Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Plant Pathology & Microbiology
Non Technical Summary
The proposed research will address one of the most important challenges in biomass processing, the use of lignin for fungible products. In particular, by complete utilization of biomass to produce fuels, chemicals, and materials, the project will uniquely enhance economic output, long-term sustainability, and overall competitiveness of US agriculture [1]. This modified project will distinctively deliver a complete lignin-based bioproduct technology portfolio to transform biomass utilization for value-added agriculture products. The U.S. Energy Independence and Security Act (EISA) bill contains provisions that increase the Renewable Fuel Standard (RFS) to 36 billion gallons by 2022, among which 22 billion gallons must be advanced biofuel derived from nonfood-based biomass. The USDA/DOE Updated Billion Ton report concluded that these goals can be met with lignocellulosic biomass including: perennial energy crops, agricultural crop and forest residues, tree farms, secondary forest industry waste materials, and other crops [2]. Despite the potential of lignocellulosic biomass, the sustainability and economic viability of lignocellulosic biorefinery have been jeopardized by the lack of a proper solution to utilize the large volume of under-utilized lignin produced by the industrial biorefinery processes.The current efforts in biorefinery development are focused on maximizing the utilization of carbohydrates including both cellulose and hemicellulose. However, the utilization of lignin for fungible products remains highly challenging, even though lignin constitutes 15 to 40 percent of lignocellulosic biomass. Moreover, lignin contains a higher carbon content (60%) as compared to carbohydrate (~40%), providing a better source for chemical and material applications. To date, commercial applications of lignin have been for polymers, adhesives, dispersants, concrete additives, emulsifiers, and binders, but proper biorefinery procedures need to be developed so that lignin chemistry can be tailored to produce quality and value-added products amenable with the carbohydrate processing. Regardless of the exact pretreatment technology employed, almost all biomass processing platforms result in the formation of a significant lignin process stream [3, 4]. Despite the need of certain amount of lignin (~30-40%) for the thermal requirements of bioethanol production, a modern biological cellulosic processing plant will often have ~60% excess lignin [5]. This excess lignin not only represents a waste of carbon source, but also generates significant issues for waste management. Lignin utilization thus offers a significant opportunity for enhancing the operational efficiency, carbon conversion rate, feedstock availability, economic viability, and sustainability of biofuel and bioproduct production from lignocellulosic biomass.Several technologies are being pursued to convert lignin to a fungible fuel including catalytic pyrolysis, hydrotreatment, alkaline fragmentation/alkylation and gasification. Even though these thermochemical or chemical processes provide a potentially viable approach to a lignin-to-fuel platform, these methods are still hindered by several factors including; the low quality fuel product that needs an up-grade, corrosive intermediates or end products, and significant cost for waste management [6]. In fact, the integration of biological and chemical biomass processing with lignin utilization thus represents a much better option, as the proper process design will allow us to tailor lignin chemistry to produce various high value products. During the past five years, with the support from USDA Hatch fund and various DOE EERE (Department of Energy, Office of Energy Efficiency and Renewable Energy) awards, the PI Yuan's group has pioneered in developing various fungible products out of lignin-containing biorefinery waste [7-16]. Moreover, his group has undertaken a holistic approach to integrate biorefinery process development and microbial strain engineering to achieve highest titer in bioplastics and lipid production from lignin [10, 11,14]. More importantly, Dr. Yuan has led a multidisciplinary and multi-institute team to advance the fundamental understanding of how lignin chemistry impacts the quality and performance of various lignin-based products including carbon fiber, asphalt binder modifier, and others [7-9, 12, 16]. The fundamental scientific understanding along with the strain and process development has opened new avenues for lignin bioproduct development. In the next five years of this hatch fund, we aim to advance new frontiers of lignin bioproducts based on the past progresses with the following aspects. First, we will further develop pretreatment and fractionation technologies to co-optimize carbohydrate output and lignin processiblity, aiming to derive lignin with tailored chemistry for carbon fiber and bioconversion to various products. Second, based on our achievement in the engineering of Rhodococcus opacus PD630 (PD630 henceafter), we will further engineer PD630, a strain with efficient growth on lignin, to produce high value products like carotenoids and Polyhydroxyalkanoates (PHA). Third, based on recent breakthroughs in quality carbon fiber from lignin, we will further advance lignin carbon fiber manufacturing and integrate the process with fractionation and bioconversion. Eventually, an integrated process for multiple high value lignin bioproducts will be developed to enable multi-product utilization of biomass.The process and microbial strain development will be integrated with the existing strength at USDA and Texas A&M Agrilife Research for sorghum biomass feedstock development. Such integration will allow us to develop an integrated agriculture system to modify the best feedstock for specific bioproduct. Lignin modification in sorghum has been studied extensively to reduce recalcitrance and increase carbohydrate processibility [17, 18]; yet very limited knowledge has been generated on how lignin modification can improve the processing of lignin into value-added bioproducts. In sorghum, brown midrib (bmr) mutants have been major assets in determining how modification of the lignin biosynthesis pathway could impact biomass conversion. The bmr mutants of sorghum have been isolated from the chemically mutagenized populations, and three bmr loci, bmr2, bmr6 and bmr12, have been characterized. The biomass from bmr6 or bmr12 plants was significantly reduced in lignin content and altered in lignin composition relative to wild- type [19-21]. Dr. Scott Sattler, our collaborator at USDA ARS, has provided us sufficient biomass for all aforementioned bmr lines, which will be integrated with processing and microbial engineering to maximize bioconversion performance. Besides the bmr mutants, Dr. Sattler has led the efforts to develop the over-expression lines for all monolignol biosynthesis genes. These lines are instrumental in understanding how lignin chemistry can impact the carbon fiber performance. Apart from the resources from USDA ARS, Dr. Bill Rooney's group has a large collection of sorghum lines with different lignin content and composition at Texas A&M Agrilife research. The diverse lines represents an important resource for dissecting how lignin modification can impact the processability of not just carbohydrate, but also lignin. Overall, the research program will integrate process development and microbial strain engineering to deliver integrated platforms to manufacture multiple value-added products from lignin-containing biorefinery waste. The biorefinery technologies will be integrated with feedstock development to enable multi-product biomass processing with high bioproduct quality, which will significantly diversify the agriculture product profile, improve economic output, and enhance overall sustainability.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
The proposed research will be carried out with three highly integrative objectives to deliver multi-product biomass processing to produce carotenoids, bioplastics, and carbon fiber from lignin-containing biorefinery waste. In Objective 1, we will develop pretreatment and fractionation technologies to improve lignin reactivity for different products. In particular, we will ensure that carbohydrate output and lignin processibility are both optimized. In Objective 2, we will design Rhodococcus opacus PD630 to convert lignin into higher value products such as PHA (Polyhydroxyalkanoates) and carotenoids. The engineered microbial strain will be used to process lignin derived from Obj. 1. In Objective 3, we will develop quality carbon fibers to integrate with biomass processing (Objective 1) and microbial fermentation (Objective 2). Overall, with the three objectives, we will well address the challenges in Problem Statement and advance integrated techniques to deliver multiple products to add value and improve sustainability of biomass processing.
Project Methods
Objective 1. Pretreatment and fractionation technologies will be developed to improve lignin reactivity for different products.Technical methods: First, we will aim to develop innovative pretreatment technologies to derive the lignin fractions for bioconversion and carbon fiber applications. Previous reports have established that different pretreatments and different severity factors impact lignin structure [10, 11, 16, 22]. In these studies, we have also shown that we can derive lignin with more fractionated aromatic compounds and can achieve co-optimization of carbohydrate output and lignin conversion [10, 11]. Based on these studies, we will examine the pretreatment chemistries that maximize the targeted structural characteristics. Pretreatment chemistries to be considered will include both dilute acid and alkaline pretreatment. In addition, various combinatorial pretreatment conditions combining acid, alkaline, and organosolv will be carried out to derive the biorefinery residues either more fractionated or with more uniform structure. Both carbohydrate output and biorefinery residues will be isolated and characterized (i.e., before and after biological conversion). Second, besides optimizing pretreatment of lignocellulosic biomass, we will also focus on biorefinery residues. We will further evaluate the potential of different fractionation and separation methods via enzymatic and chemical treatment of biorefinery residues. We have shown in previous studies that enzyme-mediator fractionation can improve lignin solubility and derive fractions for different usages [8, 9, 13]. Proper enzymatic and chemical treatment will be tested to examine if these treatments can improve lignin reactivity and processibility. The individual fractionation techniques will further be integrated with the membrane-based, centrifugation, and other separation techniques to derive different fractions of biorefinery waste streams. The lignin will be characterized before and after chemical treatment and lignin functionality will be optimized for bioconversion (Objective 2) and carbon fiber (Objective 3). Moreover, we will also aim to integrate the bioconversion with carbon fiber, where low-molecular-weight lignin and aromatic compounds can be used for bioconversion, and high-molecular-weight lignin can be used for carbon fiber. Moreover, in both pretreatment and fractionation technology development, wild type sorghum, bmr mutants and selected over-expression lines for lignin biosynthesis genes will be used, so that the bioprocess optimization and feedstock improvement can be integrated.Objective 2. Systems biology-guided biodesign will be carried out to engineer R. opacus PD630 to convert lignin into higher value products such as PHA and carotenoids.Technical Methods: We will first carry out systems biology-guided engineering of PHA production in PD630, which has been achieved in the lab. First, we will further optimize the expression of key PHA biosynthesis genes PhaC and PhaG in PD630 [25, 26]. Genes from different host sources with different enzyme kinetics will be compared and expressed at different levels. The resultant strains will be compared to optimize the carbon flux from lipid biosynthesis/degradation to PHA biosynthesis to achieve a high yield of PHA production. Second, the gene FAS I will be overexpressed with different expression levels together with PhaCGJ to drive more carbon flux for PHA production. Third, systems biology analysis as aforementioned will be carried out to analyze the selected engineered strains with high and low PHA production under biorefinery residue from Obj. 1. Both intermediate and flux analysis can be integrated with proteomics analysis to identify the key metabolic bottleneck or branching points for PHA biosynthesis, lipid production, and other metabolic pathways. Systems biology will guide the further pathway design to improve efficiency and yield for PHA from biorefinery waste. We will also carry out systems biology-guided engineering of carotenoids production in PD630. Our recent study demonstrated that PD630 can be evolved to produce higher level of carotenoids. The aforementioned systems biology analysis with proteomics and metabolomics analysis will be employed to identify the key enzymes, pathways, and regulators involved in regulating the carotenoid production. The PI has extensive experience in engineering terpene production [27, 28]. The key regulators will be over-expressed in both the wild-type strains and evolved strains to maximize the carotenoid production, especially when the strain is grown on biorefinery waste.Objective 3. Lignin-based carbon fibers will be developed to integrate with biomass processing. Technical Methods: First, we will examine how various lignin fractions from Obj. 1 will influence the miscibility, spinnability, crystallization, and ultimately fiber mechanical performance. Different lignin fractions will be subjected to lignin characterization in a similar way to our current studies: 1) lignin molecular weight and its PDI will be measured by GPC; 2) lignin aliphatic and phenolic hydroxyl groups will be measured under 31P NMR; and 3) lignin interunitary linkages will be measured with 2D HSQC NMR. The ultrastructure and mechanical properties of carbon fiber will be investigated as in previous studies [7-9]. The study will not only help to establish how different lignin fractionation technologies will derive lignin with various characteristics, but also show how different characteristics correlate with miscibility, spinnability, ultra carbon structures, and mechanical strength. Second, in combination with Obj. 1 and 2, we will use lignin derived from different fractionation methods to produce carbon fibers. The fractionated lignin with different biological and chemical fractionations will be subjected to the aforementioned lignin characterization. To investigate how fractionation optimization could lead to lignin with the structure more compatible for quality carbon fiber, carbon fiber characterization will be performed as aforementioned. We will produce quality lignin carbon fiber using lignin waste stream under the optimized pretreatment and fractionation in Obj. 1.