What are the growth kinetics and biochemical compositions of microalgae isolated from diverse aquatic ecosystems in Morocco, France, and Tunisia?

Thirty-six microalgae belonging to five taxonomic groups (Cyanobacteria, Chlorophyceae, Diatomophyceae, Euglenophyceae, and Eustigmatophyceae) were identified from six freshwater ecosystems in Morocco, two treatment stations in Etueffont landfill in France and three hot spring waters in Tunisia. Investigations on species growth kinetics and growth rates showed that the cyanobacterium Leptolyngbya gelatinosa exhibited both the highest biomass and growth rate with 4 g DW L-1 and 0.282 day-1, respectively. A significant protein production (more than 40% DW) was observed across the studied species. Cyanobacteria and chlorophytes stood out for their increased protein production with a maximum (66.63 ± 3.84% DW) attained by the cyanobacterium Leptolyngbya sp. Chlorophytes produced substantial amounts of carbohydrates (more than 20% DW). Euglenophytes including Phacus orbicularis and Euglena ehrenbergii along with the chlorophyte Graesiella sp. accumulated significant amounts of lipids (up to 31.12% DW).

Gold modification by reduction of a diazonium salt prepared from an aliphatic diamine: a new useful means to remove hazardous substances.

We studied the electrochemical reduction based on gold electrode of a diazonium salt prepared from ethylenediamine. This is the first time where the covalent functionalization on the gold electrode of an alkyldiazonium salt, 2-aminoethane-1-diazonium chloride, is demonstrated. This step requires the preparation beforehand by diazotization of one amine group from ethylenediamine. The resulting electrodeposited ethylamine film was confirmed by spectroscopic characterizations from gold surface modification monitored by electrochemical quartz crystal microbalance (EQCM) coupled to cyclic voltammetry (CV). The development of chemosensors based on such a covalent functionalization of a metal can reduce the chemical threats to human health along with drastically removing contaminants according to the green chemistry principles.

Growth, pigment changes, and photosystem II activity in the aquatic macrophyte Lemna minor exposed to bisphenol A.

As a result of its high production, bisphenol A (BPA) has become ubiquitous in aquatic and terrestrial habitats. In this study, we investigated the toxicity of BPA at 10 mg L-1 on Lemna minor after 7 days of exposure under controlled conditions according to ISO 20079. BPA statistically reduced the total frond number and frond area, while frond number per colony was significantly elevated in BPA-treated group. However, no change was recorded in root number, while root length was significantly reduced by BPA. BPA also decreased the content of Chl a, Chl b, Chl a + b, and carotenoid by 36%, 44%, 38%, and 32%, respectively, versus the control leading to a decrease in the quantum yield of photosystem II. In addition, non-photochemical quenching (NPQ) values were 2.4- and 4.5-fold higher in light than in dark conditions for control and BPA-treated plants, respectively. Thus, there is a significant activation (61.8%; p<0.01) of PSII photoprotection mechanism (NPQ) in BPA-treated plants compared to control but without removing the negative effect of BPA on PSII. The total amount of soluble sugars was reduced by 40% compared to control, and starch accumulation was mainly observed in fronds exposed to BPA. Even if the response patterns of Lemna minor based on fresh and dry weight measurements were less sensitive in our experiment conditions, further studies should be addressed since BPA represents a threat to the dynamic equilibrium governing aquatic ecosystems.

Factors inducing bryophyte growth on prehistoric pigments and effect of UV-C treatment.

In La Glacière cave (France), the touristic activity has been conducted to an environmental parameter change that has led to photosynthetic organism proliferation (microalgae, diatoms, cyanobacteria, bryophytes). The present study is focused on bryophyte development occurring in the show cave that was responsible of limestone biodeterioration. In order to understand the colonization process of limestone, we have maintained limestone blocks under optimal Lampenflora growth conditions. Moreover, some limestone blocks were painted with several pigments that were used in the prehistory (e.g., red ocher, bone char). Microorganisms and bryophyte growth were monitored during 1 year, and then, the block samples were treated using UV-C light (254 nm). Thus, obtained results were compared with in situ treatment in La Glacière cave. Results have showed dense bryophyte propagation on the several blocks. However, the growth rate was correlated with the chemical composition of the pigment. In fact, the presence of some chemical elements such as As, Cr, Ti, and Co contributed to reduce bryophyte growth. Finally, moss treatment using UV-C light has demonstrated high efficiency under in situ condition, while a fast recolonization has been observed for samples maintained in laboratory. This difference was explained by the high bryophyte density under laboratory conditions that make UV-C light penetration difficult.

Influence of hydro- and osmo-priming on sunflower seeds to break dormancy and improve crop performance under water stress.

This study explored the effects of two hydro- and osmo-priming durations (8 and 16 h) on growth and yield components of sunflowers (Helianthus annuus L.) under water stress. The pot experiment, performed under a rain shelter, consisted of 9 treatments replicated five times: unprimed seeds as control (C), hydro-primed seeds (T0), osmo-primed seeds in 10, 20, and 30% PEG-6000 (T1, T2, and T3, respectively). Severe water stress was applied for 12 days to all treatments at the beginning of the flowering stage. Statistical analysis revealed a very highly significant positive effect (p < 0.01) by all treatments on sunflower seed germination compared with the control. Moreover, primed seeds improved significantly for all growth parameters and yield components, but no significant differences were observed according to either priming technique or duration. The highest value of germination capacity, for fresh and dry biomasses, was obtained with PEG-primed seeds at 10% for 16 h. The grain number per anthodium and grain yield per plant from primed seeds were higher than those in the control (1.9- to 2.5-fold and 2.8- to 3.3-fold respectively). Under conditions of water stress, the proline content in primed plants was significantly higher than that in unprimed ones, with the exception of T3 treatment primed for 8 h. Soluble sugars and chlorophyll contents increased significantly with all applied treatments compared with the control. The study showed that the applied priming treatments improved germination characteristics in particular and increased growth and yield components for sunflowers under drought stress conditions.

Wheat biological responses to stress caused by cadmium, nickel and lead.

Several stressors like different types of heavy metals are found in the soil and can affect the growth and genomic integrity of wheat grains (Triticum aestivum L.). The aim of this study was to compare the effect of exogenous Cd (30, 60, 120 mg kg-1), Ni (50, 100 and 150 mg kg-1) or Pb (100, 200 and 300 mg kg-1) on wheat agronomic characteristics through the assessment of oxidative stress indices at protein and gene expression levels, photosynthetic pigments and genetic aberrations using RAPD analysis that were studied during two winter seasons (2015/2016 and 2016/2017). The results showed that all stressors significantly decreased the vegetative growth parameters, altered the activities of antioxidants enzymes in seedlings (after 30 days) and grains (after 5 months) and differently affected their expression levels in seedlings leaves and roots. Pb treated plants showed the poorest agronomic characteristics as it exhibited the worst affected wheat height, number of tillers, fresh and dry weight, flag leaf area as well as yield. Pb treatment caused poorest plant performance, it showed the highest proline content, least protein and chlorophyll contents, thus affects the overall plants growth followed by Cd and Ni, respectively. Furthermore, high Pb and Cd doses revealed highest degree of polymorphism and lowest degree of genome stability. Altogether, heavy metals accumulated mainly in wheat straw and induced genotoxic effect which consequently altered normal plant metabolism and pigment content which resulted in a significant reduction in wheat yield and quality. Moreover, Pb induced more genotoxic and phytotoxic effects than Cd and Ni.

Assessment of fungi proliferation and diversity in cultural heritage: Reactions to UV-C treatment.

Fungi are present in natural and non-touristic caves due to the presence of organic matter provided mainly by insects or animals such as bats. In show caves, however, tourist infrastructure and the visitors themselves are an important source of organic matter. In addition, photosynthetic biofilms provide a high amount of carbon and nitrogen sources for fungi. This study was conducted to identify the fungal communities present in caves along with the potential use of UV-C treatment against their proliferation. Thus, fungal communities proliferating in biofilms in six French and Swiss show caves were analyzed using high throughput sequencing. The results show 385 species recorded, some of them previously described in cases of fungal outbreak. This preliminary study also aimed to test the use of UV-C light as an environmentally friendly method to treat fungal proliferation. Six fungal strains, from three different sources (Lascaux cave, La Glacière cave, a church in Vicherey, France), were cultivated in an agar dish. Spores, mycelia and the entire colony were irradiated using several UV-C intensities. Results showed that four of the six fungi spores and mycelium died following a low-intensity UV-C treatment (2 kJ m-2, 160 s), though Ochroconis lascauxensis and Penicillium bilaiae spores showed higher resistance. Finally, it was demonstrated that the fungal colony could resist the UV-C light due to a shadow effect. The structure of the fungal colony was affected from the periphery to its inner part. However, after four 30 kJ m-2 treatments (39 min irradiation) all strains there definitively eradicated. Further studies will be necessary to examine the potential of UV-C light under cave conditions as a preventive and curative treatment.

Influence of different sludge compositions on understorey vegetation in an amended Pinus pinatser forest plantation.

The present study reports on the influence of sludge application on understory abundance and species richness in an eight year-old maritime pine forest. Four types of sludge (liquid, dewatered, limed and composted sludge) were applied at a rate equivalent to 3 tons dry weight per ha-1 per year-1. Understorey vegetation was monitored before treatment and for three successive years following initial sludge application. Species richness, the biodiversity index and naturally occurring plant community cover in maritime pine forests were measured. Results showed a significant increase in species richness two years following initial sludge application. In fact, compared to the unamended plot, eighteen additional species (Aira caryophyllea, Cerastium glomeratum, Conyza canadensis, Danthonia decumbens, Geranium robertianum, Gnaphalium sylvaticum, Hypericum humifusum, Hypericum perforatum ssp. perforatum, Jasione montana, Lonicera periclymenum ssp. periclymenum, Ornithopus compressus, Phytolacca americana, Rhamnus frangula, Teesdalia nudicaulis, Veronica arvensis, Vicia sativa angustifolia and Vulpia myuros) appeared after sludge application. Most of these species were observed with the solid limed or composted sludge treatments. The new species then declined the following year, highlighting the temporary effect of sludge treatment on species diversity. However, the cover by these eighteen new species was low and did not exceed 1%, except for Hypericum perforatum and Jasione montana for the limed sludge treatment and Vulpia myuros for the liquid sludge treatment. Additionally, sludge significantly reduces bare soil percentage, which ranged from 5 to 18% compared to the control (38%). According to sludge type, treatment also led to a significant change in species dominance of the understorey plant communities. Indeed, the cover of Molinia caerulea decreased in spite of an increase in Agrostis capillaris and/or Holcus lanatus following application of solid limed, liquid or composted sludge.

Biofilm biodiversity in French and Swiss show caves using the metabarcoding approach: First data.

In recent decades, show caves have begun to suffer from microorganism proliferation due to artificial lighting installations for touristic activity. In addition to the aesthetic problem, light encourages microorganisms that are responsible for physical and chemical degradation of limestone walls, speleothems and prehistoric paintings of cultural value. Microorganisms have previously been described by microscopy or culture-dependent methods, but data provided by new generation sequencing are rare. The authors identified, for the first time, microorganisms proliferating in one Swiss and in four French show caves using three different primers. The results showed that both photosynthetic and non-photosynthetic bacteria were the dominant taxa present in biofilms. Microalgae were heavily represented by the Trebouxiophyceae, Eustigmatophyceae and Chlorophyceae groups. Twelve diatoms were also recorded, with dominance of Syntrichia sp. (96.1%). Fungi were predominantly represented by Ascomycota, Zygomycota and Basidiomycota, fully half of the sampled biofilms where Fungi were detected. Comparing microbial communities from bleach-treated caves to those in untreated caves showed no significant difference except for a low-level change in the abundance of certain taxa. These findings provided by Illumina sequencing reveal a complex community structure in the 5 caves based on the assembly of bacteria, cyanobacteria, algae, diatoms, fungi and mosses.

Effects of sodium chloride salinity on ecophysiological and biochemical parameters of oak seedlings (Quercus robur L.) from use of de-icing salts for winter road maintenance.

Salt is widely used to melt snow on roads especially in mountain regions. Whether as rock salt or aerosols, spread or sprayed over road surfaces, salt may result in increased salt concentrations in soils, which, in turn, affect natural vegetation, especially tree seedlings already subjected to various other types of abiotic stress. The authors investigated the effects of salt treatment-related stress on seedling growth and certain biochemical parameters in Quercus robur to determine ion concentrations in root tips. Seedlings growing in a quartz sand/vermiculite mixture were subjected to NaCl concentrations of 0, 50, or 100 mM for 5 weeks. The results showed that high NaCl concentrations caused a marked reduction in total leaf biomass 55 and 75% for 50 and 100 mM treatments, respectively, in dry weight of stems (84%) and roots (175%) for 100 mM treatment and modified root architecture, whereas no changes appeared in leaf number. A non-significant decrease in relative water content, with changes in ion balance was recorded. Comparison of stressed to control plants show an increase in sodium (3.5-8-fold), potassium (0.6-fold), and chloride (9.5-14-fold) concentrations in the root tips while the K+/Na+ ratio decreased. In taproots, no significant biochemical differences were observed between the salt-treated and the control plants for acid invertase activity, reducing sugars, sucrose, or soluble protein contents. The significance of ion and sugar accumulations in relation to osmotic adjustment and the ability of oak seedlings to cope with salt stress are discussed.

Bleaching of biofilm-forming algae induced by UV-C treatment: a preliminary study on chlorophyll degradation and its optimization for an application on cultural heritage.

Green microalgae colonizing stone surfaces represent a major problem for the conservation of heritage monuments, since they lead to biodegradation and aesthetic issues. Previous studies in La Glacière show cave (France) have demonstrated that UV-C may have a strong effect on microalgae, thus leading to chlorophyll bleaching, which was increased when biofilms were maintained under VIS-light condition unlike to those maintained in the dark. To understand the physiological mechanisms underlying this response and in order to optimize in situ treatment, 30 kJ m-2 UV-C exposure times were applied to Chlorophyta Chlorella sp. and chlorophyll degradation kinetics were then monitored. UV-C irradiation was enough to inhibit photosynthesis and to directly kill all algal cells. Results also showed that chlorophyll a was degraded faster than chlorophyll b and that 14 h were necessary for complete degradation of all the present chlorophyll. In addition, our results highlighted the importance of visible light exposition after UV-C treatment which leading to chlorophyll bleaching. Irradiated algae cultivated in the dark were still green 5 days after treatment while cultivated samples in the light lost their green color after 14 h. An efficient UV-C treatment applicable to show caves and other heritage monuments was proposed.

Potential vulnerability of oak forests to climate change-induced flooding: effects of mild oxygen deficiency on Quercus robur and Quercus petraea seedling physiology.

Flooding is characterized by saturation of soil pores with water, leading to hypoxic conditions which affect plant root development and metabolism. We investigated the oxygen deficiency tolerance observed in Quercus robur and Quercus petraea and seek to understand whether it can be explained by enhanced efficiency in oxygen use in the roots, as estimated through radial oxygen loss visualization in relation to growth measurements and root apex respiration. The study showed that root growth, under oxygen deficiency conditions, was significantly reduced only in Q. robur seedlings. Root respiration was maintained in Q. robur, whereas it was decreased in Q. petraea. Both species set up a barrier against radial oxygen loss, though measurement of apex oxygen leakage showed greater oxygen efficiency in Q. robur seedlings. This strategy might allow Q. robur to maintain its respiration and thus to survive longer under oxygen deficiency conditions by facilitating the seedling establishment in transient flooded soils.

UV-C as an efficient means to combat biofilm formation in show caves: evidence from the La Glacière Cave (France) and laboratory experiments.

Ultra-violet C (UV-C) treatment is commonly used in sterilization processes in industry, laboratories, and hospitals, showing its efficacy against microorganisms such as bacteria, algae, or fungi. In this study, we have eradicated for the first time all proliferating biofilms present in a show cave (the La Glacière Cave, Chaux-lès-Passavant, France). Colorimetric measurements of irradiated biofilms were then monitored for 21 months. To understand the importance of exposition of algae to light just after UV radiation, similar tests were carried out in laboratory conditions. Since UV-C can be deleterious for biofilm support, especially parietal painting, we investigated their effects on prehistoric pigment. Results showed complete eradication of cave biofilms with no algae proliferation observed after 21 months. Moreover, quantum yield results showed a decrease directly after UV-C treatment, indicating inhibition of algae photosynthesis. Furthermore, no changes in pigment color nor in chemical and crystalline properties has been demonstrated. The present findings demonstrate that the UV-C method can be considered environmentally friendly and the best alternative to chemicals. This inexpensive and easily implemented method is advantageous for cave owners and managers.

UV-C as a means to combat biofilm proliferation on prehistoric paintings: evidence from laboratory experiments.

A laboratory investigation of UV-C effects was conducted over a 62-h period: a much higher dose than in classic UV-C treatment was applied to five pigments and two painting binders used by prehistoric humans. Colorimetric parameters were compared to a control to see if UV-C can change pigment and binder color. Infrared spectroscopy, scanning electron microscopy, inductively coupled plasma and X-ray crystallography were also carried out to confirm colorimetric measurement. In order to understand how microorganism may physically deteriorate paintings, limestone blocks were painted and monitored until their complete colonization by algae, cyanobacteria, fungi and/or mosses. The results show that UV-C has no effect on mineral compounds. Conversely, it is noteworthy that binder color changed under both UV-C light conditions as well as in visible light. Concerning painted blocks, a fast proliferation has been observed with deterioration of the paintings. These results show the high importance of treating biofilm as soon as possible. Moreover, these findings may be a promising avenue inducing cave managers to use friendly UV-C light to treat contaminated cave paintings and also in the prevention of biodeterioration by lampenflora.

Effects of repeated soil irrigation with liquid biological paper sludge on poplar Populus alba saplings: potential risks and benefits.

The authors explored the risks and benefits of repeated irrigation of Populus alba saplings with aqueous paper sludge (APS). Saplings were cultivated in pots of forest soil (3 L) in a greenhouse for 7 weeks and watered twice a week with differing concentrations of APS (0, 10, 20, 30, 50, 75, and 100 % v/v with deionized water). Plant growth and ecophysiological variables along with zinc and aluminum transfer were monitored. A stimulation of plant growth was observed with sludge treatments of 30 or 50 %, significantly correlated to APS input (r = 0.81). This may be explained by the easily available nitrogen as is shown with the positive correlation of CO2 assimilation and leaf nitrogen (r = 0.70). However, a significant reduction in plant growth was observed when treatments of 75 and 100 % of APS were administered, despite a high nutritional level (nitrogen and phosphorus). The study suggests that APS concentrations from 30 to 50 % may positively affect the growth of poplar saplings; however, the higher concentrations indicated a risk for plant growth and the environment.

Microbial composition and ecological features of phototrophic biofilms proliferating in the Moidons Caves (France): investigation at the single-cell level.

The authors investigated the microbial composition of phototrophic biofilms proliferating in a show cave using flow cytometry for the first time in such a context. Results are based on several biofilms sampled in the Moidons Caves (France) and concern both heterotrophic prokaryotes and autotrophic microorganisms. Heterotrophic microorganisms with low nucleic acid content were dominant in biofilms, as can be expected from the oligotrophic conditions prevailing within the cave. Analysis of the biofilm autotrophic components revealed the presence of several taxa, particularly the unicellular green algae Chlorella minutissima, specifically well adapted to this cave. Relationships between flow cytometry results and environmental variables determined in the cave were established and discussed so as to better understand biofilm proliferation processes in caves.

Effects of sewage sludge amendment on snail growth and trace metal transfer in the soil-plant-snail food chain.

Cu, Zn, Pb, and Cd concentrations in a soil plant (Lactuca sativa) continuum were measured after sewage sludge amendment. The effects of sewage sludge on growth and trace metal bioaccumulation in snails (Cantareus aspersus) were investigated in a laboratory experiment specifically designed to identify contamination sources (e.g., soil and leaves). Application of sewage sludge increased trace metal concentrations in topsoil. However, except Zn, metal concentrations in lettuce leaves did not reflect those in soil. Lettuce leaves were the main source of Zn, Cu, and Cd in exposed snails. Bioaccumulation of Pb suggested its immediate transfer to snails via the soil. No apparent toxic effects of trace metal accumulation were observed in snails. Moreover, snail growth was significantly stimulated at high rates of sludge application. This hormesis effect may be due to the enhanced nutritional content of lettuce leaves exposed to sewage sludge.

Sewage sludge application in a plantation: effects on trace metal transfer in soil-plant-snail continuum.

We studied the potential bioaccumulation of Cu, Zn, Pb and Cd by the snail Cantareus aspersus and evaluated the risk of leaching after application of sewage sludge to forest plantation ecosystems. Sewage sludge was applied to the soil surface at two loading rates (0, and 6 tons ha(-1) in dry matter) without incorporation into the soil so as to identify the sources of trace metal contamination in soil and plants and to evaluate effects on snail growth. The results indicated a snail mortality rate of less than 1% during the experiment, while their dry weight decreased significantly (<0.001) in all treatment modalities. Thus, snails showed no acute toxicity symptoms after soil amendment with sewage sludge over the exposure period considered. Additions of sewage sludge led to higher levels of trace metals in forest litter compared to control subplots, but similar trace metal concentrations were observed in sampling plants. Bioaccumulation study demonstrated that Zn had not accumulated in snails compared to Cu which accumulated only after 28 days of exposure to amended subplots. However, Pb and Cd contents in snails increased significantly after 14 and 28 days of exposure in both the control and amended subplots. At the last sampling date, in comparison to controls the Cd increase was higher in snails exposed to amended subplots. Thus, sludge spread therefore appears to be responsible for the observed bioaccumulation for Cu and Cd after 28days of exposure. Concerning Pb accumulation, the results from litter-soil-plant compartments suggest that soil is this metal's best transfer source.

Heritage materials and biofouling mitigation through UV-C irradiation in show caves: state-of-the-art practices and future challenges.

Biofouling, i.e., colonization of a given substrate by living organisms, has frequently been reported for heritage materials and particularly on stone surfaces such as building facades, historical monuments, and artworks. This also concerns subterranean environments such as show caves, in which the installation of artificial light for tourism has led to the proliferation of phototrophic microorganisms. In Europe nowadays, the use of chemicals in these very sensitive environments is scrutinized and regulated by the European Union. New and environmentally friendly processes must be developed as alternative methods for cave conservation. For several years, the UV irradiation currently used in medical facilities and for the treatment of drinking water has been studied as a new innovative method for the conservation of heritage materials. This paper first presents a review of the biofouling phenomena on stone materials such as building facades and historical monuments. The biological disturbances induced by tourist activity in show caves are then examined, with special attention given to the methods and means to combat them. Thirdly, a general overview is given of the effects of UV-C on living organisms, and especially on photosynthetic microorganisms, through different contexts and studies. Finally, the authors' own experiments and findings are presented concerning the study and use of UV-C irradiation to combat algal proliferation in show caves. Both laboratory and in situ results are summarized and synthesized from their previously published works. The application of UV in caves is discussed and further experiments are proposed to enhance research in this domain.

Factors driving epilithic algal colonization in show caves and new insights into combating biofilm development with UV-C treatments.

The proliferation of epilithic algae that form biofilms in subterranean environments, such as show caves, is a major problem for conservators. In an effort to reduce the use of chemical cleansers when addressing this problem, we proposed investigating the effects of UV-C on combating algal biofilm expansion in a cave located in northeastern France (Moidons Cave). First, the biofilms and cavity were studied in terms of their algal growth-influencing factors to understand the dynamics of colonization in these very harsh environments. Next, colorimetric measurements were used both to diagnose the initial colonization state and monitor the UV-C-treated biofilms for several months after irradiation. The results indicated that passive dispersal vectors of the viable spores and cells were the primary factors involved in the cave's algae repartition. The illumination time during visits appeared to be responsible for greater colonization in some parts of the cave. We also showed that colorimetric measurements could be used for the detection of both thin and thick biofilms, regardless of the type of colonized surface. Finally, our results showed that UV-C treatment led to bleaching of the treated biofilm due to chlorophyll degradation even one year after UV-C treatment. However, a re-colonization phenomenon was colorimetrically and visually detected 16months later, suggesting that the colonization dynamics had not been fully halted.