Bergen Mesocosm Experiment

-- Post by Ian Joint --

We have now reached the end of the experiment and I will try to summarise what we have found so far. Again it is important to stress that these are still very preliminary observations and we will need many months to complete all of the analysis. Molecular biology is quite a slow process – fictional TV forensic scientists may be able to get an instantaneous DNA profile – the rest of us have to work harder at it.

As with the first summary of our observations that we posted a week ago, the graphs show the changes that we have observed in one bag from a high CO₂ and one from a normal CO₂ treatment. The graphs show the changes that we have seen since the experiment began on 6 May, until we took the final samples on 23 May.

The experiment should be thought of as happening in two phases. In the first phase, which lasted for 10 days, we concentrated on phytoplankton – we wanted to see how they might respond in a high CO₂ world. But as the phytoplankton grew, they used up the CO2₂that we had added – so we were no longer in a high CO₂ world. In the second phase, we added more CO₂ to make the water more acidic again, so that we could study how bacteria might respond to pH change.

pH

Nutrients

Chlorophyll

Specific organisms

Hello, my name is Louise O’Sullivan and I am a postdoctoral research associate from Cardiff University. I am working on the bacterial diversity and biogeochemical processes of aquatic sediment - this is the silty mud which accumulates on the seabed. This habitat quickly becomes limited in oxygen and is home to a diverse array of microbes which carry out some of the final stages in the breakdown of organic matter on this planet. I am particularly interested in two microbial groups which play key roles in the important processes of sulphate reduction and methane generation, namely the sulphate reducing bacteria (SRBs) and the methanogenic Archaea.

As the end of this mammoth experiment approaches, it is time to collect the sediment which has accumulated at the bottom of the mesocosm bags. At the start of the experiment my colleague Andrew Weightman set up some sediment traps; these are basically plastic cylinders which are suspended at the bottom of each bag and trap any sinking material. The sediment traps have now been raised and contain relatively thick green water which is quite pungent! The next stage is to collect the sediment bacteria on a filter and extract their DNA as other researchers here at Bergen have described. Analysis of this DNA will provide an insight into the types of microbes that are living in the sediment, and how these may change from those found in today’s climate to those which may exist in a high carbon dioxide world.

( Andrew Weightman putting down the sediment traps. )

Some people might think that field science means scientists out having a jolly, spending 3-4 weeks in a beautiful country doing the odd bit of work! Nothing could be further from the truth. These kinds of experiments need many years of planning and organisation. Therefore, when it comes time to actually get out there and do it, we have to work every single day come rain or shine, sleet or snow. In fact many of us have been here for 3 weeks straight now and we have been sampling, filtering, counting, staining, calculating and never moaning every single day without fail.

All this being said we are still having a lot of fun and while we are working hard I don't think any one of us would trade this experience.

Here's to us....pushing back the frontiers of science in the Bergen rain!

Jack Gilbert(PML)

( Sampling in the rain )

( John looking happy to be here )

( Cathleen not letting the rain get her down )

( Andy's starting to crack! )

( Rachel always brightens the day )

( The sampling team is always happy )

Over the last 15 days I have spent the majority of my time filtering water (like many other good folk here). In my case once I have got home I want to use the DNA and RNA from both the prokaryotic (bacteria and archaea) and eukaryotic (phytoplankton) cells from the mesocosm to look for two specific genes. The genes I am interested in are:

1) RbcL
This gene encodes the large subunit of an enzyme called 'Ribulose bisphosphate carboxylase / oxygenase' which for obvious reasons is abbreviated to RubisCO. RubisCO is believed to be the most common protein on earth for two reasons...firstly because it plays an essential role in carbon fixation and secondly because it isn't very good. More information about RubisCO can be found here: http://en.wikipedia.org/wiki/RuBisCO

2) NifH
This gene encodes the iron protein of an enzyme called 'nitrogenase'. This enzyme is essential for the biological fixation of N₂. This biological fixation eventually results in the production of Nitrate (via nitrification). Nitrate (see Nutrient blog below)is essential for the growth of phytoplankton which, as we have seen over the duration of this experiment, can result in decreased concentrations of CO ₂. Apart from biological fixation the main source of nitrate in our mesocosm bags is the recycling of organic matter by bacteria. More on Nitrogen fixation is available here: http://en.wikipedia.org/wiki/Nitrogen_fixation

I am especially interested in organisms which are able to fix both carbon and nitrogen (clever little bugs!). The techniques I will be using at home will enable me to determine firstly, which of the tiny beasties in the mesocosm are able to fix CO₂ and N₂ and secondly, give an indication of whether those which are able to are actively producing the required enzymes. The design of the experiment will help us to anticipate what is likely to happen in the future and how the contribution of different bugs to these essential processes will change as a result of increased CO₂

Hi, my name is Cathleen Koppe, I am a PhD student at Leibniz Institute of Ecology of Freshwater and Inland Fisheries, Germany (Berlin). I have been here 14 days and now it´s time to let you all know what I am doing here in beautiful Bergen. My fascinating assignment is to analyse the composition of bacterial communities in each of our mesocosms using molecular biology. To do this I am using the method CARD-FISH. This method has nothing to do with any kind of fish! CARD-FISH stands for Fluorescence In Situ Hybridization with Catalysed Reporter Deposition. Detecting bacteria using FISH was first described more than a decade ago and was hailed as a breakthrough for microbial ecology. How does CARD-FISH work? This approach is based on hybridization with horseradish peroxidase (HRP)-labeled oligonucleotide probes and subsequent tyramide signal amplification. Tyramines are phenolic compounds, and HRP can catalyze dimerization of such compounds when they are present in high concentrations. CARD-FISH yields significantly higher signal intensities than FISH with fluorescently monolabeled oligonucleotide probes. The rRNA targeted oligonucleotide probes are designed specific to different groups of bacteria, e.g. to alpha-Proteobacteria, Roseobacteria and/or Bacteriodes (including CFB).

Every other day I take water samples from each of our 6 mesocosms. Fix these samples with formaldehyde and filter a volume between 10-30ml onto polycarbonate filters with a pore size of 0.22 µm. This traps all phytoplankton and bacteria (attached and free living bacteria). The cells trapped on the filter are broken open (lysed) with enzymes like lysozyme and achromopeptidase which disrupt the cell membranes. After sample fixation and preparation, permeabilization, hybridization with oligonucleotide probes and tyramide signal amplification I can evaluate my samples on an Epifluorescence-microscope. I utilise the tyramide Alexa 488 excited at 488 nm and with maximal emission 519 nm. That why the bacteria illuminate in a very nice green colour (see picture from Jack’s Blog earlier). I then count these little green dots. I count the bacterial cells in one microscope field (a grid) a minimum of 10 times. I also visualise the cells using a DNA stain called DAPI. At the end I will hopefully obtain enough information about the different types of bacteria present in the mesocosms. We will then look for differences between the high CO2 and normal bags.

Finally, this experiment here in Bergen is very exciting for me, because it is my first time that I am involved in a project like this one and I am the only one from Germany. However, I am enjoying being involved in the experiment and meeting so many friendly scientists from England and France.

Cathleen Koppe (Leibniz-Institute of Ecology of Freshwater and Inland Fisheries, Berlin)

( Cathleen counting her bugs )

I've been here a little while now, but as yet haven't let you all know what I'm up to. My part of the experiment is to look at the bacteria that are degrading polysaccharides. These are long chains (poly-) of sugar molecules (-saccharides) that are often found in the cell walls of algae and in other marine organisms. These long molecules are good eating for bacteria and there are large amounts of them about, but only certain bacteria have the ability to consume them. They also don't dissolve in water, unlike a lot of the other foodstuffs that bacteria eat, which means that these bacteria are important in breaking the polysaccharides down into easier to digest foods for other organisms. I'm trying to find which ones these are, and whether they change over the course of the algal bloom, or in response to the extra CO₂ that we've been adding to some of the mesocosm bags (see Ian's blog below).

But why Crabby string? The two polysaccharides that I am looking at in particular are Chitin (which is found in the shells of crabs) and cellulose, which in this case is cotton string. These two polysaccharides are the most abundant on the planet and understanding how they are degraded by bacteria in the oceans is an important part of understanding the carbon cycle. Click here for more information on the carbon cycle.

I'm using several different techniques to accomplish this. As other people are doing I'm filtering water through different sized filters and taking these back to Liverpool to analyse the DNA.

( Me, taken by surprise filtering, with balloon! )

In addition to this I am using the SIP technique that Rich described. Unlike the compounds that he uses, which can be bought from specialist suppliers, I had to make my own! We have a strain of bacteria called Acetobacter which will make cellulose when it's fed glucose, so we bought some heavy glucose and have produced heavy cellulose. I'm currently incubating this with some of the water from the bags and hopefully will start seeing some utilisation (although not a lot seems to be growing on them at the moment!).

Finally, it is possible to extract all the proteins from the organisms that are in the seawater and using a technique called proteomics, identify them. The proteins do the actual work of breaking down polysaccharides inside the bacterial cells and Jennifer Edwards, a PhD student that I work with in Liverpool, will be looking at how the levels of different proteins change and which ones seem to be working the hardest.

I head home on Sunday, just as the weather has taken a turn for the (very) wet, which is pretty good timing! Louise from Cardiff who I'm sharing my sampling with, is coming out to replace me today. Fingers crossed, she might get a bit of sunshine, or at least some clear skies! Tomorrow we'll both be off in a little boat attempting to get some sediment samples with this device (known as A Van Veen grab, apparently!). If all goes well...I'll get her to let you know how it went!

( Me with dinky Van Veen grab )

Mike Cox (University of Liverpool)

I said earlier I would write another blog to tell you all how we read bacterial DNA, so here goes. Firstly, we need lots and lots of DNA from each different bacterium in order to be able to make any sense out of it, mainly because the equipment we use is not sensitive enough to be able to read the genes within the DNA directly from the individual bacteria. So we take 30 litres of sea water and collect all the bacteria from it and then extract their DNA. We then cut this DNA up into small pieces and put each piece into a separate new bacterial cell, we call this a ‘clone’.

When we have many thousands of these clones we call it a metagenomic library. ‘Metagenomic’ means that we are looking at all the genomes from all the different types of bacteria in the sea. To explain why we call it a library I would like to use the following analogy. Say the ‘metagenome’ is ‘The Complete Works of Shakespeare’, if we were to randomly cut many thousands of copies of the Complete Works of Shakespeare into millions of 3 page bits and then re-bind those 3 page bits into their own separate books, we would have a library of many small fragments. We could then read each 3 page book and find out what it was, if we came across two books which had overlapping pages we could start to compile a single ‘scene’, then a few more books and we could compile a single ‘act’ and then finally a single ‘play’.

For a metagenomic library we take the bacterial DNA from 30 litres of sea water and cut it into small pieces, we then re-package those pieces into individual bacterial cells and then grow these cells separately. That way when we grow that bacterium we get many millions of copies of the single small piece of DNA which we can then read. As we have re-bound every small piece we cut up, we have a library of cells (instead of books) each containing a separate fragment of the metagenome. As with the analogy, if we find pieces which overlap each other we can start to reconstruct a single bacterial genome (equivalent to a play), if we had enough time and money we could find all the small pieces and all the overlaps and reconstruct the genomes of every single bacterium in the ocean! As we can only culture less than 1% of the bacteria in the sea this would help us to understand what all these marine bugs are doing.

Therefore by making a metagenomic library before we increase the carbon dioxide, and one afterwards we can determine the changes in the types of bacteria and their roles. Even though bacteria are very small and seem insignificant, without them life could not exist on this planet as they recycle all the nutrients and gases on earth, allowing us and every other animal and plant to eat and breath!

Jack Gilbert (PML)

( Jack trying to catch as many bacteria as possible with his fins! )

By Monday, it was obvious that the phytoplankton (plants) in the bags were no longer growing quickly because they had used most of the nutrients that we added at the beginning of the experiment. The bags were full of phytoplankton cells, which had grown very well in the sunny conditions of the first week. They had grown so well that they had used so most of the added CO2 – and the pH was almost back to present-day conditions. We were no longer doing an ocean acidification experiment!

We decided that we had to reduce the pH again by bubbling with CO2-enriched air – as we did at the beginning of the experiment. We needed the acidified conditions so that we could study how bacteria might be affected by lower pH in a high CO2 world. The conditions in the bags were now ideal for studying bacterial activity and breakdown of organic matter. The phytoplankton bloom had resulted in high numbers of plant cells and lots of organic matter: they were no longer growing because they had run out of nutrients: some plant cells were being infected by viruses (and we had seen a very large increase in the number of viruses in the bags) and were dying, so producing more organic matter. All these were idea conditions to study bacteria breakdown and turnover of the dying plants- but the pH was wrong.

We decide to bubble two of the bags overnight with CO2-enriched air to try to get the pH down to 7.8 or so. But we also knew that bubbles could cause problems. They can break open some of the fragile phytoplankton cells that were growing in the bags – so the process of bubbling itself would affect things. We needed a control experiment – that is, to bubble with air that was not enriched with CO2. But we were also curious to know how the bags would behave if we did not manipulate then again. So there was a need not to change all of the bags.

With these conflicting demands, we decided to do the following.

Bags 1 and 2 (original high CO2 bags) were again bubbled with high CO2.
Bag3 (also originally a high CO2 bag) was not bubbled (so we could see how the original experiment would have turned out).
Bag 4 (an original ‘present-day’ condition) was also not bubbled – again continuing the original experiment.
Bags 5 and 6 were bubbled with air (no added CO2) to see what affect the bubbling process might have on the ecology of the bags.

The next day, the pH was where we wanted it; so we stopped bubbling all the bags. Now we are sampling to see how the bacteria will change in the different conditions. The bacteria are growing very well and this morning increased to more than 10,000 million bacteria per litre in one bag. Now we need to find out which bacteria are growing and if there is any effect of low pH.

Ian Joint (PML)

( One of the bags being bubbled with CO2 )

It will be obvious from our earlier blogs that a major activity here in Bergen is ‘filtering’. It may not be obvious why this is so important and why we have a group of talented scientists who spend so much of their time doing something so boring.

I have explained that bacteria are very abundant in the sea – about 1 thousand million bacteria in every litre of seawater. If they are so abundant, why do we need to filter them out of the water? Sadly, although they may be abundant, they are very, very small and contain tiny amounts of DNA. Even the most sophisticated molecular biology methods need more DNA than you can get directly from a small sample of seawater. So we have to concentrate the bacteria – and the most effective way to do this is to filter the water.

We use filter cartridges that collect every particle that is larger than 0.2 micrometres in size (that is, particles that are a 5 thousandth of a millimetre), so we trap all the bacteria in seawater in the filter cartridge. These are then frozen quickly in liquid nitrogen and stored at -80°C until they can be worked on in the molecular biology labs back in the UK. By keeping the bacteria at these very low temperatures, we can be sure that the DNA will be stable and will not be contaminated. We can also preserve RNA – the molecule that is involved in translating the information coded in the DNA into proteins. RNA is particularly important for us because it tells us which parts of the whole bacterial genome (all of the information needed to make a new bacterial cell) are active under different conditions. This will give us clues to what will be most affected in a future high CO2 world.

Ian Joint (PML)

( Michael Maguire filtering water )

So, these all-important trace gases which Frankie has mentioned. What happens after they get produced? The vast proportion of them get gobbled up by bacteria living in the seawater. Strange as it may sound, chemicals like dimethylsulfide (DMS) and methyl chloride (CH3Cl) are actually quite exciting foodstuffs, if you’re a bacterium. In fact, 80% of DMS produced in seawater gets eaten by marine bacteria before it has a chance to get out and play its all-important role in climate control. Greedy things. One of the many burning questions that we’re trying to answer in this experiment is:

Which bacteria eat which trace gas?

Now, how do we go about that? The answer is a technique called Stable Isotope Probing (SIP).

In SIP, we make a special version of DMS in which the two carbon atoms are replaced with “heavy” carbon, which is not normally found in the environment in large quantities. Any bacteria which eat this form of DMS will end up with heavy carbon atoms in every part of their cells, including their DNA. We take a large volume of seawater, add heavy DMS and leave it for a few days for the bacteria to get eating. Once the DMS has been consumed, we extract the total DNA of all the bacteria present and use a technique far too boring to describe in detail to separate out the heavy DNA (which is the DNA from the organisms which ate the DMS) from the normal DNA (which is from the non-DMS-eaters). We can then analyse the heavy DNA with DGGE (as lovingly described by John, previously) and determine exactly which organisms are eating the trace gas.

I am doing SIP experiments with DMS (which you will all recognise as the smell of cabbages), methane (natural gas), methylamine (something fishy produced by decaying plankton) and methanol (a type of alcohol) and I’m comparing how the bacterial populations which eat these compounds change over the course of the bloom. Mike and Andy are doing SIP experiments with bicarbonate and glucose. Rach and Nick are doing a slightly different kind of SIP to study nitrogen uptake by bacteria.

And now I’ve realized what a mammoth post I’ve just written I think I’ll stop!

Rich Boden (University of Warwick)