Vol. 60: 71–83, 2010 AQUATIC MICROBIAL ECOLOGY Published online May 4 doi: 10.3354/ame01409 Aquat Microb Ecol OPENPEN ACCESSCCESS Seasonal dynamics of bacterial biofilms on the kelp Laminaria hyperborea Mia M. Bengtsson*, Kjersti Sjøtun, Lise Øvreås Department of Biology, University of Bergen, Box 7803, 5020 Bergen ABSTRACT: Seasonal variations of the cell density and bacterial community composition in biofilms growing on the surface of the kelp Laminaria hyperborea from 2 sites on the southwestern coast of Norway were investigated using total cell enumeration and denaturing gradient gel electrophoresis (DGGE) fingerprinting. The major taxonomical groups of bacteria inhabiting the biofilms were iden- tified by DGGE band sequence classification. The microbial cell density of the biofilm appeared to be highly affected by the seasonal growth cycle of the kelp and was found to be lowest on growing kelp in March (minimum 8.3 × 102 cells cm–2), while on non-growing kelp in July to February, it was around 1.0 × 107 cells cm–2 with large fluctuations. The composition of the bacterial community of the biofilm followed a continuous seasonal succession that may be explained by the influence of both biotic factors such as seasonal changes in the kelp substrate and abiotic factors such as seawater tem- perature. Planctomycetes and Alphaproteobacteria were frequently detected throughout the year, while Verrucomicrobia, Cyanobacteria, Gammaproteobacteria, Betaproteobacteria, and Bacter- oidetes were more sporadically detected. The bacterial communities of the biofilm on kelp showed little overlap with that of the surrounding seawater. KEY WORDS: Kelp · Bacteria · Biofilm · Marine · Seasonal dynamics · Seaweed · DGGE Resale or republication not permitted without written consent of the publisher INTRODUCTION bacteria as secondary producers utilizing kelp-pro- duced carbon. Norderhaug et al. (2003) showed exper- Kelps (large brown algae of the order Laminariales) imentally that kelp material that had been partially form the basis of extensive underwater ecosystems degraded by naturally associated bacteria was a better known as kelp forests, which are found on rocky coasts food source than undegraded kelp for amphipods liv- both in temperate seas and in upwelling zones in the ing in kelp forests. These studies have focused on the tropics (Graham et al. 2007). Kelps are large contribu- bacteria as a group and have not been able to identify tors to primary production in these areas (Mann 2000), the bacteria responsible for these processes. Other and the unique 3-dimensional habitats that kelp studies have aimed to describe the composition and forests constitute make them biodiversity hotspots spatial and temporal dynamics of the bacterial commu- (Christie et al. 2003, Graham 2004, Coleman et al. nities associated with kelp. Early studies (Chesters et 2007). In many countries, kelp is harvested for the al. 1956, Laycock 1974) focused on culturable bacteria extraction of industrially valuable chemicals such as associated with kelp and their degradation of kelp sub- alginate as well as for food production. stances. Laycock (1974) also found a seasonal differ- Bacteria associated with kelp are believed to be crit- ence in the culturable bacteria from kelp manifested as ically important in many processes in kelp forest a predominance of psychrophilic and mesophilic bac- ecosystems. Several studies have addressed the flow of terial strains isolated in winter and summer, respec- matter in kelp forests in South Africa (Lucas et al. 1981, tively. The application of scanning electron microscopy Stuart et al. 1981, Newell & Field 1983). Their many (SEM) and fluorescent stains enabled in situ enumera- findings highlight the important role of heterotrophic tion of bacteria growing on kelp without the need of *Email: [email protected] © Inter-Research 2010 · www.int-res.com 72 Aquat Microb Ecol 60: 71–83, 2010 culturing. Corre & Prieur (1990) studied the density hyperborea cells (Sjøtun et al. 1996) as well as exuda- and morphology of bacteria on the kelp Laminaria dig- tion of dissolved organic matter in the form of carbohy- itata using SEM. They found a change of the bacterial drates and phenolic compounds (Abdullah & Fredrik- communities both in terms of the density of bacterial sen 2004). Because the contents and exudates of the coverage and the morphology of the bacteria in kelp cells may be sources of nutrition for kelp-associ- response to the aging of the kelp tissue. In a recent ated bacteria, seasonal variations in these are likely to study, Staufenberger et al. (2008) used molecular be important factors controlling the bacterial commu- methods to investigate the bacteria associated with the nities on L. hyperborea. kelp Saccharina latissima (as Laminaria saccharina). In this study, biofilms growing on the surfaces of the They could identify several different bacterial lineages kelp Laminaria hyperborea at different times of the based on 16S ribosomal RNA gene sequences and year are described using total microbial cell enumera- found differences in the composition of the bacterial tion as well as molecular fingerprinting and classifica- communities present on different morphological parts tion based on the 16S rRNA gene sequences of bac- of the kelp. They also found indications of seasonal and teria. We aimed to reveal some of the fundamental geographical differences. factors that control the composition and dynamics of Laminaria hyperborea is the largest canopy-forming bacterial communities in kelp forests by studying them kelp in the Northern Atlantic. L. hyperborea is esti- in a seasonal context. This may lay a foundation for mated to produce approximately 50 million tonnes of increased understanding of the roles of bacteria in kelp biomass (wet weight) annually along the Norwegian forests, which can ultimately shed more light on both coast (Fredriksen 2003). It forms the raw material for the ecology of kelp forests and the dynamics of natural industrial alginate extraction presently carried out in biofilms on living surfaces. Norway, and the kelp forests are an invaluable re- source for fisheries as they serve as nursing grounds for several commercially important species of fish MATERIALS AND METHODS (Norderhaug 2003). L. hyperborea consists of a smooth blade-like structure called the lamina that is attached Sampling of kelp biofilms and seawater bacteria. to an upright and flexible stipe, which can be up to 3 m Kelp Laminaria hyperborea was collected by dredg- long. A holdfast structure at the bottom of the stipe ing from a boat at 2 sites outside Bergen, Norway attaches the kelp to the substrate. L. hyperborea has a (Stn 1: 60° 30.505’ N, 4° 49.776’ E, Stn 2: 60° 09.706’ N, pronounced seasonal growth pattern, as the lamina is 5° 02.371’ E) at a depth of 5 to 9 m. Sampling was per- renewed every year during a growth period lasting formed at approximately 2 mo intervals evenly spread from January until June (Fig. 1). The most rapid out throughout 2007, starting in early February and growth takes place from March until May (Kain 1979). ending in late November. At each site and sampling This generates fresh surface area for marine microbes occasion, 6 intact, full-grown (stipe length >40 cm) to colonize and makes an excellent system to study kelp individuals were selected from the kelp gathered natural biofilm succession and dynamics. The old lam- by the rake-shaped specialized kelp dredge. The kelp ina is shed in April and is replaced by a new lamina individuals were transported to the laboratory in an that has grown out since January. There is also a sea- insulated box and were processed within a few hours sonal pattern of the carbon and nitrogen content of L. after sampling. Two different morphological regions Fig. 1. Laminaria hyperborea. Morphology and different growth cycle stages of sampled kelp. Shading of the lamina (L) and meristem (M) indicates the age of the surface tissue (light = younger, dark = older) Bengtsson et al.: Seasonal dynamics of biofilms on kelp 73 from each kelp individual were sampled; the lower focus’ functions of the Zeiss AxioVision software. One part of the lamina (meristematic region) and the mid- ‘extended focus’ image was created for each micro- dle part of the lamina (Fig. 1). These 2 regions are scope field consisting of information from several hereafter referred to as meristem and lamina. Areas images captured at different focal planes with 1 µm that were heavily epiphytized, for example, by bry- intervals through the entire depth of the biofilm of the ozoans and seaweeds, were avoided. A surface of sample. The number of images used to create the approximately 50 cm2 from each region of every indi- extended focus image varied between 5 and about 50, vidual kelp plant (the area included both sides of the depending on the topography of the kelp surface flattened thallus) was cut out and thoroughly rinsed and/or the biofilm thickness. The DAPI-stained cells with sterile seawater to remove any loosely attached were manually counted in each extended focus image. contaminating bacteria originating from the sampling When an image contained more than approximately process. The kelp surface was scraped using a sterile 200 cells, a grid with known dimensions was digitally scalpel, as microscopic inspection proved this method overlaid on the image, and the cells within 10 prede- to be more effective in removing all attached bacteria fined squares of the grid were counted. The cell den- than wiping with a cotton swab (results not shown). sity of each sample was estimated by calculating the The scraped off material was suspended in 10 ml of average density of the 10 captured microscope fields. sterile phosphate-buffered saline (PBS, 137 mM NaCl, DNA extraction. DNA from the pellets of scraped off 2.7 mM KCl, 10 mM phosphate, pH 7.4) and mixed by material from the kelp surface layers was extracted vortexing.