CN113717882B - Geobacillus galactose BWTGW1.1 and application thereof - Google Patents
Geobacillus galactose BWTGW1.1 and application thereof Download PDFInfo
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- C05F3/00—Fertilisers from human or animal excrements, e.g. manure
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- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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Abstract
The invention discloses a geological bacillus galactose BWTGW1.1 and application thereof. Geobacillus gamastosimuseusiBWTGW1.1 was deposited at the microbiological bacterial culture Collection center (GDMCC) of Guangdong in 11.11/2020, and was assigned to building 5, post code: 510070, accession number is: GDMCCNo:61374. the galactose geological bacillus (Geobacillus galactosidasius) BWTGW1.1 has strong growth adaptability in high-temperature composting of pig manure waste, is active at a high temperature stage, can generate high-temperature resistant protease, and has great application potential in good-cultivation composting of livestock and poultry cultivation organic waste.
Description
Technical field:
the invention relates to the technical field of environmental microorganisms, in particular to a strain of geological bacillus galactose BWTGW1.1 and application thereof in treatment of livestock and poultry raising solid organic waste.
The background technology is as follows:
in recent years, the experience of domestic and foreign organic waste treatment shows that the production of high-quality organic fertilizer by high-temperature aerobic composting treatment of the cultivation waste is an important way for recycling the waste. In the aerobic composting process, on one hand, the biodegradable high molecular organic matters are rapidly propagated by microorganisms to decompose, and new stable high molecular organic matters, namely humus (an important active factor for forming soil fertility) are synthesized; on the other hand, the mass propagation of microorganisms releases heat to continuously heat the pile in a short time so as to kill various ova, pathogenic bacteria and the like in the compost.
The traditional composting method mainly utilizes indigenous microorganisms in organic wastes for fermentation, and has the defects of long natural fermentation time, serious odor pollution, low harmless degree, low fertility and the like. The existing research results show that the proper addition of microbial agents in the harmless treatment of organic wastes can accelerate the composting process and improve the composting quality. However, in exogenously added microbial agents, thermophilic high temperature fungus microorganisms are a very important class, and have decisive roles in controlling the time of the whole compost fermentation and improving the quality of the compost.
When organic waste is cultivated and composted for fermentation, mesophilic microorganisms are inhibited and even die after the heap body enters a high temperature stage, thermophilic fungus microorganisms gradually rise to become dominant microorganisms, and the thermophilic microorganisms have an important effect on further decomposition of complex organic matters such as cellulose, hemicellulose, high polymer fat, protein and complex carbohydrate in the heap body, are also main groups for pushing and maintaining the high temperature of the heap body, but are also microorganisms with relatively deficient resources. Therefore, new high-temperature or high-temperature resistant strain resources are excavated and applied to aerobic composting fermentation, which is beneficial to breaking through the traditional composting limit, accelerating the composting process, shortening the composting time and improving the composting quality. Therefore, the novel and efficient high-temperature resistant bacteria are separated from the nature, and the method has important significance for the development of recycling of the livestock and poultry raising organic wastes.
Disclosure of Invention
The invention aims to provide galactose geobacillus (Geobacillus galactosidasius) BWTGW1.1 which has a promoting effect on the good culture and composting fermentation of livestock and poultry raising organic wastes, and provides a good biological material for the resource utilization of the livestock and poultry raising organic wastes.
In order to achieve the above purpose, the invention discloses a thermophilic geological bacillus galactophilic strain which is geological bacillus galactophilic (Geobacillus galactosidasius) BWTGW1.1, wherein the strain is named BWTGW1.1, and is preserved in the microorganism strain preservation center of Guangdong province, and the address is building 5 of No. 59 of 100 university, mitsui, va.C., guangdong province, post code: 510070 with accession number GDMCC No:61374, the preservation date is 11 months and 11 days in 2020.
The galactose geological bacillus (Geobacillus galactosidasius) BWTGW1.1 is obtained by separating pig manure compost of fixed-point slaughterhouse Co., ltd. In Dongguan city in 3 months by the inventor, and the strain has the physiological and biochemical characteristics that: gram positive bacteria, the shape of the cells is short bar shape, and the size is 0.5-1.0X1.5-2.0 μm (see figure 1); the colony is white brown, has rough surface and irregular edge, and is suitable for growing at 55-65 ℃.
Extracting genome DNA of the hyperthermophilic galactose geological bacillus BWTGW1.1, amplifying a 16S rDNA gene by using a 27F/1492R primer, and sequencing to obtain a gene sequence shown in SEQ ID NO.1. The sequence was subjected to homology alignment analysis on NCBI and EzBioCloud websites to construct a phylogenetic tree, and morphological observation was combined, which revealed that the strain was geological galactose (Geobacillus galactosidasius).
The second object of the invention is to provide the application of the above-mentioned geological bacillus galactose BWTGW1.1 in the organic waste treatment of livestock and poultry raising solid.
Preferably, the method is applied to organic waste treatment of livestock and poultry raising solid such as livestock and poultry manure, livestock and poultry slaughterhouse sludge and the like.
Preferably, the galactose geological bacillus BWTGW1.1 is applied to the organic waste treatment of livestock and poultry raising solid in the form of liquid fermentation broth or powder after spray drying.
Preferably, the application of the geological bacillus galactose BWTGW1.1 in the fermentation of solid organic waste compost or tank fermentation of livestock and poultry raising.
Preferably, the weight percentage of the mixture of the bacterial liquid and the livestock and poultry breeding solid organic waste material of the galactose geological bacillus BWTGW1.1 is 0.1-0.2%.
Preferably, the weight percentage of the mixture of the galactose geological bacillus BWTGW1.1 and the livestock and poultry raising solid organic waste material in the form of bacterial powder is 0.02-0.05%.
A third object of the present invention is to provide the use of geological bacillus galactose BWTGW1.1 for the production of high temperature resistant proteases.
Compared with the prior art, the invention has the following beneficial effects:
(1) The galactose geological bacillus (Geobacillus galactosidasius) BWTGW1.1 is a thermophilic strain and can grow vigorously under the environmental condition of 65 ℃.
(2) The galactose geological bacillus (Geobacillus galactosidasius) BWTGW1.1 of the invention can generate a small amount of high Wen Danbai enzyme resistance, and the strain can form hydrolysis rings on a protein culture medium under the high temperature condition of 55 ℃.
(3) The galactose geological bacillus (Geobacillus galactosidasius) BWTGW1.1 can obviously promote the composting fermentation process and the high-temperature stage duration of pig manure waste.
In conclusion, the galactose geological bacillus (Geobacillus galactosidasius) BWTGW1.1 has strong growth adaptability in high-temperature composting of pig manure waste, is active at a high temperature stage, can generate high-temperature resistant protease, and has great application potential in good-cultivation composting of livestock and poultry cultivation organic waste.
Geobacillus galactosidasius BWTGW 1.1.1, deposited on 11 th month 11 of 2020 with the Guangdong province microbiological bacterial culture Collection center (GDMCC) under the address of building 5, post code: 510070, accession number is: GDMCC No:61374.
drawings
FIG. 1 is a photograph of a galactogeological bacterium BWTGW1.1 stained with crystal violet under an optical microscope (100X).
FIG. 2 shows the results of a plate for evaluating the ability of the galactose geological bacillus BWTGW1.1 to produce high temperature resistant protease at 60 ℃, and the presence of a hydrolytic transparent ring around a colony indicates that the strain has the ability to produce high temperature resistant protease.
FIG. 3 is a graphical representation of the effect of temperature conditions on the growth of geological bacillus galactosamine BWTGW1.1.
FIG. 4 is a graphical representation of the effect of salt concentration conditions on the growth of geological bacillus galactosamine BWTGW1.1.
FIG. 5 is a graph showing the temperature profile of a reactor for two treatments, namely, inoculation of a general microbial inoculum and inoculation of a general microbial inoculum+galactose geobacillus BWTGW1.1, in 40 tons of pig manure in tank fermentation.
FIG. 6 is a phylogenetic tree of the geological galactose BWTGW1.1 and similar strains; wherein, the NJ method is used for tree construction, and only bootstrap coefficients of more than 70% are shown in the figure (repeated 1000 times).
Detailed Description
The present invention will now be described in further detail with reference to the accompanying drawings and examples, which are given by way of illustration of the invention, but are not intended to limit the scope of the invention, and the parameters, proportions, etc. of the examples may be selected according to the circumstances without materially affecting the result. Unless otherwise indicated, all methods described in the examples are conventional and all reagents used are conventional or formulated in conventional fashion.
EXAMPLE 1 isolation and purification and preservation of geological galactose BWTGW1.1
3 middle layer samples were collected from the fixed point slaughterhouse Co., ltd at Dongguan city center 3 months in 2020 at the high temperature stage (50 ℃) of the composting fermentation of organic waste of pig manure.
Separating, purifying and preserving the high temperature bacteria: the collected 3 samples were weighed to 10g, respectively suspended with 50mL PBS (pH 7.4, concentration 0.2 mol/L), after shaking vigorously for 5min, centrifuged at 1000r/min for 3 times, each for 5min, and the supernatant was collected into a centrifuge tube. Diluting the supernatant to different concentrations to obtain 10 -3 、10 -4 、10 -5 The bacterial liquid with gradient is coated on an LB agar plate and cultured for 48 hours at the constant temperature of 60 ℃. And (3) selecting a strain with a better growth colony and a larger colony, continuing streaking and separating on an LB agar plate, and repeatedly purifying.
Dispersing the purified bacterial colony in 25% glycerin tube, and preserving at-80 deg.C for use. Thus, strain BWTGW1.1 was obtained.
EXAMPLE 2 identification of 16S rDNA of geological galactose BWTGW1.1
(1) Morphological identification: coating the strain BWTGW1.1 obtained by screening on an LB plate, culturing at 60 ℃ for 24 hours, and observing colony characteristics; single colony smears, gram stains, and cell morphology observed under a microscope are shown in FIG. 1.
(2) Identification of 16S rRNA gene molecules:
extracting genome DNA of a strain BWTGW1.1, amplifying by using a bacterial 16S rDNA gene amplification universal primer 27F/1492R (5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-TACGACTTAACCCCAATCGC-3') to obtain a PCR product, and sending the PCR product to Shanghai Meiji biological medicine science and technology Co., ltd (Guangzhou division) for sequence sequencing, wherein the length of the 16S rRNA sequence obtained after sequencing is 1447bp, and the sequence is shown in SEQ ID NO.1. The sequencing results were subjected to homology alignment analysis with 16S rDNA sequences in NCBI and EzBioCloud databases, and then, the strain BWTGW1.1 and similar strains were selected and phylogenetic tree (bootstrap was repeated 1000 times) was built by using MEGA 6.0, kimura 2-parameter model, NJ algorithm, and the obtained results of the phylogenetic tree are shown in FIG. 6.
The analysis of the results showed that the sequence similarity with the 16S rDNA gene of strain Geobacillus galactosidasius was highest (99.4%).
It was named: geological bacillus galactose (Geobacillus galactosidasius) BWTGW1.1, deposited at the cantonese province microbiological bacterial collection center (GDMCC), address: building 59 of Mitsui No. 100, guangzhou City, guangdong, with the preservation number of GDMCC No:61374.
EXAMPLE 3 evaluation of high temperature protease-producing ability of geological bacillus galactose BWTGW1.1
The high temperature resistant protease production capacity is evaluated by adopting a plate hydrolysis circle detection method:
the culture medium and the reagent mainly used are as follows:
protein medium: 40.0g of skimmed milk powder, 10.0g of soluble starch, 3.0g of yeast extract, 2.0g of potato powder and 15.0g of agar, and distilled water is added to fix the volume to 1000mL, and the pH value is 7.0-7.2. Sterilizing at 121deg.C under high temperature and pressure for 20min, and pouring into flat plate.
The activated galactose geological bacillus BWTGW1.1 is respectively inoculated on a protein culture medium plate, and is placed in a culture box at 60 ℃ for culture for 48 hours, and then whether hydrolysis transparent rings are generated around bacterial colonies is observed, so that whether the bacterial strains have the capability of secreting and producing high-temperature resistant protease can be qualitatively and directly reflected.
The observation that the strain BWTGW1.1 of the geological galactose is cultured at high temperature on a protein culture medium to generate a hydrolyzed transparent ring by the method shows that the result is shown in FIG. 2, which shows that the strain BWTGW1.1 has the capability of generating high temperature resistant protease.
EXAMPLE 4 evaluation of growth adaptability of Geobacillus galactolyticus BWTGW1.1 at temperature
(1) Preparing an LB culture medium: 5g of yeast extract, 10g of tryptone and 10g of sodium chloride, adjusting the pH to 7.0, and sterilizing at 121 ℃ for 20 min.
(2) Inoculating and culturing: inoculating Geobacillus galactolyticus BWTGW1.1 into 250mL triangular flask containing 50mL LB liquid medium, culturing at 50, 55, 60, 65, 70deg.C, culturing for 24 hr, sampling, and detecting OD 600 Values, results are shown in FIG. 3.
As shown in FIG. 3, the galactose geological bacillus BWTGW1.1 has good growth adaptability in the temperature range of 55-65deg.C, and can be grown at 60 deg.C, and OD in 24 hr culture 600 Values have exceeded 0.45; but atAt temperatures of 50 and 70 ℃, the growth of the bacterial cells is obviously inhibited.
EXAMPLE 5 evaluation of the adaptability of salinity to the growth of geological bacillus galactose BWTGW1.1
(1) Preparing LB culture media with different salinity gradients: LB was used as a basal medium, 0g, 0.5g, 1g, 1.5g, 2g, 3g and 5g of sodium chloride were added to each 100mL of basal medium, respectively, to prepare media having salinity of 0, 0.5%, 1%, 1.5%, 2%, 3% and 5%, respectively, pH was adjusted to 7.2, and the culture was autoclaved at 121 ℃.
(2) Inoculating and culturing: inoculating Geobacillus galactolyticus BWTGW1.1 into LB culture medium with different salinity gradient, culturing at 60deg.C, sampling after culturing for 0, 8, 12, 24, 48 hr, and detecting OD 600 Values, results are shown in FIG. 4.
As shown in FIG. 4, the geological galactose BWTGW1.1 has good adaptability in a certain salinity range, and can grow well under the condition of 0-2% salinity, and can grow well under the condition of 1.5% salinity, and OD is obtained when culturing for 48h 600 The value has exceeded 1.1; and after the salinity exceeds 2%, the growth of the thalli is obviously limited, and the growth rate also drops rapidly.
EXAMPLE 6 preparation of geological galactose BWTGW1.1
The invention also provides a preparation method of the geological bacillus galactose BWTGW1.1, which comprises the following steps:
(1) Culturing seed liquid: inoculating the galactose geological bacillus BWTGW1.1 into an LB culture medium, and culturing for 24 hours at 60 ℃ with a 120r/min constant temperature water bath oscillator to obtain BWTGW1.1 seed liquid;
(2) Filling a fermentation medium accounting for 60% of the total volume into a fermentation tank, inoculating a seed solution of the geological bacillus galactosis BWTGW1.1 accounting for 1% of the volume of the fermentation medium at the temperature of 60 ℃, stirring and culturing for 36h at the rotating speed of 180-200 rpm under ventilation to ensure that the bacterial count of the BWTGW1.1 strain reaches 1 multiplied by 10 9 cfu/mL；
The pH of the fermentation medium is 7.0, and the fermentation medium comprises the following components in percentage by mass: 1 to 2 percent of glucose, 0.5 to 1 percent of soybean peptone, 0.5 to 1 percent of yeast extract, 1.5 percent of sodium chloride, 0.05 percent of magnesium sulfate, 0.05 percent of potassium dihydrogen phosphate and the balance of water.
Example 7 fermentation Effect of geological galactose BWAGGW 1.1 big tank fermentation slaughterhouse pig manure
And (3) fully and uniformly mixing 40 tons of slaughterhouse pig manure with the water content of 63-65% with a starter, and putting the mixture into a 116 ton large-ventilation fermentation tank through a vertical feeding bin for ventilation fermentation. Wherein the reference starter is a common microbial inoculum with the mass content of 0.1 percent (bacillus subtilis: enterococcus faecalis: saccharomyces cerevisiae is in the mass ratio of 5:1:1), the test starter is a bacterial solution with the mass content of 0.1 percent of the common microbial inoculum and the mass content of 0.1 percent of galactose geological bacillus BWTGW1.1 (the viable count is more than or equal to 1 multiplied by 10) 8 cfu/mL). When the temperature of the stack does not reach 50 ℃, the ventilation is 15min/60min, and the stirring is synchronous; after the temperature reached 50 ℃, the ventilation was adjusted to 30min/60min while stirring was synchronized. Measuring the temperature of the pig manure pile every day, recording, fermenting for 168 hours, taking an intermediate layer sample after finishing, detecting the moisture content of the fermented pig manure, and finally measuring that the moisture content of the pig manure in the experimental group and the control group is 45% and 35% respectively; the specific temperature detection data are shown in fig. 5. The result of fig. 5 shows that the temperature of the pig manure pile body reaches 58 ℃ after 24 hours of treatment of the common bacterial agent and the galactose geological bacillus BWTGW1.1 bacterial liquid, and the highest temperature reaches 70 ℃ after the high temperature of 60-70 ℃ is continued for 108 hours. And the control treatment of the common microbial inoculum is only used, 48 hours are needed for raising the temperature of the pig manure pile to more than 50 ℃, the temperature above 50 ℃ is only 60 hours, and the highest temperature is 60 ℃. The result shows that the common microbial agent and the galactose geological bacillus BWTGW1.1 microbial fluid treatment have obvious advantages compared with the common microbial agent treatment in terms of harmless treatment and reduction.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Sequence listing
<110> Guangdong Bowalt Biotechnology Co., ltd
Microbiological institute of academy of sciences in Guangdong province (microbiological analysis and detection center in Guangdong province)
<120> a geological bacillus galactose BWTGW1.1 and its use
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1447
<212> DNA
<213> Galaxobacter (Geobacillus galactosidasius)
<400> 1
caattgacac ttcggcggct ggctccctta cgggttacct caccgacttc gggtgttgca 60
agctctcgtg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg 120
ctgatccgcg attactagcg attccggctt catgcaggcg agttgcagcc tgcaatccga 180
actgagagcg gctttttggg attcgctccc cctcgcgggt tcgcagccct ttgtaccgcc 240
cattgtagca cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac 300
cttcctccga cttttcaccg gcagtccccc tagagtgccc aactgaatgc tggcaactag 360
gggcgagggt tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac 420
aaccatgcac cacctgtcac cctgtccccc cgaaggggga acgccctgtc tccagggttg 480
tcaggggatg tcaagacctg gtaagggtct tcgcgttgct tccaattaaa ccacatgctc 540
caccgcttgg gcgggccccc gtcaattcct ttgagtttca gccttgcggc cgtactcccc 600
aggcggagtg cttaacgcgt tagctacagc actaaaggga aaacccctct aacacttagc 660
actcatcgtt tacggcgtgg actaccaggg tatctaatcc tgtttgctcc ccacgctttc 720
gcgcctcagc gtcagttaca gaccagagag ccgccttcgc cactggtgtt cctccacatc 780
tctacgcatt tcaccgctac acgtggaatt ccgctctcct cttctgcact caagtccccc 840
agtttccaat gacccctcca cggttgagcc gtgggctttc acatcagact taagggaccg 900
cctgcgcgcg ctttacgccc aataattccg gacaacgctc gccccctacg tattaccgcg 960
gctgctggca cgtagttagc cggggctttc tcgttaggta ccgtcaccgt accgccctat 1020
tcgaacggta cttcttcttc cctaacaaca gagctttacg atccgaagac cttcttcgct 1080
cacgcggcgt cgctccgtca gactttcgtc cattgcggaa gattccctac tgctgcctcc 1140
cgtaggagtc tgggccgtgt ctcagtccca gtgtggccgg tcaccctctc aggccggcta 1200
cgcatcgtcg ccttggtgag ccgttacctc accaactagc taatgcgccg cgggcccatc 1260
cgtaagtggt agcaaaagcc acctttcaac caaagaccat gcggtcttcg gtgttatccg 1320
gtattagccc cggtttcccg gagttatccc ggtcttacgg gcaggttacc cacgtgttac 1380
tcacccgtcc gccgctaacc gaacagaagc aagcttccgt tcggtccgct cgactgcatg 1440
tatagca 1447
Claims (8)
1. Geological bacillus galactoseGeobacillus galactosidasius) BWTGW1.1, accession No. GDMCC No:61374.
2. the use of the geological bacillus galactose BWTGW1.1 of claim 1 in the organic waste treatment of livestock and poultry raising solids.
3. The use according to claim 2, wherein the livestock and poultry raising solid is organic waste livestock and poultry manure, livestock and poultry slaughterhouse sludge.
4. The use according to claim 2, wherein the geological bacillus galactosis BWTGW1.1 is applied in the organic waste treatment of livestock and poultry raising solid in liquid fermentation broth or powder formulation after spray drying.
5. The use according to claim 2, characterized by the use of geological bacillus galactose BWTGW1.1 in the fermentation of organic waste compost of livestock and poultry farming solids or tank fermentation.
6. The use according to claim 4, wherein the weight percentage of the galactose geological bacillus BWTGW1.1 which is mixed with the livestock and poultry raising solid organic waste material in the form of bacterial liquid is 0.1-0.2%.
7. The use according to claim 4, wherein the ratio of the weight percentage of the mixed powder of the galactose geological bacillus BWTGW1.1 and the mixed powder of the galactose geological bacillus BWTGW are 0.02-0.05.
8. Use of the geological bacillus galactose BWTGW1.1 according to claim 1 for producing high temperature resistant proteases.
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