Sphingomonas sp. for promoting anaerobic fermentation of kitchen waste to produce carbon dioxide and application thereof

Abstract

A strain of *Sphingomonas* promoting carbon dioxide production during the anaerobic fermentation of kitchen waste and its application are disclosed. The strain, with preservation number CGMCC No. 37894 and classified as *Sphingomonas* sp., was isolated from biogas slurry from engineered anaerobic fermentation of kitchen waste. This invention significantly increased carbon dioxide production during fermentation by inoculating this strain into a kitchen waste anaerobic fermentation system, resulting in a substantial increase in the proportion of carbon dioxide in the total gas composition. This strain exhibits advantages in efficient colonization and targeted regulation of gas composition under complex fermentation conditions, providing a stable and controllable raw material guarantee for the resource utilization of carbon dioxide, a byproduct of anaerobic fermentation of kitchen waste.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a strain of Sphingomonas sp. C1A4 isolated from anaerobic fermentation slurry of kitchen waste, and its application in promoting carbon dioxide generation and resource utilization during the anaerobic fermentation of kitchen waste. Background Technology

[0002] Food waste contains a significant amount of carbohydrates, proteins, and lipids, which can be degraded through anaerobic fermentation to achieve organic matter degradation and carbon resource recovery. During fermentation, organic matter undergoes transformation under the action of microorganisms, producing fermentation gases including methane, carbon dioxide, and hydrogen. Existing anaerobic fermentation processes for food waste primarily focus on the production and utilization of methane or hydrogen, with attention mainly concentrated on the removal and separation of carbon dioxide at the later stages of fermentation. Carbon dioxide is also an important carbon-containing byproduct of anaerobic fermentation. Current processes typically employ methods such as absorption, adsorption, or membrane separation to remove or capture carbon dioxide from the already generated fermentation gases. These methods can achieve gas component separation but cannot directly regulate the amount of carbon dioxide produced during fermentation.

[0003] The carbon dioxide produced by anaerobic fermentation, after collection or necessary purification, can be used as an inorganic carbon source for microalgae cultivation, microbial synthesis, and other biotransformation processes, as well as for carbon utilization processes such as mineralization and fixation. Under conditions where electron donors such as hydrogen and corresponding functional microorganisms are available, carbon dioxide can be further converted into methane or other carbon-containing products. Therefore, increasing carbon dioxide production and its volume percentage in fermentation gases is beneficial for forming a relatively concentrated carbon-containing gas source, providing conditions for subsequent separation, collection, and conversion.

[0004] The gas production process of anaerobic fermentation of food waste can be altered by adjusting temperature, pH, substrate composition, or inoculating microorganisms. However, most existing control methods are used to increase methane or hydrogen production, with fewer microbial control methods targeting carbon dioxide generation. The composition of food waste varies depending on its source and batch, causing fluctuations in fermentation gas production and gas composition. Relying solely on adjusting conventional operating parameters or capturing gases after fermentation is insufficient to specifically increase carbon dioxide production and its volume percentage in the fermentation gases from the fermentation process itself.

[0005] Therefore, it is necessary to provide a microbial regulation method suitable for anaerobic fermentation systems of kitchen waste to promote carbon dioxide generation and increase its volume proportion in fermentation gases, thereby providing conditions for the subsequent separation, collection and conversion of carbon dioxide. Summary of the Invention

[0006] To address the aforementioned deficiencies in existing technologies, this invention provides a strain of Sphingosine Monoclonal bacteria that promotes carbon dioxide production during the anaerobic fermentation of kitchen waste and its application. This strain can effectively promote the directional generation of carbon dioxide during the anaerobic fermentation of kitchen waste, significantly increasing the proportion of carbon dioxide in the gaseous products, thereby providing a stable and controllable raw material guarantee for subsequent carbon resource utilization.

[0007] To achieve the above objectives, the present invention includes the following technical solutions: On the one hand, the present invention provides a strain of Sphingomonas that promotes the anaerobic fermentation of kitchen waste to produce carbon dioxide. It belongs to the genus Sphingomonas, with accession number CGMCC No. 37894 and classification name Sphingomonas sp.

[0008] On the other hand, the present invention provides the application of Sphingomonas as described above in promoting the anaerobic fermentation of kitchen waste to produce carbon dioxide.

[0009] As described above, preferably, the application of anaerobic fermentation of kitchen waste to produce carbon dioxide adopts the following operating steps: I. Preparation of bacterial suspension: Sphingomonas sp. C1A4 in logarithmic growth phase was inoculated into DSMZ120 liquid medium at an inoculum concentration of 1%–5% (v / v). The medium was anaerobically cultured at 30–40 °C for 5–7 days. The bacterial cells were collected by centrifugation, washed with sterile physiological saline, and then resuspended in sterile physiological saline. The bacterial suspension concentration was adjusted to 10-1. 7 ~10 10 CFU / mL, for later use; II. Mix the bacterial solution, kitchen waste, and biogas slurry prepared in step I at a volume ratio of 1:(25~120):(50~240) and put them into an anaerobic fermentation container. Purge with N2 to remove oxygen, and then cultivate at a constant temperature of 30~40℃. The total fermentation cycle is 4~10 days. Connect the anaerobic fermentation container to a gas collection device to collect the generated gas.

[0010] In the application described above, preferably, the biogas slurry is taken from the anaerobic digester of a kitchen waste treatment plant.

[0011] In another aspect, the present invention provides a method for preparing the Sphingomonas liquid inoculant as described above, the method comprising the following steps: Step I: Inoculate the Sphingomonas sp. C1A4 in the logarithmic growth phase into DSMZ120 liquid medium at an inoculation rate of 1% to 5% (v / v) and culture anaerobically at 30 to 40°C for 3 to 10 days to obtain the seed culture. Step II: Expand the culture by transferring the seed culture to DSMZ120 medium at an inoculum rate of 1% to 10% (v / v) and anaerobic culture at 30 to 40°C for 3 to 10 days; Step III: Collect bacterial cells by centrifuging the culture medium at 3000-4200 rpm for 5-20 min, washing with sterile physiological saline, and resuspending to 10⁻⁶ ppm. 7 ~10 10 CFU / mL.

[0012] The beneficial effects of this invention are reflected in the following aspects: (1) Improved the distribution of fermentation gas components.

[0013] In existing technologies, carbon dioxide is typically a byproduct of anaerobic fermentation, and its proportion in the gas phase is relatively low. This invention, through inoculation with *Sphingomonas sp.* C1A4, achieved an increase in the proportion of carbon dioxide in fermentation gas production under experimental conditions. This method provides a regulatory pathway for downstream processes requiring specific gas components, contributing to the optimization of gas production components.

[0014] (2) It improved the adaptability of the strain in complex fermentation environment.

[0015] The Sphingomonas sp. C1A4 strain selected in this invention is derived from the anaerobic fermentation slurry of kitchen waste in actual engineering. Compared with strains from non-environmental sources, it has a better colonization foundation in the complex substrate environment of the kitchen waste anaerobic fermentation system, which is conducive to maintaining the relative stability of the fermentation system.

[0016] (3) It has good process compatibility.

[0017] The bacterial strains provided by this invention can be prepared in various forms, such as liquid, solid, or immobilized bacterial agents, according to actual production needs. This flexible morphological design allows the technical solution to be well adapted to anaerobic fermentation facilities of different scales and process types, providing good convenience in application and implementation.

[0018] Preservation of biological materials Sphingosine monocytogenes strain C1A4 was deposited on March 11, 2026, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 37894, address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, telephone: 010-64807355. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0020] Example 1: Isolation and identification of Sphingosine monoclonal strain C1A4 that promotes carbon dioxide production from anaerobic fermentation of kitchen waste. 1. Strain source and sampling The sampling point was selected from the biogas slurry of the anaerobic digester at the kitchen waste treatment plant in the Fengtai District Municipal Solid Waste Recycling Economy Park, Beijing. This facility is an engineered kitchen waste treatment project with a daily processing capacity of 200 tons and an effective volume of 3000 m³ for the anaerobic digester. 3 The operating temperature was 37±1℃, pH 6.8-7.2, and hydraulic retention time (HRT) was 20 days. On October 15, 2024, 500 mL of biogas slurry was collected from the bottom of the tank using a sterile anaerobic culture bag and treated in an anaerobic environment of N2:CO2 (80:20, v / v).

[0021] 2. Culture medium preparation The components of DSMZ120 anaerobic culture medium are shown in Table 1: Table 1 Each 1000 mL of culture medium contains:

[0022] The specific preparation process is as follows: (1) Dissolve the basic culture medium components except for sodium bicarbonate, 10×Wolin vitamin solution, 50% (v / v) methanol solution, cysteine ​​hydrochloride monohydrate and sodium sulfide nonahydrate in distilled water, heat to boiling to remove dissolved oxygen, and then add cysteine ​​hydrochloride monohydrate. (2) Aerate the culture medium with a mixture of 80% N2 + 20% CO2 (v / v) for 15 minutes to establish an anaerobic environment, and add sodium bicarbonate (NaHCO3) to the anaerobic environment to adjust the pH to 6.5; (3) Dispense the obtained culture medium into anaerobic bottles, with the liquid volume controlled to be 30% of the container volume, and autoclave at 121°C for 15 minutes; (4) In a 100% N2 environment, 50% (v / v) methanol solution, reducing agent (sodium sulfide nonahydrate) and 10×Wolin vitamin solution were prepared into concentrated solutions and filtered through a 0.22 μm filter membrane for sterilization. (5) After the basic culture medium in the anaerobic bottle is sterilized, the above-filtered and sterilized stock solution is injected into the anaerobic bottle under aseptic conditions using a sterile syringe, and the final pH of the culture medium is adjusted to 6.8-7.0 and placed in the anaerobic operation box for later use.

[0023] 3. Isolation, purification, and preservation of bacterial strains (1) Sample dilution and incubation The biogas slurry samples were serially diluted, with dilution factors set at 1.8 × 10⁻⁶. 4 times, 5.4×10 4 Doubled to 1.62×10 5 The dilution was then aliquoted into 96-well cell culture plates, with 200 μL added to each well, and the plates were anaerobically incubated at 37°C in the dark for 4 weeks. (2) Strain screening Two-step PCR amplification with double-sided tags was used to amplify and identify the 16S rRNA gene of bacteria in each well of the culture plate, and to screen for target strains. (3) Strain purification The selected target strains were purified by streak plating three times. Single colonies from the third streak plate were picked and inoculated into liquid culture medium and incubated at 37°C in the dark for 7 days. The culture was continued until the bacterial suspension became turbid (OD200). 600 When the concentration is approximately 0.8-1.0, extract total DNA from 10 μL of bacterial culture.

[0024] (4) Preservation of microbial strains The 16S rRNA gene was amplified using universal primers 27F and 1492R, and the sequence was verified by Sanger sequencing. After confirming that the sequence was correct, the pure culture was added to glycerol to a final concentration of 30% (v / v) and stored in a -80°C freezer.

[0025] 4. Strain identification and preservation of biological materials (1) 16S rRNA gene sequence analysis The 16S rRNA gene sequence of the obtained strain is detailed in the "Sequence Listing". After NCBI BLAST alignment, it belongs to the genus *Sphingomonas* and is named C1A4.

[0026] (2) Preservation of biological materials Sphingosine monocytogenes strain C1A4 was deposited on March 11, 2026, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 37894, address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, telephone: 010-64807355.

[0027] Example 2: Experiment on the effect of Sphingosine Monoclonal strain C1A4 on promoting carbon dioxide production from anaerobic fermentation of kitchen waste. 1. Experimental Materials Biogas slurry: Samples were taken from the anaerobic digester of kitchen waste in the Fengtai District Municipal Solid Waste Recycling Economic Park, Beijing. Three samples were collected, namely Biogas Slurry 1, Biogas Slurry 2, and Biogas Slurry 3. After collection, the samples were passed through a 0.5 mm sieve to remove large particulate impurities, stored at 4°C, and used within 48 hours.

[0028] Simulated kitchen waste substrate: Prepared according to the mass ratio of cabbage, rice and chicken of 100:33.5:5 (dry basis mass ratio). Crush the raw materials to a particle size of less than 2 mm, adjust the solid content to 10%, seal and store in a 4°C refrigerator for later use.

[0029] 2. Experimental setup A 1 L anaerobic flask was used as the fermentation vessel, sealed with a rubber stopper and connected to an air bag to achieve fully closed anaerobic fermentation. The fermentation temperature was controlled at 37°C, and the pH value of the system was monitored using a pH meter to maintain it between 6.8 and 7.0.

[0030] 3. Experimental Methods (1) Preparation of bacterial suspension: The logarithmic growth phase strain C1A4 was inoculated into DSMZ120 liquid medium at an inoculation rate of 1% (v / v) (preparation method is shown in Example 1), and cultured under anaerobic conditions at 37°C for 5 days; after the culture was completed, the bacterial cells were collected by centrifugation at 4000 rpm for 10 minutes, washed 3 times with sterile physiological saline, and finally resuspended with sterile physiological saline to adjust the concentration of the bacterial suspension to 10. 8 CFU / mL.

[0031] (2) Experimental design: The above experimental materials, biogas slurry, kitchen waste, and bacterial solution, were mixed in accordance with the volume ratio and added to an anaerobic fermentation bottle with a volume of 500 mL. Fermentation was carried out under the fermentation parameters in the above experimental apparatus, and the total fermentation cycle was 5 days.

[0032] This experiment used three reaction systems to comprehensively evaluate the carbon dioxide production promoting effect of strain C1A4 by varying the batches of biogas slurry. Each system included a control group (CK) and an experimental group (C1A4), with three replicates per group. The specific components of each group are shown in Table 2 below: Table 2 Fermentation Experiment Reaction System Setup

[0033] (3) Fermentation process According to the above proportions, each component is placed into a 500 mL anaerobic bottle and quickly sealed with a rubber stopper. Then, N2 gas is introduced into the bottle through the gas passage interface on the rubber stopper to purge for 5-10 minutes to completely remove the air inside the bottle and create a strictly anaerobic environment. After deoxygenation is completed, the anaerobic bottle is connected to a gas bag for gas production collection to ensure that the entire fermentation process is carried out in a completely closed system. Finally, the device is placed in a constant temperature environment of 37℃ for cultivation. The total fermentation cycle is 5 days, and the gas production characteristics and metabolic patterns are monitored daily during the fermentation period.

[0034] 4. Detection Indicators and Methods Daily gas production measurement: The gas production of each group of fermentation bottles was recorded daily at regular intervals using the water displacement gas collection method.

[0035] Gas composition analysis: A Shimadzu GC-2014 gas chromatograph was used for detection. The instrument is equipped with a thermal conductivity detector (TCD) and a flame ionization detector (FID). The chromatographic column was TDX-01 (3.0 m × 3.00 mm). Chromatographic conditions: Injector, column temperature, and detector temperature were all 200°C; argon was used as the carrier gas at a flow rate of 35 mL / min; the current was 50 mA; the data acquisition time was 40 min; the injection volume was 1 mL, injected through a gas bag using a gas-tight needle.

[0036] Qualitative and quantitative analysis: Qualitative analysis was performed using the retention time of standard gases, with CO2 having a retention time of approximately 23.05 min. The target gas species were determined by comparing the retention times with those of the sample gases. Quantitative analysis was performed using the external standard method with peak area, and the volume fraction of each gas in the sample was calculated by plotting a standard curve. The actual gas production of each component was calculated using the formula: Actual gas production (mL) = Cumulative gas production × Corresponding gas volume fraction. To reduce detection error, the same sample was injected twice consecutively, and the average peak area was used in the calculation.

[0037] 5. Results Analysis The carbon dioxide production results of the three groups of experiments are shown in Table 3.

[0038] Table 3. Effects of strain C1A4 on carbon dioxide production during anaerobic fermentation of kitchen waste.

[0039] Note: Carbon dioxide production is the cumulative volume of carbon dioxide produced by each fermentation flask during the fermentation cycle, expressed in mL / flask. Data are expressed as mean ± sample standard deviation, n=3. A two-tailed Welch t-test was used to compare the strain treatment groups with the corresponding control groups within each system: *P<0.05, **P<0.01.

[0040] The carbon dioxide production results of different experimental systems are shown in Table 3. In System 1, the carbon dioxide production of the CK group was 205.52±15.35 mL / fermentation bottle, while the C1A4 treatment group increased to 704.96±8.38 mL / fermentation bottle, approximately 3.43 times that of the CK group, with a statistically significant difference (P<0.001). In System 2, the carbon dioxide production of the CK group was 252.38±10.26 mL / fermentation bottle, while the C1A4 treatment group increased to 697.35±14.60 mL / fermentation bottle, approximately 2.76 times that of the CK group, with a statistically significant difference (P<0.001). In System 3, the carbon dioxide production of the CK group was 231.43±9.01 mL / fermentation bottle, while the C1A4 treatment group increased to 697.67±15.09 mL / fermentation bottle, approximately 3.01 times that of the CK group, with a statistically significant difference (P<0.001). The results of the three experiments all showed that adding strain C1A4 could significantly increase the carbon dioxide production of the anaerobic fermentation system, and the trend of change was consistent, indicating that the strain has a relatively stable carbon dioxide production promoting effect in different experimental batches.

[0041] Table 4. Effects of strain C1A4 on the volume fraction of carbon dioxide in the anaerobic fermentation gas of kitchen waste.

[0042] Note: Carbon dioxide volume fraction is the percentage of the cumulative carbon dioxide volume produced by each fermentation flask relative to the total cumulative gas production volume during the same period. Data are expressed as mean ± sample standard deviation, n=3. A two-tailed Welch t-test was used to compare the strain treatment groups with the corresponding CK groups within each system: *P<0.05, **P<0.01.

[0043] Table 4 shows the volume fraction of carbon dioxide in the total gas phase. In System 1, the carbon dioxide volume fraction in the CK group was 21.50%±1.93%, while that in the C1A4 treatment group increased to 62.74%±1.77%, approximately 2.92 times that of the CK group, with a statistically significant difference (P<0.001). In System 2, the carbon dioxide volume fraction in the CK group was 26.25%±1.32%, while that in the C1A4 treatment group increased to 59.99%±3.73%, approximately 2.28 times that of the CK group, with a statistically significant difference (P=0.002). In System 3, the carbon dioxide volume fraction in the CK group was 24.03%±1.61%, while that in the C1A4 treatment group increased to 59.43%±5.06%, approximately 2.47 times that of the CK group, with a statistically significant difference (P=0.004). The results indicate that strain C1A4 not only increased carbon dioxide production but also increased the proportion of carbon dioxide in the gas phase products, which is beneficial for the targeted production and subsequent resource utilization of carbon dioxide.

[0044] The above experimental results show that strain C1A4 can effectively promote carbon dioxide generation and increase its proportion in the total gas in the anaerobic fermentation system of kitchen waste, demonstrating good application potential and providing a microbial basis for the targeted production and resource utilization of carbon dioxide.

Claims

1. A strain of Sphingosine monocytogenes that promotes anaerobic fermentation and carbon dioxide production from kitchen waste, characterized in that, It belongs to the genus Sphingomonas, with accession number CGMCC No. 37894 and classification name Sphingomonas sp.

2. The application of Sphingosine Monoclonal in promoting anaerobic fermentation of kitchen waste to produce carbon dioxide as described in claim 1.

3. The application as described in claim 2, characterized in that, The application of carbon dioxide production from anaerobic fermentation of kitchen waste adopts the following operational steps: I. Preparation of bacterial suspension: Sphingomonas sp. C1A4 in logarithmic growth phase was inoculated into DSMZ120 liquid medium at an inoculum concentration of 1%–5% (v / v). The medium was anaerobically cultured at 30–40 °C for 5–7 days. The bacterial cells were collected by centrifugation, washed with sterile physiological saline, and then resuspended in sterile physiological saline. The bacterial suspension concentration was adjusted to 10-1. 7 ~10 10 CFU / mL, for later use; II. Mix the bacterial solution, kitchen waste, and biogas slurry prepared in step I at a volume ratio of 1:(25~120):(50~240) and put them into an anaerobic fermentation container. Purge with N2 to remove oxygen, and then cultivate at a constant temperature of 30~40℃. The total fermentation cycle is 4~10 days. Connect the anaerobic fermentation container to a gas collection device to collect the generated gas.

4. The application as described in claim 3, characterized in that, The biogas slurry was taken from the anaerobic fermentation tank of the kitchen waste treatment plant.

5. The method for preparing the Sphingomonas liquid inoculant as described in claim 1, characterized in that, The method includes the following steps: Step I: Inoculate the Sphingomonas sp. C1A4 in the logarithmic growth phase into DSMZ120 liquid medium at an inoculation rate of 1% to 5% (v / v) and culture anaerobically at 30 to 40°C for 3 to 10 days to obtain the seed culture. Step II: Expand the culture by transferring the seed culture to DSMZ120 medium at an inoculum rate of 1% to 10% (v / v) and anaerobic culture at 30 to 40°C for 3 to 10 days; Step III: Collect bacterial cells by centrifuging the culture medium at 3000-4200 rpm for 5-20 min, washing with sterile physiological saline, and resuspending to 10⁻⁶ ppm. 7 ~10 10 CFU / mL.

Images