Bioavailability of Carbohydrate Content in Natural and Transgenic Switchgrasses for the Extreme Thermophile Caldicellulosiruptor bescii

Abstract

Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(-) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by to 15% (2.3-fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the T→M→T→M treatment was primarily glucan and inert materials (lignin and ash). While could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance. Key to a microbial process for solubilization of plant biomass is the organism's access to the carbohydrate content of lignocellulose. Economically viable routes will characteristically minimize physical, chemical, and biological pretreatment such that microbial steps contribute to the greatest extent possible. Recently, transgenic versions of plants and trees have been developed with the intention of lowering the barrier to lignocellulose conversion, with particular focus on lignin content and composition. Here, the extremely thermophilic bacterium was used to solubilize natural and genetically modified switchgrass lines, with and without the aid of hydrothermal treatment. For lignocellulose conversion, it is clear that the microorganism, plant biomass substrate, and processing steps must all be considered simultaneously to achieve optimal results. Whether switchgrass lines engineered for low lignin or natural variants with desirable properties are used, conversion will depend on microbial access to crystalline cellulose in the plant cell wall.

Lignocellulose solubilization and conversion by extremely thermophilic Caldicellulosiruptor bescii improves by maintaining metabolic activity

“…The objective was to maintain the bioreactor culture in a metabolically active state with minimal loss of feedstock. This was in contrast to batch studies, from previous reports (Zurawski et al, , ) and from this study, where C. bescii grew rapidly to reach its maximal cell density after approximately 72 to 96 hr, after which biomass was still solubilized by enzymes in the secretome but the hydrolyzed carbohydrate was not converted to fermentation products. For the continuously liquid purged bioreactor, following in situ washing of otherwise unpretreated switchgrass (four reactor volumes) at 70°C, fermentation was initiated by inoculating at 10 7 cells/ml and commencement of the fresh media flow at a volumetric turnover rate (τ) of 80 hr.…”

“…For industrial biotechnology to achieve an aspirational goal of producing bio‐based fuels and chemicals from renewable feedstocks, significant improvements must be made in the process by which lignocellulose is deconstructed, solubilized, and converted. Many strategies have been pursued along these lines, including the development of transgenic grasses and trees (Biswal et al, ; Zurawski et al, ), physical, chemical, and enzymatic pretreatment of biomass (Kim, Lee, & Kim, ), and isolation and metabolic engineering of microorganisms to enhance complex carbohydrate degradation (Conway et al, ) and fermentation to useful products (Williams‐Rhaesa et al, ). To improve process economics, feedstock‐microbe pairings that require minimal pretreatment are highly desirable.…”

“…The genus Caldicellulosiruptor contains extremely thermophilic bacteria, all of which can utilize hemicelluloses, but only some of which can utilize microcrystalline cellulose (Lee et al, ). The most studied from this genus is Caldicellulosiruptor bescii that cometabolizes five‐carbon (C5) and six‐carbon (C6) sugars (Blumer‐Schuette et al, ), thereby rapidly and extensively converting lignocellulose‐derived complex carbohydrates, such as arabinan (C5), mannan (C6), galactan (C6), xylan (C5), and cellulose (C6), into fermentation products (Zurawski et al, , ). C. bescii has already been engineered to produce ethanol from switchgrass (Chung, Cha, Guss, & Westpheling, ), and further improvements in the genetics toolbox for this bacterium (Lipscomb, Conway, Blumer‐Schuette, Kelly, & Adams, ; Williams‐Rhaesa et al, ) bode well for developing strains with enhanced carbohydrate degradation capacity (Conway et al, ) and increased levels of metabolically engineered products (Williams‐Rhaesa et al, ).…”

“…For C. bescii , in the absence of pretreatment, carbohydrate solubilization at low loadings (5 g/L) is typically below 40% (see Table ), but can be as high as 70% for transgenic switchgrass lines, if alternating and multiple hydrothermal and microbial treatments are used (Zurawski et al, ). At high loadings (50 g/L) in batch fermentations, C. bescii solubilized 60% of microcrystalline cellulose, but only 20% of unpretreated switchgrass (Basen et al, ); microbial activity was limited by the formation of fermentation products (primarily acetate), unidentified inhibitor(s) generated from the plant biomass, and transition of the culture from exponential to stationary phase.…”

Metatranscriptomic and thermodynamic insights into medium-chain fatty acid production using an anaerobic microbiome

“…S1; Supplement 5). LAC2 also had high levels of transcripts for an exo-α-L-1,5-arabinanase (GH93), predicted to release other pentose sugars from arabinan, which accounts for 3% of the sugar polymers in switchgrass (26, 27). In addition, the COR1, COR3 and LAC4 members of the community had high transcript levels for three extracellular CAZymes (GH13) that are predicted to degrade a variety of glucans that may be remaining in switchgrass conversion residue (28).…”