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Extraction and Molybdenum Blue-based Quantification of Total Phosphate and Polyphosphate in Parachlorella
拟小球藻中总磷酸盐和多磷酸盐的提取和钼蓝法定量   

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Abstract

Inorganic phosphorus is a non-renewable resource and an essential element for life on Earth. Organisms such as algae, protists, and animals can store phosphate (Pi) through uptake of Pi as polyphosphate (poly-P), which is a linear polymer of orthophosphate residues linked by high-energy phosphoanhydride bonds. Here, we describe procedures for extraction of total phosphate and poly-P from Parachlorella cells and quantification of orthophosphate based on molybdenum blue assay. The present method may be applicable for other microalgae.

Keywords: Alga(藻类), Chlorella(小球藻), Parachlorella(拟小球藻), Phosphorus(磷), Polyphosphate(多磷酸盐), Molybdenum blue reaction(钼蓝反应)

Background

Biological phosphorus recovery is a particularly attractive form of nutrient recycling. Algae can accumulate phosphate (Pi), and Pi-enriched algal biomass can be used as biofertilizer (Solovchenko et al., 2016). In a previous study, Ota et al. (2016) revealed the relationship between electron dense bodies and poly-P dynamics under sulfur-deficient (-S) conditions in Parachlorella kessleri. Parachlorella is a genus of green algae in the class Trebouxiophyceae, characterized by a rigid cell wall and an asexual, non-motile life cycle. The protocol presented here allows extraction of total Pi and polyphosphate (poly-P) from Chlorella and quantification of inorganic phosphorus based on molybdenum blue reaction, which is a standard method used to quantify orthophosphate. The theoretical background of the molybdenum blue reaction was reviewed previously by Nagul et al. (2015).

Materials and Reagents

  1. Pipette tips for 10 µl, 200 µl and 1,000 µl (Labcon, catalog numbers: 1161-965 , 1065-960 , 1168-960 )
  2. 15-ml conical centrifuge tubes (FUKAEKASEI and WATSON, catalog number: 1332-015S )
  3. 2-ml microtubes (SARSTEDT, catalog number: 72.695.500 )
  4. Aluminum foil (Mitsubishi Aluminum, 0.012 mm thick)
  5. 96-well microplates, non-treated surface (Asahi Glass, catalog number: 1860-096 )
  6. Microplate seal (qPCR seal) (4titude, catalog number: 4ti-0560 )
  7. Parachlorella kessleri (National Institute for Environmental Studies, catalog number: NIES-2152 )
  8. TAP medium (without agar; see http://mcc.nies.go.jp/02medium.html)
  9. Sodium hypochlorite (available chlorine, min. 5.0%) (Wako Pure Chemical Industries, catalog number: 197-02206 )
  10. Glass beads, acid-washed 425-600 µm (Sigma-Aldrich, catalog number: G8772 )
  11. Ethanol (99.5% v/v) (Wako Pure Chemical Industries, catalog number: 057-00456 )
  12. Potassium peroxodisulfate (K2S2O8) (Kishida Chemical, catalog number: 310-63931 )
  13. Antimony potassium tartrate trihydrate (C8H4K2O12Sb2·3H2O) (Alfa Aesar, catalog number: A13766 )
  14. L-Ascorbic acid (Wako Pure Chemical Industries, catalog number: 012-04802 )
  15. Hexaammonium heptamolybdate tetrahydrate ((NH4)6Mo7O24·4H2O) (Wako Pure Chemical Industries, catalog number: 016-06902 )
  16. Phosphate ion standard solution (NaH2PO4 in water) (Wako Pure Chemical Industries, catalog number: 168-17461 )
  17. Sulfuric acid (Wako Pure Chemical Industries, catalog number: 195-04706 )
  18. Ammonium molybdate tetrahydrate solution (see Recipes)

Equipment

  1. Micro-spatula (AS ONE, catalog number: 6-524-06 )
  2. Pipettes for 10 µl, 200 µl and 1,000 µl (Eppendorf, model: Research® plus )
  3. Microtube mixer (TOMY SEIKO, model: MT-360 )
  4. Autoclave (TOMY SEIKO, model: LSX-300 )
  5. Centrifuge, swing rotor (TOMY SEIKO, model: LC-121 )
  6. Refrigerated microcentrifuge (TOMY SEIKO, model: MX-300 )
  7. Microplate reader (BioTek Instruments, model: EPOCH )

Procedure

Note: See Figure 1 for an overview from sampling to molybdenum blue reaction.



Figure 1. Overview of the phosphate assay in Chlorella. +S, sulfur-replete medium (control culture); -S, sulfur-depleted medium (experimental culture; for details, see Ota et al., 2016).


  1. Sampling and preparing algal samples
    1. Take 10 ml of algal culture [OD595: 1-2 in TAP medium (Ota et al., 2016)] in a 15-ml conical centrifuge tube. Centrifuge for 5 min at 2,500 x g at room temperature with the swing rotor. Discard the supernatant. At this point, additional samples should be taken for dry weight and/or cell number measurement (see Data analysis below).
    2. Resuspend the pellet with 2 ml distilled water and transfer to a new 2-ml microtube. The suspended samples can be divided into halves: 1 ml for poly-P assay and 1 ml for total-P assay.
    3. Centrifuge again at room temperature for 5 min at 2,500 x g. Discard the supernatant. At this point, the pellet can be stored in a freezer at -20 °C for further analysis.

  2. Extraction of poly-P
    1. Add 1 ml of > 5% sodium hypochlorite to the cell pellet.
    2. Add 2-3 microspatulas of glass beads (approximately 50 mg) and mix vigorously using a microtube mixer for 10-15 min at 4 °C in a cold room. Centrifuge for 2 min at 14,000 x g at 4 °C. Remove the supernatant.
    3. Add 1 ml of sodium hypochlorite to the cell pellet. Centrifuge for 2 min at 14,000 x g at 4 °C. Remove the supernatant. Repeat step 3.
      Note: The high chain length of polyphosphate is virtually insoluble in alkaline sodium hypochlorite (Sutherland and Wilkinson, 1971).
    4. Add 100 µl of distilled water and incubate for 5 min at room temperature. Centrifuge for 2 min at 14,000 x g at 4 °C. Collect the supernatant (a).
    5. Add 100 µl of distilled water and incubate for 5 min. Centrifuge for 2 min at 14,000 x g at 4 °C. Collect the supernatant (b).
    6. Add 1.8 ml of ethanol to the supernatant (a + b) and centrifuge for 10 min at 14,000 x g at 4 °C. In this step, poly-Ps are precipitated as a white pellet at the bottom of the test tube.
    7. Remove the supernatant carefully, add 500 µl of distilled water, and mix vigorously (poly-P solution).
      Note: Amount of distilled water may be adjusted from 50 to 500 µl (OD880 > 0.3; see Data analysis below).
    8. Add 100 µl of 4% (w/v) potassium persulfate to the poly-P solution.
    9. For hydrolysis to orthophosphate, autoclave the poly-P solution at 121 °C for 20 min without fast exhaust option to avoid loss of samples. Leave the cap open and cover the microtube with aluminum foil when autoclaving.
    10. The autoclaved samples are now ready to use for molybdenum blue assay (see Procedure D).

  3. Extraction of total P
    1. Resuspend the cell pellet from A3 with 1 ml of distilled water.
    2. Disrupt samples. Add 2-3 microspatulas of glass beads and mix vigorously using a microtube mixer for 10-15 min at 4 °C in a cold room.
    3. Add 200 µl of 4% (w/v) potassium persulfate to the sample.
    4. Autoclave the poly-P solution at 121 °C for 20 min without fast exhaust to avoid loss of samples. Leave the cap open and cover the microtube with aluminum foil when autoclaving.
    5. The autoclaved samples are now ready to use for molybdenum blue assay (see Procedure D).
      Note: The supernatant is used for assay. Normally, there is no need for centrifugation step.

  4. Molybdenum blue reaction in a 96-well microplate
    1. Pipette 200 µl of diluted sample (196 µl of distilled water + 4 µl of poly-P/total-P samples) per well.
    2. Add 8 µl of ammonium molybdate tetrahydrate solution (see Recipes below).
    3. Add 2 µl of 7.2% (w/v) L-ascorbic acid solution.
    4. Seal with plate seal film and mix well (Invert the plate 3-5 times).
    5. Incubate for 20 min in the dark at room temperature. The mixture will turn blue in color if orthophosphate is present (Figure 2).


      Figure 2. Molybdenum blue reaction in a 96-well microplate. This is an example of the phosphate ion standard dilution series (0, 0.5, 1, 2 mg/L from left to right, n = 3).

    6. Measure the absorbance at 880 nm (OD880) using a microplate reader.
      Note: If wavelength at 880 nm is not available, select an alternative wavelength near 880 nm.

Data analysis

A calibration curve is necessary for each experiment for calculation of corresponding absolute values. A phosphate ion standard solution is diluted with distilled water ranging from 0 to 2 mg/L (e.g., 0, 0.1, 0.5, 1, 2 mg/L), where linearity between absorbance (OD880) and Pi concentration is confirmed (Figure 3). Measure OD880 at one point per well, and a mean value is calculated from at least three replicates per sample (n > 3). A calculation example is as follows: The concentration rate in poly-P extraction is 5-fold (Procedure A), 1,000/(100 + 500) - fold (Procedure B), and 4/200 - fold (Procedure D), respectively. Then, the poly-P concentration (mg/L) is calculated as follows: the corresponding value = {(OD880 - 0.05)/0.13} x1/5 x 600/1,000 x 200/4. Dry weight and/or cell number should be measured for normalization when sampling cultures. These values are used for calculating the amount of Pi per dry weight or cell.


Figure 3. Example of a calibration curve

Notes

  1. Phosphate ion standard solution can be self-produced using sodium dihydrogenphosphate (NaH2PO4).
  2. All stock solutions (potassium persulfate, ammonium molybdate tetrahydrate, L-ascorbic acid) should be prepared with distilled water (see also Recipes below).
  3. Solutions are prepared at time of use. The stock solutions can be stored up to one month at 4 °C.
  4. Samples should be diluted with distilled water, if OD880 is more than 0.3.

Recipes

  1. Ammonium molybdate tetrahydrate solution (100 ml)
    1.2 g of hexaammonium heptamolybdate tetrahydrate
    4.8 mg of potassium antimonyl tartrate sesquihydrate
    32 ml of diluted sulfuric acid (sulfuric acid:distilled water = 1:1)
    Make up with distilled water to volume

Acknowledgments

This research was supported with funding provided by the CREST program (to SK) of the Japan Science and Technology Agency (JST).

References

  1. Nagul, E. A., McKelvie, I. D., Worsfold, P. and Kolev, S. D. (2015). The molybdenum blue reaction for the determination of orthophosphate revisited: Opening the black box. Anal Chim Acta 890: 60-82.
  2. Ota, S., Yoshihara, M., Yamazaki, T., Takeshita, T., Hirata, A., Konomi, M., Oshima, K., Hattori, M., Bisova, K., Zachleder, V. and Kawano, S. (2016). Deciphering the relationship among phosphate dynamics, electron-dense body and lipid accumulation in the green alga Parachlorella kessleri. Sci Rep 6: 25731.
  3. Solovchenko, A., Verschoor, A. M., Jablonowski, N. D. and Nedbal, L. (2016). Phosphorus from wastewater to crops: An alternative path involving microalgae. Biotechnol Adv 34(5): 550-564.
  4. Sutherland, I. W. and Wilkinson, J. F. (1971). Chemical extraction methods of microbial cells. In: Norris, J. R. and Ribbons, D. W. (Eds.). Methods in Microbiology. Vol. 5B. Academic Press pp: 1-665.

简介

无机磷是一种不可再生资源,是地球上生命的基本要素。 诸如藻类,原生生物和动物之类的生物体可以通过摄取作为多磷酸(poly-P)的Pi来储存磷酸盐(poly-P),聚磷酸盐是通过高能量磷酸酐键连接的正磷酸盐残基的线性聚合物。 在这里,我们描述了从Parachlorella细胞中提取总磷酸盐和poly-P以及基于钼蓝测定定量正磷酸盐的方法。 本方法可适用于其他微藻类。
【背景】生物磷回收是一种特别有吸引力的营养物回收形式。 藻类可以积聚磷酸盐(Pi),并且富含Pi的藻类生物质可用作生物肥料(Solovchenko等,2016)。 在以前的研究中,Ota等人 (2016)揭示了Parachlorella kessleri中硫缺乏(-S)条件下的电子密实体与聚P动力学的关系。 伞藻属于Trebouxiophyceae属的绿藻属,其特征在于刚性细胞壁和无性,不运动的生命周期。 这里提出的方案允许从小球藻提取总Pi和多磷酸(poly-P),并且基于钼蓝反应定量无机磷,这是用于定量正磷酸盐的标准方法。 Nagul等人先前回顾了钼蓝反应的理论背景。(2015年)。

关键字:藻类, 小球藻, 拟小球藻, 磷, 多磷酸盐, 钼蓝反应

材料和试剂

  1. 吸取10μl,200μl和1,000μl(Labcon,目录号:1161-965,1065-960,1168-960)的提示
  2. 15毫升锥形离心管(FUKAEKASEI和WATSON,目录号:1332-015S)
  3. 2毫升微管(SARSTEDT,目录号:72.695.500)
  4. 铝箔(三菱铝,0.012mm厚)
  5. 96孔微孔板,未处理表面(Asahi Glass,目录号:1860-096)
  6. 微孔板密封(qPCR密封)(4份,目录号:4ti-0560)
  7. Parachlorella kessleri (国家环境研究所,目录号:NIES-2152)
  8. TAP培养基(不含琼脂;见 http://mcc.nies.go .jp / 02medium.html
  9. 次氯酸钠(有效氯,最少5.0%)(和光纯药公司,目录号:197-02206)
  10. 玻璃珠,酸洗425-600微米(Sigma-Aldrich,目录号:G8772)
  11. 乙醇(99.5%v / v)(Wako Pure Chemical Industries,目录号:057-00456)
  12. 过氧二硫酸钾(K 2 O 2 S 2 O 8)(Kishida Chemical,目录号:310-63931)
  13. 酒石酸锑钾三水合物(C 8 H 4 H 2 O 2 O 12 Sb 2 O 3 ·3H 2 O)(Alfa Aesar,目录号:A13766)
  14. L-抗坏血酸(和光纯药,目录号:012-04802)
  15. 四水合七钼酸铵((NH 4))6 Mo 7 O 24 H 4·4H 2 / > O)(和光纯药,目录号:016-06902)
  16. 磷酸盐离子标准溶液(NaH 2 PO 4水溶液)(Wako Pure Chemical Industries,目录号:168-17461)
  17. 硫酸(Wako Pure Chemical Industries,目录号:195-04706)
  18. 钼酸铵四水合物溶液(见配方)

设备

  1. 微铲(AS ONE,目录号:6-524-06)
  2. 移液管10μl,200μl和1,000μl(Eppendorf,型号:Research ®)
  3. Microtube搅拌机(TOMY SEIKO,型号:MT-360)
  4. 高压灭菌器(TOMY SEIKO,型号:LSX-300)
  5. 离心机,摆动转子(TOMY SEIKO,型号:LC-121)
  6. 冷冻微量离心机(TOMY SEIKO,型号:MX-300)
  7. 酶标仪(BioTek Instruments,型号:EPOCH)

程序

注意:从采样到钼蓝反应的概述见图1。



图1.小球藻中的磷酸盐测定的概述 + S,充满硫的培养基(对照培养); -S,贫硫的培养基(实验培养;详见Ota等人,2016)。


  1. 采样和制备藻类样品
    1. 在15ml锥形离心管中,在TAP培养基(Ota等人,2016)中取10毫升藻类培养物[OD 595 :1-2)。用摆动转子在室温下以2,500×g离心5分钟。丢弃上清液。此时,应采取额外的样品进行干重和/或细胞数量测量(参见下面的数据分析)。
    2. 用2ml蒸馏水重悬沉淀并转移到新的2ml微量管中。悬浮样品可以分成两半:多聚-P测定1ml,总P测定1ml。
    3. 在2,500 x g下,在室温下再次离心5分钟。丢弃上清液。此时,颗粒可以储存在-20°C的冰箱中进一步分析。

  2. 聚P的提取
    1. 加入1ml> 5%次氯酸钠至细胞沉淀。
    2. 加入2-3微玻璃珠(约50毫克),并在4℃下在冷室中使用微管混合器剧烈混合10-15分钟。在4℃下以14,000xg离心2分钟离心。去除上清液。
    3. 将1ml次氯酸钠加入细胞沉淀。在4℃下以14,000xg离心2分钟离心。去除上清液。重复步骤3.
      注意:多磷酸盐的高链长度几乎不溶于碱性次氯酸钠(Sutherland和Wilkinson,1971)。
    4. 加入100μl蒸馏水,室温孵育5 min。在4℃下以14,000xg离心2分钟离心。收集上清液(a)。
    5. 加入100μl蒸馏水,孵育5 min。在4℃下以14,000xg离心2分钟离心。收集上清液(b)。
    6. 向上清液(a + b)中加入1.8ml乙醇,并在4℃以14,000×g离心10分钟。在此步骤中,poly-Ps在试管底部以白色颗粒沉淀
    7. 小心取出上清液,加入500μl蒸馏水,并剧烈混合(poly-P溶液)。
      注意:蒸馏水的量可以调节为50至500μl(OD 880> 0.3;参见下面的数据分析)。
    8. 向聚P溶液中加入100μl4%(w / v)过硫酸钾。
    9. 为了水解成正磷酸盐,在121℃高压灭菌20分钟,无需快速排气选项,以避免样品损失。高压灭菌时,盖上盖子并用铝箔盖住微管
    10. 高压灭菌的样品现在可以用于钼蓝测定(见程序D)。

  3. 总P的提取
    1. 用A3蒸馏水重悬细胞沉淀。
    2. 破坏样品加入2-3个玻璃珠微珠,并使用微管混合器在4℃下在冷藏室中剧烈混合10-15分钟。
    3. 向样品中加入200μl4%(w / v)过硫酸钾。
    4. 在121℃高压灭菌20分钟,不用快速排气,以避免样品损失。高压灭菌时,盖上盖子并用铝箔盖住微管。
    5. 高压灭菌的样品现在可以用于钼蓝测定(参见方法D)。
      注意:将上清液用于测定。通常,不需要离心步骤。

  4. 在96孔微孔板中进行钼蓝反应
    1. 移取200μl稀释样品(196μl蒸馏水+ 4μlPoly-P / total-P样品)。
    2. 加入8μl钼酸铵四水合物溶液(见下面的配方)
    3. 加入2μl7.2%(w / v)L-抗坏血酸溶液
    4. 用板密封膜密封并充分混合(倒置板3-5次)
    5. 在室温下在黑暗中孵育20分钟。如果存在正磷酸盐,混合物会变成蓝色(图2)

      图2.在96孔微孔板中的钼蓝反应。 这是磷酸盐离子标准稀释系列(从左到右为0,0.5,1,2 mg / L,n = 3)的例子。

    6. 使用酶标仪测量880nm处的吸光度(OD 880)。
      注意:如果880 nm波长不可用,请选择880 nm附近的其他波长。

数据分析

在每个实验中需要校准曲线来计算相应的绝对值。用0至2mg / L(例如,0,0.1,0.5,1,2mg / L)的蒸馏水稀释磷酸盐离子标准溶液,其中吸光度(OD 880 ),并确认Pi浓度(图3)。在每个孔的一个点测量OD 880 ,并且从每个样品至少三次重复计算平均值(3)。计算实例如下:聚-P提取中的浓度分别为5倍(程序A),1,000 /(100 + 500)倍(程序B)和4/200倍(程序D)) 。然后,如下计算聚-P浓度(mg / l):相应值= {(OD 880 <0.05)/0.13}×1/5×600 / 1,000×200/4。当采样培养物时,应测量干重和/或细胞数量进行归一化。这些值用于计算每个干重或细胞的Pi量。


图3.校准曲线示例

笔记

  1. 磷酸盐离子标准溶液可以使用磷酸二氢钠(NaH 2 PO 4)4自身产生。
  2. 所有储备溶液(过硫酸钾,四水合钼酸铵,L-抗坏血酸)均应用蒸馏水制备(见下面的配方)。
  3. 解决方案在使用时准备就绪。库存解决方案可以在4°C下储存一个月。
  4. 如果OD 880 <0.3以上,样品应用蒸馏水稀释

食谱

  1. 钼酸铵四水合物溶液(100ml)
    1.2g七钼酸六铵四水合物
    4.8毫克酒石酸锑醇钾倍半水合物
    32ml稀硫酸(硫酸:蒸馏水= 1:1) 用蒸馏水补足量

致谢

这项研究得到了日本科学技术厅(JST)的CREST计划(SK)提供的资助。

参考

  1. Nagul,EA,McKelvie,ID,Worsfold,P.和Kolev,SD(2015)。&lt; a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/ 26347168"target ="_ blank">用于测定正磷酸盐的钼蓝反应:打开黑匣子。 890:60-82。
  2. Ota,S.,Yoshihara,M.,Yamazaki,T.,Takeshita,T.,Hirata,A.,Konomi,M.,Oshima,K.,Hattori,M.,Bisova,K.,Zachleder, Kawano,S。(2016)。解密关系磷酸盐动力学,电子密集体和绿藻中的脂质积累。 6:25731.
  3. Solovchenko,A.,Verschoor,AM,Jablonowski,ND和Nedbal,L。(2016)。从废水到作物的磷:一种涉及微藻的替代途径。生物技术Adv。34(5):550-564。
  4. Sutherland,IW和Wilkinson,JF(1971)。&lt; a class ="ke-insertfile"href ="https://books.google.com.hk/books?id=0qH9MgVTQ5EC&pg=PA346&lpg=PA346&dq=Chemical+extraction +微生物+细胞的+方法+和+微生物+细胞的方法+&+ = + =微量% >微生物细胞的化学提取方法 In:Norris,JR and Ribbons,DW(Eds。)。微生物学方法。卷。 5B。 学术出版社 pp:1-665。
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引用:Ota, S. and Kawano, S. (2017). Extraction and Molybdenum Blue-based Quantification of Total Phosphate and Polyphosphate in Parachlorella. Bio-protocol 7(17): e2539. DOI: 10.21769/BioProtoc.2539.
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