Comprehensive characterization of non-cellulosic recalcitrant cell wall carbohydrates in unhydrolyzed solids from AFEX-pretreated corn stover
- Christa Gunawan†1, 2,
- Saisi Xue†1, 2,
- Sivakumar Pattathil3, 4, 5Email author,
- Leonardo da Costa Sousa1, 2Email author,
- Bruce E. Dale1, 2 and
- Venkatesh Balan1, 2Email author
© The Author(s) 2017
Received: 17 December 2016
Accepted: 11 March 2017
Published: 29 March 2017
Inefficient carbohydrate conversion has been an unsolved problem for various lignocellulosic biomass pretreatment technologies, including AFEX, dilute acid, and ionic liquid pretreatments. Previous work has shown 22% of total carbohydrates are typically unconverted, remaining as soluble or insoluble oligomers after hydrolysis (72 h) with excess commercial enzyme loading (20 mg enzymes/g biomass). Nearly one third (7 out of 22%) of these total unconverted carbohydrates are present in unhydrolyzed solid (UHS) residues. The presence of these unconverted carbohydrates leads to a considerable sugar yield loss, which negatively impacts the overall economics of the biorefinery. Current commercial enzyme cocktails are not effective to digest specific cross-linkages in plant cell wall glycans, especially some of those present in hemicelluloses and pectins. Thus, obtaining information about the most recalcitrant non-cellulosic glycan cross-linkages becomes a key study to rationally improve commercial enzyme cocktails, by supplementing the required enzyme activities for hydrolyzing those unconverted glycans.
In this work, cell wall glycans that could not be enzymatically converted to monomeric sugars from AFEX-pretreated corn stover (CS) were characterized using compositional analysis and glycome profiling tools. The pretreated CS was hydrolyzed using commercial enzyme mixtures comprising cellulase and hemicellulase at 7% glucan loading (~20% solid loading). The carbohydrates present in UHS and liquid hydrolysate were evaluated over a time period of 168 h enzymatic hydrolysis. Cell wall glycan-specific monoclonal antibodies (mAbs) were used to characterize the type and abundance of non-cellulosic polysaccharides present in UHS over the course of enzymatic hydrolysis. 4-O-methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan were found to be the most abundant epitopes recognized by mAbs in UHS and liquid hydrolysate, suggesting that the commercial enzyme cocktails used in this work are unable to effectively target those substituted polysaccharide residues.
To our knowledge, this is the first report using glycome profiling as a tool to dynamically monitor recalcitrant cell wall carbohydrates during the course of enzymatic hydrolysis. Glycome profiling of UHS and liquid hydrolysates unveiled some of the glycans that are not cleaved and enriched after enzyme hydrolysis. The major polysaccharides include 4-O-methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan, suggesting that enzymes with glucuronidase and arabinofuranosidase activities are required to maximize monomeric sugar yields. This methodology provides a rapid tool to assist in developing new enzyme cocktails, by supplementing the existing cocktails with the required enzyme activities for achieving complete deconstruction of pretreated biomass in the future.
KeywordsRecalcitrant cell-wall glycans High-solids loading enzymatic hydrolysis Unhydrolyzed solids Glycome profiling Monoclonal antibody Non-cellulosic polysaccharides Carbohydrates linkages
Declining crude oil reserves and environmental concerns associated with greenhouse gas emissions due to petroleum products has provided an impetus to transition from the current fossil fuel scenario to a more sustainable renewable energy system . Inedible plant biomass, also known as lignocellulosic biomass, includes agricultural residues, forestry residues, herbaceous, and woody crops. These are the most abundant sources of potential feedstocks for producing renewable liquid transportation fuels . Structural carbohydrates from the plant cell walls represent a vast untapped energy source. Attempting to economically convert these carbohydrates to biofuels, particularly via the biochemical route, will be a step forward in creating a more sustainable liquid fuel for transportation. Significant research efforts have been undertaken in the field, particularly over the past few decades.
Cellulose, hemicelluloses, and pectins in the plant cell wall are embedded in a complex matrix with lignin. Plant cell walls are highly recalcitrant to biomass-degrading enzymes, which are responsible to cleave glycosidic bonds and produce monomeric sugars for fermentation [3, 4]. Obtaining high yield of monomeric carbohydrates at minimal enzyme loading is challenging and it is one of the key bottlenecks for obtaining cost-effective biofuels [5, 6]. Due to the recalcitrant nature of the cell wall, pretreatment is required for improving the access of enzymes to their substrates and improve the efficiency of biomass deconstruction [7–9]. Ammonia Fiber Expansion (AFEX™)1 is a pretreatment process in which ammonia reacts with biomass at elevated temperatures and pressures. Ammonia can be used in liquid or gaseous forms and about 97% of ammonia can be recovered and reused in the process [10–12]. The AFEX process loosens the plant cell wall ultrastructure, cleaving lignin–carbohydrate complexes (LCCs), partly relocating lignin to the surface of the cell wall, leaving behind porous structures that help to improve enzyme accessibility to carbohydrates [4, 13]. Due to their physiochemical nature, AFEX is most effective on grasses, including CS, switchgrass, sugarcane bagasse, and miscanthus. As CS is the most abundantly available feedstock in the United States, AFEX could be a promising option for biofuel production in the US [14–16].
Unlike acidic pretreatments which require a wash stream, AFEX pretreatment is a dry-to-dry process that keeps the carbohydrate composition unaltered and preserves most of the sugars intact in a single solid biomass stream [3, 8, 13, 17, 18]. The presence of hemicellulose and pectin, however, requires more complex enzyme cocktails relative to acidic pretreatments, where hemicellulases, pectinases, and other accessory enzymes must be added to cellulases to maximize overall sugar yields. Non-cellulosic polysaccharides, which account for 25–35% of plant cell walls, have branched cross-linkages with varying levels of substitution [19–22]. Thus, a higher degree of synergy between a diverse set of enzyme activities is required to completely depolymerize such complex and highly branched carbohydrate structures into monomeric sugars [23, 24]. Though enzyme cocktail complexity is increased for deconstructing ammonia-pretreated biomass, the overall enzyme loading required does not change significantly in relation to dilute acid pretreatment .
A recent study by Uppugundla et al. showed that inefficient sugar conversion is a problem for various thermochemical pretreatment technologies, including AFEX, dilute acid, and ionic liquid pretreatments . Using the advanced commercial cocktails containing Cellic Ctec2, Cellic Htec2, and Multifect Pectinase with optimized ratio, nearly 22% of total carbohydrates from AFEX-pretreated biomass were left behind as polymeric and oligomeric sugars after 7 days of hydrolysis, at high enzyme loading (20 mg protein/g glucan) and solids loading (6% glucan loading). Due to these unconverted sugars, the biofuel production potential is significantly reduced, which negatively impacts the overall economics of the biorefinery . This is a universal problem faced by researchers both for hardwood and grass substrates . Recalcitrant cell wall polysaccharides not only resist depolymerization when commercial enzymes are used, but also block the accessibility of cellulases to cellulose. Such effect further reduces overall sugar conversion and therefore, it is important to study and understand how commercial enzyme cocktails can be modified for improving hydrolysis of such recalcitrant polysaccharides.
One approach for studying this problem is to rationally design the enzyme cocktail by understanding the limiting factors that contribute to oligosaccharide and polysaccharide accumulation. For example, if some of the required biomass-degrading enzymes are not present or present at low levels in the commercial enzyme cocktail, some glycosidic linkages will tend to accumulate during the hydrolysis process. Thus, by carrying out detailed composition analysis and identifying structural features of enriched recalcitrant cell wall components, one can rationally determine the enzymes that are limiting the hydrolysis process. To facilitate such fundamental understanding of recalcitrant cell wall components, we require rapid tools that provide in-depth structural information about non-cellulosic glycans at the molecular level. One of the methods currently available is glycome profiling. Glycan profiling takes advantage of a worldwide collection of more than 200 plant cell wall glycan-directed mAbs to evaluate the glycan composition of plant cell walls. These mABs enable monitoring of carbohydrate epitopes found in most major non-cellulosic cell wall glycans . Recent studies have employed glycome profiling to better understand cell wall modifications in plant biomass during genetic modifications, biomass pretreatments, and microbial fermentations [26–34]. This information is essential to better understand the glycan linkages contributing to biomass recalcitrance and develop strategies for overcoming this problem.
Results and discussion
Time profile of unhydrolyzed solids (UHS) composition
Among the soluble sugars, hemicelluloses are generally more difficult to convert to monomeric sugars and therefore, we observed considerable levels of xylan, galactan, and arabinan containing oligomers during enzymatic hydrolysis. Nearly 25% of the total xylan was present as oligomers after 72 h, while only 9% of the glucan is present as oligomers at this time point. Likewise, a large proportion of arabinan and galactan sugars remained as oligomers, as shown in Fig. 3c and d. Most of the recalcitrant carbohydrates in hemicellulose (xylan, arabinan, and galactan) were solubilized as oligomers. It appears that the commercial enzymes used here can efficiently solubilize some of the hemicelluloses, but unable to cleave all the hemicellulose linkages to generate monomeric sugars. Cellulose is a relatively simpler structure consisting of glucan chains connected with inter- and intra- molecular hydrogen bonding. However, hemicellulose is highly branched with multiple sugars and cross-linked with other organic moieties (e.g., acetyl, feruloyl, galacturonic, glucoronyl), some of which form complexes with lignin . Multiple accessory enzymes are required to fully break down these complex hemicellulose linkages . Based on our results, it is unlikely that all those enzymes are present at sufficient quantities and/or activities in the commercial enzyme cocktails used in this study. To determine the most abundant linkages present in UHS after enzymatic hydrolysis, we have further performed glycome profiling on UHS produced during enzymatic hydrolysis of AFEX-CS. As control experiments, we have also performed glycome profiling of untreated and AFEX-CS.
Glycome profiling of untreated and AFEX-pretreated biomass
Glycome profiling and structural insights of UHS
Overall, the information provided by this work shows that specific glycans (e.g., xylans decorated with 4-O-methyl glucuronic acid residues, pectic arabinogalacta, and rhamnogalacturonan-I) are not completely digested by the commercial enzyme cocktail used in this work, even when those epitopes are completely soluble in the liquid hydrolysate and potentially free from lignin blockage. In contrast, a larger range of glycan epitopes can be detected in the UHS when they are associated with lignin, suggesting that those glycans may not be accessed by enzymes due to lignin blockage. Though these insoluble non-cellulosic carbohydrates represent less than 7% of the total carbohydrates after excessive enzyme treatment with optimized cocktails, the soluble counterpart in hydrolysate represents as much as 15% of the total carbohydrates in pretreated biomass, which is a much more significant fraction of substrate that is not converted to monomeric sugars and biofuel, ultimately. In future work, our goal is to better understand the factors that contribute to epitope accumulation in the UHS and liquid hydrolysate. Imaging techniques, such as TEM and florescent microscopy, can be applied to depict the spatial orientation of cell wall components in the UHS and understand phenomena such as lignin blockage. NMR and mass spectrometry can also be used to determine the composition, structure, and linkage patterns of purified recalcitrant carbohydrates (mostly oligosaccharides). All these studies will complement this work and help to comprehensively understand cell wall recalcitrance. The information provided by this (and future) work will help us to rationally redesign the enzyme cocktail, by adding a selection of key enzymes for improving monomeric sugar yields, with minimal enzyme usage.
The chemical nature of some of the recalcitrant carbohydrate linkages present in CS was studied by analyzing UHS and hydrolysates resulting from enzymatic hydrolysis of AFEX-CS. Samples taken at multiple time points over a period of 168 h were analyzed to understand changes in the different cell wall components as a function of time. The polysaccharides that were easier to extract after AFEX treatment were rapidly deconstructed by the enzymes, while some of the carbohydrates that required harsher alkaline extractions could not be hydrolyzed by the enzymes and accumulated in the UHS. While a wide range of polysaccharides remained in the UHS, the amount remaining in the insoluble form was relatively small (<5%) after 168 h. However, soluble polysaccharides, particularly those recognized by xylan-5 and RG-I/AG groups of mAbs, remained abundant in hydrolysate and UHS throughout the course of hydrolysis, indicating a lack of appropriate enzyme activities or severe enzyme inhibition. These results show that complete sugar conversion is not possible when using commercial enzyme cocktails (Cellic Ctec2, Cellic Htec2, and Multifect Pectinase) as used in this work, at high-solid loading enzymatic hydrolysis conditions.
Future work is needed to find enzymes that hydrolyze these recalcitrant non-cellulosic polysaccharide linkages. For example, accessory enzymes such as pectinase, α-glucuronidase, and enzyme activities targeting arabinan and galactan should be identified and added to the enzyme cocktail, so that branched linkages that block enzyme accessibility to backbone polysaccharide chains can be hydrolyzed and potentially be decoupled from the complex structure of hemicellulose. If successful, this approach could not only increase monomeric xylose yields, but may also synergistically improve cellulose hydrolysis, thus increasing glucose yields and a possible reduction in enzyme loading to lower biofuel production cost. Likewise, this approach could be adapted with other pretreatment technologies and biomass to optimize hydrolysis conditions for maximum sugar output. Moreover, since routine glycome profiling only detects large polysaccharides, more advanced techniques such as biotinylation , fluorescent-labeled antibodies studies using fluorescent microscopy and flow cytometry will be able to detect short-chain oligosaccharides that are abundant in the hydrolysate. Such studies are underway at the Great Lakes Bioenergy Center (GLBRC).
Corn stover and AFEX pretreatment
The corn (Pioneer 36H56) was planted on May 20, 2010 in field 436 of Arlington Agricultural Research Station, Wisconsin. The field was fertilized with 340 lbs/acre urea 3 days prior to planting. In October 22, 2010, the CS was harvested and milled to a particle size of 5 mm. AFEX pretreatment was performed on the CS at 100 °C for 30 min with 0.6 g ammonia and 1 g water per g biomass loading in a bench-top stainless steel batch reactor (Parr Instruments Company) [10, 11, 13]. It took 30 min for the reactor to reach 100 °C and this condition was maintained for 30 min. Then the ammonia was rapidly released, which immediately brought the biomass to room temperature. After the treatment, the biomass was transferred to aluminum tray and kept in hood overnight to remove residual ammonia, leaving behind dry material. The AFEX-CS contained 31.4% glucan, 18.7% xylan, 1.4% galactan, 3.3% arabinan, 14.3% lignin (1.23% acid soluble lignin, absorption wavelength 320 nm, absorptivity coefficient 30 L/g cm), and 13.4% ash.
Enzymatic hydrolysis was performed in duplicate using baffled Erlenmeyer flasks. The AFEX-CS was loaded at 20% dry solids in a fed-batch manner. Half of the biomass was loaded at t = 0 h, and the remaining biomass was loaded at t = 45 min. Commercial enzymes Cellic® CTec2 (Novozymes), Cellic® HTec2 (Novozymes), and Multifect Pectinase (Genencor) were loaded at 10, 5, and 5 mg protein/g glucan at t = 0 h, respectively. Flasks were incubated in a shaking incubator set at 250 rpm and 50 °C. During the sampling process, the flasks were taken out of the incubator and immediately set on ice for approximately 30 min to arrest the hydrolysis reaction at each time point (3, 6, 12, 18, 24, 48, 72, and 168 h). Separate pairs of flasks were used for each time point. The pH was adjusted to 5.0 using 12 M hydrochloric acid at the start of the hydrolysis process.
Post-hydrolysis solids recovery
The contents of the flasks were transferred into centrifuge bottles and centrifuged at 10,000×g at 4 °C for 30 min. The supernatant was decanted, the volume measured, and filtered through 0.22 μm PES membrane and stored at 4 °C for future sugar analysis. The solid was re-suspended in a known amount of water (approximately 8:1 water-to-solid ratio) and centrifuged. The supernatant was decanted to a separate tube and a ~1 mL sample was taken for sugar analysis. This process was repeated two more times to remove any residual soluble material present in the solids. The moisture content of the wet solids was measured in triplicate by drying samples at 110 °C overnight in aluminum tray.
A portion of the washed solid was treated with protease from Streptomyces griseus (Sigma Aldrich P5147) at 5% (w/v) solid loading according to the procedure by Berlin et al. . This helped to remove protein and residual enzymes bound to the cell walls prior to glycome profiling. The remaining solid was freeze-dried and stored in a refrigerator for further analysis.
Liquid and solid composition analysis
The hydrolysate supernatants were diluted and analyzed for monomeric and oligomeric sugar contents. Monomeric sugars were analyzed using an HPLC equipped with a Bio-Rad (Hercules, CA) Aminex HPX-87P column and de-ashing guard column. Column temperature was held at 80 °C, and water was used as the mobile phase flowing at 0.6 mL/min. Oligomeric sugars were determined via dilute acid hydrolysis at 121 °C according to the method of Sluiter et al. . Hydrolysis samples were neutralized and analyzed using the HPLC method given above for total sugars estimation following acid hydrolysis. The oligomeric sugars were calculated as total sugars after subtracting the monomeric sugars present in hydrolysate.
Freeze-dried solids were homogenized using mortar and pestle. Composition analysis was performed on the solids using the standard National Renewable Energy Laboratory (NREL) method for determination of structural carbohydrates and lignin according to Sluiter et al. .
Glycome profiling of untreated, AFEX™-pretreated and all unhydrolyzed biomass residues (involving preparation of sequential cell wall extracts and their mAb screenings) was carried out using the SOP previously described [29, 33]. To conduct glycome profiling, Alcohol Insoluble Residue (AIR) cell wall materials were prepared from biomass residues and were subjected to sequential extractions with increasingly harsh reagents such as ammonium oxalate (50 mM), sodium carbonate (50 mM), KOH (1 and 4 M), and acidic chlorite as explained previously . The extracts were then subjected to ELISAs against a comprehensive suite of cell wall glycan-directed mAbs  and the mAb binding responses were represented as heat maps. The amounts of different cell wall materials recovered during each extraction are depicted as bar graphs above the respective heat map panels. Plant cell wall glycan-directed monoclonal antibodies (mAbs) were received from laboratory stocks (CCRC, JIM and MAC series) maintained by the Complex Carbohydrate Research Center (available through CarboSource Services; http://www.carbosource.net) or were obtained from Bio-Supplies (Australia) (BG1, LAMP). Information on mAbs used in this study can be found in Table 2, including the link to Wall MabDB (http://www.wallmabdb.net) that provides detailed information for each antibody.
TM-AFEX is a trademark of MBI International, Lansing, Michigan.
Ammonia Fiber Expansion
CG and SX performed enzymatic hydrolysis to produce the ACSH and UHS, conducted compositional analysis and mass balance of carbohydrates in the biomass, interpreted data, drafted the manuscript, and contributed to the experimental design. CG conducted the glycome profiling on UHS solids and SX conducted the glycome profiling on hydrolysate liquor. SX also depicted and illustrated all the glycan epitopes recognized by cell-wall-specific mAbs using GWB (GlycoWorkBench). SP developed the methodology of glycome profiling, provided all antibodies for mAbs Elisa screening, and made important revisions on the manuscript critically. BED contributed on the problem identification, coordinated the collaboration work within GLBRC, and helped with the preparation of the manuscript. SP, LDSC, and VB lead the identification of the problem and troubleshooting, organized the experimental design and manuscript preparation, and coordinated this work. All authors read and approved the final manuscript.
Special thanks to Novozyme Inc. and DuPont Industrial Biosciences for the generous gift of enzymes and to Dr. Michael G Hahn and NSF for providing mAbs for glycome profiling screening. We also thank Pete Donald in BCRL for carrying out the AFEX pretreatment and producing biomass, and Sivasankari Venketachalam and Sindhu Kandemkavil at CCRC for glycome profiling and glycosyl composition analysis training given to Christa and Saisi at CCRC, University of Georgia.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article with Additional file 1: Figure S1.
Consent for publication
All authors consent for publication.
This work was funded by the DOE Great Lakes Bioenergy Research Center (http://www.greatlakesbioenergy.org) supported by the US. Department of Energy, Office of Science, Office of Biological and Environmental Research, through Cooperative Agreement DE-FC02-07ER64494 between the Board of Regents of the University of Wisconsin System and the US Department of Energy. This work was also supported and performed as part of the BioEnergy Science Center (BESC), a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. ORNL is managed by UT-Battelle, LLC, under contract DE-AC05-00OR22725 for the U.S. Department of Energy. Co-author Dale is grateful for support provided by Michigan State University AgBioResearch and the U.S. Department of Agriculture NIFA program. The generation of the plant glycan-directed monoclonal antibodies used in this study was funded by grants from the US National Science Foundation Plant Genome Program (DBI-0421683 and IOS-0923992).
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