RU2819445C1 - Method for cultivation of microalgae chlorella kessleri for use as biocomponent of fuel - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a method comprising mixing a suspension of microalgae in bioreactor for 13–17 minutes with a rotation frequency of 500 rpm every 120 minutes, wherein cultivation is carried out with continuous air blowing using a bubbling device with a flow rate of 1.2–1.8 l/min at temperature of 26–30 °C at surface illumination 2,700±200 lx, wherein the microalgae strain Chlorella kessleri is to be cultivated, the cultivation duration is 12–13 days, cultivation is carried out in a nutrient medium with concentration of KNO₃ 0.2 g/l at pH of nutrient medium 7.5–8.5.

EFFECT: method for cultivation of microalgae Chlorella kessleri enables to obtain a biomass with high lipid content of up to 22.3% under stress conditions of nitrogen deficiency in a nutrient medium and a CO₂ absorption rate during cultivation of up to 0.156 g/l per day.

1 cl, 5 dwg

Description

The invention relates to a method for cultivating microalgae and can be used in the oil refining industry to produce biofuels.

In the context of a continuous decrease in fossil fuel reserves, the task of finding new energy sources remains relevant. According to the modern classification of biofuels, fuel produced using microalgae is called third-generation biofuels. The main advantages of using microalgae to produce biofuels are their high growth rate, high lipid productivity, and their cultivation does not require high-quality agricultural land. Thus, third generation biofuel has sufficient potential as an alternative energy source.

The choice of conditions for cultivating microalgae is determined by the tasks of cultivating biomass and the quality of the resulting product.

There is a known method for producing biomass (RF patent No. 2541446, published 02/10/12015), including the cultivation of the unicellular green microalga Dunaliella salina using a quasi-continuous cultivation mode, with the culture grown on a modified Trenkenschuh nutrient medium using the method of enrichment cultures to a density of 1.5-3 g OM⋅l ^-1 , transferred to a quasi-continuous cultivation mode and further cultivation is carried out at a specific flow rate of the medium of about 0.3 day ^-1 under round-the-clock lighting with a surface illumination of 80 W⋅m ^-2 , continuous blowing with a gas-air mixture at a speed of 1 l of the mixture ⋅min ^-1 ⋅l ^-1 culture containing 3% CO ₂ and a temperature of 26-28°C, on a modified Trenkenschuh nutrient medium.

The described method allows the cultivation of biomass, which is a source of bioactive substances with antioxidant properties, such as lipids (polyunsaturated fatty acids), carotenoids (β-carotene, lutein, zeaxanthin, etc.), vitamins (tocopherol), etc. Unlike other algae , the cells of D. salina lack a cellulose or pectin shell and are surrounded only by a thin elastic protoplasmic membrane (plasmalemma), which significantly facilitates the absorption of algae biomass.

However, the cultivation method for growing the Dunaliella salina strain does not allow the absorption of gases with a high CO ₂ content.

An invention is known (patent CN 103952314) from the field of technology of biological energy sources related to the cultivation of the pond scendesmus Desmodesmus communes GS05 with conservation number SSTSS M2013609. The strain can effectively fix 0.03%-30% CO ₂ , has a CO ₂ fixation efficiency of 90-370 mg/l/day, biomass concentration (dry weight) of 700-3800 mg/l, oil content of 20%-43%, and fatty acid composition mainly consisting of short chain fatty acids C ₁₆ and C ₁₈ and is suitable for biodiesel production. The scenedesmus strain proposed in the invention can be used to reduce flue gas emissions and deep purification of industrial and municipal wastewater by conjugation; The removal rate of nitrogen and phosphorus from wastewater can reach 95%, and high yield of microalgae oil can be obtained, which greatly reduces the production cost of microalgae oil. The invention provides a good production strain of algae for efficient CO ₂ release from a high pollution source, wastewater treatment and clean energy production.

However, in the described method we are talking about a water strain, which is effectively grown mainly in open reservoirs.

There is a known method (patent KR 20020074029) for the biological fixation of carbon dioxide by Chlorella sp.NA-1 using a semi-continuous cultivation method in a photobioreactor, designed to remove large amounts of carbon dioxide from flue gases. The carbon dioxide fixation method involves the steps of feeding a photosynthetic strain into a tubular photobioreactor with internal lighting; introducing carbon dioxide into the photobioreactor and irradiating it with light to cultivate the photosynthetic strain under constant temperature conditions; and replacing an appropriate volume of the cell suspension with fresh culture medium when the microalgae growth is in the stationary phase after the exponential growth phase. However, to implement the proposed method, a high air flow rate is required when using a tubular reactor, which makes this method energy-intensive.

A known method according to RF patent No. 2508398, publ. 02/27/2014, in which the cultivation of the microalgae strain Chlorella vulgaris A1 123 is carried out in laboratory conditions at a temperature of 25°C in VVM medium pH 6.8 in nitrogen-limited conditions for 7 days, in a medium volume of 200 ml in 500 ml flasks, with continuous bubbling the suspension with sterile air at a speed of 200 ml/min, at illumination of 120 W/ ^m2 with a photoperiod of 16 hours. The disadvantage of this method is the low biomass growth.

There is a known method for cultivating the microalgae Chlorella, which involves cultivating the microalgae in a liquid nutrient medium under stirring and lighting conditions under the influence of a pulsed low-frequency electromagnetic field with a magnetic induction of 2000 Gauss at a pulse frequency of 10 Hz and a duration of 10 μs (ed. St. No. 1711734, publ. 15.02 .1992). The disadvantages of this method include low productivity and high energy consumption.

There is a known method for the artificial cultivation of microalgae and an installation for its implementation according to RF patent No. 2175013, publ. 06/10/2001. The cultivation of microalgae is carried out by photosynthesis when exposed to radioluminescent radiation and heat excited by penetrating nuclear radiation, and the spectrum of radioluminescent radiation can be selected to resonantly coincide with the spectrum of action of photosynthesis. An artificial source of energy is a source of penetrating nuclear radiation, a source of luminescent optical radiation is a radioluminophor, heat is generated in the environment of the source of nuclear radiation. A nuclear reactor is used as a source of nuclear radiation, including a breeder reactor with a uranium-thorium cycle, including in the form of a grid of nuclear radioluminescent lamps, which are surrounded on all sides by light-receiving cuvettes with a suspension of cultivated microalgae. The disadvantages are high energy consumption, the use of nuclear radiation, and the use of an expensive precursor component - radioluminescent phosphor.

There is a known method described in the patent JPH 10248553, which shows the possibility of obtaining algae of the genus Chlorella, consisting of a certain strain that reproduces in a CO ₂ -containing gas mixture at a certain temperature, useful for fixing CO ₂ in the exhaust gases of the combustion of a thermal power plant and is effective in preventing the phenomenon of global warming. The described method makes it possible to cultivate chlorella microalgae consisting of Chlorella sorokiniana strain HAK, Chlorella sorokiniana strain HFK, Chlorella sorokiniana strain YSK or Chlorella sorokiniana strain HHK and capable of proliferation in a mixed gas containing 0.03-40% CO ₂ at 25-40 °C can be obtained by cultivating chlorella with a slow supply of CO ₂ and separating the surviving strain.

However, despite the fact that in the considered method the emphasis is on the varieties of the Chlorella sorokiniana strain, the most active and characterized by a high rate of CO ₂ absorption is Chlorella vulgaris, and Chlorella kessleri contains a larger amount of lipids than other species.

The closest to the claimed method in terms of technical essence and achieved result is the method described in patent RU 2668162, publ. 09/26/2018 “Method for cultivating the microalgae Chlorella”, chosen as a prototype.

The known method is that a suspension of microalgae is placed in a photobioreactor, in which the suspension of microalgae is mixed for 13-17 minutes at a rotation speed of 500 rpm every 120 minutes, while cultivation is carried out with continuous air blowing using a bubbling device with a flow rate 1.2-1.8 l/min at a temperature of 26-30°C, continuous exposure to infrared radiation of 10900-11300 Lux and with surface illumination of 2200-2800 Lux with a photoperiod of 12 hours. The method ensures an increase in biomass growth with minimal energy costs.

However, this method does not allow obtaining the maximum possible lipid content in the biomass, which is achieved by a deficiency of nitrogen involved in the cultivation process. In addition, the proposed method is carried out under continuous exposure to IR radiation, which entails additional energy consumption.

The objective of this invention is to develop an effective method for cultivating microalgae that combine high lipid content and high dynamics of CO ₂ absorption and are suitable for use as a biocomponent of fuel.

The new technical result of the proposed method is an increase in the lipid content in microalgae, a high increase in biomass and the dynamics of CO ₂ absorption with minimal energy costs.

The claimed technical result is achieved by the proposed method of cultivating Chlorella microalgae for use as a biocomponent of fuel, including mixing a suspension of microalgae in a bioreactor for 13-17 minutes with a rotation speed of 500 rpm every 120 minutes, while cultivation is carried out with continuous air blowing using bubbling device with a flow rate of 1.2-1.8 l/min at a temperature of 26-30°C with surface illumination of 2700±200 Lux, while the microalgae strain Chlorella kessleri is subject to cultivation, the duration of cultivation is 12-13 days, cultivation is carried out in a nutrient environment with a KNO ₃ concentration of 0.2 g/l at a pH of the nutrient medium of 7.5-8.5.

The technical result achieved by the proposed method is due to its new properties discovered during research.

In the course of the research, the problem was solved by identifying optimal conditions for cultivating the microalga Chlorella kessleri (hereinafter C. kessleri), which has a high potential for fixing carbon dioxide from various sources with maximum potential for producing lipid oil, which is a biocomponent for biofuels.

The experimental results showed that the optimal conditions necessary for maximum biomass growth of the microalgae C. kessleri and, consequently, maximum CO ₂ absorption are:

- pH=8.0±0.5;

- lighting intensity - 2700±200 Lux;

- temperature 28±2°С;

- aeration of at least 1.2-1.8 l/min;

- nutrient medium with a KNO ₃ concentration of 0.2 g/l. The maximum absorption rate of CO ₂ is 0.156 g/l per day. The duration of cultivation is 12-13 days.

The maximum volume of CO ₂ utilized in 1 day is 5.17 liters at T=26-30°C and illumination 2700±200 Lux.

During the period of cultivation of the microalgae C. kessleri according to the proposed method, the maximum absorption of CO ₂ occurs during the exponential growth phase (12-13 days).

During the period of cultivation of the microalga C. kessleri according to the proposed method, the maximum absorption of CO ₂ occurs during the exponential growth phase (12-13 days).

The studied type of microalgae C. kessleri, obtained using the proposed method, is capable of absorbing gases with a high CO ₂ content (more than 25%).

Cultivation Duration

During the experiments, the dependence of the growth phase of C. kessleri was studied at constant aeration of 1.2-1.8 l/min, illumination of 2700 ± 200 Lux and temperature (26-30) ° C. It has been established that the growth curve of microalgae biomass (Fig. 1) can be divided into three stages: lag phase (0-2 days); exponential growth phase (3-9 days) and stationary phase (starting from 10 days), forming an S-shaped curve.

It has been established that the most optimal time for collecting C. kessleri biomass is on the 12-13th day of cultivation, i.e. at the beginning of the stationary phase. At the same time, maximum absorption of CO ₂ occurs.

The number of microalgae cells was determined by optical density measured on a KFK-3 KM spectrophotometer at a wavelength λ=750 nm.

Cultivation temperature

In this study, we determined the optimal temperature range for the growth of C. kessleri. Cultivation of C. kessleri was carried out for 12 days in photo bioreactors (PBR), where a certain temperature of the nutrient medium was maintained (22°C, 24°C, 26°C, 28°C, 30°C, 32°C). The suspension was bubbled with atmospheric air at a rate of 1.2-1.8 l/min under continuous illumination with fluorescent lamps at an intensity of 2700±200 Lux. The results of a study of the effect of temperature on the growth rate of biomass are presented in Fig. 2.

The highest biomass growth rate was achieved at a temperature of (30±1)°C. Taking into account the resistance of the microalgae C. kessleri to various temperature conditions, characterized by the ability to grow in the temperature range (25±5)°C, the recommended temperature values for cultivation are the temperature range of 26-30°C. At these temperatures, maximum CO ₂ absorption occurs.

This result was confirmed during experiments under the condition of bubbling speed (1.2; 1.4; 1.7; 1.8) l/min and with different lighting intensity conditions (2500, 2600, 2800, 2900) Lx.

Composition of nutrient medium for cultivation

During the experiments, a nutrient medium taken from the prototype was used for cultivating microalgae C. kessleri (Table 1).

However, when using this medium, positive results for lipid content were not obtained.

One of the most important nutrients affecting microalgae growth is nitrogen content. Lack of nitrogen causes a decrease in biomass growth. At the same time, the average dry weight of the cell increases, and the content of chlorophyll and protein in organic matter decreases, but the amount of carbohydrates and lipids related to nitrogen-free compounds increases.

During the experiments, the effect of reducing the concentration of KNO ₃ in the nutrient medium on the accumulation of lipids was studied. For this purpose, microalgae were cultivated in 5 PBS containing the following concentrations of KNO ₃ (0; 0.1; 0.2; 0.3; 0.4) g/l with constant aeration of 1.2-1.8 l/min and temperature 26-30°C. Biomass growth was assessed daily during the cultivation period (12-13) days. Stressful conditions of nitrogen deficiency have been found to promote lipid accumulation. The experiment was carried out in triplicate. The results of the effect of potassium nitrate concentration on the growth of C. kessleri are presented in Fig. 3.

The highest biomass yield was obtained at a KNO3 concentration _{of 0.3} g/l. In the absence of nitrates, the increase in biomass of C. kessleri was the smallest. Table 2 shows the effect of KNO ₃ concentration on the yield of lipids obtained from C. kessleri biomass.

According to the results obtained, a concentration of 0 g/l KNO ₃ made it possible to obtain a lipid yield of up to 25%, but with a minimal increase in biomass (Fig. 3). Maximum biomass yield was observed using a KNO ₃ concentration of 0.3 g/L, and maximum lipid content was obtained with a KNO ₃ concentration of 0.2 g/L along with a slight decrease in microalgae growth rate. Based on this, the proposed method uses a KNO ₃ concentration of 0.2 g/l.

Light level during cultivation

Light is known to play an important role in the growth and absorption of nutrients by microalgae. The study examined the effects of different light intensities on the growth of C. kessleri. To achieve this goal, a 1000 ml volume of PBS was used containing 500 ml of suspended microalgae C. kessleri in a nutrient medium at an initial cell concentration of 2 million cells/ml. The FBI was placed at different distances from the light source—fluorescent lamps (FLLs)—until effective light intensity was achieved. C. kessleri was cultivated for 12 days with constant aeration at a rate of 1.2-1.8 l/min and a temperature of 26-30°C. The results of the study are presented in Fig. 4.

According to the results, the increase in microalgae biomass was greatest at illumination of 2700 ± 200 Lux.

When cultivated for 13 days, this result was repeated.

pH of the nutrient medium used for cultivation

The pH value of the microalgae cultivation medium has a great influence on the accumulation of biomass. In order to determine the optimal pH for the growth of microalgae C. kessleri, the strain was cultivated in six parallel PBS containing 500 ml of nutrient medium with different pH values (7.0; 7.5; 8.0; 8.5; 9.0 ). The pH value in each PBS was maintained by adding solutions of sodium hydroxide NaOH 0.1 M (to increase pH) and hydrochloric acid HCl 1 M (to decrease pH). The cultures were continuously aerated and mixed by bubbling at different speeds (1.2; 1.4; 1.5; 1.7; 1.8) l/min at a temperature of 26°C. The number of cells on days 12-13 of cultivation in each medium is presented in Fig. 5.

It has been shown that pH=8.0±0.5 is most suitable for the cultivation of C. kessleri with maximum CO ₂ absorption. A shift in pH toward more acidic or alkaline values negatively affects the growth of microalgae.

Repeating the experiment at temperatures of 27°C and 30°C showed a similar result.

Dynamics of _CO2 absorption by microalgae C. kessleri

The assessment of the dynamics of _CO2 absorption by microalgae biomass in the study was carried out in sealed photobioreactor-biofilters (PBRB), with a capacity of 100 l, equipped with an aeration system, lighting, carbon dioxide supply, a system for measuring the composition of the gas mixture, a system for removing a suspension of microalgae biomass and temperature sensors.

Separately, the dynamics of CO ₂ absorption by a suspension of microalgae C. kessleri was studied when cultivated under stressful conditions of nitrogen deficiency (KNO ₃ concentration - 0.2 g/l). It was revealed that the absorption of CO ₂ by a suspension of microalgae C. kessleri under stressful conditions occurs most actively in the period up to 13 days. The phase of cell adaptation to the environment (lag phase) under stressful conditions occurs during the first hours. The optical density of the suspension increased throughout the experiment, however, rapid sedimentation of the biomass was noted.

Table 3 presents comparative characteristics of samples of the microalgae C. kessleri according to various factors of its cultivation.

During the experiment, the period from the maximum CO ₂ supply of 32% to its complete absorption was studied, which took 2.8 days. With the subsequent supply of CO ₂ at a maximum concentration of 32%, complete absorption occurred within up to 13 days from the start of the experiment, then the absorption rate decreased, which corresponded to a decrease in the growth of microalgae mass.

Thus, the inventive cultivation method makes it possible to obtain microalgae C. kessleri with a maximum absorption rate of CO ₂ - 0.156 g/l per day, at T=30°C and illumination of 2900 Lux. It is preferable to carry out the cultivation process under stressful conditions to obtain C. kessleri biomass with a high lipid content, recommended for the production of biodiesel. It has been confirmed that the maximum absorption of CO ₂ occurs during the exponential growth phase (days 12-13). Microalgae C. kessleri are capable of absorbing gases with a high CO ₂ content containing 32%.

The method of cultivating microalgae C. kessleri is implemented as follows.

A suspension of microalgae, which represents a solution of microalgae in a nutrient medium, is placed in a photobioreactor of the required volumes. Stirring of the suspension during cultivation is carried out for 13-17 minutes every 120 minutes. Mixing is carried out using a magnetic stirrer with a rotation speed of 500 rpm. Biomass cultivation is carried out with continuous air blowing using a bubbling device at surface illumination in the range from 2500 to 2900 Lux. For lighting, artificial light sources are used, such as fluorescent lamps, which create high irradiance. The temperature of the suspension solution is maintained in the range from 26 to 30°C using thermostats. The cultivation process is carried out at a pH value of 8.0±0.5.

Intensive bubbling of gases with a high CO ₂ content (15-39%) of a suspension of microalgae C. kessleri made it possible to intensify the processes of CO ₂ absorption by microalgae biomass, the pH of the solution was maintained in the range from 7.5 to 8.5.

Thus, the inventive method for cultivating the microalgae Chlorella kessleri makes it possible to obtain biomass with a high lipid content of up to 22.3% under stressful conditions of nitrogen deficiency in the nutrient medium and a CO ₂ absorption rate during the cultivation process of up to 0.156 g/l per day.

The microalga C. kessleri, cultivated by the proposed method, contains more lipids than other types of Chlorella microalgae strains and is recommended for use in the production of biodiesel.

Claims (1)

A method for cultivating the microalgae Chlorella for use as a biocomponent of fuel, including mixing a suspension of microalgae in a bioreactor for 13-17 minutes with a rotation speed of 500 rpm every 120 minutes, while the cultivation is carried out with continuous air blowing using a bubbling device with a flow rate of 1 ,2-1.8 l/min at a temperature of 26-30°C with surface illumination of 2700±200 Lux, characterized in that the microalga Chlorella kessleri is subject to cultivation, the duration of cultivation is 12-13 days, cultivation is carried out in a nutrient medium with a concentration of KNO ₃ 0.2 g/l at pH of the nutrient medium 7.5-8.5.