Heterogeneity of surface attached microbial communities from Sydney Harbour, Australia

Abstract

There is limited information on bacterial communities attached to marine surfaces. These surface attached bacterial communities can vary at a micro scale and these differences may be due to surface characteristics in marine environments. The current study investigates the heterogeneity of bacterial communities on five different marine invertebrates (Heliocidaris erythrogramma, Austrocochlea concamerata, Crassostrea gigas, Dendrilla rosea, and Actinia tenebrosa), the alga, Lobophora variegata and marine gravel from a 20 m × 20 m quadrant in Camp Cove, Sydney Harbour, Australia. Terminal restriction fragment length polymorphism (TRFLP) of 16S sequences showed that each surface contained unique combinations of TRFLP fragment lengths. Phylogenetic analysis of random clones picked from clone libraries constructed from the amplified 16S sequences revealed that 16S sequences from the communities on different surfaces clustered into distinct clades. None of the bacteria identified by 16S sequencing of the whole (uncultured) microbial communities was detected after cultivation. Overall, the study shows surface type plays a major role in shaping microbial communities in marine environments.

Introduction

Bacteria are essential components of all ecosystems, making up a high proportion of the planet's biomass and contributing to over half of its primary production (Azam et al., 1983, Fuhrman, 1992). The oceans cover 70% of the earth's surface and comprise 97% of the water on the planet (Adrianov, 2004). This suggests that uncharacterised microbial communities contribute significantly to the carbon and energy fluxes in oceans, potentially affecting predictions about the impacts of climate change (Copley, 2002, Fenchel, 2001, Karl, 2002).

Bacteria that grow on biotic and abiotic surfaces in the marine environment are heterotrophic and metabolically active (Bonin et al., 2001). They often live in complex biofilm communities that contain a diverse range of microorganisms. Biofilms are attached to a substratum and are embedded in an organic matrix of bacterial origin (Allison, 1998, Dunne, 2002, Costerton and Lewandowski, 1995). They cover the majority of tidal and subtidal habitats including abiotic surfaces, such as rocky shores, and biotic surfaces, such as marine invertebrates and algae (Dalton and March, 1998). All marine invertebrates studied to date are colonized by surface attached biofilms (Egan et al., 2008).

Until the mid 1990s, most studies on marine bacterial communities focused on the characterisation of cultured isolates. Those studies suggested most cultured surface attached communities are homogenous assemblages dominated by Alphaproteobacteria, Gammaproteobacteria, members of the Cytophaga/Flavobacterium group, or high and low GC gram positive bacteria (Lee et al., 2003). These dominant cultured groups have been identified on the surfaces of ascidians, bivalves, copepods, corals and sponges (Friedrich et al., 2001, Garland et al., 1982, Gillan et al., 1998, Hansen and Bech, 1996, Hentschel et al., 2001, Rohwer et al., 2001, Webster et al., 2001a, Wichels et al., 2006, Romanenko et al., 2001). However, the cultivation techniques used to characterise these groups generally employ nutrient rich media that are not amenable to the growth of chemo-organotrophic bacteria, which seem to make up the majority of bacteria in the marine environment (Oren, 2004). Nutrient rich culture provides a biased view of community structure by allowing some bacteria to be rapidly enriched during cultivation, even though these cultured bacteria may make up a minority of microbial ecosystems in terms of relative abundance. As a result, numerous studies have identified a large discrepancy between true bacterial diversity and the diversity evident in cultures (Eilers et al., 2000, Suzuki et al., 1997). Due to this cultivation-induced bias of microbial diversity, the majority of bacteria in the oceans, including surface attached bacteria, await discovery (Egan et al., 2008).

To counter this problem, recent studies have used molecular techniques to determine the abundance and phylogeny of all bacteria in marine environments, not just cultured isolates. The data now suggest that the most dominant groups of marine bacteria are Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Cyanobacteria (Gonzalez and Moran, 1997, Floyd et al., 2005). The recent application of molecular techniques, such as 16S rRNA gene sequencing (16S sequencing), also confirms that the majority of bacterial diversity in the marine environment remains to be discovered. These studies have revealed a wealth of unexplored diversity, including a broad array of new bacterial species, new phyla and even new bacterial kingdoms (Hugenholtz et al., 1998, DeLong and Pace, 2001). The highest estimate suggests that only 1–2% of marine bacterial species have been characterised so far, whilst other estimates of bacterial diversity suggest that only 0.1% of bacterial species have been discovered (Oren, 2004, Das et al., 2006).

Studies utilising molecular techniques have also revealed major differences between bacterial assemblages at a micro scale in communities attached to sediment, plankton and rocky shores (Falcon et al., 2008, Narvaez-Zapata et al., 2005, Scala and Kerkhof, 2000, Seymour et al., 2000, Seymour et al., 2004). Such small scale heterogeneity can only be explained by differences in environmental factors such as surface properties and biological interactions (Narvaez-Zapata et al., 2005). Some evidence also suggests that microbial assemblages may be specific to their marine eukaryotic hosts. To date, it is known that sponges and corals can harbour species-specific microbial associations (Bourne and Munn, 2005, Rohwer et al., 2002, Taylor et al., 2004, Webster and Bourne, 2007), and Taylor et al., 2005) have shown that unique species-specific associations occur on sponges at different locations. These specific associations may be driven by a number of factors, including the unique physical characteristics of each eukaryotic host surface, chemical communication between bacteria and their host, and vertical transmission of microbial communities from the eukaryotic hosts to their offspring (Egan et al., 2008).

Given the evidence for surface-specific microbial assemblages on corals and sponges and micro scale heterogeneity that can only be explained by environmental factors such as surface type, it is possible that surface specificity may be widespread on marine surfaces. The current study tests whether surface-specific associations are a common phenomenon among marine biofilms by assessing microbial diversity on 5 different classes of marine invertebrates, as well as algae and gravel, collected from a single geographic location (Camp Cove, Sydney Harbour, Australia) in a 20 m × 20 m quadrant. It also compares the microbial diversity of whole communities with cultured subsets derived from those communities to demonstrate that relying on analysis of cultured isolates may mask surface-specific associations. Initially, 16S sequence libraries were constructed using four samples from each surface, of the five marine invertebrates (Heliocidaris erythrogramma, Austrocochlea concamerata, Crassostrea gigas, Dendrilla rosea, and Actinia tenebrosa), the alga Lobophora variegate and marine gravel to determine variation in community structure between these surfaces. Random clones were then picked from different libraries constructed from these 16S sequence libraries to provide a more comprehensive view of heterogeneity of bacterial communities amongst different surfaces.