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At least 343 records · Page 19

Insights of biomass recalcitrance in natural Populus trichocarpa variants for biomass conversion

Populus has been investigated as a promising biomass feedstock for alternative fuels and chemicals. Physicochemical characteristics and genomic information of biomass feedstocks are among the essential information that can help not only advance our understanding of biomass recalcitrance but also in its efficient utilization. Here in this paper, the recalcitrance of natural Populus variants was elucidated in three aspects: (1) sugar release, (2) physicochemical properties, and (3) relative variation of gene expression within natural poplar variants. The sugar release performance of natural Populus variants was evaluated with their correlation with biomass recalcitrance-related characteristics. Among the physicochemical properties of poplar, the lignin content, lignin molecular weight, lignin S/G ratio, and cellulose accessibility were found to correlate with sugar release. The results demonstrated that the lignin content was negatively correlated with sugar release, whereas the lignin molecular weight, lignin S/G ratio, and cellulose accessibility were positively associated with poplar sugar release. The trend of differential gene expression of each variant also supports the characterization results and their effects on biomass conversion.

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Interactions with microbial consortia have variable effects in organic carbon and production of exometabolites among genotypes of Populus trichocarpa

Abstract Poplar is a short‐rotation woody crop frequently studied for its significance as a sustainable bioenergy source. The successful establishment of a poplar plantation partially depends on its rhizosphere—a dynamic zone governed by complex interactions between plant roots and a plethora of commensal, mutualistic, symbiotic, or pathogenic microbes that shape plant fitness. In an exploratory endeavor, we investigated the effects of a consortium consisting of ectomycorrhizal fungi and a beneficial Pseudomonas sp. strain GM41 on plant growth (including height, stem girth, leaf, and root growth) and as well as growth rate over time, across four Populus trichocarpa genotypes. Additionally, we compared the level of total organic carbon and plant exometabolite profiles across different poplar genotypes in the presence of the microbial consortium. These data revealed no significant difference in plant growth parameters between the treatments and the control across four different poplar genotypes at 7 weeks post‐inoculation. However, total organic carbon and exometabolite profiles were significantly different between the genotypes and the treatments. These findings suggest that this microbial consortium has the potential to trigger early signaling responses in poplar, influencing its metabolism in ways crucial for later developmental processes and stress tolerance.

59 BASIC BIOLOGICAL SCIENCES↗

Soil phosphorus drawdown by perennial bioenergy cropping systems in the Midwestern US

Abstract Without fertilization, harvest of perennial bioenergy cropping systems diminishes soil nutrient stocks, yet the time course of nutrient drawdown has not often been investigated. We analyzed phosphorus (P) inputs (fertilization and atmospheric deposition) and outputs (harvest and leaching losses) over 7 years in three representative biomass crops—switchgrass ( Panicum virgatum L.), miscanthus ( Miscanthus × giganteus ) and hybrid poplar trees ( Populus nigra × P. maximowiczii )—as well as in no‐till corn (maize; Zea mays L.) for comparison, all planted on former cropland in SW Michigan, USA. Only corn received P fertilizer. Corn (grain and stover), switchgrass, and miscanthus were harvested annually, while poplar was harvested after 6 years. Soil test P (STP; Bray‐1 method) was measured in the upper 25 cm of soil annually. Harvest P removal was calculated from tissue P concentration and harvest yield (or annual woody biomass accrual in poplar). Leaching was estimated as total dissolved P concentration in soil solutions sampled beneath the rooting depth (1.25 m), combined with hydrological modeling. Fertilization and harvest were by far the dominant P budget terms for corn, and harvest P removal dominated the P budgets in switchgrass, miscanthus, and poplar, while atmospheric deposition and leaching losses were comparatively insignificant. Because of significant P removal by harvest, the P balances of switchgrass, miscanthus, and poplar were negative and corresponded with decreasing STP, whereas P fertilization compensated for the harvest P removal in corn, resulting in a positive P balance. Results indicate that perennial crop harvest without P fertilization removed legacy P from soils, and continued harvest will soon draw P down to limiting levels, even in soils once heavily P‐fertilized. Widespread cultivation of bioenergy crops may, therefore, alter P balances in agricultural landscapes, eventually requiring P fertilization, which could be supplied by P recovery from harvested biomass.

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Colonization of Naive Roots from Populus tremula × alba Involves Successive Waves of Fungi and Bacteria with Different Trophic Abilities

ABSTRACT Through their roots, trees interact with a highly complex community of microorganisms belonging to various trophic guilds and contributing to tree nutrition, development, and protection against stresses. Tree roots select for specific microbial species from the bulk soil communities. The root microbiome formation is a dynamic process, but little is known on how the different microorganisms colonize the roots and how the selection occurs. To decipher whether the final composition of the root microbiome is the product of several waves of colonization by different guilds of microorganisms, we planted sterile rooted cuttings of gray poplar obtained from plantlets propagated in axenic conditions in natural poplar stand soil. We analyzed the root microbiome at different time points between 2 and 50 days of culture by combining high-throughput Illumina MiSeq sequencing of the fungal ribosomal DNA internal transcribed spacer and bacterial 16S rRNA amplicons with confocal laser scanning microscopy observations. The microbial colonization of poplar roots took place in three stages, but bacteria and fungi had different dynamics. Root bacterial communities were clearly different from those in the soil after 2 days of culture. In contrast, if fungi were also already colonizing roots after 2 days, the initial communities were very close to that in the soil and were dominated by saprotrophs. They were slowly replaced by endophytes and ectomycorhizal fungi. The replacement of the most abundant fungal and bacterial community members observed in poplar roots over time suggest potential competition effect between microorganisms and/or a selection by the host. IMPORTANCE The tree root microbiome is composed of a very diverse set of bacterial and fungal communities. These microorganisms have a profound impact on tree growth, development, and protection against different types of stress. They mainly originate from the bulk soil and colonize the root system, which provides a unique nutrient-rich environment for a diverse assemblage of microbial communities. In order to better understand how the tree root microbiome is shaped over time, we observed the composition of root-associated microbial communities of naive plantlets of poplar transferred in natural soil. The composition of the final root microbiome relies on a series of colonization stages characterized by the dominance of different fungal guilds and bacterial community members over time. Our observations suggest an early stabilization of bacterial communities, whereas fungal communities are established following a more gradual pattern.

59 BASIC BIOLOGICAL SCIENCES↗

Heterologous expression of Arabidopsis laccase2, laccase4 and peroxidase52 driven under developing xylem specific promoter DX15 improves saccharification in populus

Secondary cell wall holds considerable potential as it has gained immense momentum to replace the lignocellulosic feedstock into fuels. Lignin one of the components of secondary cell wall tightly holds the polysaccharides thereby enhancing the recalcitrance and complexity in the biomass. Laccases (LAC) and peroxidases (PRX) are the major phenyl-oxidases playing key functions during the polymerization of monolignols into lignin. Yet, the functions of laccase and peroxidases gene families remained largely unknown. Hence, the objective of this conducted study is to understand the role of specific LAC and PRX in Populus wood formation and to further investigate how the altered Lac and Prx expression affects biomass recalcitrance and plant growth. This study of heterologous expression of Arabidopsis Lac and Prx genes was conducted in poplar to avoid any otherwise occurring co-suppression mechanism during the homologous overexpression of highly expressed native genes. In the pursuit of optimizing lignocellulosic biomass for biofuel production, the present study focuses on harnessing the enzymatic potential of Arabidopsis thaliana Laccase2, Laccase4, and Peroxidase52 through heterologous expression. We overexpressed selected Arabidopsis laccase2 (AtLac2), laccase4 (AtLac4), and peroxidase52 (AtPrx52) genes, based on their high transcript expression respective to the differentiating xylem tissues in the stem, in hybrid poplar (cv. 717) expressed under the developing xylem tissue-specific promoter, DX15 characterized the transgenic populus for the investigation of growth phenotypes and recalcitrance efficiency. Bioinformatics analyses conducted on AtLac2 and AtLac4 and AtPrx52, revealed the evolutionary relationship between the laccase gene and peroxidase gene homologs, respectively. Transgenic poplar plant lines overexpressing the AtLac2 gene (AtLac2-OE) showed an increase in plant height without a change in biomass yield as compared to the controls; whereas AtLac4-OE and AtPrx52-OE transgenic lines did not show any such observable growth phenotypes compared to their respective controls. The changes in the levels of lignin content and S/G ratios in the transgenic poplar resulted in a significant increase in the saccharification efficiency as compared to the control plants. Overall, saccharification efficiency was increased by 35–50%, 21–42%, and 8–39% in AtLac2-OE, AtLac4-OE, and AtPrx52-OE transgenic poplar lines, respectively, as compared to their controls. Moreover, the bioengineered plants maintained normal growth and development, underscoring the feasibility of this approach for biomass improvement without compromising overall plant fitness. This study also sheds light on the potential of exploiting regulatory elements of DX15 to drive targeted expression of lignin-modifying enzymes, thereby providing a promising avenue for tailoring biomass for improved biofuel production. These findings contribute to the growing body of knowledge in synthetic biology and plant biotechnology, offering a sustainable solution to address the challenges associated with lignocellulosic biomass recalcitrance.

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Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials

Humans have been modifying plant traits for thousands of years, first through selection (i.e., domestication) then modern breeding, and in the last 30 years, through biotechnology. These modifications have resulted in increased yield, more efficient agronomic practices, and enhanced quality traits. Precision knowledge of gene regulation and function through high-resolution single-cell omics technologies, coupled with the ability to engineer plant genomes at the DNA sequence, chromatin accessibility, and gene expression levels, can enable engineering of complex and complementary traits at the biosystem level. Populus spp., the primary genetic model system for woody perennials, are among the fastest growing trees in temperate zones and are important for both carbon sequestration and global carbon cycling. Ample genomic and transcriptomic resources for poplar are available including emerging single-cell omics datasets. To expand use of poplar outside of valorization of woody biomass, chassis with novel morphotypes in which stem branching and tree height are modified can be fabricated thereby leading to trees with altered leaf to wood ratios. These morphotypes can then be engineered into customized chemotypes that produce high value biofuels, bioproducts, and biomaterials not only in specific organs but also in a cell-type-specific manner. For example, the recent discovery of triterpene production in poplar leaf trichomes can be exploited using cell-type specific regulatory sequences to synthesize high value terpenes such as the jet fuel precursor bisabolene specifically in the trichomes. By spatially and temporally controlling expression, not only can pools of abundant precursors be exploited but engineered molecules can be sequestered in discrete cell structures in the leaf. The structural diversity of the hemicellulose xylan is a barrier to fully utilizing lignocellulose in biomaterial production and by leveraging cell-type-specific omics data, cell wall composition can be modified in a tailored and targeted specific manner to generate poplar wood with novel chemical features that are amenable for processing or advanced manufacturing. Precision engineering poplar as a multi-purpose sustainable feedstock highlights how genome engineering can be used to re-imagine a crop species.

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Beneficial effect of surfactant in adsorption/desorption of lignocellulose-degrading enzymes on/from lignin with different structure

Nonionic surfactant Tween 80 is effective in alleviating the adsorption of lignocellulose-degrading enzymes onto lignin. However, the impacts of lignin structural characteristics on this mitigation effect of Tween 80 were still not clear. Herein, effects of Tween 80 on cellulase and xylanase adsorption on different enzymatic residual lignins (ERLs) and poplar samples were investigated. Besides, effects of Tween 80 on desorption of the two enzyme preparations from different ERLs and poplar samples were also evaluated. By adding Tween 80, the inhibition of ERLs to cellulase hydrolysis was offset and the positive role of Tween 80 in Avicel hydrolysis with presence of ERLs was more effective. Binding strength of cellulase with ERLs was alleviated from 236.0 – 410.9 to 43.9 – 116.6 mL/g after adding 0.5 mg/mL Tween 80. When ERLs was more hydrophobic and had more condensed phenolic OH, the alleviation effect of Tween 80 on cellulase adsorption on ERLs was intensified but its positive effect on enhancing cellulase desorption was weakened. Tween 80 was effective in increasing xylanase activity and preventing xylanase deactivation. Further, with the increase of lignin content and phenolic OH in pretreated poplar, the effect of Tween 80 in alleviating xylanase adsorption on poplar samples was intensified but the effect in enhancing xylanase desorption from poplar samples was weakened. The results provided distinct findings to understand the interaction between Tween 80 and cellulase/xylanase in enzymatic saccharification of lignocelluloses.

59 BASIC BIOLOGICAL SCIENCES↗

Field-to-Fuel Performance Testing of Lignocellulosic Feedstocks for Fast Pyrolysis and Upgrading: Techno-economic Analysis and Greenhouse Gas Life Cycle Analysis

This work shows preliminary results from techno-economic analysis and life cycle greenhouse gas analysis of the conversion of seven (7) biomass feedstocks to produce liquid transportation fuels via fast pyrolysis and upgrading via hydrodeoxygenation. The biomass consists of five (5) pure feeds (pine, tulip poplar, hybrid poplar, switchgrass, corn stover) and two blends. Blend 1 consists of equal weights of pine, tulip poplar and switchgrass, and blend 2 is 67% pine and 33% hybrid poplar. Upgraded oil product yield is one of the most significant parameters affecting the process economics, and is a function of both fast pyrolysis oil yield and hydrotreating oil yield. Pure pine produced the highest overall yield, while switchgrass produced the lowest. Interestingly, herbaceous materials blended with woody biomass performed nearly as well as pure woody feedstock, suggesting a non-trivial relationship between feedstock attributes and production yield. Production costs are also highly dependent upon hydrotreating catalyst-related costs. The catalysts contribute an average of ~15% to the total fuel cost, which can be reduced through research and development focused on achieving performance at increased space velocity (e.g., reduced catalyst loading) and prolonging catalyst lifetime. Green-house-gas reduction does not necessarily align with favorable economics. From the greenhouse gas analysis, processing tulip poplar achieves the largest GHG emission reduction relative to petroleum (~70%) because of its lower hydrogen consumption in the upgrading stage that results in a lower natural gas requirement for hydrogen production. Conversely, processing blend 1 results in the smallest GHG emission reduction from petroleum (~58%) because of high natural gas demand for hydrogen production.

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Towards a map of the Populus biomass protein-protein interaction network

Biofuels can be produced from a variety of plant feedstocks. The value of a particular feedstock for biofuels production depends in part on the degree of difficulty associated with the extraction of fermentable sugars from the plant biomass. The wood of trees is potentially a rich source fermentable sugars. However, the sugars in wood exist in a tightly cross-linked matrix of cellulose, hemicellulose, and lignin, making them largely recalcitrant to release and fermentation for biofuels production. Before breeders and genetic engineers can effectively develop plants with reduced recalcitrance to fermentation, it is necessary to gain a better understanding of the fundamental biology of the mechanisms responsible for wood formation. Regulatory, structural, and enzymatic proteins are required for the complicated process of wood formation. To function properly, proteins must interact with other proteins. Yet, very few of the protein-protein interactions necessary for wood formation are known. The main objectives of this project were to 1) identify new protein-protein interactions relevant to wood formation, and 2) perform in-depth characterizations of selected protein-protein interactions. To identify relevant protein-protein interactions, we cloned a set of approximately 400 genes that were highly expressed in the wood-forming tissue (known as secondary xylem) of poplar (Populus trichocarpa). We tested whether the proteins encoded by these biomass genes interacted with each other in a binary matrix design using the yeast two-hybrid (Y2H) method for protein-protein interaction discovery. We also tested a subset of the 400 biomass proteins for interactions with all proteins present in wood-forming tissue of poplar in a biomass library screen design using Y2H. Together, these two Y2H screens yielded over 270 interactions involving over 75 biomass proteins. For the second main objective we selected several interacting pairs or groups of interacting proteins for in-depth characterizations. Characterizations involved both in vivo and in vitro independent methods to confirm protein-protein interactions and the evaluation of novel phenotypes resulting from creation of transgenic poplar and Arabidopsis plants engineered for increased or decreased expression of the selected genes. Transgenic poplar trees were studied in growth chamber, greenhouse, and two separate replicated field trials involving over 25 distinct wood-associated proteins. In-depth characterizations yielding positive results include the following. First, a NAC domain transcription factor (NAC154) that is a promoter of stress response and dormancy in trees was discovered. Increasing expression of NAC154 caused stunted growth and premature senescence, while decreasing expression led to both delayed bud and leaf expansion in spring and delayed leaf drop (i.e., prolonged leaf retention) in fall. Second, we discovered and characterized a new connection between a negative regulator of wood formation, the NAC domain transcription factor XND1, and an important regulator of cell division and cell differentiation, RBR. Third, we identified a new network of interacting wood-associated transcription factors belonging to the MYB and HD families. One of the HD family proteins, WOX13, was used to prepare transgenic poplar for high-level expression, resulting in significantly increased lateral branch growth. Finally, we modeled and performed in vitro analyses of the insect protein rubber resilin and we prepared transgenic Arabidopsis plants for expression of resilin to test the feasibility of using resilin to modify lignin cross-linking in wood and reduce recalcitrance and improve yield of fermentable sugars for biofuels production. Analysis of these and additional transgenics created with this support is continuing.

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Sun Grant/DOE Regional Feedstock Partnership: Final Technical Report

The U.S. Department of Energy (DOE) and the Sun Grant Initiative initiated the Regional Feedstock Partnership (referred to as the Partnership) in 2007 primarily to address information gaps associated with the likelihood for realization of the sustainable and reliable production of a billion-tons of biomass annually to support the U.S. bioenergy industry by the year 2030. Publication of The Technical Feasibility of a Billion-Ton Annual Supply (Perlack et al., 2005) in 2005, with its associated yield assumptions, led to the realization that the yield goals and assumptions utilized in that analysis required careful validation. To achieve the overall goal of validating key yield assumptions, the following objectives were developed: 1. Establishment of replicated field trials across regions to determine the impact of crop residue removal (primarily corn and small grains) on future grain yields and soil health. 2. Establishment of replicated field trials of some of the most promising dedicated energy crops (herbaceous annuals and perennials, as well as woody perennials) to demonstrate the potential performance of these feedstocks across the U.S. 3. Assessment of feedstock resources to be used to estimate a sustainable national supply potential. Teams composed of representatives from Land Grant Universities, USDA ARS, DOE National Laboratories, industry, and other federal agencies were identified and assembled to address each of these objectives. For Objective 1, members of the USDA ARS’ Resilient Economic Agricultural Practices (REAP) team combined with university personnel to leverage on-going work and established new trials to determine sustainable corn residue removal rates. In addition, universities across the continental U.S. conducted surveys of published past research to define parameters for the use of cereal grain residue (primarily wheat) as a biomass feedstock. For Objective 2, and based on input from DOE, USDA, Land Grant university scientists, and others, several herbaceous and woody energy crop species were selected for new or further evaluation across wide geographic areas as cellulosic feedstock sources. Species teams were organized to assess the yield potential of candidate herbaceous feedstocks including sorghum, energycane, Miscanthus x giganteus (hereafter referred to as miscanthus), switchgrass, and mixed perennial species (primarily grasses and some legumes) on Conservation Reserve Program (CRP) land. Woody species will be critical components of the bioenergy sector; therefore, species teams were also organized for shrub willow and hybrid poplar (Populus spp.). Field trials of each species were utilized to determine yield potential across the U.S. For Objective 3, a GIS resource team was organized with representatives from each of the five Sun Grant regions. While some regional assessments were made, the multi-institutional team decided that the group from Oregon State University would utilize their PRISM ELM model to estimate national yield potential of the various species evaluated. The following are key outcomes and impacts • The Partnership demonstrated the production potential of diverse herbaceous and woody feedstocks across much of the U.S. for 5-7 consecutive growing seasons. Long-term studies such as this are not commonly possible due to limitations in funding cycles. • Long-term field trials were valuable in demonstrating the importance of agronomic management practices and genetic resources to yield of sorghum, switchgrass, miscanthus, energycane, CRP mixed perennial grasses, poplar, and willow. • National yield potential maps were developed for all species evaluated in the Partnership. • Sustainable corn stover removal rates were quantified for use by producers, harvesters, and commercial lignocellulosic biorefineries. • Data from the Partnership field trials not only validated initial Billion Ton estimates, but were instrumental in developing both the Billion Ton Update in 2011, and especially the 2016 Billion Ton Report. • The Partnership provided the opportunity to assemble and conserve a poplar germplasm collection that contains more than 20,000 clones for use in breeding programs. New clones resulting from crosses made using this germplasm collection have resulted in significantly improved cultivars that could be scaled up and deployed. • Further validation of improved yields of new varieties/cultivars of biomass sorghum, energycane, hybrid poplars, and shrub willows was produced by the Partnership. • The Partnership demonstrated the potential of energycane in northern parts of the southeastern U.S., particularly in relation to sugarcane varieties. • For certain feedstocks (e.g., CRP mixed grasses, switchgrass, willow, and poplar), production economics were ascertained through Partnership field trials. • Biomass samples collected as part of the Partnership are also being used to populate Idaho National Laboratory’s Bioenergy Feedstock Library. • Because of the size and scope of the Partnership, workforce development was a key component and outcome. Numerous undergraduate students (25), graduate students (15 MS and 8 PhD), and postdoctoral researchers (7) were trained by Partnership participants. This will help to prepare individuals for careers in the emerging fields of plant breeding, biomass production, feedstock supply logistics, and biomass conversion processes. • More than 130 scientific publications have been authored by Partnership team members. Numerous other presentations, outreach publications, book chapters, and websites have also been developed to further the continued development of the bioeconomy.

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Data from: Leaching losses of dissolved organic carbon and nitrogen from agricultural soils in the upper US Midwest

Leaching losses of dissolved organic carbon (DOC) and nitrogen (DON) from agricultural systems are important to water quality and carbon and nutrient balances but are rarely reported; the few available studies suggest linkages to litter production (DOC) and nitrogen fertilization (DON). In this study we examine the leaching of DOC, DON, NO3-, and NH4+ from no-till corn (maize) and perennial bioenergy crops (switchgrass, miscanthus, native grasses, restored prairie, and poplar) grown between 2009 and 2016 in a replicated field experiment in the upper Midwest U.S. Leaching was estimated from concentrations in soil water and modeled drainage (percolation) rates. DOC leaching rates (kg ha-1 yr-1) and volume-weighted mean concentrations (mg L-1) among cropping systems averaged 15.4 and 4.6, respectively; N fertilization had no effect and poplar lost the most DOC (21.8 and 6.9, respectively). DON leaching rates (kg ha-1 yr-1) and volume-weighted mean concentrations (mg L-1) under corn (the most heavily N-fertilized crop) averaged 4.5 and 1.0, respectively, which was higher than perennial grasses (mean: 1.5 and 0.5, respectively) and poplar (1.6 and 0.5, respectively). NO3- comprised the majority of total N leaching in all systems (59-92%). Average NO3- leaching (kg N ha-1 yr-1) under corn (35.3) was higher than perennial grasses (5.9) and poplar (7.2). NH4+ concentrations in soil water from all cropping systems were relatively low (<0.07 mg N L-1). Perennial crops leached more NO3- in the first few years after planting, and markedly less after. Among the fertilized crops, the leached N represented 14-38% of the added N over the study period; poplar lost the greatest proportion (38%) and corn was intermediate (23%). Requiring only one third or less of the N fertilization compared to corn, perennial bioenergy crops can substantially reduce N leaching and consequent movement into aquifers and surface waters.

09 BIOMASS FUELS↗

Physiochemical Characterization of Lignocellulosic Biomass Dissolution by Flowthrough Pretreatment

Comprehensive understanding of biomass solubilization chemistry in aqueous pretreatment such as water-only and dilute acid flowthrough pretreatment is of fundamental importance to achieve the goal of valorizing biomass to fermentable sugars and lignin for biofuels production. In this study, poplar wood was flowthrough pretreated by water-only or 0.05% (w/w) sulfuric acid at different temperatures (220-270 °C), flow rate (25 mL/min), and reaction times (8-90 min), resulting in significant disruption of the lignocellulosic biomass. Ion chromatography (IC), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) analysis, and solid state cross-polarization/magic angle spinning (CP/MAS) 13C nuclear magnetic resonance (NMR) spectroscopy were applied to characterize the pretreated biomass whole slurries in order to reveal depolymerization as well as solubilization mechanism and identify unique dissolution structural features during these pretreatments. Results showed temperature-dependent cellulose decrystallization in flowthrough pretreatment. Crystalline cellulose was completely disrupted, and mostly converted to amorphous cellulose and oligomers by water-only operation at 270 °C for 10 min and by 0.05 wt % H2SO4 flowthrough pretreatment at 220 °C for 12 min. Flowthrough pretreatment with 0.05% (w/w) H2SO4 led to a greater disruption of structures in pretreated poplar at a lower temperature compared to water-only pretreatment.

54 ENVIRONMENTAL SCIENCES↗

The Role of Biomass Composition and Steam Treatment on Durability of Pellets

Steam treatment has been reported to improve the durability of wood pellet likely by changing the physical and chemical structures of wood particles, but published literature is inconclusive about which structure change is the major reason for enhanced durability. Here, in this paper, steam treatment was combined either with alkaline or with SO 2 to study. The solids obtained after steam treatments along with a control sample were dried and each was compacted into pellets. The pellets were then tested for durability. Steam treatment alone dominated improvements in durability. The pellet durability increased with the amount of xylose, but xylose performed better in the pellet from raw poplar than did in the pellet from treated poplar. Water-soluble components contributed a maximum 4% of the durability of poplar pellets. The addition of lignin and sugars to substrates after steam treatment did not improve durability significantly. The surface modification that took place as a result of size reduction during steam treatment was the major reason, contributing about 50% of the durability of the pellet from steam-treated poplar. The acidity of steam treatment slightly affected the relative contributions of these structure changes on the durability. Lastly, the new knowledge helps tailor the chemical and/or mechanical pretreatment involved in pelleting biomass to durable pellets.

54 ENVIRONMENTAL SCIENCES↗

Phytoremediation of sewage sludge contaminated by trace elements and organic compounds

Highlights: • Sewage sludge reclamation by poplar, willow, eucalyptus and sunflower was evaluated. • A species-specific response in trace element (TE) extraction was observed. • Sunflower was the best candidate for As, Cu, and Zn phytoextraction. • Eucalyptus seems a promising candidate for Pb and Cr phytoextraction. • All species were able to decrease organic pollutant concentration. Phytoremediation is a green technique being increasingly used worldwide for various purposes including the treatment of municipal sewage sludge (MSS). Most plants proposed for this technique have high nutrient demands, and fertilization is often required to maintain soil fertility and nutrient balance while remediating the substrate. In this context, MSS could be a valuable source of nutrients (especially N and P) and water for plant growth. The aim of this study was to determine the capacity willow (Salix matsudana, cv Levante), poplar (Populus deltoides × Populus nigra, cv Orion), eucalyptus (Eucalyptus camaldulensis) and sunflower (Helianthus annuus) to clean MSS, which is slightly contaminated by trace elements (TEs) and organic pollutants, and to assess their physiological response to this medium. In particular, we aimed to evaluate the TE accumulation by different species as well as the decrease of TEs and organic pollutants in the sludge after one cropping cycle and the effect of MSS on plant growth and physiology. Since MSS did not show any detrimental effect on the biomass yield of any of the species tested, it was found to be a suitable growing medium for these species. TE phytoextraction rates depended on the species, with eucalyptus showing the highest accumulation for Cr, whereas sunflower exhibited the best performance for As, Cu and Zn. At the end of the trial, some TEs (i.e. Cr, Pb and Zn), n-alkanes and PCBs showed a significant concentration decrease in the sludge for all tested species. The highest Cr decrease was observed in pots with eucalyptus (57.4%) and sunflower (53.4%), whereas sunflower showed the highest Cu decrease (44.2%), followed by eucalyptus (41.2%), poplar (16.2%) and willow (14%). A significant decrease (41.1%) of Pb in the eucalyptus was observed. Zn showed a high decrease rate with sunflower (59.5%) and poplar (52%) and to a lesser degree with willow (35.3%) and eucalyptus (25.4%). The highest decrease in n-alkanes concentration in the sludge was found in willow (98.3%) and sunflower (97.3%), whereas eucalyptus has the lowest PCBs concentration (91.8%) in the sludge compared to the beginning of the trial. These results suggest new strategies (e.g. crop rotation and intercropping) to be adopted for a better management of this phytotechnology.

54 ENVIRONMENTAL SCIENCES↗

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

Some of the most damaging tree diseases are caused by pathogens that induce cankers, a stem deformation often lethal. To investigate the cause of this adaptation, we sequenced the genomes of poplar pathogens that do and do not cause cankers. We found a unique cluster of genes that produce secondary metabolites and are co-activated when the canker pathogen is grown on poplar wood and leaves. The gene genealogy is discordant with the species phylogeny, showing a signature of horizontal transfer from fungi associated with wood decay. Furthermore, genes encoding hemicellulose-degrading enzymes are up-regulated on poplar wood chips, with some having been acquired horizontally. In conclusion, we propose that adaptation to colonize poplar woody stems is the result of acquisition of these genes.

59 BASIC BIOLOGICAL SCIENCES↗

Soil carbon sequestration and land use change associated with biofuel production: Empirical evidence

Soil organic carbon (SOC) change can be a major impact of land use change (LUC) associated with biofuel feedstock production. By collecting and analyzing data from worldwide field observations with major LUCs from cropland, grassland and forest to lands producing biofuel crops (i.e., corn, switchgrass, Miscanthus, poplar and willow), we were able to estimate SOC response ratios and sequestration rates and evaluate the effects of soil depth and time scale on SOC change. Both the amount and rate of SOC change were highly dependent on the specific land transition. Irrespective of soil depth or time horizon, cropland conversions resulted in an overall SOC gain of 6-14% relative to initial SOC level, while conversion from grassland or forest to corn (without residue removal) or poplar caused significant carbon loss (9-35%). No significant SOC changes were observed in land converted from grasslands or forests to switchgrass, Miscanthus or willow. The SOC response ratios were similar in both 0-30 and 0-100 cm soil depths in most cases, suggesting SOC changes in deep soil and that use of top soil only for SOC accounting in biofuel life cycle analysis (LCA) might underestimate total SOC changes. Soil carbon sequestration rates varied greatly among studies and land transition types. Generally, the rates of SOC change tended to be the greatest during the 10 years following land conversion, and had declined to approach 0 within about 20 years for most LUCs. Observed trends in SOC change were generally consistent with previous reports. Soil depth and duration of study significantly influence SOC change rates and so should be considered in carbon emission accounting in biofuel LCA. High uncertainty remains for many perennial systems, field trials and modeling efforts are needed to determine the site- and system-specific rates and direction of change associated with their production.

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Functional Gene Discovery and Characterization of Genes and Alleles Affecting Wood Biomass Yield and Quality in Populus

Adoption of biofuels as economically and environmentally viable alternative to fossil fuels would require development of specialized bioenergy varieties. A major goal in the breeding of such varieties is the improvement of lignocellulosic biomass yield and quality. These are complex traits and understanding the underpinning molecular mechanism can assist and accelerate their improvement. This is particularly important for tree bioenergy crops like poplars (species and hybrids from the genus Populus), for which breeding progress is extremely slow due to long generation cycles. A variety of approaches have been already undertaken to better understand the molecular bases of biomass yield and quality in poplar. An obvious void in these undertakings has been the application of mutagenesis. Mutagenesis has been instrumental in the discovery and characterization of many plant traits including such that affect biomass yield and quality. In this proposal we use activation tagging to discover genes that can significantly affect biomass associated traits directly in poplar, a premier bioenergy crop. We screened a population of 5,000 independent poplar activation tagging lines under greenhouse conditions for a battery of biomass yield traits. These same plants were then analyzed for changes in wood chemistry using pyMBMS. As a result of these screens we have identified nearly 800 mutants, which are significantly (P<0.05) different when compared to wild type. Of these majority (~700) are affected in one of ten different biomass yield traits and 100 in biomass quality traits (e.g., lignin, S/G ration and C6/C5 sugars). We successfully recovered the position of the tag in approximately 130 lines, showed activation in nearly half of them and performed recapitulation experiments with 20 genes prioritized by the significance of the phenotype. Recapitulation experiments are still ongoing for many of the genes but the results are encouraging. For example, we have shown successful recapitulation for a fascilin-like gene that when overexpressed increase many biomass-yield associated traits. Genes discovered through activation tagging showed polymorphisms in P. trichocarpa association mapping population linked to the traits modified by the activation tagging. This suggests that putative alleles that are associated with improvement of the trait o interest can be discovered and used in marker associated selection. This will significantly simplify and accelerate the breeding efforts.

09 BIOMASS FUELS↗

Improved Advanced Biomass Logistics Utilizing Woody and other Feedstocks in the Northeast and Pacific Northwest (Final Report)

Willow and poplar short rotation woody crops (SRWC) have shown promise with regards to environmental benefits and rural development but wide adoption lags due to underdeveloped markets and supply systems. High costs associated with harvesting, handling and transportation (40-60% of delivered cost) have impeded expansion. A better understanding of these systems will create opportunities to improve efficiency, reduce costs, and realize environmental benefits and impacts. The project’s goal was to lower the delivered cost of hybrid poplar in the Northwest and willow in the Northeast by optimizing harvesting and logistics supply systems while maintaining or improving biomass quality along the supply chain. Over 3,400 Mg of biomass and 300 ha of willow and poplar were monitored over a range of crop and field conditions. Feedstock quality as affected by storage and preprocessing were shown to improve or maintain feedstock quality. Modeled harvesting costs ranged from $38–61 Mg -1 dry; when including delivery and preprocessing feedstock costs ranged between $79-83 Mg -1 dry for willow and $106-116 Mg -1 dry for poplar. Costs for willow minimized when hot water extraction and high-moisture densification preprocessing were used. Models also suggest that social and regional factors could further reduce costs. Results will give guidance to feedstock growers, harvesting and logistic operations, biorefinery project developers, and policy makers developing SRWC to support a growing bioeconomy.

, biomass↗