Biofilms are clusters of microorganisms that adhere to each other and a solid surface where water is present for extended periods. In nature, people often refer to photosynthetic biofilms as periphytic or algal biofilms, which consist mostly of algae, cyanobacteria, and heterotrophic bacteria living in symbiosis. In addition to these primary members, biofilms often contain additional microorganisms like flagellates and desmids and non-biological elements like silt, sand, and minerals.

Algae, which are chlorophyll-containing organisms, are widespread and lack traditional roots, stems, and leaves (Kesaano M, Sims RC et al., 2014). Biofilms are intricate communities of microorganisms that form on any surface, enclosed within a matrix of extracellular polymeric substances (EPS). These biofilms typically consist of unicellular or simple filamentous organisms, often known as microfouling or slimes. In specific conditions, such as high water flow or exposure to toxins, macroalgae like Enteromorpha and Ectocarpus can exhibit stunted growth on anti-fouling surfaces. In such cases, these algae may adapt by adopting a more compact growth form or integrating into the microfouling slime layer. The term “algal biofilms” refers to communities primarily dominated by microalgae that colonize well-lit surfaces when moisture and nutrients are present (Leadbeater BS et al., 1992, Jarvie HP, Neal C, et al., 2002).

Adherence and Attachment

Two methods can achieve microalgae immobilization: passive immobilization, accomplished through natural or induced biofilm formation, and active immobilization, achieved by trapping the cells within hydrogel polymer matrices. Researchers have utilized various immobilization media, including alginates, carrageenans, and polyacrylamide gel, for various purposes (Muñoz et al., 2009; de-Bashan and Bashan, 2010; He and Xue, 2010; He et al., 2014; Gagliano et al., 2017). Immobilization based on these polymeric matrices provides low mechanical strength and can result in restrictive diffusion of the substrate (Qureshi et al., 2005; Moreno-Garrido, 2008). This approach capitalizes on microalgae’s natural tendency to form biofilms.

Inorganic and organic compounds adhere to the surface of a substrate, creating a conducive environment for microbial growth (Qureshi et al., 2005; Stephens et al., 2015). Once microalgae and bacteria establish themselves on the surface, they secrete extracellular substances comprising nucleic acids, proteins, polysaccharides, and phospholipids. These substances serve a dual purpose: enhancing adherence to the substrate and simultaneously capturing and concentrating essential nutrients required for cell growth (Mohsenpour et al., 2021; Qureshi et al., 2005).

Algal Biofilm Habitats: Nature’s Adaptive Colonizers

In natural ecosystems such as rainforests, deserts, and ocean floors, algal biofilms are commonly present on surfaces like river stones (see Fig. 1), seashores, garden walls, tree bark, reed stems, and bamboo pipes. These microalgal biofilms thrive when they have sufficient moisture and light, and people typically collect them by scraping. They can grow on various materials, including plastics such as polyvinylchloride, polyethylene, polyurethane polymethyl methacrylate, polystyrene, polycarbonate, and polyamide; and other materials such as glass, cardboard, and ceramic tiles.

Notably, algal biofilms exhibit adaptability to environmental changes, maintaining colonies on surfaces and detaching either as single colonies or in clumps.

Composition of algal biofilms

The polysaccharide matrix called EPS embeds microorganisms in biofilms, providing cohesion and enabling component interactions (Flemming & Wingender et al., 2010).

Nutrients and dissolved gases diffuse through the boundary layer above the biofilm, then through the EPS matrix to reach biofilm cells, while waste gases and products either recycle within or diffuse outward. Surfaces concentrate charged particles and molecules, potentially serving as nutritional sources due to mineral and organic content. Various inorganic and organic molecules accumulate on surfaces, and particle-associated bacteria exhibit higher nutrient uptake rates, as shown by (Paerl and Merkel et al.,1982).

Diatoms are common early colonizers in algal biofilms, often making up the majority of associated bacteria and other unicellular organisms (Jackson & Jones, 1988). Single-celled diatoms are known for their silica frustules and their golden brown appearance, which is attributed to the fucoxanthin pigment present in their chloroplasts. They vary in size and attachment mechanisms (Daniel et al., 1987). Just a few diatom cells can start surface attachment, multiplying to form a compact biofilm.

In freshwater environments, green algae and cyanobacteria, particularly in aerial conditions, can dominate algal biofilms (Grant et al., 1982). For instance, Pleurococcus, a green unicellular alga, thrives in damp conditions on various surfaces. Trentepohlia, a filamentous green alga, can disfigure exterior surfaces in humid regions (Wee & Lee et al.,1980). Cyanobacteria, though usually minor, can become dominant due to their resilience and nitrogen-fixing abilities.

The biofilm is a dynamic community in equilibrium with its environment, undergoing growth, death, sloughing, and regeneration processes similar to bacterial biofilms but with less organization (see fig 2).

Factor affecting algal biofilm.

The development and adhesion of algal biofilms are influenced by various factors, including the physico-chemical characteristics of the substrate, operational parameters (see fig 3), the specific microalgal strain employed, and the interactions between microalgal cells, the substrate, and the liquid medium.

Light

Light is essential for algal photosynthesis and microalgal growth. Green algae dominate in high-light conditions during early colonization, while heterotrophic bacteria thrive in low-light environments. Diatoms often prevail in light-limited algal biofilms.

Increasing photon flux density (PFD) enhances growth rates in planktonic algae, but excessive PFD can result in photoinhibition and photooxidation, leading to culture stagnation or demise. Microalgae have the capacity to adjust to changing light intensities to optimize efficiency and protect against photodamage. The specific threshold for photoinhibition or light limitation varies among biofilm communities.

Under high irradiance, thick biofilms dominated by green algae resembling Scenedesmus were observed. Biofilms cultivated in a similar light range exhibited higher cellular nutrient content compared to those grown under higher light regimes. Algal Biofilms cultured under low irradiance using the same inoculum were thinner, more compact, and featured a wider variety of species, including cyanobacteria.

Hultberg et al. demonstrated that light quality has a direct impact on biofilm formation when using monochromatic illumination, suggesting the potential for enhanced cellular growth and lipid content through light quality optimization.

Temperature

Temperature plays a pivotal role in microalgal growth, biomass production, and biochemical processes. The optimal temperature range for microalgal cultivation typically spans from 20°C to 25°C. Below 16°C, growth slows down, while exceeding 35°C can be fatal for certain species. High temperatures, depending on the species, can stimulate cell growth, adhesion, and EPS production, promoting the formation of biofilms (Qureshi et al., 2005).

Claquin et al. (Claquin et al.; 2008) conducted research highlighting temperature’s significant impact on marine microalgae, influencing growth and the secretion of transparent exopolymeric particles (TEP) due to shifts in microbial and grazing activities (Honda Y, Matsumoto J, et al., 1983). Temperature also affects algal growth rates, species composition, and grazing activity within biofilm communities(Rao TS et al.; 2010). Tuchman and Blinn (1979) observed a rise in algal densities as temperature increased.

The Arrhenius relationship elucidates how temperature influences algal growth rates under consistent light and optimal nutrient conditions(Goldman JC et al.; 1974, Raven JA et al.; 1988).  Biofilms grown in thin water layers are more sensitive to temperature fluctuations compared to suspended cultures (Posadas et al., 2013).

Furthermore, temperature stress and the accumulation of calcium can have adverse effects on EPS production in algal cells (Domozych, et al., 2007). Fica and Sims (2016) determined that elevating wastewater temperature (7–27°C) and increasing organic carbon levels (300–1,200 mg L−1) significantly enhance biomass growth in algae-based biofilm systems. Temperature serves as the precise conductor orchestrating the dynamics of microalgae.

Nutrients

The abundance of algal species within biofilms is strongly influenced by nutrient concentration and light intensity, with a preference for algal dominance under conditions of high inorganic and low organic carbon levels(Unnithan VV et al.; 2014).

Nutrient availability plays a pivotal role in shaping algal growth, biofilm characteristics, succession patterns, and species composition (Nils RP et al.; 2003, Sekar R et al.; 2002). Environments enriched with biodegradable organic matter tend to favor the development of heterotrophic biofilms, whereas phototrophic biofilms thrive in response to light and the presence of inorganic nutrients (Hillebrand H et al.; 2002, Olapade OA et al.; 2006). The transport of nutrients to algal cells occurs through a concentration boundary layer via diffusion mechanisms.

Nitrogen and phosphorus availability exert distinct influences on microalgal metabolism. In instances of nitrogen starvation, microalgae pivot towards the production of lipids and/or carbohydrates as opposed to proteins (Gojkovic et al., 2020). There exists a mutual reliance between nitrogen and phosphorus uptake, with green algae initially dominating biofilms in nitrogen-deficient conditions, followed by diatoms, and ultimately cyanobacteria. The presence of phosphorus enhances nitrogen uptake, and in the presence of nitrogen, excess phosphorus can be absorbed by algae, a phenomenon known as luxury uptake (Bougaran et al., 2010)t.

Monod’s Kinetic Expression in Algal Biofilm Research

Monod’s kinetic expression finds application in algal biofilm research to elucidate growth patterns concerning nutrient concentrations and to predict rates of substrate utilization. For instance, Hill et al. 2009 established a growth saturation threshold of 25 μg/L soluble reactive phosphorus (SRP) for algal growth in stream biofilms.

Microalgae harness dissolve for in-organic carbon sources such as CO2 (aq) and HCO3 − found in wastewater, along with atmospheric CO2, and organic carbon derived from bacterial degradation (E. Posadas et al.; 2013, L.B. Christenson et al.; 2011).

Carbon, nitrogen, phosphorus, and silicon (for diatoms) are pivotal elements governing microalgae growth. The ratios of C:N:P serve as valuable indicators of nutrient limitation within algal communities, with the Redfield ratio (106:16:1 molar basis) representing a characteristic benchmark for optimally growing phytoplankton (R.S. Stelzer et al.; 2001, H. Hillebrand et al; 1999)

Elevated levels of nitrogen and phosphorus, coupled with increased inorganic carbon concentrations and heightened light intensity, augment the accumulation of photosynthetic biomass and its relative proportions in comparison to non-photosynthetic biomass. Additionally, advanced cultures and elevated temperature and nutrient loading rates contribute significantly to an upsurge in the proportions of cyanobacteria within photosynthetic biofilms.

pH

The pH level plays a crucial role in nutrient availability, including factors such as the solubility of ammonium and phosphate ions, as well as the formation of precipitates. Elevated pH values, typically at 9 or above, can trigger the formation of calcium phosphate, rendering it inaccessible to microalgae (Laliberté et al., 1997). Conversely, a decrease in pH levels can influence enzymatic activities, albeit depending on the specific microalgal species, thereby decelerating cell growth and product production.

Furthermore, pH values determine the charge of functional groups present on the surface of microalgae, consequently affecting their capacity to bind substances such as heavy metal ions.

CO2

Carbon stands as an indispensable element vital for the growth and productivity of microorganisms. Photoautotrophic organisms like microalgae possess the capacity to employ light energy to photosynthetically convert carbon dioxide (CO2) into biomass (S.B. Patwardhan et al.; 2022). Additionally, microalgae can utilize soluble carbonates as a carbon source for cellular growth, either via direct uptake or by catalyzing the conversion of carbonate into free carbon dioxide through enzymatic carboanhydrase activity.

Hence, the availability of an appropriate level of CO2 is imperative for the proper growth and metabolic activities of microalgae. Deviations from this optimum CO2 level, whether below or above, can exert adverse impacts on growth and productivity (W. Blanken et al 2014, B. Clement-Larosiere et al.; 2014). For instance, CO2 concentrations falling below the optimal level may result in carbon limitation, thereby retarding growth and productivity. Conversely, upon the introduction of an elevated concentration of CO2, microalgae assimilate a portion of the carbon through photosynthesis. Simultaneously, the excess carbon is converted into carbonic acid (H2CO3), which subsequently induces acidification of the medium.

Substratum

Algal adhesion studies primarily explore surface characteristics and material composition’s influence on biofilm formation to enhance cell attachment and biofilm growth. Ozkan and Berberoglu (2013) found differences in cell attachment between green algae and diatoms related to surface hydrophobicity, a concept emphasizing hydrophobic entities’ preference for each other to minimize water contact (Palmer et al., 2007).

Surface texture is crucial; rough or porous surfaces, with increased surface area and shear force protection, promote higher cell attachment (Gross et al., 2016; Huang et al., 2018). Increasing surface roughness, as suggested by Cao et al. (2009), creates slower flow zones that aid algal settlement. Studies by Huang et al. (2018) and Kardel et al. (2018) demonstrated weaker shear stress on grooved surfaces, influencing cell attachment based on groove shapes.

Material properties matter. Christenson and Sims (2012) found superior algal growth on cellulose-based natural polymer surfaces over synthetic polymers on various substrates. Sekar et al. (2004) observed greater attachment of certain algal species to hydrophobic surfaces (e.g., titanium, perspex, stainless steel), with exceptions for copper and its alloys, which hindered attachment due to toxicity (aluminum and admiralty brass).

Hydrophobicity

Hydrophobicity is a factor that affects the surface of the algal cell and substrate, for this reason considered an interface and a cellular factor. In diverse multi-species microbial biofilm, the presence of fimbriae, proteinaceous bacterial appendages rich in hydrophobic amino acids, can increase cell surface hydrophobicity (Barros et al., 2018). Flagellated cells show an increased ability to attach to surfaces. Flagellar motility may serve to overcome initial electrostatic surface repulsion (Bullitt and Makowski, 1995; Qureshi et al., 2005; Krasowska and Sigler, 2014). However, the microalgae biofilm on a hydrophilic surface might decrease and then increase with the increasing concentration of DOMs(dissolvable organic matter) and inorganic salts. Furthermore, many studies have shown that the DOMs can adsorb onto the substratum surface, leading to the development of surface conditioning films (Hwang et al., 2012)

Flow Velocity

The flow velocity of the liquid medium housing the algal biofilm plays a pivotal role in governing both cellular growth and attachment dynamics. This is primarily attributed to the liquid medium’s essential function as a source of vital nutrients for sustaining microalgal cells. However, it is imperative to acknowledge that elevated flow velocities can impose shear stress upon the biofilm (P. Choudhary et al.; 2017).

Furthermore, it is well-documented that turbulent flows occurring within the liquid medium have the potential to induce the detachment of cells from the biofilm, leading to a subsequent reduction in the overall thickness of the biofilm (L. Katarzyna et al.; 2015). It is noteworthy that the specific arrangement of microalgal biofilms may involve their placement within a rotational environment, resulting in periodic exposures to gaseous and liquid phases. This unique configuration introduces an added layer of complexity to the intricate interplay between flow velocity, shear stress, and the dynamics of microalgal cell growth and attachment within the biofilm milieu.

Extracellular polymeric substances (EPS)

Extracellular Polymeric Substances (EPS) in algal biofilm communities consist of polysaccharides, proteins, nucleic acids, lipids, and humic acids (A.M. Romani et al.; 2008, F. Di Pippo et al .; 2009)  influencing their physical and chemical properties. EPS serves as nutrient reservoirs, with embedded enzymes breaking down EPS and inert solids (Sutherland IW et al.; 1999), and they act as ion exchange resins, trapping nutrients through sorption (Flemming HC et al.; 2007, Wolfaardt GM  et al; 1999). EPS roles include facilitating cell movement (D.J. Smith  et al.; 1998), preventing cell desiccation, protecting against toxins (J.V. Garcia-Meza  et al.; 2005), and providing structural stability (I.W. Sutherland et al.; 2001).

Environmental Factors Influencing EPS Production

Microalgae adjust EPS production in response to environmental factors during biofilm formation. Light intensity significantly affects EPS accumulation (H. Ge et al.; 2014), e.g., Nostoc sp. produces more EPS at 206.20 mg/g DW under 80 μE.m^−2.s^−1 compared to 155.49 mg/g DW at 40 μE.m^−2.s^−1 .

Adhesion materials enhance EPS production in diatom Amphora coffaeformis (Becker K et al .; 1996). Growth materials influence EPS, as shown by Shen et al. (H. Ge et al.; 2014), and increased nutrient levels, especially nitrogen, boost EPS in diatom and green algae. Operational factors like light, temperature, nutrients, and culture density affect both biomass and EPS secretion. Li et al. noted EPS concentrations of 1.25 and 1.75 g.L^−1 with C/N ratios of 0.96 and 12.82, respectively (.H. Li, L. Ji et al.; 2020).

Young, grazed algal biofilms exhibit high EPS-to-biomass ratios for survival (C. Barranguet et al.; 2005). Cyanobacteria and diatom biomass correlate positively with EPS in wastewater-based biofilms. Light is linked to EPS production, and even in darkness, some species like Cylindrotheca closterium, Navicula perminuta, and Nitzschia sigma secrete EPS using stored glucan as a carbon source (D.J. Smith et al.; 1998).

Wastewater algal biofilms face grazing and changing conditions, necessitating species selection for EPS production and carbon management.

Species interaction

Natural biofilms encompass a diverse array of microbial constituents, including fungi, algae, protozoa, flagellates, and bacteria ( B.S.C. Leadbeater et al.; 1992, F. Di Pippo  et al.; 2009). Within photosynthetic biofilms, one finds a myriad of algae, bacteria, cyanobacteria, protozoa, and multicellular microorganisms. Notably, diatoms, green algae, and filamentous algae play substantial roles in contributing to biofilm biomass, displaying both autotrophic and heterotrophic capacities (Liang Y,Sarkany N et al; 2009). The bacterial component encompasses cyanobacteria and heterotrophic and autotrophic bacterial species.

Within algal biofilm, organisms engage in symbiotic interactions where heterotrophic bacteria serve as a source of carbon dioxide for photosynthetic organisms, thereby enabling the production of biomass and oxygen during respiration. Furthermore, excreted carbohydrates, vitamins, and organic compounds serve as vital nutrients for both algae and bacteria. This nutrient exchange is facilitated by the initial colonization of bacteria, which expedites the formation of algal biofilms (G. Roeselers et al.; 2007, É. Ács et a.; 2007).

Developmental Stages and Species Succession

The developmental stage of a biofilm exerts a discernible influence on species succession, resulting in alterations in the relative abundance and proportions of algae, bacteria, and extracellular polymeric substances (EPS) . In the early stages of photosynthetic biofilm formation, a higher proportion of EPS and bacteria is observed in comparison to algae and cyanobacteria, a phenomenon colloquially referred to as “conditioning”. Subsequently, following the establishment of the EPS matrix, algae exhibit rapid growth in the upper layers of the biofilm, thereby prompting bacteria to form aerial colonies in competition for essential nutrients.

Succession Patterns in Algal Groups

Biofilm maturity also exerts discernible effects on the dominance of specific algal groups. Diatoms are found to predominate in the early stages of biofilm development (typically within the first 15–20 days), while filamentous chlorophytes become more prevalent in later stages (Besemer K, Singer et al.; 2007, Johnson Reet al.; 1997). Some investigations have suggested a succession pattern characterized by an initial dominance of green algae during the early phase (1–4 days), followed by diatoms during the subsequent phase (5–9 days), and ultimately giving way to cyanobacteria during the third phase (10–15 days) (Sekar R et al.; 2004). Cyanobacteria are identified as late successional microorganisms (Zippel B et al.; 2005) with their distribution and prevalence being influenced by a multitude of factors (Rao TS et al.; 2010, Barranguet C et al.;2005, Sekar R et al.; 2004 ).

The presence of grazers, including Chironomids, Gastropods, Trichopteran larvae, Ephemeropteran larvae, and crustaceans, has a pronounced impact on reducing algal biomass throughout the seasons, with summer exhibiting the most significant effects (Hillebrand H et al.; 2001). Notably, filamentous species and chain-forming diatoms are more susceptible to grazing than single-celled algae species (Hillebrand H et al.;2002).

The succession of algal species within photosynthetic biofilms is governed by a complex interplay of factors, including light intensity, temperature, nutrient concentrations, and shear rates, with seasonal variations playing a pivotal role in shaping these dynamics see fig 4.

Metabolic pathway

Metabolic pathways in microalgae cultivation are influenced by the availability and form of nutrients, primarily driven by the assimilation of available carbon (Markou G et al., 2014). These pathways play a crucial role in enhancing biomass production and shaping the composition of intracellular metabolites like proteins, lipids, and pigments. They rely on inputs such as light and carbon sources (Wang J et al.,2014).

Heterotrophic cultivation of microalgae is considered the most promising approach for achieving higher biomass yield and optimizing biochemical composition (Chen F.  et al.,1996, Yang C et al.,2000). Generally, there are three distinct metabolic pathways in microalgae, determined by their nutritional requirements for growth and valuable biochemical production.

The main cultivation conditions are photoautotrophic cultivation, heterotrophic cultivation, and mixotrophic cultivation.

Photoautotrophic cultivation

The photosynthetic pathway involves converting sunlight and carbon dioxide into energy and cellular carbon dioxide, directing biochemical production in microalgae (Burkholder JM et al., 2008).

In photoautotrophic cultivation, microalgae exhibit varying lipid contents, ranging from 5% to 68%, depending on the species (Chen CY et al.; 2011). To boost lipid content during growth, nitrogen or nutrient limitations can be applied (Mata TM et al.,2010). The highest reported lipid productivity in microalgae under photoautotrophic conditions reached 179 mg/L/d using Chlorella sp. with 2% carbon dioxide and 0.25 vvm aeration (Chiu SY et al.; 2008).

Heterotrophic cultivation

Heterotrophic cultivation involves microalgae using organic compounds as both carbon and energy sources (Chojnacka K et al.; 2004). Some microalgae species can thrive in both photoautotrophic and heterotrophic conditions.

Only a few microalgae species have succeeded on a large scale in the heterotrophic mode (Lee DU et al.; 2001), but it is more efficient in terms of cell production per unit energy compared to the autotrophic mode (Yang C et al.; 2000). For instance, Chlorella protothecoides experienced a 40% increase in lipid content when shifting from photoautotrophic to heterotrophic cultivation ( Xu H et al.; 2006).

Microalgae can utilize various organic carbon sources during growth, including sucrose, glucose, lactose, galactose, glycerol, and fructose (Liang Y et al.;2009). Some microalgal species are obligate heterotrophs and are cultivated in heterotrophic mode, especially for lipid and pigment production from wastewater. This approach allows for achieving high cell densities, up to 100 g/L, facilitating microalgae biomass harvesting (Morales-Sánchez D et al.; 2015).

Mixotrophic cultivation

Mixotrophic cultivation combines autotrophic and heterotrophic modes, enhancing microalgae growth and resource utilization (Burkholder JM et al.; 2008). Microalgae use organic carbon compounds and CO2 as carbon sources, recycling CO2 during phototrophic conditions when light is available (Mata TM et al.; 2010). This approach aids global carbon dioxide reduction by utilizing carbon dioxide for microalgae growth. Industrial wastewater serves as a carbon source when combined with a light source, offering an efficient and cost-effective microalgae cultivation method (see fig 5).

Conclusion

Algal biofilms, a captivating realm within microbiology and environmental science, showcase intricate communities of microorganisms residing within a matrix of extracellular polymeric substances (EPS). Dominated by microalgae, cyanobacteria, and heterotrophic bacteria, these biofilms are versatile colonizers, populating a range of surfaces where moisture and light are abundant, from river stones to plastic surfaces and tree bark. Their ability to thrive and adapt in diverse conditions, using three primary metabolic pathways (photoautotrophic, heterotrophic, and mixotrophic cultivation), underscores their resourcefulness.

Beyond their aesthetic appeal, algal biofilms play an active ecological role. They contribute to water purification, nutrient cycling, and the production of valuable biomass and bioactive compounds, reflecting the dynamic nature of natural ecosystems. In a world grappling with environmental challenges, algal biofilms offer hope and insight, serving as a model of resilience. These microorganisms, without traditional plant structures, harness the power of photosynthesis, adapting to changing conditions, and exemplifying the ingenuity and tenacity of life.

Algal biofilms are not just subjects of scientific inquiry; they are living testaments to the intricate interplay of microorganisms in nature, demonstrating the remarkable potential for adaptation and survival. In a changing world, their adaptability and resourcefulness inspire us to explore and understand the complex relationships within ecosystems.

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