CN110791498A - Immobilization method of stenotrophomonas and composition prepared by same - Google Patents
Immobilization method of stenotrophomonas and composition prepared by same Download PDFInfo
- Publication number
- CN110791498A CN110791498A CN201911253930.7A CN201911253930A CN110791498A CN 110791498 A CN110791498 A CN 110791498A CN 201911253930 A CN201911253930 A CN 201911253930A CN 110791498 A CN110791498 A CN 110791498A
- Authority
- CN
- China
- Prior art keywords
- immobilization
- strain
- stenotrophomonas
- initial
- oxidation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 52
- 241000122971 Stenotrophomonas Species 0.000 title claims abstract description 23
- 239000000203 mixture Substances 0.000 title claims abstract description 14
- 238000001179 sorption measurement Methods 0.000 claims abstract description 47
- 239000001963 growth medium Substances 0.000 claims abstract description 24
- 238000011081 inoculation Methods 0.000 claims abstract description 24
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 21
- 238000009630 liquid culture Methods 0.000 claims abstract description 17
- 241001052560 Thallis Species 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000007254 oxidation reaction Methods 0.000 claims description 135
- 230000003647 oxidation Effects 0.000 claims description 120
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical group O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 claims description 30
- 230000008569 process Effects 0.000 claims description 25
- 239000002054 inoculum Substances 0.000 claims description 20
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 19
- 239000008103 glucose Substances 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 10
- 229910021536 Zeolite Inorganic materials 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 239000010457 zeolite Substances 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 230000003100 immobilizing effect Effects 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 67
- 230000007613 environmental effect Effects 0.000 abstract description 5
- 241000894006 Bacteria Species 0.000 description 59
- 229910052717 sulfur Inorganic materials 0.000 description 48
- 239000011593 sulfur Substances 0.000 description 47
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 45
- 230000012010 growth Effects 0.000 description 32
- 230000000694 effects Effects 0.000 description 29
- 238000002474 experimental method Methods 0.000 description 29
- 108090000623 proteins and genes Proteins 0.000 description 27
- 230000001590 oxidative effect Effects 0.000 description 26
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 24
- 230000004044 response Effects 0.000 description 21
- 230000003993 interaction Effects 0.000 description 18
- 230000037361 pathway Effects 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 15
- 108010027912 Sulfite Oxidase Proteins 0.000 description 13
- 102000043440 Sulfite oxidase Human genes 0.000 description 13
- 230000001580 bacterial effect Effects 0.000 description 12
- 239000003795 chemical substances by application Substances 0.000 description 12
- 244000005700 microbiome Species 0.000 description 12
- 230000003247 decreasing effect Effects 0.000 description 11
- 239000003344 environmental pollutant Substances 0.000 description 11
- 238000012163 sequencing technique Methods 0.000 description 11
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical compound [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 11
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 10
- 231100000719 pollutant Toxicity 0.000 description 10
- 230000002829 reductive effect Effects 0.000 description 10
- 230000006870 function Effects 0.000 description 9
- 239000002351 wastewater Substances 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 239000003054 catalyst Substances 0.000 description 8
- 230000007423 decrease Effects 0.000 description 8
- 238000005457 optimization Methods 0.000 description 8
- 230000000813 microbial effect Effects 0.000 description 7
- 238000012258 culturing Methods 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 108010057639 sulfide quinone reductase Proteins 0.000 description 6
- 238000000540 analysis of variance Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical class S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 108010022173 Thiosulfate sulfurtransferase Proteins 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 description 4
- 239000011714 flavin adenine dinucleotide Substances 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000012803 optimization experiment Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 108010026529 tetrathionate hydrolase Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000000593 degrading effect Effects 0.000 description 3
- 108091008053 gene clusters Proteins 0.000 description 3
- 238000012268 genome sequencing Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 241000589291 Acinetobacter Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 101710106383 Disulfide bond formation protein B Proteins 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 101710116318 Probable disulfide formation protein Proteins 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 229910017435 S2 In Inorganic materials 0.000 description 2
- 102100031138 Sulfide:quinone oxidoreductase, mitochondrial Human genes 0.000 description 2
- 101150116444 TST gene Proteins 0.000 description 2
- 102100034707 Thiosulfate sulfurtransferase Human genes 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 238000007323 disproportionation reaction Methods 0.000 description 2
- 229910052564 epsomite Inorganic materials 0.000 description 2
- 238000013401 experimental design Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 2
- 229940093632 flavin-adenine dinucleotide Drugs 0.000 description 2
- 101150051811 grx gene Proteins 0.000 description 2
- 101150051842 hdrA gene Proteins 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 239000010977 jade Substances 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 238000013048 microbiological method Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 150000003222 pyridines Chemical class 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000002352 surface water Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- WIGIZIANZCJQQY-UHFFFAOYSA-N 4-ethyl-3-methyl-N-[2-[4-[[[(4-methylcyclohexyl)amino]-oxomethyl]sulfamoyl]phenyl]ethyl]-5-oxo-2H-pyrrole-1-carboxamide Chemical compound O=C1C(CC)=C(C)CN1C(=O)NCCC1=CC=C(S(=O)(=O)NC(=O)NC2CCC(C)CC2)C=C1 WIGIZIANZCJQQY-UHFFFAOYSA-N 0.000 description 1
- 108700014176 ABC-type sulfate transporter activity proteins Proteins 0.000 description 1
- 101100208307 Acidithiobacillus ferrooxidans (strain ATCC 23270 / DSM 14882 / CIP 104768 / NCIMB 8455) tth gene Proteins 0.000 description 1
- PLXMOAALOJOTIY-FPTXNFDTSA-N Aesculin Natural products OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1Oc2cc3C=CC(=O)Oc3cc2O PLXMOAALOJOTIY-FPTXNFDTSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- WNBCMONIPIJTSB-BGNCJLHMSA-N Cichoriin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1c(O)cc2c(OC(=O)C=C2)c1 WNBCMONIPIJTSB-BGNCJLHMSA-N 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 102000020018 Cystathionine gamma-Lyase Human genes 0.000 description 1
- 108010045283 Cystathionine gamma-lyase Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 244000207740 Lemna minor Species 0.000 description 1
- 235000006439 Lemna minor Nutrition 0.000 description 1
- 108010048581 Lysine decarboxylase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001057811 Paracoccus <mealybug> Species 0.000 description 1
- 235000001855 Portulaca oleracea Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 101710167005 Thiol:disulfide interchange protein DsbD Proteins 0.000 description 1
- GWZOLWLJEJRQMZ-UHFFFAOYSA-N [S].S Chemical group [S].S GWZOLWLJEJRQMZ-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 229950006790 adenosine phosphate Drugs 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 101150085125 apr gene Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000007444 cell Immobilization Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 101150073875 cybB gene Proteins 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000004141 dimensional analysis Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 description 1
- 229940093496 esculin Drugs 0.000 description 1
- AWRMZKLXZLNBBK-UHFFFAOYSA-N esculin Natural products OC1OC(COc2cc3C=CC(=O)Oc3cc2O)C(O)C(O)C1O AWRMZKLXZLNBBK-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 108010084705 heterodisulfide reductase Proteins 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 229910052976 metal sulfide Inorganic materials 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 230000001089 mineralizing effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 238000003068 pathway analysis Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 101150111012 soxC gene Proteins 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 101150104699 sqr gene Proteins 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 101150056707 stp gene Proteins 0.000 description 1
- 150000004763 sulfides Chemical class 0.000 description 1
- -1 sulfur ions Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000005211 surface analysis Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 229940035936 ubiquinone Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/101—Sulfur compounds
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Inorganic Chemistry (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention belongs to the field of environmental management, and relates to an immobilization method of Stenotrophomonas sp3 and a prepared composition, wherein the immobilization method comprises the steps of inoculating Stenotrophomonas sp3 into a liquid culture medium, carrying out adsorption immobilization at the temperature of 28-32 ℃ and the rotating speed of 130-150 r/min, wherein the initial immobilized pH value is 7.0-9.0, the adsorption time is 37.5-40 h, the inoculation amount of thalli is 5.0-10.0% and the addition amount of a carrier is 0.5-2.0 g based on 200mL of the liquid culture medium; wherein the preservation number of the stenotrophomonas sp3 is CGMCC No. 18394. By the immobilization method of the present invention, it is possible toThe stenotrophomonas sp3 is efficiently immobilized, and the immobilized microbial inoculum can obviously improve the S in the stenotrophomonas black-smelly water body2‑。
Description
Technical Field
The invention belongs to the field of environmental management and relates to the immobilization of stenotrophomonas.
Background
The black and odorous phenomenon of the water body has complex causes, and relates to a plurality of processes such as physics, chemistry, biology and the like, and has more influence factors. Studies have shown S in overlying water2-Is a key factor causing the water body to turn black. It is mixed with Fe in water2+、Mn2+、Cu2+The plasma metal ions are combined to accumulate and generate metal sulfides, so that the water body is blackened. Thus, S in the black and odorous water body2-Oxidation to SO of stable valence state4 2-Can avoid the phenomenon of blackening of the water body. S2-The oxidation process of (A) is very slow in natural environment and mainly depends on indigenous Sulfur Oxidizing Bacteria (SOB) in natural water body, which makes S input from external source2-Further enrichment in black and odorous water results in deterioration of water quality. S2-The method for removing pollutants in black and odorous water mainly comprises aeration reoxygenation, sediment dredging, reinforced coagulation, microbiological method and the like. The microbiological method has the advantages of strong pertinence, no secondary pollution to the environment and the like, and has been successfully applied to the treatment of medium and small river channels. At present, some research works have been carried out on the development of microbial agents aiming at the treatment of black and odorous water, mainly including Acinetobacter (Acinetobacter), Lactobacillus (Lactobacillus), Bacillus (Bacillus) and Pseudomonas (Pseudomonas). However, these microbial agentsTakes non-sulfur oxidizing bacteria as main materials and concentrates on COD and NH in water body by microbial inoculum3Research on removal effect of pollutants such as-N and TP, and rarely relates to a key pollutant S for blackening in black and odorous water2-The removal of (3) affected the study.
Disclosure of Invention
The inventor of the present application uses inorganic sulfur (S)2)-Screening out a strain capable of efficiently oxidizing S as a target pollutant2-The indigenous sulfur-oxidizing bacteria are beneficial to the purification of the black and odorous water body. Meanwhile, the inventors have further searched for a method for immobilizing the bacterium in order to ensure the effect of the bacterium.
In a first aspect, the invention provides a strain of Stenotrophomonas sp3 with a accession number of CGMCC No. 18394.
In a second aspect, there is provided a method of immobilizing stenotrophomonas sp3 of the present invention, comprising:
inoculating stenotrophomonas sp3 into a liquid culture medium, carrying out adsorption fixation at the temperature of 28-32 ℃ and the rotating speed of 130-150 r/min, wherein the initial immobilized pH value is 7.0-9.0, the adsorption time is 37.5-40 h, the inoculation amount of the strain is 5.0% -10.0% and the addition amount of the carrier is 0.5-2.0 g, preferably 1.0-1.5 g, based on 200ml of the liquid culture medium.
Preferably, the method comprises the steps of carrying out adsorption fixation at the rotation speed of 140r/min at the temperature of 30 ℃, wherein the initial pH of the immobilization is 8.0-8.1, the adsorption time is 37.4-37.5 h, the inoculation amount of thalli is 5.0-6.0% and the addition amount of a carrier is 0.98-1.0 g in terms of 200ml of liquid culture medium; preferably, the support is a zeolite; more preferably artificial zeolite particles.
Most preferably, the initial pH of immobilization is 8.1, the adsorption time is 37.4h, the inoculum size is 6.0%, and the artificial zeolite particles are 0.98 g.
The artificial zeolite particles may be commercially available artificial zeolite particles.
Preferably, the multiple regression equation of the immobilization method is:
Y＝82.23+1.54A+0.51B+1.65C+0.096D-3.14AB-3.06AC-1.93AD-2.73 BC-2.31BD-1.66CD-4.62A2-4.05B2-5.78C2-4.75D2
wherein Y represents S2-The oxidation rate, A is the initial pH of immobilization, B is the adsorption time in hours, C is the inoculum size in grams, and D is the carrier addition.
Preferably, the liquid medium contains: 20g/L glucose, 10g/L peptone, 10g/L yeast extract powder, pH7.0.
In a third aspect, there is provided a composition prepared using the immobilization method described above. Preferably, the composition is a bacterial agent comprising a vector and stenotrophomonas sp3.
In the present invention, "%" used in reference to the glucose concentration and the amount of inoculation represents mass%, unless otherwise specified.
The stenotrophomonas can efficiently oxidize S2-Effectively reduce S in the water body2-The content of the active carbon in the black and odorous water can be improved, and the black and odorous water has obvious treatment and purification effects.
By utilizing the immobilization method, stenotrophomonas sp3 can be efficiently immobilized, and the immobilized microbial inoculum can obviously improve the S in the stenotrophomonas black-smelly water body oxidized by the stenotrophomonas2-。
The stenotrophomonas sp3 is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of Xilu No.1 of Beijing republic of south China, the preservation number is CGMCC No.18394, and the preservation time is 2019, 8 and 19 days.
Drawings
FIG. 1: strain sp3 phylogenetic tree;
FIG. 2: temperature (A), initial pH (B), initial glucose concentration (C) and initial bacterial concentration (D) for growth of strain sp3 and S2-The effect of oxidation rate;
FIG. 3: growth Curve and S of Strain sp32-An oxidation profile;
FIG. 4: change in inorganic sulfur concentration in each presence, a: experimental groups; b: a control group;
FIG. 5: strain sp3 vs S2-The major pathway of biological oxidation of (a);
FIG. 6: the removal effect of the strain sp3 on pollutants in a water sample of Beijing east Shahe is achieved;
FIG. 7: influence of immobilized initial pH value on effect of microbial inoculum on black and odorous water body treatment (A: S)2-The oxidation rate; b: COD and NH3-removal rate of N and TP);
FIG. 8: influence of immobilization adsorption time on effect of microbial inoculum on treatment of black and odorous water body (A: S)2-The oxidation rate; b: COD and NH3-removal rate of N and TP);
FIG. 9: influence of inoculum size on black and odorous water treatment effect of microbial inoculum (A: S)2-The oxidation rate; b: COD and NH3-removal rate of N and TP);
FIG. 10: influence of the addition amount of the carrier on the effect of the microbial inoculum on treating black and odorous water (A: S)2-The oxidation rate; b: COD and NH3-removal rate of N and TP);
FIG. 11: a contour map and a response surface map; wherein the content of the first and second substances,
a: interaction of two factors of pH and time on S2-A contour plot of oxidation rate effects;
b: interaction of two factors of pH and time on S2-A response surface map influenced by oxidation rate;
c: interaction of two factors of pH and inoculum size on S2-A contour plot of oxidation rate effects;
d: interaction of two factors of pH and inoculum size on S2-A response surface map influenced by oxidation rate;
e: interaction of two factors of pH and carrier addition amount on S2-A contour plot of oxidation rate effects;
f: interaction of two factors of pH and carrier addition amount on S2-A response surface map influenced by oxidation rate;
g: time and inoculum size pairwise factor interaction pair S2-A contour plot of oxidation rate effects;
h: time and inoculum size pairwise factor interaction pair S2-A response surface map influenced by oxidation rate;
i: interaction of two factors of time and carrier addition amount on S2-A contour plot of oxidation rate effects;
j: interaction of two factors of time and carrier addition amount on S2-A response surface map influenced by oxidation rate;
k: interaction of two factors of inoculation amount and carrier addition amount on S2-A contour plot of oxidation rate effects;
l: interaction of two factors of inoculation amount and carrier addition amount on S2-A response surface map influenced by oxidation rate;
FIG. 12: scanning electron micrographs of zeolite particles before and after immobilization of the microbial inoculum, A: before fixing the adsorption microbial inoculum; b: after the adsorbed microbial inoculum is fixed.
Detailed Description
The present invention will be further illustrated by the following examples, but the present invention is not limited thereto.
Unless otherwise specified, the reagents and apparatus used in the following examples are conventional in the art and are commercially available. The methods used are conventional methods, and the person skilled in the art can clearly know how to carry out the described protocol and obtain the corresponding results according to the implementation details.
The first embodiment is as follows: screening and enrichment of stenotrophomonas
1. Collecting bottom mud from Tosha river in Beijing, mixing with water sample at equal ratio, and taking the mud-water mixture as the source of microorganism enrichment and separation culture. The water sample is collected in the surface water of the east sand river, and is filtered by a membrane of 10 mu m, and large suspended particles, algae, duckweed and other impurities are removed. The sediment is collected underwater by a sediment grab bucket for 0.5-1.0 m, and impurities such as stones, animal and plant residues and the like are removed. And multi-point arrangement is carried out during sampling, so that the diversity of bacteria sources is ensured.
2. The culture media used in the experiment are respectively enrichment culture media: na (Na)2S·9H2O0.162 g/L, soluble starch 2g/L, KNO30.1g/L，K2HPO40.05g/L，NaCl 0.05g/L，MgSO4·7H2O 0.05g/L， FeSO40.001g/L, 2% of nutrient agar; separating and purifying the culture medium: na (Na)2S·9H2O 0.162g/L，KNO30.1g/L，K2HPO40.05g/L，NaCl0.05g/L，MgSO4·7H2O 0.05g/L，FeSO40.001g/L, 20g/L glucose, 10g/L peptone, 5g/L yeast extract powder and 15-20 g/L agar; liquid culture medium: 20g/L of glucose, 10g/L of peptone and 10g/L of yeast extract powder. Each medium was adjusted to pH7.0 before use and sterilized at 100MPa and 121 ℃ for 20 min.
3. Diluting 5mL of the slurry mixture prepared in step 1 in 500mL of 5% sterile NaCl solution, and inoculating the diluted solution in enrichment medium (S)2-Concentration of 21.6mg/L) and culturing at 25 ℃ for 2-3 days. After sulfur oxidizing bacteria grow in a large amount in a culture medium, selecting a single colony with good growth vigor on a separation and purification culture medium for streaking, and culturing at the constant temperature of 25 ℃ for 2-3 d. Continuously and repeatedly carrying out lineation separation until all bacterial colony characteristics on the flat plate are consistent, namely the sulfur oxidizing bacteria are obtained, and the sulfur oxidizing bacteria can efficiently oxidize the black and odorous water body blackening key pollutant S2-S in the indigenous sulfur-oxidizing bacteria of2-On a medium at a concentration of 21.6mg/L, S2-The oxidation rate can reach more than 60 percent, and the catalyst is named as sp3.
4. Preparing seed liquid and expanding culture fermentation. Selecting separated and purified sulfur oxidizing bacteria by using an inoculating loop, inoculating the sulfur oxidizing bacteria into a conical flask filled with 100mL of liquid culture medium, culturing at the constant temperature of 25 ℃ for 2 days at the rotating speed of 120r/min to serve as seed liquid, transferring the seed liquid into a fresh liquid culture medium according to the inoculation amount of 5%, culturing for 2 days in a constant-temperature shaking table at the temperature of 25 ℃ and 120r/min, and fermenting in batches for sulfur oxidizing experiments of black and odorous water bodies.
Example two: identification of strains
1. 16S rRNA sequencing: the total DNA of the sulfur oxidizing bacteria obtained in the first embodiment is extracted by using an Ezup column type bacterial genome DNA extraction kit SK8255 of Biotechnology engineering (Shanghai) GmbH, and the specific experimental steps are carried out according to the kit instructions. Primer sequences were designed 27F (AGTTTGATCTMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). The total DNA of the bacteria is used as a template, PCR amplification is carried out, and the length is 1500 bp. Finally, the sequencing work is completed by the Biotechnology engineering (Shanghai) GmbH. And (3) carrying out homology analysis on the sequencing result of the base sequence through a Blast program and the nucleotide data in GenBank to construct a phylogenetic tree.
The results showed that the 16S rRNA gene sequence of strain sp3 has a homology of 99% or more with the multi-strain Stenotrophoromonas. For this purpose, strain sp3 was identified as a genus Stenotrophoromonas. To further analyze the phylogenetic position of strain sp3, the 16S rRNA gene sequences of the 10 named pure cultured strains with the highest similarity were selected, and phylogenetic trees were constructed using Mega7 software, with the results shown in FIG. 1.
2. And (3) morphological and physiological and biochemical characteristic experiments.
(1) And (3) observing colony morphology: and (3) coating the sulfur-oxidized single strain obtained by separation and purification on a solid culture medium by adopting a plate dilution method, culturing at a constant temperature of 25 ℃ for 2-3 d, and observing and recording the characteristics of bacterial colonies such as surface morphology, size, color and the like after the bacterial colonies grow out.
The strain sp3 is a gray yellow opaque circular colony after being cultured on nutrient agar at a constant temperature of 25 ℃, the edge is smooth, the part is irregular, the texture is viscous, the colony has ammonia smell, the diameter is 0.5-1 mm, and the center is protruded.
(2) Physiological and biochemical characteristic experiment of the strain: the experiment of the physiological and biochemical characteristics of the sulfur oxidizing bacterial strain is completed by referring to the manual of identifying the common bacteria system compiled by Dongxu pearl et al and the microbiological experiment compiled by Shennu et al.
The gram-negative bacterium is observed as a red strain sp3 in gram-stain test, and the bacterium body is tiny and takes the shape of a short rod. The results of physiological and biochemical property experiments show that the strain sp3 has lysine decarboxylase activity, can reduce nitrate and utilize maltose, and can hydrolyze esculin and liquefied gelatin. It can be seen that strain sp3 has similarities with the individual morphological and physiological biochemical properties of Stenotrophormonastrapedia. And combining the sequencing identification result of 16S rRNA of the strain sp3 to preliminarily determine that the strain sp3 is stenotrophomonas. Stenotrophomonas metabolic functions are various, most of which are shown in researches on drug resistance to antibiotics, degradation of organophosphorus, degradation of DDT and the like, and few reports are made on sulfur oxidation function researches.
Example three: growth of Strain sp3 and verification of Sulfur Oxidation characteristics
1. Containing S2-Preparation of waste water
The experiment adopts artificially configured S2-And (4) waste water. According to Zhuang et al (Zhuang R, Lou Y, Qiu X, et. identification of a yeast strand able to oxidize and remove a swine high affinity [ J].Applied Microbiology&Biotechnology,2017,101(1): 391-400) describes the optimization of water distribution composition, the main components are shown in Table 1. Containing S2-And sterilizing the prepared wastewater for later use. The sterilization condition is 100MPa, and the sterilization is carried out for 20min at 121 ℃. Subsequent experiments were conducted on this wastewater basis.
TABLE 1 contains S2-Artificially prepared waste water
2. Optimization of sp3 growth conditions and oxidation conditions
2.1 inoculating sulfur oxidizing bacteria sp3 grown to stationary phase in the culture expanding fermentation experiment of step 4 of the example into a culture medium containing 200mL of S2-In a conical flask for artificially preparing wastewater, a biological oxidation reaction was carried out at 120r/min, and the results were examined for the temperature (5, 15, 20, 25, 30, 35 ℃ C., other conditions: initial pH7.0, initial glucose concentration 0.25%, initial bacteria concentration 1.00g/L), pH (4, 5, 6, 7, 8, other conditions: temperature 25 ℃, initial glucose concentration 0.25%, initial bacteria concentration 1.00g/L), glucose concentration (0.05%, 0.10%, 0.25%, 0.50%, 1.00%, other conditions: temperature 25 ℃, initial pH7.0, initial bacteria concentration 1.00g/L) and initial bacteria concentration (0.01, 0.10, 1.00, 2.00, 5.00 g/L), other conditions: temperature 25 ℃, initial pH7.0, initial glucose concentration 0.25%) versus S2-Influence of Oxidation Rate, determination of bacterial growth OD during the reaction600The sp3 pair S is determined2-The optimum biological oxidation condition of (1). At optimum S2-Under oxidizing conditions, by measuring fineness at regular intervalsAmount of growth and residual S2-Concentration, growth curve and S of sulfur oxidizing bacteria sp3 were plotted2-Oxidation profile.
2.2sp3 vs. S2-The biological oxidation pathway of (2) is studied under the optimum sulfur oxidation conditions. Experiment design 2 groups of biochemical reaction devices with the volume of 5L, and each group is provided with 3 parallel experiments. Inoculating sulfur oxidizing bacteria to S-containing bacteria at an inoculation amount of 1g/L2-Artificially preparing wastewater (experimental group), and setting up a control group without adding sulfur oxidizing bacteria. Sampling at fixed time in the reaction process, and determining S in water sample2-、S0、S2O3 2-、S4O6 2-、SO3 2-And SO4 2-Mass concentration of (2).
3. The experimental results are as follows:
(1) temperature vs. growth of the strain and S2-Influence of Oxidation Rate: as shown in A of FIG. 2, the amount of growth of the strain sp3 and the amount of growth of the strain S2-The oxidation rate of (A) increases with the temperature, and reaches the highest at 25 ℃, when S is2-The oxidation rate of the catalyst is as high as 85.2 percent. When the temperature was further increased to 35 ℃, the amount of growth of strain sp3 and the amount of para-S2-The oxidation rate of (2) is slightly decreased. It can be clearly shown that the strain sp3 is mesophilic and can carry out various metabolic activities under the condition of proper temperature, especially at 25 ℃. Thus, the temperature was controlled to 25 ℃ to S2-The oxidation of (3) is preferable.
(2) Initial pH value to Strain growth and S2-Influence of Oxidation Rate: as shown in B of FIG. 2, the amount of growth of the strain sp3 and the amount of growth of the strain S2-The oxidation rate of (A) shows a trend of increasing and then significantly decreasing with the initial pH value from strong acidity to weak alkalinity. When the initial pH value is within the range of 6-7, the growth of the strain sp3 is good, and S is2-The oxidation rate is also higher, and all the oxidation rates reach about 85 percent. The alkaline condition is not suitable for the growth of the strain sp3 and is not beneficial to S2-Biological oxidation of (2). Therefore, it is preferable that the initial pH is 7 for the growth of the strain sp3 and for S2-Biological oxidation of (2).
(3) Initial glucose concentration vs. strain growth and S2-Influence of Oxidation Rate: as shown in C of FIG. 2, when startingWhen the glucose concentration is increased from 0.05% to 0.25%, the growth amount of the strain sp3 is remarkably increased. This indicates that glucose is a carbon source available for strain sp3 and can promote the growth and propagation of bacteria. When the initial glucose concentration continued to increase to 1%, the growth of strain sp3 tended to slow, since the glucose uptake capacity of the microbial cell membrane approached saturation. S2-Shows a trend of increasing and then decreasing oxidation rate as the initial glucose concentration increases. At an initial glucose concentration of 0.25%, strain sp3 was paired with S2-Has the strongest biological oxidation ability, S2-The oxidation rate of the catalyst reaches about 83 percent. Therefore, the optimum initial glucose concentration is 0.25%.
(4) Initial bacterial concentration vs. growth of the strain and S2-Influence of Oxidation Rate: as shown in D of FIG. 2, the initial bacteria concentration is the effect S2-One of the important factors of the removal efficiency. When the initial bacteria concentration is increased from 0.01g/L to 1.00g/L, S2-The oxidation rate of (2) was highest at 81%. After continuing to increase the initial bacterial concentration, strain sp3 was paired with S2-The oxidation remains substantially stable. Therefore, the optimum initial bacteria concentration is 1.00 g/L.
To sum up, S2-The optimum conditions for oxidation are: the pH value is 7 at 25 ℃, the initial concentration of glucose is 0.25 percent, and the initial bacteria concentration is 1.00 g/L. Under these conditions, strain sp3 is paired with S2-The highest oxidation rate can reach 86.9%.
(5) Growth curve and S of Strain sp32-Oxidation curve: culturing strain sp3 under optimum conditions for 72h to obtain S2-The residual concentration and the growth amount of the strain of (A) are shown in FIG. 3. The growth curve of the strain sp3 conforms to the colony growth rule of bacteria. The first 16h is the lag phase of the strain. With addition of the strain, S2-The residual concentration begins to drop rapidly. After 16h, the strain started to grow rapidly, reaching a maximum by 35 h. Thus, this 19h is the log phase growth of the strain. During this time, the strain pair S2-The oxidation reaction of (2) is continued to promote S2-The remaining concentration of (c) continuously decreases. And the number of the strain sp3 is in a basically stable state in 35-60 h, and the growth of the strain enters a stable phase. In strain sStationary phase of p3, S2-The residual concentration of (A) slowly decreases to a minimum value of 2.9mg/L at 60h, S2-The oxidation rate is as high as 86.6%. Thereafter, the number of strains began to rapidly decrease, and strain sp3 was in the decline phase. And S2-Remains in a substantially stable state during the decay phase of strain sp3. It is evident that the strain sp3 is S2-Occurs in the first 35h, i.e. the lag phase and logarithmic growth phase of strain sp3.
(6) Strain sp3 vs S2-The biological oxidation process of (2): s 2-6 different forms of presence may be involved in the biological oxidation process, including S2-、S0、S2O3 2-、S4O6 2-、SO3 2-And SO4 2-. Therefore, it is possible to estimate S by measuring the change in the form of the 6 kinds of inorganic sulfur2-The biological oxidation process of (1). Under optimum conditions, S2-The morphological change of sulfur present during the oxidation of strain sp3 is shown in A of FIG. 4. With S2-The biological oxidation process is carried out, and inorganic sulfur with different existing forms, including S, is detected2-、S0、S2O3 2-、SO3 2-And SO4 2-And the variation of these inorganic sulfur concentrations showed significant differences throughout the reaction. Due to S2-As an electron donor, is oxidized into inorganic sulfur with other valence states, and the concentration of the inorganic sulfur rapidly decreases in the first 10 h; then slowly decreases and gradually becomes gentle, and after reacting for 30 hours, S in a water sample2-The residual concentration of (A) was 3.0mg/L, and the oxidation rate thereof reached 86.1%. S0、S2O3 2-And SO3 2-The concentration of (A) shows obvious fluctuation along with the progress of the reaction, which shows that the inorganic sulfur exists in the existing form and is generated and consumed in 2 processes simultaneously in the reaction process. S0And SO3 2-The concentrations all show a trend of increasing first, then decreasing and then increasing, and their concentrations reach maximum values at 18 and 45h respectively, which may be similar to S in the first 18h2-Is concerned with the oxidative consumption. S2O3 2-The concentration of (A) shows a trend of increasing first and then decreasing and gradually tending to be smooth, and the concentration of the (A) takes a maximum value at 8h and is 11.2 mg/L. And S2-In the opposite direction, SO4 2-The concentration of (A) was continuously increased during the reaction, and finally reached 9.2 mg/L.
The above results show that strain sp3 is S2-The oxidation process of (A) is to convert S in an unstable state2-Gradually converted into a stable SO4 2-The process of (1). With the concomitant production of a series of intermediate forms of inorganic sulfur. The final partial conversion of these inorganic sulfur into SO4 2-. Note that S was not detected in the whole reaction4O6 2-. In addition, in the control group to which the strain sp3 was not added, S2-Shows a slightly decreasing tendency, and S is detected at an extremely low concentration2O3 2-And SO4 2-Generated as shown in B of fig. 4. Other forms of inorganic sulfur present were not detected during the reaction. It can be shown that the addition of strain sp3 significantly promotes S2-Biological oxidation of (2).
Example four: sulfur oxidizing bacteria sp3 biological oxidation pathway analysis of sulfur ions
Genome preparation and sequencing: the total DNA extraction of sulfur oxidizing bacteria is performed by Omega of America
DNAkit kit is used for extracting sample DNA, and the construction of PE library adopts TruSeq of Illumina corporation of AmericaTMDNA SamplePrep Kit, bridge PCR establishment using Illumina company HiSeq PE Cluster Kit v4cBot, USA, and Illumina Hiseq sequencing process. The genome sequencing is preferably carried out by Shanghai Meiji biological medicine science and technology Limited.
Genome assembly and annotation: performing quality shearing work on the Reads obtained after the sequencing is completed to remove some low-quality Reads generated in the original sequencing process of the Illumina Hiseq, and splicing and assembling the optimized sequence by using IDBA-UD software to obtain the most preferable assembling result. Bacterial gene prediction is carried out by using Glimer 3.02 software, and the protein sequences of the predicted Genes are subjected to blastp comparison with databases such as Nr, Genes, String and Go respectively, so as to obtain annotation information of the predicted Genes.
As a result:
the genome sequencing result shows that the total base number is 1047369985bp, and the average sequencing depth is 204X. Splicing Reads into Contigs according to the cut data; contigs are then arranged together in order to form the Scafolds. The analysis and gene prediction of Scaffolds after assembly of strain sp3 were performed using Glimmer 3.02 software. The obtained indexes such as the number of genes, the total length of the genes, the GC content and the like are counted, and the results are shown in Table 2.
The study led to 2 major routes for oxidation of inorganic sulfur by SOB. One is the PSO (Paracoccus Suporus Sulfur Oxidation) pathway, i.e. from S0Oxidation to SO3 2-Reoxidized to SO4 2-Sulfur oxidation pathway (c). The other is the S4I (tetrathionate) pathway, the tetrasulfide intermediate pathway with the concomitant production of polysulfates. S4O6 2-Is only present in the S4I pathway and is an important intermediate metabolite in this pathway. The 2 pathways PSO and S4I both have 3 stages of oxidation process, including S2-To S0Low valent inorganic sulfur to SO3 2-，SO4 2-The production of (D) involves key enzymes such as sulfide quinone oxidoreductase (SQR), Sulfite Reductase (SRN), thiosulfate-sulfur transferase (TST), isodisulfide reductase (HDR), tetrathionate hydrolase (TTH), and Sulfite Oxidase (SO). Among them, TTH enzyme exists only in S4I pathway, and acts to convert S4O6 2-Conversion to SO4 2-The function of (1).
TABLE 2 test results of sp3 Gene of Strain
According to the description of related genes in sulfur oxidation metabolic pathways in public database KEGG reported in the prior literature, 31 functional genes related to inorganic sulfur biological oxidation process are detected in strain sp3 according to the genome sequencing result, and are shown in Table 3. Previous studies have shown that part of the sulfur oxidation function genes detected in strain sp3 are also present in other SOBs, such as the sufES gene encoding cysteine desulfhydrase, the hdrA gene encoding pyridine nucleotide disulfide oxidoreductase, the TST gene encoding TST enzyme, the nqor gene encoding nad (p) H-ubiquinone oxidoreductase, and the stp gene encoding sulfate ABC transporter permease. In addition, several other functional genes associated with sulfur oxidation were detected, including the cysIJ gene encoding SRN enzyme, the sox gene encoding FAD flavin adenine dinucleotide, the cybB gene encoding electron transporters, and the sox gene cluster encoding SO enzyme. Although the SQR gene encoding the SQR enzyme was not detected in the sequencing results, the hdRA gene encoding the pyridine nucleotide disulfide oxidoreductase was detected. This gene has also been shown to function as a coding SQR enzyme.
The results of the gene sequencing showed that the tetH gene encoding the tetetrasulfate hydrolase TTH was absent from the strain sp3, and that no intermediate S was detected in combination4O6 2-It can be presumed that the strain sp3 is paired with S2-The oxidation of (2) may only present the PSO pathway, as shown in figure 5. The inorganic sulfur species involved in this pathway comprises S2-、S0、S2O3 2-、SO3 2-And SO4 2-。S2-The main oxidation pathway under the action of strain sp3 is presumed as follows: a portion of the substrate S2-Oxidized to S as an electron donor0The process involves the hdrA gene encoding the SQR enzyme and the soxF gene encoding FAD flavin adenine dinucleotide; another part of the substrate S2-Direct oxidation to SO under the control of the cysIJ gene encoding the SRN enzyme3 2-(ii) a S formed during oxidation0With SO3 2-Can spontaneously react to generate S2O3 2-And S is2O3 2-Can carry out disproportionation reaction under the control of TST gene of coding TST enzyme to release SO3 2-And S0；SO3 2-Regulation in sox Gene Cluster encoding SO enzymeFurther oxidation to SO4 2-。
TABLE 3 genes related to the inorganic sulfur biological oxidation process in the genome of Strain sp3
The hdRA gene encodes an SQR enzyme which primarily catalyzes S2-Conversion to S0The function of (1). The cysIJ gene regulates S by controlling the synthesis of SRN enzymes2-To SO3 2-The process is reversible. Excess SO3 2-Can produce toxic action on microbial cells. And SO3 2-Reduction to S2-The reaction of (A) contributes to the reduction of SO3 2-Accumulation in the system and reduced risk of toxicity to the cells. S2-Direct oxidation to SO3 2-Are present in only a few SOBs. In addition, SRN enzyme pathway is also found in microorganisms with organic sulfide mineralization function and plays a role in regulating SO3 2-The function of (1). The result shows that the strain sp3 may also have the function of mineralizing organic sulfides and may become functional microorganisms for degrading odoriferous pollutants such as odoriferous mercaptans and thioethers in the black and odoriferous water body. S0Can also be directly oxidized into SO3 2-. This reaction requires the passage of the heterodisulfide reductase pathway, which is present in most SOBs. In this path, S0Is activated into a sulfane sulfur atom RSSH under the action of a thiol group RSH, and then is catalytically converted into SO by an isodisulfide reductase3 2-And RSH is newly formed. The process of RSSH production is regulated by the grx gene, while the heterologous disulfide reductase is encoded by the hdrBC gene. In the strain sp3, the grx gene was detected, but the hdrBC gene was not detected. This indicates that S may not be present in strain sp30→RSH→RSSH→SO3 2-The passage of (2). Thus, only S may be present in strain sp30With SO3 2-Spontaneous reaction therebetween, and S2O3 2-Disproportionation reaction of (1). SO (SO)3 2-Can be directly or indirectly oxidized into SO4 2-. In the direct oxidation pathway, the sox gene cluster, especially the soxC gene, plays an important role in controlling SO by regulating the synthesis of SO enzymes4 2-And (4) generating. And the indirect oxidation pathway refers to SO3 2-Oxidation to SO by the Adenosine Phosphate Sulfate (APS) pathway regulated by the Apr Gene4 2-. Since the Apr gene was not detected in the strain sp3, SO3 2-The oxidation of (a) may only be a direct oxidation route.
Example five: experiment of sulfur oxidizing bacteria on treatment of black and odorous water body
The black smelly water sample is obtained from the black smelly river reach of Tosha river in Beijing, and the specific water quality parameters are shown in Table 4. The cultured sulfur oxidizing bacteria are inoculated into a conical flask filled with 200mL of black and odorous water sample in the inoculation amount of 1 g/L. Meanwhile, a control group without adding sulfur oxidizing bacteria is set, and each group is provided with 3 parallel experiments. Reacting for 60 hours at the rotating speed of 120r/min and the temperature of 25 ℃, and measuring S2-Oxidation rate of (2), and COD, NH3-removal of N, TP and chroma.
TABLE 4 Main Water quality parameters of Black and odorous Water samples
The determination indexes in the water sample comprise S2-、S0、S2O3 2-、S4O6 2-、SO3 2-、SO4 2-、COD、NH3-N, TP, chroma and OD600。S2-And S0Determined by spectrophotometry, S2O3 2-、SO3 2-And SO4 2-Measured by ion chromatography (CIC-D120, John, Qingdao). S4O6 2-The measurement was carried out by high performance liquid chromatography (LC-10AD, Shimadzu liquid). COD and NH3N was measured using a HACH multiparameter water quality tester (DB2800,united states hash), TP was measured using an ultraviolet spectrophotometer (UV-1800, shimadzu, japan). Chroma evaluation reference dilution multiple method (State environmental protection administration Water and wastewater monitoring and analysis method, ed. Commission, Water and wastewater monitoring and analysis method [ M ]]Version 4. chinese environmental science press, 2002). The growth amount of sulfur oxidizing bacteria depends on the screening of one DDT degrading bacteria of Von-jade snow, etc. (Von-jade snow, meticulous, Lymomone) and its degrading characteristics]Determined by the bacterial count method described in environmental science, 2018, (5): 1935-1942), i.e.as OD600The value represents the amount of growth of the bacteria.
Each set of experiments was set with 3 parallel samples, and each set of experimental data was averaged over 3 measurements. Data processing and analysis were performed using OriginPro 9.0 and Microsoft Excel 2013 software, respectively.
The effect of the strain sp3 on removing main pollutants in black and odorous water samples of Tosha river, Beijing is shown in FIG. 6. The results show that the strain sp3 is applied to S in the actual black and odorous water body2-Also exhibits better oxidizing ability. S2-The initial concentration of 12.9mg/L is reduced to 5.7mg/L, and the oxidation rate reaches 55.8%. Chromaticity in water sample following S2-The concentration is reduced and the removal rate is higher than 50%. Meanwhile, strain sp3 is used for COD and NH3the-N and TP also have obvious removal effect, and the removal rate is respectively 38.9%, 45.4% and 44.1%. Control group S without addition of microbial inoculum sp32-The oxidation rate is only 5.1%, and the removal rate of pollutants such as COD is not obviously changed.
Example six: immobilization and condition optimization of sulfur oxidizing bacteria sp3
In this example, artificial zeolite particles are used as carriers, sp3 is immobilized on the artificial zeolite particles by an adsorption immobilization method, and the main factors of the immobilization process, such as the initial immobilization pH, the adsorption time, the inoculation amount, the carrier addition amount, and the like, are respectively examined for S in the black and odorous water body2-Oxidation rate, COD, ammonia nitrogen, TP removal rate and the like. Furthermore, on the basis of a single-factor experiment, main immobilization factors such as initial immobilization pH, adsorption time, inoculation amount and the like are further optimized by adopting a response surface method so as to obtain the optimal immobilization combination conditions of efficient sp3.
Experimental black odorous water sample:
the experimental black odorous water sample is collected from a typical black odorous river channel covering water in Shenyang city of Liaoning province, and is filtered by a 10-micron membrane to remove impurities and store. S2-The concentration of (A) is as high as 33.9mg/L in a black smelly water sample, while COD and NH are3N and TP are respectively 1.95, 7.8 and 4 times higher than the standard of surface water environment quality class V, and specific values of the conventional indexes are shown in Table 5.
TABLE 5 Black and odorous water sample Water quality index
Liquid culture medium:
the liquid media used in the immobilization experiments were as follows: 20g/L glucose, 10g/L peptone and 10g/L yeast extract powder. Each medium was adjusted to pH7.0 before use and sterilized at 100MPa and 121 ℃ for 20 min.
Immobilization single factor experiment:
1、pH
inoculating sp3 bacteria into 200mL liquid culture medium according to the inoculation amount of 5% (mass percent), adding 0.5g zeolite particles, adjusting the initial pH value to 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 by using HCl (1.0mol/L) or NaOH (1.0mol/L), and placing the mixture in a constant-temperature shaking incubator at the conditions of 30 ℃ and 140r/min for 48h of microorganism immobilization experiment.
2. Adsorption time
Sp3 is inoculated into 200mL of liquid culture medium according to the inoculation amount of 5%, 0.5g of zeolite particles are added into the liquid culture medium, and the liquid culture medium is placed in a constant-temperature shaking incubator to carry out microorganism immobilization experiments for 18h, 24 h, 30h, 36 h, 48h and 60h under the conditions of 30 ℃ and 140r/min rotation speed.
3. Amount of inoculation
The sp3 strain is inoculated into 200mL of liquid culture medium according to the inoculation amounts of 0.01%, 0.1%, 1.0%, 5.0%, 10.0% and 20%, 0.5g of zeolite particles are added, and the mixture is placed in a constant temperature shaking incubator at the temperature of 30 ℃ and the rotation speed of 140r/min for a microorganism immobilization experiment for 48 h.
4. Addition amount of carrier
Inoculating sp3 bacteria into 200mL of liquid culture medium according to the inoculation amount of 5%, then respectively adding 0.01, 0.1, 0.5, 1.0, 1.5 and 2.0g of zeolite particles with different masses, and placing the mixture in a constant-temperature shaking incubator at the temperature of 30 ℃ and the rotating speed of 140r/min for 48h of microbial immobilization experiment.
5. Response surface optimization experiment
On the basis of the results of the single-factor experiment, the main factors involved in 4 immobilization processes of the initial pH of immobilization, adsorption time, inoculation amount and carrier addition amount are used as independent variables, and the immobilized microbial agent is used for treating S in the black and odorous water sample2-The oxidation rate (Y) is a response value, and Box-Behnken center combined experimental Design is carried out by Design-expert 8.0.6 software, and the experimental Design factors and levels are shown in Table 6.
TABLE 6 Box-Behnken design factors and horizon table
6. Immobilization of bacteria agent for S in black and odorous water2-Oxidation experiment of
And centrifuging the immobilized bacteria liquid obtained in the single-factor experiment for 6min at 4 ℃ and 4000r/min, removing supernatant, and washing the surface of the immobilized bacteria agent for 3 times by using sterile deionized water to remove free microorganisms which are not immobilized on the surface of the zeolite particles. The obtained immobilized microbial inoculum is added into a black smelly water sample according to the mass concentration of 5.0g/L, and subjected to constant temperature oscillation reaction for 48h at the conditions of 25 ℃ and 120r/min rotation speed, and S is measured2-Oxidation rate, COD, NH3Removal rates of N and TP were determined by examining the pH, adsorption time, inoculum size, and carrier addition amount for sp3 oxidized S2-And removing COD and NH3Influence of-N and TP. The immobilized bacteria liquid treatment method obtained by the response surface optimization experiment is the same as a single-factor experiment. Optimal immobilization combination conditions for sp3 cells were obtained by optimization experiments. Each single-factor and response surface optimization experiment was repeated 3 times, and the average value was taken for data analysis.
Analytical method
Test index bag in water sampleDraw together S2-、COD、NH3-N and TP. All samples were filtered through a 0.45 μm membrane and then assayed. S2-The concentration is measured by methylene blue spectrophotometry, COD and NH3-N, TP was measured using a HACH multiparameter water quality tester. The microstructure of the immobilized microbial agent was observed by a scanning electron microscope (Quanta 200, FEI, netherlands). All data processing and analysis were performed using originPro 8.0, Design-expert 8.0.6 and SPSS 22.0 software, respectively.
Results and discussion
1. Immobilized initial pH vs S2-Influence of Oxidation Rate
pH is one of the important factors affecting the metabolic activity of microorganisms and the efficiency of pollutant treatment. Immobilized bacteria pair S prepared under different pH conditions2-The effect of the oxidation rate is shown in fig. 7A. As can be seen from the figure, when the pH was changed from strongly acidic to alkaline, the pair of immobilized bacteria S2-The oxidation rate of (a) shows a tendency of increasing first and then decreasing. When the pH value is in the range of 4.0-7.0, S2-The oxidation rate is improved from 23.9 percent to 68.9 percent; s2-The oxidation rate reached a maximum of 73.3% at a pH of 8.0. The pH value is continuously increased to 9.0, S2-The oxidation rate is significantly reduced to 61.6%. Immobilized bacteria agent pair COD and NH prepared under different pH conditions3The removal rates of-N and TP are shown in FIG. 7B. When the pH value is increased from 4.0 to 8.0, COD and NH are added3The removal rates of-N and TP are obviously improved and reach the highest values of 31.2%, 47.0% and 30.4%, respectively. However, when the pH value is increased from 8.0 to 9.0, COD and NH are present3The removal rates of-N and TP were significantly reduced to 23.1%, 38.1% and 15.2%, respectively. Immobilization of initial pH value to S2-Oxidation rate, COD, NH3The removal rates of N and TP have obvious influence, and both of them show the trend of increasing and then decreasing with the increase of pH value. Therefore, the sp3 immobilization process is suitable for an initial pH of 7.0 to 9.0, and an optimum initial pH of 8.0 to 8.1.
2. Adsorption time pair S2-Influence of Oxidation Rate
Different adsorption times in the immobilization process for oxidizing S in black and odorous water sample by sp3 microbial inoculum2-The influence of (2) is shown in FIG. 8AAs shown. As can be seen from the graph, when the immobilization adsorption time was extended from 18 hours to 37.5 hours, the microbial inoculum pair S2-The oxidation rate of (1) is increased from 43.1% to 75.9%, S2-The oxidation rate of (A) reaches the highest value; while continuing to extend the immobilization adsorption time, S2-The oxidation rate of the catalyst is reduced, and the oxidation rate is reduced to 64.1% when the catalyst is used for 60 hours. The adsorption time is used for removing COD and NH from the immobilized bacteria agent3The efficiencies of-N and TP are shown in FIG. 8B. When the immobilized adsorption time is prolonged from 18h to 45h, the removal rate of COD by the immobilized bacteria agent is remarkably improved from 4.7 percent to 27.4 percent, and NH3The removal rate of N is improved from 19.6% to 48.2%; however, as the adsorption time continues to increase, the COD and NH3The removal rate of-N shows a decreasing trend. Different from COD and NH3The removal rate of-N, TP reached the highest value at 37.5h, which was 31.9%. The results show that the proper adsorption time can enhance the removal effect of the immobilized bacteria agent on the pollutants. The growth period of the strain and the consumption rate of the substrate in the immobilization process can influence the adsorption time, and when the strain is in a stable period and the amount of the substrate to be utilized is appropriate, the activity and the amount of the bacteria attached to the carrier can be improved. In addition, the adsorption time can affect the biofilm formed by microorganisms on the carrier, and the excessively long adsorption time can cause the thallus on the outer layer of the biofilm to fall off, thereby reducing the number of bacterial strains in the zeolite particle carrier. The results in FIG. 8 show that S can be significantly influenced by the immobilization adsorption time2-Oxidation rate, and COD and NH3-removal of N and TP. With the increase of the immobilization adsorption time, S2-Oxidation rate, and COD and NH3The removal rates of-N and TP show a tendency of rising first and then falling. Therefore, the sp3 immobilization process has an appropriate adsorption time of 30-45 h, more preferably 37.5-40 h, and an optimum adsorption time of 37.4-37.5 h.
3. Inoculum size pair S2-Influence of Oxidation Rate
Immobilization procedure inoculum size pair S2-The effect of the oxidation rate is shown in FIG. 9A. As can be seen from FIG. 9A, the immobilized microbial inoculum pairs S increased with the increase in the amount of microbial inoculum2-The oxidation rate shows a change trend of firstly rising rapidly and then gradually stabilizing. When the inoculation amount of the thalli is increased from 0.01% to 5%Immobilized bacteria agent pair S2-The oxidation rate of the catalyst is rapidly improved from 7.3 percent to 76.4 percent; s when the inoculation amount of the strain continues to increase to 20%2-The oxidation rate of (A) is kept stable and even slightly reduced. COD and NH3Trend of variation of the removal rates of N and TP and S2-The oxidation rates were similar as shown in fig. 9B. When the inoculation amount of the thalli is increased from 0.01% to 5%, COD and NH are added3The removal rates of-N and TP were increased to 27.8%, 48.2% and 26.3%, respectively; with continued increase in inoculum size, their removal rates remained steady and all declined slightly. Therefore, the sp3 immobilization process is preferably carried out at an inoculum size of 5% to 10%, and more preferably at an inoculum size of 5% to 6%.
4. Amount of carrier added to S2-Influence of Oxidation Rate
Amount of immobilized carrier added to S2-The effect of the oxidation rate is shown in fig. 10A. When the addition amount of the carrier is increased from 0.01g to 1g, the pair of immobilized bacteria agent S2-The oxidation rate of the catalyst is remarkably improved from 13.0 percent to 76.8 percent; on the other hand, when the amount of the carrier added was increased from 1g to 2g, S was observed2-The oxidation rate of (a) was reduced to 68.6%. COD and NH in black and odorous water3The removal of-N and TP as a function of the amount of zeolite particles added is shown in FIG. 10B. The removal rates of COD and TP are respectively increased and then decreased with the increase of the addition amount of the carrier, and the highest removal rates are respectively 31.2 percent and 30.8 percent. NH unlike the tendency of the removal rates of COD and TP3The removal of N increased rapidly from 7.7% to 50.6% with increasing zeolite addition from 0.01g to 1 g; NH when the zeolite addition was increased to 2g3The removal of-N continued to increase slowly to 60.7%. NH (NH)3The removal rate of-N increases with the addition amount of the carrier because the artificial zeolite has adsorbed NH3-function of N. Therefore, sp3. the amount of the carrier to be added is preferably 0.5 to 2.0g, more preferably 1.0 to 1.5g, and most preferably 1.0g, in the immobilization step.
5. Response surface optimization microbial inoculum immobilization combination condition
On the basis of an immobilized single-factor experiment, determining the content range of response surface optimization adopted by each factor, and designing a 4-factor 3-level response surface optimization experimentThe design schemes and the corresponding experimental results are shown in table 7. Performing multiple regression fitting on the data in the table by using Design expert8.0.6 software to obtain S2-The multiple regression equation of the oxidation rate (Y) to the initial immobilization pH (A), the adsorption time (B), the inoculum size (C), and the carrier addition amount (D) is as follows:
Y＝82.23+1.54A+0.51B+1.65C+0.096D-3.14AB-3.06AC-1.93AD-2.73 BC-2.31BD-1.66CD-4.62A2-4.05B2-5.78C2-4.75D2
table 7 Box-Behnken test design scheme and results for response surface
The results of analysis of variance on the multiple regression model are shown in Table 8. The analysis of the integral variance of the regression model shows that the F value is 25.061, and the P value is far less than 0.01, which indicates that the model is extremely significant and has statistical significance. The analysis of variance of the misfit term shows that the F value is 8.043, and P is 0.0566 and is more than 0.05, which shows that the model is reasonable, a higher-order term equation does not need to be fitted, the interference of unknown factors to the experimental result is small, and more independent variables do not need to be introduced. In addition, the determination coefficient of the model is 0.967, which shows that the regression equation is better fitted with the actual situation and can better reflect the S in the black and odorous water body2-The oxidation rate of (a) is related to the initial pH value of immobilization, adsorption time, inoculum size and the addition amount of the carrier. Therefore, the model can be used for analyzing and predicting S in the oxidized black and odorous water body2-Sp3 cell immobilization combination conditions of (1). The regression equation coefficient significance test result shows that the first-order terms A and C are extremely significant in the model, and the second-order terms B and D are not significant; second order term A2、B2、C2、 D2And the interactive terms AB, AC, AD, BC, BD, CD are all very significant. In the regression equation, the absolute values of the regression coefficients of the first order terms are C, A, B and D in descending order, indicating that the amount of inoculation is relative to black under the immobilized combination conditionS in the body of smelly water2-The oxidation rate of (a) has the greatest effect, followed by the immobilization initial pH, followed by the adsorption time and the carrier addition. In addition, the quadratic term and the interactive term are at an extremely significant level, which shows that 4 immobilization factors of the initial pH value of immobilization, adsorption time, inoculum size and the addition amount of the carrier are applied to S2-The influence of the oxidation rate is a quadratic term effect and an interaction effect, rather than a simple linear relationship.
TABLE 8 analysis of variance of regression model for response surface
Note: p <0.05 indicates significance, p <0.01 indicates extreme significance
According to the analysis of variance results of the previous regression model, each interactive item is at a very significant level. Therefore, response surface analysis is performed on the regression equation by using software Design Expert8.0.6, and a response surface contour map and a three-dimensional analysis map of all interaction terms are obtained, as shown in fig. 11. The closer the contour lines approach to an ellipse, the more remarkable the interaction between the contour lines and the ellipse; the denser the contour is, the pair S2-The more pronounced the oxidation rate effect. In the present invention, all contour plots are elliptical, further demonstrating significant interaction between factors. As can be seen from FIG. 11, in each interactive item, S is maintained under the condition that one of the immobilization factors is kept unchanged2-The oxidation rate is increased with another immobilization factor and then shows a trend of increasing first and then slowly decreasing. However, these 6 interaction item pairs S2-The magnitude of the effect of the oxidation rate showed a difference. Wherein the interaction of the initial pH value of immobilization and the adsorption time is to S2-The influence of the oxidation rate is greatest as shown in FIGS. 11A and 11B. When the inoculation amount and the addition amount of the carrier are respectively 5.5 percent and 1.0g, S is added under the condition of keeping the initial pH value of immobilization unchanged2-The oxidation rate firstly slowly rises and then slowly falls along with the increase of the adsorption time; but while maintaining adsorptionWhen the time is not changed, S2-The oxidation rate increases and then slowly decreases with the increase of the initial pH value of the immobilization.
As can be seen from FIG. 11, the response surface plots all exhibit a downwardly opening bell-jar shape, i.e., S increases with increasing levels of the factor2-The oxidation rate tends to increase first and then decrease, and has a maximum value. Quadratic equation of fitting by Design-expert 8.0.6 software to S2-And (3) analyzing and calculating the highest oxidation rate as a target to obtain the optimal sp3 thallus immobilization combination condition: the initial pH value of immobilization was 8.14, the adsorption time was 37.35h, the inoculum size was 6.02%, and the amount of artificial zeolite carrier added was 0.98 g. Under the condition, sp3 immobilized microbial inoculum is applied to S in black and odorous water body2-The predicted oxidation rate of the catalyst reaches 82.4 percent. Based on the analysis of variance results and in combination with the feasibility of practical operation, the optimal immobilization combination conditions are determined as follows: the initial pH value of immobilization was 8.1, the adsorption time was 37.4h, the inoculum size was 6.0%, and the amount of artificial zeolite carrier added was 0.98 g. In order to further verify the result of response surface prediction, 3 parallel verification experiments are carried out under the optimal immobilization condition, and the result shows that S2-The average value of the oxidation rate reaches 81.2%, the oxidation rate is close to 82.4% of the predicted value of the model, and the fitting rate of the actual value reaches more than 99%, which shows that the predicted value and the actual value of the model have good fitting performance, and the reliability of the optimized model is high. Furthermore, the non-immobilized sp3 cells before optimization were used to determine S in the actual black and odorous water sample2-Oxidation rate (55.8%) compared to optimized S2-The oxidation rate (81.2%) is improved by 47.7%, which shows that the optimization scheme determined by the experiment is reasonable and effective in design, and the obtained sp3 immobilized microbial inoculum can obviously improve S in the black and odorous water body2-The oxidation rate of (c).
The microstructure characterization of the zeolite particles before and after immobilization was performed by scanning electron microscopy, and the results are shown in fig. 12. As can be seen from fig. 12A, the zeolite particles without adsorbing the microbial inoculum have a rough surface, a large number of cavities and pore structures in the framework, a large specific surface area, and a large number of adsorption sites, which are favorable for adsorbing and storing microorganisms. From FIG. 12B, it can be observed that the short rod-shaped bacteria adhered to the surface of the zeolite particles, and other part of the bacteria are connected to form a sheet or aggregated to form a block on the inner wall, because the microbes excrete extracellular substances through metabolism and then quickly adhere to the surface of the zeolite particles and are connected to each other to form a biofilm structure, which promotes the bacteria to be more firmly adhered to the carrier. The surface of the zeolite particles after adsorption of the microorganisms becomes smoother, the pore structure is no longer so pronounced and the number is slightly reduced, probably because the microorganisms fill and cover most of the pores of the zeolite particles, compared to zeolite particles which have not been treated with an immobilization.
Claims (8)
1. A method of immobilizing stenotrophomonas sp3, comprising:
inoculating stenotrophomonas sp3 into a liquid culture medium, performing adsorption fixation at the temperature of 28-32 ℃ and the rotating speed of 130-150 r/min, wherein the initial immobilized pH value is 7.0-9.0, the adsorption time is 37.5-40 h, the inoculation amount of thalli is 5.0% -10.0% and the addition amount of a carrier is 0.5-2.0 g, preferably 1.0-1.5 g, based on 200mL of the liquid culture medium; wherein the preservation number of the stenotrophomonas sp3 is CGMCC No. 18394.
2. The immobilization method according to claim 1, wherein the method comprises carrying out adsorption immobilization at 30 ℃ and at a rotation speed of 140r/min, wherein the initial pH of immobilization is 8.0 to 8.1, the adsorption time is 37.4 to 37.5 hours, the inoculation amount of the cells is 5.0 to 6.0% and the addition amount of the carrier is 0.98 to 1.0g based on 200mL of the liquid medium.
3. The immobilization process according to claim 1 or 2, wherein the support is a zeolite, preferably artificial zeolite particles.
4. The immobilization method according to any one of claims 1 to 3, wherein the initial pH of immobilization is 8.1, the adsorption time is 37.4 hours, the inoculum size is 6.0%, and the artificial zeolite particles are 0.98 g.
5. The immobilization method according to any one of claims 1 to 4, wherein the multivariate regression equation of the immobilization method is:
Y＝82.23+1.54A+0.51B+1.65C+0.096D-3.14AB-3.06AC-1.93AD-2.73BC-2.31BD-1.66CD-4.62A2-4.05B2-5.78C2-4.75D2
wherein Y represents S2-The oxidation rate, A is the initial pH value of immobilization, B is adsorption time in unit hours, C is the inoculum size in unit percent, and D is the carrier addition amount in unit grams.
6. The immobilization method according to any one of claims 1 to 5, wherein the liquid medium contains: 20g/L glucose, 10g/L peptone, 10g/L yeast extract powder, pH7.0.
7. A composition produced by the immobilization method according to any one of claims 1 to 6.
8. The composition of claim 7, wherein the composition is a microbial inoculum comprising a vector and stenotrophomonas sp3.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2019107691130 | 2019-08-20 | ||
| CN201910769113 | 2019-08-20 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110791498A true CN110791498A (en) | 2020-02-14 |
| CN110791498B CN110791498B (en) | 2021-09-10 |
Family
ID=69285771
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911253920.3A Expired - Fee Related CN110885773B (en) | 2019-08-20 | 2019-12-09 | Method for treating or purifying black and odorous water quality |
| CN201911253930.7A Expired - Fee Related CN110791498B (en) | 2019-08-20 | 2019-12-09 | Immobilization method of stenotrophomonas and composition prepared by same |
| CN201911253937.9A Expired - Fee Related CN110747152B (en) | 2019-08-20 | 2019-12-09 | Stenotrophomonas, compositions comprising same and uses |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911253920.3A Expired - Fee Related CN110885773B (en) | 2019-08-20 | 2019-12-09 | Method for treating or purifying black and odorous water quality |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911253937.9A Expired - Fee Related CN110747152B (en) | 2019-08-20 | 2019-12-09 | Stenotrophomonas, compositions comprising same and uses |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN110885773B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115286119A (en) * | 2022-07-20 | 2022-11-04 | 西南科技大学 | Microorganism strengthening agent using mineral/biomass as carrier for removing hexavalent chromium and preparation method thereof |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101177680B (en) * | 2006-11-10 | 2010-09-29 | 中国科学院沈阳应用生态研究所 | Method for preparing mixed bacteria immobilized particle |
| KR20130058089A (en) * | 2011-11-22 | 2013-06-04 | 수원대학교산학협력단 | Stenotrophomonas sp. cw4-y capable of reducing odor and nitrification/de nitrification on aerobic condition and odor reduction mehod using it |
| CN103305439B (en) * | 2013-05-27 | 2015-06-03 | 上海阿斯沃特企业发展有限公司 | Stenotrophomonassp. H-4 and preparation method of degrading enzyme preparation thereof as well as application |
| CN104232546A (en) * | 2014-09-18 | 2014-12-24 | 松辽流域水资源保护局松辽流域水环境监测中心 | Construction method and application of immobilized biological bacterium agent for micro-polluted water source |
| CN106145383A (en) * | 2016-07-22 | 2016-11-23 | 中国环境科学研究院 | A kind of solid union microbial bacterial agent administered for black and odorous water |
| CN107937298B (en) * | 2017-06-08 | 2020-08-21 | 博元生态修复（北京）有限公司 | Compound microbial agent for treating black and odorous water body, and obtaining method and using method thereof |
| CN108911162B (en) * | 2018-07-06 | 2021-04-20 | 华南师范大学 | Black and odorous water body sediment dry powder repairing agent and preparation method and application thereof |
| CN109536413B (en) * | 2018-12-21 | 2020-06-23 | 浙江工业大学 | Stenotrophomonas HY-2 and application thereof in degradation of organic matters |
-
2019
- 2019-12-09 CN CN201911253920.3A patent/CN110885773B/en not_active Expired - Fee Related
- 2019-12-09 CN CN201911253930.7A patent/CN110791498B/en not_active Expired - Fee Related
- 2019-12-09 CN CN201911253937.9A patent/CN110747152B/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115286119A (en) * | 2022-07-20 | 2022-11-04 | 西南科技大学 | Microorganism strengthening agent using mineral/biomass as carrier for removing hexavalent chromium and preparation method thereof |
| CN115286119B (en) * | 2022-07-20 | 2023-09-12 | 西南科技大学 | Microorganism strengthening medicament for removing hexavalent chromium by taking minerals/biomass as carrier and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110747152B (en) | 2021-09-10 |
| CN110885773A (en) | 2020-03-17 |
| CN110885773B (en) | 2021-10-15 |
| CN110747152A (en) | 2020-02-04 |
| CN110791498B (en) | 2021-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110656059B (en) | Pseudomonas strain YG8, seed liquid and preparation method and application thereof | |
| CN110655198B (en) | Method for treating nitrogen-containing wastewater by using heterotrophic nitrification-aerobic denitrification paracoccus strain | |
| CN108342339B (en) | Klebsiella strain and application thereof in river sewage and rural ammonia nitrogen-containing domestic sewage | |
| CN113174345A (en) | Heterotrophic nitrification-aerobic denitrification strain for efficient denitrification and application thereof | |
| CN108018250B (en) | Acidithiobacillus ferrooxidans and application thereof in environmental treatment | |
| CN111172061B (en) | Aerobic denitrification composite microbial inoculant and application thereof | |
| KR102564256B1 (en) | Microorganism producing sulfur and method for preparing sulfur using the same | |
| CN108865940B (en) | Heterotrophic nitrification-aerobic denitrification bacillus and composite bacterial preparation thereof | |
| CN110656057B (en) | Heterotrophic nitrification-aerobic denitrification paracoccus strain, seed liquid, preparation method and application thereof | |
| CN113151034A (en) | Bacillus subtilis strain for deodorizing livestock and poultry breeding environment | |
| CN115125164B (en) | Highly salt-resistant and heavy metal-resistant heterotrophic nitrification-aerobic denitrification self-flocculation marine bacterium and application thereof in community construction | |
| CN114890555B (en) | Solid microbial preparation for treating rural black and odorous water body and preparation method and application thereof | |
| CN115786180A (en) | Pseudomonas stutzeri BBW831 and application thereof | |
| CN110791498B (en) | Immobilization method of stenotrophomonas and composition prepared by same | |
| Yang et al. | Isolation of sulfide remover strain Thermithiobacillus tepidarius JNU-2, and scale-up bioreaction for sulfur regeneration | |
| CN110655200B (en) | Method for treating nitrogen-containing wastewater by using pseudomonas strain YG8 | |
| CN108300675B (en) | Tenecium luteum for synchronously decarbonizing, nitrogen, phosphorus and surfactant and application of Tenecium luteum in sewage treatment | |
| CN111088200B (en) | Heterotrophic nitrification aerobic denitrifying bacterium and application thereof in pig-raising wastewater treatment | |
| CN101434905A (en) | Method for screening nitrification bacteria | |
| Pol et al. | Isolation of a carbon disulfide utilizing Thiomonas sp. and its application in a biotrickling filter | |
| CN106957806B (en) | Biological composite degradation agent under anaerobic condition and application thereof | |
| CN116004459A (en) | Rhodococcus YZ-1 and application thereof in degrading organic pollutants | |
| CN111592995A (en) | Acinetobacter and culture method and application thereof | |
| CN112300961B (en) | Quinoline degrading strain and application thereof in quinoline-containing wastewater | |
| CN111826302B (en) | Iron oxidizing bacteria, and application thereof in arsenic-polluted soil remediation, product and method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210910 |