3.1 Observation of biofilm formation process on the surface of attached materials
By macroscopic observation of the growth morphology of biofilm on the surface of attachment materials, the whole process of film formation could be divided into three periods as early-stage (0-7d), middle-stage (7-15d), and late-stage (15-30d).
In the early-stage of film formation, a faint yellow water film could be observed on the material’s surface, which was sticky and slippery. It is speculated that organic matter and microorganisms had attached to the surface to form a fragile covering. The adherent substance was easy to fall off when washed with water, but there was still trace amounts of viscous substance that remained on the material’s surface. The biofilm was reversible attachment at this stage.
In the middle-stage of film formation, the surface of the attachment material became slippery and darker significantly, and the thickness also increased. Inhomogeneous yellow spots scattered on the surface of the plate. Even if washed with water, there are still plenty of sticky substances remained on the surface, indicating that a large number of organic substances were generated on the biofilm. These organics could adhere microorganisms to the material’s surface and stabilize the adhesion of biofilm, converting the adhesion process to irreversible. At this time, significant bacteria colloid could be observed under microscope in the covering layer.
At the late-stage of film formation, the surface of the attachment material has been entirely covered by grayish-brown substances. After washed with water, the sticky substances were not easy to fall off, indicating that the biofilm has completed the irreversible attachment process on the material at this time, that means the film was mature.
3.2 Effect of phosphate on the mass of biofilm
Figure 2 and Fig. 3 showed the mass change of the biofilm over time formed in water with different phosphate concentrations, including dry weight (DW) and ash free dry weight (AFDW). DW and AFDW of biofilm showed a similar trend. Organics accounted for about 70–85% of the dry weight of biofilm. The DW and AFDW of the biofilm gradually increased with the increase of immersion time of the attached material, indicating the adhere of microorganisms and microalgae on the surface of the plate. The DW and AFDW of biofilm increased slowly in the early-stage of film formation (0-7d), then grew rapidly in the middle-stage (7-15d), and subsequently maintained stable and slightly decreased in the late-phase (15-30d).
Figure 2
Figure 3
With the increase of phosphate concentrations, the mass of biofilm increased gradually and reached the maximum value at 40µg/L, then DW and AFDW reduced slightly at a higher level of 50µg/L.
3.3 Effect of phosphate on the content of Chl-a in biofilm
The accumulation of Chl-a in biofilm could indicate the adhesion process of algae. As shown in Fig. 4, when the attached material immersed in seawater, the microalgae quickly attached to the surface, and the content of Chl-a reached a certain value in 1-2d. With the extension of immersion time, the content of Chl-a in the biofilm increased slowly and reached the peak in 8-14d, after which the content of Chl-a decreased slightly. The slow increase rate of Chl-a in the middle-stage of biofilm formation may be related to the three-dimensional structure of the biofilm. With the increase of film thickness, the light intensity in the biofilm was weakened, resulting in weak photosynthesis of microalgae and lower content of Chl-a in biofilm (Liu et al. 2017). The decrease of Chl-a in the late film-forming period may be caused by the shedding or death of some microalgae.
Figure 4
The suitable phosphate levels for microalgae growth is generally 30–50 µg/L (Gruber-Brunhumer et al. 2016), and the content of Chl-a on the surface of attached materials was relatively higher at the corresponding levels.
3.4 Effect of phosphate on the total EPS in biofilms
As the main component of biofilm, EPS not only enhances the resistance of cells to the external environment, but also plays an important role in the microstructure and functional integrity of biofilm (Camacho-Chab et al. 2016). Therefore, it is the key factor for the stability of biofilms and plays a decisive role in the structure and adhesion strength of biofilm (Rasamiravaka et al. 2015). Figure 5 showed the trend of EPS in the biofilm on the surface of the attached material over time:
Figure 5
As shown in Fig. 5, the quantity of EPS amount on the surface of the attached material has similar trends with DW and AFDW. The quantity of total EPS generated was very small at the initial stage, indicating that only a few microorganisms were enriched on the carrier. EPS began to increase rapidly on the fourth day, and exceeded 70% of the organic matter in the biofilm. On the 15th day, EPS on the surface of the adhesive material increased rapidly for the second time, and then the content of EPS was relatively stable and decreased to a certain extent.
According to the formation process of biofilm, the first significant increase of EPS in the biofilm at 4-8d was caused by the attachment of microbial, and the second considerable increase of EPS at about 15 d was mainly due to the colonization of microalgae in the biofilm (Yadav et al. 2020).
With the increase of the levels of active phosphate in water, the content of EPS in the biofilms increased continually and reached the peak at 40.0 µg/L, then the content of EPS in the biofilms decreased slightly at higher phosphate level (50µg/L) (P > 0.05)
According to the above results, biofilms at day 6d, 15d, and 30d were selected in subsequent experiments to analyze changes of polysaccharides and proteins in SEPS, TB-EPS and LB-EPS.
3.5 Effects of phosphate on polysaccharide and protein in EPS of biofilms
EPS determines the structure and adhesion strength of biofilm, and its quantity could reflect the amount of biofilm to a certain extent (Borlee et al. 2010). EPSs comprise different types of biopolymers, including polysaccharides, proteins, and nucleic acids (Li et al. 2022), which can provide microbes with strong tolerance and favorable living environments (Lu et al. 2022).
Exopolysaccharides, the main component of the biofilm matrix, are complex substances consisting primarily of various polyanionic macromolecules as well as neutral and polycationic macromolecules (Fulaz et al. 2019). Recent studies demonstrated that exopolysaccharides had a significant impact on initial biofilm formation and could strengthen extracellular electron transfer (EET) with anchored c-type cytochromes (c-Cyts) (Zhuang et al. 2022).
Proteins in biofilms play an important role in their degradation and construction. Extracellular enzymes are devoted to degrading biopolymers into low-molecular-weight products that can be used as energy sources. Nonenzymatic proteins usually act in surface-associated binding processes to stabilize the matrix network. Some are indispensable parts of biofilm formation, providing microbes with biofilm forming ability, presenting a large molecular mass and being involved in bacterial infections (Lasa and Penad´eS 2006).
EPS could be divided into soluble EPS (SEPS) and bound EPS (BEPS) according to its polymerization morphology. The BEPS was consisted of tightly adhered inner layer (TB-EPS) and loosely bound outer layer (LB-EPS) (Thi et al. 2020). The effects of phosphate level on the contents of polysaccharide and protein in various EPS were shown in Fig. 6,7, and 8.
Figure 6
Figure 7
Figure 8
The quantities of various EPS, including SEPS, LB-EPS, and TB-EPS exhibited a similar trend to that of total EPS. With the increase of phosphate concentration, the qualities of various EPS in biofilm increased gradually, as well as the content of protein and polysaccharide in EPS. Once the suitable phosphate level of microorganisms and microalgae was exceeded, the qualities of various substances decreased lightly at the highest level. The protein-polysaccharide ratio (PN/PS) increased firstly and then fell slowly with the change of phosphate level.
In each type of EPS, the content of protein was higher than that of polysaccharide, which was consistent with domestic and foreign research (Dieguez et al. 2020; Hou et al. 2019). With the extension of immersion time of adhesive materials, the quantities of protein and polysaccharide in the three types of EPS all showed a tendency to increase first and then slowly decline. However, the relative contents of protein and polysaccharide in the three types of EPS showed different trends. The components of EPS exhibited a gradient change through the whole thickness of the films. The protein content was the highest in surface SEPS, which gradually decreased along the direction of biofilm depth, while polysaccharide showed the opposite. This meant that SEPS and LB-EPS contained more protein and TB-EPS contained more polysaccharide. These were because the structures of the biofilm gradually tightened along the direction of the depth of the biofilm, the metabolism of microorganisms and microalgae decreased gradually due to obstructed exchange of substances (Flemming et al. 2016). The activities of microorganisms and microalgae in the surface biofilm were higher, and more extracellular enzymes were produced, which were components of proteins in EPS, resulting in the higher protein content in the surface layer. While polysaccharides were gradually consumed by microorganisms as they metabolized. Some extracellular protein was ectoenzyme, which could hydrolyze extracellular substances, such as polysaccharides, proteins, lipids, and chitin, into small molecules, which could be absorbed by microorganisms, resulting in the reduction of polysaccharides (Wilking et al. 2013). In the three types of EPS, the values of PN/PS decreased gradually from the outermost SEPS to the innermost TB-EPS.
3.6 Effect of phosphate on microbial composition of biofilm
Figure 9 showed the changes of microbial composition in the biofilms formed on the surface of attached materials under different water temperatures. The microbial communities among these biofilms formed in different phosphate levels showed no significant difference, the microorganisms in biofilms mainly included Bacillus, Pseudomonas, Vibrio, Aeromonas, Flavobacteria, Halomonas, Photobacterium, Xanthomonas, Acinetobacter, and Alcaligenes, among which the predominant including Bacillus, Aeromonas, Pseudomonas and Vibrio. The main microalgae in biofilms were diatoms, and the dominant species were Nitzschiella and Navicula, Rhizosolenia and Pleurosigma.
Figure 9
On the 6th day, the biofilm was dominated by microorganisms including Vibrio, Bacillus, Aeromonas, and Alcaligenes, while the proportions of other microorganisms and diatoms were lower relatively. With the extension of time, the proportions of bacteria decreased, the ratios of Pseudomonas, Vibrio, Bacillus, and Alcaligenes decreased significantly (P < 0.05), while the proportion of Aeromonas slightly increased (P > 0.05). The diatoms in biofilms increased gradually over time. On the 30th day, the proportion of microorganisms in the biofilm further decreased, and the ratio of diatoms increased to more than 50%.
At the same immersing time, the proportions of Vibrio and Pseudomonas in the biofilm decreased with the increase of phosphate level, while the proportions of Bacillus and Aeromonas increased. The proportion of diatoms in the microbial population also increased with the increase of phosphate level, especially the Navicula and Nitzschiella, which changed significantly with the increase of phosphate concentration (P < 0.05).