Subaerial Biofilms Research Articles

Characterization of a biofilm and the pattern outlined by its growth on a granite-built cloister in the Monastery of San Martiño Pinario (Santiago de Compostela, NW Spain)

The upper zone of the background walls of the processional cloister of the Monastery of San Martiño Pinario (Santiago de Compostela, Galicia, Spain) is affected by a deep green, highly hydrophobic subaerial biofilm. The pattern that the biofilm follows the walls suggests that particular microclimatic conditions induce changes in the biofilm properties. To test this hypothesis, taxonomic and structural identification of the biofilm was carried out by respectively light microscopy and confocal laser scanning microscopy. In addition, the chemical composition was determined by quantification of extracellular polymeric substances in the extracellular matrix of the biofilm, and hydrophobicity was determined by contact angle and water drop penetration time of biofilm and cells. Furthermore, the bioprotective or biodeteriorative role of the biofilm on the cloister is discussed on the basis of the results of stereoscopic microscope observations, X-ray diffraction and attenuated total reflectance-Fourier transform infrared spectroscopy analysis of granite samples from uncolonized areas and from underneath the biofilm. The findings showed that Apatococcus lobatus is the predominant algae in the biofilm. The presence of this alga is favoured by water condensation and it neither damages nor protects the substrate, only causing an aesthetic impact.

The Ecology of Subaerial Biofilms in Dry and Inhospitable Terrestrial Environments.

The ecological relationship between minerals and microorganisms arguably represents one of the most important associations in dry terrestrial environments, since it strongly influences major biochemical cycles and regulates the productivity and stability of the Earth’s food webs. Despite being inhospitable ecosystems, mineral substrata exposed to air harbor form complex and self-sustaining communities called subaerial biofilms (SABs). Using life on air-exposed minerals as a model and taking inspiration from the mechanisms of some microorganisms that have adapted to inhospitable conditions, we illustrate the ecology of SABs inhabiting natural and built environments. Finally, we advocate the need for the convergence between the experimental and theoretical approaches that might be used to characterize and simulate the development of SABs on mineral substrates and SABs’ broader impacts on the dry terrestrial environment.

IR irradiation to remove a sub-aerial biofilm from granitic stones using two different laser systems: An Nd: YAG (1064 nm) and an Er:YAG (2940 nm)

A sub-aerial biofilm (SAB) developed on a granite commonly found in the built cultural heritage of the NW Iberian Peninsula was extracted with 2 different IR irradiations using an Nd:YAG laser at 1064 nm and an Er:YAG laser at 2940 nm. The methodology was based on the application of only one scan in order to evaluate the effect of the laser cleaning operated by applying different consecutive laser scanning and the suitability of these lasers as quick tools. The aim of this comparative study was twofold. The first goal was to find the most satisfactory level of extraction by comparing the results obtained by the different laser sources (IR wavelengths). The other aim was to investigate the by-effects induced by both lasers on each granite-forming mineral. Evaluations were made using stereomicroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy and scanning electron microscopy with energy-dispersive x-ray spectroscopy. The results were interpreted in terms of SAB extraction and damage induced on the granite. The results showed that the Nd:YAG laser achieved the most successful level of cleaning, because it extracted the most SAB, while causing the least amount of damage to the surfaces. Regardless of the fluence applied, the Er:YAG laser did not completely extract the SAB in only one scan; in addition, a more intense melting of biotite grains was found, producing amorphous fusion crusts and losing the distinction of the cleavage planes.

Correction to: Novel qPCR probe systems for the characterization of subaerial biofilms on stone monuments

A deep survey of biodeteriogen microorganisms reported on stone monuments in Europe has been performed based on the available literature dating back to over 30 years. The aim of the present study is to obtain accurate oligos for the characterization of subaerial biofilms on the basis of the most comprehensive collection of reports and case studies regarding subaerial biofilms, with particular regard to phototrophic and non-phototrophic bacteria, eukaryotic algae and molds. The obtained lists for eukaryotic algae, phototrophic and non-phototrophic bacteria and fungi were sorted by Genera and corresponding sequences in triplicate were downloaded by nucleotide database Genbank for a number of selected barcoding markers. On the basis of collected bibliometric diversity, multiple nucleotide alignments were produced and primers were designed for a qPCR assay. Primers were designed on conserved regions flanking a variable region, specific for each of the studied groups of microorganisms. Standard curve for absolute quantification relative to each group were determined for four markers. Then, variable regions in the alignments were used to design fluorescent internal probes for qPCR aimed for a multiplex reaction in which relative abundance can be determined. The authors propose this kind of cost-effective approach in the study of biofilms for the estimation of algae, molds and bacteria both for direct in situ analysis and in vitro simulation.

Phototrophic biofilm communities and adaptation to growth on ancient archaeological surfaces

Hypogea can be considered under-examined environments as regards microbial biodiversity. New understanding has been gained about the predominant phototrophic microorganisms forming biofilms colonising archaeological surfaces in hypogea. In fact, the description of new taxa has remained elusive until recently, as many biofilm-forming phototrophs possess a cryptic morphology with a lack of specialised cells. A multiphasic study, including cytomorphological and ecological descriptions, genetic and biochemical analysis was carried out on the biofilms colonising hypogean environments around the Maltese islands. Molecular studies were imperative because biodiversity was found to be more complex than that indicated by classical taxonomy. The dominant microbial life-form on archaeological surfaces is a compact subaerial biofilm. This study has led to new strains of the eukaryotic microalgal genus Jenufa, and the prokaryotic cyanobacteria Oculatella, Albertania and Nodosilinea being identified as the principal phototrophic biofilm-formers colonising the ancient decorated surfaces of Maltese hypogea. Complex morphologies and elaborate life cycles were eliminated as biodiversity was dictated only by the local contemporary microenvironment. The production of thick multilayered sheaths aided adherence to the substrate, concentrating microbial cells in biofilm formation. Albertania skiophila trichomes were able to glide inside the extracellular matrix. Oculatella subterranea exhibited phototaxis associated with a photosensitive apical cell containing a rhodopsin-like pigment. The biofilm provided a protective barrier and an improved chance of survival for cells growing in a low-nutrient, low-light environment. Effective strategies to prevent and control the growth of biofilms on the archaeological surface should take into consideration the adaptation of microorganisms to this particular mode of life.

Survey of relevant taxonomic groups for the design of qPCR primers and internal fluorescent probes for whole characterization of subaerial biofilm

A deep survey of biodeteriogen microorganisms reported on stone monuments in Europe has been performed based on the available literature dating back to over 30 years. The obtained lists for eukaryotic algae, phototrophic and non-phototrophic bacteria, and fungi were sorted by Genera, and corresponding sequences in triplicate were downloaded by nucleotide database Genbank for a number of selected barcoding markers. On the basis of collected bibliometric diversity, multiple nucleotide alignments were produced and primers were designed for a qPCR assay. The aim of the present study was to obtain accurate oligos for the characterization of subaerial biofilms on the basis of the most comprehensive collection of reports and case studies regarding subaerial biofilms, with particular regard to phototrophic and non-phototrophic bacteria, eukaryotic algae, and molds. Primers were designed on conserved regions flanking a variable region, specific for each of the studied groups of microorganisms. Standard curve for absolute quantification relative to each group were determined for four markers. Then, variable regions in the alignments were used to design fluorescent internal probes for qPCR aimed for a multiplex reaction in which relative abundance can be determined. The authors propose this kind of cost-effective approach in the study of biofilms for the estimation of algae, molds, and bacteria both for direct in situ analysis and in vitro simulation.

Secondary bioreceptivity of granite: effect of salt weathering on subaerial biofilm growth

Salt crystallisation is a very common and powerful weathering agent that can modify the petrophysical properties of building stone such as granite. In addition, the weathering can affect the susceptibility of the stone to biological colonisation. The aims of the present study were to examine the properties of a granite weathered by sodium chloride crystallisation and to evaluate the effects of the weathering on the secondary bioreceptivity of the stone to subaerial phototrophic biofilms. For this purpose, granite samples were subjected to a laboratory-based accelerated salt weathering test, and changes in weight, open porosity, bulk density, capillary water content, abrasion pH and surface roughness of the samples were determined. Samples of both weathered and non-weathered granite were then inoculated with a multi-species phototrophic culture derived from a natural subaerial biofilm and incubated under standardised laboratory conditions for 3 months. The weight loss produced by the weathering process was consistent with significant changes in abrasion pH and surface roughness. The bioreceptivity of the stone was also altered. According to the bioreceptivity index, the granite under study was characterised by ‘mild primary bioreceptivity’, but ‘high secondary bioreceptivity’ after the salt weathering process. Study of the secondary bioreceptivity of stone materials can provide very useful information about response to weathering effects, and the findings can be used to improve the selection of materials for building purposes.

Exposure to artificial daylight or UV irradiation (A, B or C) prior to chemical cleaning: an effective combination for removing phototrophs from granite

This study evaluated whether exposing samples of granite colonized by a natural biofilm to artificial daylight or UV-A/B/C irradiation for 48 h enhanced removal of the biofilm with a chemical product previously approved for conservation of monuments by the European Biocide Directive. Rodas granite, which is commonly found in stone-built heritage monuments in Galicia (NW Spain), was naturally colonized by a sub-aerial biofilm. The efficacy of the cleaning method was evaluated relative to uncolonized surfaces and colonized control samples without previous irradiation, treated by dry-brushing or with benzalkonium chloride. The effect of UV irradiation in the combined treatment was evident, as comparable cleaning levels were not reached in the controls. Although the biofilm was not totally removed by any of the treatments, UV-B irradiation followed by benzalkonium chloride was potentially useful for cleaning stone, with results comparable to those achieved by UV-C irradiation, which is known to have germicidal effects.

Pyrosequencing and culturing of Hawaiian corticolous biofilms demonstrate high diversity and confirm phylogenetic placement of the green alga Spongiochrysis hawaiiensis in Cladophorales (Ulvophyceae)

:Spongiochrysis hawaiiensis, a terrestrial green alga found in subaerial biofilms along the windward coast of O'ahu, was originally described as a member of the Cladophorales, a primarily marine and freshwater order, based on DNA sequence data. However, a subsequent report allied this taxon with the entirely terrestrial order Trentepohliales. Yet biofilm communities may contain multiple species of green algae, and sequences from both studies were generated from uncultured environmental samples using Sanger sequencing, rendering these results uncertain. In the current study, pyrosequencing of the SSU rDNA gene was used to determinate the algal species composition of these communities, and Sanger sequences of unialgal cultures were generated to unambiguously determine the phylogenetic placement of S. hawaiiensis. Results of the current study indicated that communities were composed primarily of ‘cladophoralean S. hawaiiensis’; numerous trentepohlialean taxa, including one that formed a clade with the previously published ‘trentepohlialean S. hawaiiensis’; and members of the Trebouxiophyceae. Abundances of the ‘cladophoralean S. hawaiiensis’ and the most abundant trentepohlialean taxon were too similar to determine which was the dominant member of the community based on pyrosequencing data alone. Phylogenetic analysis of Spongiochrysis SSU sequences from unialgal culture clearly allied the taxon within the Cladophorales, thus refuting the hypothesis that Spongiochrysis is a member of the Trentepohliales.

Roof-Inhabiting Cousins of Rock-Inhabiting Fungi: Novel Melanized Microcolonial Fungal Species from Photocatalytically Reactive Subaerial Surfaces.

Subaerial biofilms (SAB) are an important factor in weathering, biofouling, and biodeterioration of bare rocks, building materials, and solar panel surfaces. The realm of SAB is continually widened by modern materials, and the settlers on these exposed solid surfaces always include melanized, stress-tolerant microcolonial ascomycetes. After their first discovery on desert rock surfaces, these melanized chaetothyrialean and dothidealean ascomycetes have been found on Mediterranean monuments after biocidal treatments, Antarctic rocks and solar panels. New man-made modifications of surfaces (e.g., treatment with biocides or photocatalytically active layers) accommodate the exceptional stress-tolerance of microcolonial fungi and thus further select for this well-protected ecological group. Melanized fungal strains were isolated from a microbial community that developed on highly photocatalytic roof tiles after a long-term environmental exposure in a maritime-influenced region in northwestern Germany. Four of the isolated strains are described here as a novel species, Constantinomyces oldenburgensis, based on multilocus ITS, LSU, RPB2 gene phylogeny. Their closest relative is a still-unnamed rock-inhabiting strain TRN431, here described as C. patonensis. Both species cluster in Capnodiales, among typical melanized microcolonial rock fungi from different stress habitats, including Antarctica. These novel strains flourish in hostile conditions of highly oxidizing material surfaces, and shall be used in reference procedures in material testing.

Biofilm colonization of metamorphic lithotypes of a renaissance cathedral exposed to urban atmosphere

Stone architectural heritage exposed outdoor represents a challenging habitat for biological growths; nevertheless, biocolonization on heritage structure is ubiquitous and represents a major mechanism of alteration. However, the identification of specific microorganisms with known reactivity towards the stone substrate does not necessarily imply that a biodeterioration process is in progress and, in specific conditions, bioprotection effects have been highlighted as a result of colonization. The main objective of the present research is to evaluate the biofilm formation on different lithotypes exposed to similar environmental polluted conditions, and to investigate whether the presence of subaerial biofilms can be associated to an increased magnitude of deterioration of the colonized surfaces with respect to the not colonized ones. In particular, the research examines the extensive biological colonization of the stone surfaces of the façade of the Cathedral of Monza (Italy). Four metamorphic stones widely used in the façade and showing rather different compositional, mineralogical and microstructural features were studied. The state of conservation of the stones was characterized under the mineralogical and compositional point of view by X-ray diffraction and Fourier Transformed infrared analysis. The microstructure of colonized substrates and of reference not colonized ones was studied by means of optical and electron microscopy, to comparatively evaluate the damage extent and weathering patterns in both conservative conditions. The structure and the architecture of biofilms growing on different lithic surfaces were investigated by CLSM in both fluorescence and reflection modes. Captured images were analyzed for 3D reconstructions of biofilm samples. The biovolumes were also calculated to estimate the total biomass. The results indicate that the four lithotypes showed different colonization extents. However, even in presence of extensive biological growth, chemical-physical deterioration mechanisms caused by environmental exposure were largely responsible for deterioration. A relationship between compositional and surface morphological features and biocolonization was also observed.

Nodosilinea chupicuarensis sp. nov. (Leptolyngbyaceae, Synechococcales) a subaerial cyanobacterium isolated from a stone monument in central Mexico

Microorganisms of subaerial biofilms, which develop on the surface of stone monuments, have been widely studied due to their impact on Cultural Heritage deterioration. Nevertheless, these extremophile microorganisms are poorly characterised. In this study, a cyanobacterial strain isolated from an archaeological zone in north-central Mexico was characterised through a polyphasic study based on ecological, morphological, and molecular data. Phylogenetic analyses were done using sequences of the 16S rRNA gene and the 16S-23S ITS region. Based on the polyphasic data, a new species corresponding to the genus Nodosilinea is described. This alkaliphilic species has optimum growth at pH of 9. Its fatty acid profile indicates that C18:1, 9E and C16:0 are its most abundant fatty acids. Extracellular polymeric substance production was verified on this strain. Only a few species of the genus Nodosilinea have been documented and described, and this is the first report of their presence in central Mexico. Hence the name Nodosilinea chupicuarensis sp. nov. is proposed.

Corrosive extracellular polysaccharides of the rock-inhabiting model fungus Knufia petricola

Melanised cell walls and extracellular polymeric matrices protect rock-inhabiting microcolonial fungi from hostile environmental conditions. How extracellular polymeric substances (EPS) perform this protective role was investigated by following development of the model microcolonial black fungus Knufia petricola A95 grown as a sub-aerial biofilm. Extracellular substances were extracted with NaOH/formaldehyde and the structures of two excreted polymers studied by methylation as well as NMR analyses. The main polysaccharide (~ 80%) was pullulan, also known as α-1,4-; α-1,6-glucan, with different degrees of polymerisation. Αlpha-(1,4)-linked-Glcp and α-(1,6)-linked-Glcp were present in the molar ratios of 2:1. A branched galactofuromannan with an α-(1,2)-linked Manp main chain and a β-(1,6)-linked Galf side chain formed a minor fraction (~ 20%). To further understand the roles of EPS in the weathering of minerals and rocks, viscosity along with corrosive properties were studied using atomic force microscopy (AFM). The kinetic viscosity of extracellular K. petricola A95 polysaccharides (≈ 0.97 × 10−6 m2 s−1) ranged from the equivalent of 2% (w/v) to 5% glycerine, and could thus profoundly affect diffusion-dominated processes. The corrosive nature of rock-inhabiting fungal EPS was also demonstrated by its effects on the aluminium coating of the AFM cantilever and the silicon layer below.

Influence of the properties of granitic rocks on their bioreceptivity to subaerial phototrophic biofilms

As any stone substrate is susceptible to biological colonisation, the choice of lithotype used for construction is a key strategy for preventing biodeterioration. For this purpose, a comprehensive evaluation of the primary bioreceptivity to phototrophic biofilms of eleven varieties of granitic rocks, commonly used as building material, was carried out. Blocks were inoculated with a multi-species phototrophic culture and subjected to standardised growth conditions for three months. Biofilm formation was assessed by chlorophyll (chl) fluorescence, colour measurements and extracellular polymeric substances (EPS) quantification. Relationships between the biofilm growth indicators and the properties of the different rocks studied were then analysed. Results showed that the bioreceptivity of the granites is more strongly affected by the physical characteristics of the stones than by their chemical and mineralogical properties, possibly because of the similar composition of the rocks studied. Growth of phototrophic biofilms was enhanced by high open porosity, capillary water content and surface roughness, and the bioreceptivity of weathered granites was higher than that of sound granites. The results obtained can therefore help in the selection of appropriate lithotypes for building purposes. The amounts of EPS produced by subaerial biofilms primarily depended on the requirements and/or characteristics of the biofilm-forming microorganisms, rather than on the bioreceptivity of the substratum, and microorganisms produce the amounts of EPS required at the initial stage of establishment on the stone surface, independently of the subsequent biomass development. These findings are especially important from the point of view of biodeterioration, in which the EPS matrix plays a central role.

A standardized approach for co-culturing dothidealean rock-inhabiting fungi and lichen photobionts in vitro

Black, rock inhabiting fungi (RIF) are polyextremotolerant, oligotrophic organisms which colonize bare rocks and are specialized to grow in niches precluded to other microorganisms in the harshest environments. In many cases RIF share this environment with green algae and cyanobacteria forming subaerial biofilms; some of them have also been found to be associated with lichen thalli. The RIF genus Lichenothelia is of particular interest because it includes lichen parasites and species which are loosely associated with algae or grow independently on rocks. Here, in vitro culture experiments studied the development of three Lichenothelia species when co-cultured with two different lichen photobionts on growth media differing in nutrient content. The growth rates of these fungi were statistically evaluated and the structure of the mixed cultures was analyzed by light and scanning electron microscopy. The results show that the presence of algae neither influence the growth rate of fungi nor the formation of any lichen-like structure; tight contacts between the hyphae and the algal cells were also not detected. Since multiple trials were carried out on well characterized species of microcolonial fungi and the methodological procedures were established, standardized and coupled with morphological analyses, this approach proves suitable for future investigations on fungal-algal interactions in other systems.

Biofilm forming microorganisms on various substrata from greenhouse of Botanical Garden “Jevremovac”

Diversity of subaerial biofilm forming cyanobacteria, algae and fungi was investigated on 10 different substrata from greenhouse of Botanical Garden ?Jevremovac?. Out of 37 documented taxa, 16 cyanobacterial and 10 algal taxa were identified. Remaining 11 taxa belong to the Kingdom of Fungi. The highest diversity of biofilm forming microorganisms, a total of 24 taxa, was detected on the corroded metal surface, while significantly lower number of taxa was recorded on other examined substrata. Cyanobacterium Porphyrosiphon sp., diatom Achnanthes sp. and green algae Chlorella sp. and Chlorococcum minutum were the most frequently encountered photosynthetic components of biofilms. In all analyzed samples, Trichoderma sp., followed by Cladosporium sp. and Rhizopus stolonifer, were the most frequently identified fungi.

Laboratory grown subaerial biofilms on granite: application to the study of bioreceptivity

Simulated environmental colonisation of granite was induced under laboratory conditions in order to develop an experimental protocol for studying bioreceptivity. The experimental set-up proved suitable for producing subaerial biofilms by inoculating granite blocks with planktonic multi-species phototrophic cultures derived from natural biofilms. The ability of four different cultures to form biofilms was monitored over a three-month growth period via colour measurements, quantification of photosynthetic pigments and EPS, and CLSM observations. One of the cultures under study, which comprised several taxa including Bryophyta, Charophyta, Chlorophyta and Cyanobacteria, was particularly suitable as an inoculum, mainly because of its microbial richness, its rapid adaptability to the substratum and its high colonisation capacity. The use of this culture as an inoculum in the proposed experimental set-up to produce subaerial biofilms under laboratory conditions will contribute to standardising the protocols involved, thus enabling more objective assessment of the bioreceptivity of granite in further experiments.

Response surface optimization of a method for extracting extracellular polymeric substances (EPS) from subaerial biofilms on rocky substrata.

The aim of the present study was to optimize a protocol for extracting extracellular polymeric substances (EPS) from biofilms on rocky substrata, as the EPS matrix is considered key to understanding the biofilm mode of life. For this purpose, we tested the extraction efficacy of NaOH and H2SO4 at different concentrations, temperatures and times for obtaining EPS from multi-species subaerial biofilms grown on granite blocks under laboratory conditions. Two experimental designs (Box-Behnken design and full factorial design) were used in testing each extractant. The extraction efficiency was determined by analysing the carbohydrate, protein and DNA contents of the extracts obtained. H2SO4 proved unsuitable as an extractant as it caused excessive cell lysis. However, response surface optimization of NaOH-mediated extraction enabled cell lysis to be minimized. Confirmation experiments were performed under the optimal conditions established and a protocol for extracting EPS is proposed, yielding the first quantitative data on EPS extracted from subaerial biofilms developed on rocky substrata. Graphical abstract Development of a method for extracting EPS from subaerial biofilms on rocky substrata.

Subaerial biofilms on granitic historic buildings: microbial diversity and development of phototrophic multi-species cultures

Microbial communities of natural subaerial biofilms developed on granitic historic buildings of a World Heritage Site (Santiago de Compostela, NW Spain) were characterized and cultured in liquid BG11 medium. Environmental barcoding through next-generation sequencing (Pacific Biosciences) revealed that the biofilms were mainly composed of species of Chlorophyta (green algae) and Ascomycota (fungi) commonly associated with rock substrata. Richness and diversity were higher for the fungal than for the algal assemblages and fungi showed higher heterogeneity among samples. Cultures derived from natural biofilms showed the establishment of stable microbial communities mainly composed of Chlorophyta and Cyanobacteria. Although most taxa found in these cultures were not common in the original biofilms, they are likely common pioneer colonizers of building stone surfaces, including granite. Stable phototrophic multi-species cultures of known microbial diversity were thus obtained and their reliability to emulate natural colonization on granite should be confirmed in further experiments.

Laboratory Tools to Quantify Biogenic Dissolution of Rocks and Minerals: A Model Rock Biofilm Growing in Percolation Columns

Sub-aerial biofilms (SAB) are ubiquitous, self-sufficient microbial ecosystems found on mineral surfaces at all altitudes and latitudes. SABs, which are the principal causes of weathering on exposed terrestrial surfaces, are characterised by patchy growth dominated by associations of algae, cyanobacteria, fungi and heterotrophic bacteria. A recently developed in vitro system to study colonisation of rocks exposed to air included two key SAB participants - the rock-inhabiting ascomycete Knufia petricola (CBS 123872) and the phototrophic cyanobacterium Nostoc punctiforme ATCC29133. Both partners are genetically tractable and we used them here to study weathering of granite, K-feldspar and plagioclase. Small fragments of the various rocks or minerals (1 to 6 mm) were packed into flow-through columns and incubated with 0.1% glucose and 10 µM thiamine-hydrochloride (90 µL.min-1) to compare weathering with and without biofilms. Dissolution of the minerals was followed by: analysing (i) the degradation products in the effluent from the columns via Inductively Coupled Plasma Spectroscopy and (ii) by studying polished sections of the incubated mineral fragment/grains using scanning electron microscopy, transmission electron microscopy and energy dispersive X-ray analyses. K. petricola/N. punctiforme stimulated release of Ca, Na, Mg and Mn. Analyses of the polished sections confirmed depletion of Ca, Na and K near the surface of the fragments. The abrupt decrease in Ca concentration observed in peripheral areas of plagioclase fragments favoured a dissolution-reprecipitation mechanism. Percolation columns in combination with a model biofilm can thus be used to study weathering in closed systems. Columns can easily be filled with different minerals and biofilms, the effluent as well as grains can be collected after long-term exposure under axenic conditions and easily analysed.