Friday, February 28, 2020

Egos and Manganese Nodules

Manganese Nodules on the bottom of the Pacific Ocean

What’s your cell phone made of? Over forty different elements are needed for the modern phone with its touch screen and battery. Many of those elements are ones that most people have never heard of like dysprosium and terbium. Where do we find rare elements like these? Today, most of the rare earth elements, as they are called, come from third world countries that still tolerate having their citizens work in dangerous mines where rock strata known as ore deposits have accumulated over Earth’s 4 billion year history.

Economic geologists are the scientists who study how ore deposits form. They find rocks that have higher than normal concentrations of desirable elements in them (like gold or silver), bring them into a laboratory for chemical analysis, and finally determine if the ore deposit is economically feasible for mining. In the early 1980s, it was finally being recognized that microbes living in oceanic sediments were involved in precipitating minerals—some of them with economic importance--although very little was known about how bacteria did this.

I was encouraged to study the role of microorganisms in the formation of ore deposits by Carnegie Director Hatten Yoder. In 1983, I organized my first international conference on “Organic Matter in Ore Deposits”, inviting an interdisciplinary group of scientists from around the world. Ken Nealson, then at Scripps Institution of Oceanography, was working on manganese oxidizing bacteria, which related presumably to the formation of deep sea manganese nodules that carpet the bottom of certain places in the Pacific Ocean. The biogeochemistry field at this time was small enough that you bumped into people at conferences as disparate as the American Society of Microbiology (ASM) and the Geological Society of America (GSA). Ken attended my first graduate student talk at ASM. He was intrigued by my work on Rubisco and stable isotopes. When we met a few years later, we struck up a collegial friendship that has included sharing students and postdocs. Ken is a magnetic person—always optimistic, positive, innovative, and funny. I was fascinated by the possibility that stable isotopes might prove useful in studying the mechanism by which microbes turned soluble manganese into mineral precipitates. We began to discuss in greater detail a potential collaborative project.
(L-R): Wes Huntress, Doug Rumble, Marilyn, Ken Nealson, circa 2002

It was unknown if manganese oxides (MnO2—meaning one manganese atom with two oxygen atoms) were formed with molecular oxygen (O2) or with oxygen from water (H2O). Ken and his students had made manganese minerals in the laboratory catalyzed by bacteria or bacterial spores. Conversely, manganese ore deposits could also be formed at very high temperatures or spontaneously by different non-biological mechanisms. How to figure out where the oxygen atoms came from? Stable isotopes to the rescue!

The stable oxygen isotope composition of atmospheric oxygen is very different from oxygen isotopes in water. Air has significantly more of the heavy isotope (18O), while water has significantly less. Because of these basic differences in oxygen isotopes, we had a naturally occurring tracer.

I ended up working on this problem with Ken’s Ph.D. student Brad Tebo, who later replaced Ken at Scripps when he moved to the University of Wisconsin. We also worked with Brad’s first graduate student, Kevin Mandernack, who grew the microbial cells, isolated the manganese oxides, then came to Washington DC to analyze the oxygen isotope compositions of the oxides using a special metal and glass vacuum line called a fluorination line.

The work was physically demanding in several ways. You weighed out a few milligrams of sample into a nickel “bomb”--a cylinder of nickel metal about 40 cm long--and attached it to the vacuum line where a highly reactive compound—bromine pentafluoride (BrF5)--was frozen into the bomb using liquid nitrogen. We then attached furnaces to the bombs and heated them to 600°C for 20 hours. Bromine pentafluoride is explosive. We used it with extreme caution. The laboratory required a specialized ventilation system. In the old Geophysical Lab, this was a huge fan that was connected to a window built into the side of the building. When the fan was turned on, the window flipped open automatically. Often, the fittings attaching the bombs to the vacuum line leaked. The lab often had a smell like an old, indoor YMCA swimming pool.
Fluorination line with Doug Rumble and Craig Shiffries, circa 1995

This was my first and last project using bromine pentafluoride. Manganese minerals had never been analyzed before using this technique, so I had to develop methods to remove adhering water before reacting the manganese oxides. As a student, Kevin Mandernack was a good looking, blonde fellow well above 6-feet tall. Coming from UC San Diego’s Scripps Institution, he carried himself like a Southern Californian, dressed in tank tops in summer and surf clothes the rest of the year. He also sported a healthy tan and often had a wide smile and mischievous chuckle. Compared with many of the Harvard-trained postdocs, Kevin exuded self confidence to the young people of the lab. I suspect some of them were jealous.

It may be controversial to discuss appearance and demeanor of colleagues, but there is no question in my mind that these traits are important in affecting a person’s perceived standing. In the very conservative environment of the Geophysical Laboratory, people viewed as “different” sent small ripples throughout the small enclave. Speaking for myself, I feel that I was judged as being a bit of a light-weight during my career because I enjoyed and choose to work with “different” people. At times people would say to me: “You always work with tall guys” or “You always work with women”—neither of which was strictly true. Thirty years ago, it was commonplace to treat women this way. Good looking, magnetic people evoke strong opinions from others: either they become presidents and CEOs or people mumble behind their backs. I saw this repeatedly in working with the many people that I’ve mentored over my career.

I served on Kevin’s PhD committee and made several trips out to Tebo’s lab during that time to discuss the findings we’d discovered, as well as participate in Kevin’s examinations. We collaborated also with Alan Stone at Johns Hopkins University, who prepared manganese minerals of various flavors using purely chemical methods. We compared these minerals with ones that were biologically precipitated by active live, growing microbes or passive spores (Mandernack et al., 1995).
Manganese oxides formed by microbes and grown by Kevin

Oxygen isotope analysis of the manganese minerals revealed that a significant proportion (up to 50%) of the oxygen in the minerals came from molecular O2 with both purely chemical as well as biological mechanisms. Oxygen from water formed the remaining portion. This finding means that if a manganese mineral is found in an ore deposit or sediment, there probably was some atmospheric oxygen involved. In some of the Earth’s very old rocks, manganese minerals provided evidence that oxygen was present in the atmosphere at that time.

Brad Tebo, now at Oregon Health and Science University, continues to investigate bacterially-mediated manganese cycling and is a leader in that field. He has isolated many strains of bacteria, as well as studied their genomes to determine which enzymes are involved in the catalysis. Tebo’s recent work challenges the paradigm that the manganese cycle operates between soluble manganese and solid manganese phases. He's found an important intermediate phase formed by microbes in many environments that's at the heart of the manganese cycle. Tebo and his colleague George Luther (University of Delaware) have made significant strides in unraveling the complexity of manganese cycling to a much greater degree than our earlier experiments in the 1990s.
Map of location of manganese nodules

Today, Brad Tebo visited us in Mariposa with his wife Margo Haygood. We “skyped” with Kevin reminiscing about this project. Brad and I are planning retirement in a few months, while Kevin is now the Dean of Letters and Science at California State University Maritime, a small campus providing degrees in maritime transportation, business, and engineering.

Kevin’s PhD defense was a real learning experience for all of us. He had decided to invite Scripps’s most influential professors to be on his examining committee. The PhD student presents his/her findings to his/her committee typically in a one hour lecture followed by one to two hours of questioning. After this, there is a deliberation period when the student leaves the room, and the committee discusses the merits of the defense.

It didn’t take long for a fight to break out. Not a fist fight, mind you, but a battle of egos.

Those with the biggest, showiest egos trumpeted the loudest and longest. I was appalled at that behavior. Although Carnegie does not grant academic degrees, I had served on several PhD committees worldwide. I’d never seen an exam go this way. This was Brad’s first student. Tebo, a thoughtful, reflective microbiologist, is on the quiet side. When he speaks, he knows what he’s talking about. At that time, he didn’t have the stature to shut down the egos that were gobbling up the oxygen in the room.

I finally spoke up. I recognized that some of the committee members had not thoroughly read Kevin’s dissertation. It may be—and this is just a guess—that they had pre-judged him. And I think, they even pre-judged me as I heard a lot of man- and even woman-splaining about isotope fractionation and things I was an expert in. I had worked with Kevin and Brad from the get-go on this project. The work was sound and has stood the test of time. I defended Kevin’s work, his writing, and his conclusions. It felt good to stand up for both men. I was well on my way from being the shy, retiring postdoc to the outspoken scientist supporting others who need the help.

Finally, the egos subsided and the PhD was approved!

Tuesday, February 25, 2020

Isotope Blunders- Take Two

Student Quinn Roberts, Marilyn, Mat Wooller, circa 2002-always a challenge

As I’ve noted in my latest blog post, any self-respecting isotope geochemist has a closet of lab horror stories. Some have written to me--if you’re not breaking something, then it means you’re not in the lab enough! Read on for Take Two on isotope blunders.

Liquid nitrogen can freeze off your warts in a dermatologist’s office. It’s also a nearly ubiquitous liquid in many stable isotope laboratories. At -196°C, liquid nitrogen freezes water vapor and carbon dioxide into solids. We use it in the laboratory for just that purpose, to separate water vapor and carbon dioxide from nitrogen, argon, and oxygen gases. I had a high school intern in 1981 who mostly fooled around freezing various things in liquid nitrogen—rubber bands, dead flies, paper clips, soda—rather than carrying out much serious research. Today, we’d be training that student for an hour via an online course before he’d be allowed to touch the stuff.

While liquid nitrogen is useful in the laboratory, because it will also liquefy oxygen and argon, it can be dangerous if it’s used improperly. In fact, it was probably liquid nitrogen’s freezing out of oxygen from air that caused the visiting graduate student to blow up the vacuum line at the Geophysical Laboratory. If the vacuum line he was using had a crack in it, air would have been able to leak in. Oxygen in air (about 20%) would liquefy in the liquid nitrogen trap that we use to separate carbon dioxide. Liquid oxygen is a pale, eerie blue—a color that we use to describe what Earth might look like from outer space—a pale blue dot.

My first experience liquefying oxygen came from one of my “better” ideas. I was interested in studying hydrogen isotope patterns in bodily fluids—blood, saliva, and urine. Whenever any of my colleagues cut their fingers or hands, I appeared like a vampire with a capillary tube to suck their blood. My colleague Tom Hoering went even further. When he drew blood, he went into his back lab and peed into a beaker, then sealed up his urine in a tube for me to analyze. This was in the late 1970s before AIDS/HIV was an issue. I also obtained human blood samples from a friend who worked at the National Institutes of Health (NIH). I found that the hydrogen isotopes in bodily fluids had slightly more of the heavy isotope of hydrogen (2H) in them than local drinking water. My hypothesis was that water vapor in breath was the cause. I surmised that water leaving the lungs had more of the light isotope (1H) in it, leaving bodily fluids with an excess of 2H by mass balance.

Back in those days, senior staff scientists went out to lunch every day and had a martini or two at the local Hot Shoppes Cafeteria on Connecticut Ave. I had a sneaking suspicion that what I wanted to do—freeze out the water vapor from my breath—might be a slightly “dumb” thing to do, so I waited until Tom Hoering, then my postdoc advisor, went out for his martini lunch to carry out my sampling. I had a set of 6 mm (1/4”) glass tubes with one end sealed off. I stuck the sealed end into a flask of liquid nitrogen and exhaled several times into the tube. I then lit a natural gas torch and sealed off the open end with the water vapor inside. I prepared four tubes of my breath this way.
The mass spec that exploded in 1986

The tubes were resting on the lab bench, coming to room temperature. I was feeling pretty smug. I was thinking of a nice simple paper. I turned my back briefly to write in my notebook when the tubes began to explode one by one. Not only had I frozen the water vapor from my breath, but also liquefied the oxygen in my breath to that blue liquid that I could now see dancing around in the tube and expanding. All four exploded sending glass shards around the lab. Fortunately, nothing else was broken. Shaken, I quickly cleaned up the glass fragments before Tom arrived back from lunch.

I had learned a valuable lesson. If it feels risky—read about it first and think before acting.

A colleague writes that two of his postdocs accidentally trapped liquefied argon gas in a metal vacuum line. After isolating the liquefied argon between two sturdy metal valves, they removed the liquid nitrogen from the outside of the metal vacuum line. As the metal line warmed up, the argon went from the liquid phase to the gas phase and expanded, blasting open three strong, metal Swagelock valves. It then blew by the valves into their sample chamber and shattered the expensive, transparent “window” made of zinc selenide. Finally, adding insult to injury, the “window” fragments shattered the optics of a sophisticated laser associated with the vacuum line. Lasers aren’t cheap, nor are zinc selenide “windows”.

That was an error in much greater proportion than the four glass tubes in my experience. And a learning lesson for this professor and postdocs.

Another colleague writes about a new vacuum line to extract water from plants and soils. This works in the following way. The material to be extracted is placed into a glass tube, which is attached to the extraction line using a Swagelok metal fitting with a rubber O-ring in it that forms a vacuum seal when the fitting is tightened. The other side of the extraction line has an empty glass tube attached to it. The tube with the sample then gets placed in a flask of liquid nitrogen and is opened to the vacuum system to pump out all the air. Once this is done, the trap is closed off and the liquid nitrogen is moved to the empty glass tube, while the tube with the sample in it is heated to 100°C. The water from the sample boils, then condenses in the other tube that’s now in liquid nitrogen. 
Extraction line with fittings and liquid nitrogen flasks

“We were having a problem where maybe 1 in 30 tubes would violently explode when the liquid nitrogen was removed. This would send glass shards flying everywhere and would also ruin soil and plant samples. I finally solved the problem when one day I removed the liquid nitrogen flask and noticed several centimeters of liquid in the bottom of the test tube. As I realized what that meant, the tube exploded. Turns out that even though the O-rings in the Swagelok fitting were not actually in the liquid nitrogen, they were getting cold enough to contract and to allow nitrogen vapor from the liquid nitrogen flask to enter the extraction line. Since the line is under vacuum, the nitrogen vapor adiabatically cooled and liquefied inside the tube. Since the bottom of the tube was immersed in liquid nitrogen the super chilled liquid was happy to sit there until the flask was removed. After few seconds the liquid nitrogen in the tube boiled, pressurized the tube and exploded. I solved the problem by replacing all the O-rings with ones rated to as low a temperature as I could find.”

Liquid nitrogen accidents can be very powerful. There is good reason why, these days, universities take its use seriously. My high school intern, if he were in my lab today, would not be testing what rubber bands do when frozen solid.

My next liquid nitrogen story has nothing to do with a laboratory vacuum line, but does involve mini-explosions. I began a decade long study of the stable isotope biogeochemistry of mangroves, those gnarly trees that live on islands in the tropics and love salt water. I received a fellowship from the Smithsonian Institution to begin collaborative projects with their scientists. Candy Feller was studying mangroves in Fort Pierce, Florida, and invited me, postdoc Mat Wooller, and student intern Jake Waldbauer down from DC to collect samples for isotope analysis.

[Note: Mat Wooller is now a Full Professor and Director of a major isotope lab at the University of Alaska. He is a leader in Arctic stable isotope biogeochemistry. Jake Waldbauer is now an Assistant Professor of Biogeochemistry at the University of Chicago, where he is investigating the effects of viruses on organic matter cycling in the ocean. He’s measuring proteomics—a heady type of analysis that is unique to Jake.]

Mangroves are all C3 plants so we had a good idea what their carbon isotope signals would be. This wasn’t a rocket science project, however it was mid-winter and Florida beckoned with warm weather and seemed a chance to have a bit of fun. I came up with an idea to beef up the study. We were going to collect and flash freeze bits of mangrove leaves in liquid nitrogen so that we could study the Rubisco protein back in DC. [Rubisco is the plant enzyme that takes up CO2 in photosynthesis.] When we picked a leaf for isotope analysis, we took a subset, stuffed it in a small, plastic tube, and threw it into a container filled with liquid nitrogen.
Wooller and Marilyn, fish seining 2000

We also dragged a fish net through murky waters collecting samples of fish that potentially fed on decomposing mangrove leaves (i.e., mangrove detritus). Wooller and I manned the net in a narrow inlet where the tannin-laden waters reached up to our armpits. It was a creepy feeling to walk 100 meters through the mucky bottom, feeling branches and beasts bumping against your shins. We did catch fish!

Later in the day, Jake and Mat seined in clear bay water while I stood on the shore shouting, “You there! Further out!” waving them to deeper water. This phrase became one of my tag lines for “more work is always needed.”
Waldbauer and Wooller catching mangrove crabs, 2000

After a week of sampling, as a semi-important scientist, I returned to DC leaving Mat and Jake to pack up the samples and bring them to the lab. The leaf samples in liquid nitrogen were poured out on the concrete patio outside so they could be bagged up for transport. As the tubes were warming up, Jake and Mat bent down to pick them up just as the tubes began popping like fire crackers, shooting bits of plastic and mangrove leaves in every direction. Wisely they jumped back and watched the show. I had failed to realize that the plastic tubes I’d brought were not liquid nitrogen safe. Fortunately, we’d collected enough fish and mangrove leaves for isotope analysis.

Mat Wooller and I were involved in many mangrove adventures through the years. He and I worked well together in the field even though we plan our work and sampling strategies completely differently. I’m an “abstract sequential” person—rarely write down directions because they make sense to me. Operating a vacuum line with invisible gases was no problem because I always knew where they were in the line. Wooller is a “concrete spatial” person—builds things and wants things written down before proceeding. I ended up labeling our sample bags with what needed to be collected. When Mat saw them, he knew exactly what we’d be doing.

We spent several days traipsing around through the mud collecting mangrove leaves on our study sites in Belize. There was no drying oven on the island, so I had, yet again, a “better idea.” Using small, brown paper lunch bags, I put the leaves in them and hung the bags on the clothesline in the sunshine to dry the samples. At noontime, there wasn’t a hint of a breeze.
Mangrove leaves drying on the line, 2001

Again, I felt pretty smug at how resourceful I was.

I went to the field station’s lab for a couple of hours, then headed outside to check on my leaves. The wind had picked up! My heart dropped as I raced over seeing about two-thirds of the bags empty and the ground littered with mangrove leaves.

My colleagues had already noticed this, but were waiting to see what I’d do. I quickly snatched those few bags with intact samples off the clothesline, then steeled myself for the ridicule. What could any one say? They could have said, “You’re an idiot. C- sloppy work.” But they didn’t. Mat subsequently designed an oven out of an old hot plate and a Styrofoam cooler. It worked a treat for many years.
Wooller's homemade oven, Belize, 2001

Once again, I learned my lesson.

Sometimes, failure to collect the proper sample is not entirely your fault. A colleague writes the following: In early 2000’s his lab wanted to get a sample of Standard Mean Ocean Water with sodium chloride (i.e., salt) in it for their lab to measure one of the chlorine isotopes: 37Cl.  They put in a request to the Canadian Coast Guard to get a 5-liter sample, while they were out at sea, sending the appropriate request forms for the work.  A month or so later a filled 5-liter bottle of “Pacific Seawater” arrived at the isotope lab. They spent a week trying everything they knew to get the *@#$ chloride to precipitate using silver nitrate, new resins, and any other chemical trick they could think of. Finally, frustrated and tired of failure, they tested the obvious – the electrical conductivity, a measure of salinity.  It was freshwater!  In an obvious miscommunication, the Coast Guard had filled the 5 liter-bottle with the ship’s tap water, not ocean water while at sea. 

In 1996, Keith Hobson and Len Wassenar were collecting monarch butterfly samples from wintering sites in Mexico for hydrogen isotope analyses. Monarch butterflies from the United States and southern Canada migrate each winter to specific sites in Mexico, Florida, and California. Keith and Len had the idea to measure the hydrogen isotopes in the tissues of the butterflies in Mexico to determine where they had summered. It was a brilliant application of using isotopes for tracking animal migration.

They were explicitly--and in no uncertain terms--told they could only collect the many dead monarchs littering the ground. Dutifully they did so, placing the expired butterflies in paper envelopes and putting them in a bag.  Later that night at their hotel, they heard noises. Someone seemed to be scratching at the lock of the door of their room in a not exactly safe area of rural Mexico!  After listening breathlessly for a while, the sound continued.  Finally they turned on the light and got up – it was the monarchs! They were not dead—yet--and were scratching away in their paper envelopes.  Ultimately, these butterflies were naturally on their way out and gave up the ghost. The rest is now isotope history!

A last story in this series comes from a newly-minted assistant professor. He was a new, PhD candidate working late at night in his lab preparing samples for experiments, which involved filling reactors with a particular type of carbon dioxide--very expensive CO2 that his professor had obtained because of it’s exotic carbon and oxygen isotope properties.  To carry out the experiment, the student sampled an aliquot of this gas from a large, 5-foot gas cylinder, then put it into a glass vacuum line to purify it. In doing so, he accidentally snapped off a small piece of a glass valve and vented the whole line. I can imagine the vacuum pump gurgling and the student’s pounding heart.

His response was to (properly) shut off the valve to protect the pump (like a good steward) and send his professor a quick text message about what had happened. His reply, which the student can now empathize with as a junior faculty member himself, was understanding with a decided air of frustration.

It was also clearly a lesson in not working alone in the lab late at night.

When we’re inexperienced, we think working at all hours of the day shows we’re dedicated. Personally, I like folks in the lab during “business hours” so there is some cross checking.

At any rate, the frustrated student shut down the vacuum line and went home thinking this was merely annoying, right? Wrong. In his confusion from breaking the glass valve, he had totally forgot to shut off the main valve on the large, expensive, one-of-a-kind CO2 tank that had been set to vent a small stream of CO2. The next morning the professor fixed the broken glass valve with ease and helped the student get started again.

When they went to sample another aliquot of CO2 from the tank, nothing came out. Weird, they thought. Then, as they checked the tank valve and tapped the regulator gauges, they realized the student’s initial folly had cascaded into a much larger issue—losing an entire tank of calibrated, special gas. Rightly, the professor was seething with palpable anger. The next several weeks were taken to recover and rebound with two important papers ultimately being published. The professor is now an Associate Professor at a major university—doing well, while the former grad student is in his fourth year as an Assistant Professor.

Broken glass vacuum lines, lost samples, and liquid nitrogen annoyances are all a part of the stable isotope biogeochemist’s journey into a career of creativity and discovery.

Rounding Third Base and Heading Home

Cards from Franny and Flowers the Rumbles   My daughter Dana is marrying George Goryan on June 25 at our home in Mariposa...