2005 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, 2004 through September 30, 2005
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:

This project is a follow-on of R829479C016. The objective of this research 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 covert glucose and xylose to butyric acid first, and then the produced butyric acid will be converted to butanol by Clostridium 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 objectives are to: (1) study butanol production from butyric acid and glucose by C. acetobutylicum immobilized in a FBB; (2) develop a process for enhanced butyric acid production from glucose and xylose; and (3) develop and demonstrate the proposed two-step extractive fermentation process for producing butanol from glucose and xylose.

Progress Summary:

The first task of this project is to study butanol production from butyric acid and glucose by C. acetobutylicum immobilized in a FBB, with the following specific aims: (1) obtain a mutant that can tolerate a high butanol concentration (> 20 g/L); (2) evaluate operating parameters that will allow stable butyrate uptake by cells for butanol production; and (3) evaluate and optimize butanol yield from butyrate and glucose. The accomplishments to date are summarized below.

Continuous Production of Butanol by Clostridium acetobutylicum Immobilized in a Fibrous Bed Bioreactor

An investigation was undertaken to explore the influence of dilution rate and pH in continuous cultures of C. acetobutylicum. A 200-mL FBB was used to produce high cell density and butyrate concentrations at pH 5.4 and 35°C. By feeding glucose and butyrate as a co-substrate, the fermentation was maintained in the solventogenesis phase, and the optimal butanol productivity of 4.6 g L-1 hr-1 and yield of 0.42 g g-1 were obtained at the dilution rate of 0.9 hr-1 and pH 4.3. Compared to the conventional ABE fermentation, the new fermentation process greatly improved butanol yield, making butanol production from corn an attractive alternative to ethanol fermentation.

Fermentation processes using anaerobic microorganisms provide a promising path for converting biomass and agricultural wastes into chemicals and fuels. ABC fermentation with the strict anaerobic bacterium, C. acetobutylicum, was once (1917-1955) one of the largest fermentation processes ever developed in industry. With a few exceptions, however, anaerobic fermentation processes for production of fuels and chemicals, including ABE fermentation, usually suffer from a number of serious limitations including low yields, low productivity, and low final product concentrations. It is unlikely that the fermentation route will become competitive with petroleum-based solvent synthesis unless some of these limitations can be overcome. United States legislation to produce strategic chemicals, fuels, and energy from domestic renewable resources and the need to lessen the dependence on the diminishing petroleum supplies, however, have resulted in the renaissance of the fermentation process as a possible source of solvents.

Butanol has many characteristics that make it a better fuel extender than ethanol, now used in the formulation of gasohol. It can solve many problems associated with the use of ethanol. Butanol has the following advantages over ethanol: (1) butanol has 25 percent more Btu per gallon; (2) butanol is less evaporative/explosive, with a Reid vapor pressure (RVP) 7.5 times lower than ethanol; (3) butanol is safer than ethanol because of its higher flash point and lower vapor pressure; (4) butanol has a higher octane rating; and (5) butanol is more miscible with gasoline and diesel fuel but less miscible with water. Petroleum-derived butanol currently is used in food and cosmetic industries as an extractant, but there are concerns about its carcinogenic aspects associated with the residual petroleum components. Many new uses will occur in these fields as “green” butanol becomes available to the market. Other uses include current industrial applications in solvent, rubber monomers, and break fluids. Butanol has the propensity to solve some infrastructure problems associated with fuel cell use. Dispersed through existing pipelines and filling stations and then reformed onboard the fuel cell vehicle, butanol offers a safer fuel with more hydrogen.

The present research on butanol fermentation has been focused primarily on the effects of pH and dilution rate (D) in continuous cultures of the mutant strain from C. acetobutylicum ATCC 55025. To overcome the problems of low productivity and yield of butanol, cell immobilization in a convoluted FBB and feeding with dextrose and butyric acid as co-substrates to produce butanol and reduce production of ancillary byproducts were used in the fermentation. By changing the dilution rate from 0.1 hr-1 to 1.2 hr-1 at pH 4.3 and varying the pH from 3.5 to 5.5 at the dilution rate of 0.6 hr-1, the optimal conditions for high productivity and butanol yield were investigated.

This work has shown that doubling the yield of butanol to approximately 2.5 gallons/bushel of corn (0.37 L/kg) in the conventional ABE fermentation can be achieved by converting carbohydrates into mainly butanol, which can make fermentation-derived butanol economically competitive with petrochemically derived butanol. Compared to the conventional ABE fermentation (the optimum butanol yield of 0.25 g g-1 and productivity of 4.5 g L-1 hr-1), the FBB notably enhanced the yield of butanol and ABE (more than 68% and 20%, respectively) by C. acetobutylicum, making butanol production from renewable resources an attractive alternative to ethanol fermentation. Commercialization of this new technology has the propensity to reduce our nation’s dependence on foreign oil, protect our fuel generation grid from sudden disruption, develop our agricultural base, solve the hydrogen supply problem associated with fuel cells, and help reduce global warming.

The second task of this project is to develop a process for enhanced butyric acid production from glucose and xylose. The specific aims include: (1) developing a metabolically engineered C. tyrobutyricum strain; and (2) using the cells immobilized in a fibrous bed bioreactor for fermentation to achieve high butyrate yields (> 50%) and to produce butyrate at a high concentration (> 50 g/L) and high production rate (> 5 g/L/h). The accomplishments in this task are briefly summarized below.

Construction and Characterization of pta Gene Deleted Mutant of Clostridium tyrobutyricum for Enhanced Butyric Acid Fermentation

C. tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. 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 phosphotransacetylase gene (pta) 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 pta-deleted mutant (PPTA-Em), which was confirmed by Southern hybridization. Sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional protein electrophoresis results indicated that protein expressions were changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and AK in the mutant were reduced by more than 60 percent for PTA and 80 percent for AK. The mutant grew slower in batch fermentation with glucose as the substrate, but produced 15 percent more butyrate and 14 percent less acetate as compared to the wild type strain. Its butyrate productivity was approximately two-fold higher than the wild type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition and the final butyrate concentration was improved by 68 percent. Inactivation of the pta gene did not, however, completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first reported genetic engineering study demonstrating the feasibility of using a gene inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum to improve butyric acid production from glucose.

Kinetics of Butyric Acid Fermentation of Glucose and Xylose by Clostridium tyrobutyricum Wild Type and Mutant

The kinetics of butyric acid fermentation by C. tyrobutyricum at pH 6.0 and 37°C were researched with the wild-type ATCC 25755 and its mutant PPTA-Em, which was obtained from integrational mutagenesis to inactivate the chromosomal pta gene, encoding phosphotransacetylase (PTA). The potential of using this mutant to improve butyric acid production from glucose and xylose was evaluated in both free and immobilized cell fermentations. The results are summarized in Tables 1 and 2. Compared to the wild type, in freecell fermentations PPTA-Em produced 15 percent more butyrate (0.38 g/g versus 0.33 g/g) from both glucose and xylose, at much higher concentrations (37.2 g/L versus 22.9 g/L from glucose and 33.5 g/L versus 19.4 g/L from xylose). The increased butyrate production in the mutant can be attributed to the reduced acetate production as well as reduced specific growth rate. The acetate yield in the mutant was reduced by 13.5 percent (0.058 g/g versus 0.067 g/g) and 32 percent (0.045 g/g versus 0.066 g/g) from glucose and xylose, respectively. The mutant’s specific growth rate was reduced by 36 percent (0.137 hr-1 versus 0.214 hr-1) on glucose and 26 percent (0.086 hr-1 versus 0.116 hr-1) on xylose. A FBB was used to immobilize PPTA-Em mutant cells and further improve butyric acid production. The final butyric acid concentrations in fed-batch fermentations reached 49.9 g/L from glucose and 51.6 g/L from xylose, with the butyrate yield increased to 0.44 g/g glucose and 0.45 g/g xylose. As evidenced by the greatly increased butyrate/acetate ratio in the final product profile, it is concluded that the mutant’s metabolic pathway has been shifted to favor butyrate production due to the knock-out of the pta gene even though acetate production remains at a significant level. The observed metabolic shift is corroborated by the changed protein expression patterns as seen in two-dimensional protein electrophoresis and SDS-PAGE.

Table 1. Comparison of Fermentations of Glucose by C. tyrobutyricum Wild Type and PPTA- Em at 37°C, pH 6.0

 

Wild type

 

PPTA-Em

Free cell

 

Free cell

 

Immobilized cell

Cell Growth

 

 

 

 

 

 

Specific growth rate (hr-1)

 

0.214 ± 0.044

 

0.137 ± 0.032

 

0.095 ± 0.036

Biomass yield (g/g)

 

0.109 ± 0.019

 

0.141 ± 0.024

 

0.087 ± 0.008

Acid Production

 

 

 

 

 

 

Butyric acid concentration (g/L)

 

22.90 ± 4.06

 

37.22 ± 4.80

 

49.85 ± 0.51

Butyric acid yield (g/g)

 

0.33 ± 0.01

 

0.38 ± 0.01

 

0.44 ± 0.01

Acetic acid concentration (g/L)

 

4.15 ± 0.59

 

4.19 ± 0.013

 

8.74 ± 0.63

Acetic acid yield (g/g)

 

0.067 ± 0.012

 

0.058 ± 0.004

 

0.081 ± 0.005

Butyrate/Acetate ratio (g/g)

 

5.52

 

8.88

 

5.70

Gas Production

 

 

 

 

 

 

H2 yield (g/g)

 

0.017 ± 0.002

 

0.018 ± 0.001

 

0.016 ± 0.001

CO2 yield (g/g)

 

0.360 ± 0.035

 

0.389 ± 0.027

 

0.388 ± 0.005

H2/CO2 ratio (mol/mol)

 

1.05 ± 0.03

 

1.05 ± 0.03

 

0.93 ± 0.02

Note: Average ± standard deviation were calculated from two (wild type) or three (natural) batch fermentations.

Table 2. Comparison of Fermentations of Xylose by C. tyrobutyricum Wild Type and PPTA- Em at 37°C, pH 6.0

 

Wild type

 

PPTA-Em

Free cell

 

Free cell

 

Immobilized cell

Cell Growth

 

 

 

 

 

 

Specific growth rate (hr-1)

 

0.116 ± 0.009

 

0.086 ± 0.020

 

0.048 ± 0.006

Biomass yield (g/g)

 

0.095 ± 0.003

 

0.109 ± 0.013

 

0.069 ± 0.003

Acid Production

 

 

 

 

 

 

Butyric acid concentration (g/L)

 

19.42 ± 1.195

 

33.49 ± 2.89

 

51.546 ± 3.49

Butyric acid yield (g/g)

 

0.33 ± 0.02

 

0.38 ± 0.02

 

0.45 ± 0.02

Acetic acid concentration (g/L)

 

3.31 ± 0.01

 

3.89 ± 0.26

 

6.42 ± 1.60

Acetic acid yield (g/g)

 

0.066 ± 0.006

 

0.045 ± 0.001

 

0.045 ± 0.016

Butyrate/Acetate ratio (g/g)

 

5.87

 

8.61

 

8.02

Gas Production

 

 

 

 

 

 

H2 yield (g/g)

 

0.017 ± 0.001

 

0.017 ± 0.001

 

0.015 ± 0.001

CO2 yield (g/g)

 

0.365 ± 0.001

 

0.373 ± 0.031

 

0.348 ± 0.004

H2/CO2 ratio (mol/mol)

 

1.07 ± 0.02

 

1.05 ± 0.02

 

0.96 ± 0.01

Note: Average ± standard deviation were calculated from two (wild type) or three (mutant) batch fermentations.

In summary, this work was the first genetic engineering study of C. tyrobutyricum for enhanced butyric acid fermentation. In this work, the cloning procedures for C. tyrobutyricum were optimized and gene inactivation experiments were carried out to develop mutant strains of C. tyrobutyricum for butyrate production from glucose with improved productivity, yield, final product concentration, and butyrate tolerance. The manipulation of acid-forming pathways by gene inactivation proved to be feasible for obtaining metabolically advantageous mutants for butyrate production from glucose. Gene manipulations in the metabolic pathway can, however, lead to unexpected changes in protein expression pattern and other phenotypes that require further studies.

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 begin to do process optimization and scale up.


Journal Articles on this Report : 3 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 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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  • Supplemental Keywords:

    sustainable industry, waste, agricultural engineering, bioremediation, environmental engineering, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology, bioremediation, carcinogen, contamination, phytodegradation, pollutant, toxicity, heavy metal, environmental cleanup, 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

    Relevant Websites:

    http://www.cpbr.org Exit

    Progress and Final Reports:

    Original Abstract
  • 2006 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