The sea surface microlayer: a unified physicochemical and biological view of the air-ocean interface.

Michael Cunliffe1*, Anja Engel2, Sanja Frka3, Blaženka Gašparović3, Carlos Guitart4, J Colin Murrell5, Matthew Salter6, Christian Stolle7, Robert Upstill-Goddard6 & Oliver Wurl8.

1 Marine Biological Association of the United Kingdom, Plymouth, UK. 
2 Leibniz Institute of Marine Sciences (IFM-GEOMAR), Kiel, Germany. 
3 Ruđer Bošković Institute, Zagreb, Croatia.
4 Spanish Institute of Oceanography, Spain.
5 University of Warwick, Coventry, UK.
6 Newcastle University, Newcastle, UK. 
7 Leibniz Institute for Baltic Sea Research, Warnemünde, Germany.
8 Old Dominion University, Virginia, USA. 

*Corresponding author: Marine Biological Association of the United Kingdom, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK. E: micnli@mba.ac.uk 	T: +44 (0)1752 633293 F: +44 (0)1752 633102. 

Abstract
The sea surface microlayer (SML) covers more than 70% of the Earth’s surface and is the interface between the ocean and the atmosphere.  It is therefore critical to a diverse range of Earth system processes. Here we outline a SML paradigm, combining the physicochemical and biological components that are important in terms of both structure and functions. In the SML, biogenic molecules such as carbohydrates, lipids and proteinaceous material are enriched, often with distinct microbial assemblages playing defining roles in microlayer functions such as organic carbon cycling. The SML is critical to air-sea gas transfer and marine aerosol formation, processes that are affected by specific SML biochemical components and active resident microorganisms. Unfortunately the SML is also a concentrating layer and reservoir for anthropogenic pollutants, with serious implications for the health of the ocean. A progressive approach to understanding the SML and thus the ocean-atmosphere interface can only be achieved by considering together all of the key components of this complex environment.    
Introduction
The sea surface microlayer (SML) is the thin layer of the surface ocean which is in direct contact with the atmosphere, and mostly has physicochemical and biological properties that are measurably distinct from underlying waters. Natural slicks visible on the sea surface are well known in areas of high biological activity, such as coastal waters. Perhaps less well known is that the SML is almost ubiquitous and pervades most of the ocean surface, although it is mainly indiscernible with the naked eye. Because of its unique position at the air-sea interface, linking the hydrosphere with the atmosphere, the SML is central to a range of global biogeochemical and climate-related processes. Its study thus brings together several scientific disciplines with interests both in SML structure and functions. 
Early work in the Sargasso Sea established a link between visible slicks and phytoplankton blooms, and showed that slicks can have fundamental affects on the physical properties of the sea surface. Evidence of their biological origin and activity was revealed in carbohydrate enrichments in slicks relative to underlying waters (Sieburth and Conover, 1965). Such key observations have been refined during subsequent decades by researchers from diverse scientific disciplines operating in parallel but largely independent of each other. Important new findings include the enrichment of transparent exopolymer particles in the SML and compelling molecular evidence for complex SML microbial communities. 
Understanding important marine ecosystem processes and those that also impact upon the wider Earth system requires a holistic view of the ocean-atmosphere interface (Figure 1). What is clear is that SML structure and functions are intimately bound together, and that a unified physicochemical and biological perspective must be embraced and developed. We discuss the SML and highlight several important and recent scientific discoveries that improve our understanding or support previously conceived paradigms. This review is integrated across several scientific disciplines and attempts to identify the current gaps in our understanding of this fundamentally important ecosystem at the air-ocean interface. 

Sea surface microlayer thickness, sampling and the need for scientific best practice
Notwithstanding recent advances, including those outlined below, that have broadened our overall understanding of SML structure and functions, there remains a need to develop and adopt agreed scientific best practice for SML research and data reporting, following the examples set in other areas of marine research such as ocean acidification (Riebesell et al., 2010). 
One fundamentally important area where agreed best practice is essential is SML sampling and how this relates to SML thickness. The thickness of the SML is still not yet fully established. A combination of laboratory and field measurements have shown a ‘layer of sudden change’ in terms of a range of physicochemical parameters, including concentrations of organic matter, nutrients, dissolved trace metals, biochemical oxygen demand, pH and chemical oxygen demand. These studies indicate that the SML is 50 ± 10 µm thick (Zhang et al., 2003; Zhang et al., 1998). The various SML sampling devices available tend to sample distinctly different microlayer depths (Figure 2). Subsequently the SML is most often operationally defined by the chosen sampling protocol, with wide ranging implications for data analyses and interpretation (Agogue et al., 2004; Cunliffe et al., 2009a).  
Clearly, a widely agreed standardisation of SML sampling is highly desirable as it would legitimise the intercomparison of data between researchers in different regions and from different scientific disciplines. Linked to this is a need to improve and standardise analytical methods, and to agree and implement the routine measurement of standard SML variables (e.g. DOC) that could aid inter-study comparisons.  Such examples of scientific best practice would increase our understanding still further and ultimately broaden the global perspective of the SML. 

The sea surface microlayer is a physicochemically complex gelatinous film  
Historically, lipids were thought to comprise a significant fraction of SML material, forming a continuous layer above a protein-polysaccharide layer; this was the basis of an early “wet-dry” stratified model of the SML (Hardy, 1982; Hermansson, 1990; Norkrans, 1980). It is now evident that although lipid material is less abundant in the SML than previously thought, it may still be important in affecting SML physicochemical properties (Frka et al., 2009; Mazurek et al., 2008). Concentrations of both dissolved and particulate SML lipid fractions vary depending on location, season, trophic state and prevailing meteorological conditions, contributing 3-7 and 10-15% and occasionally more than 30% to the dissolved and particulate organic matter pools respectively (Gašparovic et al., 1998; Penezić et al., 2010; Saliot et al., 1991). 
The role of lipids in the formation and stability of the SML is still not clear, but it is likely that lipids influence the viscoelastic properties of microlayers (Kozarac et al., 2005). Laboratory model studies indicate that lipid monolayers efficiently accumulate bulk carbohydrates by hydrogen bonding, nonpolar and electrostatic interactions (Kozaraca et al., 2000). An important question remains unanswered: are natural SML lipids nucleation sites for hydrophilic substances in situ?
Some researchers had long considered carbohydrates to be important in the SML. Sieburth (Sieburth, 1983) proposed that the SML is a “highly hydrated loose gel of tangled macromolecules and colloids”.  Recent developments refined Sieburth’s original ideas by identifying transparent exopolymer particles (TEPs) as a dominant carbohydrate component of microlayers (Cunliffe et al., 2009b; Wurl and Holmes, 2008). 
TEPs are hydrogel particles that form from dissolved polysaccharides released during microbial production and decomposition processes (Alldredge et al., 1993). Reactive components of microbial polysaccharides such as uronic acids facilitate bonding and subsequent  micro- and macrogel aggregation (Verdugo et al., 2004). TEPs have been shown to form spontaneously in seawater (Chin et al., 2008), and their rate of formation is higher when more dissolved polysaccharides are brought into contact (Engel et al., 2004). Due to the sticky properties of TEP, they can aggregate both live and dead plankton, as well as mineral particles. Consequently they are hotspots of microbial activity in the water column (Simon et al., 2002) and they accumulate in the SML (Cunliffe et al., 2009b; Wurl and Holmes, 2008; Wurl et al., 2009).  TEP aggregation rates in the SML have been shown to be five-fold higher than in underlying water just a few tens of centimetres below (Wurl and Vagle, 2011). Compression and dilation due to capillary waves may also enhance polymer collision and further facilitate rapid gel formation within the SML. 
Amino acids and proteins in the SML have received less attention than lipids or carbohydrates. Underlying water is the main source of proteinaceous matter concentrating in the SML (Kuznetsova and Lee, 2001), and SML organisms no doubt fulfil important roles as producers, consumers and modifiers of proteins in natural films. Turnover rates of proteinaceous material in the SML can be high. Protein hydrolysis experiments have shown that concentrations of amino acids are reduced significantly in microlayer samples, often reaching lower values than those found in underlying water (Kuznetsova and Lee, 2001). Contrasting studies in the Atlantic and Mediterranean Sea have however indicated that SML amino acids are not always readily available for bacterial remineralisation (Reinthaler et al., 2008). Potentially high utilization rates of amino acids are no doubt connected to the fact that amino acids are major carbon and energy sources for marine microbes (Keil and Kirchman, 1992).  

The sea surface microlayer is stable
Wurl et al., (2011) have shown that the SML is stable enough to exist above the global average wind speed of 6.6 m s−1. Even though the SML is temporarily disrupted in the presence of breaking waves that disperse microlayer material into the underlying water, the dispersed material nevertheless rapidly adsorbs to the surface of rising bubble plumes produced by the breaking waves (Wurl et al., 2011), and these rising bubbles have been shown to also facilitate TEP aggregation (Wallace and Duce, 1978; Zhou et al., 1998). This mechanism may well be the most important means of transferring surface-active material to the SML  following wave breaking (Wurl et al., 2011). 

The sea surface microlayer is an active biofilm
The study of microbial life in aquatic surface films is not new. The Swedish botanist Einar Naumann in 1917 introduced the term neuston to identify organisms associated with the air-water interface that were ecologically distinct from plankton in underlying water (Naumann, 1917). Many studies have now shown that the diversity of microorganisms in the SML can differ from that in the underlying water even a few centimetres below, with bacterial diversity having received most attention through the utilisation of molecular ecology tools (recently reviewed by Cunliffe et al., 2011). Microlayer bacteria (bacterioneuston) assemblages are formed from underlying water recruitment (Agogue et al., 2005; Cunliffe et al., 2009a; Cunliffe et al., 2009c; Obernosterer et al., 2008; Stolle et al., 2011), often include differences in the relative abundance of cognate underlying taxa and microlayer-specific taxa (Cunliffe et al., 2009a; Cunliffe et al., 2008; Cunliffe et al., 2009c; Franklin et al., 2005; Stolle et al., 2011), show evidence of synchronicity between different sites (Cunliffe et al., 2009a; Cunliffe et al., 2009c), and are affected by prevailing meteorological conditions (Stolle et al., 2011; Stolle et al., 2010). 
Phytoneuston are also important components of SML ecosystems and are often found to be enriched in SML. As with the bacterioneuston, phytoneuston assemblages can have different structures compared to those in underlying water (De Souza Limaa and Chretiennot-Dinet, 1984; Hardy and Valett, 1981; Montes-Hugo and Alvarez-Borrego, 2007). Phytoneuston could have important biogeochemical roles especially in inshore waters (Montes-Hugo and Alvarez-Borrego, 2007; Yang et al., 2009). Autotrophic phytoneuston in combination with SML heterotrophs determine the net metabolic status of the air-sea interface, and can subsequently control CO2 flux (Calleja et al., 2005). 
SML-specific microbial diversity is not limited to bacteria and autotrophs (Hardy, 1982; Sieburth, 1983) as there is now molecular-based evidence that microlayer protist diversity is also distinct from pelagic assemblages. Components of a possible SML microbial loop have been identified, suggesting that the cycling of elements and energy through microbial trophic interactions could occur via different taxa in the microlayer compared to underlying water (Cunliffe and Murrell, 2010). 
Early attempts to understand how microbes are adapted for living in microlayers considered that individual cells must have specific cell-surface properties, such as hydrophobic surfaces, which facilitate attachment to hydrophobic moieties enriched in microlayers (Norkrans, 1980). It is now believed that many microorganisms within the SML are probably associated with the buoyant particles (e.g. TEP) that form the SML itself (Cunliffe and Murrell, 2009). Supporting this, recent studies in the southern Baltic Sea showed a strong dependence of bacterioneuston dynamics on particulate organic carbon content in the SML (Stolle et al., 2010), and that most of the distinctive bacterial taxa in the microlayer compared to the underlying water were particle-attached (Stolle et al., 2011). 
The proximity of the SML to the atmosphere means that resident microbial life could be potentially exposed to high level of ultraviolet radiation (UV). There is some evidence that SML microorganisms employ specific adaptations to minimise UV damage, such as increased UV absorbing mycosporine-like pigments (Tilstone et al., 2010). It is still not clear however what effects increased exposure to UV has on bacterioneuston assemblages. Microbial responses to UV in the SML have been discussed in detail elsewhere (reviewed by Cunliffe et al., 2011).       
Microbial life in the SML significantly influences important biogeochemical processes (Cunliffe et al., 2011). The degradation of particulate organic matter (POM) to dissolved organic matter in the water column by microbial extracellular enzymes and its substantial role in the global carbon cycle are well established (Azam, 1998). Not only is POM and TEP enriched, and likely to be formed in the SML, but it is probably also broken down there by microbial activity. The SML has already been shown to have elevated extracellular hydrolytic activity, leading to higher organic carbon turnover rates than in underlying waters (Kuznetsova and Lee, 2001). There is evidence that certain members of the bacterioneuston, e.g. Bacteroidetes, have a higher contribution to DOM turnover in the SML compared to the underlying water (Obernosterer et al., 2008), most likely due to their ability of degrading complex polymers (Kirchman, 2002). Future studies are needed to examine the relative importance of microbial POM and DOM cycling in the SML and to better understand the importance of the bacterioneuston in global carbon cycling.  

The sea surface microlayer and air-sea gas exchange
Air-sea gas exchange is a fundamental part of the Earth system and is driven by molecular and turbulent diffusion. Turbulent diffusion is defined by the length and velocity scales of turbulent eddies in bulk water and air close to the air-water interface, while molecular diffusion operates at the scale of the SML where these eddies are suppressed (Upstill-Goddard, 2006).  A “resistance” to gas exchange is thus manifested in gas concentration gradients either side of the interface, in so-called “diffusive sub-layers” through which gas transport rates constrain air-water gas exchange (Liss, 1983).  Gas transfer velocities ka and kw in air and water respectively, define these transport rates, being functions of gas diffusivity and the inverse of the diffusive sub-layer depth.  For most gases of interest however, only kw is relevant. Modification of kw occurs by various mechanisms including sea state and bubble bursting (Ho et al., 2000; Woolf, 2005a, b), and of particular relevance here, the physicochemical and biological composition of the SML (Frew, 2005; Upstill-Goddard et al., 2003)
 	Biogenic surfactants influence gas exchange in two principal ways: as a physical barrier and through modification of sea surface hydrodynamics and hence turbulent energy transfer (McKenna and McGillis, 2004; Tsai, 1996, 1998). Seawater contains both soluble and insoluble surfactants but only the former are thought to be effective in suppressing kw across a wide range of environmental wind speeds. Where slicks of insoluble surfactant form under calm conditions, they reportedly dissipate even under light winds (Bock et al., 1999; Frew et al., 1990; Goldman et al., 1988). In regions of high soluble surfactant concentration, kw parameterisations based on bulk turbulence measurements alone may therefore be erroneous. Although laboratory experiments show kw suppression in the range 5-50% for natural phytoplankton exudates (Frew et al., 1990; Goldman et al., 1988) and 60–90% for synthetic soluble surfactants (Bock et al., 1999; Frew, 2005) added to seawater, quantifying in situ kw suppression by natural surfactants remains a challenge. Consequently, the extent to which variations in biogenic surfactant activity affect air–sea gas exchange at ocean basin scales from coastal waters to oligotrophic gyres remains unknown (Salter et al., 2011).  
The direct involvement of the bacterioneuston in air-sea gas exchange has been examined experimentally in situ using free-floating gas exchange boxes (Conrad and Seiler, 1988; Frost, 1999) and with a laboratory gas exchange tank (Upstill-Goddard et al., 2003).  Early in situ work revealed significant mismatches between the invasive and evasive exchange of carbon monoxide, methane, nitrous oxide and hydrogen in the open ocean, consistent with microbial gas consumption by the bacterioneuston as the only plausible gas sink (Conrad and Seiler, 1988). This was later corroborated by Frost (1999), who found around 8% invasion–evasion mismatch for methane in coastal seawater.  Subsequent laboratory studies with added methanotrophs (Upstill-Goddard et al., 2003) confirmed the potential for active bacterioneuston control of air-sea methane exchange. Figure 3 (Upstill-Goddard et al., 2003) shows apparent gas transfer velocities of methane corrected to constant diffusivity and normalised to corresponding estimates for inert sulfur hexafluoride (SF6) derived simultaneously during invasive and evasive gas exchange experiments in a sealed laboratory tank in which surface turbulence was generated by a motor-driven baffle.  For a situation where methane behaves as an inert gas during water-air gas transfer (all gas transfer under physical control, i.e. turbulence) the ratio of the two transfer velocities would equal unity.  Clearly, this is not the case for methane exchange measured evasively in bacterial enrichment experiments, where the ratios all exceed unity (Figure 3).  This was interpreted as being due to modification of the methane concentration gradient at the air-water interface by activity of methanotrophs in the microlayer. Consistent with this, for invasive experiments the additional methane flux due to microbial consumption was not measureable due to its consumption in the microlayer; hence the ratio of the two transfer velocities measured invasively was always one (Upstill-Goddard et al., 2003). 
Molecular ecology studies of the bacterioneuston have shown, using functional gene probes, that the metabolic capability for trace gas cycling is widespread in microlayers (Cunliffe et al., 2008; Cunliffe et al., 2011). The conclusion from these studies is that the bacterioneuston is closely involved in the cycling of at least some climatically active trace gases.  Thus the bacterioneuston may potentially be both a small gas source and a small gas sink, contingent upon the prevailing microbial and biogeochemical regimes.

The sea surface microlayer and marine aerosols
Marine-derived aerosols are an important part of the global aerosol system, significantly contributing to the radiative budget, biogeochemical cycling and air quality. Primary aerosol formation at the ocean surface occurs through mechanical processes such as bubble bursting (O'Dowd and Leeuw, 2007). O’Dowd et al (2004) have shown that during phytoplankton blooms, aerosols have large organic fractions (63% of the submicrometer aerosol mass), whilst during the winter, when biological activity is minimal, the organic matter fraction of aerosols is significantly reduced (15%) (O'Dowd et al., 2004). Other studies have established that biogenic microlayer particles (including transparent gel-like particles), bacteria and viruses become airborne as aerosols (Bigg et al., 2004; Leck and Bigg, 2005), and highlighted the important contribution of carbohydrates to marine-derived aerosols (Russell et al., 2010), thus strengthening the significance of the link between the SML and marine-derived aerosols. 

The sea surface microlayer and marine pollution 
A range of anthropogenic pollutants are found in the SML (Wurl and Obbard, 2004). Pollutants such as hydrocarbons, organochlorine compounds and trace metals may typically be ten to one hundred times more concentrated in the SML than in underlying waters, increasing environmental and ecotoxicological concerns for marine ecosystems (Wurl and Obbard, 2004). Notably higher accumulations are found in the SML of coastal water close to anthropogenic sources of pollution (Cincinelli et al., 2001; Guitart et al., 2007). Both anthropogenic and natural processes including direct inputs (e.g. oil spills), coastal discharges and atmospheric deposition, are the main contributors of SML pollutants, all of which subsequently occur at inherently different temporal and spatial scales. 
SML pollutants participate in processes from the molecular scale (e.g. biochemical transformation) to the ecosystem scale (e.g. air-sea transport). For example, hydrophobic organic contaminants and metals are involved in sorption processes within the SML biogenic matrix (García-Flor et al., 2005). There is also evidence that microbial metabolism of organic pollutants such as polycyclic aromatics is important in the SML (Guerin, 1989). 
Many questions remain unresolved, including potential ecotoxicological effects in the SML and the transport of pollutants between the ocean and atmosphere. It remains unclear how the physicochemical structure of the SML, outlined above, affects pollutants from a functional perspective. Also, the occurrence and behaviour of less hydrophobic ‘emerging pollutants’, such as pharmaceutical chemical residues, in the SML remain unknown. Both laboratory experiments and field studies are required for a better understanding of the cycling and fate of pollutants in the SML in terms of the oceans’ health. 

Future research direction and perspectives
To date, study of the SML has been largely dominated by opportunistic sampling programmes that do not make multiple return visits to sample the same location. Greater understanding of the SML could be achieved by establishing a network of time-series study sites. Time-series observations of pelagic systems (e.g. Western Channel Observatory www.westernchannelobservatory.org.uk) have proven extremely effective in understanding complex and dynamic variables. Annual events, such as the affects of phytoplankton blooms on the SML, could be better understood across broader temporal and regional scales.
	Understanding the SML in its wider Earth system context must be a priority. Other processes such as atmospheric deposition (e.g. terrestrial dust deposition) need to be studied alongside the SML, including parallel atmospheric sampling during SML sampling. Future research should also consider the effects of freshwater, estuarine and coastal processes on the SML (Figure 1).  
Multidisciplinary scientific development is a fundamental challenge for SML research that requires progressive thinking from funding agencies. Key to this is an expansion of genuine and practicable multidisciplinary programmes. A holistic systems approach which details the obligate connectivity between biological diversity and ecosystem function as outlined here (Figure 1) is essential to fully understand the SML. The application of contemporary molecular biological techniques, such as metagenomics and metatranscriptomics, will help these aims to be achieved. Global-scale processes such as the role of microbes in air-sea exchange, carbon cycling and marine pollution cannot be fully evaluated without such endeavours.   

Acknowledgments
This review was born out of a multi-disciplinary meeting held at the Marine Biological Association of the United Kingdom, Plymouth (January 2011) and entitled 'What is the sea surface microlayer? Towards a unified physical, chemical and biological definition of the air-ocean interface'.  We thank the International Management Committee of the Cooperation in Science and Technology (COST) Action 735 for supporting this meeting from funds made available through the European Union. 

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Figure Legends
Figure 1. Schematic diagram showing the properties and interconnections of the sea surface microlayer (SML) discussed in this review. 

Figure  2. Three sea surface microlayer sampling devices in use, including typical sampling depths of each method. Membrane filter images reproduced from; Cunliffe et al., (2011). Glass plate and mesh screen images reproduced from; O. Wurl. 2009. Sampling and Sample Treatments. In: Practical Guidelines for the Analysis of Seawater. Ed. O. Wurl, CRC Press, Boca Ration, FL, USA.     

Figure 3.  Ratios of the apparent transfer velocities of methane and sulfur hexafluoride (SF6) normalised to a constant diffusivity, as derived from laboratory gas exchange experiments: 9232, marine Methylococcus; KO and YO, marine Methylomicrobium; Community, marine methanotrophs; OB3B freshwater Methylosinus trichosporium. Figure reproduced by permission of American Geophysical Union from Global Biogeochemical Cycles, 2003, 17, 1108, doi:10.1029/2003GB002043, 2003.

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