Rhamnogalacturonan I is a recalcitrant pectin domain during Clostridium thermocellum-mediated deconstruction of switchgrass biomass

Research
Open access
Published: 27 October 2025

Biotechnology for Biofuels and Bioproducts volume 18, Article number: 109 (2025) Cite this article

Abstract

Background

Liquid fuels from lignocellulosic feedstocks are required for transition to a sustainable bioeconomy. However, the recalcitrance of carbon-containing feedstock cell walls to deconstruction poses a barrier to cost effective biological conversion of plant biomass to biofuels. One-step consolidated bioprocessing (CBP) in which anaerobic thermophilic bacteria convert lignocellulosic biomass into liquid fuels is a platform for overcoming the recalcitrance of plant biomass.

Results

The amounts of hemicellulosic and pectic polysaccharides, two complex cell wall glycans that contribute to plant biomass recalcitrance and that are partially solubilized during CBP of switchgrass aerial biomass by Clostridium thermocellum were evaluated in the liquor, solid residues and residue washate recovered during a 120-h CBP process. After 120 h, 24% of milled switchgrass was solubilized in the C. thermocellum CBP platform. Higher concentrations of arabinose, xylose, galactose, and glucose accumulated in the CBP-fermentation liquor and washate compared to fermentation controls without C. thermocellum, indicating that C. thermocellum solubilized hemicelluloses, but did not fully metabolize them. After five days of fermentation, the relative amount of rhamnose in the solid residues increased by 16% compared to controls, and CBP solid residues had more than 23% increased reactivity against RG-I reactive monoclonal antibodies, indicating that the pectic polymer rhamnogalacturonan I (RG-I) was not effectively solubilized from switchgrass biomass by C. thermocellum CBP. Similarly, the amount of mannose (Man) in the CBP solid residues increased by 7% and reactivity against galactomannan reactive antibodies increased by greater than 14%, indicating that the hemicellulosic polymer galactomannan was also resistant to degradation by C. thermocellum during CBP fermentation.

Conclusions

These findings show that C. thermocellum is unable to effectively degrade RG-I pectic and galactomannan hemicellulosic components in switchgrass biomass. Targeting these polymers for improved solubilization could enhance the efficiency of conversion of grass biomass to biofuels.

Background

Introduction

Plants are the most abundant carbon resource on the planet for production of sustainable feed, food, energy, materials and chemicals [1,2,3]. According to the Department of Energy 2023 Billion-Ton Report [4], the perennial bioenergy crop switchgrass is the most abundant renewable carbon resource in a U.S. mature-market medium scenario with 230 million tons of biomass predicted to be available annually. The bulk of carbon in plant biomass resides in the multipolymeric and crosslinked plant cell walls [5, 6]. Extraction of carbon-rich sugars and lignin from the plant cell wall requires pretreatment of the biomass to make it accessible to enzymatic saccharification. Several effective biomass pretreatment strategies, including thermo-pressure pretreatment methods with or without alkaline catalysts and acid hydrolysis, have been developed. However, these approaches have high cost and energy demands and may cause degradation of some biomass sugars with associated generation of fermentation inhibitors [7]. An alternative microbial-based consolidated bioprocessing strategy [8] negates the need for harsh biomass pretreatment, has lower energy demands, reduces production of fermentation inhibitors, does not require environment-impacting chemicals, and produces fermentation biomass residues that retain plant cell wall polymer chemical group functionality which has value for biochemical and biomaterial production [9, 10].

Consolidated bioprocessing (CBP) is a microbial-based process in which microbes both solubilize the complex cell wall polymers of non-pretreated biomass into oligomeric or monomeric units and ferment these into fuels and/or chemical and fuel precursors [11]. The thermophilic bacterial anaerobe Clostridium thermocellum, one of the most efficient biomass solubilizing microbes known, has been extensively studied and genetically modified for CBP of bioenergy feedstock biomass [12]. However, it is not able to fully solubilize the cell wall sugars from bioenergy crops such as switchgrass, with solublization values of ~ 45% reported for non-pretreated senescenent switchgrass [9]. Clostridium thermocellulum expresses diverse glycan degrading enzymes including cellulases, hemicellulases, and pectinases in multi-enzyme complexes known as cellulosomes [13, 14]. Although these enzymes are able to solubilize many of the cell wall glycans in biomass, the complexity of the arrangement of the polysaccharides, their interactions with each other and with the polyphenolic lignin, and the difficulties associated with the multicellularity of biomass including the tight adherence between cells results in bioenergy biomass being recalcitrant to full enzymatic solubilization without prior pretreatment. The recalcitrance of bioenergy crops, such as switchgrass with its abundance of secondary walls can be reduced by genetic modification of recalcitrance factors, such as lignin [15]. The identification of additional recalcitrance factors has the potential to further improve CBP.

Pectin is the most complex polysaccharide in plant cell walls [5]. Although it is only a minor component of grass and secondary cell wall-rich bioenergy feedstock cells, pectin is required for plant cell growth and present in the middle lamella that connects cells and tissues together. Pectins are a family of complex cell wall polysaccharides that consist primarily of the pectin domains homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) [16]. All pectins have either an HG α-1,4-linked GalA backbone or α-1,4-GalA-α-1,2-Rha disaccharide repeat RG-I backbone. RG-I has galactan, arabinogalactan, and arabinan-rich side chains and is partially acetylated [5, 16]. The number, length and order of RG-I side chains in different tissues and cell types is not well understood and is an area of active research [17]. The pectic polysaccharides interact covalently and non-covalently with each other and with other wall glycans and proteoglycans, leading to a complex and not well understand cell wall architecture[16]. The most abundant pectin in secondary cell wall-rich bioenergy biomass is rhamnogalacturonan I (RG-I) [16, 18]. We recently showed in the woody bioenergy feedstock poplar, that RG-I is not effectively solubilized by Clostridium thermocellum, leading us to hypothesize that RG-I is a biomass recalcitrance factor during CBP of woody biomass [19]. To determine if RG-I might also be a recalcitrance factor for solubilization of grass biomass, which has less pectin and structurally different walls than poplar woody biomass, we analyzed the solubilization of switchgrass biomass by Closterium thermocellum CBP over a 120 h fermentation and compared that to solubiliization of the biomass during a CBP platform in the absence of the microbe. The goal was to determine how effectively C. thermocellulum solubilized a reference Bioenergy Science Center (BESC) standard switchgrass (Panicum virgatusm L.) line [18, 20] and whether the same RG-I polymer limited recalcitrance in both a grass (switchgrass) and woody bioenergy feedstock.

Results

Water-soluble components of biomass are removed during extractives washing of switchgrass with hot water prior to CBP fermentation

Washing of biomass prior to use for fuel production has been shown to remove contaminant metals and other materials and improve biomass resource performance during and after processing [21]. To determine if valuable cell wall carbon is lost in this extractives wash step, switchgrass ground biomass (75 g) was subjected to sequential hot water washes before CBP processing of the biomass and the water-soluble components were evaluated. The biomass was washed thrice with 80 °C deionized water, with supernatant samples collected after each step. The wet biomass was then dried at 60 °C and stored for further analysis. After drying, 61.4 g of dry biomass was recovered (Additional File 1: Fig. S1), corresponding to an 18% reduction in total biomass mass. The results indicate that hot water washing removes a measurable fraction of biomass which may reduce the total yield of fermentable carbohydrate in the CBP processing system. As the water-soluble components removed by the hot water wash were expected to include cell wall polysaccharides, along with salts and other extractable compounds, the hot water extract was analyzed for its sugar content.

Glycosyl residue composition analysis reveals a significant amount of cell wall carbohydrates in the hot water extractives washes of switchgrass

Samples from each extractives wash step (extractives washes 1, 2, and 3; Additional File 1: Fig. S1) were analyzed for monosaccharide composition to quantify the cell wall polysaccharides and starch removed during washing. The hot water washes solubilized significant amounts of arabinose (Ara), rhamnose (Rha), xylose (Xyl), galactose (Gal), and glucose (Glc) from the switchgrass biomass (Fig. 1; Additional File 2: Fig. S2A). All the sugars identified in the washes including Ara, Rha, fucose (Fuc), Xyl, glucuronic acid (GlcA), galacturonic acid (GalA), mannose (Man), Gal and Glc were present in significantly higher concentrations in extractives wash 1 as compared to washes 2 and 3 (Fig. 1; Additional File 2: Fig. S2A), indicating a relatively loose association with the biomass. For three of the sugars, Rha, Gal and Glc, notable amounts were also present in the second and third washing steps, suggesting a relative tighter binding of the polymers containing the sugars to the biomass. The total recovery of sugar from all three extractives washes was 513 mg (292 mg in extractives wash 1, 135 mg in wash 2, and 86 mg in wash 3) (Fig. 1A; Additional File 2: Fig. S2B). The monosaccharides solubilized in these washes, including Ara, Xyl, Gal, Rha, Man, and GalA, likely originated from loosely bound polysaccharide domains such as arabinoxylans, pectic polysaccharides [particularly RG-I and homogalacturonan, (HG)], galactomannans, and possibly residual starch. Their presence suggests that hot water washing effectively extracts a fraction of non-cellulosic wall glycans and low-molecular-weight metabolites from the switchgrass cell wall matrix.

Owing to the unexpectedly high levels of rhamnose recovered, we performed LC–MS analysis to explore the potential contribution of Rha-containing specialized metabolites. The analysis revealed multiple glycosylated flavonoids and phenolic compounds. Notably, quercetin-3-O-rhamnoside exhibited the highest total ion intensity, exceeding 4 × 10⁹, significantly surpassing all other detected rhamnose-containing compounds (Fig. 1B). Moderate levels of kaempferol-3-O-rutinose, 7-O-methoxyquercitrin, and keioside were also present, while progenin lii, nicotiflorin, and rutin were detected at lower intensities. The pronounced abundance of quercetin-3-O-rhamnoside, a known rhamnosylated flavonoid, supports the hypothesis that while some of the rhamnose detected in the extractive washes may have originated from RG-I-type pectic polysaccharides, some Rha was likely a component of soluble secondary metabolites known to be abundant in grass species [22].

Based on the types of sugars identified and the known glycan components of the switchgrass cell wall [18, 20], the results suggest that a substantial amount of hemicellulosic (e.g. xylan and mannan) and pectic sugars are removed during the extractives washing process, which may have implications for sugar yield from downstream processing steps.

CBP by C. thermocellum solubilizes more than 35% of biomass over 120 h with more than 24% solubilization requiring the action of C. thermocellum

Ground extractives-washed switchgrass was loaded at 5 g/L glucan into 0.5 L fermenters for CBP using C. thermocellum DSM1313, corresponding to 14.8 g/L total solids loading and a total of 7.39 g of switchgrass per fermenter (Additional File 3: Fig. S3). Inoculation of the CBP fermenters with C. thermocellum resulted in a total solids solubilization of approximately 14% after 24 h and 35% after 120 h (Fig. 2; Additional File 4: Fig. S4). In control reactors without microbial inoculation (fermentation controls), total solids solubilization was 10.6% at 24 h and 10.8% at 120 h, indicating that roughly 10% of biomass loss was due to abiotic process factors. The contribution of C. thermocellum to biomass solubilization was determined by subtracting the biomass loss obtained in the fermentation controls from the total biomass solubilization measured in the microbial fermentation reactors. After accounting for the solubilization due to the heat, medium, and processing conditions in the control reactors, C. thermocellum CBP was shown to solubilize 3.0% of the switchgrass biomass at 24 h and 24.4% at 120 h.

CBP of switchgrass by C. thermocellum results in accumulation of hemicelluloses in the post-fermentation liquor

The contents of the 24 h and 120 h CBP fermentations and CBP-fermentation controls were centrifuged to separate the liquor from the solid residue and these were lyophilized for five days and the dry mass recorded. Significantly more dry mass was recovered in the liquor recovered from the CBP fermentation as compared to the control (10% more at 24 h, 35% more at 120 h; Additional File 5: Fig. S5A), indicating that C. thermocellum effectively solubilized biomass during fermentation. Since the dry mass recovered from both the control and CBP liquor exceeded the total solids solubilized from the biomass (Additional Files 3 and 5: Fig. S3, Fig. S5A), dried culture medium components likely accounted for the extra mass in the control liquor, while the additional mass in CBP fermentations was due to both culture medium components and microbial biomass. To determine which components of the switchgrass biomass were solubilized by C. thermocellum and recovered in the CBP-fermentation liquor, the liquor from the control and C. thermocellum fermentations was analyzed for monosaccharide composition by GC–MS following trimethylsilyl (TMS) derivatization of glycans depolymerized by acidic methanolysis [19, 20]. Significantly more sugar was present in the liquor in C. thermocellum fermentations compared to the controls, with 294 mg xylose (Xyl) h, 53 mg arabinose (Ara), 28 mg galactose (Gal), and 26 mg glucose (Glc) recovered at 120 (Fig. 3). A total of 407 mg sugar was recovered in the liquor of 120 h C. thermocellum fermentations compared to 31 mg in the fermentation controls (Fig. 3 and Additional File 5: Fig. S5B). The accumulation of these sugars in the liquor suggests that C. thermocellum effectively solubilized from switchgrass hemicelluloses, such as arabinoxylan, but did not fully take up or metabolize them. Only trace amounts of galacturonic acid (GalA), glucuronic acid (GlcA), fucose (Fuc), and mannose (Man) were detected in the liquid fractions (Fig. 3). Interestingly, there was less rhamnose (Rha) present in the CBP-fermentation liquor at 120 h than in the fermentation controls, indicating that C. thermocellum may have assimilated Rha during fermentation. However, since Rha levels were higher in the control liquor than in the CBP liquor, it was unclear whether the Rha present in the liquor resulted from solubilization due to the process conditions, due to the action of C. thermocellum, or to both. To clarify this, the solid residues from the CBP fermentations and CBP-fermentation controls were analyzed.

CBP of switchgrass by C. thermocellum leads to the accumulation of hemicellulosic and pectic glycans in the water washate of fermentation residues

The contents of the control CBP and C. thermocellum CBP fermentations were separated by centrifugation to separate the solid residue from the supernatant (liquor). After removing the liquor, the solid residue was washed with distilled water to remove any remaining loosely bound sugars and the resulting “washate” was collected and lyophilized to dryness as described in the Materials and Methods. Approximately 10% and 30% more washate (dry mass) at 24 h and 120 h, respectively, was recovered in washate from the C. thermocellum CBP residue compared to washate from the CBP fermentation at control residue (Additional File 6: Fig. S6A), indicating that the washing steps removed a significant amount of sugar that had been solubilized from the biomass residue by C. thermocellum. To determine which components of the switchgrass biomass were present in the residue washate, both the residue washates from the C. thermocellum CBP and from the CBP fermentation control were analyzed for monosaccharide composition using the trimethylsilyl (TMS) derivatization and GC–MS method [19, 20]. Multiple sugars were detected in the washates including Xyl, Ara, Gal and Glc and lesser amounts of Rha, GalA, GlcA, Fuc and Man with a total of 385 mg sugar in the washates from the C. thermocellum 120 h CBP residue as compared to 46 mg sugar in the washate of the 120 h CBP control residue (Fig. 4 and Additional File 6: Fig. S6B). These monosaccharides are derived from non-cellulosic polysaccharides, which in this context refer primarily to hemicelluloses (e.g., glucuronoarabinoxylan, galactomannan) and pectins (e.g., RG-I and HG). The elevated sugar levels in the CBP washates likely reflect solubilized or weakly bound components of these polymers that were released from the cell wall matrix during microbial hydrolysis but not fully metabolized by C. thermocellum. Interestingly, as observed in the liquor, there was a reduction in the amount of Rha in the CBP fermentation residue washate compared to the CBP process control residue. The relative amounts of the sugars in the washate were comparable to the levels detected in the liquor samples.

Time-dependent changes in lignin composition reveal selective solubilization of lignin during CBP of switchgrass by C. thermocellum

To assess the composition of lignin, cellulose, and non-cellulosic polysaccharides remaining in the C. thermocellum CBP fermentation residues compared to the CBP fermentation control residues, alcohol-insoluble residue (AIR) which primarily consists of plant cell wall material was isolated from the post-CBP fermentation and control biomass [19, 20]. The AIR was treated with α-amylase for 48 h to remove residual starch, and the de-starched AIR was analyzed to quantify the content of lignin, cellulose, and non-cellulosic sugars [19, 20]. The amount and composition of lignin in the solid residues from the C. thermocellum CBP fermentation and the CBP fermentation control was assessed as total lignin content and as lignin monomer guaiacyl (G), p-hydroxyphenyl (H), and syringyl (S) subunits using pyrolysis molecular beam mass spectrometry (py-MBMS) [18, 19]. Additionally, the lignin S/G ratio was analyzed. A decrease in the total lignin and lignin monomers present in the 24 h and 120 h CBP fermentation solid residues and in the fermentation control residues compared to the starting switchgrass biomass was observed, indicating that some lignin-containing wall material was solubilized during the CBP process (Fig. 5A). Curiously, after 24 h of fermentation of switchgrass by C. thermocellum, the total lignin content in the 24 h CBP solid residues was 12% greater than in the fermentation control (Fig. 5A–D and Additional File 7: Fig. S7), suggesting that C. thermocellum action during the CBP process reduced the amount or rate of lignin solubilization. After 120 h, however, the CBP solid residues contained less total lignin than the fermentation control, likely due to the solublization of cell wall carbohydrates, such as hemicellulose, to which some of the lignin was bound. The lignin S/G ratio in the 120 h residue was 9% greater than in the fermentation control (Fig. 5E), consistent with prior reports showing that after CBP and thermochemical deconstruction S/G ratio in woody and grass biomass is increased [19, 23,24,25]. One possible reason for the increased S/G ratio in the CBP residue is that C. thermocellum preferentially solubilizes hemicellulose or other carbohydrate associated with G-lignin, leading to a higher S/G ratio.

C. thermocellum efficiently solubilizes crystalline cellulose in non-pretreated switchgrass during CBP

The Saeman hydrolysis [26] method, which quantifies glucose from both cellulosic and non-cellulosic polysaccharides, was used to determine the cellulose content in post-fermentation solid residues. First, the amount of glucose present in non-crystalline polysaccharides and non-cellulosic cell wall glycans was measured by GC–MS of TMS-derivatized methyl glycosides. This value was then subtracted from the total glucose content (cellulosic + non-cellulosic) obtained through Saeman hydrolysis to determine the crystalline cellulose content [19, 26] (Additional File 8: Fig. S8A–C). CBP of the switchgrass biomass by C. thermocellum for five days resulted in a 54% reduction in crystalline cellulose content (mg Glc/starting biomass) based on the Saeman hydrolysis method. After accounting for the CBP fermentation control, this corresponded to a 51% net reduction (i.e. solubilization) of crystalline cellulose by C. thermocellum (Fig. 6, Additional File 8: Fig. S8C, Table 1). Previous studies [25, 27] reported that C. thermocellum achieved 49% glucan solubilization from non-pretreated switchgrass within five days of fermentation, consistent with the findings reported here and confirming that this microbe can effectively solubilize a significant portion of cellulose in non-pretreated switchgrass biomass.

Table 1 Mass of lignin, cellulosic, and non-cellulosic sugars in the starting biomass and in the residual solids after 120 h of CBP by C. thermocellum

Full size table

Non-cellulosic sugar composition of CBP solid residues reveals incomplete solubilization of RG-I and Galactomannan by C. thermocellum

The solubilization of non-cellulosic sugars from the switchgrass biomass was assessed by comparing the monosaccharide composition of solid residues present after 24 and 120 h of microbial solubilization of biomass by C. thermocellum CBP to the solubilization occurring during CBP with no microbe (i.e. CBP fermentation control) (Fig. 7). A significant reduction in the mass of five sugars was observed in the biomass residue remaining following C. thermocellum CBP, with 6% to 20% reduced Ara, 9% to 26% reduced Xyl, 10% to 19% reduced Gal, 9% to 20% reduced Glc, and 14% to 51% reduced GalA at 24 h and 120 h, respectively (Fig. 7). The decreased GalA content in the solid residue, with minimal amounts of GalA in the liquor, indicates that C. thermocellum actively metabolized pectic polymers. Noteably, in contrast, Rha and Man levels increased by 10% to 20% and 6% to 14%, respectively, in the 24 h and 120 h CBP residues compared to CBP fermentation controls, indicating that polymers containing Rha (e.g., RG-I) and Man (e.g. galactomannan) were largely not solubilized by C. thermocellum and thus became enriched in the residue. Taken together, the total mass of non-cellulosic sugars in the C. thermocellum CBP residue was reduced 9% to 24% at 24 and 120 h, respectively, compared to the CBP fermentation control residue (Fig. 7; Additional File 9: Fig. S9). The results are consistent with the solubilization by C. thermocellum of the non-cellulosic wall polymers arabinoxylan, the major hemicellulose in switchgrass, and of some of the pectic polysaccharides such as homogalacturonan. A comparison of the remaining cellulose, lignin, and non-cellulosic sugars in the solid residues after 120 h of fermentation showed that 51% of the cellulose, 14% of the lignin, and 24% of the non-cellulosic sugars were solubilized microbially by C. thermocellum CBP (Fig. 8A–C, Table 1). Analysis of the specific non-cellulosic sugars solubilized showed that Rha increased by 16% in the solid residue compared to its relative amount in the starting non-processed BESC standard switchgrasss and 20% compared to the CBP fermentation control, and that Man increased 7% compared to the starting non-processed biomass and 14% compared to the CBP fermentation control (Fig. 8D; Table 1). These results suggested that Rha-containing polymers, such as RG-I and Man-containing polymers, such as galactomannan may not have been effectively solubilized from the switchgrass biomass by C. thermocellum during the CBP process.

Enzyme-linked immunosorbent assays (ELISA) reveal that RG-I and galactomannan epitopes are enriched in C. thermocellum CBP solid residues

We hypothesized that the increased relative amounts of Rha and Man present in residues remaining after CBP fermentation of switchgrass biomass were components of the rhamnose-rich pectin RG-I and the mannose-rich hemicellulose galactomannan. To test this, the relative abundance of RG-I and galactomannan in starting biomass and in residues remaining after 24 h and 120 h of C. thermocellum CBP and in corresponding CPB fermentation controls were assayed by enzyme-linked immunosorbent assays (ELISAs) with antibodies specific to RG-I [28, 29] and to galactomannan [28,29,30]. Alcohol-insoluble residues (AIR) prepared from CBP fermentation residue and CBP control residue were sequentially extracted with 50 mM ammonium oxalate and 4 M KOH, as previously described [18, 19], to provide pectin-enriched and hemicellulose-enriched fractions, respectively. Given the relative increase in Rha and Man in the CBP solid residues (Figs. 7, 8D), we hypothesized that RG-I and galactomannan signals would be stronger in the CBP residues compared to the CBP fermentation control. To test this, RG-I was detected using three RG-I-specific antibodies (CCRC-M14, CCRC-M35, CCRC-M72) previously shown to recognize an unsubstituted RG-I hexasaccharide backbone oligosaccharide [→ 2)-α-l-rhamnose-(1 → 4)-α-d galacturonic acid −(1 →]₃ (DP = 6, R₃U₃) [28, 29, 31]. For detection of galactomannan, three galactomannan-specific antibodies were used: CCRC-M74 which recognizes plant galactomannans containing mannose to galactose ratios of ~ 1.5:1, and CCRC-M174/CCRC-M175, which recognize plant galactomannan with mannose to galactose ratios of 1.5 to 4:1 [28, 29, 31]. The ELISA signals for each of these antibodies in ammonium oxalate and 4 M KOH extracts of switchgrass cell walls (Fig. 9A, C) confirmed the presence of RG-I and galactomannan in switchgrass biomass. As shown in Fig. 9B, ELISA signals for all three RG-I-specific antibodies significantly increased in C. thermocellum CBP solid residues compared to CBP fermentation control solid residues. RG-I ELISA signals were 39% to 86% increased in the ammonium oxalate extract of 120 h CBP residues compared to 120 h CBP fermentation control residues, while the RG-I ELISA signal in 4 M KOH extracts increased 23% to 55% in the 120 h CPB residues compared to the fermentation control. Similarly, ELISA signals for the galactomannan-specific antibodies increased 16% to 29% in 120 h CBP residue ammonium oxalate extracts and 14% to 33% in the 4 M KOH extracts (Fig. 9D). The unexpected enrichment of galactomannan signals led us to review previously published glycome profiling results of C. themocellum processed switchgrass, through which we found confirmatory evidence of a significant enrichment of galactomannan epitopes in the residue remaining following C. thermocellum CBP of switchgrass biomass compared to starting non-pretreated switchgrass [25].

Inspection of the monosaccharide composition of starting switchgrass biomass versus the residue remaining after CBP fermentation (Fig. 7) showed a significant decrease in xylose in the CBP solid residues, providing evidence of effective xylan solubilization. To validate this, we used three xylan-specific antibodies: CCRC-M149 which recognizes short β-1,4-linked xylosyl disaccharides [28, 30,31,32], CCRC-M137, which targets unsubstituted xylan backbone regions of DP ≥ 5, and CCRC-M152, which requires at least four contiguous β-1,4-linked xylosyl residues [31]. Using these antibodies, strong signals were detected in the ammonium oxalate and 4 M KOH extracts of switchgrass biomass (Additional File 10: Fig. S10A). As expected, all three xylan-specific antibodies had significantly reduced ELISA signals in solid residues following CBP, with 12% to 18% decreased signal in ammonium oxalate extracts and 22% to 26% reduced signal in 4 M KOH extracts (Additional File 10: Fig. S10B). These findings, consistent with the monosaccharide composition results (Fig. 7), confirmed that C. thermocellum effectively solubilized xylan, but did not efficiently degrade RG-I and galactomannan in switchgrass biomass.

Discussion

The goal of this study was to characterize the solubilization of cellulosic and non-cellulosic sugars in non-pretreated switchgrass biomass by C. thermocellum at low solids loadings. To accomplish this, we analyzed the initial biomass, post-fermentation liquors, and solid residues recovered during the CBP process. Our goal was to identify recalcitrant glycans that may limit the solublization of switchgrass biomass by C. thermocellum.

We first evaluated how much, if any, cell wall sugars were removed by hot water extractives washing of the switchgrass biomass prior to CBP processing. Supernatant samples from three sequential washes (Extractive Washes 1, 2, and 3) were analyzed for monosaccharide composition (Fig. 1A; Additional File 1 and 2: Figs. S1, S2A, B). The extractive washes released a substantial quantity of soluble sugars, with the majority recovered in the first wash. Extractive Wash 1 contained the highest levels of arabinose, rhamnose, xylose, galactose, and glucose, along with significant amounts of fucose, glucuronic acid, galacturonic acid, and mannose compared to subsequent washes. In total, the three washes yielded 513 mg of monosaccharides. Interestingly, we observed relatively high levels of rhamnose across all three washes. Given that rhamnose is primarily found in the backbone of rhamnogalacturonan I (RG-I) and pectin constitutes a minor component of switchgrass cell walls [18, 20], we hypothesized that some of the water extractable rhamnose might represent rhamnose-containing metabolites known to be present in grasses [22]. These include rhamnose-containing specialized plant metabolites such as the C-glycosylflavones maysin and violanthin, the flavonol rutin, quercetin, and kaempferol, the anthocyanin lobelinin A, the triterpenoid saponin α-hederin, and the steroidal alkaloid khasianine [33]. LC–MS analysis supported this hypothesis, as quercetin-3-O-rhamnoside was identified as an abundant compound in all water extract samples, with a total ion intensity exceeding 4 × 10⁹ (Fig. 1B). Other glycosylated flavonoids, including kaempferol-3-O-rutinose and 7-O-Methoxyquercitrin, were also present in high abundance (Fig. 1B). The presence of such rhamnosylated flavonoids suggests that rhamnose in the extractive washes likely represented a combination of pectic polysaccharides and soluble metabolic products. Although the origin of rhamnose from specialized metabolites is beyond the scope of this study, these findings underscore the need for future studies of the metabolic contributions of diverse sugar pools in biomass deconstruction studies. These findings indicate that a considerable portion of easily solubilizable hemicellulosic and pectic sugars may be removed during the initial wash step, potentially reducing the pool of accessible glycans available for microbial deconstruction during CBP. This is an important consideration when interpreting downstream solubilization data, as some labile wall components may be lost prior to fermentation.

Clostridium thermocellum deconstructs lignocellulosic biomass using a highly specialized cellulosome—a complex of over 70 glycoside hydrolases with broad substrate specificities. The organism modulates this enzymatic system in response to biomass type, enabling cellulose degradation through diverse catalytic mechanisms [12, 34,35,36]. Recent discoveries, including a unique double-dockerin module, point to additional regulatory complexity in enzyme assembly, while adaptive evolution and comparative genomics highlight C. thermocellum’s metabolic flexibility and its placement within a broader clade of cellulolytic bacteria [37,38,39]. Despite these capabilities, C. thermocellum cannot fully depolymerize untreated plant cell walls, particularly in woody and mature grass feedstocks. Although it can efficiently solubilize 93–95% of crystalline cellulose (Avicel) at both low and high loadings [40, 41], its performance on unprocessed lignocellulose with its ~ 30% cellulose content embedded within a complex hemicellulosic, pectic and lignin-rich architecture is limited. In our previous work, we reported 14% total solids solubilization of Populus at low solids loading, aligning with other studies showing 12–20% solubilization across different poplar variants [19, 24]. To build on these findings, we extended our investigation to switchgrass, a grass feedstock with a markedly different cell wall composition than poplar wood [18]. Prior studies showed that C. thermocellum’s solubilization efficiency in switchgrass declines with increasing biomass loading, despite complete utilization of Avicel under the same conditions [42, 43]. Reported solubilization rates for switchgrass vary widely, ranging from 76% at 9.2 g L⁻¹ to 37% at 50 g L⁻¹ solids loading [25, 42,43,44,45]. In the present study, we achieved 24% total solids and 49% cellulose solubilization of switchgrass at 14.8 g L⁻¹ solids loading after 5 days of fermentation (Fig. 2, 6; Additional File 3: Fig. S3). These findings, together with prior reports, confirm that while C. thermocellum can moderately solubilize non-pretreated biomass, achieving high conversion at elevated solids loading will require additional pretreatment or targeted biomass modifications [25, 42]. Complementary metaproteomic studies further emphasize the need for cooperative enzymatic strategies within microbial consortia to enhance deconstruction under industrially relevant conditions [46].

The efficiency of C. thermocellum in deconstructing switchgrass biomass during CBP is strongly influenced by the quantity and composition of lignin. Analysis of post-fermentation solid residues from BESC standard switchgrass revealed time-dependent changes in lignin abundance and monomeric composition. At 24 h, the CBP residues showed enrichment in lignin content, relative to the fermentation controls (Table 1; Fig. 5; Additional File 7: Fig. S7). This enrichment likely reflects preferential solubilization of carbohydrates early in fermentation. However, by 120 h, CBP resulted in the solubilization of 128 mg of total lignin, equivalent to a 14% net reduction compared to the controls (Table 1; Fig. 5A). Among lignin monomers, G-lignin showed the greatest absolute and relative solubilization (73 mg; 16% reduction), followed by H-lignin (4 mg; 19% reduction) and S-lignin (26 mg; 8% reduction) (Table 1; Fig. 5B–D). These results demonstrate that C. thermocellum is capable of partial lignin deconstruction over extended fermentation, especially of the more condensed and cross-linked G-lignin. These results indicate that C. thermocellum is capable of partial lignin solubilization over extended fermentation. These findings align with previous studies identifying lignin as a major barrier to efficient biomass deconstruction. For example, variation in switchgrass cell wall composition, including lignin content, monomer distribution, and S/G ratio, have been shown to affect digestibility by C. thermocellum under both thermochemical pretreatment and CBP conditions [25]. Similarly, total lignin content in natural Populus variants was negatively correlated with sugar release following enzymatic hydrolysis after pretreatment [47], and genetically modified switchgrass lines with altered lignin content exhibited enhanced conversion under both SSF (simultaneous saccharification and fermentation) and CBP [48, 49]. Notably, in this study, solid residues from C. thermocellum-mediated CBP had a 9% increased S/G ratio after 5 days of microbial hydrolysis (Fig. 5E), compared to fermentation controls. This observation is significant, as hydrothermal and dilute alkali pretreatments are known to preferentially remove guaiacyl (G) units, thereby elevating the S/G ratio and improving cellulose accessibility [25]. The 14% net reduction in total lignin also correlates with a 24% solubilization of total solids and 49% solubilization of cellulose, supporting the conclusion that lignin removal contributes to enhanced substrate accessibility. This underscores the importance of lignin monomer composition in governing biomass digestibility and highlights the value of designing feedstocks or microbial systems tailored to overcome lignin-related barriers in CBP.

The hemicellulose content in switchgrass species has been reported to range from 21% to 31% [25, 44, 50,51,52,53]. The secondary cell walls of grasses typically contain acetylated glucuronoarabinoxylans (AcGAXs), which are characterized as having a β-1,4-xylosyl (Xylp) backbone. This backbone is commonly substituted at the O-2 position with either glucuronic acid (GlcA) or 4-O-methyl-α-d-glucuronic acid ((Me)GlcA), and at the O-3 position with α-l-arabinofuranose (Araf) residues. These α-l-Araf side chains are frequently further modified at their O-2 position with either α-l-Araf or β-d-Xylp residue [54,55,56]. In primary walls, AcGAXs may be singly or doubly substituted on the backbone with α-1,2- and/or α-1,3-linked arabinosyl residues [57]. In switchgrass, xylan is the dominant hemicellulose [20, 25, 53, 58] and plays a central role in shaping the structure and recalcitrance of the plant cell wall. In addition to cellulases, C. thermocellum cellulosomes contain various hemicellulose-degrading enzymes, including xylanases, xyloglucanase, mannanases, and accessory enzymes such as β-1,3-1,4-glucanase and endochitinase [19, 59,60,61]. Despite this hydrolytic capability, C. thermocellum lacks the ability to ferment hemicellulose-derived sugars from lignocellulosic biomass [36, 62], which limits its efficiency in utilizing all carbohydrate fractions. In this study, we show that the concentrations of non-cellulosic sugars derived from hemicelluloses in the liquor during C. thermocellum CBP fermentation are significantly higher than those in the fermentation controls. Specifically, the liquor from CBP fermentations contained between 419% and 18.3-fold more Xyl (974% to 2.5x fold more mg recovered/total solubilized liquor), 347% to 923% more Ara (824% to 12.9x-fold more mg recovered /total solubilized liquor), 123% to 1340% more Gal (361% to 18.6 × mg recovered/total solubilized liquor), and 218% to 716% more Glc (562% to 10.2x-fold mg recovered/total solubilized liquor), compared to the fermentation control over the 120-h incubation period (Fig. 3 and Additional File 5: Fig. S5B). As expected, the washate, representing the distilled water rinse of solid residues after liquor removal, reflected a similar pattern of increased Ara, Xyl, Gal, and Glc in the C. thermocellum CBP samples compared to the control (Fig. 4 and Additional File 6: Fig. S6B). The total non-cellulosic sugar content in the post-fermentation CBP solid residues from 24 to 120 h in C. thermocellum CBP residue also was reduced by 9% to 24% compared to the fermentation control (Fig. 7; Additional File 9: Fig. S9), including a 6 to 26% mg/starting material reduction in Ara, Xyl, Gal, and non-cellulosic Glc (Fig. 7). The presence of significant amounts of the solubilized Ara, Xyl, Gal, and non-cellulosic Glc in the fermentation liquor supports the hypothesis that C. thermocellum cannot grow on pentose sugars and also is not able to ferment hemicellulosic oligosaccharides containing Ara, Xyl, Gal, and non-cellulosic Glc from switchgrass bomass. This is consistent with the known metabolic limitations of C. thermocellum, which lacks the native ability to utilize most pentose sugars and some hemicellulosic oligosaccharides [63, 64]. These accumulated sugars likely reflect solubilization of hemicellulose and pectin without subsequent microbial metabolism. The hemicellulose-solubilizing enzymes are suggested to function by cleaving hemicelluloses from the biomass in order to expose to C. thermocellum the preferred cellulose substrate for effective cellulosomal solubilization [36, 65, 66].

In contrast to other sugars, Man levels increased by 6% to 14% in C. thermocellum CBP solid residues compared to the CBP fermenetation controls. This suggests an enrichment of galactomannan, a hemicellulose with a β-1,4-linked mannose backbone, in the CBP residual biomass. The persistence of galactomannan in the CBP residues indicates that C. thermocellum has limited enzymatic capacity to degrade this polysaccharide. It has also previously been shown that galactomannan inhibits cellulase activity [67]. Although C. thermocellum encodes mannan-degrading enzymes such as Man26A and Man26B [68, 69], their expression appears to be relatively low and likely insufficient for effective galactomannan degradation in complex lignocellulosic matrices during the CBP process. For example, metaproteomic data from anaerobic microbiomes under high-solids conditions revealed generally low expression of mannosidases, particularly in the supernatant and substrate-bound fractions [46]. Consistent with this, Kothari and coworkers [25] demonstrated through glycome profiling that galactomannan-specific monoclonal antibodies, particularly Galactomannan-2, bound much more strongly to CBP residues than to unfermented switchgrass fractions, confirming the higher concentration of galactomannan epitopes in post-CBP biomass. In this study, ELISA data also showed a similar trend, with galactomannan antibody signals increasing by 16% to 29% in ammonium oxalate extracts and 14% to 33% in 4 M KOH extracts of C. thermocellum-treated switchgrass compared to untreated biomass (Fig. 9D). Evidence of galactomannan’s inhibitory role comes from Kumar and Wyman [67], who showed that galactomannan significantly suppresses fungal cellulase activity, with inhibition increasing with the degree of galactose substitution of the galactomannan. Their findings demonstrated that high concentrations of galactomannan substantially reduce cellulose conversion efficiency, likely by binding directly to cellulase enzymes or cellulose surfaces and obstructing enzymatic access to cellulose. Similarly, Beri and coworkers [62] reported that galactomannan moderately inhibited corn stover solubilization, and that glucomannan had an even stronger inhibitory effect. Together, these findings suggest that galactomannan is both resistant to microbial breakdown and interferes with cellulolytic enzyme activity, either by blocking access to cellulose or through competitive binding. The accumulation of galactomannan in the CBP residues reported here confirms it as a recalcitrant plant biomass polymer and underscores the potential value of enzyme supplementation or use of a microbial consortia with galactomannan degrading enzymes to improve polysaccharide accessibility in grassy feedstocks like switchgrass.

Although pectin is a minor component in the secondary cell walls of grasses including switchgrass, it contributes to biomass recalcitrance through its roles in cell wall architecture, wall integrity, and cell:cell adhesion by limiting access of enzymes to cellulosic and hemicellulosic glycan regions [18, 19]. Recent studies have identified multiple pectin-degrading enzymes as components of the C. thermocellum cellulosome complex [61, 70], including three endo-pectin lyases (PL1A, PL1B, and PL9) and a rhamnogalacturonan I (RG-I) lyase (RGLf) [70, 71]. Proteomic analyses of the C. thermocellum cellulosome have further identified additional pectic enzymes, such as galactanases and rhamnogalacturonan lyases [61]. All of these enzymes are modular, featuring carbohydrate-binding modules (CBMs) and functions as components of the cellulosome. Notably, PL1A contains a CBM6 domain, which binds to cellulose or hemicellulose. In contrast, PL1B, PL9, and RGLf contain CBM35 domains which have recently been shown to bind to RG-I [72]. The presence of this domain architecture indicates that pectin solubilization is genetically encoded within the C. thermocellum enzymatic system as part of its broad strategy for biomass deconstruction. The presence of these enzymes in the C. thermocellum enzyme arsenal is confirmed by our cell wall sugar composition data which reveal a 14% to 51%, reduction in galacturonic acid (GalA) in CBP biomass residues compared to the CBP fermentation controls (Fig. 7; Additional File 9: Fig. S9; Table 1). These results suggest that C. thermocellum efficiently utilizes polygalacturonic acid (homogalacturonan, HG) present in switchgrass biomass as a carbon source, consistent with prior observations in sugar beet and poplar [19, 73]. Supporting this, a metaproteomic study of anaerobic methanogenic microbiomes under high-solids loading showed a significant increase in two pectin lyases that cleave the α-1,4-linkages of HG, particularly within the planktonic cell and substrate-bound fractions [46]. Together, these structural features and functional outcomes emphasize the role of pectin-degrading enzymes in biomass solubilization and highlight their contribution to the overall deconstruction strategy employed by C. thermocellum.

Our earlier work demonstrated that C. thermocellum is unable to efficiently degrade the rhamnogalacturonan I (RG-I) component of pectin in poplar, potentially limiting overall biomass solubilization and identifying RG-I as a key target for improving CBP efficiency in woody biomass [19]. Here we extended our studies to grass biomass, specifically switchgrass, to compare woody and grass feedstock RG-I deconstruction by C. thermocellum during CBP. Interestingly, we observed a significant increase in Rha, a major sugar in the RG-I backbone, ranging from 10 to 20% in the solid residues after five days of C. thermocellum fermentation compared to CBP fermentation controls (Figs. 7 and 8), suggesting that RG-I in both woody and grass feedstocks is not efficiently solubilized by C. thermocellum during CBP. ELISA analysis further supported this observation, revealing enriched signals for RG-I-specific epitopes. In particular, monoclonal antibodies CCRC-M14, CCRC-M35, and CCRC-M72 showed increased binding, with RG-I signals elevated by 48% to 86% in ammonium oxalate extracts and by 23% to 55% in 4 M KOH extracts relative to fermentation controls (Fig. 9B). Although C. thermocellum encodes a putative RG-I lyase (RGLf) [71], the increase in RG-I epitopes suggests that this enzyme is either poorly expressed during CBP or catalytically insufficient for effective degradation of the branched RG-I domains in switchgrass [18]. This interpretation is reinforced by metaproteomic data from Chirania and coworkers [46], which identified numerous pectin-degrading CAZymes, classified into four functional groups: galacturonan lyases, rhamnogalacturonan lyases, rhamnosidases, and pectinesterases. Notably, pectinesterases and rhamnosidases showed decreased abundance at higher solids loadings, suggesting a limited microbial capacity to deconstruct complex RG-I structures under these fermentation conditions. Although galactosidase and arabinosidase are classified as hemicellulose-degrading CAZymes, they also contribute to pectin breakdown. Galactosidase exhibited a slight increase in the substrate-bound fraction at higher solids loadings, while arabinosidase displayed consistent and significant upregulation across all three fractions with increasing solids loading [46], suggesting a broad and active role in hemicellulose and potentially pectin deconstruction under high-substrate conditions.

C. thermocellum hydrolysis of switchgrass biomass removed only 13% more of the total non-cellulosic sugars after 5 days of fermentation compared to the fermentation control, indicating that the residual biomass retained most of its non-cellulosic wall polysaccharides (Additional File 9: Fig. S9B). Given that total solids solubilization reached only 24% (Fig. 1), and that monosaccharides associated with galactomannan and RG-I were enriched in the post-fermentation solids, we hypothesize that these polymers are resistant to C. thermocellum-mediated hydrolysis and contribute to limited biomass deconstruction. This idea is further supported by recent findings in poplar wood, where RG-I has been shown to play a structural role in maintaining cell–cell adhesion and tissue integrity [74], and by similar evidence from blueberry fruit showing that RG-I, although present in low abundance, contributes significantly to cell wall firmness and rheological properties [75]. These findings reinforcing the notion that RG-I may hinder microbial access and solubilization in both woody and grassy feedstocks. However, the structural and functional role of galactomannan in grass cell walls, particularly in relation to cell wall integrity and its impact on microbial deconstruction, remain poorly understood and warrant further investigation.

Together, our findings highlight structural complexity of switchgrass cell walls as a critical barrier to efficient solubilization by C. thermocellum. While moderate levels of total solids and cellulose solubilization were achieved, the persistence and selective enrichment of lignin, galactomannan, and RG-I in post-CBP residues suggest that these components act as recalcitrant barriers that limit microbial access and enzyme activity. The ELISA and sugar compositional data reveal that C. thermocellum can partially deconstruct homogalacturonan but lacks sufficient activity against structurally more complex polymers such as RG-I and galactomannan. These limitations may be due to low expression levels or insufficient catalytic efficiency of relevant enzymes, including RG-I lyases and mannanases and sidebranch removing enzymes. To validate the role of RG-I in limiting biomass deconstruction, future studies could explore microbial consortia that include RG-I-degrading fungi such as Aspergillus niger and Aspergillus aculeatinus, both known to secrete a broad array of pectinolytic enzymes [76, 77]. Such co-culture strategies could complement the enzymatic repertoire of C. thermocellum and enhance the solubilization of structurally complex pectic domains. In addition, the inability of C. thermocellum to utilize solubilized hemicellulosic sugars restricts its overall carbon conversion efficiency. Overcoming these challenges by introducing microbial consortia, supplementing with accessory enzymes, or engineering strains with enhanced pectinolytic and mannanolytic activity will be critical for improving CBP performance on non-pretreated grass feedstocks like switchgrass.

Materials and methods

Switchgrass biomass preparation

The lowland switchgrass (Panicum virgatum L.) genotype Alamo used was the reference standard biomass line from the BESC (BioEnergy Science Center) collection [18, 78]. The aerial biomass of 5-month-old plants was harvested, air-dried, and milled to a particle size of 20 mesh (0.85 mm). The ground material was used for all the reported analyses.

Consolidated bioprocessing (CBP)

Biomass washing steps before CBP

An initial 75 g of ground switchgrass aerial biomass (see methods) was washed with 750 mL of 80 °C deionized water. Hot water was transferred into a 1 L centrifuge bottle containing the dry biomass and incubated for 15 min at 80 °C. The mixture was centrifuged for 20 min at 8000 RPM, and the supernatant extracted using an electronic pipette and referred as “wash 1”. A second wash was done with 400 mL of 80 °C deionized water (400 mL corresponds to the volume that could be removed as supernatant from the wet pellet). Following a 15-min incubation time the mixture was centrifuged and the aqueous extract pipetted and labeled “wash 2”. The pellet was again washed with 400 mL of 80 °C deionized water, centrifuged and the supernatant collected as “wash 3”. The wet biomass was dried for five days at 60 °C in a convection oven. A net final mass of 61.4 g of biomass was recovered, indicating that 18% of total mass was lost during the washing steps (Additional File 1: Fig. S1).

C. thermocellum culture medium, growth medium and growth conditions

Fermentations were conducted using C. thermocellum strain DSM1313 grown in 1 × MTC (Medium for Thermophilic Clostridia) solution made as described [79]. The MTC stock solutions were diluted with H₂O and the desired carbon source added to reach a 1 × concentration. The medium was prepared as described by Holwerda and coworkers [79] and Biswal et al. [19], using the BESC standard ‘Alamo’ switchgrass line [18] as the carbon source (5 g/L glucan loading) in place of Avicel or cellobiose. For seed train cultures, 5 g/L cellobiose was used as the primary carbon source in initial serum bottle seeds, while 5 g/L Avicel was used in seed bioreactors. All growth media were prepared and maintained at pH 7.0 in bioreactor experiments.

Bioreactors were prepared with a filter-sterilized 2 × MTC solution (without carbon source), stored in an anaerobic chamber (85% N₂, 10% CO₂, 5% H₂) until reactor sterilization. This 2 × stock was aseptically added to autoclaved reactors containing water and BESC standard switchgrass inside a biological safety cabinet to reach a final volume to a 1 × MTC concentration.

Serum bottles containing 50 mL of MTC solution containing 5 g/L cellobiose were inoculated with 0.5 mL of C. thermocellum DSM1313 glycerol stocks. Serum bottle seeds were grown on cellobiose to OD₆₀₀ ≈ 0.6 and then transferred at 10% v/v to 500 mL Avicel seed reactors. C. thermocellum cultures grown in Avicel seed reactors to the exponential growth phase were transferred at 10% v/v to switchgrass-containing reactors. The optimal transfer phase of Avicel-grown seed cultures was determined by monitoring base consumption and cell morphology prior to subculturing into the switchgrass reactor.

Fermentations

Four sartorius vessels (0.5 L total working volume) were set up identically for fermentation. Each reactor was loaded with 7.39 g of BESC switchgrass (80/20 mesh sieved) to achieve a glucan loading of 5 g/L (0.5% w/v). The reactors, biomass, and water were autoclaved at 121 °C for 30 min. Post-autoclaving, filter-sterilized MTC media components were added as described above. Cultures in fermenters were maintained at pH 7.0 using 2N HCl and 2N KOH as the acid and base, respectively. The temperature was maintained at 60 °C, with agitation at a minimum of 150 rpm to prevent biomass accumulation on the bottom of the reactor. Anaerobic conditions were maintained by sparging 50 ccm of N₂ through the reactor headspace. Each reactor was inoculated with a 10% (v/v) inoculum of DSM1313 Clostridium thermocellum from a common seed culture containing 5 g/L Avicel. At 24 and 120 h, two reactors were shut down and harvested (Additional File 3: Fig. S3). Control reactors, which were not inoculated with C. thermocellum, were prepared by replacing the inoculum with an equal volume of medium to reach the same final volume. These reactors were held under the same process conditions for 120 h as those inoculated with the microbe.

Separations and quantification of total biomass solubilization

To separate the undigested solids from the liquor, the reactor contents were transferred to centrifuge bottles and centrifuged at 10,000 RPM for 15 min. The supernatant was removed by pipetting, frozen for storage, and this fraction was referred to as ‘liquor’. The solids residue was washed with distilled water and centrifuged again under the same conditions. The wash fraction was removed by pipetting and this fraction was referred to as ‘washate’. The residual solids were dried for 5 d at 60 °C. The dried biomass was weighed, and the total solids solubilization was calculated by dividing the total recovered biomass after fermentation by the initial biomass loaded into the reactors at the start of fermentation.

Cell wall analysis

The preparation of alcohol-insoluble residues (AIR) from post-fermentation solid residues was as previously described [18,19,20]. To remove starch, AIR samples were treated with alpha-amylase (0.47 U per mg biomass, Sigma Cat # A6255) in 100 mM ammonium formate (pH 6.0) at 25 °C for 48 h, followed by three ddH₂O and two acetone washes. Samples were then dried in a fume hood for 72 h. Glycosyl residue composition analysis of the AIR (~ 2 mg), liquor (300 µg), and washate (300 µg) was performed by GC–MS of trimethylsilyl (TMS) derivatized methyl glycosides produced from the sample by acidic methanolysis as previously described [20, 80, 81]. In brief, samples were hydrolyzed in 1 M methanolic–HCl at 80 °C for 18 h, followed by derivatization with 200 μL of TriSil and heated to 80 °C for 20 min. The derivatized samples were then filtered through packed glass wool, resuspended in 150 μL hexane, and 1 μL of sample was injected into the GC–MS for analysis as described earlier [20].

Saeman hydrolysis method of cellulose quantification

Cellulose content was determined by first quantifying the total glucose (Glc) content (cellulosic glucose + non-cellulosic glucose) in samples subjected to Saeman hydrolysis [26, 82] for hydrolysis of both cellulosic and non-cellulosic polymers and then measuring total glucose content via glycosyl residue composition analysis. The amount of non-cellulosic glucose was determined separately by TMS GC–MS. The cellulose content was taken as the difference between total glucose and non-cellulosic glucose content. The Saeman hydrolysis method hydrolyzes crystalline cellulose. Approximately 2 mg of AIR along with 20 μg of inositol as an internal standard, was hydrolyzed in 72% H₂SO₄ for 1 h at room temperature, with vortexing every 15 min. Samples were then diluted to 1 M H₂SO₄ and heated at 121 °C for 1 h. After cooling, they were neutralized with 0.25 M Ba(OH)₂, centrifuged at 3600 g for 20 min, and the supernatant lyophilized. Glucose was quantified using GC–MS of TMS-derivatized methyl glycosides, as previously described above [20]. This provided the total Glc content, including both cellulosic and non-cellulosic glucose. The fibrillary cellulose content was determined by subtracting the non-cellulosic Glc quantified in a separate TMS GC–MS analysis, from the total Glc value.

Lignin quantification

Lignin content and the S/G-lignin monomer ratio of post-fermentation solid residues was determined using the Complex Carbohydrate Research Center (CCRC) high-throughput pyrolysis molecular beam mass spectrometry (MBMS) method [83]. Samples (1.5–3.0 mg) were prepared in duplicate and placed into stainless steel cups for single-shot pyrolysis (Frontier Lab) at 500 °C, to produce volatile compounds. These compounds were analyzed for lignin content using a molecular beam mass spectrometer (Extrel Core Mass Spectrometers). Raw data were processed with UnscramblerX 10.1 software to obtain principal components and raw lignin data.

Preparation of cell wall fractions and total sugar estimation

Sequential fractionation of AIR from post-fermentation solid residues was performed to isolate fractions enriched in different cell wall components, as previously described [18, 80]. AIR (200 mg) was first suspended in 20 mL of 50 mM ammonium oxalate (pH 5.0) and incubated at 25 °C for 24 h with constant shaking (100 rpm). After centrifugation (4000g, 15 min), the supernatant was collected as the ammonium oxalate (AO) extract. The pellet was washed with 30 ml ddH₂O, resuspended in 20 mL of 4 M KOH with 1% (w/v) sodium borohydride, and incubated under the same conditions. After centrifugation, the supernatant was collected as the 4 M KOH extract, neutralized with glacial acetic acid, and stored at 4 °C. Both fractions were dialyzed for 72 h, lyophilized, and dissolved in deionized water (200 μg/mL). Total sugar content was determined using the phenol–sulfuric acid assay [84, 85]. Each extract (100 μL) was mixed with 100 μL of 5.0% phenol and 500 μL of H₂SO₄, vortexed, and incubated for 20 min. Absorbance was measured at 490 nm using an ELISA plate reader. d-Glucose standard curves were used to quantify total sugar in the AO and 4 M KOH extracts.

Metabolomics by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Samples of each extractives wash (extractives washes 1, 2, and 3; Additional File 1: Fig. S1) were lyophilized using a Labconco system (USA) and prepared for metabolite extraction via a biphasic approach. Equal volumes of ice-cold hydrated ethyl acetate and HPLC-grade water were mixed to separate polar and non-polar metabolites. The organic (ethyl acetate) fraction was air-dried and subsequently resuspended in 70% acetonitrile containing 0.1% formic acid. Aqueous extracts were then filtered through a 10 kDa molecular weight cutoff spin column (Sartorius Vivaspin 2 Centrifugal Concentrator, Polyethersulfone membrane), lyophilized, and resuspended in a solution of 98% LC–MS-grade water and 2% acetonitrile with 0.1% formic acid. All prepared samples were stored at 4 °C prior to analysis by liquid chromatography–nanoelectrospray ionization–tandem mass spectrometry (LC-nESI-MS/MS). Chromatographic separation was carried out on a Vanquish LC coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific), using a linear gradient from 5 to 100% organic solvent (70% acetonitrile, 0.1% formic acid) over 30 min at a flow rate of 250 nL/min. Mass spectra were acquired over a range of 135 to 2000 m/z at a resolution of 70,000. A top-N data-dependent acquisition method (N = 5) was applied for MS/MS fragmentation using stepped collision energies (10, 20, and 40 eV), with a 10-s dynamic exclusion to prevent oversampling of abundant metabolites.

Raw data from the aqueous (polar) and ethyl acetate (non-polar) fractions were processed separately for spectral matching using Compound Discoverer (CD) version 3.3.1 (Thermo Fisher Scientific). Compounds were detected using default settings, identifying ions such as [M+H], [M+2H], [M+3H], [M+H–H₂O], [M+K], and [M+Na]. Compound grouping was performed with a 5 ppm mass tolerance and a 0.2-min retention time window, with integrated peak areas based on the most common ion. Peaks ratings required a threshold of 4 across a minimum of three files to be considered for further analysis. Spectral matching was performed using mzVault, with a precursor mass tolerance of 10 ppm. The HighChem HighRes algorithm was used to search against the NIST 2020 MS/MS High-Resolution library and the GNPS library (accessed December 2023). Additional compound annotation was conducted using SIRIUS v6.0.7 [86] to interrogate molecular formulas and structures. For each targeted compound, the.mzML file with the highest-quality MS/MS spectrum was selected for annotation, and results were filtered using confidence thresholds of CD score > 0.8 and SIRIUS score > 0.64. The abundance of each metabolite was manually curated using Skyline [87].

Enzyme-linked immunosorbent assay (ELISA)

Ammonium oxalate and 4 M KOH extracts (50 μL of 20 μg/mL) were applied to 96-well plates and dried overnight at 37 °C [29]. Plates were blocked with 200 μL of 1% (w/v) nonfat dry milk in 0.1 M Tris-buffered saline (TBS, pH 7.6) for 1 h at room temperature. Washing and aspiration were performed using an ELx405 microplate washer (Bio-Tek Instruments). In the next step, 50 μL of primary monoclonal antibodies (mAbs) were added and incubated for 1 h, followed by three washes with 300 μL of 0.1% (w/v) nonfat dry milk in TBS. Then, 50 μL of peroxidase-conjugated anti-mouse IgG (1:5000 dilution, Sigma-Aldrich A4416) was added and incubated for 1 h. Wells were washed five times before adding 50 μL of TMB substrate. After 20 min, reactions were stopped with 50 μL of 0.5 N sulfuric acid. Absorbance at 450 nm was measured, with background subtraction at 655 nm, using a Bio-Rad Model 680 microplate reader. The primary antibodies used were the anti-RG-I (CCRC-M14, CCRC-M35, CCRC-M72) [28, 29], anti-xylan (CCRC-M137, CCRC-M149, CCRC-M152) [28,29,30], and anti-galactomannan (CCRC-M74, CCRC-M174, CCRC-M175) [28,29,30]. A full list of antibodies, their recognized epitopes, polymer class, and supporting references is provided in Additional File 11: Fig. S11.

Data availability

No datasets were generated or analyzed during the current study.

Abbreviations

AIR:: Alcohol-insoluble residue
Ara:: Arabinose
CBP:: Consolidated bioprocessing
Fuc:: Fucose
Gal:: Galactose
GalA:: Galacturonic acid
GC–MS:: Gas chromatography–mass spectrometry
Glc:: Glucose
GlcA:: Glucuronic acid
Man:: Mannose
MBMS:: Molecular beam mass spectrometer
Rha:: Rhamnose
RG-I:: Rhamnogalacturonan I
TMS:: Trimethylsilyl
Xyl:: Xylose

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Acknowledgements

We thank CCRC Analytical Services for lignin analysis.

Funding

This work was supported by the Center for Bioenergy Innovation (CBI), U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number ERKP886 and partially by U.S. Department of Energy, Office of Science, Basic Energy Sciences, grant number DE-SC0015662 to Parastoo Azadi at the Complex Carbohydrate Research Center.

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Xiaochun Cheng

Present address: Department of Pharmacology and Cancer Biology, Duke University School of Medicine, 308 Research Drive, LSRC Building, Durham, NC, 27710, United States

Authors and Affiliations

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA

Ajaya K. Biswal, Melani A. Atmodjo & Debra Mohnen
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, USA

Ajaya K. Biswal, Melani A. Atmodjo, Sushree S. Mohanty, Ian M. Black, Xiaochun Cheng, David Ryno, Parastoo Azadi & Debra Mohnen
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA

Paul E. Abraham, Nancy L. Engle & Timothy J. Tschaplinski
Biosciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA

Neal N. Hengge & Yannick J. Bomble
Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, United States

Ajaya K. Biswal, Neal N. Hengge, Melani A. Atmodjo, Paul E. Abraham, Nancy L. Engle, Sushree S. Mohanty, David Ryno, Timothy J. Tschaplinski, Yannick J. Bomble & Debra Mohnen

Authors

Ajaya K. Biswal
Neal N. Hengge
Melani A. Atmodjo
Paul E. Abraham
Nancy L. Engle
Sushree S. Mohanty
Ian M. Black
Xiaochun Cheng
David Ryno
Parastoo Azadi
Timothy J. Tschaplinski
Yannick J. Bomble
Debra Mohnen

Contributions

AKB, YJB, and DM conceived and designed the research. AKB, NNH, MAA, PEA, NLE, SSM, IMB, XC, DR, PA, and TJT conducted the experiments and evaluated the results. AKB, NNH, PEA, TJT, YJB, and DM performed data analysis. AKB, NNH, YJB, and DM wrote the manuscript. All authors reviewed and approved the final version of the manuscript.

Corresponding authors

Correspondence to Ajaya K. Biswal or Debra Mohnen.

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Biswal, A.K., Hengge, N.N., Atmodjo, M.A. et al. Rhamnogalacturonan I is a recalcitrant pectin domain during Clostridium thermocellum-mediated deconstruction of switchgrass biomass. Biotechnol. Biofuels Bioprod. 18, 109 (2025). https://doi.org/10.1186/s13068-025-02703-9

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Received: 13 June 2025
Accepted: 07 September 2025
Published: 27 October 2025
DOI: https://doi.org/10.1186/s13068-025-02703-9

Abstract

Background

Results

Conclusions

Background

Introduction

Results

Water-soluble components of biomass are removed during extractives washing of switchgrass with hot water prior to CBP fermentation

Glycosyl residue composition analysis reveals a significant amount of cell wall carbohydrates in the hot water extractives washes of switchgrass

CBP by C. thermocellum solubilizes more than 35% of biomass over 120 h with more than 24% solubilization requiring the action of C. thermocellum

CBP of switchgrass by C. thermocellum results in accumulation of hemicelluloses in the post-fermentation liquor

CBP of switchgrass by C. thermocellum leads to the accumulation of hemicellulosic and pectic glycans in the water washate of fermentation residues

Time-dependent changes in lignin composition reveal selective solubilization of lignin during CBP of switchgrass by C. thermocellum

C. thermocellum efficiently solubilizes crystalline cellulose in non-pretreated switchgrass during CBP

Non-cellulosic sugar composition of CBP solid residues reveals incomplete solubilization of RG-I and Galactomannan by C. thermocellum

Enzyme-linked immunosorbent assays (ELISA) reveal that RG-I and galactomannan epitopes are enriched in C. thermocellum CBP solid residues

Discussion

Materials and methods

Switchgrass biomass preparation

Consolidated bioprocessing (CBP)

Biomass washing steps before CBP

C. thermocellum culture medium, growth medium and growth conditions

Fermentations

Separations and quantification of total biomass solubilization

Cell wall analysis

Saeman hydrolysis method of cellulose quantification

Lignin quantification

Preparation of cell wall fractions and total sugar estimation

Metabolomics by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Enzyme-linked immunosorbent assay (ELISA)

Data availability

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Additional information

Publisher’s Note

Supplementary Information

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About this article

Cite this article

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