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.

<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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+evaluation+of+biofuel+potential+from+microalgae.">Global evaluation of biofuel potential from microalgae.</a> Proceedings of the National Academy of Sciences. 111 (23), 8691-8696 (2014).</li><li>Pinto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heavy+metal-induced+oxidative+stress+in+algae.">Heavy metal-induced oxidative stress in algae.</a> J Phycol. 39 (6), 1008-1018 (2003).</li><li>Gupta, A., Lutsenko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+copper+transporting+ATPases+in+eukaryotic+organisms.">Evolution of copper transporting ATPases in eukaryotic organisms.</a> Curr Genomics. 13 (2), 124-133 (2012).</li><li>Perales-Vela, H. V., Pe&#241;a-Castro, J. M., Ca&#241;izares-Villanueva, R. 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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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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.

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

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

Evaluation of the Productivity of Social Wasp Colonies (Vespinae) and an Introduction to the Traditional Japanese Vespula Wasp Hunting Technique

For vespine wasps, colony productivity is typically estimated by counting the number of larval cells. This paper presents an improved method that enables researchers to estimate more accurately the number of adults produced, counting the number of meconia (the stools left in the cells by wasp larvae when pupating into adults, per 100 cells) in each comb. This method can be applied before or after colony collapse (i.e., in active or inactive nests). The paper also describes how to locate wild Vespula wasp colonies by &#34;flagging&#34; wasp baits and chasing the wasp collecting them, using a method traditionally performed by local people in central Japan (as illustrated in the associated video). The Vespula chasing method described has several advantages: it is easy to reinitiate the chase from a point where the forager flying back to the nest was lost, and it is easy to pinpoint the nest location as marked wasps often lose their flag at the nest entrance. These methods for estimating colony productivity and collecting nests can be valuable for researchers studying social wasps.

Evaluation of the Productivity of Social Wasp Colonies Vespinae and an Introduction to the Traditional Japanese Vespula Wasp Hunting Technique

This methodological paper evaluates the productivity of a social wasp colony by examining the number of meconia per 100 cells of comb, to estimate the total number of adults the wasps produced. The associated video describes how to search for Vespula wasp nests, a method developed by amateur wasp chasers.

For vespine wasps, colony productivity is typically estimated by counting the number of larval cells. This paper presents an improved method that enables ...

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...

A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal toxicity test is frequently used in regulatory risk assessment of chemicals. In order to achieve high reliability and reproducibility the development of standardized guidelines is vital. For algal toxicity testing, the guidelines require stable and uniform conditions of parameters such as pH, temperature, carbon dioxide levels and light intensity. Nanomaterials and other so-called difficult substances can interfere with light causing a large variation in results obtained hampering their regulatory acceptance. To address these challenges, we have developed LEVITATT (LED Vertical Illumination Table for Algal Toxicity Tests). The setup utilizes LED illumination from below allowing for a homogenous light distribution and temperature control while also minimizing intra-sample shading. The setup optimizes the sample volume for biomass quantification and does at the same time ensure a sufficient influx of CO2 to support exponential growth of the algae. Additionally, the material of the test containers can be tailored to minimize adsorption and volatilization. When testing colored substances or particle suspensions, the use of LED lights also allows for increasing the light intensity without additional heat generation. The compact design and minimal equipment requirements increase the possibilities for implementation of the LEVITATT in a wide range of laboratories. While compliant with standardized ISO and OECD guidelines for algal toxicity testing, LEVITATT also showed a lower inter-sample variability for two reference substances (3,5-Dicholorophenol and K2Cr2O7) and three nanomaterials (ZnO, CeO2, and BaSO4) compared to Erlenmeyer flasks and microtiter plates.

We demonstrate algal toxicity testing for difficult substances (e.g., colored substances or nanomaterials) using a setup illuminated vertically with an LED.

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal ...

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.