Name: bioprospecting of extremophilic microorganisms to address environmental pollution
Uploaded: 2021-12-30T16:00:00
Description: Watch this Scientific Journal Video about Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution at JoVE.com

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 s...

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.

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		<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>
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			<td>Bacitracin</td>
			<td>Sigma Aldrich</td>
			<td>B0125</td>
			<td></td>
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			<td>Cadmium chloride</td>
			<td>Sigma Aldrich</td>
			<td>202908</td>
			<td></td>
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			<td>Chloramphenicol</td>
			<td>Sigma Aldrich</td>
			<td>C0378</td>
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			<td>Ciprofloxacin</td>
			<td>Sigma Aldrich</td>
			<td>17850</td>
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			<td>Cobalt chloride</td>
			<td>Sigma Aldrich</td>
			<td>C8661</td>
			<td></td>
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			<td>Copper chloride</td>
			<td>Sigma Aldrich</td>
			<td>224332</td>
			<td></td>
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			<td>Erythromycin</td>
			<td>Sigma Aldrich</td>
			<td>E5389</td>
			<td></td>
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		<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>
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		<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>
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		<tr>
			<td>Mercury chloride</td>
			<td>Sigma Aldrich</td>
			<td>215465</td>
			<td></td>
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			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Nickel chloride</td>
			<td>Sigma Aldrich</td>
			<td>654507</td>
			<td></td>
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		<tr>
			<td>Orion Star A221 Portable pH Meter</td>
			<td>Thermo Scientific</td>
			<td>STARA2218</td>
			<td></td>
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		<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>
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		<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>
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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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