Method for producing target product from biomass waste

Abstract

A method for producing a target product from biomass waste, comprising the following steps: (A) putting bacteria A into a first culture medium made from biomass waste and performing degrading fermentation, the bacteria A being organic matter degrading bacteria, inactivating the materials after the reaction, and performing separation to obtain cell debris of the bacteria; and (B) using as a second culture medium the cell debris obtained in the step (A), putting transgenic bacteria B into the second culture medium to carry out heterologous gene expression, and performing separation to obtain a target product.

Description

Method for producing target products based on biomass waste Technical Field

[0001] The present invention relates to a method for producing a target product based on biomass waste, and in particular to a method for producing lactic acid based on biomass waste. Background Art

[0002] Biomass waste is a major source of solid waste, which mainly includes kitchen waste, feces, plant straw (tree pruning residues), etc. In view of the many environmental problems caused by the direct discharge of biomass waste, there are currently methods for recycling biomass waste. For example, patent document CN102794294A discloses a biomass waste treatment process, the process steps of which are: dry and wet separation of residents' biomass waste to obtain dry garbage and wet garbage; dry garbage is manually sorted and then sent for recycling; wet garbage is treated and crushed, and the wet garbage is sent to a crusher for crushing; steaming, the crushed material is sent to a steamer for steaming to achieve sterilization and oil dissolution effect; pressing, the steamed material is sent to a press for pressing to obtain residue and oil and water; and then the oil and water and residue are treated separately. However, these methods have the problems of complicated processes, low waste treatment efficiency and low production efficiency of target products, and cannot take into account both the efficient metabolic treatment of biomass waste and the acquisition of target products.

[0003] Summary of the Invention

[0004] In response to the above problems, the purpose of the present invention is to provide a method for producing target products based on biomass waste, which uses mixed bacteria A to metabolize biomass waste, and the metabolized bacterial cell debris is used as a culture medium for a single bacteria B to produce the target product, thereby significantly metabolizing organic waste and efficiently expressing heterologous genes. The degradation and fermentation of metabolic biomass waste and the production reaction environment of using the host to express heterologous genes are separated, and different strains are used to exert their characteristics, taking into account the two goals of metabolizing organic solid waste and expressing heterologous genes to produce target products.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for producing a target product based on biomass waste comprises the following steps:

[0007] (A) adding bacteria A to a first culture medium made from biomass waste for degradation and fermentation, wherein bacteria A is an organic matter-degrading bacterium, inactivating the reacted material, and separating and obtaining bacterial cell debris;

[0008] (B) The cell debris obtained in step (A) is used as a second culture medium, and the transgenic bacteria B is added to the second culture medium to express the heterologous gene, and the target product is obtained after separation.

[0009] Preferably, in step (A), the reacted material is inactivated and centrifuged, the supernatant is discarded, and the slurry layer is removed as the second culture medium. The second culture medium consists solely of the slurry, without any other substances added. The slurry provides the necessary carbon and nitrogen sources for the growth of bacteria B, and optionally also provides trace elements, including but not limited to metal cations such as sodium and magnesium.

[0010] Preferably, bacteria A comprises one or more combinations of fungi and / or bacteria that are capable of metabolizing the biomass waste.

[0011] More preferably, the fungus A comprises a combination of one or more fungi from the genera Synechiochytrium, Achyranthes, Rhizopus, Trichoderma, Aspergillus and Mucor.

[0012] In a specific embodiment, fungus A is an artificially domesticated fungus.

[0013] Furthermore, fungus A includes a combination of one or more of Mucor pusillus, Rhizopus arrhizus and Aspergillus niger.

[0014] Preferably, the source of the biomass waste includes one or more combinations of kitchen waste, feces and plant straw.

[0015] Preferably, the biomass waste comprises one or more combinations of proteins, lipids, and carbohydrates.

[0016] Preferably, the target product includes protein, lipid or organic acid, and the heterologous gene transferred from bacteria B into the host cell is selected from genes expressing protein, lipid or organic acid.

[0017] More preferably, the target product comprises lactic acid, bio-oil or elastin collagen.

[0018] In a specific and preferred embodiment, the target product is lactic acid, and the strain B is Rhizopus oryzae transformed with the lactate dehydrogenase gene. The lactic acid is used as a raw material for producing polylactic acid plastic masterbatch.

[0019] Preferably, in step (A), bacteria A and the first culture medium are degraded and fermented in the first reactor, the degraded and fermented materials are inactivated and centrifuged to obtain a water-insoluble product, which is used as the second culture medium.

[0020] Preferably, in step (B), bacteria B and the second culture medium are reacted in a second reactor, the reaction mixture is inactivated, centrifuged, and the separated water-soluble product is purified.

[0021] In a specific and preferred embodiment, the method is implemented as follows:

[0022] mixing biomass waste and a nitrogen source to form a first culture medium;

[0023] One or more bacteria from the genera Synechiochytrium, Achyranthes, Rhizopus, Trichoderma, Aspergillus, and Mucor are added to the first culture medium for degradation and fermentation;

[0024] The above-mentioned degradation and fermentation products are subjected to high-temperature inactivation treatment, centrifuged, the supernatant is discarded, and the pulpy substance at the bottom layer is taken, which is the cell debris of the bacteria mentioned above;

[0025] Adding genetically modified bacteria B into the slurry and culturing;

[0026] Inactivate and separate to obtain the target product.

[0027] In a specific and preferred embodiment, a method for producing lactic acid based on biomass waste is specifically implemented as follows:

[0028] Mixing biomass waste and a nitrogen source to form a first culture medium, and further adding salt ions; the biomass waste may contain starch or not;

[0029] One or more bacteria from the genera Synechiochytrium, Achyranthes, Rhizopus, Trichoderma, Aspergillus, and Mucor are added to the first culture medium for degradation and fermentation;

[0030] The above-mentioned degradation and fermentation products are subjected to high temperature inactivation treatment, centrifuged, the supernatant is discarded, and the pulpy substance at the bottom layer is taken;

[0031] Adding genetically modified bacteria B into the slurry to carry out fermentation culture;

[0032] When the pH value drops to the set value, a neutralizer is added to react, the reaction is followed by centrifugation, and the supernatant is taken, heated, and esterified and hydrolyzed to obtain lactic acid.

[0033] The neutralizing agent is (NH2)2CO-(NH4)HCO3-NH3·H2O or calcium hydroxide. The set value of the pH value is less than 6.

[0034] The present invention adopts the above solution and has the following advantages compared with the prior art:

[0035] The method of the present invention adopts a two-stage fermentation reaction. In the first stage fermentation reaction, bacteria A is first used to degrade and ferment biomass waste, ensuring that the biomass waste can be efficiently degraded and treated and a good treatment effect is obtained. Then, the material after the first stage fermentation reaction is inactivated, and the fungal / bacterial cell debris therein is used as a culture medium for the second stage fermentation reaction. Bacteria B (designed for the target product and a single bacterium into which a heterologous gene expressing the target product is transferred) is added to perform efficient expression of the heterologous gene to obtain the target product, such as lactic acid. Bacteria A and bacteria B respectively exert their respective characteristics to achieve the two goals of metabolizing organic solid waste and expressing the heterologous gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] FIG1 is a schematic diagram of a transparent circle on a simulated flat plate.

[0038] Figure 2 is a photograph of a plate after a protein degradation experiment with Mucor microsporus.

[0039] Figure 3 is a photograph of a plate subjected to a lipid degradation experiment by Mucor microsporus.

[0040] FIG4 is a photograph of a plate after the starch degradation experiment by Rhizopus arrhizus.

[0041] FIG5 is a photograph of a plate after the cellulose degradation experiment by Rhizopus arrhizus.

[0042] FIG6 is a photograph of a plate after the starch degradation experiment by Aspergillus niger.

[0043] FIG7 is a photograph of a plate after the Aspergillus niger degradation experiment. DETAILED DESCRIPTION

[0044] The following is a detailed description of the preferred embodiments of the present invention so that the advantages and features of the present invention can be more easily understood by those skilled in the art. It should be noted that the description of these embodiments is intended to help understand the present invention, but does not constitute a limitation of the present invention.

[0045] To meet the needs of the Bihuan Project, efficient metabolism of organic solid waste (kitchen waste) is achieved while efficiently expressing implanted heterologous genes. The number of heterologous genes that each host cell can effectively carry is limited. Too many heterologous gene implants will not necessarily kill the host, but will affect the host's viability. The present invention adopts a two-stage fermentation model to separate the degradation fermentation of metabolic organic solid waste and the production reaction environment of utilizing the host to express heterologous genes. Different strains are used to exert their characteristics, taking into account the two goals of metabolic organic solid waste and expression of heterologous genes, and the two reaction environments are combined through technology to form a production closed loop.

[0046] (A) The first fermentation reaction includes the following steps: adding a slurry culture medium and bacteria A into the first bioreactor A, wherein the carbon source of the slurry culture medium is formed by crushing biomass waste (mainly kitchen waste, and may also include feces and plant residues), and then adding a nitrogen source (such as agar, gelatin, etc.), or further adding an inorganic salt (such as sodium salt, etc.). The reaction conditions are controlled to allow bacteria A to metabolize the biomass waste. Then, the reacted materials in the first bioreactor A are collected, the reacted materials are inactivated as a whole (for example, high temperature and high pressure inactivation), and centrifuged (at high speed); the water-soluble products (mostly organic matter such as fungal protein residues) are treated according to regulations; the fungal / bacterial cell debris in the water-insoluble products are collected and used as the culture medium for the second reaction.

[0047] Furthermore, bacteria A has been artificially domesticated or genetically modified in advance. The details are as follows:

[0048] 101. Construction of heterologous plasmid of engineered bacteria A: This heterologous plasmid is used to enhance the parasitic nature of fungi / bacteria.

[0049] 102. Screening host cells for engineered bacteria A: Select host cells with strong parasitic fungi / bacteria. Fungi are selected from the genera Synchytrium, Achlya, Rhizopus, Trichoderma, Aspergillus, and Mucor; bacteria are selected from enteric bacteria, including but not limited to Escherichia coli and methanogens.

[0050] 103. Constructing engineered bacteria A: Implant the constructed heterologous plasmid gene into the selected mixed bacteria using the CaCl2 / PEG method.

[0051] 104. Cultivate the engineered bacteria A to stimulate the growth activity of strongly parasitic fungi / bacteria, expand the base number of fungi / bacteria, and then put them into the first bioreactor A.

[0052] The bacteria A and the slurry culture medium may be continuously added to the first bioreactor A to perform continuous fermentation.

[0053] (B) The second fermentation reaction comprises the following steps:

[0054] 201. Construction of heterologous telomeres in engineered bacteria B: The heterologous telomeres can be ldhA-AmpRes telomeres.

[0055] 202. Amplify telomere mass: Amplify the mass of ldhA-AmpRes telomeres by PCR and detect it using a Nanodrop ultra-micro instrument.

[0056] 203. Constructing engineered bacteria B: Implanting a heterologous telomere gene into a host cell. The host cell can be Rhizopus oryzae. Implanting the lactate dehydrogenase gene into Rhizopus oryzae using the CaCl2 / PEG method to obtain transgenic Rhizopus oryzae.

[0057] 204. Cultivate the engineered bacteria B to stimulate the growth activity of strongly parasitic fungi / bacteria, expand the fungi / bacteria base, and then add them to the second bioreactor B. Genetically modified Rhizopus oryzae can be added to the second bioreactor B.

[0058] 205. The fungal / bacterial cell debris after the first stage reaction is used as a second culture medium and added into a second bioreactor B, and engineered bacteria B (genetically modified Rhizopus oryzae) is added to carry out a fermentation reaction.

[0059] Alternatively, the engineered bacteria B and the slurry culture medium may be continuously added to the second bioreactor B for continuous fermentation.

[0060] 206. Collect the reacted material in the second bioreactor B.

[0061] 207. The entire reacted material is inactivated under high temperature and high pressure, and then centrifuged (at high speed); the water-soluble product (i.e., the cell-free supernatant after centrifugation) is collected and purified by esterification and distillation to obtain the desired product. In this embodiment, the desired product is lactic acid, which can be used as a raw material for the production of polylactic acid and further polymerized to produce polylactic acid plastic masterbatch.

[0062] In step 205, when the pH drops to a predetermined value, ammonia (e.g., (NH2)2CO-(NH4)HCO3-NH3·H2O) or calcium hydroxide is added to the second bioreactor B as a neutralizer, and the generated lactic acid reacts with the neutralizer.

[0063] In step 207, the esterification distillation purification specifically includes: adding n-butanol to the water-soluble product, performing an esterification reaction between ammonium lactate and n-butanol, and releasing the desired lactic acid through hydrolysis of the generated n-butyl lactate.

[0064] It should also be noted that Engineered B can express a wide range of heterologous genes, including proteins, lipids, and organic acids, which can be used as raw materials for bio-based energy or materials. However, the host cell is not necessarily Rhizopus oryzae; the host fungal cell is selected based on the target product. Engineered B utilizes a parasitic strain to metabolize the fungal carcasses in the first reaction. The target products are primarily raw materials for bio-energy and materials, such as lactic acid, bio-oil (biodiesel feedstock), and elastin collagen (organoid feedstock).

[0065] Typically, biomass waste, such as kitchen waste, consists primarily of proteins, lipids, carbohydrates (such as starch), and cellulose. Therefore, a culture plate was used to simulate a waste source, and a clearing zone test was performed to calculate the degradation efficiency. For details, see the various samples in Example 1 below.

[0066] Example 1

[0067] 1. Production of biowaste flat panels

[0068] Prepare culture media according to Table 1 below and make waste source simulation plates to simulate protein waste source, fat waste source, cellulose waste source, and starch waste source, respectively. Round plastic culture dishes with a diameter of 90 mm were used for all experiments.

[0069] Table 1

[0070] 2. Garbage decomposition operation

[0071] The garbage simulation plate is a single color as a whole (such as white or red). After bacteria A decomposes the protein, fat, starch or cellulose in it, the color will fade, forming one or several circles close to the color of agar, as shown in Figure 1. The experiment uses a round plastic culture dish with a diameter of 90mm. The volume of each garbage simulation plate is 20ml. The time for determining the diameter of the transparent circle is 72h after inoculation. Each small point in the experiment uses a 10-microliter standard strain of uniform concentration from a homemade bacterial library. All strains were put into the experiment after self-domestication. The calculation of the transparent circle and the consumption of the plate components are shown in the following formula:

[0072] Wherein, M represents the mass of the culture medium, m represents the mass of the degraded culture medium, and the degradation amount of the target substance can be obtained according to m and the concentration value in the culture medium.

[0073] 3. Experiment

[0074] 3.1 Mucor microsporus

[0075] (a) Protein degradation experiment by Mucor microsporus

[0076] Prepare a protein waste source simulation plate according to Table 1. Add water to skim milk powder to make the whole plate white. Inoculate Mucor microti (10 μL, concentration 1×10 7 CFU) and incubated at 35°C for 72 hours. As Mucor microplus degrades protein, the white color fades, forming a circle on the plate that resembles the color of agar. The average circle diameter from three replicates was measured to be: (3.32 cm + 3.36 cm + 3.12 cm) / 3 = 3.27 cm. Using the above formula, protein consumption was calculated to be 132 mg.

[0077] (b) Lipid degradation experiment by Mucor microsporus

[0078] Prepare a fat waste source simulation plate according to Table 1. Add water to lard to make the whole milky white. Inoculate Mucor microsporus (10 μL, concentration 1×10 7 CFU) and incubated at 35°C for 72 hours. As Mucor microsporus breaks down fat, the milky white color fades, forming a circle on the plate that resembles the color of agar. The average circle diameter from three replicate experiments was measured to be: (5.01 cm + 4.91 cm + 5.11 cm) / 3 = 5.01 cm. Using the above formula, the consumption of lard was calculated to be 0.124 ml.

[0079] 3.2 Rhizopus arrhizus

[0080] (a) Starch degradation experiment of Rhizopus arrhizus

[0081] Prepare a starch waste source simulation plate according to Table 1. After adding water to the soluble starch, the whole plate will turn white. Inoculate Rhizopus arrhizus (10 μL, concentration 1×10 7 CFU) and incubated at 35°C for 72 hours. As Rhizopus arrhizus degrades starch, the white color fades, forming a circle on the plate that resembles the color of agar. In three replicate experiments, the plates were completely transparent after 72 hours of incubation, indicating complete starch consumption and at least 400 mg of protein.

[0082] (b) Cellulose degradation experiment by Rhizopus arrhizus

[0083] Prepare a cellulose waste source simulation plate according to Table 1. After adding water to cellulose and Congo red, the whole plate will turn dark red. Inoculate Rhizopus arrhizus (10 μL, concentration 1×10 7 CFU) and incubated at 35°C for 72 hours. As Rhizopus arrhizus breaks down fat, the deep red color fades to a lighter red, forming a light red circle on the plate. The average circle diameter from three replicate experiments was measured to be: (3.32 cm + 3.35 cm + 3.09 cm) / 3 = 3.25 cm. Using the above formula, 19 mg of cellulose was consumed.

[0084] 3.3 Aspergillus niger

[0085] (a) Aspergillus niger starch degradation experiment

[0086] Prepare a starch waste source simulation plate according to Table 1. The soluble starch turns white after adding water. Inoculate Aspergillus niger (10 μL, concentration 1×10 7CFU) and incubated at 35°C for 72 hours. As Aspergillus niger degrades starch, the white color fades, forming a circle on the plate that resembles the color of agar. The average circle diameter from three replicate experiments was measured to be: (2.81 cm + 2.62 cm + 2.23 cm) / 3 = 2.55 cm. Using the above formula, 12 mg of cellulose was consumed.

[0087] (b) Cellulose degradation experiment by Aspergillus niger

[0088] Prepare a cellulose waste source simulation plate according to Table 1. After adding water to cellulose and Congo red, the whole plate will turn dark red. Inoculate Aspergillus niger (10 μL, concentration 1×10 7 CFU) and incubated at 35°C for 72 hours. As Aspergillus niger decomposes fat, the deep red color fades to a lighter red, forming a light red circle on the plate. The average circle diameter from three replicate experiments was measured to be: (0.25 cm + 0.32 cm + 0.20 cm) / 3 = 0.26 cm. Using the above formula, cellulose consumption was calculated to be 0.0016 g.

[0089] Example 2

[0090] Step (1): The system in the plate after each experiment in Example 1 was inactivated by high temperature and high pressure at a temperature of 121°C and a pressure of 0.12 MPa. The system was centrifuged, the upper water layer was discarded, and the slurry in the lower layer was removed. The collected slurry after each experiment was mixed and inoculated with Rhizopus oryzae that had been transferred to the ldhA-AmpRes telomere without mixing with other substances.

[0091] Step (2): Cultivate at 32°C for 72 hours in a 100 mL culture scale; when the pH of the system drops to 5.5, add (NH2)2CO-(NH4)HCO3-NH3·H2O as a neutralizer; centrifuge, filter, and collect the supernatant; heat the supernatant and add n-butanol to carry out an esterification reaction at 120°C, a pressure of 270 mmHg, and a reaction time of 1 hour to produce lactic acid. The final lactic acid yield of three parallel experiments was (1.680 + 1.600 + 1.427) / 3 = 1.569 g.

[0092] Comparative Example

[0093] A culture medium containing protein, fat and cellulose was prepared according to Table 1, and Rhizopus oryzae with ldhA-AmpRes telomeres was inoculated into the culture medium. The culture was cultured according to step (2) of Example 2. No lactic acid was produced, and the content of protein, fat and cellulose did not decrease significantly.

[0094] Example 2: Using a variety of bacteria capable of metabolizing biomass waste, biomass waste from various sources and compositions can be converted into a culture medium for transgenic Rhizopus oryzae to produce lactic acid. The method of this example can be directly applied to biomass waste with complex compositions (typically, kitchen waste) and can produce lactic acid. For biomass fertilizers that do not contain starch, transgenic Rhizopus oryzae rarely decomposes and produces very little lactic acid.

[0095] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. If a definition used herein conflicts or is inconsistent with a definition in other published documents, the definition used herein shall prevail.

[0097] Unless the context clearly indicates otherwise, the indefinite articles "a", "an" and "an" preceding an element or component herein are intended to specify the number of instances (i.e., the number of occurrences) of the element or component without limitation. Therefore, "a", "an" and "an" should be understood to include one or at least one, and the singular form of the element or component also includes the plural form.

[0098] The above embodiment is intended only to illustrate the technical concepts and features of the present invention and is a preferred embodiment. Its purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. It is not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the principles of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A method for producing a target product based on biomass waste, characterized in that: The steps include: (A) adding bacteria A to a first culture medium made from biomass waste for degradation and fermentation, wherein bacteria A is an organic matter-degrading bacterium, inactivating the reacted material, and separating and obtaining bacterial cell debris; (B) The cell debris obtained in step (A) is used as a second culture medium, and the transgenic bacteria B is added to the second culture medium to express the heterologous gene, and the target product is obtained after separation.

2. The method for producing target products based on biomass waste according to claim 1, characterized in that: In step (A), the reacted material is inactivated and then centrifuged, the supernatant is discarded, and the slurry material at the bottom layer is taken as the second culture medium.

3. The method for producing target products based on biomass waste according to claim 1 or 2, characterized in that: Bacteria A includes one or more combinations of fungi and / or bacteria that can metabolize the biomass waste.

4. The method for producing target products based on biomass waste according to claim 3, characterized in that: The fungus A includes one or more of the genera Synechiochytrium, Aspergillus, Rhizopus, Trichoderma, Aspergillus and Mucor.

5. The method for producing target products based on biomass waste according to claim 3 or 4, characterized in that: Fungus A includes one or more of Mucor pusillus, Rhizopus arrhizus and Aspergillus niger.

6. The method for producing target products from biomass waste according to any one of the preceding claims, characterized in that The source of the biomass waste includes one or more combinations of kitchen waste, excrement and plant straw.

7. The method for producing target products from biomass waste according to any one of the preceding claims, characterized in that The biomass waste includes one or more combinations of proteins, lipids, and carbohydrates.

8. Method for producing target products from biomass waste according to any one of the preceding claims, characterized in that The target product includes protein, lipid or organic acid.

9. Method for producing target products from biomass waste according to any one of the preceding claims, characterized in that The heterologous gene transferred into the host cell of bacteria B is selected from genes expressing proteins, lipids or organic acids.

10. Method for producing target products from biomass waste according to any one of the preceding claims, characterized in that The target product includes lactic acid, biological oil or elastic collagen.

11. The method for producing target products from biomass waste according to claim 10, characterized in that: The target product is lactic acid, and the bacterium B is Rhizopus oryzae into which the lactate dehydrogenase gene is transferred.

12. Method for producing target products from biomass waste according to any one of the preceding claims, characterized in that In step (A), bacteria A and a first culture medium are degraded and fermented in a first reactor, and the degraded and fermented materials are inactivated and centrifuged to obtain a water-insoluble product, which is used as the second culture medium. In step (B), bacteria B and the second culture medium are reacted in a second reactor, and the reaction mixture is inactivated as a whole and centrifuged, and the separated water-soluble product is purified.

13. Method for producing target products from biomass waste according to any one of the preceding claims, characterized in that The method is specifically implemented as follows: mixing biomass waste and a nitrogen source to form a first culture medium; One or more bacteria from the genera Synechiochytrium, Achyranthes, Rhizopus, Trichoderma, Aspergillus, and Mucor are added to the first culture medium for degradation and fermentation; The above-mentioned degradation and fermentation products are subjected to high temperature inactivation treatment, centrifuged, the supernatant is discarded, and the pulpy substance at the bottom layer is taken; Adding genetically modified bacteria B into the slurry and culturing; The target product was isolated and obtained.