Thermophilic microorganisms are the Holy Grail in Biotechnology. Thermophilic microbes are extensively used in processes such as thermophilic anaerobic digestion, composting as well as other fermentation processes and the cellular components of such microorganisms (i.e. enzymes «thermozymes», proteins and nucleic acids) have considerable potential for many industrial applications (Bruins et al. 2001, Haki 2003). However, the real biotope of this unique microbial resource is still poorly known and in fact, the resource is under-exploited.

The average temperature of the earth is 15°C. The temperature, however, varies from 7000°C (centre of earth) to −89°C (Vostok Station in Antarctica). Over this huge range of temperatures, microbes can grow between -18°C and 113°C and can encounter four sources of heat: geothermal energy, self-heating (the organisms’ metabolism), human/animal activity and solar radiation.

The first one is considered as the true ‘thermophile biotope’, but geothermally-heated regions are rare, scattered, far removed from laboratories and to find them, the “hunter” of these microbes must travel to some of the most remote and inhospitable regions of our planet.

The heat from organisms’ metabolism concerns mainly mesophilic microbes (through homeothermic animals), but it concerns also thermophilic ones through aerobic fermentation where microbial metabolic activity generates its own heat with temperatures up to 65°C (Diaz 1999). Known examples in nature have not been fully documented, except for their use by humans and certain animals which drive such thermophilic fermentations (Hechenleitner et al. 2015, see Table 1). All the self-heated fermentation observed has involved plant biomass.

Table 1. Download as CSV 

List of known thermophilic habitats.

Habitat	Origin of the heat	Temperature	Type	Ref
hot springs	geothermy	50 to 120°C	natural	Chaban et al. 2006
deep sea hydrothermal vents	geothermy	50 to 120°C	natural	Chaban et al. 2006
oil reservoir	geothermy	50 to 120°C	natural	Chaban et al. 2006
ocean crust	geothermy	50 to 120°C	natural	Chaban et al. 2006
deep marine sediment	geothermy	50 to 120°C	natural	Chaban et al. 2006
continental deep subsurface	geothermy	50 to 120°C	natural	Chaban et al. 2006
some cheese making (pressed cooked cheese family)	human heating	45°C	animal created	Tansey 1973
anaerobic digestion reactor	human heating	45 to 60°C	animal created	van Lier et al. 2001
fermentation of tobacco leaves	self-heating	>40°C	animal created	English et al. 1967
fermentation of cacao beans	self-heating	>40°C	animal created	Beg 2017
post-fermented Chinese teas	self-heating	50°C	animal created	Lv et al. 2013
mounds for egg incubation (Australasian megapodes, crocodiles)	self-heating	+/- 40°C	animal created	Hechenleitner et al. 2015, Whitehead et al. 1993
bird nests	animal heating	+/- 40°C	animal created	Korniłłowicz-Kowalska and Kitowski 2012
garden compost heaps	self-heating	50 to 70°C	animal created	Ryckeboer et al. 2003
composting plants	self-heating	50 to 70°C	animal created	Diaz 1999

Mankind has developed various types of high-temperature biological processes where the heat is artificially produced. Such technological environments, inhabited by thermophilic microbes (optimal growing conditions between 50°C-65°C) are various or ancient, notably: (i) some cheese making processes (pressed cooked cheeses), (ii) anaerobic digestion reactors (van Lier et al. 2001, Table 1).

Finally, the sun is the main source of heat on Earth, providing 3680 times more energy than the geothermal energy. Surfaces which directly receive this energy must provide a thermophilic environment but paradoxically, it does not define any known thermophilic biotopes.

We deal here with the ubiquity of thermophilic microbes but, as highlighted, there are only four origins for heat and three known biotopes, with two of them being artificial. Moreover, the only natural habitat (geothermal energy) is located in few very specific places. Thus, the biotope for the majority of thermophiles still remains unknown.

To resolve this discrepancy between presence without the known biotope, we made the assumption that the leaf surface could well be the best candidate for natural biotopes of thermophilic microbes because they are widespread all over the planet.

Several observations allow us to make the hypothesis of the phyllosphere as a thermophilic habitat. First of all, we are looking for a biotope for thermophilic organisms that are present in abundance on mesophilic environments of the Earth. Secondly, we need to find a biotope for thermophilic organisms that is not linked to the Earth’s geothermal energy. Finally, we need to find a biotope for thermophilic organisms, isolated from mesophilic environments which are directly or indirectly related to plant material and can utilise and grow on polysaccharides. This ability is incompatible with the scarcity of such polysaccharides in thermophilic biotopes, such as hot springs or deep sediments.

On the other hand, the phyllosphere ticks all the boxes as the main habitat for thermophilic microbes: firstly, leaf surfaces are everywhere: plants covert 90% of planet's land surface and the phyllosphere is estimated to exceed 10⁸ km² (Morris and Kinkel 2002). Moreover, plant leaves harbour a large number of microorganisms: the estimate is about 10²⁴ to 10²⁶ bacterial cells (Morris and Kinkel 2002) of the 10³⁰ that live on Earth (Whitman et al. 1998). Up to 10⁷ bacteria per cm² are present on leaf surfaces (Morris and Kinkel 2002, Carotenuto 2017, Lindow and Brandl 2003, Bulgarelli 2013). Secondly, leaf surfaces are heated by the sun and plants have a very low albedo and absorb a large amount of the energy from solar radiation. Plant albedo values vary widely, ranging from 0.05 in coniferous forests to 0.25 in grasslands (Coakley 2003). The main feature of heat from the sun is its variability. The heat source stops each night and its intensity during the day depends on the latitude, seasons and climate. Finally, leaves are plant material and are rich in polysaccharides.

The aim of this paper is to provide arguments in support of this hypothesis and to answer the following questions: (i) Can leaf surface provide the main biotope for thermophilic microbes? (ii) Are thermophilic microbes present on leaves? (iii) What are the ecology and the life cycle of microbes living on leaf surface hot biotope?

The thermophilic identification, based on the ability to grow at temperatures higher than 45°C, was checked in the bibliography. By using this method, 44 out of 582 OTUs identified on fresh leaf samples were close to known thermophilic bacterial species, 40/368 OTUs after aerobic incubation at 55°C and 20/168 OTUs after anaerobic incubation at 55°C (Fig. 1). Comparison of the abundances showed a complete shift of the microbial communities, with a large increase in the abundance of the identified thermophilic phylotypes from 3% on fresh leaves to 45% and 10% on aerobic incubation at 55°C and on anaerobic incubation at 55°C, respectively (Fig. 1). In fact, simply, thermophiles present, but rare (not detected), on fresh leaves grow at 55°C. Anyway, these values are only the default minimum values. The trend is stronger if we consider species described as non-thermophilic, but belonging to genera containing certain thermophilic species, from 10 to 74 and 72%, respectively (Fig. 1).

Figure 1.

Phylogenetic, geographical, environmental parameters of leaf samples and thermophilic state of OTU identification. ¹, average annual sum of solar potential; ², average temperature corresponding to the month of sampling. ³, abundance of identified OTUs are shown for each leaf sample: T+; correspond to closest species described as thermophilic or thermo-resistant; T?; correspond to closest species described as non-thermophilic but belonging to genus containing some thermophilic or thermo-resistant species; T-; correspond to closest species described as non-thermophilic or thermo-resistant or thermophilic status unknown due to a distant phylogeny or low abundance of the OTU.

These results confirm data in literature. Indeed, 16S rDNA corresponding to bacteria described as thermophilic are often found on leaf surface communities (Gandolfi 2017). Thermophilic fermentation of plant products leads to an increase in thermophiles on fermented products such as tea (Lv et al. 2013) or compost. A comparison between leaf bacteria after thermophilic incubation with bacteria found in compost (Silva et al. 2016) reveals a great similarity between the microbial compositions of compost plant and the leaf surface. Thus, leaf surface is a biotope for many thermophiles, but all microbes that inhabit leaf surfaces are not thermophiles or cannot be identified as thermophiles.

The biotope

The temperature of leaf surfaces varies with the local meteorological parameters and regularly fluctuates in accordance with the day/night alternation. In addition, even for heliophytes, only one side of the leaf is generally exposed to the sun and thanks to the sun's location, all the leaves are not exposed at once. For living leaves, the highest temperature reported on the surface was 53.1°C for a succulent plant (agave) and 50.4°C for a non-succulent (liriodendron) (Kuraishi and Nito 1980). These temperatures are highly dependent on the plant type (conifer, evergreen, herbaceous etc.) (Kuraishi and Nito 1980), as well as on environmental conditions (i.e. solar radiation, wind speed, air temperature and humidity) (Pincebourde and Casas 2015). Evapotranspiration enables plants to keep leaves below 50°C. Leaves have complex structures covered by a cuticle. The cuticle, composed of wax, reflects radiation and regulates the temperature. Furthermore, the leaves 3D structure is complex, with trichomes and stomatal cavities which, by diffusion, increase the protection against solar radiation. At the microbial scale, the actual temperature remains unknown. To sum up, temperatures on the phyllosphere are basically very variable, ranging from cold to hot and often even reaching 50°C, but in the absence of evapotranspiration on dead leaves or partially-dead leaves, the temperature may increase.

Which foods can microbes find on the leaf surface? Living plant cells are well protected and only a few specialised pathogens can penetrate to feed inside the cells. At the microbial level, however, leaf surfaces can provide water, exudates or volatile compounds (terpenoids). The vast surface area of foliage facilitates access to compounds in the air such as O₂, CO₂, CO, CH₄, volatile organic compounds or alkanes in urban pollution (Gandolfi 2017). The life cycle of leaves is, however, from living cells to dead cells. Microbes can feed from dead cells on plants (e.g. yellow dots) or dead leaves on the soil (see section: Transportation and cycle of life and Fig. 2). As examples, the production of phytase from thermophilic fungi (Singh and Satyanarayana 2009) or of cellulose from thermophilic bacteria (Acharya and Chaudhary 2012) indicates, unambiguously, the substrate of these organisms.

Figure 2.

Life cycle of thermophilic microbes associated with leaf surfaces.

The inhabitants

Candidate which can belong to the thermophilic phyllosphere?

Identification of thermophiles by experiment

Our observations show the presence of live thermophilic bacteria on the surfaces of many different leaves. However, this does not prove the hypothesis of the phyllosphere as a thermophilic biotope.

Consequently, rather than characterising a physiology (thermophilic) or a biotope (leaf surfaces), it is necessary to consider the cycle life of these organisms. Their cycle is the same as the leaf biomass, with three phases: (i) the living leaf, (ii) the decomposing leaf and (iii) the air (Fig. 2).

First, leaves are non-perennial and non-contiguous biotopes; each new leaf must be colonised by new microbes. This colonisation from old leaves or from other environments is done mainly by airborne transportation. Atmospheric depositing must be considered as essential in the ecology of these thermophilic microbes. To survive the stress of transportation (UV radiation, desiccation, cold), many thermophiles are spore-forming organisms (firmicutes, actinomyces, dycarya) (Setlow 2014).

The presence of thermophilic micro-organisms in the air can be explained by the carrying off of their putative biotope from their leaf surfaces. Thus, thermophilic microbes are present in air (Marchant et al. 2008) and transportation by air can explain the presence of thermophiles in many unexpected environments, for example, in the Arctic or Antarctic (Hubert 2009, Marchant et al. 2008). For microbes, air is an extreme environment (UV, dry, cold), but not a thermophilic environment since, due to the size of microbes, radiation from sun diffuses rapidly. Moreover, air and the phyllosphere have microbial communities with different structures (Vokou et al. 2012) and leaves cannot be just a convenient landing surface for airborne microbes. Indeed, airborne bacteria can land on leaf surfaces, but they can also land on other surfaces everywhere (Ottesen et al. 2016). Thus thermophiles can be found everywhere.

The thermophilic inhabitants of the phyllosphere must be able to cope with high temperatures coming from two origins: the sun on the surface of the leaves, but also in the soil during self-heating compost-type fermentation. These microorganisms must also be able to withstand low temperatures: (i) on the surface of the leaves during daily and seasonal variations; and (ii) in the air during their dissemination. This may explain the resistance to low and high temperatures observed in certain bacteria whose biotope is still poorly defined (Hubert 2009).

Responses to stresses such as UV radiation, reactive oxygen species (ROS) and desiccation have been identified as important functional traits of the leaf-colonising bacteria (Vorholt 2012) and metaproteomic analysis of these phyllosphere bacteria confirms this assertion (Delmotte 2009). Such stresses are similar to stresses undergone during an air journey (Potts 2005). Known thermophiles present physiological responses to various stress conditions which include the stresses within the phyllosphere (Ranawat and Rawat 2017).

The optimal growth temperature for microbes covers a broad range, from -18°C to 113°C, but not as a continuum. Two temperatures seem to be most favoured: one around 30°C-40°C (mesophilic range), a second around 50°C-60°C (thermophilic range). This observation may be biased by the sampling. However, unbiased measurements, based on cold Arctic seabed populations and on sulphate-reduction activity, also reveals mesophilic and thermophilic optima (Hubert 2009). The mesophilic range can be easily explained by the temperature prevailing in the bodies of homeothermic animals (birds and mammals). This present study provides an explanation for the thermophilic range which may correspond to the maximum temperature which occurs on leaf surfaces exposed to the sun.

The central interest in our hypothesis is that it reconsiders thermophilic microbes through their biotope and their life cycle. The phyllosphere is a carbon-rich habitat (heterotroph) which must cope with day/night variations, strong radiation and water-related stresses; this habitat is completely different from the widely-known thermophilic habitat which is stable in temperature, poor in carbon and undergoes chemical stresses (chemolithotroph), but exhibits no stresses due to water deficit or radiation. Moreover, the phyllosphere is a discontinuous habitat and the life cycle of leaves goes from living plant (from less than 6 months for annual plants or deciduous trees to several years for evergreen plants) to decayed plant on the ground. This involves for associated microbes: self-seeding by air, colonisation and resistance to the associated stresses (UV radiation and desiccation). Thus, thermophilic organisms or thermophilic enzymes from the phyllosphere will, in all probability, be more suitable for use in biotechnology (Blumer-Schuette 2012) than those counterparts from the geothermal energy habitat.

The living phyllosphere and the dead phyllosphere on the ground cover the majority of the Earth's land surface and this surface is warmed up by the sun. Physiology of many thermophilic microbes corresponds to a life associated with plant material. Literature and our experiments show the presence of thermophiles within the phyllosphere microbiota. Despite these converging facts, final proof that the phyllosphere is a major thermophilic habitat cannot be proved because: (i) the temperature conditions at leaf surfaces are not really known; (ii) the thermophilic status of the majority of environmental microbes remains unknown; (iii) thermophiles are present in the air and they are spread everywhere by the air. Nevertheless, our hypothesis does not simply offer a newly-discovered habitat for homeless thermophiles, but it also allows a different interpretation of data related to the physiology of our neighbour's thermophilic microbes and, in this way, can improve their use for biotechnology purposes.

We acknowledge the support of the PHC SIAM project (27512XJ 2012-13).

JJG: concept, acquisition, interpretation, drafting; AG: acquisition, interpretation, drafting; EL: concept, interpretation; PO: acquisition, interpretation; JPS: interpretation, drafting.

No conflicts of interest