Rob Mitchell[1]‘* and Marty Schmer[2]

This chapter provides an overview of switchgrass and its development into a biomass energy crop. It is not our intent to provide an exhaustive review of a specific topic, but to introduce numerous topics that will be covered in detail in later chapters.

Switchgrass is native to the North American tallgrass prairie and to habitats east of the Rocky Mountains and south of 55°N latitude (Stubbendieck et al. 1997). Switchgrass is a warm-season perennial grass that has the characteristic C4 physiology and anatomy (Parrish et al. 2012). Switchgrass is broadly-adapted to soil types, fertility, and moisture conditions throughout North America. Switchgrass plants are generally caespitose or with short rhizomes and reproduce both sexually and asexually. The main taxonomic subdivision is at the ecotype level named largely for phenotypic differentiation based on habitat (Casler 2012). Switchgrass has two primary ecotypes (upland and lowland) and two primary ploidy levels (tetraploid and octoploid) (Vogel et al. 2011). Switchgrass genotypes are largely self-incompatible and seed production results from cross-pollination by wind (Vogel 2004). Switchgrass generally grows 1 to 3 m tall depending on environment and genetic background and can develop extensive root systems that occupy most of the soil profile to a depth of 3 m (Weaver 1954; Vogel 2004). The extensive fibrous and deep perennial root system protects soil from erosion and sequesters large quantities of carbon (C) in the soil profile (Liebig et al. 2005).

Although interest escalated after switchgrass was mentioned in the 2006 State of the Union Address by President Bush, switchgrass is not a new crop and switchgrass research is not a new undertaking. The USDA location in Lincoln, Nebraska, USA has conducted switchgrass research continually since 1936. As a result, switchgrass was seeded on thousands of hectares of marginally-productive cropland as part of the Conservation Reserve Program (CRP) as well as in pastures and vegetative filter strips throughout the eastern half of the USA. The biomass accumulation and root structure make switchgrass well-suited for both bioenergy production and conservation plantings. For example, switchgrass grown in vegetative filter strips has removed 47% to 76% of the total reactive phosphorus in surface runoff water in areas treated with manure (Sanderson et al. 2001). Although the first 50-years of research and use focused on switchgrass for livestock and conservation, the research since 1990 has emphasized bioenergy (Vogel 2004; Vogel et al. 2011; Parrish et al. 2012).

An important consideration in growing perennial energy crops such as switchgrass is the type and amount of land that will be required to grow an adequate feedstock supply. Switchgrass is well suited to marginally — productive or difficult to farm parcels (Mitchell et al. 2012a, b). Most switchgrass production for bioenergy likely will occur on marginally productive land currently planted to other crops and from areas enrolled in CRP. The ability of these marginally productive sites to provide long-term sustainable production of maize (Zea mays L.) and soybeans (Glycine max L. Merr) is in question. A 5-year study conducted on marginally productive land that qualified for CRP in Nebraska demonstrated that the potential ethanol yield of switchgrass was equal to or greater than the potential ethanol yield of no-till maize grown on similar sites (Varvel et al. 2008). Switchgrass provides environmental advantages compared to traditional annual crops such as reduced inputs, reduced erosion on marginal cropland, and enhanced wildlife habitat (Mitchell et al. 2010a). However, there are concerns about converting CRP to switchgrass. For example, the historic loss of grassland habitat has reduced many grassland nesting birds. The establishment of more than 12 million ha of perennial grasslands under CRP has mitigated this grassland habitat loss and has been a highly successful program for grassland bird species recovery in the Great Plains and Midwest. The response of grassland birds to the conversion of CRP, which is typically floristically and structurally diverse, to switchgrass, which is more uniform, is uncertain (Robertson et al. 2010). In the end, switchgrass must be productive, protective of the environment, and profitable for the farmer to be adopted on a large scale (Mitchell et al. 2012b).

Mitchell et al. (2012b) addressed supplying feedstock to a commercial scale biorefinery. They made some basic assumptions (i. e., an ethanol yield of 334 liters from each Mg of switchgrass dry matter (DM) using SSF, switchgrass yield of 11 to 22.4 Mg DM ha-1, 40 km transport distance) and reported that a 189 million liter (50 million gallon) per year biorefinery requires about 567,000 Mg of feedstock each year. Given these parameters, the total land area required in switchgrass production can range from 5 to 50% of the cropland in the 40-km radius around the biorefinery, depending on the biomass production of the feedstocks.

One viable land resource may be non-irrigated center pivot corners (Mitchell et al. 2012b). A center pivot located on a quarter section (~64 ha, 160 acres) typically irrigates only 53 ha (132 acres), leaving 11 ha (28 acres) of rainfed cropland in the four corners. Consequently, the pivot corners are marginally productive relative to the irrigated land because they receive no supplemental water. For example, a single fuelshed in eastern Nebraska that is heavily irrigated with center pivots could grow 50,500 ha of switchgrass in pivot corners alone, enough for one 189.3 million liter (50 million gallon) per year ethanol plant at 11.2 Mg ha-1 (5 tons acre-1), or two 189.3 million liter per year plants at 22.4 Mg ha-1 (10 tons acre-1) (Mitchell et al. 2012b). Managing switchgrass as a hay crop is not foreign to most farmers and the economic opportunities presented by switchgrass for small, difficult to farm, or poorly-productive fields will provide an economic incentive for many farmers to grow switchgrass (Mitchell et al. 2012b). Due to escalating interest in switchgrass for bioenergy, in-depth evaluations of switchgrass for bioenergy are increasingly available (Vogel et al. 2011; Sanderson et al. 2012).

Commercially available harvesting technologies are in use on farms to harvest and package forages for livestock and, in most cases, can be used on high-yielding (> 12 Mg ha-1) switchgrass fields (Mitchell and Schmer 2012). Harvest machines with rotary heads are superior to those with sicklebars. Use of the latter likely will be limited to low-yield or small farm operations where harvesting switchgrass can take advantage of existing hay-making equipment but where energy cropping is not the primary enterprise.

With large-scale commercial bioenergy production, rotary mowers will be required to efficiently handle the volume and coarse stems typical of bioenergy switchgrass production fields and will be facilitated by self- propelled harvesters (Mitchell and Schmer 2012). For independent mowing and baling operations, a cutting height of 10 to 15 cm will keep the windrows elevated above the soil surface; this facilitates air movement under the swath and speeds drying to less than 20% moisture prior to baling (Vogel et al.

2011) . However, senesced switchgrass often can be dry enough for one-pass (i. e., essentially simultaneous) cutting and baling operations in which the baler is pulled behind the swather (Fig. 4). Such operating systems likely will be commonplace in well-developed harvest systems.

While it is often assumed that switchgrass will be packaged for storage and transportation in large round or rectangular bales (Mitchell et al. 2010; Vogel et al. 2011), there is some question about the best methods of switchgrass collection. Chopping with direct hauling or chopping and pressing into modules has been suggested as an alternative to baling (Popp and Hogan Jr. 2007; Bransby et al. 2008; Sokhansanj et al. 2009) and some analyses suggest this will be the preferred method of harvest (Larson et al.

2010) . Advantages and disadvantages are evident for each system, and we discuss these briefly below.

In bale-based systems, the baling step densities and bundles switchgrass or other biomass; this eases handling, transport and storage needs, in part because baling breaks the link between in-field harvest and hauling. Because bales can be dropped on the ground and recovered later, this provides an important advantage over a chopping-based system, which will require at least two laborers at any time—one to harvest and one to haul away the material. Bales also are easy to handle with front end loaders or forklifts and require less storage infrastructure.

When comparing bale types, large round bales have a storage advantage in that they have fewer losses than large rectangular bales when stored outside (Cundiff and Marsh 1996); this may have greater importance in the humid east. In contrast, large rectangular bales will be the bundling method of choice in regions with larger field sizes and lower amounts of precipitation. This is because large square balers can achieve higher bale density and the bales tend to be easier to handle and to load on a truck for transport without road width restrictions (Mitchell et al. 2010).

While baling can reduce labor needs on the harvest-end of a biomass system, moving bales from fields to storage locations (and at any other stage in the process) can significantly increase the harvest system’s labor and equipment costs. Bales also will need to be size reduced (for flowability and improved conversion) at a satellite storage or process site. Thus, although the added labor and infrastructure of chopping at harvest has been noted, there is significant interest in using in-field chopping to eliminate a downstream process step. Some analyses suggests these systems are most cost effective (Larson et al. 2010) but actual head-to-head comparisons are few and the outcomes depend on several assumptions about equipment costs (e. g., new vs. existing), use (hours/day) and field site accessibility among others.

Laura Bartley,* Tao Xu, Chengcheng Zhang,
Hoang Nguyen and Jizhong Zhou

Introduction

Compared with current commercial biofuel production from sugars, starch, and oils in food commodities, future generation biofuel production has potential to improve energy yields and reduce green house gas emissions (Farrell et al. 2006; Fargione et al. 2008; Schmer et al. 2008). The polysaccharides and lignin in plant vegetative tissues, i. e., leaves and stems, are an abundant source of chemical energy for biofuel production. This so-called lignocellulosic material includes dedicated energy crops as well as agricultural and industrial waste products, such as corn stover, wheat straw, and paper mill waste. Economists estimate that grass biomass represents ~55% of the biomass that can be sustainably produced in the United States, with about half of this from agricultural residues and half from perennial crops (US-DOE 2011). Among candidate bioenergy grasses, switchgrass (Panicum virgatum L.) has received the most attention due to its relatively high yield even under low-input conditions (Thomason et al. 2004), among other factors. The two major approaches for production

Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019.

Corresponding author: lbartley@ou. edu

of biofuels from lignocellulosic biomass are biochemical conversion and thermochemical conversion. With ground-breaking on a handful of lignocellulosic biochemical conversion production plants in the U. S. in 2012, biochemical conversion is close to commercialization and will be the main focus of this chapter. Thermochemical conversion is briefly discussed here and thoroughly covered in other chapters of this volume.

Since associations between genetic markers and quantitative traits were first reported (Sax 1923), much attention has been given to the potential uses of markers in breeding programs. A DNA molecular marker is an identifier (sometimes called a "tag" or "flag") of a particular genotype variant in the DNA sequence. Compared to morphological and biochemical markers representing phenotypic and biochemical variations (e. g., flower color, flowering time, seed size, seed coat color and sugar content), DNA molecular markers have several advantages: 1) not subject to environmental influence and usually more objective; 2) widely distributed in the whole genome; 3) can be detected in any stages of life including early growth stages of plants; 4) more abundant and less expensive than morphological markers, especially when they can be genotyped in high-throughput, which is greatly facilitated by the next-generation sequencing technologies. Molecular markers have been widely used to study genetic diversity, decipher evolution and phylogenetic relationships, characterize inheritance of plant traits, and assist in selective breeding in switchgrass.

miRNAs in switchgrass have recently been studied using both bioinformatics and experimental approaches (Matts et al. 2010; Xie et al. 2010), providing a first glimpse of the miRNA components and their possible target genes in switchgrass.

Using bioinformatics approaches, Matts et al. (2010) identified a total of 16 conserved miRNA families in switchgrass, among which 12 families are conserved between monocotyledonous and dicotyledounous, whereas 3 families (miR437, miR444, and miR528) are conserved only among monocotyledonous plants. The predicted fold-back structures of these switchgrass miRNAs are also conserved (Matts et al. 2010). With different criteria for computational strategies, Xie et al. discovered 121 conserved miRNAs belonging to 44 miRNA families (Xie et al. 2010). Unlike Matts et al. who only discovered one member in miR444 family similar to that in wheat (Yao et al. 2007) and Brachypodium distachyon (Unver and Budak

2009) , Xie et al. identified 13 members in miR444 family. Another interesting discovery from Xie et al. study is that they identified miR414 family, which had previously been discovered only in Arabidopsis, rice and moss (Wang et al. 2004; Fattash et al. 2007), but supposedly should exist in all plant species because of its existence in both dicot (Arabidopsis) and monocot (rice) as well as moss (Xie et al. 2010). Xie et al. also discovered one miRNA cluster (including miR2118a and miR2188b), and miR164 with an antisense miRNA in switchgrass (Xie et al. 2010).

Using experimental approaches, Matts et al. identified 34 conserved miRNAs from 16 families in switchgrass. Based on frequencies of different miRNAs in the library, miR172 family and miR156 family are the most abundant (Matts et al. 2010). Expression analysis of these miRNA families in different organs and developmental stages demonstrated that most miRNA families are expressed ubiquitously, whereas a few showed a distinct tissue-specific pattern. They also discovered that unlike other plant species in which miR395 and miR399 are induced in sulfate and phosphate deficit conditions (Jones-Rhoades and Bartel 2004; Fujii et al. 2005; Jagadeeswaran et al. 2009; Matts et al. 2010), miR395 and miR399 are detected to be expressed in relatively high basal level in switchgrass under optimal growth conditions, and their expression levels are only slightly changed under low sulfate and phosphate conditions, indicating the potential of switchgrass adaptation to sulfate — and phosphate-deficit soil (Matts et al. 2010).

Based on high complementarities between plant miRNAs and their targets, Matts et al. predicted 37 targets for conserved miRNAs in switchgrass, most of which are transcription factors (including SBP, MYB, TCP, NAC, ARFs, Scarecrow-like, AP2, MADS and CBF families), whereas others are transport inhibitor response 1 protein, Argonaute 1-like protein, plant acyanin and ubiquitin conjugating enzyme (Matts et al.

2010) , indicating a diverse role switchgrass miRNAs play in development, reproduction and stress response through regulating different targets. Of the predicted targets, only 4 (NAC for miR164, HD-zip for miR166, SPL for miR156 and AP2—like for miR172) are confirmed by modified 5’RACE (Matts et al. 2010). Xie et al. (2010) identified a total of 839 potential targets for switchgrass miRNAs. They also conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to predict biological processes and metabolic pathways these miRNAs-targets may be involved in (Xie et al. 2010). Their analysis indicated that 19 miRNAs might play a role in biofuel-related metabolic pathways and have the potential to contribute to enhancing biofuel production from switchgrass in the future (Xie et al. 2010).

Syngas is converted into alcohols using microbial or chemical catalysts. Syngas fermentation research using micro-organisms such as strains of Clostridium ljungdahli, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium ragsdalei and Alkalibaculum bachi yielded ethanol, butanolisopropanol and acetic acid. Recent advances in the syngas fermentation include developing new strains of microorganisms, improved reactor design and optimized conditions such as temperature, pH, buffer presence and media to increase yield and reduce the cost for production of alcohols (Kundiyana et al. 2010, 2011a, b; Maddipati et al. 2011; Liu et al. 2012).

Chemical catalysts have also been used to convert syngas into mixed alcohols. The process takes places at high pressure and low temperature in presence of catalysts with the function of hydrogenation, C-O bond breaking and CO insertion. Catalysts based on both noble and non-noble metals have been used for synthesis of mixed alcohols. Noble metal based catalysts containing Rh, Ru or Re and supported on oxides such as SiO2 and Al2O3 have high alcohol selectivity but are not economical for commercial applications. Major non-noble metal based catalysts for mixed-alcohol synthesis contain MoS2, Cu-Co, Cu-Zn-Al and Zn-Cr-K (Fang et al. 2009). Recent advances in mixed alcohols production using chemical catalysts include synthesis and development of new catalysts, optimization of reaction conditions to increase yield and reduce cost of alcohols production. However, the alcohol synthesis process still suffers from low yield and poor selectivity of the desired alcohol product (Subramani and Gangwal 2008). Syngas is also considered building block for many chemicals, such as aldehydes and acetic acid, produced through catalytic and microbial conversions. Hydrogen can also be separated from syngas for producing ammonia or refining hydrocarbon fuels.

Biofuel production (such as ethanol) is a complex process using lignocellulosic feedstock (such as switchgrass, sweet sorghum bagasse, pine wood chips, etc.) compared to sugarcane syrup or corn. The carbohydrates in lignocellulosic feedstock are much more problematic in terms of both solubility and utilizing their different component sugars (mainly glucose, xylose and arabinose) compared to starch in corn or sucrose in sugarcane syrup. The complexity of lignocellulosic feedstock provides different routes for fermentation including direct microbial conversion (DMC), separate hydrolysis and fermentation (SHF) and simultaneous saccharification and co-fermentation (SSCF).

Note:

ag ethanol gA xylose consumed

bThe relevant genotype is given in parantheses. pdc, pyruvate decarboxylase; pfl, pyruvate formate lyase; adhB, alcohol dehydrogenase II;/rd, fumarate reductase, pZB5 carries the genes for xylose isomerase, xylulokinase, transketolase and transalolase cMaximum volumetric productivity

dFigures in parentheses denote number of strains investigated (if more than one)

eThe relevant genotype is given in parenthese. XYL 1, xylose reductase; XYL 2, xylitol dehydrogenase; xyl A, xylose isomerase

Note:

SSF, Simultaneous saccharification and fermentation SSCF, Simultaneous saccharification and co-fermentation SHCF, Separate hydrolysis and co-fermentation

Direct Microbial Conversion (DMC) or Consolidated Bio-Processing (CBP)

Direct Microbial Conversion (DMC) is a consolidated process of production of cellulolytic enzymes (cellulase and xylanase mixture), hydrolysis of lignocellulosic biomass and fermentation into bioproducts such as ethanol in a single vessel. Clostridium phytofermentans would be an ideal microorganism for DMC ethanol production. However, C. phytofermentans has been reported to produce low ethanol yields (less than 0.2% (w/v)) with several by-products such as hydrogen, acetic acid, and formic acid that ultimately lowers ethanol productivity (Warnick et al. 2002).

Second generation lignocellulosic bioenergy crops are often viewed as environmentally sustainable relative to first generation and non-renewable alternatives in the transportation fuel sector (Dale et al. 2011). In addition to the carbon emissions-affiliated metrics discussed above, there are a number of indicators that can provide information on the environmental quality of bioenergy systems (McBride et al. 2011). These include indicators affiliated with soil quality, water quality (and quantity), biodiversity, air quality, and productivity. Here we concentrate on providing a brief overview of effects of agronomic production of switchgrass on (a) soil and water quality, (b) biodiversity, and (c) invasiveness issues. In some contexts more than others, the fact that it would likely be grown in large monocultures at expansive spatial scales is highly relevant.

Switchgrass can be established with conventional planting techniques— broadcasting or drilling into a well-till seedbed—or with no-till planters that can drill seed into un-worked bare soil, killed sod or crop stubble. Aside from environmental considerations associated with tillage, perhaps the biggest issue with planting method is the ability to achieve proper planting depth. As noted above, seed depth is critical for successful establishment, and poor seed placement has been a common cause of stand failure with both conventional drilling or broadcast and no-till seeding methods.

Broadcast seeding is the least preferred of planting methods and typically only successful when the seedbed is rolled or compacted after the seed are broadcast (Evers and Butler 2000; Monti et al. 2001). Rolling or packing likely pushes some seed to the appropriate depth, allowing for establishment and survival.

Comparisons of switchgrass establishment by drilling into tilled or non-tilled seedbeds are often confounded and inconsistent (Parrish and Fike

2005) with outcomes dependent on year, location and presence of residues (e. g., see King et al. 1989). Success with no-till seeding methods have been possible for some time (e. g., see Wolf et al. 1989) and Rehm (1990) reported no yield differences between the two planting methods. Some studies have reported greater seedling numbers with no-till planting methods (Harper et al. 2004) but seedling numbers may or may not have long-term effects on stand productivity (see section on row spacing below).

Row Spacing

Although less critical than planting depth and timing, appropriate row spacing allows plants to optimize resource capture with decreased seeding rates. Reducing competition can allow more efficient resource use and support increased yields. Results in Texas from row spacing experiments during establishment years indicate that switchgrass produces more robust plants with greater tiller density and mass as row spacing increases (Muir et al. 2001). Increasing row spacing from 20 to 80 cm also increased yields in Alabama without N fertility (Ma et al. 2001). Similar data are reported by Foster et al. (2012) who found that switchgrass planted at row spacings of 17.8 to 88.9 cm had similar DM yield. These data indicate row spacing may not be a major consideration to successful establishment of switchgrass as a biomass energy crop at low latitude locations. Although there is little information to guide such planting decisions at greater latitudes, we would predict similar outcomes. Potential for success with various row widths offers greater flexibility for farmers with different types of drills and who might apply mechanical tools for weed control.

Plant growth-promoting bacterial endophytes can affect growth directly by providing bacterium-synthesized compounds, often plant hormones, and by facilitating the acquisition of compounds from the environment, including atmospheric nitrogen fixation. Endophytes may also act indirectly by decreasing or preventing the colonization or the deleterious effects of pathogenic organisms (Lodewyckx et al. 2002) by producing antibiotics to outcompete plant pathogens (Bibi et al. 2012).

One of the most well-studied bacterial endophyte associations is atmospheric nitrogen fixation by specific endophytes. This symbiosis is well known in leguminous plants (Stacey et al. 2006) where the soil bacteria Rhizobia infect the roots of the host plants, inducing the formation of nodules where they fix atmospheric nitrogen and provide it to the host plant in exchange for carbon compounds (Lodewyckx et al. 2002). Additionally mutualistic associations through the fixation of nitrogen can also be observed in non-leguminous plants, such as rice (Mattos et al.

2008) , maize (Montanez et al. 2009), sugarcane (Oliveira et al. 2009), wheat (Webster et al. 1997), strawberries (de Melo Pereira et al. 2012), and grasses (Reinhold-Hurek et al. 1993; Kirchhof et al. 2001).

Nitrogen-fixing bacteria have been studied extensively in the bioenergy crop sugarcane, and include Gluconacetobacter spp., Azospirillum spp., Herbaspirillum spp. and Burkholderia spp. (James and Olivares 1998; James et al. 2001; Suman et al. 2005; de Carvalho et al. 2011). In fact, in Brazil, the cultivation of sugarcane uses only a small amount of fertilizer (de Carvalho et al. 2011) without showing nitrogen deficiency symptoms (Rosenblueth and Martinez-Romero 2006), and there is evidence that a significant amount of nitrogen is obtained from plants associated with bacterial endophytes (de Carvalho et al. 2011). To date, there are no reports on nitrogen-fixing bacterial endophytes in switchgrass, and screening for diazotrophic bacteria that inhabit switchgrass is under way in our laboratory.

There have been numerous publications on plant growth promotion by bacterial endophytes (see review in Berg 2009; Mei and Flinn 2010). In switchgrass, young seedlings of the cultivar Alamo inoculated with Burkholderia phytofirmans strain PsJN, isolated from onion roots (Frommel et al. 1991), showed significant growth promotion with an increase of root and shoot length of 35.6% and 32.8%, respectively, as well as an increase of fresh weight of 83.6% compared with control plants (non-inoculated) after one month under in vitro conditions (Kim et al. 2012). The same pattern was observed under growth chamber and greenhouse conditions, where plants inoculated with the B. phytofirmans strain PsJN showed persistent growth vigor with significant increases in fresh and dry weights, and an increase in the number of early tillers (Kim et al. 2012). Also, results showed that B. phytofirmans strain PsJN has potential in the development of a low input and sustainable switchgrass feedstock production system on marginal lands as higher biomass yields were observed under sub-optimal growth conditions with PsJN inoculated plants over control (Kim et al. 2012). However, PsJN growth promotion is genotype specific in switchgrass as the upland cultivar Cave-in-Rock did not respond to inoculation. We are currently isolating bacterial endophytes from switchgrass tissues and have made progress in screening and selecting beneficial bacterial endophytes which have a broad spectrum of growth promotion in various switchgrass cultivars.