Optimization of xylitol production from xylose by a novel arabitol limited co-producing Barnettozyma populi NRRL Y-12728.

Xylitol is a widely marketed sweetener with good functionality and health-promoting properties. It can be synthetized by many yeast species in a one-step reduction of xylose. Arabinose is a common contaminant found in xylose and there is ongoing interest in finding biocatalysts that selectively produce xyltiol. From a screen of 99 yeasts, Barnettozyma populi Y-12728 was found to selectively produce xylitol from both mixed sugars and corn stover hemicellulosic hydrolysate. Here, fermentation conditions for xylitol production from xylose by B. populi were optimized. The medium for xylitol production was optimized through response surface methodology. The yeast produced 31.2 ± 0.4 g xylitol from xylose (50 g L-1) in 62 h using the optimized medium. The optimal pH for xylitol production was 6.0. Glucose (10 g L-1), acetic acid (6.0 g L-1), HMF (4 mM) and ethanol (2.0 g L-1) inhibited the xylitol production. The glucose inhibition was entirely mitigated by using a 2-stage aeration strategy, indicating that the yeast was inhibited by ethanol produced from glucose under low aeration. This culture strategy will greatly benefit xylitol production from hemicellulosic hydrolysates, which often contain glucose. This is the first report on optimization of xylitol production by a Barnettozyma species.

Efficient itaconic acid production by Aspergillus terreus: Overcoming the strong inhibitory effect of manganese.

Itaconic acid (IA), a building block platform chemical, is produced industrially by Aspergillus terreus utilizing glucose. Lignocellulosic biomass can serve as a low cost source of sugars for IA production. However, the fungus could not produce IA from dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate even at 100-fold dilution. Furfural, hydroxymethyl furfural and acetic acid were inhibitory, as is typical, but Mn2+ was particularly problematic for IA production. It was present in the hydrolyzate at a level that was 230 times over the inhibitory limit (50 ppb). Recently, it was found that PO43- limitation decreased the inhibitory effect of Mn2+ on IA production. In the present study, a novel medium was developed for production of IA by varying PO43- , Fe3+ and Cu2+ concentrations using response surface methodology, which alleviated the strong inhibitory effect of Mn2+ . The new medium contained 0.08 g KH2 PO4 , 3 g NH4 NO3 , 1 g MgSO4 ·7H2 O, 5 g CaCl2 ·2 H2 O, 0.83 mg FeCl3 ·6H2 O, 8 mg ZnSO4 ·7H2 O, and 45 mg CuSO4 ·5H2 O per liter. The fungus was able to produce IA very well in the presence of Mn2+ up to 100 ppm in the medium. This medium will be extremely useful for IA production in the presence of Mn2+ . This is the first report on the development of Mn2+ tolerant medium for IA production by A. terreus.

Valorization of egg shell as a detoxifying and buffering agent for efficient polymalic acid production by Aureobasidium pullulans NRRL Y-2311-1 from barley straw hydrolysate.

Stepwise formulation of a versatile and cost-effective medium based on barley straw hydrolysate and egg shell for efficient polymalic acid production by A. pullulans NRRL Y-2311-1 was carried out for the first time. The strain did not grow and produce polymalic acid when dilute acid pretreated barley straw hydrolysate (total fermentable sugars: 94.60 g/L; furfural: 1.01 g/L; hydroxymethylfurfural: 0.55 g/L; acetic acid: 5.06 g/L) was directly used in medium formulation without detoxification (e.g. charcoal pretreatment). When CaCO3 in the medium formulation was substituted with egg shell powder, efficient production of polymalic acid was achieved without a detoxification step. Utilization of 40 g/L of egg shell powder led to 43.54 g polymalic acid production per L with the productivity of 0.30 g/L/h and yield of 0.48 g/g. The bioprocess strategy used in this study can also be utilized for mass production of several other industrially important microbial organic acids and biomaterials.

Factors Affecting Production of Itaconic Acid from Mixed Sugars by Aspergillus terreus.

Itaconic acid (IA; a building block platform chemical) is currently produced industrially from glucose by fermentation with Aspergillus terreus. In order to expand the use of IA, its production cost must be lowered. Lignocellulosic biomass has the potential to serve as low-cost source of sugars for IA production. It was found that the fungus cannot produce IA from dilute acid pretreated and enzymatically saccharified wheat straw hydrolysate even at 100-fold dilution. The effects of typical compounds (acetic acid, furfural, HMF and Mn2+, enzymes, CaSO4), culture conditions (initial pH, temperature, aeration), and medium components (KH2PO4, NH4NO3, CaCl2·2H2O, FeCl3·6H2O) on growth and IA production by A. terreus NRRL 1972 using mixed sugar substrate containing glucose, xylose, and arabinose (4:3:1, 80 g L-1) mimicking the wheat straw hydrolysate were investigated. Acetic acid, furfural, Mn2+, and enzymes were strong inhibitors to both growth and IA production from mixed sugars. Optimum culture conditions (pH 3.1, 33 °C, 200 rpm) and medium components (0.8 g KH2PO4, 3 g NH4NO3, 2.0 g CaCl2·2H2O, 0.83-3.33 mg FeCl3·6H2O per L) as well as tolerable levels of inhibitors (0.4 g acetic acid, < 0.1 g furfural, 100 mg HMF, < 5.0 ppb Mn2+, 24 mg CaSO4 per L) for mixed sugar utilization were established. The results will be highly useful for developing a bioprocess technology for IA production from lignocellulosic feedstocks.

Butanol production from sweet sorghum bagasse with high solids content: Part I-comparison of liquid hot water pretreatment with dilute sulfuric acid.

In these studies, we pretreated sweet sorghum bagasse (SSB) using liquid hot water (LHW) or dilute H2 SO4 (2 g L-1 ) at 190°C for zero min (as soon as temperature reached 190°C, cooling was started) to reduce generation of sugar degradation fermentation inhibiting products such as furfural and hydroxymethyl furfural (HMF). The solids loading were 250-300 g L-1 . This was followed by enzymatic hydrolysis. After hydrolysis, 89.0 g L-1 sugars, 7.60 g L-1 acetic acid, 0.33 g L-1 furfural, and 0.07 g L-1 HMF were released. This pretreatment and hydrolysis resulted in the release of 57.9% sugars. This was followed by second hydrolysis of the fibrous biomass which resulted in the release of 43.64 g L-1 additional sugars, 2.40 g L-1 acetic acid, zero g L-1 furfural, and zero g L-1 HMF. In both the hydrolyzates, 86.3% sugars present in SSB were released. Fermentation of the hydrolyzate I resulted in poor acetone-butanol-ethanol (ABE) fermentation. However, fermentation of the hydrolyzate II was successful and produced 13.43 g L-1 ABE of which butanol was the main product. Use of 2 g L-1 H2 SO4 as a pretreatment medium followed by enzymatic hydrolysis resulted in the release of 100.6-93.8% (w/w) sugars from 250 to 300 g L-1 SSB, respectively. LHW or dilute H2 SO4 were used to economize production of cellulosic sugars from SSB. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:960-966, 2018.

High solid fed-batch butanol fermentation with simultaneous product recovery: Part II-process integration.

In these studies, liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed-batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level of toxic chemicals, in particular acetic acid released from SSB during the hydrolytic process. To be able to ferment the hydrolyzate I obtained from 250 g L-1 SSB hydrolysis, a fed-batch reactor with in situ butanol recovery was devised. The process was started with the hydrolyzate II and when good cell growth and vigorous fermentation were observed, the hydrolyzate I was slowly fed to the reactor. In this manner the culture was able to ferment all the sugars present in both the hydrolyzates to acetone butanol ethanol (ABE). In a control batch reactor in which ABE was produced from glucose, ABE productivity and yield of 0.42 g L-1 h-1 and 0.36 were obtained, respectively. In the fed-batch reactor fed with SSB hydrolyzates, these productivity and yield values were 0.44 g L-1 h-1 and 0.45, respectively. ABE yield in the integrated system was high due to utilization of acetic acid to convert to ABE. In summary we were able to utilize both the hydrolyzates obtained from LHW pretreated and enzymatically hydrolyzed SSB (250 g L-1 ) and convert them to ABE. Complete fermentation was possible due to simultaneous recovery of ABE by vacuum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:967-972, 2018.

Ninety six well microtiter plate as microbioreactors for production of itaconic acid by six Aspergillus terreus strains.

Itaconic acid (IA) is a building block platform chemical that is currently produced industrially from glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has the potential to serve as low cost source of sugars for production of IA. Previously, 100 A. terreus strains were evaluated for production of IA from pentose sugars in shake-flasks. Six selected strains were then investigated for IA production in shake-flasks. But none of the strains grew and produced IA using biomass hydrolyzates. In order to study the factors inhibiting fungal growth and IA production, we have evaluated these six strains for sugar utilization and IA production from glucose, xylose, arabinose, mixed sugars, and both dilute acid and liquid hot water pretreated wheat straw hydrolyzates in microtiter plate (MTP) microbioreactors at 100µL scale. The results clearly indicate that MTP is very useful as a convenient, reliable and affordable platform to investigate the reasons for inhibition of growth and IA production by the A. terreus strains and should greatly aid in strain development and optimization of IA production by the fungal strains.

Production of itaconic acid from pentose sugars by Aspergillus terreus.

Itaconic acid (IA), an unsaturated 5-carbon dicarboxylic acid, is a building block platform chemical that is currently produced industrially from glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has potential to serve as low-cost source of sugars for production of IA. Research needs to be performed to find a suitable A. terreus strain that can use lignocellulose-derived pentose sugars and produce IA. One hundred A. terreus strains were evaluated for the first time for production of IA from xylose and arabinose. Twenty strains showed good production of IA from the sugars. Among these, six strains (NRRL strains 1960, 1961, 1962, 1972, 66125, and DSM 23081) were selected for further study. One of these strains NRRL 1961 produced 49.8 ± 0.3, 38.9 ± 0.8, 34.8 ± 0.9, and 33.2 ± 2.4 g IA from 80 g glucose, xylose, arabinose and their mixture (1:1:1), respectively, per L at initial pH 3.1 and 33°C. This is the first report on the production of IA from arabinose and mixed sugar of glucose, xylose, and arabinose by A. terreus. The results presented in the article will be very useful in developing a process technology for production of IA from lignocellulosic feedstocks. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1059-1067, 2017.

Emerging biotechnologies for production of itaconic acid and its applications as a platform chemical.

Recently, itaconic acid (IA), an unsaturated C5-dicarboxylic acid, has attracted much attention as a biobased building block chemical. It is produced industrially (>80 g L-1) from glucose by fermentation with Aspergillus terreus. The titer is low compared with citric acid production (>200 g L-1). This review summarizes the latest progress on enhancing the yield and productivity of IA production. IA biosynthesis involves the decarboxylation of the TCA cycle intermediate cis-aconitate through the action of cis-aconitate decarboxylase (CAD) enzyme encoded by the CadA gene in A. terreus. A number of recombinant microorganisms have been developed in an effort to overproduce it. IA is used as a monomer for production of superabsorbent polymer, resins, plastics, paints, and synthetic fibers. Its applications as a platform chemical are highlighted. It has a strong potential to replace petroleum-based methylacrylic acid in industry which will create a huge market for IA.

Biological pretreatment of corn stover with Phlebia brevispora NRRL-13108 for enhanced enzymatic hydrolysis and efficient ethanol production.

Biological pretreatment of lignocellulosic biomass by white-rot fungus can represent a low-cost and eco-friendly alternative to harsh physical, chemical, or physico-chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid-state cultivation of corn stover with Phlebia brevispora NRRL-13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed-batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed-batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL-13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365-374, 2017.

Production of xylitol by a Coniochaeta ligniaria strain tolerant of inhibitors and defective in growth on xylose.

In conversion of biomass to fuels or chemicals, inhibitory compounds arising from physical-chemical pretreatment of the feedstock can interfere with fermentation of the sugars to product. Fungal strain Coniochaeta ligniaria NRRL30616 metabolizes the furan aldehydes furfural and 5-hydroxymethylfurfural, as well as a number of aromatic and aliphatic acids and aldehydes. Use of NRRL30616 to condition biomass sugars by metabolizing the inhibitors improves their fermentability. Wild-type C. ligniaria has the ability to grow on xylose as sole source of carbon and energy, with no accumulation of xylitol. Mutants of C. ligniaria unable to grow on xylose were constructed. Xylose reductase and xylitol dehydrogenase activities were reduced by approximately two thirds in mutant C8100. The mutant retained ability to metabolize inhibitors in biomass hydrolysates. Although C. ligniaria C8100 did not grow on xylose, the strain converted a portion of xylose to xylitol, producing 0.59 g xylitol/g xylose in rich medium and 0.48 g xylitol/g xylose in corn stover dilute acid hydrolysate. 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016 © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:606-612, 2016.

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates.

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.

Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production.

Effects of substrate-selective inoculum prepared by growing on glucose, xylose, arabinose, GXA (glucose, xylose, arabinose, 1:1:1) and corn stover hydrolyzate (dilute acid pretreated and enzymatically hydrolyzed, CSH) on ethanol production from CSH by a mixed sugar utilizing recombinant Escherichia coli (strain FBR5) were investigated. The initial ethanol productivity was faster for the seed grown on xylose followed by GXA, CSH, glucose and arabinose. Arabinose grown seed took the longest time to complete the fermentation. Delayed saccharifying enzyme addition in simultaneous saccharification and fermentation of dilute acid pretreated CS by the recombinant E. coli strain FBR5 allowed the fermentation to finish in a shorter time than adding the enzyme simultaneously with xylose grown inoculum. Use of substrate selective inoculum and fermenting pentose sugars first under glucose limited condition helped to alleviate the catabolite repression of the recombinant bacterium on ethanol production from lignocellulosic hydrolyzate.

Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.

Pilot scale conversion of wheat straw to ethanol via simultaneous saccharification and fermentation.

The production of ethanol from wheat straw (WS) by dilute acid pretreatment, bioabatement of fermentation inhibitors by a fungal strain, and simultaneous saccharification and fermentation (SSF) of the bio-abated WS to ethanol using an ethanologenic recombinant bacterium was studied at a pilot scale without sterilization. WS (124.2g/L) was pretreated with dilute H2SO4 in two parallel tube reactors at 160°C. The inhibitors were bio-abated by growing the fungus aerobically. The maximum ethanol produced by SSF of the bio-abated WS by the recombinant Escherichia coli FBR5 at pH 6.0 and 35°C was 36.0g/L in 83h with a productivity of 0.43gL(-1)h(-1). This value corresponds to an ethanol yield of 0.29g/g of WS which is 86% of the theoretical ethanol yield from WS. This is the first report on the production of ethanol by the recombinant bacterium from a lignocellulosic biomass at a pilot scale.

Dilute sulfuric acid pretreatment of corn stover for enzymatic hydrolysis and efficient ethanol production by recombinant Escherichia coli FBR5 without detoxification.

A pretreatment strategy for dilute H2SO4 pretreatment of corn stover was developed for the purpose of reducing the generation of inhibitory substances during pretreatment so that a detoxification step is not required prior to fermentation while maximizing sugar yield. The optimal conditions for pretreatment of corn stover (10%, w/v) were: 0.75% H2SO4, 160°C, and 0-5 min holding time. The conditions were chosen based on maximum glucose release after enzymatic hydrolysis, minimum loss of pentose sugars and minimum formation of sugar degradation products such as furfural and hydroxymethyl furfural. The pretreated corn stover after enzymatic saccharification generated 63.2 ± 2.2 and 63.7 ± 2.3 g total sugars per L at 0 and 5 min holding time, respectively. Furfural production was 0.45 ± 0.1 and 0.87 ± 0.4 g/L, respectively. The recombinant Escherichia coli strain FBR5 efficiently fermented non-detoxified corn stover hydrolyzate if the furfural content is <0.5 g/L.

Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production.

Dilute H(3)PO(4) (0.0-2.0%, v/v) was used to pretreat corn stover (10%, w/w) for conversion to ethanol. Pretreatment conditions were optimized for temperature, acid loading, and time using central composite design. Optimal pretreatment conditions were chosen to promote sugar yields following enzymatic digestion while minimizing formation of furans, which are potent inhibitors of fermentation. The maximum glucose yield (85%) was obtained after enzymatic hydrolysis of corn stover pretreated with 0.5% (v/v) acid at 180°C for 15min while highest yield for xylose (91.4%) was observed from corn stover pretreated with 1% (v/v) acid at 160°C for 10min. About 26.4±0.1g ethanol was produced per L by recombinant Escherichia coli strain FBR5 from 55.1±1.0g sugars generated from enzymatically hydrolyzed corn stover (10%, w/w) pretreated under a balanced optimized condition (161.81°C, 0.78% acid, 9.78min) where only 0.4±0.0g furfural and 0.1±0.0 hydroxylmethyl furfural were produced.

Synthetic resin-bound truncated Candida antarctica lipase B for production of fatty acid alkyl esters by transesterification of corn and soybean oils with ethanol or butanol.

A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomyces cerevisiae. Western blot analysis detected five truncated CALB variants, suggesting multiple translation starts from the six in-frame ATG codons. The longest open reading frame, which corresponds to amino acids 35-317 of the mature lipase, appeared to be expressed in the greatest amount. The truncated CALB was immobilized on Sepabeads® EC-EP resin and used to produce ethyl and butyl esters from crude corn oil and refined soybean oil. The yield of ethyl esters was 4-fold greater from corn oil than from soybean oil and was 36% and 50% higher, respectively, when compared to a commercially available lipase resin (Novozym 435) using the same substrates. A 5:1 (v/v) ratio of ethanol to corn oil produced 3.7-fold and 8.4-fold greater yields than ratios of 15:1 and 30:1, respectively. With corn oil, butyl ester production was 56% higher than ethyl ester production. Addition of an ionic catalytic resin step prior to the CALB resin increased yields of ethyl esters from corn oil by 53% compared to CALB resin followed by ionic resin. The results suggest resin-bound truncated CALB has potential application in biodiesel production using biocatalysts.

Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF.

Sugarcane bagasse was characterized as a feedstock for the production of ethanol using hydrothermal pretreatment. Reaction temperature and time were varied between 160 and 200°C and 5-20 min, respectively, using a response surface experimental design. The liquid fraction was analyzed for soluble carbohydrates and furan aldehydes. The solid fraction was analyzed for structural carbohydrates and Klason lignin. Pretreatment conditions were evaluated based on enzymatic extraction of glucose and xylose and conversion to ethanol using a simultaneous saccharification and fermentation scheme. SSF experiments were conducted with the washed pretreated biomass. The severity of the pretreatment should be sufficient to drive enzymatic digestion and ethanol yields, however, sugars losses and especially sugar conversion into furans needs to be minimized. As expected, furfural production increased with pretreatment severity and specifically xylose release. However, provided that the severity was kept below a general severity factor of 4.0, production of furfural was below an inhibitory concentration and carbohydrate contents were preserved in the pretreated whole hydrolysate. There were significant interactions between time and temperature for all the responses except cellulose digestion. The models were highly predictive for cellulose digestibility (R (2) = 0.8861) and for ethanol production (R (2) = 0.9581), but less so for xylose extraction. Both cellulose digestion and ethanol production increased with severity, however, high levels of furfural generated under more severe pretreatment conditions favor lower severity pretreatments. The optimal pretreatment condition that gave the highest conversion yield of ethanol, while minimizing furfural production, was judged to be 190°C and 17.2 min. The whole hydrolysate was also converted to ethanol using SSF. To reduce the concentration of inhibitors, the liquid fraction was conditioned prior to fermentation by removing inhibitory chemicals using the fungus Coniochaeta ligniaria.

Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars.

Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 was mutagenized using UV-C irradiation to produce yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol. UV-C irradiation potentially produces large numbers of random mutations broadly and uniformly over the whole genome to generate unique strains. Wild-type cultures of S. stipitis NRRL Y-7124 were subjected to UV-C (234 nm) irradiation targeted at approximately 40% cell survival. When surviving cells were selected in sufficient numbers via automated plating strategies and cultured anaerobically on xylose medium for 5 months at 28°C, five novel mutagenized S. stipitis strains were obtained. Variable number tandem repeat analysis revealed that mutations had occurred in the genome, which may have produced genes that allowed the anaerobic utilization of xylose. The mutagenized strains were capable of growing anaerobically on xylose/glucose substrate with higher ethanol production during 250- to 500-h growth than a Saccharomyces cerevisiae yeast strain that is the standard for industrial fuel ethanol production. The S. stipitis strains resulting from this intense multigene mutagenesis strategy have potential application in industrial fuel ethanol production from lignocellulosic hydrolysates.