Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

1. Growth system

Figure 1. Microalgae growth system. (A) air rotometer, (B) CO₂ rotometer, (C) pH controller with solenoid, (D) data logger, (E) in-line air filters, (F) air distribution header, (G) fluorescent light bank, (H) pH meters, (I) cooling system, (J) water bath, (K) thermocouple wire, (L) air lift photobioreactor, (M) heater, (N) walk-in fume hood, (O) vent, (P) air delivery capillary tube, (Q) air filters, (R) sampling tube, (S) PBR silicone lid, and (T) pH well in silicone lid. Please click here to view a larger version of this figure.

1. Build the following microalgae experimental growth system (Figure 1).
   1. Acquire twelve airlift PBRs consisting of glass tube reactors 4.5 cm in diameter and 80 cm in height with a cultivation capacity of 1.1 L with silicone lids. Acquire pre-cut glass capillary tubes (5 mm external diameter and 1 mm internal diameter) of 10 cm (3 per PBR) and 85 cm (1 per PBR) in length.
   2. Freeze silicone lids in a -80 °C freezer. Lubricate a drill bit with glycerol and while lids are frozen drill 3 holes to host the vent, sampling and gas delivery capillary tubes, and 1 hole of 17 mm diameter to host a pH probe.
   3. Insert the 3 capillary tubes in place with the longest tube extending 2 cm from the bottom of the PBR. In the other capillary tube add a silicon tube with a capillary tube attached to the other end extending to a desired sampling point. Cover the hole for the pH meter with a silicone stopper size 21D.
   4. Humidify ambient air by bubbling it through water and deliver the humidified air to the air distribution header. Pass the gas through a 0.2 µm filter and deliver it to the algal suspension through the longest glass delivery capillary tube.
   5. Deliver compressed CO₂ into the humidified air stream in order to maintain a neutral pH of 7.0 ± 0.1 in the culture suspension. Control the rate of CO₂ delivery with an automated CO₂ dispensary system (pH controller) that opens a magnetic solenoid when the algal culture reaches pH 7.1 and closes at pH 6.9.
   6. Provide light using 24 T5 fluorescent lamps that result in an average illumination of 984 µmol m^-2 sec^-1 similar to peak outdoor conditions.
   7. Immerse the PBRs in a water bath in order to maintain a constant temperature of approximately 25 °C. Control the temperature of the system by using a recirculating chiller and an automated heating recirculating water bath unit control.
   8. Monitor temperature and pH in real-time and record with a data logger.
   9. Ensure that all components of the microalgae growth system are properly working, especially before harvesting microalgae inoculum or preparing inorganic contaminants as they cannot be preserved.

2. Lab Ware Preparation

1. Wash volumetric flasks, PBRs, carboys and any containers, with soap and tap water. Rinse with deionized water (DW).
2. Acid rinse the lab ware in order to eliminate any traces of inorganic contaminants. This can be done by one of two ways:
   1. Soak lab ware O/N in 10% trace metal grade nitric acid (CAUTION: Do not breath fumes, concentrated nitric acid can produce severe burning and toxic fumes, work in a fume hood using nitrile gloves, goggles and lab coat).
   2. Soak lab ware for 15 min in 50% trace metal grade nitric acid.
   3. Rinse the lab ware with DW thoroughly at least 3 times making sure all acid is removed. It is critical that PBRs are thoroughly rinsed, especially the sampling tubes and the capillary tubes. Failure to do this will produce acidification of the medium and possible inhibition of growth. Test the pH of the rinse water to verify all acid has been removed.
   4. Sterilize PBRs, containers and flasks by autoclaving them at 120 °C and standard atmospheric pressure for at least 30 min.

3. N. salina Medium Preparation

1. Preparation of solution A: Partially fill a 1 L volumetric flask with DW. Insert a magnetic stir bar and add the chemicals shown in Table 1 one after the other. Ensure that each ingredient dissolves before the addition of the next constituent. Remove the magnet and fill the flask to the 1 L volume mark.

Table 1: Solution A recipe. Quantities are amounts needed in the preparation of 1 L of concentrated solution. 

2. Preparation of vitamin solution: In three separate volumetric flasks add the vitamins as shown in Table 2. Filter each vitamin solution through a sterile 0.2 µm syringe filter to a sterile container. Preserve vitamins at -4 °C in the dark.

Table 2: Vitamin solution recipe. Quantities are amounts needed for the preparation of concentrated solution. 

3. Partially fill a 20 L autoclavable container with DW and insert a magnetic stir bar. Place the container on top of a magnetic stirrer plate and add the chemicals shown in Table 3 (except the vitamins), adding them one after the other and after each fully dissolves. Fill the container to reach 20 L.

Table 3: N. salina medium recipe. Quantities are amounts needed in the preparation of 20 L of nutrient-rich medium. 

4. Sterilize the medium by autoclaving for 30 min at 120 °C and atmospheric pressure. Let the medium cool down to RT.
5. Place the container on a magnetic stirrer plate. Add the vitamins prepared in step 3.2 and let the medium mix thoroughly.

4. Inorganic contaminants stock preparation

1. Partially fill the volumetric flasks indicated in Table 4 with DW and add the individual salt listed. Fill with DW to the required final volume and mix thoroughly. Do not preserve these stocks as some elements adsorb to flask walls
   CAUTION: Several inorganic contaminants used in this protocol are carcinogenic, teratogenic and mutagenic, wear a face mask, gloves and lab coat when handling salts.

Table 4: Concentrated inorganic contaminants stock preparation. Addition of 1 ml of this concentrated stock to the 1.1 L PBR medium produces the final concentration shown in the last column. 

2. Sterilize the inorganic contaminant stocks by passing the solution through a sterile 0.2 µm syringe filter and collect the filtrate in a sterile tube.

5. N. salina Inoculum Production

1. In a 500 ml Erlenmeyer flask add 200 ml of medium prepared in step 3 and then add 3 g of agar. Cover the flask with aluminum foil and autoclave for 20 min at 120 °C. Pour the solution into sterile petri-dishes and let it cool until it solidifies. This should be completed be a sterile hood or at least near a flame in a clean environment to reduce risk of contamination.
2. Streak N. salina cells in sterile petri-dishes prepared in step 5.1 using a sterile seeding loop. Place the petri-dish cultures on a table illuminated with T12 lights maintained at RT. Let microalgae grow until colonies are visible.
3. Transfer colonies to sterile baffled Erlenmeyer flasks containing 200 ml of nutrient rich medium prepared in step 3 and keep them on an illuminated shaker table (1,000 RPM). Let the culture grow until medium becomes green.
4. Transfer the microalgae to a 1.1 L sterile PBR. Place the PBR in an inoculum water bath illuminated at 200 µmol m^-2 sec^-1 with T8 fluorescent lights and maintained at 23 °C by a recirculating chiller and an automated heating recirculating water bath control. Adjust the air and CO₂ rotometers to 2.5 L min^-1 and 25 cc min^-1, respectively.
5. After a week of growth split biomass into new 1 .1 L PBRs containing new medium and let it grow until a total of at least 28 g of dry weight biomass are obtained between the two reactors which can be determined through optical density.
6. Harvest the inoculum biomass by centrifugation at 2,054 × g for 15 min at 10 °C using sterile centrifuge bottles and sterile techniques to avoid contamination. Dispose of the supernatant and continue cell concentration as needed.
7. Once all biomass is centrifuged, re-suspend the cells in 300 ml of fresh sterile medium.
8. Dilute 0.1 ml of microalgal culture in 3 ml of DW and then dilute 0.1 ml of this new solution in 3 ml of DW. Ensure the sample is thoroughly mixed. Measure the optical density (OD) of the microalgae concentrate at 750 nm () immediately using a spectrophotometer.
9. Use equation (1) to determine the amount of biomass in the concentrate.
   Note: Equation (1) was obtained from the linear regression between versus total suspended solids ( in g/L^-1) for N. Salina (R²=0.9995). Equation 1 was developed for the spectrophotometer model in the Materials Table, generate a new calibration if using another spectrophotometer model.
   1. Using equation (2) calculate the volume of microalgae concentrate (in L) needed to obtain a 4 g/L^-1 culture density in a PBR of 1.1 L volume (in L).

10. Using sterile techniques, add the volume of microalgae concentrate found in the step 5.9 to an autoclaved PBR to reach an initial culture density of 4 g/L^-1. Fill PBR with medium to 1.1 L. Repeat this step until 6 PBRs are inoculated. Place the PBRs in the inoculum water bath.
11. Let the microalgae in the PBRs grow for 8 days and then harvest the biomass (by repeating steps 5.6 to 5.7). Repeat step 5.8 to calculate the initial inoculum volume for an initial culture density of 1 g/L^-1.

6. Experimental Reactors

1. Using sterile techniques add approximately 1 L of medium prepared in step 3 to each of the 12 acid-rinsed sterile PBRs. Place the PBRs in the water bath of the experimental growth system. Turn sparge air on at 1.5 L min^-1.
2. Sterilize a calibrated pH meter by cleaning it with 70% ethanol. Measure the pH of the medium in the PBR and ensure pH is approximately 7.0; if not, repeat step 2 to remove acid leached from the acid rinsing step.
3. Calibrate each pH controller using buffer pH 7, disinfect the probes using ethanol (70%) and then insert them in the PBRs lids.
4. To each PBR (except the control PBRs) add 1 ml of each of the sterile inorganic contaminants stocks prepared in step 4. Let the contaminants thoroughly mix in the PBR. The final concentration of the inorganic contaminants in the PBRs are shown in the last column in Table 4, and are the estimated maximum concentrations expected from a coal-fired power plant integration.
5. Add 14 ml of sterile DW to the control PBRs.
6. Add the concentrated microalgae inoculum obtained in step 5.11 to the experimental PBRs in order to obtain an initial culture density of 1 g/L^-1. Let biomass mix thoroughly.
7. Turn high light intensity lights (of 984 µmol m^-2 sec^-1) and pH controllers on and adjust CO₂ to 30 cc min¹. Increase the CO₂ flow to 50 cc min^-1 from day 3 afterwards. Initial low CO₂ flow rate is critical in order to avoid large changes in pH due to delays in gas/liquid transfer and pH measurement.
8. Measure and take samples as needed. Make sure to mark the water level after sampling. (CAUTION: some inorganic contaminants in the PBR are carcinogenic, teratogenic and mutagenic; use gloves and capped containers when handling samples).
9. Add sterile DW daily to the PBRs in order to compensate for losses due to evaporation.
10. After 7 days of growth, harvest the biomass by centrifugation at 9,936 × g and preserve both, biomass and supernatant medium, at -80 °C.
11. Freeze dry the biomass at 0.1 mbar and -50 °C O/N. Powder the biomass (use a spatula to powder biomass inside the centrifuge tube). Preserve freeze dried biomass at -80 °C.

7. Microwave Assisted Digestion of Samples

The digestion of the biomass samples is required as a pre-processing step for ICP-MS analysis.
Note: These steps use a closed vessel microwave digestion system with controlled pressure relief. (CAUTION: High pressures develop during acid digestion, inspect the physical integrity of the digestion vessels and shields, and reshape the microwave digestion vessel lids before every use).

1. Wash Teflon microwave digestion vessels with soap and water, rinse with DW and let vessels air dry. To remove trace metal contamination in the vessels digest acid as described in the following steps.
2. Reshape the microwave digestion vessel lids and close the vials tightly.
3. Add 10 ml of nitric acid to each.
4. Introduce the vessel in the safety shield. Ensure that no biomass, water or any reagents are left on the walls of the safety shield or in the outer walls of the digestion vessels in order to avoid damage to the safety shield. Cap the safety shield with the safety valve making sure the spring in the vial is flush. Locate the shield on the rotor with the cap vents pointing outward in the outer row and inwards in the inner row.
5. On vessel number one, insert the ceramic thermowell and the temperature sensor. This thermometer monitors the actual internal temperature in the vial and serves as the controlling parameter to execute the digestion program. Ensure that vial number one contains the same sample and reagent amounts as the other vials.
6. Input the digestion parameters shown in Table 5 and start digestion. When the program has finished, air cool the vials until they reach RT.

Table 5: Parameters used in the microwave digestion program.

7. Inside a fume hood, insert the pressure relief tool on the shield cap with the cap vents point away from you. Once pressure is released open the cap (CAUTION: Always open digested vials inside fume hood since biomass digestion using acid produces toxic fumes).
8. Dispose of the acid. Rinse the Teflon vessels with DW 3 times. Let vials air dry.
9. To digest biomass, add 50 mg of freeze dried biomass to microwave digestion vessels. For quality control (QC) prepare the following vials: in two different vials add either 5 ml of Level 7 ICPMS or 5 ml of Level 7 Hg CVAAS standard prepared in steps 9.1 and 10.1 (the digested solution from this vial is called the laboratory fortified blank (LFB)), leave another vial empty (the digested solution from this vial is called the laboratory reagent blank (LRB)).
10. To digest medium, add 10 ml supernatant medium to dry acid rinsed microwave digestion vessels. For quality control (QC) prepare the following vials: In two different vials add 5 ml of Level 7 ICPMS or CVAAS metal standard prepared in step 9.1 and 10.1 (the digested solution from this vial is called the LFB), to another vial add 10 ml of DW (the digested solution from this vial is called the LRB).
11. Reshape the microwave digestion vessel lids and close the vials tightly.
12. Add 7 ml of concentrated trace metal grade nitric acid and 3 ml hydrogen peroxide to each vial. Homogenize the contents by gently swirling the solution. Digest the contents of the vials by repeating steps 7.4 to 7.7 (use the microwave digestion parameters for sample digestion in Table 5).
13. Add digested sample to a 25 ml volumetric flask, rinsing the vessels with DW for increased recovery. Fill the volumetric flask with DW to the mark.
14. Transfer digested samples to a capped container. Preserve samples at 4 °C until analysis can be completed. For this study analysis is done the same day for Hg and within three days for the other elements.

8. Quality Control (QC) Samples

Note: Analyze QC samples in order to assure reliability of the results from experimental samples.

1. Partially fill an acid rinsed 1 L volumetric flask with DW. Add 280 ml of concentrated trace metal grade nitric acid and mix thoroughly (this solution is also called the blank solution) (CAUTION: always add acid to water, never add water to acid as the exothermic reaction can be violent). Let solution cool to RT.
2. In addition to QC samples prepared in steps 7.9 and 7.10, prepare the following QC samples.
   1. For the continuing calibration verification (CCV): Fill a polystyrene tube with calibration standard (for preparation see step 9.2 and 10.1). Put the Hg standard solution on the CVAAS rack and the ICPMS standard solution in the ICPMS autosampler.
   2. For the continuing calibration blank (CCB): Fill two polystyrene tubes (16 ml) with the blank (solution prepared in step 8.1). Place one sample in the CVAAS rack and the other sample in the ICPMS autosampler.
   3. For the laboratory-fortified matrix (LFM): Randomly choose 1 sample of every 12 samples for each type of sample (i.e., biomass or medium) and use it to prepare a LFM. For ICPMS, add 0.5 ml of ICPMS standard Level 7 and 3 ml of digested experimental sample (from either biomass or medium) to a polystyrene tube.
   4. Mix contents and place the vials on the ICPMS autosampler. For CVAAS, add 2 ml Hg standard Level 7 and 6 ml of digested experimental sample (from either biomass or medium) to a polystyrene tube. Mix contents and place vials on the CVAAS rack.
   5. For the duplicate samples: Randomly choose 1 sample of every 12 samples for each type of matrix (e.g., biomass, medium, LFM or any diluted matrix) and duplicate the vial. Place the repeated vials in the ICPMS autosampler or the CVAAS rack.
   6. For the duplicate samples: Randomly choose 1 sample of every 12 samples for each type of matrix (e.g., biomass, medium, LFM or any diluted matrix) and duplicate the vial. Place the repeated vials in the ICPMS autosampler or the CVAAS rack.
3. Define the data quality criteria for the study. For the present study duplicate the quality criteria established by Eaton, Clesceri, Rice and Greenberg ²⁵. The parameters established for the QC are: percent difference (%D) for CCV within ± 10%²⁵ (with exception of Pb and Sb, see discussion), LFB percent recovery (%R) within ± 70-130%²⁵, LFM percent recovery (%R) within 75-125%²⁵, and relative percent difference (RPD) within ± 20%²⁵, and a continuing calibration blank (CCB) below method reporting limit (MRL)²⁵. See calculation equations in step 9.7.

9. Quantification by Inductively Coupled Plasma Mass Spectrometry (ICPMS)

1. On the day of analysis, transfer approximately 5 ml of digested sample to polystyrene tubes and place them in the ICPMS autosampler. Add approximately 15 ml of digested samples to polystyrene tubes and place them in the CVAAS rack.
2. The same day of analysis prepare the calibration standards. Add purchased ICPMS standard solution and refill with blank (solution prepared in step 8.1) as described in Table 6 (see standard solution description in Material Table) to acid-rinsed volumetric flasks.

 Table 6: Concentration of calibration standards. Levels 1 to 7.

3. Remove the cones from the ICPMS and sonicate them for 1 min in DW. Dry the cones and put them back in the instrument.
4. Turn on the water chiller, gasses (Ar, H₂, He), the ICPMS, plug lines to internal standard, and fill auto-sampler rinse containers (DW, 10% nitric acid, 1% nitric acid + 0.5% hydrochloric acid).
5. Open the Masshunter Workstation software and turn on the plasma, tune the ICPMS and load the method set to parameters in Table 7.

 Table 7: ICPMS operating conditions.

6. Place calibration standard, QC samples and experimental samples in the autosampler. In the ICPMS software add the analysis sequence and analyze samples. Aspirate the sample inside the instrument to the plasma where the elements are ionized. Then a vacuum withdraws the ions to a counter. The ions will separate depending on their atomic weight from the lightest to the heaviest.
   CAUTION: Collect ICPMS waste in hazardous containment and handle appropriately for disposal.
7. Ensure that the correlation coefficient (R) value for the calibration curve for each metal or metalloid is greater than 0.995²⁴.
8. During sample analysis, calculate %R, %D and RPD as described in equations 3 to 6²⁶ and compare the results to the project data quality criteria in 8.3.
   1. Calculate percent recovery (%R) to determine losses/gaining from the laboratory fortified blank (LFB) and matrix interference from laboratory-fortified matrix (LFM).

2. Calculate percent difference (%D) to determine instrument performance changes with time when running CCV samples.

3. Calculate relative percent difference (RPD) to determine changes in method precision with time when running experimental samples.

9. To reduce matrix interference (%R out of acceptable range), dilute the samples for poor %R to a ratio 1:3 (sample:DW).

10. Hg quantification by Cold Vapor Atomic Absorption Spectrophotometer (CVAAS)

1. Prepare calibration standards the same day of analysis. Dilute purchased Hg standard by adding 1 ml of purchased Hg standard solution to a 100 ml volumetric flask and fill with the solution prepared in step 8.1.
   1. Add 2.5 ml of this solution into a 100 ml volumetric flask and fill with the solution prepared in step 8.1 (this new solution is Level 7 Hg standard). Add diluted Level 7 Hg standard to volumetric flasks and fill with blank (solution prepared in step 8.1.) as described in Table 8 (see purchased Hg standard solution description in Material Table).

 Table 8: Concentration of Hg calibration standard. Levels 1 to 6.

2. Open the Ar gas and air valve, turn on the Atomic Absorption Spectrophotometer and the Flow Injection Atomic Spectroscopy (FIAS). Open the CVAAS Winlab software, turn on the Hg lamp and let it warm up until the software’s energy parameter reaches 79. Load the program for Hg analysis with the parameters in Table 9. Adjust the light path in the instrument to give the maximum transmittance.

 Table 9: CVAAS operating conditions.

3. Plug the line to the carrier solution made of 3% trace metal grade hydrochloric acid.
4. Plug the line to the reducing agent solution made of 10% stannous chloride (suitable for Hg analysis) in 3% trace metal grade hydrochloric acid. Prepare this solution the same day of analysis as it is prone to atmospheric oxidation (CAUTION: Stannous chloride is very hazardous, use protective wear when working with it. Collect CVAAS waste in hazardous containment and properly dispose).
5. Place the Hg standards, QC samples and experimental samples in the CVAAS rack and input the sequence in the CVAAS Winlab software. Run standards and generate the calibration equation.
6. Run QC samples and experimental samples. The CVAAS draws approximately 5 ml of sample into the instrument, reduces the Hg present in the sample to elemental Hg (Hg⁰) gas and purges the gas from solution with a carrier gas (Ar) in a closed system. The Hg vapor passes through a cell in the Hg lamp light path. A detector determines the light absorbed at 253.7 nm and correlates it to concentration. (CAUTION: Hg vapor is toxic, ensure instrument exhaust hood is in place).
7. Calculate %R, %D and RPD in step 9.7 during analysis and compare the results to the project data quality criteria.