WO2024039857A1

WO2024039857A1 - Cascade continuous fermentation of cellulosic biomass via consolidated bioprocessing

Info

Publication number: WO2024039857A1
Application number: PCT/US2023/030593
Authority: WO
Inventors: Lee R. Lynd
Original assignee: The Trustees Of Dartmouth College
Priority date: 2022-08-18
Filing date: 2023-08-18
Publication date: 2024-02-22

Abstract

A system for converting biomass to ethanol and other desired products is disclosed. The system comprises a plurality of bioreactors connected in series with mills between each bioreactor. This configuration leads to shorter reaction times, which is beneficial to industrial processes. The present disclosure relates to systems and methods for converting biomass efficiently using consolidated bioprocessing (CBP) for conversion of cellulosic biomass into fuels and/or chemicals without added enzymes and without thermochemical pretreatment.

Description

CASCADE CONTINUOUS FERMENTATION OF CELLULOSIC BIOMASS VIA CONSOLIDATED BIOPROCESSING CROSS-REFERENCE TO RELATED APPLICATION This application claims benefit of priority to United States Provisional Patent Application No. 63/399,133 filed on August 18, 2022, the content of which is incorporated herein by reference in its entirety. GOVERNMENT RIGHTS This invention was made with government support provided by the Center for Bioenergy Innovation (CBI), which is a U.S. Department of Energy (DOE) Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. The government has certain rights in the invention. BACKGROUND I. Field of the Invention [0001] The disclosure relates to a system for the conversion of biomass to biofuel or other useful products. II. Description of the Related Art [0002] Biomass is a relatively inexpensive, renewable and abundant material that can be used to generate fuels, chemicals, fibers, and energy. However, large-scale utilization of plant biomass is hindered, at least in part, by the lack of technologies capable of efficiently converting the biomass into component fractions or reactive intermediates at a low cost. For example, most plant biomass is resistant to digestion by cellulase. In addition, production of biofuels from non- food biomass has been slow to develop due to expensive enzyme and bioprocess equipment required to access the sugars in lignocellulose. SUMMARY [0003] The present disclosure relates to systems and methods for converting biomass efficiently using a system comprising a plurality of bioreactors connected in series and using consolidated bioprocessing (CBP) for conversion of cellulosic biomass into fuels and/or chemicals without added enzymes and without thermochemical pretreatment. [0004] Thermophilic, lignocellulose-femennting bacteria have the potential to dramatically lower the costs of renewable biofuel production from cellulosic biomass. However, challenges remain in order for thermophilic bacteria to be practical. Robust industrial biocatalysts that possess the following characteristics are needed to improve the process: undiminished solubilization at high solids, complete utilization of hemicellulose solubilization products, production of high product titers. Energy-efficient means to augment accessibility to biological attack would also help improve the economics of biomass utilization. [0005] To this end, consolidated bioprocessing (CBP) using thermophilic bacteria need to be carried out in bioreactor(s) which need to be configured for maximum effectiveness in order to be cost-effective. In certain embodiments, the present disclosure provides a system having multiple continuous, well-mixed bioreactors connected in series — a configuration referred to as "cascade continuous." [0006] In certain embodiments, the cascade continuous design helps reduce reaction time, which, in turn, helps reduce processing cost. Continuous processing offers particular advantages for lignocellulose processing via CBP. Some of these advantages over more conventional processing strategies involving added cellulase and related enzymes are surprising and unexpected. In certain embodiments, first-order kinetics have been observed for many microbial systems mediating lignocellulose deconstruction, including defined cultures of thermophilic bacteria. Analysis based on first-order kinetics indicates that reaction times to reach likely feasible extents of substrate solubilization is about 4.5 days in a single well-mixed continuous bioreactor. This reaction time is very similar to the reaction times observed for batch cultures of thermophilic bacteria fermenting lignocellulose. By contrast, based on the same analysis used to predict a reaction time of 4.5 days in a single CSTR, the reaction time required for three bioreactors arranged in series is only 1.8 days (a 2.5-fold reduction). [0007] In some embodiments, a system for converting biomass into a liquid fuel is disclosed, the system comprising a) a reservoir; and b) a plurality of reactors arranged in series. In one aspect, each reactor has an inlet, and an overflow outlet, wherein the inlet of first reactor of the plurality of reactors is connected to the reservoir. In another aspect, the plurality of reactors comprise a defined culture, which contains at least one living organism capable of producing at least one carbohydrate-active enzyme (CAZyme). [0008] Here, the biomass moves from one reactor to the next through the overflow outlet, and the steps of CAZyme production, substrate hydrolysis and fermentation by the defined culture without added enzymes all occur within each of the plurality of reactors. The enzymes, i.e., CAZyme, is produced by the living organism. No other enzyme is added into the series of reactors. This feature is in contrast to batch operation via the conventional processing paradigm where enzyme is typically added to boost the enzyme:substrate ratio during the process. [0009] In some embodiments, the configuration of the system allows the overflow from the first reactor in the series feeds the second reactor immediately downstream of the first reactor, and overflow from the N reactor feeds N+1 reactor immediately downstream of the N reactor, and so on, wherein N is an integer ranging from 1 to n, and wherein n is an integer, and n+1 is equal to the total number of reactors in the plurality of reactors. [0010] In some embodiments, the system may further comprise one or more mills placed between any two neighboring reactors to further process the moving biomass. [0011] In some embodiments, the system may comprise a starting biomass, wherein the starting biomass is loaded into the reservoir, and the biomass flows one-way from the reservoir to the first reactor. [0012] In some embodiments, the plurality of reactors may contain 3 or more reactors, or at least 4 reactors, or at least 5 reactors, and so on. [0013] In some embodiments, the system runs continuously or semi-continuously. In one aspect, in a CBP process using the disclosed system, the initial enzyme concentration is high and accessible substrate concentration is high in the initial bioreactors in the cascade, which is advantageous over batch or single CSTR configurations. In another aspect, the series of reactors running continuously or semi-continuously significantly shortens reaction time from the biomass to the liquid fuel as compared to reaction time in a batch or single continuous bioreactor. [0014] In some embodiments, the ratio between the CAZyme and the substrate in the first reactor is at least 0.1, or at least 1 unit, or at least 10 units of CAZyme per pmol of the substrate. In some embodiments, the ratio between the CAZyme and the substrate in each reactor of the system is at least 0.1, or at least 1 unit, or at least 10 units of CAZyme per pmol of the substrate. In some embodiments, the CAZyme is in excess of the substrate to ensure substrate access and fast reaction time. [0015] In some embodiments, the liquid fuel is ethanol, or other chemicals. [0016] In some embodiments, the system may include a defined culture comprising a living organism such as Clostridium thermocellum and a hemicellulose-fermenting microorganism selected from the group consisting of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium saccharolyticum. [0017] In some embodiments, the starting biomass is a member selected from the group consisting of corn stover, sugar cane, wheat or rice straw, miscanthus, switchgrass, energy cane and combination thereof. [0018] In certain embodiments, cascade continuous system is applied to CBP, but not to simultaneous saccharification and fermentation (SSF), because the benefits of the cascade continuous configuration are specific to CBP, and not to systems with added enzymes including but not limited to SSF. While traditional CBP process typically has low initial enzyme concentration, cascade continuous system with CBP achieve high initial enzyme concentration which helps speed up the conversion of biomass to biofuel. [0019] In certain embodiments, a method is also disclosed for using the system described above to produce a liquid fuel from biomass. The method may include, a) loading a starting biomass into the reservoir; b) allowing the biomass to move from the reservoir into the first reactor where at least a portion of the biomass is converted into alcohol, c) allowing the system to run continuously or semi-continuously wherein the reservoir feeds the first reactor and overflow from the N reactor moves to N+1 reactor immediately downstream of the N reactor, d) repeating steps (a)-(c) above to allow the biomass to move from the N reactor to the N+1 reactor through the series of reactors until it reaches the last reactor, e) allowing the system to run continuously or semi-continuously, wherein steps of CAZyme production, substrate hydrolysis and fermentation by the defined culture without adding enzymes all occur within each of the plurality of reactors, and f) collecting the liquid fuel from the plurality of reactors. [0020] In certain embodiments, the solids loading in the system is about 5-30 wt.%, or about 10-25 wt.% solids, or about 15-25 wt.% solids. BRIEF DESCRIPTION OF THE DRAWINGS [0021] FIG. 1 shows a preferred embodiment of the bioreactor configuration described herein. [0022] FIG. 2 shows a comparison of solubilization data using fungal cellulase SSF and C. thermocellum. [0023] FIG. 3 shows an exemplary graph for the kinetic characterization of continuous culture. DETAILED DESCRIPTION [0024] Described herein are systems and methods for converting biomass to ethanol or other desired products. In a preferred embodiment, for example as shown in FIG. 1, the system of the present invention comprises: a) a reservoir; b) a first bioreactor, a second bioreactor, and a third bioreactor; and c) a first mill coupled to the first bioreactor and the second bioreactor and a second mill coupled to the second bioreactor and the third bioreactor. In other embodiments, the system comprises: a) a reservoir; b) a plurality of bioreactors, with each bioreactor in series; and c) a plurality of mills, wherein each mill is coupled to two bioreactors. [0025] The configuration of the systems described herein confer several important advantages: 1) Conditions are conducive to microbial growth and rapid reaction in the initial stage and high fractional carbohydrate solubilization and product titer in later stages; 2) Reaction times are several-fold shorter as compared to batch or single-stage continuous processing. This benefit is specific to CBP and not realized by more conventional processes featuring added enzymes; 3) Continuous fermentation is advantageous for C-CBP because it avoids mixing and mass transfer challenges at high solids and is a preferred inoculum preparation mode for obligate anaerobes. [0026] Embodiments of the present disclosure are further illustrated by the following items. [0027] Item 1. A system for converting biomass into a liquid fuel, comprising: a) a reservoir; and b) a plurality of reactors arranged in series, each reactor having an inlet, and an overflow outlet, the inlet of first reactor of the plurality of reactors being connected to the reservoir, wherein the plurality of reactors comprise a defined culture comprising at least one living organism capable of producing at least one carbohydrate-active enzyme (CAZyme) , wherein steps of CAZyme production, substrate hydrolysis and fermentation by the defined culture without added enzymes all occur within each of the plurality of reactors. [0028] Item 2. The system of Item 1, wherein overflow from a first reactor in the series feeds a second reactor immediately downstream of the first reactor, and overflow from the N reactor feeds N+1 reactor immediately downstream of the N reactor, wherein N is an integer ranging from 1 to n, and wherein n is an integer, and n+1 is equal to the total number of reactors in the plurality of reactors. [0029] Item 3. The system of any preceding Items, further comprising one or more mills placed between two neighboring reactors. [0030] Item 4. The system of any preceding Items, further comprising a starting biomass, wherein the starting biomass is loaded into the reservoir, and the biomass flows one- way from the reservoir to the first reactor. [0031] Item 5. The system of any preceding Items, wherein the plurality of reactors comprises 3 or more reactors. [0032] Item 6. The system of any preceding Items, wherein the system runs continuously or semi-continuously. [0033] Item 7. The system of any preceding Items, wherein the series of reactors running continuously or semi-continuously shortens reaction time from the biomass to the liquid fuel. [0034] Item 8. The system of any preceding Items, wherein the reaction time from the biomass to the liquid fuel is shorter as compared to reaction time in a batch or single continuous bioreactor. [0035] Item 9. The system of any preceding Items, wherein ratio between the CAZyme and the substrate in the first reactor is at least one unit of CAZyme per pmol of the substrate. [0036] Item 10. The system of any preceding Items, wherein the liquid fuel is ethanol. [0037] Item 11. The system of any preceding Items, wherein the defined culture comprises Clostridium thermocellum and a hemicellulose-fermenting microorganism selected from the group consisting of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium saccharolyticum. [0038] Item 12. The system of any preceding Items, wherein the starting biomass is a member selected from the group consisting of corn stover, sugar cane, wheat or rice straw, miscanthus, switchgrass, energy cane and combination thereof. [0039] Item 13. A method of using the system of claim 1 to produce a liquid fuel from biomass, comprising a) loading a starting biomass into the reservoir, b) allowing the biomass to move from the reservoir into the first reactor where at least a portion of the biomass is converted into alcohol, c) allowing the system to run continuously or semi-continuously wherein the reservoir feeds the first reactor and overflow from the N reactor moves to N+1 reactor immediately downstream of the N reactor, d) repeating steps (a)-(c) above to allow the biomass to move from the N reactor to the N+1 reactor through the series of reactors until it reaches the last reactor, e) allowing the system to run continuously or semi-continuously, wherein steps of CAZyme production, substrate hydrolysis and fermentation by the defined culture without adding enzymes all occur within each of the plurality of reactors, and f) collecting the liquid fuel from the plurality of reactors [0040] Item 14. The method of Item 13, wherein the liquid fuel is ethanol. [0041] Item 15. The method of any one of Items 13-14, wherein one or more mills positioned between a reactor and an immediately downstream reactor processes the overflow mechanically. [0042] Item 16. The method of any one of Items 13-15, wherein the system runs in a continuous or semi-continuous mode, not a batch mode. [0043] Item 17. The method of any one of Items 13-16, wherein the solids loading is about 10-25 wt.% solids. [0044] Item 18. The method of any one of Items 13-17, wherein the starting biomass is selected from the group consisting of corn stover, sugar cane, wheat or rice straw, miscanthus, switchgrass, energy cane and combination thereof. [0045] Item 19. The method of any one of Items 13-18, wherein ratio between the CAZyme and the substrate in the first reactor is at least one unit of CAZyme per pmol of the substrate. [0046] For purpose of this disclosure, the term “biomass” refers to cellulosic biomass. In one embodiment of this disclosure, biomass refers to plant biomass which includes any plant-derived matter (woody or non-woody) that is available on a sustainable basis. Plant biomass may include, but is not limited to, agricultural crop wastes and residues such as corn stover, wheat straw, rice straw, sugar cane bagasse and the like, grass crops, such as switch grass, alfalfa, winter rye, and the like. Plant biomass may further include, but is not limited to, woody energy crops, wood wastes and residues such as trees, softwood forest thinnings, barky wastes, sawdust, paper and pulp industry residues or waste streams, wood fiber, and the like. In urban areas, plant biomass may include yard waste, such as grass clippings, leaves, tree clippings, brush, etc., vegetable processing waste, as well as recycled cardboard and paper products. EXAMPLE [0047] Kinetics and Reactor Design for Rapid Processing via C-CBP [0048] In batch cultures, 5 to 7 days are typically required to achieve complete fermentation via CBP as indicated by cessation of gas production. For industrial processes, shorter reaction times are highly desirable. Bioconversion requires many, large bioreactors (e.g., 15 to 20 one million gallon reactors) for a medium sized facility producing 50 million gallons of ethanol per year. Thus, bioreactors represent a substantial fraction of overall capital investment for any lignocellulose conversion process and are the single largest cost contributor for consolidated bioprocessing with cotreatment (C-CBP). [0049] Reactions times have been widely observed to be much longer for lignocellulose conversion compared to other plant-based feedstocks, leading to larger bioreactor volume and investment. Table 1 summarizes reaction times for lignocellulose conversion compared to other plant-based feedstocks. Table 1.

2 Stowers et al., 2009. Confirmed by communication with industrial producers 3 Values are typical of literature for both the conventional and thermochemical pretreatment/fungal cellulase processing paradigm as well as C-CBP [0050] For lignocellulose, the upper limit for ethanol titer is about 50 g/L due to materials handling constraints. Shorter reaction times is the most promising path to decreased bioreactor investment. However, this has received little attention likely because it is assumed to be constrained by recalcitrance. [0051] Continuous (or semi-continuous) processing of lignocellulose is a solution to this problem. Although unreacted lignocellulose at solids at required loadings (> 15 wt.%) are impractical to mix, biologically-mediated deconstruction results in dramatic liquefaction (viscosity reduced by orders of magnitude). Additionally, continuous lignocellulose-grown cultures, have marked advantages over batch culture for kinetic characterization. Industrial preparation of inoculum is also more easily done with facultative aerobes (e.g. yeast, E. coli) than with the obligate anaerobes envisioned for C-CBP.

[0052] However, there are several challenges to continuous (or semi-continuous) processing of lignocellulose. Very few potential contaminants will be able to compete with C. thermocellum (and similar microbes) for access to the surface of lignocellulose. Additionally, Wild-type thermophiles are not able to tolerate the high ethanol concentrations that need to be produced by engineered strains in order to be commercially viable.

[0053] The framework for describing the rate of lignocellulose solubilization applicable to mixed enrichments and defined cultures is described by the following equations:

_[0054]

[0055] Where: is the carbohydrate loading fed to the bioreactor (g/L); C is the

residual carbohydrate in the steady-state bioreactor (g/L); 0 is the residence time (day); r is the rate of carbohydrate solubilization k is the solubilization rate constant , equal

to the slope of FIG. 3; and is the fraction of recalcitrant carbohydrate, equal to the intercept of

FIG. 3.

[0056] The implications of continuous conversion of cellulosic biomass was explored with a rate constant of 2 The steady-state material balance on carbohydrate, with a first

order rate constant, and equal flows in and out:

[0057]

[0058] Where

is the residence time (bioreactor working volume/volumetric flow rate.

[0059] The total carbohydrate concentration, C, is equal to the sum of the accessible concentration , plus the inaccessible concentration

[0060]

[0061] Substituting into the material balance, the following equation is obtained:

[0062]

[0063] This may be solved fo

[0064] [0065]

[0066] Consider continuous lignocellulose fermentation with kinetics as described above and parameters as follows:

Given these values, consider the residence time to solubilize 90% of the accessible carbohydrate: [0067] [0068] [0069] [0070]

[0071] For each reactor , the results

are summarized in Table 2 below: [0072] Table 2. Summary of results for 5 CSTRs in series

[0073] For an infinite CSTR limiting case: [0074]

[0075] results are summarized

below in Table 3: [0076] Table 3. Summary of results for infinite CSTRs

[0077] The configuration of the bioreactors matter a lot and is a high-impact target for innovation. There is potential for microbial deconstruction to be much faster than current technology, which is 5 to 7 days. This analysis did not consider a few key factors including: the kinetic, physiological impacts of high product, solids; and the possible accelerating impact of cotreatment. Below is a summary of the kinetic benefit of cascade continuous operation, which appears to be specific to CBP: [0078] Table 4. Summary of benefits of cascade continuous operation

[0079] It will be readily apparent to those skilled in the art that the systems and methods described herein may be modified and substitutions may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. [0080] The contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited throughout this application are hereby expressly incorporated by reference in their entirety for any purpose into the present disclosure. The disclosure may employ, unless otherwise indicated, conventional techniques of microbiology, molecular biology and cell biology, which are well known in the art. [0081] The disclosed methods and systems may be modified without departing from the scope hereof. It should be noted that the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense.

Claims

CLAIMS What is claimed is: 1. A system for converting biomass into a liquid fuel, comprising: a) a reservoir; and b) a plurality of reactors arranged in series, each reactor having an inlet, and an overflow outlet, the inlet of first reactor of the plurality of reactors being connected to the reservoir, wherein the plurality of reactors comprise a defined culture comprising at least one living organism capable of producing at least one carbohydrate-active enzyme (CAZyme) , wherein steps of CAZyme production, substrate hydrolysis and fermentation by the defined culture without added enzymes all occur within each of the plurality of reactors.

2. The system of claim 1, wherein overflow from a first reactor in the series feeds a second reactor immediately downstream of the first reactor, and overflow from the N reactor feeds N+1 reactor immediately downstream of the N reactor, wherein N is an integer ranging from 1 to n, and wherein n is an integer, and n+1 is equal to the total number of reactors in the plurality of reactors.

3. The system of claim 2, further comprising one or more mills placed between two neighboring reactors.

4. The system of claim 2, further comprising a starting biomass, wherein the starting biomass is loaded into the reservoir, and the biomass flows one-way from the reservoir to the first reactor.

5. The system of claim 1, wherein the plurality of reactors comprises 3 or more reactors.

6. The system of claim 1, wherein the system runs continuously or semi-continuously.

7. The system of claim 6, wherein the series of reactors running continuously or semi- continuously shortens reaction time from the biomass to the liquid fuel.

8. The system of claim 7, wherein the reaction time from the biomass to the liquid fuel is shorter as compared to reaction time in a batch or single continuous bioreactor.

9. The system of claim 1, wherein ratio between the CAZyme and the substrate in the first reactor is at least one unit of CAZyme per pmol of the substrate.

10. The system of claim 1, wherein the liquid fuel is ethanol.

11. The system of claim 1, wherein the defined culture comprises Clostridium thermocellum and a hemicellulose-fermenting microorganism selected from the group consisting of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium saccharolyticum.

12. The system of claim 11, wherein the starting biomass is a member selected from the group consisting of corn stover, sugar cane, wheat or rice straw, miscanthus, switchgrass, energy cane and combination thereof.

13. A method of using the system of claim 1 to produce a liquid fuel from biomass, comprising a) loading a starting biomass into the reservoir, b) allowing the biomass to move from the reservoir into the first reactor where at least a portion of the biomass is converted into alcohol, c) allowing the system to run continuously or semi-continuously wherein the reservoir feeds the first reactor and overflow from the N reactor moves to N+1 reactor immediately downstream of the N reactor, d) repeating steps (a)-(c) above to allow the biomass to move from the N reactor to the N+1 reactor through the series of reactors until it reaches the last reactor, e) allowing the system to run continuously or semi-continuously, wherein steps of CAZyme production, substrate hydrolysis and fermentation by the defined culture without adding enzymes all occur within each of the plurality of reactors, and f) collecting the liquid fuel from the plurality of reactors

14. The method of claim 13, wherein the liquid fuel is ethanol.

15. The method of claim 13, wherein one or more mills positioned between a reactor and an immediately downstream reactor processes the overflow mechanically.

16. The method of claim 14, wherein the system runs in a continuous or semi-continuous mode, not a batch mode.

17. The method of claim 13, wherein the solids loading is about 10-25 wt.% solids.

18. The method of claim 13, wherein the starting biomass is selected from the group consisting of corn stover, sugar cane, wheat or rice straw, miscanthus, switchgrass, energy cane and combination thereof.

19. The method of claim 13, wherein ratio between the CAZyme and the substrate in the first reactor is at least one unit of CAZyme per pmol of the substrate.