The authors are grateful for the financial support provided by Instituto Polit&#233;cnico Nacional grants 20120110, 20130399 and 20140239 SIP and also by Instituto de Ciencia y Tecnolog&#237;a del Distrito Federal Mexico (PICS 08-79, ICYT-DF, 2009-2012). Thanks also to CONACYT &#8211; Mexico for the graduate scholarship (225806) awarded to Selene Montserrat Garc&#237;a-Solares and for the financial support provided by grant 82627.

<ol>
	<li>Lens, P., Esposito, M. V. G., Zandvoort, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+of+sulfate+reducing+bioreactors+in+environmental+biotechnology.">Perspectives of sulfate reducing bioreactors in environmental biotechnology.</a> ReViews Environmental Science and Biotechnology. 1 (4), 311-325 (2002).</li><li>Omil, F., Lens, P., Hulshoff, P., Lettinga, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+biomass+from+a+sulfidogenic,+volatile+fatty+acid-degrading+granular+sludge+reactor.">Characterization of biomass from a sulfidogenic, volatile fatty acid-degrading granular sludge reactor.</a> Enzyme and MicrobialTechnology. 20, 229-236 (1997).</li><li>Lopes, S. I. C., Wang, X., Capela, M. I., Lens, P. N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfate+reduction+during+the+acidification+of+sucrose+at+pH+5+under+thermophilic+(55+&#176;C)+conditions.II:+Effect+of+sulfide+and+COD/SO4-2+ratio.">Sulfate reduction during the acidification of sucrose at pH 5 under thermophilic (55 &#176;C) conditions.II: Effect of sulfide and COD/SO4-2 ratio.</a> Bioresource Technology. 101, 4278-4284 (2010).</li><li>Alfonso, P., Prol-Ledesma, R. M., Canet, C., Melgarejo, J. C., Fallick, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfur+isotope+geochemistry+of+the+submarine+hydrothermal+coastal+vents+of+Punta+Mita,+Mexico.">Sulfur isotope geochemistry of the submarine hydrothermal coastal vents of Punta Mita, Mexico.</a> Journal of Geochemical Exploration. 78-79, 301-304 (2003).</li><li>Valdemarsen, T., Kristensen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+dissolved+organic+monomers+and+short+chain+fatty+acids+in+sandy+marine+sediment+by+fermentation+and+sulfate+reduction.">Degradation of dissolved organic monomers and short chain fatty acids in sandy marine sediment by fermentation and sulfate reduction.</a> Geochimica et Cosmochimica Acta. 74, 1593-1605 (2010).</li><li>Quistad, S. D., Valentine, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+propane+oxidation+in+marine+hydrocarbon+seep+sediments.">Anaerobic propane oxidation in marine hydrocarbon seep sediments.</a> Geochimica et Cosmochimica Acta. 75, 2159-2169 (2011).</li><li>Futagami, T., Morono, Y., Terada, T., Kaksonen, A. H., Inagaki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dehalogenation+activities+and+distribution+of+reductive+dehalogenase+homologous+genes+in+marine+subsurface+sediments.">Dehalogenation activities and distribution of reductive dehalogenase homologous genes in marine subsurface sediments.</a> Applied and Environmental Microbiology. 75 (21), 6905-6909 (2009).</li><li>U.S. Environmental Protection Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=List+of+priority+pollutants.">List of priority pollutants.</a> Clean Water Methods. , (2014).</li><li>Ozdemir, C., Dursun, S., Karatas, M., Sen, N., Sahinkaya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+trichloroethylene+(TCE)+in+upFlow+anaerobic+sludge+blanket+reactors+(UASB).">Removal of trichloroethylene (TCE) in upFlow anaerobic sludge blanket reactors (UASB).</a> Biotechnology and Biotechnological Equipment. 21 (1), 107-112 (2007).</li><li>Zhang, Y., Wang, X., Hu, M., Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hydraulic+retention+time+(HRT)+on+the+biodegradation+of+trichloroethylene+wastewater+and+anaerobic+bacterial+community+in+the+UASB+reactor.">Effect of hydraulic retention time (HRT) on the biodegradation of trichloroethylene wastewater and anaerobic bacterial community in the UASB reactor.</a> Applied Microbiology and Biotechnology. 99, 1977-1987 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard Methods for the Examination of Water and Wastewater. , (1998).</li><li>Guerrero-Barajas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sulfate+reduction+and+trichloroethylene+(TCE)+biodegradation+in+a+UASB+reactor+operated+with+sludge+developed+from+hydrothermal+vents+sediments:+process+and+microbial+ecology.">Enhanced sulfate reduction and trichloroethylene (TCE) biodegradation in a UASB reactor operated with sludge developed from hydrothermal vents sediments: process and microbial ecology.</a> International Biodeterioration and Biodegradation. 94, 182-191 (2014).</li><li>Tr&#252;per, H. G., Schlegel, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulphur+metabolism+in+Thiorhodaceae+I.+Quantitative+measurements+on+growing+cells+of+Chromatium+okenii.">Sulphur metabolism in Thiorhodaceae I. Quantitative measurements on growing cells of Chromatium okenii.</a> Antoine van Leeuwenhoek. 30, 225-238 (1964).</li><li>Gallegos-Garc&#237;a, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biological processes of sulfate reduction in biofilms for metals precipitation [Ph D thesis]. , (2009).</li><li>Guerrero-Barajas, C., Garibay-Orijel, C., Rosas-Rocha, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfate+reduction+and+trichloroethylene+biodegradation+by+a+marine+microbial+community+from+hydrothermal+vents+sediments.">Sulfate reduction and trichloroethylene biodegradation by a marine microbial community from hydrothermal vents sediments.</a> International Biodeterioration and Biodegradation. 65, 116-123 (2011).</li></ol>

The authors Selene Montserrat Garc&#237;a-Solares, Claudio Garibay-Orijel and Claudia Guerrero Barajas submitted (as inventors) in 2012 the application to obtain a patent entitled: &#8220;Process for the anaerobic treatment of industrial wastewater with high content of sulfate and chlorinated compounds&#8221;. This submission has been approved to the stage of the formatting. The submission has Instituto Polit&#233;cnico Nacional as the rights owner.

Il existe plusieurs applications de sulfidogenesis dans la biotechnologie environnementale, l&#39;une des applications les plus utilisées du métabolisme des bactéries réductrices de sulfate dans des consortiums avec la fermentation des bactéries est en traitement des eaux usées. Réacteurs UASB sont parmi les principales approches conçus pour le traitement des eaux usées industrielles avec des concentrations élevées de sulfate. Dans ce travail, nous présentons un protocole pour obtenir des boues sulfurogènes...

Une des contributions les plus importantes à la biotechnologie environnementale a été la conception de bioréacteurs dans lequel la boue utilisée (inoculum) a été en mesure d&#39;effectuer dans des conditions de sulfate de réduction. Sulfate de réduction (SR) permet le traitement de flux d&#39;eaux usées qui contiennent des concentrations élevées de sulfate en plus de l&#39;élimination simultanée de la DCO, des métaux lourds et des polluants organiques, ce qui rend une caractéristique souhaitable SR de la boue 1. Quelques exemples d&#39;effluents contaminés avec du sulfate proviennent de tannerie, papier, des produits pharmaceutiques et de fabrication de produits chimiques industries 1. Cependant, la plupart de la littérature se réfère à sulfurogènes boues lorsque la boue granulaire méthanogène a été adapté pour sulfidogenesis 2. Cette adaptation est souvent atteint par la manipulation de la COD / SO 4 2- rapport dans le bioréacteur et ajout de produits chimiques pour inhiber les bactéries méthanogènes dans le 2,3 de boues. En plus de la longue période que may exiger la formation des granules sulfurogènes, la concurrence entre les méthanogènes et réducteurs de sulfate et de la tolérance de la boue à de fortes concentrations de sulfure sont quelques-uns des principaux problèmes qui pourraient survenir si la boue sulfurogènes utilisé dans le bioréacteur est obtenu à partir de l&#39;adaptation de principalement boues méthanogène en sulfate des conditions réductrices. Dans ce travail, nous décrivons la procédure pour obtenir une boue principalement sulfurogènes des évents hydrothermaux sédiments (Punta Mita, Nayarit, Mexique) dans un réacteur anaérobie à lit de boues à flux ascendant (UASB), puis nous évaluons son sulfate réduction de l&#39;activité au fil du temps et menons une expérience d&#39;évaluer son application sur la déchloration réductrice. L&#39;emplacement des sédiments a été choisi car il a été rapporté que, dans ce site il ya formation de sulfures en raison de l&#39;activité de sulfate réduire manifestée par la communauté microbienne qui habitent cet endroit particulier 4. Il ya Severavantages al dans l&#39;obtention de ces boues sulfurogènes des sédiments sur l&#39;adaptation de boue granulaire méthanogène à sulfidogenesis. Certains de ces avantages sont les suivants: (1) il est inutile de former des granulés pour le bioréacteur de fonctionner, (2) des boues tolère des concentrations relativement élevées de sulfure par rapport aux autres UASB qui fonctionnent avec des boues méthanogène adapté, et (3) il est pas de compétition pour le substrat avec de l&#39;acétate méthanogènes même si on utilise dans le mélange des acides gras volatils qui est inclus dans le milieu de culture pour favoriser la formation de la boue. Cette procédure a été suivie de promouvoir sulfidogenesis parce sédiments marins sont une piscine naturelle d&#39;une grande variété de micro-organismes tels que le sulfate de réduire les bactéries, la fermentation des bactéries et déshalogénante bactéries pour ne citer que quelques-uns de 5,6. Le type de consortium développé à partir de sédiments marins en utilisant ce protocole peut présenter l&#39;efficacité dans la réduction de sulfate et donc grand s<html> ulfate réduction de l&#39;activité au fil du temps et une plus grande tolérance au sulfure à des concentrations supérieures à la signalé comme toxique pour les bactéries méthanogènes et sulfatoréductrices bactéries. D&#39;autre part, il est probable que la capacité de déshalogénation est également représenté dans les sédiments en suivant le protocole proposé ici, mais il peut dépendre de la communauté microbienne d&#39;origine. Cette hypothèse est fait sur ​​la base du fait que la déchloration réductrice peut se produire soit par la respiration ou cométabolisme, deux conditions qui peuvent être promus dans la communauté microbienne marine 7. La culture des sédiments pour obtenir la boue a été réalisée en utilisant un mélange d&#39;acétate, propionate et butyrate en tant que substrat, parce que ces acides gras volatils sont utilisés par plusieurs souches de bactéries sulfato-réductrices. Ces acides sont également le type de composés de carbone fréquemment trouvés dans les sédiments marins, selon plusieurs rapports de la littérature sur la matière carbonée dans les sédiments de la mer 5,6. contenu &quot;&gt; Enfin, certains des composés les plus toxiques que l&#39;on retrouve dans les corps de l&#39;eau souterraine et d&#39;autres l&#39;eau partout dans le monde sont les solvants chlorés tels que le trichloroéthylène (TCE) et de perchloroéthylène (PCE). Ces composés sont toxiques non seulement à l&#39;être humain, mais également des microorganismes, en particulier TCE, qui est toujours considéré comme un polluant prioritaire par l&#39;Environmental Protection Agency des États-Unis 8. Dans ce travail, nous avons proposé une expérience dans laquelle la boue sulfurogènes est testé sur sa capacité de réduire le TCE à des concentrations qui sont dans la fourchette indiquée pour les composés chlorés biodégradation dans des conditions méthanogènes 9,10. Il est à noter que la plupart des recherches sur la biodégradation des composés chlorés a été menée dans des conditions méthanogènes 9,10. Nous considérons que l&#39;expérience avec TCE proposé dans ce protocole est un bon exemple des applications potentielles de la boue. L&#39;objectif de cette expérience était de evaloriser la tolérance de la boue au TCE et l&#39;effet de TCE sur la réduction de l&#39;activité du sulfate. Tenant compte du fait que la plupart des recherches sur la biodégradation des composés chlorés est effectuée dans des conditions méthanogènes, ce protocole suggère la formation d&#39;une boue peut être utilisé pour simultanément: (1) retirer le sulfate, (2) enlever la DCO et (3) supprimer des composés chlorés. Une autre étape pourrait consister à évaluer la boue sur l&#39;élimination simultanée de TCE et de métaux lourds (en plus de sulfate et de la DCO), deux conditions qui ne peuvent pas être évalués dans des conditions méthanogènes.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>trichloroethylene&#160;</td>
			<td>sigma Aldrich</td>
			<td align="right">251402</td>
			<td></td>
		</tr>
		<tr>
			<td>cis- 1,2 dichlorotehylene</td>
			<td>sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>trans-1,2 dichloroethylene</td>
			<td>sigma Aldrich</td>
			<td>D-62209</td>
			<td></td>
		</tr>
		<tr>
			<td>vinyl chloride scotty standard</td>
			<td>supelco</td>
			<td></td>
			<td>1000 ppm v/v in nitrogen</td>
		</tr>
		<tr>
			<td>ethene scotty standard</td>
			<td>supelco</td>
			<td></td>
			<td>99% purity</td>
		</tr>
		<tr>
			<td>pump</td>
			<td>Masterflex</td>
			<td></td>
			<td>Model 7553-75</td>
		</tr>
		<tr>
			<td>spectrophotometer</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gas tight syringes&#160;</td>
			<td>any</td>
			<td></td>
			<td>100 and 200 microliters</td>
		</tr>
		<tr>
			<td>UASB glass reactor</td>
			<td>any</td>
			<td></td>
			<td>under design</td>
		</tr>
		<tr>
			<td>gas chromatograph&#160;</td>
			<td>any</td>
			<td></td>
			<td>FID detector</td>
		</tr>
		<tr>
			<td>capillary column SPB-624</td>
			<td>supelco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>viton tubing</td>
			<td>Masterflex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>basal medium reagents</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>trace metals reagents</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vitamins solution reagents</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfate</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>volatile fatty acids</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>COD determination kit</td>
			<td>HACH</td>
			<td></td>
			<td>range 0-15000 mg/L</td>
		</tr>
		<tr>
			<td>TOPO-TA cloning kit pCR&#174;4.0&#160;</td>
			<td>Invitrogen, US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S.N.A.P. TM Miniprep Kit&#160;</td>
			<td>Invitrogen, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pure link TM Quick Plasmid Miniprep kit</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Un comportement typique de la réduction de sulfate dans le bioréacteur est représenté sur la Figure 5. Il est important de noter que pendant les premières semaines de la réduction de sulfate de fonctionnement sera lente. Cependant lente, la consommation de plus de 90% de sulfate au fil du temps indique que l&#39;inoculum développe une communauté microbienne capable de réduire le sulfate et donc, enrichi en sulfate bactéries réductrices. Les différentes périodes de la figure indiquent que la...

Watch this Scientific Journal Video about Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor at JoVE.com

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

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.

Développement de sulfurogènes boues de sédiments marins et la réduction de trichloroéthylène dans une couverture Réacteur à refoulement de boue anaérobie

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

development-sulfidogenic-sludge-from-marine-sediments

Research

JoVE Journal

Environment

9.6K vues.  Instituto Politécnico Nacional. 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.

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

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Un poisson-alimentation Laboratoire d&#39;essais biologiques pour évaluer l&#39;activité antiprédatrices des métabolites secondaires dans les tissus d&#39;organismes marins

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Recherche

Environnement

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VACUSIP, une méthode Inex améliorée pour<em&gt; In Situ</em&gt; Mesure des particules et composés dissous transformés par suspensivores actifs

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na2CO3) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca2+). From the six extraction methods used, only the Na2CO3 extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca2+. The Ca2+-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.

The protocol provides a methodology to solubilize aerobic granular sludge in order to extract alginate-like extracellular polymers (ALE).

Extraction des substances Structural extracellulaires polymériques de Aerobic Granular Sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

Un système d&#39;exposition accréditives pour l&#39;évaluation de suspension Sédiments effets sur la vie aquatique

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

Préparatifs de Pyrite authigènes de méthane-sédiments In Situ analyse isotopique du soufre à l’aide de SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

Développement de nouvelles méthodes de quantification de la densité des poissons à l’aide d’outils de stéréo-vidéo sous-marine

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

Un appareil ultra propre multicouche pour la collecte de plancton marin taille fractionné et particules en suspension

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

In Situ Techniques d’hybridation pour échantillons de corail adultes paraffine

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

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.

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.

Un dosage anaérobie biocapteurs pour la détection du mercure et du Cadmium

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

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

Mesurer la forme et la taille des particules de boues activées immobilisées dans la gélose avec un Pipeline de logiciels Open Source

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...