in vitro culturing of environmental sediment sample for the growth of anaerobic microorganisms

Método anaeróbico simulado in situ para cultura de micróbios ambientais

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 &#34;Great Plate Count Anomaly&#34;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 &#34;microbial dark matter&#34;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 &#34;microbial dark matter.&#34; Many members of the &#34;microbial dark matter&#34; 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 &#34;microbial dark matter,&#34; 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 &#34;microbial dark matter,&#34; 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&#160;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&#39;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 na&#239;ve 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&#39;s environment and microorganisms of interest.

Autor em destaque: Desvendando os mistérios dos microrganismos anaeróbicos terrestres em ambientes desconhecidos por cultura in situ

Um conjunto de formatos de meios simulados informados in situ para cultivo de microrganismos anaeróbios ambientalmente adquiridos

Our research focuses on terrestrial surface and subsurface microorganisms, culturing them and investigating their genomes and phenotypes. Of particular focus are microorganisms in environments that are not well understood, like those in the deep subsurface and in certain wetlands. Most of the microorganisms we culture are anaerobic, which means the culturing process generally requires more time and attention than processes for more commonly-used aerobic microorganisms.We hope the audiovisual presentation of anaerobic medium preparation will help researchers new to the technique learn it relatively quickly and easily. This medium preparation is based on the modified Hungate technique of Miller and Wolin. Culturing fastidious microbes is tedious and problematic.Having more tools to grow them is very welcome to the scientific community. Future research in general will be similar to our current research of understudied terrestrial microorganisms. There are still many new microbes to discover and study.

Cultivo in vitro de amostra de sedimento ambiental para o crescimento de microrganismos anaeróbios

Para começar, coloque um frasco estéril de nitrogênio e os frascos de cultura, e um frasco de coleta de amostras. Pinge 100% de etanol nas rolhas e ateie fogo nelas usando um isqueiro. Quando as rolhas não estiverem mais em chamas, insira uma seringa de um mililitro equipada com uma agulha calibre 23 no frasco de nitrogênio e retire aproximadamente um mililitro de nitrogênio.Extraia a agulha e pressione lentamente o êmbolo para liberar o nitrogênio. Insira imediatamente a agulha no frasco de coleta de amostra e retire 0,5 mililitros da amostra ambiental. Inocular a amostra colhida no frasco de cultura contendo o meio.Gire suavemente o frasco e coloque-o em uma incubadora na temperatura desejada. As contagens diretas de células antes da inoculação estavam frequentemente abaixo do limite de detecção. Após a inoculação, a contagem de células aumentou mais de 10 milhões de células por mililitro nos dias 30, 37 e 44.Embora a contagem de células estivesse abaixo do limite de detecção antes da adição do inóculo, NGS indicou a presença basal de microrganismos pré-inóculo no sedimento e o gênero majoritário geobacillus foi estável nos dias oito, 15 e 22. Após a inoculação com o inóculo BLM-1 no dia 23, geobacillus não dominou mais, e um novo gênero principal surgiu juntamente com uma infinidade de gêneros menos presentes.

in-vitro-culturing-environmental-sediment-sample-for-growth-anaerobic

Research

JoVE Journal

Environment

In Vitro Culturing of Environmental Sediment Sample for the Growth of Anaerobic Microorganisms

89 visualizações.  Southern Illinois University Carbondale. Para começar, coloque um frasco estéril de nitrogênio e os frascos de cultura, e um frasco de coleta de amostras. Pinge 100% de etanol nas rolhas e ateie fogo nelas usando um isqueiro. Quando as rolhas não estiverem mais em chamas, insira uma seringa de um mililitro equipada com uma agulha calibre 23 no frasco de nitrogênio e retire aproximadamente um mililitro de nitrogênio.Extraia a agulha e pressione lentamente o êmbolo para liberar o nitrogênio. Insira imediatamente a agulh...

Vídeo: Cultivo in vitro de amostra de sedimento ambiental para o crescimento de microrganismos anaeróbios

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.

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 ...