Environmental switching during biofilm development in a cold seep system and functional determinants of species sorting

The functional basis for species sorting theory remains elusive, especially for microbial community assembly in deep‐sea environments. Using artificial surface‐based biofilm models, our recent work revealed taxonomic succession during biofilm development in a newly defined cold seep system, the Thuwal cold seeps II, which comprises a brine pool and the adjacent normal bottom water (NBW) to form a metacommunity via the potential immigration of organisms from one patch to another. Here, we designed an experiment to investigate the effects of environmental switching between the brine pool and the NBW on biofilm assembly, which could reflect environmental filtering effects during bacterial immigration to new environments. Analyses of 16S rRNA genes of 71 biofilm samples suggested that the microbial composition of biofilms established in new environments was determined by both the source community and the incubation conditions. Moreover, a comparison of 18 metagenomes provided evidence for biofilm community assembly that was based primarily on functional features rather than taxonomic identities; metal ion resistance and amino acid metabolism were the major species sorting determinants for the succession of biofilm communities. Genome binning and pathway reconstruction of two bacterial species (Marinobacter sp. and Oleispira sp.) further demonstrated metal ion resistance and amino acid metabolism as functional traits conferring the survival of habitat generalists in both the brine pool and NBW. The results of this study shed new light on microbial community assembly in special habitats and bridge a gap in species sorting theory.     

Biofilm—“City of microbes” or an analogue of multicellular organisms?

The definition of the term “biofilm” and the validity of the analogy between these structured microbial communities and multicellular organisms are discussed in the review. The mechanisms of biofilm formation, the types of interrelations of the components of biofilms, and the reasons for biofilm resistance to biocides and stress factors are considered in detail. The role of biofilms in microbial ecology and in biotechnology is discussed.     

Biofilm--"City of microbes" or an analogue of multicellular organisms?

The definition of the term "biofilm" and the validity of the analogy between these structured microbial communities and multicellular organisms are discussed in the review. The mechanisms of biofilm formation, the types of interrelations of the components of biofilms, and the reasons for biofilm resistance to biocides and stress factors are considered in detail. The role of biofilms in microbial ecology and in biotechnology is discussed.     

Metagenomic and metaproteomic analyses of Accumulibacter phosphatis‐enriched floccular and granular biofilm

Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory‐scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high‐stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter‐enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.     

Mineral‐hosted biofilm communities in the continental deep subsurface,Deep Mine Microbial Observatory,SD, USA

Deep subsurface biofilms are estimated to host the majority of prokaryotic life on Earth, yet fundamental aspects of their ecology remain unknown. An inherent difficulty in studying subsurface biofilms is that of sample acquisition. While samples from marine and terrestrial deep subsurface fluids have revealed abundant and diverse microbial life, limited work has described the corresponding biofilms on rock fracture and pore space surfaces. The recently established Deep Mine Microbial Observatory (DeMMO) is a long‐term monitoring network at which we can explore the ecological role of biofilms in fluid‐filled fractures to depths of 1.5 km. We carried out in situ cultivation experiments with single minerals representative of DeMMO host rock to explore the ecological drivers of biodiversity and biomass in biofilm communities in the continental subsurface. Coupling cell densities to thermodynamic models of putative metabolic reactions with minerals suggests a metabolic relationship between biofilms and the minerals they colonize. Our findings indicate that minerals can significantly enhance biofilm cell densities and promote selective colonization by taxa putatively capable of extracellular electron transfer. In turn, minerals can drive significant differences in biodiversity between fluid and biofilm communities. Given our findings at DeMMO, we suggest that host rock mineralogy is an important ecological driver in deep continental biospheres.     

Antimicrobial activity of surface attached marine bacteria in biofilms

Relatively little is known about the microbial ecology of biofilm communities or the diversity of antimicrobial molecules that they produce to regulate these communities. This study tested whether the production of antimicrobial activity in biofilm cultures is enhanced towards competing bacteria found in those biofilms. First, the production of antimicrobial activity of marine bacteria grown in biofilms was tested. Fourteen of the 105 marine isolates tested were found to produce antimicrobial factors when grown in biofilms. The antimicrobial activity produced by these isolates in biofilms was more potent and inhibited a broader range of target bacteria grown in biofilms compared to shaken liquid cultures. In a separate experiment, we found that cultivation in biofilms containing produced metabolites from an ‘inducer’ bacterium stimulated the production of antimicrobial molecules by ‘producer’ bacteria that were active against the ‘inducer’ bacterium. Overall, the study suggests that surface attached marine bacteria can target their antimicrobial activity towards competing bacteria in biofilms.     

Biophysical controls on cluster dynamics and architectural differentiation of microbial biofilms in contrasting flow environments

Ecology, with a traditional focus on plants and animals, seeks to understand the mechanisms underlying structure and dynamics of communities. In microbial ecology, the focus is changing from planktonic communities to attached biofilms that dominate microbial life in numerous systems. Therefore, interest in the structure and function of biofilms is on the rise. Biofilms can form reproducible physical structures (i.e. architecture) at the millimetre‐scale, which are central to their functioning. However, the spatial dynamics of the clusters conferring physical structure to biofilms remains often elusive. By experimenting with complex microbial communities forming biofilms in contrasting hydrodynamic microenvironments in stream mesocosms, we show that morphogenesis results in ‘ripple‐like’ and ‘star‐like’ architectures – as they have also been reported from monospecies bacterial biofilms, for instance. To explore the potential contribution of demographic processes to these architectures, we propose a size‐structured population model to simulate the dynamics of biofilm growth and cluster size distribution. Our findings establish that basic physical and demographic processes are key forces that shape apparently universal biofilm architectures as they occur in diverse microbial but also in single‐species bacterial biofilms.     

High bacterial diversity in nearshore and oceanic biofilms and their influence on larval settlement by Hydroides elegans (Polychaeta)

Settlement of many benthic marine invertebrates is stimulated by bacterial biofilms, although it is not known if patterns of settlement reflect microbial communities that are specific to discrete habitats. Here, we characterized the taxonomic and functional gene diversity (16S rRNA gene amplicon and metagenomic sequencing analyses), as well as the specific bacterial abundances, in biofilms from diverse nearby and distant locations, both inshore and offshore, and tested them for their ability to induce settlement of the biofouling tubeworm Hydroides elegans, an inhabitant of bays and harbours around the world. We found that compositions of the bacterial biofilms were site specific, with the greatest differences between inshore and offshore sites. Further, biofilms were highly diverse in their taxonomic and functional compositions across inshore sites, while relatively low diversity was found at offshore sites. Hydroides elegans settled on all biofilms tested, with settlement strongly correlated with bacterial abundance. Bacterial density in biofilms was positively correlated with biofilm age. Our results suggest that the localized distribution of H. elegans is not determined by ‘selection’ to locations by specific bacteria, but it is more likely linked to the prevailing local ecology and oceanographic features that affect the development of dense biofilms and the occurrence of larvae.     

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the calgary biofilm device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.     

Neutral Models of Microbiome Evolution

There has been an explosion of research on host-associated microbial communities (i.e.,microbiomes). Much of this research has focused on surveys of microbial diversities across a variety of host species, including humans, with a view to understanding how these microbiomes are distributed across space and time, and how they correlate with host health, disease, phenotype, physiology and ecology. Fewer studies have focused on how these microbiomes may have evolved. In this paper, we develop an agent-based framework to study the dynamics of microbiome evolution. Our framework incorporates neutral models of how hosts acquire their microbiomes, and how the environmental microbial community that is available to the hosts is assembled. Most importantly, our framework also incorporates a Wright-Fisher genealogical model of hosts, so that the dynamics of microbiome evolution is studied on an evolutionary timescale. Our results indicate that the extent of parental contribution to microbial availability from one generation to the next significantly impacts the diversity of microbiomes: the greater the parental contribution, the less diverse the microbiomes. In contrast, even when there is only a very small contribution from a constant environmental pool, microbial communities can remain highly diverse. Finally, we show that our models may be used to construct hypotheses about the types of processes that operate to assemble microbiomes over evolutionary time.     

Characterisation of the bacteria associated with barnacle,Balanus amphitrite,shell and their role in gregarious settlement of cypris larvae

The acorn barnacle Balanus amphitrite (syn. Amphibalanus amphitrite) is a model organism to investigate pelago-benthic transitions in marine invertebrates. A driver for larval settlement in this organism is the need to attach close to conspecifics, to allow reproduction to take place. Adult barnacles are covered by microbial biofilms and the contribution of these biofilms to conspecific recognition is not fully understood. Little information is available on microbial communities associated with B. amphitrite. We compared biofilm communities from the barnacle shell surface with those from the surrounding rocks using the culture-independent methods of quantitative PCR and denaturing gradient gel electrophoresis. Quantification of the relative abundances of higher bacterial taxa showed that barnacles hosted a greater proportion of α-Proteobacteria compared to rock-associated biofilms (p < 0.01). Differences in relative abundances of other taxa were not observed but DGGE profiling suggested that differences were present at lower taxonomic levels. The capacity of these communities to influence larval settlement was assessed by growing multispecies biofilms on artificial medium, obtained by extracting nutrients from adult barnacles. Biofilms composed of shell-associated bacteria were capable of promoting conspecific settlement by 67% compared to control surfaces (p < 0.05), while rock-associated communities showed contrasting effects. A taxonomic comparison of settlement-stimulating and -inhibiting bacteria was performed by DGGE and band sequencing. All partial 16S rRNA genes sequenced were similar to members of the Vibrio and Pseudoalteromonas genera, suggesting that larvae can detect and respond to variations in the composition of microbial biofilms at low taxonomic levels. Our results indicate that barnacle larvae may be able to detect parentally-associated biofilms and use this information to settle close to members of its own species.     

Exoenzyme accumulation in epilithic biofilms

Although exoenzyme accumulation is often proposed as an explanation for the high metabolic activity of biofilms, little is known about the abundance, distribution and turnover rates of exoenzymes within these communities. To assess accumulation, epilithic biofilm samples were collected from a fourth-order boreal river and homogenized. The resulting particles were fractionated by size and each fraction was assayed for nine exoenzyme activities, chlorophyll, and ATP. In general, carbohydrase activities were not correlated with microbial biomass indicators; the largest pool of activity was in the aqueous phase (< 0.2 µm). Phenol oxidase, peroxidase, and phosphatase activities were largely particle-bound and often correlated with microbial biomass distribution. It was concluded that the epilithic biofilm matrix was effective at accumulating carbohydrase activity and that this accumulation may partially account for the metabolic resistance of epilithic biofilms to dissolved organic matter fluctuations.     

Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin activity

Due to the refractory nature of pathogenic microbial biofilms, innovative biofilm eradication strategies are constantly being sought. Thus, this study addresses a novel approach to eradicate Pseudomonas aeruginosa biofilms. Magnetic nanoparticles (MNP), ciprofloxacin (Cipro), and magnetic fields were systematically evaluated in vitro for their relative anti-biofilm contributions. Twenty-four-hour biofilms exposed to aerosolized MNPs, Cipro, or a combination of both, were assessed in the presence or absence of magnetic fields (Static one-sided, Static switched, Oscillating, Static + oscillating) using changes in bacterial metabolism, biofilm biomass, and biofilm imaging. The biofilms exposed to magnetic fields alone exhibited significant metabolic and biomass reductions (p < 0.05). When biofilms were treated with a MNP/Cipro combination, the most significant metabolic and biomass reductions were observed when exposed to static switched magnetic fields (p < 0.05). The exposure of P. aeruginosa biofilms to a static switched magnetic field alone, or co-administration with MNP/Cipro/MNP + Cipro appears to be a promising approach to eradicate biofilms of this bacterium.     

Influence of an Oyster Reef on Development of the Microbial Heterotrophic Community of an Estuarine Biofilm

We characterized microbial biofilm communities developed over two very closely located but distinct benthic habitats in the Pensacola Bay estuary using two complementary cultivation-independent molecular techniques. Biofilms were grown for 7 days on glass slides held in racks 10 to 15 cm over an oyster reef and an adjacent muddy sand bottom. Total biomass and optical densities of dried biofilms showed dramatic differences for oyster reef versus non-oyster reef biofilms. This study assessed whether the observed spatial variation was reflected in the heterotrophic prokaryotic species composition. Genomic biofilm DNA from both locations was isolated and served as a template to amplify 16S rRNA genes with universal eubacterial primers. Fluorescently labeled PCR products were analyzed by terminal restriction fragment length polymorphism, creating a genetic fingerprint of the composition of the microbial communities. Unlabeled PCR products were cloned in order to construct a clone library of 16S rRNA genes. Amplified ribosomal DNA restriction analysis was used to screen and define ribotypes. Partial sequences from unique ribotypes were compared with existing database entries to identify species and to construct phylogenetic trees representative of community structures. A pronounced difference in species richness and evenness was observed at the two sites. The biofilm community structure from the oyster reef setting had greater evenness and species richness than the one from the muddy sand bottom. The vast majority of the bacteria in the oyster reef biofilm were related to members of the γ- and δ-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium -Bacteroides cluster, and the phyla Planctomyces and Holophaga-Acidobacterium. The same groups were also present in the biofilm harvested at the muddy sand bottom, with the difference that nearly half of the community consisted of representatives of the Planctomyces phylum. Total species richness was estimated to be 417 for the oyster reef and 60 for the muddy sand bottom, with 10.5% of the total unique species identified being shared between habitats. The results suggest dramatic differences in habitat-specific microbial diversity that have implications for overall microbial diversity within estuaries.     

Influence of an oyster reef on development of the microbial heterotrophic community of an estuarine biofilm

We characterized microbial biofilm communities developed over two very closely located but distinct benthic habitats in the Pensacola Bay estuary using two complementary cultivation-independent molecular techniques. Biofilms were grown for 7 days on glass slides held in racks 10 to 15 cm over an oyster reef and an adjacent muddy sand bottom. Total biomass and optical densities of dried biofilms showed dramatic differences for oyster reef versus non-oyster reef biofilms. This study assessed whether the observed spatial variation was reflected in the heterotrophic prokaryotic species composition. Genomic biofilm DNA from both locations was isolated and served as a template to amplify 16S rRNA genes with universal eubacterial primers. Fluorescently labeled PCR products were analyzed by terminal restriction fragment length polymorphism, creating a genetic fingerprint of the composition of the microbial communities. Unlabeled PCR products were cloned in order to construct a clone library of 16S rRNA genes. Amplified ribosomal DNA restriction analysis was used to screen and define ribotypes. Partial sequences from unique ribotypes were compared with existing database entries to identify species and to construct phylogenetic trees representative of community structures. A pronounced difference in species richness and evenness was observed at the two sites. The biofilm community structure from the oyster reef setting had greater evenness and species richness than the one from the muddy sand bottom. The vast majority of the bacteria in the oyster reef biofilm were related to members of the gamma- and delta-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium -Bacteroides cluster, and the phyla Planctomyces and Holophaga-Acidobacterium. The same groups were also present in the biofilm harvested at the muddy sand bottom, with the difference that nearly half of the community consisted of representatives of the Planctomyces phylum. Total species richness was estimated to be 417 for the oyster reef and 60 for the muddy sand bottom, with 10.5% of the total unique species identified being shared between habitats. The results suggest dramatic differences in habitat-specific microbial diversity that have implications for overall microbial diversity within estuaries.     

Nitrotoga is selected over Nitrospira in newly assembled biofilm communities from a tap water source community at increased nitrite loading

Community assembly is a central topic in microbial ecology: how do assembly processes interact and what is the relative contribution of stochasticity and determinism? Here, we exposed replicate flow‐through biofilm systems, fed with nitrite‐supplemented tap water, to continuous immigration from a source community, present in the tap water, to determine the extent of selection and neutral processes in newly assembled biofilm communities at both the community and the functional guild (of nitrite‐oxidizing bacteria, NOB) levels. The community composition of biofilms assembled under low and high nitrite loading was described after 40 days of complete nitrite removal. The total community assembly, as well as the NOB guild assembly were largely governed by a combination of deterministic and stochastic processes. Furthermore, we observed deterministic enrichment of certain types of NOB in the biofilms. Specifically, elevated nitrite loading selected for a single Nitrotoga representative, while lower nitrite conditions selected for a number of Nitrospira. Therefore, even when focusing on ecologically coherent ensembles, assembly is the result of complex stochastic and deterministic processes that can only be interrogated by observing multiple assemblies under controlled conditions.     

Growth of phototrophic biofilms from limestone monuments under laboratory conditions

In the current study, five phototrophic biofilms from different Southern Europe limestone monuments were characterised by molecular techniques and cultivated under laboratory conditions. Phototrophic biofilms were collected from Orologio Tower in Martano (Italy), Santa Clara-a-Velha Monastery and Ajuda National Palace, both in Portugal, and Seville and Granada Cathedrals from Spain. The biofilms were grown under laboratory conditions and periodically sampled in order to monitor their evolution over a three-month period. Prokaryotic communities from natural samples and cultivated biofilms were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments in conjunction with clone sequencing and phylogenetic analysis. DNA-based molecular analysis of 16S rRNA gene fragments from the natural green biofilms revealed complex and different communities composition with respect to phototrophic microorganisms. The biofilms from Orologio Tower (Martano, Italy) and Santa Clara-a-Velha Monastery (Coimbra, Portugal) were dominated by the microalga Chlorella. The cyanobacterium Chroococcidiopsis was the dominating genus from Ajuda National Palace biofilm (Lisbon, Portugal). The biofilms from Seville and Granada Cathedrals (Spain) were both dominated by the cyanobacterium Pleurocapsa. The DGGE analysis of the cultivated biofilms showed that the communities developed differently in terms of species establishment and community composition during the three-month incubation period. The biofilm culture from Coimbra (Portugal) showed a remarkable stability of the microbial components of the natural community in laboratory conditions. With this work, a multiple-species community assemblage was obtained for further stone colonisation experiments.     

Unraveling assembly of stream biofilm communities

Microbial biofilms assemble from cells that attach to a surface, where they develop into matrix-enclosed communities. Mechanistic insights into community assembly are crucial to better understand the functioning of natural biofilms, which drive key ecosystem processes in numerous aquatic habitats. We studied the role of the suspended microbial community as the source of the biofilm community in three streams using terminal-restriction fragment length polymorphism and 454 pyrosequencing of the 16S ribosomal RNA (rRNA) and the 16S rRNA gene (as a measure for the active and the bulk community, respectively). Diversity was consistently lower in the biofilm communities than in the suspended stream water communities. We propose that the higher diversity in the suspended communities is supported by continuous inflow from various sources within the catchment. Community composition clearly differed between biofilms and suspended communities, whereas biofilm communities were similar in all three streams. This suggests that biofilm assembly did not simply reflect differences in the source communities, but that certain microbial groups from the source community proliferate in the biofilm. We compared the biofilm communities with random samples of the respective community suspended in the stream water. This analysis confirmed that stochastic dispersal from the source community was unlikely to shape the observed community composition of the biofilms, in support of species sorting as a major biofilm assembly mechanism. Bulk and active populations generated comparable patterns of community composition in the biofilms and the suspended communities, which suggests similar assembly controls on these populations.