Bacteriophage-mediated extracellular DNA release is important for the structural stability of aerobic granular sludge
Graphical abstract
Introduction
Aerobic granular sludge (AGS) is a promising wastewater biotechnology that has advantages due to its compact and layered structure, which results in increased biomass concentration, as well as providing greater resistance to adverse conditions (Tay et al., 2005). The stability of this type of aggregated microbial structure is affected by many factors, such as shear force, dissolved oxygen level, pH, extracellular polymeric substances (EPS), salinity and so on (Lee et al., 2010). Among them, EPS plays a major role in structural stability because it can affect cell surface characteristics, such as cell surface hydrophobicity or surface charge density (Yuan et al., 2018). The major constituents of this extracellular matrix are typically polysaccharides (PS), proteins (PN) and extracellular DNA (exDNA) but these vary in type and amount depending on the bacterial species present as well as variation in growth conditions such organic loading or ionic strength (Flemming and Wingender, 2010; Montanaro et al., 2011; Rusanowska et al., 2019; Hou et al., 2019). Moreover, many studies have reported that both the amount and the ratio of PN and PS play crucial roles in microbial granulation (Corsino et al., 2017; Halverson et al., 2015; Sarma et al., 2017). However, despite significant advances in understanding the role of PN and PS (Yu et al., 2018), relevant studies on the source and function of the exDNA in microbial aggregation are lacking.
The importance of exDNA for surface attachment and biofilm strengthening has been demonstrated only in pure culture biofilms, including those of Pseudomonas aeruginosa (Steinberger and Holden, 2005; Whitchurch et al., 2002), Staphylococcus epidermidis (Qin et al., 2007) and Bacillus cereus (Vilain et al., 2009). Additional studies have suggested exDNA is more important for structural stability during the early stages of biofilm formation than that in mature stages (Qin et al., 2007; Seper et al., 2011). Like biofilms, the life cycle of AGS has been proposed to go through various stages which include growth followed by breakup before subsequent reformation of mature granules (Barr et al., 2010; Verawaty et al., 2012). Studies have suggested granule size is an important factor involved in granule formation and disintegration. As the size increases gradually, diffusion of compounds to the center of the granule decreases eventually leading to aggregate fragmentation (Liu et al., 2014). The crushed granule acts as a seed to which microaggregates or smaller crushed granules attach to form larger granules again under environmental stress (Verawaty et al., 2012).
Since the exDNA is considered to be an important polymer for the stability of biofilms, it is likely to be structurally important in AGS as well. However, its role in AGS integrity is often overlooked (He et al., 2019; Adav et al., 2007). Therefore, the mechanism of its production should be better understood to aid in efforts to facilitate the development of new strategies to implement AGS-based biotechnology. Over the last two decades several studies with pure culture biofilms have concluded that DNA release from cells occurs in two ways. One mechanism is cell lysis which can occur as a result of infection by prophage, actions of autolysins or reactive oxygen species (ROS) (Das et al., 2013). Autolysis, including programmed cell death (altruistic suicide) and killing of sister cells (fratricide) can be triggered by various environmental and physiological signals, such as cell density, the type of substrate, changes in pH, or the presence of antibiotics, all of which lead to lysis of some cells which can contribute to exDNA release (Beltrame et al., 2015; Ikryannikova et al., 2019). ROS disrupt membrane-bound proteins in bacterial cell membranes resulting in lysis of cells. The other mechanism is via active secretion which is usually carried out by the type IV secretion apparatus (Jakubovics et al., 2013). The mechanism related to exDNA release in AGS has yet to be definitively established due to lack of appropriate methods for high efficiency extraction and analysis of exDNA. Recently, a method for efficient extraction of exDNA from mixed biofilm samples has been developed (Alawi et al., 2014; Torti et al., 2018), making it possible to study the microbial characteristics of exDNA in AGS using a metagenomic approach.
In summary, the aims of this study were as follows: 1) to investigate the microbial characteristics (GC content, source and mechanism of production) of exDNA in different size AGSs, using metagenomic-based techniques; 2) to investigate the structural role of exDNA in different size AGSs. Findings from this work will provide new insights into AGS aggregation and structural stability which are critical for the successful implementation of AGS technology for wastewater treatment.
Section snippets
AGS sampling
AGSs used in the experiments were collected from a 3 L lab-scale sequential batch reactor (SBR) as described in Wu et al. (2019). The influent concentrations of glucose and NH4+-N were about 1000 mgCOD/L and 50 mgN/L, respectively. The reactor was operated in a 6 h cycle: 5 min of filling, 20 min of anoxia, 325 min of aeration, 5 min of settling and 5 min of effluent discharge. The COD and NH4+-N removal efficiency were kept at 95.0% and 90.0%, respectively. The collected granules were sieved
Source of exDNA in AGS
A comparison of MASH distances (Ondov et al., 2016) done using PCA showed that the exDNA and inDNA reads separated along dimension 1, which accounted for most of the variance (Fig. S2). This indicates the DNA sources from which the reads are derived are distinct. The separation of the exDNA and inDNA reads was consistent with their statistically significant (p < 0.05) differences in GC content (Table S3), ~46.0% and ~65.0%, respectively. These types of differences showed the same trend as
Conclusion
This study elucidates the specific characteristics of extracellular DNA in aerobic granular sludge. The data indicated exDNA in all tested size AGSs was mainly derived from members of the phylum Bacteroidetes. CRISPR arrays with many spacer elements as well as associated Cas encoding genes were found in almost all the Bacteroidetes MAGs The high occurrence of CRISPR spacers as well as associated Cas encoding genes in this group suggests that bacteriophage-dependent lysis is an important
CRediT authorship contribution statement
Yi-qiao Wang: Conceptualization, Investigation, Methodology, Writing - original draft, Formal analysis. Wei Li: Resources, Writing - review & editing, Supervision, Formal analysis, Validation. Jin-long Zhuang: Validation, Formal analysis, Software. Yong-di Liu: Writing - review & editing, Supervision, Validation. James P. Shapleigh: Writing - review & editing, Formal analysis, Validation.
Declaration of competing interest
The authors declare that there is no conflict of interest.
Acknowledgements
This work was supported by National Natural Science Foundation of China (51808223, 51578240), Fundamental Research Funds for the Central Universities (JKB012014053, 222201817009), National Key Research and Development Program of China (2019YFC0408202).
References (66)
- et al.
Aerobic granular sludge: recent advances
Biotechnol. Adv.
(2008) - et al.
A procedure for separate recovery of extra- and intracellular DNA from a single marine sediment sample
J. Microbiol. Methods
(2014) - et al.
Genomic insights into Candidatus Amarolinea aalborgensis gen. nov., sp. nov., associated with settleability problems in wastewater treatment plants
Syst. Appl. Microbiol.
(2019) - et al.
Rapid reformation of larger aerobic granular sludge in an internalcirculation membrane bioreactor after long-term operation: effect of shorttime aeration
Bioresour. Technol.
(2019) - et al.
Physical properties and extracellular polymeric substances pattern of aerobic granular sludge treating hypersaline wastewater
Bioresour. Technol.
(2017) - et al.
Performance and bacterial characteristics of aerobic granular sludge in response to alternating salinity
Int. Biodeterior. Biodegradation
(2019) - et al.
Microbial community structures in different wastewater treatment plants as revealed by 454-pyrosequencing analysis
Bioresour. Technol.
(2012) - et al.
Flame retardant property of flax fabrics coated by extracellular polymeric substances recovered from both activated sludge and aerobic granular sludge
Water Res.
(2020) - et al.
Biocontrol of biomass bulking caused by Haliscomenobacter hydrossis using a newly isolated lytic bacteriphage
Water Res.
(2011) - et al.
Advances in aerobic granule formation and granule stability in the course of storage and reactor operation
Biotechnol. Adv.
(2010)
Regulation of aerobic granular sludge reformulation after granular sludge broken: effect of poly aluminum chloride (PAC)
Bioresour. Technol.
Aerobic granular sludge technology: mechanisms of granulation and biotechnological applications
Bioresour. Technol.
Changes in extracellular polymeric substances (EPS) content and composition in aerobic granule size-fractions during reactor cycles at different organic loads
Bioresour. Technol.
Finding knowledge gaps in aerobic granulation technology
Trends Biotechnol.
Rapid cultivation of stable aerobic phenol-degrading granules using acetate-fed granules as microbial seed
J. Biotechnol.
Determining the mechanisms for aerobic granulation from mixed seed of floccular and crushed granules in activated sludge wastewater treatment
Water Res.
Enhanced adsorption of Zn2+ by salinity-aided aerobic granular sludge: performance and binding mechanism
J. Environ. Manag.
Extracellular DNA enhances the adsorption of Sulfobacillus thermosulfidooxidans strain ST on chalcopyrite surface
Hydrometallurgy
Effects of aeration intensity on formation of phenol-fed aerobic granules and extracellular polymeric substances
Appl. Microbiol. Biotechnol.
The RAST server: rapid annotations using subsystems technology
BMC Genomics
Granule formation mechanisms within an aerobic wastewater system for phosphorus removal
Appl. Environ. Microbiol.
Inactivation of the autolysis-related genes lrgB and yycI in Staphylococcus aureus increases cell lysis-dependent eDNA release and enhances biofilm development in vitro and in vivo
PLoS One
Phage-bacteria relationships and CRISPR elements revealed by a metagenomic survey of the rumen microbiome
Environ. Microbiol.
Whole-community metagenomics in two different anammox configurations: process performance and community structure
Environ. Sci. Technol.
Bacterial extracellular DNA forming a defined network-like structure
FEMS Microbiol. Lett.
Fast and sensitive protein alignment using DIAMOND
Nat. Methods
Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments
Appl. Environ. Microbiol.
Viruses control dominant bacteria colonizing the terrestrial deep biosphere after hydraulic fracturing
Nat. Microbiol.
Role of extracellular DNA in initial bacterial adhesion and surface aggregation
Appl. Environ. Microbiol.
Role of eDNA on the adhesion forces between Streptococcus mutans and substratum surfaces: influence of ionic strength and substratum hydrophobicity
Langmuir
The roles of extracellular DNA in the structural integrity of extracellular polymeric substance and bacterial biofilm development
Environ. Microbiol. Rep.
Role of extracellular polymeric substances (EPS) production in bioaggregation: application to wastewater treatment
Appl. Microbiol. Biotechnol.
Extracellular DNA is abundant and important for microcolony strength in mixed microbial biofilms
Environ. Microbiol.
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