A low-temperature-tolerant Bacillus subtilis and its application

Abstract

The present invention belongs to the field of microorganisms, and particularly relates to a low-temperature-tolerant Bacillus subtilis and its application. The technical solution is as follows: A strain of Bacillus subtilis N3-3 was deposited at the Guangdong Microbial Culture Collection Center on December 12, 2022, with the deposit number: GDMCC NO: 62537. The present invention provides a new cellulose-degrading bacterium that can maintain good activity and cellulose-degrading ability even under cold conditions. Based on this microorganism, a microbial composition is provided, and the microorganisms in the composition play a synergistic role with each other, which can further improve the degradation ability of various organic substances under cold conditions.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a low-temperature-resistant Bacillus subtilis and applications thereof. Background Art

[0002] In arid, cold, and impoverished areas, dry latrines are the predominant form of toilets. The harmless treatment and resource utilization of feces and sewage are low, often limited to storage and limited degradation. Feces contain numerous pathogens and harmful substances. Direct discharge into the environment poses serious risks to natural water bodies, the atmosphere, and human health. Furthermore, feces also contain a variety of organic substances, such as fat, protein, and cellulose, which are difficult for a single microorganism to effectively degrade.

[0003] Currently, biological treatment is the primary method for treating feces. This involves using microorganisms to degrade the organic matter in feces, accelerating the humification process and rapidly reducing the volume. The degraded feces can then be used as farmland fertilizer. However, in cold environments, microorganisms generally have difficulty adapting to the harsh environment, and their growth and reproduction are inhibited, making them unable to rapidly degrade organic matter.

[0004] Therefore, screening strains that can tolerate low-temperature environments and degrade organic matter, and fixing them on carriers to prepare microbial agents, is particularly important for reducing fecal pollution and promoting resource utilization. Summary of the Invention

[0005] The purpose of the present invention is to provide a low-temperature-resistant Bacillus subtilis and application thereof.

[0006] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a strain of Bacillus subtilis N3-3, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 12, 2022, with the deposit number: GDMCCNO: 62537, and the address of the deposit unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] Correspondingly, a strain of Bacillus subtilis N3-3 has a 16S rDNA sequence as shown in SEQ ID NO: 1.

[0008] Correspondingly, the bacterial preparation comprises the Bacillus subtilis.

[0009] Preferably, the bacterial preparation further comprises oil-degrading bacteria.

[0010] Preferably, the oil-degrading bacteria is Pseudomonas mandalayi with a deposit number of CGMCC NO: 1.6426.

[0011] Preferably, the bacterial preparation further comprises protein-degrading bacteria.

[0012] Preferably, the protein-degrading bacterium is Pseudomonas azotofixans with the preservation number of CGMCC NO: 1.6428.

[0013] Correspondingly, the application of the Bacillus subtilis or the bacterial preparation in degrading cellulose.

[0014] Correspondingly, the application of the Bacillus subtilis or the bacterial preparation in composting.

[0015] Correspondingly, the application of the Bacillus subtilis or the bacterial preparation in degrading feces.

[0016] The present invention has the following beneficial effects: The present invention provides a new cellulose-degrading bacterium, which can maintain good activity and cellulose-degrading ability even under cold conditions. And based on this microorganism, a microbial composition is provided, and the microorganisms in the composition play a synergistic role with each other, which can further improve the degradation ability of various organic matters under cold conditions. Detailed implementation manners

[0017] The present invention provides a new Bacillus subtilis N3-3, which was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on December 12, 2022, with the deposit number: GDMCC NO: 62537. The Bacillus subtilis N3-3 has strong cellulose-degrading ability and can still achieve good cellulose-degrading effect even at 10°C or even below 10°C, and can survive well at 4°C and achieve certain cellulose-degrading effect.

[0018] The present invention further provides a microbial bacterial preparation. The microbial bacterial preparation includes a cellulose-degrading bacterium, an oil-degrading bacterium and a protein-degrading bacterium. The cellulose-degrading bacterium is N3-3. The oil-degrading bacterium is preferably Pseudomonas mandelii, and a more preferred scheme is: Pseudomonas mandelii with the preservation number of CGMCC NO: 1.6426. The protein-degrading bacterium is preferably Pseudomonas azotofixans, and a more preferred scheme is: Pseudomonas azotofixans with the preservation number of CGMCC NO: 1.6428.

[0019] A preferred scheme is: in the bacterial preparation, the viable concentration of microorganisms ≥ 10 9 CFU / mL. Oil-degrading bacterium: Protein-degrading bacterium: Cellulose-degrading bacterium = (4 - 8):(0.5 - 1.5):(1 - 3), and a more preferred one is: Oil-degrading bacterium: Protein-degrading bacterium: Cellulose-degrading bacterium = 6:0.5:3.

[0020] Those skilled in the art can prepare the bacterial preparation by conventional technical means in the industry. Here, an optional preparation method is provided, including the following steps: inoculate each microorganism into a culture medium, activate and expand the culture to obtain a seed solution. Adjust the concentration of each seed solution to OD 600 = 1.5 or the viable bacteria count is 10 9 CFU / mL, mix the microbial culture solutions according to the required volume ratio to obtain a mixed bacterial solution. The mixed bacterial solution can be used directly, or the mixed bacterial solution can be mixed with rice husks or other carriers at a mass ratio of 1-5:1-5. After mixing, it can be used directly or can be used after being placed at room temperature and naturally dried.

[0021] When using the bacterial preparation, the usage method can be selected according to the actual situation. If the bacterial preparation is liquid, the bacterial preparation can be sprayed on the object to be degraded or the object to be degraded can be mixed with the bacterial preparation; if the bacterial preparation is solid, the bacterial preparation can be mixed evenly with the object to be degraded for treatment.

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well-known to those skilled in the art. The data obtained are all the averages obtained after at least 3 repetitions, and all the repetitions obtained are valid data.

[0023] The culture medium and solution formulas involved in the embodiments are as follows:

[0024] 1. The nutrient agar solid medium is: peptone 10 g / L, beef extract 3 g / L, NaCl 5 g / L, agar 20 g / L, pH 7.0-7.2, sterilized at 121 °C for 20 min.

[0025] 2. LB solid medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 5 g / L, agar 20 g / L, pH 7.0-7.2, sterilized at 121 °C for 20 min.

[0026] 3. Oil-degrading bacteria screening medium: beef extract 5 g / L, peptone 10 g / L, soybean oil 10 g / L, NaCl 5 g / L, 0.6% neutral red aqueous solution 2 mL / L, agar 20 g / L, pH 7.0-7.2, sterilized at 121 °C for 20 min.

[0027] 4. Oil-degrading bacteria fermentation medium: (NH4)2SO4 5 g / L, K2HPO4 2 g / L, KH2PO4 2 g / L, NaCl 2 g / L, MgSO4·7H2O 0.5 g / L, edible oil 5 g / L, pH 7.0-7.2, sterilized at 121 °C for 20 min.

[0028] 5. Screening medium for protein-degrading bacteria: Beef extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, skim milk powder 15 g / L, agar 20 g / L, pH 7.0 - 7.2, sterilized at 121 °C for 20 min.

[0029] 6. Fermentation medium for protein-degrading bacteria: KH2PO4 0.36 g / L, Mg2SO4·7H2O 0.5 g / L, ZnCl2 0.014 g / L, Na2HPO4 1.07 g / L, NaCl 0.16 g / L, CaCl2 0.002 g / L, FeSO4·7H2O 0.002 g / L, casein 4 g / L, tryptone 0.05 g / L, pH 7.0 - 7.2, sterilized at 121 °C for 20 min.

[0030] 7. Screening medium for cellulose-degrading bacteria: (NH4)2SO4 2 g / L, Mg2SO4·7H2O 0.5 g / L, K2HPO4 1 g / L, NaCl 0.5 g / L, CMC-Na 2 g / L, Congo red 0.4 g / L, agar 20 g / L, pH 7.0 - 7.2, sterilized at 121 °C for 20 min.

[0031] 8. Fermentation medium for cellulose-degrading bacteria: (NH4)2SO4 2 g / L, Mg2SO4·7H2O 0.5 g / L, K2HPO4 1 g / L, NaCl 0.5 g / L, CMC-Na 2 g / L, pH 7.0 - 7.2, sterilized at 121 °C for 20 min.

[0032] 9. Mixed fermentation medium: Edible oil 15 g, CMC-Na 5 g, casein 2.5 g, Na2HPO4 1 g, KH2PO4 1 g, MgSO4 0.5 g, distilled water 1000 mL, pH 7.0 - 7.2, sterilized at 121 °C for 20 min.

[0033] The liquid medium corresponding to the solid medium does not add agar, and the plate refers to the solid medium.

[0034] Example 1: Screening and identification of strains

[0035] 1. Screening and identification of low-temperature cellulose-degrading strains. Take 10 g of soil sample from a park in Yinchuan City and fecal sample from Hongyuan County, Aba Prefecture, add it to a 250 mL conical flask containing 90 mL of sterile water and small glass beads, and culture it in a shaker at 10 °C and 180 rpm for 3 days. Take it out and let it stand for 30 min, then use a pipette to aspirate 0.5 mL and dilute it to 10 -1 、10 -2 、10 -3 、10 -4 and 10-5 Concentration gradient. 0.5 mL of each gradient was separately aspirated and spread on nutrient agar medium, and incubated upside down in an incubator at 10 °C for 3 days. Strains with different morphologies were picked from the plate and streaked in four zones, then incubated upside down in an incubator at 10 °C for 3 days. Single colonies were picked and streaked twice repeatedly for the purification culture of the strains.

[0036] The isolated and purified single bacteria were inoculated into nutrient broth liquid medium, and cultured on a shaker at 10 °C and 180 rpm for 3 days. After adjusting the biomass of the strains to be consistent, they were spot-inoculated on the screening plate for cellulose-degrading bacteria and cultured in an incubator at 10 °C, and whether a transparent circle appeared around the colonies was observed.

[0037] The strains with a transparent circle around the colonies were inoculated into nutrient broth liquid medium, and cultured on a shaker at 10 °C and 180 rpm for 3 days. After adjusting the biomass of the strains to be consistent, they were inoculated into the cellulose-degrading bacteria fermentation medium at 5% (v / v) and cultured on a shaker at 10 °C and 180 rpm. The fermentation broth at 1 day, 3 days and 5 days of culture was taken respectively, and the cellulase activity was determined by the DNS method. Definition of enzyme activity unit: Under the condition of 50 °C, the amount of enzyme required for 1 mL of enzyme solution to catalyze the substrate to produce 1 μg of glucose in 1 min is one enzyme activity unit, expressed as 1 U / mL.

[0038] There were 4 strains with a transparent circle around the colonies, numbered N3-3, N3-4, N3-6 and N3-7 respectively. The CMC enzyme activity and FPA enzyme activity of the 4 strains at 1, 3 and 5 days are shown in Table 1.

[0039] Table 1 Results of determination of cellulase activity of strains

[0040]

[0041] According to the results of the above table, the strain numbered N3-3 was selected. The results of the physiological and biochemical experiments of the strain are shown in Table 2.

[0042] Table 2 Physiological and biochemical characteristics of strain N3-3

[0043]

[0044]

[0045] The 16S rDNA of N3-3 (the sequence is shown in SEQ ID NO: 1) was PCR amplified using the bacterial universal primers 1492R (sequence: 5'-CTACGGCTACCTTGTTACGA-3') and 27F (sequence: 5'-AGAGTTTGATCCTGGCTCAG-3'). The PCR amplification product was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that N3-3 was Bacillus subtilis.

[0046] 2. Screening of low-temperature oil-degrading bacteria. Five strains of bacteria that may have the ability to degrade oil were purchased commercially and were respectively inoculated into beef extract peptone liquid medium, and cultured on a shaker at 10 °C and 180 rpm for 3 days. After adjusting the biomass of the strains to be the same, they were spot-inoculated onto the screening plate for oil-degrading bacteria and cultured in an incubator at 10 °C. Observe whether red spots appear around the colonies.

[0047] The strains with red spots appearing around the colonies, labeled as YJ1, YJ2, and YJ3, were inoculated into beef extract peptone liquid medium, and cultured on a shaker at 10 °C and 180 rpm for 3 days. After adjusting the biomass of the strains to be the same, they were respectively inoculated at 5% (v / v) into the fermentation medium for oil-degrading bacteria and cultured on a shaker at 10 °C and 180 rpm. The oil degradation rate of the strains in 3 days was determined by the petroleum ether extraction and weighing method. The specific operation was as follows: Add 20 mL of petroleum ether to the fermentation broth, shake well and pour it into a separatory funnel. Then add another 10 mL of petroleum ether to extract the residual oil on the bottle wall and pour it into the separatory funnel as well. Let it stand for 3 min; drain the aqueous layer, and make the oil layer flow through a sintered glass funnel containing anhydrous sodium sulfate into a weighed disposable cup. Place the cup containing the oil and petroleum ether mixture in a fume hood, and weigh it again after the petroleum ether has completely volatilized to obtain the weight of the remaining oil in the fermentation broth. The original weight of the oil in the fermentation medium is denoted as W0, and the weight of the remaining oil obtained after petroleum ether extraction is denoted as W1. The oil degradation rate can be calculated according to the following formula. The results are shown in Table 3.

[0048]

[0049] Table 3 Oil degradation rate of strains

[0050] Number 3-day oil degradation rate (%) YJ1 45.48±0.74 YJ2 34.65±0.54 YJ3 33.89±0.89

[0051] According to the results in Table 3, YJ1, namely Pseudomonas mandelii (CGMCC NO: 1.6426), was selected as the candidate oil-degrading bacterium.

[0052] 3. Screening of protein-degrading bacteria. Five strains of bacteria that may have the ability to degrade proteins were purchased commercially and were respectively inoculated into beef extract peptone liquid medium, and cultured on a shaker at 10 °C and 180 rpm for 3 days. After adjusting the biomass of the strains to be the same, they were spot-inoculated onto the screening plate for protein-degrading bacteria and cultured in an incubator at 10 °C. Observe whether a clear zone appears around the colonies.

[0053] The strains with clear zones around the colonies were numbered DJ1, DJ2, and DJ3, and were respectively inoculated into beef extract peptone liquid medium, and cultured on a shaker at 10°C and 180 rpm for 3 days. After adjusting the biomass of the strains to be consistent, they were respectively inoculated at 5% (v / v) into the protein-degrading bacteria fermentation medium, and cultured on a shaker at 10°C and 180 rpm. The fermentation broth at 1 day, 3 days, and 5 days of culture was taken, and the proteinase activity was measured using the protease determination method in the "General Experimental Methods for Industrial Enzyme Preparations" (QB / T 1803 - 1993). Definition of enzyme activity unit: Under the condition of 40°C, the amount of enzyme required for 1 mL of enzyme solution to hydrolyze casein to produce 1 μg of tyrosine in 1 minute is one enzyme activity unit, expressed as 1 U / mL. The measurement results are shown in Table 4.

[0054] Table 4 Proteinase Activity of Strains

[0055]

[0056] According to the results in Table 6, DJ3, namely Pseudomonas azotofixans (CGMCC NO: 1.6428), was selected as the alternative protein-degrading bacterium.

[0057] 4. Survival test in ultra-low temperature and extreme pH environment.

[0058] When the temperature was 0°C, the culture medium and feces froze, and it was impossible to conduct tests and treatments. At the same time, combined with the actual situation of feces treatment in most regions, 4°C was the lowest temperature that might be encountered during actual treatment. N3-3 and each alternative protein-degrading bacterium and oil-degrading bacterium were respectively inoculated into LB solid medium at 5% (v / v). Under the conditions of pH = 7 and temperature of 4°C, the growth of each microorganism was regularly observed every 12 h, and the OD of the strain was measured 600nm and the corresponding enzyme activity and degradation rate were tested. The results are shown in Table 5. The enzyme activity unit is U / mL.

[0059] Table 5 Enzyme Activity of Each Microorganism in Extreme Environment

[0060]

[0061] According to the results in Table 5, N3-3, oil-degrading bacterium YJ1 (hereinafter referred to as N3-1), and protein-degrading bacterium DJ3 (hereinafter referred to as N3-2) were selected as the components of the feces-degrading bacterium agent. In addition, the pH tolerance of cellulose-degrading bacterium N3-3, oil-degrading bacterium N3-1, and protein-degrading bacterium N3-2 was tested, and they could all grow well within the range of 5 - 9, covering the feces pH range.

[0062] Example 2: Preparation and Effect Display of Compound Bacterium Agent

[0063] The activated N3-1, N3-2, and N3-3 were respectively inoculated into LB liquid medium and cultured at 10°C and 180 rpm for 48 h to obtain seed solutions. The concentration of each group of seed solutions was adjusted to OD 600 = 1.5, and then they were mixed according to the volume ratios in Table 6 to obtain mixed bacterial solutions. Each group of mixed bacterial solutions was inoculated into the mixed fermentation medium at 5% respectively, and cultured on a shaker at 4°C. The oil degradation rate, protease activity, and cellulase activity on the 2nd, 3rd, and 4th days after inoculation were measured. The results are shown in Table 7. The enzyme activity unit is U / mL, and the degradation rate unit is %. In addition, a control group was set up: under the same other conditions, Pseudomonas mandelii (M1) with the number CCTCC S2017018 was used to replace N3-1 as Group 10, Pseudomonas azotofixans (G1) with the number CGMCC NO: 1.1792 was used to replace N3-2 as Group 11, and Bacillus subtilis (K1) with the number CGMCC NO: 1.821 was used to replace N3-3 as Group 12. It should be noted that the inventors did not only conduct the following group tests. Only for reasons of space, some representative groups were selected for display.

[0064] Table 6 Compound control table of each group of compound bacterial agents

[0065] Group Oil-degrading bacteria: Protein-degrading bacteria: Cellulose-degrading bacteria 1 N3-1:N3-2:N3-3 = 4:0.5:1 2 N3-1:N3-2:N3-3 = 4:1:3 3 N3-1:N3-2:N3-3 = 4:1.5:2 4 N3-1:N3-2:N3-3 = 6:0.5:3 5 N3-1:N3-2:N3-3 = 6:1:2 6 N3-1:N3-2:N3-3 = 6:1.5:1 7 N3-1:N3-2:N3-3 = 8:0.5:2 8 N3-1:N3-2:N3-3 = 8:1:1 9 N3-1:N3-2:N3-3 = 8:1.5:3 10 M1:N3-2:N3-3 = 6:0.5:3 11 N3-1:G1:N3-3 = 6:0.5:3 12 N3-1:N3-2:K1 = 6:0.5:3

[0066] Table 7 Effect comparison table of each group of compound bacterial agents

[0067]

[0068]

[0069] The above-described embodiments are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A bacterial preparation, characterized in that: The bacterial preparation includes Bacillus subtilis ( Bacillus subtilis ) N3-3, the Bacillus subtilis was deposited in Guangdong Provincial Microbial Culture Collection Center on December 12, 2022, with a deposit number of GDMCC NO: 62537; the bacterial preparation also includes oil-degrading bacteria, and the oil-degrading bacteria is Pseudomonas meningitidis with a deposit number of CGMCC NO: 1.6426 ( Pseudomonas mandelii The bacterial preparation also includes protein-degrading bacteria, and the protein-degrading bacteria is a nitrogen-producing Pseudomonas sp. with a deposit number of CGMCC NO: 1.6428 ( Pseudomonas azotoformans ).

2. Use of the bacterial preparation according to claim 1 in degrading cellulose.

3. Use of the bacterial preparation according to claim 1 in composting.

4. Use of the bacterial preparation according to claim 1 in degrading feces.

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