The authors would like to acknowledge funding from the National Science Foundation (award # 1335550), Utah Water Research Laboratory, Professor Joan McLean and Tessa Guy for their help during the metal/metalloids analysis. The authors also thank Laura Birkhold for her support with the data collection and Danna Olbright.

1. Growth system
<img alt="Figure 1" src="/files/ftp_upload/52936/52936fig1.jpg" />
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</strong...

<ol>
	<li>Dismukes, G. C., Carrieri, D., Bennette, N., Ananyev, G. M., Posewitz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatic+phototrophs:+efficient+alternatives+to+land-based+crops+for+biofuels.">Aquatic phototrophs: efficient alternatives to land-based crops for biofuels.</a> Curr Opin Biotechnol. 19 (3), 235-240 (2008).</li><li>Moody, J. W., McGinty, C. M., Quinn, J. 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C., Wood, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Impact of Heavy Metals from Flue Gas Integration with Microalgae Production. , (2015).</li><li>Eaton, A. D., Clesceri, L. S., Rice, E. W., Greenberg, A. E. 3. 1. 2. 5. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductively+coupled+plasma/mass+spectrometry+(ICP/MS)+method.">Inductively coupled plasma/mass spectrometry (ICP/MS) method.</a> Standard methods for the examination of water and wastewater. , (2005).</li><li>Smith, M., Compton, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Matrix effects in the ICP-MS analysis of selenium in saline water samples. , (2004).</li><li>Mehta, S. K., Gaur, J. 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The authors declare that they have no competing financial interests.

Saline microalgae N. salina can be successfully grown in the designed growth system with repeatable results and high biomass yields. Airlift mixing allowed for a well-mixed suspended culture with minimal settling or biofouling over the 7 day growth periods. The minimal light variability across the fluorescent light bank is also shown to not produce noticeable differences in growth.
The study shows heavy metal contaminated media at concentrations representative of integration with coal...

Compared to traditional terrestrial crops microalgae have been shown to achieve higher biomass and lipid yields due to inherent higher solar conversion efficiencies1,2. Cultivation of microalgae at high productivity rates requires the supply of various nutrients including an external carbon source. It is expected that large-scale growth facilities will be integrated with industrial waste streams such as industrial flue gas in order to minimize production costs and at the same time provide environmental remediation. Industrial waste carbon is typically in the form of gaseous carbon dioxide and can contain contaminants that have the potential to negatively impact microalgae production. Specifically, flue gas derived from coal will have a variety of contaminants including but not limited to combustion products water and carbon dioxide, as well as oxides of sulphur and nitrogen, fine dust, organic contaminants such as dioxins and furans, and inorganic contaminants such as heavy metals. The impact of the majority of these contaminants including inorganics with some of them known as heavy metals on microalgae productivity have not been explored. Some of these elements can be nutrients at appropriate concentrations, however at higher concentrations they can produce cell dysfunction and even death3.
The integration of microalgae with industrial flue gas has the potential to directly introduce inorganic contaminants into growth media. Coal based flue gas has a variety of inorganic elements (e.g., As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) at various concentrations some of which, in low concentration, represent nutrients for microalgae growth. Inorganic contaminants have a high affinity to bind to microalgae and further be sorbed internally through nutrient transporters. Some inorganic contaminants (i.e., Co, Cu, Zn and Mn) are nutrients that form part of enzymes involved in photosynthesis, respiration and other functions3,4. However, in excess metals and metalloids can be toxic. Other elements, such as Pb, Cd, Sn, Sb, Se, As and Hg, are not known to support cell function in any concentration and represent non-nutrient metals which could negatively impact culture growth3,5,6. The presence of any of these contaminants has the potential to produce negative effects on microalgae cell function. Furthermore, the interaction of multiple metals with microalgae complicates growth dynamics and has the potential to impact growth.
Large-scale economics have been directly linked to the productivity of the cultivation system7-19. Moreover, medium recycle in the microalgae growth system for either open raceway ponds (ORP) or photobioreactors (PBR) is critical as it represents 99.9 and 99.4% of the mass, respectively20. The presence of inorganic contaminants in the media could ultimately limit microalgae productivity and the recycling of media due to contaminant build up. This study experimentally determined the impact of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn), at concentrations expected from the integration of microalgae cultivation systems with coal derived flue gas, on the productivity of N. salina grown in airlift PBRs. The contaminants used in this study have been shown to not only be present in coal-based flue gas but municipal waste-based flue gas, biosolids-based flue gas, municipal wastewater, produced water, impaired groundwater and seawater21-23. The concentrations used in this study are based on what would be expected if microalgae growth systems were integrated with a coal based CO2 source with an uptake efficiency demonstrated in commercial PBR systems20. Detailed calculations supporting the concentrations of the heavy metals and inorganic contaminants are presented in Napan et al.24 Analytical techniques were used to understand the distribution of the majority of the metals in the biomass, media and environment. The methods presented enabled the assessment of productivity potential of microalgae under inorganic contaminant stress and quantification of their end fate.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">Chemicals</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium meta silicate nonahydrate&#160;</td>
			<td>Fisher Scientific</td>
			<td>S408-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate&#160;</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium nitrate</td>
			<td>EMD Chemical</td>
			<td>PX1520-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic&#160;</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium ferric citrate</td>
			<td>Fisher Scientific</td>
			<td>I72-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Fisher Scientific</td>
			<td>A73-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium molybdate, dihydrate</td>
			<td>EMD Chemical</td>
			<td>SX0650-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese chloride tetrahydrate</td>
			<td>Fisher Scientific</td>
			<td>M87-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>Z68-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cupric sulfate pentahydrate</td>
			<td>Fisher Scientific</td>
			<td>C489-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin&#160;</td>
			<td>Acros Organics</td>
			<td>230090010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine&#160;</td>
			<td>Acros Organics</td>
			<td>148990100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin B12&#160;</td>
			<td>Acros Organics</td>
			<td>405920010</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper (II) chloride dihydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>221783-100G</td>
			<td>Irritant, Dangerous to the Environment</td>
		</tr>
		<tr>
			<td>Lead (II) chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>268690-250G</td>
			<td>Toxic, Dangerous to the Environment</td>
		</tr>
		<tr>
			<td>Sodium dichromate dihydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>398063-100G</td>
			<td>Oxidizing, Highly Toxic, Dangerous to the Environment</td>
		</tr>
		<tr>
			<td>Cobalt (II) chloride hexahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>255599-100G</td>
			<td>Toxic, Dangerous to the Environment</td>
		</tr>
		<tr>
			<td>Nickel (II) chloride hexahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>223387-500G</td>
			<td>Toxic, Dangerous to the Environment</td>
		</tr>
		<tr>
			<td>Sodium (meta) arsenite&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>71287</td>
			<td>Toxic, Dangerous to the Environment</td>
		</tr>
		<tr>
			<td>Cadmium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>202908-10G</td>
			<td>Highly Toxic, Dangerous to the Environment</td>
		</tr>
		<tr>
			<td>Mercury (II) chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>215465-100G</td>
			<td>Toxic, Dangerous to the Environment</td>
		</tr>
		<tr>
			<td>Tin (II) chloride dihydrate</td>
			<td>Fisher Scientific</td>
			<td>T142-500</td>
			<td>Corrosive. Suitable for Hg analysis. Very hazardous.</td>
		</tr>
		<tr>
			<td>Manganese chloride tetrahydrate</td>
			<td>Fisher Scientific</td>
			<td>M87-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vanadium (V) oxide</td>
			<td>Acros Organics</td>
			<td>206422500</td>
			<td>Dangerous to the Environment</td>
		</tr>
		<tr>
			<td>Carbon dioxide&#160;</td>
			<td>Air Liquide</td>
			<td>I2301S-1</td>
			<td>Compressed</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>H325-500</td>
			<td>Fisher Scientific</td>
			<td>30% in water</td>
		</tr>
		<tr>
			<td>ICP-MS standard</td>
			<td>ICP-MS-6020</td>
			<td>High Purity Standards</td>
			<td></td>
		</tr>
		<tr>
			<td>Mercury standard</td>
			<td>CGHG1-1</td>
			<td>Inorganic Ventures</td>
			<td>1000&#177;6 &#181;g/mL in 5% nitric acid</td>
		</tr>
		<tr>
			<td>Argon</td>
			<td>Air Liquide</td>
			<td></td>
			<td>Compressed</td>
		</tr>
		<tr>
			<td>Helium</td>
			<td>Air Liquide</td>
			<td></td>
			<td>Compressed, ultra high purity</td>
		</tr>
		<tr>
			<td>Hydrogen</td>
			<td>Air Liquide</td>
			<td></td>
			<td>Compressed, ultra high purity</td>
		</tr>
		<tr>
			<td>Nitric acid</td>
			<td>Fisher Scientific</td>
			<td>A509-P212</td>
			<td>67-70% nitric acid, trace metal grade. Caution: manipulate under fume hood.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A508-P212</td>
			<td>35% hydrochloric acid, trace metal grade. Caution: manipulate under fume hood.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>Scientific prevacuum sterilizer</td>
			<td>Steris</td>
			<td>31626A</td>
			<td>SV-120</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher</td>
			<td>46910</td>
			<td>RC-6 Plus</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Shimadzu</td>
			<td>1867</td>
			<td>UV-1800</td>
		</tr>
		<tr>
			<td>pH controller</td>
			<td>Hanna</td>
			<td>BL981411</td>
			<td>X4</td>
		</tr>
		<tr>
			<td>Rotometer, X5</td>
			<td>Dwyer</td>
			<td>RMA-151-SSV</td>
			<td>T31Y</td>
		</tr>
		<tr>
			<td>Rotometer, X5</td>
			<td>Dwyer</td>
			<td>RMA-26-SSV</td>
			<td>T35Y</td>
		</tr>
		<tr>
			<td>Water bath circulator</td>
			<td>Fisher Scientific</td>
			<td>13-873-45A</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact chiller</td>
			<td>VWR</td>
			<td>13270-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>Labconco</td>
			<td>7752020</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td>Fisher Scientific</td>
			<td>11-100-49S</td>
			<td></td>
		</tr>
		<tr>
			<td>pH lab electrode</td>
			<td>Phidgets Inc</td>
			<td>3550</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma mass spectrometer</td>
			<td>Agilent Technologies</td>
			<td>7700 Series ICP-MS</td>
			<td>Attached to autosampler CETAC ASX-520</td>
		</tr>
		<tr>
			<td>FIAS 100</td>
			<td>Perkin Elmer Instruments</td>
			<td>B0506520</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic absorption spectrometer</td>
			<td>Perkin Elmer Instruments</td>
			<td>AAnalyst 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell heater (quartz)</td>
			<td>Perkin Elmer Instruments</td>
			<td>B3120397</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Milestone</td>
			<td></td>
			<td>Programmable, maximum power 1200 W</td>
		</tr>
		<tr>
			<td>Microwave rotor</td>
			<td>Milestone</td>
			<td></td>
			<td>Rotor with 24 75 mL Teflon vessels for closed-vessel microwave assisted digestion.</td>
		</tr>
		<tr>
			<td colspan="4">Materials</td>
		</tr>
		<tr>
			<td>0.2 micron syringe filter</td>
			<td>Whatman</td>
			<td>6713-0425</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 micron syringe filter</td>
			<td>Whatman</td>
			<td>6713-1650</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 micron syringe filter</td>
			<td>Thermo Fisher</td>
			<td>F2500-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene tubes</td>
			<td>Evergreen</td>
			<td>222-2094-050</td>
			<td>17x100 mm w/cap, 16 mL, polysteryne</td>
		</tr>
		<tr>
			<td>Octogonal magnetic&#160;stir&#160;bars</td>
			<td>Fisher scientific</td>
			<td>14-513-60</td>
			<td>Magnets encased in PTFE fluoropolymer</td>
		</tr>
	</tbody>
</table>

quantification of heavy metals and other inorganic contaminants on the productivity of microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Biomass yields
Production of N. salina in the PBR system used in this study grew from 1 g/L-1 to 8.5 &#177; 0.19 g/L-1 (N = 12) for control reactors and 4.0 &#177; 0.3 g/L-1 (N = 12) for the multi-metal contaminated in 7 days. The experiments produced repeatable data across triplicate reactors and multiple batches. Figure 2A shows the average culture density with very small standard error based on sampling from three independent PBRs. ...

Watch this Scientific Journal Video about Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae at JoVE.com

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

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

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JoVE Journal

Environment

15.8K Views.  Utah State University. Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.