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PROJECT NARRATIVE

Carbon sequestration and greenhouse gas emissions associated with cellulosic bioenergy feedstock production on marginal agricultural lands in the Lower Mississippi Alluvial

Valley  

INTRODUCTION Current Research

The long-term goal of our research is to develop ecologically and economically viable agroforest cropping systems for producing cellulosic bioenergy feedstocks on marginal agricultural land in the Lower Mississippi Alluvial Valley (LMAV). We established a study in 2009 to evaluate the ability of eastern cottonwood (Populus deltoides L.) and switchgrass (Panicum virgatum L) to produce cellulosic bioenergy feedstocks as well as important ecosystems services such as wildlife habitat and nitrogen retention. Individual stands of cottonwood and switchgrass as well as alley cropped switchgrass and cottonwood agroforests (15-20 m alleys) were established at three sites (Figure 1) on poorly drained agricultural land that had been in row crop production. In addition a regionally common row crop rotation (soybean-grain sorghum) for these poorly drained soils is being grown at each site. Biomass production, biofuel quality, production economics, nitrogen loss, and small mammal populations of these cropping systems are being determined and will be compared among the various cropping systems. To facilitate our ability to determine carbon sequestration of these various cropping systems, we sampled soil prior to establishing all crop types to provide initial baseline soil carbon pools for these sites and the study. Analyses indicated that prior to study establishment C pools in the surface 30 cm were respectively 25.6, 31.9, and 44.6 Mg ha-1 at the Pine Tree, Rohwer, and Archibald study sites, respectively. Current research efforts are funded through June 2012, allowing us to observe biomass and ecosystem service characteristics through the establishment phase of the study. These cottonwood and switchgrass crops will mature and be harvested in upcoming years, providing an opportunity to expand the scope of our research. Switchgrass will begin to reach maturity for annual harvesting in 2011, and eastern cottonwood will reach merchantable size and potentially maximum production in 2013.

Figure 1. Study site locations

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Objectives

We propose to enhance this project by more fully focusing on the potential for these systems to sequester C in biomass and soil as well as to quantify the fluxes of C associated with producing feedstocks and fuels from these cropping systems. We plan to extend our research beyond the establishment phase of the switchgrass and cottonwood by monitoring key aspects (biomass, biofuel feedstock production, greenhouse gas emission) of the cropping systems past the first scheduled harvesting date (the fifth growing season) of the cottonwood. To accomplish these goals, our objectives are to: (1) quantify net total above- and below-ground biomass C production and above-ground biofuel production, (2) determine total C accumulation within the soil profile, (3) measure gaseous emissions of CO2, N2O, and CH4, (4) perform life cycle assessment of C associated with the production of these biofuel feedstocks and conversion to biofuel, and (5) elucidate soil-plant physical and biological processes that impact C cycling. These components will then be compared among the crops and systems to assess the sustainability of cellulosic bioenergy feedstock production for drop-in liquid biofuel production. Background

The LMAV has a high potential for bioenergy crop production due to its long growing season and well-developed agricultural infrastructure (Trip, Powell, and Nelson 2009). However, for escalation of biofuels production of the region to be sustainable, feedstock alternatives to conventional biofuel crops, notably corn and soybean, are needed to mitigate the environmental, economic, and social constraints to conventional biofuel production. Reliance on conventional agricultural crops for producing transportation fuels can be associated with increased greenhouse gas emissions if land is converted from forests or grasslands into intensive agriculture (Sedjo and Sohngen 2009). Conversion of forest to intensive agriculture also reduces cover and mast production important for sustaining wildlife habitat productivity. Crops currently used as biofuels also require high levels of resource inputs (fertilizer, water, pesticides, and/or machinery) to provide acceptable levels of feedstock production. Furthermore, corn and soybean are vitally important to global food security, so the ethics and feasibility of diverting increasing proportions of these crops to biofuels production are uncertain (Pimentel and Patzek 2005).

Growing crops dedicated as energy feedstocks could increase biofuel production capacity with relatively minimal adverse environmental impacts. Key required traits for energy crops include the ability to be converted into virtually any energy form and higher biomass yields per unit of land area than conventional agricultural crops. High biomass growth capacity of energy crops can minimize land requirements, agricultural chemical use, and transportation costs (European Commission Biofuels Research Advisory Council 2006). Energy crop production can be a closed-loop process in which the entire production process from planting to conversion of biomass to energy results in small positive net CO2 emissions, with the only emission sources being fuel consumption associated with harvesting, transportation, and feedstock preparation operations (Haq 2002). Switchgrass and cottonwood are among the most promising species for use as dedicated energy feedstocks, particularly on poor soils (Thorton et al. 1998; Stanturf et al. 2000). Switchgrass, a perennial C4 grass, has been identified by the U.S. Department of Energy as a model energy crop species after a comprehensive evaluation of potential feedstocks (Vogel 1996). Switchgrass is native to much of North America, with the exception of areas west of the

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Rocky Mountains and north of 55° north latitude (Vogel 2004). Switchgrass biomass yields range between 11 to 36 Mg ha-1 yr-1 (Comis 2006). Switchgrass can be established from seed at low cost, grown with minimal fertilization, produces high yields even on marginal or suboptimal soils, and tolerates both drought and flooding (BFDP 2006). Switchgrass has several traits that foster relatively high sequestration of atmospheric CO2. It has an extensive deep root system, a low fertilizer requirement, and high biomass growth rates (Ma et al. 2000, Frank et al. 2004). Johnson et al. (2007) found that switchgrass root density is an order of magnitude greater than that of soybean and more than 3-fold greater than that of corn, suggesting a greater capacity for switchgrass to sequester C in soil relative to these conventional biofuel feedstocks. Carbon accumulation rates of 2.4 to 4.0 Mg ha-1 yr-1 within the top 90 cm of soils have been observed in switchgrass grown in conservation easements in the northern portion of its range (Lee et al. 2007).

Similar to switchgrass, the U.S. Department of Energy identified cottonwood as a model energy crop species in its comprehensive Short Rotation Woody Crops Program, later renamed the Bioenergy Feedstock Development Program (Kszos et al. 2001). Short rotation woody crop management entails use of improved genetic stock, competition suppression, and fertilization to produce fast-growing trees. Woody biomass produced from these systems has high density and low ash content, is easy to store and handle, and mixes easily with other woody feedstock such as mill wastes and forest harvest residues (Johnson et al. 2007). Cottonwood is one of the fastest-growing native trees in the southern portion of the United States and attains its highest growth rates on wet soils in the Mississippi Valley. On these soils cottonwood can grow 1.5 to 2 meters in height each year, with production rates ranging from 5 to 20 Mg ha-1 yr-1 (Johnson et al. 2007). Carbon in soils in which short rotation woody crops are grown has been shown to increase relative to that of agricultural soils. As such, fields used to grow short rotation woody crops can also be substantial carbon sinks (Johnson et al. 2007). Agroforest systems that grow alleys of switchgrass between rows of cottonwood trees could be an ecologically and economically superior alternative to conventional agricultural cropping systems for producing biofuel feedstock on marginal agricultural land in the LMAV. Agroforest systems are among the most productive and environmentally benign agricultural systems. Agroforests are designed to optimize the use of growing space, water, light, and nutrients. As such, agroforests are associated with numerous economic and environmental benefits, including: (1) greater total yields, (2) risk mitigation, (3) product diversity, (4) low fertilizer and herbicide costs, (5) improved soil nutrient usage and recycling, (6) improved soil and water quality, (7) enhanced plant, animal, and microbial biodiversity, and (8) greater sequestration of atmospheric carbon dioxide than conventional agricultural cropping systems and forests (Best et al. 1990, Garrett and McGraw 2000, Jose et al. 2004, Sharrow and Ismail 2004).

Agroforests have been developed to provide an assortment of management alternatives for combining trees and grasses (e.g., silvopasture systems in which trees are managed within pastures) and for combining trees and annual or perennial crops (e.g., alley cropping systems where crops are planted between rows of trees)(Gold et al. 2000). However, agroforest systems for combining cottonwood and switchgrass for biofuel production have not been fully developed or evaluated. Switchgrass has been shown to grow more favorably under shade of longleaf pine (Pinus palustris Mill.) than in open sunlight in natural ecosystems of the mid-South U.S. and under shade of loblolly pine in an alley cropping system (Pitman 2000, Blazier 2009). Accordingly, switchgrass may have a high potential for incorporation into an alley cropping system with cottonwood. An alley cropping system combining switchgrass and cottonwood

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would be unique in that it would combine two dedicated energy crops with high biomass production potential. Carbon sequestration potential of switchgrass grown in an alley cropping system may be greater than that of switchgrass grown in pure stands because higher levels of carbon storage have been found in agroforests such as silvopastures than in forests and pastures (Sharrow and Ismail 2004). RATIONALE AND SIGNIFICANCE

Switchgrass and cottonwood have high potential for sequestering C when managed as cellulosic biofuel feedstocks. Relative to conventional agricultural crops used for biofuel production, switchgrass and cottonwood have low nutrient and agricultural chemical requirements, which foster minimal fossil fuel expenditures associated with farm equipment. Additionally, switchgrass and cottonwood have high annual biomass growth rates, which promote C sequestration in biomass. Due to the deep, dense rooting habit of switchgrass and expansive rooting of cottonwood, these species may also foster long-term C storage in soils (Harlow et al. 1991, Ma et al. 2000, Frank et al. 2004). For a comprehensive understanding of the potential of switchgrass and cottonwood management systems to sequester C, fluxes of C associated with these cropping/management systems need to be monitored throughout multiple harvesting cycles on a variety of sites. Monitoring of C sequestration and fluxes necessitates monitoring of : (1) above- and below-ground biomass C, (2) long-term storage forms of C in soil, (3) greenhouse gas emissions, and (4) “carbon costs” of feedstock production associated with fossil fuel emissions of equipment used in management and harvest activities. The objective of this proposed project is to monitor these phenomena throughout several harvesting cycles of switchgrass and the first harvesting cycle of cottonwood grown as monocultures and agroforests. This study will address the Carbon Sequestration and Sustainable Bioenergy Production Priority of the Sustainable Bioenergy Research Program because we will quantify the effect of producing cottonwood and switchgrass biomass on C sequestration in both soil and standing biomass as well as production effects on greenhouse gas emissions. We will also compare C sequestration and greenhouse gas emission of switchgrass and cottonwood cropping systems to that associated with a soybean-grain sorghum rotation in order to elucidate the potential impacts of the conversion of conventional cropping systems to biofuel feedstock production systems on C sequestration. This study would likely provide needed information for the development of agricultural policies associated with biofuel feedstock production in the LMAV. Our research focuses on switchgrass and cottonwood production for cellulosic biofuel feedstocks in the LMAV because this region is uniquely suited for biofuel production, particularly drop-in biofuels such as biogasoline. The LMAV is centrally located within the U.S. and has a long growing season and precipitation patterns capable of growing a wide array of crops. The LMAV has a substantial agricultural infrastructure, with approximately 16 million acres currently being used for conventional agricultural production (Gardiner and Oliver 2005). This infrastructure can foster the emergence of a biogasoline industry due to the widespread availability of farmers and crop consultants with interest in innovative cropping techniques and investment opportunities, farming and forestry equipment, crop storage facilities, and close access to railways, the Mississippi River, and the Gulf of Mexico to facilitate biomass transport (Trip, Powell, and Nelson 2009). The LMAV also has an extensive infrastructure for producing and transporting fuels. Louisiana ranks fourth among all states in crude oil production and second among states in refining capacity. Louisiana supplies much of the Southeast U.S. with

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motor gasoline via the Plantation Pipeline originating near Baton Rouge (EIA 2010). Due to this refining and pipeline infrastructure, a biogasoline industry would be regionally appropriate because biogasoline could readily be blended and/or transported via this infrastructure. Switchgrass and short-rotation woody crops such as cottonwood have been identified as the most promising feedstocks for biogasoline production (Huber and Dale 2009). Switchgrass and cottonwood are native to the LMAV region and may achieve high growth on soils of marginal quality for conventional crops.

A novel biomass production facet of this research is the integration of switchgrass and cottonwood management in an agroforestry alley cropping system. There are currently no agroforestry management systems that combine woody and perennial grass together as a dedicated energy cropping system. Combining cottonwood and switchgrass in such systems can diversify farm production capacity of biogasoline feedstocks and improve farm C sequestration potential. Both species produce high biomass yields and have high below-ground biomass growth. The species differ in the portions of the soil profile exploited by their root systems, with switchgrass having deep, dense rooting and cottonwood having shallow, expansive rooting (Harlow et al. 1991, Ma et al. 2000, Frank et al. 2004). This diversity in belowground biomass may increase overall long-term storage of C in soil when these crops are grown as agroforests and thus improve the C sequestration potential of these biofuel feedstocks.

This project has the potential to yield several improvements in the long-term sustainability of U.S. agriculture and food systems. Development of viable management systems for growing cottonwood and switchgrass crops for biofuel production on marginal soils of the LMAV will allow productive soils to remain in conventional agriculture to sustain food production capability of the region. Additionally, producing switchgrass and cottonwood in the LMAV could reduce nutrient transport in surface and subsurface waters due to the relatively low fertilizer needs of these crops and the lack of annual tillage and reestablishment that occurs with the annual crops currently grown on these soils. The LMAV contributes a relatively high amount of P/watershed area to the Mississippi River and Gulf of Mexico (Goolsby et al. 1999). Nitrogen fertilization of agricultural crops has been shown to lead to NO3-N contamination of waterways, which can prompt eutrophication that reduces fish populations (Burkhart and James 2001). A prevalent example of fish reductions from agriculture-derived nutrient transport to waterways is the seasonal 18,000-km2 hypoxic zone that forms each year in the commercially significant fisheries of the Gulf of Mexico (National Ocean Service 2003). Adding switchgrass and cottonwood to the landscape of the LMAV can also contribute to restoring C sequestration capacity of the region because switchgrass and cottonwood have higher C sequestration potential than conventional agricultural crops. Native forest cover in the LMAV was reduced by 26% from the 1700’s through the 20th century as land was cleared for agriculture, thereby reducing the region’s long-term C sequestration potential (Gardiner and Oliver 2005). APPROACH

We will utilize an ongoing cottonwood/switchgrass agroforest study to evaluate the

impact of the biofuel production of these cropping systems on carbon sequestration. With the addition of this proposed project, carbon accumulation, carbon losses, greenhouse gas (GHG) emission, and life cycle analyses will be performed from data collected during the first seven years following establishment of the agroforest project. Our proposed methods are described in this section, and an illustrated timeline of the activities to be pursued is provided in the

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Figure 2. Schematic of plots, three replications at each study site.

“Timeline of Study Activities” subsection on page 17 of this project narrative. Overall Study Design

Agroforest systems have been established on three sites: 1) Pine Tree Branch Experiment Station, UA Division of Agriculture, Colt, AR- “Pine Tree” 2) the Southeast Research and Extension Center, UA Division of Agriculture at Rohwer, AR-“Rohwer”, and 3) the Stevenson Farm in Archibald, LA-“Archibald”. Locations of the three study sites are shown in Figure 1. At each study site, three blocks were established in early 2009, each with plots receiving one of five treatments; 1) 100% cottonwood (W), 2) 100% switchgrass (S) 3) 67% cottonwood and 33% switchgrass (WS), 4) 33% cottonwood and 67% switchgrass (SW), and 5) a control consisting of a conventional (C) soybean-grain sorghum rotation (Figure 2). Cottonwood and switchgrass in the WS and SW treatments were established in alternating 15 and 20 m alleys to provide the appropriate composition. The blocks are located at each site in such a way to capture potential soil variation. Two blocks have plots that are 30 x 90 m in size and the other block is established with 90 x 90 m plots. The larger plots are used to support ongoing small mammal monitoring (funded by previous grants) as well as to estimate harvest costs at a more “operational size” scale. In each of the plots in all three blocks a 17 x 45 m measurement subplot has been located to monitor vegetation biomass as well as other biotic and abiotic parameters. Vegetation outside these plots is used for destructive sampling when needed.

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Figure 3. Cottonwood plot, Rowher study site, August 2009.

Figure 4. Switchgrass plot, Archibald study site, April 2010.

Treatment Establishment

Cottonwood establishment consisted of planting 40 cm cuttings of three clones (ST-66, S7C20, and mix of clones from a LA Dept. of Agriculture and Forestry nursery) in February 2009 at a density of 4,495 cuttings/ha following mid-winter site preparation (subsoiling) and pre-emergent herbicide application. Survival rates of the three cottonwood clones at the end of the first growing season were between 80 and 85%. Cottonwood trees will be grown for five seasons and harvested in fall 2013. After harvest, the trees will be allowed to re-sprout for a second rotation. A FLD Biobaler WB55 or similar harvesting technology will be used to remove and bundle the cottonwood trees or trees will be harvested and chipped and hauled to a processing facility. A cottonwood plot is illustrated in Figure 3.

Switchgrass was drilled in the soil at a rate of 11.2 kg ha-1 between late April and mid-May 2009 following application of fertilizer where needed. Switchgrass establishment was unsuccessful (<40% coverage at the end of the first growing season) at one site and replanted in May 2010 along with supplemental plantings where needed at the other two sites. Switchgrass will be harvested annually beginning in October 2010 by mowing with a commercial forage-grass cutter, field curing to less than 20% moisture content (wet basis), and baling with a large baler. A switchgrass plot is illustrated in Figure 4.

The row crop planted and harvested in 2009 was soybeans using typical agricultural practices for the local area of each site. Grain sorghum was planted in 2010; soybeans will be grown in the next two consecutive years (2011-2012) before grain sorghum is again planted in 2013.

All material inputs, labor, and machine times associated with establishment and maintenance of all cropping systems are recorded annually.

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Objective 1: Quantify net total above- and below-ground biomass C production

Aboveground: Current funds (USDA SARE and Sun Grant) are being utilized to monitor aboveground biomass production and biofuel quantity and quality (Q&Q) of the various cropping systems and vegetation during the 2009-2011 growing seasons. The requested funds will extend this monitoring into the 2016 growing season, two years following the first planned harvest of the cottonwood trees. Switchgrass and soybean or grain sorghum biomass is determined by harvesting a set area within each treatment annually. Tree biomass will be determined by developing allometric equations from destructively sampled trees and applying these estimates to tree diameter and heights which are measured annually. The first destructive sampling to develop these allometric equations occurred outside the measurement subplots in February 2010; a total of 90 trees (1/3 from each clone) were destructively sampled at each site. Destructive sampling will occur again at age 3 (using current funding) and again at age 5 at the time of the first cottonwood harvest (using proposed funding). Carbon concentrations and biofuel QQ will be determined for the harvested samples from the switchgrass and soybean-grain sorghum crops annually while these parameters will be determined for the cottonwood trees from the destructive samples taken at ages 1, 3 (using current funding) and 5 (using proposed funding). Below is a summary of the methods used to measure biomass production and biofuel Q&Q analysis:

Cottonwood Trees: A total of 90 trees/plot (30/clone) have been selected for annual measurements at each site. Only trees growing in the measurement plots were selected. Trees were selected in the WS and SW plots to incorporate any edge effects created by switchgrass/cottonwood borders. For each tree, ground line diameter (GLD), total height (HT), and crown ratio (CR; i.e. ratio live crown length to total height) will be measured. For trees greater than 1.4 m in height, diameter at breast height (DBH) will also be measured.

To develop allometric equations, destructive sampling of 30 trees per study site (10 per clone) from the large treatment plots outside the measurement plots will occur in September 2011 (age 3) and again in September 2013 (age 5) prior to harvest. Each tree’s GLD, DBH, CR, and HT will be measured and individual tree components (stem, branches, and foliage) will be weighed with a load cell to record green weight. Three 10 cm stem (butt, mid-stem, and start of tree crown) and 3 branch samples (lower, mid, and upper crown) and foliage samples will be collected from each tree. One portion of these samples will be used for C analysis. Another portion of each sample will be weighed, dried at 60oC and weighed again to determine moisture content. These values will be utilized to determine a dry component weight and dry total tree weight for each destructively sampled tree. A portion of the dried samples will be used for biofuel Q&Q analysis. Allometric equations will be fit to dimensional and biomass data from the destructively sampled trees. These equations will be used to predict green and dry mass of each permanent measurement tree, which then can be used to estimate total amount of cottonwood biomass for a given cottonwood cropping system. This information along with C concentrations will be used to determine aboveground C content and biofuel production of the for each annual tree measurement. Switchgrass: Switchgrass stand counts (basal ground cover by the base of the plant, or crown) will be performed twice yearly, in spring when there is 15-20 cm of new growth, and fall after biomass has been removed. One count consists of counting the number of cells in a 25-cell grid quadrat (Vogel and Masters, 2001) containing at least one switchgrass crown or crown portion each time the quadrat is placed on the ground. The counts will be done along two

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diagonal transects in the measurement subplot of each plot with switchgrass. The number of counts in each measurement subplot depends on the size of the switchgrass area to maintain the same sampling density (one count per 21.25 m2), with 12 counts taken in treatment WS, 24 counts in treatment SW, and 36 counts in treatment S.

Biomass yield will be determined beginning in fall 2010 and then again annually to the end of the proposed study in 2016. A self-propelled forage chopper will be used to cut two 1.2 x 4.9 m strips per plot (10 cm stubble height), and these will be summed. Biomass will be sub-sampled (~1 kg) for moisture determination to correct plot harvested mass to per-hectare dry matter yield. A second 20 kg subsample will be collected, dried, and ground for biofuel Q&Q and C analysis. The product of biomass yield and C concentration or biofuel yield/unit weight will provide the aboveground estimate of C or biofuel content. Unharvested stubble and dropped leaves and seeds on the soil surface will be considered part of the soil C.

Soybean-Grain Sorghum: In the soybean-grain sorghum (C) treatment plots, biomass and biofuel Q&Q samples are manually collected in five 1 m2 plots or, where available, samples are collected by a plot harvester within a 9.2 m2 plot. When collected manually the entire plant (soybean or sorghum) is removed and the grain is separated from the remainder of the plant. With the plot harvester the grain and plant residues are removed from the plot harvester. Materials are dried (60 oC) and weighed. A sample from the collected grain and plant material is taken for C and biofuel Q&Q analysis. Samples are ground to pass a 2 mm sieve prior to C analysis.

Biofuel Q&Q and C Analysis: The biofuel analysis will be done using a BioMax 25 Biopower System housed at a USDA Forest Service Southern Research Station in central Louisiana. An approximate 20 kg sample is needed for this analysis. Thus, individual vegetation or vegetation components sample will be composited either on a plot or treatment basis to provide adequate amount of samples for analysis. The BioMax 25 Biopower system is equipped with a gas analyzer and will determine gas production (CO2, CO, H2, and CH4), tar, particulate matter, ash, moisture content, potential energy production (kjoules) and potential fuel production (biogasoline and syndiesel) generated via biomass gasification. Values will be determined on a per-unit mass basis for each biomass sample. Biomass yields and the values obtained from the BioMax 25 Biopower System will be used to determine total annual biofuel quantity production on a per unit area for each cropping system. C analysis will be done by combustion with a Vario Max CN analyzer (Elementar, Inc., Philadelphia, PA). Replicate sample analysis will be performed for all samples. As with biofuel Q&Q, total C content for a component or crop will be determined by multiplying yield by C concentration. Belowground: Belowground biomass samples will be collected to a depth of 30 cm during the 2011-12 dormant season (using current funding) and to a depth of 60 or 150 cm (using proposed funding) in the dormant season of 2013-14 following cottonwood harvesting. During the 2011-12 sampling period only biomass of live fine roots (< 2 mm and 2-5 mm) will be determined while during 2013-2014 biomass of live fine and coarse roots (>5 mm) will be quantified. Typically belowground biomass in this region is at its maximum during the dormant season (Coleman 2007) and thus belowground biomass and C estimates should be at their seasonal maximums at our selected time of sampling. Fine Root: Fine root samples during both sampling periods will be collected using 5 cm diameter cores. A total of 8 sampling locations will be located along a transect running the length of each measurement plot in the 100% cottonwood (W), 100% switchgrass (S), and soybean-grain sorghum (C) plots. The samples will be taken at 5-m intervals along a transect.

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In the SW and WS treatments we expect that belowground biomass and C will be more variable at the interface between the cottonwood and switchgrass alleys but similar to what we find in the W and S treatments in the interior of each of the respective allies. Thus we will take samples at a distance of 1.5 m, 4.0 m, and 6.5 m from the border of the cottonwood/switchgrass alleys. A total of 6 samples will be taken, 3 in each alley (3 in the cottonwood and 3 in the switchgrass alley) along a transect running the length of the measurement plot. Samples will be taken (using current funding) to a depth of 30 cm using an impact core sampler during the 2011-12 sampling period. Generally the greatest amounts and variation of root biomass, length, and density of switchgrass and woody species such as cottonwood occurs in the surface soil, especially in the upper 30 cm (Tufekcioglu et al. 1999, Rytter 1999, Ma et al. 2000, Hendricks et al. 2006). All samples collected during the 2013-2014 dormant season will be collected to a depth of 60 cm except 4 of the 8 samples collected in the W, S, and C plots, which will be collected to a depth of 150 cm. All samples during the 2013-14 dormant period will be taken using a tractor-mounted Giddings sampler. Samples will be stratified by 30 cm depth increments. Fine roots will be extracted from each 30 cm increment. All cores will be stored at <5°C until samples can be processed. Roots will be extracted from soil using a hydropneumatic root elutriator (Gillison’s Variety Fabrication, Inc., Benzonia, MI) and sorted into size classes, dried to constant mass at 60°C and weighed for root mass determination. Coarse Roots (>5mm): Coarse root sampling will only occur in the W treatment since previous studies have shown no or minimal development of coarse roots in switchgrass or soybeans (Tufekcioglu et al. 1999). Cottonwood coarse roots will be sampled to a depth of 60 cm following fine root sampling in 2013-14. Coarse roots are spatially more variable than fine roots (Coleman 2007). Consequently, larger sample volumes are typically used to quantify coarse root biomass. Roots will be sampled using 75 x 75 cm pits dug to a depth of 60 cm. The corner of the pit will be centered on a tree stump. This represents 25% of the area allocated to each planted tree. Three pits will be excavated per plot. The pits will be located in such a way to represent the range of tree sizes and densities in a given plot. Samples will be stored in containers at <5°C until they can be processed. These samples will be wet sieved, rinsed, dried at 60°C and weighed. Carbon Analysis: Dried root samples will be composited by sampling period, depth increment, size and plot. Subsamples of each composite will be milled and then analyzed for C concentrations by combustion with a Vario Max CN analyzer (Elementar, Inc., Philadelphia, PA). Root C content will be calculated using root mass and C concentration data. Objective Evaluation: Differences among treatments in all variables measured for Objective 1 will be determined using analyses of variance (ANOVAs) with site, block, treatments, and their interactions as independent variables in the model. It is expected that aboveground biomass C will rank as agroforest mixtures > cottonwood plantation > switchgrass plantation > conventional agriculture at the end of the cottonwood rotation. However, aboveground biomass C of switchgrass will likely exceed that of cottonwood over the course of this project due to the repeated harvests of the switchgrass. Total belowground biomass C and fine root mass will likely be greatest for switchgrass. The soybean-sorghum rotation will likely have lower belowground biomass C relative to switchgrass and cottonwood. Possible pitfalls that may be encountered in this research are unfavorable precipitation patterns (drought, flooding) that hinder growth of all crops. Additionally, pests such as diseases and army worms could damage all crops if control measures are unsuccessful, particularly the soybean-sorghum rotation and cottonwood. Pest damage to cottonwood could alter its growth

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form, resulting in poor model fit that would lead to under- or over-estimation of biomass C. In addition this may slow the maturity time of the cottonwood thus requiring a later date for harvesting. The Biomax gasification unit may encounter drawbacks in processing switchgrass biomass and/or the soybean-sorghum materials. The unit is fed by a conveyor system that has been used successfully with wood chips, but finely ground biomass may not travel efficiently along the conveyor. If it does not feed into the gasification effectively it may be necessary to pelletize the biomass. Belowground core sampling may prove difficult at the sites with heavy clay soil. It may be necessary to perform the collection of these cores during moderately moist conditions to promote more efficient sample collection. Objective 2: Determine Total C Accumulation in the Soil Profile

The impact of different cropping systems and vegetation species on C accumulation will be determined in three ways 1) the amount of C in the surface soil (30 cm depth) five years following the study initiation will be compared to that prior to switchgrass or cottonwood establishment, 2) soil C contents of the soils to a depth of 150 cm five years after treatment establishment will be compared among treatments (S, W, and C) and 3) soil carbon contents (60 cm depth) along the cottonwood-switchgrass borders five years after establishment of in the WS and SW treatments will be compared to that of the measurement plots in the W and S treatments. Soil Sampling Prior to Treatment Establishment: A total of 10 (2.5cm diameter) core samples along two transects running the length of the measurement plots was taken to a depth of 30 cm in each plot for C analysis. In addition, a total of six 5-cm core samples were taken on the two transects for bulk density. Samples for C analysis were composited by transect and then air-dried. These composited soils were sieved to pass a 2 mm mesh and then analyzed for C content by the combustion method with a Vario Max CN analyzer (Elementar, Inc., Philadelphia, PA). Each bulk density sample was dried at 105 oC and weighed to determine bulk density.

Soil Sampling Fifth Year: Soil samples will be taken from the cores utilized for root sampling conducted as described above in pursuit of Objective 1. Roots will be removed by sieving from a subsample collected from each depth increment and core collected for belowground biomass sampling. This soil will be composited by depth increment for each plot. Soils will be processed and analyzed in the same manner indicated for the samples take prior to treatment establishment. Three additional 5 cm diameter cores to a depth of 150 cm will be taken along the transect utilized for the belowground biomass cores for bulk density in the W, S, and C treatment plots. Each 30 cm depth increment will be extracted, sieved to pass a 2 mm mesh, dried at 105 oC, and weighed to determine bulk density. C concentrations and bulk density will be used to calculate total C at each depth. Objective Evaluation: Differences among treatments in variables assessed in pursuit of Objective 2 will be elucidated with ANOVA as mentioned above for Objective 1. It is expected that total soil C and total C accrual will be greatest at the switchgrass-cottonwood borders and that switchgrass will have greater soil C deeper into the soil profile than cottonwood and the soybean-sorghum rotation. If there is high variability in crop growth or in soil texture in our plots, it will be more difficult to accurately sample and assess soil C.

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Objective 3: Measure Gaseous Emissions of CO2, N2O, and CH4

CO2 Emission: Soil surface CO2 flux measurements will be conducted at all sites in the 100% cottonwood (W), 100% switchgrass (S), and the soybean-grain sorghum rotation treatments using a procedure similar to that used by Brye et al. (2006). A minimum of five 10-cm-diameter PVC collars will be randomly located inside each 17 by 45 m measurement plot. Each collar will be inserted approximately 2 cm into the soil. A minimum total of 45 measurements (total from all 3 treatments) will be recorded once a month for three years beginning in 2012 at each site. This will allow us to monitor pre-and post-harvest responses in all treatments as well as seasonal variation of soil CO2 fluxes. Soil respiration measurements will be made using a LI-6400 or LI-8100 CO2 analyzer and a LI6400-09 or a LI8100-102 soil respiration chamber (Li-Cor, Inc., Lincoln, NE). Both systems provide similar estimates of CO2 flux in our cropping systems. Measurements will be taken between 1000 and 1400 hours during each measurement date. The instruments will measure the ambient CO2 concentration and perform the soil respiration measurements at the measured ambient concentration on each sample date. Green vegetation will be removed from inside each collar prior to conducting measurements so as to avoid any effects of photosynthesis on the soil respiration measurement. Collars will be relocated in the plots after every two or three consecutive measurement dates.

In addition to soil respiration measurements, soil temperature will be recorded at 2- and 10-cm depths adjacent to each collar while respiration measurements are being made using a probe-type thermometer. Following each respiration measurement, a Theta Probe (model TH2O, Dynamax, Inc., Houston, TX) will be used to measure the volumetric soil water content in the top 6 cm from inside each collar. The soil temperature and moisture data will be used along with soil respiration measurements in a multiple regression approach to develop predictive equations for soil respiration based on soil temperature and moisture for each treatment. Methodologies utilized for this equation development will be similar to that done by Brye and Riley (2009) in a chronosequence of grassland restorations in northwest Arkansas.

CH4 and N2O Emissions: Soil emissions of CH4 and N2O will be measured monthly at the Archibald and Rohwer sites (which were selected for this effort due to their dissimilarity in soil texture) from January 2012 through December 2013 to ascertain the efflux of these greenhouse gases from mature switchgrass and cottonwood plantations relative to the conventional cropping system. As with CO2 emissions measurements, CH4 and N2O emissions will be measured in the W, S, and C treatments. To do so, a vented chamber method similar to that described by Venterea et al. (2005) will be used for soil flux measurement of CH4 and NO2. This method is reliable and well-tested (Rochette and Eriksen-Hamel, 2008). Four chambers which measure 54 x 32 x 10 cm will be constructed and installed in each plot at all sites. Chambers will be positioned along two transects within the measurement subplot area of each plot. The top of each chamber will have a sampling port and venting port and its horizontal edge is lined with a rubber gasket to aid in sealing the base. The base will be inserted into soils at least 5 cm (Rochette and Eriksen-Hamel, 2008), and the top will secured with metal clamps. Gas samples for measuring gas fluxes will be taken monthly throughout the experiment. Chamber gas samples during each collection date will be taken at a regular interval of 0, 30, and 60 min by inserting the needle of a syringe through the septum at the sample port after the top is placed on the base of chamber. Soil temperature and volumetric moisture will be determined concurrent with gas sampling as described above for CO2 gas sampling protocol. The drawn gas samples will be stored in vacutainers and transported to the laboratory. The CH4 and NO2 in samples will

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be analyzed by gas chromatography equipped with a methanizer and flame ionization detector and electron capture detector. The CH4 and N2O flux will be calculated from the rate of change in chamber concentration, chamber volume, and soil surface area using the formulation described by Hutchinson and Mosier (1981). Objective Evaluation: Differences among treatments will be elucidated by ANOVA with a repeated measures model with site, date, block, and treatment as independent variables in the model. In addition, equation parameters from the fitted equations for the soil CO2 flux will be compared among treatments with an ANOVA to determine if CO2 flux rates for similar ambient conditions differ by treatment. Despite the high frequency of sampling on greenhouse gas emissions, it is possible that we may still miss opportunity to capture all variability of emissions due to the nature of the fluxes, which are heavily influenced by microsite variation in soil texture and moisture. Objective 4: Life Cycle Assessment of C Life cycle assessment consists of four major stages, scoping, life-cycle inventory, life-cycle impact assessment, and life-cycle interpretation (Curran 2006). This study will focus on carbon life-cycle inventory (LCI) of carbon and energy balances for cottonwood-switchgrass agroforest systems in the LMAV. The LCI will use actual measurements of carbon and other greenhouse gasses sequestered and emitted in this study rather than modeled data. Methane and nitrous oxide will be converted into CO2 equivalents using standards set by the U.S. Environmental Protection Agency (EPA 2005a). Since energy consumption and energy substitution is so critical to carbon balance (Robertson and Grace 2004), the life-cycle inventory will include energy consumption in the production of all inputs into the cropping systems as well as the energy replacement value of the biomass produced. This will permit a net-carbon or net greenhouse balance value for all crops and cropping systems to be determined. Switchgrass seed production, cottonwood cutting stock production inputs in energy and transportation will be accounted for at source nurseries used in this study. Maintenance inputs on the crops will be monitored to determine carbon releases from tractors and other equipment used to maintain the cropping systems. Harvest equipment and transportation fuel values will be measured. Fertilizer and other soil additives, as well as herbicide and insecticide use will be recorded for the LCI. All mechanical/fuel consumption figures will be converted into carbon emissions values using standard values (EPA 2005b). Aboveground biomass carbon will be determined from sampled trees at ages 3 and 5 as part of Objective 1, the year 5 data being collected just prior to harvest. Belowground biomass as determined in Objective 1 and soil fluxes of CO2, CH4, and N2O measured annually in Objective 3 along with their carbon equivalents will be included in the inventory. Biomass production will be converted into energy values for substitute natural gas (SNG) and Fischer-Tropsch (FT) liquids (Larson and Jin 1999). Both can be rapidly converted to or used as transportation fuels. Indeed, there have been recent commercial proposals for establishing biomass-to-SNG and biomass-to-FT conversion facilities in southern Arkansas. Most previous studies have converted harvested energy values into ethanol (Adler et al. 2007), electrical generation (Heller et al. 2003), or co-firing (Keoleian and Volk 2005). Because the technologies for non-ethanol biofuels are rapidly developing, we expect that conversion values will likely change and be more precise in 2-3 years than those available today. Fuel equivalents will permit the calculation of fossil fuel energy replacement and subsequent carbon emission reductions. Producer-gas yield data from direct gasification of

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biomass in this study will be used to estimate both FT-to-liquid and SNG production from the differing cropping systems. Finally, a comparison of the greenhouse gas balance will be made with previous studies that were based on biogeochemistry models (Adler et al. 2007). It is expected that switchgrass and cottonwood will reduce greenhouse gas emissions because switchgrass and hybrid poplar were found to reduce GHC emissions by 115% in northeast agroforest systems when utilizing ethanol and biodiesel as the replacement fuels. A weakness of this analysis is the relative deficiency in published energy conversion values for non-ethanol biofuels. Objective 5: Elucidate Soil-Plant Physical and Biological Processes that Impact C Cycling.

Labile C: Soil labile C has been defined as the fraction of soil organic carbon that has a degradation time of less than a few years as opposed to the recalcitrant portion that has a turnover time of several thousand years (Parton et al. 1987). Soil labile carbon is thus the most active fraction of soil organic carbon and is highly sensitive to changes in land management (Harrison et al. 1993). The oxidation of labile C is a predominant driver of the flux of CO2 between soils and atmosphere (Zou et al. 2005). Due to this importance of soil labile C, labile C samples will be randomly collected to a 30-cm soil depth using a punch auger in all plots at all sites in early September in 2011 through 2015.

Labile C samples will be collected along two transects within the measurement subplot of all plots, with eight samples collected per transect and composited. All samples will be collected in September because above- and below-ground biomass of all crops will be at the seasonal maximum, which provides the optimum opportunity to observe changes in labile C. Labile soil C will be determined using a sequential fumigation-incubation (SFI) procedure (Zou et al. 2005). The method is based on the assumption that soil labile C is decayed during these cycles according to the negative exponential equation as widely reported in plant decomposition studies, so Cmic accumulated during the cycles is estimated from a first-order kinetics model. During the fumigation-incubation cycles, fumigation creates relatively ideal conditions for microbial growth by eliminating soil available pore space and predation activities that typically limit microbial growth under field conditions. Microbial growth during the cycles is limited solely by carbon availability in terms of total quantity and its lability. This method thus provides an index of the pool size of soil labile organic C provided by the Cmic trends during the fumigation-incubation cycles. The potential turnover rate of labile C (a parameter for the quality of labile organic C available to soil microbes) is also determined from this method as the inverse of k in the first-order kinetics model multiplied by 10 due to the 10-day incubation cycle of this method (Zou et al. 2005). Samples will be sieved and then undergo eight cycles of fumigation with chloroform followed by a 10-day incubation (30 oC) in which respired CO2 is collected in a NaOH solution. Titration will be used to quantify the flux of CO2-C associated with each individual incubation period. Labile C will be estimated from the accumulated CO2-C amounts using a first-order kinetics model. Individual equations will be fitted to the data using non-linear least squares regression. Microbial biomass and activity: Microbial biomass C (Cmic) and activity will be measured quarterly in 2012 through 2015 to determine the influences of mature switchgrass and cottonwood as well as harvesting of switchgrass and cottonwood on soil quality. Soil microbial biomass and activity are highly sensitive to changes amounts and forms of soil organic carbon (Powlson et al. 1987; Fauci and Dick 1994; Harris 2003), so measuring these phenomena in this

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study will foster understanding of the potential of these cropping systems to alter forms of soil organic carbon and concomitantly the soil microbial biomass essential in converting organic matter into bioavailable nutrients. Soil samples will be collected to a 30 cm depth along transects as described above for labile C soil sampling. The chloroform fumigation-incubation method will be used to determine Cmic (Jenkinson and Powlson 1976). Procedures include a 10-day pre-incubation of soil samples at 25°C followed by fumigation with alcohol-free CHCl3 vapor for 24 h. Respired CO2 is collected with 2 M NaOH, and CO2 is quantified by titration with 0.1 N HCl. The equation developed by Horwath et al. (1996) will be used to convert CO2-C to Cmic due to the equation’s standardization against direct microscopy and its close correlation with Cmic observed by direct microscopy on a broad range of soils. Microbial activity will be estimated by determining dehydrogenase activity (Lenhard 1956; Alef 1995). Dehydrogenase, which is only active in viable living cells, serves as an indicator of total microbial metabolic activity (Tabatabai 1994; Camiña et al. 1998). To quantify dehydrogenase, triphenyltetrazolium chloride (TTC) is used as an artificial electron acceptor. Dehydrogenase reduces TTC to red-colored triphenyl formazan (TPF) that can be extracted with methanol and quantified colorimetrically (Thalmann 1968). Metabolic activity of microbial communities will be determined by quantifying ratios of intracellular enzymatic activities to soil Cmic (Landi et al. 2000; Deng et al. 2006).

C Fractionation and Organic Matter Composition: Subsamples of soil from the cores taken five years after stand establishment as part of Objective 2 (described above in the Soil Sampling Fifth Year subsection) will be subjected to physical fractionation to determine silt- and clay-associated soil organic carbon (SOC) using a method described by Brunn et al. (2008). These forms of SOC are relatively slow to decompose, so land use practices that increase these forms of soil organic carbon increase long-term residence time of carbon in soil. Thus, exploration of these variables in this study will provide an indication of the capacity for these cropping systems to increase C sequestration in 30-cm soil depth increments to a depth of 150 cm as the crops mature as well as the differences in soil C sequestration among these cropping systems.

For quantification of silt- and clay-bonded SOC, aggregates larger than 20 µm will be disrupted by shaking 50 g of soil with 120 ml water and five 10 mm glass beads in an end-over-end shaker for 18 hours. After centrifugation at 1400 g for 20 minutes, the floating fraction will be removed and the residual soil will be mixed with a polytungstate suspension (density 1.8 g cm-3) and centrifuged at 1400 g for 20 minutes. The floating fraction will be removed and combined with the fraction floating on water to produce the light fraction. The remaining material will be subjected to particle size fraction by allowing particles > 20 µm to settle for a time specified by Stokes’s law. The clay and silt particles will be siphoned off. This process will be repeated until the water above the sedimentation front is clear. Subsequently, the clay and silt particles will be flocculated with a small amount of CaCl2 and dried at 70°C. Total organic carbon in the clay and silt fraction will be analyzed by a CN analyzer. Subsamples of soils from the silt- and clay-bonded SOC procedure will be subjected to further analysis for molecular composition of soil organic matter (SOM) using pyrolysis gas chromatography/mass spectrometer (Py-GC/MS). This procedure provides measurement of shifts in the forms of C molecular moieties in soil in response to land use changes, ranging from molecules that are readily decomposed to recalcitrant forms. Subsamples will be drawn from soils of each cropping system at each site for both silt- and clay-bonded SOC sampling periods. Pyrolysis-GC/MS will be performed using a method similar to that described by Wang et al.

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(2010). Specifically, pyrolysis will be carried out at 620 oC for 20 s using a CDS 5000 pyroprobe platinum heated filament pyrolyser, directly connected to a Varian 3900 gas chromatograph coupled to a Varian Saturn 2100T ion trap mass spectrometer. A Varian factor FOUR VF-5MS capillary column coated with poly (5% diphenyl / 95%dimethyl) siloxane stationary phase (30 m, 0.25mm i.d., 0.25 μm film thicknesses) will be used for separation of pyrolysis products and the temperature of GC column oven will be ramped from 40oC to 300 oC at 5 oC min−1. Mass spectra will be recorded in the electron impact mode (70 eV) at 1 scan s−1 in the 40–650 m/z range. The identification of pyrolysis products will be based on a comparison of their mass spectra with those of standard compounds and NIST mass spectral library, literature data and GC–MS characteristics. The relative quantities of the products will be estimated using the peak areas of the total ion current (TIC) pyrograms. Besides examining the specific bio-indicator molecules, the identified compounds in the Py- GC/MS analysis will be classified based on the expected origin into alkyl compounds, lignin-derived compounds, N-containing compounds, polyphenols/other aromatic compounds, and polysaccharides. Pyrolysis-GC/MS has been successively used for characterizing the nature and complex SOM (White, 2004), describing the relations between vegetation shifts and aerobic/anaerobic decomposition of SOM in peatlands (Schellekens et al., 2009), and SOM composition under different climatic regions (Vancampenhout et al., 2009). By characterization of these different pools of C, we expect to better understand C sequestration and stability in switchgrass and cottonwood bioenergy feedstock production systems.

Objective Evaluation: Differences among treatments in all variables measured for Objective 5 will be determined using an ANOVA with site, block, treatments, and their interactions as independent variables in the model. Further characterization of the effects of these cropping systems on soil C forms will be possible through correlation analyses of labile C data of 2011 and 2013 with long-term C forms assayed in the same years. It is expected that switchgrass and cottonwood will be associated with greater labile C, Cmic, and microbial activity than conventional agriculture due to their greater root mass that serves as a source of root exudates, as seen in a recent study of a loblolly pine and switchgrass alley cropping system by Blazier et al. (In press). In the Blazier et al. (In press) study, the sequential fumigation-incubation and dehydrogenase procedures were precise enough to detect differences among land use types after one year of switchgrass growth, so these procedures should be sensitive enough for this proposed project. A possible limitation of the procedures is the ability to determine differences in labile C and activity among sites if the sites differ dramatically in soil moisture at the time of sampling because soil moisture will affect activity of the soil microbes. It is likely that switchgrass and cottonwood will have greater long-term forms of C in soil relative to conventional cropping due to their perennial nature. It is also expected that switchgrass will be associated with more long-term forms of C deeper into the soil profile than cottonwood due to its deep, dense rooting habit relative to the shallow rooting habit of cottonwood. A limitation of the procedures used to determine long-term C in soil is the ability to detect differences among treatments if there is high microsite variability in rooting and soil texture. Use of Results

This study’s results will be synthesized in a series of publications for scientific outlets, including peer-reviewed journal articles, graduate student theses and a dissertation, and conference proceedings. To extend the study’s results beyond the scientific community, the

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research team will incorporate the study’s results into their ongoing extension programming effort affiliated with this project. On a yearly basis, the team is hosting field tours of the research sites targeted to prominent farmers, forest owners, natural resource management professionals, land investment professionals, biofuel investors, environmental groups, and local and state elected officials. Research activities and results are shared with these groups. Additionally, the research team publishes research results in university extension bulletins, websites, and popular press outlets such as farm organization magazines. They also publicize research results through university press releases via television, radio, and newspaper outlets. These activities will continue throughout this proposed project in order to foster public engagement and potential investment in these cropping systems. Hazardous Materials No hazardous materials will be used in the procedures of this proposed project. To assure safety of all workers in this project, all investigators perform routine safety training procedures and have proper safety gear available for all laboratory and field work. Timeline of Study Activities:

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Timeline for Objective Attainment and Project Deliverables Objective attainment is highlighted in italics, and deliverables are underlined. 2011:

- Aboveground cottonwood destructive sampling - Belowground biomass sampling - Begin annual labile C sampling

2012: - Begin monthly CO2, CH4, and N2O emission sampling - Begin quarterly soil microbial biomass C and activity sampling

2013: - Operational cottonwood harvest - Cottonwood destructive sampling, C analysis & biofuel QQ - Belowground biomass sampling - Sampling of long-term forms and total C in soil – Objective 2 attainment - End monthly CH4 and N2O emission sampling - Ph.D. dissertation on CH4 and N2O emission

2014: - End monthly CO2 emission sampling – Objective 3 attainment - M.S. thesis on soil CO2 emission - Journal article on effects of land use change on long-term soil C - Journal article on CH4 and N2O emission - Journal article on rotation-length above- and below-ground C - Journal article on long-term soil C in mature agroforests and monocultures of

switchgrass, cottonwood, and soybean-sorghum 2015

- End aboveground biomass measurement of all crops – Objective 1 attainment - End quarterly soil microbial biomass C and activity sampling – Objective 5 attainment - Begin life cycle analysis - Journal article on CO2 emission

2016 - End life cycle analysis – Objective 4 attainment - Journal article on life cycle analysis