This work was co-funded by BRGM, a postdoctoral grant from the Conseil G&#233;n&#233;ral du Loiret and the Carnot Institute. We also gratefully acknowledge the financial support provided to the PIVOTS project by the R&#233;gion Centre - Val de Loire.

1. Experimental Preparation
<ol>
	<li>Acid-wash all materials (glass, polytetrafluoroethylene (PFTE)) in contact with samples (5 days in 20% nitric acid (HNO3) v/v) followed by 5 days in hydrochloric acid (HCl) 10% v/v). Rinse several times with ultra-pure water and dry under a laminar flow hood prior to use.</li>
	<li>Use polyethylene gloves (or similar) and a fume hood for all steps involving chemicals.</li>
</ol>
2. Prepare Hg and As Spiked Amorphous Iron Oxides
<ol>
	<li>Prepare approximately 20 g of ferryhydrate (Fe(OH)3): dissolve 50 g of FeCl3-6H2O in 500 mL of ultra-pure water (resistivity &#62;18 M&#937; cm-1) under agitation in a glass reactor with a stainless-steel impeller or magnetic stirrer. Initial pH is &#60;2.</li>
	<li>Manually add a solution of 10 M NaOH to precipitate ferryhydrate. 
	NOTE: Approximately 50 mL will be required to precipitate all the iron (oxy)hydroxides. Adjust the pH to 6 and maintain agitation for 1 h to stabilize.
	<ol>
		<li>For Hg-spiked (oxy)hydroxides: prepare 10 mL of HgNO3 at 10 g L-1 and add 350 &#181;L to the (oxy)hydroxide solution. 
		NOTE: This will yield a final Hg content in the wet (oxy)hydroxides of ~4 &#181;g g-1 (oxy)hydroxides.</li>
		<li>For As-spiked (oxy)hydroxides: prepare 100 mL of As2O325 at 10 g L-1 and add 70 mL to the iron-oxide solution. This will yield a final As(III) content of ~70 mg/g (oxy)hydroxides.</li>
	</ol>
	</li>
	<li>Leave under agitation with a stainless-steel impeller or magnetic stirrer for 3 h and then centrifuge for 20 min at 2,000 x g. Remove supernatant and re-suspend the (oxy)hydroxides in 500 mL of ultra-pure water. Repeat the centrifugation and rinsing steps twice. Recover the humid (oxy)hydroxides (solids have a moisture content of 85-90% wt.) and store at 4 &#176;C until use.</li>
	<li>Sterilize humid Hg or As-spiked iron oxides by gamma radiation, with a minimum absorbed radiation dose of 25 kGy.</li>
	<li>Control Hg and As (oxy)hydroxides contents
	<ol>
		<li>Determine Hg contents of the pellet26. 
		NOTE: We found 3.90 &#177; 0.08 &#181;g Hg g-1 solid. Thus, the total amount of mercury added to each column in the 18.3 g of iron oxides was 71.4 &#177; 1.51 &#181;g.</li>
		<li>Determine As contents in the pellet. Use hot acid mineralization (8 mL of 5 N HCL for 4 h at 50 &#176;C) and analyze by Atomic Adsorption Spectrometry (AAS). 
		NOTE: We found 70 mg As g-1 solid. Thus, the total amount of As added to the column in the 18.3 g of iron oxides was ~1.3 g.</li>
	</ol>
	</li>
</ol>
3. Prepare Silica Gel and Sand Matrix
NOTE: A loose silica gel matrix was used to stop the fine iron oxides migrating from the sand/iron oxide mixture under the water flow. The final gel matrix was 6% silica gel so as not to form a block but just to loosely aggregate the oxides.
<ol>
	<li>Prepare a 10% silica gel mixture by heating 4 g of silica gel in 40 mL of a solution of 7% KOH on a hot plate, stirring with a magnetic stir bar until dissolved.</li>
	<li>Add 60 mL of ultra-pure water then cool the solution to ~20 &#176;C. Quickly titrate with diluted phosphoric acid (20 %) to pH 7.5. Then quickly mix the liquid silica gel with 320 g of sterile sand and the previously added 18.3 g of Hg-spiked or As-spiked iron oxides before it solidifies.</li>
	<li>Break up the &#34;jellified&#34; mixture by mixing with a spatula and keep sterile before use in step 4.</li>
</ol>
4. Setup the Column
<ol>
	<li>Use glass columns with a water jacket cooling system (internal volume = 400 mL, height = 30 cm, diameter 3.5 cm) and five silica septa set regularly (every 5 cm) along the columns to enable you to sample all along the column.</li>
	<li>Cut PTFE tubing (PTFE int &#216; 3 mm) to ensure sufficient length at the column inlet and outlet. Connect the inflow to peristaltic tubing, which in turn is connected to the water/medium supply.</li>
	<li>Sterilize all materials (glass, tubing) by autoclaving (1 h at 110 &#176;C).</li>
	<li>Attach column vertically.</li>
	<li>Connect the water jacket to a water cooling system to maintain an average temperature of 20 &#176;C.</li>
	<li>Fill column from the top as follows: 
	- A layer of damp rock wool to avoid solid loss; 
	- 320 g of sterile sand (Fontainebleau sand, D50 = 209 &#181;m); 
	- 320 g of sterile sand mixed with 18.3 g hydrated amorphous iron oxides spiked either with Hg or As (see step 1) and fixed in a 6% silica gel matrix (see step 2).</li>
	<li>Attach column vertically and connect ascendant flow of continuously N2 bubbled ultrapure sterile water at low velocity (~2 mL h-1).</li>
	<li>Cover the column with aluminum foil to protect from the light.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56240/56240fig1v2.jpg" /> 
Figure 1: Sketch and photo of the column setup. <a href="//cloudfront.jove.com/files/ftp_upload/56240/56240fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

The experimental column setup proved to be a convenient laboratory device to study anaerobic biogeochemical processes in continuous conditions. Continuous column systems allow working in conditions closer to those of real aquifers than slurry batch systems or microcosms. Continuous systems can simulate the movement of groundwater through aquifer sediments.
The most critical step within the protocol is preparing the TE-iron (oxy)hydroxides and the mixture with silica gel and sand, which needs t...

Understanding and predicting trace element (TE) mobility and biogeochemistry in the environment is essential in order to monitor, develop, and apply appropriate management decisions for polluted sites. This especially applies in the case of toxic TEs such as arsenic (As) and mercury (Hg). The fate and speciation of these TEs in soil or aquifers are closely related to physico-chemical conditions, such as Eh and pH, but also to microbial activities that can play either a direct role on speciation or an indirect role on mobility.
Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). This affects As toxicity, since As(III) is the most toxic form of As, and mobility, since As(III) is more mobile than As(V), which can readily adsorb to iron (oxy)hydroxides or organic matter1,2. Likewise, bacteria are strongly involved in mercury cycling, either through its methylation, mainly by sulfate and iron reducing bacteria3,4, forming the neurotoxin monomethyl mercury (readily bioaccumulated in the food chain), or through its reduction to volatile elementary Hg (Hg&#176;)5.
Both As and Hg fates are also strongly linked to soil or aquifer composition, since compounds such as organic matter or iron (oxy)hydroxides can influence their sequestration and bioavailability. As(V) adsorbs well to iron (oxy)hydroxides6, whereas Hg has a very high affinity for organic matter (OM; mainly for thiol groups) but also for colloidal iron or manganese (oxy)hydroxides in OM depleted environments7,8,9,10,11.
Bacterial activities can then influence the fate of TEs adsorbed to (oxy)hydroxides or organic matter through the reduction of iron (oxy)hydroxides or the mineralization of organic matter. Direct iron reduction by bacteria is the dominant pathway for iron reduction in sulfur depleted zones12,13, Fe(III) being used as a terminal electron acceptor, whereas indirectly, Fe(III) can be reduced to Fe(II) by sulfide formed by a bacterial sulfate reduction14. Moreover, the presence of sulfate can also modify Hg and As speciation through the formation of complexes such as thio-arsenates15 with As or metacinnabar with Hg.
Thus, a better understanding of the impact of iron and sulfate cycling on the fate of TE, such as Hg and As, could help us to better manage contaminated sites and maintain soil and water quality. Data could also contribute to reinforcing existing metal-mobility models. Microbial Fe(III)-reduction16,17,18 can cause the desorption of TE. Theoretically, the indirect reduction of iron (oxy)hydroxides by sulfide produced by the microbial reduction of sulfate could also impact TE mobility. However, the extent and kinetics of these reactions are generally studied in batch homogenous systems or batch microcosms16,18,19,20. The drawback of batch experiments is the lack of dissociation of the occurring phenomena; indeed, activity is based on and limited by the resources present in the batch and only gives a final result of the shifts in speciation and adsorption. Using a column approach enables the renewal of inflowing media and the monitoring of the fate of TE over time and space. These conditions are more realistic when compared to an aquifer, where real phenomena are closely linked to continuous percolation conditions. Moreover, heterogeneous iron (oxy)hydroxide occurrence in aquifer sediments is common21,23, and the spatial changes in the mineralogical and chemical composition of the solid phases certainly drives microbial activities.
To elucidate the influence of these heterogeneities on geo-microbial phenomena and the fate of iron-associated TE, we developed a laboratory, a continuously-fed column representing a simplified model aquifer. The column is filled to create an iron-depleted zone at the column entrance and an iron-rich zone at the top. Regular sampling ports enable us to study each zone individually as well as interface-associated phenomena. An example of the application of this experimental device for the study of Hg fate and speciation is already available24. Here we give a detailed description of the experimental setup and a second example of its application focused on the behavior of As in contaminated aquifers.

<table><tbody><tr><td>Glass columns</td><td>Beaucaverre, France</td><td>Specific request</td><td>columns were composed of 3 separate pieces, the main column core with the cooling jacket and the 5 sampling ports (size GL14 with olive) and a top and bottom piece that fits to the main column body and is held in place with a silicone joint and screw (RIN F 40x38 &amp;amp; SVL 42). note: this design was discussed directly with the company. We recommend to find a local glazier.</td></tr><tr><td>Septa PTFE/silicone diameter 20 mm</td><td>Sigma-Aldrich</td><td>508608</td><td></td></tr><tr><td>PTFE tubing ID 3 mm</td><td>VWR</td><td>228-0745</td><td></td></tr><tr><td>Peristaltic pump</td><td>Dominique Dutsher SAS</td><td>66493</td><td></td></tr><tr><td>Peristaltic pump tubing LMT 55</td><td>VWR</td><td>224-2250</td><td>Tygon&amp;reg; LMT 55&amp;nbsp;</td></tr><tr><td>Fontainbleau sand D50=209 &amp;micro;m</td><td>SIBELCO, France</td><td></td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt; for bubbling</td><td>Air Liquide, France</td><td></td><td></td></tr><tr><td>Gamma irradiation</td><td>Ionisos, Dagneux, France</td><td></td><td></td></tr><tr><td>Automatic Mercury Analyzer (AMA 254)</td><td>Courtage Analyses, France</td><td></td><td></td></tr><tr><td>Varian SpectrAA 300 Zeeman</td><td>Agilent</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HNO&lt;sub&gt;3&lt;/sub&gt; Supra pur</td><td>VWR</td><td>1.00441.1000</td><td>Manufacturer: Merck</td></tr><tr><td>HCL 30% Supra pur</td><td>VWR</td><td>1.00318.1000</td><td>Manufacturer: Merck</td></tr><tr><td>Hg(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>516953</td><td></td></tr><tr><td>As&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck</td><td>202673</td><td></td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;-6H2O</td><td>Merck</td><td>207926</td><td></td></tr><tr><td>silica gel</td><td>Sigma-Aldrich</td><td>336815-500G</td><td></td></tr></tbody></table>

experimental column setup for studying anaerobic biogeochemical interactions between iron (oxy)hydroxides, trace elements, and bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Example 1. Impact of iron reduction of As mobility and speciation 
The As column was directly inoculated with groundwater from a site presenting an As concentration higher than the drinking standards (Bracieux, Loire et Cher, France). Groundwater was sampled in sterile bottles, and stored at 5 &#176;C until use. The column was fed from the bottom with this water containing the natural endogenous microbial commun...

Watch this Scientific Journal Video about Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria at JoVE.com

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

experimental-column-setup-for-studying-anaerobic-biogeochemical

Research

JoVE Journal

Environment

7.8K Views.  BRGM. Lot en speciatie van arseen en kwik in watervoerende lagen zijn nauw verbonden aan fysio-chemische voorwaarden en microbiële activiteit. Hier presenteren we een oorspronkelijke experimentele kolominstelling die bootst een aquifer en maakt een beter begrip van het spoorelement biogeochemie in zuurstofvrije omstandigheden. Twee voorbeelden worden gepresenteerd, combinatie van geochemische en microbiologische werkwijzen.

Video: Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 &#176;C) and thermophilic (55 &#176;C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 &#176;C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 LCH4/kgVS&#160;(VS: volatile solids) and 121 L CH4/kgVS&#160;at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kgdry_biochar was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.

A novel Upflow Anaerobic Solid State (UASS) reactor was used for biogas production from fibrous feedstock. Digestate from UASS reactor was hydrothermally carbonized into HTC biochar in a pressurized batch reactor. The integration of the two bioenergy concepts was applied in this study to increase overall bioenergy production.

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be critical for microbial energy production and persistence in many subsurface environments, especially those lacking soluble terminal electron acceptors. While EET-capable microbes have been successfully isolated from various environments, the diversity of bacteria capable of EET is still poorly understood, especially in difficult-to-sample, low energy or extreme environments, such as many subsurface ecosystems. Here, we describe an on-site electrochemical system to enrich EET-capable bacteria using an anode as a respiratory terminal electron acceptor. This anode is connected&#160;to a cathode capable of&#160;catalyzing abiotic oxygen reduction. Comparing this approach with electrocultivation methods that use a potentiostat for poising the electrode potential, the two-electrode system does not require an external power source. We present an example of our on-site enrichment utilized in an alkaline pond at the Cedars, a terrestrial serpentinization site in Northern California. Prior attempts to cultivate mineral reducing bacteria were unsuccessful, which is likely due to the low-biomass nature of this site and/or the low relative abundance of metal reducing microbes. Prior to implementing our two-electrode enrichment, we measured the vertical profile of dissolved oxygen concentration. This allowed us to place the carbon felt anode and platinum-electroplated carbon felt cathode at depths that would support anaerobic and aerobic processes, respectively. Following on-site incubation, we further enriched the anodic electrode in the laboratory and confirmed&#160;a distinct microbial community compared to the surface-attached or biofilm communities normally observed at the Cedars. This enrichment subsequently led to the isolation of the first electrogenic microbe from the Cedars. This method of on-site microbial enrichment has the potential to greatly enhance the isolation of EET-capable bacteria from low biomass or difficult to sample habitats.

On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET).

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be ...

In Situ Simulated Anaerobic Method for Culturing Environmental Microbes

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

Author Spotlight: Unraveling the Mysteries of Terrestrial Anaerobic Microorganisms in Uncharted Environments by In Situ Culturing

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Using Flexible Gold-Titanium Reaction Cells to Simulate Pressure-Dependent Microbial Activity in the Context of Subsurface Biomining

Laboratory studies investigating subsurface microbial processes, such as metal leaching in deep ore deposits (biomining), share common and challenging obstacles, including the special environmental conditions that need to be replicated, e.g., high pressure and in some cases acidic solutions. The former requires an experimental setup suitable for pressurization up to 100 bar, while the latter demands a fluid container with high chemical resistance against corrosion and unwanted chemical reactions with the container wall. To meet these conditions for an application in the field of in situ biomining, a special flexible gold-titanium reaction cell inside a rocking high-pressure reactor was used in this study. The described system allowed simulation of in situ biomining through sulfur-driven microbial iron reduction in an anoxic, pressure-controlled, highly chemically inert experimental environment. The flexible gold-titanium reaction cell can accommodate up to 100 mL of sample solution, which can be sampled at any given time point while the system maintains the desired pressure. Experiments can be performed on timescales ranging from hours to months. Assembling the high-pressure reactor system is fairly time consuming. Nevertheless, when complex and challenging (microbiological) processes occurring in the earth&#39;s deep subsurface in chemically aggressive fluids have to be investigated in the laboratory, the advantages of this system outweigh the disadvantages. The results found that even at high pressure the microbial consortium is active, but at significantly lower metabolic rates.

This protocol describes microbial experiments under elevated pressures to study in situ biomining processes. The experimental approach employs a rocking high-pressure reactor equipped with a gold-titanium reaction cell containing a microbial culture in an acidic, iron-rich medium.

Laboratory studies investigating subsurface microbial processes, such as metal leaching in deep ore deposits (biomining), share common and challenging ...

Engineering

Enhancing CO2 Sequestration: A Soil Biota Approach to Boost Mineral Weathering

Design and Construction of an Experimental Setup to Enhance Mineral Weathering through the Activity of Soil Organisms

Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies on accelerating the natural process of mineral weathering in soils by manipulating the abiotic variables that govern this process, in particular mineral grain size and exposure to acids dissolved in water. EW mainly aims at reducing atmospheric CO2 concentrations by enhancing inorganic carbon sequestration. Until now, knowledge of EW has been mainly gained through experiments that focused on the abiotic variables known for stimulating mineral weathering, thereby neglecting the potential influence of biotic components. While bacteria, fungi, and earthworms are known to increase mineral weathering rates, the use of soil organisms in the context of EW remains underexplored. 
 This protocol describes the design and construction of an experimental setup developed to enhance mineral weathering rates through soil organisms while concurrently controlling abiotic conditions. The setup is designed to maximize weathering rates while maintaining soil organisms' activity. It consists of a large number of columns filled with rock powder and organic material, located in a climate chamber and with water applied via a downflow irrigation system. Columns are placed above a fridge containing jerrycans to collect the leachate. Representative results demonstrate that this setup is suitable to ensure the activity of soil organisms and quantify their effect on inorganic carbon sequestration. Challenges remain in minimizing leachate losses, ensuring homogeneous ventilation through the climate chamber, and avoiding flooding of the columns. With this setup, an innovative and promising approach is proposed to enhance mineral weathering rates through the activity of soil biota and disentangle the effect of biotic and abiotic factors as drivers of EW.

Author Spotlight: Unraveling the Role of Earthworms in Enhancing Mineral Weathering for CO2 Removal 

Here we present the construction and operation of an experimental setup to enhance mineral weathering through the activity of soil organisms while concurrently manipulating abiotic variables known to stimulate weathering. Representative results from the functioning of the setup and sample analyses are discussed together with points for improvement.

Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies ...

Biochemistry

Investigating Bacterial-Fungal Interactions using Fungal Highway Columns in Diverse Environments and Substrates

Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and microbial dispersal. Mycelial networks created by filamentous fungi or other filamentous microorganisms (e.g., Oomycetes) act as 'fungal highways' that can be utilized by bacteria for transport throughout heterogeneous environments, greatly facilitating their mobility and granting them access to regions that may be challenging or impossible to reach on their own (e.g., due to air pockets within the soil). Several devices and experimental protocols have been created to study these fungal highways, including fungal highway columns. The fungal highway column designed by our group can be used for a variety of in situ or in vitro applications, as well as with diverse environmental and host-associated sample types. Herein, we describe the methods for performing experiments with these columns, including designing, printing, sterilizing, and preparing the devices. The options for analyzing data obtained from the use of these devices are also discussed here, and troubleshooting advice regarding potential pitfalls associated with experiments using fungal highway columns is offered. These devices can be used to gain a more comprehensive understanding of the diversity, mechanisms, and dynamics of fungal highway BFIs to provide valuable insights into the structural and functional dynamics within complex environments (e.g., soils) and across diverse habitats in which bacteria and fungi co-exist.

This protocol provides detailed instructions on how to construct, sterilize, assemble, utilize, and reuse the fungal highway columns to enrich bacterial-fungal pairs interacting through fungal highways from diverse environmental substrates.

Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and ...

Biology

Creating a Winogradsky Column: A Method to Enrich the Microbial Species in a Sediment Sample

Source: Elizabeth Suter1, Christopher Corbo1, Jonathan Blaize1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
The Winogradsky column is a miniature, enclosed ecosystem used for enriching sediment microbial communities, especially those involved in sulfur cycling. The column was first used by Sergei Winogradsky in the 1880s and has since been applied in the study of many diverse microorganisms involved in biogeochemistry, such as photosynthesizers, sulfur oxidizers, sulfate reducers, methanogens, iron oxidizers, nitrogen cyclers, and more (1,2).
The majority of microorganisms on Earth are considered unculturable, meaning that they cannot be isolated in a test tube or on a petri dish (3). This is due to many factors, including that microorganisms depend on others for certain metabolic products. The conditions in a Winogradsky column closely mimic a microorganism&#39;s natural habitat, including their interactions with other organisms, and allows for them to be grown in a lab. Therefor, this technique permits scientists to study these organisms and understand how they are important to Earth&#39;s biogeochemical cycles without having to grow them in isolation.
Earth&#39;s environments are full of microorganisms which thrive in all types of habitats, such as soils, ocean water, clouds, and deep-sea sediments. In all habitats, microorganisms depend on each other. As a microorganism grows, it consumes particular substrates, including carbon-rich fuels like sugars as well as nutrients, vitamins, and respiratory gases like oxygen. When these important resources run out, different microorganisms with different metabolic needs can then bloom and thrive. For example, in the Winogradsky column, microbes first consume the added organic material while depleting the oxygen in the bottom layers of the column. Once the oxygen is used up, anaerobic organisms can then take over and consume different organic materials. This consecutive development of different microbial communities over time is called succession (4). Microbial succession is important in a Winogradsky column, where microbial activity changes the chemistry of the sediment, which then affects activity of other microbes and so on. Many microorganisms in soils and sediments also live along gradients, which are transitional zones between two different types of habitats based on the concentrations of substrates (5). At the correct spot in the gradient, a microbe can receive optimal amounts of different substrates. As a Winogradsky column develops, it begins to mimic these natural gradients, particularly in oxygen and sulfide (Fig. 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10506/10506fig1.jpg" /> 
Figure 1: A representation of the oxygen (O2) and sulfide (H2S) gradients that develop in a Winogradsky column.
In a Winogradsky column, mud and water from a pond or wetland are mixed in a transparent column and allowed to incubate, typically in the light. Additional substrates are added to the column to give the community sources of carbon, usually in the form of cellulose, and sulfur. Photosynthesizers typically start to grow in the top layers of the sediment. These photosynthetic microorganisms are largely composed of cyanobacteria, which produce oxygen and appear as a green or red-brown layer (Fig. 2, Table 1). While photosynthesis produces oxygen, oxygen is not very soluble in water and it decreases below this layer (Fig. 1). This creates a gradient of oxygen, ranging from high concentrations of oxygen in the top layers to zero oxygen in the bottom layers. The oxygenated layer is called the aerobic layer and the layer without oxygen is called the anaerobic layer.
In the anaerobic layer, many different microbial communities can proliferate depending on the type and amount of substrates that are available, the source of the initial microbes, and the porosity of the sediment. At the bottom of the column, organisms that anaerobically break down organic matter can thrive. Microbial fermentation produces organic acids from the breakdown of cellulose. These organic acids can then be used by sulfate reducers, which oxidize those organics using sulfate, and produce sulfide as a byproduct. The activity of sulfate reducers is indicated if the sediment turns black, because iron and sulfide react to form black iron-sulfide minerals (Fig. 2, Table 1). The sulfide also diffuses upward, creating another gradient in which sulfide concentrations are high in the bottom of the column and low in the top of the column (Fig. 1).
Near the middle of the column, sulfur oxidizers take advantage of the supply of oxygen from above and sulfide from below. With the right amount of light, photosynthetic sulfur oxidizers can develop in these layers. These organisms are known as green and purple sulfur bacteria, and often appear as green, purple, or purple-red filaments and blotches (Fig. 2, Table 1). Green sulfur bacteria have a higher tolerance for sulfide and usually develop in the layer directly below purple sulfur bacteria. Above the purple sulfur bacteria, purple nonsulfur bacteria may also develop. These organisms photosynthesize using organic acids as electron donors instead of sulfide and often appear as a red, purple, orange, or brown layer. Nonphotosynthetic sulfur oxidizers can develop above the purple nonsulfur bacteria, and these usually appear as white filaments (Fig. 2, Table 1). Additionally, bubbles may also form in the Winogradsky column. Bubbles in the aerobic layers indicates the production of oxygen by the cyanobacteria. Bubbles in the anaerobic layers are likely due to the activity of methanogens, organisms which anaerobically break down organic matter and form methane as a byproduct.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Position in Column</td>
			<td>Functional group</td>
			<td>Organism Examples</td>
			<td>Visual Indicator</td>
		</tr>
		<tr>
			<td>Top</td>
			<td>Photosynthesizers</td>
			<td>Cyanobacteria</td>
			<td>Green or reddish-brown layer. Sometimes bubbles of oxygen.</td>
		</tr>
		<tr>
			<td rowspan="5"></td>
			<td>Nonphotosynthetic sulfur oxidizers</td>
			<td>Beggiatoa, Thiobacilus</td>
			<td>White layer.</td>
		</tr>
		<tr>
			<td>Purple nonsulfur bacteria</td>
			<td>Rhodomicrobium, Rhodospirilum, Rhodopseuodmonas</td>
			<td>Red, purple, orange, or brown layer.</td>
		</tr>
		<tr>
			<td>Purple sulfur bacteria</td>
			<td>Chromatium</td>
			<td>Purple, or purple-red layer.</td>
		</tr>
		<tr>
			<td>Green sulfur bacteria</td>
			<td>Chlorobium</td>
			<td>Green layer.</td>
		</tr>
		<tr>
			<td>Sulfate Reducing Bacteria</td>
			<td>Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfuromonas</td>
			<td>Black layer.</td>
		</tr>
		<tr>
			<td>Bottom</td>
			<td>Methanogens</td>
			<td>Methanococcus, Methanosarcina</td>
			<td>Sometimes bubbles of methane.</td>
		</tr>
	</tbody>
</table>
Table 1: The main groups of bacteria that may appear in a classical Winogradsky column, from top to bottom. Examples of organisms from each group are given, and the visual indicators of each layer of organisms are listed. Based on Perry et al. (2002) and Rogan et al. (2005).

Winogradsky Column to Enrich Microbial Species in a Sample

Source: Elizabeth Suter1, Christopher Corbo1, Jonathan Blaize1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 
...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

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