Location: Sustainable Biofuels and Co-products Research
2024 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: Corn fiber was fractionated and recovered following ethanol fermentation of degermed corn. The recovered corn fiber was enriched in cellulose and hemicellulose content exceeding 33% and 20% on a dry weight basis, respectively. Using alkaline sodium carbonate pretreatment, the corn fiber was pretreated and utilized for downstream biochemical conversion to ethanol. Enzymatic hydrolysis was conducted using a reduced loading of cellulase and hemicellulase enzymes for optimal sugar release. Total sugar (i.e. glucose and xylose) conversion reached over 70% for the pretreated corn fiber. The resulting sugars from pretreated corn fiber were used as supplemental biomass for ethanol fermentation with corn. When mixed with varying ratios of corn solids the production of ethanol remained consistent, but xylose content increased with fermentation confirms that recovered corn fiber can be co-fermented with corn. Additionally, sodium carbonate pretreated corn fiber was used as a biomass supplement with thin stillage for astaxanthin production. Low astaxanthin yields and biomass growth by Phaffia rhodozyma cultivated on pretreated corn fiber hydrolysate with thin stillage were exhibited. This suboptimal growth was determined to be caused by fluctuation to higher pH from using the sodium carbonate pretreated corn fiber. Improved biomass and astaxanthin yields were confirmed by switching to ammoniated corn stover hydrolysate instead of sodium carbonate pretreated corn fiber.
Objective 2: Switchgrass (SG) biomass was pretreated using a sodium carbonate solution formed by the reaction of carbon dioxide (ethanol fermentation by-product) in a 5 M sodium hydroxide solution. The milled SG was loaded in a stainless-steel reactor at a 5% (weight-to-volume) solid dried biomass loading and heated to 150 Celsius for 90 minutes. Solids were vacuum filtered, and water washed to achieve neutral pH and dried to 55 Celsius. Pretreated SG underwent enzymatic hydrolysis at a 10% (weight-to-volume) solids loading and hydrolyzed with a cellulase/hemicellulase enzyme mixture using a 50 mM citric acid buffer. Sugar release was determined over a 72-hour time interval. In addition, two samples were also used to compare the carbonate treatments with the control (untreated) and low moisture anhydrous ammonia treatments (LMAA). Total sugar concentration was determined for recovered carbonate pretreated SG, LMAA SG, and untreated SG to be 64.6 g/L, 49.5 g/L, and 12.3 g/L, respectively. Additionally, lignin fractions were extracted and recovered from SG and corn stover (CS) biomass using an organic solvent pretreatment conducted at 150 Celsius for 60 minutes with an ethanol/water mixture and 0.05 M sulfuric acid. The organic fractions were recovered using a separatory funnel and by rotary evaporation for solvent removal. The organic fraction was freeze-dried to collect the organic-rich phase of lignin. The samples were subjected to characterization to identify aromatic units in lignin as well as side aliphatic chains, methoxy groups, and sugars that are still existent in the lignin. Preliminary results show in switchgrass a strong signal of the three monomers (G (guaiacyl), S (syringyl), and a stronger signal of H (p-hydroxyphenyl) units).
Objective 3: The resulting bio-based 2,3-butanediol (2,3-BDO) produced via fermentation of biomass sugars was catalytically upgraded to compounds that are utilized in sustainable aviation fuel (SAF). Following fermentation 2,3-BDO was recovered using phase separation with organic solvents and inorganic salts. The top layer containing 2,3-BDO was evaporated to remove organic solvent and the 2,3-BDO reconstituted in a known volume of water. The recovered 2,3-BDO underwent dehydration to produce an intermediate chemical methyl ethyl ketone (MEK), which can serve as a building block molecule for producing hydrocarbons for SAF. MEK generation was tested using both homogenous and heterogeneous catalysis. Sulfuric acid was utilized for homogeneous catalysis. Heterogeneous catalysis was conducted with a commercially available zeolite (ZSM-5) prepared in two separate acidic forms with and without transition metal surface modification. Using a six-vessel stirred parallel reactor, multiple dehydration reaction conditions such as temperature, time, and catalyst loading were tested and varied to determine overall MEK yields. A control dehydration reaction was also completed using cell-free fermentation broth to further evaluate if 2,3-BDO dehydration to MEK could be performed without upstream separation and recovery. Further research is being performed on the prepared heterogeneous catalysts to characterize catalyst structures and catalyst recyclability efficiency for continued use.
Accomplishments
1. Cryo-milled hemp for bio-based chemical production. Restrictions around cultivating industrial hemp in the United States have been loosened allowing more farmers to grow this crop. This increase in hemp production allows for more access to plant biomass for biochemical conversion to bio-based products to support sustainability efforts. ARS scientists in Wyndmoor, Pennsylvania, have collaborated with researchers at the University of Virginia (UVA) to understand the effects of hemp pre-processing on downstream biochemical conversion capability. Field grown hemp mixed with decorticated fiber, i.e. fiber separated by mechanical action, was pre-processed using cryogenic milling with liquid nitrogen (LN2). Aside from particle size reduction, this also produced lignin removal around the plant cell wall. The effect of cryo-milling further showed that a reduction in alkaline chemical pretreatment severity (lower temperature and alkaline loading) could be used and still maintain high sugar yields following enzymatic hydrolysis. The resulting hydrolysates were fermented with Paenibacillus polymyxa under anoxic conditions producing upwards of 19 g/L 2,3-butanediol (2,3-BDO), a valuable platform chemical. This research demonstrated the potential of industrial hemp as a bioenergy feedstock, and a particle size reduction process that would reduce downstream pretreatment severity with potential savings on processing costs.
2. Oxygen limited fermentation condition for 2,3-Butanediol production. Oxygen limitation improves 2,3-butanediol generation from using fermentable sugars from agricultural feedstocks subjected to sodium carbonate pretreatment. The bacteria strain Paenibacillius 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 (SAF). 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., Garcia-Negron, V., Latona, R.J., Toht, M.J. 2023. Limiting acetoin generation during 2,3-butanediol fermentation with Paenibacillus polymyxa using lignocellulosic hydrolysates. Bioresource Technology. https://doi.org/10.1016/j.biortech.2023.130053.
Stoklosa, R.J., Latona, R.J., Berger, B.W., Timko, M.P., Shlanta, A.V., Himes, M.R. 2024. Hemp cryo-milling and the impact of alkaline pretreatment on biochemical conversion. ACS Sustainable Resource Management. https://doi.org/10.1021/acssusresmgt.4c00005.