Monitoring and Analyzing Process Streams Towards Understanding Ionic Liquid Pretreatment of Switchgrass (Panicum virgatum L.)
- 1.7k Downloads
Fundamental understanding of biomass pretreatment and its influence on saccharification kinetics, total sugar yield, and inhibitor formation is essential to develop efficient next-generation biofuel strategies, capable of displacing fossil fuels at a commercial level. In this study, we investigated the effect of residence time and temperature during ionic liquid (IL) pretreatment of switchgrass using 1-ethyl-3-methyl imidazolium acetate. The primary metrics of pretreatment performance are biomass delignification, xylan and glucan depolymerization, porosity, surface area, cellulase kinetics, and sugar yields. Compositional analysis and quantification of process streams of saccharides and lignin demonstrate that delignification increases as a function of pretreatment temperature and is hypothesized to be correlated with the apparent glass transition temperature of lignin. IL pretreatment did not generate monosaccharides from hemicellulose. Compared to untreated switchgrass, Brunauer–Emmett–Teller surface area of pretreated switchgrass increased by a factor of ∼30, with a corresponding increase in saccharification kinetics of a factor of ∼40. There is an observed dependence of cellulase kinetics with delignification efficiency. Although complete biomass dissolution is observed after 3 h of IL pretreatment, the pattern of sugar release, saccharification kinetics, and total sugar yields are strongly correlated with temperature.
KeywordsBiomass Cellulase kinetics Delignification Ionic liquid Pretreatment Porosity Surface area Switchgrass
With the prospect of diminishing fossil fuel supplies and estimated high levels of atmospheric carbon dioxide projected to reach 600 ppm by year 2035 (http://www.occ.gov.uk/activities/stern.htm, Office of Climate Change, UK), there is a great urgency for developing carbon neutral and renewable sources of transportation fuels. Advanced biofuels derived from lignocellulosic biomass are a potential source of renewable transportation fuel, with significantly reduced carbon emissions. Lignocellulosic biomass is composed mainly of cellulose, hemicellulose, and lignin. Cellulose is the most abundant polymer on earth and is composed of fermentable glucose that can be readily converted into biofuel . However, the glucose is hard to liberate cost-effectively due to extensive intermolecular and intramolecular hydrogen bonding of the β-1,4-glycan chains in crystalline cellulose . Hemicellulose is rich in xylose and interacts with cellulose and lignin to strengthen the cell wall. The interactions are mostly noncovalent, but in grasses the arabinoxylan can be covalently linked to lignin through oxidative coupling via ferulate esters . The lignin is a polymer of monolignols, which are phenolic alcohols derived from coumaric acid. Lignin plays an essential role in plants by giving strength to stems and by making tracheary elements watertight and able to withstand the negative pressure in the xylem. Lignin is very difficult to break down and makes polysaccharides inaccessible to enzymes.
Before enzymatic saccharification, lignocellulosic biomass must be pretreated to increase the accessibility of the polysaccharides to hydrolytic enzymes . After pretreatment, enzyme cocktails are capable of hydrolyzing the polysaccharides into simple sugars (C6 and C5)  but are currently very expensive. It is therefore crucial that pretreatment methods enable the saccharification to take place without excessive amounts of enzyme. The bond energies of the massive hydrogen bonding network present in cellulose can reach more than 23–25 kJ/mol and render traditional solvents unsuitable for effective biomass dissolution [6, 7]. Various pretreatment technologies are currently being tested and show some promise but face significant commercialization challenges due to an incremental enhancement in saccharification kinetics, requisite high temperature and/or pressure, and overall process economics [8, 9, 10].
Recently, IL pretreatment has been shown to be a promising pretreatment technology due to its ability to solubilize biomass by overcoming the hydrogen bonding within cellulose [10, 11, 12, 13, 14]. Other benefits of IL pretreatment include efficient precipitation and recovery of dissolved polysaccharides upon addition of antisolvent and desirable solvent attributes like low volatility, nonflammability, and thermal stability [10, 11, 12, 13, 14]. In order for ILs to develop beyond these initial positive results into an effective and commercial biomass pretreatment, three main conditions must be met: (1) solubilization of bioenergy crops at high biomass loading (20–30 wt.%), (2) solvent recovery and recycling, and (3) minimal generation of inhibitory by-products that may render cellulolytic enzymes and fermentation microbes inactive.
Detailed parametric studies of temperature and residence time were carried out for a promising IL for biomass solubilization, 1-ethyl-3-methyl imidazolium acetate (abbreviated as [C2mim][OAc]) . We have selected a potential dedicated energy crop switchgrass (Panicum virgatum L.). Switchgrass is native to North America; it is drought resistant and can grow over 1.8 m tall. It is presently used for forage production and soil conservation and has shown potential for biofuel production [24, 25, 26, 27, 28]. The recovered biomass upon antisolvent addition was analyzed in terms of delignification, porosity, surface area, total sugar yield after saccharification, and initial saccharification kinetics. The liquid from the process stream was analyzed for total monomeric sugar yields of cellulose, hemicellulose, xylose, arabinose, glucuronic acid, and other minor C5 and C6 sugars.
Materials and Methods
Switchgrass was obtained from Ken Vogel at the US Department of Agriculture, Lincoln, NE, USA. It was milled with a Thomas-Wiley Mini Mill fitted with a 40-mesh screen (Model 3383-L10 Arthur H. Thomas Co., Philadelphia, PA, USA).
Preparation of Alcohol-Insoluble Residue
The plant material (50 mg) was treated with 95% ethanol (1:4 w/v) at 100°C for 30 min. After the treatment, sample was centrifuged (10,000×g, 10 min), and the residue was subsequently washed five times with 70% ethanol and dried at 32°C under vacuum. The dried powder obtained after 70% ethanol wash is designated as alcohol-insoluble residue (AIR). The AIR was destarched essentially as described by Obro et al. . AIR was incubated with heat-stable amylase from Bacillus licheniformis (Megazyme, Bray, Ireland) at 0.3 U per 10 mg AIR in 3-(N-morpholino) propanesulfonic acid buffer (50 mM, pH 7.0) at 85°C for 1 h. Subsequently, the sample was incubated with amyloglucosidase from Aspergillus niger (0.33 U per 10 mg AIR) and pullulanase from B. licheniformis (0.04 U/10 mg AIR) in 200 mM sodium acetate (pH 4.5), for 2 h at 50°C. Amyloglucosidase and pullulanase were purchased from Megazyme. The reaction was stopped by adding three volumes of 95% ethanol, vortexed and centrifuged at 10,000×g for 10 min. The residue obtained after centrifugation was washed ten times with 70% ethanol and dried at 32°C under vacuum. The destarched AIR was used as biomass in the pretreatment experiments.
Ionic Liquid Pretreatment of Biomass
Biomass (destarched AIR) was treated with 1-ethyl-3-methylimidazolium acetate (Sigma-Aldrich, St Louis, MO, USA) at a loading of 3% at 110–160°C for 3 h in an oven (Thelco Laboratory oven). Upon cellulose regeneration with water, the pretreated material was washed with deionized hot water. Samples were centrifuged at 10,000×g for 20–25 min, and washes were continued until a colorless supernatant was obtained to ensure complete washing of regenerated biomass. This indicated the absence of ionic liquid in wash which was further confirmed with Fourier transform infrared measurement. The infrared spectrum of supernatant showed no ionic liquid peaks. The pooled washes were concentrated under vacuum for further analysis. For residence time optimization, biomass at 3% loading was treated with 1-ethyl-3-methylimidazolium acetate. The temperature was varied from 110°C to 160°C at increments of 10°C for 3, 6, 24 h, 2, 3, 4, and 5 days in an oven. Pretreated material was washed and pooled as described above.
After IL pretreatment and precipitation of cellulose by water, all the supernatants from the washing steps were collected and concentrated. Three-hundred microliters of solution was treated with 150 μl of trifluoroacetic acid (TFA) at 120°C for 1 h. The supernatant was placed in a CentriVap Vacuum Concentrator (Labconco Corp, Kansas City, MO, USA) at 32°C. Monosaccharides produced from untreated and pretreated samples both before and after TFA hydrolysis were analyzed by high-performance anion-exchange chromatography (HPAEC) on an ICS-3000 system equipped with an electrochemical detector and a 4 × 250 mm CarboPac PA™ 20 column (Dionex, Sunnyvale, CA, USA), according to Obro et al. . The monosaccharides including fucose, arabinose, rhamnose, galactose, mannose, xylose, glucose, glucuronic acid, and galacturonic acid used as the external standards for HPAEC were obtained from Sigma-Aldrich and Alfa Aesar (Ward Hill, MA, USA).
Porosimetry of Biomass
Nitrogen porosimetry (Micromeritics ASAP 2020, Norcross, GA, USA) was used to measure the surface area, pore size distribution, and pore volume of the untreated and IL pretreated switchgrass. Samples were degassed at 100°C for 15 h and were cooled in liquid nitrogen, allowing nitrogen gas to condense on the surfaces and within the pores. Each data point along the isotherm was taken with a minimum equilibration time of 100 s to allow the pressure in the sample holder to stabilize. The quantity of gas that condensed could be inferred from the pressure decrease after the sample was exposed to the gas.
Lignin Quantification Using Acetyl Bromide
The lignin content of both untreated and regenerated biomass was determined with a modified acetyl bromide method [30, 31]. Switchgrass powder (5 mg) was treated with 25% (w/w) acetyl bromide in glacial acetic acid (0.2 ml). The tubes were sealed and incubated at 50°C for 2 h at 1,050 rpm on a thermomixer. After digestion, the solutions were diluted with three volumes of acetic acid, and then 0.1 ml was transferred to 15-ml centrifuge tubes and 0.5 ml acetic acid was added to it. The solutions were mixed well and 0.3 M sodium hydroxide (0.3 ml) and 0.5 M hydroxylamine hydrochloride (0.1 ml) were added to it. The final volume was made to 2 ml with the addition of acetic acid. The UV spectra of the solutions were measured against a blank prepared using the same method. The lignin content was determined with the absorbance at 280 nm and calculated with an averaged extinction coefficient of 18.1951 l g−1 cm−1 for grass samples . The reagents used were from Alfa Aesar.
The untreated samples and regenerated biomass from various conditions were hydrolyzed in a batch system. The total batch volume was 10 ml of 50 mM sodium citrate buffer (pH 4.8) with 80 mg glucan contents, cellulase (cellulase from Trichoderma reesei, Worthington Biochemical Corporation, Lakewood, NJ, USA) with a loading of 12.5 IU/ml, and β-glucosidase (Novozyme 188, Novozymes Corporation, Davis, CA, USA) with a loading of 5.1 IU/ml. The digestion vials were incubated in a rotary shaker under the conditions of 150 rpm and 50°C. Experiments were conducted in triplicate for 72 h. The reaction was monitored by periodically taking evenly mixed slurry samples, centrifuging at 16,100×g for 10 min, and measuring the release of soluble reducing sugars by using 3,5-dinitrosalicylic acid (DNS, Sigma-Aldrich) colorimetric assay with d-glucose as a standard . The supernatants (60 μl) were mixed with DNS solution (60 µl) and heated at 95°C for 5 min. After cooling down, their absorbances were taken at 540 nm . The initial rates of formation of total soluble reducing sugars were calculated based on the sugar released in the first 30 min of hydrolysis .
Results and Discussion
Hemicellulose Disposition and Pattern of Sugar Release Using HPAEC
Pattern of saccharide release measured by HPAEC and its dependence on pretreatment temperature
Saccharides (μg/mg sample)
Effect of IL Pretreatment on Porosity and Surface Area of Biomass
Effect of Pretreatment Temperature
Effect of Residence Time
Mass Balance for the Ionic Liquid Process
Detailed parametric studies of IL pretreatment of switchgrass have been carried out to understand the disposition of cellulose, hemicellulose, and lignin in order to optimize the [C2mim][OAc] pretreatment process. Our findings indicate that efficient depolymerization of hemicellulose occurs regardless of residence time or temperature. The hemicellulose is converted to oligosaccharides, and only trace amounts of monomeric sugars (xylose and glucose) are detected in the IL hydrolysates. Quantification of xylose, glucose, arabinose, galactose, rhamnose, and other minor C6 and C5 sugars after TFA digestion of IL-pretreated and regenerated biomass as a function of temperature and residence time shows very different patterns of sugar release. Temperature studies show that three times as much oligomeric hemicellulose are released at 160°C when compared to 120°C. IL pretreatments conducted at 100–140°C show similar total sugar yields in the IL hydrolysate supernatant.
Three hour IL pretreatment delignified switchgrass by 73.5% at 160°C. An interesting observation was significant enhancement in delignification at 150°C. This is consistent with the reported process temperatures of acid and ammonia fiber expansion pretreatment technologies. These results suggest softening or melting of lignin to be primarily responsible for observed increase in enzymatic hydrolysis kinetics of pretreated biomass. In addition, the temperature variation of IL pretreatment from 110°C to 160°C resulted in lignin removal efficiency that is monotonically related to the increase of enzymatic hydrolysis.
It is also observed that the BET surface area increased ∼30-fold after IL treatment at 160°C. Pore volume (BJH absorption) also increased ∼30-fold after IL pretreatment with average measured pore size of 10–15 nm for [C2mim][OAc]-pretreated switchgrass. Porosimetry data of untreated switchgrass indicate a nonporous matrix with minimal surface accessibility for cellulolytic enzymes.
Time-series experiments show that, for 120°C pretreatment, 3 h IL pretreatment is sufficient since 3 h and 5 day IL pretreatment show no difference in sugar yields. However, pretreatment residence time has a significant effect on switchgrass pretreated at 160°C with the optimum residence time found to be 3 h, whereas there was very little sugar production for the 24 h sample with the yield even lower than the untreated switchgrass. The biomass was completely solubilized for 160°C pretreated sample with a residence time of 5 days and resulted in no recovery of biomass. It is interesting that 160°C and 5 day IL pretreatment resulted in IL hydrolysate which showed presence of oligosaccharides and only trace amounts of monosaccharides by HPAEC measurements. Saccharification kinetics were ∼3.8 times faster for 160°C pretreated switchgrass than 120°C pretreated switchgrass. However, at 160°C, the hydrolysis kinetics increased ∼6.1 times when compared to 110°C pretreatment showing doubling of kinetics for 10°C increase in pretreatment temperature and reached to a maximum of ∼39 times higher than the untreated switchgrass. This observed enhancement of enzymatic hydrolysis kinetics is significant and substantiates the importance of pretreatment technologies for rapid advancement of biofuel production from lignocellulosic biomass.
This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. The authors thank Drs. Patanjali Varanasi and Anthe George from the Joint BioEnergy Institute for their help with manuscript proofreading and their valuable comments.
- 2.Blanch HW, Wilke CR (1982) Sugars and chemicals from cellulose. Rev Chem Eng 1:71–119Google Scholar
- 11.Swatloski RP, Spear SK, Holbrey JD, Rogers RD (2003) Ionic liquids as green solvents for the dissolution and regeneration of cellulose. Abstr Pap Am Chem Soc 225:U288–U288Google Scholar
- 19.Ramos LP (2003) The chemistry involved in the steam treatment of lignocellulosic materials. Quim Nova 26(6):863–871Google Scholar
- 37.Zhu Z, Sathitsuksanoh N, Vinzant T, Schell DJ, McMillan JD, Zhang YH (2009) Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: enzymatic hydrolysis, supramolecular structure, and substrate accessibility. Biotechnol Bioeng 103(4):715–724CrossRefPubMedGoogle Scholar