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[This corrects the article DOI: 10.1186/s13068-019-1353-7.].

BACKGROUND: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. RESULTS: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. CONCLUSION: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.

Abstract Background Grasses are relatively recalcitrant to genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass (Panicum virgatum L.) has previously been reported after cocultivation of explants with Agrobacterium and biolistics of embryogenic calli. Experiments to increase transient gene expression in planta may lead to stable transformation methods with increased efficiency. Results A high-throughput Agrobacterium-mediated transient gene expression system has been developed for in planta inoculation of germinating switchgrass seedlings. Four different Agrobacterium strains were compared for their ability to infect switchgrass seedlings, and strain AGL1 was found to be the most infective. Wounding pretreatments such as sonication, mixing by vortex with carborundum, separation by centrifugation, vacuum infiltration, and high temperature shock significantly increased transient expression of a reporter gene (GUSPlus, a variation of the β-glucuronidase (GUS) gene). The addition of L-cysteine and dithiothreitol in the presence of acetosyringone significantly increased GUS expression compared with control treatments, whereas the addition of 0.1% surfactants such as Silwet L77 or Li700 decreased GUS expression. 4-Methylumbelliferyl beta-D-galactopyranoside (MUG) assays showed a peak of β-glucuronidase (GUS) enzyme activity 3 days after cocultivation with Agrobacterium harboring pCambia1305.2, whereas MUG assays showed a peak of enzyme activity 5 days after cocultivation with Agrobacterium harboring pCambia1305.1. Conclusion Agrobacterium strains C58, GV3101 and EHA105 are less able to deliver transfer DNA to switchgrass seedlings (cultivar Alamo) compared with strain AGL1. Transient expression was increased by double or triple wounding treatments such as mixing by vortex with carborundum, sonication, separation by centrifugation, and heat shock. The addition of thiol compounds such as L-cysteine and dithiothreitol in combination with acetosyringone during cocultivation also increased transient expression. The combination of multiple wounding treatments along with the addition of thiol compounds during cocultivation increased transient expression levels from 6% to 54%. There were differences in temporal GUS expression induced by pCambia1305.1 and pCambia1305.2.

Summary: Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate‐expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The native recalcitrance of plants hinders the biomass conversion process using current biorefinery techniques. Down-regulation of the caffeic acid O-methyltransferase (COMT) gene in the lignin biosynthesis pathway of switchgrass reduced the thermochemical and biochemical conversion recalcitrance of biomass. Due to potential environmental influences on lignin biosynthesis and deposition, studying the consequences of physicochemical changes in field-grown plants without pretreatment is essential to evaluate the performance of lignin-altered plants. We determined the chemical composition, cellulose crystallinity and the degree of its polymerization, molecular weight of hemicellulose, and cellulose accessibility of cell walls in order to better understand the fundamental features of why biomass is recalcitrant to conversion without pretreatment. The most important is to investigate whether traits and features are stable in the dynamics of field environmental effects over multiple years. Results: Field-grown COMT down-regulated plants maintained both reduced cell wall recalcitrance and lignin content compared with the non-transgenic controls for at least 3 seasons. The transgenic switchgrass yielded 35-84% higher total sugar release (enzymatic digestibility or saccharification) from a 72-h enzymatic hydrolysis without pretreatment and also had a 25-32% increase in enzymatic sugar release after hydrothermal pretreatment. The COMT-silenced switchgrass lines had consistently lower lignin content, e.g., 12 and 14% reduction for year 2 and year 3 growing season, respectively, than the control plants. By contrast, the transgenic lines had 7-8% more xylan and galactan contents than the wild-type controls. Gel permeation chromatographic results revealed that the weightaverage molecular weights of hemicellulose were 7-11% lower in the transgenic than in the control lines. In addition, we found that silencing of COMT in switchgrass led to 20-22% increased cellulose accessibility as measured by the Simons' stain protocol. No significant changes were observed on the arabinan and glucan contents, cellulose crystallinity, and cellulose degree of polymerization between the transgenic and control plants. With the 2-year comparative analysis, both the control and transgenic lines had significant increases in lignin and glucan contents and hemicellulose molecular weight across the growing seasons. Conclusions: The down-regulation of COMT in switchgrass resulting in a reduced lignin content and biomass recalcitrance is stable in a field-grown trial for at least three seasons. Among the determined affecting factors, the reduced biomass recalcitrance of the COMT-silenced switchgrass, grown in the field conditions for two and three seasons, was likely related to the decreased lignin content and increased biomass accessibility, whereas the cellulose crystallinity and degree of its polymerization and hemicellulose molecular weights did not contribute to the reduction of recalcitrance significantly. This finding suggests that lignin down-regulation in lignocellulosic feedstock confers improved saccharification that translates from greenhouse to field trial and that lignin content and biomass accessibility are two significant factors for developing a reduced recalcitrance feedstock by genetic modification. [ABSTRACT FROM AUTHOR]