Untreated GNS was purchased from local farmers in Puttaparthi, Anantapur, A.P, India. NaOH (Merck, >97%), Potato dextrose agar (Himedia), Proteose peptone (Himedia), (NH₄)₂SO₄ (SDFCL), KH₂PO₄ (Merck), Urea (Merck), MgSO₄・7H₂O (SDFCL), CaCl₂・2H₂O, (SDFCL), ZnSO₄・7H₂O (SDFCL), FeSO₄ (SDFCL, 99.5%), MnSO₄ (SDFCL, >98%), ZnCl₂ (SDFCL, >98%), Dinitro-salicylic acid (Sigma Aldrich), Cellulose (Sigma ALdrich), filter paper (Whatman No.1), Cellulase (Sigma Aldrich), β-glucosidase (Sigma Aldrich), double distilled water is used in all experiments. AO (NCIM1212) purchased from NCIM, Pune. PS from Indian Institute of Horticulture Research, Bangalore and TH, isolated from Aegle marmelos, courtesy department of Biosciences, SSSIHL.

GNS was subjected to physicochemical treatments before being used for production of the cellulolytic enzyme system. GNS was washed thoroughly to remove dirt by repeating the steps such as soaking in water for an hour and filtering. It was then air dried and subsequently in hot air oven at 80˚C initially to remove the water. The samples were ground and sieved (BSS 25 - 16 mesh) to obtain a particle size of 600 - 1000 µm. GNS (200 g) was treated in 3000 ml of 1% (w/w) (0.25 M) Sodium hydroxide (NaOH) in boiling hot water bath. Biomass was allowed to cool followed by repeated washing in tap water to neutralize pH and rinsed in distilled water, after which it was air-dried. Pretreated GNS was stored in airtight containers at room temperature until used.

The composition of the GNS was analysed for extractives, lignin, glucan, xylan, and arabinan content by following the NREL standard protocols. In brief, sample was extracted first with deionized water for 24 h, and then with 95% ethanol for 24 h using a Soxhlet Extraction. The sum of water-soluble extractives and acid-soluble extractives was reported as total extractives content of the biomass. The extracted sample was dried in hot-air oven at 45˚C overnight. The extraction-free sample was hydrolysed using 72% (w/w) sulphuric acid at 30˚C for one hour, followed by dilution of the slurry with DI water to make the acid concentration of 4% (w/w) and autoclaved at 121˚C for 60 minutes. The completely hydrolysed biomass slurry was filtered using a medium porosity porcelain filtering crucible. The filtrate was used to measure glucose, xylose and arabinose content using HPLC, and calculated glucan, xylan and arabinan content. The filtrate was also used to measure acid-soluble lignin content using UV-VIS spectrophotometer. The solid residue in the filtering crucible was first dried in hot-air over at 105˚C and then in muffle furnace at 575˚C to measure acid-insoluble lignin. The sum of acid-soluble and acid-insoluble lignin was reported as total lignin content of the biomass [24].

The Mandel and Reese medium used for enzyme production had composition in g/L: Urea, 0.3; MgSO₄. 7H₂O, 0.3; peptone, 0.75; KH₂PO₄, 2.0; (NH₄)₂SO₄, 1.4; yeast extract, 0.25; CaCl₂・2H₂O, 0.4; and trace elements (mg/L): FeSO₄・7H₂O, 5.0; MnSO₄・7H₂O, 1.6; ZnSO₄・7H₂O, 1.4. The extracellular cellulases were extracted by adding 30 ml of citrate buffer (pH 5, 50 mM) to each stationary shake flask in rotary shake incubator (N-Biotek, NB-205V) at 4˚C, 60 RPM for 30 minutes. The contents were then filtered and analysed for filter paper activity (FPU/gds), CMCase (IU/gds) and β-glucosidase (IU/gds). Enzymatic assays were carried out using standard protocols as described in Brijwani et al. [9]

Three cultures Aspergillus oryzae (AO, NCIM1212, micro fungi), Pycnoporous sanguineus (PS, wood decaying macro fungi) and Trichoderma harzianum (TH, endophytic fungi) were used in this study. Cultures were used as mono, binary (1:1) and ternary combinations (1:1:1). Pretreated GNS (2 g) was maintained at 70% moisture content (wet basis) by using Mandel and Reese media [25]of pH 5.0 and sterilization was done in vertical top load autoclave (121˚C/15 psi gauge) for 20 minutes. Cultures were added as disks with Pasteur pipette for non-sporulating fungi and 10⁸ spores/ml suspension for sporulating fungi. The generation, propagation and maintenance of spore suspension are described in our earlier work. The conditions of temperature (30˚C), pH, moisture (70%), and incubation days of the SSF process used in this study were optimized in our earlier research [25]. SmF study was performed with same conditions as in SSF with respect to microorganisms used, temperature conditions and incubation period. However, the water content in SmF is at about 10 ml per 2 g of biomass along with 5 ml of Mandel and Reese media.

The enzyme treated biomass was washed thoroughly with de-ionised water and sterilized in autoclave for 30 minutes. The residual substrate was oven dried overnight at 80˚C. Physico chemical changes in the biomass substrate were studied using thermal, spectroscopic and surface morphological studies.

Under both SSF and SmF conditions, there is a continuous increase in FPU and CMCase until 6^th day; increase in β-glucosidase until the 4^th day, for all the culture combinations undertaken in this study. After the 6^th and 4^th day, the activity either stayed stable or declined, respectively. The authors had observed a similar trend in their previous study involving cellulase production from biomass under SSF and SmF conditions for individual and binary cultures [26]. The reason behind this trend could be attributed to the exhausting of available monosaccharide sugars, which are released by fungi during saccharification of oligosaccharides derived from cellulose. Decline of these sugars, especially glucose will signal the fungi to release more hydrolytic enzymes resulting in more sugars formation; however, the time interval at which the optimum enzyme activity occurs for these different classes of enzymes varies. This cycle would continue until all the sugars are consumed completely.

This explains why we observe rise in enzyme activity on 6^th day and again on 10^th day for CMCase and β-glucosidase. Cellulase (FPU/gds), CMCase (IU/gds) and β-glucosidase (IU/gds) of 40.5 ± 4.84, 192.8 ± 8.1 and 177.3 ± 6.9 are produced respectively for mono-culture (AO) on 2^nd day which is higher than reported [27]. Cellulase, CMCase and β-glucosidase activity in the range 74.0 - 154.2 (FPU/gds), 180.9 - 471.5 (IU/gds), and 180.6 - 956.6 (IU/gds), respectively, for combination of cultures in 6 - 10 days were observed in SSF (Figure 1). With mixed fungal cultures, we observed higher productivity over individual cultures. Earlier studies on agro wastes also established that mixed cultures (A. niger and T. reesei) exhibit synergism over mono cultures in SSF conditions [28].

Cellulase activity: In SSF condition (Figure 1(a)), highest activity of 154.2 ± 1.6 FPU/gds and 123.8 ± 3.7 FPU/gds was observed on the 6^th day for AO + PS and AO + PS + TH combinations respectively. In SmF conditions (Figure 1(b)), highest activity of 154.27 ± 4.7 FPU/gds and 139.05 ± 11.8 FPU/gds was observed on the 6^th day for AO + PS combination and monoculture of PS, respectively.

CMCase activity: The increase in CMCase activity was significant on 6^th day for SSF across mono and combination of cultures whereas independent PS released greater amounts of CMCase on 2^nd day that decreased gradually on 10^th day. The CMC activity of SSF (Figure 1(c)) for ternary combination of AO + PS + TH was maximum on 6^th day at 352.4 ± 2.1 IU/gds; and 10^th day at 471.5 ± 1.7 IU/gds for TH + PS. In SmF conditions (Figure 1(d)), the activity was only 179.9 ± 1.0 IU/gds (6^th day) for ternary combination of AO + PS + TH and 342.12 ± 34.5 IU/gds for TH + PS suggesting that SSF conditions are more favourable over SmF conditions. The reason could be the presence of glucose in fermentation medium in higher concentration thus causing catabolic repression. Although CMCase activity declined for PS after 48 hours, an increase in CMCase was observed in combination with AO and ternary combination of AO and TH. The increase was significant for ternary combination of fungi on 6^th day compared to other mono cultures.

β-glucosidase activity: β-glucosidase production was maximum on 4^th day for all the combinations of fungi for both SSF and SmF conditions. Highest value of 797.6 ± 46.5 IU/gds and 876.7 ± 19.8 IU/gds was observed for 10^th day in AO + PS + TH and AO + PS combination, respectively, in SSF condition (Figure 1(e)). In SmF condition (Figure 1(f)), 875.63 ± 26.4 IU/gds and 987.0 ± 64.2 IU/gds was observed for AO + PS + TH and AO + PS combination, respectively; suggesting that β-glucosidase production is promising in both SSF and SmF conditions.

From our studies (Table 1), we can deduce that to attain high activity of cellulase enzyme a ternary combination of AO + PS + TH cultures under SmF condition is conducive, whereas, monoculture of PO gave high activity of CMCase under SSF condition. Further, binary combination of AO + PS under SmF condition provided very high β-glucosidase activity. Our promising approach for targeted enzymes production is summarized in Table 1. Thus, we can conclude that depending on the particular class of enzyme required for production, an appropriate combination of fungal consortia and the method of fermentation will provide the desired high activity.

The FT-IR spectra of GNS samples indicated chemical changes in the biomass upon pretreatment and enzyme saccharification. Variation in the absorbance values provided understanding of changes in biomass substrate composition and partial removal of lignin. Absorbance values of the analysed peaks displayed a close trend of variation, which corresponds to the cellulose, hemicellulose and lignin content as shown by a vertical line in Figure 3 whose corresponding peaks assigned to functional groups are displayed in Table 4. The glycosidic linkage in hemicelluloses and lignin display a peak at 850 cm⁻¹ while the 917 cm⁻¹ peak was attributed to cellulose, hemicellulose and lignin’s glycosidic linkage. C-O valence vibration of cellulose displays a peak at 993 cm⁻¹. Peak at 1072 cm⁻¹ is due to glycosidic linkage C-O, C-C-O stretching of three polymers.

Asymmetric stretching of C-O-C in cellulose and hemicellulose could possibly result as peak at 1111 cm⁻¹. Intense peak at 1220 cm⁻¹ is due to C-O stretch in lignin due to alcohol groups. Syringyl ring of lignin displays a peak at 1322 cm⁻¹ due to C-O stretch clearly demonstrates decrease in lignin content from untreated GNS. Cellulose and hemicellulose in GNS showed an intense peak at 1400 cm⁻¹ and 1384 cm⁻¹ due to aliphatic C-H band. Dominant peak at 1590 cm⁻¹ is attributed to aromatic ring C = C stretch of lignin which changed after alkali treatment due to removal of lignin. The broad absorbance peak in 3400 - 3000 cm⁻¹ is due to surface hydroxyl groups stretching vibration and increased remarkably after alkali treatment indicating the exposure of cellulose and hemicellulose. Summary of functional groups present in the biomass and the corresponding absorbance values are given in Table 4. In our previous study, we observed that changes in the functional group intensities of the biomass help in comprehending the structural variation of biopolymers [35].

TGA analysis of the raw, alkali-treated and fungal-treated substrates was performed to test their thermal decomposition. Thermogravimetry curve which records weight change of the biomass as a function of temperature i.e., the thermal behaviour of biomass on pyrolysis. First derivative of TGA known as differential thermogravimetry curve (DTG) gives the rate at which the reaction is undergoing. The DTG thermogram in Figure 4 revealed prominently three peaks in the exothermic regions in agreement with previous studies [26].

Degradation of GNS happened in three stages. In stage one, at around 85˚C - 120˚C in which, bound water desorption occurs along with weakening of hydrogen bonds between the carbohydrates [36]. In stage two, degradation occurs around 200˚C - 350˚C where decomposition of low volatile carbohydrates such as cellulose and hemicellulose is observed. Hemicellulose starts to decompose earlier due to its linear polymer chain structure and short side chains and at about 325˚C, relatively more stable crystalline cellulose starts to degrade between 325˚C - 400˚C.

In Figure 4(a), raw GNS contain great amounts of lignin covering the cellulose and hemicellulose, whereas alkali treatment disrupts the lignin bonding with holocellulose. It is observed in Figure 4(b) where the exposed cellulose is resulting in a peak corresponding to cellulose degradation. In Figure 4(c), the effect of fungal treatment is observed with the loss of cellulose peak due to the action of cellulases on the alkali treated GNS. In stage three, above 400˚C, reveals higher volatiles degradation such as lignin which is relatively more stable than cellulose and hemicellulose. The process displays exothermic effect till 735˚C, corresponding to the char aggregation and the heat produced is the result of the degradation of biomass [37]. This is in correlation with the data reflected in biomass composition (Table 2) and XRD data.

Microbe fortified biomass consisting of the various combinations of fungal cultures in both SSF and SmF conditions were analyzed for surface morphological changes using SEM. Untreated GNS display high roughness with hollow cavities and after alkali treatment the surface smoothened indicating deconstruction of lignocellulosic linkages in Figure 5. A & B thereby suggesting that the cellulosic fibers are more exposed.

Further on enzymatic treatment with binary (AO + PS) and ternary cultures (AO + PS + TH) it was observed that the texture exhibited more porosity indicating bioconversion of cellulosic fibres as observed in Figure 5(c) and Figure 5(d). During alkali treatment, the cell walls collapses and the fibres are detached along with formation of pores, which is evident in AO + PS + TH combination of Figure 5(e). In ternary culture of GNS with AO + PS + TH Figure 5(f), the result of enzymatic action on GNS disturbed the integrity of surface, forming pronounced pits on the surface; thereby indicating that the ternary consortia was more efficient in the systemic breakdown of the heteropolymers present in the biomass.

Cellulase and CMCase production improved significantly till 6^th day in mixed culture fermentation whereas β-glucosidase production was produced in greater quantities. The alteration in the physico-chemical attributes in biomass composition due to alkali treatment and enzyme treatment was thoroughly studied using characterization techniques and found to be culture specific. The FT-IR spectroscopy along with X-ray crystallography studies of alkali treated and enzyme treated substrates established the changes in nature of holocelluloses. DSC and TGA revealed the changes in the structural integrity of biomass.