CN114958931B

CN114958931B - Method for increasing yield of xanthomonas oil and palmitoleic acid

Info

Publication number: CN114958931B
Application number: CN202210421754.9A
Authority: CN
Inventors: 张虎; 张成武; 高保燕
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2023-03-31
Anticipated expiration: 2042-04-21
Also published as: CN114958931A

Abstract

The invention discloses a method for improving yields of yellow algae grease and palmitoleic acid, which comprises the following steps: step 1, obtaining algae seeds from an algae seed chamber, transferring the algae seeds into a 500mL culture bottle filled with a liquid culture medium, standing and culturing for 4-5 days, wherein the illumination is 30-40 mu mol photons m ^‑2 s ^‑1 Then, continuously transferring the algae seeds to a 1000mL culture bottle filled with the liquid culture medium for standing culture for 5-7 days; step 2, inoculating the algae strains in the step 1 into a light cylindrical glass bioreactor, wherein the light intensity is 70-100 mu mol phosns m ^‑2 s ^‑1 By introducing 1% CO ₂ The air is blown to culture for 5 to 7 days; and 3, washing the algae seeds obtained in the step 2 by using a fresh liquid culture medium, and transferring the algae seeds into an 800mL cylindrical photobioreactor according to the initial inoculation density of 0.5 g/L. The method improves the biomass concentration and the contents of grease and palmitoleic acid of the hyphomycete by transferring to red light culture after green light culture for 24 hours, thereby improving the yield of the grease and the palmitoleic acid.

Description

Method for increasing yield of xanthomonas oil and palmitoleic acid

Technical Field

The invention relates to the technical field of microalgae biological resources and biology, in particular to a method for improving the biomass concentration and the contents of grease and palmitoleic acid of hypusillosis schneideriana by culturing hypusillosis with an LED (light-emitting diode) and transferring the hypusillosis to red light culture after culturing for 24 hours by green light so as to improve the yield of the grease and palmitoleic acid.

Background

Palmitoleic acid (POA) is a hexadecanoic unsaturated fatty acid containing 1 double bond, which is one of the omega-7 monounsaturated fatty acids. It has important application value in the fields of medicine, health products, aquatic feed, chemical products, biological energy and the like. However, the traditional method for producing POA by using wild plants such as sea buckthorn fruit and macadamia nut as raw materials is not only limited by factors such as season and geographical distribution, but also has low oil content in seeds, poor agronomic characters, low yield, difficulty in harvesting and the like, and is difficult to meet the large demand of POA for human consumption and industrial use. Researches find that filamentous microalgae, namely chrysophyceae parvulus (Tribonema minus), can accumulate a large amount of grease under specific culture conditions, and the POA content in the total fatty acids can reach more than 30%. Meanwhile, the xanthoceras fulva also has the advantages of high growth speed, high biomass concentration, strong protozoan predation resistance and easy harvesting, so that the xanthoceras fulva becomes an ideal raw material for producing grease and POA, and is expected to meet the huge demand of the market through large-scale culture. As a photoautotrophic organism, light conditions such as light quality, light intensity, light cycle and the like can all influence growth of the chrysosporium parvum and accumulation of oil and fat and POA, wherein a light quality conversion strategy has the characteristics of simplicity in operation, automation control and easiness in industrial production, but no method for improving yield of the chrysosporium parvum oil and palmitoleic acid through light quality conversion exists in current reports.

Disclosure of Invention

In order to improve the yield of grease and POA of the yellow-brown algae and reduce the production cost of the yellow-brown algae, the invention discovers that the yellow-brown algae is cultured by green light for 24 hours to ensure that the algae filament is completely divided into the daughter cells, and then the yellow-brown algae is cultured under red light, so that the growth of the yellow-brown algae can be obviously promoted, the yield of the grease and the POA can be improved, and the technical support is provided for the high-efficiency production of the grease and the POA by utilizing the microalgae.

The invention discovers that when the yellow-light algae is cultured in the columnar photobioreactor, the white light, the red light and the blue light of the LED are all beneficial to the rapid growth of the yellow-light algae and accumulate the biomass with higher concentration; the growth rate of the yellow-light and green-light algae is slower, and the concentration of the accumulated biomass is lower (fig. 1). In addition, the total lipid and POA content accumulated by the yellow-light alga was highest under white light and red light conditions, and was lower under green light conditions and yellow light and blue light conditions (FIG. 2). But under green and yellow light the phycofilaments rapidly divided into multiple subcells (fig. 3). We have taken the example of algal cells under green light and found that algal filaments completely divide into daughter cells within 24 hours of culture (FIG. 4). The degree of mixing of algal fluids, light transmittance and the number of photons received by each cell change after the large algal filament is divided into small daughter cells. We adopted the light-quality transformation strategy, first cultured the chrysophyceae in green light for 24 hours to make the mycelium completely split into daughter cells, then transferred to white light, red light and blue light to culture, found that the biomass concentration in red light (GR) is significantly higher than the results in white light (GW) and blue light (GB) (fig. 5A). In addition, the grease and POA contents under GR condition were higher than those under GW and GB conditions (FIGS. 5B-C). In addition, the yields of biomass, grease and POA of the yellow croaker under the several light qualities are compared, and the results under GR conditions are the highest (Table 2), so that the light quality conversion culture strategy is an effective method for improving the yields of the yellow croaker grease and the POA and has important application prospects.

The purpose of the invention is realized by the following technical scheme:

to achieve the above object, the algal species required for the present invention was purchased from the algae culture Collection center (SAG) of the university of Gettingen, germany (http:// www.uni-goettingen.de/en/45175. Html). The algae species are preserved in optimized mBBM medium (1) Table 1mBBM medium formula

Adding the above components into 1L deionized water, and sterilizing at 121 deg.C for 20 min.

The specific culture process is as follows:

step 1, after obtaining algae seeds from an algae seed chamber, transferring the algae seeds into a 500mL culture flask filled with a liquid culture medium, standing and culturing for 4-5 days, wherein the illumination is 30-40 mu mol photons m ^-2 s ^-1 Then, continuously transferring the algae seeds to a 1000mL culture bottle filled with the liquid culture medium for standing culture for 5-7 days, and carrying out next step of seed expansion;

step 2, inoculating the algae strains in the step 1 into a light column-shaped glass bioreactor with the diameter of 6cm, wherein the culture medium is the liquid culture medium in the step 1, and the light intensity is 70-100 mu mol phosns m ^-2 s ^-1 Introduction of a gas containing 1% of CO ₂ The air is blown to culture for 5 to 7 days;

step 3, washing the algae seeds obtained in the step 2 by using a fresh liquid culture medium, transferring the algae seeds into an 800mL cylindrical photobioreactor according to an initial inoculation density of 0.5g/L, wherein the culture medium is the liquid culture medium, culturing the algae seeds under green light or yellow light for 24h, performing light-to-mass conversion culture, transferring the algae seeds to white light, red light or blue light for culture, and introducing CO with the content of 1% according to 0.2vvm in the culture process ₂ Is aerated and mixed.

In the step 3, the culture is firstly carried out for 24 hours under green light, and then the culture is transferred to red light to produce the grease and the POA.

The liquid culture medium is mBBM liquid culture medium.

Compared with the prior art, the invention has the following advantages and effects:

(1) The invention provides a method for efficiently producing grease and POA (pre-oxidized lipoid) by utilizing chrysophyceae, wherein the highest yield of biomass, grease and POA after light-quality conversion is improved by more than 30% compared with the result without light-quality conversion.

(2) The method for producing POA by using the Aphanizomenon flavum has the advantages of greenness and safety, and the raw material source is not limited by geography; meanwhile, the method has no season limitation, can be used for culturing and continuously harvesting in an indoor photobioreactor all year round, and can meet huge raw material gaps required by oil and POA production.

(3) The invention provides a method for efficiently producing grease and POA (pre-oxidized acrylic acid), which can remarkably improve the yield of the grease and the POA by converting the light quality of an LED (light-emitting diode). The method is simple, efficient, easy for industrial production and suitable for large-scale production.

Drawings

FIG. 1 is a spectrum (A) of five LED light qualities and a growth curve of Sphaerotheca miniata under the 5 light qualities.

FIG. 2 shows the total lipid changes (A) of the Sphaerotheca parvula in 5 light qualities and the comparison (B) of the POA content at the end of the culture.

FIG. 3 shows the cell microscopic morphology change of the yellow croaker algae of the present invention under 5 different light qualities.

FIG. 4 shows the cell morphology change of Sphaerotheca parvula in the present invention when transferred to white, red and blue light cultures, respectively, after 24 hours of green light culture.

FIG. 5 is a graph showing the growth curve and the contents of fats and POA of the strain of the present invention after the conversion of light quality.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The specific culture process is as follows:

step 1, obtaining algae seeds from an algae seed chamber, transferring the algae seeds into a culture bottle (500 mL) filled with 250mL mBBM liquid culture medium, standing and culturing for 4-5 days, wherein the illumination is 30-40 mu mol photons m ^-2 s ^-1 (ii) a Then, continuously transferring the algae seeds to a 1000mL culture bottle filled with an mBBM liquid culture medium for standing culture for 5-7 days, and carrying out next step of seed expansion;

step 2, inoculating the algae strain in the step 1 into a light column-shaped glass bioreactor (length =60 cm) with the diameter of 6cm, wherein the culture medium is mBBM liquid culture medium, and the light intensity is 70-100 mu mol photons m ^-2 s ^-1 Introduction of a gas containing 1% of CO ₂ The air is blown to culture for 5 to 7 days;

step 3, washing the algae seeds obtained in the step 2 by using a fresh mBBM liquid culture medium, transferring the algae seeds into the 800mL cylindrical photobioreactor according to the initial inoculation density of 0.5g/L, wherein the culture medium is the mBBM liquid culture medium, and the light quality is set to be full-spectrum white light and red light (lambda) _max =660 nm), yellow light (λ) _max (= 595 nm), green light (. Lamda.) _max =520 nm) and blue light (λ) _max =460 nm) light intensity 300. + -. 25. Mu. Mol photons m ^-2 s ^-1 . In the light-quality conversion experiment, after being cultured for 24 hours under green light, the culture is directly transferred to white light, red light and blue light respectively. The content of CO during the culture was 1% by passing through the medium at 0.2vvm ₂ Is aerated and mixed.

And 4, regularly measuring the biomass concentration every day in the culture process, reserving samples every 3 days, collecting algae cells by centrifugation (3500g, 5min), and then carrying out vacuum freeze-drying to obtain freeze-dried algae powder.

Example 1: the cell growth and the change of grease and POA accumulation are compared when the yellow-brown algae is cultured under five different LED light qualities, and the experimental materials are as follows: and (4) yellow silk algae (which is subjected to seed expansion culture).

Preparation of culture medium (formula see table 1): adding the above components into 1L deionized water, and sterilizing at 121 deg.C for 20 min.

The test method comprises the following steps:

washing the obtained algae seeds with a fresh mBBM liquid culture medium, transferring the algae seeds into the 800mL cylindrical photobioreactor according to an initial inoculation density of 0.5g/L, wherein the culture medium is the mBBM liquid culture medium, and the light quality is set to be full-spectrum white light and red light (lambda) _max =660 nm), yellow light (λ) _max (= 595 nm), green light (. Lamda.) _max =520 nm) and blue light (λ) _max =460 nm), corresponding to an intensity of 300. + -.25. Mu. Mol photons m ^-2 s ^-1 . The content of CO during the culture was 1% by passing through the medium at 0.2vvm ₂ Aeration and mixed culture was performed for 12 days with three replicates per set of experiments. The biomass concentration was measured periodically every day, samples were kept every 3 days, and algal cells were collected by centrifugation (3500g, 5min). And (4) after the algae are subjected to vacuum freeze drying, determining the contents of grease and POA in the algae powder.

The total lipid content is measured by adopting a differential weight method, accurately weighing 45-55mg of freeze-dried algae powder (marked as M0), putting the freeze-dried algae powder into a screw glass centrifuge tube with the volume of 15mL, putting a magnetic rotor, adding 2mL of dimethyl sulfoxide-methanol mixed solution with the volume ratio of 1. And adding 4mL of a n-hexane-ether mixed solution with the volume of 1. Adding 4mL of deionized water into a glass vial containing the combined supernatant, standing overnight for layering, carefully transferring the upper organic phase into another clean glass centrifuge tube, centrifuging at 3000r/min for 5min, sucking the supernatant into a labeled vial, concentrating with a nitrogen blower, dissolving the oil with a small amount of diethyl ether, transferring into a weighed 1.5mL Eppendorf tube (the tube is recorded as M1), and drying in a nitrogen blower until the weight is constant (recorded as M2). The calculation formula of the total lipid content is as follows: the total lipid content (% DW) = (M2-M1)/M0 × 100%.

The fatty acid composition and content are measured as follows: accurately weighing 25mg of freeze-dried algae powder, putting into a screw glass centrifuge tube with a volume of 15mL, and putting into a magnetic rotor2mL portions of 2% H ₂ SO ₄ The method comprises the following steps of (1) taking 100 mu L of 0.25% heptadecanoic acid (C17: 0) as an internal reference, filling inert gas argon, sealing, magnetically stirring at 80 ℃ for 1.5h to perform fatty acid methyl esterification, cooling, adding 1mL of n-hexane and 1mL of deionized water, centrifuging at 3000r/min for 5min, taking 200 mu L of an upper-layer organic phase, transferring the upper-layer organic phase into a gas chromatography sample bottle, and storing in a refrigerator at-20 ℃ for testing. Detecting a sample by using a high performance gas chromatograph, wherein the model of a chromatographic column is CD-2560, the specification is 100m multiplied by 0.25mm multiplied by 0.20 mu m, and the GC conditions are as follows: keeping the column temperature at 140 ℃ for 20min;4 ℃ for min ^-1 Heating to 240 deg.C, and maintaining for 20min; the sample injection amount is 1 mu L, and the injection port temperature of the FID detector is 250 ℃; the detection port temperature was 260 ℃. The composition and the content of the fatty acid in the sample can be calculated according to the peak-appearing time and the corresponding peak area of the internal reference fatty acid (heptadecanoic acid).

It was found that the red, green and blue light have narrower spectra, the yellow light has a slightly broader spectrum, the white light is the widest spectrum (fig. 1A), and the white light, the blue light, the green light, the yellow light and the red light are sequentially arranged at the peak of the line in fig. 1A from left to right. The yellow-brown algae can grow rapidly after inoculation in white light, red light and blue light culture, the culture starts to enter a plateau stage after 7 days, the biomass concentration increases slowly, and the biomass concentration under the three culture conditions is about 4.5g L when the culture is finished ^-1 (p>0.05 ); the growth speed of the chrysophyceae minitans is slower when the chrysophyceae minitans are cultured under the conditions of yellow light and green light, the biomass concentration is slightly increased, the biomass concentration increased under the green light after the chrysophyceae minitans are cultured for 7 days is higher than the increased amount under the yellow light, and the biomass concentration under the green light is 3.56g L when the culture is finished ^-1 Significantly higher than the culture result under yellow light (2.98 g L) ^-1 )(p<0.05). These results indicate that white, red and blue light are suitable for fast growth of the microcystis fulvellus, and green and yellow light can also sustain the growth of the microcystis fulvellus, but the biomass concentration obtained is low.

As shown in FIG. 2A, in white light and red light cultures, the yellow-brown algae can rapidly accumulate a large amount of oil after 3 days of culture, and the total lipid content can reach 48% DW at the end of culture. The lipid accumulation amount of Sphaerotheca fuliginea is reduced in the blue, yellow and green light cultures, wherein the total lipid content is the lowest in the blue and yellow light cultures, and only 33% DW (p < 0.05) at the end of the culture. The total lipid content in the green light was significantly increased compared to the yellow and blue light cultures, up to 39% DW at the end of the culture (p < 0.05). The change trends of the POA content and the total lipid content of the yellow-light alga in different light qualities are basically consistent, namely the POA content in white light and red light cultivation is highest (more than 18% DW), the POA content in green light cultivation is lower (16.8% DW), and the POA content in yellow light and blue light is lowest (about 12 DW) (p < 0.05) (FIG. 2B).

As shown in FIG. 3, in white light cultivation, the mycelium of the Boerhavia fulvescens is filamentous in the whole cultivation period, the algae cells gradually change from deep yellow green to light yellow green along with the increase of the cultivation process, the oil droplets in the cells gradually grow, and the large oil droplets in the algae cells are clearly visible (shown by red arrows) at the end of the cultivation. The change in cell morphology of the yellow alga in red light culture was similar to that in white light, and a large number of oil droplets were observed at the end of the culture. In the blue light culture, the microcystis fulvescens also appeared filamentous throughout the culture period, but a smaller number of oil droplets was observed. During the yellow light and green light cultivation, the phycofilaments of the chrysophyceae fulvidraco are broken at the H-shaped sleeve (shown by blue arrows) and are divided into a plurality of small sub-cells, and the sub-cells are gradually grown into short phycofilaments (the length is about 100-300 mu m) with 2-10 connected sub-cells along with the increase of the cultivation time, but no obvious large oil drop is seen in the phycofilaments in the late cultivation period. The results show that different light qualities have great influence on the morphology of the microcystis fulvellus, white light, red light and blue light can maintain the filament to be in a filament shape, and yellow light and green light promote the division of the filament into daughter cells.

Example 2: the light-to-mass conversion promotes production of oil and POA by Aphanizomenon flavedo.

Sphaerotheca miniata (expanded culture)

The test method comprises the following steps:

the obtained algal species were washed with fresh mBBM liquid medium and then used as an initial 0.5g/L mediumInoculating density is transferred to the 800mL columnar photobioreactor, a culture medium is mBBM liquid culture medium, the algal filaments are completely divided into daughter cells after being cultured for 24 hours under green light, and then the algal filaments are directly transferred to white light, red light and blue light to be cultured respectively. The content of CO during the culture was 1% by passing through the medium at 0.2vvm ₂ The culture was performed for 12 days with aeration and mixing by compressed air, and three replicates were set up for each set of experiments. The biomass concentration was measured periodically every day, samples were kept every 3 days, and algal cells were collected by centrifugation (3500 g,5 min). And (4) after the algae are subjected to vacuum freeze drying, determining the contents of grease and POA in the algae powder. The result shows that after the yellow-brown algae are inoculated for 4 hours, a large number of zooblast cells can be observed, the algae filament body is obviously shortened (shown by a black arrow) and is broken at the H-shaped sleeve sheet; after 8H of culture, the phycofilaments are further reduced, and the visual field is filled with daughter cells and broken H-shaped nest plates; the phycofilaments are further reduced after 12h of culture, and the residual phycofilaments are empty shells; after 24h of culture, all the daughter cells were observed in the visual field, and the volume of the daughter cells began to increase (FIG. 4). These results indicate that the filament morphology of the xanthomonas campestris responds very rapidly to green light, and that the filament is almost completely converted into daughter cells after 24h of culture.

As shown in FIG. 5A, there was less increase in biomass concentration in the green culture of Sphaerotheca fuliginea for 24h (day 1); the green light is transferred to white light (GW), red light (GR) and blue light (GB) respectively, and when the green light is cultured to the 2 nd day, the growth has obvious lag phase; thereafter algal cells can grow rapidly, but the growth rate is fastest in GR culture, the next time GW is, and the slowest in GB. The culture reaches a plateau after 9 days, and the biomass concentration under GR can reach 5.98g L after the culture is finished ^-1 Significantly higher than the results in GW and GB (4.58 and 4.20g L, respectively) ^-1 )(p<0.05 And also higher than the results obtained without light-to-mass conversion in white, red and blue light (4.62, 4.51 and 4.26g L, respectively) ^-1 )(p<0.05 (FIG. 5B). This shows that green light is used to induce the division of algae cells first and then red light is used to culture algae to promote the accumulation of biomass.

The change in total lipid content of the yellow-brown algae after the light-quality transition is shown in fig. 5B. The microcystis fulvelloides after photoperiod transformation can accumulate a large amount of oil and fat under GW and GR, and the total lipid content can reach 46% or more DW by the end of culture. There was no significant difference between this and the total lipid content in the absence of light-to-mass conversion under white-and red-light culture conditions, respectively (p > 0.05). Furthermore, when Sphaerotheca fuliginea was cultured in GB, the total lipid content increased slowly, with the total lipid content being the lowest at the end of the culture, 34.4% DW, comparable to the previous result in blue light culture alone (32.5% DW). These results show that the final oil content is not affected by further culturing with other light quality after 24h of culturing with green light. In addition, the accumulation amount of POA at the end of the culture and the tendency of the total lipid content also agreed, i.e., the POA content was high at GW and GR culture, and reached 18% DW or more, and the POA content was lowest at GB culture, and only 12.48% DW (FIG. 5C) (p < 0.05). This was also consistent with the accumulated POA content of Sphaerotheca fuliginea cultured under each light quality when no light quality change was previously performed (p > 0.05), indicating that the light quality change did not significantly affect the POA accumulation amount of algal cells.

We analyzed the size of the above-mentioned 3 indexes of the Sphaerotheca fuliginea under different light qualities (Table 2), and found that the biomass yield of the Sphaerotheca fuliginea under GR condition is the highest, and can reach 456.3mg L ^-1 d ^-1 Significantly higher than the results (p) in other light qualities<0.05 GR) shows that GR can effectively promote the growth of chrysophyceae minitans. In addition, the total lipid yield and POA yield of the yellow-brown algae are highest under GR condition and reach 231.5 mg L and 97.9mg L respectively ^-1 d ^-1 The total lipid and POA yield of the yellow-brown algae can be effectively improved under the GR condition. Therefore, the method for efficiently producing the grease and the POA by adopting the light-quality transformation strategy is beneficial to the yellow-brown algae by culturing for 24 hours under green light, dividing the algae filament into the daughter cells and then transferring the algae filament into red light for culturing.

TABLE 2 comparison of Biomass, total lipid and POA yields of Sphaerotheca parvula in different photoplasmic cultures

The foregoing is illustrative of the preferred embodiments of the present invention, and it will be appreciated by those skilled in the art that various changes and modifications may be made without departing from the principles of the invention, which are to be considered within the scope of the invention.

Claims

1. A method for improving the yield of yellow algae grease and palmitoleic acid is characterized in that: the method comprises the following steps:

step 1, obtaining algae seeds from an algae seed chamber, transferring the algae seeds into a 500mL culture flask filled with a liquid culture medium, standing and culturing for 4-5 days, wherein the illumination is 30-40 mu moloptonsm ^-2 s ^-1 Then, continuously transferring the algae to a 1000mL culture flask filled with the liquid culture medium, standing and culturing for 5-7 days, and carrying out next step of expanding;

step 2, inoculating the algae seeds in the step 1 into a light columnar glass bioreactor with the diameter of 6cm, wherein the culture medium is the liquid culture medium in the step 1 and the light intensity is 70-100 mu moloptonsm ^-2 s ^-1 Introduction of a gas containing 1% of CO ₂ The air is blown to culture for 5 to 7 days;

step 3, washing the algae seeds obtained in the step 2 by using a fresh liquid culture medium, transferring the algae seeds into an 800mL cylindrical photobioreactor according to the initial inoculation density of 0.5g/L, wherein the culture medium is the liquid culture medium, culturing the algae seeds under green light for 24 hours, then transferring the algae seeds to red light for culturing and producing grease and palmitoleic acid, and introducing 1-percent CO according to 0.2vvm in the culture process ₂ Is aerated and mixed.

2. The method for increasing production of oil and palmitoleic acid from filaggrin as claimed in claim 1, wherein: the liquid culture medium is mBBM liquid culture medium.