TWI762145B - Chlorella sorokiniana, method of treating wastewater using the same and bioagent including the same - Google Patents

Abstract

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

Description

Chlorella, its method for wastewater treatment, and biological preparations containing the same

The present invention relates to a kind of Chlorella and its application, in particular to a kind of Chlorella algae strain AK-1, its method for wastewater treatment and its biological preparation.

Human activities are accompanied by pollution, which not only destroys the environment, but also affects human health. For example, industrial and animal husbandry wastewater contains high levels of organic compounds, total nitrogen, and total phosphorus, which easily lead to eutrophication of water bodies, causing microorganisms such as algae to proliferate due to excessive nutrients. The rapid increase in the number of microorganisms affects the light transmittance of water bodies, resulting in the death of plants and/or animals in the water. Dead animals and/or plant carcasses and the decomposition of the above organic compounds consume a large amount of oxygen, resulting in a decrease in the oxygen content of the water body, and the generation of greenhouse gases and odors.

Small (1 μm to 10 μm) single-celled algae, also known as microalgae, characterized by large biomass, short growth cycle and/or high tolerance to pollutants. Second, the growth and/or reproduction of microalgae requires a large amount of carbon, nitrogen and/or phosphorus, so microalgae can be used to remove pollutants from wastewater and generate additional products products, such as biomass oils and/or nutritional supplements. However, the tolerance of microalgae to pollutants and the conversion rate of bio-products has limitations, and for some wastewater (such as phosphorus content can be as high as 50mg/L to 230mg/L, and nitrogen content can be as high as 800mg/L to 2300mg/L Swine wastewater) is not effective in removing pollutants, and it is difficult to grow and/or reproduce in such an extreme environment.

Therefore, there is an urgent need to provide an algae and a method for treating wastewater to solve the above problems.

Therefore, one aspect of the present invention provides a method for wastewater treatment, which comprises 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 can contain the above-mentioned Chlorella algal strain AK-1 as an active ingredient.

According to the above aspect of the present invention, a method for treating wastewater is provided, which may include performing a first treatment step and at least one second treatment step. The first treatment step may include inoculating the Chlorella algal strain AK-1 as an inoculum 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 above-mentioned Chlorella algal strain AK-1 can, for example, be deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331, Shichi Road, Hsinchu, Taiwan on January 5, 2021, and the deposit number is BCRC 980057. The above-mentioned diluted wastewater is made by diluting 100 weight percent (wt %) of the wastewater stock solution with pure water to 30 wt % to 50 wt %. The above inoculation amount is 0.2g to 0.5g of Chlorella per liter of diluted wastewater Algal strain AK-1.

The above-mentioned second treatment step may include the discharge volume of the first treated water, and then inject the diluted waste water of the added volume, and carry out the second reaction step at 27°C for 6 to 8 days to obtain the second treated water, Wherein based on the total volume of the first treated water being 100 volume percent (vol%), the discharge volume can be 50vol% to 90vol%, and the added volume is the same as the discharge volume. 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% relative to the wastewater stock solution.

According to an embodiment of the present invention, the COD content of the wastewater stock solution may be, for example, greater than 4000 mg/L, the total nitrogen content may be, for example, greater than 450 mg/L and/or the total phosphorus content may 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 into the diluted wastewater, wherein the porous carrier may include sponge and/or activated carbon, and the porous carrier supports Inoculum source for Chlorella algal strain AK-1.

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

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

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

According to an embodiment of the present invention, after the first treatment step and/or the second treatment step is performed, the treatment of the first treatment water and/or the second treatment step may optionally be included. The solid-liquid separation step was carried out to obtain the algal body of the Chlorella algal strain AK-1.

According to the above aspect of the present invention, a biological preparation is proposed, wherein the biological preparation can comprise the above-mentioned Chlorella strain AK-1 as an active ingredient, wherein the accession number of the Chlorella strain AK-1 can be, for example, BCRC 980057 .

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

According to an embodiment of the present invention, the above-mentioned biological agent may selectively include at least one of Chlorella vulgaris, Scenedesmus, Bacillus, Pseudomonas, Enterobacter, Nitrosomonas and Nitrobacter.

Using the Chlorella algae strain AK-1 of the present invention to treat wastewater can effectively remove pollutants in the wastewater, and increase the biomass, protein and/or lutein content of the Chlorella algae strain AK-1, and the obtained algae It can also be used as an active ingredient in biological preparations.

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:

In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the detailed description of the accompanying drawings is as follows: [FIG. 1A] to [FIG. 1C] illustrate an embodiment of the present invention. Bar graphs of biomass concentration (FIG. 1A), protein content (FIG. 1B) and lutein content (FIG. 1C) of different algal strains and their yields.

[FIG. 2A] to [FIG. 2C] illustrate different second Bar graphs of biomass concentration (FIG. 2A), protein content (FIG. 2B), and lutein content (FIG. 2C) of dilution concentrations and their yields.

[FIG. 3A] to [FIG. 3C] show the biomass concentration (FIG. 3A), protein content (FIG. 3B) and lutein content (FIG. 3C) of different BG-11 medium concentrations according to an embodiment of the present invention and a histogram of its yield.

[ FIG. 4A ] to [ FIG. 4C ] are plots of biomass concentration ( FIG. 4A ), protein content ( FIG. 4B ) and lutein content ( FIG. 4C ) of different carriers and their yields according to an embodiment of the present invention Histogram.

[FIG. 5A] to [FIG. 5C] show the biomass concentration (FIG. 5A), protein content (FIG. 5B) and lutein content (FIG. 5B) of the first treated water and the second treated water according to an embodiment of the present invention 5C) and histograms of their yields.

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

The above-mentioned Chlorella, also known as green algae, is a common unicellular alga in the Chlorella genus of the Chlorophyta phylum. Different chlorella strains have different adaptability to the environment, and some of them have better adaptability to the above-mentioned wastewater, which can not only effectively remove pollutants such as 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 algal strain AK-1 can, for example, be deposited in the Biological Resource Center of the Food Industry Development Research Institute of Taiwan Hsinchu Foundation on January 5, 2021 (BCRC), accession number BCRC 980057.

The above-mentioned wastewater is, for example, industrial wastewater and/or livestock wastewater, and the livestock wastewater can 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 a general term for phosphorus-containing compounds such as phosphides, phosphates, and phosphites. The aforementioned wastewater can generally be defined by conditions such as 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 total removal rate refers to the difference between the pollutants in the wastewater before treatment and the discharge water after wastewater treatment divided by the percentage of 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 comprises inoculating the Chlorella algal strain AK-1 as an inoculation source into the diluted wastewater, and then inoculating the diluted wastewater at the first treatment step. The first reaction step is carried out at 20°C to 30°C for 6 days to 8 days, preferably at 27°C for 6 days to 8 days, to obtain the first treated water. The above-mentioned dilution wastewater can be, for example, 100 weight percent (wt%) wastewater stock solution diluted with pure water to 30wt% to 50wt%, so that the COD content in the diluted wastewater is less than 3000mg/L. The above-mentioned inoculum size may be, for example, 0.2 g to 0.5 g of Chlorella algal strain AK-1 per liter of diluted wastewater. It is worth noting that if the concentration of the diluted effluent is greater than 50wt%, the turbidity in the diluted effluent is too high and/or there are too many microorganisms competing for resources (eg: space, nutrients and/or oxygen), thus affecting Chlorella Photosynthesis of algal strain AK-1, Causes the growth of Chlorella algae strain AK-1 to be inhibited. If the concentration of the diluted wastewater is less than 30wt%, the removal rate of pollutants by Chlorella algae strain AK-1 is not good. Specific examples of the above pure water include distilled water and/or secondary water. If the inoculum of Chlorella algal strain AK-1 is too small, the amount of algae in the first reaction step is insufficient. If the inoculum of Chlorella algae strain AK-1 is too large, the removal effect of pollutants will not be significantly improved.

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

The above-mentioned second treatment step may include, based on the total volume of the first treated water being 100 vol%, after the discharge volume of the first treated water flows out, then the added volume of diluted wastewater is injected, and the second reaction is performed at 20°C to 30°C The step is for 6 to 8 days, preferably the first reaction step is carried out at 27°C for 6 to 8 days to obtain the second treated water, wherein the discharge volume can be, for example, 50vol% to 90vol%, and the added volume and The discharge volume is the same. 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 50vol%, the removal efficiency of biological oxygen demand is low. If the discharge volume is greater than 90 vol%, it is easy to lose more algal bodies of the Chlorella algal strain AK-1, resulting in insufficient algal body of the Chlorella algal strain AK-1 as the inoculum source in the second treatment step .

The wastewater treatment method of the present invention can selectively treat the wastewater raw liquid, the first The first treated water and/or the second treated water is subjected to a solid-liquid separation step, wherein the solid-liquid separation step can be performed by conventional solid-liquid separation methods, such as filtration, natural precipitation and/or centrifugation. The solid-liquid separation of the wastewater stock solution can remove solids (such as feces, sediment and/or other microorganisms), so as 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 the algal body of the Chlorella algal strain AK-1. The algal biomass, protein per unit weight and/or lutein of the Chlorella strain AK-1 were increased compared to the inoculum source of the Chlorella strain AK-1. The algal body of the above-mentioned Chlorella algal strain AK-1 can be used as an inoculation source for the second reaction step. In one embodiment, the algal bodies obtained from the solid-liquid separation step of the first treated water can be used as an inoculum 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 into the diluted wastewater, wherein the porous carrier may include sponge and/or activated carbon, and the porous carrier supports small The inoculum source of the coccus algal strain AK-1. In one embodiment, based on 100wt% of the diluted wastewater, the dry content of the sponge (ratio of dry weight to the weight of the diluted wastewater) can be 0.1wt% to 0.3wt% of sponge, preferably 0.2wt%. 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, each 100 g of diluted waste water 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 can 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 confirmed that the above-mentioned wastewater treatment method can effectively remove the pollutants in the wastewater stock solution. The total removal rate of various pollutants in the second treated water compared to 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 strain AK-1 can be increased by 0.52g/L per day, and the yields of protein and lutein can reach 0.28g/L and 3.10mg/L per day . The above protein is one of the three major nutrients for calories, which 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 the growth, reproduction and/or pigment formation of animals, and increase the color of animal skin, meat and/or eggs.

It can be seen from the above that the use of Chlorella algae strain AK-1 to treat wastewater can remove pollutants in the wastewater and increase the biomass, protein and lutein of Chlorella algae algae strain AK-1, and the algae are properly treated. After the conventional process treatment (such as cleaning, solid-liquid separation, etc.), it can be used as a biological preparation, wherein the biological preparation can, for example, use Chlorella algae strain AK-1 as an active ingredient to remove COD, total nitrogen in wastewater and/or total phosphorus. In one embodiment, the biological preparation can selectively include sponge, wherein the dry weight ratio of sponge and inoculation source of Chlorella algae strain AK-1 is not particularly limited, and can refer to the conventional usage amount, but in some specific examples. , the dry weight ratio of sponge to Chlorella algal strain AK-1 may 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 At least one of 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 intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. retouch.

Example 1. Microbiological properties of algal strain AK-1

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

The DNA of the algal strain AK-1 was purified, and then the PCR upstream and downstream primers of 23S rDNA as shown in SEQ ID NOs. 1 and 2 were used to carry out polymerase chain reaction (PCR) , to obtain the 23S rDNA of the algal strain AK-1 (as shown in SEQ ID NO. 3). The above DNA purification and PCR It is well known to those skilled in the art to which the present invention pertains, and details are not described herein again. The 23S rDNA sequences of the algal strain AK-1 were aligned using the Basic Local Alignment Search Tool (BLAST), and it was confirmed that the algal strain AK-1 was Chlorella. The above-mentioned algal strain AK-1 has been deposited in 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 980057.

It is supplemented that Chlorella algal strain MS-C1 and Chlorella algal strain TJ5 (hereinafter referred to as algal strain MS-C1 and algal strain TJ5 respectively) were isolated from the same batch of pond water samples. -C1 and strain TJ5 and strain AK-1 are different strains of the same species (Chlorella).

Embodiment 2. Sewage treatment method and screening of algal strain AK-1

In a photobioreactor (PBR), algal strain AK-1, algal strain MS-C1 and algal strain TJ5 were used as inoculation sources to perform the first reaction step on the diluted wastewater for 15 days to obtain the first treated water. , wherein the diluted wastewater is obtained after filtering and diluting the stock solution of the above-mentioned livestock wastewater, wherein the composition of the livestock wastewater is as shown in Table 1 [mean ± standard error of the mean (SEM)], and The pH of livestock wastewater is between 7 and 8.

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

The detection steps are respectively performed on the diluted waste water and the first treated water. First, the water samples were randomly sampled from the diluted waste water and the first treated water, and the water samples were centrifuged at 6000 rpm for 5 minutes to obtain a supernatant liquid and a sediment, wherein the sediment contained the algal body of the algal strain. The content of COD, total nitrogen (TN) and total phosphorus (TP) in the supernatant was detected by using the AL450 photometer of the German company Aqualytic, and the migration of pollutants by different algae strains was calculated. Removal rate, wherein 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-mentioned detection methods of COD, total nitrogen and total phosphorus are carried out according to the instruction manual of AL450 photometer detection, and will not be repeated here.

Dry the sediment at a temperature of 120 ° C until the weight of the sediment does not change, 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 algal 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 (beginning of the first reaction step) divided by the time for the first reaction step to obtain biomass Yield (unit: mg/L/day).

The pellet was lyophilized to obtain a lyophilisate. Then, the weights of amino acids and lutein in the lyophilisate were quantified by conventional detection methods to calculate the content and yield of protein and lutein. The quantification method was published in Biotechnology Journal in 2015 by Dr. Chen Junyan's team. The papers in Volume 10, Issue 6, pages 905 to 914 and the methods published in Journal of Bioscience and Bioengineering in 2018, pages 1 to 7, will not be repeated here. The protein content and the lutein content are the percentage of the sum of the weight of amino acids divided by the weight of the lyophilized powder (unit: %) and the weight ratio of lutein divided by the biomass (unit: mg/g). The yields of protein and lutein are the product of protein content and lutein content over biomass yield, respectively (unit: mg/L/day).

The stock solution of livestock wastewater is diluted with the conventional blue-green algae medium BG-11 to obtain the first dilution solution, wherein the concentration of the first dilution solution is 10wt% based on the concentration of the stock solution being 100wt%. The aforementioned BG-11 culture medium contains 1.5g NaNO ₃ , 0.04g K ₂ HPO ₄ , and 0.075g MgSO ₄ per liter. 7H ₂ O, 0.006g citric acid, 0.02g Na ₂ CO ₃ , 0.036g CaCl ₂ , 0.001g 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 the balance of water. Next, the algal strain AK-1, the algal strain MS-C1 and the algal strain TJ5 (0.4 g of the algal strain per liter of the first dilution solution) were inoculated into the first dilution solution, respectively, to carry out the above-mentioned first reaction step, thereby obtaining The first treated water. The detection steps are respectively performed on the first diluent (initial) and the first treated water (end), and the removal rate of pollutants in the first treated water relative to the first diluent is calculated. The results are reported in Table 2 and Figures 1A-1C.

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

1A to 1C are graphs showing biomass concentration ( FIG. 1A ), protein content ( FIG. 1B ) and lutein of different algal strains according to an embodiment of the present invention A bar graph of the content (FIG. 1C) and its yield. The horizontal axis of FIG. 1A represents algal strains, the left vertical axis and bar 111 represent biomass concentration, and the right vertical axis and bar 113 represent biomass yield. The horizontal axis of FIG. 1B represents algal strains, the left vertical axis and bar 121 represent protein content, and the right vertical axis and bar 123 represent protein yield. The horizontal axis of FIG. 1C represents the algal strain, the left vertical axis and bar 131 represent the lutein content, and the right vertical axis and 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±0.20g/L) and biomass yield (0.29±0.01g/L/day) of the algal strain AK-1 were significant higher than algal strain MS-C1 (3.90±0.22g/L and 0.24±0.02g/L/day, respectively) and algal strain TJ5 (4.18±0.22g/L and 0.26±0.02g/L/day, respectively) ( n=3, P<0.05). Secondly, compared with the algal strain MS-C1 and the algal strain TJ5, the protein yield and the lutein yield of the algal strain AK-1 were higher.

From the above results, it can be seen that the algae strain AK-1 has a better metabolic effect on the pollutants in livestock wastewater, and the yield of biomass, protein and lutein in the livestock wastewater is higher, indicating that the algae strain AK-1 has a relatively high yield. The best adaptability of livestock wastewater.

Example 3. Assessing the effect of diluted wastewater concentration on metabolic pollutants of algal strain AK-1

The stock solution of livestock wastewater is diluted with distilled water to obtain a second dilution, wherein the concentration of the stock solution based on livestock wastewater is 100wt%, and the concentrations of the second dilution are 10wt%, 30wt%, 50wt%, 70wt% and 90wt%, respectively. Next, use 0.4 g of algae per liter of the second dilution The inoculation source of the strain AK-1 was inoculated with the inoculum amount, and then the above-mentioned first reaction step was carried out for 15 days to obtain the first treated water. The above detection steps were performed on the second dilution solution (initial) and the first treated water (end), and the results were recorded in Table 3 and FIGS. 2A to 2C .

As shown in Table 3, when the concentration of the second diluent is 30wt% to 90wt%, 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, for example, be in excess of 95%. It is worth noting that the COD content of the second dilution with a concentration of 90wt% is 3600mg/L to 5400mg/L, but under these conditions, the removal rate of COD and total nitrogen by algae strain AK-1 can reach 80% %, and the removal rate of total phosphorus can reach more than 95%, which shows that the algae strain AK-1 has good adaptability to livestock wastewater, and the high COD content does not affect the efficiency of the algae strain AK-1 to remove pollutants.

FIGS. 2A to 2C are plots of biomass concentration ( FIG. 2A ), protein content ( FIG. 2B ) and lutein content ( FIG. 2C ) of different second dilution concentrations and their yields according to an embodiment of the present invention Histogram. The horizontal axis table of FIG. 2A The concentration of livestock wastewater is shown, the left vertical axis and bar 211 represent biomass concentration, and the right vertical axis and bar 211 represent biomass yield. The horizontal axis of FIG. 2B represents the concentration of livestock wastewater, the left vertical axis and bar 221 represent protein content, and the right vertical axis and bar 223 represent 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 FIGS. 2A to 2C , compared with the second dilution of 70 to 90 wt %, the biomass, protein and The yield of lutein is higher. Preferably, in the 50wt% second dilution, the content and yield of the algal biomass are 5.45±0.55g/L and 0.36±0.04g/L/day, respectively, and the protein content and yield are respectively 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 light transmittance of the second dilution solution with high COD concentration is low, thus reducing the photosynthetic 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 contaminants in the second dilution of 30wt% to 90wt%, but the algal body of the algae strain AK-1 is in the second dilution of 10wt% to 50wt%. The yield of the biomass was higher, indicating that it is better to carry out the first reaction step with 30wt% to 50wt% of the second dilution liquid, and it is better to carry out the first reaction step with 50wt% of the second dilution liquid.

Embodiment 4. Assess the concentration of BG-11 culture solution

The third dilution solution was prepared by using stock solution of livestock wastewater, BG-11 culture solution and distilled water respectively, wherein the concentration of livestock waste water in the third dilution solution was 50wt%, and the concentration of BG-11 culture solution was 0wt%, 25wt% and 50wt% respectively. and 100wt%. Next, the first reaction step is performed on the above-mentioned third dilution liquid using the algal strain AK-1 to obtain the first treated water. The detection steps were performed on the third dilution solution (initial) and the first treated water (end), and the results were recorded in Table 4 and FIGS. 3A to 3C .

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

3A to 3C show the biomass concentration ( FIG. 3A ), protein content ( FIG. 3B ) and lutein content ( FIG. 3C ) of different BG-11 medium concentrations and their yields according to an embodiment of the present invention histogram. The horizontal axis of FIG. 3A represents the concentration of the BG-11 medium, 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 medium, 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. picture The horizontal axis of 3C represents the concentration of BG-11 broth, the left vertical axis and bar 331 represent the lutein content, and the right vertical axis and bar 333 represent the lutein yield.

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

In conclusion, considering the removal rate of pollutants and the yield of algal biomass, protein and/or lutein, the concentration of algal strain AK-1 in the culture medium without BG-11 and the concentration of stock solution in livestock wastewater It is preferable to perform the first reaction step with 50 wt% of the third diluent (ie, 50 wt% of the second diluent).

Example 5. Carriers for evaluating algal bodies

The carrier was added to 50 wt % of the second dilution, and the first reaction step was performed to obtain the first treated water, wherein the stirring rate of the first reaction step was 150 rpm. The above-mentioned carrier includes activated carbon and sponge of porous material [material: polyurethane (polyurethane)], and sodium alginate crystal ball and clay block of non-porous material. The above activated carbon is a cube with a diameter and 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 the clay block are spheres with a diameter of 1 cm. based on dilution waste The water was 100 wt %, the dry content of activated carbon, clay blocks and sodium alginate crystal balls was 2.0 g/100 mL, and the dry content of the sponge was 0.2 wt %. Next, the detection step was performed on the 50 wt% second dilution solution (initial) and the first treated water (end), and the results were recorded in Table 5 and FIGS. 4A to 4C .

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

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

As shown in Figure 4A to Figure 4C, compared with the control group, using activated carbon and sponge as carriers, the yields of biomass, protein and lutein increased, wherein using sponge as carrier, the biomass of algal strain AK-1 The concentration can reach 8.08±0.39g/L, the biomass yield can reach 0.52±0.03g/L/day, the protein yield can reach 0.28±0.02g/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 decreased. It can be seen that using porous materials such as sponge or activated carbon as a carrier can increase the removal rate of pollutants and the yield of biological products by the algal strain AK-1.

Embodiment 6, evaluate the second reaction step condition

Using the semi-batch method for the second reaction step, livestock wastewater can be treated continuously. In detail, the first reaction step was performed on 50wt% of the second dilution water with algal strain AK-1 for 6 to 8 days to obtain the first treated water, and then the first treated water was 100vol% based on the weight of the first treated water, and the effluent was discharged. Volume of the first treated water. Next, inject the second dilution water of the added volume, and carry out the second reaction step at 27° C. for 6 to 8 days to obtain the second treated water, wherein the volume of the second dilution water is added and the first treated water is discharged. The volume is the same.

After adding the diluted wastewater and after the second reaction step, samples were taken respectively, and the COD, biochemical oxygen demand (BOD), total phosphorus and total nitrogen of the water samples before and after the second reaction step were measured. content, and calculate the removal rate of pollutants in the second reaction step to evaluate the range of the discharge volume, wherein the measurement method of BOD is not limited, and can be performed by conventional methods. Specifically, for example, it is carried out according to the method of No. NIEA W510.55B published by the Environmental Inspection Institute of the Environmental Protection Agency, Taiwan Executive Yuan. The measurement results show that when the discharge volume is 50vol%, compared with the diluted wastewater, 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 removal rate is 83.9±3.2%. 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 70vol%, compared with the diluted wastewater, the removal rate of COD of the second treatment water is 86.1±3.1%, the removal rate of BOD is 94.4%±2.3, and the removal rate of total nitrogen is 79.8 ±8.7%, and the removal rate of total phosphorus was 93.6±4.3%. When the discharge volume is 90vol%, compared with the diluted wastewater, the removal rate of COD of the second treatment water is 88.2±1.2%, the removal rate of BOD is 94.5±2.1%, and the removal rate of total nitrogen is 88.2±1.2%. 84.3±4.2%, and the removal rate of total phosphorus was 89.5±5.5%. According to the results of BOD, the discharge volume of the first treated water is selected as 50 vol% to carry out the second reaction step.

It is worth noting that the first reaction step and the second reaction step include using a sponge as a carrier, and filtration is required before the first treated water or the second treated water flows out, so as to avoid the loss of the sponge and the algal strain AK-1. 50wt% of the second dilution water (initial) and the second treated water (end) were respectively subjected to the detection steps, and the results were recorded in Table 6.

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 removal rate of pollutants by the algae strain AK-1 is better.

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

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

It is added that by comparing the pollutant content of the second treated water and the stock solution of livestock wastewater (the initial content of COD is 4920mg/L, the initial content of total nitrogen is 460mg/L and the initial content of total phosphorus is 96mg/L), the first The total removal rate of COD, total nitrogen and total phosphorus in the two treatment steps relative to the stock solution of livestock wastewater. The results show that 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% compared to the raw wastewater.

5A to 5C are diagrams showing the biomass concentration ( FIG. 5A ), protein content ( FIG. 5B ), and lutein content ( FIG. 5C ) of the first treated water and the second treated water according to an embodiment of the present invention and their A bar graph of productivity, where the horizontal axis of FIG. 5A represents the first and second treated water, the left vertical axis and bar 511 represent biomass concentration, and the right vertical axis and bar 513 represent 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 bar 531 represent the lutein content, and the right vertical axis and the bar 533 represent the lutein yield.

As shown in Fig. 5A, in the first treated water, the growth of algal strain AK-1 The mass concentration was 2.48±0.13g/L, and the biomass concentrations of the first to fourth second treated water were 3.24±0.24g/L, 3.36±0.20g/L, 3.32±0.20g/L and 2.96 ±0.28g/L. The average biomass concentration and average biomass yield of the above process were 3.22±0.15g/L and 0.55±0.22g/L/day, respectively, indicating that the sponge could maintain the biomass concentration of the algal strain AK-1. However, as shown in Figure 5B and Figure 5C, with the increase of the number of the second reaction step, the yield of protein and lutein decreased, but still within the acceptable range, indicating that the sponge can effectively adsorb and immobilize the algal strain AK-1, and the algal strain AK-1 can continuously treat livestock wastewater.

It is added that after performing the second reaction step for 4 times, the algal body of the algal strain AK-1 can be observed to adhere to the surface of the sponge using a scanning electron microscope, which confirms 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 has more algal bodies, so as to save The time required for the initial growth of the algae strain AK-1, thereby improving the overall wastewater treatment efficiency.

In conclusion, since the sponge can carry the algal strain AK-1 with a large absorption capacity, more biomass can be generated without adding additional nutrients in the process of wastewater treatment.

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

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

According to the above embodiment, using the Chlorella algae strain AK-1 of the present invention to treat livestock wastewater can not only effectively remove COD, total nitrogen and total phosphorus in the livestock wastewater, but also increase the amount of Chlorella algae strain AK-1. Biomass, protein and lutein content, so it can be used as an active ingredient in biological preparations, feed additives and/or microbial fertilizers and other compositions.

To sum up, although the present invention takes specific algal strains, specific conditions or specific evaluation methods as examples, the Chlorella algae strain AK-1 of the present invention is described. The total removal rate of pollutants and the yield of biological products, but anyone with ordinary knowledge in the technical field to which the present invention pertains can know, the present invention is not limited to this, and the present invention also does not deviate from the spirit and scope of the present invention. Other algal strains, other conditions, or other assessment methods can be used.

Although the present invention has been disclosed above with several embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs, without departing from the spirit and scope of the present invention, can make various Therefore, the scope of protection of the present invention should be determined by the scope of the appended patent application.

【Biological Material Deposit】 Domestic storage information

Chlorella algal strain AK-1 is deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan, where the deposit date is January 5, 2021, and the deposit number is BCRC 980057.

511, 513: Straight

Claims (7)

A method for wastewater treatment, comprising: performing a first treatment step, wherein the first treatment step comprises inoculating Chlorella algal strain AK-1 as an inoculation source into a diluted wastewater, and performing a first treatment at 27° C. A reaction step for 6 to 8 days to obtain a first treated water, the Chlorella algae strain AK-1 was deposited at the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan on January 5, 2021 Biological Resource Center (BCRC), the deposit number is BCRC 980057, the diluted wastewater is one of 100 weight percent (wt%) wastewater stock solution diluted with pure water to 30wt% to 50wt%, and the COD content of the wastewater stock solution is greater than 4000mg/ L, the total nitrogen content is greater than 450 mg/L, and the inoculation amount is 0.2 g to 0.5 g of the Chlorella algae strain AK-1 per liter of the diluted wastewater; and at least one second treatment step is performed, wherein The second treatment step comprises that after a discharge volume of the first treatment water flows out, an added volume of the diluted wastewater is injected, and a second reaction step is performed at 27° C. for 6 to 8 days to obtain a The second treated water, wherein based on a total volume of the first treated water is 100 volume percent (vol%), the discharge volume is 50 vol% to 90 vol%, the addition volume is the same as the discharge volume, and the same Compared with the wastewater stock solution, 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%, and wherein The first treatment step and/or the second treatment step includes adding a porous carrier to the diluted wastewater, and the porous carrier supports the pellets This inoculation source of algal strain AK-1. The method for treating wastewater according to claim 1, wherein the total phosphorus content of the wastewater stock solution is greater than 70 mg/L. The method for treating wastewater as claimed in claim 1, wherein the porous carrier comprises a sponge or an activated carbon. The method for treating wastewater according to claim 3, wherein based on 100 wt % of the diluted wastewater, a dry content of the sponge is 0.2 wt %. The method for wastewater treatment 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 as claimed in claim 3, wherein the wastewater treatment comprises performing the second treatment step at least four times. The method for wastewater treatment according to claim 1, wherein after the first treatment step and/or the second treatment step is performed, further comprising performing a solid-liquid separation on the first treated water and/or the second treated water step to obtain one algal body of the Chlorella algal strain AK-1.

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