Ciliates as engineers of phototrophic biofilms

1. Phototrophic biofilms consist of a matrix of phototrophs, non‐photosynthetic bacteria and extracellular polymeric substances (EPS) which is spatially structured. Despite widespread exploitation of algae and bacteria within phototrophic biofilms, for example by protozoans, the ‘engineering’ effects of these ciliates on the spatial heterogeneity of phototrophic biofilms are poorly studied. 2. We studied the potential engineering effects of two ciliates, Urostyla sp. and Paramecium bursaria, on the spatial heterogeneity of synthetic multispecies biofilms. Biomass of phototrophic organisms, EPS and bacteria was analysed three dimensionally using confocal laser scanning microscopy. Spatial heterogeneity and cover of the phototrophs, bacteria and EPS were determined at several depths within the biofilm. 3. Ciliate species did not interfere with the overall development of phototrophic microorganisms, because the thickness of the biofilm was equal whether the ciliates were present or not, even though their abundance did affect spatial heterogeneity of biofilm components. When Urostyla was present, it reduced aggregation in EPS and bacteria and increased EPS biovolume. This implies a local facilitating effect of ciliates on photosynthetic activity. Biofilms to which Paramecium was added did not differ from controls in terms of phototrophs, EPS cover and biovolume. Nevertheless, ciliates affected the spatial heterogeneity of these components as phototrophs and EPS became more evenly distributed. 4. This study shows that ecosystem engineering by organisms does not only occur at large spatial scales, as in grasslands and estuaries, but also plays a role at the microscopic scale of biofilms. This effect on spatial heterogeneity was not driven by substantial exploitation of biofilm components, but via the subtle engineering effects of ciliates.     

Capsular polysaccharides of cultured phototrophic biofilms

Phototrophic biofilm samples from an Italian wastewater treatment plant were studied in microcosm experiments under varying irradiances, temperatures and flow regimes to assess the effects of environmental variables and phototrophic biomass on capsular exopolysaccharides (CPS). The results, obtained from circular dichroism spectroscopy and High Performance Liquid Chromatography, suggest that CPS have a stable spatial conformation and a complex monosaccharide composition. The total amount present was positively correlated with the biomass of cyanobacteria and diatoms, and negatively with the biovolume of green algae. The proportion of uronic acids showed the same correlation with these taxon groups, indicating a potential role of cyanobacteria and diatoms in the removal of residual nutrients and noxious cations in wastewater treatment. While overall biofilm growth was limited by low irradiance, high temperature (30°C) and low flow velocity (25 l h^?1) yielded the highest phototrophic biomass, the largest amount of CPS produced, and the highest proportion of carboxylic acids present.     

Diversity and biomass accumulation in cultured phototrophic biofilms

In the present study, biomass development and changes in community composition of phototrophic biofilms grown under different controlled ambient conditions (light, temperature and flow) were examined. Source communities were taken from a wastewater treatment plant and used to inoculate growth surfaces in a semi-continuous-flow microcosm. We recorded biofilm growth curves in cultures over a period of 30 days across 12 experiments. Biovolume of phototrophs and community composition for taxonomic shifts were also obtained using light and electron microscopy. Species richness in the cultured biofilms was greatly reduced with respect to the natural samples, and diversity decreased even further during biofilm development. Diadesmis confervacea, Phormidium spp., Scenedesmus spp. and Synechocystis spp. were identified as key taxa in the microcosm. While a significant positive effect of irradiance on biofilm growth could be identified, impacts of temperature and flow rate on biofilm development and diversity were less evident. We discuss the hypothesis that biofilm development could have been subject to multistability, i.e. the existence of several possible stable biofilm configurations for the same set of environmental parameters; small variations in the species composition might have been sufficient to switch between these different configurations and thus have contributed to overwriting the original effects of temperature and flow velocity.     

Quinones of phototrophic purple bacteria

Abstract The quinone composition of the recognized species of the phototrophic purple nonsulfur bacteria, the Ectothiorhodospiraceae, and some Chromatiaceae species has been determined. Altogether more than 50 strains of 33 species have been investigated. Some of the purple nonsulfur bacteria have Q-10 as sole quinone component, while others have Q-10, Q-9, or Q-8, respectively, together with menaquinones of the same isoprenoid chain length as the major components. Rhodoquinone is present in Rhodospirillum rubrum and Rhodospirillum photometricum . The Ectothiorhodospira species have either Q-8 and MK-8, like the Chromatiaceae species, or Q-7 and MK-7 as the major components.     

Photosynthetic performance of phototrophic biofilms in extreme acidic environments

Photosynthesis versus irradiance curves and their associated photosynthetic parameters from different phototrophic biofilms isolated from an extreme acidic environment (Río Tinto, SW, Spain) were studied in order to relate them to their species composition and the physicochemical characteristics of their respective sampling locations. The results indicated that the biofilms are low light acclimated showing a photoinhibition model; only floating communities of filamentous algae showed a light saturation model. Thus, all the biofilms analysed showed photoinhibition over 60 μmol photon m(-2) s(-1) except in the case of Zygnemopsis sp. sample, which showed a light-saturated photosynthesis model under irradiations higher that 200 μmol photon m(-2) s(-1). The highest values of compensation light intensity (I(c)) were showed also by Zygnemosis sp. biofilm (c. 40 μmol photon m(-2) s(-1)), followed by Euglena mutabilis and Chlorella sp. samples (c. 20 μmol photon m(-2) s(-1)). The diatom sample showed the lowest I(c) values (c. 5 μmol photon m(-2) s(-1)). As far as we know this is the first attempt to determine the photosynthetic activity of low pH and heavy metal tolerant phototrophic biofilms, which may give light in the understanding of the ecological importance of these biofilms for the maintenance of the primary production of these extreme and unique ecosystems.     

Evaluation of DNA extraction methods from complex phototrophic biofilms

Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 ± 12 to 1893 ± 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol–chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol–chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms.     

Application of phototrophic biofilms: from fundamentals to processes

Bioprocess and Biosystems Engineering - Biotechnological production of valuables by microorganisms is commonly achieved by cultivating the cells as suspended solids in an appropriate liquid medium....     

Active transport in phototrophic bacteria

Phototrophic bacteria utilize light-driven, cyclic electron flow to pump protons out of their cytoplasm, creating an electrochemical proton gradient, _H+, outside acid and positive. These bacteria exchange external protons for internal cations (Na⁺, K⁺ and Ca⁺²), allowing the cells to maintain a nearly constant internal pH while maintaining the electrical component of _H+. Na⁺/H⁺ exchange also establishes an electrochemical Na⁺ gradient. Phototrophic bacteria are able to utilize these electrochemical gradients as energy sources for the uptake of a wide variety of metabolites (e.g., sugars, organic acids and amino acids) via metabolite/cation symports.     

A flow-lane incubator for studying freshwater and marine phototrophic biofilms

Phototrophic biofilms are defined as interfacial microbial communities mainly driven by light as energy source and are studied for both ecological and technological reasons. Field investigations of biofilms usually do not offer the opportunity to study the effects of a large number of external parameters. In order to investigate the temporal development of phototrophic communities a laboratory flow-lane incubator for cultivation of freshwater and marine biofilms was developed. The incubator has four lanes which accommodate microscope slides used as substratum and for sampling. The slides can be of different material and may be employed for characterisation of phototrophic biofilms by means of gravimetry, microscopy, taxonomy, molecular biology and chemical analysis. The design allows control of irradiance, temperature and flow velocity. Furthermore, on-line control of biomass accumulation via specially adapted light sensors was proved to be a suitable indicator of temporal developmental stages (initial adhesion, active growth and mature stage). Spatial heterogeneity of the cultivated phototrophic biofilms along the flow direction within each flow-lane was low. Biofilm growth characteristics (e. g. lag time, net accrual rate, peak biomass) recorded in dependency from external conditions may be used as input data for training of artificial neural networks (ANN) and mechanistic modelling. The material and devices used in combination with low maintenance costs and ease of handling suggests the flow-lane incubator as a useful tool for studying the influence of abiotic and biotic factors on the development of freshwater and marine phototrophic biofilms.     

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.     

Phosphate regime structures species composition in cultured phototrophic biofilms

1. The effect of phosphate on species composition in biofilms was studied under three different phosphate regimes (0.5, 5 and 50 μm ) in two different multi species communities: one composed of the four diatom species Melosira varians, Nitzschia perminuta, Navicula trivialis and Achnanthes lanceolata and one containing these diatom species plus the two cyanobacterial species Leptolyngbya foveolarum and Cylindrospermum stagnale. 2. Algal growth in monocultures and mixtures was measured as chlorophyll a and PAM fluorimetry was applied to document density and physiological condition of the two main groups of photosynthetic organisms in mixed cultures. 3. In phosphate‐replete communities, a single species dominated the community (N. perminuta in the diatom mixture and L. foveolarum in the all species mixture), while in the phosphate‐deprived communities several species persisted, in spite of severe phosphate limitation. 4. We conclude that high supply of phosphate enables the species L. foveolarum, and to a lesser extent N. perminuta, to overgrow biofilm consortia, facilitated by their filamentous growth form, motility or the excretion of inhibitors. The persistence of several species under a low phosphate regime is explained by a less intense interspecific interaction in low‐density biofilms. This clarifies field observations published previously.     

Influence of local climate and climate change on aeroterrestrial phototrophic biofilms

Aeroterrestrial phototrophic biofilms colonize natural and man-made surfaces and may damage the material they settle on. The occurrence of biofilms varies between regions with different climatic conditions. The aim of this study was to evaluate the influence of meteorological factors on the growth of aeroterrestrial phototrophs. Phototrophic biomass was recorded on roof tiles at six sites within Germany five times over a period of five years and compared to climatic parameters from neighboring weather stations. All correlating meteorological factors influenced water availability on the surface of the roof tiles. The results indicate that the frequency of rainy days and not the mean precipitation per season is more important for biofilm proliferation. It is also inferred that the macroclimate is more important than the microclimate. In conclusion, changed (regional) climatic conditions may determine where in central Europe global change will promote or inhibit phototrophic growth in the future.     

Organic nitrogen metabolism of phototrophic bacteria

Recent reviews dealing with phototrophic bacteria are concerned with bioenergetics, nitrogen fixation and hydrogen metabolism, synthesis of the photosynthetic apparatus and phylogeny/taxonomy. The organic N-metabolism of these phylogenetically diverse bacteria has last been reviewed in 1978. However, amino acid utilization and biosynthesis, ammonia assimilation, purine and pyrimidine metabolism and biosynthesis of -aminolevulinic acid as precursor of bacteriochlorophylls and hemes are topics of vital importance. This review focusses on utilization of amino acids as N- and C/N-sources, the pathways of purine and pyrimidine degradation, novel aspects of amino acid biosynthesis (with emphasis on branched-chain amino acids and -aminole-vulinic acid) and some aspects of ammonia assimilation and glutamate synthesis by purple bacteria, green sulfur bacteria and Chloroflexus aurantiacus.Abbreviations R Rhodospirillum - Rhb Rhodobacter - Rc Rhodocyclus - Rp Rhodopila - Rps Rhodopseudomonas     

Diversity of phototrophic components in biofilms from Piperno historical stoneworks

We investigated phototrophic microorganisms dwelling on stone walls made of Piperno, a volcanic rock frequently used as construction material in historical buildings in Naples, Italy. Biofilms from three historical buildings in the center of the city and from a natural Piperno quarry located in a suburban area were examined. Light and electron microscopy, and molecular biology techniques allowed the identification of 17 species belonging to Cyanobacteria, Rhodophyta, Bacillariophyta, and Chlorophyta. Cyanobacteria were the dominant components in all the biofilms. No significant differences in microbial composition were observed for biofilms collected in autumn and spring, with minor exceptions for the quarry samples, where environmental conditions were relatively more stable than in the city. Results are discussed in comparison with information on microbial communities dwelling on other kinds of substrata commonly used in historical buildings in the Neapolitan area.     

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.     

Cysteine and S-sulfocysteine biosynthesis in phototrophic bacteria

Forteen species (17 strains) of phototrophic bacteria as well as one strain of Thiobacillus denitrificans were tested for cysteine synthase and S-sulfocysteine synthase. All strains contain cysteine synthase active with O-acetylserine; only the Chromatiaceae, two species of the Rhodospirillaceae and T. denitrificans contain S-sulfocysteine synthase. In six species repression by different sulfur compounds in the medium was studied. In Chromatium vinosum, cysteine synthase was found to be constitutive, while in the Rhodospirillaceae tested the enzyme is repressed by sulfide. Thiosulfate had a derepressive effect in Rhodopseudomonas globiformis but strongly repressed cysteine synthase in R. sulfidophila and R. palustris. Cysteine had only moderate effects with the species tested.     

True marine and halophilic anoxygenic phototrophic bacteria

Anoxygenic phototrophic bacteria are widely distributed in marine sediments and shallow waters of the coastal zone, where they often form intensely colored mass developments. The phototrophic bacteria have adapted to the whole spectrum of salt concentrations, from freshwater to saturated brines, and it is apparent that individual species have adapted well to particular habitats and mineral salts compositions, both qualitatively and quantitatively. This adaptation is reflected not only in the demand for defined ranges of salt concentrations, but also in the phylogenetic relationships of these bacteria, as established by 16S rDNA sequences. Major phylogenetic branches of purple sulfur bacteria are represented by: (1) marine and extremely halophilic Ectothiorhodospiraceae, (2) truly marine and halophilic Chromatiaceae and (3) freshwater Chromatiaceae, some of which are tolerant to low salt concentrations and are successful competitors in brackish and marine habitats. Quite similarly, salt-dependent green sulfur bacteria form distinct phylogenetic lines. In addition, also among the phototrophic alpha-Proteobacteria (purple nonsulfur bacteria), distinct phylogenetic lines of salt-dependent species are recognized. Available data give rise to the assumption that salt concentrations of natural habitats are an important selective factor that determines the development of a selected range of phototrophic bacteria in an exclusive way. As a consequence, the salt responses of these bacteria are reflected in their phylogenetic relationships.     

1H-NMR analysis of water mobility in cultured phototrophic biofilms

The present work reports on the first attempt to study water mobility in phototrophic biofilms, applying the ¹H-NMR relaxometry technique to closely monitored microbial communities grown in a microcosm under controlled ambient conditions. Longitudinal water proton relaxation times exhibited a bi-exponential behavior in all biofilm samples, indicating two types of water molecules with diverging dynamic properties, confined to different compartments of the biofilm. The fast-relaxing component can be attributed to water molecules tightly bound to the intracellular matrix, while the slow-relaxing component could reflect the behavior of water embedded in the biopolymer matrix, confined into matrix pores and channels. The results are discussed with respect to a possible key role of exopolysaccharides and uronic acids in water binding in phototrophic biofilms.     

1H-NMR analysis of water mobility in cultured phototrophic biofilms

The present work reports on the first attempt to study water mobility in phototrophic biofilms, applying the (1)H-NMR relaxometry technique to closely monitored microbial communities grown in a microcosm under controlled ambient conditions. Longitudinal water proton relaxation times exhibited a bi-exponential behavior in all biofilm samples, indicating two types of water molecules with diverging dynamic properties, confined to different compartments of the biofilm. The fast-relaxing component can be attributed to water molecules tightly bound to the intracellular matrix, while the slow-relaxing component could reflect the behavior of water embedded in the biopolymer matrix, confined into matrix pores and channels. The results are discussed with respect to a possible key role of exopolysaccharides and uronic acids in water binding in phototrophic biofilms.