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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** 2021 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: Initial evaluation of low-moisture anhydrous ammonia (LMAA) pretreated biomass in corn to ethanol process was done, and a manuscript was published. Ethanol production and sugar conversion yields of the pretreated biomass were evaluated and studied at different levels of biomass addition and different levels of corn incorporation. Results showed that the biomass could be enzymatically hydrolyzed simultaneously with the corn starch and fermented. At higher levels of corn incorporation, observing the contribution of the biomass addition was difficult to quantify as the biomass contributes significantly less fermentable sugar by weight relative to corn. The release of sugars (arabinose and xylose) from the biomass was still quantifiable under these conditions and indicated efficient biomass conversion. Additionally, the residual levels of non-fermented sugars indicated that  additional ethanol could be produced with an alternative organism capable of co-fermenting xylose and arabinose. At lower corn incorporation levels, the contribution of the pretreated biomass to ethanol production was readily determined. The distillers' dried grains with solubles (DDGS) were altered in nutrient composition and significantly darker in color  than the DDGS from corn fermentation alone. Additional work is needed to confirm the feed value of the mixed DDGS; however, it is unlikely that the corn portion would be negatively impacted.

Objective 2: A process for the absorption of carbon dioxide from fermentation has been developed and evaluated. Carbon dioxide released as a by-product from fermentations was absorbed in a packed column containing a high molarity sodium hydroxide solution. The absorbed carbon dioxide reacts with sodium hydroxide to form sodium carbonate in the solution. The recovered sodium carbonate solution can be recovered and recycled for utilization as a less severe chemical pretreatment for agricultural feedstocks. Ethanol fermentation using sweet sorghum juice (SSJ) showed a 92% absorption efficiency for carbon dioxide using the current process setup. The resulting solution contained 2.3 M sodium carbonate at a pH of 11.2. Chemical pretreatment using the recovered sodium carbonate solution could preserve the entire fraction of glucan while only losing around 8% of the xylan component in sweet sorghum bagasse.  The pretreatment also provided up to 40% delignification of the feedstock. The minimized polysaccharide loss coupled with lignin removal allow this pretreatment to maximize fermentable sugars available for downstream fermentation. Research is continuing with this process in order to determine carbon dioxide absorption efficiency using 2,3-butanediol fermentation. Additional work is in the planning stages to identify different process configurations to optimize carbon dioxide absorption from fermentation off-gas. 

Objective 3: The microorganism Paenibacillus (P.) polymyxa was cultivated in synthetic media to determine conditions that impact biomass growth and 2,3-butnaediol output. The synthetic media was prepared to closely mimic the sugar concentrations present in hydrolysates from pretreated agricultural feedstocks (around 40 g/L glucose and 20 g/L xylose). It was observed the 2,3-butanediol, along with the side product of acetoin, could be generated in a supplemented synthetic media. In order to determine other cultivation requirements, P. polymyxa was then fermented in enzymatic hydrolysate obtained from pretreated sweet sorghum bagasse. Even with organic nitrogen supplementation, cultivation of P. polymyxa in this hydrolysate could not be achieved, most likely due to the presence of inhibitor compounds from dissolved lignin. To limit the presence of these compounds a 1:1 hydrolysate mixture containing raw hydrolysate with detoxified hydrolysate and organic nitrogen supplementation was prepared. This hydrolysate showed successful cultivation of P. polymyxa along with 2,3-butanediol generation up to 15 g/L after 48 hours. However, as fermentation time progressed, 2,3-butanediol yield decreased as the organism converted the product to acetoin. Current research is underway to limit the production of acetoin during fermentation and to cultivate P. polymyxa in enzymatic hydrolysates at the bioreactor scale. **Accomplishments** **Review Publications**
                            *Nghiem, N.P., Toht, M.J. 2019. Pretreatment of sweet sorghum bagasse for ethanol production using Na2CO3 obtained by NaOH absorption of CO2 generated in sweet sorghum juice ethanol fermentation. Fermentation. 5(4)91:1-10. https://doi.org/10.3390/fermentation5040091.*
                                *Stoklosa, R.J., Moore, C., Latona, R.J., Nghiem, N.P. 2021. Butyric acid generation by clostridium tyrobutyricum from low moisture anhydrous ammonia (LMAA) pretreated sweet sorghum bagasse. Applied Biochemistry and Biotechnology. 193(3):761-776. https://doi.org/10.1007/s12010-020-03449-w.*


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