Bacteriophage-mediated extracellular DNA release is important for the structural stability of aerobic granular sludge

https://doi.org/10.1016/j.scitotenv.2020.138392Get rights and content

Highlights

  • GC content of the exDNA was very different from that of inDNA.

  • ExDNA in all sizes of AGSs was mainly derived from the phyla Bacteroidetes.

  • CRISPR assays suggest phage may lyse Bacteroidetes cells and release exDNA.

  • The structural role of exDNA is dependent on the size of the granule in AGS.

  • Acid–base interactions are responsible for exDNA-mediated structural effects.

Abstract

The aim of this study was to investigate the microbial characteristics and the structural role of exDNA in different size AGSs. Metagenomic results showed that exDNA has a significantly lower GC content, ~46.0%, than the ~65.0% GC of intracellular DNA (inDNA). Taxonomic predictions showed most of the reads from the exDNA that could be taxonomically assigned were from members of the phyla Bacteroidetes (55.0–64.2% of the total exDNA reads). Assigned inDNA reads were mainly from Proteobacteria (50.9–57.8%) or Actinobacteria (18.0–28.0%). Reads mapping showed that exDNA read depths were similar across all predicted open reading frames from assembled genomes that were assigned as Bacteroidetes which is consistent with cell lysis as a source of exDNA. Enrichment of CRISPR-CAS proteins in exDNA reads and CRISPR spacers in Bacteroidetes associated draft genomes suggested that bacteriophage infection may be an important cause of lysis of these cells. A critical role for this exDNA was found using DNase I digestion experiments which showed that the exDNA was vital for the structural stability of relatively small sized AGS but not for the larger sized AGS. The characteristics of exDNA in AGSs revealed in this work provide a new perspective on AGS components and structural stability.

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).

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