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.     

On the reproducibility of microcosm experiments - different community composition in parallel phototrophic biofilm microcosms

Phototrophic biofilms were cultivated simultaneously using the same inoculum in three identical flow-lane microcosms located in different laboratories. The growth rates of the biofilms were similar in the different microcosms, but denaturing gradient gel electrophoresis (DGGE) analysis of both 16S and 18S rRNA gene fragments showed that the communities developed differently in terms of species richness and community composition. One microcosm was dominated by Microcoleus and Phormidium species, the second microcosm was dominated by Synechocystis and Phormidium species, and the third microcosm was dominated by Microcoleus- and Planktothrix- affiliated species. No clear effect of light intensity on the cyanobacterial community composition was observed. In addition, DGGE profiles obtained from the cultivated biofilms showed a low resemblance with the profiles derived from the inoculum. These findings demonstrate that validation of reproducibility is essential for the use of microcosm systems in microbial ecology studies.     

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.     

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.     

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.     

Short-term response of monospecific and natural algal biofilms to copper exposure

The effect of copper additions (Cu ranging from 0 to 30?µM) on the photosynthesis of three different microalgal biofilms was studied to identify the factors that cause sensitivity differences between benthic and pelagic algae. The response of biofilms which colonized artificial substrata in the River Meuse was compared with those of two laboratory-grown monospecific biofilms, one consisting of the diatom Synedra ulna, and the other composed of a filament-forming cyanobacterium, Oscillatoria sp. The photosynthetic yield Φ_II (quantum efficiency of photosystem II) was studied with PAM (Pulse Amplitude Modulated) fluorimetry. S. ulna biofilms appeared to be the most sensitive to Cu, followed by the cyanobacteria, while natural biofilms, dominated by supposedly very sensitive diatom species such as Melosira varians and Diatoma vulgare, were the most resistant to Cu. In the highly productive biofilms, pH is suggested to play a role in lowering toxicity by helping the precipitation of cupric ions. Cu accumulation by the biofilms during the exposure period followed a linear relationship with Cu concentration, saturation not being observed; natural biofilms had an accumulation factor of 1–2.5?×?10³ relative to the concentrations in the water, while the diatoms growing unattached to the substratum had a higher concentration factor, up to 4.9?×?10³. It was concluded that the physical structure of the biofilm (package of cells and thickness), and not the species composition, was the main factor regulating the sensitivity of the biofilm to Cu toxicity during short-term exposures.     

Associating particulate essential fatty acids of the ω3 family with phytoplankton species composition in a Siberian reservoir

1. We studied variation in the composition of fatty acids in the seston of a small freshwater reservoir with changes in phytoplankton composition during four growth seasons. We focused on the dynamics of the ω3 fatty acids because of their potential importance for zooplankton nutrition. 2. Total diatoms were related to the 20:5ω3 fatty acid (eicosapentaenoic, EPA) content in seston. Among two dominant diatom genera, Cyclotella was not associated with EPA content. In contrast, there was a significant correlation between Stephanodiscus and the percentage contribution and content of EPA throughout the study. Hence, freshwater diatoms can differ strongly in content of the essential EPA. 3. We considered abundant cyanobacteria as a potential source of 18:3ω3 fatty acid (linolenic, ALA) to aquatic food webs. Among four dominant cyanobacteria species, two (Anabaena flos‐aquae and Planktothrix agardhii) showed significant correlation with the ALA content of the seston, while the other two (Aphanizomenon flos‐aquae and Microcystis aeruginosa) did not. 4. Dinophyta had a relatively high level of 22:6ω3 (docosahexaenoic, DHA) for freshwater species and can be also a source of EPA to aquatic food webs. 5. Our results show that various species of diatoms as well as cyanobacteria can be of contrasting nutritional value for zooplankton because of their different content of the essential PUFAs. Diatoms, which are low in EPA, could not be considered as a valuable food, while some field populations of cyanobacteria might be valuable sources of essential ALA.     

Chlorophyll f distribution and dynamics in cyanobacterial beachrock biofilms

Chlorophyll (Chl) f, the most far‐red (720–740 nm) absorbing Chl species, was discovered in cyanobacterial isolates from stromatolites and subsequently in other habitats as well. However, the spatial distribution and temporal dynamics of Chl f in a natural habitat have so far not been documented. Here, we report the presence of Chl f in cyanobacterial beachrock biofilms. Hyperspectral imaging on cross‐sections of beachrock from Heron Island (Great Barrier Reef, Australia), showed a strong and widely distributed signature of Chl f absorption in an endolithic layer below the dense cyanobacterial surface biofilm that could be localized to aggregates of Chroococcidiopsis‐like unicellular cyanobacteria packed within a thick common sheath. High‐pressure liquid chromatography‐based pigment analyses showed in situ ratios of Chl f to Chl a of 5% in brown‐pigmented zones of the beachrock, with lower ratios of ~0.5% in the black‐ and pink‐pigmented biofilm zones. Enrichment experiments with black beachrock biofilm showed stimulated synthesis of Chl f and Chl d when grown under near‐infrared radiation (NIR; 740 nm), with a Chl f to Chl a ratio increasing 4‐fold to 2%, whereas the Chl d to Chl a ratio went from 0% to 0.8%. Enrichments grown under white light (400–700 nm) produced no detectable amounts of either Chl d or Chl f. Beachrock cyanobacteria thus exhibited characteristics of far‐red light photoacclimation, enabling Chl f ‐containing cyanobacteria to thrive in optical niches deprived of visible light when sufficient NIR is prevalent.     

Cyanobacteria-containing biofilms from a Mayan monument in Palenque,Mexico

Surfaces of buildings at the archaeological site of Palenque, Mexico, are colonized by cyanobacteria that form biofilms, which in turn cause aesthetic and structural damage. The structural characterization and species composition of biofilms from the walls of one of these buildings, El Palacio, are reported. The distribution of photosynthetic microorganisms in the biofilms, their relationship with the colonized substratum, and the three-dimensional structure of the biofilms were studied by image analysis. The differences between local seasonal microenvironments at the Palenque site, the bioreceptivity of stone and the relationship between biofilms and their substrata are described. The implications for the development and permanence of species capable of withstanding temporal heterogeneity in and on El Palacio, mainly due to alternating wet and dry seasons, are discussed. Knowledge on how different biofilms contribute to biodegradation or bioprotection of the substratum can be used to develop maintenance and conservation protocols for cultural heritage.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

Modelling epilithic biofilms combining hydrodynamics,invertebrate grazing and algal traits

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In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength

We studied the laser ablation of laboratory-developed biofilm on titanium and glass surfaces. Specifically, Pseudoalteromonas carrageenovora, a marine biofilm forming bacterium was used to generate laboratory biofilm. Two fluences, 0.05 and 0.1 J/cm(2) and three durations of irradiation, 30 s, 5 min, and 10 min were tested using an Nd;YAG laser of 532 nm wavelength (in the green light area). Nonirradiated coupons with biofilm served as control. The biofilm removal efficiency increased with the increase in laser fluence and duration of irradiation. The maximum biofilm area cover on control coupons of glass and titanium was 62.5 and 76.0%, respectively. Upon irradiation with fluence 0.1 J/cm(2) for the very short duration of 30 s, this reduced to 5.6 and 12.4% and at 10 min to 2.17 and 0.7% on glass and titanium coupons, respectively, while the controls did not show any reductions (62.5 and 76.0% respectively, for glass and titanium coupons). The biofilm TRC (Total Resuscitated Cells) reduction during this period was even more prominent than the area cover, indicating that the remaining biofilm portions on coupons after irradiation were largely composed of dead bacterial cells. The TRC in the irradiation chamber medium for short durations of irradiation showed a significant increase, indicating that the laser irradiation removed live bacteria from the biofilm. The re-growth of the resuscitated cells showed they could grow like the control cells but with a significant lag. The laser's efficiency in the removal of biofilm was better seen on titanium coupons than on glass. Our results showed that a low-power pulsed laser irradiation could be used to remove biofilm formed on hard surfaces.     

Subaerial biofilms on granitic historic buildings: microbial diversity and development of phototrophic multi-species cultures

Microbial communities of natural subaerial biofilms developed on granitic historic buildings of a World Heritage Site (Santiago de Compostela, NW Spain) were characterized and cultured in liquid BG11 medium. Environmental barcoding through next-generation sequencing (Pacific Biosciences) revealed that the biofilms were mainly composed of species of Chlorophyta (green algae) and Ascomycota (fungi) commonly associated with rock substrata. Richness and diversity were higher for the fungal than for the algal assemblages and fungi showed higher heterogeneity among samples. Cultures derived from natural biofilms showed the establishment of stable microbial communities mainly composed of Chlorophyta and Cyanobacteria. Although most taxa found in these cultures were not common in the original biofilms, they are likely common pioneer colonizers of building stone surfaces, including granite. Stable phototrophic multi-species cultures of known microbial diversity were thus obtained and their reliability to emulate natural colonization on granite should be confirmed in further experiments.     

Protection of cells from salinity stress by extracellular polymeric substances in diatom biofilms

Diatom biofilms are abundant in the marine environment. It is assumed (but untested) that extracellular polymeric substances (EPS), produced by diatoms, enable cells to cope with fluctuating salinity. To determine the protective role of EPS, Cylindrotheca closterium was grown in xanthan gum at salinities of 35, 50, 70 and 90 ppt. A xanthan matrix significantly increased cell viability (determined by SYTOX-Green), growth rate and population density by up to 300, 2,300 and 200%, respectively. Diatoms grown in 0.75% w/v xanthan, subjected to acute salinity shock treatments (at salinities 17.5, 50, 70 and 90 ppt) maintained photosynthetic capacity, F_q′/F_m′, within 4% of pre-shock values, whereas F_q′/F_m′ in cells grown without xanthan declined by up to 64% with hypersaline shock. Biofilms that developed in xanthan at standard salinity helped cells to maintain function during salinity shock. These results provide evidence of the benefits of living in an EPS matrix for biofilm diatoms.     

Bacterial composition of biofilms formed on dairy-processing equipment

Abstract

Polymicrobial biofilms often form on the surfaces of food-processing machinery, causing equipment damage and posing a contamination risk for the foods processed by the system. The composition of the microbial communities that make up these biofilms is largely unknown, especially in the dairy industry. To address this deficit, we investigated the bacterial composition of biofilms that form on the surfaces of equipment during dairy processing using Illumina MiSeq sequencing and culture-dependent methods. Illumina sequencing identified eight phyla, comprising six classes, ten orders, fifteen families, eighteen genera, and eighteen species. In contrast, only eight species were isolated from the same samples using the culture-based method. To determine the ability of the identified bacteria to form biofilms, biofilm formation analysis via crystal violet staining was performed. Five of the eight culturable species, Acinetobacter baumannii, Acinetobacter junii, Enterococcus faecalis, Corynebacterium callunae, and Stenotrophomonas maltophilia, were able to form biofilms. Since most of the identified bacteria are potential food-borne or opportunistic pathogens, this study provides guidance for quality control of products produced in dairy processing facilities.     

Characterization of cyanobacteria isolated from biofilms on stone monuments at Santiniketan,India

Cyanobacterial biofilms occurring on the exterior of three stone monuments at Santiniketan, India were analyzed. Species of Scytonema and Tolypothrix were the major components of these biofilms. Identification was obtained by morphometric procedures and 16S rRNA gene sequencing. Biofilms cultured for prolonged periods revealed the presence of several other cyanobacteria belonging to 14 different genera. Cyanobacteria on stone in the tropical environment of India formed a distinct cluster that was quite different from that of cyanobacteria reported for a similar substratum in temperate regions. Absorption spectra of the organisms from Santiniketan showed a high quantity of scytonemin, mycosporine-like amino acids, and carotenoids. All of the organisms survived in a desiccated state and rapidly revived after wetting. The organisms were heterocystous and nitrogenase activity was reactivated within 24?h of wetting by which time heterocysts in their filaments had also appeared.     

Genetic diversity in cultured and wild marine cyanomyoviruses reveals phosphorus stress as a strong selective agent

Viruses that infect marine cyanobacteria–cyanophages–often carry genes with orthologs in their cyanobacterial hosts, and the frequency of these genes can vary with habitat. To explore habitat-influenced genomic diversity more deeply, we used the genomes of 28 cultured cyanomyoviruses as references to identify phage genes in three ocean habitats. Only about 6–11% of genes were consistently observed in the wild, revealing high gene-content variability in these populations. Numerous shared phage/host genes differed in relative frequency between environments, including genes related to phosphorous acquisition, photorespiration, photosynthesis and the pentose phosphate pathway, possibly reflecting environmental selection for these genes in cyanomyovirus genomes. The strongest emergent signal was related to phosphorous availability; a higher fraction of genomes from relatively low-phosphorus environments–the Sargasso and Mediterranean Sea–contained host-like phosphorus assimilation genes compared with those from the N. Pacific Gyre. These genes are known to be upregulated when the host is phosphorous starved, a response mediated by pho box motifs in phage genomes that bind a host regulatory protein. Eleven cyanomyoviruses have predicted pho boxes upstream of the phosphate-acquisition genes pstS and phoA; eight of these have a conserved cyanophage-specific gene (PhCOG173) between the pho box and pstS. PhCOG173 is also found upstream of other shared phage/host genes, suggesting a unique regulatory role. Pho boxes are found upstream of high light-inducible (hli) genes in cyanomyoviruses, suggesting that this motif may have a broader role than regulating phosphorous-stress responses in infected hosts or that these hlis are involved in the phosphorous-stress response.