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. 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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><tbody><tr><td>Chemicals</td><td></td><td></td><td></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&amp;nbsp;</td><td>Fisher Scientific</td><td>S408-500</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Acros Organics</td><td>230090010</td><td></td></tr><tr><td>Thiamine&amp;nbsp;</td><td>Acros Organics</td><td>148990100</td><td></td></tr><tr><td>Vitamin B12&amp;nbsp;</td><td>Acros Organics</td><td>405920010</td><td></td></tr><tr><td>Copper (II) chloride dihydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>221783-100G</td><td>Irritant, Dangerous to the Environment</td></tr><tr><td>Lead (II) chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>268690-250G</td><td>Toxic, Dangerous to the Environment</td></tr><tr><td>Sodium dichromate dihydrate&amp;nbsp;</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&amp;nbsp;</td><td>Sigma-Aldrich</td><td>255599-100G</td><td>Toxic, Dangerous to the Environment</td></tr><tr><td>Nickel (II) chloride hexahydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>223387-500G</td><td>Toxic, Dangerous to the Environment</td></tr><tr><td>Sodium (meta) arsenite&amp;nbsp;</td><td>Sigma-Aldrich</td><td>71287</td><td>Toxic, Dangerous to the Environment</td></tr><tr><td>Cadmium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>202908-10G</td><td>Highly Toxic, Dangerous to the Environment</td></tr><tr><td>Mercury (II) chloride&amp;nbsp;</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&amp;nbsp;</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&amp;plusmn;6 &amp;micro;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>Equipment</td><td></td><td></td><td></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 1,200 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>Materials</td><td></td><td></td><td></td></tr><tr><td>0.2 &amp;#956;m syringe filter</td><td>Whatman</td><td>6713-0425</td><td></td></tr><tr><td>0.2 &amp;#956;m syringe filter</td><td>Whatman</td><td>6713-1650</td><td></td></tr><tr><td>0.45 &amp;#956;m 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>17 x 100 mm w/cap, 16 ml, polysteryne</td></tr><tr><td>Octogonal magnetic&amp;nbsp;stir&amp;nbsp;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. ...

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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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15.9K 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

Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ammonia removal, nitrogen removal and algal growth are compared over an 8-week period in paired sets of small (100 L) and large (1,000 L) reactors designed for algal remediation of landfill wastewater. Contents of the small and large scale reactors were mixed before the beginning of each weekly testing interval to maintain equivalent initial conditions across the two scales. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted to better equalize conditions occurring at both scales. During the short 8-week representative time period, starting ammonia and total nitrogen concentrations ranged from 3.1-14 mg NH3-N/L, and 8.1-20.1 mg N/L, respectively. The performance of the treatment system was evaluated based on its ability to remove ammonia and total nitrogen and to produce algal biomass. Mean &#177; standard deviation of ammonia removal, total nitrogen removal and biomass growth rates were 0.95&#177;0.3 mg NH3-N/L/day, 0.89&#177;0.3 mg N/L/day, and 0.02&#177;0.03 g biomass/L/day, respectively. All vessels showed a positive relationship between the initial ammonia concentration and ammonia removal rate (R2=0.76). Comparison of process efficiencies and production values measured in reactors of different scale may be useful in determining if lab-scale experimental data is appropriate for prediction of commercial-scale production values.

An experimental methodology is presented to compare the performance of small (100 L) and large (1,000 L) scale reactors designed for algae remediation of landfill wastewater. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted based on application.

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ...

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 ...

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to seasonality variation in pH and temperature, fluctuation in element composition is periodically observed within these extreme environments, influencing the environmental microbial communities. Extremophilic microorganisms that thrive in volcanic thermal vents have developed resistance mechanisms to handle several metal ions present in the environment, thus taking part to complex metal biogeochemical cycles. Moreover, extremophiles and their products have found an extensive foothold in the market, and this holds true especially for their enzymes. In this context, their characterization is functional to the development of biosystems and bioprocesses for environmental monitoring and bioremediation. To date, the isolation and cultivation under laboratory conditions of extremophilic microorganisms still represent a bottleneck for fully exploiting their biotechnological potential. This work describes a streamlined protocol for the isolation of thermophilic microorganisms from hot springs as well as their genotypical and phenotypical identification through the following steps: (1) Sampling of microorganisms from geothermal sites ("Pisciarelli", a volcanic area of Campi Flegrei in Naples, Italy); (2) Isolation of heavy metal resistant microorganisms; (3) Identification of microbial isolates; (4) Phenotypical characterization of the isolates. The methodologies described in this work might be generally applied also for the isolation of microorganisms from other extreme environments.

The isolation of heavy metal-resistant microbes from geothermal springs is a hot topic for the development of bioremediation and environmental monitoring biosystems. This study provides a methodological approach for isolating and identifying heavy metal tolerant bacteria from hot springs.

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to ...

Microalgal Cultivation for Biomethane Obtention from Swine Waste Biogas

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

Author Spotlight: Scaling Microalgal Biotechnology for Enhanced Biomethane Production 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

Enhancing Chlorophyll Production in Microalgae via Growth Factor Optimization

Evaluation of the Effect of Growth Factors on Chlorophylls a and b Production from Microalgae

Microalgae contain two main groups of pigments: chlorophylls and carotenoids. Chlorophyll is a green pigment that absorbs light energy and transforms it into chemical energy to facilitate the synthesis of organic compounds. This pigment serves as a valuable primary source for biotechnological input products in the food, pharmaceutical, and cosmetic industries due to its high antioxidant properties and coloring capabilities. The objective of this research was to evaluate the effect of growth factors (CO2 concentration, light color, and light intensity) through a Taguchi L4 experimental design on cell growth and the cellular content of chlorophyll a and b in Chlorella sorokiniana, followed by validation of the method using Haematococcus pluvialis microalgae as an additional study model. Cell growth was quantified using the optical density spectrophotometric technique at a wavelength of 550 nm. For the quantification of chlorophylls, a cell extract was obtained using a 90% pure acetone solution, and subsequently, the concentrations of chlorophylls a and b were quantified using spectrophotometric techniques at wavelengths of 647 nm and 664 nm, according to the method described by Jeffrey and Humphrey. The experimental results indicated that controlling conditions of low CO2 addition, purple light, and low light intensity increases both cell growth and the concentration of chlorophylls a and b within the cells. The implementation of this chlorophyll quantification method allows for quick, simple, and precise determination of chlorophyll content, as the wavelengths used are at the absorbance peaks of both types of chlorophylls, making this technique easily reproducible for any microalgae under study.

Author Spotlight: Optimizing Growth Factors for Production of Biotechnologically Relevant Secondary Metabolites

The present study highlights the advantages of employing the method developed by Jeffrey and Humphrey for extracting and quantifying fat-soluble pigments from microalgae. This method serves as a valuable tool for assessing the influence of growth factors on chlorophyll production and cellular content in these organisms.

Microalgae contain two main groups of pigments: chlorophylls and carotenoids. Chlorophyll is a green pigment that absorbs light energy and transforms it ...

Biochemistry

High-Throughput Metabolic Profiling for Model Refinements of Microalgae

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a systems-level. Genome-scale metabolic models may include gaps as well as reactions that are unverified experimentally. Reconstructed models of newly isolated microalgal species will result in weaknesses due to these gaps, as there is usually sparse biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology is an effective, high-throughput method that functionally determines cellular metabolic activities&#160;in response to a wide array of entry metabolites. Combining the high throughput phenotypic assays with metabolic modeling can allow existing metabolic network models to be rapidly reconstructed or optimized by providing biochemical evidence to support and expand genomic evidence. This work will show the use of PM assays for the study of microalgae by using the green microalgal model species Chlamydomonas reinhardtii as an example. Experimental evidence for over 254 reactions obtained by PM was used in this study to expand and refine a genome-scale C. reinhardtii metabolic network model, iRC1080, by approximately 25 percent. The protocol created here can be used as a basis for functionally profiling the metabolism of other microalgae, including known microalgae mutants and new isolates.

This protocol demonstrates the use of a phenotype microarray (PM) technology platform to define metabolic requirements of Chlamydomonas reinhardtii, a green microalga, and refine an existing metabolic network model.

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a ...

Biology

Operation of Laboratory Photobioreactors with Online Growth Measurements and Customizable Light Regimes

The laboratory study of microalgae can be experimentally challenging. In addition to the cultivation requirements of non-photosynthetic microorganisms, phototrophs also require illumination. Routinely, researchers seek to provide custom light supplies, i.e., vary the light intensity and time over which it is delivered. Such flexibility is difficult with standard benchtop lights. Usually, cultivation studies also require growth comparisons between experimental treatments. Frequently, growth is assessed over an extended duration, e.g., multiple times a day over a week-long trial. Manual measurements can be time-consuming and lack data resolution. Therefore, photobioreactors (PBRs) with automatic growth monitoring and customizable light supply are useful for replicated experiments with multiple treatments. The current work presents the design, construction, and operation of laboratory PBRs. The materials are easily sourced and relatively inexpensive. The design could be duplicated with moderate skill. Each structure has a footprint of ~40 cm2 and hosts three 1 L glass bottles for triplicate replication. Bottles rest upon platforms containing magnetic stirrers and are arranged vertically within a 1 m high and 15 cm diameter polyvinyl chloride (PVC) pipe. The pipe interior is lined with light-emitting diodes (LEDs). These LEDs produce continuous light intensities from 0&#8211;2400 &#181;mol photons m-2 s-1 of photosynthetically active radiation (PAR). Users design a custom lighting program. The light intensity can be adjusted each second or held constant for longer durations. Oxygen produced from photosynthesis exits each bottle via a one-way volumetric gas sensor. Software is used to record gas sensor data. The amount of oxygen produced can be correlated to biomass growth. If biomass samples are required, a syringe can be used to extract culture. The method is suited for microalgae grown with bicarbonate as the carbon source. These PBRs are valuable to a laboratory that requires replicated experiments, light regime flexibility, and continuous high-resolution growth data.

This publication describes the design of laboratory photobioreactors (PBRs) with customizable light regimes. The growth of cyanobacteria or microalgae, using bicarbonate as their carbon source, is monitored continuously by measuring volumetric oxygen production. These PBRs facilitate rapid, replicated laboratory growth comparisons with little user intervention during experiments.

The laboratory study of microalgae can be experimentally challenging. In addition to the cultivation requirements of non-photosynthetic microorganisms, ...

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

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