CN106947718A - It is a kind of that the technology that rare-earth tailing improves microbial inoculum is prepared by raw material culture plant-growth promoting rhizobacteria of piggery wastewater - Google Patents
It is a kind of that the technology that rare-earth tailing improves microbial inoculum is prepared by raw material culture plant-growth promoting rhizobacteria of piggery wastewater Download PDFInfo
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- 239000002351 wastewater Substances 0.000 title claims abstract description 45
- 239000002994 raw material Substances 0.000 title claims abstract description 18
- 230000008635 plant growth Effects 0.000 title claims abstract description 16
- 229910052761 rare earth metal Inorganic materials 0.000 title claims abstract description 15
- 150000002910 rare earth metals Chemical class 0.000 title claims abstract description 15
- 238000005516 engineering process Methods 0.000 title claims abstract description 13
- 241000589157 Rhizobiales Species 0.000 title claims abstract 12
- 230000001737 promoting effect Effects 0.000 title claims abstract 12
- 239000002068 microbial inoculum Substances 0.000 title claims abstract 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 16
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 11
- 241000194103 Bacillus pumilus Species 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 9
- 230000006872 improvement Effects 0.000 claims abstract description 7
- 239000002154 agricultural waste Substances 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims abstract 2
- 241000894006 Bacteria Species 0.000 claims description 50
- 239000003795 chemical substances by application Substances 0.000 claims description 26
- 230000001580 bacterial effect Effects 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims 2
- 239000012530 fluid Substances 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 239000002689 soil Substances 0.000 abstract description 14
- 239000002699 waste material Substances 0.000 abstract description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 abstract description 2
- 238000001704 evaporation Methods 0.000 abstract description 2
- 230000035558 fertility Effects 0.000 abstract description 2
- 239000003337 fertilizer Substances 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 241000233866 Fungi Species 0.000 abstract 1
- 230000015556 catabolic process Effects 0.000 abstract 1
- 239000003638 chemical reducing agent Substances 0.000 abstract 1
- 238000006731 degradation reaction Methods 0.000 abstract 1
- 230000008020 evaporation Effects 0.000 abstract 1
- 230000012010 growth Effects 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 238000012856 packing Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 34
- 238000004321 preservation Methods 0.000 description 8
- 229910001385 heavy metal Inorganic materials 0.000 description 5
- 238000009630 liquid culture Methods 0.000 description 4
- 239000010865 sewage Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 238000003723 Smelting Methods 0.000 description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 239000010871 livestock manure Substances 0.000 description 2
- 238000005065 mining Methods 0.000 description 2
- 239000005416 organic matter Substances 0.000 description 2
- 101000765244 Bacillus pumilus Anthranilate synthase component 1 Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- -1 change the physical Substances 0.000 description 1
- 238000005056 compaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 239000003673 groundwater Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002688 soil aggregate Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
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Abstract
Description
技术领域technical field
本发明涉及修复离子型稀土尾矿的技术，具体的说是一种以农业废物猪场废水为原料培养植物促生细菌制备稀土尾矿改良菌剂的技术。属于生物技术领域。The invention relates to a technology for repairing ionic rare earth tailings, in particular to a technology for cultivating plant growth-promoting bacteria with agricultural waste pig farm wastewater as raw materials to prepare rare earth tailings improving bacterial agents. Belongs to the field of biotechnology.
背景技术Background technique
土壤的重金属污染大多是由人类的活动引起的。其中，重金属的开采及冶炼对周边土壤环境产生的危害较大。而重金属的开采冶炼过程中产生的尾矿，由于处理不及时、长期堆积及雨水浸泡，导致重金属向周边土壤迁移，对其所处的生态环境可造成十分严重的重金属污染。因受污染的土壤面积大，传统的物理和化学的修复方法均因经济或技术原因难以实施。近年来，植物修复技术普遍被认为具有物理化学修复方法所无法比拟的费用低廉、不破坏场地结构、不造成地下水的二次污染、美化环境等优点，受到有关方面的广泛重视。所以，超积累植物的筛选是植物修复技术的研究基础,具有现实意义。Heavy metal pollution in soil is mostly caused by human activities. Among them, the mining and smelting of heavy metals are more harmful to the surrounding soil environment. The tailings produced during the mining and smelting of heavy metals, due to untimely treatment, long-term accumulation and rainwater immersion, lead to the migration of heavy metals to the surrounding soil, which can cause very serious heavy metal pollution to the ecological environment in which they are located. Due to the large area of contaminated soil, traditional physical and chemical remediation methods are difficult to implement due to economic or technical reasons. In recent years, phytoremediation technology is generally considered to have the advantages of low cost, no damage to site structure, no secondary pollution of groundwater, and beautification of the environment, which cannot be compared with physical and chemical restoration methods, and has been widely valued by relevant parties. Therefore, the screening of hyperaccumulator plants is the research basis of phytoremediation technology and has practical significance.
本专利涉及到的保藏编号为：CCTCC M 2013283的Bacillus amyloliquefaciensH47的解淀粉芽孢杆菌有很好的植物促生特性。根际植物促生细菌是能够定植在植物根系，并对植物生长发育有利的细菌。市售地衣芽孢杆菌和市售短小芽孢杆菌与Bacillusamyloliquefaciens H47组成混合菌剂可协助Bacillus amyloliquefaciens H47促进植物生长。目前培养植物促生细菌的原料成本高昂，不利于植物促生菌剂的大规模应用。因此研究和建立以猪场废水为原料培养植物促生细菌制备稀土尾矿改良菌剂的技术有重要意义。The preservation number involved in this patent is: Bacillus amyloliquefaciens H47 of CCTCC M 2013283 has good plant growth-promoting properties. Rhizosphere plant growth-promoting bacteria are bacteria that can colonize plant roots and are beneficial to plant growth and development. The commercially available Bacillus licheniformis and the commercially available Bacillus pumilus and Bacillus amyloliquefaciens H47 form a mixed bacterial agent, which can assist Bacillus amyloliquefaciens H47 to promote plant growth. At present, the cost of raw materials for cultivating plant growth-promoting bacteria is high, which is not conducive to the large-scale application of plant growth-promoting bacteria agents. Therefore, it is of great significance to study and establish the technology of cultivating plant growth-promoting bacteria to prepare rare earth tailings improving bacterial agent by using pig farm wastewater as raw material.
猪场污水主要包括猪尿、部分猪粪和猪舍冲洗水，属高浓度有机污水，而且悬浮物和氨氮含量大。这种未经处理的污水进入自然水体后，使水中固体悬浮物、有机物和微生物含量升高，改变水体的物理、化学和生物群落组成，使水质变坏。污水中还含有大量的病原微生物将通过水体或通过水生动植物进行扩散传播，危害人畜健康。Pig farm sewage mainly includes pig urine, some pig manure and pig house flushing water, which is a high-concentration organic sewage with a large content of suspended solids and ammonia nitrogen. After this untreated sewage enters the natural water body, it will increase the content of suspended solids, organic matter and microorganisms in the water, change the physical, chemical and biological community composition of the water body, and deteriorate the water quality. Sewage also contains a large number of pathogenic microorganisms that will spread through water bodies or through aquatic animals and plants, endangering the health of humans and animals.
我国的绝大部分规模化养猪场是采用水冲式工艺。这种工艺比较方便快捷，但是同时会把猪粪、饲料残渣与猪排泄物等一起冲洗到废水中，因此废水中含有大量的固体悬浮物等，而其他的污染物浓度也很高。养猪废水是一种高氨氮、高有机物浓度、高固体悬浮物浓度的有机废水，规模化猪场废水的处理对于节能减排具有重大意义。Most of the large-scale pig farms in my country use water flushing technology. This process is more convenient and fast, but at the same time, pig manure, feed residue and pig excrement will be washed together into the wastewater, so the wastewater contains a large amount of suspended solids, etc., and the concentration of other pollutants is also high. Pig wastewater is a kind of organic wastewater with high ammonia nitrogen, high organic matter concentration, and high suspended solid concentration. The treatment of large-scale pig farm wastewater is of great significance for energy saving and emission reduction.
发明内容Contents of the invention
本发明以猪场废水为原料培养植物促生细菌制备稀土尾矿改良菌剂的技术。以保藏编号为：CCTCC M 2013283的Bacillus amyloliquefaciens H47的解淀粉芽孢杆菌、市售地衣芽孢杆菌和市售短小芽孢杆菌为植物促生菌剂，通过对猪场废水预处理后加入植物促生菌进行扩大培养，即得到大量具有改善土壤理化状况、促进植物生长的离子型稀土尾矿原位修复的植物促生菌剂。该方法对植物在离子型尾矿的生长有明显的促进作用。猪场废水得到合理利用，变废为宝，价格低廉且简便易行。The invention uses pig farm wastewater as raw material to cultivate plant growth-promoting bacteria to prepare rare earth tailings improving bacteria agent. With the preservation number: Bacillus amyloliquefaciens H47 of CCTCC M 2013283, Bacillus amyloliquefaciens H47, commercially available Bacillus licheniformis and commercially available Bacillus pumilus as plant growth-promoting bacteria agents, and adding plant growth-promoting bacteria after pretreatment of pig farm wastewater. By expanding the cultivation, a large number of plant growth-promoting bacteria agents that can improve the physical and chemical conditions of the soil and promote the growth of plants can be obtained. The method can obviously promote the growth of plants in the ionic tailings. The waste water from pig farms can be rationally utilized, turning waste into treasure, which is cheap and easy to implement.
本发明的技术方案：Technical scheme of the present invention:
用2013年6月24日保存于中国典型培养物保藏中心的保藏编号为：CCTCC M2013283的Bacillus amyloliquefaciens H47的解淀粉芽孢杆菌，市售地衣芽孢杆菌，市售短小芽孢杆菌混合植物促生菌剂，以猪场废水为原料培养植物促生细菌，制备稀土尾矿改良菌剂的技术。工艺为：The preservation number preserved in the China Type Culture Collection Center on June 24, 2013 is: Bacillus amyloliquefaciens H47 of CCTCC M2013283, commercially available Bacillus licheniformis, commercially available Bacillus pumilus mixed plant growth-promoting bacteria agent, The technology of cultivating plant growth-promoting bacteria with pig farm wastewater as raw material to prepare rare earth tailings improving bacterial agent. The process is:
1)将保藏编号为CCTCC M 2013283的Bacillus amyloliquefaciens H47、市售地衣芽孢杆菌、市售短小芽孢杆菌等菌种活化并制备成植物促生菌剂：1) Bacillus amyloliquefaciens H47 with preservation number CCTCC M 2013283, commercially available Bacillus licheniformis, commercially available Bacillus pumilus and other strains are activated and prepared into plant growth-promoting bacteria:
(1)在三种菌种斜面分别取一环在PDA平板上划线接菌丝，在培养箱中30℃培养18小时；(1) Take a ring on the slope of the three bacterial strains, draw a line on the PDA plate to inoculate the hyphae, and cultivate in an incubator at 30°C for 18 hours;
(2)在PDA平板上挑取三种菌种单菌落分别接入装有LB液体培养基的三角瓶中，准备若干份，摇床培养，培养条件：转速200r/min，温度30℃，恒温培养30小时，即得到植物促生菌剂扩大培养的种子。(2) Pick single colonies of the three strains on the PDA plate and put them into the Erlenmeyer flasks equipped with LB liquid medium, prepare several copies, and cultivate them on a shaking table. The culture conditions: rotation speed 200r/min, temperature 30°C, constant temperature After cultivating for 30 hours, the seeds of the expanded cultivation of the plant growth-promoting bacterium agent are obtained.
2)猪场废水预处理：调节猪场废水含水量至50—70％，调节PH至7.0，装在耐高温的塑料袋中，121℃灭菌30分钟。2) Pig farm wastewater pretreatment: adjust the water content of pig farm wastewater to 50-70%, adjust the pH to 7.0, pack it in a high-temperature-resistant plastic bag, and sterilize at 121°C for 30 minutes.
3)将上述发酵菌液混合后按比例1:10(v/w)接入处理好的猪场废水培养基中，保持水分50—60％，在25—30℃条件下增值2—3天，即得扩大培养的植物促生细菌改良菌剂。3) Mix the above-mentioned fermented bacteria liquid into the treated pig farm wastewater medium at a ratio of 1:10 (v/w), keep the water content at 50-60%, and add value at 25-30°C for 2-3 days , that is, the plant growth-promoting bacterium improving bacterial agent obtained by expanding cultivation.
本发明的有益效果：Beneficial effects of the present invention:
1)本发明以猪场废水为原料培养植物促生细菌制备的稀土尾矿改良菌剂，可以利用植物促生菌解铵增加土壤肥力。还可作为一种保水剂，促进土壤团粒结构形成，防止土壤板结；破坏土壤毛细现象，防止土壤水分蒸发。1) The present invention uses pig farm wastewater as a raw material to cultivate plant growth-promoting bacteria to prepare the rare earth tailings improving bacteria agent, which can use plant growth-promoting bacteria to decompose ammonium to increase soil fertility. It can also be used as a water-retaining agent to promote the formation of soil aggregate structure and prevent soil compaction; destroy soil capillarity and prevent soil moisture from evaporating.
2)本发明用猪场废水为原料培养植物促生细菌，来源广泛价格低廉，菌种增殖速度快，而且还可作为肥料，为植物提供各种营养物质。2) The present invention uses pig farm wastewater as a raw material to cultivate plant growth-promoting bacteria, which has a wide range of sources and low price, and has a fast proliferation rate of bacteria, and can also be used as fertilizer to provide various nutrients for plants.
3)本发明使得猪场废水得到合理利用，变废为宝。3) The present invention enables rational utilization of pig farm waste water, turning waste into treasure.
4)本发明的工艺简单，生产成本低，易于推广。4) The process of the present invention is simple, the production cost is low, and it is easy to popularize.
具体实施方式detailed description
下面将结合本发明实施例，对本发明的技术方案进行清楚、完善地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动的前提下所获得的其他实施例，都属于本发明保护的范围。The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by persons of ordinary skill in the art without making creative efforts all belong to the protection scope of the present invention.
实施例1Example 1
1)将保藏编号为CCTCC M 2013283的Bacillus amyloliquefaciens H47、市售地衣芽孢杆菌、市售短小芽孢杆菌等菌种活化并制备成植物促生菌剂：1) Bacillus amyloliquefaciens H47 with preservation number CCTCC M 2013283, commercially available Bacillus licheniformis, commercially available Bacillus pumilus and other strains are activated and prepared into plant growth-promoting bacteria:
(1)在三种菌种斜面分别取一环在PDA平板上划线接菌丝，在培养箱中30℃培养18小时；(1) Take a ring on the slope of the three bacterial strains, draw a line on the PDA plate to inoculate the hyphae, and cultivate in an incubator at 30°C for 18 hours;
(2)在PDA平板上挑取三种菌种单菌落分别接入装有50ml LB液体培养液的250ml三角瓶中，每个菌接种12个，摇床培养，培养条件：转速180r/min，温度28℃，恒温培养36小时，即得到植物促生菌剂扩大培养的种子。(2) On the PDA plate, single colonies of three kinds of bacteria were picked and inserted into 250ml Erlenmeyer flasks equipped with 50ml LB liquid culture solution respectively, 12 of each bacteria were inoculated, cultured on a shaking table, culture conditions: rotating speed 180r/min, The temperature is 28° C., and the constant temperature is cultivated for 36 hours to obtain the seeds of the expanded cultivation of the plant growth-promoting bacterium agent.
2)猪场废水预处理：称取3千克猪场废水调节含水量至60％，调节PH至7.0，装在耐高温的塑料袋中，121℃灭菌30分钟。2) Pretreatment of pig farm wastewater: Weigh 3 kg of pig farm wastewater, adjust the water content to 60%, adjust the pH to 7.0, pack in a high-temperature-resistant plastic bag, and sterilize at 121° C. for 30 minutes.
3)将上述发酵菌液按1:1:1混合后按比例1:10(v/w)接入处理好的猪场废水培养基中，保持水分65％，在30℃条件下增值2—3天，即得扩大培养的植物促生细菌改良菌剂。3) Mix the above-mentioned fermented bacteria liquid at a ratio of 1:1:1, then insert it into the treated pig farm wastewater medium at a ratio of 1:10 (v/w), keep the water content at 65%, and increase the value by 2- at 30°C After 3 days, the plant growth-promoting bacterium-improving bacterial agent obtained by expanding the culture can be obtained.
实施例2Example 2
1)将保藏编号为CCTCC M 2013283的Bacillus amyloliquefaciens H47、市售地衣芽孢杆菌、市售短小芽孢杆菌等菌种活化并制备成植物促生菌剂：1) Bacillus amyloliquefaciens H47 with preservation number CCTCC M 2013283, commercially available Bacillus licheniformis, commercially available Bacillus pumilus and other strains are activated and prepared into plant growth-promoting bacteria:
(1)在三种菌种斜面分别取一环在PDA平板上划线接菌丝，在培养箱中30℃培养20小时；(1) Take one ring respectively on the slope of the three strains, draw a line on the PDA plate to inoculate the hyphae, and cultivate in an incubator at 30°C for 20 hours;
(2)在PDA平板上挑取三种菌种单菌落分别接入装有50ml LB液体培养液的250ml三角瓶中，每个菌接种12个，摇床培养，培养条件：转速200r/min，温度26℃，恒温培养32小时，即得到植物促生菌剂扩大培养的种子。(2) On the PDA flat plate, pick three kinds of bacteria single colonies and insert them into 250ml Erlenmeyer flasks equipped with 50ml LB liquid culture solution respectively, inoculate 12 of each bacteria, shake table culture, culture conditions: rotating speed 200r/min, The temperature is 26° C., and the constant temperature is cultivated for 32 hours, and the seeds of the expanded cultivation of the plant growth-promoting bacteria agent are obtained.
2)猪场废水预处理：称取3千克猪场废水调节含水量至70％，调节PH至7.0，装在耐高温的塑料袋中，121℃灭菌30分钟。2) Pretreatment of pig farm wastewater: Weigh 3 kg of pig farm wastewater, adjust the water content to 70%, adjust the pH to 7.0, pack it in a high-temperature-resistant plastic bag, and sterilize at 121° C. for 30 minutes.
3)将上述发酵菌液按1:2:1混合后按比例1:10(v/w)接入处理好的猪场废水培养基中，保持水分65％，在30℃条件下增值2—3天，即得扩大培养的植物促生细菌改良菌剂。3) Mix the above-mentioned fermented bacteria liquid at a ratio of 1:2:1 and then insert it into the treated pig farm wastewater medium at a ratio of 1:10 (v/w), keep the water content at 65%, and increase the value by 2- at 30°C After 3 days, the plant growth-promoting bacterium-improving bacterial agent obtained by expanding the culture can be obtained.
实施例3Example 3
1)将保藏编号为CCTCC M 2013283的Bacillus amyloliquefaciens H47、市售地衣芽孢杆菌、市售短小芽孢杆菌等菌种活化并制备成植物促生菌剂：1) Bacillus amyloliquefaciens H47 with preservation number CCTCC M 2013283, commercially available Bacillus licheniformis, commercially available Bacillus pumilus and other strains are activated and prepared into plant growth-promoting bacteria:
(1)在三种菌种斜面分别取一环在PDA平板上划线接菌丝，在培养箱中30℃培养22小时；(1) Take a ring respectively on the slope of the three bacterial strains, draw a line on the PDA plate to inoculate the hyphae, and cultivate in an incubator at 30°C for 22 hours;
(2)在PDA平板上挑取三种菌种单菌落分别接入装有50ml LB液体培养液的250ml三角瓶中，每个菌接种12个，摇床培养，培养条件：转速160r/min，温度32℃，恒温培养28小时，即得到植物促生菌剂扩大培养的种子。(2) On the PDA flat plate, pick three kinds of bacteria single colonies and insert them into 250ml Erlenmeyer flasks equipped with 50ml LB liquid culture solution respectively, inoculate 12 of each bacteria, shake table culture, culture conditions: rotating speed 160r/min, The temperature is 32° C., and the constant temperature is cultivated for 28 hours to obtain the seeds of the expanded cultivation of the plant growth-promoting bacterium agent.
2)猪场废水预处理：称取3千克猪场废水调节含水量至65％，调节PH至7.0，装在耐高温的塑料袋中，121℃灭菌30分钟。2) Pretreatment of pig farm wastewater: Weigh 3 kg of pig farm wastewater, adjust the water content to 65%, adjust the pH to 7.0, pack in high temperature resistant plastic bags, and sterilize at 121°C for 30 minutes.
3)将上述发酵菌液按1:1:2混合后按比例1:10(v/w)接入处理好的猪场废水培养基中，保持水分60％，在30℃条件下增值2—3天，即得扩大培养的植物促生细菌改良菌剂。3) Mix the above-mentioned fermented bacteria liquid at a ratio of 1:1:2, then insert it into the treated pig farm wastewater medium at a ratio of 1:10 (v/w), keep the water content at 60%, and increase the value by 2- at 30°C After 3 days, the plant growth-promoting bacterium-improving bacterial agent obtained by expanding the culture can be obtained.
实施例4Example 4
1)将保藏编号为CCTCC M 2013283的Bacillus amyloliquefaciens H47、市售地衣芽孢杆菌、市售短小芽孢杆菌等菌种活化并制备成植物促生菌剂：1) Bacillus amyloliquefaciens H47 with preservation number CCTCC M 2013283, commercially available Bacillus licheniformis, commercially available Bacillus pumilus and other strains are activated and prepared into plant growth-promoting bacteria:
(1)在三种菌种斜面分别取一环在PDA平板上划线接菌丝，在培养箱中30℃培养16小时；(1) Take a ring on the slope of the three bacterial strains, draw a line on the PDA plate to inoculate the hyphae, and cultivate in an incubator at 30°C for 16 hours;
(2)在PDA平板上挑取三种菌种单菌落分别接入装有50ml LB液体培养液的250ml三角瓶中，每个菌接种12个，摇床培养，培养条件：转速200r/min，温度25℃，恒温培养36小时，即得到植物促生菌剂扩大培养的种子。(2) On the PDA flat plate, pick three kinds of bacteria single colonies and insert them into 250ml Erlenmeyer flasks equipped with 50ml LB liquid culture solution respectively, inoculate 12 of each bacteria, shake table culture, culture conditions: rotating speed 200r/min, The temperature is 25° C., and the constant temperature is cultivated for 36 hours, and the seeds of the expanded cultivation of the plant growth-promoting bacteria agent are obtained.
2)猪场废水预处理：称取3千克猪场废水调节含水量至65％，调节PH至7.0，装在耐高温的塑料袋中，121℃灭菌30分钟。2) Pretreatment of pig farm wastewater: Weigh 3 kg of pig farm wastewater, adjust the water content to 65%, adjust the pH to 7.0, pack in high temperature resistant plastic bags, and sterilize at 121°C for 30 minutes.
3)将上述发酵菌液按2:1:1混合后按比例1:10(v/w)接入处理好的猪场废水培养基中，保持水分55％，在30℃条件下增值2—3天，即得扩大培养的植物促生细菌改良菌剂。3) Mix the above-mentioned fermented bacteria liquid at a ratio of 2:1:1 and then insert it into the treated pig farm wastewater medium at a ratio of 1:10 (v/w), keep the water content at 55%, and increase the value by 2- at 30°C After 3 days, the plant growth-promoting bacterium-improving bacterial agent obtained by expanding the culture can be obtained.
本发明中，混合菌的比例、接种量、猪场废水培养基含水量等等不限于实施例所举的组合，可根据实际情况选择不同的组合。In the present invention, the ratio of mixed bacteria, inoculum amount, water content of pig farm wastewater medium, etc. are not limited to the combinations mentioned in the examples, and different combinations can be selected according to actual conditions.
以上所述仅为本发明的较佳实例而已，并不用以限制本发明，凡在本发明的思路和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred examples of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the ideas and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.
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