A review of quorum sensing regulating heavy metal resistance in anammox process: Relations, mechanisms and prospects

Abstract Quorum sensing (QS) is a crucial process of intercellular communication in bacteria that synchronizes their gene expression and physiological behaviors. This ability assists bacteria in surviving under environmental stressors. One such prevalent environmental stressor is heavy metals, which can significantly impair bacterial function, with anaerobic ammonium oxidation (anammox) consortia being particularly susceptible. Herein, we systematically and critically review the key information about QS regulation to enhance heavy metal resistance of anammox consortia. This review first discusses the interaction between QS and heavy metal, then elaborates on QS regulations for anammox consortia behaviors, revealing the ternary relations among QS, heavy metal and anammox. Furthermore, the underlying mechanisms of QS regulation to heavy metal resistance in anammox consortia are analyzed, including how QS regulates extracellular polymeric substances biosorption, antioxidant defense, electron transfer, resistance to bacterial infection and microbial interaction. This work deepens the understanding of the QS regulatory mechanisms that enable bacterial survival in response to environmental stressors, providing a fundamental basis for applying QS regulation to enhance bacterial resistance in the environmental engineering. Graphical abstract

QS has gained extensive attention as a promising regulation approach for microbial activity enhancement and biofilm formation to remove contaminants in the environment (Tang et al., 2018b;Sun et al., 2022).The exogenous addition of AHL extracts was reported to increase the activity of Pseudomonas aeruginosa to facilitate phenol biodegradation (Yong & Zhong, 2010).AHL add-back to municipal wastewater also increased the biofilm thickness by 206% after 24 h (Wang et al., 2020).Further, QS also works as an environmental sensing system to regulate more biochemical behaviors to resist stress, which is not noticed in the field of environmental science and engineering.QS has been demonstrated to allow bacteria to survive better and maintain healthy cooperative societies under environmental stresses, such as oxidative, osmotic, thermal, and heavy metal stress in the medicinal field (García-Contreras et al., 2015).For example, exogenous AHL at 5 μM could completely restore the survival ratio of Deinococcus radioduran under the exposure of H 2 O 2 , indicating that AHL-mediated QS enhance the resistance to oxidative stress induced by H 2 O 2 (Lin et al., 2016).As a prevalent environmental stressor, heavy metals threaten the survival of environmental microorganisms due to their acute and persistent toxicity.
Anaerobic ammonium oxidation (anammox) bacteria, utilizing nitrite as electron acceptor and then transforming ammonium to nitrogen, are highly susceptible to environmental factors, particularly heavy metals (e.g., Cu, Zn, Pb and Cr) in wastewaters (Tang et al., 2011;Xu et al., 2021;Feng et al., 2023b).Hg(II), Cd(II), Cr(VI) and Pb(II) severely inhibited the anammox activity with the half maximal inhibitory concentration (IC 50 ) values of 2.33, 7.00, 9.84 and 10.40 mg•L −1 , respectively (Yu et al., 2016;Qu et al., 2023).Due to the growing focus on recovering anammox performance from heavy metal inhibition, several approaches for removing heavy metal has been produced, such as dosing chelating agents (e.g., ethylenediamine tetraacetic acid EDTA) and precipitants (e.g., sodium sulfide) (Zhang et al., 2015;Zhang et al., 2017).However, these approaches cannot remove heavy metals completely, and residual heavy metals at low concentrations still threaten anammox performance.Enhancing the resistance to heavy metals may be an efficient strategy to address the limitation.The heavy metal resistance is associated with cellular signaling pathways, and QS as a prominent signal system probably has a role in bacterial susceptibility to metal toxicity (Prabhakaran et al., 2016).Thaden et al. reported that QS system activates a Cu resistance system composed of 11 genes via the transcriptional regulator PA4778, increasing the tolerance of Pseudomonas aeruginosa to Cu (Thaden et al., 2010).Thus, it is worth exploring whether QS can be utilized to regulate the heavy metal resistance of anammox consortia.
In this review, we focus on the role of QS regulation in heavy metal resistance.The ternary relations among QS, heavy metal and anammox were expounded firstly.Based on their close correlations, the underlying mechanisms of QS regulation for enhancing heavy metal resistance in anammox consortia were highlighted.Finally, the perspectives on future research needs for QS regulation in enhancing heavy metal resistance were discussed.The study sheds light on the guideline for enhancing heavy metal resistance of anammox consortia based on QS regulation to support the practical application of anammox-based process in the environmental engineering.

Current potential applications of QS
The current potential applications of QS involve the construction of engineered QS systems, microbial biosensors, biological remediation, biofilm development and sludge granulation in the field of environmental science and engineering.
First, engineered QS systems could be constructed using engineered biological devices (e.g., QS signal synthases, receptors, and cognate promoter elements), and overexpressing the genes encoding QS regulons (e.g., the rhlI and rhlR genes) (Choudhary & Schmidt-Dannert, 2010;Yong et al., 2011).Engineered QS systems were valuable in the production of biochemical substances, bioenergy generation, and mixed-species fermentations.Second, biosensors with a responsive transcriptional regulator for signaling molecule and a cognate promoter could be used to recognize pathogenic microbes present in the contaminated environment, and increased resistance to bacterial infection (Choudhary & Schmidt-Dannert, 2010;Mangwani et al., 2012).Third, QS signaling molecules (i.e., AHLs, AI-2 and AIPs) were involved in the production of biochemicals (e.g., biosurfactant) to remove heavy metals from contaminated sites, and increase microbial activity to facilitate the biodegradation of organic matters (e.g., nicotine and phenol) in the water and soil (Yong & Zhong, 2010;Mangwani et al., 2012).Fourth, QS signaling molecules regulated the production of EPS to accelerate the formation of biofilm and granular sludge in biological wastewater treatments (Huang et al., 2016).However, it remains unclear whether QS is applicable to help environmental microorganisms resist environmental stress.

Interaction between QS and heavy metal
As environmental stressors, heavy metal and metal-based nanoparticles disrupt the QS system due to their acute toxicity, but the QS system is of great significance for bacteria to survive under heavy metal stress.First, the investigation of the relations between QS and heavy metal was performed to evaluate the possibility of QS regulation to microbial resistance (Table S1).On the one hand, heavy metals disrupted QS system under some circumstances.For example, titanium dioxide nanoparticles (TiO 2 NPs) and silver nanoparticles (Ag NPs) hindered the biosynthesis of QS signals, while zinc oxide nanoparticles (ZnO NPs) interfered the signal perception (Gómez-Gómez et al., 2019).However, heavy metals also induce the generation of QS signals (Xu et al., 2019).Xu et al. (2019) demonstrated that nano-cerium oxide (CeO 2 NPs) increased the secretion of AHL and AI-2 signals thus the biofilm development, where resistant bacteria (e.g., Pseudomonas and Citrobacter) were enriched.The prevalence of QS function in the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was notably higher in areas with high levels of heavy metal contamination as compared to those with low levels of contamination (Gao et al., 2022c).These studies indicated that the close link of QS with heavy metal resistance is evident though there is antagonism between QS and heavy metals.

Potential role of QS in heavy metal resistance
QS is a vital regulatory system for developing resistance to heavy metals.The survival rate of wild type Pseudomonas aeruginosa with QS system showed 3.2-6.5 times higher than the parental QS-deficient bacteria under Cd(II) stress (García-Contreras et al., 2015).Similarly, with the treatment of a QS inhibitor (i.e., furanone), the viability of Pseudomonas aeruginosa decreased by 87.8% due to the disrupted QS system (García-Contreras et al., 2015).The disrupted QS system resulted in the reduction of gene expression involved in Cu homeostasis in Acidithiobacillus ferrooxidans, which indicated that bacterial resistance to Cu(II) significantly decreased (Wenbin et al., 2011).These studies indicated that the effect of QS system on developing heavy metal resistance becomes predominant over the disruption of heavy metal on the QS system.
To enhance heavy metal resistance, signaling molecules generally are added to trigger QS in the biological system.The addition of AHLs (i.e., C6-HSL, 3-OC12-HSL) increased live/dead cell ratio and exoelectrogen Geobacter abundance in the anode biofilm, enhancing the tolerance and recovery capability after high toxicity shock of Pb(II) and Cu(II) (Pan et al., 2020).Alternatively, AHL-mediated QS regulation promoted cellular viability and superoxide dismutase (SOD) activity of Nitrosomonas europaea, benefiting to resist CeO 2 nanoparticle stress (Gao et al., 2022a).Therefore, these studies strongly indicate that the QS system regulates heavy metal resistance to survive under heavy metal stress, and its regulatory mechanisms are diverse.An important question regarding this process is: how the QS system regulates gene expression and biochemical behaviors to enhance heavy metal resistance in the complex community?

Regulation of QS to anammox behaviors
The three QS signals drive multiple QS systems to regulate collective behaviors of anammox consortia.First, interactions between AHL, DSF and AI-2 induced the production of extracellular polymeric substances (EPS), facilitating biofilm formation.Second, microbiological and metabolomics analyses uncovered that AHL influenced bacterial activity by regulating electron shuttles, enhanced bacterial g r o w t h b y m o d u l a t i n g g l y c e r o p h o s p h o l i p i d m e t a b o l i s m , a n d promoted floc aggregation by altering particle size distribution.Based on these metabolic regulations triggered by exogenous AHL, the specific anammox activity, nitrogen removal rate, and growth rate of anammox consortia significantly enhanced in the anammox process (Tang et al., 2018b).Third, anammox bacteria communicate with nitrifiers or heterotrophs to regulate nitrogen and amino acid recycling via the multiple QS systems.Therefore, QS exerts efficient regulation to biochemical behaviors of anammox consortia, achieving better nitrogen removal performance.

EPS production and biosorption
EPS constitute the first defense line against heavy metals into cells, which are produced through bacterial secretion, cell surface substance shedding, and cell lysis (Ni et al., 2009;Flemming & Wingender, 2010).Previous studies indicated that QS involved the regulation of EPS production in the anammox process (Tan et al., 2014;Tang et al., 2018b).Exogenous addition of 2 µM AHLs significantly increased the contents of extracellular proteins by 21-34% (p < 0.05) and polysaccharides by 6-17% (p < 0.05) in EPS of anammox consortia (Tang et al., 2018b).Further investigation found that AHL regulated the synthesis of amino acids (i.e., alanine, valine, glutamicacid, asparticacid and leucine) to produce proteins, and regulated the N-acetylmannosamine biosynthetic pathway and uridine diphosphate-Nacetylgalactosamine pathway to produce polysaccharides (Chavez-Dozal et al., 2015;Tang et al., 2018b).The increased EPS regulated by QS facilitated the biosorption for heavy metals.A single-metal biosorption study showed that the adsorption capabilities of EPS extracted from anammox consortia were up to 84.9, 52.8, 21.7 and 7.4 mg•gTS EPS −1 for Pb(II), Cu(II), Ni(II) and Zn(II), respectively, demonstrating the strong biosorption ability of EPS to heavy metals (Pagliaccia et al., 2022).
Further, previous studies indicated that the anammox performance can be improved with the enhanced EPS adsorption by QS.For example, the increased secretion of tryptophan and protein in EPS was conducive to enhance the anammox performance (specific anammox activity increased by approximately 15%) under 2 mg•L −1 Cd(II).Further, Ma et al. revealed that the half maximal inhibitory concentration (IC 50 ) of As(III) on anammox sludge decreased from 408 mg•L −1 to 41.97 mg•L −1 when EPS was exfoliated, indicating that EPS biosorption strengthen the tolerance of anammox bacteria to As(III).The study suggested that anammox performance is better with EPS biosorption under heavy metal stress.

Interaction between EPS and heavy metal
EPS interacts with heavy metals through complexation, ion exchange, and surface precipitation, which largely depend on the components and properties of the EPS (Li & Yu, 2014).Figure 2 shows the compositions and structures of EPS, which consist primarily of polysaccharides, proteins, and phospholipids with abundant functional groups, such as hydroxyl, carboxyl, and amine.First, the functional groups present in EPS form stable complexes with aqueous metal cations through electrostatic and covalent interactions (Wei et al., 2017;Chen et al., 2018).Li et al. confirmed that carboxyl groups of proteins were preferentially bound to Cu(II) compared to polysaccharides and hydrocarbons in anammox consortia (Li et al., 2020).Second, heavy metals with high adsorption affinity can compete with those that have low adsorption affinity (e.g., Ca(II), Mn(II) and Na(I)) for binding sites through ion exchange in EPS (Comte et al., 2006;Mikutta et al., 2012).Third, surface precipitation supports the adsorption of heavy metals onto EPS at neutral or alkaline pH.As the pH increased, heavy metal would transfer from hydrated metal cations to crystalline oxide precipitates (Kushwaha et al., 2012).Fourth, the hydrophobic/hydrophilic polarity of EPS also affect the binding of heavy metals with EPS.
The extracellular proteins exhibited complex secondary structures in anammox consortia, such as α-helix, β-turn, β-sheet and random coil.The twisted and pleated sheet structure of the β-sheet exposes a significant number of inner hydrophobic amino acid groups, which increased the hydrophobicity of anammox sludge (Jia et al., 2017).Hydrophobic EPS was more efficient in adsorbing Cu(II) and Zn(II) than hydrophilic EPS (Wei et al., 2017).
In addition to the metal-binding capability and hydrophobicity, double-layer microstructure of EPS also contributes to resist heavy metals.The protein and living cells occupied the inner layer, but the polysaccharide and dead cells dominated the out layer under CeO 2 NPs stress (Xu et al., 2019).The outer polysaccharide and dead cells provided biosorption sites to form metal precipitates or chelates, thereby reducing microbial susceptibility to toxic metals (Harrison et al., 2007).Although the direct evidence is lacking, the indirect observation strongly recommends that QS regulates EPS production and microstructure distributions to enhance the resistance to heavy metals.

QS increases antioxidant defense against reactive oxygen species induced by heavy metal
Toxic heavy metals into cells would induce toxic reactions to liberate toxic reactive oxygen species (ROS), such as superoxide anion ( O 2

•−
) and hydrogen peroxide (H 2 O 2 ) (Harrison et al., 2007).The elevated level of ROS induces oxidative stress in cells, resulting in the damage to DNA, lipids, and proteins.To defend against oxidative stress, antioxidant enzymes are activated to participate in ROS detoxification, such as SOD, catalase (CAT), glutathione peroxidase and peroxiredoxin (Somasundaram et al., 2018).

Activities of antioxidant enzymes
QS increases the activities of antioxidant enzymes to relieve oxidative stress induced by heavy metals.A correlation analysis showed that AHL concentrations positively correlated with both SOD and CAT activities (r > 0.62, p < 0.01) under ZnO NP stress (Gao et al., 2022b).Further, exogenous addition of different AHLs (i.e., C6-HSL, C10-HSL and C14-HSL) observably increased the SOD activities of N. europaea by 8.0%-13.9%,and decreased intracellular ROS level by 5.0%-20.6%under CeO 2 NP stress (Gao et al., 2022a).Coincidentally, both SOD and CAT activities of the wild-type strain with the normal QS system were 2 times higher than that of mutant strain with the defective QS system (García-Contreras et al., 2015).Mechanistically, QS increases the activities of SOD and CAT activities.SOD catalyzes the dismutation of O 2 •− to H 2 O 2 and O 2 (Ighodaro & Akinloye, 2018).Then, CAT catalyzes the degradation or reduction of H 2 O 2 to H 2 O and O 2 , achieving the ROS detoxification (Góth et al., 2004).

Expression of antioxidant genes
QS regulates the expression of antioxidant genes to enhance antioxidative responses against heavy metals.Previous studies reported that wild type Escherichia coli depend on the QS system to activate a series of antioxidant genes, achieving higher cell survival rates after exposure to heavy metals (Lemire et al., 2013).These antioxidant genes encode Mn cofactored superoxide dismutase (sodA), Fecofactored superoxide dismutase (sodB), glutathione oxidoreductase (gor), thioredoxin (trxA), glutathione synthetase (gshA) and glutaredoxin (grxA) (Harrison et al., 2009).The upregulation of antioxidant genes notably increased the resistance of Escherichia coli to toxicity from Cd(II), Ni(II), Co(II), Cu(II) and Zn(II), and Pseudomonas aeruginosa resistance to As(III) (Inaoka et al., 1999;Geslin et al., 2001;Parvatiyar et al., 2005).In the genome of anammox bacteria (Candidatus Kuenenia), abundant antioxidant genes were identified, whose detailed information was shown in Table 1.Putative antioxidant genes encoding SOD, CAT, thioredoxin, thioredoxin reductase, glutathione synthase and superoxide reductase indicated the potential for antioxidation in anammox bacteria (Strous et al., 2006).The putative antioxidant genes sod and kat were transcribed in anammox bacteria, indicating the existence of an antioxidant defense system (Wang et al., 2021).Moreover, the function verification of sod and kat genes showed that these two genes are responsible for the production of SOD and CAT by heterologous expression in Escherichia coli (Wang et al., 2021).Therefore, QS regulates the production and activities of antioxidant enzymes to reduce the production of ROS, relieving the oxidative stress induced by heavy metals in anammox consortia.

QS accelerates electron transfer coupled with the redox of heavy metal
The toxicity of heavy metals is primarily determined by their chemical species, which is often characterized by the difference in valence states.For instance, Cr(VI) is approximately 100 times more toxic than Cr(III), while V(V) is 5 times more toxic than V(IV) (Huang et al., 2019).Anammox consortia have been observed to reduce Cr(VI) to Cr(III) both extracellularly and intracellularly, with intracellular reduction accounting for 98.7% of the total Cr(VI) reduction (Yu et al., 2019;Qu et al., 2023b).Similarly, the reduction of V(V) to V(IV) was achieved with a high efficiency of 87% using immobilized mixed anaerobic consortia (Shi et al., 2020).These findings suggested that microbial oxidation and reduction of heavy metals have a significant impact on their toxicity.The redox transformation of heavy metals involves electron transfer, which is the movement of electrons between atoms or molecules.Electron shuttles transfer electrons among multiple redox reactions both within and between cells, communities and ecosystems through their reversible oxidation and reduction (Stams & Plugge, 2009).Cell-excreted electron shuttles typically include but are not limited to cytochrome c, nicotinamide adenine dinucleotide (NAD) and Fe-S protein in anammox consortia (Feng et al., 2023a).

Synthesis of electron shuttles
QS enhances the synthesis of electron shuttles to mediate the electron transfer during the redox transformation of heavy metals.The cytochrome c protein containing heme c approximately accounts for 20% of the cellular proteins in anammox cells, which participates in electron transfer of anammox respiratory chain (Wang & Zheng, 2017;Feng et al., 2022).AHL addition significantly upregulated the expression of genes encoding cytochrome P450 and cytochrome c precursor, resulting in an increased production of these cytochrome.The cytochrome facilitated the electron transfer from intracellular ubiquinone to the cytoplasmic space (Whiteley et al., 1999).Additionally, AHL increased the NAD level of anammox consortia in the metabolite analysis, suggesting that AHL-mediated QS could increase the amount of electron shuttles (Tang et al., 2018b).Subsequently, cytochrome c and NADH (the reduction state of NAD) were reported to catalyze V(V) and Cr(VI) reduction to V(IV) and Cr(III), respectively (Shi et al., 2020).

Expression of electron transfer gene
QS regulates the expression of genes encoding key protein in the anammox respiration chain to enhance electron transfer.More than 200 genes related to anammox respiration were identified in the genome of anammox bacteria (Candidatus Kuenenia), which indicated the presence of diverse electron transfer pathways (as shown in Fig. 3).These genes encode complex I (proton and sodium pumping NADH: quinon oxidoreductase), complex II (succinate dehydrogenase), complex III (bc1, quinol: cytochrome c oxidoreductase), small electron carrier cytochromes, ferredoxins and iron sulfur proteins, some of which are involved in iron and manganese respiration and other metal metabolism (Strous et al., 2006;Suarez et al., 2022).Overall, QS regulates the synthesis of electron shuttles, the abundance of electrochemically active bacteria, and the expression of genes related to anammox respiration to enhance electron transfer coupled with the redox of heavy metal for detoxication in anammox consortia.

QS enhances bacterial resistance to heavy metal
Heavy metal resistance genes (HMRGs) play a crucial role in bacterial resistance to heavy metals, as they encode heavy metal-sensitive transcriptional regulators, binding proteins, efflux pumps, and detoxification enzymes (Jung et al., 2016).For instance, the copA gene encodes a Cu(I)-translocating P-type ATPase that is closely associated with Cu resistance, while the czcA gene encodes a heavy metal efflux pump that increases resistance to Co, Zn, and Ca (Rensing & Grass, 2003).In response to heavy metal stress, the abundance of HMRGs in anammox bacteria is significantly increased to enhance their resistance to heavy metals.Previous studies revealed that anammox bacteria increased the transcription of efflux genes encoding for the resistance nodulation cell division (RND) family, cation diffusion facilitators (CDF family) and P-type ATPase to export Zn(II), and upregulated cysteine synthesis genes to chelate Zn(II) (Ma et al., 2020).Genes involved in heavy metal homeostasis, mercuric resistance and chromate efflux were more abundant in the Cr and Hg contaminant sediment, which worked for Cr and Hg detoxification (Hemme et al., 2010;Yin et al., 2015b).Further, QS as a gene-to-phenotype regulation strategy was reported to involve the expression of HMRGs.Under Cu stress, AHL signal was recognized by the LasR regulator and directly binds to the cueR homolog to trigger the expression of Cu efflux gene (Thaden et al., 2010).Therefore, QS system is potential to regulate the HMRGs expression of anammox bacteria and symbiotic bacteria to enhance resistance to heavy metals.

QS promotes microbial interaction for survival to heavy metal
Biodiversity, shift of microbial community and interaction networks might increase resistance to heavy metals.There are complicated microbial compositions and interactions in anammox FIG. 3. diverse electron transfer pathways in anammox bacteria (Kartal & Keltjens, 2016).
consortia.In addition to anammox bacteria belonging to Planctomycete, the amounts of Chloroflexi, Chlorobi, Proteobacteria, Acidobacteria and Bacteroidetes account for 30-70% (Bhattacharjee et al., 2017;Feng et al., 2018).The mixed consortia are significantly more resistant to heavy metals than pure cultures, indicating that the biodiversity is beneficial for heavy metal resistance (Mejias Carpio et al., 2018).

Microbial community structure
QS alters the structure of microbial community in response to heavy metal stress.The bacteria highly resistant to heavy metals, such as Firmicutes, Chloroflexi, and Crenarchaeota, were found to be more abundant compared to those with relatively low resistance to heavy metals, such as Proteobacteria and Actinobacteria (Yin et al., 2015).Additionally, a predominant genera Halomonas was found to survive and work under no or low Cr(VI) stress, whereas Thauera was tolerant to high Cr(VI) stress (Miao et al., 2015).These studies indicated different bacteria employed varying strategies to resist heavy metal stress in the complicated microbial community.Tang et al. demonstrated that Chloroflexi (e.g., Anaerolinea, Chloroflexus, Ca. Chloracidobacterium), Proteobacteria (e.g., Nitrosomonas and Pseudomonas), Firmicutes (e.g., Bacilli), and Thauera had close interactions for their cooperation, forming a co-occurrence pattern in anammox consortia (Tang et al., 2018a).The cooperation of bacteria in the anammox community has also been reported relative to micro-environment paragenesis (Liu et al., 2015a).Thus, anammox bacteria and symbiotically resistant bacteria work together to cope with heavy metal stress.Applying QS regulation to optimize the proportion of resistant bacteria can be beneficial for increasing the resistance of the microbial community.

Microbial interaction network
Based on the group characteristic, QS is involved in intra-and interspecific communication, cooperation, competition and mutualistic interactions in the microbial community (Barra Caracciolo & Terenzi, 2021).Anammox bacteria used QS signals to communicate with symbiotic bacteria for mutual benefit (Lawson et al., 2017;Zhao et al., 2018).Microbial cross-feeding is a typical and common interaction in anammox consortia.For instance, anammox bacteria produce nitrate to support the growth of Chlorobi, Acidobacteria, and Omnitrophica.These species produce nitrite via denitrification to provide subtract for anammox bacteria (Speth et al., 2016).Further, the enhancement of QS improved microbial interaction in anammox consortia.Exogenous addition of DSF could bridge bacterial interactions through regulating public goods (i.e., EPS and amino acids) for metabolic cross-feedings (Guo et al., 2021).AHL-mediated QS system could control microbial interactions among anammox bacteria, nitrifiers and heterotrophs to achieve their balances in the partial nitritation and anammox system (Feng et al., 2019).The positive interactions among microorganisms promotes microbial cooperation to cope with heavy metal stress, while negative interactions make them less tolerant to heavy metals.For instance, Acidobacteria GP6 was demonstrated more cooperative interactions with other microorganisms such as Firmicute and Chloroflexi, whereas the ecological connections of Janthinobacterium decreased in heavy metal-contaminated sediments.As a result, the relative abundance of Acidobacteria GP6 increased, whereas that of Janthinobacterium decreased (Yin et al., 2015).More importantly, microbial interaction networks affect migration and transformation of heavy metals for their detoxication.The autotrophs (e.g., Sulfuricurvum) provided volatile fatty acids for heterotrophic reducers (e.g., Geobacter) for V(V) and/or Cr(VI) reduction (Jiang et al., 2018;Shi et al., 2020).The interactions between microorganisms involved in As, Fe and nitrogen metabolism promotes nitrate-dependent Fe(II) oxidation, and anaerobic ammonium oxidation coupled to Fe(III) reduction (Feammox), which ultimately facilitated the mobilization of As (Xiu et al., 2021;Xiu et al., 2022).Therefore, diverse biodiversity and dynamic microbial community might provide the foundation for the QS regulation to microbial interaction under heavy metal stress.The interactions facilitated by QS regulation can mediate heavy metal transformation, leading to an enhancement in the integral resistance of the microbial community.

Conclusions and prospects
Recent advances in QS research revealed the potential role of QS regulation in heavy metal resistance of anammox consortia.First, relations of QS system with heavy metals were elucidated, indicating the role of QS in the regulation of heavy metal resistance.Second, the existence of QS system and its function in anammox consortia suggested that QS was an effective regulatory method to anammox behaviors under heavy metal stress.Thus, the tripartite relationship among QS, heavy metals and anammox sheds light on the possibility of QS regulation to enhance heavy metal resistance in anammox consortia.Further, the review provides a comprehensive analysis of QS regulatory mechanisms for enhancing heavy metal resistance in anammox consortia.Generally, bacteria activated QS system to change gene expression and biochemical behaviors in response to heavy metal stress.The QS-mediated heavy metal resistance mechanisms typically include EPS biosorption, antioxidant defense, electron transfer of redox reactions, bacterial resistance and microbial interaction (Fig. 4).Therefore, QS regulation serves as a potential strategy for enhancing nitrogen removal from ammonium-rich wastewater contaminated with heavy metals.Based on the current researches, several notable directions for future work below are proposed to be considered seriously.
1. Current evidence indirectly supports the feasibility of QS-based regulation approaches for the enhancement of heavy metal resistance.Further verification and elucidation of the actual role and mechanism of QS regulation in heavy metal resistance hold practical significance in the anammox process.2. The effectiveness of QS regulation to heavy metal resistance is affected by environmental conditions, such as operational parameters and wastewater compositions.For example, landfill leachate not only contains nitrogenous pollutants and heavy metals, but also coexists with large amounts of dissolved organic matter, salts, and other organic compounds (e.g., Five regulatory mechanisms of Qs are illustrated as follows.First, Qs induces extracellular polymeric substances (ePs) to adsorb and sequester heavy metals.second, Qs increases antioxidant defense against reactive oxygen species (ros) induced by heavy metals.third, Qs accelerates electron transfer coupled with the redox of heavy metals for their detoxification.Fourth, Qs regulates the transcription of heavy metal resistance genes to enhance bacterial resistance.Fifth, Qs promotes microbial interaction to facilitate cooperation for survival against heavy metals.

Figure 1 .
Figure 1. the regulatory mechanisms of quorum sensing (Qs) for the transcription of downstream genes and biochemical behaviors in microbial community.

Figure 2 .
Figure2. the composition and structure of extracellular polymeric substances of anammox consortia(Flemming & wingender, 2010;Jia et al., 2017).the red ovals, blue rectangles and purple squares on the top left represent different bacteria in the microbial community.

FIG. 4 .
FIG. 4.Proposed regulatory mechanisms of quorum sensing (Qs) for enhancing heavy metal resistance of anammox consortia.Five regulatory mechanisms of Qs are illustrated as follows.First, Qs induces extracellular polymeric substances (ePs) to adsorb and sequester heavy metals.second, Qs increases antioxidant defense against reactive oxygen species (ros) induced by heavy metals.third, Qs accelerates electron transfer coupled with the redox of heavy metals for their detoxification.Fourth, Qs regulates the transcription of heavy metal resistance genes to enhance bacterial resistance.Fifth, Qs promotes microbial interaction to facilitate cooperation for survival against heavy metals.

Table 1 .
antioxidant genes identified in the reference genome of anammox bacteria (Candidatus Kuenenia) from the national center for biotechnology information (nCBi) database.