A Complete Guidebook on Biofilm Study

A Complete Guidebook on Biofilm Study has emphasized the biofilm-related issues in the present context related to research and development. For this purpose, experimental design and relevant experimental protocols for the biofilm studies have been highlighted here. In addition to that, inhibitors from natural or synthetic sources against microbial biofilm development have been addressed. This approach has been further substantiated by bioinformatics as well as nanotechnology-based reports. Both, the image processing related to biofilm study and the characters of substratum associated with biofilm development have also been included for a better understanding of the beginners in this field. Further, how biofilm helps and/or hampers in food processing and waste management system, that discussion has been considered in this book. Similarly, human benefits from biofilm and reverse of it have also been included considering host-pathogen interaction, immunity aspects, and others.

• Carrying huge resources/information/ideas in a compiled manner for biofilm study/work
• Has highlighted how biofilm-related experiment has to be designed based on protocols
• This book has focused majorly about biofilm-related gene regulation along with the development of different inhibitors for therapeutic aspects. This paradigm has been further discussed based on the nanotechnology and bioinformatics approach
• Biofilm studies related to waste management, food processing, and image processing, which are newly upcoming have been emphasized in this book

• Language: English
• Publisher: Academic Press
• Release date: Jun 22, 2022
• ISBN: 9780323884815

Book preview

Chapter 1

Biofilm: Design of experiments and relevant protocols

Susmita Dattaa, Soma Naga, Dijendra N. Royb

aDepartment of Chemical Engineering, National Institute of Technology, Agartala, Tripura, India

bDepartment of Bio Technology, National Institute of Technology, Raipur, Chhattisgarh, India

1.1 Introduction

The rapid population growth, their standard of living in the society, progressive socialization, and energy consumption have certainly served as the driving force to expanded anthropography and thus, effect on the environment, eventually, this has an immense impact on human health as well. A slow but serious threat of advance and the modern drug is biofilm formation by microorganisms and associated infections [1,2]. It's been more than 75 years of the first report on biofilms [3], since then Biofilm formation by microbes and their antibiotic-resistant efficacy is gradually increasing and emanating as the great menace to global well-being. Biofilm formation was evolved into a pervasive event for human infections as well as for nonbiological conditions [4,5]. Antonie van Leuwenhoek in 1674 first discovered assemble of animalcules or a coat-like aggregate of cells and cellular particles adhered on human tooth surfaces with the use of his primitive microscope and termed it as biofilm [6].

Biofilm illustrates as any group of microbes whither an organized community of each microbial cell adhere with others, and then attach to various surfaces [7] to form microbial communities by staying independently or associate together within the self-produced matrix of extracellular polymeric substances (EPS) [8,9]; also the biofilm-forming microbial population has maintained the environment for interchanging genetic materials among microbial cells [10,11]. Biofilms can be either single or multilayered, may form on both biotic and abiotic surfaces [12], on living and nonliving surfaces, [13] can also be formed on liquid surfaces as a floating mat and in the submerged state as well [14]. Biofilms are either homogenous or heterogeneous structural communities of bacteria or other microbes enclosed with a matrix of extracellular polymeric substances (EPS) and can be present in natural, domestic, industrial and hospital environment [13]. EPS consists of polysaccharides, although, biomolecules like proteins, lipids, and nucleic acids are nested in EPS as well [12]. Glycopeptides, lipids, and lipopolysaccharides polymers establish an arena and keep the biofilms united [15]. Bacterial cells of biofilm have formed a matrix of extracellular polymeric substances to adhere in that matrix, consequently expressing a different phenotype related to gene transcription and growth rate [16]. Within a biofilm, bacteria interact among themselves through the formation of chemotactic particles or pheromones, and this phenomenon is called quorum sensing [17,18]. The structure and composition of biofilms are constantly changing along with the maturation process due to environmental conditions such as growth conditions, nature of fluid movements, physicochemical properties of the substrate, availability of nutrition, etc. [19]. Bacteria with rising hydrophobicity have decreased repulsion amidst the extracellular matrix and bacterium, thus hydrophobicity performs a vital role to ordain the potentiality of bacteria to produce biofilm [20].

Biofilms must possess few characteristics such as the capability to self-organize or autopoiesis, can resist natural, disruption or Homeostasis, be efficient in correlation than in segregation or synergy, and can reciprocate to climatic changes unitedly instead of individually or communality. For example, dental plaque is perfect for demonstrating biofilms that possess all of these four characteristics [21]. Biofilms are generally hovering in nature and play vital roles in various activities; amidst them some are beneficial and some are harmful. Within biofilms microbes are usually well-protected against disinfectants, antibiotics [22], and the host immune system; consequently, it's highly challenging to eradicate biofilms [23]. It has been reported in the literature that biofilms are associated with 65% of nosocomial infections [24]. Biofilm-associated microorganisms become resistant to conventional antibiotics and gradually reduce their sensitivity toward antimicrobial agents; thence, exhibit treatment failure. They are associated with various medical diagnoses, such as dental caries, gastric ulcers, vascular catheters, urinary catheters, and artificial joints, like, implanted medical devices, keratitis, kidney stones, meningitis, osteomyelitis, pneumonia, sinusitis, tonsillitis, gallstones, chronic wound infections [25,26], vaginitis [27], colitis [28], conjunctivitis [29], gingivitis [30], urethritis [31], and otitis [32]. Biofilms also endure a few beneficial impacts on nature including production, degradation of organic matter, remediation of environmental recalcitrant pollutants, cycling of nitrogen, sulfur, and many metals. Purification of sewage [33], treatment of groundwater contaminated with petroleum [34], nitrification [35], soil fertility, plant growth [36], and can enhance the degradation of polymer efficiently [37,38]. Furthermore, biofilms have been employed in water treatment plants, wastewater treatment plants, septic systems associated with private homes, and utilized for removing pathogens and reducing organic matter present in the water or wastewaters [39]. There are several methods are present to detect biofilm formation and development such as tissue culture plate (TCP), tube method (TM), Congo red agar (CRA) method, bioluminescent assay, piezoelectric sensors, and fluorescent microscopic examination [40,41]. In recent times, few new methodologies have been adapted to study biofilm formation and development. In this chapter, we have tried to put light on experiments and protocols of biofilm development.

1.2 Biofilm: Formation, development, and its consequences

1.2.1 Formation of biofilm

Biofilms are widely investigated for the last 40 years and a variety of information has lightened regarding the mechanism of microbial adhesion and initial biofilm formation. The characteristics of both the substratum and cell surface should be studied comprehensively to perceive the microbial attachment which is the first step of biofilm formation. The properties of the substratum can influence and elevate the momentum and amount of microbial attachment. Conventionally, more harsh and highly hydrophobic components will enhance the faster biofilm growth [42,43]. Hydrophobicity of cell surface exhibited its crucial role in microbial attachment [44]. Biofilms can adhere and survive on any surface including plastic materials, metals, glass, soil substances, wooden medical implant products, tissues, and food items. The free-floating microbes are attached to a surface and initiate biofilm formation. After the microbial attachment, microbes develop colonization by weak, reversible adhesion through Vander Waals forces [26]. Most microbes are potent to adhere to any surfaces on their own; however, some are not capable to attach directly to the surface but they can attach themselves to the matrix. The motile bacteria can easily identify the surfaces and assemble whereas nonmotile bacteria are inept [45] (Fig. 1.1).

Figure 1.1 Different stages of biofilm formation: (1) Reversible attachment, (2) Irreversible attachment, (3) Maturation-I, (4) Maturation-II, (5) Dispersion.

1.2.2 Development of biofilm

The microbial cells attach inevitably to the surfaces and initiate cell division, forming microcolonies and extracellular polymers. The water channels transport vital nutrients and oxygen to the growing microbial cells within the biofilms [46]. Biofilms tend to perform as a filter for entangling different substances such as minerals, fibrin, RBCs, and platelets [47]. It is a complex, dynamic, and heterogeneous stepwise mechanism along with the constantly growing by external and internal processes. Biofilm development consists of five main stages that include reversible attachment, irreversible attachment, maturation stage I, maturation stage II and dispersion [7,10,26].

In the first phase, biofilm growth was initiated by the interaction between planktonic microbial cells and substrate surface. Either the physical forces or bacterial appendages such as Pilli or flagella cause the attachment of the planktonic cells to the surface [138]. Van der Waals forces, steric interactions, and electrostatic interactions like physical forces nurture bacterial adhesion [6]. This phase is termed reversible adsorption as few bacteria adhere to the substrate surface for a short period and lately detached [26]. The microbial cells' attachment to the surface is defined as adhesion and attachment amidst microbial cells is defined as cohesion. Bacterial adhesion was influenced significantly by distinct factors such as temperature, pressure, and surface potentiality [7]. Gene expression does not change in this stage and consequently, bacteria can effortlessly reappear to the planktonic environment. The duration of this stage is a couple of minutes [8].

The second phase is known as the irreversible attachment phase where a few reversible adhered cells become rigid on the substrate surface and evade their mobility; consequently, convert into irreversible adhered cells. In this stage, microbial cells initiate micro colonization, and participate significantly in biofilm development as whenever a microbial cell attaches to a hydrophobic nonpolar surface it causes hydrophobic interaction amid the surface and the microbe, thus the repulsive force amidst them has decreased. Hence, hydrophobicity of cell surface exhibits its vital part in biofilm development and this stage winds up within two hours [48].

In the third phase, microbial cells initiate interaction with each other by producing autoinducer signals [14], thus it affects the biofilm-specific gene expression. Microbes begin to form the matrix of extracellular polysaccharide substances (EPS) for maintaining the biofilm structure. In this stage the number of microcolonies has elevated and developed more layers, thus it becomes more thick and dense and has reached to 10µm. Thence, this phase is termed maturation-1 and the duration is three days [49].

In this stage, microcolonies extent and their thickness reach 100 µm. These microcolonies generally contain distinct microbial communities. The various microorganisms within biofilms often possess complicated properties and integrated appearances. This stage is termed maturation-II and this stage ends after six days. Investigation on protein expression of biofilms exhibits a considerable amount of diversity amid maturation I and maturation II stages [49]. Therefore, the maturation-I and maturation-II phases retain the accumulation of microbial cells, form microcolonies, development, and maturation of attached cells. In the maturation phase, biofilms adapt to the extrinsic factors through the manipulation of their structure, physiology, and metabolism [7].

In the fifth or the final stage, biofilm disperses and immigrate to a new surface. The architecture of microcolonies within biofilms altered as the microbial cells placed in their middle section become mobilized again and detach from the existing well-organized biofilm network. Thus this stage is defined as dispersion. In this stage, microbes within the biofilm secrets various saccharolytic enzymes for splitting the biofilm stabilizing polysaccharides, and consequently discharge the microbes that exist on the head of biofilm architecture so that the free-floating microbe can form a new colony on a new surface. This phase may be more convenient for nutrient accessibility and the water channels are produced in between the microcolonies. This stage has ended up in twelve days [26].

1.3 Requirement of biofilm in microbial community

The earlier sections illustrate an overview of biofilms and the driving force behind biofilm formation and development. This section will provide a précis on the requirements of biofilms in the microbial community. In the very beginning microbes that are hovered in nature, industrial and clinical environment, it has adhered to a surface for their growth and development and is dispersed in the environment after their growth and maturity. Biofilms are an optimal haven for microbial colonies because they constantly influence the formation of chemical inclines and structural heterogeneity of distinctive cell communities [50]. Biofilm formation by microbial communities could be an act of their defense mechanism. Microbes within the biofilms are resistant to phagocytosis, physical stress like shear forces formed by blood flow, and the rinsing activity of saliva; they also resist pH fluctuation, nutrient distress, oxygen radicals, disinfectants, antibiotics. Exopolysaccharide or EPS plays a vital role in both early and late stages of biofilm formation, as well as participate in defense mechanisms, helps to withstand shear forces, phagocytosis by inflammatory cells [19] and also associate with the tolerance of biofilms to antimicrobial agents [51]. Biofilm formation by microbes was required in the microbial community for their survival in a favorable niche. Bacteria and other biofilm-forming microorganisms have been living within the host organism's body as an act of commensalisms [52]. Bacteria have several approaches to make sure that they stay rigid and settled in the host body. Surface proteins of bacteria bind to host extracellular matrix proteins or microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) like fibronectin, fibrinogen, vitronectin, and elastin which are frequently taken a vital part in bacterial initial adherence to solid surfaces within the host. For example, Streptococcus pyogenes have the genes for fibronectin (prtF) and fibrinogen (emm) binding proteins [53]. If there is any nutrient deficiency has occurred at the site of biofilm development, the microbial cells get detached and become planktonic cells, consequently looking for favorable habitat for their growth. Bacteria and other microorganisms form biofilms as an act of defense mechanism and emerge to depict the bloodstream's uplifted glucose levels so that they can separate themselves from circulation and defend themselves from the immune system. Polysaccharide production may serve as a procedure of glucose cache and/or as a process to improve the accumulation phase, thus whenever the organism is exposed to a nutrient-rich environment they conquer that habitat [52].

Biofilms have a promising and significant role ecologically and serve in industrial wastewater treatment. It was reported in the literature that bacterial biofilm can degrade chemical pollutants of industries that are treated as recalcitrant and serve as a carbon source [54]. Some researchers also stated that biofilm is an essential component of the aquatic food chain such as rivers and streams because they have been providing foods to the invertebrates, eventually, it can be ingested by fishes living in the aquatic environment [55,56]. Biofilms also play a significantly in the sewage purification system. Sewage water treatment can be achieved by few phases and amidst them, one phase affirms the productive goal of biofilms by allowing the contaminated water to discharge through the filters which are composed of microbial biofilms' layer, consequently, nutrients present in flowing water are elicited by filters made up with microbial biofilms and thus eliminate the organic pollutants from contaminated water [57,58]. The surface area enhances the water treatment process by biofilms. Besides that, microbial biofilms were also required for ecological balance. Microbial biofilms can cut down the deadwood junk produced from dead fish and aquatic plants and simultaneously they can consume heavy ions from the water without decreasing the oxygen matter [59]. Microorganisms such as Bacillus cereus, B. licheniformis, Arthrobacter species, Pseudomonas species, Candida albicans, etc. are renowned strains that can form biofilms on the various substrates and has industrial importance [60–62]. Microbial biofilms can decrease the toxic effect of phenols that were released into the environment as an aftereffect of industrial operations. Microbes associate with the aerobic degradation of phenols is included Arthrobacter species, Pseudomonas species, Acinetobacter, Candida sp., etc. [63–66]. Primarily biofilm helps microorganisms to survive and enhance their spread within the host body. Matrix of biofilm provides a protecting shelter to pathogenic microbes and defends the highly concentrated antibiotics. However, biofilms increase the capability of microbial communities for adapting the hostile surroundings, and microbes of biofilm become inactive to let the pathogens endure within the adverse environment. These inactive microbial cells within biofilms are extremely secure and bypass the antimicrobial activity but whenever the surroundings turn benign these cells can perform as initiator cells to facilitate the procedure and these microbial cells of biofilms are called persister cells [67]. The extra polymeric matrix of biofilm is the basic and flexible element that protects the adherent microbial cells from adverse environmental components. The components of biofilm such as polysaccharides, DNA, RNA, proteins, lipids, and peptidoglycan are required for metabolic activities, additionally, irons help microbial biofilms for their survival. Environmental factors including pH, temperature and ionic concentration can regulate the proficiency of microbial biofilms. Literature also exhibits that the growth and development of microbial biofilms were related to mutations and quorum sensing. Genetic studies on microbial biofilms have demonstrated the mechanism of horizontal gene transfer within the microbial biofilm, consequently, evolve into new strains which can combat the potency of antimicrobial agents [14,52]. In recent times, biofilms have been practiced in wastewater treatment, bioremediation and nurtured as a promising source of global energy. Biofilms have either a positive effect or negative effect on natural bioresources, for example, biofilms work in river systems and edible water circulation operations or work as a bioresource by energy generation in various ways including direct electricity formation, hydrocarbons production, or as an originator of biomass for biofuel. Biofilm formation by cyanobacteria is a potent source to generate electrical current. So it can be concluded that biofilms have diverse characteristics and are very much required in the microbial community [68].

1.4 Purpose of various biofilm experiments

The science and technology behind biofilm has been emerging as an enthusiastic terrain for study and currently, it was entrenched that most of the microorganisms that are hovering in nature are survived by adhering to various surfaces within organized biofilm environs, not as free-floating microbes. Over the last decades, the concept of biofilms has evolved significantly, thence; new technology has adapted and developed such as new imaging technologies, biochemical methods, and molecular ecosystem biology tools for studying biofilm science. So now the 3-D structure of biofilm can be studied and gathered more knowledge to nanoscale level [69], additionally, can also acquire a profound knowledge of the physiology of biofilms' microbial cells, phenotypic-genotypic variation amidst the biofilm microbial community, and the metabolome, proteome, transcriptome of biofilm [70]. The biofilm formation technique has changed for the improvement of biofilm related-devices which enhance resemble of existing environmental situations. The profound knowledge of biofilms either as an entity or as a single cell and their interactions with the neighboring environment can help to establish the adequate approaches that can either curb detrimental biofilms including clinical biofilms, food contaminants, biofouling on industrial equipment and ship hulls or elevate and regulate the constructive biofilms for waste-water treatment, bioremediation, generation of electricity and production of biofiltration [71].

The biofilm mechanism emerges as a major threat to public health and industrial applications. Bacteria as planktonic microbial cells are less protected as compared to microbial cells within a mature biofilm structure that are finely secured from a hostile environment and antimicrobial agents [72]. Control of biofilm formation possesses immense concern to the industrial practice because their aggregation is responsible for economic losses; a few examples of economic losses through biofilms include equipment degradation by inducing corrosion [73] or elevating fluid resistance [74], biofilm contamination in energy and chemical industries [75]. Furthermore, biofouling in paper industries causes a deleterious impact on the final product's condition or the aggregation of biofilms beneath waterline on ships' hulls can lead to significant losses for shipping industries [75]. Contrary to creating damage, biofilm development by a few nonpathogenic microbes has been utilized in many industrial applications to inhibit the growth of pathogenic microbes [76], to prevent fungal-associated food decomposition [77]. Nonpathogenic microbes within Biofilms can also be applied in the area of engineering biofuels [78], in wastewater treatment [79], in fuel spills clean-up [80], and electricity production [81]. Interaction between biofilms and industrial operation is a continuing phenomenon, and thus, the biofilm-forming microbial community drowns attention due to economic importance. Microbial contamination has occurred due to the biofilm development in food, medical and agricultural field, consequently, biofilm control is essential to restrain the disruption of human disease and decrease the quantity of food debris but it's very difficult to regulate biofilm formation because microbes within biofilm seize various characteristics for their growth and survival such as growing resistance against antimicrobials [82], and alliance with other microorganisms [83] through quorum sensing (QS) [84]. Therefore studies and experiments on biofilms are gradually increasing to recognize the characteristics of biofilm which aid microbes to defeat different treatments [22,85]. Several studies were performed to quantify biofilm structure and its relation with biofilm activity and one example is the biofilm reactor which was designed to know to relations between biofilm structure and performance of biofilm reactors, furthermore, this information could be fruitful for the water treatment system to reduce the harmful impact of biofilms on membrane processes. However, a definite understanding has required of biofilm structure and how it reacts to an adverse condition as without proper knowledge it is not possible to manifest a procedure for exploiting biofilm structure to obtain the desire results. Few studies stated that biofilm structure needs to be quantified and accordingly several experiments have been formulated to observe the structure of biofilms and counting the parameters that characterize the biofilm structure; however, these parameters include [86] analysis of biofilm structures [87], examine the ability to reproduce the structure of biofilms [88], observation of physical changes that occurred in the structure of biofilms [87,89], verifying the impacts of different compounds like antimicrobial agents on the structure of biofilms [90,91], quantification of environmental conditions which influence the biofilm structure [89], and computation of the parameters that represent the properties of biofilm structure and utilized for biofilm modeling [92]. To operate a biofilm experiment requires an experimental platform and these platforms were selected based on what type of information needs to be elicited. Biofilms formed on various devices and these devices were chosen to achieve the desired result but every selected device has a few benefits and drawbacks [71]. In order to aid achieve the physical procedure of microorganisms' communication within biofilms and with nature, medical, and industrial environment; simultaneously focused on the characterization of biofilm mass, thickness, morphology, and the related gene expression through different biofilm-related microbial species and substrata materials. Additionally, biofilm experiments will not only help to evaluate of potent variation that occurred in biofilm structure also help to test the parameters that make microbes within biofilm more resistant to oxidative stress, hostile environment, and antimicrobial agents. However, with the help of biofilm experiments, we can employ biofilm in positive ways such as water treatment, wastewater treatment and eradicate the negative effects such as antimicrobial resistance, contamination in food, chemical, or medical industries. Certain biofilm-forming prevalent bacteria such as Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa[41] are the major bacterial pathogens of humans that cause various diseases. Biofilm formation is a major virulence factor in the pathogenicity of bacteria, and bacterial biofilms are hard to eradicate due to their very high antibacterial resistance. Consequently, research into the pathogenicity of bacteria has focused on the prevention and management of biofilm development, their architecture, and antibacterial resistance. As time passes by, researchers gradually understand the vital role of biofilm in treatment procedures, and the development status has been intensely investigated for their immense impact on the treatment process potentiality which added temperature, nutrients, extracellular polymeric substances (EPS), the interaction of species, and light for algal-based processes [93]. The biofilm experimental studies are significant to draw and calibrate the potent treatment process; hence, optimal surroundings must be given to the microbial communities to obtain the desired results through the treatment process [94]. Although studies have shed some light, molecular mechanisms that influence biofilm formation and pathogenicity remain at the center of the microbiologist's attention.

1.5 Reports on model microbes for biofilm experiments

During the past few decades, plentiful in-vitro, in-vivo models of biofilms have been developed to recognize the morphology of biofilm and to observe the impacts of exoteric factors like change in pH or temperature or nutrient's effects or effect of oxygen, enzyme, or exposure to antimicrobials on biofilm formation [26]; even though many things still await for elucidation and definitive methods are yet to be created to characterize the biofilm [95]. Microbes including Enterococcus faecium, S. epidermidis, K. pneumoniae, Acinetobacter baumannii, P. aeruginosa, Enterobacter spp, E. faecalis, E. coli, methicillin-resistant S. aureus (MRSA), and Streptococcus spp. are the most frequently occurring life-threatening pathogens of biofilm-associated infections which are responsible for infections associate with urinary catheters to prostheses, chronic, persistent infections and indwelling medical devices' infections [96]. After the rigorous literature studies, it can be said that pseudomonas sp. is the most common microbes for biofilm experiments, and jillion reports in literature illustrate that pseudomonas sp. is the model organism for various biofilm associate experiments [72,97].

A phase‐field (PF) continuum model and the Oldroyd‐B constitutive equation were conjugated to develop a model by a few researchers to exert its influence for simulating biofilm deformation. The efficiency of the model was estimated through two classes of biofilms: a synthetic biofilm that was made from alginate mixed with bacterial cells, and a P. aeruginosa PAO1 biofilm. To demonstrate the mechanical parameters for each biofilm shear rheometry was employed as inputs for this model and optical coherence tomography was used for experimental observation of biofilm deformation under fluid flow. This Experiment not only validates a Phase-Field computational model with the Oldroyd‐B constitutive equation for biofilms by utilizing independently demonstrated biofilm mechanical properties but also gives a significant path to gather and predict biofilm viscoelastic behavior. This model gives a vital weapon to predict biofilm viscoelastic deformation along with the advantage to design and control of biofilms in engineering systems [98]. Extracellular products of P. aeruginosa PAO1 were investigated to prevent the staphylococcal growth and agitate biofilm architecture formed by S. epidermidis[99]. Interaction between P. aeruginosa and S. epidermidis was studied in this experiment as P. aeruginosa is a model organism to study biofilm formation, quorum-sensing and extracellular virulence factors [72].This experiment exhibited that P. aeruginosa can serve quorum-sensing independent extracellular products (generally polysaccharides) to break down biofilm architecture formed by S. epidermidis. Although, the outcomes of this study demonstrate that the polysaccharides produced through two operons named pel and psl were potent for efficiently interrupting the developed biofilm of S. epidermidis biofilms. It was already reported in the literature that microbial biofilms were used for water, wastewater treatment systems; however, biofilm fouling is one of the main hurdles that disrupt the application of membranes in the water-based treatment system. One study was done by a few researchers to investigate the impact of ultrafiltration membrane surface characteristics on P. aeruginosa biofilm initiation to reduce biofouling. Initial adherence of microbes to the surfaces is considered the most vital and significant concern of biofouling. Hence, they concluded that biofilm generation by P. aeruginosa enhances, consequently, surfaces turn into rougher and highly hydrophobic meanwhile, less fouling has occurred if the surface charge was reduced but it was raised with escalating charge (positive/negative) [100]. Another group of researchers isolates 500 urine samples from catheterized patients to study the comparison of three different methods of detection of biofilm production by uropathogens in tropical catheterized patients and for this biofilm experiment, they used three bacterial strains as reference strain that are S. aureus ATCC 35556, P. aeruginosa ATCC 27853, and E. coli ATCC 35218. TAM, TCPM, and modified CRA (MCRA) methods were applied to detect biofilm production [41]. From their investigation, they conclude that TCPM is a quantitative and reliable method to detect biofilm-forming microorganisms when compared to TAM and MCRA methods and TCPM can be suggested as a regular screening method for the detection of biofilm-producing bacteria in laboratories. TCPM method has good reproducibility and good specificity. This method can be used routinely in the microbiology laboratory to detect biofilm formation, especially, when the causative organism is resistant E. coli. A research group had carried out a biofilm experiment to design and evaluate a polyspecies biofilm model with bacteria that caused severe infections in burn patients. The bacterial strains used for this experiments are E. faecalis OMZ 422 (ATCC 29212), E. coli OMZ 56 (ATCC 25922), Streptococcus intermedius OMZ 871 (SK57, ATCC 9895), P. aeruginosa OMZ 154 (ATCC 27853), and S. aureus OMZ 1122 (ATCC 25923) [101]. S. aureus, P. aeruginosa, E. faecalis, and E. coli were the most frequently isolated microbial strains for various biological experiments especially for burn-patients related investigations [102]. From their literature review and clinical investigation, they concluded that P. aeruginosa and E. coli are the most prevalent pathogens. Additionally, they stated that polyspecies burn-biofilm models are vital to investigate the potentiality of current strains. Diverse microbial communities in biofilm pose discrete metabolic pathways which endow biofilm to either individually or concurrently break down various contaminants [103] and these microbial communities are served to dorm biofilm for wastewater treatment such as fungi [104], algae [105], bacteria [106], and yeast [107]. Although few researchers found that the application of algae into the biofilm reactors is an infliction because of its clogging flows but contrary to that, few researchers considered it as an origin of oxygen that provides huge amounts of oxygen toward the better development of other microbial species within biofilms [93]. Biofilm treatment systems are beneficial for the particular growth of passive-developing microbes like nitrogen-oxidizing bacteria and phosphorus-acquiring microbes through the nurturing of high biomass age that decrease the erosion from the system [108]. Various biofilm-forming microorganisms were applied in biofilm-based treatment systems like bioreactors and these microbes are supported on a variety of materials. A few examples of the biofilm-based treatment system with employed microorganisms, targeted contaminants, supported materials, types of bioreactors used for and treatment efficacy has exhibited in Table 1.1[94].

Table 1.1

Plenty of experimental reports are present in the literature on the application of various microorganisms for the biofilm-based experiment, among them, some are prevalent microbes and some are not. From the rigorous literature analysis of published papers, it can be stated that bacterial biofilms are not only used for the biofilm-based experiment but fungal biofilm was also utilized for the biofilm-based experiment. Fungi like Candida albicans associated with bacteria named S. aureus and S. epidermidis, thus produce polymicrobial biofilm [109–111]. A researcher group studied polymicrobial biofilm architecture formed by Candida albicans fungi and this investigation illustrates the efficacy of the denture model for evaluating the effect of gene materials, analyzing the biofilm architecture, and investigating the biofilm drug resistance. Besides that, this model is significantly beneficial for examining the mixed microbial biofilm, the effect of individual host components, and the interaction between bacteria and fungi [109]. Microalgal biofilms are also utilized for wastewater treatment and bioenergy production [112]. Natural microalgal biofilms were isolated from freshwater, saline lakes, and marine habitats. These established biofilms possess cyanobacteria, microalgae, diatoms, bacteria, and fungi. The collected photosynthetic components of biofilms were applied to treat reverse osmosis concentrate types wastewater system; applied for concentration of commercial microalgal species, working as bioflocculating agents; used for highly efficient and minimal energy utilized treatment system such as commercial wastewater treatments; and serve as novel sustainable feedstocks that carry the compositions suitable to form renewable bioenergy: biohydrogen and biodiesel. Few prevalent microorganisms were utilized for biofilm-based experiments has summarized in the Table 1.2.

Table 1.2

1.6 Standard protocols for various biofilm-associated experiments

Immense and promising advantages, as well as the menace of biofilms, draw attention; hence, researchers from various disciplines have been studying biofilm properties and anti-biofilm activities for developing propitious operations and inhibition strategies. Despite this, researchers find it very difficult to make an analytical decision to formulate an experimental protocol for developing, characterizing, and quantifying biofilms that will be applied to their research problem. However, few biofilm-based experimental protocols that are published in various research papers are highlighted in this