A method for rapidly enriching and isolating purple non-sulfur bacteria from sewage treatment systems

Abstract

The present invention discloses a method for rapidly enriching and isolating purple non-sulfur bacteria from sewage treatment systems. This method optimizes the PNSB enrichment medium and uses infrared anaerobic methods to rapidly enrich high concentrations of PNSB from complex sewage environments. Simultaneously, a single PNSB strain is isolated using the optimized isolation medium and a simple light anaerobic device. The method provided by the present invention enables rapid enrichment and isolation of PNSB from complex environments, resulting in higher bacterial purity and simpler isolation methods, thus possessing important practical applications in microbial culture technology.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial enrichment and cultivation, and particularly relates to a method for rapidly enriching and separating purple non-sulfur bacteria from a sewage treatment system. Background Art

[0002] Purple non-sulfur bacteria (PNSB) are a class of anoxic microorganisms that use light as their energy source. They possess flexible metabolic pathways that adapt to diverse growth environments, utilizing diverse carbon sources for growth and metabolism and producing high-value products. In recent years, they have been widely used in wastewater treatment and resource recovery. PNSB are primarily found in various water bodies and soils in nature, and are often found in anaerobic environments within sewage systems and sediments.

[0003] Currently, photosynthetic microorganisms are commonly grown and propagated in a light incubator to provide appropriate light and temperature conditions. PNSB are then enriched and screened from mixed microbial samples using enrichment culture media. However, this method has a long culture cycle, slow PNSB growth, and requires at least three passages. Furthermore, the enriched PNSB content is low. Furthermore, the effects of the content and ratio of carbon sources and other components in the culture medium on the growth of enriched PNSB are unknown. Furthermore, the extremely high microbial diversity in sewage systems makes it difficult to find a single selective culture medium that is universally compatible with the growth of all PNSB, which also poses challenges to PNSB enrichment and screening.

[0004] The selection and isolation of PNSB is also more challenging than for most anaerobic microorganisms because they require both oxygen exclusion and light during the culture process. Anaerobic jar and glove box cultivation methods are costly, while traditional pyrogallic acid and biological oxygen consumption methods are cumbersome. High-density agar culture significantly reduces light availability, extending isolation and culture time. Summary of the Invention

[0005] In view of the problems in the prior art of long enrichment period and difficult separation conditions of purple non-sulfur bacteria, the purpose of the present invention is to provide a method that is simple to operate and can quickly enrich and separate purple non-sulfur bacteria from complex sewage environments, and the obtained PNSB bacterial community has high purity.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for rapidly enriching and separating purple non-sulfur bacteria from a sewage treatment system comprises the following steps:

[0008] Step 1: Take a sample from the sewage treatment system and place it in an enrichment medium, and culture it in a closed environment under infrared light;

[0009] Step 2: Dip the culture solution obtained in step 1 and streak it on the separation medium, and culture it under light in an anaerobic environment. After a single colony grows, pick a single colony and continue streaking and separating it until a single morphology of bacteria appears under microscopy.

[0010] The enrichment medium has a pH of 7.0 and specifically includes: 1-2 g / L sodium acetate, 0.5-1.3 g / L NH4Cl, 2.2-4.4 g / L KH2PO4, 1.7-3.4 g / L K2HPO4, 0.5-1 g / L NaHCO3, 0.01-0.1 g / L CaCl2·2H2O, 0.2-0.3 g / L MgCl2·6H2O, 0.25-0.5 g / L NaCl, 0.39-0.78 g / L KCl and 1 mL / L of trace element solution A.

[0011] In the above technical solution, the sample taken from the sewage treatment system is preferably anaerobic activated sludge or anoxic activated sludge.

[0012] In the above-mentioned enrichment culture medium, sodium acetate, as a cheap and readily available carbon source, has been proven to be more universal for PNSB enrichment than other small molecule organic acids (such as sodium succinate and potassium citrate); at the same time, when the sodium acetate concentration range is 1-2 g / L, the bacterial biomass increases rapidly with the increase of concentration, and the PNSB enrichment time is significantly shortened and the effect is better, but the gain effect is very small after the concentration exceeds 2 g / L; in addition, KH2PO4, K2HPO4 and NaHCO3 are used as buffers to regulate the pH of the enrichment process. In addition, it was found during the study that when the buffer concentration is too high, the PNSB enrichment time increases, and low-salt conditions are more conducive to PNSB enrichment, and PNSB can occupy a dominant position in about 3-4 days.

[0013] Furthermore, in the above-mentioned enrichment medium, the composition of trace element solution A is specifically as follows: per 1000 mL of distilled water, add FeSO4·7H2O 1g, ZnCl2 70mg, MnCl2·4H2O 100mg, H3BO3 6mg, CaCl2·6H2O 130mg, CuCl2·2H2O 2mg, NiCl2·6H2O 24mg, Na2MoO4·2H2O 36mg, and CoCl2·6H2O 238mg.

[0014] In the above technical solution, the separation medium in step 2 is a solid culture medium, specifically made of a liquid culture medium added with agar, and the added amount of agar is 1.5-2.0 wt%.

[0015] The liquid culture medium is preferably prepared by adding 3 g of sodium acetate, 0.9 g of K2HPO4, 0.6 g of KH2PO4, 0.2 g of MgSO4·7H2O, 0.075 g of CaCl2·2H2O, 0.0118 g of FeSO4·7H2O, 0.02 g of ethylenediaminetetraacetic acid, 15 μg of biotin, 0.5 g of NH4Cl and 1 mL of trace element solution B per 1000 mL of distilled water; and the pH of the culture medium is 7.0.

[0016] Furthermore, the composition of trace element solution B is as follows: 280 mg H3BO3, 188 mg MnCl2·4H2O, 75 mg Na2MoO4·2H2O, 24 mg ZnSO4·7H2O, and 5.5 mg CuSO4·H2O are added per 100 mL of distilled water.

[0017] In the above technical solution, the closed culture temperature in step 1 is 27-33°C and the time is 5-7 days. Preferably, the specific operation of step 1 can be: placing the sample in a serum bottle containing enrichment medium, first flushing the medium with N2 to provide anaerobic conditions, then sealing the bottle, and irradiating it with a halogen tungsten lamp to provide the light and temperature required for PNSB growth. During irradiation, an infrared filter is placed on the side of the serum bottle exposed to the light source to block the visible light and allow only infrared light to pass.

[0018] In addition, in practical applications, after the first closed culture is completed, part of the bacterial liquid can be added to a new enrichment medium for repeated enrichment as needed.

[0019] In the above technical solution, the anaerobic environment in step 2 is provided by an anaerobic separation device. Preferably, the anaerobic separation device comprises a transparent container and a lid that seals the opening of the transparent container. The lid is provided with a vacuum valve, which is capped with a gas collection bag filled with inert gas. In a specific embodiment, both the transparent container and the lid are made of glass, and the sealing portion of the transparent container and the lid are frosted.

[0020] Furthermore, in the aforementioned anaerobic separation device, a permeable partition can be installed within the transparent container, dividing the interior of the transparent container into upper and lower compartments. The compartment above the permeable partition is used to accommodate a culture dish, while the compartment below the permeable partition contains a desiccant and an anaerobic gas production bag. In a specific embodiment, the permeable partition is a ceramic plate with through holes, and the desiccant is color-changing silica gel.

[0021] The principle of using the above-mentioned anaerobic separation device to create an anaerobic environment is specifically as follows: a culture dish containing the scribed separation culture medium is placed in a transparent container, a lid is sealed on the opening of the transparent container (for example, when both are made of glass and the lid is frosted, vaseline can be applied to the frosted surface to keep the container airtight), the enclosed space in the container is evacuated to a vacuum through a vacuum valve, and then a gas collection bag filled with inert gas (such as N2) is placed on the vacuum valve to allow the inert gas to enter the enclosed space of the container to keep the internal and external pressures consistent. In addition, the desiccant and anaerobic gas production bag are more conducive to maintaining the dryness and oxygen-deficient state of the enclosed space. Moreover, since the container is transparent, the culture dish in the enclosed space can still be kept in a light environment.

[0022] The beneficial effects of the present invention are:

[0023] The enrichment culture medium provided by the present invention has a good enrichment and culture effect, and can overcome the difficulty in obtaining a high-purity PNSB bacterial community due to the complex sewage environment; combined with the infrared light anaerobic environment, it provides a good growth environment for PNSB. After 3-4 days of enrichment, a high-purity PNSB bacterial community (>60%) can be obtained from a complex sewage environment, and the color of the bacterial solution gradually deepens to a deep red solution; and when the PNSB obtained after one week of enrichment using the present invention is used for sewage treatment, the pollutant (COD) removal rate is increased by 30%, and the COD removal rate is also significantly improved.

[0024] At the same time, given that PNSB separation has high requirements for culture medium and environment, the separation culture medium and anaerobic separation device provided by the present invention can quickly and cost-effectively screen out bacteria of a single form; and the anaerobic separation device provided by the present invention can be obtained by modifying a common dryer in the laboratory, which further saves costs and has practical application significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic structural diagram of an anaerobic separation device used in an embodiment of the present invention;

[0026] Figure 2 The characteristic peak scanning change diagram after enrichment in Examples 1 and 2;

[0027] Figure 3 The COD change diagram during the enrichment process of Example 1 and Example 2;

[0028] Figure 4 This is a graph showing the color change of the culture medium obtained from enriching PNSB in Example 1 and Example 2 over a two-week period.

[0029] Figure 5 This is a community diversity analysis diagram after one week of enrichment of PNSB in Example 1 and Example 2. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0031] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0032] Example 1

[0033] In this example, anaerobic sludge P from the A2O process section of an industrial park in Wuhan was selected as a sample for enrichment and separation of purple non-sulfur bacteria. The specific process is as follows:

[0034] (1) Preparation of enrichment medium: distilled water 1000 mL, sodium acetate 1.0 g, KH2PO4 4.4 g, K2HPO4 3.4 g, NaHCO3 1 g, CaCl2·2H2O 0.0146 g, MgCl2·6H2O 0.2 g, NH4Cl 1.3 g, NaCl 0.5 g, KCl 0.78 g, trace element solution 1 mL; adjust the pH to 7.0 with 12 M NaOH. The ratio of the trace element solution is: FeSO4·7H2O1g, ZnCl2 70mg, MnCl2·4H2O 100mg, H3BO3 6mg, CaCl2·6H2O 130mg, CuCl2·2H2O 2mg, NiCl2·6H2O 24mg, Na2MoO4·2H2O 36mg, CoCl2·6H2O 238mg, and distilled water 1000mL.

[0035] (2) PNSB enrichment process:

[0036] An appropriate amount of anaerobic sludge P was transferred to a serum bottle containing 400 mL of fresh artificial wastewater (~0.3 g VSS / L). The culture medium was flushed with nitrogen for 5 minutes before sealing. A 100W tungsten-halogen lamp was used to provide the required light and temperature (30 ± 3°C) for PNSB growth. An infrared filter was placed on the side of the serum bottle exposed to the light source, filtering out visible light and allowing only infrared light to pass. PNSB were then incubated and screened. After 5 days of culture, the bacterial culture turned noticeably red. On the 7th day, 40 mL of the culture was sampled and enriched again.

[0037] During the enrichment process, 2.5 mL of sample was collected from the bottle every 24 h, and the supernatant was taken after passing through a 0.22 μm filter membrane for analysis of COD and ammonia nitrogen concentrations. At the end of each enrichment cycle (7 days), samples were collected for characteristic peak scanning to confirm the presence of obvious carotenoid and bacteriochlorophyll characteristic peaks, which could preliminarily determine that the enrichment contained PNSB.

[0038] (3) Separation process of PNSB:

[0039] The isolation medium consists of: 1000 mL of deionized water, 3 g of sodium acetate, 0.9 g of K2HPO4, 0.6 g of KH2PO4, 0.2 g of MgSO4·7H2O, 0.075 g of CaCl2·2H2O, 0.0118 g of FeSO4·7H2O, 0.02 g of EDTA, 15 μg of biotin, 0.5 g of NH4Cl, and 1 mL of trace element solution; the pH is adjusted to 7.0 with 12 M NaOH. The trace element solution consists of: 280 mg of H3BO3, 188 mg of MnCl2·4H2O, 75 mg of Na2MoO4·2H2O, 24 mg of ZnSO4·7H2O, and 5.5 mg of CuSO4·H2O per 100 mL of deionized water.

[0040] Add 1.5% agar to the above liquid culture medium, heat to a boil while stirring, sterilize with high-pressure steam at 121°C for 15 minutes, and pour it onto a plate when it cools to about 50°C to make a solid separation medium. Wait for the medium to solidify and use it as a sterile operating table. Use an inoculating loop to dip the mixed PNSB culture medium into the plate and streak it for separation.

[0041] The anaerobic separation device used in this embodiment is a modification directly performed on the basis of a glassware dryer. Figure 1 As shown, the device comprises a transparent container 1, a lid 2, a vacuum valve 3 mounted on the lid, a gas collection bag 4 filled with inert gas, and a breathable baffle 5. During use, a thin layer of vaseline is applied to the ground edge of the desiccator to maintain airtightness. A desiccant, color-changing silica gel 6, and an anaerobic gas-generating bag (not shown) are placed beneath the breathable baffle 5. A scribed plate of culture medium 8 is placed on the desiccator's porcelain plate. A vacuum pump is used to evacuate the interior of the desiccator, followed by a gas collection bag filled with nitrogen. The valve is then opened to maintain consistent pressure inside and outside the desiccator. This device effectively removes oxygen from the enclosed space, maintaining an anaerobic / anoxic state for the culture medium.

[0042] Finally, the device was incubated for several days under 3000 lux illumination with a halogen tungsten lamp (which also provided temperature). Once a single colony grew on the plate, it was streaked out. After streaking three or more times, a single bacterial morphology was observed under a microscope, thus obtaining the PNSB strain. 16S rRNA analysis confirmed that the strain was Rhodopseudomonas palustris, a typical PNSB bacterium.

[0043] Example 2

[0044] Unlike Example 1, the sample used in this example was sludge Q from the anoxic section of the Wuhan Tangxun Lake Wastewater Treatment Plant.

[0045] The PNSB strain isolated in this example was identified as a typical PNSB bacterium, Rhodopseudomonas palustris, after 16S rRNA detection.

[0046] The data monitored in the above two embodiments are analyzed, specifically including the following aspects:

[0047] Figure 2 The characteristic peak scanning results of the sludge samples in the enrichment process of Example 1 and Example 2 are shown. Figure 2 It can be seen that the peak lines of the sludge samples in Example 1 and Example 2 were stable in the initial stage. After one week of enrichment, the sludges in Examples 1 and 2 showed characteristic absorption peaks of bacteriochlorophyll (500nm, 538nm, 590nm) and carotenoids (806nm, 866nm). After two weeks of enrichment, the characteristic absorption peaks of bacteriochlorophyll and carotenoids were observed to be clearer and more obvious, further confirming the presence of PNSB.

[0048] Figure 3 The COD variation diagram during the enrichment process of Example 1 and Example 2 is shown in FIG. Figure 3 It can be seen that after the first week of enrichment, the COD removal rates reached 51.39% and 52.71% respectively, and the removal rates were 0.053g / L / d and 0.048g / L / d respectively; after the second week of enrichment, the COD removal rates increased significantly, reaching 81.38% and 89.86% respectively, and the removal rates also increased to 0.060g / L / d and 0.067g / L / d respectively.

[0049] Figure 4 The color change diagram of the culture medium during the enrichment process of Example 1 and Example 2 shows that the enrichment medium and enrichment conditions provided by the present invention enable the sludge to quickly turn into a deep red bacterial liquid in the first week (left figure). Very few black bacteria can be observed with the naked eye. After the second week (right figure), the color of the bacterial liquid gradually turns into pure purple-red.

[0050] Figure 5 This is a community diversity analysis diagram of Example 1 and Example 2 after enrichment of PNSB for one week. It can be seen from the figure that high-purity Rhodopseudomonas palustris was detected in the enrichment culture fluid of both examples, with abundances reaching 69.61% and 52.71%, respectively, which is consistent with the phenomenon observed with the naked eye.

[0051] In summary, the culture medium and separation and enrichment method provided by the present invention can quickly enrich and separate PNSB from complex sewage environments, effectively solving the problems of the existing technology of long PNSB enrichment cycle from activated sludge, non-universality of culture medium, low bacterial content and high separation cost.

[0052] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for rapidly enriching and isolating Rhodopseudomonas palustris from a sewage treatment system, characterized in that: The following steps are involved: Step 1: Take a sample from the sewage treatment system and place it in an enrichment medium, and culture it in a closed environment under infrared light; after the closed culture is completed, take part of the bacterial liquid and add it to a new enrichment medium to repeat the enrichment once; Step 2: Dip the culture solution obtained in step 1 and streak it on the separation medium, and culture it under light in an anaerobic environment. After a single colony grows, pick a single colony and continue streaking and separating it until a single morphology of bacteria appears under microscopy; The enrichment medium has a pH of 7.0 and specifically comprises: 1-2 g / L sodium acetate, 0.5-1.3 g / L NH4Cl, 2.2-4.4 g / L KH2PO4, 1.7-3.4 g / L K2HPO4, 0.5-1 g / L NaHCO3, 0.01-0.1 g / LCaCl2∙2H2O, 0.2-0.3 g / LMgCl2·6H2O, 0.25-0.5 g / L NaCl, 0.39-0.78 g / L KCl and 1 mL / L of trace element solution A; The sample is anaerobic activated sludge or anoxic activated sludge; The composition of the trace element solution A is as follows: per 1000 mL of distilled water, add 1 g of FeSO4∙7H2O, 70 mg of ZnCl2, 100 mg of MnCl2∙4H2O, 6 mg of H3BO3, 130 mg of CaCl2∙6H2O, 2 mg of CuCl2∙2H2O, 24 mg of NiCl2∙6H2O, 36 mg of Na2MoO4∙2H2O, and 38 mg of CoCl2∙6H2O.

2. The method for rapidly enriching and isolating Rhodopseudomonas palustris from a sewage treatment system according to claim 1, characterized in that: The separation medium is made of a liquid culture medium supplemented with agar, the pH of the liquid culture medium is 7.0, and the specific composition is: 3 g sodium acetate, 0.9 g K2HPO4, 0.6 g KH2PO4, 0.2 g MgSO4·7H2O, 0.075 g CaCl2·2H2O, 0.0118 g FeSO4·7H2O, 0.02 g ethylenediaminetetraacetic acid, 15 μg biotin, 0.5 g NH4Cl and 1 mL of trace element solution B are added per 1000 mL of distilled water.

3. The method for rapidly enriching and isolating Rhodopseudomonas palustris from a sewage treatment system according to claim 2, characterized in that: The composition of the trace element solution B is as follows: 280 mg H3BO3, 188 mg MnCl2·4H2O, 75 mg Na2MoO4·2H2O, 24 mg ZnSO4·7H2O, and 5.5 mg CuSO4·H2O are added to every 100 mL of distilled water.

4. The method for rapidly enriching and isolating Rhodopseudomonas palustris from a sewage treatment system according to claim 1, characterized in that: The closed culture time in step 1 is 5 to 7 days.

5. The method for rapidly enriching and isolating Rhodopseudomonas palustris from a sewage treatment system according to claim 1, characterized in that: The anaerobic environment in step 2 is provided by an anaerobic separation device, and the anaerobic separation device includes a transparent container and a lid tightly covering the opening of the transparent container. The lid is provided with a vacuum valve, and the vacuum valve is covered with a gas collection bag filled with inert gas.

6. The method for rapidly enriching and isolating Rhodopseudomonas palustris from a sewage treatment system according to claim 5, characterized in that: A breathable partition is provided in the transparent container, and a desiccant and an anaerobic gas production bag are provided below the breathable partition.

7. The method for rapidly enriching and isolating Rhodopseudomonas palustris from a sewage treatment system according to claim 5, characterized in that: The transparent container and the lid are both made of glass, and the sealed joints between the two are frosted.

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