Measurement of biogenic silica from plankton

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 -20°C

Filter plankton sample in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg)

Equipment

Value	Label
Filter forceps	NAME
blunt end, stainless steel	TYPE
Millipore	BRAND
XX6200006P	SKU
http://www.emdmillipore.com/	LINK

Rinse filter funnel with filtered artificial seawater without macronutrients

Transfer filter with sample into 2 mL cryogenic vial

Flash freeze and store at -20°C

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.

Equipment

Value	Label
Forced air oven	NAME
VWR	BRAND
89511-410	SKU

Equipment

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

Molybdate reagent stock solution

Ammonium paramolybdate:

[(NH4)6Mo7O24.4H2O]

CAS: 12054-85-2

Add 1.6g 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

Use graduated cylinder, measure 95 mL MilliQ and transfer into a 125 mL plastic bottle.

In the fume hood, add 5mL 12 N HCl into the bottle, mix well.

Metol-sulfite solution

Require:

(1) 50 mL syringe

(2) Syringe filter

Equipment

Value	Label
Syringe filter	NAME
0.2 um PES	TYPE
VWR	BRAND
28145-501	SKU

In a 100 to 250 mL plastic beaker, add 0.6g sodium sulphite.

Add 1g 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

In a 125 mL plastic bottle, add 6g 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%)

Mix 3 part concentrated sulphuric acid into 7 part of MilliQ

Cool down to room temperature

Primary silica standard solution (~ 1 mM Si)

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 ~0.1881g (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.

2M Na₂CO₃(18.69%)

Each sample requires 10 mL 2 M Na₂CO₃

Weigh 186.9 g Na₂CO₃in a weighing dish.

(CAS: 497-19-8, FW 105.99)

Tare a 1 L plastic erlenmeyer flask

Transfer Na₂CO₃into 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 85°C

In the fume hood, transfer diatomaceous into a 5 mL plastic tube for weighing convenience (the original package is 1 kg).

Weigh 100~200 ug diatomaceous into 50 mL falcon tube, in triplicate. Log the actual weight.

Prepare one empty 50 mL falcon tube as the reagent blank for diatomaceous.

Add 10mL 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).

Volume of 12 N HCl required:

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 30µL Methyl orange into the tube.

Add MilliQ until the volume of solution in the falcon tube is 10 mL.

Dropwise add 3mL 12 N HCl by using 1000 uL pipet.

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).

Transfer resulted solution from falcon tube to 25 or 50 mL polypropylene volumetric flask.

Use MilliQ to rinse the tube three times and transfer all samples into the volumetric flask.

Use transfer pipet, top final volume to 25 or 50 mL with Milli-Q.

Shake and thoroughly mix the solution.

Transfer solution from volumetric flask to a clean and labelled Falcon tube.

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)

A	B	C	D
S1	0	500	0
S2	5	495	1
S3	10	490	2
S4	20	480	4
S5	40	460	8
S6	60	440	12
S7	80	420	16
S8	100	400	20

Vortex and then transfer 50µL from (1) blank for check standards, (2) check standards, (3) blank for samples, and (4) samples into labelled 2 mL microtubes.

Add 450µL MilliQ into each tube to obtain a 10% dilution.

Molybdate working solution

1 part Molybdate stock reagent

1 part HCl stock reagent

Add 200µL Molybdate reagent into each tube.

Vortex each tube and then shake at Room temperature for 0h 15m 0s for the formation of silicomolybdic acid.

Reducing solution

1 part Metol-sulfite solution

1 part oxalic acid solution

1 part sulphuric acid solution

Add 300µL reducing solution into each tube.

Vortex each tube and then shake at Room temperature for 3h 0m 0s

Measure pH of each sample (in the Falcon tube)

A	B	C
Blank for check standards
Check standard 1
Check standard 2
Check standard 3
Blank for samples
Sample 1
Sample 2

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

Value	Label
96-Well Microplates, Polystyrene, Clear,	NAME
Greiner Bio-One	BRAND
655101	SKU

Setup the layout.

Setup the program

A	B
Shake duration	00:00:05
Shaking type	Continuous
Shaking force	High
Shaking speed [rpm]	600
Wavelength [nm]	812
Use transmittance	No
Pathlength correction	No
Measurement Time [ms]	100

Equipment

Value	Label
Varioskan LUX Multimode Microplate Reader	NAME
Thermo Fisher	BRAND
VL0L00D0	SKU

Read the samples.

Export data sheet to excel.

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.

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

A	B	C
Si per sample	umol	element Si in the sample collected
Si	uM	silicate concentration calculated from the standard curve
V	mL	volume of volumetric flask
DF		From 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)

A	B	C
% Diatomaceous recovery		percentage recovery of diatomaceous
Si	uM	silicate concentration calculated from the standard curve
V	mL	volume of volumetric flask
MW	ug/umol	molecular mass of SiO2, i.e. 60.08
DF		From volumetric flask to the microtube, DF=10
M	ug	actual mass of diatomaceous
Purity		purity of SiO2 in Celite S diatomaceous earth (06858) is 90.2%