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Final Report: Pretreatment of Agricultural Residues Using Aqueous Ammonia for Fractionation and High Yield Saccharification
EPA Grant Number: R831645Title: Pretreatment of Agricultural Residues Using Aqueous Ammonia for Fractionation and High Yield Saccharification
Investigators: Lee, Y. , Elander, Richard
Institution: Auburn University Main Campus , National Renewable Energy Laboratory
EPA Project Officer: Richards, April
Project Period: June 1, 2004 through May 31, 2006 (Extended to December 31, 2006)
Project Amount: $190,156
RFA: Technology for a Sustainable Environment (2003)
Research Category: Pollution Prevention/Sustainable Development
Description:
Objective:The overall objective of this project is to develop a pretreatment process suitable for enzymatic conversion of agricultural residues into fermentable sugars. The proposed process uses aqueous ammonia (a non-polluting substance) as the pretreatment reagent. Use of ammonia offers significant economic and environmental merits since it is easily recovered and leaves no residual effect on the environment. The proposed pretreatment is a part of the integral biomass-to-fuels process that does not generate net CO2 (a green energy process). When it is incorporated into the biomass saccharification processes, it can accomplish a near complete fractionation of biomass into the three major constituents (cellulose, hemicellulose, and lignin). The goal of this project is to expand the fundamental knowledge base of this method and advance it to a point where it can be evaluated as a process technology.
A pretreatment process is applied before the biomass is subjected to the biological processing. The proposed method has been proven to be highly effective in delignification of agricultural residues and herbaceous feedstocks. Delignifying biomass at the early phase of the process is beneficial for a number of reasons. Low lignin in the solid substrate improves the digestibility and the overall enzyme efficiency thus lowering the enzyme dosage. The low-lignin carbohydrates are less toxic to microorganisms. Early removal of lignin also eliminates the complications in the downstream processing including the cell separation in the bioreactor and the distillation. The lignin separated by the proposed process is clean and free of contaminants. It is a high-grade fuel with no known environmental problems. It is also a potential feedstock very much amenable for further conversion into value-added chemicals. In addition to the clean lignin, the proposed process can produce high-grade cellulosic material that has broader market value than the saccharification feedstock. It is a short-chain cellulose fiber with high α-glucan content. Its potential market includes filler-fiber in papermaking and microcrystalline cellulose.
Summary/Accomplishments (Outputs/Outcomes):The first part of this project was focused on fractionation of corn stover. Utilization efficiency of lignocellulosic biomass is significantly improved by fractionation of the biomass. A two-stage percolation process was applied for pretreatment and fractionation of corn stover. This process is composed of hot water treatment followed by treatment with aqueous ammonia, both applied in a flow-through (percolation) reactor. The first stage processing is intended for hemicellulose removal whereas the second stage is intended for delignification. Ammonia is easily recoverable, and it poses no environmental problem in aqueous solution. The treated end-product was found to be very high in cellulose content and also highly susceptible for enzymatic digestion. The conditions that achieve satisfactory level of biomass fractionation and acceptable enzymatic hydrolysis were identified in terms of reaction temperature, flow rate (retention time), and reaction time for each stage. With proper operation of the two-stage treatment, fractionation of biomass was achieved to the extent that the xylan fraction was hydrolyzed with 92–95% conversion, recovered with 83–86% yields, and the lignin removal was 75–81%. The remaining solid after two-stage treatment contained 78–85% cellulose. The two-stage treatments enhanced the enzymatic digestibility to 90–96% with 60 filter paper units (FPU)/g of glucan, and 87–89% with 15 FPU/g of glucan. The composition and digestibility data of treated samples indicate that the lignin content in the biomass is one of the major factors controlling the enzymatic digestibility.
In the second phase of this work, a new pretreatment method, soaking in aqueous ammonia (SAA), was investigated. In this method, a feedstock is soaked in aqueous ammonia over an extended period (1–10 days) at room temperature. This is done without agitation under atmospheric pressure. Treatment of corn stover by SAA removes 55–74% of the lignin, but retains nearly 100% of the glucan and 85% of the xylan. The SAA treatment of corn stover achieved enzymatic digestibilities comparable to those of high temperature aqueous ammonia treatments such as Ammonia Recycle Percolation (ARP). The xylan remaining in the corn stover after SAA was hydrolyzed, along with glucan, by cellulase enzyme due to the presence of xylanase in “cellulase.” The SAA-treated corn stover was further evaluated by simultaneous saccharification and fermentation (SSF) and by simultaneous saccharification and co-fermentation (SSCF). In the standard SSF test using Saccharomyces cerevisiae (NREL-D5A), an ethanol yield of 73% was obtained on the basis of the glucan content in the treated corn stover. Xylose accumulation in the SSF appears to inhibit the cellulase activity of glucan, limiting the yield of ethanol. In the SSCF test using recombinant Escherichia coli (KO11), both the glucan and xylose were effectively utilized, giving an overall ethanol yield of 77% of the theoretical maximum based on glucan and xylan. With the SSCF results, the fact that the xylan fraction is retained is a desirable feature in pretreatment since the overall bioconversion can be carried out in a single step without separate recovery of xylose from the pretreatment liquid.
In order to reduce the treatment time in the SAA, increase of treatment temperature was attempted. In the modified SSA, corn stover was soaked in 15–30 weight % aqueous ammonia at 40–90°C for 6–24 hours. The optimum treatment conditions of this process were 15 wt.% of NH3, 60°C, 1:6 solid-to-liquid ratio, and 12 hours of treatment time. The modified SAA retained 85% of the xylan and removed 62% of the lignin. Under optimum treatment conditions, the enzymatic digestibility of glucan was enhanced from 17 to 85% for glucan with 15 FPU/g-glucan enzyme loading, and 78% of the xylan in the treated biomass was also hydrolyzed by cellulase enzyme. The SSCF test of the modified SAA samples (3% weight/volume glucan loading, recombinant E. coli [KO11])has shown highly effective glucan and xylan utilization for eventual conversion into ethanol. The overall ethanol yield of the SSCF was 77% of the maximum theoretical yield based on glucan and xylan. The maximum ethanol concentration reached 19.2 g/L, and it occurred at 96 hours.
In the third phase of this project, effective utilization of hemicellulose was explored investigating the production of value-added chemicals other than ethanol from the pretreated corn stover. The end-products sought were xylooligosaccharides (high-value food additive) and lactic acid. SAA treatment of corn stover and corn cobs resulted in “clean” and xylan-rich substrates, which were susceptible for xylanolytic hydrolysis but poorly reactive for autohydrolysis. The products from the enzymatic hydrolysis using endoxylanase consisted of primarily xylooligosaccharides. Fractionation and refining of xylooligosaccharides were accomplished by charcoal adsorption followed by ethanol elution. An effective product purification method was also developed whereby all of xylose monomer and color forming substances were removed, yet the majority of xylooligosaccharides was recovered. The digestion of xylan in SAA-treated corn stover caused a slight decrease in the cellulose digestibility but still achieved above 80% digestion.
Corn stover treated by SAA was further investigated as the substrate for lactic acid production by SSCF. A commercial cellulase (Spezyme-CP) and Lactobacillus pentosus ATCC 8041 (CECT-4023) were employed in the SSCF. In batch operation of the SSCF, the carbohydrates in the treated corn stover were efficiently converted to lactic acid. The maximum lactic acid yield reached 92% of the stoichiometric maximum based on total fermentable carbohydrates (glucose, xylose, and arabinose). A small amount of acetic acid was also produced in the process from pentoses through the phosphoketolase (PK) pathway. Among the major process variables of the SSCF, the enzyme loading and amount of yeast extract were found to be the key factors affecting lactic acid production. Further tests on nutrients indicate that corn steep liquor could be used as a nitrogen source in place of yeast extract without adversely affecting the lactic acid yield. Fed-batch operation of the SSCF was beneficial in raising the concentration of lactic acid, the maximum value reaching 75 g/L.
Conclusions:Fractionation of corn stover into three main components of biomass was achieved by successive processing of hot water treatment followed by aqueous ammonia treatment. The first stage processing is intended for hemicellulose removal, whereas the second stage is intended for delignification. With operation of two-stage treatment under optimum conditions, fractionation of biomass was achieved to the extent that the xylan fraction was hydrolyzed with 92–95% conversion, recovered with 83–86% yields, and the lignin removal was 75–81%. The remaining solid after two-stage treatment contained 78–85% cellulose. The two-stage treatments enhanced the enzymatic digestibility of the remaining cellulose to 90–96% with 60 FPU/g of glucan and 87–89% with 15 FPU/g of glucan.
The ammonia pretreatment was further modified from high temperature processing in a flow-through reactor (ARP) to SAA that employs low-to-moderate temperature treatment. In this method, a feedstock is soaked in aqueous ammonia over an extended period (1–10 days) at room temperature. Treatment of corn stover by SAA removes 55–74% of the lignin, but retains nearly 100% of the glucan and 85% of the xylan. The SSA-treated corn stover was further evaluated by SSF and by SSCF. In the SSCF test using cellulase (Genencor, Spezyme CP) and recombinant E. coli (KO11), both the glucan and xylose were effectively utilized, giving an overall ethanol yield of 77% of the theoretical maximum based on glucan and xylan.
Effective utilization of hemicellulose was explored investigating the production of value-added chemicals other than ethanol from the pretreated corn stover. The end-products sought were xylooligosaccharides (high-value food additive) and lactic acid. SAA treatment of corn stover and corn cobs resulted in “clean” and xylan-rich substrates, which were susceptible for xylanolytic hydrolysis but poorly reactive for autohydrolysis. The products from the enzymatic hydrolysis using endoxylanase consisted of primarily xylooligosaccharides. Fractionation and refining of xylooligosaccharides were accomplished by charcoal adsorption followed by ethanol elution. The digestion of xylan in SAA-treated corn stover caused a slight decrease in the cellulose digestibility but still achieved above 80% digestion.
Corn stover treated by SAA was further investigated as the substrate for lactic acid production by SSCF. A commercial cellulase (Spezyme-CP) and L. pentosus ATCC 8041 (CECT-4023) were employed in the SSCF. From this process, the carbohydrates in the treated corn stover were efficiently converted to lactic acid. The maximum lactic acid yield reached 92% of the stoichiometric maximum based on total fermentable carbohydrates (glucose, xylose, and arabinose). A small amount of acetic acid was also produced in the process from pentoses through the phosphoketolase (PK) pathway. Fed-batch operation of the SSCF was beneficial in raising the concentration of lactic acid, the maximum value reaching 75 g/L.
Journal Articles on this Report: 2 Displayed | Download in RIS Format
| Other project views: | All 8 publications | 5 publications in selected types | All 5 journal articles |
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Kim TH, Lee YY. Fractionation of corn stover by hot-water and aqueous ammonia treatment. Bioresource Technology 2006;97(2):224-232. |
R831645 (Final) |
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Zhu Y, Lee YY, Elander RT. Optimization of dilute-acid pretreatment of corn stover using a high-solids percolation reactor. Applied Biochemistry and Biotechnology 2005;124(1-3):1045-1054. |
R831645 (2005) R831645 (Final) |
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pretreatment, corn stover, aqueous ammonia, simultaneous saccharification and fermentation, simultaneous saccharification and co-fermentation, SSF, SSCF, xylo-oligosaccharides, ethanol, lactic acid,
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INTERNATIONAL COOPERATION, Sustainable Industry/Business, Scientific Discipline, RFA, Technology for Sustainable Environment, Sustainable Environment, Chemicals Management, Environmental Chemistry, biomass, biowaste, green chemistry, environmentally friendly transportation fuel, fermentation of sugars, aqueous ammonia, agricultural byproducts, alternative energy source, alternative materials, enzyme transformations, alternative fuel
Progress and Final Reports:
2004 Progress Report
2005 Progress Report
Original Abstract