Life on the rocks

Biofilms are interface micro-habitats formed by microbes that differ markedly from those of the ambient environment. The term 'subaerial biofilm' (SAB) was coined for microbial communities that develop on solid mineral surfaces exposed to the atmosphere. Subaerial biofilms are ubiquitous, self-sufficient, miniature microbial ecosystems that are found on buildings, bare rocks in deserts, mountains, and at all latitudes where direct contact with the atmosphere and solar radiation occurs. Subaerial biofilms on exposed terrestrial surfaces are characterized by patchy growth that is dominated by associations of fungi, algae, cyanobacteria and heterotrophic bacteria. Inherent subaerial settlers include specialized actinobacteria (e.g. Geodermatophilus), cyanobacteria and microcolonial fungi. Individuals within SAB communities avoid sexual reproduction, but cooperate extensively with one another especially to avoid loss of energy and nutrients. Subaerial biofilm metabolic activity centres on retention of water, protecting the cells from fluctuating environmental conditions and solar radiation as well as prolonging their vegetative life. Atmospheric aerosols, gases and propagatory particles serve as sources of nutrients and inoculum for these open communities. Subaerial biofilms induce chemical and physical changes to rock materials, and they penetrate the mineral substrate contributing to rock and mineral decay, which manifests itself as bio-weathering of rock surfaces. Given their characteristic slow and sensitive growth, SAB may also serve as bioindicators of atmospheric and/or climate change.     

Characteristics and role of the exocellular polysaccharides produced by five cyanobacteria isolated from phototrophic biofilms growing on stone monuments

Three coccoid and two filamentous cyanobacterial strains were isolated from phototrophic biofilms exposed to intense solar radiation on lithic surfaces of the Parasurameswar Temple and Khandagiri caves, located in Orissa State, India. Based on to their morphological features, the three coccoid strains were assigned to the genera Gloeocapsosis and Gloeocapsa, while the two filamentous strains were assigned to the genera Leptolyngbya and Plectonema. Eleven to 12 neutral and acidic sugars were detected in the slime secreted by the five strains. The secretions showed a high affinity for bivalent metal cations, suggesting their ability to actively contribute to weakening the mineral substrata. The secretion of protective pigments in the polysaccharide layers, namely mycosporine amino acid-like substances (MAAs) and scytonemins, under exposure to UV radiation showed how the acclimation response contributes to the persistence of cyanobacteria on exposed lithoid surfaces in tropical areas.     

Characteristics and role of the exocellular polysaccharides produced by five cyanobacteria isolated from phototrophic biofilms growing on stone monuments

Three coccoid and two filamentous cyanobacterial strains were isolated from phototrophic biofilms exposed to intense solar radiation on lithic surfaces of the Parasurameswar Temple and Khandagiri caves, located in Orissa State, India. Based on to their morphological features, the three coccoid strains were assigned to the genera Gloeocapsosis and Gloeocapsa, while the two filamentous strains were assigned to the genera Leptolyngbya and Plectonema. Eleven to 12 neutral and acidic sugars were detected in the slime secreted by the five strains. The secretions showed a high affinity for bivalent metal cations, suggesting their ability to actively contribute to weakening the mineral substrata. The secretion of protective pigments in the polysaccharide layers, namely mycosporine amino acid-like substances (MAAs) and scytonemins, under exposure to UV radiation showed how the acclimation response contributes to the persistence of cyanobacteria on exposed lithoid surfaces in tropical areas.     

Ultraviolet Radiation-Absorbing “Humic Pigments” of Cyanobacteria in Microbial Mats: Their Presumptive Photoprotective Function and Relevance to Early Precambrian Microbial Ecology and Evolution

Cyanobacteria that form the primary components of microbial mats in freshwater bogs and intertidal marine environments in the Bahamas produce water-soluble brown pigments whose spectral properties imply that they are a type of humic acid. These “humic pigments” are produced by vital processes of living cyanobacteria, not by decomposition of dead ones, as shown by decreases in the concentrations of humic pigments, ultraviolet (UV) radiation-absorbing photoprotective mycosporine-like amino acids (MAAs), and chlorophyll from upper to lower layers of the mats, and by the occurrence of humic pigments in cyanobacterial cultures. Unlike MAAs, which absorb UV radiation only within limited ranges of wavelengths, humic pigments absorb radiation spanning the entire UV spectrum, and absorbance increases with decreasing wavelength. These observations suggest that the biosynthesis of humic pigments originated as a photoprotective adaptation in the early Precambrian, enabling cyanobacteria to colonize shallow-water and terrestrial environments even though the atmosphere was virtually devoid of O₂ and O₃ and therefore transparent to all solar radiation in the UV region of the spectrum. Moreover, the evolution of this photoprotective mechanism may have been linked to the evolution of photosynthesis.     

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.     

Generation of reactive oxygen species upon red light exposure of cyanobacteria from Roman hypogea

Subterranean archaeological sites in Rome (Italy) are threatened by phototrophic biofilms predominated by cyanobacteria and associated microorganisms. They damage the frescoes, mortar, marble, and tufa rock wherever artificial lighting is installed. During the past two decades, the conservation strategies have evolved gradually; rather than restricting the illumination time and intensity, the latest approach is to use strong light to reduce their growth. Since cyanobacterial cells are abundant in phycobilisomes and chlorophyll a, which produce reactive oxygen species (ROS) upon irradiation, strong red light (620–650 nm) was applied to generate high amount of ROS in a rate beyond the quenching capacity of the organism. After 25 h of irradiation, the photosystem II quantum yields of seven cyanobacterial isolates in culture were reduced by 65–94%. Conversely, blue light (460–480 nm) promoted photosystem II activity by up to 35%. δ-Aminolevulinic acid (D-ALA) was introduced to enhance the treatment, as it can be transformed into protochlorophyllide by cyanobacteria and then excited by red light to generate ROS inside the cells. Since the natural photosynthetic pigments as well as the endogenous protochlorophyllide exist only within the cyanobacterial cells, they are unlikely to contaminate or damage the underlying stone substrata. Electron spin resonance spectroscopy confirmed that D-ALA treatment caused the formation of ROS; spin trap experiments indicated that radicals were produced in the system.     

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.     

Laboratory grown subaerial biofilms on granite: application to the study of bioreceptivity

Simulated environmental colonisation of granite was induced under laboratory conditions in order to develop an experimental protocol for studying bioreceptivity. The experimental set-up proved suitable for producing subaerial biofilms by inoculating granite blocks with planktonic multi-species phototrophic cultures derived from natural biofilms. The ability of four different cultures to form biofilms was monitored over a three-month growth period via colour measurements, quantification of photosynthetic pigments and EPS, and CLSM observations. One of the cultures under study, which comprised several taxa including Bryophyta, Charophyta, Chlorophyta and Cyanobacteria, was particularly suitable as an inoculum, mainly because of its microbial richness, its rapid adaptability to the substratum and its high colonisation capacity. The use of this culture as an inoculum in the proposed experimental set-up to produce subaerial biofilms under laboratory conditions will contribute to standardising the protocols involved, thus enabling more objective assessment of the bioreceptivity of granite in further experiments.     

Phycoerythrin synthesis is induced by solar UV-B in the cyanobacterium Nostoc

Solar UV-B (280–315 nm) induces the synthesis of phycoerythrin (PE) in a Nostoc species isolated from the Andean high altitude lake Yanaqocha. The outdoor experiments were carried out in a small lake in Erlangen, Germany, using natural conditions. After 2- and 4-h exposure to solar radiation, the immunodetection signal using monoclonal antibodies anti-PE was lower in control cells (exposed to PAR + UV-A) than in cells exposed to total solar radiation (PAR + UV-A + UV-B). Cells exposed at depths in which no UV-B penetrated showed no differences from control cells regarding PE content. When exposed to monochromatic radiation of 280, 300 or 360 nm, purified PE was photodegraded in a wavelength dependent manner resulting in different polypeptide fragments carrying chromophore groups. Immunodetection revealed active synthesis of PE in parallel to photodamage by solar UV-B indicating that PE is important for photoadaptation to shorter wavelengths in the cyanobacterium Nostoc sp.     

Life in the dark: far-red absorbing cyanobacteria extend photic zones deep into terrestrial caves

Chlorophyll (Chl) f and d are the most recently discovered chlorophylls, enabling cyanobacteria to harvest near-infrared radiation (NIR) at 700–780 nm for oxygenic photosynthesis. Little is known about the occurrence of these pigments in terrestrial habitats. Here, we provide first details on spectral photon irradiance within the photic zones of four terrestrial cave systems in concert with a detailed investigation of photopigmentation, light reflectance and microbial community composition. We frequently found Chl f and d along the photic zones of caves characterized by low light enriched in NIR and inhabited by cyanobacteria producing NIR-absorbing pigments. Surprisingly, deeper parts of caves still contained NIR, an effect likely attributable to the reflectance of specific wavelengths by the surface materials of cave walls. We argue that the stratification of microbial communities across the photic zones of cave entrances resembles the light-driven species distributions in forests and aquatic environments.     

Diversity and function of Chloroflexus-like bacteria in a hypersaline microbial mat: phylogenetic characterization and impact on aerobic respiration

We studied the diversity of Chloroflexus-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum Chloroflexi resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family Chloroflexaceae. Photopigment analysis revealed the presence of bacteriochlorophyll c (BChlc), BChld, and gamma-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of Chloroflexus aurantiacus. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.     

Mechanisms of amplification of photosynthetically active radiation in the symbiotic deep-water coral Leptoseris fragilis

The symbiotic coral Leptoseris fragilis lives in the Red Sea at depths of 95–145 m. Symbiotic dinoflagellates (zooxanthellae) themselves possess well known adaptations to low light intensities. In L. fragilis we found indications that light amplifying mechanisms of the host improve photosynthesis of the symbionts. Light of short wavelengths is absorbed by host pigments which transform short into longer wavelengths. The transformed light is more efficient for photosynthesis. Action spectra measurements of photosynthesis demonstrated the amplification of photosynthetically active radiation. Monochromatic light of 387 nm (outside the main absorption maxima of the algal pigments) at subsaturation photon flux densities was as effective photosynthetically as polychromatic light of 415–490 nm, which fits the absorption maxima of the zooxanthellae.     

Fungal melanins as a sun screen for symbiotic green algae in the lichen<Emphasis Type="Italic"> Lobaria pulmonaria</Emphasis>

The mycobiont of the high-light-susceptible forest lichen Lobaria pulmonaria was shown to deposit brown, melanic compounds in the outer layer of the upper cortex, depending on the long-term level of solar radiation in its natural habitat. Furthermore, pale thalli from a shady habitat produced melanic compounds after transplantation to a sunny habitat. This browning of the cortex appeared to be a physiologically active process, taking place only during periods with frequent hydration. Melanin production was slow. After transplantation, more than 1 year was needed for a shade-adapted thallus to reduce the cortical transmittance (230-1000 nm) to a similar level to that of naturally sun-exposed specimens. Melanic compounds acted as a sun screen, especially reducing UVB and UVA wavelengths, but also visible wavelengths, at the photobiont level. In the near infrared range, there was only a small difference in transmittance between shade- and sun-adapted cortices. A negative correlation was found between the natural light level and the cortical transmittance of wavelengths below 700 nm. However, previous studies have shown that even photobionts of melanic L. pulmonaria thalli are relatively susceptible to high-light exposure. Since melanins also increase the absorbance of solar energy for the whole thallus, it appears that what is gained in terms of UV- and light protection in melanic L. pulmonaria specimens may be offset by increased exposure to excess temperatures for this highly heat-susceptible lichen.     

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.     

Genetic Characterization of Epilithic Cyanobacteria and Their Associated Bacteria

Epilithic phototrophic biofilms develop inside Roman Necropolis and Catacombs on rock surfaces exposed to artificial light sources and are composed by a microbial consortium dominated by cyanobacteria. In this work, six non-axenic cultures of Leptolyngbya sp. strains isolated from biofilms from different Roman hypogea and maintained in cultures from 11 to 20 years were analysed along with their associated bacteria isolated in culture. The employment of PCR-fingerprinting techniques, using HIP1 and ERIC derived primers, allowed the clustering in three groups of the six Leptolyngbya strains and the typing of their isolated bacteria. The bacterial fingerprinting patterns were in agreement with the 16S rRNA gene sequencing and showed the presence in Leptolyngbya isolates of Pseudomonas, Stenotrophomonas, Agrobacterium and Bacillus representatives that were detected also in biofilms sampled from catacombs.     

Interaction of Photoprotective and Acclimation Mechanisms in Ulva rigida (Chlorophyta) in Response to Diurnal Changes in Solar Radiation in Southern Chile

Species of the genus Ulva (Chlorophyta) are regarded as opportunistic organisms, which efficiently adjust their metabolism to the prevailing environmental conditions. In this study, changes in chlorophyll‐a fluorescence‐based photoinhibition of photosynthesis, electron transport rates, photosynthetic pigments, lipid peroxidation, total phenolic compounds, and antioxidant metabolism were investigated during a diurnal cycle of natural solar radiation in summer (for 12 h) under two treatments: photosynthetically active radiation (PAR: 400–700 nm) and PAR+ ultraviolet (UV) radiation (280–700 nm). In the presence of PAR alone, Ulva rigida showed dynamic photoinhibition, and photosynthetic parameters and pigment concentrations decreased with the intensification of the radiation. On the other hand, under PAR+UV conditions a substantial decline up to 43% was detected and an incomplete fluorescence recovery, also, P‐I curve values remained low in relation to the initial condition. The phenolic compounds increased their concentration only in UV radiation treatments without showing a correlation with the antioxidant activity. The enzimatic activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased over 2‐fold respect at initial values during the onset of light intensity. In contrast, catalase (CAT) increased its activity rapidly in response to the radiation stress to reach maxima at 10 a.m. and decreasing during solar. The present study suggests that U. rigida is capable of acclimating to natural radiation stress relies on a concerted action of various physiological mechanisms that act at different times of the day and under different levels of environmental stress.     

Diversity and Function of Chloroflexus-Like Bacteria in a Hypersaline Microbial Mat: Phylogenetic Characterization and Impact on Aerobic Respiration

We studied the diversity of Chloroflexus-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum Chloroflexi resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family Chloroflexaceae. Photopigment analysis revealed the presence of bacteriochlorophyll c (BChlc), BChld, and γ-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of Chloroflexus aurantiacus. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.     

Microbial rhodopsins on leaf surfaces of terrestrial plants

The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface between the terrestrial biosphere and solar radiation. It is estimated to host up to 10(26) microbial cells that may intercept part of the photon flux impinging on the leaves. Based on 454-pyrosequencing-generated metagenome data, we report on the existence of diverse microbial rhodopsins in five distinct phyllospheres from tamarisk (Tamarix nilotica), soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), clover (Trifolium repens) and rice (Oryza sativa). Our findings, for the first time describing microbial rhodopsins from non-aquatic habitats, point towards the potential coexistence of microbial rhodopsin-based phototrophy and plant chlorophyll-based photosynthesis, with the different pigments absorbing non-overlapping fractions of the light spectrum.     

Fungal tissue per se is stronger as a UV-B screen than secondary fungal extrolites in Lobaria pulmonaria

To test the hypotheses that (1) protective mycobiont tissues and/or (2) medullary UV-B-absorbing carbon-based secondary compounds (CBSCs) protect lichen photobionts against UV-B radiation, we quantified cortical UV-transmittance and ran a three-way factorial lab experiment with (1) three UV radiation regimes, (2) photobiont layers with/without a screening cortex, and (3) with natural/reduced CBSC-concentration. We used melanin-deficient Lobaria pulmonaria from shaded forests. Maximum photochemical efficiency of photosystem II (Fv/Fm) in photobionts inside thalli with natural CBSC-concentrations was not affected by any UV-regime, consistent with close to 0% measured cortical transmittance of wavelengths <325 nm. Exposing photobiont layers to direct radiation strongly aggravated photoinhibition (P < 0.001), as did an increase in UV-exposure (P < 0.001). The effect of CBSC-removal was weaker (yet significant at P = 0.001), mainly affecting exposed photobiont layers given short-wavelength UV radiation. Based on these findings, we conclude that the primary role of extrolites in L. pulmonaria is not to screen excess solar radiation.