Abstract
A successful conversion of lignocellulosic biomass into biofuel could be achieved through an ionic liquids (ILs)-pretreatment and an enzyme saccharification. In this study, the fungal strains with high cellulase productions were isolated and identified as Penicillium janthinellum FHH1 and P. oxalicum FLY4. A high cellulase production was obtained at pH 4.0 and 30 °C for P. janthinellum FHH1 with corn stalk and beef extract for 9 days and for P. janthinellum FHH1 with corn stalk and peptone for 7 days. A pH range between 4.0 to 6.0 and a temperature range from 55 °C to 60 °C were applicable for cellulase with high activities during hydrolysis. A high NaCl-stability and a relatively high ILs-stability were found for cellulase obtained from P. oxalicum FLY4. High yields of reducing sugar were obtained by enzymatic hydrolysis of the original corn stalk and ILs-pretreated corn stalk. P. oxalicum FLY4 has a great potential for ILs-assisted fungi in the degradation of lignocellulosic biomass into value-added products.
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Screening of Highly Efficient Fungi for the Degradation of Lignocelluloses by Ionic Liquids-assisted Cellulase
Xiao-Mei Hu,a,c,1 Shi-Jia Dong,a,b,1,* Hui-Ying Han,c Yun-Fei Gao,d and Bi-Xian Zhang d
A successful conversion of lignocellulosic biomass into biofuel could be achieved through an ionic liquids (ILs)-pretreatment and an enzyme saccharification. In this study, the fungal strains with high cellulase productions were isolated and identified as Penicillium janthinellum FHH1 and P. oxalicum FLY4. A high cellulase production was obtained at pH 4.0 and 30 °C for P. janthinellum FHH1 with corn stalk and beef extract for 9 days and for P. janthinellum FHH1 with corn stalk and peptone for 7 days. A pH range between 4.0 to 6.0 and a temperature range from 55 °C to 60 °C were applicable for cellulase with high activities during hydrolysis. A high NaCl-stability and a relatively high ILs-stability were found for cellulase obtained from P. oxalicum FLY4. High yields of reducing sugar were obtained by enzymatic hydrolysis of the original corn stalk and ILs-pretreated corn stalk. P. oxalicum FLY4 has a great potential for ILs-assisted fungi in the degradation of lignocellulosic biomass into value-added products.
DOI: 10.15376/biores.17.1.355-368
Keywords: Lignocelluloses; Ionic liquids; Fungi; Cellulase; Degradation
Contact information: a: Harbin University, Harbin, 150086, P.R. China; b: Heilongjiang Province Key Laboratory of Cold Region Wetland Ecology and Environment Research; c: Northeast Agricultural University, Harbin, 150030, P.R. China; d: Heilongjiang Academy of Agricultural Sciences, Harbin, 150086, P.R. China; *Corresponding author: huxiaomei1982@163.com; 1These authors contributed equally to this work.
INTRODUCTION
Lignocellulosic biomass is one of the most abundant and renewable resources on earth. The focus on the bioconversion of lignocellulosic biomass into biofuels has increased (Taha et al. 2015; Jatoi et al. 2021). Cellulose and hemicellulose, which comprise two-thirds of lignocelluloses, are known as the main feedstock for biofuel production. The recalcitrant nature of lignocelluloses and the high crystallinity of cellulose limit the effective utilization of cellulosic biomass. An effective approach to convert cellulose into a valuable product is enzymatic saccharification (Pandey et al. 2000; Kirk et al. 2002). However, the low efficiency and the high cost of cellulase are still the main challenges faced (Baba et al. 2015).
Cellulase plays an important role in the release of monomeric sugars from cellulosic biomass. A complete cellulase system includes endoglucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (BGL). These enzymes hydrolyze the β-1,4-D-glucan bonds in the cellulose to release glucose, cellobiose, and oligosaccharides (Paramjeet et al. 2018). Numerous microbes, such as Trichoderma sp., Aspergillus sp., and Penicillium sp. are described as cellulase producers (Verma et al. 2010). Nevertheless, the lack of the complete cellulase reduces the hydrolysis efficiency; for instance, the lack of β-glucosidase of T. reesei lowers the enzymatic hydrolysis. Researchers have focused on the isolation of microbes with a high production of the complete cellulase to achieve the efficient cellulose digestibility (Dashtban and Qin 2012).
Due to the recalcitrant structure of lignocelluloses, the utilization of steam explosion, acid, alkaline conditions, or ionic liquids (ILs) as a pretreatment have been a requirement. The ILs have been considered as an efficient pretreatment solvent (Fitzpatrick et al. 2010). This solvent can decompose the complex structure of lignocelluloses and reduce the crystallinity of cellulose to improve the subsequent enzymatic hydrolysis. The combination of ILs-pretreatment and cellulase hydrolysis has potential to greatly improve the bioconversion of lignocelluloses.
In this study, a series of microbes were isolated and identified with two dominant cellulase-producing strains. Fermentation factors, including carbon, nitrogen, initial pH, and fermentation time, were evaluated to improve the cellulase production. The NaCl stability and ILs stability of both microbes and cellulase were determined. The optimal pH and temperature of cellulase were studied for enzymatic hydrolysis. Enzymatic saccharification was investigated for corn stalk and ILs-pretreated corn stalk.
EXPERIMENTAL
Sample Collection
Soil samples were collected from the Greater Khingan Mountains (124.15951090674938E, 50.444505204846436N) in Heilongjiang province, China. Biomass was dried, ground, and passed through 40-mesh sieves. The chemicals pyridine (99%), 1,4-dichlorobutane (99%), 3-chloropropionic acid (99%), and potassium hexafluorophosphate (99%) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).
Screening of Cellulase-producing Microbes
Each isolate was incubated on carboxyl methyl cellulose (CMC) medium including K2HPO4 (0.1%, weight per volume), NaCl (0.05%, weight per volume), (NH4)2SO4) (0.2%, weight per volume), MgSO4 (0.05%, weight per volume), sodium carboxymethyl cellulose (CMC-Na) (1.0%, weight per volume), and agar (1.0%, weight per volume) at 30 °C for 5 days (Wood et al. 1988). After that, the plates were stained with Congo-red solution (0.1%, weight per volume) and were discolored with 1 mol/L NaCl. One of the strains with a large hydrolysis zone was cultured in potato dextrose agar (PDA) medium including potato (0.2%, weight per volume), glucose (0.02%, weight per volume), and agar (1.0%, weight per volume) at 30 °C for 3 days (Xi et al. 2020).
A 1 cm agar piece was inoculated into potato dextrose (PD) medium including potato (0.2%, weight per volume), and glucose (0.02%, weight per volume). The solution was cultured at 30 °C and 150 rpm for 3 days. A total of 1 mL of the suspension (2%, volume per volume) was added into 100 mL of Hutchinson medium including NaNO3 (0.25%, weight per volume), KH2PO4 (0.10%, weight per volume), MgSO4 (0.03%, weight per volume), NaCl (0.01%, weight per volume), CaCl2 (0.01%, weight per volume), FeCl3 (0.001%, weight per volume) with corn stalk (1%, weight per volume), and peptone (0.2%, weight per volume). The medium was incubated at 30 °C and pH 7.0 and 150 rpm for 5 days. The solution was centrifuged at 4 °C with 10528 g for 10 min to provide the crude cellulase (Xi et al. 2020)
Determination of Cellulase Activity
The CMCase activity was measured with 1.5 mL of sodium carboxymethyl cellulose (1%, weight per volume) and 0.5 mL of the crude cellulase at 50 °C for 30 min. β-glucosidase activity was determined with 1.5 mL of salicin solution (0.5%, weight per volume) and 0.5 mL of the crude cellulase at 50 °C for 30 min. Filter paper (FPase) activity was measured with 50 mg of Whatman No. 1 filter paper (1.0 cm × 6.0 cm) and 1.5 mL of sodium citrate buffer (pH 4.8) at 50 °C for 60 min. The reducing sugars were determined by the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959; Papa et al. 2017). One unit (U) of the cellulase activity was defined as the amount of the enzyme that released 1 mmol of reducing sugar per min under the conditions indicated.
Molecular Identification
The DNA was extracted using a DNA kit based on the manufacturer’s instructions (Omega BIO-TEC, Norcross, GA, USA). The universal primers ITS1 (5’-AGAAGTCGT-AACAAGGTTTCCGTAGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGCTTAA-3’) were used for the polymerase chain reaction. The products were sequenced at the Huada Gene Company (Beijing, China). The phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) program (version 11, Mega Limited, Auckland, New Zealand).
Determination of Fermentation Factors
The carbon source (1%, w/v) and nitrogen source (0.2%, w/v) were determined at pH 7.0 for 5 days at 30 °C and 150 rpm with 2.0% (volume per volume) of inoculation of strains. The fermentation time was examined at pH 7.0 with corn stalk and peptone for the strain FLY4, and with corn stalk and beef extract for the strain FHH1. The initial pH was measured with corn stalk and peptone after 7 days for the strain FLY4. The initial pH was measured with corn stalk and beef extract after 9 days for the strain FHH1. The supernatant was obtained by centrifugation at 10,000 rpm for 10 min for the determination of cellulase activity.
Synthesis of Ionic Liquids
Ionic liquids were synthesized according to Han et al. (2019). Pyridine (1.0 mmol) and 1,4-dichlorobutane (1.0 mmol) were reacted at 60 °C for 4 days. The solid was purified by silica column chromatography (CH2Cl2/MeOH, 3/1) and was dried at 60 °C under vacuum for 4 h. After that, the product (1.0 mmol) was reacted with KPF6 (1.0 mmol) in deionized water at room temperature for 3 h. The resulting product was dried in a vacuum for 12 h. Following that, the product (0.1 mmol) was reacted with pyridine (0.12 mmol) at 80 °C for 24 h. The resulting product was recrystallized with acetonitrile/ethyl acetate (1/10) and was dried in a vacuum at 60 °C for 4 h to give hexafluorophosphate [1-(1-pyridinium-yl- butyl)-4-pyridinium] chloride ([PF6][(PYR)C4(PYR)][Cl]).
Pyridine (0.2 mol) was reacted with 3-chloropropionic acid (0.2 mol) at 70 °C for 24 h. After that, the product (0.1 mmol) was reacted with hexafluorophosphate (0.1 mmol) in 30 mL of deionized water at room temperature for 3 h. The resulting solid was filtered and dried at 50 °C for 10 h to obtain [1-(2-carboxylpropyl)- pyridinium] hexaufluorophosphate ((PYR)C2COOH][PF6]).
Determination of NaCl Stability and ILs Stability
The optimal pH and temperature for the crude cellulase were measured in citrate buffer at pH values in a range of 3.0 to 11.0 and temperatures in a range of 30 to 70 °C for 1 h. The stability of cellulase was measured in citrate buffer with the addition of NaCl or ILs at the optimal pH and temperature for different cellulase for 30 min.
Enzymatic Hydrolysis of Corn Stalk
Corn stalk (200 mg) was treated with 1.0 g of 1-(2-carboxylpropyl)-1-methylimida-zolium hexafluorophosphate ([MIMC2COOH][PF6]) and 3.0 g of hexafluorophosphate [1-(1-pyridinium-yl-butyl)-4-methylimidazolium] chloride ([PF6][PYRC4MIM][Cl]) at 100 °C for 3 h, followed by the addition of 1.5 mL of deionized water for 3 h (Han et al. 2019). The residue was filtered and washed with water. Following that, the residue was dried in a vacuum at 50 °C for 6 h. The enzymatic reaction was performed with 100 mg of the original corn stalk or ILs-pretreated corn stalk and 5 mL of the crude enzyme in 15 mL of citrate buffer with the addition of 200 μ of NaN3 (10 mg/μ L) for 24, 48, 72, and 96 h. The reducing sugar was measured by the DNS method (Miller 1959; Papa et al. 2017). The yields of reducing sugar were determined as the ratio of the mass of reducing sugar in the mass of corn stalk or ILs treated corn stalk that was subjected to enzymatic hydrolysis according to Eq. 1.
(1)
Statistical Analyses
Each experiment was performed in triplicate. The results are presented as mean values of three replicates with standard deviations (SD) shown as error bars. Statistical significance was determined by Student’s t-test at significance levels indicated by asterisks as follows: *p ≤ 0.05, **p < 0.01, ***p < 0.001
RESULTS AND DISCUSSION
Identification of Cellulase-producing Microbes
A preliminary isolation of microbes was based on a Congo-red staining method. The isolates exhibiting clear hydrolytic zones were referred to as the potential cellulase-producing microbes. Two predominant strains exhibited high enzymatic activities, indicating their strong cellulase-producing capability.
The ITS sequence of DNA was used for the identifications of fungal strains. The isolate FHH1 was identified as Penicillium janthinellum FHH1, as it showed 99% similarity with Penicillium janthinellum (Fig. 1). The isolate FLY4 was identified as Penicillium oxalicum FLY4, as it showed 100% similarity with Penicillium oxalicum. Numbers of strains of Penicillium sp., such as Penicillium janthinellum EMS-UV-8, Penicillium oxalicum JU-A10-T, and Penicillium oxalicum GZ-2 were reported as good cellulase producers (Singhania et al. 2014; Liao et al. 2014; Yao et al. 2015; Agrawal et al. 2017). The obtained nucleotide sequences were submitted to NCBI GeneBank under accession no. OK310849 for P. janthinellum FHH1 and OK310850 for P. oxalicum FLY4.
Fig. 1. The phylogenetic tree of the strains
Enzyme Activity in Different Carbon Sources
Carbon is a crucial nutrient for the growth and the reproduction of microbes. As shown in Fig. 2, corn stalk and rice straw were the effective carbon sources for P. janthinellum FHH1 and P. oxalicum FLY4. In particular, high productions of CMCase and β-glucosidase were observed with corn stalk as the carbon source. A high FPase production was obtained with rice straw as the carbon source. Nevertheless, low cellulase production was found with the utilization of CMC-Na and cellulose. As cellulase-producing activity of the strain could be inducible by cellulosic biomass, high cellulase productions were obtained for P. janthinellum FHH1 and P. oxalicum FLY4 using corn stalk as enzymatic substrates.
Agricultural wastes exhibit cellulolytic enzyme inducible properties. As reported, higher cellulase activities were obtained for P. oxalicum GZ-2 when lignocellulosic biomass was used instead of commercial cellulose (Liao et al. 2015). Similarly, congress grass was used as good carbon source for Parthenium sp. with high xylanase production (Dwivedi et al. 2009). Rice straw and wheat straw were found to be the most effective for FPase production (Liao et al. 2015). Corn cob and oat husk were the best inducers of xylanase for Penicillium janthinellum (Oliveira et al. 2006). Therefore, agricultural residue is more efficient than purified cellulose at inducing lignocellulolytic enzyme production (Liao et al. 2014). The use of agricultural wastes has both economic and environmental advantages.
Fig. 2. Effect of carbon source on cellulase production: a) P. janthinellum FHH1 and b) P. oxalicum FLY4
Enzyme Activity in Different Nitrogen Sources
Nitrogen is a necessary nutrient for the growth of microbes, and it has an impact on their metabolic process. As presented in Fig. 3, high productions of CMCase, β-glucosidase, and FPase were observed with beef extract as the nitrogen source for P. janthinellum FHH1. Low cellulase productions were found with the utilization of NH4NO3 and urea.
In addition, peptone and yeast extracts were the effective nitrogen sources for P. oxalicum FLY4. Specifically, high productions of β-glucosidase and FPase were observed with peptone as the nitrogen source. A high production of CMCase was observed with yeast extract as the nitrogen source. Organic nitrogen on enzyme production was better than inorganic nitrogen for both strains. Therefore, beef extract was used for P. janthinellum FHH1 and peptone was used for P. oxalicum FLY4.
Fig. 3. Effect of nitrogen source on cellulase production: a) P. janthinellum FHH1 and b) P. oxalicum FLY4
Enzyme Activity after Different Fermentation Time
Cellulase production was improved with the increase of fermentation time (Fig. 4). A high yield of cellulase was obtained for P. janthinellum FHH1 after 9 days and for P. oxalicum FLY4 after 7 days. As the fermentation time increased, cellulase production was decreased due to the reduction and the depletion of nutrients. Moreover, the higher ratio of β-glucosidase and FPase of P. janthinellum FHH1 and P. oxalicum FLY4 was capable of improving the inhibition of enzymatic hydrolysis due to cellobiose accumulation.
Fig. 4. Effect of fermentation time on cellulase production. a) P. janthinellum FHH1 and b) P. oxalicum FLY4
Enzyme Activity at Different Initial pH
Filamentous fungi have good growth over a broad range from pH 2 to 9 with an optimal range from 3.8 to 6.0 (Gowthaman et al. 2001). P. janthinellum FHH1 preferred acidic pH conditions (3.0 to 5.0). Particularly, this strain possessed a high cellulase production at pH 9.0. In addition, a broad pH range from 3.0 to 11.0 was applicable for P. oxalicum FLY4 with pH 5.0 being optimal (Fig. 5). High production of CMCase and β-glucosidase was obtained at pH 7.0 and pH 5.0, respectively.
Fig. 5. Effect of initial pH value on cellulase production: a) P. janthinellum FHH1 and b) P. oxalicum FLY4
pH Value on Cellulase Activity during Hydrolysis
The pH value influences the stability of cellulase during the hydrolysis. Inappropriate pH may cause the destruction of the spatial structure and the dissociation state of the enzyme (Adsul et al. 2007). A pH value between 3.0 and 6.0 was applicable for cellulase of P. janthinellum FHH1 (Fig. 6). High activities of CMCase and β-glucosidase were obtained at pH 4.0. A high FPase activity was observed at pH 5.0. Meanwhile, a pH value from 4.0 to 6.0 was applicable for P. oxalicum FLY4 with pH 4.0 being optimal for CMCase, β-glucosidase, and FPase. The CMCase and β-glucosidase of P. oxalicum GZ-2 showed remarkable stability in the pH range 4.0 to 9.0 and pH 3.0 to 7.0 with 80% of the maximum activity (Liao et al. 2015). Optimum pH for activity of the crude enzyme of Penicillium oxalicum IODBF-5 were pH 5 (Saini et al. 2015). Thus, an acidic condition was preferred for P. janthinellum FHH1 and P. oxalicum FLY4 in this study.
Fig. 6. Effect of pH value on cellulase activity: a: P. janthinellum FHH1; b: P. oxalicum FLY4
Hydrolysis Temperature on Cellulase Activity
A low temperature may decrease the rate of enzymatic reaction, while a high temperature may denature the protein leading to the loss of enzyme activity (Larsson et al. 2001). As the temperature was increased, the activities of β-glucosidase and CMCase for P. janthinellum FHH1 were improved with the maximum activity at 60 and 50 °C, respectively (Fig. 7).
Fig. 7. Effect of hydrolysis temperature on cellulase activity: a) P. janthinellum FHH1 and b) P. oxalicum FLY4
No obvious reduction of FPase activity was found with the temperature variation. As reported, the optimal temperatures for CMCase and β-glucosidase activity were at 50 and 60 °C, respectively (Liao et al. 2015). The optimum temperature for activity of the crude enzyme of Penicillium oxalicum IODBF-5 were 50 °C (Saini et al. 2015). A high temperature was found for CMCase and β-glucosidase of P. janthinellum FHH1. Meanwhile, when the temperature was increased, the activities of β-glucosidase and FPase for P. oxalicum FLY4 were improved with the maximum activity at 55 and 50 °C, respectively. However, the optimal temperature was found at 35 °C for CMCase of P. oxalicum FLY4. Thus, CMCase activity was reduced with the increase of the temperature.
NaCl Stability of Cellulase
When NaCl concentration was increased from 2.5% to 7.5% (weight per volume), NaCl stability of CMCase was improved for P. janthinellum FHH1 (Fig. 8). No obvious reduction of FPase stability was found. Nevertheless, the stability of β-glucosidase was reduced with the increase of NaCl concentration.
Moreover, an improved CMCase activity was achieved for P. oxalicum FLY4 in a range from 25% to 35% (w/v) of NaCl concentration with the maximum CMCase activity at 15% (w/v). NaCl precipitated out of the solution at 40% (w/v). A slight reduction of the stability of β-glucosidase and FPase was found with the increase of NaCl concentration. Therefore, a high NaCl stability was achieved for P. oxalicum FLY4.
Fig. 8. NaCl-stability of cellulase. a: P. janthinellum FHH1; b: P. oxalicum FLY4
ILs Stability of Cellulase
A higher ILs stability was achieved with cellulase obtained from P. oxalicum FLY4 than that of P. janthinellum FHH1 (Fig. 9). A total of 66% and 63% of the CMCase activity was retained for P. oxalicum FLY4 with the addition of 2.5% (w/v) of [(PYR)C2COOH][PF6], and [PF6][(PYR)C4(PYR)][Cl], respectively. A total of 57% and 51% of the CMCase activity was retained for P. janthinellum FHH1 with [(PYR)C2COOH][PF6], and [PF6][(PYR)C4(PYR)][Cl], respectively. The stability of CMCase was higher than β-glucosidase. It was reported that β-glucosidase was more stable than CMCase for P. janthinellum mutants, as CMCase showed 90% of its initial activity in 10% of 1-butyl-3-methylimidazolium chloride ([bmim]Cl) and β-glucosidase retained 85% of its original activity in 30% of ionic liquid (Adsul et al. 2009). In addition, CMCase that was obtained from P. oxalicum FLY4 retained 86% of the original activity with [EMIM]CH3COOH. A higher stability was found with [EMIM]CH3COOH for both strains.
Fig. 9. ILs stability of cellulase: a) P. janthinellum FHH1 and b) P. oxalicum FLY4; L1: [(PYR)C2COOH][PF6], L2: [PF6][(PYR)C4(PYR)][Cl] and L3: [EMIM]CH3COOH
Enzymatic Hydrolysis of Corn Stalk
The enzymatic reaction was performed with 100 mg of the original corn stalk and 5 mL of the crude cellulase for 24, 48, 72, and 96 h at pH 4.0 for P. janthinellum FHH1. The temperature conditions varied at 60 °C for FHH1 and 35 °C for P. oxalicum FLY4. As the hydrolysis time was increased from 24 to 96 h, the reducing sugars yield was improved for both of strains. After 96 h, the reducing sugars yield was 18% for P. janthinellum FHH1 and 33.75% for P. oxalicum FLY4. A higher performance of cellulase of P. oxalicum FLY4 was achieved than that of P. janthinellum FHH1 (Fig. 10). Compared with acid pretreated and steam exploded wheat straw, the untreated wheat straw could be a good carbon source for the production of quality cellulolytic enzymes by P. janthinellum EMS-UV-8 mutant (Sharma et al. 2015). This is different with P. janthinellum FHH1 and P. oxalicum FLY4, because high hydrolysis efficiency was observed with ILs-pretreated corn stalk.
Fig. 10. Enzymatic hydrolysis of corn stalk: a) P. janthinellum FHH1 and b) P. oxalicum FLY4
Enzymatic Hydrolysis of ILs-pretreated Corn Stalk
During the treatment by the mixture of [PF6][(PYR)C4(PYR)][Cl] and [(PYR)C2COOH][PF6] at 100 °C, a high reducing sugar yield for corn stalk was achieved at 18.80% (Han et al. 2019). The residue was obtained at 68.75% after ILs treatment. The enzymatic reaction was conducted with 100 mg of ILs-pretreated corn stalk and 5 mL of the crude cellulase for 24, 48, 72, and 96 h for P. janthinellum FHH1 at pH 4.0 and 60 °C and for P. oxalicum FLY4 at pH 4.0 and 35 °C. The reducing sugar yield increased with the increase of hydrolysis time (Fig. 11). After 96 h, the reducing sugar yield was 30.93% for P. janthinellum FHH1. A high yield was achieved of 52.37% for P. oxalicum FLY4 after 72 h. Meanwhile, the yield of the reducing sugar was 42.21% for [EMIM]CH3COOH-pretreated corn stalk. Hydrolysis of acid or alkali-pretreated rice straw using cellulase enzyme preparations from P. janthinellum and T. reesei released 37% and 43% glucose, respectively (Sreeja‑Raju et al. 2020). A crude enzyme solution of P. oxalicum Z1-3 was able to hydrolyze 92.5% of KOH-pretreated sugarcane bagasse to glucose within 96 h (Jing et al. 2015). A crude enzyme solution of Penicillium oxalicum EU2106 was able to hydrolyze 94.3 ± 1.5% of NaOH–H2O2-pretreated cassava residue to glucose under solid-state fermentation for 96 h (Su et al. 2017). In the present work, a high production of reducing sugar was achieved with the hydrolysis of [PF6][(PYR)C4(PYR)][Cl] / [(PYR)C2COOH][PF6]-pretreated corn stalk by cellulase obtained from P. oxalicum FLY4.
Fig. 11. Enzymatic hydrolysis of ILs-pretreated corn stalk: a) P. janthinellum FHH1 and b) P. oxalicum FLY4
Herein, cellulases from P. janthinellum FHH1 and P. oxalicum FLY4 have been successfully used for saccharification of corn stalk. Both of the strains from Penicillium species exhibit remarkable saccharification performance for supplementation of β-glucosidase-deficient Trichoderma cellulases (Gusakov and Sinitsyn 2012). Agricultural wastes act as non-food resource to save costs of the enzyme production process. In addition, the acidic group (COOH–) of ILs assists the hydrolysis of hemicellulose and cellulose of corn stalk. The anion (Cl–) of ionic liquids made it more effective at disrupting inter- and intra-molecular hydrogen bonding in lignocelluloses. Thus, the effective ILs-treatment of lignocelluloses contributed to achieve the high enzymatic digestibility. These cellulases from P. janthinellum FHH1 and P. oxalicum FLY4 are potential candidates to be utilized in hydrolyzing the lignocellulosic biomass.
CONCLUSIONS
- The fungal strains with high cellulase productions were identified as P. janthinellum FHH1 and P. oxalicum FLY4. Corn stalk was considered as the optimal carbon source for both strains. Beef extract and peptone were the optimal nitrogen source for P. janthinellum FHH1 and P. oxalicum FLY4, respectively.
- High cellulase activities were obtained in a pH range of 4.0 to 6.0 and a temperature between 55 and 60 °C during hydrolysis. A higher NaCl stability and ILs stability was observed for cellulase obtained from P. oxalicum FLY4 than P. janthinellum FHH1.
- High reducing sugar yields were achieved by enzymatic hydrolysis of ILs-pretreated corn stalk. A combination of ILs pretreatment and cellulase saccharification will have a successful utilization for the degradation of lignocellulosic biomass into value-added products.
ACKNOWLEDGMENTS
This work was financially supported by Heilongjiang Province Key Laboratory of Cold Region Wetland Ecology and Environment Research (201901), and Young Doctoral Research Initiation Fund Project of Harbin University (HUDF2021110), and Natural Science Foundation of Heilongjiang Province (YQ2020C001).
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Article submitted: July 18, 2021; Peer review completed: September 5, 2021; Revised version received and accepted: November 3, 2021; Published: November 18, 2021.
DOI: 10.15376/biores.17.1.355-368