CN109811013B - Application of Rhodococcus pyridinivorans in methane production through anaerobic fermentation - Google Patents
Application of Rhodococcus pyridinivorans in methane production through anaerobic fermentation Download PDFInfo
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- CN109811013B CN109811013B CN201910219351.4A CN201910219351A CN109811013B CN 109811013 B CN109811013 B CN 109811013B CN 201910219351 A CN201910219351 A CN 201910219351A CN 109811013 B CN109811013 B CN 109811013B
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Abstract
The invention discloses an application of Rhodococcus pyridinivorans in methane production through anaerobic fermentation. Experiments show that after Rhodococcus pyridinivorans (HR-1) is added into a raw material for anaerobic fermentation, the gas production rate of the biogas is obviously increased, and the methane content in the biogas is obviously increased, so that Rhodococcus pyridinivorans (HR-1) can be applied to methane production through anaerobic fermentation. The Rhodococcus pyridinivorans (Rhodococcus pyridinovorans) HR-1 belongs to facultative anaerobes, can survive in the presence of oxygen, is easy to culture and is relatively simple to operate. The invention develops a new application field for rhodococcus pyridinovorus and provides a new microbial strain with stronger adaptability for an anaerobic fermentation methane production system.
Description
The technical field is as follows:
the invention belongs to the technical field of microbial system classification and environmental biology, and particularly relates to application of rhodococcus pyridinivorans in methane production through anaerobic fermentation.
Background art:
anaerobic fermentation mainly refers to a process of decomposing organic substances to generate methane and carbon dioxide under anaerobic conditions, is a common treatment process of sludge sewage, kitchen waste, household garbage, livestock and poultry manure and the like at present, and how to improve the performance of anaerobic fermentation for generating methane is one of current research hotspots.
Earlier researches show that the anaerobic fermentation efficiency of the system can be effectively promoted and the methane yield can be improved by adding iron additives, graphene, extracellular polymers and the like into the anaerobic fermentation system. However, the addition of the substances generally has the problems of increasing the operation cost, influencing the resource utilization of biogas slurry and biogas residues and the like. Research shows that methanogens play a key role in the anaerobic fermentation process and have a more direct effect on improving the yield of methane.
Rhodococcus pyridinivorans is a microbial strain capable of degrading azacyclo-aromatic hydrocarbon compounds such as p-o-xylene, pyridine, phenol, iminodiacetonitrile and the like, and the invention patent application with the application number of 201710363708.7 discloses Rhodococcus pyridinivorans which can be used as an anode catalyst of a microbial fuel cell for treating landfill leachate and has a wider electron donor utilization spectrum. Through literature and patent search, no report about the promotion of anaerobic fermentation and methane production of rhodococcus pyridinivorans is found.
The invention content is as follows:
the invention aims to overcome the defects in the prior art and provides the application of rhodococcus pyridinivorans in anaerobic fermentation for methane production.
Experiments show that after Rhodococcus pyridinovorans (HR-1) is added into the anaerobic fermentation raw material, the gas production of the biogas is obviously increased, and the methane content in the biogas is obviously increased.
Therefore, the invention aims to provide the application of Rhodococcus pyridinovorans (Rhodococcus pyridinovorans) HR-1 in methane production through anaerobic fermentation.
The Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) HR-1 electron donor disclosed by the invention has a wide spectrum, and can grow by taking one or more of citric acid, succinic acid, glucose, sucrose, yeast powder and sodium acetate as an electron acceptor. The morphological characteristics are as follows: the colony morphology is circular and red, the diameter of a single colony is about 2mm, and the edge is regular and regular; the cells are short rod-shaped, have a size range of 1.0-2.0 μm × 0.2-0.4 μm, and are facultative anaerobic.
Preferably, the Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) HR-1 is applied to the anaerobic fermentation of excrement or kitchen waste serving as a raw material to produce methane.
The manure is preferably pig manure or chicken manure.
Compared with the prior art, the invention has the following beneficial effects:
(1) rhodococcus pyridinivorans HR-1 is used as an active strain of an anaerobic fermentation methane production system, belongs to facultative anaerobes, is separated and identified from garbage percolate, is easy to culture, cannot be inactivated in the presence of oxygen, does not need too severe operation in the culture and operation processes, and is simple to operate and relatively low in operation cost.
(2) The raw material utilization spectrum of the rhodococcus pyridinivorans HR-1 is relatively wide, and the invention patent with the application number of 201710363708.7 shows that the rhodococcus pyridinivorans can grow and propagate by taking one or more organic matters of citric acid, succinic acid, glucose, sucrose, yeast powder and sodium acetate as raw materials, can completely oxidize and decompose the organic matters, and has relatively high electron recovery rate.
(3) Experiments show that Rhodococcus pyridinivorans HR-1 can significantly improve the biogas yield and the methane content in biogas in anaerobic fermentation, develop a new application field for Rhodococcus pyridinivorans, and provide a new microbial strain with stronger adaptability for an anaerobic fermentation methane production system.
Rhodococcus pyridinivorans (Rhodococcus pyridinovorans) HR-1 of the present invention is deposited in China general microbiological culture Collection center (CGMCC), address: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and Beijing, the preservation number is: CGMCC NO. 14021. The bacterium is disclosed in patent No.: 201710363708.7, title of the invention: a Rhodococcus pyridinivorans and its application in microbial fuel cells.
Description of the drawings:
FIG. 1 is a schematic diagram of a pilot plant system for anaerobic fermentation for methane production; wherein, 1, an anaerobic fermentation reaction device; 11. a first rubber plug; 12. a first triangular flask; 13. an exhaust pipe; 14. a sample adding pipe; 15. a first valve; 2. a constant temperature control device; 3. a gas collection device; 31. a second rubber plug; 32. a second triangular flask; 33. an air inlet pipe; 34. a drain pipe; 4. a measuring device 4; 5. a first hose; 51. a second valve; 6. a second hose; 7. a glass tube.
FIG. 2 shows the change of methane content in the biogas generated by a pilot plant system for anaerobic fermentation of pig manure as the raw material for anaerobic fermentation.
FIG. 3 shows the change of methane content in the biogas generated by a small-scale system for anaerobic fermentation of chicken manure as the anaerobic fermentation raw material.
FIG. 4 shows the change of methane content in the biogas generated by a small-scale anaerobic fermentation methane generation system using kitchen waste as an anaerobic fermentation raw material.
The specific implementation mode is as follows:
the following is a further description of the invention and is not intended to be limiting.
The experimental procedures described in the following examples can be carried out with reference to conventional techniques for process parameters not specifically noted; the reagents and materials, unless otherwise indicated, are commercially available.
Example 1: verification of performance of Rhodococcus pyridinivorans in promoting anaerobic fermentation for producing methane
(1) Constructing a small test system for producing methane by anaerobic fermentation:
as shown in figure 1, the small test system for producing methane by anaerobic fermentation comprises an anaerobic fermentation reaction device 1, a constant temperature control device 2, a gas collecting device 3 and a measuring device 4. Anaerobic fermentation reaction unit 1 includes the triangular flask 12 sealed with first rubber buffer 11, and first triangular flask 12 is 2000mL triangular flask, is equipped with blast pipe 13 and application of sample pipe 14 on the first rubber buffer 11, and the application of sample end of application of sample pipe 14 is equipped with first valve 15. The constant temperature control device 2 is a constant temperature water tank, and the anaerobic fermentation reaction device 1 is placed in the constant temperature water tank when anaerobic fermentation is used for producing methane, so that the temperature of the anaerobic fermentation can be controlled. Gas collecting device 3 includes the sealed second triangular flask 32 that is equipped with water with second rubber buffer 31, and second triangular flask 32 is 2000mL triangular flask, is equipped with intake pipe 33 and drain pipe 34 on the second rubber buffer 31, and blast pipe 13 and intake pipe 33 are communicated by first hose 5, are equipped with second valve 51 on the first hose 5. The measuring device 4 is a glass bottle with an opening at the upper end, a plurality of metering scales are arranged on the glass bottle, the water outlet end of the water discharge pipe 34 is connected with a second hose 6, the other end of the second hose 6 is connected with a glass tube 7, and the glass tube 7 is placed into the glass bottle during anaerobic fermentation.
(2) Preparing a preliminary inoculum containing Rhodococcus pyridinivorans bacterial liquid as a small test system for producing methane by anaerobic fermentation:
firstly, inoculating Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) HR-1 into a conventional LB liquid culture medium, culturing for 18h under anaerobic condition at 30 ℃, centrifuging to collect thalli, resuspending the thalli in a fresh LB liquid culture medium, wherein the concentration of a bacterial suspension is 106cfu/mL, and a preliminary inoculum was obtained after incubation again for 6h under the same conditions.
Wherein, the LB liquid culture medium comprises the following components: each liter contains 5g of yeast extract, 5g of NaCl and 10g of tryptone, and the balance is water, and the pH is 7; the preparation method comprises the following steps: weighing 5g of yeast extract, 5g of NaCl and 10g of tryptone, dissolving in a small amount of water, adding water to a constant volume of 1L, adjusting the pH value to 7, and sterilizing for later use.
(3) Domesticating the primary inoculum obtained in the step (2):
domesticating the primary inoculum obtained in the step (2), diluting biogas slurry with an LB liquid culture medium to obtain fermentation raw materials containing biogas slurry with different concentrations, gradually domesticating Rhodococcus pyridinivorans HR-1 by increasing the concentration of the biogas slurry in the fermentation raw materials, wherein the volume ratio of the LB liquid culture medium to the biogas slurry in the fermentation raw materials is as follows in sequence: 10:1, 8:1, 5:1, 2:1, 1:1 and 0: 1. And finally, when the fermentation raw materials are completely changed into biogas slurry and the methane content in the biogas reaches over 65 percent, and the biogas is maintained for a period of time, the acclimation process is considered to be completed, and the inoculum is obtained.
The specific domestication steps are as follows: inoculating the primary inoculum according to the inoculation amount of 5% by volume to a fermentation raw material 1 (the fermentation raw material 1 is prepared by mixing an LB liquid culture medium and biogas slurry according to the volume ratio of 10: 1), culturing at 30 ℃ under an anaerobic condition, obtaining a fermentation liquid 1 after the fermentation liquid 1 grows to a logarithmic growth phase, inoculating the fermentation liquid 1 according to the inoculation amount of 5% by volume to a fermentation raw material 2 (the fermentation raw material 2 is prepared by mixing the LB liquid culture medium and the biogas slurry according to the volume ratio of 8: 1), culturing at 30 ℃ under an anaerobic condition, obtaining a fermentation liquid 2 after the fermentation liquid grows to the logarithmic growth phase, inoculating the fermentation liquid 2 according to the inoculation amount of 5% by volume to the fermentation raw material 3 (the fermentation raw material 3 is prepared by mixing the LB liquid culture medium and the biogas slurry according to the volume ratio of 5: 1), culturing at 30 ℃ under an anaerobic condition, obtaining the fermentation liquid 3 after the fermentation liquid grows to the logarithmic growth phase, inoculating the fermentation liquid 3 according to the inoculation amount of 5% by volume to the fermentation raw material 4 (the fermentation The culture medium and the biogas slurry are mixed according to the volume ratio of 2: 1), the culture is carried out under the anaerobic condition at 30 ℃, the fermentation liquid 4 is obtained after the fermentation liquid grows to the logarithmic growth phase, the fermentation liquid 4 is inoculated into the fermentation raw material 5 (the fermentation raw material 5 is formed by mixing the LB liquid culture medium and the biogas slurry according to the volume ratio of 1: 1) according to the inoculation amount of 5 percent of the volume ratio, the culture is carried out under the anaerobic condition at 30 ℃, the fermentation liquid 5 is obtained after the fermentation liquid grows to the logarithmic growth phase, the fermentation liquid 5 is inoculated into the fermentation raw material 6 (the fermentation raw material 6 is the biogas slurry) according to the inoculation amount of 5 percent of the volume ratio, the culture is carried out under the anaerobic condition at 30 ℃ until the logarithmic growth phase, the biogas yield reaches 400mL/g.VS, the methane content in the biogas is measured to 65 percent, namely the domestication process is completed. Wherein the biogas slurry is prepared from pig manure as fermentation raw material.
(4) The domesticated inoculum containing the Rhodococcus pyridinivorans is connected into a small test system for methane production by anaerobic fermentation for methane production detection:
500g of pig manure is put into a first triangular flask 12 of a small test system for producing methane by anaerobic fermentation shown in figure 1, the temperature of a constant temperature control device 2 is adjusted to 30 ℃, a first valve 15 on a sample adding pipe 14 is closed, a second valve 51 on a first hose 5 is opened, the pig manure is cultured under the anaerobic condition at 30 ℃, bacteria carried by the pig manure are fermented under the anaerobic condition to generate biogas, the biogas sequentially passes through an exhaust pipe 13, a first hose 5 and an air inlet pipe 33 and enters a second triangular flask 32 of a gas collecting device 3, water in the second triangular flask 32 passes through a drain pipe 34, a second hose 6 and a glass pipe 7 and flows into a measuring device 4, the gas production amount of the biogas is read and recorded at any time through a metering scale (the gas volume is measured by a liquid discharge method), after 160min of anaerobic fermentation, the first valve 15 on the sample adding pipe 14 is opened, the second valve 51 on the first hose 5 is closed, inoculating 100mL of domesticated inoculum containing rhodococcus pyridinovorus into pig manure through a sample adding pipe 14, closing a first valve 15 on the sample adding pipe 14, opening a second valve 51 on a first hose 5, culturing at 30 ℃ under anaerobic conditions, and measuring the gas production of methane. As a result, after the domesticated inoculum containing the Rhodococcus pyridinivorans is added, the gas production rate of the biogas is obviously increased from 270-300 mL/g.VS to 400 +/-30 mL/g.VS, and the methane content in the biogas is obviously increased from 53.3% to 68.3% (see figure 2).
Example 2:
the specific operation process is the same as that of the embodiment 1, except that:
the chicken manure is used as the raw material in the small test system for producing methane through anaerobic fermentation, the methane yield of the original anaerobic fermentation methane of the chicken manure is 320-350 mL/g.VS, the methane content in the produced methane is about 57.3%, after the domesticated inoculum containing rhodococcus pyridinivorans is added, the methane yield is increased to 420 +/-30 mL/g.VS, and the methane content in the methane is increased to 73.1%, as shown in figure 3.
Example 3:
the specific operation process is the same as that of the embodiment 1, except that:
the kitchen waste is used as the raw material in the small-scale system for producing methane through anaerobic fermentation, the methane production rate of the original kitchen waste through anaerobic fermentation is 230-250 mL/g.VS, the methane content in the produced methane is about 48.3%, after the domesticated inoculum containing rhodococcus pyridinivorans is added, the methane production rate of the methane is increased to 320 +/-30 mL/g.VS, and the methane content in the methane is increased to 67.3%, as shown in figure 4.
The above detailed description is specific to possible embodiments of the present invention, and the embodiments are not intended to limit the scope of the present invention, and all equivalent implementations or modifications that do not depart from the scope of the present invention should be included within the scope of the present invention.
Claims (3)
1. Rhodococcus pyridinivorans (A)Rhodococcus pyridinovorans) The application of HR-1 in the anaerobic fermentation methane production is characterized in that the Rhodococcus pyridinivorans (R) (A)Rhodococcus pyridinovorans) HR-1 has the accession number: CGMCC NO. 14021.
2. The use of claim 1, wherein Rhodococcus pyridinivorans (R), (B)Rhodococcus pyridinovorans) HR-1 is applied to the anaerobic fermentation of excrement or kitchen waste as a raw material to produce methane.
3. Use according to claim 2, wherein the manure is pig manure or chicken manure.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102439160A (en) * | 2009-03-09 | 2012-05-02 | 诺维信公司 | Improved method for methane generation |
| CN106967653A (en) * | 2017-05-22 | 2017-07-21 | 中国科学院广州能源研究所 | A kind of thermophilic pyridine Rhodococcus sp and its application in microbiological fuel cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102439160A (en) * | 2009-03-09 | 2012-05-02 | 诺维信公司 | Improved method for methane generation |
| CN106967653A (en) * | 2017-05-22 | 2017-07-21 | 中国科学院广州能源研究所 | A kind of thermophilic pyridine Rhodococcus sp and its application in microbiological fuel cell |
Non-Patent Citations (1)
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| 小试厌氧混合处理糖蜜酒精废液及不同阶段活性污泥中可培养微生物的群落结构分析;耿三春等;《基因组学与应用生物学》;20161231;第35卷(第12期);摘要、第3441页左栏第1段至第3443页右栏第4段 * |
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