Concluding Impressions

The time has come. The elevator is empty, Jason is stowed, and the lab is slowly being separated into boxes. The screens show a quiet deck and the wake of the ship as we steam towards Auckland.

The end of a cruise is a bittersweet time – everyone looks forward to their return home (and to solid ground), but the family gained while on board is something to be missed. We have created both intellectual and personal relationships with everyone, whether during van shifts, meal times, or simple down time. We return to port for one last celebration of time between shipmates, between new friends and old colleagues, and of the science that brought us all together.

Jason team and science watch the semi-final ping pong match

Niya dilutes metal samples to create vials for SiO₂ samples from the fluids gained at each vent. “It was a great learning experience,” she said. “This was my first cruise, and I didn’t know how things worked. I loved being in the control van and actually seeing the vents in real time. I hadn’t appreciated how small the vent sites really were. Jason gives off this tiny halo of light, and you realize just how much you could be missing. We learned that on this cruise with the Eccentric Gardens site – it was on the edge of the map, and no one had ever found it before. It was great seeing the critters at each vent and learning what they were, writing down ‘galatheids’ and other names I’d never heard of before.” She grinned. 

Neya finishing her analysis

Others had similar opinions. For many it was their first cruise, and expectations varied across the board. “I didn’t expect to be so seasick,” Alex jokes, and the rest of the lab laughs. “Nonetheless, it was a very interesting experience. For example, doing PCR without a PCR box in such rocky conditions, but still getting good results. It was interesting to see all the work that goes into piloting Jason, and I’d definitely come back, but it’ll be good to stand on steady ground again.”

When asked, everyone agreed that they would come back in a heartbeat. It is such a wonderful experience and we all learned so much. It isn’t until you’re on a research cruise that you realize just how much work goes into sampling, collecting, and processing. In the end, however, it’s all worth it. When it comes to the best part of the cruise? That varies:

·      Being in the control van and seeing things many only read about

·      Finding the new vent sites

·      Getting good samples!

Alex setting up one last qPCR experiment

·      Seeing unique structures and finding the microbial communities that live within them

·      The teamwork, meeting new people, and playing (and losing) board games in the downtime

·      Watching pilots attempt to collect chimneys far bigger than expected

The one thing no one agreed upon was the favorite site. From Toilet Bowl to Mothership to ABE and back, the opinions varied - and rightly so. Every site we visited had a special significance to someone and provided a sample that could potentially give science a new insight into hydrothermal vents and their microbes, chemistry, and communities.

It has been an unforgettable experience. Someday we will return to sample again, but for now this cruise – and this blog – have ended. Thank you to all science members, pilots, engineers, and ship crew. We couldn’t have done this without you.

The "Mothership" flange complex at Tui Malila

Tubeworms seen at Mariner

Towards the end of our dive at Mariner, we spotted some tubeworms.  The most obvious invertebrates in the Lau Basin are snails (Ifremeria and Alvinichoncha) and mussels, shrimp and crabs. So seeing tubeworms is quite unusual. We did see some in our research cruise to this area in 2006, but never at Mariner.  A very healthy community was spotted this time on a huge hydrothermal pillar near our "toilet bowl" sampling area. Crabs were happily munching away on a tube or 2 about 2 cm (200mm, ~ 1 inch) long.   There were thick communities of this tiny little tubeworm. They appear to be Arcovestia ivanovi, found in the Lau and Manus hydrothermal basin areas.

Tubeworms have an interesting lifestyle.  The have no mouth or digestive system like other worms. They are essentially a sack (trophosome) of bacteria or endosymbionts, and a plume (the red structure) that captures hydrogen sulfide, carbon dioxide and oxygen.  These gases are transported from the plume to the trophosome, where they are food (energy and carbon sources) for the bacteria. The chemolithoautotrophic bacteria oxidize the sulfide, and use the energy in that redox reaction to fix carbon dioxide into organic carbon. The tubeworm is then sustained by the endosymbionts organic carbon.  Sulfide levels are quite low at Mariner relative to other deep-sea vent sites where tubeworms thrive, so it is indeed curious that we found them here.

Notice the crabs eating the tubes

SinD Lopper

We dive down with a bright yellow light

Chief Sci says when you gonna sample it right

Oh Anna dear we're not decision-making ones

And microbes, they want to have fun

Oh microbes just want to have fun

Jimmy sits in the middle of the row

He yells out we're gonna do this thing right

Oh Jimmy dear you know you're Jason pilot one

 But microbes, they want to have fun

Oh microbes just want to have

That's all they really want

Some fun

When the science is all done

Microbes - they want to have fun

Oh microbes just want to have fun

Some say all you need is CTD

But we say you need more creativity

We want to be the ones to bring them up in the sun

Oh microbes they want to have fun

Oh microbes just want to have

That's all they really want

Some fun

When the science is all done

Microbes - they want to have fun

Oh microbes just want to have fun, 

They want to have fun,

They want to have fun....

Science isn't all technobabble and pipetting. Sometimes we have to get creative in our sampling methods, and at sea, that's where engineers come in. See our post on its origins from a previous cruise here: http://tinyurl.com/FST3000.   After Anna-Louise challenged our scientists to come up with something that, unlike the strong arms of Jason, wouldn't crush fragile chimney and beehive structures, creativity came to life.

We began with the FST3000, whose tin bottom was tragically too weak to cut through the chimneys all the way. After some experimenting, Jimmy came up with a new contraption - the SinD Lopper. Made out of a coffee can with the bottom removed, the SinD Lopper acts as a guillotine, sending a metal bottom slicing across the chimney and beehives to remove them from their bases. Not only is this less forceful than Jason's grip, the reduced surface area allows the chimney structures to stay more or less intact for transfer to Jason's deck. It is still undergoing beta testing, but SinD Lopper is so far doing better than we could have imagined, and we look forward to exploring future realms of the deep with the new invention!
Contributed by Morgan Haldeman 

Back in the water!

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

Growing ELSC heat-loving microbes

In addition to our geochemical and metagenomic research aims on this expedition, we also seek to isolate novel microbial species.  To achieve this, we use data from our geochemical and metagenomic experiments to direct traditional cultivation-dependent microbiological techniques.  Studying an isolated microorganism in culture allows us to test a range of physiological conditions and can help elucidate the microbe's metabolism beyond what one might learn from genomics alone.

Growing and isolating select microorganisms is very difficult and often proves impossible.  Scientists estimate less than 2% of environmental microbes may be easily isolated and cultured in a laboratory setting.  While some bacteria thrive easily –and grow like “weeds” – on media rich in nutrients, others require atypical growth nutrients.  Interspecies microbial relationships that involve one microbe providing a vital growth factor for another may exist –making it impossible for one species to grow without the other present.

There are billions of microbes colonizing hydrothermal deep-sea vent chimneys waiting to be isolated (see photo below).  Working with the Jason II team, great care is taken to preserve the integrity of chimneys as they are sampled and brought to the surface for study.  Once on board and processed, microbial biofilms on the  outer layer of the chimney are harvested by scraping and used in enrichment and isolation experiments.

Scanning electron micrograph of microbial biofilm on a hydrothermal vent chimeny

On this cruise one group we're targeting is the isolation of a thermoacidophilic deep-sea hydrothermal vent-dwelling Euryarchaeota (Aciduliprofundum species). In this case we use an acidic culture media and incubate it at temperatures between 60 ºC and 90ºC.  We hope these conditions will promote growth of these thermoacidophiles, but if it does grow, it won't be the only microbe.

enrichment cultures to extinction.  This is to say – we dilute cultures in series until the dilution is sufficient to reduce enrichment cultures down to a single species.

If after all of these steps we're lucky enough to get our new isolate, we still have the challenge of maintaining it, getting a good stock, and preserving it for future characterization studies or to share with other labs. In Dr. Reysenbach's lab at Portland State University we have been lucky to get tricky thermophilic microbes to grow and thrive. We're a part of the Center for Life in Extreme Environments and maintain an Extremophile Culture Collection – think of it as a library (or zoo) of interesting microbes, many of them yet to be fully characterized!

Jessica checking some of the cultures with her shrunken head friends

Contributed by Jessica Hardwicke (undergrad student at PSU)

The Importance of Coffee

Winds are meant to ease over the next day or so... and hopefully we'll get to dive again. In the meantime we have time for a cup of coffee. 

 

Jeff and Sean's morning Jo'

Jeff and Sean take their coffee very seriously. Somewhere packed among the crates of scientific equipment is an espresso machine. By the time we leave Auckland, ten bags of coffee beans are set to accompany us. We have also managed to pick up a few New Zealand-themed espresso cups, rainbow-colored sheep and kiwis. Should the espresso machine break due to some unforeseen tragedy, a rarely used French press stands ready to fill the gap.

Sean jokes about how they made sure to calibrate the thermocouple (a fancy thermometer) on the espresso machine before leaving Jeff's lab in Woods Hole. Thermocouples are something close to Jeff and Sean's scientific work as well, as is the handling of hot, pressurized fluids. Besides the espresso machine, the center-point of their laboratory is a set of isobaric gas-tight fluid samplers (IGTs) with thermocouples attached to the nozzles . These highly specialized and custom-built pieces of equipment are able to collect hydrothermal fluids at the bottom of the ocean and return them, at seafloor pressure, to the surface. Keeping the fluid pressurized is essential for measuring dissolved gases that are an important part of the fluid’s chemistry. However, releasing these fluids without losing the gases and measuring them accurately enough to be useful is also tricky, and requires an elaborate, mobile chemical laboratory. It is also important to keep the IGTs well serviced in order for them to function properly. Like a two-man pit crew, Jeff and Sean routinely assemble and disassemble the equipment. With IGTs going in and fluid samples coming out of the water at all times of day, the espresso machine sure gets a workout.   Contributed Guy Evans

Jeff and Sean’s collection of espresso cups (upper right) and coffee beans (lower left). IGT samplers 5 and 6 ready for servicing (above center) and being serviced (right).

Geomicrobiology in the Taupō Volcanic Zone (TVZ)

The wind speeds are up, and the swell --big!

Extending SSE from the Lau basin, and part of the "Ring of Fire", we encounter New Zealand. Here, plate tectonic movement, and consequently spreading and fracturing in the Earth's crust, manifests as geothermal springs. Just prior to our research expedition to Lau, several of us visited this geothermal area and sampled some hot springs in collaboration with our colleagues at GNS Science to find and grow a very unusual microbial group; a parasitic/symbiotic group of Archaea called the Nanoarchaeota.

Just three hours south of Auckland within a geothermally active area known as the Taupō Volcanic Zone (TVZ) is the town Taupō-nui-a-Tia. Translated from the Māori language as "The great cloak of Tia", both the town and lake are named after Tia, the Māori chief who discovered the area.   Some hotsprings in the TVZ are a gentle 25°C with neutral pH and others boil to the surface, more acidic than battery-acid. 

Carlo and Karen, on our research expedition, are members of the GNS Extremophiles laboratory,  and are actively studying the geomicrobiology of the New Zealand hot springs. They have helped collect microbiological and geochemical samples for the “1000 Springs Project”; a comprehensive bio-inventory of the microbial diversity of New Zealand’s geothermal ecosystems. To date the Extremophiles laboratory has collected over 42,000 geochemical data points with corresponding microbial community information for each hot-spring. All this information is publicly available in an impressive user friendly and educational website: 1000springs.org.nz.

Inferno crater

During our stay we visited Waimangu, New Zealand’s youngest geothermal field.  Waimangu was created in 1886 following the eruption of Mt. Tarawera- An eruption that also created Lake Rotomohana and flooded the fabled ‘Pink and White terraces’.   Inferno crater is particularly impressive.  This turquoise feature oscillates between 35°C and 80°C every 30 days, a fluctuation the resident microbes need to adapt to!  At low temperatures, microbial communities are dominated by non-spore forming Bacteria while at high temperatures, heat-loving Archaea dominate. 

Waiotapu (Māori for “sacred waters”) is located 52 km north of Taupō in the Okataina Volcanic Centre.  This geothermal area is high in concentrations of sulfur, arsenic and antimony, and produce colorful minerals such as orpiment  (As₂S₃) and stibnite (Sb₂S₃).  Two of New Zealand’s most impressive geothermal hot-springs, the neon-green “Devil's Bath” and bright orange “Champagne Pool”are in this area.

Champagne pool

"Devils bath"

 Contributions from Guy Evans, Jessica Hardwicke and Carlo Carere