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.
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.
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