Grantee Research Project Results
Final Report: Investigation of Solvent Toxicity in Bacterial Strains Involved in Butanol Production
EPA Contract Number: EPD07030Title: Investigation of Solvent Toxicity in Bacterial Strains Involved in Butanol Production
Investigators: Bhattacharyya, Anamitra
Small Business: Integrated Genomics, Inc.
EPA Contact: Richards, April
Phase: I
Project Period: March 1, 2007 through August 31, 2007
Project Amount: $68,525
RFA: Small Business Innovation Research (SBIR) - Phase I (2007) RFA Text | Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , SBIR - Pollution Prevention
Description:
Reducing the U.S. dependency on petroleum by increasing the use of renewable biomass-based resources is a primary goal in the development of a sustainable industrial society and for effective control of greenhouse gas emissions. Butanol is a better biofuel than ethanol, which currently is used in gasohol formulations. Butanol can be produced from crops using acetone-butanol (AB) fermentation by butanolagenic microbes, such as Clostridium spp. To make the AB fermentation economically viable, however, a number of limitations must be addressed. Currently, there are two major shortcomings of the AB fermentation process that uses Clostridium acetobutylicum. First, butanol is toxic to the clostridial cells at quite low concentrations meaning that the solvent concentration in the final fermentation broth is 2 percent or less. Second, the inefficient regulation of electron flow and distribution in C. acetobutylicum leads to loss of reducing equivalents and to an incorrect redox balance that limits the efficiency of butanol production.
The purpose of this research effort is to perform a complete and thorough in silico gene annotation and metabolic reconstruction of the complete genomes of the butanolagenic microorganisms C. acetobutylicum and Clostridium beijerinckii. Specifically, this phase of the work is designed to develop a thorough understanding of the physiology and biochemistry of these organisms as it pertains to butanol production and butanol resistance. In silico findings will facilitate the design of metabolic strain engineering approaches applicable to Clostridium species. This work will help create improved butanol resistance and yields, leading to the creation of the next generation of microbial butanol producers.
The tasks performed successfully under this project include:
- Identified open-reading frames (ORFs) in Clostridium spp. genomes, generated similarity tables, and loaded them into the ERGO™ Genome Analysis Suite;
- Developed a metabolic reconstruction from the complete genome sequences of C. acetobutylicum and C. beijerinckii using the ERGO™ Genome Analysis Suite applying automated and manual approaches;
- Compared these Clostridium spp. genomes using tools in the ERGO™ Genome Analysis Suite to identify attributes of Clostridium spp. and other genomes of solvent tolerant microorganisms useful for developing microbial solvent resistance; and
- Analyzed organic solvent exporters, bioenergetics (e.g., anaerobic respiratory chain, electron transport, anaerobic stress responses, ATP generation), and aspects of carbohydrate metabolism of these organisms regarding butanol tolerance, production, and anaerobic growth.
Summary/Accomplishments (Outputs/Outcomes):
The major research findings from this Phase I study relate directly to the basic biochemistry, metabolism, and physiology of butanolagenic Clostridium spp. and the roles they play in butanol production and resistance to this solvent. Specifically, some of these findings include:
- Both Clostridium spp. show that glycolysis is a major mechanism of ATP generation and a source of substrates for butanol production. A non-PTS-based glucose transporter was identified in this study that probably allows for glucose import into the cell.
- Clostridium spp. demonstrates anaerobic metabolism that reveals poor diversity and representation of enzymes to replenish reducing equivalents inside the cell. In addition the production of hydrogen is directly coupled to the generation of membrane potential and ATP biosynthesis.
- This study identified a proton-translocating pyrophosphatase in the genomes of these Clostridium spp. that provides an additional source energy generation without additional expenditure of ATP.
- Solvent efflux proteins found in other Gram-positive bacteria responsible for export of alcohols have been identified and are in the process of being cloned and expressed in Clostridium spp.
- Experimentation directed toward understanding the physiological butanol tolerance behavior of other bacteria (including Gram-positive and Gram-negative clades) indicates levels of butanol resistance equal to or better than those displayed by wild-type Clostridium spp. strains at some levels of butanol.
- There are multiple mechanisms for oxygen detoxification that allow these anaerobic microbes to be experimentally tractable in the laboratory under aerobic conditions.
Conclusions:
The availability of a fully annotated and reconstructed set of butanolagenic microorganisms is an invaluable in silico resource because it provides a foundation for designing and planning strain development strategies to increase butanol yields. Thus for some of the research findings summarized above, there are strategies that can be implemented to further improve butanol production. For example, if glycolysis plays a major role in ATP generation, then mutants that increase glycolytic flux can be isolated, which will lead to higher levels of intermediates in the butanol biosynthetic pathway. Similarly, strain engineering approaches more suitable for Phase II of this research effort will be directed toward improving the regeneration of reducing equivalents in the cell by cloning in suitable genes (already identified) from other bacteria that have this capability.
In addition, other sets of genes, such as specific solvent efflux genes (identified in this study), are being introduced directly into Clostridium cells to determine their relative efficacy to butanol resistance and butanol yield improvements. Regarding commercialization of the technology, the publicity from the Phase I proposal (e.g., press releases, market research) has been invaluable in attracting potential commercialization partners. Publicity also has been aided by internal business development efforts and previous patent disclosures on this subject to generate an avenue of co-development and venture capitalist partnership opportunities that are being pursued actively. For example, regarding cloning of the putative solvent exporter, after successful completion of the laboratory work, Integrated Genomics expects that the transporter will substantially improve the butanol resistance of microorganisms into which it is cloned and expressed.
Integrated Genomics will commercialize the technology by forming collaborations with companies that are engineering better butanol production microorganisms. The company expects to receive milestone and royalty payments from the success of the projects as the engineered butanol production systems are commercialized in the next generation of butanol biorefineries. Integrated Genomics currently is discussing such commercial collaborations with several other companies.
Supplemental Keywords:
small business, SBIR, biofuels, butanol, patent, venture capital, co-development, market research,, RFA, Scientific Discipline, Sustainable Industry/Business, Environmental Chemistry, Sustainable Environment, Technology for Sustainable Environment, bacteria, alternative fuel, butanol, alternative energy source, biofuel, bio-based energyThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.