Hyperthermophilic aerobic fermentation inoculant prepared by using municipal sewage sludge and its method

Abstract

The present disclosure discloses a preparation method for hyperthermophilic aerobic fermentation inoculant prepared by using sewage sludge, the method includes the following steps: carrying out fermentation after the activation of hyperthermophilic aerobic bacteria, removing the supernatant from the fermentation products, and adding the protective agent and stirring until uniform, drying to obtain a product, pulverizing the product by a pulverizer, and sieving the product before sub-packing. The solution of the present disclosure has the following advantages.

Claims

1. A preparation method for a hyperthermophilic aerobic fermentation inoculant prepared by using sewage sludge, comprising: fermenting hyperthermophilic aerobic bacteria to obtain a fermentation broth, removing supernatant from the fermentation broth, mixing the fermentation broth with protective agent to obtain a mixture, drying the mixture, pulverizing the dried mixture, and sieving the pulverized mixture for packing; wherein a culture medium for the fermentation is prepared by: taking sewage sludge and adding water to the sewage sludge to regulate a solid content of the sewage sludge; adding antifoaming agents of polyethers, yeast powder, ammonium chloride, sucrose, soluble starch, and potassium dihydrogen phosphate to the sewage sludge; regulating pH of the sewage sludge; adding composite carrier into the sewage sludge, and stirring the sewage sludge for uniformly mixing the composite carrier and the sewage sludge; sterilizing the mixture of the composite carrier and the sewage sludge; and cooling the sewage sludge to obtain the culture medium for the fermentation.

2. The preparation method according to claim 1, wherein the protective agent is composed of non-fat milk powder, soluble starch, glycerin, and water.

3. The preparation method according to claim 1, wherein the protective agent comprises 2 to 3 g non-fat milk powder, 1 to 2 g soluble starch, 10 mL glycerin, and 85 to 87 mL water.

4. The preparation method according to claim 1, wherein the composite carrier is composed of at least one selected from the group consisting of kaolin, biochar, and wheat bran.

5. The preparation method according to claim 1, wherein the content of the composite carrier is 50 to 100 g/L.

6. The preparation method according to claim 1, wherein the solid content of the sludge is regulated to 2 to 3% by the addition of the water.

7. The preparation method according to claim 1, wherein the composite carrier is composed of biochar and wheat bran, and a mass ratio of the biochar to the wheat bran is 1.5:1 to 3:1.

8. The preparation method according to claim 1, wherein the hyperthermophilic aerobic bacteria is at least one selected from the group consisting of Thermus thermophilus, Calditerricola yamamurae, Calditerricola satsumensis, Thermaerobacter composti, Geobacillus thermocatenulatus or Thermaerobacter subterraneus.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1 is a flow diagram showing a preparation method for a hyperthermophilic aerobic fermentation inoculant prepared by using sewage sludge.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(2) Referring to FIG. 1 which shows a preparation method for a hyperthermophilic aerobic fermentation inoculant prepared by using sewage sludge, comprising the following steps: S1, fermentation of activated hyperthermophilic aerobic bacteria; S2, removing the supernatant from the fermentation products, and S3, mixing with protective agent, drying, pulverizing, and sieving before sub-packing.

(3) In the following detailed embodiments, the present disclosure will be further clarified.

Embodiment 1

(4) Production of Calditerricola satsumensis Fermentation Inoculant

(5) (1) Sample of Sewage Sludge

(6) The concentrated sludge is taken from a sewage treatment plant in Beijing. See Table 1 for the physicochemical properties of the sludge.

(7) TABLE-US-00001 TABLE 1 Physicochemical properties of the sludge Total Total Total Moisture carbon content nitrogen phosphorus pH content (%) (%) content (%) content (%) 7.5 87.5 45.7 3.5 1.0

(8) (2) Preparation of Sludge Medium for Fermentation

(9) Adding water to adjust the solid content of sludge to 2%, adding 0.2% antifoaming agents of polyethers, 1 g/L yeast powder, 0.5 g/L ammonium chloride, 0.3 g/L sucrose, 0.2 g/L soluble starch, 0.3 g/L potassium dihydrogen phosphate, regulating the pH to 7.0, adding composite carrier of 50 g/L biochar/wheat bran (w/w=2/1), mixing evenly, sterilizing at 100 C. for 45 minutes, cooling to 70-80 C. for further use.

(10) (3) Calditerricola satsumensis FAFU012 colony was picked from solid medium, and inoculated into 100 mL of CYS liquid medium in 300 mL conical flask, cultured at 80 C., 180 rpm (Revolution(s) Per Minute) for 12 hours. The components of CYS medium are: yeast extract 4.0 g, hydrolyzed casein protein 6.0 g, soluble starch 3.0 g, NaCl 30.0 g, MgCl.sub.2.Math.6H.sub.2O 0.27 g, CaCl.sub.2 0.025 g, FeSO.sub.4.Math.7H.sub.2O 0.01 g, trace element 100 L (Na.sub.2MoO.sub.4.Math.2H.sub.2O 12.0 g/L, VOSO.sub.4.Math.xH.sub.2O 1.0 g/L, MnCl.sub.2 5.0 g/L, ZnSO.sub.4.Math.7 H.sub.2O 0.6 g/L, CuSO.sub.4.Math.5H.sub.2O 0.15 g/L, CoCl.sub.2.Math.6H.sub.2O 8.0 g/L, NiCl.sub.2.Math.6H.sub.2O 0.2 g/L), and the initial value of pH is 7.5, 0.15 MPa, sterilized at 121 C. for 30 min.

(11) (4) Fermentation of Seed

(12) The above cultures was inoculated into a small fermenter containing a sludge fermentation medium at an inoculation amount of 2%, fermented at 75 C. for 16 hours with ventilation volume of 100 m.sup.3/h and stirring speed of 180 rpm.

(13) (5) Large-Scale Fermentation

(14) The seed solution of Calditerricola satsumensis FAFU012 was inoculated into the fermentation tank at an inoculation amount of 2%, and fermented at 75 C. for 24 hours with ventilation volume of 100 m.sup.3/h and stirring speed of 180 rpm.

(15) (6) Preparation of Fermentation Inoculant

(16) The supernatant of fermentation products was removed by pressure filtration, and a 1% protective agent was added. The protective agent consisted of non-fat milk powder, soluble starch, glycerin, and water. Wherein, non-fat milk powder:soluble starch:glycerin:water=3:2:10:85 (w/w/v/v), the unit of mass is g and the unit of volume is mL. Mixing them at high speed, drying at 90 C., pulverizing by a pulverizer, and sieving through a 80 mesh sieve before sub-packing.

(17) (7) Quality Inspection of the Inoculants

(18) The hyperthermophilic aerobic inoculant produced by fermentation of sludge medium were randomly sampled and tested in accordance with Microbial inoculants in agriculture (GB 20287-2006) of the national standard of the People's Republic of China. After testing, the number of live bacteria was 2.810.sup.9 cfu/g.

Embodiment 2

(19) Production of Calditerricola yamamurae Fermentation Inoculant

(20) (1) Sample of Sewage Sludge

(21) The concentrated sludge is taken from Zhengzhou City, Henan Province. See Table 2 for the physicochemical properties of the sludge.

(22) TABLE-US-00002 TABLE 2 Physicochemical properties of the sludge Total Total Total Moisture carbon content nitrogen phosphorus pH content (%) (%) content (%) content (%) 7.6 90.2 38.9 2.7 0.8

(23) (2) Preparation of Sludge Medium for Fermentation

(24) Adding water to adjust the solid content of sludge to 3%, adding 0.2% antifoaming agents of polyethers, 1.5 g/L yeast powder, 1.0 g/L ammonium chloride, 0.5 g/L sucrose, 0.5 g/L soluble starch, 0.5 g/L potassium dihydrogen phosphate, regulating the pH to 7.0, adding composite carrier of 80 g/L biochar/wheat bran (w/w=1.5/1), mixing evenly, sterilizing at 100 C. for 45 minutes, cooling to 70-80 C. for further use.

(25) (3) Calditerricola yamamurae UTM801 colony was picked from solid medium, and inoculated into 100 mL of CYS liquid medium in 300 mL conical flask, cultured at 80 C., 180 rpm (Revolution(s) Per Minute) for 12 hours. The components of CYS medium are: yeast extract 4.0 g, hydrolyzed casein protein 6.0 g, soluble starch 3.0 g, NaCl 30.0 g, MgCl.sub.2.6H.sub.2O 0.27 g, CaCl.sub.2 0.025 g, FeSO.sub.4.Math.7 H.sub.2O 0.01 g, trace element 100 L (Na.sub.2MoO.sub.4.Math.2H.sub.2O 12.0 g/L, VOSO.sub.4.Math.xH.sub.2O 1.0 g/L, MnCl.sub.2 5.0 g/L, ZnSO.sub.4.Math.7 H.sub.2O 0.6 g/L, CuSO.sub.4.Math.5H.sub.2O 0.15 g/L, CoCl.sub.2.Math.6H.sub.2O 8.0 g/L, NiCl.sub.2.Math.6H.sub.2O 0.2 g/L), and the initial value of pH is 7.5, 0.15 MPa, sterilized at 121 C. for 30 min.

(26) (4) Fermentation of Seed

(27) The above cultures was inoculated into a small fermenter containing a sludge fermentation medium at an inoculation amount of 2%, fermented at 80 C. for 16 hours with ventilation volume of 100 m.sup.3/h and stirring speed of 180 rpm.

(28) (5) Large-Scale Fermentation

(29) The seed solution of Calditerricola yamamurae UTM801 was inoculated into the fermentation tank at an inoculation amount of 2%, and fermented at 80 C. for 24 hours with ventilation volume of 100 m.sup.3/h, and stirring speed of 180 rpm.

(30) (6) Preparation of Fermentation Inoculant

(31) The supernatant of fermentation products was removed by pressure filtration, and a 1% protective agent was added. The protective agent consisted of non-fat milk powder, soluble starch, glycerin, and water. Wherein, non-fat milk powder:soluble starch:glycerin:water=3:2:10:85 (w/w/v/v), the unit of mass is g and the unit of volume is mL. Mixing them at high speed, drying at 90 C., pulverizing by a pulverizer, and sieving through a 80 mesh sieve before sub-packing.

(32) (7) Quality Inspection of the Inoculants

(33) The hyperthermophilic aerobic inoculant produced by fermentation of sludge medium were randomly sampled and tested in accordance with Microbial inoculants in agriculture (GB 20287-2006) of the national standard of the People's Republic of China. After testing, the number of live bacteria was 4.510.sup.9 cfu/g.

Embodiment 3

(34) Production of Thermus thermophilus Fermentation Inoculant

(35) (1) Sample of Sewage Sludge

(36) The concentrated sludge is taken from a sewage treatment plant in Fuzhou City, Fujian Province. See Table 3 for the physicochemical properties of the sludge.

(37) TABLE-US-00003 TABLE 3 Physicochemical properties of the sludge Total Total Total Moisture carbon content nitrogen phosphorus pH content (%) (%) content (%) content (%) 7.3 90.0 29.4 2.5 1.0

(38) (2) Preparation of Sludge Medium for Fermentation

(39) Adding water to adjust the solid content of sludge to 3%, adding 0.2% antifoaming agents of polyethers, 1.5 g/L yeast powder, 0.5 g/L ammonium chloride, 0.5 g/L sucrose, 0.5 g/L soluble starch, 1 g/L potassium dihydrogen phosphate, regulating the pH to 7.0, adding composite carrier of 100 g/L biochar/wheat bran (w/w=1.5/1), mixing evenly, sterilizing at 100 C. for 45 minutes, cooling to 70-80 C. for further use.

(40) (3) Thermus thermophilus HB8 colony was picked from solid medium, and inoculated into 100 mL of CYS liquid medium in 300 mL conical flask, cultured at 75 C., 180 rpm (Revolution(s) Per Minute) for 12 hours. The components of CYS medium are: yeast extract 4.0 g, hydrolyzed casein protein 6.0 g, soluble starch 3.0 g, NaCl 30.0 g, MgCl.sub.2.Math.6H.sub.2O 0.27 g, CaCl.sub.2 0.025 g, FeSO.sub.4.Math.7H.sub.2O 0.01 g, trace element 100 L (Na.sub.2MoO.sub.4.Math.2H.sub.2O 12.0 g/L, VOSO.sub.4.Math.xH.sub.2O 1.0 g/L, MnCl.sub.2 5.0 g/L, ZnSO.sub.4.Math.7 H.sub.2O 0.6 g/L, CuSO.sub.4.Math.5H.sub.2O 0.15 g/L, CoCl.sub.2.Math.6H.sub.2O 8.0 g/L, NiCl.sub.2.Math.6H.sub.2O 0.2 g/L), and the initial value of pH is 7.5, 0.15 MPa, sterilized at 121 C. for 30 min.

(41) (4) Fermentation of Seed

(42) The above cultures were inoculated into a small fermenter containing a sludge fermentation medium at an inoculation amount of 2%, fermented at 75 C. for 16 hours with ventilation volume of 100 m.sup.3/h and stirring speed of 180 rpm.

(43) (5) Large-Scale Fermentation

(44) The seed solution of Thermus thermophilus HB8 was inoculated into the fermentation tank at an inoculation amount of 2%, and fermented at 75 C. for 24 hours with ventilation volume of 100 m.sup.3/h and stirring speed of 180 rpm.

(45) (6) Preparation of Fermentation Inoculant

(46) The supernatant of fermentation products was removed by pressure filtration, and a 1% protective agent was added. The protective agent consisted of non-fat milk powder, soluble starch, glycerin, and water. Wherein, non-fat milk powder:soluble starch:glycerin:water=(2-3):(1-2):10:(85-87) (w/w/v/v), the unit of mass is g and the unit of volume is mL. Mixing them at high speed, drying at 90 C., pulverizing by a pulverizer, and sieving through a 80 mesh sieve before sub-packing.

(47) (7) Quality Inspection of the Inoculants

(48) The hyperthermophilic aerobic inoculant produced by fermentation of sludge medium were randomly sampled and tested in accordance with Microbial inoculants in agriculture (GB 20287-2006) of the national standard of the People's Republic of China. After testing, the number of live bacteria was 2.310.sup.9 cfu/g.

Embodiment 4

(49) Effect of Different Treatments on Calditerricola Fermentation and Inoculant Quality

(50) (1) Sample of Sewage Sludge

(51) The concentrated sludge was taken from a sewage treatment plant in Fuzhou, Fujian Province. The physicochemical properties of the sludge are shown in Table 4.

(52) TABLE-US-00004 TABLE 4 Physicochemical properties of the sludge Total Total Total Moisture carbon content nitrogen phosphorus pH content (%) (%) content (%) content (%) 7.5 90.3 35.4 3.1 1.0

(53) (2) Activation of Fermentation Strains

(54) Calditerricola satsumensis FAFU012 colony was picked from solid medium, and inoculated into CYS liquid medium, and cultured at 75 C., 180 rpm (Revolution(s) Per Minute) for 16 hours.

(55) (3) Fermentation and Preparation of the Inoculant

(56) According to different treatments in Table 5, the activated Calditerricola satsumensis FAFU012 was subjected to seed fermentation, large-scale fermentation, and microbial inoculant preparation, respectively. Wherein, in seed fermentation, the activated bacterial solution was inoculated into a small fermenter containing a sludge fermentation medium at an inoculation amount of 2%, fermented at 75 C. for 16 hours with ventilation volume of 100 m.sup.3/h and stirring speed of 180 rpm; in Large-scale fermentation, the seed solution was inoculated into a fermentation tank at an inoculation amount of 2%, fermented at 75 C. for 24 hours with the pressure of 0.06 Mpa, the ventilation volume of 100 m.sup.3/h, and stirring speed of 180 rpm. The supernatant of fermentation products was removed by pressure filtration after fermentation, and the corresponding protective agent was added. Mixing them at high speed evenly, drying at 90 C. to constant weight, pulverizing by a pulverizer, and sieving through a 80 mesh sieve before sub-packing.

(57) TABLE-US-00005 TABLE 5 Fermentation and preparation of Calditerricola satsumensis FAFU012 inoculant with different schemes Batch (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) Fermen- Solid content 4 3 3 3 3 3 3 3 3 3 tation of sludge (%) medium pH 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 Polyether (%) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 yeast powder 1.5 1.5 1.5 1.5 1.5 1.5 1.5 (g/L) ammonium 2.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 chloride (g/L) Sucrose (g/L) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 soluble starch 1.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (g/L) potassium 1 1 1 1 1 1 1 1 dihydrogen phosphate (g/L) Carrier Kaolin (g/L) 50 50 biochar/wheat bran 50 50 50 (1.5/1, g/L) Protective Glycerin (%) 25 25 1 1 Agent non-fat milk 1 1 1 powder: soluble starch: glycerin (3:2:10, %) Preservation temperature 20 normal normal normal normal normal normal normal normal normal ( C.) temper- temper- temper- temper- temper- temper- temper- temper- temper- ature ature ature ature ature ature ature ature ature

(58) (4) Quality Inspection of Fermentation Products and the Inoculants

(59) TABLE-US-00006 TABLE 6 Effect of different schemes on the fermentation and quality of Calditerricola satsumensis FAFU012 inoculant Effective live bacteria in Effective live bacteria fermentation in the inoculants Batch products 0 day 15 day (1) 2.3 10.sup.5 cfu/mL 1.0 10.sup.5 cfu/mL 0 (2) 2.3 10.sup.5 cfu/mL 1.0 10.sup.5 cfu/mL 3.1 10.sup.2 cfu/mL (3) 3.6 10.sup.6 cfu/mL 3.6 10.sup.6 cfu/mL 2.6 10.sup.2 cfu/mL (4) 2.4 10.sup.9 cfu/mL 2.4 10.sup.9 cfu/mL 3.6 10.sup.3 cfu/mL (5) 3.6 10.sup.9 cfu/mL 2.6 10.sup.8/g 1.6 10.sup.6/g (6) 1.2 10.sup.9 cfu/mL 2.1 10.sup.9/g 3.6 10.sup.6/g (7) 3.4 10.sup.9 cfu/mL 1.2 10.sup.8/g 2.6 10.sup.8/g (8) 3.8 10.sup.9 cfu/mL 2.3 10.sup.9 cfu/mL 3.3 10.sup.5 cfu/mL (9) 3.4 10.sup.9 cfu/mL 2.8 10.sup.9/g 2.6 10.sup.7/g (10) 2.8 10.sup.9 cfu/mL 3.1 10.sup.9/g 2.7 10.sup.9/g

(60) The above results show that compared with other schemes, the Calditerricola inoculant prepared in the present disclosure has high effective live bacteria and good preservation effect.