Introduction
Salt from the Cervia salterns has been extracted since pre-Roman times (Guarnieri, Reference Guarnieri2019). Included in the Po Delta Regional Park, the largest wetland zone in Italy, these salterns represent an excellent example of the coexistence of historical heritage, economical interest and nature. The high naturalistic interest of the area hosting this man-made system of ponds is evidenced by its inclusion in the Ramsar Convention as a wetland of international importance. Therefore, since 1979 the Salina di Cervia has been declared a natural repopulation reserve for a number of water bird species that nest here.
The considerable extent of the Cervia salt pans makes the site unique along this area of the northern Adriatic Sea, where systems of coastal dunes represent the main physiographic features (Caruso et al., Reference Caruso, Giambastiani, Gabbianelli and Diani2005) partitioning the sea from the wide lowlands of the Po River delta and its neighbouring areas (Gambolati et al., Reference Gambolati, Giunta, Putti, Teatini, Tomasi, Betti, Morelli, Berlamont, De Backer, Decouttere, Monbaliu, Yu, Brøker, Christensen, Elfrink, Dante, Gonella and Gambolati1998).
With the transition to an industrial production system of saltworks in Cervia, today only one area, the Salina Camillone (Fig. 1a), remains as a document of the ancient artisan production system of salt. In this area, which is currently included in the Salina di Cervia Nature Reserve, a single salt evaporation pond (Fig. 1b and c) is still preserved in its natural condition and where, therefore, no evaporation and salt collection takes place. This provides an opportunity of analysing the relationships between a physical surface environment and its microbiological component.
Fig. 1. (a) Location map of the study area, white star locates the Salina Camillone, next to the town of Cervia, approx. 2 km away from the Adriatic Sea. (b) The Salina Camillone pond still preserved in its natural state as it appears in the sampled site. (c) Thickened, subrounded margins (arrows) of the polygonal cracks that make up the surface of the shallow pond. Note water trapped by curb-shaped margins of the polygons.
The objective of this study is to examine the anatomy of surficial macro- and microscopic structures determined by the interactions between cohesive microbial mats and salt precipitation and clastic sediments in a temperate, saline confined habitat. The ultimate goal is to assess the bio-sedimentary origin of cm- to mm-scale morphologies and their preservation during the first steps of their formation in an intermediate environmental context between emerged and submerged conditions largely controlled by recurrent desiccation processes.
Saline environments in a temperate climate zone and their function as habitats for microbial mats
In temperate coastal regions, aquatic environments typically consist of salt marshes developed in sheltered intertidal areas or lagoons. These saltwater wetlands produce a variety of habitats that sustain a remarkable biodiversity (e.g., Adam, Reference Adam1990), including phototrophic microorganisms. For example, diatoms such as Nitzschia are producers of phototrophic biofilm and efficient early colonizers (Anderson, Reference Anderson2016) of substrates through their abundant secretion of extracellular polymeric substances (EPS). Filamentous cyanobacteria belonging to halotolerant form-genera, such as Microcoleus and Phormidium, also typify these environments and largely contribute to the formation of microbial slimes (e.g., Tkavc et al., Reference Tkavc, Gostinčar, Turk, Visscher, Oren and Gunde-Cimerman2010). EPS can constitute up to 90% of biofilm composition (Nielsen et al., Reference Nielsen, Jahn and Palmgren1997), and their ability to store large amounts of water can protect cell colonies against moisture deficiency in temporarily emerged areas that may occur in such environmental settings. Conditions of high salinity and evaporation comparable to those inducing salt crusts precipitation in arid environments, however, are rarely found in temperate areas. Due to the climatic conditions of temperate regions, salt pan environments – and associated interactions between mudflats, saline crusts and microbial communities – exist only in exceptional cases. The Cervia salterns, together with those from Slovenia (Tkavc et al., Reference Tkavc, Gostinčar, Turk, Visscher, Oren and Gunde-Cimerman2010), are the northernmost salterns of the Mediterranean and can be included within these exceptional areas.
The abundance of microbial mats and specific interactions with their immediate environment enables the formation of microbial-induced sedimentary structures (MISS, Noffke et al., Reference Noffke, Gerdes, Klenke and Krumbein2001a), a discrete class of sedimentary structures defined as the result of the interaction between sediments and microbial mat-induced products (Davies et al., Reference Davies, Liu, Gibling and Miller2016; Callefo et al., Reference Callefo, Ricardi-Branco, Amaral Cataldo, Noffke, Alderton and Elias2021). These surface morphologies and textures were described from different climatic conditions, such as the coastal areas of the North Sea (Noffke et al., Reference Noffke, Gerdes, Klenke and Krumbein1997; Noffke, Reference Noffke1999) and of southern Tunisia (Noffke et al., Reference Noffke, Gerdes, Klenke and Krumbein2001b), and they have not changed significantly over 3.5 billion years (Noffke et al., Reference Noffke, Decho and Stoodley2013b). In undisturbed environments, such as a natural salt-pan pond, the extremely low and fine-grained sedimentary supply enables microbial mat fabrics to grow and bind the fall-out of sedimentary particles through the biofilm they secrete. In this regard, it is noteworthy that most of the EPS secreted by cyanobacteria, including the filamentous genera Microcoleus and Phormidium, contain uronic acids (Stal, Reference Stal, Krumbein, Paterson and Stal1994; Moreno et al., Reference Moreno, Vargas, Olivares, Rivas and Guerrero1998; Rashmi et al., Reference Rashmi, Shylajanaciyar, Prabaharan, Uma, Das and Dash2017), carbohydrates that increase their adhesive, ‘sticky’ capacity towards the materials they enwrap. This has been experimentally documented for the case of loose fine-grained sediments, where enhanced erosion resistance of sedimentary particles, through an increase of boundary shear stress, takes place in the presence of cyanobacterial binders capable of synthesizing uronic acids (Dade et al., Reference Dade, Davis, Nichols, Nowell, Thistle, Trexler and White1990; Chen et al., Reference Chen, Zhang, Paterson, Townend, Jin, Zhou, Gong and Feng2019).
Additionally, diatoms have strong adhesive properties due to both intrinsic cell adhesion abilities, as in the case of certain Nitzschia species (Laviale et al., Reference Laviale, Beaussart, Allen, Quilès and El-Kirat-Chatel2019), and mucilage strand properties leading to efficient sediment stabilization (e.g., Vos et al., Reference Vos, Misdorp, De Boer, de Boer, van Gelder and Nio1988). In the latter case, similarly to bacterial mucilage, high amounts of uronic acids in EPS appear to be the primary factor promoting adhesiveness in diatoms (Sutherland, Reference Sutherland2001; de Brouwer et al., Reference de Brouwer, Wolfstein, Ruddy, Jones and Stal2005; Poulsen et al., Reference Poulsen, Kröger, Harrington, Brunner, Paasch and Buhmann2014). The end result is that some microbial mat and associated morphologies may leave records in sediments and, at last, in the sedimentary record (e.g., Gerdes, Reference Gerdes, Schieber, Bose, Eriksson, Banerjee, Sarkar, Altermann and Catuneanu2007).
At Cervia, recurrent desiccation and moisturizing may also offer a means of evaluating microorganisms in terms of their contribution to the formation of distinguishable micromorphologies under such stressful conditions.
Cervia salt-pans
Environmental context and site description
As the largest salt pan habitat along the upper Adriatic coast, the Cervia salterns make up a selective environment inhabited by a wealth of species able to survive high salt concentrations and water depths not exceeding a few centimetres. This wetland ecosystem spans 785 hectares (Ramsar area) less than 2 km away from the sea (Fig. 1a). Coastal deposits accumulating south of the Po River delta are primarily composed of sands and sandy/silty clays; their deposition is strongly dependent on the anti-clockwise circulation regime of the northern Adriatic Sea that distributes fine-grained sediments transported from the Po River mouth to the south (Franco et al., Reference Franco, Jeftić, Malanotte Rizzoli, Michelato and Orlić1982). This type of sedimentary regime also takes place in the Cervia salterns area. As in other hyperaline areas, the water becomes seasonally reddened by bloom of Dunaliella salina, a halotolerant, unicellular green alga able to accumulate carotenoids to as much as 13% of its dry weight (Abd El-Baky et al., Reference Abd El-Baky, El Baz and El-Baroty2004). Dunaliella salina is in turn the food for Artemia salina, a micro-crustacean, which is a favourite food of the flamingos and pink herons whose presence typifies the man-made shallow ponds of the Cervia salterns. For the preservation of habitats and species, as well as salt production, recurrent seawater circulation is necessary; this explains the presence of euryhaline species, such as the benthic foraminifer Ammonia beccarii.
After the industrialization of salt extraction, that began at the end of the 1950s, only in a very limited area – the Salina Camillone – have traditional salt-production methods been preserved. Next to this area and along the provincial road SP 254, connecting the hinterland to Cervia (Fig. 1a), is the only pond not involved in the extraction activity that, since it lacks a direct human disturbance, has been preserved in a rather natural condition (Fig. 1b). This shallow pond, while maintaining a necessary connection with the circulation of the open seawater, reaches a maximum depth of approximately one metre and an extent of approx. 5000 square metres. Sampling was conducted at its SW margin (coordinates: 44°15′15″N, 12°20′03″E). Most of its clayey surface (mudflat) is permanently covered by seawater, the same water that feeds the salt production system. If surface layers of the mudflat are removed, a distinct smell of hydrogen sulphide can be detected, a by-product of the anaerobic decay of organic matter.
Maximum flooding in the pond occurs in the autumn season (average monthly rainfall: 68 mm), and the average air temperature ranges from 22.7°C in July to 3.1°C in January.
Due to the seasonal variation of the water level within the pond, some parts, especially towards the margins, repeatedly emerge. Because of recurrent subaerial exposure, polygonal oscillation cracks, ranging in size from a couple of metres down to a few centimetres, are formed at the surface of clayey sediments and develop a distinct morphology throughout the surface of the pond (Fig. 1b and c). Even where polygons are completely inundated, wide open cracks persist. This is because on top of the polygonal oscillation cracks crusts made up of multiple microbial mats interbedded with laminae of clayey (prevailing) and sandy mud and saline precipitates are present and produce further shrinkage morphologies (see also McMahon et al., Reference McMahon, Van Smeerdijk Hood, McIlroy, Brasier, McIlroy and McLoughlin2017); the final result is the formation of compact and cohesive, although still elastic, surfaces (Fig. 2a), similar to those described from tidal flat and coastal sabkha areas (e.g., Cameron et al., Reference Cameron, Cameron, Richard and Curran1985; Noffke et al., Reference Noffke, Gerdes, Klenke and Krumbein1996; Gerdes, Reference Gerdes, Schieber, Bose, Eriksson, Banerjee, Sarkar, Altermann and Catuneanu2007; Porada et al., Reference Porada, Bouougri, Ghergut, Schieber, Bose, Eriksson, Banerjee, Sarkar, Altermann and Catuneanu2007; Bouougri and Porada, Reference Bouougri, Porada, Reitner, Quéric and Arp2011; Chen et al., Reference Chen, Zhang, Paterson, Townend, Jin, Zhou, Gong and Feng2019), with overfolded margins of the desiccation polygons (Fig. 1b and c).
Fig. 2. (a) Effect of a strongly cohesive microbial mat: bio-sedimentary sample collected in a submerged (approx. 10 cm water depth) area where the mucilaginous component (slime) gives a surface stabilization such as to favour the preservation of different superficial micro-morphologies. In the background, slight polygonal cracks document previous phases of emersion. (b) Close-up of a typically irregular bio-sedimentary surface composed of a mix of salt efflorescence (whitish), clayey sediment (grey) and slime-producing microbes, such as diatoms (whitish) and cyanobacteria (brownish). Width of view 7 cm.
The mode of formation of the overgrowth around the polygons has been previously described in a southern Tunisia lagoon (Noffke et al., Reference Noffke, Gerdes, Klenke and Krumbein2001a) as a consequence of repeated upturning of microbial layers leading to the development of curb-shaped thickenings that exceptionally emphasize their margins (Fig. 1c). The leathery surface produced by the recurrent subaerial exposure of the polygons has enabled their recovery even in fossilized microbialites of up to Neoproterozoic age (Eriksson et al., Reference Eriksson, Porada, Banerjee, Bouougri, Sarkar, Bumby, Schieber, Bose, Eriksson, Banerjee, Sarkar, Altermann and Catuneau2007; Noffke et al., Reference Noffke, Beukes, Bower, Hazen and Swift2008).
Close (hand lens) observation on the field of the microbial mat (Fig. 2a and b) shows that they exhibit amorphous, bumpy surfaces draping plant remnants and other microbial crust portions produced during previous desiccation phases. The colours of the crusty microbial surfaces include shades of brown and green produced by clayey components and photosynthetic pigments, together with light spots derived from saline exudation locally mixed with dense concentrations of diatom frustules (Fig. 2b).
Mucilage biofilm producers in a saline clayey substrate of temperate climate
Being ubiquitous in natural hydrated environments, microbial biofilms have a strong influence on sedimentary surfaces, such that sediment particles coated with the biofilm – for which the term bio-sediment has been proposed (Fang et al., Reference Fang, Fazeli, Cheng, Huang and Hu2015) – can determine the properties of sediment and the formation of surface micromorphologies. In the Cervia salterns, the sedimentary substrate on which the microbial communities secrete their mucilage biofilm is composed of clayey minerals associated with evaporitic salts. As in many other areas, these photosynthetically active microbial mats are thin (less than 1 millimetre in thickness), strongly cohesive and placed above fine-grained sediments of low-energy environments in which their black colour and strong hydrogen sulphide smell indicate persistent anoxic conditions (Skyring and Bauld, Reference Skyring, Bauld and Marshall1990). Thin biological crusts observed in ephemerally emerged areas of the Cervia pond that still retains its natural state derive from the ability of biofilm producers to trap and bind loose mineral or skeletal particles for stabilizing a sediment surface (Neumann et al., Reference Neumann, Gebelein and Scoffin1970; Paterson et al., Reference Paterson, Aspden, Visscher, Consalvey, Andres, Decho, Stolz and Reid2008). This ability may determine surface micromorphological features derived from the interactions between physical environments and microbes (Viles, Reference Viles2012), which can be preserved through the deep time (Noffke et al., Reference Noffke, Decho and Stoodley2013b).
Although interspecific interactions between different slime-producing microbial components (e.g., between prokaryotes and unicellular eukaryotes) have often been detected, they are still poorly known. Evidence of interactions has been observed in diatoms from different species (Roubeix and Coste, Reference Roubeix and Coste2017) and between diatoms and bacteria (Amin et al., Reference Amin, Parker and Armbrust2012), and may provide mutual benefits via symbiotic relationships. Regarding the latter case, surface morphologies potentially preservable as fossil biosignatures have been observed as the result of the interactions between diatoms and filamentous cyanobacteria in continental sabkha environments (Barbieri and Cavalazzi, Reference Barbieri and Cavalazzi2018). Even in the present case study, the leading players are filamentous cyanobacteria – which can form dense mats in saltern ponds (Oren, Reference Oren2015) and dominate the surface part of the microbial mats of hypersaline (or other harsh) environments (Stal, Reference Stal, Whitton and Potts2000) – and the benthic diatom Nitzschia, a widespread genus living in freshwater and marine habitats all over the world, where it can accumulate in great numbers in undisturbed surface sediments (Kociolek et al., Reference Kociolek, Spaulding, Lowe, Wehr, Sheath and Kociolek2015). Communities belonging to this diatom genus have also been described from more challenging conditions, such as geothermal (e.g., Owen et al., Reference Owen, Renaut and Jones2008; Stavreva-Veselinovska and Todorovska, Reference Stavreva-Veselinovska and Todorovska2010) and hypersaline environments (e.g., DasSarma and DasSarma, Reference DasSarma and DasSarma2017; Balakrishnan et al., Reference Balakrishnan, Santhanam, Jeyanthi, Divya and Srinivasan2019) where they behave as extremophiles. Nitzschia secretes abundant EPS for locomotion (e.g., de Brouwer and Stal, Reference de Brouwer and Stal2002), which may significantly contribute to the stabilization of loose sediment in low-energy environments (Stal and de Brouwer, Reference Stal, de Brouwer and Rullkötter2003). Although some active competition has been observed between filamentous cyanobacteria and Nitzschia species in muddy habitats (Watermann et al., Reference Watermann, Hillebrand, Gerdes, Krumbein and Sommer1999), their adhesive mucilages may both contribute to the biostabilisation of a loose substrate.
Sampling and analytical methods
This study is based on samples collected in May 2016 from a lateral margin of the only pond preserved in natural condition at the Cervia salt-pans (Fig. 1b; Fig. 3a). Samplings of the surficial crusts and sediment portions, with a mean thickness of 1 cm, involved three different types of materials collected in contiguous areas: (i) wet (submerged) and then naturally dried samples; (ii) moderately crustose samples (from a recently desiccated part of the pond); and (iii) completely dried and crustose samples (from a permanently dry part of the pond). These materials include a range of still active (living) and desiccated surface and subsurface microbial mats variously associated with clayey sediment and saline precipitates (Fig. 3). Sampling was performed at the beginning of the dry season (end of May). In the samples collected from the submerged part, a 10 to 20 cm thick layer of water covered a still active microbial mat at the water-sediment interface. Samples were then placed in a box to avoid any deformations during transport from the field to the laboratory.
Fig. 3. (a) Google Earth view of the Salina Camillone, the broken line delimits the permanently submerged area. Letters position the sampled sites in submerged – recently emerged (b, c) and permanently emerged (d) areas. (b–d) Examples (ESEM micrographs) of what the sampled sedimentary surface looks like: trichome masses of filamentous cyanobacteria (b, see details in Fig. 8); diatom valves accumulation (c, see details in Fig. 9); clumps of halite ribbons protruding from a diatom-rich surface (d, see details in Figs. 6 and 9).
Whereas the samples taken in emerged parts were then investigated under the binocular microscope as they were at the time of sampling, samples collected from submerged parts were first observed with a field microscope immediately after the collection and before removing the surface water, to avoid changes to the surface features, especially to the microbial mat component. Subsequently, they were air-dried at room temperature for further investigation.
The samples of living microbial mats and organo-sedimentary mixed samples were investigated through freshly, randomly broken mat portions, dissected in points chosen ad hoc by using a scalpel, in order to observe both the surface morphology and the millimetre- to micrometre-scale microstratigraphy. A combination of transmitted-light and environmental scanning electron microscopy (ESEM) analyses with both backscattered-electron (BSE) and secondary electrons imaging was used. For the evaluation of the surface microscopic morphologies and of the microenvironmental context with respect to the different components, reflected light observations were performed by using a Wild M8 stereomicroscope and a Zeiss Axioplan binocular optical microscope equipped with a Nikon digital camera Head DS-Fi2. ESEM observations were performed on uncoated samples using an FEI Quanta 200, under low vacuum conditions, with an accelerating voltage of 20–25 kV. The elemental compositions were performed with an OXFORD-SATW light elements X-ray spectroscope (SEM-EDX).
In the field, water characteristics including electrical conductivity, pH and temperature, were measured by using an XS Instruments pH 70 portable laboratory.
Optical microscopy analysis
From submerged to occasionally or permanently emerged areas
Recurrent field observations have shown that wide areas of the pond are permanently emerged, whereas other areas have periodic (occasional) emersion connected with the water exchanges of the salt pan. Optical microscope observations were performed in samples from both permanently submerged and emerged (occasionally or permanently) areas. They enabled an evaluation of the microenvironmental context where the data collected during the ESEM investigations are framed, and to make comparative observations between the active (submerged or still wet at the time of sampling) and (permanently emerged) areas.
On-site observations with a field microscope of samples from submerged parts revealed that at the top of the mucilaginous mat growing over the clayey substrate a grey, soft microlayer (less than one millimetre thick) represents the actual sediment-water interface and consists of clay particles, amorphous fragments of biofilm, diatoms thecae and single strands of cyanobacteria (Fig. 4). At its topmost portion the microlayer consists of a watery suspension where loose cyanobacteria filaments mimic a phototactic behaviour, and a denser lower portion where tangles of filamentous cyanobacteria are immersed in a water, biofilm- and granules-rich medium that fall-out to the substrate.
Fig. 4. Transmitted light micrograph from a sample of the soft microlayer at the sediment-water interface from submerged parts of the pond (see text for further details). At its topmost portion, this watery suspension contains clay particles (c), amorphous fragments of biofilm (b), diatoms thecae (d) and single strands of cyanobacteria (s).
Beneath the surficial soft microlayer, microbial communities are organized in thick, planar to undulose bundles of filamentous cyanobacteria densely aligned parallel to the sedimentary surface (Fig. 5a). The bright and dark green-blue colour of this packed surface microlayer remains preserved even after its complete desiccation (Fig. 5b). The thickness of this pigmented horizon can reach one millimetre with dense bundles that lie on surfaces made of silt-clay material, salt precipitates and organic remnants. Filamentous bundles appear overall clean from clay sediment, thereby contrasting with the sediment-microbe interactions that typify certain MISS structures, such as in modern sandy tidal flats. In permanently desiccated areas, the bundles are masked by salt precipitation that barely enables their dark colour to be detected.
Fig. 5. (a) Dark blue bundles of filamentous cyanobacteria aligned parallel to the surface of a sample from a just emerged (still wet at the time of sampling) area. The typical dark colour was maintained by the cyanobacterial colonies even after drying. (b, c) Vertical cross section with normal stratigraphic orientation of the bio-sedimentary interval (microbial + sediment) in the top 5–10 mm of the muddy substratum. Note the dark laminae (cyanobacteria) alternating with the light layers composed of a mix of clayey sediments and saline precipitates. Whereas in B the top of the section entirely consists of filamentous cyanobacteria that envelope growing salt crystals (arrows), in C a thin siliceous felt made up of diatom frustules partially masks the underlying dark green cyanobacterial layer. Arrow in C shows the upwelling of sediment and the development of evaporitic material leading to the surface micromorphology observable in D. (d) Bulges and lumps produced by ongoing desiccation and evaporitic precipitation on a just emerged (still wet at the time of sampling) surface almost completely covered by diatom frustules. Width of view: B and C: 1 cm; D: 2 cm.
Along with the cyanobacterial strands, dense accumulations of the needle-shaped diatom Nitzschia cover rather irregular and knobbed surfaces (Fig. 5c and d). The siliceous felt produced by diatom frustules may completely drape some surface portions. When viewed in a microstratigraphic section, just below the thin diatom microlayer are filaments of cyanobacteria organized as dark laminae alternating with light layers composed of a mix of clayey sediments and saline precipitates (Fig. 5c).
In samples collected from permanently or temporary emerged (still wet) areas, a readily observable feature are the whitish, siliceous felts consisting of thin needles associated to microcrystalline aggregates of evaporation salt and the seeming ‘fluid weaving’ of the dark green bundles of filamentous cyanobacteria. Sub-millimetre thick siliceous felty layers produced by the accumulation of Nitzschia thecae, associated with cyanobacterial laminae, overlay irregular surfaces produced by bulges and lumps making up a recurrent micromorphological feature in the salt-pan surface of Cervia (Fig. 5d). The combined action of the drying of the clayey sediment and the salts evaporation is the possible agent responsible for these irregular surfaces.
Overall, in the top centimetre, the microstratigraphy exhibits a biolaminite organization developed just beneath the sediment-water interface, with an irregular alternation of millimetre-scale layering of dark laminae of microbial mats, and lighter silty/clayey/evaporite layers (Figs. 5b and c and 6a and b). This microstratigraphic organization has some lateral continuity, and it is observable in both recently and permanently emerged areas. Depending on their location, however, the thickness of laminae differs considerably for adapting to the ground micromorphology and to some local disruptions by salt efflorescence.
Fig. 6. (a, b) Section across the bio-sedimentary (microbial + sediment) surface crust of a permanently emerged area. The organization and thickness of the thin strata adapt to the surface morphology and local disruptions produced by salt efflorescence. Dark laminae (cyanobacteria) irregularly alternate with sediment and salt (whitish patches). Width of view: A: 1 cm; B: 0.5 cm. (c, d) Bread crust-like surface morphologies in an emerged area from which twisted or curled ribbons of fibrous halite grow (arrows). (E) ESEM micrograph where halite ribbons (arrows) protrude from a recently emerged surface; bulges, lumps and salt crystals are covered by diatom frustules and their biofilm remnants.
On recently emerged areas, still wet at the time of sampling, surfaces covered with microcrystalline salt precipitates allow to glimpse dark patches that reveal the presence of cyanobacterial bundles. In some portions the salt precipitation has covered filaments and biofilm remnants that previously had contributed to trap loose sedimentary and skeletal grains, such as hydrobiid shells, smooth and thin-shelled ostracod valves and benthic foraminiferal tests (e.g., Ammonia beccarii).
Emerged areas are also typified by saline crinkled crusts with long, twisted or curled ribbons of fibrous halite with a characteristic silky appearance (Fig. 6c and d). First described, with several centimetres long ribbons, from the Fayum Depression, in the Western Desert, Egypt (Phillips and Rigby, Reference Phillips and Rigby1991), at Cervia the salt ribbons are remarkably smaller and attain a maximum size of a few millimetres. They are the likely product of salt efflorescence growth during evaporation. A sort of capillary migration through silty-clayey deposits takes place so that salt ribbons protrude from bread crust-like surface morphologies (Fig. 6c). Similar ribbons were also recovered from samples collected in submerged areas soon after desiccation (Fig. 6e).
Environmental scanning electron microscope analysis
In the surficial sediments covering the Cervia pond the microbiotic components act as inter-particle binders and make up the main framework of the bio-sediment. This is well detectable by ESEM analysis on wet or partially hydrated and unconsolidated bio-sediment samples that, thanks to the flexible structure of the microbial component, still retain their original three-dimensional architecture. Abundant remnants of adhesive mucilage have also been detected in both the samples dried at room temperature after collection and in those collected in naturally dried (permanently emerged) areas. Overall, they contribute to document the transition from a taphonomically active phase – in which surface microbially-controlled features rapidly degrade – to a more stable and potentially preservable bio-sedimentary frame.
Microbe-evaporite surface structures
Both diatom frustules and cyanobacterial filaments concur to the formation of microbial-induced surface morphologies. A common feature is the light-coloured accumulation of Nitzschia thecae settled on a clayey and sandy substrate or on pre-existing dark green microbial laminations. These thin diatom-rich surfaces are typified by a strongly iso-oriented organization of frustules (Fig. 7a), which gives the surface a fluid flow-like look. We attribute this organization to gliding motility, a benthic pennate diatom movement performed on the substratum of aquatic environments and based on the secretion of extracellular material through the raphe slit (e.g., Wetherbee et al., Reference Wetherbee, Lind and Burke1998; Heintzelman, Reference Heintzelman2006). Some Nitzschia species were also observed to glide in epibenthic (muddy) habitats (e.g., Gupta and Agrawal, Reference Gupta and Agrawal2007). Extremely thin (a few tens of micrometres) layers have also been observed in active (submerged) surfaces, where iso-orientation of diatom thecae may change from layer to layer (Fig. 7b). Since diatom cells require adhesion to a substrate during the horizontal gliding process, they need to maintain direct contact with a physical interface and this could be the factor that determined diatom micro-layers having a very few up to single-cell thickness. Despite the minimal thickness of diatom micro-layers typified by a flow-like look (Fig. 7c), a potential delivery of the effects of gliding to the fossil record can be favoured by the abundance of adhesive mucilage and the stabilizing effect of saline precipitation.
Fig. 7. ESEM micrographs of diatom (Nitzschia) frustules of a submerged (approx. 10 cm water depth) area. (a) Fluid flow look produced by the accumulation of iso-oriented frustules. (b) Remnants of mucilage (arrows) secreted by diatoms. (c) Minimal thickness (less than 50 μm) of a surface diatomaceous layer (arrows) from a cross section. At the top right (arrow) a hydrobiid gastropod shell.
Masses of slender filaments (average diameter: 10 micrometres) commonly aligned and locally coiled, tangled or twisted, make up cyanobacteria colonies (Fig. 8a), which result in long trichome masses (from several hundred micrometres to several millimetres for each single trichome). Similar to diatoms, they display a distinct iso-orientation (Fig. 8b) that gives a fluid flow-like appearance to cyanobacteria masses. Their surface organization in an orientation parallel to the trichome axis takes place in a body of almost still water, keeping direct contact with a muddy substrate. Again similar to diatoms, iso-orientation is interpreted as the product of gliding motility, a trichomes well-known function (Buchard, Reference Buchard1981; Hoiczyk, Reference Hoiczyk2000; Ali et al., Reference Ali, Asghar, Anwar Bég and Sajid2016). In filamentous cyanobacteria that lie on a firm substrate, gliding movements lead to their iso-orientation and is associated to the steady secretion of mucilage (Hoiczyk, Reference Hoiczyk2000). Such an abundant production of mucilage sheath leads to the increase the amorphous surfaces in which the cyanobacteria trichomes are often completely plunged. The spiral fashion of the trichomes (Fig. 8c) should be an additional gliding effect (e.g., Tamulonis et al., Reference Tamulonis, Postma and Kaandorp2011).
Fig. 8. ESEM micrographs of trichome masses of filamentous cyanobacteria from the surface of a still wet and emerged (at the time of sampling) area. Note the abundant production of mucilage secretion in which the cyanobacteria trichomes (some of them coiled or stretched) are plunged.
At the microscale, the combined effect of a coarse (sandy grains) component of the sedimentary surface, the salt evaporation, with some early formation of fibrous halite ribbons, and several generations of overlapping microbial slime produced by the symbiotic joint action of filamentous cyanobacteria and diatoms lead to an irregular morphology of the substrate surface. A typical result is the knobbed surface in submerged or just emerged parts where felts of diatom frustules drape the substrate surfaces (Fig. 9a). The thin felt coverage consists of a minimal thickness (less than 50 μm) of a single or very few frustule layers (Fig. 7b and c) where biofilm is only present as membranes remnants. Escaping from the knobs as the efflorescence product of the salt, fibrous ribbons disrupt their surface leaving the sedimentary and diatom top layers permanently deformed (Fig. 9b and c).
Fig. 9. ESEM micrographs in a submerged (approx. 10 cm water depth) knobbed area (a) covered by diatom (Nitzschia) frustules. (b, c) The diatomaceous and sedimentary surface layer has been permanently deformed by the knobs from which salt ribbons have been extruded (arrows).
Bio-sedimentary ‘packaging’
The firm crusts of the Cervia pond mainly consist of clay mineral clots, some sand-sized grains, and various organic components (Fig. 10a) and primarily derive from the binding action of filamentous living microbiota, organized as dense and compact single to multi-layered systems (Fig. 10b), and its mucilage. Since cyanobacteria have a documented desiccation tolerance (Holzinger and Karsten, Reference Holzinger and Karsten2013 cum bibl.), they can efficiently contribute as sedimentary binders (Fig. 10b and c) before their dehydration following water emersion and subsequent destruction. Together with their mucilage protection, also cell wall thickness may have contributed to delay the desiccation of cyanobacteria trichomes at Cervia (Fig. 10d). Especially in temporarily to permanently emerged areas along the pond margins, salt precipitation has also favoured the substrate stabilization. Salt precipitation can be either diffused in the crusts of the emerged areas or associated to a roughly laminated organization capable of enveloping salt crystals during their growth (Fig. 11a and b). Just beneath the substrate (or water-sediment interface in the submerged parts), this laminated bio-sedimentary architecture combines clayey particles, biofilm and trichome remnants and residual porosities (Fig. 11b), whereas at places it consists of an organic alveolar texture (Fig. 11b and c) derived from diatom/cyanobacteria mucilage trapped between laminae during a phase of its biodegradation process. Likewise, the framework of the bio-sediment, that associates trichomes of filamentous cyanobacteria, diatom thecae, sediment and slime remnants, enables the formation of a stable and tight bio-sedimentary ‘packaging’, which appears as an early stage of a bio-sediment fossilization process (Fig. 11d).
Fig. 10. ESEM micrographs of bio-sedimentary surface crusts from recently and permanently emerged areas. (a) Firm surface crust mainly consisting of sand grains and clayey sediments held together by microbial mucilage produced by diatoms and cyanobacteria filaments as they appear during the early fossilization (‘packaging’) phase. (b, c) Cyanobacteria trichomes as sedimentary binders from a recently emerged area. Note the cell division pattern. (d) Locally collapsed (arrows) cyanobacteria trichome from a permanently emerged area, with cell division pattern no longer visible.
Fig. 11. ESEM micrographs of bio-sedimentary surface sections from a permanently emerged area as they appear during the early fossilization (‘packaging’) phase. Section across a bio-sedimentary surface crust where salt crystals (arrows in A) appear enveloped by the bio-laminated (arrow at the top in B) consisting of sediment particles, biofilm and trichome remnants, residual porosities and organic alveolar texture of the crust (arrow at the bottom in B, magnified in C) during a phase of their biodegradation process. (d) Bio-sedimentary ‘packaging’ topped with a thin diatom level (arrow at the top) and where trichomes of filamentous cyanobacteria (other arrows) make up the framework. Samples with normal stratigraphic orientation.
The Cervia hypersaline environment from an astrobiological perspective
The exploration of the extreme environments on Earth has long revealed an unexpected diversity and opened new frontiers on the existence and adaptation of life (e.g., Horikoshi and Grant, Reference Horikoshi and Grant1998; Rothschild and Mancinelli, Reference Rothschild and Mancinelli2001; Hallsworth et al., Reference Hallsworth, Mancinelli, Conley, Dallas, Rinaldi, Davila, Benison, Rapoport, Cavalazzi, Selbmann, Changela, Westall and Yakimov2021). Although particularly stressful and challenging for biotic communities, some of the environments where evaporitic salts accumulate, such as coastal lagoons, salt pans and playas, may harbour diverse, salt-tolerant microbial communities (Oren, Reference Oren2002, Reference Oren2015; Ley et al., Reference Ley, Harris, Wilcox, Spear, Miller, Bebout, Maresca, Bryant, Sogin and Pace2006), and in ancient evaporite deposits microbial cells and DNA have been successfully extracted (Vreeland et al., Reference Vreeland, Rosenzweig and Powers2000, Reference Vreeland, Jones, Monson, Rosenzweig, Lowenstein, Timofeeff, Satterfield, Cho, Park, Wallace and Grant2007; Panieri et al., Reference Panieri, Lugli, Manzi, Roveri, Schreiber and Palinska2010; Stivaletta et al., Reference Stivaletta, Barbieri and Billi2012). With due caution required by both the harsh environment of Mars, unfavourable to a biological context, and the still absence of unambiguous traces of life, the case of terrestrial evaporitic sulphates is particularly illustrative of the role played by environments with chemical precipitation even in searching for life on Mars (e.g., Rothschild, Reference Rothschild1990; Barbieri, Reference Barbieri, De Vera and Seckbach2013). It is in fact starting the discovery of extensive sulphate (gypsum) deposits on Mars – some of them interpreted as of evaporitic origin (e.g., Bibring et al., Reference Bibring, Langevin, Gendrin, Gondet, Poulet, Berthé, Soufflot, Arvidson, Mangold, Mustard and Drossart2005; McLennan et al., Reference McLennan, Bell, Calvin, Christensen, Clark, de Souza, Farmer, Farrand, Fike, Gellert, Ghosh, Glotch, Grotzinger, Hahn, Herkenhoff, Hurowitz, Johnson, Johnson, Jolliff, Klingelhofer, Knoll, Learner, Malin, McSween, Pocock, Ruff, Soderblom, Squyres, Tosca, Watters, Wyatt and Yen2005; Tosca and McLennan, Reference Tosca and McLennan2006) – that a primary astrobiological relevance has been attributed to evaporitic sulphates as well as to their palaeontological potential, which was previously largely ignored. From evaporitic sulphates permineralized microfossils and other types of preserved microbial signatures were detected (Barbieri et al., Reference Barbieri, Stivaletta, Marinangeli and Ori2006; Schopf et al., Reference Schopf, Farmer, Foster, Kudryavtsen, Gallardo and Espinosa2012; Allwood et al., Reference Allwood, Burch, Rouchy and Coleman2013) confirming their excellent ability to preserve biosignatures.
Their being primary target for astrobiological missions also relies on the habitability potential (past and present) of evaporitic salts, which is based on the salts capacity to retain water and generate liquid water brines. In addition to the morphologic biosignatures rapidly incorporated in evaporitic minerals or deposits, the rapid precipitation in salt flats facilitates primary fluid inclusions, a potential tool, not yet developed, for Martian life detection (Benison, Reference Benison2019). The primary relevance of evaporitic environments as a potential target for the forthcoming astrobiological programs has also benefited from the proven ability of halophilic archaea and halotolerant bacteria to survive when exposed to surface Mars conditions (Leuko et al., Reference Leuko, Rettberg, Pontifex and Burns2014; Oren, Reference Oren2014) or to those that hypothetically existed on the early Mars (Bryanskaya et al., Reference Bryanskaya, Berezhnoy, Rozanov, Serdyukov, Malup and Peltek2019). Also the recognized thermochemical stability in Martian conditions of evaporitic minerals (Benison, Reference Benison2019) has further increased their potential of storing biosignatures, if any.
In this context, the easily accessible Cervia site enables the investigation of a range of MISS produced by the interactions between physical and chemical (siliciclastic and evaporitic) sediments and the products of epibenthic microbial activity during the early taphonomic phases. Similar to the Earth of the early Achaean, when certain MISS already existed (Noffke et al., Reference Noffke, Christian, Wacey and Hazen2013a), Late Noachian and Hesperian Mars had aquatic palaeoenvironments where structures and textures potentially having the same origin may have formed (Noffke, Reference Noffke2021). At Cervia these morphologies derive from sediment and microbe accumulations with finely laminated near surface textures, where, despite the sedimentary ‘packaging’, microbial morphologies are still recognizable. Also in desiccation morphologies, such as polygons and their curb-shaped margins, the microbiotic component is recognizable. Morphologies at the scale of those described here will likely be investigated by the analytical technologies of future Martian missions.
Concluding remarks
The Cervia pond enabled the evaluation of features developed on clastic sediment surfaces where biofilms found suitable conditions (i.e., sufficient time span of non-burial) for growing up to microbial mats and develop their strongly cohesive nature by binding and trapping the sedimentary grains. These mats have gone through successive stages in a sedimentary context where repeated subaerial exposures have left their traces during the sediment-microbe interplay, which allowed the formation of irregular leathery crusts up to their bio-sedimentary ‘packaging’. Comparable environmental combinations have been observed in MISS features from different climatic conditions, such as the coastal areas of the North Sea and southern Tunisia. The Cervia pond also shares some geomicrobiological features with other confined habitats of warmer and arid conditions – such as the inland sabkhas from Western Sahara, where diatom and cyanobacteria-dominated mats interact each other (Barbieri and Cavalazzi, Reference Barbieri and Cavalazzi2018) – suggesting a relative independence from temperature as long as the availability of saline water and a relatively dry season, albeit temporary, is ensured.
Evaporation in the Cervia pond led to the formation of curled ribbons of fibrous halite, a distinctive mineral morphology, however ephemeral, which implies emersion and/or arid conditions. Aside from extremely arid areas, in fact, these morphologies lack a real preservation potential. Instead, a different matter regarding preservation are the deformation structures (knobs) associated with ribbons and produced on the substrate during their extrusion. Since they involve a biological component (thin layers of diatom frustules deformed in the knobs from which the salt ribbons are extruded) having a fossilization potential, some preservation in the stratigraphic record might be hypothesized.
The top-most cm in partially stabilized siliciclastic sediments provides an example of biolaminite, where a sub-millimetre-scale accretion is governed by mat-producing communities growing on sediment surfaces. Some biostabilization of the sediment surface allows persistence of the accretion process and the consequent formation of a microstratigraphic record.
In this type of deposit, where some sediment starvation makes easier the growth of microbial slime, phases of desiccation take place. Based on seasonal variations of the water levels in the pond, polygonal oscillation cracks have widened, shrivelled and eventually thickened their margins by differential development of microbial mats at the edges of the polygons as a consequence of repeated desiccation and moistening phases. Exceptionally developed in the Cervia pond, these thickened curb-shaped morphologies, known since the Neoproterozoic, can represent palaeoclimatic indicators capable of suggesting subtle changes in water levels and, therefore, seasonality.
In the Cervia pond, with an extremely low hydrodynamic regime, dark green laminae originating from monolayered microbial mats are set on clayey substrates that may result from minimal clay deposition derived from seawater input or wind action. This energy regime may have allowed the iso-oriented organization and a fluid flow-like appearance of the needle-shaped diatom frustules and cyanobacteria, both attributed to gliding motility, with the potential of leaving traces of a biological function in the geological record.
Acknowledgements
Thanks are due to the Salina di Cervia Nature Reserve for repeated permission to visit the site, Veronica Menna (Arpae Emilia-Romagna) for diatoms identification, Massimo Tonelli (University of Modena and Reggio Emilia) for ESEM assistance and the former students Antonio D'Alonzo and Jacopo Vujovic for their assistance during field samplings. The careful work of two anonymous reviewers was much appreciated. This study was funded by the University of Bologna (RFO 2017-2018).
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