Thomas W. Jeffries

Biodegradation of lignin-carbohydrate complexes

Covalent lignin-carbohydrate (LC) linkages exist in lignocellulose from wood and groups herbaceous plants. In wood, they consist of ester and ether linkages through sugar hydroxyl to the α-carbanol of phenylpropane subunits in lignin. In grasses, ferulic and p-coumaric acids are esterified to hemicelluloses and lignin, respectively. Hemicelluloses also contain substitutents and side groups that restrict enzymatic attack. Watersoluble lignin-carbohydrate complexes (LCCs) often precipitate during digestion with polysaccharidases, and the residual sugars are more diverse than the bulk hemicellulose. A number of microbial esterases and hemicellulose polysaccharidases including acetyl xylan esterase, ferulic acid esterase, and p-coumaric esterase attack hemicellulose side chains. Accessory hemicellulases include α-l-arabinofuranosidase and α-methyl-glucuranosidase. Both of these side chains are involved in LC bonds. β-Glucosidase will attach sugar residues to lignin degradation products and when carbohydrate is attached to lignin, lignin peroxidase will depolymerize the lignin more readily.

Efficiencies of acid catalysts in the hydrolysis of lignocellulosic biomass over a range of combined severity factors

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

Fueling the future with fungal genomics

Fungi play important roles across the range of current and future biofuel production processes. From crop/feedstock health to plant biomass saccharification, enzyme production to bioprocesses for producing ethanol, higher alcohols, or future hydrocarbon biofuels, fungi are involved. Research and development are underway to understand the underlying biological processes and improve them to make bioenergy production efficient on an industrial scale. Genomics is the foundation of the systems biology approach that is being used to accelerate the research and development efforts across the spectrum of topic areas that impact biofuels production. In this review, we discuss past, current, and future advances made possible by genomic analyses of the fungi that impact plant/feedstock health, degradation of lignocellulosic biomass, and fermentation of sugars to ethanol, hydrocarbon biofuels, and renewable chemicals.

Conversion of xylose to ethanol under aerobic conditions by Candida tropicalis

SummaryCandida tropicalis converts xylose to ethanol under aerobic, but not anaerobic, conditions. Ethanol production lags behind growth and is accelerated by increased aeration. Adding xylose to active cultures stimulates ethanol production as does serial subculture in a medium containing xylose as a sole carbon source.

Production of ethanol from wood hydrolyzate by yeasts.

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

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

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

The roles of xylan and lignin in oxalic acid pretreated corncob during separate enzymatic hydrolysis and ethanol fermentation.

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

Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology.

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

Cloning and disruption of the b-isopropylmalate dehydrogenase gene (LEU2 ) of Pichia stipitis with URA3 and recovery of the double auxotroph

Abstract Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5′-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ“pop-out” cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu− Ura+ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5′-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu−Ura− phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids.

Comparison of corn steep liquor with other nutrients in the fermentation of D-xylose by Pichia stipitis CBS 6054

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