Consolidated bioprocessing of Populus using Clostridium (Ruminiclostridium) thermocellum: a case study on the impact of lignin composition and structure

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Background Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Although much of the work-to-date has centered on characterizing this microbe’s growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand its metabolism on more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol.ResultsThe most striking feature of the metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum’s cellular membrane as the culture progresses. This is undoubtedly a response to the gradual accumulation of lignocellulose-derived inhibitory compounds as the organism deconstructs the switchgrass to access the embedded cellulose. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in various enzymes, including those involved in the interconversion of branched amino acids valine, leucine, and isoleucine to iso- and anteiso-fatty acid precursors. Additionally, the metabolic accumulation of hemicellulose-derived sugars and sugar alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism/pentose phosphate pathway indicates that C. thermocellum shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux in response to C5 sugar metabolites that increase during lignocellulose deconstruction.ConclusionsIntegrated omic platforms provided complementary systems biological information that highlight C. thermocellum’s specific response to cytotoxic inhibitors released during the deconstruction and utilization of switchgrass. These additional viewpoints allowed us to fully realize the level to which the organism adapts to an increasingly challenging culture environment—information that will prove critical to C. thermocellum’s industrial efficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0697-5) contains supplementary material, which is available to authorized users.

Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass

Poudel

Giannone

Rodríguez

et al. 2017

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“…The measured A O adsorption was significantly smaller on unpretreated biomass than the pretreated biomass reported in [22] likely due to the cell wall matrix in pretreated biomass being disrupted by pretreatment leading to increased cellulose exposure. Significantly lower DO adsorption was also reported on unpretreated poplar [24, 25]. In addition, the switchgrass adsorbed more orange than blue and the O/B ratio was higher in the transgenic (1.35 and 1.09) relative to the control (1.02 and 0.98) group.…”

Section: Resultsmentioning

confidence: 65%

Study of traits and recalcitrance reduction of field-grown COMT down-regulated switchgrass

Yoo

et al. 2017

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BackgroundThe 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.ResultsField-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 weight-average 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.ConclusionsThe 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 dow...

“…1 filter paper 317.15.3ND20[26]CS8Milled Whatman No. 1 filter paper 318.07.2ND20[26]JN4Avicel 56.27.61360[59]YSAvicel 2036.012.0ND–[27]ATCC 35609MN300 831.222.5ND120[60]JW20Avicel 1025.413.09.416[28]YM4Avicel 1044.726.520.496[61]BC1Whatman No. 1 filter paper 10102ND144[40]DBT-IOC-C19Avicel 1032.5518.675.1496This study

ND not detected, metabolite yield reported as g L −1 converted to millimolar using a molecular weight of 46.07 g mol −1 for ethanol, 60.05 g mol −1 for acetate, and 90.08 g mol −1 for lactate

…”

Section: Resultsmentioning

confidence: 99%

Cellulosic ethanol production via consolidated bioprocessing by a novel thermophilic anaerobic bacterium isolated from a Himalayan hot spring

Singh

Mathur

Tuli

et al. 2017

BackgroundCellulose-degrading thermophilic anaerobic bacterium as a suitable host for consolidated bioprocessing (CBP) has been proposed as an economically suited platform for the production of second-generation biofuels. To recognize the overall objective of CBP, fermentation using co-culture of different cellulolytic and sugar-fermenting thermophilic anaerobic bacteria has been widely studied as an approach to achieving improved ethanol production. We assessed monoculture and co-culture fermentation of novel thermophilic anaerobic bacterium for ethanol production from real substrates under controlled conditions.ResultsIn this study, Clostridium sp. DBT-IOC-C19, a cellulose-degrading thermophilic anaerobic bacterium, was isolated from the cellulolytic enrichment cultures obtained from a Himalayan hot spring. Strain DBT-IOC-C19 exhibited a broad substrate spectrum and presented single-step conversion of various cellulosic and hemicellulosic substrates to ethanol, acetate, and lactate with ethanol being the major fermentation product. Additionally, the effect of varying cellulose concentrations on the fermentation performance of the strain was studied, indicating a maximum cellulose utilization ability of 10 g L−1 cellulose. Avicel degradation kinetics of the strain DBT-IOC-C19 displayed 94.6% degradation at 5 g L−1 and 82.74% degradation at 10 g L−1 avicel concentration within 96 h of fermentation. In a comparative study with Clostridium thermocellum DSM 1313, the ethanol and total product concentrations were higher by the newly isolated strain on pretreated rice straw at an equivalent substrate loading. Three different co-culture combinations were used on various substrates that presented two-fold yield improvement than the monoculture during batch fermentation.ConclusionsThis study demonstrated the direct fermentation ability of the novel thermophilic anaerobic bacteria on various cellulosic and hemicellulosic substrates into ethanol without the aid of any exogenous enzymes, representing CBP-based fermentation approach. Here, the broad substrate utilization spectrum of isolated cellulolytic thermophilic anaerobic bacterium was shown to be of potential utility. We demonstrated that the co-culture strategy involving novel strains is efficient in improving ethanol production from real substrate.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0756-6) contains supplementary material, which is available to authorized users.