This work was supported by ERA-NET Cofund MarTERA: &#34;FLAshMoB: Functional Amyloid Chimera for Marine Biosensing&#34;, PRIN 2017-PANACEA CUP:E69E19000530001 and by GoodbyWaste: ObtainGOOD products-exploit BY-products-reduce WASTE, MIUR 2017-JTNK78.006, Italy. We thank Dr. Monica Piochi and Dr. Angela Mormone (Istituto Nazionale di Geofisica e Vulcanologia, Sezione di Napoli Osservatorio Vesuviano, Italy) for the identification and characterization of geothermal site.

1. Sampling of microorganisms from geothermal sites
<ol>
	<li>Choose the site for sampling using as criterion places with desired temperature and pH. Measure the physical parameters through a digital thermocouple probe, inserting it into the selected pools or muds.</li>
	<li>Collect 20 g of soils samples (in this case, from mud in the hydrothermal site of Pisciarelli Solfatara), picking them up with a sterilized spoon. Take at least two samples for each site chosen.</li>
	<li>Put the samples in 50 mL sterile polypropylene tubes and immediately close.</li>
	<li>Measure pH and temperature with a digital thermocouple probe by directly inserting it into the sampling site. After use, rinse the probe carefully with deionized water.</li>
</ol>
2. Isolation of heavy metal resistant microorganisms
NOTE: Perform steps 2.1-2.7 under a sterile biological hood.
<ol>
	<li>Inoculate 2 g of each collected sample into 50 mL of freshly prepared Luria-Bertani medium (LB), in which the pH has been adjusted to 4 or 7 through the addition of HCl or NaOH.</li>
	<li>Incubate the samples at the same temperature of the sampling site and at &#177;5 &#176;C (55 &#176;C and 60 &#176;C for Pisciarelli samples) in a temperature-controlled orbital shaker for 24 h with a shaking rate of 180 rpm.</li>
	<li>Plate 200 &#181;L of the grown samples on LB agar (pH 4 or pH 7) and incubate in static condition for 48 h at 55 &#176;C or 60 &#176;C.</li>
	<li>Isolate single colonies and repeat streak-plating cycles (steps 2.3 and 2.4) at least three times.</li>
	<li>To prepare -80 &#176;C frozen cell stocks, grow the cultures overnight (ON) and add to the grown cells 20% glycerol (in a final volume of 1 mL); use a mixture of acetone and dry ice for fast freezing.</li>
	<li>To prepare an inoculum from a glycerol stock, inoculate 50 &#956;L in 50 mL of LB (pH 4 or pH 6) and incubate at 55 &#176;C or 60 &#176;C in the orbital shaker at 180 rpm ON.</li>
	<li>To obtain a growth profile, dilute a preculture (obtained from step 2.6) to 0.1 OD600 nm in 10 mL of LB (pH 4 or pH 6), grow the cells at 55 &#176;C or 60 &#176;C for 16 h in the orbital shaker, and measure the OD600 nm at 30 min intervals.</li>
	<li>Construct a growth curve from the data obtained in step 2.7 with time (min) on X-axis and OD600 nm on Y-axis.</li>
	<li>Realize the same growth curve described in steps 2.7 and 2.8 but varying the pH (&#177; 1 unit) of the culture medium (e.g., pH 3 and 5 for samples grown at pH 4) to determine the optimal pH for laboratory conditions.</li>
</ol>
3. Identification of microbial isolates
<ol>
	<li>Preparation of genomic DNA
	<ol>
		<li>Inoculate the isolate streaked from the glycerol stock in 50 mL of LB medium (pH 4 or pH 6) and grow in an orbital shaker at 55 &#176;C or 60 &#176;C at 180 rpm ON.</li>
		<li>Harvest the ON culture by centrifugation for 10 min at 5000 x g. Discard the supernatant.</li>
		<li>Prepare 10 mL of bacteria lysis buffer composed by: 20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1.2% Triton X-100, and lysozyme (20 mg/mL) immediately before use.</li>
		<li>Resuspend the pellet in 180 &#181;L of bacteria lysis buffer. Incubate for 30 min at 37 &#176;C.</li>
		<li>Follow the guidelines indicated by a Genomic DNA Purification kit (Table of Materials) to extract genomic DNA.</li>
		<li>Quantify the extracted genomic DNA and its purity by UV-Vis measurement. For purity determine ratios-OD 260/280 nm and OD 260/230 nm.</li>
		<li>Assess the integrity of the genomic DNA by loading 200 ng of each sample on a 0.8% agarose gel and comparing the size distribution to a high-weight molecular marker.</li>
		<li>Commission to an external service the 16S rRNA fragment preparation, sequencing, and comparative analysis of the sequence obtained (1000 bp) with those present in the nucleotide database of the US National Center for Biotechnology Information (NCBI)33.</li>
	</ol>
	</li>
	<li>To corroborate data of 16S rRNA sequencing, also perform automated ribotyping on the digested chromosomal DNA (external service, Table of Materials).</li>
	<li>In the case in which the specie identification cannot be determined only with ribotyping data, commission a MALDI-TOF MS analysis for fatty acid identification.</li>
	<li>To perform a phylogenetic analysis of the genus identified, analyze the 16S rRNA sequence of the isolate with BLASTn34. Sequences with identities from 99% to 97% must be used to build a multiple sequence alignment using CLUSTAL Omega35. Construct a neighbor-joining tree using the default option of ClustalW2 (Simple Phylogeny).</li>
</ol>
4. Heavy-metals and antibiotics susceptibility
<ol>
	<li>Inoculate the isolate from a glycerol stock (see step 2.5) and grow it in 200 mL of LB under the optimal pH and temperature conditions previously determined.</li>
	<li>Dilute each preculture at 0.1 OD600 nm in 5 mL of LB medium (at the appropriate pH) containing increasing concentrations of heavy metals. The concentrations vary from 0.01-120 mM for heavy metals [As(V), As(III), Cd(II), Co(III), Cr(VI), Cu(II), Hg(II), Ni(II), V(V)] or 0.5-1 mg/mL for the antibiotics [Ampicillin, Bacitracin, Chloramphenicol, Ciprofloxacin, Erythromycin, Kanamycin, Streptomycin, Tetracycline, and Vancomycin].</li>
	<li>Perform heavy metal and antibiotic treatments separately. Use a 50 mL polypropylene tube and grow the cells in a temperature-controlled orbital shaker with a shaking rate of 180 rpm at 55 &#176;C or 60&#176;C for 16 h for each condition/treatment.</li>
	<li>Calculate Minimum Inhibitory Concentration (MIC) either for antibiotics or heavy metals by identifying the concentration values in the tubes where microbial growth does not occur, i.e., determining the values that completely inhibit cell growth after 16 h.</li>
	<li>Check that the concentration is inhibitory and not lethal for the cells by plating 200 &#956;L of the culture grown at the value that is considered as MIC on LB-agar plates (at the appropriate pH and temperature) and verifying the presence of colonies after ON incubation. 
	NOTE: Since the culture on LB agar plate is viable at 4 &#176;C only for a few weeks, in order to preserve the isolates for a longer time, glycerol stocks were prepared and stored at -80 &#176;C. For MIC determination, at least three independent replicates using independent cultures were carried out. The standard deviation was calculated among triplicate experiments.</li>
</ol>

The authors declare that they have no conflicts of interest.

Hot springs contain an untapped diversity of microbiomes with equally diverse metabolic capacities12. The development of strategies for the isolation of microorganisms that can efficiently convert heavy metals into less toxic compounds10 represents a research area of growing interest worldwide. This paper aims to describe a streamlined approach for the screening and isolation of microbes with the ability to resist toxic chemicals. The method described can be easily modified...

The extreme environments on our planet are excellent sources of microorganisms capable of tolerating harsh conditions (i.e., temperature, pH, salinity, pressure, and heavy metals)1,2, being Iceland, Italy, USA, New Zealand, Japan, Central Africa and India, the best-recognized and studied volcanic areas3,4,5,6,7,8,9. Thermophiles have evolved in harsh environments in a range of temperatures from 45 &#176;C to 80 &#176;C10,11,12. Thermophilic microorganisms, either belonging to the archaeal or bacterial kingdoms, are a reservoir for the study of biodiversity, phylogenesis, and the production of exclusive biomolecules for industrial applications 13,14,15,16. Indeed, in the last decades, the continuous industrial demand in the global market has encouraged the exploitation of extremophiles and thermozymes for their diversified applications in several biotechnological fields 17,18,19.
Hot springs, where organisms live in consortia, are rich sources of biodiversity, thus representing an attractive habitat to study microbial ecology20,21. Moreover, these&#160;volcanic metal-rich areas are commonly colonized by microorganisms that have evolved tolerance systems to survive and adapt to the presence of heavy metals22,23 and are therefore actively involved in their biogeochemical cycles. Nowadays, heavy metals are considered priority pollutants for humans and the environment. The heavy-metal-resistant microorganisms are able to solubilize and precipitate metals by transforming them and remodeling their ecosystems24,25. The comprehension of the molecular mechanisms of heavy-metal resistance is a hot topic for the urgency to develop novel green approaches26,27,28. In this context, the discovery of new tolerant bacteria represents the starting point for developing new strategies for environmental bioremediation24,29. In accompanying the efforts to explore hydrothermal environments through microbiological procedures and increase knowledge on the role of the gene(s) underpinning heavy metal tolerance, a microbial screening was conducted in the hot-spring area of Campi Flegrei in Italy. This heavy metal-rich environment shows a powerful hydrothermal activity, fumarole, and boiling pools, variable in pH and temperature in dependence of seasonality, rainfall, and underground geological movements30. In this perspective, we describe an easy-to-apply and efficacious way to isolate bacteria resistant to heavy metals, for example, Geobacillus stearothermophilus GF1631 (named as isolate 1) and Alicyclobacillus mali FL1832&#160;(named as isolate 2) from Pisciarelli area of Campi Flegrei.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>A9393</td>
			<td></td>
		</tr>
		<tr>
			<td>Aura Mini</td>
			<td>bio air s.c.r.l.</td>
			<td></td>
			<td>Biological hood</td>
		</tr>
		<tr>
			<td>Bacitracin</td>
			<td>Sigma Aldrich</td>
			<td>B0125</td>
			<td></td>
		</tr>
		<tr>
			<td>Cadmium chloride</td>
			<td>Sigma Aldrich</td>
			<td>202908</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma Aldrich</td>
			<td>C0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>Sigma Aldrich</td>
			<td>17850</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt chloride</td>
			<td>Sigma Aldrich</td>
			<td>C8661</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper chloride</td>
			<td>Sigma Aldrich</td>
			<td>224332</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Sigma Aldrich</td>
			<td>E5389</td>
			<td></td>
		</tr>
		<tr>
			<td>Exernal Service</td>
			<td>DSMZ</td>
			<td></td>
			<td>Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH</td>
		</tr>
		<tr>
			<td>Genomic DNA Purification Kit</td>
			<td>Thermo Scientific</td>
			<td>#K0721</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin sulphate</td>
			<td>Sigma Aldrich</td>
			<td>60615</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQTM 4000 Benchtop Orbital Shaker</td>
			<td>Thermo Scientific</td>
			<td>SHKE4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mercury chloride</td>
			<td>Sigma Aldrich</td>
			<td>215465</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel chloride</td>
			<td>Sigma Aldrich</td>
			<td>654507</td>
			<td></td>
		</tr>
		<tr>
			<td>Orion Star A221 Portable pH Meter</td>
			<td>Thermo Scientific</td>
			<td>STARA2218</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium (meta) arsenite</td>
			<td>Sigma Aldrich</td>
			<td>S7400</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium arsenate dibasic heptahydrate</td>
			<td>Sigma Aldrich</td>
			<td>A6756</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma Aldrich</td>
			<td>87128</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone BioChemica</td>
			<td>Applichem Panreac</td>
			<td>A1553</td>
			<td></td>
		</tr>
		<tr>
			<td>Vancomycin</td>
			<td>Sigma Aldrich</td>
			<td>PHR1732</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract for molecular biology</td>
			<td>Applichem Panreac</td>
			<td>&#160;A3732</td>
			<td></td>
		</tr>
	</tbody>
</table>

bioprospecting of extremophilic microorganisms to address environmental pollution

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

Sampling site 
This protocol illustrates a method for the isolation of heavy metal-resistant bacteria from a hot spring. In this study, the Pisciarelli area, an acid-sulfidic geothermal environment, was used as a sampling site (Figure 1). This ecosystem is characterized by the flow of aggressive sulfurous fluids derived from volcanic activities. It has been demonstrated that the microbial communities in acid-sulfidic geothermal systems are subjected to ...

Watch this Scientific Journal Video about Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution at JoVE.com

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

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

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

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3.5K Views.  University of Naples Federico II. Het protocol beschrijft een gestroomlijnde aanpak voor het screenen en isoleren van zware metaalresistente microben uit geothermische bronnen. Deze aanpak vertegenwoordigt een onderzoeksgebied van groeiende interesse wereldwijd voor toepassing op het gebied van biosensing en bioremediatie. De daarin beschreven methode kan eenvoudig worden gewijzigd om microben te isoleren uit verschillende omgevingsbronnen zoals water, voedsel, bodem of sediment.De minimale remmende concentratie om res...

Video: Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

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

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

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

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

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may be treated with simple systems designed to promote sorption of contaminants to vegetation and soils and promote infiltration. Two example systems are described: a bioswale treatment system for urban stormwater treatment, and a vegetated drainage ditch for treating agriculture irrigation runoff. Both have similar attributes that reduce contaminant loading in runoff: vegetation that results in sorption of the contaminants to the soil and plant surfaces, and water infiltration. These systems may also include the integration of granulated activated carbon as a polishing step to remove residual contaminants. Implementation of these systems in agriculture and urban watersheds requires system monitoring to verify treatment efficacy. This includes chemical monitoring for specific contaminants responsible for toxicity. The current paper emphasizes monitoring of current use pesticides since these are responsible for surface water toxicity to aquatic invertebrates.

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

A Dual-Functional Electroactive Filter Towards Simultaneously Sb(III) Oxidation and Sequestration

We have designed a facile method to synthesize a dual-functional electrochemical filter consisting of two 1-D materials: titanate nanowires and carbon nanotubes. The hybrid titanate-CNT filter was prepared by a sonication coupled with a post-filtration route. Due to the synergistic effects of the increased number of exposed sorption sites, electrochemical reactivity, small pore size of the titanate-CNT network coupled with a flow-through design, simultaneous Sb(III) oxidation and sequestration can be readily achieved. Atomic fluorescence spectrometer technology demonstrated that the applied electrical field accelerates the Sb(III) conversion rate and the as-obtained Sb(V) were adsorbed effectively by the titanate nanowires due to their Sb specificity. This protocol provides a practical solution for the removal of highly toxic Sb(III) and other similar heavy metal ions.

A Dual-Functional Electroactive Filter Towards Simultaneously SbIII Oxidation and Sequestration

A protocol for the rational design of a dual-functional electroactive filter consisting of carbon nanotubes and titanate nanowires is reported and their environmental applications towards Sb(III) oxidation and sequestration is presented.

We have designed a facile method to synthesize a dual-functional electrochemical filter consisting of two 1-D materials: titanate nanowires and carbon ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues and organs. Ecotoxicological effects of microplastics on the development of bird embryos are not known. The bird egg is a complete development and nutrition system, and the entire embryo development occurs in the eggshell. Therefore, a direct record of bird embryo development under the stress of pollutants such as microplastics is highly limited by the opaque eggshell in traditional hatching. In this study, the effects of microplastics on quail embryo development were visually monitored by hatching without an eggshell. The main steps include the cleaning and disinfection of fertilized eggs, the incubation before exposure, the short-term incubation after exposure, and the sample extraction. The results show that compared with the control group, the wet weight and body length of the microplastics-exposed group displayed a statistical difference and the liver proportion of the whole exposed group significantly increased. Additionally, we evaluated external factors that affect the incubation: temperature, humidity, egg rotation angle, and other conditions. This experimental method provides valuable information on the ecotoxicology of microplastics and a novel way to study the adverse effects of pollutants on the development of embryos.

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues ...

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

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