Efficient Bottom-Improving Bacillus and Compound Bottom-Improving Microbial Agent Prepared From the Same and Applications Thereof

Abstract

The applicant provides a strain of Bacillus tequilensis ZSGD5, with accession number of the deposit of CCTCC NO.: M 2014004, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan City, China on Jan. 6, 2014. The applicant also provides a compound microecological Bacillus agent. The compound microbial agent comprises the following bacterial species: Bacillus tequilensis, Type I Bacillus subtilis, Type II Bacillus subtilis, Type I Bacillus licheniformis, Type II Bacillus licheniformis, Bacillus pumilus, and Bacillus megaterium. The Bacillus tequilensis provided in the present invention has an efficient water bottom cleaning capability, and can rapidly decompose and decrease the sludge at the bottom of a pond and effectively improve environmental waters for such phenomena as black water, and turbid water, so the Bacillus tequilensis can be used for water environment treatment in aquaculture and for urban wastewater treatment.

Claims

1. A strain of Bacillus tequilensis (Bacillus tequilensis) ZSGD5, with accession number of the deposit of CCTCC NO.: M 2014004, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan City, China on Jan. 6, 2014.

2. A compound microecological Bacillus agent, wherein, the compound microbial agent comprises the following bacterial species: Bacillus tequilensis, Type I Bacillus subtilis, Type II Bacillus subtilis, Type I Bacillus licheniformis, Type II Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium; the Bacillus tequilensis, Type I Bacillus subtilis, and Type I Bacillus licheniformis, respectively numbered as CCTCC NO.: M 2014004, CCCTC NO.: M 2011443, and CCTCC NO.: M 2012458, are deposited at the China Center for Type Culture Collection, and all of other bacterial species are commercially available.

3. The compound microecological Bacillus agent according to claim 2, wherein, the effective viable count of the compound microecological Bacillus agent is 5.0108-2.01010 cfu/g, the dosage is 20-100 g/mu, and the bacterial species composition is as follows: Bacterial species viable count of bacterial species per gram of product, in cfu/g Bacillus tequilensis 1.0108-5.0109 Type I Bacillus subtilis 1.010 8-5.010 9 Type II Bacillus subtilis 1.010 8-5.0109 Type I Bacillus licheniformis 5.010 7-1.0109 Type II Bacillus licheniformis 0.810 7-5.010 8 Bacillus pumilus 0.210 7-1.010 8 Bacillus megaterium 5.010 7-1.0109 Total viable count 5.010 8-210 10.

4. The compound microecological Bacillus agent according to claim 2, wherein, the compound microecological Bacillus agent is used for improving the quality of the bottom water in a pond for aquaculture.

5. The compound microecological Bacillus agent according to claim 4, wherein, the compound microecological Bacillus agent effectively control the pollution caused by the bait residue and the excrement of culture objects in culture water to maintain the increase in dissolved oxygen, suppress the moss at the bottom of a pond and reduce the contents of ammonia nitrogen, nitrite and hydrogen sulfide.

6. The compound microecological Bacillus agent according to claim 2, wherein, the compound microecological Bacillus agent is used for treatment of black water and turbid water in urban rivers.

7. The compound microecological Bacillus agent according to claim 6, wherein, the compound microecological Bacillus agent improve the status of dead black water in rivers and decrease the COD in water in rivers to 40%.

Description

DETAILED DESCRIPTION OF THE INVENTION

Embodiment

[0035] 1. Screening of efficient bottom-improving bacterial species

[0036] The microbial strain suitable for water quality improvement is separated from the bottom slurry and water in an aquaculture pond, and the genus of the bacterial species is effectively and accurately determined through such means for the strain as morphological observation, physiological & biochemical experiments, and 16S rDNA analysis.

[0037] 2 g of bottom slurry sample taken from a crab culture pond in Jiangyan, Taizhou, Jiangsu is weighed. 98 mL of sterile physiological saline is added. The mixture is oscillated on a shaking table at a temperature of 37 C. and at a speed of 120 rpm for 20 min. 1 mL of supernatant is pipetted into the liquid with the bottom slurry taken from a polluted river in Wuxi City as matrix. The obtained liquid is oscillated for 6 hr. After the enriched liquid is diluted by a factor of 10,000, an applying rod is used to apply the diluted liquid onto a solid plate with the bottom sludge taken from the river as a unique matrix, and the solid plate is cultured in a thermostatic incubator at a temperature of 37 C. for 40-48 hr. Then, three strains growing on the plate are obtained, which are numbered as ZSGD-1, ZSGD-5, and ZSGD-9, respectively. Single colonies are picked for enlarged culturing. The bacterial bodies obtained from the enlarged culturing are used for the processing test of the polluted river water. A strain with a relatively good effect, that is, ZSGD-5, is picked out. The identification and test results of 16S rDNA and physiological & biochemical properties show that this bacterial species belongs to Bacillus tequilensis, and this Bacillus can grow rapidly at a low oxygen concentration and consume the nutrients in polluted waters.

[0038] n-1 classification and naming is Bacillus tequilensis, deposited at the China Center for Type Culture Collection on Jan. 6, 2014, and with accession number of the deposit of CCTCC M 2014004.

[0039] 2. Enlarged culture of strain and collection of product

[0040] According to the above-mentioned culture and enlarged culture methods for the strain, the enlarged culture of bevel Bacillus tequilensis CCTCC M 2014004 is carried out to obtain the spore solution of Bacillus subtilis with a certain concentration; the spore solution are centrifuged, spray-dried, etc. to obtain spore powder product.

[0041] 3. Preparation method for the compound bottom-improving microbial agent (namely, compound microecological Bacillus agent)

[0042] The screened Bacillus tequilensis and other effective strains are mixed according to the viable count ratio in respective solid bacterial powder; the viable bacteria adding ratios (based on ratio to total viable count of product) of the solid bacterial powder of various effective strains are as follows:

[0043] Bacterial species Viable count of bacterial species per gram of product, in cfu/g

[0044] Bacillus tequilensis (CCCTC M 2014004) 1.0109

[0045] Type I Bacillus subtilis (CCCTCM 2011443) 1.0109

[0046] Type II Bacillus subtilis 1.0109

[0047] Type I Bacillus licheniformis (CCTCC M 2012458) 1.0108

[0048] Type II Bacillus licheniformis 0.810 8

[0049] Bacillus pumilus 0.2108

[0050] Bacillus megaterium 1.0108

[0051] Total viable count 5.0109

[0052] All bacterial species numbered above are deposited at the China Center for Type Culture Collection, whereas all of Type II Bacillus subtilis, Type II Bacillus licheniformis, Bacillus pumilus, and Bacillus megaterium are provided by Taizhou LvShengYuan Biological Co., Ltd.

[0053] After the above various kinds of bacterial powder are evenly mixed at predetermined ratios, one or more carriers (bran powder, glucose, corn powder, rice bran powder, zeolite powder, diatomite, activated carbon, sericite powder) are added to supplement the total weight to 100% (with Bacillus as effective component. Carriers are added so that the total weight of product conforms to that the final viable count reaches 5.0109 cfu/g). Granulation is carried out to get microbial agent granules, and then the microbial agent granules are put into use.

[0054] Each kind of effective single bacterial powder is prepared strictly according to the above method, and then, the compound bottom-improving microbial agent described in the present invention, also called compound microecological Bacillus agent, can be obtained. This product is used to conduct effective bottom improvement on waters polluted by various organic matters so that the bottom slurry turns from black to yellow, and can reduce residual baits and excrement in the bottom of a culture water pond, and effectively eliminate such substances as ammonia nitrogen, nitrite, and hydrogen sulfide at the bottom of a pond.

[0055] In the above whole processing process, it shall be ensured that the environment is clean to avoid contamination. The environmental humidity is controlled to be below 70%, and the moisture content in each raw material to be below 15%. This product prepared can be stored for 18 months at normal temperature; the shelf life can reach 36 mouths at a temperature of 18 C. and the viable count is stable within the shelf life.

[0056] 4. Test example 1: The compound microecological Bacillus agent was used for improvement of Carassius auratus culture water.

[0057] Taking water sample: The water sample was taken at a position 30 cm below water surface and 1.5 m away from bank in a culture pond in Taicang, Suzhou. The water sample was processed according to the Specification for Marine Monitoring. In the test, the culture water temperature was 22-27 C., and the result in the control pond was obtained without using the compound bottom-improving microbial agent.

[0058] The microbial agent granules were spread into the pond at a dose of 500 g per mu.Math.m.

[0059] The processing results are shown in Table 1.

TABLE-US-00001 TABLE 1 Total Ammonia inorganic Dissolved nitrogen Nitrite nitrogen oxygen Phosphate (ppm) (ppm) (ppm) (ppm) (ppm) Control Test Control Test Control Test Control Test Control Test pond pond pond pond pond pond pond pond pond pond Before 2.4 2.5 0.15 0.14 0.249 0.241 3.2 3.1 0.065 0.067 processing 2 days 2.3 1.9 0.13 0.09 0.252 0.185 3.1 3.5 0.093 0.043 after processing 3 days 2.3 0.08 0.16 0.07 0.247 0.102 3.4 4.5 0.087 0.038 after processing 4 days 2.4 0.07 0.15 0.06 0.253 0.103 3.6 5.2 0.087 0.029 after processing

[0060] According to the data in table 1, it can be seen that, the compound microbial agent can effectively decrease the ammonia nitrogen, nitrite, total inorganic nitrogen, and phosphate in water and can increase the content of dissolved oxygen in water.

[0061] 5. Test example 2: The compound microecological Bacillus agent was used for treatment of black water in river.

[0062] Two polluted water samples were taken at different positions of a polluted river in Wuxi. Treatment tests were conducted with this microbial agent respectively: the control group was not processed. The test scale was 5 L, and COD was tested according to the corresponding China national standard. The microbial agent granules were added at a dose of 500 g per mu.Math.m. The processing results are shown in Table 2.

TABLE-US-00002 TABLE 2 Total Ammonia inorganic Dissolved nitrogen Nitrite nitrogen oxygen Phosphate (ppm) (ppm) (ppm) (ppm) (ppm) Control Test Control Test Control Test Control Test Control Test pond pond pond pond pond pond pond pond pond pond Before 2.4 2.5 0.15 0.14 0.249 0.241 3.2 3.1 0.065 0.067 processing 2 days after 2.3 1.9 0.13 0.09 0.252 0.185 3.1 3.5 0.093 0.043 processing 3 days after 2.3 0.08 0.16 0.07 0.247 0.102 3.4 4.5 0.087 0.038 processing 4 days after 2.4 0.07 0.15 0.06 0.253 0.103 3.6 5.2 0.087 0.029 processing

[0063] According to the data in table 2, it can be seen that, when this microbial agent is used to process the polluted water in river, it can effectively decrease the COD in the polluted water in the river.