2006 Progress Report: A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass

EPA Grant Number: R829479C023
Subproject: this is subproject number 023 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
Investigators: Yang, Shang-Tian
Institution: The Ohio State University - Main Campus
EPA Project Officer: Lasat, Mitch
Project Period: October 1, 2004 through September 30, 2007 (Extended to December 31, 2007)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

The goal of this project is to develop a new two-step fermentation process for economical production of butanol from agricultural commodities and food processing wastes containing carbohydrates as an inexpensive feedstock. In this new process, a butyric acid bacterium (Clostridium tyrobutyricum) will be used to convert glucose and xylose to butyric acid first, and then the produced butyric acid will be converted to butanol by C. acetobutylicum in a separate bioreactor. By separating acidogenesis and solventogenesis in two sequential bioreactors, one can better control the metabolic pathway and direct more carbon source to butanol instead of other byproducts (acetone, ethanol, acetate, and butyrate) commonly found in the acetone-butanol-ethanol (ABE) fermentation, thus increasing butanol yield and productivity. Also, a novel immobilized fibrous bed bioreactor (FBB) will be used for the fermentation to adapt cells to tolerate high butanol concentration. The specific tasks include:

  1. To study butanol production from butyric acid and glucose by C.acetobutylicum immobilizedin a FBB.
  2. To develop a process for enhanced butyric acid production from glucose andxylose.
  3. To develop and demonstrate the proposed two-step extractive fermentationprocess for producing butanol from glucose and xylose.

Progress Summary:

Conventional ABE fermentation involves two phases (acidogenesis and solventogenesis), is difficult to control, and suffers from low butanol productivity and yield. A novel approach uses two sequential fermentation steps to short-cut the complex ABE fermentation pathway by directing glucose fermentation to butyric acid and then to butanol. By separating the acid (butyric acid) production (using C. tyrobutyricum) from solvent (butanol) formation (using C. acetobutylicum), more glucose carbon can be used for butanol production, and a higher butanol yield of up to 40 percent (w/w) can be expected, which is 100 percent higher than that from conventional ABE fermentations. The new process also allows both bacteria to work under their respective optimal pH and temperature conditions, and thus can increase reactor productivity. In this work, a stable continuous fermentation for butanol production from glucose was developed by co-feeding the bioreactor with butyrate, the precursor for butanol formation, so that the fermentation would shift to and stay in the solventogenesis phase, thus producing more butanol from glucose and reducing the byproducts (e.g., ethanol and acetone). The enhanced butyrate uptake rate and sustained culture stability with high butanol productivity and yield by using a novel FBB and butyrate as a co-substrate with glucose should provide an economic and energy-efficient process for butanol production from corn.

Butyric Acid Production Form Glucose and Xylose

C. tyrobutyricum is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products (Zhu, et al., 2005). In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A non replicative integrational plasmid containing acetate kinase gene (ack) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the ack-deleted mutant (PAK-Em). This mutant was used in fermentations to produce butyric acid and hydrogen from glucose, xylose, and sugars (glucose and fructose) present in the waste grape juice from wine manufacturing. Free-cell fermentations at pH 6.0 and 37°C produced ~40 g/L of butyric acid with high yields of butyric acid (0.42 g/g) and hydrogen (~0.024 g/g) from sugars. To further improve the fermentation, a FBB was used to immobilize and adapt PAK-Em cells, which increased the butyric acid production to 50 g/L at pH 6.0 and 80 g/L at pH 6.3, which is the highest concentration ever produced in the fermentation. The butyric acid yield from glucose also increased to 0.45 g/g, mainly because of the reduced cell growth in the immobilized-cell fermentation. Compared to the wild type, PAK-EM produced much more butyric acid (0.42 g/g vs. 0.34 g/g) under the same fermentation conditions (pH 6.0, 37°C, glucose).

We have also started the functional genomics study of C. tyrobutyricum and have constructed the genomic library, developed microarray gene chips for transcriptomics study, and are working on proteomics as well.

Continuous Production of Butanol From Butyric Acid and Glucose

Continuous fermentation of glucose and butyrate by C. acetobutylicum in an FBB was carried out at 35°C, pH between 3.5 and 5.5, and dilution rate between 0.1 and 1.2 h-1 to study the effects of butyric acid concentration, pH, and dilution rate on butyrate uptake rate and butanol production. In general, increasing the butyrate concentration (up to 7.4 g/L) also increased butyrate uptake rate (to 2.4 g/L·h) and butanol production. High butanol productivity of ~5.6 g/L·h and yield of ~0.30 g/g glucose (~0.43 g/g for total solvents) were obtained at pH 4.3 and 0.6 h-1 (see Figure 1). The fermentation was stable and did not show any significant degeneration for the entire period of 2 months studied. We are continuing this study with the goal to adapt the organism to produce butanol at a higher concentration and productivity. This work has shown that doubling the yield of butanol to ~2.5 gal/bushel of corn (0.37 L/kg) in the conventional ABE fermentation can be achieved by converting carbohydrates into butyrate and then butanol, which can make fermentation -derived butanol economically competitive with petrochemically derived butanol. Compared to the conventional ABE fermentation with butanol yield of 0.20 g/g and productivity of 4.5 g/ L·h, the FBB notably enhanced the yield of butanol by more than 50 percent, making butanol production from renewable resources an attractive alternative to ethanol fermentation.

Product Yields From Glucose in a Continuous Butanol Fermentation Process With Glucose and Butyrate as the Substrates.

Figure 1. Product Yields From Glucose in a Continuous Butanol Fermentation Process With Glucose and Butyrate as the Substrates

Future Activities:

We will continue to work on both butanol fermentation and butyric acid fermentation by improving the cultures through in-process adaptation as well as genetic and metabolic engineering. Once we have reached our goals on the product yield and concentration, we will develop the proposed two-step process, evaluate its economic feasibility, and do process optimization and scale up. Currently, we are scaling up the bioreactor from the laboratory size (less than 1 liter) to about 100 gallons. We are also working on process optimization based on industrial feedstocks.


Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other subproject views: All 10 publications 4 publications in selected types All 4 journal articles
Other center views: All 211 publications 48 publications in selected types All 44 journal articles
Type Citation Sub Project Document Sources
Journal Article Liu X, Zhu Y, Yang S-T. Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production. Biotechnology Progress 2006;22(5):1265-1275. R829479 (2006)
R829479C016 (Final)
R829479C023 (2006)
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  • Journal Article Liu XG, Zhu Y, Yang S-T. Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mutants. Enzyme and Microbial Technology 2006;38(3-4):521-528. R829479 (2006)
    R829479C016 (Final)
    R829479C023 (2005)
    R829479C023 (2006)
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  • Journal Article Liu X, Yang S-T. Kinetics of butyric acid fermentation of glucose and xylose by Clostridium tyrobutyricum wild type and mutant. Process Biochemistry 2006;41(4):801-808. R829479C023 (2005)
    R829479C023 (2006)
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  • Journal Article Zhu Y, Liu XG, Yang ST. Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation. Biotechnology and Bioengineering 2005;90(2):154-166. R829479 (2006)
    R829479C016 (Final)
    R829479C023 (2005)
    R829479C023 (2006)
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  • Abstract: Wiley-Abstract
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  • Supplemental Keywords:

    Clostridium acetobutyricum, C. tyrobutyricum, butanol, butyric acid, fibrous bed bioreactor, gene inactivation, ack, pta,, RFA, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Environmental Chemistry, Sustainable Environment, Technology, Technology for Sustainable Environment, Biochemistry, waste vegetable oil, plant biomass sugars, agricultural byproducts, biomass, biotechnology, butanol, environmentally benign alternative

    Progress and Final Reports:

    Original Abstract
  • 2005 Progress Report
  • 2007
  • Final

  • Main Center Abstract and Reports:

    R829479    The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829479C001 Plant Genes and Agrobacterium T-DNA Integration
    R829479C002 Designing Promoters for Precision Targeting of Gene Expression
    R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
    R829479C005 Development of Novel Plastics From Agricultural Oils
    R829479C006 Conversion of Paper Sludge to Ethanol
    R829479C007 Enhanced Production of Biodegradable Plastics in Plants
    R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
    R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
    R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
    R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
    R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
    R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
    R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
    R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
    R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
    R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
    R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
    R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
    R829479C025 Chemical Induction of Disease Resistance in Trees
    R829479C026 Development of Herbicide-Tolerant Hardwoods
    R829479C027 Environmentally Superior Soybean Genome Development
    R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
    R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
    R829479C030 Engineered Plant Virus Proteins for Biotechnology