Introduction

Biofilms are complex biostructures which adhere to surfaces of carriers (George et al. 2000). A biofilm consists of mixed microbes such as yeasts, fungi, and protozoa, and associated deposits enclosed in a self-produced extracellular polymeric substances (EPS). The presence of biofilms may have a harmful impact on a broad range of areas, specifically in the food, environmental, and biomedical fields (Flint et al. 1997; Maukonen et al. 2003; Sihorkar and Vyas 2001); however, it can be used beneficially in biological wastewater treatment. The biofilm provides structural integrity, bacterial protection of critical and sensitive microorganisms, intercellular communication, formation and maintenance of the microcolony, and capturing and consumption of nutrients, and is of vital importance for the performance of biofilm processing system (Boltz et al. 2017).

Biofilm process (also known as attached-growth process) has been widely used in biological wastewater treatment in the past few decades. In the biofilm wastewater treatment process, biofilms are attached to biocarriers and substrates such as biochemical oxygen demand (BOD), ammonia nitrogen, nitrate, dissolved O2, and so on, and are delivered from bulk liquid to the interface. And then, the nutrients supplied are exploited to synthesize new generations of microbes and for metabolic consumption; thus, contaminants in the wastewater are removed. A wide variety of biofilm reactors have been developed and applied to deal with domestic sewage and a variety of industrial wastewater (Andreottola et al. 2002; Odegaard et al. 1993), such as biological contact oxidation tank (Zhang et al. 2015), biological rotating disc (Visscher et al. 2013), biological aerated filter (BAF), biological fluidized bed, moving bed biofilm reactor (MBBR), and integrated fixed-film activated sludge reactor (IFAS) (Boltz et al. 2017). In general, biofilm reactors have several advantages such as strong adaptability, high removal rate of organics and nitrogen, low excess sludge production, and convenient operation management, resulting in an increasing need for application in the upcycling of wastewater treatment plants around the world (Escudie et al. 2011). In recent years, with the development of anaerobic ammonium oxidation (ANNAMMOX), autotrophic denitrification and other biofilm-based technologies (Augusto et al. 2018; Jiang et al. 2018; Li et al. 2016), and gradual improvement of wastewater discharge standards in many countries and regions, the biofilm process has become as a hot spot in the aspects of advanced wastewater treatment, water reuse, and the upgrading and reconstruction of wastewater treatment plants. However, there are few literatures on summarizing wastewater biofilm during the life cycle. In particular, there is a vacancy on characterization at various stages of biofilm and its regulation.

In order to fill knowledge gaps in the above fields and offer effective guidance for the innovative application of biofilm process, this review provided a whole look at biofilm formation and its affecting factors, development of biofilm accompanied by microbial physiology, ecology and activity especially emphasized on the initialization formation, and characterization of biofilm. Recent studies about biofilm physio-ecology analysis and methods on evaluating microbial activity were summarized, and the concept of aging biofilm was also presented to outline the entire life cycle of biofilm. Furthermore, regulation methods of biofilm were reviewed, and future research trends on biofilm control were prospected, aiming at offering guidelines on biofilm control in biofilm-based wastewater treatment.

Biofilm formation and its affecting factors

Biofilm formation process

Formation and development of biofilm usually can be summarized into the following four stages based on the previous studies (Fig. 1): initial adsorption of macromolecules (e.g., protein, polysaccharide) to surfaces of carriers, microbes adhesion, biofilm development and maturity, and biofilm aging (Derlon et al. 2013a; Huang et al. 2014; Simões et al. 2010). Initial adsorption of macromolecules to surfaces triggers the whole process of biofilm formation and creates conditions for bacterial cells colonization on biocarrier surfaces. On awareness of this, we systematically investigated deposition behaviors of soluble pollutants (prepared by real and synthetic wastewaters with different configurations of model macromolecules) on model carriers by using a quartz crystal microbalance with dissipation monitoring (QCM-D). Moderate concentrations of calcium ion and rhamnolipid were proved to have a promoting effect on macromolecular deposition which has important implications for regulating biofilm formation (Huang et al. 2015, 2018a). Microbe adhesion stage includes two circumstances: One is nonselective adhesion includes adhesion and aggregation of bacteria to carriers and other bacteria mediated by high-affinity adhesion factors (membrane transport protein, viscous polysaccharide, extracellular DNA) or accessory structure (i.e., flagellum, pili) of bacteria surface; the other is specific adhesion that is adhesion triggered by the recognition of specific adhesion protein on the surface of bacteria to surface receptors (i.e., glycoprotein and glycolipid) (Jefferson 2004; Verstraeten et al. 2008). Development and maturity of biofilm mainly include the growth and accumulation of microbes, with the operation of biofilm system, the biofilm gets thicker and thicker and then the nutrient transfer is hindered, leading to decrease of biofilm activity and treatment efficiency, which is called aging biofilm state. The detachment of aging biofilm caused by the erosion and sloughing is vital to the recovery of biofilm activity.

Fig. 1
figure 1

Formation, development, and aging of biofilm in wastewater treatment

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Affecting factors

Biofilm formation is a very complex process, and a variety of factors contribute an impact to this process which can be concluded as three types: biocarrier surface properties, interface fluid characteristics, and cell properties (Jefferson 2004; Shen et al. 2015; Simões et al. 2010) as illustrated in Table 1.

Table 1 Main affecting factors in biofilm formation
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In general, attachment of microorganisms occurs more commonly on surfaces that are rougher, more hydrophobic, and coated by conditioning films (Shen et al. 2015). There is an electrostatic repulsion between negative organic molecules of carriers and the bacteria which makes it difficult for bacteria to attach to the surfaces. Increasing the interface fluid velocity which is below the critical velocity or the nutrient concentrations properly can also promote bacteria adhesion (Paul et al. 2012; Simões et al. 2010).

As studies have shown, pH value can influence bacterial surface charge characteristics. When pH value in liquid phase is higher than isoelectric point of bacteria, bacterial surfaces show electronegativity due to amino acids’ ionization. Otherwise, bacterial surfaces show electropositivity; pH-induced changes of bacterial surface electrical behavior influence the dynamics of bacterial adhesion directly. Liu (Liu 1995) applied the colloidal stability theory to explain influences of liquid pH to nitrobacteria fixed rate. Stable electric double layer or solvation structures are formed around the electriferous bacteria in the presence of Zeta potential and hinders the effective contact between bacteria and surfaces. Besides, the solvation structures can lead to steric hindrance among bacteria which will do harm to bacterial adherence to carriers. Zeta potential of bacterial surfaces tends to be zero, and surface solvation structures almost disappear when bacteria are in the isoelectric-point environment. Microorganisms in liquid phase are in an extreme stable state under this circumstance and would adhere to the carriers or gather together to decrease surface free energy and reach a new stable state.

Bacterial extracellular appendants (i.e., flagella and pilus) are also necessary for their adhesion and aggregation (Jefferson 2004; Sauer and Camper 2001; Simões et al. 2010). In general, there will be a repulsion between bacteria and surfaces when the contact distance is 10–20 nm. If bacteria can use the mentioned appendants to overcome this repulsion and make the distance lower than 1 nm, a strong attraction will exist between the surfaces of carriers and bacteria which could promote the adhesion (Sauer and Camper 2001). Extracellular polymeric substances (EPS) secreted by microbials can aid in the adhesion of microorganisms to carrier surface. EPS can bind the free cells and ions in the wastewater together and protect microbes from toxic heavy mental and organic pollutants (Cao et al. 2011; Lai et al. 2018). Thus, the production of EPS has a significant impact on the formation of biofilm.

In recent years, bacterial quorum sensing (QS) gradually attracts much attention in wastewater treatment field. For example, Shrout and Nerenberg (2012) summarized bacterial quorum sensing theory and its regulation in wastewater treatment biofilm. Ren et al. (2013) verified that N-acy-l-homoserine lactones (AHLs) produced by aerobic granular sludge have effects in the formation of Escherichia coli K12 biofilm. They found AHL degrading enzyme activity in the activated sludge and concluded that quorum quenching and quorum sensing exist at the same time. In a recent study, we also found that distribution of QS signaling molecules displayed significant positive relationship with the concentration of EPS, providing a potential method for improving biofilm formation (Wang et al. 2018a).

As a kind of second messenger, intracellular c-di-GMP also plays a critical role in biofilm formation and its shedding, which was first identified to be an important factor that participates in the biosynthesis of cellulose (Ross et al. 1987). Then, it was discovered that c-di-GMP was associated with the phenotypes regulation in some bacteria (Tischler and Camilli 2004). It was also reported that extracellular matrix components such as polysaccharides, pili, adhesins (e.g., LapA is a kind of large adhesive protein that promotes microorganisms attachment and biofilm formation in Pseudomonas putida), and extracellular DNA can be regulated by c-di-GMP (Hinsa et al. 2003; Irie et al. 2012; Jain et al. 2012; Ueda and Wood 2010) through specific targets, while all these substances contribute to biofilm formation and its three-dimensional structure. In the meantime, c-di-GMP also take part in the dispersal of biofilm, for example, increased c-di-GMP levels enable BalA protein activation which was identified to be necessary for the biofilm dispersion of Pseudomonas aeruginosa (Petrova and Sauer 2012). Generally, increased concentration of intracellular c-di-GMP promotes surface attachment and the formation of biofilm, while the decreasing concentration of intracellular c-di-GMP can cause biofilm dispersal. However, research also showed that high level of c-di-GMP can promote the production of polysaccharide, while it may suppress the quorum sensing-dependent biofilm formation (Schmid et al. 2017). And, a relatively high concentration (200 μm) of extracellular c-di-GMP inhabits intercellular interactions and reduces biofilm formation of Staphylococcus aureus (Karaolis et al. 2005). The regulation of biofilm by regulating the content of c-di-GMP may be a promising research direction in the field of wastewater biofilm treatment, while a more precise relationship between biofilm formation and c-di-GMP remains to be ascertained.

Based on the knowledge of quorum sensing, the effect of exogenous AHLs on microbial adhesion of high ammonia nitrogen wastewater was investigated in our recent study with the help of QCM-D. Interesting results were obtained, which indicated that the addition of exogenous AHLs, especially N-octanoyl-l-homoserine lactone, improved microbial adhesion to surfaces of carriers, deposition amount, and thus the formation of biofilm, suggesting that exogenous AHLs might be potential in accelerating the startup process of biofilm formation in high ammonia nitrogen wastewater treatment systems (Peng et al. 2018b).

Biofilm physic-ecology and its characterization

In the field of microbial physiological and ecological research, with the help of microelectrodes (He et al. 2017; Zhou et al. 2011), electron microscopy (Fu et al. 2011; Zhou et al. 2011), atomic force microscope (AFM) (Zhang et al. 2011; Zhu et al. 2015), and modern molecular biology techniques, such as PCR-DGGE (He et al. 2017; Zhang et al. 2011), FISH (Persson et al. 2014) and high-throughput sequencing (Lu et al. 2014; Peng et al. 2014), biofilm morphology, internal mass transfer, and microbial community, etc., have been effectively characterized.

Biofilm structure

The macrostructure of biofilm is the common result of biofilm growth and hydraulic shear. Two typical biofilm structure models are “heterogeneous Mosaic structure” model and “mushrooms or tulip” model (Wimpenny et al. 2000; Zhou et al. 2011), both formed by the random combination and attachment of independent accumulations or communities. The “mushroom or tulip model” is a structure resembling a mushroom or tulip shaped by a micro colony and the bottom of which is narrower than the top. Furthermore, there are water channels around these colonies to transport nutrients, enzymes, metabolic products, and discharged wastes since the aqueous solution can continuously flow and circulate in the channels.

During the process of substrates utilization by microbes, the thickness of biofilm increases. Since dissolved oxygen can only spread to a certain area of biofilm, the anaerobic zone is formed in the inner biofilm close to the carriers. According to the diffusion of substrates, biofilm can be divided into two parts in functional structure: One is the substrates utilization area that directly exposed to wastewater, and the other is microbial hunger area that close to the surface of carriers since the substrates are almost utilized by the microbes in the outer layer. Microorganisms in the hunger area have to take advantages of energy from metabolism of their own cells to maintain their biological activities and usually lose the ability of adhering to carriers which lead to the shedding from the surfaces (Jefferson 2004; Paul et al. 2012; Verstraeten et al. 2008).

The team led by Paul Etienne is one of the most representative group focusing on the structure of biofilms (Coufort et al. 2007; Derlon et al. 2008; Derlon et al. 2013a; Marcato-Romain et al. 2012; Ochoa et al. 2007; Paul et al. 2012; Ras et al. 2011). They studied the effects of hydrodynamic and growth conditions (electron donor and receptor, C/N ratio, etc.) on the physical and chemical properties of biofilms by using Couette-Taylor reactors, and put forward the hierarchical (stratification) model of biofilm. It is believable that biofilm is composed of the basal layer and the outer layer under the action of the shear stress, where the base is compacted and the bonding layer is easy to fall off. Furthermore, the effect of specific centrifugal forces on biofilm structures was also reported in our group that three different fractions of biofilms could be divided under different centrifugal forces (Wang et al. 2018a).

Biofilm biophase

Biofilm is mainly composed of biophase and surrounding EPS. Biophase in biofilm is very abundant, and it forms a complex ecosystem composed of bacteria, fungi, algae, protozoa, and metazoan (Sudo 1988). As a functional organism, the distribution of biophase is not a simple combination among microbes, but an organic configuration based on the optimization principle of the whole metabolism function of organism, and can serve as biological indicators to inspect and judge operation conditions and wastewater treatment effects of the biofilm reactor (Derlon et al. 2013a).

Bacteria are the principal part of the biophase in biofilm and EPS produced by them establishes the foundation for biofilm structure. The presence and dominance of bacteria are usually related to their growth rates, wastewater qualities, and environmental conditions, such as nutrition, attachment growth conditions, dissolved oxygen supply, and temperature. Heterotrophic bacteria are the main type of bacteria in biofilm, who can gain sufficient energy substrates from water flowing through the biofilm surface. According to the demand of oxygen, heterotrophic bacteria can be divided into aerobic heterotrophic bacteria, anaerobic respiration heterotrophic bacteria, anaerobic heterotrophic bacteria, and facultative anaerobes. The common species of heterotrophic bacteria in biofilm include Sphaerotilus, Zoogloeas, Thiobacillus, Alcaligenes, Pseudomonas, Nocardias, Sarcinas, Streptococcus faecalis, Escherichia coli, Nitrosations, and Bacillus (Kim et al. 2015b; Tang et al. 2018; Wang et al. 2018b). In addition, fungi such as filamentous bacteria will appear in biofilm under specific circumstances (i.e., composition change of organic matter in sewage, increase of load, decrease of temperature, etc.) and common species of which include Subbaromyces splendens and Trichosporon cutaneum. Algae are not the main microorganism population in biofilm; thus, their function of purifying wastewater is little. The common species of algae in biofilm include Chlorella, Chlorococcum, Oscillatoria, Stigeoclonium, and Circumfili. Protozoa are the lowest unicellular animals in the Animalia. In the mature biofilm, protozoa feed on bacteria, playing a positive role on the physical activity state of biofilm. The common species of protozoa include Flagellates (i.e., Oikomonas termo), Sarcodina (i.e., Amoeba, Vahlkampfia, and Arcella) and Ciliates (i.e., Opercularia microdiscum, Vorticella convallaria, and Opercularia coarctata) (Dopheide et al. 2011). Metazoan are multicellular animals, which belong to the invertebrate. The common species of metazoan in biofilm include Rotifera, Nemata, Oligochaeta, and insects and their larva (Derlon et al. 2013b).

Generally, the morphology and chemical constituents of biofilm can be characterized using microscopy, spectroscopy, and microelectrode technology (details are shown in Table 1). Furthermore, emerging in situ monitoring techniques such as ultrasonic time-domain reflectometry (UTDR) have been developed for biofilm monitoring so that they can provide more information about the actual and dynamic process about the absorption and accumulation of biofilm. Initial adherence, reversible adhesion, and irreversible adhesion during the initial biofilm formation process could be successfully distinguished by the UTDR measurement in our recent study (Wang et al. 2018a). Biotechnologies have been also widely used in this research area. The currently and commonly used combined applications of quantitative polymerase chain reaction (qPCR), fluorescent in situ hybridization (FISH), advanced 2-D microscopy, and micro-scale chemical sensors have facilitated researchers to obtain a better vision of biofilm composition including both the cellular matter and their excretions than ever before (Boltz et al. 2017). To explore the microbial composition of biofilms, phospholipid fatty acid (PLFA) analysis has been employed in different environmental samples such as soil and wastewater treatment, for PLFAs can be biomarkers to characterize microorganisms (Amir et al. 2008), for instance, fungus, protozoa, Gram-negative, and Gram-positive bacteria can be identified through this method. With the development of biological analysis technology, 16s rRNA sequence analysis gradually becomes the main analytical tool for applications since it can make systematic classification of bacteria in biofilm (Zhu et al. 2015). Meanwhile, it is foreseeable that the proteomics and functional genomics technologies, describing structures, functions, and interactions of specific genes and proteins, will play an important role in biofilm research in the near future (Herschend et al. 2017; Hu et al. 2016; Tang et al. 2016).

In the future, research on biofilm formation and its structure or biophase will remain the focus in biofilm studies. In situ and real-time monitoring methods, such as QCM-D and OCT, will attract more attention. Especially for OCT, it is easy to obtain physical characteristics on macro- and mesoscales in a visual way (Wagner et al. 2010). Furthermore, multiple parameter monitoring such as a combination of optical, electrochemical, acoustic, and microbial community information will be a trend in biofilm analysis.

Biofilm activity

Biofilm activity is the objective basis of wastewater purification and the basic guarantee for the normal operation of biofilm process, leading to the degradation of pollutants through physical absorption, biochemical actions, and classified predations of biophase in biofilm (de Assis et al. 2017; Laureni et al. 2015). Currently, biofilm activity indicators are mainly composed of total solid and volatile solid content, oxygen uptake rate, adenosine triphosphate content, dehydrogenase activity, deoxyribonucleic acid content, etc. Table 2 compares various methods for estimation of biofilm activity.

Table 2 A comparison of various methods for estimation of biofilm activity
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Constrained by testing conditions in the previous studies, biofilm activity evaluation methods generally lack biological response. Along with the advance of modern biotechnologies, bio-driven biofilm activity from different scales (i.e., molecular level, bacterial populations, and biological communities) can be well characterized. Additionally, the study of aging biofilm evaluation is inadequate. According to the literatures, generation and consumption of ATP correspond to the internal energy charge state of the cells (Blagodatskaya and Kuzyakov 2013; Huenken et al. 2005; Xiao et al. 2015), i.e., the ATP generation process is suppressed and the utilization of ATP is stimulated under a high energy charge state, while the effect is the opposite when the energy charge state is relatively low. The content of ATP directly reflects the activity of biofilm communities. In actual wastewater treatment, aging biofilm may decrease the efficiency of biofilm treatment and collapse the whole system. Therefore, the effective regulation of aging biofilm on carriers is an important and urgent issue in the field of biofilm-based wastewater treatment (Yu et al. 2016). Some preliminary results on basic characteristics, chemical and enzymatic treatments, and regenerations towards aging biofilm have also been conducted in our previous studies (Hu et al. 2013a, b; Huang et al. 2014, 2018b). Even so, biofilm activity evaluation based on biological response and the activation of aging biofilm still need to be further explored in the context of biological wastewater treatment.

Regulation methods of biofilm

The formation and aging of biofilm in wastewater treatment is a series of complex processes, and effective regulation towards the processes according to the actual situation is the only way to achieve the best performance of biofilm reactors.

In the early stages of biofilm formation, it is needed to create a good adhesion condition which can promote the formation of biofilm and subsequent activity. Except for the conventional affecting factors list in Table 3, quorum sensing caused by AHLs also can be employed to regulation the biofilm formation. Once the biofilm is developed and mature, the biophase is synergistic to achieve the metabolic transformation of the pollutants. For the biofilm that reaches the aging state, it is necessary to be removed and activated. There are many approaches reported which are mainly composed of physical, chemical, and biological methods. The conditions, objectives, and effects of different approaches are shown in Table 4. It was worth noting that rhamnolipids can not only be used for activity recovery of aging biofilm but can also be used to promote biofilm formation and improve the treatment efficiency of biofilm process under low concentration (i.e., 20 and 50 mg/L), as reported in our recent study (Peng et al. 2018a). Generally speaking, related researches on aging biofilm control were mainly focused on traditional hydraulic shear method and chemical sterilization method. There are few studies on the application of economically efficient and environmentally friendly methods for activity recovery of aging biofilm in wastewater treatment systems, which should be paid more attention to in the future research.

Table 3 Common analysis methods for biofilm characterization
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Table 4 Recovery methods and rapid formation of biofilm activity
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Conclusions and prospects

This article reviewed wastewater biofilm from four main aspects: formation and its affecting factors, characterization, activity, and regulation. Further investigations are still needed: (1) specific and feasible methods for shortening biofilm formation in refractory wastewater treatment; (2) revealing the characteristics of biofilm from a more microscopic point of view through molecular biology technologies (i.e., macroproteomics and metagenomic approaches) and in situ monitoring techniques; (3) recovery of aging biofilm by cost-effective and environmentally friendly regulation methods. Moreover, novel biofilm reaction principles and technologies are continuing to inspire people’s interest to meet the increasing requirements of pollutants removal and lower energy consumption.