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)
 

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

SECTION: Day 1: Dissolution
17. 2 M Na2CO3 (18.69%)

 

SECTION: Day 1: Dissolution
17.1. Each sample requires 10 mL 2 M Na2CO3
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17.3. Tare a 1 L plastic erlenmeyer flask
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17.4. Transfer Na2CO3 into the flask

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17.5. Top to 1000 g with MilliQ and shake until all salt is completely dissolved.
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18. Turn on airforce oven to 85 °C 
SECTION: Day 1: Dissolution
19. 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
19.1. Weigh 100~200 ug diatomaceous into 50 mL falcon tube, in triplicate. Log the actual weight.

 


 

SECTION: Day 1: Dissolution
19.2. Prepare one empty 50 mL falcon tube as the reagent blank for diatomaceous.
SECTION: Day 1: Dissolution
20. 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
22. 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
11. Molybdate reagent stock solution

 

SECTION: Standard primary solution and reagents
11.1.   

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

SECTION: Standard primary solution and reagents
11.2. 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
11.3. Store out of direct sunlight. Discard if white precipitation forms.
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12. HCl stock solution
 

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

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

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

SECTION: Standard primary solution and reagents
13.5. Top to 50 g with MilliQ water.
SECTION: Standard primary solution and reagents
13.6. 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
13.7. Prepare fresh every month.
SECTION: Standard primary solution and reagents
14. Oxalic acid solution
 

SECTION: Standard primary solution and reagents
14.1.   
SECTION: Standard primary solution and reagents
14.2. In a 125 mL plastic bottle, add 6 g oxalic acid and top to 100 g.
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14.3. Let the solution stand at room temperature overnight.
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14.4. Decant the solution from the crystals into a plastic bottle.
SECTION: Standard primary solution and reagents
14.5. Keep at room temperature.
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15. Sulphuric acid (30%)
SECTION: Standard primary solution and reagents
15.1. 
 

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

SECTION: Standard primary solution and reagents
16. Primary silica standard solution (~ 1 mM Si)
SECTION: Standard primary solution and reagents
16.1.   

SECTION: Standard primary solution and reagents
16.2. Transfer 1 g sodium fluorosilicate in a plastic vial 
SECTION: Standard primary solution and reagents
16.3. Keep the vial in a vacuum desiccator overnight to remove excess water (do not heat or fuse)
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16.4. 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
16.5. It takes about 30 min to complete the dissolution. This cannot be rushed.
SECTION: Standard primary solution and reagents
16.6. Store in a plastic bottle at room temperature.
SECTION: Day 2: Acidification
23. Volume of 12 N HCl required:
about 3.5 mL X N
SECTION: Day 2: Acidification
30. Transfer resulted solution from falcon tube to 25 or 50 mL polypropylene volumetric flask.
 

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

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

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

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

SECTION: Day 2: Molybdate reaction
39.1. 1 part Molybdate stock reagent 
1 part HCl  stock reagent
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43. Reducing solution
 

SECTION: Day 2: Molybdate reaction
43.1. 1 part Metol-sulfite solution
1 part oxalic acid solution
1 part sulphuric acid solution
SECTION: Day 2: Molybdate reaction
41. Add 200 µL Molybdate reagent into each tube.
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44. Add 300 µL reducing solution into each tube.
SECTION: Day 2: Molybdate reaction
45. Vortex each tube and then shake at Room temperature for 180 min 
SECTION: Day 2: Colorimetric measurement
47. 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
49. Setup the program
   AB1Shake duration00:00:052Shaking typeContinuous3Shaking forceHigh4Shaking speed [rpm]6005Wavelength [nm]8206Use transmittanceNo7Pathlength correctionNo8Measurement Time [ms]100

 

SECTION: Waste disposal
52. Collect all solution with paramethylaminophenol sulphate and sodium fluorosilicate into the waste container.
SECTION: Day 2: Calculation
54. 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
55. 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
56. 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
57. 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
58. Si per sample = Si X V X (0.001) X DF

 

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

SECTION: Standard primary solution and reagents
12.1. Use graduated cylinder, measure 95 mL MilliQ and transfer into a 125 mL plastic bottle.
SECTION: Standard primary solution and reagents
12.2. In the fume hood, add  5 mL 12 N HCl into the bottle, mix well.
SECTION: Standard primary solution and reagents
13.1.   
  
SECTION: Day 2: Molybdate reaction
40. 
 

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


SECTION: Day 2: Molybdate reaction
37. 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
46. ....Measure pH of each sample (in the Falcon tube)

 

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

SECTION: Day 2: Acidification
29. 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
26. In the fume hood, add 30 µL Methyl orange into the tube.   
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25. Work on one tube at a time, and leave other tubes in the oven. 
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24. Transfer 12 N HCl into a 50 mL Falcon tube in the fume hood. 
SECTION: Day 2: Molybdate reaction
38. Add 450 µL MilliQ into each tube to obtain a 10% dilution.

SECTION: Day 2: Colorimetric measurement
48. Setup the layout. 
SECTION: Waste disposal
53. Rinse microtubes and microplate with tap water, dispose in blue recycling bin.
SECTION: Day 2: Colorimetric measurement
50. Read the samples. 
SECTION: Day 2: Colorimetric measurement
51. Export data sheet to excel.
SECTION: Day 2: Acidification
27. Add MilliQ until the volume of solution in the falcon tube is 10 mL. 
 

SECTION: Sample collection
7. Fold the filter with two tweezers:
(1) Fold in half along its diameter, creating a semicircular shape;
(2) Fold once more in the same direction, resulting in a long strip;
(3) Fold once more, halving its length, so that sample is secured.
SECTION: Sample collection
10. 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. Prior to transferring:

(1) For filters folded into half-strip, unfold once to return to a strip.
(2) For filters folded into quarter-circles, unfold once to return to a half-circle shape, then fold once along the dimension to form a strip. 
(2) For filters haphazardly into a compact mass, carefully unfold with two tweezers (avoiding losing biomass), fold once into a half-cricle shape, then fold once more along the dimension to form a strip