Bacterial Biofilms: Gram Staining, Cell Wall Structure, and Surface Charge Dynamics

A microbial biofilm is a structured community of microorganisms adhering to a surface and encased within a self-produced protective matrix of extracellular polymeric substances (EPS). This matrix, composed of polysaccharides, proteins, lipids, and DNA, forms a complex three-dimensional architecture that acts as a biological "glue." Biofilms are ubiquitous in nature and can incorporate inorganic components like mineral particles, which provide structural support. Their presence can significantly alter the chemical and physical properties of their environment, influencing factors such as surface charge, particle aggregation, and local chemistry.

Since many biofilms are composed of bacteria, a fundamental understanding of the bacterial cell wall is essential for elucidating the initial processes of biofilm formation and adhesion. Bacteria are broadly categorized into two major groups based on their cell wall structure: Gram positive and Gram negative. This critical classification originates from the Gram stain, a differential staining technique developed in 1884 by the Dutch bacteriologist Hans Christian Joachim Gram. The distinct staining result—a blue-violet color for Gram-positive cells versus a red color for Gram-negative cells under light microscopy—is a direct consequence of fundamental structural differences in their cell envelopes.

The architectural divergence between the two groups dictates their surface properties. Both Gram-negative and Gram-positive bacterial cell walls possess components that typically impart an overall electronegative charge to the cell surface. This negative charge is crucial as it promotes the adsorption of metal cations and facilitates attachment to positively charged mineral surfaces. The Gram-positive cell wall is characterized by a thick, dominant layer of peptidoglycan (PG), which is rich in carboxylate groups. Secondary polymers like teichoic acids or teichuronic acids embedded within the peptidoglycan can contribute additional negatively charged carboxyl or phosphoryl groups.

In contrast, the Gram-negative cell wall features a more complex structure. It consists of a thin layer of peptidoglycan sandwiched between two distinct membranes: the inner plasma membrane and a unique outer membrane (OM). This outer membrane contains lipopolysaccharide (LPS) molecules in its outer leaflet, which are highly negatively charged and play a significant role in the cell's interaction with its environment, including its susceptibility to antimicrobial agents and its adhesion capabilities.

While the majority of bacteria exhibit a net negative surface charge, exceptions exist, particularly under specific environmental conditions. Research has documented net positively charged environmental bacterial surfaces at pH levels below 5, a phenomenon likely related to the protonation of amino groups. Furthermore, studies have identified specific bacterial strains, such as one described by Jucker et al., that remain positively charged at physiological pH and can cause serious infections by attaching to medical implants. A bacterium's surface charge is not static; it can be influenced by metabolic processes, meaning that metabolizing and non-metabolizing cells may present different surface properties.

The complexity of the bacterial surface extends beyond its net charge. Even in a non-metabolizing state, the charge is not concentrated in a single plane but is distributed across a complex, three-dimensional structure. This distribution presents significant challenges for accurately modeling processes like bacterial surface protonation/deprotonation and metal adsorption behavior. To address this, scientists employ techniques such as surface titrations to determine the acidity constant (pKa) values of functional sites on viable, but non-metabolizing, bacterial surfaces, providing critical data for predictive models.

Visualizing Biofilm Complexity in Extreme Environments. Biofilms are present in a wide array of environments, and they can be both complex and colorful. For example, Figure 1 is a photograph of a biofilm in an extreme environment at Yellowstone National Park. The image shows an extensive microbial mat featuring conical, columnar, and lilypad stromatolites shaped by cyanobacteria (Leptolyngbya) that form in some of the quiet hydrothermal pools. Silicate precipitates are present in association with the biofilm, and this image represents the intricate 3-dimensional architecture that can be associated with these microbial communities. The biofilms in Yellowstone vary according to temperature, water chemistry, and geologic conditions, and they have been studied for decades as models of early life and microbial adaptation.

Biofilm Formation: Microbial Attachment, Architecture, and Genetic Regulation. Microbial attachment to surfaces, spanning natural materials like clays and man-made structures such as concrete and medical implants, is a critical area of scientific inquiry. This process is a primary driver of biologically influenced corrosion in metals and concrete, as well as the development of dental caries on tooth enamel. The investigation of microbial biofilms has advanced significantly with the evolution of sophisticated imaging and genetic characterization techniques. These methodologies have revealed the complex, community-based nature of microbial life on surfaces, moving beyond the historical focus on planktonic, or free-swimming, cells.

Initial Attachment and Biofilm Development. The pioneering work of Marshall et al. [13] established a two-stage model for bacterial adhesion. Initially, cells undergo reversible adsorption to a solid surface, primarily mediated by electrostatic interactions. In a subsequent phase, bacteria transition to irreversible adsorption through the production of extracellular polymeric fibrils that anchor the cell firmly [14]. During initial adhesion, if both the bacterium and the substrate possess negative surface charges, attachment requires overcoming electrostatic repulsion with stronger attractive forces. These forces include van der Waals forces and hydrophobic interactions, which facilitate the first contact.

Supporting this, research by Ams et al. [15] demonstrated that viable but nonmetabolizing bacteria adsorbed more extensively to Fe-oxide-coated quartz sand than to uncoated quartz. Under experimental conditions, the pristine quartz surface is negatively charged, while the Fe oxide coatings impart a positive charge. Consequently, the results from Ams et al. [15] align with the principle that initial attachment is a reversible process governed largely by electrostatic forces. This stage is crucial as it determines whether a transient interaction will progress to stable colonization.

The irreversible phase of attachment is marked by increased cell division and the active secretion of Extracellular Polymeric Substances (EPS), forming the foundational matrix of the biofilm. As EPS accumulates and the initial colonizers replicate, they form microcolonies that often begin as a monolayer. Over time, this structure matures into a complex, three-dimensional arrangement. Other microbial species are frequently recruited, leading to a diverse and synergistic community. Ultimately, a dispersal phase occurs where microorganisms detach from the biofilm to colonize new surfaces, completing the lifecycle.

Biofilm Architecture. Biofilm architecture is inherently complex and heterogeneous, characterized by clusters of microbial cells separated by interstitial voids or water channels [16]. These channels are not incidental; they are functional components that permit the convective flow of nutrients and the removal of metabolic waste products throughout the biofilm structure [17]. The creation of these distinct microenvironments allows microorganisms within biofilms to establish conditions that differ significantly from the bulk solution. These microenvironments can exhibit unique pH levels and concentrations of dissolved oxygen, nutrients, and metals [18].

This capacity to modify their immediate surroundings enables biofilms to act as sites for enhanced mineral corrosion or precipitation. The open, heterogeneous structure is crucial for the survival of the community, as bacteria depend on the efficient transport of essential compounds. Biofilm thickness can vary dramatically, from thin films of a few tens of microns to extensive structures several centimeters thick, depending on environmental conditions. This structural variability is a direct response to the local habitat.

Even for a single bacterial strain grown under controlled laboratory conditions, biofilm architecture can exhibit significant plasticity. Factors such as the type of carbon source, stir rate, dissolved oxygen distribution, and temperature can cause structures to range from open, filamentous networks to dense, compact clumps. For instance, Shrout et al. [19] demonstrated that Pseudomonas aeruginosa biofilms develop distinctly different architectures when the carbon source is supplied as glucose, glutamate, or succinate. This underscores how subtle chemical variations in the environment can profoundly influence the physical form of a biofilm.

Gene Regulation and Quorum Sensing in Biofilms. Research has demonstrated that the initial attachment of cells to a substratum triggers the up-regulation and down-regulation of various genes. Studies using pure cultures have further revealed that when specific genes are mutated, biofilms may fail to form entirely, develop defectively, or exhibit substantially altered characteristics [20]. Investigations into gene expression within complex, multi-species biofilm communities have yielded intricate and sometimes contradictory results. This complexity highlights that the genetic regulation of biofilm development is a topic requiring extensive further research.

A key process governing biofilm formation is quorum sensing, a sophisticated cell-cell communication system. Bacteria coordinate their behavior, both within a species and between different species, by producing, releasing, and detecting small, diffusible signaling molecules. The concentration of these autoinducers is proportional to the local cell density, and upon reaching a critical threshold, they trigger coordinated changes in gene expression. This process can regulate behaviors such as EPS production, virulence factor secretion, and swarming motility.

The discovery that microbes within biofilms engage in this high degree of coordinated behavior has led researchers to analogize these structures to "cities" of microorganisms [21]. In natural environments, a mature biofilm is often highly organized, with different microbial species occupying specific layers or niches. This organization creates a structured ecosystem where the metabolic activities of one group of organisms can provide nutrients or alter chemical parameters, such as oxygen tension or pH, thereby making the environment more or less habitable for other members of the community.