Light‐responsive current generation by phototrophically enriched anode biofilms dominated by green sulfur bacteria

The objective of this study was to employ microbial electrochemical cells (MXCs) to selectively enrich and examine anoxygenic photosynthetic bacteria for potential anaerobic respiration capabilities using electrodes. In the process, we designed a novel enrichment strategy that manipulated the poised anode potential, light, nitrogen availability, and media supply to promote growth of phototrophic bacteria while minimizing co‐enrichment of non‐phototrophic anode‐respiring bacteria (ARB). This approach resulted in light‐responsive electricity generation from fresh‐ and saltwater inocula. Under anoxic conditions, current showed a negative light response, suggesting that the enriched phototrophic consortia shifted between phototrophic and anaerobic respiratory metabolism. Molecular, physical, and electrochemical analyses elucidated that anode biofilms were dominated by green sulfur bacteria, and biofilms exhibited anode respiration kinetics indicative of non‐mediated electron transfer, but kinetic parameters differed from values previously reported for non‐phototrophic ARB. These results invite the utilization of MXCs as microbiological tools for exploring anaerobic respiratory capabilities among anoxygenic photosynthetic bacteria. Biotechnol. Bioeng. 2013; 110: 1020–1027. © 2012 Wiley Periodicals, Inc.     

Heterotrophic Pioneers Facilitate Phototrophic Biofilm Development

Phototrophic biofilms are matrix-enclosed microbial communities, mainly driven by light energy. In this study, the successional changes in community composition of freshwater phototrophic biofilms growing on polycarbonate slides under different light intensities were investigated. The sequential changes in community composition during different developmental stages were examined by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments in conjugation with sequencing and phylogenetic analysis. Biofilm development was monitored with subsurface light sensors. The development of these biofilms was clearly light dependent. It was shown that under high light conditions the initial colonizers of the substratum predominantly consisted of green algae, whereas at low light intensities, heterotrophic bacteria were the initial colonizers. Cluster analysis of DGGE banding patterns revealed a clear correlation in the community structure with the developmental phases of the biofilms. At all light intensities, filamentous cyanobacteria affiliated to Microcoleus vaginatus became dominant as the biofilms matured. It was shown that the initial colonization phase of the phototrophic biofilms is shorter on polycarbonate surfaces precolonized by heterotrophic bacteria.     

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.     

Light availability affects stream biofilm bacterial community composition and function,but not diversity

Changes in riparian vegetation or water turbidity and browning in streams alter the local light regime with potential implications for stream biofilms and ecosystem functioning. We experimented with biofilms in microcosms grown under a gradient of light intensities (range: 5–152 μmole photons s^?1 m^?2) and combined 454‐pyrosequencing and enzymatic activity assays to evaluate the effects of light on biofilm structure and function. We observed a shift in bacterial community composition along the light gradient, whereas there was no apparent change in alpha diversity. Multifunctionality, based on extracellular enzymes, was highest under high light conditions and decoupled from bacterial diversity. Phenol oxidase activity, involved in the degradation of polyphenolic compounds, was twice as high on average under the lowest compared with the highest light condition. This suggests a shift in reliance of microbial heterotrophs on biofilm phototroph‐derived organic matter under high light availability to more complex organic matter under low light. Furthermore, extracellular enzyme activities correlated with nutrient cycling and community respiration, supporting the link between biofilm structure–function and biogeochemical fluxes in streams. Our findings demonstrate that changes in light availability are likely to have significant impacts on biofilm structure and function, potentially affecting stream ecosystem processes.     

Effect of light regimes on the utilisation of an exogenous carbon source by freshwater biofilm bacterial communities

This study reports the novel use of nucleic acid stable isotope probing (NA-SIP) to identify metabolically active ([¹³C]-acetate assimilating) bacteria in freshwater biofilms. Currently, a little is known of the factors affecting the structure and activity of these complex microbial biofilm communities, although it is likely that they are influenced by riparian vegetation through attenuation of light and alteration of allochthonous inputs of carbon. NA-SIP was used to investigate the effect of varying light regimes on [¹³C]-acetate assimilating bacteria within laboratory biofilm microcosms. Differences in clone libraries of 16S rRNA and rRNA genes from ¹³C-labelled and unlabelled nucleic acids indicated differential uptake of acetate and the rapid transfer of ¹³C to organisms at a higher trophic level. Biofilm communities incubated in the dark changed least over time and retained a significant fraction of phototrophic organisms. Incubation under elevated light caused the greatest change in bacterial community structure. Contrary to expectation, a complete loss of chlorophyll containing organisms occurred within this treatment, challenging current thinking that elevated light promotes communities dominated by photoautotrophs in nutrient enriched environments.     

Phototrophic biofilms and their potential applications

Phototrophic biofilms occur on surfaces exposed to light in a range of terrestrial and aquatic environments. Oxygenic phototrophs like diatoms, green algae, and cyanobacteria are the major primary producers that generate energy and reduce carbon dioxide, providing the system with organic substrates and oxygen. Photosynthesis fuels processes and conversions in the total biofilm community, including the metabolism of heterotrophic organisms. A matrix of polymeric substances secreted by phototrophs and heterotrophs enhances the attachment of the biofilm community. This review discusses the actual and potential applications of phototrophic biofilms in wastewater treatment, bioremediation, fish-feed production, biohydrogen production, and soil improvement.     

Rare but active taxa contribute to community dynamics of benthic biofilms in glacier‐fed streams

Glaciers harbour diverse microorganisms, which upon ice melt can be released downstream. In glacier‐fed streams microorganisms can attach to stones or sediments to form benthic biofilms. We used 454‐pyrosequencing to explore the bulk (16S rDNA) and putatively active (16S rRNA) microbial communities of stone and sediment biofilms across 26 glacier‐fed streams. We found differences in community composition between bulk and active communities among streams and a stronger congruence between biofilm types. Relative abundances of rRNA and rDNA were positively correlated across different taxa and taxonomic levels, but at lower taxonomic levels, the higher abundance in either the active or the bulk communities became more apparent. Here, environmental variables played a minor role in structuring active communities. However, we found a large number of rare taxa with higher relative abundances in rRNA compared with rDNA. This suggests that rare taxa contribute disproportionately to microbial community dynamics in glacier‐fed streams. Our findings propose that high community turnover, where taxa repeatedly enter and leave the ‘seed bank’, contributes to the maintenance of microbial biodiversity in harsh ecosystems with continuous environmental perturbations, such as glacier‐fed streams.     

Electroactivity of phototrophic river biofilms and constitutive cultivable bacteria

Electroactivity is a property of microorganisms assembled in biofilms that has been highlighted in a variety of environments. This characteristic was assessed for phototrophic river biofilms at the community scale and at the bacterial population scale. At the community scale, electroactivity was evaluated on stainless steel and copper alloy coupons used both as biofilm colonization supports and as working electrodes. At the population scale, the ability of environmental bacterial strains to catalyze oxygen reduction was assessed by cyclic voltammetry. Our data demonstrate that phototrophic river biofilm development on the electrodes, measured by dry mass and chlorophyll a content, resulted in significant increases of the recorded potentials, with potentials of up to +120 mV/saturated calomel electrode (SCE) on stainless steel electrodes and +60 mV/SCE on copper electrodes. Thirty-two bacterial strains isolated from natural phototrophic river biofilms were tested by cyclic voltammetry. Twenty-five were able to catalyze oxygen reduction, with shifts of potential ranging from 0.06 to 0.23 V, cathodic peak potentials ranging from -0.36 to -0.76 V/SCE, and peak amplitudes ranging from -9.5 to -19.4 μA. These isolates were diversified phylogenetically (Actinobacteria, Firmicutes, Bacteroidetes, and Alpha-, Beta-, and Gammaproteobacteria) and exhibited various phenotypic properties (Gram stain, oxidase, and catalase characteristics). These data suggest that phototrophic river biofilm communities and/or most of their constitutive bacterial populations present the ability to promote electronic exchange with a metallic electrode, supporting the following possibilities: (i) development of electrochemistry-based sensors allowing in situ phototrophic river biofilm detection and (ii) production of microbial fuel cell inocula under oligotrophic conditions.     

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.     

Loss of Rare Fish Species from Tropical Floodplain Food Webs Affects Community Structure and Ecosystem Multifunctionality in a Mesocosm Experiment

Experiments with realistic scenarios of species loss from multitrophic ecosystems may improve insight into how biodiversity affects ecosystem functioning. Using 1000 L mesocoms, we examined effects of nonrandom species loss on community structure and ecosystem functioning of experimental food webs based on multitrophic tropical floodplain lagoon ecosystems. Realistic biodiversity scenarios were developed based on long-term field surveys, and experimental assemblages replicated sequential loss of rare species which occurred across all trophic levels of these complex food webs. Response variables represented multiple components of ecosystem functioning, including nutrient cycling, primary and secondary production, organic matter accumulation and whole ecosystem metabolism. Species richness significantly affected ecosystem function, even after statistically controlling for potentially confounding factors such as total biomass and direct trophic interactions. Overall, loss of rare species was generally associated with lower nutrient concentrations, phytoplankton and zooplankton densities, and whole ecosystem metabolism when compared with more diverse assemblages. This pattern was also observed for overall ecosystem multifunctionality, a combined metric representing the ability of an ecosystem to simultaneously maintain multiple functions. One key exception was attributed to time-dependent effects of intraguild predation, which initially increased values for most ecosystem response variables, but resulted in decreases over time likely due to reduced nutrient remineralization by surviving predators. At the same time, loss of species did not result in strong trophic cascades, possibly a result of compensation and complexity of these multitrophic ecosystems along with a dominance of bottom-up effects. Our results indicate that although rare species may comprise minor components of communities, their loss can have profound ecosystem consequences across multiple trophic levels due to a combination of direct and indirect effects in diverse multitrophic ecosystems.     

Kinetic modeling of phototrophic biofilms: the PHOBIA model

A kinetic model for mixed phototrophic biofilms is introduced, which focuses on the interactions between photoautotrophic, heterotrophic, and chemoautotrophic (nitrifying) functional microbial groups. Biofilm-specific phenomena are taken into account, such as extracellular polymeric substances (EPS) production by phototrophs as well as gradients of substrates and light in the biofilm. Acid-base equilibria, in particular carbon speciation, are explicitly accounted for, allowing for the determination of pH profiles across the biofilm. Further to previous models reported in literature, the PHOBIA model combines a number of kinetic mechanisms specific to phototrophic microbial communities, such as internal polyglucose storage under dynamic light conditions, phototrophic growth in the darkness using internally stored reserves, photoadaptation and photoinhibition, preference for ammonia over nitrate as N-source and the ability to utilize bicarbonate as a carbon source in the absence of CO(2). The sensitivity of the PHOBIA model to a number of key parameters is analyzed. An example on the potential use of phototrophic biofilms in wastewater polishing is discussed, where their performance is compared with conventional algal ponds. The PHOBIA model is presented in a manner that is compatible with other reference models in the area of water treatment. Its current version forms a theoretical base which is readily extendable once further experimental observations become available.     

Biodiversity and ecosystem functioning in food webs: the vertical diversity hypothesis

One challenge in merging community and ecosystem ecology is to integrate the complexity of natural multitrophic communities into concepts of ecosystem functioning. Here, we combine food‐web and allometry theories to demonstrate that primary production, as measured by the total nutrient uptake of the multitrophic community, is determined by vertical diversity (i.e. food web's maximum trophic level) and structure (i.e. distributions of species and their abundances and metabolic rates across trophic levels). In natural ecosystems, the community size distribution determines all these vertical patterns and thus the total nutrient uptake. Our model suggests a vertical diversity hypothesis (VDH) for ecosystem functioning in complex food webs. It predicts that, under a given nutrient supply, the total nutrient uptake increases exponentially with the maximum trophic level in the food web and it increases with its maximum body size according to a power law. The VDH highlights the effect of top–down regulation on plant nutrient uptake, which complements traditional paradigms that emphasised the bottom–up effect of nutrient supply on vertical diversity. We conclude that the VDH contributes to a synthetic framework for understanding the relationship between vertical diversity and ecosystem functioning in food webs and predicting the impacts of global changes on multitrophic ecosystems.     

OptCom: a multi-level optimization framework for the metabolic modeling and analysis of microbial communities

Microorganisms rarely live isolated in their natural environments but rather function in consolidated and socializing communities. Despite the growing availability of high-throughput sequencing and metagenomic data, we still know very little about the metabolic contributions of individual microbial players within an ecological niche and the extent and directionality of interactions among them. This calls for development of efficient modeling frameworks to shed light on less understood aspects of metabolism in microbial communities. Here, we introduce OptCom, a comprehensive flux balance analysis framework for microbial communities, which relies on a multi-level and multi-objective optimization formulation to properly describe trade-offs between individual vs. community level fitness criteria. In contrast to earlier approaches that rely on a single objective function, here, we consider species-level fitness criteria for the inner problems while relying on community-level objective maximization for the outer problem. OptCom is general enough to capture any type of interactions (positive, negative or combinations thereof) and is capable of accommodating any number of microbial species (or guilds) involved. We applied OptCom to quantify the syntrophic association in a well-characterized two-species microbial system, assess the level of sub-optimal growth in phototrophic microbial mats, and elucidate the extent and direction of inter-species metabolite and electron transfer in a model microbial community. We also used OptCom to examine addition of a new member to an existing community. Our study demonstrates the importance of trade-offs between species- and community-level fitness driving forces and lays the foundation for metabolic-driven analysis of various types of interactions in multi-species microbial systems using genome-scale metabolic models.     

Trophic Niche Differentiation and Microhabitat Utilization in a Species‐rich Montane Forest Small Mammal Community of Eastern Madagascar

Unraveling the mechanisms facilitating species coexistence in communities is a central theme in ecology. Species‐rich tropical mammal communities provide excellent settings to explore such mechanisms as they often harbor numerous congeneric species with close phylogenetic relationships. Explicit tests for the mechanisms that allow syntopic occurrence in these assemblages, however, is often hampered because of the difficulty in obtaining detailed ecological data on the organisms making up the community. Using stable nitrogen and carbon ratios of hair samples, we examine whether trophic niche differentiation and microhabitat segregation explain the coexistence of 21 small mammal species at a montane humid forest site in eastern Madagascar. Overall, the community was trophically diverse and covered wide isotopic space. This diversity was based on: (1) a multi‐layered trophic community structure with mainly frugivorous‐granivorous rodents (subfamily Nesomyinae) as primary consumers and insectivorous tenrecs (family Tenrecidae) as secondary and tertiary consumers; (2) trophic segregation of rodents and tenrecs with the latter occupying different microhabitats; and (3) a dense and regular packing of species in the community. The 12 locally occurring Microgale shrew tenrecs (subfamily Oryzorictinae) showed high trophic redundancy, but were maximally spaced from each other within the trophic space covered by the genus. Results of stable isotope analysis suggest that in combination the differentiation of microhabitats and trophic niches explain the coexistence of small mammals in this community. Congeneric species appeared to be under more intense competition compared with non‐congeneric species and their coexistence can only partly be explained by trophic and microhabitat niche segregation.     

With or without you: Effects of the concurrent range expansion of an herbivore and its natural enemy on native species interactions

Global climatic changes may lead to the arrival of multiple range‐expanding species from different trophic levels into new habitats, either simultaneously or in quick succession, potentially causing the introduction of manifold novel interactions into native food webs. Unraveling the complex biotic interactions between native and range‐expanding species is critical to understand the impact of climate change on community ecology, but experimental evidence is lacking. In a series of laboratory experiments that simulated direct and indirect species interactions, we investigated the effects of the concurrent arrival of a range‐expanding insect herbivore in Europe, Spodoptera littoralis, and its associated parasitoid Microplitis rufiventris, on the native herbivore Mamestra brassicae, and its associated parasitoid Microplitis mediator, when co‐occurring on a native plant, Brassica rapa. Overall, direct interactions between the herbivores were beneficial for the exotic herbivore (higher pupal weight than the native herbivore), and negative for the native herbivore (higher mortality than the exotic herbivore). At the third trophic level, both parasitoids were unable to parasitize the herbivore they did not coexist with, but the presence of the exotic parasitoid still negatively affected the native herbivore (increased mortality) and the native parasitoid (decreased parasitism rate), through failed parasitism attempts and interference effects. Our results suggest different interaction scenarios depending on whether S. littoralis and its parasitoid arrive to the native tritrophic system separately or concurrently, as the negative effects associated with the presence of the parasitoid were dependent on the presence of the exotic herbivore. These findings illustrate the complexity and interconnectedness of multitrophic changes resulting from concurrent species arrival to new environments, and the need for integrating the ecological effects of such arrivals into the general theoretical framework of global invasion patterns driven by climatic change.     

Phototrophic microbes form endolithic biofilms in ikaite tufa columns (SW Greenland)

Marine tufa‐columns, formed by the hydrated carbonate mineral ikaite, present a unique alkaline microbial habitat only found in Ikka Fjord (SW‐Greenland). The outermost parts of the ikaite columns exhibit a multitude of physico‐chemical gradients, and the porous ikaite is colonized by endolithic phototrophic biofilms serving as a substrate for grazing epifauna, where scraping by sea urchins affects overall column‐topography. We present a detailed study of the optical microenvironment, spatial organization, and photosynthetic activity of endolithic phototrophs within the porous ikaite crystal matrix. Cyanobacteria and diatoms formed distinctly coloured zones and were closely associated with ikaite‐crystals via excretion of exopolymers. Scalar‐irradiance measurements showed strong attenuation of visible light (400–700 nm), where only ～1% of incident irradiance remained at 20 mm depth. Transmission spectra showed in vivo absorption signatures of diatom and cyanobacterial photopigments, which were confirmed by HPLC‐analysis. Variable‐chlorophyll‐fluorescence‐imaging showed active photosynthesis with high‐light acclimation in the outer diatom layer, and low‐light acclimation in the underlying cyanobacterial part. Phototrophs in ikaite thus thrive in polymer‐bound endolithic biofilms in a complex gradient microhabitat experiencing constant slow percolation of highly alkaline phosphate‐enriched spring water mixing with cold seawater at the tufa‐column‐apex. We discuss the potential role of these biofilms in ikaite column formation.     

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.