In natural aquatic environments bacteria predominantly grow as multi-species communities called biofilms, attached to submerged surfaces. Better Knowledge of the development of biofilms is essential for studying their ecological role and also for devising effective strategies to control detrimental biofilms.

The present study is aimed to understanding the molecular aspects of biofilm formation on surfaces exposed to seawater. Here, bacteria were isolated from early stage biofilms developed on surfaces exposed to seawater at Kalpakkam (east coast of India) and phenotypically different strains were tested for their ability to form biofilms as monocultures.

Table-1 describes the number of isolates obtained from early stage marine biofilms associated with metal surfaces. These bacterial isolates were characterized by colony morphology, colony colour, Gram staining behaviour, cell motility, and biochemical tests. Biofilm formation potential of the bacterial strains was assessed by a microtiter plate assay and by direct microscopy coupled with image analysis. The results were further confirmed by studies using confocal laser scanning microscopy. Preliminary results indicate significant variability in biofilm forming potential among the biofilm bacterial strains isolated (Fig.1).

Fig 1. Biofilm formation by bacteria isolated from natural marine biofilms. The bars show the average A570±SD for triplicate wells of polystyrene multi-well plate. Biofilm formation potential of selected isolates is shown.

Only about 30% of the strains could form thick or moderately thick biofilms as monocultures, while the rest were poor biofilm formers. Isolate MA-S15 exhibited maximum biofilm forming ability and confocal laser scanning microscopic view of its biofilm on a glass substratum is shown in Fig.2. These bacterial strains are referred to as primary biofilm formers. It is presumed that these pioneer colonisers play an important role in the initiation of biofilm development in seawater and the subsequent colonizers co-adhere to them, leading to the eventual development of multi-species microbial communities on material surfaces.

Fig 2 Biofilm of a marine isolate MA-S15 grown on a glass substratum in mineral medium, stained with acridine orange and imaged using confocal laser scanning microscopy (Leica TCS SP2, Germany). A) Maximum projection of 30-xy optical sections of each 1 µm thickness. B) 1 µm xy- optical section taken at 5 µm from the substratum-biofilm interface. Voids are clearly seen within the microcolony of this bacterial biofilm.

Table- 1: Bacteria isolated from early stage biofilms developed on metal surfaces exposed to seawater (Kalpakkam, east coast of India)

• Isolates - Number of isolates
  Gram positive isolates - 11
  Gram negative isolates - 55
  Total number of isolates - 6

Interaction between biofilm bacteria was studied by a co-aggregation assay. Coaggregation refers to physical cell-to-cell interaction by which genetically distinct bacteria become attached to one another with the help of specific molecules. Present understanding suggests that such cell-to-cell interactions influence the development of complex multi-species biofilms.

In this context, the isolated bacterial strains were tested for their ability to coaggregate with one another in pair-wise manner using a simple visual coaggregation assay. Assay results showed that all the bacterial isolates demonstrate coaggregation with at least one other isolate (Table.2). Little correlation between the extent of coaggregation and any particular cellular characteristics of the isolates, such as motility, colony color, colony morphology or Gram stain, could be discerned.

Present study has revealed for the first time that coaggregation does take place among marine biofilm bacteria. The fraction of heterotrophic bacteria from marine environmental sample culturable on a general medium has been reported to be less than 1% of the total number of bacteria present. Therefore, the bacteria used in the present study possibly represent only a small proportion of the total biofilm bacteria. Even within these culturable bacteria coaggregation appears to be a significant phenomenon and is expected to play a role in the development of multispecies communities. Further studies are required to understand the role of co-aggregation and co-adhesion on the development of complex microbial biofilms on material surfaces exposed to the marine environment.

From- http://www.nio.org/; Y.V. Nancharaiah, Rajesh Kumar and V.P. Venugopalan, Water & Steam Chemistry Laboratory, Tamil Nadu