112 ACTA BOT. CROAT. 80 (1), 2021 Acta Bot. Croat. 80 (1), 112–116, 2021 CODEN: ABCRA 25 DOI: 10.37427/botcro-2020-031 ISSN 0365-0588 eISSN 1847-8476 Short communication Phototrophic biofilms and microbial mats from the marine littoral of the central Mediterranean Gabrielle Zammit1,2*, Sarah Schembri1,2, Mark Fenech1,2 1 Laboratory of Applied Phycology, Centre for Molecular Medicine and Biobanking, University of Malta, Msida, Malta 2 Microbiology Lab, Department of Biology, Faculty of Science, University of Malta Abstract – Phototrophic biofilm and microbial mat communities grow along the rocky coastline of the Maltese is- lands. This research involved studying phototrophs from the mediolittoral and supralittoral zones over a two-year period and seasonal changes were observed. Attachment of pioneer microorganisms to the porous eroded limestone bedrock was facilitated via a gelatinous matrix composed of exopolymeric substances (EPS). In submerged areas, such as undisturbed rock pools, these progressively formed green or brown compact biofilms, some of which thickened over the spring to form microbial mats via the production of more extensive EPS layers. Microbial mats gradually attained a lighter colouration due to the presence of ultraviolet (UV) screening pigments. In full summer, they were observed to shrink, detach from the exposed substrate, harden and progressively calcify. Biofilm microorganisms survived the harsh summer months in sheltered areas. The major biofilm formers were filamentous non-heterocytous cyanobacteria belonging to the Leptolyngbyaceae, Pseudanabaenaceae and Oscillatoriaceae. Their sheaths were thick, lamellated and often confluent. A higher biodiversity of phototrophs was observed in late autumn and winter, when tufts of heterocytous Calothrix sp. grew on thin compact biofilms of Nodosilinea sp., Toxifilum sp. and Phormidesmis spp., while Lyngbya spp. trichomes were surrounded by thick brown sheaths. Germlings of green and brown macroalgal species belonging to Ulva, Cladophora and Sphacelaria were embedded in biofilms and microbial mats and gradually grew to form extensive macroalgal covers submerged in rock pools. Erythrotrichia sp. filaments colonised the medio- littoral zone and were confined to areas that were exposed to wave action and submerged intermittently. Over the summer, macroalgal coverage diminished and microalgal biofilms and microbial mats prevailed in rock pools. Keywords: biofilm, cyanobacteria, marine littoral, microalgae, microbial mat, Mediterranean Introduction Phototrophic biofilms and microbial mats grow on coastal rocky shores around the Maltese islands. They con- stitute under-explored communities as there is an overall lack of research about the biotic assemblages of Maltese rocky shores (Schembri et al. 2005) and of the Mediterra- nean area in general, as evidenced bythe lack of scientific literature published on the subject. A biofilm can be defined as a self-sustaining community of microorganisms associated with a substrate that is sev- eral millimetres thick (Dang and Lovell 2015). On the other hand, microbial mats (also referred to as biomats) are strat- ified microbial communities, commonly associated with aquatic habitats, which for the purpose of this study were over one centimetre in thickness. Many of the microbial associations within biofilms and microbial mats are sym- biotic, which confers a selective advantage on the commu- nity as a whole. In phototrophic communities, a significant proportion of the microorganisms are photosynthetic and highly dependent on the presence of light. Research inter- est in these communities is related to the discovery of new taxa that produce novel enzymes with high biotechnological potential in green environmental solutions and biomedical applications (Prieto-Barajas et al. 2018). This study consists of a structural exploration of photo- trophic biofilms and microbial mats occurring on the rocky shores of the Maltese islands and a morphological descrip- * Corresponding author e-mail: gabrielle.zammit@gmail.com MARINE BIOFILMS AND BIOMATS IN THE CENTRAL MEDITERRANEAN ACTA BOT. CROAT. 80 (1), 2021 113 tion of the cyanobacterial and algal biodiversity comprising these communities. The main objectives were to identify any seasonal variations and to attempt to further knowl- edge about the strategies adopted by these communities that enable them to form on hostile surfaces and thus persist throughout different seasons. Areas of the Maltese coastline colonised by phototroph- ic communities were visually observed and documented over a two-year period, and representative biofilms and mi- crobial mats were then seasonally sampled and directly ob- served by light and electron microscopy. To our knowledge, this is the first such study of marine phototrophic biofilms and microbial mats growing on rocky shores in the central Mediterranean region. Materials and methods Phototrophic microbial communities growing in the mediolittoral and supralittoral zones of different coastal lo- cations around the Maltese islands were studied over a two- year period starting in autumn 2017 and seasonal changes were visually observed and documented. Representative biofilms and microbial mats were then sampled from the rocky shoreline around St Julian’s Tow- er in Sliema (35°55’05.6” N, 14°29’57.8” E) and from the coastline of the village of Baħar iċ-Ċagħaq (35°55’06.4” N, 14°29’58.0” E) in Malta during autumn 2017, spring and autumn 2018 and again during spring 2019. Sampling was carried out during autumn and spring, as these were the seasons in which visible changes in biofilm and biomat structure and extent of growth were recorded. Sampling took place during November 2017, May and December 2018 and again during April 2019. Since the growth of endoliths was not expected, sampling of the phototrophic communi- ties was carried out using techniques that were non-invasive to the underlying rock. The biofilms and microbial mats were transferred to en- riched sea water (Provasoli 1968) in Petri dishes and incu- bated at 20 °C with a photoperiod of 10 hours. The sampled phototrophic communities and the same phototrophic communities growing in culture were ob- served by means of light microscopy using an Olympus BX 51 microscope equipped with an Olympus DP-71 digi- tal camera. For transmission electron microscopy (TEM), biofilm microsamples were fixed in 2.5% glutaraldehyde, postfixed in a 1% osmium tetroxide solution, dehydrated in a graded ethanol series, and embedded in epoxy resin (Epoxy 812 Resin Kit, Multilab Supplies, Newcastle upon Tyne, UK). Thin sections were collected on copper grids, stained with uranyl acetate and lead citrate (Reynolds 1963) and were observed using an H-7100 TEM (Hitachi, Tokyo, Japan) operating at 100 kV. Identification of phototrophic biofilm and biomat-form- ing organisms was carried out using morphological keys for Cyanobacteria (Komárek and Anagnostidis 1999, 2005, Komárek 2013), Chlorophyta (Ettl and Gärtner 2013, Cor- maci et al. 2014, Lange-Bertalot et al. 2017, Škaloud et al. 2018), Phaeophyceae (Cormaci et al. 2012) and Rhodophyta (Cormaci et al. 2017). Results and discussion Seasonal changes in biofilm and biomat structure Different phototrophic communities grew as biofilms and microbial mats in the marine littoral zone (Fig. 1). In submerged areas, such as undisturbed rock pools or salt pans, microorganisms formed green or brown coloured compact biofilms (Fig. 1a). During spring and summer, similar phototrophic communities prevailed. These were characterised by an abundance of microbial mats located in rock pools found in the mediolittoral and supralittoral zones. Towards the end of spring, the warm weather and re- stricted wave action led to biofilms becoming restricted to rock pools in the mediolittoral zone. Seasonality was a crucial factor affecting ecological suc- cession. In fact, some microbial communities thickened over spring to form microbial mats via the production of more extensive EPS layers. These gradually attained a light- er colouration, probably due to the presence of EPS and UV screening pigments such as carotenoids and scytonemins in the upper layers. In full summer, both biofilms and microbi- al mats survived only close to the shore, submerged in rock pools. On the other hand, exposed biofilms appeared shriv- elled, fragmented and progressively detached from the rock surface (Fig. 1b). The mediolittoral zone became reduced during summer due to high average temperatures above 28 °C, a UV index above 9 and low average wind speeds of 2.9 km/h. This led to biofilms being restricted to the area of the mediolittoral zone closest to the shore. Biofilm and microbi- al mat organisms survived only in rock crevices and shaded areas (Fig. 1c). Dry biomats hardened and became progres- sively calcified. During autumn, succession occurred, when the rock surfaces previously conditioned by biofilm growth, became colonised by macroalgae. Composition of the phototrophic communities Microscopic observations showed that these photo- trophic biofilms and biomats were highly diverse commu- nities composed of both phototrophic and heterotrophic microorganisms. Each phototrophic community had a dis- tinctive morphology and species composition which de- pended on the respective microhabitat. The predominant microorganisms in communities that were submerged from autumn to spring were filamentous cyanobacteria including fine Leptolyngbyaceae filaments belonging to Leptolyngbya sp., Nodosilinea sp., Pseudan- abaenaceae of Toxifilum sp. and non-heterocytous Oscil- latoria spp., Phormidium spp. and Lyngbya spp. strains. Dense networks of these filamentous cyanobacteria provid- ed structural strength to the biofilms and microbial mats due to their ability to grow intertwined in sheets or bun- dles of filaments (Fig. 2), in which other organisms, such ZAMMIT G., SCHEMBRI S., FENECH M. 114 ACTA BOT. CROAT. 80 (1), 2021 Fig. 1. Dark biofilms grow submerged in man-made salt pans or natural rock pools along the Maltese coastline (a), dry biofilms shrink and completely detach from dry horizontal rock surfaces in full summer (b), biofilm microorganisms survive in crevices and shaded areas on vertical rock faces (c). Fig. 2. Biofilm and biomat - forming cyanobacteria Lyngbya sp. trichomes surrounded by dark brown sheaths (a), Calothrix sp. filaments with hyaline hair (b), arrows indicate profuse trichome fragmentation and formation of hormogonia (c), release of spherical propagules from the open ends of sheaths (d). TEM micrographs of biofilm-forming Pseudanabaenaceae and Leptolyngbyaceae. A biofilm consist- ing exclusively of fine filaments of Toxifilum sp., arranged both longitudinally and horizontally, each surrounded by thick polysaccharide sheaths (e). A different biofilm composed of a more diverse community of Nodosilinea sp. filaments with heterotrophic bacteria embedded in a dense EPS matrix (f ). Scale bars: 25 µm (a, b), 20 µm (c), 10 µm (d), 5 µm (e, f ). as the filamentous heterocytous cyanobacteria Nunduva sp. and Calothrix sp. (Fig. 2b) and diverse coccal microalgae became embedded. Coccal cyanobacteria included species of Aphanocapsa sp. and Chroococcus sp., while microalgal strains belonged to species of Chlamydomonas, Chlorella, Coelastrella and Navicula. A higher biodiversity of photo- trophs was observed in autumn and winter, mainly due to the occurrence of macroalgal germlings in addition to the microbial taxa. The included the filamentous macroalgae Ulva croatica, Ulva flexuosa, Cladophora ruchingeri and Sphacelaria sp. Filaments of Erythrotrichia sp. colonised the mediolittoral zone and were mostly confined to areas that were exposed to wave action and submerged intermit- tently in autumn and winter. Streptomycetes, micronem- atodes, ciliates and microcrustaceans were also observed living and feeding within the biofilm and biomat commu- nities. Adaptation and survival strategies These biofilms and microbial mats formed in response to the prevailing stressful environmental conditions related to temperature, solar irradiation, dehydration and salinity prevalent in the Maltese islands. The layered 3-D structure gave better overall protection than that of constituent mi- croorganisms, such as for instance, the UV protection con- ferred individually by the cyanobacterium Lyngbya and the thermotolerant microalga Coelastrella. In such microorgan- MARINE BIOFILMS AND BIOMATS IN THE CENTRAL MEDITERRANEAN ACTA BOT. CROAT. 80 (1), 2021 115 isms, salt and light stress also accelerate carotenoid pigment and oil production (Hu et al. 2013). Lyngbya aestuarii was common in microbial mat struc- ture. Trichomes in the upper layers of microbial mats were surrounded by a thick brown lamellated sheath (Fig. 2a), which probably contributed to protection against UV radi- ation (280-400 nm). The filaments formed several separa- tion discs and frequently fragmented into short filaments, which have the potential to grow under favourable condi- tions (Rath and Adhikary 2007). The species is known to grow within microbial mats present in the marine intertid- al zone, in which it performs roles vital to the community and connected to photosynthesis, protection, and hydrogen production, which is considered a key metabolite (Kothari et al. 2013). Tufts of heterocytous Calothrix and Nunduva spp. fil- aments provided for nitrogen (N) fixation. Calothrix fila- ments were heteropolar, with a heterocyte at the basal part of the filament that provided for N fixation and the end of the trichome narrowed into a long hyaline hair to facili- tate the uptake of phosphorus (P) (Fig. 2b). Each of these survival strategies provided a competitive advantage which contributed to the success of the biofilm or microbial mat community as a whole. TEM revealed that the attachment of pioneer microor- ganisms to the porous eroded limestone bedrock was facili- tated via a gelatinous polymeric matrix, in which the whole community ultimately became embedded (Fig. 2e, f ). In- dividual trichomes and cells were surrounded by thick ge- latinous sheaths that were often confluent (Fig. 2) and that enabled cells and trichomes to glide in a protected micro- environment that retained the moisture around them (Fig 2f). The production and subsequent release of propagules were frequent (Fig. 2c, d). The microorganisms were also observed to alter their morphology as a survival strategy to adapt to fluctuating seasonal environmental conditions. One such change was fragmentation observed in Lyngbya sp., in which filaments became fragmented into several smaller pieces allowing faster adaptation. Other filaments, especially those belong- ing to the Leptolyngbyaceae and Oscillatoriaceae were ob- served to coil and compress, allowing the organism to self- shade within biofilm or biomat structure (Wu et al. 2005). Microbial mats provided an adequate survival strategy capable of withstanding the harsh environmental condi- tions prevailing towards the end of spring and summer. In fact, the top protective EPS layer became thicker, allow- ing microorganisms living in the lower layers to survive. The biomats formed to counteract water shortage, since the larger volume of EPS retains more water via hydro- gen bonding, facilitating water absorption and preventing its quick loss, thus allowing the communities to survive in the supralittoral zone, where they are only hydrated by sea spray. The EPS also acted as a buffer preventing rapid micro-environmental changes from occurring within the microbial mat. Lyngbya aestuarii was often found in the top layers of microbial mats, where it provided UV protection due to its thick lamellated pigmented sheaths that are known to con- tain scytonemin (Rath and Adhikary 2007). Other micro- organisms which required UV protection, such as Leptolyn- gbyaceae and Rivulariaceae filaments, were found in the bottom layers closer to the substrate. In fact, another survival strategy involved the migration of filaments, where microorganisms such as Leptolyngbya- ceae were able to glide and migrate to the bottom layer of the microbial mat, especially during spring and summer. This strategy has also been recorded and studied in other genera, for instance, Synechococcus isolates from Octopus Spring, Yellowstone National Park were observed to migrate away from a strong light source via cell gliding (Ramsing et al. 1997). The biofilms and microbial mats of the marine littoral constitute unexplored microbial communities in the central Mediterranean region. In fact, they often contain under- studied taxa such as those belonging to the genera Toxifilum sp. and Nunduva sp. that were identified also in this study and have only been recently described (Zimba et al. 2017, González-Resendiz et al. 2018). These microorganisms are currently being investigated via a polyphasic approach, in- volving the application of molecular genetics and bioinfor- matics tools to identify relevant species. An improved un- derstanding of the biodiversity and survival adaptations of such phototrophic biofilms and biomats would contribute greatly to our understanding of the role of microbial com- munities within the coastal ecosystem as a whole. 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