Microbial dark matter

Short description: Microbiological non-culture dependent methods

Microbial dark matter (MDM) comprises the vast majority of microbial organisms (usually bacteria and archaea) that microbiologists are unable to culture in the laboratory, due to lack of knowledge or ability to supply the required growth conditions.[1][2]

Microbial dark matter is analogous to the dark matter of physics and cosmology due to its elusiveness in research and importance to our understanding of biological diversity. Microbial dark matter can be found ubiquitously and abundantly across multiple ecosystems, but remains challenging to study due to difficulties in detecting and culturing these species, posing obstacles to research efforts.[3] It is difficult to estimate its relative magnitude, but the accepted gross estimate is that as little as one percent of microbial species in a given ecological niche are culturable.

In recent years, more effort has been directed towards deciphering microbial dark matter by means of recovering genome DNA sequences from environmental samples via culture independent methods such as single cell genomics[4] and metagenomics.[5] These studies have enabled insights into the evolutionary history and the metabolism of the sequenced genomes,[6][7] providing valuable knowledge required for the cultivation of microbial dark matter lineages. Other methods like culturomics have been developed to learn how to culture microbial dark matter alongside techniques like gene sequencing to identify species of previously undiscovered microbes.[8][9] However, microbial dark matter research remains comparatively undeveloped and is hypothesized to provide insight into processes radically different from known biology, new understandings of microbial communities, and increasing understanding of how life survives in extreme environments.[10]

Many members of the microbial dark matter have a wide diversity of habitats, ways of surviving and thriving in those habitats, and even novel products made through biochemical pathways.[11][12] This diversity includes microbes that live in very harsh environments, work together to support survival of the population, and even make new antibiotics to compete with other microbes.[11][13][14]

## Contents

* 1 History of the term
* 2 Methods for studying microbial dark matter
* 2.1 Metagenomics
* 2.2 Single-cell genomics
* 2.3 Improved culturing techniques
* 2.4 Computational tools
* 2.5 Culturomics
* 3 Microbes with highly unusual DNA
* 4 Microbial Dark Matter Diversity
* 4.1 Extremophiles
* 4.2 Symbionts
* 4.3 Metabolic Products
* 5 See also
* 6 References
* 7 External links

## History of the term

Our contemporary understanding of microbial dark matter was born from a field that still faced constraints with the cultivation of traditional microbes. One of the main constraints of this time was an over dependence on the use of culturing methods. This over reliance meant that a large amount of microbial diversity remained yet to be discovered. However in the late 20th century new developments in molecular techniques led to a surge in discovery of uncultured microbes. Despite this newfound diversity, a large majority of microbial species remain uncharacterized.[15] This fact was further proven by the development of advanced genomic sequencing techniques in the early 21st century which uncovered a larger amount of microbial diversity than previously thought.[10]

## Methods for studying microbial dark matter

### Metagenomics

Metagenomics is a technique in the field of microbial studies that enables us to sequence DNA directly from samples of microbial environments. This innovative technique allows us to identify the genetic material of unknown microbes and avoid overreliance on the use of culturing. The use of metagenomics differs from other microbial methods in that it uses a broad description through its use of bulk samples. This technique has expanded our understanding of microbial functions in ecosystems through the discovery of new genes and metabolic pathways.[16]

### Single-cell genomics

Methods of single-cell genomics have shown promise in supporting metagenomics approaches by allowing the study of individual microbial cells isolated from their natural environments, a method which has been employed to uncover the genomic and functional diversity within microbial communities, particularly those that cannot be cultured. Single-cell techniques have also successfully identified numerous new branches on the tree of life, providing insight into the gaps of current phylogenetic understanding and metabolic potential of these organisms.[17]

### Improved culturing techniques

Despite the rise of culture-independent methods as successful methods for dark matter research, improvements in culturing techniques remain both relevant and necessary to further current understanding of MRM microbes. To this point, developments in methods such as highly specific growth media to mimic natural microbial environments and co-culturing of synergistic microbial species have shown success in studying previously unculturable microbes. These advancements also serve to facilitate the application of MRM research into biotechnological and physiological uses.[18]

### Computational tools

Genomic studies produce vast amounts of data to be analyzed. This analysis requires the use of advanced computational components. The scientific subdiscipline of bioinformatics used computational technology to collect genomes and conduct analysis on metabolic pathways. In recent years, research on artificial intelligence and machine learning has produced new ways to increase our ability to predict the behavior of microbial species using their genetic data.[19] Metagenome-assembled genomes (MAGs) are one example of the emerging technology, which from the combined genomic material of diverse microbial communities group assembled DNA sequence fragments; enabling the reconstruction of genomes by using computational binning methods.[20][21] These new developments in the world of computational tools have allowed us to further understand the structure and dynamics present in microbial communities.

### Culturomics

Limitations imposed by both culture-independent methods and traditional culturing methods have caused the rise of a new strategy to improve recovery and identification of microbial dark matter.[8] Culturomics aims to create environments that simulate the natural conditions in which microbes can be found so they can be isolated, identified, and analyzed.[9] Culturomics relies on high-throughput culture conditions varying in media, pH, temperature, and atmospheric conditions to enable the culturing and isolation of large and diverse samples simultaneously.[8] This is used in conjunction with matrix-assisted laser desorption mass spectrometry (MALDI-ToF) for identification of isolated species.[9] This can be assisted by 16S rRNA gene sequencing in order for further taxonomic identification if needed.[22] Culturomics has already yielded significant results in culturing microbial dark matter, with a singular study resulting in the discovery of 31 new bacterial species and genera, as well as a total of 416 previously unculturable phylotypes identified.[23]

## Microbes with highly unusual DNA

It has been suggested that certain microbial dark matter genetic material could belong to a new (i.e., fourth) domain of life,[24][25] although other explanations (e.g., viral origin) are also possible, which has ties with the issue of a hypothetical shadow biosphere.[26]

## Microbial Dark Matter Diversity

### Extremophiles

Molecular markers and sequencing techniques have played a key role in the discovery of extremophiles and expanded the study of microbial dark matter.[27] Many members of the microbial dark matter remain unculturable due either to their preference or requirement to grow in harsh, extreme environments, and it is likely that a vast majority have unique adaptations to living in these environments.[11][28]

One member of the extremophiles, known as acidophiles, thrive in acidic, low pH conditions. In addition to acidic conditions, acidophiles are also capable of surviving in environments with increased levels of heavy metals, temperature, chloride-related stressors, and/or osmotic stress.[29] One major adaptation that acidophiles have made is the ability to gain energy through the oxidation of sulfur in their environment by creating sulfur oxidoreductase enzymes.[30]

Halophiles, which are bacteria, archaea, and some fungi that thrive in environments of very high salt concentrations, seem to have a unique strategy that involves building up salts and solutes from their environments within the cell itself, which helps to maintain osmotic pressure.[28][30] This helps protect the halophiles from drying out (desiccation), and from the cell shrinking due to the desiccation of the cytoplasm.[30]

### Symbionts

Through fairly recent metagenomic studies, it has been shown that many unculturable organisms may participate in symbiotic relationships with other living things in their environment, relying on one another to survive.[13] One such example of unculturable microbes acting in symbiosis comes from microbes that live in radioactive environments, wherein they make peroxidases to prevent highly reactive oxygen molecules from damaging themselves and other microbes in the environment, thus mutually protecting each other.[13] Some acidophiles may even encode for necessary metabolic pathways for other members of its genus, performing things like glycolysis and oxidation for other acidophiles.[31]

### Metabolic Products

Members of the microbial dark matter group seem to have notably unique biochemical pathways, allowing for the creation of many new products, such as antibiotics, enzymes, and more.[12][14] A notable example of a unique metabolic product from an uncultured organism is teixobactin, which seems to be a new class of antibiotic that may have wide-spectrum effects against many bacteria.[14] As with the symbionts mentioned before, other metabolic products are used by microbial dark matter to combat reactive radical oxygen species, such as peroxidases and other general antioxidants.[32][12] Many more unique metabolic products are likely to be found as more members of the microbial dark matter continue to be sequenced and studied, despite the difficulty in studying their genomes and their lack of cultivability.[14][12]

## See also

* Biological dark matter
* Microbial ecology
* Microbiology

## References

1. ↑ Filee, J.; Tetart, F.; Suttle, C. A.; Krisch, H. M. (2005). "Marine T4-type bacteriophages, a ubiquitous component of the dark matter of the biosphere". Proceedings of the National Academy of Sciences 102 (35): 12471–12476. doi:10.1073/pnas.0503404102. ISSN 0027-8424. PMID 16116082. Bibcode: 2005PNAS..10212471F.
2. ↑ University of Tennessee at Knoxville (25 September 2018). "Study: Microbial dark matter dominates Earth's environments". Eurekalert! (Press release). Retrieved 26 September 2018. `{{cite press release}}`: Cite has empty unknown parameter: `|1=` (help)
3. ↑ Dorminey, Bruce. "Microbial 'Dark Matter' Still Eludes Earth's Astrobiologists" (in en). https://www.forbes.com/sites/brucedorminey/2023/11/23/microbial-dark-matter-still-eludes-earths-astrobiologists/.
4. ↑ Rinke, Christian (2018). "Single-Cell Genomics of Microbial Dark Matter". in Robert G. Beiko. Microbiome Analysis: Methods and Protocols. Methods in Molecular Biology. 1849. New York: Springer New York. pp. 99–111. doi:10.1007/978-1-4939-8728-3_7. ISBN 978-1-4939-8728-3.
5. ↑ Jiao, Jian-Yu; Liu, Lan; Hua, Zheng-Shuang; Fang, Bao-Zhu; Zhou, En-Min; Salam, Nimaichand; Hedlund, Brian P; Li, Wen-Jun (2021-03-01). "Microbial dark matter coming to light: challenges and opportunities". National Science Review 8 (3): –280. doi:10.1093/nsr/nwaa280. ISSN 2095-5138. PMID 34691599.
6. ↑ Hedlund, Brian P.; Dodsworth, Jeremy A.; Murugapiran, Senthil K.; Rinke, Christian; Woyke, Tanja (2014). "Impact of single-cell genomics and metagenomics on the emerging view of extremophile "microbial dark matter"". Extremophiles 18 (5): 865–875. doi:10.1007/s00792-014-0664-7. ISSN 1431-0651. PMID 25113821.
7. ↑ Rinke, Christian; et, al. (2013). "Insights into the phylogeny and coding potential of microbial dark matter". Nature 499 (7459): 431–437. doi:10.1038/nature12352. PMID 23851394. Bibcode: 2013Natur.499..431R.
8. ↑ 8.0 8.1 8.2 Li, Zheng-Hui; Li, Shuang; Chen, Hai-Bo; Ji, Yi-Zhi; Zhang, Chong (2026-03-01). "The advances of strategies and technologies in high-throughput microbial culturomics". Biotechnology Advances 87. doi:10.1016/j.biotechadv.2025.108783. ISSN 0734-9750. https://www.sciencedirect.com/science/article/pii/S0734975025002691.
9. ↑ 9.0 9.1 9.2 "Error: no `|title=` specified when using {{Cite web}}". https://mah.bioscientifica.com/view/journals/mah/1/1/MAH-23-0001.xml.Error:+no+`|title=`+specified when+using+{{[[Template:Cite+web|Cite+web]]}}&rft.atitle=&rft_id=https://mah.bioscientifica.com/view/journals/mah/1/1/MAH-23-0001.xml&rfr_id=info:sid/en.wikibooks.org:Biology:Microbial_dark_matter">
10. ↑ 10.0 10.1 Bernard, Guillaume; Pathmanathan, Jananan S; Lannes, Romain; Lopez, Philippe; Bapteste, Eric (2018). "Microbial Dark Matter Investigations: How Microbial Studies Transform Biological Knowledge and Empirically Sketch a Logic of Scientific Discovery". Genome Biology and Evolution 10 (3): 707–715. doi:10.1093/gbe/evy031. PMID 29420719.
11. ↑ 11.0 11.1 11.2 Ji, Minhui; Ma, Bingda; Dong, Jiayu; Liu, Shan; Shi, Ying; Bu, Meiting; Wang, Luoyi; Liu, Ling (2025-10-20). "Mining Microbial Dark Matter: Advanced Cultivation Techniques for Bioactive Compound Discovery". Pharmaceuticals (Basel, Switzerland) 18 (10): 1583. doi:10.3390/ph18101583. ISSN 1424-8247. PMID 41155696. PMC 12566907. https://pmc.ncbi.nlm.nih.gov/articles/PMC12566907/.
12. ↑ 12.0 12.1 12.2 12.3 Alanzi, Abdullah R. (2024-11-18). "Exploring Microbial Dark Matter for the Discovery of Novel Natural Products: Characteristics, Abundance Challenges and Methods". Journal of Microbiology and Biotechnology 35: e2407064. doi:10.4014/jmb.2407.07064. ISSN 1738-8872. PMID 39639495. PMC 11813339. https://pmc.ncbi.nlm.nih.gov/articles/PMC11813339/.
13. ↑ 13.0 13.1 13.2 Marzban, Gorji; Tesei, Donatella (2025-04-13). "The Extremophiles: Adaptation Mechanisms and Biotechnological Applications". Biology 14 (4): 412. doi:10.3390/biology14040412. ISSN 2079-7737. PMID 40282277. PMC 12024854. https://pmc.ncbi.nlm.nih.gov/articles/PMC12024854/.
14. ↑ 14.0 14.1 14.2 14.3 Ji, Minhui; Ma, Bingda; Dong, Jiayu; Liu, Shan; Shi, Ying; Bu, Meiting; Wang, Luoyi; Liu, Ling (2025-10-20). "Mining Microbial Dark Matter: Advanced Cultivation Techniques for Bioactive Compound Discovery". Pharmaceuticals (Basel, Switzerland) 18 (10): 1583. doi:10.3390/ph18101583. ISSN 1424-8247. PMID 41155696. PMC 12566907. https://pmc.ncbi.nlm.nih.gov/articles/PMC12566907/.
15. ↑ Amann, R. I., Ludwig, W., & Schleifer, K. H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 59(1), 143-169.
16. ↑ Quince, Christopher; Walker, Alan W.; Simpson, Jared T.; Loman, Nicholas J.; Segata, Nicola (2017). "Shotgun metagenomics, from sampling to analysis". Nature Biotechnology 35 (9): 833–844. doi:10.1038/nbt.3935. PMID 28898207.
17. ↑ Stepanauskas, Ramunas (2012). "Single cell genomics: An individual look at microbes". Current Opinion in Microbiology 15 (5): 613–620. doi:10.1016/j.mib.2012.09.001. PMID 23026140.
18. ↑ Zengler, Karsten; Toledo, Gerardo; Rappé, Michael; Elkins, James; Mathur, Eric J.; Short, Jay M.; Keller, Martin (2002). "Cultivating the uncultured". Proceedings of the National Academy of Sciences 99 (24): 15681–15686. doi:10.1073/pnas.252630999. PMID 12438682. Bibcode: 2002PNAS...9915681Z.
19. ↑ Nayfach, Stephen; Roux, Simon; Seshadri, Rekha; Udwary, Daniel; Varghese, Neha; Schulz, Frederik; Wu, Dongying; Paez-Espino, David et al. (2021). "A genomic catalog of Earth's microbiomes" (in en). Nature Biotechnology 39 (4): 499–509. doi:10.1038/s41587-020-0718-6. PMID 33169036.
20. ↑ Jiao, Jian-Yu; Liu, Lan; Hua, Zheng-Shuang; Fang, Bao-Zhu; Zhou, En-Min; Salam, Nimaichand; Hedlund, Brian P; Li, Wen-Jun (2021-03-19). "Microbial dark matter coming to light: challenges and opportunities" (in en). National Science Review 8 (3). doi:10.1093/nsr/nwaa280. ISSN 2095-5138. PMID 34691599. PMC 8288357. https://academic.oup.com/nsr/article/doi/10.1093/nsr/nwaa280/6027462.
21. ↑ Vollmers, John; Wiegand, Sandra; Lenk, Florian; Kaster, Anne-Kristin (2022-07-22). "How clear is our current view on microbial dark matter? (Re-)assessing public MAG & SAG datasets with MDMcleaner" (in en). Nucleic Acids Research 50 (13): e76–e76. doi:10.1093/nar/gkac294. ISSN 0305-1048. PMID 35536293. PMC 9303271. https://academic.oup.com/nar/article/50/13/e76/6583244.
22. ↑ Chen, Lingxi; An, Yujie; Huang, Kailang; Wang, Junhong; Zhang, Yuanyuan; Wang, Jiakun (January 2024). "Culturomics: Bringing culture back to the forefront for effective gastrointestinal bacterial capture" (in en). Animal Nutriomics 1: e12. doi:10.1017/anr.2024.17. ISSN 2977-3776. https://www.cambridge.org/core/journals/animal-nutriomics/article/culturomics-bringing-culture-back-to-the-forefront-for-effective-gastrointestinal-bacterial-capture/B9BEB4900D14E84ED2A14BE16F02DB41.
23. ↑ Lagier, J.-C.; Armougom, F.; Million, M.; Hugon, P.; Pagnier, I.; Robert, C.; Bittar, F.; Fournous, G. et al. (2012-12-01). "Microbial culturomics: paradigm shift in the human gut microbiome study" (in English). Clinical Microbiology and Infection 18 (12): 1185–1193. doi:10.1111/1469-0691.12023. ISSN 1198-743X. PMID 23033984. https://www.clinicalmicrobiologyandinfection.org/article/S1198-743X(14)60804-1/fulltext.
24. ↑ "Stalking the fourth domain in metagenomic data: searching for, discovering, and interpreting novel, deep branches in marker gene phylogenetic trees". PLOS ONE 6 (3). March 2011. doi:10.1371/journal.pone.0018011. PMID 21437252. Bibcode: 2011PLoSO...618011W.
* Colin Barras (18 March 2011). "Biology's 'dark matter' hints at fourth domain of life". New Scientist. https://www.newscientist.com/article/dn20265-biologys-dark-matter-hints-at-fourth-domain-of-life/.
25. ↑ "Highly divergent ancient gene families in metagenomic samples are compatible with additional divisions of life". Biology Direct 10. October 2015. doi:10.1186/s13062-015-0092-3. PMID 26502935.
* Colin Barras (11 November 2015). "Mystery microbes in our gut could be a whole new form of life". New Scientist. https://www.newscientist.com/article/mg22830474-800-mystery-microbes-in-our-gut-could-be-a-whole-new-form-of-life/.
26. ↑ Schulze-Makuch, Dirk (Feb 28, 2017). "Could Alien Life Be Hidden All Around Us?". Smithsonian Institution. Smithsonian Magazine. https://www.smithsonianmag.com/air-space-magazine/could-alien-life-be-hidden-all-around-us-180962328/.
27. ↑ Bernard, Guillaume; Pathmanathan, Jananan S.; Lannes, Romain; Lopez, Philippe; Bapteste, Eric (2018-03-01). "Microbial Dark Matter Investigations: How Microbial Studies Transform Biological Knowledge and Empirically Sketch a Logic of Scientific Discovery". Genome Biology and Evolution 10 (3): 707–715. doi:10.1093/gbe/evy031. ISSN 1759-6653. PMID 29420719. PMC 5830969. https://pmc.ncbi.nlm.nih.gov/articles/PMC5830969/.
28. ↑ 28.0 28.1 Hedlund, Brian P.; Dodsworth, Jeremy A.; Murugapiran, Senthil K.; Rinke, Christian; Woyke, Tanja (2014-09-01). "Impact of single-cell genomics and metagenomics on the emerging view of extremophile "microbial dark matter"" (in en). Extremophiles 18 (5): 865–875. doi:10.1007/s00792-014-0664-7. ISSN 1433-4909.
29. ↑ Aliyu, Godwin O.; Ezugworie, Flora N.; Onwosi, Chukwudi O.; Nnamchi, Chukwudi I.; Ekwealor, Chito C.; Igbokwe, Victor C.; Sani, Rajesh K. (2024-12-01). "Multi-stress adaptive lifestyle of acidophiles enhances their robustness for biotechnological and environmental applications". Science of The Total Environment 954. doi:10.1016/j.scitotenv.2024.176190. ISSN 0048-9697. https://www.sciencedirect.com/science/article/pii/S0048969724063460.
30. ↑ 30.0 30.1 30.2 Marzban, Gorji; Tesei, Donatella (2025-04-13). "The Extremophiles: Adaptation Mechanisms and Biotechnological Applications". Biology 14 (4): 412. doi:10.3390/biology14040412. ISSN 2079-7737. PMID 40282277. PMC 12024854. https://pmc.ncbi.nlm.nih.gov/articles/PMC12024854/.
31. ↑ Hedlund, Brian P.; Dodsworth, Jeremy A.; Murugapiran, Senthil K.; Rinke, Christian; Woyke, Tanja (2014-09-01). "Impact of single-cell genomics and metagenomics on the emerging view of extremophile "microbial dark matter"" (in en). Extremophiles 18 (5): 865–875. doi:10.1007/s00792-014-0664-7. ISSN 1433-4909.
32. ↑ Marzban, Gorji; Tesei, Donatella (2025-04-13). "The Extremophiles: Adaptation Mechanisms and Biotechnological Applications". Biology 14 (4): 412. doi:10.3390/biology14040412. ISSN 2079-7737. PMID 40282277. PMC 12024854. https://pmc.ncbi.nlm.nih.gov/articles/PMC12024854/.

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## External links

* Microbial Dark Matter project site

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1. ↑ Quince, C., et al. (2017). Shotgun metagenomics, from sampling to analysis. Nature Biotechnology, 35(9), 833-844. https://doi.org/10.1038/nbt.3935

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