CN110819554B - Extreme thermophilic bacteria and application thereof in high-temperature compost fermentation - Google Patents
Extreme thermophilic bacteria and application thereof in high-temperature compost fermentation Download PDFInfo
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Abstract
The invention relates to the field of microbial technology and environmental engineering, and discloses an extreme thermophilic bacterium and application thereof in high-temperature compost fermentation, wherein the extreme thermophilic bacterium is named as GW-2, the preservation number of the extreme thermophilic bacterium is CGMCC 18012, and the classification name of microorganisms isChelativorans composti. The strain can normally metabolize and grow at the temperature of 40-105 ℃, the pH value of 5-11 and the salinity of 0-7 percent, can grow by utilizing various carbon sources and nitrogen sources, and has a very broad substrate spectrum. The strain can be used for performing high-temperature aerobic composting treatment on a plurality of organic matters such as kitchen waste, agricultural product tailings and slaughterhouse waste by matching with the special high-temperature composting process, so that the organic matters are completely humified, dehydrated and dried, and reduced in weight by 70-92%, and finally the organic fertilizer with excellent quality is formed.
Description
Technical Field
The invention relates to the fields of microbial technology and environmental engineering, in particular to an extreme thermophilic bacterium and application thereof in high-temperature compost fermentation.
Background
Solid wastes containing a large amount of organic matters such as kitchens and the like are generated by human activities, if the solid wastes are not treated, serious pollution is caused to the environment, and the solid wastes can be effectively stabilized by a high-temperature composting technology which is gradually developed in recent years. High temperature composting is a process of decomposing organic matter into humus by utilizing the growth activity of microorganisms under high temperature conditions. According to the difference of the used microorganism, the method can be divided into aerobic high-temperature compost and anaerobic high-temperature compost, and the aerobic high-temperature compost technology is generally adopted at home and abroad at present. Compared with the anaerobic, aerobic high-temperature composting has the following advantages: firstly, aerobic high-temperature composting degradation is more complete for the same organic matters, namely the removal rate is higher. Secondly, in the same time, the amount of organic matters which can be degraded by the aerobic high-temperature compost is far larger than that of the anaerobic high-temperature compost, namely, the degradation rate of the aerobic high-temperature compost is faster. And thirdly, the aerobic high-temperature composting is convenient for mechanical operation, and the labor cost and the operation difficulty can be effectively reduced in the aspect of engineering. In addition, the heat energy released by the aerobic high-temperature compost can not only maintain the temperature required by the reaction without external heat supply, but also effectively kill partial bacteria and viruses and reduce the toxic and harmful effects of the garbage.
The high-temperature composting technology is also called as fermentation technology, the traditional fermentation temperature is generally maintained at about 60 ℃, and is not suitable to exceed 70 ℃, and the reaction time can last for about 10 days. When the temperature is too high, the microorganisms are inactivated due to heat intolerance and die in a large amount, resulting in a slow fermentation rate. The fermentation can be divided into three stages according to the reaction temperature of the stack: the temperature rise stage, the high temperature stage and the temperature reduction stage, and the reaction is mainly carried out at the high temperature stage.
From the above, the current high temperature composting technology is mainly limited to the lack of highly efficient extreme thermophilic functional microorganisms, resulting in the difficulty of reaching higher fermentation temperatures. Therefore, it is necessary to select more desirable extreme thermophilic functional microorganisms.
Disclosure of Invention
In order to solve the technical problems, the invention provides an extreme thermophilic bacterium and application thereof in high-temperature compost fermentation, wherein the bacterial strain GW-2 can endure a high temperature of 105 ℃ and maintain certain activity, and can survive in a wider pH (5-11) and salinity (0-7%) environment and maintain higher activity. The invention can greatly improve the composting temperature, accelerate the decomposition of organic solid wastes and save the time cost.
The specific technical scheme of the invention is as follows: the invention provides an extreme thermophilic bacterium named GW-2, which is preserved in China general microbiological culture Collection center (CGMCC) at 21.6.2019, the preservation number is CGMCC 18012, and the classified name of the microorganism isChelativorans composti(ii) a The 16s ribosome subunit gene sequence of GW-2 is shown in SEQ ID NO. 1.
The strain is separated from the high-temperature hot spring environment of the hot spring of Nanjing Tangshan, and the strain with the most similar gene sequence of the 16s ribosomal subunit of the GW-2 strain is the bacterium Nis 3 (a) of the compost chelating agentChelativorans composti Nis 3). The characteristics are as follows:
(1) and screening and subculturing by using a TM culture medium. (Na is contained per liter of TM medium:. RTM.)2HPO4·12H2O 0.48g、KH2PO4 60.0mg、MgCl2·6H2O 0.24g、CaSO4·2H245.0mg of O, 0.6mL of 0.01M ferric citrate solution, 1.0mL of trace element solution, 0.12g of nitrilotriacetic acid, 2.7g of tryptone, 2.7g of yeast extract, 18g of agar (prepared with a solid culture medium) and pH7.5, wherein each liter of the 1000-fold trace element solution contains: cocl (cobalt carbide)2·6H2O 48.0mg、Na2MoO4·2H2O 27.0mg、CuSO4·5H2O 27.0mg、H3BO3 0.54g、ZnSO4·7H2O 0.54g、MnSO4·H2O 2.4g、Srcl238.0mg、Na2SiO34.2mg, NaF 2.5mg, concentrated sulfuric acid 0.6 mL).
(2) And the strain GW-2 is extreme thermophilic bacteria, and the growth conditions in a TM culture medium are as follows: the temperature is 40-105 deg.C (optimally 90 deg.C), pH is 5-11 (optimally 8), and salinity is 0-7% (w/v, optimally 1.5-3.5%).
(3) And the bacterial strain GW-2 is gram-positive bacteria with the size of 1-3 mu m.
(4) And the generation time of the strain GW-2 in the TM culture medium is 20 min.
(5) And the bacterial strain GW-2 is a compost chelon genus and has no harm to crops, human beings and animals.
The traditional fermentation temperature is 60 ℃ generally, and can not exceed 70 ℃, otherwise, the microorganism is killed massively. The strain GW-2 can endure the high temperature of 105 ℃ and maintain certain activity, and can survive in wider pH (5-11) and salinity (0-7%) environment and maintain higher activity. The invention can greatly improve the composting temperature, accelerate the decomposition of organic solid wastes and save the time cost.
Preferably, the hyperthermophiles may be a culture of the aforementioned hyperthermophiles GW-2 or a culture after passaging.
The invention also provides a mutant of the extreme thermophilic bacteria, which is obtained by carrying out mutagenesis, domestication, gene recombination or natural mutation on the extreme thermophilic bacteria GW-2.
The invention also provides a bacterial culture containing the extreme thermophilic bacteria or the mutant.
The invention also provides a bacterial culture which is a bacterial liquid, a microbial inoculum or activated sludge and the like.
The invention also provides an enzyme preparation or a single-cell protein containing the extreme thermophilic bacterium or the mutant.
The invention also discloses the application of the extreme thermophilic bacteria or the mutant in sewage treatment, garbage treatment, composting, enzyme preparation production and single-cell protein production.
The invention also provides a high-temperature compost fermentation method, which comprises the following steps:
1) and (4) pretreating the first batch of materials.
2) And (3) putting strains: and (3) fully mixing fermentation liquor obtained after fermentation of the extreme thermophilic bacteria of claim 1 or 2 or the mutant of claim 3 with wood chips, drying at 60-85 ℃ to form a microbial inoculum, and putting the microbial inoculum into the first batch of materials.
3) And (4) heating the materials.
4) High-temperature fermentation: the fermentation temperature is 70-100 ℃.
5) And (5) semi-continuously feeding and discharging.
Preferably, step 1) is specifically: cutting and crushing the first batch of materials to form particles with the radius of 3-5cm, adding quicklime accounting for 3-6% of the mass of the materials, and controlling the water content of the obtained mixture to be 70-85%; after stirring evenly, 0.3-1.0g of esterase, 0.8-2.0g of cellulase, 0.3-1.0g of alkaline amylase and 0.2-1.2g of alkaline protease are added into each ton of material in turn, mixed evenly again and kept stand for 0.5-1 h.
The invention adopts special pretreatment means such as physical cutting, heating, chemical hydrolysis, enzymatic hydrolysis and the like to quickly heat the compost to above 60 ℃, so that the compost quickly enters a high-temperature stage, GW-2 strains quickly become absolute dominant strains in a compost system, and on the basis of the absolute dominant strains, a continuous compost fermentation process capable of feeding and discharging materials every day is realized.
In the pretreatment of the present invention, the reason for the physical cutting is that the smaller the particle size of the material, the more easily it is decomposed, but considering that while the surface area of the material is increased, a certain degree of porosity must be maintained in order to allow ventilation so that the material can obtain a sufficient supply of oxygen, the organic material is cut and pulverized here into pieces of 3 to 5 cm. 3-6% of quicklime is added into the material, and the quicklime can react with excessive moisture in the material to release heat, so that the material is rapidly heated, and meanwhile, the moisture content of the material can be controlled at the optimal reaction humidity of 70-85%. Esterase, cellulase, alkaline amylase and alkaline protease are added into the materials. The method can help to hydrolyze starch, protein and lipid substances in the materials into small molecules which are easy to be utilized by high-temperature strains in advance, so that the compost can enter a high-temperature stage quickly.
Preferably, in the step 2), the fermentation liquor and the wood chips are mixed according to the volume ratio of 1:0.8-1.0, and the microbial inoculum prepared by drying is added according to 25-35% of the mass of the first batch of materials.
Preferably, step 3) is specifically: heating the first batch of materials to above 45 ℃, then keeping the heating state for 2-4h until the temperature of the stack reaches 60-65 ℃, and stopping heating.
Preferably, the step 4) is specifically: controlling the humidity of the fermentation environment to be 85-90%, and timely supplementing water or garbage leachate if the humidity is insufficient; the fermentation time lasts for 48-60 h.
After the microbial inoculum is activated at the temperature of 40 ℃, the microbial inoculum rapidly breeds and decomposes organic matters to generate a large amount of heat, so that the temperature of materials is raised to 70-100 ℃, and the high temperature can be maintained until the materials are humified and most of water is gasified. After fermentation is completed, the weight of the material can be reduced by 70-92%. In the fermentation process, the equipment controls the air humidity in the fermentation container to be 80-90%, and if the humidity is insufficient, the equipment replenishes water by spraying water. The GW-2 bacteria can inhibit the growth of harmful bacteria, and does not produce foul smell, rancidness, etc.
Preferably, the step 5) is specifically as follows: after high-temperature fermentation, the material is converted into humus, and then the material starts to enter a repeated feeding and discharging stage; controlling the discharging proportion to be below 20wt%, directly adding the fed material without carrying out pretreatment again, wherein the adding amount is not more than 20wt% of the material obtained in the step 4); in the repeated feeding and discharging stage, the interval of each time is controlled to be 12-24 h.
Preferably, after each batch of microbial inoculum is added, the requirement of 30-50 times of discharging feeding operation can be met, and then the fermentation period is restarted according to the steps 2) -5).
Compared with the prior art, the invention has the beneficial effects that:
1) the strain can normally metabolize and grow at the temperature of 40-105 ℃, the pH value of 5-11 and the salinity of 0-7% (w/v), various monosaccharides, oligosaccharides, polysaccharides, organic acids and alcohols can be used as carbon sources, various composite nitrogen sources such as tryptone, fish peptone and beef extract can be used for growing, and the substrate spectrum is very wide.
2) The strain can be used for performing high-temperature aerobic composting treatment on a plurality of organic matters such as kitchen waste, agricultural product tailings and slaughterhouse waste by matching with the special high-temperature composting process, so that the organic matters are completely humified, dehydrated and dried, and reduced in weight by 70-92%, and finally the organic fertilizer with excellent quality is formed.
3) The high-temperature composting process also relates to special pretreatment means such as physical cutting, heating, chemical hydrolysis, enzymatic hydrolysis and the like, so that the compost can be heated quickly to be more than 60 ℃, the high-temperature composting stage is quickly started, the GW-2 strain is quickly changed into the absolute dominant strain in the compost system, and on the basis of the absolute dominant strain, the continuous composting fermentation process capable of feeding and discharging materials every day is realized.
Drawings
FIG. 1 is a graph showing the growth of hyperthermophile extreme GW-2 at different temperatures.
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
An extreme thermophilic bacteria named GW-2, which has been preserved in China general microbiological culture Collection center (CGMCC) at 21.6.2019 with the preservation number of CGMCC 18012, and the classification name of the microorganism isChelativorans composti(ii) a The 16s ribosome subunit gene sequence of GW-2 is shown in SEQ ID NO. 1.
Preferably, the hyperthermophiles may be a culture of the above-mentioned GW-2 or a culture after passaging.
A mutant of extreme thermophilic bacteria, said mutant is obtained by mutagenesis, domestication, gene recombination or natural mutation of the above-mentioned GW-2.
A cell culture comprising the above-mentioned hyperthermophilic extremophile or the above-mentioned mutant.
A bacterial culture is a bacterial liquid, a bacterial agent or activated sludge.
An enzyme preparation or a single-cell protein comprising the above hyperthermophiles or the above mutants.
The invention also discloses the application of the extreme thermophilic bacteria or the mutant in sewage treatment, garbage treatment, composting, enzyme preparation production and single-cell protein production.
FIG. 1 shows the growth curves of strain GW-2 at different temperatures (70 deg.C, 80 deg.C, 90 deg.C, 100 deg.C). As can be seen from the figure, the bacterial activity of GW-2 decreases with increasing temperature at 70 ℃, 80 ℃ and 90 ℃, and decreases with increasing temperature at 100 ℃. Therefore, the optimum temperature of GW-2 is around 90 ℃. And in the range of 60-100 ℃, the activity of the strain fluctuates, but the whole strain is still at a higher level.
A high-temperature compost fermentation method comprises the following steps:
1) pretreatment of first batch of materials: cutting and crushing an original organic garbage material as a first batch of material into fragments with the diameter of 3-5cm, mixing the fragments with 3-6% of quicklime by mass, and controlling the water content of the obtained mixture to be 70-85%; 0.3-1.0g of esterase, 0.8-2.0g of cellulase, 0.3-1.0g of alkaline amylase and 0.2-1.2g of alkaline protease are added into each ton of original organic garbage material, fully mixed and kept stand for 0.5-1 h.
2) And (3) putting strains: and fully mixing fermentation liquor obtained after the fermentation of the extreme thermophilic bacteria or the mutant is finished with wood chips, drying at 55-65 ℃ to form a microbial inoculum, and putting the microbial inoculum into the first batch of materials. Wherein the volume ratio of the fermentation liquor to the wood chips is 1: 0.8-1.0; the addition amount of the microbial inoculum is at least 30 percent of the weight of the first batch of materials.
3) Heating materials: putting the first batch of materials and strains into a fermentation device with electric heating, stirring, ventilating and water replenishing functions, heating the materials to over 45 ℃ within 1 hour by adopting an electric heating mode, maintaining the heating for 2-4 hours, and stopping the electric heating after the temperature of the materials rises to 60-65 ℃.
4) High-temperature fermentation: the fermentation temperature is 70-100 ℃, the air humidity of the fermentation environment is controlled to be 80-90%, and if the humidity is insufficient, water supplement or garbage leachate is added; the high-temperature fermentation time is 48-60 h.
5) Semi-continuous feeding and discharging: completing humification after the high-temperature fermentation in the step 4), and starting discharging and feeding; the discharging proportion is not more than 20%, then new organic garbage materials are put in, the new materials can be directly put in without pretreatment or water proportion control, and the putting proportion is not more than 20% of the original weight of the fertilizer pile. And the material can be discharged and supplemented for the second time within 18-24h after the new material is supplemented, and the semi-continuous fermentation can be realized by repeated operation. Wherein, after each batch of microbial inoculum is added, the requirement of 30-50 times of discharging and feeding operations can be met, and then the fermentation period is restarted according to the steps 2) -5).
Example 1: kitchen waste composting treatment
5 tons of kitchen waste is taken, cut and stirred to be broken blocks with the diameter of about 3 cm. Putting the fragments into a fermentation device, adding 3% of quicklime, 0.5g of esterase, 0.8g of cellulase, 0.4g of alkaline amylase and 0.5g of alkaline protease, controlling the water content of the materials to be about 80%, uniformly stirring, and standing for 0.5 h. Then 1.5 tons of GW-2 microbial inoculum (the volume ratio of fermentation liquor to dry wood chips is 1:0.9 in the preparation process) is added into the fermentation tank, the fermentation tank is started to heat after being uniformly stirred, and the temperature in the fermentation tank reaches 45 ℃ after 0.5 hour. After maintaining heating for 3h, the temperature rises to 60 ℃, and the heating is stopped, and then the high-temperature composting stage is carried out. After 20h, the temperature was raised to 100 ℃ and the humidity was reduced to 75%. After 24h, the temperature in the fermentation tank begins to slowly decrease, and after 44h, the temperature is decreased to 60 ℃, at this time, 20wt% of fermented materials are taken out, and 1 ton of kitchen waste which is not pretreated is added again. And continuously increasing the temperature of the fertilizer pile, maintaining the temperature between 70 and 100 ℃ according to the organic matter content condition of fed materials, and discharging and feeding materials in the same way again after feeding for 45 hours. After discharging and feeding for 10 times, 1.8 tons of organic fertilizer are obtained totally, the water content is 35.8 percent, and the weight of the material is reduced by 88.1 percent.
Example 2: solid waste compost for cultivation
Adding 4.5% of quicklime into 10 tons of pig farm manure, uniformly stirring, controlling the water content of the materials to be 82%, adding 0.8g of esterase, 1.3g of cellulase, 0.3g of alkaline amylase and 0.8g of alkaline protease, continuously mixing and stirring, starting electric heating, heating to 45 ℃, and maintaining for 1 hour. 4 tons of GW-2 microbial inoculum wood chips are added, (during preparation, the volume ratio of fermentation liquor to dry wood chips is 1: 0.8) and fully stirred, the electric heating power is increased to raise the temperature to 45 ℃, and high-temperature composting is started. After starting for 4 hours, the temperature of the fertilizer pile is increased to 65 ℃, the temperature is gradually increased within 24 hours of starting, finally the temperature reaches 85-100 ℃, and the humidity is gradually reduced from about 80% initially, and finally about 53%. The temperature then slowly dropped, falling back to 65 ℃ within 48 hours of start-up, at which time 20% by volume of old material was discharged, and 2 tons of fresh material were replenished and fermentation continued. And continuously increasing the temperature of the fertilizer pile, maintaining the temperature between 70 and 100 ℃ according to the organic matter content condition of fed materials, and discharging and feeding materials in the same way again after feeding for 24 hours. After discharging and feeding for 10 times, the total amount of the obtained organic fertilizer is 4.2 tons, the water content is 37.5 percent, and the weight of the material is reduced by 86.0 percent.
Example 3: compost of agricultural and sideline products
Taking 5 tons of straws, adding the straws into a fermentation device with cutting and stirring functions, continuously cutting and stirring the straws into fragments of about 6cm, adding 5 percent of quicklime, uniformly stirring the mixture, controlling the water content of the materials to be 85 percent, adding 0.7g of esterase, 1.3g of cellulase, 0.6g of alkaline amylase and 0.5g of alkaline protease, continuously mixing and stirring the mixture, starting electric heating, raising the temperature to 45 ℃ and maintaining the temperature for 2 hours. 2.5 tons of GW-2 microbial inoculum wood chips are added, (the volume ratio of fermentation liquor to dry wood chips is 1:1 during preparation), the mixture is fully stirred, the electric heating power is increased, the temperature is raised to 45 ℃, and high-temperature composting is started. After starting for 4 hours, the temperature of the fertilizer pile is increased to 60 ℃, the temperature is gradually increased within 24 hours of starting, finally, the temperature reaches 85-100 ℃, and the humidity is gradually reduced from initial 87 percent to final about 58 percent. The temperature was then slowly reduced and dropped back to 65 ℃ over 60 hours of start-up, at which time 25% of the volume of old material was discharged and 1.25 tonnes of fresh material was replenished and fermentation continued. And continuously increasing the temperature of the fertilizer pile, maintaining the temperature between 70 and 100 ℃ according to the organic matter content condition of fed materials, and discharging and feeding materials in the same way again after feeding for 24 hours. After discharging and feeding for 10 times, 2.36 tons of organic fertilizer are obtained in total, the water content is 35 percent, and the weight of the material is reduced by 81.1 percent.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Sequence listing
<110> Hangzhou Xichuan science and technology Co Ltd
<120> extreme thermophilic bacteria and application thereof in high-temperature compost fermentation
<130> 2019
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
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gagtgatgaa tgccttaggg ttgtaaagcg ctttcagcgg ggaagataat gacggtacct 360
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ttgttcggaa ttactgggcg taaagggcgc gtaggcggac tgttaagtca ggggtgcaat 480
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gcggctcact ggcccgctac tgacgctgag gcgcgaaagc gtggggagca aacaggatta 660
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agtggcgccg ctaacgcatt aagctctccg cctggggagt acggtcgcaa gattaaaact 780
caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggcctaatt cgaagcaacg 840
cgcagaacct taccagccct tgacatcccg gtcgcggtta ccagagatgg tttccttcag 900
ttcggctgga ccggagacag gtgctgcatg gctgtcgtca gctcgtgtcg ttaggtgttg 960
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ttgtacacac cgcccgtcac accatgggag ttgggtttgc ccgaagacgg tgcgctaacc 1320
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Claims (8)
1. An extreme thermophilic bacterium, characterized in that: the extreme thermophilic bacteria is named as GW-2, and has been preserved in the China general microbiological culture Collection center of China general microbiological culture Collection center (CGMCC) at 21.6.2019, the preservation number is CGMCC 18012, and the classification of the microorganisms is named asChelativorans composti(ii) a The 16s ribosome subunit gene sequence of GW-2 is shown in SEQ ID NO. 1.
2. A bacterial culture comprising the hyperthermophilic bacterium according to claim 1.
3. The microbial culture according to claim 2, which is a bacterial solution, a microbial inoculum or an activated sludge.
4. An enzyme preparation or single-cell protein comprising the hyperthermophiles as described in claim 1.
5. Use of the hyperthermophilic extremophile of claim 1 in sewage treatment, garbage treatment, composting, enzyme preparation production, single cell protein production.
6. The application of extreme thermophilic bacteria in high-temperature compost fermentation is characterized by comprising the following steps:
1) preprocessing a first batch of materials;
2) and (3) putting strains: fully mixing fermentation liquor obtained after the fermentation of the extreme thermophilic bacteria of claim 1 with wood chips, drying at 60-85 ℃ to form a microbial inoculum, and putting the microbial inoculum into a first batch of materials;
3) heating the materials;
4) high-temperature fermentation: the fermentation temperature is 70-100 ℃;
5) and (5) semi-continuously feeding and discharging.
7. The use of claim 6, wherein:
the step 1) is specifically as follows: cutting and crushing the first batch of materials to form particles with the radius of 3-5cm, adding quicklime accounting for 3-6% of the mass of the materials, and controlling the water content of the obtained mixture to be 70-85%; after stirring evenly, 0.3-1.0g of esterase, 0.8-2.0g of cellulase, 0.3-1.0g of alkaline amylase and 0.2-1.2g of alkaline protease are added into each ton of materials in turn, and then the materials are mixed evenly again; and/or
In the step 2), the fermentation liquor and the wood chips are mixed according to the volume ratio of 1:0.8-1.0, and the dried microbial inoculum is added according to 25-35% of the mass of the first batch of materials; and/or
The step 3) is specifically as follows: heating the first batch of materials to above 45 ℃, then keeping the heating state for 2-4h until the temperature of the stack reaches 60-65 ℃, and stopping heating; and/or
The step 4) is specifically as follows: controlling the humidity of the fermentation environment to be 85-90%, and timely supplementing water or garbage leachate if the humidity is insufficient; the fermentation time lasts for 48-60 h; and/or
The step 5) is specifically as follows: after high-temperature fermentation, the material is converted into humus, and then the material starts to enter a repeated feeding and discharging stage; controlling the discharging proportion to be below 20wt%, directly adding the fed material without carrying out pretreatment again, wherein the adding amount is not more than 20wt% of the material obtained in the step 4); in the repeated feeding and discharging stage, the interval of each time is controlled to be 12-24 h.
8. The use of claim 7, wherein: and adding the microbial inoculum again after 30-50 times of discharging and feeding operations.
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