Enrichment and culture method of polyethylene glycol degrading flora

Abstract

The invention relates to the technical field of enrichment and culture of a polyethylene glycol degrading flora, and discloses an enrichment and culture method of the polyethylene glycol degrading flora, which comprises the following steps: in the first stage, PEG100 and PEG800 are taken as substrates in influent water, the COD (Chemical Oxygen Demand) concentration is controlled to be 1000mg / L and 3000mg / L, the substrates are alternately changed according to a period, and the concentration is controlled to be 20-40mg / L; in the second stage, PEG100, PEG800 and PEG1000 are used as substrates in inlet water, the COD (Chemical Oxygen Demand) concentrations are controlled to be 3000mg / L and 5000mg / L and are alternately changed according to a period, and the concentrations are controlled to be 40-60mg / L; in the third stage, PEG100, PEG800, PEG1000 and PEG4000 are used as substrates in inlet water, the COD (Chemical Oxygen Demand) concentrations are controlled to be 5000 mg / L and 7000 mg / L and alternately changed according to a period, and the concentrations are controlled to be 60-80 mg / L; in the fourth stage, PEG100, PEG800, PEG1000, PEG4000 and PEG6000 are used as substrates in inlet water, the COD (Chemical Oxygen Demand) concentration is controlled to be 7000 mg / L and 9000 mg / L and alternately changed according to a period, and the concentration is controlled to be 80-100 mg / L; according to the invention, through four stages of enrichment culture, the flora contains obligate degrading bacteria aiming at various PEG molecular weights, and has strong tolerance to polyethylene glycol concentration and molecular weight fluctuation.

Description

Technical Field

[0001] This invention relates to the field of enrichment and cultivation of polyethylene glycol-degrading microbial communities, specifically a method for enriching and cultivating polyethylene glycol-degrading microbial communities. Background Technology

[0002] Polyethylene glycol (PEG) is a water-soluble polymer polymerized from ethylene oxide, with the chemical formula HO(CH2CH2O). n H, commonly used as a solvent in industries such as pharmaceuticals, cosmetics, textiles, chemicals, and new energy, leads to the accumulation of polyethylene glycol (PEG) in industrial wastewater, posing a potential environmental hazard. PEG can be classified according to molecular weight into PEG100, PEG200, PEG400, PEG1000, PEG4000, and even those with molecular weights exceeding 20,000. Statistics show that global annual PEG production exceeds 1 million tons, with approximately 30% entering wastewater systems. Current treatment methods for PEG-containing wastewater mainly include physical, chemical, and biological methods. Traditional physicochemical treatment methods, such as membrane filtration, activated carbon adsorption, iron-carbon micro-electrolysis oxidation, advanced oxidation, Fenton oxidation, and membrane separation technology, while capable of partial removal and simple to operate, suffer from incomplete treatment, high costs, high energy consumption, and the potential for secondary pollution. In contrast, biodegradation methods centered on activated sludge offer advantages such as environmental friendliness, low cost, and sustainability.

[0003] However, the current activated sludge method for treating polyethylene glycol (PEG) wastewater has the following problems: (1) the microbial community in ordinary activated sludge can remove conventional organic pollutants, but it is not specific to the degradation of PEG; (2) ordinary activated sludge has limited effect on treating high-concentration PEG wastewater; (3) it has poor effect on treating high molecular weight PEG; (4) the concentration and molecular weight of PEG fluctuate greatly, making the treatment effect unstable; (5) in addition, the existing methods for enriching PEG-degrading bacteria use a single type of PEG molecular weight, and the obtained degrading bacteria are not effective in treating PEG wastewater with other molecular weights. Therefore, in order to solve the above technical problems, a method for enriching and cultivating PEG-degrading bacteria is provided. Summary of the Invention

[0004] This invention provides a method for enriching and cultivating polyethylene glycol (PEG) degrading bacteria. Through four stages of enrichment and cultivation, the obtained PEG degrading bacteria can adapt to PEG wastewater with COD concentrations ranging from 1000 mg / L to 9000 mg / L and PEG molecular weights from 100 to 6000. Because the bacteria contain specific degrading bacteria targeting various PEG molecular weights, they have strong tolerance to fluctuations in PEG concentration and molecular weight.

[0005] This invention provides the following technical solution:

[0006] A method for enriching and cultivating polyethylene glycol-degrading bacteria includes the following steps: Step 1: In the first stage, PEG100 and PEG800 are used as substrates in the influent, and the COD concentration is controlled at 1000 mg / L and 3000 mg / L, respectively, alternating periodically. The concentration is controlled at 20-40 mg / L; Step 2: In the second stage, PEG100, PEG800, and PEG1000 are used as substrates in the influent, and the COD concentration is controlled at 3000 mg / L and 5000 mg / L, alternating periodically. The concentration is controlled at 40-60 mg / L; Step 3: In the third stage, PEG100, PEG800, PEG1000, and PEG4000 are used as substrates in the influent, and the COD concentration is controlled at 5000 mg / L and 7000 mg / L, alternating periodically. The concentration is controlled at 60-80 mg / L; Step 4: In the fourth stage, PEG100, PEG800, PEG1000, PEG4000, and PEG6000 are used as substrates in the influent, and the COD concentration is controlled at 7000 mg / L and 9000 mg / L, alternating periodically. The concentration should be controlled at 80-100 mg / L.

[0007] As a preferred embodiment of the present invention, polyethylene glycol of various molecular weights in each stage provides an equal amount of COD, and the polyethylene glycol substrate loading alternates between the two stages.

[0008] As a preferred embodiment of the present invention, the polyethylene glycol substrate loading increases in each stage of the enrichment process, and the molecular weight of the polyethylene glycol used increases.

[0009] As a preferred technical solution of the present invention, the first stage in step one consists of 1-6 cycles.

[0010] As a preferred embodiment of the present invention, the second stage in step two is a cycle of 7-11.

[0011] As a preferred embodiment of the present invention, the third stage in step three is a cycle of 12-17.

[0012] As a preferred embodiment of the present invention, the fourth stage in step four is a cycle of 18-30.

[0013] As a preferred embodiment of the present invention, one cycle lasts for 24 hours.

[0014] Compared with existing technologies, this invention provides a method for enriching and cultivating polyethylene glycol-degrading bacteria, which has the following beneficial effects:

[0015] 1. The enrichment and cultivation method of polyethylene glycol degrading bacteria overcomes the limitation of low treatment effect caused by the lack of specificity in the traditional activated sludge method for treating polyethylene glycol wastewater, and can be used for polyethylene glycol wastewater treatment in various practical scenarios.

[0016] 2. The enrichment and cultivation method of the polyethylene glycol degrading bacteria makes the enriched polyethylene glycol bacteria highly adaptable to fluctuations in polyethylene glycol concentration.

[0017] 3. In the enrichment and cultivation method of polyethylene glycol degrading bacteria, by gradually increasing the molecular weight of polyethylene glycol, low-molecular-weight and high-molecular-weight polyethylene glycol degrading bacteria can be obtained in a step-by-step manner, which has strong adaptability to fluctuations in the types of polyethylene glycol.

[0018] 4. In the enrichment and cultivation method of polyethylene glycol degrading bacteria, by gradually increasing the PEG concentration, the obtained bacteria can not only treat low-concentration polyethylene glycol wastewater, but also directly treat high-concentration polyethylene glycol wastewater without dilution, which helps to reduce the time and space costs of treatment.

[0019] 5. The enrichment and cultivation method for this polyethylene glycol degrading microbial community requires an acclimatization and enrichment time of approximately 20-30 days to obtain a mature and stable polyethylene glycol degrading microbial community. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0021] Figure 1 This is a schematic diagram illustrating the changes in COD settings during the enrichment process of polyethylene glycol-degrading microbial communities in this invention.

[0022] Figure 2 This is a schematic diagram illustrating the change in COD removal rate during the enrichment process of polyethylene glycol-degrading microbial communities in this invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example:

[0024] Reference Figure 1 and Figure 2The enrichment and cultivation method for polyethylene glycol (PEG) degrading bacteria employs an alternating and gradual substrate loading approach. The enrichment process includes four stages:

[0025] Phase 1: Cycles 1-6. PEG100 and PEG800 are used as substrates in the influent. PEG100 is low molecular weight polyethylene glycol with short molecular chains, easily degraded by microorganisms, and can serve as a basic carbon source to rapidly support bacterial growth and proliferation. PEG800 is medium molecular weight polyethylene glycol with longer molecular chains, making it more difficult to degrade. This allows for the screening of functional strains capable of degrading medium molecular weight PEG. Mixing the two avoids the problems of a single low molecular weight substrate enriching only low molecular weight polyethylene glycol-degrading bacteria, or a single medium molecular weight substrate inhibiting bacterial growth. Specifically, there is no specific ratio requirement, but neither should be less than 30%. This ratio is to avoid substrate imbalance leading to biased bacterial selection. COD concentrations are controlled at 1000 mg / L and 3000 mg / L, alternating periodically to enhance the bacterial population's load tolerance and degradation stability. The concentration should be controlled between 20-40 mg / L;

[0026] Phase Two: Cycles 7-11. PEG100, PEG800, and PEG1000 are used as substrates in the influent. PEG1000 is a high molecular weight polyethylene glycol with a longer molecular chain and more complex spatial structure, making it far more difficult to degrade than PEG100 (low molecular weight) and PEG800 (medium molecular weight). Phase One has already enriched bacterial communities capable of degrading low and medium molecular weight PEG. The addition of PEG1000 in Phase Two applies new selective pressure, directionally screening for specific strains capable of degrading high molecular weight PEG. This upgrades the bacterial community from a low- and medium molecular weight degrader to a broad-spectrum degrader of low, medium, and high molecular weight PEGs. COD concentrations are controlled at 3000 mg / L and 5000 mg / L, alternating periodically. The concentration should be controlled at 40-60 mg / L;

[0027] Phase 3: Cycles 12-17. PEG100, PEG800, PEG1000, and PEG4000 are used as substrates in the influent. PEG4000 is a higher molecular weight polyethylene glycol with a long molecular chain and significant steric hindrance, making it far more difficult to degrade than PEG100, PEG800, and PEG1000. The first two phases have already cultivated bacterial communities capable of degrading low and medium molecular weight PEG. The addition of PEG4000 in Phase 3 aims to create a more challenging selection process, selectively identifying specific strains capable of degrading higher molecular weight PEG. These strains can break the ether bonds of PEG4000, breaking it down into smaller molecular fragments (such as PEG1000 and PEG800). These fragments are then further mineralized by existing functional strains within the bacterial community. Ultimately, this results in a leap in the community's ability to degrade PEG from low, medium, and high molecular weight PEG to low, medium, and high PEG. COD concentrations are controlled at 5000 mg / L and 7000 mg / L, alternating between these two values ​​in the cycle. The concentration should be controlled at 60-80 mg / L;

[0028] Phase Four: Cycles 18-30. PEG100, PEG800, PEG1000, PEG4000, and PEG6000 are used as substrates in the influent. PEG6000 is an ultra-high molecular weight polyethylene glycol with extremely long molecular chains and significant steric hindrance, making its ether bonds much more difficult to break than PEG4000. It is the most challenging substrate in the acclimation process. The first three phases have acclimated a broad-spectrum bacterial community capable of degrading PEG100 / 800 / 1000 / 4000. Phase Four introduces PEG6000 to apply a higher level of selective pressure, targeting and screening for strains capable of degrading ultra-high molecular weight PEG. These strains can target and break the long-chain ether bonds of PEG6000, breaking it down into smaller molecular fragments such as PEG4000 and PEG1000. These fragments are then mineralized stepwise by existing functional strains in the bacterial community, ultimately achieving a breakthrough in the community's ability to degrade all molecular weights, from the broad spectrum to extreme molecular weights. COD concentrations are controlled at 7000 mg / L and 9000 mg / L, alternating periodically. The concentration should be controlled at 80-100 mg / L.

[0029] In stages one through four, polyethylene glycol of various molecular weights provides equal amounts of COD in each cycle. The polyethylene glycol substrate load alternates between two cycles. The polyethylene glycol substrate load gradually increases throughout the enrichment process, and the molecular weight of the polyethylene glycol used gradually increases. A batch process is adopted, and each cycle in the influent is 24 hours.

[0030] The working principle of this invention is as follows: The core of the conventional activated sludge method for degrading polyethylene glycol (PEG) is that organic matter-degrading bacteria break down PEG molecules into carbon dioxide and water. However, while the organic matter-degrading bacteria naturally present in activated sludge can remove conventional organic pollutants, they lack specificity for PEG degradation. Furthermore, conventional activated sludge typically has low tolerance to organic loads, resulting in reduced treatment efficiency for high-concentration PEG-containing wastewater. Additionally, conventional activated sludge can only treat low-molecular-weight PEG, exhibiting low decomposition efficiency and long treatment times for high-molecular-weight PEG. The large fluctuations in the molecular weight and concentration of actual PEG wastewater also lead to decreased treatment efficiency and increased costs. In summary, using activated sludge as seed sludge and employing alternating PEG loads and gradually increasing concentrations and molecular weights to enrich the PEG-degrading bacteria community offers a wide applicable concentration range, broad molecular weight range, and strong tolerance to fluctuations.

[0031] In some embodiments, during Phase 1, heterotrophic microorganisms in the sludge capable of degrading PEG100 and PEG800 break down the large polyethylene glycol molecules into smaller molecules, which are then converted into carbon dioxide and water. Supported by other nutrients, these microorganisms gradually multiply and become enriched. Simultaneously, because the PEG concentration alternates between 1000 mg / L and 3000 mg / L between the two cycles during this phase, microorganisms that can adapt to changes in substrate concentration are enriched, while those that cannot are eliminated.

[0032] In some embodiments, during Phase 2, heterotrophic microorganisms in the sludge capable of degrading PEG100, PEG800, and PEG1000 break down the large polyethylene glycol molecules into smaller molecules, which are then converted into carbon dioxide and water. Supported by other nutrients, these microorganisms gradually multiply and become enriched. Simultaneously, because the PEG concentration alternates between 3000 mg / L and 5000 mg / L between the two cycles during this phase, microorganisms that can adapt to changes in substrate concentration are enriched, while those that cannot are eliminated.

[0033] In some embodiments, during Phase 3, heterotrophic microorganisms in the sludge capable of degrading PEG100, PEG800, PEG1000, and PEG4000 break down the large polyethylene glycol molecules into smaller molecules, which are then converted into carbon dioxide and water. Supported by other nutrients, these microorganisms gradually multiply and become enriched. Simultaneously, because the PEG concentration alternates between 5000 mg / L and 7000 mg / L between the two cycles during this phase, microorganisms that can adapt to changes in substrate concentration are enriched, while those that cannot are eliminated.

[0034] In some embodiments, during stage four, heterotrophic microorganisms in the sludge capable of degrading PEG100, PEG800, PEG1000, PEG4000, and PEG6000 break down the large polyethylene glycol molecules into smaller molecules, which are then converted into carbon dioxide and water. Supported by other nutrients, these microorganisms gradually multiply and become enriched. Simultaneously, because the PEG concentration alternates between 7000 mg / L and 9000 mg / L between the two cycles during this stage, microorganisms that can adapt to changes in substrate concentration are enriched, while those that cannot are eliminated.

[0035] Through the above four stages of enrichment culture, the obtained polyethylene glycol-degrading bacterial community is adaptable to polyethylene glycol wastewater with COD concentrations ranging from 1000 mg / L to 9000 mg / L and PEG molecular weights from 100 to 6000. Because the community contains specific degrading bacteria targeting various PEG molecular weights, it exhibits strong tolerance to fluctuations in polyethylene glycol concentration and molecular weight.

[0036] This invention effectively addresses the problem that while ordinary activated sludge microorganisms can remove conventional organic pollutants, they lack specificity for the degradation of high-molecular-weight polyethylene glycol (PEG). Through a substrate regulation strategy involving progressively increasing and alternating loading, the invention enhances the targeted degradation of PEG by the microorganisms, improving their treatment efficiency for high-concentration PEG wastewater and high-molecular-weight PEG. This ensures reduced fluctuations in PEG concentration and molecular weight, stabilizing the treatment effect, increasing the diversity of PEG molecular weights used, and improving the treatment efficiency of degrading bacteria for PEG wastewater with other molecular weights.

[0037] Components not described in detail in this article are existing technologies.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for enriching and culturing polyethylene glycol-degrading microbial communities, characterized in that, Includes the following steps: Step 1: In the first stage, PEG100 and PEG800 are used as substrates in the influent, and the COD concentration is controlled at 1000 mg / L and 3000 mg / L, respectively, alternating periodically. The concentration should be controlled between 20-40 mg / L; Step Two: In the second stage, PEG100, PEG800, and PEG1000 are used as substrates in the influent, with COD concentrations controlled at 3000 mg / L and 5000 mg / L, alternating periodically. The concentration should be controlled at 40-60 mg / L; Step 3: In the third stage, PEG100, PEG800, PEG1000, and PEG4000 are used as substrates in the influent, and the COD concentration is controlled at 5000 mg / L and 7000 mg / L, alternating periodically. The concentration should be controlled at 60-80 mg / L; Step Four: In the fourth stage, PEG100, PEG800, PEG1000, PEG4000, and PEG6000 are used as substrates in the influent, with COD concentrations controlled at 7000 mg / L and 9000 mg / L, alternating periodically. The concentration should be controlled at 80-100 mg / L.

2. The method for enriching and culturing polyethylene glycol-degrading bacteria according to claim 1, characterized in that, Polyethylene glycol of various molecular weights in each stage provides equal amounts of COD, and the polyethylene glycol substrate loading alternates between the two stages.

3. The method for enriching and culturing polyethylene glycol-degrading bacteria according to claim 1, characterized in that, The polyethylene glycol substrate loading and the molecular weight of the polyethylene glycol used increase in each enrichment stage.

4. The method for enriching and culturing polyethylene glycol-degrading bacteria according to claim 1, characterized in that, The first stage in Step One consists of cycles 1-6.

5. The method for enriching and culturing polyethylene glycol-degrading bacteria according to claim 4, characterized in that, The second stage in step two consists of a 7-11 cycle.

6. The method for enriching and culturing polyethylene glycol-degrading bacteria according to claim 5, characterized in that, The third stage in step three consists of 12-17 cycles.

7. The method for enriching and culturing polyethylene glycol-degrading bacteria according to claim 6, characterized in that, The fourth stage in step four consists of 18-30 cycles.

8. The method for enriching and culturing polyethylene glycol-degrading bacteria according to claim 7, characterized in that, One cycle lasts 24 hours.

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