Biofilms are microbial aggregations growing at interfaces. They are involved in a number of environmental processes and are widely employed in biotechnological applications. They also cause substantial revenue loss in various industries by way of biofouling and biocorrosion.

Various studies have indicated that multispecies biofilms have characteristic architecture and behave very much like 'quasi-tissues', exhibiting remarkable metabolic co-operativity among constituent microcolonies and a primitive type of homoeostasis (Costerton et al, 1995; Palmer and White, 1997; Davies et al, 1998). New understanding regarding the complex behavioural and survival strategies of biofilms have led to the emergence of the concept of 'biofilm ecosystem' (Palmer and White, 1997), with biofilms being referred to as “cities of microbes” (Watnick and Kolter, 2000). Results of various studies show that the three-dimensional distribution of microbial species within the biofilm matrix is a very important factor determining various functional aspects of the biofilms such as substrate removal and conversion, nutrient and oxygen flux as well as interactions between constituent species (Rickard et al, 2003). Nevertheless, the temporal and often sequential development of such complex architecture, wherein the physical, chemical and biological structure of the biofilms determine their functional role in environmental and biotechnological applications, is not well understood. Attempts have been made to gain clear understanding of the dynamic structural and compositional changes during the development of mixed-population biofilms, occurring in different phases of their formation, especially with respect to cell adhesion, exopolymer generation, microcolony formation, spatial arrangement of microcosortia and their interactions among themselves (Neu et al, 2002). Such data would be helpful in better understanding of the significance of biofilm architecture in the functional efficiency and survival strategies of mixed population biofilms growing in natural and industrial environments and us help design better bioreactors and more effective means of controlling undesirable biofilms.

Studies on biofilm architecture received an impetus with the advent of confocal laser scanning microscope (CLSM). CLSM allows non-destructive optical sectioning of biofilms. Combined with multiple staining, multichannel flow cells, time lapse imaging and digital image processing, confocal microscopy offers a powerful suite of technique for the elucidation of the development of architecture in biofilms and its influence on biofilm structure and function (Palmer and Sternberg, 1999).

Numerous studies have shown that biofilms consist of columns of cell aggregations, interspersed with water channels that extend up to the biofilm-substratum interface. Structural and functional heterogeneity in biofilms can be studied using techniques such as fluorescence in situ hybridization, reporter genes (e.g., gfp ) and fluorogenic redox indicators like 5-cyano-2,3-ditolyl tetrazolium chloride ((Errampalli et al, 1999; Jansson, 2003; Wuertz et al, 1998). Temporal changes in void fraction and water channels vis-à-vis cell aggregates in the biofilm have been elucidated using diffusion of fluorescently tagged dextrans or fluorescent dyes such as fluorescein and phycoerythrin, and following their movement through the biofilm matrix using CSLM. Particle velocimetry have been employed to study liquid flow through biofilm channels.

The development of characteristic architecture in nascent bacterial biofilms was studied using a gfp -tagged derivative of Sphingomonas sp. strain L126 grown in flow cells. The method could show the development of microcolonies under varying flow conditions, distribution of biofilm biomass and EPS in space and time and changes in diffusion length as a fucntion of EPS production. Analysis of the confocal stacks showed that biomass and EPS distribution in the biofilm were maximal at some distance above the substratum. Consequently, the higher void fraction near the substratum may allow environmental fluids greater access to the biofilm base. Early stages of biofilm formation were characterised by dynamic fluctuations in biovolume, indicating growth/accretion and dislodgement of cells. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform; adjacent microcolonies exhibited significant differences in growth rate. The microcolonies had the ability to move across the attachment surface, irrespective of fluid flow direction. Though the p resent understanding suggests that microcolony formation is the outcome of a coordinated, adaptive response, the key processes that regulate multicellular differentiation inside biofilms are poorly understood. Factors such as signal molecules (e.g., acylated homoserine lactones) and programmed cell death have been reported to be involved (Miller and Bassler, 2001).

Biofilms are formed because of the innate ability of bacteria to attach to surfaces and entrap themselves in their own EPS. Biofilms and other such immobilised microbial systesm are extensively used in biotechnological applications (Wilderer et al, 2000). Nevertheless, bacteria also have the ability to attach to one another and form self-immobilised granules, by a process known as biogranulation. These granules are dense, compact aggregates of microorganisms and consist of a consortium of different microorganisms held together in a polymer matrix. Microbial granulation under aerobic conditions is a recently described phenomenon (Beun et al, 1999). Aerobic granules have great potential to be used for degradation and removal of wastes. They have several advantages as compared to conventional activated sludge flocs, such as compact and dense microbial structure, good settling ability, high biomass retention in bioreactors, good volumetric exchange ratio and the ability to withstand fluctuating substrate loading rates. There are very few studies on the architecture of aerobic granules and its significance in substrate removal. Mixed culture microbial granules were successfully cultivated in laboratory scale sequencing batch reactors and used for degradation of synthetic metal complexants. Conofcal microscopy based techniques are being used to study the architecture of the granules. Salient features of the results and problems encountered are presented.

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