TITLE: Measurement of biogenic silica from plankton
AUTHORS: Ying-Yu Hu, Nuwanthi Samarasinghe, Zoe V. Finkel

[DESCRIPTION]
Here, we present a method for measuring biogenic silica from plankton. Biogenic silica is digested using a wet-alkaline method, in which 2 M sodium carbonate is used to hydrate and depolymerize amorphous silica, resulting in the production of monosilicic acid. The molybdate measurement technique is based on the method described by Shemesh et al. (1988) and follows the JGOFS protocols outlined by UNESCO (1994).
To ensure the accuracy of the measurement, Celite S diatomaceous earth is used as a check standard for the recovery of biogenic silica. Our method yields a recovery rate of 85% to 90%.
 




[STEPS]
SECTION: Sample collection
1. Estimation:
The low limit of detection is approximately 0.6 uM silicate in the molybdate method. For siliceous plankton, sample requires no less than 4 ug PON (particulate organic nitrogen) per filter when using a 50 mL volumetric flask, or 2 ug PON per filter when using a 25 mL volumetric flask. The sampling volume for biogenic silica samples is approximately 10% of the PON sample volume. For seawater samples, the sampling volume for biogenic silica samples should be determined based on the community composition.
SECTION: Sample collection
5. Filter plankton sample in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg)
 

SECTION: Sample collection
6. Rinse filter funnel with filtered artificial seawater without macronutrients
SECTION: Sample collection
7. Transfer filter with sample into 2 mL cryogenic vial
SECTION: Sample collection
2. Filter blank media (without cells, same volume as plankton samples) through polycarbonate filter as blank
SECTION: Sample collection
8. Flash freeze and store at -20 °C 

SECTION: Day 1: Dissolution
16. 2 M Na2CO3 (18.69%)

 

SECTION: Day 1: Dissolution
16.1. Each sample requires 10 mL 2 M Na2CO3
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16.3. Tare a 1 L plastic erlenmeyer flask
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16.4. Transfer Na2CO3 into the flask

SECTION: Day 1: Dissolution
16.5. Top to 1000 g with MilliQ and shake until all salt is completely dissolved.
SECTION: Day 1: Dissolution
17. Turn on airforce oven to 85 °C 
SECTION: Day 1: Dissolution
18. In the fume hood, transfer diatomaceous into a 5 mL plastic tube for weighing convenience (the original package is 1 kg).
 

 
  
SECTION: Day 1: Dissolution
18.1. Weigh 100~200 ug diatomaceous into 50 mL falcon tube, in triplicate. Log the actual weight.

 


 

SECTION: Day 1: Dissolution
18.2. Prepare one empty 50 mL falcon tube as the reagent blank for diatomaceous.
SECTION: Day 1: Dissolution
19. Add 10 mL  2 M Na2CO3 to each tube, including:
reagent blank for check standards
check standards
blank for samples
samples
SECTION: Day 1: Dissolution
21. Loose the caps and place all tubes into the airforce oven overnight (for example, from 5 pm to 9 am).
SECTION: Standard primary solution and reagents
10. Molybdate reagent stock solution

 

SECTION: Standard primary solution and reagents
10.1.   

Ammonium paramolybdate: 
[(NH4)6Mo7O24.4H2O]
CAS: 12054-85-2

SECTION: Standard primary solution and reagents
10.2. Add 1.6 g ammonium paramolybdate into a 125 mL plastic bottle and top to 100 g with MilliQ.

SECTION: Standard primary solution and reagents
10.3. Store out of direct sunlight. Discard if white precipitation forms.
SECTION: Standard primary solution and reagents
11. HCl stock solution
 

SECTION: Standard primary solution and reagents
12. Metol-sulfite solution
 

SECTION: Standard primary solution and reagents
12.2. Require:
(1) 50 mL syringe
(2) Syringe filter
 

SECTION: Standard primary solution and reagents
12.3. In a 100 to 250 mL plastic beaker, add 0.6 g sodium sulphite.
SECTION: Standard primary solution and reagents
12.4. Add 1 g 4-(methyl amino)phenol hemisulfate.

SECTION: Standard primary solution and reagents
12.5. Top to 50 g with MilliQ water.
SECTION: Standard primary solution and reagents
12.6. Fill syringe with Metol-sulfite solution, filter through the syringe filter, collect filtrate into four 15 mL falcon tubes wrapped with foil, keep at room temperature. 
SECTION: Standard primary solution and reagents
12.7. Prepare fresh every month.
SECTION: Standard primary solution and reagents
13. Oxalic acid solution
 

SECTION: Standard primary solution and reagents
13.1.   
SECTION: Standard primary solution and reagents
13.2. In a 125 mL plastic bottle, add 6 g oxalic acid and top to 100 g.
SECTION: Standard primary solution and reagents
13.3. Let the solution stand at room temperature overnight.
SECTION: Standard primary solution and reagents
13.4. Decant the solution from the crystals into a plastic bottle.
SECTION: Standard primary solution and reagents
13.5. Keep at room temperature.
SECTION: Standard primary solution and reagents
14. Sulphuric acid (30%)
SECTION: Standard primary solution and reagents
14.1. 
 

SECTION: Standard primary solution and reagents
14.2. Mix 3 part concentrated sulphuric acid into 7 part of MilliQ
Cool down to room temperature
 

SECTION: Standard primary solution and reagents
15. Primary silica standard solution (~ 1 mM Si)
SECTION: Standard primary solution and reagents
15.1.   

SECTION: Standard primary solution and reagents
15.2. Transfer 1 g sodium fluorosilicate in a plastic vial 
SECTION: Standard primary solution and reagents
15.3. Keep the vial in a vacuum desiccator overnight to remove excess water (do not heat or fuse)
SECTION: Standard primary solution and reagents
15.4. 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: Standard primary solution and reagents
15.5. It takes about 30 min to complete the dissolution. This cannot be rushed.
SECTION: Standard primary solution and reagents
15.6. Store in a plastic bottle at room temperature.
SECTION: Day 2: Acidification
22. Volume of 12 N HCl required:
3.5 mL X N
SECTION: Day 2: Acidification
29. Transfer resulted solution from falcon tube to 25 or 50 mL polypropylene volumetric flask.
 

SECTION: Day 2: Acidification
30. Use MilliQ to rinse the tube three times and transfer all samples into the volumetric flask. 
 

SECTION: Day 2: Acidification
31. Use transfer pipet, top final volume to 25 or 50 mL with Milli-Q.
SECTION: Day 2: Acidification
32. Shake and thoroughly mix the solution.
 

SECTION: Day 2: Molybdate reaction
34. Secondary standard solution (Freshly prepared prior to the assay)

50 uL primary stock solution
450 uL MilliQ
SECTION: Day 2: Molybdate reaction
38. Molybdate working solution
 

SECTION: Day 2: Molybdate reaction
38.1. 1 part Molybdate stock reagent 
1 part HCl  stock reagent
SECTION: Day 2: Molybdate reaction
42. Reducing solution
 

SECTION: Day 2: Molybdate reaction
42.1. 1 part Metol-sulfite solution
1 part oxalic acid solution
1 part sulphuric acid solution
SECTION: Day 2: Molybdate reaction
40. Add 200 µL Molybdate reagent into each tube.
SECTION: Day 2: Molybdate reaction
43. Add 300 µL reducing solution into each tube.
SECTION: Day 2: Molybdate reaction
44. Vortex each tube and then shake at Room temperature for 180 min 
SECTION: Day 2: Colorimetric measurement
46. In the fume hood, vortex each tube and then load 250 µL of the sample into one well of the microplate. Vortex again and load the same sample into another well of the microplate as replicate.
 

SECTION: Day 2: Colorimetric measurement
48. Setup the program
   AB1Shake duration00:00:052Shaking typeContinuous3Shaking forceHigh4Shaking speed [rpm]6005Wavelength [nm]8206Use transmittanceNo7Pathlength correctionNo8Measurement Time [ms]100

 

SECTION: Waste disposal
51. Collect all solution with paramethylaminophenol sulphate and sodium fluorosilicate into the waste container.
SECTION: Day 2: Calculation
53. 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: Day 2: Calculation
54. 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: Day 2: Calculation
55. Prepare a standard curve by plotting the average blank-corrected 812 nm absorbance for each standard working solution versus its concentration in uM.
  
SECTION: Day 2: Calculation
56. Use the standard curve to determine the silicate concentration of each unknown sample by using its blank-corrected 812 nm absorbance.
SECTION: Day 2: Calculation
57. Si per sample = Si X V X (0.001) X DF

 

SECTION: Day 2: Calculation
58. % Diatomaceous recovery = 100 X Si X V X (0.001) X MW X DF /(M_ug X Purity X 0.01)

 

 

SECTION: Sample collection
4. Flash freeze and store at -20 °C 
SECTION: Sample collection
3. Transfer filter into 2 mL cryogenic vial
SECTION: Sample collection
9. Transfer sample to 50 mL falcon tube with clean filter forceps (rinsed by 95% ethanol and air-dried), dry at 90 °C  in the airforce oven.
 

 

SECTION: Day 2: Acidification
33. Transfer solution from volumetric flask to a clean and labelled Falcon tube. 
SECTION: Day 2: Molybdate reaction
41. Vortex each tube and then shake at Room temperature for 15 min for the formation of silicomolybdic acid. 
SECTION: Day 2: Molybdate reaction
35. Standard working solutions (Freshly prepared prior to the assay)
 

SECTION: Standard primary solution and reagents
11.1. Use graduated cylinder, measure 95 mL MilliQ and transfer into a 125 mL plastic bottle.
SECTION: Standard primary solution and reagents
11.2. In the fume hood, add  5 mL 12 N HCl into the bottle, mix well.
SECTION: Standard primary solution and reagents
12.1.   
  
SECTION: Day 2: Molybdate reaction
39. 
 

SECTION: Day 1: Dissolution
20. Vortex
SECTION: Day 2: Acidification
27. Dropwise add 3 mL 12 N HCl by using 1000 uL pipet.
 


SECTION: Day 2: Molybdate reaction
36. Vortex and then transfer 50 µL from (1) blank for check standards, (2) check standards, (3) blank for samples, and (4) samples into labelled 2 mL microtubes. 
SECTION: Day 2: Molybdate reaction
45. Measure pH of each sample (in the Falcon tube)

 

SECTION: Day 1: Dissolution
16.2. Weigh 186.9 g Na2CO3 in a weighing dish.
(CAS: 497-19-8, FW 105.99)
SECTION: Day 1: Dissolution
16.6. Aliquot the solution into four 250 mL plastic bottles.

SECTION: Day 2: Acidification
28. Switch to a 100 uL pipette, add 100 uL at a time. Near the equivalence point, when the colour starts to change to pink more markedly but after mixing the orange colour returns, it is necessary to add HCl drop by drop. The first drop that causes a permanent colour change to pink determines the equivalence point. Stop adding HCl. Cap the tube, hold tube horizontally, gently invert the tube to wash residue at the inner side of the cap down to the solution. The color may change back to orange, add more drops of HCl until the color turns to permanent pink again (See the color of the left tube).
  
 


SECTION: Day 2: Acidification
25. In the fume hood, add 30 µL Methyl orange into the tube.   
SECTION: Day 2: Acidification
24. Work on one tube at a time, and leave other tubes in the oven. 
SECTION: Day 2: Acidification
23. Transfer 12 N HCl into a 50 mL Falcon tube in the fume hood. 
SECTION: Day 2: Molybdate reaction
37. Add 450 µL MilliQ into each tube to obtain a 10% dilution.
SECTION: Day 2: Colorimetric measurement
47. Setup the layout. 
SECTION: Waste disposal
52. Rinse microtubes and microplate with tap water, dispose in blue recycling bin.
SECTION: Day 2: Colorimetric measurement
49. Read the samples. 
SECTION: Day 2: Colorimetric measurement
50. Export data sheet to excel.
SECTION: Day 2: Acidification
26. Add MilliQ until the volume of solution in the falcon tube is 10 mL.