Recipient Organization
BIO-MISSIONS LLC
7666 DEER PARK WAY
REYNOLDSBURG,OH 43068
Performing Department
(N/A)
Non Technical Summary
This project is to develop a novel bioprocess to produce chemicals and fuels from low-cost agricultural residues such as corn stalk economically and sustainably with no CO2 emission. We aim to produce n-butanol, an industrial solvent and advanced biofuel, from agricultural residues and fermentation waste gases (CO2 and H2) in an integrated bioprocess using engineered clostridia in a "Linear Immobilized Bioreactor" (LIBR) with in-situ gas stripping for butanol recovery. Biofuel production has been limited by low product yield, productivity, and titer; whereas biorefinery using lignocellulosic feedstocks suffers from high capital and operational costs associated with pretreatments and cellulose hydrolysis, which also generate inhibitors negatively affecting fermentation performance. We have engineered a clostridia strain as a superior cell factory that not only can produce n-butanol from both glucose and xylose simultaneously with high yield and productivity, but also has a high tolerance to hydrolysate inhibitors and can use biomass hydrolysates directly without detoxification. This novel cell factory is robust for butanol production but has not been used in industrial fermentation. In addition, a novel mixotrophic fermentation with acetogen to reassimilate formate derived from CO2 released from sugars will also be developed to further increase butanol production. Phase I studies will assess the feasibility of the proposed bioprocess involving two key fermentation steps: 1) mixotrophic fermentation for converting glucose and formate to acetate with the co-cultures of a homolactic acid bacterium and acetogen, and 2) butanol production from glucose, xylose, and acetate by engineered clostridia immobilized in LIBR with in-situ gas stripping. Fermentation kinetics and process performance data (titer, rate, and yield) will be collected and used in techno-economic analysis (TEA) of the integrated bioprocess, which will also include biomass pretreatment/hydrolysis and pervaporation for final product purification. The proposed fermentation can achieve a 50% increase in butanol yield, >10% (w/v) product titer for energy efficient purification, and substantial cost reduction to less than $2.5/gal, competitive for applications as industrial solvent and advanced biofuel.Biobutanol is an advanced liquid fuel with an enormous potential to compete with ethanol if its production cost can be reduced to less than $2.5/gal by using low-cost, renewable agricultural residues such as corn stalk. The proposed process can produce n-butanol at a high yield of ~0.5 g/g from lignocellulose sugars (glucose and xylose). The proposed technology thus can provide an economically competitive and superior biofuel to replace ethanol for blending in gasoline. This advanced biobutanol process can be readily adopted by the biofuels industry by retrofitting or adding onto existing bioethanol and/or corn-based acetone-butanol-ethanol (ABE) fermentation plants. It will thus enhance the economic viability of the rural area, where substantial amounts of lignocellulosic wastes are generated, and reduce the burden from waste biomass disposal. Successfully developing the proposed biobutanol fermentation technology will also satisfy the public interests, especially in providing a safe, renewable energy, protecting natural resources and the environment, and enhancing energy security, economic opportunity and quality of life.
Animal Health Component
70%
Research Effort Categories
Basic
20%
Applied
70%
Developmental
10%
Goals / Objectives
This project in Phase I will study and demonstrate the technical feasibility of the proposed integrated bioprocess involving the following two key fermentation steps:Mixotrophic fermentation for converting glucose and formate to acetate with the co-cultures of LAB and acetogenButanol production from glucose, xylose, and acetate by engineered C. tyrobutyricum immobilized in LIBR with in-situ gas stripping for product recoveryFermentation kinetics and process performance data (titer, rate, and yield) will be collected and used in techno-economic analysis (TEA) of the integrated bioprocess, which will also include biomass pretreatment/hydrolysis and pervaporation for final product purification, to confirm that biobutanol can be produced from lignocellulose at the competitive cost of $2.5/gal.
Project Methods
A. Mixotrophic fermentation for acetate production from formate and glucose The mixotrophic fermentation with an acetogen (to be selected from C. formicoaceticum, A. woodii, C. ljungdahlii, etc.) will be evaluated in serum bottles/flasks and then followed with more detailed studies with controlled parameters (pH, Temperature, etc.) in stirred-tank bioreactors to find the optimal conditions with maximum carbon conversion and acetate production rates. Initially, media with different lactate?to?formate molar ratios between 0.5 and 2.0 will be screened for their effects on cell growth and acetate production and to verify our hypothesis that formate and lactate can be effectively converted to acetate at a high yield of >0.9 g/g substrate. The results will be used to guide the design and optimization of cocultured fermentation with homolactic acid bacteria (e.g., L. lactis) with glucose and formate as co-substrates. The coculture will be evaluated for their interactions at different population ratios in the inoculum in batch fermentation under various culture conditions (pH, substrate concentration, nutrient level, etc.). Substrate (glucose and formate) consumption, production (acetate) production, and total cell density will be monitored using established HPLC and cell dry weight methods. The interactions between LAB and acetogen and their effects on community (commensal and symbiotic) growth under optimized conditions should enhance acetate production from glucose and formate to achieve a high yield of >0.9 g/g substrate.B. Butanol production from glucose/xylose and acetate by C. tyrobutyricumVarious engineered C. tyrobutyricum strains overexpressing adhE2 (selected from WT-adhE2, Δcat1::adhE2, Δack-adhE2, and Δack-adhE2-hbd) will be studied for their fermentation kinetics and performance in fermentation with glucose/xylose and acetate as co-substrates. Our prior studies showed that both butyrate and butanol were produced at considerable amounts for strains WT-adhE2 and Δack-adhE2, whereas the strain Δcat1::adhE2 with cat1 knockout produced little butyrate but considerable amounts of acetate and butanol. The strain Δack-adhE2-hbd co-expressing adhE2 and hbd produced the most butanol with little acetate and only a moderate amount of butyrate, because overexpressing hbd in the strain increased its intracellular NAD(P)H pool favoring butanol biosynthesis over acids production. Butanol production also increased significantly with acids production decreased in the presence of an artificial electron carrier such as methyl viologen (MV) for all four strains (butanol yield >0.32 g/g glucose; OSU confidential data). It should be noted that cat1 knockout in the strain Δcat1::adhE2 disabled its ability to reassimilate acetate. Also, strains co-expressing a heterologous aldehyde oxidoreductase (AOR) were able to convert acetate and butyrate to corresponding aldehydes and then to alcohols via alcohol dehydrogenase (ALD). We expect that the best strain would convert both glucose and acetate to butanol at a yield of >0.4 g/g under optimal conditions.Continuous butanol production from lignocellulose hydrolysates in LIBRThe selected (best) strain from the afore-mentioned kinetics/process screening study will be further evaluated with lignocellulose hydrolysates containing glucose, xylose, and acetate in (semi-)continuous fermentation carried out in an FBB. We will demonstrate/evaluate the feasibility of producing butanol from corn stover hydrolysate sugars and acetate produced by acetogen in the mixotrophic fermentation (Task A). Corn stover will be pretreated with dilute acid or alkali followed with hydrolysis with commercially available cellulases. Non-detoxified hydrolysates will be used as already demonstrated in our previous studies.Corn steep liquor (CSL) will be used to supplement the hydrolysate for additional nutrients needed in the fermentation. We expect that cells immobilized in the fibrous bed bioreactor will reach a high cell density of >25 g/L.Once the reactor has reached a high cell density and pseudo-steady state, gas sparging from the bottom of the LIBR will be studied for its effects on butanol production and recovery from the fermentation broth. With gas stripping, we expect to reach a productivity >2 g/L?h and butanol titer >150 g/L in the condensate collected in the product tank.C. Techno-economic and life cycle analysesUS is the largest corn producer in the world, generating huge amount of corn stover as a low-value agriculture byproduct currently used mainly in animal feed. DOE estimates ~271 million dry tons of corn stover can be supplied by 2030. A techno-economic analysis (TEA) for butanol production from corn stover (lignocellulose) will be performed for the integrated bioprocess involving unit operation steps of acid/alkali pretreatment, hydrolysis, two-stage fermentation, and final product separation by pervaporation. No hydrolysate detoxification to remove inhibitorsis necessary as expected based on our previous studies, thus reducing the costs of hydrolysate sugars to be competitive with corn and sugarcane sugars.Process economics will be analyzed based on the fermentation data obtained in the study and available literature data for corn stover hydrolysis, electrochemical reduction of CO2, and pervaporation, which is considered most energy-efficient and scalable technology for butanol separation from fermentation broth.We will also determine the energy balance and associated GHG emissions. A well-to-wheels life cycle analysis (LCA) will be performed using Argonne National Laboratory's GREET Model (greet.es.anl.gov/). Reduction in GHG emissions will be compared with corn-based butanol from ABE fermentation and petroleum-derived butanol as the benchmark. We expect that butanol produced from the proposed process can reduce GHG emissions by more than 50%.We expect that TEA will show that butanol can be produced at <$2.5/gal using the proposed process. Process scale-up and optimization issues will be identified for further studies in Phase II.