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Ingram, Lonnie O.

Publications and source records attributed to Ingram, Lonnie O..

Principles and practice of designing microbial biocatalysts for fuel and chemical production

Abstract The finite nature of fossil fuels and the environmental impact of its use have raised interest in alternate renewable energy sources. Specifically, nonfood carbohydrates, such as lignocellulosic biomass, can be used to produce next generation biofuels, including cellulosic ethanol and other nonethanol fuels like butanol. However, currently there is no native microorganism that can ferment all lignocellulosic sugars to fuel molecules. Thus, research is focused on engineering improved microbial biocatalysts for production of liquid fuels at high productivity, titer, and yield. A clear understanding and application of the basic principles of microbial physiology and biochemistry are crucial to achieve this goal. In this review, we present and discuss the construction of microbial biocatalysts that integrate these principles with ethanol-producing Escherichia coli as an example of metabolic engineering. These principles also apply to fermentation of lignocellulosic sugars to other chemicals that are currently produced from petroleum.

09 BIOMASS FUELS↗

A Combined Thermochemical and Microbial Process for Recycling Polylactic Acid Polymer to Optically Pure L-Lactic Acid for Reuse

Polylactic acid polymer (PLA) produced from renewable resources can be recycled at the end of life to constituent monomer, optically pure lactic acid (LA), by a combination of chemical and biological processes. Efficient application of this closed loop of LA-PLA plastics-LA can minimize accumulation of plastics waste that pollute land and oceans. Temperature-dependent hydrolysis of PLA in water to LA follows apparent first order decay kinetics after a short lag. A modified Gompertz equation can explain the overall hydrolysis process. Alkali increased the rate of hydrolysis of PLA and reduced the length of lag period compared to water alone. The stoichiometry of base added to LA released was 1.0. The highest lactic acid yield was 0.95 g g -1 of PLA. D-LA in the syrup obtained after hydrolysis of PLA-plastics was removed using an engineered Escherichia coli to produce a L-LA syrup with an optical purity ≥ 99%. Here, these results show that thermochemical hydrolysis of PLA-based plastics to LA with optimum amount of base followed by bio-based purification to L-LA is an effective method of recycling PLA-plastics for reuse.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Chromosomal mutations in Escherichia coli that improve tolerance to nonvolatile side–products from dilute acid treatment of sugarcane bagasse

Lignocellulosic biomass provides attractive non-food carbohydrates for production of ethanol and dilute acid pretreatment is a biomass-independent process for access to these carbohydrates. However, this pretreatment also releases volatile and nonvolatile inhibitors of fermenting microorganisms. To identify unique gene products contributing to sensitivity/tolerance to non- volatile inhibitors, ethanologenic Escherichia coli strain LY180 was adapted for growth in vacuum-treated sugarcane bagasse acid hydrolysate (VBHz) lacking furfural and other volatile inhibitors. A mutant, strain AQ15, obtained after about 500 generations of growth in VBHz, grew and fermented the sugars in a medium with 50% VBHz. Comparative genome sequence analysis of strains AQ15 and LY180 revealed 95 mutations in strain AQ15. Six of these mutations were also found in strain SL112, an independent inhibitor-tolerant derivative of strain LY180. Among these six mutations, null mutations in mdh and bacA were identified as contributing factors to VBHz tolerance in strain AQ15, based on genetic and physiological analysis. Deletion of either gene in strain LY180 increased tolerance to VBHz from about 30% to 50% (v/v). Here, considering the location and physiological role of the two enzymes in the cell, it is likely that the two enzymes contribute to VBHz sensitivity of ethanologenic E. coli by different mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗

US-India Consortium for Development of Sustainable Advanced Lignocellulosic Biofuel Systems

This multi-institutional project had as it main objectives to develop a fully integrated platform for the production of advanced biofuels, specifically butanol, from sustainably produced switchgrass and biomass sorghum and to develop economic models that could guide the establishment of a crop to fuel value chain. The project was organized into three work packages: (1) WP1 Feedstock Development and Supply. Activities in this WP resulted in the development of improved biomass sorghum cultivars and biomass hybrids that generate high yields of biomass with limited inputs of water, fertilizer and agri-chemicals and that display less recalcitrance to enzymatic saccharification at the biorefinery; the identification of physiological responses and genes associated with sorghum’s tolerance to water logging as the basis for the development of biomass sorghums that can be cultivated on land prone to flooding; the identification of water-logging tolerant switchgrass genotypes among existing germplasm; the identification of management practices that maximize switchgrass biomass yield (fertilizer applications, harvest regimens) on commercial farms on different types of soil in Missouri. (2) WP2 Biorefinery Technologies. This WP included the development of a recombinant E. coli strain able to convert fermentable sugars derived from biomass sorghum and switchgrass to butyrate as a precursor for the advanced biofuel butanol. Butanol is toxic to most microbes, so that yields tend to be low. In contrast, butyrate can be produced in high titer and high yield by several naturally occurring bacteria, but these species are strictly anaerobic, which complicates commercial production. Our strategy involved developing a recombinant strain of E. coli able to produce high yields and high titers of butyrate. The butyrate can then be reduced to butanol via chemical conversion. In addition, a techno-economic analysis of converting sorghum and switchgrass biomass to ethanol was conducted based on data obtained at a pilot biorefinery. These data combined with small-scale butanol production data were used as the basis for a techno-economic analysis of biomass-to-butanol conversion. Furthermore, the biorefinery stillage was evaluated as a source of fertilizer or soil amendment. In contrast to the use of biochar as a soil amendment, stillage resulted in enhanced microbial activity in the soil and release of CO2. Stillage was determined to be a suitable feedstock for biogas production in an anaerobic digester. Biorefinery lignin can be blended with polymers to create novel UV-resistant materials promising for application in outer space, and as a source of organic acids for use in plastics. These applications offer opportunities to create value from the biorefinery residues. (3) WP3. This WP focused on development of economic models that can help define the most successful strategy for implementing biofuel production from bioenergy crops. Southeastern Missouri was determined to be the best location for a bio-butanol facility that processes biomass crops, due to the abundance of feedstock within a small radius of the facility. The most effective transportation method is by rail car. Feedstock availability was considered a more important driver for commercial success than the availability of a butanol market. The average cost of producing switchgrass biomass was calculated to be $65/ton assuming a biomass yield of 8 tons per hectare (the upper end of the yield in the commercial trials in WP2). Lower yields drive up costs, but improved genotypes will conversely reduce costs. Based on farmer surveys, conducting university extension activities was determined to positively influence farmers’ willingness to consider switchgrass. Other factors included experience with pasture land and forest land, and the availability of policies that provide a safety net to the farmers in case of instability in the biofuels market. This instability can be mitigated in part by ensuring adequate biomass supplies through diversification. The project supported the training of 20 graduate students and resulted in 57 peer-reviewed journal articles (with eight additional in review or in progress), eight book chapters and five patents.

09 BIOMASS FUELS↗

Metabolic engineering of Escherichia coli for the production of butyric acid at high titer and productivity

Background: Several anaerobic bacteria produce butyric acid, a commodity chemical with use in chemical, pharmaceutical, food and feed industries, using complex media with acetate as a co-product. Butyrate titer of various recombinant Escherichia coli did not exceed 10 g l -1 in batch fermentations in any of the media tested. Results: A recombinant E. coli (strain LW393) that produced butyrate as the major fermentation product was constructed with genes from E. coli, Clostridium acetobutylicum and Treponema denticola. Strain LW393 produced 323±6 mM (28.4±0.4 g l -1 ) butyric acid in batch fermentations in mineral salt medium with glucose as C source at a yield of 0.37±0.01 g (g glucose consumed) -1 . Butyrate accounted for 90% of the total products produced by the culture. Supplementing this medium with yeast extract further increased butyric acid titer to 375±4 mM. Average volumetric productivity of butyrate with xylose as C source was 0.89±0.07 g l -1 h -1 . Conclusions: The butyrate titer reported in this study is about 2.5–3-times higher than the values reported for other recombinant E. coli and this is achieved in mineral salt medium with an expectation of lower purifcation and production cost of butyrate.

09 BIOMASS FUELS↗

Techno-Economic Evaluation of Cellulosic Ethanol Production Based on Pilot Biorefinery Data: a Case Study of Sweet Sorghum Bagasse Processed via L+SScF

Replacing fossil fuels with renewable fuels derived from lignocellulosic biomass can contribute to the mitigation of global warming and the economic development of rural communities. This will require lignocellulosic biofuels to become price competitive with fossil fuels. Techno-economic analyses can provide insights into which parts of the biofuel production process need to be optimized to reduce cost or energy use. Here, we used data obtained from a pilot biorefinery to model a commercial-scale biorefinery that processes lignocellulosic biomass to ethanol, with a focus on the minimum ethanol selling price (MESP). The process utilizes a phosphoric acid-catalyzed pre-treatment of sweet sorghum bagasse followed by liquefaction and simultaneous saccharification and co-fermentation (L+SScF) of hexose and pentose sugars by an engineered Escherichia coli strain. After validating a techno-economic model developed with the SuperPro Designer software for the conversion of sugarcane bagasse to ethanol by comparing it to a published Aspen Plus model, six different scenarios were modeled for sweet sorghum bagasse Under the most optimistic scenario, the ethanol can be produced at a cost close to the energy-equivalent price of gasoline. Aside from an increase in the price of gasoline, the gap between ethanol and gasoline prices could also be bridged by either a decrease in the cost of cellulolytic enzymes or development of value-added products from lignin.

Biofuel↗

Fermentation of dihydroxyacetone by engineered Escherichia coli and Klebsiella variicola to products

Methane can be converted to triose dihydroxyacetone (DHA) by chemical processes with formaldehyde as an intermediate. Carbon dioxide, a by-product of various industries including ethanol/butanol biorefineries, can also be converted to formaldehyde and then to DHA. DHA, upon entry into a cell and phosphorylation to DHA-3-phosphate, enters the glycolytic pathway and can be fermented to any one of several products. However, DHA is inhibitory to microbes due to its chemical interaction with cellular components. Fermentation of DHA to d -lactate by Escherichia coli strain TG113 was inefficient, and growth was inhibited by 30 g⋅L −1 DHA. An ATP-dependent DHA kinase from Klebsiella oxytoca (pDC117d) permitted growth of strain TG113 in a medium with 30 g⋅L −1 DHA, and in a fed-batch fermentation the d -lactate titer of TG113(pDC117d) was 580 ± 21 mM at a yield of 0.92 g⋅g −1 DHA fermented. Klebsiella variicola strain LW225, with a higher glucose flux than E. coli , produced 811 ± 26 mM d -lactic acid at an average volumetric productivity of 2.0 g −1 ⋅L −1 ⋅h −1 . Fermentation of DHA required a balance between transport of the triose and utilization by the microorganism. Using other engineered E. coli strains, we also fermented DHA to succinic acid and ethanol, demonstrating the potential of converting CH 4 and CO 2 to value-added chemicals and fuels by a combination of chemical/biological processes.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolic engineering of Bacillus subtilis for production of D-lactic acid

Poly lactic acid (PLA) based plastics is renewable, bio–based, and biodegradable. Although present day PLA is composed of mainly L–LA, an L– and D– LA copolymer is expected to improve the quality of PLA and expand its use. To increase the number of thermotolerant microbial biocatalysts that produce D–LA, a derivative of Bacillus subtilis strain 168 that grows at 50°C was metabolically engineered. Since B. subtilis lacks a gene encoding D–lactate dehydrogenase (ldhA), five heterologous ldhA genes (B. coagulans ldhA and gldA101, and ldhA from three Lactobacillus delbrueckii) were evaluated. Corresponding D–LDHs were purified and biochemically characterized. Among these, D–LDH from L. delbrueckii subspecies bulgaricus supported the highest D…LA titer (about 1M) and productivity (2 g h –1 g cells –1 ) at 37°C (B. subtilis strain DA12). The D–LA titer at 48°C was about 0.6 M at a yield of 0.99 (g D–LA g–1glucose consumed). Strain DA12 also fermented glucose at 48°C in mineral salts medium to lactate at a yield of 0.89 g g –1 glucose and the D–lactate titer was 180 ± 4.5 mM. Furthermore, these results demonstrate the potential of B. subtilis as a platform organism for metabolic engineering for production of chemicals at 48°C that could minimize process cost.

59 BASIC BIOLOGICAL SCIENCES↗

Plasmidic Expression of nemA and yafC * Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate

Hydrolysate-resistant Escherichia coli SL100 was previously isolated from ethanologenic LY180 after sequential transfers in AM1 medium containing a dilute acid hydrolysate of sugarcane bagasse and was used as a source of resistance genes. Many genes that affect tolerance to furfural, the most abundant inhibitor, have been described previously. To identify genes associated with inhibitors other than furfural, plasmid clones were selected in an artificial hydrolysate that had been treated with a vacuum to remove furfural. Two new resistance genes were discovered from Sau3A1 libraries of SL100 genomic DNA: nemA (N-ethylmaleimide reductase) and a putative regulatory gene containing a mutation in the coding region, yafC *. The presence of these mutations in SL100 was confirmed by sequencing. A single mutation was found in the upstream regulatory region of nemR ( nemRA operon) in SL100. This mutation increased nemA activity 20-fold over that of the parent organism (LY180) in AM1 medium without hydrolysate and increased nemA mRNA levels >200-fold. Addition of hydrolysates induced nemA expression (mRNA and activity), in agreement with transcriptional control. NemA activity was stable in cell extracts (9 h, 37°C), eliminating a role for proteinase in regulation. LY180 with a plasmid expressing nemA or yafC * was more resistant to a vacuum-treated sugarcane bagasse hydrolysate and to a vacuum-treated artificial hydrolysate than LY180 with an empty-vector control. Neither gene affected furfural tolerance. The vacuum-treated hydrolysates inhibited the reduction of N -ethylmaleimide by NemA while also serving as substrates. In conclusion, expression of the nemA or yafC * plasmid in LY180 doubled the rate of ethanol production from the vacuum-treated sugarcane bagasse hydrolysate.

59 BASIC BIOLOGICAL SCIENCES↗

Techno-economic analysis of ethanol production from sugarcane bagasse using a Liquefaction plus Simultaneous Saccharification and co-Fermentation process

A techno-economic analysis was conducted for a simplified lignocellulosic ethanol production process developed and proven by the University of Florida at laboratory, pilot, and demonstration scales. Data obtained from all three scales of development were used with Aspen Plus to create models for an experimentally-proven base-case and 5 hypothetical scenarios. The model input parameters that differed among the hypothetical scenarios were time of L+SScF, enzyme loading, enzymatic conversion, solids loading, and overall process yield. The minimum ethanol selling price (MESP) varied between 50.38 and 62.72 US cents/L. The feedstock and the capital cost were the main contributors to the production cost, comprising between 23-28% and 40-49% of the MESP, respectively. A sensitivity analysis showed that overall ethanol yield had the greatest effect on the MESP. Here, these findings suggest that future efforts to increase the economic feasibility of a cellulosic ethanol process should focus on optimization for highest ethanol yield.

09 BIOMASS FUELS↗