Microbial agent and method for repairing cadmium-polluted rice field soil by microbial agent

Abstract

The invention discloses a microbial agent and a method for repairing cadmium-polluted rice field soil by using the microbial agent, relates to the technical field of repairing cadmium pollution of rice field soil, and solves the problem that the cadmium metal enrichment capacity of duckweed is limited due to the fact that the duckweed has certain endurance capacity to cadmium stress. The microbial agent comprises bacillus subtilis ATCC6633 and pseudomonas aeruginosa ATCC27853, and the mass ratio of the bacillus subtilis ATCC6633 to the pseudomonas aeruginosa ATCC27853 is 2: 1, and the method comprises the following steps: step 1, spreading the microbial agent on the surface layer of cadmium-polluted rice field soil, then mixing the microbial agent with the rice field soil, raking the rice field soil, adjusting the water-holding capacity of the rice field soil, applying a base fertilizer, and irrigating the rice field. According to the method, the cadmium tolerance of the duckweed can be promoted, so that the enrichment capacity of the duckweed on cadmium is promoted, the remediation capacity of the duckweed on cadmium-polluted soil is improved, the cadmium content in the soil can be reduced, and the content of metal cadmium in rice is also reduced through a competitive cadmium absorption way.

Description

Technical Field

[0001] The invention relates to the technical field of repairing cadmium-contaminated paddy soil, and specifically relates to a microbial agent and a method for repairing cadmium-contaminated paddy soil with the microbial agent. Background Art

[0002] In recent years, due to the irrational application of chemical fertilizers and improper management of high-intensity planting, the soil quality problems in my country's main grain-producing areas have become increasingly serious, especially the intensification of heavy metal pollution. The application of large amounts of chemical fertilizers has not brought about a significant increase in grain yields, but has led to a decrease in soil organic matter content and a continuous decline in farmland fertility. Cadmium exposure is a serious hazard to human health, especially its long-term toxicity to the kidneys, liver, and central nervous system. After chronic cadmium exposure enters the human body through food, water, and air, it accumulates in the liver and kidneys, increasing the risk of cancer and osteoporosis and impairing the function of these organs. Cadmium can also cause neurotoxicity through oxidative stress and mitochondrial dysfunction, manifested as cognitive impairment and behavioral abnormalities, especially affecting children, pregnant women, and the elderly. Reducing cadmium exposure in the environment and taking measures such as nutritional intervention are key strategies for preventing and controlling cadmium toxicity.

[0003] Phytoremediation is the use of plant metabolic activities to reduce the content of heavy metals or their toxicity in the soil by absorbing, enriching, degrading or transforming heavy metals. Therefore, bioremediation technology has attracted increasing attention due to its advantages such as good remediation effect, small investment, low cost, easy management and operation, and no secondary pollution. It has become a research hotspot for the remediation of heavy metal contaminated soil.

[0004] In the prior art, researchers have found that duckweed can absorb some heavy metals, but because duckweed has a certain tolerance to cadmium stress, its ability to enrich cadmium metal is limited; therefore, it does not meet existing needs. In this regard, we propose a microbial agent and a method for using a microbial agent to repair cadmium-contaminated paddy soil. Summary of the invention

[0005] The purpose of the present invention is to provide a microbial agent and a method for repairing cadmium-contaminated paddy soil with a microbial agent, so as to solve the problem raised in the above background technology that duckweed has a certain tolerance to cadmium stress, resulting in limited ability of duckweed to enrich cadmium metal.

[0006] To achieve the above object, the present invention provides the following technical solution: a microorganism, comprising, by weight: 80 to 100 parts of Bacillus subtilis ATCC6633 and 40 to 50 parts of Pseudomonas aeruginosa ATCC27853.

[0007] Preferably, the mass ratio of Bacillus subtilis ATCC6633 to Pseudomonas aeruginosa ATCC27853 is 2:1.

[0008] Preferably, the method for culturing Bacillus subtilis ATCC6633 comprises the following steps:

[0009] Bacillus subtilis ATCC6633 was selected and placed in a sterilized growth medium for culture. The formula of the growth medium was: beef extract 4 g / L, yeast extract powder 9 g / L, sodium chloride 3 g / L, and the balance was deionized water;

[0010] Under the conditions of pH 7.0, temperature 36°C, and rotation speed 140 r / min, the number of Bacillus subtilis ATCC6633 reached 1.0×10 9 -10 10 cfu / Ml;

[0011] After the growth process is completed, the culture fluid of the strain is harvested and extracted, the bacteria and solid matter are separated by filtration or centrifugation, and the culture fluid is processed by concentration, purification and spray drying to obtain high-purity and high-activity Bacillus subtilis ATCC6633 powder products.

[0012] Preferably, the method for culturing Pseudomonas aeruginosa ATCC27853 comprises the following steps:

[0013] Pseudomonas aeruginosa ATCC27853 was selected and placed in a sterilized nutrient broth medium for culture. The formula of the nutrient broth medium was: 3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, and the balance was deionized water;

[0014] Under the culture conditions of pH 7.0, temperature 35°C, and rotation speed 200 r / min, the number of Pseudomonas aeruginosa ATCC27853 reached 1.0× 10 9 -10 10 cfu / M1;

[0015] After the growth process is completed, the culture fluid of the strain is harvested and extracted, the bacteria and solid matter are separated by filtration or centrifugation, and the culture fluid is processed by concentration, purification and spray drying to obtain a high-purity and high-activity Pseudomonas aeruginosa ATCC27853 product.

[0016] A method for repairing cadmium-contaminated paddy soil using a microbial agent comprises the following steps:

[0017] Step 1: Spread the microbial agent on the surface of the cadmium-contaminated paddy field soil, then mix the microbial agent with the paddy field soil, harrow the paddy field soil, adjust the water holding capacity of the paddy field soil, apply base fertilizer, and irrigate the paddy field;

[0018] Step 2: Transplant rice seedlings and duckweed, and flood the field until the rice is ripe.

[0019] Preferably, after adjusting the water holding capacity of the paddy field soil, basal fertilizer is applied after 7-15 days, and the paddy field is irrigated after 3-5 days of application of the basal fertilizer, and transplanting rice seedlings and placing duckweed after 3-5 days of irrigation.

[0020] Preferably, after the duckweed is placed in the paddy field, excess duckweed is removed every 7-15 days, and the remaining duckweed covers 50%-70% of the water surface.

[0021] Preferably, the inoculation amount of the duckweed is 30kg-50kg per mu of rice field, and the addition amount of the microbial agent is 1-5kg / mu.

[0022] Preferably, the duckweed includes one or more of Spirodendrum oligorrhiza, Spirodendrum chlororaphis, and Spirodendrum polyrrhiza.

[0023] Preferably, the duckweed consists of Spirodina paucirhiza, Spirodina affine and Spirodina polyrhiza, and the ratio of Spirodina paucirhiza, Spirodina affine and Spirodina polyrhiza is 1:1:1 by mass.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention combines two microorganisms, Bacillus subtilis and Pseudomonas aeruginosa, in a specific ratio. First, by adjusting the pH value of paddy field soil, the heavy metal cadmium is maintained in a stable state in the soil, and the migration and transformation of the heavy metal cadmium in the soil to the edible parts of rice are blocked, thereby helping to reduce the content of metal cadmium in rice; second, it can also produce plant growth regulators (plant hormones) and increase the secretion of plant heavy metal transport proteins (iron carrier proteins, etc.) to promote plant growth; third, it can promote the cadmium tolerance of duckweed, thereby promoting the cadmium enrichment ability of duckweed, and improving the duckweed's ability to repair cadmium-contaminated soil, which can not only reduce the cadmium content in the soil, but also reduce the content of metal cadmium in rice by competing for cadmium absorption;

[0026] 2. The method of the present invention not only achieves the increase in rice field yield and the restoration of soil cadmium pollution, but also the cadmium content of rice produced in rice fields meets national standards. By constructing a microorganism-high cadmium adsorption duckweed variety-rice symbiotic ecosystem, duckweed competitively adsorbs cadmium to reduce the absorption of cadmium by rice from the source, while continuously collecting duckweed and constantly "taking away" cadmium, gradually realizing the restoration of cadmium-contaminated soil. The final cadmium content of rice produced in rice fields is 0.021 mg / kg, which meets the national standard-GB2762-2022 "National Food Safety Standard Limits of Contaminants in Food". BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A data bar graph of cadmium content in duckweed in a test field set up in Example 1 of the present invention;

[0028] Figure 2 A bar graph showing the cadmium content in paddy soil of the test field set up in Example 1 of the present invention and the comparative example;

[0029] Figure 3 This is a bar chart comparing the effects of microbial agents on duckweed's tolerance to cadmium;

[0030] Figure 4 It is a bar chart comparing the cadmium content in the test field set up in Example 1 and the comparative example rice;

[0031] Figure 5 It is a bar graph comparing the cadmium content in the roots of rice in the test field set up in Example 1 and the comparative example. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0033] See also Figures 1 to 5 , an embodiment provided by the present invention: a microbial agent, calculated by weight, includes: Bacillus subtilis ATCC6633: 80-100 parts, Pseudomonas aeruginosa ATCC27853: 40-50 parts, and the mass ratio of Bacillus subtilis ATCC6633 to Pseudomonas aeruginosa ATCC27853 is 2:1.

[0034] The above-mentioned culture method of Bacillus subtilis ATCC6633 comprises the following steps:

[0035] Bacillus subtilis ATCC6633 was selected and placed in a sterilized growth medium for culture. The formula of the growth medium was: 4 g / L beef extract, 9 g / L yeast extract powder, 3 g / L sodium chloride, and the balance was deionized water;

[0036] Under the conditions of pH 7.0, temperature 36°C, and rotation speed 140 r / min, the number of Bacillus subtilis ATCC6633 reached 1.0× 10 9 -10 10 cfu / Ml;

[0037] After the growth process is completed, the culture fluid of the strain is harvested and extracted, the bacteria and solid matter are separated by filtration or centrifugation, and the culture fluid is processed by concentration, purification and spray drying to obtain high-purity and high-activity Bacillus subtilis ATCC6633 powder products.

[0038] The above-mentioned method for culturing Pseudomonas aeruginosa ATCC27853 comprises the following steps:

[0039] Pseudomonas aeruginosa ATCC27853 was selected and placed in a sterilized nutrient broth medium for culture. The formula of the nutrient broth medium was: 3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, and the balance was deionized water;

[0040] Under the culture conditions of pH 7.0, temperature 35°C, and rotation speed 200 r / min, the number of Pseudomonas aeruginosa ATCC27853 reached 1.0×10 9 -10 10 cfu / Ml;

[0041] After the growth process is completed, the culture fluid of the strain is harvested and extracted, the bacteria and solid matter are separated by filtration or centrifugation, and the culture fluid is processed by concentration, purification and spray drying to obtain a high-purity and high-activity Pseudomonas aeruginosa ATCC27853 product.

[0042] The method for repairing cadmium-contaminated paddy soil with the above-mentioned microbial agent comprises the following steps:

[0043] Step 1: Spread the microbial agent on the surface of the cadmium-contaminated paddy field soil, and then mix the microbial agent with the paddy field soil. The microbial agent is mixed with the paddy field soil by plowing or rotary tillage, raking the paddy field soil, adjusting the water holding capacity of the paddy field soil, and applying basal fertilizer after 7-15 days after adjusting the water holding capacity of the paddy field soil. After applying the basal fertilizer, wait for 3-5 days to irrigate the paddy field, wait for 3-5 days after irrigation to transplant rice seedlings and put in duckweed. After putting in the duckweed, remove the excess duckweed in the paddy field every 7-15 days, and the remaining duckweed covers 50%-70% of the water surface. The inoculation amount of duckweed is 30kg-50kg per mu of paddy field, and the addition amount of the microbial agent is 1-5kg / mu. Apply basal fertilizer and irrigate the paddy field.

[0044] Adjusting the water holding capacity of paddy field soil is to ensure the normal growth of microbial agents in paddy field soil. Therefore, the water holding capacity of paddy field soil can be adjusted according to actual conditions;

[0045] Flooding management is the process of flooding paddy field soil with water.

[0046] The application of the method of using microbial agents to repair cadmium-contaminated paddy soil includes application in repairing cadmium metal-contaminated soil.

[0047] Step 2: Transplant rice seedlings and duckweed, and flood the field until the rice is ripe.

[0048] The duckweeds mentioned above include one or more of Spirodendrum oligorrhiza, Spirodendrum affine and Spirodendrum polyrrhiza;

[0049] When the duckweed is composed of Spirodendrum sparserhizome, Spirodendrum affine and Spirodendrum multirhizome, the ratio of Spirodendrum sparserhizome, Spirodendrum affine and Spirodendrum multirhizome is 1:1:1 by mass.

[0050] The test field is set up in the above embodiment 1:

[0051] Use an agricultural four-wheel self-propelled sprayer or drone to evenly spread the microbial agent on the surface of the cadmium-contaminated paddy field soil, and then plow or rotary tillage with a plowing depth of 15-20cm. Fully mix the microbial agent with the paddy field soil, harrow the paddy field soil, and adjust the soil water holding capacity to ensure the growth of the composite bacteria in the soil; apply base fertilizer after 7-15 days, and irrigate the field 3-5 days after applying the base fertilizer. The amount of microbial agent added is 4kg / mu, and the microbial agents are Bacillus subtilis ATCC6633 and Pseudomonas aeruginosa ATCC27853; among them, the mass ratio of the bacterial powder of Bacillus subtilis ATCC6633 and the bacterial powder of Pseudomonas aeruginosa ATCC27853 is 2:1;

[0052] 3-5 days after applying the base fertilizer, transplant the rice seedlings and inoculate duckweed. During the growth of rice, the soil is managed by long-term flooding, and the flooding height is fixed between 5-15cm from the surface soil. The duckweed used is the few-rooted duckweed, green duckweed, and multi-rooted duckweed. The inoculation ratio is 1:1:1, and the inoculation amount is 30kg per mu. After inoculating duckweed, remove the excess duckweed in the rice field every 7-15 days, and the remaining duckweed continues to grow and reproduce. Repeat the previous steps to ensure that the duckweed covers 50%-70% of the water surface.

[0053] Apply base fertilizer and adjust the amount of fertilizer according to actual conditions.

[0054] Comparative Example 1 is set as a control field: the same as Example 1, but no microbial agent is added in Comparative Example 1, and the other methods are the same as Example 1.

[0055] The embodiment and comparative example were tested: the cadmium content in duckweed and paddy soil was tested: 0.2g to 0.5g of the sample was weighed into a microwave digestion inner tank, 5mL-10mL nitric acid was added, the cover was placed for 1h, the tank cover was tightened, and digestion was performed according to the standard operating procedures of the microwave digestion instrument (heating for 5min to 120℃ and keeping the temperature for 5min, heating for 5min to 150℃ and keeping the temperature for 10min, heating for 5min to 190℃ and keeping the temperature for 20min). After cooling, take it out, slowly open the tank cover to vent, rinse the inner cover with a small amount of water, place the digestion tank on a temperature-controlled electric hot plate or in an ultrasonic water bath, heat at 100℃ for 30min, dilute to 25mL with water, mix well for use, and do a blank test at the same time;

[0056] Content calculation: Calculate the cadmium content in the sample from the concentration obtained from the ICP-MS analysis, in units (mg / kg), taking into account the dilution factor and the initial sample weight or volume. (V: 25mL; f = 1) The cadmium content in the sample is low, and the calculation formula is as follows:

[0057]

[0058] Where:

[0059] X is the content of the element to be tested in the sample, in milligrams per kilogram or milligrams per liter (mg / kg);

[0060] ρ is the mass concentration of the element being measured in the sample solution, in micrograms per liter (kg / L);

[0061] ρo is the mass concentration of the element being measured in the sample blank solution, in micrograms per liter (μ g / L);

[0062] V is the fixed volume of the sample digestion solution, in milliliters (mL);

[0063] f-sample dilution multiple;

[0064] m is the initial sample weight or volume, in grams or milliliters (g or mL);

[0065] 1000-conversion factor; the calculation result shall be rounded to three significant figures.

[0066] Results and analysis:

[0067] Through the method of the present invention, firstly, the soil contaminated by cadmium can be repaired, secondly, the yield of rice can be increased, and thirdly, the cadmium content in rice can be reduced, and the planted rice meets the national standards.

[0068] In the present invention, the yield of the test field set in the above-mentioned embodiment 1 is 1049 jin / mu, while the yield of the comparative example 1 is 930 jin / mu. It can be seen that the method of the present invention can achieve the improvement of rice yield. The cadmium content of rice output from the paddy field in the embodiment 1 is 0.021 mg / kg, which meets the national standard (GB 2762-2022 "National Food Safety Standard Limits of Contaminants in Food" The cadmium content of rice shall not exceed 0.20 mg / kg"), while the cadmium content of rice in the paddy field of the comparative example 1 is 0.28 mg / kg, which exceeds the national limit standard;

[0069] like Figure 1 As shown, the cadmium content in duckweed in the rice field where the test field is set up in the above embodiment 1 is detected, and it is found that with the increase of time, duckweed can achieve the enrichment of cadmium;

[0070] like Figure 2As shown, the cadmium content in the paddy field soil of the test field set up in the above embodiment 1 and the comparative example 1 was detected, and it was found that the cadmium content in the paddy field soil of the embodiment 1 was significantly reduced;

[0071] like Figure 3 As shown, the duckweed in the test field set up in the first embodiment of the present invention was placed in cadmium-containing water for cadmium enrichment, wherein the mass concentration of cadmium was 5 mg / L, and the culture was carried out for 7 days, and then the test was carried out. Bacillus subtilis (i.e., Bacillus subtilis W16 in the present invention) and sulfate-reducing bacteria (Shanghai Ruichu Biotechnology Co., Ltd., strain number: SHMCC (SHBCC) D25136) were also used for comparison, and the results are as follows Figure 3 As shown, it can be found that when duckweed grows in cadmium-containing water, it is found that the chlorophyll content of duckweed treated with cadmium is significantly decreased compared to that of duckweed grown normally. However, after being treated with the microbial agent of the present invention, the chlorophyll content in duckweed increases, proving that the microbial agent of the present invention can improve the cadmium resistance of duckweed, reduce the impact of cadmium on the growth of duckweed, and is also beneficial to improving the cadmium enrichment capacity of duckweed. On this basis, it is found that the effects of different duckweeds treated with microbial agents are different, and the improvement effects of different combinations of microorganisms on duckweed are also different, and the microbial agent of the present invention has a better improvement effect on the duckweed of the present invention. It can be seen that under the action of the microbial agent of the present invention, the chlorophyll content in duckweed increases, proving that the microbial agent can improve the cadmium resistance of duckweed, thereby allowing duckweed to grow normally in a cadmium-containing solution;

[0072] like Figure 4 As shown, the cadmium content in the rice in the test field set in the above embodiment 1 is significantly lower than that in the comparative example 1, and the cadmium content of the rice in the test field meets the national standard, while the cadmium content of the rice in the comparative example exceeds the national standard as the growth cycle is extended. The rice harvested after maturity was tested, and the cadmium content of the rice produced by the paddy field in which the test field is set in the above embodiment 1 is 0.021 mg / kg, which meets the national standard (GB 2762-2022 "National Food Safety Standard Limits of Contaminants in Food" The cadmium content of rice shall not exceed 0.20 mg / kg"), while the cadmium content in the rice in the paddy field of the comparative example 1 is 0.28 mg / kg, which exceeds the national limit standard;

[0073] The cadmium content of rice roots in the experimental field set up in the above embodiment 1 and the comparative example 1 at different time periods was tested. The results are as follows: Figure 5 As shown, the cadmium content in the rice roots in the test field set up in the above Example 1 is significantly lower than that in the comparative example 1;

[0074] In summary, the method of the present invention can not only repair cadmium-contaminated soil, but also improve the cadmium tolerance of duckweed, thereby facilitating the enrichment of cadmium by duckweed; in addition, the cadmium content of rice in the present invention meets national standards and the yield is improved.

[0075] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A microbial agent, characterized in that: Calculated by weight, the invention comprises: 80 to 100 parts of Bacillus subtilis ATCC6633 and 40 to 50 parts of Pseudomonas aeruginosa ATCC27853.

2. A microbial agent according to claim 1, characterized in that: The mass ratio of Bacillus subtilis ATCC6633 to Pseudomonas aeruginosa ATCC27853 is 2:

1.

3. A microbial agent according to claim 2, characterized in that: The culture method of Bacillus subtilis ATCC6633 comprises the following steps: Bacillus subtilis ATCC6633 was selected and placed in a sterilized growth medium for culture. The formula of the growth medium was: beef extract 4 g / L, yeast extract powder 9 g / L, sodium chloride 3 g / L, and the balance was deionized water; Under the conditions of pH 7.0, temperature 36°C, and rotation speed 140 r / min, the number of Bacillus subtilis ATCC6633 reached 1.0×10 9 -10 10 cfu / Ml; After the growth process is completed, the culture fluid of the strain is harvested and extracted, the bacteria and solid matter are separated by filtration or centrifugation, and the culture fluid is processed by concentration, purification and spray drying to obtain high-purity and high-activity Bacillus subtilis ATCC6633 powder products.

4. A microbial agent according to claim 3, characterized in that: The culturing method of Pseudomonas aeruginosa ATCC27853 comprises the following steps: Pseudomonas aeruginosa ATCC27853 was selected and placed in a sterilized nutrient broth medium for culture. The formula of the nutrient broth medium was: 3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, and the balance was deionized water; Under the culture conditions of pH 7.0, temperature 35°C, and rotation speed 200 r / min, the number of Pseudomonas aeruginosa ATCC27853 reached 1.0×10 9 -10 10 cfu / M1; After the growth process is completed, the culture fluid of the strain is harvested and extracted, the bacteria and solid matter are separated by filtration or centrifugation, and the culture fluid is processed by concentration, purification and spray drying to obtain a high-purity and high-activity Pseudomonas aeruginosa ATCC27853 product.

5. A method for repairing cadmium-contaminated paddy soil using any one of the microbial agents of claims 1 to 4, characterized in that: The steps include: Step 1: Spread the microbial agent on the surface of the cadmium-contaminated paddy field soil, then mix the microbial agent with the paddy field soil, harrow the paddy field soil, adjust the water holding capacity of the paddy field soil, apply base fertilizer, and irrigate the paddy field; Step 2: Transplant rice seedlings and duckweed, and flood the field until the rice is ripe.

6. The method for repairing cadmium-contaminated paddy soil with a microbial agent according to claim 5, characterized in that: The microbial agent is mixed with the paddy field soil by plowing or rotary tillage. After adjusting the water holding capacity of the paddy field soil, basal fertilizer is applied after 7-15 days. After applying the basal fertilizer, the paddy field is irrigated after 3-5 days. After irrigation, rice seedlings are transplanted and duckweed is placed after 3-5 days.

7. The method for repairing cadmium-contaminated paddy soil with a microbial agent according to claim 6, characterized in that: After the duckweed is placed in the rice field, excess duckweed is removed every 7-15 days, and the remaining duckweed covers 50%-70% of the water surface.

8. The method for repairing cadmium-contaminated paddy soil with a microbial agent according to claim 7, characterized in that: The inoculation amount of the duckweed is 30kg-50kg per mu of rice field, and the addition amount of the microbial agent is 1-5kg / mu.

9. The method for repairing cadmium-contaminated paddy soil with a microbial agent according to claim 8, characterized in that: The duckweed includes one or more of Spirodendrum oligorrhiza, Spirodendrum affine and Spirodendrum polyrrhiza.

10. The method for repairing cadmium-contaminated paddy soil with a microbial agent according to claim 8, characterized in that: The duckweed consists of Spirodina paucirhiza, D. edulis and Spirodina multirhiza. Based on mass, the ratio of Spirodina paucirhiza, D. edulis and Spirodina multirhiza is 1:1:1.

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