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Jan 6, 2015 - Eight fungal strains were isolated from compost and forest litter of an Indo-Burma Biodiversity hotspot and were evaluated for their lig...
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Rice Straw Based Evaluation of Lignolytic and Cellulolytic Capabilities of Novel Strains of Saprophytic Fungi from Indo-Burma Biodiversity Hotspot Yogesh B. Chaudhari,† Narayan C. Talukdar,† Nirab C. Adhikary,‡ Mohan C. Kalita,§ and Mojibur R. Khan*,† †

Life Sciences Division, Institute of Advanced Study in Science and Technology (IASST), Guwahati, 781035, India Physical Sciences Division, Institute of Advanced Study in Science and Technology (IASST), Guwahati, 781035, India § Department of Biotechnology, Gauhati University, Guwahati, 781014, India ‡

S Supporting Information *

ABSTRACT: Superior lignocellulolytic microbes are useful for efficient bioconversion of plant biomass. Eight fungal strains were isolated from compost and forest litter of an Indo-Burma Biodiversity hotspot and were evaluated for their lignolytic and cellulolytic capabilities. X-ray diffraction (XRD) study was performed to test their effect on cellulose crystallinity of rice straw. Scanning electron spectroscopic (SEM) and Fourier transform-infrared (FT-IR) spectroscopic analysis were performed to study the surface morphology and chemical changes occurred in the fungi treated rice straw, respectively. The fungus Fusarium equiseti strain TWRF-10 showed endoglucanase, exoglucanase, and endoxylanase activity of 99.19, 87.39, and 166.81 IU g−1 rice straw, respectively, whereas Penicillium simplicissimum strain TRF-27 showed laccase and Mn peroxidase activity of 248.53 and 168.5 IU g−1 rice straw, respectively. The strain TRF-27 caused a 65% reduction in cellulose crystallinity. These strains should be further tested for industrial use.

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search is still on to find efficient microbes for bioconversion of lignocellulosic biomass. The diversity of lignocellulolytic microbes from North-East India (Indo-Burma biodiversity hotspot) are still to be explored. This study aimed to isolate efficient cellulolytic fungal strains from compost and forest litter of North-East India and to test their efficacy in degradation of rice straw. Eight fungal strains of three different genera and five species were evaluated based on their lignolytic and cellulolytic enzymes. The fungi treated rice straw samples were further evaluated by scanning electron microscopy (SEM), X-ray diffraction (XRD) analysis, and Fourier transform-infrared (FTIR) spectroscopy to study the structural and chemical changes.

INTRODUCTION Lignocellulosic biomass is composed of cellulose and hemicellulose, two major structural components of plant cell wall, which are connected by aromatic polymers of lignin.1 Efficient degradation of plant biomass requires efficient enzymes to act on lignin and crystalline cellulose. A set of lignolytic enzymes, namely, laccases (EC 1.10.3.2), manganese peroxidases (EC 1.11.1.3), lignin peroxidases (EC 1.11.1.4), versatile peroxidases (EC 1.11.1.16), and other accessory enzymes (aryl alcohol oxidase, glyoxal oxidase, etc.) are required for degradation of lignin.2 The enzymes belonging to glycosyl hydrolase family (EC 3.2.1) hydrolyze the complex cellulose into simple sugar. The enzymatic hydrolysis of lignocellulose requires the synergetic action of cellulase complex which includes endo(1,4)-β-D-glucanase (EC 3.2.1.4), exo-(1,4)-β-D-glucanase (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21). Endoglucanases act on the internal β-1,4-glycosidic linkage randomly along the cellulose fiber to release protruding ends for the action of exoglucanase to release two sugar molecules (cellobiose) for the action of β-glucosidase to release glucose units.3 Hemicelluloses with basic structural component, mainly xylans are branched heteropolysaccharide of β-1,4-xylopyranosyl units that can be substituted with arabinosyl, glucuronyl acetyl residues. The hydrolysis of hemicelluloses is accomplished by endoxylanases, β-xylosidases, arabinofuranosidases, α-glucuronidases, and acetyl esterases.4 Many microorganisms, including fungi and bacteria such as Trichoderma, Aspergillus, Penicillum, Fusarium verticillioides, Sclerotinia homoeocarpa, Chrysoporthe cubensis, Aquitalea sp., Bacillus sp., Burkholderia sp., Cupriavidus sp., Gordonia sp., etc. are known to produce cellulolytic enzymes.5−11 However, the © XXXX American Chemical Society

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MATERIALS AND METHODS

Substrates (Carboxymethyl cellulose, birch wood xylan, bovine serum albumin) were purchased from Himedia Pvt. Ltd., India. Avicel PH101, ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), manganese sulfate, and veratryl alcohol were purchased from SigmaAldrich. Glucose and cellobiose were purchased from Merck Millipore. Filter paper (Whatman no. 1 for determination of cellulase activity) was purchased from Whatman (Whatman Limited, England). The proven cellulolytic fungus Fusarium solani strain MTCC 10158 was procured from Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India, and used in this research as a reference strain. Rice straw (Oryza sativa L. cultivar Ranjit) was collected from a local farmer near Guwahati, Assam. Isolation of Fungi. Forest litter and compost samples were collected from different sites of North-East India during April 2012− Received: September 19, 2014 Revised: January 2, 2015

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Energy & Fuels Table 1. Fungal Strains with Their Accession Numbers and Origins fungal strains Fusarium equiseti strain MNP-C Fusarium solani strain ONP13512 Fusarium equiseti strain TWRF-10 Mucor circinelloides strain BLRF-115 Fusarium equiseti strain MBL115 Penicillium citrinum strain RWS-30 Penicillium simplicissimum strain TRF-27 Penicillium citrinum strain BLPNP-17

Microbial Repository Centre (MRC) accession no.

NCBI accession no.

MRC 9722

KJ774560

compost

MRC 9723

KJ801775

MRC 9725

KJ801777

forest litter compost

MRC 9726

KJ801778

MRC14702

KJ801779

MRC 9822

KJ801781

MRC 9732

KJ801784

MRC 9733

KJ801785

origin

forest litter compost forest litter compost forest litter

location Manas National Park (MNP), Assam, India (26°45′3.93″N 91°0′3.94″E) Orang National Park (ONP), Assam, India (26°33′1.49″N 92°19′57.37″E) Tawang Reserve Forest (TWRF), Arunachal Pradesh, India (27°34′14.06″N 91°52′28.52″E) Bomdila Reserve Forest (BLRF), Arunachal Pradesh, India (27°15′39.16″N 92°25′15.10″E) Bomdila Reserve Forest (BLRF), Arunachal Pradesh, India (27°15′39.16″N 92°25′15.10″E) Rowa Wildlife Sanctuary (RWS), Tripura, India (24°17′35.48″N 92°9′55.14″E) Tura Reserve Forest (TRF), Meghalaya, India (25°31′11.00″N 90°13′12.00″E) Balphakram National Park (BLPNP), Meghalaya, India (25°14′35.68″N 90°54′15.21″E)

8 days. Treatment with minimal medium without conidia served as negative controls. Culture supernatant was extracted by adding 10 mL of sterile double distilled water. The tubes were incubated at 28 °C at 150 rpm for 1 h and centrifuged at 10 000 rpm for 20 min. The clear supernatant was collected and used for enzyme assays and protein analysis. The total fungal biomass was determined as described earlier by Augustine et al.18 Three replicate flasks were used for each treatment, and the experiment was conducted twice. Enzyme Assays. The crude supernatant was used for the enzyme assays. Total protein content in the supernatant was determined by the Bradford method.19 Laccase activity was determined by using 0.5 mM 2.2-azino-bisethylbenthiazolina (ABTS) in 0.1 M sodium acetate buffer, pH 4.5 (ε = 29 300 M−1 cm−1) as described earlier by Munoz et al.20 Manganese peroxidase activity was determined by using 1 mM MnSO4 in 50 mM sodium malonate buffer (pH 4.5) as described earlier by Wariishi et al.21 Lignin peroxidase activity was determined by using 4 mM veratryl alcohol in 20 mM sodium succinate buffer (pH 4.5) as described earlier by Tien et al.22 The activity of total cellulase (filter paper activity, FPA), endoglucanase, exoglucanase, β-glucosidase, and endoxylanase in the supernatant was determined according to the methods recommended by the International Union of Pure and Applied Chemistry (IUPAC) commission on biotechnology.23 One unit enzyme activity was defined as 1 μmol product formed per min under the assay conditions and expressed in units per gram of rice straw (IU g−1 rice straw). Enzyme analysis was carried out twice for each of the three replicate samples. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis. Crude supernatant was subjected to SDSPAGE in a 10% resolving and 5% stacking gel.24 After electrophoresis, the gel was stained with Coomassie brilliant blue R-250. The molecular weight of the proteins was determined using protein marker (New England Biolabs, U.K.). Scanning Electron Spectroscopy (SEM). SEM was performed to study the morphological changes in the rice straw upon SSF with the fungi. Fungi treated dried straw samples were coated with gold− palladium25 and then observed under FE-SEM (ΣIGMA-VP, Carl Zeiss, Germany) at a voltage of 5 kV. X-ray Diffraction (XRD) Analysis of Fungi Treated Rice Straw. The fungal conidial suspension was produced in mung bean broth and was resuspended in the minimal medium at a concentration of 106 conidia mL−1. A volume of 100 μL of the conidial suspension was inoculated onto a piece of rice straw of size 2 cm × 2 cm and incubated at 28 °C for fungal growth. After 7 days of incubation, the samples were air-dried at room temperature for 3 days. The fungal mycelia from the air-dried rice straw was removed gently with the help of a brush. Rice straw was mounted on a glass slide with the help of adhesive tape along the borders so that it adheres uniformly on the glass slide. The samples were subjected to XRD analysis (Bruker-D8 Advance, Bruker, Germany). The radiation was with the goebel mirror in the primary optic at a wavelength of 0.1541 nm. The X-ray unit was

June 2012 (Table 1). The samples were sieved through 4 mm mesh to remove plant debris and stored at 4 °C until use. Cellulolytic fungi were isolated from the samples by plating their serial dilutions onto agar plates containing M-9 minimal medium with 0.2% carboxymethyl cellulose (CMC) of composition (%; wv−1): Na2HPO4·7H2O 1.28, KH2PO4 0.3, NaCl 0.05, NH4Cl 0.1, MgSO4 0.0492, CaCl2 0.0014, CMC 0.2, agar 1.5 (Himedia Pvt. Ltd., India). The pH was adjusted to 7.2. The plates were incubated at 28 °C. Single colonies were subcultured onto CMC-agar plates, and mycelia were stored in 10% glycerol at −80 °C. Pure cultures were deposited in the Microbial Repository Centre (MRC), Institute of Bioresources and Sustainable Development (IBSD), Imphal, India, and the accession numbers were obtained (Table 1). Isolation of DNA, PCR Amplification of Internal Transcribed Spacer (ITS), and Sequencing. Fungal DNA was extracted using the CTAB method described earlier by Jasalavich et al.12 The internal transcribed spacer (ITS) region of fungal DNA was amplified using the universal ITS primers, ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′).13 PCR reaction was performed in a 25 μL volume in a thermal cycler (Mastercycler Nexus gradient, Eppendorf, Germany). Each PCR reaction contained a final concentration of 1× standard buffer, 1.5 mM MgCl2, 0.2 μM each primer, 0.2 mM dNTPs, and 0.25 U Taq DNA polymerase (SigmaAldrich) and 25 ng of template DNA. The PCR program consisted of 95 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s, and finally at 72 °C for 7 min. PCR products were separated in a 1.2% agarose gel along with 100 bp DNA ladder and visualized under BioDoc-It Imaging System (UVP). PCR products were purified using a PCR clean up kit (Sigma-Aldrich). Purified PCR products were sequenced in both the directions with commercial service provider Xcelris Genomics (Ahmedabad, India). ITS sequences were subjected to BLAST analysis14 using the National Centre for Biotechnology Information (NCBI) blast resource (www.ncbi.nlm.nih. gov). The eight fungal strains under study were identified as Fusarium equiseti strains MNP-C, TWRF-10, and MBL-115, Fusarium solani strain ONP-13512, Mucor circinelloides strain BLRF-115, Penicillium citrinum strains RWS-30 and BLPNP-17, and Penicillium simplicissimum strain TRF-27. The phylogenetic relationship among the fungi was determined using a neighbor-joining method described earlier by Saitou and Nei.15 ITS sequences were submitted to NCBI and the accession numbers were obtained (Table 1). Solid State Fermentation of Rice Straw with the Fungi. Rice straw was ground in a mixer grinder (HL1632, Philips, India) and passed through a sieve (2 mm pore size). One gram of ground straw was mixed with a 3 mL of minimal medium16 and autoclaved at 121 °C for 15 min in a 50 mL conical flask. Fungal conidia was produced in mung bean broth as described by Brennan et al.17 and was resuspended in the minimal medium at a concentration of 106 conidia mL−1. The conidial suspension was mixed with the sterilized rice straw at a final 91% moisture (vw−1) content and was incubated at 28 °C for B

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Energy & Fuels Table 2. Activity of the Lignocellulolytic Enzymes of the Fungal Strainsa enzyme activity (IU g‑1 rice straw)

Mn peroxidase

endoglucanase

exoglucanase

β-glucosidase

endoxylanase

0.00

141.1 ± 4.60

61.23 ± 12.51

28.22 ± 0.22

76.67 ± 3.97

7.23 ± 1.43

25.75 ± 0.47

25.12 ± 1.06

127.86 ± 7.69

0

0

33.55 ± 0.79

32.32 ± 1.75

6.61 ± 0.72

18.28 ± 0.56

113.16 ± 5.75

13.5 ± 9.74

133.2 ± 5.90

0

26.70 ± 1.74

26.31 ± 1.37

41.60 ± 3.18

17.27 ± 1.19

62.44 ± 6.04

42.32 ± 7.03

51.2 ± 5.90

0

16.73 ± 0.46

99.16 ± 0.93

87.39 ± 0.11

0

166.81 ± 2.0

0

99.0 ± 5.90

0

28.73 ± 0.34

41.52 ± 2.04

7.28 ± 0.70

6.43 ± 0.58

40.08 ± 4.40

8.10 ± 1.56

160.5 ± 25.60

0

29.87 ± 3.42

49.46 ± 1.67

8.75 ± 1.06

33.38 ± 0.36

96.41 ± 6.35

11.71 ± 7.03

122.9 ± 15.80

0

31.90 ± 1.62

58.17 ± 3.80

7.36 ± 1.27

8.77 ± 0.97

100.69 ± 6.93

248.53 ± 14.29

168.5 ± 11.50

0

29.11 ± 1.59

79.71 ± 4.00

6.10 ± 0.19

10.80 ± 0.13

60.28 ± 3.68

3.6 ± 0.90

161.6 ± 18.60

0

29.09 ± 1.11

36.01 ± 4.05

42.14 ± 2.84

16.94 ± 0.49

28.64 ± 1.62

fungal strain Fusarium solani MTCC 10158 Fusarium equiseti strain MNP-C Fusarium solani strain ONP-13512 Fusarium equiseti strain TWRF-10 Mucor circinelloides strain BLRF-115 Fusarium equiseti. strain MBL-115 Penicillium citrinum strain RWS-30 Penicillium simplicissimum strain TRF-27 Penicillium citrinum strain BLPNP-17 a

FPase (filter paper) activity

lignin peroxidase

laccase

Values are mean of three replicates ± standard error.

(248.53 and 168.5 IU g−1 rice straw, respectively). None of the tested fungal strains had lignin peroxidase activity except the reference cellulolytic fungus F. solani strain MTCC 10158. Total cellulase (FPase) activity was 16.73−33.55 FPU g−1 rice straw (Table 2). Endoglucanase, exoglucanase, β-glucosidase, and endoxylanase activity of the fungal strains was 26.31− 99.16, 6.1−87.39, 6.43−33.38, and 25.12−166.81 IU g−1 rice straw, respectively (Table 2). The F. equiseti strain MNP-C had the highest total cellulase activity (33.55 FPU g−1). The F. equiseti strain TWRF-10 had highest exoglucanase (87.39 IU g−1), endoglucanase (99.19 IU g−1), and endoxylanase (166.81 IU g−1) activity. The F. equiseti strain MBL-115 showed the highest β-glucosidase activity (33.38 IU g−1). Interestingly, the F. equiseti strain TWRF-10 had a higher activity of laccase, endoglucanase, exoglucanase, and endoxylanase, compared to the reference cellulolytic fungus F. solani strain MTCC 10158 (Table 2). Genetic relatedness of the fungal strains was not reflected on their enzyme activities (Figure 1 and Table 2). Activity of the enzymes of the fungal strains was higher when compared to earlier reports on fungal conversion of rice straw. In a similar study on rice straw, Dar et al.27 reported that the fungus Thermoascus aurantiacus strain MTCC 375 had laccase,

operated at 40 kV and 40 mA. The samples were scanned over the angular 2θ range from 10 to 40°, under lock coupled mode. The data was analyzed using DIFFRAC.EVA software package (Bruker, Germany). The crystallinity of cellulose was calculated by peak height method earlier described by Segal et al.26 CrI(%) = [(I002 − Iam)/I002] × 100 where CrI is the crystalline index, I002 is the maximum intensity at 2θ = 22.5°, and Iam is the minimum intensity at 2θ = 18°. XRD analysis was carried out twice for each of the three replicate samples. The relative changes in the CrI of the fungi treated rice straw have been presented as % reduction of CrI compared to untreated rice straw (control). Fourier Transform-Infrared (FT-IR) Spectroscopic Analysis of Fungi Treated Rice Straw. The fungi treated rice straw samples were dried at room temperature and finely ground for FT-IR analysis. Spectra were obtained on a Nicolet 6700 FT-IR (Thermo Scientific) using a KBr pellet containing finely ground samples of fungi treated and untreated rice straw. A total 32 scans were recorded in the range of 4000−400 cm with a resolution of 4 cm−1. The data (% absorbance) of fungi treated rice straw of the selected wavenumbers corresponding to lignin, cellulose, and hemicellulose were noted. The data were corrected by subtracting the % absorbance of pure fungi (based on their total biomass) from raw fungi treated rice straw to avoid the interference from fungal mycelia (Table S1 in the Supporting Information). Statistical Analysis. The FT-IR data (% absorbance) was used for prinicipal component analysis (PCA). The data was non-normally distributed and was transformed to normal distribution within Minitab (Minitab 17.0, Minitab, Ltd. U.K.). The transformed data was subjected to principal component analysis (PCA) within the Statistical Package for the Social Sciences (SPSS 18.0, SPSS Inc., Chicago, IL). The principal components were extracted based on eigenvalue (value > 1) and rotated in Varimax (for orthogonal plotting). PCA was performed on the data sets to assess the degree of relatedness among the samples and to analyze the probable similarity in the actions of the fungal strains on rice straw.

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RESULTS AND DISCUSSION Lignolytic and Cellulolytic Enzymes of the Fungal Strains. Lignolytic and cellulolytic activities of the fungal strains are presented in Table 2. The P. simplicissimum strain TRF-27 had the highest laccase and Mn peroxidase activity

Figure 1. Phylogenetic relatedness of the fungal strains constructed based on DNA sequence of internal transcribed spacer (ITS) regions. C

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Figure 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the crude protein extracts obtained from solid state fermentation of rice straw.

Figure 3. Scanning electron microscopic (SEM) images of fungal colonization of rice straw. Untreated rice straw (A), and treated with F. equiseti strain MNP-C (B), F. solani strain ONP-13512 (C), F. equiseti strain TWRF-10 (D), M. circinelloides strain BLRF-115 (E), F. equiseti strain MBL-115 (F), P. citrinum strain RWS-30 (G), P. simplicissimum strain TRF-27 (H), and P. citrinum strain BLPNP-17 (I) were photographed in a scanning electron microscope under 1000× magnification.

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Energy & Fuels manganese peroxidase, and lignin peroxidase activity of 65, 125, and 500 IU g−1 rice straw, respectively. Similarly, Pasha et al.28 reported FPase, endoglucanase, exoglucanase, and β-glucosidase activity of the fungus Aspergillus niger strain CP1 to be 15, 140, 45, 24 IU g−1 rice straw. The fungi P. simplicissimum strain TRF-27 and F. equiseti strain TWRF-10 should be further studied to characterize their efficient lignolytic and cellulolytic enzymes and to clone the corresponding genes. Such genes may be used for genetic improvement of commercial microbial strains for lignocellulosic bioconversion. SDS-PAGE Analysis. The SDS-PAGE profiles of the secretory proteins of the fungal strains are presented in Figure 2. The protein bands with sizes 110 kDa and ∼38−40 kDa were found to be common for all the samples. Relatively faint protein bands were seen in some of the samples (strain TWRF10, RWS-30, and TRF-27). Though belonging to F. solani, the secretory proteins of the strain MTCC 10158 were different from ONP-13512 but similar to the P. citrinum strain BLPNP17. On the other hand, the closely related strains MNP-C and MBL-115 belonging to F. equiseti had similar secretory proteins. Despite low secretory proteins of the strain TWRF-10, the fungus had higher cellulolytic enzyme activity. Analysis of the Fungal Action on the Treated Rice Straw. SEM Analysis. The fungal colonization and resulting structural changes in rice straw were evaluated by SEM analysis. Characteristic mycelial growth and attachments of the fungal mycelia on the rice straw were observed for all the fungal strains (Figure 3). At 1000× magnification, cellulose microfibrils became visible in the fungi treated rice straw (Figure 3). Changes in the surface morphology of the fungi treated rice straw is caused by enzymatic hydrolysis due to production of extracellular cellulolytic enzymes by fungi and also due to the penetration of fungal mycelia.29,30 Liu et al.31 reported the occurrence of pores on fungal treatment of switchgrass. Phutela et al.32 reported that cellulose microfibrils were exposed in the fungi treated paddy straw due to formation of pores. Similarly Sun et al.33 also reported that gaps and many pores were observed in between the cellulose fibers of Trametes hirsuta yj9 pretreated corn stover. XRD Analysis. The effect of fungal action on the crystal structure of the cellulose of rice straw was studied by XRD analysis. In lignocellulosic biomass, cellulose is the crystalline component, while hemicellulose and lignin forms the amorphous component and XRD analysis provide the qualitative and semiquantitative measurements.34 XRD profiles of the fungi treated rice straw has been presented in Figure 4. The major peaks were observed at 15.4°, 16.2°, and 22.5°, which correspond to 101, 101̅, and 002 crystallographic planes of cellulose I, respectively.35 The crystallinity index (CrI) of the untreated rice straw was 59.84% (Figure 4). On fungal treatment, CrI was reduced by 19−65%. Treatment with the P. simplicissimum strain TRF-27 caused the highest reduction of CrI (65%), followed by P. citrinum strain RWS-30 (62%). The decrease in CrI in the fungi treated rice straw indicates degradation of crystalline cellulose, after readily available amorphous components, including lignin and hemicellulose.36 FT-IR Analysis. FT-IR spectroscopy was performed to investigate the chemical changes occurred in the fungi treated rice straw after SSF. The FT-IR spectra of the fungi treated and untreated rice straw has been presented in Figure 5. The peaks which are related to lignin and cellulose can be summarized as follows. The broad absorption at 3420 cm−1 is due to the stretching of H-bonded OH groups.37 The band at 2920 cm−1

Figure 4. X-ray diffraction (XRD) spectra of fungal colonized rice straw. Crystallinity index (CrI) was calculated as earlier described by Segal et al.26

is associated with C−H aliphatic axial deformation in CH2 and CH3 groups of cellulose, hemicellulose, and lignin.38 The peaks at 1735 and 1642 cm−1 are of an unconjugated carbonyl group of hemicelluloses39 and of hydroxyl bending of the xylan,40 respectively. The peak at 1512 cm−1 is assigned for CC stretching of the aromatic ring of lignin. The peaks at 1425 cm−1 and 1375 cm−1 are due to CH2 and CH bending mode of cellulose, respectively.39 The peak at 1318 cm−1 is ascribed to the CH2-wagging vibrations in cellulose and hemicellulose.41 The peaks at 1163 and 897 cm−1 are assigned for hydroxyl bending of amorphous cellulose.39 The FT-IR spectra of the fungi treated and untreated rice straw were different (Figure 5). This shows the changes in the functional groups of lignin and celluloses of rice straw after fungal treatment. The broad peak at 3420 cm−1 which is associated with the stretching of H-bonded OH groups increased in absorbance with narrowing due to fungal treatment which indicates the changes in hydrogen bonding energy occurred in enzymatic hydrolysis.41 The peak intensity at 2920 cm−1 reduced in the MNP-C, MBL-115, and TWRF-10 treated rice straw which was due to combined degradation of the lignocellulosic content of rice straw.42 An interesting change was that the distinct absorption peak at 1735 cm−1 associated with the hemicellulose disappeared in the fungi treated rice straw compared to untreated straw. The disappearance of 1735 cm−1 was due to the removal of a large portion of hemicelluloses and pectin content during SSF43 which is attributed to their higher endoxylanase activities (Table 2). A peak at 1512 cm−1 due to lignin was significantly enhanced in the fungi treated rice straw samples compared to the untreated sample due to the removal of hemicelluloses and/or may be redeposition of degraded lignin onto rice straw.43 Significant changes were seen at bands 1375, 1318, 897, and 670 cm−1 E

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Figure 5. Fourier transform-infrared (FT-IR) spectra of degraded rice straw after treatment with the fungi. Rice straw was treated with (A) Fusarium solani strain MTCC 10158 (MTCC 10158), (B) F. equiseti strain MNP-C (MNP-C), (C) F. solani strain ONP-13512 (ONP-13512), (D) F. equiseti strain TWRF-10 (TWRF-10), (E) Mucor circinelloides strain BLRF-115 (BLRF-115), (F) F. equiseti strain MBL-115 (MBL-115), (G) Penicillium citrinum strain RWS-30 (RWS-30), (H) P. simplicissimum strain TRF-27 (TRF-27), and (I) P. citrinum strain BLPNP-17 (BLPNP-17) and subjected to FT-IR analysis. Changes in the selected bands corresponding to lignocellulosic material were observed (J).

5). On the other hand, the strains of Penicillium and Mucor showed similar effects on rice straw irrespective of their species. A PCA analysis based on the normalized data of the absorbance values of the selected wavenumbers corresponding to lignin, cellulose, and hemicellulose reveals that the characteristic action

which are associated with the typical cellulose and hemicellulose regions of rice straw. Overall, the reference strain MTCC 10158 treatment caused most structural changes in the rice straw followed by TWRF-10 treatment. The three strains of F. equiseti had similar effect on rice straw degradation (Figure F

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Figure 6. Principal component analysis (PCA) based on FT-IR data of fungi treated rice straw. Rice straw was subjected to solid state fermentation with the fungi, Fusarium solani strains MTCC 10158 and ONP-13512, Penicillium citrinum strains RWS-30 and BLPNP-17, P. simplicissimum strain TRF-27, Mucor circinelloides strain BLRF-115, F. equiseti strains MNP-C, MBL-115, and TWRF-10. The degraded rice straw was subjected to FT-IR analysis and the normalized data after correction of interference due to fungal biomass was subjected to PCA analysis.

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of the fungal strains on rice straw is linked to their genetic relatedness (Figures 1 and 6). Two components with high variance (79% and 14%) were extracted (Figure 6). Two distinct clusters, one among the strains of F. equiseti and another among the strains of Penicillium were formed. These strains, though originated from geographically isolated places had similar action on rice straw as revealed by FT-IR analysis.

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Corresponding Author

*E-mail: [email protected]. Phone: +91-361-2273058. Fax: +91-361-2273062. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by the Department of Biotechnology, Government of India under Institutional Level Biotech Hub project (Grant BT/22/NE/2011 Sl. No. 74) and Ramalingaswami fellowship (Grant BT/RLF/Re-entry/29/2010). The authors thank Mr. Bedanta Gogoi and Dr. Devasish Chowdhury, IASST for their help in the FT-IR and SEM analysis.

CONCLUSION

Eight saprophytic fungal strains from composts and forest litters of North-East India were evaluated for lignocellulolytic activity. The fungi F. equiseti strain TWRF-10 and P. simplicissimum strain TRF-27 had high cellulase and ligninase activity, respectively. The overall chemical changes caused in rice straw due to the fungal treatment could not be explained by the conventional enzyme assays as revealed by XRD and FT-IR analysis. Degradation of lignocellulosic biomass is a complex process and, therefore, selection of efficient fungal strains may require methods for evaluation of their individual enzymes, synersism between them, and any other factor which may involve.

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AUTHOR INFORMATION

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

Figures showing Fourier transform-infrared (FT-IR) spectra of pure fungal mycelia, X-ray diffraction (XRD) spectra of representative pure fungal mycelia, and table showing fungal biomass after solid state fermentation with rice straw. This material is available free of charge via the Internet at http:// pubs.acs.org. G

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DOI: 10.1021/ef502119r Energy Fuels XXXX, XXX, XXX−XXX