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Fungal pretreatment of non-sterile miscanthus for enhanced enzymatic hydrolysis
Bioresource Technology 203 (2016) 118–123
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Fungal pretreatment of non-sterile miscanthus for enhanced enzymatic hydrolysis Juliana Vasco-Correa, Xumeng Ge, Yebo Li ⇑ Department of Food, Agricultural and Biological Engineering, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Ave., Wooster, OH 44691-4096, USA
h i g h l i g h t s Successful fungal pretreatment of non-sterile miscanthus by white rot fungus. Inoculation with at least 30% pre-colonized miscanthus was crucial for the success. Enzymatic digestibility and glucose yield was 3–4 times higher than raw miscanthus. Comparable glucose yield to that of fungal pretreated miscanthus with sterilization.
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Article history: Received 30 October 2015 Received in revised form 3 December 2015 Accepted 9 December 2015 Available online 14 December 2015 Keywords: Miscanthus Fungal pretreatment Sterilization Ceriporiopsis subvermispora White rot fungi
a b s t r a c t Miscanthus was pretreated with the fungus Ceriporiopsis subvermispora under non-sterile conditions, using sterile miscanthus that had been previously colonized with the fungus as the inoculum. Inoculum ratios equal to or greater than 30% yielded a successful pretreatment, enhancing the enzymatic digestibility of miscanthus by 3- to 4-fold over that of raw miscanthus, which was comparable with the fungal pretreatment under sterile conditions. This enhanced digestibility was linearly correlated with lignin degradation. Although cellulose loss of up to 13% was observed for the successful non-sterile pretreatments, the final glucose yield was 3–4 times higher than that of raw miscanthus and comparable to that of the sterile pretreated miscanthus. A time course study showed that maximum glucose yield can be achieved with a pretreatment time of 21 days. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The increasing need for alternative renewable sources of energy has prompted the development of biomass-based biorefineries with the potential of replacing a significant portion of the petroleum that is currently used for the production of fuels and chemicals (Cherubini, 2010). Biorefineries’ success depends on the availability of renewable, consistent, high volume feedstock supplies, which can be provided by dedicated energy crops, such as switchgrass or miscanthus (U.S. Department of Energy, 2011). Miscanthus is a tall perennial grass that has high annual biomass yields, low nutrients and water requirements, and the ability to adapt to a wide variety of climate and soil conditions (Jones and Walsh, 2001). Therefore, miscanthus has been identified as one of the most promising energy crops (Heaton et al., 2010).
⇑ Corresponding author. Tel.: +1 330 263 3855; fax: +1 330 263 3670. E-mail address: [email protected] (Y. Li). http://dx.doi.org/10.1016/j.biortech.2015.12.018 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Miscanthus, a lignocellulosic biomass, is mainly composed of cellulose, hemicellulose, and lignin. Biological transformation of lignocellulosics into bioenergy utilizes only the sugars present in the cellulose (and sometimes in the hemicellulose) for the production of biofuels, while the presence of lignin is undesirable (Zeng et al., 2014). Cellulose and hemicellulose are polysaccharides composed of 5- and 6-carbon sugars that can be fermented by microorganisms for the production of biofuels and bioproducts. Lignin is an aromatic heteropolymer that protects the polysaccharides from microbial degradation, besides giving support and stiffness to the plant structure (Hendriks and Zeeman, 2009). Miscanthus has a recalcitrant structure and pretreatment is required to enhance the accessibility of cellulose to hydrolytic enzymes for sugar release (Brosse, 2009). Commonly used pretreatment methods often employ strong chemicals and/or high temperatures, and require large amounts of water (Kim, 2013). Alternatively, biological pretreatment exploits the ligninolytic capacity of some microorganisms (fungi and bacteria) to reduce the recalcitrance of the lignocellulose (Chen et al., 2010). For fungal
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pretreatment, rotting fungi, such as Ceriporiopsis subvermispora, are grown in the lignocellulosic biomass to breakdown lignin, resulting in a feedstock that is easier to digest (Wan and Li, 2012). Compared to other pretreatment methods, fungal pretreatment can be operated at room temperature with minimal use of water and chemicals, and without generation of waste streams (Saritha et al., 2012). Fungal pretreatment is usually performed via solid-state cultivation of the fungus in the feedstock. But because fungi cannot use lignin as a sole carbon and energy source, parts of the cellulose and/or hemicellulose are also degraded during this process, which is detrimental for the purpose of pretreatment (Sánchez, 2009). C. subvermispora is a white rot basidiomycetes that degrades lignin selectively over cellulose. It has been used to successfully pretreat lignocellulosic biomass, resulting in significant lignin degradation and enhanced enzymatic digestibility. Although some parts of the hemicellulose are also degraded, most of the cellulose remains intact (Wan and Li, 2011). Glucose yields between 24% and 69% have been obtained for fungal pretreatment with C. subvermispora of feedstocks such as hardwood, corn stover, and switchgrass, but this pretreatment has been unsuccessful with other feedstocks such as soybean and wheat straw (Cianchetta et al., 2014; Ge et al., 2015; Salvachúa et al., 2011; Wan and Li, 2010, 2011). Sterilization of the feedstock, a costly step, is usually required for fungal pretreatment, because the indigenous microorganisms may outcompete the introduced fungi. C. subvermispora has been reported to be especially sensitive to the presence of other microorganisms (Akin et al., 1995). A promising alternative strategy is to grow the fungus on sterile feedstock first, and then use the colonized feedstock as an inoculum for fungal pretreatment of non-sterile feedstock (Zhao et al., 2014). However, this strategy has only been studied in woody biomass, and it is not clear how it would behave in herbaceous crops, such as miscanthus. White rot fungi naturally colonizes wood, but it has been shown that its growth in herbaceous biomass can be more difficult (Vasco-Correa and Li, 2015; Wan and Li, 2011), probably because of differences in the cell wall structure and composition. However, herbaceous biomass has more potential as a bioenergy feedstock because it is less recalcitrant and grows faster than wood, producing higher biomass yields (Wan and Li, 2011). It is unknown how much previously colonized feedstock would be needed as an inoculum for successful pretreatment of herbaceous biomass, how the moisture content of the feedstock–inoculum mixture would affect this process, and how this strategy would affect the pretreatment time. Therefore, systematic research on using this strategy for fungal pretreatment of non-sterile herbaceous crops is needed. In this study, fungal pretreatment of non-sterile miscanthus, using miscanthus colonized with C. subvermispora as the inoculum, was evaluated. Effects of inoculum ratio and moisture content on glucose yield, enzymatic digestibility, lignin degradation, and sugar loss were investigated. Also, a time course study of the fungal pretreatment process was performed, monitoring the degradation of different components during 28 days and effects on the glucose yield.
2. Methods
Table 1 Characteristics of miscanthus.
Total solids (%) Extractivesa (%) Cellulosea (%) Hemicellulosea (%) Lignina (%) a
Raw miscanthus
Inoculum (fungal colonized miscanthus)
93.8 ± 0.5 8.9 ± 0.2 38.0 ± 0.2 18.5 ± 0.4 20.9 ± 0.2
47.9 ± 0.5 15.33 ± 0.1 43.6 ± 0.9 17.4 ± 0.3 17.1 ± 0.2
Based on total solids.
2.2. Inoculum preparation Ceriporiopsis subversmipora (ATCC 96608) was obtained from the American Type Culture Collection (Manassas, VA, USA) and kept on 2% malt extract agar at 4 °C. Sterile miscanthus colonized with C. subversmipora was used as the inoculum for the fungal pretreatment experiments. To prepare this inoculum, 130 g (dry basis) of raw miscanthus were placed in 2 L reactors, which were supplemented with deionized water to adjust moisture to 60%, and then covered with cotton. Reactors were autoclaved (121 °C, 15 min) and cooled down. Then, C. subversmipora mycelium, which was grown in 50 ml of 2% malt extract liquid medium (7 days, 28 °C, static conditions), was added to each reactor and then incubated for 28 days at 28 °C. At the end, the fungal-colonized miscanthus was taken out of the flask and mixed well before using it as inoculum for the successive fungal pretreatment of non-sterile miscanthus. Composition of the inoculum is shown in Table 1. 2.3. Fungal pretreatment of non-sterile miscanthus Non-sterile raw miscanthus and inoculum (fungal-colonized miscanthus) were mixed in different proportions and added to 1 L reactors. Deionized water was added to adjust moisture content and reactors were incubated at 28 °C. First, a full factorial design with three replicates was performed, with the following factors: inoculum ratio (0%, 10%, 20%, 30%, 40%, and 50%, dry basis) and moisture content (60% and 75%). Reactors were incubated for 28 days. The inoculum ratio 0% (no inoculation) was considered to be the negative control. The positive control was sterile miscanthus inoculated with mycelium (as described in Section 2.2), which was incubated along with the treatments. At the end of the 28 days, samples were taken out of the reactors and mixed well. Part of the fresh material was used for enzymatic hydrolysis and the rest was dried at 40 °C in a convection oven for 24 h, then milled and passed through a 1 mm screen (Model 4 Wiley Mill, Thomas Scientific, Swedesboro, NJ, USA) for subsequent cellulose, hemicellulose, and lignin content determination. Second, an experiment to evaluate the degradation over time of components of miscanthus during fungal pretreatment was performed at 50% inoculum ratio and 60% moisture content. Reactors were prepared in the same manner as in the first experiment and stopped at different times (0, 7, 14, 21, and 28 days). At the end of the specific incubation time, samples were processed as in the first experiment, for subsequent enzymatic hydrolysis, and cellulose, hemicellulose, and lignin content determination.
2.1. Feedstock collection and storage 2.4. Enzymatic hydrolysis Miscanthus giganteus (hereafter miscanthus) was harvested from a field in Ashtabula, OH, in spring 2013. The moisture content was around 6% and its composition is shown in Table 1. The feedstock was milled to pass through a 12 mm screen using a hammer mill (The C.S Bell Co., Tiffin, OH, USA) and stored under dry conditions.
Enzymatic digestibility of raw and pretreated miscanthus was determined according to Selig et al., using the enzyme mix Cellic CTec2 (Novozymes, Bagsværd, Denmark), at a cellulase concentration of 10 FPU/g dry substrate (Selig et al., 2008). About 2.5 g (dry basis) of feedstock were added to 250 ml flasks along with citrate
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%Enzymatic Digestibility ¼
%Glucose yield ¼
½Glucose þ 1:053½Cellobiose 100% 1:111f p ½Biomass
½Glucose þ 1:053½Cellobiose 100% 1:111f r ½Biomass
where [Glucose] and [Cellobiose] are the residual glucose and cellobiose concentrations (g/L), [Biomass] is the dry biomass concentration at the beginning of the enzymatic hydrolysis (g/L), and fp and fr are the fraction of cellulose in the pretreated and original (raw) dry biomass (g/g), respectively (Wang et al., 2014). Thus, the sugar yield accounted for the cellulose loss that could occur during the fungal pretreatment. 2.5. Analytical methods Cellulose, hemicellulose, and lignin contents were determined according to the NREL protocol (Sluiter et al., 2012), using a twostep acid hydrolysis. Sugars liberated after the acid hydrolysis were quantified by HPLC (LC-20AB, Shimadzu, Kyoto, Japan) with an Aminex HPX-87P column (Bio-Rad, Inc., Hercules, CA, USA) and a refractive index detector. Acid soluble lignin was determined by UV–vis spectroscopy (BioMate 3, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and acid-insoluble lignin was quantified gravimetrically. Dry matter was determined gravimetrically, by drying triplicate samples of the miscanthus at 105 °C. Cellulose, hemicellulose, lignin, and dry matter degradations were calculated based on that of the initial mixture (raw material plus inoculum). To measure the miscanthus extractives, an Accelerated Solvent Extraction (ASE 350, Thermo Fisher Scientific, Inc., Waltham, MA, USA) with water and ethanol was used, following the NREL analytical procedure (Sluiter et al., 2008). Then, the solvents were removed from the extract by evaporation (Rocket Evaporator, Genevac, Ipswich, England), and the evaporated extract was dried at 40 °C before measurement of the dry weight. 2.6. Data analysis Statistical significance was evaluated by analysis of variance (ANOVA) (a = 0.05), and mean comparisons were performed by Tukey–Kramer test with a 95% significance, using the Software JMPÒ. Error bars in the figures represent the standard error. 3. Results and discussion 3.1. Cellulose, hemicellulose, and dry matter loss Cellulose, hemicellulose, and dry matter losses after 28 days of the fungal pretreatment of non-sterile miscanthus strongly depended on the inoculum ratio, but not on the moisture content, for the conditions evaluated (Fig. 1). Besides, the losses of cellulose (5–18%), hemicellulose (3–24%), and dry matter (4–18%) were comparable with those obtained previously in fungal pretreatment of non-sterile yard trimmings (Zhao et al., 2014).
(a)
40%
Cellulose loss (%)
35%
Moisture 60% Moisture 75%
30% 25% 20% 15% 10% 5% 0% 0
(b)
40% 35%
Hemicellulose loss (%)
buffer (0.05 M, pH 5.0), 1 ml of 2% sodium azide solution, and the enzyme, for a total working volume of 100 ml. Enzymatic hydrolysis was performed at 50 °C and a pH of 5.0 for 72 h, and then the hydrolysate was centrifuged (12,000 g, 10 min), and the supernatant was passed through a 0.2 lm nylon filter into glass vials. Free sugars were analyzed by high-performance liquid chromatography (HPLC) (LC-20AB, Shimadzu, Kyoto, Japan) with an Aminex HPX-87P column (Bio-Rad, Inc., Hercules, CA, USA) and a refractive index detector. Enzymatic digestibility and glucose yield were calculated as follow:
10
20 30 40 Inoculum ratio (%)
50
+
50
+
50
+
Moisture 60% Moisture 75%
30% 25% 20% 15% 10% 5% 0% 0
(c)
40% 35%
Dry matter loss (%)
120
10
20 30 40 Inoculum ratio (%)
Moisture 60% Moisture 75%
30% 25% 20% 15% 10% 5% 0% 0
10
20 30 40 Inoculum ratio (%)
Fig. 1. Components loss after 28 days of fungal pretreatment of miscanthus. (a) Percentage of cellulose loss. (b) Percentage of hemicellulose loss. (c) Percentage of dry matter loss. ‘‘+”: positive control (sterilized miscanthus).
Cellulose loss was between 7% and 13% for non-sterile pretreatments with inoculum ratios of 30–50%, which was comparable with that in the miscanthus incubated without inoculation (0%). On the contrary, cellulose loss was negligible for the pretreated sterile miscanthus (positive control) (Fig. 1a). This suggests that the cellulose loss in the non-sterile pretreatment was due to the presence of other microorganisms (contaminants) that prefer cellulose as carbon and energy sources. Cellulose is the main source of sugars for the subsequent processes and its degradation during the pretreatment is undesirable. However, the cellulose loss in the non-sterile pretreatment was still similar to those observed for other pretreatment methods. For example, alkaline pretreatment of miscanthus with 1 M NaOH at 150 °C for 60 min caused a cellulose loss of about 20% (Cha et al., 2014). Aqueous ammonia pretreatment showed a cellulose loss of 4–12% (Liu et al., 2013). Organosolv pretreatment with formic and acetic acid led to a cellulose loss of about 0–25%, depending on pretreatment temperature, time, and solvent concentration (Vanderghem et al., 2012). The hemicellulose loss was probably mainly caused by the growth of C. subvermispora, which uses these sugars as its main carbon source (Wan, 2011), but other microorganisms present in the reactor could also have consumed it at a small rate, as can be
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3.2. Lignin degradation and enzymatic digestibility Lignin loss was almost negligible for pretreatments with inoculum ratios of 10% and 20%, as well as for the miscanthus without inoculation. On the other hand, lignin loss of around 25–35% was achieved with inoculum ratios of 30–50%, as well as with the sterilized miscanthus (Fig. 2a), which was significantly higher than that obtained using a similar pretreatment strategy for nonsterile yard trimmings (Zhao et al., 2014). An analysis of variance indicated that both inoculum ratio and moisture content had a significant effect (p < 0.05) on lignin degradation (Fig. 2a). At inoculum ratios between 30% and 50%, higher lignin degradation was observed at a moisture content of 75%.
Lignin degradation (%)
(a) 40%
Moisture 60% Moisture 75%
30%
20%
10%
0% 0
Enzymatic digestibility (%)
(b) 60%
10
20 30 40 Inoculum ratio (%)
50
+
50
+
Moisture 60% Moisture 75%
50% 40% 30% 20% 10% 0%
0
(c) Enzymatic digestibility (%)
seen in the hemicellulose degradation in the miscanthus incubated without inoculation (negative control) (Fig. 1b). There was a significant difference between the hemicellulose loss in the pretreated sterilized miscanthus (positive control) and non-sterilized miscanthus with inoculum ratios of 30–50% (p < 0.05). That difference was equivalent to the hemicellulose loss obtained in the miscanthus without inoculation, which suggests that the higher degradation in the non-sterile treatments compared to sterilized miscanthus was due to hemicellulose consumption by other microorganisms present in the reactor. Hemicellulose loss can be beneficial to enhance cellulose digestibility, but if the subsequent fermentation processes use microorganisms that are able to utilize xylan, hemicellulose degradation is detrimental (Alvira et al., 2010). Hemicellulose degradation for the successful non-sterile pretreatment was between 16% and 24%, while the hemicellulose loss in the sterile pretreatment was about 16–17%. These values were still smaller than those obtained with other pretreatments. Alkaline pretreatment usually degrades a large percentage of the hemicellulose. For example, miscanthus treated with 1 M NaOH at 150 °C for 60 min resulted in a hemicellulose loss of about 79% (Cha et al., 2014). Aqueous ammonia pretreatment of miscanthus caused a hemicellulose loss of 23–61% (Liu et al., 2013). Organosolv pretreatment with formic/acetic acid led to a loss of hemicellulose of 9–79%, depending on the pretreatment temperature, time, and solvent concentration (Vanderghem et al., 2012). Autohydrolysis of miscanthus (pretreatment with water under pressure at 130– 150 °C) generated a hemicellulose loss of about 50%, with no cellulose or lignin degradation (El Hage et al., 2010). Dry matter loss was 13–18% for the non-sterile pretreatments with inoculum ratios of 30–50%, which was significantly higher than that of the sterilized miscanthus (7–9%) and the miscanthus without inoculation (4–7%) (Fig. 1c). For the non-sterile pretreatments with 30–50% inoculum ratios, 40–50% of the dry matter loss was due to lignin degradation, and the rest was equally distributed between cellulose and hemicellulose loss. For the sterile pretreatment, about 70–80% of the dry matter loss was due to lignin degradation, and the rest was hemicellulose loss because, as mentioned previously, cellulose loss was negligible. For the miscanthus without inoculation, 70–80% of the dry matter loss was due to loss in cellulose and the rest to hemicellulose loss, with practically no lignin degraded in this case. Therefore, the difference in dry matter loss between the non-sterile pretreatments with 30–50% inoculum ratios and the sterile pretreatment (positive control) was due primarily to the loss in cellulose in the former. The main disparity between the non-sterile pretreatments with 30–50% inoculum ratios and the miscanthus without inoculation (negative control) was due to the absence of lignin degradation in the latter. For the non-sterile pretreatments with 10% and 20% inoculum ratios, the dry matter loss proportions were similar to those of the miscanthus without inoculation, since the lignin degradation was very low in these cases (0–5%).
10
20 30 40 Inoculum ratio (%)
60% 50%
R² = 0.963
40% 30% 20% 10% 0% 0%
10% 20% 30% Lignin degradation (%)
40%
Fig. 2. (a) Enzymatic digestibility of fungal pretreated miscanthus after 28 days. (b) Percentage of lignin loss after 28 days of fungal pretreatment of miscanthus. (c) Correlation between lignin loss and enzymatic digestibility of fungal pretreated miscanthus after 28 days. ‘‘+”: positive control (sterilized miscanthus).
The enzymatic digestibility was significantly affected by the inoculum ratio (p < 0.05), but not by the moisture content (p > 0.05), according to the analysis of variance (Fig. 2b). This suggests that moisture content can vary between 60% and 75% without significantly affecting enzymatic digestibility, which is advantageous because moisture content can be a parameter difficult to control in solid-state fermentation (Robinson et al., 2001). The enzymatic digestibility and lignin degradation of the reactors with inoculum ratios of 30%, 40%, and 50% were comparable with those of the sterilized miscanthus, showing that at an inoculum ratio between 30% and 50%, fungal pretreatment of non-sterile miscanthus was feasible. The enzymatic digestibility and lignin degradation of miscanthus with inoculum ratios of 10% and 20% were equivalent to those of the miscanthus without inoculation (0% inoculum ratio) and the raw miscanthus, which had an enzymatic digestibility of about 12% (Fig. 2a and b). This suggests that the pretreatment was unsuccessful for inoculum ratios less than 20%. When the inoculum ratio was increased to 30–50%, a 2-fold increase in enzymatic digestibility was obtained compared with the initial mixture, which was equivalent to a 3- to 4-fold increase compared to the raw miscanthus. These results were similar to
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maintenance. Glucose yield generally increased with the increase of lignin degradation, especially in the first 7 days when half of the lignin was degraded. Wan and Li (2010) studied the time course of fungal pretreatment of corn stover during 42 days of incubation under sterile conditions. In that case, most of the lignin degradation occurred during the first 15 days, and then the degradation rate decreased significantly, which was not seen in the present case. However, hemicellulose and dry matter loss occurred continuously during the pretreatment of sterile corn stover, similar to the non-sterile miscanthus. Glucose yield showed a similar tendency for sterile corn stover and non-sterile miscanthus, with a high initial increase and a later stationary phase. The main difference was that the glucose yield curve for sterile corn stover showed a lag phase of about 7 days, which was not present in that of the non-sterile miscanthus. This difference was probably due to the inoculum. For the miscanthus, the inoculum probably had a higher concentration of the fungus and the fungus was better adapted to the feedstock, which could have shortened the lag phase.
those obtained by fungal pretreatment of sterile switchgrass in previous research (Wan and Li, 2011). Enzymatic digestibility was also analogous to that obtained after a 28-days fungal pretreatment of non-sterile corn stover with Irpex lacteus, even though this feedstock underwent a disinfection process with lime prior to fungal inoculation (Song et al., 2013). Strong correlation between lignin loss and enzymatic digestibility was observed (Fig. 2c), suggesting that lignin was probably one of the main factors that reduced the accessibility of structural sugars to the enzyme, as described by other authors (Chen et al., 2010; Wan, 2011), and that its removal positively and directly contributed to enhancement of enzymatic digestibility. This result confirmed that the main effect of fungal pretreatment on the lignocellulosic structure is lignin degradation, which makes the cellulose more accessible to hydrolytic enzymes (Saritha et al., 2012). Glucose yield for effective inoculum ratios (30%, 40%, and 50%) ranged between 35% and 48%, which represented an increase of 3to 4-fold, compared with the raw miscanthus. Compared with the initial mixture of inoculum (fungal colonized miscanthus) and raw miscanthus, the glucose yield was increased by a factor of 2. The glucose yields for sterilized miscanthus (positive control) were between 35% and 39%, which was comparable with the effective pretreatments with inoculum ratios of 30–50%. These results suggest that even though cellulose degradation during fungal pretreatment of non-sterile miscanthus was higher than that with sterilized miscanthus, the overall glucose yield was not affected drastically. Fungal pretreatment of miscanthus could be performed under non-sterile conditions, yielding similar results as sterilized miscanthus, using the process described in this study.
4. Conclusions Fungal pretreatment of non-sterile miscanthus achieved 3- to 4-fold increases in enzymatic digestibility and glucose yield, compared to the raw feedstock. This pretreatment can be performed in 21 days, using 30–50% fungal colonized miscanthus as the inoculum and a moisture content of 60–75%, to obtain a glucose yield comparable with that of fungal pretreatment of sterile miscanthus, despite having a higher cellulose loss. Sterilization was still required for preparing the inoculum that was used for this single batch pretreatment of non-sterile miscanthus. Future research would explore consecutive batches of fungal pretreatment in order to further reduce the cost.
3.3. Time course of the non-sterile fungal pretreatment During solid-state fungal pretreatment of non-sterile miscanthus with 50% inoculum, the glucose yield increased significantly until day 21. There was no significant increment in the glucose yield from day 21 to day 28 (p > 0.05) (Fig. 3). There was also no significant cellulose loss between those dates; in fact, all the cellulose degradation seemed to occur at the beginning of the process (between day 0 and 7) and it did not increase after that. Therefore, the pretreatment could be reduced to 21 days without affecting the glucose yield, while enhancing the productivity of the process. In contrast, there was continuous degradation of hemicellulose throughout the process, which suggests that the hemicellulose was acting as a carbon and energy source for fungal growth and
35%
This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2012-10008-20302. The authors want to thank Fulbright-Colombia for providing funding for the doctoral student Juliana Vasco-Correa and Mrs. Mary Wicks (Department of Food, Agricultural and Biological Engineering, OSU) for reading through the manuscript and providing useful suggestions.
45%
Cellulose loss Hemicellulose loss Lignin loss Dry matter loss Glucose yield
30%
40% 35%
25% 30% 20%
25%
15%
20% 15%
Glucose yield (%)
Component loss (%)
Acknowledgements
10% 10% 5%
5%
0%
0% 0
5
10
15
20
25
30
Time (days) Fig. 3. Time course study of fungal pretreatment of non-sterile miscanthus during 28 days at 50% inoculum ratio and 60% moisture content.
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Sánchez, C., 2009. Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnol. Adv. 27, 185–194. Saritha, M., Arora, A., Lata, 2012. Biological pretreatment of lignocellulosic substrates for enhanced delignification and enzymatic digestibility. Indian J. Microbiol. 52, 122–130. Selig, M., Weiss, N., Ji, Y., 2008. Enzymatic Saccharification of Lignocellulosic Biomass. Laboratory Analytical Procedure (LAP), Golden, Colorado. Sluiter, A., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008. Determination of Extractives in Biomass. Laboratory Analytical Procedure (LAP), Golden, Colorado. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2012. Determination of Structural Carbohydrates and Lignin in Biomass. Laboratory Analytical Procedure (LAP), Golden, Colorado. Song, L.L., Yu, H.B., Ma, F.Y., Zhang, X.Y., 2013. Biological pretreatment under nonsterile conditions for enzymatic hydrolysis of corn stover. Bioresources 8, 3802– 3816. U.S. Department of Energy, 2011. U.S. Billion-Ton Update: Biomass Supply for a Bioenergy and Bioproducts Industry. Oak Ridge National Laboratory, Oak Ridge. Vanderghem, C., Brostaux, Y., Jacquet, N., Blecker, C., Paquot, M., 2012. Optimization of formic/acetic acid delignification of Miscanthusgiganteus for enzymatic hydrolysis using response surface methodology. Ind. Crops Prod. 35, 280–286. Vasco-Correa, J., Li, Y., 2015. Solid-state anaerobic digestion of fungal pretreated Miscanthus sinensis harvested in two different seasons. Bioresour. Technol. 185, 211–217. Wan, C., 2011. Microbial Pretreatment of Lignocellulosic Biomass with Ceriporiopsis Subvermispora for Enzymatic Hydrolysis and Ethanol Production. The Ohio State University. Wan, C., Li, Y., 2010. Microbial delignification of corn stover by Ceriporiopsis subvermispora for improving cellulose digestibility. Enzyme Microb. Technol. 47, 31–36. Wan, C., Li, Y., 2011. Effectiveness of microbial pretreatment by Ceriporiopsis subvermispora on different biomass feedstocks. Bioresour. Technol. 102, 7507– 7512. Wan, C., Li, Y., 2012. Fungal pretreatment of lignocellulosic biomass. Biotechnol. Adv. 30, 1447–1457. Wang, W., Meng, X., Min, D., Song, J., Jin, Y., 2014. Effects of green liquor pretreatment on the chemical composition and enzymatic hydrolysis of several lignocellulosic biomasses. BioResources 10, 709–720. Zeng, Y., Zhao, S., Yang, S., Ding, S.-Y., 2014. Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels. Curr. Opin. Biotechnol. 27, 38–45. Zhao, J., Ge, X., Vasco-Correa, J., Li, Y., 2014. Fungal pretreatment of unsterilized yard trimmings for enhanced methane production by solid-state anaerobic digestion. Bioresour. Technol. 158, 248–252.
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Fungal pretreatment of non-sterile miscanthus for enhanced enzymatic hydrolysis Juliana Vasco-Correa, Xumeng Ge, Yebo Li ⇑ Department of Food, Agricultural and Biological Engineering, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Ave., Wooster, OH 44691-4096, USA
h i g h l i g h t s Successful fungal pretreatment of non-sterile miscanthus by white rot fungus. Inoculation with at least 30% pre-colonized miscanthus was crucial for the success. Enzymatic digestibility and glucose yield was 3–4 times higher than raw miscanthus. Comparable glucose yield to that of fungal pretreated miscanthus with sterilization.
a r t i c l e
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Article history: Received 30 October 2015 Received in revised form 3 December 2015 Accepted 9 December 2015 Available online 14 December 2015 Keywords: Miscanthus Fungal pretreatment Sterilization Ceriporiopsis subvermispora White rot fungi
a b s t r a c t Miscanthus was pretreated with the fungus Ceriporiopsis subvermispora under non-sterile conditions, using sterile miscanthus that had been previously colonized with the fungus as the inoculum. Inoculum ratios equal to or greater than 30% yielded a successful pretreatment, enhancing the enzymatic digestibility of miscanthus by 3- to 4-fold over that of raw miscanthus, which was comparable with the fungal pretreatment under sterile conditions. This enhanced digestibility was linearly correlated with lignin degradation. Although cellulose loss of up to 13% was observed for the successful non-sterile pretreatments, the final glucose yield was 3–4 times higher than that of raw miscanthus and comparable to that of the sterile pretreated miscanthus. A time course study showed that maximum glucose yield can be achieved with a pretreatment time of 21 days. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The increasing need for alternative renewable sources of energy has prompted the development of biomass-based biorefineries with the potential of replacing a significant portion of the petroleum that is currently used for the production of fuels and chemicals (Cherubini, 2010). Biorefineries’ success depends on the availability of renewable, consistent, high volume feedstock supplies, which can be provided by dedicated energy crops, such as switchgrass or miscanthus (U.S. Department of Energy, 2011). Miscanthus is a tall perennial grass that has high annual biomass yields, low nutrients and water requirements, and the ability to adapt to a wide variety of climate and soil conditions (Jones and Walsh, 2001). Therefore, miscanthus has been identified as one of the most promising energy crops (Heaton et al., 2010).
⇑ Corresponding author. Tel.: +1 330 263 3855; fax: +1 330 263 3670. E-mail address: [email protected] (Y. Li). http://dx.doi.org/10.1016/j.biortech.2015.12.018 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Miscanthus, a lignocellulosic biomass, is mainly composed of cellulose, hemicellulose, and lignin. Biological transformation of lignocellulosics into bioenergy utilizes only the sugars present in the cellulose (and sometimes in the hemicellulose) for the production of biofuels, while the presence of lignin is undesirable (Zeng et al., 2014). Cellulose and hemicellulose are polysaccharides composed of 5- and 6-carbon sugars that can be fermented by microorganisms for the production of biofuels and bioproducts. Lignin is an aromatic heteropolymer that protects the polysaccharides from microbial degradation, besides giving support and stiffness to the plant structure (Hendriks and Zeeman, 2009). Miscanthus has a recalcitrant structure and pretreatment is required to enhance the accessibility of cellulose to hydrolytic enzymes for sugar release (Brosse, 2009). Commonly used pretreatment methods often employ strong chemicals and/or high temperatures, and require large amounts of water (Kim, 2013). Alternatively, biological pretreatment exploits the ligninolytic capacity of some microorganisms (fungi and bacteria) to reduce the recalcitrance of the lignocellulose (Chen et al., 2010). For fungal
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pretreatment, rotting fungi, such as Ceriporiopsis subvermispora, are grown in the lignocellulosic biomass to breakdown lignin, resulting in a feedstock that is easier to digest (Wan and Li, 2012). Compared to other pretreatment methods, fungal pretreatment can be operated at room temperature with minimal use of water and chemicals, and without generation of waste streams (Saritha et al., 2012). Fungal pretreatment is usually performed via solid-state cultivation of the fungus in the feedstock. But because fungi cannot use lignin as a sole carbon and energy source, parts of the cellulose and/or hemicellulose are also degraded during this process, which is detrimental for the purpose of pretreatment (Sánchez, 2009). C. subvermispora is a white rot basidiomycetes that degrades lignin selectively over cellulose. It has been used to successfully pretreat lignocellulosic biomass, resulting in significant lignin degradation and enhanced enzymatic digestibility. Although some parts of the hemicellulose are also degraded, most of the cellulose remains intact (Wan and Li, 2011). Glucose yields between 24% and 69% have been obtained for fungal pretreatment with C. subvermispora of feedstocks such as hardwood, corn stover, and switchgrass, but this pretreatment has been unsuccessful with other feedstocks such as soybean and wheat straw (Cianchetta et al., 2014; Ge et al., 2015; Salvachúa et al., 2011; Wan and Li, 2010, 2011). Sterilization of the feedstock, a costly step, is usually required for fungal pretreatment, because the indigenous microorganisms may outcompete the introduced fungi. C. subvermispora has been reported to be especially sensitive to the presence of other microorganisms (Akin et al., 1995). A promising alternative strategy is to grow the fungus on sterile feedstock first, and then use the colonized feedstock as an inoculum for fungal pretreatment of non-sterile feedstock (Zhao et al., 2014). However, this strategy has only been studied in woody biomass, and it is not clear how it would behave in herbaceous crops, such as miscanthus. White rot fungi naturally colonizes wood, but it has been shown that its growth in herbaceous biomass can be more difficult (Vasco-Correa and Li, 2015; Wan and Li, 2011), probably because of differences in the cell wall structure and composition. However, herbaceous biomass has more potential as a bioenergy feedstock because it is less recalcitrant and grows faster than wood, producing higher biomass yields (Wan and Li, 2011). It is unknown how much previously colonized feedstock would be needed as an inoculum for successful pretreatment of herbaceous biomass, how the moisture content of the feedstock–inoculum mixture would affect this process, and how this strategy would affect the pretreatment time. Therefore, systematic research on using this strategy for fungal pretreatment of non-sterile herbaceous crops is needed. In this study, fungal pretreatment of non-sterile miscanthus, using miscanthus colonized with C. subvermispora as the inoculum, was evaluated. Effects of inoculum ratio and moisture content on glucose yield, enzymatic digestibility, lignin degradation, and sugar loss were investigated. Also, a time course study of the fungal pretreatment process was performed, monitoring the degradation of different components during 28 days and effects on the glucose yield.
2. Methods
Table 1 Characteristics of miscanthus.
Total solids (%) Extractivesa (%) Cellulosea (%) Hemicellulosea (%) Lignina (%) a
Raw miscanthus
Inoculum (fungal colonized miscanthus)
93.8 ± 0.5 8.9 ± 0.2 38.0 ± 0.2 18.5 ± 0.4 20.9 ± 0.2
47.9 ± 0.5 15.33 ± 0.1 43.6 ± 0.9 17.4 ± 0.3 17.1 ± 0.2
Based on total solids.
2.2. Inoculum preparation Ceriporiopsis subversmipora (ATCC 96608) was obtained from the American Type Culture Collection (Manassas, VA, USA) and kept on 2% malt extract agar at 4 °C. Sterile miscanthus colonized with C. subversmipora was used as the inoculum for the fungal pretreatment experiments. To prepare this inoculum, 130 g (dry basis) of raw miscanthus were placed in 2 L reactors, which were supplemented with deionized water to adjust moisture to 60%, and then covered with cotton. Reactors were autoclaved (121 °C, 15 min) and cooled down. Then, C. subversmipora mycelium, which was grown in 50 ml of 2% malt extract liquid medium (7 days, 28 °C, static conditions), was added to each reactor and then incubated for 28 days at 28 °C. At the end, the fungal-colonized miscanthus was taken out of the flask and mixed well before using it as inoculum for the successive fungal pretreatment of non-sterile miscanthus. Composition of the inoculum is shown in Table 1. 2.3. Fungal pretreatment of non-sterile miscanthus Non-sterile raw miscanthus and inoculum (fungal-colonized miscanthus) were mixed in different proportions and added to 1 L reactors. Deionized water was added to adjust moisture content and reactors were incubated at 28 °C. First, a full factorial design with three replicates was performed, with the following factors: inoculum ratio (0%, 10%, 20%, 30%, 40%, and 50%, dry basis) and moisture content (60% and 75%). Reactors were incubated for 28 days. The inoculum ratio 0% (no inoculation) was considered to be the negative control. The positive control was sterile miscanthus inoculated with mycelium (as described in Section 2.2), which was incubated along with the treatments. At the end of the 28 days, samples were taken out of the reactors and mixed well. Part of the fresh material was used for enzymatic hydrolysis and the rest was dried at 40 °C in a convection oven for 24 h, then milled and passed through a 1 mm screen (Model 4 Wiley Mill, Thomas Scientific, Swedesboro, NJ, USA) for subsequent cellulose, hemicellulose, and lignin content determination. Second, an experiment to evaluate the degradation over time of components of miscanthus during fungal pretreatment was performed at 50% inoculum ratio and 60% moisture content. Reactors were prepared in the same manner as in the first experiment and stopped at different times (0, 7, 14, 21, and 28 days). At the end of the specific incubation time, samples were processed as in the first experiment, for subsequent enzymatic hydrolysis, and cellulose, hemicellulose, and lignin content determination.
2.1. Feedstock collection and storage 2.4. Enzymatic hydrolysis Miscanthus giganteus (hereafter miscanthus) was harvested from a field in Ashtabula, OH, in spring 2013. The moisture content was around 6% and its composition is shown in Table 1. The feedstock was milled to pass through a 12 mm screen using a hammer mill (The C.S Bell Co., Tiffin, OH, USA) and stored under dry conditions.
Enzymatic digestibility of raw and pretreated miscanthus was determined according to Selig et al., using the enzyme mix Cellic CTec2 (Novozymes, Bagsværd, Denmark), at a cellulase concentration of 10 FPU/g dry substrate (Selig et al., 2008). About 2.5 g (dry basis) of feedstock were added to 250 ml flasks along with citrate
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%Enzymatic Digestibility ¼
%Glucose yield ¼
½Glucose þ 1:053½Cellobiose 100% 1:111f p ½Biomass
½Glucose þ 1:053½Cellobiose 100% 1:111f r ½Biomass
where [Glucose] and [Cellobiose] are the residual glucose and cellobiose concentrations (g/L), [Biomass] is the dry biomass concentration at the beginning of the enzymatic hydrolysis (g/L), and fp and fr are the fraction of cellulose in the pretreated and original (raw) dry biomass (g/g), respectively (Wang et al., 2014). Thus, the sugar yield accounted for the cellulose loss that could occur during the fungal pretreatment. 2.5. Analytical methods Cellulose, hemicellulose, and lignin contents were determined according to the NREL protocol (Sluiter et al., 2012), using a twostep acid hydrolysis. Sugars liberated after the acid hydrolysis were quantified by HPLC (LC-20AB, Shimadzu, Kyoto, Japan) with an Aminex HPX-87P column (Bio-Rad, Inc., Hercules, CA, USA) and a refractive index detector. Acid soluble lignin was determined by UV–vis spectroscopy (BioMate 3, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and acid-insoluble lignin was quantified gravimetrically. Dry matter was determined gravimetrically, by drying triplicate samples of the miscanthus at 105 °C. Cellulose, hemicellulose, lignin, and dry matter degradations were calculated based on that of the initial mixture (raw material plus inoculum). To measure the miscanthus extractives, an Accelerated Solvent Extraction (ASE 350, Thermo Fisher Scientific, Inc., Waltham, MA, USA) with water and ethanol was used, following the NREL analytical procedure (Sluiter et al., 2008). Then, the solvents were removed from the extract by evaporation (Rocket Evaporator, Genevac, Ipswich, England), and the evaporated extract was dried at 40 °C before measurement of the dry weight. 2.6. Data analysis Statistical significance was evaluated by analysis of variance (ANOVA) (a = 0.05), and mean comparisons were performed by Tukey–Kramer test with a 95% significance, using the Software JMPÒ. Error bars in the figures represent the standard error. 3. Results and discussion 3.1. Cellulose, hemicellulose, and dry matter loss Cellulose, hemicellulose, and dry matter losses after 28 days of the fungal pretreatment of non-sterile miscanthus strongly depended on the inoculum ratio, but not on the moisture content, for the conditions evaluated (Fig. 1). Besides, the losses of cellulose (5–18%), hemicellulose (3–24%), and dry matter (4–18%) were comparable with those obtained previously in fungal pretreatment of non-sterile yard trimmings (Zhao et al., 2014).
(a)
40%
Cellulose loss (%)
35%
Moisture 60% Moisture 75%
30% 25% 20% 15% 10% 5% 0% 0
(b)
40% 35%
Hemicellulose loss (%)
buffer (0.05 M, pH 5.0), 1 ml of 2% sodium azide solution, and the enzyme, for a total working volume of 100 ml. Enzymatic hydrolysis was performed at 50 °C and a pH of 5.0 for 72 h, and then the hydrolysate was centrifuged (12,000 g, 10 min), and the supernatant was passed through a 0.2 lm nylon filter into glass vials. Free sugars were analyzed by high-performance liquid chromatography (HPLC) (LC-20AB, Shimadzu, Kyoto, Japan) with an Aminex HPX-87P column (Bio-Rad, Inc., Hercules, CA, USA) and a refractive index detector. Enzymatic digestibility and glucose yield were calculated as follow:
10
20 30 40 Inoculum ratio (%)
50
+
50
+
50
+
Moisture 60% Moisture 75%
30% 25% 20% 15% 10% 5% 0% 0
(c)
40% 35%
Dry matter loss (%)
120
10
20 30 40 Inoculum ratio (%)
Moisture 60% Moisture 75%
30% 25% 20% 15% 10% 5% 0% 0
10
20 30 40 Inoculum ratio (%)
Fig. 1. Components loss after 28 days of fungal pretreatment of miscanthus. (a) Percentage of cellulose loss. (b) Percentage of hemicellulose loss. (c) Percentage of dry matter loss. ‘‘+”: positive control (sterilized miscanthus).
Cellulose loss was between 7% and 13% for non-sterile pretreatments with inoculum ratios of 30–50%, which was comparable with that in the miscanthus incubated without inoculation (0%). On the contrary, cellulose loss was negligible for the pretreated sterile miscanthus (positive control) (Fig. 1a). This suggests that the cellulose loss in the non-sterile pretreatment was due to the presence of other microorganisms (contaminants) that prefer cellulose as carbon and energy sources. Cellulose is the main source of sugars for the subsequent processes and its degradation during the pretreatment is undesirable. However, the cellulose loss in the non-sterile pretreatment was still similar to those observed for other pretreatment methods. For example, alkaline pretreatment of miscanthus with 1 M NaOH at 150 °C for 60 min caused a cellulose loss of about 20% (Cha et al., 2014). Aqueous ammonia pretreatment showed a cellulose loss of 4–12% (Liu et al., 2013). Organosolv pretreatment with formic and acetic acid led to a cellulose loss of about 0–25%, depending on pretreatment temperature, time, and solvent concentration (Vanderghem et al., 2012). The hemicellulose loss was probably mainly caused by the growth of C. subvermispora, which uses these sugars as its main carbon source (Wan, 2011), but other microorganisms present in the reactor could also have consumed it at a small rate, as can be
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3.2. Lignin degradation and enzymatic digestibility Lignin loss was almost negligible for pretreatments with inoculum ratios of 10% and 20%, as well as for the miscanthus without inoculation. On the other hand, lignin loss of around 25–35% was achieved with inoculum ratios of 30–50%, as well as with the sterilized miscanthus (Fig. 2a), which was significantly higher than that obtained using a similar pretreatment strategy for nonsterile yard trimmings (Zhao et al., 2014). An analysis of variance indicated that both inoculum ratio and moisture content had a significant effect (p < 0.05) on lignin degradation (Fig. 2a). At inoculum ratios between 30% and 50%, higher lignin degradation was observed at a moisture content of 75%.
Lignin degradation (%)
(a) 40%
Moisture 60% Moisture 75%
30%
20%
10%
0% 0
Enzymatic digestibility (%)
(b) 60%
10
20 30 40 Inoculum ratio (%)
50
+
50
+
Moisture 60% Moisture 75%
50% 40% 30% 20% 10% 0%
0
(c) Enzymatic digestibility (%)
seen in the hemicellulose degradation in the miscanthus incubated without inoculation (negative control) (Fig. 1b). There was a significant difference between the hemicellulose loss in the pretreated sterilized miscanthus (positive control) and non-sterilized miscanthus with inoculum ratios of 30–50% (p < 0.05). That difference was equivalent to the hemicellulose loss obtained in the miscanthus without inoculation, which suggests that the higher degradation in the non-sterile treatments compared to sterilized miscanthus was due to hemicellulose consumption by other microorganisms present in the reactor. Hemicellulose loss can be beneficial to enhance cellulose digestibility, but if the subsequent fermentation processes use microorganisms that are able to utilize xylan, hemicellulose degradation is detrimental (Alvira et al., 2010). Hemicellulose degradation for the successful non-sterile pretreatment was between 16% and 24%, while the hemicellulose loss in the sterile pretreatment was about 16–17%. These values were still smaller than those obtained with other pretreatments. Alkaline pretreatment usually degrades a large percentage of the hemicellulose. For example, miscanthus treated with 1 M NaOH at 150 °C for 60 min resulted in a hemicellulose loss of about 79% (Cha et al., 2014). Aqueous ammonia pretreatment of miscanthus caused a hemicellulose loss of 23–61% (Liu et al., 2013). Organosolv pretreatment with formic/acetic acid led to a loss of hemicellulose of 9–79%, depending on the pretreatment temperature, time, and solvent concentration (Vanderghem et al., 2012). Autohydrolysis of miscanthus (pretreatment with water under pressure at 130– 150 °C) generated a hemicellulose loss of about 50%, with no cellulose or lignin degradation (El Hage et al., 2010). Dry matter loss was 13–18% for the non-sterile pretreatments with inoculum ratios of 30–50%, which was significantly higher than that of the sterilized miscanthus (7–9%) and the miscanthus without inoculation (4–7%) (Fig. 1c). For the non-sterile pretreatments with 30–50% inoculum ratios, 40–50% of the dry matter loss was due to lignin degradation, and the rest was equally distributed between cellulose and hemicellulose loss. For the sterile pretreatment, about 70–80% of the dry matter loss was due to lignin degradation, and the rest was hemicellulose loss because, as mentioned previously, cellulose loss was negligible. For the miscanthus without inoculation, 70–80% of the dry matter loss was due to loss in cellulose and the rest to hemicellulose loss, with practically no lignin degraded in this case. Therefore, the difference in dry matter loss between the non-sterile pretreatments with 30–50% inoculum ratios and the sterile pretreatment (positive control) was due primarily to the loss in cellulose in the former. The main disparity between the non-sterile pretreatments with 30–50% inoculum ratios and the miscanthus without inoculation (negative control) was due to the absence of lignin degradation in the latter. For the non-sterile pretreatments with 10% and 20% inoculum ratios, the dry matter loss proportions were similar to those of the miscanthus without inoculation, since the lignin degradation was very low in these cases (0–5%).
10
20 30 40 Inoculum ratio (%)
60% 50%
R² = 0.963
40% 30% 20% 10% 0% 0%
10% 20% 30% Lignin degradation (%)
40%
Fig. 2. (a) Enzymatic digestibility of fungal pretreated miscanthus after 28 days. (b) Percentage of lignin loss after 28 days of fungal pretreatment of miscanthus. (c) Correlation between lignin loss and enzymatic digestibility of fungal pretreated miscanthus after 28 days. ‘‘+”: positive control (sterilized miscanthus).
The enzymatic digestibility was significantly affected by the inoculum ratio (p < 0.05), but not by the moisture content (p > 0.05), according to the analysis of variance (Fig. 2b). This suggests that moisture content can vary between 60% and 75% without significantly affecting enzymatic digestibility, which is advantageous because moisture content can be a parameter difficult to control in solid-state fermentation (Robinson et al., 2001). The enzymatic digestibility and lignin degradation of the reactors with inoculum ratios of 30%, 40%, and 50% were comparable with those of the sterilized miscanthus, showing that at an inoculum ratio between 30% and 50%, fungal pretreatment of non-sterile miscanthus was feasible. The enzymatic digestibility and lignin degradation of miscanthus with inoculum ratios of 10% and 20% were equivalent to those of the miscanthus without inoculation (0% inoculum ratio) and the raw miscanthus, which had an enzymatic digestibility of about 12% (Fig. 2a and b). This suggests that the pretreatment was unsuccessful for inoculum ratios less than 20%. When the inoculum ratio was increased to 30–50%, a 2-fold increase in enzymatic digestibility was obtained compared with the initial mixture, which was equivalent to a 3- to 4-fold increase compared to the raw miscanthus. These results were similar to
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maintenance. Glucose yield generally increased with the increase of lignin degradation, especially in the first 7 days when half of the lignin was degraded. Wan and Li (2010) studied the time course of fungal pretreatment of corn stover during 42 days of incubation under sterile conditions. In that case, most of the lignin degradation occurred during the first 15 days, and then the degradation rate decreased significantly, which was not seen in the present case. However, hemicellulose and dry matter loss occurred continuously during the pretreatment of sterile corn stover, similar to the non-sterile miscanthus. Glucose yield showed a similar tendency for sterile corn stover and non-sterile miscanthus, with a high initial increase and a later stationary phase. The main difference was that the glucose yield curve for sterile corn stover showed a lag phase of about 7 days, which was not present in that of the non-sterile miscanthus. This difference was probably due to the inoculum. For the miscanthus, the inoculum probably had a higher concentration of the fungus and the fungus was better adapted to the feedstock, which could have shortened the lag phase.
those obtained by fungal pretreatment of sterile switchgrass in previous research (Wan and Li, 2011). Enzymatic digestibility was also analogous to that obtained after a 28-days fungal pretreatment of non-sterile corn stover with Irpex lacteus, even though this feedstock underwent a disinfection process with lime prior to fungal inoculation (Song et al., 2013). Strong correlation between lignin loss and enzymatic digestibility was observed (Fig. 2c), suggesting that lignin was probably one of the main factors that reduced the accessibility of structural sugars to the enzyme, as described by other authors (Chen et al., 2010; Wan, 2011), and that its removal positively and directly contributed to enhancement of enzymatic digestibility. This result confirmed that the main effect of fungal pretreatment on the lignocellulosic structure is lignin degradation, which makes the cellulose more accessible to hydrolytic enzymes (Saritha et al., 2012). Glucose yield for effective inoculum ratios (30%, 40%, and 50%) ranged between 35% and 48%, which represented an increase of 3to 4-fold, compared with the raw miscanthus. Compared with the initial mixture of inoculum (fungal colonized miscanthus) and raw miscanthus, the glucose yield was increased by a factor of 2. The glucose yields for sterilized miscanthus (positive control) were between 35% and 39%, which was comparable with the effective pretreatments with inoculum ratios of 30–50%. These results suggest that even though cellulose degradation during fungal pretreatment of non-sterile miscanthus was higher than that with sterilized miscanthus, the overall glucose yield was not affected drastically. Fungal pretreatment of miscanthus could be performed under non-sterile conditions, yielding similar results as sterilized miscanthus, using the process described in this study.
4. Conclusions Fungal pretreatment of non-sterile miscanthus achieved 3- to 4-fold increases in enzymatic digestibility and glucose yield, compared to the raw feedstock. This pretreatment can be performed in 21 days, using 30–50% fungal colonized miscanthus as the inoculum and a moisture content of 60–75%, to obtain a glucose yield comparable with that of fungal pretreatment of sterile miscanthus, despite having a higher cellulose loss. Sterilization was still required for preparing the inoculum that was used for this single batch pretreatment of non-sterile miscanthus. Future research would explore consecutive batches of fungal pretreatment in order to further reduce the cost.
3.3. Time course of the non-sterile fungal pretreatment During solid-state fungal pretreatment of non-sterile miscanthus with 50% inoculum, the glucose yield increased significantly until day 21. There was no significant increment in the glucose yield from day 21 to day 28 (p > 0.05) (Fig. 3). There was also no significant cellulose loss between those dates; in fact, all the cellulose degradation seemed to occur at the beginning of the process (between day 0 and 7) and it did not increase after that. Therefore, the pretreatment could be reduced to 21 days without affecting the glucose yield, while enhancing the productivity of the process. In contrast, there was continuous degradation of hemicellulose throughout the process, which suggests that the hemicellulose was acting as a carbon and energy source for fungal growth and
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This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2012-10008-20302. The authors want to thank Fulbright-Colombia for providing funding for the doctoral student Juliana Vasco-Correa and Mrs. Mary Wicks (Department of Food, Agricultural and Biological Engineering, OSU) for reading through the manuscript and providing useful suggestions.
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Cellulose loss Hemicellulose loss Lignin loss Dry matter loss Glucose yield
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Glucose yield (%)
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Acknowledgements
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Time (days) Fig. 3. Time course study of fungal pretreatment of non-sterile miscanthus during 28 days at 50% inoculum ratio and 60% moisture content.
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