Device and method for strengthening lignocellulose degradation by micro-aerobic regulation and recruitment of biological membrane

Abstract

The invention provides a device and a method for strengthening lignocellulose degradation by micro-aerobic regulation and recruitment of a biological membrane, the device comprises a main body reaction bottle, a membrane assembly is arranged in the main body reaction bottle, two ends of the membrane assembly respectively extend out of screw ports at two sides of the main body reaction bottle, and one end of the membrane assembly is connected with an aeration pump through a gas flowmeter; a dissolved oxygen probe, a sampling needle and an air bag of the dissolved oxygen instrument are inserted into the bottle body through a bottle opening in the top of the main body reaction bottle; the magnetic stirrer is arranged at the lower part of the main body reaction bottle, and a rotor of the magnetic stirrer is positioned on the inner bottom surface of the main body reaction bottle and rotates to realize stirring. The method comprises the steps of inoculum acquisition, reactor inoculation and starting, directional domestication and community structure analysis. According to the method, the multi-species biological membranes are recruited on the surfaces of the micro-aerobic membrane filaments, the biological diversity of the biological membranes recruited under the intermittent aeration condition with straw as a substrate is the highest, and the best straw degradation effect reaches 57.78 + / -2.28%. The invention relates to the technical field of microorganisms.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a micro-oxygen-regulated biofilm recruitment method for enhancing lignocellulose degradation. Apparatus and methods. Background Technology

[0002] Lignocellulose is the most abundant renewable biomass energy source on Earth, with a global annual production exceeding 200 billion tons. This natural polymer composite is an ideal raw material for biofuels and chemicals and a key contributor to carbon neutrality goals. However, its inherent structural inertia remains a major obstacle to bioconversion. This complex lignin-carbohydrate complex structure hinders efficient contact between microorganisms and enzymes and lignincellulose, thus necessitating the development of efficient and low-consumption strategies.

[0003] In natural ecosystems, diverse microorganisms efficiently degrade lignocellulose by forming multi-species biofilms. In soil ecosystems, biofilms play a central role in anchoring microorganisms, extracellular enzymes, and substrates. Aerobic fungi and bacteria work synergistically, secreting various enzymes to degrade lignocellulose into H2O and CO2. A typical example of anaerobic multi-species biofilms for lignocellulose degradation in nature is the rumen. Bacteria, fungi, and archaea in the rumen of ruminants form multi-species biofilms on the rumen wall and the surface of lignocellulose. They secrete cellulase, hemicellulase, and ligninase, synergistically driving lignocellulose degradation and fully converting plant fiber into volatile fatty acids, CH4, and H2O. The synergistic multi-species interaction and complete enzyme systems in these ecosystems are key to the efficient degradation of lignocellulose. However, in nature or in the laboratory, lignocellulose-degrading bacteria are typically enriched under aerobic or anaerobic conditions, making it challenging to cultivate multi-species biofilms for the synergistic degradation of lignocellulose. If aerobic, anaerobic, and facultative anaerobic lignocellulose-degrading bacteria can be recruited simultaneously into biofilms, the efficiency of lignocellulose degradation will be further improved.

[0004] In a membrane-aerated biofilm reactor (MABR), oxygen is transported to the biofilm through a hydrophobic membrane, establishing an aerobic-anaerobic gradient and providing a controllable microaerobic environment for the recruitment of multi-species biofilms. The bubble-free aeration of the MABR prevents biofilm structural damage and significantly improves oxygen transfer efficiency. This feature is particularly crucial for aerobic biofilm formation. The permeated oxygen is exhausted through the aerobic layer, while an anaerobic layer forms on the outer layer of the aerobic biofilm. This indicates that the MABR has the potential to simultaneously enrich aerobic, anaerobic, and facultative anaerobic lignocellulosic bacteria, serving as a platform for constructing multi-species biofilms that degrade lignocellulosic bacteria. Summary of the Invention This invention overcomes limitations by enriching multiple species of lignocellulose-degrading bacteria that possess aerobic, anaerobic, and facultative anaerobic properties. Based on this, a device and method for recruiting biofilms under micro-oxygen regulation to enhance the degradation of lignocellulose are proposed.

[0005] The technical solution adopted by the present invention to solve the above problems is as follows: This invention proposes a device for micro-oxygen-regulated recruitment of biofilm to enhance the degradation of lignocellulose, comprising a main reaction bottle, inside which a membrane assembly is provided. Both ends of the membrane assembly extend from screw ports on both sides of the main reaction bottle, with one end connected to an aeration pump via a gas flow meter. The dissolved oxygen probe, sampling needle, and gas bag of a dissolved oxygen meter are all inserted into the bottle body through the top opening of the main reaction bottle. A magnetic stirrer is placed at the bottom of the main reaction bottle, with its rotor located on the bottom surface of the main reaction bottle, rotating to achieve stirring.

[0006] Furthermore, the membrane module is composed of multiple hollow fiber membranes, each with a length of 18 cm and an effective membrane area of ​​120 cm². 2 .

[0007] This invention proposes a method for enhancing lignocellulose degradation by micro-oxygen regulation and recruitment of biofilms, based on the aforementioned device for enhancing lignocellulose degradation by micro-oxygen regulation and recruitment of biofilms. The method includes the following steps: Step 1: Inoculum Acquisition: Activated sludge and paper mill wastewater are screened separately to remove impurities, and then mixed in proportion to serve as inoculum. Step 2, Reactor Inoculation and Start-up: After preparing the acclimatization culture medium, add inoculum in a certain proportion, aerate the reactor to remove oxygen, and then seal it; adjust the aeration mode, aeration rate, reaction temperature, and stirring speed to start the reaction; Step 3, directional domestication: After a period of cultivation, the culture medium in the reactor is replaced and the aeration parameters are gradually adjusted. After 6 generations of domestication cultivation, a multi-species biofilm that degrades lignocellulose is obtained. Step 4: Community structure analysis: Samples of biofilm on the surface of membrane filaments were taken and 16S rRNA sequencing analysis was performed.

[0008] Furthermore, in step one, activated sludge and paper mill wastewater are mixed at a volume ratio of 1:1 after passing through a 100-mesh sieve and used as inoculum.

[0009] Furthermore, in step two, the acclimatization culture medium consists of: Na2HPO4 1.86 g / L, KH2PO4 1.03 g / L, NaCl 8 g / L, KCl 0.2 g / L, (NH4)2SO4 1.4 g / L, vitamins 1 mL / L, trace elements 10 mL / L, and microcrystalline cellulose or corn stalks 5-10 g / L.

[0010] Furthermore, in step two, the reactor is started with an inoculation ratio of 10% (v / v), and the aeration is achieved by introducing high-purity nitrogen into the culture medium for 15 minutes to remove oxygen.

[0011] Furthermore, in step two, the aeration modes include no aeration, intermittent aeration, and continuous aeration. No aeration refers to natural oxygen permeation; intermittent aeration involves 12 hours of cyclic aeration followed by 12 hours of aeration followed by cessation; and continuous aeration refers to uninterrupted aeration. The aeration rate for both intermittent and continuous aeration is 1-50 mL / min.

[0012] Furthermore, the reaction temperature of the mixture in the main reaction flask is 35°C, and the rotor speed is 300 rpm.

[0013] Furthermore, in step four, the sequencing region for 16S rRNA sequencing is the V3-V4 region, and the amplification primers are 338F and 806R.

[0014] The beneficial effects of this invention are: This invention uses a mixture of activated sludge and paper mill wastewater as inoculum, and employs a micro-oxygen regulation device for targeted acclimatization to recruit a multi-species biofilm that degrades lignocellulose. The biofilm contains... Bacteroides , Pseudobacteroides and Lentimicrobium Anaerobic bacteria and Desulfovibrio , Sulfurovum , Sulfuricurvum , Moheibacter , Halothiobacillus and Azovibrio Both aerobic and facultative anaerobic bacteria were included. Microorganisms in multi-species biofilms significantly improved the degradation of lignocellulose through interspecific interactions. The complex composition of straw resulted in higher biodiversity in biofilms enriched using straw as a substrate (Examples 4-6). Under intermittent aeration conditions using straw as a substrate, the highest degradation rate of straw by multi-species biofilms reached 57.78 ± 2.28%. Furthermore, experiments verified that the degradation capacity of multi-species biofilms for lignocellulose exhibited good stability.

[0015] This invention relates to a device and method for recruiting lignocellulose-degrading bacteria based on micro-oxygen regulation to enhance biofilm degradation. This method overcomes oxygen limitations in laboratory and natural conditions, obtaining multi-species biofilms that degrade lignocellulose and significantly improving degradation efficiency. This invention provides a new approach for the efficient enrichment of microorganisms that degrade lignocellulose and other recalcitrant organic matter, and has significant value for the treatment and resource recovery of recalcitrant pollutants. Attached Figure Description

[0016] Figure 1 A schematic diagram of a device for micro-oxygen regulation to recruit biofilms and enhance lignocellulose degradation; In the diagram: 1-Main reaction flask; 2-Membrane module; 3-Dissolved oxygen meter; 4-Dissolved oxygen probe; 5-Sampling needle; 6-Gas bag; 7-Aeration pump; 8-Gas flow meter; 9-Magnetic stirrer; 10-Rotor.

[0017] Figure 2 The diagram shows the composition of multi-species biofilms on the surface of membrane filaments in Examples 1-6 and Comparative Examples 1-2 of this invention. Detailed Implementation

[0018] Specific implementation method one: As follows Figure 1 As shown, this embodiment proposes a device for micro-oxygen regulation to recruit biofilms to enhance the degradation of lignocellulose, including a main reaction bottle 1, which is a GL45 screw-top bottle with an effective volume of 250 mL and two GL14 screw-top interfaces on both sides for installing membrane module 2. Membrane module 2 is located inside the main reaction bottle 1. Both ends of membrane module 2 are connected to the two screw ports on both sides of the main reaction bottle 1 through threaded joints. One end of membrane module 2 is connected to aeration pump 7 through gas flow meter 8. Gas flow meter 8 and aeration pump 7 are connected in series through pump pipe. Under the drive of aeration pump 7, the gas flows into the hollow fiber membrane from one end of membrane module 2, and the flow rate is controlled by flow meter 8. Some oxygen diffuses through the membrane wall, and the remaining gas flows out from the other end of membrane module 2. Preferably, the membrane module is composed of multiple (50) hollow fiber membranes, each hollow fiber membrane having a length of 18 cm and an effective membrane area of ​​120 cm². 2 .

[0019] The dissolved oxygen probe 4, sampling needle 5 and gas bag 6 of the dissolved oxygen meter 3 are all inserted into the main reaction bottle 1 through the top opening of the main reaction bottle 1; the dissolved oxygen meter 3 is used for dissolved oxygen monitoring; the sampling needle 5 is used for liquid sampling, and the gas bag 6 is used for collecting and discharging gas. The magnetic stirrer 9 is placed at the bottom of the main reaction flask 1, and its rotor 10 is located on the bottom surface inside the main reaction flask 1. Stirring is achieved by rotating.

[0020] Specific Implementation Method Two: This implementation method proposes a method for enhancing lignocellulose degradation by recruiting biofilms under micro-oxygen regulation, which is accomplished using the device for enhancing lignocellulose degradation by recruiting biofilms under micro-oxygen regulation described in Specific Implementation Method One. The method includes the following steps: Step 1, Inoculum Acquisition: The activated sludge and paper mill wastewater were sieved separately (100 mesh sieve) to remove impurities, and then mixed at a volume ratio of 1:1 as inoculum. Step 2, reactor inoculation and start-up, specifically includes: 2.1 Preparation of acclimatization culture medium: The acclimatization culture medium consists of: Na2HPO4 1.86 g / L, KH2PO4 1.03 g / L, NaCl 8 g / L, KCl 0.2 g / L, (NH4)2SO4 1.4 g / L, vitamins 1 mL / L, trace elements 10 mL / L, and microcrystalline cellulose or corn straw 5-10 g / L.

[0021] Preferably, the substrate reactor uses microcrystalline cellulose (MCC) with a substrate concentration of 5 g / L for batches 1-5 and straw with a substrate concentration of 10 g / L for batch 6; the substrate reactor uses straw with a substrate concentration of 5 g / L for batches 1-3 and straw with a substrate concentration of 10 g / L for batches 4-6.

[0022] Preferably, the vitamin components are: vitamin B7 20 mg / L, vitamin B9 20 mg / L, vitamin B6 100 mg / L, vitamin B1 50 mg / L, vitamin B2 50 mg / L, vitamin B3 50 mg / L, vitamin B5 50 mg / L, vitamin B12 1 mg / L, para-aminobenzoic acid 50 mg / L, and lipoic acid 50 mg / L.

[0023] Preferably, the trace element composition is as follows: 1.5 g / L N-triacetic acid, 3 g / L MgSO4·7H2O, 0.5 g / L MgSO4·7H2O, 1 g / L NaCl, 0.1 g / L FeSO4·7H2O, 0.1 g / L CaCl2·2H2O, 0.1 g / L CoCl2·6H2O, 0.1 g / L ZnSO4·7H2O, 0.01 g / L CuSO4·5H2O, 0.01 g / L H3BO4, 0.01 g / L NiCl2·6H2O, 0.01 g / L Na2WO4·2H2O, 0.01 g / L Na2MO4·2H2O, 0.01 g / L Na2SeO4·5H2O, and 0.01 g / L AlK(SO4)2·12H2O. g / L.

[0024] 2.2 Add inoculum at a certain ratio: The reactor start-up inoculum ratio is 10% (v / v). 2.3 The reactor is sealed after aeration and deoxygenation; the aeration is to introduce high-purity nitrogen into the culture medium for 15 min to remove oxygen.

[0025] 2.4 Adjust the aeration mode, aeration rate, reaction temperature, and stirring speed to start the reaction; the aeration modes include: no aeration, intermittent aeration, and continuous aeration; no aeration means gas is released through natural oxygen permeation; intermittent aeration means circulating aeration for 12 hours and then stopping for 12 hours; continuous aeration means uninterrupted aeration. The aeration rate for intermittent and continuous aeration is 1-50 mL / min.

[0026] Step 3, Targeted Domestication: After a period of cultivation, the culture medium in the reactor was replaced and the aeration parameters were gradually adjusted. After six generations of domestication and cultivation, a multi-species biofilm that degrades lignocellulose was obtained. Preferably, the acclimatization period is 12 days, and in each subculture, all MCCs or straw and 80% liquid medium are replaced. The aeration rates of the substrate reactor batches 1, 2, 3 and 4-6 are 1 mL / min, 5 mL / min, 20 mL / min and 50 mL / min, respectively.

[0027] Preferably, the aeration rates of batches 1-2, 3-4, 5 and 6 of the straw-based reactor are 1 mL / min, 5 mL / min, 20 mL / min and 50 mL / min, respectively.

[0028] Step 4: Community Structure Analysis Biomembrane samples were taken from the surface of the membrane filaments and 16S rRNA sequencing analysis was performed. The sequencing region of the 16S rRNA was V3-V4, and the amplification primers were forward primer 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′).

[0029] The present invention will be described below with reference to specific embodiments. Unless otherwise specified, the methods, reagents, and equipment used in the present invention are all conventional methods, reagents, and equipment.

[0030] In the following examples, the MCC degradation rate is calculated using formula (1):

[0031] In the following embodiments, the straw degradation rate is calculated using formula (2):

[0032] Example 1: A method for micro-oxygen regulation to recruit biofilms and enhance lignocellulose degradation includes the following steps: (1) Reactor assembly: according to Figure 1Assemble the reactor, which consists of a main reaction bottle 1, a membrane module 2, a dissolved oxygen meter 3, a dissolved oxygen probe 4, a sampling needle 5, an air bag 6, a magnetic stirrer 9, and a rotor 10.

[0033] (2) Inoculum acquisition: The activated sludge and paper mill wastewater were respectively passed through a 100-mesh sieve to remove impurities and then mixed at a volume ratio of 1:1 to be used as inoculum.

[0034] (3) Reactor inoculation and start-up: The acclimatization medium contained 1.86 g / L Na2HPO4, 1.03 g / L KH2PO4, 8 g / L NaCl, 0.2 g / L KCl, 1.4 g / L (NH4)2SO4, 1 mL / L vitamins, 10 mL / L trace elements, and 5 g / L MCC. The medium was inoculated at a 10% (v / v) inoculation ratio, and then aerated with high-purity nitrogen for 15 min to remove oxygen. The aeration mode was non-aeration, and the reaction temperature and stirring rate were 35℃ and 300 rpm, respectively.

[0035] (4) Directional acclimatization: After 12 days of cultivation, all MCC and 80% of the liquid in the reactor were replaced. After acclimatization for 6 generations, a multi-species biofilm degrading lignocellulose was obtained. The substrate concentration, type, aeration mode, and aeration rate of batches 1-6 are shown in Table 1.

[0036] (5) Community structure analysis: Biomembrane samples were taken from the surface of the membrane filaments and 16S rRNA sequencing analysis was performed on the V3-V4 region.

[0037] Example 2, Example 3: The differences between Examples 2 and 3 and Example 1 are that the reactor includes an aeration pump and a gas flow meter, and the aeration mode and aeration rate are also different, as shown in Table 1. All other reaction conditions and operating procedures are the same.

[0038] Comparative Example 1: The difference between Comparative Example 1 and Example 1 lies in the use of a conventional anaerobic reactor and the differences in aeration mode and aeration rate, as shown in Table 1. All other reaction conditions and operating procedures are the same.

[0039] Example 4: The difference between Example 4 and Example 1 lies in the substrate type and concentration, as shown in Table 1. All other reaction conditions and operating procedures are the same.

[0040] Example 5 and Example 6: The differences between Examples 5 and 6 and Example 4 are that the reactor includes an aeration pump and a gas flow meter, and the aeration mode and aeration rate are also different, as shown in Table 1. All other reaction conditions and operating procedures are the same.

[0041] Comparative Example 2: The difference between Comparative Example 2 and Example 4 lies in the use of a conventional anaerobic reactor and the differences in aeration mode and aeration rate, as shown in Table 1. All other reaction conditions and operating procedures are the same.

[0042] Table 1. Substrate type, substrate concentration, aeration mode, and aeration rate of Comparative Examples 1-2 and Examples 1-6 in batches 1-6

[0043] Table 2 shows the MCC degradation rate and straw degradation rate in batches 1-6 of Examples 1-6 and Comparative Examples 1-2.

[0044] Table 2. Degradation rates of MCC and straw in Comparative Examples 1-2 and Examples 1-6 in batches 1-6

[0045] The microbial community structures of Examples 1-6 and Comparative Examples 1-2 are as follows: Figure 2 As shown.

[0046] As demonstrated by the above examples, recruiting lignocellulose-degrading bacteria using a micro-oxygen regulation system can significantly increase the species diversity of the microbial community. Biodiversity is less affected by aeration mode when using MCC as a substrate, while biodiversity increases with increasing aeration rate when using straw as a substrate. The membrane filament biofilm contains... Bacteroides , Pseudobacteroides and Lentimicrobium Anaerobic bacteria and Desulfovibrio , Sulfurovum , Sulfuricurvum , Moheibacter , Halothiobacillus and Azovibrio Aerobic and facultative anaerobic bacteria were included. Multiple species enhanced the degradation of lignocellulose through interspecific interactions, with the bacterial community enriched by intermittent aeration using straw as a substrate achieving the highest straw degradation rate of 57.78 ± 2.28% (Example 5).

[0047] In summary, this invention overcomes the oxygen limitations of laboratory and natural conditions, disclosing a device and method for enhancing lignocellulose degradation by recruiting biofilms under micro-oxygen regulation. This results in the acquisition of multi-species biofilms that degrade lignocellulose, significantly improving lignocellulose degradation efficiency. This invention provides a new approach for the efficient enrichment of multi-species biofilms for the degradation of lignocellulose and other recalcitrant organic matter, and can be extended to other fields.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A device for micro-oxygen-regulated recruitment of biofilms to enhance lignocellulose degradation, characterized in that: The system includes a main reaction bottle (1), which contains a membrane assembly (2). The membrane assembly (2) extends from the two ends of the main reaction bottle (1) through the screw holes on both sides. One end is connected to the aeration pump (7) through a gas flow meter (8). The dissolved oxygen probe (4), sampling needle (5), and gas bag (6) of the dissolved oxygen meter (3) are all inserted into the bottle body through the top opening of the main reaction bottle (1). A magnetic stirrer (9) is placed at the bottom of the main reaction bottle (1), and its rotor (10) is located on the bottom surface inside the main reaction bottle (1). Stirring is achieved by rotating the rotor.

2. The device for micro-oxygen-regulated recruitment of biofilm to enhance lignocellulose degradation according to claim 1, characterized in that: The membrane module (2) consists of 50 hollow fiber membranes, each with a length of 18 cm and an effective membrane area of ​​120 cm². 2 .

3. A method for enhancing lignocellulose degradation by micro-oxygen-regulated recruitment of biofilms, implemented using the apparatus for enhancing lignocellulose degradation by micro-oxygen-regulated recruitment of biofilms as described in claim 1 or 2, characterized in that: The method includes the following steps: Step 1: Inoculum Acquisition: Activated sludge and paper mill wastewater are screened separately to remove impurities, and then mixed in proportion to serve as inoculum. Step 2, Reactor Inoculation and Start-up: After preparing the acclimatization culture medium, add inoculum in a certain proportion, aerate the reactor to remove oxygen, and then seal it; adjust the aeration mode, aeration rate, reaction temperature, and stirring speed to start the reaction; Step 3, directional domestication: After a period of cultivation, the culture medium in the reactor is replaced and the aeration parameters are gradually adjusted. After 6 generations of domestication cultivation, a multi-species biofilm that degrades lignocellulose is obtained. Step 4: Community structure analysis: Samples of biofilm on the surface of membrane filaments were taken and 16S rRNA sequencing analysis was performed.

4. The method for micro-oxygen regulation-controlled recruitment of biofilm to enhance lignocellulose degradation according to claim 3, characterized in that, In step one, activated sludge and paper mill wastewater are mixed at a volume ratio of 1:1 after passing through a 100-mesh sieve and used as inoculum.

5. The method for micro-oxygen regulation-controlled recruitment of biofilm to enhance lignocellulose degradation according to claim 3, characterized in that, In step two, the acclimatization culture medium consists of: Na2HPO4 1.86 g / L, KH2PO4 1.03 g / L, NaCl 8 g / L, KCl 0.2 g / L, (NH4)2SO4 1.4 g / L, vitamins 1 mL / L, trace elements 10 mL / L, and microcrystalline cellulose or corn stalks 5-10 g / L.

6. The method for micro-oxygen regulation-induced recruitment of biofilm to enhance lignocellulose degradation according to claim 3, characterized in that, In step two, the reactor is started with an inoculation ratio of 10% (v / v), and the aeration is achieved by introducing high-purity nitrogen into the culture medium for 15 minutes to remove oxygen.

7. The method for micro-oxygen-regulated recruitment of biofilms to enhance lignocellulose degradation according to claim 3, characterized in that, In step two, the aeration mode includes: No aeration: relies on natural oxygen infiltration; Intermittent aeration: circulate aeration for 12 hours, then stop aeration for 12 hours; Continuous aeration: Uninterrupted aeration; The aeration rate for intermittent and continuous aeration is 1-50 mL / min.

8. The method for micro-oxygen-regulated recruitment of biofilms to enhance lignocellulose degradation according to claim 3, characterized in that: The reaction temperature of the mixture in the main reaction flask (1) is 35℃, and the rotation speed of the rotor (10) is 300 rpm.

9. The method for micro-oxygen regulation-induced recruitment of biofilm to enhance lignocellulose degradation according to claim 3, characterized in that, In step three, the acclimatization period is 12 days, and all MCC or straw and 80% liquid culture medium in the culture medium are replaced each time a subculture is performed.

10. The method for micro-oxygen-regulated recruitment of biofilms to enhance lignocellulose degradation according to claim 3, characterized in that, In step four, the sequencing region for 16S rRNA sequencing is the V3-V4 region, and the amplification primers are 338F and 806R.

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