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ARS Home Northeast Area Wyndmoor, Pennsylvania Eastern Regional Research Center Sustainable Biofuels and Co-products Research Research





- Stoklosa, Ryan
- Garcia-Negron, Valerie
- Johnston, David
- 2023
- 2022
- 2021
- Publications
- Product Quality and New Uses (306)

Research Project: Integrated Biological/Chemical Biorefining for Production of Chemicals and Fuels **Location:
                            Sustainable Biofuels and Co-products Research** 2023 Annual Report **Objectives**
                                

Objective 1 Develop integrated processes to enable commercial production of value-added products in existing corn ethanol biorefineries. 
This includes co-conversion of biomass (corn stover, corn fibers) with corn starch for production of value-added products to enhance biorefinery profitability and stability.

Sub-objective 1-A. Develop technologies that enable the integrated processing of pretreated biomass with grains at existing biofuels production facilities for cellulosic ethanol production.
Sub-objective 1-B. Utilize advanced enzymatic fractionation processes to separate fiber and starch prior to fermentation to generate a fiber rich streams for pretreatment and biomass sugar production studies.
Sub-objective 1-C. Produce and utilize sugars from biomass/grain mixtures for the fermentation of value-added coproducts.

Objective 2 Develop self-sustained biomass pretreatment and conversion processes to enable commercial production of fermentable sugars and lignin-derived products.
This includes but is not limited to the use of Na2CO3, which can be generated by NaOH absorption of CO2, a co-product of aerobic/anaerobic fermentation, for biomass pretreatment. Both lignin fractions obtained prior to pretreatment and after sugar extraction will be investigated as feedstocks for conversion to high-value products or fuels. 

Sub-objective 2-A. Develop a process for pretreatment of biomass using the Na2CO3 solutions made by absorption of CO2 from ethanol and 2,3-butanediol (2,3-BDO) fermentations in NaOH.
Sub-objective 2-B. Develop a process using the Na2CO3-pretreated biomass as feedstocks for production of fermentable sugar solutions suitable for use as substrates in industrial fermentations.
Sub-objective 2-C. Develop lignin recovery and conversion processes that generate advanced biofuels, high-value chemicals, or renewable materials.

Objective 3 Develop hybrid biocatalytic/chemical processing technologies to enable the commercial conversion of lignocellulosic sugars to advance biofuels or chemicals.
Fermentable sugars from biomass feedstocks will be utilized to produce a microbial source of 2,3-BDO. The recovered 2,3-BDO will then be investigated by downstream chemical upgrading technologies to produce advance biofuels or high-value chemicals.

Sub-objective 3-A. Develop fermentation processes for the production and recovery of 2,3-butanediol.
Sub-objective 3-B. Develop chemical conversion process to upgrade 2,3-butanediol to an advanced biofuel. **Approach**
                                

The first objective will establish technologies that integrate sugars derived from biomass feedstocks that are not directly connected to corn grains (e.g. corn stover and switchgrass) with corn starch-derived sugars for the production of ethanol and/or industrial chemicals. Pretreated biomass will be co-converted with corn or other starch-containing feedstocks for production of cellulosic ethanol within existing biorefineries. The primary focus will investigate additional ethanol production in dry-grind corn facilities. Other investigations will be made into process technologies whereby the fiber isolated from corn kernels prior to ethanol fermentation can be utilized in conjunction with other pretreated biomass to produce additional fermentable sugars. The fermentable sugars will then be utilized in fermentation processes for production of value-added co-products, such as the carotenoid astaxanthin, in existing ethanol biorefineries. 

The second objective will develop a self-sustainable pretreatment process for corn stover and switchgrass using sodium carbonate solutions generated by the absorption of carbon dioxide produced from simulated industrial fermentations. This pretreatment process will be optimized in order to maintain at least 85% of the orginal cellulose and at least 70% of the original hemicellulose in corn stover and switchgrass. Following sodium carbonate pretreatment the pretreated biomass will be hydrolyzed to generate fermentable sugars using commercially available enzymes. The enzymatic hydrolysis process will be optimized to maintain at least 50 g/L of total sugars in the hydrolysate and greater than 75% theoretical sugar yields. The residual insoluble solids obtained after pretreatment or enzymatic hydrolysis will also be utilized to develop a process for lignin recovery. The recovered lignin will be utilized as a separate feedstock for the generation of advanced biofuels via biochemical conversion, or in material applications for preparing biobased epoxy resins.  

The third objective will investigate fermentation processes for the production of 2,3-butanediol (2,3-BDO) from fermentable sugars of pretreated corn stover or switchgrass. The 2,3-BDO produced by fermentation will be catalytically upgraded to  an advanced biofuel. Fermentation processes will be developed and optimized to generate 2,3-BDO at high titers and yields. Additional process development will focus on separation and recovery processes following fermentation in order to obtain a high purity yield of 2,3-BDO for downstream chemical upgrading. The recovered 2,3-BDO will then be upgraded via a multi-step chemical conversion route utilizing dehydration, aldol condensation, and hydrodeoxygenation to generate a hydrocarbon fuel. Both chemical catalyst selection will be identified and process parameters optimized as the upgrading process is investigated. **Progress Report**
                                

Objective 1: Mixed fermentations utilizing sodium bicarbonate pretreated fiber fractions, that were isolated from corn kernels, and ground corn are nearly completed. Fermentations utilizing only the pretreated fiber are complete and show near theoretical ethanol production. Mixed corn and pretreated fiber fermentation are underway. The composition of the distillers dried grains with soluble from these fermentations are being analyzed and the results will be included in the manuscript outlining these results. Utilizing the results from the fiber sugar compositional analysis, a fermentation system to produce astaxanthin using Phaffia rhodozyma has been outlined. Preliminary experiments are underway.

Objective 2: Recovered sodium carbonate solution formed from the reaction of carbon dioxide (fermentation by-product) and sodium hydroxide solution was used to pretreat corn stover and switchgrass biomass. Improvements on the column reaction design were evaluated using different packing material and changing the direction of the solution. Also, the recovered sodium carbonate solution was improved during the collection of it and reducing the presence of precipitation during the absorption of carbon dioxide. Biomass was pretreated at 5% solids loading using the recovered solution at 150 degrees C for 90 minutes in a sealed stainless-steel reactor. The pretreated biomass was washed with water until a neutral pH was obtained followed by vacuum filtration and drying the biomass at 55 degrees C. The pretreated biomass was then subjected to enzymatic hydrolysis to generate fermentable sugars. For both corn stover and switchgrass the enzymatic hydrolysis was conducted to obtain the high yields of glucose and xylose by studying the sugar release up to 72 hours. Glucose yield exceeded 70% for both pretreated samples while the yield of xylose was greater than 85%. Lignin fractions from the pretreatment liquor and after enzymatic hydrolysis have been recovered. From the pretreatment liquor the dissolved lignin was precipitated by adjusting the pH to acidic levels while the enzymatic hydrolysis residue was subjected to further alkaline extraction followed by precipitation. Both fractions have been freeze-dried and will undergo analytical characterization to determine chemical composition. 

Objective 3: Sodium carbonate pretreated corn stover and switchgrass was further deconstructed using enzymes. The resulting hydrolysates were comprised of monomeric sugars in the form of glucose and xylose. Following nutrient supplementation, the hydrolysates were utilized as media to cultivate Paenibacillus polymyxa (P. polymyxa) to produce 2,3-butanediol (2,3-BDO). It was previously determined that by eliminating oxygen supply after 24 hours of fermentation with molasses media more favorable conditions for the generation of 2,3-BDO were achieved. This processing parameter was then applied to fermentations conducted using pretreated corn stover and switchgrass hydrolysates. Within 24 hours of fermentation P. polymyxa grew exponentially and consumed all sugars present in the hydrolysates. Between 24 and 48 hours of fermentation 2,3-BDO production maxed out at 43 g/L and 34 g/L for corn stover and switchgrass hydrolysates, respectively. The fermentations continued up to 120 hours, but this resulted in a 2,3-BDO output decrease as the microbe consumed the 2,3-BDO and converted it into the byproduct acetoin. The fermentation broth was also found to be amenable to 2,3-BDO separation and recovery. The broth was mixed with an organic solvent and aqueous salt solution to force a biphasic phase separation between the top organic layer and the bottom aqueous layer. This resulted in an 87% recovery of 2,3-BDO from the fermentation broth into the organic solvent. The processing conditions used for fermentation and separation/recovery are currently undergoing additional experiments to optimize both 2,3-BDO generation and downstream recovery. **Accomplishments**
                                

1. Oxygen limitation improving 2,3-butanediol generation from lignocellulosic hydrolysates. The bacteria strain Paenibacillius polymyxa (P. polymyxa) naturally produces the platform chemical 2,3-butanediol (2,3-BDO), a starting material to produce several value added products including sustainable aviation fuel. In order to be economically feasible to produce via this method, high concentrations of 2,3-BDO must be achieved. ARS scientists in Wyndmoor, Pennsylvania, have shown that utilizing fully aerobic fermentation conditions within the first 24 hours can provide exponential biomass growth along with substantial sugar consumption. Upon reaching 24 hours of fermentation the addition of oxygen (via air) can be terminated for the remainder of the processing time stimulating production of 2,3-BDO. Controlling the fermentation oxygen level via this process achieves much higher concentrations (nearly 40 g/L 2,3-BDO) than a control fermentation where oxygen is kept constant. Continuing research studies are ongoing by incorporating fed-batch processing to produce concentrations of 2,3-BDO greater than 40 g/L that will allow for more efficient downstream separation and recovery of the product. **Review Publications**
                            *Stoklosa, R.J., Latona, R.J., Johnston, D. 2022. Assessing oxygen limiting fermentation conditions for 2,3-butanediol production from paenibacillus polymyxa. Frontiers in Chemical Engineering. 4:1038311. https://doi.org/10.3389/fceng.2022.1038311.*
                                *Garcia-Negron, V., Toht, M.J. 2022. Corn stover pretreatment with Na2CO3 solution from absorption of recovered CO2. Fermentation. https://doi.org/10.3390/fermentation8110600.*


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