Applications and Use

Over the past decade, we applied these cultivation platforms to a variety of environments, and a variety of questions, as summarized in the list of our publications:

D Jung, EY Seo, SS Epstein, Y Joung, J Han, VV Parfenova, OI Belykh, (2014) Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of Lake Baikal, Russia. FEMS microbiology ecology 90: 417-423

MV Sizova, PA Muller, D Stancyk, NS Panikov, M Mandalakis, A Hazen, T Hohmann, SN Doerfert, W Fowle, AM Earl, KE Nelson, SS Epstein (2014) Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov., obligately anaerobic bacteria from the human oral cavity, and emended description of the genus Oribacterium. International journal of systematic and evolutionary microbiology 64: 2642-2649

MV Sizova, P Muller, N Panikov, M Mandalakis, T Hohmann, A Hazen, T Hohmann, A Hazen, W Fowle, T Prozorov, DA Bazylinski, SS Epstein (2013) Stomatobaculum longum gen. nov., sp. nov., an obligately anaerobic bacterium from the human oral cavity. International journal of systematic and evolutionary microbiology 63: 1450-1456

D Jung, EY Seo, SS Epstein, Y Joung, JH Yim, H Lee, TS Ahn (2013) A new method for microbial cultivation and its application to bacterial community analysis in Buus Nuur, Mongolia. Archiv für Hydrobiologie 182: 171-181

SD Brown, AV Palumbo, N Panikov, T Ariyawansa, DM Klingeman, CM Johnson, ML Land, SM Utturkar, SS Epstein (2012) Draft genome sequence for Microbacterium laevaniformans strain OR221, a bacterium tolerant to metals, nitrate, and low pH. Journal of bacteriology 194 :3279-3280

MV Sizova, T Hohmann, A Hazen, BJ Paster, SR Halem, CM Murphy, NS Panikov, SS Epstein (2012) New approaches for isolation of previously uncultivated oral bacteria Applied and environmental microbiology 78: 194-203

Bollmann, A, Palumbo A.V., Lewis K., and Epstein, S.S. (2010). Isolation and physiology of bacteria from contaminated subsurface sediments. Appl. Eviron. Microbiol. 76: 7413–7419

Nichols,, D., Cahoon,N., Trakhtenberg, E.M., Pham, L, Mehta, A., Belanger, A., Kanigan, T.,  Lewis, K., and Epstein, S.S. (2010). Ichip for high-throughput in situ cultivation of “uncultivable” microbial species.  Appl. Environ. Microbiol., 76: 2445-2450

D’Onofrio, A., Crawford, J.M., Stewart, E.J., Witt, K., Gavrish, E., Epstein, S., Clardy, J., Lewis, K. (2010). Siderophores from neighboring organisms promote the growth of uncultured bacteria.  Chem. Biol. 17: 254-264

Gavrish, E., Bollmann, A., Epstein, S. S., and Lewis, K. (2008) A trap for in situ cultivation of filamentous actinobacteria. J. Microbiol. Methods 72: 257–262.

Nichols D., Lewis, K., Orjala, J., Mo, S., Ortenberg, R., O’Connor, P., Zhao, C., Vouros, P., Kaeberlein, T., and Epstein, S.S. (2008) Short peptide induces an “uncultivable” microorganism to grow in vitro. Appl. Environ. Microbiol., 74: 4889-4897

Bollmann, A., K.Lewis, and Epstein, S.S. (2007). Incubation of Environmental Samples in a Diffusion Chamber Increases the Diversity of Recovered Isolates. Appl. Environ. Microbiol. 73: 6386–6390.

Giovannoni, S. J, R. Foster, M. S. Rappé, and Epstein, S.S. (2007). New cultivation strategies bring more microbial plankton species into the laboratory. J. Oceanog. Soc. 20: 24-30

Current projects include large-scale investigation of ecology of Arctic microbial communities and of microbial assemblages forming on catheters, both in collaboration with C. Venter Institute. We are also capitalizing on having a large culture collection of formerly uncultivated microorganisms from various environments, including human body.  In particular, we are investigating the chemical nature of interactions among such microorganisms, and also between them and well known species. We are also continuing a former description of the most novel species we have isolated.

In spite of much progress achieved in understanding biology of the “uncultivables,” the key question remains unresolved: how to explain the Great Plate Count Anomaly? Why “uncultivable” microorganisms are “uncultivable”? We have learned how to grow at least some of them but the underlying reasons for this success are still unclear.  Resolution of the mechanisms of the phenomenon is one of the most important directions in the lab. We advanced new hypotheses on the nature of the Anomaly, and tested some of the hypotheses advanced by others.

One commonly held view is that the standard media are insufficient to support growth of the majority of microbial species.  The fact of our cultivation of many formerly uncultivated species in situ, and their successful domestication on such standard media in the lab speaks against this view.

Some argue that “uncultivables” may be slow growers missed by cultivation in standard Petri dishes.  We tested this hypothesis directly, and found no empirical evidence in its support (S Buerger, A Spoering, E Gavrish, C Leslin, L Ling, SS Epstein (2012) Microbial scout hypothesis and microbial discovery. Applied and environmental microbiology 78: 3229-3233; S Buerger, A Spoering, E Gavrish, C Leslin, L Ling, SS Epstein (2012) Microbial scout hypothesis, stochastic exit from dormancy, and the nature of slow growers. Applied and environmental microbiology 78: 3221-3228).

To address the apparent failure of these and other ideas on the nature of microbial “uncultivability,” we advanced a new one termed “microbial scout hypothesis”  (Epstein, S.S. (2009) Microbial awakenings. Nature 457: 1083). It posits that most microorganisms are in dormancy most of the time, and exit from dormancy in a stochastic fashion, not in response to environmental cues. This explains why perfectly healthy and viable cells would not grow in otherwise sufficient nutrient environment in the lab: if dormant, they could resume activities way beyond standard incubation time, and be masked or inhibited by growth of “weed” strains.  We tested this hypothesis directly and found significant support for the idea of infrequent, stochanstic awakening in both model (Escherichia coli, Mycobacterium smegmatis) and environmental species (S Buerger, A Spoering, E Gavrish, C Leslin, L Ling, SS Epstein (2012) Microbial scout hypothesis and microbial discovery. Applied and environmental microbiology 78: 3229-3233; S Buerger, A Spoering, E Gavrish, C Leslin, L Ling, SS Epstein (2012) Microbial scout hypothesis, stochastic exit from dormancy, and the nature of slow growers. Applied and environmental microbiology 78: 3221-3228). A detailed analysis of these and other findings are in our recent review papers (SS Epstein  (2013) The phenomenon of microbial uncultivability. Current opinion in microbiology 16: 636-642; Epstein, S.S., (2009) Uncultivated Microorganisms. Series: Microbiology Monographs, Vol. 10; Springer Berlin / Heidelberg, 208 pp.; Epstein, S.S. (2009) General model of microbial uncultivability, in Uncultivated Microorganisms, (Ed. S.S. Epstein). Series: Microbiology Monographs (Series Ed. Alexander Steinbuchel), Vol. 10; Springer Berlin / Heidelberg, 131-150.).

However successful the microbial scout hypothesis is in explaining some aspects of the behavior of “uncultivable” species, it cannot explain all of them.  For example, explicit in this hypothesis is that actively growing and well represented in the sample species should be the first to grow and be detected in Petri dish.  However, the empirical observations are just the opposite: strains represented well in a sample are often the last ones to grow in the lab.

We are determined to resolve the nature of microbial “uncultivability” and of the Great Plate Count Anomaly. Most recently, we came up with a brand new idea we term The Four Seasons of Microbial Life.  We argue that this is the only hypothesis that accommodates ALL known to us facts about “uncultivables”, and is singularly promising to be THE explanation we are looking for.  The hypothesis is being tested in the lab and remains unpublished at the present time … so stay tuned :) 

 

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