The thermophilic, anaerobic bacterium Thermoanaerobacterium saccharolyticum digests hemicellulose and utilizes the major sugars present in biomass. It was previously engineered to produce ethanol at yields equivalent to yeast. While saccharolytic anaerobes have been long studied as potential biomass-fermenting organisms, development efforts for commercial ethanol production have not been reported.

BACKGROUND:Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation.RESULTS:Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results.CONCLUSION:These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum.

The liberation of acetate from hemicellulose negatively impacts fermentations of cellulosic biomass, limiting the concentrations of substrate that can be effectively processed. Solvent-producing bacteria have the capacity to convert acetate to the less toxic product acetone, but to the best of our knowledge, this trait has not been transferred to an organism that produces ethanol at high yield.

Background: A previously developed mathematical model of low solids thermophilic simultaneous saccharification and fermentation (tSSF) with Avicel was unable to predict performance at high solids using a commercial cellulase preparation (Spezyme CP) and the high ethanol yield Thermoanaerobacterium saccharolyticum strain ALK2. The observed hydrolysis proceeded more slowly than predicted at solids concentrations greater than 50 g/L Avicel. Factors responsible for this inaccuracy were investigated in this study.Results: Ethanol dramatically reduced cellulase activity in tSSF. At an Avicel concentration of 20 g/L, the addition of ethanol decreased conversion at 96 hours, from 75% in the absence of added ethanol down to 32% with the addition of 34 g/L initial ethanol. This decrease is much greater than expected based on hydrolysis inhibition results in the absence of a fermenting organism. The enhanced effects of ethanol were attributed to the reduced, anaerobic conditions of tSSF, which were shown to inhibit cellulase activity relative to hydrolysis under aerobic conditions. Cellulose hydrolysis in anaerobic conditions was roughly 30% slower than in the presence of air. However, this anaerobic inhibition was reversed by exposing the cellulase enzymes to air.Conclusion: This work demonstrates a previously unrecognized incompatibility of enzymes secreted by an aerobic fungus with the fermentation conditions of an anaerobic bacterium and suggests that enzymes better suited to industrially relevant fermentation conditions would be valuable. The effects observed may be due to inactivation or starvation of oxygen dependent GH61 activity, and manipulation or replacement of this activity may provide an opportunity to improve biomass to fuel process efficiency.

This project addressed the need for economical technology for the conversion of lignocellulosic biomass to fuels, specifically the conversion of pretreated hardwood to ethanol. The technology developed is a set of strains of the bacterium Thermoanaerobacterium saccharolyticum and an associated fermentation process for pretreated hardwood. Tools for genetic engineering and analysis of the organism were developed, including a markerless mutation method, a complete genome sequence and a set of gene expression profiles that show the activity of its genes under a variety of conditions relevant to lignocellulose conversion. Improved strains were generated by selection and genetic engineering to be able to produce higher amounts of ethanol (up to 70 g/L) and to be able to better tolerate inhibitory compounds from pretreated hardwood. Analysis of these strains has generated useful insight into the genetic basis for desired properties of biofuel producing organisms. Fermentation conditions were tested and optimized to achieve ethanol production targets established in the original project proposal. The approach proposed was to add cellulase enzymes to the fermentation, a method called Simultaneous Saccharification and Fermentation (SSF). We had reason to think SSF would be an efficient approach because the optimal temperature and pH for the enzymes and bacterium are very close. Unfortunately, we discovered that commercially available cellulases are inactivated in thermophilic SSF by a combination of low redox potential and ethanol. Despite this, progress was made against the fermentation targets using bacterial cellulases. Thermoanaerobacterium saccharolyticum may still prove to be a commercially viable technology should cellulase enzyme issues be addressed. Moreover, the organism was demonstrated to produce ethanol at approximately theoretical yield from oligomeric hemicellulose extracts, an ability that may prove to be uniquely valuable in pretreatment configurations in which cellulose and hemicellulose are separated.

Response surface methodology (RSM), based on a 22 full factorial design, evaluated the moisture effects in recovering xylose by diethyloxalate (DEO) hydrolysis. Experiments were carried out in laboratory reactors (10 mL glass ampoules) containing corn stover (0.5 g) properly ground. The ampoules were kept at 160 °C for 90 min. Both DEO concentration and corn stover moisture content were statistically significant at 99% confidence level. The maximum xylose recovery by the response surface methodology was achieved employing both DEO concentration and corn stover moisture at near their highest levels area. We amplified this area by using an overlay plot as a graphical optimization using a response of xylose recovery more than 80%. The mathematical statistical model was validated by testing a specific condition in the satisfied overlay plot area. Experimentally, a maximum xylose recovery (81.2%) was achieved by using initial corn stover moisture of 60% and a DEO concentration of 4% w/w. The mathematical statistical model showed that xylose recovery increases during DEO corn stover acid hydrolysis as the corn stover moisture level increases. This observation could be important during the harvesting of corn before it is fully dried in the field. The corn stover moisture was an important variable to improve xylose recovery by DEO acid hydrolysis.

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono- and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a d -xylulokinase mutant of Pichia stipitis, FPL-YS30 ( xyl3 -∆1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic acids used for neutralization and urea or ammonium sulfate used as nitrogen sources. Phosphoric acid improved color and removal of phenolic compounds. d -Gluconic acid enhanced cell growth. Ammonium sulfate increased cell yield and maximum specific cell growth rate independently of the acid used for neutralization. The highest xylitol yield (0.61 g xylitol /g xylose ) and volumetric productivity (0.18 g xylitol /g xylose l) were obtained in hydrolysate neutralized with phosphoric acid. However, when urea was the nitrogen source the cell yield was less than half of that obtained with ammonium sulfate.

Autohydrolysis is a simple, green method of recovering sugars from biomass, using only hot water. One potential drawback is that byproducts are formed during the autohydrolysis process that could interfere with subsequent hydrolysis and fermentation to ethanol. In the present work, autohydrolysis prehydrolyzate from mixed hardwood chips was detoxified with activated carbon and the removal efficiency of byproducts as well as the loss of sugars determined. The resulting detoxified prehydrolyzate was evaluated for the fermentation to ethanol with a thermophilic anaerobic bacterium. Activated carbon at a 2.5 wt% level on the prehydrolyzate was able to remove 42% of formic acid, 14% of acetic acid, 96% of hydroxymethylfurfural (HMF) and 93% of the furfural. However, 8.9% of sugars were also removed. The removal of HMF and furfural follow expected adsorption isotherms but formic acid, acetic acid, and sugars did not. Autohydrolysis prehydrolyzates from mixed hardwood detoxified with activated carbon can be fermented with Thermoanaerobacterium saccharolyticum strain MO1442 with an essentially 100% yield. T. saccharolyticum strain MO1442 is able to metabolize the glucose, xylose, and arabinose in the hydrolyzate. The results showed the detoxification process with activated carbon improved the ethanol yields by the removal of toxic compounds, mainly HMF and furfural, with moderate loss of fermentable sugars. Published by Elsevier Ltd.

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative β-1–4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H 2 O 2 . These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H 2 O 2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.

Oxalate from partially decayed southern pine wood was analyzed by HPLC or colorimetric assay. Oxalate extraction efficiency, assessed by comparing analysis of whole wood cubes with ground wood, showed that both wood geometries could be extracted with comparable efficiency. To differentiate soluble oxalate from total oxalate, three extraction methods were assessed, using phosphate buffer, sulphuric acid or sodium hydroxide. Oxalate values from phosphate buffer extracts were two to three times higher than those from sulphuric acid extracts by both methods of analysis. Results for HPLC analysis were not significantly different from results for colorimetric analysis within each method of extraction. Results from sodium hydroxide extraction were inconsistent. The presence of oxalate in phosphate and sulphuric acid extracts was confirmed with oxalate decarboxylase but could not be confirmed for NaOH extracts. HPLC and the colorimetric assay remain valid methods of reporting soluble oxalic acid from decayed wood and are particularly useful for comparing relative determinations between treatments or fungal isolates.

AbstractDiethyl oxalate (DEO) was injected into a digester containing wood chips (pine, spruce, or aspen) preheated to 130–140°C and held for 30 min at the same temperature. When mechanical pulps were produced from these pretreated chips, savings in electrical refiner energy could be achieved. For southern yellow pine (Pinus taeda), the electrical refiner energy required to produce pulps with Canadian standard freeness (CSF) of 100 ml was 38– 55% less than for an untreated control. Energy savings were also observed for aspen (Populusspp.) and spruce (Piceaspp.). DEO-treated pine handsheets showed a 26% improvement in tear index compared with a control at 100 ml CSF. Under the pretreatment conditions de-scribed, DEO rapidly vaporizes, reacts with water already present in the chip, and forms an oxalic acid (OA) solution. Reported handsheet properties and energy savings for pine chips treated with a solution of OA are similar to DEO-treated handsheets. It can be concluded that the effect of DEO treatment is due to reactions catalyzed by OA.

Wood chips of pine, spruce, aspen, and maple were treated at 135-140 degrees C with diethyl oxalate (DEO) and analyzed for extractable and residual carbohydrates. Under these conditions, DEO hydrolyzes to ethanol and oxalic acid (OA). The amount and identity of carbohydrates released from the chips were species-dependent. For all wood species, increasing the amount of chemical, time, or temperature resulted in an increment in carbohydrates released. Approximately 50% (by wt) of extracted carbohydrates were monosaccharides. In addition, acetic acid was detected in the water extracts. When extracts were subsequently alkali ne-treated, more acetate was released, indicating the presence of acetyl esters. The composition of water extracts and of wood chips after treatment indicates that these treatments primarily affect hemicelluloses. In summary, treatment of wood chips with DEO or OA releases carbohydrates suitable for fermentation, with no evidence of cellulose degradation.

Biorefining wood into paper and chemicals is not as easy as making a single traditional paper product. Paper is made from the cellulose-containing fractions of wood and processing may remove lignin and hemicellulose components. The yield and composition of the product depend upon the type of paper being produced. The paper process often alters the non-cellulose fractions, making them less suitable for conversion to other valuable products. This chapter will review methods of obtaining hemicellulosic sugars and investigate the potential for obtaining sugars by a wood pretreatment before it is made into pulp. As an example of hemicellulose removal the results of a new pretreatment using diethyl oxalate are also described. Hemicellulose removal by this system provides other benefits to the pulping process.

L'invention concerne un procede de production de pâte a partir d'un materiau ou d'une source lignocellulosique fibreuse. Ledit procede comprend une etape de traitement ou de pretraitement au cours de laquelle on expose le materiau a des derives d'acide oxalique, en particulier a des derives d'ester dyalkyle, en particulier en phase vapeur. Apres le traitement, il est possible d'affiner le materiau en utilisant plusieurs methodes de reduction en pâte, de maniere a obtenir un produit de pâte finale. La methode de production du produit permet d'obtenir une meilleure resistance du papier produit a partir de la pâte et de faire des economies d'energie dans la fabrication de la pâte. Le traitement ou pretraitement permettent egalement de produire une source de carbohydrate soluble ainsi que d'autres composants (p. ex. de l'acide acetique, autres composants du bois) en vue d'un developpement ulterieur du produit. Dans certains modes de realisation, on ne fabrique pas un produit de pâte et tous les hydrates de carbone presents dans la lignocellulose sont transformes en sucres solubles.

We investigated how biopulping modifies chemical and physical properties of wood and how these changes affect the properties of the resulting fiber. Mechanical and chemical testing revealed wood cell changes during 2 weeks of colonization by Ceriporiopsis subvermispora. Typical mechanical properties, such as modulus of elasticity and maximum load, tracked reductions in energy needed for mechanical refining to pulp. The data indicate the fiber saturation point of spruce increased from 29% to 42% during biopulping. At the same time, titratable acid groups increased up to 62%. Chemical analysis showed that oxalic acid esters were produced in the wood during biopulping in sufficient amounts to account for the increase in acid groups. The benefits of biopulping - energy savings and increased handsheet strength - as well as other physical property changes are consistent with the mechanism we propose: biopulping increases the acid group content of wood.