preparation of anaerobic liquid medium in a carboy for culturing environmental anaerobic microorganisms in a bioreactor

환경 미생물 배양을 위한 현장 시뮬레이션 혐기성 방법

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.

저자 스포트라이트: In Situ Culturing을 통해 미지의 환경에서 육상 혐기성 미생물의 미스터리 풀기

환경적으로 획득한 혐기성 미생물을 배양하기 위한 In Situ Informed Simulated Medium 형식 세트

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.

생물 반응기에서 환경 혐기성 미생물을 배양하기 위해 Carboy에서 혐기성 액체 배지의 준비

Carboy 설정을 준비하려면 6.4cm 호스 미늘 볼의 밸브를 엽니다. 2cm 길이의 실리콘 튜브를 호스 미늘 볼 밸브의 양쪽 끝에 부착합니다. 7.9-6.4mm 호스 바브 어댑터 피팅을 어셈블리의 한쪽 끝에 부착합니다.어셈블리의 다른 쪽 끝을 8리터 Carboy의 호스 미늘에 부착합니다. 지퍼 타이를 사용하여 Carboy의 호스 바브와 튜브 사이의 연결부와 호스 바브 어댑터 피팅과 튜브 사이의 연결을 고정합니다. 필요한 미디어 구성 요소를 측정하고 8리터 Carboy에 추가합니다.필요한 pH를 조정한 후 핫 플레이트에서 저어주어 성분을 균질하게 혼합하고 액체를 30분 동안 끓입니다. 다음으로, 멸균 10밀리리터 유리 피펫을 질소 가스 매니폴드 설정에 부착합니다. 매니폴드를 열어 산소를 배출하고 질소 가스가 흘러나오도록 합니다.불을 끈 후 피펫 팁을 액체에 넣고 질소 가스로 30분 동안 적당히 격렬하지만 넘치지 않도록 씻어냅니다. 그런 다음 피펫 팁을 Carboy의 헤드스페이스로 당깁니다. 중탄산나트륨 2g과 황화나트륨 비수화물 4.8g을 넣고 레사주린이 무색으로 변할 때까지 기다립니다.레사주린이 무색이 되면 유리 피펫을 제거하고 즉시 카보이를 10번 스토퍼로 덮습니다. 스토퍼 위와 Carboy 가장자리 주위에 와이어를 비틀어 스토퍼를 고정합니다.

preparation-anaerobic-liquid-medium-carboy-for-culturing

Research

JoVE Journal

Environment

Preparation of Anaerobic Liquid Medium in a Carboy for Culturing Environmental Anaerobic Microorganisms in a Bioreactor

84 조회수.  Southern Illinois University Carbondale. Carboy 설정을 준비하려면 6.4cm 호스 미늘 볼의 밸브를 엽니다. 2cm 길이의 실리콘 튜브를 호스 미늘 볼 밸브의 양쪽 끝에 부착합니다. 7.9-6.4mm 호스 바브 어댑터 피팅을 어셈블리의 한쪽 끝에 부착합니다.어셈블리의 다른 쪽 끝을 8리터 Carboy의 호스 미늘에 부착합니다. 지퍼 타이를 사용하여 Carboy의 호스 바브와 튜브 사이의 연결부와 호스 바브 어댑터 피팅과 튜브 사이의 ?...

비디오: 생물 반응기에서 환경 혐기성 미생물을 배양하기 위해 Carboy에서 혐기성 액체 배지의 준비

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 ...