Coastal microbial mats: the physiology of a small-scale ecosystem

Coastal inter-tidal sandy sediments! salt marshes and mangrove forests often support the development of microbial mats. Microbial mats are complex associations of one or several functional groups of microorganisms and their formation usually starts with the growth of a cyanobacterial population on a solid substrate. They are considered as analogues of fossil Precambrian stromatolites. Primary production by the cyanobacteria fuels the metabolism of sulfate reducing


Introduction
In their natural environment , microorganisms may occur essentially in one of the following 3 ways: free-living. associated with an d often inside other organisms or fo rm ing multicell ular, mono-or muftispecies aggregates. In the latter form they may occur as colon ies, biofilms or microbial mats. I will not attempt to give an all-embracing definition of any of the 3 forms of microbial aggregates, because this seems to be a nearly impossible tas k and also the borderlines between the diffe rent forms are in fact continuous. Nevertheless, it is important to indicate in a more general way of what is understood in Ihe framework of th is review by a microbial mat. Microorganisms th at grow on a solid surface may eventually form microbial mats. Microbial mats have been the subject of 3 international meetings of which th e results have been published , Cohen and Rosenberg 1989, Stal and Caumette 1994. A mat diffe rs fro m a biofilm mainly because of size and the coherent structure typical for the former. In its ultimate form, microbial mats resemb le something like a doormat, which can be peeled from the surface as a whole. This explains the origin of the concept of a microbial mat. Before this te rm became common property, microbial mats were known as 'laminated microbial ecosystems' or recent or potential stromatolites (Krumbein 1983) . Stromatolites are fossil laminated rock formations of biogenic origin . The oldest stromatolites date back to more than 3.5 Gyr B.P. and represe nt the earliest indications of life on earth. It is generally accepted that stro-bacteria and the sulfide that they produce is oxidised by anoxygenic phototrophic bacteria and by colorless SUlfur bacteria. Growth and metabolism of these microorganisms result in markedly fluctuating vertical gradients of oxygen and sulfide that shift during a day-night cycle. This review discusses the metabolic contribut ions of the different functional groups of microorganisms and how their joint effort results in the formation of the mat. matolites have been built th rough the growth and metabolic activity of microbial mals that lithilied through calcification and subsequently by silicification and other diagenetic processes (Walter 1976). The lamination in the rock rep resents the seasonal or erratic growth pattern of the microbial mats, comparable to the growth rin gs in the trunk of a tree . In stromatolites fossi l remnants of microorganisms have been found Ihat morphologically resemble modern cyanobacteria, which also today are common builders of microbial mats (Schopf 2000) . Since there is little doubt that many of these strom atolites were lormed through au totrophic metabolism , it is tempting to believe that they were built by cyanobacteria. However, the recent discovery that cyanobacteria are an evolutionary relatively young group within the Proteobacteria, argues against this hypothesis (Gupta 2000).
Modern microbial mats have been termed recent stromato li tes in order to distinguish them from th e fossil ones. However, the majority of the modern microbial mats do not lithify and therefore th e term 'stromatolite' was considered not appropriate, except in the few examples that are known to calcify. The same is true for 'potential stromatolites' because it is uncertain whether non-calcifying microbial mats in fact possess the potential of lithification. Similar as stromatolites, modern microbial mats retain a lamination, representing older, partly degrad ed mats. However, the term 'laminated microbial ecosystems' usually does not refer to this historical lamination but rather to a vertical zonation of different functional groups of microorganisms , which is often visible to the naked eye because of the different colors. T hus, the actual active microbial mat is laminated through differen t groups of organisms. However, neither the historical nor the instantaneous lamination is the law of the Medes and Persians . Microbial mats , after growlh has ceased, may be decomposed complete ly, not leaving a visible trace and the substrate is co lonised every season anew. Likewise, a microbial mat may be composed of one species or different species may nol be separated into differenl strata or they may not be dislinguished as such . Such systems are obviously not 'laminated' but Ihey do nol principally differ from Ihe 'laminated microbial ecosystems' and can all be em braced by the term 'microbial mat'.
Theorelically, microb ial mats may be composed of one particu lar species. However, as a ru le a complex microbial ecosystem forms, in which a variety of different functional groups of microorganisms rep resent a structu ral and physiological unit (Van Gemerden 1993). II has been proposed that microbial mats are structural and physiological equivalents of tissues (Wachendorfer 1991).

In this review I wi ll describe the processes and organisms
that are involved in Ihe formation of one Iype of microbial mal Ihat is buill by cyanobacteria Ihal is frequently found in coastal inteHida1 sediments, mangrove forests and salt marshes all around Ihe globe. Microbial mats developing in extreme environments such as hypersaline ponds , thermal sp rings or hot or cold deserts, nor non-phototrophic systems such as mals of the gliding sulfur bacleri um Beggiatoa or Thioploca (Larkin and Strohl 1983) are nol discussed here.
The de velopment of a cyanobacterial mat: prim ary production is the motor of the ecosystem

Colonisation
Inter-tidal sandy sediments are high energy environments , exposed to strong hydrodynamic cond itions . Sediment part icies of small grain size such as silt and cl ays will not deposit here , or they will be eroded away. On the most exposed a reas only the heavier quartz sand grains a re deposited. Inter-tidal sand flats can be consi dered as ex treme environments because besides Ihe physical forces they are low in nutrients and the periodic inundation causes desiccation and strong variations in salinity and temperature. Few organisms are capable of colon ising these environments. Cyanobacteria have remarkably few nutritional requirements. T heir main way 01 life is photoautotrophic, which means Ihat they use light as the source of energy, water as the electron donor and C02 as the sole source 01 carbon (Stal 1995). Moreover, many species are capable of fixing atmospheric nit rogen , which makes them independent on sources of com bi ned nitrogen such as nit rate or ammonium or organic nitrogen , which are generally in low supply in the marine environmenl (Paerl et al. t 996, Bergman et al. 1997). Hence, the only cri lical nulri ent fo r these cyanobacteria is phosphate. Cyanobacteria are well-known for their high affinity towards this important nutrient , which they can store inlracellularly as polyphosphale (Ri egman and Mur 1985).
Cyanobacteria are therefore excellent cand idates to Sial colon ise low-nutrient environments. In inter-tida l coastal sedimen ts they prefer fine sandy sedim ents , which com bine moderately strong hydrodynamics and low sedimentation rales (Yallop et al. 1994). Moreover, the quartz sand grains allow excellent Iransmission of lighl into Ihe sediments (Stal et al. 1985, Kuhl et al. 1994. Initial coloni sation of the sediment by Ihe usually fi lamentous cyanobacte ria is Ihrough adhesion to sand grains probably by sticky extracellu lar polymers. This property allow cyanobacteria to colon ise environme nts with relat ively high energy, Without being washed away. Some species are particularly well equipped to settle in high-energy environments. For instance, in the Bahama's Schizothrix spp. is known to coloni se envi ronments with strong wave currents where other organisms a re unable to sellie (Reid and Browne 1991 ). These organi sms give rise to the formation of modern stromatolites .

Photorespiration
Once a successful colon isation has occurred, the cyanobacteria th rough growlh and their pholosynlhetic aclivity enrich the sediment with organic matter, which becomes available to other microorganisms. T here are a number of different mechanisms by which the pholosynthetically fixed carbon is liberated into the environment. An important mechan ism cou ld be Ihrough pholorespiralion . The cyanobacterial mal is characte rised by a high concentrat ion of biomass. The oxygen thai is produced through pholosynthesis accumulates in the mal and can only leave it through diffusion. Although Ihe diffusion coefficienls of the polysaccharide matrix of the cyanobacterial mat is not much different from that of wate r, the medium is stagnant and no turbu lence can aid the exchange of gas with the overlying water or air. Hence, the cyanobacterial mat may become supersaturated with oxygen (Revsbech et al. 1983). Two to three-fold oxygen supersaturation in cyanobacterial mats is not exception~ al. At the same time the inverse is true for C02. Carbon dioxide is fixed during photosynthesis and depleted Irom Ihe mat. It can only be repl enished by diffusion from the overlying medium . Rlbulose-l ,5-bisphosphale ca rboxylase/oxygenase (Rubisco) , the key enzyme of the reductive pen tose phosphate cycl e (Calvin) an d Ihe enzyme responsible for the fixation of CO.!, possesses also oxygenase activity, Le . uses 0 2 for the oxidative cleavage of ribu l ose~ 1 ,5-bisphos· phate (Tabila 1994) . In facl, Rubisco has a much beller affinily for 0 , than fo r CO, and in order 10 be able to effectively fix CO" its concenlration musl be much higher Ihan 0 ,. In an aerobic environment, many cyanobacteria (and other microalgae) achieve th is requ irement by concentraling CO, (Carbon Concenlraling Mechanism, CCM) and Rubisco in carboxysomes (Price andBadger 1991 , Reinhold et a/. 1991). Howeve r, when both the ambient CO, concentration is low and the 0 , level is high, the GGM may nol be able 10 prevenl the oxygenalion of ribulose-l ,5-bisphosphate, whi ch evenlually will lead to the formalion of glycolale Ihat is excreted inlo Ihe medium (Renslrom and Bergman 1989).
Hence, the oxygenation reaction leads to a loss of fixed ca r· bon . This photorespiration might be a major source of organic carbon lor the microbial community associated with the cyanobacterial mat.

Fermentation
Another major source of organic matter is the fermentative metabolism of cyanobacte ri a. Whereas in th e light the cyanobacte rial mat may be su persaturated wilh oxygen , in the dark not on ty no oxygen is produced but also there is a high demand for oxygen. Initially, the cyanobacte ria will mobilise their storage carbohydrate (glycogen) and resptre it aerobically in order to generate bIOch emi cal energy.
However, this will deplete the oxygen in the cyanobacterial mat quickly and diffu sion from the overlying water or air is slow. This will result in th e factual anoxic conditions in the mat. Measurements of oxygen dynamics in microbia l mats have repeated ly show n th at an oxic conditions are often establ ished withi n minutes after darkening, meaning that the cyanobacteria in the mat are confronted with anoxia during virtually the who le night (Revsbech et al. 1983). In order to cover their energy demands during dark anoxic cond itions th es e cyanobacteria switch to a fermentative metabol ism (Stal and Moezel aar 1997) . The reserve co mpound glycogen and sometimes also energy-rich com patible so lutes (osmotica) are mobilised and fermented to a variety of lowmolecular compounds that are excreted into the med ium . Depending on the species, cyanobacteria have been shown to possess homo-and heterolactic ferm entation, mixed acid Fermentation and homoacetic fermentation. A considerable part of the glycoge n is fe rm ented and has to be replenished by pho tosy nthetic CO, Itxation during the subsequent day period. Hence , an important part of this fixed CO? end s up as low-molecular ferm entation products in the medium and become s available to other microorganisms.

Compatible so/utes
Although fenmentation and photo respiration are probably the major mechanisms by wh ich organic matter becomes available in the cyanobacterial mat, thei r exist also other mechani sms, that may be important under certain conditions. Marine cyanobacteria accumulate compatible solutes that serve as osmoprotectants (Reed et a/. 1986). Th e most common osmoprotectant in marine cyanobacteria is glycosyl glycerol, but also the disaccharides trehalose and to a tesser extent sucrose can be found. Glycine-betaine is more common as compatible solute in cyanobacteria in hypersaline environments. Particul arly the disaccharides and glucosyl glycerol are energy rich compounds and it has been shown that they may be used in addition to the storag e compounds as energy reserve in the dark, particu larly under anoxic conditions. Cyanobacterial mats in inter-tidal sediments a.n d on rocky sh ores may be exposed to dramatic changes in salinity. This is the case when during exposure th e ma ts experien ce a salinity down-shock when it ra ins.
The only way for cyanobacteria to protect them from such a sudden osmotic down shock is to quickly excrete the compa tible sol ute into th e environment where it subsequently becomes available to other microorgani sms. Th is mechanism of transfe r of organ ic matter to the community is rather erratic.

Extracellular polymeric substances
Mat-bUilding cya nobacteria also excrete large amou nts of polymeric su bstances (extracellular polym eric su bstances, EPS) , wh ich are largely composed of polysaccharides and with minor componenls of protein and lipidS (Stal 1994, Decho 1994. One may distinguish roughly two types of EP S: one is more or less intimately associ ated with the organism (cells or trichomes) and is usually design ated as the sheath (De Philippis and Vincenzini 1998) . This is a more or less structural cell component, albeit that it is outside the cell wall. Depending on th e organ ism this sheath may be thick and may in fact be wider than the trich om e itself, or it may be vanishing th in or even absent. Some unice llular cyano bact eria such as Gloeolhece spp . or Gloeocapsa spp. form colonies of which the cells are embedded in a polysaccharide sheath (Tease et al. 1991).
The trichomes of the cosmopoli tan mat-forming cyanobacte rium Microcoleus chthonoplastes form bundles that are enclosed by a common sheath (Garcia-Pichel et al. 1996) . Cyanobacteria such as Lyngbya aestuarii that produce very thick sheaths are usually not motile (Rippka et al. 1979). The sheath may serve d i~erent functions. The highly hydrated polysaccharide s are an effective protectIon from desiccation that may occur when the mat is exposed to the air. The sheath is also effective as an adhesive that attac hes the organism to the su bstrate . It may further scave nge rare mi cro nutri ents and protect the organism from a variety of exte rnal th reats, including grazing. The sheath is always produced in the cyanobacteri a that have one, bu t its prope rties may vary with the envi ronmental condition s in which the organism thrives. Th e other type of EPS produced by cyanobacteria is not inti mately associated with th e organism and is excreted into the medium as mucilage. In many cases this type of EPS seems to be produced as th e result of unbalanced growth . This occurs when growth of the organism is timited by a nu tri ent (o~en N) whi le its photosynthetic and COr fixing capac ity are not impaired. Under such conditions. cyanobacteria initially produce more of the intracellular storage compound glycogen, but the space for this inside the cells is lim ited (Lehmann and W6b er 1976) .
Cyanobacteria rarely produce more than 50% of dry weight as glycogen. Excess of fixed CO, is subsequently excreted as mucilage into the medium. This seems a waste but in this way, cyan ob acteria dissipate the light energy that they harvest through their pigments. This EPS forms a gel matrix in which the cyanobacteria are embedded and it may have similar advantages for the community as a whole as the sheath has for individuats. In addition , EPS excretion may occur as part of the mechani sm of gliding motility of some cyanobacte ria (Ca stenholz 1982).
Although EPS represents a high quality and energy-rich substrate for microorganisms, it must be hydrolysed extracel lularly before it can be taken up and utilised. Sh eath EPS, but also the mucilage, can be considered as rather recalcitrant compoun ds. In many coastal microbial mats the amount of mucilage is low as compared to microbial mats in hypersaline environments. In the latter, the degradation of complex organ ic molecules appears to be very slow probably because of high salinity. In the well-investigated hyper-saline microbial mats 01 Solar Lake (Sinai, Egypt) and the salterns of Guerrero Negro (Baja California, Mexico), the top layer of 1 and 0.1 m, res pectivel y, is purely organic in nature and formed through the accu mulation of the successive active cyanobacterial mats. The mat (Pond 5) of Guerrero Negro grows at a rate of approximately 1 em y 1, close to the rate of decomposition of the older layers, keeping the system more or less at a thickness of 10cm (Des Marais 1995). Howeve r, some net accumulation mus t have occu rred in order to produce the layer of 10em of organic matter. The Solar Lake microbial mat system has been estimated to be 2000 years old (Krumbe in ef al. 1977). An average yearly net accumulation rate of O.5mm wou ld have yielded the present day thickness. This IS only a fraction of the yearly gross accumulation, which is in the same order of magnitude as in the Gue rrero Negro mat. This means that 99.5% of the organic matter produced is degraded in these hypersaline mats and that only th e most recalcitrant molecu les remain as refractory matter in the system. In coasta l inter-tidal microbial mats this net accumulation is usually not observed (Stal ef al. 1985, Stal 1994). This may be attributed to the fact that the degradation of recalcitrant organic matter may be easier at lower salinity, bu t it is more likely that these systems are exposed to dynamic conditions, causing erosion or oxygenation . The occasionally in troduction of oxygen would facilitate the deg radation of recalcitrant organic molecu les.

Grazing
After having discussed photo respira tion , fermentation , osmotic down-shock and EPS exudation as major mechanisms by which photosynthetic fi xed CO, is liberated as organic matter in the e nvironment, a few othe r mechanisms should be mention ed here as well. Obviously, organi sms and that includes cyanobacteria, have a limited life. Cell s may stop dividing and eventually die and disintegrate. In coastal micro bial mats grazing may be more important as a cause 01 cell death (Fenchel 1998, Fenchel andKuhI 2000).

T he possibility of vira l or bac terial attack has a cause of cell lysis in microbi al mats has received only little attention in lit-
erature (Margulis ef al. 1990), but may prove to be an important process. However, conSide ring the fact that the total amount of fixed CO? that is liberated as non-structural compou nds may exceed 90%, the con tribution of structural cyanobacterial cell material to the chemotrophic community is comparatively smal l.

Secondary metabolites
Other orga nic com pou nds that may be produced a nd excreted by mat-form ing cya nobacteria are the resu lt of secondary metabolism (Jultner 1987, Carmichael 1992. The amounts of these compounds a re usually small and do not contri bute to the organic substrate that is available for the microbi al community, but they may be of great importance for the ecosystem functioning . Examples of products of secondary metabolism include geosmines and othe r volatile organi c odorous com pou nds. Not much is known of th ese compounds from microbial mats. The same is true for cyanobacterial toxins . They are well-known from certain plankton ic, Sial b l oom~forming cyanobacte ria, but benthic cyanobacteria have not been investigated in th is respect. Recently, it has been suggested that cyanobacteria, including mat-forming species, may be produce com pounds with antibiotic activities or substances that are involved in cell-to-cell Signaling (Kreitlow et al. 1999). All these subjects are still in their infancy.

Oxygen dynamics in microbial mats
Cyanobacteri a are oxygenic phototrophic organisms. Therefore . highly dynamic vertical oxygen profi les are an important phenomenon in microbial mats (J0rgensen et a/. 1979). These are in the lirst place produced as a result from the daily light curve. During the night oxygen is consumed by respiration. The oxyg en demand is usually high so that the entire mat may become anoxic. Only the mat surface may receive oxygen, particu larly when it is exposed directly to air.
During the day oxygenic photosynthesis is only possibl e in the upper part of the mat in which sufficient ligh t penetrate s.
The euphotic zone of the mat is defined as the layer in which gross oxygenic photosynthesis occu rs . This is not the same as the depth at which oxygen may penetrate. In the light a typical oxygen profile shows a concentration maximum at a depth of 0.1-0.3mm . This peak usually coincides with th e maximum photosynthetic activity or a concentrat ion of photosynthetic biomass or both . Cl oser to the surface the higher irradiation may be sub-optimal, resu lting in lower photosynthetic rates or even in negative values when photooxidalion prevails. Below the oxygen optimum, irradi ance levels a re not satu rating , tikewise resulting in lower oxygen production rates. Obviously, the actual lotal oxygen production rate at any place in the mat is a function of the active ph otosynth etic biomass and the level of irradiance. The latte r varies during a diurnal cycle and the oxygen maximum is therefo re pushed from the surface down to reach its maxImum depth at noon, when the level of irradiance has become maximal. On the other hand, in some microbial mats the standing stock of photosynthetic biomass is also not constant at a particular place during a diurnal cycle.
Many mat-bUilding cyanobacteria are motile by gliding and may constantly move in order to position themselves at optimum light conditions. H ence, the cyanobacte rial mat optimizes its photosynthetic pertorm ance continuou sly and this is reflected in the oxygen profile in the mat. In addition, light impinges rare ly as a pure sinus curve and may fluctuate tremendously during a diurnal cycl e du e to clo uds or to water covering the mat. Photosynthesis respo nds immediately to any fluctuation in light intensity, consequently changing th e oxygen profile.

The actual oxygen concentration at any location in the mat
is the resu lt of its production by photosyn thesis, its consumption by respiratory an d chemical oxidat ion processes and diffusion. This is the basis of a poputar method to measure photosynthes is in microbial mats using micro-electrodes: the decrease of oxygen concentration in the first couple of seconds after the darkening of the mat equals the photosynthetic rate (changing the minus sig n to positive) (Revsbech ef al. 1983). Downward diffusion of oxygen may result in aerobic or micro-aerobic conditions below the euphotic zone. On the other hand respiration and chemical oxidations may exceed the photosynthetic oxygen production, resulting in anoxic conditions within part of the euphotic zone. In this latter case, the method for measuring photosynthesis as described above, does not work.
Microbial mats: a joint venture of several functional groups of micro-organisms As we have seen, much of the photosynthetic lixed CO" becomes readily available to the microbial community. A large portion of this organic matter is easily degradable lowmolecular compoun ds. All 01 this material is produced in the photosynth etically active cyanobacteria l mat and it can be anticipated that is also decomposed there. In the presence of oxygen it can be assumed that aerobic processes predominate in the decomposition of this organic matter. During the night, oxygen is depleted often within minutes, which is mostly attributed to the respiratory activity of the cya nobacteria them selves, subsequently switching to fermentation. Th e major fermentation products are acetate, formic acid, lactate, ethanol , H, and CO,. These are excellent substrates for the obligate anaerobic sulfate-reducing bacteria (Hansen 1994). Methan ogenic or acetogeni c bacteria could also be involved in the degradation 01 these compounds but since sulfate-reducing bacteria are superior in their affinity for these substrates and sui late reduction yie lds more energy. these bacteria do not playa role of importance in coastal microbial mats as long as sulfate is present (Raskin et al. 1996). Seawater contains 28mM 01 sullate and its depletion in marine microbial mats is only envisaged at extraordinary high productivity or when th e supply of seawater lags behind. In hypersaline microbial mats methanogenic bacteri a may be important because of the presence of non-competitive substrates (Orem land and Polci n 1982, Cytryn ef al. 2000).

Sulfate reduction
We may assume that a substantial part of the fermentation products excreted by the cyanobacteria is metabolised by sulfate-reducing bacteria. It should be noted that these bacteria should occur in the Vicin ity of whe re their substrate is produced, I.e. in the cyanobacterial mat. This is remarkable , since sulfate-reducing bacteri a are consid ered as obligate anaerobic organisms and the cyanobacterial mat may be even supersaturated with oxygen during daytime. This seems contradictory. Although it cou ld be conceived that either the sulfate-reducing bacteri a move up and down in the mat with the shifting oxygen profiles or that the fe rme ntation products diffuse into the lower permanent anoxic part of the mat, it has been clearly demonstrated that large numbers of sulfate-reducing bacteria are perm an ently present in th e cyanobacte rial mat and that the highest rates 01 sulfate reduction indeed are found in th is layer (Visscher ef a/. 1992, Tes ke ef a/. 1998). More recent work has shown that many sulfate-reducing bacteria tolerate oxygen and that some are able even to perform limited aerobic respiration (Dilling and Cypionka 1990). The vertical distribution 01 sulfate-reduc ing bacteria revea ls that typica l oxygen-tOlerant species dominate the top layer of the mat, wh ereas those that can not tolerate oxygen are found in the permanently anoxic layers 403 (Risatt i ef al. 1994). Sullate reduction has been demonstrated even in the light in the fully oxygenated cyanobacterial mat. Since a search for anoxic micro-niches in this ma t was unsuccessful, it was conceived that sulfate-reduction wou ld take place under fully aerobic conditions (Canfietd and Des Marais 1991) . Attempts to isolate sulfate-reducing bacte ria that carry out sulfate reduct ion under aerobic conditions have failed so far. The substrate for aerobic sulfate reduction co uld be glycolate, excreted by th e cyanobacteria in the light as the result 01 photorespi ration . Glycolate has been shown to be an important substrate for chemotroph ic bacteria in microbi al mats and sulfate-reducing bacteria using glycolate have been isolated from cyano bacte rial mats (FrD nd and Cohen 1992, Nold andWard 1996). These observations question the importance 01 aerobic degradation 01 the bulk of the organic matter in these microbial mats. Ae robic metabolism may be limited to the oxidative attack of complex, recalcitrant compounds.

Sulfide
Sulfate reduction is a form of anaerobic respiration, using sulfate as the term inal electron acceptor an d producing SUlfide. Next to oxygen, sulfide is a major compound determining microb ial activities and the vertical stratification of different functional groups of microorganisms tha t is typical for marine microbial mats. It should be noted that sulfate reduction is not the only process that results in the fo rmation of sulfide. For ins tance, cyanobacteria under anaerobic conditions may use zero-valence 'elemental' sulfur as electron sink fo r fermentation, which also results in the formation of sulfide (Stal 1991). A variety of other bacteria use zerovalence sulfur as electron acceptor for anaerobic respiration , but it is not known how important these organisms are in marine microbial mats. Sul fide is chemically and biologicalty very reactive and in addition it is toxic to almost all organisms, including those who produce it or depend on it as substrate. In the lirst place, sulfide reacts instantan eously with iron. It is oxidised to zero-valence sulfur by ferric iron, wh ich itself is reduced to ferrous iron . Sulfide preci pitates with ferrous iron forming the virtually insoluble FeS, which produces the characteristic intense black sediment. FeS may subsequently react with zero-valence sulfur to form the very stable pyrite, which is a relatively slow process (Howarth andJergensen 1984, Thode-Andersen andJ0rgensen 1989) .
Sulfide also readily reacts chemically with oxygen and therefore both species can not co -exist at high concentrations. Co-existence of low concentrations of oxygen and sulfide has been shown to occur in microbial mats, but more Irequently th is is hardly measurable because the ra te 01 biological oxidation of sulfide is much faste r than the chem ical oxidation (Kru mbein ef al. 1979, Revsbech et al. 1983. Two important functional groups of microorganisms are involved in sulfide oxidation in microbial mats. The colorless sulfur bacteria are chemosynthetic organisms that oxidise sulfide to sulfate using oxygen as electron acceptor. Many of these species can live autotrophic, Le. are capable of fixing C02. Very high numbers (up to 10' cm' ) of these bacteria have been lound in microbial mats (Visscher et al. 1992). Some species are facultative anaerobic and capable of denitrifica· lion , using nitrate as the te rminal electron acceptor, but since marine microbial mats are usually nitrogen·depleted, this mode of metabolism is considered unimportant. The colorless sulfur bacteria are more or less homogeneously distributed in the mat and are probably only active at the sui· fide-oxygen interlace , thereby generating an almost perlect separation between these two. Becau se the oxygen gradient is moving up and down during a diurna l cycle, the sulfide-oxygen interlace follows it. It seems that the color· less sulfur bacteria would have only a very limited time to be metabolically active, unless they move with the sulfide-oxy· gen interlace. It is not known wh ich of these options apply in microbial mats.

Anoxygenic, purple sulfur, phototrophic bacteria
The other important functional group of microorganism s in microbial mats thai are involved in the oxidation of sulfide are the anoxygenic phototrophic bacteria (De . These are essentially obligate anaerobic organisms that perlorm photosyn thesis and fix CO,. In stead of water they use sulfide as the electron donor. Most com· monly found in marine microbial mats are the purple sulfur bacteria. Because they need light, they are found immedi· ately below the cyanobacterial mat. Although the cyanobac· teria filter out most of the visible light. purple sulfur bacteria use a different part 01 the spectrum, particularly in the infrared, which penetrates the mat extremely well. Moreover. purple sutfur bacteria are low·light adapted organisms that perlorm photosynthesis at light levels as low as 0 .1 % of full sunlight (Overmann et al. 1992). Another important reason why purple sulfur bacteria are found in microbial mats is that these organisms are also very oxygen-tolerant or are even capable of aerobic metabolism (De Wit and Van Gemerden 1990). This is important, because of their occurrence immediately below the cyanobacterial mat, where oxygen may be present. In the presence of oxygen. purple sutfur bacteria continue photosynthesis but they are unable to synthesise the major lighl·harvesting pigment: bacteriochlorophyll a. After prolonged exposure to oxygen . they wilt eventually end up as colorless organisms. not capab le anymore of photosynthesis. Although these cells are capable of a chemosyn· thetic mode of metabolism, oXidising sulfide with oxygen . identical to the colorless sulfur bacteria, they can not compete WIth the latter organisms because of poor substrate affinity.
Purple sulfur bacteria oxidise sulfide in two major steps. They first oxidise it to ze ro-valence sulfur, which is stored Intra cellularly (in fact outside the cytoplasm membrane). This reaction is relatively quick. by which the sulfide avail· able is rapidly depleted. The oxidation of sulfide to sulfur produces only 2 electrons with which only y, CO, can be fIxed. The subsequent oxidation of sulfur to sulfate occurs after the sulfide has been depleted , is slower, and because it yields 6 electrons it allows for the fixat ion of 3 times as much CO,. This strategy is ecologically advantageous when different species compete for sulfide. Once the sulfur is stored inside the cells . it is not available to oth er organisms (Van Gemerden 1983).

SIal
The intracellular stored sulfur serves another important function in purple sulfur bacteria. As is the case with cyanobacteria, the purple sulfur bacteria switch to fermentation in the dark, using elemental sulfur as electron sink. In this way, purple sulfur bacteria also contribute to the production of sulfide in the microbial mat (Van Gemerden 1968).
Whereas the anaerobic purple sulfur bacteria display aero obic metabolism when exposed to oxygen, most ma t-fo rming cyanobacteria are capable of ano xygenic photosynthesis when exposed to sulfide (Cohen et al. 1986). Sulfide is a potent inhibitor of oxygenic photosynthesis, but it may donate electrons to photosystem 1 and in this manner allow C02 fixation . In some species th is property must be induced and requires de novo protein synthesis. Th is is for example the case with the cyanobacterium Oscillatoria limnetica from the sulfide· rich hypolimnion of the hypersaline Solar Lake (Sinai) (Arieli et al. 1989). However, oxygen and sulfide gra· dients in microbial mats are strongly fluctuating and th erefore mat-forming cyanobacte ria such as Microcoleus chthonoplastes usually possess the capacity of anoxygenic photosynlhesis constitutively and perlorm oxygenic and anoxygenic photosynthesis simultaneously at sulfide concentrations lower than 1 mM, with the relative importance of oxygenic photosynthesis decreasing with increasing sulfide concentration (De Wit et a/. 1988). Above 1 mM sulfide oxy· genic photosynlhesis is complelely inhibited. It is likely th at in this case growth of the cyanobacterium becomes impossible, since M. chthonoplastes has an indispensable requirement lor oxygen.

Vertical stratification of funct ion al groups of microorganisms in microbial mats
Four major functional groups of microorganisms have been distinguished so far. These are: the oxygenic cyanobacteria, the anoxygenic phototrophic bacteria, the sulfate·reducing bacteria and the colo rl ess sullur bacteria. Only the first 2 . groups lorm clearty stratified layers. visible with naked eye. The latter 2 groups are distributed throughout the microbial mat, allhough Ihe sullate reducing bacteria may be parti· tioned into a more oxygen·tolerant population in the top lay· ers and the truly Obligate an ae robic species in the permanent anoxic layers.
This vertical stratification of microorganisms may be more complex. In some microbial mats a layer of green sulfur bacteria is found beneath the purple sullur bacteria (Nicholson et al. 1987). The green sutfur bacteria are another group of anoxygenlc phototro phic bacteria. Althoug h they share part of the light spectrum with the cyanobacleria, their light require· ments are extremely low and when sufficient light impinges on the mat it can be anticipated that these organisms may live phototrophically. Green sulfur bacteria are extremely oxygen sensitive but resist high sulfide concentrations.
The occurrence of a green layer beneath the purple sulfur bacteria does not necessanly indicate the presence of green sulfur bacteria. Sometimes a second layer of cyanObacteria can be found below the layer of purple sullur bacteria. Measurements of oxygen and photosynthesis have shown that these cyanobacteria periorm predominantly an oxy· genic mode 01 photosynthesis (Krumbein et al. t 9771. The activity of the purp le sulfur bacteria takes care of the elimination of sulfide that otherwise would prevent the occurrence of oxygenic photosynthesis. The oxygen profile as measured by micro -electrodes shows then 2 peaks, separated by an anoxic layer. Although the species composition of the deep layer of cyanobacteria differs from th e surface mat, it seems likely that it represents the original surface mat that has been overgrown by a new one and that successively shifts in species composition have occurred. In other occasions 'inverted mats' are encountered where the purple sulfur bacteria form the top layer and the cyanobacteria occur beneath them (Van Gemerden et al. 1989, Van Gemerden et al. 1989. Such mats form on sediments that rec eive a high load of exogenous produced organic malter, for instance algae or sea grasses deposited on the beach. lis decomposition result in the production of large amounts of sulfide, preventing growth of cyanobacteria. Mats of purple sulfur bacteria may develop that scavenge the sulfide, allowing cyanobacteria to grow below them.

Layer of ferric iron
The separation of the oxygenic and anoxygenic phototrophic bacteria l commun ities is clearly the resu lt of the opposite gradients of oxygen and sulfide and of course of light.
Although the separation appears perfect to the naked eye, there is overlap between the two when observed at the micro-meter scale, giving rise to competitive interactions and the exposure to sulfide and oxygen in the aerobic and anaerobic communities, respectively. In some microbial mats an additional layer can be distinguished between the cyanobacteria and the purple sulfur bacteria. This layer has a rusty color and presumably is composed of iron hydroxides (Stal 1994). This would represent an ideal barrier between the aerobic cyanobacteria and the anaerobic purple sulfur bacteria (Figure 1). Any sulfide diffusing upwards will react with ferric or fe rrous iron before it reaches the cyanobacteria l mat. Vice versa, any oxygen diffusing downwards will react with ferrous iron or FeS and be unable to interfere with the purple sulfur bacteria. II is presumed that this layer will tend to reduce at night and oxidise during daytime. This hypothesis so far has not been proven experimentally. Apart from being a pure chemical barrier, this rusty layer may also represent a community of an oxygenic phototrophic (purple) bacteria that uses ferrous iron as electron donor, oxidising it to ferric iron (Widdel et al. 1993). Such bacteria have been isolated from a variety of environments but it is not known whether th ey are important in microbial mats. It has also been proposed that cyanobacteria may be capable of irondependent anoxygenic photosynthesis but experimental proof for this hypothesis is lacking (Cohen 1984). The involvement of chemosynthetic bacteria in the oxidation of iron in microbial mats is less likely. Some colorless sulfur bacteria are capable of oxidising ferrous iron aerobically, but this process occurs only at extremely low pH (-2), which does not occur in coastal microbial mats. Species such as Gallionella ferruginea or Sphaerotilus natans which oxidise ferrous iron at neutral pH are unlikely to be able to compete with the chemical oxidation of iron in microbial mats (Emerson and Revsbech 1994). Sulfide subsequently reacts with ferrous iron (Fe 2 . ) to produce the black insoluble FeS. During the day, when the cyanobacteria evolve O~ in the cou rse of photosynthesis, FeS is oxidised. This prevents OJ from diffusing into the layer of the anaerobic, anoxygenic bacteria. The sulfide produced by the SRB reacts with the Fe" and prevents it from reaching the cyanobacteria.
Mat-forming cyanobacteria are protected by an iron coat The rusty layer that sometimes separates the green and purple communities may prevent sulfide diffusing from below into the cyanobacterial mat but it does not help against sulfide which is produced inside the cyanobacterial mat by sul-fate~reducing bacteria or by the cyanobacteria themselves. The mat-forming cyanobacterium M. chthonoplastes has been shown to accumulate iron in its polysaccharide sheath (Stal 1994). Th is layer of iron may serve a similar function as the rusty layer of the mat (Figure 2). When during fe rmentation in the dark M. chthonoplasles reduces sulfur to sulfide, the latter reacts with fe rric iron, oxidising the sulfide back to zero valence 'elemen tal' sulf.ur, and producing ferrous iron. The net result of this fermentation is the reduction of iron . Another process coup led to fermentation by which iron is reduced is the oxidation of formic acid to COl. During the subsequent light period the ferrous iron is oxidised back to ferric iron by the oxygen produced during photosynthesis. This wrll keep the oxygen partial pressure low in the cell and in its immediate vicinity which is beneficial for the organism because it reduces losses of photosynthate by photorespiration.
Nitrogen fixation: without it coastal microbial mats would not develop Nitrogen comprises 7-1 0% of cell dry weight matter and represents therefore the second most important element. In the In order to prevent 0 2 from accu mulation, ferrous iron in the sheath is oxidised. B. During the night, gl ycogen and the compati ble solute is fermented and concomitantly su lfur is reduced to sulfide . The fermentation products are end-oxidised by sulfate-reducing bacteria (SRB) and sulfide is formed. Th e sulfide reacts with ferric iron, preventing th e accumulation of sulfide to toxic levers while regenerating ferrous iron.
cell it is mainly present in its redu ced form . Ironically, in its most ubiquitous form, atmospheric dinitrogen (N,) it is not accessible to most organisms and the bound forms such as nitrate, ammonia or organiC nitrogen are usually in low supply, particularly in the marine environment. A limited group of, excl usively, prokaryotic organ isms possesses the ability to reduce the extre me stable triple bond between the two molecules of N in N2. These organisms all co ntain the en zyme co mplex nitrogenase which catalyses the redu ction of N, to NH, at the expense of a large amount of energy and low-potential electrons (ferredoxin) (Peters ef al. 1995) . Among cyanobacteria many species are known to be capable of lixing nitrogen. As oxygenic photo-autotrophic organisms they are particularly we ll equipped to meet the energy and electron demands of nitrogen fixation . On the other hand, nitrogenase is extremely sensitive to oxygen and diazotrophic cyanobacteria the refore developed strategies to circumvent this problem (Gallon 1992). Clearly, the best ad aptation has been evolved by the heterocystous cyanobacte ria . These filamentous cyanobacteria differentiate special cells, the heterocysts, which have lost the ability of oxygenic photosynthesis and CO, fixation and wh ich have become the sites of nitrogen fixation. Nitroge n fixation in these organisms is strong ly light dependent although som e activity can be sustained in the dark, driven by aerobic respiration. Heterocystous cyanobacteria are remarkably rare in coastal microbial mats (Stal ef al. 1994). More frequently, non-heterocystous filamentous cyanobacteria have been shown to fix nitrogen in these environments. These organ-isms can be distinguished with resp ect to their strategy to fix nitrogen in two major groups (Stal 1995, Bergman ef al. 1997. One grou p is capable of fixing nitrogen only under anaerobic conditions and its strategy can be described as 'avoidance of oxygen'. There are a number of ways by which this can be achieved. First of all, nitrogen fixa tion ca n be confined to the dark period, whe n the mat has become anoxic. The disadvantage of this strategy is that only a limited nitrogenase activity can be sustain ed under such conditions be cause 01 the low energy yield of fermentalion . Experiments and calcu lations have unequivocally demonstrated that some nitrogen fixation can be supported by fermentation by mat-forming cyanobac teria such as Oscil/aforia Iimosa (Stal and Moezelaar 1997). Another way is to realise a spatial separation of oxygenic photosynthesis and nitrogen fixation in Ihe mat. At the bottom of the cyanobacterial mat oxygenic photosynthesis may not be possible because only lar red light (>700nm) predominates here, which is specilically harvested by the anoxygenic photosystem I (Stal ef al. 1985). Moreover, sulfide may al so reach higher concentrations in this part of the mat and this is a potent inhibitor of oxygenic photo synthesis. It has indeed been shown that the specific (chlorophyll-normalised) nitrogenase activity increased with depth in the mat. Sulfide has been shown to induce nitrogenase in mat-forming cyanobacteria in the light (Villbrandt and Stal 1996). The problem with this strategy is that the nitrogen-fixing cyanobacteria do not fix CO, (except when sulfide-dependent anoxygenic photosynthesis occurs) and the cyano bacteria in the suriace lay-ers are still devoid of nitrogen. Tra nsport or fixed nitrogen outside the cells seems ineHicient and would rather favor non-diazotroph ic organisms in the mat. It seems more likely that in this case th e cyanobacteria move up and down between the two sites. Motility of mat-rorming cyanobacteria has been shown in several occasions but these were all controlled by light (Garcia-Pichel et al. 1994, Kruschel andCastenholz 1998). Motility controlled by the nitrogen status of cyanobacteria still awaits experimental proof. The second group of non-heterocystous diazotroph ic cyanobacte ria is capable of fi xing nitrogen under fu lly aerobic conditions (Bergman et al. 1997). It is not precisely know n by wh ich mechanism these organisms protect nitrogenase from oxygen inactivation. Although they are capable of di azot rophic grow th in culture under fu lly aerobic co nditions and under continuous illumination , they fix nitrogen excluSively during the dark when grown under an alternating light-dark cycle. The sam e day-night pattern of nitrogen fixation can be found in most microbial mats. Presu mably, oxygen concentrations reach too high levels in the mat during daytim e. Som etimes , two peaks of nitrogenase activity can be observed during a day-night cycle in a mat of aerobic nitrogen-fixing cyanobacteri a, on e each at sunris e and sunset an d low or zero activity at night and during th e day (Villbrandt et at. 1990) .
In the few exampl es of microbial mats formed by heterocys tous cyanobacteri a, nitrogen fixation occurs during daytirne (Stal 1995). It is not precisely known why these organisms are excluded in many microbial mats. It has been suggested that heterocystous cyanobacteria are more sensitive to sulfide tha n non-heterocystous species (Howsley and Pearson 1979) . However, it is H,S which freely diffuses into the cell and exerts is tox icity and because of Ihe alkalin e con di tions in coastal microbial mats the concentrations of this gas remains extrem ely low. Another suggestion is that heterocystou s cyanobacteria do not tolerate anoxic conditions in the dark. Indeed, mats of heterocystous and nonheterocystous cyanobacteria occurring close to each other on an inter-tidal flat in Baja California , Mexico, differed markedly in their oxygen dynamics (Stal 1995). The mat of the heterocystous cyanobacterium Calothrix sp. did not turn anoxic during the night and lacked the black layer of FeS and purple sulfur bacteria. This also hinted to the absence of sulfide in th is mat. During daytime oxygen concentrations in the Calothrix mat did not reach excessive high levels. This, and th e availability of oxygen during the dark could have been critical for the heterocystous cyanobacterium to proliferate.
The rarity of heterocystous cyanobacteria is not only the case in marine microbial mats but extends to the whole marine environment. The most important nitrogen-fixing cyanobacterium in the marine plankton is the non-heterocystous Trichodesmium , whereas in fres hwater and brackish environments only heterocystous species occur (Paerl 1990, Moisander and Paerl 2000). Hence, it could also be that high salinity selects again st heterocystous species , although the precise mechanism remains to be elucidated. The mats of Calothrix in Baja California occur high in the inter-tidal sedimen ts and are rarely submersed and may be are therefore not strongly influenced by the seawater.
Whatever may be the reason, the fact th at nitrogen fixa-407 tion in most microbial mats is accomplished by non-heterocystous cyanobacteria and that th is occurs predominantly under anaerobic conditions implies that th is process is far from eHldent. It can be anticipated that growth of the cyanobacteria is strongly nitrogen-limited in these mats.
Is nitrogen-limited growth of the cyanobacterial mat preventing the formation of a stromatolite?
Wh en nitrogen is limiting growth of the mat-building cyanobacteria these organisms will divert th e fixed CO! to non-nitrogenous compounds, mainly carbohydrates. This mode of growth is termed 'unbalanced'. Du ring balanced growth all cell components are synthesised in the right propo rtion s, but when nitrogen is unavailable, proteins , nucleic acids and cell walls can not be produced. However, photosynthetic COl fixation is not impaired and carbohydrates are synlhesised. Intracellu lar, glycoge n is stored, but since limited space is available polysaccharides are also excreted into the medium as mucilage and sheath materia l. Many marine microbial mats are the refore embedded in a th ick gelatinous polysaccharide matrix. Photosynthetic C02 fixation res ults in the formation of carbonate ion (CO..'). It has therefore been hypothesised th at cya nobacterial photosynthesis would promo te calcification (Krum bein and Giele 1979). However, in most marine micrabi al mats , calcification was not spatially associated with the cyanobacteria (Lyons et al. 1984, Chafetz andBuczynski 1992) . Th is is rem arkable because with the concentration of ca lcium ion in seawater it would easily exceed the solubility product of calcium carbon ate minerals such as aragonite and calcite. Thu s. it was obvious to suppose a mechanism that would prevent calcification. Since it is known that polysaccharides can bind Ca~· and/or Mg2. one possible mechanism that has be en proposed is that the extracellular polysaccharides produced by the cya nobacteria in the mat would lower the activity of these ions to such extend that calcium carbonate precipitation does not occur (Borman e t al. 1982al. , Westbroek et al. 1994. Alternatively, polysaccharides may interact with crystallisation nuclei , preven ting their growth, or a com bination of both mechanisms.
Hence, a chronic nitrogen depletion occurring in many marine microb ial mats, leading to excessive extracellul ar polysaccharide prod uction , may offer an explanation for the fact that th e majority of these systems do not lith ify and produce stromatolites.

Concluding remarks
The classic example of a microbial mat is built by cyanobacte ria and these oxygenic phototrophiC microo rganisms lulfill a key role in the systems function and metabolism. Most, if not all . of the primary production of these cyanobacleria is more or less directly transferred to the micro bial mat system and not primarily used for the synthesis of structural cell material. Probably the most important mechanism is through the excretion of fermentation products. But also other mechanisms are responsible for the transfer of fixed carbon to the microbial comm unity that is fueled in this way. Th e solar en ergy fixed by the cyanobacteria is utilised by th e sulfate reducing bacteria and the sulfide they produce, is utilised by anoxygenic phototrophic bacteria and colorless sulfur bacteria. In this way, the microbial mat functions as an internally closed system , driven by solar energy. The mal behaves like a lissue, displaying its own physiology. The nel exchange with the atmosphere and geosphere is limited because the cycles of the elements are essentially closed. In many microbial mats, decomposition is complete but in a few other systems a slow accumulation of organic matter may occur. Rarely, microbial mats calcify and lithification results in the formation of stromatolites.