Stanley C.K. Lau

Marine Biofilms as Mediators of Colonization by Marine Macroorganisms: Implications for Antifouling and Aquaculture

In the marine environment, biofilms on submerged surfaces can promote or discourage the settlement of invertebrate larvae and macroalgal spores. The settlement-mediating effects of biofilms are believed to involve a variety of biofilm attributes including surface chemistry, micro-topography, and a wide range of microbial products from small-molecule metabolites to high-molecular weight extracellular polymers. The settled organisms in turn can modify microbial species composition of biofilms and thus change the biofilm properties and dynamics. A better understanding of biofilm dynamics and chemical signals released and/or stored by biofilms will facilitate the development of antifouling and mariculture technologies. This review provides a brief account of 1) existing knowledge of marine biofilms that are relevant to settlement mediation, 2) biotechnological application of biofilms with respect to developing non-toxic antifouling technologies and improving the operation of aquaculture facilities, and 3) challenges and future directions for advancing our understanding of settlement-mediating functions of biofilms and for applying this knowledge to real-life situations.

Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (2 and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea.

A distinctive epibiotic bacterial community on the soft coral Dendronephthya sp. and antibacterial activity of coral tissue extracts suggest a chemical mechanism against bacterial epibiosis.

Abstract Different bacterial community profiles were observed on the soft coral Dendronephthya sp. and an inanimate reference site using terminal restriction fragment length polymorphism analysis of bacterial community DNA. To correlate the observation with a chemical defense mechanism against bacterial epibiosis, antibacterial effects of coral tissue extracts and waterborne products of coral-associated bacterial isolates (11 morphotypes) were tested against indigenous benthic bacterial isolates (33 morphotypes) obtained in the vicinity of the coral colonies. The coral tissue extracts and waterborne products of coral-associated bacteria inhibited growth and attachment of indigenous bacterial isolates, suggesting an endogenous chemical and an exogenous biological mechanism against bacterial epibiosis in this soft coral.

Inhibitory effect of phenolic compounds and marine bacteria on larval settlement of the barnacle Balanus amphitrite amphitrite Darwin

This study examined the inhibitory effect of 3 phenolic compounds and 12 strains of marine bacteria on the larval settlement of Balanus amphitrite amphitrite. The phenolic compounds used were phlorotannins, phloroglucinol and tannic acid. Phlorotannins are polymers of phloroglucinol (1,3,5‐trihydroxybenzene) known only from brown algae. Tannic acid, which exists in terrestrial plants, is composed of oligomers of phloroglucinol attached to a sugar molecule. The bacterial strains used were isolated from a natural biofilm. The following were investigated: 1) the toxicity of the phenolic compounds to B. a. amphirite in three different larval stages, viz. nauplius II, nauplius V and cyprid; 2) the potency of the compounds as inhibitors of larval settlement and the possible mechanism involved in settlement inhibition; and 3) the effects of the bacteria on larval settlement. The level of toxicity of the phenolic compounds varied widely for the larvae. Phlorotannins were most toxic, having LC50 values ranging from 9.47 to 40.35 μg ml‐1; phloroglucinol was least toxic, having LC50 values of 235.12 to 368.28 μg ml‐1. In general, nauplii were more sensitive to the toxicity of the phenolic compounds than cyprids. The greater sensitivity of nauplii may be due to their active feeding behavior, which exposes the interior of their bodies to the compounds by active intake. Phloroglucinol was the most potent settlement inhibitor, having an EC50 value of 0.02 μg ml‐1. Phlorotannins and tannic acid had EC50 values of 1.90 μg ml‐1 and 14.05 μg ml‐1, respectively. Phloroglucinol appeared to inhibit larval settlement through a relatively non‐toxic mechanism as its LC50 value was four orders of magnitude higher than its EC50 value. The high potency of phloroglucinol indicates that a simple constituent of a complex natural compound can be more effective than the natural compound itself. Larval settlement bioassays with monospecies bacterial films indicated that some of the bacterial species were inhibitory to larval settlement while the others showed no effect. None of the bacterial strains in this study induced larval settlement.

The bioactivity of bacterial isolates in Hong Kong waters for the inhibition of barnacle (Balanus amphitrite Darwin) settlement

Abstract Three bacterial isolates ( Micrococcus sp., Rhodovulum sp., and Vibrio sp.) from natural biofilms were investigated for their effects on cyprid settlement of Balanus amphitrite in laboratory bioassays. The inhibitive effect of these bacteria was clearly demonstrated by using a choice assay, in which cyprids settled preferentially on surfaces without bacterial pretreatment over those possessing a monospecies bacterial film. This result suggested that the inhibitive effect was mediated by direct larval contact with bacterial film surface rather than the perception of diffusible bacterial products. In a no-choice assay, monospecies bacterial films of different cell densities reduced cyprid settlement in a density-dependent manner. Vibrio sp. was the most potent inhibitor among the three isolates as it effectively inhibited cyprid settlement by relatively low-density films. The cells of Vibrio sp. were the smallest among the three isolates, suggesting that the correlation between bacterial cell density and cyprid settlement might not be merely due to the reduction of free-space availability. For all three isolates, films that were killed by formaldehyde or UV treatment were as potent as untreated, live films. These films remained inhibitive even in the presence of a strong promoter for cyprid settlement, namely conspecific settlement factor (SF), obtained from adult B. amphitrite . However, SF reverted the inhibitive effect of natural biofilms developed in the intertidal region.

Isolation of bacterial metabolites as natural inducers for larval settlement in the marine polychaete Hydroides elegans (Haswell)

The bacterial component of marine biofilms plays an important role in the induction of larval settlement in the polychaete Hydroides elegans. In this study, we provide experimental evidence that bacterial metabolites comprise the chemical signal for larval settlement. Bacteria were isolated from biofilms, purified and cultured according to standard procedures. Bacterial metabolites were isolated from spent culture broth by chloroform extraction as well as by closed-loop stripping and adsorption of volatile components on surface-modified silica gel. A pronounced biological activity was exclusively observed when concentrated metabolites were adsorbed on activated charcoal. Larvae did not respond to waterborne metabolites when prevented from contacting the bacterial film surface. These results indicate that an association of the chemical signal with a sorbent-like substratum may be an essential cofactor for the expression of biological activity. The functional role of bacterial exopolymers as an adsorptive matrix for larval settlement signals is discussed.

Induction of larval settlement in the serpulid polychaete Hydroides elegans (Haswell): role of bacterial extracellular polymers.

Larval settlement in the marine polychaete Hydroides elegans is effectively mediated upon contact with the surface of marine bacterial films. Using the bacterium Roseobacter litoralis as a model strain, the effect of bacterial extracellular polymers (exopolymers) on larval settlement of H. elegans was investigated. Bioassays with exopolymer fractions dissociated from bacterial films evoked the initial stages of the larval settlement process, i.e. larvae slowed down, secreted a mucous thread and crawled over the surface. This response is typical of larvae that encounter an attractive bacterial film. In contrast, bioassays with exopolymers in association with UV‐irradiated, metabolically inactive bacterial films evoked complete settlement. However, the percentage of responding larvae was negatively correlated with the magnitude of UV‐dosage. Since UV energy crosslinks both intra‐ and extracellular proteinaceous components, it could not be distinguished whether the decrease in larval settlement was due to a modification of proteinaceous components of exopolymers or due the elimination of cellular activity. Nevertheless, the results ascribe bacterial exopolymers the role of an indicator of substratum suitability and provide evidence that the polysaccharide moiety of exopolymers does not complement this effect.

Hydrothermally generated aromatic compounds are consumed by bacteria colonizing in Atlantis II Deep of the Red Sea

Hydrothermal ecosystems have a wide distribution on Earth and many can be found in the basin of the Red Sea. Production of aromatic compounds occurs in a temperature window of ∼60–150 °C by utilizing organic debris. In the past 50 years, the temperature of the Atlantis II Deep brine pool in the Red Sea has increased from 56 to 68 °C, whereas the temperature at the nearby Discovery Deep brine pool has remained relatively stable at about 44 °C. In this report, we confirmed the presence of aromatic compounds in the Atlantis II brine pool as expected. The presence of the aromatic compounds might have disturbed the microbes in the Atlantis II. To show shifted microbial communities and their metabolisms, we sequenced the metagenomes of the microbes from both brine pools. Classification based on metareads and the 16S rRNA gene sequences from clones showed a strong divergence of dominant bacterial species between the pools. Bacteria capable of aromatic degradation were present in the Atlantis II brine pool. A comparison of the metabolic pathways showed that several aromatic degradation pathways were significantly enriched in the Atlantis II brine pool, suggesting the presence of aromatic compounds. Pathways utilizing metabolites derived from aromatic degradation were also significantly affected. In the Discovery brine pool, the most abundant genes from the microbes were related to sugar metabolism pathways and DNA synthesis and repair, suggesting a different strategy for the utilization of carbon and energy sources between the Discovery brine pool and the Atlantis II brine pool.

A bacterial culture-independent method to investigate chemically mediated control of bacterial epibiosis in marine invertebrates by using TRFLP analysis and natural bacterial populations

It has been postulated that a variety of physically undefended marine invertebrates have evolved strategies to control microbial epibiosis chemically. Ecologically meaningful experiments that demonstrate chemically mediated antibacterial effects are difficult due to the small number of cultivable bacteria. Based on the example of three sponges, this study introduces a culture-independent methodology to investigate chemically mediated control of bacterial epibiosis by analyzing the natural bacterial consortia. Organic extracts of sponges were immobilized in hydrogels at tissue level concentrations and exposed to the same source of natural seawater for bacterial colonization. Terminal restriction fragment length polymorphism analysis of polymerase chain reaction-amplified bacterial community DNA obtained from these gels was shown to be a useful tool to study bacterial community shifts in response to sponge metabolites by comparing bacterial ribotypes obtained from the gel surfaces. Several terminal restriction fragments were absent relative to the control suggesting that settlement of specific bacteria was prevented. On the other hand, additional fragments occurred in some treatments, coinciding with higher bacterial abundance evidenced by DAPI counts of bacterial cells, indicating the bacterial utilization of sponge extract components. The advantages of this method are (1) a culture-independent approach, i.e. the assessment of antimicrobial activities against natural bacterial communities, (2) no restriction to particular modes of microbial colonization, i.e. antibiotic and repellant, and (3) the in situ assessment of antimicrobial compounds under flow conditions.

Response of bacterioplankton community structures to hydrological conditions and anthropogenic pollution in contrasting subtropical environments

Bacterioplankton community structures under contrasting subtropical marine environments (Hong Kong waters) were analyzed using 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of predominant bands for samples collected bimonthly from 2004 to 2006 at five stations. Generally, bacterial abundance was significantly higher in the summer than in the winter. The general seasonal variations of the bacterial community structure, as indicated by cluster analysis of the DGGE pattern, were best correlated with temperature at most stations, except for the station close to a sewage discharge outfall, which was best explained by pollution-indicating parameters (e.g. biochemical oxygen demand). Anthropogenic pollutions appear to have affected the presence and the intensity of DGGE bands at the stations receiving discharge of primarily treated sewage. The relative abundance of major bacterial species, calculated by the relative intensity of DGGE bands after PCR amplification, also indicated the effects of hydrological or seasonal variations and sewage discharges. For the first time, a systematic molecular fingerprinting analysis of the bacterioplankton community composition was carried out along the environmental and pollution gradient in a subtropical marine environment, and it suggests that hydrological conditions and anthropogenic pollutions altered the total bacterial community as well as the dominant bacterial groups.