CN114854648A - 能够耐受高光强的集胞藻驯化菌株的驯化方法 - Google Patents
能够耐受高光强的集胞藻驯化菌株的驯化方法 Download PDFInfo
- Publication number
- CN114854648A CN114854648A CN202210686467.0A CN202210686467A CN114854648A CN 114854648 A CN114854648 A CN 114854648A CN 202210686467 A CN202210686467 A CN 202210686467A CN 114854648 A CN114854648 A CN 114854648A
- Authority
- CN
- China
- Prior art keywords
- synechocystis
- illumination intensity
- strain
- intensity
- acclimatization
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000192584 Synechocystis Species 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000005286 illumination Methods 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- 238000002965 ELISA Methods 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 238000002372 labelling Methods 0.000 claims abstract description 6
- 230000003287 optical effect Effects 0.000 claims abstract description 4
- 241000192593 Synechocystis sp. PCC 6803 Species 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 4
- 238000002474 experimental method Methods 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- 238000000424 optical density measurement Methods 0.000 claims description 2
- 241000894007 species Species 0.000 claims 1
- 229930002868 chlorophyll a Natural products 0.000 abstract description 7
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 abstract description 7
- 229920002527 Glycogen Polymers 0.000 abstract description 6
- 235000021466 carotenoid Nutrition 0.000 abstract description 6
- 150000001747 carotenoids Chemical class 0.000 abstract description 6
- 229940096919 glycogen Drugs 0.000 abstract description 6
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 241000192700 Cyanobacteria Species 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 7
- 230000029553 photosynthesis Effects 0.000 description 5
- 238000010672 photosynthesis Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 4
- 230000000243 photosynthetic effect Effects 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 description 3
- ALRHLSYJTWAHJZ-UHFFFAOYSA-N 3-hydroxypropionic acid Chemical compound OCCC(O)=O ALRHLSYJTWAHJZ-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 108010060806 Photosystem II Protein Complex Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- XMGQYMWWDOXHJM-UHFFFAOYSA-N limonene Chemical compound CC(=C)C1CCC(C)=CC1 XMGQYMWWDOXHJM-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000008832 photodamage Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010081996 Photosystem I Protein Complex Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- FRHBOQMZUOWXQL-UHFFFAOYSA-L ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229960004642 ferric ammonium citrate Drugs 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 235000000011 iron ammonium citrate Nutrition 0.000 description 1
- 239000004313 iron ammonium citrate Substances 0.000 description 1
- 229940087305 limonene Drugs 0.000 description 1
- 235000001510 limonene Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 210000001916 photosynthetic cell Anatomy 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000009528 severe injury Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种获得耐受高光强集胞藻菌株的定向驯化方法,步骤:确定集胞藻野生型菌株的初始敏感光照强度;在培养基中接种集胞藻野生型菌液使酶标仪测定OD750nm为0.04,在初始敏感光照强度下,培养至平台期;转接入培养基,继续驯化,以此连续传代培养至集胞藻在平台期的酶标仪测定OD750nm≥0.8;逐步提高光照条件,继续驯化,以此连续传代培养至光照强度到2000μmol光子m‑2s‑1时集胞藻在平台期的酶标仪测定OD750nm≥0.8,本发明获得的驯化菌株能在2000μmol光子m‑2s‑1高光条件下生长7天:OD750nm、叶绿素a、类胡萝卜素、干重和糖原含量高于野生型菌株。
Description
技术领域
本发明属于工业微生物领域,具体涉及能够耐受高光强的集胞藻驯化菌株的驯化方法。
背景技术
化石燃料是不可再生的资源,过多使用会释放大量的温室气体到大气中,导致全球变暖。因此,探索可再生和绿色的生物能源以替代传统的化石能源并减轻环境污染是我们迫切需要解决的问题。经由光合作用,每年约2580亿吨CO2被固定为有机物,而这其中陆地植物贡献约为30%,其余70%由海洋光合微生物贡献。其中,蓝细菌是唯一可以进行光合作用并释放氧气的原核生物,其可贡献全球固碳总量的25%左右。除了作为初始生产者的传统角色之外,近几十年来,借助先进的合成生物学技术,蓝细菌已被成功地改造为“光驱动细胞工厂”,可直接利用二氧化碳生产数十种可再生燃料和化学物质,包括乙醇、丁醇、丁二醇、丙二醇、丙酮、蔗糖、柠檬烯和3-羟基丙酸等。以上研究充分证明了基于光合蓝细菌开发可持续生产系统的可行性。其中模式蓝细菌集胞藻PCC 6803是蓝藻门、集胞藻属,繁殖方式为二分裂形式的一种单细胞非固氮球形蓝藻,在1968年首次从淡水湖中分离得到。作为模式菌株,集胞藻6803的全基因组测序在1996年已经完成,其基因组全长为3573kb,GC含量为47.7%,约含3264个蛋白编码基因。
光作为蓝细菌进行光合作用的能量来源,在蓝细菌的生长过程中是必不可少的。但是,早期研究表明,过多的光能会破坏蓝细菌的能量供应和消耗之间的平衡,从而导致细胞内的毒性活性氧(ROS)积累,包括过氧化氢(H2O2)、超氧阴离子(O2 -)、羟基自由基(OH·)和单线态氧(1O2)。过多的ROS进一步破坏其他关键细胞成分,如核酸、脂质、色素和蛋白质,最终导致细胞死亡。具体来说,光合作用可以被认为是一系列有序的氧化还原反应。当光能供应等于消耗时,光合作用的氧化还原平衡得以维持,但当收集过多的光时,平衡将被破坏,从而导致电子传递链的过度还原。此外,光系统II比光系统I更容易受到高光强度的影响,高光对光系统II反应中心极易产生光损伤。而高光造成的光损伤无疑将会限制蓝细菌的光合作用效率及碳固定能力。另外,为了具有成本效益,蓝细菌的工业或大规模培养必须基于户外培养利用天然太阳能。对于户外培养,保持持续光照成本很高,而且蓝细菌在自然界中不可避免地遭受高光胁迫。在自然环境中,太阳光最强可达2000μmol光子m-2s-1,对细胞造成严重的损伤并大大降低其光合效率。因此,为了促进高效光合细胞工厂的建设及其工业应用,构建高光耐受底盘以及解析蓝细菌的高光耐受机制是我们亟待解决的问题。
发明内容
本发明的目的是克服现有技术的不足,提供能够耐受高光强的集胞藻驯化菌株的驯化方法。
本发明的技术方案概述如下:
能够耐受高光强的集胞藻驯化菌株的驯化方法,包括如下步骤:
(1)通过体外敏感实验确定集胞藻野生型菌株的初始敏感光照强度;
(2)在液体BG11培养基中接种集胞藻野生型菌液使酶标仪测定OD750 nm为0.04,在初始敏感光照强度下,培养至平台期;
(3)将步骤(2)培养至平台期的细胞转接入液体BG11培养基,在初始敏感光照强度下继续驯化,以此连续传代培养至集胞藻在平台期的酶标仪测定OD750 nm大于或等于0.8;
(4)逐步提高光照条件,继续驯化,以此连续传代培养至光照强度到2000μmol光子m-2s-1时集胞藻在平台期的酶标仪测定OD750 nm大于或等于0.8,得到能够耐受高光强的集胞藻驯化菌株。
菌种优选:集胞藻(Synechocystis sp.PCC 6803)ATCC 27184。
逐步提高光照条件为光照强度从初始敏感光照强度至2000μmol光子m-2s-1。
初始敏感光照强度为100μmol光子m-2s-1。
本发明的优点:
本发明的方法获得的集胞藻驯化菌株能够耐受2000μmol光子m-2s-1的光强。本发明的方法获得的能够耐受高光强的集胞藻驯化菌株的能够在2000μmol光子m-2s-1高光条件下生长7天:OD750 nm可达到野生型的2.55倍,叶绿素a含量可达到野生型的1.61倍,类胡萝卜素含量可达到野生型的3.87倍,干重可达到野生型的2.13倍,糖原含量可达到野生型的1.82倍。
附图说明
图1为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的生长曲线。
图2为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的叶绿素a含量。
图3为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的类胡萝卜素含量。
图4为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的干重。
图5为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的糖原含量。
具体实施方式
本发明各实施例使用集胞藻(Synechocystis sp.PCC 6803),该菌购自美国菌株保藏中心ATCC,菌株代码ATCC 27184。菌株购买于2012.5.30。
下面的实施例是为了使本领域的技术人员更好地理解本发明但并不用于限制本发明。下面结合具体实施例对本发明作进一步说明:
配制液体BG11培养基配方:NaNO3 1.5g,K2HPO4·3H2O 0.04g,MgSO4·7H2O0.075g,EDTA 0.001g,Na2CO3 0.02g,H3BO3 2.86g,MnCl2·4H2O 1.81g,ZnSO4·7H2O0.222g,NaMoO4·5H2O 0.390g,CuSO4·5H2O 0.079g,Co(NO3)2·6H2O 0.0494g,CaCl2·2H2O0.036g,柠檬酸铁铵0.006g,加水至1L而成,pH为7.5,121℃,20min高压锅灭菌;
实施例1
能够耐受高光强的集胞藻驯化菌株的驯化方法,包括如下步骤:
(1)通过体外敏感实验确定集胞藻野生型菌株的初始敏感光照强度;具体步骤是:在25mL液体BG11培养基中接种集胞藻野生型菌液(集胞藻PCC 6803菌液)使酶标仪测定OD750nm为0.04,在50-2000μmol光子m-2s-1光照条件下(分别为:50、100,再以100为一个梯度,至2000μmol光子m-2s-1)培养集胞藻野生型菌株,确定集胞藻野生型菌的初始敏感光照强度为100μmol光子m-2s-1;
(2)在25mL液体BG11培养基中接种集胞藻野生型菌液使酶标仪测定OD750nm为0.04,在100μmol光子m-2s-1光照条件下,培养至平台期;
(3)将步骤(2)培养至平台期的细胞转接入液体BG11培养基,在100μmol光子m-2s-1光照条件下继续驯化,以此连续传代培养至集胞藻在平台期的酶标仪测定OD750nm大于0.8(也可以等于0.8);
(4)在100μmol光子m-2s-1至2000μmol光子m-2s-1范围逐步提高光照条件,继续驯化,以此连续传代培养至光照强度到2000μmol光子m-2s-1时集胞藻在平台期的酶标仪测定OD750nm大于0.8(也可以等于0.8),得到能够耐受高光强的集胞藻驯化菌株。
检测:
1.实施例1获得的能够耐受高光强的集胞藻驯化菌株(简称驯化菌株)和集胞藻野生型(集胞藻PCC 6803,简称野生型菌株)生长曲线:
在25mL液体BG11培养基中接种驯化菌株和野生型菌株使酶标仪测定OD750nm为0.04,每组设置三个平行。在2000μmol光子m-2s-1光照条件下培养,摇床参数为160rpm,30℃,每隔24h取200μL菌液使用酶标仪测定OD750nm,并绘制生长曲线,见图1。
2.驯化菌株和野生型菌株干重特性的分析:
在25mL液体BG11培养基中接种驯化菌株和野生型菌株使酶标仪测定OD750nm为0.04,每组设置三个平行,摇床参数为160rpm,30℃。在2000μmol光子m-2s-1条件下培养培养至7天,取20mL细胞液,7500rpm,5min离心收集菌体,使用真空冷冻干燥机冻干2-3天,称取对应菌体的重量,得到的菌体重量除以体积即对应菌株的干重数据;见图4。
3.驯化菌株和野生型菌株的叶绿素a含量和类胡萝卜素含量的测定:
在25mL液体BG11培养基中接种驯化菌株和野生型菌株使酶标仪测定OD750nm为0.04,每组设置三个平行,摇床参数为160rpm,30℃,在2000μmol光子m-2s-1条件下培养7天。取1mL OD750nm为0.4的细胞培养液,7500rpm,5min离心收集菌体,加入1mL的纯甲醇(4℃,预冷)重悬菌体,4℃萃取20min。12000,5min离心后取1-2mL上清液,使用紫外分光光度计测定OD665nm、OD470nm和OD720nm,通过公式叶绿素a含量[μg/ml]=稀释倍数*12.9447*(OD665nm-OD720nm);类胡萝卜素含量[μg/ml]=稀释倍数*[1,000*(OD470nm-OD720nm)-2.86*(叶绿素a含量)]/221计算叶绿素a和类胡萝卜素含量;见图2和图3。
4.驯化菌株和野生型菌株糖原含量的测定:在25mL液体BG11培养基中接种驯化菌株和野生型菌株使酶标仪测定OD750nm为0.04,每组设置三个平行,摇床参数为160rpm,30℃,在2000μmol光子m-2s-1条件下培养7天。取1mL酶标仪测定OD750nm为0.4的细胞培养液,按照biobox的糖原测定试剂盒说明书进行糖原含量的测定,见图5。
Claims (4)
1.能够耐受高光强的集胞藻驯化菌株的驯化方法,其特征是包括如下步骤:
(1)通过体外敏感实验确定集胞藻野生型菌株的初始敏感光照强度;
(2)在液体BG11培养基中接种集胞藻野生型菌液使酶标仪测定OD750nm为0.04,在初始敏感光照强度下,培养至平台期;
(3)将步骤(2)培养至平台期的细胞转接入液体BG11培养基,在初始敏感光照强度下继续驯化,以此连续传代培养至集胞藻在平台期的酶标仪测定OD750nm大于或等于0.8;
(4)逐步提高光照条件,继续驯化,以此连续传代培养至光照强度到2000μmol光子m-2s-1时集胞藻在平台期的酶标仪测定OD750nm大于或等于0.8,得到能够耐受高光强的集胞藻驯化菌株。
2.根据权利要求1所述的驯化方法,其特征在于所述菌种为集胞藻PCC 6803。
3.根据权利要求1所述的驯化方法,其特征在于所述逐步提高光照条件为光照强度从初始敏感光照强度至2000μmol光子m-2s-1。
4.根据权利要求1或3所述的驯化方法,其特征在于所述初始敏感光照强度为100μmol光子m-2s-1。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210686467.0A CN114854648A (zh) | 2022-06-17 | 2022-06-17 | 能够耐受高光强的集胞藻驯化菌株的驯化方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210686467.0A CN114854648A (zh) | 2022-06-17 | 2022-06-17 | 能够耐受高光强的集胞藻驯化菌株的驯化方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114854648A true CN114854648A (zh) | 2022-08-05 |
Family
ID=82624732
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210686467.0A Pending CN114854648A (zh) | 2022-06-17 | 2022-06-17 | 能够耐受高光强的集胞藻驯化菌株的驯化方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114854648A (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100003739A1 (en) * | 2008-02-08 | 2010-01-07 | Ulf Duhring | Selection of ADH In Genetically Modified Cyanobacteria For The Production Of Ethanol |
JP2015073469A (ja) * | 2013-10-08 | 2015-04-20 | 国立大学法人神戸大学 | 微細藻の生育機能を増強する方法 |
CN109642192A (zh) * | 2016-04-04 | 2019-04-16 | 国家信息及自动化研究院 | 用于微藻的选择性生物反应器 |
CN114196547A (zh) * | 2022-02-16 | 2022-03-18 | 广东省科学院生态环境与土壤研究所 | Dcmu在提高微藻混养发酵时叶绿素产量或强光耐受性中的应用 |
-
2022
- 2022-06-17 CN CN202210686467.0A patent/CN114854648A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100003739A1 (en) * | 2008-02-08 | 2010-01-07 | Ulf Duhring | Selection of ADH In Genetically Modified Cyanobacteria For The Production Of Ethanol |
JP2015073469A (ja) * | 2013-10-08 | 2015-04-20 | 国立大学法人神戸大学 | 微細藻の生育機能を増強する方法 |
CN109642192A (zh) * | 2016-04-04 | 2019-04-16 | 国家信息及自动化研究院 | 用于微藻的选择性生物反应器 |
CN114196547A (zh) * | 2022-02-16 | 2022-03-18 | 广东省科学院生态环境与土壤研究所 | Dcmu在提高微藻混养发酵时叶绿素产量或强光耐受性中的应用 |
Non-Patent Citations (2)
Title |
---|
KATSUNORI YOSHIKAWA等: "Mutations in hik26 and slr1916 lead to high-light stress tolerance in Synechocystis sp. PCC6803", 《COMMUNICATIONS BIOLOGY》, vol. 4, no. 343, pages 1 - 11 * |
季祥: "《生物质能源及废物利用新技术》", 吉林大学出版社, pages: 9 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Eroglu et al. | Microalgal hydrogen production research | |
Shokrkar et al. | Bioethanol production from acidic and enzymatic hydrolysates of mixed microalgae culture | |
Skjånes et al. | BioCO2–A multidisciplinary, biological approach using solar energy to capture CO2 while producing H2 and high value products | |
Shukla et al. | Algal growth in photosynthetic algal microbial fuel cell and its subsequent utilization for biofuels | |
Rizza et al. | Bioprospecting for native microalgae as an alternative source of sugars for the production of bioethanol | |
Ho et al. | Characterization of photosynthetic carbon dioxide fixation ability of indigenous Scenedesmus obliquus isolates | |
Melis | Green alga hydrogen production: progress, challenges and prospects | |
El-Dalatony et al. | Metabolic pathways for microalgal biohydrogen production: Current progress and future prospectives | |
Work et al. | Improving photosynthesis and metabolic networks for the competitive production of phototroph-derived biofuels | |
de Morais et al. | Isolation and selection of microalgae from coal fired thermoelectric power plant for biofixation of carbon dioxide | |
Chi et al. | Selection of microalgae and cyanobacteria strains for bicarbonate-based integrated carbon capture and algae production system | |
Prince et al. | The photobiological production of hydrogen: potential efficiency and effectiveness as a renewable fuel | |
Sarma et al. | Cyanobacteria: A metabolic power house for harvesting solar energy to produce bio-electricity and biofuels | |
Kirst et al. | The chloroplast signal recognition particle (CpSRP) pathway as a tool to minimize chlorophyll antenna size and maximize photosynthetic productivity | |
Buitrón et al. | Biohydrogen production from microalgae | |
Tanvir et al. | Harnessing solar energy using phototrophic microorganisms: A sustainable pathway to bioenergy, biomaterials, and environmental solutions | |
CN103215190B (zh) | 一株淡水小球藻及其在固定co2和生产微藻油脂中的应用 | |
Hellingwerf et al. | Alternative routes to biofuels: light-driven biofuel formation from CO2 and water based on the ‘photanol’approach | |
Taikhao et al. | Dark fermentative hydrogen production by the unicellular halotolerant cyanobacterium Aphanothece halophytica grown in seawater | |
Miura | Hydrogen production by biophotolysis based on microalgal photosynthesis | |
CN103952312B (zh) | 一株淡水小球藻Chlorella sorokiniana GS03及其应用 | |
Sukrachan et al. | Enhanced hydrogen production by Nostoc sp. CU2561 immobilized in a novel agar bead | |
Dudek et al. | The effect of biomass separation method on the efficiency of hydrogen production by Platymonas subcordiformis | |
CN114854648A (zh) | 能够耐受高光强的集胞藻驯化菌株的驯化方法 | |
Kumar et al. | A review on the contribution of nanotechnology for biofuel production from algal biomass: a bridge to the reduction of carbon footprint |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |