Clocking the Microbes

by Forest Rohwer, head researcher on the Microbe Team

Microbial soup above an algae-carpeted reef at Christmas Atoll. Photograph by Jen Smith.

Microbes are major players in the health or decline of a coral reef. On the 2005 Line islands Expedition, we found that there were ten times as many microbes in each milliliter of reef water on the degraded, algae-covered reefs at Christmas Island as compared to pristine Kingman. Not only were there more, but they were more active and growing faster, and more of them were of the pathogenic kind. These observations combined with aquarium experiments indicated that what the microbes were doing was interrelated with other happenings on the benthos.

When a reef is not grazed, there is a lot more of the large fleshy algae that are commonly called seaweeds. Some of the sugars they make by photosynthesis are released into the seawater. These dissolved sugars make up a big part of the dissolved organic carbon pool—the DOC pool. Microbes eat DOC. More algae, more DOC, more microbes and more rapidly growing microbes. Too many microbes growing too fast are bad news for the corals. Normally there are the right numbers and the right kinds of microbes living in the coral’s surface mucus. There they pay their rent by protecting the coral from invading pathogens and by supplying it with usable nitrogen compounds. But when overfed, they can grow so fast that they consume all the local oxygen, and suffocate the coral.

One of the goals on the 2010 expedition is to measure the growth rate of the microbes at different locations and determine how it relates to the productivity of the other main members of the reef community (the corals, the algae, and the fish) and to the health of the reef (the reef’s CHI).

An effective and quick way to measure how fast the microbes on a reef are growing is to monitor their respiration by clocking the rate at which they use oxygen. Faster respiration means more rapid growth.

 

 

In principle, measuring microbial respiration is straight-forward. Put a water sample containing the microbes in question in a gas-impermeable jar, close the lid, and monitor oxygen usage over time. The devil is in the details.

First, microbes like to be locked up. This is called the bottle effect. Just putting the water sample in a jar makes the microbes grow faster (presumably because of the extra surfaces, which they like). So any experiment using a closed vessel will overestimate microbial respiration. That is not a major problem in this study because we will be comparing rates of microbial respiration from different sites, all measured under the same bottle conditions.

Second, even though there are collectively lots of microbes, there are relatively few in a water sample (about a million per ml). This means that the microbes in our samples use a relatively small amount of oxygen. to measure this we need particularly sensitive devices. If you put a small fish in a sealed 1 liter bottle to measure its oxygen usage, the fish would use up all the oxygen and die relatively quickly. If instead you fill that 1 liter bottle with reef water, the microbes therein could respire happily for some time.

On this cruise, to measure the oxygen uptake by the microbes we will be using both microelectrodes and optodes, the former being the established method and the later still a relatively new technique. The microelectrodes are our on-board choice to measure respiration in bottles, the optodes are what we will be using to measure the rates within the benthic tents deployed on the reefs. (For more about the benthic tents, visit  The Science page.)

Giant clams nestled among Caulerpa (a fast-growing, upright green algae) on Kingman Reef. Photograph by Jen Smith.

The downside for all this sophisticated equipment is that commercially available optode (and microelectrode) systems cost $2,000-$15,000 apiece. As a group, we have only six underwater optode systems. However, for this cruise Allison Gregg, an SDSU undergraduate, traveled to Ronnie Glud’s lab to learn how to make optodes. The Glud lab is one of the world leaders in this field. Notably, they adapted the system for two-dimensional on-site mapping of oxygen levels for deep-sea studies. Morten Larsen, also from their lab, invented a technique that allows one to use a simple CCD camera to measure the oxygen-quenching of the fluorescent optode sheet—a strategy that reduces the cost dramatically. We now have five of these systems, as well.

The advantage of making our own optodes is that we can also use them for profiling microbial production, i.e., their production of new biomass. A major challenge to measuring production on the reef is radiation, or the lack there of. The standard method used is to feed the microbes small amounts of tagged thymidine (a compound that is a building block used to make DNA) and then measure how much of the thymidine is now incorporated into new DNA. The reasonable assumption here is that the new DNA represents new cells. Again, because there are few microbes in a milliliter of seawater, the amount of thymidine incorporated into DNA by the microbes in a milliliter is very small. The easiest way to measure the thymidine incorporated is to use radio-labeled thymidine. This, of course, is fine in the lab but much harder in the field because you need permission to use any radioactive materials on a reef. Getting said permission can be nearly impossible when traveling between countries. And the Republic of Kiribati in particular has reasons to not want any of our radiation.

So we are trying a new, non-radioactive method to measure the production of new cells, a method developed by Invitrogen called Click-iT EdU. Instead of using a radio-labeled DNA precursor, this method uses a compound that is incorporated into newly synthesized DNA and that can be detected using fluorescent flow cytometry. Thus no radiation, and quick visualization of newly synthesized cells.

Clocking oxygen usage and new cell production by the microbes at multiple reef locations on six atolls equates to a lot of hours, and that’s not all our Microbe Team has planned for these short weeks.