Method for reducing resistance genes in penicillin mushroom dreg aerobic compost

Abstract

The invention discloses a method for reducing resistance genes in penicillin mushroom residue aerobic compost, relates to the technical field of solid waste disposal and resource utilization, and aims to solve the problems of ARGs increase caused by antibiotic residues in the earlier stage of penicillin mushroom residue composting and ARGs rebound in the later stage of composting. And in the cooling period of the compost, the reduction of resistance genes in the penicillin mushroom dreg aerobic compost is realized by increasing the water content. According to the method, the moisture content is regulated and controlled, the void ratio of a compost body is changed, microbial oxygen supply is reduced, and a local micro-aerobic or anaerobic environment is created, so that microbial communities in the later period of composting are changed, meanwhile, the number of facultative microorganisms and anaerobic microorganisms is increased, the structure of the microbial communities is changed, the living space of drug-resistant bacteria is reduced, and the abundance of ARGs in the mushroom dregs is reduced. Theoretical basis and technical support are provided for harmless treatment and resource utilization of the penicillin mushroom residues, and antibiotic waste residue treatment is promoted to develop towards the green and sustainable direction.

Description

Technical Field

[0001] This invention belongs to the field of solid waste disposal and resource utilization technology, specifically involving antibiotic removal methods, aerobic composting process of bacterial residue, and resistance gene control technology. Background Technology

[0002] Penicillin is one of the most widely used β-lactam antibiotics, used extensively in human medicine and animal health. Its industrial production generates a large amount of byproducts, namely penicillin fermentation residue. Approximately 9 tons of wet fermentation residue are produced for every 1 ton of penicillin manufactured. The main components of penicillin fermentation residue include unreacted culture medium, organic acids, inorganic salts such as nitrogen and phosphorus, fermentation microbial mycelium, and a large amount of residual penicillin antibiotics. Due to its rich organic matter and nitrogen source, penicillin fermentation residue has resource potential and is often used as organic fertilizer or soil conditioner. However, if the nutrient-rich wet fermentation residue is not properly treated, it will rot and deteriorate, seriously affecting the ecological environment. Furthermore, extensive research has found that penicillin fermentation residue not only produces large quantities but also poses certain drug residues and the risk of inducing drug resistance. Penicillin fermentation residue contains high levels of resistance genes (ARGs) and the microbial communities carrying these genes. ARGs have strong environmental persistence and can spread between different species through horizontal gene transfer. Their main vectors include mobile genetic elements such as plasmids, transposons, and integrons. Related studies have shown that these ARGs not only exist within the microbial cells of fermentation residues but can also be adsorbed or encapsulated on organic particles, making them difficult to completely remove under conventional treatment processes. The environmental release of ARGs can pose numerous environmental risks. Therefore, how to reduce ARGs in penicillin fermentation residues has become a crucial issue.

[0003] Aerobic composting is a bio-fermentation process that utilizes microorganisms to convert organic matter into humus. It can reduce the abundance of aerobic glutamate (ARGs) to some extent. ARGs generally have high abundance in the early stages of composting, decrease significantly during the high-temperature period, and rebound to varying degrees in the later stages. This dynamic change reflects the impact of microbial community succession on ARGs during composting. Furthermore, due to the drastic temperature changes during composting, the composition of the microbial community within the compost pile changes, consequently altering the host bacteria of ARGs and significantly affecting their abundance. While traditional aerobic composting can reduce some ARGs, a rebound phenomenon occurs later. Therefore, developing new technologies for reducing ARGs through penicillin-containing compost is extremely necessary.

[0004] Penicillin is an unstable antibiotic that decomposes completely within 2-5 days during composting. However, high concentrations of penicillin exposed to the composting environment can easily lead to the selection of drug-resistant bacteria, increasing the risk of ARGs (antibiotic-resistant bacteria). Therefore, pre-treating the bacterial residue to remove antibiotics is a crucial step in reducing ARGs. Currently, there are very limited reports on removing penicillin from the bacterial residue before composting, and reports on preventing the resurgence of host microbial communities carrying ARGs in the later stages of composting are also scarce. Summary of the Invention

[0005] In order to simultaneously address the problems of increased ARGs due to antibiotic residues in the early stage of penicillin bacterial residue composting and the rebound of ARGs in the later stage of composting, this invention provides a method for reducing resistance genes in aerobic composting of penicillin bacterial residue.

[0006] The present invention provides a method for reducing resistance genes in aerobic composting of penicillin bacterial residue. This method involves pre-treating the penicillin bacterial residue to remove it, then composting it, and increasing the moisture content during the cooling period of the compost to reduce the resistance genes in the aerobic composting of penicillin bacterial residue.

[0007] Furthermore, the cooling-off period is 10 to 20 days.

[0008] Furthermore, increasing the moisture content during the cooling period means increasing the moisture content to 50%~60%.

[0009] Furthermore, the penicillin-removed bacterial residue is removed through a pretreatment method, which may be heat treatment or acid / alkali treatment.

[0010] Furthermore, the pretreatment is a heat treatment, with the heat treatment conditions being heating at 95℃~100℃ for more than 3 hours.

[0011] Furthermore, the penicillin-removed bacterial residue composting pile is obtained by pre-treating the penicillin-removing bacterial residue to remove penicillin, and then mixing it with sawdust for composting.

[0012] Furthermore, the dry weight ratio of the penicillin bacterial residue to sawdust is 1:3 to 1:5.

[0013] Furthermore, the initial moisture content during composting is 50%~60%, and the C / N ratio is 25:1~30:1.

[0014] This study found that even after penicillin was removed, the amount of ARGs in the bacterial residue compost still increased. This may be because some host bacteria carrying ARGs revived in the later stages of composting and eventually became the dominant bacteria, leading to the increase in ARGs.

[0015] Changes in ARGs and microbial communities can often be achieved by modulating environmental factors in composting. Host microorganisms evolve in response to changes in temperature, pH, organic matter content, and other environmental factors, thus influencing the fate of ARGs. Therefore, exploring the behavior of ARGs during composting by altering environmental factors is particularly important. Among the many influencing factors, moisture content remains a crucial factor controlling microbial growth. Generally, the initial moisture content of compost is 50%–60%, gradually decreasing as composting progresses, reaching approximately 40% after the cooling period. Increasing the moisture content at this point may alter the composting environment, leading to changes in some physicochemical properties and the microbial community. Higher moisture content increases microbial activity and metabolic capacity; however, for compost, moisture content also affects the porosity and temperature of the compost pile. When the compost moisture content exceeds 60%, porosity decreases by approximately 10%–30%, creating a localized anaerobic environment within the pile, leading to changes in the microbial community. When the microbial community changes, the host bacteria carrying ARGs may decrease accordingly. Currently, research on reducing ARGs by altering moisture content during microbial composting is very limited, and no reports indicate that adjusting moisture content alone can achieve the effect of reducing ARGs. This invention is the first to discover that even after penicillin is removed from the microbial compost, ARGs rebounded in the later stages of composting. Therefore, this invention is also the first to attempt to reduce the abundance of ARGs in microbial compost by adjusting only the single factor of moisture content in the later stages of composting (10-20 days).

[0016] The present invention has the following beneficial effects:

[0017] This invention modifies the microbial community in the later stages of composting by adjusting the moisture content and changing the porosity of the compost pile, thereby reducing the oxygen supply to microorganisms and creating localized microaerobic or anaerobic environments. Localized anaerobic conditions reduce the number of aerobic microorganisms while increasing the number of facultative and anaerobic microorganisms, altering the microbial community structure, reducing the living space for drug-resistant bacteria, and thus lowering the abundance of ARGs in the compost residue. This provides a theoretical basis and technical support for the harmless treatment and resource utilization of penicillin compost residue, promoting the green and sustainable development of antibiotic waste residue treatment. Attached Figure Description

[0018] Figure 1 Schematic diagram of reactor composting;

[0019] Figure 2 The graph shows the changes in the physicochemical properties of compost: (a) temperature; (b) pH value; (c) moisture content; (d) ammonia nitrogen; (e) nitrate nitrogen; (f) nitrite nitrogen.

[0020] Figure 3Figure 10: Abundance changes of ARGs during penicillin bacterial residue composting (logarithm to base 10); (a) relative abundance of blaTEM gene; (b) absolute abundance of blaTEM gene; (c) relative abundance of ampC1 gene; (d) absolute abundance of ampC1 gene; (e) relative abundance of blaL1 gene; (f) absolute abundance of blaL1 gene; (g) absolute abundance of 16S rRNA gene. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0022] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0023] Example 1

[0024] The penicillin-containing bacterial residue was pretreated to remove penicillin (a hydrothermal method at 100℃ for 3 hours can be used, but this is not the only option), then mixed with a composting agent (sawdust or straw) (dry weight ratio 1:4), adjusting the initial moisture content to 55% and the C / N ratio to approximately 25:1. The composting time was one month. Composting was conducted in a laboratory using an automatic temperature-controlled water bath or spontaneous heating, with an air pump supplying aeration at a rate of 1.5 L / min. -1 ·kg -1 Dry weight. The moisture content was increased from 40% to 55% during the cooling period (10-20 days). There were three treatments: untreated bacterial residue compost (CK), pretreated bacterial residue compost (HT), and pretreated bacterial residue compost + cooling period to increase moisture content (HT+HM). Untreated bacterial residue compost (CK) refers to compost without pretreatment to remove penicillin and without increasing the moisture content from 40% to 55% during the cooling period (10-20 days), otherwise the same as in Example 1. Pretreated bacterial residue compost (HT) refers to compost where only penicillin was removed through pretreatment of the penicillin bacterial residue, without increasing the moisture content from 40% to 55% during the cooling period (10-20 days), otherwise the same as in Example 1. Pretreated bacterial residue compost + cooling period to increase moisture content (HT+HM) involves pretreatment of the penicillin bacterial residue to remove penicillin, followed by increasing the moisture content from 40% to 55% during the cooling period (10-20 days), otherwise the same as in Example 1. The aerobic composting process is simulated using a water bath (e.g.) Figure 1 As shown in the figure, the system investigated the effects of different treatments on the reduction of ARGs.

[0025] Table 1. The multiplication rate of ARGs relative to the control group in different treatment groups of bacterial residue compost (only the detected genes are shown, and the multiplication rate is calculated as 2^(relative abundance of genes in the treatment group - relative abundance of genes in the control group). A multiplication rate > 1 indicates gene amplification.

[0026]

[0027] Table 1 also shows that, compared to the CK group, most data in the HT and HT+HM groups decreased, indicating that pretreatment of the bacterial residue was beneficial for ARG removal. However, at 18 and 30 days, the HT group showed a significant increase in values, indicating that even after hydrothermal removal of antibiotics, bacterial residue composting still carries the risk of ARG rebound. This may be because some drug-resistant strains present in the bacterial residue became dominant bacteria in the later stages of composting. The HT+HM group was the most favorable for ARG removal, with all multipliers less than 1, indicating that the combined effect of bacterial residue pretreatment and increased moisture content during the cooling period can reduce ARGs during bacterial residue composting.

[0028] Depend on Figure 2 As shown, the three composting groups exhibited relatively small differences in temperature and pH, and the temperature changes met the composting requirements, indicating that the pretreatment of the microbial residue or the increase in moisture content during the cooling period did not significantly affect normal composting. Regarding moisture content changes, the moisture content of the CK and HT groups decreased from an initial 55% to 40%, while in the HT+HM group, the moisture content increased to 55% between 10 and 20 days, a significant difference from the other groups. In terms of nitrogen changes, the pretreatment of the microbial residue had a greater impact on ammonia nitrogen release, possibly due to the hydrothermal treatment initiating the decomposition of organic matter in the residue. The increase in moisture content during the cooling period (HT+HM) had a certain impact on nitrate formation, decreasing compared to the HT group.

[0029] Figure 3 This study demonstrates the changes in ARG abundance during penicillin-containing bacterial residue composting under three treatment conditions. High-throughput real-time quantitative PCR was used to detect 15 β-lactam ARGs, and three ARGs were detected: blaTEM, ampC1, and blaL1. These genes are very common ARGs in bacterial residue, likely inherent to the residue itself, as no additional bacterial strains were added during composting, resulting in no ARG input. Since the control group (CK) did not undergo pretreatment, it retained more penicillin, leading to higher levels of blaTEM, ampC1, and blaL1 before day 8. In the pretreated bacterial residue compost (HT), these three detected ARGs significantly decreased in the early stages compared to the CK group, indicating that penicillin removal through pretreatment does not increase ARG content in the early composting stage. This may be because, although penicillin is easily decomposed, it can screen for drug-resistant bacteria in the early stages of composting, leading to an increase in the abundance of ARG-containing bacterial communities.

[0030] In the later stages of composting (after 12 days), the ARGs in the HT group showed a significant increase, increasing by up to 2.5 orders of magnitude compared to the CK group, indicating a rebound in ARGs and proving that hydrothermal pretreatment of the microbial residue compost alone is insufficient to reduce ARGs. However, in the HT+HM group, compared to the HT group, the three detected ARGs showed significant differences, all exhibiting a decreasing trend, indicating that increasing the moisture content during the compost cooling period is beneficial for ARG removal. From the performance of the CK and HT groups, normal composting has a certain effect on ARG removal, but it does not control ARG abundance. However, after adjusting the moisture content according to this invention, ARG abundance was significantly controlled.

Claims

1. A method for reducing resistance genes in aerobic composting of penicillin bacterial residue, characterized in that, The method involves pre-treating the penicillin residue to remove it, then composting it, and increasing the moisture content during the cooling period of the compost to reduce resistance genes in the aerobic compost of the penicillin residue.

2. The method for reducing resistance genes in aerobic composting of penicillin bacterial residue according to claim 1, characterized in that, The cooling-off period is 10 to 20 days.

3. The method for reducing resistance genes in aerobic composting of penicillin bacterial residue according to claim 1, characterized in that, The increase in moisture content during the cooling period is to increase the moisture content to 50%~60%.

4. The method for reducing resistance genes in aerobic composting of penicillin bacterial residue according to claim 1, characterized in that, The penicillin-removed bacterial residue is removed through a pretreatment method, which may be heat treatment or acid / alkali treatment.

5. The method for reducing resistance genes in aerobic composting of penicillin bacterial residue according to claim 4, characterized in that, The pretreatment is heat treatment, and the heat treatment conditions are: heating at 95℃~100℃ for more than 3 hours.

6. The method for reducing resistance genes in aerobic composting of penicillin bacterial residue according to claim 1, characterized in that, The penicillin-removed bacterial residue is obtained by pre-treating the penicillin-removed bacterial residue to remove penicillin, and then mixing it with sawdust for composting.

7. The method for reducing resistance genes in aerobic composting of penicillin bacterial residue according to claim 6, characterized in that, The dry weight ratio of the penicillin bacterial residue to sawdust is 1:3 to 1:

5.

8. The method for reducing resistance genes in aerobic composting of penicillin bacterial residue according to claim 6, characterized in that, The initial moisture content during composting is 50%~60%, and the C / N ratio is 25:1~30:1.

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