Low-temperature composting composite microbial inoculant for toilet feces and preparation method of low-temperature composting composite microbial inoculant

Abstract

The invention discloses a toilet excrement low-temperature composting complex microbial inoculant and a preparation method thereof, and relates to the technical field of microorganisms. In order to solve the problem that the composting process is difficult to start under the low-temperature condition (less than or equal to 15 DEG C), and the toilet excrement treatment effect is seriously influenced, the toilet excrement low-temperature composting complex microbial inoculant disclosed by the invention is prepared from stenotrophomonas pavanii (Stennotrophomonas pavanii) R3, pseudomonas pseudomonas (Massiella pseudomonas R4 and pseudomonas kackmussii) R6. The invention further discloses a preparation method of the toilet excrement low-temperature composting complex microbial inoculant, and the preparation method of the toilet excrement low-temperature composting complex microbial inoculant disclosed by the invention has the advantages that the problem that the toilet excrement treatment effect is seriously influenced is solved; the complex microbial inoculant can promote smooth temperature rise starting of the toilet manure compost at low temperature, can accelerate and thoroughly biodegrade pollutants in the toilet manure compost, accelerates the fermentation process of the toilet manure compost at low temperature, improves the quality of compost products, and is applied to the field of low-temperature compost.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a low-temperature composting composite bacterial agent and a preparation method thereof. Background Art

[0002] The safe disposal and resource utilization of a large amount of toilet feces are related to environmental ecology and human health. In some remote and underdeveloped areas, dry toilets are still used to collect feces. For the comprehensive resource disposal of dry toilet feces, fermentation or composting methods are mostly adopted. Among them, aerobic composting has the advantages of fast and thorough degradation of organic matter, high harmlessness level, simple operation and good fertilizer efficiency, and thus has received extensive attention. However, composting is easily affected by temperature. In the autumn and winter seasons in some high-altitude areas, the environmental temperature is low and lasts for a long time, and the efficiency of aerobic composting is thus greatly limited. Especially under low-temperature conditions (≤15°C), the composting process is difficult to start, seriously affecting the treatment effect of toilet feces. Summary of the Invention

[0003] The purpose of the present invention is to solve the problem that the composting process is difficult to start under low-temperature conditions (≤15°C), seriously affecting the treatment effect of toilet feces, and to provide a low-temperature composting composite bacterial agent for toilet feces and a preparation method thereof.

[0004] A low-temperature composting composite bacterial agent for toilet feces of the present invention is composed of Stenotrophomonas pavanii R3, Massilia suwonensis R4, and Pseudomonas knackmussii R6.

[0005] The Stenotrophomonas pavanii R3 is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is February 25, 2025, and the preservation number is CGMCC No: 33647. The Massilia suwonensis R4 is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is February 25, 2025, and the preservation number is CGMCC No: 33648. The Pseudomonas knackmussii R6 is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is February 25, 2025, and the preservation number is CGMCC No: 33649.

[0006] The specific steps for preparing a low-temperature composting composite bacterial agent for toilet feces and sewage are as follows:

[0007] 1. Dip the bacterial liquid of Stenotrophomonas pavanii R3 and streak it on an LB solid plate. After culturing at room temperature, pick single colonies and transfer them to a sterilized and cooled LB liquid medium, and shake-culture to obtain a seed liquid; then transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2%, and continue to shake-culture to obtain a bacterial suspension of Stenotrophomonas pavanii R3.

[0008] 2. Dip the bacterial liquid of Massilia aquatica R4 and streak it on an LB solid plate. After culturing at room temperature, pick single colonies and transfer them to a sterilized and cooled LB liquid medium, and shake-culture to obtain a seed liquid; then transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2%, and continue to shake-culture to obtain a bacterial suspension of Massilia aquatica R4.

[0009] 3. Dip the bacterial liquid of Pseudomonas kribbensis R6 and streak it on an LB solid plate. After culturing at room temperature, pick single colonies and transfer them to a sterilized and cooled LB liquid medium, and shake-culture to obtain a seed liquid; then transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2%, and continue to shake-culture to obtain a bacterial suspension of Pseudomonas kribbensis R6.

[0010] 4. Mix the bacterial suspensions of Stenotrophomonas pavanii R3, Massilia aquatica R4, and Pseudomonas kribbensis R6 according to a ratio of viable bacteria content of 3:2:1 to obtain a low-temperature composting composite bacterial agent.

[0011] Advantages of the present invention:

[0012] The low-temperature composting liquid composite bacterial agent for toilet feces and sewage prepared by the present invention can promote the smooth temperature rise and start of toilet feces and sewage composting at low temperatures, more quickly and thoroughly biodegrade the pollutants therein, and achieve the resource utilization and harmless treatment of toilet feces and sewage in these areas. Through high-throughput sequencing and analysis, it is found that adding the prepared low-temperature composting liquid composite bacterial agent for toilet feces and sewage can increase the community diversity and the abundance of functional bacteria in low-temperature composting, and the community structure gradually converges with that of normal-temperature composting. This explains from the perspective of microbial community ecology the reason why the low-temperature composting effect can be comparable to that of normal-temperature composting after adding the low-temperature composting liquid composite bacterial agent prepared by the present invention.

[0013] The present invention has developed a composite bacterial agent suitable for toilet feces and sewage composting in low-temperature environments. After inoculation, it can not only promote the smooth temperature rise and start of toilet feces and sewage composting at low temperatures, but also more quickly and thoroughly biodegrade the pollutants therein, accelerate the fermentation process of toilet feces and sewage composting at low temperatures, improve the quality of compost products, and ultimately achieve the resource utilization and harmless treatment of toilet feces and sewage in these areas. Description of the Drawings

[0014] Figure 1 Phylogenetic tree of strain R3;

[0015] Figure 2 Phylogenetic tree of strain R4;

[0016] Figure 3 Phylogenetic tree of strain R6;

[0017] Figure 4 Temperature change process of the compost;

[0018] Figure 5 Moisture content change process of the compost;

[0019] Figure 6 pH change process of the compost;

[0020] Figure 7 Organic matter content change process of the compost;

[0021] Figure 8 C / N ratio change process of the compost;

[0022] Figure 9 Seed germination index change process of the compost;

[0023] Figure 10 NMDS analysis chart and hierarchical clustering analysis chart of the compost;

[0024] Figure 11 Species composition at the genus level of the compost. Specific implementation manners

[0025] Specific implementation manner 1: The low-temperature composting composite bacterial agent for toilet feces in this implementation manner is composed of Stenotrophomonas pavanii R3, Massilia suwonensis R4, and Pseudomonas knackmussii R6.

[0026] Specific implementation manner 2: The difference between this implementation manner and specific implementation manner 1 is that the Stenotrophomonas pavanii R3 is preserved in the China General Microbiological Culture Collection Center, the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is February 25, 2025, and the preservation number is CGMCC No: 33647. Others are the same as specific implementation manner 1.

[0027] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that the Massilia suwonensis R4 is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is February 25, 2025, and the preservation number is CGMCC No: 33648. Other steps are the same as those in Embodiment 1 or 2.

[0028] Embodiment 4: The difference between this embodiment and any one of Embodiments 1 to 3 is that the Pseudomonas knackmussii R6 is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is February 25, 2025, and the preservation number is CGMCC No: 33649. Other steps are the same as those in any one of Embodiments 1 to 3.

[0029] Embodiment 5: The difference between this embodiment and any one of Embodiments 1 to 4 is that Stenotrophomonas pavanii R3, Massilia suwonensis R4, and Pseudomonas knackmussii R6 are formed into a compound microbial agent according to the ratio of viable bacteria content of 3:2:1. Other steps are the same as those in any one of Embodiments 1 to 4.

[0030] Embodiment 6: The specific steps for preparing a compound microbial agent for low-temperature composting of toilet feces are as follows:

[0031] I. Dip the bacterial liquid of Stenotrophomonas pavanii R3 and streak it on an LB solid plate. After culturing at room temperature, pick a single colony into a sterilized and cooled LB liquid medium and shake-culture to obtain a seed liquid; then transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2%, and continue to shake-culture to obtain a bacterial suspension of Stenotrophomonas pavanii R3;

[0032] II. Dip the bacterial liquid of Massilia suwonensis R4 and streak it on an LB solid plate. Culture at room temperature, pick a single colony into a sterilized and cooled LB liquid medium and shake-culture to obtain a seed liquid; then transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2%, and continue to shake-culture to obtain a bacterial suspension of Massilia suwonensis R4;

[0033] III. Dip the bacterial liquid of Pseudomonas kribbensis R6 and streak it on an LB solid plate, then culture it at room temperature. Pick a single colony and transfer it into a sterilized and cooled LB liquid medium, and shake-culture to obtain a seed liquid; then transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2%, and continue to shake-culture to obtain a bacterial suspension of Pseudomonas kribbensis R6.

[0034] IV. Mix the bacterial suspensions of Stenotrophomonas pavanii R3, Massilia hydrotropica R4, and Pseudomonas kribbensis R6 according to the ratio of viable bacteria content of 3:2:1 to obtain a low-temperature composting composite bacterial agent.

[0035] Specific Embodiment VII: The difference between this embodiment and Specific Embodiment VI is that the effective viable bacteria counts of the bacterial suspensions of Stenotrophomonas pavanii R3, Massilia hydrotropica R4, and Pseudomonas kribbensis R6 are all ≥1×10 8 CFU / mL. Other steps are the same as those in Specific Embodiment VI.

[0036] Specific Embodiment VIII: The difference between this embodiment and Specific Embodiment VI or VII is that the shake-culture is carried out at 10±2°C and 180 r / min. Other steps are the same as those in Specific Embodiment VI or VII.

[0037] Specific Embodiment IX: The difference between this embodiment and any one of Specific Embodiments VI to VIII is that the inoculation amount of the composite bacterial agent accounts for 5% of the total mass of the low-temperature composting of toilet feces. Other steps are the same as those in any one of Specific Embodiments VI to VIII.

[0038] Specific Embodiment X: The difference between this embodiment and any one of Specific Embodiments VI to IX is that the environmental temperature of the low-temperature composting of toilet feces is 10±2°C. Other steps are the same as those in any one of Specific Embodiments VI to IX.

[0039] The following examples are used to verify the beneficial effects of the present invention:

[0040] Example 1 Screening of functional strains of the composite bacterial agent

[0041] Source of strains: Toilet feces, taken from a dry toilet in a rural area near Harbin.

[0042] Culture medium: The basic information of the main culture medium used is shown in Table 1. Among them, if an LB solid culture medium plate needs to be prepared, 20 g / L of agar needs to be additionally added.

[0043] Table 1 Composition table of the main culture medium

[0044]

[0045] The strains used to prepare the composite bacterial agent of the present invention are obtained by the following method:

[0046] 1.1 Isolation and purification of low-temperature degrading strains

[0047] Weigh 10 g of toilet fecal sewage and put it into a conical flask, add 90 mL of sterile normal saline and a few glass beads. Place the conical flask in a shaker at a rotation speed of 150 r / min and shake for 30 min. Let it stand for 10 min to obtain the dilution. Perform gradient dilution according to the ten-fold dilution method, select the dilution with an appropriate concentration and spread it on the LB plate, culture at 10 ± 2 °C, pick single colonies and repeat streaking on a new plate until purification. A total of 16 strains were obtained, including 8 bacteria and 8 fungi.

[0048] 1.2 Primary screening of low-temperature degrading strains

[0049] Inoculate the strains obtained by isolation and purification on the protein, starch, and fat medium plates, culture at 10 ± 2 °C, and observe whether the strains can utilize the substrates. The primary screening results show that there are 8 strains that can degrade protein, 9 strains that can degrade starch, and 4 strains that can degrade fat.

[0050] 1.3 Re-screening and molecular biology identification of low-temperature degrading strains

[0051] Inoculate the strains obtained by primary screening on the protein, starch, and fat medium plates respectively, culture at 10 ± 2 °C, use the degradation circle method to screen out the strains with strong degradation activity for each substrate, and select 3 strains with the strongest degradation ability for protein, starch, and fat as the candidate strains for the compound microbial agent for species identification.

[0052] Determine the 16S rRNA gene sequences of the 3 strains, use primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) for PCR amplification. The sequence of strain R3 is shown as SEQ ID NO: 1. Select gene sequences with high homology and construct a phylogenetic tree through MEGA11.0 software, and conduct comparative analysis according to the phylogenetic tree, as Figure 1 shown. The sequence of strain R4 is shown as SEQ ID NO: 2, and the phylogenetic tree is as Figure 2 shown. The sequence of strain R6 is shown as SEQ ID NO: 3, and the phylogenetic tree is as Figure 3 shown. Finally, it is determined that R3 is Stenotrophomonas pavanii R3, R4 is Massiliasuwonensis R4, and R6 is Pseudomonas knackmussii R6.

[0053] The compound microbial agent for low-temperature composting of toilet fecal sewage described in the present invention is prepared from these 3 strains.

[0054] The Stenotrophomonas pavanii R3 described above is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 25, 2025, and the deposit number is CGMCC No: 33647. Massilia suwonensis R4 is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 25, 2025, and the deposit number is CGMCC No: 33648. Pseudomonas knackmussii R6 is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 25, 2025, and the deposit number is CGMCC No: 33649.

[0055] Example 2 Preparation of a composite bacterial agent for low-temperature composting of toilet feces

[0056] The specific steps for the preparation method of the composite bacterial agent in this example are as follows:

[0057] (1) Take the cryopreserved Stenotrophomonas pavanii R3, dip the bacterial liquid and streak it on an LB solid plate, incubate at room temperature for 24 h, pick a single colony into a sterilized and cooled LB liquid medium, and incubate at 10 ± 2 °C and 180 r / min with shaking for 24 h to obtain a seed liquid. Transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2% and incubate at 10 ± 2 °C and 180 r / min with shaking for 36 h to obtain a bacterial suspension of Stenotrophomonas pavanii R3;

[0058] (2) Take the cryopreserved Massilia suwonensis R4, dip the bacterial liquid and streak it on an LB solid plate, incubate at room temperature for 24 h, pick a single colony into a sterilized and cooled LB liquid medium, and incubate at 10 ± 2 °C and 180 r / min with shaking for 24 h to obtain a seed liquid. Transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2% and incubate at 10 ± 2 °C and 180 r / min with shaking for 36 h to obtain a bacterial suspension of Massilia suwonensis R4;

[0059] (3) Take the cryopreserved Pseudomonas knackmussii R6, dip the bacterial liquid and streak it on an LB solid plate, incubate at room temperature for 24 h, pick a single colony into a sterilized and cooled LB liquid medium, and incubate at 10 ± 2 °C and 180 r / min with shaking for 24 h to obtain a seed liquid. Transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2% and incubate at 10 ± 2 °C and 180 r / min with shaking for 32 h to obtain a bacterial suspension of Pseudomonas knackmussii R6;

[0060] (4) Mix the bacterial suspensions of Stenotrophomonas pavanii R3, Massilia hydrothermale R4, and Pseudomonas kribbensis R6 according to the ratio of viable bacteria content of 3:2:1 to prepare a composite microbial agent for low-temperature composting of toilet feces and sewage.

[0061] Example 3: Experiment on the promotion of low-temperature composting of toilet feces and sewage by the composite microbial agent

[0062] The application of the composite microbial agent for low-temperature composting of toilet feces and sewage prepared in Example 2 in low-temperature composting of toilet feces and sewage is as follows:

[0063] Composting raw materials: The raw materials for low-temperature composting are toilet feces and crushed corn straw. The toilet feces used in the experiment were taken from a dry toilet in a rural area near Harbin, and the crushed corn straw was purchased from a Taobao store, with a size of about 5 mm. The physical and chemical properties of the two are shown in Table 2.

[0064] Table 2 Physical and chemical properties of composting raw materials

[0065]

[0066] A total of 3 treatments were set up in this experiment, namely a low-temperature control group (placed in an environment of 10±2°C, without adding microbial agent, denoted as 10°C without microbial agent), a normal-temperature control group (placed in an environment of 20±2°C, without adding microbial agent, denoted as 20°C without microbial agent), and a low-temperature experimental group (placed in an environment of 10±2°C, adding the composite microbial agent in Example 2, denoted as 10°C + microbial agent). Each group of composting was about 12 catties in weight. Among them, the dosage of the composite microbial agent in Example 2 in the low-temperature experimental group was 5% (V / W).

[0067] The sampling time for the physical and chemical index samples of composting was at 0, 7, 14, 21, 28, 35, and 42 d, and the sampling time for high-throughput sequencing samples was before composting and after the end of composting for each group.

[0068] 3.1 Effect of the composite microbial agent on composting temperature

[0069] Measure the temperature of the compost piles of the three groups at 10 o'clock and 20 o'clock every day. Take the average value of the temperatures at all sampling points as the overall temperature of the compost pile and record it. The results are as Figure 4 shown:

[0070] All three groups of composting processes repeatedly experienced a process of first warming up and then cooling down. Compared with the composting without microbial agents at 10°C, the composting without microbial agents at 20°C and the composting with microbial agents at 10°C reached the first high-temperature period 1 day earlier. The high-temperature durations were 5 days and 6 days respectively, and the highest temperatures were 62.3°C and 62°C respectively. The first high-temperature period of the composting without microbial agents at 10°C only lasted for 3 days, and the highest temperature was 47.8°C. After turning the pile, the composting without microbial agents at 20°C and the composting with microbial agents at 10°C could re-warm up and enter the second high-temperature period, while the temperature of the composting without microbial agents at 10°C only remained between 20 and 30°C. In addition, only the composting with microbial agents at 10°C among the three groups of composting maintained above 50°C for 10 days, meeting the temperature requirements for harmless treatment. It can be seen that compared with the low-temperature control group, adding a compound microbial agent can enable the low-temperature composting of toilet feces in a low-temperature environment to quickly warm up and start, enter the high-temperature period in advance, increase the overall temperature and extend the high-temperature time during the high-temperature period, increase the number of occurrences of the high-temperature period, and achieve an effect comparable to that of normal-temperature composting.

[0071] 3.2 Effects of compound microbial agent on the moisture content of compost

[0072] The samples were dried to a constant weight at 105°C, and the moisture content was calculated. The results are as Figure 5 shown:

[0073] After about 42 days of aerobic composting process, the moisture content of each group of compost decreased gradually with the increase of temperature. The moisture contents of the composting without microbial agents at 20°C and the composting with microbial agents at 10°C decreased from the initial 68.90% and 70.48% to 47.91% and 46.77% respectively, with a decrease of 20.99% and 23.71% respectively. However, the composting without microbial agents at 10°C only maintained a high temperature briefly in the early stage, and the temperature remained between 20 and 30°C in the later stage. The moisture content did not decrease significantly, only decreasing by 6.31%. It can be seen that compared with the low-temperature control group, adding a compound microbial agent can promote the decrease of the moisture content of the low-temperature composting of toilet feces in a low-temperature environment.

[0074] 3.3 Effects of compound microbial agent on the pH of compost

[0075] The compost samples were mixed with distilled water at a ratio of 1:10 (m / V), shaken at a speed of 150 r / min at room temperature for 1 h, left to stand for 20 min, and then filtered through gauze. The pH of the supernatant was measured with a pH meter. The results are as Figure 6 shown:

[0076] The pH values of the three groups of compost all showed a trend of first decreasing and then increasing, but always fluctuated between 7.5 and 9. However, the pH values of the composting without microbial agents at 20°C and the composting with microbial agents at 10°C changed earlier than those of the composting without microbial agents at 10°C. It can be seen that compared with the low-temperature control group, adding a compound microbial agent can accelerate the change process of the pH of the low-temperature composting of toilet feces in a low-temperature environment.

[0077] 3.4 Effects of the composite microbial agent on the organic matter content of the compost

[0078] The compost samples dried to a constant weight were calcined at 525 ± 10 °C for 6 h, and the organic matter content was calculated. The results are as Figure 7 shown below:

[0079] The organic matter content of the compost without the microbial agent at 20 °C decreased by 18.69%, which was the most obvious. The reduction amounts of the organic matter in the compost with the microbial agent at 10 °C and the compost without the microbial agent at 10 °C were 15.48% and 11.72% respectively. When the compost without the microbial agent at 20 °C and the compost with the microbial agent at 10 °C were in the high-temperature period, the reduction rate of the organic matter content was the fastest.

[0080] 3.5 Effects of the composite microbial agent on the C / N ratio of the compost

[0081] The dried compost samples were ground and passed through a 200-mesh sieve. The TC and TN contents in the samples were measured with an elemental analyzer, and the C / N ratio was calculated. The results are as Figure 8 shown below:

[0082] Generally, when the C / N ratio of the compost pile drops below 16:1, the compost is considered basically mature. At the end of the composting, the C / N ratios of the compost without the microbial agent at 20 °C, the compost with the microbial agent at 10 °C, and the compost without the microbial agent at 10 °C were 13.74:1, 15.13:1, and 16.59:1 respectively. It can be seen that compared with the low-temperature control group, adding the composite microbial agent can promote the reduction of the C / N ratio of the toilet feces low-temperature composting in the low-temperature environment, making it reach a maturity level equivalent to that of the normal-temperature composting.

[0083] 3.6 Effects of the composite microbial agent on the seed germination index of the compost

[0084] The compost samples were mixed with distilled water at a ratio of 1:10 (m / V), shaken at a speed of 150 r / min at room temperature for 2 h, and filtered through gauze to remove larger solids. The supernatant was used as the compost extract for the seed germination index experiment. Small Chinese cabbage seeds with uniform size and no obvious spoilage were selected, with 20 seeds in each group. 5 mL of the compost extract was added to each group, and after culturing at room temperature for 48 h, the seed germination rate and seed root length of each group were measured, and the GI value was calculated. The results are as Figure 9 shown below:

[0085] The seed germination index (GI) is the most persuasive indicator for judging the maturity of compost. Generally, it is considered that when GI ≥ 50%, the compost is initially mature; when GI ≥ 85%, the compost is completely mature. The 20°C compost without inoculant and the 10°C compost with inoculant reached maturity first within 2 weeks of composting, and the GIs of their final products reached 118.5% and 112.6% respectively; while the 10°C compost without inoculant did not mature until 5 weeks after the start of composting, and the final GI reached 93.5%. After the composting ended, the maturity order of the three groups of composts was 20°C compost without inoculant > 10°C compost with inoculant > 10°C compost without inoculant. It can be seen that compared with the low-temperature control group, adding a composite inoculant can accelerate the increase of GI in the low-temperature composting of toilet feces in a low-temperature environment, accelerate the composting process, and promote maturity.

[0086] 3.7 Effects of composite inoculant on the compost community structure and composition

[0087] Samples without inoculant before composting were taken and named BC. Then samples were taken from the three compost piles after the composting ended and named CK10 (10 ± 2°C, without inoculant), CK20 (20 ± 2°C, without inoculant), and MA10 (10 ± 2°C, adding the composite inoculant obtained in Example 2). Shanghai Personalbio was entrusted to conduct high-throughput sequencing, and primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used for RCR amplification of bacterial 16S rRNA. The data was processed through the Personalbio Gene Cloud platform to analyze the similarity relationship and community composition of microorganisms in each group of compost samples.

[0088] Beta diversity analysis of the four groups of compost samples:

[0089] Beta diversity analysis is mainly used to explore the degree of difference in the microbial community composition among multiple samples. NMDS analysis and hierarchical clustering analysis were performed on the four groups of compost samples. In the calculations of both analyses, the presence and relative abundance of species in the community were considered simultaneously, and the results are as Figure 10 shown.

[0090] The NMDS analysis diagram shows the distances between samples under the Bray-Curtis algorithm. Points of different colors represent different samples. The closer the distance between two points, the smaller the difference between the two samples and the more similar the community structures. The hierarchical clustering analysis diagram shows the distances between samples under the weighted Unifrac algorithm. If two samples are on the same branch, it indicates that the microbial communities in these two samples are more similar. In the NMDS analysis diagram, only the two points representing sample CK20 and sample MA10 are relatively close, and the distances between any other two points are far. This shows that the addition of the composite microbial agent obtained in Example 2 can promote the effect of low-temperature composting to approach that of normal-temperature composting at the community level, indicating that the addition of the composite microbial agent obtained in Example 2 plays a positive role in promoting the smooth progress of the composting process and the maturity of the compost pile at low temperatures by affecting the microbial community structure; the result that sample CK20 and sample MA10 are on the same branch in the hierarchical clustering analysis diagram also confirms this point.

[0091] Analysis of the genus composition of four groups of compost samples:

[0092] The species composition of the four groups of compost samples was analyzed at the genus level, and the results are as Figure 11 shown. Only the genera ranked in the top 50 in total abundance can be shown in the figure, and the genera with too small total abundance are grouped into the Others item.

[0093] The dominant bacterial communities at the genus level in the four groups of compost samples were different. The top five genera with their relative abundances in the BC sample were Aequorivita (11.14%), Cellvibrio (6.92%), Arenibacter (5.68%), Proteiniphilum (4.39%), and Pseudomonas (3.96%). The top five genera with their relative abundances in the CK10 sample were BIrii41 (15.68%), Cellvibrio (9.10%), Luteimonas (7.04%), Aequorivita (6.99%), and Parapedobacter (5.50%). The top five genera with their relative abundances in the CK20 sample were Muricauda (9.26%), Nonomuraea (7.59%), Luteimonas (7.24%), Longimicrobiaceae (6.66%), and MWH-CFBk5 (5.69%). In the MA sample, the top five genera with their relative abundances were Pseudomonas (14.61%), Cellvibrio (6.13%), NS9 marinegroup (5.96%), Muricauda (5.3%), and Nonomuraea (4.11%). It is not difficult to see that Muricauda and Nonomuraea are the dominant microorganisms during the composting process at normal temperature. After adding the composite bacterial agent obtained in Example 2 during low-temperature composting, although the abundances are lower than those in normal-temperature composting, these two types of functional bacterial communities can still be enriched. Both CK10 and CK20 contain a certain amount of Luteimonas, indicating that there may be some functional bacteria that are more common during composting under natural conditions in this genus, and they may have stronger adaptability to low temperatures. Cellvibrio is a kind of cellulose-decomposing bacterium, which is commonly found in humus soil and exists in all compost samples. However, its abundance is relatively high in CK10 and MA10, and only 2.54% in CK20. It is speculated that this type of functional bacteria may be more adaptable to low-temperature environments. In addition, it is worth mentioning that Pseudomonas is the dominant microorganism in the MA10 sample, and the functional bacterium R6 in the composite bacterial agent obtained in Example 2 also belongs to this genus. The addition of the composite bacterial agent may promote the enrichment of the functional bacteria in this genus.It can be seen that, compared with the low-temperature control group, adding a compound microbial agent to the low-temperature composting of toilet feces and sewage can improve the community diversity and the abundance of functional flora in the low-temperature composting, and gradually make its community structure converge with that of the normal-temperature composting. As a result, the effect of the low-temperature composting of toilet feces and sewage after adding the compound microbial agent is comparable to that of the normal-temperature composting.

Claims

1. A composite microbial agent for low-temperature composting of toilet fecal sewage, characterized in that, The composite bacterial agent is composed of Stenotrophomonas pavanii R3, Massilia suwonensis R4, and Pseudomonas knackmussii R6.

2. The composite bacterial agent for low-temperature composting of toilet fecal sewage according to claim 1, characterized in that, The Stenotrophomonas pavanii R3 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 25, 2025, and the deposit number is CGMCC No: 33647.

3. A composite bacterial agent for low-temperature composting of toilet fecal sewage according to claim 1, characterized in that, The Massilia suwonensis R4 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 25, 2025, and the deposit number is CGMCC No: 33648.

4. The composite microbial agent for low-temperature composting of toilet feces and sewage according to claim 1, wherein The Pseudomonas knackmussii R6 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 25, 2025, and the deposit number is CGMCC No: 33649.

5. A composite bacterial agent for low-temperature composting of toilet fecal sewage according to claim 1, characterized in that, Stenotrophomonas pavanii R3, Massilia suwonensis R4, and Pseudomonas knackmussii R6 form a composite bacterial agent according to the ratio of viable bacteria content of 3:2:

1.

6. The preparation method of a composite microbial inoculum for low-temperature composting of toilet feces and sewage according to claim 1, characterized in that, The specific steps of the preparation method are as follows:

1. Dip the bacterial liquid of Stenotrophomonas pavanii R3 and streak it on an LB solid plate. After culturing at room temperature, pick a single colony and transfer it to a sterilized and cooled LB liquid medium, and shake-culture to obtain a seed liquid; then transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2%, and continue to shake-culture to obtain a bacterial suspension of Stenotrophomonas pavanii R3.

2. Dip the bacterial liquid of Massilia suwonensis R4 and streak it on an LB solid plate. After culturing at room temperature, pick a single colony and transfer it to a sterilized and cooled LB liquid medium, and shake-culture to obtain a seed liquid; then transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2%, and continue to shake-culture to obtain a bacterial suspension of Massilia suwonensis R4.

3. Dip the bacterial liquid of Pseudomonas knackmussii R6 and streak it on an LB solid plate. After culturing at room temperature, pick a single colony and transfer it to a sterilized and cooled LB liquid medium, and shake-culture to obtain a seed liquid; then transfer the seed liquid to a sterilized and cooled LB liquid medium at a volume ratio of 2%, and continue to shake-culture to obtain a bacterial suspension of Pseudomonas knackmussii R6.

4. Mix the bacterial suspensions of Stenotrophomonas pavanii R3, Massilia suwonensis R4, and Pseudomonas knackmussii R6 according to the ratio of viable bacteria content of 3:2:1 to obtain a low-temperature compost composite bacterial agent.

7. The preparation method of a composite bacterial agent for low-temperature composting of toilet fecal sewage according to claim 6, characterized in that, The viable cell counts of the bacterial suspensions of Stenotrophomonas pavanii R3, Massilia aquatica R4, and Pseudomonas kribbensis R6 are all ≥ 1×10 8 CFU / mL.

8. The preparation method of a composite microbial inoculum for low-temperature composting of toilet fecal sewage according to claim 6, characterized in that, The shaking culture is carried out under the conditions of 10±2°C and 180 r / min.

9. The preparation method of a composite bacterial agent for low-temperature composting of toilet fecal sewage according to claim 6, characterized in that, The inoculation amount of the composite bacterial agent accounts for 5% of the total mass of the low-temperature composting of toilet fecal sewage.

10. The preparation method of a composite microbial inoculum for low-temperature composting of toilet feces and sewage according to claim 9, characterized in that, The environmental temperature of the low-temperature composting of toilet fecal sewage is 10±2°C.

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