Biofilm, a complex assembly of microbial cells anchored to surfaces within a polysaccharide matrix, forms a harmonious and organized microbial community. This strategy enables microorganisms to thrive in favorable conditions, avoiding displacement by currents. Comprising cells and extracellular polymeric substances, biofilms exhibit dynamic equilibrium with their surroundings, cycling through growth, death, and regeneration.

Microorganisms gain remarkable advantages from biofilm cultivation, including protection from harsh conditions, enhanced resilience to stress, and cooperative metabolic and gene expression adjustments. Microorganisms thriving in biofilms exhibit an astonishing ability to adapt, demonstrating a captivating collective and synchronized behavior that intrigues both scientists and enthusiasts. (Donlan et al., 2002; Chmielewski and Frank et al., 2004; Van Houdt and Michiels et al., 2010). Multispecies biofilms are prevalent in nature, significantly impacting ecosystems (Donlan et al., 2002; Hall-Stoodley et al., 2004).

1. Microbial Biofilm Formation: A Complex Developmental Process in Bacteria

Biofilm formation is a captivating process wherein bacteria transition from single cells to structured multicellular communities. These parallels other bacterial developmental processes like sporulation (Dunny GM, Leonard BA. Et al.,1997), fruiting body formation (Plamann L et al.,1995, Shimkets et al.;1999, Wall D et al.,1999), and stalked-cell formation (Fukuda AK et al.,1977, Hecht GB et al., 1995, Trun NJ et al.,1990, Quon KC et al., 1996, Wu J et al.,1997). In nature, biofilms exist as diverse microbial communities, with bacteria joining, departing, exchanging genetic material, and occupying specific niches, resembling complex cities rather than developed organisms.

1.1 Biofilm Formation in Non-Motile Species

In non-motile species, during favorable conditions for biofilm formation, bacteria increase adhesin expression, enhancing stickiness for cell-to-cell and cell-to-surface adherence (Gotz, 2002). Surface proteins like Bap(biofilm-associated protein)  in staphylococci aid cell interactions and matrix creation (Lasa and Penades et al., 2006). Similar proteins exist in other species, often with repeated domains undergoing recombination within the bap gene, yielding variable-length proteins (Latasa et al., 2006). Additionally, species that lack motility also generate exopolysaccharides (EPS), which subsequently become crucial constituents of the extracellular matrix. An illustrative instance of this is the production of PIA(Polysaccharide intercellular adhesion )/PNAGS (Poly-N-acetylglucosamine )EPS by the gene products originating from the ica operon in staphylococcal species. Thus, altered surface proteins and EPS production are key in initiating biofilm formation in non-motile bacteria.

1.2 Biofilm Formation in Motile Species

In motile species, conducive conditions trigger bacteria to adhere to a surface, lose motility, and form biofilms. An extracellular matrix binds them together. Flagella are vital for biofilm initiation; flagella-minus mutants show reduced biofilm formation (Pratt and Kolter et al., 1998; Watnick and Kolter 1999; Lemon et al., 2007). Listeria monocytogenes recover initial adhesion with directed movement, indicating motility’s role in overcoming repulsion for biofilm formation (Lemon et al., 2007). The initial surface encounter leads to transient adherence, determining stable biofilm development or return to planktonic state.

2. Genetically Distinct Stages of Biofilm Formation

Biofilm formation can be divided into five genetically distinct stages:

1. Initial surface attachment

2. Monolayer formation

3. Migration to form multilayered microcolonies

4. Production of extracellular matrix

5. Biofilm maturation with characteristic three-dimensional architecture (O’Toole et al., 2000)(fig 1).

3. Regulatory Variations in Biofilm Formation

In numerous motile bacteria, initial surface attachment relies on flagella-mediated motility. However, specific Gram-negative bacteria require type IV pili-associated surface motility for microcolony and three-dimensional architecture formation. Notably, this motility is absent in Gram-positive bacteria, except for Clostridia ssp. (Varga et al., 2006; O’Toole et al., 2000)

4. Extensive Cellular Differentiation in Biofilm Environment

Once the bacteria have successfully adhered to the surface, they begin producing an extracellular matrix. This matrix serves as a crucial organizational element, enabling the formation of structured communities within the biofilm. As a result, extensive cellular differentiation can occur within the biofilm environment.

5. Biofilm Composition and Architecture

Its composition hinges on the inoculum’s characteristics, while external factors like substrate, nutrients, competition, and grazing shape colonization and growth (Baier et al., 1980; Characklis & Cooksey et al., 1983; Marshall et al., 1985).

The architecture of biofilm is shaped by hydrodynamics, nutrients, bacterial motility, communication, exopolysaccharides, and proteins. Altered biofilm morphology in mutants lacking components of extracellular polymeric substances (EPS) illustrates their impact. Exopolysaccharides in Vibrio cholerae and colanic acid in Escherichia coli influence three-dimensional biofilm formation.

5.1 Biofilm Architecture in Bacillus subtilis

In the case of Bacillus subtilis biofilm, the matrix consists of an exopolysaccharide and the secreted protein Tas A, both of which are essential for maintaining the structural integrity of the matrix and facilitating the development of biofilm architecture resembling fruiting body-like structures (Fig 2). 

6. Factors affecting the formation of biofilm.

Biofilms form at interfaces of aqueous or gaseous phases and substratum surfaces in diverse systems. Metabolic substrates required for growth must be accessible in the aqueous phase (e.g., medical catheters) or between aqueous and solid phases (e.g., minerals). Nutrient quantity and ratios in the liquid phase impact growth-limiting factors (van Loosdrecht et al., 2002).

Biofilm development is influenced by factors like temperature, pH, Oxygen levels, hydrodynamics, osmolarity, ions, nutrients, and biotic elements, collectively shaping bacterial behavior as mentioned in Fig 3 (van Loosdrecht et al., 2002).

6.1 The Relevence of EPS in Biofilm Composition

In biofilms, microorganisms make up less than 10% of the dry mass, with over 90% being the EPS matrix. EPS facilitates cell adhesion, cohesion, and interactions, including cell-cell communication, promoting the formation of micro consortia. It acts as an external digestive system, retaining enzymes to sequester nutrients from the water phase. Biofilm morphology varies, from smooth and flat to rough, fluffy, or filamentous, even forming mushroom-shaped colonies surrounded by water-filled voids. The structure adapts in response to nutritional changes, supporting diverse habitats and the coexistence of mixed-species consortia (Klausen et al., 2003).

Table 1 Function of EPS for Biofilm Formation ( Flemming HC, Wingender J et al., 2010)

6.2 Surface factor

Interactions between bacterial cell walls and surfaces are influenced by interfacial electrostatic and van der Waals forces (McClaine JW et al.,2002, Vigeant MA et al.,2002). Other factors, such as hydration forces, hydrophobic interactions, and steric forces, also impact cell attachment (Findenegg GH. JN Israelachvili et al., 1985). Hydrophobic interactions (low surface energy) and electrostatic interactions charge have been extensively studied.

6.2.1 Interactions Driving Bacterial Cell Attachment to Surfaces: Electrostatic Force

Electrostatic forces drive bacterial cell attachment to surfaces, given bacteria’s net negative charge (Soni KA et al., 2008, Katsikogianni MG et al 2010). Positively charged surfaces facilitate rapid attachment, while negative surfaces hinder it. Extracellular organelles aid in overcoming repulsion (Bullitt E et al., 1995). High ionic strength reduces charge effects. Bacterial cell walls expose functional groups interacting with substrates (Hong Y et al.,2008). Adsorbed molecules influence surface chemistry and charge, aiding adsorption and biofilm growth. Hydrophobic groups and organelles stabilize interactions after repulsion.

Biofilms form on various materials in contact with bacteria-containing fluids, influenced by surface roughness, chemistry, and conditioning films (Donlan et al., 2002). Hydrophobic surfaces promote faster attachment through interactions with flagella, fimbriae, and pili (Donlan et al., 2002; Donlan and Costerton et al., 2002). However, exceptions like Listeria monocytogenes prefer hydrophilic surfaces (Chavant et al., 200). Clinical isolates of Staphylococcus epidermidis favor biofilm formation on hydrophobic substrates (Cerca et al., 2005). Hydrophilic surfaces generally exhibit higher bacterial attachment than hydrophobic surfaces (Donlan, 2002).

6.2.2 High energy surface

Thermodynamic analysis of surface energies reveals insights into bacterial adhesion (Absolom DR 1983). Hydrophilic surfaces enhance bacterial adhesion when cell wall surface tension exceeds the surrounding liquid (Absolom DR et al.,1983). Fluorinated materials with large contact angles indicate low-energy surfaces (Absolom DR et al., 1983). Oxidation of fluorinated surfaces reduces initial bacterial attachment due to altered hydrophilic properties (Davidson CA et al., 2004). Bacterial attachment depends on surface free energy; high surface free energy surfaces like stainless steel and glass are more hydrophilic (Davidson CA et al., 2004). Biofilm growth depends on various factors, including nutrient concentrations and light availability, once bacterial cells adhere and achieve confluence.

6.3 Role of Biosurfactants in Bacterial Attachment and Biofilm Formation

Biosurfactants with antibacterial and antifungal properties are crucial for bacterial attachment to and detachment from oil droplets (Kim HJ et al., 2011 ). Research interest in environmentally friendly chemicals has grown, including biosurfactants (Kim HJ, Boedicker JQ, Choi JW, Ismagilov RF et al., 2008, Eun YJ et al.,2009 ). Microorganisms produce biosurfactants at the air-water interface, influencing surface tension and gas exchange in surface waters (Flickinger ST et al.,2011). Rhamnolipids, found in the EPS matrix of P. aeruginosa, act as surfactants, contributing to microcolony formation, bacterial migration, and biofilm dispersion (Boedicker JQ et al.,2009) (Vincent ME et al.,2010, Renner LD, Weibel DB et al.,2011) (Harmsen M et al., 2010).

6.4 Influence of Quorum Sensing (QS) on Biofilm Formation

Quorum sensing (QS) is a crucial regulatory mechanism for biofilm formation (Parsek and Greenberg 2005). Microorganisms release auto-inducers (AIs) that induce or repress QS-controlled genes, impacting biofilm structure and 3-dimensional organization (Steinmoen et al. 2002). QS also influences population size and dispersion in biofilms (Lewis et al., 2001; Davies et al., 2003; Jesaitis et al. 2003). Additionally, QS can induce behaviors and control group activities, affecting biofilm development and resistance to stress (Camilli and Bassler et al.,2006). Cell properties like hydrophilicity, fimbriae, and flagella also influence microbial attachment in biofilms.

6.5 Chemotaxis triggers biofilm formation

Chemotaxis is vital for biofilm formation in bacteria like Pseudomonas aeruginosa, E. coli, and Vibrio cholerae (O’Toole GA et al., 1998, Pratt LA et al.,1998, Watnick PI et al.,1999). Bacteria sense chemical stimuli, use flagella to swim toward nutrients, and attach to the substrate. Flagella and/or type IV pili are essential for initial cell adhesion, leading to microcolony formation (Stelmack PL et al.,1999). E. coli also utilizes type I pili for initial attachment. Bacterial chemotaxis aids biofilm growth and spread as it develops.

6.6 Importance of Horizontal Gene Transfer (HGT) in Microbial Communities

Horizontal gene transfer (HGT) is crucial for microbial evolution, facilitated by mobile       genetic elements like plasmids, transposons, and bacteriophages (Koonin EV et al., 200) Bacterial biofilms show distinct gene expression compared to planktonic counterparts, influenced by the surface they settle on (Keyhani NO et al.,1996) HGT occurs within biofilms, allowing gene transfer between bacteria (Roberts AP et al.,1999).HGT also plays a significant role in animal evolution, diseases, and intercellular processes, like Agrobacterium transferring genetic material to plants (Guo M et al.,2019). Chemotaxis influences HGT, seen in Agrobacterium’s DNA transfer to plant cells (Guo M et al.,2019)., Niehus R et al.,2015). Other pathogenic bacteria also employ chemotaxis-derived HGT within host cells (Dougherty K et al.,2014 , Desmond E et al.,2007).

6.7 Interspecies Interactions in Bacterial Adhesion: Implications for Biofilm Stabilization and Commensal Relationships

McEldowney and Fletcher (McEldowney S, Fletcher M et al.,1987) showed that bacterial adhesion to a surface can affect other species differently, with negative, positive, or neutral outcomes. Inhibitory interactions may involve cell blockage or secretion of inhibitory macromolecules (Belas MR, Colwell RR et al.,1982).

6.7.1 Positive Interactions during Adhesion

Positive interactions during adhesion can result from bacterial products modifying the conditioning film or direct cell-to-cell contact. Dental plaque exemplifies direct interactions, where ligand-receptor interactions cause interspecies coaggregation (Kolenbrander PE et al., 1989). Hydroxyapatite attachment enhances the adhesion of co-aggregating pairs (Ciardi JE, McCray GF et al.,1987, Schwarz SU, Ellen RP 1987 ). Other interactions, like mutans streptococci adhering to immobilized oral bacteria, play a role in biofilm formation (Lamont RJ et al.,1990). Interspecies interactions are crucial during the initial stages of biofilm formation.

6.7.2 Neutral Adhesion Interactions

The attachment of one species to a substrate may not always be influenced by another species’ attachment (Cowan MM et al.,1991, McEldowney S et al.,1987). Neutral interactions could result from separate binding sites on the substrate for each species, including multiple high- and low-affinity sites within species (Gibbons RJ et al.,1983, Korber DR et al.,1994). For example, a nonmotile mutant strain of Pseudomonas fluorescens attached to glass independently of the parent strain, suggesting different binding sites (, Korber DR et al.,1994). Adhesion interactions depend on the surface and species involved (McEldowney S, Fletcher M et al.,1987), leading to variations in adhesion mechanisms for different substrates. Vibrio proteolytica’s adhesion to hydrophobic substrates was inhibited by proteases, while hydrophilic substrates were unaffected (Paul JH et al.,1985).

6.8 Commensal Interactions in Biofilm Stabilization

One intriguing aspect of biofilm stabilization is that it can be seen as a commensal interaction, where one species benefits from another’s ability to form a stable film.

6.8.1 Types of Commensal Interactions in Biofilms

Commensal interactions in biofilms occur when one population benefits without affecting the other. Oxygen consumption by aerobic/facultative microorganisms creates oxygen gradients, enabling the growth of obligate anaerobes (Costerton JW et al.,1994,De Beer D et al.,1994, Lewandowski Z et al.,1994). This interaction is crucial for sulfate-reducing bacteria growth in anaerobic microniches, contributing to microbially-induced corrosion (Hamilton WA. et al.,1985, Lee W, Lewandowski Z et al.,1993).

6.8.2 Commensalism in Dental Plaque Development

In dental plaque development, commensalism involving oxygen consumption occurs, with aerobic bacteria declining, and anaerobic Veillonella spp. predominating in deeper layers, while aerobic species dominate upper layers (Ritz HL et al.,1967 1969). Also in waste-water treatment biofilms, layering of aerobic and anaerobic bacterial species illustrates commensal interactions (Alleman JE, Veil JA et al.,1982). Other commensal interactions like substrate provision may be common in biofilms but require further research (Ritz HL et al.,1967).

7. Symbiotic Relationships and Enhanced Growth in Bacterial-Associated Algal Biofilms

Bacteria initiate EPS matrix formation in algal biofilms, leading to symbiotic relationships and competition with algae (Davis LS, Hoffmann JP et al.,1990, Mack WN et al., 1975). Studies show significant benefits of bacterial presence for algal recruitment and growth (Irving TE et al.,2011, Hodoki Y et al.,2005, Sekar R et al., 2004, Holmes PE et al., 1986). Algal biomass increases with rising pre-conditioned bacterial biofilm concentrations (Hodoki Y et al.,2005). Mixed community biofilms exhibit higher algal growth compared to pure algal cultures (Holmes PE et al., 1986).

Algae biofilms in non-sterile wastewater show increased thickness, switch to a sessile state, and resist shear stress better due to bacteria and EPS presence (Irving TE et al.,2011).Irving and Allen (Irving TE et al.,2011) demonstrated a nine-fold increase in algae biofilm thickness when grown on non-sterile secondary wastewater effluent compared to sterile wastewater. They also found that in the presence of unsterile wastewater, algae cells had a greater tendency to switch from a planktonic (free-floating) to a sessile (attached) state and exhibited enhanced resistance to shear stress.

8. Impactful Roles of Biofilm in Chemistry and Environmental Restoration

Biofilms are valuable in chemical synthesis processes, including ethanol, poly-3-hydroxybutyrate, and benzaldehyde production (Kunduru and Pometto, 1996; Li et al., 2006; Zhang et al., 2004). They also play essential roles in wastewater treatment, phenol bioremediation, and the degradation of dinitrophenols and toxic metals in environmental remediation (Lendenmann et al., 1998; Luke and Burton, 2001; Nicolella et al., 2000; Singh and Cameotra, 2004). Biofilms are highly resilient and serve as valuable tools for beneficial purposes in various chemical processes and environmental applications.

9. Conclusion

Biofilms represent dynamic and well-organized microbial communities that play a vital role in various ecological and industrial processes. They form through a complex developmental process, transitioning bacteria from a free-floating state to sedentary, multi-layered communities. The formation of biofilms involves various factors, such as surface characteristics, chemotaxis, and horizontal gene transfer, which contribute to the unique three-dimensional architecture and stability of these communities.
Interspecies interactions within biofilms further enhance their complexity, leading to commensal relationships and symbiotic associations that benefit different microbial species within the community. Bacteria initiate biofilm formation, and their presence fosters the recruitment and growth of algae, creating symbiotic relationships within mixed community biofilms.
The significance of biofilms extends beyond microbial interactions, as they also find applications in chemical synthesis and environmental restoration. Biofilms play crucial roles in wastewater treatment, bioremediation of contaminated sites, and the production of various chemicals, showcasing their potential as valuable tools for beneficial purposes.
Overall, the study of biofilms provides valuable insights into the intricacies of microbial communities, their interactions, and their adaptability to diverse environments. Understanding biofilm formation and behaviour can lead to the development of innovative strategies for improving industrial processes, environmental management, and human health. As researchers delve deeper into the world of biofilms, they uncover the fascinating intricacies of these dynamic microbial communities and their impact on the ecosystem and human activities.

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