Structural evaluation and bioethanol production by simultaneous saccharification and fermentation with biodegraded triploid poplar
© Wang et al.; licensee BioMed Central Ltd. 2013
Received: 30 October 2012
Accepted: 14 February 2013
Published: 21 March 2013
Pretreatment is a key step to decrease the recalcitrance of lignocelluloses and then increase the digestibility of cellulose in second-generation bioethanol production. In this study, wood chips from triploid poplar were biopretreated with white rot fungus Trametes velutina D10149. The effects of incubation duration on delignification efficiency and structural modification of cellulose were comparably studied, as well as the digestibility of cellulose by simultaneous saccharification and fermentation (SSF).
Although microbial pretreatments did not significantly introduce lignin degradation, the data from SSF exhibited higher cellulose conversion (21-75% for biopretreated samples for 4–16 weeks) as compared to the untreated poplar (18%). In spite of the essential maintain of crystallinity, the modification of lignin structure during fungal treatment undoubtedly played a key role in improving cellulose bioconversion rates. Finally, the ethanol concentration of 5.16 g/L was detected in the fermentation broth from the cellulosic sample biodegraded for 16 weeks after 24 h SSF, achieving 34.8% cellulose utilization in poplar.
The potential fungal pretreatment with Trametes velutina D10149 was firstly explored in this study. It is found that the biopretreatment process had a significant effect on the digestibility of substrate probably due to the removal and unit variation of lignin, since the crystallinities of substrates were rarely changed. Additional investigation is still required especially to improve the selectivity for lignin degradation and optimize the digestibility of cellulose.
KeywordsBiodegradation Lignocellulose Simultaneous saccharification and fermentation Bioethanol Trametes velutina
Lignocellulose is the major component of biomass, consisting of three types of polymers, cellulose, hemicelluloses and lignin that are strongly intermeshed and chemically bonded by non-covalent forces and by covalent cross-linkages . Many scientific challenges remain in understanding the recalcitrancy of lignocelluloses, and numerous chemical and physical methods have been attempted to unlock lignin polymers from the cell wall complex [2–6]. However, these pretreatment processes are often limited by the lack of selectivity to the target component, together with high energy requirement, economical feasibility and environmental unfriendliness. Thus, effective, low-cost, and green biopretreatment under mild conditions and low energy consumption has manifested the superiority over the aforementioned chemical means .
Nature has created a mixture of enzymatic complexes, which are capable of opening the complex structure of lignin molecules by selectively cleaving the chemical bonds between the lignin units without using/releasing any environmentally harsh chemicals . Thereby, it is considered to be an environmentally friendly process with its own advantages, including no chemicals, no required special reactor, no waste and no inhibitor to fermentation. In fact, biological pretreatment has long been studied in the pulping process to save energy, increase pulp quality and reduce environmental impacts . White-rot fungi, as the most promising microorganisms for selective lignin degradation, have been receiving extensive attention for biodelignification of lignocellulosic biomass. It has the ability to produce lignolytic enzymes, including manganese peroxidases (MnPs), lignin peroxidases (LiPs) and esterases (ESTRs) , as well as H2O2 generating enzyme systems (copper radical oxidases, aryl alcohol oxidases, glyoxal oxidases etc.), which play key roles during the lignin degradation/modification process [11, 12]. Nowadays, several basidiomycetes such as Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Phlebia subserialis, and Pleurotus ostreatus have been examined on different lignocellulosic substrate to evaluate their delignification efficiency. As known, most white-rot fungi simultaneously degrade carbohydrates (cellulose and hemicelluloses) and lignin, resulting in the homogeneous decay of the cell wall. Meanwhile, some species preferentially degrade lignin and part of hemicelluloses, dissolving the middle lamella and then creating the defibrillation effect. Due to the deconstruction of the impact matrix in biomass, the accessibility of cellulose for cellulase is improved and the production of sugars during bioconversion process is significantly enhanced [13–15]. Baba et al. reported that biopretreatment with white-rot fungi resulted in significantly higher sugar yield (>35%) than untreated softwood (10.2%) . It was also reported that the enzymatic digestibilities of corn stover, which had been pretreated with Cyathus stercoreus and P. chrysosporium, were 3.75 and 1.26 times greater than that of untreated sample, respectively . Different white rot fungus varies greatly in the relative rates, where the degradation of lignin and carbohydrates occur in lignocelluloses. Zhang et al. screened 34 kinds of white rot fungi and found only two isolates were suitable for the biopretreatment process . Recently, a new fungus, Trametes velutina D10149, was isolated and identified in Institute of Microbiology, Beijing Forestry University. Thereby, it is necessary to investigate the biodegradation pattern and evaluate the pretreatment efficiency on bio-ethanol production by Trametes velutina D10149, in order to fully exploit the potential of this fungus.
In the present study, biological delignification of triploid poplar (Populus tomentosa Carr.) using white-rot fungus T. velutina D10149 was attempted. The methods of wet chemistry, X-ray diffraction (XRD), Fourier transform infrared (FTIR), and scanning electron microscopy (SEM) were applied to characterize and gain insights on the delignification process and structural modification of cellulose in lignocelluloses. The biological pretreatment was further evaluated by ethanol yield from bioconversion process, simultaneous saccharification and fermentation (SSF), which will better meet the requirement for 2nd generation bio-ethanol production.
Results and discussion
Delignification process evaluation
The content of lignin (ABSL, wt%), infrared ratios and crystallinity indices of untreated and biopretreated cellulosic samples after different incubation time
Yields of monolignol derivatives (w/w, μg/mg) a of untreated and biopretreated cellulosic samples, obtained from 1N NaOH extraction at room temperature
Yields of monolignol derivatives (w/w, μg/mg) a of untreated and biopretreated cellulosic samples, obtained from 4N NaOH extraction at 170°C
Yields of phenolic acids and aldehydes (w/w, μg/mg) a from alkaline nitrobenzene oxidation of untreated and biopretreated cellulosic samples
Crytallinity and morphologic analysis
Products in SSF
Selective white rot fungus has shown potential for lignocelluloses pretreatment. In this study, a new fungal isolate, Trametes velutina D10149, was used in the biological pretreatment to enhance the digestibility of triploid poplar. Although no unique and significant selectivity for lignin degradation was observed in biodegradation process, the ethanol production was achieved 5.16 g/L in the fermentation broth after 24 h SSF from fungal-pretreated poplar substrate. Based on wet-chemical analysis of remaining lignin, the structural variation in lignin macromolecules was important for improving lignocelluloses bioconversion efficiency.
Raw materials and chemicals
Triploid poplar (Populus tomentosa Carr.) of 3-year-old was cut from the suburb of Beijing, China. After being processed through a combination of chipping and milling, the fraction passing 40-mesh was collected and used throughout the study. The main components of the wood were determined as: glucose 44.4±0.6%, xylose 23.0±0.5%, Klason lignin 19.4±0.3%, and acid soluble lignin 4.9±0.1% (weight % of starting material). All chemicals are of analytical grade unless otherwise mentioned.
Fungal strains and biopretreatment process
The white rot fungus Trametes velutina D10149 was isolated from Jilin province in China, and preserved on 2% (w/v) malt-extract agar (MEA) plates at 4°C in Institute of Microbiology, Beijing Forestry University. A plug of the fungi activated in 100 mL basic medium (containing glucose 20 g/L, yeast extract 5 g/L, KH2PO4 1 g/L, MgSO4 0.5 g/L, and VB1 0.01 g/L). After been cultured on a rotary shaker at 28°C with a speed of 150 rpm, mycelial pellets were harvested after 5 d and mixed with 100 ml distilled water. This suspension would act as inoculums.
The biological pretreatment with T. velutina D10149 was carried out in a 250-mL Erlenmeyer flask with 5 g of air-dried poplar and 12.5 mL of distilled water. The samples were sterilized in the autoclave for 20 min at 121°C and inoculated with 5 mL inoculums. The culture was incubated statically in 28°C for 4–16 week. The non-inoculated sample was served as the control. All experiments were performed in triplicate.
Enzyme and yeast
The Cellulast 1.5L (cellulase) and Novozyme 188 (β-glucosidase) were kindly provided by Novozymes Investment Co. Ltd. (Beijing, China). The microorganism used for fermentation was Saccharomyces cerevisiae in the form of dry yeast (thermal resistant) (Angel Yeast Company Ltd, Yichang, China). Dry yeast was activated in 2% glucose solution at 40°C for 20 minutes, then at 34°C for 2 hour.
Evaluation of delignification and monolignol
The associated lignin was analyzed through spectrophotometric method, which is easy and rapid for determining the total lignin concentration of a cell wall sample . The procedure was described in detail in a previous paper , and the concentration of ABSL was calculated by adopting the appropriated absorption coefficient, 18.21 mL mg-1 cm-1. The ester-bound, ether-bound and non-condensed phenolics were analyzed with 1N NaOH at room temperature, 4N NaOH at 170°C and alkaline nitrobenzene oxidation processes, respectively. The separation and identification of phenolics was achieved with a HPLC system (1200 series, Agilent Technologies, USA) on a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm) by comparison of retention times and UV spectra (DAD, diode array detector) of the eluting peaks and the authentic standard compounds (Sigma–Aldrich Corp.; St. Louis, MO, USA) .
FTIR spectra of the cellulosic samples were recorded from an FTIR spectrophotometer (Tensor 27, Bruker, Germany) in the range 4000 to 800 cm-1, using a KBr disc containing 1% finely ground samples. Shimadzu XRD-6000 instrument (Japan) was used to examine the crystallinity, scanning from 5° to 35° 2θ by a goniometer at a scanning speed of 5°/min. The relative crystallinity is commonly measured as a ratio between the diffraction portion from the crystalline part of the sample and the total diffraction from the same sample. The surface characteristics of fungal treated substrates were observed by SEM, which was conducted with an S-3400N instrument (HITACHI, Japan) at acceleration voltages of 10 kV. Samples were firstly coated with gold-palladium in a sputter coater (E-1010, HITACHI, Japan).
Simultaneous saccharification and fermentation
The SSF experiments were performed under non-sterile conditions in 50 mL Erlenmeyer flasks sealed with a rubber stopper fitted with a one-way air valve to maintain an anaerobic environment. Each fermentation flask was composed of 0.5 g of untreated and fungal-treated substrates and 9 mL of nutrient medium, containing 10 g/L yeast extract and 20 g/L peptone in 50 mM sodium acetate buffer (pH 4.8). The insoluble substrates and the fermentation medium were sterilized at 121°C for 20 min. Then, 30 FPU/g substrate of cellulase (Celluclast 1.5L), 60 IU/g substrate of β-glucosidase (Novozyme 188) and 3 g/L yeast were added. Fermentation was carried out at 40°C for 24 h with shaking at 120 rpm. Aliquots of 0.5 mL were withdrawn and centrifuged at 10000 rpm for 5 min, and the supernatants were subjected to fermentation products analysis. All fermentations were performed in triplicate .
Each sample was filtered through a 0.22 μm filter and diluted appropriately with deionized water. Quantitative analysis for ethanol, glucose and xylose was performed on above HPLC system (1200 series, Agilent Technologies, USA) equipped with a refractive index detector. The separation was achieved using an Aminex HPX-42H column (300×7.8 mm i.d.; Bio-Rad Labs, Richmond, CA, USA) at 65°C with 4 mM H2SO4 as eluent at a flow rate of 0.6 mL/min. The cellulose conversion was calculated using the following formula : 1.
This work was supported by the grants from the Ministry of Science and Technology (973-2010CB732204) and the Fundamental Research Funds for the Central Universities (TD2011-11). Special thanks to Novozyme (China) Investment Co. Ltd. (Beijing) for their generous gift of cellulase and β-glucosidase. We also thank our colleagues for their valuable suggestions during the course of this work.
- Perez J, Munoz-Dorado J, De-la-Rubia T, Martinez J: Biodegradation and biological treatment of cellulose, hemicellulose and lignin: an overview. Int Microbiol 2002, 5: 53-63. 10.1007/s10123-002-0062-3View ArticleGoogle Scholar
- Agbor VB, Cicek N, Sparling R, Berlin A, Levin DB: Biomass pretreatment: fundamentals toward application. Biotechnol Adv 2011, 29: 675-685. 10.1016/j.biotechadv.2011.05.005View ArticleGoogle Scholar
- Wang K, Jiang JX, Xu F, Sun RC: Influence of steaming explosion time on the physic-chemical properties of cellulose from Lespedeza stalks ( Lespedeza crytoborya ). Bioresour Technol 2009, 100: 5288-5294. 10.1016/j.biortech.2009.05.019View ArticleGoogle Scholar
- Yang B, Wyman CE: Effect of xylan and lignin removal by batch and flow through pretreatment on enzymatic digestibility of corn stover cellulose. Biotechnol Bioeng 2004, 86: 88-95. 10.1002/bit.20043View ArticleGoogle Scholar
- Zavrel M, Bross D, Funke M, Buchs J, Spiess AC: High-throughput screening for ionic liquids dissolving (ligno-)cellulose. Bioresour Technol 2009, 100: 2580-2587. 10.1016/j.biortech.2008.11.052View ArticleGoogle Scholar
- Zhu ZG, Sathitsuksanoh N, Vinzant T, Shell DJ, McMillan JD, Zhang Y-HP: Comparitive study of corn stover preteated by dilute acid and cellulose solvent-based lignocellulose fractionation: enzymatic hydrolysis, supramolecular structure, and substrate accessibility. Biotechnol Bioeng 2009, 103: 715-724. 10.1002/bit.22307View ArticleGoogle Scholar
- Kumar P, Barrett DM, Delwiche MJ, Stroeve P: Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind Eng Chem Res 2009, 48: 3713-3729. 10.1021/ie801542gView ArticleGoogle Scholar
- Singh D, Zeng J, Laskar DD, Deobald L, Hiscox WC, Chen S: Investigation of wheat straw biodegradation by Phanerochaete chrysosporium . Biomass Bioenerg 2011, 35: 1030-1040. 10.1016/j.biombioe.2010.11.021View ArticleGoogle Scholar
- Dorado J, Van Beek TA, Claassen FW, Sierra-Alvarez R: Degradation of lipophilic wood extractive constituents in Pinus sylvestris by the white-rot fungi Bjerkandera sp. and Trametes versicolor . Wood Sci Technol 2001, 35: 117-125. 10.1007/s002260000077View ArticleGoogle Scholar
- Martinez D, Larrondo LF, Putnam N, Gelpke MDS, Huang K, Chapman J, Helfenbein KG, Ramaiya P, Detter JC, Larimer F, Coutinho PM, Henrissat B, Berka R, Cullen D, Rokhsar D: Genome sequence of the lignocelluloses degrading fungus Phanerochaete chrysosporium strain RP78. Nat Biotechnol 2004, 22: 695-700. 10.1038/nbt967View ArticleGoogle Scholar
- Singh D, Chen S: The white-rot fungus Phanerochaete chrysosporium: conditions for the production of lignin-degrading enzymes. Appl Microbiol Biotechnol 2008, 81: 399-417. 10.1007/s00253-008-1706-9View ArticleGoogle Scholar
- Tien M, Kirk TK: Lignin-degrading enzyme from Phanerochaete chrysosporium: Purification, characterization, and catalytic properties of a unique H 2 O 2 -requiring oxygenase. Proc Natl Acad Sci U S A 1984, 81: 2280-2284. 10.1073/pnas.81.8.2280View ArticleGoogle Scholar
- Chen F, Dixon RA: Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotechnol 2007, 25: 759-761. 10.1038/nbt1316View ArticleGoogle Scholar
- Dias AA, Freitas GS, Marques GSM, Sampaio A, Fraga IS, Rodrigues MAM, Evtuguin DV, Bezerra RMF: Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi. Bioresour Technol 2010, 101: 6045-6050. 10.1016/j.biortech.2010.02.110View ArticleGoogle Scholar
- Gupta R, Mehta G, Khasa YP, Kuhad RC: Fungal delignification of lignocellulosic biomass improves the saccharification of cellulosics. Biodegradation 2011, 22: 797-804. 10.1007/s10532-010-9404-6View ArticleGoogle Scholar
- Baba Y, Tanabe T, Shirai N, Watanabe T, Honda Y, Watanabe T: Pretreatment of Japanese cedar wood by white rot fungi and ethanolysis for bioethanol production. Biomass Bioenerg 2011, 35: 320-324. 10.1016/j.biombioe.2010.08.040View ArticleGoogle Scholar
- Keller FA, Hamilton JE, Nguyen QA: Microbial pretreatment of biomass: Potential for reducing severity of thermochemical biomass pretreatment. Appl Biochem Biotechnol 2003, 105–108: 27-41.View ArticleGoogle Scholar
- Zhang X, Yu H, Huang H, Liu Y: Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo culms. Int Biodeterior Biodegrad 2007, 60: 159-164. 10.1016/j.ibiod.2007.02.003View ArticleGoogle Scholar
- Fukushima RS, Hatfield RD: Comparison of the acetyl bromide spectrophotometric method with other lignin methods for determining lignin concentration in forage samples. J Agric Food Chem 2004, 52: 3713-3720. 10.1021/jf035497lView ArticleGoogle Scholar
- Zhang L, Li D, Wang L, Wang T, Zhang L, Chen XD, Mao Z: Effect of steam explosion on biodegradation of lignin in wheat straw. Bioresour Technol 2008, 99: 8512-8515. 10.1016/j.biortech.2008.03.028View ArticleGoogle Scholar
- Dinis MJ, Bezerra RMF, Nunes F, Dias AA, Guedes CV, Ferreira LMM, Cone JW, Marques GSM, Barros ARN, Rodrigues MAM: Modification of wheat straw lignin by solid state fermentation with white-rot fungi. Bioresour Technol 2009, 100: 4829-4835. 10.1016/j.biortech.2009.04.036View ArticleGoogle Scholar
- Chua MGS, Wyman M: Characterization of autohydrolysis aspen ( P. tremuloides ) lignin. Part 1. Composition and molecular weight distribution of extracted autohydrolysis lignin. Can J Chem 1979, 57: 1141-1149. 10.1139/v79-187View ArticleGoogle Scholar
- Ke J, Laskar DD, Chen S: Biodegradation of hardwood lignocellulosics by the western poplar clearwing borer, Paranthrene robiniae (Hy. Edwards). Biomacromolecules 2011, 12: 1610-1620. 10.1021/bm2000132View ArticleGoogle Scholar
- Tai D, Terazawa M, Chen CL, Chang HM: Lignin biodegradation products from birch wood by Phanerochaete chrysosporium . Part 1. Fractionation of methanol-extractive and characterization of ether-insoluble low-molecular-weight fraction. Holzforschung 1990, 44: 185-190. 10.1515/hfsg.1922.214.171.124View ArticleGoogle Scholar
- Tai D, Terazawa M, Chen CL, Chang HM: Lignin biodegradation products from birch wood decayed by Phanerochaete chrysosporium . Part 2. The constituents of ether-soluble low-molecular-weight fractions . Holzforschung 1990, 44: 257-262. 10.1515/hfsg.19126.96.36.1997View ArticleGoogle Scholar
- O’Conner RT, DuPer EF, Mitcham D: Application of infrared absorption spectroscopy to investigations of cotton and modified cottons. Part I: physical and crystalline modifications and oxidation. Text Res J 1958, 28: 382-392. 10.1177/004051755802800503View ArticleGoogle Scholar
- Nelson ML, O’Connor RT: Relation of certain infrared bands to cellulose crystallinity and crystal lattice type. Part II. A new infrared ratio for estimation of crystallinity in cellulose I and II. J Appl Polym Sci 1964, 8: 1325-1341. 10.1002/app.1964.070080323View ArticleGoogle Scholar
- Yoon JJ, Kim YK: Degradation of Crystalline Cellulose by the Brown-rot Basidiomycete Fomitopsis palustris . J Microbiol 2005, 43: 487-492.Google Scholar
- Wingren A, Galbe M, Zacchi G: Techno-economic evaluation of producing ethanol from softwood: a comparison of SSF and SHF and identification of bottlenecks. Biotechnol Prog 2003, 19: 1109-1117.View ArticleGoogle Scholar
- Linde M, Jakobsson EL, Galbe M, Zacchi G: Steam pretreatment of dilute H 2 SO 4 -inpregnated wheat straw and SSF with low yeast and enzyme loading for bioethanol production. Biomass Bioenerg 2008, 32: 326-332. 10.1016/j.biombioe.2007.09.013View ArticleGoogle Scholar
- Chang VS, Holtzapple MT: Fundamental factors affecting biomass enzymatic reactivity. Appl Biochem Biotechnol 2000, 84: 5-37. 10.1385/ABAB:84-86:1-9:5View ArticleGoogle Scholar
- Moilanen U, Kellock M, Galkin S, Viikari L: The laccase-catalyzed modification of lignin for enzymatic hydrolysis. Enzyme Microb Technol 2011, 49: 492-498. 10.1016/j.enzmictec.2011.09.012View ArticleGoogle Scholar
- Yang HY, Wang K, Wang W, Xu F, Sun RC: Improved bioconversion of poplar by synergistic treatments with white-rot fungus Trametes velutina D10149 pretreatment and alkaline fractionation. Bioresour Technol 2012. http://dx.doi.org/10.1016/j.biortech.2012.12.103Google Scholar
- Wang K, Yang HY, Guo SH, Tang Y, Jiang JX, Xu F, Sun RC: Organosolv fractionation process with various catalysts for improving bioconversion of triploid poplar. Process Biochem 2012, 47: 1503-1509. 10.1016/j.procbio.2012.06.002View ArticleGoogle Scholar
- Foster CE, Martin TM, Pauly M: Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) Part I: Lignin. J Vis Exp 2010, 37: 1745. 10.3791/1745Google Scholar
- Wang K, Yang HY, Yao X, Xu F, Sun RC: Structural transformation of hemicelluloses and lignin from triploid poplar during acid-pretreatment based biorefinery process. Bioresour Technol 2012, 116: 99-106.View ArticleGoogle Scholar
- Sasaki C, Takada R, Watanabe T, Honda Y, Karita S, Nakamura Y, Watanabe T: Surface carbohydrate analysis and bioethanol production of sugarcane bagasse pretreated with the white rot fungus, Ceriporiopsis subvermispora and microwave hydrothermolysis. Bioresour Technol 2011, 102: 9942-9946. 10.1016/j.biortech.2011.07.027View ArticleGoogle Scholar
- Dowe N, McMillan J: SSF experimental protocols — lignocellulosic biomasshydrolysis and fermentation. National Renewable Energy Laboratory: Laboratory Analytical Procedure (LAP); 2008. http://www.nrel.gov/docs/gen/fy13/42618.pdfGoogle Scholar
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