Name: comparison of scale in a photosynthetic reactor system for algal remediation of wastewater
Uploaded: 2017-03-06T14:00:00
Description: Watch this Scientific Journal Video about Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater at JoVE.com

The authors would like to thank the Sandtown Landfill in Felton, DE for sharing their knowledge and leachate.

1. System Setup
Note: A &#39;paired system&#39; refers to one aquarium tank and one raceway pond, run in parallel.
<ol>
	<li>For one paired system, use one 100 L aquaria tanks (AT), with an overhead mixer for the small-scale vessel, and one 1,000 L raceway pond (RWP), with a paddle wheel mixer for the large-scale vessel. Vessels used in this system are pictured in Figure 1.</li>
	<li>Inoculate all vessels with the same algae culture. Use a high density of the inoculation, resulting in a final de...

<ol>
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Biotechnol. 160 (1), 3-9 (2012).</li><li>Sauer, M., Porro, D., Mattanovich, D., Branduardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+production+of+organic+acids:+expanding+the+markets.">Microbial production of organic acids: expanding the markets.</a> Trends in Biotechnol. 26 (2), 100-108 (2008).</li><li>Junker, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale-up+methodologies+for+Escherichia+coli+and+yeast+fermentation+processes.">Scale-up methodologies for Escherichia coli and yeast fermentation processes.</a> J. Biosci. Bioeng. 97 (6), 347-364 (2004).</li><li>Brennan, L., Owende, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofuels+from+microalgae-a+review+of+technologies+for+production,+processing,+and+extractions+of+biofuels+and+co-products.">Biofuels from microalgae-a review of technologies for production, processing, and extractions of biofuels and co-products.</a> Renewable Sustainable Energy Rev. 14 (2), 557-577 (2010).</li><li>Van Den Hende, S., Beelen, V., Bore, G., Boon, N., Vervaeren, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-scaling+aquaculture+wastewater+treatment+by+microalgal+bacterial+flocs:+from+lab+reactors+to+an+outdoor+raceway+pond.">Up-scaling aquaculture wastewater treatment by microalgal bacterial flocs: from lab reactors to an outdoor raceway pond.</a> Bioresour. Technol. 159, 342-354 (2014).</li><li>Hewitt, C. J., Nienow, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Scale-Up+of+Microbial+Batch+and+Fed-Batch+Fermentation+Processes.">The Scale-Up of Microbial Batch and Fed-Batch Fermentation Processes.</a> Adv Appl Microbiol. 62, 105-135 (2007).</li><li>Downton, W., Bishop, D., Larkum, A., Osmond, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+Inhibition+of+Photosynthetic+Oxygen+Evolution+in+Marine+Plants.">Oxygen Inhibition of Photosynthetic Oxygen Evolution in Marine Plants.</a> Funct Plant Biol. 3 (1), 73-79 (1976).</li><li>Pholchan, M. K., Baptista, J. d. C., Davenport, R. J., Curtis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+study+of+the+effect+of+operating+variables+on+reactor+performance+and+microbial+diversity+in+laboratory-scale+activated+sludge+reactors.">Systematic study of the effect of operating variables on reactor performance and microbial diversity in laboratory-scale activated sludge reactors.</a> Water Res. 44 (5), 1341-1352 (2010).</li><li>Richmond, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of microalgal culture: biotechnology and applied phycology. , (2008).</li><li>Clesceri, L. S., 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=.">.</a> Standard Methods for the Examination of Water and Wastewater. , (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Statistics for Macintosh v.23.0. , (2015).</li><li>Devore, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Probability and Statistics for Engineering and the Sciences. , (2015).</li><li>Sniffen, K. D., Sales, C. M., Olson, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+removal+from+raw+landfill+leachate+by+an+algae-bacteria+consortium.">Nitrogen removal from raw landfill leachate by an algae-bacteria consortium.</a> Water Sci. Technol. 73 (3), 479-485 (2015).</li><li>Paerl, H. W., Fulton, R., Moisander, P. H., Dyble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harmful+freshwater+algal+blooms,+with+an+emphasis+on+cyanobacteria.">Harmful freshwater algal blooms, with an emphasis on cyanobacteria.</a> Scientific World J. 1, 76-113 (2001).</li><li>Abeliovich, A., Azov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+Ammonia+to+Algae+in+Sewage+Oxidation+Ponds.">Toxicity of Ammonia to Algae in Sewage Oxidation Ponds.</a> Appl. Environ. Microbiol. 31 (6), 801-806 (1976).</li><li>Azov, Y., Goldman, 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=Free+ammonia+inhibition+of+algal+photosynthesis+in+intensive+cultures.">Free ammonia inhibition of algal photosynthesis in intensive cultures.</a> Appl. Environ. Microbiol. 43 (4), 735-739 (1982).</li><li>Adamsson, M., Dave, G., Forsberg, L., Guterstam, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+identification+evaluation+of+ammonia,+nitrite+and+heavy+metals+at+the+Stensund+Wastewater+Aquaculture+Plant,+Sweden.">Toxicity identification evaluation of ammonia, nitrite and heavy metals at the Stensund Wastewater Aquaculture Plant, Sweden.</a> Water Sci. Technol. 38 (3), 151-157 (1998).</li><li>Quinn, J. C., Davis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potentials+and+challenges+of+algae+based+biofuels:+a+review+of+the+techno-economic,+life+cycle,+and+resource+assessment+modeling.">The potentials and challenges of algae based biofuels: a review of the techno-economic, life cycle, and resource assessment modeling.</a> Bioresour. Technol. 184, 444-452 (2015).</li><li>Liu, X., 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=Pilot-scale+data+provide+enhanced+estimates+of+the+life+cycle+energy+and+emissions+profile+of+algae+biofuels+produced+via+hydrothermal+liquefaction.">Pilot-scale data provide enhanced estimates of the life cycle energy and emissions profile of algae biofuels produced via hydrothermal liquefaction.</a> Bioresour. Technol. 148, 163-171 (2013).</li><li>Van Den Hende, S., 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=Treatment+of+industrial+wastewaters+by+microalgal+bacterial+flocs+in+sequencing+batch+reactors.">Treatment of industrial wastewaters by microalgal bacterial flocs in sequencing batch reactors.</a> Bioresour. Technol. 161, 245-254 (2014).</li><li>Rawat, I., Kumar, R. R., Mutanda, T., Bux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiesel+from+microalgae:+A+critical+evaluation+from+laboratory+to+large+scale+production.">Biodiesel from microalgae: A critical evaluation from laboratory to large scale production.</a> Appl. Energy. 103, 444-467 (2013).</li><li>Cloern, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relative+importance+of+light+and+nutrient+limitation+of+phytoplankton+growth:+a+simple+index+of+coastal+ecosystem+sensitivity+to+nutrient+enrichment.">The relative importance of light and nutrient limitation of phytoplankton growth: a simple index of coastal ecosystem sensitivity to nutrient enrichment.</a> Aquat Ecol. 33 (1), 3-15 (1999).</li></ol>

System performance:
Over the course of an 8-week study, the productivity of the small- and large-scale vessels in a system were compared. In this study nitrogen and ammonia removal rates and biomass growth rates were used as measures of productivity of the treatment system. The system was operated as a semi-batch reactor, where each week was operated under discrete conditions. Representative results account for the first 8 weeks of system operation, however a full study would extend for much long...

Translation of bench-scale data to larger scale applications is a key step in the commercialization of bioprocesses. Production efficiencies in small-scale reactor systems, particularly those focusing on the use of microorganisms, have been shown to consistently over predict efficiencies occurring in commercial-scale systems 1,2,3,4. Challenges also exist in scaling up photosynthetic cultivation of algae and cyanobacteria from the laboratory scale to larger systems for the purpose of manufacturing high-value products, such as cosmetics and pharmaceuticals, for production of biofuels, and for the treatment of wastewater. The demand for large-scale algal biomass production is growing with the emerging industry for algae in biofuel, pharmaceuticals/nutraceuticals, and livestock feed 5. The methodology described in this manuscript aims to evaluate the influence of increasing scale of a photosynthetic reactor system on biomass growth rate and nutrient removal. The system presented here uses algae to remediate landfill leachate wastewater but can be adapted for a variety of applications.
Production efficiencies of large scale systems are often predicted using smaller scale experiments; however, several factors must be considered to determine the accuracy of these predictions, as scale has been shown to affect the performance of bioprocesses. For example, Junker (2004) presented results from a comparison of eight different-sized fermentation reactors, ranging from 30 L to 19,000 L, which showed that actual productivity at pilot- or commercial-scales was almost always lower than the values predicted using small-scale studies 4. Inequalities in vessel dimension, mixing power, agitation type, nutrient quality, and gas transfer were predicted to be the major causes for the decreased productivity 4. Similarly, it has been shown in algae growth reactors that biomass growth and biomass related products are nearly always reduced when scale is increased 6.
Biological, physical, and chemical factors change with the size of a reactor, with many of these factors influencing microbial activity at small scales differently than at larger scales 2,7. Since most full-scale systems for algae, such as raceway ponds, exist outdoors, one biological factor to consider is that microbial species and bacteriophages can be introduced from the surrounding environment, which may alter the microbial species present and thus the microbial function of the system. The activity of the microbial community will also be sensitive to environmental factors, such as light and temperature. Mass transfers of gasses and fluid motion are examples of physical factors that are influenced in the scale up of microbial processes. Achieving ideal mixing in small reactors is easy; however, with increasing scale, it becomes a challenge to engineer ideal-mixing conditions. At larger scales, reactors are more likely to have dead zones, non-ideal mixing, and reduced efficiencies in mass transfer 2. Since algae are photosynthetic organisms, commercial growth must account for changes in light exposure due to changes in water depth and surface area when increasing volume. High biomass density and/or low mass transfer rates can cause decreased CO2 concentrations and increased O2 concentrations, both of which may result in inhibition of biomass growth 8. Chemical factors in an algae growth system are driven by pH dynamics of the aquatic environment 2, which is consequently affected by changes in pH buffering compounds such as dissolved CO2 and carbonate species. These factors are compounded by complex interactions among the biological, physical, and chemical factors, often in unpredictable ways 9.
This study presents a paired reactor system designed to regulate and compare growth conditions in vessels of two different scales. The experimental protocol focuses on quantifying leachate treatment and algae growth; however, it could be adapted to monitor other metrics such as changes in the microbial community over time or the CO2 sequestration potential of algae. The protocol presented here is designed to evaluate the effect of scale on algal growth and nitrogen removal in a leachate treatment system.

<table border="0" cellpadding="0" cellspacing="0" width="1148">
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Aquarium Tank</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:612px;">Any 100+L aquarium tank with optically clear glass can be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">RW 3.5</td>
			<td style="width:135px;">MicroBio Engineering</td>
			<td style="width:119px;"></td>
			<td>Raceway Pond</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Eurostar 100 digital</td>
			<td style="width:135px;">IKA</td>
			<td style="width:119px;">4238101</td>
			<td style="width:612px;">Overhead mixers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Leachate</td>
			<td style="width:135px;">Sandtown Landfill</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Sampling Bottles</td>
			<td style="width:135px;">Nalgene</td>
			<td style="width:119px;"></td>
			<td>Plastic or glass, lab grade, 125-200mL&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Transfer Pumps</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td>Garden type pump with drinking water quality hoses will be suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">AmVer Salicylate Test &#39;N Tube</td>
			<td style="width:135px;">Hach</td>
			<td style="width:119px;">2606945</td>
			<td>High Range Ammonia Tests</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">NitraVer X Nitrogen - Nitrate Reagent Set&#160;</td>
			<td style="width:135px;">Hach</td>
			<td style="width:119px;">2605345</td>
			<td>High Range Nitrate Tests</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">NitriVer 2 Nitrite Reagent Powder Pillows</td>
			<td style="width:135px;">Hach</td>
			<td style="width:119px;">2107569</td>
			<td>High Range Nitrite Tests</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Hach DR2400 Spectrophotmeter</td>
			<td style="width:135px;">Hach</td>
			<td style="width:119px;"></td>
			<td>The DR2400 was discontinued, but any DR series Hach spectrophotometer can be used in this application.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">EMD Microbiological Analysis Membrane Filters</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:119px;">HAWG047S6</td>
			<td style="width:612px;">0.45&#181;m filters&#160;</td>
		</tr>
	</tbody>
</table>

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.

The aim of this study is to compare the biomass growth and nutrient removal capabilities of algal cultures grown in small- and large-scale reactors. This study uses two paired systems, referred to as System 1 and System 2, to duplicate its findings. These representative results are from an 8-week period, February through April, 2016. The first raceway pond was inoculated with algae originally sourced from an outdoor pond in Philadelphia, PA 14. This culture was gro...

Watch this Scientific Journal Video about Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater at JoVE.com

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

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, ...

The overall goal of this procedure is to evaluate the effect of scale on algal growth and nitrogen removal in a leachate treatment system. This method can help determine how increasing the scale of a system will impact the treatment efficiency. The main advantage of this technique is that it can be adapted for a variety of microbial and wastewater treatment systems.For this protocol, a paired system refers to one 100 liter aquarium tank and one 1, 000 liter raceway pond run in parallel. Start with a high density inoculum, which may take weeks to months to grow enough algae for. Inoculate all vessels with the same algae culture, ensuring that the final density is no less than 0.1 grams per liter once diluted to the full volume in the tank or pond.Untreated landfill leachate serves as the nutrient source for this system. Use leachate taken from a landfill that accepts primarily domestic waste and has low levels of toxins. The amount of leachate used in each tank or pond can vary depending on the strength of the wastewater.Start the aquarium tank with one liter of leachate in 59 liters of water for a 60 liter working volume. Likewise, start the raceway pond with 10 liters of leachate in 590 liters of water for a 600 liter working volume. Week to week, operate the aquarium tank and raceway pond as semi-batch reactors with hydraulic retention times of three weeks.Monitor environmental conditions, such as air temperature and solar radiation, using a commercial weather station. Also monitor tank or pond conditions, such as water temperature, pH, and dissolved oxygen, using commercial probes and a data logger. At the beginning of each week, take a 125 milliliter sample from each vessel to be tested for biomass density, as well as ammonia-N, nitrate-N, and nitrite-N.Measure the biomass by a standard total suspended solids protocol using 0.45 micron filters. After weighing the filter paper, filter 20 to 40 milliliters of sample using a vacuum filtration system. Dry the biomass and filter paper in an oven at 105 degrees Celsius for one hour.After drying, calculate the biomass density. Next, measure ammonia, nitrate, and nitrite using a spectrophotometer and commercial method kits according to the manufacturer's protocol. After one week, take 125 milliliter samples from each vessel and analyze the samples as before.This is the end of the first week. To prepare for the next week's cycle, pump the entire volume of the aquarium tank into the raceway pond. Remove 1/3 of the volume from the raceway pond.Replace the volume removed with water and untreated leachate. Then, transfer approximately 60 liters from the raceway pond back into the aquarium tank. This ensures that the aquarium tank and the raceway pond are starting with the same nutrient and biological conditions each week.In this study, two paired systems were used, referred to as system one and system two, to duplicate the findings. Ammonia removal rate, total nitrogen removal rate, and biomass growth rate were used as metrics to evaluate the treatment system. The rates from system one and system two are shown here for the representative study period.Data from all vessels over the representative study period showed that ammonia removal trended positively with increasing starting ammonia concentration. Once the system is up and running, reactor parameters, such as biomass density, length of time between mixing, working volume, retention time, and the amount of wastewater added, can be adjusted to better test a customized system. Ultimately, results from this procedure can be used to inform feasibility studies of full scale treatment processes.For example, the effect of scale on important reactor parameters, such as growth rate and nutrient removal, can be used to verify the accuracy of input data used to simulate full scale production processes and lifecycle assessments and technoeconomic analyses. This is a versatile methodology which can be adapted to study the treatment of a variety of wastewaters.

comparison-scale-photosynthetic-reactor-system-for-algal-remediation

Research

JoVE Journal

Environment

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the production of synthetic PM for laboratory studies have become a widespread necessity. Herein, experimental protocols demonstrate approaches to produce aerosolized organic PM by &#945;-pinene ozonolysis in a flow tube reactor. Methods are described for measuring the size distributions and morphology of the aerosol particles. The video demonstrates basic operations of the flow tube reactor and related instrumentation. The first part of the video shows the procedure for preparing gas-phase reactants, ozonolysis, and production of organic PM. The second part of the video shows the procedures for determining the properties of the produced particle population. The particle number-diameter distributions show different stages of particle growth, namely condensation, coagulation, or a combination of both, depending on reaction conditions. The particle morphology is characterized by an aerosol particle mass analyzer (APM) and a scanning electron microscope (SEM). The results confirm the existence of non-spherical particles that have grown from coagulation for specific reaction conditions. The experimental results also indicate that the flow tube reactor can be used to study the physical and chemical properties of organic PM for relatively high concentrations and short time frames.

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Techniques for Investigating the Anatomy of the Ant Visual System

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest.

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

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 ...

A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis

Harmful algae blooms (HABs) monitoring has been implemented worldwide, and Chile, a country famous for its fisheries and aquaculture, has intensively used microscopic and toxin analyses for decades for this purpose. Molecular biological methods, such as high-throughput DNA sequencing and bacterial assemblage-based approaches, are just beginning to be introduced in Chilean HAB monitoring, and the procedures have not yet been standardized. Here, 16S rRNA and 18S rRNA metabarcoding analyses for monitoring Chilean HABs are introduced stepwise. According to a recent hypothesis, algal-bacterial mutualistic association plays a critical synergetic or antagonistic relationship accounting for bloom initiation, maintenance, and regression. Thus, monitoring HAB from algal-bacterial perspectives may provide a broader understanding of HAB mechanisms and the basis for early warning. Metabarcoding analysis is one of the best suited molecular-based tools for this purpose because it can detect massive algal-bacterial taxonomic information in a sample. The visual procedures of sampling to metabarcoding analysis herein provide specific instructions, aiming to reduce errors and collection of reliable data.

This protocol introduces steps of metabarcoding analysis, targeting 16S rRNA and 18S rRNA genes, for monitoring harmful algal blooms and their associated microbiome in seawater samples. It is a powerful molecular-based tool but requires several procedures, which are visually explained here step-by-step.

Harmful algae blooms (HABs) monitoring has been implemented worldwide, and Chile, a country famous for its fisheries and aquaculture, has intensively used ...