Feb 28, 2024

Public workspaceMeasurement of biogenic silica from plankton V.3

DOI
dx.doi.org/10.17504/protocols.io.8epv5jjzjl1b/v3
  • 1Dalhousie University
Ying-Yu Hu
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5jjzjl1b/v3
Protocol Citation: Ying-Yu Hu, Nuwanthi Samarasinghe, Zoe V. Finkel 2024. Measurement of biogenic silica from plankton. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jjzjl1b/v3Version created by Ying-Yu Hu
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 27, 2023
Last Modified: February 28, 2024
Protocol Integer ID: 88443
Keywords: biogenic silica, dissolution, silicomolybdic acid
Funders Acknowledgement:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 723789
Abstract
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%.
CITATION
Shemesh, Aldo; Mortlock, Richard A; Smith, R J; Froelich, Philip N (1988). Determination of Ge/Si in marine siliceous microfossils: separation, cleaning and dissolution of diatoms and radiolaria. Marine Chemistry.
LINK
https://doi.org/10.1016/0304-4203(88)90113-2



Protocol materials
ReagentAmmonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G
Step 11.1
ReagentOxalic acid dihydrateVWR InternationalCatalog #BDH4556-500G
Step 14.1
ReagentSodium hexafluorosilicateMerck MilliporeSigma (Sigma-Aldrich)Catalog #250171
Step 16.1
ReagentSodium sulfiteFisher ScientificCatalog #S430-500
Step 13.1
Reagent4-(methylamino)phenol hemisulfate saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #320013
Step 13.1
ReagentMethyl orangeMerck MilliporeSigma (Sigma-Aldrich)Catalog #1013230250
Step 26
ReagentCelite S diatomaceous earthMerck MilliporeSigma (Sigma-Aldrich)Catalog #06858
Step 19
Sample collection
Sample collection
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.
Filter blank media (without cells, same volume as plankton samples) through polycarbonate filter as blank
Transfer filter into 2 mL cryogenic vial
Flash freeze and store at Temperature-20 °C
Filter plankton sample in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg)
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU

Rinse filter funnel with filtered artificial seawater without macronutrients
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.
Transfer filter with sample into 2 mL cryogenic vial
Flash freeze and store at Temperature-20 °C

Transfer sample to 50 mL falcon tube with clean filter forceps (rinsed by 95% ethanol and air-dried), dry at Temperature90 °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

Equipment
Forced air oven
NAME
VWR
BRAND
89511-410
SKU

Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 50 mL
TYPE
Corning®
BRAND
352070
SKU

Standard primary solution and reagents
Standard primary solution and reagents
Molybdate reagent stock solution

Note
Require 100 uL per sample

ReagentAmmonium molybdateVWR InternationalCatalog #09878-100G

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

Add Amount1.6 g ammonium paramolybdate into a 125 mL plastic bottle and top to 100 g with MilliQ.

Store out of direct sunlight. Discard if white precipitation forms.
HCl stock solution
Note
Require 100 uL per sample

Use graduated cylinder, measure 95 mL MilliQ and transfer into a 125 mL plastic bottle.
In the fume hood, add Amount5 mL 12 N HCl into the bottle, mix well.
Metol-sulfite solution
Note
Require 100 uL per sample

Reagent4-(methylamino)phenol hemisulfate saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #320013
ReagentSodium sulfiteFisher ScientificCatalog #S430-500
Require:
(1) 50 mL syringe
(2) Syringe filter
Equipment
Syringe filter
NAME
0.2 um PES
TYPE
VWR
BRAND
28145-501
SKU

In a 100 to 250 mL plastic beaker, add Amount0.6 g sodium sulphite.
Add Amount1 g 4-(methyl amino)phenol hemisulfate.

Top to 50 g with MilliQ water.
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.
Prepare fresh every month.
Oxalic acid solution
Note
Require 100 uL per sample.

ReagentOxalic acid dihydrateVWR InternationalCatalog #BDH4556-500G
In a 125 mL plastic bottle, add Amount6 g oxalic acid and top to 100 g.
Let the solution stand at room temperature overnight.
Decant the solution from the crystals into a plastic bottle.
Keep at room temperature.
Sulphuric acid (30%)

Note
Require 100 uL per sample

Mix 3 part concentrated sulphuric acid into 7 part of MilliQ
Cool down to room temperature
Note
This can be prepared on Day 2 prior to molybdate reaction

Primary silica standard solution (~ 1 mM Si)
ReagentSodium hexafluorosilicateVWR InternationalCatalog #250171

Transfer 1 g sodium fluorosilicate in a plastic vial
Keep the vial in a vacuum desiccator overnight to remove excess water (do not heat or fuse)
In a one litre plastic volumetric flask, dissolve ~Amount0.1881 g (log the actual mass) of dry sodium fluorosilicate in MilliQ water and top to 1 L with MilliQ water.

It takes about 30 min to complete the dissolution. This cannot be rushed.
Store in a plastic bottle at room temperature.
Day 1: Dissolution
Day 1: Dissolution
Concentration2 Mass Percent Na2CO3 (18.69%)

Note
Need to be freshly prepared.
The old reagent can yield high blank possibly by leaching silicate from plastic material.


Each sample requires 10 mL 2 M Na2CO3
Weigh 186.9 g Na2CO3 in a weighing dish.
(CAS: 497-19-8, FW 105.99)
Tare a 1 L plastic erlenmeyer flask
Transfer Na2CO3 into the flask

Top to 1000 g with MilliQ and shake until all salt is completely dissolved.
Aliquot the solution into four 250 mL plastic bottles.

Turn on airforce oven to Temperature85 °C
In the fume hood, transfer diatomaceous into a 5 mL plastic tube for weighing convenience (the original package is 1 kg).
Note
  1. Diatomaceous is used as a check standard for the recovery of biogenic silica
  2. Diatomaceous is hygroscopic, it needs to be stored in the vacuum desiccator

Safety information
Diatomaceous:
Upper respiratory irritant. May cause coughing or throat irritation. Breathing dust containing crystalline silica over a long period may cause lung damage.
ReagentCelite S diatomaceous earthMerck MilliporeSigma (Sigma-Aldrich)Catalog #06858
Weigh 100~200 ug diatomaceous into 50 mL falcon tube, in triplicate. Log the actual weight.

Safety information
Do not open the container until the static charge of diatomaceous powder has been neutralized by ionization blower.


Note
Less than 100 ug sample might introduce more error amongst the replicates in recovery.

Prepare one empty 50 mL falcon tube as the reagent blank for diatomaceous.
Add Amount10 mL 2 M Na2CO3 to each tube, including:
  • reagent blank for check standards
  • check standards
  • blank for samples
  • samples
Vortex
Loose the caps and place all tubes into the airforce oven overnight (for example, from 5 pm to 9 am).
16h
Day 2: Acidification
Day 2: Acidification
Volume of 12 N HCl required:
about 3.5 mL X N
Transfer 12 N HCl into a 50 mL Falcon tube in the fume hood.
Work on one tube at a time, and leave other tubes in the oven.
In the fume hood, add Amount30 µL Methyl orange into the tube. ReagentMethyl orangeMerck MilliporeSigma (Sigma-Aldrich)Catalog #1013230250
Add MilliQ until the volume of solution in the falcon tube is 10 mL.
Note
The original volume of Na2CO3 is reduced due to evaporation of water during 20-h dissolution.

Dropwise add Amount3 mL 12 N HCl by using 1000 uL pipet.
Safety information
Do it slowly. Swirl the tube until reaction stops and then add the next drop. The most vigorous reaction is at about 3 mL 12 N HCl.


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).

Note
We have found that the optimal pH for the reaction between silicate and molybdate to form silicomolybdic acid is 3 to 4. Too low or too high pH decreases recovery of biogenic silica.
The acidified solution yields pH at 2 to 3. It is diluted to 10% in the molybdate assay, which gives pH at 3 to 4.


Transfer resulted solution from falcon tube to 25 or 50 mL polypropylene volumetric flask.
Note
Be careful while transferring the solution and ensure that the filter does not fall out of the tube, which spills the solution and causes sample loss.

Use MilliQ to rinse the tube three times and transfer all samples into the volumetric flask.
Note
If a 50 mL volumetric flask is used, rinse the falcon tube with 5 mL of MilliQ at a time.
If a 25 mL volumetric flask is used, rinse the falcon tube with 1 mL of MilliQ at a time.

Use transfer pipet, top final volume to 25 or 50 mL with Milli-Q.
Shake and thoroughly mix the solution.
Note
Before mixing, check the cap to avoid leaking

Transfer solution from volumetric flask to a clean and labelled Falcon tube.
Day 2: Molybdate reaction
Day 2: Molybdate reaction
3h
Secondary standard solution (Freshly prepared prior to the assay)

50 uL primary stock solution
450 uL MilliQ
Standard working solutions (Freshly prepared prior to the assay)
StandardsSecondary (uL)MilliQ (uL)Conc. (uM)
S105000
S254951
S3104902
S4204804
S5404608
S66044012
S78042016
S810040020

Vortex and then transfer Amount50 µL from (1) blank for check standards, (2) check standards, (3) blank for samples, and (4) samples into labelled 2 mL microtubes.
Add Amount450 µL MilliQ into each tube to obtain a 10% dilution.

Molybdate working solution
Note
Require 200 uL per sample

1 part Molybdate stock reagent
1 part HCl stock reagent

Safety information
The addition of reagent must be operated in the fume hood. Acidified sodium fluorosilicate may contain some hydrofluoric acid.

Add Amount200 µL Molybdate reagent into each tube.
Vortex each tube and then shake at TemperatureRoom temperature for Duration00:15:00 for the formation of silicomolybdic acid.
15m
Reducing solution
Note
Require 300 uL per sample

1 part Metol-sulfite solution
1 part oxalic acid solution
1 part sulphuric acid solution
Add Amount300 µL reducing solution into each tube.
Vortex each tube and then shake at TemperatureRoom temperature for Duration03:00:00
3h
....Measure pH of each sample (in the Falcon tube)

Sample code (example)Sample codepH
Blank for check standards
Check standard 1
Check standard 2
Check standard 3
Blank for samples
Sample 1
Sample 2
...

Day 2: Colorimetric measurement
Day 2: Colorimetric measurement
3h
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.
Equipment
96-Well Microplates, Polystyrene, Clear,
NAME
Greiner Bio-One
BRAND
655101
SKU

Setup the layout.
Setup the program
AB
Shake duration00:00:05
Shaking typeContinuous
Shaking forceHigh
Shaking speed [rpm]600
Wavelength [nm]812
Use transmittanceNo
Pathlength correctionNo
Measurement Time [ms]100

Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU

Read the samples.
Export data sheet to excel.
Waste disposal
Waste disposal
3h
Collect all solution with paramethylaminophenol sulphate and sodium fluorosilicate into the waste container.
Rinse microtubes and microplate with tap water, dispose in blue recycling bin.
Day 2: Calculation
Day 2: Calculation
3h
Subtract the average absorbance at 812 nm of the blank standard replicates from the absorbance at 812 nm of all other standard working solutions.
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.
Prepare a standard curve by plotting the average blank-corrected 812 nm absorbance for each standard working solution versus its concentration in uM.

Use the standard curve to determine the silicate concentration of each unknown sample by using its blank-corrected 812 nm absorbance.
Si per sample = Si X V X (0.001) X DF

VariableUnitDefinition
Si per sampleumolelement Si in the sample collected
SiuMsilicate concentration calculated from the standard curve
VmLvolume of volumetric flask
DFFrom volumetric flask to the microtube, DF=10

% Diatomaceous recovery = 100 X Si X V X (0.001) X MW X DF /(M_ug X Purity X 0.01)

VariableUnitDefinition
% Diatomaceous recoverypercentage recovery of diatomaceous
SiuMsilicate concentration calculated from the standard curve
VmLvolume of volumetric flask
MWug/umolmolecular mass of SiO2, i.e. 60.08
DFFrom volumetric flask to the microtube, DF=10
Mugactual mass of diatomaceous
Puritypurity of SiO2 in Celite S diatomaceous earth (06858) is 90.2%

Note
The recovery should be around 85 to 90%.

Citations
Shemesh, Aldo; Mortlock, Richard A; Smith, R J; Froelich, Philip N. Determination of Ge/Si in marine siliceous microfossils: separation, cleaning and dissolution of diatoms and radiolaria
https://doi.org/10.1016/0304-4203(88)90113-2