Shifts in microbial community structure and function in light‐ and dark‐grown biofilms driven by warming

Biofilms are dynamic players in biogeochemical cycling in running waters and are subjected to environmental stressors like those provoked by climate change. We investigated whether a 2°C increase in flowing water would affect prokaryotic community composition and heterotrophic metabolic activities of biofilms grown under light or dark conditions. Neither light nor temperature treatments were relevant for selecting a specific bacterial community at initial phases (7‐day‐old biofilms), but both variables affected the composition and function of mature biofilms (28‐day‐old). In dark‐grown biofilms, changes in the prokaryotic community composition due to warming were mainly related to rotifer grazing, but no significant changes were observed in functional fingerprints. In light‐grown biofilms, warming also affected protozoan densities, but its effect on prokaryotic density and composition was less evident. In contrast, heterotrophic metabolic activities in light‐grown biofilms under warming showed a decrease in the functional diversity towards a specialized use of several carbohydrates. Results suggest that prokaryotes are functionally redundant in dark biofilms but functionally plastic in light biofilms. The more complex and self‐serving light‐grown biofilm determines a more buffered response to temperature than dark‐grown biofilms. Despite the moderate increase in temperature of only 2°C, warming conditions drive significant changes in freshwater biofilms, which responded by finely tuning a complex network of interactions among microbial populations within the biofilm matrix.     

Biophysical Controls on Community Succession in Stream Biofilms

Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as “ecosystem engineers,” modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms.     

Relevance of Polymeric Matrix Enzymes During Biofilm Formation

Extracellular polymeric substances (EPS) contribute to biofilm stability and adhesion properties. The EPS matrix might also be a site for free extracellular enzyme activity; however, little is known about participation of enzyme activity in EPS during biofilm formation. In this study, we analyzed the activities of beta-glucosidase, leu-aminopeptidase, and beta-glucosaminidase during the colonization of artificial substrata (glass tiles) in a stream distinguishing enzyme activity in EPS matrix (matrix-enzymes) and total biofilm extracellular enzyme activity. The 1-h incubation of a biofilm suspension and cation-exchange resin followed by centrifugation seems appropriate to extract the matrix fraction (supernatant) and measure matrix enzymes (including free and linked to EPS) in freshwater biofilms, although there is a methodological limitation for using a biofilm suspension instead of an undisrupted biofilm. Total biofilm activities and matrix-enzyme activities showed similar capabilities to decompose organic matter compounds, with a greater capacity for peptide decomposition (leu-aminopeptidase) than for polysaccharides (beta-glucosidase), and a low decomposition of chitin and peptidoglycan (beta-glucosaminidase). Matrix-enzyme activity increased with colonization time, but more slowly than that of total enzyme activity. At the beginning of the colonization experiment (days 1-4) matrix enzymes accounted for 65-81% of total biofilm enzyme activity. Higher proportion of polysaccharides in EPS versus total biofilm, and higher matrix-enzyme activities per microgram of polysaccharides in the EPS were measured during the first 1-3 days of biofilm formation, indicating a high rate of enzyme release into the matrix during this period. Relative contribution of matrix-enzyme activities decreased as biofilm matures, but was maintained at 13-37% of total enzyme activity at the 42- to 49-day-old biofilm. These enzymes, retained and conserved in the EPS, may contribute to community metabolism. When analyzing extracellular enzymes in biofilms, the contribution of matrix enzymes must be considered, especially for young biofilms.     

Coordinating environmental genomics and geochemistry reveals metabolic transitions in a hot spring ecosystem

We have constructed a conceptual model of biogeochemical cycles and metabolic and microbial community shifts within a hot spring ecosystem via coordinated analysis of the "Bison Pool" (BP) Environmental Genome and a complementary contextual geochemical dataset of ~75 geochemical parameters. 2,321 16S rRNA clones and 470 megabases of environmental sequence data were produced from biofilms at five sites along the outflow of BP, an alkaline hot spring in Sentinel Meadow (Lower Geyser Basin) of Yellowstone National Park. This channel acts as a >22 m gradient of decreasing temperature, increasing dissolved oxygen, and changing availability of biologically important chemical species, such as those containing nitrogen and sulfur. Microbial life at BP transitions from a 92 °C chemotrophic streamer biofilm community in the BP source pool to a 56 °C phototrophic mat community. We improved automated annotation of the BP environmental genomes using BLAST-based Markov clustering. We have also assigned environmental genome sequences to individual microbial community members by complementing traditional homology-based assignment with nucleotide word-usage algorithms, allowing more than 70% of all reads to be assigned to source organisms. This assignment yields high genome coverage in dominant community members, facilitating reconstruction of nearly complete metabolic profiles and in-depth analysis of the relation between geochemical and metabolic changes along the outflow. We show that changes in environmental conditions and energy availability are associated with dramatic shifts in microbial communities and metabolic function. We have also identified an organism constituting a novel phylum in a metabolic "transition" community, located physically between the chemotroph- and phototroph-dominated sites. The complementary analysis of biogeochemical and environmental genomic data from BP has allowed us to build ecosystem-based conceptual models for this hot spring, reconstructing whole metabolic networks in order to illuminate community roles in shaping and responding to geochemical variability.     

Interactions between Bdellovibrio and like organisms and bacteria in biofilms: beyond predator–prey dynamics

Bdellovibrio and like organisms (BALOs) prey on Gram-negative bacteria in the planktonic phase as well as in biofilms, with the ability to reduce prey populations by orders of magnitude. During the last few years, evidence has mounted for a significant ecological role for BALOs, with important implications for our understanding of microbial community dynamics as well as for applications against pathogens, including drug-resistant pathogens, in medicine, agriculture and aquaculture, and in industrial settings for various uses. However, our understanding of biofilm predation by BALOs is still very fragmentary, including gaps in their effect on biofilm structure, on prey resistance, and on evolutionary outcomes of both predators and prey. Furthermore, their impact on biofilms has been shown to reach beyond predation, as they are reported to reduce biofilm structures of non-prey cells (including Gram-positive bacteria). Here, we review the available literature on BALOs in biofilms, extending known aspects to potential mechanisms employed by the predators to grow in biofilms. Within that context, we discuss the potential ecological significance and potential future utilization of the predatory and enzymatic possibilities offered by BALOs in medical, agricultural and environmental applications.     

人工纳米材料与生物膜相互作用机制及影响因素研究进展

人工纳米材料在水体中的环境行为与生物环境安全问题成为环境科学领域研究的热点,人工纳米材料与生物膜相互作用机制和影响因素是其中迫切需要研究解决的关键科学问题。本文主要探讨了人工纳米材料释放进入到水体中后对生物膜细菌活性、微生物群落结构、净污活性等的毒性效应,分析了人工纳米材料对生物膜的毒性作用机制及其影响因素,同时探讨了生物膜对人工纳米材料的吸附作用及机理,为深入研究人工纳米材料与生物膜的相互作用机制提供了重要的理论基础。     

Plastics select for distinct early colonizing microbial populations with reproducible traits across environmental gradients

Little is known about early plastic biofilm assemblage dynamics and successional changes over time. By incubating virgin microplastics along oceanic transects and comparing adhered microbial communities with those of naturally occurring plastic litter at the same locations, we constructed gene catalogues to contrast the metabolic differences between early and mature biofilm communities. Early colonization incubations were reproducibly dominated by Alteromonadaceae and harboured significantly higher proportions of genes associated with adhesion, biofilm formation, chemotaxis, hydrocarbon degradation and motility. Comparative genomic analyses among the Alteromonadaceae metagenome assembled genomes (MAGs) highlighted the importance of the mannose-sensitive hemagglutinin (MSHA) operon, recognized as a key factor for intestinal colonization, for early colonization of hydrophobic plastic surfaces. Synteny alignments of MSHA also demonstrated positive selection for mshA alleles across all MAGs, suggesting that mshA provides a competitive advantage for surface colonization and nutrient acquisition. Large-scale genomic characteristics of early colonizers varied little, despite environmental variability. Mature plastic biofilms were composed of predominantly Rhodobacteraceae and displayed significantly higher proportions of carbohydrate hydrolysis enzymes and genes for photosynthesis and secondary metabolism. Our metagenomic analyses provide insight into early biofilm formation on plastics in the ocean and how early colonizers self-assemble, compared to mature, phylogenetically and metabolically diverse biofilms.     

Availability of glucose and light modulates the structure and function of a microbial biofilm

We have studied the differences in the organic matter processing and biofilm composition and structure between autoheterotrophic and heterotrophic biofilm communities. Microbial communities grown on artificial biofilms were monitored, following incubation under light and dark conditions and with or without the addition of glucose as a labile organic compound. Glucose addition greatly affected the microbial biofilm composition as shown by differences in 16S rRNA gene fingerprints. A significant increase in β-glucosidase and peptidase enzyme activities were also observed in glucose-amended biofilms incubated in the dark, suggesting an active bacterial community. Light enhanced the algal and bacterial growth, as well as higher extracellular enzyme activity, thereby indicating a tight algal–bacterial coupling in biofilms incubated under illumination. In these biofilms, organic compounds excreted by photosynthetic microorganisms were readily available for bacterial heterotrophs. This algal–bacterial relationship weakened in glucose-amended biofilms grown in the light, probably because heterotrophic bacteria preferentially use external labile compounds. These results suggest that the availability of labile organic matter in the flowing water and the presence of light may alter the biofilm composition and function, therefore affecting the processing capacity of organic matter in the stream ecosystem.     

Fungal-bacterial biofilms: their development for novel biotechnological applications

The attachment of microbes on biotic or abiotic surfaces to form biofilm structures has a great impact on biodegradation and biosynthesis in nature. Various interactions in such biofilms and their extracellular polymeric substances (EPS) layer make them considerably different in physiology and action, compared to that of their individual microbes in planktonic (free swimming) mode of growth. Expression of new genes is up-regulated in the biofilm cells, due in part to the cellular interactions, compared with the planktonic cells. Formation of fungal-bacterial biofilms (FBB) by bacterial colonization on biotic fungal surface gives the biofilm enhanced metabolic activities compared to monocultures, and perhaps multi-species bacterial or fungal biofilms on abiotic surfaces. Incorporation of a N₂-fixing rhizobial strain to the FBB to form fungal-rhizobial biofilms (FRB) has been shown to improve potential biofilm applications in N-deficient settings and in the production of biofilmed inocula for biofertilizers and biocontrol in plants. Their applications in agricultural and environmental settings, enzyme technology, drug discovery studies and energy research are being investigated. Thus, it has already been shown that the use of the FBB is a promising technology for many applications. This review deals with the different areas in which FBB/FRB have been seen to be applied with successful results as well as the numerous emerging avenues in which they show promising potential.     

The CprS sensor kinase of the zoonotic pathogen Campylobacter jejuni influences biofilm formation and is required for optimal chick colonization

Campylobacter jejuni , a prevalent cause of bacterial gastroenteritis, must adapt to different environments to be a successful pathogen. We previously identified a C. jejuni two-component regulatory system (Cj1226/7c) as upregulated during cell infections. Analyses described herein led us to designate the system CprRS ( C ampylobacter p lanktonic growth r egulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The Δ cprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. Δ cprS also displayed a striking dose-dependent defect for colonization of chicks and was modestly enhanced for intracellular survival in INT407 cells. Proteomics analyses identified changes consistent with modulation of essential metabolic genes, upregulation of stress tolerance proteins, and increased expression of MOMP and FlaA. Consistent with expression profiling, we observed enhanced motility and secretion in Δ cprS , and decreased osmotolerance and oxidative stress tolerance. We also found that C. jejuni biofilms contain a DNase I-sensitive component and that biofilm formation is influenced by deoxycholate and the metabolic substrate fumarate. These results suggest that CprRS influences expression of factors important for biofilm formation, colonization and stress tolerance, and also add to our understanding of C. jejuni biofilm physiology.     

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.     

Food or just a free ride? A meta-analysis reveals the global diversity of the Plastisphere

It is now indisputable that plastics are ubiquitous and problematic in ecosystems globally. Many suggestions have been made about the role that biofilms colonizing plastics in the environment—termed the “Plastisphere”—may play in the transportation and ecological impact of these plastics. By collecting and re-analyzing all raw 16S rRNA gene sequencing and metadata from 2,229 samples within 35 studies, we have performed the first meta-analysis of the Plastisphere in marine, freshwater, other aquatic (e.g., brackish or aquaculture) and terrestrial environments. We show that random forest models can be trained to differentiate between groupings of environmental factors as well as aspects of study design, but—crucially—also between plastics when compared with control biofilms and between different plastic types and community successional stages. Our meta-analysis confirms that potentially biodegrading Plastisphere members, the hydrocarbonoclastic Oceanospirillales and Alteromonadales are consistently more abundant in plastic than control biofilm samples across multiple studies and environments. This indicates the predilection of these organisms for plastics and confirms the urgent need for their ability to biodegrade plastics to be comprehensively tested. We also identified key knowledge gaps that should be addressed by future studies.Subject terms: Microbial ecology, Soil microbiology, Water microbiology, Microbial ecology, Microbiome     

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.     

A semi-quantitative approach to assess biofilm formation using wrinkled colony development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis, and Gram-negative bacteria, such as Vibrio cholerae, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Vibrio fischeri. The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect, while strains exhibiting increased biofilm phenotypes are enhanced for colonization. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization. In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.     

Poly-beta-hydroxy alkanoate and the support of river biofilm metabolism following radical changes in environmental conditions

Endogenous carbon reserves such as poly-beta-hydroxy alkanoate (PHA) can sustain microbial viability during conditions of nutrient deprivation. Microbial extracellular enzyme activities under one set of environmental conditions might be wholly inappropriate for another, and thus PHA might also serve as an energy source as the biofilm acclimates to a changed environment. In order to test this hypothesis, radical changes in environmental conditions were imposed upon river biofilms by transferring them between three rivers of acid, circum-neutral and alkaline pH. The findings supported the hypothesis; each of the transfers resulted in reduced PHA levels, while the physiology of the biofilm (metabolic activity, population density, phosphatase & glucosidase activities) acclimated to the environmental conditions of the recipient site. The greatest PHA depletion was observed when the magnitude of the imposed change resulted in an inability of phosphatase enzyme to respond to the change. The implicit greater dependence on the reserves of PHA, is similarly consistent with the hypothesis.     

Comparison of the Phenotypes and Genotypes of Biofilm and Solitary Epiphytic Bacterial Populations on Broad-Leaved Endive

The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety. Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor. However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies. Broad-leaved endive (Cichorium endivia var. latifolia) harbors biofilms containing fluorescent pseudomonads. These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads. To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive. Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation. The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains. However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms. We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states. This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.     

Molecular Analysis of Shower Curtain Biofilm Microbes

Households provide environments that encourage the formation of microbial communities, often as biofilms. Such biofilms constitute potential reservoirs for pathogens, particularly for immune-compromised individuals. One household environment that potentially accumulates microbial biofilms is that provided by vinyl shower curtains. Over time, vinyl shower curtains accumulate films, commonly referred to as “soap scum,” which microscopy reveals are constituted of lush microbial biofilms. To determine the kinds of microbes that constitute shower curtain biofilms and thereby to identify potential opportunistic pathogens, we conducted an analysis of rRNA genes obtained by PCR from four vinyl shower curtains from different households. Each of the shower curtain communities was highly complex. No sequence was identical to one in the databases, and no identical sequences were encountered in the different communities. However, the sequences generally represented similar phylogenetic kinds of organisms. Particularly abundant sequences represented members of the α-group of proteobacteria, mainly Sphingomonas spp. and Methylobacterium spp. Both of these genera are known to include opportunistic pathogens, and several of the sequences obtained from the environmental DNA samples were closely related to known pathogens. Such organisms have also been linked to biofilm formation associated with water reservoirs and conduits. In addition, the study detected many other kinds of organisms at lower abundances. These results show that shower curtains are a potential source of opportunistic pathogens associated with biofilms. Frequent cleaning or disposal of shower curtains is indicated, particularly in households with immune-compromised individuals.