拟小球藻中总磷酸盐和多磷酸盐的提取和钼蓝法定量

Shuhei Ota; Shigeyuki Kawano

doi:10.21769/BioProtoc.2539

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Peer-reviewed

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

SO Shuhei Ota email

SK Shigeyuki Kawano

发布: 2017年09月05日第7卷第17期 DOI: 10.21769/BioProtoc.2539 浏览次数: 10573

评审: Maria SinetovaAnonymous reviewer(s)

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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

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

Equipment

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

Procedure

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如何引用

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.