CN110452937B

CN110452937B - Method for improving microalgae grease yield

Info

Publication number: CN110452937B
Application number: CN201910845622.7A
Authority: CN
Inventors: 高锋; 李晨; 杨红丽; 陆苗苗
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2023-05-05
Anticipated expiration: 2039-09-02
Also published as: CN110452937A

Abstract

The invention provides a method for improving the yield of microalgae grease, which belongs to the field of bioengineering and bioenergy, and the microalgae is cultivated by light energy heterotrophic culture, wherein the microalgae is chlorella, and the initial concentration of neohesperidin in a culture medium used by the heterotrophic culture medium is 0.75-1.1g/L. The method has no obvious toxic or side effect on the growth of microalgae, can promote the expression of ME coding genes ME g4297 and ME g6562 while ensuring higher biomass of the microalgae, increases the supply of intracellular electron energy, and provides more NADPH for fat synthesis; promoting the expression of DGAT coding genes DGAT g3280 and DGAT g7566, improving the synthesis amount of TAG, improving the content of grease and improving the yield of grease.

Description

Method for improving microalgae grease yield

Technical Field

The invention belongs to the field of bioengineering and bioenergy, and particularly relates to a method for improving microalgae grease yield.

Background

Microalgae were used as an alternative fuel to fossil fuels during the first petroleum crisis in the early 70 s of the 20 th century, however, because of the excessive cost, no intensive research has been done at that time. Later, microalgae are widely paid attention to as oil-producing raw materials again due to the problems of energy shortage and the like. The united states was the earliest country in which algae research was formally initiated and the large-scale cultivation of open sea was successfully completed. Subsequently, studies on oleaginous microalgae have raised a hot spot worldwide. Related researches are also conducted in the countries such as the united kingdom, japan and china, and the way of industrialization of microalgae biodiesel is being explored from various aspects. The main interesting index of microalgae used for biodiesel production is the oil content of the microalgae cells. Microalgae are various, and the oil content of microalgae is generally 5% -75% due to different algae species and different culture modes, and known microalgae with high oil content include eukaryotic algae such as Chlorella (Chlorella sp.), dunaliella sp.), and Nannochloropsis sp. The heterotrophic culture technique of microalgae starts later, and in 1953, lewis et al first found heterotrophic microalgae, thereby pulling open a prelude to the study of heterotrophic culture of microalgae. In the 90 s of the 20 th century, students studied and improved several heterotrophic culture medium formulas, which lay a foundation for later study. Compared with autotrophic culture, the heterotrophic culture process needs to additionally provide organic matters as a carbon source, does not need to manually increase illumination, can save space, improve the growth speed and the yield per unit volume by utilizing an industrial fermentation method, is convenient to manage through automatic control, reduces the cost of technologies such as harvesting and the like, and overcomes the defects of an autotrophic system.

The invention discloses a method for rapidly accumulating pH-regulated microalgae grease in the prior art, such as Chinese patent publication No. CN 103757064B, which comprises the following steps: (1) microalgae biomass preculture: culturing microalgae in a reactor until growth of the microalgae enters a stationary phase; (2) CO2 culture: introducing CO 2-containing waste gas into the reactor, and culturing the microalgae obtained in the step (1) until the microalgae enter a stabilization period, and continuing culturing for 2-3 days; (3) microalgae grease rapid accumulation: the CO2 gas is stopped from being introduced into the reactor, and the air is introduced until the pH value of the culture solution is higher than 10. The method solves the problem of low microalgae grease content under the condition of high concentration CO2, and realizes the rapid accumulation of grease under the condition of sufficient nitrogen source; has the advantages of high biomass density, rapid grease accumulation, low operation cost, convenience, rapidness, low energy consumption and the like.

Disclosure of Invention

The invention aims to provide a method for improving the yield of microalgae grease, which has no obvious toxic or side effect on the growth of microalgae, can promote the expression of ME coding genes ME g4297 and ME g6562 while ensuring higher biomass of the microalgae, increases the supply of intracellular electron energy and provides more NADPH for fat synthesis; promoting the expression of DGAT coding genes DGAT g3280 and DGAT g7566, improving the synthesis amount of TAG, improving the content of grease and improving the yield of grease.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a method for improving oil yield of microalgae comprises heterotrophic culture of microalgae, wherein the microalgae is Chlorella, and the initial concentration of neohesperidin in culture medium used for heterotrophic culture is 0.75-1.1g/L. The main component of microalgae oil is Triglyceride (TAG), and the biosynthesis of the microalgae oil takes fatty acid as a precursor, and the process is divided into three stages of fatty acid synthesis, TAG biosynthesis and oil body formation. The enzyme related to oil synthesis in the metabolism of central carbon, i.e. Malic Enzyme (ME), catalyzes the conversion of malic acid into pyruvic acid, and the generated pyruvic acid can enter into the fatty acid synthesis pathway, and simultaneously one molecule of NADP is added ⁺ Reduced to NADPH. Fatty acid synthetases in microalgae metabolic pathways can almost exclusively utilize the production of NADPH by malic enzymes. The neohesperidin has no obvious toxic or side effect on the growth of microalgae, and by adding the neohesperidin, the expression of ME coding genes ME g4297 and ME g6562 is promoted, the supply of intracellular electronic energy is increased, more NADPH is provided for fat synthesis, and the activity of relevant enzymes on a TAG synthesis path is improved, so that the synthesis and accumulation of intracellular grease are promoted, and the higher grease yield is obtained.

In some embodiments, the nitrogen source in the heterotrophic medium is nitrate nitrogen. When nitrate nitrogen is used as a nitrogen source, the final biomass and the grease content are high.

In some embodiments, the heterotrophic medium has a carbon to nitrogen ratio of 4.5-5.5:1. When the C/N ratio is less than 4.5, the chlorella biomass increases with increasing C/N; when the C/N ratio is greater than 4.5, the Chlorella biomass is rather decreased. When the C/N ratio is less than 5.5, the fat content increases with an increase in the C/N ratio; when the C/N ratio is more than 5.5, the fat content decreases with an increase in the C/N ratio. When the C/N ratio is 4.5-5.5:1, the biomass and the grease content are higher.

In some embodiments, fe is added at the initial stage of the heterotrophic culture ³⁺ Adding Fe at the end of heterotrophic culture index growth to a concentration of 1.5-2.5mg/L ³⁺ To a concentration of 5-7mg/L. Adding Fe with proper concentration at the initial stage of culture ³⁺ The obtained microalgae has high biomass, and high concentration of Fe is added at the end of index ³⁺ The unit grease accumulation amount obtained by the ions is higher, so that higher grease yield is obtained.

In some embodiments, tea polyphenols are added to the medium to a concentration of 1.2-1.6g/L at the end of the heterotrophic culture index growth. The bioactive molecular tea polyphenol has no obvious toxic or side effect on the growth of microalgae, and the fatty acid composition of the microalgae can be changed by adding the tea polyphenol at the end of exponential growth.

In some embodiments, the polyunsaturated fatty acid content of the microalgae fatty acid is 14-22%. The grease used for producing the biodiesel needs to contain a large amount of microalgae containing saturated fatty acid and monounsaturated fatty acid, which is an ideal raw material for preparing the biodiesel, the oxidation rate of the unsaturated fatty acid is closely related to the number and the positions of double bonds of the unsaturated fatty acid, and when the content of polyunsaturated fatty acid is higher, the oxidation stability is lower.

In some embodiments, salvianolic acid b is added to the culture medium to a concentration of 1.2-1.6g/L at the end of the heterotrophic culture index growth. Diacylglycerol acyltransferase (DGAT) is a key enzyme in the last step of Triacylglycerol (TAG) biosynthesis, and also a rate-limiting enzyme in the synthesis of TAG by acetyl coa, and plays a key role in the lipid synthesis pathway. The salvianolic acid b has no obvious toxic or side effect on the growth of microalgae, and the addition of the salvianolic acid b can promote the expression of the coding genes DGAT 3280 and DGAT 7566 of diacylglycerol acyltransferase (DGAT), improve the synthesis amount of TAG, stimulate the biosynthesis of fatty acid, facilitate the metabolic flow to the synthesis direction of TAG, improve the content of grease and improve the yield of grease.

In some embodiments, the microalgae has a grease yield of ∈ 1.13g/L. Through gene regulation and control of microalgae, synthesis and accumulation of TAG are promoted, so that intracellular grease content is improved, grease yield is improved, and the highest grease yield of the microalgae can reach 1.13g/L under the preferable condition.

In some embodiments, the heterotrophic culture is a photoautotrophic culture, the culture conditions being: the illumination intensity is 60-85 mu mol/(m) ² S) at 25-30deg.C and light-dark period of 12-14h/d. The culture in the light energy heterotrophic growth mode is not limited by light, has larger growth density and higher substrate conversion rate, and is favorable for realizing automatic culture of microalgae.

In some embodiments, the microalgae recovery method is a flocculation precipitation method, wherein the flocculant used in the flocculation precipitation method is chitosan. The chitosan is used for harvesting microalgae, and has the advantages of good flocculation effect, large flocculate volume, high flocculation rate and no secondary pollution.

The beneficial effects of the invention are as follows:

1) The invention carries out heterotrophic culture on chlorella by light energy and carries out heterotrophic culture on nitrogen, carbon nitrogen ratio and Fe of a culture medium ³⁺ The concentration is optimized to obtain higher biomass and grease content, and higher grease yield is achieved;

2) According to the invention, neohesperidin and salvianolic acid b are added at the end of the heterotrophic culture index growth of the chlorella, so that the expression of ME coding gene ME g6562 is promoted, the supply of intracellular electronic energy is increased, more NADPH is provided for fat synthesis, the expression of diacylglycerol acyltransferase (DGAT) coding gene DGAT g7566 is promoted, the synthesis of TAG is promoted, the fat content is improved, the fat yield of the microalgae is improved, and the fat yield of the microalgae is not less than 1.13g/L under the preferable condition;

3) According to the invention, tea polyphenol is added at the end of the heterotrophic culture index growth of the chlorella, so that the fatty acid composition of the microalgae is changed, the content of polyunsaturated fatty acid in the fatty acid of the microalgae is 14-22%, the oxidation stability is improved, and the quality of biodiesel is improved.

Drawings

FIG. 1 is a graph showing the growth of Chlorella according to the present invention;

FIG. 2 is an expression abundance diagram of the chlorella ME isozyme gene ME g4297, ME g6562 of the invention;

FIG. 3 is a graph showing the expression abundance of the chlorella DGAT isozyme gene DGAT 3280, DGAT g7566 of the present invention;

FIG. 4 shows the algae density-OD of the present invention ₆₈₀ Standard curve and linear regression equation;

FIG. 5 shows the oil content-OD of the present invention ₅₃₀ Is a standard curve and a linear regression equation of (2);

FIG. 6 is a graph showing the biomass, oil yield and oil content of Chlorella according to the present invention.

Detailed Description

The invention is described in further detail below with reference to examples:

example 1:

a method for improving microalgae grease yield comprises the following steps:

the microalgae are chlorella obtained by sampling and screening in a laboratory.

A heterotrophic culture medium was prepared and the pH was adjusted to 7-7.8, the formulation of which is shown in table 1.

TABLE 1 heterotrophic culture medium formulation

Inoculating sterile algae into large test tube containing 20ml heterotrophic culture medium, culturing for one week, transferring the culture solution for one week into culture bottle containing 200ml culture solution, and shaking at 120rpm under illumination of 80 μmol/(m) ² S) at 26℃with a photoperiod of 12h/d and a cultivation period of 18d, the absorbance of which was measured at 680nm by a spectrophotometer every 1d, to prepare a growth curve of Chlorella, see FIG. 1. From FIG. 1, it can be seen that Chlorella reached the stationary phase at 13 days of cultivation.

Inoculating the culture solution to heterotrophic culture medium at 10% (v/v) of 10d (logarithmic growth phase) under illumination of 80 μm ^-2 s ^-1 The culture temperature is 26 ℃, the photoperiod is 12h/d,culturing on a culture rack, shaking culture solution for 3-5 times every day to prevent uneven light receiving of algae solution due to adherence, and continuously adding FeCl when the culture is at the end of exponential growth ₃ ·6H ₂ O to Fe ³⁺ The concentration is 6mg/L, tea polyphenol is added to the concentration of 1.4g/L, salvianolic acid b is added to the concentration of 1.2g/L, and the culture period is 14d.

Taking out the algae liquid, regulating the pH of the algae liquid to 6 by using acetic acid, adding 10mg of chitosan into each liter of the algae liquid, stirring for 3min at 300r/min by using a mechanical stirrer, standing for 10min, and filtering and separating to obtain the precipitated algae.

Comparative example 1:

the heterotrophic medium was free of neohesperidin and the remainder was identical to example 1.

Comparative example 2:

salvianolic acid b was not fed at the end of exponential growth, and the remainder was identical to example 1.

Comparative example 3:

the heterotrophic medium contained no neohesperidin, no salvianolic acid b was added at the end of exponential growth, and the remainder was completely identical to example 1.

Test example 1:

detection of expression levels of Chlorella Malatase (ME) isozyme genes ME g4297, ME g6562 and diacylglycerol acyltransferase (DGAT) isozyme genes DGAT g3280, DGAT g 7566:

referring to the SV Total RNA Isolation Kit operating manual of Promega, total RNA from the harvested Chlorella was extracted, 10. Mu.L of the dissolved RNA was added to a 0.2mL RNase-free PCR tube, and the first step of reverse transcription was performed in a PCR apparatus at 65℃for 5min and cooled at 4℃for use. The reverse transcription reaction system using the random primer as the reverse transcription primer is shown in Table 2. The reaction conditions are as follows: after the reaction is completed, the mixture is stored at the temperature of minus 20 ℃ for 15min and 95 min.

TABLE 2 reverse transcription reaction system

Reagent(s)	Volume (mu L)
		RNase-free water	18
Mixed primer	2
		5×Buffer	8
RT-E Mix	2
		RNA	10

Taking the reverse transcription product obtained in the steps as a template of RT-PCR, wherein PCR amplification primers comprise:

primer of me g4297 gene:

F：GGACACAAGTGAGCGCAACCG

R：GGATGGGTCGTTGGGCAGAA

primer of me g6562 gene:

F：CGTCGTGGTTCGCATCTTTG

R：GTGTGGAACTGCTACTTAAGG

primer of dgat g3280 gene:

F：GGCTCATAGAATTCGACGT

R：ACATTCAAGAGGTCGGTG

primer of dgat g7566 gene:

F：GGGGTTCCACACCCTAGAT

R：AGCATCACAGTCGGCTGTGG

the reaction system of fluorescent quantitative PCR is shown in Table 3. The reaction conditions are as follows: pre-denaturation at 95℃for 4min, denaturation at 95℃for 15s, annealing at 60-63℃for 15s, extension at 72℃for 30s for 40 cycles. The annealing temperature of each copy gene is as follows: me g4297 is 60.4 ℃; me g6562 and dgat g7566 are 62.3 ℃; dgat g3280 was 61.6 ℃. Dissolution profile analysis: 65℃for 10s,0.5℃per second, to 95 ℃.

Ct value obtained by fluorescence quantitative PCR is 2 ^-△△Ct And (5) analyzing by a method. The expression abundance of ME isozyme gene ME g4297 and ME g6562 is shown in figure 2, and the expression abundance of DGAT isozyme gene dgat 3280 and dgat 7566 is shown in figure 3.

TABLE 3 fluorescent quantitative PCR reaction System

Reagent(s)	Volume (mu L)
		cDNA	0.2
10 mu mol/L upstream primer	0.5
		10 mu mol/L downstream primer	0.5
2×Taq PCR Master Mix	10
		ddH ₂ O	8.8

As can be seen from FIG. 3, the expression abundance of the ME g4297 and ME g6562 genes in example 1 and comparative example 2 is obviously higher than that in comparative example 1 and comparative example 3, which shows that the addition of neohesperidin to the heterotrophic culture medium can promote the expression of the chlorella ME encoding genes ME g4297 and ME g 6562.

As can be seen from FIG. 4, the expression abundance of the DGAT g3280 and DGAT g7566 genes in example 1 and comparative example 1 is significantly higher than that in comparative example 2 and comparative example 3, which shows that the addition of salvianolic acid b at the end of exponential growth can promote the expression of DGAT encoding genes DGAT g3280 and DGAT g 7566.

Test example 2:

measurement of chlorella growth:

diluting Chlorella stock solution by 2,4,8, 16 times, measuring absorbance at 680nm, weighing appropriate amount of the stock solution, centrifuging, oven drying at 120deg.C to constant weight, weighing again, calculating to obtain concentration of the stock solution at each dilution, measuring absorbance at 680nm, and drawing algae density-OD ₆₈₀ Is the standard curve of algae density-OD ₆₈₀ The standard curve and linear regression equation are shown in FIG. 4.

Determination of Chlorella oil content:

the color development method of vanillin phosphate is adopted. Preparing a vanillin phosphate solution, and measuring a sample: taking 100 mu L of algae liquid, adding 2ml of concentrated sulfuric acid, carrying out water bath at 100 ℃ for 10min, cooling for 5min in an ice water bath, adding 5ml of vanillin phosphate solution, carrying out water bath at 37 ℃ for 15min, cooling to room temperature, shaking uniformly, taking distilled water as a blank control, and measuring absorbance at 530 nm. Standard curve: oil tea seed oil is dissolved in chloroform to be used as a sample to determine the content of grease, and the content of grease-OD is drawn ₅₃₀ Standard curve of (d) oil content-OD ₅₃₀ The standard curve and linear regression equation of (2) are shown in FIG. 5. The biomass, oil yield and oil content of chlorella were calculated and the results are shown in FIG. 6.

As can be seen from fig. 6, the biomass of example 1, comparative example 2 and comparative example 3 is not significantly different, which indicates that adding neohesperidin into heterotrophic culture medium and adding salvianolic acid b at the end of exponential growth have no obvious toxic effect on chlorella; the grease content (60.4%) and the grease yield (1.13 g/L) of example 1 were significantly higher than those of comparative example 1 (42.9%, 0.79 g/L), comparative example 2 (36.7%, 0.68 g/L), and the grease content and the grease yield of comparative example 1, comparative example 2 were significantly higher than those of comparative example 3 (30.1%, 0.57 g/L), as can be confirmed by the combination of test example 1: the addition of neohesperidin in the heterotrophic culture medium can promote the expression of the chlorella ME coding genes ME g4297 and ME g6562, provide more NADPH for fat synthesis, promote the synthesis of intracellular grease and improve the grease yield; the salvianolic acid b is added at the end of exponential growth, so that the expression of DGAT coding genes DGAT g3280 and DGAT g7566 can be promoted, the synthesis amount of TAG is increased, the biosynthesis of fatty acid is stimulated, the metabolic flow is assisted to the synthesis direction of TAG, the oil content is increased, and the oil yield is increased.

The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.

The above embodiments are merely for illustrating the present invention and not for limiting the same, and various changes and modifications may be made by one of ordinary skill in the art without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions are also within the scope of the present invention, which is defined by the claims.

Claims

1. A method for improving the yield of microalgae grease is characterized by comprising the following steps: the microalgae culture is heterotrophic culture, wherein the microalgae is chlorella, and the initial concentration of neohesperidin a culture medium used for the heterotrophic culture is 0.75-1.1g/L; fe is added in the initial stage of heterotrophic culture ³⁺ Adding Fe to the final stage of exponential growth until the concentration is 1.5-2.5mg/L ³⁺ To a concentration of 5-7mg/L; adding tea polyphenol to the culture medium at the end of the heterotrophic culture index growth until the concentration is 1.2-1.6g/L, and adding salvianolic acid b to the culture medium at the end of the heterotrophic culture index growth until the concentration is 1.2-1.6g/L; the heterotrophic culture is light energy heterotrophic culture, and the culture conditions are as follows: the illumination intensity is 60-85 mu mol/(m) ² S) at 25-30deg.C with a light-dark period of 12-14h/d.

2. The method according to claim 1, characterized in that: the nitrogen source in the heterotrophic culture medium is nitrate nitrogen.

3. The method according to claim 1, characterized in that: the carbon-nitrogen ratio of the heterotrophic culture medium is 4.5-5.5:1.

4. The method according to claim 1, characterized in that: the polyunsaturated fatty acid content in the fatty acid of the microalgae is 14-22%.

5. The method according to claim 1, characterized in that: the oil yield of the microalgae is not less than 1.13g/L.

6. The method according to claim 1, characterized in that: the microalgae harvesting method is a flocculation precipitation method, wherein a flocculant used in the flocculation precipitation method is chitosan.