Biofilm Populations Research Articles

Metabolism Interactions Promote the Overall Functioning of the Episymbiotic Chemosynthetic Community of Shinkaia crosnieri of Cold Seeps.

ABSTRACTRemarkably diverse bacteria have been observed as biofilm aggregates on the surface of deep-sea invertebrates that support the growth of hosts through chemosynthetic carbon fixation. Growing evidence also indicates that community-wide interactions, and especially cooperation among symbionts, contribute to overall community productivity. Here, metagenome-guided metatranscriptomic and metabolic analyses were conducted to investigate the taxonomic composition, functions, and potential interactions of symbionts dwelling on the seta of Shinkaia crosnieri lobsters in a methane cold seep. Methylococcales and Thiotrichales dominated the community, followed by the Campylobacteriales, Nitrosococcales, Flavobacteriales, and Chitinophagales Metabolic interactions may be common among the episymbionts since many separate taxon genomes encoded complementary genes within metabolic pathways. Specifically, Thiotrichales could contribute to detoxification of hydroxylamine that is a metabolic by-product of Methylococcales. Further, Nitrosococcales may rely on methanol leaked from Methylococcales cells that efficiently oxidize methane. Elemental sulfur may also serve as a community good that enhances sulfur utilization that benefits the overall community, as evidenced by confocal Raman microscopy. Stable intermediates may connect symbiont metabolic activities in cyclical oxic-hypoxic fluctuating environments, which then enhance overall community functioning. This hypothesis was partially confirmed via in situ experiments. These results highlight the importance of microbe-microbe interactions in symbiosis and deep-sea adaptation.IMPORTANCE Symbioses between chemosynthetic bacteria and marine invertebrates are common in deep-sea chemosynthetic ecosystems and are considered critical foundations for deep-sea colonization. Episymbiotic microorganisms tend to form condensed biofilms that may facilitate metabolite sharing among biofilm populations. However, the prevalence of metabolic interactions among deep-sea episymbionts and their contributions to deep-sea adaptations are not well understood due to sampling and cultivation difficulties associated with deep-sea environments. Here, we investigated metabolic interactions among the episymbionts of Shinkaia crosnieri, a dominant chemosynthetic ecosystem lobster species in the Northwest Pacific Ocean. Meta-omics characterizations were conducted alongside in situ experiments to validate interaction hypotheses. Furthermore, imaging analysis was conducted, including electron microscopy, fluorescent in situ hybridization (FISH), and confocal Raman microscopy (CRM), to provide direct evidence of metabolic interactions. The results support the Black Queen Hypothesis, wherein leaked public goods are shared among cohabitating microorganisms to enhance the overall adaptability of the community via cooperation.

VxrB Influences Antagonism within Biofilms by Controlling Competition through Extracellular Matrix Production and Type 6 Secretion.

ABSTRACTThe human pathogen Vibrio cholerae grows as biofilms, communities of cells encased in an extracellular matrix. When growing in biofilms, cells compete for resources and space. One common competitive mechanism among Gram-negative bacteria is the type six secretion system (T6SS), which can deliver toxic effector proteins into a diverse group of target cells, including other bacteria, phagocytic amoebas, and human macrophages. The response regulator VxrB positively regulates both biofilm matrix and T6SS gene expression. Here, we directly observe T6SS activity within biofilms, which results in improved competition with strains lacking the T6SS. VxrB significantly contributes to both attack and defense via T6SS, while also influencing competition via regulation of biofilm matrix production. We further determined that both Vibrio polysaccharide (VPS) and the biofilm matrix protein RbmA can protect cells from T6SS attack within mature biofilms. By varying the spatial mixing of predator and prey cells in biofilms, we show that a high degree of mixing favors T6SS predator strains and that the presence of extracellular DNA in V. cholerae biofilms is a signature of T6SS killing. VxrB therefore regulates both T6SS attack and matrix-based T6SS defense, to control antagonistic interactions and competition outcomes during mixed-strain biofilm formation.

Combatting Planktonic and Biofilm Populations of Carbapenem-Resistant Acinetobacter baumannii with Polymyxin-Based Combinations.

Carbapenem-resistant Acinetobacter baumannii (CRAB) can cause serious infections that are associated with high mortality rates. During the course of an infection, many CRAB isolates are able to form biofilms, which are recalcitrant to several antibiotics and can be difficult to treat. Polymyxin-based regimens are a first-line treatment option for CRAB infections, but they have not been optimized against both planktonic and biofilm phases of growth. The objective of this study was to identify polymyxin-based combinations that are active against planktonic and biofilm populations of CRAB. Four CRAB isolates (meropenem MICs: 8–256 mg/L) capable of forming biofilms were used in each experiment. The activities of polymyxin B alone and in combination with ampicillin/sulbactam, meropenem, minocycline, and rifampin were assessed using time-kill assays, with the CRAB isolates grown in planktonic and biofilm phases. Viable colony counts were used to detect the bactericidal activity and synergy of the antibiotic combinations. Against the planktonic populations, polymyxin B combined with meropenem, minocycline, ampicillin/sulbactam, and rifampin caused 3.78, −0.15, 4.38, and 3.23 mean log10 CFU/mL reductions against all isolates at 24 h, respectively. Polymyxin B combined with meropenem, ampicillin/sulbactam, or rifampin was synergistic against 75–100% (3/4 or 4/4) of CRAB isolates. Against biofilms, polymyxin B combined with meropenem, minocycline, ampicillin/sulbactam, and rifampin caused 1.86, 1.01, 0.66, and 3.55 mean log10 CFU/mL reductions against all isolates at 24 h, respectively. Only the combination of polymyxin B and rifampin retained bactericidal activity or synergy against any of the isolates when grown as biofilms (50% of isolates). The combination of polymyxin B and rifampin may be promising for CRAB infections that have planktonic and biofilm populations present.

A Noninvasive Method for Time-Lapse Imaging of Microbial Interactions and Colony Dynamics.

ABSTRACTComplex interactions between microbial populations can greatly affect the overall properties of a microbial community, sometimes leading to cooperation and mutually beneficial coexistence, or competition and the death or displacement of organisms or subpopulations. Interactions between different biofilm populations are highly relevant in diverse scientific areas, from antimicrobial resistance to microbial ecology. The utilization of modern microscopic techniques has provided a new and interesting insight into how bacteria interact at the cellular level to form and maintain microbial biofilms. However, our ability to follow complex intraspecies and interspecies interactions in vivo at the microscopic level has remained somewhat limited. Here, we detailed BacLive, a novel noninvasive method for tracking bacterial growth and biofilm dynamics using high-resolution fluorescence microscopy and an associated ImageJ processing macro (https://github.com/BacLive) for easier data handling and image analysis. Finally, we provided examples of how BacLive can be used in the analysis of complex bacterial communities.IMPORTANCE Communication and interactions between single cells are continuously defining the structure and composition of microbial communities temporally and spatially. Methods routinely used to study these communities at the cellular level rely on sample manipulation which makes microscopic time-lapse experiments impossible. BacLive was conceived as a method for the noninvasive study of the formation and development of bacterial communities, such as biofilms, and the formation dynamics of specialized subpopulations in time-lapse experiments at a colony level. In addition, we developed a tool to simplify the processing and analysis of the data generated by this method.

In vitro interactions within a biofilm containing three species found in bacterial vaginosis (BV) support the higher antimicrobial tolerance associated with BV recurrence.

Bacterial vaginosis (BV), the most common cause of vaginal discharge, is characterized by the presence of a polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp., but also other anaerobic species. Interactions between bacteria in multi-species biofilms are likely to contribute to increased virulence and to enhanced antimicrobial tolerance observed in vivo. However, functional studies addressing this question are lacking. To gain insights into the role that interactions between BV-associated species in multi-species BV biofilms might have on antimicrobial tolerance, single- and triple-species biofilms formed by Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae and Peptostreptococcus anaerobius were characterized, before and after metronidazole or clindamycin treatment. Total biofilm biomass, total cells and cfu counts prior to and after antibiotic treatment were first determined. In addition, bacterial populations in the triple-species biofilms were also quantified by quantitative PCR (qPCR) and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH). Despite the effect observed in single-species biofilms, neither metronidazole nor clindamycin was effective in reducing triple-species biofilm biomass. Similar results were obtained when evaluating the number of total or culturable cells. Interestingly, despite differences between strain susceptibilities to antibiotics, the composition of the triple-species biofilms was not strongly affected by antibiotics. Taken together, these results strengthen the idea that, when co-incubated, bacteria can interact synergistically, leading to increased tolerance to antimicrobial therapy, which helps explain the observed clinically high BV recurrence rates.

Time after time: detecting annual patterns in stream bacterial biofilm communities.

SummaryTo quantify the major environmental drivers of stream bacterial population dynamics, we modelled temporal differences in stream bacterial communities to quantify community shifts, including those relating to cyclical seasonal variation and more sporadic bloom events. We applied Illumina MiSeq 16S rRNA bacterial gene sequencing of 892 stream biofilm samples, collected monthly for 36‐months from six streams. The streams were located a maximum of 118 km apart and drained three different catchment types (forest, urban and rural land uses). We identified repeatable seasonal patterns among bacterial taxa, allowing their separation into three ecological groupings, those following linear, bloom/trough and repeated, seasonal trends. Various physicochemical parameters (light, water and air temperature, pH, dissolved oxygen, nutrients) were linked to temporal community changes. Our models indicate that bloom events and seasonal episodes modify biofilm bacterial populations, suggesting that distinct microbial taxa thrive during these events including non‐cyanobacterial community members. These models could aid in determining how temporal environmental changes affect community assembly and guide the selection of appropriate statistical models to capture future community responses to environmental change.

The interplay of active energy harvesting and wastewater organic loading regulates fermentation products and microbiomes in microbial fuel cells

Fruit juice wastewater (FJW) contains high strength and acidic organics that are difficult to remove. We developed microbial fuel cells (MFCs) to treat FJW and employed new energy harvesting methods to investigate how different operation conditions influence system performance and microbial community assembly. Specifically, we found both energy harvesting regime and organic loading affected current production, organic matter decomposition, product distribution, and microbial community. The power output under maximum power harvesting (MFC-MPP) condition showed the most stable output of 300 mW/m2 under different organic loadings. The MFC reactors maintained good organic removal (82.10–94.65%) at low loading of 1500–3000 mg/L, but the removal decreased with high loadings (7500–15,000 mg/L), presumably due to insufficient retention time, acid accumulation, and reduced degradability. Accordingly, higher VFA concentrations were observed in high loading reactors with the dominant products, formic acid and acetic acid. Electrofermentation coefficients (0.02–23.85%) of MFCs suggested that microaerobic electrofermentation (MEF) occurred in all reactors. MFCs under active harvesting demonstrated lower community diversity of anode biofilms at high organic loadings. The majority of dominant populations in the anode biofilm was affiliated with Lactococcus, Veillonella, and Bacteroides. This work demonstrates active energy harvesting and organic loading had interplay effects on system performance and microbiome, and system operation should consider such impacts to improve performance. AEH also provides a new alternative to a poised potential strategy for electrofermentation controlling.

Viral diversity is linked to bacterial community composition in alpine stream biofilms

Biofilms play pivotal roles in fluvial ecosystems, yet virtually nothing is known about viruses in these communities. Leveraging an optimized sample-to-sequence pipeline, we studied the spatiotemporal turnover of dsDNA viruses associated with stream biofilms and found an astounding diversity to be structured by seasons and along the longitudinal gradient in the stream. While some vOTUs were region- or season-specific, we also identified a large group of permanent biofilm phages, taxonomically dominated by Myoviridae. Comparison of the observed viral distribution with predictions based on neutral community assembly indicated that chance and dispersal may be important for structuring stream biofilm viral communities. Deviation from neutral model predictions suggests that certain phages distribute efficiently across distant locations within the stream network. This dispersal capacity appears to be linked to EPS depolymerases that enable phages to efficiently overcome the biofilm barrier. Other phages, particularly vOTUs classified as Siphoviridae, appear locally overrepresented and to rely on a lysogenic life cycle, potentially to exploit the spatial distribution of bacterial populations in stream biofilms. Overall, biofilm viral and bacterial community turnover were significantly coupled. Yet, viral communities were linked to the presence of the most abundant bacterial community members. With this work, we provide a foundational ecological perspective on factors that structure viral diversity in stream biofilms and identify potentially important viral traits related to the biofilm mode of life.

Bacterial metataxonomic analysis of industrial Spanish-style green table olive fermentations

In this work, metataxonomic-compositional data analysis was applied to study the influence of inoculation, type of ecosystem (brine or fruit), and time of fermentation (0, 24, and 83 days) on the bacterial composition dynamic during industrial Manzanilla Spanish-style green table olive fermentations. Differences in the physicochemical and microbiological characteristics between inoculated and spontaneous fermentations were scarce, except for the high lactic acid bacteria population (>10 million CFU/g) in the inoculated olive biofilms. The metataxonomic analysis showed eight relevant taxa (Lactiplantibacillus, Vibrio, Halolactibacillus, Aerococcus, Catenococcus, Salinivibrio, Alkalibacterium, and Marinilactibacillus) and absence of pathogens, but the microbiota composition markedly changed throughout fermentation. At the process onset, the main genera were Vibrio, Halolactibacillus, Marinilactibacillus, and Alkalibacterium, while Lactiplantibacillus was almost absent. Regardless of inoculation, Lactiplantibacillus became the predominant genus as the fermentation progressed, albeit Vibrio, Marinilactibacillus, Alkalibacterium, and Halolactibacillus also persisted until the end of the process (but did not cause spoilage). Ferrimonas genus was detected for the first time in table olives, being found in 71% of the samples. Besides, there was more bacterial biodiversity in the brine ecosystem than olive biofilms. Overall, inoculation led to more homogenous and equilibrated bacterial populations at the end of the process. This study expands the knowledge on the bacterial population changes occurring during industrial table olive fermentation, the differences between populations in brines and biofilms, and the beneficial effects of inoculation for using table olives as a carrier of beneficial microorganisms.

Modular 3D-Printed Peg Biofilm Device for Flexible Setup of Surface-Related Biofilm Studies.

Medical device-related biofilms are a major cause of hospital-acquired infections, especially chronic infections. Numerous diverse models to study surface-associated biofilms have been developed; however, their usability varies. Often, a simple method is desired without sacrificing throughput and biological relevance. Here, we present an in-house developed 3D-printed device (FlexiPeg) for biofilm growth, conceptually similar to the Calgary Biofilm device but aimed at increasing ease of use and versatility. Our device is modular with the lid and pegs as separate units, enabling flexible assembly with up- or down-scaling depending on the aims of the study. It also allows easy handling of individual pegs, especially when disruption of biofilm populations is needed for downstream analysis. The pegs can be printed in, or coated with, different materials to create surfaces relevant to the study of interest. We experimentally validated the use of the device by exploring the biofilms formed by clinical strains of Escherichia coli and Klebsiella pneumoniae, commonly associated with device-related infections. The biofilms were characterized by viable cell counts, biomass staining, and scanning electron microscopy (SEM) imaging. We evaluated the effects of different additive manufacturing technologies, 3D printing resins, and coatings with, for example, silicone, to mimic a medical device surface. The biofilms formed on our custom-made pegs could be clearly distinguished based on species or strain across all performed assays, and they corresponded well with observations made in other models and clinical settings, for example, on urinary catheters. Overall, our biofilm device is a robust, easy-to-use, and relevant assay, suitable for a wide range of applications in surface-associated biofilm studies, including materials testing, screening for biofilm formation capacity, and antibiotic susceptibility testing.

Effects of Methyl, Ester, and Amine Surface Groups on Microbial Activity and Communities in Nitrifying Biofilms.

The objective of this study was to determine how different attachment surface chemistries affected the initial and long-term performance and microbial populations of nitrifying biofilms under well-controlled hydrodynamic mixing conditions. While much previous research has focused on the effects of surface properties such as hydrophobicity on bacterial attachment in pure cultures, this study evaluated the effects of specific functional groups on mixed culture composition and functional behavior. Three surfaces with varying hydrophobicity and charge were evaluated for biofilm community development and performance: unmodified poly(dimethylsiloxane) (PDMS), which included terminal methyl groups and was relatively hydrophobic (P-Methyl), PDMS silanized with ester groups (P-Ester), which was uncharged and relatively hydrophilic, and PDMS modified with amine groups (P-Amine), which possessed a positive charge and was the most hydrophilic. The surface chemistries of the three attachment surfaces were characterized by contact angle goniometry, Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). These surfaces were inoculated with dilute activated sludge, and biofilms were grown in rotating annular bioreactors for 80 days, with experimental triplicates. Nitrification rates increased most rapidly in P-Amine biofilm reactors, and their biofilm communities contained significantly more Nitrosomonas (p < 0.05) than those on the other surfaces in early growth stages (days 40-50). From days 50-60, the P-Amine surface biofilm had significantly higher nitrate production rates than the P-Methyl and P-Ester biofilms. The biofilms grown on the P-Amine and P-Methyl surfaces were significantly (p < 0.05) more diverse than the P-Ester biofilms, containing higher relative abundances of the order Rhizobiales, including a significantly higher abundance of the nitrifying genus Nitrobacter (p < 0.05), which coincided with higher rates of nitrate generation. Conversely, biofilms grown on the uncharged hydrophilic P-Ester surface were consistently less productive and had lower diversity than biofilms on the other surfaces. These results indicate that surface chemistry may be a useful design parameter to improve the performance of nitrifying biofilm systems for wastewater treatment and that surface chemistry affects mixed biofilm community composition.

Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity

Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial, for example, in agricultural as well as in food technology contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity, the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level; however, the molecular structures and elemental distribution across whole biofilms, as well as possible linkages between them, remain unexplored. Mapping X-ray diffraction across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores, and water. By simultaneously following the X-ray fluorescence signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese, and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and may possibly lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution, and sporulation across whole, intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.

Collective bacterial disinfection by opto-chemical treatment on mature biofilm in clinical endoscope.

The present study proposes an innovative opto-chemical treatment using a basket-integrated optical device (BIOD) to disinfect mature bacterial biofilm on endoscope channels. A BIOD was designed to position an optical diffuser on the central axis of an endoscope channel and to distribute laser light concentrically to the bacterial biofilm on the channel surface. To apply thermal damage and oxidative stress to the bacterial biofilm, a low concentration of a crosslinking agent (glutaraldehyde ~0.5%) was combined with 808nm infrared (IR) and 405nm blue (BL) laser lights. The applied irradiances of IR and BL were 10W/cm2 and 1.6W/cm2 for Teflon channel model and 20W/cm2 and 3.2W/cm2 for a clinical model, respectively. Individual irradiation of either IR or BL for 180s induced the maximum temperatures of 62±2°C and 53±3°C on the biofilm, respectively. The simultaneous opto-chemical treatment reduced a significant population of the bacterial biofilms (7.5-log10 for Staphylococcus aureus and 7.1-log10 for Pseudomonas aeruginosa), which were 2.9-fold and 3.9-fold higher than that of the standard treatment with 2% glutaraldehyde (GA) solution, respectively. The proposed opto-chemical disinfection method can help reduce multi-drug resistant bacteria and prevent cross-infection during the clinical usage of a flexible endoscope.

Microbial growth and attachment of Salmonella and enterohemorrhagic and enteroaggregative Escherichia coli strains on cress microgreens grown in peat soil system

PurposeThis study aimed to investigate how enteric pathogens and their biofilm populations on fresh produce survive according to time that contamination has occurred on leaves and contamination route: seed irrigation water.Design/methodology/approachCress was contaminated in two different ways: contamination of seeds and irrigation water with 8-log MPN/mL bacterial load, Salmonella Newport, Escherichia coli O157:H7, O104:H4 or O78:H2. While contaminated seeds were cultivated for seed contamination, contaminated irrigation was applied at the end of each week to separate groups of samples obtained from cultivated surface-sterile seeds to understand how long these pathogens could survive until harvest.FindingsThe results indicated these pathogens survived until harvest, and formed biofilms on cress leaves grown using both contaminated seeds and irrigation water. No significant difference was observed among populations of Salmonella and E. coli groups in terms of survival (∼4.5–6.0 log MPN/g) and biofilm formation (∼4.4–5.7 log MPN/g) for contamination by seed. Also, SEM images revealed biofilm-like structures, the proofs of the attachment of these pathogens on leaf surfaces.Originality/valueFrom our knowledge this is the first study focusing on the survival and biofilm formation of one Salmonella serotype (Newport) and three E. coli serotypes (O157:H7, O104:H4, and O78:H2), representing enterohemorrhagic and enteroaggregative E. coli pathogenic subgroups, under the same irrigation and growth schemes. Furthermore, this study mimics the contamination of seeds and irrigation water with sewage or wastewater and may shed light on contamination of fresh produce grown using poor wastewater treatment.

Phosphorus Concentration in Water Affects the Biofilm Community and the Produced Amount of Extracellular Polymeric Substances in Reverse Osmosis Membrane Systems.

Biofouling is a problem that hinders sustainable membrane-based desalination and the stratification of bacterial populations over the biofilm’s height is suggested to compromise the efficiency of cleaning strategies. Some studies reported a base biofilm layer attached to the membrane that is harder to remove. Previous research suggested limiting the concentration of phosphorus in the feed water as a biofouling control strategy. However, the existence of bacterial communities growing under phosphorus-limiting conditions and communities remaining after cleaning is unknown. This study analyzes the bacterial communities developed in biofilms grown in membrane fouling simulators (MFSs) supplied with water with three dosed phosphorus conditions at a constant biodegradable carbon concentration. After biofilm development, biofilm was removed using forward flushing (an easy-to-implement and environmentally friendly method) by increasing the crossflow velocity for one hour. We demonstrate that small changes in phosphorus concentration in the feed water led to (i) different microbial compositions and (ii) different bacterial-cells-to-EPS ratios, while (iii) similar bacterial biofilm populations remained after forward flushing, suggesting a homogenous bacterial community composition along the biofilm height. This study represents an exciting advance towards greener desalination by applying non-expensive physical cleaning methods while manipulating feed water nutrient conditions to prolong membrane system performance and enhance membrane cleanability.

Insights into the antibiotic resistance in Biofilms – A Review

The biofilms comprise a population of bacteria with a varying variety of polysaccharides, proteins and DNA. Bacterial multi-phase defence consists of low antibiotic absorption, sluggish replication in the biofilm, and adaptive stress response. This antibiotic resistance produced by biofilm makes it challenging to deal with bacteria with effective antibiotic dosages in planktonic forms. A crucial component in the virulent colonisation of live tissues or medical equipment is having favourable situation for bacteria to create biofilms. The high level of recalcitrance in biofilm populations is due to several molecular pathways. As the stock of effective antibiotics is depleting, bacterial resistance is becoming an increasing risk to public health. As a result, new antibiotics are urgently needed. This review includes current empirical findings related to antibiotic resistance in biofilms and summarises the biofilm resistance and tolerance mechanisms.

Low strength wastewater anammox start-up by stepwise decrement in influent nitrogen: Biofilm formation mechanism and mathematical modelling

The application of mainstream anammox process is hampered by its overlong start-up and instability under disturbance. A lab-scale mainstream anammox moving bed biofilm reactor (MBBR) was successfully started in 120 days with stepwise decrement in influent nitrogen concentration from sidestream to mainstream condition. The initial colonization by Candidatus Jettenia and filamentous fermenter Anaerolineaceae were potentially mediated by hydrophobic interaction and type IV pilus. Ca. Kuenenia with higher substrate affinity outcompeted Ca. Jettenia, and the predominant fermenters shifted to fermentative Ignavibacteriaceae in the mature biofilm. A novel mainstream anammox biofilm development (MABD) model was constructed to describe biofilm growth, population dynamics, and nitrogen removal performance. The simulation results suggested that higher inocula biomass (460–690 mgVSS·L−1), relative abundance of low-affinity AnAOB in the inocula (e.g., Ca. Jettenia, 1.3–2%), and the early-stage solids retention time (45–68 days) were desired to form thicker biofilm and improve effluent quality during 120-day mainstream anammox MBBR start-up. The mechanistic insights into biofilm formation and predictive power of the newly developed MABD model are of importance to the design and operation of mainstream anammox processes towards more biofilm biomass and higher nitrogen removal efficiency.

Enrichment of Clostridia enhances Geobacter population and electron harvesting in a complex electroactive biofilm

Current research on microbial fuel cell or microbial electrolysis cell dealt with finding new electroactive bacteria and understanding the mechanisms of electronic exchange. Complex consortia allowed to obtain better performances than pure cultures in part thanks to inter-species cooperation. However, the role of each bacterium in a complex biofilm in the electron harvest on an electrode remains unclear. Thus, we combined electrochemical monitoring of electron exchange and high throughput sequencing analysis in order to describe the bacterial composition and the electroactive performance of mangrove mud biofilms. In this study, secondary electroactive biofilms were formed on carbon electrodes from Desulfuromonas-dominated inoculum of pre-formed bioanodes. The performances and the Desulfuromonas-dominated profile were the same as those of primary bioanodes when the planktonic community was conserved. However, a Clostridium enrichment allowed to restore the performance in maximal current densities promoting an increase of Geobacter population, becoming the most dominant group among the Deltaproteobacteria, replacing Desulfuromonas. These results highlight a positive collaboration between Clostridium and Geobacter spp. helping a bacterial population to achieve with the depletion of their environment. Our study provides new insight into relationships between dominant electroactive bacteria and other bacteria species living in an organic matter-rich environment as mangrove sediments.

Effects of dispersal on species distribution, abundance, diversity and interaction in a bacterial biofilm metacommunity.

Dispersal effects on biofilms have not been adequately studied despite their strong potential impacts on biofilm development. We investigated the effects of dispersal on biofilm metacommunity. A bacterial consortium was allowed to form biofilms on 12glass beads attached to disposable plates (compartmentalized or not), and biofilms were scrutinized on days 5, 10 and 15 using quantitative PCR and MiSeq sequencing. Biofilm population density was lesser by 2 orders of magnitude on day 5 when dispersal was allowed (p<0.05). Then, the population rapidly increased by 4.4 orders with dispersal (p<0.05) but did not change without dispersal. Community analyses revealed that dispersal increased the species diversity at all sampling times (p<0.05). Dispersal affected the community structure and increased the homogeneity of local communities (p<0.05). Distance-decay analysis showed that dispersal reduced the dissimilarity among local communities at all distance levels. Furthermore, dispersal reduced the variability of diversity, population and community structure. Network analysis revealed that dispersal increased the clustering coefficient, network density and connectivity. Dispersal increased the species diversity, population and interaction and reduced the variability of the diversity, population and structure among local communities. Our results suggest that dispersal can induce the niche complementarity and mass effects.

Genomic Evolution of the Marine Bacterium Phaeobacter inhibens during Biofilm Growth.

Phaeobacter inhibens 2.10 is an effective biofilm former on marine surfaces and has the ability to outcompete other microorganisms, possibly due to the production of the plasmid-encoded secondary metabolite tropodithietic acid (TDA). P. inhibens 2.10 biofilms produce phenotypic variants with reduced competitiveness compared to the wild type. In the present study, we used longitudinal, genome-wide deep sequencing to uncover the genetic foundation that contributes to the emergent phenotypic diversity in P. inhibens 2.10 biofilm dispersants. Our results show that phenotypic variation is not due to the loss of the plasmid that carries the genes for TDA synthesis but instead show that P. inhibens 2.10 biofilm populations become rapidly enriched in single nucleotide variations in genes involved in the synthesis of TDA. While variants in genes previously linked to other phenotypes, such as lipopolysaccharide production (i.e., rfbA) and cellular persistence (i.e., metG), also appear to be selected for during biofilm dispersal, the number and consistency of variations found for genes involved in TDA production suggest that this metabolite imposes a burden on P. inhibens 2.10 cells. Our results indicate a strong selection pressure for the loss of TDA in monospecies biofilm populations and provide insight into how competition (or a lack thereof) in biofilms might shape genome evolution in bacteria. IMPORTANCE Biofilm formation and dispersal are important survival strategies for environmental bacteria. During biofilm dispersal, cells often display stable and heritable variants from the parental biofilm. Phaeobacter inhibens is an effective colonizer of marine surfaces, in which a subpopulation of its biofilm dispersal cells displays a noncompetitive phenotype. This study aimed to elucidate the genetic basis of these phenotypic changes. Despite the progress made to date in characterizing the dispersal variants in P. inhibens, little is understood about the underlying genetic changes that result in the development of the specific variants. Here, P. inhibens phenotypic variation was linked to single nucleotide polymorphisms (SNPs), in particular in genes affecting the competitive ability of P. inhibens, including genes related to the production of the antibiotic tropodithietic acid (TDA) and bacterial cell-cell communication (e.g., quorum sensing). This work is significant as it reveals how the biofilm lifestyle might shape genome evolution in a cosmopolitan bacterium.