Cellulose is a polymer of glucose, linked by beta-1,4-glycosidic bonds. Annual production of cellulose of about 180 billion tons makes this polysaccharide as a huge organic carbon reservoir on earth (1). It is used in different industries including production of paper, clothes, food additives, and ruminant nutrients. Recently, due to identification of cellulose-degrading enzymes, or cellulases, cellulose has emerged as a renewable source of energy with enormous potential (2). The cellulose-degrading enzymes play the key role in the biological conversion of cellulosic biomass to valuable biofuels and chemicals (3).
Cellulase is a complex family comprising of three groups of hydrolytic enzymes including endo-(1,4)-β-D-glucanase [endoglucanase (EG), endocellulase, CMCase (EC 126.96.36.199)], exo-(1,4)-β-D-glucanase [exoglucanase, cellobiohydrolase, exocellulase, microcrystalline cellulase, avicelase (EC 188.8.131.52)], and finally, β-glucosidase [cellobiase, (EC 184.108.40.206)]. Endoglucanases act randomly on soluble and insoluble cellulose chains while exoglucanases act to remove cellobiose from the reducing and non-reducing ends of cellulose chains, and glucosidases remove glucose from cellobiose (2). The synergistic action of these three enzyme groups with different functions is required for the complete hydrolysis of cellulose (4). The cellulases make us possible to utilize cellulosic biomass in an environmentally friendly manner (5).
CMCases have a broad variety of applications in detergent and animal feed production production (6), in the textile industry, pulp and paper industry, starch, grain alcohol fermentation, malt and beer production, extraction of fruit and vegetable juices (7), production of organic solvents (8). However, in many bioconversion strategies, the cellulases required for biomass conversion may still account for as much as 40% of the total process cost (9). Thus, large-scale and low-cost production of cellulase is a significant key for the overall process economics of lignocellulosics bioconversion.
A large number of microorganisms including bacteria, actinomycetes and fungi (10, 11) are capable to produce CMCases. Among fungi, many basidiomycetes including Volvariella volvacea (12, 13), Fomitosis sp. (14-16), Lentinus edodes (17), Irpex lacteus (18), Bjerkandera adusta and Pycnoporus sanguineus (19) have been studied for the CMCase production. In this study, for the first time, we described the identification of a novel basidiomycete isolate NDVN01 and optimization of culture conditions and medium components for the CMCase production by the basidiomycete Peniophora sp. NDVN01 under liquid state fermentation.
In this study, for the first time, we described the identification of a novel basidiomycete isolate NDVN01 and optimization of culture conditions and medium components for the CMCase production by the basidiomycete Peniophora sp. NDVN01 under liquid state fermentation.
3. Materials and Methods
3.1. Strains and Culture Conditions
Five basidiomycete strains Peniophora sp. NDVN01, Pleurotus sajor-caju, Pleurotus ostreatus, Ganoderma lucidum, Flammulina velutipes were collected from the Culture Collection of the Laboratory of Biology, Department of Life Science, College of Sciences, Thainguyen University, Vietnam. Peniophora sp. NDVN01 was identified based on morphology and on the sequence variation region containing 18S ribosomal RNA (partial sequence), internal transcribed spacer 1, 5.8S ribosomal RNA, internal transcribed spacer 2 (complete sequence), and 28S ribosomal RNA (partial sequence).
Peniophora sp. NDVN01 was maintained on PDA slant at 4°C and sub-cultured on fresh sterile PDA slant and incubated for 7 days. The strain was grown in 100 mL Erlenmeyer flasks containing 30 mL of basal medium at 30°C and on an orbital shaker with 200 rpm for CMCase production. The basal medium contained 20% (v/v) potato infusion and 0.6% (w/v) pulp, adjusted to pH 6. Two hundreds gram of peeled and sliced potatoes were boiled in 1 liter of distilled water for 30 minutes and then filtered through cheesecloth to obtain potato infusion. The culture supernatant harvested by centrifugation at 10000 rpm and 4°C for 10 minutes, was used for determination of CMCase activity.
Carboxymethyl cellulose from Biochemika (Sigma Aldrich Co., St. Luis, USA), peptone and yeast extract from Bio Basic Inc. (Ontario, Canada), 3,5-dinitrosalicyclic acid (DNS) from Fluka (Sigma Aldrich Co., St. Luis, USA). Escherichia coli DH10B, pJET1.2/blunt vector, T4 ligase, and Taq polymerase (Fermentas, Thermo Fisher Scientific Inc., Waltham, USA) were used for DNA manipulations and amplification. All other reagents were of analytical grade unless otherwise stated.
3.3. Identification of the Fungus
Isolation of genomic DNA: Fungal isolate NDVN01 was grown on PDA at 30°C with agitation of 200 rpm for 5 days. 30 mL of the culture was harvested by centrifugation at 10000 rpm and 4°C for 10 minutes. The fungal mycelium was ground in liquid nitrogen to produce a fine powder, it was transferred to a fresh 2 mL tube, added with 600 μL of cell lysis buffer and 50 μL protease K (200 mg.mL -1) and incubated at 56°C for 3 hours with slightly hand shaking. 200 μL 5 M potassium acetate was added to the mixture and incubated on ice bath for 10 minutes. After centrifugation at 10000 rpm and 4°C for 10 minutes, the supernatant (700 μL) containing genomic DNA was extracted using 700 μL chloroform: isoamyl alcohol (24 : 1) to destroy proteins and the upper phase (500 μL) was added to 500 μL100% isopropanol to precipitate DNA. The DNA pellet was dissolved in TE buffer at pH 8.
ITS sequence of the fungal isolate NDVN01 was amplified by PCR using primer pairs as follow: ITS1F (5'-CTT GGT CAT TTA GAG GAA GTA A-3') and NL4 (5'-GGT CCG TGT TTC AAG ACG G-3'). The PCR mixture contained 2.5 µL 10X PCR buffer; 2 µL of 2 mM dNTP; 2 µL of 25 mM MgCl2; 1.5 µL genomic DNA (50 - 100 ng); 0.25 µL 5 unit Taq polymerase and 1 µL each primer (10 pmol), supplemented in 14.75 µL distilled water adjusted to a final volume of 25 µL. The thermocycler conditions were as follows: 95°C/5’; 30 cycles of (95°C/1’, 55°C/1’, 72°C/1’); 72°C/10’. The PCR products amplified from the genomic DNA with both primer ITS1F and NL4 were inserted into the cloning vector pJET1.2/blunt, resulting in pJITS and then sequenced. DNA sequencing was performed on ABI PRISM 3100 Avant Genetic Analyzer. Sequence obtained was compared with ITS sequences available in GenBank, using Clustal W method and a dendrogram was constructed to establish the taxonomic rank of the fungus (MegAlign DNAStar, Madison, WI, USA).
3.4. Enzyme Assay
CMCase activity was examined relatively by the halo radius of enzyme diffusion on agar plates containing 0.5% (w/v) CMC. After 120 hours of growth in the basal medium containing 0.5% (w/v) CMC, 50 µL of the culture supernatant was dropped into small holes (0.8 cm diameter) on agar plates and incubated at 4°C for overnight for enzyme diffusion then at 37°C for 6 - 8 hours and stained with 1% (w/v) Lugol’s solution.
The CMCase activity towards CMC was measured in terms of release of reducing sugars by DNS method (Miller 1959). The reaction mixture containing 0.25 mL of diluted enzyme and 0.5 mL of 1% (w/v) CMC in 100 mM sodium acetate buffer at pH 5 was incubated at 50°C for 10 minutes and stopped by adding 0.75 mL of 1% (w/v) DNS reagent, followed by boiling for 10 minutes. The mixture was cooled down at room temperature and developed color was read at 575 nm on a UV-2500 spectrophotometer (Labomed Inc., Culver City, California, USA). The blanks were prepared as the samples but DNS was added before CMC. Glucose was used as the standard reduced sugar for concentration estimation. One unit of CMCase activity is deﬁned as the amount of enzyme that remove 1 μmol glucose.min -1 under the standard assay conditions.
3.5. Native Polyacrylamide Gel Electrophoresis
To detect molecular mass and CMCase activity, native polyacrylamide gel electrophoresis (PAGE) was performed using 12.5% polyacrylamide gel (Laemmli 1970) containing 0.2% CMC (w/v) using Biometra equipment (Göttingen, Germany). The native gel was submerged in sterile distilled water for 30 minutes and then in 1% (w/v) Triton X-100 for 1 hour at room temperature. The gel was then transferred into 50 mM sodium acetate buffer at pH 4.5 for 30 minutes and incubated overnight at room temperature. The gel was stained in a solution of 1% (w/v) Congo Red for 30 minutes, and destained in 1 M sodium chloride for 15 minutes. The activity band was clearly visible as yellowish clearances against a deep red background after 10 minutes of destaining.
3. 6. Time Course of CMCase Production
Time course of the CMCase production was varied from 24 to 216 hours at 28°C.
3. 7. Initial Medium pH
To determine initial medium pH optimum for the CMCase production, Peniophora sp. NDVN01 was grown in a 100 mL ﬂask containing 30 mL of the basal medium at different pH ranges from 4 to 10 which was adjusted by using 1 N HCl or 1 N NaOH.
3.8. Culture Temperature
To determine optimum temperature for the CMCase production, Peniophora sp. NDVN01 was grown at different temperature from 22 to 37 °C on basal medium at pH 7.
3.9. Inducer Source and Concentration
The effect of substrates as inducer including CMC, coffee outerskin, coir, corncob, peanut shells, rice straw, sawdust, and sugarcane bagasse at the concentration of 0.6% (w/v) on CMCase production was studied. The concentration of the substrate as inducer for the highest CMCase production by Peniophora sp. NDVN01 was varied from 0.1 to 1% (w/v).
3.10. Potato Infusion Concentration
To determine the influence of potato infusion on the CMCase production, Peniophora sp. NDVN01 was grown in 100 mL shaking flasks containing 30 mL of the medium with potato infusion at various concentrations from 10 to 100% (v/v), at 28°C and pH 7, with agitation of 200 rpm for 120 hours.
3.11. Carbon Source and Concentration
The effect of various additional carbon sources including CMC, coffee outerskin, coir, corncob, peanut shells, rice straw, sawdust, sugarcane bagasse, galactose, glucose, lactose, mannose, sucrose, and xylose at the concentration of 0.2% (w/v) on the CMCase production was studied. Peniophora sp. NDVN01 was grown in 100 mL shaking flasks containing 30 mL of the medium with 80% of potato infusion, 0.5% paper pulp (w/v) and 0.2% (w/v) of different carbon sources. The concentration of the carbon source giving the highest CMCase production varied from 0.1 to 1.4% (w/v).
3.12. Nitrogen Source and Concentration
The effect of various additional nitrogen sources including beef extract, fish meal, meat extract, peptone, soybean meal, yeast extract, ammonium hydrogen phosphate, ammonium nitrate, ammonium sulphate, and potassium nitrate at the concentration of 0.2% (w/v) was investigated. The concentration of the nitrogen giving the highest CMCase production varied from 0.1 to 1% (w/v).
3.13. Mineral Source and Concentration
The effect of various mineral sources including BaCl2, CaCO3, FeSO4, KCl, and MgSO4 at the concentration of 0.05 to 0.2% (w/v) was studied.
3.14. Statistical Analysis
All measurements were carried out in triplicate with the resulting values being the mean of the cumulative data obtained.
4.1. Identiﬁcation of the Fungus Peniophora sp. NDVN01
Among 5 basidiomycetes, Peniophora sp. NDVN01 showed the highest CMCase production rate on agar plate containing 0.5% of CMC (Figure 1) and was selected for optimization of culture conditions and medium components for the CMCase production.
The basidiomycete isolate NDVN01 was identified based on the sequence variation region containing 18S ribosomal RNA gene (partial sequence), internal transcribed spacer 1, 5.8S ribosomal RNA gene, internal transcribed spacer 2 (complete sequence), and 28S ribosomal RNA gene (partial sequence). The ITS sequence consisted of 1255 bp from the basidiomycete isolate NDVN01 showed maximum identity of 93.7 to 99.2% with those from Peniophora strains (Figure 2).
Based on the morphological analyzes (data not shown) and the sequence variations present in internal transcribing spacer (ITS) region, the basidiomycete NDVN01 was identified as Peniophora and named as Peniophora sp. NDVN01. The sequence was deposited in GenBank with an accession number of JF925333 for Peniophora sp. NDVN01.
4.2. Time Course of CMCase Production
The CMCase production by Peniophora sp. NDVN01 increased slowly from 0.27 ± 0.06 U.mL -1 (12%) at 24 hours to 0.71 ± 0.03 U.mL -1 (31%) at 72 hours, then linearly to the maximum of 2.32±0.07 U.mL -1 (100%) at 120 hours (5 days) of cultivation (Figure 3) and gradually decreased to 1.44±0.13 U.mL -1 (62%) at 216 hours. On the native PAGE, the supernatant of Peniophora sp. NDVN01 showed a CMCase-activity stained band with a molecular mass of 32-33 kDa (Figure 4).
4.3. Effect of Physiological Parameters on CMCase Production
4.3.1. Effect of Initial pH
Initial medium pH is one of the most critical physical factors affecting both the mycelial growth and production of extracellular enzymes by microbial strains. The CMCase production by Peniophora sp. NDVN01 was influenced by the initial pH of the LSC medium, that gradually increased from 1.17±0.06 U.mL -1 (45%) to the maximum of 2.62±0.17 U.mL -1 (100%) at the initial pH of 7 (Figure 5 A). Increasing the initial medium pH from 7 to 10 showed a rapid decrease in the CMCase production to 0.39±0.05 U.mL -1 (15%) (Figure 5 A).
4.3.2. Effect of Temperature
As initial medium pH, temperature is one of the most important physical variable affecting both the mycelial growth and production of extracellular enzymes by microbial strains. The CMCase production by Peniophora sp. NDVN01 gradually increased from 2.25±0.14 U.mL -1 (75%) to the maximum amount of 2.99±0.13 U.mL -1 (100%) at 28°C and dramatically decreased to 0.61±0.10 U.mL -1 (20%) at 33°C (Figure 5 B).
The maximal CMCase production was obtained at least within 5 days by Peniophora sp. NDVN01 (in this study) and V. volvacea (12), within 6 days by Bjerkandera adusta UAMH 8258 (19) and V. volvacea (20), even longer within 8 days by Pycnoporus sanguineus CEIBMD01 (19). In contrast, under solid-substrate culture conditions, basidiomycetes produced maximum amount of CMCase within 7 days by Peniophora sp. NDVN01 (21) or 11 days by Fomitopsis sp. RCK2010 (14).
The optimal pH for the CMCase production by Peniophora sp. NDVN01 was coincident with that in previous studies (21). Peniophora sp. NDVN01 produced maximum CMCase in solid-substrate culture at the initial pH of 7 (21), and two isolates of wood-decay basidiomycetes from the Sonoran Desert produced alkaline-active cellulases at optimal level when cultivated at pH 7. But other basidiomycetes B. adusta, P. sanguineus (19), V. volvacea (12) and Fomitopsis sp. RCK2010 (14) showed optimal CMCase production at lower initial medium pH of 5-6.
Most basidiomycetes including B. adusta, P. sanguineus (19), wood-decay basidiomycetes (22), Fomitopsis sp. RCK2010 (14) and Peniophora sp. NDVN01 (21) and also in this study produced maximum CMCase at a moderate temperature from 27 to 30°C. But the fungus V. volvacea showed maximum CMCase production at higher temperature, specifically at 37°C (12).
In this study, amongst all tested inducers, pulp was found to be the inducer for the maximum amount of CMCase production. In contrast, paper pulp did support neither growth nor enzyme production by two Sonoran Desert basidiomycetes (22). Other studies reported that Avicel microcrystalline cellulose showed the highest induction of CMCase production by V. volvacea (12).
Different basidiomycetes used different carbon sources for maximum CMCase production. Under solid state fermentation, Peniophora sp. NDVN01 (21) and Fomitopsis sp. RCK2010 (14) produced maximum CMCase using corncob and wheat bran as carbon source, respectively. Under liquid state fermentation, for the highest CMCase production, Peniophora sp. NDVN01 (in this study), two basidiomycetes from the Sonoran Desert (22), Phlebia gigantea (23), Fomes fomentarius IBB, Pleurotus ostreatus IBB 10, Pseudotremella gibbosa IBB 22 (24) used rice straw, wheat bran, carboxymethyl cellulose, residue of ethanol production from wheat grains, peach pomace, and mandarin peels as inducers, respectively. In general, sugars as carbon sources decreased the CMCase production by Peniophora sp. NDVN01 (in this study) and P. gigantea (23) since in presence of these simple carbohydrates, the cellulose production was repressed.
Peniophora sp. NDVN01 showed the maximum CMCase production in the medium containing ammonium hydrogen phosphate under liquid state fermentation (in this study) as well as under solid state fermentation (21). Fomitopsis sp. RCK2010 (14) produced maximum CMCase in the medium containing urea under solid state fermentation.
Metal ions K+ and Ca2+ seemed to be important for the CMCase production by Peniophora sp. NDVN01 but Mg2+, Fe2+ and Ba2+ repressed the enzyme production which was also found for enzyme production by Rhizopus oryzae (25).
The basidiomycete isolate NDVN01 was identified as Peniophora sp. NDVN01 and produced a CMCase with a molecular mass of 32-33 kDa at maximum level when grown at 28°C, with the initial pH of 7 within 120 hours of cultivation in the optimal medium containing 80% (v/v) of potato infusion, 0.6% rice straw as additional carbon source and 0.2% (w/v) (NH4)2HPO4 as additional nitrogen source, and 0.5% (w/v) pulp as inducer. Under the optimal conditions, the amount of CMCase produced by Peniophora. NDVN01 (24.65±0.37 U.mL -1) was 8.6 times higher than the amount before optimization (2.87±0.28 U.mL -1).
There are no acknowledgments.
Implication for health policy/practice/research/medical education: Cellulases have a broad application in biodegradation of natural celluloses (rice straw, sugarcane bagasse, corn cob, stalk, etc.) to produce bioethanol, thus to produce novel cellulases with better properties are the aim of many studies. This study focused on identification of a new basidiomycte isolate NDVN01 and optimization of the culture conditions and medium components for the CMCase production by the Basidiomycete peniophora sp. NDVN01 under liquid state fermentation.
Authors’ Contribution: DTQ designed, provided consultation, supervised the study, analyzed data and wrote the manuscript. DKT performed the experiments, analyzed data and wrote the manuscript. TTHN performed experiments. TTD and NMN provided consultation.
Financial Disclosure: There is no financial disclosure.
Funding/Support: The study was supported by the Master Program of Development and Application of Biotechnology in Agriculture and Rural Development Towards 2020, Vietnam Ministry of Agriculture and Rural Development (Project: Production and application of highly qualitative multienzyme products by recombinant microbes to improve the effective use of animal feed, 2007-2010).