CN103571759B - Method for screening obligate aerobic denitrification fungi - Google Patents
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- CN103571759B CN103571759B CN201310545337.6A CN201310545337A CN103571759B CN 103571759 B CN103571759 B CN 103571759B CN 201310545337 A CN201310545337 A CN 201310545337A CN 103571759 B CN103571759 B CN 103571759B
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Abstract
The invention relates to a method for screening denitrification fungi, and in particular relates to a method for screening obligate aerobic denitrification fungi. The method comprises the steps of preparation of an enrichment obligate aerobic denitrification fungi culture medium, coating culture, purification culture, and identification including aerobic denitrification capacity identification, obligate aerobic bacteria identification and molecular biological identification. Through repeated culture and screening, three obligate aerobic denitrification fungi are obtained and are applied to removal of nitrate in a water body, and the removal rate of nitrate within 28 hours reaches 85.25 percent, 88.51 percent and 89.54 percent respectively. According to the invention, the species of aerobic denitrification bacteria are supplemented, and a wide space can be provided for denitrification application of an aerobic denitrification technology to an actual water environment.
Description
Technical field
The present invention relates to the method for screening denitrification fungi, a kind of particularly method of screening obligate aerobic denitrification fungi.
Background technology
Remove the nitrogen in water body, control body eutrophication is played a key effect.And in water body denitrification technology, biological denitrificaion is a kind of denitride technology of efficient, low cost.Traditional biological denitrificaion relies on the denitrification of the nitrated activity of nitrobacteria and denitrifying bacterium movable.
Along with Aerobic Denitrification Phenomenon obtain science confirm after, aerobic denitrification technology obtains continuous further investigation, and aerobic denitrification microorganism can carry out denitrification for Removing Nitrogen under aerobic conditions, this makes nitrification and denitrification can carry out under substantially identical condition, and this denitrogenates than traditional nitrification-denitrification and has obvious advantage can to reduce processing cost.Finding and cultivate aerobic denitrification flora is one of emphasis of research aerobic denitrification technology, still based on bacterium in the aerobic denitrification bacterial classification of current discovery, and aerobic denitrification fungal studies is little, but aerobic denitrifying bacteria is higher unsatisfactory in actual applications to environmental requirement.
But compare with aerobic denitrifying bacteria, aerobic denitrification fungi has stronger environmental compatibility and tolerance than bacterium.Therefore screen and identify aerobic denitrification fungi and the denitrogenation that water body is particularly difficult to realize the open river of anaerobism is applied to for aerobic denitrification technology provides new approach, there is larger practical significance.The method of current screening aerobic denitrification fungi, the aerobic denitrification fungi nitrate removal rate filtered out is lower.
Summary of the invention
The present invention is directed to deficiency of the prior art, a kind of method of screening obligate aerobic denitrification fungi is provided.
Technical scheme of the present invention is:
Screen a method for obligate aerobic denitrification fungi, comprise the following steps:
Step (1): the preparation of enrichment obligate aerobic denitrification fungi substratum: described substratum often rises and comprises dibromothymolsulfonphthalein 0.01-0.02 gram, agar 18-25 gram, Sunmorl N 60S is 5-10 gram, SODIUMNITRATE and Sodium Nitrite mixture are 1.8-2.4 gram, described SODIUMNITRATE: Sodium Nitrite=1:0.5, SODIUM PHOSPHATE, MONOBASIC 1-2.0 gram, calcium chloride 0.01-0.03 gram, magnesium sulfate 0.01-0.02 gram, sodium bicarbonate 0.2-0.5 gram, ferrous sulfate 0.01-0.02 gram, manganous sulfate 0.01-0.02 gram, zinc sulfate 0.01-0.03 gram, cupric chloride 0.01-0.03 gram, rose vitriol 0.01-0.02 gram, each composition except dibromothymolsulfonphthalein is joined respectively together and mixes, sterilizing, dibromothymolsulfonphthalein is dissolved in dehydrated alcohol, is aseptically joined in the substratum after above-mentioned sterilizing and also again mix, make enrichment obligate aerobic denitrification fungi culture medium flat plate,
Step (2): coating is cultivated: denitrification fungi mixed solution to be screened is applied to uniformly on the flat board of the enrichment obligate aerobic denitrification fungi substratum of preparation in step (1); Under the condition keeping oxygen abundance, be inverted at 30 DEG C and cultivate 7-10 days, until substratum color is blue from green change;
Step (3): become blue fine hair shape bacterial strain by green in picking step (2) substratum, line is separated on the substratum prepared by step (1) again, under the condition keeping oxygen abundance, is inverted at 30 DEG C and cultivates 7-10 days; Repeat said process, until the blue bacterial strain that enrichment obligate aerobic denitrification fungi substratum occurs is clear, profile is consistent, without concomitance bacterium and miscellaneous bacteria, till.
On the basis of above scheme, the preparation of the enrichment obligate aerobic denitrification fungi substratum described in step (1): described substratum often rises and comprises dibromothymolsulfonphthalein 0.015 gram; 22 grams, agar; Sunmorl N 60S is 8 grams; SODIUMNITRATE and Sodium Nitrite mixture are 2.0 grams, described SODIUMNITRATE: Sodium Nitrite=1:0.5; SODIUM PHOSPHATE, MONOBASIC 1.8 grams; 0.02 gram, calcium chloride; 0.01 gram, magnesium sulfate; Sodium bicarbonate 0.4 gram; 0.015 gram, ferrous sulfate, manganous sulfate 0.015 gram, 0.02 gram, zinc sulfate, cupric chloride 0.01 gram, rose vitriol 0.01 gram.
On the basis of above scheme.Also comprise the authentication method of step 4 obligate aerobic denitrification fungi, comprise the qualification of aerobic denitrification ability, the obligate aerobic dientification of bacteria, molecular biology identification:
Steps A: aerobic denitrification ability is identified
Prepare aerobic denitrification capability and identify that substratum often rises aerobic denitrification capability and identifies that substratum is composed as follows: SODIUMNITRATE 1-1.5 gram; Potassium primary phosphate 1-1.5 gram; Sunmorl N 60S 5-10 gram; Water 90-120 milliliter; Calcium chloride 0.01-0.03 gram; Magnesium sulfate 0.01-0.02 gram; ; Sodium bicarbonate 0.2-0.5 gram; Trace element mixed solution: ferrous sulfate 0.01-0.02 gram, manganous sulfate 0.01-0.02 gram, zinc sulfate 0.01-0.03 gram, cupric chloride 0.01-0.03 gram, rose vitriol 0.01-0.02 gram; Above-mentioned composition is joined in Erlenmeyer flask successively, 121 DEG C of sterilizings 20 minutes; Prepare indicator, described indicator moiety is as follows: 4-aminobenzene sulfonamide 20g, N-1-naphthodiamide hydrochloride 1g, phosphoric acid 50mL, and water 250mL, is diluted to 500mL; By inoculation in described aerobic denitrification capability qualification substratum, under the condition of oxygen abundance, be inverted cultivation at 30 DEG C and add indicator in backward substratum in 3-5 days, as substratum reddens, then show that test strains has aerobic denitrification ability;
Step B: the obligate aerobic dientification of bacteria
By step (A) identify after inoculation on obligate aerobic denitrification fungi substratum, and transfer to pour nitrogen in anaerobism bottle after seal; Cultivate 1 month at 30 DEG C; If bacterial strain is without growth sign, illustrate that this bacterial classification is obligate aerobic bacterium;
Step C: molecular biology identification
By step (B) identify after bacterial strain carry out respectively DNA extraction go forward side by side performing PCR amplification, then check order; The gene database in GenBank in blast program and MycoBank is used to compare with sequencing result respectively, and reference Fungal identification handbook, obtain the kind of bacterial strain.
On the basis of above scheme, the method of described screening obligate aerobic denitrification fungi, it is composed as follows that the aerobic denitrification capability qualification substratum described in step 4 often rises described substratum: SODIUMNITRATE 1.3 grams, potassium primary phosphate 1.3 grams, Sunmorl N 60S 8 grams, 110 milliliters, water, 0.02 gram, calcium chloride, 0.01 gram, magnesium sulfate, sodium bicarbonate 0.4 gram; Trace element mixed solution: 0.015 gram, ferrous sulfate, manganous sulfate 0.015 gram, 0.01 gram, zinc sulfate, cupric chloride 0.015 gram, rose vitriol 0.01 gram.
The invention has the beneficial effects as follows: the present invention by gather alternative bacterium source by being inoculated on enrichment obligate aerobic denitrification fungi substratum, by repeatedly repeating to cultivate, screen, obtain three kinds of obligate aerobic denitrification fungi, the bacterium application obtained is removed the nitrate in water body, the nitrate removal rate in 28 hours reaches 85.25%, 88.51% and 89.54% respectively.This is not only supplementing aerobic denitrification bacteria kind, and can provide more wide space for the application of denitrogenating of aerobic denitrification technology in actual water surrounding.
Accompanying drawing explanation
Fig. 1 is the schema of the method for screening obligate aerobic denitrification fungi of the present invention;
Fig. 2 is to nitrate removal rate design sketch under the different substrate of three kinds of fungies that the present invention screens;
Fig. 3 is that three kinds of fungies screening of the present invention are to the degradation rule design sketch of nitrate;
Fig. 4 is that three kinds of fungies screening of the present invention are to the degradation rule design sketch of nitrite.
Embodiment
The specific embodiment of the present invention is as follows:
Embodiment 1:
Basin, Jiaozhou Bay nitrate pollution is comparatively serious, and multiple-microorganism is rich in this basin, therefore adopts the bed mud in contaminated river, basin, Jiaozhou Bay as bacterium source to be selected.
The method of screening obligate aerobic denitrification fungi, comprises the following steps:
Step 1: the preparation of enrichment obligate aerobic denitrification fungi substratum
Whether the quality of enrichment obligate aerobic denitrification fungi substratum directly affects can filter out obligate aerobic denitrification fungi well.Consisting of of often liter of obligate aerobic denitrification fungi substratum: dibromothymolsulfonphthalein 0.015 gram; 22 grams, agar; Sunmorl N 60S is 8 grams; SODIUMNITRATE and Sodium Nitrite mixture are 2.0 grams, described SODIUMNITRATE: Sodium Nitrite=1:0.5; SODIUM PHOSPHATE, MONOBASIC 1.8 grams; 0.02 gram, calcium chloride; 0.01 gram, magnesium sulfate; Sodium bicarbonate 0.4 gram; Trace element mixed solution: 0.015 gram, ferrous sulfate, manganous sulfate 0.015 gram, 0.02 gram, zinc sulfate, cupric chloride 0.01 gram, rose vitriol 0.01 gram etc.
Preparation process: each composition except dibromothymolsulfonphthalein is joined respectively together and mixes, the substratum this obtained afterwards carries out sterilizing, then dibromothymolsulfonphthalein is dissolved in dehydrated alcohol, also again mix in substratum after aseptically being joined above-mentioned sterilizing afterwards, then be poured into dull and stereotyped upper placement while hot to solidify, what obtain is enrichment obligate aerobic denitrification fungi substratum.
Step 2: dilution and coating
Getting 0.1 milliliter of mixed bacterium source to be screened is placed in multiple container respectively, adopts aqua sterilisa to dilute 10 respectively
8doubly, the dilution bacterium liquid diluted separately is placed in airbath shaking table and keeps temperature to be that 30 DEG C of constant temperature oscillations shake up for 15 minutes; Mix rear get respectively 0.1-0.5 milliliter be applied to uniformly enrichment obligate aerobic denitrification fungi substratum is housed flat board on.Enrichment obligate aerobic denitrification fungi substratum after coating is inverted in constant incubator and cultivates at 30 DEG C.Under the condition keeping oxygen abundance, cultivate more than 7-10 days substratum colors after green change basket, represent that obligate aerobic denitrification fungi occurs.
Step 3: become blue fine hair shape bacterial strain by green in picking step (2) substratum,: aseptically adopt in this substratum of transfering loop picking and on the unused obligate aerobic denitrification fungi substratum of another one, carry out line separation by the green fine hair shape bacterial strain becoming indigo plant, after line, be inverted into constant incubator.Under the condition keeping oxygen abundance, thermostat container keeps 30 DEG C to cultivate 7 days-10 days.
Rule more than repeating, cultivate this process, namely each enrichment obligate aerobic denitrification fungi substratum after thermostat container cultivation is by after green change basket, again aseptically adopt in this substratum of transfering loop picking and on new obligate aerobic denitrification fungi substratum, carry out line separation by the green fine hair shape bacterial strain becoming indigo plant, under the condition keeping oxygen abundance, keep 30 DEG C in constant incubator and cultivate 7-10 days.
Rule more than repeating, cultivate this process 5 times, namely obtain purebred denitrification fungi three kinds, respectively called after HYFXHZJ-1, HYFXHZJ-2 and HYFXHZJ-3.
Step 4: obtained 3 kinds of obligate aerobic denitrification fungi identified further, comprises the qualification of aerobic denitrification ability, the obligate aerobic dientification of bacteria, molecular biology identification:
Steps A: aerobic denitrification ability is identified
Prepare aerobic denitrification capability and identify that substratum often rises aerobic denitrification capability and identifies that substratum is composed as follows: SODIUMNITRATE 1.3 grams; Potassium primary phosphate 1.3 grams; Sunmorl N 60S 8 grams; 110 milliliters, water; 0.02 gram, calcium chloride; 0.01 gram, magnesium sulfate; Sodium bicarbonate 0.4 gram; Trace element mixed solution: 0.015 gram, ferrous sulfate, manganous sulfate 0.015 gram, 0.01 gram, zinc sulfate, cupric chloride 0.015 gram, rose vitriol 0.01 gram; Above-mentioned composition is joined in Erlenmeyer flask successively, 121 DEG C of sterilizings 20 minutes.Prepare indicator, described indicator moiety is as follows: 4-aminobenzene sulfonamide 20g, N-1-naphthodiamide hydrochloride 1g, phosphoric acid 50mL, and water 250mL, is diluted to 500mL; By inoculation in described aerobic denitrification capability qualification substratum, under the condition of oxygen abundance, be inverted cultivation at 30 DEG C and add indicator in backward substratum in 3-5 days, substratum reddens, and shows that test strains all has aerobic denitrification ability;
Step B: the obligate aerobic dientification of bacteria
By step (A) identify after inoculation on obligate aerobic denitrification fungi substratum, and transfer to pour nitrogen in anaerobism bottle after seal; Cultivate 1 month at 30 DEG C, bacterial strain, without growth sign, illustrates that this bacterial classification is obligate aerobic bacterium;
Step C: molecular biology identification
By step (B) identify after bacterial strain carry out respectively DNA extraction go forward side by side performing PCR amplification, then check order; The gene database in GenBank in blast program and MycoBank is used to compare with sequencing result respectively, and reference Fungal identification handbook, obtain the kind of bacterial strain.
The aerobic denitrification capability qualification of three kinds of obligate aerobic denitrification fungi:
Above-mentioned three kinds of obligate aerobic denitrification fungi screening obtained are inoculated in artificial distribution according to the inoculum size of 15%, water distribution adopts sucrose to be substrate, nitrate concentration is 50mg/L, nitrite 35mg/L, after at 30 DEG C, shaking table cultivates 28 hours, the clearance of NO3-N and NO2-N the results are shown in Figure 2.The removal ability of three kinds of obligate aerobic denitrification fungi is shown in Fig. 3, Fig. 4.
In Fig. 2, three kinds of obligate aerobic denitrification fungi NO3-N and NO2-N degradation effect under different substrate is obvious, and the nitrate removal rate in 28 hours reaches 85.25%, 88.51% and 89.54% respectively; Nitrite reaches 83.72%, 76.54% and 81.21% respectively.
In Fig. 3, Fig. 4, three kinds of obligate aerobic denitrification fungi under sucrose is substrate in time to the degradation rule of NO3-N and NO2-N, degraded after 28 hours NO3-N and NO2-N reach higher clearance, along with continuation degraded, the clearance change of NO3-N and NO2-N is little.
Claims (4)
1. screen a method for obligate aerobic denitrification fungi, it is characterized in that, comprise the following steps:
Step (1): the preparation of enrichment obligate aerobic denitrification fungi substratum: described substratum often rises and comprises dibromothymolsulfonphthalein 0.01-0.02 gram, agar 18-25 gram, Sunmorl N 60S is 5-10 gram, SODIUMNITRATE and Sodium Nitrite mixture are 1.8-2.4 gram, described SODIUMNITRATE: Sodium Nitrite=1:0.5, SODIUM PHOSPHATE, MONOBASIC 1-2.0 gram, calcium chloride 0.01-0.03 gram, magnesium sulfate 0.01-0.02 gram, sodium bicarbonate 0.2-0.5 gram, ferrous sulfate 0.01-0.02 gram, manganous sulfate 0.01-0.02 gram, zinc sulfate 0.01-0.03 gram, cupric chloride 0.01-0.03 gram, rose vitriol 0.01-0.02 gram, each composition except dibromothymolsulfonphthalein is joined respectively together and mixes, sterilizing, dibromothymolsulfonphthalein is dissolved in dehydrated alcohol, is aseptically joined in the substratum after above-mentioned sterilizing and also again mix, make enrichment obligate aerobic denitrification fungi culture medium flat plate,
Step (2): coating is cultivated: denitrification fungi mixed solution to be screened is applied to uniformly on the flat board of the enrichment obligate aerobic denitrification fungi substratum of preparation in step (1); Under the condition keeping oxygen abundance, be inverted at 30 DEG C and cultivate 7-10 days, until substratum color is blue from green change;
Step (3): become blue fine hair shape bacterial strain by green in picking step (2) substratum, line is separated on the substratum prepared by step (1) again, under the condition keeping oxygen abundance, is inverted at 30 DEG C and cultivates 7-10 days; Repeat said process, until the blue bacterial strain that enrichment obligate aerobic denitrification fungi substratum occurs is clear, profile is consistent, without concomitance bacterium and miscellaneous bacteria, till.
2. the method for screening obligate aerobic denitrification fungi according to claim 1, is characterized in that the preparation of the enrichment obligate aerobic denitrification fungi substratum described in step (1): described substratum often rises and comprises dibromothymolsulfonphthalein 0.015 gram; 22 grams, agar; Sunmorl N 60S is 8 grams; SODIUMNITRATE and Sodium Nitrite mixture are 2.0 grams, described SODIUMNITRATE: Sodium Nitrite=1:0.5; SODIUM PHOSPHATE, MONOBASIC 1.8 grams; 0.02 gram, calcium chloride; 0.01 gram, magnesium sulfate; Sodium bicarbonate 0.4 gram; 0.015 gram, ferrous sulfate, manganous sulfate 0.015 gram, 0.02 gram, zinc sulfate, cupric chloride 0.01 gram, rose vitriol 0.01 gram.
3. the method for screening obligate aerobic denitrification fungi according to claim 1, it is characterized in that, also comprise the authentication method of step (4) obligate aerobic denitrification fungi, comprise the qualification of aerobic denitrification ability, the obligate aerobic dientification of bacteria, molecular biology identification:
Steps A: aerobic denitrification ability is identified
Prepare aerobic denitrification capability and identify that substratum often rises aerobic denitrification capability and identifies that substratum is composed as follows: SODIUMNITRATE 1-1.5 gram; Potassium primary phosphate 1-1.5 gram; Sunmorl N 60S 5-10 gram; Water 90-120 milliliter; Calcium chloride 0.01-0.03 gram; Magnesium sulfate 0.01-0.02 gram; Sodium bicarbonate 0.2-0.5 gram; Trace element mixed solution: ferrous sulfate 0.01-0.02 gram, manganous sulfate 0.01-0.02 gram, zinc sulfate 0.01-0.03 gram, cupric chloride 0.01-0.03 gram, rose vitriol 0.01-0.02 gram; Above-mentioned composition is joined in Erlenmeyer flask successively, 121 DEG C of sterilizings 20 minutes; Prepare indicator, described indicator moiety is as follows: 4-aminobenzene sulfonamide 20g, N-1-naphthodiamide hydrochloride 1g, phosphoric acid 50mL, and water 250mL, is diluted to 500mL; By inoculation in described aerobic denitrification capability qualification substratum, under the condition of oxygen abundance, be inverted cultivation at 30 DEG C and add indicator in backward substratum in 3-5 days, as substratum reddens, then show that test strains has aerobic denitrification ability;
Step B: the obligate aerobic dientification of bacteria
By step (A) identify after inoculation on obligate aerobic denitrification fungi substratum, and transfer to pour nitrogen in anaerobism bottle after seal; Cultivate 1 month at 30 DEG C; If bacterial strain is without growth sign, illustrate that this bacterial classification is obligate aerobic bacterium;
Step C: molecular biology identification
By step (B) identify after bacterial strain carry out respectively DNA extraction go forward side by side performing PCR amplification, then check order; The gene database in GenBank in blast program and MycoBank is used to compare with sequencing result respectively, and reference Fungal identification handbook, obtain the kind of bacterial strain.
4. the method for screening obligate aerobic denitrification fungi according to claim 3, it is characterized in that, it is composed as follows that the aerobic denitrification capability qualification substratum described in step 4 often rises described substratum: SODIUMNITRATE 1.3 grams, potassium primary phosphate 1.3 grams, Sunmorl N 60S 8 grams, 110 milliliters, water, 0.02 gram, calcium chloride, 0.01 gram, magnesium sulfate, sodium bicarbonate 0.4 gram; Trace element mixed solution: 0.015 gram, ferrous sulfate, manganous sulfate 0.015 gram, 0.01 gram, zinc sulfate, cupric chloride 0.015 gram, rose vitriol 0.01 gram.
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Inventor after: Xue Jianliang Inventor after: Li Weisi Inventor after: Cui Qinqin Inventor after: Cheng Jianguang Inventor after: Yu Hao Inventor before: Xue Jianliang Inventor before: Li Weisi Inventor before: Zhao Chaocheng Inventor before: Cheng Jianguang Inventor before: Yu Hao Inventor before: Zhao Dongfeng |
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