This work was funded by the Government of Canada&#8217;s Federal Economic Development Agency (FedDev) Applied Research and Commercialization Extension to Queen&#8217;s University (Dr. Allison Rutter and Dr. Darko Matovic). Sincerest thank you to Burt&#8217;s Greenhouses (Odessa, ON) for providing the biochars. Special thanks to Yuxing Cui of the CBRN Protection Group at RMC and staff of the ASU and Zeeb Lab for their ongoing support.

The authors declare that they have no competing financial interests.

All of the methods listed in the protocol have been carefully validated and extensively used for soils. As biochar characterization is still in its infancy, the effectiveness of these methods for the carbon-rich substrate was largely unknown. Hence, although these methods themselves are not novel, their application to routinely characterize biochar is. In terms of quality assurance/ quality control, there were no issues among any of the methods with respect to the blanks being below detection limits or the recoveries bei...

Biochar is a carbon-rich by-product produced during the pyrolysis of organic matter 1. Interest, both publicly and academically, in adding biochar to soils, stems from its ability to improve soil quality and plant growth 2, 3, sustainably sequester carbon 4, and sorb harmful contaminants 2, 3, 5-7 whilst simultaneously offering alternatives for waste management and energy production by pyrolysis.
Biochars are being produced by numerous companies and organizations worldwide via different pyrolysis systems. Materials used for biochar production include (but are not limited to) woodchips, animal manure and construction wastes1. These differences are expected to alter the biochars&#8217; physical and chemical properties and thus their ability to improve substrates, promote long-term stability and increase sorption capabilities. Additionally, during the pyrolysis process the biochar may become unintentionally contaminated with metals, PAHs and PCBs as a result of contaminated feedstocks or inappropriate pyrolysis conditions. Therefore, before biochar can be applied on a large scale to the environment as a soil amendment, careful characterization of the biochar for contaminants, specific surface area, cation exchange capacity, earthworm avoidance and germination and others suggested by the International Biochar Initiative (IBI) must be conducted. In 2013, the first Standardized Product Definition and Product Testing Guidelines for Biochar, which sets standards for biochar physical and chemical characteristics, was published and made publically available.
Research has shown that biochar produced at a commercial greenhouse in Odessa, ON, Canada has the ability to significantly improve plant growth in intensely degraded soils and sorb persistent organic pollutants (POPs) such as PCBs 2, 3. This biochar has been produced from three different feedstocks (i.e. organic matter sources) via a boiler system where the heat generated is used to warm their greenhouse operation during winter months.
This study provides characterization data pertinent to the production of biochar in a biomass boiler, and the use of biochar as a soil amendment. The objective of this study is to thoroughly characterize the physical, chemical and biological characteristics of six biochars according to standards set by the IBI in their Standardized Product Definition and Product Testing Guidelines (Version 1.1) (2013). These characteristics will be linked, where possible, to the performance of each biochar as agricultural amendments and their ability to sorb contaminants.

<table border="0" cellpadding="0" cellspacing="0" width="2073">
	<tbody>
		<tr height="30">
			<td height="30" style="height:30px;width:343px;">Name of Material/ Equipment</td>
			<td style="width:359px;">Company</td>
			<td style="width:208px;">Catalog Number</td>
			<td style="width:1163px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Biochar</td>
			<td style="width:359px;">Burt&#39;s Greenhouses</td>
			<td style="width:208px;"></td>
			<td>All six biochars were produced at Burt&#39;s Greenhouses via BlueFlame Boiler system</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:343px;">NaOAc</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">E124-4</td>
			<td>Dissolving 136.08 g of NaOAC.3H2O in 750mL distilled, deionized&#160; water (DDI water)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Acetic Acid</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">A38-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium Hydroxide</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td>SS284-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Isopropanol</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">A416P4</td>
			<td>80% IPA- 800 mL IPA with 200 mL DDI water.&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:343px;">NH4Cl</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">A649500</td>
			<td>Dissolving 5.35 g NH4Cl into 1 L DDI water.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Alumminum Drying Pan</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td>08-732-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Drying Oven</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">508N0024</td>
			<td>200&#176;C for 2 hours.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Desiccator</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td>08-595A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Balance</td>
			<td style="width:359px;">Mettler</td>
			<td style="width:208px;">1113032410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Saturating Solution</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">06-664-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Vortex</td>
			<td style="width:359px;">Barnstead/Thermolyne</td>
			<td style="width:208px;">871000536389&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Centrifuge</td>
			<td style="width:359px;">International Equipment Company</td>
			<td style="width:208px;">24372808</td>
			<td>3000 g for 5 mins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Rinsing Solution</td>
			<td style="width:359px;">Fisher Scientific (Ricca Chemistry Company)</td>
			<td style="width:208px;">06-664-24</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Conductivity Meter</td>
			<td style="width:359px;">WESCAN</td>
			<td style="width:208px;">88298</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Replacing Solution</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">06-664-24</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">ICP-AES</td>
			<td style="width:359px;">Varian</td>
			<td style="width:208px;">EL00053841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">ASAP 2000 Surface Area Analyser&#160;</td>
			<td style="width:359px;">Cavlon</td>
			<td style="width:208px;">885</td>
			<td>Degassing at 120&#176;C for a minimum of 2 hours.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Muffle Furnace</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">806N0024</td>
			<td>Heat for 16 hours covering at 420&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">pH Meter</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">1230185263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sieve</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">2288926</td>
			<td>4.7 mm sieve being at the top.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sieve Skaker</td>
			<td style="width:359px;">Meinzer II</td>
			<td style="width:208px;">0414-02</td>
			<td>Shake for 10 min.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium Sulphate</td>
			<td style="width:359px;">VWR</td>
			<td style="width:208px;">EM-SX0761-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Ottawa Sand</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">S23-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Soxhlet Apparatus</td>
			<td style="width:359px;">Fisher Scientific (Pyrex)</td>
			<td style="width:208px;">09-557A</td>
			<td>4&#160;hours at 4&#8211;6&#160;cycles per hour.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DCBP</td>
			<td style="width:359px;">Suprlco Analytical</td>
			<td style="width:208px;">48318&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Dichloromethane</td>
			<td style="width:359px;">Sigma Aldrich</td>
			<td style="width:208px;">40042-40855-U</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">6890 Plus Gas Chromatograph Micro 63 Ni ECD</td>
			<td style="width:359px;">Agilent</td>
			<td style="width:208px;">US00034778</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Helium</td>
			<td style="width:359px;">AlphaGaz</td>
			<td style="width:208px;">SPG-NIT1AL50SMART</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Nitrogen</td>
			<td style="width:359px;">AlphaGaz</td>
			<td style="width:208px;">SPG-HEL1AL50SMART</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Mortor and Pestle</td>
			<td style="width:359px;">Fisher Scientific (CoorsTeh)</td>
			<td>12-948G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Nitric Acid</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">351288212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">No. 40 Filter Paper</td>
			<td style="width:359px;">Fisher Scientific (Whatman)</td>
			<td>09-845A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Quartz/Nickel weigh boats</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td>11-474-210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DMA-80</td>
			<td style="width:359px;">ATS Scientific</td>
			<td style="width:208px;">5090264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">98-99% Formic Acid</td>
			<td style="width:359px;">Sigma Aldrich</td>
			<td style="width:208px;">33015-1L</td>
			<td>1L volumetric filled to 750 mL with DDI water add 20 mL formic acid and fill to volume with DDI water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sonicator</td>
			<td style="width:359px;">Fisher Sientific</td>
			<td>15338284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Rotating Shaker</td>
			<td style="width:359px;">New Brunswick Scientific (Innova 2100)</td>
			<td style="width:208px;">14-278-108</td>
			<td>1 hour at 200 rpm.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">No. 42 Filter Paper</td>
			<td style="width:359px;">Fisher Scientific (Whatman)</td>
			<td style="width:208px;">09-855A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">WhirlPacks</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">R55048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Potassium Dihydrogen Orthophospahte</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">181525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">2M KCl</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">P282100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plastic Vials</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">03-337-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Ammonium Chloride</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">PX05115</td>
			<td>Allow to warm up to room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Colour Reagent</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">361028260</td>
			<td>Allow to warm up to room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Colorimeter</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">13-642-400</td>
			<td>Turn on to let the lamp warm up and run for 5 minutes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">ASEAL Auto Analyzer 2</td>
			<td style="width:359px;">SEAL</td>
			<td style="width:208px;">4723A12068</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Liquified Phenol</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">MPX05115</td>
			<td>Alkaline Phenol- Measure 87 mL of liquefied phenol into 1-L volumetric filled 2/3 with DDI water.&#160; Add 34 g NaOH, make up to volume with DDI water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">NaOH</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">S318-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Commercial Bleach</td>
			<td style="width:359px;">Retail Store</td>
			<td style="width:208px;"></td>
			<td>Hypochlorite Solution- using 100-mL graduated cylinder measure 31.5 mL of commercial bleach and fill to 100 mL with DDI water.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">NaOH Pellets</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">S320-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Disodium EDTA</td>
			<td style="width:359px;">Sigma Aldrich</td>
			<td style="width:208px;">E5124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium Hyprchlorite</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">SS290-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Triton (10%)</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">BP151-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium Nitroprusside</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">S350-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Ammonium Salts</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">A637-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Phenoxide</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td>AC388611000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Eisenia Fetida</td>
			<td>The Worm Factory</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Spade</td>
			<td style="width:359px;">Retail Store</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Bucket</td>
			<td style="width:359px;">Retail Store</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Potting Soil</td>
			<td style="width:359px;">Retail Store</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Avoidance Wheel</td>
			<td style="width:359px;">Environment Canada</td>
			<td style="width:208px;"></td>
			<td>Constructed by a modified design from Environment Canada&#8217;s Acute Avoidance Test.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Alumminum Foil</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">01-213-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Petri Dishes</td>
			<td style="width:359px;">Fisher Scientific</td>
			<td style="width:208px;">08-757-11</td>
			<td>8.5 cm in diameter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Pumpkin Seeds</td>
			<td style="width:359px;">Ontario Seed Company (OSC)</td>
			<td align="right" style="width:208px;">2055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Alfalpha Seeds</td>
			<td style="width:359px;">Ontario Seed Company (OSC)</td>
			<td align="right" style="width:208px;">6675</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Centrifuge Tubes (30mL)</td>
			<td style="width:359px;">Fisher Scientific&#160;</td>
			<td style="width:208px;">22-038-906</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:343px;">Beakers (50mL)</td>
			<td style="width:359px;">Fisher Scientific (Pyrex)</td>
			<td style="width:208px;">02-540G</td>
			<td>Oven dry at 105oC.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Beakers (30mL)</td>
			<td style="width:359px;">Fisher Scientific (Pyrex)</td>
			<td style="width:208px;">20-540C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Erlenmeyer Flasks (125mL)</td>
			<td style="width:359px;">Fisher Scientific (Pyrex)</td>
			<td style="width:208px;">S76106C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Volumetric Flask (100mL)</td>
			<td style="width:359px;">Fisher Scientific (Pyrex)</td>
			<td style="width:208px;">10-211C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Estuarine Sediment</td>
			<td style="width:359px;">National Insititute of Standards</td>
			<td style="width:208px;">1546A</td>
			<td>Standard Reference Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Bleach</td>
			<td style="width:359px;">Clorox Ultra (5-10% sodium hypochlorite)</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A summary of all results including a comparison to the criteria set by the IBI 13 can be found in Tables 1 (summary), 2 (New, High, Low, Third Feedstock and High-2 biochars) and 3 (Old biochar). All biochars and feedstocks used in 2012 and 2013 (Table 2) were well within the criterion set by the IBI and there were little differences among biochars. Old biochar (Table 3), the first biochar submitted for testing, was made from u...

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

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 ...

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35.0K Views.  Royal Military College of Canada. 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.

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

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Growth of Medicago truncatula plants in symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti in individual, sterile microcosms made from standard laboratory plates permits frequent examination of root systems and nodules without compromising sterility. Plants can be maintained in these growth chambers for up to 9 weeks.

Rhizobial  bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host  legume plants.  One  of the most well-developed model systems ...

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

A Bending Test for Determining the Atterberg Plastic Limit in Soils

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a considerable subjective judgment from the operator that carries out the test is involved during its performance, which may affect the final result significantly. Different alternative methods have been put forward, but they cannot compete with the standard rolling test in speed, simplicity and cost.
In an earlier study by the authors, a simple method with a simple device to determine the PL was presented (the &#34;thread bending test&#34; or simply &#34;bending test&#34;); this method allowed the PL to be obtained with minimal operator interference. In the present paper a version of the original bending test is shown. The experimental basis is the same as the original bending test: soil threads which are 3 mm in diameter and 52 mm long are bent until they start to crack, so that both the bending produced and its related moisture content are determined. However, this new version enables the calculation of PL from an equation, so it is not necessary to plot any curve or straight line to obtain this parameter and, in fact, the PL can be achieved with only one experimental point (but two experimental points are recommended).
The PL results obtained with this new version are very similar to those obtained through the original bending test and the standard rolling test by a highly experienced operator. Only in particular cases of high plasticity cohesive soils, there is a greater difference in the result. Despite this, the bending test works very well for all types of soil, both cohesive and very low plasticity soils, where the latter are the most difficult to test via the standard thread rolling method.

The traditional standardized test for determining the plastic limit in soils is performed by hand, and the result varies depending on the operator. An alternative method based on bending measurements is presented in this study. This allows the plastic limit to be obtained with a clear and objective criterion.

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

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 ...

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, ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

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 ...

Thermal Scanning Conductometry (TSC) as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to follow the dynamically changing state of the ionogels, and to deliver more information and details about the subtle change of conductive properties with an increase or decrease in the temperature. Moreover, the method allows the performance of long term (i.e. days, weeks) measurements at a constant temperature to investigate the stability and durability of the system and the aging effects. The main advantage of the TSC method over classical conductometry is the ability to perform measurements during the gelation process, which was impossible with the classical method due to temperature stabilization, which usually takes a long time before the individual measurement. It is a well-known fact that to obtain the physical gel phase, the cooling stage must be fast; moreover, depending on the cooling rate, different microstructures can be achieved. The TSC method can be performed with any cooling/heating rate that can be assured by the external temperature system. In our case, we can achieve linear temperature change rates between 0.1 and approximately 10 &#176;C/min. The thermal scanning conductometry is designed to work in cycles, continuously changing between heating and cooling stages. Such an approach allows study of the reproducibility of the thermally reversible gel-sol phase transition. Moreover, it allows the performance of different experimental protocols on the same sample, which can be refreshed to initial state (if necessary) without removal from the measuring cell. Therefore, the measurements can be performed faster, in a more efficient way, and with much higher reproducibility and accuracy. Additionally, the TSC method can be also used as a tool to manufacture the ionogels with targeted properties, like microstructure, with an instant characterization of conductive properties.

Thermal Scanning Conductometry TSC as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

The kinetics of the cooling process defines the properties of ionic gels based on low molecular weight gelators. This manuscript describes the usage of thermal scanning conductometry (TSC), which obtains full control over the gelation process, along with in situ measurements of the samples' temperature and conductivity.

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to ...

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.

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

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Chapters in this video

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

A Bending Test for Determining the Atterberg Plastic Limit in Soils

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

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

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

Techniques for Investigating the Anatomy of the Ant Visual System

Thermal Scanning Conductometry (TSC) as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

A Precise and Autonomous System for the Detection of Insect Emergence Patterns