In response to certain environmental signals, bacteria will differentiate from an independent free-living mode of growth and an interdependent surface-attached existence. These surface-attached microbial communities are known as biofilm. Biofilms are three-dimensional functional consortia of microbial cells, bound to, and growing at an interface ( e.g. solid-liquid; solid-air; liquid-air, liquid-liquid) enveloped within extracellular polymeric substances ( EPS) produced by the microorganisms. The biofilms can be found on stones in rivers, on-pipes in industrial waste water systems, in paper and paint manufacturing plants, in health-care settings and cooling water systems, inner part of food packaging, and in heat exchangers. In dairy and food industry, biofouling causes serious problems such as impeding the flow of heat across the surface, increase in the fluid frictional resistance at the surface and increase in the corrosion rate at the surface leading to energy and product losses. Stubborn biofilm can be produced by Pseudomonas aeruginosa, since this bacteria produce rhamnolipid and autoinducer. It is N-acyl homoserine lactone, which can produce signal if the amount of microorganism achieve certain number of growth. The aims of this research is to study the effect of autoinducer(PAI2) in biofilm development, since this compound has the ability to retard the growth of the bacteria at certain level of concentration. In this work, the study was extended to evaluate the effect of PAI2 and rhamnolipid on biofilm development using the wild type and rhlI(-)mutant. The presence of rhamnolipid may be able to maintain open channels by afflecting cell-cell interaction and the attachment of bacterial to surfaces.

It is shown that the addition of autoinducer ( 10 microM) gave impact to the thickness of biofilm and also increase rhamnolipid production. The PAI2 can give two effects, the first is to slow down the growth of the bacteria, and the second is to make the biofilm more porous due to the rhamnolipid production. There is no rhamnolipid production in rhlI(-)mutant , since there is no PAI2 is produced , but there is small amount of rhamnolipid production in rhlI(-)mutant which is added with PAI2. There is a different in the biofilm thickness on rhlI(-)mutant which is fed by PAI2 compare to that wasn't fed by PAI2 . The results showed that the effect of PAI2 addition in the wild type can be seen after the day 4 (Fig2a). The concentration of PAI2 does not always reflect what is occurring in the biofilm ( Table 1), however we notice that the growth is slightly slow when the PAI2 occurred which can be seen in the thickness of biofilm in wild type compare to mutant, the thickness of biofilm is greater when the production of rhamnolipid is also greater. The concentration of PAI2 increased for the first 24 hours due to it is synthesized and then decrease due to it is degraded naturally ( Table 1). Since we change the culture medium every day with the fresh medium, hence the most significance period to observe the trend of rhamnolipid concentration, the autoinducer (PAI2) concentration was at the first 24 hours

Table.1. Concentration of PAI2 in wild type . Time (hours) PAI2 concentration (Wild type) PAI2 concentration (Wild type , PAI2 addition ) 0 0 1.99 8 3.34 9.58 11 7.95 9.8 Change to new medium 0 0.53 2.3 9 7.45 2.69

Fig.2.a. The thickness of biofilm in wild type , PAI2 addition compare to no addition of PAI2

In conclusion, at the beginning formation of biofilm , the PAI2 will slow down the development of the biofilm, then once the rhamnolipid production increase, which is induced by rhlI, the biofilm becomes more porous . To control the biofilm development , we need to destroy the autoinducer either using enzyme or other approach, since the PAI2 is responsible for the production of rhamnolipid which relates to the thickness of biofilm as well.

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