Process evaluation of electron beam irradiation-based biodegradation relevant to lignocellulose bioconversion
© Bak; licensee Springer. 2014
Received: 1 July 2014
Accepted: 26 August 2014
Published: 29 August 2014
In order to solve the inefficient problem of long-term biodegradation by wood-decaying fungus, rice straw (RS) was depolymerized using electron beam irradiation-based biodegradation (EBIBB). This environment-friendly program without the use of inhibitory byproducts significantly increased the digestibility and fermentability of RS. Specifically, when irradiated RS was simultaneously biodegraded by Phanerochaete chrysosporium for 10 days, the sugar yield was 65.5% of the theoretical maximum. This value was on the same level as the 64.8% (for 15 days) measured from unirradiated RS. In case of fermentability, similarly, EBIBB program had an effect on time/energy saving. Furthermore, the transcriptomic profiles under different biosystem were analyzed in order to verify possible substrate-specific regulation based on change of lignocellulosic components. Interestingly, the overall correlation based on the bias (upregulation or downregulation) was reasonably analogous, especially lignocellulolysis-related genes.
Fuel bioethanol from lignocellulosic plant biomass is being explored as an alternative energy source due to exhaustion of oil resources and environmental concerns, especially global warming. To commercialize bioethanol process, the effective pretreatment of the biomass are essential, due to the inaccessibility from cellulose crystallinity, in the bioconversion of recalcitrant substrates into fermentable sugars (Sanderson, 2011). Recent trends of pretreatment process have been studied on environmentally friendly biodegradations using wood-rotting fungi instead of general processes using physicochemical tools and evaluated by various indexes (Menon and Rao, 2012; Wan and Li, 2012). However, the use of only biodegradation to enhance the hydrolysis yield of lignocellulosic substrates has not been sufficient for commercial programs yet. More importantly, it is hard for useful programs to hydrolyze the substrates due to the inevitable necessity of long-term treatment.
Electron beam technology have been broadly studied to extend the range of applications in the properties of the polymeric materials (Hamm and Hamm, 2012). Particularly, the mechanism of chain scission (by electron attacks) focus on change (or degradation) in structural crystallinity of substrates (e.g., lignocellulose; Bak, 2014b). Therefore, to address the weak points in the fungal biodegradation, such as the low yield and the long-term process, an irradiation-treated substrate was used in this biodegradation program. This study was conducted to verify the feasibility and efficiency of electron beam irradiation-based biodegradation (EBIBB) program. Its impact was evaluated based on various bioprocessing properties of pretreated substrate, such as digestibility yield and fermentation efficiency. Furthermore, in order to understand the mainstream of fungal lignocellulolytic system in EBIBB program, the pattern of gene expression profiles was analyzed using whole genome microarray-based approach at transcriptome level.
Materials and methods
Electron beam irradiation-based biodegradation system
Rice straw (RS), harvested from Korea University Farm (Deokso, Korea), was used as the lignocellulose model compound. After the preprocessing procedures (Supporting Information), processed RS was used as the starter substrate for the fungal biodegradation. Prior to the biodegradation, RS was irradiated by using a linear electron accelerator (Korea Atomic Energy Research Institute, Daejeon, Korea) in order to enhance the effects of substrate pretreatment. The optimized condition (1 Mev and 80 kGy at 0.12 mA) of irradiation was based on a previously reported methodology (Bak et al., 2009b).
Next, based on previously optimized fungal cultivation (Bak et al., 2009a), after the addition of irradiated RS (4.4 g), Phanerochaete chrysosporium (ATCC 32629) was cultured in 200 mL of optimized medium containing 1% (w/v) of glucose (as an initial carbon source) at 29°C and 150 rpm for 15 days. No substrate was added to the control cultures. Further details are provided in Additional file 1.
Simultaneously, the concentration of inhibitory byproducts (hydroxymethylfurfural and furfural) and theoretical maximum yields (digestibility and fermentability) of the EBIBB-pretreated RS were analyzed following the public biomass analytical protocols (http://www.nrel.gov/biomass/capabilities.html). Further details are provided in Additional file 1. At the same time, according to the public biomass protocols, the change of 3 components (lignin, cellulose, and hemicellulose) of RS were confirmed based on a dry weight basis. Based on generally accepted methods (Additional file 1), the extracellular activities of well-known enzymes involved in lignocellulose degradation were assayed during the biodegradation. Further details are provided in Additional file 1. All experiments were conducted in triplicate.
After EBIBB pretreatment, the microstructural changes of substrates was determined using a Hitachi S-4700 scanning electron microscope (Tokyo, Japan). Especially, diffraction spectra of substrates was performed with a powder X-ray diffractometer (Bruker D5005, Karlsruhe, Germany) to identify crystallinity index on EBIBB-pretreated RS. The signals were analyzed in triplicate using the previously confirmed θ-2θ method (Bak et al., 2009b).
Under different biodegradation condition (whether irradiation-based system or not), the complementary relationship between the lignocellulolytic targets and % theoretical yields was analyzed by transcriptomic expression analysis. After six biological replicates of the biodegradations, cDNA hybridization of targets was performed with Custom Array 12 K microarray (CombiMatrix Corporation, Mukilteo, WA). The significance of the array was confirmed with the quantitative real-time PCR data. Further details are provided in Additional file 1. After the data processing (Additional file 1), hierarchical clustering was performed to reorganize genes into functional categories (Eisen et al., 1998). In order to graphically present the genetic expression, PermutMatrix ver. 1.9.3 software (http://www.atgc-montpellier.fr/permutmatrix/) was used in this study (Caraux and Pinloche, 2005).
Results and discussion
Theoretical yields of EBIBB system
Downstream evaluation for scale-up in advanced EBIBB program
Hydrolysis indexd(per 100 g biomass)
Fermentation indexe(per 100 g biomass)
EBIBB for 5 days
EBIBB for 10 days
EBIBB for 15 days
EBIBB after 10 days
≤ 55.2% (17.3 ± 0.2 g glucose)
≤ 65.5% (20.7 ± 0.1 g glucose)
≤ 61.2% (19.1 ± 0.2 g glucose)
≤ 62.9% (10.0 ± 0.2 g ethanol) at 10 day
≤ 48.3% (15.4 ± 0.2 g glucose)
≤ 58.4% (18.7 ± 0.2 g glucose)
≤ 64.8% (20.8 ± 0.2 g glucose)
≤ 62.5% (9.9 ± 0.2 g ethanol) at 15 day
≤ 27.1%b (8.7 ± 0.1 g glucose)
≤ 29.5%b (4.7 ± 0.1 g ethanol)
Overall, the hydrolysis yield of EBIBB program, which is reflected in fermentable sugars, was lower than those of plant biomass (70-85%) pretreated using conventional chemical program (Kim et al., 2002; Ko et al., 2009). Furthermore, the ethanol productivity (0.52 g/L/h) of chemical programs (especially ammonia-soaking treatment) are greater than the productivity (0.40 g/L/h) observed in the present study. However, we speculate that this program is superior to others, especially alkaline (30-80%) and fungal-based program (<50% after 14 days), in terms of the% yield and time effectiveness (Keller et al., 2003; Shi et al., 2009; Zhao et al., 2008). Additionally, the fermentability (<0.03 g ethanol/g lignocellulosic biomass) in previous P. chrysosporium-based system was finally obtained after 144 h of fermentation (Shi et al., 2009; Shrestha et al. 2008), which was not more than the EBIBB-level (0.10 g ethanol/g RS after 72 h). More importantly, unlike previous system, the EBIBB approach secured a bridgehead for energy/time saving in conventional competitiveness.
Change of lignocellulosic structure
Unlike the smooth structure of untreated lignocellulosic surfaces, EBIBB-pretreated surfaces had randomly degraded cracks and non-spherical protrusions (Figure 2). We speculate that the exposure of crystalline structures may be further accelerated by inducible cell-wall disruption due to EBI-based preprocess. However, when compared to non-EBI biodegradation (Bak et al., 2009a), the change in the crystalline (or amorphous) structures were hard to distinguish by EBIBB within the significant difference.
Analysis of the main components of RS following EBIBB pretreatment
Total external substratec (dry wt. basis)
Change of RS components
Lignin (g lignin/L) Before/After
Cellulose (g glucan/L) Before/After
Hemicellulose (g xylan/L) Before/After
EBIBB (at 10 days)
21.5 g RSd (97.7%)
4.1 g/≤ 2.6 g
7.8 g/≤ 6.1 g
2.3 g/≤ 1.4 g
NCa (at 15 days)
22.0 g RS (100.0%)
4.4 g/≤ 3.4 g
7.9 g/≤ 6.5 g
2.4 g/≤ 1.9 g
22.0 g RS (100.0%)
4.4 g/4.4 g
7.9 g/7.9 g
2.4 g/2.4 g
Transcriptomic evaluation of irradiation-based fungal biosystem
In the biodegradation system of lignocellulosic biomass, cooperation and harmony of genetic factors is an indispensable feature for evolutionary survival tactic (Cullen and Kersten, 2004). Furthermore, it means that the effective yields of biodegradation may well have involved the systematic regulation of upstream signals (especially lignocellulolytic genes).
Extracellular activity of well-known linocellulolytic targets in optimal EBIBB system
Ligninolytic enzymes (U/L)
Cellulolytic enzymes (U/L)
Ligninolytic oxalate (g/L)
EBIBB (at 10 days)
NCa (at 15 days)
Based on the results of above mentioned similarity (here transcriptomic expression), we can predict that the abundance (or presence) of opened (or modified) biodegradable substrates (by directly oxidative attack of electrons) is a key to the understanding of P. chrysosporium metabolism. In other words, an important determinant of mainstream (or substream) in fungal biodegradation mechanism is really a matter of substrate style (structure and component; Figure 2 and Table 2) rather than just recalcitrant substrate. Furthermore, we confirmed that the combined program containing the irradiation treatment help to enhance the functional metabolic uniformity in the bioconversion process (or regulatory network).
Based on mass balance, the EBIBB-pretreated RS after 10 days showed significant increases in industrial yields compared to the untreated RS. Particularly, the reduction of a lengthy time in advanced EBIBB-program had a strong advantage in downstream bioprocess. Although the production yields of this program was lower than those of substrate pretreated by physicochemical programs, the inhibitory byproducts was rarely generated. Microfibril composition analysis revealed that physical (or chemical) changes in substrate surfaces were likely a result of EBIBB. Lastly, the profiling of intracellular genes involved in lignocellulolytic cascades during the optimal EBIBB-treatment could help the understanding of mainstream system.
This work was supported by the Ministry of Education, Science and Technology, Republic of Korea.
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