TITLE: Sample Preparation for Elemental Analysis of Auxenochlorella protothecoides (UTEX 250) Cells and Spent Media by Inductively Coupled Plasma Mass Spectrometry (ICP-MS/MS) and Total Organic Carbon (TOC).
AUTHORS: Dimitrios Camacho, Charles Perrino, Sabeeha Merchant

[DESCRIPTION]
This protocol describes a method for quantifying the elemental composition of Auxenochlorella protothecoides (UTEX 250) cells and the spent HP medium (Camacho, 2024). Elements measured include C (as total organic C), Na, Mg, P, S, K, Ca, Mn, Fe, Cu, Zn, Se, and Mo. Samples are collected, washed, digested, and analyzed for total organic carbon (Shimadzu) and other elements by ICP-MS/MS (Agilent 8900). Elemental measurements can be normalized to volume of cultures, cell number, sulfur, or total organic carbon. 

[BEFORE_START]
Acid wash all stock bottles with fresh (unused) 6 Molarity (M). Do not use glass containers as glass may leach metals. Use high density polyethylene (HDPE) bottles instead.
 
     1.1. Dilute fresh (unused) 12 Molarity (M) HCl to 6 Molarity (M). Remember to add acid to water and never water to acid. 
 
     1.2. Add 100 mL of 6 Molarity (M) HCl to 1 L stock bottles. Swirl 6 Molarity (M) HCl in the bottle to clean every internal surface. Transfer the used 6 Molarity (M) HCl to the next bottle and repeat. 
 
     1.3. Rinse with ICP-MS grade ultra-pure H2O at least seven times. See next section for a detailed description of the Milli-Q H2O.
 
     1.4. Neutralize the used 6 Molarity (M) HCl. 
 
          1.4.1. Add 1 L of H2O to a 4 L beaker placed in a larger, chemically compatible secondary container, such as an autoclavable polypropylene 12 L tray (Cat. no. S37253, Fisher Scientific). 

           1.4.2. Slowly and carefully add 1 L of 6 Molarity (M) HCl to water. Always add acid to water. The solution may heat up, so take your time. 
 
          1.4.3. Neutralize 3 Molarity (M) HCl by adding NaHCO3 (Arm & Hammer pure baking soda) slowly, scoop by scoop until no foam is formed. 
 
          1.4.4. Use a pH indicator strip to verify that the acid is safely neutralized (pH 7).
 
          1.4.5. Neutralized acid may be discarded down the drain.
 
2. The ICP-MS/MS is extremely sensitive and contaminants may confound your measurements. Use trace metal grade reagents and ICP-MS grade ultra-pure Milli-Q H2O. The production of ICP-MS grade ultra-pure Milli-Q H2O relies on a Q-POD‱ Element containing a Quantum‱ ICP filter (REF QTUM00ICP). 
 
     2.1. ICP-MS grade ultrapure Milli-Q H2O will be referred to as Milli-Q H2O throughout the rest of this protocol.
 
     2.2. Before using the Milli-Q H2O, verify that the resistivity is 18.2 MΩ • cm and the total organic carbon (TOC) reading is <10 ppb.
 
     2.3. Keep 1 L of Milli-Q H2O in an acid washed 1 L high density polyethylene (HDPE) bottle.
 
     2.4. Keep the bottle closed to avoid dust from entering. 
 
     2.5. Replace your Milli-Q H2O before each experiment.
 
3. Be mindful of metal contamination from tubes, plastic bottles, pipets, and dust.
 
     3.1. Ensure all work areas are thoroughly wiped to be free of dust. If a clean room is not available, work can be carried out in a clean fume hood and a room where the paint is not deteriorated.
 
     3.2. Keep tubes and reagents closed to avoid metal contamination from dust. Zn is abundant in dust. 
 
     3.3. Most 15 mL and 50 mL conical tubes contain residual metal contamination. We recommend acid washing tubes with 12 Molarity (M) HCl (step 5 in this section). Tube manufacturers may change their sources of materials over time, so make sure to check that the tubes you are using are metal free using the ICP-MS. Record the lot numbers for all tubes. 
 
          3.3.1. Metal free 15 mL tubes - Globe Scientific Inc. Catalog No. 6295 were specially chosen because they consistently contained less metals and can withstand a force of 17,000 ×g, which is needed to collect Auxenochlorella cells. These tubes however contain a large amount of Ca and therefore need to be washed if you are interested in the Ca content of cells and supernatants. 
 
3.4. Disposable 10 mL and 25 mL serological pipets Cat. No. 13-676-10J and 13-676-10K, respectively, will introduce a detectable amount of Zn contamination to your samples. We advise rinsing each newly opened pipet by pipetting Milli-Q H2O to the maximum volume and disposing the water before pipetting any samples or reagents.
 
4. Pre-label a set of 15 mL tubes for cell pellets and another set for spent media (optional). 
 
     4.1. Label tubes with the date, your initials, sample name, and experiment ID. 
 
     4.2. If measuring - Fe, - Cu, or - Zn media, prepare five 15 mL tubes for each supernatant sample. 
 
     4.3. Include six or more method blanks for cell pellets and three method blanks for supernatant sample processing. Treat the method blanks as if they contained cells or supernatants, using the same reagents for digestion and dilution. Use a different set of three method blanks for each digestion method.
 
5. Acid-wash all 15 mL tubes with 5 mL of fresh 12 Molarity (M) in the fume hood. Cap and invert the tube gently. Re-use the 5 mL of 12 Molarity (M) HCl for the next 15 tubes. 
 
     5.1. Neutralize used 12 Molarity (M) HCl by diluting to 3 Molarity (M) HCl in the fume hood. Refer to steps 1.4 to 1.4.5 but adjust volumes for 12 Molarity (M) instead of 6 Molarity (M) HCl. 
 
     5.2. Rinse tubes with ICP-MS grade Milli-Q H2O at least seven times. Rinse the exterior of the capped tube to prevent exposure to residual HCl. It is very important to ensure there is no residual HCl. HCl will discolor some plastics and corrode metals. 
 
     5.3. Dry the exterior of the tube with a KimwipeTM and centrifuge the empty tubes at 3000 x g, 2 min, 22 °C using the 5810 R centrifuge fitted with a swinging bucket rotor (rotor A-4-81) and 15 mL tube adapters. Rinse the rotors and buckets with Milli-Q H2O when you are finished.
 
     5.4. Use a P200 pipette to carefully remove remaining water in the tube. Avoid touching the inner wall of the tube with the pipette. 
 
 6. Prepare the centrifuges, rotors, and adapters.
 
     6.1. Set the JXN-26 centrifuge to 17000 x g, 2 min, 22 °C, rotor A-4-81 with 15 mL tube adapters.
 
     6.2. Set the 5810 R centrifuge to 3220 x g, 2 min, 22 °C, rotor JA-14.50 with 15 mL tube adapters.
 
7. Determine the density of the culture by counting cells with a hemocytometer (Camacho & Merchant, 2024). Calculate and record the volume of culture required to collect 1 × 108 - 3 × 108 total cells. 

[STEPS]
SECTION: Procedure
1.  Collect 1 × 108 - 3 × 108 cells at 17000 x g, 2 min, 22 °C in red screw cap 15 mL tubes using the JXN-26 centrifuge. Do not disturb the pellet. The pellet will partially adhere to the side of the tube at a 45-degree angle. 

If cells are sticking to the side of the tube and not collecting to the bottom of the tube, centrifuge for an additional 5 min - 10 min. 

SECTION: Procedure
14. Cell pellet samples are now ready for the ICP-MS/MS analysis.

Samples in capped tubes may be stored at room temperature in a cabinet equipped with an exhaust system to remove HNO3 fumes.
SECTION: Procedure
13. Fill to 5 mL with Milli-Q H2O if 143 µL 70% HNO3 was used. Fill to 10 mL if 286 µL of HNO3 was used. Final HNO3 concentration should be 2%. Record the volume of 70% HNO3 and Milli-Q H2O added.

If filled to 5 mL, the dilution factor is 5. 

If filled to 10 mL, the dilution factor is 10. 

 


SECTION: Procedure
12.2. 
 
The mixture should be clear with absolutely no solids present. If cell pellets are not fully digested, you may take either one (or a combination) of the following actions until the mixture is clear with no precipitates.

1.  Incubate for an additional 120 min at 80 °C while shaking at 175 RPM.

2. Incubate samples in a 65 °C ultra-sonic water bath for 60 min at maximum power. Use floats or a strainer tray to make sure that nothing touches the bottom of the ultra-sonic bath tank. The cavitation effect will be diminished and the transducers may break if an object is placed on the bottom of the tank or if the water level is too low. Ensure that the water level is maintained precisely at the operating level. 

3. Add fresh 50 µL of 30% hydrogen peroxide to the pellet and incubate at 85 °C for 120 min with shaking at 175 RPM.

4. Use a dedicated microwave (800 W) in a fume hood to denature samples. Heat samples in increments of 15 s and slightly twist open tubes to avoid fume pressurization. Let the tubes cool before placing back in the microwave. Do not exceed 90 s total. 

Tubes may leach material and metals as they encounter HNO3, H2O2, are heated, sonicated, and/or microwaved. Use a different set of method blanks for each digestion method or combination of digestion methods. Only analyze blanks that have undergone the same digestion method(s) as the samples.

SECTION: Procedure
12.1. Cell pellets that appear larger than 100 µL will require an additional 143 µLof HNO3. For instance, fatty mixotrophic or heterotrophic cells grown with 2% glucose are larger in diameter so although the number of cells is the same, the biomass of these cells will be greater than that of photoautotrophic cells. Lipid rich cells are also harder to digest.

SECTION: Procedure
12. Digest cells with 143 µL of 70% Optima® grade or similar ICP-MS grade HNO3 and incubate at 65 °C while shaking at 175 RPM (setting 5 on New Brunswick Gyrotory Water Bath Shaker, Model G76) for 960 min. Fill the water bath before leaving to ensure that the water does not fully evaporate.

SECTION: Procedure
11. (Optional) Freeze the pellet and supernatant samples at -20 °C until 1 day before the ICP-MS/MS analysis.

SECTION: Procedure
10. Carefully remove the supernatant with a P200 pipette. Do not take up any cells. If you accidentally take up cells into the tip, dispense the liquid back into the tube and centrifuge again.

Add bleach to the liquid waste to make a 10% bleach and 90% waste solution. Wait 60 min and dispose down the drain. Do not mix bleach with ammonia, acids, alcohol, hydrogen peroxide, or other household cleaners. 

SECTION: Procedure
9. Transfer the tubes to the swinging bucket rotor on the Eppendorf 5810 R centrifuge and pellet the cells at 3220 x g, 2 min, 22 °C

SECTION: Procedure
8. Remove the supernatant by slowly decanting in one motion. Do not disturb the pellet.
SECTION: Procedure
7. Centrifuge again at 17000 x g, 2 min, 22 °C.

SECTION: Procedure
6. Resuspend the cell pellet in 1 mL of Milli-Q H2O by closing the cap and swinging the tube in a circular motion.

SECTION: Procedure
5. Centrifuge the mixture 17000 x g, 2 min, 22 °Cand discard the supernatant by carefully decanting, again with one motion and the pellet to the bottom.

SECTION: Procedure
4. Pipette 1 mL of 1 millimolar (mM) Na2EDTA into the tube containing the pellet. Resuspend the pellet by closing the cap and swinging the tube in a circular motion until the pellet is fully resuspended.

 

SECTION: Procedure
3. Slowly and carefully decant the remaining supernatant into a liquid waste container in one motion. Keep the side with the pellet to the bottom and make sure that no cells are decanted. You may keep about 100 µL of liquid to avoid decanting cells. 

Do not mix liquid waste with ammonia, acids, alcohol, hydrogen peroxide, or other household cleaners. You will add bleach to the liquid waste later. 

 

SECTION: Procedure
2. Carefully transfer 0.5 mL of supernatant to each prelabeled 15 mL tube without disturbing the pellet. If measuring Fe, Cu, or Zn deficient media, then aliquot 0.5 mL into each of the five tubes.

SECTION: Procedure
16. To prepare - Fe, - Cu, or - Zn supernatant samples for measurement, perform a standard addition of Fe, Cu, and/or Zn to each 0.5 mL sample.

SECTION: Procedure
15. To prepare spent media from cultures grown in replete conditions, add 9.5 mL of 2.1% HNO3 to each tube containing 0.5 mL of sample, for a final HNO3 concentration of 2%. The dilution factor is 20.

SECTION: Procedure
16.4. Execute a serial dilution by transferring 1 mL of 1x solution into the tube labeled 0.1x which contains 9 mL of Milli-Q H2O. Invert the tube to mix and transfer 1 mL of the 0.1x solution to 9 mL of Milli-Q H2O in the tube labeled 0.01x. Repeat for 0.001x using the 0.01x solution.

Label the five supernatant samples 0, 1x, 0.1x, 0.01x, and 0.001x

Do not add any standard into the tube labeled 0. Aliquot 100 µL of the standard addition stocks into their corresponding supernatant samples. Repeat for all supernatant samples.

Add 9.5 mLof 2.1% HNO3 to all supernatant samples without standard additions. 

Add 9.4 mL of 2.1% HNO3 to all supernatant samples with standard additions.

The dilution factor for all supernatant samples is 20x.


 


SECTION: Procedure
16.3. Pipette 1 mL of 2 millimolar (mM) Cu, 20 millimolar (mM)Fe, and 10 millimolar (mM) Zn stocks from the HP medium into the 1x tube.

SECTION: Procedure
18. For measurement of ICP-MS/MS supernatants from step 15 – 16, transfer 500 µL of 2% HNO3 sample to a TOC vial containing 14.4 mL of Milli-Q H2O and 135 µL of 3 Molarity (M) HCl. The total dilution factor is 600.

If you are measuring ICP-MS/MS supernatants which originally contained 2% glucose, then transfer 180 µL  of ICP-MS/MS samples to a vial containing 14.7 mL of Milli-Q H2O and 135 µL of 3 Molarity (M) HCl. The total dilution factor is 1666.67

SECTION: Procedure
17. To obtain total organic carbon measurements from ICP-MS/MS cell samples, transfer 500 µL of digested 2% HNO3 sample to a TOC vial containing 14.4 mL of Milli-Q H2O and 135 µL of 3 Molarity (M) HCl. Measure carbon only and include a set of method blanks from step 12.2. Samples are now ready for TOC measurement. The dilution factor for cell pellets is 30 × ICP-MS/MS dilution factor.

SECTION: Procedure
16.2. Aliquot 7 mL of Milli-Q H2O into the tube labeled SAS1x and 9 mL into each of the remaining tubes.

SECTION: Procedure
16.1. Prepare 4 acid washed 15 mL tubes. Label the tubes standard addition stock (SAS) 1x, SAS 0.1x, SAS 0.01x, and SAS 0.001x.