Field of the invention:
The present invention relates to the process of production of biofuel precursors that are useful in preparation of biofuels. This present invention particularly relates to the production of biofuel precursors from Sunn hemp.
Background and prior Art:
The depleting sources of fossil fuels added to their harmful effects led to the requirement to probe into renewable sources of fuel and efficient technologies to produce biofuels from such sources. Biofuels are environment friendly and are expected to meet the rapidly increasing requirements for transportation fuels, reduce dependence on import of fossil fuels, provide high energy security, address global concerns about containment of carbon emissions in the environment, reduce trade deficits and create jobs in rural areas. A target of 20% blending of biofuel in fossil fuel is specified in the Indian Government’s bioenergy roadmap by 2025.
Sunn hemp (Crotalaria juncea L.) is an annular summer legume and is the most widely grown green manure in the tropics and sub tropics, where it is used as a fiber and does not qualify as a food source. It is an ideal cover crop because of its fast growth and nitrogen fixation capacity. It is also capable of suppressing weed growth and soil root nematodes. It is grown in almost all states of India like Bihar, Orissa, Rajasthan, Madhya Pradesh, Maharashtra, Uttar Pradesh and west Bengal that cover about 87% of the total area under cultivation of Sunn hemp crop. It prefers to grow on fairly light, well drained soil having sandy loam or loamy textured soil that retains sufficient moisture. Sunn hemp must have ample sunlight to grow and the growing areas of India range from latitude 1-30 N. The stem of the plant is approximately 2 cm in diameter, leaves are simple and petioles are about 5cm long. The shape of the blades are linear elliptical to oblong. It is approximately 4-12 cm long and 0.5-3 cm broad and bright green in color. The plant grows about 103 meter within a year. The crop is an open pollinated species and was obtained from the National Plant Germplasm System in the several countries. They are classified as PI 207657 in Sri Lanka, PI 322377 in Brazil, PI391567 in South Africa and PI 426626 in Pakistan.
Characterization of Sunn hemp fiber gives 75.6% cellulose, 10.32% lignin, 10.05% hemicelluloses, 3.6% moisture and 0.43% ash contents. It can be used as an inexpensive source of bioethanol production for its high cellulose content.
Previous patents by other researchers suggest that Sunn hemp is suitable for making various paper pulp products.US4889591 provides the process of subjecting the fibers to field drying to reduce moisture content to less than 30% by wt, shredding and depithing, contacting with Sodium Hydroxide (8% by wt) at temperature more than 180 C to produce paper pulps.
US8551747describes a process of separating lignin from lignocellulosic material, comprising the successive steps of: a) deconstructing a lignocellulosic plant raw material by contacting the lignocellulosic plant raw material with a mixture containing formic acid and water at a temperature between 95°C and 110° C; b) separating the deconstructed lignocellulosic plant raw material at atmospheric pressure into a solid phase mainly composed of said cellulose constituting a first substrate, and a liquid phase containing in aqueous solution, hemicelluloses, lignins and the formic acid, constituting a second substrate; c) hydrolysing the first and second substrates to produce a hydrolysed material; d) fermenting the hydrolysed material to produce a fermented material containing ethanol; and e) distilling ethanol from the fermented material to producebioethanol.
US6022419Adescribes a process of producing bioethanol in continuous shrinking bed reactor.
US 8883469 provides a method of producing bioethanol characterized in that crushed lignocelluloses biomass is treated with an alkanolamine as a solvent for extracting the lignin therein for at least 10 minutes at a temperature of about 80° C to 150° C andwherein before the extraction of the lignin, the lignocellulose biomass is treated with ammonia gas or aqueous ammonia solution.
CN200910094892 discloses a method for hydrolyzing lignocelluloses, where lignocellulose is pulverized into particles, and the particles are mixed with water to form even seriflux; the seriflux is preheated to 60-150 C by using ultrasonic waves or microwaves; the preheated even seriflux is quickly heated to 120-300 C, solid alkali is added and used as a catalyst. However such processes are lengthy and involve use of solid catalysts, which in turn involves further separation steps for removal of the catalysts.
Even though microwave assisted processes for hydrolyzing lignocellosic fibres are known in the art, there is a need for improving such methods in terms of lesser reaction time, simpler reactions and use of environment friendly catalysts. Further as discussed above, the prior art processes involve steps of pretreatment which results in rendering the processes lengthy and cumbersome.
Accordingly, there is a need in the art for simpler and convenient processes having desirable yields of biofuel precursors.
Object of invention
Accordingly, it is an object of the present invention to overcome the drawbacks of the prior arts and provide a cost-efficient, simple and rapid process of production of biofuel precursors.

It is a further object of the present invention to provide an efficient microwave-assisted, ionic liquid based process for rapid catalytic conversion of non-edible, lignocellulose fibers of Sunn Hemp to biofuel precursors.

It is another object of the present invention to provide an efficient microwave-assisted, ionic liquid based process for rapid catalytic conversion of Sunn hemp fibres to biofuel precursors such as glucose, 5-hydroxymethylfurfural (HMF), Levulinic Acid and Formic Acid.

It is yet another object of the present invention to recycle the catalyst for use in catalytic hydrolysis process.

It is yet another object of the present invention to ferment the biofuel precursors to produce biofuels.

Brief description of The Accompanying Figures:

Figure 1. Characterization of the non-edible lignocellulosic substrate Sunn hemp (Crotalaria juncea) fibre: (A) XRD Analysis for untreated and reacted Sunn hemp fibre (B) FTIR Analysis for untreated and reacted Sunn hemp fibre (C) FESEM Analysis for untreated Sunn hemp fibre (D) FESEM Analysis for untreated Sunn hemp fibre
Figure 2. Effect of pre- treatment on glucose yield at (A) 160°C (B) 180°C (C) 200°C; Effect of pre-treatment on HMF yield at (D) 160°C (E) 180°C (F) 200°C ( With pre-treatment, Without pre-treatment)
Figure 3. Effect of pre- treatment on Levulinic Acid yield at (A) 160°C (B) 180°C (C) 200°C; Effect of pre-treatment on Formic Acid yield at (D) 160°C (E) 180°C (F) 200°C ( With pre-treatment, Without pre-treatment)
Figure 4. Effect of reaction time in microwave reactor on glucose yield at (A) 160°C (B) 180°C (C) 200°C; Effect of reaction time in microwave reactor on HMF yield at (D) 160°C (E) 180°C (F) 200°C (Time 36 minutes, 41 minutes, 46 minutes and 51 minutes
Figure 5. Effect of reaction time in microwave reactor on Levulinic Acid yield at (A) 160°C (B) 180°C (C) 200°C; Effect of reaction time in microwave reactor on FA yield at (D) 160°C (E) 180°C (F) 200°C (Time 36 minutes, 41 minutes, 46 minutes and 1 51 minutes)
Figure 6. Effect of glucose yield for different amount of water addition at various temperatures for different catalyst loadings per unit of substrate (w/w): (A) 6% (B) 7% (C) 8% (D) 10% (E)12% (F) 14% (G) 16% (H) 20% (Temperature 160°C, 180°C, 2 200°C)
Figure 7. Effect of HMF yield for different amount of water addition at various temperatures for different catalyst loadings per unit of substrate (w/w): (A) 6% (B) 7% (C) 8% (D) 10% (E) 12% (F) 14% (G) 16% (H) 20% (Temperature 160°C, 180°C, 200°C)
Figure 8. Effect of Levulinic Acid yield for different amount of water addition at various temperatures for different catalyst loadings per unit of substrate (w/w): (A) 6% (B) 7% (C) 8% (D) 10% (E) 12% (F) 14% (G) 16% (H) 20% (Temperature 160°C, 180°C, 200°C)
Figure 9. Effect of Formic Acid yield for different amount of water addition at various temperatures for different catalyst loadings per unit of substrate (w/w): (A) 6% (B) 7% (C) 8% (D) 10% (E) 12% (F) 14% (G) 16% (H) 20% (Temperature 160°C, 180°C, 200°C)
Figure 10. Effect of substrate to Ionic Liquid ratio on the yields of glucose ( A, B & C) and HMF (D, E & F) for different amount of water addition at various temperatures (w/w): (A) and (D) 160°C, (B) and (E) 180°C, (C) and (F) 200°C ( 2.5% substrate to Ionic Liquid ratio, 5% substrate to Ionic Liquid ratio, 7.5% substrate to Ionic Liquid ratio 10% substrate to Ionic Liquid ratio)

Figure 11. Effect of substrate to Ionic Liquid ratio on the yields of Formic Acid ( A, B & C) and Levulinic Acid (D, E & F) for different amount of water addition at various temperatures (w/w): (A) and (D) 160°C, (B) and (E) 180°C, (C) and (F) 200°C ( 2.5% substrate to Ionic Liquid ratio, 5% substrate to Ionic Liquid ratio, 7.5% substrate to Ionic Liquid ratio 10% substrate to Ionic Liquid ratio
Figure 12. Effect of substrate loadings per unit of Ionic Liquid recycle on glucose and HMF yield at various temperatures for different amounts of water addition: (A) and (B) 1.25%, (C) and (D) 2.5%, (E) and (F) 3.75% (G) & (H) 5%: ( 160°C, Recycled IL; × 160°C, IL; 180°C, Recycled IL; 180°C, IL; 200°C,Recycled IL; 200°C, IL)
Figure 13. Effect of substrate loadings per unit of Ionic Liquid recycle on Levilinic Acid and Formic Acid yield at various temperatures for different amounts of water addition: (A) and (B) 1.25%, (C) and (D) 2.5%, (E) and (F) 3.75% (G) & (H) 5%: ( 160°C, Recycled IL; × 160°C, IL; 180°C, Recycled IL; 180°C, IL; 200°C, Recycled IL; 200°C, IL)
Figure 14. Effect of Glucose and HMF separation on the yields of (A) Glucose, (B) HMF (C) Levulinic Acid, (D) Formic Acid
Figure 15. Temporal dynamics of yeast-mediated glucose fermentation to Bioethanol for various glucose sources: (A) Increase of Yeast ((Saccharomyces cerevisae) Cell concentration with time (B) Decrease in Glucose concentration with time (C) Variation in Bioethanol concentration with time (D) Variation of Bioethanol yield with time .
Summary of the invention
In one aspect the present invention provides a rapid process of producing biofuel precursors from Sunn hemp fibers, said process comprising steps of:
(i) microwave heating assisted, catalytic hydrolysis of said fibers in presence of an ionic liquid based catalyst;
(ii) separation of at least one precursor as and when required; and
(iii) optionally recycling the ionic liquid catalyst to be used in step (i)
wherein the precursors are selected from glucose, hydroxymethyl furfural (HMF), levulinic acid and formic acid.
In another aspect the present invention provides a process comprising recycling of the ionic liquid based catalyst.
In a further aspect the present invention provides biofuel precursors like glucose which can be used in the production of bioethanol.

Detailed Description of the invention
As discussed above, there is need for alternative fuels, which apart from being renewable in nature are also environment friendly. The present invention relates to a process of production of precursors from lignocellulose fibers of Sunn Hemp using the cost-effective, rapid, microwave-assisted, alkali metal catalyst based hydrolysis. The process leads to the formation of precursors such as glucose that is fermented to bioethanol and high-value platform chemicals such as 5-hydroxymethylfurfural (HMF), Levulinic Acid and Formic Acid. These precursors to gasoline and other C8-C20 alkenes, which are alternative biofuels.
The present invention provides a rapid process of producing biofuel precursors from Sunn hemp fibers, said process comprising step of:
(i) microwave heating assisted, catalytic hydrolysis of said fibersin presence of an ionic liquid based catalyst;
(ii) separation of at least one precursor as and when required; and
(iii) optionally recycling the ionic liquid catalyst to be used in step (i)
wherein the precursors are selected from glucose, hydroxymethylfurfural (HMF), levulinic acid and formic acid.
Microwaves are the form of electromagnetic energy that falls at the lower frequency end of the electromagnetic spectrum with the wavelength ranging from 1m to 1mm, which corresponds to a frequency range of 300 MHz to 300 GHz.
In the present invention the catalyst can be alkali metal chloride based catalysts. Said catalysts can be selected fromCuCl2, CrCl3, AlCl3, ZnCl3, and FeCl3. The most preferred catalyst being CuCl2.
The present inventors surprisingly found that microwave heating surprisingly enhances the rate of the reaction, thus reducing the total time for the reaction from 5-10 hours as reported by A. Gaikwad and S. Chakraborty, “Mixing and temperature effects on the kinetics of alkali metal catalyzed, ionic liquid based batch conversion of cellulose to fuel products,” Chemical Engineering Journal, vol. 240, pp. 109-115, 2014; to only 36-51 minutes and significantly increasing the product yields. In a preferred embodiment the reaction time is about 46 minutes.
In the present process non-edible lignocellulosic substrate Sunn hemp having particle size of 230-250 µm, preferably 250 µm is used for the microwave-assisted Ionic Liquid based catalytic conversion to fuel precursors.
In the present invention, four biofuel precursors, namely, glucose, hydroxymethylfurfural (HMF), levulinic acid and formic acid are formed. The overall reaction schemes/ major reactions/ gross reactions are as following

The conversion of lignocellulose to glucose and HMF (5-hydroxymethylfurfural) to Levulinic Acid and Formic Acid need more amount of water whereas conversion of glucose to HMF (5-hydroxymethylfurfural) needs scarcity of water in the system. Reaction (A) and (C) are hydrolysis reaction whereas Reaction (B) is dehydration.The present invention provides a process which maximizes the glucose, Levulinic Acid and Formic Acid yields from hydrolysis and HMF (5-hydroxymethylfurfural) yield from dehydration from the same starting material.

In the present invention the concentrations of water (w/w) can be varied from 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% and 90%.The preferable water concentration (w/w) that varied from isabout 25% to 40% for maximizing the production of glucose, Levulinic Acid and Formic Acid, HMF yield. When the preferred precursors are glucose, Levulinic Acid and Formic Acid, then the preferred water concentration should be 40%.When the preferred precursor is HMF, then the preferred water concentration should be 25%.
The above mentioned concentrations (w/w) are based on 100mg of Ionic Liquid such as 5% (w/w) of water means 0.1 gm of water/ 2 gm of Ionic Liquid.

In the present invention the catalyst to substrate ratios (w/w) varies from 6%, 7%, 8%, 10%, 12%, 14%, 16% to20%. The above mentioned concentrations are based on 100 mg of substrate such as 6% of catalyst to substrate ratio means 0.06 mg of catalyst per mg of substrate i.e. 6mg/100 mg. The preferable concentration is about 16% for maximizing the yield of glucose, HMF, Levulinic Acid and Formic Acid.

The reaction temperature is varied from 160°C to 200°C with 20°C increase at a time. The preferable temperature was found to be 1600C for maximizing glucose, 1800C for maximizing HMF and 2000C for maximizing Levulinic Acid and Formic Acid. The preferred temperature range for performing the process is 1600C to 2000C, depending on the biofuel precursor to be separated. When the glucose is to be separated the preferred reactor temperature is 160°C; whenHMF is to be separated, then the preferred reactor temperature is180°C; when the preferred precursors are LA and FA, then the preferred reactor temperature is200°C.

The substrate to Ionic Liquid ratios (w/w) are varied as 1.25%, 2.5%, 5%, 7.5% and 10%. 1.25% of substrate to Ionic Liquid ratio means 0.0125 mg of substrate per mg of Ionic Liquid i.e. 25 mg/2000mg. The preferred Sunn hemp fibre to Ionic Liquid ratio is about 2.5% for maximizing the production of glucose, HMF, Levulinic Acid and Formic Acid.
In the present invention a catalyst to substrate ratio is defined as the ratio between the catalyst weight and the substrate weight for a constant ionic liquid loading of 2 gm. Similarly, substrate to ionic liquid ratio is defined as the ratio between the substrate weight and the ionic liquid weight for a constant catalyst loading of 8 mg.
In an embodiment involving the optional step (iii) of the present invention, the ionic liquid is recycled to allow for its repetitive use in the process. In order to recycle the ionic liquid from the reaction mixture, a base solution is added to the reaction mixture and the solution is stirred at about 100 rpm for about 10 minutes to about 20 minutes, preferably 15 minutes. While stirring, the temperature of the solution is maintained between 600C and 900C, preferably at about 700C. Then the samples are cooled to room temperature and centrifuged. Three different phases are formed in the solution after centrifugation. The upper portion is the catalyst rich phase. The middle portion is the solid phase containing reacted substrate, and the bottom portion is base salt rich phase. The catalyst phase is separated and it is evaporated at about 1100C to about 1300C, preferably about0 1200C for about 15 minutes to about 30 minutes, preferably about 20 minutes to remove the water from that phase. The reacted substrate present in the solid phase is washed with water and then dried. The findings of the present invention demonstrate that the activity of the catalyst is not significantly reduced by recycling. Consequently, the process of the present invention is economically significant yet having comparable yields.
In said embodiment the bases can be selected from 40 wt% K3PO4, K2HPO4, K2CO3, KHPO4 and the like. The preferred base is 40 wt% K3PO4.
As understood from the foregoing the preparation and separation of the at least one precursor as and when required can be controlled by adjusting the reaction temperature and water concentration. For example when the preparation of HMF is desired, the reaction temperature is maintained at 180°C and the water concentration is about 25% (w/w). This resultant product may contain slight amounts of the other precursors, which can be further separated using methods known to a person skilled in the art such as Ionic Liquid immiscible organic solvent.
The biofuel precursors so obtained and separated using the present invention are converted to the respective biofuels using methods known to a person skilled in the art. For instance glucose is separated from the reaction mixture using Ionic Liquid immiscible organic solvent. The organic phase and aqueous (Ionic liquid) phase are separated. HMF gets dissolved in organic phase while glucose, which does not dissolve in organic phase, is left behind in aqueous phase.
In a particular embodiment, the present invention provides a process for production of bioethanol from Sunn hemp fibres involving production of bioethanol precursor glucose by the present process. The glucose so obtained is fermented to bioethanol using Saccharomyces cerevisiae.
The present inventors compared the yields of bioethanol obtained from commercial glucose, glucose produced present process using the Ionic Liquid medium and glucose produced by present processusing the recycled Ionic Liquidmedium.Commercially available glucose was purchased from Sigma Aldrich (USA). The yields of bioethanol from the glucose obtained by present process was comparable to that obtained using the commercially available glucose.
Examples
Example 1:Characterization
Characterization of Sunn hemp fibre gives 75.6% cellulose, 10.05% hemicelluloses, 10.32% lignin, 3.6% moisture, and 0.43% ash contents. We measured the crystallinity of untreated and reacted samples. The crystallinity of untreated sample and reacted sample are 63% and 54%, respectively. (Fig 1 A). Fig 1B presents the FTIR spectra for the various bonds in pure Sunn hemp as well as in reacted Sunn hemp with 25%, 30% and 40%. Field Emission Scanning Electron Microscope (FESEM) was used to study the morphology of the sample and the substrate is observed to be not highly porous. (Fig. 1 (C-D)).
Example 2:Pre-treatment of the substrate
The effect of pre-treatment with Ionic Liquid medium for 30 minutes is studied on the yields of the products. It can be observed from Fig.2 and Fig.3 that the product yields decrease with pre-treatment. Therefore, pre-treatment is not required for this microwave-assisted catalytic conversion.
Example 3: Reaction time
The effect of reaction time was studied with different amount of water addition while keeping all other parameters constant. The total reaction times of the process are varied as 36 minutes, 41 minutes, 46 minutes and 51 minutes. The yields of the products are most preferred at 46 minutes (Fig.4 and Fig. 5).
Example 4: Catalysis and recycling process
The present reactionwas carried out by mixing catalyst, ionic liquid, water and the substrate (Sunn hemp fibres) together in a microwave reactor. After that, the mix was heated to 1200C with microwave transmitting power of 900 W and microwave frequency of 2.45 GHz for five minutes. Then the temperature ramp to 1600C - 2000C under a pressure of 5 bar for 15 minutes with increasing the microwave transmitting power from 900 W to 1200 W. The total reaction times of the process was varied as 36 minutes, 41 minutes, 46 minutes and 51 minutes. The concentrations of water (w/w) was varied as 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, in order to observe its effects on the yield of the products (glucose, HMF, Levulinic Acid and Formic Acid). The above mentioned concentrations (w/w) are based on 100mg of Ionic Liquid such as 5% (w/w) of water means 0.1 gm of water/ 2 gm of Ionic Liquid. The reaction temperature is varied from 160°C to 200°C with 20°C increase at a time. The catalyst to substrate ratios (w/w) of 6%, 7%, 8%, 10%, 12%, 14%, 16% and 20% are used in our experiments. 6% of catalyst to substrate ratio means 0.06 mg of catalyst per mg of substrate i.e. 6mg of catalyst/100 mg of substrate. The substrate to Ionic Liquid ratios (w/w) are varied as 1.25%, 2.5%, 5%, 7.5% and 10%. 1.25% of substrate to Ionic Liquid ratio means 0.0125 mg of substrate per mg of Ionic Liquid i.e. 25 mg of substrate/2000mg of ionic liquid.
For recycling the ionic liquid from the reaction mixture 5 ml of 40 wt% K3PO4 solution is added to the reaction mixture and the solution is stirred at 100 rpm for 15 minutes. While stirring, the temperature of the solution is maintained at 700C. Then the samples are cooled to room temperature. After that, the samples are centrifuged. Three different phases are formed in the solution after centrifugation. Upper portion is the ionic liquid rich phase. Middle portion is the solid phase containing reacted substrate and the bottom portion is K3PO4 salt rich phase. The ionic liquid phase is separated and then the phase is evaporated at 1200C for 20 minutes to remove the water from that phase. The recovery rate of recycle ionic liquid is 84%. The reacted substrate present in the solid phase is washed with water and then dried.
Example 5: Addition of water
With the addition of water in the reaction mixture, the yields of glucose, HMF, Levulinic Acid and Formic Acid increase initially, reach corresponding maximum values, beyond which they decrease.Now, the maximum yields of glucose, Levulinic Acid and Formic Acid are found at 40% water content whereas the maximum yield of HMF has been found at 25% water content. This relatively lower requirement of water addition for maximum production of HMF is due to the fact that the reaction (B) is a dehydration reaction. On the other hand, reactions [(A) & (C)] are hydrolysis reactions that require the presence of water in the system. Hence, the maximum values of glucose, Levullinic Acid and Formic Acid are found at higher value of 40% water content. The reason for the decrease of product yields below their optimum values may be attributed to the fact that the excessive addition of water precipitates the cellulose in the reaction. This eventually reduces the yield of glucose as well as that of HMF, Levulinic Acid and Formic Acid.
Example 6: Influence of catalyst loadings per unit substrate
Fig. 6 (A-H), Fig. 7 (A-H), Fig. 8 (A-H) and Fig. 9 (A-H) show the variation of yields of Glucose, HMF, Levullinic Acid and Formic Acid, respectively, for different catalyst loadings per unit substrate (w/w) of 6%, 7%, 8%, 10%, 12%, 14%, 16% and 20%. From the plots in Fig. 6, it is observed that for 160oC, the maximum glucose yields increases by 7.56% as catalyst loadings per unit substrate varied from 6% to 16%. Similarly, the plots in Fig. 7, Fig. 8 and Fig. 9 illustrate the maximum yields of HMF, Levullinic Acid and Formic Acidincrease by 4.75% at 180oC, 4.225% at 200oC and 1.68% at 200oC, respectively, within the same range (6%-16%) of catalyst loadings per unit substrate.
Example 7: Influence of temperature
In the temperature range of 160-180oC, for optimum catalyst loading per unit substrate and for optimum water content, glucose yield decreases by 45.54% while HMF, Levulinic Acid and Formic Acid yields increase by 23.99%, 15.89% and 6.32% respectively. In the temperature increase of 180-200oC, for the same optimum catalyst loading per unit substrate and water content, glucose and HMF yields decrease by 14.88% and 6.87% respectively whileLevulinic Acid and Formic Acid yield increase by 66% and 7.4% respectively. Thus, it is seen that lower temperature (160°C) favoursglucose production, higher temperature (200°C) favoursLevulinic Acid, Formic Acid while intermittent temperature (180°C) favours for HMF.
Example 8: Influence of substrate loadings per unit of Ionic Liquid
The substrate loadings per unit of Ionic Liquid loading also play a crucial role in determining the yields of Glucose, HMF, Levulinic Acid and Formic Acid. Different concentration levels of substrate loadings/IL such as 1.25%, 2.5%, 5%, 7.5% and 10% have been chosen, keeping the catalyst loadings constant. Figure 10 (A-C) displays the variation of glucose yields with respect to water addition along with the variation of substrate loadings/IL for all three reaction temperatures. It is inferred from the figures that the yield of glucose is maximum at 2.5% substrate loadings/IL loading and gradually decreases as substrate loadings/IL increases from 2.5 % to 10%. Similarly, the yields of HMF, Levulinic Acid and Formic Acid also decrease with increasing substrate loadings/IL concentrations (Fig. 10(D-F) and Fig. 11(A-F)). The negative effect of substrate loadings/IL is due to the fact that at particular catalyst dosage, additional presence of substrate inhibits the product feedback i.e. conversion of lignocellulose to glucose. As a result, the formation of the other products also diminished sequentially.

Example 8-A
The Sunn hemp fibres comprise cellulose, hemicelluloses, lignin, moisture, and ash contents. (Ex1). [Bmim]Cl (1-Butyl 3- methyl imidazolium chloride) with a purity of 97% to 99%, preferably 98%, used as Ionic Liquid is purchased from HIMEDIA Labs, Mumbai, India. Catalyst CuCl2 is purchased from S.D. Fine Chemicals Limited, Mumbai, India.
The experiments are conducted in a Titan MPSTM 16 vessel position microwave sample preparation system (Perkin Elmer, Waltham, USA) by mixing the non-edible lignocellulosic substrate with catalyst, Ionic Liquid and water according to the process exemplified in example 4.

Measurement of glucose: Samples of 0.1 ml are collected from the reaction mixture after complete dissociation of the substrate at the specified time, temperature and pressure in the microwave digester. 0.9 ml of HPLC grade water is used to dilute the samples and arrest the reaction. Then the tubes are centrifuged at 12000 rpm for 15 minutes to precipitate all the insoluble solids. GOD-POD test kit purchased from Accurex Biomedical Pvt. Ltd., Mumbai, India is used for the measurement of glucose concentration. 40µl of the diluted sample is mixed with 4 ml. of GOD-POD solution and is kept for 4 hours at 40°C. Glucose oxidase (GOD) converts glucose to Gluconic Acid and hydrogen peroxide, following which the peroxide oxidatively coupled with 4-ammineantipyrene and phenol in the presence of peroxidise to produce red Quinoeimine dye that absorbs light at 505 nm wavelength in UV Spectrophotometer (Agilent Technologies). The absorbance is directly proportional to the concentration of glucose in the sample.
Glucose + O2 + H2O Gluconic Acid + H2O2
H2O2 + 4-AAP + Phenol Red Quinoeimine dye + H2
Validation with HPLC: The concentrations of glucose found in the UV spectrophotometer are validated with a HPLC system (Agilent Technologies). ZORBAX Carbohydrate Analysis Column (5µm, 4.6×150mm2) equipped with Refractive Index Detector is used for analyzing the concentration of glucose. A solution of 75:25 (v/v) Acetonotrile / Water is used as the mobile phase with the flow rate of 1.5 ml / min. at operating temperature of 250C for the measurement of glucose concentration in HPLC. HPLC grade glucose (purchased from Sigma Aldrich, St. Louis, USA) is used to establish a standard curve in order to determine the concentrations and retention times of each unknown samples of glucose. Standard samples of 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 40 and 50 mg/ml of glucose are injected into the system. These known glucose concentrations correspond to a peak area and an equation is developed using those HPLC peak area to calculate the concentration of glucose for each unknown sample. Then the concentration of glucose obtained from HPLC is plotted against the concentration of glucose obtained from UV spectrophotometer; the points fall on the 45º line passing through the origin, suggesting that the UV spectrophotometer readings are accurate.
Measurement of HMF, Formic Acid and Levulinic Acid: A HPLC system (Perkin Elmer, USA) is used to determine the concentrations of HMF (5-hydroxymethylfurfural), Levulinic Acid and Formic Acid. Wakosil II 5C18 column (5µm, 4.6×150mm2) is used for analyzing these compounds. A solution of 90:10 (v/v) methanol / water is used as the mobile phase with the flow rate 1 ml / min at 282 nm wavelength for the measurement of HMF (5-hydroxymethylfurfural) concentration. Aqueous solution of H2SO4 (5mM) is used as the mobile phase with a volumetric flow rate of 0.6 ml / min at 210 nm wavelength at operating temperature of 30°C for the measurement of Levulinic Acid and Formic Acid.
A high purity (97%) HMF (5-hydroxymethylfurfural) (purchased from HIMEDIA Labs, Mumbai, India), HPLC grade Levulinic Acid and Formic Acid (purchased from Sigma Aldrich, St. Louis, USA) are used to establish the standard curves to determine the concentrations and retention times of HMF (5-hydroxymethylfurfural), Levulinic Acid and Formic Acid of each unknown samples. Standard samples of 5, 10, 20, 30, 50 and 100 ppm HMF (5-hydroxymethylfurfural), 1000, 3000, 5000, 6000 and 9000 ppm of Levulinic Acid and 300, 400, 500, 750, 1000, 2000, 3000, 4000 and 5000 ppm of Formic Acid are injected into the HPLC system. All known HMF (5-hydroxymethylfurfural), Levulinic Acid and Formic Acid concentrations correspond to a peak area and the equations are developed using the HPLC peak areas to calculate the HMF (5-hydroxymethylfurfural), Levulinic Acid and Formic Acid concentrations of unknown samples. The yields of each unknown samples is calculated by the formulae: % Yield = (concentration at the end of the reaction (mg/ml)/ substrate loadings (mg/ml)) × 100.

Example 9: Effect of recycling of Ionic Liquids
Fig. 12 (A, C, E and G) presents a comparative study of the effect of substrate loadings/Ionic Liquid (IL) and substrate loadings/Recycled Ionic Liquid (RIL) on the yields of glucose at three different temperatures and different water additions for fixed catalyst loadings. The maximum yields of glucose at 160ºC are 71.04%, 66.48%, 63.43% and 61.55% for substrate loadings/IL of 1.25%, 2.5%, 3.75% and 5%, respectively, whereas for the same concentrations of substrate loadings/RIL the maximum yields become 64.21%, 61.28%, 58.8% and 57.15%. It can be inferred from the data that the use of recycled Ionic Liquids does not significantly affect the product yields. Thus, we can conclude that activity of the Ionic Liquid is not significantly reduced by recycling. Similarly, Fig. 12 (B, D, F and H), Fig. 13 (A, C, E and G) and 13 (B, D, F and H) present the same features for the yields of HMF, Levulinic Acid and Formic Acid, respectively. We find that the maximum yields of HMF at 180ºC are 23.25%, 22.14%, 21.12% and 20.25% for substrate loadings/IL of 1.25%, 2.5%, 3.75% and 5%, respectively, while for the same concentrations of substrate loadings/RIL the maximum yields become 21.56%, 20.46%, 19.45% and 18.59%. For the case of Levulinic Acid at 200oC, the maximum yields are observed to be 35.19%, 32.84%, 31.01% and 28.67% for substrate loadings/IL of 1.25%, 2.5%, 3.75% and 5%, respectively, whereas for the same concentrations of substrate loadings/RIL the maximum yields become 33.28%, 30.99%, 29.15% and 26.89%. The maximum yields of Formic Acid at 200oC are 14.05%, 13.02%, 12.33% and 11.51% for substrate loadings/IL of 1.25%, 2.5%, 3.75% and 5%, respectively while for the same concentrations of substrate loadings/RIL the maximum yields become 13.21%, 12.30%, 11.57% and 10.68%. The corresponding data shows that the Ionic Liquid can be recycled for further usage in the process without reducing much of its effectiveness. When the Ionic Liquid is recycled without any replenishment, the reduction in the product yield is restricted to 1-3%. Hence, we can proceed for using recycled Ionic Liquid in our experimental processes instead of using fresh Ionic Liquids for the sake of cost-effective biofuel production. This slight reduction in the product yields may also be avoided by replenishing the Ionic Liquid in the amount it has been depleted during the separation process.
Example 10: Separation of glucose and HMF
An Ionic Liquid immiscible organic solvent (V1-Butanol: VethylIso Butyl ketone = 3: 7, Morganic solvent: MIL=3:1) is added to the reaction mixture, following which the aqueous phase (Ionic Liquid phase) and the organic phase are separated. The upper layer of organic phase is removed for analysis of HMF and the glucose, which does not dissolve in the organic phase, is left only in the Ionic Liquid phase. The glucose concentration is measured from the aqueous phase. HMF concentration is measured both in the organic phase and Ionic Liquid phase. The separation efficiency for glucose is obtained as 85%, while the separation efficiencies of HMF, Levulinic Acid and Formic Acid are 99.8%, 98.9% and 91.8%, respectively.

Example 11: Bioethanol formation from the hydrolysed product

Fermentation experiments are carried out using three different glucose sources. First one is commercially available glucose purchased from Sigma Aldrich (USA), second one is glucose produced from the experiments in the Ionic Liquid medium and the third one is glucose produced from the experiments in the recycled Ionic Liquid medium. Substrate is made moisture free before starting the inoculums. 20 ml of Malt extract, Glucose, Yeast extract and Peptone [MGYP] media in distilled water is prepared using 240 mg of dried glucose as a sole carbon source. Other nutrients, malt and yeast extract, amounting 72 mg of each (Sisco Research Laboratories Pvt. Ltd., India) and 120 mg of peptone (Sisco Research Laboratories Pvt. Ltd., India) are added in the solution. Commercially available brewing yeast, wild-type yeast strain Saccharomyces cerevisiae (MTCC 170) is purchased from Microbial Type Culture Collection and Gene Bank, Chandigarh. The microorganisms are grown in the culture media on the Petri dish at room temperature and stored at 40C.

The yeast inoculum is started 35 hours before the beginning of each batch run. Three different types of mixtures are prepared for three different types of glucose sources. For pure glucose source, 20 ml of MGYP media in distilled water is prepared using 240 mg of dried glucose as a sole carbon source. Other nutrients, malt and yeast extract, amounting 72 mg of each (Sisco Research Laboratories Pvt. Ltd., India) and 120 mg of peptone (Sisco Research Laboratories Pvt. Ltd., India) are added in the solution. Again, for the glucose source, which produced from the experiments in the Ionic Liquid medium, 20 ml of total MGYP media is prepared using 12 ml of 20 mg/ml glucose with Ionic Liquid as a sole carbon source. Other nutrients, malt and yeast extract, amounting 72 mg of each (Sisco Research Laboratories Pvt. Ltd., India) and 120 mg of peptone (Sisco Research Laboratories Pvt. Ltd., India) are added to 8 ml of distilled water. Now, for the glucose source, which produced from the experiments in the recycled Ionic Liquid medium, 20 ml of total MGYP media is prepared using 15 ml of 16 mg/ml glucose with Ionic Liquid as a sole carbon source. Other nutrients, malt and yeast extract, amounting 72 mg of each (Sisco Research Laboratories Pvt. Ltd., India) and 120 mg of peptone (Sisco Research Laboratories Pvt. Ltd., India) are added to 5 ml of distilled water. Thus, 20 ml of three different media are prepared. Then the mixtures are autoclaved for 30 minutes. Yeast cells (S.cerevisiae) grown in the Petri dish are added into the autoclaved solution (pH 4.8-5.4) and then incubated for 32 h to grow yeast cells at no mixing condition. Cell growth is monitored by measuring the optical density (OD) at 600 nm using a UV-spectrophotometer (PerkinElmer). The inoculum containing grown cells with OD 0.62, i.e., after the mid-log phase is attained, is taken for glucose fermentation. Before the start of the fermentation experiment, the inoculum is centrifuged and washed thrice with the fermentation media to avoid ethanol contamination. 1 ml of inoculum containing thoroughly washed cells is added into the fermentation media.
The experiments are carried out using the initial cell concentration of 0.56 mg/ml to 0.58 mg/ml, preferably 0.57 mg/ml and glucose concentration of 12 mg/ml, under anaerobic conditions at 25-350C, preferably at 300C for 60 hours at 30-50 rpm, preferably 40 rpm. The samples are collected at regular intervals of time to analyse the dry cell, glucose and ethanol concentrations. The bioethanol concentration in the distillates of the fermentation broth samples is determined by gas chromatography, using a system of Chemito GC 8610 equipped with a flame ionization detector (FID). 10% OV–17 column is operated isothermally at 90°C to detect ethanol. Injector and detector temperatures are maintained at 110°C and 150°C, respectively. Nitrogen is used as a carrier gas. Standard aqueous solutions of ethanol in the concentration range of 2–10 µl/ml are used for the calibration curve for ethanol estimation.
(Fig 15.A) shows that maximum cell concentrations of 5.01 mg/ml, 4.69 mg/ml and 4.48 mg/ml are obtained after 60 hours of incubation when the substrate is fresh glucose, glucose obtained from Ionic Liquid mediated hydrolysis, and glucose obtained from Recycled Ionic Liquid mediated hydrolysis. It can be seen from (Fig 15.B) glucose gradually decreases for all three cases with incubation time. The maximum bioethanol yields are obtained after 15 hours of fermentation, as 87.89%, 75.59% and 71.68% when the substrate is fresh glucose, glucose obtained from Ionic Liquid mediated hydrolysis, and glucose obtained from Recycled Ionic Liquid mediated hydrolysis, respectively. (Fig.15 D).

Claims:

1. A rapid process of producing biofuel precursors from Sunn hemp fibers, said process comprising steps of:
(iv) microwave heating assisted, catalytic hydrolysis of said fibers in presence of an ionic liquid based catalyst;
(v) separation of at least one precursor as and when required; and
(vi) optionally recycling the ionic liquid catalyst to be used in step (i)
wherein the precursors are glucose, hydroxymethyl furfural (HMF), levulinic acid and formic acid.
2. The process as claimed in claim 1, wherein said ionic based liquid catalyst is an alkali metal chloride catalyst.
3. The process as claimed in claim 2, wherein said alkali metal chloride is selected from copper chloride (CuCl2), chromium chloride (CrCl3), aluminium chloride (AlCl3), zinc chloride (ZnCl3),iron chloride (FeCl3).
4. The process as claimed in claim 3, wherein said alkali metal chloride is preferably copper chloride (CuCl2).
5. The process as claimed in claim 1, wherein the particle size of Sunn hemp fibre is in the range of 240 to 260 µm.
6. The process as claimed in claim 5, wherein the particle size of Sunn hemp fibre is preferably 250 µm.
7. The process as claimed in claim 1, wherein water concentration for the hydrolysis varies from 5% to 90%.
8. The process as claimed in claim 7, wherein water concentration for the hydrolysis preferably varies from 25% to 40%.
9. The process as claimed in claims 1 and 7, wherein the biofuel precursors are predominantly a mix of glucose, levulinic acid and formic acid when the water concentration is about 40%.
10. The process as claimed in claims 1 and 7, wherein the biofuel precursor is predominantly hydroxymethyl furfural (HMF) when the water concentration is about 25%.
11. The process as claimed in claim 1, wherein the catalyst to Sunn hemp fibre ratio varies from 6% to 20% (w/w).
12. The process as claimed in claim 9, wherein the catalyst to Sunn hemp fibre ratio preferably varies from 6% to 16% (w/w).
13. The process as claimed in claim 1, wherein heating is carried out at a temperature varying from about 1600C to about 2000C.
14. The process as claimed in claims 1 and 13, wherein the biofuel precursor is predominantly glucose when heating is carried out at a temperature of about 160°C.
15. The process as claimed in claims 1 and 13, wherein the biofuel precursor is predominantly hydroxymethyl furfural (HMF) when heating is carried out at a temperature of about 180°C.
16. The process as claimed in claims 1 and 13, wherein the biofuel precursor is predominantly a mix of levulinic acid and formic acid when heating is carried out at a temperature of about 200°C.
17. The process as claimed in claim 1, wherein said Sunn hemp fibre to Ionic Liquid ratio varies from 1.25% to 10%.
18. The process as claimed in claim 12, wherein said Sunn hemp fibre to Ionic Liquid ratio is preferably 2.5%.
19. The process as claimed in claim 1, wherein said optional step (iii) of recycling comprises steps of:
(i) reacting the ionic liquid based catalyst with 40 % wt K3PO4 and stirring at about 100 rpm for about 10 minutes to about 20 minutes between 600C and 900C;
(ii) cooling the mixture to room temperature followed by centrifugation;
(iii) separating the phases formed upon centrifugation; wherein the upper phase is the catalyst rich phase;
(iv) evaporating said upper phase at about 1100C to about 1300C for about 15 minutes to about 30 minutes to obtain ionic liquid based catalyst wherein said ionic based catalyst is reused in step (i) of claim 1.
20. A process for preparation of bioethanol comprising steps of:
a. hydrolyzing Sunn hemp fibres by the process as claimed in claim 1 to obtain glucose;
b. fermenting the glucose from step (a) using Saccharomyces cerevisiaee in MGYP media under anaerobic conditions at a temperature of about 25°C to 350C and mixing at a speed of 30 to 50 rpm for about 10 hours to about 20 hours.
21. A process for preparation of bioethanol as claimed in claim 16, wherein fermentation is preferably performed at 300C and at a mixing speed of 40 rpm for about 15 hours.