We're back at Mariner, at >1900 m as of noon today! Yesterday, with the the weather situation, Stephane L'Haridon made use of the time to run a different experiment... he explains below...
Ocean covered 70 % of the Earth's surface, and the microbial diversity of the oceans is still not well understood. The number of microbes in the sea has been estimated around 1029 cells, the number of microbial species is still under debate but estimates suggest the presence of at least 106 different microbial species in the ocean. Finding out who these species were, was greatly aided by the technical revolution that started in the 1980s in the field of molecular biology. Microbiologists were able to identify microorganisms without first needing to cultivate them based on the sequence of the small-subunit ribosomal RNA (16S rRNA) gene. Recently, a new generation of DNA sequencers have been developed that allows us to sequence full genomes of uncultured microbes and has led to the development of “Omics-based” approaches to microbiology. Most of the organisms detected by this approach have never been described, even after 120 years of trying to get them to grow. These microbes and are the so called “uncultivable” species; less than half of the known phyla of microorganisms have a cultured representative, and more than 99% of all microbes remain “uncultivable”. It is these microbes that we are trying to grow from samples collected on this expedition.
|Stephane, Brett, and Bud watch the CTD computer display|
Mainly two strategies are employed to isolate pure strains, after a first step of enrichment, microbiologists isolated colonies by streaking on medium solidified by a gelling agent or they performed serial dilution in liquid medium until they obtained a pure culture.
Claude Zobell, who was a pioneer in the study of marine microbes, developed the marine agar 2116 medium (Marine Broth Medium) in1941. This nutrient-rich medium has allowed the isolation of many heterotrophic bacteria belonging principally to a fairly limited number of genera, including Vibrio, Pseudomonas, Oceanospirillum, Aeromonas, Deleya, Flavobacterium, Alteromonas, and Marinomonas. Nutrient rich-media at the step of enrichment on liquid or solid medium favor the growth of faster-growing bacteria, referred to as “r”-strategists at the expense of slow growing bacteria,”K”-strategists, the latter represent the relevant microorganisms in the pelagic environment. Mimicking the natural sea water that microbes thrive in, is a challenge for microbiologists. The Marine Broth 2216 medium has 170- fold more dissolved organic carbon than natural sea water which clearly inhibits the growth of oligotrophs (microbes that live on a very little 'food') which represent the majority of the microbes in the ocean. Only recently, in 1993, did D. K. Button and colleagues used the natural sea water with small inocula for isolating marine bacteria. This concept of natural sea water as medium and low concentration of cells as inocula was improved in the Giovannoni laboratory at Oregon State University (USA) two decades later by using high throughtput cultivation (HTC). The HTC permits the isolation of members of the Sar11 group of microbes, one of the most dominant groups of microbes in the ocean.
|CTD profile showing sensors such as oxygen, depth, temperature|
In the framework of the European Program, MaCuMBA, coordinated by Dr Lucas Stal, in which we are one of the 23 partners, we are developing new approaches, new devices to cultivate the “uncultivable” microbes by applying innovative methods, and the use of automated high throughput procedures. To isolate new key players microbes from the Tongan sea water, we deployed a CTD cast to a depth of 500meters and collected samples from 5, 95, 200, 300 and 500 meters depth. To perform the HTC method, after sterilizing some of the seawater we collected, I added nitrogen, phosphate, carbon and vitamin sources at very low concentrations. I then put the medium into the 96 wells of the microplate, and added about 2 cells from the samples we collected to each well. It’s a challenge for microbiologist to try to work under sterile conditions on a vessel without a PSM (Microbial sterile cabinet)! Only 7 hours after the recovery of water sampling, 34 microplates containing the cultures were incubated at room temperature (20°C).
|My microtiter plates cultures incubating at room temperature|
After patiently waiting, in two months, when I am back in France and in my laboratory, I will check to see if anything grew in these 96 X 34 little wells. From past experience, there will be lots of novel bacteria happily living in these culture wells!
Contributed by Stephane L'Haridon