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

EPA Grant Number: R829479C016
Subproject: this is subproject number 016 , 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, 2002 through September 30, 2004
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation

Objective:

The goal of this research project was to develop a process for economical production of butyric acid from glucose and xylose. The specific goals included:

  1. developing a metabolically engineered Clostridium tyrobutyricum strain and
  2. using the cells immobilized in a fibrous bed bioreactor for fermentation to achieve high butyrate yields and to produce butyrate at a high concentration and high production rate.

Summary/Accomplishments (Outputs/Outcomes):

C. tyrobutyricum is a gram-positive, rod-shaped, spore-forming, obligate anaerobic bacterium capable of fermenting a wide variety of carbohydrates to butyric and acetic acids. There has been increasing interest in the production of butyric acid from agricultural commodities and processing wastes using C. tyrobutyricum. Butyric acid has many applications in chemical, food, and pharmaceutical industries. A conventional butyric acid fermentation process is not yet economically competitive because it produces butyric acid at a relatively low concentration, yield, and rate. To improve the economics of the fermentation process, butyrate production should increase while acetate production decreases, which also reduces the product separation cost.

In this research project, novel metabolic engineering approaches, at both the molecular biology and process engineering levels, were developed for enhanced butyric acid production by C. tyrobutyricum. Recombinant DNA technology was used to knock out genes in the acetate formation pathway and to overexpress genes in the butyrate formation pathway in mutant strains with improved butyrate production as compared to the wild-type strain. A novel fibrous-bed bioreactor (FBB) also was used for fermentation of xylose and glucose to produce butyrate with enhanced reactor productivity, product concentration, and yield. Cells in the FBB grew into high density and adapted to a higher butyrate concentration, which was not achievable in conventional fermentation systems.

To decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt genes associated with the acetate formation pathway in C. tyrobutyricum. The basic approach was to first amplify gene coding regions and then construct integrative plasmids for transformation into C. tyrobutyricum to inactivate the homologous genes on the chromosome. Cloning and sequencing of acetate kinase (ack) and phosphotransacetylase (pta) were carried out first. Based on known sequences of cloned ack and pta from several microorganisms in the genome database, partial coding regions of gene ack (560 bp) and pta (740 bp) have been polymerase chain reaction (PCR) amplified, cloned, and sequenced. The sequences from six different clones for each gene were analyzed (using molecular analysis tools DNASIS Max) to obtain the correct DNA sequence. The deduced protein sequence alignments were used to identify the conserved regions throughout the cloned sequences and to demonstrate the validity of these genes.

Inactivation of ack and pta and determining gene expression pattern were then carried out. Integrational plasmid technology was used to disrupt metabolic pathways leading to acetate formation in C. tyrobutyricum. Non-replicative plasmids pAK-Em and pPTA-Em were constructed to contain a DNA fragment of either ack or pta from the host C. tyrobutyricum and a genetic marker erythromycin-resistance gene (Emr) for which selection can be made. These plasmids did not carry the clostridial origin of replication, but they were established by integrating into the homologous region on the host chromosome resulting in disruption of the gene on the chromosome and the loss of function, producing a mutant phenotype. A negative control plasmid without the gene fragment was used to confirm the integration event by homologous recombination. After obtaining the mutants, they were studied for their fermentation kinetics and physiology in a standard stirred-tank fermentor with pH and temperature controls. The fermentation rate and product yield were monitored by taking samples at regular time intervals and analyzed with a high performance liquid chromatography (HPLC) following well-established protocol. Gas production, both carbon dioxide and hydrogen, also was monitored with a gas analyzer (respirometer), which was calibrated with a standard gas mixture and automated with a computer control and data acquisition system.

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

A nonreplicative integrational plasmid containing the 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. 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 phosphotransacetylase (PTA) and acetate kinase (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 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 pta gene, however, did not 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 gene inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum to improve butyric acid production from glucose.

Butyric Acid and Hydrogen Production by C. tyrobutyricum and Mutants

C. tyrobutyricum ATCC 25755 produces butyric acid, acetic acid, hydrogen, and carbon dioxide as its main fermentation products. In this work, mutants with inactivated pta gene, encoding PTA, and ack gene, encoding AK, were studied for their potential to improve butyric acid production in the fermentation. PTA and AK are two key enzymes in the acetate-producing pathway. PTA and AK activities in the pta-deleted mutant (PPTA-Em) were reduced by 44 percent and 91 percent, respectively, whereas AK activity in the ack-deleted mutant (PAK-Em) decreased by 50 percent. Meanwhile, the activity of butyrate kinase (BK) in PPTA-Em increased by 44 percent and hydrogenase activity in PAK-Em increased by 40 percent. As compared with the wild type, the specific growth rate of the mutants decreased by 32 percent (from 0.28 h-1 to 0.19 h-1) because of the impaired PTA-AK pathway. Meanwhile, butyric acid production by these mutants was improved greatly, with higher butyric acid yield (> 0.4 g/g vs. 0.34 g/g) and final concentration (43 g/L vs. 29 g/L), which also indicated that the mutants had better tolerance to butyric acid inhibition. Acetate production in the mutants, however, was not significantly reduced even though more butyrate was produced from glucose, suggesting the existence of an additional acetate forming pathway in C. tyrobutyricum. Hydrogen production by PAK-Em mutant also increased significantly, with higher hydrogen yield (2.61 vs. 1.35 mol/mol glucose) and H2/CO2 ratio (1.43 vs. 1.08). The SDS-PAGE also showed significantly different expression levels of proteins with molecular weights around 32 kDa and 70 kDa. These results suggested that integrational mutagenesis resulted in global metabolic shift and phenotypic changes, which also improved production of butyric acid and hydrogen from glucose in the fermentation. Table 1 summarizes the comparison between the mutants and wild type in fed-batch fermentations with glucose as the substrate.

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

 

PPTA-Em

 

PAK-Em

 

Cell Growth

Specific growth rate, µ (h-1 )

0.28±0.03

 

0.19±0.02

 

0.19±0.02

 

Cell biomass yield (g/g)

0.13

 

0.15

 

0.11

 

Acid Production

Final butyric acid conc. (g/L)

28.6

 

42.1

 

43.0

 

Butyric acid yield (g/g)

0.34±0.01

 

0.40±0.02

 

0.47±0.03

 

Final Acetic acid conc. (g/L)

9.7

 

12.2

 

11.9

 

Acetic acid yield (g/g)

0.12±0.01

 

0.13±0.01

 

0.12±0.01

 

Butyrate/Acetate ratio (g/g)

2.96

 

3.19

 

3.74

 

Gas Production

H 2 yield (g/g)

0.015

 

0.012

 

0.029

 

CO 2 yield (g/g)

0.305

 

0.251

 

0.446

 

H2/CO2 ratio (mol/mol)

1.08

 

1.06

 

1.43

 

Adaptation of C. tyrobutyricum for Enhanced Tolerance to Butyric Acid in a Fibrous-Bed Bioreactor

By immobilization in a FBB, we succeeded in adapting and selecting an acid-tolerant strain of C. tyrobutyricum that can produce high concentrations of butyrate from glucose and xylose. This mutant grew well under high butyrate concentrations (> 30 g/L) and had better fermentative ability as compared to the wild-type strain used to seed the bioreactor (see Table 2). Kinetic analysis of butyrate inhibition on cell growth, acid-forming enzymes, and ATPase activity showed that the adapted cells from the FBB are physiologically different from the original wild type. Compared to the wild type, the adapted culture’s maximum specific growth rate increased by 2.3-fold and its growth tolerance to butyrate inhibition increased by 29-fold. The key enzymes in the butyrate-forming pathway, phosphotransbutyrylase (PTB) and butyrate kinase (BK), also were more active in the mutant, with 175 percent higher PTB and 146 percent higher BK activities. The mutant’s ATPase also was less sensitive to inhibition by butyric acid, as indicated by a 4-fold increase in the inhibition rate constant, and was more resistant to the enzyme inhibitor N,N’-dicyclohexylcarbodiimide (DCCD). The lower ATPase sensitivity to butyrate inhibition might have contributed to the increased growth tolerance to butyrate inhibition, which also might be attributed to the higher percentage of saturated fatty acids in the membrane phospholipids (74% in the mutant vs. 69% in the wild type). This study shows that cell immobilization in the FBB provides an effective means for in-process adaptation and selection of a mutant with higher tolerance to inhibitory fermentation product.

Table 2. Comparison Between Free-Cell and Immobilized-Cell Fermentations at pH 6.0, 37°C
Substrate Free-Cell Fermentation Immobilized-Cell Fermentation
Glucose Xylose Glucose Xylose
Specific growth rate (h-1) 0.063 ± 0.004 0.055 ± 0.004 0.094 ± 0.008 0.057 ± 0.002
Butyric acid
Final concentration (g/L) 16.3 19.2 43.4 37.3
Yield (g/g) 0.34 ± 0.01 0.17 – 0.47 0.42 ± 0.02 0.28 – 0.54
Productivity (g/L_h) 1 0.19 ± 0.08 0.14 – 0.27 6.77 ± 0.23 1.45 – 3.3
Acetic acid
Final concentration (g/L) 3.6 0.7 8.4 4.9
Yield (g/g) ~0.06 ~0.02 ~0.095 0.06 – 0.11

1Productivity for immobilized-cell fermentation was based on the fibrous bed bioreactor volume of 400 mL, instead of the total liquid medium volume of 2 L in the reactor system.

Butyric Acid and Hydrogen Production by ack-Deleted Mutant of C. tyrobutyricum Immobilized in a FBB

In this work, one metabolic engineered mutant with an inactivated ack gene, encoding AK associated with the acetate formation, was created by integrational mutagenesis to improve butyric acid and hydrogen production from fermentation. The ack fragment cloned from C. tyrobutyricum was used to construct the integrational plasmid pAK-Em containing an erythromycin-resistance cassette. The nonreplicative pAK-Em was introduced into C. tyrobutyricum by electroporation, inactivated the target ack gene by homologous recombination on the chromosome, and produced the PAK-Em. The enzyme assay showed that the AK activity in PAK-Em decreased by approximately 50 percent, meanwhile, the PTA and hydrogenase activities increased by approximately 40 percent, respectively. Fed-batch fermentations with free cells of the mutant were carried out at pH 6.0 and 37 oC to evaluate its ability to produce butyric acid and hydrogen from glucose and xylose. As compared with the wild type, the specific growth rate of the mutant from glucose decreased by 42 percent, from 0.24 h-1 to 0.14 h-1, caused by the impaired PTA-AK pathway. The butyric acid production by the mutant, however, was improved greatly with higher butyric acid yield 0.42 g/g and final concentration 41.65 g/L, which suggested higher tolerance to butyric acid inhibition. Hydrogen production by PAK-Em mutant also increased significantly, with higher hydrogen yield 0.024 g/g glucose and H2/CO2 ratio 1.44. The butyric acid and hydrogen production were similar for glucose and xylose.

Fed-batch fermentations were then carried out with cells immobilized in FBBs to further evaluate their abilities to produce butyric acid and hydrogen from various sugar sources, including glucose, xylose, and fructose. Through adaptation in the FBB, a high butyric acid concentration of 82 g/L was obtained at pH 6.3 with PAK-Em. This concentration is the highest ever attained to date in butyric acid fermentation. The butyrate yield also was increased to approximately 0.5 g/g caused by reduced cell growth in the immobilized-cell fermentation. A new mutant that produced even more hydrogen with a H2/CO2 ratio of approximately 2 also was discovered from the FBB adaptation. Metabolic flux analysis showed that the global metabolic flux distributions were altered in these mutants. These results suggested that further enhancements in butyric acid and hydrogen production from sugar wastes by C. tyrobutyricum can be achieved by metabolic engineering via integrational mutagenesis and cell adaptation in the FBB.

Conclusions:

Butyric acid has wide applications in food and pharmaceutical industries. Its production by fermentation from natural resources has become an increasingly attractive alternative to the petroleum-based production route currently used in the chemical industry. This study demonstrated that both genetic engineering and culture adaptation in the FBB can be used to develop mutant strains of C. tyrobutyricum and improve the process economics for butyrate fermentation. The adapted culture from the FBB is different physiologically from the original culture used to seed the bioreactor. This important finding never has been reported in conventional fermentation systems. The high productivity and high product concentration obtained in the FBB are either comparable or better than those reported in the literature. The manipulation of acid-forming pathways by gene inactivation and overexpression proved to be feasible for obtaining metabolically advantageous mutants for butyrate production from sugars. The fermentation kinetic studies of these mutants also provided valuable information about gene function in cellular metabolism, which can guide future effort to engineer novel super-producing strains of C. tyrobutyricum for industrial applications. The increased productivity and selectivity by mutant strains of C. tyrobutyricum immobilized in the FBB should lower the cost of bio-based butyrate and allow it to compete favorably in the market.


Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other subproject views: All 13 publications 6 publications in selected types All 5 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 Zhu Y, Yang S-T. Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum. Journal of Biotechnology 2004;110(2):143-157. R829479 (2006)
    R829479C016 (Final)
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  • Journal Article Zhu Y, Yang S-T. Adaptation of Clostridium tyrobutyricum for enhanced tolerance to butyric acid in a fibrous-bed bioreactor. Biotechnology Progress 2003;19(2):365-372. R829479 (2006)
    R829479C016 (Final)
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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:

    clostridium Clostridium tyrobutyricum, butyric acid, acetic acid, hydrogen, gene inactivation, ack, pta, metabolic engineering, fibrous bed bioreactor , sustainable industry, innovative technology, bioeng ineering, biotechnology, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Genetics, Geochemistry, Technology, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, agrobacterium, fermentation process, bioengineering, transgenic plants, biomass crop plants, plant genes, biotechnology, remediation, plant biotechnology, butanol production

    Relevant Websites:

    http://www.cpbr.org exit EPA

    Progress and Final Reports:

    Original Abstract
  • 2003

  • 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