CN111423256A - Organic garbage pyrolysis method - Google Patents

Abstract

The invention provides an organic garbage pyrolysis method, which comprises the steps of adding modified transgenic bacteria as an exogenous bacterial preparation when compost enters a high-temperature stage, and adding thermophilic microorganisms to improve the abundance of a microbial system of the compost and increase the number of bacteria in the high-temperature composting process.

Description

Organic garbage pyrolysis method

Technical Field

The invention relates to the field of organic garbage treatment, in particular to an organic garbage pyrolysis method.

Background

The composting technology of solid organic waste refers to a biochemical process that relies on microorganisms such as bacteria, actinomycetes, fungi, etc. to controllably promote the conversion of organic matter that is degradable by microorganisms to stable humus. According to the temperature change and the microorganism growth condition in the stacking process, the stacking process is divided into a heating stage (heat generation stage), a high temperature stage and a cooling stage.

Mesophilic microorganisms in the compost pile at the initial stage of composting utilize soluble and easily degradable organic matters as nutrition and energy sources to quickly proliferate and release heat energy, so that the temperature of the compost is continuously increased, and when the temperature of the compost pile is increased to be more than 45 ℃, the high-temperature stage is started. Usually, from the beginning of stacking fermentation, the temperature of the compost can be rapidly increased to 55 ℃ within 2-3 days, the temperature can reach the maximum value (the maximum value can reach 80 ℃) within 1 week, and besides the residual and newly formed soluble organic matters in the previous stage are continuously decomposed and converted, complex organic matters such as hemicellulose, cellulose, protein and the like are also intensively decomposed. In the high temperature stage, mesophilic microorganisms are inhibited and thermophilic microorganisms are gradually replaced.

The biological strengthening technology is to add microorganisms with specific functions into a biological treatment system to improve the treatment effect of the original treatment system, and the added microorganisms can come from the original treatment system. Researchers have applied this technology to the treatment of non-degradable toxic and harmful substances in industrial wastewater, surface water and underground water or to improve the wastewater treatment effect, and can significantly improve the microbial activity.

In the use process of the biological strengthening technology, how to fully exert the potential of microorganisms, improve the treatment effect of refractory organic matters and how to inhibit the microorganisms in high-temperature treatment materials is an urgent problem to be solved in industrial production.

Disclosure of Invention

In order to solve the technical problem, the invention provides an organic garbage pyrolysis method.

The invention is realized by the following technical scheme:

a method for pyrolyzing organic waste comprises adding transformed transgenic bacteria as exogenous bacterial preparation when compost enters a high-temperature stage.

Further, the engineered transgenic bacteria comprise:

(a) has a nucleotide sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2;

(b) a nucleotide sequence obtained by substituting and/or deleting and/or adding one or more nucleotides to the nucleotide sequence in (a) and encoding the same amino acid sequence as in (a);

(c) a nucleotide sequence which has more than 95 percent of homology with the nucleotide sequence shown by SEQ ID NO. 1 or SEQ ID NO. 2 and can be expressed in host cells and ensure that the host cells have thermophilic property.

Further, the expression cassette comprises the above nucleotide sequence under the control of an operably linked regulatory sequence.

Further, the recombinant vector comprises the above nucleotide sequence or the above expression cassette.

Further, the method comprises the step of constructing a hot spring sample thermophilic gene library, preferably, constructing a hot spring sample high temperature resistant gene library by using an Escherichia coli expression system.

Further, respectively culturing escherichia coli inserted with exogenous genes at different environmental temperatures, sequencing and identifying the length and specific sequence of an inserted fragment by using common general primers and archaea specific primers, removing a carrier from the obtained sequence to obtain a high-temperature-resistant related gene, and then randomly inserting bacillus to perform gene modification on the bacillus.

Further, the recombinant cloning vectors are respectively transformed into bacillus subtilis competent cells by a heat shock method, white colonies are picked, plasmids are extracted after culture, positive clones are subjected to sequencing verification after enzyme digestion identification, and the results show that the nucleotide sequences are respectively and correspondingly inserted into the recombinant cloning vectors.

The invention has the following beneficial effects:

1) the gene sequence of the thermophilic microorganism has significant differences from the known microbial homology, and the sequences of nearly half of the functional genes have not been confirmed. The gene resource of the hyperthermophilic archaea is fully utilized, and the function of the unknown gene of the hyperthermophilic archaea is discovered and identified, so that the method has important theoretical significance for determining the molecular mechanism and the enzymological characteristics of the hyperthermophilic enzyme and exploring the biogenesis and life evolution.

2) The added thermophilic microorganism improves the abundance of a composting microbial system and increases the number of bacteria in the high-temperature composting process.

3) The method has the advantages of simple operation, low cost and no pollutant generation in the treatment process, can avoid the problem that the traditional microbial inoculant is easily influenced by composting conditions or inhibits the growth of the traditional microbial inoculant in the high-temperature composting process, and obviously improves the composting efficiency and the quality of compost products.

4) The method for promoting compost maturity of livestock and poultry manure through microbial pretreatment has good application value, and compost enterprises can utilize the technology to carry out reduction treatment on livestock and poultry manure and crop straws and organic fertilizer production.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below, and the following reagents or microbial agents are commercially available reagents or microbial agents unless otherwise specified.

As is well known to those skilled in the art, DNA typically exists in a double stranded form. In this arrangement, one strand is complementary to the other strand, and vice versa. Other complementary strands of DNA are produced as the DNA replicates in plants.

The present invention includes the use of the polynucleotides exemplified in the sequence listing and their complementary strands. The "coding strand" as commonly used in the art refers to the strand to which the antisense strand is joined. To express a protein in vivo, one strand of DNA is typically transcribed into the complementary strand of an mRNA, which serves as a template for translation of the protein. mRNA is actually transcribed from the "antisense" strand of DNA.

The "sense" or "coding" strand has a series of codons (a codon is three nucleotides, three of which at a time can yield a particular amino acid) that can be read as an Open Reading Frame (ORF) to form a protein or peptide of interest. The present invention also includes RNAs that are functional equivalent to the exemplified DNA.

In one aspect, the present invention provides a DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least 85% sequence identity to either of SEQ id nos 1, 2; (b) a sequence comprising any one of SEQ ID NOs 1, 2; and (c) a fragment of any one of SEQ ID NOs: 1, 2, wherein said fragment has gene regulatory activity, wherein said sequence is operably linked to a heterologous transcribable polynucleotide molecule.

In one embodiment, the DNA molecule has at least about 90% sequence identity to the DNA sequence of any one of SEQ ID NO 1, 2. In another embodiment, the DNA molecule has at least 95% sequence identity with the DNA sequence of any one of SEQ ID NO 1, 2.

A substantially homologous sequence is a nucleic acid molecule that specifically hybridizes under highly stringent conditions to the complementary strand of a matching nucleic acid molecule suitable stringent conditions that promote DNA hybridization, e.g., about 45 ℃ with 6.0 × sodium chloride/sodium citrate (SSC) followed by 50 ℃ with 2.0 × SSC, which are well known to those skilled in the art, e.g., the salt concentration in the washing step can be selected from about 2.0 × SSC for low stringency conditions, about 50 ℃ to about 0.2 × SSC for high stringency conditions, 50 ℃ in addition, the temperature conditions in the washing step can be varied from about 22 ℃ at room temperature for low stringency conditions to about 65 ℃ for high stringency conditions, the temperature conditions and salt concentration can both be varied, one can be held constant while the other is varied, preferably, the stringent conditions of the invention can be varied from about 6 × SSC, 0.5% SDS solution at 65 ℃ with SEQ ID: 1, SEQ ID: 461, SEQ ID: 29% and SEQ ID: 29% SSC, and then 0.591% membrane specific hybridization with SEQ ID NO: 29 SSC.

Recombinant nucleotides or recombinant DNA constructs comprising the coding sequences can be delivered into host cells by vectors such as plasmids, baculoviruses, artificial chromosomes, virosomes, cosmids, phagemids, bacteriophages or viral vectors. Such vectors may be used to achieve stable or transient expression of a coding sequence in a host cell; and, if possible, subsequently expressed as a polypeptide. An exogenous recombinant polynucleotide or recombinant DNA construct comprising a coding sequence and introduced into a host cell is also referred to herein as a "transgene".

Thus, transgenic bacteria expressing any one or more coding sequences comprising any recombinant polynucleotide (i.e., a transgene) are also provided. "bacterial cells" or "bacteria" may include, but are not limited to, Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas, or Rhizobium cells.

Modification of genes and easy construction of gene variants can be achieved using standard techniques. For example, techniques for making point mutations are well known in the art. Another example is U.S. patent No. 5605793, which describes methods for generating other molecular diversity using DNA reassembly after random fragmentation. Fragments of the full-length gene can be made using commercial endonucleases, and exonucleases can be used following standard procedures.

Due to the redundancy of the genetic code, a plurality of different DNA sequences may encode the same amino acid sequence. It is well within the skill of the art to generate such alternative DNA sequences encoding the same or substantially the same protein. These different DNA sequences are included in the scope of the present invention. The "substantially identical" sequence refers to a sequence having amino acid substitutions, deletions, additions or insertions which do not substantially affect pesticidal activity, and also includes fragments which retain pesticidal activity.

In the present invention, there is a certain homology with the amino acid sequence expressed by SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3. These sequences typically have a similarity/identity of greater than 78%, preferably greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, and may be greater than 99% to the sequences of the present invention.

Preferred polynucleotides and proteins of the invention are defined according to a more specific range of identity and/or similarity. For example 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity and/or similarity to a sequence exemplified herein.

Example 1:

specifically, the sample is taken from the water body and the surrounding soil of the Duzhong hot spring, the water body is sampled for 1000m L, the soil sampling amount is 1000g, the temperature environment of 55 ℃ is kept in the transportation process, after the water body and the soil are fully mixed, the supernatant is taken and placed in a liquid enrichment culture medium, the culture is carried out for 5 days at 55 ℃, the cell is cracked by adopting a freeze-thaw method, the total DNA of microorganisms is extracted, and the DNA sample is purified.

The DNA sample obtained by purification is sequenced, an escherichia coli expression system (purchased from Shanghai Pongjing industries, Ltd.) is used for constructing a hot spring sample high-temperature resistant gene library, specifically, total genome DNA is randomly broken into fragments with the lengths of 50bp, 100bp, 150bp, 200bp, 250bp, 500bp, 1.0kb, 1.5kb and 2.0kb, the broken DNA fragments are connected with a connector, then short sequence libraries (sequences of 50bp, 100bp, 150bp, 200bp, 250bp and 500 bp) are obtained by sequencing, a large fragment library (sequences of 1.0kb, 1.5kb and 2.0 kb) is constructed by adopting Cre-L ox library construction technology, kyotope sequencing is carried out after connecting L oxP connectors at two ends of the large fragment, and genome sequencing is completed by an SO L EXA sequencing platform (Nanjinsrui).

Example 2:

specifically, the Escherichia coli having the foreign gene inserted therein of example 1 was inoculated into L B medium (solid medium) and subjected to a high temperature resistance test at 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ for 3 days, 3 dishes were placed for each temperature condition, and after 3 days, 30 to 40 vigorous colonies were picked for each dish and sequenced.

Common universal primers and archaea specific primers are used for sequencing and identifying the length and specific sequence of the insert, after a carrier is removed from the obtained sequence, high temperature resistant related genes Rq-01, Rq-02, Rq-03, Rq-04, Rq-05, Rq-06 and Rq-07 are obtained, codon modification is carried out on the Rq-05 and the Rq-06 with higher homology with bacillus through B L AST comparison, and then the bacillus is randomly inserted for carrying out gene modification on the bacillus.

Example 3:

the nucleotide sequence of the obtained modified gene Rq-05 is shown as SEQ ID NO:1, the nucleotide sequence of Rq-06 is shown as SEQ ID NO:2, the 5 'end of the sequence is also connected with an NcoI enzyme cutting site, the 3' end of the sequence is also connected with a SwaI enzyme cutting site, the nucleotide sequences of Rq-05 and Rq-06 are synthesized by Nanjing Kinry, the synthesized Rq-05 and Rq-06 nucleotide sequences are respectively connected in a cloning vector pGEM-T (purchased from Promega), and the connection process refers to an instruction book to obtain recombinant cloning vectors pGEM-05 and pGEM-Rq-06 (the vector structure: Amp represents ampicillin resistance penicillin gene, f1 represents the replication origin of phage f1, L acZ is an initiation codon L acZ, SP6 is an SP6RNA polymerase promoter, T7 is a T7RNA polymerase promoter, Rq-05/Rq-06 is Rq-05/Rq-06 nucleotide sequence, and MCS is a multiple cloning site).

The recombinant cloning vectors pGEM-Rq-05 and pGEM-Rq-06 were transformed into Bacillus subtilis competent cells (commercially available) by a heat shock method, respectively. And selecting a white colony, culturing, extracting a plasmid, carrying out enzyme digestion identification, and carrying out sequencing verification on the positive clone, wherein the result shows that the Rq-05/Rq-06 nucleotide sequence is correspondingly inserted into the recombinant clone vectors pGEM-Rq-05 and pGEM-Rq-06 respectively.

Example 4: high temperature resistance test of transgenic bacillus subtilis

Respectively inoculating the bacillus subtilis inserted with the exogenous gene into 50ml L B liquid culture medium, respectively inserting Rq-05 nucleotide sequence into 21 bottles, dividing into 7 groups, inoculating 3 bottles in each group, inserting Rq-06 nucleotide sequence into 21 bottles, dividing into 7 groups, inoculating 3 bottles in each group, inoculating unmodified bacillus subtilis into 50ml L B liquid culture medium, dividing into 8 groups, and 1 bottle in each group, wherein the inoculation amount of the bacillus subtilis is 1g, respectively culturing at 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and 75 ℃ for 2h, and measuring OD value, and the measurement result is shown in Table 1:

TABLE 1 determination of OD values at different temperatures for 2 hours

The result shows that the bacillus subtilis transformed with the Rq-05 nucleotide sequence has faster proliferation at 44-65 ℃ and vigorous growth at 55 ℃; the bacillus subtilis transferred with the Rq-06 nucleotide sequence can keep a higher proliferation speed at 50-70 ℃ and has vigorous vigor at 60 ℃; the bacillus subtilis transferred with the Rq-05 nucleotide sequence obviously shows that the high temperature has an inhibiting effect on the growth and development of the bacillus subtilis at the temperature of 75 ℃.

Example 5:

and testing the treatment effect of the bacillus subtilis with the Rq-05 nucleotide sequence and the bacillus subtilis with the Rq-06 nucleotide sequence on the mixed perishable organic garbage. Sorting the mixed perishable organic garbage to be treated, removing plastics and the like. Adding bacillus subtilis with Rq-05 nucleotide sequence transferred and bacillus subtilis with Rq-06 nucleotide sequence transferred into the mixed perishable organic garbage according to the mass ratio of 1:1 to prepare a solid microbial inoculum, wherein the mass ratio of the solid microbial inoculum to the mixed perishable organic garbage is 1: 200, the total amount is 1kg, the mixture is stirred uniformly and then is subjected to aerobic fermentation at the temperature of 55 ℃, the same mass of mixed perishable organic garbage is taken as a control CK group, the mixture is fermented under the conditions of the same temperature and humidity, and the ventilation is kept in the process. And continuously monitoring the mass of the mixture, and weighing every 12h to obtain the wet weight reduction data of the mixed perishable organic garbage, wherein the reduction data are shown in a table 2.

TABLE 2 blending perishable organic waste reduction data

Duration of time	12h(g)	24h(g)	36h(g)	48h(g)	60h(g)	72h(g)
							Test set	1000	546	182	173	171	122
CK group	1000	876	565	576	556	544

The wet weight reduction rate of the mixed perishable organic garbage mixed with the bacillus subtilis with the Rq-05 nucleotide sequence and the bacillus subtilis with the Rq-06 nucleotide sequence in 48 hours is up to 87.8 percent, and is close to the maximum value after 36 hours, while the wet weight reduction rate of a control group without inoculation of strains is 45.6 percent. Compared with a control group, the strain FH-JW1 improves the wet weight loss rate of the mixed perishable organic waste by 42.2%. Therefore, the bacillus subtilis transformed with the Rq-05 nucleotide sequence and the bacillus subtilis transformed with the Rq-06 nucleotide sequence can improve the wet weight reduction rate of the mixed perishable organic garbage under aerobic conditions, and have high application value and wide application prospect.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.

Sequence listing

<110> Shanghai environmental science and technology Limited

<120> organic garbage pyrolysis method

<130>CZGZhuanC-sanhuan-2020042303

<141>2020-04-23

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<170>SIPOSequenceListing 1.0

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cgtagcggcc gttttgaaag ccaggcgccg gtgagcccgg tgctggtgga tacccgtgcg 180

gcgcagccga gcgcgccgaa agtgctgcgt tataccaccg gcagcgtgta taaaattgcg 240

ctgaacaccg gcccggcgtg gagcacctgg accgtgtata ccgaagcgcg tgtgacccgt 300

tatagcgata tttatagcga acgtggccgt gaatgcagcg gcgcgaccac ccgtcgtgaa 360

agcgtgcaga gcgatctgac ctttcgttat cgtgtgcgtc tggatcgtat tccgggctat 420

aaaggcatgc cgcagaacgc gaccctgcgt cgtattaaaa gccgtgaaat ggaacattat 480

catcagctga gcgcgtttct gtgccgtggc acctatgaaa gcagcggcgc gcgtcagcag 540

cgtgcgattg cgggcgtgag cgcgaccagc gaagcggtgg atgatagccg tgcgggcatt 600

ctgtttagca gcctgcgtta tggcatgatt ccgccgagcg aagataacct gccggatgcg 660

agcgtgtata ttccgctgcc gagcccggat ctgctgcgtc cgctggtgat tcgtaccacc 720

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ctggcgctgt ttaacgtgga ttgcgatccg cagggcaaag cgtgcgcgag caccgtggtg 840

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Claims (7)

1. A method for pyrolyzing organic waste, which comprises adding a modified transgenic bacterium as an exogenous bacterial preparation when the compost enters a high-temperature stage.

2. The method of claim 1, wherein the engineered transgenic bacterium comprises:

(a) has a nucleotide sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2;

(b) a nucleotide sequence obtained by substituting and/or deleting and/or adding one or more nucleotides to the nucleotide sequence in (a) and encoding the same amino acid sequence as in (a);

(c) a nucleotide sequence which has more than 95 percent of homology with the nucleotide sequence shown by SEQ ID NO. 1 or SEQ ID NO. 2 and can be expressed in host cells and ensure that the host cells have thermophilic property.

3. An expression cassette comprising the nucleotide sequence of claim 2 under the control of an operably linked regulatory sequence.

4. A recombinant vector comprising the nucleotide sequence of claim 2 or the expression cassette of claim 3.

5. The method according to claim 1, wherein the method comprises constructing a thermophilic gene library of the hot spring sample, preferably a thermophilic gene library of the hot spring sample by using an escherichia coli expression system.

6. The method as claimed in claim 5, wherein the Escherichia coli with the inserted foreign gene is cultured at different environmental temperatures, the common general primers and the archaea specific primers are used for sequencing to identify the length and specific sequence of the inserted fragment, the vector is removed from the obtained sequence to obtain the high temperature resistant related gene, and then the Bacillus is randomly inserted to modify the Bacillus.

7. The method as claimed in claim 6, wherein the recombinant cloning vectors are transformed into competent cells of Bacillus subtilis by a heat shock method, white colonies are selected, plasmids are extracted after culture, and positive clones are subjected to sequencing verification after enzyme digestion identification, wherein the results show that the nucleotide sequences as claimed in claim 2 are respectively and correspondingly inserted into the recombinant cloning vectors.