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







 

Keeping up Moral

The weather forecast has been accurate and the seas are still too rough to launch Jason. As we wait for better weather to arrive, we are keeping busy by processing samples that were collected during our dives to Mariner and ABE vents. Our cultures are growing in the incubators and our gas samples have been measured, but life on board the ship isn't all work.

Carlo and Nick in training for the big tournament

Caption contest starring Styro Foam trying on a survival suit

Ping pong has a long and storied history among seagoing scientists and there is no greater honor than winning the expedition ping pong tournament. While ping pong is an official game of the Olympics, playing ping pong on a heaving ship requires the skill and coordination of a gaming master. Our great tournament has begun with tournament underdog John defeating his lab predecessor Gilbert in the first round. Board games are also popular on the cruise, and another pastime is the nightly Settlers of Catan game which has been dominated by Carlo thus far on the cruise. Finally, a “caption this picture” contest has emerged on our common whiteboard where the funniest caption wins the author fame and glory (and more!). All of these activities help keep moral high on the ship while we wait out the weather and hopefully we'll be diving again by the end of the week.

Contributed by Rick Davis

The Pressure Effects....

Today' weather forecast: "Big high pressure (1033 mb) near North Is, NZ to drift slowly to the E with little change in strength during the next 2-3 days. This high may begin to weaken slowly during the middle of... Appears worst conditions may develop during Mon and continue..."

Pressure also has a big impact in the deep ocean. The bottom of the ocean is an inhospitable place. It is dark, cold, and under extreme pressures. Imagine you are swimming in a pool that is ten feet deep, or just about three meters. You dive to the bottom to pick up a pool toy or a penny and your ears pop. You feel the pressure on your body and on your head. Now imagine increasing that pressure over 100 times. With every ten meters of seawater, add one atmosphere of pressure. Now imagine traveling to 1000m, or 100x10m. Since we already exist at 1bar of pressure, the total pressure would be about 101bar.

Jason can dive to depths of 6000 meters. The ROV is specially reinforced to deal with the extreme pressures at depth so that it doesn’t implode, or compress explosively. The areas we work in the Lau Basin are at depths of 1700 to 2600m, but the pressures are no less damaging. 

Styrofoam cups and heads before their descent to our deep-sea research sites.

To demonstrate the pressures at depth, we sent down several Styrofoam heads that Allie, Joy, Francis and Piper illustrated, and cups that middle school teachers attending a science (STEM) workshop at the University of Nevada illustrated.   The cups and heads dropped to depths between 1900m and 2400m. Simply dropping to the bottom of the ocean allowed the pressures of nearly 200bars, or around 2500 pounds per square inch, to do its work.

The cups, once a full 10oz and over 3 inches tall, shrank down to around 2 to 3oz and around 2 inches tall. We stuffed wads of paper inside to allow them to keep their shape, but that did not necessarily ensure they were perfectly round at the end. Instead, they crumpled as the pressure increased, crushing them on one side or another depending on which way they were oriented. 

The extreme left scale bar was 1 inch. This illustrates nicely how much the cup shrunk!

Styrofoam is made of foam called polystyrene, which is full of air pockets. When put under pressure the air is squeezed out, but the foam retains its shape. This is why Styrofoam makes such a good example of pressure at depth – it does not warp the material past recognition, but it shrinks to a size that seems unrealistic to our 1bar atmosphere. 

 This extreme pressure at deep-sea vents also allows the very hot hydrothermal fluid to remain in a liquid state in most cases, except when the temperature is so high the fluid will boil as we saw in the Mariner blog post.  Many of the deep-sea invertebrates; snails, mussels, shrimp, crabs, do not have gas-filled organelles, such as humans or other land-dwellers, which would be compressed under pressure, and so these creatures are not nearly as affected by pressure as we are.

the normal head

the shrunken head

The bacteria we bring up to our lab are not necessarily barophiles (piezophiles), or pressure-loving, but they do thrive under those conditions nonetheless. Even the fluid samples we bring up show evidence of pressure – there are no bubbles emerging from the vents, but when we remove the fluid from its pressure-tight sampler (the IGT see "hot water" post), it may erupt with bubbles as the gases escape the solution.

Much of the deep ocean is as yet unexplored. And how pressure affects life in this deep ocean biosphere will no doubt lead to a new understanding of the extent of life on Earth.

Contributed Morgan Haldeman, Nick Rhoades and John Kelley.  

Rough Seas Ahead

Despite the best planning, problems arise while at sea. The ship can have a malfunction, Jason needs to be repaired, scientists forget an important chemical back home in the lab. These are problems that we can usually fix or work around to get the job done and continue with our exploration of the Lau Basin. Unfortunately, one factor that we cannot plan for and have no control over is the weather at sea. Our ship, the R/V Roger Revelle has no problem in rough seas, however the ROV Jason II needs relatively calm seas to be safely launched and recovered. This is because Jason must be craned from the side of the ship and high wind and waves will make the ROV swing on the crane, which is dangerous because Jason weighs over 4000 kg out of the water which is about three times heavier than an average automobile. 

Rough seas prevent Jason from diving

Weather conditions such as wave height are monitored in the computer lab.

We are currently experiencing 20 knot winds and an average wave height of 3.5 meters. The weather forecast calls for more wind in the next couple of days so our dives are likely to be on hold for at least that long. Luckily we have lots of good samples from ABE and Mariner Vent Sites and we are still busy at work measuring the water chemistry and grow microorganisms from these samples. There is always plenty to do when out at sea, even when Jason is sitting on the deck.

Contributed by Rick Davis

Taming wild microbes

Karen Houghton checking growth  of acidophiles in her culture tubes

On the surface of Earth, most biological communities are fueled by organisms that use light energy to convert inorganic carbon (CO₂) into organic compounds (i.e. biomass) in a process known as photosynthesis. This is what plants do. In deep-sea hydrothermal environments, there is no sunlight so instead these communities are supported by chemosynthesis – a process by which microorganisms use chemical energy to convert CO₂ into biomass. The chemical energy for chemosynthesis comes in the form of reduced chemical compounds (H₂, Fe²⁺, H₂S, CH₄) that are relatively abundant in hydrothermal fluids. Organisms that perform chemosynthesis are known as chemolithoautotrophs.  Both aerobic microorganisms, which require oxygen (O₂) to grow and anaerobic microorganisms, which use alternative electron acceptors (NO₃^-, S⁰, SO₃^-2), are found in hydrothermal environments.  Within the Lau Basin and other deep-sea vent fields, microbial communities are heavily influenced by steep chemical, pH and temperature gradients.  For this reason, microbiologists on board the Revelle hope to isolate several novel acid-loving (acidophiles), high-temperature (thermophile) species on this expedition.

90 degree Celsius oven filled with enrichment cultures from samples collected at ABE and Mariner

The isolation of chemolithoautotrophs is driven by the principles of growth selection.  In practice, synthetic media is prepared that mimics the conditions in the deep-sea but only the necessary energy (e.g. reduced inorganic chemical), carbon (e.g. CO₂) and electron acceptors (O₂ or NO₃^-, S⁰, SO₃^-2) required for a specific type of chemosynthetic metabolism are supplied.  By restricting alternative metabolisms and by exposure to stringent pH and temperatures, microorganisms with desirable traits out-compete undesirable species and become enriched.  Over time, with the application of sufficiently selective growth conditions, it is possible to remove all undesired microorganisms; resulting in a “pure” isolate.

In our lab on the Revelle, microbiologists are working hard to isolate high temperature, acid-loving hydrogen-oxidizers and methane oxidizing species; in addition to heterotrophic bacteria capable of eating organic compounds (amino acids, peptides, chitin).  In the weeks to come, both microbial community and metagenomic data will be used to refine the selective media used in isolations.    

Contributed by Carlo Carere and Gilbert Flores