CN113388552B - Thermophilic cellulose degradation strain and application thereof - Google Patents
Thermophilic cellulose degradation strain and application thereof Download PDFInfo
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Images
Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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- Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention particularly relates to a salt-tolerant thermophilic efficient strain thermophilic aerothiobacillus thiobacillus Aneurinibacillus thermoaerophilus SX-2 capable of simultaneously degrading cellulose and grease in solid wastes, and the preservation number of the strain thermophilic aerothiobacillus thiobacillus is preferably CGMCC No.22533. The strain is innovative in that the strain has good growth characteristics under thermophilic (50 ℃) and acidic (pH 5) conditions, and simultaneously has the capability of degrading cellulose and grease. Most notably, in shake flask pilot applications, its ability to degrade grease increases with increasing grease content. The degradation rate is as high as 69.6% when the initial grease concentration is 30 g/L. The microbial strain disclosed by the invention can be used for efficiently degrading cellulose and degrading grease, and has very important application value for affecting the current carbon emission reduction policy and realizing harmless treatment of wastes.
Description
Technical Field
The invention belongs to the technical field of degradation treatment of solid wastes, and particularly relates to a salt-tolerant thermophilic strain capable of simultaneously degrading cellulose and grease in solid wastes and application thereof.
Background
With the continuous development of urban treatment and the rapid increase of population quantity in China, the volume of urban solid waste is obviously increased. In addition, the content of the urban green plants, kitchen wastes and other wastes accounting for the content of the urban total solid wastes is continuously increased, so that the degradation of cellulose and grease in the urban solid wastes becomes the key of garbage disposal.
The urban green planting and the large accumulation of kitchen wastes are great challenges and pressures for urban development, and have great influence on human health. The strict management of such solid waste also shows the urgency and necessity of the national disposal of solid waste. Therefore, with the increasing prominence of these problems, there is an urgent need for an environmentally friendly technology for efficiently degrading such solid waste.
Disclosure of Invention
The invention aims to provide a thermophilic cellulose degrading strain and application thereof, and the provided strain has the capability of efficiently degrading cellulose and kitchen waste grease, thereby overcoming the defects of the prior art.
The invention provides thermophilic aerophilic thiobacillus Aneurinibacillus thermoaerophilus SX-2 which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 22533 in the year of 2021 and the month of 05 and 17;
the thermophilic aerophilic thiobacillus SX-2 strain provided by the invention is used for degrading and treating grease;
the thermophilic aerophilic thiobacillus SX-2 strain provided by the invention can also be used for degrading and treating cellulose;
the invention also provides a bacterial product for degrading grease and/or cellulose, which comprises the thermophilic bacillus thiobacillus SX-2 strain.
The strain provided by the invention has the characteristics of simultaneously degrading cellulose and grease, can resist high temperature, and still has higher grease degradation efficiency in a high-oil environment.
Drawings
Fig. 1: a screening result diagram of cellulose degrading bacteria;
fig. 2: culture morphology of the screened SX-2;
fig. 3: a growth condition diagram of the strain SX-2 under different salinity gradients;
fig. 4: growth status diagrams of strain SX-2 at different pH values;
fig. 5: the change of the oil degradation rate of the strain SX-2 under different oil concentrations is compared with a graph.
Detailed Description
The strain screening, environmental pressure resistance effect and application effect thereof in degrading cellulose and grease of the present invention will be described in detail with reference to examples.
Example 1: screening of thermophilic cellulose degrading strains
The main culture medium comprises:
cellulose degrading bacteria culture medium (CMC, g/L): CMC-Na 10.0, ammonium sulfate 4.0, magnesium sulfate heptahydrate 0.5, potassium dihydrogen phosphate 2.0, peptone 1.0, agar 20.0, ph=6.5.
Congo red cellulose medium: directly purchasing for standby.
Luria-Bertani (LB) medium (g/L): sodium chloride 10.0, tryptone 10.0, yeast extraction 5.0, distilled water to 1L, and pH to 7.2.
Grease degrading bacteria screening culture medium (g/L): 5.0 ml of NaCl, 0.3% of monopotassium phosphate, 0.1% of magnesium sulfate heptahydrate, 1.5% of dipotassium phosphate, 1.0% of ammonium sulfate, 5ml of soybean oil, 20.0% of agar and pH=7.2-7.4.
In the present invention, unless otherwise specified, the medium was autoclaved at 120℃for 20min.
The specific experimental method comprises the following steps:
taking 10g of soil sample from the kitchen waste stacking area of Tianjin industrial biotechnology institute of China academy of sciences in a 250ml triangular flask, adding 90ml of sterile water into a 180r/min shaking table at 30 ℃ for full shaking for 30min to obtain suspension, and carrying out gradient dilution to 10 -7 Select 10 -5 -10 -7 Dilutions were applied to LB solid medium plates, CMC solid medium plates and Congo red fibers, respectivelyIn the plates of the solid culture medium of the vitamin, 3 plates are coated on each gradient, 200 mu L of diluted sample suspension is coated on each plate, the plates are sealed and marked, then are placed in a 50 ℃ incubator for culturing for 48 hours, different types of single bacteria are picked from different plates, separated and purified by a three-area lineation method on corresponding screening plates, single bacteria are selected and inoculated in LB liquid culture medium, single bacteria bacterial liquid is obtained by shaking culture for 24 hours at 50 ℃ at 180r/min, and the single bacteria bacterial liquid is stored in 25% glycerol at-80 ℃ for standby.
Screening cellulose degrading bacteria: the separated single bacteria are placed in LB culture medium, shake flask culture is carried out to obtain bacterial liquid, 20 mu L of bacterial liquid is taken to fully wet filter paper sheets placed on Congo red cellulose culture medium plates, 3 filter paper sheets are placed on each plate, and the plates are set to be 3 parallel. Sealing the flat plate, placing the flat plate in a 50 ℃ incubator, culturing for 1-2 hours in a normal position, and culturing in an inverted mode after the filter paper is dried. After culturing for 72 hours, the degradation capacity of the strain to cellulose is primarily judged according to the hydrolysis circle of the flat plate.
Screening of grease degrading bacteria: and (3) respectively connecting each single bacterial spot to a corresponding screening flat plate, uniformly connecting 3 drops of 10 mu L bacterial liquid to each flat plate, namely 3 parallel bacterial liquids, after the bacterial liquids are dried, inversely culturing the bacterial liquids in a 50 ℃ incubator for 72 hours, and preliminarily estimating the degradation capacity of the bacterial strain to various substances by comparing the ratio (D/D) of the diameter (D) of the transparent ring to the diameter (D) of the bacterial colony, wherein the bacterial strain with stronger degradation capacity, namely the larger ratio, is selected as a screening result.
Identification of strains: identifying species of the purified strain, extracting DNA of the strain obtained by screening, performing PCR amplification by utilizing bacterial 16S rRNA, performing agarose gel electrophoresis on PCR stock solution, sending the stock solution with the bands to a sequencing company for sequencing to obtain a 16S rRNA sequence of the target strain, and identifying the genus of the strain by performing similarity comparison on a BLAST database and combining morphological characteristics and physiological and biochemical characteristics of the strain.
Experimental results:
1. cellulose degrading bacterium screening result
The cellulose degrading bacteria are single bacteria separated from soil samples by using different culture medium plates, 8 single bacterial strains are obtained by co-separation, the single bacteria are inoculated to Congo red cellulose culture medium for preliminary screening, and the degradation capability of the bacterial strains can be preliminarily judged according to the color depth of red precipitation rings (figure 1) generated around bacterial colonies, and the degradation results are shown in table 1.
Table 1: primary screening result table for grease degrading bacteria
2. Screening result of grease degrading bacteria
7 single strains are obtained by screening culture medium plates through grease degrading bacteria, 7 strains are subjected to primary separation from soil samples, degradation capacity of the 7 strains is tested, the diameter of a hydrolysis ring and the diameter of bacterial colonies are measured, 5 strains with stronger capacity are obtained by comparing the ratio, and degradation results are shown in table 2.
Table 2: primary screening result table for grease degrading bacteria
3. Identification result of strain
Based on the comparison of the sequencing results and the morphological characteristics comprehensive analysis of the strain (FIG. 2), the genus of the strain obtained by screening was determined, and strain CG-3 was identified as Aneurinibacillus thermoaerophilus and named: aneurinibacillus thermoaerophilus SX-2.
The screened thermophilic bacillus thiobacillus Aneurinibacillus thermoaerophilus SX-2 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 22533 in the year 05 and the day 17 of 2021.
Example 2: effects of salinity and pH on thermophilic cellulose degrading bacteria Aneurinibacillus thermoaerophilus SX-2
The main culture medium comprises:
Luria-Bertani (LB) medium (g/L): sodium chloride 10.0, tryptone 10.0, yeast extraction 5.0, distilled water to 1L, and pH to 7.2.
In the present invention, unless otherwise specified, the medium was autoclaved at 120℃for 20min.
The specific experimental method is as follows:
tolerance test of salinity: inoculating target strain into LB liquid culture medium with NaCl concentration of 10, 50, 100, 150, 200g/L respectively at 10%, arranging 3 strains in parallel, shake culturing at 50deg.C for 24 hr at 180r/min, and measuring OD of bacterial liquid 600 And judging the growth condition of the target strain in the culture media with different salt concentration gradients.
Acid-base resistance test: inoculating target strain into LB liquid culture medium with pH of 3, 5, 9, 11 respectively at 10%, setting 3 parallel, shake culturing at 50deg.C for 24 hr at 180r/min, and measuring OD of bacterial solution 600 And judging the tolerance degree of the target strain to different pH values.
Statistical processing all statistical analyses were performed using the SPSS 20.0 statistical software package. The metering data are expressed as mean.+ -. Standard deviation (x.+ -. S).
Experimental results:
1. growth conditions of Aneurinibacillus thermoaerophilus SX-2 at different salinity gradients
The cellulose degrading bacteria Aneurinibacillus thermoaerophilus SX-2 obtained by the screening are subjected to salinity tolerance test and OD 600 As an index of strain growth, the strain was tested for tolerance to high salinity environments. As shown in FIG. 3, the strain grew well at a NaCl concentration of 10g/L and had an OD at 24 hours 600 The value can reach 1.428.
2. Growth conditions of Aneurinibacillus thermoaerophilus SX-2 at different pH values
Culturing cellulose degrading bacteria Aneurinibacillus thermoaerophilus SX-2 obtained by the above experiment in LB liquid culture medium with different pH values for 24 hr, and according to OD 600 And judging the tolerance degree of the strain to the pH value. As shown in fig. 4. Through the research, the strain can grow well in the environment with the pH value of 5 and grow better than that of the strain with the pH value of 7. At a pH of 5 and 7, an OD of 24 hours 600 The values are 1.650 and 1.428, respectively. At a pH of 3, 5The strain also showed signs of growth, but the growth was not good, OD for 24 hours 600 The mean values were 0.297 and 0.287, respectively.
Example 3: the thermophilic cellulose degrading bacterium Aneurinibacillus thermoaerophilus SX-2 has the effect of small test application on cellulose and grease degradation
The main culture medium comprises:
filter paper medium (g/L): ammonium sulfate 4.0, magnesium sulfate heptahydrate 0.5, potassium dihydrogen phosphate 2.0, peptone 1.0, filter paper 1%, pH=6.5-7.0.
Lipid degrading bacteria re-screening culture medium (g/L): peptone 1.0, ammonium nitrate 0.2, dipotassium hydrogen phosphate 0.5, potassium dihydrogen phosphate 0.5, magnesium sulfate heptahydrate 0.1, soybean oil 3.0ml, ph=7.2 to 7.4.
In the present invention, unless otherwise specified, the medium was autoclaved at 120℃for 20min.
The specific experimental method is as follows:
small test of cellulose degrading bacteria: inoculating the bacterial liquid Aneurinibacillus thermoaerophilus SX-2 to a filter paper culture medium, and quantitatively determining the degradation capacity of the bacterial strain by calculating the degradation rate of the bacterial strain to the filter paper, so as to select high-efficiency degradation bacteria. Inoculated to filter paper culture medium shake flask culture, set three parallel and one group of non-inoculated control group CK, under identical conditions at 180r/min, after 3d culture at 50 ℃ to determine the change of filter paper weight.
Filter paper degradation rate = (CK filter paper weight-treated filter paper weight)/CK filter paper weight
Determination of the mass of the filter paper: spreading gauze and covering the gauze on a 1L beaker mouth, pouring the culture solution into the beaker through the gauze, filtering filter paper scraps, keeping the filter paper scraps on the gauze, washing the triangular flask for 1-2 times by using distilled water, slowly pouring the washing solution from the filter paper scraps on the gauze, washing bacterial liquid of the culture solution remained in the filter paper scraps, and enabling filter residues to be basically white. The remaining water of the filter paper residue was gently squeezed out, placed in a petri dish and dried under vacuum at 105 ℃ in a vacuum drying oven until the mass was no longer changed, and the mass of the remaining filter paper was measured.
Small test of oil degradation effect: the screened strain Aneurinibacillus thermoaerophilus SX-2 is inoculated to a small oil degradation test shake flask culture medium as a microbial inoculum treatment group, a group of CK is arranged as a blank control, the initial oil concentration is 3g/L, and simultaneously the two treatment groups are placed in the identical environment for 180r/min, are cultured at 50 ℃ and are used for 48 hours to measure the oil content change.
Oil degradation rate = (CK soybean oil concentration-treated soybean oil concentration)/CK soybean oil concentration
Manufacturing a kitchen waste grease standard curve: the research shows that the maximum absorption peak of kitchen waste grease is 225nm, so 7 volumetric flasks of 50ml are taken, petroleum ether is taken as solvent to prepare solution of soybean oil of 7 concentrations of 0.1,0.2,0.4,0.5,0.6,0.8 and 1.0mg/ml, and OD is measured 225 And (3) drawing a standard curve by taking the soybean oil concentration as an abscissa and the absorbance value as an ordinate. Three replicates of each concentration were obtained according to this method, with standard oil concentration on the abscissa and absorbance OD 225 And (3) taking the ordinate as the standard curve for kitchen waste grease. The subject group has previously obtained a standard curve: y=0.58893x+0.00548, r 2 ＝0.99375。
Determination of the oil content in the Medium: the shake flask medium was transferred to a 250ml separating funnel, acidified by adding 1ml of concentrated hydrochloric acid, and then added with about 2% (m/v) NaCl, the shake flask was washed with 5ml of petroleum ether, the wash solution was poured into the separating funnel, and the washing with 5ml of petroleum ether was repeated once. And (5) sufficiently oscillating the separating funnel, and standing to separate the separating funnel. After delamination, the aqueous layer was transferred to a triangular flask, the petroleum ether layer was transferred to a 100ml ceramic crucible and capped to reduce petroleum ether evaporation (or transferred to a small beaker covered with a petri dish). The aqueous layer in the flask was transferred again to a separatory funnel and the extraction was repeated once with 10ml petroleum ether. Adding proper amount of anhydrous sodium sulfate into the recovered petroleum ether, stirring, covering with a cover, and standing for more than half an hour to ensure full dehydration. Transferring petroleum ether into 25ml volumetric flask, adding petroleum ether dropwise to scale mark, mixing thoroughly, and measuring OD 225 The oil content of the medium was determined from the standard curve.
Degradation efficiency of different initial oil contents: preparing oil degradation culture mediums with initial concentration of 3, 9, 15, 30 and 45g/L of soybean oil respectively, inoculating target strain into the oil degradation culture medium with 10% of inoculum size, shake culturing at 50deg.C for 48h at 180r/min, measuring soybean oil content in the culture medium, and calculating degradation rate.
Statistical processing all statistical analyses were performed using the SPSS 20.0 statistical software package. The metering data are expressed as mean.+ -. Standard deviation (x.+ -. S).
The experimental results are as follows:
1. the cellulose degradation strain is used for filter paper degradation test
And (3) inoculating the strain to a filter paper culture medium to measure the degradation rate of the filter paper, and quantitatively judging the degradation capacity of the strain on cellulose. According to the experimental results, the strain Aneurinibacillus thermoaerophilus SX-2 is subjected to shaking culture at 50 ℃ and 180r/min for 3d, and then the residue is collected to obtain filter paper, and the degradation rate is calculated to be about 32.9% by drying and weighing the residue mass.
2. The strain is subjected to shake flask small test under the condition of 3g/L grease concentration
According to a drawn standard curve of the grease petroleum ether solution, carrying out shake flask small test on the strain Aneurinibacillus thermoaerophilus SX-2 obtained by preliminary screening, measuring the small-scale degradation rate of the strain on grease in 48 hours, quantitatively judging the degradation capacity of the strain, and obtaining the degradation rate of the strain on the soybean grease in 48 hours through preliminary calculation, wherein the degradation rate is as follows: 53.4%.
3. The degradation rate of the degradation bacteria under different initial oil contents
Strain Aneurinibacillus thermoaerophilus SX-2 was cultured in different soybean oil concentrations, and after 48 hours, the soybean oil content was measured, and the soybean oil degradation rate was calculated. The degradation rate of the strain in the different soybean oil concentration media is shown in fig. 5. Notably, the results show that the degradation rate of the strain Aneurinibacillus thermoaerophilus SX-2 is in the concentration range of 3-30g/L of the initial oil content, the degradation rate is increased along with the increase of the oil concentration, and the degradation rate is as high as 69.6% when the initial oil content is 30 g/L. Even at a concentration of 45g/L, the degradation rate of soybean oil still maintains the effect of the degradation rate of 22.6%.
In conclusion, the strain Aneurinibacillus thermoaerophilus SX-2 of the present invention has cellulose degrading ability and excellent lipid degrading ability, and has excellent growth ability in an acidic environment (pH 5). The degradation rate of the strain is increased along with the increase of the concentration of the grease, and the strain still keeps higher degradation rate of the grease when the content of the grease is up to 30 g/L. The results show that the strain Aneurinibacillus thermoaerophilus SX-2 provided by the invention has excellent effects in degrading cellulose and kitchen grease, and has good application and popularization values.
Claims (4)
1. The thermophilic bacillus thiobacillus is characterized in that the preservation number of the strain is CGMCC No.22533.
2. The use of the bacillus stearothermophilus according to claim 1 for degrading grease.
3. Use of the bacillus stearothermophilus according to claim 1 for degrading cellulose.
4. A bacterial preparation for degrading grease and/or cellulose, wherein the preparation comprises the bacillus stearothermophilus according to claim 1.
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| CN111662853A (en) * | 2020-07-17 | 2020-09-15 | 中国科学院成都生物研究所 | Kitchen waste biological drying stabilizing microbial agent and preparation method and application thereof |
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| CN111662853A (en) * | 2020-07-17 | 2020-09-15 | 中国科学院成都生物研究所 | Kitchen waste biological drying stabilizing microbial agent and preparation method and application thereof |
| CN112175877A (en) * | 2020-10-19 | 2021-01-05 | 山东碧蓝生物科技有限公司 | Cellulose degrading strain with sewage ammonia nitrogen degrading capability and application thereof |
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