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Measurement of Chlorophyll a and Carotenoids Concentration in Cyanobacteria
蓝藻细菌中叶绿素a和类胡萝卜素浓度的测量   

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本实验方案简略版
Journal of Biotechnology
Nov 2012

Abstract

This is a protocol for precise measurement of chlorophyll a and total carotenoid concentrations in cyanobacteria cells. Cellular chlorophyll concentration is one of the central physiological parameters, routinely followed in many research areas ranging from stress physiology to biotechnology. Carotenoids concentration is often related to cellular stress level; combined pigments assessment provides useful insight into cellular physiological state. The current protocol was established to minimize time and equipment requirements for the routine pigments analysis. It is important to note that this protocol is suitable only for cyanobacteria containing chlorophyll a, and is not designed for species containing other chlorophyll molecules.

Keywords: Pigments (颜料), Photosynthesis (光合作用), Methanol extraction (甲醇提取), Spectrophotometry (分光光度法)

Materials and Reagents

  1. Cyanobacteria culture (Note 1)
  2. Methanol ≥99.9% (GC) (Sigma-Aldrich)

Equipment

  1. Eppendorf safe-lock tubes (1.5 ml)
  2. Centrifuge with relative centrifugal force (RCF) of 15,000 x g and cooling option to +4 °C (Sigma-Aldrich, model: 1-16 K )
  3. Pipette 100 µl -1,000 µl + pipette tips (RAININ, Mettler-Toledo)
  4. Fridge (+4 °C)
  5. Spectrophotometer with slit width 1 nm (Shimadzu, model: UV-2600 )
  6. Spectrophotometric plastic or glass VIS/UV-VIS semi-micro 0.75-1.5 ml cuvettes
  7. Mixing device (Silamat S6, Ivoclar Vivadent) or vortex (IKA MS3 digital, IKA®)
  8. Aluminum foil
  9. Holder for Eppendorf tubes

Procedure

  1. Work under modest irradiance [up to 5 µmol (photons) m-2 s-1 of white light or 10 µmol (photons) m-2 s-1 of green light] in order to prevent degradation of extracted pigments.
  2. Harvest 1 ml of cyanobacterial culture suspension (Note 2).
  3. Centrifuge cells at 15,000 x g at laboratory temperature for 7 min and thoroughly discard supernatant. If necessary repeat the centrifugation (Note 3).
  4. Add 1 ml of methanol, precooled to +4 °C.
  5. Homogenize the sample by mixing (Silamat S6, 2 sec), vortexing (2,000 rpm, 4 sec), or by gentle pipetting up and down.
  6. Cover the samples with aluminum foil. Incubate at +4 °C for 20 min in order to extract the pigments from the cells (Note 4).
  7. Centrifuge at 15,000 x g, +4 °C for 7 min and visually check pellet; it should be ranging between bluish and purple (Figure 1) with no green color. If the pellet is green, repeat steps 5-6.


    Figure 1. Colors of cyanobacteria pellets before addition of methanol (A) and after chlorophyll a and carotenoids extraction (B). The color of pellets after the methanol extraction will be ranging from bluish (Synechocystis sp. PCC 6803) to purple (Lyngbya sp. IPPAS B-1204), depending on particular combination of phycobiliproteins.

  8. Calibrate spectrophotometer using methanol as blank.
  9. Measure pigments concentration by spectrophotometer with slit width 1 nm.
    1. Measure absorbance of sample and blank at 470 nm, 665 nm and 720 nm (Note 5).
    2. Calculate concentration of chlorophyll a content according to equations:
      Chla [µg/ml] = 12.9447 (A665 − A720) (Ritchie, 2006)
      Chla [µM] = 14.4892 (A665 − A720); for Chla molar mass = 893.4890 g/mol
      Carotenoids [µg/ml] = [1,000 (A470 − A720) − 2.86 (Chla [µg/ml])] / 221 (Wellburn, 1994).
  10. Perform the analysis at least in triplicates as necessary for calculations of averages and standard deviations from each pigments assessment.

Representative data



Figure 2. Representative measurements of chlorophyll a and carotenoids concentrations in cell culture of cyanobacterium Synechocystis sp. PCC 6803. Dense cyanobacteria culture (chlorophyll a: 22 µg/ml, carotenoids: 7 µg/ml) was gradually diluted by half up to pigments concentration 0.1 µg/ml. Each measurement was performed in triplicates; the error bars represent standard deviations.

Notes

  1. The authors are not aware of any restrictions of this protocol usage for cyanobacterial strains containing only chlorophyll a. The protocol was successfully applied to Cyanothece sp. ATCC 51142, Synechococcus elongatus sp. PCC 7942, Cyanobacterium sp. IPPAS B-1200, Synechocystis sp. PCC 6803, Arthrospira platensis IPPAS B-256, Anabaena sphaerica IPPAS B-404, Chroococcus sp. IPPAS B-1203, Lyngbya sp. IPPAS B-1204, Aphanocapsa sp. IPPAS B-1205, Anabaena sp. IPPAS B-1206.
  2. The amount of cell suspension required for analysis can vary with the culture density. With very diluted cultures it is recommended to harvest bigger culture volume; for cultures with chlorophyll a density around 10 ng(Chl) mlculture-1 or lower even up to 5 ml. On the contrary, for very dense cultures lower sample volume is recommended – with high cellular densities, some pigments can remain in cells after extraction.
  3. If some supernatant remains in the tube after the first centrifugation, the extraction will not take place in pure methanol and the pigment concentrations will not be measured properly.
  4. The pigment extraction time can be prolonged up to 2 h with no significant pigment degradation.
  5. The final absorbance at each wavelength should be in linear absorbance range. For spectrophotometer UV-2600 (Shimadzu) this linear absorbance range is 0.01 - 2.5. If necessary, dilute the sample with methanol to fit in the spectrophotometer linear absorbance range.
  6. In case of using different volumes of cyanobacteria samples and/or methanol than 1 ml, the final pigment concentration should be calculated according to following equation:

Acknowledgments

The protocol was adopted from publication “On the dynamics and constraints of batch culture growth of the cyanobacterium Cyanothece sp. ATCC 51142” (Sinetova et al., 2012). T. Z. and J. C. were supported by the MEYS CR within CzechGlobe Centre, reg. no. CZ.1.05/1.1.00/02.0073 484, the National Sustainability Program I (NPU I), grant number LO1415 and by EC OP project, reg. no. CZ.1.07/2.3.00/20.0256. S. M. A. was supported by grant from Russian Scientific Foundation no. 14-24-00020).

References

  1. Balizs, G., Benesch-Girke, L., Borner, S. and Hewitt, S. A. (1994). Comparison of the determination of four sulphonamides and their N4-acetyl metabolites in swine muscle tissue using liquid chromatography with ultraviolet and mass spectral detection. J Chromatogr B Biomed Appl 661(1): 75-84.
  2. Ritchie, R. J. (2006). Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents. Photosynth Res 89(1): 27-41.
  3. Sinetova, M. A., Červený, J., Zavřel, T. and Nedbal, L. (2012). On the dynamics and constraints of batch culture growth of the cyanobacterium Cyanothece sp. ATCC 51142. J Biotechnol 162(1): 148-155.
  4. Wellburn, A. R. (1994). The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313.

简介

这是用于精确测量蓝细菌细胞中叶绿素a和总类胡萝卜素浓度的方案。 细胞叶绿素浓度是中心生理参数之一,在从应激生理学到生物技术的许多研究领域中常规地遵循。 类胡萝卜素浓度通常与细胞应激水平相关; 组合颜料评估提供了有用的洞察细胞生理状态。 建立了当前方案以使对常规色素分析的时间和设备要求最小化。 重要的是注意,该方案仅适用于含有叶绿素a的蓝细菌,并且不被设计用于含有其他叶绿素分子的物种。

关键字:颜料, 光合作用, 甲醇提取, 分光光度法

材料和试剂

  1. 蓝藻培养(注1)
  2. 甲醇≥99.9%(GC)(Sigma-Aldrich)

设备

  1. Eppendorf安全锁管(1.5 ml)
  2. 以15,000×g的相对离心力(RCF)和+4℃的冷却选择(Sigma-Aldrich,型号:1-16K)离心,
  3. 移液器100μl-1,000μl+移液器吸头(RAININ,Mettler-Toledo)
  4. 冰箱(+4°C)
  5. 具有狭缝宽度1nm(Shimadzu,型号:UV-2600)的分光光度计
  6. 分光光度法塑料或玻璃VIS/UV-VIS半微量0.75-1.5毫升比色皿
  7. 混合装置(Silamat S6,Ivoclar Vivadent)或涡流(IKA MS3 digital,IKA )
  8. 铝箔
  9. Eppendorf管支架

程序

  1. 在适度的辐照度下[高达5μmol(光子)m -1 s -1的白光或10μmol(光子)m <-2d/sup] >绿色光的 -1 ],以防止所提取的颜料的降解。
  2. 收获1ml蓝藻培养悬浮液(注2)。
  3. 在实验室温度下将细胞以15,000×g离心7分钟,并彻底丢弃上清液。 如果必要,重复离心(注3)
  4. 加入1ml甲醇,预冷至+ 4℃
  5. 通过混合(Silamat S6,2秒),涡旋(2,000rpm,4秒)或通过上下轻轻吹吸使样品均质化。
  6. 用铝箔覆盖样品。在+ 4°C孵育20分钟,以从细胞中提取颜料(注4)
  7. 在15,000×g/min,+ 4℃下离心7分钟,目视检查沉淀;它应该在蓝绿色和紫色之间(图1),没有绿色。如果颗粒是绿色的,重复步骤5-6

    图1.在添加甲醇(A)之前和在叶绿素a和类胡萝卜素萃取(B)之后蓝细菌颗粒的颜色。甲醇萃取后的颗粒的颜色将是从蓝藻( Synechocystis sp。PCC 6803)到紫色( Lyngbya sp。IPPAS B-1204),取决于藻胆蛋白的特定组合。
  8. 使用甲醇作为空白校准分光光度计
  9. 通过分光光度计,狭缝宽度1 nm测量颜料浓度。
    1. 在470nm,665nm和720nm测量样品和空白的吸光度(注5)
    2. 根据方程计算叶绿素浓度 a :
      (Ritchie,2006)。
      [μg/ml] = 12.9447(A sub 665-A 720) Chl a [μM] = 14.4892(A 665 720 ); 对于Chl a摩尔质量= 893.4890g/mol 类胡萝卜素[μg/ml] = [1,000(A 470 -A 720)-2.86(Chl a [μg/ml] 221(Wellburn,1994)。
  10. 根据每个颜料评估的平均值和标准偏差的计算,至少进行三次重复分析

代表数据



图2.在蓝细菌集胞藻属的细胞培养物中叶绿素a和类胡萝卜素浓度的代表性测量。 PCC 6803。将浓密蓝细菌培养物(叶绿素:22μg/ml,类胡萝卜素:7μg/ml)逐渐稀释一半直到颜料浓度为0.1μg/ml。每次测量一式三份;误差棒表示标准偏差。

笔记

  1. 作者不知道对于仅含有叶绿素a的蓝藻菌株,该方案用法的任何限制。该协议已成功应用于Cyanothece sp。 ATCC 51142,细长聚球藻(Synechococcus elongatus) PCC 7942,Cyanobacterium IPPAS B-1200, Synechocystis PCC 6803,慢性节旋藻 IPPAS B-256,Anabaena sphaerica IPPAS B-404,Chroococcus IPPAS B-1203, Lyngbya IPPAS B-1204, aphanocapsa IPPAS B-1205, IPPAS B-1206。
  2. 分析所需的细胞悬浮液的量可随培养物密度而变化。对于非常稀释的培养物,建议收获更大的培养体积;对于具有大约10ng(Ch1)ml培养物〜sup-1或更低,甚至高达5ml的叶绿素密度的培养物。相反,对于非常密集的培养物,推荐更低的样品体积 - 具有高细胞密度,一些颜料可以在提取后留在细胞中。
  3. 如果在第一次离心之后一些上清液留在管中,则提取不会在纯甲醇中进行,并且不能适当地测量颜料浓度。
  4. 颜料萃取时间可以延长至2小时,没有明显的颜料降解。
  5. 每个波长的最终吸光度应在线性吸光度范围内。对于分光光度计UV-2600(Shimadzu),该线性吸光度范围为0.01-2.5。如果需要,用甲醇稀释样品以适应分光光度计线性吸光度范围
  6. 在使用不同体积的蓝细菌样品和/或甲醇的情况下,1ml以上,最终的颜料浓度应按下式计算:

致谢

该方案采用自出版物"On the dynamics and constraints of batch culture growth of the cyanobacterium Cyanothece sp。 ATCC 51142"(Sinetova等人,2012)。 T.Z.和J.C.由MEYS CR在CzechGlobe Center,reg。没有。 CZ.1.05/1.1.00/02.0073 484,国家可持续计划I(NPU I),授权号LO1415和EC OP项目,没有。 CZ.1.07/2.3.00/20.0256。 S. M. A.得到俄罗斯科学基金会资助。 14-24-00020)。

参考文献

  1. Balizs,G.,Benesch-Girke,L.,Borner,S.and Hewitt,S.A。(1994)。 使用液相色谱与猪肌肉组织中四种磺胺类药物及其N4-乙酰基代谢物的测定比较紫外和质谱检测。 66 Chromatogr B Biomed Appl 661(1):75-84。
  2. Ritchie,R.J。(2006)。 一致的丙酮,甲醇和乙醇溶剂的分光光度法叶绿素方程组。 Photosynth Res 89(1):27-41。
  3. Sinetova,M.A.,Červený,J.,Zavřel,T。和Nedbal,L。(2012)。 关于蓝细菌Cyanothece sp的分批培养生长的动力学和约束 。 ATCC 51142. J Biotechnol 162(1):148-155
  4. Wellburn,A.R。(1994)。 使用各种溶剂,使用不同分辨率的分光光度计,对叶绿素a和b以及总类胡萝卜素进行光谱测定。 J Plant Physiol 144:307-313。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zavřel, T., Sinetova, M. A. and Červený, J. (2015). Measurement of Chlorophyll a and Carotenoids Concentration in Cyanobacteria. Bio-protocol 5(9): e1467. DOI: 10.21769/BioProtoc.1467.
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