Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2‐year comparative analysis of field‐grown lines modified for target gene or genetic element expression

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4‐KD, miRNA156‐OE, MYB4‐OE, COMT‐KD and FPGS‐KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second‐ versus the first‐year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second‐year growth of transgenics targeted for wall modification, GAUT4‐KD, MYB4‐OE, COMT‐KD and FPGS‐KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next‐generation bio‐feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.     

Impacts of land use change due to biofuel crops on carbon balance,bioenergy production,and agricultural yield,in the conterminous United States

Growing concerns about energy and the environment have led to worldwide use of bioenergy. Switching from food crops to biofuel crops is an option to meet the fast‐growing need for biofuel feedstocks. This land use change consequently affects the ecosystem carbon balance. In this study, we used a biogeochemistry model, the Terrestrial Ecosystem Model, to evaluate the impacts of this change on the carbon balance, bioenergy production, and agricultural yield, assuming that several land use change scenarios from corn, soybean, and wheat to biofuel crops of switchgrass and Miscanthus will occur. We found that biofuel crops have much higher net primary production (NPP) than soybean and wheat crops. When food crops from current agricultural lands were changed to different biofuel crops, the national total NPP increased in all cases by a range of 0.14–0.88 Pg C yr^?1, except while switching from corn to switchgrass when a decrease of 14% was observed. Miscanthus is more productive than switchgrass, producing about 2.5 times the NPP of switchgrass. The net carbon loss ranges from 1.0 to 6.3 Tg C yr^?1 if food crops are changed to switchgrass, and from 0.4 to 6.7 Tg C yr^?1 if changed to Miscanthus. The largest loss was observed when soybean crops were replaced with biofuel crops. Soil organic carbon increased significantly when land use changed, reaching 100 Mg C ha^?1 in biofuel crop ecosystems. When switching from food crops to Miscanthus, the per unit area croplands produced a larger amount of ethanol than that of original food crops. In comparison, the land use change from wheat to Miscanthus produced more biomass and sequestrated more carbon. Our study suggests that Miscanthus could better serve as an energy crop than food crops or switchgrass, considering both economic and environmental benefits.     

Opportunities and roadblocks in utilizing forages and small grains for liquid fuels

This review focuses on the potential advantages and disadvantages of forages such as switchgrass (Panicum virgatum), and two small grains: sorghum (Sorghum bicolor), and wheat (Triticum aesitvum), as feedstocks for biofuels. It highlights the synergy provided by applying what is known from forage digestibility and wheat and sorghum starch properties studies to the biofuels sector. Opportunities therefore, exist to improve biofuel qualities in these crops via genetics and agronomics. In contrast to cereal crops, switchgrass still retains tremendous exploitable genetic diversity, and can be specifically improved to fit a particular agronomic, management, and conversion platform. Combined with emerging studies on switchgrass genomics, conversion properties and management, the future for genetic modification of this species through conventional and molecular breeding strategies appear to be bright. The presence of brown-midrib mutations in sorghum that alter cell wall composition by reducing lignin and other attributes indicate that sorghum could serve as an important model species for C₄-grasses. Utilization of the brown-midrib traits could lead to the development of forage and sweet sorghums as novel biomass crops. Additionally, wheat crop residue, and wheat and sorghum with improved starch content and composition represent alternate biofuel sources. However, the use of wheat starch as a biofuel is unlikely but its value as a model to study starch properties on biofuel yields holds significant promise. JIMB-2008: BioEnergy—Special issue.     

Using satellite vegetation and compound topographic indices to map highly erodible cropland buffers for cellulosic biofuel crop developments in eastern Nebraska,USA

Cultivating annual row crops in high topographic relief waterway buffers has negative environmental effects and can be environmentally unsustainable. Growing perennial grasses such as switchgrass (Panicum virgatum L.) for biomass (e.g., cellulosic biofuel feedstocks) instead of annual row crops in these high relief waterway buffers can improve local environmental conditions (e.g., reduce soil erosion and improve water quality through lower use of fertilizers and pesticides) and ecosystem services (e.g., minimize drought and flood impacts on production; improve wildlife habitat, plant vigor, and nitrogen retention due to post-senescence harvest for cellulosic biofuels; and serve as carbon sinks). The main objectives of this study are to: (1) identify cropland areas with high topographic relief (high runoff potentials) and high switchgrass productivity potential in eastern Nebraska that may be suitable for growing switchgrass, and (2) estimate the total switchgrass production gain from the potential biofuel areas. Results indicate that about 140,000 hectares of waterway buffers in eastern Nebraska are suitable for switchgrass development and the total annual estimated switchgrass biomass production for these suitable areas is approximately 1.2 million metric tons. The resulting map delineates high topographic relief croplands and provides useful information to land managers and biofuel plant investors to make optimal land use decisions regarding biofuel crop development and ecosystem service optimization in eastern Nebraska.     

Using species distribution models to identify suitable areas for biofuel feedstock production

The 2007 Energy Independence and Security Act mandates a five‐fold increase in US biofuel production by 2022. Given this ambitious policy target, there is a need for spatially explicit estimates of landscape suitability for growing biofuel feedstocks. We developed a suitability modeling approach for two major US biofuel crops, corn (Zea mays) and switchgrass (Panicum virgatum), based upon the use of two presence‐only species distribution models (SDMs): maximum entropy (Maxent) and support vector machines (SVM). SDMs are commonly used for modeling animal and plant distributions in natural environments, but have rarely been used to develop landscape models for cultivated crops. AUC, Kappa, and correlation measures derived from test data indicate that SVM slightly outperformed Maxent in modeling US corn production, although both models produced significantly accurate results. When compared with results from a mechanistic switchgrass model recently developed by Oak Ridge National Laboratory (ORNL), SVM results showed higher correlation than Maxent results with models fit using county‐scale point inputs of switchgrass production derived from expert opinion estimates. However, Maxent results for an alternative switchgrass model developed with point inputs from research trial sites showed higher correlation to the ORNL model than the corresponding results obtained from SVM. Further analysis indicates that both modeling approaches were effective in predicting county‐scale increases in corn production from 2006 to 2007, a time period in which US corn production increased by 24%. We conclude that presence‐only methods are a powerful first‐cut tool for estimating relative land suitability across geographic regions in which candidate biofuel feedstocks can be grown, and may also provide important insight into potential land‐use change patterns likely to be associated with increased biofuel demand.     

Biocontrol potential varies with changes in biofuel–crop plant communities and landscape perenniality

We examined the potential local‐ and landscape‐level impacts of different biofuel production systems on biocontrol, an important service provided by arthropod natural enemies. Specifically, we sampled natural enemies with sweep nets and measured predation of sentinel pest eggs in stands of corn, switchgrass and mixed prairie in Michigan and Wisconsin (total n=40 for natural enemy sampling, n=60 for egg predation), relating them to crop type, forb cover and diversity, and the composition and heterogeneity of the surrounding landscape. Grasslands with intermediate levels of forb cover and flower diversity supported two‐orders of magnitude more natural enemy biomass, fourfold more natural enemy families, and threefold greater rates of egg predation than corn. Data suggest this was in part due to a general increase in biomass, richness and predation in perennial grasslands compared with corn, combined with a positive effect of intermediate levels of forb cover and flower diversity. Specifically, natural enemy biomass and family richness showed hump‐shaped relationships to forb cover that peaked in sites with 5–25% forbs, while egg predation increased with floral diversity. At the landscape scale, both natural‐enemy biomass and egg predation increased with the area of forest in the landscape, and egg predation almost doubled as the area of herbaceous, perennial habitats within 1.5 km of study sites increased. Our results suggest that floristically diverse, perennial grasslands support diverse and abundant predator communities that contribute to natural pest suppression. In addition, large‐scale production of biofuel crops could positively or negatively affect biocontrol services in agricultural landscapes through associated changes in the area of perennial habitats. Biofuel landscapes that incorporate perennial grasslands could support a variety of beneficial organisms and ecosystem services in addition to producing biomass.     

Bioenergy crops Miscanthus x giganteus and Panicum virgatum reduce growth and survivorship of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Large-scale cultivation of plants used as biofuels is likely to alter the ecological interactions of current agricultural crops and their insect pests in a myriad of ways. Recent evidence suggests many contemporary maize pests will be able to use potential biofuel crops such as switchgrass, Panicum virgatum L., and miscanthus as hosts. To determine how suitable these biofuels are to the maize, Zea mays L., pest and generalist graminivore, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), we examined host plant preference and larval performance on foliage grown for commercial biofuel production. Larvae fed leaf tissue from both field- and greenhouse-grown switchgrass and miscanthus were monitored for survival, development, and food use relative to field-grown maize. Survivorship on biofuel crops was high on greenhouse-grown leaf tissue but severely reduced for field-grown switchgrass, and no larvae survived on field-grown miscanthus. Larvae fed field-grown tissue had larger head capsules yet achieved lower pupal weights because the increased toughness of the leaf tissue prevented the assimilation of nitrogen. Given that larvae overwhelmingly preferred maize to other biofuel crop species and that survival and performance were dramatically reduced on biofuel crop species, it is likely that biofuel crops, as grown for field cultivation, will suffer reduced damage from maize pests such as S. frugiperda because of reduced suitability.     

Expression of ZmGA20ox cDNA alters plant morphology and increases biomass production of switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is considered a model herbaceous energy crop for the USA, for its adaptation to marginal land, low rainfall and nutrient‐deficient soils; however, its low biomass yield is one of several constraints, and this might be rectified by modulating plant growth regulator levels. In this study, we have determined whether the expression of the Zea mays gibberellin 20‐oxidase (ZmGA20ox) cDNA in switchgrass will improve biomass production. The ZmGA20ox gene was placed under the control of constitutive CaMV35S promoter with a strong TMV omega enhancer, and introduced into switchgrass via Agrobacterium‐mediated transformation. The transgene integration and expression levels of ZmGA20ox in T0 plants were analysed using Southern blot and qRT‐PCR. Under glasshouse conditions, selected transgenic plants exhibited longer leaves, internodes and tillers, which resulted in twofold increased biomass. These phenotypic alterations correlated with the levels of transgene expression and the particular gibberellin content. Expression of ZmGA20ox also affected the expression of genes coding for key enzymes in lignin biosynthesis. Our results suggest that the employment of ectopic ZmGA20ox and selection for natural variants with high level expression of endogenous GA20ox are appropriate approaches to increase biomass production of switchgrass and other monocot biofuel crops.     

Bioenergy crop productivity and potential climate change mitigation from marginal lands in the United States: An ecosystem modeling perspective

Growing biomass feedstocks from marginal lands is becoming an increasingly attractive choice for producing biofuel as an alternative energy to fossil fuels. Here, we used a biogeochemical model at ecosystem scale to estimate crop productivity and greenhouse gas (GHG) emissions from bioenergy crops grown on marginal lands in the United States. Two broadly tested cellulosic crops, switchgrass, and Miscanthus, were assumed to be grown on the abandoned land and mixed crop‐vegetation land with marginal productivity. Production of biomass and biofuel as well as net carbon exchange and nitrous oxide emissions were estimated in a spatially explicit manner. We found that, cellulosic crops, especially Miscanthus could produce a considerable amount of biomass, and the effective ethanol yield is high on these marginal lands. For every hectare of marginal land, switchgrass and Miscanthus could produce 1.0–2.3 kl and 2.9–6.9 kl ethanol, respectively, depending on nitrogen fertilization rate and biofuel conversion efficiency. Nationally, both crop systems act as net GHG sources. Switchgrass has high global warming intensity (100–390 g CO₂eq l^?1 ethanol), in terms of GHG emissions per unit ethanol produced. Miscanthus, however, emits only 21–36 g CO₂eq to produce every liter of ethanol. To reach the mandated cellulosic ethanol target in the United States, growing Miscanthus on the marginal lands could potentially save land and reduce GHG emissions in comparison to growing switchgrass. However, the ecosystem modeling is still limited by data availability and model deficiencies, further efforts should be made to classify crop‐specific marginal land availability, improve model structure, and better integrate ecosystem modeling into life cycle assessment.     

Field‐grown transgenic switchgrass (Panicum virgatum L.) with altered lignin does not affect soil chemistry,microbiology, and carbon storage potential

Cell wall recalcitrance poses a major challenge on cellulosic biofuel production from feedstocks such as switchgrass (Panicum virgatum L.). As lignin is a known contributor of recalcitrance, transgenic switchgrass plants with altered lignin have been produced by downregulation of caffeic acid O‐methyltransferase (COMT). Field trials of COMT‐downregulated plants previously demonstrated improved ethanol conversion with no adverse agronomic effects. However, the rhizosphere impacts of altering lignin in plants are unknown. We hypothesized that changing plant lignin composition may affect residue degradation in soils, ultimately altering soil processes. The objective of this study was to evaluate effects of two independent lines of COMT‐downregulated switchgrass plants on soils in terms of chemistry, microbiology, and carbon cycling when grown in the field. Over the first two years of establishment, we observed no significant differences between transgenic and control plants in terms of soil pH or the total concentrations of 19 elements. An analysis of soil bacterial communities via high‐throughput 16S rRNA gene amplicon sequencing revealed no effects of transgenic plants on bacterial diversity, richness, or community composition. We also did not observe a change in the capacity for soil carbon storage: There was no significant effect on soil respiration or soil organic matter. After five years of establishment, δ¹³C of plant roots, leaves, and soils was measured and an isotopic mixing model used to estimate that 11.2 to 14.5% of soil carbon originated from switchgrass. Switchgrass‐contributed carbon was not significantly different between transgenic and control plants. Overall, our results indicate that over the short term (two and five years), lignin modification in switchgrass through manipulation of COMT expression does not have an adverse effect on soils in terms of total elemental composition, bacterial community structure and diversity, and capacity for carbon storage.     

Long-term variability of root production in bioenergy crops from ingrowth core measurements

用内生长法测定的生物能源作物根生产的长期变化 对于土地用途转为种植生物燃料作物后的根系产量,还很少进行过长期的测定。为了评价此前的土地用途对地下生物量积累的影响,我们在“美国休耕保护项目”(Conservation Reserve Program, CPR)下生长了22年的草地(CRP草地)和使用期超过50年的农业用地(AGR农地)转为种植生物燃料作物玉米(Zea mays, Corn, C)、柳枝稷(Panicum virgatum, Switchgrass, Sw)和恢复性草原植被(Prairie, Pr)。我们将一块CPR草地维持为对照。我们的假设是土地利用历史和作物类型对根系密度有显著的影响;其中,原CRP草地上种植的多年生作物具有较高的根系生产力,而在原农业用地上种植的玉米的根系生产力最低。通过内生长土芯法对内生长根系生物量进行了原位测定,同时对地上净初级生产力(ANPP)进行了测量。包括气温、生长季长度和降水量在内的辅助测量则被用来考查它们对根系生产量的影响。根系生产力在未转变的CRP草地最高(1716 g m⁻² yr⁻¹),而在玉米田中最低(526 g m⁻² yr⁻¹)。由CRP草地和AGR农田转变而来的多年生作物种植地在第一年都具有较低的根系生物量和ANPP,但柳枝稷在2011年达到峰值,恢复后的草原植被也在一年后达到峰值。恢复后的草原生态系统稳定性较高(AGR-Pr: 4.3 ± 0.11; CRP-Pr: 4.1 ± 0.10),而仅种植单一作物的生态系统稳定性都较低。根系生产量与ANPP呈正相关性(R² = 0.40)。总体而言,对生物燃料作物大规模种植过程中的根系生物量积累应予以重视,因为这是固碳的一种主 要来源。     

Genetic Engineering of Energy Crops: A Strategy for Biofuel Production in China Free Access

Biomass utilization is increasingly considered as a practical way for sustainable energy supply and long‐term environment care around the world. In concerns with food security in China, starch or sugar‐based bioethanol and edible‐oil‐derived biodiesel are harshly restricted for large scale production. However, conversion of lignocellulosic residues from food crops is a potential alternative. Because of its recalcitrance, current biomass process is unacceptably expensive, but genetic breeding of energy crops is a promising solution. To meet the need, energy crops are defined with a high yield for both food and biofuel purposes. In this review, main grasses (rice, wheat, maize, sorghum and miscanthus) are evaluated for high biomass production, the principles are discussed on modification of plant cell walls that lead to efficient biomass degradation and conversion, and the related biotechnologies are proposed in terms of energy crop selection.     

Impact of agronomic uncertainty in biomass production and endogenous commodity prices on cellulosic biofuel feedstock composition

This study evaluates the effect of agronomic uncertainty on bioenergy crop production as well as endogenous commodity and biomass prices on the feedstock composition of cellulosic biofuels under a binding mandate in the United States. The county‐level simulation model focuses on both field crops (corn, soybean, and wheat) and biomass feedstocks (corn stover, wheat straw, switchgrass, and Miscanthus). In addition, pasture serves as a potential area for bioenergy crop production. The economic model is calibrated to 2022 in terms of yield, crop demand, and baseline prices and allocates land optimally among the alternative crops given the binding cellulosic biofuel mandate. The simulation scenarios differ in terms of bioenergy crop type (switchgrass and Miscanthus) and yield, biomass production inputs, and pasture availability. The cellulosic biofuel mandates range from 15 to 60 billion L. The results indicate that the 15 and 30 billion L mandates in the high production input scenarios for switchgrass and Miscanthus are covered entirely by agricultural residues. With the exception of the low production input for Miscanthus scenario, the share of agricultural residues is always over 50% for all other scenarios including the 60 billion L mandate. The largest proportion of agricultural land dedicated to either switchgrass or Miscanthus is found in the southern Plains and the southeast. Almost no bioenergy crops are grown in the Midwest across all scenarios. Changes in the prices for the three commodities are negligible for cellulosic ethanol mandates because most of the mandate is met with agricultural residues. The lessons learned are that (1) the share of agricultural residue in the feedstock mix is higher than previously estimated and (2) for a given mandate, the feedstock composition is relatively stable with the exception of one scenario.     

Implications of Three Biofuel Crops for Beneficial Arthropods in Agricultural Landscapes

Production of biofuel feedstocks in agricultural landscapes will result in land use changes that may have major implications for arthropod-mediated ecosystem services such as pollination and pest suppression. By comparing the abundance and diversity of insect pollinators and generalist natural enemies in three model biofuel crops: corn, switchgrass, and mixed prairie, we tested the hypothesis that biofuel crops comprised of more diverse plant communities would support increased levels of beneficial insects. These three biofuel crops contained a diverse bee community comprised of 75 species. Overall, bees were three to four times more abundant in switchgrass and prairie than in corn, with members of the sweat bee (Halictidae) and small carpenter bee (Ceratina spp.) groups the most abundant. Switchgrass and prairie had significantly greater bee species richness than corn during the July sampling period. The natural enemy community at these sites was dominated by lady beetles (Coccinellidae), long-legged flies (Dolichopodidae), and hover flies (Syrphidae) which varied in their response to crop type. Coccinellids were generally most abundant in prairie and switchgrass, with the exception of the pollen feeding Coleomegilla maculata that was most abundant in corn. Several rare or declining coccinellid species were detected in prairie and switchgrass sites. Dolichopodidae were more abundant in prairie and switchgrass while Syrphidae showed no significant response to crop type. Our results indicate that beneficial insects generally responded positively to the increased vegetational diversity of prairie and switchgrass sites; however, when managed as a dedicated biofuel crop, plant and arthropod diversity in switchgrass may decrease. Our findings support the hypothesis that vegetationally diverse biofuel crops support higher abundance and diversity of beneficial insects. Future policy regarding the production of biofuel feedstocks should consider the ecosystem services that different biofuel crops may support in agricultural landscapes.     

Influence of spatially dependent,modeled soil carbon emission factors on life‐cycle greenhouse gas emissions of corn and cellulosic ethanol

Converting land to biofuel feedstock production incurs changes in soil organic carbon (SOC) that can influence biofuel life‐cycle greenhouse gas (GHG) emissions. Estimates of these land use change (LUC) and life‐cycle GHG emissions affect biofuels' attractiveness and eligibility under a number of renewable fuel policies in the USA and abroad. Modeling was used to refine the spatial resolution and depth extent of domestic estimates of SOC change for land (cropland, cropland pasture, grassland, and forest) conversion scenarios to biofuel crops (corn, corn stover, switchgrass, Miscanthus, poplar, and willow) at the county level in the USA. Results show that in most regions, conversions from cropland and cropland pasture to biofuel crops led to neutral or small levels of SOC sequestration, while conversion of grassland and forest generally caused net SOC loss. SOC change results were incorporated into the Greenhouse Gases, Regulated Emissions, and Energy use in Transportation (GREET) model to assess their influence on life‐cycle GHG emissions of corn and cellulosic ethanol. Total LUC GHG emissions (g CO₂eq MJ^?1) were 2.1–9.3 for corn‐, ?0.7 for corn stover‐, ?3.4 to 12.9 for switchgrass‐, and ?20.1 to ?6.2 for Miscanthus ethanol; these varied with SOC modeling assumptions applied. Extending the soil depth from 30 to 100 cm affected spatially explicit SOC change and overall LUC GHG emissions; however, the influence on LUC GHG emission estimates was less significant in corn and corn stover than cellulosic feedstocks. Total life‐cycle GHG emissions (g CO₂eq MJ^?1, 100 cm) were estimated to be 59–66 for corn ethanol, 14 for stover ethanol, 18–26 for switchgrass ethanol, and ?7 to ?0.6 for Miscanthus ethanol. The LUC GHG emissions associated with poplar‐ and willow‐derived ethanol may be higher than that for switchgrass ethanol due to lower biomass yield.     

Influence of biofuel crops on mosquito production and oviposition site selection

The proposed expansion of biofuels production may cause unintended land‐use changes and potentially alter ecosystem services. This study evaluated the impact of first‐generation (corn) and second‐generation (switchgrass and Miscanthus) biofuel crops on production and oviposition site selection by two vector mosquitoes, the yellow fever mosquito Aedes aegypti and the Asian tiger mosquito Aedes albopictus. Larvae of the two species were reared at varying conspecific and heterospecific densities in senescent leaf infusions prepared from one of the three biofuel crops and their survival and development time to adulthood determined. The effects of the three leaf infusions on water chemistry and oviposition site selection by the two mosquito species were also determined. Ae. albopictus females deposited significantly fewer eggs in Miscanthus than in corn infusion while Ae. aegypti females deposited significantly fewer eggs in Miscanthus than in both corn and switchgrass infusion. Survival to adulthood for both mosquito species was significantly lower in corn than in switchgrass and Miscanthus infusions; was consistently lower at high‐ (0:40 and 20:20) than at low density treatments in both switchgrass and Miscanthus infusions; and significantly lower at high intraspecific density (40:0 and 0: 40) than at high interspecific density (20:20) in Miscanthus infusion. Development time to adulthood was positively related to larval density, but was not influenced by biofuel leaf treatment. Corn infusion had lower pH values and higher salinity, conductivity, total dissolved solids (TDS), and temperature values than switchgrass and Miscanthus infusions. These findings demonstrate the potential for biofuel crops to modify the chemistry of aquatic habitats in ways that may influence mosquito production and thereby the risk of exposure to mosquito‐borne diseases.