阅读说明：本技术 一种硫酸盐还原菌对硝化纤维素脱硝的应用 (Application of sulfate reducing bacteria to denitration of nitrocellulose ) 是由 陈可泉 陈乔煜 张阿磊 丁亚军 肖忠良 欧阳平凯 于 2021-01-21 设计创作，主要内容包括：本发明公开了一种硫酸盐还原菌对硝化纤维素脱硝的应用,所述硫酸盐还原菌为硫酸盐还原菌Desulfosporosinus meridieiDSM 13257,由美国菌种保藏中心保藏,其保藏编号为ATCC NO.BAA-275。本发明提供的硫酸盐还原菌,在厌氧条件下可以完成硝化纤维素的脱硝,并且菌株可以将生成的硝酸盐及亚硝酸盐作为营养物质利用,减少污染。该菌株与硝化纤维素共培养14d后,脱去一部分硝基,使原氮含量13.01%的硝化纤维素氮含量降至12.58%。(The invention discloses application of sulfate reducing bacteria to denitration of nitrocellulose, wherein the sulfate reducing bacteria are sulfate reducing bacteria Desulfosporosinus meridieiDSM 13257 Deposited by the American type culture Collection under accession number ATCC No. BAA-275. The sulfate reducing bacteria provided by the invention can complete the denitration of the nitrocellulose under the anaerobic condition, and the bacterial strain can utilize the generated nitrate and nitrite as nutrient substances, thereby reducing pollution. After the bacterial strain is co-cultured with nitrocellulose for 14 days, a part of nitro groups are removed, so that the nitrogen content of the nitrocellulose with the original nitrogen content of 13.01 percent is reduced to 12.58 percent.)

1. The application of sulfate reducing bacteria in denitration of nitrocellulose is characterized in that the sulfate reducing bacteria are sulfate reducing bacteriaDesulfosporosinus meridiei13257Deposited at the American type culture Collection with the deposit number of ATCC NO. BAA-275.

2. The use of sulfate-reducing bacteria for denitration of nitrocellulose according to claim 1, comprising the steps of:

step 1, preparing an anaerobic bacteria culture medium

Weighing 1.0g/L of glucose, 2.5g/L of yeast powder, 7.5g/L of peptone, 15.0g/L of meat extract and 2.5g/L, NaCL 5.0.0 g/L of acid hydrolyzed casein according to the following mass, mixing the components, adding 1% of resazurin as an oxygen indicator and 2.5% of L-cysteine reducer, uniformly stirring, and performing high-temperature high-pressure steam sterilization at 115 ℃ for 20min to obtain an anaerobic bacteria culture medium;

step 2, adding a culture solution into an anaerobic bottle in an aseptic environment, sealing and transferring into an anaerobic box for later use; the inoculation amount of the culture solution is not more than half of the capacity of the anaerobic bottle;

step 3, deoxidizing the anaerobic bottle in an anaerobic box until the oxygen indicator resazurin turns colorless, inoculating the strain DSM13257 into the anaerobic bottle according to the inoculation amount of 10 percent, and transferring the anaerobic bottle into an incubator to cultivate the blank OD₆₀₀When the content is 0.6-0.8, the product is ready for use;

step 4, adding a substrate of 4g/L nitrocotton into the culture solution in an anaerobic box, and culturing together with the strain;

step 5, after the culture is finished, centrifuging the culture solution at 8000rpm, taking the precipitate, washing the precipitate by using ultrapure water and 1M NacL solution respectively, and repeatedly washing for three times; after washing, 8000rpml of precipitate is centrifuged, acetone solution is added into the precipitate according to the proportion that 1g of precipitate is added into 40mL of acetone, the mixture is dissolved for 2 hours at 40 ℃, and supernatant is centrifuged; and then dropwise adding water and ethanol into the supernatant according to a volume ratio of 2: 1, mixing the solution until the nitrocellulose is not separated out any more; and drying the separated nitrocellulose, weighing, calculating the loss rate, and analyzing the N content.

3. The use of sulfate-reducing bacteria for denitration of nitrocellulose according to claim 2, wherein casein is hydrolyzed by adding a small molecular nitrogen source during co-culture in step 2.

4. The use of sulfate-reducing bacteria for denitration of nitrocellulose according to claim 2, wherein the co-culture temperature in step 2 is 37 ℃ and the system pH is 7.0-7.5.

Technical Field

The invention belongs to the fields of environmental engineering and biology, and particularly relates to application of sulfate reducing bacteria to denitration of nitrocellulose.

Background

Nitrocellulose is an excellent material and can be applied to various fields. After the hydroxyl groups in the cellulose molecule are all replaced by nitro groups, each monomer C₆H₇O₂(ONO₂)₃The nitrogen content was 14.1%. The nitrogen content of the explosive nitrocotton is usually between 12.0 and 13.4 percent. Nitrocellulose with too high nitrogen content is unstable in performance and extremely easy to explode, and a large amount of nitrate and nitrite are generated during degradation, so that the nitrocellulose pollutes the environment and has certain toxicity. Therefore, it is very significant to find a safe and green method for degrading nitrocellulose.

During denitrification by a chemical method, a large amount of nitrite and nitrate are generated, and both of the nitrite and the nitrate have toxicity and great destructive power to the environment; and the reaction degree of chemical nitrogen reduction is extremely difficult to control, and the main chain structure of the nitrocellulose can be damaged or depolymerized, so that the performance of the nitrocellulose is influenced.

Compared with chemical denitrification, biological denitrification is more and more emphasized as a novel pollution-free treatment technology, the biological treatment technology has the advantages of no secondary pollution, green cleanness, flexible treatment mode and the like, and the development of the biological denitrification technology becomes a novel hotspot for the denitrification treatment of the nitrocellulose in recent years.

When studying the microbial biodegradability of nitrocellulose, it was earlier often treated with a mixed culture of microorganisms rather than a single microorganism system. For example, the microbial flora in the sludge is utilized to perform chemical pretreatment (alkaline hydrolysis) on the nitrocellulose to generate a hydrolysate, and the hydrolysate can be removed from nitrate groups in the presence of other carbon sources to promote the degradation of the nitrocellulose by activated sludge microorganisms. This process can be summarized as: alkaline pretreatment-denitrification-activated sludge treatment-further denitrification-produces the desired effluent. The pretreatment method is extremely difficult to control the degree of alkaline hydrolysis, the structure of cellulose molecules can be damaged, the expected effect cannot be achieved, and the generated nitrate and nitrite pollute the environment. In combination with the above analysis, a single microbial strain is used to perform the denitrification of nitrocellulose, and if the pretreatment of chemical alkaline hydrolysis is not required, the structure of the carbon main chain of nitrocellulose is not destroyed, which means that the denitrification of nitrocellulose can be controlled more simply and effectively, and the microbial strain utilizes nitrate and nitrite generated by denitrification as a nitrogen source, thereby realizing energy-cleaning treatment. Therefore, a single microbial strain is urgently needed to be found to meet the production requirement, and a green and clean nitrogen reduction mode is realized in the process.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of sulfate reducing bacteria to the denitration of the nitrocellulose, the sulfate reducing bacteria DSM13257 can have denitrification performance under anaerobic conditions to realize the application of the nitrocellulose, and the bacterial strain can utilize nitrate and nitrite degraded on the nitrocellulose as self nutrient substances under the anaerobic conditions, thereby avoiding the secondary pollution of the nitrate and nitrite to the environment.

Application of sulfate reducing bacteria to denitration of nitrocellulose, wherein the sulfate reducing bacteria are sulfate reducing bacteriaDesulfosporosinus meridiei （DSM）13257Deposited at the American type culture Collection with the deposit number of ATCC NO. BAA-275.

By culturing the obtained strain, the strain has the following characteristics:

(1) colony characteristics: the colony surface is smooth, the edge is neat, and the colony is light yellow; short rod shape, single colony; growth is only under strict anaerobic conditions;

(2) TIANGEN DNA KIT is adopted to extract original bacteria genome DNA, and the 16S rDNA gene sequence characteristics of the strain are as follows: the 16S rDNA has a nucleotide sequence shown in the sequence table, the length of the 16S rDNA is 1472 bp, the measured sequence is compared with the known bacteria 16S rDNA sequence in a GenBank database by using BLAST software, and the improved strain is found to be the sulfate reducing bacteriaDesulfosporosinus meridiei DSM 13257；

(3) And (3) constructing a 16S rDNA phylogenetic tree by applying MEGA software and adopting a Neighbor-Joining method, determining the evolution position of the phylogenetic tree, and combining morphological characteristics and physiological and biochemical characteristics of the phylogenetic tree.

As an improvement, the application comprises the following steps:

step 1, preparing an anaerobic bacteria culture medium

Weighing 1.0g/L of glucose, 2.5g/L of yeast powder, 7.5g/L of peptone, 15.0g/L of meat extract and 2.5g/L, NaCL 5.0.0 g/L of acid hydrolyzed casein according to the following mass, mixing the components, adding 1% of resazurin as an oxygen indicator and 2.5% of L-cysteine reducer, uniformly stirring, and performing high-temperature high-pressure steam sterilization at 115 ℃ for 20min to obtain an anaerobic bacteria culture medium;

step 2, adding a culture solution into an anaerobic bottle in an aseptic environment, sealing and transferring into an anaerobic box for later use; the inoculation amount of the culture solution is not more than half of the capacity of the anaerobic bottle;

step 3, deoxidizing the anaerobic bottle in an anaerobic box until the oxygen indicator resazurin turns colorless, and inoculating the strain DSM13257 into the anaerobic bottle according to the inoculation amount of 10%;

step 4, adding a substrate of 4g/L nitrocotton into the culture solution in an anaerobic box, and culturing together with the strain;

step 5, after the culture is finished, centrifuging the culture solution at 8000rpm, taking the precipitate, washing the precipitate by using ultrapure water and 1M NacL solution respectively, and repeatedly washing for three times; after washing, 8000rpml of precipitate is centrifuged, acetone solution is added into the precipitate according to the proportion that 1g of precipitate is added into 40mL of acetone, the mixture is dissolved for 2 hours at 40 ℃, and supernatant is centrifuged; and then dropwise adding water and ethanol into the supernatant according to a volume ratio of 2: 1, mixing the solution until the nitrocellulose is not separated out any more; and drying the separated nitrocellulose, weighing, calculating the loss rate, and analyzing the N content.

In the improvement, during the co-culture in the step 2, a small molecular nitrogen source is added to hydrolyze casein.

As a modification, the temperature of co-cultivation in step 2 is 37 ℃ and the pH of the system is 7.0 to 7.5.

Has the advantages that:

compared with the prior art, the method has the advantages that,the invention discloses an application of sulfate reducing bacteria in nitrocellulose denitration, and the sulfate reducing bacteriaDesulfosporosinus meridiei （DSM）13257Preliminary experiments on the denitrification and denitrogenation of the nitrocellulose show that the strain can effectively reduce the nitrogen content of the nitrocellulose at 37 ℃ under strict anaerobic conditions, and nitrate nitrogen and nitrite nitrogen generated by the nitro group removal of the nitrocellulose are converted into carbon sources for utilization, so that the strain grows better, better denitrification and denitrogenation effects are achieved, the strain is circulated and recycled, no harmful nitrate or nitrite is produced, and green cleanness is realized.

The concrete advantages are as follows:

(1) the sulfate-reducing bacteria provided by the inventionDesulfosporosinus meridiei（DSM）13257Before denitration, alkaline hydrolysis pretreatment is not needed, and the main chain of cellulose is not damaged;

(2) the sulfate-reducing bacteria provided by the inventionDesulfosporosinus meridiei（DSM）13257Strictly anaerobic in the denitrification and denitration treatment process of the nitrocellulose, and no nutrient is required to be fed; the nitrate nitrogen and nitrite nitrogen after denitrification in the culture medium can be used as nitrogen sources to realize the growth of the bacterial strain so as to better realize denitrification and denitrification without causing secondary pollution, and the nitrogen content of the nitrocellulose with 13.01 percent of original nitrogen content can be reduced to 12.58 percent after 14 days;

(3) the sulfate-reducing bacteria provided by the inventionDesulfosporosinus meridiei （DSM）13257The denitrification treatment and the denitrification treatment of the nitrocellulose do not produce harmful nitrate and nitrite and do not pollute water quality and soil.

Drawings

FIG. 1 shows sulfate-reducing bacteria on different days of cultureDesulfosporosinus meridiei （DSM）13257A comparison graph of denitrification results of nitrocellulose;

FIG. 2 shows sulfate-reducing bacteria on different days of cultureDesulfosporosinus meridiei （DSM）13257FTIR comparison graph after denitrification of nitrocellulose;

FIG. 3 shows sulfate-reducing bacteriaDesulfosporosinus meridiei（DSM）13257A flow chart of the denitrification of the nitrocellulose is shown in the specification, wherein A is test tube inoculation, B is flat plate activation, C is substrate addition, D is anaerobic culture, E is acetone dissolution extraction, F is weighing, and G is element analysis.

Detailed description of the preferred embodiments

The sulfate-reducing bacteria of the present inventionDesulfosporosinus meridiei （DSM）13257Deposited by the American type culture Collection, the collection numbers are ATCC NO:DSM 13257 [NCIMB 13706, S10]the sulfate reducing bacteria strain is given by Nanjing university of agriculture and the date of the giving is No. 9/9 of 2020.

Example 1: activation of the Strain

Firstly, preparing an anaerobic bacteria culture medium

Weighing 1.0g/L of glucose, 2.5g/L of yeast powder, 7.5g/L of peptone, 15.0g/L of meat extract and 2.5g/L, NaCL 5.0.0 g/L of acid hydrolyzed casein according to the following mass, mixing the components, adding 1% of resazurin as an oxygen indicator and 2.5% of L-cysteine reducer, uniformly stirring, and performing high-temperature high-pressure steam sterilization at 115 ℃ for 20min to obtain an anaerobic bacteria culture medium;

secondly, adding 50mL of sterilized culture solution into an anaerobic bottle with the capacity of 100mL in a super-clean workbench, covering a plug and a cover of the anaerobic bottle after the culture solution is finished, and transferring the anaerobic bottle into an anaerobic box;

subsequently, the cap and stopper of the anaerobic bottle were opened in the anaerobic chamber, oxygen was removed until the oxygen indicator resazurin became colorless, and subsequent inoculation was performed in the anaerobic chamber.

Finally, in an anaerobic chamber, to the oxygen-purged culture broth, the strain DSM13257 was inoculated into an anaerobic flask at an inoculum size of 10% (since it is generally difficult to utilize a macromolecular carbon-nitrogen source at the initial stage of growth of anaerobic microorganisms, the biomass was shortened to OD by increasing the initial inoculum size₆₀₀Time of (d);

after inoculation is finished, placing the inoculated anaerobic bottle in an incubator at 37 ℃ for culture; after 5 days of growth, the concentration of the cells in the culture solution was significantly increased. 2mL of the solution was aspirated in an anaerobic incubator, and OD was measured₆₀₀Is 0.653.

Example 2: application process of sulfate reducing bacteria DSM13257 to nitrocellulose

Strain culture: for details, see example 1

Substrate conversion: preparation of 4g/L nitrocellulose: weighing 0.2g of nitrocellulose, adding into 10mL of deionized water, sterilizing at 121 ℃ for 20min by high-temperature high-pressure steam, and drying the nitrocellulose under the aseptic condition to obtain 0.2g of aseptic nitrocellulose after sterilization. ② transferring 0.2g of sterile nitrocellulose into OD₆₀₀In the culture solution of the strain at 0.6, the final system of nitrocellulose in the anaerobic flask was set to 4g/L, and the strain was co-cultured with the substrate.

Substrate re-extraction: centrifuging reaction liquid cultured for 7d at 8000rpm for 15min to obtain a mixture, and drying at 60 deg.C; the dried precipitate was dried, weighed, added with acetone (40 mL acetone added to 1g precipitate) and reacted at 40 ℃ for 2 h. After the reaction was completed, 8000rpm was centrifuged for 15min, the supernatant was collected, and a mixture of water and ethanol (water: ethanol = 2: 1) was added to the supernatant to precipitate nitrocotton until the precipitation of nitrocotton was stopped. And drying the separated nitrocotton at 60 ℃, weighing, and testing the N content by using an element analyzer. The flow chart is shown in figure 3.

Example 3: influence of small molecular nitrogen source addition and non-addition on sulfate reducing bacteria in culture medium

The culture medium A comprises the following components: 1.0 of glucose; 2.5 of yeast powder; peptone 7.5; 15.0 parts of meat extract; NaCL 5.0, unit g/L.

The components of the culture medium B are as follows: 1.0 of glucose; 2.5 of yeast powder; peptone 7.5; 15.0 parts of meat extract; acid hydrolysis casein 2.5; NaCL 5.0, unit g/L.

1 percent of resazurin is added as an oxygen indicator and 2.5 percent of L-cysteine salt reducing agent respectively. After the preparation of the culture medium is finished, the culture medium is sterilized by steam at the high temperature and the high pressure for 20min at the temperature of 115 ℃. And adding a culture solution into the anaerobic bottle in a super-clean workbench, wherein the inoculation amount of the culture solution is not more than half of the capacity of the anaerobic bottle, covering a plug and a cover of the anaerobic bottle after the culture solution is completely inoculated, and transferring the culture solution into an anaerobic box. Opening the cover and the plug of the anaerobic bottle in an anaerobic box, deoxidizing until the oxygen indicator resazurin becomes colorless, and performing subsequent inoculation in the anaerobic box.

Inoculating in anaerobic box, culturing at 37 deg.C for one week, continuously measuring OD to determine the growth of strain, and finding out that the strain added with small molecular nitrogen source (acid hydrolyzed casein) can reach OD within 32 hr₆₀₀0.8, medium without addition of small molecular nitrogen source, required 64h to reach OD₆₀₀0.8. It can be seen from the data that the strain added with the small molecular nitrogen source grows much faster, so that the growth of the strain can be obviously improved by adding the small molecular carbon source into the culture medium.

Example 4: sulfate reducing bacteria on different daysDesulfosporosinus meridiei DSM 13257Effect on Nitrogen reduction of Nitrocellulose

Firstly, preparing an anaerobic bacteria culture medium

The components of the culture medium are as follows: mixing glucose 1.0g/L, yeast powder 2.5g/L, peptone 7.5g/L, meat extract 15.0g/L, acid hydrolyzed casein 2.5g/L, NaCL 5.0g/L, and adding 1% resazurin as oxygen indicator and 2.5% L-cysteine salt reducer. After the preparation of the culture medium is finished, the culture medium is sterilized by steam at the high temperature and the high pressure for 20min at the temperature of 115 ℃.

Then, 50mL of culture solution was added to a 100mL anaerobic bottle in a clean bench, and after completion, the stopper and the lid of the anaerobic bottle were closed, and the medium was transferred to an anaerobic tank.

Subsequently, the cap and stopper of the anaerobic bottle were opened in the anaerobic chamber, oxygen was removed until the oxygen indicator resazurin became colorless, and subsequent inoculation was performed in the anaerobic chamber.

Finally, in an anaerobic tank, the strain DSM13257 was inoculated into an anaerobic bottle at an inoculum size of 10% to the culture solution depleted of oxygen.

Inoculated strain OD₆₀₀To a concentration of 0.6 to 0.8, 4g/L nitrocellulose was added as a substrate, the mixture was cultured for 7 days, and the cultured substrate nitrocellulose was re-extracted by a re-extraction method, specifically, as in example 2, the nitrogen content of the nitrocellulose after 7 days of treatment with the original nitrocellulose and the sulfate-reducing bacteria was measured using an element analyzerChanges are found to be obviously reduced, and no literature reports exist beforeDesulfosporosinus meridiei （DSM）13257Since the culture medium has a nitrogen-reducing effect, repeated experiments are carried out to verify whether the culture medium has the nitrogen-reducing effect, and the culture time is prolonged to see whether the culture medium can further reduce nitrogen with the time. After the completion of the culture for 7 days and 14 days, elemental analysis was carried out on the re-extracted nitrocellulose, and it was found thatDesulfosporosinus meridiei （DSM）13257The nitrogen-reducing effect on the nitrocellulose is really achieved, and the nitrogen-reducing effect is more obvious along with the increase of time. See figure 1 for details.

And (3) comparing the nitrocellulose treated by the sulfate reducing bacteria on different days with the original nitrocellulose by Fourier infrared spectra. 1635 cm^-1Is formed by-ONO₂Asymmetric stretching vibration, 1274 cm^-1Is formed by-ONO₂Symmetric telescopic vibration, 1061 cm^-1Is in C-O-C telescopic vibration at 996 cm^-1Is subjected to C-O-C telescopic vibration of 824 cm^-1C-O-C stretching vibration is adopted. It was found that the main groups of nitrocellulose did not disappear and no new groups were produced, and the absorption peak of nitro groups was significantly reduced after the treatment with sulfate-reducing bacteria, and the reduction was more significant with longer time, as shown in fig. 2.

The sulfate reducing bacteria DSM13257 firstly carry out denitrification and denitration treatment on the nitrocellulose, and find that the sulfate reducing bacteria DSM13257 can really carry out denitrification on the nitrocellulose; after the sulfate reducing bacteria are added with the micromolecular carbon source acid to hydrolyze casein, the time for reaching the same biomass in the same time is shortened by one time; the sulfate reducing bacteria can utilize nitrate and nitrite generated by the denitration of the nitrocellulose as nutrient substances under the anaerobic condition, thereby reducing the generation of pollutants.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.