitis NRRL Y-7124 to evaluate the effect of biotransformation of aromatic compounds on ethanol production, and better fermentation results were obtained in this case compared to untreated ones. The untreated hemicellulose hydrolysates were not able to be fermented when they were incubated with Pichia stipitis. However, in RSHH treated hydrolysates, ethanol (Y(P/S)) and biomass (Y(X/S)) yields and volumetric ethanol productivity (Q(P)) were 0.17 g g(-1), 0.15 g g(-1) and 0.09 g L(-1) h(-1), respectively. The OTHH-treated hydrolysates showed less favorable results compared to RSHH, but the fermentation process was favored with regard to untreated hydrolysate. These results showed that the fermentation by P. stipitis in untreated hydrolysates was strongly inhibited by toxic compounds present in the media and that treatment with S. cerevisiae promoted a significant reduction in their toxicities.

Immobilization of Scheffersomyces stipitis cells with calcium alginate beads: A sustainable method for hemicellulosic ethanol production from sugarcane bagasse hydrolysate

Lignocellulosic ethanol has shown promising alternative to gasoline however expensive and cumbersome bioprocessing limits the commercialization of biofuels. The major impediment toward the economic ethanol production is the bioconversion of sugars into ethanol via microbial fermentation. Application of immobilized cells in fermentation of hemicellulosic hydrolysate could minimize the ethanol production cost. This study evaluated the conditions for cell immobilization for the yeast Scheffersomyces stipitis NRRL Y-7124 by the method of entrapment in calcium alginate gel. A full factorial design (23) was designed to investigate the effect of three process variables i.e. sodium alginate concentration (1.0, 1.5 and 2.0%), calcium chloride concentration (0.1, 0.15 and 0.2 M) and conditioning time (12, 18 and 24 h). Twelve numbers of experiments were performed with central points in quadruplicates. The range of ethanol production in all experiments was observed from 4.88 g/L (Yp/s, 0.16 g/g) to 9.9 g/L ethanol (Yp/s, 0.29 g/g). Statistical analysis revealed that immobilization conditions (2.0% sodium alginate concentration, 0.1M calcium chloride concentration and 12 h conditioning time) were responsible for high stability of immobilized cells which in-turn enabled the maximum ethanol production (7.2 g/L, Yp/s, 0.26 g/g) from hemicellulosic hydrolysate of sugarcane bagasse