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[This corrects the article DOI: 10.1186/s13068-019-1353-7.].
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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.
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BACKGROUND: Milling during fermentation, termed cotreatment, has recently been proposed as an alternative to thermochemical pretreatment as a means to increase the accessibility of lignocellulosic biomass to biological attack. A central premise of this approach is that partial solubilization of biomass changes the slurry's physical properties such that milling becomes more impactful and more feasible. A key uncertainty is the energy required to mill partially fermented biomass. To inform both of these issues, we report rheological characterization of small-particle, corn stover slurries undergoing fermentation by Clostridium thermocellum. RESULTS: Fermented and unfermented corn stover slurries were found to be shear-thinning and well described by a power law model with an exponent of 0.10. Plastic viscosity of a slurry, initially at 16 wt.% insoluble solids, decreased as a result of fermentation by a factor of 2000, with the first eightfold reduction occurring in the first 10% of carbohydrate conversion. Large amplitude oscillatory shear experiments revealed only minor changes to the slurry's rheological fingerprint as a result of fermentation, with the notable change being a reduction in the critical strain amplitude needed for the onset of nonlinearity. All slurries were found to be elastoviscoplastic, with the elastic/viscous crossover at roughly 100% strain amplitude. CONCLUSIONS: Whereas prior biomass rheology studies have involved pretreated feedstocks and solubilization mediated by fungal cellulase, we report results for feedstocks with no pretreatment other than autoclaving and for solubilization mediated by C. thermocellum. As observed in prior studies, C. thermocellum fermentation results in a dramatic decrease in viscosity. The magnitude of this decrease, however, is much larger starting with unpretreated feedstock than previously reported for pretreated feedstocks. LAOS measurements provide a detailed picture of the rheological fingerprint of the material. Viscosity measurements confirm the hypothesis that the physical character of corn stover slurries changes dramatically during fermentation by C. thermocellum, and indicate that the energy expended on overcoming slurry viscosity will be far less for partially fermented corn stover than for unfermented corn stover.
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BACKGROUND: Clostridium thermocellum has been the subject of multiple metabolic engineering strategies to improve its ability to ferment cellulose to ethanol, with varying degrees of success. For ethanol production in C. thermocellum, the conversion of pyruvate to acetyl-CoA is catalyzed primarily by the pyruvate ferredoxin oxidoreductase (PFOR) pathway. Thermoanaerobacterium saccharolyticum, which was previously engineered to produce ethanol of high yield (> 80%) and titer (70 g/L), also uses a pyruvate ferredoxin oxidoreductase, pforA, for ethanol production. RESULTS: Here, we introduced the T. saccharolyticum pforA and ferredoxin into C. thermocellum. The introduction of pforA resulted in significant improvements to ethanol yield and titer in C. thermocellum grown on 50 g/L of cellobiose, but only when four other T. saccharolyticum genes (adhA, nfnA, nfnB, and adhEG544D ) were also present. T. saccharolyticum ferredoxin did not have any observable impact on ethanol production. The improvement to ethanol production was sustained even when all annotated native C. thermocellum pfor genes were deleted. On high cellulose concentrations, the maximum ethanol titer achieved by this engineered C. thermocellum strain from 100 g/L Avicel was 25 g/L, compared to 22 g/L for the reference strain, LL1319 (adhA(Tsc)-nfnAB(Tsc)-adhEG544D (Tsc)) under similar conditions. In addition, we also observed that deletion of the C. thermocellum pfor4 results in a significant decrease in isobutanol production. CONCLUSIONS: Here, we demonstrate that the pforA gene can improve ethanol production in C. thermocellum as part of the T. saccharolyticum pyruvate-to-ethanol pathway. In our previous strain, high-yield (~ 75% of theoretical) ethanol production could be achieved with at most 20 g/L substrate. In this strain, high-yield ethanol production can be achieved up to 50 g/L substrate. Furthermore, the introduction of pforA increased the maximum titer by 14%.