TWI614019B - Alkali resistant chlorella sp. and method for reducing and recycling co2 - Google Patents

Abstract

The present disclosure relates to an alkali-resistant microalgae strain screened by a N-methyl-N-nitro-N-methylnitrosoguanidine mutation. Furthermore, the present disclosure relates to a method for reducing and reusing carbon dioxide using the alkali-resistant microalgae strain, comprising the steps of: providing the aforementioned alkali-resistant microalgae strain; and cultivating the alkali-resistant microalgae strain in an alkaline culture solution, The alkali-resistant microalgae strain is cultured under the condition of introducing carbon dioxide.

Description

Alkali-resistant microalgae strain and method for reducing and reusing carbon dioxide

The present disclosure relates to an alkali-resistant microalgae strain and a method for reducing and reusing carbon dioxide thereof, and more particularly to an alkali-resistant microalgae strain which can be grown in an alkaline culture solution and a method for reducing and reusing carbon dioxide.

With industrial development, climate warming has become one of the issues of concern to all walks of life. Among them, one of the causes of climate warming is carbon dioxide emissions. In particular, in industrially developed countries, carbon dioxide emissions are increasing year by year. Therefore, all walks of life are actively seeking a way to reduce carbon dioxide emissions, in order to reduce climate warming.

In the past, research on microalgae biomass energy technology focused on the growth of microalgae and the total amount of carbon dioxide capture. It is less focused on whether the carbon dioxide introduced into the microalgae culture plant is fully utilized. The practical problem is to access the microalgae culture device. If the carbon dioxide in the medium is not fully dissolved in the liquid or absorbed by the microalgae, it will cause the carbon dioxide to be released into the atmosphere again. Not only will the carbon dioxide loss be lost, but the carbon dioxide that escapes to the surface may be a serious environmental problem. In addition, in order to use industrial waste gas in microalgae cultivation, it is necessary to set up the two in the same geographical location. This has difficulties in practice. Therefore, physical or chemical methods are used to absorb carbon dioxide in the exhaust gas and then transport it to the algae cultivation site. The use of this is a viable strategy, so the efficient use of carbon dioxide is important.

In order to effectively reduce carbon dioxide, it is necessary to increase the amount of carbon dioxide dissolved in the liquid; and one of the feasible methods is to alkalize the culture solution. In view of this, there is an urgent need to develop a microalgae strain which has an alkaline environmental tolerance and can be cultured in an alkaline culture solution.

The main object of the present disclosure is to provide an alkali-resistant microalgae strain ( Chlorella sp.) and a method for reducing and reusing carbon dioxide, which can achieve the purpose of utilizing carbon dioxide in waste gas or waste water.

Wherein, through the present disclosure based nitro-N- methyl -N- -N- methyl nitrosoguanidine (NTG; N -methyl- N '-nitro- N -nitrosoguanidine) wild type Chlorella (Chlorella sp .) A mutation is made to obtain a mutant strain of alkali-resistant microalgae. Among them, the mutant alkali-resistant microalgae strain provided by the present disclosure is deposited in the Food Industry Development Research Institute of the Republic of China, and the registration number is BCRC980041.

In addition, the method for reducing and reusing carbon dioxide according to the present disclosure includes the steps of: providing the aforementioned alkali-resistant microalgae strain; and cultivating the alkali-resistant microalgae strain in an alkaline culture solution under the condition of introducing carbon dioxide. The alkali-resistant microalgae strain is cultured.

In the method of reducing the amount of carbon dioxide and the method of reusing carbon dioxide, the pH of the alkaline medium for culturing the alkali-resistant microalgae strain is not particularly limited as long as it is tolerable in the range of the alkali-resistant microalgae strain. For example, the alkaline culture solution may have a pH greater than 7 and less than or equal to 11; preferably a pH greater than 8 and less than 11; and more preferably a pH between 9 and 10. The range of plus or minus 0.5 falls within the scope of this disclosure. Here, the pH of the alkaline culture solution can be adjusted with NaOH, KOH, NaHCO ₃ or a mixture thereof.

In the method of reducing and reusing carbon dioxide disclosed in the present disclosure, it is possible to selectively illuminate while introducing carbon dioxide. Among them, the equipment for illuminating is not particularly limited, and equipment commonly used in the art, such as fluorescent lamps, fluorescent lamps, natural light, and the like can be used. In addition, the illuminance and time of the illumination are not particularly limited as long as it is tolerable in the alkali-resistant microalgae strain. For example, an alkali-resistant microalgae strain can be cultured under the illumination of 10-500 μmol ∙m ^-2 ∙s ^-1 using a white fluorescent lamp; or the alkali-resistant microalgae strain can be cultured under outdoor natural light conditions.

In the method for reducing the amount of carbon dioxide and the method for reusing carbon dioxide, the carbon dioxide concentration of the alkali-resistant microalgae strain is not particularly limited as long as it can be tolerated by the alkali-resistant microalgae strain. For example, the alkali-resistant microalgae strain can be cultured at a carbon dioxide concentration of 0.03-50% (v/v); and the alkali-resistant microalgae strain is cultured at a carbon dioxide concentration of preferably 1-10% (v/v).

In the method of reducing and reusing carbon dioxide disclosed in the present disclosure, the manner of introducing carbon dioxide is not particularly limited, and for example, carbon dioxide may be introduced in a continuous or intermittent manner. Preferably, carbon dioxide is introduced in a batch manner. Among them, when carbon dioxide is introduced in an indirect manner, there is no particular limitation on the interval between the introduction of carbon dioxide and the passage time. For example, carbon dioxide can be introduced for 10 to 60 minutes every 1-24 hours.

Further, in the method of reducing the amount of carbon dioxide and the method of reusing carbon dioxide, the temperature of the alkali-resistant microalgae strain is not particularly limited as long as it is tolerable in the alkali-resistant microalgae strain. For example, the alkali-tolerant microalgae strain can be cultured at an indoor or outdoor ambient temperature.

In addition, in the method of reducing and reusing carbon dioxide disclosed in the present disclosure, the microalgae biomass can be collected after cultivating the alkali-resistant microalgae strain.

In the method of reducing and reusing carbon dioxide disclosed in the present disclosure, the mutant alkali-resistant microalgae strain provided by the present disclosure can be cultured in an alkaline culture solution as compared with the original algal strain Chlorella sp. Therefore, by using an alkaline culture solution, the amount of carbon dioxide dissolved in the culture solution can be increased, thereby increasing the rate of carbon dioxide reduction. Thereby, the amount of carbon dioxide in various exhaust gases can be effectively reduced; or, it can also be applied to the treatment of various waste waters and discharged water to achieve the effect of purified water.

The embodiments of the present disclosure are described by way of specific examples, and those skilled in the art can readily appreciate the other advantages and advantages of the disclosure. The disclosure may also be implemented or applied by other different embodiments. The details of the present specification may also be applied to various aspects and applications, and various modifications and changes may be made without departing from the spirit of the present invention.

Alkali resistance Chlorella Screening of microalgae strains

Chlorella sp. is a wild type freshwater algae strain from Taiwan. Based on this algae strain, the mutagenic agent N-methyl-N'-nitro-N-nitrosoguanidine (N-methyl-N) was used. After treatment with '-nitro-N-nitrosoguanidine, NTG), an alkali-resistant Chlorella sp. microalgae strain was obtained by screening on an alkaline solid medium, and named Chlorella sp. AT1.

The mutation screening procedure for Chlorella sp. AT1 is as follows. In the culture, the microalgae cells growing in log phase are about 1 x 10 ⁶ (in this case, the OD ₆₈₂ nm value of Chlorella sp. broth is about 1), and 0.1, 0.5, 1, 5, 10, 50, 100 and 500 μg/mL NTG was treated for 60 minutes, and then washed three times with PB buffer solution. The microalgae cells were plated on alkaline solid medium and cultured at 28 ⁰ C, 100 mmol/m ² /s for 3 days. The number of cells that survived. The results showed that when the concentration of NTG was lower than 5 μg/mL, the mortality of Chlorella sp. was positively correlated with the dose of NTG treatment, as shown in Figure 1A. When the concentration of NTG was 5 μg/mL, the lethality of Chlorella sp. was about 76%, and when the NTG treatment concentration was increased to 50 and 500 μg/mL, the Chlorella sp. lethality was only about 90% and 95%, respectively, as shown in Figure 1B.

Based on the above results, this example tested the lethality of Chlorella sp. at different treatment times with 5 μg/mL NTG. The results showed that after 10 minutes of treatment with 5 μg/mL NTG, the lethality of Chlorella sp. was about 50%, and within 60 minutes of NTG treatment, the cell death rate of Chlorella sp. was positively correlated with NTG treatment time, but prolonged. The treatment time of NTG was 120 minutes, and the mortality rate of Chlorella sp. was only increased from 80% of 60 minutes of NTG treatment to about 90%, as shown in Fig. 2.

According to the present embodiment and the processing time of the concentration effect of NTG effects on Chlorella sp. Lethality of the result, a 5 μg / mL NTG for 60 minutes conditions, Chlorella sp. Mutation treatment, and at a pH of 10 or 11 The solid medium was screened for a microalgae strain that could grow and grow faster. The strains screened in the first round were transferred to a solid medium of pH 11.5, and their alkali resistance and growth rate were continuously tested. In this example, an alkali-resistant microalgae strain was finally obtained, named Chlorella sp. AT1; and the mutant strain was deposited in the Food Industry Development Research Institute of the Republic of China, and the accession number was BCRC980041.

Chlorella Sp. AT1 Growth test in alkaline medium

In this experiment, the microalgae culture solution of pH 6, 7, 8, 9, 10 and 11 was prepared with NaOH (the composition of the culture solution was 1.25 g KNO ₃ , 1.25 g KH ₂ PO ₄ , 1 g MgSO ₄ ∙7H ₂ O per liter). , 83.5 mg CaCl ₂ ∙2H ₂ O, 0.1142 g H ₃ BO ₃ , 49.8 mg FeSO ₄ ∙7H ₂ O, 88.2 mg ZnSO ₄ ∙7H ₂ O, 14.4 mg MnCl ₂ ∙4H ₂ O, 10 mg CuSO ₄ , 7.1 Mg Na ₂ MoO ₄ and 4 mg CoCl ₂ ∙6H ₂ O) were used to test the growth of Chlorella sp. AT1 in alkaline medium. The experimental microalgae were cultured in a 1-L photoreactor (6 cm in diameter and 80 cm in height) under the conditions of artificial light of 300 mmol/m ² /s, temperature of 28 ± ¹⁰ ° C, and air gas. The aeration rate was 0.2 vvm (0.2 liters of gas per liter of culture broth per minute, volume/volume/minute). When Chlorella sp. AT1 was cultured at pH 6, 7, 8, 9 and 10, the growth rate increased with the increase of the pH value of the medium, and the growth at pH 10 was the best, and the growth rate at pH 9 was the second. When Chlorella sp. AT1 was cultured in pH 11 medium, it grew well in the first 3 days, and then grew flat, as shown in Fig. 3A. Chlorella sp. AT1 has an alkali resistance compared to its original strain Chlorella sp., as shown in Figure 3B. Chlorella sp. is significantly inhibited in the pH > 9 medium, at pH 11 medium. Then it does not grow. Chlorella sp. AT1 is compared with Chlorella sp., the main progress is that in alkaline medium, Chlorella sp. AT1 can maintain its growth ability and produce microalgal biomass, especially at pH 10, Chlorella sp. AT1 The advantage of alkali resistance is the most significant, as shown in Figure 4.

Example 1: Growth test of Chlorella sp. AT1 in alkaline medium absorbing carbon dioxide

In this example, a pH 11 microalgae culture solution was prepared with NaOH for testing the growth condition of Chlorella sp. AT1 in an alkaline culture solution, and the other culture conditions were the same as those of the above experiment except that the gas conditions were introduced. The gas conditions for the culture were respectively continuous air, 10% (v/v) carbon dioxide gas, and 10% (v/v) carbon dioxide for 30 minutes at intervals of 3, 6, and 12 hours. The carbon dioxide introduced in this embodiment is a purified compressed carbon dioxide in a steel bottle. During the 7-day culture, the microalgae yield of continuous air introduction was 0.073 g/L/day, and the microalgae yield of 10% (v/v) carbon dioxide continued to be 0.801 g/L/day. The microalgae yields of 10% (v/v) carbon dioxide for 30 minutes at intervals of 3, 6, and 12 hours were 0.775, 0.682, and 0.603 g/L/day, respectively, as shown in Fig. 5A. The experimental results show that carbon dioxide is introduced into the alkaline culture solution at intervals, so that the alkaline culture solution can adsorb saturated carbon dioxide to provide self-supporting growth of the microalgae. Although the interval of carbon dioxide in the alkaline culture solution treatment group has a lower microalgae yield, its utilization of carbon dioxide is significantly improved, as shown in Fig. 5B, which is very suitable for carbon dioxide source limitation, or does not want to A microalgae culture strategy that allows carbon dioxide to escape.

Example 2: Aquaculture test using Chlorella sp. AT1 in alkaline farms

In the present embodiment, the microalgae are cultured by using the pig farm to discharge water. The discharged water from the pig farm in Taiwan is treated by solid-liquid separation, anaerobic fermentation, aeration and other processes. The total nitrogen is about 500 ~ 600 ppm (about 80 ~ 90% ammonia nitrogen) and about 20-30 total phosphorus. The ppm and chemical oxygen demand (COD) are about 400 ~ 500 ppm. The gas conditions of Chlorella sp. AT1 culture are continuous air, continuous gas with 10% (v/v) carbon dioxide, and 10% (v/v) carbon dioxide for 30 minutes at intervals of 3, 6, and 12 hours. . During the 7-day culture, the microalgae yield of continuous air introduction was 0.086 g/L/day, and the microalgae yield of 10% (v/v) carbon dioxide continued to be 0.974 g/L/day. The microalgae yields of 10% (v/v) carbon dioxide for 30 minutes at intervals of 3, 6, and 12 hours were 0.954, 0.797, and 0.712 g/L/day, respectively, as shown in Fig. 6A. The experimental results show that compared with the culture liquid, the pig farm discharge water is more suitable for the cultivation of microalgae, while the alkalized pig farm discharge water, and the medium of carbon dioxide can be used to promote the growth of microalgae to produce micro. Algae biomass. In addition, in the 7-day culture, the total nitrogen in the discharged water of the farm can be reduced by about 70-80%, and the total phosphorus can be reduced by more than 90%. The effect of purifying the water is remarkable.

In the results of the present embodiment, the microalgae cultured in the pig farm can be used to re-use water, purify sewage, and reduce the cost of the medium.

In addition, in the present embodiment, the alkalized pig farm discharge water is also used to absorb the carbon dioxide in the exhaust gas of the boiler combustion (fuel is natural gas) to breed Chlorella sp. AT1, and the carbon dioxide content in the exhaust gas of the boiler is about 8 to 10% (v /v). In this embodiment, the alkalized pig farm discharge water of pH 11 is prepared by using NaOH, and the combustion exhaust gas of the boiler is directly introduced into the discharge water of the alkalized pig farm, and the saturated concentration of carbon dioxide absorbed is about 10-15% higher than that of the alkalized culture liquid. Chlorella sp. AT1 culture conditions As in the above experiment, the yield of microalgae continuously flowing into the air was 0.089 g/L/day in the 7-day culture, and the microalgae yield of the exhaust gas continuously flowing into the boiler was 0.982 g/ L/day, the microalgae yields of the boiler exhaust gas for 30 minutes at intervals of 3, 6, and 12 hours were 0.961, 0.837, and 0.706 g/L/day, respectively, as shown in Fig. 6B. The experimental results show that compared with the effect of aquaculture with pure carbon dioxide (as shown in Fig. 6A), there is a better growth trend of microalgae in the boilers that burn the exhaust gas, which may pass into the exhaust gas with the alkalized pig farm discharge water. There is a higher concentration of carbon dioxide saturation.

Therefore, the foregoing results prove that microalgae cultivation can utilize carbon dioxide in the exhaust gas to achieve the effect of biological carbon reduction.

Example 3: Semi-continuous long-term Chlorella sp. AT1 culture

The experiment was carried out in an indoor microalgae culture chamber in a 1-L photobioreactor, and Chlorella sp. AT1 was incubated with a NaOH-adjusted pH-alkali pig farm draining water, and the boiler was burned for 30 minutes every 12 hours. The algae liquid is replaced by half every 3 days. The replacement method is to recover half of the algae liquid, and then add half of the freshly prepared alkalinized pig farm release water. The semi-continuous microalgae culture is carried out for 7 cycles (21 days in total), each of which The growth of Chlorella sp. AT1 is maintained in a stable state. As shown in Fig. 7, the concentration of Chlorella sp. AT1 microalgae before the discharge of the alkalized pig farm is about 4.4 ~ 4.8 g / L, and the average microalgae production The rate is 0.798 g/L/day.

This embodiment demonstrates that the direct and intermittent introduction of the burned off gas into the boiler by the alkalized pig farm discharge water can be continuously used for the cultivation of the alkali-resistant microalgae Chlorella sp. AT1.

Example 4: Large-scale outdoor field Chlorella sp. AT1 culture

This embodiment is carried out in an outdoor solid farm system, which is an upright 60-L photobioreactor (18 cm in diameter and 250 cm in height), which can be connected in series, in parallel, or each photobioreactor can be Independently operated outdoor farming system. The experiment was conducted from October to November 2015. The climate during the 85% experiment was sunny, the rest was cloudy, the average sunshine time was 12.3 hours, and the daytime light intensity on sunny days was greater than 1,000 mmol/m ² /s. The average daily temperature is 27 ⁰ C, the average night temperature is 23 ⁰ C, and the rate of gas introduction is 0.2 vvm. The effluent from the alkalized pig farm with pH 11 was adjusted with NaOH, and the exhaust gas was burned into the boiler for 30 minutes every day. The time of the exhaust gas was taken every morning, and the concentration of Chlorella sp. AT1 was 0.3 g/L. The culture continued for 7 days. After the day, half of the freshly configured alkalinized farms were discharged. The results showed that Chlorella sp. AT1 grew faster in the first four days, the average yield was 0.264 g/L/day, and the average yield in the last three days was 0.099 g/L/day, as shown in Figure 8, the last three. The decrease in the yield of the day is due to the increase in the density of the microalgae and the significant increase in the shadowing effect. After 7 days of culture, the Chlorella sp. AT1 concentration was approximately 2 g/L. In two semi-continuous cultures, the growth of microalgae was similar, and the experimental results showed that the Chlorella sp. AT1 of the present disclosure can be cultured in an outdoor field.

The results of the foregoing examples show that the Chlorella sp. AT1 alkali-resistant microalgae strain provided by the present invention can be efficiently cultured in an alkaline environment. In addition, the carbon dioxide gas used in the cultivation, in addition to the purified carbon dioxide gas, may be an industrial waste gas generated by combustion, an exhaust gas generated by burning natural gas as a fuel, a gas generated after a biological fermentation reaction, or A carbon dioxide-containing gas that is captured after physical or chemical capture. Thereby, when the Chlorella sp. AT1 alkali-resistant microalgae strain of the present disclosure is cultured in an environment of an alkaline culture solution, the purpose of effectively reducing and reusing carbon dioxide can be achieved.

The above-mentioned embodiments are merely examples for convenience of description, and the scope of the claims is intended to be limited to the above embodiments.

no.

Figure 1A is a graph of cell lethality after 60 minutes of low concentration NTG treatment. Figure 1B is a graph of cell lethality after 60 minutes of treatment with high concentrations of NTG. Figure 2 is a graph showing the cell lethality rate after NTG treatment for different times. Figure 3A is a graph showing the growth curve of the mutant strain Chlorella sp. AT1 under different pH alkaline medium. Fig. 3B is a graph showing the growth curve of the original algal strain Chlorella sp. under different pH alkaline medium. Fig. 4 is a graph showing the growth ability of the mutant strain Chlorella sp. AT1 and the original strain Chlorella sp. under different pH alkaline medium. Figure 5A is a graph showing the growth curve of Chlorella sp. AT1 cultured in an alkaline culture solution using different aeration methods. Fig. 5B is a graph showing the results of carbon dioxide utilization efficiency of Chlorella sp. AT1 cultured in an alkaline culture solution by different aeration methods. Fig. 6A is a graph showing the growth curve of Chlorella sp. AT1 cultured in an alkaline culture solution using purified carbon dioxide gas and different aeration methods. Fig. 6B is a graph showing the growth curve of Chlorella sp. AT1 cultured in an alkaline culture solution by using exhaust gas from a boiler and different ventilation methods. Figure 7 is a graph showing the results of indoor semi-continuous long-term cultivation of Chlorella sp. AT1. Figure 8 is a graph showing the results of a large-scale semi-continuous culture of Chlorella sp. AT1 in an outdoor field.

TW Republic of China Foundation of Food Industry Development 2016/10/27 BCRC980041

Claims (13)

An alkali-resistant microalgae strain, which is deposited in the Food Industry Development Research Institute of the Republic of China, with the registration number BCRC980041. A method for reducing and reusing carbon dioxide, comprising: providing an alkali-resistant microalgae strain, which is deposited in the Food Industry Development Research Institute of the Republic of China, with the registration number BCRC980041; and cultivating the alkali-resistant microalgae strain in a base The culture medium is cultured, and the alkali-resistant microalgae strain is cultured under the condition of introducing carbon dioxide. The method of claim 2, wherein the alkaline culture solution has a pH greater than 7 and less than or equal to 11. The method of claim 3, wherein the alkaline culture solution has a pH greater than 8 and less than 11. The method of claim 4, wherein the alkaline culture solution has a pH between 9 and 10. The method of claim 2, wherein the alkali-resistant microalgae strain is cultured at a carbon dioxide concentration of 0.03-50% (v/v). The method of claim 6, wherein the alkali-resistant microalgae strain is cultured at a carbon dioxide concentration of 1-10% (v/v). The method of claim 2, wherein the alkali-resistant microalgae strain is cultured under the conditions of passing carbon dioxide and simultaneously illuminating. The method of claim 2, wherein the carbon dioxide is introduced in a batch manner. The method of claim 9, wherein the carbon dioxide is introduced every 10 to 60 minutes every 1 to 24 hours. The method of claim 2, wherein the alkali-resistant microalgae strain is cultured at an indoor or outdoor ambient temperature. The method of claim 2, wherein the alkaline broth is prepared by NaOH, KOH, NaHCO ₃ or a mixture thereof. The method of claim 2, wherein the microalgae biomass is harvested after the alkali-resistant microalgae strain is cultured.