The oceans are rich in microorganisms, with approximately 104–107 cells/mL in seawater and 103–1010 cells/cm3 in sediments.1 The culture and isolation of marine microorganisms are difficult in the laboratory. Modifying existing culture methods and using advanced culture-independent techniques can overcome this barrier.
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Introduction
A decline in the discovery of novel metabolites using terrestrial microorganisms and antibiotic resistance has led researchers to find bioactive substances in marine microorganisms. Marine microorganisms grow in unique, diverse, and extreme habitats, such as hot springs (thermophiles), cold arctic water (psychrophiles), acidic water (acidophiles), hypersaline water (halophiles), alkaline water (alkalophiles), and under high pressure (barophiles) and have adapted themselves to survive in inhospitable conditions.
For example, psychrophiles have a membrane lipid composition containing unsaturated fatty acids, and cold shock proteins are bound to the RNA structure for stability. Halophiles produce unique proteins that are stable and active in high-salt environments.2
Approximately 0.001%–2% of marine microorganisms form colonies using standard culture methods, and 90%–99% of microorganisms are “viable but not culturable” (VBNC) due to a lack of suitable growth conditions, reduced growth rates, need for metabolites produced by other microbes, and dormant cells.3
Advancements in conventional culture media and cultivation methods and the development of advanced techniques have promoted the growth of VBNC species.
Techniques for Marine Microbe Isolation
Enrichment Culture
Low-abundance microorganisms can be isolated using an enrichment culture wherein specific nutrients and environmental conditions are provided for the growth of a particular microbe.
As in nature, organisms compete for low substrate concentrations with low specific growth rates; chemostats using sterile growth media with one particular limiting substrate are used for enrichment.4
Mimicking the Natural Environment
Culture media modifications to mimic the natural environment include changes in the composition and concentration of nutrients, pressure, or pH. Filtered seawater with different percentages of agar, organic substrates (casein, laminarin, etc.), and inorganic substrates have been used.
For example, seawater with 1.5% agar for solidification and ammonium chloride has successfully isolated Alphaproteobacteria.5
Dilution-to-Extinction
The sample is diluted with filtered seawater obtained from the same site as the sample until 1–10 cells are left. The cells are inoculated in a physically separate well. Cells are isolated from a mixed population by reducing competition with other bacteria. SAR11 was isolated using this method.4
It is crucial to practice aseptic techniques, including personal protective equipment, equipment sterilization, aseptic transfer techniques, and a controlled environment to avoid contamination of samples or cultures to maintain the reliability and reproducibility of the research outcomes.
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Beyond the Plate: Unveiling the "Unculturable" Majority
A major limitation of culture-dependent techniques is that 99% of marine microorganisms resist growth on standard media. Culture-independent methods have been developed to overcome the challenge of microbial uncultivability.
Single-cell Approach
A single-cell approach relates a microorganism's identity to its function. Identity probes, such as fluorescence in situ hybridization (FISH) probes, are used to identify the microbe before microscopy or fluorescence-activated cell sorting.
Functional probes target the structure or physiology of the cell, such as propidium iodide, which provides information on the condition of the cell membrane. Activity probes include radioactivity-labeled nutrients and will indicate the cells taking up the substrate when combined with micro autoradiography.
Stable-isotope probing (SIP) involves introducing an isotope-labeled substrate into a microbial community to identify substrate uptake by fatty acid or nucleic acid extraction.
This determines the molecules where the substrate is incorporated. A DNA-stable-SIP with 13C-labelled substrates, such as amino acids and cyanobacteria lysates, was used to determine the bacteria and archaea lineages responsible for substrate incorporation.4
Metagenomics
Metagenomics analyzes the microbial diversity of an environmental sample and has shown high species diversity in marine samples. The metagenomic library construction includes the isolation of environmental DNA, shotgun cloning into a vector, transformation, and screening for positive clones.
High throughput screening of metagenomic libraries enables the identification of individual clones containing DNA regions encoding potentially novel genetic clusters. PCR amplification of the DNA segment within a specific clone enables the identification of the gene responsible for the observed phenotype.
The bacterial populations associated with marine sponges have been identified using metagenomic analysis. The drawback to this approach is the absence of reference genomes and sequences and the availability of suitable hosts for the heterologous expression of these unknown DNA sequences.4
Obstacles to Marine Microbe Isolation
The challenges associated with culturing marine microorganisms are as follows:
- Microorganisms interact with their natural environment, which may be lost during laboratory culture. For example, the fast-growing microbial species will dominate those that are slow-growing, thereby creating an imbalance in cell-cell communication. Some microbes may produce inhibitory compounds causing the inactivation of cells in their immediate vicinity.
- As few marine microbes are in culture, limited knowledge exists on the substrates used or their concentrations in the ocean. This prevents microbial growth on the substrates used in the laboratory setting.
- Marine bacteria have evolved under oligotrophic conditions; thus, the high concentrations of substrates used in the laboratory may prove toxic.
- Patience is key as seen with the SAR11 clade, a dominant bacterioplankton community, wherein it took >10 years to identify the first strain of SAR11.5
Strategies to Overcome the Cultivation Barrier
Consortia
Traditional culture methods disrupt cell-cell communication, and microbes whose growth depends on signal exchanges with other microbes will fail to grow. Diffusion chambers can target a consortium.
A semipermeable membrane separates the bacterial assemblage placed in the diffusion chamber from the nutrient source. The nutrients diffuse through the chamber, and the inhibitory metabolic end-products diffuse out, increasing the cell density.5
Microencapsulation
Microencapsulation is a method where gel microdroplets are prepared by dispersing agarose, mixed with bacteria-containing water from the natural environment, into an oil phase to prepare an emulsion. Rapid cooling of the emulsion solidifies the molten agarose, and continuous stirring causes microdroplet formation, some of which contain single bacterial cells.
The advantage of encapsulation is the physical separation of a single cell from other cells. Agarose is porous and allows nutrients to diffuse in and waste products to diffuse out; thus, the growth is unaffected despite the physical constraints. Many bacteria belonging to the 16s rRNA clade have been isolated via microencapsulation. 5
Coculture
Coculture is a strategy where unknown biotic factors may contribute to cultivating uncultured microbes. Microorganisms forming colonies in a diffusion chamber could grow on a Petri dish only in the presence of other species from the same environment.
Short peptides, siderophores, quinones, and γ-aminobutyric acid are crucial biotic factors. Helper microbes also enhance the growth of unculturable microorganisms. For example, they secrete catalase to scavenge reactive oxygen species to remove toxic compounds from other microorganisms.6
Resuscitation
Dormant bacteria can be revived using a resuscitation-promoting factor, Rpf. Rpf likely remodels the peptidoglycan in cell walls by generating muropeptides, which act as a wake-up call for dormant bacteria and stimulate their growth.6
The Future of Marine Microbe Isolation: A Collaborative Effort
Improvements in the conventional culture-based methods and culture-independent methods have drastically improved microbe cultivation. The combination of culture-dependent and culture-independent techniques will not only overcome the individual drawbacks but also provide a clear picture of the microbial diversity, functions, and interactions.
Steps to strengthen research partnerships between local and international communities and between researchers (in the fields of microbiology, molecular biology, and biochemistry) and industry will help eliminate the bottlenecks in marine microbe culture and isolation.
References
- Wang F, Li M, Huang L, Zhang XH. (2021) Cultivation of uncultured marine microorganisms. Marine Life Science & Technology. May;3(2):117-20. https://doi.org/10.1007/s42995-021-00093-z
- Poli A, Finore I, Romano I, Gioiello A, Lama L, Nicolaus B. Microbial diversity in extreme marine habitats and their biomolecules. Microorganisms. 2017 May 16;5(2):25. https://doi.org/10.3390/microorganisms5020025
- Rocha-Martin J, Harrington C, Dobson AD, O’Gara F. Emerging strategies and integrated systems microbiology technologies for biodiscovery of marine bioactive compounds. Marine drugs. 2014 Jun 10;12(6):3516-59. https://doi.org/10.3390/md12063516
- European Science Foundation. Marine microbial diversity and its role in ecosystem functioning and environmental change. 2012 May. Available from: http://archives.esf.org/fileadmin/Public_documents/Publications/MarineBoard_PP17_microcean.pdf
- Joint I, Mühling M, Querellou J. Culturing marine bacteria–an essential prerequisite for biodiscovery. Microbial biotechnology. 2010 Sep;3(5):564-75. https://doi.org/10.1111%2Fj.1751-7915.2010.00188.x
- Mu DS, Ouyang Y, Chen GJ, Du ZJ. Strategies for culturing active/dormant marine microbes. Marine Life Science & Technology. 2021 May;3:121-31. https://doi.org/10.1007%2Fs42995-020-00053-z
Further Reading
Innovative Static Culture Method for Microbial Growth and Analysis