CN111100796B

CN111100796B - Scenedesmus rich in oil and culture application thereof

Info

Publication number: CN111100796B
Application number: CN201811255609.8A
Authority: CN
Inventors: 师文静; 张霖; 樊亚超; 李晓姝; 廖莎
Original assignee: China Petroleum and Chemical Corp; Sinopec Dalian Research Institute of Petroleum and Petrochemicals
Current assignee: Sinopec Dalian Petrochemical Research Institute Co ltd; China Petroleum and Chemical Corp
Priority date: 2018-10-26
Filing date: 2018-10-26
Publication date: 2021-07-09
Anticipated expiration: 2038-10-26
Also published as: CN111100796A

Abstract

The invention relates to a scenedesmus rich in oil and culture application thereof, the algae strain is scenedesmus HY-D3 which is classified and named as scenedesmus (Scenedesmus)Scenedesmus acutus) The strain has been preserved in China general microbiological culture Collection center (CGMCC) at 2018, 2 and 5, with the preservation number of CGMCC No. 15298. The scenedesmus provided by the invention can tolerate high-concentration SO₂Can use CO-containing₂And SO₂The flue gas is irradiated for autotrophic growth to obtain biomass, the carbon fixation efficiency is high, and the obtained biomass is rich in grease and can be used for producing biodiesel.

Description

Scenedesmus rich in oil and culture application thereof

Technical Field

The invention belongs to the fields of biotechnology and biological energy, and particularly relates to scenedesmus rich in oil and culture application thereof.

Background

As fossil energy is decreasing and the greenhouse effect is increasing due to the use of fossil energy, more and more researchers are focusing on the development and utilization of renewable energy. Biomass can be the most important renewable energy on earth, and comprises forestry biomass, crops, aquatic plants, agricultural wastes and the like. Among the many biomass energy sources, microalgae are important renewable resources. They have the characteristics of wide distribution, large biomass, high photosynthesis efficiency, strong environment adaptability, short growth period, high biomass yield and the like. The cells contain unique primary or secondary metabolites and are chemically complex. The solar energy conversion efficiency of the microalgae can reach 3.5 percent, the microalgae is a potential resource for producing medicines, fine chemicals and novel fuels, and fatty acid obtained from the microalgae can be converted into fatty acid methyl ester, namely biodiesel.

With the development of the world economy, the use and consumption of a large amount of fossil energy, resulting in the shortage of energy and the increasing deterioration of the environment, particularly CO₂The greenhouse effect is more and more serious due to the sharp increase of the carbon dioxide, and besides, the industrial waste gas from the petroleum and chemical industry contains high-concentration CO₂The concentration of acid gases such as SOx and NOx is high in addition to the gas (15 v% to 30 v%). Short growth period of microalgae, high photosynthetic efficiency, and CO₂High fixing efficiency which is more than 10 times of that of terrestrial plants under certain conditions, and can reduce CO₂The discharge and the culture cost are reduced; CO removal₂Besides, some SOx, NOx and other components in the exhaust gas are purified along with the metabolism of the microalgae, so that the emission of harmful gases is effectively reduced. Therefore, biodiesel produced using microalgal oil as a feedstock is currently the most likely renewable energy source for fuels needed for world transportation.

At present, more researches are carried out on oil-producing microalgae such as chlorella and scenedesmus. CN20110144545.6 discloses a ScenedesmusScenedemus sp.) The algae strain can grow by using artificial culture medium or properly treated waste water, and is characterized by that its oil yield is higher than that of most of existent algae-separating strains, and its application field includes CO₂Immobilization, purification of waste water, oils, proteins,Production of pigment, starch, polysaccharide and nucleic acid. CN201110427579.6 discloses a Scenedesmus strain (Scenedesmus sp.Koch)Desmodesmus sp.) ENN2203A, the scenedesmus has the characteristics of high temperature resistance and easy collection, has strong environmental adaptability, is suitable for high-density culture, can be used for treating sewage containing nitrogen and phosphorus, can be used for reducing carbon dioxide emission, and can be used for producing biodiesel or used as aquatic product bait or animal feed and the like. However, in the course of culturing, the algal strain was cultured by introducing a mixed gas of carbon dioxide and air at a concentration of 1.5 to 2% into the culture medium until the 16 th day, and the final dry weight was 10.93 g/L. But in practical application, when CO is in the environment₂When the volume fraction is more than 5v%, the growth of most microalgae is inhibited, and the carbon fixation efficiency is influenced. And when the introduced fossil fuel waste gas contains gases such as SOx, NOx and the like with high concentration, the growth of microalgae can be inhibited and the carbon fixation efficiency can be reduced.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides scenedesmus rich in grease and culture application thereof. The scenedesmus provided by the invention can tolerate high-concentration SO₂Can use CO-containing₂And SO₂The flue gas is irradiated for autotrophic growth to obtain biomass, the carbon fixation efficiency is high, and the obtained biomass is rich in grease and can be used for producing biodiesel.

The invention relates to a Scenedesmus HY-D3 which is classified and named as Scenedesmus (Scenedesmus sp.) (Scenedesmus acutus) The strain has been preserved in China general microbiological culture Collection center (CGMCC) in 2018, 2 and 5, with the preservation number of CGMCC 15298.

The scenedesmus HY-D3 rich in oil provided by the invention is green in algae cells under a microscope, and is often gathered into groups, the groups are usually composed of 4-8 cells, and the cells of the groups are arranged in a straight line. The single cells are oblong, oval, smooth in cell wall, containing melanosomes, each cell containing a protein nucleus, and are about 3-6 μm long and about 2-3 μm wide. The upper and lower ends of the cells on both sides of the population have 1 long or straight or slightly curved spikes, and both ends of the cells in the middle and the side free parts of the cells on both sides have no spikes. 4 cells with a population width of 10-24 microns and a puncture length of 10-13 microns.

The Scenedesmus HY-D3 rich in oil provided by the invention can tolerate CO₂Has a concentration of 40v%, and is tolerant to SO₂The concentration of (A) can reach 400 x 10^-6（v/v），SO₂The removal rate of the catalyst reaches more than 50 percent.

The 18S rDNA gene sequencing analysis result of the oil-rich scenedesmus HY-D3 provided by the invention is shown in a sequence table. According to the sequence alignment, the Scenedesmus HY-D3 of the invention has a difference with the published 18S rDNA data of Scenedesmus strains.

The culture method of the Scenedesmus HY-D3 rich in oil comprises culturing in a photobioreactor with fresh water culture medium such as BG11, SE, etc. under the conditions of illumination intensity of 1500-₂And (3) culturing the gas with the content of 5-40 v% until the stationary phase is finished, and harvesting the microalgae cells. Through detection, the dry weight of the algae cells reaches more than 8g/L, and the total lipid content of the cells accounts for more than 40 percent of the dry weight of the cells.

The scenedesmus HY-D3 of the invention is used for fixing CO₂The use of (1). The strain can tolerate CO up to 40v%₂Higher concentration of CO₂The efficiency is fixed.

The scenedesmus HY-D3 is applied to production of microalgae grease. Under the appropriate growth conditions, the total lipid content of the cells of the scenedesmus strain can account for more than 40% of the dry weight of the cells, and the production of biodiesel can be carried out.

The scenedesmus HY-D3 is used for purifying substances containing CO₂And SO₂In exhaust gases or flue gases. The strain can utilize the strain containing CO₂And SO₂The waste gas or the smoke is subjected to illumination autotrophic growth to obtain biomass rich in grease, and CO in the waste gas or the smoke₂Not more than 40% by volume of SO₂The content is not more than 400 × 10^-6(v/v). Specifically, fresh water culture medium such as BG11, SE, etc. is used for culture, and CO is introduced from the bottom of the reactor₂And SO₂The illumination intensity of the culture is 1500-.

The scenedesmus HY-D3 can also tolerate low temperature, can normally grow at the temperature as low as 10-15 ℃, and keeps higher biomass yield.

Compared with the prior art, the invention can bring the following beneficial effects:

(1) the scenedesmus HY-D3 bred by the method can tolerate high-concentration SO₂Can utilize CO in the exhaust gas₂Autotrophic growth and CO fixation₂The problem of greenhouse effect brought by the current industrial society is solved; at the same time can resist SO in the waste gas₂Avoiding the high concentration of SO when the microalgae grows by utilizing the waste gas₂Inhibiting photosynthesis of microalgae and maintaining normal growth of microalgae.

(2) The strain can be used for fixing CO in flue gas₂Meanwhile, part of SO in the flue gas can be utilized₂The sulfur is converted into biomacromolecules of algae cells through a sulfur metabolism way, and the smoke can be purified.

(3) The algal strain has the advantages of high growth rate, short growth cycle (no more than 10 days), high total lipid content in algal cells, suitability for producing biodiesel and capability of solving the problem of lipid source in biodiesel production.

Biological material preservation instructions

Scenedesmus (A) and (B) provided by the inventionScenedesmus acutus) HY-D3, deposited in China general microbiological culture Collection center; address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing; the preservation number is: CGMCC number 15298; the preservation date is as follows: year 2018, month 2 and day 5.

Detailed Description

The present invention will be described in further detail by way of examples. In the present invention, v% is a volume fraction.

Example 1 isolation and acclimatization screening of Scenedesmus HY-D3

(1) Obtaining starting algae strains: taking 150mL of water sample from Channa Argus Ming lake in 2013 in 10 months, filtering the water sample with gauze to remove large impurities, taking 50mL of the filtered water sample, inoculating into 200mL of BG11 culture medium for enrichment culture, wherein the illumination intensity of the culture is 5000Lux, the temperature is 25 ℃, the illumination period is 24h, the light-dark time ratio is 14:10, and after about half a month of culture, the culture medium is greenAnd (4) color. Diluting the water sample of enrichment culture to 10^-5And coating the mixture on a BG11 solid plate under aseptic conditions for culture, wherein the illumination intensity of the culture is 5000Lux, the temperature is 25 ℃, green single algae colonies appear on the plate after about 10 days of culture, selecting the single algae colonies to culture on a shake flask, the culture temperature is 25 ℃, the illumination intensity is 5000Lux, after 10 days of culture, observing and determining whether the single algae colonies are pure algae strains by a microscope, and repeating the steps until the pure culture algae strains are determined. The pure algae strain is numbered HY-3 after repeated culture.

（2）CO₂The domestication culture: inoculating the pure algae cultured in the shake flask in the step (1) into a microalgae aeration culture device for acclimatization culture, wherein the illumination intensity is 5000Lux, the temperature is 25 ℃, and CO in the introduced gas₂The content of (A) was gradually increased from 5v% to 40v%, each time increased by 5v%, and the culture was terminated after 10 days, and the acclimation culture was repeated 3 times.

(3) And (3) culturing the algae liquid domesticated and cultured in the step (2) in a plate streaking mode to obtain pure algae seeds, wherein the culturing step is the same as the step (1).

（4）SO₂The domestication culture: introducing CO into the algae liquid in the logarithmic growth phase of the algae strains in the step (3)₂20v% of mixed gas, culturing at 25 deg.C and illumination of 5000Lux, and injecting SO into the mixed gas₂Gas SO of algae species₂The tolerance of the culture medium is cultured, and SO in the mixed gas is generated in the culture process₂In an amount of from 50X 10^-6(v/v) is increased to 400 x 10 step by step^-6(v/v), increase by 100X 10/day during the cultivation^-6(v/v), after 10 days of culture, repeated acclimation and culture were carried out 3 times to harvest SO-tolerance₂The algal solution.

(5) And (3) obtaining pure algae colonies by adopting a plate streaking mode for the algae liquid obtained by domestication in the step (4), wherein the culture step is the same as the step (1), and after the culture is finished, selecting larger algae colonies for shake flask culture to obtain a target algae strain named HY-D3.

Example 2 identification of algal strains

Extracting DNA of HY-D3 algae cell by CTAB method, cloning 18S rDNA gene, and introducing the obtained 3 positive clones to ShanghaiCompany sequencing. The sequencing analysis result of the 18S rDNA gene is shown in a sequence table. The 18S rDNA sequence is logged into a Genbank database for Blast comparison, and the result is displayed and compared withScenedesmus acutusHas the greatest similarity, the BLASTn value is 2614, the Max index value is 99 percent, and HY-D3 can be determined as scenedesmus (Scenedesmus obliquus) (AScenedesmus acutus）。

Example 3 application of Scenedesmus HY-D3

Inoculating HY-D3 algae solution in logarithmic growth phase into BG11 culture medium (formula of BG11 shown in tables 1 and 2), culturing in photobioreactor, and inoculating OD of the culture solution₆₉₀Is 0.2. Introducing CO from the bottom of the reactor₂Flue gas with a content of 30v%, wherein SO₂The content is 400 multiplied by 10^-6(v/v). In the culture process, the illumination intensity is 5000Lux, the culture temperature is 28 ℃, the pH value is controlled between 7 and 9, the illumination period is 24 hours, the light-dark time ratio is 14:10, and the culture is in a stable period for 7 days. After the culture is finished, centrifuging and collecting algae liquid, carrying out vacuum freeze drying at the temperature of-60 ℃ to constant weight, measuring the dry weight of algae powder, and calculating the biomass yield; and adopting n-hexane: the total lipid content was determined by ethyl acetate method. Through detection, the biomass yield of the HY-D3 algal strain is 8.1237g/L, and the total lipid content of the cells accounts for 41.56% of the dry weight of the cells.

TABLE 1 BG11 culture Medium

Table 2 composition of a5+ Co solution in table 1

Example 4 comparison of growth effects of HY-D3 and HY-3

Inoculating HY-D3 and HY-3 algae solution in logarithmic growth phase into BG11 culture medium, culturing in photobioreactor, and adjusting density to OD₆₉₀Is 0.2. According to the test requirements, the CO with different contents is prepared₂And SO₂Mixing the gases, then fromThe bottom of the reactor is introduced. In the culture process, the illumination intensity is 5000Lux, the culture temperature is 28 ℃, the pH value is controlled between 7 and 9, the illumination period is 24 hours, the light-dark time ratio is 14:10, the culture time is 10 days, the algae cells are collected after the culture is finished, the dry weight of the cells is measured, and the results are shown in Table 3. As can be seen from the results, the domesticated and screened HY-D3 was CO-resistant as compared with the original algal strain HY-D3₂And SO₂Has better tolerance, good biomass stability and SO resistance₂Also has higher removal rate.

TABLE 3 different CO₂And SO₂Comparison of Effect of contents

Examples 5 HY-D3 and HY-3 Low temperature resistance

Inoculating HY-D3 and HY-3 algae solution in logarithmic growth phase into BG11 culture medium, culturing in photobioreactor, and culturing to OD₆₉₀Is 0.2. Introducing CO from the bottom of the reactor₂Flue gas with a content of 10v%, wherein SO₂Content of 200X 10^-6(v/v). In the culture process, the illumination intensity is 8000Lux, the pH value is controlled to be 7-8, the illumination period is 24h, the light-dark time ratio is 14:10, and the culture time is 7 days. After the completion of the culture, algal cells were collected and the dry weight of the cells was measured, and the results are shown in Table 4.

TABLE 4 comparison of HY-D3 and HY-3 culture results

As can be seen from Table 4, HY-D3 screened by the present invention has better resistance to low temperature than the original algal strain HY-3.

Sequence listing

<110> China petrochemical Co., Ltd

China Petroleum & Chemical Corporation Dalian Petrochemical Research Institute

<120> Scenedesmus rich in oil and culture application thereof

<130> New patent application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1418

<212> DNA

<213> Scenedesmus acutus

<400> 1

gtagtcatat gcttgtctca aagattaagc catgcatgtc taagtataaa ctgcttatac 60

tgtgaaactg cgaatggctc attaaatcag ttatagttta tttggtggta ccttactact 120

cggataaccg tagtaattct agagctaata cgtgcgtaaa tcccgacttc tggaagggac 180

gtatatatta gataaaaggc cgaccgagct ttgctcgacc cgcggtgaat catgatatct 240

tcacgaagcg catggccttg tgccggcgct gttccattca aatttctgcc ctatcaactt 300

tcgatggtag gatagaggcc taccatggtg gtaacgggtg acggaggatt agggttcgat 360

tccggagagg gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgcaaat 420

tacccaatcc tgatacgggg aggtagtgac aataaataac aataccgggc attttatgtc 480

tggtaattgg aatgagtaca atctaaatcc cttaacgagg atccattgga gggcaagtct 540

ggtgccagca gccgcggtaa ttccagctcc aatagcgtat atttaagttg ttgcagttaa 600

aaagctcata gttggatttc gggtgggttc tagcggtccg cctatggtga gtactgctat 660

ggccttcctt tctgtcgggg acgggcttct gggcttcact gtccgggact cggagtcgac 720

gtggttactt tgagtaaatt agagtgttca aagcaggctt acgccagaat actttagcat 780

ggaataacac gataggactc tggcctatct tgttggtctg taggaccgga gtaatgatta 840

agagggacag tcgggggcat tcgtatttca ttgtcagagg tgaaattctt ggatttatga 900

aagacgaact actgcgaaag catttgccaa ggatgttttc attaatcaag aacgaaagtt 960

gggggctcga agacgattag ataccgtcgt agtctcaacc ataaacgatg ccgactaggg 1020

attggcgaat gtttttttaa tgacttcgcc agcaccttat gagaaatcaa agtttttggg 1080

ttccgggggg agtatggtcg caaggctgaa acttaaagga attgacggaa gggcaccacc 1140

aggcgtggag cctgcggctt aatttgactc aacacgggaa aacttaccag gtccagacat 1200

agtgaggatt gacagattga gagctctttc ttgattctat gggtggtggt gcatggccgt 1260

tcttagttgg tgggttgcct tgtcaggttg attccggtaa cgaacgagac ctcagcctgc 1320

taaatagtct cagttgcttt ttgcagctgg ctgacttctt agagggacta ttggcgttta 1380

gtcaatggaa gtatgaggca ataacaggtc tgtgatgc 1418

Claims

1. A Scenedesmus Alopecuroides H.H-D3 rich in oil is classified and named Scenedesmus Alopecuroides H (C.Scenedesmus) H.H. (C.Scenedesmus)Scenedesmus acutus) The strain has been preserved in China general microbiological culture Collection center (CGMCC) in 2018, 2 and 5, with the preservation number of CGMCC 15298.

2. The method for culturing Scenedesmus HY-D3 as claimed in claim 1, which comprises: culturing in photobioreactor with BG11 and SE fresh water culture medium at illumination intensity of 1500-₂A gas content of 5-40 v%.

3. The scenedesmus of claim 1 for CO fixation₂The use of (1).

4. Use of scenedesmus as claimed in claim 1 for the production of microalgae lipid.

5. The scenedesmus as claimed in claim 1 for purifying CO₂And SO₂In exhaust gases or flue gases.

6. Use according to claim 5, characterized in that: the strain utilizes the strain containing CO₂And SO₂The waste gas or the smoke is subjected to illumination autotrophic growth to obtain biomass rich in grease, and CO in the waste gas or the smoke₂Not more than 40% by volume of SO₂The content is not more than 400 × 10^-6（v/v）。

7. Use according to claim 5 or 6, characterized in that: culturing the strain with BG11 and SE fresh water culture medium, introducing CO from the bottom of the reactor₂And SO₂The illumination intensity of the culture is 1500-.