Organization Of Microbial Communities Research Articles

Chemorepulsion from the Quorum Signal Autoinducer-2 Promotes Helicobacter pylori Biofilm Dispersal.

ABSTRACTThe gastric pathogen Helicobacter pylori forms biofilms on abiotic and biotic surfaces. We have shown previously that H. pylori perceives the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report here that H. pylori chemorepulsion from endogenous AI-2 influences the proportions and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (∆luxS strains) or are defective for chemotaxis (∆cheA strains) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wild-type biofilms. Reciprocally, a strain that overproduced AI-2 (luxSOP) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified AibA and AibB, two novel periplasmic binding proteins that are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ∆luxS mutant. Furthermore, exogenous administration of AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis-dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal.

Atmospheric N deposition alters connectance, but not functional potential among saprotrophic bacterial communities.

The use of co-occurrence patterns to investigate interactions between micro-organisms has provided novel insight into organismal interactions within microbial communities. However, anthropogenic impacts on microbial co-occurrence patterns and ecosystem function remain an important gap in our ecological knowledge. In a northern hardwood forest ecosystem located in Michigan, USA, 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This ecosystem-level response occurred concomitantly with compositional changes in saprophytic fungi and bacteria. Here, we investigated the influence of experimental N deposition on biotic interactions among forest floor bacterial assemblages by employing phylogenetic and molecular ecological network analysis. When compared to the ambient treatment, the forest floor bacterial community under experimental N deposition was less rich, more phylogenetically dispersed and exhibited a more clustered co-occurrence network topology. Together, our observations reveal the presence of increased biotic interactions among saprotrophic bacterial assemblages under future rates of N deposition. Moreover, they support the hypothesis that nearly two decades of experimental N deposition can modify the organization of microbial communities and provide further insight into why anthropogenic N deposition has reduced decomposition, increased soil C storage and accelerated phenolic DOC production in our field experiment.

Carbonate fabrics in the modern microbialites of Pavilion Lake: two suites of microfabrics that reflect variation in microbial community morphology, growth habit, and lithification.

Modern microbialites in Pavilion Lake, BC, provide an analog for ancient non-stromatolitic microbialites that formed from insitu mineralization. Because Pavilion microbialites are mineralizing under the influence of microbial communities, they provide insights into how biological processes influence microbialite microfabrics and mesostructures. Hemispherical nodules and micrite-microbial crusts are two mesostructures within Pavilion microbialites that are directly associated with photosynthetic communities. Both filamentous cyanobacteria in hemispherical nodules and branching filamentous green algae in micrite-microbial crusts were associated with calcite precipitation at microbialite surfaces and with characteristic microfabrics in the lithified microbialite. Hemispherical nodules formed at microbialite surfaces when calcite precipitated around filamentous cyanobacteria with a radial growth habit. The radial filament pattern was preserved within the microbialite to varying degrees. Some subsurface nodules contained well-defined filaments, whereas others contained only dispersed organic inclusions. Variation in filament preservation is interpreted to reflect differences in timing and amount of carbonate precipitation relative to heterotrophic decay, with more defined filaments reflecting greater lithification prior to degradation than more diffuse filaments. Micrite-microbial crusts produce the second suite of microfabrics and form in association with filamentous green algae oriented perpendicular to the microbialite surface. Some crusts include calcified filaments, whereas others contained voids that reflect the filamentous community in shape, size, and distribution. Pavilion microbialites demonstrate that microfabric variation can reflect differences in lithification processes and microbial metabolisms as well as microbial community morphology and organization. Even when the morphology of individual filaments or cells is not well preserved, the microbial growth habit can be captured in mesoscale microbialite structures. These results suggest that when petrographic preservation is extremely good, ancient microbialite growth structures and microfabrics can be interpreted in the context of variation in community organization, community composition, and lithification history. Even in the absence of distinct microbial microfabrics, mesostructures can capture microbial community morphology.

Ammonia and temperature determine potential clustering in the anaerobic digestion microbiome

Anaerobic digestion is regarded as a key environmental technology in the present and future bio-based economy. The microbial community completing the anaerobic digestion process is considered complex, and several attempts already have been carried out to determine the key microbial populations. However, the key differences in the anaerobic digestion microbiomes, and the environmental/process parameters that drive these differences, remain poorly understood. In this research, we hypothesized that differences in operational parameters lead to a particular composition and organization of microbial communities in full-scale installations. A total of 38 samples were collected from 29 different full-scale anaerobic digestion installations, showing constant biogas production in function of time. Microbial community analysis was carried out by means of amplicon sequencing and real-time PCR. The bacterial community in all samples was dominated by representatives of the Firmicutes, Bacteroidetes and Proteobacteria, covering 86.1 ± 10.7% of the total bacterial community. Acetoclastic methanogenesis was dominated by Methanosaetaceae, yet, only the hydrogenotrophic Methanobacteriales correlated with biogas production, confirming their importance in high-rate anaerobic digestion systems. In-depth analysis of operational and environmental parameters and bacterial community structure indicated the presence of three potential clusters in anaerobic digestion. These clusters were determined by total ammonia concentration, free ammonia concentration and temperature, and characterized by an increased relative abundance of Bacteroidales, Clostridiales and Lactobacillales, respectively. None of the methanogenic populations, however, could be significantly attributed to any of the three clusters. Nonetheless, further experimental research will be required to validate the existence of these different clusters, and to which extent the presence of these clusters relates to stable or sub-optimal anaerobic digestion.

Scaling microbial biomass, metabolism and resource supply

The microbiome concept has drawn attention to the complex signal and syntrophic networks that underlie microbial community organization. This self-organization may lead to patterns in the allometric scaling of microbial community metabolism that differ from those of macrobial communities. Using meta-analyses, we analyzed the power scaling relationships between community production, respiration, extracellular enzyme activity and biomass for bacteria and fungi across aquatic and terrestrial ecosystems. The scaling exponents for community production versus biomass for fungi and bacteria were 0.85 ± 0.06 (95 % CI) and 0.72 ± 0.07, respectively. The scaling exponent for fungal respiration versus production was 0.61 ± 0.06. Previous studies reported exponents of 0.41, 0.44 and 0.58 for bacterial respiration versus production. Carbon use efficiency increased with biomass for both fungi and bacteria with an exponent of 0.27 ± 0.06. The potential activities of four widely measured extracellular enzymes were directly related to community production with power scaling exponents of 1.0–1.2. The frequency distribution of biomass turnover times (median 112 h for bacteria and 1,128 h for fungi) overlapped substantially with those for environmental substrate turnover, presented in a prior analysis of extracellular enzyme kinetics. These metabolic relationships, which have scaling exponents of 0.5, are linked by the ratio of assimilation to carbon use efficiency. This connection ties ecological stoichiometry and metabolic theory to microbial community homeostasis. At the ecosystem scale, allometry of microbial communities has similarities to that of eusocial insects but differs from that of plant communities, perhaps as a result of proto-cooperative processes that contribute to microbial community organization.

Fluvial network organization imprints on microbial co-occurrence networks.

Recent studies highlight linkages among the architecture of ecological networks, their persistence facing environmental disturbance, and the related patterns of biodiversity. A hitherto unresolved question is whether the structure of the landscape inhabited by organisms leaves an imprint on their ecological networks. We analyzed, based on pyrosequencing profiling of the biofilm communities in 114 streams, how features inherent to fluvial networks affect the co-occurrence networks that the microorganisms form in these biofilms. Our findings suggest that hydrology and metacommunity dynamics, both changing predictably across fluvial networks, affect the fragmentation of the microbial co-occurrence networks throughout the fluvial network. The loss of taxa from co-occurrence networks demonstrates that the removal of gatekeepers disproportionately contributed to network fragmentation, which has potential implications for the functions biofilms fulfill in stream ecosystems. Our findings are critical because of increased anthropogenic pressures deteriorating stream ecosystem integrity and biodiversity.

Three-Dimensional Stratification of Bacterial Biofilm Populations in a Moving Bed Biofilm Reactor for Nitritation-Anammox

Moving bed biofilm reactors (MBBRs) are increasingly used for nitrogen removal with nitritation-anaerobic ammonium oxidation (anammox) processes in wastewater treatment. Carriers provide protected surfaces where ammonia oxidizing bacteria (AOB) and anammox bacteria form complex biofilms. However, the knowledge about the organization of microbial communities in MBBR biofilms is sparse. We used new cryosectioning and imaging methods for fluorescence in situ hybridization (FISH) to study the structure of biofilms retrieved from carriers in a nitritation-anammox MBBR. The dimensions of the carrier compartments and the biofilm cryosections after FISH showed good correlation, indicating little disturbance of biofilm samples by the treatment. FISH showed that Nitrosomonas europaea/eutropha-related cells dominated the AOB and Candidatus Brocadia fulgida-related cells dominated the anammox guild. New carriers were initially colonized by AOB, followed by anammox bacteria proliferating in the deeper biofilm layers, probably in anaerobic microhabitats created by AOB activity. Mature biofilms showed a pronounced three-dimensional stratification where AOB dominated closer to the biofilm-water interface, whereas anammox were dominant deeper into the carrier space and towards the walls. Our results suggest that current mathematical models may be oversimplifying these three-dimensional systems and unless the multidimensionality of these systems is considered, models may result in suboptimal design of MBBR carriers.

The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules

Quorum sensing (QS) signalling has been extensively studied in single species populations. However, the ecological role of QS in complex, multi-species communities, particularly in the context of community assembly, has neither been experimentally explored nor theoretically addressed. Here, we performed a long-term bioreactor ecology study to address the links between QS, organization and composition of complex microbial communities. The conversion of floccular biomass to highly structured granules was found to be non-random, but strongly and positively correlated with N-acyl-homoserine-lactone (AHL)-mediated QS. Specific AHLs were elevated up to 100-fold and were strongly associated with the initiation of granulation. Similarly, the levels of particular AHLs decreased markedly during the granular disintegration phase. Metadata analysis indicated that granulation was accompanied by changes in extracellular polymeric substance (EPS) production and AHL add-back studies also resulted in increased EPS synthesis. In contrast to the commonly reported nanomolar to micromolar signal concentrations in pure culture laboratory systems, QS signalling in the granulation ecosystem occurred at picomolar to nanomolar concentrations of AHLs. Given that low concentrations of AHLs quantified in this study were sufficient to activate AHL bioreporters in situ in complex granular communities, AHL mediated QS may be a common feature in many natural and engineered ecosystems, where it coordinates community behaviour.

Metabolic proximity in the order of colonization of a microbial community.

Microbial biofilms are often composed of multiple bacterial species that accumulate by adhering to a surface and to each other. Biofilms can be resistant to antibiotics and physical stresses, posing unresolved challenges in the fight against infectious diseases. It has been suggested that early colonizers of certain biofilms could cause local environmental changes, favoring the aggregation of subsequent organisms. Here we ask whether the enzyme content of different microbes in a well-characterized dental biofilm can be used to predict their order of colonization. We define a metabolic distance between different species, based on the overlap in their enzyme content. We next use this metric to quantify the average metabolic distance between neighboring organisms in the biofilm. We find that this distance is significantly smaller than the one observed for a random choice of prokaryotes, probably reflecting the environmental constraints on metabolic function of the community. More surprisingly, this metabolic metric is able to discriminate between observed and randomized orders of colonization of the biofilm, with the observed orders displaying smaller metabolic distance than randomized ones. By complementing these results with the analysis of individual vs. joint metabolic networks, we find that the tendency towards minimal metabolic distance may be counter-balanced by a propensity to pair organisms with maximal joint potential for synergistic interactions. The trade-off between these two tendencies may create a “sweet spot” of optimal inter-organism distance, with possible broad implications for our understanding of microbial community organization.

Extracellular enzymes in terrestrial, freshwater, and marine environments: perspectives on system variability and common research needs

Extracellular enzymes produced by het- erotrophic microbial communities are major drivers of carbon and nutrient cycling in terrestrial, freshwater, and marine environments. Although carbon and nutrient cycles are coupled on global scales, studies of extracellular enzymes associated with terrestrial, freshwater, and marine microbial communities are not often compared across ecosystems. In part, this disconnect arises because the environmental parame- ters that control enzyme activities in terrestrial and freshwater systems, such as temperature, pH, and moisture content, have little explanatory power for patterns of enzyme activities in marine systems. Instead, factors such as the functional diversity of microbial communities may explain varying patterns of enzyme activities observed in the ocean to date. In any case, many studies across systems focus on similar issues that highlight the commonalities of microbial community organization. Examples include the effec- tive lifetime of enzymes released into the environ- ment; the extent to which microbial communities coordinate enzyme expression to decompose complex organic substrates; and the influence of microbial community composition on enzyme activities and kinetics. Here we review the often-disparate research foci in terrestrial, freshwater, and marine environ- ments. We consider the extent to which environmental factors may regulate extracellular enzyme activities within each ecosystem, and highlight commonalities and current methodological challenges to identify

Development of a high throughput cross-flow filtration system for detailed investigation of fouling processes

Fouling, more precisely biofouling, remains one of the main obstacles for a wider application of membrane technology. One of the most significant problems limiting membrane fouling control is the inadequate understanding of the basic mechanisms governing adsorption/deposition of foulants in and on membranes. A detailed knowledge with regard to fouling characteristics and development would be clearly advanced if a reliable and repeatable, non-invasive study tool would be available. For this purpose, a direct visual observation system, containing multiple separated cross-flow filtration units, coupled with either an optical or a confocal laser scanning microscope was developed. This approach allows the online, non-destructive monitoring of the fouling process while filtering and the high number of parallel filtrations makes it possible to either analyze different membranes, to apply statistical evaluations, run different feed solutions or to sample and perform membrane autopsies at different time points. This technique allows for the investigation of microbial ecology with respect to microbe–membrane attachment, site-specific associations of micro-organisms, deposition of foulants on the membrane surface and spatial organization of microbial communities on membrane surfaces through the use of fluorescent microscopy techniques. This manuscript illustrates the reproducibility of the developed research tool with respect to membrane flux decline and fouling characterization. Two membrane types, a polyvinylidene fluoride microfiltration (under filtering conditions) and a polyamide reverse osmosis membrane (in non-permeating mode), with respectively a bacterial foulant, Pseudomonas aeruginosa and tap water, were chosen as model systems.

Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members

Many studies of molecular microbial ecology rely on the characterization of microbial communities by PCR amplification, cloning, sequencing, and phylogenetic analysis of genes encoding rRNAs or functional marker enzymes. However, if the established clone libraries are dominated by one or a few sequence types, the cloned diversity is difficult to analyze by random clone sequencing. Here we present a novel approach to deplete unwanted sequence types from complex nucleic acid mixtures prior to cloning and downstream analyses. It employs catalytically active oligonucleotides containing locked nucleic acids (LNAzymes) for the specific cleavage of selected RNA targets. When combined with in vitro transcription and reverse transcriptase PCR, this LNAzyme-based technique can be used with DNA or RNA extracts from microbial communities. The simultaneous application of more than one specific LNAzyme allows the concurrent depletion of different sequence types from the same nucleic acid preparation. This new method was evaluated with defined mixtures of cloned 16S rRNA genes and then used to identify accompanying bacteria in an enrichment culture dominated by the nitrite oxidizer "Candidatus Nitrospira defluvii." In silico analysis revealed that the majority of publicly deposited rRNA-targeted oligonucleotide probes may be used as specific LNAzymes with no or only minor sequence modifications. This efficient and cost-effective approach will greatly facilitate tasks such as the identification of microbial symbionts in nucleic acid preparations dominated by plastid or mitochondrial rRNA genes from eukaryotic hosts, the detection of contaminants in microbial cultures, and the analysis of rare organisms in microbial communities of highly uneven composition.

Metagenomics and its connection to microbial community organization

Microbes dominate most global biogeochemical cycles, and microbial metagenomics (studying the collective microbial genomes) provides invaluable new insights into microbial systems, independent of cultivation. Metagenomic approaches targeting specific genes, e.g. small subunit (ssu) ribosomal RNA (rRNA), can be used to investigate microbial community organization by efficiently showing which taxa of organisms are present, while shotgun approaches show all genes and can indicate what functions the organisms are capable of. But collecting and organizing comprehensive shotgun data is extremely challenging and costly, and, in theory, predicting functionalities from microbial identities alone would save immense effort. However, we don’t yet know to what extent such predictions are applicable.

Anaerobic digestibility of marine microalgae Phaeodactylum tricornutum in a lab-scale anaerobic membrane bioreactor

The biomass of industrially grown Phaeodactylum tricornutum was subjected in a novel way to bio-methanation at 33°C, i.e., in an anaerobic membrane bioreactor (AnMBR) at a hydraulic retention time of 2.5 days, at solid retention times of 20 to 10 days and at loading rates in the range of 2.6-5.9 g biomass-COD L(-1) day(-1) with membrane fluxes ranging from 1 to 0.8 L m(-2) h(-1). The total COD recovered as biogas was in the order of 52%. The input suspension was converted to a clear effluent rich in total ammonium nitrogen (546 mg TAN L(-1)) and phosphate (141 mg PO(4)-P L(-1)) usable as liquid fertilizer. The microbial community richness, dynamics, and organization in the reactor were interpreted using the microbial resource management approach. The AnMBR communities were found to be moderate in species richness and low in dynamics and community organization relative to UASB and conventional CSTR sludges. Quantitative polymerase chain reaction analysis revealed that Methanosaeta sp. was the dominant acetoclastic methanogen species followed by Methanosarcina sp. This work demonstrated that the use of AnMBR for the digestion of algal biomass is possible. The fact that some 50% of the organic matter is not liquefied means that the algal particulates in the digestate constitute a considerable fraction which should be valorized properly, for instance as slow release organic fertilizer. Overall, 1 kg of algae dry matter (DM) could be valorized in the form of biogas ( euro 2.07), N and P in the effluent (euro 0.02) and N and P in the digestate (euro 0.04), thus totaling about euro 2.13 per kilogram algae DM.

N 2O emission from a partial nitrification–anammox process and identification of a key biological process of N 2O emission from anammox granules

Emission of nitrous oxide (N 2O) during biological wastewater treatment is of growing concern. The emission of N 2O from a lab-scale two-reactor partial nitrification (PN)–anammox reactor was therefore determined in this study. The average emission of N 2O from the PN and anammox process was 4.0 ± 1.5% (9.6 ± 3.2% of the removed nitrogen) and 0.1 ± 0.07% (0.14 ± 0.09% of the removed nitrogen) of the incoming nitrogen load, respectively. Thus, a larger part (97.5%) of N 2O was emitted from the PN reactor. The total amount of N 2O emission from the PN reactor was correlated to nitrite (NO 2 −) concentration in the PN effluent rather than DO concentration. In addition, further studies were performed to indentify a key biological process that is responsible for N 2O emission from the anammox process (i.e., granules). In order to characterize N 2O emission from the anammox granules, the in situ N 2O production rate was determined by using microelectrodes for the first time, which was related to the spatial organization of microbial community of the granule as determined by fluorescence in situ hybridization (FISH). Microelectrode measurement revealed that the active N 2O production zone was located in the inner part of the anammox granule, whereas the active ammonium consumption zone was located above the N 2O production zone. Anammox bacteria were present throughout the granule, whereas ammonium-oxidizing bacteria (AOB) were restricted to only the granule surface. In addition, addition of penicillin G that inhibits most of the heterotrophic denitrifiers and AOB completely inhibited N 2O production in batch experiments. Based on these results obtained, denitrification by putative heterotrophic denitrifiers present in the inner part of the granule was considered the most probable cause of N 2O emission from the anammox reactor (i.e., granules).

Effects of poly-β-hydroxybutyrate (PHB) on Siberian sturgeon (Acipenser baerii) fingerlings performance and its gastrointestinal tract microbial community

Poly-β-hydroxybutyrate (PHB) is a natural polymer that can be depolymerized into water-soluble short-chain fatty acid monomers. These monomers can act as microbial control agents. In this study, the effects of partially replacing the diet of Siberian sturgeon fingerlings with 2% and 5% PHB were investigated. Replacing 2% of the diet with PHB improved weight gain, specific growth rate (SGR) and survival in the sturgeon fingerlings during the 10-week experimental period. Community-level physiological profiling and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) were used to analyze the microbial community diversity and community organization in the sturgeon gastrointestinal tract. DGGE analysis revealed that PHB affected the intestinal microbial species richness and diversity. The highest species richness was observed with 2% PHB. DNA sequencing of the dominant bands in 2% and 5% PHB treatments revealed that PHB stimulated bacteria belonging to the genera Bacillus and Ruminococcaceae. Principal component analysis, Lorenz curves and the Shannon index of Biolog Ecoplate data revealed that aerobic metabolic potential of the bacterial community was different in the PHB-treated fishes as compared with the control situation. Overall, our results indicate that PHB act as microbial control agents and replacement of 2% of Siberian sturgeon fingerling diet with PHB has beneficial effects.

Correlative microscopy for phylogenetic and ultrastructural characterization of microbial communities

Transmission electron microscopy (TEM) can provide ultrastructural information for cells in microbial community samples and phylogenetic information can be recovered via molecular surveys. Here we report an approach to link these data sets by coupling fluorescence in situ hybridization (FISH) with either conventional biological or cryogenic TEM. The method could fundamentally improve our understanding of the organization and functioning of microbial communities in natural systems.

Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging

Microbes in nature frequently function as members of complex multitaxon communities, but the structural organization of these communities at the micrometer level is poorly understood because of limitations in labeling and imaging technology. We report here a combinatorial labeling strategy coupled with spectral image acquisition and analysis that greatly expands the number of fluorescent signatures distinguishable in a single image. As an imaging proof of principle, we first demonstrated visualization of Escherichia coli labeled by fluorescence in situ hybridization (FISH) with 28 different binary combinations of eight fluorophores. As a biological proof of principle, we then applied this Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) strategy using genus- and family-specific probes to visualize simultaneously and differentiate 15 different phylotypes in an artificial mixture of laboratory-grown microbes. We then illustrated the utility of our method for the structural analysis of a natural microbial community, namely, human dental plaque, a microbial biofilm. We demonstrate that 15 taxa in the plaque community can be imaged simultaneously and analyzed and that this community was dominated by early colonizers, including species of Streptococcus, Prevotella, Actinomyces, and Veillonella. Proximity analysis was used to determine the frequency of inter- and intrataxon cell-to-cell associations which revealed statistically significant intertaxon pairings. Cells of the genera Prevotella and Actinomyces showed the most interspecies associations, suggesting a central role for these genera in establishing and maintaining biofilm complexity. The results provide an initial systems-level structural analysis of biofilm organization.

Biogeographical patterns of soil molecular microbial biomass as influenced by soil characteristics and management

Aim The spatial organization of soil microbial communities on large scales and the identification of environmental factors structuring their distribution have been little investigated. The overall objective of this study was to determine the spatial patterning of microbial biomass in soils over a wide extent and to rank the environmental filters most influencing this distribution.

Modeling Diverse Communities of Marine Microbes

Biogeochemical cycles in the ocean are mediated by complex and diverse microbial communities. Over the past decade, marine ecosystem and biogeochemistry models have begun to address some of this diversity by resolving several groups of (mostly autotrophic) plankton, differentiated by biogeochemical function. Here, we review recent model approaches that are rooted in the notion that an even richer diversity is fundamental to the organization of marine microbial communities. These models begin to resolve, and address the significance of, diversity within functional groups. Seeded with diverse populations spanning prescribed regions of trait space, these simulations self-select community structure according to relative fitness in the virtual environment. Such models are suited to considering ecological questions, such as the regulation of patterns of biodiversity, and to simulating the response to changing environments. A key issue for all such models is the constraint of viable trait space and trade-offs. Size-structuring and mechanistic descriptions of energy and resource allocation at the individual level can rationalize these constraints.