Forming biofilms is a complex process influenced by various factors, such as growth conditions, growth media, and the presence of specific matrix components. Recent research conducted by Sauer et al. [82] has revealed that the structure of biofilms in P. aeruginosa may vary depending on the growth circumstances and medium used. It has also been observed that bacterial biofilms can develop pillars and mushroom-like structures, indicating that biofilm architecture is diverse and intricate [83].

According to research by Hartmann et al. [84], mechanical contacts between cells are responsible for developing three-dimensional organization and structure in expanding biofilms. This process can be regulated by controlling the production of specific matrix components. Yan et al. [85] have also suggested that global mechanical instabilities play a role in biofilm structure formation, emphasizing the mechanisms’ complexity. Bacterial biofilm structure is significantly impacted by eDNA, which contributes to the formation, composition, and function of biofilms [86].

The intricate relationship between mechanical instability and the development of biofilm architecture highlights the dynamic and adaptable nature of microbial communities [85]. During the initial stages, shear stress in fluid environments is necessary to facilitate the attachment of microbial cells to surfaces. Fluctuations in fluid dynamics create a microenvironment where bacteria can either adhere effectively or encounter difficulties in making initial contact. This essential first step sets the foundation for subsequent biofilm formation [87, 88].

As biofilms progress in their development, mechanical forces play a crucial role in facilitating microbial attachment and shaping the structure of these communities. The movement of fluids can produce distinct patterns within the biofilm matrix, including streamers and pillars [82, 89]. This dynamic response underscores the biofilm’s remarkable ability to adapt and reorganize itself in response to an array of mechanical stimuli, revealing the intricate nature of biofilm architecture. It has been noted that mechanical shear stress significantly influences biofilm properties such as thickness, density, permeability, and the spatial distribution of live cells within the biofilm [90].

Mechanical cues are critical in regulating the extracellular matrix comprising polysaccharides, proteins, and eDNA. Elevated shear stresses prompt bacteria to increase their EPS production. The heightened production of biofilm matrix strengthens its stability, creating a defensive barrier that protects the microbial community from external stimuli. The control of EPS generation demonstrates the biofilm’s ability to respond and adapt to the mechanical properties of its environment [84, 91].

Additionally, varying oxygen levels are recognized as a crucial element in forming the final structure of Aspergillus fumigatus biofilms, highlighting the ever-changing process of biofilm development [83]. As A. fumigatus biofilms mature, oxygen gradients naturally occur and play a critical role in shaping their architecture. As studies have shown, these low-oxygen environments promote the growth of filamentous fungal biofilms and enhance their resistance to antifungal treatment [92]. The biofilm structure of A. fumigatus is regulated by a gene family known as biofilm architectural factor A (bafA) which is produced heterogeneously. This particular gene family affects the growth of the fungus in low oxygen conditions and the appearance of colonies, ultimately impacting its ability to form biofilm and cause disease [83].

The development and structure of biofilms are complex processes influenced by various factors, such as microbial composition, components of the EPS, and environmental conditions [86]. Biofilms are organized communities of microorganisms surrounded by an EPS, which protects cells from external and chemical threats, maintains the integrity of the biomass, and facilitates hydration and nutrient uptake [86].

Understanding biofilms’ spatial arrangement and structural changes is crucial for comprehending bacterial interactions and the collective functional abilities of biofilms that surpass those of individual cells. The formation and characteristics of biofilms are determined by the bacterial composition and the surrounding environmental conditions [17]. To gain a thorough understanding of bacterial interactions and the emergent functional capabilities of biofilms, it is essential to comprehend the spatial organization and structural dynamics of biofilms. General composition of biofilm matrix in common microbial communities are explained through Fig. 3.

The process of biofilm formation involves a series of distinct stages, namely initial adhesion, microcolony creation, maturation, and dispersion [93, 94].

The EPS matrix is an essential component of biofilms, and studies have shown that it plays a significant role in antibiotic resistance [131]. Comprised of proteins, eDNA, and polysaccharides, the EPS matrix has a complex chemical composition that creates a barrier, hindering the diffusion of antibiotics into the biofilm [132]. This barrier reduces the speed at which antibiotics penetrate the biofilm, making it more resistant to antibiotic therapy [133, 134]. The EPS matrix not only prevents the infiltration of antibiotics but also resists phagocytosis by immune cells, further enhancing the resistance of biofilms against antibiotics [135]. The limited ability of antibiotics to permeate the biofilm matrix and the extended duration required for antibiotics to infiltrate the biofilm has been identified as contributing factors to the relationship between antibiotic resistance and biofilm formation [136].

The microenvironments created by biofilms have a notable impact on the efficacy of antibiotics. These habitats contain varying pH levels, oxygen, and nutrients that can influence how antibiotics interact with and penetrate biofilms. Altered conditions within biofilms, such as lowered pH and oxygen levels, can hinder the ability of antibiotics to enter and remain within the biofilm, ultimately promoting antibiotic resistance [137, 138, 139].

The slow growth and dormancy of bacteria that form biofilms contribute significantly to their increased resistance to antibiotics. According to Pu et al. [140], inactive bacteria within biofilms are associated with reduced metabolic and reproductive rates, making them more resistant to antibiotics. This sluggish growth hinders the ability of antibiotics to penetrate the biofilm, resulting in decreased effectiveness of antibiotic [141, 142]. Additionally, the varied control of metabolic activity and growth rate of cells within biofilms, including inactive and dead bacteria, further enhances the resistance of biofilms to antibiotics [142].

Efflux pumps play a critical role in both the development of biofilms and the resistance to antimicrobial agents. These pumps can regulate the internal conditions of biofilms, protect cells from antibiotic attacks, and contribute to multi-drug resistance [143]. Efflux pump inhibitors have been identified as a promising strategy to disrupt biofilm formation and enhance the efficacy of antibiotics, thereby reversing pathogen resistance to these drugs [144]. Understanding the functions of efflux pumps in biofilm formation can pave the way for innovative therapeutic approaches that target their activity, dismantle biofilms, and improve the treatment of infections [145]. Numerous studies have demonstrated the efficacy of efflux pump inhibitors in reducing biofilm production in a variety of bacteria, including E. coli, P. aeruginosa, S. aureus, and Klebsiella pneumoniae [145, 146]. Efflux pumps not only mediate antibiotic resistance but also contribute to biofilm development and several other physiological processes [147]. Moreover, research suggests that efflux pump inhibitors can impede biofilm production by regulating drug efflux pumps in S. saprophyticus [73].

Studies have shown that bacteria forming biofilms exhibit higher levels of antibiotic resistance due to genes associated with biofilm formation [148]. Biofilms are also related to the spread of antibiotic-resistant genes across bacterial species and the promotion of resistance transmission [149, 150]. The dense cellular population and extracellular matrix present in biofilms facilitate the genetic mutations and transfer of antibiotic-resistance genes in biofilms [151, 152]. The increased cellular density also leads to higher levels of exogenous DNA, aiding the acquisition of mobile genetic elements and the horizontal transfer of genes, which contributes significantly to antibiotic resistance dissemination [152]. In addition, the genetic and physical heterogeneity within biofilms plays a crucial role in enabling bacteria to withstand the effects of antibacterial medications [153]. B. subtilis researchers have also examined the genetic variants and gene expression levels of essential genes responsible for biofilm formation and matrix creation in ionizing-radiation-resistant bacteria [154], providing insight into the genetic factors behind biofilm development and resistance. Furthermore, bacteria capable of building biofilms possess specific genes related to biofilm formation, which enhance their resistance, and the development of biofilms is linked to a more extraordinary occurrence of genetic factors that confer resistance to quinolone antibiotics [150, 155].

Biofilms respond to stress in various ways, including shear dynamics, electrical oscillations, and metabolic and electric processes. According to Kurz et al.’s study [156], shear stress induces intermittent behavior in preferential flow channels within biofilms in porous media, indicating a non-Newtonian response. Electrical oscillations in biofilms may help them resist light-induced stress [157]. Meanwhile, Martinez-Corral et al.’s research [158] reveals that biofilms react to nutrient-limitation-driven stress from the core to the periphery through metabolic and electric processes.

Furthermore, research has shown that environmental stress can cause existing biofilms to restructure, highlighting the significant impact of stress on the arrangement of biofilm structures [159]. Stress reactions play a crucial role in developing biofilms and acquiring antibiotic resistance in pathogenic bacteria. The c-di-GMP molecule regulates various aspects of biofilm formation, including resistance to antimicrobial medicines and other stress-induced reactions [160]. The extracellular matrix and changes in the physiological state of cells within the biofilm help biofilms withstand stress [161]. Moreover, genes associated with biofilm development are activated in response to stress, further supporting their contribution to stress resistance [162].

Persister cells are essential in conferring resistance to antimicrobial agents in bacterial biofilms. Persister cells have been discovered in different types of bacteria, including Mycobacteria, Borrelia, S. epidermidis, P. aeruginosa, Acinetobacter baumannii, and C. albicans [110, 163, 164]. Persister cells inside biofilms trigger toxin/antitoxin systems, suppressing protein synthesis, inducing dormancy, and developing antibiotic tolerance [164]. In addition, studies have demonstrated that the creation of biofilms is associated with a significant increase in resistance to antifungal treatments and the facilitation of the development of persister cells that are tolerant to antifungal agents [165]. Furthermore, the prolonged survival of the biofilm community when exposed to antibiotics enables the development of persister cells through mechanisms associated with inherent protection against antibiotic-induced harm [166]. Biofilm persister cells have been proposed to exhibit resistance to antibiotics up to 1000 times higher than the most minor inhibitory concentrations [167]. Persister cells are formed within biofilms to increase the tolerance of biofilm cells. These cells are a tiny subset called “transiently resistant” cells linked to biofilms’ unique growth pattern [168].

Crystal violet staining has become a fundamental technique in biofilm research, recognized for its effectiveness in measuring biofilm biomass and evaluating biofilm formation. Its versatility has been demonstrated in various investigations, such as the classification of Stenotrophomonas maltophilia strains based on their biofilm production ability [181] and the quantification of biofilm biomass in high-throughput workflows [182].

Crystal violet staining is also applied to examine the effects of biofilm removal, including the quantification of biomolecules in bacterial cells and extracellular polymeric compounds [183, 184] and the evaluation of the inhibitory impacts on biofilm formation [185, 186].

In addition to these applications, crystal violet staining has also been crucial in other scientific fields. For instance, one study utilized it to evaluate biofilm formation capacity in canine cystitis [187]. Crystal violet staining has proven to be a valuable technique in studying Pseudomonas syringae and assessing biofilm biomass [188]. The method has also played a crucial role in evaluating the impact of various substances on biofilm formation, including silver nanoparticles [189], propolis [190], and norspermidine [191]. The adaptability and dependability of this method in providing both quantitative and qualitative information about biofilm biomass and formation make it an essential tool in biofilm research. Its extensive utilization in diverse settings, from examining bacterial varieties to testing antibiotic substances, underscores its fundamental contribution to advancing our knowledge about biofilms.

Scientists have extensively researched ConA, a protein that binds to carbohydrates, for its ability to visualize biofilms. It is well-known for its strong attraction to mannose and glucose molecules [192]. When used in conjunction with other fluorescent stains in Confocal Laser Scanning Microscopy (CLSM), ConA has been proven effective in examining live bacterial biofilms and observing the structural elements of the biofilm matrix [193, 194]. Moreover, researchers have successfully utilized ConA to modify mesoporous silicon-based biosensors for label-free optical detection of bacteria in real-time mode [195]. In addition, ConA has been identified as a critical element in developing nanosystems for treating infections, underscoring its crucial role in biofilm research [196].

SYTO 9 and propidium iodide (PI) use in biofilm research has been extensively documented in scientific literature. The LIVE/DEAD BacLight™ Bacterial Viability Kit frequently employs these fluorescent dyes to differentiate between live and dead cells by assessing the integrity of their membranes [197, 198, 199]. SYTO 9 and PI staining have been utilized in a variety of research endeavors, including investigating biofilm production in different strains of A. baumannii [199], evaluating the antibacterial and antibiofilm activities against S. mutans [200], and assessing the impacts of antiseptic agents on S. aureus biofilm [201]. Additionally, SYTO 9 and PI staining have been used to examine the implications of eugenol, trans-cinnamaldehyde, citronellol, and terpineol on the management of E. coli biofilm [202]. The SYTO 9 and PI combination has also been used to determine the viability of S. mutans biofilm [203], evaluate the effectiveness of antiseptic agents against staphylococcal biofilm [204], and assess the bacterial viability of chlorine- and quaternary ammonium compounds-treated Lactobacillus cells [205]. However, it is worth noting that PI staining, despite its widespread use, has been found to underestimate the vitality of adhering bacterial cells [206]. Fig. 4 described different methods of staining biofilms.

DAPI staining, which uses 4${}^{\prime }$,6-diamidino-2-phenylindole, is commonly used for visualizing biofilms. This method helps identify EPS constituents in biofilms, enabling microscopic examination [109, 207]. Structural and biochemical characteristics of the biofilm matrix, such as surface adhesion, social interactions, and antimicrobial resistance, play a crucial role in the unique qualities of biofilms. DAPI staining can reveal these properties [208]. DAPI staining has proven to be a valuable tool for assessing cellular components and evaluating the effectiveness of decellularization in tissue engineering [209]. Fluorescence microscopy, which employs DAPI staining, has been instrumental in investigating the process of biofilm formation, motility, and adhesion, providing valuable insights into microbial community behavior [210, 211]. Furthermore, the dye has been used to measure microbial adherence and assess the biofilm life cycle, highlighting its importance in microbiological research [212, 213].

Acridine orange staining is a widely accepted process for visualizing and measuring biofilms. Its application spans multiple studies, such as one conducted by Hamdoon et al et al. [214], which analyzed biofilm formation on different orthodontic retainer materials. In another study, Hrynchuk et al. [215] utilized acridine orange fluorescence staining to assess the survival of bacterial cells within fully formed biofilms of S. aureus, while investigating the effects of an adamantane derivative. Acridine orange staining is a crucial tool for investigating biofilm kinetics and composition. Monmeyran et al. [216] explored the efficacy of the inducible chemical-genetic fluorescent marker FAST to monitor bacterial biofilm dynamics, in contrast to classical fluorescent proteins. Schiessl et al. [217] highlighted the importance of understanding biofilm metabolism and its impact on antibiotic resistance. Their findings demonstrated the contribution of phenazine synthesis to antibiotic tolerance and metabolic variability in P. aeruginosa biofilms [217]. These results suggest that integrating acridine orange staining, fluorescent markers, and metabolic profiling can provide valuable insights into the intricate mechanisms underlying bacterial biofilms and their antibiotic resistance.

Researchers have discovered that lectins can be practical tools for studying the composition of biofilm matrices, as they selectively bind to extracellular matrices [218]. Lectins have been used to identify polysaccharides in biofilms of P. aeruginosa [219], and to visualize and analyze carbohydrate-containing EPS in biofilms of Sulfolobus acidocaldarius [220]. They have also been employed to investigate changes in biofilm composition by labeling extracellular proteins and polysaccharides, indicating that lectins can potentially explore the dynamics of biofilm matrices [221].

However, it is vital to recognize the limitations of lectin staining. A study by Domingue et al. [222] cautions that while lectins can label biofilms formed under non-living conditions, they cannot distinguish between carbohydrates from microbes and those from humans. This limitation makes it difficult to accurately determine the molecular source of a biofilm matrix in living organisms. As macromolecules, lectins have limited ability to penetrate biofilms, which could impact their effectiveness in certain situations [223]. It is crucial to consider these limitations when interpreting results obtained from lectin staining of biofilms.

Numerous papers have extensively reported on using calcofluor white stain in biofilm research. This fluorochrome specifically attaches to $\beta$-1,3 or $\beta$-1,4 polysaccharides like chitin and cellulose, which are frequently found in the EPS of biofilms [224, 225, 226]. Using this staining technique, researchers can observe the EPS of biofilms and target the staining of polysaccharides present in the biofilm structure [227, 228]. Moreover, calcofluor white has also been used to detect chitin, a $\beta$-polysaccharide in fungal cell walls. It has been recognized as a straightforward and expeditious technique for identifying specific pathogenic components [229].

The use of Fluorescein diacetate (FDA) staining is a well-established technique in assessing microbial activity and the formation of biofilms. Through FDA staining, researchers can observe biofilm bacteria attached to surfaces and quantify their activity level [230]. The adaptability of this staining method is demonstrated by its application in investigating the biofilm formation of pathogens, including P. aeruginosa and S. mutans [231, 232, 233]. Moreover, using FDA staining alongside other dyes for dual viability staining has increased its effectiveness in evaluating the viability of microorganisms [234]. The staining technique has also been refined to facilitate a quantitative assessment of biofilm formation, highlighting its significance in measuring the extent of biofilm growth [235].

Recent studies have shown a growing interest in using peptide nucleic acid (PNA) probes for investigating biofilms. PNA fluorescence in situ hybridization (FISH) has proven effective in differentiating bacterial populations within biofilms, providing insights into their spatial arrangement and metabolic processes [236, 237]. This technique offers a more comprehensive understanding of biofilm structure and functional progression [238, 239]. PNA probes have also been utilized to quantify cystic fibrosis multispecies biofilms, demonstrating their capacity to evaluate specific populations within complex biofilms of multiple microbial species [240]. Moreover, PNA-FISH has been successfully employed to examine biofilms in diverse settings, including septic arthritis models and wound care, highlighting its versatility across various research domains [241, 242, 243]. PNA probes exhibit a strong affinity for complementary nucleic acids and boast biological stability due to their lack of charge and peptide bond-linked backbone [239], making them highly useful for investigating biofilms. PNA-FISH probes have been specifically designed to detect pathogens such as Gardnerella vaginalis [244] and Atopobium vaginae [245] in the context of bacterial vaginosis. These probes confirm the presence and impact of these bacteria within biofilms. Biofilm stains and their applications are presented in Table 1 (Ref. [187, 188, 192, 196, 207, 215, 217, 218, 219, 227, 228, 230, 231, 237, 240, 246, 247, 248]).

The application of microtiter plate-based model systems in biofilm research has gained significant attention due to its adaptability and relevance in diverse research domains. Microtiter plate-based techniques have been utilized to investigate the development of biofilms in various microorganisms, including A. baumannii, S. aureus, P. aeruginosa, and E. coli [430, 431, 432, 433]

The efficacy of these methodologies in examining biofilm formation, assessing the impact of different conditions on biofilm growth, and screening antimicrobial drugs has been demonstrated [434, 435]. Microtiter plate-based tests have been employed with other techniques, such as confocal laser scanning microscopy, to assess biofilm formation comprehensively [436].

Furthermore, microtiter plate-based model systems have been utilized to study the inhibitory effects of different chemicals on biofilm growth. For instance, the effectiveness of gold nanoparticles in combating biofilm formation by A. baumannii was evaluated using this technique [437]. Additionally, the microtiter plate technique has been employed to examine the influence of antibiotics on biofilm, thereby demonstrating its usefulness in investigating the efficacy of antimicrobial medicines [432]. Moreover, microtiter plate-based assays have been utilized to study the biofilm inhibitory effects of natural products and synthetic drugs [433, 435]. A schematic representation for the different techniques of common in vitro models for use in biofilm studies are presented in Fig. 6.

The CDC biofilm reactor is an invaluable tool for investigating the complex process of biofilm formation and evaluating the effectiveness of antimicrobial drugs. It recreates the environmental conditions observed in clinical settings, including continuous nutrition supply and shear stress, allowing for controlled biofilm cultivation [438]. The reactor has been extensively utilized to examine the impact of biomaterial characteristics on biofilm growth and investigate the formation of biofilms on various surfaces by different pathogens [439, 440, 441, 442]. Additionally, it has been employed to cultivate a diverse range of oral microbiota for scientific investigation purposes [443]. The CDC biofilm reactor has been confirmed as a reliable and consistent system for studying several aspects of biofilm physiology, morphology, growth dynamics, and antibiotic susceptibility profiles [248, 444]. Computational fluid dynamics has also been utilized to examine the shearing stresses within the reactor [445].

Furthermore, the reactor has been observed to facilitate the development of more intricate and resilient biofilm formations compared to alternative techniques, making it a valuable instrument for investigating biofilm architecture and antimicrobial resistance [438, 446].

Capillary biofilm reactors (CBRs) have shown potential in cultivating high-density biofilms due to their high surface area-to-volume ratio and exceptional cell densities [447]. Nevertheless, one major challenge in implementing CBRs is the extended period required for biofilm formation, which can take up to 5 weeks [448]. Additionally, research indicates that the continuous operation of CBRs promotes microbial growth, resulting in denser biofilms and higher current densities [449].

Moreover, the use of CBRs for the continuous oxidation of cyclohexane to cyclohexanol has been explored, revealing the potential of employing specific microorganisms at high cell densities [450]. This study emphasizes the importance of evaluating the technique in expanding biofilm-based capillary reactor systems [451]. This reactor can assess biofilm development, including biofilm dispersion, where individual cells exit the biofilm and return to a planktonic state [129]. This highlights the fluid nature of biofilms and the significance of comprehending their life cycle for efficient reactor design and operation.

Extensive research has been conducted on using flow cell reactors for cultivating biofilms, focusing on investigating key factors such as biofilm development, biomass production, and reactor performance. For instance, a recent study employed light-sheet microscopy to reveal a coordinated flow, akin to a fountain, that promotes the sideways spread of the biofilm by transferring cells toward its leading edge [262]. This discovery highlights the critical role flow dynamics play in the growth and spread of biofilms. Moreover, it was found that hydrodynamic conditions significantly impact the plasmid copy number in both planktonic and biofilm cells, underscoring the importance of fluid dynamics in genetic expression within biofilms [452]. Other studies have focused on using drip-flow biofilm reactors to simulate the growth of biofilms under consistent laminar flow conditions. These studies have emphasized the need to maintain controlled flow conditions in biofilm research [453, 454]. A study examining the flow dynamics of a reverse fluidized bed biofilm reactor has provided valuable insights into how the speed of air near the surface affects the behavior of the biofilm [32].

The potential of rotating disk reactors has been widely explored across various fields, including biofilm research, photocatalysis, and wastewater treatment [455]. One study investigated the formation of drinking water biofilms in a rotating annular reactor, highlighting the impact of turbulence on biofilm development [455]. Similarly, another study emphasized the importance of rotation speed in a photocatalytic degradation process using a photochemical rotor-stator spinning disk reactor [456]. These findings underscore the crucial role of rotation in regulating biofilm formation and mass transfer within the reactor.

Furthermore, researchers have examined the effects of mechanical stresses on biofilm elimination and cyclohexanone conversion in rotating biofilm reactors. They have found that high shear rates produced by rotational speed are crucial for biofilm growth and developing a structure suitable for biocatalytic applications [454, 457]. Another study presented a rotating disk bioelectrochemical reactor (RDBER) capable of cultivating both cathodic and anodic biofilms, with the added benefit of being autoclavable and allowing for observation of biofilm development through optical coherence tomography [458].

A drip-flow biofilm reactor is an essential tool for exploring the behavior and development of biofilms in various scenarios. This reactor model has been utilized in numerous studies to examine biofilm formation in chronic wound infections [459], investigate oral biofilms [460], evaluate the efficacy of biofilm management in wound infections [461], and monitor treatment responses in models of periodontal multispecies biofilms [453]. It has also been used for cultivating, treating, collecting, and examining P. aeruginosa biofilms [462]. The unique feature of the drip-flow biofilm reactor is that it allows for the growth of biofilms at the interface between air and liquid while maintaining a constant and steady flow. This creates a model that resembles a problematic wound environment [453, 461]. Additionally, it has been utilized to study the reactions of anaerobic biofilms to various electron acceptors, which affect the effectiveness of selenium removal and the characteristics of the biofilm [463]. The reactor has also cultivated single and mixed flow-cell biofilms on various metal surfaces at the air-liquid interface [464].

The widely used Robbins method has been updated to provide a more effective way of studying biofilm development and evaluating antibiofilm agents. The technique involves using a specialized device that can rapidly create and mold biofilms in a liquid, offering promising results for antibiotic lock treatment in removing biofilms from colonized surfaces [465]. Among the several biofilm reactors used in biofilm labs, the modified Robbins device is crucial to biofilm research [466].

The laminar flow chamber of the modified Robbins device comes equipped with suspended substrates to examine biofilm growth under controlled experimental settings. It is highly promising and indicative of in vivo conditions [182, 467]. Moreover, it has been used to recreate biofilms of P. aeruginosa and MRSA, demonstrating its adaptability in studying different biofilm varieties [468]. The updated Robbins device now comes with more sampling ports for analysis, allowing for the simultaneous collection of multiple biofilm samples at different time points throughout biofilm growth [467].

The modified Robbins method’s significance is further emphasized by its inclusion in many biofilm models, such as the Calgary biofilm device and the CDC rotating biofilm reactor. This indicates its broad adoption and utilization in biofilm research [469, 470]. Additionally, this technique has been used to investigate bacterial attachment to various surfaces in static and dynamic environments, demonstrating its versatility in different experimental scenarios [471, 472].

The annular biofilm reactor has proven to be an invaluable tool in a wide range of research, demonstrating its significance in studying biofilm formation, microbial activity, and the impact of various substrates and environmental factors on biofilm growth. Lee et al. [473] utilized this method to investigate the effect of pipe material on biofilm formation and structure in drinking water distribution systems.

Moreover, it has been utilized to replicate the shear stress experienced by sewer biofilms during flow designed for self-cleaning purposes, making it highly relevant to environmental science and wastewater treatment [474]. The lab-scale rotating annular reactor has also been used to assess the spatial arrangement of antibiotic resistance genes and integrons in biofilms, underlining the importance of studying microbial behavior and biofilm resistance [475].

In addition, a rotating annular reactor has been employed to investigate biofilm growth in diverse flow patterns, demonstrating its effectiveness in comprehending the process of biofilm formation under distinct circumstances [455]. Furthermore, the reactor has been used to assess the endurance of naturally occurring biofilms, highlighting its significance in evaluating the longevity and stability of biofilms [476]. Table 4 (Ref. [129, 432, 434, 435, 440, 446, 452, 453, 455, 456, 460, 461, 468, 471, 472, 473, 475, 477, 478]) is a summery of these reactors.

Dealing with biofilms is a challenging task, especially when it comes to combating antibiotic resistance. These films are notorious for their ability to confer resistance to multiple antibiotics, creating a significant obstacle in clinical settings [512]. Their resilient structure, restricted antibiotic penetration, and enhanced resistance transfer all contribute to the difficulties associated with antibiotic therapy [513]. Moreover, biofilm cells exhibit rapid microevolution in response to antibiotics, resulting in resistance levels far surpass those observed in planktonic cultures [337]. The bacteria’s ability to acquire resistance against conventional antimicrobial treatments that rely solely on antibiotics only exacerbates the issue [514]. The significant resistance of biofilms to various antimicrobials worsens the ongoing antibiotic crisis [515]. Protective EPS molecules encasing biofilms create microenvironments that pose challenges in dispersing them with conventional antibiotics [516].

Biofilms significantly enhance microbial cell antibiotic resistance, yet the specific mechanisms that facilitate this ability are not fully known [517]. Alternative therapies are urgently needed due to the lack of effective treatment options for antibiotic-resistant bacteria in biofilms [518].

Numerous factors contribute to the resistance of biofilms to antibiotic treatment, including their multicellular nature, EPS barrier, activation of efflux pumps, synthesis of enzymes that degrade antibiotics, heterogeneous modulation of metabolic activity and growth rate, and presence of highly drug-tolerant persister cells [519, 520]. Additionally, the varying occurrence of resistance resulting from biofilm penetration could help explain why some antibiotic treatments with limited ability to penetrate biofilms may depend on bacterial management to inhibit resistance development [521]. Effectively addressing biofilm-associated illnesses and antibiotic resistance requires a comprehensive understanding of the mechanisms involved in antibiotic resistance in biofilms. Comprehending and controlling biofilms, particularly concerning antibiotic resistance, presents complex challenges that require a thorough understanding of these mechanisms.

The comprehension and control of biofilms present a complex challenge due to their many components and nuanced attributes. Sauer et al. [82] proposed an extended conceptual framework that offers a unified basis for enhancing our understanding of biofilm formation and devising customized antibiofilm approaches. However, the intricate nature of biofilms poses a substantial obstacle in developing efficient antibiofilm techniques, perpetuating their persistence as a problem across various domains [82].

To comprehend biofilms, it is essential to understand the spatial and temporal arrangement of the biofilm matrix [522], which is crucial for their perception and control. However, this continues to be a significant obstacle due to the complexity of the samples and the limited range of methods currently available. Additionally, the wide range of glycoside hydrolases found in biofilms, as examined by Ellis et al. [523], creates challenges in anticipating or understanding the enzymes that may have potential therapeutic uses in the future. The intricate nature of these enzymes adds to the challenge of creating effective therapies for illnesses associated with biofilms.

The characteristics of biofilm-associated infections, such as their resilience and endurance, have become a significant obstacle to public health [524]. The presence of such resistance adds complexity to managing these infections. Suarez et al.’s research [119] further emphasizes the importance of biofilm thickness in shaping microbial communities, making it challenging to predict their structure accurately. Additionally, Jana et al.’s investigation [525] into the non-linear rheological properties of bacterial biofilms consisting of a single species demonstrates variability, potentially due to the diversity of ingredients in the extracellular matrix. Understanding the connection between phenotypic traits, diverse development settings, and community characterization is crucial in establishing structure-property relationships in oral biofilms [526].

The extent of variation in different strains of biofilms and their vulnerability to EPS-breaking enzymes [527] still needs to be better understood, underscoring the need for further investigation. Staats et al. [528] stress the significance of thorough comprehension of biofilms at a molecular level in a shared environment to counteract biofilm development in periprosthetic joint infections. Overall, these issues highlight the necessity for continuous investigation and innovative strategies to gain comprehensive comprehension and control of biofilms.

Dealing with biofilms in the context of chronic infections is a complex and persistent challenge in modern medicine. Biofilms are renowned for their resilience and ability to resist traditional treatment approaches, making biofilm-associated infections an ongoing and formidable obstacle [524]. One specific challenge is the limited effectiveness of certain therapeutic approaches, such as the restricted ability of photosensitizers to penetrate biofilms coupled with high levels of biofilm glutathione that consume reactive oxygen species. This reduces the efficacy of photodynamic therapy for treating biofilm infections [529]. Exploring alternate or more efficient therapeutic approaches to combat biofilms effectively is crucial.

Biofilm-related infections, especially in chronic wounds, pose a significant global challenge. Developing tailored antimicrobial therapies to improve wound healing is essential [530, 531, 532, 533]. These infections require specific care due to unique challenges, such as bacteria associated with biofilms having increased resistance to antibiotics. The notion of “biofilm tolerance”, which refers to the heightened resistance provided by biofilm lifestyles, adds complexity to disease management [148, 164, 534]. Biofilm presence in various therapeutic scenarios, such as infections related to orthopedic devices, poses significant therapy challenges [535, 536]. The correlation between microorganisms that create biofilms and infections in prosthetic joints affects all aspects of infection management, including diagnosis, treatment, and prevention techniques.

It is important to consider the resilience of biofilms to cleaning agents, disinfectants, and antibiotics. This knowledge can help us develop better strategies to effectively address diseases caused by biofilms and mitigate their impact [537]. Additionally, the variation within biofilm communities of the same species, which enhances individual biofilm populations’ ability to survive and thrive, adds an extra layer of complexity to biofilm management [538]. Biofilms present long-lasting challenges in understanding and controlling diseases. Due to their tenacious nature, increased resistance to traditional treatment approaches, and correlation with diverse clinical disorders, they pose a difficult challenge in the healthcare field. To overcome these problems, it is crucial to develop customized antimicrobial strategies, enhance our understanding of biofilm production, and explore alternative treatment methods to combat illnesses associated with biofilms effectively.

Identifying and evaluating biofilms present complex challenges across numerous fields, particularly in modern medicine and clinical practice. One of the biggest hurdles in clinical settings is accurately detecting biofilm infections. Silva et al. [539] noted that the absence of a universally accepted diagnostic protocol poses significant difficulties in pinpointing illnesses related to biofilms. This lack of standardized diagnostic procedures can lead to inconsistencies and complications regarding treatment.

Traditional methods of detecting biofilms, such as culture, microscopy, and biochemical assays, have notable limitations. These techniques may need to be more accurate in the total number of biofilm populations due to the presence of viable but non-culturable (VBNC) cells [540, 541]. In addition, the methods commonly used to tackle biofilm-related issues often result in inaccurate adverse outcomes and lack precision and accuracy [542, 543]. Addressing the complexities associated with comprehending and managing biofilms involves many challenges, such as diagnosis, identification, evaluation, and the development of effective treatment strategies. A multidisciplinary approach, standardized protocols, and advanced assessment methodologies are required to manage biofilm-related concerns effectively. The mysterious attributes of EPS further complicate the intricate nature of biofilms. Seviour et al. [544] have highlighted that these compounds are essential for biofilm formation and operation. The limited knowledge of EPS compounds contributes to the difficulties of accurately targeting and controlling biofilms in various environments [544].

Moreover, ongoing research has found that pinpointing the bacteria responsible for biofilm formation remains challenging [545]. Effective biofilm management is paramount due to their propensity to develop in various environments. However, assessing biofilms in clinical settings requires intricate techniques, as Magana et al. [249] emphasize the need for comprehensive approaches to characterize, monitor, and quantify biofilms while also creating efficient imaging and sensing tools.

Additionally, discovering new drugs, particularly antifungal agents, to counteract biofilm production in clinical care is challenging [546]. Even preventing and disrupting biofilms in settings such as contact lens cases presents methodological difficulties [547]. The resistance of bacteria residing in biofilms to antibiotics and disinfectants, demonstrated by species like Salmonella, makes it challenging to determine the effectiveness of these treatments [548].

The practical application of antibiofilm peptides (ABPs) for therapeutic purposes and their evaluation using murine biofilm models pose considerable challenges [549]. Furthermore, evaluating biofilm formation on diverse surfaces, ranging from marine to dental surfaces, requires a comprehensive approach to assess the impact of surface attributes on biofilm growth [550, 551].

The management of biofilms in environmental settings is challenging due to the resilient nature of these microbial communities. Biofilms exhibit exceptional resistance to antimicrobial agents and ecological stresses, which can pose significant risks to public health and environmental sustainability [149, 552].

Within water distribution networks and aquatic ecosystems, biofilms are essential in managing microbial communities and pharmaceutical contaminants, as evidenced by numerous studies [553, 554]. However, the adaptability of biofilms to their surroundings adds further complexity to understanding and controlling these organisms in the environment [369, 500].

Controlling biofilm infections presents a significant challenge due to their resilience to various antimicrobial drugs, including antifungal therapies. The development of low-oxygen microenvironments within the fungal biofilm structure highlights the complex nature of these infections [92, 555]. Moreover, biofilms can adapt and shield themselves against stress factors, underscoring their durability and presenting additional obstacles to the creation of effective control strategies [1].

Biofilms significantly impact both human health and environmental processes, including biogeochemical cycling and the breakdown of environmental pollutants—however, their ability to form organized colonies and withstand environmental stress challenges ecological systems. To fully grasp the environmental risks associated with biofilms and their effects on complex populations, it is crucial to understand their impact on microbial communities and behavior in aquatic environments [556].

Addressing the impact and management of biofilms requires a multidisciplinary approach. This approach should include improved detection techniques, a thorough understanding of how biofilms behave in natural environments and developing and implementing effective control strategies. Addressing these intricate issues can decrease biofilms’ adverse effects on environmental systems, human health, and industrial processes.

The persistence of biofilms presents a significant challenge in both medical and environmental contexts due to their inherent toughness and resilience to conventional antimicrobial therapies. Extensive research, as discussed earlier [557, 558], has demonstrated the resistance of biofilms to antibiotics and antimicrobials, underscoring the difficulty of eliminating infections caused by these structures. This resistance makes biofilm control more challenging and raises serious concerns about the efficacy of current treatment approaches.

Another study highlighted the restricted availability of efficacious therapeutic choices as a crucial element in the difficulty of eliminating biofilms [558]. This limitation contributes to difficulties in early detection, inadequate distribution of traditional medications within the biofilm structure, altered conditions within biofilms, and the rapid adaptation and emergence of resistance in bacterial populations [171].

A comprehensive approach has been explored to tackle the challenge of biofilms. A proactive strategy has been developed to create novel antibacterial techniques to hinder biofilm production [559, 560]. A more holistic approach also involves integrating biological, physical, and chemical methods to manage and eliminate biofilms [561]. By utilizing combination therapies that specifically target critical proteins involved in biofilm development and control, there is promising potential to disrupt the entire lifecycle of biofilms [562].

Moreover, a study on developing new materials and techniques to eliminate biofilms suggests a shift towards more targeted and efficient treatment approaches. Nonthermal plasmas have shown promise in laboratory settings for combating bacteria associated with biofilms, providing a unique physical method for controlling biofilms [563, 564, 565]. To effectively eliminate biofilms, a thorough and multifaceted strategy is necessary. Addressing the issue of biofilm elimination requires the development of new antibacterial techniques, combination medications, and innovative bio-remedial procedures. The resilience and flexibility of biofilms necessitate the improvement of existing treatments and the creation of new methods that can successfully dismantle biofilm structure and resistance mechanisms. This comprehensive strategy exemplifies the collaborative effort of academics and healthcare professionals in their pursuit to address and control biofilm-related illnesses and environmental consequences efficiently.

The study of biofilms is a complex and interdisciplinary field that poses significant research gaps. Due to biofilm activity’s intricate nature and its wide-ranging effects on sectors like healthcare, environmental management, and industrial practices, collaborative research efforts spanning multiple disciplines are crucial for advancing our comprehension and control of biofilms.

In the medical realm, biofilms can have significant consequences for human health, particularly in the context of medical device-related infections. A recent study underscores the importance of collaboration between medical researchers and engineers to develop efficient approaches for detecting, managing, and eliminating biofilms in intricate infections [566, 567]. The manipulation of materials and surfaces is crucial in preventing or facilitating biofilm growth, highlighting the need for interdisciplinary cooperation.

Biofouling is a similarly complex problem in environmental research and industrial applications, affecting water treatment systems, shipping industries, and other sectors. Thorough research is necessary to develop detection methods that can be universally applied across different fields of study [567]. Microbiologists, environmental scientists, and materials engineers must collaborate due to the intricate nature of biofouling to create inventive and environmentally friendly, efficient, and long-lasting solutions.

The study of biofilms from an interdisciplinary perspective can yield new insights into their spatial arrangement. Collaborative studies on biofilm structure have enhanced our understanding of microbial interactions and survival strategies within these communities [568]. This knowledge is critical for developing effective strategies to break down or utilize biofilms, depending on the intended objective.

Biofilm engineering has opened up exciting possibilities in the field of material science. In their research, Ng et al. [569] demonstrate the potential of biofilms to generate catalytic devices, highlighting the promising intersection of microbiology and materials science for innovative applications. The convergence of biofilms and technology presents unique opportunities but requires a deep understanding of biofilm biology and engineering principles.

Integrating machine learning into biofilm research has been suggested to harness the possibilities of interdisciplinary collaboration [570]. Machine learning has the potential to provide powerful tools for identifying patterns and predicting the development and behavior of biofilms. However, achieving this goal requires a strong partnership between biofilm and data science experts to translate complex biological mechanisms into computer models.

Despite advancements in the biofilm field, significant shortcomings remain areas for improvement, particularly in translating academic research into practical applications in industries. Highmore et al. [44] highlighted the disparity between experimental results obtained in a controlled laboratory setting and their implementation in real-life scenarios. To bridge this gap, it is essential to foster collaborations between academics and industry to expedite the application of biofilm research findings into tangible solutions.

In veterinary medicine, there has been a noticeable lack of direct research on the therapeutic significance of microbial biofilms [571]. This presents an interdisciplinary area for investigation that has yet to be fully explored. This underscores a broader issue: understanding human biofilm infections could be more effectively utilized in veterinary settings, representing a missed opportunity for transferring knowledge between different fields.

Furthermore, interdisciplinary research is necessary to explore the economic dimensions of biofilms [572]. The economic impact of biofilms spans several industries, requiring a comprehensive approach that integrates scientific research and financial analysis.

There are numerous multidisciplinary gaps in biofilm research, including machine learning, veterinary medicine, biochemical engineering, and economic analysis. Collaborative research addressing these knowledge gaps is crucial to developing a deeper understanding of biofilms and using scientific findings practically. Incorporating multiple disciplinary perspectives will be essential in formulating groundbreaking solutions to the complex issues presented by biofilms.