TW202126588A - Chlorella sorokiniana, method of treating wastewater using the same and bioagent including the same - Google Patents

Abstract

The present invention relates to Chlorella sorokinian, a method of treating wastewater using the same and a bioagent including the same. After treating the wastewater using Chlorella sorokiniana strain AK-1, pollutant in the wastewater can be removed efficiently, and protein amount and/or lutein amount of Chlorella sorokiniana strain AK-1 increase. The obtained algae of Chlorella sorokiniana strain AK-1 can be used as an effective ingredient of the bioagent.

Description

Chlorella, its method for wastewater treatment and biological preparation containing it

The present invention relates to a kind of chlorella and its application, in particular to a kind of chlorella strain AK-1, a method for treating wastewater and biological preparations containing the same.

Human activities are accompanied by pollution, which not only destroys the environment, but also affects human health. For example, the high content of organic compounds, total nitrogen, and total phosphorus in wastewater from industry and animal husbandry can easily cause eutrophication of water bodies, causing microorganisms such as algae in the water bodies to multiply due to excessive nutrients. The rapidly increasing number of microorganisms affects the light transmittance of the water body, causing the death of plants and/or animals in the water. The corpses of dead animals and/or plants and the decomposition of the above-mentioned organic compounds consume a large amount of oxygen, which leads to a decrease in the oxygen content of the water body, and produces greenhouse gases and malodors.

Small (1 μm to 10 μm) single-celled algae, also known as microalgae, have the characteristics of large biomass, short growth cycle and/or high tolerance to pollutants. Secondly, the growth and/or reproduction of microalgae requires a large amount of carbon, nitrogen and/or phosphorus. Therefore, microalgae can be used to remove pollutants in wastewater and produce additional products, such as bio-oil and/or nutritional supplements. However, the tolerance of microalgae to pollutants and the conversion rate of biological products has a limit. For some wastewater (such as phosphorus content can be as high as 50 mg/L to 230 mg/L, and nitrogen content can be as high as 800 mg/L to 2300 The pollutant removal effect of mg/L pig farming wastewater is not good, and it is difficult to grow and/or reproduce in such an extreme environment.

Therefore, it is urgent to provide an algae and a method for treating wastewater to solve the above-mentioned problems.

Therefore, one aspect of the present invention provides a wastewater treatment method, which includes using a Chlorella sorokiniana strain AK-1 to remove pollutants in wastewater, which can be applied to wastewater treatment.

Another aspect of the present invention is to provide a biological preparation, which may contain the above-mentioned Chlorella sp. strain AK-1 as an active ingredient.

According to the above aspect of the present invention, a wastewater treatment method is proposed, which may include performing a first treatment step and at least one second treatment step. The first treatment step may include inoculating the Chlorella sp. strain AK-1 as an inoculation source into the diluted wastewater, and performing the first reaction step at 27° C. for 6 to 8 days to obtain the first treated water. The aforementioned Chlorella sp. strain AK-1 can be deposited at the Bioresource Center (BCRC), Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan, for example, on January 5, 2021, and the deposit number is BCRC. The above-mentioned diluted wastewater is to dilute 100 weight percent (wt%) of the wastewater stock solution with pure water to 30 wt% to 50 wt%. The above-mentioned inoculation amount is 0.2 g to 0.5 g of Chlorella sp. strain AK-1 per liter of diluted wastewater.

The above-mentioned second treatment step may include flowing out the discharged volume of the first treated water, then injecting the added volume of diluted wastewater, and performing the second reaction step at 27°C for 6 to 8 days to obtain the second treated water , Where the total volume of the first treated water is 100% by volume (vol%), the discharge volume can be 50 vol% to 90 vol%, and the added volume is the same as the discharge volume. Compared with the original wastewater solution, the total removal rate of COD of the obtained second treated water is at least 95%, the total removal rate of total nitrogen is at least 97%, and the total removal rate of total phosphorus is at least 90%.

According to an embodiment of the present invention, the COD content of the wastewater stock solution can be, for example, greater than 4000 mg/L, the total nitrogen content can be, for example, greater than 450 mg/L, and/or the total phosphorus content can be, for example, greater than 70 mg/L.

According to an embodiment of the present invention, the first treatment step and/or the second treatment step may optionally include adding a porous carrier to the diluted wastewater, wherein the porous carrier may include sponge and/or activated carbon, and the porous carrier carries The inoculation source of Chlorella sp. strain AK-1.

According to an embodiment of the present invention, based on the dilution wastewater being 100 wt%, the dry content of the sponge may be, for example, 0.2 wt%.

According to an embodiment of the present invention, based on the diluted wastewater being 100 wt%, the content of activated carbon may be 2.0 wt%, for example.

According to an embodiment of the present invention, wastewater treatment includes at least four second treatment steps.

According to an embodiment of the present invention, after performing the first treatment step and/or the second treatment step, it may optionally include a solid-liquid separation step on the first treatment water and/or the second treatment water to obtain Chlorella The algae of strain AK-1.

According to the above aspect of the present invention, a biological preparation is proposed, wherein the biological preparation can include the above-mentioned Chlorella sp. strain AK-1 as an active ingredient, and the deposit number of the Chlorella sp. strain AK-1 can be, for example, BCRC.

According to an embodiment of the present invention, the above-mentioned biological preparation may optionally include a sponge, wherein the dry seed ratio of the sponge to the inoculation source of the Chlorella sp. strain AK-1 is 1:2.

According to an embodiment of the present invention, the above-mentioned biological agent may optionally include at least one of Chlorella vulgaris, Scenedesmus, Bacillus, Pseudomonas, Enterobacter, nitrosating bacteria, and nitrifying bacteria.

The application of the Chlorella sp The body can also be used as an active ingredient in biological preparations.

Based on the above, the present invention provides a Chlorella sorokiniana strain AK-1. The use of chlorella strain AK-1 to treat wastewater can effectively increase the total removal rate of pollutants in the wastewater, and increase the biomass, protein and/or lutein content of the chlorella strain AK-1.

The above-mentioned chlorella is also called green algae, which is a common single-celled algae in the Chlorella genus Chlorella. Different Chlorella algae strains have different adaptability to the environment. Some Chlorella algae strains have better adaptability to the above-mentioned wastewater. Not only can they effectively remove COD, total nitrogen and total phosphorus in wastewater, but also It can grow rapidly in wastewater treatment and synthesize more protein and/or lutein content. The above-mentioned Chlorella sp. strain AK-1 can be deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, Hsinchu, Taiwan, for example, on January 5, 2021, and the deposit number is BCRC.

The above-mentioned wastewater is, for example, industrial wastewater and/or livestock wastewater, and livestock wastewater may come from livestock farms, such as pig farms, cattle farms, sheep farms, horse farms, and/or poultry farms. The aforementioned pollutants may include, but are not limited to, chemical oxygen demand (COD), total nitrogen, and total phosphorus. The aforementioned total nitrogen is a general term for nitrogen-containing compounds such as nitrides, nitrates, and nitrites. The aforementioned total phosphorus is the general term for phosphorus-containing compounds such as phosphide, phosphate, and phosphite. The aforementioned wastewater can generally be defined by COD greater than 4000 mg/L, total nitrogen content greater than 450 mg/L, and/or total phosphorus content greater than 70 mg/L. The above-mentioned total removal rate refers to the difference between the pollutants in the wastewater before treatment and the discharged water after wastewater treatment divided by the percentage of the pollutants in the wastewater before treatment.

The above-mentioned method for treating wastewater may include, but is not limited to, performing a first treatment step and at least one second treatment step, wherein the first treatment step includes inoculating the chlorella strain AK-1 as an inoculation source into the diluted wastewater, and The first reaction step is performed at 20°C to 30°C for 6 to 8 days, and the first reaction step is preferably performed at 27°C for 6 to 8 days to obtain the first treated water. For the above-mentioned diluted wastewater, for example, 100 weight percent (wt%) of the wastewater stock solution can be diluted with pure water to 30 wt% to 50 wt%, so that the COD content in the diluted wastewater is less than 3000 mg/L. The above-mentioned inoculation amount can be, for example, 0.2 g to 0.5 g of Chlorella sp. strain AK-1 per liter of diluted wastewater. It is worth noting that if the concentration of the diluted wastewater is greater than 50 wt%, the turbidity in the diluted wastewater is too high and/or there are too many microorganisms competing for resources (such as space, nutrients and/or oxygen), which will affect the pellets. The photosynthesis of the algae strain AK-1 caused the growth of the Chlorella algae strain AK-1 to be inhibited. If the concentration of the diluted wastewater is less than 30 wt%, the removal rate of pollutants by Chlorella sp. strain AK-1 is poor. Specific examples of the above-mentioned pure water include distilled water and/or secondary water. If the inoculation amount of Chlorella sp. strain AK-1 is too small, the amount of algae in the first reaction step is insufficient. If the inoculation amount of Chlorella sp. strain AK-1 is too much, the removal effect of pollutants will not be significantly improved.

The first reaction step can be carried out under conventional conditions. Specifically, the rotation speed of the agitation can be 150 to 300 rpm (revolution per minute, rpm), and the ventilation conditions can be 0.1 vvm ventilation with 2 volume percent (vol%) CO ₂ , and light conditions It can be 100 μ ^-2 s ^-1 to 200 μ ^-2 s ^-1 .

The above-mentioned second treatment step may include 100 vol% based on the total volume of the first treated water, after flowing out the discharged volume of the first treated water, then injecting the added volume of diluted wastewater, and performing it at 20°C to 30°C The second reaction step is up to 6 days to 8 days, preferably the first reaction step is performed at 27°C for 6 days to 8 days to obtain the second treated water, where the discharge volume can be, for example, 50 vol% to 90 vol% , And the added volume is the same as the discharged volume. The first reaction step and the second reaction step can be carried out under the same conventional conditions. It should be noted that if the discharge volume is less than 50 vol%, the efficiency of removing biological oxygen demand is low. If the discharge volume is greater than 90 vol%, more algae of Chlorella sp. AK-1 will be easily lost, resulting in the amount of algae of Chlorella sp. AK-1 used as the inoculation source in the second treatment step. insufficient.

The waste water treatment method of the present invention can selectively perform a solid-liquid separation step on the raw waste water, the first treated water and/or the second treated water, wherein the solid-liquid separation step can be performed by a conventional solid-liquid separation method, such as filtration , Natural precipitation and/or centrifugation. The solid-liquid separation of the raw wastewater can remove solids (such as feces, sediment and/or other microorganisms) to prevent the solids from reducing the light transmittance of the wastewater. The solid-liquid separation step is performed on the first treated water and/or the second treated water to obtain algal bodies of the Chlorella sp. strain AK-1. Compared with the inoculation source of Chlorella sp. strain AK-1, the biomass, protein and/or lutein per unit weight of the algae body of Chlorella sp. strain AK-1 increased. The algal body of the Chlorella sp. strain AK-1 can be used as the inoculation source for the second reaction step. In one embodiment, the algae obtained by the solid-liquid separation step of the first treatment water can be used as the inoculation source for the second treatment step.

In one embodiment, the first treatment step and/or the second treatment step may optionally include adding a porous carrier to the diluted wastewater, wherein the porous carrier may include sponge and/or activated carbon, and the porous carrier may carry a small amount of water. The source of inoculation of S. vulgaris strain AK-1. In one embodiment, based on 100 wt% of the diluted wastewater, the dry content of the sponge (the ratio of the dry weight to the weight of the diluted wastewater) may be 0.1 wt% to 0.3 wt% of the sponge, preferably 0.2 wt%. The material and shape of the sponge are not limited. In a specific example, the material of the sponge can be, for example, a natural material or a synthetic material such as Polyurethane, and the shape of the sponge can be, for example, a three-dimensional structure with a side length of 1 cm to 3 cm. . In one embodiment, every 100 g of diluted wastewater may contain, for example, 1.0 g to 2.0 g of activated carbon. The shape of the activated carbon is not limited. In a specific example, the shape of the activated carbon may be, for example, a cylinder with a diameter and a length of 3 mm to 4 mm and 0.5 cm to 1.5 cm, respectively.

Experiments have proved that the above-mentioned wastewater treatment method can effectively remove pollutants in the raw wastewater. Compared with the raw wastewater, the total removal rate of various pollutants in the second treated water (that is, the difference between the pollutant content of the raw wastewater and the pollutant content of the second treated water divided by the percentage of the pollutant content of the raw wastewater) Listed as follows: the total removal rate of COD of the second treated water is at least 95%, the total removal rate of total nitrogen is at least 97%, and the total removal rate of total phosphorus is at least 90%.

In addition, in the first reaction step, the biomass of Chlorella sp. strain AK-1 can increase by 0.52 g/L per day, and the yield of protein and lutein can reach 0.28 g/L and 3.10 mg/L per day. . The above-mentioned protein is one of the three major nutrients of calories. It not only provides calories, but also builds new tissues, repairs existing tissues, maintains acid-base balance and/or water balance in the body, assists in nutrient transport, and constitutes enzymes, hormones and / Or antibodies, etc. The above-mentioned lutein is a kind of carotenoid, which can help animal growth, reproduction and/or pigment formation, and increase the color of animal skin, meat and/or eggs.

It can be seen from the above that the use of Chlorella sp After the conventional process treatment (such as cleaning, solid-liquid separation, etc.), it can be used as a biological agent. The biological agent can, for example, use Chlorella sp. strain AK-1 as an active ingredient to remove COD and total nitrogen in wastewater. And/or total phosphorus. In one embodiment, the biological preparation may optionally include sponge, wherein the dry weight ratio of the inoculation source of the sponge and the Chlorella sp. strain AK-1 is not particularly limited, and the conventional usage amount can be referred to, but in some specific examples In, the dry weight ratio of the sponge to the Chlorella sp. strain AK-1 can be, for example, 1:2. In one embodiment, the biological agent may optionally include but is not limited to other microorganisms, such as: Chlorella vulgaris , Scenedesmus spp., Bacillus spp., Pseudomonas spp. ( Pseudomonas spp.), Enterobacter spp., Nitrosomonas spp., and Nitrobacter spp.

Several embodiments are used below to illustrate the application of the present invention, but they are not used to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various modifications and changes without departing from the spirit and scope of the present invention. Retouch. Example 1. Microbiological properties of algae strain AK-1

Chlorella sorokiniana (Chlorella sorokiniana) algae strain AK-1 (hereinafter referred to as algae strain AK-1) was isolated from a sample taken from a pond area in southern Taiwan. Under an optical microscope, the algal strain AK-1 cells are round or oval, with a diameter of 3 μm to 10 μm, but the diameter can reach 23 μm during reproduction, and belongs to a monocytic green algae. In addition, the cells of the algae strain AK-1 have a large grass-green pigment body, which is cup-shaped, flake-shaped or perennial. The photosynthetic pigments of the algae strain AK-1 include chlorophyll a, chlorophyll b, lutein and carotene.

Purify the DNA of the algae strain AK-1, and then use the PCR upstream primers and downstream primers of 23S rDNA as shown in SEQ ID NOs: 1 and 2 to perform polymerase chain reaction (PCR) To obtain the 23S rDNA of the algal strain AK-1 (as shown in SEQ ID NO. 3). The above-mentioned DNA purification and PCR are well known to those with ordinary knowledge in the technical field to which the present invention belongs, and will not be repeated here. The 23S rDNA sequence of the algal strain AK-1 was aligned using the Basic Local Alignment Search Tool (BLAST) to confirm that the algal strain AK-1 was Chlorella. The above-mentioned algae strain AK-1 has been deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan on January 5, 2021, and the deposit number is BCRC.

It is added that the Chlorella sp. strain MS-C1 and Chlorella sp. strain TJ5 (hereinafter referred to as the algae strain MS-C1 and the algae strain TJ5 respectively) were separately isolated from the same batch of pond water samples. The algae strain MS has been confirmed -C1 and the algae strain TJ5 and the algae strain AK-1 are different algae strains of the same species (Chlorella). Example 2: Sewage treatment method and screening of algae strain AK-1

In the photobioreactor (PBR), using the algae strain AK-1, the algae strain MS-C1, and the algae strain TJ5 as the inoculation source, the diluted wastewater is subjected to the first reaction step for 15 days to obtain the first treated water , Where the diluted wastewater is obtained by filtering and diluting the raw liquid of the above-mentioned livestock wastewater, and the composition of the livestock wastewater is as shown in Table 1 [Standard error of the mean (SEM)], and The pH value of livestock wastewater is between 7 and 8.

Table 1 Element Content (mg/L) Element Content (mg/L) Cod 5200±900 In 0.0035±0.0005 Total nitrogen (TN) 510±10 B 0.342±0.074 Total Phosphorus (TP) 76.1±5.0 Mn 0.183±0.112 NH ⁴⁺ -N 450±15 Fe 0.728±0.542 NO ^3- -N 1.19±0.24 Cr 0.01±0.001 PO ₄ ^2- 36.7±7.3 Ga 0.009±0.001 Cl ^- 154.7±9.5 Cu 0.0015±0.0005 SO ₄ ^2- 143.5±29.8 Ag 0.001±0.001 K 229.9±55.7 Al 0.2095±0.0585 Na 66.24±9.60 Sr 0.3625±0.1995 Ca 63.88±36.67 Ba 0.003±0.002 Mg 37.49±20.26 Ti 0.155±0.026 Bi 0.0075±0.0005 Zn 0.087±0.035 Co 0.0175±0.0045 Pb 0.0425±0.0015 Cd 0.003±0.001 Ni 0.2805±0.0745

The above photobioreactor can provide constant temperature (27°C), light [intensity of 150 μmol m ^-2 s ^-1 (TL5 fluorescent tube 14W; supplier: Philips of the Netherlands)], air flow [contains 2 vol% of CO ₂ , where the aeration rate is 0.1 photobioreactor volume/inlet gas volume/minute (vvm)] and agitation (300 rpm).

Perform detection steps on the diluted wastewater and the first treated water respectively. First, random samples were taken from the diluted wastewater and the first treated water to obtain a water sample, and the water sample was centrifuged at 6000 rpm for 5 minutes to obtain a supernatant and a sediment, where the sediment contains the algae of the algae strain. Use the AL450 photometer of German Aqualytic to detect COD, total nitrogen (TN) and total phosphorus (TP) content in the supernatant, and calculate the migration of pollutants by different algae strains. The removal rate, where the removal rate is the percentage of the difference between the COD, total nitrogen, or total phosphorus content of the first treated water and the diluted wastewater sample to the COD, total nitrogen, or total phosphorus content of the diluted wastewater, respectively. The above COD, total nitrogen and total phosphorus detection methods are based on the AL450 photometer detection instructions, and will not be repeated here.

Dry the sediment at a temperature of 120°C until the weight of the sediment no longer changes to measure the dry weight of the sediment and calculate the ratio of the weight of the sediment to the volume of the water sample to obtain the biomass concentration of the algae strain (Unit: g/L). Next, calculate the difference between the biomass concentration of the first treated water (end of the first reaction step) and the biomass concentration of the diluted wastewater (the beginning of the first reaction step) divided by the time of the first reaction step to obtain the biomass Yield (unit: mg/L/day).

The precipitate was lyophilized to obtain a lyophilized product. Then, the conventional detection method was used to quantify the weight of amino acid and lutein in the lyophilized substance to calculate the content and yield of protein and lutein. The quantitative method was published in Biotechnology Journal by Dr. Chen Junyan’s team in 2015. The papers on pages 905 to 914 of Volume 10, Issue 6, and the methods published on pages 1 to 7 of the Journal of Bioscience and Bioengineering in 2018, will not be repeated here. The protein content and the lutein content are the percentage of the total weight of amino acids divided by the weight of the freeze-dried powder (unit: %) and the weight ratio of lutein divided by the biomass (unit: mg/g). The yield of protein and lutein is the product of protein content and lutein content and biomass yield (unit: mg/L/day).

The stock solution of livestock wastewater is diluted with the conventional blue-green algae culture medium BG-11 to obtain the first dilution solution, wherein the concentration based on the stock solution is 100 wt%, and the concentration of the first dilution solution is 10 wt%. The BG-11 medium containing per liter _{1.5 g NaNO 3, 0.04 g K} 2 HPO 4, 0.075 g MgSO 4 · 7H 2 O, 0.006 g citric _{acid, 0.02 g Na 2 CO 3,} 0.036 g CaCl 2, 0.001 g EDTA ·2Na, 2.8 × 10 ^-3 g H ₃ BO ₃ , 2.2 × 10 ^-4 g ZnSO ₄ ·7H ₂ O, 1.8 × 10 ^-3 g MnCl ₂ ·4H ₂ O, 7.9 × 10 ⁻⁵ g CuSO ₄ ·5H ₂ O and a balanced amount of water. Next, the algae strain AK-1, the algae strain MS-C1, and the algae strain TJ5 (0.4 g of the algae strain per liter of the first dilution were inoculated) were respectively inoculated into the first dilution solution to perform the above-mentioned first reaction step to obtain The first treatment water. Perform detection steps on the first diluent (initial) and the first treated water (end) respectively, and calculate the removal rate of pollutants in the first treated water relative to the first diluent. Record the results in Table 2 and Figures 1A to 1C.

Table 2 Algae Strain AK-1 MS-C1 TJ5 COD Initial content (mg/L) 892±47 End content (mg/L) 100±13 265±21 192±15 Removal rate (%) 88.8±0.9 70.3±1.4 78.5±0.6 Total nitrogen Initial content (mg/L) 308±36 End content (mg/L) 67±12 84±10 84±12 Removal rate (%) 78.3±1.4 72.7±0.1 72.7±0.7 Total phosphorus Initial content (mg/L) 8.5±2.5 End concentrated content (mg/L) <0.2 <0.2 <0.2 Removal rate (%) ＞97.7 ＞97.7 ＞97.7

As shown in Table 2, relative to the first dilution, the removal rate of the algal strain AK-1 on COD, total nitrogen and total phosphorus was higher than that of the algal strain MS-C1 and the algal strain TJ5 on COD, total nitrogen and total phosphorus. Removal rate.

Figures 1A to 1C are bar graphs showing the biomass concentration (Figure 1A), protein content (Figure 1B), lutein content (Figure 1C) and yields of different algae strains according to an embodiment of the present invention . The horizontal axis of FIG. 1A represents algae strains, the left vertical axis and the bar 111 represent the biomass concentration, and the right vertical axis and the bar 113 represent the biomass yield. The horizontal axis of FIG. 1B represents the algae strain, the left vertical axis and the bar 121 represent the protein content, and the right vertical axis and the bar 123 represent the protein yield. The horizontal axis of FIG. 1C represents the algae strain, the left vertical axis and the straight bar 131 represent the lutein content, and the right vertical axis and the straight bar 133 represent the lutein yield.

As shown in Figure 1A to Figure 1C, using student's t-test for statistics, the biomass concentration (4.70±0.20 g/L) and biomass yield (0.29±0.01 g/L/day) of the algae strain AK-1 are significant Higher than the algal strain MS-C1 (3.90±0.22 g/L and 0.24±0.02 g/L/day respectively) and the algal strain TJ5 (respectively 4.18±0.22 g/L and 0.26±0.02 g/L/day) ( n=3, P<0.05). Secondly, compared with the algal strain MS-C1 and the algal strain TJ5, the algal strain AK-1 has a higher protein yield and lutein yield.

It can be seen from the above results that the algal strain AK-1 has a better metabolic effect on the pollutants of livestock wastewater, and the biomass, protein and lutein yields in the livestock wastewater are higher, indicating that the algal strain AK-1 has a higher Good adaptability to livestock wastewater. Example 3: Assess the influence of the concentration of the diluted wastewater on the metabolic pollutants of the algal strain AK-1

Dilute the stock solution of livestock wastewater with distilled water to obtain a second dilution solution, wherein the concentration of the stock solution based on livestock wastewater is 100 wt%, and the concentration of the second dilution solution is 10 wt%, 30 wt%, 50 wt%, 70 wt, respectively % And 90 wt%. Then, use the inoculation source of the algae strain AK-1 containing 0.4 g per liter of the second dilution as the inoculum for inoculation, and then perform the above-mentioned first reaction step for 15 days to obtain the first treated water. Perform the above detection steps on the second diluent (initial) and the first treated water (finished), and record the results in Table 3 and Figures 2A to 2C.

table 3 Concentration of stock solution of livestock wastewater (wt%) 10 30 50 70 90 COD Initial content (mg/L) 624±208 1120±90 3625±595 1985±245 2915±95 End content (mg/L) 112±12 226±11 536±16 313±58 562±138 Removal rate (%) 83.7±7.3 79.9±0.7 85.2±2.0 84.3±4.9 80.7±4.1 Total nitrogen Initial content (mg/L) 281±26 380±40 658±52 467±23 629±41 End content (mg/L) 66±2 55±11 122±88 46±9 108±74 Removal rate (%) 76.7±1.6 85.4±4.4 81.5±12.0 90.1±1.4 82.9±10.6 Total nitrogen Initial content (mg/L) 9.5±1.0 30.8±3.8 93.0±5.0 45.0±3.5 68.5±2.5 End content (mg/L) 0.6±0.4 1.0±0.4 4.5±3.0 <0.2 2.1±0.5 Removal rate (%) 94.2±3.2 96.8±1.7 95.2±3.5 ＞99.6 97.0±0.6

As shown in Table 3, when the concentration of the second diluent is 30 wt% to 90 wt%, the removal rate of COD of the first treated water can be, for example, 80% to 85%, and the removal rate of total nitrogen can be, for example, 80% to 90%, and the removal rate of total phosphorus may be, for example, more than 95%. It is worth noting that the COD content of the second diluent with a concentration of 90 wt% is 3600 mg/L to 5400 mg/L, but under this condition, the algal strain AK-1 has a removal rate of COD and total nitrogen It can reach 80%, and the removal rate of total phosphorus can reach more than 95%, indicating that the algal strain AK-1 has good adaptability to livestock wastewater, and the high COD content does not affect the efficiency of the algal strain AK-1 in removing pollutants.

Figures 2A to 2C illustrate the biomass concentration (Figure 2A), protein content (Figure 2B), and lutein content (Figure 2C) and yields of different second dilution solutions according to an embodiment of the present invention Bar graph. The horizontal axis of FIG. 2A represents the concentration of livestock wastewater, the left vertical axis and the bar 211 represent the biomass concentration, and the right vertical axis and the bar 211 represent the biomass yield. The horizontal axis of FIG. 2B represents the concentration of livestock wastewater, the left vertical axis and the bar 221 represent the protein content, and the right vertical axis and the bar 223 represent the protein yield. The horizontal axis of FIG. 2C represents the concentration of livestock wastewater, the left vertical axis and the bar 231 represent the lutein content, and the right vertical axis and the bar 233 represent the lutein yield.

As shown in Figures 2A to 2C, compared with the second dilution of 70 wt% to 90 wt%, in the second dilution of 10 wt% to 50 wt%, the organism of the algae strain AK-1 The yield, protein and lutein are higher. Preferably, in the second dilution of 50 wt%, the biomass content and yield of the algae are 5.45±0.55 g/L and 0.36±0.04 g/L/day, respectively, and the protein content and yield are respectively They were 56.08±1.69% and 0.27±0.01 g/L/day, and the content and yield of lutein were 6.41±0.48 mg/g and 2.20±0.39 mg/L/day, respectively. This may be because the second dilution with a high COD concentration has a lower light transmittance, thus reducing the photosynthesis efficiency of the algal strain AK-1, thereby affecting the growth of the algae.

The above results show that the algal strain AK-1 has a high removal rate of pollutants in the second dilution of 30 wt% to 90 wt%, but the algal body of the algal strain AK-1 is in the 10 wt% to 50 wt% The yield of biomass in the second diluent is relatively high, indicating that it is better to perform the first reaction step with the second diluent of 30 wt% to 50 wt%, and the second diluent of 50 wt% is better for the first reaction step. good. Example 4: Evaluate the concentration of BG-11 culture solution

A third dilution was prepared using the stock solution of livestock wastewater, BG-11 culture solution and distilled water. The concentration of livestock wastewater of the third dilution solution was 50 wt%, and the concentration of BG-11 culture solution was 0 wt% and 25 wt%, respectively. , 50 wt% and 100 wt%. Then, the algal strain AK-1 is used to perform the first reaction step on the third dilution solution to obtain the first treated water. Perform detection steps on the third diluent (initial) and the first treated water (finished), and record the results in Table 4 and Figures 3A to 3C.

Table 4 BG-11 culture solution concentration (wt%) 0 25 50 100 COD Initial content (mg/L) 2345±42 2285±57 2445±35 2095±355 End content (mg/L) 361±61 475±37 345±12 345±25 Removal rate (%) 84.6±2.9 79.2±1.1 85.9±0.3 83.5±4.1 Total nitrogen Initial content (mg/L) 180±25 240±20 268±26 497±53 End content (mg/L) ＜10 12±2 19±13 49±11 Removal rate (%) ＞94.4 95.0±0.4 92.9±4.2 90.2±1.3 Total phosphorus Initial content (mg/L) 37.5±1.3 39.0±0.9 41.5±1.3 50.0±8.5 End content (mg/L) 0.2±0.0 0.3±0.1 0.3±0.1 0.3±0.1 Removal rate (%) 99.5±0.0 99.2±0.3 99.3±0.2 99.4±0.1

As shown in Table 4, when the third dilution does not contain the third dilution of the BG-11 culture solution (ie, the second dilution with a concentration of 50 wt%), the removal rate of COD and total phosphorus is higher.

Figures 3A to 3C show the biomass concentration (Figure 3A), protein content (Figure 3B) and lutein content (Figure 3C) and yields of different BG-11 culture solution concentrations according to an embodiment of the present invention Histogram. The horizontal axis of FIG. 3A represents the concentration of the BG-11 culture solution, the left vertical axis and the bar 311 represent the biomass concentration, and the right vertical axis and the bar 313 represent the biomass yield. The horizontal axis of FIG. 3B represents the concentration of the BG-11 culture solution, the left vertical axis and the bar 321 represent the protein content, and the right vertical axis and the bar 323 represent the protein yield. The horizontal axis of FIG. 3C represents the concentration of the BG-11 culture solution, the left vertical axis and the bar 331 represent the lutein content, and the right vertical axis and the bar 333 represent the lutein yield.

As shown in FIGS. 3A to 3C, when the concentration of the BG-11 culture solution in the third dilution water is 25 wt%, the algal strain AK-1 has a better biomass concentration and yield. However, compared with the concentration of the BG-11 culture solution in the third dilution water of 50 wt% and 100 wt%, the algal strain AK-1 in the third dilution water without BG-11 culture solution (equivalent to 50 wt% The second dilution water), biomass concentration and yield (6.52±0.28 g/L and 0.41±0.02 g/L/day, respectively) are higher. The concentration of the BG-11 culture solution has little effect on the protein and lutein concentration and yield of the algae strain AK-1, but the algae strain AK-1 in the diluted wastewater without the BG-11 culture solution has better lutein Yield.

In summary, considering the removal rate of pollutants and the yield of algae biomass, protein and/or lutein, the concentration of algal strain AK-1 on the broth without BG-11 and the stock solution of livestock wastewater It is better to perform the first reaction step with the 50 wt% third diluent (that is, the 50 wt% second diluent). Example 5 Carriers for evaluating algae

The carrier was added to the second dilution of 50 wt%, and the first reaction step was performed to obtain the first treated water, wherein the agitation rate of the first reaction step was 150 rpm. The carrier includes activated carbon and sponge made of porous materials [material: polyurethane (polyurethane)], and sodium alginate crystal balls and clay blocks made of non-porous materials. The above activated carbon is a cube with a diameter and a length of 4 mm and 1 cm, respectively, the sponge is a cube with a side length of 2 cm, and the sodium alginate crystal ball and clay block are spheres with a diameter of 1 cm. Based on 100 wt% of diluted wastewater, the dry content of activated carbon, clay block and sodium alginate crystal ball is 2.0 g/100 mL, and the dry content of sponge is 0.2 wt%. Then, the 50 wt% second dilution liquid (initial) and the first treated water (end) were tested, and the results were recorded in Table 5 and FIGS. 4A to 4C.

table 5 Carrier Control group Activated carbon sponge Sodium Alginate Crystal Ball Clay block COD Initial content (mg/L) 2108±197 End content (mg/L) 335±93 235±40 177±20 542±35 338±15 Removal rate (%) 84.1±4.0 88.9±1.7 91.6±0.2 74.3±2.0 84.0±0.2 Total nitrogen Initial content (mg/L) 219±23 End content (mg/L) 25±20 15±3 13±3 18±2 24±6 Removal rate (%) 88.6±10.0 93.1±2.0 94.1±1.1 92.0±1.3 89.0±2.3 Total phosphorus Initial content (mg/L) 39.8±4.3 End content (mg/L) 0.3±0.1 <0.2 <0.2 <0.2 <0.2 Removal rate (%) 99.3±0.3 ＞99.5 ＞99.5 ＞99.5 ＞99.5

As shown in Table 5, compared to the control group (without carrier), the removal rate of COD and total nitrogen by the algae strain AK-1 increased compared to the second dilution with activated carbon and/or sponge as the carrier. Liquid, the removal rate of total phosphorus in the first treated water is greater than 99%. It can be seen that using sponge or activated carbon as a carrier can improve the removal rate of pollutants by the algae strain AK-1, but the sponge as a carrier for the algae strain has a better effect on removing pollutants.

4A to 4C are bar graphs showing the biomass concentration (FIG. 4A ), protein content (FIG. 4B ), lutein content (FIG. 4C) and yield of different carriers according to an embodiment of the present invention. The horizontal axis of FIG. 4A represents the carrier, the left vertical axis and the bar 411 represent the biomass concentration, and the right vertical axis and the bar 413 represent the biomass yield. The horizontal axis of FIG. 4B represents the vector, the left vertical axis and the bar 421 represent the protein content, and the right vertical axis and the bar 423 represent the protein yield. The horizontal axis of FIG. 4C represents the carrier, the left vertical and axis bars 431 represent the lutein content, and the straight bars 433 and the right vertical axis represent the lutein yield.

As shown in Figures 4A to 4C, compared with the control group, the biomass, protein and lutein yields increased with activated carbon and sponge as the carrier. Among them, the biomass of the algae strain AK-1 was increased with sponge as the carrier. The concentration can reach 8.08±0.39 g/L, the biomass yield can reach 0.52±0.03 g/L/day, the protein yield can reach 0.28±0.02 g/L/day, and the lutein yield can reach 3.10±0.75 mg/L/day. However, with sodium alginate crystal balls and clay blocks as carriers, the yield of biological products decreases instead. It can be seen that using porous materials such as sponge or activated carbon as a carrier can increase the removal rate of pollutants by the algal strain AK-1 and the yield of biological products. Example 6 Evaluation of the conditions of the second reaction step

Using the semi-batch method to carry out the second reaction step can continuously treat livestock wastewater. In detail, first use the algae strain AK-1 to perform the first reaction step with 50 wt% of the second dilution water for 6 to 8 days to obtain the first treated water, and then 100 vol% based on the weight of the first treated water, The first treated water that flows out of the discharge volume. Then, inject the volume of the second dilution water, and perform the second reaction step at 27°C for 6 to 8 days to obtain the second treated water, where the volume of the second dilution water is added and the first treated water is discharged The volume is the same.

After adding diluted wastewater and after the second reaction step, samples were taken separately, and then the COD, biological oxygen demand (BOD), total phosphorus and total nitrogen content of the water samples before and after the second reaction step were measured, and the second reaction step was calculated. The removal rate of pollutants in the reaction step can be used to evaluate the range of the discharged volume. The BOD measurement method is not limited and can be performed in a conventional method. Specifically, for example, it is carried out according to the number NIEA W510.55B announced by the Environmental Inspection Institute of the Environmental Protection Agency of the Executive Yuan of Taiwan. The measurement results show that when the discharge volume is 50 vol%, the COD removal rate of the second treated water is 83.9±3.2%, the BOD removal rate is 96.1±1.1%, and the total nitrogen The removal rate was 68.3±7.7%, and the removal rate of total phosphorus was 89.0±7.6%. When the discharge volume is 70 vol%, the COD removal rate of the second treated water is 86.1±3.1%, the BOD removal rate is 94.4%±2.3, and the total nitrogen removal rate is 86.1±3.1% compared to the diluted wastewater 79.8±8.7%, and the removal rate of total phosphorus is 93.6±4.3%. When the discharge volume is 90 vol%, the COD removal rate of the second treated water is 88.2±1.2%, the BOD removal rate is 94.5±2.1%, and the total nitrogen removal rate is 88.2±1.2% compared to the diluted wastewater. It is 84.3±4.2%, and the removal rate of total phosphorus is 89.5±5.5%. According to the results of BOD, the discharge volume of the first treated water is selected as 50 vol% for the second reaction step.

It is worth noting that the first reaction step and the second reaction step include the use of sponge as a carrier, and filtration is required before the first treated water or the second treated water flows out to prevent the sponge and the algae strain AK-1 from being lost. The 50 wt% of the second dilution water (initial) and the second treated water (finished) were tested respectively, and the results were recorded in Table 6.

Table 6 Days (days) 4 6 8 COD Initial content (mg/L) 2348±89 2132±243 2115±186 End content (mg/L) 261±13 208±27 186±24 Removal rate (%) 88.9±0.6 90.1±1.9 91.1±1.5 Total nitrogen Initial content (mg/L) 266±34 259±36 258±20 End content (mg/L) 15±7 7±4 8±3 Removal rate (%) 93.9±3.2 97.0±2.1 96.9±1.1 Total phosphorus Initial content (mg/L) 61.0±5.4 58.0±5.8 65.0±7.3 End content (mg/L) 13.8±2.6 4.4±3.6 <0.2 Removal rate (%) 77.6±2.8 92.8±5.5 ＞99.7

As shown in Table 6, when the number of days is 8 days, the removal rate of COD and total phosphorus is the highest, and when the number of days is 6 days, the removal rate of total nitrogen is the highest. It can be seen that when the number of days is 6 to 8 days, the algal strain AK-1 has a better removal rate of pollutants.

After the first reaction step was performed for 6 days, the second reaction step was performed for another 6 days up to 4 times. Add 50 wt% of the second dilution liquid (the beginning of the first reaction step), the first treated water (the end of the first reaction step), the first treated water or the second treated water, and then add 50 wt% of the second dilution The liquid mixture (the beginning of the second reaction step) and the second treated water (the end of the second reaction step) are subjected to the detection step, and the results are recorded in Table 7, Figs. 5A to 5C.

Table 7 The first reaction step Second reaction step 1 2 3 4 COD Initial content (mg/L) 2460±123 2500±150 2314±93 1942±97 1846±55 End content (mg/L) 269±81 164±7 212±8 119±6 110±3 Removal rate (%) 89.2±2.7 93.4±0.1 90.8±0.0 93.9±0.0 94.0±0.0 TN Initial content (mg/L) 230±12 220±13 310±12 250±13 265±8 End content (mg/L) 14±4 14±1 ＜5 ＜5 ＜5 Removal rate (%) 94.0±1.5 93.6±0.1 ＞98.4 ＞98.0 ＞98.1 TP Initial content (mg/L) 48.0±2.4 60.0±3.6 58.0±2.3 49.0±2.5 30.0±0.9 End content (mg/L) 1.2±0.4 9.6±0.4 5.8±0.2 0.6±0.0 0.8±0.0 Removal rate (%) 97.5±0.6 84.0±0.3 90.0±0.0 98.8±0.0 97.3±0.0

As shown in Table 7, after each second reaction step, the removal rate of COD and total nitrogen in the second treated water is at least 90%, and the removal rate of total phosphorus exceeds 90%, indicating that the sponge can effectively carry Algae strain AK-1, and after the second reaction step is performed 4 times, it does not affect the removal of pollutants by the algae strain AK-1, which means that the sponge can be used as a carrier to continuously treat livestock wastewater.

The supplementary explanation is to compare the pollutant content of the second treated water and the stock solution of livestock wastewater (the initial content of COD is 4920 mg/L, the initial content of total nitrogen is 460 mg/L, and the initial content of total phosphorus is 96 mg/L). Calculate the total removal rate of COD, total nitrogen and total phosphorus in the second treatment step relative to the stock solution of livestock wastewater. The results showed that compared with the original wastewater, the total removal rate of COD in the second treated water was at least 95%, the total removal rate of total nitrogen was at least 97%, and the total removal rate of total phosphorus was at least 90 mg/ L.

Figures 5A to 5C show the biomass concentration (Figure 5A), protein content (Figure 5B), and lutein content (Figure 5C) of the first treated water and the second treated water according to an embodiment of the present invention. A bar graph of the yield, where the horizontal axis of FIG. 5A represents the first treated water and the second treated water, the left vertical axis and the bar 511 represent the biomass concentration, and the right vertical axis and the bar 513 represent the biomass yield. The horizontal axis of FIG. 5B represents the first treated water and the second treated water, the left vertical axis and the bar 521 represent the protein content, and the right vertical axis and the bar 523 represent the protein yield. The horizontal axis of FIG. 5C represents the first treated water and the second treated water, the left vertical axis and the straight bar 531 represent the lutein content, and the right vertical axis and the straight bar 533 represent the lutein yield.

As shown in Figure 5A, the biomass concentration of the algae strain AK-1 in the first treatment water is 2.48±0.13 g/L, and the biomass concentration of the first to fourth second treatment water is 3.24±0.24 g, respectively /L, 3.36±0.20 g/L, 3.32±0.20 g/L and 2.96±0.28 g/L. The average biomass concentration and average biomass yield of the above process were 3.22±0.15 g/L and 0.55±0.22 g/L/day, respectively, indicating that the sponge can maintain the biomass concentration of the algae strain AK-1. However, as shown in Figure 5B and Figure 5C, as the number of the second reaction step increases, the yield of protein and lutein decreases, but it is still within an acceptable range, showing that sponges can effectively adsorb and immobilize algae strains AK-1, and the algae strain AK-1 can continuously treat livestock wastewater.

It is supplemented that after performing the second reaction step 4 times, a scanning electron microscope (scanning electron microscope) can be used to observe the algae body of the algae strain AK-1 attached to the surface of the sponge, confirming the absorption capacity of the sponge. Larger, it can provide more attachment and growth space for the algae strain AK-1, so that the algae strain AK-1 in the initial stage of the first reaction step and/or the second reaction step can have more algae bodies, so as to save The time required for the growth of the algae strain AK-1 during the initial growth period, thereby improving the overall wastewater treatment efficiency.

In summary, because the sponge can carry the algae strain AK-1 with a larger absorption capacity, in the process of treating wastewater, no additional nutrients are needed to produce more biomass.

On-site experiments were carried out on livestock farms, including the use of sponges to carry the inoculation source of the algae strain AK-1, the concentration of the diluted wastewater was 50 wt%, the first reaction step and the second reaction step 4 times, the COD content could be increased from 803.43± Reduce 83.01 mg/L to 178.21±20.69 mg/L, reduce the total nitrogen content from 315.21±45.77 mg/L to 26.43±6.80 mg/L, and reduce the total phosphorus content from 150.00±16.92 mg/L to 0.02±0.01 mg/L. This shows that the algae strain AK-1 can indeed effectively remove pollutants in livestock wastewater.

The above-mentioned Chlorella sp. strain AK-1 can be used as an effective ingredient of biological preparations. In this specific example, the aforementioned biological agent may optionally include, but is not limited to, for example, at least one of Chlorella vulgaris, Scenedesmus, Bacillus, Pseudomonas, Enterobacter, nitrosating bacteria, and nitrifying bacteria. In other examples, the dry weight ratio of the aforementioned biological agent to the sponge is not particularly limited, and the conventional use amount can be considered. However, in some specific examples, the dry weight ratio of the sponge to the Chlorella sp. strain AK-1 can be, for example, It is 1:2. In other examples, using the aforementioned biological agents to perform the above-mentioned first treatment step on the diluted wastewater with a concentration of 50 wt%, the COD content in the diluted wastewater can be reduced from 2108±197 mg/L to 711±20 mg/L (shift The removal rate is 91.6±0.2%), the total nitrogen content is reduced from 219±23 mg/L to 13±3 mg/L (removal rate is 94.1±1.1%), and the total phosphorus content is reduced from 39.8±4.3 mg/L L is reduced to less than 0.2 mg/L (removal rate is greater than 99.5%).

According to the above-mentioned embodiment, the treatment of livestock wastewater by the Chlorella sp The content of biomass, protein and lutein, so it can be used as the effective ingredients of biological preparations, feed additives and/or microbial fertilizers.

In summary, although the present invention takes a specific algae strain, a specific condition or a specific evaluation method as an example, it illustrates the total removal rate of pollutants and the yield of biological products of the Chlorella sp. strain AK-1 of the present invention. However, anyone with ordinary knowledge in the technical field of the present invention can know that the present invention is not limited to this. Without departing from the spirit and scope of the present invention, the present invention can also use other algae strains, other conditions or other evaluations. Way to proceed.

Although the present invention has been disclosed in several embodiments as above, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs can make various modifications without departing from the spirit and scope of the present invention. Modifications and modifications, therefore, the scope of protection of the present invention shall be subject to those defined by the attached patent application scope.

111,113,121,123,131,133,211,213,221,223,231,233,311,313,321,323,331,333,411,413,421,423,431,433,511,513,521,523,531,533: Straight

In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious and understandable, the detailed description of the attached drawings is as follows: [Figure 1A] to [Figure 1C] show the biomass concentration (Figure 1A), protein content (Figure 1B), lutein content (Figure 1C) and yields of different algae strains according to an embodiment of the present invention Histogram. [Fig. 2A] to [Fig. 2C] show the biomass concentration (Fig. 2A), protein content (Fig. 2B), and lutein content (Fig. 2C) of different second dilution solutions according to an embodiment of the present invention. Histogram of its yield. [Figure 3A] to [Figure 3C] show the biomass concentration (Figure 3A), protein content (Figure 3B) and lutein content (Figure 3C) of different BG-11 culture solution concentrations according to an embodiment of the present invention Bar graph of its yield. [Figure 4A] to [Figure 4C] show the biomass concentration (Figure 4A), protein content (Figure 4B) and lutein content (Figure 4C) and yields of different carriers according to an embodiment of the present invention Bar graph. [Figure 5A] to [Figure 5C] show the biomass concentration (Figure 5A), protein content (Figure 5B) and lutein content (Figure 5A) of the first treated water and the second treated water according to an embodiment of the present invention 5C) Bar graph of its yield.

Domestic hosting information The Chlorella sp. strain AK-1 is deposited at the Bioresource Center (BCRC), Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan. The deposit date is January 5, 2021, and the deposit number is BCRC.

511,513: straight

Claims (10)

A method of wastewater treatment, including: Perform a first treatment step, where the first treatment step includes inoculating the Chlorella sp. strain AK-1 as an inoculation source into a diluted wastewater, and performing a first reaction step at 27°C for 6 days to 8 days to obtain a first treated water. The Chlorella sp. strain AK-1 was deposited at the Bioresource Center (BCRC), Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan on January 5, 2021. The deposit number is BCRC. The diluted wastewater is 100% by weight (wt%) of the original wastewater diluted with pure water to 30 wt% to 50 wt%, and the inoculation amount is 0.2 g to 0.5 g per liter of the diluted wastewater. Of the Chlorella sp. strain AK-1; and Perform at least one second treatment step, wherein the second treatment step includes flowing out a discharge volume of the first treated water, and then injecting a volume of the diluted wastewater added, and performing a second treatment at 27°C The reaction step takes 6 to 8 days to obtain a second treated water, wherein based on a total volume of the first treated water 100% by volume (vol%), the discharge volume is 50 vol% to 90 vol%, The added volume is the same as the discharge volume, and compared to the wastewater stock solution, the total removal rate of COD of the second treated water is at least 95%, and the total removal rate of total nitrogen is at least 97%, and The total removal rate of total phosphorus is at least 90%. The method for treating wastewater according to claim 1, wherein the COD content of the raw wastewater is greater than 4000 mg/L, the total nitrogen content is greater than 450 mg/L, and/or the total phosphorus content is greater than 70 mg/L. The method for treating wastewater according to claim 1, wherein the first treatment step and/or the second treatment step further comprises adding a porous carrier to the diluted wastewater, the porous carrier comprising a sponge or an active Carbon, and the porous carrier carries the inoculation source of the Chlorella sp. strain AK-1. The method for treating wastewater according to claim 3, wherein based on the diluted wastewater being 100 wt%, a dry content of the sponge is 0.2 wt%. The method for treating wastewater according to claim 3, wherein based on the diluted wastewater being 100 wt%, a content of the activated carbon is 2.0 wt%. The method for wastewater treatment according to claim 3, wherein the wastewater treatment includes the second treatment step performed at least four times. The method for treating wastewater according to claim 1, wherein after performing the first treatment step and/or the second treatment step, it further comprises performing a solid-liquid separation on the first treated water and/or the second treated water Steps to obtain one of the algae bodies of the Chlorella sp. strain AK-1. A biological preparation comprising a chlorella strain AK-1 as an active ingredient, wherein the deposit number of the chlorella strain AK-1 is BCRC. The biological preparation according to claim 8, further comprising a sponge, wherein the dry weight ratio of the sponge to the inoculation source of the Chlorella sp. strain AK-1 is 1:2. The biological preparation according to claim 8, further comprising at least one of Chlorella vulgaris, Scenedesmus, Bacillus, Pseudomonas, Enterobacter, nitrosating bacteria, and nitrifying bacteria.

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