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

Mariner vent field

We have been working at the Mariner vent field for the past 48 hours or so. For some of us this is one of our favorite places on the bottom of the ocean. Because this deep-sea vent field is very close to the island arc volcanoes, the hydrothermal vent fluids are greatly influenced by very acidic fluids released by the magma chamber below the seafloor.  These acidic fluids mix with the seawater-derived hydrothermal fluids to create highly acidic hydrothermal fluids that leach metals from the oceanic crust, such copper.  Because these fluids are more acidic (~pH 2.5) than the vent fluids to the North (e.g. ABE and previously Kilo Moana) they contain much higher concentrations of metals like copper, zinc and iron. When these fluids form hydrothermal chimneys, they are quite tall spindly structures, venting high temperatures fluids over 360 degrees C. We also found some fluids that were boiling,which causes phase separation and results in rather unusual fluid chemistry.

Copper and iron rich hydrothermal chimneys at Mariner

The bright white areas at the right, that look a little like white flames are fluids that are being emitted and are boiling.

The microbial biofilm on the "Toilet bowl" rock

The 'Toilet bowl' structure

 From a microbial perspective, Mariner is also very interesting. Samples from here, collected in 2005, resulted in the first truly acid and heat-loving ('thermoacidophile') microbe from deep-sea vents. But also the microbial communities associated with these hydrothermal chimneys are dramatically different from those in the North.  One area that has yielded DNA sequences of heat-loving microbes never before found at deep-sea vents is where a structure we call the 'toilet bowl' is located.  So this vent field has great potential for discovery of new microbes and providing deeper insights into the diversity and extent of life on Earth.

Samples are placed in 'bioboxes' on the Jason.  Note sample still 'smoking'

Genomes from Smokers

We’ve got samples! Now comes the really fun part-- discovering new microbes and finding out how they survive and grow in such extreme environments. We are examining these communities using two different approaches. Many of the microbiologists on board are developing new culturing techniques to get novel Bacteria and Archaea to grow as isolated cultures in a lab. Another approach is to look at the communities using molecular techniques to determine the ecology and biochemical mechanisms of microbes in the environment. One of the most exciting developments in environmental microbiology is the ability to perform genomic analysis of environmental samples, known as metagenomics.

Hydrothermal vent chimneys about to be sampled

Metagenomic analysis of a sample should give us information about who is living at these vents as well as how they survive there, but the process is long and difficult. The method begins with extracting and purifying total DNA from sulfide chimneys. Once we break open the cells, we need to purify the DNA to remove any traces of metals or cell debris which inhibit our future sequencing steps. This purified DNA consists of a mixture of genomes from each microorganism that is living in the sample. We then sequence the fragments using a next-generation sequencer that gives us hundreds of millions of fragments of DNA sequences. We then rebuild the genomes from these small fragments using powerful computer algorithms so we can study them in more detail. Once these genomes are reconstructed, it will help us understand how the genomes of these microbes differ between the different deep-sea vents sites here in the Lau Basin, and help guide our culturing efforts in new directions to try and get these microbes to grow in a test tube. We’ll follow this post with more information about how we grow microbes on the ship. 

Contributed by Rick Davis

Inside view of a hydrothermal sulfide chimney sample from ABE. The yellowish mineral is
chalcopyrite, a copper, sulfide, iron mineral CuFeS₂

Hot water!

Last night Jason successfully landed at the ABE hydrothermal vent field, at a depth of about 2140m. Jason touched down directly next to a pair of smaller spires, glimmering white with sulfur and microbes. Shimmering water rose from the tops of the chimneys, where shrimp and a particularily feisty crab had made its dwelling. Several other towers loomed out of the black as Jason's lights touched their bases.

Hydrothermal vents occur primarily in areas where magma is close to the surface such as around tectonic plate boundaries. Seawater sinks into the crust through fractures and pore spaces where it comes close to the magma below and becomes superheated and buoyant. This heated water then moves back through the crust and is expelled as hydrothermal fluid. Vent chimneys are formed when the metals in the superheated fluid precipitate as they are ejected from the subsurface and mix with cold seawater. Initially the minerals are amorphous, and the structures that form are soft and friable, often referred to as "beehives".

"Beehive" chimney from 2140 m in the ABE vent fields

With time, iron, copper and zinc minerals -pyrite, chalcopyrite and sphalerite, become crystalline and harden, making large structures like we saw today. Heat-loving (thermophiles) microbes colonize these structures and then very quickly other invertebrates appear in places that are not too hot (more soon on the microbes and biology of the vents).

Today we collected numerous chimney samples to explore the microbes associated with these rocks. We also took temperature measurements and samples of the hydrothermal fluids being emitted from the chimney structures. To do this, we use an Isobaric Gas-Tight (IGT) sampler that is designed specifically to fill slowly and maintain the fluid samples at seafloor pressure (which is more than 200 times atmospheric pressure. Just think what that would do to a styrofoam cup if you took it down that deep! We'll do that experiment, and show you the result in an upcoming post).

IGT sampling fluid of about 280 degrees Celsius

After 8 IGT samplers-full, several sulfides and more, we are all ready for bed. Tomorrow. we'll give you a glimpse into some of our ABE scientific treasures.

Contributed by Morgan Haldeman

Geology of Lau

Today, another weather day, so we are on hold above ABE. There is a brisk wind, and the swell is building. So here is a bit about the geology of backarc basins.  Check the sidebar for more on basins, arcs etc.

A subduction zone is a place where physics, chemistry, and geology come alive in the best of ways. It is a place where a dense oceanic plate, often old and water-rich from its millions of years beneath the waters of the Pacific or other oceans, sinks beneath another younger or more buoyant plate. The dense plate is pushed from behind by a spreading center thousands of miles away, and pulled down by ultra-dense minerals and the sheer pressure of the depths to which it drops.

As the plate descends, the temperatures and pressures of the overlying mantle wedge can cause melting of the hydrated crustal slab. This melting forms magma, rich in incompatible elements such as sodium and potassium, which rises to the surface and erupts from the volcanic arc.

This arc grants the Lau Basin its geologic title of ‘back-arc basin’ – a descriptive name, meaning exactly as it says (see sidebar for more details). Behind the arc of subduction zone volcanoes, a center of spreading emerges and creates a basin. This tends to preferentially occur behind island arc systems, but may happen under continental crust as well – see the basin and range province in North America. One must also note that not all subduction zones form basins: indeed, some areas, like the central Andes, actually undergo compression behind the arc.

A back arc basin is an extensional province behind a subduction zone. At first thought it seems implausible to have an area of extension behind an area of convergent stresses, but consider: as a plate subducts, it creates a trench between the two plates. Over time, this trench begins to undergo ‘rollback’, which means it begins to move seaward as the overlying plate converges.  This rollback motion creates tensional forces, which pull at the mantle material beneath the overlying plate. These tensional forces meet tensional forces from the other direction, and thus the crust above begins to pull apart at a spreading center behind the arc. This is known as a back-arc basin.

Back-arc basins are curious in many ways: biologically, chemically, and physically. Some, like the Lau Basin, are extremely seismically active, meaning that earthquakes occur frequently. They tend to spread in an asymmetric manner compared to mid-ocean ridge spreading centers, meaning that some areas spread much faster or slower compared to others on the same center.

Each basin is a little different. The Lau Basin was actually formed from two separate spreading centers that joined together through an extensional transform zone. Currently, the spreading rate varies based on whether one is in the north or south of the center. The North is faster, up to 9 cm/year, compared to the 4 cm/year in the South. This rate means that the Lau Basin is a relatively fast spreading center.

Keep an eye on the blog for further information on the Lau Basin’s chemistry and biology as the cruise goes on!  

Contributed Morgan Haldeman

Kilo Moana: A big surprise.

Jason is in the water!
Our first dive began with much excitement of seeing the deep-sea vents at Kilo Moana. Our pre-dive work involved getting our samplers cleaned and placed on Jason. Since this was a late dive, the work had to be done in the dark which is not the easiest thing to do. The microbiologists needed to clean and disinfect their samplers, while the chemists needed to prepare their gas-tight water samplers (IGTs) for installing onto Jason or the elevator.

Nick, Gilbert, and Karen prep Jason for the first dive with help from Scott (left). Gilbert washes out a biobox (right)

Jeff, Morgan, Sean, and Vivian get ready to launch the elevator under Brett's supervision.

Jason being launched for the first dive

Routinely, the elevator is filled with samplers that can then be exchanged with their used counterparts on Jason during the dive. The elevator can then be raised to the surface and we can get our samples without needing to bring Jason to the surface. This allows Jason to continue working on the sea floor enabling us to work for days without stopping.

Once on the bottom, some 2600m below the ship, we returned to sites we visited in 2005 and 2009, where there had been active deep-sea hydrothermal vents.  

Elevator going in the water

Our first site yielded: extinct sulfide structures, lots of mussels, many dead mussels, occasional crabs (see below)... and as we proceeded in hope for some active deep-sea vents, our hopes diminished. The entire area was no longer active; snuffed out between 2009 and now. There are many possible explanations for this. Most likely the heat source that fueled the hydrothermal vents at Kilo Moana just waned and finally shut off.

Extinct chimney structure

Brisingid asteroid covering a hydrothermal marker

Hydrothermal mussels and Galatheid crabs still remain on the vent chimneys

We're headed to Kilo Moana

This morning we arrived on site, -22.180S, -176.601 W; Mariner vent 1900 m below us.  There is a storm somewhere to the west of us, and is generating occasional large swells. Tito, the ROV Jason expedition leader, has decided we need to wait till the swell diminishes some, so we decided instead of waiting at Mariner, we will head up North to our northern most site, Kilo Moana. Ultimately we'd need to make this transit at some time, and why not now while we wait for the seas to subside a bit?  Kilo Moana is about 240 km away. We'll pass to the west of Tongatapu and hopefully spot an island or two on our way up (as we did this morning).  Karen also spotted her first Albatross this morning, which was a special treat for an early morning rise.

Small island welcomed our morning sunrise today

This is one of the most seismically active areas in the world, and so new islands form relatively frequently. The most recent was just this January (see http://www.abc.net.au/news/2015-01-16/tonga-volcano-creates-large-new-land-mass/6022094, or google Tongan Volcano). We are very curious to visit our deep-sea vent sites to see if they have changed at all from the last time we were here in 2009.  At that time, the hydrothermal fluids at Kilo Moana had changed from when they were first collected in 2005, so what does Kilo Moana have in store for us this year?!  We will have some clues after we do our first dive there, hopefully tomorrow.

ROV Jason Debrief

Tito explaining how Medea functions

Today the research team spent the afternoon becoming familiar the remotely operated vehicle (ROV) Jason. Jason II will be our eyes, ears and hands at the bottom of the ocean while we explore the Eastern Lau Spreading Center for the next 15 days. This sophisticated ROV is capable of diving to great depths (over 6000m), is equipped with multiple high definition cameras, powerful LED lights, thrusters for maneuverability and two fully-articulated robotic arms. As Jason collects samples for analysis we use a device called a sample elevator to bring up samples and re-equip the ROV with fresh equipment. Using the elevator, we maximize our time at depth and can do more science. Jason is tethered to another vehicle, Medea, which serves as an intermediary to the ship on the surface. Because of Medea, the motion of the ocean above won’t bother Jason while it is busy working.

Tito giving a tour of Jason

The Jason team operates inside a double-wide shipping container outfitted with the equipment necessary to control not only Jason, but also Medea and the ship. At all times during a dive there will be a pilot, a navigator, an engineer, a science leader, a data logger and a video recorder on watch. Teamwork is key and so the Jason team spent some time today (thanks Tito and Scotty!) explaining, to the uninitiated, everyone’s responsibilities.

The Jason elevator brings samples to
the surface so Jason can keep working

Inside the Jason control van

With less than 20 hours to go until we reach our first dive site –Mariner- all aboard are getting excited and finalizing preparations for our first dive tomorrow. Stay tuned!

Contributed by Carlo Carere

Why Lau Basin?

Our journey on this cruise takes us to the middle of the Lau Basin, just west of Tongatapu, the main island of the Kingdom of Tonga. We are particularly interested in the 240km-long Eastern Lau Spreading Center (ELSC) and Valu Fa Ridge (VFR). Here, the crust splits apart and magma wells up from the mantle, spilling lava onto the seafloor. This is also an area of unusually high heat flow, where concentrated geothermal energy powers deep-sea hydrothermal vents.

Seafloor hydrothermal systems form when geothermal heat interacts with seawater that has percolated into the seafloor. Water and rocks chemically react at high temperatures, forming hydrothermal fluids. I like to think of it like making coffee. Hot water poured into coffee grounds dissolves certain compounds and forms a new substance. The type and flavor of coffee you get depends on what type of grounds you use and the ratios of water, heat, and coffee beans. Similarly, the type and chemistry of hydrothermal fluid depends on the types of rocks in the seafloor and the ratio of water and heat added in the process. 

It is a peculiar characteristic of the ELSC that its direction forms a 17-degree angle with that of the nearby Tonga Subduction Zone. As a consequence, the distance between the ELSC and the volcanic arc that forms above the subduction zone decreases from ~100 km in the north to ~50 km in the south. At about ~70 km, there is a rapid change in the rock composition. Far to the south, at Mariner vent field, magmatic fluids such as water, carbon dioxide (CO₂ ) and sulfur dioxide (SO₂) mix directly with the hydrothermal fluids, a sort of super-charge mixture of high heat and low pH (acid) that extracts additional material from the rocks.

All of these differences in rock type, temperature, and fluid chemistry provide an excellent natural laboratory for exploring, in depth, the factors that influence the diversity and relationships of microbial communities at actively forming deep-sea hydrothermal deposits.  

Contributed Guy Evans.   

Welcome to Laugeomicro2015!

R/V Revelle docked in Auckland

Today we set off on our research expedition on the R/V Revelle from Auckland harbor in New Zealand. We are headed to our deep-sea vent study sites along the Eastern Lau Spreading Center (ELSC) in the Southwestern Pacific. We will be studying the microbiology and geochemistry of several of the deep-sea vents sites using the remotely operated vehicle (ROV) Jason II that is designed, built, and operated by engineers and scientists from the Deep Submergence Laboratory at the Woods Hole Oceanographic Institute, Woods Hole, USA. Our first site will be the southern most site, the Mariner vent field, at -22°S, -179.6°W and at a depth of about 1900 m.

Science and crew gather on deck as we begin our expedition

Anna-Louise and Jessica saying goodbye to Auckland

Top clockwise: Morgan avoiding the glare, Jeff, Sean and Morgan setting up lab, Auckland skyline as we leave the harbor

We will update this blog often as the cruise progresses with more detailed descriptions of our dive sites, the methods we will use to study them, and our new discoveries. We hope you follow along with us on our cruise and feel to use the comments section in our blog to interact with our scientists and ask questions as we go-- we would love to hear from you!