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

The overall goal of this procedure is to quantify the impact and end fate of heavy metals introduced into microalgae growth systems as a result of carbon dioxide from coal power plants being used as the carbon source for growth. This is accomplished by first understanding the potential contamination levels based on integrating coal, flu gas. With microalgae cultivation, these levels are used as baseline contamination and stocks are made.

Next, the required inoculum is cultivated and the large scale growth platform is set up. Then the system is inoculated and samples are periodically taken for processing. Finally, the biomass is isolated and the end fate of the contaminants is quantified.

Ultimately, inductively coupled plasma mass spectrometry or I-C-P-M-S is used to determine the metals that are absorbed by the biomass, and combined with optical density measurements enables the impact of the contaminants on the system to be understood. The main advantage of this technique over existing methods is the ability to directly quantify the end fate of a multi-meter system. Do this method can provide inside into the impact of heavy metals from flu gas.

It can also be applied to other systems. This method can help answer key questions in the microalgae field, such as the impact of heavy metals on productivity, and the ability of microalgae to be used for bioremediation. Generally, individuals new to this method will struggle because of the potential for contamination, the required precision.

In preparing samples and analysis with an IC PMS, We first had the idea for this method. We were trying to understand the potential impacts of metals and algae from flu gas integration. It was through discussions with fellow researchers at the water research laboratory.

We were made aware of this method After building the micro algae experimental growth system and preparing LabWare according to the text protocol, partially fill a 20 liter autoclavable container with deionized water and insert a magnetic stir bar. Place the container on top of a magnetic stir plate and one at a time. Add the chemicals shown here, allowing each to fully dissolve before adding the next autoclave, the medium at 120 degrees Celsius for 30 minutes, and allow it to cool to room temperature before adding the vitamins.

To prepare inorganic contaminant stock solutions with distilled water partially fill volumetric flasks and add the individual salt listed here. Then fill with distilled water to the required volume and mixed thoroughly. Sterilize the inorganic contaminant stocks by passing the solutions through a sterile 0.2 micron syringe filter and collect the filtrates in sterile tubes to prepare and Salina inoculum after growing colonies in sterile Petri dishes, according to the text protocol, inoculate 200 milliliters of previously prepared nutrient-rich medium in baffled erlenmeyer flasks with en Salina colonies and incubate them on an illuminated shaker table.

Allow the culture to grow until the medium becomes green. Transfer the microalgae to a 1.1 liter sterile photo bioreactor or PBR Place the PBR in an inoculums water bath. Illuminated at 200 micromoles per meter square per second with T eight fluorescent lights, and use a recirculating chiller and automated heating recirculating water bath control to maintain at 23 degrees Celsius.

Adjust the air and carbon dioxide rotor meters to 2.5 liters per minute and 25 cubic centimeters per minute. Respectively grow and harvest the microalgae according to the text protocol. Calibrate the pH meter and pH controller using 70%ethanol.

Sterilize the calibrated pH meter to each previously prepared PBR. Add one milliliter of each of the sterile inorganic contaminant stocks to reach a final concentration shown in this table. Next, add the concentrated microalgae inoculum to the experimental PBRs at an initial culture density of one gram per liter.

Then turn on the high intensity lights and pH controllers and adjust carbon dioxide to 30 cubic centimeters per minute. After growing the cultures for seven days, according to the text protocol, harvest the biomass by centrifugation at 9, 936 times G.Collect both the biomass and supernatant medium and preserve at minus 80 degrees Celsius. Then freeze dry the biomass at 0.1 millibars and minus 50 degrees Celsius overnight.

Use a spatula inside the centrifuge tube to powder the biomass to prepare samples for inductively coupled plasma mass. Spectrometry or I-C-P-M-S begin by using soap and water to wash Teflon microwave digestion vessels and let them air dry. After removing trace metals, according to the text protocol, to digest the biomass, add 50 milligrams of freeze dried biomass to the microwave digestion vessels.

For quality control. Prepare two laboratory fortified blank or LFB vials containing either five milliliters of level seven I-C-P-M-S standard, or five milliliters of level seven mercury cold vapor atomic absorption spectrometry or CVAS standard. Leave another vial empty to be used as the laboratory reagent blank or LRB.

Next to each vial, add seven milliliters of concentrated trace metal grade nitric acid, and three milliliters of hydrogen peroxide. Then swirl the solution to homogenize before digesting the contents of each vial and analyzing according to the text protocol. This figure shows the average Lina seven day culture densities for control reactors and multi metal contaminated reactors with very small standard error based on sampling from three independent PBRs.

To ensure this was not an isolated result, three more batches were grown, and the combined results for all four batches are illustrated Here. This study shows that there is a statistically different and negative impact of inorganic contaminants on Lina production from two days onwards compared to controls. 12 of the 14 elements analyzed were fully recoverable after digestion, as shown by the LFB percent, R with percent R near 100%indicating no losses, no gains, and no cross-contamination of analytes during digestion.

The concentrations of heavy and inorganic contaminants found in the biomass for the 12 elements analyzed are shown here. Combining data from triplicate PBRs four batches consistently shows that inorganic contaminants are present in the biomass Results from triplicate. PBRs for batch number one, show that most contaminants were localized to the biomass rather than the supernatant.

With several sample concentrations close to the MRL of the instrument. Results for all four batches are presented here While attempting this procedure, it's important to remember to be meticulous in maintaining a clean system as contaminants can be detrimental to the results Results following this procedure. Other growth experiments can be performed in order to answer additional questions like the impact of ladder nutrients on growth systems contaminated with heavy metals, or the potential for microalgae to be used for bioremediation.

After watching this video, you should have a good understanding of how to not only cultivate algae, but also prepare samples for I-C-P-M-S. Don't forget that working with heavy metals can be extremely hazardous and precautions such as glove eye protection and the use of a fem hood should be used while performing this procedure.