CN106754389B - A method of cultivating microalgae - Google Patents

Abstract

The invention discloses a kind of methods for cultivating microalgae.It is that microalgae is inoculated into culture medium to cultivate, and stream adds or adds Phos to culture in batches and terminates during the cultivation process.The present invention is by largely studying; fully consider micro algae growth and oil and fat accumulation characteristic; it is proposed a kind of method that low-phosphorous fed-batch cultivation microalgae obtains grease; while capable of guaranteeing that microalgae grease content quicklys increase; obtain higher Biomass yield; efficiently solve the contradiction between microalgae cell oil content, biomass and lipid-producing; greatly improve the production efficiency of microalgae grease; can should be during the large-scale cultivation of microalgae, the algal gel of acquisition can be used in the fields such as health care of food, feed, bioenergy and cosmetics.

Description

A method of cultivating microalgae

Technical field:

The invention belongs to the field of oil preparation by microalgae, and particularly relates to a method for culturing microalgae.

Background technique:

Microalgae are a class of lower aquatic plants with diverse phylogenies, small individuals, usually unicellular or multicellular populations, capable of photosynthesis. Many types of microalgae can accumulate a large amount of storage oils. Studies have shown that microalgae storage oils have broad application prospects in the fields of bioenergy, cosmetics, medicines, functional foods, and feed additives.

Microalgae cannot accumulate too much storage oil under the conditions of sufficient nutrition and suitable environment. Only under stress conditions (environmental stress, nutrient stress), cells can accumulate storage oil in large quantities and quickly. The above-mentioned characteristics of microalgae limit its inability to obtain microalgal biomass with high oil content through the complete nutrient culture mode. A large number of studies have shown that although microalgae can be cultivated in a complete nutrient culture mode, although a higher biomass yield can be obtained, the oil content of the biomass is low, which is difficult to meet the needs of oil extraction. The stress conditions that can increase the lipid content of microalgae mainly include: low nitrogen stress, low phosphorus stress, high salt stress, high temperature stress, pH stress, hormones, oxidants, high light intensity stress, etc. Among the above stress conditions, nitrogen stress is the most widely studied. It is used by research institutions to study the mechanism of microalgae and obtain a small amount of oil-containing algal powder, and there is no example found in large-scale aquaculture.

There are two ways to realize low nitrogen stress: "initial low nitrogen stress" and "nitrogen sufficiency-nitrogen starvation two-step method". The initial low nitrogen stress is to add a low concentration of nitrogen elements at the beginning of the culture, and no nitrogen elements are added during the whole culture process. The improvement is limited. At the same time, due to the influence of nitrogen deficiency, the overall metabolic process of microalgae cells is disordered, which is not conducive to the long-term and continuous cultivation of microalgae. Nitrogen sufficiency-nitrogen starvation two-step method firstly uses a complete nutrient medium to cultivate microalgae to rapidly proliferate, and then collects algal cells by centrifugation or filtration, and then re-inoculates the collected algal cells into nitrogen-free or low-nitrogen medium Among them, nitrogen starvation or nitrogen limitation culture induces the accumulation of microalgal lipids, and the process of collecting algal cells for re-inoculation consumes a lot of energy, which is difficult to achieve after large-scale scale-up. Therefore, the lipid induction method under low nitrogen stress cannot be applied to the large-scale production of microalgae so far.

Phosphorus is one of the indispensable elements for the normal growth of microalgae. It is the main element that constitutes cell genetic material (DNA and RNA), energy currency (ATP), reducing power (NADPH), cell membrane and certain proteins. The lipid accumulation of algae also has a significant impact. Reducing the phosphorus source can promote the decomposition of carbohydrates and convert them into lipids, which can significantly increase the lipid content. Phosphorus is the main element of cell genetic material (DNA, RNA), and low phosphorus inhibits cell division. Because the low phosphorus condition has little effect on the metabolism of proteins and pigments, the fed addition under the low phosphorus condition can significantly increase the oil content and yield while ensuring the normal growth of cells, and at the same time, it can significantly improve the long-term and continuous culture of microalgae. Less affected. At present, there is no relevant report on the improvement of microalgae oil accumulation by low phosphorus flow addition.

How to effectively cultivate microalgae to obtain higher oil yield, several related technological inventions have been announced at home and abroad, but these technologies cannot be well promoted and used.

(1) A low-phosphorus medium for the heterotrophic culture of Chlorella (CN200710047684.0), the invention relates to a low-phosphorus medium for the heterotrophic culture of Chlorella, and the KH ₂ PO ₄ concentration in the medium is 10-1000mg/L, the dosage of KH ₂ PO ₄ is only 1.6%-16% of the original Basal formula, but the inoculum amount, specific growth rate, biomass yield, growth trend and culture time of Chlorella culture reach the original Basal culture. quite level. This patent belongs to the heterotrophic culture mode, and there is a big difference in nutritional requirements between heterotrophic culture and photoautotrophic culture. There is no conflict between this patent and the patent of the present invention in terms of technology and protection scope.

(2) A cultivation method for improving oil production of microalgae (CN201410386845.9), which discloses a cultivation method for improving oil production of microalgae. Hydrogen peroxide (H ₂ O ₂ ) at a final concentration of 0.35-12 mM was added to the microalgal medium to induce rapid accumulation of microalgal lipids. The method can shorten the cultivation period, reduce the production cost, is environmentally friendly, has convenient operation and obvious effect, and is suitable for the large-scale cultivation of microalgae for oil production. The safety of the microalgae powder obtained by this patent needs to be further evaluated, and there is no conflict with the patent of the present invention in terms of technology and protection scope.

(3) A method for increasing the biomass and oil accumulation of oil-producing microalgae by using a two-stage cultivation strategy of concurrent nutrition and nitrogen enrichment-nitrogen deficiency (CN201110192159.4), the invention adds a certain concentration of organic carbon source glucose to the autotrophic medium , and CO ₂ as an autotrophic carbon source for co-cultivation. At the same time, under nitrogen-enriched conditions, after culturing the microalgae at a high density to a stable stage, the algal liquid was transferred to a nitrogen-deficient medium for continued cultivation. This method is difficult to scale up, and there is no conflict with the patent of the present invention in terms of technology and protection scope.

(4) A two-stage pH adjustment culture method for improving microalgae oil production (CN201310492806.2), the invention discloses a pH two-stage adjustment culture method for improving microalgae oil production, the first stage makes the pH of the medium It is fixed at the most suitable value for algal growth to obtain the maximum biomass accumulation; in the second stage, the pH of the medium is fixed at the most suitable value for the oil accumulation of the algae to obtain the maximum oil accumulation, so that the maximum oil accumulation can be achieved. On the basis of biomass, strive for the maximum oil accumulation, that is, to obtain the maximum total oil content and increase the production of biodiesel. This technology may have the problem of adaptability of algae species, the cost of increasing and fixing pH control is high, it is difficult to promote, and there is no conflict with the patent of the present invention in terms of technology and protection scope.

(5) A method for improving oil yield of microalgae (CN201410441942.3), firstly, microalgae are subjected to photoautotrophic culture in an improved medium with plant hormones, and the algal bodies obtained by centrifugation are used as seeds after the stable period Light stress and nitrogen stress culture were carried out in stress medium. The safety of algal sludge obtained by this technology needs to be further evaluated. At the same time, there is a high-cost process of centrifugally harvesting algal bodies, which is difficult to enlarge and popularize, and there is no conflict with the patent of the present invention in terms of technology and protection scope.

(6) A culture method for promoting the accumulation of neutral lipids in autotrophic microalgae (CN201310331939.1), in which the microalgae in the late stage of proliferation and with the ability to accumulate neutral lipids are subjected to light wave conditions of 400-550 nm for neutral lipid accumulation It is difficult to carry out large-scale outdoor application of this technology, and there is no conflict with the patent of the present invention in terms of technology and protection scope.

(7) a kind of method (CN201510657796.2) of high temperature stress microalgae accumulating grease rapidly, in the reactor, cultivate the microalgae until the growth of the microalgae enters the end of the logarithmic growth phase, and the above-mentioned microalgae is placed in a high temperature environment. Stress cultivation for 1-2 days and centrifugation to obtain microalgae with high oil content. This technology has high high temperature control costs, technical difficulties, and is difficult to carry out outdoor large-scale application, and there is no conflict with the patent of the present invention in terms of technology and protection scope .

(8) A method (CN201410649618.0) for promoting the rapid accumulation of oil in microalgae, by adding hormones to increase the oil content, and a culture method for promoting oil accumulation in microalgae (CN201510219454.2) by adding N-cyclohexyl- 2-Aminoethanesulfonic acid improves the oil yield of microalgae. The safety and environmental safety of algal flour obtained by these two methods need to be further evaluated, and there is no conflict with the patent of the present invention in terms of technology and protection scope.

(9) The method for cultivating microalgae and the method for producing oil (CN201310229352.X), the algal liquid after inoculation adopts ultra-high nitrogen source to cultivate, the nitrogen fertilizer concentration in terms of nitrogen atom is greater than 30mmol/L, and the photoautotrophic culture is more than 10 days Harvest, this patent may have problems of adaptability of algae species, high cost and environmental safety, and there is no conflict with the patent of the present invention in terms of technology and protection scope.

It can be seen that the disadvantages of the prior art are as follows

(1) The oil production is low. Although the initial low nitrogen stress can obtain higher oil content, it cannot obtain high biomass yield. oil production.

(2) The cost is high and the realization is difficult. Although the "nitrogen sufficiency-nitrogen starvation two-step method" solves the contradiction between high oil content and low oil production in microalgae cells step by step, it requires a huge energy-consuming process of collecting algal cells between the two steps. The method is feasible in small-scale studies, but difficult to achieve in large-scale farming.

(3) Initial low phosphorus stress can also increase oil content, but phosphorus deficiency will also seriously affect the growth of microalgae. The technical limitations of low phosphorus stress are consistent with low nitrogen stress, and it is difficult to solve the oil content, biomass and oil production of microalgae cells. Therefore, there are difficulties in large-scale application.

Invention content:

The purpose of the present invention is to provide a method for culturing microalgae that can ensure the rapid increase of the oil content of the microalgae and obtain a higher biomass yield.

The method for culturing microalgae of the present invention is characterized in that the microalgae is inoculated into a medium for cultivation, and inorganic phosphorus is fed or added in batches during the cultivation process until the cultivation is completed.

The microalgae are, but are not limited to, unicellular green algae and euglena.

The medium is a seawater medium or a freshwater medium for culturing microalgae.

The culture mode of microalgae can be Erlenmeyer flask, open pond or photobioreactor. Preferably, when culturing in a flask or an open pool, the medium, calculated as NaH ₂ PO ₄ ·2H ₂ O, contains an initial phosphorus concentration of 0-4 mg L ^-1 , more preferably 0-2 mg L ^{- 1.} When it is a photobioreactor, the initial phosphorus concentration is 0-16 mg L ^-1 , and the optimal concentration is 0-8 mg L ^-1 ; the microalgae are inoculated into the culture medium by OD ₇₀₀ at 0.1 The microalgae liquid between -1.0 is inoculated into the medium for cultivation; during the cultivation process, inorganic phosphorus is added or added in batches to the end of the cultivation, and the microalgae is inoculated into the medium for 2-4 days and then added to the medium for cultivation. Add or add inorganic phosphorus in batches.

For the batch supplement of inorganic phosphorus, the amount of phosphorus added is calculated as NaH ₂ PO ₄ ·2H ₂ O, and the added amount is 0.1-3 mg L ^-1 per liter of culture medium per day.

The inorganic phosphorus is preferably any one or a mixture of inorganic phosphorus-containing compounds such as disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and phosphoric acid.

When the culture is finished, the confirmation of the time is that when the total lipid content and biomass of the microalgae no longer continue to increase, the supplementation of inorganic phosphorus is stopped. The culture period of the reactor is 10-16 days, and the optimal open-pond culture time is 10-20 days.

Preferably, when the microalgae are unicellular green algae, the medium initially contains 2 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O, and from the second day, 1 mg L ^-1 NaH ₂ PO ₄ ·2H is added every day ₂ O until the end of the incubation.

Preferably, when the microalgae is S. eustoma, the medium initially contains 8 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O, and from the 4th day, 2 mg L ^-1 NaH ₂ PO ₄ ·2H is added every day ₂ O until the end of the incubation.

After the cultivation, the algal mud with high oil content is obtained through the process of harvesting and cleaning. The algal cells are harvested by centrifugation, filtration, flocculation sedimentation, etc., and after washing, algal mud with high oil content is obtained, and the obtained algal mud can be used in the fields of food health care, feed, bioenergy and cosmetics.

Through a large number of studies, the present invention fully considers the growth and oil accumulation characteristics of microalgae, and proposes a method for obtaining oil by low-phosphorus fed-batch cultivation of microalgae, which can ensure that the oil content of the microalgae increases rapidly and at the same time obtains higher biomass production It can effectively solve the contradiction between the oil content, biomass and oil yield of microalgae cells, and greatly improve the production efficiency of microalgae oil. It can be used in the large-scale cultivation of microalgae, and the obtained algal mud can be used in food. Health care, feed, bioenergy and cosmetics and other fields.

Detailed ways:

In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

Example 1: Low-phosphorus flow additive culture of unicellular green algae improves cell oil yield

The following 10 phosphorus treatment groups were set, of which treatment groups 1-4 were one-time phosphorus addition groups, and treatment groups 5-10 were low-phosphorus flow addition groups, as follows:

Group 1: 0.5 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O was added to the culture medium at one time at the beginning of the culture;

Group 2: 1 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O was added to the culture medium at one time at the beginning of the culture;

Group 3: 2mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O was added to the medium at one time at the beginning of the culture;

Group 4: 50 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O was added to the culture medium at one time at the beginning of the culture;

Group 5: The concentration of NaH ₂ PO ₄ ·2H ₂ O in the initial medium was 1 mg L ^-1 , and from the second day, 1 mg L ^{- 1} NaH ₂ PO ₄ · 2H ₂ O was supplemented every day until the end of the culture (specifically, The initial medium contains 1 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O. From the 2nd day, NaH ₂ PO ₄ ·2H ₂ O is supplemented once a day to make the NaH ₂ PO ₄ ·2H ₂ in the medium The O concentration was increased by 1 mg L ^-1 every day until the end of the culture);

Group 6: NaH ₂ PO ₄ ·2H ₂ O concentration in the initial medium was 1 mg L ^-1 , from the second day, 1 mg L ^{- 1} NaH ₂ PO ₄ · 2H ₂ O was added every 2 days until the end of the culture ( Specifically, the initial medium contains 1 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O, and from the 2nd day, NaH ₂ PO ₄ ·2H ₂ O is supplemented every 2 days to make the NaH ₂ PO 4 in the medium _4. The 2H ₂ O concentration was increased by 1 mg L ^-1 every 2 days until the end of the culture);

Group 7: NaH ₂ PO ₄ ·2H ₂ O concentration in the initial medium was 1 mg L ^-1 , from the second day, 1 mg L ^{- 1} NaH ₂ PO ₄ · 2H ₂ O was added every 3 days until the end of the culture ( Specifically, the initial medium contains 1 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O, and from the 2nd day, NaH ₂ PO 4 ·2H 2 O is supplemented every 3 days to make the NaH ₂ PO ₄ · 2H ₂ O in the medium _4. The 2H ₂ O concentration was increased by 1 mg L ^-1 every 3 days until the end of the culture);

Group 8: NaH ₂ PO ₄ ·2H ₂ O concentration in the initial medium was 2 mg L ^-1 , from the second day onwards, 1 mg was added every day

L ^-1 NaH ₂ PO ₄ · 2H ₂ O to the end of the culture (specifically, the initial medium contains 2 mg L ^-1 NaH ₂ PO ₄ · 2H ₂ O, from the second day onwards, NaH ₂ PO ₄ is supplemented once a day · 2H ₂ O, increase the NaH ₂ PO ₄ · 2H ₂ O concentration in the medium by 1 mg L ^-1 per day until the end of the culture);

Group 9: The initial concentration of NaH ₂ PO ₄ ·2H ₂ O in the medium was 2 mg L ^-1 , from the second day onwards, 1 mg L ^{- 1} NaH ₂ PO ₄ · 2H ₂ O was added every 2 days until the end of the culture ( Specifically, the initial medium contains 2 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O, and from the 2nd day, NaH ₂ PO 4 ·2H 2 O is supplemented every 2 days to make the NaH ₂ PO ₄ · 2H ₂ O in the medium _4. The 2H ₂ O concentration was increased by 1 mg L ^-1 every 2 days until the end of the culture);

10 groups: the initial concentration of NaH ₂ PO ₄ ·2H ₂ O in the medium was 2 mg L ^-1 , from the second day onwards, 2 mg L ^-1 NaH ₂ PO ₄ · 2H ₂ O was added every 3 days until the end of the culture ( Specifically, the initial medium contains 2 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O, and from the third day, NaH ₂ PO ₄ · 2H ₂ O is supplemented every 3 days to make the NaH ₂ PO 4 in the medium _4. The 2H ₂ O concentration was increased by 2 mg L ^-1 every 3 days until the end of the culture);

Except for phosphorus element (NaH ₂ PO ₄ ·2H ₂ O), the medium composition is: 3g L ^-1 NaHCO ₃ ; 0.5g L ^-1 NaNO ₃ ; 0.02g L ^-1 CaCl ₂ ; 0.05g L ^-1 MgSO ₄ · 7H ₂ O; 0.1g L ^-1 KCl; 1mL L ^-1 A ₅ solution; 1mL L ^-1 EDTA-Fe ³⁺ , water is the solvent, and the above components can be uniformly mixed and sterilized according to the content of each component; A ₅ solution : 2.86g L ^-1 H ₃ BO ₃ , 1.80g L ^{- 1} MnCl ₂ ·4H ₂ O, 0.22g L ^-1 ZnSO ₄ ·7H ₂ O, 0.08g L ^-1 CuSO ₄ ·5H ₂ O, 0.39g L ^-1 Na ₂ MoO ₄ ·2H ₂ O, water is the solvent, and the above components can be uniformly mixed and sterilized according to the content of each component.

The OD ₇₀₀ was inoculated into the medium with 0.5 microalgae liquid, the culture temperature was 24±1℃, the light intensity was about 120μmol·m ^-2 ·s ^-1 , the light was continuous for 24 hours, and the static culture time of the triangular flask was 9 days (when the total lipid content and biomass of microalgae no longer continue to increase).

It can be seen from Table ₁ that the lipid yield of the phosphorus addition group (treatment group 5-10) was significantly higher _than that of the phosphorus one ^- time addition group ( _1-4 ). Adding 1 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O, the oil yield reached 0.35 g L ^-1 , which was significantly improved compared to the initial batch addition group.

Table 1

Example 2 Low-phosphorus flow additive cultivation of S. eucalyptus improves cell oil yield

The following 5 phosphorus treatment groups were set, of which treatment group 1 and treatment group 2 were batch phosphorus addition groups, and treatment groups 3, 4, and 5 were low phosphorus flow addition groups, as follows:

Group 1: 8 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O was added to the medium at one time at the beginning of the culture;

Group 2: 40 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O was added to the culture medium at one time at the beginning of the culture;

Group 3: The initial concentration of NaH ₂ PO ₄ ·2H ₂ O in the medium was 8 mg L ^-1 , and from the 4th day, 1 mg L ^{- 1} NaH ₂ PO ₄ · 2H ₂ O was supplemented every day until the end of the culture (specifically, The initial medium contains 8 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O. From the 4th day, NaH ₂ PO ₄ ·2H ₂ O is supplemented once a day to make the NaH ₂ PO ₄ ·2H ₂ in the medium The O concentration was increased by 1 mg L ^-1 every day until the end of the culture);

Group 4: The initial concentration of NaH ₂ PO ₄ ·2H ₂ O in the medium was 8 mg L ^-1 , and from the 4th day, 2 mg L ^{- 1} NaH ₂ PO ₄ · 2H ₂ O was supplemented every day until the end of the culture (specifically, The initial medium contains 8 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O. From the 4th day, NaH ₂ PO ₄ ·2H ₂ O is supplemented once a day to make the NaH ₂ PO ₄ ·2H ₂ in the medium The O concentration was increased by 2 mg L ^-1 every day until the end of the culture);

Group 5: The concentration of NaH ₂ PO ₄ ·2H ₂ O in the initial medium was 8 mg L ^-1 , and from the 4th day, 3 mg L ^{- 1} NaH ₂ PO ₄ · 2H ₂ O was supplemented every day until the end of the culture (specifically, The initial medium contains 8 mg L ^-1 NaH ₂ PO ₄ ·2H ₂ O. From the 4th day, NaH ₂ PO ₄ ·2H ₂ O is supplemented once a day to make the NaH ₂ PO ₄ ·2H ₂ in the medium The O concentration was increased by 3 mg L ^-1 every day until the end of the culture);

The medium is an improved f/2 medium, which is cultivated in a column-type photosynthetic bioreactor. The improved f/2 medium is prepared by using the upper seawater of the South China Sea. Except for phosphorus elements, the medium is composed of: 0.5 g L ^-1 NaHCO ₃ , 0.1 g L ^{- 1} NaNO ₃ , 4.36 mg L ^-1 Na ₂ EDTA, 3.16 mg L ^-1 FeCl ₃ .6H ₂ O, 0.01 mg L ^-1 CuSO ₄ .5H ₂ O, 0.023 mg L ^-1 ZnSO ₄ ·7H ₂ O ^, 0.012 mg L ^-1 CoCl ₂ ·6H ₂ O, 0.18 mg L ^-1 MnCl · 4H ₂ O, 0.07 mg L ^-1 Na ₂ MoO ₄ ·2H ₂ O, the solvent was The upper seawater in the South China Sea can be sterilized by mixing the above-mentioned components uniformly according to the above-mentioned components and contents.

The OD ₇₀₀ was inoculated into the culture medium with 0.5 microalgae liquid, the culture system was 300mL, 24 hours of continuous light culture, the culture period was 16d (when the total lipid content and biomass of microalgae no longer continued to increase), the light intensity was 300μmol ·m ^-2 ·s ^-1 . The salinity was controlled at 25‰, the temperature was controlled at 24±1°C, and the pH of the medium was controlled between 7.5-8.0 by continuously supplementing 1.0% _CO2 compressed gas.

As can be seen from Table 2, the lipid yield of the phosphorus feeding group (treatment groups 3-5) was significantly higher than that of the phosphorus one-time feeding group ( ¹ , 2). Adding 2 mg L ^-1 of NaH ₂ PO ₄ ·2H ₂ O can give lipid yields up to 2.03 g L ^-1 .

Table 2

Claims (2)

1. a method for cultivating microalgae, is characterized in that, microalgae is inoculated in substratum and cultivates, in cultivating process, add inorganic phosphorus in batches to cultivate and finish; The inoculation of microalgae into the culture medium is to inoculate the microalgae liquid with an OD ₇₀₀ of 0.1-1.0 into the culture medium; The cultivation mode is the flask, open pool or photobioreactor mode cultivation; When the microalgae are unicellular green algae, the medium initially contains 2 mg/L NaH ₂ PO ₄ ·2H ₂ O, and from the second day, 1 mg/L NaH ₂ PO ₄ ·2H ₂ O is added every day to the culture Finish; When the microalgae is S. eustoma, the medium initially contains 8 mg/L NaH ₂ PO ₄ ·2H ₂ O, and from the 4th day, 2 mg/L NaH ₂ PO ₄ ·2H ₂ O is added every day to the culture Finish. 2 . The method according to claim 1 , wherein, when the culture is finished, the confirmation of the time is when the total lipid content and biomass of the microalgae no longer continue to increase, and the addition of inorganic phosphorus is stopped. 3 .