A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms
Summary
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.”
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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 th…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
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