3. PREAMBLE TO THE DESCRIPTION

The embodiments of the present disclosure relates a novel process for delignification of biomass, especially lignocellulosic biomass and more particularly woody biomass involving a steam treatment, followed by laccase enzymatic treatment in presence of organic solvent. In the second embodiment this present invention includes generation of lignin derived feed stock chemicals from the lignin rich wastes. Such novel process is designed with core biocatalysts as laccase developed by us. Laccases (benzenediol: oxygen oxidoreductase EC 1.10.3.2) are multicopper containing enzymes, which have received much attention of researchers in last decades due to their ability to oxidize of broad range of compounds including phenols, polyphenols, aromatic amines, and non phenolic substrates. Laccases have also been used in variety of other important industrial applications including but not limited to

1. Delignification of pulp,

2. Bleaching of textile and carcinogenic dyes,

3. Detoxification of water and soils and

4. Removal of phenolics from wines etc.,

These plethora of industrial applications makes laccases one of the "greenest enzymes" of 21st century(Mate et al., 2013).

Laccases, albeit used in different industrial applications, this present invention pertains to the use of laccase particularly LccH secreted by a novel basidiomycetes fungi from the previous work of the inventors (Patent ref 645 CHE2015.CBR NO 2547) for delignification of lignocellulosic biomass and simultaneous generation of lignin derived high value products. Lignocellulosic biomass is the most abundant renewable resources on the earth consisting mostly of agricultural wastes, forestry residues and energy crops. The major composition of lignocellulosic biomass is cellulose (40-50%), hemicellulose (30-40%) and lignin (20-30%) (Blaschek et al., 2010). Cellulose is polymer of glucose and linked by IS1-4 glycosidic linkage and hemicelluloses are highly branched short polymer consisting of xylose, arabinose, glucose, galactose and mannose (Chandra et al., 2007). Lignin is the most complex polymer made of phenyl proponoid units and in the lignocellulosic biomass it is covalently linked with hemicelluloses and cellulose. Thus, to obtain hydrolysis of cellulose and hemicelluloses into fermentable sugars, the covalently bound lignin needs to be removed (Andreu and Vidal, 2013). In addition, lignin the most underutilized resource is abundantly available and is generated annually around 50 million tonnes worldwide (Ragauskas et al., 2014). Traditional industries interested in polysaccharides have burnt lignin and used for power generation. However, there is a growing interest in valorization of lignin which can be used to produce potential high-value products such as low cost carbon fiber, engineering plastics and thermoplastic elastomers, polymeric foams and membranes, and a variety of fuels and chemicals which are all currently sourced from petroleum (Ragauskas et al., 2014). Therefore, Bioreflnery research geared towards valorization of lignin for generation of variety of chemicals which can be a feedstock for variety of industries. In order to produce high value chemicals, either the lignin needs to be isolated in pure form or depolymerised into low molecula weight compounds.

In the recent years, several strategies of pre-treatment methods are being followed for delignification (removal of lignin) of lignocellulosic biomass. Although several physical, chemical, mechanical and biological methods are available, combinations of more than one method works better for recovery of higher sugars and removal of lignin. For instance, efficient delignification can be achieved using alkaline pretreatment, organosolv, wet oxidation and Ammonia Fibre Explosion (AFEX). Among the above methods, commercial pilot plants are available for most of the methods except for AFEX and wet oxidation due to their high operational costs(Alvira et al., 2010). Organosolv pretreatment can efficiently remove high quality lignin which can be valorized for generation of higher value products (Zhao et al, 2009). Although significant delignification was achieved from the above methods, use of chemicals (chlorine and alkaline extraction) are harsh in nature it causes significant degradation of cellulose besides releasing highly corrosive chlorinated lignin breakdown products which are mutagenic, carcinogenic and toxic to environment (Anwar et al., 2014). Similarly, the sulfite process preserves cellulose in a non reactive form, degrades some hemicelluloses into reactive by-products and sulfonates all lignin. The Kraft (sulphate) process doesn't not fractionize lignin, cellulose and hemicellulose. On the other hand, organic solvent (organosolv) produces non reactive cellulose and doesn't hydrolyze hemicelluloses, besides that, the temperature and pressure used in organosolv process is significantly higher than conventional pulping process. One of the most common and so-called mild pretreatment, dilute inorganic acid and inorganic solvent has a disadvantage of hydrolyzing both cellulose and hemicelluloses (Heap et al., 2014).

Therefore, it is highly imperative to use mild operational conditions, shorter residence time and economic as well as environmentally sound pretreatment methods. Enzymatic pretreatment of lignocellulosic biomass using laccases, recently receiving much attention on delignification of lignocellulosic biomass as they are much safer to the environment (Ammann et al., 2013). Laccase when applied alone can oxidize the non phenolic part of lignin and in the presence of mediator it can significantly remove phenolic portion which is of 80% of total lignin. Similarly, laccases combined with xylanases can improve the delignification of pulp. Significant delignification can also be obtained by synergistic application of laccase, lignin peroxidase and Mn peroxidases. Recently, ionic liquids are reported to enhance the laccases activity, which could increase lignin removal and enhance the saccharification of lignocellulosic biomass (Andreu and Vidal, 2011).

Nevertheless, there are no embodiments related to process of delignification of lignocellulosic biomass involving steam treatment and laccase supplemented with solvent. Moreover, when the above novel process is applied on lignocellulosic biomass it also generates lignin derived products which are of high value feed stock chemicals for various industries.

Back Ground and Prior Art

It is known that removal of lignin from biomass not only opens way for production of fuels (from cellulose and hemicellulose) and other industrial products, but also degradation of lignin is one of the key steps in recycling the carbon dioxide fixed by the photosynthesis. When such biological degradation/conversion/transformation of lignin occurs it also generates high value chemicals from the lignocellulosic biomass. Traditionally, potential neucleophiles (sufite, chlorine and sulphate) were used to cleave the lignin. Due to harsh nature of the chemicals and growing environmental concerns, the central aspect of lignin degradation is resolving around the application of biocatalysts. Lignin peroxidases (UP), Mangnese Peroxidases(MnP) are two lignin degrading enzymes from white rot basidiomycetes most extensively studied. High redox potential of these enzymes enable them to directly oxidize the benzene ring and opens it invariable of its degree of methylation (Hammel and Cullen, 2008). Similarly, Versatile Peroxidases(VP) are more recently described having the same role as LiP and MnP with some advantages of attacking the non phenolic part of lignin. Although, effective lignin removal can be achieved using the above enzymes, they posses some drawbacks such as, contiouous requirement of H202for their activity and difficulty of heme cofactor in penetration to the internal lignin units, hence these enzymes oxidize only on the surface of lignin.

While laccases having significat advatageous over other enzymes are recently been recogonised as an industrially significant enzyme in pulp and textile manufacture, beverage and food processing and pollutant detoxification. Although laccases have lower redox potential than peroxidases it can oxidize varitety of phenolic, non phenolic, anilines, and aromatic thiols substares. Unlike peroxidases.they donot reqire co facor (heme in case of peroxidases) and contionous supply of H2O2 for its activity. Additionaly, in the presence of small aromatic mediators their catalytic efficency is improved as the mediators can diffuse into cell walls and form stable radicals which then can further accelarate the oxidation of both phenolic and non phenolic parts of the lignin. Hence, laccase mediated delignification is of particular interest to scientific and industrial communtiy.

Several embodiments have been disclosed the use of laccase or ligninolytic enzymes in delignification of biomass. The patent no 2014/0302567A1 describes the method of reduction or reformation of lignin in lignocellullosic biomass such as switch grass, sweet sorghum, miscanthus, pine wood and cornstover as biomass for delignification. Initially, solid state fermentation was carried out with Pycnoporus Sp SYBC-L3 to simultaneously remove the lignin and produce ligninilytic and hydrolytic enzymes. The fermentation lasted for 36 days, and a maximum of 30% delignification of sweet sorghum biomass was observed without altering cellulose and hemicelluloses content of the biomass besides producing a considerable amount of laccase 6.3U.g by the fungus. In addition to that, due to penetration of mycelium into the biomass, porosity of the biomass was increased which resulted increased hydrolysis of the biomass.

In the same embodiment (2014/0302567A1), sweet sorghum, was enzymatically delignified with laccase (10U.ml-1) which resulted in 5-8% lignin removal. Whereas, in the presence of 1.8mM HBT mediator the catalytic acitivity was improved recording 25.5% delignification. Similarly in the presence of 0.63mM of vaioluric acid, the laccase could delignify switch grass biomass to a tune of 28%.

The patnet no EP0406 617 A2 relates to the plural stage application of enzymes (Xylanase, Laccase, UP and MnP) on northern kraft wood pulp for efficent delignifiaction, laccase treatment alone on oxygenated kraft pulp resulted in 28% lignin removal, similarly xylanase alone application resluted in 15% delignification. The delignification percent have been greatly improved to 44% when sequential treatment of xylanase, lignin peroxidase, manganese peroxidasae (166.7 U.g-1 of biomass for xylanase / (48.3 U.g-1of biomass Lip & 57.3 MnP U.g-1 of biomass) respectively was applied on northern soft wood pulp.

Intrestingly, a method of delignification with xylanase and a mediator HBT alone without laccase was described in patent WO 2009069143 A2 where in the kraft pulp was treated with xylanase and HBT for 3h at 65° C. A total of 8 % delignification was observed.

A novel protein rLDM™ sequence from mutant strain of Phanerochaete chrysosporium, (SC26) having delignification ability was described in patent (CA1268132 A1). In the embodiment/a portion (lOXIO^to 20X10-6) of protein was applied on soft wood kraft pulp which resulted in 30% delignification.

Enzymatic delignification method involving laccase for treatment of palm froads and seaweed with laccase (20 U.mM with 1mM HBT) resulted in 9 and 28% delignificaion respectively. Similarly, the report also describes application of Ionic liquids (C2mim)(OAc) at room temprature to the biomass resulted in swelling of the biomass and improved catalytic activty of the laccase which eventually resulted in increased saccharification (12.6%). The reason might be due to the easy accesiblity of enzymes to the biomass.

The US patent No. 008414660B2 describes the first report of a novel laccase (LccA) from halophilic archaeon, Haloferax volcanii with properties of thermal, salt and solvent stablity. This organism and enzyme LccA are ideal candidates for biotechnology advancements. LccA was useful in phenolic remvoal of acid and steam exploded pretreated sugarcane bagasse.

However, in the prior art there was no evidence that process of delignification of lignicellulosic biomass with steam treatment followed by application of laccase and solvent. This embodiment as in, uses the solvent and thermostable crude laccase (LccH) 50 U.mM for delignification of woody biomass of Melia dubia. The process removed 48% of lignin form biomass which is almost higher than many cases reported (see tablel). The advantage of the process over the other delgifnciation process is that, this process firstly used crude laccase enzyme 50U.mM wherein all the embodiments either used commerical enzyme or purified product of the reported enzyme. Secondly, in majority of the disclosures to improve delignification two aspects were considered; one is the use of sequential application of xylanse prior to laccase, and the other is addition of phenolic mediators in combination with laccase. Whereas, in the present embodiment no such additions were used which might indicate the cost significance of the present process. Moreover, use of solvent to imporve the catalytic performance of laccase in delignification is the first attmept. Above all the concentration of solvent used (2%) in this process is significantly minimal and could yield hihger delignifaction (48%).

Field of the Invention

A Process for removal of lignin from lignocellulosic biomass using laccase (LccH) is provided and disclosed. Specifically steam pre-treated woody biomass is delignified using LccH and solvent (ethanol) under mild operating conditions was described. Upon delignification, the cellulose rich biomass is used for saccharification and fermentation, the hydrolysate containing low molecular weight phenols/lignin derived compounds are used for generation of high value products.

4.Detailed description of the Invention

In the present investigation, in order to accelerate the delignification of lignocellulosic complex a new process of pretreatment was developed involving steam, laccasse (LccH) and a solvent (ethanol) called Enzolv. The reason behind using ethanol with LccH is that the crude LccH showed improved activity in the presence of various solvents particularly ethanol.

Activity and stability of LccH under different solvents

The effect of different solvents (ethanol, methanol, acetone and DMSO) on LccH activity and stability was evaluated by incubating crude enzyme (50 U.mH) in solvents (ethanol, methanol, acetone and DMSO) at concentrations ranging from 10 to 100 %, while LccH in buffer (citrate phosphate, pH 3.4) served as a control.

Initial activity of LccH on solvents

Increased initial LccH activity of 127, 125, 123 and 104 % for ethanol and 120, 120, 117 and 117 % for methanol, respectively was observed upto 40% concentration. (Fig.2). On the other hand initial activity showed a drastic reduction of 82% in 50% ethanol, whereas in methanol concentration above 40% followed a gradual decrease retaining 40 and 20% activity for 50 and 60% concentration.

LccH maintained 100% initial activity in acetone until 20% and thereafter declined, while DMSO showed a reducing trend from 10% concentration onwards. The initial LccH activity was reduced to 20% with 20% DMSO and then gradually decreased to 73, 63 and 41% respectively for 30, 40 and 50% concentration (Fig.2). In general, solvent concentrations above 60% were not favourable for initial LccH activity. Our initial work with an archaeal laccase recorded enhanced enzyme activity in the presence of organic solvents (Uthandi et al., 2010)

Stability of LccH on solvents

In order to be useful in biotechnological applications, an enzyme should be stable for extended periods of time. We evaluated the effect of LccH stability on solvents (ethanol, methanol, acetone and DMSO) used in a variety of industrial applications. Solvent concentrations ranging from 10 to 100 % was added to crude LccH (50 U.mM) and incubated for 17 hat 40 °C.

The enzyme stability varied considerably with respect to different solvents (Fig.3). Ethanol and acetone at 10 % improved LccH stability to a maximum of 122 and 117%, while at 20 %; stability was reduced to 111 and 100%, respectively. Further, 16 % of the LccH activity was recovered at 40% ethanol whereas activity was completely lost at 30% acetone.

Set aside ethanol and acetone, LccH stability decreased from 10% onwards for methanol and DMSO. However, it is interesting to note that LccH retained 80% of its activity in DMSO upto 30% and its activity was halved at 40% concentration. In general, >40% concentration was detrimental to LccH stability in all the solvents tested wherein no or less activity was observed on these concentrations.

In an attempt to reduce the concentration of ethanol, from 10% being used in the newly developed Enzolv process, LccH initial activity and stability was measured using various concentrations starting from 2% to 10% ethanol. The result shows that, increased LccH activity of above 20% over control (with buffer only) was observed for both initial activity as well as stability from ethanol concentration of 2% until 10%. Further, initial activity and stability showed similar trend for the different ethanol concentrations used. It is also noted that, there was no significant change in the activity (both initial and stability) of LccH with respect to different concentrations of ethanol although slight improvement in initial activity was observed than stability (26% over 21%) the difference is statistically negligible (Fig 4). Since the lowest concentration of ethanol (2%) was sufficient for enhanced activity considering the cost and amount of solvent needed the concentration 2% ethanol was selected to develop the process and the LccH stability was monitored using the same 2% concentration.

These results suggest that, LccH can be used for variety of industrial applications involving laccase and solvent. Moreover, the added advantage of using LccH is that it can maintain (stable) the enhanced activity up to 17 hours and this property makes LccH a potential candidate for many industrial applications.

The reason for enhanced activity and stability of LccH on solvents can be explained as follows. Laccases being the most versatile oxido-reductases requires oxygen for its activity, fortunately oxygen is more soluble in organic solvents than water and hence the accessibility of electrons due to oxidation form the reduced substrate is much easier for oxygen in solvents when compared to water. In contrast, when water/buffer is used instead of solvents the traffic of electrons becomes much tighter between the water insoluble reducing substrate and oxygen. Besides this, ethanol itself was proved to be an inducer for laccase activity as observed during growth as well as assay conditions (Lomascolo et al. 2003).

However, the concentration of solvents to certain limit and prolonged incubation leads to denaturation or inactivation of biocatalysts. Additionally concentration of solvents changes the equilibrium of non covalent interactions that determine the stability of enzyme when the enzymes is exposed to higher concentrations of solvents instead of water and eventually makes the enzyme unfolded.

Enzolv- novel process of delignification of biomass

This process was tested on biomass of wood {Melia dubia). Milled and dried biomass (2.5 g) was subjected to steam treatment at 121 °C for 1 h. After cooling, the biomass was saturated with buffer (citrate phosphate, pH 3.4), enzyme (LccH) and solvent (2% ethanol) to attain a 5% final consistency with enzyme loading of 50 U.g-1 of dry biomass. The flasks were kept overnight (17h) at 40 °C, 120 rpm. After incubation, the biomass was filtered and the filtrate and biomass was subjected to compositional analysis. Through this new Enzolv process, a maximum lignin removal of 48.1 (Fig.5) was achieved in wood and. This removal efficiency is much higher when compared to crude LccH treatment alone which removed 29.7%.

The results of individual process and its combinations are presented in Table 2 which shows that when the biomass was subjected to steam pretreatment there was no significant change in cellulose content where as non-negligible amount of hemicelluloses and lignin were removed. It is known that steam pretreatment generally removes hemicellulose and alter the lignin structure and enhances biomass for better accessible to ligninolytic and cellulolytic enzymes. Furthermore, individual effect of solvent at 2% concentration resulted in minimal removal of hemicellulose (5% over control) but had a significant effect on delignification where in 24% reduction of lignin was achieved. This is not surprising because organic solvents (process known as Organosolv) such as ethanol, methanol, dioxane, pyridine and DMSO were known to remove lignin from lignocellulosic biomass either alone or in combinations with catalyst such as organic acids. It is reported that above 90% delignification can be achieved when pure organic solvents or in combinations of catalysts was used; the lignin thus removed would be of high quality and can be valorized for further production of high value products. However, the organosolv process operates at higher temperatures at 200 °C for 10 to 15 min to achieve higher lignin removal. The current enzolv process was operated at 37 °C for 17 h. Additionally, when steam treated woody biomass was subjected to solvent treatment significantly higher (p<0.01) removal of hemicelluloses (15%) and lignin (28%) was achieved compared to biomass treated with steam alone (Table 2). The reason behind achieving higher removal efficiency was due to steam pretreatment which might have altered the structural components of biomass for solvent to react better.

Similarly, when crude LccH was applied alone it resulted in 30.57% reduction of lignin. Likewise, when steam treated biomass was subjected to LccH crude enzyme, LccH performed well on steam pretreated biomass and removed significantly (p<0.01) higher lignin (34.29%) over LccH alone. This indicates the superiority of delignification efficiency of crude LccH over solvent. Further, contribution of steam altering lignin structure of the biomass for enhanced lignin removal could not be ignored. In contrast, when the enzyme and steam were used the hemicelluloses removal was reduced to 15.49% this shows that LccH might have only laccase activity. Finally, when steam treated biomass was subjected to solvent and enzyme treatment (Enzolv) at 1:3 ratio highest lignin was removed (48.17%) (p<0.01) leaving behind the higher cellulose content of 52.12% for further fermentation process. The reason for achieving highest lignin removal could be explained due to the enhanced LccH activity (26%) and stability (22%) on 2% ethanol might have influenced higher removal rate.

Morphological changes of woody biomass of M.dubia under different process combinations observed by Scanning Electron Microscopy (SEM)

Morphological changes of the biomass after Enzolv process were observed by Scanning Electron Microscopy (SEM). Fig 6 and 7 shows the SEM images of biomass obtained from individual components and its combinations of Enzolv process respectively. Morphology of untreated biomass (a) was intact and smooth without any structural modification which implies that the lignin and hemicellulose covering on the surface. The surface and internal structure of the biomass was loosened and the bundles become visible on the morphology of steam treated biomass (b). Very less struetural changes such as widening as shown in fig 6 c, formation of small ridges in the surface of the biomass were noticed on solvent treated biomass. In contrast, when the biomass was subjected to enzyme (LccH) treatment, a dramatic change in the structure of the biomass was observed, where in enzyme caused several pores on the biomass fig 6 (d), which might be due to the oxidation of lignin in the biomass by the laccase enzyme. Pore size of the biomass was also measured and it ranged from 3pm to 7pm, the increase in porosity of biomass indicates the catalytic efficiency of the enzyme and this structural changes attribute to the removal of lignin (30.57% reduction) form biomass as estimated by klason lignin.

Similarly, structural changes caused by Enzolv process and its combinations were depicted in fig 7. The SEM image of the steamed biomass with solvent is showed in fig 7 e. The disintegration of bundles becomes more apparent and modifications such as ribs widening, loose arrangements of fibers were noticed (e). Structural changes were more evident in the case of steamed biomass treated with enzyme, higher disintegration of bundles along with increase in separation of vascular bundles were visible which reason out the effect of enzyme removing lignin in the steam altered biomass, loosening of biomass by steam enabled the enzyme to reach the lignin molecules present in the middle lamella and oxidized the lignin (f). Biomass structural changes were slightly improved as bundles and ridges formation were apparently higher incase of biomass treated with enzyme and solvent (g). Pronounced effect on lignin removal and separation of vascular bundles in combination with cellulose enrichment was obvious when all three combinations were employed to the biomass (h) in addition to that improving the catalytic efficiency of laccase by supplementation 2% ethanol which evidently increased the number of pores on the biomass structure (fig.7h).

Fourier Transformation Infrared (FT-IR) analysis of wood biomass from M.dubia subjected to Enzolv The changes in the compositions of woody biomass after the Enzolv treatment were evaluated using ATR-FT-IR from the wavelength range of 400 -4000 cm-1. Results of the FT-IR spectral data and its corresponding polymer are presented in Table3. FT-IR spectra of individual treatments such as steam, enzyme and solvent are presented in Fig 8.

Spectral intensities corresponding to lignin, hemicellulsoe and cellulose was markedly reduced at the wave number 3338 cm-1' 1725 and1028 for enzyme and solvent compared to steam. The change in vibration of C-H stretch at 2883 cm-1 of lignin and simultaneous reduction in intensity at the same wavenumber was observed due to LccH enzymatic treatment when compared to solvent and steam treated biomass. No change in the intensity at the regions at 1724 and 1245cm1 comprising the C-0 stretch of hemicelluloses was observed for steam and enzyme treatment, this attributes to the non hemicellulolytic nature of enzyme (fig 8). While a relatively reduced intensity of C-0 stretch region was noticed for solvent treatment indicating a minimum removal of hemicelluloses. LccH and solvent treatment reduced the aromatic lignin content as can be evident by the reduction in the intensity of aromatic ring region at 1598 cm-1 and 1592 cm-1 compared to steam treatment (Fig 8). The availability of cellulose was higher in steam followed by solvent which is apparent by the reduction in intensities at the region 1029 cm-1 of C-0 stretch. The reason for the increased availability of cellulose might be steam treatment loosened the biomass.

FT-IR spectra of combinational treatments of Enzolv process are presented in Fig 8a where, it is clearly evident in the reduction of intensities at the region 3338 cm -1 indicating the lignin reduction was higher in the combination of steam and enzyme followed by solvent and enzyme than solvent and steam. Similar trend in intensity was observed for lignin reduction for combinational treatments at the region 2833 cm-1 comprising C-H stretch. LccH activity was enhanced when it was applied to steam treated biomass where the LccH significantly removed lignin at the regions 1594 and 1502 cm -1 as noticeable by the reduction in intensity of the aromatic ring vibration. Whereas LccH cannot accomplish this removal when applied alone (fig.8) Another interesting fact in removal of lignin by steam and enzyme was observed in the regions 1465,1425 and 1323 cm-1 where in discernible reduction in intensities were experienced when compared to LccH alone (fig.8). Intensity of C=0 stretch was reduced in steam and enzyme combination where as less change in intensity was noticed for solvent and enzyme and steam and solvent. As a result of lignin and hemicelluose removal, the availability of cellulose was higher in steam and enzyme combinations followed by solvent and enzyme than solvent and stealn.

Finally, the FT-IR spectra of the Enzolv treated biomass (cumulative effects of the steam, solvent and enzyme) treatment of the biomass was compared with untreated (fig 8b). It is obvious form the spectra that the intensity of O-H spectra was higher than all the treatments indicating significant lignin removal as well as higher intensity reduction in the lignin regions such as 1465, 1425 and 1323 cm-1 was perceived. It is noteworthy that the intensity was far reduced than the combinations indicating the significance of the process. As a consequence of the removal of lignin, intensity of cellulose at 1029 C-0 cm-1 stretching was remarkably higher in the Enzolv treated process.

Hence from the FT-IR spectra, it can be concluded that the developed novel process (Enzolv) due to its combinations significantly reduces lignin and makes cellulose more available for the further process. XRD analysis of Biomass Xray Diffraction analysis of M.dubia biomass after enzolv process was performed and the crystalinity index (Cl%) was calculated using the relative 002 peak for cellulose and the minimum dip. between the 002 and the 101 peaks, which are assigned to the amorphous region. A relationship between cellulose content and crystalinity index was calculated and depicted in fig 9. It was found that the crystalinity of untreated biomass was 58%. When subjected to Enzolv process a linear relationship of crystalinity was established. Individual treatments of Enzolv such as steam, solvent and enzyme had a crystalinity of 58, 59 and 60% with a corresponding cellulose content of 33, 33.07 and 38% respectively. When the combination of Enzolv process was evaluated; the crystalinity was increased and reached a maximum of 78.39% in Enzolv process which corresponds to the cellulose content 52.12%. One of the objective of the pretreatmentjs to provide the cellulose without much modifying its native structure. Thus the Enzolv process increases the crystalinity of the cellulose which might be useful in producing high-strength composite materials.

Generation of lignin derived products using LccH

The biomass hydrolysate after the enzolv process was derivitized using BSFTA and subjected to GC-MS for identification of different compounds released during delignification. Variety of products were generated and detected, simultaneously control samples samples without Enzolv process was also analysed for comparison.

The biomass hydrolysate after the enzolv process was analysed by GC-MS . BSFTA derivitized samples were run on GC and the resultant products were identified by MS. Table 4 summarizes the major derived products obtained by untreated LccH and ensolv treated biomass. Phenol and fatty alcohols were detected form the hydrolysate of untreated biomass. LccH alone treated biomass had several compounds of interest. Phenolics compounds and their derivatives were obtained apart form few sugars. Benzaldehyde, one of the industrially useful phenolic compounds used as flavoring reagent in food, pharma and cosmetic industries. Their price costs around 360 US dollars per litre which can be obtained by treating the woody biomass with LccH alone. Apart from phenolic compounds, thiozolic group of compounds were identified for example isothiazole an aromatic thiozolic group used in drug discovery. Similarly, few aromatic carboxylic acid such as propyonic acid, benzene dicarboxylic acid was also detected in the LccH treated hydrolysate.

Comparing LccH alone, Enzolv treated biomass had highly useful chemicals such as vanillin and isolvanillin form the hydrolysates which are of significant industrial interest. They are widely been used in food industry as a flavoring agent. Some phenolic derivatives such as trizaine derivatives find useful applications in drug production are also present in Enzolv process. Simple sugars, aromatic polymers and fatty acids were also found as a degraded product of biomass when Enzolv was operated.

Thus using this process it can be possible to generate industrially useful green chemicals such as benzaldehyde.vanillin and isovanillin etc. This process not only delignifies the biomass but generates high value chemicals as well.

Figures Attached

Figure 1. Enzolv process on M.dubia woody biomass for efficient delignification and simultaneous generation of valuable chemicals

Figure 2.Dependance of Initial Laccase activity on solvents at different concentrations

Figure 3.Stablity of LccH on solvents at different concentrations

Figure 4. LccH initial activity and stability under different concentrations of ethanol

Figure 5 Effect of Enzolv Process on delignifaction of Melia dubia wood biomass

Figure 6. Scanning Electron Microscopic images of biomass subjected to the Enzolv process

Figure 7.Scanning Electron Microscopic images of biomass subjected to the Enzolv process combinations

Figure 8. FT-IR spectra of M.dubia biomass subjected to Enzolv process (individual treatments) 1. Enzyme treatment, 2. Solvent treatment, 3. Steam treatment

Figure 8a. FT-IR spectra of M.dubia biomass subjected to Enzolv process (combinational treatments) 1. Solvent and Enzyme, 2. Steam and Enzyme, 3. Solvent and steam

Figure 8b. FT-IR spectra of M.dubia biomass of Enzolv process and untreated biomass.1. Enzolv process

2.Untreated Biomass Figure 9. Relationship between crystalinity index and cellulose content of M.dubia biomass of Enzolv process

Tables Attached

Table-1 Delignification of lignocellulosic Biomass by enzymatic means

Table 2. Effect of Enzolv process on cellulose, hemicellulose and lignin content of M.dubia

Table 3. FT-IR spectra of functional groups and corresponding polymer of Melia dubia biomass subjected to Enzolv process Table 4. GC-MS analysis of biomass hydrolysates of Enzolv process

References

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5.CLAIMS

MADE IN THE INVENTION

We declare that

1 .Novel Enzolv process for enzymatic delignification of lignocellulosic biomass

2.The Enzyme Laccase (LccH) used in this process is solvent tolerant upto 25%

3. This process mentioned in claim 1 also simultaneously generates lignin derived products and some high value chemicals synthesis is claimed.