BACTERIAL BIOFILM IN THE ENVIRONMENT

CHAPTER ONE

INTRODUCTION

A biofilm is defined as a community of bacteria enclosed in a self- produced exopolysaccharide

matrix that adheres to a biotic or abiotic surface (Chew and Yang, 2016). More elaborately,
biofilms are surface-attached, structured microbial communities containing sessile cells (bacteria
and/or fungi) embedded in a self-produced extracellular matrix composed of polysaccharides,
DNA, and other components (Coenye, 2013), and that can show new character with respect to
gene expression, protein synthesis, growth rate, and metabolic activities (Oxaram et al, 2018). In
comparison to planktonic cells, which are just cells in suspension, sessile cells are often much
more resistant to antimicrobial agents. Biofilm formation is an important adaptation and survival
strategy commonly employed by bacteria. Bacteria in the biofilm are protected from adverse
environmental factors and immune response by the exopolysaccharide (EPS). Chemical
gradients generated throughout the biofilm enable bacteria to exist in a wide range of
physiological states, thereby providing insurance effects in changing environments. Biofilms can
be composed of multiple species that interact with each other (Chew and Yang, 2016).

HISTORY OF BACTERIAL BIOFILM RESEARCH

The study of biofilm is relatively recent and arose as a result of observations within the food
industry. At the beginning of the research, there were debates concerning several issues
surrounding the phenomenon. One was concerning the definition of the term ‘biofilm. According
to some authors, bacterial biofilm are embedded in a matrix of extracellular polymeric substances
(EPS). According to others, bacteria form a biofilm even if EPS are absent or below a detectable
level. This debate was due to the observation that the high resistance to antimicrobial agents,
commonly associated with biofilms is acquired within a few hours following the adhesion of the
bacteria, and before any embedment in an EPS matrix can occur, even with a food environment.
Therefore, the definition of a biofilm for the food hygienist could be, ‘a microbial community,
adhering to a surface and frequently embedded in an extracellular matrix’ (Briggith, 1999).

A second area of debate concerns the design of biofilms and the factors which influence it. On
surfaces common in the food industry, biofilms frequently do not form a continuous film, but
rather distant microcolonies, which adhere onto surfaces. In other situations, biofilms may be
large mushroom-shaped microbial clusters, separated by voids or water channels that can be
observed under scanning confocal laser microscope. Another design that has also been described
is of dense confluent biofilms. The latter two biofilm structures are unlikely to be found on
surfaces that are regularly cleaned and disinfected, but rather on immersed surfaces that are not
subjected to frequent hygiene operations (e.g. heat exchangers in power plants, bioreactors,
catheters) (Brigitte, 1999).

CHAPTER TWO

BACTERIAL BIOFILM FORMATION

Bacteria can transition from the planktonic to sessile stage, and when they are in the sessile
stage, they form biofilms (Molobela and Ilunga, 2011). Biofilm formation is a natural
phenomenon which occurs wherever there are microorganisms and surfaces – whether only
slightly wet or immersed, and whether surrounded by a high or a low level of nutrients (even the
low concentration of nutrients in ultra-pure water is sufficient to support biofilm formation).
Biofilms can build up on any surface, in natural, industrial, and clinical environments.

Bacterial cell growth within biofilm is very slow and produces persistent cells that can survive
hostile conditions such as exposure to antibiotics and other biocides. Within the food industry,
the most contaminated surfaces are floors, drains, and some conveyor belts. Less contaminated
surfaces are walls, ceilings, and the inside and outside of equipment. Recesses and crevices,
where cleaning is difficult, are most prone to biofilm accumulation (Brigitte, 1999). In dairy
manufacturing plants, the presence of biofilms can be detected in pipelines, around seals, and on
product contact surfaces in processing equipment such as heat exchangers and evaporators. In
medical setting microbial biofilms have been detected on medical indwelling devices and on
human tissues (Mack et al. 2006), leading to the cause of various diseases in humans such as
cystic fibrosis (CF), native valve endocarditis, otitis media (OM), periodontitis, wound
infections, superficial skin infections and chronic bacterial prostatitis (Agarwal et al. 2010; Otto
2010; Thornton et al. 2013;). Many bacteria are know that forms biofilm on plants roots, shoots
and on leaves and help in plant growth promotion and pest control (Yadav, 2017).

No matter the environment or surface of biofilm formation, there is a general process that has
been observed in the formation of biofilm. This process of biofilm formation can be summarized
under the following stages: i) Reversible attachment of bacterial cells to pre-conditioned surface
(biotic or abiotic) immersed in aqueous medium (Prakash et al. 2003; Ghannoum and O’Toole
2004). ii) Irreversible attachment of cells, iii) formation of microcolonies, a step mediated mainly
by the production of extracellular polymeric substances (EPS), iv) loss of flagella-driven motility
by cells (O’Toole et al. 2000; Kumar and Prasad 2006). v) Maturation of biofilms, the process
whereby biofilm cells communicate through the exchange of genetic materials and other
processes taking place during biofilm growth and development (An et al. 2000; Rachid et al.
2000). vi) Dispersion or detachment stage during which single motile cells disperse from the
microcolonies; the detachment process can be through erosion or sloughing (Dunne 2002; Li et
al. 2007).

A more detailed examination of the bacterial biofilm formation reveals several stages.

1. Initial or Reversible Attachment: The development of bacterial surface attachment

represents a transitional process from planktonic life to the biofilm mode [56]. Reversible
attachment has to do with the interaction of planktonic microorganisms that has developed a
conditioned surface [57–59]. At the initial stage, the interaction is very weak and involves van
der Waals, electrostatic forces and hydrophobic interactions. It has been reported that the
attachment will be best on surfaces that are rough, hydrophobic, and coated with different
organic substances [44]. Bacterial structures such as the fimbriae, pili and flagella enhances the
interaction between bacteria and the surface of attachment [60]. Generally, cell appendages
involved in the reversible attachment and bacteria at this stage commit to the biofilm lifestyle or
leave the surface and return to the planktonic lifestyle [56].

2. Irreversible Attachment: At this stage, loosely bound organisms reinforce the attachment
process by secreting extracellular polymeric substances (EPS) that form a complex binding with
surface materials and/or receptor-specific ligands located on pili, fimbriae, and fibrillae or both
[57–59]. After microorganisms are attached on preconditioned and permissive surfaces, then the
cell starts an irreversible attachment and aggregates as clusters of multilayered cells [61]. As
some studies revealed biofilm formation starts with a layer of polymeric substances (EPS) in
which microbial cells are swarming on the surface as the growth of the biofilm continues (Jang et
al., 2017). During this step, the cells undergo a number of physiological and structural changes,
such as non-motility of the attached cells [58].

3. Microcolony Formation: Microbial cells existing within the extracellular matrix undergo
coordinated community growth that leads to the formation of microcolonies. According to
Dunne (2002), microcolony formation results from simultaneous aggregation and growth of
microorganisms and is accompanied by the production of EPS [57]. Microcolonies which are
basic units of biofilm are segmented by channels, each having different distinct
microenvironments[29](Figure1). The final stage of microcolony formation is the secretion of
extracellular polymeric substances by numerous microorganisms that can act as a “glue” to fix
microorganisms on different surfaces. This happens after cells are firmly attached to conducive
surfaces, leading to the production of microcolonies.

4. Biofilm Maturation: Under suitable conditions for sufficient growth and differentiation, a
biofilm may further develop into spatially well-arranged, three-dimensional mature biofilm
structures [61] such as mushroom or tower-like structures interspersed with fluid filled channels
in which nutrients, oxygen, and essential substances can be diffused and circulate in each
microenvironment [51]. The biofilm development is a cooperative group behavior coordinated by
density-dependent chemical signals released by bacterial populations embedded in a self-
produced extracellular matrix [63]. This signaling mechanism known as quorum sensing is used
to communicate and organize group behaviors, such as virulence factor secretion and biofilm
formation [64, 65]. Quorum sensing activates the maturation and disassembly of the biofilm in a
coordinate manner [63]. Generally, cell-to-cell signaling plays a tremendous role in cell
attachment and detachment from biofilm [66].

5. Biofilm Dispersal: Biofilm formation is a cyclical process in which bacterial cells are
detached from the mature biofilm and enter into their previous mode of life, i.e., planktonic state.
Detached bacterial cells will look for new suitable surfaces to attach and start up a new round of
biofilm formation. In this step, microbial cells will decide based on the environmental conditions
whether they live together or “fall apart” [46]. From a food contamination point of view, this step
is important to disseminate microorganisms into food products. Biofilm cells can be detached
from actively growing cells or from the deprived environment, communication, or removal of
aggregates. It has been reported that nutrient limitation forces microorganisms to seek new
environments [29, 46].

2.1 Extracellular polymeric substance

The extracellular polymeric substances provide the structural integrity of the biofilm (Yongqin et
al. 2010). Biofilms produce different EPS, and the structural composition of these EPS determine
the conditions in which a biofilm will grow (Dignac et al. 1998; Flemming et al. 2000). EPS are
composed of high-molecular-weight compounds, including polysaccharides, proteins, nucleic
acids, lipids and DNA, with proteins and carbohydrates representing the major components
(Donlan 2002; Liu et al. 2004; Cheng et al. 2007; Vu et al. 2009). Humic substances are also
found in some EPS (Hoyle 1992). The ionisable functional groups, such as the carboxylic,
phosphoric amino and hydroxyl groups, play a key role in the formation of biofilm EPS (Pan et
al. 2010). Figure 3 illustrates different components of extracellular polymeric substances (EPS).
Published studies on the composition of biofilm EPS in terms of the ratio of carbohydrates to
proteins have shown that biofilms produce unequal amounts of EPS depending on the

growth conditions. Some researchers have found that certain biofilm EPS have a higher
concentration of proteins than polysaccharides, while others have found that in some instances
polysaccharides were the dominant component of the biofilm EPS (Sutherland 1994; Liu et al.
2004, Girbal-Neuhauser 2011). In the study of Ras et al. (2008), carbohydrates and proteins were
the two major constituents relative to other components (lipid, amino acids, DNA) of the biofilm
EPS. Similar observations were reported by Liu and Fang (2002) regarding the protein and
carbohydrate ratio except that DNA components were also found in the wastewater. Flemming et
al. (2007) found protein components to be more dominant than carbohydrate components, while
Jiao et al. (2010) found the reverse. These results imply that the EPS structural composition is
variable amongst bacterial strains. Nonetheless, the quantity and quality and the biofilm EPS is
dependent on a number of factors, including microbial species, nutrients, the type of limiting
substrate (carbon, nitrogen and phosphorus), oxygen limitation, temperature and shear force
(Zhang and Fang 2001; Fang et al.2002; Liu and Fang 2002; Liu et al.2004; Bhaskar and Bhosle
2005). Due to structural complexity and other influential factors, including parameters that
contribute to the formation of biofilm EPS, the study and analysis of biofilm structures, biomass
and thickness may be quite difficult and demanding.

2.2 Factors affecting biofilm formation