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.

<img alt="Figure 1" src="/files/ftp_upload/52956/52956fig1highres.jpg" width="450" /> 
Figure 1. Scheme for the steps of the protocol. <a href="https://www.jove.com/files/ftp_upload/52956/52956fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Collect Marine Sediments for the Formation of the Sludge
<ol>
	<li>Identify an approachable subsea area either close to hydrothermal vents (due to the presence of sulfides, which may ...

<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.

There are several applications of sulfidogenesis in environmental biotechnology, one of the most used applications of the metabolism of sulfate reducing bacteria in consortia with fermenting bacteria is in wastewater treatment. UASB reactors are among the main engineered approaches to industrial wastewater treatment with high sulfate concentrations. In this work, we present a protocol to obtain sulfidogenic sludge from marine sediments in a UASB reactor. The critical steps within the protocol to obtain a sulfidogenic slu...

One of the most important contributions to environmental biotechnology was the design of bioreactors in which the sludge used (inoculum) was able to perform under sulfate reducing conditions. Sulfate reduction (SR) allows the treatment of wastewater streams that contain high concentrations of sulfate in addition to the simultaneous removal of COD, heavy metals and organic pollutants, a fact that makes SR a desirable characteristic of the sludge 1. Some examples of effluents contaminated with sulfate come from tannery, paper, pharmaceutical and chemical manufacturing industries 1. However, most of the literature refers to sulfidogenic sludge when methanogenic granular sludge has been adapted to sulfidogenesis 2. This adaptation is commonly attained by manipulating the COD/SO42- ratio in the bioreactor and adding chemicals to inhibit methanogens in the sludge 2,3. In addition to the long time that may require the formation of the sulfidogenic granules, the competition between methanogens and sulfate reducers and the tolerance of the sludge to high concentrations of sulfide are some of the main problems that may arise if the sulfidogenic sludge used in the bioreactor is obtained from the adaptation of predominantly methanogenic sludge to sulfate reducing conditions. In this work, we describe the procedure to obtain a predominantly sulfidogenic sludge from hydrothermal vents sediments (Punta Mita, Nayarit, Mexico) in an upflow anaerobic sludge blanket reactor (UASB), then we evaluate its sulfate reducing activity over time and conduct an experiment to evaluate its application on reductive dechlorination. The location of the sediments was chosen because it has been reported that in that site there is formation of sulfides due to the sulfate reducing activity exhibited by the microbial community inhabiting that particular place4.
There are several advantages in obtaining this sulfidogenic sludge from sediments over adapting methanogenic granular sludge to sulfidogenesis. Some of these advantages are: (1) it is not necessary to form granules for the bioreactor to operate, (2) the sludge tolerates relatively high concentrations of sulfide compared to others UASB that operate with adapted methanogenic sludge, and (3) there is no competition for substrate with methanogens even if acetate is used in the mixture of volatile fatty acids that is included in the culture medium to promote the formation of the sludge.
This procedure was followed to promote sulfidogenesis because marine sediments are a natural pool of a wide variety of microorganisms such as sulfate reducing bacteria, fermenting bacteria and dehalogenating bacteria just to mention a few 5,6. The type of consortium developed from marine sediments by using this protocol may exhibit efficiency in sulfate reduction and therefore, high sulfate reducing activity over time and higher tolerance to sulfide at concentrations higher than the reported as toxic to methanogens and sulfate reducing bacteria. On the other hand, it is likely that the dehalogenating capability is also shown in the sediments by following the protocol proposed here but it may depend on the original microbial community. This assumption is done based on the fact that reductive dechlorination can occur either by respiration or cometabolism, both conditions that may be promoted in the marine microbial community 7. The cultivation of the sediments to obtain the sludge was conducted by using a mixture of acetate, propionate and butyrate as substrate because these volatile fatty acids are used by several strains of sulfate reducing bacteria. These acids are also the type of carbon compounds frequently found in marine sediments, according to several reports in literature on carbonaceous material in sea sediments5,6.
Finally, some of the most toxic compounds that are found in groundwater and other water bodies around the world are the chlorinated solvents such as trichloroethylene (TCE) or perchloroethylene (PCE). These compounds are toxic not only to the human being but also to microorganisms, particularly TCE, which is still considered a priority pollutant by the Environmental Protection Agency in the US8. In this work we proposed an experiment in which the sulfidogenic sludge is tested on its capability of reducing TCE at concentrations that are in the range reported for chlorinated compounds biodegradation under methanogenic conditions9,10. It is worth mentioning that most of the research on biodegradation of chlorinated compounds has been conducted under methanogenic conditions 9,10. We consider that the experiment with TCE proposed in this protocol is a good example of the potential applications of the sludge. The objective of this experiment was to evaluate the tolerance of the sludge to the TCE and the TCE effect on the sulfate reducing activity. Taking into account that most of the research on biodegradation of chlorinated compounds is carried out under methanogenic conditions, this protocol suggests the formation of a sludge may be used to simultaneously: (1) remove sulfate, (2) remove COD and (3) remove chlorinated compounds. A further step could be to evaluate the sludge on the simultaneous removal of TCE and heavy metals (in addition to sulfate and COD), two conditions that cannot be evaluated under methanogenic conditions.

<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.

A typical behavior of the sulfate reduction in the bioreactor is shown in Figure 5. It is important to notice that during the first weeks of operation sulfate reduction will be slow. However slow, the consumption of over 90% of sulfate over time indicates that the inoculum is developing a microbial community capable of reducing sulfate and therefore, enriched in sulfate reducing bacteria. The different periods in the figure indicate that sulfate reduction was increasing its rate over time. At the beginni...

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.

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

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9.6K Views.  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.