The present patent application is a patent of addition of the main Indian Patent Application No. 201821008982 of Filing date Mar. 12, 2018, and Publication date Jan. 3, 2020. The present application comprises an improvement in or a modification of the invention claimed in the specification of the main patent applied for in the Indian Patent Application No. 201821008982.
The present invention relates to a process for continuous production of second-generation ethanol from lignocellulosic biomass in simultaneous saccharification and co-fermentation (SSCF) process. More particularly, the process achieves overall ethanol yield up to 70% for both C5 and C6 sugars from pretreated biomass. C5 utilization exceeds 95% after SSCF. This involves using three separate vessels/fermentors for the fermentation process based on the temperature changes required.
Simultaneous Saccharification and fermentation/co-fermentation (SSF/SSCF) removes sugar inhibition on enzymatic hydrolysis thus increasing the hydrolysis sugar yield and reducing contamination risk. Moreover, SSF/SSCF reduces the overall reaction time and reactor volume (Kristensen et al., 2009). SSF/SSCF sacrifices the optimal conditions for both enzymatic hydrolysis and fermentation. Typically, in enzymatic hydrolysis and fermentation in SSF system the temperature is kept at 37-42° C. as a compromise for better enzymatic hydrolysis and fermentation (Dien et al., 2003b). In addition, SSF/SSCF introduces a new inhibitor (ethanol) for enzymatic hydrolysis. But the inhibitory effect from ethanol is much lower compared to cellobiose or glucose (Taherzadeh & Karimi, 2007).
Continuous approach of SSCF is more economical and practical at demo and production level because it makes the process more economical and less labor-intensive approach. There is a report regarding continuous SSCF by Jin et al. 2013 for ethanol production from Ammonia Fiber Expansion (AFEX™) pretreated corn stover. In this study, the author represented enhanced ethanol production from pretreated corn stover by following pre-hydrolysis by fermentation. In the approach of continuous SSCF, pre-hydrolysis is followed for 24 hours and the then the fermentation is followed in three conjugative fermentors arranged in a bioreactor train fashion. The flow rate in the reactors is similar.
Furthermore, the main Indian Patent Application No. 201821008982 disclosed a batch SSCF process, in which fermentation time reduced significantly with application of low dose of cellulase enzyme in comparison to the conventional SSCF process. However, the present invention discloses a process in a continuous mode which has several advantages over the main Indian Patent Application No. 201821008982 as described below.
Accordingly, the present invention provides a process which overcomes the aforesaid drawback of the prior arts. In the present invention, overall ethanol yield was achieved upto 70% for both C5 and C6 sugars from pretreated biomass. C5 utilization exceeded 95% after SSCF. In the current practice the C5 and C6 sugars were targeted for fermentation in a sequence manner to achieve higher ethanol titer at short time of fermentation and low dose of enzyme.
Present invention relates to a process for continuous production of second-generation ethanol from a lignocellulosic biomass, wherein the process includes a first fermentor vessel for fermenting mainly C5 sugars and continuous transferring the fermented biomass to a second fermentor vessel for hydrolyzing the fermented biomass and then continuous transferring the hydrolysate to a third fermentor vessel for selectively fermenting C6 sugars to obtain ethanol. The C5 and C6 sugars are targeted for fermentation in a sequential manner to achieve higher ethanol titer at short time of fermentation (56 hours) and low dose of enzyme. Further, in the present invention of continuous SSCF process, overall ethanol yield up to 70% was achieved for both C5 and C6 sugars from pretreated biomass; and C5 utilization exceeded 95% after SSCF. Therefore, the C5 fermentation, hydrolysis and C6 fermentation is performed in three separate vessels at the required temperature. The present invention ultimately reduces vessel numbers, reduction of continuous yeast dosing to fermentation vessels, low concentration of the xylose maintained in both fermentation vessels throughout the process which reduces the C5 fermentation time and initial higher viscosity problem in batch process is reduced up to 90%, which ultimately saves overall energy input for stirring and no additional filling or emptying time in steady state.
Accordingly, present invention provides a process for continuous production of a second-generation ethanol from a lignocellulosic biomass comprising;
In one of the features of the present invention, the C5 sugar is selected from xylose and C6 sugar is selected from glucose.
In another feature of the present invention, the concentration of the cellulase enzyme in a range of 1.8-2.5 FPU/TS is employed for the fermentation process.
In yet another feature of the present invention, the fermentation of C5 sugar is carried out at a temperature in a range of 33-35° C. for 16-20 hours.
In still another feature of the present invention, the fermentation of C6 sugar is carried out at a temperature in a range of 35-37° C. for 8-10 hours.
In yet another feature of the present invention, the pre-treated lignocellulosic biomass slurry is added in the first fermenting vessel of the fermentor system of step (i) without any detoxification. In another feature of the present invention, the pH of the slurry of step (i) to 5-5.5 is adjusted with a pH adjuster. The pH adjuster is selected from aqueous ammonium solution, NaOH, KOH, CaCO3, or a substance which is alkaline in nature and increases pH.
In still another feature of the present invention, the nutrient is MgSO4 or any other magnesium salt. Nitrogen source such as urea, ammonium sulfate etc is required in case pH adjuster is other than aqueous ammonia.
In still another feature of the present invention, the cellulase enzyme is from fungal or bacterial origin, composed of cellobiohydrolase (I &II), endo-glucanase and β-glucosidase along with other accessory enzyme, wherein the other accessory enzyme is selected from xylanase, β-xyloxidase, arabinofuranosidase, and pectinse or any other enzyme which hydolyyze glucan and/or xylan.
In yet another feature of the present invention, the co-fermenting (C6 and C5 sugar) microorganism is selected from Saccharomyces cerevisiae, or any ethanogenic co-fermenting microorganism such as Pichia sp., Candida sp., Zymomonas mobilis and E. coli.
In still another feature of the present invention, the lignocellulosic biomass is selected from straw, wheat straw, rice straw, sugarcane bagasse, cotton stalk, barley stalk, bamboo or any agriculture residues which contain cellulose or hemicellulose or both.
In one of the features, the present invention provides a process for continuous production of a second-generation ethanol from a lignocellulosic biomass comprising:
In one of the preferred features, the present invention provides a system for continuous production of a second-generation ethanol from a lignocellulosic biomass, said system comprising: a first fermentor vessel with size of 16000 M3 and hydraulic reaction time (HRT) of 16 hours and dilution rate maintained at 0.0625 h−1; a second fermentor vessel with size of 30000 M3 and HRT of 30 hours and dilution rate maintained at 0.033 h−1; and a third fermentor vessel with size of 10000 M3 and HRT of 10 hours; wherein three fermentor vessels are arranged in a sequential manner; and wherein in-out flow rate to all the fermentor vessels is maintained at a constant rate of 1000M3 to achieve steady state.
While the invention is susceptible to various modifications and alternative forms, specific embodiment thereof will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the scope of the invention as defined by the appended claims.
For the purposes of this invention, the following terms will have the meaning as specified therein: “Pre-treated biomass” or “Pretreatment of biomass” used herein clears away physical and chemical barriers that make native biomass recalcitrant and exposes cellulose for better enzymatic hydrolysis. In most of the pretreatment, chemical (acid or alkali) and physical (high temperature or pressure) parameters are used individually or in mixed manner to remove barriers for enzymatic hydrolysis and improve the enzymatic digestibility.
“Detoxification” used herein is the process where the inhibitors (toxic compound such hydroxymethyl furfural, furfural, acetic acids, formic acids, etc.) produced during the pretreatment process are removed or neutralized from pre-treated biomass by chemical, physical, or biological process.
“Cellulase enzyme” used herein is a mixed form of enzyme which is mostly composed of cellobiohydrolase (I &II), endo-hydrolase and β-glucosidase. This enzyme was mostly produced from fungal sources. Cellulase breaks down the cellulose molecule into monosaccharide and shorter polysaccharides or oligosaccharides. In the present invention the cellulase enzyme is selected from commercially available cellulase enzymes which are suitable for the purposes.
“C5 sugars” used herein represents xylose.
“C5 fermentation” used herein is xylose fermentation into ethanol.
“C6 sugar” used herein represents glucose.
“C6 fermentation” used herein is glucose fermentation into ethanol.
“Nutrient” used herein is MgSO4. In the salt MgSO4 used in fermentation where, Mg+2 acts as an essential enzyme cofactor and acts as key structural component of most biological pathways. During fermentation Mg+2 plays a major role for proper functioning of fermenting enzymes in yeast.
Simultaneous saccharification and co-fermentation (SSCF) is a promising strategy for obtaining high ethanol yield. This process operates in a single fermentor vessel where the required temperature keeps changing during the fermentation practice. But shifting of temperature from higher to lower in a batch process makes the process more energy intensive at higher operation level (demo or production scale). Also, large scale plant reactors would require transferring volume from one vessel to another vessel of different temperatures, which would require additional emptying and filling time. This would result in higher capital and operational cost to the process. To overcome this issue, in the present invention, the temperature changes (33° C. for C5 fermentation, 50° C. for hydrolysis and 35° C. for C6 fermentation) for fermentation process are performed in three separate fermentor vessels sequentially at their desired temperatures. This makes the process easier, however, at large scale operation two bioreactor train system (i.e., three vessels in each train in a parallel manner, in total six vessels required) arrangements are required to make the process continuous. Continuous fermentation process reduces vessel number, reduction of continuous yeast dosing to fermentation vessel, low concentration of xylose maintained in both fermentation vessels throughout the process which reduces the C5 fermentation time and initial higher viscosity problem in batch process is reduced up to 90%, which ultimately saves overall energy input for stirring. In the process of the present invention, the C5 and C6 sugars targeted for fermentation in sequence manner to achieve higher ethanol titer at short time of fermentation and low dose of enzyme. An overall ethanol yield up to 70% was achieved for both C5 and C6 sugars from pretreated biomass. C5 utilization also exceeded 95% after SSCF.
The present invention discloses a process for continuous production of second-generation ethanol from lignocellulosic biomass in SSCF process using three separate vessels/fermentors for the fermentation process based on the temperature changes required.
A continuous SSCF process as disclosed in the present invention when compared to a batch SSCF process (see
A process for continuous production of a second-generation ethanol from a lignocellulosic biomass (see
In accordance with the present invention, steady state ethanol production achieved using dilute acid pretreated rice straw. In this process, three reactors of different volume are in series with constant flow of slurry. However, different HRT maintained by different volume size of reactor. First reactor utilized for preferential xylose fermentation at 33° C. followed by hydrolysis in second reactor at 50° C. Thereafter, slurry moved to third reactor for mainly glucose fermentation at 37° C. This resulted into several advantages which are tabulated in Table-1.
In another feature of the present invention, when all vessels in the described continuous SSCF process reached to the designated volume then the flow rate to the entire reactor maintained in a constant rate. All the flow and the dilution in the fermentor vessel are maintained as described in the
Process for Continuous Production of Second-Generation Ethanol from a Lignocellulosic Biomass:
Pretreated biomass (slurry, TS approximately 20-22%) without any detoxification is introduced directly to the first fermentor vessel of the fermentor system. The pH of the slurry was adjusted to 5-5.5 with aqueous ammonium solution (25% initial concentration). The pH adjusted slurry was fortified with MgSO4 (0.5%), cellulase enzyme (in-house enzyme/Ctec, 2.3 FPU/TS) and co-fermenting ethanologenic yeast Saccharomyces cerevisiae (1 g dry cell biomass/100 gTS, xylose utilizing genetically modified yeast). Required amount of water was added to the process to maintain the final biomass concentration to 15%. The whole process was incubated at 33° C. for 16 hours for the xylose fermentation. The fermented broth is then transferred to second fermentor vessel of the fermentor system and is allowed for hydrolysis at 50° C. for 30 hours. The volume of the reactor is maintained at 1.87 times higher as compared to pentose sugar utilizing fermentor vessel for giving hydraulic reactor time of 30 hours. After the hydrolysis, the hydrolysate is transferred to third fermentor vessel of a fermenting system and for the hexose sugar fermenting vessel for 10 hours. All reactors have 1000 M3 flow. When all vessels in the described continuous SSCF process reached to the designated volume then the flow rate to the entire reactor was maintained in a constant rate. At final fermentation, 32 g/L ethanol concentration was produced in third fermentor vessel and continued in steady state. At the same time in other fermentor (First and second) vessels, sugar release and ethanol concentration are in steady state. This steady state was achieved after 56 hours of the fermentation. The results of this experiment are represented by
Process for Batch Production of Second-Generation Ethanol from a Lignocellulosic Biomass (Main Indian Patent Application No. 201821008982)
The pH of the pretreated slurry was adjusted to 5.5 with aqueous ammonium solution (25% initial concentration). The pH adjusted slurry was fortified with 3 g/l MgSO4, cellulase enzyme (Commercial enzyme, 3.3 FPU/TS) and co-fermenting Saccharomyces cerevisiae (1 g dry cell biomass/litre, xylose and glucose utilizing yeast). Required amount of water was added to the process to adjust the final biomass concentration to 20%. The whole process was incubated at 30° C. for 16 h for the fermentation with 200 rpm. When the free xylose concentration in the slurry comes near to 6-7 g/1, the temperature of the process was increased to 33° C. and 35° C., incubated for 2 h in each temperature for better hydrolysis and fermentation. After that temperature increased to 48° C. This step mainly required for rapid releases of glucose sugar from cellulose which converted simultaneously with hydrolysis to ethanol by yeast biomass. As the temperature was reached at desired target the process was allowed to maintain the required temperature (48° C.) for 23 h for better enzymatic hydrolysis. After this incubation the system was allowed to cool down to temperature 35° C. A second dose of co-fermenting S. cerevisiae (1 g dry cell biomass/liter) was inoculated to the system for the second stage of fermentation. The second fermentation was stopped after 6 h of fermentation. This process took 46 h incubation including fermentation and enzymatic hydrolysis.
In the present invention, the continuous SSCF approach is considered as advantageous over the conventional SSCF and batch SSCF due to several reasons as described in Table-1.
Number | Date | Country | Kind |
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201821008982 | Mar 2018 | IN | national |
Number | Date | Country | |
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Parent | 17075486 | Oct 2020 | US |
Child | 17212528 | US | |
Parent | 16351045 | Mar 2019 | US |
Child | 17075486 | US |