CN107400645A - Composite bacteria agent for repairing polluted by nitrogen soil and preparation method thereof - Google Patents

Composite bacteria agent for repairing polluted by nitrogen soil and preparation method thereof Download PDF

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CN107400645A
CN107400645A CN201710642918.XA CN201710642918A CN107400645A CN 107400645 A CN107400645 A CN 107400645A CN 201710642918 A CN201710642918 A CN 201710642918A CN 107400645 A CN107400645 A CN 107400645A
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bacterium
denitrification
nitrogen
soil
synchronous nitration
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高太忠
庞会从
李秀荣
张灿灿
沈洪艳
赵志瑞
刘亚鑫
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Hebei University of Science and Technology
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Hebei University of Science and Technology
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Abstract

The invention discloses a kind of composite bacteria agent for being used to repair polluted by nitrogen soil and preparation method thereof, including synchronous nitration and denitrification bacterium and denitrifying bacteria, the synchronous nitration and denitrification bacterium is at least one of Aeromonadaceae Zobellella bacterial strains, Micrococcaceae Arthrobacter bacterial strains, Sinorhizobium/Ensifer group Ensifer bacterial strains；The denitrifying bacteria is YY12 bacterial strains, and its preserving number is CGMCC No.6924；The bacterium number proportioning of the synchronous nitration and denitrification bacterium and denitrifying bacteria is 1: 1.The composite bacteria agent of acquisition, it is ammonia nitrogen, nitrate nitrogen and the excellent degradation effect of nitrite nitrogen to the nitrogen of soil three to have, and especially has efficient degradation rate to nitrate nitrogen, can effectively remove the polluted by nitrogen of soil, the improvement for soil is significant.The preparation of composite bacteria agent, simple to operate, microbial inoculum activity, the stability of acquisition are high, and common heterotrophic nitrification and the denitrification culture medium of current generally use is abandoned in preparation, screening and enrichment of the selection suitable for the culture medium, more conducively synchronous nitration and denitrification bacterium of synchronous nitration and denitrification bacterium.

Description

Composite bacteria agent for repairing polluted by nitrogen soil and preparation method thereof
Technical field
The present invention relates to microbial bacterial agent technical field, the composite bacteria agent of polluted by nitrogen soil is more particularly, to repaired, together When, further relate to the preparation method of the microbial inoculum.
Background technology
Soil polluted by nitrogen, causes nitrates accumulation in soil, not only deteriorates the physico-chemical property of soil, and soil In nitrate generate nitrous oxide through denitrification, into Sudden warming in stratosphere, chemical reaction can occur with ozone and consume The ozone in ozone layer is damaged, turns into one of major reason for the trace gas that damages the ozone layer.The main source of soil polluted by nitrogen has： The discharge of sewage or solid waste and the use of agrochemical, nitrogen by dosing to soil layer, Initial pollution surface water in nitrogen Release, nitrogen enter soil etc. with rainwater；Wherein, with the first pollution sources the most extensively, seriously.
In the repair mode to soil, microbial method is using microorganism species under suitable environmental condition, is promoted Or the degradation function of enhancement microbiological, reduce the biological prosthetic skill of toxic pollutant activity or degraded in innocuous substance so as to reach Art, compared with physical chemistry, plant soil recovery technique, have：(1) its physics of the soil environment of process reparation, chemistry and life Thing property is held essentially constant, or even can be better than the property of original soil；(2) concentration of pollutant is reduced to greatest extent； (3) processing form is various, can treatment in situ；(4) environment influences small；(5) rehabilitation cost expense is low；(6) application limits smaller etc. Advantage, it is widely used in the reparation of contaminated soil.In recent years, the denitrogenation of simultaneous nitrification-denitrification (SND) is found by studying Mode is most economical denitrogenation means compared with traditional biological denitrificaion mode, but is applied to synchronous nitration denitrification denitrogenation mode Bacterial screening, culture and using limiting the development of the soil remediation method.
The content of the invention
To solve the shortcomings of the prior art, the invention provides one kind by synchronous nitration and denitrification bacterium and denitrification The composite bacteria agent for being used to repair polluted by nitrogen soil that bacterium is formed, the composite bacteria agent is ammonia nitrogen, nitrate nitrogen and Asia to the nitrogen of soil three The degraded of nitrate nitrogen is respectively provided with good effect, can effectively remove polluted by nitrogen, rehabilitating soil.
To achieve the above object, provided by the present invention for the composite bacteria agent of reparation polluted by nitrogen soil, including synchronous nitration Denitrifying bacteria and denitrifying bacteria, the synchronous nitration and denitrification bacterium be Aeromonadaceae Zobellella bacterial strains, In Micrococcaceae Arthrobacter bacterial strains, Sinorhizobium/Ensifer group Ensifer bacterial strains extremely Few one kind；The denitrifying bacteria is YY12 bacterial strains, and its preserving number is CGMCC No.6924；The synchronous nitration and denitrification is thin The bacterium number of bacterium and denitrifying bacteria proportioning is 1: 1.
The microbial inoculum of the present invention, choose synchronous nitration and denitrification bacterium and form composite bacteria agent with denitrifying bacteria, using synchronization The mode of nitration denitrification, for the reparation to polluted by nitrogen soil, wherein, can be simultaneously with oxygen using synchronous nitration and denitrification bacterium With advantage of the nitric acid as electron acceptor, Seedling height rate is obtained, is enriched with high efficiency nitrification denitrification short-cut denitrification bacterium, to improve nitrification The efficiency of denitrification denitrogenation process, while the growth for aerobic denitrifying bacteria, enrichment and control provide advantage, solve In biological denitrification process, because the major microorganisms nitrifier for participating in nitrification is autotrophic bacterium, causes the slow-growing of appearance and limit The problem of denitrification effect processed；Degraded of the microbial inoculum of acquisition to the nitrogen of soil three i.e. ammonia nitrogen, nitrate nitrogen and nitrite nitrogen is set to be respectively provided with very Good effect, especially has efficient degradation rate to nitrate nitrogen, can effectively remove polluted by nitrogen, complete the reparation of soil.For synchronization Nitration denitrification bacterium and the screening of denitrifying bacteria, with Aeromonadaceae Zobellella bacterial strains or Micrococcaceae Arthrobacter bacterial strains or Sinorhizobium/Ensifer group Ensifer bacterial strains, with YY12 bacterial strains (belonging to Rhodococcus sp. bacterial strains), which coordinate, forms composite bacteria agent, and two kinds of bacterial strains are mutually auxiliary in soil remediation process Coordinate, cooperate, more conducively the enrichment of bacterium number and nitric efficiency, improve the removal ability to polluted by nitrogen.
As the restriction to above-mentioned technical proposal, the composite bacteria agent is the immobilized bacterium using diatomite or haydite as carrier Agent, synchronous nitration and denitrification bacterium bacterium number is 10 in the immobilized microbial inoculum7~108CFU/g, denitrifying bacteria bacterium number are 107~ 108CFU/g。
As the restriction to above-mentioned technical proposal, the composite bacteria agent is Sinorhizobium/Ensifer group The bacterium solution that Ensifer strain culturings 24h is obtained is fixed on the immobilized microbial inoculum that carrier diatomaceous earth obtains, with YY12 strain culturings The bacterium solution that 24h is obtained is fixed on the immobilized microbial inoculum that carrier diatomaceous earth obtains, the composite bacteria agent being mixed to get.
Composite bacteria agent is further limited as immobilized microbial inoculum type, and optimal synchronous nitration and denitrification bacterium bacterial strain and Fixation support, to obtain the composite bacteria agent for being used to repair polluted by nitrogen soil that performance is more stable, denitrification ability is more superior.
Meanwhile present invention also offers the as described above preparation method for being used to repair the composite bacteria agent of polluted by nitrogen soil, Comprise the following steps：
A, synchronous nitration and denitrification bacterium bacterial strain and denitrifying bacteria bacterial strain are obtained；
B, culture obtains synchronous nitration and denitrification bacterial solution and denitrifying bacteria bacterium solution respectively：
B1, the culture medium with synchronous nitration and denitrification bacterium, under the conditions of 30 DEG C, 150r/min constant-temperature shaking culture 24~ 48h, bacterium number is obtained as 107~108CFU/g synchronous nitration and denitrification bacterial solution；The training of the synchronous nitration and denitrification bacterium Supporting base includes following raw material components：Sodium citrate 3.7g/L, ammonium sulfate 2.0g/L, CaCO35.0g/L, K2HPO41.0g/L FeSO4·7H2O 0.4g/L, MgSO4·7H2O0.5g/L, NaCl 2.0g/L；
B2, the enriched medium with denitrifying bacterium, 24~48h of constant-temperature shaking culture, is obtained under the conditions of 30 DEG C, 150r/min It is 10 to bacterium number7~108CFU/g denitrifying bacteria bacterium solution；
C, composite bacteria agent is prepared：Synchronous nitration and denitrification bacterial solution, denitrifying bacteria bacterium solution are mixed with carrier respectively Being fixed, the mixed proportion for adding 15g carriers by 35mL bacterium solutions stir, then by immobilization synchronous nitration and denitrification Bacterium microbial inoculum mixes with immobilization denitrifying bacteria microbial inoculum, obtains composite bacteria agent, is preserved under normal temperature in valve bag, during use Incorporate in surface water, through being sprayed to soil, for repairing polluted by nitrogen soil.
In the preparation method of microbial inoculum, the culture medium of synchronous nitration and denitrification bacterium is limited, abandons current generally use Common heterotrophic nitrification and denitrification culture medium are i.e. with conventional organic matter (sucrose, glucose, butanedioic acid, citric acid, acetic acid) for carbon Source, ammonia nitrogen or nitrate nitrogen are nitrogen source, culture medium of the selection suitable for synchronous nitration and denitrification bacterium, more conducively synchronous nitration and denitrification The screening and enrichment of bacterium, beneficial to the high performance microbial inoculum of acquisition.
As the restriction to above-mentioned technical proposal, synchronous nitration and denitrification bacterium bacterial strain is from North China Plain moisture soil in step a Soil sample in enrichment isolation obtain, the screening process comprises the following steps：
A1, the soil sample that 15g chooses is weighed, added into the synchronous nitration and denitrification bacteria culture media of sterilizing, in 30 DEG C, Constant-temperature shaking culture 7 days under the conditions of 150r/min, the supernatant 50mL of nutrient solution is then taken to be transferred to fresh 150mL synchronization nitre Change in denitrifying bacteria culture medium, constant-temperature shaking culture 7 days under the conditions of 30 DEG C, 150r/min, then culture of transferring, continuous sampling Switching 3 times, obtains the enrichment culture liquid of synchronous nitration and denitrification bacterium；
A2,10 μ L, 100 μ L and 200 μ L enrichment cultures liquid are taken to carry out flat board coating culture respectively, 30 DEG C are inverted culture 5 days, Then picking individual colonies, continuous plate streaking culture 4-6 times, the strain screened, and it is stored in 4 DEG C of ice with inclined-plane preservation method It is stand-by in case；The flat board is that synchronous nitration and denitrification bacteria culture media adds 2% agar with culture medium.
In summary, using technical scheme, the composite bacteria agent for being used to repair polluted by nitrogen soil of acquisition, be with Aeromonadaceae Zobellella bacterial strains, Micrococcaceae Arthrobacter bacterial strains or Sinorhizobium/ The synchronous nitration and denitrification bacterium of Ensifer group Ensifer bacterial strains, with the denitrifying bacteria of YY12 bacterial strains, being matched somebody with somebody Close, for the reparation of polluted by nitrogen soil, it is ammonia nitrogen, nitrate nitrogen and the excellent degradation effect of nitrite nitrogen to the nitrogen of soil three to have, Especially there is efficient degradation rate to nitrate nitrogen, can effectively remove the polluted by nitrogen of soil, the improvement for soil is significant. The preparation of composite bacteria agent, it is simple to operate, and the microbial inoculum activity and stability that obtain are high, it is thin for synchronous nitration and denitrification in preparation Bacterium culture medium, abandons common heterotrophic nitrification and the denitrification culture medium of current generally use, and selection is suitable to synchronous nitration and denitrification The screening and enrichment of the culture medium of bacterium, more conducively synchronous nitration and denitrification bacterium, beneficial to the high performance microbial inoculum of acquisition.
Brief description of the drawings
Fig. 1, be the 24h and 48h of the synchronous nitration and denitrification bacterium that are screened in the embodiment of the present invention ammonia nitrogen degradation rate figure Spectrum；
Fig. 2, it is the synchronous nitration and denitrification bacterium 24h and 48h nitrate nitrogen degradation rate figure screened in the embodiment of the present invention Spectrum；
Fig. 3, it is the synchronous nitration and denitrification bacterium 24h and 48h nitrite nitrogen synthetic ratio screened in the embodiment of the present invention Collection of illustrative plates.
Fig. 4, it is the bacterium activity change figure of RT2 immobilized microbial inoculums screened in the embodiment of the present invention；
Fig. 5, it is the bacterium activity change figure of FT2 immobilized microbial inoculums screened in the embodiment of the present invention；
Fig. 6, it is the bacterium activity change figure of FT5 immobilized microbial inoculums screened in the embodiment of the present invention；
Fig. 7, it is the bacterium activity change figure of YY12 immobilized microbial inoculums chosen in the embodiment of the present invention；
Wherein, T represents carrier as haydite, and G represents carrier as diatomite；
The nitrogen figure of degraded three of immobilized microbial inoculum prepared by Fig. 8, the RT2 strain culturings 24h to be screened in the embodiment of the present invention Spectrum；
The nitrogen figure of degraded three of immobilized microbial inoculum prepared by Fig. 9, the RT2 strain culturings 48h to be screened in the embodiment of the present invention Spectrum；
The nitrogen figure of degraded three of immobilized microbial inoculum prepared by Figure 10, the FT2 strain culturings 24h to be screened in the embodiment of the present invention Spectrum；
The nitrogen figure of degraded three of immobilized microbial inoculum prepared by Figure 11, the FT2 strain culturings 48h to be screened in the embodiment of the present invention Spectrum；
The nitrogen figure of degraded three of immobilized microbial inoculum prepared by Figure 12, the FT5 strain culturings 24h to be screened in the embodiment of the present invention Spectrum；
The nitrogen figure of degraded three of immobilized microbial inoculum prepared by Figure 13, the FT5 strain culturings 48h to be screened in the embodiment of the present invention Spectrum；
The nitrogen collection of illustrative plates of degraded three of immobilized microbial inoculum prepared by Figure 14, the YY12 bacterial strains to be chosen in the embodiment of the present invention；
Wherein, AJ represents ammonia nitrogen degradation rate, and XJ represents nitrate nitrogen degradation rate, and YH represents nitrite nitrogen synthetic ratio.
Figure 15, it is that FT5 microbial inoculums and YY12 microbial inoculums in the embodiment of the present invention using diatomite as carrier is respectively applied to little Bai The nitrogen content of dish experiment in cultivation three changes over time collection of illustrative plates；
Figure 16, the composite bacteria agent for acquisition of the embodiment of the present invention are applied to the nitrogen content of pakchoi experiment in cultivation three anaplasia at any time Change collection of illustrative plates.
Embodiment
Below in conjunction with embodiment, technical scheme is clearly and completely retouched referring to 1~Figure 16 of accompanying drawing State, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based on the present invention In embodiment, the every other implementation that those of ordinary skill in the art are obtained under the premise of creative work is not made Example, belongs to the scope of protection of the invention.
Embodiment one
The present embodiment is related to composite bacteria agent and its preparation for repairing polluted by nitrogen soil.
A, synchronous nitration and denitrification bacterium bacterial strain and denitrifying bacteria bacterial strain are obtained；
A1, most representational three kinds of soil samples (R, N, F) in the moisture soil of North China Plain are chosen, wherein the physics and chemistry of three kinds of soil samples It is characterized as：R soil samples ammonia nitrogen, nitrate nitrogen are sub-, nitrate nitrogen content and total plate count are respectively 0.0173g/kg, 0.1158g/kg, 0.3059g/kg and 2.5 × 106CFU/g；N soil samples ammonia nitrogen, nitrate nitrogen are sub-, nitrate nitrogen content and total plate count are respectively 0.0645g/kg, 0.3267g/kg, 1.0533g/kg and 4.8 × 106CFU/g；F soil samples ammonia nitrogen, nitrate nitrogen are sub-, nitrate nitrogen content It is respectively 0.0975g/kg, 0.1072g/kg, 1.4066g/kg and 2.7 × 10 with total plate count7CFU/g。
Enrichment isolation synchronous nitration and denitrification bacterium is distinguished from each soil sample, operation is as follows：Each soil sample weighs 15g respectively (i.e. R soil samples 15g, N soil sample 15g, F soil sample 15g), it is added separately to fill the synchronous nitration and denitrification bacterium training of 100mL sterilizings In the conical flask for supporting base, in 30 DEG C, constant-temperature shaking culture 7 days under the conditions of 150r/min, the supernatant of nutrient solution is then taken respectively Liquid 50mL is transferred in fresh 150mL synchronous nitration and denitrification bacteria culture medias, and in 30 DEG C, constant temperature shakes under the conditions of 150r/min Culture 7 days, then culture of transferring are swung, continuous sampling is transferred 3 times, obtains the enrichment culture liquid of synchronous nitration and denitrification bacterium,
Different soil samples carry out 3 groups of parallel laboratory tests；
The culture medium of the synchronous nitration and denitrification bacterium includes following raw material components：Sodium citrate 3.7g/L, ammonium sulfate 2.0g/L, CaCO35.0g/L, K2HPO41.0g/L, FeSO4·7H2O 0.4g/L, MgSO4·7H2O 0.5g/L, NaCl 2.0g/L；
A2,10 μ L, 100 μ L and 200 μ L enrichment cultures liquid are taken to carry out flat board coating culture respectively, 30 DEG C are inverted culture 5 days, Compare the clump count of each soil sample, therefrom choose suitable flat board picking individual colonies, continuous plate streaking culture 4 times, confirm pure bacterium And it is stored in inclined-plane preservation method stand-by in 4 DEG C of refrigerators；The flat board is synchronous nitration and denitrification bacteria culture media with culture medium Add 2% agar.
Separation screening, the 4 kinds of simultaneous nitrification and denitrification nitre that will be sifted out in R soil samples are carried out to the synchronous nitration and denitrification bacterium in soil sample Change bacterium and be designated as RT1, RT2, RT3 and RT4；By sifted out in N soil samples 3 kinds of synchronous nitration and denitrification bacteriums be designated as NT1, NT2 and NT3；Sifted out in F soil samples 5 kinds of synchronous nitration and denitrification bacteriums are designated as FT1, FT2, FT3, FT4 and FT5；Each bacterial strain shape is described State and Gram's staining identification, the results are shown in Table 1：
The colonial morphology of the synchronous nitration and denitrification bacterium of 1 different soil samples of table
The measure of bacterial strain denitrification effect：It is i.e. same that the bacterium colony picking individual colonies filtered out are inoculated in fresh enriched medium Walk in nitration denitrification bacteria culture media, 150r/min vibrations, ammonia nitrogen, Asia in each nutrient solution are determined respectively at 0h, 24h and 48h The content of nitrate and nitrate, as a result as shown in Figures 1 to 3, comprehensive three nitrogen (ammonia nitrogen, nitrate nitrogen, nitrite nitrogen) changes of contents, It is efficient denitrification bacterial strain to determine bacterial strain RT2, FT2 and FT5.In detection, ammonia nitrogen is determined using Nessler's reagent photometer, nitrite Using diazonium-azo method measure, nitrate uses determined by ultraviolet spectrophotometry.
The 16S rDNA sequencings of bacterial strain：By the most strong bacterial strain of filter out 3 plants of denitrification abilities, Beijing Bo Maide is sent to Gene technology Co., Ltd carries out Sequence Identification, and sequencing identification result is：Bacterial strain RT2 is accredited as Aeromonadaceae Zobellella, FT2 are accredited as Micrococcaceae Arthrobacter, and FT5 is accredited as Sinorhizobium/ Ensifer group Ensifer；Bacterial strain RT2, FT2 and FT5 gene code are shown in txt texts.
Synchronous nitration and denitrification bacterium chooses bacterial strain RT2, FT2 and FT5；
Denitrifying bacteria chooses YY12 bacterial strains, category Rhodococcus sp (Rhodococcus sp.), deposit number CGMCC No.6924。
B, culture obtains synchronous nitration and denitrification bacterial solution and denitrifying bacteria bacterium solution respectively：
B1, the culture medium with synchronous nitration and denitrification bacterium, under the conditions of 30 DEG C, 150r/min constant-temperature shaking culture 24~ 48h, bacterium number is obtained as 107~108CFU/g synchronous nitration and denitrification bacterial solution；
B2, the enriched medium with denitrifying bacteria, 24~48h of constant-temperature shaking culture under the conditions of 30 DEG C, 150r/min, Bacterium number is obtained as 107~108CFU/g denitrifying bacteria bacterium solution；
C, composite bacteria agent is prepared：
C1, immobilized microbial inoculum preparation：Respectively using diatomite and haydite as the fixation support of microbial inoculum, each bacterium solution is distinguished The mixed proportion of 15g carriers is added to stir being fixed by 35mL bacterium solutions with carrier, then normal temperature is put in valve bag, And leave the osculum of two cms.
The initial bacterium colony concentration of each bacterium solution is determined, took 1.0g immobilized microbial inoculum sample comparative analysis microbial inoculums every 10 days afterwards Bacterium colony concentration and to three nitrogen degradation efficiency, further pick out most suitable microbial inoculum, and determine its quality guarantee period.
The measure of immobilized microbial inoculum activity：
(1) immobilized microbial inoculum 1.0g is weighed to be put into 9mL sterilized waters (121 DEG C of sterilizing 25min), concussion 30min (120~ 130r/min), 30s is stood；
(2) inoculating gun Aspirate supernatant 1mL is used on super-clean bench, is added in the test tube for filling 9mL sterilized waters, that is, dilutes For 10-2, test tube is vibrated, changes pipette tips, 1mL liquid is therefrom drawn and is added in next test tube for filling 9mL sterilized waters, i.e., it is dilute It is interpreted as 10-3, the like be diluted to required concentration, drawing 0.1mL respectively from the test tube of different gradients afterwards is inoculated into bacterium Culture medium, put upside down after being coated with uniformly with spreader in biochemical cultivation case, it is i.e. countable after about 3 days；
(3) measurement result：Each immobilized microbial inoculum shelf-life is at least three months；RT2, FT2, FT5 and YY12 bacterial strain are consolidated Surely microbial inoculum activity change as Fig. 4 is changed to shown in 7, its general activity changes for the immobilized microbial inoculum of synchronous nitration and denitrification bacterium Scope is：Clump count using diatomite as the microbial inoculum of carrier can be by initial 9 × 107Cfu/g drop to three months after 1 × 106Cfu/g, the clump count using haydite as the microbial inoculum of carrier can be by initial 3.4 × 107Cfu/g drop to three months after 1.3 × 105cfu/g；For the immobilized microbial inoculum of denitrifying bacteria, its activity change scope is：Bacterium using diatomite as the microbial inoculum of carrier Falling number can be by initial 2.3 × 107Cfu/g drop to three months after 2.3 × 106Cfu/g, the bacterium using haydite as the microbial inoculum of carrier It is 1.6 × 10 to fall number6Cfu/g drop to three months after 1.1 × 106cfu/g.From the activity result of variations of bacterium：Diatomite bacterium Agent activity ﹥ haydites microbial inoculum activity, it is thus determined that diatomite is the carrier for being more suitable for fixing microbial inoculum.
The measure of microbial inoculum degradation efficiency：
Each immobilized microbial inoculum samples of 1.0g are taken in new enriched medium, 150r/min vibrations, and respectively at 0h, 24h The content of ammonia nitrogen, nitrite and nitrate in each nutrient solution is measured by sampling with 48h, obtain RT2 as shown in Fig. 8 to 14, The nitrogen performance of degraded three of the immobilized microbial inoculum of FT2, FT5 and YY12 bacterial strain.
The degradation results of immobilized microbial inoculum prepared by culture 24h and 48h synchronous nitration and denitrification bacterium are shown, with diatom Soil is that the microbial inoculum of carrier is 6.4%~46.1% to the degradation rate of ammonia nitrogen, to the degradation rate of nitrate nitrogen for -161.9%~ 246.9%, the synthetic ratio to nitrite nitrogen is -14.2%~855.8%；It is the microbial inoculum of carrier to the degradation rate of ammonia nitrogen using haydite For -2.54%~50%, the nitrogen degradation rate to nitre state is -125.7%~249.9%, to the synthetic ratio of nitrite nitrogen for - 42.6%~543.5%.Integrated comparative, it is higher as the ammonia nitrogen degradation rate of the FT5 bacteria immobilization microbial inoculums of carrier using diatomite, and ammonia Nitrogen degradation rate almost appears at the 50th day of culture 48h microbial inoculums, the relatively good control of application time with nitrate nitrogen degradation rate. Therefore, the immobilized microbial inoculum for selecting the FT5 bacterium using diatomite as the culture 48h of carrier to prepare carries out the polluted by nitrogen soil of next step Reparation application.
Using diatomite as the immobilized microbial inoculum of the denitrifying bacteria YY12 bacterium of carrier be 4.4% to the degradation rate of ammonia nitrogen~ 42.1%, be -34.2%~228.6% to the degradation rate of nitrate nitrogen, to the synthetic ratio of nitrite nitrogen for -10.6%~ 470.4%；Using haydite as the immobilized microbial inoculum of the denitrifying bacteria YY12 bacterium of carrier be 12% to the degradation rate of ammonia nitrogen~ 47.2%, the nitrogen degradation rate to nitre state is 4.8%~93.2%, and the synthetic ratio to nitrite nitrogen is 22.8%~610.7%.Two Fluctuation of the microbial inoculum of kind carrier for nitrite nitrogen synthetic ratio is larger, and compares, the ammonia using diatomite as carrier microbial inoculum The degradation rate of nitrogen and nitrate nitrogen is more slightly higher for the degradation rate of carrier than haydite, therefore selects diatomite to make YY12 as carrier The immobilized microbial inoculum of bacterial strain carries out the degraded application of the polluted by nitrogen soil of next step.
C2, composite bacteria agent preparation：By immobilization synchronous nitration and denitrification bacterium microbial inoculum and immobilization denitrifying bacteria bacterium Agent mixes, and obtains composite bacteria agent, is preserved under normal temperature in valve bag, incorporates in surface water during use, through being sprayed to soil In, for repairing polluted by nitrogen soil.
Embodiment two
The present embodiment is related to using diatomite as the immobilized microbial inoculum of the culture 48h of carrier FT5 bacterium preparation and with diatomite The reparation application of the blended obtained composite bacteria agent of immobilized microbial inoculum as the YY12 bacterial strains of carrier in polluted by nitrogen soil
Application test：By pakchoi plantation into flowerpot, cultivating soil is F soil, and flowerpot is placed in the greenhouse of balcony, periodically Dig nursing, ensure illumination, but avoid strong light and heavy rain；Test period be pakchoi growth cycle, i.e., nearly three months.Will Immobilized microbial inoculum prepared by the FT5 bacterium using diatomite as the culture 48h of carrier, YY12 bacterial strains using diatomite as carrier are consolidated Surely change microbial inoculum, and the composite bacteria agent of two kinds of immobilized microbial inoculums, incorporate respectively in surface water, spray arrives plantation pakchoi not With, (per spraying once every other day), soil sampling carries out the test of three nitrogen contents, every sampling in 10 days once, altogether in trial zone soil Take 9 times, per sub-sampling from afternoon 4：00 starts；Using 10 basin pakchois as test group, test result is averaged.
Shown in application test result as Figure 15,16, synchronous nitration and denitrification bacterium microbial inoculum and denitrifying bacteria microbial inoculum are to ammonia Nitrogen, nitrate nitrogen, nitrite nitrogen have obvious degradation effect, and composite bacteria agent has compared to the application effect of two kinds of independent microbial inoculums There is significant synergistic function.
It can be determined that according to above-mentioned application experiment, composite bacteria agent of the invention is preferable to three nitrogen degradation effects in soil, has Ammonia nitrogen and nitrate nitrogen are eliminated to effect, avoids the accumulation of nitrite；And above two flora is suitable to be mixed and made into fixation Change microbial inoculum to be used to improve Hebei Plain moisture soil nitrogen environment, have a good application prospect.
In summary, composite bacteria agent of the invention, it is that ammonia nitrogen, nitrate nitrogen and nitrite nitrogen are excellent to the nitrogen of soil three to have Degradation effect, especially there is efficient degradation rate to nitrate nitrogen, can effectively remove the polluted by nitrogen of soil, the improvement for soil has Important meaning.

Claims (5)

  1. A kind of 1. composite bacteria agent for being used to repair polluted by nitrogen soil, it is characterised in that：Including synchronous nitration and denitrification bacterium and instead Nitrobacteria, the synchronous nitration and denitrification bacterium are Aeromonadaceae Zobellella bacterial strains, Micrococcaceae At least one of Arthrobacter bacterial strains, Sinorhizobium/Ensifer group Ensifer bacterial strains；The anti-nitre Change bacterium is YY12 bacterial strains, and its preserving number is CGMCC No.6924；The synchronous nitration and denitrification bacterium and denitrifying bacteria Bacterium number proportioning is 1: 1.
  2. 2. the composite bacteria agent according to claim 1 for being used to repair polluted by nitrogen soil, it is characterised in that：The composite bacteria agent For the immobilized microbial inoculum using diatomite or haydite as carrier, synchronous nitration and denitrification bacterium bacterium number is in the immobilized microbial inoculum 107~108CFU/g, denitrifying bacteria bacterium number are 107~108CFU/g。
  3. 3. the composite bacteria agent according to claim 2 for being used to repair polluted by nitrogen soil, it is characterised in that：The composite bacteria agent Carrier diatomaceous earth is fixed on for the obtained bacterium solutions of Sinorhizobium/Ensifer group Ensifer strain culturings 48h to obtain The immobilized microbial inoculum arrived, the immobilized microbial inoculum that carrier diatomaceous earth obtains is fixed on the obtained bacterium solutions of YY12 strain culturings 24h, is mixed Close obtained composite bacteria agent.
  4. A kind of 4. preparation method as claimed in claim 1 for being used to repair the composite bacteria agent of polluted by nitrogen soil, it is characterised in that Comprise the following steps：
    A, synchronous nitration and denitrification bacterium bacterial strain and denitrifying bacteria bacterial strain are obtained；
    B, culture obtains synchronous nitration and denitrification bacterial solution and denitrifying bacteria bacterium solution respectively：
    B1, the culture medium with synchronous nitration and denitrification bacterium, 24~48h of constant-temperature shaking culture under the conditions of 30 DEG C, 150r/min, Bacterium number is obtained as 107~108CFU/g synchronous nitration and denitrification bacterial solution；The culture medium of the synchronous nitration and denitrification bacterium Including following raw material components：Sodium citrate 3.7g/L, ammonium sulfate 2.0g/L, CaCO35.0g/L, K2HPO41.0g/L FeSO4·7H2O 0.4g/L, MgSO4·7H2O 0.5g/L, NaCl 2.0g/L；
    B2, the enriched medium with denitrifying bacterium, 24~48h of constant-temperature shaking culture, obtains bacterium under the conditions of 30 DEG C, 150r/min Number is 107~108CFU/g denitrifying bacteria bacterium solution；
    C, composite bacteria agent is prepared：Synchronous nitration and denitrification bacterial solution, denitrifying bacteria bacterium solution are mixed into progress with carrier respectively Immobilization, the mixed proportion for adding 15g carriers by 35mL bacterium solutions stir, then by immobilization synchronous nitration and denitrification bacterium Microbial inoculum mixes with immobilization denitrifying bacteria microbial inoculum, obtains composite bacteria agent, is preserved under normal temperature in valve bag, incorporates during use In surface water, through being sprayed to soil, for repairing polluted by nitrogen soil.
  5. 5. the preparation method according to claim 4 for being used to repair the composite bacteria agent of polluted by nitrogen soil, it is characterised in that：Step Synchronous nitration and denitrification bacterium bacterial strain enrichment isolation from the soil sample of North China Plain moisture soil obtains in rapid a, the screening process bag Include following steps：
    A1, the soil sample that 15g chooses is weighed, added into the synchronous nitration and denitrification bacteria culture media of sterilizing, in 30 DEG C, 150r/ Constant-temperature shaking culture 7 days under the conditions of min, the supernatant 50mL of nutrient solution is then taken to be transferred to fresh 150mL simultaneous nitrification and denitrifications In nitrobacteria culture medium, constant-temperature shaking culture 7 days under the conditions of 30 DEG C, 150r/min, then culture of transferring, continuous sampling switching 3 times, obtain the enrichment culture liquid of synchronous nitration and denitrification bacterium；
    A2,10 μ L, 100 μ L and 200 μ L enrichment cultures liquid are taken to carry out flat board coating culture respectively, 30 DEG C are inverted culture 5 days, then Picking individual colonies, continuous plate streaking culture 4-6 times, the strain screened, and be stored in inclined-plane preservation method in 4 DEG C of refrigerators It is stand-by；The flat board is that synchronous nitration and denitrification bacteria culture media adds 2% agar with culture medium.
CN201710642918.XA 2017-07-31 2017-07-31 Composite bacteria agent for repairing polluted by nitrogen soil and preparation method thereof Pending CN107400645A (en)

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CN109897804A (en) * 2019-03-26 2019-06-18 广东省微生物研究所（广东省微生物分析检测中心） One plant has Zhuo Beier Salmonella and its application of nitrification and denitrification function simultaneously
CN111647530A (en) * 2020-05-29 2020-09-11 微米环创生物科技(北京)有限公司 Compound microbial agent for treating black and odorous substrate sludge and application thereof
CN114525271A (en) * 2022-02-16 2022-05-24 湖北茂盛生物有限公司 Microbial remediation microbial inoculum for soil in phosphorus-potassium mining area, preparation device and method
CN116024123A (en) * 2022-09-26 2023-04-28 河南科技大学 Application of sword bacteria S2_8_1 in promoting soil nitrification
CN116267387A (en) * 2023-03-08 2023-06-23 沈阳农业大学 Application of heterotrophic nitrifying bacteria microbial inoculum combined with nitrogenous fertilizer to improving soil habitat and promoting growth of apple sapling

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Cited By (9)

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Publication number Priority date Publication date Assignee Title
CN108940224A (en) * 2018-08-08 2018-12-07 北京东方园林环境股份有限公司 A kind of preparation method of compound immobilized biological adsorbent
CN109897804A (en) * 2019-03-26 2019-06-18 广东省微生物研究所（广东省微生物分析检测中心） One plant has Zhuo Beier Salmonella and its application of nitrification and denitrification function simultaneously
CN109897804B (en) * 2019-03-26 2022-07-26 广东省微生物研究所（广东省微生物分析检测中心） Zoebelia with nitrification and denitrification functions and application thereof
CN111647530A (en) * 2020-05-29 2020-09-11 微米环创生物科技(北京)有限公司 Compound microbial agent for treating black and odorous substrate sludge and application thereof
CN111647530B (en) * 2020-05-29 2020-12-04 微米环创生物科技(北京)有限公司 Compound microbial agent for treating black and odorous substrate sludge and application thereof
CN114525271A (en) * 2022-02-16 2022-05-24 湖北茂盛生物有限公司 Microbial remediation microbial inoculum for soil in phosphorus-potassium mining area, preparation device and method
CN116024123A (en) * 2022-09-26 2023-04-28 河南科技大学 Application of sword bacteria S2_8_1 in promoting soil nitrification
CN116024123B (en) * 2022-09-26 2024-06-04 河南科技大学 Application of sword bacteria S2_8_1 in promoting soil nitrification
CN116267387A (en) * 2023-03-08 2023-06-23 沈阳农业大学 Application of heterotrophic nitrifying bacteria microbial inoculum combined with nitrogenous fertilizer to improving soil habitat and promoting growth of apple sapling

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