Method for jointly treating biogas slurry by using anabaena and trichoderma and application

Abstract

The invention belongs to the field of biological treatment of pollutants, and particularly relates to a method for jointly treating biogas slurry by using anabaena and trichoderma and application. The anabaena and the trichoderma viride are simultaneously cultured in the pig farm biogas slurry, so that ammonia nitrogen, phosphorus and COD in the pig farm biogas slurry can be effectively reduced. Meanwhile, the harvested compound of the anabaena and the trichoderma viride can be applied to rice fields and has application potential in the aspect of crop disease resistance.

Description

Technical Field

[0001] This invention belongs to the field of biological treatment of pollutants, and specifically relates to a method and application of using Anabaena and Trichoderma to treat biogas slurry. Background Technology

[0002] The scale of livestock and poultry farming is constantly expanding, and large-scale farms generate a large amount of excrement every day. If not properly treated, this can cause serious environmental pollution. Anaerobic digestion technology, as an important means of realizing the resource utilization of manure, has effectively alleviated some environmental pressure, but the resulting biogas slurry brings new pollution challenges. Anaerobic fermentation is used for treatment, but the biogas slurry produced is rich in nitrogen, phosphorus, and organic matter, and contains pollutants such as antibiotics and heavy metals. Traditional treatment methods are energy-intensive and costly. This problem is particularly prominent in China, where the lack of sufficient farmland around many farms to absorb the biogas slurry leads to a high risk of pollution to water bodies, soil, and air (Wang et al., 2020). Direct discharge of biogas slurry into water bodies can cause eutrophication, resulting in the death of fish and other aquatic organisms.

[0003] To address this challenge, emerging algae-microbe co-processing technology exhibits unique advantages: filamentous microalgae are easier to collect than traditional single-celled microalgae and can be directly used as biofertilizer in paddy fields; fungi such as Trichoderma, with their efficient organic matter degradation and biocontrol capabilities, are ideal microbial strains for algae co-processing. The combined treatment system not only recovers nitrogen and phosphorus from biogas slurry and removes pollutants, but the harvested algae and fungi can also be used in agricultural production, exhibiting green, low-carbon, and sustainable characteristics. Our prior research, namely Chinese invention patent ZL2021111957007, discloses a method and application for treating pig farm biogas slurry with nitrogen-fixing cyanobacteria and Trichoderma to produce agricultural microbial agents, which utilizes Nostoc commune. Nostoc The combined treatment of pig farm biogas slurry with *Sp. SCAU13* and *Trichoderma viride* under shake flask conditions showed the effects on TP, TN, and NH4+. + The removal rates of -N were 92.0%, 71.0%, and 75.4% on day 8, respectively. However, the removal rates of TP, COD, and NH4 were significantly lower. + The treatment effect of biogas slurry with higher N-concentration or larger-scale biogas slurry treatment volume was not satisfactory, according to the technical solution provided in Chinese invention patent ZL2021111957007. Analysis suggests this may be related to the fact that *Nostoc* sp. SCAU13, when treated with high concentrations of TP, COD, and NH4+,... + The mutualistic interaction between -N and Trichoderma in large-scale biogas slurry cultures is unstable. Therefore, further research is needed to develop more suitable bacterial-algae composite formulations and methods for treating biogas slurry in large-scale cultures. Summary of the Invention

[0004] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and provide a method for treating biogas slurry by using anabaena and trichoderma.

[0005] Another purpose of the present application is to provide the use of the method for treating biogas slurry by using anabaena and trichoderma.

[0006] The purpose of the present application is achieved by the following technical solutions: A method for treating biogas slurry by using anabaena and trichoderma, comprising the following steps: (1) Anabaena is inoculated into an algal culture medium and cultured to the logarithmic growth phase to obtain anabaena algal liquid; then the anabaena algal liquid is transferred to biogas slurry for domestication to obtain domesticated anabaena algal liquid; (2) Trichoderma viride is inoculated into a fungal culture medium for culture to obtain trichoderma viride bacterial liquid; (3) The domesticated anabaena algal liquid obtained in step (1) and the trichoderma viride bacterial liquid obtained in step (2) are selected to remove the culture medium and added to the biogas slurry for culture to obtain treated biogas slurry and bacterial-algal co-culture.

[0007] The anabaena in step (1) is preferably anabaena azotica (Anabaena azotica) SCAU26. Anabaena

[0008] The algal culture medium in step (1) is preferably BG110 liquid culture medium.

[0009] The culture conditions in step (1) are preferably 25-30°C, illumination 6000-10000lx, light-dark time 14-18h:6-10h; more preferably 28°C, illumination 8000lx, light-dark time 16h:8h.

[0010] The culture time in step (1) is preferably 6-8 days; more preferably 7 days.

[0011] The biogas slurry in step (1) and step (3) is preferably pig farm biogas slurry; its composition is preferably NH4 + -N 180-190 mg / L, TP 60-70 mg / L, COD 1150-1250 mg / L, pH=8.0-9.0; more preferably NH4 + -N 189 mg / L, TP 68 mg / L, COD 1210 mg / L, pH=8.6.

[0012] The transfer inoculation amount in step (1) is preferably 5-15% v / v; more preferably 10% v / v.

[0013] ​The acclimation conditions in step (1) are preferably 25-30°C, light 6000-10000 lx, light and dark time 14-18h:6-10h; more preferably 28°C, light 8000 lx, light and dark time 16h:8h.

[0014] The acclimation time in step (1) is preferably 6-8 days; more preferably 7 days.

[0015] The acclimation times in step (1) are preferably 1-3 times; more preferably 2 times.

[0016] The Trichoderma viride in step (2) is preferably Trichoderma viride (GDMCC 3.443). Trichoderma viride

[0017] The fungal culture medium in step (2) is preferably PDB liquid medium.

[0018] The culture conditions in step (2) are preferably 25-30°C, 90-110 rpm; more preferably 28-29°C, 100 rpm.

[0019] The culture time in step (2) is preferably 2-3 days; more preferably 2 days.

[0020] The Trichoderma viride liquid and the Anabaena liquid in step (3) are preferably mixed at a volume ratio of 4-8:1; more preferably at a volume ratio of 8:1.

[0021] The Trichoderma viride and the Anabaena in step (3) are preferably mixed at a biomass ratio of 320-640:39; more preferably at a biomass ratio of 640:39.

[0022] The selective removal medium in step (3) refers to a medium that can remove the Anabaena liquid and / or the Trichoderma viride liquid, and the obtained algal bodies and / or bacterial bodies are added to the biogas slurry; or the medium is not removed, and the Anabaena liquid and / or the Trichoderma viride liquid is directly added to the biogas slurry.

[0023] The culture in step (3) is preferably ordinary culture or culture using a bubbling column reactor.

[0024] The ordinary culture refers to culture using an ordinary container.

[0025] The ordinary container includes a bottle, a basin, a tank, and a pool.

[0026] ​The general culture condition is preferably 25-30 DEG C, illumination 6000-10000 lux, light and dark time 14-18h:6-10h, and the mixing operation is carried out in intervals.

[0027] The mixing operation is preferably shaking, stirring or bubbling.

[0028] The bubbling column reactor is preferably a 10L bubbling column reactor.

[0029] The culture condition using the bubbling column reactor is preferably 25-30 DEG C, illumination 6000-10000 lux, light and dark time 14-18h:6-10h, and the bubbling rate is 40-50 L / min; more preferably 28 DEG C, illumination 8000 lux, light and dark time 16h:8h, and the bubbling rate is 45 L / min.

[0030] The method for treating biogas slurry by using anabaena and trichoderma can utilize the nutrients in the biogas slurry of a pig farm, effectively treat the biogas slurry of the pig farm, and obtain a microbial agent, i.e., a bacteria-algae compound.

[0031] The biogas slurry is preferably biogas slurry of a pig farm, and the composition is preferably NH4 + -N 180-190 mg / L, TP 60-70 mg / L, COD 1150-1250 mg / L, and pH=8.0-9.0; more preferably NH4 + -N 189 mg / L, TP 68 mg / L, COD 1210 mg / L, and pH=8.6.

[0032] The present application has the following advantages and effects compared with the prior art. The method for treating biogas slurry by using anabaena and trichoderma can make trichoderma viride and anabaena form a complex, and the biomass and the removal rates of nitrogen, phosphorus and COD of the complex are higher than those of anabaena alone; and when a 10L bubbling column reactor is used to treat the biogas slurry in an enlarged volume, the removal rates of ammonia nitrogen, TP and COD are 97.8%, 72.8% and 72.5% respectively. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure 1 is a preliminary test result graph of treating biogas slurry of a pig farm by using anabaena and trichoderma in different bacteria-algae ratios.

[0034] Figure 2is the 2L flask experimental result figure of the pig farm biogas slurry treated by the combined green trichoderma of different bacteria and algae ratio of fishy algae.

[0035] Figure 3 is the experimental result figure of the comparison of the biogas slurry treated by the combined algae and bacteria and the biogas slurry treated by single algae in the 10L columnar culture device. DETAILED DESCRIPTION

[0036] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0037] The composition of BG110 liquid medium is as follows: 0.04 g / L K2HPO4·3H2O, 0.075 g / L MgSO4·7H2O, 0.036 g / L CaCl2·2H2O, 0.006 g / L citric acid, 0.006 g / L ferric ammonium citrate, 0.001 g / L EDTA, 0.02 g / L Na2CO3, 1 mL trace element A5, and the balance is water; The composition of trace element A5 is as follows: H3BO3 2.860 g / L, NaMoO4·2H2O 0.021 g / L, ZnSO4·7H2O 0.222 g / L, CuSO4·5H2O 0.079 g / L, MnCl2·4H2O 1.810 g / L, NiSO4·6H2O 0.479 g / L, and the balance is water.

[0038] The composition of PDB liquid medium is as follows: potato infusion powder 5 g / L, glucose 20 g / L, pH value 6.0±0.2, and the balance is water.

[0039] The PDA solid culture medium is the basis of PDB liquid medium plus 1.4% (m / V) agar.

[0040] The pig farm biogas slurry used in the experiment: NH4 + -189 mg / L, TP 68 mg / L, COD 1210 mg / L, pH=8.6.

[0041] Example 1: Bacteria and algae inoculation ratio preliminary screening experiment The growth of algae and bacteria under 5 different ratios of bacteria and algae treatment and single algae treatment was evaluated by using 500 mL bottles containing 200 mL of biogas slurry.

[0042] (1) Algae activation, culture and biogas slurry domestication Fishy algae Anabaenasp. SCAU26 (disclosed in our group's paper "Zhou YW, Bao JQ, Zhang DH, Li Y, Li HS, He HZ*. Effect of heterocystous nitrogen-fixing cyanobacteria against rice sheathblight and the underlying mechanism. Applied Soil Ecology, 2020, 153: 103580") was deposited on sterile BG110 solid agar plates. When needed, a small amount was taken from the plate and added to a 50 mL glass Erlenmeyer flask containing 20 mL of sterile BG110 liquid medium. The flask was then incubated for 7 days in a light incubator at a controlled temperature of 28℃, a light intensity of 8000 lx, and a light-dark cycle of 16 h:8 h. Then, the algae solution was transferred to the biogas slurry at an inoculation ratio of 10% v / v for cultivation and acclimatization. The acclimatization conditions were controlled in a light incubator with a temperature of 28℃, a light intensity of 8000 lx, and a light-dark time of 16h:8h. The algae solution was transferred to fresh biogas slurry every 7 days, and the acclimatization was carried out twice.

[0043] (2) Activation and culture of Trichoderma viride Trichoderma viride ( Trichoderma viride GDMCC 3.443 was purchased from Guangdong Provincial Microbial Culture Collection Center and preserved on PDA solid agar plates. A small amount of *Trichoderma viride* spores were scraped from the PDA plates and placed into PDB liquid medium, and incubated at 28.5℃ and 100 rpm for 48 h on a shaker.

[0044] (3) Initial screening of bacterial and algal inoculation ratio The *Trichoderma viride* cultured for 48 hours was mixed with *Anabaena globulus* cultured at different volume ratios (1:1, 2:1, 4:1, 8:1, and 1:2). Specifically, the cultured algae cultured in step (1) was inoculated into the biogas slurry at 10% v / v. Then, the *Trichoderma viride* cultured for 48 hours was thoroughly dispersed, and bacterial cultures were taken at volume ratios of 1:1, 2:1, 4:1, 8:1, and 1:2. The culture medium was removed by centrifugation, and the bacteria were added to the biogas slurry. Taking 1:1 as an example, the initial biomass of *Anabaena globulus* and *Trichoderma viride* in the final biogas slurry was 39 mg / L and 80 mg / L, respectively. The biomass of *Anabaena globulus* remained unchanged with different treatment ratios, while the biomass of *Trichoderma viride* increased proportionally. Simultaneously, a control group was set up by inoculating the pig farm biogas slurry with 10% cultured algae cultured at the same ratio. The chlorophyll was cultured continuously for 10 days in an incubator with a temperature of 28℃, a light intensity of 8000 lux, and a light-dark ratio of 16h:8h, with the mixture shaken three times a day. OD680 values, pigment content, and chlorophyll fluorescence parameters were recorded and measured every other day.

[0045] The experimental results are shown in Figure 1 From the figure, it can be seen that the algal-bacterial body growth of the 8:1 treatment group is the best overall, and the state of the algae is also the best.

[0046] Example 2: 2L large bottle re-screening experiment of algal-bacterial inoculation ratio According to the above test results, three algal-bacterial ratios with larger differences, 1:2, 2:1 and 8:1, were selected for testing, and 800 mL of biogas slurry was treated in a 2L bottle. The specific experimental method is the same as in Example 1. The experimental results show that the algae in each treatment can basically grow, and the OD and chlorophyll a concentration increase continuously with time extension ( Figure 2 ). Consistent with the preliminary screening results, the algal-bacterial body growth of the 8:1 treatment group is the best, and the state of the algae is also the best. By the eighth day, the OD value, chlorophyll a, and carotenoid of the 8:1 treatment group are higher than those of other treatment groups. At the same time, the removal rates of ammonia nitrogen, TP and COD are also higher, reaching 81.9%, 54.6% and 55.9% respectively by the eighth day, which are better than those of other treatment groups. In addition, the total biomass of the 8:1 treatment group is 0.76±0.03 g / L, which is also significantly higher than that of other treatment groups (Table 1).

[0047] Table 1: Biomass of different algal-bacterial inoculation ratios under biogas slurry culture

[0048] Note: Different letters in the same column represent significant differences between treatments ( P <0.05).

[0049] Example 3: Effect of biogas slurry on growth and physiological characteristics of algal-bacterial body in columnar culture device According to the results of the 2L large bottle experiment, the algal-bacterial ratio of 8:1 was selected for further magnification experiment using a 10L bubbling column reactor (model: PZ10L, Shanghai Bintu Organic Glass Co., Ltd.). The volume of biogas slurry treated was about 8.5 L. The column reactor was a cylindrical hard acrylic container with an outer diameter of 19 cm and a height of 40 cm. The bottom had a gas outlet connected to a gas pump with a brand of Hai Li and a power of 25W, and the air blowing rate was 45L / min. The culture conditions were temperature 28℃, light intensity 8000lux, light-dark ratio 16h:8h, and continuous culture for 10 days in a light incubator.

[0050] The experimental results show that, compared with the TS treatment group alone, the growth, physiological state of microalgae and pollutant treatment effect of the Trichoderma-TS algal-bacterial ratio 8:1 treatment group are significantly better than those of the shake flask culture ( Figure 3The removal rates of ammonia nitrogen, TP and COD reached 97.8%, 72.8% and 72.5% respectively, which were significantly higher than those of the algal control group and the aforementioned shake flask experiment. At the same time, the final total biomass reached 1.64±0.03 g / L, which was significantly higher than that of the single algal treatment (Table 2) and the aforementioned 2 L shake flask experiment.

[0051] Table 2 Biomass of microalgae and bacteria-algae in the columnar culture device under biogas slurry culture

[0052] Note: * indicates significant difference between treatments p <0.05).

[0053] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A method for treating biogas slurry by combined treatment of Anabaena and Trichoderma, characterized in that It comprises the following steps: (1) inoculate Anabaena into algal culture medium, culture to logarithmic growth phase, obtain Anabaena algal liquid; then transfer Anabaena algal liquid into biogas slurry for domestication, obtain domesticated Anabaena algal liquid; (2) inoculate Trichoderma viride into fungal culture medium for culture, obtain Trichoderma viride bacterial liquid; (3) selectivity remove culture medium from the domesticated Anabaena algal liquid obtained in step (1) and the Trichoderma viride bacterial liquid obtained in step (2), and add into biogas slurry for culture, obtain treated biogas slurry and bacterial-algal co-culture.

2. The method for treating biogas slurry by using Anabaena and Trichoderma viride combination according to claim 1, characterized in that: The anabaena mentioned in step (1) is anabaena ( Anabaena sp.)SCAU26; The Trichoderma viride in step (2) is Trichoderma viride (ATCC 56765). Trichoderma viride ) GDMCC 3.

443.

3. The method for treating biogas slurry by using Anabaena and Trichoderma viride combination according to claim 1, characterized in that: the algal culture medium in step (1) is BG110 liquid culture medium; the fungal culture medium in step (2) is PDB liquid culture medium.

4. The method for treating biogas slurry by using Anabaena and Trichoderma viride combination according to claim 1, characterized in that: the culture condition in step (1) is to culture at 25-30℃, illumination 6000-10000lx, light-dark time 14-18h:6-10h; the inoculation amount for transfer in step (1) is 5-15% v / v; the domestication condition in step (1) is to domesticate at 25-30℃, illumination 6000-10000lx, light-dark time 14-18h:6-10h; the culture condition in step (2) is to culture at 25-30℃, 90-110 rpm.

5. The method for treating biogas slurry by using Anabaena and Trichoderma viride combination according to claim 4, characterized in that: the culture time in step (1) is 6-8 days; the domestication time in step (1) is 6-8 days; the domestication frequency in step (1) is 1-3 times; the culture time in step (2) is 2-3 days.

6. The method for treating biogas slurry by using Anabaena and Trichoderma viride combination according to claim 1, characterized in that: the ratio of Trichoderma viride bacterial liquid to Anabaena algal liquid in step (3) is 4-8:

1.

7. The method for treating biogas slurry by using Anabaena and Trichoderma viride combination according to claim 1, characterized in that: the culture in step (3) is ordinary culture or culture using bubbling column reactor.

8. The method for treating biogas slurry by using Anabaena and Trichoderma viride combination according to claim 7, characterized in that: the ordinary culture condition is to culture at 25-30℃, illumination 6000-10000lx, light-dark time 14-18h:6-10h, and to mix evenly during the culture; the culture condition using bubbling column reactor is to culture at 25-30℃, illumination 6000-10000lx, light-dark time 14-18h:6-10h, and the air blowing rate is 40-50 L / min.

9. Use of the method for treating biogas slurry with combined treatment of Anabaena and Trichoderma as claimed in any one of claims 1 to 8 in the treatment of biogas slurry and / or in the preparation of a bacteria-algae composite.

10. Use according to claim 9, characterized in that: The biogas slurry is pig farm biogas slurry.

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