Substrate dependent microalgal biofilm cultivation system for the blue and green economy: formation, mechanism, and applications in environmental engineering and biotechnology

Substrate dependent microalgal biofilm cultivation system for the blue and green economy: formation, mechanism, and applications in environmental engineering and biotechnology | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Substrate dependent microalgal biofilm cultivation system for the blue and green economy: formation, mechanism, and applications in environmental engineering and biotechnology Maruthanayagam Veerabadhran, Jyothi Kanagaraj, Aravinth Annamalai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8649144/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Microalgae and cyanobacteria biofilm have simple physiology, fast photosynthetic growth using wastewater (WW) (primary nutrients removal, N, P), and can provide feedstock for microalgae-based biorefinery attempts. Suspended or planktonic cultures of photosynthetic algae have been widely developed for producing industrial biochemical by improving metabolic engineering via synthetic biology. Planktonic cultures can experience low algal biomass productivity, high operating costs and energy consumption when cultivating both indoor and outdoor cultivations. Algal biofilm or immobilized cells have drawn a lot of attention recently due to their prospective to improve the sustainability of livestock production, implications for carbon sequestration and climate change resilience, value-added products, and blue and green economies. Microalgal biofilm (MAB) or attached growth can be beneficial for effective removal and degradation of micropollutants, heavy metals, and hazardous chemicals, reducing harvesting costs, and transforming bioactive compounds and livestock for downstream operation. Non-axenic MABs are a smart choice for energy-alternative animal, municipal, and mining WW treatment (WWT), but they have not yet been developed as industrial options for deployment in sewage water. Besides, numerous aspects of MAB including biofilm formation mechanisms, stability, and bioreactor design, cell-cell interaction, extracellular polymeric substances (EPS), signaling molecules, and renewable products deserve deeper investigation. This article's goal is to provide a thorough review of MAB communities, their presence in the biosphere, their expansion in industrialized uses and the bio-based economy. Microalgae Circular economy Resource recovery Biofilms Wastewater treatment Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Microalgal biofilms in dominant and widespread distribution in nature composed of prokaryotic and eukaryotic microalgae ( Fig 1 ). The growth of these phototrophic biofilms is crucial for purifying the aquatic ecosystem and influencing external nutrient changes (Cheah and Chan 2021). Biofilm cultivation of algae is considered beneficial for eco-friendly technology because it is simply concentrated by scraping, which eliminates the costly concentrating techniques employed in suspension-based concentrating such as flocculation and centrifugation. The immobilized growth enhanced the biomass production through a higher absorb rate of solar energy and carbon dioxide (CO 2 ) fixation (Gross et al. 2015). This types of attached cultivation is a sustainable strategy over the conventional aqua-suspend or planktonic cultivation methods for dynamic ecosystems, autotrophic and heterotrophic components in the synthesis of high-value bio-products and WWT (Wang et al. 2017). In recent years, plentiful research has been accomplished on boosting the concentration of microalgal biomass for applications in agriculture, as well as for the production of bioenergy, bioremediation, and requiring less quantity of water (Singh et al. 2021; Masudi et al. 2023). However, harvesting rates for 20-30 % of the overall budget of protein mass production. By observing nature, energy savings in harvesting microalgae can be achieved by cultivating algae in biofilm (Ali et al. 2025). In natural, autotrophic microorganisms (microalgae and cyanobacteria), they commonly live on the artificial substrate or surface of a natural environment in the form of biofilms. This kind of algal mode of growth significantly produces EPS that can help to resist stress in photic aquatic environments (Ugya et al. 2020). Among these, attached growth has become popular for fuel applications because of its economic feasibility, production costs that are eight to ten times lesser than those of a liquid-based culturing method, an efficient and affordable way of immobilizing cells, and production of densely packed microalgal cells (Ennaceri et al. 2023). A biofilm-based growth system can be more efficient than conventional planktonic microalgae growth systems due to advantages such as increased increase to colonization, biomass thickening, barrier against toxins, and grazers (Miranda et al. 2017). Besides removing pollutants and increasing biomass, photosynthetic microorganisms also help remove of nutrients from industrial, domestic, and agricultural runoffs ( Table 1 and 2 ). Furthermore, MAB aids in the enhancement of algal recovery in WW and contributes to an ecological interest and a circular economy (Hu et al. 2021). Still, there are challenges in cultivating algal biofilm, optimizing growth under various environmental parameters of biofilm, and scaling up for industrial applications. Previous research showed that numerous parameters, including shear stress, thickness, attachment material properties, and the algal species, can strongly influence the development of biofilms (Mantzorou and Ververidis, 2019; Novoveská et al. 2023). The objective of this review article is to give a broad outline of MAB communities, industrial applications, and recent developments in MAB research. Different aspects of MAB, such as their development, interactions, bio-based products, and biotechnological applications, are covered. Table 1. Different microalgal biofilm cultivation systems utilized in the treatment of domestic wastewater (DWW) Wastewater Nature of biofilm/ culture system Products or activity Reference synthetic (modified BBM) Scenedesmus vacuolatus and C. vulgaris /PBR removal of PO₄³⁻, NO 3 -N, and nutrients 20-40 mg L −1 , 60-240 mg L −1 and 90% Moreno Osorio et al. 2019 municipal Halochlorella rubescens /Twin-Layer PBR microalgal growth of 6.3 g m -2 d -1 , efficiently reduced the concentrations of P (70%) and N (99 %) in the effluents Shi et al. 2014 seawater-diluted anaerobically digested effluent Chlorella SDEC-18/ inclined algal biofilm PBR high biomass productivity -5.66 g m -2 d -1 , removal of TP- 0.25 g m -2 d -1 , TN - 0.65 g m -2 d -1 , COD- 3.31 g m -2 d -1 Yu et al. 2020 synthetic S. obliquus , C. vulgaris and O. tenuis / vertical-enhanced algal-biofilm raceway TN, TP, and COD reduction of over 73.68%, 89.85%, and 86.61%, high biomass production (6.95 - 8.11 g m -2 day -1 ) Zhang et al. 2018a synthetic municipal non-axenic C. sorokiniana and Staphylococcus sp. / polystyrene culturing plates residual N, P, and TOC concentrations were respectively 70.1 %, 50.3 %, and 32.3 % Zhang et al. 2025a municipal C. vulgaris -microorganisms/ pilot-scale tubular PBR effectively remove the carbon and nitrogen Li et al. 2024 synthetic microalgae-biofilm anammox system/ up-flow anaerobic sludge bed reactor at 25 °C, TN and P were removed by 89.9 % and 94.2 % respectively Chen et al. 2024b low-carbon C. vulgaris -activated sludge biofilm / continuous-flow algal-bacterial system 31.95% and 9.56% reduction in CO 2 emissions and an improvement in COD removal efficiency Wang et al. 2025c high-saline Dunaliella salina /Microalgae biofilm PBR removal efficiency of 91.0 %, 84.3 %, and 81.6 %, for PO 4 3— P, COD, and TN Fan et al. 2023 Table 2. Treatment of industrial wastewater using different microalgal biofilms cultivation systems Wastewater Nature of biofilm/ culture system Products or activity Reference brewery Leptolyngbya sp. and Chroococcus -like population/flat-plate PBR high removal efficacy (over 65%) of the WW’s pollutants (total Kjeldahl nitrogen, orthophosphate, ammonium, nitrite, NO 3 -N, and COD), biomass 392and406 mg L −1 for polyurethane andglass (Papadopoulos et al. 2020) marine aquaculture C .. vulgaris /microalgae biofilm membrane PBR biomass productivity 22.03 mg L −1 day −1 , reduction in SMZ, sulfamethazine, sulfadiazine, dissolved inorganic P, and dissolved inorganic nitrogen were found to range from 91.0-99.6%, 60.8-82.1%, 50.0-76.7%, 61.0-79.2%, and 92.1-98.4%, correspondingly Peng et al. 2020 contaminated petrochemical stream Microalgae high reduction efficiency of turbidity (71%), sulfate (37.5%), alkalinity (62.5%), TSS (66.7%), Ni (74.0%), Cd (70.0%), and Pb (71.0%) Ugya et al. 2021b hog manure C. vulgaris / algal biofilm PBR high (7.37 g m -2 ) biomass production and removal rates of the NH 4 –N (91.24%), TN + (69.55%), COD (95.67%), and TP (64.40%) Wu et al. 2019 P-enriched nickel (Ni) refinery tailings microalga/pertri dish biofilm system high total carbohydrates (40.0%), increased lipids (6.7% - 19.5%), and removal of heavy metals (strontium, cobalt, manganese, Ni) from the WW with 26.4%, 10.5%, 24.8% and 24.8% Palma et al. 2017 swine microalgal-bacterial COD, NH 4 + -N, and TP removal efficiency 99, 92, and 98 %, respectively, high content (63 %) of fatty acids (C18:2n6c, C18:1n9c, C18:0 and C16:0 ) appropriate for biofuel generation Zhang et al. 2024b recirculating aquaculture system effluent water Phaeodactylum tricornutum biomass production of 15.28 g m −2 d −1 Böpple et al. 2024 landfill leachate cathodic algal 76.7% of TN, 89.4% of NH 4 + -N, and 86% of COD removal; produced the highest biomass and voltage production of 1.23 and g L −1 d −1 of 0.39 V Elmaadawy et al. 2020 marine AWW Chlorella sp. TOC, NH 4 + , and PO 4 3− removal efficiencies up to 80%, 96%, 99% Zhang et al. 2024c 2. Microalgal biofilm derived from nature In aquatic ecosystems, algae epiphytic biofilms (submerged macrophytes) play a significant contributor in biogeochemical cycles, migration, and transformation of contaminants. The biofilms are important for influencing ecological function, community composition, diversity, and microbial abundance in the marine and freshwater environments (Xia et al. 2020). Chlorella sorokiniana exhausted the total available ammonia nitrogen (NH₄⁺-N) by excluding it under low nutrient conditions, which promoted the growth of Scenedesmus pectinatus between algal and bacterial members in high-rate algal ponds (Krichen et al. 2019). The formation of Haematococcus lacustris biofilms in coastal rock ponds in Kandalaksha Bay of the White Sea is a natural phenomenon. Microalgae have adapted and formed dry biofilms to cope with the highly adverse stressful conditions, and preserving the ecosystem of the supralittoral zone of Subarctic and Arctic seas (Kublanovskaya et al. 2020). This kind of association between heterotrophic bacteria and microalgae biofilms in a metal-contaminated aquatic habitat helps to provide supportive metabolites for the bacterial growth and mediates the remediation of acid mine drainage (Abinandan et al. 2017). In benthic ecosystems, Phormidium autumnale frequently grows as biofilms at a site on the Hutt River (New Zealand). These microalgae-bacterial communities dominated by cyanobacterial blooms are causing increased neurotoxin production (Brasell et al. 2015). Microflora and micro-meiofauna in aquatic biofilms influence the provision of food resources, primary production, and nutrient cycling in rivers. Ingestion of a high amount of microalgae by micro-meiofauna significantly impacts the qualitative and quantitative composition of periphyton (Neury-Ormanni et al. 2016). Diatoms frequently develop biofilm on plastics in the aphotic and photic zones of a freshwater reservoir, but with subsequent growth in the environment, they fragment plastic and generate microplastics (Smith et al. 2021). Exposure of nickel and temperature to a biofilm dominated by natural green algae and cyanobacteria cultured in mesocosms were found to increase a fatty acid synthesis, which can be an indicator of environmental anxiety (Fadhlaoui et al. 2020). Altered bacterial communities, microbial respiration, and algal growth in stream biofilms are inhibited by pharmaceutical compounds in surface waters. In the combination or alone chemicals suppressed the biofilm respiration affecting higher trophic levels and important ecosystem processes (Rosi-Marshall et al. 2013). Solid mats are formed in the lake in Chungcheongnam-do by the dominant Halomicronema sp and Chlamydomonas sp. These ecological biomimetic MAB mats are formed by the attachment of microalgae to EPS produced by biofilm algal mats that mimic the natural algal mat system (Lee et al. 2023). Oscillatoria sp., Leptolyngbya sp., and Chroococcus sp., were intertidal cyanobacterial biofilms from the Sundarbans mangrove forest and can be utilized to produce maximum chlorophyll a , EPS, and bioactive metabolites (Veerabadhran et al. 2018). Copper (Cu) plays important roles such as reducing microalgal biomass, shifting the population from diatoms to cyanobacteria, and changing the grazing activity in artificial stream channels (Barranguet et al. 2003). Thorium (Th) exposed to periphytic biofilm was collected from Cap Rouge River and cultivated in a laboratory. This Th can modify the diatom community, resulting in a significant decrease in Shingopyxis and Pseudanabaena genera (Doose et al. 2021). Municipal wastewater (MWW) on the periphyton biofilms and influences the both prokaryotic and eukaryotic communities. The biofilm is made up of a significant amount of benthic microalgae, but there are low levels of parasites (Blifernez-Klassen et al. 2023). Microalgal colonization on plastic surfaces was observed in a field of lentic biospheres containing diverse environmental conditions (e.g., macrophyte coverage, conductivity, and nutrient concentration) and a broad geographical gradient. Polyethylene terephthalate showed higher biomass attachment than high-density polyethylene as plastic substrate surfaces (Nava et al. 2022). 3. Microalgal biofilms MAB is generally stated to as photosynthetic or autotrophic, and it consists of dominant microalgae, bacteria, and axenic culture (Wang et al. 2022a). However, various biofilm communities have been reported for varying growth and harvesting conditions. Primary productivity by photosynthesis was observed high on the surface of biofilms and proportional to the intensity of light and CO 2 concentration in MABs (Mkpuma et al. 2023). MABs growth and appropriate biomass production depend on appropriate surface in aquatic ecosystems ( Fig 2 ). The suitable substrate accounts for cultivation and affirms the relative abundance and microbial community properties (Kim et al. 2014). Biofilm aggregation and surface attachment in the substratum are addressed to the surface area of carriers. Concentrated biomass yields are maximized due to the design and material optimization of the carriers on an industrial scale (Berner et al. 2014). Attached biofilms are formed by the agglomeration of planktonic cells on a carrier material. Suspended biofilms that grow without attachment material or are detached, are called flocs or granules. The mode of biofilm growth is dependent on different internal gradients of environmental and nutritional conditions (Aqeel et al. 2019). Development of microalgae biofilms is based on different parameters such as substrates, light intensity, bioreactor configuration, biocarriers, and operating conditions such as hydraulic retention time (HRT) and shear stress. 3.1. Operating conditions and formation mechanisms of biofilm MAB is a type of green microalgae or cyanobacteria that can be rendered immobile through artificial or natural techniques. (1) Adsorption, which is the passive immobilization of algae on carrier materials, and (2) microalgal cell entrapment, which is the active immobilization of algae by chemical attachment, flocculant agents, or gel entrapment (Moreno Osorio et al. 2021). Various factors including hydrophobicity, pH, and hydrophilicity, surface properties of the substrate, photoperiod, and light intensity influence the growth of attached microalgal species (Rosmahadi et al. 2021). Stainless steel acts carrier for adhering to the unicellular cyanobacteria Microcystis aeruginosa , on a hydrophilic surface, and nutrient stress that triggers biofilm formation (Barros et al. 2019). Pseudochlorococcum sp. is known to accumulate in cellulose nitrate/cellulose acetate (CA) membranes, and high light intensity plays a part in the faster growth of biofilm (Ji et al. 2014). Chlorella vulgaris planktonic growth can be shifted into sessile growth in batch and continuous cultivation systems due to non-sterile conditions (Irving and Allen 2011). The biofilms of Scenedesmus obliquus were more attached to V-grooves than U-grooves. High shear stress resistance was observed on U-grooved surfaces. MABs showed resistance to hydraulic shear stress and a high surface area, which indicates the significance of microgrooves in biofilm attachment from 14.29% to 165.84 g m −2 on the surface (Huang et al. 2018). A temperature-responsive gel material on the inner surface of the 3D porous substrate (N-isopropylacrylamide) with a cooling temperature enhances the detachment capability of C. sorokiniana compared to high temperatures for developing the harvesting technology. Growth of microalgal species and secretion of EPS improves biofilm adhesion and reduces the cell detachment of unicellular microalgae (Wang et al. 2024a). C. vulgaris attached cultivation was favored on capillary ceramic membrane substrate surfaces, increasing biomass productivity more than other attached cultivation methods. Centrifugation resulted in a biomass harvest equal to the moisture content of the biofilm (Dalirian et al. 2021). Polyvinyl chloride substrate is responsible for the biofilm yield and maximum adhesion of C. vulgaris , succeeded by polypropylene, polyethylene, and stainless steel in a rotating flat plate photobioreactor (PBR) (Melo et al. 2018). The growth of unicellular and filamentous microalgae, attachment, and biomass productivity are influenced by membrane and fabric fiber substrates. The biomass production of Tribonema minus and Scenedesmus dimorphus increased by 7.81 and 10.24 g m -2 day -1 with the use of nitrate cellulose/CA membrane (Ji et al. 2023). The rough surfaces on the 30 kDa polyethersulfone membrane enhance the higher degree of cell attachment for benthic diatoms Cylindrotheca fusiformis , followed by Amphora coffeaeformis and Navicula incerta (Tong and Derek 2022a). On the contrary, biofilm reactors based on carriers or attachments reveal the requirement for less energy and ease of biomass harvesting (Hoh et al. 2016). Pine saw dust was proven to have better MAB production (10.92 g m −2 day −1 after 16 days of cultivation) in a cost-effective manner. Other lignocellulosic materials like oak saw dust, sugarcane bagasse and rice husk did not perform optimally. Pine saw dust showed to be effective for S. obliquus , C. vulgaris , and Oscillatoria tenuis (Zhang et al. 2017). Cotton coated carriers showed greater protein synthesis than uncoated carriers due to nutrient absorption and immobilization. Scenedesmus LX1 and associated EPS coating with cotton showed prominent results (Zhuang et al. 2016). C. vulgaris attachment is largely influenced by the chemical functional groups, micro-texture, and hydrophobicity. Cells cultured on Terrazzo had a higher carbohydrate pool, photosynthetic capacity (1.32-fold), and biomass productivity (3.20-fold) than those grown on cotton (Li et al. 2023). Walnut shell, a cheap substrate, is responsible for the cultivation of C. vulgaris and S. obliquus biofilm in a self-permeating biofilm reactor. Microalgae's biofilm production increased when the CO 2 concentration was 2%, the walnut shell size was 0.8-1.2 mm, and the light intensity was 150 mmol m -2 s -1 (Zouet al. 2021). Pine sawdust was used as a biocarrier to accelerate the biofilm growth and energy conversion properties of Diplosphaera sp. Furthermore, rick husk and sugarcane bagasse leachate inhibited the growth of C. vulgaris while also increasing the methyl ester content of saturated fatty acids and modifying the chemical compositions of algal cells (Zhang et al. 2019). The production of C. sorokiniana biofilm is highly influenced by the efficiency of CO 2 utilization. The biofilm biomass was increased by 96% of the flue gas CO 2 usage (Blankenet al. 2017). The rotating algal biofilm cultivating system proved effective in accelerating the mixing of nutrients and aeration. Thus, MA biofilms showed augmented growth and were easy to harvest (Gross et al. 2013). The arrayed attached culture systems in the MAB were enhanced to expand their utilization from the laboratory to outdoor systems (Huang et al. 2021). In contrast to the other bubbling conditions, the application of a bubble (1.4 mm diameter) to C. vulgaris cultivation in the thin-gap bubble column PBRs led to limited biofilm development, constrained flow, and low culture thickness (Thobie et al. 2022). Rotating biological contactor-based PBRs are more suitable for cultivation of C. sorokiniana because the algadisk is partially submerged in a growth medium for biofilm formation. Productivity and biomass yield of microalgae have been improved in the lab-scale algadisk reactor (Blanken et al. 2014). Ankistrodesmus falcatus with a long stripe with S. obliquus at a volume ratio of 1-4 resulted in a shortening the inoculation time of the biofilm by bridging and twining involved in the co-flocculation microalgae cells (Zheng et al. 2019). The wall shear stress during the cultivation of C. vulgaris biofilm with 6 Pa did not disturb stability and helped to choose the operational settings of the MAB cultivation systems (Belohlav et al. 2020). Chlorella pyrenoidosa , Chlorella sp., and Nannochloris oculata biofilm structure was affected by surface energy (SE). Furthermore, the cells formed a biofilm that was flat and homogeneous, with a porosity below 20% when the cell SEs were 50 to 65 mJ m -2 (Zhang et al. 2020a). The white light influences the structure of S. obliquus biofilm, which is a heterogeneous biofilm with roughness, many voids, and high porosity (Yuan et al. 2021). A light intensity of 300 and 100 μmol m −2 s −1 developed a biofilm with uniform cell distribution, which contributed to the high final cell density of C. vulgaris (Gao et al. 2024). Flashing light is a key parameter that regulates photosynthetic activity of a mixture of chlorophyceae dominated by Scenedesmus spp. and Chlorella spp. A 10 kHz light flash at a 5% light fraction resulted in a seven fold increase in the photosynthetic activity of microalgae biofilm compared to continuous exposure (51 µmol photons m -2 s -1 ) (Martín-Girela et al. 2017). With a high light intensity at a nitrate (NO 3 -N) concentration of 2 mg L -1 , C. pyrenoidosa resulted in a granular and very rough biofilm morphology, while at low light intensities, the biofilm was smooth and flat (Luo and Long 2022). 3.2. The function of EPS and signaling molecules in formation The mode of growth of the biofilm has a substantial effect on the production of concentrated biomass, with the largest productivity of Chlorella and Scenedesmus in the industry, which is the linear increase with culture time (Zhuang et al. 2018). The cyanobacterium Synechocystis sp. PCC 6803, a Type IV pili, is likely mediating more effective cellular aggregation than Wza dependent EPS and the S-layer in wild type, as determined by mutational analysis (Allen et al. 2019). Microalgae biofilm that generates tryptophan-like protein has a significant impact on the production of loosely bound EPS (LB-EPS) and soluble EPS. The composition of EPS and molecule weight decreased hydrophobic alkanes and recalcitrant aromatics were responsible for the mitigation of the membrane fouling (Liu et al. 2024a). The biofilm was developed by the indigenous microalgal-bacterial consortium by using high light intensity. A tightly interconnected EPS matrix was formed by exoprotein annotation and exopolysaccharide chains, and the structural stability of the biofilm landscape was significantly enhanced by increased branding degree, width, and height (Chen et al. 2024a). Algal-bacterial biofilms were developed by adding microplastic particles with sizes ranging from 0.065 μm to 5 μm. The biofilm was damaged, rough and loose, and the maximum algal recovery was achieved using microplastic particles with a size of 5 μm. Protein-rich EPS can be secreted by biofilm to protect it from the stress of microplastic particles (Gong et al. 2023). Novosphingobium sp. EPS had an advantage over M. aeruginosa in enhancing the faster growth of cyanobacteria. Exogenous aggregated substances, which are responsible for affecting biofilm formation, alter EPS composition ratios, and the production of EPS (Wang et al. 2022b). Marine microalgae biofilm adhesion is enhanced, hydraulic resistance is improved, and hydrophobic protein levels are raised by pre-coating modified microporous membranes with EPS (Tong et al. 2023). The accumulation of proline and alanine contents in EPS when cultivated under a glycerol carbon source helps algae to enhance attached biomass and biofilm formation of the C. pyrenoidosa by increasing hydrophobicity (Qian et al. 2023). Submerged commercial polyvinylidene fluoride (PVF) was more colonized by A. coffeaeformis and N. incerta than by C. fusiformis . The adhesion of algae to form biofilm is influenced by the ratio of polysaccharide to protein (Tong and Derek, 2022b). Li et al (2021) found that increasing the released polysaccharide in the cyanobacterium Synechocystis sp. can inhabit biofilm formation in cells. Cell hydrophobicity is reduced in the mutants (Δslr1076, Δsll5043 and Δslr1063) cells treated by the wild-type RPS. Cyanobacteria’s aggregation was changed by the physicochemical properties of RPS, cell surface charge, and cell hydrophobicity. The metal-ion-binding protein and algal cell-adhesion molecules promote the cell adhesion, self-flocculation, and bridging of Chlorella sp. The addition increased the abundance and distribution of hydrophilic α-helix in the EPS matrix, which facilitates microalgal separation from MWW (Chen et al. 2021). Soluble extrapolymeric substances-coated microporous PVF membranes, marine diatom N. incerta had an increase in cell adhesion due to a surface roughness and hydrophobicity (Tonget al. 2023). Signaling molecules can respond to the growth of microalgae or bacteria in biofilms by activating N-acyl-homoserine lactones (AHLs), which are characterized by their facilitation of ecological reconfiguration and frequent interspecies communication towards biofilm growth. The regulation of algal-bacterial symbiosis in the biofilm is affected by dominant genera such as Candidatus _Brocadia, Nitrosomonas , and Geitlerinema (Liu et al. 2025). C. vulgaris cultivated in AHL supplemented media modulates the surface electron donor of the cells. Quorum sensing of AHL-like substances in the algae varied the protein secondary structure, amino acid composition proteins in EPS, and extracellular protein secretion, contributing to the adhesion of microalgal cells (Ou et al. 2023). Cylindrotheca sp. grown in AHLs supplemented f/2 medium, the diatoms showed a significantly higher cell growth and EPS secretion. The thickness of biofilms and the length of carbon chains increased after the addition of AHL (Yang et al. 2023). The Cylindrotheca sp. grown with bacterial signaling molecules of AHLs (3-OH-C10-HSL, C10-HSL and 3-OXO-C10-HSL,) was revealed. The fouling diatom-biofilm growing under AHL has promoted Ca 2+ efflux, which has a positive effect on the formation of biofilm (Yang et al. 2016). The addition of exogenous C8-HSL enhanced the production of EPS, contributing to the formation of biofilm and the efficiency of phosphorus (P) and nitrogen (N) removal of heterotrophic nitrification-aerobic denitrification bacteria and the Chlorella symbiotic system (Qin et al. 2025). Exposure of the low concentrations (0.1 mg L -1 ) of indole-3-acetic acid (IAA) effects has an impact on the adhesion of C. vulgaris biofilm due to its influence on electron donor properties. IAA upregulates genes that are involved in the Calvin cycle, secretes protein, and promotes the production of hydrophobic amino acids from MABs (Xie et al. 2024). Tightly-bound EPS (TB-EPS) and LB-EPS increased P and N removal under semi-continuous operation at a HRT of 2 days. Soluble-EPS, has the capability to favor the growth of biofilms and the synthesis of chlorophyll in C. sorokiniana (Zhang et al. 2025b). The concentration of c-di-GMP is correlated with the dominant genera Brevundimonas , Devosia , and Comamonas as evidenced by research. The dominant bacteria have the capability to modulate microalgal-bacterial biofilm maturation when subjected to continuous lighting (24 hours) conditions (Wang et al. 2025a). C. vulgaris biofilms attachment is greatly promoted by multiple applications of low concentration IAA than by a single high-concentration application. The hydrophobic amino acid pathway has been upregulated by the low level of IAA's concentration, resulting in a reduction in the energy barrier between the receptive surface and the microalgae (Tang et al. 2026). The genes wza1 , wzt , luxR-05665, TetR/AcrR and LysR are responsible for the production of EPS and the formation of biofilm in the cyanobacterium Halomicronema sp (Caldara et al. 2025). 3.3. Strategies for improved formation of MAB Cell-surface properties induce the productivity of biofilms, and a higher microalgal biofilm formation is notably correlated with the surface hydrophobicity (Roostaei et al. 2018). To successfully form biofilm, it is essential to maintain the surface tension of WW to promote the growth of unicellular microalgae Chlorella sp. The surface tension of the liquid medium in the community increased, resulting in the formation of biofilm (Zhang et al. 2018b). The growth of the hybrid microalgae biofilm system was supported by a moderate NH₄⁺-N concentration of 25 mg L -1 . Furthermore, filamentous algae formed a web-like structure and attached to the carrier, attracting bacteria and non-filamentous algae to promote the development of biofilm. The high molecular weight, tryptophan, N ≡ N functional group, and protein of EPS initiated the colonization (Liu et al. 2023). C. vulgaris - Escherichia coli biofilm growth was enhanced by bio-coating derived microalgals on hydrophilic PVF membranes. Stimulating the extra-and intra-organic metabolites as a defensive response by increasing the deposition of organic substances in the bio-coating cultivation (Tong et al. 2024). The biofilm attached cultivation of Chromochloris zofingiensis and mixotrophic conditions was attained higher biomass productivity than the suspended mode of cultivation. The cultivation strategy is accountable for the elevated accumulation of chemical substances, larger cell size, a considerably greater energy supply efficiency, and an upregulated carbon fixation pathway (Liu et al. 2024b). The polyethylenimine -crosslinked PVF membrane with a higher pattern height is capable of speeding up biofilm development and biomass accumulation. A low-shear area was created in the biofilm of the membrane due to an increase in the active area for microalgal attachment (Zhao et al. 2021). The yield of S. obliquus - C. pyrenoidosa cocultured biofilm biomass is enhanced by uniform microstructure and small cell clusters. It is a reliable selection of microalgae species for manipulating biofilm microstructures and boosting yields as contrasted to other cultivation methods (Wang et al. 2024b). Attachment is facilitated and is ten times higher than the initial cell adhesion degree of N. incerta , A. coffeaeformis , and C. fusiformis when EPS derived from microalgae is pre-deposited as a thin conditioning layer on microporous PVF membranes with a higher cell finding affinity (Tong et al. 2022). Sugarcane bagasse and pine sawdust were used as a biocarriers to accelerate medium holding capacity and Brunauer-Emmett-Teller specific surface area and biofilm productivity of Chlorella sp. biofilm. Furthermore, to enhance algal biomass production, it is necessary to select a laying quantity of various biocarriers and a mixture of particle sizes (Lu et al. 2025). The biofilm formation and adhesion of microalgae cells to the roughness and materials of membrane surfaces effectively control the development of C. vulgaris biofilm. This strategy increases the amount of biofilm and cell adhesion by reducing the synthesis of EPS and salt bridging or charge neutralization mechanisms of the zeta potential of membranes (Liao et al. 2023). 4. Biotechnological applications Immobilized microalgae have been effectively employed to accelerate treatment efficiency, reduce carbon footprint, and improve commercial value in the agricultural and chemical industries ( Fig 3 ). Also, it is preferred to upgrade sustainable biological WWT systems and remediate the aqueous environment (Han et al. 2023). The Scenedesmus biofilm growth is very more effective for biodiesel, lipid, biomass, and cell density generation than suspension growth (Bagheri et al. 2024). The use of biofilm reactors and high-rate algal ponds is considered a sustainable way to efficiently treat WW from domestic and fruit-based juice production industries (Abrantes Silva et al. 2024). Biofilm-based microalgae are also improving in clam larvae’s survival and settlement rates (Azirar et al. 2025). Artificial, non-sterile, MABs are also promising for the generation of biofuels, valuable biomass, biochemical, and photosynthesis carbon capture (Paquette et al. 2020). The Microalgal-bacterial biofilm removes or recovers the pollutants, sequesters CO 2 , and cost-effectively separates biomass and water, and synthesizes value-added products through use of natural sunlight. In comparison, the microalgae-rich biofilm mode of growth resists sudden substantial changes in pollutants concentration and helps them achieve the economic benefits of WWT (Zhang et al. 2024a). 4.1. Fine chemicals and biomass production The marine phototrophic dinoflagellate Symbiodinium voratum was immobilized on a Twin-Layer PBR in order to increase the production of the anticancerogenic carotenoid peridinin by 24 mg m−2 day−1 at a light intensity of 74 μmol m −2 s −1 (Benstein et al. 2014). The biomass production from Revolving C. vulgaris biofilm cultivation system increased 302% than standard raceway pond. The biofilm was produced 18.9 g m -2 day -1 as the efficient cultivation system for commercial scale (Gross and Wen 2014). The MAB system has been utilized to successfully remediate WW, remove heavy metals, and provide a promising source of metabolites for medicinal product synthesis (Ugya et al. 2024). The cultivation of C. vulgaris biofilm involves the use of urea and glycerol as N and carbon sources. In order to produce lipids that comprise a considerable amount of polyunsaturated fatty acids, which grow better in extremely low light and use less water (Rincon et al. 2017). Biofilm was cultivated by heterotrophic-assisted photoautotrophic biofilm (HAPB) growth mode to examine the influence on biomass and lipid generation. C. vulgaris formed HAPB with the maximum lipid content (120%) and biomass (78%) at a ratio of 1: 20 total organic carbon (TOC) (glucose) to total inorganic carbon (CO 2 ) and 72:1 total carbon to total N (Ye et al. 2018). S. quadricauda biofilm has been cultivated in synthetic MWW and achieved a significant increase in biomass productivity of 9.11 g m −2 d −1 than other biofilm systems. The lipids ranged from 25.14 to 30.40 %, with a superior quality of biodiesel and a higher percentage of C18:1 content (Xu et al. 2025). Attached biofilm was produced by the inoculation of microalgae in multi-layer PBRs. The productivity and quality of lipid production increased in the biofilm of Botryococcus braunii by 42.3 % - 51.3 %, and by 48.17 % -55.44 % of hexadecanoic acid (16:0), respectively (Shen et al. 2015). Similarly, by growing S. obliquus in nickel foam using the modified surface as the carrier, the protein synthesis rate of biofilm, protein concentration, and CO 2 biofixation rate were induced in the range of 43.11 g m −3 h −1 , 0.892 g L −1 , and 4465.6 µmol m −3 s −1 , correspondingly. (Guo et al. 2018). The quality and biosynthesis of polyhydroxyalkanoate are enhanced by salinity stress, which alters the bioavailability and precipitation of P, triggering in sole microbial groups in the microalgae biofilm. In the biofilms of salinity stress, there was a boost in photosystem II and metabolic flux towards carbon storage in the system due to the alterations in water chemistry (Ugya et al. 2025). The stratified microalgal-bacterial biofilm experienced a rise in CO 2 fixation as a result of biomass production. The SMBB cultivation system enhances photosynthesis activity, total EPS, protein and polysaccharide concentrations over hybrid microalgal-bacterial biofilm (Li et al. 2025). 4.2. Carbon and nutrient removal MAB cultivation integrated with bioreactors ensures efficient utilization of the nutrients from municipal or agricultural WW and high biomass production (Ugya et al. 2023). N and P input into the aquatic ecosystem causes harmful algal blooms and eutrophication (Lan et al. 2024). The reactive oxygen species generated by freshwater microalgae in the biofilm can remove turbidity, NO 3 -N, phosphate (PO₄³⁻), and zinc (Zn) from petroleum-contaminated water. The absorption efficiency of the EPS enhances the formation of microalgae biofilm through larger surface areas and microspores present in the cells (Ugya et al. 2021a). A method for removing microbe-derived dissolved organic nitrogen (mDON) using algae biofilm comprised cyanobacteria Limnothrix and Kamptonema spp. has been revealed. The biofilm reduced the concentration of mDON formation in DON-free WW and fed with activated sludge effluent by 83% and 72%, respectively (Lin et al. 2021). The efficient reduction of ammonia (NH 3 ) was achieved by the mycoalgae biofilm, which consists of Mucor indicus and C. vulgaris , in simulated aquaculture wastewater (AWW). Biofilm developed under a range of NH 3 conditions (6.14 mg L −1 to 124 mg L −1 ), with different compositions of fungi and algae (14.8 % to 0.5% algae) in the biofilm (Barnharst et al. 2023). Light irradiance is a significant parameter that influences the growth of microalgae in the phototrophic biofilms in outdoor large-scale cultivation and nutrient removal. A day time favored removal of N and P overnight time (Boelee et al. 2014). Mixed culture of MAB cultivation in rotating algae biofilm reactors has shown advantages over open pond lagoon systems. Efficient removal of suspended solids, N, and P from petroleum refining WW is also important for biomass production (Hodges et al. 2017). The solid-surface culture system for Chlorella kessleri - increases the cell densities, and CO 2 capture, respectively, by 120 mg chlorophyll m −2 and 110 tons ha −1 year −1 from 1/5 concentration liquid Gamborg's B5 medium under continuous illumination (Miyauchi et al. 2020). Oxidation ponds were observed to face challenges in the removal of nutrients. Ponds contain hydrogen-fed indigenous bacterial-algal biofilm, which assists in the higher removal of 97.1 % for total phosphorus (TP) and 97.9 % for total nitrogen (TN) than control systems (Li et al. 2024). 4.3. Pollutant biodegradation C. vulgaris exhibited high phenol degradation by increasing the metabolic activity and thickness of the biofilm (191−222 μm) (Zhong et al. 2019). Lowering the concentration of residual linear alkylbenzene sulfonates can be mitigated in aeration-free greywater by increasing the levels of density, highest biofilm concentration, and specific oxygen consumption and generation rates by adding an organic load. The ability of non-aeration Synechocystis -bacteria biofilm to remove pollutants was investigated in a push-flow microalgal-bacteria biofilm reactor. A complete removal of pollutants and a reduction of dissolved organic nitrogen was achieved through high nitrogen assimilation by microalgae (Wu et al. 2023). Fresh water-derived biofilm of Pediastrum sp., Oscillatoria sp., Nitzschia sp., Cymbella sp., and Merismopedia sp. demonstrated higher adsorption and absorption capabilities for removing bisphenol A (Vanniaraj et al. 2025). Utilizing synthetic wastewater (SWW) with polyacrylamide (PAM) to cultivate biofilm in revolving algae biofilm reactors will increase the removal rates of the PAM, the N source, TN, chemical oxygen demand (COD), and TOC concentration (Zhang et al. 2021). The symbiotic bacterial-algal-fungal biofilm in the moving bed bacterial-algal biofilm reactor system with a phenol degrader reduced the phenol content by 2.3-fold and converted NH 4 + -N by 85 %, from phenolic-laden coal chemical WW. The biofilm had a higher EPS matrix and functional taxa ( Flavihumibacter , Hydrogenophaga , Fusarium ) than conventional systems (Yang et al. 2025). Increased production of glutathione reductase, superoxide dismutase (SOD), catalase, and peroxidase caused the microalgae biofilm to degrade the phenol-containing textile WW, while the reactive oxygen species counteracted. The alkalinity (47%), chloride (52%), COD (83%), and biochemical oxygen demand (68%) may be removed by the biofilm activities of enzyme release or adsorption of the pollutants (Ugya 2021). The seawater Chlorella sp. biofilm was responsible for removing a significant amount of tetracycline (74.5-85.2 %), clarithromycin (60.8-69.5 %), and sulfamethoxazole (SMZ) (13.5-44.1 %) from the combined antibiotic mixture. Antibiotic removal from marine water has a potential to protect the blue economy from the persistent xenobiotics (Yu et al. 2024a). The production of aminopyrine N-demethylase (54%) and aniline hydroxylase (48%) in periphytic biofilm ( Serendipita , Thioalkalivibrio , Azoarcus , Acidibacter , Achnanthidium , and Sellaphora ) may aid Iris pseudacorus in increasing the expression of cytochrome P450 enzymes to alter or degrade the SMZ and doxylamine succinate from the urban WW (Yadav et al. 2021). 4.4. Metal(loid) removal Axenic Nostoc muscorum 's biofilm was effective in removing cadmium (Cd) from a fresh medium containing 0.05 to 100 ppm cadmium chloride at pH 5-9. Sequestration mechanisms such as chemisorption and Langmuir's adsorption isotherm confer on the removal of Cd(II) (Raghavan et al. 2020). Iron (Fe), Zn, and Cd were removed from mine effluent through binding and absorption mechanisms by green microalgae biofilms from with 85%, 95%, and 99%, respectively (Makhanya et al. 2021). Banana stem-based microalgae biofilm transformed hazardous arsenic (As) into less toxic arsenate and dimethylarsenic acid with an efficiency of 97.6% within 5 days of removal; this may be increased to 20% more than microalgae biofilm cultured on nylon mesh. Exposure of biofilm As increased the proteins and lipids which were also facilitated for the binding of As (Kumar et al. 2022). The green algae Stichococcus bacillaris produced more biofilm, thus removing 2-3 mg L −1 of Zn from real mine dump leachate and SWW, which contributed to biofilm resistance (Li et al. 2015). The effectiveness of S. obliquus and C. vulgaris in removing heavy metals is affected by mixed biofilm culture in aqueous medium. Mix culture efficiently increased the uptake of Fe (91.67-97.85%), NO 3 -N and PO₄³⁻ (Yousefi et al. 2023). Table 3 displays the removal of heavy metals by microalgal biofilm. Table 3. Heavy metals removal from environments or laboratory conditions using immobilized or biofilm-forming microalgae Nature of biofilm heavy metal removal/ activity Sources of Contaminated water/ culture medium Reference Chlorella sp. antimony, As, beryllium, Se, and Zn at concentrations of 0.07546, 0.05709, 0.09326, 0.4618, and 0.00979 mg L -1 Dhiba port Alhumairi et al. 2022 P. autumnale and Nitzschia palea higher production of EPS with protein-like polymers induced by metal 600 μg L −1 of Zn various metal concentrations are present in BG-11 medium Loustau et al. 2019 algal-bacterial Biofilm 21.8 ± 3.4 mg kg -1 of selenium (Se) was present in the biofilm fertilizer Se-rich AWW Han et al. 2020 Phormidium bigranulatum -dominated mat ( Lyngbya sp. and O. tenuis ) removed 80% to 94% Cu 2+ 10 to 100 µM Cu 2+ supplemented to BG-11 medium Kumar and Gaur 2014 Euglena mutabilis samarium, europium, ytterbium, and lutetium adsorbed with an adsorption capacity of 0.035, 0.033, 0.033, and 0.031 mmol g -1 through elevated EPS synthesis Ashbridges Bay Wastewater Treatment Plant (WWTP) Zak et al. 2025 periphytic microalgae on shells and glass Cu (89%), lead (Pb) (97%), and Zn (97%), for mussel shells; Cu (73%), Pb (96%), and Zn (86%) for recycled glass heavy metal contained synthetic stormwater Bremner et al. 2020 microalgae biofilm As accumulation in fish muscle gets reduced by 41-63 % by increasing the organic arsenic species fish exposed to As-contaminated water Kumar et al. 2025 biogenic aqua crust ( Cytophagales sp., Hyphomonadaceae sp., and Leptolyngbyaceae sp.) biofilm formation (e.g. and LuxS, OmpR and CRP), genes encoding extracellular peptidase (e.g. family S1, S9) and CAZymes (e.g. GT2, CBM50), thus enhance the capability of metal (loid) removal tailing wetland Wang et al. 2023 S. obliquus highest cadmium (II) removal rate is 91.27% Cd 0, 1, 5, 10, 15, and 20 mg L -1 in BG-11 medium Ma et al. 2021 5. Biofilm treatment of real wastewaters Different microalgal cultivation technologies such as PBRs, planktonic or suspended, mixotrophic, and heterotrophic cultivation methods have been conventionally used for treating WW ( Fig 4 ). The most common sewage treatment technologies include the planktonic cultivation technology (Daneshvar et al. 2019; Sathinathan et al. 2023). Some of the drawbacks of these technologies, for example a land requirements, biomass valorization, and low scattered density in the culture media, environmental and operational conditions are not addressed in the bottleneck of the suspended cultivation technology (Molinuevo-Salces et al. 2019). Because of its efficiency in ground usage and increasing harvest per ground area, carbon sequestration, producing potential therapeutic compounds, and WWT efficacy, biofilm is currently being evaluated as a feasible cultivation technology (Patwardhan et al. 2024). Increasing the P and N content while improving loading rates results in a significantly reduced internal N to P ratio of MWW effluent. MAB can increase the efficiency of removing residual NO 3 − -N and PO 4 3− -P, and observe the maximum uptake capacity (Boelee et al. 2011). Cyanobacteria, coccal green algae and diatom biofilm cultivation in the municipal WW allows efficient P removal and maximum biomass production in horizontal flat panel PBR (Sukačová et al. 2015). Scenedesmus quadricauda , Chlorobia and Deltaproteobacteria biofilm microbial fuel cells (MFC) were fed with DWW originating from the sewer. The microalgae and bacteria in the biofilm cells effectively remove COD, TN, and TP which were higher in the algae biofilm (AB) MFC than MFC or AB alone (Yang et al. 2018). C. vulgaris biofilm was cultivated on the piggery farm WW. The removal of NH 4 + -N and TN from WW can be achieved by using a pine sawdust biocarriers, which removes 95.54% and 96.10%. Additionally, biocarrier in cultivation were noted to support robust growth of algal biofilm (Zhang et al. 2020b). Real dye textile WW used cathodic C. vulgaris biofilm to enhance the electrochemical performance of MFCs, substituting the platinum cathode and removing Cr (54-80%), Zn (98%), and COD (92-98%) (Logroño et al. 2017). The cultivation of Chlorella proteinucleus -bacteria symbiotic biofilm was utilized by sequential batches to treat municipal and soybean soaking WW. Enhanced biomass productivity, proteins, carbohydrate, and lipid levels, increased removal rate of NH₄⁺-N, TP, TN, and COD (Yu et al. 2024b). The microalgal biofilm treatment can increase the production of lipids, protein and polysaccharides. It was shown that the concentration levels of sulfamethazine, COD, TP, NH 4 + -N, and TN in the swine digestion effluent were 0.99, 367.28, 4.11, 9.32, and 22.65 mg L −1 , correspondingly. This meets the discharge standards (Mou et al. 2023). The co-cultivation of Isochrysis galbana biofilm can effectively increase the self-sedimentation rate by synthesizing TB-EPS content. This immobilization strategy increases the removal rate of an inorganic nitrogen and NO 3 -N in AWW (Wang et al. 2025b). MAB had a high degree of efficacy in removing P residues and recovering P from simulated WW under small WWTP (Sukačová et al. 2020). Co-culture of C. pyrenoidosa and Phormidium sp., in livestock WW enhanced their biofilm adhesion through the synthesis of TB-EPS and its polysaccharides and proteins (Liu et al. 2024c). Cultivation of non-immersed zigzag microalgae biofilm in digested piggery WW exhibited a maximum growth rate of the biofilm and enhanced the NH₄⁺-N removing properties of the biofilm (Huang et al. 2023). 6. Conclusion Even though the attention that studies on microalgal-based cultivation have been published for many years, this absorption property is becoming increasingly popular due to the cost-effective development of biofilm bioreactor systems. By advancing in knowledge and expertise, more potential benefits can be gained from the natural tendency to attach to surfaces and grow. The attached or clump growth in substrate and bioreactor can be optimized by exploring the selection of biocarriers or surface materials. Highly effective support materials for MABs were discovered to include stainless steel, cotton, sawdust, sugarcane bagasse, nickel foam, rice husk, N-isopropylacrylamide, polyethylene terephthalate, polyethersulfone, and polyvinylidene fluoride membrane. In the future, researchers may create low-cost cultivation systems that produce concentrated algal biomass to increase yields and recover products. This also means that regulating biofilm growth with known microalgal species is an integral part of the future path for building specific integrated biofilm systems to produce desired products or potential effects. The technique can be employed to increase microalgal harvest, but it still has the potential to form biofilms and immobilize microalgae. Smart biofilm cultivation of microalgae can be achieved by implementing an integrated multi-technology framework to optimize interaction effectiveness and address the pollutant characteristics and operational constraints of the microalgae engineering. However, research to elucidate the underlying physiological mechanisms is still necessary, especially when it involves interaction between cells and the pattern of gene expression in biofilms. A better thoughtful of the mechanisms elaborated in designing the bioreactors is essential. The focus of microalgal immobilization is receiving much recognition from the research community in terms of sustainable real WWT and recovery. In any case, research on this issue can exploit the metagenomics, adhesion, aggregates and biofilm formation for bioremediation, EPS synthesis, algal growth modulation, and signaling molecules. Some current works report the application of this mode of growth to alleviate xenobiotics, remove COD, N, P, and metal contaminants. In natural ecosystems, there has been an investigation into the mechanism of biofilm formation between microbes and microalgae. The operating parameters are complex and underdeveloped to bring biofilm growth to the industrial level. Declarations The authors state that there are no potential financial or other interests that could influence the results of this research. Funding The preparation of this review did not have any external financial support. Availability of data and material The data generated or analyzed during this study has been published and can be made available upon request. Competing interests The authors state that there are no competing interests. Author Contributions MV : Conceptualization, writing, Methodology, Investigation, Review, Editing & Formal Analysis, JK : Formal Analysis, Data Curation, AA: Editing &Review. CR : Figure and art work. AK : Original Draft, Data Curation, and Review & Editing. References Abinandan S, Subashchandrabose SR, Venkateswarlu K, et al (2018) Microalgae-bacteria biofilms: a sustainable synergistic approach in remediation of acid mine drainage. Appl Microbiol Biotechnol 102:1131–1144. Abrantes Silva T, Pereira ASAP, Ferreira J, et al (2024) Enhancing microalgae biomass production: Exploring improved scraping frequency in a hybrid cultivation system. J Environ Manage 355:120505. 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Zou X, Xu K, Chang W, et al (2021) A novel microalgal biofilm reactor using walnut shell as substratum for microalgae biofilm cultivation and lipid accumulation. Renewable Energy 175:676e685. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 12 May, 2026 Reviews received at journal 23 Mar, 2026 Reviewers agreed at journal 12 Mar, 2026 Reviewers agreed at journal 02 Mar, 2026 Reviewers invited by journal 09 Feb, 2026 Editor assigned by journal 04 Feb, 2026 Submission checks completed at journal 02 Feb, 2026 First submitted to journal 20 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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12:37:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8649144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8649144/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102745854,"identity":"9373ba4a-d233-46a1-82b5-636280682347","added_by":"auto","created_at":"2026-02-16 08:54:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":327971,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of microalgal biofilm\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8649144/v1/89b6f614259e6ca5096e06f4.png"},{"id":102443394,"identity":"83034207-7c9b-44f6-b8dc-49ca6ee7cec4","added_by":"auto","created_at":"2026-02-11 17:19:51","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":238073,"visible":true,"origin":"","legend":"\u003cp\u003eMicroalgal biofilm formation and their applications\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8649144/v1/ee0f423eaae8d5ab602d1bf1.jpeg"},{"id":102745646,"identity":"916232ee-4720-475e-a207-746286fd1005","added_by":"auto","created_at":"2026-02-16 08:53:04","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127117,"visible":true,"origin":"","legend":"\u003cp\u003eIndustrial and biotechnological applications of wastewater treatment and pollutants removal\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8649144/v1/0feeb1e35fe993b00c1945f8.jpeg"},{"id":102443396,"identity":"c3d5f887-a1ce-4c32-950c-5c80034d2adf","added_by":"auto","created_at":"2026-02-11 17:19:51","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148699,"visible":true,"origin":"","legend":"\u003cp\u003eMicroalgal biofilm treatment of real wastewater treatment\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8649144/v1/31f8d3a51c3e27cf82377c58.jpeg"},{"id":102750583,"identity":"1a0757a6-8dff-4d3d-b9b9-115675ae4649","added_by":"auto","created_at":"2026-02-16 09:20:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1900953,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8649144/v1/cb74faae-ac29-451e-92b6-31c0ad947243.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Substrate dependent microalgal biofilm cultivation system for the blue and green economy: formation, mechanism, and applications in environmental engineering and biotechnology","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicroalgal biofilms in dominant and widespread distribution in nature composed of prokaryotic and eukaryotic microalgae (\u003cstrong\u003eFig 1\u003c/strong\u003e). The growth of these phototrophic biofilms is crucial for purifying the aquatic ecosystem and influencing external nutrient changes (Cheah and Chan 2021). Biofilm cultivation of algae is considered beneficial for eco-friendly technology because it is simply concentrated by scraping, which eliminates the costly concentrating techniques employed in suspension-based concentrating such as flocculation and centrifugation. The immobilized growth enhanced the biomass production through a higher absorb rate of solar energy and carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) fixation (Gross et al. 2015). This types of attached cultivation is a sustainable strategy over the conventional aqua-suspend or planktonic cultivation methods for dynamic ecosystems, autotrophic and heterotrophic components in the synthesis of high-value bio-products and WWT (Wang et al. 2017). In recent years, plentiful research has been accomplished on boosting the concentration of microalgal biomass for applications in agriculture, as well as for the production of bioenergy, bioremediation, and requiring less quantity of water (Singh et al. 2021; Masudi et al. 2023). However, harvesting rates for 20-30 % of the overall budget of protein mass production. By observing nature, energy savings in harvesting microalgae can be achieved by cultivating algae in biofilm (Ali et al. 2025).\u003c/p\u003e\n\u003cp\u003eIn natural, autotrophic microorganisms (microalgae and cyanobacteria), they commonly live on the artificial substrate or surface of a natural environment in the form of biofilms. This kind of algal mode of growth significantly produces EPS that can help to resist stress in photic aquatic environments (Ugya et al. 2020). Among these, attached growth has become popular for fuel applications because of its economic feasibility, production costs that are eight to ten times lesser than those of a liquid-based culturing method, an efficient and affordable way of immobilizing cells, and production of densely packed microalgal cells (Ennaceri et al. 2023). A biofilm-based growth system can be more efficient than conventional planktonic microalgae growth systems due to advantages such as increased increase to colonization, biomass thickening, barrier against toxins, and grazers (Miranda et al. 2017). Besides removing pollutants and increasing biomass, photosynthetic microorganisms also help remove of nutrients from industrial, domestic, and agricultural runoffs (\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;2\u003c/strong\u003e). Furthermore, MAB aids in the enhancement of algal recovery in WW and contributes to an ecological interest and a circular economy (Hu et al. 2021). Still, there are challenges in cultivating algal biofilm, optimizing growth under various environmental parameters of biofilm, and scaling up for industrial applications. Previous research showed that numerous parameters, including shear stress, thickness, attachment material properties, and the algal species, can strongly influence the development of biofilms (Mantzorou and Ververidis, 2019; Novovesk\u0026aacute; et al. 2023). The objective of this review article is to give a broad outline of MAB communities, industrial applications, and recent developments in MAB research. Different aspects of MAB, such as their development, interactions, bio-based products, and biotechnological applications, are covered.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Different microalgal biofilm cultivation systems utilized in the treatment of domestic wastewater (DWW)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWastewater\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNature of biofilm/ culture system\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProducts or activity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003esynthetic (modified BBM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cem\u003eScenedesmus vacuolatus\u003c/em\u003e and \u003cem\u003eC. vulgaris\u003c/em\u003e/PBR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eremoval of PO₄\u0026sup3;⁻, NO\u003csub\u003e3\u003c/sub\u003e-N, and nutrients 20-40 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e, 60-240 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 90%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eMoreno Osorio et al. 2019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003emunicipal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cem\u003eHalochlorella rubescens\u003c/em\u003e/Twin-Layer PBR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003emicroalgal growth of 6.3 g m\u003csup\u003e-2\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e, efficiently reduced the concentrations of P (70%) and N (99 %) in the effluents\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eShi et al. 2014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003eseawater-diluted anaerobically digested effluent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cem\u003eChlorella\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eSDEC-18/ inclined algal biofilm PBR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003ehigh biomass productivity -5.66 g m\u003csup\u003e-2\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e, removal of TP- 0.25 g m\u003csup\u003e-2\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e, TN - 0.65 g m\u003csup\u003e-2\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e, COD- 3.31 g m\u003csup\u003e-2\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eYu et al. 2020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003esynthetic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. obliquus\u003c/em\u003e, \u003cem\u003eC. vulgaris\u003c/em\u003e and \u003cem\u003eO. tenuis\u003c/em\u003e/ vertical-enhanced algal-biofilm raceway\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eTN, TP, and COD reduction of over 73.68%, 89.85%, and 86.61%, high biomass production (6.95 - 8.11 g m\u003csup\u003e-2\u003c/sup\u003e day\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eZhang et al. 2018a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003esynthetic municipal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003enon-axenic \u003cem\u003eC. sorokiniana\u0026nbsp;\u003c/em\u003eand \u003cem\u003eStaphylococcus\u003c/em\u003e sp. / polystyrene culturing plates\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eresidual N, P, and TOC concentrations were respectively 70.1 %, 50.3 %, and 32.3 %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eZhang et al. 2025a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003emunicipal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. vulgaris\u003c/em\u003e-microorganisms/ pilot-scale tubular PBR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eeffectively remove the carbon and nitrogen\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eLi et al. 2024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003esynthetic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003emicroalgae-biofilm anammox system/ up-flow anaerobic sludge bed reactor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eat 25 \u0026deg;C, TN and P were removed by 89.9 % and 94.2 % respectively\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eChen et al. 2024b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003elow-carbon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. vulgaris\u003c/em\u003e -activated sludge biofilm / continuous-flow algal-bacterial system\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e31.95% and 9.56% reduction in CO\u003csub\u003e2\u003c/sub\u003e emissions and an improvement in COD removal efficiency\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eWang et al. 2025c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003ehigh-saline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cem\u003eDunaliella salina\u003c/em\u003e/Microalgae biofilm PBR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eremoval efficiency of 91.0 %, 84.3 %, and 81.6 %, for PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026mdash;\u003c/sup\u003eP, COD, and TN\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eFan et al. 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Treatment of industrial wastewater using different microalgal biofilms cultivation systems\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWastewater\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNature of biofilm/ culture system\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProducts or activity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003ebrewery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eLeptolyngbya\u003c/em\u003e sp. and \u003cem\u003eChroococcus\u003c/em\u003e-like population/flat-plate PBR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003ehigh removal efficacy (over 65%) of the WW\u0026rsquo;s pollutants (total Kjeldahl nitrogen, orthophosphate, ammonium, nitrite, NO\u003csub\u003e3\u003c/sub\u003e-N, and COD), biomass 392and406 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e for polyurethane andglass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e(Papadopoulos et al. 2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003emarine aquaculture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eC\u003c/em\u003e\u003cem\u003e..\u0026nbsp;\u003c/em\u003e\u003cem\u003evulgaris\u003c/em\u003e /microalgae biofilm membrane PBR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003ebiomass productivity 22.03 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;1\u003c/sup\u003e, reduction in SMZ, sulfamethazine, sulfadiazine, dissolved inorganic P, and dissolved inorganic nitrogen were found to range from 91.0-99.6%, 60.8-82.1%, 50.0-76.7%, 61.0-79.2%, and 92.1-98.4%, correspondingly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003ePeng et al. 2020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003econtaminated petrochemical stream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003eMicroalgae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003ehigh reduction efficiency of turbidity (71%), sulfate (37.5%), alkalinity (62.5%), TSS (66.7%), Ni (74.0%), Cd (70.0%), \u0026nbsp;and Pb (71.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eUgya et al. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 2021b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003ehog manure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eC.\u0026nbsp;\u003c/em\u003e\u003cem\u003evulgaris\u003c/em\u003e/ algal biofilm PBR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003ehigh (7.37 g m\u003csup\u003e-2\u003c/sup\u003e) biomass production and removal rates of the NH\u003csub\u003e4\u003c/sub\u003e\u0026ndash;N (91.24%), TN + (69.55%), COD (95.67%), and TP (64.40%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eWu et al. 2019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003eP-enriched nickel (Ni) refinery tailings\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003emicroalga/pertri dish biofilm system\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003ehigh total carbohydrates (40.0%), increased lipids (6.7% - 19.5%), and removal of heavy metals (strontium, cobalt, manganese, Ni) from the WW with 26.4%, 10.5%, 24.8% and 24.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003ePalma et al. 2017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003eswine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003emicroalgal-bacterial\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003eCOD, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, and TP removal efficiency 99, 92, and 98 %, respectively, high content (63 %) of fatty acids (C18:2n6c, C18:1n9c, C18:0 and C16:0 ) appropriate for biofuel generation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eZhang et al. 2024b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003erecirculating aquaculture system effluent water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003ebiomass production of 15.28 g m\u003csup\u003e\u0026minus;2\u003c/sup\u003e d\u003csup\u003e\u0026minus;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eB\u0026ouml;pple et al. 2024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003elandfill leachate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003ecathodic algal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e76.7% of TN, 89.4% of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, and 86% of COD removal; produced the highest biomass and voltage production of 1.23 and g L\u003csup\u003e\u0026minus;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;1\u0026nbsp;\u003c/sup\u003eof 0.39 V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eElmaadawy et al. 2020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003emarine AWW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eChlorella\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003eTOC, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e removal efficiencies up to 80%, 96%, 99%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eZhang et al. 2024c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"2. Microalgal biofilm derived from nature","content":"\u003cp\u003eIn aquatic ecosystems, algae epiphytic biofilms (submerged macrophytes) play a significant contributor in biogeochemical cycles, migration, and transformation of contaminants. The biofilms are important for influencing ecological function, community composition, diversity, and microbial abundance in the marine and freshwater environments (Xia et al. 2020). \u003cem\u003eChlorella sorokiniana\u003c/em\u003e exhausted the total available ammonia nitrogen (NH₄⁺-N) by excluding it under low nutrient conditions, which promoted the growth of \u003cem\u003eScenedesmus pectinatus\u003c/em\u003e between algal and bacterial members in high-rate algal ponds (Krichen et al. 2019). The formation of \u003cem\u003eHaematococcus lacustris\u003c/em\u003e biofilms in coastal rock ponds in Kandalaksha Bay of the White Sea is a natural phenomenon. Microalgae have adapted and formed dry biofilms to cope with the highly adverse stressful conditions, and preserving the ecosystem of the supralittoral zone of Subarctic and Arctic seas (Kublanovskaya et al. 2020). This kind of association between heterotrophic bacteria and microalgae biofilms in a metal-contaminated aquatic habitat helps to provide supportive metabolites for the bacterial growth and mediates the remediation of acid mine drainage (Abinandan et al. 2017). In benthic ecosystems, \u003cem\u003ePhormidium autumnale\u003c/em\u003e frequently grows as biofilms at a site on the Hutt River (New Zealand). These microalgae-bacterial communities dominated by cyanobacterial blooms are causing increased neurotoxin production (Brasell et al. 2015).\u003c/p\u003e\n\u003cp\u003eMicroflora and micro-meiofauna in aquatic biofilms influence the provision of food resources, primary production, and nutrient cycling in rivers. Ingestion of a high amount of microalgae by micro-meiofauna significantly impacts the qualitative and quantitative composition of periphyton (Neury-Ormanni et al. 2016). Diatoms frequently develop biofilm on plastics in the aphotic and photic zones of a freshwater reservoir, but with subsequent growth in the environment, they fragment plastic and generate microplastics (Smith et al. 2021). Exposure of nickel and temperature to a biofilm dominated by natural green algae and cyanobacteria cultured in mesocosms were found to increase a fatty acid synthesis, which can be an indicator of environmental anxiety (Fadhlaoui et al. 2020). Altered bacterial communities, microbial respiration, and algal growth in stream biofilms are inhibited by pharmaceutical compounds in surface waters. In the combination or alone chemicals suppressed the biofilm respiration affecting higher trophic levels and important ecosystem processes (Rosi-Marshall et al. 2013).\u003c/p\u003e\n\u003cp\u003eSolid mats are formed in the lake in Chungcheongnam-do by the dominant \u003cem\u003eHalomicronema\u003c/em\u003e sp and \u003cem\u003eChlamydomonas\u003c/em\u003e sp. These ecological biomimetic MAB mats are formed by the attachment of microalgae to EPS produced by biofilm algal mats that mimic the natural algal mat system (Lee et al. 2023). \u003cem\u003eOscillatoria\u003c/em\u003e sp., \u003cem\u003eLeptolyngbya\u003c/em\u003e sp., and \u003cem\u003eChroococcus\u003c/em\u003e sp., were intertidal cyanobacterial biofilms from the Sundarbans mangrove forest and can be utilized to produce maximum chlorophyll \u003cem\u003ea\u003c/em\u003e, EPS, and bioactive metabolites (Veerabadhran et al. 2018). Copper (Cu) plays important roles such as reducing microalgal biomass, shifting the population from diatoms to cyanobacteria, and changing the grazing activity in artificial stream channels (Barranguet et al. 2003). Thorium (Th) exposed to periphytic biofilm was collected from Cap Rouge River and cultivated in a laboratory. This Th can modify the diatom community, resulting in a significant decrease in \u003cem\u003eShingopyxis\u003c/em\u003e and \u003cem\u003ePseudanabaena\u003c/em\u003e genera (Doose et al. 2021). Municipal wastewater (MWW) on the periphyton biofilms and influences the both prokaryotic and eukaryotic communities. The biofilm is made up of a significant amount of benthic microalgae, but there are low levels of parasites (Blifernez-Klassen et al. 2023). Microalgal colonization on plastic surfaces was observed in a field of lentic biospheres containing diverse environmental conditions (e.g., macrophyte coverage, conductivity, and nutrient concentration) and a broad geographical gradient. Polyethylene terephthalate showed higher biomass attachment than high-density polyethylene as plastic substrate surfaces (Nava et al. 2022).\u003c/p\u003e"},{"header":"3. Microalgal biofilms","content":"\u003cp\u003eMAB is generally stated to as photosynthetic or autotrophic, and it consists of dominant microalgae, bacteria, and axenic culture (Wang et al. 2022a). However, various biofilm communities have been reported for varying growth and harvesting conditions. Primary productivity by photosynthesis was observed high on the surface of biofilms and proportional to the intensity of light and CO\u003csub\u003e2\u003c/sub\u003e concentration in MABs (Mkpuma et al. 2023). MABs growth and appropriate biomass production depend on appropriate surface in aquatic ecosystems (\u003cstrong\u003eFig 2\u003c/strong\u003e). The suitable substrate accounts for cultivation and affirms the relative abundance and microbial community properties (Kim et al. 2014). Biofilm aggregation and surface attachment in the substratum are addressed to the surface area of carriers. Concentrated biomass yields are maximized due to the design and material optimization of the carriers on an industrial scale (Berner et al. 2014). Attached biofilms are formed by the agglomeration of planktonic cells on a carrier material. Suspended biofilms that grow without attachment material or are detached, are called flocs or granules. The mode of biofilm growth is dependent on different internal gradients of environmental and nutritional conditions (Aqeel et al. 2019). Development of microalgae biofilms is based on different parameters such as substrates, light intensity, bioreactor configuration, biocarriers, and operating conditions such as hydraulic retention time (HRT) and shear stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1. Operating conditions and formation mechanisms of biofilm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMAB is a type of green microalgae or cyanobacteria that can be rendered immobile through artificial or natural techniques. (1) Adsorption, which is the passive immobilization of algae on carrier materials, and (2) microalgal cell entrapment, which is the active immobilization of algae by chemical attachment, flocculant agents, or gel entrapment (Moreno Osorio et al. 2021). Various factors including hydrophobicity, pH, and hydrophilicity, surface properties of the substrate, photoperiod, and light intensity influence the growth of attached microalgal species (Rosmahadi et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStainless steel acts carrier for adhering to the unicellular cyanobacteria \u003cem\u003eMicrocystis aeruginosa\u003c/em\u003e, on a hydrophilic surface, and nutrient stress that triggers biofilm formation (Barros et al. 2019). \u003cem\u003ePseudochlorococcum\u003c/em\u003e sp. is known to accumulate in cellulose nitrate/cellulose acetate (CA) membranes, and high light intensity plays a part in the faster growth of biofilm (Ji et al. 2014). \u003cem\u003eChlorella vulgaris\u0026nbsp;\u003c/em\u003eplanktonic growth can be shifted into sessile growth in batch and continuous cultivation systems due to non-sterile conditions (Irving and Allen 2011). The biofilms of \u003cem\u003eScenedesmus obliquus\u003c/em\u003e were more attached to V-grooves than U-grooves. High shear stress resistance was observed on U-grooved surfaces. MABs showed resistance to hydraulic shear stress and a high surface area, which indicates the significance of microgrooves in biofilm attachment from 14.29% to 165.84 g m\u003csup\u003e\u0026minus;2\u003c/sup\u003e on the surface (Huang et al. 2018). A temperature-responsive gel material on the inner surface of the 3D porous substrate (N-isopropylacrylamide) with a cooling temperature enhances the detachment capability of \u003cem\u003eC. sorokiniana\u003c/em\u003e compared to high temperatures for developing the harvesting technology. Growth of microalgal species and secretion of EPS improves biofilm adhesion and reduces the cell detachment of unicellular microalgae (Wang et al. 2024a). \u003cem\u003eC. vulgaris\u003c/em\u003e attached cultivation was favored on capillary ceramic membrane substrate surfaces, increasing biomass productivity more than other attached cultivation methods. Centrifugation resulted in a biomass harvest equal to the moisture content of the biofilm (Dalirian et al. 2021). Polyvinyl chloride substrate is responsible for the biofilm yield and maximum adhesion of \u003cem\u003eC. vulgaris\u003c/em\u003e, succeeded by polypropylene, polyethylene, and stainless steel in a rotating flat plate photobioreactor (PBR) (Melo et al. 2018). The growth of unicellular and filamentous microalgae, attachment, and biomass productivity are influenced by membrane and fabric fiber substrates. The biomass production of \u003cem\u003eTribonema minus\u003c/em\u003e and \u003cem\u003eScenedesmus dimorphus\u003c/em\u003e increased by 7.81 and 10.24 g m\u003csup\u003e-2\u003c/sup\u003e day\u003csup\u003e-1\u003c/sup\u003e with the use of nitrate cellulose/CA membrane (Ji et al. 2023). The rough surfaces on the 30 kDa polyethersulfone membrane enhance the higher degree of cell attachment for benthic diatoms \u003cem\u003eCylindrotheca fusiformis\u003c/em\u003e, followed by \u003cem\u003eAmphora coffeaeformis\u003c/em\u003e and \u003cem\u003eNavicula incerta\u003c/em\u003e (Tong and Derek 2022a).\u003c/p\u003e\n\u003cp\u003eOn\u0026nbsp;the\u0026nbsp;contrary,\u0026nbsp;biofilm\u0026nbsp;reactors\u0026nbsp;based\u0026nbsp;on\u0026nbsp;carriers\u0026nbsp;or\u0026nbsp;attachments reveal\u0026nbsp;the\u0026nbsp;requirement\u0026nbsp;for\u0026nbsp;less\u0026nbsp;energy\u0026nbsp;and\u0026nbsp;ease\u0026nbsp;of\u0026nbsp;biomass\u0026nbsp;harvesting (Hoh et al. 2016). Pine saw dust was proven to have better MAB production (10.92 g m\u003csup\u003e\u0026minus;2\u003c/sup\u003eday\u003csup\u003e\u0026minus;1\u003c/sup\u003e after 16 days of cultivation) in a cost-effective manner. Other lignocellulosic materials like oak saw dust, sugarcane bagasse and rice husk did not perform optimally. Pine saw dust showed to be effective for \u003cem\u003eS. obliquus\u003c/em\u003e, \u003cem\u003eC. vulgaris\u003c/em\u003e, and \u003cem\u003eOscillatoria tenuis\u003c/em\u003e (Zhang et al. 2017). Cotton coated carriers showed greater protein synthesis than uncoated carriers due to nutrient absorption and immobilization. \u003cem\u003eScenedesmus\u0026nbsp;\u003c/em\u003eLX1 and associated EPS coating with cotton showed prominent results (Zhuang et al. 2016). \u003cem\u003eC. vulgaris\u003c/em\u003e attachment is largely influenced by the chemical functional groups, micro-texture, and hydrophobicity. Cells cultured on Terrazzo had a higher carbohydrate pool, photosynthetic capacity (1.32-fold), and biomass productivity (3.20-fold) than those grown on cotton (Li et al. 2023). Walnut shell, a cheap substrate, is responsible for the cultivation of \u003cem\u003eC. vulgaris\u003c/em\u003e and \u003cem\u003eS. obliquus\u003c/em\u003e biofilm in a self-permeating biofilm reactor. Microalgae\u0026apos;s biofilm production increased when the CO\u003csub\u003e2\u003c/sub\u003e concentration was 2%, the walnut shell size was 0.8-1.2 mm, and the light intensity was 150 mmol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e (Zouet al. 2021). Pine sawdust was used as a biocarrier to accelerate the biofilm growth and energy conversion properties of \u003cem\u003eDiplosphaera\u003c/em\u003e sp. Furthermore, rick husk and sugarcane bagasse leachate inhibited the growth of \u003cem\u003eC. vulgaris\u003c/em\u003e while also increasing the methyl ester content of saturated fatty acids and modifying the chemical compositions of algal cells (Zhang et al. 2019).\u003c/p\u003e\n\u003cp\u003eThe production of \u003cem\u003eC. sorokiniana\u003c/em\u003e biofilm is highly influenced by the efficiency of CO\u003csub\u003e2\u003c/sub\u003e utilization. The biofilm biomass was increased by 96% of the flue gas CO\u003csub\u003e2\u003c/sub\u003e usage (Blankenet al. 2017). The rotating algal biofilm cultivating system proved effective in accelerating the mixing of nutrients and aeration. Thus, MA biofilms showed augmented growth and were easy to harvest (Gross et al. 2013). The arrayed attached culture systems in the MAB were enhanced to expand their utilization from the laboratory to outdoor systems (Huang et al. 2021). In contrast to the other bubbling conditions, the application of a bubble (1.4 mm diameter) to \u003cem\u003eC. vulgaris\u003c/em\u003e cultivation in the thin-gap bubble column PBRs led to limited biofilm development, constrained flow, and low culture thickness (Thobie et al. 2022). Rotating biological contactor-based PBRs are more suitable for cultivation of \u003cem\u003eC. sorokiniana\u003c/em\u003e because the algadisk is partially submerged in a growth medium for biofilm formation. Productivity and biomass yield of microalgae have been improved in the lab-scale algadisk reactor (Blanken et al. 2014). \u003cem\u003eAnkistrodesmus falcatus\u0026nbsp;\u003c/em\u003ewith a long stripe with \u003cem\u003eS. obliquus\u003c/em\u003e at a volume ratio of 1-4 resulted in a shortening the inoculation time of the biofilm by bridging and twining involved in the co-flocculation microalgae cells (Zheng et al. 2019). The wall shear stress during the cultivation of\u003cem\u003e\u0026nbsp;C. vulgaris\u003c/em\u003e biofilm with 6 Pa did not disturb stability and helped to choose the operational settings of the MAB cultivation systems (Belohlav et al. 2020). \u0026nbsp;\u003cem\u003eChlorella pyrenoidosa\u003c/em\u003e, \u003cem\u003eChlorella\u003c/em\u003e sp., and \u003cem\u003eNannochloris oculata\u003c/em\u003e biofilm structure was affected by surface energy (SE). Furthermore, the cells formed a biofilm that was flat and homogeneous, with a porosity below 20% when the cell SEs were 50 to 65 mJ m\u003csup\u003e-2\u003c/sup\u003e (Zhang et al. 2020a).\u003c/p\u003e\n\u003cp\u003eThe white light influences the structure of \u003cem\u003eS. obliquus\u003c/em\u003e biofilm, which is a heterogeneous biofilm with roughness, many voids, and high porosity (Yuan et al. 2021). A light intensity of 300 and 100 \u0026mu;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;1\u003c/sup\u003e developed a biofilm with uniform cell distribution, which contributed to the high final cell density of \u003cem\u003eC. vulgaris\u003c/em\u003e (Gao et al. 2024). Flashing light is a key parameter that regulates photosynthetic activity of a mixture of chlorophyceae dominated by \u003cem\u003eScenedesmus\u0026nbsp;\u003c/em\u003espp. and \u003cem\u003eChlorella\u003c/em\u003e spp. A 10 kHz light flash at a 5% light fraction resulted in a seven fold increase in the photosynthetic activity of microalgae biofilm compared to continuous exposure (51 \u0026micro;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e) (Mart\u0026iacute;n-Girela et al. 2017). With a high light intensity at a nitrate (NO\u003csub\u003e3\u003c/sub\u003e-N) concentration of 2 mg L\u003csup\u003e-1\u003c/sup\u003e, \u003cem\u003eC. pyrenoidosa\u003c/em\u003e resulted in a granular and very rough biofilm morphology, while at low light intensities, the biofilm was smooth and flat (Luo and Long 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. The function of EPS and signaling molecules in formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mode of growth of the biofilm has a substantial effect on the production of concentrated biomass, with the largest productivity of \u003cem\u003eChlorella\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e in the industry, which is the linear increase with culture time (Zhuang et al. 2018). The cyanobacterium \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803, a Type IV pili, is likely mediating more effective cellular aggregation than Wza dependent EPS and the S-layer in wild type, as determined by mutational analysis (Allen et al. 2019). Microalgae biofilm that generates tryptophan-like protein has a significant impact on the production of loosely bound EPS (LB-EPS) and soluble EPS. The composition of EPS and molecule weight decreased hydrophobic alkanes and recalcitrant aromatics were responsible for the mitigation of the membrane fouling (Liu et al. 2024a). The biofilm was developed by the indigenous microalgal-bacterial consortium by using high light intensity. A tightly interconnected EPS matrix was formed by exoprotein annotation and exopolysaccharide chains, and the structural stability of the biofilm landscape was significantly enhanced by increased branding degree, width, and height (Chen et al. 2024a). Algal-bacterial biofilms were developed by adding microplastic particles with sizes ranging from 0.065 \u0026mu;m to 5 \u0026mu;m. The biofilm was damaged, rough and loose, and the maximum algal recovery was achieved using microplastic particles with a size of 5 \u0026mu;m. Protein-rich EPS can be secreted by biofilm to protect it from the stress of microplastic particles (Gong et al. 2023). \u003cem\u003eNovosphingobium\u003c/em\u003e sp. EPS had an advantage over \u003cem\u003eM. aeruginosa\u003c/em\u003e in enhancing the faster growth of cyanobacteria. Exogenous aggregated substances, which are responsible for affecting biofilm formation, alter EPS composition ratios, and the production of EPS (Wang et al. 2022b).\u003c/p\u003e\n\u003cp\u003eMarine microalgae biofilm adhesion is enhanced, hydraulic resistance is improved, and hydrophobic protein levels are raised by pre-coating modified microporous membranes with EPS (Tong et al. 2023). The accumulation of proline and alanine contents in EPS when cultivated under a glycerol carbon source helps algae to enhance attached biomass and biofilm formation of the \u003cem\u003eC. pyrenoidosa\u003c/em\u003e by increasing hydrophobicity (Qian et al. 2023). Submerged commercial polyvinylidene fluoride (PVF) was more colonized by \u003cem\u003eA. coffeaeformis\u003c/em\u003e and \u003cem\u003eN. incerta\u003c/em\u003e than by \u003cem\u003eC. fusiformis\u003c/em\u003e. The adhesion of algae to form biofilm is influenced by the ratio of polysaccharide to protein (Tong and Derek, 2022b). Li et al (2021) found that increasing the released polysaccharide in the cyanobacterium \u003cem\u003eSynechocystis\u003c/em\u003e sp. can inhabit biofilm formation in cells. Cell hydrophobicity is reduced in the mutants (\u0026Delta;slr1076, \u0026Delta;sll5043 and \u0026Delta;slr1063) cells treated by the wild-type RPS. Cyanobacteria\u0026rsquo;s aggregation was changed by the physicochemical properties of RPS, cell surface charge, and cell hydrophobicity. The metal-ion-binding protein and algal cell-adhesion molecules promote the cell adhesion, self-flocculation, and bridging of \u003cem\u003eChlorella\u003c/em\u003e sp. The addition increased the abundance and distribution of hydrophilic \u0026alpha;-helix in the EPS matrix, which facilitates microalgal separation from MWW (Chen et al. 2021). Soluble extrapolymeric substances-coated microporous PVF membranes, marine diatom \u003cem\u003eN. incerta\u003c/em\u003e had an increase in cell adhesion due to a surface roughness and hydrophobicity (Tonget al. 2023).\u003c/p\u003e\n\u003cp\u003eSignaling molecules can respond to the growth of microalgae or bacteria in biofilms by activating N-acyl-homoserine lactones (AHLs), which are characterized by their facilitation of ecological reconfiguration and frequent interspecies communication towards biofilm growth. The regulation of algal-bacterial symbiosis in the biofilm is affected by dominant genera such as \u003cem\u003eCandidatus\u003c/em\u003e_Brocadia, \u003cem\u003eNitrosomonas\u003c/em\u003e, and \u003cem\u003eGeitlerinema\u003c/em\u003e (Liu et al. 2025). \u003cem\u003eC. vulgaris\u003c/em\u003e cultivated in AHL supplemented media modulates the surface electron donor of the cells. Quorum sensing of AHL-like substances in the algae varied the protein secondary structure, amino acid composition proteins in EPS, and extracellular protein secretion, contributing to the adhesion of microalgal cells (Ou et al. 2023). \u003cem\u003eCylindrotheca\u003c/em\u003e sp. grown in AHLs supplemented f/2 medium, the diatoms showed a significantly higher cell growth and EPS secretion. The thickness of biofilms and the length of carbon chains increased after the addition of AHL (Yang et al. 2023). The \u003cem\u003eCylindrotheca\u003c/em\u003e sp. grown with bacterial signaling molecules of AHLs (3-OH-C10-HSL, C10-HSL and 3-OXO-C10-HSL,) was revealed. The fouling diatom-biofilm growing under AHL has promoted Ca\u003csup\u003e2+\u003c/sup\u003e efflux, which has a positive effect on the formation of biofilm (Yang et al. 2016). The addition of exogenous C8-HSL enhanced the production of EPS, contributing to the formation of biofilm and the efficiency of phosphorus (P) and nitrogen (N) removal of heterotrophic nitrification-aerobic denitrification bacteria and the \u003cem\u003eChlorella\u003c/em\u003e symbiotic system (Qin et al. 2025). Exposure of the low concentrations (0.1 mg L\u003csup\u003e-1\u003c/sup\u003e) of indole-3-acetic acid (IAA) effects has an impact on the adhesion of \u003cem\u003eC. vulgaris\u003c/em\u003e biofilm due to its influence on electron donor properties. IAA upregulates genes that are involved in the Calvin cycle, secretes protein, and promotes the production of hydrophobic amino acids from MABs (Xie et al. 2024).\u003c/p\u003e\n\u003cp\u003eTightly-bound EPS (TB-EPS) and LB-EPS increased P and N removal under semi-continuous operation at a HRT of 2 days. Soluble-EPS, has the capability to favor the growth of biofilms and the synthesis of chlorophyll in \u003cem\u003eC. sorokiniana\u003c/em\u003e (Zhang et al. 2025b). The concentration of c-di-GMP is correlated with the dominant genera \u003cem\u003eBrevundimonas\u003c/em\u003e, \u003cem\u003eDevosia\u003c/em\u003e, and \u003cem\u003eComamonas\u003c/em\u003e as evidenced by research. The dominant bacteria have the capability to modulate microalgal-bacterial biofilm maturation when subjected to continuous lighting (24 hours) conditions (Wang et al. 2025a). \u003cem\u003eC. vulgaris\u003c/em\u003e biofilms attachment is greatly promoted by multiple applications of low concentration IAA than by a single high-concentration application. The hydrophobic amino acid pathway has been upregulated by the low level of IAA\u0026apos;s concentration, resulting in a reduction in the energy barrier between the receptive surface and the microalgae (Tang et al. 2026). The genes \u003cem\u003ewza1\u003c/em\u003e, \u003cem\u003ewzt\u003c/em\u003e, luxR-05665, TetR/AcrR and LysR are responsible for the production of EPS and the formation of biofilm in the cyanobacterium \u003cem\u003eHalomicronema\u003c/em\u003e sp (Caldara et al. 2025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Strategies for improved formation of MAB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell-surface properties induce the productivity of biofilms, and a higher microalgal biofilm formation is notably correlated with the surface hydrophobicity (Roostaei et al. 2018). To successfully form biofilm, it is essential to maintain the surface tension of WW to promote the growth of unicellular microalgae \u003cem\u003eChlorella\u003c/em\u003e sp. The surface tension of the liquid medium in the community increased, resulting in the formation of biofilm (Zhang et al. 2018b). The growth of the hybrid microalgae biofilm system was supported by a moderate NH₄⁺-N concentration of 25 mg L\u003csup\u003e-1\u003c/sup\u003e. Furthermore, filamentous algae formed a web-like structure and attached to the carrier, attracting bacteria and non-filamentous algae to promote the development of biofilm. The high molecular weight, tryptophan, N \u0026equiv; N functional group, and protein of EPS initiated the colonization (Liu et al. 2023). \u003cem\u003eC. vulgaris\u003c/em\u003e- \u003cem\u003eEscherichia coli\u003c/em\u003e biofilm growth was enhanced by bio-coating derived microalgals on hydrophilic PVF membranes. Stimulating the extra-and intra-organic metabolites as a defensive response by increasing the deposition of organic substances in the bio-coating cultivation (Tong et al. 2024). The biofilm attached cultivation of \u003cem\u003eChromochloris zofingiensis\u003c/em\u003e and mixotrophic conditions was attained higher biomass productivity than the suspended mode of cultivation. The cultivation strategy is accountable for the elevated accumulation of chemical substances, larger cell size, a considerably greater energy supply efficiency, and an upregulated carbon fixation pathway (Liu et al. 2024b).\u003c/p\u003e\n\u003cp\u003eThe polyethylenimine -crosslinked PVF membrane with a higher pattern height is capable of speeding up biofilm development and biomass accumulation. A low-shear area was created in the biofilm of the membrane due to an increase in the active area for microalgal attachment (Zhao et al. 2021). The yield of \u003cem\u003eS. obliquus\u003c/em\u003e- \u003cem\u003eC. pyrenoidosa\u0026nbsp;\u003c/em\u003ecocultured biofilm biomass is enhanced by uniform microstructure and small cell clusters. It is a reliable selection of microalgae species for manipulating biofilm microstructures and boosting yields as contrasted to other cultivation methods (Wang et al. 2024b). Attachment is facilitated and is ten times higher than the initial cell adhesion degree of \u003cem\u003eN. incerta\u003c/em\u003e, \u003cem\u003eA. coffeaeformis\u003c/em\u003e, and \u003cem\u003eC. fusiformis\u003c/em\u003e when EPS derived from microalgae is pre-deposited as a thin conditioning layer on microporous PVF membranes with a higher cell finding affinity (Tong et al. 2022). Sugarcane bagasse and pine sawdust were used as a biocarriers to accelerate medium holding capacity and Brunauer-Emmett-Teller specific surface area and biofilm productivity of \u003cem\u003eChlorella\u003c/em\u003e sp. biofilm. Furthermore, to enhance algal biomass production, it is necessary to select a laying quantity of various biocarriers and a mixture of particle sizes (Lu et al. 2025). The biofilm formation and adhesion of microalgae cells to the roughness and materials of membrane surfaces effectively control the development of \u003cem\u003eC. vulgaris\u003c/em\u003e biofilm. This strategy increases the amount of biofilm and cell adhesion by reducing the synthesis of EPS and salt bridging or charge neutralization mechanisms of the zeta potential of membranes (Liao et al. 2023).\u003c/p\u003e"},{"header":"4. Biotechnological applications ","content":"\u003cp\u003eImmobilized microalgae\u0026nbsp;have\u0026nbsp;been\u0026nbsp;effectively\u0026nbsp;employed\u0026nbsp;to accelerate treatment efficiency, reduce carbon footprint, and improve commercial value in the agricultural and chemical industries (\u003cstrong\u003eFig 3\u003c/strong\u003e). Also, it is preferred to upgrade sustainable biological WWT systems and remediate the aqueous environment (Han et al. 2023). The \u003cem\u003eScenedesmus\u003c/em\u003e biofilm growth is very more effective for biodiesel, lipid, biomass, and cell density generation than suspension growth (Bagheri et al. 2024). The use of biofilm reactors and high-rate algal ponds is considered a sustainable way to efficiently treat WW from domestic and fruit-based juice production industries (Abrantes Silva et al. 2024). Biofilm-based microalgae are also improving in clam larvae\u0026rsquo;s survival and settlement rates (Azirar et al. 2025). Artificial, non-sterile, MABs are also promising for the generation of biofuels, valuable biomass, biochemical, and photosynthesis carbon capture (Paquette et al. 2020). The Microalgal-bacterial biofilm removes or recovers the pollutants, sequesters CO\u003csub\u003e2\u003c/sub\u003e, and cost-effectively separates biomass and water, and synthesizes value-added products through use of natural sunlight. In comparison, the microalgae-rich biofilm mode of growth resists sudden substantial changes in pollutants concentration and helps them achieve the economic benefits of WWT (Zhang et al. 2024a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1. Fine chemicals and biomass production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe marine phototrophic dinoflagellate \u003cem\u003eSymbiodinium voratum\u003c/em\u003e was immobilized on a Twin-Layer PBR in order to increase the production of the anticancerogenic carotenoid peridinin by 24 mg m\u0026minus;2 day\u0026minus;1 at a light intensity of 74 \u0026mu;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;1\u003c/sup\u003e (Benstein et al. 2014). The biomass production from Revolving \u003cem\u003eC. vulgaris\u003c/em\u003e biofilm cultivation system increased 302% than standard raceway pond. The biofilm was produced 18.9 g m\u003csup\u003e-2\u003c/sup\u003e day\u003csup\u003e-1\u003c/sup\u003e as the efficient cultivation system for commercial scale (Gross and Wen 2014). The MAB system has been utilized to successfully remediate WW, remove heavy metals, and provide a promising source of metabolites for medicinal product synthesis (Ugya et al. 2024). The cultivation of \u003cem\u003eC. vulgaris\u003c/em\u003e biofilm involves the use of urea and glycerol as N and carbon sources.\u003cem\u003e\u0026nbsp;\u003c/em\u003eIn order to produce lipids that comprise a considerable amount of polyunsaturated fatty acids, which grow better in extremely low light and use less water (Rincon et al. 2017). Biofilm was cultivated by heterotrophic-assisted photoautotrophic biofilm (HAPB) growth mode to examine the influence on biomass and lipid generation. \u003cem\u003eC. vulgaris\u003c/em\u003e formed HAPB with the maximum lipid content (120%) and biomass (78%) at a ratio of 1: 20 total organic carbon (TOC) (glucose) to total inorganic carbon (CO\u003csub\u003e2\u003c/sub\u003e) and 72:1 total carbon to total N (Ye et al. 2018). \u003cem\u003eS. quadricauda\u003c/em\u003e biofilm has been cultivated in synthetic MWW and achieved a significant increase in biomass productivity of 9.11 g m\u003csup\u003e\u0026minus;2\u003c/sup\u003e d\u003csup\u003e\u0026minus;1\u003c/sup\u003e than other biofilm systems. The lipids ranged from 25.14 to 30.40 %, with a superior quality of biodiesel and a higher percentage of C18:1 content (Xu et al. 2025). Attached biofilm was produced by the inoculation of microalgae in multi-layer PBRs. The productivity and quality of lipid production increased in the biofilm of \u003cem\u003eBotryococcus braunii\u003c/em\u003e by 42.3 % - 51.3 %, and by 48.17 % -55.44 % of hexadecanoic acid (16:0), respectively (Shen et al. 2015). Similarly, by growing \u003cem\u003eS. obliquus\u003c/em\u003e in nickel foam using the modified surface as the carrier, the protein synthesis rate of biofilm, protein concentration, and CO\u003csub\u003e2\u003c/sub\u003e biofixation rate were induced in the range of 43.11 g m\u003csup\u003e\u0026minus;3\u003c/sup\u003e h\u003csup\u003e\u0026minus;1\u003c/sup\u003e, 0.892 g L\u003csup\u003e\u0026minus;1\u003c/sup\u003e, and 4465.6 \u0026micro;mol m\u003csup\u003e\u0026minus;3\u003c/sup\u003e s\u003csup\u003e\u0026minus;1\u003c/sup\u003e, correspondingly. (Guo et al. 2018). The quality and biosynthesis of polyhydroxyalkanoate are enhanced by salinity stress, which alters the bioavailability and precipitation of P, triggering in sole microbial groups in the microalgae biofilm. In the biofilms of salinity stress, there was a boost in photosystem II and metabolic flux towards carbon storage in the system due to the alterations in water chemistry (Ugya et al. 2025). The stratified microalgal-bacterial biofilm experienced a rise in CO\u003csub\u003e2\u003c/sub\u003e fixation as a result of biomass production. The SMBB cultivation system enhances photosynthesis activity, total EPS, protein and polysaccharide concentrations over hybrid microalgal-bacterial biofilm (Li et al. 2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. Carbon and nutrient removal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMAB cultivation integrated with bioreactors ensures efficient utilization of the nutrients from municipal or agricultural WW and high biomass production (Ugya et al. 2023). N and P input into the aquatic ecosystem causes harmful algal blooms and eutrophication (Lan et al. 2024). The reactive oxygen species generated by freshwater microalgae in the biofilm can remove turbidity, NO\u003csub\u003e3\u003c/sub\u003e-N, phosphate (PO₄\u0026sup3;⁻), and zinc (Zn) from petroleum-contaminated water. The absorption efficiency of the EPS enhances the formation of microalgae biofilm through larger surface areas and microspores present in the cells (Ugya et al. 2021a). A method for removing microbe-derived dissolved organic nitrogen (mDON) using algae biofilm comprised cyanobacteria \u003cem\u003eLimnothrix\u003c/em\u003e and \u003cem\u003eKamptonema\u003c/em\u003e spp. has been revealed. The biofilm reduced the concentration of mDON formation in DON-free WW and fed with activated sludge effluent by 83% and 72%, respectively (Lin et al. 2021).\u003c/p\u003e\n\u003cp\u003eThe efficient reduction of ammonia (NH\u003csub\u003e3\u003c/sub\u003e) was achieved by the mycoalgae biofilm, which consists of \u003cem\u003eMucor indicus\u003c/em\u003e and \u003cem\u003eC. vulgaris\u003c/em\u003e, in simulated aquaculture wastewater (AWW). Biofilm developed under a range of NH\u003csub\u003e3\u003c/sub\u003e conditions (6.14 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e to 124 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e), with different compositions of fungi and algae (14.8 % to 0.5% algae) in the biofilm (Barnharst et al. 2023). Light irradiance is a significant parameter that influences the growth of microalgae in the phototrophic biofilms in outdoor large-scale cultivation and nutrient removal. A day time favored removal of N and P overnight time (Boelee et al. 2014). Mixed culture of MAB cultivation in rotating algae biofilm reactors has shown\u0026nbsp;advantages\u0026nbsp;over open\u0026nbsp;pond\u0026nbsp;lagoon\u0026nbsp;systems. Efficient removal of suspended solids, N, and P from petroleum refining WW is also important for biomass production (Hodges et al. 2017). The solid-surface culture system\u003cem\u003e\u0026nbsp;\u003c/em\u003efor \u003cem\u003eChlorella kessleri\u0026nbsp;\u003c/em\u003e- increases the cell densities, and CO\u003csub\u003e2\u003c/sub\u003e capture, respectively, by 120 mg chlorophyll m\u003csup\u003e\u0026minus;2\u003c/sup\u003e\u0026nbsp; and 110 tons ha\u003csup\u003e\u0026minus;1\u003c/sup\u003e year\u003csup\u003e\u0026minus;1\u003c/sup\u003e from 1/5 concentration liquid Gamborg\u0026apos;s B5 medium under continuous illumination (Miyauchi et al. 2020). Oxidation ponds were observed to face challenges in the removal of nutrients. Ponds contain hydrogen-fed indigenous bacterial-algal biofilm, which assists in the higher removal of 97.1 % for total phosphorus (TP) and 97.9 % for total nitrogen (TN) than control systems (Li et al. 2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3. Pollutant biodegradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. vulgaris\u003c/em\u003e exhibited high phenol degradation by increasing the metabolic activity and thickness of the biofilm (191\u0026minus;222 \u0026mu;m) (Zhong et al. 2019). Lowering the concentration of residual linear alkylbenzene sulfonates can be mitigated in aeration-free greywater by increasing the levels of density, highest biofilm concentration, and specific oxygen consumption and generation rates by adding an organic load. The ability of non-aeration \u003cem\u003eSynechocystis\u003c/em\u003e-bacteria biofilm to remove pollutants was investigated in a push-flow microalgal-bacteria biofilm reactor. A complete removal of pollutants and a reduction of dissolved organic nitrogen was achieved through high nitrogen assimilation by microalgae (Wu et al. 2023). Fresh water-derived biofilm of \u003cem\u003ePediastrum\u003c/em\u003e sp., \u003cem\u003eOscillatoria\u003c/em\u003e sp., \u003cem\u003eNitzschia\u003c/em\u003e sp., \u003cem\u003eCymbella\u003c/em\u003e sp., and \u003cem\u003eMerismopedia\u003c/em\u003e sp. demonstrated higher adsorption and absorption capabilities for removing bisphenol A (Vanniaraj et al. 2025). Utilizing synthetic wastewater (SWW) with polyacrylamide (PAM) to cultivate biofilm in revolving algae biofilm reactors will increase the removal rates of the PAM, the N source, TN, chemical oxygen demand (COD), and TOC concentration (Zhang et al. 2021). The symbiotic bacterial-algal-fungal biofilm in the moving bed bacterial-algal biofilm reactor system with a phenol degrader reduced the phenol content by 2.3-fold and converted NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N by 85 %, from phenolic-laden coal chemical WW. The biofilm had a higher EPS matrix and functional taxa (\u003cem\u003eFlavihumibacter\u003c/em\u003e, \u003cem\u003eHydrogenophaga\u003c/em\u003e, \u003cem\u003eFusarium\u003c/em\u003e) than conventional systems (Yang et al. 2025). Increased production of glutathione reductase, superoxide dismutase (SOD), catalase, and peroxidase caused the microalgae biofilm to degrade the phenol-containing textile WW, while the reactive oxygen species counteracted. The alkalinity (47%), chloride (52%), COD (83%), and biochemical oxygen demand (68%) may be removed by the biofilm activities of enzyme release or adsorption of the pollutants (Ugya 2021).\u003c/p\u003e\n\u003cp\u003eThe seawater \u003cem\u003eChlorella\u003c/em\u003e sp. biofilm was responsible for removing a significant amount of tetracycline (74.5-85.2 %), clarithromycin (60.8-69.5 %), and sulfamethoxazole (SMZ) (13.5-44.1 %) from the combined antibiotic mixture. Antibiotic removal from marine water has a potential to protect the blue economy from the persistent xenobiotics (Yu et al. 2024a). The production of aminopyrine N-demethylase (54%) and aniline hydroxylase (48%) in periphytic biofilm (\u003cem\u003eSerendipita\u003c/em\u003e, \u003cem\u003eThioalkalivibrio\u003c/em\u003e, \u003cem\u003eAzoarcus\u003c/em\u003e, \u003cem\u003eAcidibacter\u003c/em\u003e, \u003cem\u003eAchnanthidium\u003c/em\u003e, and \u003cem\u003eSellaphora\u003c/em\u003e) may aid \u003cem\u003eIris pseudacorus\u003c/em\u003e in increasing the expression of cytochrome P450 enzymes to alter or degrade the SMZ and doxylamine succinate from the urban WW (Yadav et al. 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4. Metal(loid) removal\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAxenic \u003cem\u003eNostoc muscorum\u003c/em\u003e\u0026apos;s biofilm was effective in removing cadmium (Cd) from a fresh medium containing 0.05 to 100 ppm cadmium chloride at pH 5-9. Sequestration mechanisms such as chemisorption and Langmuir\u0026apos;s adsorption isotherm confer on the removal of Cd(II) (Raghavan et al. 2020). Iron (Fe), Zn, and Cd were removed from mine effluent through binding and absorption mechanisms by green microalgae biofilms from with 85%, 95%, and 99%, respectively (Makhanya et al. 2021). Banana stem-based microalgae biofilm transformed hazardous arsenic (As) into less toxic arsenate and dimethylarsenic acid with an efficiency of 97.6% within 5 days of removal; this may be increased to 20% more than microalgae biofilm cultured on nylon mesh. Exposure of biofilm As increased the proteins and lipids which were also facilitated for the binding of As (Kumar et al. 2022). The green algae \u003cem\u003eStichococcus bacillaris\u003c/em\u003e produced more biofilm, thus removing 2-3 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e of Zn from real mine dump leachate and SWW, which contributed to biofilm resistance (Li et al. 2015). The effectiveness of \u003cem\u003eS. obliquus\u003c/em\u003e and \u003cem\u003eC. vulgaris\u003c/em\u003e in removing heavy metals is affected by mixed biofilm culture in aqueous medium. Mix culture efficiently increased the uptake of Fe (91.67-97.85%), NO\u003csub\u003e3\u003c/sub\u003e-N and PO₄\u0026sup3;⁻ (Yousefi et al. 2023). \u003cstrong\u003eTable 3\u003c/strong\u003e displays the removal of heavy metals by microalgal biofilm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Heavy metals removal from environments or laboratory conditions using immobilized or biofilm-forming microalgae\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNature of biofilm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eheavy metal removal/ activity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSources of Contaminated water/ culture medium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eChlorella\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eantimony, As, beryllium, Se, and Zn at concentrations of 0.07546, 0.05709, 0.09326, 0.4618, and 0.00979 mg L\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eDhiba port\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eAlhumairi et al. 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eP. autumnale\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNitzschia palea\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003ehigher production of EPS with protein-like polymers induced by metal 600 \u0026mu;g L\u003csup\u003e\u0026minus;1\u003c/sup\u003e of Zn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003evarious metal concentrations are present in BG-11 medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eLoustau et al. 2019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003ealgal-bacterial Biofilm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003e21.8 \u0026plusmn; 3.4 mg kg\u003csup\u003e-1\u003c/sup\u003e of selenium \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(Se) \u0026nbsp;was present in the biofilm fertilizer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eSe-rich AWW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eHan et al. 2020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003ePhormidium\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ebigranulatum\u003c/em\u003e-dominated mat ( \u003cem\u003eLyngbya\u003c/em\u003e sp. and \u003cem\u003eO.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003etenuis\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eremoved 80% to 94% Cu\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e10 to 100 \u0026micro;M Cu\u003csup\u003e2+\u003c/sup\u003e supplemented to BG-11 medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eKumar and Gaur 2014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eEuglena mutabilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003esamarium, europium, ytterbium, and lutetium adsorbed with an adsorption capacity of 0.035, 0.033, 0.033, and 0.031 mmol g\u003csup\u003e-1\u003c/sup\u003e through elevated EPS synthesis \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eAshbridges Bay Wastewater Treatment\u003c/p\u003e\n \u003cp\u003ePlant (WWTP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eZak et al. 2025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003eperiphytic microalgae on shells and glass\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003e\u0026nbsp;Cu (89%), lead (Pb) (97%), and \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Zn (97%), for mussel shells; Cu (73%), \u0026nbsp;Pb (96%), and Zn (86%) \u0026nbsp;for recycled glass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eheavy metal contained synthetic stormwater \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eBremner et al. 2020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003emicroalgae biofilm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eAs accumulation in fish muscle gets reduced by 41-63 % by increasing the organic arsenic species\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003efish exposed to As-contaminated water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eKumar et al. 2025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003ebiogenic aqua crust (\u003cem\u003eCytophagales\u003c/em\u003e sp., \u003cem\u003eHyphomonadaceae\u0026nbsp;\u003c/em\u003esp., and \u003cem\u003eLeptolyngbyaceae\u0026nbsp;\u003c/em\u003esp.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003ebiofilm formation (e.g. and LuxS, OmpR and CRP), genes encoding extracellular peptidase (e.g. family S1, S9) and CAZymes (e.g. GT2, CBM50), thus enhance the capability of metal (loid) removal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003etailing wetland\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eWang et al. 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. obliquus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003ehighest cadmium (II) removal rate is 91.27%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eCd 0, 1, 5, 10, 15, and 20 mg L\u003csup\u003e-1\u003c/sup\u003e in BG-11 medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eMa et al. 2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"5. Biofilm treatment of real wastewaters ","content":"\u003cp\u003eDifferent microalgal cultivation technologies such as PBRs, planktonic or suspended, mixotrophic, and heterotrophic cultivation methods have been conventionally used for treating WW (\u003cstrong\u003eFig 4\u003c/strong\u003e). The most common sewage treatment technologies include the planktonic cultivation technology (Daneshvar et al. 2019; Sathinathan et al. 2023). Some of the drawbacks of these technologies, for example a land requirements, biomass valorization, and low scattered density in the culture media, environmental and operational conditions are not addressed in the bottleneck of the suspended cultivation technology (Molinuevo-Salces et al. 2019). Because of its efficiency in ground usage and increasing harvest per ground area, carbon sequestration, producing potential therapeutic compounds, and WWT efficacy, biofilm is currently being evaluated as a feasible cultivation technology (Patwardhan et al. 2024). Increasing the P and N content while improving loading rates results in a significantly reduced internal N to P ratio of MWW effluent. MAB can increase the efficiency of removing residual NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e-P, and observe the maximum uptake capacity (Boelee et al. 2011). Cyanobacteria, coccal green algae and diatom biofilm cultivation in the municipal WW allows efficient P removal and maximum biomass production in horizontal flat panel PBR (Sukačov\u0026aacute; et al. 2015). \u003cem\u003eScenedesmus quadricauda\u003c/em\u003e, \u003cem\u003eChlorobia\u003c/em\u003e and \u003cem\u003eDeltaproteobacteria\u003c/em\u003e biofilm microbial fuel cells (MFC) were fed with DWW originating from the sewer. The microalgae and bacteria in the biofilm cells effectively remove COD, TN, and TP which were higher in the algae biofilm (AB) MFC than MFC or AB alone (Yang et al. 2018).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. vulgaris\u003c/em\u003e biofilm was cultivated on the piggery farm WW. The removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and TN from WW can be achieved by using a pine sawdust biocarriers, which removes 95.54% and 96.10%. Additionally, biocarrier in cultivation were noted to support robust growth of algal biofilm (Zhang et al. 2020b). Real dye textile WW used cathodic \u003cem\u003eC. vulgaris\u003c/em\u003e biofilm to enhance the electrochemical performance of MFCs, substituting the platinum cathode and removing Cr (54-80%), Zn (98%), and COD (92-98%) (Logro\u0026ntilde;o et al. 2017). The cultivation of \u003cem\u003eChlorella proteinucleus\u003c/em\u003e-bacteria symbiotic biofilm was utilized by sequential batches to treat municipal and soybean soaking WW. Enhanced biomass productivity, proteins, carbohydrate, and lipid levels, increased removal rate of NH₄⁺-N, TP, TN, and COD (Yu et al. 2024b). The microalgal biofilm treatment can increase the production of lipids, protein and polysaccharides. It was shown that the concentration levels of sulfamethazine, COD, TP, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, and TN in the swine digestion effluent were 0.99, 367.28, 4.11, 9.32, and 22.65 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e, correspondingly. This meets the discharge standards (Mou et al. 2023). The co-cultivation of \u003cem\u003eIsochrysis galbana\u003c/em\u003e biofilm can effectively increase the self-sedimentation rate by synthesizing TB-EPS content. This immobilization strategy increases the removal rate of an inorganic nitrogen and NO\u003csub\u003e3\u003c/sub\u003e-N in AWW (Wang et al. 2025b). MAB had a high degree of efficacy in removing P residues and recovering P from simulated WW under small WWTP (Sukačov\u0026aacute; et al. 2020). Co-culture of \u003cem\u003eC. pyrenoidosa\u003c/em\u003e and \u003cem\u003ePhormidium\u003c/em\u003e sp., in livestock WW enhanced their biofilm adhesion through the synthesis of TB-EPS and its polysaccharides and proteins (Liu et al. 2024c). Cultivation of non-immersed zigzag microalgae biofilm in digested piggery WW exhibited a maximum growth rate of the biofilm and enhanced the NH₄⁺-N removing properties of the biofilm (Huang et al. 2023).\u003c/p\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003eEven though the attention that studies on microalgal-based cultivation have been published for many years, this absorption property is becoming increasingly popular due to the cost-effective development of biofilm bioreactor systems. By advancing in knowledge and expertise, more potential benefits can be gained from the natural tendency to attach to surfaces and grow. The attached or clump growth in substrate and bioreactor can be optimized by exploring the selection of biocarriers or surface materials. Highly effective support materials for MABs were discovered to include stainless steel, cotton, sawdust, sugarcane bagasse, nickel foam, rice husk, N-isopropylacrylamide, polyethylene terephthalate, polyethersulfone, and polyvinylidene fluoride membrane. In the future, researchers may create low-cost cultivation systems that produce concentrated algal biomass to increase yields and recover products. This also means that regulating biofilm growth with known microalgal species is an integral part of the future path for building specific integrated biofilm systems to produce desired products or potential effects. The technique can be employed to increase microalgal harvest, but it still has the potential to form biofilms and immobilize microalgae. Smart biofilm cultivation of microalgae can be achieved by implementing an integrated multi-technology framework to optimize interaction effectiveness and address the pollutant characteristics and operational constraints of the microalgae engineering. However, research to elucidate the underlying physiological mechanisms is still necessary, especially when it involves interaction between cells and the pattern of gene expression in biofilms. A better thoughtful of the mechanisms elaborated in designing the bioreactors is essential. The focus of microalgal immobilization is receiving much recognition from the research community in terms of sustainable real WWT and recovery. In any case, research on this issue can exploit the metagenomics, adhesion, aggregates and biofilm formation for bioremediation, EPS synthesis, algal growth modulation, and signaling molecules. Some current works report the application of this mode of growth to alleviate xenobiotics, remove COD, N, P, and metal contaminants. In natural ecosystems, there has been an investigation into the mechanism of biofilm formation between microbes and microalgae. The operating parameters are complex and underdeveloped to bring biofilm growth to the industrial level.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors state that there are no potential financial or other interests that could influence the results of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of this review did not have any external financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated or analyzed during this study has been published and can be made available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMV\u003c/strong\u003e: Conceptualization, writing, Methodology, Investigation, Review, Editing \u0026amp; Formal Analysis, \u003cstrong\u003eJK\u003c/strong\u003e: Formal Analysis, Data Curation, AA: Editing \u0026amp;Review. \u003cstrong\u003eCR\u003c/strong\u003e: Figure and art work. \u003cstrong\u003eAK\u003c/strong\u003e: Original Draft, Data Curation, and Review \u0026amp; Editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbinandan S, Subashchandrabose SR, Venkateswarlu K, et al (2018) Microalgae-bacteria biofilms: a sustainable synergistic approach in remediation of acid mine drainage. Appl Microbiol Biotechnol 102:1131\u0026ndash;1144.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbrantes Silva T, Pereira ASAP, Ferreira J, et al (2024) Enhancing microalgae biomass production: Exploring improved scraping frequency in a hybrid cultivation system. J Environ Manage 355:120505.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlhumairi A, Hamouda R, Saddiq A (2022) Comparative study between immobilized and suspended \u003cem\u003eChlorella\u003c/em\u003e sp in treatment of pollutant sites in Dhiba port Kingdom of Saudi Arabia. Heliyon 8(9):e10766.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAli SS, Al-Tohamy R, Al-Zahrani M, et al (2025) Advancements and challenges in microalgal protein production: A sustainable alternative to conventional protein sources. 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Renewable Energy 175:676e685.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Microalgae, Circular economy, Resource recovery, Biofilms, Wastewater treatment","lastPublishedDoi":"10.21203/rs.3.rs-8649144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8649144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroalgae and cyanobacteria biofilm have simple physiology, fast photosynthetic growth using wastewater (WW) (primary nutrients removal, N, P), and can provide feedstock for microalgae-based biorefinery attempts. Suspended or planktonic cultures of photosynthetic algae have been widely developed for producing industrial biochemical by improving metabolic engineering via synthetic biology. Planktonic cultures can experience low algal biomass productivity, high operating costs and energy consumption when cultivating both indoor and outdoor cultivations. Algal biofilm or immobilized cells have drawn a lot of attention recently due to their prospective to improve the sustainability of livestock production, implications for carbon sequestration and climate change resilience, value-added products, and blue and green economies. Microalgal biofilm (MAB) or attached growth can be beneficial for effective removal and degradation of micropollutants, heavy metals, and hazardous chemicals, reducing harvesting costs, and transforming bioactive compounds and livestock for downstream operation. Non-axenic MABs are a smart choice for energy-alternative animal, municipal, and mining WW treatment (WWT), but they have not yet been developed as industrial options for deployment in sewage water. Besides, numerous aspects of MAB including biofilm formation mechanisms, stability, and bioreactor design, cell-cell interaction, extracellular polymeric substances (EPS), signaling molecules, and renewable products deserve deeper investigation. This article's goal is to provide a thorough review of MAB communities, their presence in the biosphere, their expansion in industrialized uses and the bio-based economy.\u003c/p\u003e","manuscriptTitle":"Substrate dependent microalgal biofilm cultivation system for the blue and green economy: formation, mechanism, and applications in environmental engineering and biotechnology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 17:19:46","doi":"10.21203/rs.3.rs-8649144/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"213005417850616910216200082882520154848","date":"2026-05-12T17:05:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T23:53:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293246627166065901734889499150157061789","date":"2026-03-12T22:56:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284285963767341308660665761498326297030","date":"2026-03-02T12:05:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-09T10:46:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-04T07:48:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-03T04:57:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2026-01-20T11:37:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"037f67b2-c92b-4541-be73-f99ac790b09c","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"213005417850616910216200082882520154848","date":"2026-05-12T17:05:15+00:00","index":32,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-11T17:19:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 17:19:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8649144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8649144","identity":"rs-8649144","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}