CN115109725A - Method for enriching homoacetogenic bacteria at normal temperature and application thereof - Google Patents
Method for enriching homoacetogenic bacteria at normal temperature and application thereof Download PDFInfo
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- 230000003403 homoacetogenic effect Effects 0.000 title claims abstract description 90
- 241000894006 Bacteria Species 0.000 title claims abstract description 87
- 238000000034 method Methods 0.000 title claims abstract description 50
- 239000010802 sludge Substances 0.000 claims abstract description 113
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 63
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 61
- 239000004280 Sodium formate Substances 0.000 claims abstract description 56
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical compound [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 claims abstract description 56
- 235000019254 sodium formate Nutrition 0.000 claims abstract description 56
- 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 abstract description 39
- 239000008103 glucose Substances 0.000 claims abstract description 39
- 230000007704 transition Effects 0.000 claims abstract description 34
- 239000010865 sewage Substances 0.000 claims abstract description 12
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 6
- 230000000789 acetogenic effect Effects 0.000 claims abstract description 5
- 238000000855 fermentation Methods 0.000 claims description 38
- 230000002829 reductive effect Effects 0.000 claims description 7
- 239000002028 Biomass Substances 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 12
- 239000003112 inhibitor Substances 0.000 abstract description 6
- 230000000696 methanogenic effect Effects 0.000 abstract description 4
- 238000004064 recycling Methods 0.000 abstract description 2
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- 235000011054 acetic acid Nutrition 0.000 description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 16
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- 235000019253 formic acid Nutrition 0.000 description 9
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 244000005700 microbiome Species 0.000 description 8
- 229910052757 nitrogen Inorganic materials 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 7
- 229910052698 phosphorus Inorganic materials 0.000 description 7
- 239000011574 phosphorus Substances 0.000 description 7
- 235000013619 trace mineral Nutrition 0.000 description 6
- 239000011573 trace mineral Substances 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000589220 Acetobacter Species 0.000 description 4
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 235000014113 dietary fatty acids Nutrition 0.000 description 4
- 238000005265 energy consumption Methods 0.000 description 4
- 229930195729 fatty acid Natural products 0.000 description 4
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- 150000004665 fatty acids Chemical class 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
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- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 4
- 230000002572 peristaltic effect Effects 0.000 description 4
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- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 3
- 229910003208 (NH4)6Mo7O24·4H2O Inorganic materials 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 2
- 241000589651 Zoogloea Species 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- 241001468161 Acetobacterium Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 150000001243 acetic acids Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000001569 carbon dioxide Substances 0.000 description 1
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- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000002401 inhibitory effect Effects 0.000 description 1
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- 230000014759 maintenance of location Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- ZNCPFRVNHGOPAG-UHFFFAOYSA-L sodium oxalate Chemical compound [Na+].[Na+].[O-]C(=O)C([O-])=O ZNCPFRVNHGOPAG-UHFFFAOYSA-L 0.000 description 1
- 229940039790 sodium oxalate Drugs 0.000 description 1
- 229940005605 valeric acid Drugs 0.000 description 1
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Abstract
The invention relates to a method for enriching homoacetogenic bacteria at normal temperature and application thereof, belonging to the technical field of sludge biological treatment and recycling. The method for enriching homoacetogenic bacteria at normal temperature comprises an initial stage, a transition stage and an enrichment stage; the initial stage utilizes glucose; in the transition stage, glucose and sodium formate are used as carbon sources, and the adding amount of the sodium formate is gradually increased along with the increase of the culture days; in the enrichment stage, sodium formate is used as a carbon source to realize enrichment of homoacetogenic bacteria in the inoculated sludge; the culture temperature of the initial stage, the transition stage and the enrichment stage is 23-25 ℃. The method has the advantages that additional BES methanogenesis inhibitor is not needed to be added in the process of enriching acetogenic bacteria to inhibit methanogenic bacteria in the sludge and external temperature control measures are not needed, the method can be applied to actual resource utilization of excess sludge and sewage treatment on a large scale, and the applicability is strong.
Description
Technical Field
The invention relates to the technical field of sludge biological treatment and recycling, in particular to a method for enriching homoacetogenic bacteria at normal temperature and application thereof.
Background
At present, a large amount of excess sludge is generated in the sewage treatment process, the excess sludge is an aggregate consisting of zoogloea formed by a plurality of microorganisms and organic matters and inorganic matters adsorbed by the zoogloea, and the organic content is high; the resource utilization of the excess sludge is carried out, and the excess sludge is developed to be used as an organic carbon source in the processes of biological phosphorus removal and biological denitrification nitrogen removal in sewage treatment and a raw material in industrial synthesis, so that the method becomes a new research hotspot.
Through anaerobic fermentation of the excess sludge, organic matters in the excess sludge are converted into short-chain fatty acids which are used as carbon sources for biological nitrogen and phosphorus removal. The anaerobic fermentation process of the sludge can be divided into a series of stages of hydrolysis, acidification, acetic acid production, methane production and the like; among them, acetic acid is an important biosynthetic raw material and also an important intermediate product in the anaerobic digestion process, so that the generation of methane is inhibited and acetic acid is enriched in the anaerobic fermentation process, more carbon sources for biological nitrogen and phosphorus removal are generated, and better resource utilization of the excess sludge is realized.
Homoacetogens are a class of acetogens that can pass through the anaerobic acetyl-CoA pathway, with CO 2 The enrichment of the anaerobic microorganisms which are used as a terminal electron acceptor to reduce the anaerobic microorganisms into acetic acid is beneficial to the accumulation of short-chain fatty acids mainly comprising acetic acid in the anaerobic fermentation process, and has great significance for the resource utilization of excess sludge.
At present, glucose, sodium formate, sodium oxalate and the like are mostly used as carbon sources in the conventional method for enriching homoacetogenic bacteria; meanwhile, a BES methanogenesis inhibitor is required to be added to inhibit methanogens in sludge in the process of enriching acetogenic bacteria, and an external temperature control measure is required to control the enrichment temperature to be about 35 ℃, so that the enrichment cost is high, the energy consumption is high, the enrichment condition requirement is high, the large-scale application to the practical resource utilization of residual sludge and sewage treatment is difficult, and the applicability is poor.
Disclosure of Invention
The invention aims to overcome the defects that a BES methanogenesis inhibitor is required to be added to inhibit methanogens in sludge in the conventional process of enriching acetogenic bacteria, an external temperature control measure is required to control the enrichment temperature to be 35 ℃, the cost is high, the energy consumption is high, the enrichment condition requirement is high, the method cannot be applied to actual residual sludge resource utilization and sewage treatment on a large scale, and the applicability is poor.
In order to solve the above-mentioned purpose, the invention provides the following technical scheme:
in a first aspect, the present application provides a method for enriching homoacetogenic bacteria at normal temperature, which adopts the following technical scheme:
a method for enriching homoacetogenic bacteria at normal temperature comprises three culture stages, namely an initial stage, a transition stage and an enrichment stage;
in the initial stage, glucose is used as a carbon source to carry out amplification culture on the seed sludge;
in the transition stage, glucose and sodium formate are used as carbon sources to perform selective culture on the inoculated sludge subjected to the enlarged culture, and the adding amount of the sodium formate is gradually increased along with the increase of the culture days;
in the enrichment stage, sodium formate is used as a carbon source, and the inoculated sludge subjected to selective culture is further subjected to enrichment culture to obtain homoacetogenic bacteria-enriched sludge;
the culture temperature of the initial stage, the transition stage and the enrichment stage is 23-25 ℃.
By adopting the technical scheme, all strains in the inoculated sludge are subjected to amplification culture by using glucose as a carbon source in an initial stage, so that the number of homoacetogenic bacteria in the inoculated sludge is increased, and the subsequent selectivity and enrichment culture are facilitated;
in the transition stage, glucose is still used as a part of carbon source, sodium formate is added as the carbon source, the microorganisms in the inoculated sludge are gradually adapted to the environment using sodium formate as the carbon source by increasing the adding amount of sodium formate, homoacetogenic bacteria can be selectively cultured, the competitiveness of homoacetogenic bacteria in the inoculated sludge is increased, and the enrichment of homoacetogenic bacteria by only using sodium formate as the carbon source in the follow-up process is facilitated;
in the enrichment stage, sodium formate is used as a carbon source to enrich homoacetogenic bacteria in the inoculated sludge;
at present, homoacetogenic bacteria are generally enriched under the conditions of inhibiting methanogenesis and medium temperature, the adaptability of microorganisms to the environment with sodium formate as a carbon source is improved by gradually reducing the input amount of glucose while adding sodium formate, and homoacetogenic bacteria are enriched by only using sodium formate as a carbon source; in the technical scheme, the adding amount of the sodium formate is gradually increased, the homoacetogenic bacteria can simultaneously take the glucose and the sodium formate as carbon sources, and the degradation of the homoacetogenic bacteria on the glucose is favorable for the degradation of the sodium formate; the method is used for feeding the carbon source at 23-25 ℃, so that the homoacetogenic bacteria have higher competitiveness than methanogen and other strains, and a better enrichment effect of the homoacetogenic bacteria can be realized without additionally adding a methanogen inhibitor;
in the technical scheme, the enrichment temperature is 23-25 ℃, the temperature is close to the room temperature, so that extra temperature control measures are not needed, the energy consumption is reduced, an inhibitor is not needed to be additionally added to inhibit methanogens, the enrichment cost is low, the method can be applied to resource utilization of excess sludge and sewage treatment in reality on a large scale, and the method has strong applicability and good popularization and application prospects.
Preferably, the inoculated sludge is prepared by filtering and pretreating excess sludge with a 50-mesh-ratio sieve and heating and boiling the excess sludge.
By adopting the technical scheme, the inoculated sludge is prepared after the residual sludge is filtered and heated to boil, and the dissolution of organic matters in the sludge is accelerated, so that the hydrolysis in the anaerobic fermentation process can be shortened, and the subsequent enrichment of homoacetogenic bacteria is facilitated.
Preferably, in the initial stage, the amount of glucose to be added is gradually increased as the number of days of culture increases.
Preferably, in the transition stage, the initial dosage of the sodium formate is 0.8-1.2g/L, and the dosage of the sodium formate is increased by 1.0-2.0g/L every 15-20 days.
Preferably, in the transition stage, the initial dosage of the sodium formate is 1.0g/L, and the dosage of the sodium formate is increased by 1.0 to 1.3g/L every 18 to 20 days.
Preferably, in the transition stage, the dosage of the glucose is gradually reduced along with the increase of the culture days, and the dosage of the glucose is 3.0-4.0 g/L.
By adopting the technical scheme, the glucose is gradually reduced along with the increase of the culture days, but the adding amount is still kept at 3.0-4.0g/L, the initial adding amount of the sodium formate is 0.8-1.2g/L, the glucose and the sodium formate are used as carbon sources in the transition stage, the adding amount of the sodium formate is increased according to the increasing range of 1.0-2.0g/L every 15-20 days, the adding amount of the sodium formate in the transition stage is increased in a gradient manner, so that homoacetogenic bacteria in inoculated sludge gradually adapt to the environment taking the sodium formate as the carbon source, the competitiveness of the homoacetogenic bacteria in inoculated sludge strains is strong, the occupation ratio of the acetogenic bacteria in the inoculated sludge is gradually increased, the selective culture of the homoacetogenic bacteria in the inoculated sludge is realized, and the homoacetogenic bacteria in the inoculated sludge become dominant strains.
Preferably, in the enrichment phase, the amount of sodium formate fed is gradually increased with the number of days of cultivation.
By adopting the technical scheme, the input amount of the carbon source in the system is correspondingly increased along with the increase of the culture time, and the normal life activity of homoacetogenic bacteria in the inoculated sludge is ensured.
In a second aspect, the present application provides a homoacetogenic bacteria-enriched sludge prepared by any one of the above methods for enriching homoacetogenic bacteria at normal temperature, which adopts the following technical scheme:
homoacetogenic bacteria-enriched sludge, wherein the absolute abundance of homoacetogenic bacteria in homoacetogenic bacteria-enriched sludge is 1.24 multiplied by 10 14 Copy number/gram wet sludge, the proportion of homoacetogenic flora is 38-40%.
By adopting the technical scheme, the sludge prepared by the method is analyzed to obtain: the absolute abundance of the key functional gene fhs in the homoacetogenic pathway is 1.24X 10 14 Copy number/g wet sludge, compared to 1.09X 10 before enrichment 12 Copy number/gram wet sludge, the enrichment effect is good, the percentage of homoacetogenic flora is 39.32%, and the homoacetogenic flora is dominant in all flora of sludge.
In a third aspect, the present application provides an anaerobic fermentation apparatus, which adopts the following technical scheme:
an anaerobic fermentation device comprises the homoacetogenic bacteria-enriched sludge.
By adopting the technical scheme, the inoculated sludge is treated by using a method for enriching homoacetogenic bacteria at normal temperature, so that homoacetogenic bacteria in the inoculated sludge are enriched, short-chain fatty acids such as acetic acid biosynthesized by homoacetogenic bacteria in an anaerobic fermentation device are used as carbon sources for removing nitrogen and phosphorus of microorganisms, and resource utilization of excess sludge is realized; the anaerobic fermentation device containing inoculated sludge enriched with homoacetogenic bacteria is used for carrying out anaerobic fermentation on the biomass, so that the yield of acetic acid is greatly improved, and the anaerobic fermentation device has a great application prospect in the aspects of carbon dioxide resource utilization and carbon emission reduction.
In a fourth aspect, the application provides an application of the homoacetogenic bacteria-enriched sludge in sewage treatment or biomass resource utilization.
Has the advantages that:
(1) culturing the treated inoculated sludge in stages, and firstly, carrying out amplification culture on strains in the inoculated sludge by using glucose as a carbon source in an initial stage; then, in the transition stage, under the condition of keeping the glucose input amount within a certain range, gradually increasing the input amount of sodium formate, so that the strains in the inoculated sludge gradually adapt to the environment using sodium formate as a carbon source, and simultaneously selectively culturing homoacetogenic bacteria, wherein the homoacetogenic bacteria have higher competitiveness than strains such as methanogenic bacteria and the like in the inoculated sludge under the culture condition and become dominant strains in the inoculated sludge; finally, sodium formate is used as a carbon source in the enrichment stage to realize further enrichment culture of methanogens of the same type, so that acetic acid is a main fermentation product in anaerobic fermentation, the acetic acid can be used as a carbon source for biological nitrogen removal and oxygen removal and can also be used as a raw material for industrial synthesis, and resource utilization of excess sludge is realized;
(2) in the transition stage, a certain glucose adding amount is kept within a certain range, the adding amount of sodium formate is increased in a staged manner, and the feeding is carried out according to the mode, so that the competitive power of homoacetogenic bacteria at 23-25 ℃ is higher than that of methanogenic bacteria, and therefore, an additional methanogenic inhibitor is not required to be added in the enrichment process, and an additional temperature control measure is not required to heat and preserve the temperature of an enrichment system, the energy consumption is lower, and the practical application prospect is large.
Drawings
In order to illustrate the technical solution of the present invention more clearly, the drawings will be briefly described below, and it is apparent that the drawings in the following description relate only to some embodiments of the present invention and are not intended to limit the present invention.
FIG. 1 is a schematic view of the construction of an anaerobic fermentation apparatus according to example 7 of the present invention;
FIG. 2 is a graph showing the results of the generation of volatile acids in an anaerobic fermentation apparatus according to an example of the present invention;
FIG. 3(a) is one of the carbon flux results of the anaerobic fermentation device provided in the embodiment of the present invention in the P3 stage and the P10 stage;
FIG. 3(b) is one of the carbon flux results of the anaerobic fermentation apparatus provided in the embodiment of the present invention at the P3 stage and the P10 stage.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. In the following examples, "%" means weight percent, unless otherwise specified.
The application provides a method for enriching homoacetogenic bacteria at normal temperature, which comprises an initial stage, a transition stage and an enrichment stage;
in the initial stage, the inoculation sludge is subjected to amplification culture by using glucose as a carbon source;
in the transition stage, glucose and sodium formate are used as carbon sources to perform selective culture on the inoculated sludge subjected to the enlarged culture, and the adding amount of the sodium formate is gradually increased along with the increase of the culture days;
in the enrichment stage, sodium formate is used as a carbon source to further enrich the inoculated sludge subjected to selective culture to obtain homoacetogenic bacteria enriched sludge;
the culture temperature of the initial stage, the transition stage and the enrichment stage is 23-25 ℃;
preferably, the culture temperature is 24 ℃.
In the invention, the Mixed Liquor Suspended Solid (MLSS) inoculated with the sludge and the Mixed Liquor Volatile Suspended Solid (MLVSS) are respectively 42.4g/L and 13.9 g/L.
In the invention, trace elements and minerals are also added into the enrichment system to provide nutrients necessary for the growth and reproduction of microorganisms, and the nutrients comprise the following substances: trace elements (mg/L): FeCl 2 ·H 2 O,2；H 3 BO 3 ，0.05；ZnCl 2 ，0.05；CuCl 2 ·2H 2 O，0.038；MnCl 2 ·4H 2 O，0.05；(NH 4 ) 6 Mo 7 O 24 ·4H 2 O，0.05；AlCl 3 ,0.05；CoCl 2 ·6H 2 O，0.05；NiCl 2 ·6H 2 O，0.092；Na 2 WO 4 ·2H 2 O, 0.05; minerals (mg/L): CaCl 2 ，0.05；MgCl 2 ·6H 2 O，0.1；NaCl,0.1。
In one embodiment, the inoculated sludge is prepared by subjecting the excess sludge to a filtration pretreatment using a 50 mesh sieve and boiling the pretreated excess sludge under heating.
In the present invention, after impurities are removed by filtering excess sludge using a 50-mesh sieve, the filtered sludge is boiled at a temperature of 100 ℃ for 2 hours. In one embodiment, the amount of glucose added in the initial phase is gradually increased with the number of days of culture.
In one embodiment, the initial amount of sodium formate dosed is 0.8-1.2g/L and the amount of sodium formate dosed is increased by 1.0-2.0g/L every 15-20 days in the transition phase.
In one embodiment, the initial dosage of sodium formate during the transition phase is 1.0g/L, and the dosage of sodium formate is increased by 1.0-1.3g/L every 18-20 days.
In one embodiment, the glucose is added in an amount decreasing with the number of days of cultivation in the transition period, and the glucose is added in an amount of 3.0 to 4.0 g/L.
In the invention, in the transition stage, although the adding amount of the glucose is gradually reduced, the adding amount of the glucose is still kept within the range of 3.0-4.0g/L, the adding amount of the sodium formate is gradually increased so as to ensure that the strain in the inoculated sludge is suitable for the environment taking the sodium formate as a carbon source, and meanwhile, homoacetogenic bacteria are selectively cultured.
In one embodiment, the sodium formate dosage in the enrichment phase increases with the number of days of cultivation.
In the invention, the number of the strains in the inoculated sludge is increased along with the increase of the culture days, so the input amount of the carbon source is required to be increased, and the normal life activity of the strains in the inoculated sludge is ensured.
The application also provides homoacetogenic bacteria enriched sludge prepared by the homoacetogenic bacteria enriching method at normal temperature, wherein the absolute abundance of homoacetogenic bacteria in the homoacetogenic bacteria enriched sludge is 1.24 multiplied by 10 14 Copy number/gram wet sludge, the proportion of homoacetogenic flora is 38-40%.
The application also provides an anaerobic fermentation device, which comprises the homoacetogenic bacteria-enriched sludge.
The application also provides the application of the homoacetogenic bacteria enriched sludge in sewage treatment or biomass resource utilization.
In the invention, the anaerobic fermentation technology is used for treating biomass such as sewage treatment, organic waste and the like, wherein sludge enriched with homoacetogenic bacteria is added into an anaerobic fermentation system to improve the hydrolysis acidification efficiency of organic matters in the anaerobic fermentation process, so that more carbon flows to volatile short-chain fatty acids such as formic acid, acetic acid and the like in the anaerobic fermentation process, and the carbon can be used as a carbon source for biological nitrogen and phosphorus removal and can also be used as a raw material for industrial production.
Example 1 method for enrichment of homoacetogenic bacteria at Normal temperature
In the embodiment, a 50-mesh sieve is used for filtering residual sludge generated in the sewage treatment process to remove impurities, the filtered sludge is heated and boiled at 100 ℃ for 2 hours to obtain inoculated sludge, and Mixed Liquor Suspended Solids (MLSS) and Mixed Liquor Volatile Suspended Solids (MLVSS) in the inoculated sludge are 42.4g/L and 13.9g/L respectively.
Glucose is used as a carbon source in the initial stage; in the transition stage, under the condition of keeping the adding amount of glucose unchanged, adding sodium formate, and taking glucose and sodium formate as carbon sources; in the enrichment stage, sodium formate is independently used as a carbon source; in the whole enrichment process, ammonium chloride and potassium dihydrogen phosphate/dipotassium hydrogen phosphate are used as a nitrogen source and a phosphorus source, and trace elements and mineral substances are added to provide nutrients necessary for growth and reproduction of microorganisms, wherein the specific components and concentrations are as follows: trace elements (mg/L): FeCl 2 ·H 2 O,2；H 3 BO 3 ，0.05；ZnCl 2 ，0.05；CuCl 2 ·2H 2 O，0.038；MnCl 2 ·4H 2 O，0.05；(NH 4 ) 6 Mo 7 O 24 ·4H 2 O，0.05；AlCl 3 ,0.05；CoCl 2 ·6H 2 O，0.05；NiCl 2 ·6H 2 O，0.092；Na 2 WO 4 ·2H 2 O, 0.05; minerals (mg/L): CaCl 2 ，0.05；MgCl 2 ·6H 2 O, 0.1; 0.1 parts of NaCl; and adding a suitable amount of NaHCO 3 So that the pH of the enrichment system is maintained between 6.8 and 7.2.
The substrates added during the initial, transition and enrichment phases are shown in table 1:
TABLE 1 substrate addition Table for EXAMPLE 1
Example 2 method for enriching homoacetogenic bacteria at Normal temperature
This example differs from example 1 in that the culture temperature in the initial stage, the transition stage and the enrichment stage was 23 ℃.
Example 3 method for Normal temperature enrichment of homoacetogenic bacteria
This example differs from example 1 in that the culture temperature in the initial stage, the transition stage and the enrichment stage was 25 ℃.
Example 4 method for enrichment of homoacetogenic bacteria at Normal temperature
The difference between this example and example 1 is that the substrate addition amount in the transition phase is different, as shown in table 2:
TABLE 2 TABLE of substrate addition for EXAMPLE 4
Example 5 method for Normal temperature enrichment of homoacetogenic bacteria
The difference between this example and example 1 is that the substrate addition amount in the transition phase is different, as shown in table 3:
TABLE 3 TABLE 5 TABLE FOR SUBSTRATE ADDITION OF EXAMPLE 5
Example 6 homoacetogenic bacteria-enriched sludge
The homoacetogenic bacteria-enriched sludge provided in this example is prepared by the method provided in examples 1 to 5; wherein the absolute abundance of the key functional gene fhs in the homoacetogenic pathway in the sludge is 1.24 multiplied by 10 14 Copy number/g wet sludge, 1.09X 10 to that before enrichment 12 The copy number/gram of wet sludge is obviously improved. The proportion of the homoacetogenic flora is 39.32%, which is the dominant flora in the sludge.
EXAMPLE 7 an anaerobic fermentation apparatus
The anaerobic fermentation device provided by the embodiment has a structure as shown in fig. 1, and comprises a sample inlet barrel, a reactor and a gas collecting bag, wherein a peristaltic pump is further arranged between the sample inlet barrel and the reactor, and the sample inlet barrel, the peristaltic pump and the peristaltic pump are communicated with the reactor through pipelines in sequence; the reactor is filled with homoacetogenic bacteria-enriched sludge provided by embodiment 6, the reactor is also internally provided with a stirring paddle, the joint of the stirring paddle and the reactor is provided with a silica gel pad to prevent air leakage, and the gas collection bag is communicated with the upper part of the reactor through a pipeline.
Before the anaerobic fermentation device is formally operated, the homoacetogenic bacteria-enriched sludge is obtained by treating the inoculated sludge by using the method for enriching homoacetogenic bacteria at normal temperature provided in the embodiment 1-5, or the homoacetogenic bacteria-enriched sludge provided in the embodiment 6 is directly added into a reactor; waste water and other anaerobic fermentation substrates are placed in a sample feeding barrel and enter a reactor at a constant flow rate through a peristaltic pump; when anaerobic fermentation is carried out in the reactor, the wastewater and the sludge are mechanically stirred by the stirring paddle, so that more carbon in the anaerobic fermentation carried out in the anaerobic fermentation device flows to low-carbon volatile fatty acids such as formic acid, acetic acid and the like, and the low-carbon volatile fatty acids are used as organic carbon sources in the processes of biological phosphorus removal and biological denitrification nitrogen removal in sewage treatment and raw materials in industrial synthesis, and the resource utilization of the residual sludge is better realized.
Examples of the experiments
Carrying out normal-temperature enrichment of homoacetogens by using the anaerobic fermentation device provided in embodiment 7 and the homoacetogenic bacteria enrichment method provided in embodiment 1; wherein, the water inlet of the anaerobic fermentation device is artificial inlet water, the pH value of the inlet water is regulated to 7, and the hydraulic retention time is 8 h; after the enrichment culture of the sludge homoacetogenic bacteria is finished, continuously taking glucose as a carbon source to carry out anaerobic fermentation, wherein the adding amount of the glucose is 4.0 g/L; and the Oxidation Reduction Potential (ORP) of water, the volume of gas, the composition of gas, and the composition and content of Volatile Fatty Acids (VFAs) in effluent were measured in the above-described process, and the results are as follows.
1. Generation of volatile acid
FIG. 2 is a graph showing the results of the generation of volatile acids in an anaerobic fermentation apparatus, from which it can be seen that the concentration of VFAs in the apparatus gradually increases at the initial stage (P1-P3), and that the OLR is high; in the transition stage (P4-P6), the concentration of OLR and total VFAs is close to the stage P3, and the concentration of acetic acid in the VFAs is increased along with the increase of the dosage of sodium formate and the decrease of the dosage of glucose, so that the yield of the acetic acid is obviously improved, and the addition of the sodium formate realizes the selective culture of homoacetogenic bacteria and promotes the generation of the acetic acid; in the enrichment stage (P7-P9), the yield of acetic acid is better, and the yield of acetic acid is obviously improved along with the increase of the dosage of sodium formate, reaches about 1000mg HAc/L in the P8 stage, and reaches about 1500mg HAc/L in the P9 stage.
P10 is a stage of continuing anaerobic fermentation by taking glucose as a carbon source after the enrichment culture of the sludge homoacetogenic bacteria is finished; at P10, using only glucose as a carbon source, acetic acid and n-butyric acid were the major VFAs produced in the plant; and comparing the P3 stage with the P10 stage, the yield of acetic acid in the P10 stage reaches more than 700mg HAc/L, which is improved by about 21% compared with the P3 stage, and the yield of formic acid in the P10 stage is about 60-70mg HAc/L, the sludge treated by the method provided by the embodiment 1 is applied to anaerobic fermentation, so that more carbon flows to formic acid and acetic acid in the anaerobic fermentation process, the pressure of mutual degradation of two or more carbon volatile acids is reduced, and the risk of system acidification caused by the accumulation of VFAs (volatile organic acids) of two or more carbon is also reduced.
2. In-plant carbon flow analysis
The results are shown in FIGS. 3(a) and 3(b), where 0.02% of carbon flows to biogas and 31.39% of carbon flows to VFAs in the P3 stage, where 19.3% of carbon flows to propionic acid, butyric acid and valeric acid, and only 0.22% and 11.72% of carbon flows to formic acid and acetic acid, indicating that homoacetogenesis is weak in the initial stage. After enrichment, the carbon flow to biogas in the P10 stage is only 0.02% as compared to the P1-P9 stage, but only 0.02% of CO is produced in P10 2 The carbon flow to the VFAs increased significantly to 34.46% of the total VFAs, with 2.26% and 16.82% for the six carbons to formic and acetic acids, respectively, and 15.39% for propionic, butyric and valeric acids, indicating that anaerobic fermentation using inoculated sludge enriched with homoacetogens allowed more carbon flow to low carbon volatile fatty acids such as formic, acetic and the like.
3. Sludge colony analysis
With formic acid or H 2 /CO 2 The substrate homoacetobacter acetobacter and glucose homoacetogen Clostridium sensu stricoto _1 were enriched. In the P3 stage before enrichment, the relative abundance of Acetobacter and Clostridium _ sensu _ stricoto _1 is 0.22% and 30.15%, respectively, and the absolute abundance of the key functional gene fhs in the homoacetogenic pathway is 1.09 × 10 12 Copy number per gram wet sludge. In the stage from P7 to P9, the relative abundance of Acetobacter is gradually increased to 37.52 percent along with the continuous increase of the concentration of formic acid in the inlet water, which shows that the Acetobacter is in the normal temperature environmentThe addition of formic acid has obvious enrichment effect on homoacetogenic bacteria. The relative abundance of Acetobacterium and Clostridium _ sensu _ stricto _1 at P10 after completion of enrichment was 13.38% and 25.93%, respectively. The absolute abundance of the key functional gene fhs in the homoacetogenic pathway was 1.24X 10 14 Copy number/g wet sludge, indicating that homoacetogenic flora gradually becomes dominant flora in an acid production system after enrichment.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A method for enriching homoacetogenic bacteria at normal temperature is characterized in that: comprises three culture stages of an initial stage, a transition stage and an enrichment stage;
in the initial stage, the inoculation sludge is subjected to amplification culture by using glucose as a carbon source;
in the transition stage, glucose and sodium formate are used as carbon sources to perform selective culture on the inoculated sludge subjected to the enlarged culture, and the adding amount of the sodium formate is gradually increased along with the increase of the culture days;
in the enrichment stage, sodium formate is used as a carbon source to further enrich the inoculated sludge subjected to selective culture to obtain homoacetogenic bacteria enriched sludge;
the culture temperature of the initial stage, the transition stage and the enrichment stage is 23-25 ℃.
2. The method for normal temperature enrichment of homoacetogenic bacteria according to claim 1, wherein: the inoculated sludge is prepared by filtering and pretreating excess sludge with a 50-mesh-ratio sieve and heating and boiling.
3. The method for normal temperature enrichment of homoacetogenic bacteria according to claim 1, wherein: in the initial stage, the amount of glucose added was gradually increased as the number of days of culture increased.
4. The method for normal temperature enrichment of homoacetogenic bacteria according to claim 1, wherein: in the transition stage, the initial adding amount of the sodium formate is 0.8-1.2g/L, and the adding amount of the sodium formate is increased by 1.0-2.0g/L every 15-20 days.
5. The method for normal-temperature enrichment of homoacetogenic bacteria according to claim 4, wherein in the transition stage, the initial dosage of sodium formate is 1.0g/L, and the dosage of sodium formate is increased by 1.0-1.3g/L every 18-20 days.
6. The method for normal-temperature enrichment of homoacetogenic bacteria according to claim 1, wherein the glucose dosage is gradually reduced with the increase of the culture days in the transition stage, and the glucose dosage is 3.0-4.0 g/L.
7. The method according to claim 1, wherein the amount of sodium formate fed during the enrichment phase is gradually increased with the number of days of culture.
8. The homoacetogenic bacteria-enriched sludge prepared by the method for enriching homoacetogenic bacteria at normal temperature according to any one of claims 1 to 7, which is characterized in that: said enriched homoproductionThe absolute abundance of homoacetogenic bacteria in the sludge of acetogenic bacteria is 1.24 multiplied by 10 14 Copy number per gram wet sludge; the proportion of the homoacetogenic flora is 38-40%.
9. An anaerobic fermentation apparatus comprising the homoacetogenic bacteria-enriched sludge according to claim 8.
10. The use of homoacetogenic bacteria-enriched sludge according to claim 8 in sewage treatment or biomass resource utilization.
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| CN104263764A (en) * | 2014-09-15 | 2015-01-07 | 常州大学 | Process for high-efficiency anaerobic production of acetic acid with homoacetogenic bacteria-rich seed sludge |
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| CN102433278A (en) * | 2011-12-13 | 2012-05-02 | 江南大学 | Enrichment culture method of homoacetogenic bacteria |
| CN104263764A (en) * | 2014-09-15 | 2015-01-07 | 常州大学 | Process for high-efficiency anaerobic production of acetic acid with homoacetogenic bacteria-rich seed sludge |
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