CN111996135B

CN111996135B - High-temperature-resistant high-carbohydrate-yield hot spring cyanobacteria and application thereof

Info

Publication number: CN111996135B
Application number: CN202010713348.0A
Authority: CN
Inventors: 吕雪峰; 朱涛; 尼丁·克什李; 李辉; 谢玉曼; 齐凤霞
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2022-04-08
Anticipated expiration: 2040-07-22
Also published as: CN111996135A

Abstract

The invention relates to Sphaeranthus thermocellus NK1-22 for high yield of carbohydrate, wherein the Sphaeranthus thermocellus is preserved in China general microbiological culture Collection center (CGMCC NO. 19155) in 2019, 12 months and 27 days; also relates to the application of the Sphingomonas thermocellus in producing carbohydrate; also relates to a method for culturing the Sphingomonas thermocellus and a culture obtained by the method. The sphingomyelina thermocellum of the invention has a soluble sugar content of 57% and can secrete a higher concentration of soluble sugar into the culture environment. Although the strain is separated from a hot spring environment, the temperature tolerance interval is very wide, the strain can grow well in a conventional environment at 30 ℃ and a high-temperature environment at 50 ℃, has the capability of producing carbohydrate at high yield, has strong adaptability to high light intensity, provides high-temperature high light intensity during culture, can keep high-speed growth, and can reduce the possibility of polluting other algae and bacteria.

Description

High-temperature-resistant high-carbohydrate-yield hot spring cyanobacteria and application thereof

Technical Field

The invention relates to the field of microalgae application, and more particularly relates to a high-temperature-tolerant high-carbohydrate-yield hot spring cyanobacterium and application thereof.

Background

The microalgae biotechnology can get rid of the dilemma of 'competing for grains with people' and 'competing for land with grains' in the traditional green chemical industry, and is a photosynthesizing route which is very concerned. However, with the rapid development of large-scale microalgae cultivation techniques, bottleneck problems of microbial, viral, protozoan contamination and the like are increasingly highlighted, which have not been discovered or predicted in laboratories or at intermediate scales. The problems of breeding excellent algae and developing a novel culture process to develop high-quality products meeting the requirements of the national civilian are urgently solved in the industry.

Cyanobacteria is the oldest photosynthetic prokaryotic microorganism on earth, can perform plant-type oxygen production photosynthesis, and is widely distributed on various water bodies, soil and even rock surfaces. Has extremely strong vitality, can also find the trace of some extreme environments (such as hot spring, ice source, desert and salt lake), and is called pioneer creatures. Cyanobacteria can synthesize phycocyanin, polysaccharide, pigment and a plurality of secondary metabolites in the life processes of carbon fixation, oxygen release, nitrogen fixation and the like, and is a green microbial cell factory with great development potential.

The temperature is an important environmental factor for highlighting the growth advantages of target algae species and controlling the pollution of the algae species in the large-scale culture of the microalgae. Thermophilic cyanobacteria are important primary producers in high temperature environments such as hot springs, with optimal growth temperatures around 45 ℃ or higher. Thanks to various omics studies and the rapid development of isotope labeling technology, people have a certain degree of cognition on the resource diversity and metabolic characteristics of cyanobacteria in hot spring habitats. However, due to the relatively late research on the separation and purification of hot spring microorganisms, the development of "temperature-type" microalgae resources in terrestrial hot springs still has room for development. The microalgae resources with wide temperature tolerance range and rapid growth are discovered, the regional limitation of microalgae culture can be further overcome, the efficiency of large-scale microalgae culture is improved, the biological pollution is controlled by the temperature tolerance characteristics, and the development prospect is wide.

Disclosure of Invention

The inventor collects algae samples from the field, and obtains an algae strain capable of producing carbohydrates with high yield through culture and separation. The form of the gene is that a plurality of cells are strung into a single-row cell filament body which is blue-green, has NO cell nucleus, has the same width from top to bottom and has a colloidal sheath outside, presents a typical form of the coleus thermocementeri, is sequenced to obtain the 16S rRNA gene sequence which is shown as SEQ ID NO:1, is compared in an ncbi database, has higher similarity with the group of the coleus thermocementeri, is named as the coleus thermocementeri NK1-22, and has the Latin name of thermoleptylngbya sp.NK1-22.

Based on the findings, the invention provides high-temperature-resistant high-carbohydrate-yield Sphingomonas thermocellus, which is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 12 months and 27 days, and the preservation number is CGMCC NO. 19155.

The invention also provides the application of the coleus thermocemenus in producing carbohydrate.

In a specific embodiment, the carbohydrate is a soluble sugar.

In a specific embodiment, the carbohydrate is one or a combination of sucrose, mannose, lactose and maltose.

The invention also provides a method for culturing the sphingomyelina thermocellum, which comprises the step of inoculating the sphingomyelina thermocellum into a liquid culture medium and carrying out photosynthetic culture.

In a preferred embodiment, the liquid medium is BG11 medium supplemented with 0-300mM sodium chloride.

In a preferred embodiment, the cultivation temperature is 30-50 ℃.

In a preferred embodiment, the cultivation is carried out by passing a liquid medium containing 3% CO₂Air culture of (2).

In a preferred embodiment, the light intensity during the cultivation is 100-500. mu. mol photons m^-2s^-1。

The invention also provides a sphingomyelina thermocellum culture obtained by the culture method.

The sphingomyelina thermocellum of the invention has a soluble sugar content of 57% and can secrete a higher concentration of soluble sugar into the culture environment. In addition, although the strain is separated from a hot spring environment, the strain is different from common thermophilic bacteria in that the temperature tolerance interval is very wide, the strain can grow well in a conventional environment of 30 ℃ and a high-temperature environment of 50 ℃, simultaneously has the capability of producing carbohydrate at a high yield, and has strong adaptability to high light intensity, so that the high-temperature high light intensity is provided during culture, high-speed growth can be maintained, and the possibility of polluting miscellaneous algae and bacteria can be reduced.

Preservation of microorganisms

The strain related to the invention is obtained by separating from hot spring of Yunnan province in China, and through 16S rRNA gene sequencing and morphological identification, the microalgae belongs to the genus coleus thermocemenus of the oscillaceae of the cyanophyta, and the similarity of the 16S rRNA gene sequence and the coleus thermocemenus (thermolytolyngbya sp.) is higher. The microalgae is preserved in China general microbiological culture Collection center (CGMCC) of microbiological research institute of China academy of sciences, No. 3, West Lu 1 institute of Western Xilu, Beijing, and the area of Tokyo, 12 months and 27 days in 2019, the preservation number is CGMCC NO.19155, the microalgae is named as Sphaeranthus thermocellus NK1-22, and the Latin is named as Thermometoningbya sp.NK1-22. The taxonomy is named as: thermoleponyngbya oregonensis.

Drawings

FIG. 1 is an optical micrograph of Sphingomonas thermocellus NK 1-22;

FIG. 2 is a phylogenetic tree constructed by comparing the 16S rRNA gene of Sphingomonas thermocellus NK1-22 with other microalgae;

FIG. 3 is a statistical graph of the carbohydrate, protein and lipid content in cells of Sphingomonas thermocellus NK1-22 algae;

FIG. 4 is a statistical distribution of the different components in the carbohydrates of Sphingomonas thermocellus NK 1-22;

FIG. 5 shows the growth curve of Sphingomonas thermocellum NK1-22 in a conical flask at different temperatures with shaking (fixed light intensity 30. mu. mol phosns m)^-2s^-1)；；

FIG. 6 shows the growth curve of Sphingomonas thermocellum NK1-22 in a column photobioreactor with aeration culture at different temperatures (fixed light intensity 100. mu. mol photons m)^-2s^-1)；

FIG. 7 is a graph showing the growth of Sphingomonas thermocellum NK1-22 in a column photobioreactor cultured with aeration at 40 ℃ under different light intensities;

FIG. 8 shows Sphingomonas thermocellus NK1-22 in Erlenmeyer flasks at 40 ℃ and 30. mu. mol phosns m under salt stress (with different concentrations of NaCl)^-2s^-1OD after 8 days of shaking culture under light intensity₇₃₀And (6) carrying out statistical graph.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

1. Obtaining of Sphaerotheca thermocellum NK1-22

The algal strain (thermolysis bya sp. NK1-22) of the present invention was isolated from a bio-pad sample from Yunan Hot spring. The cell morphology observed by an optical microscope and a scanning electron microscope is shown in figure 1, and is a filament of a single line of cells formed by a plurality of cells in series, which is blue-green, has no cell nucleus, has the same width of the upper part and the lower part of the filament, and has an obvious colloidal sheath outside, and is a typical sphingomyelina thermocellum morphology. The extracted genome DNA amplified 16S rRNA gene is sequenced as shown in SEQ ID NO 1, and is compared with microalgae 16S rRNA genes in a database to construct an evolutionary tree, which shows that the similarity of the 16S rRNA gene sequence and the group of the genus Sphingomonas thermocellus is higher (figure 2). Combining morphology and molecular evidence, the strain was classified into the genus Sphingomonas thermocellum, named Sphingomonas thermocellus NK 1-22. The microalgae is preserved in China general microbiological culture Collection center (CGMCC) at 27.12.2019, the preservation number is CGMCC NO.19155, the microalgae is named as Sphaerotheca thermosiphon NK1-22, and the Latin chemical name is Thermoleptolyngbya sp.NK1-22.

2. Carbohydrate composition of Sphingomonas thermocellus NK1-22

Culturing Sphaeranthus lanuginosus NK1-22 for 8 days, centrifuging to collect algae cells, freeze drying, and determining total lipid, total protein and total carbohydrate (saccharide) content by chloroform-methanol method combined with GC-MS determination, Kjeldahl method, and phenol-sulfuric acid method. The results are shown in FIG. 3, where the carbohydrate content is 57% of the dry cell weight, which is much higher (about 20-40%) than that reported for conventional cyanobacteria. Further analysis of the carbohydrate composition revealed that the carbohydrate in Sphingomonas thermocellus NK1-22 is substantially soluble sugar, mainly sucrose, mannose, lactose and maltose, as shown in FIG. 4.

3. Culture optimization of Sphingomonas thermocellus NK1-22

3.1 temperature optimization

Sphingomonas thermocellus NK1-22 was inoculated at the initial inoculation concentration OD₇₃₀About 0.1 is inoculated into a triangular flask containing BG11, and the culture is carried out by shaking at different temperatures, the shaking speed is 150rpm, the light intensity is 30 mu mol photons m^-2s^-1Daily sampling for OD measurement₇₃₀As shown in FIG. 5, the growth curve was plotted, and Sphingomonas thermocellus NK1-22 was able to grow at about 30-50 ℃ (30 + -2 ℃ to 50 + -2 ℃), and entered logarithmic phase after 3 days of culture, and OD value after 8 days of culture was about 1.5-2. Around 55 ℃, the growth of the Sphingomonas thermocellus NK1-22 is severely inhibited, and even the algal cells are killed. Therefore, the strain can adapt to high temperature like thermophilic cyanobacteria, can also tolerate the conventional temperature of 30 ℃ or below, and shows a wide temperature adaptation range.

To further optimize the culture conditions of Sphaeranthus thermocellus NK1-22, the strain was cultured in a 100mL column photobioreactor at different temperatures (the rest of the culture conditions refer to the reactor's conventional parameters, 3% CO₂Aeration culture with light intensity of 100. mu. mol photons m^-2s^-1) Daily sampling for OD measurement₇₃₀The growth curve is plotted as shown in FIG. 6, Sphingomonas thermocellus NK1-22 grows well at 30-50 deg.C, and preferably at about 45 deg.C (45 + -2 deg.C), enters logarithmic phase after two days of culture, and OD 6 days after culture₇₃₀Reaching 6 deg.C or so, and culturing at 30 deg.C, 35 deg.C and 40 deg.C for 6 days to obtain OD₇₃₀Between 5 and 6, 50 ℃ earlier entered the plateau phase, OD 6 days after culture₇₃₀About 4, OD at each temperature₇₃₀All are much higher than shaking culture.

The results show that the Sphingomonas thermocellus NK1-22 can grow well at 30-50 ℃ under both low light intensity and high light intensity, and has a very wide temperature adaptation range.

3.2 optimization of illumination intensity

According to the above experimental results, 40 ℃ was selected as the culture temperature to optimize the light intensity during the culture. Inoculating Sphingomonas thermocellus NK1-22 into a column type photobioreactor containing BG11, culturing under different light intensities, introducing gas containing 3% CO₂Culturing at 40 + -2 deg.C, sampling daily for measuring OD₇₃₀. The results are shown in FIG. 7, where the strains were 250 and 500. mu. mol phosns m^-2s^-1Under light intensity, the light enters logarithmic phase on the same day, and enters plateau phase after 3-4 days of culture, the time for entering plateau phase is shortest, and OD is OD₇₃₀About 8. 100 μ E/m²The growth state under the light intensity/s is similar to the experimental results, the logarithmic phase is entered after 2 days of culture, the plateau phase is entered after 4 days, and the OD is entered after 5 days₇₃₀Is about 5. Therefore, the strain has a wide temperature adaptation interval and has good adaptability to high light intensity.

3.3 salt concentration optimization

Inoculating Sphingomonas thermocellus NK1-22 into a triangular flask containing BG11 (added with NaCl of different concentrations), and culturing at 40 deg.C under shaking with light intensity of 20-30 μmol photons m^-2s^-1OD determination after 8 days of culture₇₃₀As shown in FIG. 8, 100mM and 300mM of sodium chloride inhibited the growth of Sphingomonas thermocellus NK1-22, and 600mM inhibited the growth of algal cells severely, even leading to the death of algal cells. Thus, Sphingomonas thermocellus NK1-22 was able to grow in BG11 medium at 0-300mM sodium chloride, but the growth of algal cells was inhibited by the increase in salt concentration.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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<120> high-temperature-resistant high-carbohydrate-yield hot spring cyanobacteria and application thereof

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gcagcagtgg ggaattttcc gcaatgggcg caagcctgac ggagcaagac cgcgtgcggg 240

aggacggcct attggttgta aaccgctttt gatagggaag aagctctgac ggtacctatc 300

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gaattcccag tgtagcggtg aaatgcgtag atattgggaa gaacaccggt ggcgaaagcg 540

cctgactgga cctgcactga cactgaggga cgaaagctag gggagcgaaa gggattagat 600

acccc 605

Claims

1. A high temperature tolerant high carbohydrate yield Sphaeranthus lanuginosus (C. lanuginosus)Thermoleptolyngbya oregonensis) The Sphaerotheca thermocellum is preserved in the China general microbiological culture Collection center in 2019, 12 months and 27 days, and the preservation number is CGMCC number 19155.

2. Use of Sphingomonas thermocellum according to claim 1 for the production of carbohydrates.

3. Use according to claim 2, wherein the carbohydrate is a soluble sugar.

4. The use according to claim 3, wherein the carbohydrate is one or more of sucrose, mannose, lactose and maltose in combination.

5. A method for culturing the sphingomyelina thermocellum according to claim 1, comprising the step of inoculating the sphingomyelina thermocellum in a liquid medium and performing photosynthetic culture.

6. The method according to claim 5, wherein the liquid medium is BG11 medium supplemented with 0-300mM sodium chloride.

7. The method according to claim 5, wherein the culture temperature is 30 to 50 ℃.

8. The method of claim 5, wherein the culturing is performed by introducing a gas containing 3% CO₂Air culture of (2).

9. The method as claimed in claim 5, wherein the light intensity during the cultivation is 100-^-2s^-1。