Effect of N-acyl homoserine lactone on biofilm characteristics and formation in a Poly-hydroxybutyrate-hydroxyvalerate-supported solid phase denitrification system

doi:10.21203/rs.3.rs-4760516/v1

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Effect of N-acyl homoserine lactone on biofilm characteristics and formation in a Poly-hydroxybutyrate-hydroxyvalerate-supported solid phase denitrification system

https://doi.org/10.21203/rs.3.rs-4760516/v1

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Quorum sensing (QS) plays an important role in biofilm formation, and its involvement in biofilm formation during heterotrophic denitrification has remained underexplored. This study investigated the impact of N-acyl homoserine lactone (AHLs) on biofilm characteristics and formation in a Poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV)-supported SPD system. The effects of AHLs from SPD system on biofilm formation were conducted by batch experiments. The results revealed that exogenous addition of N-(3-Oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL) resulted in the highest concentration of TB-EPS, reaching 254.2 mg/L. 3-oxo-C14-HSL has a more pronounced effect on TB-PN synthesis. However, exogenous addition of 3-oxo-C12-HSL increased the negatively charged LB-EPS content, resulting in the electrostatic exclusion and reduced cell attachment capacity and a lower degree of microbial adhesion. Furthermore, the presence of exogenous 3-oxo-C14-HSL AHLs promoted the formation of tryptophan-like proteins and humic acids. The biofilm thickness and adhesion force in EPS were 47.75 uM and 4.0 uN, respectively, when adding 3-oxo-C14-HSL. Confocal laser scanning microscopy (CLSM) studies demonstrated that the 3-oxo-C14-HSL-mediated QS system enhanced the formation of bioaggregates and increased biofilm thickness in biological denitrification. These findings confirm the involvement of AHLs-mediated QS in the regulation of biofilm characteristics and formation in SPD systems. The insights gained from this study contribute to the theoretical understanding of QS and provide practical guidance for biofilm acclimation in SPD systems.

Solid-phase denitrification

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Quorum sensing

Extracellular polymeric substances

Biofilm

A cellular communication system, also known as quorum sensing (QS), is utilized by bacteria to coordinate the expression of specific genes based on the density of their population [1,2](Marketon et al., 2003; Shrout and Nerenberg, 2012). QS is an extensively researched mechanism of bacterial cell-to-cell communication. Bacteria regulate their physiological behavior and various phenotypes, such as biofilm formation, virulence, sporulation, and luminescence, by producing and responding to specific signaling molecules that accumulate [3-5](Li et al., 2022; Nicolas et al., 2019; Sun et al., 2018). Since the discovery of QS in 1979, researchers have investigated numerous bacteria and identified a soluble small molecule signal substance as the carrier of intercellular information transmission [6](Nealson and Hastings, 1979). This signal substance is referred to as QS signal molecules or 'autoinducers'. The population sensing system is regulated by the sensing signal molecule [7,8](Miller and Bassler, 2001; Fuqua and Winans, 1994). Various signaling molecules have been identified in bacterial communities, which can be broadly classified into three main types: acylhomoserine lactones (AHLs) produced by Gram-negative bacteria, modified oligopeptides or self-inducible peptides (AIP) commonly used by Gram-positive bacteria, and autoinducer-2 (AI-2) used for interspecies communication by both Gram-negative and Gram-positive bacteria [9-11](Withers et al., 2001; Taga and Bassler, 2003; Waters and Bassler, 2005).

In recent years, researchers have recognized the importance of population sensing in the field of environmental engineering. Wastewater biological nitrogen removal treatment system contains numerous dense microbial communities, which exist in the form of flocculating constituents, biofilms, or granular sludge. With the progress of molecular biology techniques, and the analysis and detection methods, QS signal molecules were detected in various wastewater treatment systems [12-14](Panchavinin et al., 2019; Liu et al., 2021; Fu et al., 2022). Valle et al. (2004) identified the presence of AHLs sensing signaling molecules in activated sludge systems [15]. Ren et al. (2010) investigated the impact of QS on aerobic granular sludge formation and observed that sensing signaling molecules influence cell attachment growth, facilitating sludge granulatio [16]. Among the various functions of QS, biofilm formation has been extensively studied due to its significant role.The first study on the relationship between QS and biofilm formation was reported by Davies et al. (1998) [17]. It was also found that the roles of the AHLs-mediated QS system in biological nitrogen removal can be effectively improved bacteria activity. Therefore, the AHLs-mediated QS system plays an important regulatory role in the biological nitrogen removal of wastewater treatment systems.

AHL-mediated quorum sensing (QS) has been found to have an impact on biofilm adhesion and thickness, as well as the regulation of microbial community structure. This, in turn, ensures the activity and stability of the biofilm, creating a suitable environment for AHL secretion and enhancing sewage treatment effectiveness. Feng et al. (2019) confirmed the significant role of N-Octanoyl-L-homoserine lactone (C8-HSL), N-Decanoyl-L-homoserine lactone(C10-HSL), and N-dodecanoyl-L-homoserine lactone (C12-HSL) in influencing microbial growth, aggregation, extracellular polymeric substances (EPS), and particle formation, ultimately leading to enhanced anaerobic ammonia oxidation and reduced ammonia nitrogen concentrations [18]. The presence of AHLs, particularly long-chain AHLs like C10-HSL and C12-HSL, has been detected throughout biofilm formation in systems using aerobic granular sludge for ammonia and low concentration organic wastewater treatment [19,20](Chen et al., 2019; Wang et al., 2019). These findings have sparked further research on the impact of QS in various wastewater treatment systems. Additionally, studies suggest that N-Hexanoyl-L-homoserine lactone (C6-HSL) and C12-HSL may be involved in the ammonium oxidation metabolism of ammonia-oxidizing bacteria [21](Liu et al., 2019). It has been indicated that AHL-mediated QS plays a crucial role in the entire process of biofilm formation in water treatment, ultimately affecting the efficacy of wastewater treatment [22](Li et al., 2015). Overall, the AHLs-mediated QS system promotes microbial attachment, making it essential for biofilm formation.

Adding AHLs to nitrifying biofilm can increase the biomass and rapidly accelerate damaged biofilm recovery [23,24](Batchelor et al., 1997; Gamage et al., 2011). Moreover, exogenous AHLs can significantly increase bacteria activity [25,26](De Clippeleir et al., 2011; Tang et al., 2015). According to Li et al. (2015), an autotrophic nitrifying sludge community may be regulated using select AHL molecules [22]. Adding AHLs into the system facilitated cell adhesion, nitrification and sludge granulation. These studies indicate that the performance of a wastewater treatment bioreactor can be tuned through selectively manipulating AHL activity. Although previous researches have indicated that bacteria might be affected by AHL-mediated QS [26,27](Zhang et al., 2012; Tang et al., 2015), all studies are mostly concerned with the AHLs that impact the nitrification process but rarely consider the effect of heterotrophic denitrification system. In other words, the literature provides no clear information on the effect of QS for the heterotrophic denitrification process. Therefore, deeply understanding whether a heterotrophic denitrification system can operate stably through the effects of AHLs is essential.

The biological nitrogen removal process using insoluble biodegradable polymers as a carbon source is referred to as solid-phase denitrification (SPD) [28](Hiraishi and Khan, 2003). The SPD system offers easy regulation and control, preventing carbon source overdosing commonly seen in traditional processes, thus promoting stable operation of water treatment systems [29](Aslan and Turkman, 2006). However, the slow denitrification rate remains a major drawback of SPD, limiting its widespread use in wastewater treatment. In this context, we propose a novel strategy for improving the efficiency of SPD system. Whether accelerate biofilm formation by adding AHLs to influence the behaviors of microbial consortia for higher nitrogen removal efficiency. To test this hypothesis, we utilized biodegradable polymers, specifically poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), as both a carbon source and biofilm carrier for the denitrification system. Our study comprehensively investigates two main aspects: (1) the feasibility and performance analysis of QS regulation in SPD for nitrogen removal, and (2) the biofilm formation and characteristics of the SPD system with exogenous input AHLs. The findings of this study might be a feasible strategy for improving reactor efficiency though coordinating the bacterial response to environmental challenges, which provides a new perspective for facilitating pollutant removal and decreasing the reactor start-up period.

2.1 Carbon source, inoculum and wastewater characteristics

The PHBV pellets, which served as both the carbon source and carrier, were obtained from Ningbo Tian an Biomaterials Co., Ltd. (Zhejiang, China). These milky white cylindrical particles have a diameter of 2 mm and a height of 3 mm, with a specific surface area of 0.015 m²/g.

The inoculum for the experiments was obtained from the effluent of running PHBV-supported SPD reactors in our laboratory. This inoculum is more effective in reducing the enrichment time of denitrifying bacteria compared to activated sludge as an inoculum. The wastewater was prepared artificially using sodium nitrate as the nitrogen source. The initial nitrate concentration was 50 mg/L. PHBV pellets were added to the wastewater as a carbon source for the denitrification process. Additionally, other ingredients such as 10 mg/L of Mg²⁺ (MgSO₄.7H₂O), 10 mg/L of PO₄^3--P (KH₂PO₄), 10 mg/L of Ca²⁺ (CaCl₂), 5 mg/L of Fe²⁺ (FeSO₄.7H₂O), and 1 mL/L of a trace element solution were included in the synthetic wastewater. The trace element solution was prepared following the procedure outlined by Zhang et al. (2017)[30].

2.2 Shake flask experimental

The experiments were conducted in 1000 mL conical flasks, which contained synthetic wastewater and 20,000 mg (wet weight) of denitrification sludge taken from a laboratory-operated SPD reactor. The experiment was terminated until the nitrate concentration was decreased below 3 mg/L in the experiment. Four kinds of standard AHL reagents, namely C8-HSL, N-tetradecanoyl-L-homoserine lactone (C14-HSL), N-(3-Oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL), and N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), were purchased from Sigma Aldrich (USA). These four most endogenous AHLs are released during the steady state phase of operation of the PHBV-supported SPD reactor (Fig.S1). To avoid the inhibitory effect of organic solvents on microorganisms, stock solutions were prepared in methanol at millimolar concentrations. The stock solution is stored at -20^oC and further diluted in sterile water to achieve the desired concentration. At the beginning of the reaction, C8-HSL, C14-HSL, 3-oxo-C14-HSL, and 3-oxo-C12-HSL were added to the conical flasks (R1-R4). The final concentration of AHLs in the reactor was 500 mmol/L. R5 represents a mixture of the four AHLs (C8-HSL, C14-HSL, 3-oxo-C14-HSL, and 3-oxo-C12-HSL) in equal volumes, and an equal volume of methanol was added to the R6 reactor as a control group. The experiment was carried out at room temperature (25^oC). Samples were taken weekly and filtered through 0.45-um membrane before analysis, followed by determination of nitrate and nitrite concentrations. Nitrate and nitrite levels were determined using the Ultraviolet spectrophotometric method and N-(1-naphthyl)-ethylene diamine photometry, respectively. The experimental procedure was conducted following the previously described method [31](Zhang et al., 2018).

2.3 Extraction, analysis of EPS content and components

The loosely-bound EPS (LB-EPS) and tightly-bound EPS (TB-EPS) were extracted using the method provided by Malamis and Andreadakis [32](2009). Firstly, a certain amount of biofilm was manually scrubbed off the surface of PHBV to obtain a biofilm suspension. The suspension was then centrifuged at 5000 r/min for 15 min at 4^oC, and the collected supernatant represented the LB-EPS fraction. The TB-EPS fraction was extracted using a heating method. The biofilm samples from the previous step were mixed with deionized water and heated in a 100^oCwater bath for 1 hour. After cooling to room temperature, the mixture was centrifuged again under the same conditions, and the collected supernatant represented the TB-EPS fraction. The concentrations of extracellular polysaccharides (PS) and protein (PN) in both LB-EPS and TB-EPS were determined using the methods described by Lowry et al. (1951) and Dubois et al. (1956), respectively [33,34]. The TB-EPS concentration was calculated as the sum of the concentrations of tightly bound PN (TB-PN) and tightly bound PS (TB-PS), while the LB-EPS content was the sum of loosely bound PN (LB-PN) and loosely bound PS (LB-PS).

The fluorescence peaks in the three-dimensional fluorescence spectrum were analyzed to determine the main fluorescent component substances in EPS. The fluorescence spectrophotometer (F-2700, Hitachi High-Tech Corporation, Japan) was used to analyze the excitation emission matrix (EEM) of the EPS, following the method described by Luo et al. (2014) [35]. The scanning test conditions were as follows: a xenon lamp was used as the light source, with the excitation wavelength (Ex) ranging from 250 to 450 nm in 5-nm increments, and the emission wavelength (Em) ranging from 300 to 600 nm in 5-nm increments. The slit width was set to 5 nm and the scanning speed was 12000 nm/min.

2.4 Analysis of the biofilm properties

Biofilm thickness was assessed using an inverted microscope (Nikon Ti2000, Japan) and recorded via a CCD camera. Adhesins in EPS of biofilms were stained using Thioflavin T according to the method of Colowick et al. (2002), and confocal laser scanning microscopy (CLSM) (Nikon Corporation, Japan) was used to record digital images for quantification [36]. CLSM was used to observe the distribution of cells and biofilm thickness as described by [37]Wu et al. (2021).

2.5 Biofilm formation ability test

Biofilm morphology was visualized using confocal laser scanning microscopy (CLSM, Ultra View VOX, PE, USA). Labelling was performed using isothiofluorescein-knife bean protein A (FITC-ConA) (Sigma Aldrich, USA). The PHBV particles were rinsed with 1×PBS buffer solution to remove surface salts. Then, the particles were incubated with 100 ug/mL of FITC-ConA for 10 minutes at room temperature, followed by three washes with 1×PBS buffer solution. Subsequently, the stained PHBV particles were placed in blood cell culture dishes and observed using an instrument. Imaris software was utilized for image reconstruction, while Image J software was employed for image processing.

2.6 Data analysis

The average and standard deviation of water quality and EPS contents were calculated based on triplicate tests. Pearson's correlation analysis was conducted using Origin 8.5 software to examine the correlations between different indices. A p-value < 0.05 was considered statistically significant.

3.1 Effects of AHLs on NO₃^--N removal and system performance

The effects of QS on SPD system investigated by using the different AHLs to eliminate in this study. The nitrate removal performance and nitrite accumulate were shown in Fig.1. In the SPD system, the removal of NO^3--N was examined in Fig.1(a). In the shake-flask experiment, the addition of exogenous 3-oxo-C14-HSL (R3) fraction exhibited a best effect on nitrogen removal performance. The removal rate of NO^3--N to 95%, which was significantly better than the blank control group (R6) (p<0.05). At the same time, nitrite concentration was less than 0.1 mg/L, in which almost no accumulation Fig.1(b). However, 3-oxo-C12-HSL (R4), which has a more similar structure to 3-oxo-C14-HSL, had a nitrate removal rate of 63.7%. At the end of the reaction, small amounts of nitrite were accumulated. It should be noted that these two AHLs, both of which contain a carbonyl substituent at the 3-carbon position, showed different results for nitrate removal. AHLs with a carbonyl substituent at the 3-carbon position were associated with nitrogen removal, but exhibit different denitrogenation effects. The data in Fig.1(a) also show that the addition of exogenous C8-HSL had a very significant inhibitory effect on nitrate removal (p<0.05). The nitrate removal rate was and 48.4%. The variation of nitrite showed that exogenous addition of C8-HSL, C14-HSL, 3-oxo-C12-HSL and Mix in SPD system resulted in nitrite accumulation of 0.10, 0.20, 0.13, and 0.15, respectively Fig.1(b). All of them were higher than the accumulation of exogenously added 3-oxo-C14-HSL. Previous studies have shown that exogenous AHLs can enhance the microbial degradation of refractory organic pollutants [22,38-40](Li et al., 2015; Wu et al., 2017; Lv et al., 2021; Maddela et al., 2019). Since SPD involves the degradation of carbon sources and denitrification by microorganisms, it is a complex nitrogen removal process that requires the combined action of multiple microorganisms [41](Zhang et al., 2022). Therefore, the effect of QS on SPD is also relatively more complex compared to the conventional denitrification process. These results suggest that the intervention of AHLs is beneficial for denitrifying bacteria to utilize biodegradable carbon sources more efficiently and achieve better nitrogen removal in the SPD system.

3.2 Impact of AHLs on EPS characteristics

3.2.1 Analysis of EPS production

The concentration of TB-EPS and LB-EPS varied when different AHLs were added, as shown in Fig.2. TB-EPS is the main component of biofilm EPS. PS and PN are the primary components of extracellular polymers, which are crucial for promoting biofilm adhesion, microbial structural development, and biofilm formation. PS enables bacterial cells to cluster together, enhancing their adhesion to solid media and facilitating the formation of early biofilm microcolonies [31](Zhang et al., 2018). Previous studies have confirmed that PS serves as the biofilm skeleton and provides the framework structure for embedded microbial cells. PN, which mainly consists of cell surface appendages, also plays a significant role in biofilm formation and stability. These cell surface appendages influence bacterial migration and attachment to solid surfaces, thus contributing to biofilm formation [42](Qi et al., 2019). Furthermore, PN can alter cell surface charge and hydrophobicity, leading to improved cell adhesion [43-45](Song et al., 2021; Wei et al., 2017; Xiong et al., 2020).

The effects of AHLs on EPS are shown in Fig.2. Fig.2(a) demonstrates that the exogenous addition of 3-oxo-C14-HSL resulted in the highest concentration of TB-EPS, reaching 254.2 mg/L. Among this, TB-PN concentration accounted for more than 73.7%, at 187.4 mg/L, while TB-PS concentration was only 66.8 mg/L. In other words, 3-oxo-C14-HSL has a more pronounced effect on TB-PN synthesis. The concentrations of TB-PN in the biofilm samples were 107.4 mg/L when 3-oxo-C12-HSL was added externally. These findings suggest that TB-PN was the primary component of TB-EPS. And the addition of 3-oxo-C14-HSL effectively promotes EPS production, particularly TB-EPS(p<0.05). TB-EPS is the main form of EPS, which is consistent with previous studies. In a study by Li et al (2015), it was observed that the addition of AHLs with a carbonyl group at the 3-carbon position substituent promoted the formation of EPS in attached autotrophic nitrifying sludge [22]. The data in Fig. 2(a) also illustrate that there was a tendency for microbial secretion of EPS to increase in the presence of AHLs, with 3-oxo-C14-HSL having a significantly better effect on TB-EPS than the other AHLs. TB-EPS possesses numerous hydroxyl groups and some carboxyl groups, which provide ample cation binding sites. Additionally, the relatively loose protein secondary structure in TB-EPS promotes its aggregation, making it crucial for sludge agglomeration [18](Feng et al., 2019). The results of this experiment demonstrate that the addition of 3-oxo-C14-HSL AHLs enhances biofilm aggregation and stability. Combined with the data in Fig. 1 (a), the exogenous addition of 3-oxo-C14-HSL showed the highest removal rate of nitrate. It seems possible to conclude that exogenous addition of 3-oxo-C14-HSL favors TB-EPS secretion, which promotes biofilm formation and further improves nitrate removal.

These findings align with the results of our study, where the concentration of TB-EPS was significantly higher than LB-EPS in all biofilm samples. Exogenous addition of 3-oxo-C12-HSL resulted in a nearly three-fold increase in LB-EPS, as compared to the Control group. LB-EPS levels remained very low, the concentration was only 5.75 mg/L (Fig.2(b)). Previous research by Shi et al. (2017) has suggested that excessive LB-EPS negatively impacts cell attachment and aggregation stability [46]. This is because LB-EPS is typically negatively charged, leading to electrostatic repulsion and reduced cell attachment capacity. Therefore, a higher LB-EPS content corresponds to lower adhesion of microbial cells. This explains the lower nitrate removal rate of exogenous 3-oxo-C12-HSL than the Control group.

3.2.2 Analysis of 3D-EEM spectroscopy

Excitation-emission matrix (3D-EEM) was also applied to analyze the EPS contents in the different biofilms. According to the study by Chen et al. (2003), the fluorescence regions were divided into five sections labeled from Ⅰ to Ⅴ, representing fulvic acid-like, humic acid-like, microbial by-products, and aromatic proteins, respectively [47]. The types as well as the positions of the EPS peaks were similar for each group of samples, but the fluorescence intensities were different. In the presence of different AHLs, there were two main peaks observed for the EPS (Fig. 3). The peak (A), with Ex/Em of 275-300 nm/300-375 nm, suggested the presence of microbial by-products such as proteins (e.g., tyrosine-like, tryptophan-like, and protein-like substances) (Zhang et al., 2018) [31]. The other peak (B), with excitation/emission wavelengths (Ex/Em) of 325-400 nm/425-475 nm, indicated the presence of humic acid-like substances [48](He et al., 2020). The fluorescence intensity of the A peak was higher than that of the B peak, which was mainly composed of proteins, which was consistent with the fact that proteins accounted for the major part of the measured TB-EPS. The microbial by-product-like proteins represented by the A peak were mainly dominated by tryptophan-like proteins. The experimental results show that AHLs have the potential to change the chemical structure of EPS by promoting the production of microbial by-product-like proteins. Tryptophan-like proteins are hydrophobic substances, which can promote the formation of tighter structures in the biofilm, and can work together with the aromatic ring amino acid structure in EPS to improve the structural stability of the biofilm [49](Dong et al., 2017). Exogenous addition of 3-oxo-C14-HSL sample had the highest fluorescence intensity of the A peak among all experimental groups. It was concluded that 3-oxo-C14-HSL could promote the secretion of tryptophan-like proteins in TB-EPS, so that strong fluorescence peaks of tryptophan-like proteins could be detected in R3, and the intensity of the peaks was significantly higher than that of the Control group. Notably, for the fluorescence intensity of the B peak, the experiments presented similar findings. It suggests that the exogenous addition of 3-oxo-C14-HSL can not only promote the secretion of tryptophan-like proteins, but also facilitate the generation of humic acids. Humic acids play an important role in adhesion and provision of electron donors or acceptors, and a minor role in flocculation and biosorption of EPS, the effects of which depend strongly on their properties and concentration [46](Shi et al., 2017). Consequently, the addition of 3-oxo-C14-HSL AHLs also led to an increase in EPS content, which was consistent with the findings shown in Fig. 2. The presence of proteins and humic acids enables efficient electron transfer, leading to high electrochemical activity in EPS biofilms that contain abundant protein-like and humic acid-like substances[50,51] (Klüpfel et al., 2014; Kumar et al., 2017). Other researchers have suggested that exogenous AHLs contribute to the increased conductive contents in the EPS of electroactive biofilms [52,53](Chen et al., 2017; Fang et al., 2018). This is an important reason for the higher denitrification performance of exogenously added 3-oxo-C14-HSL in denitrification systems.

3.3 Physico-chemical characterisation of biofilms

3.3.1 Analysis of biofilm thickness and adhesion force

Biofilm properties in the presence of AHLs in the SPD system were determined, including biofilm thickness and adhesion force in EPS. The reversible adhesion phase of biofilm formation is defined based on the cooperative behavior of multiple microorganisms in the community, rather than the behavior of individual cells (Liu et al., 2003)[54]. Biofilm thickness is generally used as a physical indicator to quantitatively analyze the initial process of biofilm formation (Bruchmann et al., 2013)[55]. As shown in Fig. 4a, the biofilm thicknesses were 15.75 um (R1, C8-HSL), 12.15 um (R2, C14-HSL), 47.75 um (R3, 3-oxo-C14-HSL), 10.75 um (R4, 3-oxo-C12-HSL), and 22.50 um (R5, Mix). It was clear that AHLs do not have the same effect on biofilm thickness. Particularly, with the addition of 3-oxo-C14-HSL, biofilm thickness was significantly (p<0.05) increased as compared to the Control group (Fig.4a). Biofilm thickness increased by 131.68% compared to the Control group (20.61 um). This indicates that active QS induction during the formation of the biofilm. In contrast, C8-HSL, C14-HSL and 3-oxo-C12-HSL showed inhibition of the increase in biofilm thickness, significantly (p<0.05). Additionally, the biofilm thicknesses obtained from the analysis of CLSM images are shown in Table S1, which align with the findings in Fig.4(a). Fu et al. (2022) demonstrated that C6-HSL, C12-HSL and C14-HSL promote the growth of multifunctional microorganisms in MBBR processes at low temperatures, which differs from the results of the present study [14]. No obvious differences were shown between the mixed AHLs and the Control group (p>0.05). Different AHLs that can affect microbial QS have different effects. In addition, SPD was a complex water treatment process, including the degradation of PHBV particles into a soluble carbon source; a process of denitrification using a soluble carbon source. In other words, the role of QS in environmental applications is complex, and the structure of AHLs can have varying effects on the QS of the system.

Adhesion force is a crucial surface property of cell membranes, playing a vital role in maintaining effective attachment and biofilm formation (Feng et al., 2021)[56]. In Fig. 4b, exogenous C14-HSL caused no significant difference in adhesion force from the Control group (p>0.05). On the contrary, addition of C8-HSL, 3-oxo-C12-HSL, 3-oxo-C14-HSL and Mix, respectively, resulted in a significant enhancement of biofilm adhesion compared to the Control group (p<0.05). The biofilm adhesion force was significantly higher in R3 (3-oxo-C14-HSL) (P<0.05) than in the other reactors. Microorganisms, as the subjects in the adhesion process, have a significant influence on the adhesion process by their surface free energy and electrification. Most microorganisms were negatively charged and have a relatively stable effect on adhesion; whereas the variability in the surface free energy of microorganisms was a source of influence on the outcome of microbial adhesion (Zhang et al.,2014)[57]. Differences in the cell surface substances of microorganisms, such as lipopolysaccharides, EPS, extracellular DNA and proteins, all affect the surface free energy of microorganisms (Sharma et al.,2002). Thus, exogenous addition of 3-oxo-C14-HSL prompted the microorganisms to secrete more EPS. which in turn resulted in higher adhesion.

3.3.2 Analysis of biofilm morphology

Biofilm is a survival mechanism in which microorganisms attach to body tissues or carrier surfaces during growth to adapt to changes in their environment. This adaptation allows microorganisms to exhibit greater environmental adaptability. In this study, the effect of QS molecules on biofilm formation in SPD systems was analyzed using confocal laser scanning microscopy (CLSM), as shown in Fig. 5. Fig. 5(a) exhibits the strongest green fluorescence and the largest range, indicating the presence of a stable biofilm with a significant polysaccharide matrix. Fig. 5(b) and Fig. 5(c) show slightly less polysaccharide matrix in the biofilm. These results suggest that 3-oxo-C14-HSL promotes microbial density and the secretion of exocellular polysaccharides. Polysaccharides, which act as the "skeleton" of the biofilm structure, play an adhesive role in the aggregation of microorganisms. Fig. 5(d) and Fig. 5(e) display the green fluorescence in the minimum area, indicating that the exogenous addition of C8-HSL and 3-oxo-C12-HSL reduces biofilm activity and loosens the biofilm structure compared to the control group. The results in Fig. 2 have demonstrated that exogenous addition of 3-oxo-C12-HSL increased the negatively charged LB-EPS content, resulting in the electrostatic exclusion and reduced cell attachment capacity and a lower degree of microbial adhesion. This conclusion is consistent with the small extent of the biofilm matrix presented by Fig.5 (d) in this section.

3.4 Future research perspectives

Biofilm formation is a complex process regulated by QS. The control of biofilm formation has received extensive attention in wastewater treatment. To improve and optimize the control strategies of biofilms, it is important to understand the factors that strongly influence biofilm formation. This article summarizes the significant role of AHLs-mediated QS on biofilm formation in suspended growth (SPD) systems, specifically in terms of bacterial activity, EPS production, and microbial aggregation. However, there are still several unresolved issues. The QS system in SPD processes remains poorly understood, necessitating systematic studies on the QS system in related microorganisms using emerging biotechnology. Genetic engineering techniques, such as metabolic engineering, omics-based approaches, genome editing, and bioinformatics approaches, hold promise for advancing biofilm-related wastewater treatment research. Additionally, further research is needed to understand the metabolism, distribution, and fate of QS signaling molecules produced during SPD system wastewater treatment, as well as changes in the microbial community at the molecular level. From an economic perspective, considering the cost and continuous effect of exogenous AHLs addition, it is necessary to screen bacteria that secrete AHLs and apply them to the biological nitrogen removal process, enabling low-cost, low-energy consumption, and environmentally friendly wastewater treatment.

This study comprehensively investigated the role of AHL-mediated QS and biofilm characteristics in the SPD system. The study found that the exogenous addition of 3-oxo-C14-HSL AHLs in the PHBV-supported SPD system led to a higher nitrogen removal efficiency. Additionally, the SPD system produced more TB-PN and TB-PS when 3-oxo-C14-HSL was added, compared to other signaling factors and controls. This suggests that 3-oxo-C14-HSL AHLs promote the secretion of biofilm EPS, which has a significant effect on the formation and composition of biofilm EPS. The AHLs promote the formation of tryptophan-like proteins and humic acids. Overall, these findings contribute to a better understanding of QS regulation in the formation of biofilms in the SPD system.

Author Contributions

Shusong Zhang, Supervision. Simeng Zhou, Writing- Reviewing and Editing. Wenting Shen, Data curation Visualization, Investigation. Peng Xu, Supervision. Yueting Fan, Manuscript Revision.

Funding

This work was supported by the National Natural Science Foundation of China (32201427); Major Basic Research Project of the Natural Science Foundation of the Jiangsu Higher Education Institutions (22KJD61005); Joint Project of Industry-University-Research of Jiangsu Province (BY2022441); Basic Research Programme of Yancheng City(YCBK2023012); the Key Laboratory Open Projects of Yancheng Teachers University (JKLBS2019009); Transversal Research Project of Jiangsu (202232007000240).

Data availability

The datasets generated and/or analyzed during the current study are not publicly available because the project is not finished but are available from the corresponding author on reasonable request.

Acknowledgements:

We would also like to thank Editage (www.editage.cn) for English language editing during the preparation of this article.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Effect of N-acyl homoserine lactone on biofilm characteristics and formation in a Poly-hydroxybutyrate-hydroxyvalerate-supported solid phase denitrification system

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