Process for the production of ethanol by an electroactive escherichia coli using cellulosic pyrolysis liquids

Abstract

The application discloses a process for preparing ethanol by using cellulose pyrolysis liquid through electroactive escherichia coli, and belongs to the technical field of electrochemistry. In the application, waste cellulose is pyrolyzed and then pH is adjusted, a salt solution suitable for the growth of escherichia coli is prepared, and an antibiotic is added into a reaction system for fermentation and electrochemical conversion, and then the prepared escherichia coli reaction system is connected to an electrochemical system under sterile fermentation conditions to provide a micro-voltage. Through the above process, most of the internal ether sugar can be converted into ethanol, and the inhibitor in the pyrolysis liquid can be reduced by using an electric current, so that the degradation rate of the inhibitor is improved.

Description

Technical Field

[0001] This invention relates to the field of electrochemical technology, and more specifically to a process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution. Background Technology

[0002] With dwindling fossil fuel supplies, rising CO2 emissions, and soaring fuel prices, it is essential to find environmentally sustainable and economically viable renewable alternatives. The National Energy Administration points out that biomass energy is the world's fourth largest energy source after oil, coal, and natural gas, and a crucial force in international energy transition. Biomass energy primarily uses agricultural materials such as corn stalks, sugarcane bagasse, rice straw, and wheat straw as raw materials, with a massive annual output. It boasts advantages such as being renewable, inexpensive, abundant reserves, high raw material diversity, and strong adaptability to a wide range of land resources. Most importantly, biomass energy is a zero-carbon fuel, facilitating carbon neutrality and recycling; therefore, it has attracted significant attention as a renewable energy source. In my country, biomass-based biofuel production has become a major way to alleviate energy problems. Bioethanol is the most useful biofuel that can replace fossil fuels. Currently, bioethanol is widely used. Cellulose materials, after being treated by pyrolysis technology, are then fermented by E. coli to convert into clean energy bioethanol, which is of great significance for waste resource utilization, environmental protection, and energy security. However, acids and aldehydes in the pyrolysis solution have a strong inhibitory effect on E. coli, which is the biggest obstacle to the downstream conversion and utilization of lignocellulose pyrolysis solutions. Constructing a microbial electrolysis cell (MEC) system within a microbial electrochemical system enhances its resistance to or conversion of inhibitors and its ethanol fermentation capacity, thereby significantly reducing raw material processing costs and further improving the substrate range available to E. coli and the ethanol production efficiency. This enables one-step fermentation from cellulose pyrolysis liquid to bioethanol, which has significant academic value and practical implications. Ultimately, it provides a new pathway for the production of bioethanol from cellulosic materials and offers a scientific basis and technological demonstration for its industrial application.

[0003] However, at present, microbial electrolyzers are still in their infancy and far from practical application. Many challenges facing microbial electrolyzers need to be addressed before considering scaling up and real-world implementation. Most previous studies have used model electroactive cultures and pure substrates, rather than real waste. This invention directly utilizes cellulose pyrolysis wastewater for fermentation. Real cellulose pyrolysis wastewater has complex physicochemical properties and variable composition, making it directly applicable for industrial-scale wastewater treatment.

[0004] In summary, how to provide a microbial electrolysis cell system for directly preparing ethanol from cellulose pyrolysis solution is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a process for preparing ethanol from cellulose pyrolysis using electroactive Escherichia coli. This invention connects a bioreactor to an electrochemical system, which can stimulate the activity of Escherichia coli in the system, accelerate the electron transfer rate, shorten the reaction cycle, and also solve the problem that the ether sugars cannot be fully utilized under the condition of inhibitors in the pyrolysis solution.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution includes the following steps:

[0008] (1) Cellulose is pretreated to obtain cellulose pyrolysis solution;

[0009] (2) Pre-place carbon brushes in the culture medium, and then culture genetically engineered Escherichiacoli LGE until E. coli biofilms are attached to the carbon brushes.

[0010] Only the growth of vegetative cells is allowed during this culture process; spore germination is not permitted.

[0011] (3) A carbon brush with Escherichia coli biofilm attached was used as the bioanode of the microbial electrolysis cell, and a stainless steel mesh and carbon cloth were used as the cathode. The pyrolysis solution that had been filtered and sterilized by a 0.22 μm membrane was added to the sterilized M9 basic salt culture medium as the substrate for fermentation and electrochemical conversion to construct a microbial electrolysis cell system.

[0012] (4) Adjust the temperature of the reaction system to 30±1℃ and apply an external voltage of 0.4~0.8V.

[0013] The beneficial effects achieved are as follows: Through the above process, most of the ether sugars can be converted into ethanol. Due to the different redox potentials of different end products (bioenergy), the pyrolysis solution contains a large number of acids, phenols and aldehydes, which act as electron acceptors in the microbial electrolysis cell system. Under different potential conditions, they are reduced through electron transfer, thereby producing different bioenergy.

[0014] Furthermore, the specific operation of step (1) is as follows: a pyrolysis solution is obtained by preparing pyrolysis material derived from waste cotton using a pyrolysis device, and the pH is adjusted to 7.

[0015] Furthermore, the method for constructing the genetically engineered Escherichiacoli LGE described in step (2) is as follows: based on the synthetic metabolic pathway of the heterologous genes encoding L-glucan kinase, pyruvate decarboxylase and alcohol dehydrogenase, this pathway is introduced into Escherichia coli to produce the genetically engineered Escherichiacoli LGE for the production of ethanol from L-glucan.

[0016] Furthermore, the culture medium in step (2) comprises the following components in mass concentrations: tryptone: 10 g / L, yeast extract: 5 g / L and NaCl: 10 g / L.

[0017] Furthermore, the culture conditions in step (2) are: cultured at 30°C with shaking at 160 rpm.

[0018] Furthermore, the bioanode and cathode are connected by titanium wire.

[0019] Furthermore, the M9 basic salt culture medium described in step (3) comprises the following components in mass concentration: Na2HPO4: 7.10 g / l, KH2PO4: 3.00 g / l, NaCl: 0.50 g / l, NH4Cl: 1.00 g / l, MgSO4: 0.49 g / l, and CaCl2: 14.70 mg / l.

[0020] Furthermore, in step (3), a stainless steel mesh is passed through the carbon cloth and fixed to the inner wall of the reactor.

[0021] Furthermore, in step (3), the volume ratio of the pyrolysis solution to the M9 basic salt culture medium is 1:9.

[0022] Furthermore, in step (3), antibiotics are added to the substrates for fermentation and electrochemical conversion, wherein the antibiotics are 100 μg / ml ampicillin and 34 μg / ml chloramphenicol.

[0023] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by this invention are as follows: This invention can convert most of the ether sugars into ethanol, and can utilize electric current to reduce inhibitors in the pyrolysis solution, thereby improving the degradation rate of the inhibitors. Furthermore, it utilizes electrochemical techniques, such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), to monitor the activity and growth of *E. coli*. This invention's process opens up the possibility of utilizing electricity for bioproduction in various other cell factories in engineering. This invention provides a new process for converting cellulose into clean energy, promoting the resource utilization of cellulose. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0025] Figure 1 The attached figure is a technical roadmap of the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0028] Example 1

[0029] (1) A pyrolysis solution was prepared from untreated waste cotton using a pyrolysis apparatus, and then the pH of the pyrolysis solution was adjusted to 7 with 10% (w / v) NaOH.

[0030] (2) The genetically engineered Escherichiacoli LGE (based on heterologous genes encoding L-glucan kinase (lgk), pyruvate decarboxylase (pdc), and alcohol dehydrogenase (adh) constructed in the laboratory earlier, which utilizes ether sugars, the main component of pyrolysis solution, to produce ethanol, was modified into the pET-21a vector. The synthesized lgk gene fragment was cloned into the pET-21a vector using EcoRI and XhoI restriction endonucleases to generate the pET-lgk vector. Competent E. coli BL21(DE3) cells were prepared by the CaCl2 method. At least 1 μg of pET-lgk vector DNA was added to 100 μl of competent cells. The mixture was then transferred to an electroporation cuvette with a 0.2 cm gap and pulsed in a Scientz-2C gene pulse generator. The transformed cells were reset in LB medium and thawed at 37°C for 1 day. Recombinant cells were cultured on LB agar plates supplemented with appropriate antibiotics for 11 hours. An ethanol production pathway was introduced by cloning the pyruvate decarboxylase gene *pdc* and the alcohol dehydrogenase gene *adh* from the genomic DNA of *Fermentomonas motilityis* using primers. The sequenced *adh* DNA fragment was then digested with BamHI / EcoRI and cloned into the pZBC vector to produce the pZBC-adh vector. Subsequently, the sequenced *pdc* DNA fragment with NdeI and BamHI restriction sites at both ends was further cloned into the pZBC-adh vector to generate the pZBC-adh-pdc vector. To ensure efficient translation of the relevant genes, the ribosome binding site sequence of the T7lac promoter was added upstream of 50 of *lgk* and *adh*. The isolated and purified recombinant strain was verified by PCR amplification and sequencing. See the article "Conversion efficiency of bioethanol from..." for details. The *E. coli* was cultured in LB medium (tryptone: 10 g / L, yeast extract: 5 g / L, NaCl: 10 g / L) pre-filled with carbon brushes required for constructing the microbial electrolysis system. The culture was continuously incubated at 30°C with shaking at 160 rpm until a biofilm of *E. coli* adhered to the surface of the carbon brushes (only vegetative cell growth was allowed during this process; spore germination was not permitted).

[0031] (3) In a sterile operating table, the carbon brush with the E. coli biofilm attached and part of the culture medium were removed and used as the bioanode of the microbial electrolysis cell. The cathode was a stainless steel mesh and a carbon cloth (the stainless steel mesh was passed through the carbon cloth and fixed to the inner wall of the reactor). The pyrolysis solution that had been filtered and sterilized by a 0.22 μm membrane was added to the sterilized M9 basic salt culture medium as the substrate for fermentation and electrochemical conversion. The volume ratio of the pyrolysis solution to the M9 basic salt culture medium was 1:9. Antibiotics were also added, namely 100 μg / ml ampicillin and 34 μg / ml chloramphenicol, to construct the microbial electrolysis cell system.

[0032] (4) The temperature of the reaction system is adjusted to 30±1℃ using a constant temperature water bath. The anode and cathode of the microbial electrolysis cell are connected by titanium wire. An external voltage is applied to accelerate the reaction. The voltage is adjusted to a small value of 0.4~0.8V to keep the voltage of the system stable within the range of variation. The external voltage is provided by an electrochemical workstation, which can record the current changes in the circuit within a set time interval. When the current drops to a small value (~1mA), it represents the end of a cycle.

[0033] The M9 basic salt medium formula is as follows: Na2HPO4: 7.10 g / l, KH2PO4: 3.00 g / l, NaCl: 0.50 g / l, NH4Cl: 1.00 g / l, MgSO4: 0.49 g / l, CaCl2: 14.70 mg / l.

[0034] Example 2

[0035] To investigate the effect of microbial electrolysis on ethanol fermentation, conventional fermentation was carried out in a reactor without an external voltage. The presence or absence of voltage was the only variable, and all other reaction conditions were the same as in Example 1.

[0036] Example 3

[0037] The growth rate of *E. coli*, ether sugars, ethanol, formic acid, acetic acid, furfural, voltage, and current were measured during the preparation processes of Examples 1 and 2. Measurements were taken every 4 hours, in triplicate.

[0038] Detection method:

[0039] (1) The growth rate of Escherichia coli was determined by measuring the optical density (600 nm) in a 10 mm glass cuvette using a spectrophotometer (UV 759 Yoke Increument).

[0040] (2) The analysis of ether sugars and ethanol was performed by high performance liquid chromatography using an ICSep ICE-ION-300 column (300×78mm) liquid chromatographic instrument (HPLC, LC-20AT, Shimadzu Corporation, KYOTO, Japan).

[0041] (3) The concentrations of formic acid, acetic acid, and furfural were determined using a C18 column (250 mm × 4.6 mm, 5 μm particle size, YOSOH Corporation). The mobile phase flow rate was 1 ml / min, and the injection volume was 20 μl.

[0042] (4) Use the Student's t test function in Origin 9.0 software to analyze the statistical differences among the data obtained from the above three parallel experiments.

[0043] Experimental data:

[0044] Table 1. Growth status of Escherichia coli (OD) 600 )

[0045] Example 1 (Connected to an electrochemical system) Example 2 (without connection to an electrochemical system) 0h 0.38 0.19 4 0.39 0.23 8 0.52 0.29 12 0.53 0.31 16 0.62 0.37 20 0.79 0.49 24 0.86 0.56 28 1.24 0.63 32 1.45 0.69 36 1.58 0.73 40 1.73 0.79 44 1.76 0.83 48 1.71 0.96 52 1.72 1.10

[0046] Table 2. Consumption of ether sugars (g / L)

[0047]

[0048]

[0049] Table 3 Ethanol Production (g / L)

[0050] Example 1 (Connected to an electrochemical system) Example 2 (without connection to an electrochemical system) 0h 0 0 4 0.34 0.17 8 0.49 0.26 12 0.86 0.36 16 1.26 0.49 20 1.55 0.72 24 1.83 1.10 28 2.25 1.67 32 3.11 2.12 36 4.10 2.91 40 5.09 3.45 44 5.20 4.06 48 5.24 5.10 52 5.25 5.25

[0051] Table 4 Formic acid concentration (g / L)

[0052] Example 1 (Connected to an electrochemical system) Example 2 (without connection to an electrochemical system) 0h 1.96 1.92 52 0.26 0.48

[0053] Table 5 Acetic acid concentration (g / L)

[0054] Example 1 (Connected to an electrochemical system) Example 2 (without connection to an electrochemical system) 0h 2.98 2.93 52 0.83 0.89

[0055] Table 6. Furfural concentration (g / L)

[0056] Example 1 (Connected to an electrochemical system) Example 2 (without connection to an electrochemical system) 0h 0.001 0.001 52 0 0

[0057] Experimental data analysis:

[0058] (1) The growth of Escherichia coli without access to the electrochemical system was lower than that with access to the electrochemical system, even though both strains reached the stable growth period at 40h.

[0059] (2) After connecting to the electrochemical system, the maximum ethanol yield obtained by using electroactive Escherichia coli to produce ethanol is 0.13 g / L / h, that is, fermentation for 40 h is optimal, and the ether sugar in the pyrolysis solution is almost completely utilized. Under the condition of 10 g / L ether sugar, the theoretical yield of ethanol is 5.68 g / L, reaching 93% of the theoretical yield.

[0060] (3) The ethanol production rate when connected to the electrochemical system is 0.13 g / l / h, which is much higher than the 0.11 g / l / h when not connected to the electrochemical system.

[0061] (4) All furfural connected to the electrochemical system is metabolized by Escherichia coli, with a maximum conversion rate of 99% and a maximum conversion rate of 72% for acid.

[0062] in conclusion:

[0063] In the control experiment without the electrochemical system, *E. coli* converted ether sugars in the cellulose pyrolysis solution into ethanol. However, organic acids, aldehydes, and phenols in the pyrolysis solution strongly inhibited the growth and fermentation of *E. coli*, resulting in lower ethanol yield and a longer reaction cycle in the control group without the electrochemical system. In contrast, the experimental group with the electrochemical system, under the influence of a weak voltage, was able to convert ether sugars into ethanol more quickly and accelerate the degradation of inhibitors into less toxic substances, which were then converted into ethanol, thus enriching the ethanol synthesis pathway. Furthermore, the electrochemical system stimulated the activity of *E. coli* in the reaction system, accelerated electron transfer, shortened the reaction cycle, and promoted the transformation of cellulose into a resource-utilizable resource.

[0064] Comparison with other research findings:

[0065] This invention significantly improves the tolerance or transformation ability of *E. coli* to complex inhibitors, building upon previous research, thereby greatly increasing the range of substrates available to *E. coli* and the final ethanol yield. For example, Chi and Rover et al. used *E. coli* KO11+lgk to detoxify and ferment the pyrolysis broth, resulting in very low ethanol yields, approximately 0.028 and 0.05 g ethanol / g ether sugar, respectively.

[0066] In this invention, after the electrochemical system was connected, the maximum ethanol yield obtained by electroactive Escherichia coli from cellulose pyrolysis was 5.25 g / L, reaching 93% of the theoretical yield. The conversion rate of inhibitors was also greatly improved, with the maximum conversion rate of furfural at 99% and the maximum conversion rate of acid at 72%. This indicates that the MEC technology can promote the conversion of these inhibitors by Escherichia coli, thereby indirectly improving the stress resistance of Escherichia coli.

[0067] Summarize:

[0068] This invention provides a process for producing ethanol from cellulose pyrolysis using electroactive Escherichia coli. Compared with existing research, the yield of ethanol is significantly increased. At the same time, the inhibitors in the pyrolysis solution can be oxidized or reduced to low-toxicity substances. Using this invention can promote the resource utilization process of converting cellulose into clean energy.

[0069] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0070] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution, characterized in that, Includes the following steps: (1) Cellulose is pretreated to obtain cellulose pyrolysis solution; (2) Pre-place carbon brushes in the culture medium, and then culture the genetically engineered organism. Escherichia coli LGE, until an E. coli biofilm adheres to the carbon brush; (3) A carbon brush with Escherichia coli biofilm attached was used as the bioanode of the microbial electrolysis cell, and a stainless steel mesh and carbon cloth were used as the cathode. The pyrolysis solution that had been filtered and sterilized by 0.22 μm membrane was added to the sterilized M9 basic salt culture medium as the substrate for fermentation and electrochemical conversion to construct a microbial electrolysis cell system. (4) Adjust the temperature of the reaction system to 30±1℃ and apply an external voltage of 0.4~0.8 V; The specific operation of step (1) is as follows: use a pyrolysis device to prepare pyrolysis material derived from waste cotton to obtain pyrolysis solution, and adjust the pH to 7; The genetic engineering Escherichia coli The LGE is constructed as follows: L-glucan was modified into the pET-21a vector. The synthesized lgk gene fragment was cloned into the pET-21a vector using EcoRI and XhoI restriction endonucleases to generate the pET-lgk vector. Competent E. coli BL21(DE3) cells were prepared using the CaCl2 method. pET-lgk vector DNA was added to the competent cells, and the mixture was then transferred to an electroporation cuvette for pulsed transformation. The transformed cells were then revived in LB medium and cultured on LB plates supplemented with antibiotics. An ethanol production pathway was introduced. The pyruvate decarboxylase gene pdc and the alcohol dehydrogenase gene adh were cloned from the genomic DNA of *Fermentosum motilityis* using primers. The sequenced adh DNA fragment was then digested with BamHI / EcoRI and cloned into the pZBC vector to produce the pZBC-adh vector. Subsequently, the sequenced pdc fragment with NdeI and BamHI restriction sites at both ends was... The DNA fragment was further cloned into the pZBC-adh vector to generate the pZBC-adh-pdc vector. The ribosome binding site sequence of the T7lac promoter was added upstream of 50 of lgk and adh. The isolated and purified recombinant strain was verified by PCR amplification and sequencing.

2. The process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution as described in claim 1, characterized in that, The culture medium in step (2) comprises the following components in mass concentrations: tryptone: 10 g / L, yeast extract: 5 g / L and NaCl: 10 g / L.

3. The process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution as described in claim 1, characterized in that, The culture conditions in step (2) are: cultured at 30°C with shaking at 160 rpm.

4. The process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution as described in claim 1, characterized in that, The bioanode and cathode are connected by titanium wire.

5. The process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution as described in claim 1, characterized in that, The M9 basic salt culture medium described in step (3) comprises the following components in mass concentration: Na2HPO4: 7.10 g / l, KH2PO4: 3.00 g / l, NaCl: 0.50 g / l, NH4Cl: 1.00 g / l, MgSO4: 0.49 g / l, CaCl2: 14.70 mg / l.

6. The process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution as described in claim 1, characterized in that, In step (3), a stainless steel mesh is passed through carbon cloth and fixed to the inner wall of the reactor.

7. The process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution as described in claim 1, characterized in that, In step (3), the volume ratio of the pyrolysis solution to the M9 basic salt culture medium is 1:

9.

8. The process for preparing ethanol from electroactive Escherichia coli using cellulose pyrolysis solution as described in claim 1, characterized in that, In step (3), antibiotics are added to the substrates for fermentation and electrochemical conversion, namely 100 μg / ml ampicillin and 34 μg / ml chloramphenicol.

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