Engineering of the hypoxia-induced Pichia stipitis ADH2 promoter to construct a promoter library for Pichia pastoris.

Hypoxia-inducible promoters of a wide range of activities are desirable for fine-tuning gene expression in response to oxygen limitation, especially for the Crabtree negative yeast Pichia pastoris (Komagataella phaffii) with a high oxygen consumption rate in large-scale fermentations. Here we constructed a hypoxia-inducible promoter library for P. pastoris through error-prone PCR of Pichia stipitis ADH2 promoter (PsADH2). The library of 30 selected promoters showing 0.4- to 5.5-fold of the PsADH2 activity was obtained through high-throughput screening in microplates using the reporter yeast-enhanced green fluorescent protein. Two strong promoters, AM23 and AM30, were further characterized in shake flask cultures at high and low dissolved oxygen levels. They responded more sensitively to the low dissolved oxygen level, achieving a 4.6-, 7.9-fold and 3.6-, 7.7-fold higher fluorescence intensity and transcript level, respectively, than the wild-type PsADH2. Their hypoxia-inducible properties were confirmed with two additional reporters: ß-galactosidase and Vitreoscilla hemoglobin, to demonstrate the broad applicability of the promoter library. During the typical fermentation process in shake flasks, the promoter AM30 showed strong expression with cell growth and decreased oxygen levels, without any additional chemical inducers or operations. Since the potent industrial host P. pastoris is recognized as an easy to scale-up system, it is reasonable to expect that the obtained hypoxia-inducible promoter library may have great potential to enable convenient regulation of gene expression under industrial fermentations which are usually run under oxygen limitation due to high cell density cultivations.

Comprehensive Characterization of Mutant Pichia stipitis Co-Fermenting Cellobiose and Xylose through Genomic and Transcriptomic Analyses.

The development of a yeast strain capable of fermenting mixed sugars efficiently is crucial for producing biofuels and value-added materials from cellulosic biomass. Previously, a mutant Pichia stipitis YN14 strain capable of co-fermenting xylose and cellobiose was developed through evolutionary engineering of the wild-type P. stipitis CBS6054 strain, which was incapable of cofermenting xylose and cellobiose. In this study, through genomic and transcriptomic analyses, we sought to investigate the reasons for the improved sugar metabolic performance of the mutant YN14 strain in comparison with the parental CBS6054 strain. Unfortunately, comparative wholegenome sequencing (WGS) showed no mutation in any of the genes involved in the cellobiose metabolism between the two strains. However, comparative RNA sequencing (RNA-seq) revealed that the YN14 strain had 101.2 times and 5.9 times higher expression levels of HXT2.3 and BGL2 genes involved in cellobiose metabolism, and 6.9 times and 75.9 times lower expression levels of COX17 and SOD2.2 genes involved in respiration, respectively, compared with the CBS6054 strain. This may explain how the YN14 strain enhanced cellobiose metabolic performance and shifted the direction of cellobiose metabolic flux from respiration to fermentation in the presence of cellobiose compared with the CBS6054 strain.

Physiological responses of Pichia stipitis to imidazolium chloride ionic liquids with different carbon chain length.

Ionic liquids (ILs) are used as detoxication agents for fermentation of lignin into ethanol because of their good applicability. However, the residual ILs may be toxic to the yeast. In order to improve the use of ILs for fermentation and protected environment, the toxicity of ILs with different carbon chain length to Pichia stipitis was studied in this paper. Four kinds of common imidazolium chloride ILs ([C4mim]Cl, [C6mim]Cl, [C8mim]Cl and [C10mim]Cl) were selected. ILs can inhibit the proliferation of Pichia stipitis and increase their mortality. Oxidative stress reaction occurred in the cells, and the activities of antioxidant enzymes are affected. Comparing with the integrated biomarker response (IBR) index, it was found that the toxicity increases with increasing chain length. ILs may enter cells by damaging cell membranes and reduce ethanol production by damaging organelles such as mitochondria. ILs caused wrinkles and dents on the surface of cells up to cell deformation and even rupture. The toxicity sequence was as follows: [C10mim]Cl> [C8mim]Cl>[C6mim]Cl>[C4mim]Cl. Due to this toxicity to Pichia stipitis, these compounds should be used carefully in the fermentation process and also to avoid toxic effects on other organisms in the environment.

Yeast-Based Biosynthesis of Natural Products From Xylose.

Xylose is the second most abundant sugar in lignocellulosic hydrolysates. Transformation of xylose into valuable chemicals, such as plant natural products, is a feasible and sustainable route to industrializing biorefinery of biomass materials. Yeast strains, including Saccharomyces cerevisiae, Scheffersomyces stipitis, and Yarrowia lipolytica, display some paramount advantages in expressing heterologous enzymes and pathways from various sources and have been engineered extensively to produce natural products. In this review, we summarize the advances in the development of metabolically engineered yeasts to produce natural products from xylose, including aromatics, terpenoids, and flavonoids. The state-of-the-art metabolic engineering strategies and representative examples are reviewed. Future challenges and perspectives are also discussed on yeast engineering for commercial production of natural products using xylose as feedstocks.

Bioethanol Production from Azolla filiculoides by Saccharomyces cerevisiae, Pichia stipitis, Candida lusitaniae, and Kluyveromyces marxianus.

Ethanol was produced by separate hydrolysis and fermentation using Azolla filiculoides as a biomass. Thermal acid hydrolysis and enzymatic saccharification were used as pretreatment methods to produce monosaccharides from Azolla. The optimal content for thermal acid hydrolysis of 14% (w/v) Azolla weed slurry produced 16.7-g/L monosaccharides by using 200 mM H2SO4 at 121 °C for 60 min. Enzymatic saccharification using 16 U/mL Viscozyme produced 61.6 g/L monosaccharide at 48 h. Ethanol productions with ethanol yield coefficients from Azolla weed hydrolysate using Kluyveromyces marxianus, Candida lusitaniae Saccharomyces cerevisiae, and Pichia stipitis were 26.8 g/L (YEtOH = 0.43), 23.2 g/L (YEtOH = 0.37), 18.2 g/L (YEtOH = 0.29), and 13.7 g/L (YEtOH = 0.22), respectively. Saccharomyces cerevisiae produces the lowest yield as it utilized only glucose. Bioethanol from Azolla weed hydrolysate can be successfully produced by using Kluyveromyces marxianus because it consumed the mixture of glucose and xylose completely within 60 h.

Encapsulation enhances protoplast fusant stability.

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.

Improved ethanol productivity and ethanol tolerance through genome shuffling of Saccharomyces cerevisiae and Pichia stipitis.

Genome shuffling by recursive protoplast fusion between Saccharomyces cerevisiae and Pichia stipitis also known as Scheffersomyces stipitis resulted in a promising yeast hybrid strain with superior qualities than those of the parental strains in enhancing biofuel production. Our study focused on the substrate utilization, ethanol fermentation, and ethanol tolerance of the hybrids and the parental strains. The parental strain S. cerevisiae is limited to utilize only hexose sugars, and this leads to decrease in the ethanol yield when they are subjected to ethanol production from lignocellulosic biomass which is rich in pentose sugars. To overcome this limitation, we constructed a hybrid yeast strain through genome shuffling which can assimilate all the sugars present in the fermentation medium. After two rounds of recursive protoplast fusion, there was a higher increase in substrate utilization by hybrid SP2-18 compared to parental strain S. cerevisiae. SP2-18 was able to consume 34% of xylose sugar present in the fermentation medium, whereas S. cerevisiae was not able to utilize xylose. Further, the hybrid strain SP2-18 was able to reach an ethanol productivity of 1.03 g L-1 h-1, ethanol yield 0.447 g/g, and ethanol concentration 74.65 g L-1 which was relatively higher than that of the parental strain S. cerevisiae. Furthermore, the hybrid SP2-18 was found to be stable in the production of ethanol. The random amplified polymorphic DNA profile of the yeast hybrid SP2-18 shows the polymorphism between the parental strains indicating the migration of specific sugar metabolizing genes from P. stipitis, while the maximum similarity was with the parent S. cerevisiae.

Existence of a scaling relation in continuous cultures of Scheffersomyces stipitis: the steady states are completely determined by the ratio of carbon and oxygen uptake rates.

BACKGROUND: Recently, we showed that steady-state continuous cultures of S. stipitis follow the principles of growth on mixture of two complementary substrates. More precisely, when such cultures are fed with progressively higher concentrations of glucose s f at fixed dilution rate D = 0.1 h-1, oxygen mass-transfer coefficient k l a = 50 h-1, and oxygen solubility c o ∗ , they transition from glucose- to oxygen-limited growth through an intermediate dual-limited regime in which both glucose and oxygen are limiting, and ethanol is produced without loss of glucose. It is, therefore, of considerable interest to characterize the dual-limited regime. We found that the dual-limited regime occurs precisely when the operating parameters D, s f, k l a, and c o ∗ satisfy the relation Y os < D s f / k l a · c o ∗ < Y os ' , where Y os and Y os ' denote g of glucose consumed per g of oxygen consumed in the carbon- and oxygen-limited regimes. In this work, our goal was to determine if the above characterization of the dual-limited regime holds over a wider range of D, k l a, and to understand why the dual-limited regime is determined by the dimensionless ratio D s f / k l a · c o ∗ . RESULTS: To this end, we performed the foregoing experiments at three additional dilution rates (D = 0.07, 0.15, and 0.20 h-1) and one additional mass-transfer coefficient (k l a = 100 h-1). We find that the above characterization of the dual-limited regime is valid for these conditions as well. Furthermore, the boundaries of the dual-limited regime are determined by the dimensionless ratio D s f / k l a · c o ∗ , because the steady-state concentrations are completely determined by this ratio. More precisely, if the steady-state concentrations of biomass, glucose, oxygen, and ethanol are suitably scaled, they collapse into a single curve with D s f / k l a · c o ∗ as the independent variable. CONCLUSION: The dual-limited regime is characterized by the relation Y os < D s f / k l a · c o ∗ < Y os ' over the entire range of operating condition 0.07 h-1 ≤ D ≤ 0.20 h-1 and 50 h - 1 ≤ k l a ≤ 100 h - 1 . Since the effect of all operating parameters is embedded in the single parameter D s f / k l a · c o ∗ , the dimensionless plot provides a powerful tool to compare, with only a handful of data, various ethanol-producing strains over a wide range of operating conditions.

Application of the Severity Factor and HMF Removal of Red Macroalgae Gracilaria verrucosa to Production of Bioethanol by Pichia stipitis and Kluyveromyces marxianus with Adaptive Evolution.

Gracilaria verrucosa, red seaweed, is a promising biomass for bioethanol production due to its high carbohydrate content. The optimal hyper thermal (HT) acid hydrolysis conditions are 12% (w/v) G. verrucosa with 0.2 M H2SO4 at 130 °C for 15 min, with a severity factor of 1.66. This HT acid hydrolysis produces 50.7 g/L monosaccharides. The maximum monosaccharide concentration of 58.0 g/L was achieved with 96.6% of the theoretical monosaccharide production from 120 g dry weight/L G. verrucosa slurry after HT acid hydrolysis and enzymatic saccharification. Fermentation was carried out by removing an inhibitory compound and via yeast adaptation to galactose. Both Pichia stipitis and Kluyveromyces marxianus adapted to galactose were excellent producers, with the ethanol yield (YEtOH) of 0.50 and 29.0 g/L ethanol production. However, the bioethanol productivity with Pichia stipitis adapted to galactose is higher than that with Kluyveromyces marxianus adapted to galactose, being 0.81 and 0.35 g/L/h, respectively. The results from this study can be applied to industrial scale bioethanol production from seaweed.

Transcriptome and metabolome analysis of Pichia stipitis to three representative lignocellulosic inhibitors.

During the bioconversion of xylose to ethanol, Pichia stipitis cells are often inhibited by substances generated in the lignocellulosic hydrolysate. However, the response mechanism of P. stipitis to inhibitors has not been completely understood till date. With this aim, integrated transcriptomic and metabolomic analyses were performed on P. stipitis to investigate the interactive effects of three representative inhibitors [vanillin, 5-hydroxymethylfurfural (5-HMF), and acetic acid] present in lignocellulosic hydrolysates. The genes involved in carbohydrate metabolism were observed to significantly down-regulated in the presence of the three combined inhibitors in both lag and middle exponential phases. In addition, inhibitor addition induced amino acid metabolism (e.g., glutamine and asparagine syntheses), since the yeast cells required more amino acids in stressful conditions. The metabolomic analysis yielded similar results, particularly those related with the analysis of metabolic biomarkers including fatty acids, amino acids, and sugars. 70 intracellular metabolites were detected by gas chromatography coupled with mass spectrometry (GC-MS), and samples from different phases were clearly separated by principal component analysis (PCA). The large amount of specific responsive genes and metabolites highlighted the complex regulatory mechanisms involved in the fermentation process in the presence of the three combined inhibitors.

Quantifying the parametric sensitivity of ethanol production by Scheffersomyces (Pichia) stipitis: development and verification of a method based on the principles of growth on mixtures of complementary substrates.

Under aerobic conditions, Crabtree-negative yeasts grow but do not ferment, and under anaerobic conditions, they ferment but do not grow. It is therefore believed that fermentation by these yeasts is sensitive to small variations of the operating parameters, e.g. dilution rate D, mass transfer coefficient kla and oxygen solubility co*. However, this parametric sensitivity has never been quantified. Here, we present a method to quantify the parametric sensitivity of ethanol production in the Crabtree-negative yeast Scheffersomycesstipitis. The method is based on our experimental observation that S. stipitis cultures follow the principles of growth on mixtures of complementary substrates. Specifically, if a chemostat operating at fixed D, kla and co* is fed with progressively increasing glucose feed concentrations sf, the culture passes through three regimes. (1) At low sf, the culture is carbon-limited and no ethanol is produced. (2) At high sf, the culture is oxygen-limited and ethanol is produced, but unused glucose is lost with the effluent. (3) At intermediate sf, both glucose and oxygen are limiting, and ethanol is produced without loss of glucose. Ethanol must therefore be produced in this dual-limited regime. The dual-limited regime can be predicted by simple unstructured models. It is characterized by the relation Yos

Evaluation and Optimization of Organic Acid Pretreatment of Cotton Gin Waste for Enzymatic Hydrolysis and Bioethanol Production.

This paper investigates the efficiency of the organic acids on the pretreatment of an industrially generated cotton gin waste for the removal of lignin, thereby releasing cellulose and hemicellulose as fermentable sugar components. Cotton gin waste was pretreated with various organic acids namely lactic acid, oxalic acid, citric acid, and maleic acid. Among these, maleic acid was found to be the most efficient producing maximum xylose sugar (126.05 ± 0.74 g/g) at the optimum pretreatment condition of 150 °C, 500 mM, and 45 min. The pretreatment efficiency was comparable to the conventional dilute sulfuric acid pretreatment. A lignin removal of 88% was achieved by treating maleic acid pretreated biomass in a mixture of sodium sulfite and sodium chlorite. The pretreated biomass was further evaluated for the release of sugar by enzymatic hydrolysis and subsequently bioethanol production from hydrolysates. The maximum 686.13 g/g saccharification yield was achieved with maleic acid pretreated biomass which was slightly higher than the sulfuric acid (675.26 g/g) pretreated waste. The fermentation of mixed hydrolysates(41.75 g/l) produced 18.74 g/l bioethanol concentration with 2.25 g/l/h ethanol productivity and 0.48 g/g ethanol yield using sequential use of Saccharomyces cerevisiae and Pichia stipitis yeast strains. The production of bioethanol was higher than the ethanol produced using co-culture in comparison to sequential culture. Thus, it has been demonstrated that the maleic acid pretreatment and fermentation using sequential use of yeast strains are efficient for bioethanol production from cotton gin waste.

High-activity production of xylanase by Pichia stipitis: Purification, characterization, kinetic evaluation and xylooligosaccharides production.

As an industrially important biological macromolecule, xylanase hydrolyzes xylan to produce xylooligosaccarides (XOS). XOS, with a degree of polymerization (DP) 2 to 4, are important prebiotics used as food ingredients. In this study, xylanase (5536 U/g substrate) was produced by Pichia stipitis using corncob and wheat bran mixture under solid state fermentation. Crude xylanase were purified and biochemically characterized. XOS hydrolyzed by crude and purified xylanases were quantified. Molecular weight of the purified enzyme was around 31.6 kDa on SDS-PAGE. Enzyme kinetics showed Km and Vmax values of 4.52 mg/mL and 9.17 µmol/min/mL, respectively. The optimal conditions were pH 6.0 and 50 °C. Xylanase was stable at pH 5-8 for 60 min by retaining 57% activity and at 50 °C for 80 min by retaining 65% activity. Cooper and potassium had no inhibitory effect on xylanase activity. Xylan hydrolysates produced by purified xylanase contained 92% XOS consisting of 14% xylotetroase (DP 4), 49% xylotriose (DP 3) and 29% xylobiose (DP 2). These findings indicate the potential of applying purified xylanase for industrial XOS production.

Quantitative lipidomic insights in the inhibitory response of Pichia stipitis to vanillin, 5-hydroxymethylfurfural, and acetic acid.

To obtain a global view of the dynamic phospholipids in Pichia stipitis during the ethanol fermentation in the presence of three representative inhibitors (vanillin, 5-hydroxymethylfurfural, and acetic acid). Considerable efforts have been expended to elucidate the biochemical mechanisms of inhibitors interaction with phospholipids. In this study, a comparative lipidomic analysis was performed using liquid chromatography-mass/mass spectrometry (LC-MS/MS) on P. stipitis. Partial least squares-discriminate analysis (PLS-DA) was used to deal with the large quantity of data generated using the systematic methods. PLS-DA revealed that phosphatidylinositol (PI) (PI34:1, PI34:2 and PI34:6), phosphatidylserine (PS) (PS34:1 and PS34:2), phosphatidylethanolamine (PE) (PE34:1 and PE34:2), and phosphatidylcholine (PC) (PC34:1, PC34:2, and PC34:3) were the predominant biomarkers. Further analysis of different classes of phospholipids indicated that: (a) the samples from three combined inhibitors condition during the lag phase possessed the lowest PI/PS value 1.4%, (b) alterations in PC/PE ratios with changes in inhibitors were coincident with the changes in xylose utilization rates, and (c) the levels of unsaturated and the relatively long chain phospholipids increased in the inhibitor-plus condition. These findings suggest that regulation of membrane properties with inhibitors might offer a way of self-protection of yeast to inhibitors stress.

The Mesomeric Effect of Thiazolium on non-Kekulé Diradicals in Pichia stipitis Transketolase.

It is theoretically plausible that thiazolium mesomerizes to congeners other than carbene in a low effective dielectric binding site; especially given the energetics and uneven electronegativity of carbene groups. However, such a phenomenon has never been reported. Nine crystal structures of transketolase obtained from Pichia stipitis (TKps) are reported with subatomic resolution, where thiazolium displays an extraordinary ring-bending effect. The bent thiazolium congeners correlate with non-Kekulé diradicals because there is no gain or loss of electrons. In conjunction with biophysical and biochemical analyses, it is concluded that ring bending is a result of tautomerization of thiazolium with its non- Kekulé diradicals, exclusively in the binding site of TKps. The chemophysical properties of these thiazolium mesomers may account for the great variety of reactivities carried out by thiamine-diphosphate-containing (ThDP) enzymes. The stability of ThDP in living systems can be regulated by the levels of substrates, and hydration and dehydration, as well as diradical-mediated oxidative degradation.

Evaluation of fermentation kinetics of acid-treated corn cob hydrolysate for xylose fermentation in the presence of acetic acid by Pichia stipitis.

The efficient utilization of lignocellulosic biomass for ethanol production depends on the fermentability of the biomass hydrolysate obtained after pretreatment. In this work we evaluated the kinetics of ethanol production from xylose using Pichia stipitis in acid-treated corn cob hydrolysate. Acetic acid is one of the main inhibitors in corn cob hydrolysate that negatively impacts kinetics of xylose fermentation by P. stipitis. Unstructured kinetic model has been formulated that describes cell mass growth and ethanol production as a function of xylose, oxygen, ethanol, and acetic acid concentration. Kinetic parameters were estimated under different operating conditions affecting xylose fermentation. This is the first report on kinetics of xylose fermentation by P. stipitis which includes inhibition of acetic acid on growth and product formation. In the presence of acetic acid in the hydrolysate, the model accurately predicted reduction in maximum specific growth rate (from 0.23 to 0.15 h-1) and increase in ethanol yield per unit biomass (from 3 to 6.2 gg-1), which was also observed during experimental trials. Presence of acetic acid in the fermentation led to significant reduction in the cell growth rate, reduction in xylose consumption and ethanol production rate. The developed model accurately described physiological state of P. stipitis during corn cob hydrolysate fermentation. Proposed model can be used to predict the influence of xylose, ethanol, oxygen, and acetic acid concentration on cell growth and ethanol productivity in industrial fermentation.

Structural and biochemical interrogation on transketolase from Pichia stipitis for new functionality.

In the development of new functionalities of transketolase for the industrial strain Pichia stipitis (TKps) the structural information of TKps would allow us to gain insight into the enzyme's reaction mechanisms, substrates selectivity and reaction directionality to help reach the goal. We here report seven TKps crystal structures of wild type (WT) and mutants in complex with various physiological ligands. These complexes were refined to resolutions at 1.6-1.03 Å. Both biochemical and mutagenic analyses concluded that residues His27, His66, His100, His261, His478, Asp473, Arg356 and Arg525 play important roles in coenzyme binding and substrates recognition. In general, His66 and His261 hold thiamine diphosphate in place; Arg356 and Arg525 serve as gatekeepers interacting with the terminal phosphate group of sugar-phosphates. His27, His66, His100, His478 and Asp473 are critical for sugars recognition/binding, in which His27 is relatively more important in interaction with sedoheptulose-7-phosphate (S7P) than xylulose-5-phosphate (X5P) in terms of molecular recognition/binding affinity. Kinetically, the reactions with X5P (forward) which were catalyzed by WT or H27A are indistinguishable, while in the reactions with S7P (backward) H27A exhibits weaker activity relative to WT. As a result, given TKps(H27A) as the biocatalyst the overall reactivity reverses from the backward reaction preference to forward, thus facilitating net xylose assimilation.

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

BACKGROUND: Lignocellulosic 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. RESULTS: Repetitive 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. CONCLUSIONS: Robust 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.

Effect of four pretreatments on enzymatic hydrolysis and ethanol fermentation of wheat straw. Influence of inhibitors and washing.

Pretreatment is essential in the production of alcohol from lignocellulosic material. In order to increase enzymatic sugar release and bioethanol production, thermal, dilute acid, dilute basic and alkaline peroxide pretreatments were applied to wheat straw. Compositional changes in pretreated solid fractions and sugars and possible inhibitory compounds released in liquid fractions were analysed. SEM analysis showed structural changes after pretreatments. Enzymatic hydrolysis and fermentation by Pichia stipitis of unwashed and washed samples from each pretreatment were performed so as to compare sugar and ethanol yields. The effect of the main inhibitors found in hydrolysates (formic acid, acetic acid, 5-hydroxymethylfurfural and furfural) was first studied through ethanol fermentations of model media and then compared to real hydrolysates. Hydrolysates of washed alkaline peroxide pretreated biomass provided the highest sugar concentrations, 31.82g/L glucose, and 13.75g/L xylose, their fermentation yielding promising results, with ethanol concentrations reaching 17.37g/L.

Unraveling the structure of sugarcane bagasse after soaking in concentrated aqueous ammonia (SCAA) and ethanol production by Scheffersomyces (Pichia) stipitis.

BACKGROUND: Fuel ethanol production from sustainable and largely abundant agro-residues such as sugarcane bagasse (SB) provides long term, geopolitical and strategic benefits. Pretreatment of SB is an inevitable process for improved saccharification of cell wall carbohydrates. Recently, ammonium hydroxide-based pretreatment technologies have gained significance as an effective and economical pretreatment strategy. We hypothesized that soaking in concentrated aqueous ammonia-mediated thermochemical pretreatment (SCAA) would overcome the native recalcitrance of SB by enhancing cellulase accessibility of the embedded holocellulosic microfibrils. RESULTS: In this study, we designed an experiment considering response surface methodology (Taguchi method, L8 orthogonal array) to optimize sugar recovery from ammonia pretreated sugarcane bagasse (SB) by using the method of soaking in concentrated aqueous ammonia (SCAA-SB). Three independent variables: ammonia concentration, temperature and time, were selected at two levels with center point. The ammonia pretreated bagasse (SCAA-SB) was enzymatically hydrolysed by commercial enzymes (Celluclast 1.5 L and Novozym 188) using 15 FPU/g dry biomass and 17.5 Units of ß-glucosidase/g dry biomass at 50°C, 150 rpm for 96 h. A maximum of 28.43 g/l reducing sugars corresponding to 0.57 g sugars/g pretreated bagasse was obtained from the SCAA-SB derived using a 20% v/v ammonia solution, at 70°C for 24 h after enzymatic hydrolysis. Among the tested parameters, pretreatment time showed the maximum influence (p value, 0.053282) while ammonia concentration showed the least influence (p value, 0.612552) on sugar recovery. The changes in the ultra-structure and crystallinity of native SCAA-SB and enzymatically hydrolysed SB were observed by scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid-state (13)C nuclear magnetic resonance (NMR) spectroscopy. The enzymatic hydrolysates and solid SCAA-SB were subjected to ethanol fermentation under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) by Scheffersomyces (Pichia) stipitis NRRL Y-7124 respectively. Higher ethanol production (10.31 g/l and yield, 0.387 g/g) was obtained through SSF than SHF (3.83 g/l and yield, 0.289 g/g). CONCLUSIONS: SCAA treatment showed marked lignin removal from SB thus improving the accessibility of cellulases towards holocellulose substrate as evidenced by efficient sugar release. The ultrastructure of SB after SCAA and enzymatic hydrolysis of holocellulose provided insights of the degradation process at the molecular level.