Bacterial communities are responsible for biological nutrient removal and flocculation in engineered systems such as activated floccular sludge. Predators such as bacteriophage and protozoa exert ...significant predation pressure and cause bacterial mortality within these communities. However, the roles of bacteriophage and protozoan predation in impacting granulation process remain limited. Recent studies hypothesised that protozoa, particularly sessile ciliates, could have an important role in granulation as these ciliates were often observed in high abundance on surfaces of granules. Bacteriophages were hypothesized to contribute to granular stability through bacteriophage-mediated extracellular DNA release by lysing bacterial cells. This current study investigated the bacteriophage and protozoan communities throughout the granulation process. In addition, the importance of protozoan predation during granulation was also determined through chemical killing of protozoa in the floccular sludge.
Four independent bioreactors seeded with activated floccular sludge were operated for aerobic granulation for 11 weeks. Changes in the phage, protozoa and bacterial communities were characterized throughout the granulation process. The filamentous phage, Inoviridae, increased in abundance at the initiation phase of granulation. However, the abundance shifted towards lytic phages during the maturation phase. In contrast, the abundance and diversity of protozoa decreased initially, possibly due to the reduction in settling time and subsequent washout. Upon the formation of granules, ciliated protozoa from the class Oligohymenophorea were the dominant group of protozoa based on metacommunity analysis. These protozoa had a strong, positive-correlation with the initial formation of compact aggregates prior to granule development. Furthermore, chemical inhibition of these ciliates in the floccular sludge delayed the initiation of granule formation. Analysis of the bacterial communities in the thiram treated sludge demonstrated that the recovery of 'Candidatus Accumulibacter' was positively correlated with the formation of compact aggregates and granules.
Predation by bacteriophage and protozoa were positively correlated with the formation of aerobic granules. Increases in Inoviridae abundance suggested that filamentous phages may promote the structural formation of granules. Initiation of granules formation was delayed due to an absence of protozoa after chemical treatment. The presence of 'Candidatus Accumulibacter' was necessary for the formation of granules in the absence of protozoa.
Mature Pseudomonas aeruginosa biofilms undergo specific developmental events. Using a bacteriophage mutant, generated by deletion of the entire filamentous Pf4 prophage, we show that the phage is ...essential for several stages of the biofilm life cycle and that it significantly contributes to the virulence of P. aeruginosa in vivo. Here, we show for the first time that biofilms of the Pf4 phage-deficient mutant did not develop hollow centres or undergo cell death, typical of the differentiation process of wild-type (WT) P. aeruginosa PAO1 biofilms. Furthermore, microcolonies of the Pf4 mutant were significantly smaller in size and less stable compared with the WT biofilm. Small colony variants (SCVs) were detectable in the dispersal population of the WT biofilm at the time of dispersal and cell death, whereas no SCVs were detected in the effluent of the Pf4 mutant biofilm. This study shows that at the time when cell death occurs in biofilms of the WT, the Pf4 phage converts into a superinfective form, which correlates with the appearance of variants in the dispersal population. Unexpectedly, mice infected with the Pf4 mutant survived significantly longer than those infected with its isogenic WT strain, showing that Pf4 contributes to the virulence of P. aeruginosa. Hence, a filamentous prophage is a major contributor to the life cycle and adaptive behaviour of P. aeruginosa and offers an explanation for the prevalence of phage in this organism.
There has been an increasing interest in the novel integration of forward osmosis (FO) and biological process known as the osmotic membrane bioreactor (OMBR). However, little operating experience is ...available to adequately assess the feasibility of the technology for larger scale application. The goal of this study is to provide fundamental information on the technology. In this study, an OMBR system was continuously operated for 73
days. It was found that the high retention property of the FO membrane and salt transmission from the draw solution resulted in increasing mixed liquor salinity until a stable state was reached. In spite of the elevated salinity, the water flux was relatively stable at around 3
L
m
−
2
h
−
1
. Post-experiment analyses indicated mild membrane fouling, and its effect on water permeability was insignificant. An ion analysis check indicated that scaling did not occur. The SEM examination detected a thin gel-like secondary layer on the membrane surface. It was deduced that this secondary layer could have an influencing role on salt transmission during the experiment, and moderated salt concentration in the bioreactor.
► Salt accumulation occurs in an OMBR due to feed retention and draw solute transmission. ► Stable flux (~3
Lm
-2h
-1) could be achieved in the elevated salt environment. ► Post-experiment analyses indicated mild membrane fouling. ► Gel-like secondary layer on membrane could have influenced salt transmission.
Recent discoveries regarding the regulation of the biofilm life cycle by bacterial signaling systems have identified novel strategies for manipulation of biofilm development to control the biofouling ...of membrane-based water purification systems. Nitric oxide (NO) signaling has been shown to induce dispersal of a wide range of single- and multi-species biofilms. However, the impact of NO-mediated biofilm dispersal on the taxa composition of natural communities as well as the potential selection for non-responding community members have rarely been addressed. Here, we investigated the effect of diethylenetriamine (DETA) NONOate, an NO donor with a long half-life, on biofilm dispersal of a bacterial community responsible for membrane biofouling to address this question. The biofilm of a complex community from a fouled industrial reverse osmosis (RO) membrane was dispersed over 50% by 500μM of DETA NONOate treatment in a continuous flow system. Once-daily treatment with DETA NONOate in a laboratory-scale RO system demonstrated its anti-biofouling effect by delaying the transmembrane pressure increase during constant-flux filtration. Characterization of the bacterial communities of dispersed cells and remaining biofilm cells using a 16S Illumina MiSeq metabarcoding approach demonstrated that biofilm dispersal by DETA NONOate had no selection bias in the community.
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•DETA NONOate showed the highest biofilm dispersal among the NO donors tested.•NO treatment by DETA NONOate mitigated biofouling in a lab-scale RO system.•Biofilm dispersal by NO treatment had no selection bias in the bacterial community.
In both natural and artificial environments, bacteria predominantly grow in biofilms, and bacteria often disperse from biofilms as freely suspended single-cells. In the present study, the formation ...and dispersal of planktonic cellular aggregates, or 'suspended biofilms', by Pseudomonas aeruginosa in liquid batch cultures were closely examined, and compared to biofilm formation on a matrix of polyester (PE) fibers as solid surface in batch cultures. Plankton samples were analyzed by laser-diffraction particle-size scanning (LDA) and microscopy of aggregates. Interestingly, LDA indicated that up to 90% of the total planktonic biomass consisted of cellular aggregates in the size range of 10-400 microm in diameter during the growth phase, as opposed to individual cells. In cultures with PE surfaces, P. aeruginosa preferred to grow in biofilms, as opposed to planktonicly. However, upon carbon, nitrogen or oxygen limitation, the planktonic aggregates and PE-attached biofilms dispersed into single cells, resulting in an increase in optical density (OD) independent of cellular growth. During growth, planktonic aggregates and PE-attached biofilms contained densely packed viable cells and extracellular DNA (eDNA), and starvation resulted in a loss of viable cells, and an increase in dead cells and eDNA. Furthermore, a release of metabolites and infective bacteriophage into the culture supernatant, and a marked decrease in intracellular concentration of the second messenger cyclic di-GMP, was observed in dispersing cultures. Thus, what traditionally has been described as planktonic, individual cell cultures of P. aeruginosa, are in fact suspended biofilms, and such aggregates have behaviors and responses (e.g. dispersal) similar to surface associated biofilms. In addition, we suggest that this planktonic biofilm model system can provide the basis for a detailed analysis of the synchronized biofilm life cycle of P. aeruginosa.
Persistence of the opportunistic bacterial pathogen Vibrio cholerae in aquatic environments is the principal cause for seasonal occurrence of cholera epidemics. This causality has been explained by ...postulating that V. cholerae forms biofilms in association with animate and inanimate surfaces. Alternatively, it has been proposed that bacterial pathogens are an integral part of the natural microbial food web and thus their survival is constrained by protozoan predation. Here, we report that both explanations are interrelated. Our data show that biofilms are the protective agent enabling V. cholerae to survive protozoan grazing while their planktonic counterparts are eliminated. Grazing on planktonic V. cholerae was found to select for the biofilm-enhancing rugose phase variant, which is adapted to the surface-associated niche by the production of exopolymers. Interestingly, grazing resistance in V. cholerae biofilms was not attained by exopolymer production alone but was accomplished by the secretion of an antiprotozoal factor that inhibits protozoan feeding activity. We identified that the cell density-dependent regulator hapR controls the production of this factor in biofilms. The inhibitory effect of V. cholerae biofilms was found to be widespread among toxigenic and non-toxigenic isolates. Our results provide a mechanistic explanation for the adaptive advantage of surface-associated growth in the environmental persistence of V. cholerae and suggest an important contribution of protozoan predation in the selective enrichment of biofilm-forming strains in the out-of-host environment.
Seawalls are important in protecting coastlines from currents, erosion, sea-level rise, and flooding. They are, however, associated with reduced biodiversity, due to their steep orientation, lack of ...microhabitats, and the materials used in their construction. Hence, there is considerable interest in modifying seawalls to enhance the settlement and diversity of marine organisms, as microbial biofilms play a critical role facilitating algal and invertebrate colonization. We assessed how different stone materials, ranging from aluminosilicates to limestone and concrete, affect biofilm formation. Metagenomic assessment of marine microbial communities indicated no significant impact of material on microbial diversity, irrespective of the diverse surface chemistry and topography. Based on KEGG pathway analysis, surface properties appeared to influence the community composition and function during the initial stages of biofilm development, but this effect disappeared by Day 31. We conclude that marine biofilms converged over time to a generic marine biofilm, rather than the underlying stone substrata type playing a significant role in driving community composition.
Carbon starvation has been shown to induce a massive dispersal event in biofilms of the opportunistic pathogen Pseudomonas aeruginosa; however, the molecular pathways controlling this dispersal ...response remain unknown. We quantified changes in the proteome of P. aeruginosa PAO1 biofilm and planktonic cells during glucose starvation by differential peptide-fingerprint mass-spectrometry (iTRAQ). In addition, we monitored dispersal photometrically, as a decrease in turbidity/opacity of biofilms pre-grown and starved in continuous flow-cells, in order to evaluate treatments (e.g. inhibitors CCCP, arsenate, chloramphenicol, L-serine hydroxamate) and key mutants altered in biofilm development and dispersal (e.g. nirS, vfr, bdlA, rpoS, lasRrhlR, Pf4-bacteriophage and cyaA). In wild-type biofilms, dispersal started within five minutes of glucose starvation, was maximal after 2 h, and up to 60% of the original biomass had dispersed after 24 h of starvation. The changes in protein synthesis were generally not more than two fold and indicated that more than 100 proteins belonging to various classes, including carbon and energy metabolism, stress adaptation, and motility, were differentially expressed. For the different treatments, only the proton-ionophore CCCP or arsenate, an inhibitor of ATP synthesis, prevented dispersal of the biofilms. For the different mutants tested, only cyaA, the synthase of the intracellular second messenger cAMP, failed to disperse; complementation of the cyaA mutation restored the wild-type phenotype. Hence, the pathway for carbon starvation-induced biofilm dispersal in P. aeruginosa PAO1 involves ATP production via direct ATP synthesis and proton-motive force dependent step(s) and is mediated through cAMP, which is likely to control the activity of proteins involved in remodeling biofilm cells in preparation for planktonic survival.
Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not ...completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation.
In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain.
This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.
It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in
PAO1. These behaviours are linked to ...the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the
bacteriophage P2 RepC protein. A mutation, Pf4r*, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r* protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r's immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in
biofilm development.
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