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 overall goal of the following experiment is to develop a sulfide genic sludge from marine sediments in A-U-A-S-B reactor and to evaluate its performance on reduction of TRICHLOROETHYLENE or TCE. This is achieved by collecting marine sediments to obtain a pool of a great variety of microorganisms that are enriched in sulfate reducing bacteria when in an environment of the appropriate mineral, medium vitamins, and volatile fatty acids to serve as electron donors. As a second step, the marine sediments are used as the inoculum in A-U-A-S-B reactor, which is set and maintained under sulfate reducing conditions for several weeks until it reaches steady sulfate reducing activity.

Next, the consortium in the sulf Cytogenic, UASB reactor is evaluated for TCE reduction in order to assess the capability of the sludge to transform organic pollutants while sulf agenesis is still active. The results show that sulfide agenesis was established in the bioreactor and that the sludge was able to reduce TCE under sulfate reducing conditions based on the analysis of the microbial community, suggesting that the reduction of TCE was carried out in a consortium formed by sulfate reducing and fermentative bacteria. The main advantage of this procedure over existing methods like adaptation of methanogens lodges to Tosis is that this lodge obtained is tolerant to higher sulfate concentrations, and it does not present competition for substrate with methanogens.

Generally, individuals new to this method will struggle because they expect immediate formation of the slot. They may tend to add the nutrients and sulfide to the bioreactor, either less or more frequently than required. These approaches will only lead to a system imbalance.

It is convenient to carry out periodical analysis for COD sulfate and sulfate to know the course of the reaction and the right timing for the next fifth. We first had the idea for this method because we wanted a highly active sulf photogenic lodge to combine organic matter sulfate and pollutants removal, something that cannot be accomplished under metagenic conditions, particularly when some of the pollutants are mixed with heavy methods. We used a general method with good equipment for the identification of bacteria in the consortium.

The 16 S-R-R-N-A gene region was the target of analysis, and we found interesting general of bacteria To begin this procedure. Collect the marine samples as described in the text protocol. Once in the lab, take a large portion of the sediment sample and use an appropriate mesh to eliminate the large debris of carbonaceous material from the sediments.

After passing the sediment through the mesh, mix the selected portion to ensure that it is homogeneous. For the purpose of this work, use an upflow anaerobic sludge blanket glass reactor with a total working volume of three liters. 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.

Prepare a stock solution of basal medium containing phosphate chloride nitrogen, magnesium salts, trace metals and vitamins with a bicarbonate buffer solution, taking into account the working volume of the reactor. Next, prepare a stalk solution of volatile fatty acids composed of acetate, propionate, and butyrate. In a 2.5 to one to one chemical oxygen demand or COD proportion, the final COD concentration in the reactor must be 2.7 grams per liter.

Finally, prepare a stock solution of sodium sulfate in an appropriate concentration to deliver to the reactor a final concentration of 4, 000 milligrams per liter of the sulfate ion. Once the solutions have been prepared, place the sediments in the reactor mixed with a portion of the basal medium to make sure they reach the bottom of the reactor. Mix the rest of the basal medium and buffer solution with the volatile fatty acid solution and the sulfate solution.

Make sure that the solution of volatile fatty acids is poured into the liquid. Then add the combined solutions to the reactor. Set the connections and pipelines of the reactor to the recycling pump.

Then set the recycling flow rate at 60 milliliters per minute. Set the bioreactor in the temperature chamber to 34 degrees Celsius regularly. Check that the temperature variations are small.

Finally, set the connections to the gas displacement column. After one week of incubation, take a sample of five to seven milliliters of the liquid to conduct analysis for COD sulfate and sulfide content and pH. Following standard methods, analyze the sulfide in the liquid using the methylene blue method.

First place five milliliters of a zinc acetate solution in a 25 milliliter volumetric flask. And quickly add 200 microliters of the sample to the zinc acetate solution. Then add 2.5 milliliters of A DMP solution and 125 microliters of an iron three ammonium sulfate solution.

Complete the 25 milliliters of the volumetric flask with distilled water. Wait 30 minutes for the reaction to occur so that the blue color is stabilized. Once the reaction is complete, wait at least 15 minutes, but not more than 60 minutes.

To test the samples in the spectrophotometer, conduct the reading of the final blue solution in the spectrophotometer at a wavelength of 670 nanometers. Quantify sulfate as barium sulfate by using a turbo metric method. First place five milliliters of a conditioning solution in a 25 milliliter volumetric flask.

Then add one milliliter of the sample that has been previously centrifuged at 11, 320 times G.Complete the 25 milliliters of the volumetric flask with distilled water and add one gram of barium chloride. Mix the solution for one minute in a vortex. Wait for four minutes for the barium sulfate to form and read the sample in a spectrophotometer at a wavelength of 420 nanometers.

To prepare for the COD determination, centrifuge the sample thoroughly to remove the remaining sulfide that may interfere in the COD determination. Following centrifugation, add two milliliters of the 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 two milliliters of distilled water to another reaction vial and homogenize the mixture. Place the vials in the digestion reactor at 150 degrees Celsius for two hours. Then 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 nanometers. Next, obtain the gas volume from the gas displacement column. Once sulfate is consumed, supply fresh, medium, and new nutrients for each batch.

As done before when sulfate consumption is over 80%in less than 24 hours, 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 or HRT to 24 hours by adjusting the flow in the pump and maintain the sulfate concentration at four grams per liter and the COD at 10 grams per liter at any given day. Stop the reactor after one HRT cycle and supply fresh, medium, and new nutrients for each batch as done before, using a COD concentration of 10 grams per liter.

Once the bioreactor is fed, take five to seven milliliter samples of the liquid and perform analysis for COD sulfate, sulfide and pH every hour. Also, record the gas volume produced for the TCE test. Prepare a stock solution of TCE taking into account that the final concentration of this compound in the liquid phase of the bioreactor must be 300 micromolar.

Consider the partitioning of the compound to the headspace by using the Henry's law Dimensionless constant for TCE at 34 degrees Celsius. Next, prepares standard curves on the gas chromatograph for each of the compounds to be analyzed using the methods referenced in the text protocol at any given day. Stop the reactor after one HRT cycle and supply fresh, medium, and new nutrients for each batch as done before, using a COD concentration of 10 grams per liter.

Once the bioreactor is fed, add the TCE directly to the liquid in the bioreactor from the stock solution. The final TCE concentration in the liquid phase of the bioreactor must be 300 micromolar. Set the HRT to 12 hours at the end of one HRT cycle.

Take samples of the liquid and conduct analysis for COD sulfate and sulfide. Also take samples of the headspace and conduct analysis in the gas chromatograph. A typical behavior of the sulfate reduction in the bioreactor is shown here.

It is important to notice that during first weeks of operation sulfate reduction will be slow. The different periods indicate that sulfate reduction was increasing its rate over time until 4, 000 milligrams per liter of sulfate were consumed in less than 24 hours. Then the reactor was operating under continuous regime.

The development of the sludge is shown in the reactor representative results on sulfate reduction. Sulfide concentration, COD consumption and pH variations over time are shown here. These results were obtained in experiments conducted after the bioreactor was under continuous regime for several weeks.

For the experiment in which the sludge was tested on the capacity to reduce TCE, the results obtained are shown here. The sulfate reducing activity obtained was slightly lower than that obtained before the TCE edition. The gas chromatograph revealed that approximately 80%of the TCE was reduced to et Ethan sulfate, reducing bacteria, fermenting bacteria and de halogenating bacteria were identified in the sludge developed by using this protocol.

The genera of bacteria such as sulfa vibrio de sulfa, microbial des sulfide bacterium, clostridium deha backer, and SUL fossum have been related to sulfate reduction and biodegradation of chlorinated compounds Once mastered, this technique can be done in a similar way with different marine sediments if it is performed properly. The span of time for this lodge to develop may depend on the source of the sediments While attending this procedure. Remember that the most important thing to do is to periodically check the mass balance on the COD sulfate and sulfate.

The more active the lodge obtained, the higher the possibility to use it for the reduction of TCE and eventually for the degradation of any other toxic compound that may be degraded under sulfate reducing conditions. After watching this video, you should have a good understanding on how to develop Genics Lodge from marine sediments in a USB reactor and to evaluate its performance on TC reduction. You should understand how to inoculate the reactor, how to follow the reduction reaction over time, and how to conduct a test on TCE reduction Following this procedure.

Other methods like period sequencing of other gene analysis can be performed in order to answer an additional questions like how many bacteria we have per general, or how the consortium can degradate TCE Insul photogenic environment.