CN115725476B - A strain of Pseudomonas marginale and its application - Google Patents

本发明属于污水生物脱氮技术领域，具体涉及一株边缘假单胞菌及其应用。边缘假单胞菌(Pseudomonas marginalis)，其特征在于，所述菌株为WS45，于2021年7月22日被保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.22943。在1‑40℃条件下，所述菌株WS45具有异养硝化‑好氧反硝化代谢能力，对于污水中硝态氮、亚硝态氮和铵态氮具有较好的去除效果。该菌株适应低温能力佳，可用于我国寒冷地区的中高浓度生活污水、畜禽养殖废水或工业有机废水等多种水体无机氮污染治理应用，具有环境友好、适用温度范围宽等优点。The present invention belongs to the technical field of biological denitrification of sewage, and specifically relates to a strain of Pseudomonas marginalis and its application. Pseudomonas marginalis (Pseudomonas marginalis), characterized in that the strain is WS45, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on July 22, 2021, with a deposit number of CGMCC No.22943. Under 1-40°C conditions, the strain WS45 has heterotrophic nitrification-aerobic denitrification metabolic capabilities, and has a good removal effect on nitrate nitrogen, nitrite nitrogen and ammonium nitrogen in sewage. The strain has good adaptability to low temperatures and can be used for inorganic nitrogen pollution control in various water bodies such as medium and high concentration domestic sewage, livestock and poultry breeding wastewater or industrial organic wastewater in cold areas of my country. It has the advantages of being environmentally friendly and having a wide applicable temperature range.

Pseudomonas marginalis and application thereof

Technical Field

The invention belongs to the technical field of biological denitrification of sewage, and particularly relates to pseudomonas marginalis and application thereof.

Background

Inorganic nitrogen pollution in water bodies frequently occurs in many countries around the world, and has posed a potential threat to the environmental quality of the ecosystem and human health. Evidence shows that the ammonia nitrogen and nitrate nitrogen concentration in the earth surface and near-shore water body of China are worth noting. How to economically and effectively remove inorganic nitrogen pollution in water is an important content for improving the quality of ecological environment and guaranteeing the health of an ecological system.

The sewage denitrification technology mainly comprises a chemical method, a physical method and a biological method. The microbial denitrification technology is widely valued because of the advantages of wide application range, environmental friendliness, economy, feasibility and the like. The core of the microbial denitrification technology is the efficient operation of processes such as microbial autotrophic nitrification, anaerobic denitrification metabolism and the like, and the metabolic pathways can promote the degradation of ammoniacal nitrogen, nitrate nitrogen and nitrite nitrogen in sewage. Through many years of development, a mature biological denitrification process such as A ² O, SBR and the like is applied to the engineering practice of sewage denitrification. In the microbial denitrification process, maintaining the metabolic activity of denitrifying microorganisms in sludge under complex and variable environmental conditions is a key to maintaining denitrification effect.

Many studies have demonstrated that low temperature is one of the major factors affecting biological denitrification efficiency. Most of denitrifying microorganism strains such as nitrifying bacteria and denitrifying bacteria or the most of denitrifying process technologies have moderate temperature range (20-35 ℃), and nitrifying and denitrifying microorganism strain resources capable of tolerating low temperature conditions (below 20 ℃) are relatively lacking. On the other hand, most of northern areas in China have long winter time, and the water temperature is often lower than 15 ℃. Under the low-temperature condition, the metabolic activity of denitrifying microorganisms in the sludge is obviously inhibited, and the quality of effluent water of a sewage treatment plant is difficult to reach the standard. The low-temperature denitrification microbial strain resource has a realistic technical demand.

In recent years, the screening of low temperature resistant denitrifying bacteria has been increasingly emphasized and some progress has been made. However, since environmental conditions (such as water temperature, salinity, inorganic nitrogen pollution concentration, etc.) of nitrogen-contaminated water bodies are often complex and variable, the existing medium-temperature denitrification strains cannot exhibit high-efficiency denitrification activity under low-temperature conditions, and the denitrification effect is often unsatisfactory.

Disclosure of Invention

Aiming at the current situations of poor effect and low-temperature-resistant denitrification strain resource deficiency of the current sewage biological denitrification technology under complex environmental conditions, particularly at low temperature, the invention aims to provide Pseudomonas marginalis (Pseudomonas marginalis) WS45 and application thereof in removing inorganic nitrogen pollutants in water.

In order to achieve the above purpose, the invention adopts the following technical scheme:

Pseudomonas marginalis (Pseudomonas marginalis), which is characterized in that the strain is WS45 and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 22943 in the year 7 and the month 22 of 2021.

The application of the strain is that the strain is used for removing inorganic nitrogen pollutants in water body at the temperature of 1-40 ℃.

The strain removes one or more of ammoniacal nitrogen, nitrate nitrogen and nitrite nitrogen in water at the temperature of 1-40 ℃.

The nitrogen is one or more of ammoniacal nitrogen, nitrate nitrogen and nitrite nitrogen, and the total concentration is not higher than 600mg/L. The concentration of ammonia nitrogen in the sewage is 0.1-250mg/L, the concentration of nitrate nitrogen is 0.1-600mg/L, and the concentration of nitrite nitrogen is 0.1-80mg/L.

A microbial inoculum for removing inorganic nitrogen pollutants in water body contains the strain.

The microbial inoculum is prepared by activating the strain.

The application of the microbial inoculum is that the microbial inoculum is used for removing inorganic nitrogen pollutants in water under the condition of wide temperature range.

A method for removing inorganic nitrogen pollutants in water body comprises the steps of adding activated strain or microbial inoculum into the water body environment to be treated according to the inoculation amount of 0.1-20% (v/v), so as to further remove the inorganic nitrogen pollutants in the water body, wherein the cell concentration of strain WS45 in the water body is more than or equal to 10 ⁴/ml.

The concentration of dissolved oxygen in the water body to be treated is above 5mg/L, the ratio of C/N in the water is 3-30, the pH of the water body is 6-9, the water temperature is 1-40 ℃, the salinity (calculated by NaCl concentration) is 0.2-30g/L, the hydraulic retention time is above 5 days, and the inorganic nitrogen pollutants in the water body can be removed.

The strain is activated by inoculating strain WS45 into a culture medium at a rotating speed of 120-220rpm and at a temperature of 15-35 ℃ for 10-72 hours, wherein the culture medium comprises 0.2-30.0% of NaCl, 0.5-20.0% of peptone, 0.5-10.0% of yeast extract and 1000mL of distilled water, and the pH value is regulated to 7.0-8.0.

The invention has the beneficial effects that:

(1) The Pseudomonas marginalis (Pseudomonas marginalis) strain WS45 can well withstand low-temperature conditions and can effectively remove ammoniacal nitrogen, nitrate nitrogen and nitrite nitrogen in water at the temperature of 1-40 ℃;

(2) The strain of the invention keeps good denitrification activity in a wide temperature range of 1-40 ℃, and can be used for sewage denitrification treatment in geographic areas with large annual average temperature difference change in north of China;

(3) The culture medium for activating and expanding the strain has simple components, and the preparation process of the bacterial liquid is relatively easy, thereby being beneficial to industrial production and subsequent application.

Detailed Description

The invention is further illustrated by the following examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The examples provided below are intended as references for further optimization of the development by those skilled in the art and are not in any way to be construed as limiting the present invention. The present invention is not limited to the following examples.

The Pseudomonas marginalis strain WS45 has the capability of effectively removing nitrate nitrogen, nitrite nitrogen and ammonium nitrogen in sewage at the temperature of 1-40 ℃, can resist low temperature, has the metabolic activity of heterotrophic nitrification and aerobic denitrification, and has a good potential application value in the treatment of water body nitrogen pollution in cold areas of China.

The strain WS45 separating screen is selected from activated sludge of a domestic sewage treatment plant of Heilongjiang, is primarily identified as Pseudomonas marginalis through 16S rRNA gene sequence comparison, and has obvious degradation and removal effects on inorganic nitrogen pollution under wider temperature conditions.

The experimental methods in the following examples, unless specifically indicated, are conventional or standard methods and may be carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The materials required in the following examples:

1) Activated sludge is collected from an activated sludge tank of a sewage treatment plant in a city of Heilongjiang.

2) Culture medium:

The denitrifying bacteria enrichment culture medium (g.L ^-1) comprises NaCl 1.0, peptone 12.0, yeast extract 6.0, distilled water 1L and pH 7.0-8.0.

1ML of denitrification culture medium (g·L^-1)：CH₃COONa 0.6;NH₄Cl 0.2、KNO₃ 0.4、NaNO₂ 0.2、KH₂PO₄0.05;CaCl₂·2H₂O 0.15;MgSO₄·7H₂O 0.06; microelement liquid, 1L of distilled water, pH 7.2-8.0, and 2% of agar added into a solid culture medium.

Microelement liquid (g/L)：EDTA0.35;ZnSO₄·7H₂O 0.2;CuSO₄·5H₂O 0.1;MnSO₄·7H₂O 0.1;Co(NO₃)₂·7H₂O 0.09;H₃BO₃ 0.1;Na₂MoO₄ 0.1.

After the culture medium is prepared, the culture medium needs to be sterilized for 20-25min under the conditions of 103.4kPa and 121 ℃ and is used after being cooled.

3) Main experimental instrument:

constant temperature biochemical incubator, constant temperature shaking table, ultra-clean bench, pressure steam sterilizer, miniature ultraviolet spectrophotometer, PCR instrument, electrophoresis apparatus, etc.

Example 1

Screening, preliminary identification and preservation of strain WS45

Taking 30g of activated sludge, transferring the sediment into a 500mL conical flask preloaded with 100mL denitrifying bacteria enrichment medium in a sterile ultra-clean workbench, and then shake-culturing for 15d on a constant-temperature shaking table at 2 ℃ and 180 rpm;

taking a proper amount of bacterial liquid after shake culture, carrying out gradient dilution by 10 times, then coating the bacterial liquid in a solid denitrification culture medium plate, and then culturing for more than 10 days at the temperature of 2 ℃;

After bacterial colonies appear on the plate culture medium, a plate streak separation method is adopted to pick the bacterial colonies, the streak separation culture process is repeated until the bacterial colonies are single in form, microscopic examination and denitrification effect measurement are carried out, and the bacterial strain with the largest inorganic nitrogen removal rate is selected and numbered WS45. WS45 is inoculated on a solid slant culture medium and is preserved at 4 ℃ for one month temporary storage period for strain identification and denitrification performance evaluation.

The bacterial genome DNA extraction kit was used and the genome DNA of the extracted strain WS45 was subjected to PCR detection and DNA quality evaluation, and then sent to Beijing Liuhua macrogene technologies Co., ltd for 16S rRNA gene sequencing with reference to the instructions for use. Sequence alignment of the measured gene sequences in NCBI database revealed 99% sequence identity between WS45 and Pseudomonas marginalis (DSM 13124), suggesting that strain was closest to Pseudomonas marginalis. The 16S rRNA gene sequence of strain WS45 is:

WS45

Assembly of 2sequences into 1contig(s)

the strain WS45 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22943.

EXAMPLE 2 evaluation of denitrification Effect of Strain WS45 at 1 ℃

And (one) activating the strain. WS45 strain inclined plane is taken, inoculated into a 500mL conical flask filled with 150mL denitrifying bacteria enrichment medium in a sterile workbench, and then placed in a constant temperature shaking table for activation for 48h at 25 ℃ and 180 rpm.

And (II) evaluating denitrification performance of the strain. The experiments were divided into 2 groups, namely a inoculation group and a control group. Inoculating a proper amount of activated bacterial liquid, inoculating the activated bacterial liquid into a denitrification culture medium according to an inoculating proportion of 2% by volume, and culturing for 6d at a constant temperature of 1 ℃ and 180 rpm. The control group is inoculated with an equal volume of WS45 bacterial liquid sterilized by high-temperature steam, and the rest conditions are completely the same as those of the inoculation group. Each treatment was set to 3 parallels. After the 6d culture period is finished, filtering the water sample in the bottle by using a 0.22 mu m filter membrane, and taking filtrate to measure the concentration of ammoniacal nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 1.

TABLE 1 Denitrification Effect of Strain WS45 at 1 ℃

As can be seen from Table 1, after 6d, the WS45 strain in the treatment group had ammonia nitrogen removal rates of 30.2%, 35.7% and 24.4% respectively at a low temperature of 1℃and the control group had little denitrification effect, and the three inorganic nitrogen removal rates were only 0.04%, 0.02% and 0.05% respectively. The experimental result shows that the strain WS45 has better denitrification capability under the low-temperature condition of 1 ℃.

EXAMPLE 3 evaluation of denitrification Effect of Strain WS45 at 5 °C

And (one) activating the strain. The strain activation is described in example 2.

And (II) evaluating denitrification performance of the strain. The experiment was carried out with bacterial groups and control groups. The brief process of inoculation group is that a proper amount of activated bacterial liquid is inoculated into a denitrification culture medium according to an inoculation proportion of 2 percent, and the culture is carried out for 6 days at a constant temperature of 5 ℃ and 180 rpm. The control group is inoculated with an equal volume of WS45 bacterial liquid sterilized by high-temperature steam, and the rest conditions are completely the same as those of the inoculation group. Each treatment was set to 3 parallels. After the 6d culture period is finished, filtering the water sample in the bottle by using a 0.22 mu m filter membrane, and taking filtrate to measure the concentration of ammoniacal nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 2.

TABLE 2 denitrification Effect of Strain WS45 at 5 ℃

As can be seen from Table 2, after 6d, the WS45 strain in the treatment group had ammonia nitrogen removal rates of 52.5%, 61.4% and 45.2% respectively at a low temperature of 5℃and the nitrite nitrogen removal rates of 0.01%, 0.10% and 0.12% respectively in the control group, while the control group had little denitrification effect. The experimental result shows that WS45 has better denitrification capability under the condition of 5 ℃.

EXAMPLE 4 evaluation of denitrification Effect of Strain WS45 at 10 ℃

And (one) activating the strain. The strain activation is described in example 2.

And (II) evaluating denitrification performance of the strain. The experiment was carried out with bacterial groups and control groups. The brief process of inoculation group is that a proper amount of activated bacterial liquid is inoculated into a denitrification culture medium according to an inoculation proportion of 2 percent, and the culture is carried out at a constant temperature of 6 d under the conditions of 10 ℃ and 180 rpm. The control group is inoculated with an equal volume of WS45 bacterial liquid sterilized by high-temperature steam, and the rest conditions are completely the same as those of the inoculation group. Each treatment was set to 3 parallels. After the culture period of 6 d is finished, the water sample in the bottle is filtered by a 0.22 mu m filter membrane, and the filtrate is taken to determine the concentration of ammoniacal nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 3.

TABLE 3 denitrification Effect of Strain WS45 at 10 ℃

As can be seen from Table 3, after 6 d, the WS45 strain in the treatment group had ammonia nitrogen removal rates of 77.2%, 84.5% and 68.7% respectively at a low temperature of 10℃and the control group had little denitrification effect, and the three inorganic nitrogen removal rates were only 0.10%, 0.10% and 0.11% respectively. The experimental result shows that WS45 has good denitrification capability under the condition of low temperature of 10 ℃.

EXAMPLE 5 evaluation of denitrification Effect of Strain WS45 at 20 °C

And (one) activating the strain. The strain activation is described in example 2.

And (II) evaluating denitrification performance of the strain. The experiment was carried out with bacterial groups and control groups. The brief process of inoculation group is that a proper amount of activated bacterial liquid is inoculated into a denitrification culture medium according to an inoculation proportion of 1%, and the culture is carried out at a constant temperature of 5d under the conditions of 20 ℃ and 180 rpm. The control group is inoculated with an equal volume of WS45 bacterial liquid sterilized by high-temperature steam, and the rest conditions are completely the same as those of the inoculation group. Each treatment was set to 3 parallels. After the culture period of 5d is finished, the water sample in the bottle is filtered by a 0.22 mu m filter membrane, and the filtrate is taken to determine the concentration of ammoniacal nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 4.

TABLE 4 denitrification Effect of Strain WS45 at 20 ℃

As can be seen from Table 4, after 5d, the WS45 strain in the treatment group had ammonia nitrogen removal rates of 92.4%, nitrate nitrogen removal rates of 93.6% and nitrite nitrogen removal rates of 90.4% at 20℃respectively, whereas the control group had little denitrification effect, and the three inorganic nitrogen removal rates were only 0.11%, 0.14% and 0.10% respectively. The results of this experiment show that WS45 has good denitrification capability at 20 ℃.

EXAMPLE 6 evaluation of denitrification Effect of Strain WS45 at 35 ℃

And (one) activating the strain. The strain activation is described in example 2.

And (II) evaluating denitrification performance of the strain. The experiment was carried out with bacterial groups and control groups. The brief process of inoculation group is that a proper amount of activated bacterial liquid is inoculated into a denitrification culture medium according to an inoculation proportion of 1%, and the culture is carried out at constant temperature of 35 ℃ and 180 rpm for 3 d. The control group is inoculated with an equal volume of WS45 bacterial liquid sterilized by high-temperature steam, and the rest conditions are completely the same as those of the inoculation group. Each treatment was set to 3 parallels. After the 3d culture period is finished, filtering the water sample in the bottle by using a 0.22 mu m filter membrane, and taking filtrate to measure the concentration of ammoniacal nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 5.

TABLE 5 denitrification Effect of Strain WS45 at 35 ℃

As can be seen from Table 5, after 3d, the WS45 strain in the treatment group had ammonia nitrogen removal rates of 99.1%, nitrate nitrogen removal rates of 99.4% and nitrite nitrogen removal rates of 97.2% at 35℃respectively, while the control group had little denitrification effect, and the three inorganic nitrogen removal rates were only 0.24%, 0.06% and 0.01% respectively. The results of this experiment show that WS45 has excellent denitrification capability at 35 ℃.