A protocol is described for preparing a simple model ecosystem that recreates the methane-oxygen counter gradient found in the natural habitat of aerobic methane-oxidizing bacteria, enabling the study of their physiology in a spatially resolved context. Modifications to common biochemical assays for use with the agarose-based model ecosystem are also described.
Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental niche within methane-oxygen counter gradients found in soils and sediments, which influences their behavior on an individual and community level. However, conventional methods to study the physiology of these greenhouse gas-mitigating microorganisms often use homogeneous planktonic cultures, which do not accurately represent the spatial and chemical gradients found in the environment. This hinders scientists’ understanding of how these bacteria behave in situ. Here, a simple, inexpensive model ecosystem called the gradient syringe is described, which uses semi-solid agarose to recreate the steep methane-oxygen counter gradients characteristic of methanotrophs’ natural habitats. The gradient syringe allows for the cultivation of methanotrophic strains and the enrichment of mixed methane-oxidizing consortia from environmental samples, revealing phenotypes only visible in this spatially resolved context. This protocol also reports various biochemical assays that have been modified to be compatible with the semi-solid agarose matrix, which may be valuable to researchers culturing microorganisms within other agarose-based systems.
Microorganisms living at an anoxic-oxic interface often serve important ecological roles1. One example is aerobic methane-oxidizing bacteria (methanotrophs), which exist in counter gradients of methane and oxygen in soils and sediments2. These microorganisms possess unique metabolic and physiological characteristics that enable them to exploit the gas gradients present in their environments and have been the subject of ongoing research for decades3,4,5. Currently, most published research about methanotrophs and methane-oxidizing communities is based on work with homogenous planktonic cultures that often fail to capture the spatial and chemical gradients that are inherent to their natural microbial habitats. This limitation hinders our understanding of microbial physiology and our ability to link genomic information to phenotypic traits.
This protocol reports a simple, laboratory-based model ecosystem that creates reproducible conditions for studying both specific methanotrophs, such as Methylomonas sp. strain LW13, and methane-oxidizing communities directly from environmental soil samples. Importantly, cultivation in the gradient syringe results in counter gradient-specific phenotypes that are not present in homogenous planktonic cultures6, highlighting the system's ability to unveil new aspects of methanotroph physiology. Inspired by previously published model ecosystems7,8,9, the gradient syringe is a simplified method that can be used to collect chemical and molecular information from microorganisms cultured using this approach.
The reported procedures for genetic, chemical, and molecular analyses have been modified to work reliably on microbial cultures grown within a semi-solid agarose matrix. These procedures may also be useful for analyzing bacteria grown in other semi-solid agarose-based systems, such as those used for bacterial soft agar swimming assays. Adapting these analyses to spatially resolved contexts may open new avenues for studying microbial life in more ecologically relevant environments.
The details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Preparation and extrusion of gradient syringes
NOTE: Gradient syringe preparation should be performed using sterile technique.
2. Determining counter gradient gas concentrations
3. Counting cells in the gradient syringe
4. Biomolecule detection assays
5. RNA extraction
Here, the gradient syringe model ecosystem was used to cultivate a single strain (the methanotroph Methylomonas sp. strain LW13) (Figure 1A)6, but it can also be used to enrich for a methane-oxidizing microbial community by direct soil inoculation (Figure 1B). The presence of a methane-oxygen counter gradient was validated by measuring the concentration of methane and oxygen across cell-free and inoculated syringes (Figure 1C). For LW13-inoculated gradient syringes, a counter gradient formed within one day of flushing the syringe, which steepened over three days of incubation. Over the same time period, a horizontal band formed at the same depth at which both gas substrates reached their lowest concentrations (Figure 1A). The steep gas gradient and depletion of methane and oxygen past the depth of the horizontal band showed that LW13 aerobically metabolized methane and produced a phenotype not observed in homogenous planktonic culture. This phenotype is also produced by other methanotrophic bacteria isolated from the same environmental sample as LW136. Strain-dependent variation in the timing and depth of the horizontal band development among different methanotrophic strains suggested that the horizontal band was affected by the specific behavior of each microbe when cultivated in a spatially resolved context6.
The number of cells in the entire agarose plug was measured using flow cytometry and colony counts (CFU/mL) (Figure 2A). This method was used to compare the cell distribution and survival of wild-type LW13 to a mutant strain of LW13 containing a deletion in the fucose 4-O-acetyltransferase (OAT) gene, which was previously shown to influence horizontal band development6. The ΔOAT mutant of LW13 had lower overall growth in the gradient syringe over 6 days compared to the wild-type, an effect that was not observed in homogenous planktonic cultures of the same strains6. The mutant strain did not form the same distinct horizontal band as the LW13 wild type when cultured in the gradient syringe (Figure 2B). Cell numbers and horizontal band appearance were restored to levels similar to the wild type upon gene complementation in the mutant strain. These results demonstrate that the gradient syringe can be used to link genes to specific phenotypes only present in the methane-oxygen counter gradient.
A variety of genetic, chemical, and molecular techniques were adapted for use with bacteria grown within a semi-solid agarose matrix. The gradient syringe model ecosystem can be readily used for standard biomolecular quantification assays with the inclusion of uninoculated agarose as the negative control. The concentration of three different biomolecules commonly found in extracellular polymeric substances and biofilms was measured: polysaccharides, protein, and extracellular DNA15 (Figure 3). In LW13-inoculated syringe segments, the horizontal band had significantly more polysaccharides than other segments, with no significant increase in protein or extracellular DNA.
RNA-seq was used to measure transcriptional differences in LW13 growing at different depths of the syringe. Robust RNA extraction was achieved using a CTAB-based extraction buffer followed by conventional phenol: chloroform extraction and precipitation steps. The results from the RNA-seq analysis were later used to identify genes implicated in the production of the horizontal polysaccharide band. These results indicate that the semi-solid agarose essential for the creation of a spatially resolved model ecosystem does not prevent further biochemical analyses that are generally reserved for planktonic and plate-based cultures.
Figure 1: The gradient syringe model ecosystem. (A) The gradient syringe inoculated with the methanotroph Methylomonas sp. LW13. A distinct horizontal band (arrowhead) develops within two days of flushing the syringe with 100% methane. (B) Close-up photos of gradient syringes inoculated with soil diluted 10-1 and 10-4 and incubated for two weeks. Gradient syringes containing more dilute soil resulted in spherical colonies throughout the agarose, whereas more concentrated soil inocula resulted in a distinct band. (C) Characterization of the methane-oxygen counter gradient in LW13-inoculated and sterile gradient syringes after three days of incubation. The gray bar indicates the range of depths at which the polysaccharide band was located; data show the mean ± SD of three independent experiments with three technical replicates each. Panels (A) and (C) were modified from Beals et al.6. Please click here to view a larger version of this figure.
Figure 2: Quantification of LW13 wild type and mutant after incubation in the gradient syringe. (A) The total number of LW13 cells per mL extruded agarose recovered from gradient syringes on Day 0 and Day 7 measured by flow cytometry. ΔOAT contains a deletion of the fucose 4-O-acetyltransferase gene, which was found to be highly expressed in cells located at the depth of the polysaccharide band. ΔOAT+OAT contains the OAT gene inserted at a distal location of the ΔOAT genome. *, significantly different (two-tailed heteroscedastic t-test, α = 0.05); n.s., not significantly different. Data show the mean ± SD of three independent experiments with two technical replicates each. (B) Horizontal band development in gradient syringes inoculated with LW13 wild type, ΔOAT, or ΔOAT+OAT after seven days of incubation. This figure was modified from Beals et al.6. Please click here to view a larger version of this figure.
Figure 3: Quantification of biomolecules at increasing depths of the gradient syringe. Relative polysaccharide content (%), protein concentration (µg/mL), and DNA concentration (ng/µL) in eight sections of LW13-inoculated gradient syringes incubated for seven days (filled circles; open circles show values from sterile gradient syringes). Data show the mean ± SD of three independent experiments with three technical replicates each. For relative polysaccharide content, * indicates a significant difference from sterile control at equivalent depth (two-tailed heteroscedastic t-test, α = 0.05). For protein and DNA concentrations, * indicates a significant difference from the section containing the horizontal band (one-way ANOVA with Tukey-Kramer post hoc analysis); n.s., not significant. The figure was modified from Beals et al.6. Please click here to view a larger version of this figure.
Methods for methanotroph cultivation
Methanotrophs have been studied for decades to understand their physiology, their individual and community behavior in the natural environment, and their potential for methane mitigation in industrial applications. Throughout these studies, much of the research conducted has been performed using homogenous planktonic cultures where spatial context is lost. The gradient syringe model ecosystem was developed to replicate the methane-oxygen counter gradient characteristic of natural methanotroph habitats in the lab, allowing researchers to study methanotrophs growing in an environment that more closely resembles where these organisms evolved.
Over the past 30 years, researchers have recreated the methane-oxygen counter gradient in the lab using a variety of methods, often with the primary goal of isolating and classifying methanotrophs from mixed methane-oxidizing consortia. These methods can be divided into two approaches, both involving the use of opposing chambers of methane and oxygen: suspending relatively undisturbed soil on a membrane16,17,18, or inoculating small amounts of soil or pure bacterial culture into a minimal medium in agarose7,8,19. The gradient syringe method described here combines the syringe-based approach of Dedysh and coworkers9 with the cultivation of methanotrophs from previous work by Amaral and Knowles8, and Schink and coworkers7. The latter of these methods laid the foundation for cultivating methanotrophs in a methane-oxygen counter gradient and used a continuous flow of methane and oxygen on either side of the agarose plug. While this provides a more constant environment, this approach adds complexity to the experimental setup and necessitates dedicated gas sources.
In contrast, the gradient syringe described here relies on daily flushing of the syringe to provide fresh methane, a process that takes less than a minute per syringe, while providing continuous access to atmospheric oxygen through a sterile PTFE filter tip. This simpler method may enable wider adoption of this model ecosystem for studying methanotrophs in a spatially resolved context. The described protocol also details chemical and molecular-level analyses that can be performed directly on bacteria incubated in the semi-solid agarose. As a result, bacteria do not need to be excised and cultured outside the agarose matrix before analysis, preserving the gas gradient conditions at the time of sampling.
Remarks on the protocol
Because the bacteria are cultured within a polypropylene volumetric syringe, researchers can use the accompanying syringe plunger to accurately and reproducibly segment the agarose plug while maintaining the spatial integrity of the agarose matrix that still remains in the syringe barrel. Without the air-tight design inherent to the syringe, agarose plugs would need to be removed from the syringe barrel and sliced, introducing uncertainty in the volume of agarose segments, and releasing unquantifiable amounts of methane and dissolved oxygen into the atmosphere. Agarose extrusion through a sterile needle simplifies sample preparation and helps homogenize extruded segments without shearing bacterial cells. This method allows researchers to divide each inoculated gradient syringe into at least eight agarose segments and perform parallel experiments on methanotrophs growing in a range of oxygen and methane concentrations.
In optimizing RNA extraction from high-polysaccharide content agarose, it was found that common reagents like guanidium thiocyanate and TRIzol led to agarose gelation, which obstructed purification columns and resisted pelleting by centrifugation. Low RNA yields and quality were also a concern as large polysaccharide molecules can trap nucleic acids while small polysaccharides can co-precipitate with RNA20. Instead, an extraction buffer containing the cationic surfactant CTAB was used, which solubilizes lipid membranes20; and NaCl, which prevents CTAB-nucleic acid complexes from forming and allows nucleic acids to precipitate but keeps polysaccharides in solution21. RNases were denatured by the inclusion of β-mercaptoethanol in the CTAB buffer. For the RNA-seq experiment, an optional column-based purification step was included to exclude small (<200 nucleotides) RNAs before library preparation.
Limitations and considerations
While the NMS and agarose provide a minimal medium matrix for cultivating methanotrophic bacteria, the gradient syringe as described here only recreates the gas gradients of methanotrophic habitats, but not other gradients present in those environments such as trace metals22, salinity23, or other nutrients24. It is possible these gradients can be added to a similar system in the future. Additionally, the volume of the syringe (8 mL agarose) limits the total biomass per syringe, necessitating pooling multiple syringes for some analyses (as described in step 5.16). Although the handheld syringe conveniently segments the agarose into 1 mL aliquots, its size also limits the headspace to approximately 4 mL, limiting the amount of bulk methane that can be stored for the cultivated microbes. Since methane oxidation rates are proportional to the growth rate of aerobic methanotrophs25, daily replenishment of headspace methane is recommended. While this may still result in periods of methane limitation, these periods are reproducible in the laboratory and likely mimic situations found in natural environments.
While using the gradient syringe, the presence of the agarose polysaccharides necessitates some adjustments to the assays used to analyze methanotrophs grown in this system. For example, protocols requiring the transfer of small volumes of extruded agarose need multiple dilution steps with thorough homogenization between each dilution for accurate pipetting. Additionally, in cases such as the polysaccharide assay where the polysaccharides inherent to the agarose matrix will react with the sulfuric acid-phenol reagent, the inclusion of a sterile, cell-free agarose negative control is essential. Early attempts to mitigate these issues by including the agarose-hydrolyzing enzyme β-agarase were unsuccessful and introduced an unknown variable to the biological experiments. The use of multiple technical replicates, thorough dilution, homogenization, and the inclusion of controls can be used to mitigate most of the challenges inherent to the agarose matrix.
Applications
In addition to single-strain studies, the gradient syringe can support the co-culture of multiple strains, and soil can be used as the inoculum in place of pure bacterial culture. The simple design of the gradient syringe model ecosystem is amenable to the culture of other types of microorganisms that exist at the interface between anoxic and oxic environments by using a different gas substrate, such as H2 or CO, in place of methane. In summary, the use of a simple, spatially resolved model ecosystem allows researchers to study the unique physiology and metabolic adaptations of anoxic-oxic microorganisms and can be used to link genes with organismal phenotypes.
The authors have nothing to disclose.
This work was supported by startup funding from the University of Utah Department of Chemistry and NSF CAREER Award #2339190. We thank members of the Puri Lab for helpful discussions. We thank Rachel Hurrell (University of Utah) for initial guidance with the flow cytometry experiment.
1% Gas mix analytical standard | Supelco | 22561 | 1% each component in nitrogen: carbon monoxide, carbon dioxide, hydrogen, methane and oxygen |
100% Methane | Airgas | ME CP300 | chemically pure grade |
15 ppm Gas mix analytical standard | Supelco | 23470-U | 15 ppm each component in nitrogen: methane, ethane, ethylene, acetylene, propane, propylene, propyne, and n-butane |
1x Nitrate mineral salts | see CAS numbers below | Dissolve the following in Mili-Q water and autoclave: 0.2 g/L MgSO4·7H2O, 0.2 g/L CaCl2·6H2O, 1 g/L KNO3, and 30 μM LaCl3. Before use, add trace elements to a 1X final concentration and phosphate buffer (pH 6.8) to a final concentration of 5.8 mM. | |
23 G needle | BD Biosciences | 305194 | sterile, Luer-Lok |
500x Trace elements | see CAS numbers below | Dissolve the following in Milli-Q water: 1.0 g/L Na2-EDTA, 2.0 g/L FeSO4·7H2O, 0.8 g/L ZnSO4·7H2O, 0.03 g/L MnCl2·4H2O, 0.03 g/L H3BO3, 0.2 g/L CoCl2·6H2O, 0.6 g/L CuCl2·2H2O, 0.02 g/L NiCl2·6H2O, and 0.05 g/L Na2MoO·2H2O. | |
96 Well plate | CELLTREAT | 229596 | sterile |
Acid phenol:chloroform:IAA (125:24:1) | Invitrogen | AM9720 | pH 4.5 |
Agarose | Fisher Scientific | BP160 | molecular biology grade, CAS 9012-36-6 |
Aluminum crimp seals | VWR | 30618-460 | 20 mm |
Bead beater | Qiagen | 9003240 | TissueLyser III |
Butyl rubber stopper | Chemglass Life Science | 50-143-854 | 20 mm, blue |
Chloroform:isoamyl alcohol (24:1) | Millipore Sigma | 25666 | BioUltra, for molecular biology |
Clark-type O2 microelectrode | Unisense | OX-500 | |
DEPC-treated water | Thermo Scientific | R0601 | |
DNase I (Ambion) | Invitrogen | AM2222 | |
Flow cytometer | Beckman Coulter | CytoFLEX | |
Gas chromatograph (flame ionization detection) | Agilent | 6890N | |
Gastight analytical syringe | Hamilton | 81220 | 1750 TLL |
Gastight analytical syringe needle | Hamilton | 7729-07 | 22 G, metal hub needle, 2 in, point style 5 |
Gas-tight vials | Labco | 938W | Exetainer vial: 12 mL, round bottom |
Glass culture tubes | Bellco Glass | 2048-00150 | 18 x 150 mm |
LiCl precipitation solution (7.5 M) | Invitrogen | AM9480 | |
One-way stopcock | VWR | MFLX30600-00 | inlet port: female luer, outlet port: male luer lock |
Petri dish, square | Fisher Scientific | FB0875711A | 100 x 100 mm |
Phosphate buffer, 0.2 M (pH 6.8) | see CAS numbers below | Dissolve the following in Milli-Q water and autoclave: 12.24 g/L KH2PO4, 26.29 g/L Na2HPO4 · 7H2O | |
Pierce BCA Protein Assay Kit | Thermo Scientific | 23225 | |
PTFE syringe filter tip | Thermo Scientific | 03-050-469 | hydrophobic, pore size: 0.2 µm, diameter: 4 mm |
Qubit 1x dsDNA High Sensitivity Assay Kit | Invitrogen | Q33230 | |
Qubit 4 Fluorometer | Invitrogen | Q33238 | |
RNA Clean & Concentrator-5 | Zymo Research | R1013 | |
Serum stopper | Fisher Scientific | 03-340-302 | 20 mm |
Syringe | BD Biosciences | 302995 | Luer-Lock, 10 mL, single use, sterile |
Syringe pump | New Era Pump Systems Inc. | 1000-US | NE-1000 one channel programmable |
SYTO9, propidium iodide, microspheres | Invitrogen | L34856 | LIVE/DEAD BacLight Bacterial Viability Kit |
Zirconia/silica beads | BioSpec Products | 11079101z | 0.1 mm diameter |
Chemical reagents | CAS number | ||
CaCl2·6H2O | 7774-34-7 | ||
CoCl2·6H2O | 7791-13-1 | ||
Concentrated sulfuric acid | 7664-93-9 | ||
CTAB, cetrimonium bromide | 57-09-0 | ||
CuCl2·2H2O | 10125-13-0 | ||
Ethanol | 64-17-5 | ||
FeSO4·7H2O | 7782-63-0 | ||
H3BO3 | 10043-35-3 | ||
Isopropanol | 69-63-0 | ||
KH2PO4 | 7778-77-0 | ||
KNO3 | 7757-79-1 | ||
LaCl3 | 10099-58-8 | ||
MgSO4·7H2O | 10034-99-8 | ||
MnCl2·4H2O | 13446-34-9 | ||
Na2CO3, sodium carbonate | 497-19-8 | ||
Na2-EDTA | 139-33-3 | ||
Na2HPO4 · 7H2O | 7782-85-6 | ||
Na2MoO·2H2O | 10102-40-6 | ||
NaCl, sodium chloride | 7647-14-5 | ||
NiCl2·6H2O | 7791-20-0 | ||
Phenol (90% solution in water) | 108-95-2 | ||
PVP40, polyvinylpyrrolidone | 9003-39-8 | ||
Tris-HCl | 1185-53-1 | ||
ZnSO4·7H2O | 7446-20-0 | ||
β-Mercaptoethanol | 60-24-2 |
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