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 indicate a higher sulfate reducing activity) or to an area where debris of organic matter are detectable.</li>
	<li>For the purpose of this work, take approximately 3 or 4 kg of sediment and drain the water off the samples. Place the samples in dark plastic bags. No refrigeration is needed.</li>
	<li>Once in the lab, keep the bags with the samples in the refrigerator if they are not going to be used immediately. For the purpose of this work, samples can be in the refrigerator for weeks or months before using them.</li>
	<li>Take a large portion of the sediment sample (i.e., 1 or 2 kg) and use an appropriate mesh (0.2 cm) to eliminate from the sediments the large debris of carbonaceous material that may be found or some rocks that may be present. 
	Note: In this case a mesh of 0.20 cm diameter (0.0767 in) was used but it may be of a different size according to the size of the particles in the sample.
	<ol>
		<li>After passing the sediment through the mesh, mix the portion selected to promote that the portion is homogeneous.</li>
		<li>Take separated smaller samples (i.e., 2 to 3 g) to determine the volatile suspended solids (VSS) content by following the standard methods 11 . 
		Note: See Figure 2 for steps 1.2 to 1.4.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52956/52956fig2.jpg" /> 
Figure 2. Photographs of the sediment samples. (A) Sediment samples just after being taken. (B) Sediment sample after passing through the mesh. (C) Sample taken for weighing prior to volatile suspended solids (VSS) determination. The Petri dish does not need to be sterilized. <a href="https://www.jove.com/files/ftp_upload/52956/52956fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Bioreactor Set Up
<ol>
	<li>For the purpose of this work, use a UASB glass reactor with a total working volume of 3 L. Alternatively, use a 1 or 2 L volume glass reactor.</li>
	<li>Based on the VSS content of the sediments calculate the amount of sediment to be used as inoculum to obtain 5 g of VSS in 1 L.</li>
	<li>Take into account that if the amount of sediment after calculation is too large, then approximately 25% to 30% volume of the bioreactor should be occupied by the sediments instead.
	<ol>
		<li>Record the VSS content since it will change when the microbial community is enriched in the bioreactor. The VSS content is needed for the calculations of sulfate reducing activity in the bioreactor.</li>
	</ol>
	</li>
	<li>Ensure that the final concentration of the basal medium and buffer solution in the bioreactor is similar to the reported by Guerrero-Barajas et al. (2014)12.
	<ol>
		<li>Ensure that the final volumes of the sediments, basal medium, buffer solution and volatile fatty acids are equal to the final working volume of the reactor. The basal medium recipe12 contains the appropriate concentrations for the trace metals and vitamins solution.</li>
		<li>Prepare a stock solution of basal medium and buffer solution in an appropriate concentration for the working volume of the reactor used (i.e., 2, 3 or 4 fold more concentrated than the reported in step 2.4) to ensure that when it is diluted, it is at the concentration reported by Guerrero-Barajas et al. (2014)12). 
		Note: The stock solution for the basal medium is always necessary, however, the buffer solution is only needed at the start up. It is not necessary to add buffer solution after this time.</li>
		<li>Prepare a stock solution of volatile fatty acids: acetate, propionate and butyrate in a 2.5:1:1 COD proportion. Take into account for the calculations the sodium acetate included in the basal medium. The final COD concentration in the reactor must be 2.7 g/L. 
		Caution: Prepare this solution in a fume hood. Wear nitrile gloves and goggles for the preparation of this solution. Take into account the stoichiometry of the reactions of sulfate with the volatile fatty acids that is shown in Figure 3.</li>
		<li>Prepare a stock solution of sodium sulfate (Na2SO4) in an appropriate concentration to deliver to the reactor a final concentration of 4,000 mg/L of the sulfate ion (SO42-). Alternatively, include the amount of sulfate required in the basal medium instead of adding it from a stock solution as long as the final sulfate (SO42-) concentration is right.</li>
	</ol>
	</li>
	<li>Place the sediments in the reactor mixed with a portion of the basal medium to make sure they reach the bottom of the reactor.
	<ol>
		<li>Add the rest of the basal medium and buffer solution mixed with the volatile fatty acids solution and the sulfate solution. Make sure that the solution of volatile fatty acids is poured into the liquid. Note: Conduct this step in a fume hood.</li>
		<li>Set the connections and pipelines of the reactor to the recycling pump. Set the recycling flow rate at 60 ml/min. Set the bioreactor in the temperature chamber at 34 &#176;C. Regularly check that the temperature variations are small (i.e., 34 &#177; 1.7 &#176;C)</li>
		<li>Set the connections to the gas displacement column. 
		Note: See Figure 4 for steps 2.1 to 2.5.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/52956/52956fig3highres.jpg" width="600" /> 
Figure 3. Stoichiometry of sulfate reduction with VFA (acetate, propionate and butyrate). <a href="https://www.jove.com/files/ftp_upload/52956/52956fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52956/52956fig4highres.jpg" width="400" /> 
Figure 4. UASB reactor.&#160;(A) Initial time. (B) Continuous regime after 300 days of operation. <a href="https://www.jove.com/files/ftp_upload/52956/52956fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Operation of the Reactor to Promote Sulfidogenesis and Growth of the Microorganisms
Note: Allow for the inoculum to consume the volatile fatty acids and sulfate. For this purpose, wait for one week to carry out the first analysis for sulfate, sulfide and COD consumption.
<ol>
	<li>After one week of incubation take a sample of 5 to 7 ml of the liquid to conduct analysis for COD, sulfate and sulfide content and pH following standard methods 11, 13.
	<ol>
		<li>Analyze sulfide in the liquid spectrophotometrically (at a wavelength (&#955;) of 670 nm) by following the methylene blue method 13.
		<ol>
			<li>Place 5 ml of a zinc acetate solution (2% w/w) in a 25 ml volumetric flask, add quickly 200 &#181;l of the sample to the zinc acetate solution.</li>
			<li>Add 2.5 ml of an N,N-dimethyl-p-phenylenediamine oxalate (DMP) solution (0.2% w/w in 20% H2SO4) and 125 &#181;l of the iron (III) ammonium sulfate solution (10% w/w in 2% H2SO4) and complete with distilled water the 25 ml in the volumetric flask. Wait 30 min for the reaction to occur, time at which the blue color is stabilized. 13. 
			Note: Wait at least 15 min, but not more than 60 min to test the samples in the spectrophotometer. Conduct the reading of the blue final solution in the spectrophotometer.</li>
		</ol>
		</li>
		<li>Analyze sulfate according to standard methods 11. Here, quantify sulfate as barium sulfate by using a turbidimetric method.
		<ol>
			<li>Place 5 ml of a conditioning solution (hydrochloric acid HCl 1:1) in a volumetric flask of 25 ml, add 1 ml of the previously centrifuged sample (at 11,320 &#215; g), complete the 25 ml of the volumetric flask with distilled water and add 1 g of barium chloride.</li>
			<li>Mix the solution for 1 min in a vortex. Wait for 4 min for the barium sulfate to form and read the sample in the spectrophotometer at a wavelength (&#955;) of 420 nm 11.</li>
		</ol>
		</li>
		<li>Analyze COD according to standard methods 11. Alternatively, use a COD determination kit.
		<ol>
			<li>Prior to the COD determination, centrifuge the sample thoroughly (at 11,320 x g) to remove the remaining sulfide that may interfere in the determination. If necessary, centrifuge twice: the first time immediately after taking the sample and the second time wait 6 or 8 hr and then conduct the COD analysis.</li>
			<li>Add 2 ml of sample to a reaction vial of the COD determination kit, seal the vial and homogenize the mixture by gentle agitation. Prepare a blank by adding 2 ml of distilled water to another reaction vial and homogenize the mixture.</li>
			<li>Place the vials in the digestion reactor at 150 &#176;C for 2 hr. Remove the vials and let them cool down in the dark. Take the readings of the vials in the spectrophotometer at a wavelength of 620 nm.</li>
		</ol>
		</li>
		<li>Obtain the gas volume from the gas displacement column.</li>
	</ol>
	</li>
	<li>Wait for up to another 5 to 7 days until the sulfate is consumed. Sulfate and COD must be consumed in approximately 85% to 90% before a new fed batch is started.</li>
	<li>Once sulfate (and COD) are consumed, completely repeat step 2.4. Supply fresh medium and new nutrients for each batch.</li>
	<li>Repeat steps 3.1 and 3.2. At this point each batch should last between 7 and 10 days.</li>
	<li>When 3 to 4 batches have been completed, repeat step 2.4 but increase the COD concentration to 4 g/L.</li>
	<li>Repeat step 3.1 and step 3.2.
	<ol>
		<li>Repeat step 3.3 but increase the COD concentration to 6 g/L.</li>
		<li>Repeat 3.6 and 3.6.1 gradually increasing COD concentration until it is 10 g/L. 
		Note: Make the graph that presents the sulfate concentration (mg/L) versus time (d).</li>
	</ol>
	</li>
	<li>When sulfate consumption is over 80% in less than 24 hr and this occurs for more than one week, switch the operation of the reactor to continuous mode. For the continuous mode set the hydraulic retention time (HRT) at 24 hr and maintain the sulfate concentration at 4 g/L and the COD at 10 g/L. 
	Note: Over time the sulfate consumption should be faster.</li>
</ol>
4. Sulfate Reducing Activity Test
<ol>
	<li>Prior to this test make sure that the bioreactor under continuous regime presents less than 10% variation in the sulfate concentration remaining.</li>
	<li>At any given day, stop the reactor after one HRT cycle and conduct step 2.4. For step 2.4.3 use a COD concentration of 10 g/L.</li>
	<li>Once the bioreactor is fed, take 5 to 7 ml samples of the liquid and perform analysis for COD, sulfate, sulfide (step 3.1) and pH every hour. Record the gas volume produced.</li>
	<li>Calculate the sulfate reducing activity according to the literature 14. 
	&#160; 
	<img alt="Equation 1" src="/files/ftp_upload/52956/52956eq1.jpg" /></li>
</ol>
SRA = sulfate reducing activity (mg COD-H2S)/gVSS*d
m H2S = sulfide concentration expressed as mg COD-H2S
VSS = volatile suspended solids concentration
t = time (d or hr)
<ol start="5">
	<li>Make the corresponding graphs that show the percentage of sulfate consumption versus sulfide concentration over time in mg/L. Make the graphs that show percentage of COD consumption over time. Make the graphs that show pH variation over time.</li>
</ol>
5. Trichloroethylene (TCE) Reduction Test
<ol start="1">
	<li>Prior to this test make sure that the bioreactor is working under continuous regime and presents less than 10% variation in the sulfate concentration remaining. Do not start this test if sulfate reduction in the bioreactor is less than 90%.</li>
	<li>Prepare a stock solution of trichloroethylene (TCE) taking into account that the final concentration of this compound in the liquid phase of the bioreactor must be 300 &#956;M. Consider the partitioning of the compound to the headspace by using the Henry&#180;s Law dimensionless constant (H&#180;) for TCE at 34 &#176;C. H&#180;at 34 &#176;C for TCE is 0.4722. 
	<img alt="Equation 2" src="/files/ftp_upload/52956/52956eq2.jpg" /> 
	Caution: Prepare this solution in a fumehood and wear gloves and goggles.
	<ol>
		<li>For example, for a 5,000 &#956;M stock solution, calculate as followed: 
		<img alt="Equation 3" src="/files/ftp_upload/52956/52956eq3.jpg" /> 
		TCE gas phase concentration = (0.4722)*(5,000) = 2,139 &#956;M. Include this concentration in the preparation of the stock solution since this amount of TCE will be in the headspace. 
		Then in the liquid (water) of the stock solution, the actual TCE concentration will be: 5,000 + 2,139 = 7,139 &#956;M. TCE density = 1.43 g/ml. Convert the 7139 &#956;M to mg and then by using the density of TCE calculate the volume of TCE for the stock solution. 
		Note: The concentration of the TCE stock solution may be lower than 5,000 &#956;M, i.e., 3,000 or 1,000 &#956;M, this depends on how much volume of this solution may be delivered to the bioreactor according to its liquid phase volume.</li>
	</ol>
	</li>
	<li>Prepare standard curves in the gas chromatograph for TCE, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, vinyl chloride and ethene. Prepare the cis-1,2-dichloroethylene and trans-1,2-dichloroethylene standard curves from a stock solution of these compounds by following the same procedure described in 5.2 for the TCE stock solution. Prepare the standard curves for vinyl chloride and ethene by diluting the concentration of each gas from the standards (gas cylinders).
	<ol>
		<li>Prepare the standard curves of these compounds in a range of 20 to 300 &#956;M. Use the method reported by Guerrero-Barajas et al. (2011)15 for the analysis of these compounds in the gas chromatograph. 
		Caution: Prepare these standard solutions in a fumehood and wear gloves and goggles.</li>
	</ol>
	</li>
	<li>At any given day, stop the reactor after one HRT cycle and conduct step 2.4. For step 2.4.3 use a COD concentration of 10 g/L.</li>
	<li>Once the bioreactor is fed, add the TCE directly to the liquid in the bioreactor from the stock solution prepared in 5.2, the final TCE concentration in the liquid phase of the bioreactor must be 300 &#956;M. Set the HRT to 12 hr.
	<ol>
		<li>At the end of one HRT cycle take samples of the liquid (500 to 1,000 &#956;l) and conduct analysis for COD, sulfate and sulfide (steps 3.1.1, 3.1.2 and 3.1.3). Take samples of the headspace (100 to 250 &#956;l) and conduct analysis for TCE, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, vinyl chloride and ethene in the gas chromatograph.</li>
	</ol>
	</li>
	<li>Repeat step 2.4. For step 2.4.3 use a COD concentration of 10 g/L.</li>
	<li>Do not repeat any TCE reduction test until the bioreactor presents over 90% sulfate reduction and less of 10% variation in both, sulfate reduction and sulfate remaining in the bioreactor.</li>
	<li>Repeat 5.4, 5.5 and 5.6 two or three times more.</li>
	<li>Take sediment samples (0.5 g) to conduct identification of the microorganisms just after a TCE reduction test has finished. Do this after 2 or 3 TCE reduction tests.</li>
</ol>
6. Sulfate Reducing Activity Test after TCE Reduction Experiment
<ol start="1">
	<li>Repeat step 4 completely.</li>
</ol>
7. Identification of the Microorganisms
<ol>
	<li>Take samples of sludge of approximately 0.5 g each and conduct RNA total extraction according to standard method 12.</li>
	<li>Amplify the 16S rRNA gene with reverse transcription and conduct the polymerase chain reaction (RT-PCR) amplification one step 12.</li>
	<li>Design the primers to amplify or use as an initial approach the ones suggested in literature 11. Follow the amplification procedure suggested in literature 12.</li>
	<li>Construct the 16S rRNA libraries. PCR amplicons can be cloned by using a cloning-kit 11. Typically, 10 colonies from each plate (each colony representing one PCR product) can be cloned. Prepare the plasmid DNA for sequencing according to the procedure suggested in literature 12.</li>
	<li>Conduct the sequencing of fragments. Re-amplify approximately 1,400 bp of the PCR external products with the protocol for PCR amplification previously described (step 7.4) and clone according to the procedure suggested in literature 12. Isolate the recombinant plasmid from E. coli colonies as suggested in literature 12. Do conduct the partial procedure for sequencing with M13 universal primers 12.</li>
	<li>Conduct the sequences analysis. Align the nucleotide sequences by using the Clustal X and manually adjust in the text editor. Perform BLAST searches of the NCBI database. (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi) 12.</li>
	<li>Obtain the nucleotide sequence accession numbers. Deposit the nucleotide sequences of the clones identified in the EMBL nucleotide sequence database (Gen-Bank/EMBL/DDBJ) under the corresponding accession numbers (i.e., JQ713915eJQ713925 for sequences from amplicons) 12.</li>
</ol>

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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 ...

development-sulfidogenic-sludge-from-marine-sediments

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JoVE Journal

Environment

9.6K vistas.  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.

Video: 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.

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

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.

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).

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.

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.

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.

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.

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.

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.

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.

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