Aim of the project

Despite being the smallest components of the plankton community, viruses and bacteria play a major role in pelagic food chains and in biogeochemical cycles. Aquatic microbiology is a rapidly developing field, thanks to new methods for species identification, enumeration and measurement of rates of multiplication and turnover of carbon and other elements. There are roughly one million bacteria and 10 million viruses per ml in temperate marine water and fresh water in summer, which are far higher numbers than earlier estimates (Azam et al., 1983 and Bergh et al., 1989). Determination of bacterial diversity has become possible due to the development of DNA-based methods. However, knowledge in this field is still rudimentary, and there is no consensus among scientists with respect to the total number of species and their geographical distribution. In the marine environment, several thousands to millions of species have been anticipated (Giovannoni et al., 1990 and Pace, 1996). A cosmopolitan distribution with a low number of species raises the question of how such homogeneity is maintained, considering the large geographical differences among other components of the plankton community.

Much less is known about bacterial and virus diversity and abundance in the polar region compared to lower latitudes. Nielsen and Hansen (1999) found that bacterial abundance varied between 0.5 and 1.5 million per ml in water from the western coast of Greenland during summer, i.e. not different from conditions in temperate seas. Although exposed to extreme environmental conditions, e.g. sub-zero temperatures and low inputs of organic carbon through phytoplankton production, there is still controversy about what are the main factors controlling bacterial abundance and activity in the Arctic Ocean: temperature, nutrients, phytoplankton production, flagellate grazing or virus infection.

Our aim in joining the expedition with the ice-breaker Oden to the Arctic Ocean in 2002 was to describe, quantitatively and qualitatively, the structure of the microbial community in the East Greenland Current during a winter/spring situation. We estimated microbial abundance and bacterial species richness with flow cytometry and molecular biology methods, techniques hitherto seldom used in the Arctic seas.

Our study was based on the following hypotheses:

1. There is a gradient in bacterial and viral abundance, with increasing numbers from north to south and from east to west (i.e. away from Greenland towards more open water).
2. There is a positive correlation between bacterial abundance, bacterial activity and abundance of phytoplankton.
3. Geographic variation in bacterial abundance and activity will be large only in the first 100 m of the water column, while at greater depths bacteria are uniformly few.
4. Key microbial groups are widespread in the Arctic zone and are similar to groups found in other seas of the earth.

Work on board

Sampling was made mainly in transects from the east Greenland coast and eastward. The route, transects and sampling stations were mainly determined by the needs of the oceanographers, and as no prior knowledge of viruses and bacteria was available for the area, we choose to cover as large a geographical area and as many depths as possible. We sampled 32 stations in all for bacterial and viral abundance. By using water from the CTD rosette, we could sample through the water column down to the bottom at a depth of 3 000-meters on the deeper stations. Epifluorescence microscopy was used on board to quantify viruses, bacteria and flagellates. In addition bacterial numbers were routinely measured on board with flow cytometry in very small volumes (

Samples were taken from the non-toxic running seawater system of the ship (representing surface water from an approximate depth of 5 metres) for chlorophyll analyses and for determination of stable C and N isotope ratios. Large volumes (up to 20 l) were filtered through glass fibre filters for subsequent chlorophyll and isotope ratio analyses. Plankton was also collected by letting large volumes of the running seawater pass through nylon nets of various mesh sizes, concentrating mainly on zooplankton or phytoplankton. Chlorophyll was used as a measure of phytoplankton biomass and activity. Stable carbon and nitrogen isotope ratio analyses can allow an identification of carbon sources for bacteria and organisms higher up in the food chain (e.g. terrestrial sources versus organic carbon from phytoplankton) as well as an estimation of the trophic level of the organisms (e.g. grazers on phytoplankton versus predators on other animals). Phytoplankton samples were also collected for measurements of carbon uptake and parasitic infection as well as for genetic studies.

Common bacterial culturing techniques in combination with up to date molecular biology approaches were applied on 42 discrete water samples to assess marine microbial diversity. An aliquot of each sample was spread on a Petri dish with solid bacterial growth medium (in agar). If bacteria can grow on the medium they will form visible colonies. After 30 days’ growth each colony was picked up and transferred to a storage medium for later determination of species identity. Moreover, a larger volume of water was centrifuged to precipitate community DNA and frozen in liquid nitrogen for further analyses of bacterial species richness.

Preliminary results

Results presented in this report are preliminary, and data treatment and analysis are still ongoing.

We found a clear variation in bacterial numbers in surface water from north to south and from west to east. The lowest numbers of bacteria were found at the “ice stations”, i.e. stations with a solid 100% ice cover (e.g. section 1 and stations close to the Greenland coast). On the other hand, variation in bacterial abundance among stations was small for deep water, e.g. ranging from 0.04 to 0.15 million cells per ml at 1000 m. At the surface, bacterial abundance was an order of magnitude larger, and ranged from 0.1 to 1.5 million cells per ml. Increase in bacterial abundance was due mainly to an increase in the abundance of high DNA bacteria, suggesting a tight coupling between bacterial abundance and activity, i.e. the more bacteria the more active they were. In general, our results show that bacterial abundance and activity (expressed as the proportion of high DNA cells) were closely associated with high amounts of phytoplankton (expressed as chlorophyll a), high flagellate abundance (grazers of bacteria) and a low ratio of viruses to bacteria.

Concerning bacterial diversity, we were able to isolate 61 bacterial strains on Petri dishes. After comparing the small ribosomal DNA units (16S rDNA) of each isolate, 30 distinct strains remained (the rest were duplicates). The DNA of these 30 strains was sequenced at the Department of Molecular Biology, Umeå University. Unfortunately, results from this sequencing were not reliable (DNA not clean enough). Cleaning of DNA from the cultures is in progress, whereafter new sequencing attempts will be made. Each bacterial strain was also tested for sugar degradation ability (BiOLOG test) and total enzymatic activity (ApiZym test). These tests are often used in food and clinical microbial studies to determinate what bacterial species the isolate represents. All information related to each isolate is stored in our database, which will allow comparison with previous studies on bacterial diversity.

An integrated analysis of our microbial data, together with oceanographic data (e.g. salinity, temperature, nutrients, light) and phytoplankton data (e.g. species composition, abundance, pigments) will allow some of the questions put forward in the “Aims of the project” section above to be answered: What is the relationship between microbial abundance/activity and physical/chemical variables? Could differences between water masses, based on physical and chemical characteristics, explain the spatial variation in bacterial diversity? What is the main biological factor controlling bacterial abundance and activity in the Arctic Ocean (e.g. primary production, flagellate grazing or virus infection)? Answers to these questions will contribute to a better understanding of the mechanisms controlling the “microbial loop” (phytoplankton exudates – bacteria – flagellates – larger zooplankton) in cold waters. This is important as a large part of the biogenic elements in the sea involved in global change processes, e.g. carbon, are channelled through and controlled by bacteria, and probably by the viruses infecting them. Current knowledge of marine bacterial diversity is still very rudimentary, as many bacteria cannot be cultivated with standard methods, and as no assessment covering all seas using up to date methods has been done. The Arctic Ocean 2002 expedition will help to fill this gap with data from a rather extreme environment.

Phytoplankton samples from seven stations containing the toxin-producing dinoflagellate Dinophysis acuta were collected and fixed for later genetic and parasitic infection analysis. Single cells of Dinophysis acuta have already been processed for genetic analysis. The purpose of this was to explore the relationships between the plastids (chloroplasts) inside  Dinophysis acuta  with those from Dinophysis spp. from the North Sea and Baltic Sea. The plastids inside Dinophysis differ in pigment composition from those of other dinoflagellates. This means that Dinophysis might have replaced their original plastid, or that they feed phagotrophically on other algae. Sequences of the photosynthesis gene psbA (encoding the core protein D1 in photosystem II) have been determined from eight individual cells (Janson and Granéli, submitted manuscript). The results confirmed the hypothesis that the plastids have been replaced with plastids from a cryptomonad species, which belongs to another phytoplankton group. These data are important to understand the evolutionary history of the dinoflagellates and how they maintain their photosynthetic capacity.