TITLE: Biogenic silica measurement from diatom
AUTHORS: Ying-Yu Hu, Nuwanthi Samarasinghe, Zoe V. Finkel

[DESCRIPTION]
Here we describe the measurement of biogenic silica from diatoms. Biogenic silica is digested by wet-alkaline method,  using 2 M sodium carbonate to hydrate and depolymerize amorphous silica and yield monosilicic acid. Diatomaceous earth is used as a check standard for the recovery of biogenic silica. The molybdate measurement is adopted from Mortlock et al. (1989) and JGOFS protocols (UNESCO 1994).

Digestion: 
 

Molybdate measurement:
 


[STEPS]
SECTION: Sample collection
1. Estimation:
Low limit of detection of this assay is ~3 uM silicate per sample in the molybdate assay, equivalent to about 1 ug PON per filter.
SECTION: Sample collection
2. Filter micro algae in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg)
 

SECTION: Sample collection
3. Rinse filter funnel with filtered artificial seawater without macronutrients to avoid sample loss 
SECTION: Sample collection
4. Transfer filter with sample into 2 mL cryogenic vial
SECTION: Sample collection
5. Filter blank media (without cells) through polycarbonate filter as blank
SECTION: Sample collection
6. Flash freeze and store at -20 °C 

SECTION: Dissolution
7. 2 M Na2CO3

SECTION: Dissolution
7.1. Each sample requires 10 mL 2 M Na2CO3
SECTION: Dissolution
7.2. In a 1 L polypropylene volumetric flask, add 500 mL Milli-Q
SECTION: Dissolution
7.3. Add and dissolve 212 g Na2CO3 (CAS: 497-19-8, FW 105.99)

SECTION: Dissolution
7.4. Top final volume to 1 L
SECTION: Dissolution
8. Turn on airforce oven to 85 °C 
 

SECTION: Dissolution
9. Transfer sample to 50 mL falcon tube with clean filter forceps (rinsed by 95% ethanol and air-dried)
 

SECTION: Dissolution
11. Add 10 mL  2 M Na2CO3 into each tube with sample or diatomaceous and vortex.
SECTION: Acidification
20. 1 Molarity (M) HCl

SECTION: Acidification
20.1. 12 N HCl : Milli-Q = 1 : 11 (v/v)
SECTION: Acidification
20.2. Require 5 mL 1 M HCl per sample
SECTION: Acidification
21. Gradually add 5 mL 1 M HCl to each tube (diatomaceous references, blank for diatomaceous reference, diatom samples and diatom sample blanks), mix well before adding the next portion of acid to avoid vigorous reaction.

SECTION: Dissolution
13. Remove tubes from the oven and cool to room temperature

 

SECTION: Acidification
22. Transfer resulted solution from falcon tube to 25 mL polypropylene volumetric flask

SECTION: Acidification
23. Fill empty falcon tube with about 2 mL Milli-Q, cap tightly and vortex. Transfer all liquid to the volumetric flask.
SECTION: Acidification
24. Fill empty falcon tube with about 2 mL Milli-Q, cap tightly and vortex. Transfer all liquid to the volumetric flask.
SECTION: Acidification
25. Fill empty falcon tube with about 2 mL Milli-Q, cap tightly and vortex. Transfer all liquid to the volumetric flask.
SECTION: Acidification
26. Top final volume to 25 mL with Milli-Q.
SECTION: Acidification
27. Shake and thoroughly mix the solution.
SECTION: Molybdate reaction
29. Standard working solutions
 

SECTION: Dissolution
10. Diatomaceous 
 

SECTION: Dissolution
10.1. Weigh ~1000 ug diatomaceous into 50 mL falcon tube, in triplicate.
  
SECTION: Dissolution
12. Loose the caps and place all samples into the airforce oven overnight (for example, from 5 pm to 9 am).
SECTION: Prepare standard primary solution and reagents
14. Molybdate reagent stock solution

 

SECTION: Prepare standard primary solution and reagents
14.1.   

Ammonium paramolybdate: 
[(NH4)6Mo7O24.4H2O]
CAS: 12058-85-2

SECTION: Prepare standard primary solution and reagents
14.2. In the fume hood, add 0.8 g ammonium paramolybdate and 2.4 mL 12 N HCl into a 100 mL plastic volumetric flask.

SECTION: Prepare standard primary solution and reagents
15. HCl stock solution
In a fume hood, add 4.8 mL 12 N HCl into a 100 mL plastic volumetric flask and top to 100 mL. 
 

SECTION: Prepare standard primary solution and reagents
14.3. Top to 100 mL with MilliQ water.
SECTION: Prepare standard primary solution and reagents
16. Metal-sulfite solution
 

SECTION: Prepare standard primary solution and reagents
14.4. Store in a brown polyethylene bottle out of direct sunlight. Discard if white precipitation forms.
SECTION: Prepare standard primary solution and reagents
16.1.   
  

SECTION: Prepare standard primary solution and reagents
16.2. Require:
(1) No.1 Whatman filter paper
(2) Swinnex filter holder
(3) 50 mL syringe
SECTION: Prepare standard primary solution and reagents
16.3. In the fume hood, add 60 mL MilliQ into a 100 mL plastic volumetric flask. 
SECTION: Prepare standard primary solution and reagents
16.4. Add 1.2 g sodium sulphite and mix to dissolve.

SECTION: Prepare standard primary solution and reagents
16.5. Add 2 g 4-(methyl amino)phenol hemisulfate, mix to dissolve.

SECTION: Prepare standard primary solution and reagents
16.6. Top to 100 mL with MilliQ water.
SECTION: Prepare standard primary solution and reagents
16.7. Cut No. 1 Whatman filter paper into 25 mm disk and load it into Swinnex filter holder.
SECTION: Prepare standard primary solution and reagents
16.8. Fill syringe with Metal-sulfite solution, filter through the filter paper, collect filtrate into an amber or dark plastic bottle, keep at room temperature. 
SECTION: Prepare standard primary solution and reagents
16.9. Prepare fresh every month.
SECTION: Prepare standard primary solution and reagents
17. Oxalic acid solution
 

SECTION: Prepare standard primary solution and reagents
17.1.   

SECTION: Prepare standard primary solution and reagents
17.2. In a 100 mL plastic volumetric flask, dissolve 6 g oxalic acid in MilliQ and top to 100 mL.

SECTION: Prepare standard primary solution and reagents
17.3. Let the solution stand at room temperature overnight.
SECTION: Prepare standard primary solution and reagents
17.4. Decant the solution from the crystals into a plastic bottle.
SECTION: Prepare standard primary solution and reagents
17.5. Keep at room temperature.
SECTION: Prepare standard primary solution and reagents
18. Sulphuric acid (0.5 M)
If following Stanford protocol, it shall be 50%. Then prepare as 1 part concentrated sulphuric acid into 1 part of MilliQ
Wait for Nuwanthi's confirmation
SECTION: Prepare standard primary solution and reagents
18.1. 
 

SECTION: Prepare standard primary solution and reagents
18.2. In a 100 mL plastic volumetric flask, add 50 mL MilliQ
SECTION: Prepare standard primary solution and reagents
18.3. Slowly add 2.778 mL concentrated sulphuric acid

SECTION: Prepare standard primary solution and reagents
18.4. Allow the mixture cool to room temperature and then top to 100 mL with MilliQ water.
SECTION: Prepare standard primary solution and reagents
18.5. Store in a plastic bottle at room temperature. 
SECTION: Prepare standard primary solution and reagents
19. Primary silica standard solution (~ 1 mM Si)
SECTION: Prepare standard primary solution and reagents
19.1.   

SECTION: Prepare standard primary solution and reagents
19.2. Transfer 1 g sodium fluorosilicate in a plastic vial 
SECTION: Prepare standard primary solution and reagents
19.3. Keep the vial in a vacuum desiccator overnight to remove excess water (do not heat or fuse)
SECTION: Prepare standard primary solution and reagents
19.4. In a one litre plastic volumetric flask, dissolve ~0.1881 g (log the actual mass) of dry sodium fluorosilicate in MilliQ water and top to 1 L with MilliQ water.

SECTION: Prepare standard primary solution and reagents
19.5. It takes about 30 min to complete the dissolution. This cannot be rushed.
SECTION: Prepare standard primary solution and reagents
19.6. Store in a plastic bottle at room temperature.
SECTION: Molybdate reaction
30. Molybdate working solution
 

SECTION: Molybdate reaction
30.1. 1 part Molybdate stock reagent 
1 part HCl  stock reagent
SECTION: Molybdate reaction
31. Reducing solution
 

SECTION: Molybdate reaction
31.1. 1 part Metol-sulfite solution
1 part oxalic acid solution
1 part sulphuric acid solution
SECTION: Molybdate reaction
34. Diatom samples and blanks:
Transfer 50 µL solution from volumetric flask into 2 mL microtube, and add 450 µL MilliQ water

SECTION: Molybdate reaction
32. Standard working solutions:

Transfer 50 µL standard working solution into 2 mL microtube, and add 450 µL MilliQ water

SECTION: Molybdate reaction
35. Add 200 µL Molybdate reagent into each tube.

SECTION: Molybdate reaction
36. After yellow color is developed in 15 min, add 300 µL reducing solution into each tube.

SECTION: Molybdate reaction
37. Vortex each tube and then shake at Room temperature for 180 min 

SECTION: Colorimetric measurement
38. Load microplate with 250 µL reactant from each tube, duplicate.
 


SECTION: Colorimetric measurement
39. Read plate in microplate reader
   AB1Shake duration00:00:052Shaking typeContinuous3Shaking forceHigh4Shaking speed [rpm]6005Wavelength [nm]8206Use transmittanceNo7Pathlength correctionNo8Measurement Time [ms]100

 

SECTION: Calculation
41. Subtract the average absorbance at 812 nm of the blank standard replicates from the absorbance at 812 nm of all other standard working solutions.
SECTION: Calculation
42. Subtract the average absorbance at 812 nm of the blank sample (i.e. blank filter) replicates from the absorbance at 812 nm of all other individual samples.
SECTION: Calculation
43. Prepare a standard curve by plotting the average blank-corrected 812 nm absorbance for each standard working solution versus its concentration in uM.
SECTION: Calculation
44. Use the standard curve to determine the silicate concentration of each unknown sample by using its blank-corrected 812 nm absorbance.
SECTION: Calculation
45. (Si per sample)_umol = Si_uM X (25)_mL X (0.001)
SECTION: Calculation
46. Diatomaceous recovery = 100 X Si_uM X DF X (25)_mL X (0.001) X (60.1_ug/umol)/(M_ug X Purity X 0.01)
Where, DF is the dilution factor (DF=10), M is the mass of diatomaceous, purity of SiO2 in Celite S diatomaceous earth (06858) is 90.2%.
 

SECTION: Dissolution
10.2. Prepare one empty 50 mL falcon tube as the reagent blank for diatomaceous.
SECTION: Molybdate reaction
33. Diatomaceous reference and blank:
SECTION: Molybdate reaction
33.1. Transfer 50 µL solution from volumetric flask to a 2 mL microtube, add 450 µL MilliQ.

SECTION: Molybdate reaction
33.2. Transfer 50 µL diluted solution into a new 2 mL microtube, add 450 µL MilliQ. 

SECTION: Waste disposal
40. Collect all solution with paramethylaminophenol sulphate in waste container.
SECTION: Acidification
28.