Adaptation of Saccharomyces cerevisiae in a concentrated spent sulphite liquor waste stream for increased inhibitor resistance.

The fermentation of spent sulphite liquor (SSL) from the pulping of hardwoods is limited by the combination of xylose, the primary fermentable sugar and high concentrations of microbial inhibitors that decrease the yeast fermentation ability. The inhibitor resistance phenotypes of xylose-capable Saccharomyces cerevisiae strains were therefore enhanced by combining rational engineering for multi-inhibitor tolerance, with adaptation in concentrated hardwood SSL as selective pressure. The adapted strains were assessed in fermentations with 60-80% v/v concentrated SSL under industrially relevant fermentation conditions. During adaptation, strains produced ethanol concentrations between 11.0 and 15.4 g/L in the range of that reported in literature. The adapted TFA40 and TP50 strains displayed enhanced inhibitor resistance phenotypes and were able to ferment xylose-rich SSL at pH below 5, exhibiting improved ethanol yields relative to the reference strain. Using yeast extract and peptone as nitrogen source in concentrated SSL fermentations further improved ethanol yields. However, strains exhibited a trade-off between resistance and ethanol productivity, indicating a carbon/energy cost for the expression of this inhibitor tolerance phenotype. KEY POINTS : • Achieved fermentation of xylose-rich hardwood spent sulphite liquor at pH below 5.0 • Adaptation of xylose-capable S. cerevisiae in concentrated spent sulphite liquor • Adapted strains exhibited enhanced inhibitor resistance phenotypes.

The effect of growth rate on the production and vitality of non-Saccharomyces wine yeast in aerobic fed-batch culture.

Non-Saccharomyces wine yeasts are of increasing importance due to their influence on the organoleptic properties of wine and thus the factors influencing the biomass production of these yeasts, as starter cultures, are of commercial value. Therefore, the effects of growth rates on the biomass yield (Yx/s) and fermentation performance of non-Saccharomyces yeasts at bench and pilot scale were examined. The fermentative performance and (Yx/s) were optimised, in aerobic fed-batch cultivations, to produce commercial wine seed cultures of Lachancea thermotolerans Y1240, Issatchenkia orientalis Y1161 and Metschnikowia pulcherrima Y1337. Saccharomyces cerevisiae (Lalvin EC1118) was used as a benchmark. A Crabtree positive response was shown by L. thermotolerans in a molasses-based industrial medium, at growth rates exceeding 0.21 h-1 (µcrit), resulting in a Yx/s of 0.76 g/g at 0.21 h-1 (46% of µmax) in the aerobic bioreactor-grown fed-batch culture at bench scale. At pilot scale and 0.133 h-1 (36% of µmax), this yeast exhibited ethanol concentrations reaching 10.61 g/l, as a possible result of substrate gradients. Crabtree negative responses were observed for I. orientalis and M. pulcherrima resulting in Yx/s of 0.83 g/g and 0.68 g/g, respectively, below 32% of µmax. The Yx/s of M. pulcherrima, I. orientalis and L. thermotolerans was maximised at growth rates between 0.10 and 0.12 h-1 and the fermentative capacity of these yeasts was maximised at these lower growth rates.

Investigating the effect of different inulin-rich substrate preparations from Jerusalem artichoke (Helianthus tuberosus L.) tubers on efficient inulooligosaccharides production.

Commercial production of inulooligosaccharides (IOS) relies largely on chicory roots. However, Jerusalem artichoke (JA) tubers provide a suitable alternative due to their high inulin content and low cultivation requirements. In this study, three inulin-rich substrate preparations from JA were investigated to maximize IOS production, namely powder from dried JA tuber slices (Substrate 1), solid residues after extracting protein from the JA powder (Substrate 2) and an inulin-rich fraction extracted from protein extraction residues (Substrate 3). The preferred temperature, pH and inulin substrate concentration were determined after which enzyme dosage and extraction time were optimized to maximize IOS extraction from the three substrates, using pure chicory inulin as benchmark. Under the optimal conditions, Substrate 3 resulted in the highest IOS yield of 82.3% (w/winulin). However, IOS production from the Substrate 1 proved more efficient since it renders the highest overall IOS yield (mass of IOS per mass of the starting biomass). In the case of co-production of protein and IOS from the JA tuber in a biorefinery concept, IOS production from the Substrate 2 is preferred since it reduces the inulin losses incurred during substrate preparation. For all the inulin-rich substrates studied, an enzyme dosage of 14.8 U/ginulin was found to be optimal at reaction time less than 6 h. JA tuber exhibited excellent potential for commercial production of IOS with improved yield and the possible advantage of a reduced biomass cost.

Sequential extraction of protein and inulin from the tubers of Jerusalem artichoke (Helianthus tuberosus L.).

An increase in inulin and plant-protein based nutraceutical demand ultimately puts pressure on available resources. Therefore, there is a need to prospect for supplementary feedstocks and sustainable ways to exploit them. The aim of this study was to explore the technical feasibility of sequential extraction of inulin and protein from Jerusalem artichoke tubers and understand the interrelationships between processes and product functional properties. The response surface methodology was used to determine the optimal parameters for sequential extraction. Protein functional properties analysis was done to identify the effects of the extraction process. The extraction approach adopted in this study was preceded by mechanical pressing of the tuber to yield a protein-rich juice. However, only 40.8% of the protein was recovered from the juice, therefore a subsequent solvent extraction step followed to extract the residual protein and inulin retained in the solids. Selective extraction was achieved when protein was solubilised in the first step of solvent extraction. The overall protein and inulin yields from pressing and both sequential extraction steps were 71.88 and 67.6%, respectively. The inulin yields were substantially higher than the maximum overall yields when inulin extraction, from the pressed tuber, was performed first thus improving yields from 57.3 to 67.6%. Consequently, mechanical pressing improved the overall protein yield. Sequential extraction resulted in an inulin extract with minimal protein contamination compared to the conventional method. Therefore, sequential extraction was efficient in yielding extracts with reduced impurities and good functional properties.

Expression of unique chimeric human papilloma virus type 16 (HPV-16) L1-L2 proteins in Pichia pastoris and Hansenula polymorpha.

Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two prophylactic vaccines being commercially available, they are unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1-L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Muts ) and GS115 (Mut+ ), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV-16 capsid L1 protein with an L2 peptide. Two different feeding strategies in fed-batch cultures of P. pastoris Muts were evaluated: a predetermined feed rate vs. feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Muts strains than in the Mut+ strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg L-1 culture while P. pastoris Muts only produced 23.61 mg L-1 . H. polymorpha showed greater potential for the expression of HPV-16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high-level producer of heterologous proteins.

Expression of rotavirus VP6 protein: a comparison amongst Escherichia coli, Pichia pastoris and Hansenula polymorpha.

During this study. we successfully expressed a codon-optimized gene for rotavirus VP6 protein intracellularly in two methylotrophic yeasts, Pichia pastoris and Hansenula polymorpha, during methanol induction. Expressions were performed in shake flasks and subsequently scaled-up to 1.3 L bioreactors. The yields obtained in the yeasts were compared with that observed in Escherichia coli. Despite producing the lowest biomass levels of all the expression systems in shake flasks, the highest VP6 concentration was obtained with E. coli. In shake flasks, P. pastoris yielded higher volumetric levels of VP6 than H. polymorpha, but specific production of VP6 was approximately similar in both yeasts. In the controlled environment of bioreactors, yeast strains attained typical high cell densities, but also increased VP6 production compared to all shake flask cultures. Unlike in shake flask expressions, H. polymorpha outperformed both P. pastoris as well as E. coli during bioreactor cultivation. VP6 production was in all three expression systems growth-associated. In contrast to yeast expressions, bacterial expressed VP6 protein was found to be insoluble upon analysis. This is the first report of VP6 expressed in methylotrophic yeast and holds the promise for the inexpensive production of VP6 as a possible vaccine candidate or drug delivery mechanism.

Engineering Saccharomyces cerevisiae for direct conversion of raw, uncooked or granular starch to ethanol.

The production of raw starch-degrading amylases by recombinant Saccharomyces cerevisiae provides opportunities for the direct hydrolysis and fermentation of raw starch to ethanol without cooking or exogenous enzyme addition. Such a consolidated bioprocess (CBP) for raw starch fermentation will substantially reduce costs associated with energy usage and commercial granular starch hydrolyzing (GSH) enzymes. The core purpose of this review is to provide comprehensive insight into the physiological impact of recombinant amylase production on the ethanol-producing yeast. Key production parameters, based on outcomes from modifications to the yeast genome and levels of amylase production, were compared to key benchmark data. In turn, these outcomes are of significance from a process point of view to highlight shortcomings in the current state of the art of raw starch fermentation yeast compared to a set of industrial standards. Therefore, this study provides an integrated critical assessment of physiology, genetics and process aspects of recombinant raw starch fermenting yeast in relation to presently used technology. Various approaches to strain development were compared on a common basis of quantitative performance measures, including the extent of hydrolysis, fermentation-hydrolysis yield and productivity. Key findings showed that levels of α-amylase required for raw starch hydrolysis far exceeded enzyme levels for soluble starch hydrolysis, pointing to a pre-requisite for excess α-amylase compared to glucoamylase for efficient raw starch hydrolysis. However, the physiological limitations of amylase production by yeast, requiring high biomass concentrations and long cultivation periods for sufficient enzyme accumulation under anaerobic conditions, remained a substantial challenge. Accordingly, the fermentation performance of the recombinant S. cerevisiae strains reviewed in this study could not match the performance of conventional starch fermentation processes, based either on starch cooking and/or exogenous amylase enzyme addition. As an alternative strategy, the addition of exogenous GSH enzymes during early stages of raw starch fermentation may prove to be a viable approach for industrial application of recombinant S. cerevisiae, with the process still benefitting from amylase production by CBP yeast during later stages of cultivation.

Direct, simultaneous quantification of fructooligosaccharides by FT-MIR ATR spectroscopy and chemometrics for rapid identification of superior, engineered ß-fructofuranosidases.

Fructooligosaccharides (FOS) are popular components of functional foods produced by the enzymatic transfer of fructose units to sucrose. Improving ß-fructofuranosidase traits by protein engineering is restricted by the absence of a rapid, direct screening method for the fructooligosaccharide products produced by enzyme variants. The use of standard high-performance liquid chromatography (HPLC) methods involves time-consuming sample preparation and chromatographic and data analysis steps. To overcome these limitations, this work presents a rapid method for screening ß-fructofuranosidase variant libraries using Fourier transform mid-infrared attenuated total reflectance (FT-MIR ATR) spectroscopy and calibration using partial least squares (PLS) regression. The method offers notable improvements in terms of sample analysis times and cost, with the added benefit of the absence of toxic eluents. Wavenumber interval selection methods were tested to develop optimised PLS regression models that were successfully applied to quantify of glucose, fructose, sucrose, 1-kestose and nystose, the substrates and products of ß-fructofuranosidase activity. To the best of our knowledge, this is the first report on the use of infrared spectroscopy and PLS calibration for the quantification of 1-kestose and nystose. Independent test set-validated results indicated that optimal wavenumber selection by interval PLS (iPLS) served to provide the best models for all sugars, bar glucose. Application of this screening method will facilitate the engineering of ß-fructofuranosidases and other glycosyltransferase enzymes by random mutagenesis strategies, as it provides, for the first time, a rapid, direct assay for transferase products that may be adapted to a high-throughput set-up.

Simultaneously improving xylose fermentation and tolerance to lignocellulosic inhibitors through evolutionary engineering of recombinant Saccharomyces cerevisiae harbouring xylose isomerase.

BACKGROUND: Yeasts tolerant to toxic inhibitors from steam-pretreated lignocellulose with xylose co-fermentation capability represent an appealing approach for 2nd generation ethanol production. Whereas rational engineering, mutagenesis and evolutionary engineering are established techniques for either improved xylose utilisation or enhancing yeast tolerance, this report focuses on the simultaneous enhancement of these attributes through mutagenesis and evolutionary engineering of Saccharomyces cerevisiae harbouring xylose isomerase in anoxic chemostat culture using non-detoxified pretreatment liquor from triticale straw. RESULTS: Following ethyl methanesulfonate (EMS) mutagenesis, Saccharomyces cerevisiae strain D5A⁺ (ATCC 200062 strain platform), harbouring the xylose isomerase (XI) gene for pentose co-fermentation was grown in anoxic chemostat culture for 100 generations at a dilution rate of 0.10 h⁻¹ in a medium consisting of 60% (v/v) non-detoxified hydrolysate liquor from steam-pretreated triticale straw, supplemented with 20 g/L xylose as carbon source. In semi-aerobic batch cultures in the same medium, the isolated strain D5A(+H) exhibited a slightly lower maximum specific growth rate (µ(max) = 0.12 ± 0.01 h⁻¹) than strain TMB3400, with no ethanol production observed by the latter strain. Strain D5A(+H) also exhibited a shorter lag phase (4 h vs. 30 h) and complete removal of HMF, furfural and acetic acid from the fermentation broth within 24 h, reaching an ethanol concentration of 1.54 g/L at a yield (Y(p/s)) of 0.06 g/g xylose and a specific productivity of 2.08 g/gh. Evolutionary engineering profoundly affected the yeast metabolism, given that parental strain D5A+ exhibited an oxidative metabolism on xylose prior to strain development. CONCLUSIONS: Physiological adaptations confirm improvements in the resistance to and conversion of inhibitors from pretreatment liquor with simultaneous enhancement of xylose to ethanol fermentation. These data support the sequential application of random mutagenesis followed by continuous culture under simultaneous selective pressure from inhibitors and xylose as primary carbon source.

Enhanced ethanol production from paper sludge waste under high-solids conditions with industrial and cellulase-producing strains of Saccharomyces cerevisiae.

Reported ethanol titres from hydrolysis-fermentation of the degraded fibres in paper sludge (PS) waste, generally obtained under fed-batch submerged conditions, can be improved through fermentation processes at high solids loadings, as demonstrated in the present study with two industrial PS wastes at enzyme dosages appropriate for solids loadings up to 40% (w/w). The industrial yeast,Saccharomyces cerevisiaestrain Ethanol Red®, was compared to two genetically engineeredS. cerevisiaestrains, namely Cellusec® 1.0 and Cellusec® 2.0, capable of xylose utilisation, and xylose utilisation and cellulase production, respectively. High-solids batch fermentations were conducted in 3 L horizontal rotating reactors and ethanol titres of 100.8 and 73.3 g/L were obtained for virgin pulp and corrugated recycle PS, respectively, at 40% (w/w) solids loading using Ethanol Red®. Xylose utilisation by Cellusec® 1.0 improved ethanol titres by up to 10.3%, while exogenous cellulolytic enzyme requirements were reduced by up to 50% using cellulase-producing Cellusec® 2.0.

The metabolic burden of cellulase expression by recombinant Saccharomyces cerevisiae Y294 in aerobic batch culture.

Two recombinant strains of Saccharomyces cerevisiae Y294 producing cellulase using different expression strategies were compared to a reference strain in aerobic culture to evaluate the potential metabolic burden that cellulase expression imposed on the yeast metabolism. In a chemically defined mineral medium with glucose as carbon source, S. cerevisiae strain Y294[CEL5] with plasmid-borne cellulase genes produced endoglucanase and ß-glucosidase activities of 0.038 and 0.30 U mg dry cell weight(-1), respectively. Chromosomal expression of these two cellulases in strain Y294[Y118p] resulted in no detectable activity, although low levels of episomally co-expressed cellobiohydrolase (CBH) activity were detected. Whereas the biomass concentration of strain Y294[CEL5] was slightly greater than that of a reference strain, CBH expression by Y294[Y118p] resulted in a 1.4-fold lower maximum specific growth rate than that of the reference. Supplementation of the growth medium with amino acids significantly improved culture growth and enzyme production, but only partially mitigated the physiological effects and metabolic burden of cellulase expression. Glycerol production was decreased significantly, up to threefold, in amino acid-supplemented cultures, apparently due to redox balancing. Disproportionately higher levels of glycerol production by Y294[CEL5] indicated a potential correlation between the redox balance of anabolism and the physiological stress of cellulase production. With the reliance on cellulase expression in yeast for the development of consolidated bioprocesses for bioethanol production, this work demonstrates the need for development of yeasts that are physiologically robust in response to burdens imposed by heterologous enzyme production.

Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast.

The development of a yeast that converts raw starch to ethanol in one step (called consolidated bioprocessing) could yield large cost reductions in the bioethanol industry. The aim of this study was to develop an efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production. A native and codon-optimized variant of the Aspergillus awamori glucoamylase gene were expressed in the S. cerevisiae Y294 laboratory strain. Codon optimization resulted to be effective and the synthetic sequence sGAI was then δ-integrated into a S. cerevisiae strain with promising industrial fermentative traits. The mitotically stable recombinant strains showed high enzymatic capabilities both on soluble and raw starch (2425 and 1140 nkat/g dry cell weight, respectively). On raw corn starch, the engineered yeasts exhibited improved fermentative performance with an ethanol yield of 0.42 (g/g), corresponding to 75 % of the theoretical maximum yield.

Influence of codon optimization, promoter, and strain selection on the heterologous production of a ß-fructofuranosidase from Aspergillus fijiensis ATCC 20611 in Pichia pastoris.

Fructooligosaccharides (FOS) are compounds possessing various health properties and are added to functional foods as prebiotics. The commercial production of FOS is done through the enzymatic transfructolysation of sucrose by ß-fructofuranosidases which is found in various organisms of which Aureobasidium pullulans and Aspergillus niger are the most well known. This study overexpressed two differently codon-optimized variations of the Aspergillus fijiensis ß-fructofuranosidase-encoding gene (fopA) under the transcriptional control of either the alcohol oxidase (AOX1) or glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters. When cultivated in shake flasks, the two codon-optimized variants displayed similar volumetric enzyme activities when expressed under control of the same promoter with the GAP strains producing 11.7 U/ml and 12.7 U/ml, respectively, and the AOX1 strains 95.8 U/ml and 98.6 U/ml, respectively. However, the highest production levels were achieved for both codon-optimized genes when expressed under control of the AOX1 promoter. The AOX1 promoter was superior to the GAP promoter in bioreactor cultivations for both codon-optimized genes with 13,702 U/ml and 2718 U/ml for the AOX1 promoter for ATUM and GeneArt®, respectively, and 6057 U/ml and 1790 U/ml for the GAP promoter for ATUM and GeneArt®, respectively. The ATUM-optimized gene produced higher enzyme activities when compared to the one from GeneArt®, under the control of both promoters.

Rational engineering of Saccharomyces cerevisiae towards improved tolerance to multiple inhibitors in lignocellulose fermentations.

BACKGROUND: The fermentation of lignocellulose hydrolysates to ethanol requires robust xylose-capable Saccharomyces cerevisiae strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial S. cerevisiae strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability. The strategy consisted of multiple sequential delta-integrations of double-gene cassettes containing one gene conferring broad inhibitor tolerance (ARI1, PAD1 or TAL1) coupled with an inhibitor-specific gene (ADH6, FDH1 or ICT1). The performances of the transformants were compared with the parental strain in terms of biomass growth, ethanol yields and productivity, as well as detoxification capacities in a synthetic inhibitor cocktail, sugarcane bagasse hydrolysate as well as hardwood spent sulphite liquor. RESULTS: The first and second round of delta-integrated transformants exhibited a trade-off between biomass and ethanol yield. Transformants showed increased inhibitor resistance phenotypes relative to parental controls specifically in fermentations with concentrated spent sulphite liquors at 40% and 80% v/v concentrations in 2% SC media. Unexpectedly, the xylose fermentation capacity of the transformants was reduced compared to the parental control, but certain combinations of genes had a minor impact (e.g. TAL1 + FDH1). The TAL1 + ICT1 combination negatively impacted on both biomass growth and ethanol yield, which could be linked to the ICT1 protein increasing transformant susceptibility to weak acids and temperature due to cell membrane changes. CONCLUSIONS: The integration of the selected genes was proven to increase tolerance to pretreatment inhibitors in synthetic or industrial hydrolysates, but they were limited to the fermentation of glucose. However, some gene combination sequences had a reduced impact on xylose conversion.

Intensification of Xylo-oligosaccharides Production by Hydrothermal Treatment of Brewer's Spent Grains: The Use of Extremely Low Acid Catalyst for Reduction of Degradation Products Associated with High Solid Loading.

Brewers' spent grains (BSG) make up to 85% of a brewery's solid waste, and is either sent to landfill or sold as cheap animal feed supplement. Xylo-oligosaccharides (XOS) obtained from BSG are antioxidants and prebiotics that can be used in food formulations as low-calorie sweeteners and texturisers. The effect of extremely low acid (ELA) catalysis in liquid hot water (LHW) hydrothermal treatment (HTT) was assessed using BSG with dry matter contents of 15% and 25%, achieved by dewatering using a screw press. Batch experiments at low acid loadings of 5, 12.5 and 20 mg/g dry mass and temperatures of 120, 150 and 170 °C significantly affected XOS yield at both levels of dry mass considered. Maximum XOS yields of 76.4% (16.6 g/l) and 65.5% (31.7 g/l) were achieved from raw BSG and screw pressed BSG respectively, both at 170 °C and using 5 mg acid/g dry mass, after 15 min and 5 min, respectively. These XOS yields were obtained with BSG containing up to 63% less water and temperatures more than 20 °C lower than that reported previously. The finding confirms that ELA dosing in LHW HTT allows lowering of the required temperature that can result in a reduction of degradation products, which is especially relevant under high solid conditions. This substantial XOS production intensification through higher solid loadings in HTT not only achieved high product yield, but also provided benefits such as increased product concentrations and decreased process heat requirements.

Effect of dimorphic regulation on heterologous glucose oxidase production by Mucor circinelloides.

The production of Aspergillus niger glucose oxidase (GOX) and native amylase by the recombinant M. circinelloides KFA199 strain under conditions of dimorphic growth was investigated. The recombinant KFA199 strain was compared to its parental ATCC 1216b strain and a wild-type CBS 232.29 strain under similar morphology-controlled conditions. Cultivation in Vogel's medium supplemented with ergosterol/Tween-80 and sparged with nitrogen gas was most suitable for yeast-like biomass production under anaerobic conditions. Anaerobic growth was characterized by high levels of ethanol formation and linear growth rates of 0.24-0.05/h, indicating metabolic stress. Subsequent to anaerobic growth, cultures were shifted to aerobic conditions to induce aerobic mycelial growth. GOX produced by the recombinant KFA199 after the shift to aerobic conditions was poorly secreted and accumulated intracellularly to 0.56 U/ml(culture). Amylase production by the KFA199, ATCC12b and CBS 232.29 strains was determined during growth on starch after the shift to aerobic culture. Growth-associated amylase production by the ATCC 1216b (0.63 U/ml(culture)) and wild-type CBS 232.29 (0.33 U/ml(culture)) strains was substantially higher than by the recombinant KFA199 strain (0.07 U/ml(culture)), which may be related to the leucine auxotrophy of the transformation host, or genetic changes induced during transformation of the KFA199 strain.

Techno-economic analysis of chemically catalysed lignocellulose biorefineries at a typical sugar mill: Sorbitol or glucaric acid and electricity co-production.

Global concerns about depletion of fossil reserves has driven countries towards bio-economies utilising mostly first generation feedstocks. The economic viability of energy self-sufficient biorefineries processing sugarcane lignocelluloses into sorbitol or glucaric acid and electricity was investigated. Aspen Plus® simulations represented glucose conversion processes via SO2-steam explosion or dilute acid pre-treatment, followed by enzymatic hydrolysis. The most economically viable sorbitol scenario using dilute acid pretreatment with a capital investment cost per litre of US$ 3.96/L was marginally profitable having a selling price 5% below the US$ 655/t market price. To secure private investment, the sorbitol selling price should reach US$ 1283/t.

Steam explosion pre-treatment of alkali-impregnated lignocelluloses for hemicelluloses extraction and improved digestibility.

The application of steam explosion pre-treatment to extract xylan-rich biopolymers from alkali-impregnated lignocelluloses, while simultaneously increasing the enzymatic digestibility of cellulose, was investigated. Steam-enhanced extraction of xylan from sugarcane trash (SCT) and aspen wood (AW) was performed at varying temperatures (176-204 °C) and retention times (3-17 min) after the impregnation of biomass samples with sodium hydroxide at 1:20 (w/w) solid loading ratio. Xylan extraction and cellulose digestibility results were statistically analysed to fix the condition/s for significantly enhanced values. Accordingly, maximum xylan yields of 51 and 24%, and highest cellulose digestibility of 92 and 81%, were attained for SCT and AW respectively following their pre-treatment at 204 °C for 10 min. At this most-severe condition, neither xylose nor furfural - a degradation product from xylose - were observed in the hemicellulose extract, indicating steam explosion pre-treatment with alkali impregnation of lignocelluloses as viable biorefinery approach to co-produce xylan biopolymers and bioethanol.

Amberlite IRA 900 versus calcium alginate in immobilization of a novel, engineered ß-fructofuranosidase for short-chain fructooligosaccharide synthesis from sucrose.

The immobilization of ß-fructofuranosidase for short-chain fructooligosaccharide (scFOS) synthesis holds the potential for a more efficient use of the biocatalyst. However, the choice of carrier and immobilization technique is a key to achieving that efficiency. In this study, calcium alginate (CA), Amberlite IRA 900 (AI900) and Dowex Marathon MSA (DMM) were tested as supports for immobilizing a novel engineered ß-fructofuranosidase from Aspergillus japonicus for scFOS synthesis. Several immobilization parameters were estimated to ascertain the effectiveness of the carriers in immobilizing the enzyme. The performance of the immobilized biocatalysts are compared in terms of the yield of scFOS produced and reusability. The selection of carriers and reagents was motivated by the need to ensure safety of application in the production of food-grade products. The CA and AI900 both recorded impressive immobilization yields of 82 and 62%, respectively, while the DMM recorded 47%. Enzyme immobilizations on CA, AI900 and DMM showed activity recoveries of 23, 27, and 17%, respectively. The CA, AI900 immobilized and the free enzymes recorded their highest scFOS yields of 59, 53, and 61%, respectively. The AI900 immobilized enzyme produced a consistent scFOS yield and composition for 12 batch cycles but for the CA immobilized enzyme, only 6 batch cycles gave a consistent scFOS yield. In its first record of application in scFOS production, the AI900 anion exchange resin exhibited potential as an adequate carrier for industrial application with possible savings on cost of immobilization and reduced technical difficulty.

Incorporating anaerobic co-digestion of steam exploded or ammonia fiber expansion pretreated sugarcane residues with manure into a sugarcane-based bioenergy-livestock nexus.

The co-digestion of pretreated sugarcane lignocelluloses with dairy cow manure (DCM) as a bioenergy production and waste management strategy, for intensive livestock farms located in sugarcane regions, was investigated. Ammonia fiber expansion (AFEX) increased the nitrogen content and accelerated the biodegradability of sugarcane bagasse (SCB) and cane leaf matter (CLM) through the cleavage of lignin carbohydrate crosslinks, resulting in the highest specific methane yields (292-299 L CH4/kg VSadded), biogas methane content (57-59% v/v) and biodegradation rates, with or without co-digestion with DCM. To obtain comparable methane yields, untreated and steam exploded (StEx) SCB and CLM had to be co-digested with DCM, at mass ratios providing initial C/N ratios in the range of 18 to 35. Co-digestion with DCM improved the nutrient content of the solid digestates, providing digestates that could be used as biofertilizer to replace CLM that is removed from sugarcane fields during green harvesting.