McLane Pumps

Hello my readers! I apologize for not posting yesterday, but, to be honest, I was exhausted and fell asleep before I had a chance to write anything. Yesterday was just an extremely busy day – a ton of samples were collected and the nutrient analyses run.

We did have an excellent surprise though – a group of pilot whales decided to hang out and swim alongside the ship for a while! They were beautiful.

 

Pilot whales swimming alongside the Endeavor (photo courtesy of Jimmy Ji)

Pilot whales swimming alongside the Endeavor (photo courtesy of Jimmy Ji)

 

Today was our third day at the Process Station. As I mentioned previously, we are spending time here because we want to study the biological processes occurring in this part of the ocean more thoroughly. One of the ways we do that is with McLane pumps.

These pumps allow for large volume filtration (around 300L) in situ, meaning that seawater gets pumped through while the pump is still in the ocean, concentrating lots of particles onto the filter for analysis. So, instead of collecting water then filtering (like I explained in the spotlight on Dr. Fawcett), she can collect much more suspended particles directly from the ocean.

 

Recovery of the McLane Pumps (photo courtesy of Jimmy Ji)

Recovery of the McLane Pumps (photo courtesy of Jimmy Ji)

 

The McLane pump is truly an interesting piece of electrical equipment. It’s controlled by a computer, and the researchers can program the McLane what time to start and stop and the flow rate at which to pump (how fast the water moves through the filters). The McLane also records data (flow rate, depth, etc.) that can be accessed when next connected to the computer. Timing is very important, as we need to ensure that the pump is at the right depth before it starts pumping. We usually deploy the pumps about an hour before they are scheduled to being their work.

McLane Pump

At the upper left is a round black casing – this houses the filters and is where the water flows into the machine. The actual pump is the smaller silver cylinder in the center of the machine; this is what allows for the movement of water. The large, brownish-gray cylinder at the bottom contains the electronics and batteries that control the machine and transmit information to a computer. Water flows out of the pump at the bottom right – through a flow meter and out the white spigot.   We have two pumps on board and they are both deployed simultaneously, but go to different depths. The housing for the pumps (the metal case surrounding the whole thing) has notches and a clamp that secure it to a very strong line so they can be safely and securely deployed to whatever depth we want to study.

The pump can hold up to three filters, depending on what is going to be studied and the filters’ various sizes (i.e. 100µm, 5µm, 0.4µm, etc.) allow us to collect different particles/organisms. Filter size refers to space between the pores – larger spaces capture larger particles (for example, phytoplankton) whereas smaller filters will collect smaller cells, like bacteria. Using multiple filters to collect particles of different sizes is called size fractionation – things on each filter are similar in size to each other. This is the first step in identifying the various particles that are present in the seawater.

Once the McLane pumps are recovered, the first step is to remove any excess water from the filters to ensure that particles are not lost when the filter is taken out of the pump. To do this, the filter and its casing are connected to a small vacuum pump that sucks out the water. Once this is complete, the filter is removed and then stored for further processing (often in the freezer until we return to Princeton).

 

The filter from one of the McLane pumps, following its time in the ocean.  The filter starts out white - all that greenish stuff is particulates to be studied.

The filter from one of the McLane pumps, following its time in the ocean. The filter starts out white – all that greenish stuff is particulates to be studied.

There are numerous analyses that can be performed from the filters – measurements of carbon and nitrogen content, isotope ratios (13C/12C and 15N/14N), DNA, natural abundance, and flow cytometry.

 

Researchers Dr. Sarah Fawcett (right) and doctoral candidate Jimmy Ji (left) prepare a McLane filter for further analysis back at Princeton

Researchers Dr. Sarah Fawcett (right) and doctoral candidate Jimmy Ji (left) prepare a McLane filter for further analysis back at Princeton

 

Flow cytometry is very cool. First, the filters, and therefore the cells, are fixed in formalin (a solution of formaldehyde in water) to retain their shape and structure. When the filters are put into a flow cytometry machine, the cells are essentially lined up, then cut with lasers. Based on how the cells scatter the light, the machine is able to determine size, complexity, and pigmentation of the cells. What this does is allow researchers to look at different populations based on their morphology (size and shape) and pigmentation.

We have another day at the Process Station, then we continue our journey – there’s only a week left of the expedition!

Until next time,

Ms. B

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The Wonderful World of Water

Today was a beautiful day! The seas were calm, the skies were clear, and we started around-the clock analyses. OK, so that last one wasn’t really beautiful, but it does mean we have hita major milestone for the expedition. Today we arrived at our first process station, which is an area of the ocean we want to study more thoroughly. We’ll spend about four days at this location, collecting lots of samples and performing numerous tests and analyses.   For me, this means more time with my buddy, the NOx Box, and making a bunch of solutions and standards.

As I look around, all I see beyond the ship is water, the inspiration for today’s topic. Water is one of the most abundant molecules on Earth. The world ocean comprises 71% of the Earth’s surface area and is completely essential for life on our planet (this isn’t the total amount of water on Earth – there’s also ice and other smaller bodies of water).

If you’ve ever tasted seawater (intentionally or accidentally), the first thing you probably noticed is that it was salty. Unlike pure water (H2O only), seawater contains a lot of salt. This salinity, which is the concentration of all the chemicals in the ocean, gives seawater some different properties than freshwater. Two of these are density and freezing point. Density, as my students should remember, is the amount of mass per unit of volume (usually mL, L, or cm3). The density of all water depends on temperature and pressure. At 20°C, the density of water is about 1g/mL (1kg/L). When you decrease the temperature, the density also decreases. Think about a glass of ice water – are the ice cubes at the top or bottom of the glass? From experience, you know that ice floats. This is due to its density being less than that of the liquid it’s in – less dense will always float on top of a denser substance (this is actually a unique property of water; for most substances, the solid form is more dense than the liquid).

Seawater’s density, like that of fresh water, also depends on temperature and pressure but also on salinity. As the salinity of water increases, the density also increases. This partially explains why, at the sea’s surface, the density of water is about 1.027 kg/L. It also helps explain an interesting difference between pure water and seawater in their maximum densities. Pure water is at its densest at 4°C, whereas seawater is at its densest right before freezing.

Another difference between fresh water and seawater that’s caused by salt ions is the freezing point, the temperature at which a substance becomes solid. Water’s freezing point is normally 0°C (32°F), but seawater has a freezing point of -1.9°C (28.58°F). This decrease, called a freezing point depression, is one of the colligative properties (a property that depends on the ratio of solute molecules to solvent molecules) of all solutions – increasing the amount of dissolved solutes (in this case, salt ions) increases the molality of the solution, which changes the freezing point of the solution. Molality is the mass of solute (what is dissolved in a solution) per kilogram of solvent (what is doing the dissolving). In seawater, the solutes are the salt ions whereas the solvent is water.

 By mass, approximately 86% of the salt in seawater is NaCl. The other main ionic components are magnesium, calcium, potassium, and sulfate. Scientists have experimentally determined the molality of the each of the major ions in the ocean. From that, we can calculate the molar concentration of each salt component in the ocean.

Let’s do some math!

The molality of sodium ions in the ocean is 10.76 g/kg. What is the molarity (molar concentration) of sodium? Assume the density of seawater is 1.027 kg/L.

To solve this, we need to convert g/kg to mol/L – both of which are simple to do!

Step 1: Write down what you know

Solute = sodium

Solvent = water

Molality = 10.76 g/kg (this means 10.76 g Na+ / 1 kg water)

Density = 1.027 kg/L

Molar mass of sodium (from your Periodic Table!) = 23 g/mol

 

Step 2: Convert the mass of sodium to moles

10.76 g Na x (1 mol Na / 23 g Na) = 0.4678 mol Na+ 

Step 3: Convert mass of water to liters (use density!)

D= 1.027 kg/L

Mass = 1 kg (from the molality)

The equation for density is D = mass/volume, if we rearrange that to solve for volume, we get the equation V = mass/D

V = 1 kg / (1.027 kg/L) = 0.973 L

Step 4: Solve for Molarity!

M = mol/L

M = 0.4678 mol / 0.973 L = 0.481 M

This means that in every liter of water, there are 0.481 moles of sodium ions.

Now, I want you to calculate the molar concentration of chlorine: molality = 19.35 g/kg, molar mass of Cl = 35.45 g/mol, density = 1.027 kg/L. Just follow the same steps! (you should get 0.561 M Cl)

Until next time,

Ms. B.

On-Board Entertainment

Yesterday’s post was really heavy on the science, so I’m going to give all of my readers a bit of a break and talk again about life at sea. We’ve now been aboard the R/V Endeavor for two weeks – time is certainly flying! We are essentially sailing in the middle of nowhere and are together almost 24/7, working very hard to ensure our experiments are running smoothly and gathering data (to say nothing of the amazing crew, who work around the clock). It’s very easy to go a little stir crazy being in a confined space all the time, so in order to keep up morale and maintain our sanity, we formulate our own on-board entertainment. We do have a lot of movies and TV shows that we can watch, as well as a library with hundreds of books, but over the last couple days, we ramped up the amusements.

The first was a haiku contest (haikus are Japanese poems with seventeen syllables grouped in three lines: five syllables – seven syllables – five syllables) in which all the scientists and Erich, our Marine Tech (he keeps everything running for us) submitted poems anonymously. The haikus were then grouped into categories and judged by the First Mate, Shanna. To my surprise, I won for the most “HAI-gooeyest” haiku (i.e. the cheesiest). Here is my submission:

Gentle seas ahead
The NOx Box hums gleefully
Science is awesome

My Hai-gooeyest medal (made by Dr. Bess Ward, our Chief Scientist)

My Hai-gooeyest medal (made by Dr. Bess Ward, our Chief Scientist)

I am quite proud of it  ☺

This morning I awoke to another of the science party’s entertainments – a shrunken Styrofoam head (like the Styrofoam cups from a few days ago). I call her Frida, after Frida Kahlo, the Mexican painter.

Frida, the Shrunken Head

Frida, the Shrunken Head

When I went to get ice for the NOx Box, I opened the freezer and there, encased in ice, was a shrunken head staring back at me. It freaked me out…not quite what I expect to see at 3:45 in the morning! The rest of the science team had frozen her to surprise Andrew and he left it for me to see. Andrew and I are now in possession of Frida and have to figure out where (and for whom) to hide her. I can’t wait!

Finally, today is dedicated to our mothers. The science team and I wish all of our moms a very happy Mother’s Day. We love and miss you.

On a personal note, a very, very big thank you to my mom who is my staunchest ally, biggest supporter, and most trusted confidante – thank you for always believing in me and pushing me to be my best (I certainly wouldn’t be on this expedition without your support and encouragement). I love you! XO

Get ready for lots of science tomorrow!
Until next time,
Ms. B.

Isotopes

One of my favorite, and in my opinion one of the most important, topics in chemistry is isotopes.  Isotopes are atoms of the same element with different masses.  This means that the atoms have the same number of protons (which is what determines the element’s identity) but different numbers of neutrons – thereby giving a different atomic mass because

atomic mass = # protons + # neutrons

Every element has naturally-occurring isotopes.  For example, Carbon exists as 3 isotopes: Carbon-12 (6 protons + 6 neutrons and by far the most abundant of the C isotopes), Carbon-13 (6 protons + 7 neutrons) and Carbon-14 (6 protons + 8 neutrons) while Nitrogen’s two main isotopes are Nitrogen-14 (7 protons + 7 neutrons) and Nitrogen-15 (7 protons + 8 neutrons). Additionally, some isotopes, like C-14, are radioactive, whereas others like N-15 are stable.

What’s really interesting about isotopes, and how we’re using them in the research, is that they can be used to determine the rates and amounts of different biological processes.  Two examples of this that will be conducted during the expedition are uptake tracer experiments and natural abundance nitrogen isotope ratios.  Today’s post will discuss the uptake tracer experiment; I will talk about natural abundance in a later post.

Uptake Tracer Experiments

uptake tracer experiments.  the labeled N-15 nutrients that are taken up by the phytoplankton and analyzed by a mass spectrometer

uptake tracer experiments. the labeled N-15 nutrients that are taken up by the phytoplankton and analyzed by a mass spectrometer

Ammonium (15NH4+) and nitrate (15NO3) stocks containing N-15 instead of the vastly more abundant N-14 are injected into our seawater samples.  The phytoplankton take in these nutrients to build up their biomass and are left to grow in incubators (water temperature remains constant to allow phytoplankton to continue to grow) that are on deck.  After some time has elapsed, the phytoplankton and seawater are put through a very small filter to remove all the water; therefore, only phytoplankton are left on the filter.  A machine called a mass spectrometer then determines the amount of N-15 on the filter.  A mass spec combusts and ionizes a sample and separates the ions based on mass.  This allows us to see the mass profile of the phytoplankton and, consequently, how much 15NH4and 15NO3–  the phytoplankton consumed.

Because we know how much time elapsed during the incubation, and how much ammonium and nitrate the phytoplankton took up, we can use the final amounts to determine the rate of nutrient incorporation into the phytoplankton biomass.

We don’t have a mass spec onboard the R/V Endeavor.  In order to fully perform these analyses, the phytoplankton filters are immediately frozen after the filtration to preserve them.  The researchers studying them will run the mass spec analysis once we get back to Princeton.

Until next time,

Ms. B.

Spotlight Series: Jeff Hoffman

“Spotlight Series” is a group of posts designed to introduce you to the other members of the science team on the research expedition.  Each scientist and his/her research will be featured – I provided the prompts and the scientists added their own information.  Feel free to ask questions about what they are studying and how they became interested in science!

Jeff Hoffman

Jeff Hoffman

Jeff Hoffman

Jeff Hoffman works for the J. Craig Venter Institute (www.jcvi.org) and travels the world sampling water and extracting microbial DNA.  He is originally from New Orleans.

What is your field of study?  The samples I take are for extracting DNA – I take water from wherever we are and extract DNA, looking at different organisms around the world.  The information goes into a piblic database that is accessible by everyone and is used for various research purposes, for example, alternative energy, cancer research, and synthetic biology.  I mainly look at microbial diversity in waters around the world.  The DNA is extracted from the water samples then goes to the sequencing facility, where different scientists look at it.

 

Where have you been sampling?  It’s easier to list where I haven’t been.  From 2004-2006, we did a circumnavigation around the world on Craig Venter’s boat, Sorcerer II.  We mostly stayed on the equator and went around the world – including the Panama Canal, French Polynesia, Vanuatu, Australia, South Africa, and the Caribbean.

From 2006-2009, I traveled up and down the East and West Coasts of the US.  On the East Coast, we went as far north at Nantucket, and on the West, up to Juneau, Alaska.

After 2009, we did a couple summers in Europe, in the Baltic and Mediterranean.  I’ve also been to Antarctica 5 times and, in February, I went to the Amazon River.

 

How did you become interested in science?  I never wanted to wear a suit and tie, and science was the way to go.  It’s always interested me in high school and college.  I was pre-med but preferred to be in the lab.

 

Educational background:  I did my undergraduate degree at LSU (Louisiana State University) in microbiology with a minor in psychology.  I also completed my masters at LSU in microbiology.

 

Plans for the future:  I have another trip planned to the Amazon River in September and Antarctica in December.

Back to Ms. B:

How cool is Mr. Hoffman’s job?

Mr. Hoffman’s work with the J. Craig Venter Institute becomes public knowledge – demonstrating that scientific achievement and knowledge should always be shared.  It doesn’t do much good to keep scientific information a secret!

Until next time,

Ms. B.

Nitrite

As you may recall from my earlier posts, I analyze three nutrients (nitrate, nitrite, and ammonium).  I’ve already talked in detail about nitrate, so today’s post is dedicated to our nitrite experiment.  Get ready for a lot of chemistry 🙂

Nitrite, like nitrate, is a polyatomic ion made up of nitrogen and oxygen, but it has only 2 oxygen atoms instead of the three of nitrate.  Therefore, nitrite’s formula is NO2.  We’re analyzing nitrite concentrations in the seawater because nitrite is an intermediate (it’s both a product and a reactant) in many biological processes in the nitrogen cycle.

What I find really cool is that we use a completely different method to test for each of the nutrients.  For NO2 we measure absorbance of light to tell us a solution’s concentration.  Absorbance is, quite simply, how much light is absorbed by a solution.  Think of it like blocking the sun with curtains.  Dark curtains will absorb a lot of the light, and therefore stop it from entering your room ,while light curtains will allow much more light through  Solutions work in the exact same way – deeply colored solutions will absorb light while colorless solutions allow light to pass through.  We can measure how much light passes through a solution with a spectrophotometer.

the Spectrophotometer

the spectrophotometer (properly secured, of course!)

Seawater (which contains nitrite) is naturally colorless, so in order to produce color, we need to react it with compounds that will cause a color change.  To do this, we add sulfanilamide and napthylethylenediamine (NED) to 10mL of our nitrite standards and all of our samples.  These compounds, when reacted with nitrite, form a magenta-colored product.  Standards and samples with greater nitrite concentrations produce the most color, and are therefore the deepest, while the most colorless standards and samples have the least amount of nitrite.  The reaction occurs according to the following mechanism:

The mechanism for the nitrite, sulfanilamide, and NED reaction (adapted from Promega)

The mechanism for the nitrite, sulfanilamide, and NED reaction (adapted from Promega)

I know this looks a lot more complicated that what you’re used to, but that’s because sulfanilamide and NED are organic compounds, so they’re drawn using their structure, not just their formulas.  The nitrite (circled in red) first reacts with the sulfanilamide (underlined in green).  Then, the product of that reaction (underlined in gray) reacts with NED (underlined in blue) to make an azo compound (boxed in magenta), which is a double-bonded nitrogen compound.  It’s this azo compound that provides the color for the absorbance analysis – and the color is magenta.

Cuvettes (increasing concentration from left to right)

Cuvettes containing nitrite standards (increasing nitrite concentration from left to right)

Once we’ve added the sulfanilamide and NED to the samples, we can start analyzing them.  We fill a cuvette with the standard or sample we want to test, then put it in the machine.  The spectrophotometer will shine light at a particular wavelength through the sample and measures how much passes through, then displays how much is absorbed.  We use 543nm for the nitrite analysis because this is the where the azo compound absorbs the most light, as seen from the graph below (adapted from Promega).  543nm is green light, which is best absorbed by our magenta solution (blue and red light go right through the solution, so they are not helpful).

Peak absorbance graph for the reaction.  This shows the best wavelength of light to measure absorbance (adapted from Promega)

Peak absorbance graph for the reaction. This shows the best wavelength of light to measure absorbance (adapted from Promega)

The reason a spectrophotometer works is due to the Beer-Lambert Law, which states that absorbance (A) is directly correlated to concentration (C) when multiplied by a constant (k) and the path length (L, the distance the light must travel) – A = k × L × C.  Since the constant and path length are unchanged, the only factor that affects the absorbance is the concentration.  If you look at the picture of the cuvettes, the darkest magenta on the right will have the highest absorbance, while the lightest one on the left will have the smallest absorbance.

Just like with the nitrate analysis, we use the absorbance values from the standards solutions to make a standard curve; then, when we plot the samples’ absorbance values to that curve, we can determine the nitrite concentration of each of our samples.  This gives the researchers on the expedition an idea of the biological processes happening at each depth sampled.

Science is awesome. 🙂

Until next time,

Ms. B.

4000m

What a day. Today was, by far, the busiest one I’ve had. The reason is quite simple: we did two casts, which meant double the preparation and double the number of samples to analyze. Most of our casts are sent to 1000m but we occasionally want to sample even deeper water to measure the chemical, physical, and biological properties of the water that makes up a lot of the deep ocean. I can imagine you saying “Wait a second. Isn’t water just water?” The answer to that is, not quite. Although all water has the same basic chemical makeup (H2O), its other properties can vary quite a bit. By testing water at various depths, we can see how its properties change.

Besides being busy, however, today was also quite fun – we decorated Styrofoam cups!

Think about what happens when you swim down in the ocean – if you go deep enough, you feel the pressure around you change (your ears may pop or feel clogged). This is because water pressure increases with depth. We use this to our advantage. When we send Styrofoam cups to 4000m, it drastically alters their appearance.

Here is a picture of my cup when I colored it:

My Periodic-Table inspired cup design (it's my last name: BaBBiN!)

My Periodic-Table inspired cup design (it’s my last name: BaBBiN)

And now here is a picture after it came back up (next to a blank of it its original size for reference):

My cup, next to one of the original size.  Water is amazing!

My cup, next to one of the original size. Water is amazing!

There’s a huge difference in the cup’s size!

Here’s your challenge – Why does the water pressure at 4000m shrink the cup? Don’t worry about answering this now, we’ll discuss this when I come visit in June (but feel free to message me via Edmodo with your ideas!).

Tomorrow is certainly going to be interesting.  We’ve been sailing in some rough weather for the past few hours and will continue to do so.  Therefore, we won’t have a pre-dawn cast with the Niskins – it’s just too dangerous to be out on deck.  We’re completely safe inside though!

Until next time,

Ms. B.