Recipient Organization
OHIO STATE UNIVERSITY
PLANT BIOTECHNOLOGY CENTER
COLUMBUS,OH 43210
Performing Department
Chemical and Biomolecular Eng
Non Technical Summary
The goal of this project is to produce advanced biofuel and specialty chemicals (n-butanol, butyrate, and butyl butyrate) from agricultural residues in an integrated bioprocess. Butanol is an industrial solvent and also an attractive biofuel with superior fuel properties (higher energy density, lower volatility, etc.) than ethanol. Butyric acid is a specialty chemical widely used in food, chemical and pharmaceutical industries. The use of butyrate in animal feed to replace antibiotics is also expanding rapidly in the animal feed market. Both butyric acid and butanol can be produced biologically from biomass, which, however, is not economically viable because conventional fermentation processes are limited by severe product inhibition, low product yield and titer, and difficulty in product recovery and purification.To solve these problems, we have engineered Clostridium tyrobutyricum for butyrate and butanol production from glucose and xylose present in lignocellulosic biomass hydrolysates. Also, adaptive evolutionary engineering was applied to increase cell tolerance to toxic compounds present in the fermentation broth. In this project, fermentation processes will be optimized and integrated with in situ product separation to alleviate product inhibition and further increase productivity and yield. In addition, we will also develop a novel immobilized enzyme reactor and an extractive fermentation-esterification process for converting fermentation-produced butyric acid and butanol to butyl butyrate, a higher-value flavoring compound used in the food industry. It can also be used as a biofuel and oxygenator to enrich biodiesel. The engineered C. tyrobutyricum can produce butyric acid and butanol at high titers, yields, and productivities for economical production of these chemicals, and are superior to conventional solventogenic clostridia widely used in the ABE fermentation. Also, in situ esterification with exogenous lipase in extractive fermentation can further increase reaction rate and conversion due to the removal of inhibitory products. These process concepts have not been investigated for industrial applications before. By applying these innovative technologies, butyric acid, n-butanol, and butyl butyrate can be produced economically in an integrated biorefinery.
Animal Health Component
70%
Research Effort Categories
Basic
20%
Applied
70%
Developmental
10%
Goals / Objectives
To achieve our goal of developing an economical process for butyl butyrate production from abundant low-cost agricultural residues, such as corn stover, we will pursue the specific objectives in the following areas to be performed by research partners at OSU and SIUE:A. Engineering C. tyrobutyricum for butyric acid and n-butanol production (OSU)Metabolic and evolutionary engineering will be used to develop robust strains that can efficiently produce butyric acid and butanol from both glucose and xylose present in lignocellulose hydrolysates. The study initially will focus on the overexpression of known genes in the butyric acid and butanol biosynthesis pathways (A.1) and the heat shock proteins that may enhance stress tolerance (A.2), as well as knockout of the Spot0A and/or orphan histidine kinase genes that regulate sporulation (A.3). Effects of these gene manipulations will be evaluated in fermentation kinetics studies (A.4) and the results will be used to identify targeted genes for knock-in/knock-out on the genome to create stable mutant strains (A5) for use in long-term continuous fermentation (see Task C).B. Cloning and production of lipase for esterification (SIUE)We will clone and express several known lipases in P. pastoris (B.1). These lipases will be characterized for their reaction rate, specificity, and stability under different pHs and temperatures (B.2). The best lipase will be used for esterification of butyric acid and butanol to produce butyl butyrate in a bioreactor with simultaneous solvent extraction in a two-phase system (B.3) as well as with a membrane extractor (B.4). Finally, selected lipase gene will also be cloned and expressed in C. tyrobutyricum (B.5), which will be used for simultaneous fermentation and esterification without adding an exogenous lipase (see Task C).C. Extractive fermentation-esterification process development and analysis (OSU & SIUE)The integrated process including fermentation, esterification, and extraction for butyl butyrate production from hydrolysate sugars will be optimized at a bench scale. The studies include preparing hydrolysate sugars from corn stover with acid pretreatment and enzymatic hydrolysis using commercial cellulases (C.1), process integration and optimization (C.2), techno-economic analysis (TEA) and life cycle analysis (LCA) to evaluate the feasibility and advantages of the proposed technology (C.3). The results will be used to guide further process optimization, scale up, and commercial development with potential commercialization partners.
Project Methods
A. Engineering C. tyrobutyricum for butyric acid and n-butanol production A.1 Overexpressing genes in butyric acid and butanol synthesis pathwaysWe will clone fnr from C. acetobutylicum into C. tyrobutyricum CtΔack-adhE2 to form CtΔack-adhE2-fnr and replace adhE2 with separate ald and adh genes to form CtΔack-ald-adh-fnr, which will be evaluated for their ability to produce butanol from glucose in fermentation kinetics studies. These mutant strains should have enhanced butanol biosynthesis from butyryl-CoA via the ALD-ADH pathway and a higher butanol yield of >0.35 g/g glucose.A.2 Overexpressing heat shock proteins to enhance stress toleranceTo increase stress tolerance to hydrolysate inhibitors and inhibitory metabolites (butyric acid and butanol), class I heat shock proteins (including dnaJK, grpE, groESL, and htpG) will be introduced in C. tyrobutyricum to improve stress tolerance to butanol and hydrolysate inhibitors, which should enhance butanol/butyrate production from lignocellulose hydrolysates.A.3 Knockout Spot0A and histidine kinase genes to down-regulate sporulationThe strain CtΔack-adhE2 will be engineered to knock out/down spo0A involved in regulating sporulation and orphan histidine kinases involved in the phosphorylation of Spo0A, the global regulator in clostridia, to enhance its robustness.A.4 Fermentation kinetics and gene manipulation effectsFermentation kinetics will be studied in serum bottles and stirred-tank bioreactors with pH control to evaluate the effects of gene over-expressions and knockouts in C. tyrobutyricum. Metabolic flux analysis (MFA) will be used to evaluate carbon flux distributions based on well-established model of stoichiometric equations. High performance liquid chromatography (HPLC) will be used to analyze organic compounds, including acetate, butyrate, ethanol, butanol, and sugars (glucose and xylose) present in the fermentation broth, and gas chromatography (GC) will be used to analyze volatile compounds in the fermentation broth. Gene expression levels will be analyzed with RT-PCR as well as enzyme activity assays.A.5 Genome engineering to create stable/robust mutant strainsOnce the beneficial effects of gene over-expression/knockout are confirmed in A.4, the target genes will be knocked in or out of the genome using established CRISPR-Cas9 systems to establish genetically stable strains for process studies in Task C.B. Cloning and production of lipase for esterificationB.1 Production of lipases in recombinant P. pastorisThe expression of heterologous lipase in P. pastoris will be optimized by engineering the expression cassettes, including screening different promoters and optimizing gene-copy number. The lipase production will be further improved by host engineering, such as improving secretion signal related to co-translational translocation. Then, a suitable fermentation strategy such as fed batch will be developed for the yeast fermentation. The effects of temperature, pH, feeding time on lipase production will be studied. The fermentation will be optimized, and the kinetics data will be used to scale up the fermentation process.Enzyme immobilization. Lipase in the fermentation broth will be partially purified by fractional precipitation and then immobilized on cotton cloth, which not only can improve the stability of biocatalyst, but also ease separation of lipase from reaction mixtures. The immobilization method will be optimized and scaled up. Factors to be optimized include the enzyme loading rate and PEI concentration and pH to be used in the process. The reactor packing method will also be optimized to increase the reaction (mass transfer) efficiency and minimize pressure drop.B.2 Characterization of lipaseLipases obtained from recombinant P. pastoris fermentation will be characterized for their catalytic activities in both forward (esterification) and reverse (hydrolysis) directions, substrate affinity or specificity (ethanol vs. butanol; acetate vs. butyrate), and thermal stability under different pHs (4-7) and temperatures (25-60oC). The effects of trace amounts of water on the enzyme activity and reaction equilibrium will also be evaluated in an organic solvent containing various amounts of water.B.3 Esterification in a two-phase systemEsterification of butanol and butyric acid to butyl butyrate will be studied in an aqueous-organic two-phase system with lipase present in the aqueous phase. The organic phase (solvent) with a high partition coefficient or selectivity for butyl butyrate will be used to facilitate the esterification reaction to attain a high conversion. Three solvents (hexadecane, dodecane, and decane) will be evaluated. The reaction kinetics and distribution of butyl butyrate, butanol and butyric acid in the two-phase system will be studied to select the best solvent.B.4 Esterification with simultaneous solvent extractionEsterification with simultaneous solvent extraction will be carried out with a hollow-fiber membrane extractor (HFME). The HFME provides intimate contacts between the two phases within the membrane pores to facilitate efficient extraction without mixing the two phases. The esterification reaction kinetics and extraction performance will be studied.B.5 Cloning and expressing lipase in C. tyrobutyricumThe best lipase for esterification identified in B.2 will be cloned and expressed in C. tyrobutyricum. To optimize the gene expression, the lipase gene will be codon-optimized to fit with the low G/C content of the host cells. In addition, the original ribosome binding site will be replaced with the consensus sequence "AGGAGG" to optimize gene expression in C. tyrobutyricum. The lipase-producing mutant strain will be tested for simultaneous fermentation and esterification.C. Extractive fermentation-esterification process development and analysis The integrated bioprocess consisting of extractive fermentation and esterification will be studied and demonstrated at a bench scale following the successful completion of Tasks A and B. Data obtained will be used in TEA and LCA, which will guide final process design and optimal production strategy for the multi-product biorefinery.C.1 Butanol and butyric acid production from corn stover hydrolysateWe will first demonstrate the feasibility of producing butyric acid and butanol from corn stover hydrolysate sugars using the engineered C. tyrobutyricum. Corn stover will be pretreated with dilute acid followed with hydrolysis with commercially available cellulases. Corn steep liquor (CSL) will be used to supplement the hydrolysate for additional nutrients needed in the fermentation. The fermentation will be carried out with either cell recycle or immobilization in a fibrous-bed bioreactor (FBB) to reach a high viable cell density and productivity.C.2 Process integration and optimizationA bench-scale demonstration model consisting of a fibrous-bed bioreactor for fermentation, a lipase reactor for esterification, and a HFME for solvent extraction will be designed based on the data obtained in Tasks A and B and tested initially with a synthetic medium containing glucose and xylose at a 3:1 ratio and then with corn stover hydrolysate. We will optimize the feed and recirculation rates and operate the system continuously for at least two weeks to check the process performance and stability under each condition studied.C.3 Techno-economic and life cycle analyses A techno-economic analysis (TEA) for butyl butyrate production from corn stover using the proposed process will be performed. Process economics will be analyzed using Aspen Plus. Costs for feedstock and chemical, capital equipment, energy, labor etc. will be based on market prices and data in NREL reports and our previously published economic analysis. Process scale-up and optimization issues will be identified for future commercial development consideration.