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 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 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.
Most microorganisms remain uncultured; this is supported by the great disparity between cells observed through microscopy contrasted by the few microorganisms successfully cultured using agar plates. Staley and Konopka named this disparity the "Great Plate Count Anomaly"1. The estimated unaccounted diversity is supported by metagenomic and metatranscriptomic data showing many novel genera distributed in rank abundance curves from several different environments2. Microorganisms that have been observed (generally by random shotgun sequencing of a microbial community) but have not been cultured have been referred to as "microbial dark matter"3,4.
In the age of -omics, culturing microorganisms remains imperative to fully evaluate genomic data and verify the function/phenotype of genes present. Sequencing cultured microorganisms is still the only way to confidently obtain complete genomes until technologies such as shotgun metagenomics and metagenome-assembled genomes from the environment become admissibly infallible5. Genomic evaluations coupled with cultured microorganisms provide strong inferences for understanding "microbial dark matter." Many members of the "microbial dark matter" perform crucial functions that impact the cycling of nutrients and other elements and the production of valuable natural products, support ecological systems, and perform ecological services. From the medical perspective, about half of all currently marketed pharmaceuticals are products and derivatives of products from bacteria, and profiling uncultured species is suspected to reveal the antibiotics of the future. To gain access to this uncultured majority, a variety of culturing methodologies must be increased6. Among the members of the "microbial dark matter," anaerobic oligotrophic microorganisms are largely underreported and likely hold ecologically and industrially valuable biochemical pathways7, making them important targets of culturing. However, anaerobic oligotrophic microorganisms are more difficult to culture than their aerobic and copiotrophic counterparts due to often-required longer incubation times, fastidious conditions (e.g., particular non-standard in vitro temperatures), and the use of specialized media recipes.
Current developing techniques to culture members of the "microbial dark matter," including novel anaerobic oligotrophic microorganisms, have greatly improved our understanding and increased the representation of these microorganisms within the phylogenetic tree. Current techniques using informed media for culturing novel microorganisms (i.e., media which is derived using knowledge of the microorganism/s of interest) can be separated into three distinct methods. The first of the methods entails the direct removal of a discrete section of the environment for transfer into an in vitro growth chamber that already contains the microorganisms of interest within a membrane. The discrete section (e.g., seawater) acts to provide the microorganisms of interest with the geochemical habitat they use in situ, while the membrane arrests the movement of cells across (cells of interest will remain within; extraneous cells that arrived with the discrete section will remain without). By including compounds naturally available to target microorganisms in their natural habitat, such microorganisms can be cultured8. The second method utilizes metatranscriptomics or genomics to elucidate metabolic capabilities, providing clues to narrow culturing parameters for a targeted medium design. This approach provides an eco-physiological profile that can be used to target the enrichment of specific types of microorganisms out of an environment. The medium's provisions are catered to the identified genes present that are presumed to support the targeted microorganism(s) to reduce enrichment diversity,9,10. One caveat is that genomic information does not directly infer the expression of genes, while transcriptomic information does.
The third method encompasses environmentally informed and simulated media, distinct from the first method, which does not simulate the media but rather uses the environment directly as a source of media. This third method requires environmental reconnaissance of the geochemistry of a field site containing microorganisms of interest. With this knowledge, primary components and physical parameters are identified to produce an environmentally informed simulated medium. The medium then receives a direct infusion of microorganism-containing sediment or liquid from the environment into the medium. This method is of particular value in cases where the culturing microbiologist does not have access to sufficient quantities of source environment (as needed for the first method) nor appropriate metatranscriptomic or genomic data (as needed for the second).
The following protocol is an example of the third method; it is informed by and aims to simulate environments of interest. Three naïve media recipes targeting different anaerobic microorganism cultures acquired in the field are presented in parallel within the protocol. The three cultures represented are mixed cultures originating from soil (hereafter, soil mixed culture), mixed cultures originating from within a borehole (hereafter, borehole mixed culture), and an isolated methanogen originating from within a borehole (hereafter, borehole isolated methanogen). The compound identities and amounts in the media recipes shared here are meant as a beginning guide; they are able and encouraged to be customized to the reader's environment and microorganisms of interest.
1. Production of customizable anaerobic liquid medium
2. in situ acquisition of environmental anaerobic microorganisms
3. in vitro growth of anaerobic microorganisms acquired in the field
Here we show results from a bioreactor study using a borehole mixed culture medium preparation method and a bioreactor setup method as described herein. The borehole mixed culture medium was modified to contain as a carbon source a slurry of corn cobs processed by Oxidative Hydrothermal Dissolution (OHD)13,14. Modified borehole mixed culture medium was pumped into the bioreactor for 44 days at a rate of 0.4 mL/min. On day 23, an inoculum sourced from borehole BLM-1 in the Death Valley region of Nevada, USA, was added15. Liquid samples from the bioreactor were collected in a sampling catch bottle on days 1, 8, 15, 22, 23 (after inoculation), 30, 37, and 44.
To track the general status of the inoculum culture (and pre-inoculum autoclave-surviving denizens of the bioreactor sediment), bioreactor liquid samples were observed by direct cell counts. Direct cell counts of sampled liquid were done with a Petroff-Hauser hemocytometer. To learn the identity of microbial members of the bioreactor, DNA was extracted from bioreactor liquid samples, and the 16S rRNA genes were analyzed by Next Generation Sequencing (NGS). The NGS data were processed in-house using mothur following the MiSeq SOP16.
Before inoculation, direct cell counts were often below the detection limit (b.d.l.) of 1.0 × 106 cells/mL. Day 23 (after inoculation) had a low cell count of 2.6 × 106 cells/mL, while the following days 30, 37, and 44 had cell counts over 1.0 × 107 cells/mL (Figure 3). Although cell counts were b.d.l. on days 1, 8, and 22, DNA was extracted for NGS from every time point indicating a basal presence of pre-inoculum microorganisms in the sediment even after autoclaving. The major genus present (determined by the number of reads in each Operational Taxonomic Unit (OTU)) was stable on days 8, 15, and 22, and was identified as Geobacillus. After inoculation with the BLM-1 inoculum on day 23, Geobacillus no longer dominated, and new major genera arose along with a multitude of minorly present genera, which for the most part, maintained a presence to the end of the study (Figure 4). From these, we conclude the BLM-1 inoculum was an active and dynamically shifting culture within the bioreactor.
An additional study of the NGS data revealed members of the "microbial dark matter." OTUs with assigned taxonomic names containing the words "unclassified" or "uncultured" were used as a proxy for OTUs containing members of the "microbial dark matter." NGS data were gathered for day 44 (the final day of post-inoculation with microorganisms from borehole BLM-1) and filtered to not contain OTUs that had reads in pre-inoculation data (days 1, 8, 15, or 22) as these OTUs were not likely to have been in the inoculum. The filtered day 44 NGS data contained 1,844 OTUs and 3,396 reads. Of these, 366 OTUs (20%) and 925 reads (27%) were unclassified at the phylum level, and 1252 OTUs (68%) and 2357 reads (69%) were unclassified or uncultured at the genus level. Though short-term, by this proxy, this pilot study supports the potential of a bioreactor with environmentally informed media to culture members of the "microbial dark matter."
Figure 1: Schematic of custom-designed borosilicate bioreactor used for anaerobic culturing. (A) Bioreactor view from one side. (B) Rotating A by 90° (clockwise) about the z-axis gives the view). (C) A top view of the bioreactor. The jacketed design allows for temperature control with water or oil. The two jacket ports can be seen on the right side of the view in B. The inner chamber can be filled with sediment of any type or can be left empty of sediment for planktonic culturing. Three sampling ports (seen on the right side of the view in A) make it possible to remove material at different depths of the bioreactor column. Standard 45, 18, and 14 GL openings can be sealed using Teflon or butyl septa that will hold a gastight seal for 1-6 months. Please click here to view a larger version of this figure.
Figure 2: Active bioreactor assembly. Glassware presented in photo from left to right are (A) 8 L carboy, (B) bioreactor (see Figure 1), and (C) non-sampling catch bottle. Please click here to view a larger version of this figure.
Figure 3: Direct cell counts from bioreactor study. Microbial populations within the bioreactor were monitored using a 0.02 mm Petroff-Hauser hemocytometer cell counting chamber. Inoculum was added at the start of day 23. Cell counts for days 1, 8, and 22 were below the detection limit. The effective detection limit of direct cell counting is 1 × 106 cells/mL. Abbreviation: b.d.l. = below the detection limit. Please click here to view a larger version of this figure.
Figure 4: Bioreactor Next Generation Sequencing. OTUs are represented in stacked bar graphs indicating the percentage of reads belonging to unique OTUs from each time point of the bioreactor study. Unique OTUs were assigned to their associated taxonomy at a genus cutoff. Genus 'Other' is defined as all genera less than 10% of reads in each time point. The total number of DNA reads represented is 506,358. The number of reads for each time point is listed at the top of the graph. Inoculum was added at the start of day 23. Abbreviation: OTU = Operational Taxonomic Unit. Please click here to view a larger version of this figure.
Compound | Amount for soil mixed culture | Amount for borehole mixed culture | Amount for borehole isolated methanogen |
Distilled water | 1000.0 mL | 1000.0 mL | 1000.0 mL |
HEPES | 3.600 g | – | – |
MOPS | – | – | 2.000 g |
MgCl2 ∙6 H2O | 0.400 g | 0.400 g | 0.400 g |
KCl | 0.500 g | 0.250 g | 0.500 g |
NH4Cl | 0.268 g | 0.268 g | 0.268 g |
Na2SO4 | – | 0.284 g | 1.500 g |
Na2HPO4 ∙2 H2O | – | – | 0.356 g |
1 M KH2PO4 at pH 6 | 1.0 mL | 1.0 mL | – |
1 M H3BO3 at pH 9.4 | 1.0 mL | 1.0 mL | 1.0 mL |
Trace minerals | 1.0 mL | – | 1.0 mL |
Vitamins | – | – | 1.0 mL |
1 mg/mL sodium resazurin | 0.4 mL | 0.3 mL | 0.4 mL |
pH adjustment to | – | 7.0 | 7.0 |
pH adjustment by | – | NaOH | KOH |
Incubation temperature | 25 °C | 60 °C | 47 °C |
Table 1: Media composition. List of compounds and physical parameters for each medium. The presented media are naïve; they do not contain carbon and energy sources as these can be specific to and should be informed by the reader's microorganisms and environment of interest. See the discussion section for ideas of possible carbon and energy source additions.
Supplemental File 1: Gas manifold setup. Please click here to download this File.
The medium production section of this protocol (section 1) owes its structure to the modified Hungate technique of Miller and Wolin17, which has been widely used since its publication. The practicality of this expanded protocol comes from its descriptive nature and pairing with the in situ acquisition of microorganisms. Culture bottles containing environmentally informed and simulated media have been used to successfully culture the following in situ-acquired former members of the "microbial dark matter": anaerobic subsurface bacteria Thermoanaerosceptrum fracticalcis strain DRI1318, Caldiatribacterium inferamans strain SIUC1 (accession number MT023787; manuscript in preparation), and Anaerothermus hephaesti strain SIUC3 (accession number MK618647; manuscript in preparation), and anaerobic subsurface bacterium uncharacterized strain DRI14 (accession number KR708540).
One focus of this protocol is that it is made to be highly adaptable to surface and subsurface anaerobic environments and microorganisms. First, the medium composition is encouraged to be altered to reflect the composition of the environment of interest. For example, 3,000 mg/L of NaCl can be added to the recipe when simulating an environment with ionic concentrations of 3,000 mg/L for both Na+ and Cl–; or 650 mL of sand and 650 mL of shale can be placed in the bioreactor when simulating an environment with a 1:1 volume ratio of sandstone to shale. Second, the medium composition can be modified to encourage the growth of specific microorganisms of interest. Particularly, naïve media like the three media in this paper are primed for and dependent upon such modifications. Examples of carbon and energy sources include fumarate18, peptone18, cellulose-containing organic matter19, acetate20, yeast extract19,20, xylitol (strain SIUC1), formate (strain SIUC3), and other potential sources such as H2/CO2, citrate, and glucose. Finally, the medium composition can be changed depending on the desired study. For example, the main interest of the study described in the representative results section was to discover how representative subsurface anaerobic microorganisms would respond to a unique mixed organic slurry (a slurry of corn cobs processed by OHD13,14); thus, its recipe contained this carbon source instead of more conventional options. Other potential amendments for specific studies include the addition of plastics, acids, and heavy metals, or other xenobiotics for bioremediation studies; the addition of recalcitrant carbon polymers such as cellulose and plastics for value-added depolymerized product studies; and the addition of manure and plant wastes for small-scale compost and other agricultural studies.
If post-reductant analysis of a medium is desired, such as measurements of pH, Oxidation Reduction Potential (ORP), and Dissolved Oxygen (DO), then this may be accomplished indirectly by sacrificing a representative number of medium bottles to confidently understand the character of that batch. Upon exposing a sealed medium bottle to air, it is strongly recommended that ORP and DO readings are taken under 1 min upon opening. Non-oxygen-dependent measurements (e.g., pH) can be taken non-urgently after the initial opening. Fastidious anaerobic microorganisms typically require ORP values < -200 mV for growth. If the medium results in an ORP value of > -200 mV and/or DO values >0.40 mg/L, additional Na2S (0.1-0.2 g/L) or another reductant (e.g., titanium citrate, L-cysteine, ascorbic acid) can be used to reduce the medium further.
Resazurin is often used as an oxygen indicator (as in this protocol); however, it is more accurately a redox indicator21, indicating the presence of oxygen indirectly as the oxygen changes the redox potential of a solution. Resazurin in solution initially appears blue in color. Upon exposure to reducing agents, the solution shifts irreversibly to a pink color. When the solution is further reduced, it reversibly shifts to colorless and returns to pink upon oxidization. The authors have observed instances where reduced media containing resazurin shifted from blue to pink but failed to shift to colorless. However, the authors have also observed that such media often shift to colorless after autoclaving, and that increased time spent boiling and flushing the media generally allows resazurin to shift to its colorless form after adding reductants. If desired, the concentration of oxygen in the medium can be verified with a dissolved oxygen probe.
All materials needed to run these bioreactors and the custom-made glass bioreactors themselves did not exceed ~$5K (this assumes support equipment is already available such as water baths, pumps, and incubators). These anaerobic cultivation techniques are economically friendly, as compared to commercial automated bioreactors which can start at >$10K. Additionally, many commercial bioreactors are switching to one-time-use reactor containers, which changes the dynamic of comparison and is not cost-effective for smaller research laboratories/universities to continually replace. Additionally, commercial systems may have manufacturing chemical biases (from the plastics used, grease on gaskets/fittings, or chemically treated water) that will affect the success of growing fastidious microorganisms. The low cost, customizability, and versatility to collect an anaerobic environmental sample, in combination with an informed media that can be inoculated, which results in generating enrichments for study, greatly enhances the chances of acquiring unique microorganisms.
The authors have nothing to disclose.
The authors would like to acknowledge the lineage of information and mentorship that has influenced/evolved these techniques over the years. Dr. Hamilton-Brehm as a former graduate student, postdoc, and current professor owes a debt of gratitude to those who took the time to teach anaerobic techniques: Dr. Mike Adams, Dr. Gerti Schut, Dr. Jim Elkins, Dr. Mircea Podar, Dr. Duane Moser, and Dr. Brian Hedlund. The Nature Conservancy and American Rivers supported this work through grants G21-026-CON-P and AR-CE21GOS373, respectively. Any opinions, findings, conclusions, or recommendations expressed in this paper are those of the authors and do not necessarily reflect the views of the Nature Conservancy or American Rivers. This work was supported by a grant from the SIU Advanced Energy Institute, which gratefully acknowledges funding awarded through the Advanced Energy Resource Board. NGS was performed by LC Sciences.
General Materials | |||
1 L borosillicate bottle | Fisher Scientific | ||
1 mL syringe with slip tip | Fisher Scientific | ||
10 mL glass pipette | Fisher Scientific | ||
100 mL culture bottle | Fisher Scientifc | ||
20 mm hand crimper | Fisher Scientifc | ||
23 G needle | Fisher Scientifc | ||
500 mL borosilicate bottle | Fisher Scientific | ||
Aluminum seal | Fisher Scientifc | ||
Cannula, 31.5 cm length | Fisher Scientific | ||
Cannula, 6 cm length | Fisher Scientifc | ||
Corer | Giddings Machine Company | Assembled from company parts | |
Gas manifold | Swagelok | Assembled from many different parts | |
Lighter | Lowe's | ||
N2 gas | Airgas | ||
Nitrile gloves | Fisher Scientific | ||
Rubber stopper (for GL45 bottles) | Glasgeratebau OCHS | ||
Rubber stopper (for culture bottles) | Ace Glass | ||
Stirring hot plate | Corning | ||
Trace minerals | ATCC | ||
Vitamins | ATCC | ||
Bioreactor-specific Materials | |||
#10 rubber stopper | Ace Glass | ||
#7 rubber stopper | Fisher Scientifc | ||
1 mL syringe with luer lock tip | Fisher Scientifc | ||
1/4" hose barb ball valve | Amazon | ||
10 mL syringe with luer lock tip | Fisher Scientifc | ||
3.5 L borosilicate bottle | Fisher Scientific | ||
5/16" – 1/4" hose barb adapter fitting | Amazon | ||
60 mL syringe with luer lock tip | Fisher Scientifc | ||
8 L borosillicate carboy | Allen Glass | ||
Angled hose connector for GL14 open top cap | Ace Glass | 7623-20 | |
Balloon | Party City | ||
Borosillicate bioreactor | Allen Scientific Glass | Custom made upon request | |
Drill | Lowe's | ||
Female luer lock adapter coupler | Amazon | ||
GL14 open top cap | Ace Glass | 7621-04 | |
GL18 open top cap | Ace Glass | 7621-08 | |
GL45 open top cap | Ace Glass | ||
PTFE faced silicone septum for GL14 open top cap | Ace Glass | 7625-06 | |
PTFE faced silicone septum for GL18 open top cap | Ace Glass | 7625-07 | |
Ring stand | Fisher Scientific | ||
Ring stand chain clamp | Amazon | ||
Ring stand clamp | Fisher Scientific | ||
Silicone tubing; 1/4" id, 1/2" od | Grainger | 55YG13 | |
Silicone tubing; 3/16" id, 3/8" od | Grainger | ||
Straight hose connector for GL14 open top cap | Ace Glass | 7623-22 | |
Three-way stopcock | Amazon | ||
Two-way stopcock | Amazon | ||
Ultra low flow variable flow mini-pump | VWR | ||
Water bath | Fisher Scientifc | ||
White rubber septum for 13-18 mm od tubes | Ace Glass | 9096-49 | |
Wire | Lowe's | ||
Zip tie | Lowe's |