We would like to thank comments from two anonymous reviewers as well members of the Poulain Lab for insightful discussion on the development of the anaerobic biosensor. An Early Researcher Award from the Province of Ontario and a Discovery Grant and an accelerator supplement from the Natural Sciences and Engineering Research Council of Canada to A.J.P. funded this study.

The authors have nothing to disclose.

Microbial biosensors are useful tools to identify mechanisms by which metals are interacting with microbes. Here, we describe a method that can anaerobically quantify HgII and CdII bioavailability to a Gram-negative host cell (E. coli) and give a quantifiable result within a few hours. One of the major strengths of this protocol is that it allows extensive control of metal speciation in the exposure medium by avoiding strong binding ligands or components that may lead to metal precipitation. Metal speciation has been modelled and tested in this exposure medium using PHREEQC17, however other metal speciation software may be deployed. In the case of no added ligands, Hg speciation is expected to be present as 97% Hg(OH)2 and 3% Hg(NH3)22+, while Cd speciation will be present as 59% Cd-&#946;-Glycerophosphate, 25% Cd2+, and 16% CdSO4. Using a simple thermodynamic modelling software, the user can design exposure media and test for the bioavailability of the metal of interest. In addition, the biosensor host cell (E. coli NEB5&#945;) is viable over a wide range of pH (5-8.5) and NaCl concentration (0-0.55 M)17.
Hg methylation is an anaerobic process, and the protocol outlined in this study does not have a requirement for oxygen, allowing for more accurate description of anaerobic metabolism on metal bioavailability. This is important because the presence of oxygen alters gene expression profiles48,49&#160;and hence, potential transport pathways; therefore this method presents an advantage over currently exising aerobic alternatives. The biosensing construct presented here can potentially be used with other anaerobic hosts that may be more relevant for mercury methylation (e.g., Geobacter, Desulfovibrio), but maybe less tractable than E. coli. One current limitation of the approach presented here is that our limit of detection has not yet reached pM levels, contrary to existing aerobic systems4,19,37. It is however important to note that to achieve these low detection limits several steps need to be taken44: i) ligand addition is required to ensure that Hg remains in solution and does not adsorb onto the microbial cell wall (Hg will be irreversible bound to cell surface thiols preventing its bioavailability25,27; see the threshold response for Hg in Figure 3A), ii) modifications to cell density, or iii) modify the genetic construct to include transport proteins of the mer-operon (namely merT and merP), increasing Hg flux inside the cell50,51. These modifications would be beneficial in detecting low concentrations of Hg, but not necessarily ideal when assessing environmentally relevant situations. Whereas previous cadmium biosensors primarily exist as a &#34;proof of principle&#34;, they were designed in complex media that do not allow the investigator to assess the role of speciation on bioavailability41,42,43,44.
The biosensor is an incredibly useful tool in determining mechanisms in which metal species are bioavailable. Because the host organism is not a Hg-methylator, it may only be used to develop a model for how Hg may enter Gram-negative bacteria and not a definitive rationale for how Hg-methylators acquire Hg. Other methods exist for determining Hg bioavailability, such as methylation, as an outcome of uptake or the use of a mass balance approach10,15,20,45,46. That being said, the method presented here offers the advantage of quasi real time bioavailability data in viable cells. We do not recommend that this method be used to quantify total Hg or Cd levels in an environmental matrix. Despite the proposed use of biosensors to determine metal concentrations in the environment36, many more readily available standard methods are available such as ICP-MS, FAAS (for Cd analysis) or Cold vapor atomic absorption spectroscopy (for Hg analysis). The biosensor can however be used to determine if a given environmental matrix has the potential to enhance or hamper bioavailability; this is achieved by performing standard additions.
The pH of the exposure media may be altered to anywhere within the range of 5 and 8.5, provided the MOPS Free acid (buffer) is exchanged with an alternate free acid of a buffer with the appropriate pKa (please see list of appropriate buffers (Ferreira et al. (2015)47) and adjusted with KOH to the appropriate pH when making the exposure media. In addition, the method is not limited to Hg and Cd, but could be extended to other metals using other transcription regulators.
The exposure assay may be modified to explore the influence of other electron acceptors such as O2 and fumarate on Hg or Cd bioavailability. Minor modifications to the method to utilize both O2 and fumarate as electron acceptors are available upon request.
In summary we would like to emphasize the following points: I) It is imperative that the concentrations of Cd or Hg stocks are known in step 2, as these will be used to calibrate the biosensor. II) On the exposure day, the growth of cells must be stopped at an OD600 of 0.6 (&#177; 0.1) and that care is taken when resuspending the cell cultures, as the biosensor is calibrated to this cell density. III) Lastly, it is important that the exposure medium is made meticulously on the exposure day. To ensure the success of the protocol, multiple cultures should be grown simultaneously (to circumvent the possibility of growth failure) and the growth medium should be remade weekly (to circumvent the metastability of the media and possible contamination). It should also be noted that biological replicates (multiple cell cultures) express variability when it comes to signal production. Although the fluorescent responses may vary from culture to culture, the fluorescent trends in response to a given variable should remain the same throughout numerous biological replicates.

Mercury (Hg) is a global pollutant and its bioavailability to Hg methylating microbes is the first step towards its biomagnification through food webs and its possible neurotoxic effects in human and wildlife1. It is currently thought that microbial Hg methylation is an intracellular process that requires: i) the species of Hg to be bioavailable2,3,4,5,6,7 and ii) for the cell to be physiologically capable of methylating Hg8,9,10. Cadmium (Cd) bioaccumulates in organisms, but does not biomagnifies in foodwebs and is widely used in industrial and commercial processes that commonly cause acute exposures in people and the environment11. Although microbes play several key roles in the fate of Hg in the environment, most studies on Cd geochemistry and ecotoxicology focus on microbial eukaryotes12. Consumption of agricultural crops (e.g., rice) is the main source of direct exposure to cadmium; in this case, the bioavailability of Cd to microbes of the rhizosphere directly influences the amount plants can accumulate13.
Hg transport pathways are complex and possibly involve an active transport step14. When a transporter is involved, recent work suggested that HgII uses a ZnII or MnII transporter7,15,16,17. Whereas CdII is hypothesized to be accidentally transported into the cytosol through divalent metal transport pathways (particularly MnII or ZnII), mechanisms of CdII transport inside the cells remain speculative, and no Cd-specific transport pathway has been identified13,18. Regardless of the nature of the transporters involved, three mechanistic factors ultimately determine the ability of metals to enter a cell: i) the metal speciation in solution2,6,15,16,17,19,20,21,22,23, ii) the biophysiochemical properties of the cell membrane17,24,25,26,27,28,29,30,31, and iii) the ability for the metal to access a transport site7,32. Cd and Hg are unlikely to exists as free ions under microbial physiologically relevant conditions due to their high affinity for Dissolved Organic Matter (DOM), chelating contaminants (e.g., EDTA), or reduced sulfur moieties33,34,35 (CdII can exist as a free ion or form ion-pairs in the absence of these ligands). There is a lack of efficient methods in determining how these metal species are bioavailable under conditions relevant to their fate in the environment. For instance, Hg is methylated under anaerobic conditions14, and both cadmium and Hg are soft metals (or class B cations), requiring that their speciation be investigated under conditions that maintain the integrity of reduced sulfur groups.
Microbial biosensors are bacterial cells that emit a quantifiable signal in response to the intracellular concentrations of a metal, in this case Hg or Cd. As such, they are ideal tools to understand how metals enter a cell36, provided that exposure conditions are carefully controlled for. Hg biosensors typically contain gene fusions between the regulatory circuitry of the mer-operon (including genes encoding for the transcription regulator MerRas well as the operator and promoter regions of the operon), and reporting genes (e.g., lux, gfp, lac genes). When mercury is present in the cytoplasm, it will bind to MerR, resulting in transcription of the reporting genes and subsequent signal production19,37. Specific Cd biosensors are usually designed using the cadC, cadAC, zntA or zntR encoded transcription regulators38, but it is worth noting that MerR has a lower, yet quantifiable affinity to Cd5. Due to aerobic restriction of most commonly used luminescent or fluorescent reporter proteins, until recently microbial biosensors remained unable to offer insights into the biotransformation of metals under anoxic conditions. This makes anaerobic detection of metals bioavailability very difficult over a range of conditions relevant to their environmental fate, specifically in the presence of redox sensitive ligands (e.g., sulfide and thiols)4,5,39.
To alleviate the methodological hurdle of live imaging in the absence of oxygen, Drepper et al. (2007) have developed a flavin-based fluorescent protein (FbFp), based on light oxygen voltage domain of SB2 protein from P. putida. This protein family is able to fluoresce in the absence of oxygen40. Building on the work of Drepper et al., our lab designed an anaerobic biosensor allowing for the study of Hg bioavailability under oxic and anoxic conditions and over a wide range of salinity 17. In the current paper, we describe how to prepare and use the biosensor to test environmental variables&#39; influence on Hg or Cd bioavailability. Although we developed the biosensor for HgII, we chose to perform experiments with CdII as a means to draw the reader&#39;s attention to the fact that biosensors may also respond to multiple stressors that are likely to co-occur in environmental matrices; in this case CdII was investigated because it is known to bind to the transcriptional regulator MerR5. Here, we show representative calibration and functional linear ranges with respect to either metal. We also give an example when the biosensor&#39;s results are conclusive (MgII and MnII on Hg bioavailability) and inconclusive (ZnII on Hg bioavailability).

<table>
	<tbody>
		<tr>
			<td>7 ml standard vial, rounded interior</td>
			<td>Delta Scientific</td>
			<td>200-007-20</td>
			<td>34 recommended</td>
		</tr>
		<tr>
			<td>Vial tray, 21 mm openings</td>
			<td>Delta Scientific</td>
			<td>730-2001</td>
			<td>4 for (4 x 8 grid)</td>
		</tr>
		<tr>
			<td>24 mm Closure</td>
			<td>Delta Scientific</td>
			<td>600-024-01</td>
			<td>32 recommended</td>
		</tr>
		<tr>
			<td>LID CORNER NOTCH BLK STR CS/50</td>
			<td>Corning</td>
			<td>Corning 3931</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96- well clear-bottom nonbinding surface microplate</td>
			<td>Corning</td>
			<td>Corning 3651</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerobic Chamber (glovebox)</td>
			<td></td>
			<td></td>
			<td>The air in the anaerobic chamber should be (97 % N2 &#177; 2 % and 3 % H2 &#177; 2 %)</td>
		</tr>
		<tr>
			<td>Palladium catalyst</td>
			<td></td>
			<td></td>
			<td>Converts O2 to H2O in the anaerobic chamber. Not required but recommended.</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>450 (&#177;10) nM filter for the microplate reader</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 (&#177;10) nM filter for the microplate reader</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerobic culture vial (balch tubes) + rubber stoppers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>Modified for anaerobic culture tubes</td>
		</tr>
		<tr>
			<td>MA-3000 (mercury analyzer)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH probe</td>
			<td></td>
			<td></td>
			<td>Any pH probe will work</td>
		</tr>
		<tr>
			<td>50 mL conical sterile polypropylene centrifuge tubes.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m polyethersulfone syringe filter + syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile/clean glass bottles</td>
			<td></td>
			<td></td>
			<td>For growth media and standards</td>
		</tr>
		<tr>
			<td>Sterile/clean plastic or PTFE bottles</td>
			<td></td>
			<td></td>
			<td>For alkali solutions (NaOH/KOH)</td>
		</tr>
		<tr>
			<td>Reagents powders: Na2MoO4, Na2SeO4, H3BO3, NaOH, KOH, MnSO4, ZnSO4, CoCl2, NiCl2, ampicillin sodium salt, Difco M9 Minimal Salts, Glucose, MgSO4, Thiamine HCl, NaNO3, l-leucine, l-isoleucine valine, EDTA sodium salt, FeSO4, Sodium Beta-Gylcerophosphate, Mops free acid, (NH4)2SO4, Hg(NO3)2, CdCl2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Milli-Q water</td>
			<td></td>
			<td></td>
			<td>Autoclaved or filter sterilized is fine.</td>
		</tr>
		<tr>
			<td>Lysogeny Broth</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical grade H2SO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an anaerobic biosensor assay for the detection of mercury and cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Once the fluorescence peaks have been quantified according to step 5.3, the result of the fluorescence peaks can be graphed according to the variable concentration illustrating how that variable affects the relative bioavailability of either Hg or Cd. For example, the calibration curve of fluorescence over [HgII] from Figure 2 will yield the inducible data presented in Figure 3A. For HgII calibration, the curve will always contain 3 components for the Hg-inducible strain; a threshold response of about 1-2 nM HgII before fluorescence signal production is linearly proportional to [HgII], the linear range where 5 nM HgII will reliably always be in the center of that range, and a plateau where increasing [HgII] will no longer increase fluorescence signal. No change in signal production on the constitutive strain shows that toxicity from [HgII] does not affect signal production. For CdII in Figure 3B, there are always 2 components for the inducible strain; a linear range where 200-300 nM CdII will reliably always be in the center of the linear range and a plateau. A decrease in fluorescence signal with increasing [CdII] shows that higher Cd concentrations are toxic to the cells and can explain a decrease in the inducible fluorescence production after the plateau at 1000 nM CdII. Therefore, when testing Hg or Cd bioavailability with respect to an environmental variable, we suggest using 5 nM for HgII and 300 nM for CdII.
In some instances, signal production can be properly attributed to Hg or Cd bioavailability, but in other cases, signal production can be affected by variation in the physiological state of the biosensor cell host (e.g., the metal of interest or environmental conditions tested are toxic). In Figure 4A, 5 nM Hg bioavailability was tested over a gradient of Zn (0-10 &#181;M). In both Hg-inducible and constitutive strains, there is a similar decrease in signal with increasing Zn concentrations. Therefore, one cannot discriminate whether the signal results from lowered bioavailability or is a result of Zn toxicity. In Figure 4B and 4C, 5nM Hg bioavailability was tested over a gradient of MgII (0-10 mM) and MnII (0-10 &#181;M). Increasing MgII and MnII concentrations decreased the fluorescence signal of the inducible strain. On the other hand, the constitutive strain did not show a decrease in fluorescence with increasing MgII and MnII concentrations (MgII and MnII are beneficial for the cells in the production of the FbFp, as demonstrated by an increase in the fluorescence signal). This demonstrates that the cells are viable and the fluorescence decrease of the Hg-inducible strain results from a decrease in Hg bioavailability. This data emphasizes how important it is for all biosensor assays to also provide constitutive measurements of overall cell fitness.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58324/58324fig3.jpg" /> 
Figure 3: Linear ranges of the biosensor with Mercury and Cadmium. Maximum fluorescence measured as relative fluorescence units (RFU) &#177; 1 Standard Deviation emitted by E. coli NEB5&#945; harboring the pUC57merR-Pp (Hg-Inducible) and pUC19Balch-Pp (Constitutive) with the addition of A) HgII (0-15 nM) and B) CdII (0-1,000 nM) under anaerobic conditions. Fluorescence was the average of 3 technical replicates at 37 &#730;C. <a href="https://www.jove.com/files/ftp_upload/58324/58324fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58324/58324fig4.jpg" /> 
Figure 4: Example of an inconclusive result with Zinc and a conclusive result with Magnesium and Manganese. Maximum fluorescence measured as relative fluorescence units (RFU) &#177; 1 Standard Deviation emitted by E. coli NEB5&#945; harboring the pUC57merR-Pp (Hg-Inducible) and pUC19Balch-Pp (Constitutive) with the addition of A) ZnII (0-10 &#181;M), B) MgII (0-10 mM), and C) MnII (0-10 &#181;M) under anaerobic conditions. [HgII] was set to 5 nM for all treatments and fluorescence was the average of 3 technical replicates at 37 &#730;C. <a href="https://www.jove.com/files/ftp_upload/58324/58324fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium at JoVE.com

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

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10.1K Views.  University of Ottawa. Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Video: An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 &#176;C) and thermophilic (55 &#176;C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 &#176;C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 LCH4/kgVS&#160;(VS: volatile solids) and 121 L CH4/kgVS&#160;at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kgdry_biochar was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.

A novel Upflow Anaerobic Solid State (UASS) reactor was used for biogas production from fibrous feedstock. Digestate from UASS reactor was hydrothermally carbonized into HTC biochar in a pressurized batch reactor. The integration of the two bioenergy concepts was applied in this study to increase overall bioenergy production.

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal ...

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide. 
This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.

Microbial sulfate reduction is a process of great importance in environmental biotechnology. The success of the sulfidogenic reactors depends among other factors on the microbial composition of the sludge. Here, we present a protocol to develop sulfidogenic sludge from hydrothermal vents sediments in a UASB reactor for reductive dechlorination purposes.

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate ...

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate the content of kerosene in diesel/kerosene blends, a wide-spread activity to adulterate fuel. In solvents with low viscosity, the dyes rapidly deactivate via a so-called twisted intramolecular charge transfer state, efficiently quenching the fluorescence. Measurements of diesel/kerosene blends revealed a good linear correlation between the decrease in fluorescence and the increase of the fraction of the less viscous kerosene in diesel/kerosene blends. Immobilization of the hydroxy derivative 4-DNS-OH in cellulose paper yielded test strips that preserve the fluorescent indicator&#39;s behavior. Combination of the strips with a reader based on a smartphone and a controlling app allowed to create a simple field test. The method can reliably detect the presence of kerosene in diesel from 7 to 100%, outperforming present standard methods for diesel adulteration.

Here, we present a protocol to detect the adulteration of diesel with kerosene using test strips coated with a fluorescent viscosity probe together with a smartphone-based analysis system.

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

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.