The following specification particularly describes the invention and the manner in which it is to be performed.
CYCLIC TANNIC ACID MEDIATED INTENSIFIED BIO-PRETREATMENT FOR LIGNOCELLULOSE ETHANOL PRODUCTION
Field of invention
This present disclosures relates to novel cyclic biological pretreatment for ethanol production using lignocellulosic biomass.
Background of the invention and the prior art
Biological pretreatment is an efficient way for demineralization of lignocellulosic biomass except prolonged incubation/process time. Biological agents like fungus takes some time for their growth and attachment on the biomass and then releasing potential delignification substances such as enzymes and acids. During this process, fungi are utilizing cellulose, which is an essential sugar polymer for fermentation. To reduce the process time, cost and increase the availability of cellulose, Cyclic tannic acid Mediated intensified Bio-pretreatment (CTMB) technology was studied. CTMB technology using tannic acid in grape leaf as Cellulase Inhibitor was studied on paddy straw. Pretreatment was carried out under solid-state condition in 5kg capacity/unit. Here in this, 8% tannic acid supplementation in pretreatment medium improves fermentable sugar recovery and delignification by 1.2 and 1.5-fold respectively, compared to conventional bio-pretreatment. It was observed 61% of inhibition of cellulase activity in CTMB. Further increase in tannic acid concentration in pretreatment medium showed negative effect on Pleurotusflorida growth and suppress the laccase productivity by 1.1-fold. Laccase activity in CTMB was found to be 2.0 U/mL on 19th day, which is higher than (1.02 U/mL) conventional bio-pretreatment. According to these findings, the GTMB technology can be a viable eco-friendly technology for sustainable production of bioethanol. We have done various field visits and surveys to validate the technology up to pilot scale level. This has been designed to work in cyclic system to improve the process efficiency as well as minimizing time. In CTMB, produced biomolecules were processed in various conditions and then introduced in to the next cycle with biomass alone.
> Methods for producing extracted and digested products from pretreated lignocellulosic biomass, ShishirChundawat, Leonardo Da Costa Sousa, Albert M. Cheh, VenkateshBalan, Bruce Dale US9650657B2.
o Methods for producing extracted and digested products from pretreated lignocellulosic biomass are provided. The methods include converting native

cellulose 1(3 to cellulose IIII by pretreating the lignocellulosic biomass with liquid ammonia under certain conditions, and performing extracting or digesting steps on the pretreated/converted lignocellulosic biomass.
> Alkaline treatment of lignocellulosic biomass, Praveen Paripati, Ananthram Prasad
DADI, WO2014144588A1
o Method and apparatus for enhanced production of sugars and lignin via fractionation of lignocellulosic biomass through ionic liquid pretreatment and mild alkaline treatment. The resulting biomass is easily fractionated and amenable to efficient and rapid enzymatic hydrolysis or acid hydrolysis and catalytic conversion to valuable products with high recovery of the enzymes used in the hydrolysis.
> Improvements in a process for delignifyinglignocellulosic pulp by means of oxygen -
EP0524127A2 European Patent Office.
o The present invention refers to an improvement for delignifying a lignocellulosic pulp by means of oxygen wherein ethanol is used as an additive and the process is carried out at high temperature thereby obtaining a higher reduction of lignin content of the lignocellulosic pulp, that is, a higher reduction of the Kappa Number in comparison with conventional processes without, however, prejudicing the pulp quality. The use of the present invention provides a pulp which, when subjected to subsequent bleaching sequences, will require a lower amount of chloro compounds in order to achieve the desired whiteness and will also exhibit a high viscosity thus rendering a product with good resistance properties.
> Improvements introduced in the cycling pre-treatment equipment of electronically
commanded fuel, special application to internal combustion motors, Jose Manuel
Esteban Calvo ES2264344B1
o Improvements in cyclic pretreatment equipment for internal combustion engine engines are described, through which the performance and duration of the engine are greatly improved, and maintenance operations thereof are reduced. The fuel passes through a preunit that includes a brass bushing, a copper coil and an electrical resistor, encapsulated in epoxy resin; a unit that includes a bushing, a coil with an insertion resistance inside, and a piece of zamak that hugs the coil; a set of sensors with which both the temperature of the fuel at the fuel pump inlet, and the temperature of the outside environment are measured, and the information is sent to a control center, and means in said control center, to set management, and remote, such as a mobile phone, by sending short messages.

Brief description of the drawing
Figure -1 describes process diagram of Novel Cyclic Inhibitor Mediated intensified Bio-pretreatment (CIMB) system and contains the following parts as mentioned in the figure.
1: Biomass milling
2: Presoaking
3: Inhibitor mediated biological pretreatment
4: Blending pretreated biomass
5: Solid liquid separation
6: Biomass collection
7: Liquor collection
8: Phenolic compound removal
9: Solid liquid separation
10: Isolation of phenols
11: Buffering
12: Enzyme concentration
13: Hydrolysis
14: Effluent collection
15: Hydrolysate collection
16: Fermentation
17: Distillation
18: Cyclic bio-pretreatment
19: Fiber separation
20: Ethanol storage

Detailed description of the drawing
Cyclic tannic acid Mediated intensified Bio-pretreatment (CTMB) is a viable technology as like mushroom cultivation technology with negligible power requirement and effluent generation. This technology can be categorized into two stages, first stage involves growing microbes on biomass and second stage involves isolation and application of biomolecules for the next cycle. Successful biological pretreatment studies with optimum process conditions will be transformed to next level with cyclic metabolite flow on corresponding LCB residues. The efficiency of this process will be analysed by kinetics of lignin to Carbohydrate degradation ratio in each cycle under specified condition. Here the mobile phase is the secreted metabolite from preliminary biomass pretreatment followed by addition of inducers in further. Our, invention has designed in such a way to get rid of this issue with the use of endogenously produced biomolecules in cyclic system. To breakdown complex bio-molecular structure in lignocellulosic biomass requires very specific and active biocatalysts. Here, we have designed the cyclic pretreatment process as mentioned in figure -1 in order to recover the specific biocatalyst secreted by fungus growing on used biomass and activating again for next phase of pretreatment in queue. Recirculating endogenously produced biocatalysts on feedstock does not require any microbes to breakdown the complex structure. Hence, this will reduce major part of time and cost taken for pretreatment.
STAGES IN NOVEL CYCLIC BIOLOGICAL PRETREATMENT SYSTEM
Stage 1: Biomass milling - Collected raw lignocellulosic biomass will chopped to the size of 5 to 7 cm using pulverizer and washed thoroughly with water.
Stage 2: Presoaking - Chopped and washed biomass will be soaked in a water for 4 h to remove adhered particulates and to enhance water absorption.
Stage 3: Inhibitor mediated biological pretreatment - Pretreatment of preprocessed biomass using white rot fungi with 8% of natural cellulase inhibitor compounds for 28 d.
Stage 4: Blending pretreated biomass - using proportional feeder biomass is soaked and blended with buffer solutions at 10% (w/v) concentration to extract potential enzymes.
Stage 5: Solid liquid separation - blended mixture is passed to filter press at 200bar to separate liquid content with enzymes and solid content with broken sugar molecules.
Stage 6: Biomass collection - enzyme extracted, water free pretreated biomass will be collected for further process.
Stage 7: Liquor collection - filter pressed enzyme extract with other molecules are collected in a gravity settling vessel.
Stage 8: Phenolic compound removal - Collected biomass will be treated with hot water and Ti02 scrubbers to remove phenolic compounds.

Stage 9: Solid liquid separation - Hot water and Ti02 treated biomass mixture will be filter pressed to remove breakdown lignin products from pretreated biomass under 200bar pressure.
Stage 10: Isolation of phenols - Phenolic compounds from extracted liquids are isolated and valorized.
Stage 11: Buffering - Collected liquid fraction are buffered accordingly with 50 mM phosphate and citrate buffer with pH 4.8 to enhance endogenously produced enzyme activities.
Stage 12: Enzyme concentration - Enzyme molecules present in the buffered liquid fraction will be concentrated by NH2S04 precipitation at 75% saturation.
Stage 13: Hydrolysis - Enzymatic hydrolysis of pretreated solid biomass will be carried out with 10% solid loading and cocktail mixture of enzymes for 48h.
Stage 14: Effluent collection - Hydrolysate free slurry will be collected as effluent after gravity settling for 2h.
Stage 15: Hydrolysate collection - After enzymatic hydrolysis, hydrolysate containing hexose and pentose sugars will be collected for further process.
Stage 16: Fermentation - Sugar solution will be added with peptone and other nutrients for ethanol fermentation by yeast culture (S. cerevisiae) and ethanol fermentation will be carried out for 72h.
Stage 17: Distillation - Distillation of fermentation broth followed by gravity settling will be carried by three different columns.
Stage 18: Cyclic bio-pretreatment - Preprocessed biomass will be treated with endogenously produced fungal enzyme in multiple cycles with 10% (W/v) concentration.
Stage 19: Fiber separation - Followed by phenols extraction the biomass containing structural fibers will be transferred to the enzymatic hydrolysis vessel.
Stage 20: Ethanol storage - Produced 97% ethanol will be stored in good condition.
We claim
1. A method for biopretreatment of lignocellulosic biomass using Pleurotusfiorida comprising, Cyclic tannic acid Mediated Intensified Bio-pretreatment (CTMB) technology using tannic acid present in grape leaf as cellulase inhibitor.
2. The method of claim I, wherein said CTMB comprises 8 % tannic acid supplementation to inhibit the 61 % cellulase activity.

3. The method of claim 1, wherein lignocellulosic packed bed is washed using during every cycle and the spent wash is recycled limiting to 13% total moisture content.
4. The method of claim 1, wherein the grape leaves are placed 3- 5 g at 5cm equal distance into the lignocellulosic packed bed for 5 - 10 kg capacity / unit.
5. The method of claim 3, wherein said spent wash containing laccase reduces the cost and time in successive cycles with increase of 1.2 and 1.5-fold of fermentable sugar recovery and delignification and cellulose respectively
6. The method of claim 5, wherein the cellulase recovered from spent wash during every cycle is utilized for saccharification.
7. The method of any one of claim 2-6, wherein said the spent wash from the
lignocellulosic packed bed is concentrated using salt precipitation technique provided
during the recycling process.