Chapter 2

Chapter 4

Figure 4. Schematic model of the regulatory network of secondary cell wall biosynthesis based primarily on studies in Arabidopsis. Peach circles represent transcription factors known to function in Arabidopsis. The red circles demarcate the transcription factors whose function has been studied in grasses. The yellow octagons represent enzymes. The grey squares represent secondary cell wall polymers. The green triangle represents a property of the cell wall, saccharification. Arrows signify positive regulation; whereas, dashed edges with T ends indicate negative regulation. Cis-elements are labeled on the edges as follows: Secondary wall NAC Binding Element (SNBE), Tracheary Element Responsive Element (TERE), Secondary wall MYB Responsive Element (SMRE), and the AC-rich elements found in lignin biosynthesis gene promoters (AC). See text for references and further discussion. For simplicity, not all known or suspected interactions are shown. Abbreviations are as follows: Lig Bios Enz, lignin biosynthesis enzymes; SCW Enz, secondary cell wall biosynthesis enzymes the specific identity of which has not been specified; PAL1, Phenylalanine Ammonium Lyase 1; 4CL1, 4-Coumaroyl Ligase 1; COMT, Caffeic acid O-MethylTransferase; C4H, Cinnamate 4-Hydroxylase; CESA, Cellulose Synthase A; SHN, shine/wax inducer 1; VND, Vasculature-related NAC-Domain; SND, Secondary wall-associated NAC-Domain protein; NST, NAC Secondary wall Thickening factor; VNI2, VND-interacting 2 NAC protein 2.

4RN, UK

[1]Research Agronomist, USDA-ARS, Grain, Forage, and Bioenergy Research Unit, 137 Keim Hall, University of Nebraska East Campus, Lincoln, NE 68583-0937.

[2]Research Agronomist, USDA-ARS, Agroecosystem Management Research Unit, 131 Keim Hall, University of Nebraska East Campus, Lincoln, NE 68583-0937.

Email: marty. schmer@ars. usda. gov *Corresponding author: rob. mitchell@ars. usda. gov

Virginia Tech, 365 Smyth Hall, Blacksburg, VA 24061.

[4]Noble Foundation, Ardmore OK 73402.

Email: tjbutler@noble. org

[5]USDA-ARS, 137 Keim Hall, Lincoln, NE 68583. Email: rob. mitcheU@ars. usda. gov ^Corresponding author: jfike@vt. edu

[6]The Institute for Sustainable and Renewable Resources, The Institute for Advanced Learning and Research, Danville, VA, USA; Departments of Horticulture and Forest Resources and Environmental Conservation, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.

Email: barry. ftinn@ialr. org

[7]The Institute for Sustainable and Renewable Resources, The Institute for Advanced Learning and Research, Danville, VA, USA.

Email: alejandra. lara@ialr. org

[8]The Institute for Sustainable and Renewable Resources, The Institute for Advanced Learning and Research, Danville, VA, USA; Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.

Email: scott. lowman@ialr. org

Corresponding author: chuansheng. mei@ialr. org

department of Plant and Soil Science, Oklahoma State University, 368 AG Hall, Stillwater, OK74078-6028, USA.

Email: yanqi. wu@okstate. edu

2National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China.

[10]Corresponding author: liulinglong@njau. edu. cn

[11]Clemson University Genomics Institute, Clemson University, Biosystems Research Complex, Clemson, SC 29634.

Email: Saski@clemson. edu

[12]Department of Genetics and Biochemistry, Clemson University, 110 Biosystems Research Complex, Clemson, SC 29634.

Corresponding author: hluo@clemson. edu

[13]State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

Email: dyli@genetics. ac. cn

[14]Department of Genetics and Biochemistry, Clemson University, 110 Biosystems Research Complex, Clemson, SC 29634.

^Corresponding author: hluo@clemson. edu

[15]228 Agricultural Hall, Biosystems and Agricultural Engineering Department, Oklahoma State University, Stillwater, OK 74078.

[16]223 Agricultural Hall, Biosystems and Agricultural Engineering Department, Oklahoma State University, Stillwater, OK 74078.

Email: raymond. huhnke@okstate. edu *Corresponding author: ajay. kumar@okstate. edu

department of Environmental Engineering and Earth Sciences, Clemson University, Clemson, SC 29634.

aEmail: arpanj@clemson. edu; arpanjain0211@gmail. com bEmail: walker4@clemson. edu

2Clemson Department of Economic Development, Clemson University, Clemson, SC 29634. Email: karl@clemson. edu

[18]Corresponding authors

[19]USDA-ARS, Grassland, Soil and Water Research Laboratory, 808 East Blackland Road, Temple, TX 76502.

“Email: Jim. Kiniry@ars. usda. gov

[20]Texas AgriLife Blackland Research and Extension Center, 720 East Blackland Road, Temple, TX 76502.

Email: nmeki@brc. tamus. edu

[21]Oklahoma State University, Department of Plant and Soil Sciences, 368 Agricultural Hall, Stillwater, OK 74078.

Email: yanqi. wu@okstate. edu

^Corresponding author: Kate. Behrman@ars. usda. gov

department of Agricultural Economics, Oklahoma State University, Stillwater, Oklahoma, USA 74078.

Email: f. epplin@okstate. edu

department of Agricultural and Resource Economics, University of Tennessee, 314B Morgan Hall, 2621 Morgan Circle, Knoxville, Tennessee, USA 37996.

Email: agriff14@utk. edu

[24]The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma, USA 73401.

Email: mhaque@noble. org *Corresponding author

Using appropriate herbicides reduces the time required to establishment and maximum biomass yields (Vogel et al. 2011). This approach is similar to the preferred method of establishing cool-season perennial grasses such as tall fescue when winter annual grass weeds are present. Butler et al. (2008) reported that sequential applications of glyphosate, one application in the spring to prevent annual grass weed production followed by second application in the autumn after rainfall and first flush of weed emergence, was very effective in increasing stand establishment. However, this technique is not well documented on switchgrass, therefore future research is needed.

Iron, one of the most abundant minerals on the planet, is not readily available to bacteria because its most commonly found form, ferric iron (Fe+3), is only slightly soluble and tightly bound to many particles in the soil. To gather iron needed for growth, bacteria and fungi secrete low molecular weight compounds called siderophores. Bacterial siderophores generally act to inhibit pathogenic fungi as a result of having higher affinity to iron than fungal siderophores (Ordentlich et al. 1988). Like many mechanisms of action in bacteria and fungi, environmental factors such as pH, nutrient levels including iron may affect synthesis of siderophores. Siderophore secretion has been confirmed in a number of bacterial taxa including Bacillus, Pseudomonas, Rhodococcus, Serratia, Obesumbacterium and Lysinibacillus (Czajkowski et al. 2012) as well as the fungal endophyte actinomycetes (Nimnoi et al. 2010). Genes encoding siderophores may be more difficult to introduce to other plant growth promoting endophytes since studies have shown that they are located in multiple loci (Osullivan et al. 1990) and have complex control mechanisms (Ovadis et al. 2004).

In addition to those described above, another intriguing approach to achieve process consolidation and cost reduction is in planta expression of lignocellulolytic enzymes. This approach fuses the discovery and development of advanced biocatalysts with engineering higher quality crop biomass. Indeed, expression of GHs and other hydrolytic enzymes in planta offer several advantages, including the following: (1) Growing transgenic plants in the field requires less energy than microbial production of the same enzymes; (2) Proteins can benefit from eukaryotic post-translational processing that can increase stability and activity; (3) Proteins can be targeted to subcellular compartments to prevent damage and allow high accumulation of the protein in the cell; and (4) Expression of proteins in planta juxtaposes the enzyme and the substrate, reducing enzyme demand needs during deconstruction due to inefficient mass transfer (Taylor et al. 2008; Sainz 2009). Reviews by Sticklen et al. (2006), Taylor et al. (2008), and Sainz et al. (2009) summarize numerous studies over expressing hydrolases in dicots and of greater relevance to switchgrass, in other grasses, including tall fescue, barley, maize, and rice. Here, we highlight key goals of, and new results related to, this approach.

Through the resources described above, switchgrass is becoming well — positioned for functional genomic studies. A major tool for functional genomic studies is genetic engineering of targeted genes facilitating a one to one tool relating DNA sequence with function. There are a large number of genome sequences currently available with many in progress allowing researchers a rich source of genes for potential manipulation for tolerance to abiotic stresses (Mittler and Blumwald 2010). A potential novel source of transgenes can be found by looking to sequences from organisms inhabiting extreme environments from desert adapted plants, to freeze tolerant fish, to even the diverse metagenomic projects being assessed for traits of functional interest (Mittler and Blumwald 2010). However, switchgrass is still considered recalcitrant for genetic modification, but significant progress has been made in optimizing transformation conditions and efficiency (see more detail in Chapter 9). There have been reports for Agrobacterium — mediated transformation with a range of efficiencies depending on the genetic background or genotype of the target (Somleva et al. 2008; Fu et al. 2011; Li and Qu 2011; Ramamoorthy and Kumar 2012). Particle bombardment of calli for switchgrass transformation has also been reported (Richards et al. 2001; Mann et al. 2011), but Agrobacterium infection of plants appears to be the method of choice for switchgrass transformation. A recent study suggests that a high-throughput and reproducible transformation system for the cultivar Alamo has been developed (Casler et al. 2011; Li and Qu 2011) reaching up to 90% efficiency. Other reports note that genetic manipulation of single genes can have a large genomic effect on biomass and conversion properties (Fu et al. 2011; Saathoff et al. 2011a; Xu et al.

2011) . The convergence of DNA sequence analysis and functional genomics demonstrate a trend toward understanding and manipulating gene function for desired traits.

Another approach to unraveling and targeting important pathways in switchgrass is to look directly at the protein and small molecule (metabolite) profiles through global proteomics and metabolomics. For example, the process of lignification is critical in biomass quality and the identification and characterization of the key enzymes involved is important in the identification of targets for manipulation. Through a proteomic approach, key enzymes such as cinnamyl alchol dehydrogenases have been identified (Saathoff et al. 2011b). Global studies into the switchgrass proteome have yet to be deployed, but unraveling the entire compliment of proteins and their modifications will lend key insight into the cellular physiology surrounding key traits. Metabolomics efforts are designed to study and characterize the unique chemical fingerprints that remain from specific cellular processes (Daviss 2005) and these approaches and cellular signatures are excellent tools in a functional genomics perspective for determining phenotype caused by genetic manipulation. Metabolomic analysis of switchgrass is still very few, but prospects to applying these techniques to identify elite lines for biofuel production are on the horizon.

Thermochemical technologies employ high temperature, and use of oxidizing agents or catalysts to break down the biomass polymers into liquid or gaseous fuels. These include combustion, gasification, pyrolysis, liquefaction, and hydrogenation. The most noteworthy difference among these processes is the target products that these processes are used to produce. Heat (or power) is the main direct product of combustion. Gaseous fuel (synthesis gas or producer gas) is the direct product of gasification; whereas, the direct product of pyrolysis, liquefaction and hydrogenation is a liquid (bio-oil) or solid (char) depending on the process operating conditions. In some situations, two or more biomass thermochemical processes are applied in series to increase conversion efficiency, obtain desired chemicals, or reduce environmental emissions. For example, biomass gasification followed by combustion of syngas (or producer gas) provides an opportunity to remove contaminants from the gaseous fuels and to use a gas turbine and other gaseous fuel-based technologies.

Organosolv pretreatment processing involves the use of aqueous organic solvents such as ethanol, methanol, hexane, acetone and inorganic acid catalysts such as hydrochloric acid (HCl) or sulfuric acid (H2SO4) to break the internal lignin and hemicellulose bonds. Organosolv pretreatments, as with other chemical pretreatments, often produce microbial inhibitory agents because it is usually performed at higher temperature (above 180°C) and can require large amounts of pretreatment reagents (Zhu et al. 2010). The organic solvents dissolve lignin and hemicellulose and leave the biomass residue with high cellulose content. The lignin and hemicellulose recovered from the organic solvent have a potential market value (see Table 2).

A plan or listing of activities for establishing and harvesting switchgrass is an essential prerequisite for preparing a switchgrass enterprise budget. The most economical method for establishing stands of switchgrass will differ across regions and soil types. The plan that follows is appropriate for cropland in the U. S. Southern Plains that is harvested in the summer or fall or for cropland pasture.

1. Conduct primary tillage in the fall prior to the spring in which the crop is to be planted.

2. Test the soil and if necessary apply the appropriate levels of phosphorus and potassium fertilizer and agricultural lime.

3. Conduct secondary tillage in late winter and use a cultipacker to firm the seedbed.

4. Wait for rainfall to germinate annual weeds.

5. If weeds are present, apply glyphosate within three days after planting. In some regions some pre-emerge selective herbicides may be registered for use. For example, if registered, s-metolachlor may be applied to fields prior to switchgrass emergence if the seed has been safened with fluxofenin (Vogel et al. 2002). Vogel et al. (2002) also report using atrazine as a pre-emerge herbicide. Mitchell et al. (2012) recommend a combination of quinclorac and atrazine as pre-emerge herbicides for switchgrass establishment.

6. In April, without additional tillage, plant 5.6 kg/ha pure live switchgrass seed 0.6 to 1.3 cm deep in the firm seedbed.

7. If broadleaf weeds are present, apply a labeled post-emerge herbicide.

8. In the summer, if weeds are excessive, a mowing activity with a rotary mower may be warranted before the weeds start to canopy the switchgrass. Clipping the weeds at the top of the switchgrass may increase the probability that sunlight can reach the young switchgrass plants.

9. Exercise patience and permit the young plants to become firmly established. Do not harvest during the establishment year.

10. In late winter after the establishment year, a prescribed burn may be conducted to facilitate new growth.

11.In year 2 and all subsequent years, fertilize with an appropriate level of nitrogen at spring green up and harvest once per year. Late in the growing season, nutrients (including nitrogen, phosphorus, and potassium) translocate from the above ground foliage to the plant’s crown and rhizomes. If harvest is delayed until after the first frost and the initiation of senescence, biomass yield will be maximized and nutrients will have translocated, reducing the quantity of fertilizer needed for biomass production in subsequent years (Madakadze et al. 1999; Sanderson et al. 1999; Reynolds et al. 2000; Vogel et al. 2002; Adler et al. 2006; Guretzky et al. 2011).

Weed control is an important factor in crop production and especially in switchgrass establishment. Weed competition can be reduced by using labeled herbicides or by tilling in the fall and again in the spring prior to planting. Broadleaf selective herbicides may be used to control most broadleaf weeds in stands of young switchgrass. However, grassy weeds can be more problematic. As noted in step 8, if weeds are excessive, a summer mowing activity with a rotary mower may be warranted before the weeds start to canopy the switchgrass. Clipping the weeds at the top of the switchgrass is a strategy designed to enable sunlight to reach the young switchgrass plants. Table 1 includes a listing of the field operations budgeted for switchgrass establishment with conventional tillage. Steps for establishing switchgrass in fields previously used to produce winter annuals such as wheat, barley, or rye are also included in Table 1.

Table 2 includes a listing of field operations that may be used to establish switchgrass without tillage. Adequate soil fertility, weed control, and an effective no-till drill are critical components of successful no-till switchgrass establishment.

Dedicated to

My beloved wife and colleague,
Phullara

Preface

While facing the global fossil energy crisis, switchgrass, a warm-season C4 perennial species has been identified as one of the most important candidate grasses for development into an herbaceous energy crop providing cellulosic feedstock for bioenergy production. A better understanding of this bioenergy crop will greatly facilitate efforts in managing large-scale cultivation, developing novel agronomic, genetic, genomic and molecular as well as chemical and bioengineering tools for enhanced biomass production and bioconversion. This book contains the most comprehensive reviews on the latest development of switchgrass research including the agronomy of the plant, use of endophytes and mycorrhizae for biomass production, genetics and breeding of bioenergy related traits, molecular genetics and molecular breeding, genomics, transgenics, processing, bioconversion, biosystem and chemical engineering, biomass production modeling, and economics of switchgrass feedstock production.

The Chapter 1 by Rob Mitchell and Marty Schmer provides an excellent overview of switchgrass and its development into a biomass energy crop. This is followed by Chapter 2 by John Fike and co-authors discussing the agronomic considerations and potential associated with switchgrass use as a bioenergy crop. In Chapter 3, Chuansheng Mei and co-authors describe beneficial plant-microbe interactions that offer practical ways to improve plant growth and disease resistance, pointing out the great potential of endophytes and mycorrhizae for use in the development of a low-input and sustainable switchgrass production system. The Chapter 4 by Laura Bartley and co-authors gives a thorough and in-depth review of switchgrass biomass content, synthesis and biochemical conversion to biofuels. This chapter provides information about primary and secondary cell wall compositions, and biomass content variation with environment and genotype, discussions on switchgrass cell wall synthesis, regulation and molecular genetics approaches for cell wall modifications as well as biochemical conversion of biomass to biofuels including pretreatment, enzymatic digestion and fuel synthesis. In Chapter 5, Yanqi Wu describes and discusses the progress in switchgrass improvement using classic genetics and breeding, focusing on target bioenergy traits, basic information of inheritance and cytogenetics,

germplasm pools and collections, and breeding and selection methods and the potential to develop hybrid cultivars in switchgrass. In parallel to this, Chapter 6 by Linglong Liu and Yanqi Wu focuses on switchgrass molecular genetics including the development of molecular markers, construction of linkage maps, and application of molecular breeding. Chapter 7 by Christopher Saski and Hong Luo summarizes the recent advances in switchgrass genomics research focusing on structural genomics resources development and their important applications. This is followed by Chapter 8, in which Dayong Li and co-authors provide an overview of the research on switchgrass small RNA molecules, microRNAs, and discuss their potential applications in switchgrass genetic improvement. Chapter 9 by Bingyu Zhao and co-authors is a thorough review on research on tissue culture, genetic transformation, trait modifications using transgenic approaches in switchgrass. The chapter also discusses strategies for future switchgrass improvement. In Chapter 10, Ajay Kumar and Raymond Huhnke provide an overview of major thermochemical conversion processes for conversion of biomass into fuels, chemicals and power, which is followed by Chapter 11 from Terry Walker and co-authors reviewing biological and biosystems engineering for switchgrass feedstocks processing and biofuel production. In Chapter 12, Kathrine D. Behrman and co-authors highlight five applications of process-oriented models of switchgrass growth and show how they can be used to generate a better understanding of large-scale switchgrass biomass production, pointing out the effectiveness of crop simulation models for assessing the sustainability and long-term impacts of converting land to bioenergy crops in a timely and cost-effective manner. Chapter 13 by Francis M. Epplin and co-authors discusses economics of switchgrass feedstock production for the emerging cellulosic biofuel industry, focusing on identifying practical issues related to the economics of developing switchgrass as a dedicated energy crop and providing estimates of the price for delivered switchgrass biomass that would be required to compensate for the cost of inputs used to produce and deliver it to a biorefinery. Chapter 14 by Charles Kwit and co-authors highlights advantages, concerns, and future prospects of developing switchgrass as a bioenergy crop, discussing the role of switchgrass in avoiding biomass for energy and biomass for food dilemma, the impact of switchgrass on climate change and the effects of switchgrass on environmental sustainability and pointing out that improvement efforts in the bioenergy crop switchgrass on multiple fronts present numerous challenges.

The 14 chapters of this book volume contributed by 39 internationally reputed scientists will be of interest and great value to the bioenergy research communities in academia and industry as well as government agencies. It would be an important handbook for agronomists, geneticists, breeders, molecular biologists, physiologists, biosystems engineers and chemical engineers.

We would like to thank all the contributing authors for their excellent work and enthusiastic support and cooperation during the preparation of this volume. Financial support from the USDA National Institute of Food and Agriculture as well as the USDA-CSREES for research on genetic improvement of perennial grasses at Clemson University is gratefully acknowledged.

Hong Luo, Ph. D.

Clemson University

Yanqi Wu, Ph. D.

Oklahoma State University

Chittaranjan Kole

Bidhan Chandra Agricultural University

Switchgrass has an extensive perennial root system which protects soil from erosion and sequesters carbon (C) in the soil profile (Liebig et al.

2005) . Soil organic carbon (SOC) typically increases rapidly when annual cropland is converted to switchgrass (Schmer et al. 2011; Mitchell et al. 2012). Switchgrass grown and managed for bioenergy on three cropland sites that qualified for CRP in Nebraska resulted in an average SOC increase of 2.9 Mg C ha-1 yr-1 in the top 1.2 m of soil in just 5 years (Liebig et al. 2008). In South Dakota, switchgrass grown in former cropland enrolled in CRP stored SOC at a rate of 2.4 to 4.0 Mg ha-1 yr-1 at the 0 to 90 cm depth (Lee et al. 2007). Switchgrass managed for bioenergy on multiple soil types in the Northern Plains stored 4.42 Mg C ha-1 yr-1 into the soil profile (Frank et al. 2004), whereas switchgrass stored an average of 1.7 Mg C ha-1 yr-1 sequestered in the Southeast USA (McLaughlin et al. 2002).