Genome-scale resources for Thermoanaerobacterium saccharolyticum

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. Electronic supplementary material The online version of this article (doi:10.1186/s12918-015-0159-x) contains supplementary material, which is available to authorized users

Development of an advanced, continuous mild gasification process for the production of coproducts

Research continued on the production of coproducts from continuous mild gasification. During the third quarter of 1990, work focused on start-up and operation of the 50 pound/hour char-to-carbon (CTC) process research unit (PRU). Start-up procedures have been finalized for the methane production reactor, and the design temperature has been achieved. Flows and pressures for the overall process have been balanced and optimized. We have achieved temperatures above 1500{degree}F in the carbon formation reactor. Upgrading experiments on mild gasification pitch have also continued on a pitch produced in run MG-122. Results of heat treating and catalytic treating tests are reported

Development of an advanced continuous mild gasification process for the production of coproducts. Task 4, System integration studies: Char upgrading

This document describes the results of Task 4 under which a 50 pound/hour char-to-carbon (CTC) process research unit (PRU) was designed in the second half of 1989, with construction completed in June 1990. The CTC PRU at Golden was operated for nearly one year during which 35 runs were completed for a total of nearly 800 hours of operation. Char methanation and carbon production reactor development activities are detailed in this report, as well as the results of integrated runs of the CTC process. Evaluation of the process and the carbon product produced is also included. It was concluded that carbon could be produced from mild gasification char utilizing the CTC process. Char methanation and membrane separation steps performed reasonably well and can scaled up with confidence. However, the novel directly heated reactor system for methane cracking did not work satisfactorily due to materials of construction and heat transfer problems, which adversely affected the quantity and quality of the carbon product. Alternative reactor designs are recommended

Development of an advanced, continuous mild gasification process for the production of co-products. Task 4.6, Economic evaluation

The principal finding of this study was the high capital cost and poor financial performance predicted for the size and configuration of the plant design presented. The XBi financial assessment gave a disappointingly low base-case discounted cash flow rate of return (DCFRR) of only 8.1% based on a unit capital cost of $900 per ton year (tpy) for their 129,000 tpy design. This plant cost is in reasonable agreement with the preliminary estimates developed by J.E. Sinor Associates for a 117,000 tpy plant based on the FMC process with similar auxiliaries (Sinor, 1989), for which a unit capital costs of$938 tpy was predicted for a design that included char beneficiation and coal liquids upgrading--or about $779 tpy without the liquid upgrading facilities. The XBi assessment points out that a unit plant cost of$900 tpy is about three times the cost for a conventional coke oven, and therefore, outside the competitive range for commercialization. Modifications to improve process economics could involve increasing plant size, expanding the product slate that XBi has restricted to form coke and electricity, and simplifying the plant flow sheet by eliminating marginally effective cleaning steps and changing other key design parameters. Improving the financial performance of the proposed formed coke design to the level of a 20% DCFRR based on increased plant size alone would require a twenty-fold increase to a coal input of 20,000 tpd and a coke production of about 2.6 minion tpy--a scaling exponent of 0.70 to correct plant cost in relation to plant size

Development of an advanced process for drying fine coal in an inclined fluidized bed

The objective of this research project was to demonstrate a technically feasible and economically viable process for drying and stabilizing high-moisture subbituminous coal. Controlled thermal drying of coal fines was achieved using the inclined fluidized-bed drying and stabilization process developed by the Western Research Institute. The project scope of work required completion of five tasks: (1) project planning, (2) characterization of two feed coals, (3) bench-scale inclined fluidized-bed drying studies, (4) product characterization and testing, and (5) technical and economic evaluation of the process. High moisture subbituminous coals from AMAX Eagle Butte mine located in the Powder River Basin of Wyoming and from Usibelli Coal Mine, Inc. in Healy, Alaska were tested in a 10-lb/hr bench-scale inclined fluidized-bed. Experimental results show that the dried coal contains less than 1.5% moisture and has a heating value over 11,500 Btu/lb. The coal fines entrainment can be kept below 15 wt % of the feed. The equilibrium moisture of dried coal was less than 50% of feed coal equilibrium moisture. 7 refs., 60 figs., 47 tabs

Final Report on Development of Thermoanaerobacterium saccharolyticum for the conversion of lignocellulose to ethanol

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

Development of pyrF-Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant ▿ †

We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes the enzyme phosphotransacetylase. The C. thermocellum Δpta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources