Potential of cyanobacterial secondary metabolites as biomarkers for paleoclimate reconstruction

Potential of cyanobacterial secondary metabolites as biomarkers for paleoclimate reconstruction

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Contents lists available at ScienceDirect

Catena journal homepage: www.elsevier.com/locate/catena

Potential of cyanobacterial secondary metabolites as biomarkers for paleoclimate reconstruction ⁎

Dijana Lalića, , Jussi Meriluotoa,b, Miroslav Zorićc, Tamara Dulića,b, Milan Mirosavljevićc, Milan Župunskia, Zorica Svirčeva,b a

LAPER, University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland c Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia b



Keywords: Cyanobacteria Scytonemin Mycosporine-like amino acids Double-centered interaction matrix analysis Biomarkers Loess Paleoclimate reconstruction

Loess is the most important archive of Quaternary palaeoclimate evolution, with more thorough and systematic investigations carried out in the past two decades in the Carpathian Basin. Application of novel proxies in loess research could improve the state of knowledge of the past climatic changes. In order to examine the feasibility of cyanobacterial pigments to be used as biomarkers in paleoclimate reconstruction and thereby substantiate the presence of cyanobacterial community during loess accumulation, geochemical evidence of cyanobacteria-specific biomarkers in the sediment is required. In this study structurally different cyanobacterial metabolites were examined for their potential to be used as biomarkers. These compounds included scytonemin wavelength equivalent (SWE) and mycosporine wavelength equivalent (MWE) compounds. The effect of various physico-chemical factors (pH value, temperature and light source) on the production of SWE and MWE compounds in correlation with the nitrogen content of the growth medium was studied. SWE compounds were observed in 8 out of 15 soil and aquatic cyanobacterial strains, while MWE compounds were found in all 15 strains. The results show that exposure to UV light induced a higher synthesis of both pigments. Moreover, the presence of SWE compounds was confirmed in cyanobacterial cultures isolated from biological loess crusts (BLCs) as well as in BLC and loess sediment samples. The potential application of these pigment groups as biomarkers in paleoenvironmental and paleoclimatic reconstruction is discussed.

1. Introduction Reconstructions of past environmental changes are critical for understanding the natural variability of global climatic conditions and for providing a context of present and future global change. Understanding past changes of climate conditions requires the reconstruction of climatic patterns reflected in lacustrine/marine sediments, as well as in terrestrial sediments (e.g. loess deposits). Organic matters preserved in geologic materials are increasingly being utilized to reconstruct past environmental conditions as they provide a direct indicator of environmental conditions at the time of deposition (Castañeda and Schouten, 2011). Lacustrine/marine sediments are sensitive recorders of climatic change and they are characterized by relatively high sedimentation rates, offering continuous and high-resolution climate

archives. On the other hand, proxies obtained from loess provide more direct information on paleoclimatic and paleoenvironmental conditions, since the very sediment is in a direct contact with atmospheric and the climatic conditions at the time of sediment formation (Sheldon and Tabor, 2009). Loess sediments cover approximately 10% of the Earth’s surface and are therefore among the most important terrestrial archives of paleoenvironmental changes during the Quaternary (Pye, 1995; Muhs, 2013). Loess deposits are among the best preserved and most continuous land-based archives of the global climate (Kukla et al., 1990; Pye, 1995; Muhs, 2013) which reach up to 22 Ma in the past (Guo et al., 2002). They are mainly considered deposits of aeolian origin, however, the biogenic contribution to the formation of loess deposits is recognized in several recent studies (e.g. Smalley et al., 2011; Dulić et al.,

Abbreviations: ANOVA, one-way analysis of variance; BLC, biological loess crusts; BLOCDUST model, biological loess crusts dust trapping model; d.w., dry weight; HPLC-DAD, high-performance liquid chromatography with diode-array detection; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MAAs, mycosporine-like amino acids; MWE, mycosporine wavelength equivalent; SWE, scytonemin wavelength equivalent; UV, ultraviolet irradiation ⁎ Corresponding author. E-mail address: [email protected] (D. Lalić). https://doi.org/10.1016/j.catena.2019.104283 Received 30 October 2018; Received in revised form 31 August 2019; Accepted 20 September 2019 0341-8162/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Dijana Lalić, et al., Catena, https://doi.org/10.1016/j.catena.2019.104283

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can serve as their diagnostic biomarker (e.g. Garcia-Pichel and Castenholz, 1991; Proteau et al., 1993; Balskus et al., 2011; Wada et al., 2013; Pathak et al., 2017). Its relative stability is another reason that makes it an important potential biomarker (Vítek et al., 2014). Scytonemin has been found in the sediment of archaic origin (Leavitt et al., 1997; Vítek et al., 2014), and a scytonemin-like compound was found preserved on the silicified Proterozoic stromatolites (Golubic and Hofmann, 1976). This finding clearly suggests that UV protection by extracellular pigments is an ancient adaptation (Abed et al., 2008). Cyanobacteria are regarded as the most primitive organisms capable of synthesizing MAAs (Häder et al., 2007; Singh et al., 2008). Except for cyanobacteria, MAAs have been identified in some marine heterotrophic bacteria, fungi, and phytoplankton exposed to high levels of UV irradiation (Arai et al., 1992; Häder et al., 1998; Sommaruga, 2001; Sinha and Häder, 2002; Sinha et al., 2007). MAAs are found from tropical to polar regions (Sinha et al., 2007). These compounds have high molar extinction coefficients (e = 28,100–50,000 M−1 cm−1) and photostability in water which support the idea that MAAs have a photoprotective role (Whitehead and Hedges, 2005). According to literature data (Cockell and Knowland, 1999; Edwards et al., 2005; Svirčev et al., 2013) MAAs can be determined in fossil records. Unlike in lake/marine sediments, biomarkers in loess deposits are quite rare or very degraded (Guthrie, 1990; Carter, 2000; Muhs et al., 2014) which makes reconstructing dynamics of the change of biota from loess sediments more difficult (Wells and Stewart, 1987; Rousseau and Kukla, 1994; Muhs, 2013). There is thus a clear need of implementating some new paleoclimate proxies. This manuscript provides crucial insights into the role of cyanobacterial secondary metabolites and aims to fill up the gaps and inconsistencies of traditionally used proxies for paleoclimatic reconstruction in loess deposits. The use of cyanobacteria-specific compounds, combined with other complementary proxies, will permit a more accurate interpretation of paleoenvironmental research. Cyanobacteria are one of the earliest life forms known to have existed on Earth (Archaean rocks of western Australia, dated 3.5 billion years old) (Sugitani et al., 2007; Chaurasia, 2015). The chemical composition of cyanobacteria is sensitive to changes in the environment, therefore their metabolites can be used as indicators of changes

2017; Sprafke and Obreht, 2016; Svirčev et al., 2016). Svirčev et al. (2013) developed the BLOCDUST hypothesis (Biological LOess Crusts DUSt Trapping model) which advocates the partly biogenic origin of loess and suggests a model of stabilization and deposition of dust particles through specialized extremophilic microbial communities formed on the loess sediment surface. In these important micro-environments in arid and semi-arid regions, called Biological Loess Crusts (BLC), the cyanobacterial component plays a major role. As highly productive microenvironments, biocrusts establish and control some basic physicochemical and geomorphological processes such as some pedological, hydrological and aeolian processes. In this regard, the sticky exopolymer synthesized by cyanobacteria in BLC is considered to play a major role in the accumulation of dust particles and further promote the formation of loess sediment. Cyanobacteria are the largest group of Gram-negative photosynthetic prokaryotes. A high variability of metabolic strategies provides them with tolerance to unfavorable environmental factors, enabling them to occupy most ecological niches. Cyanobacterial metabolic strategies against intensive UV irradiation include the synthesis of UV sunscreen compounds (Garcia-Pichel and Castenholz, 1991; Sinha et al., 1998; Oren and Gunde-Cimerman, 2007): extracellular sheath pigment scytonemin (Fig. 1) (Garcia-Pichel and Castenholz, 1991; Sinha et al., 1998) and intracellular mycosporine-like amino acids (MAAs) (Fig. 1) (Garcia-Pichel and Castenholz, 1991; Häder et al., 2007; Singh et al., 2008). Compounds that can be traced to source organisms from aquatic, terrestrial, and sedimentary components can be considered biomarkers (Simoneit, 2002) and they reflect environmental conditions (Pancost and Boot, 2004; Sachs et al., 2013). It is very important that biomarker candidates are specific and highly stable, so that they can persist in nature for a long time (unchanged or modified in predictable ways). Due to the fact that cyanobacteria are highly adaptive to environmental changes, their adapted chemotypes could be used as indicators of environmental changes and for paleoenvironmental reconstruction (Dachs et al., 1998; Svirčev et al., 2013). High amounts of scytonemin are often characteristic of the upper layer of terrestrial biological mats and crusts (Garcia-Pichel and Castenholz, 1991). Scytonemin is exclusive to cyanobacteria and thus

Fig. 1. Structures of scytonemin and some representative mycosporine-like amino acids (drawn according to Sinha et al., 1998). 2

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in environmental conditions (Florian et al., 2015; Jiménez et al., 2015; Cook et al., 2016), and if they leave behind fossil records, their biomarkers can be very useful in paleoclimatic reconstruction. Biomarker analyses in combination with paleosol geochemistry promotes the understanding of the paleoclimate. This approach will offer the possibility of a descriptive interpretation of the climatic proxies that prevailed in the past, contrary to some methods which provide precise numerical values for paleoclimate proxies. This means that the observed abundance of a biomarker does not define the absolute, numerical values of paleoecological and paleoclimatic proxies, but rather makes it possible to compare the tested parameters from different time periods and thus enhance the interpretation of climate conditions. Analyses of new potential biomarkers from cyanobacteria can be considered a significant piece of progress in paleoclimatic reconstruction and paleoecological interpretation of records from loess sediments (Pantelić, 2017). The main goal of this study was to investigate newly developed proxies that could be used to reconstruct paleoenvironmental conditions from Quaternary loess deposits. The present work explores the usefulness of scytonemin and MAAs as environment-sensitive biomarkers by analyzing the abundance of these compounds in aquatic and terrestrial cyanobacterial strains under various environmental stressors important from an ecological perspective (pH, temperature, light source and nitrogen content). This study presents double-centered interaction matrix analysis and heat-map visualization as convenient statistical tools for understanding the production of specific metabolites/biomarkers under various treatment conditions.

Table 1 NSCCC cyanobacterial strains used in this study.

2. Material and methods The most natural method of analysis of pigments including the UV protecting pigments scytonemin and MAAs is spectrophotometry with corrections applied for other pigments co-absorbing (Section 2.3.). Due to the lack of specificity of spectrophotometric methods, new terms for the compounds tested in this study were introduced: scytonemin wavelength equivalent (SWE) and mycosporine wavelength equivalent (MWE). Sample preparation, extraction and quantification of SWE and MWE compounds were performed according to the procedures previously established for scytonemin and MAAs (Garcia-Pichel and Castenholz, 1991, 1993; Dillon and Castenholz, 1999).

Cyanobacterial cultures



Soil, solonetz Soil, chernozem Soil, solonetz Soil, solonetz Mrtva Tisa Krivaja Zobnatica Bačko Gradište Tamiš Palić Zobnatica Krivaja Tavankut DTD Bečej PCC 7806 BLC-Serbia (Titel) BLC-Serbia (Titel) BLC-Serbia (Titel) BLC-Serbia (Titel) BLC-Serbia (Titel) BLC-Serbia (Titel)

S1 S2 S3 S4 W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 L20 L21 L22 L23 L24 L25


Calothrix sp. Anabaena sp. Nostoc sp. Nostoc sp. Anabaena sp. Aphanizomenon sp. Nostoc sp. Nostoc sp. Nostoc sp. Phormidium sp. Phormidium sp. Oscillatoria sp. Oscillatoria sp. Oscillatoria sp. Microcystis aeruginosa Leptolyngbya sp. NA NA Leptolyngbya sp. NA Nostoc sp., Leptolyngbya sp., Nodosilinea sp. Leptolyngbya sp.


Tolypothrix sp., Scytonema sp.


Leptolyngbya sp.


Leptolyngbya sp.


Leptolyngbya sp.


Leptolyngbya sp., Tolypothrix sp.




Leptolyngbya sp., Lyngbya sp.

BLC 1 (BG11 (Ruma) BLC 1 (BG11 (Ruma) BLC 2 (BG11 (Ruma) BLC 2 (BG11 (Ruma) BLC 3 (BG11 (Ruma) BLC 3 (BG11 (Ruma) BLC 4 (BG11 (Ruma) BLC 4 (BG11 (Ruma)

+N)-Serbia −N)-Serbia +N)-Serbia −N)-Serbia +N)-Serbia −N)-Serbia +N)-Serbia −N)-Serbia

*BLC – biological loess crust; NA – not available. Table 2 Origin of loess samples and BLCs for SWE analyses.

2.1. Material used in this study


The survey on the effects of different physico-chemical factors on the production of SWE and MWE compounds was conducted on 15 cyanobacterial strains (Table 1). SWE and MWE concentrations were analyzed in cyanobacterial strains isolated from freshwater (10 samples) in the Vojvodina region of Serbia and from chernozem (1) and solonetz type of soil (3) collected in the Province of Vojvodina (Northern Serbia). The Microcystis aeruginosa PCC 7806 was purchased from Pasteur Culture Collection (http://www.pasteur.fr/bio/PCC). The strains were cultivated as a part of the Novi Sad Cyanobacterial Culture Collection (NSCCC) at the Department of Biology and Ecology, University of Novi Sad, Serbia. The SWE concentrations were also analyzed in cyanobacterial cultures isolated from BLCs collected from open loess surfaces in Ruma (old brickyard, 45°00′43,8″N; 19°51′28,8″E) and Mošorin (Titel Loess Plateau, 45°17′46″N; 20°11′16″E) in 2006 (Table 1), corresponding BLCs collected at the Ruma sampling site (Northern Serbia) (Table 2) and loess sediment samples collected at the Titel Loess Plateau sampling site (45° 16,51″N; 20° 14,53″E) (Northern Serbia) (Table 2). Samples from loess and BLCs were collected from the loess localities after a preliminary visual inspection of the area. BLCs were sampled from the open vertical loess profiles in Mošorin and from a horizontal open loess surface in Ruma, which became uncovered during brickyard manufacture work. The loess sediment samples were collected from a vertical profile by first removing the top 10–20 cm of the sediment, in


T1 T5 T10 T15 T20 T25


Sampling details

Loess-Serbia (Titel) Loess-Serbia (Titel) Loess-Serbia (Titel) Loess-Serbia (Titel) Loess-Serbia (Titel) Loess-Serbia (Titel) BLC-Serbia (Ruma, old BLC-Serbia (Ruma, old BLC-Serbia (Ruma, old BLC-Serbia (Ruma, old

in or near the top layer 25 cm deep 50 cm deep 75 cm deep 100 cm deep 125 cm deep brickyard) brickyard) brickyard) brickyard)

order to avoid the contamination by recent environmental factors. From the purged sediment profile, samples were taken at every 5 cm with a spatula and blades. Upon returning to the laboratory, the samples were kept in dry and dark conditions. The isolation of cyanobacteria from BLCs was performed by a series of purification steps in the liquid and solid (with agar) BG11 −N and BG11 +N (supplemented with nitrate as a source of nitrogen) growth media. The purified strains were kept under 50 μmol m−2 s−1 (12:12 light/dark cycle) illumination at room temperature. Specific codes for the samples were given for easier interpretation of the results (Tables 1 and 2), along with the determined genera (Table 1). In addition to the code NSCCC, every culture has been given a number and a letter designating origin, so all freshwater strains are marked with the letter W, soil strains with S and the strains isolated 3

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absorbance. The concentration of SWE compounds was calculated using the trichromatic equation established for scytonemin by Garcia-Pichel and Castenholz (1991):

from BLC with L. Isolated cyanobacterial cultures with the codes NSCCC L20 to NSCCC L25 are from BLCs sampled at Titel Loess Plateau that were not analyzed in this study. Isolated cyanobacterial cultures with the codes NSCCC L26 to NSCCC L33 are from the BLCs sampled in Ruma section that were used in this study (BLC 1 to BLC 4), cultivated in BG11 +N, BG11 −N (Table 1).

Aλ = 1.04 A384 − 0.79 A663 − 0.27 A490 Aλ – the measured absorbance at λ; A384 – the absorbance at 384 nm; A663 – the absorbance at 663 nm; A490 – the absorbance at 490 nm.

2.2. Effects of physico-chemical conditions on the synthesis of SWE and MWE compounds Prior to the treatments, all cyanobacterial strains were cultivated for 21 days under optimal laboratory conditions (23 ± 2 °C, pH 7.5) in the liquid synthetic mineral medium BG-11 (Rippka et al., 1979). To examine the effects of diverse physico-chemical factors (pH, temperature, light) on the synthesis of SWE and MWE compounds, the cultures were exposed to different treatment procedures. All experimental cultures were shaken occasionally during the exposure to avoid self-shading effects. To examine the influence of pH on the synthesis of SWE and MWE compounds, cyanobacterial strains were cultivated at pH 6.0, 7.5, and 9.0. At the beginning of experiment, the pH of the medium was adjusted using hydrochloric acid or sodium hydroxide. All samples were incubated for 21 days at a constant temperature of 23 ± 2 °C, under natural solar light through south-facing window. The influence of temperature on the synthesis of SWE and MWE compounds was studied by incubating samples at different temperatures (15 °C, 25 °C, 35 °C). All experimental cultures were grown for 21 days in a medium with pH 7.5, under natural solar light through a south-facing window. Some cultures were exposed to different light sources (natural solar light, white light, UV light) for 60 h. The exposure of cultures to natural solar light and white light (50 μmol m−2 s−1; 12:12 light/dark) was performed in Erlenmeyer flasks. The UV irradiation of cultures was performed inside a specially designed UV light-chamber fitted with cool white fluorescent lamps as well as with UV lamps (Philips (ultraviolet) TUV 36 W/G36 T8 LONGLIFE). The cultures in open glass Petri dishes (diameter = 9 cm, depth = 1.5 cm) were irradiated using only UV lamps as a light source at a constant temperature of 23 ± 2 °C and pH 7.5. After 60 h of continuous exposure, the concentrations of SWE and MWE compounds were analyzed. To examine the effect of nitrogen on the synthesis of SWE and MWE compounds, cyanobacterial cultures were cultivated in the medium with nitrogen (with code +N) and in medium with low nitrogen content (with code -N). The BG-11 (+N) medium contained 1.5 g l−1 NaNO3 as the source of nitrogen, while the BG-11 (−N) medium contained only traces of nitrogen in the forms of Co(NO3)2·6H2O (0.0494 g l−1) and Ferric ammonium citrate (0.006 g l−1). The effect of the nitrogen content was examined in the cultures grown in media with different pH value (6.0, 7.5, 9.0), under different temperatures (15 °C, 25 °C, 35 °C) and under different sources of light (natural solar light, white light, UV light). All these combinations were applied in total amount of 18 different conditions.

The unit represents the absorbance of 1 mg (dry weight) material extracted in 1 ml acetone in a quartz cuvette with 1 cm pathlength. The specific content is expressed as A × mg (d.w.)−1 (Garcia-Pichel and Castenholz, 1991). To extract SWE compounds from loess sediment and BLCs, the samples were ground with mortar and pestle and suspended in 90% acetone. The samples were extracted overnight at 4 °C followed by bath sonication (Bransonic 220) for 10 min (with cycles of 30 sec/30 sec). The samples were then centrifuged (12,000g for 10 min) and the separated supernatants were filtrated through GF/C (Whatman) filters. The absorbances of the supernatants were measured on the UV–visible spectrophotometer. MWE compounds were extracted following the protocol for the extraction of MAAs in phytoplankton (Garcia-Pichel and Castenholz, 1993), with minor modifications. Cyanobacterial cell suspension was harvested by filtration on GF/C (Whatman) filters, ground and then freeze-dried for 2 h. The biomass was suspended and homogenized in 20% (vol/vol) aqueous methanol at 45 °C in a water bath for 2 h. After that, repeated freezing/thawing steps were performed, followed by bath sonication (10 min with cycles of 30 sec/30 sec). The cell debris was separated by centrifugation at 12,000g for 10 min. The supernatants were then separated and the absorbances were determined spectrophotometrically on the Beckman DU-65 UV/Visible spectrophotometer (Beckman Instruments). The measurements were performed at the wavelength of maximum absorbance of MAAs (value was fixed based on several extracts), and corrections were made according to the following expression (Garcia-Pichel and Castenholz, 1993):

Aλ = Aλ − A260 (1.85 − 0. 005λ ) Aλ* – corrected value of absorbance at the maximum; Aλ – measured value of absorbance at the maximum; λ – wavelength (nm) of maximal absorbance. The unit represents the absorbance of 1 mg (dry weight) material extracted in 1 ml of solvent in a quartz cuvette with 1 cm pathlength. The specific content is expressed as A × mg (d.w.)−1 (Garcia-Pichel and Castenholz, 1993). 2.4. Statistical analysis The experimental data are presented as mean values ± SD (standard deviation) of three replicates. The statistical analysis was done by one-way analysis of variance (ANOVA) (DSAASTAT version 1.101.). The means were separated by Fisher's multiple comparison test (level of significance p < 0.01). In order to illustrate the effect of each environmental treatment on the SWE and MWE compound production in the strains, the doublecentered interaction matrix of strain by treatment data was used. The matrix is organised as row × column table, in which the values for the interaction of the strain and treatment are displayed. It is calculated by subtracting the mean value of the row and column from any value in the table, and by adding afterwards to this number the mean value of all the values in the table. In this way we obtain a table of interaction values between the various strains and treatment (a negative value represents a negative interaction and vice versa). This matrix was

2.3. Extraction and measurement of SWE and MWE compounds To extract SWE compounds, cyanobacterial cell suspension was harvested by filtration on GF/C (Whatman) filters, ground and then freeze-dried. The biomass was suspended in 90% acetone, and kept overnight at 4 °C in darkness. After that repeated freezing/thawing steps were performed, followed by sonication (Bransonic 220) (10 min with cycles of 30 sec/30 sec). Separation of cell debris was accomplished by centrifugation at 12,000g for 10 min. The absorbance of the separated supernatant was measured on the Beckman DU-65 UV/ Visible spectrophotometer (Beckman Instruments) at 384 nm (scytonemin maximum), 490 nm (pooled carotenoid), 663 nm (Chl a). The value of absorbance at 750 nm was subtracted from all measured 4

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In the experiment with different light sources (Fig. 4), the lowest production of SWE compounds was observed when the strains were cultivated under natural day light. With cultivation under white fluorescent light a slightly higher production of SWE compounds was obtained when compared to the cultivation under natural day light. It was observed that the treatment with the UV light led to a compelling increase (over 7 times higher in NSCCC S1) in the production of SWE compounds in all analyzed strains. The production of MWE compounds was observed in all analyzed cyanobacterial strains (Figs. 5–7) in all tested conditions. An increased production of MWE compounds was observed in all analyzed strains cultivated in the +N medium under all treatments, with prominent increase in all soil strains. The nitrogen content had less influence on the production of MWE compounds in all aquatic strains. In the treatment with different pH values (Fig. 5), the lowest production of MWE compounds was observed in the medium with pH 6.0. The concentration of MWE compounds was increased somewhat at pH 7.5 and even more at the pH 9.0. Besides the treatment at pH 6.0, the growth at 15 °C resulted in the lowest production of MWE compounds among all experimental conditions in almost all the strains (Fig. 6). Along with an increase in temperature, an increase in the MWE compound production was observed in all analyzed strains. The highest synthesis of MWE compounds was observed in the treatment with UV light (Fig. 7). Cool white light gave the lowest concentration of MWE compounds among the applied light conditions.

constructed from two-way table of the strain by treatment data values for SWE and MWE compounds. This double-centred table contains only the interaction effects or interaction residuals. The interpretation of the interaction effects will be shown by heat-map graphical display. The estimation and visualization of the interaction effects was accomplished within R computing environment (R Core Team, 2017). The model helps to visualize the increased production of certain secondary metabolites under certain environmental conditions and thus contributes to choosing appropriate biomarkers in paleoclimatic reconstructions.

3. Results 3.1. Effects of different physico-chemical conditions on the production of SWE and MWE compounds The concentrations of SWE and MWE compounds varied to a great extent depending on the examined strain and also on the physico-chemical conditions. In order to determine a possible relationship between the compound production and the origin of the studied cyanobacteria, a comparison of SWE and MWE compounds yield was made between soil and aquatic strains. The synthesis of SWE and MWE compounds was analyzed at different pH values, temperatures, and light source, in a medium BG-11 +N and −N. SWE compounds were found in 8 out of 15 investigated cyanobacterial strains in all tested physico-chemical conditions (Figs. 2–4). Production was observed in all soil strains and in 4 out of 11 aquatic strains. A lower production of SWE compounds was observed in the +N medium for most of the analyzed strains (6). Soil strains showed a more prominent production of SWE compounds in all analyzed conditions as compared to aquatic strains. In the treatment with different pH values (Fig. 2), the lowest production of SWE compounds was observed in the medium with pH 6.0, slightly increased production was observed in the medium with pH 7.5, and the most prominent production in a medium with pH 9.0. In the temperature treatment (Fig. 3), the production of SWE compounds was clearly more pronounced at the temperature of 35 °C, when compared to lower temperatures.

3.2. Interpretation of the interaction effects The influence of selected treatment variables on the production of SWE compounds is presented on the heat-map graph (Fig. 8). The graph presents a very low variety of achieved interactions, with the most number of the ‘strain by treatment’ combinations having interaction residuals close to zero. This indicates a stable and a similar reaction of the strains to the treatment conditions which is characterized by an absence, or a rather low production of SWE compounds. The highest ‘strain by treatment’ interaction was observed in strains

Fig. 2. The effect of different pH values on SWE concentrations in the examined cyanobacterial strains. The error bars show the standard deviation (n = 3). 5

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Fig. 3. The effect of temperature on SWE concentrations in the examined cyanobacterial strains. The error bars show the standard deviation (n = 3).

Fig. 4. The effect of different light source on SWE concentrations in the examined cyanobacterial strains. The error bars show the standard deviation (n = 3). 6

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Fig. 5. The effect of different pH values on MWE concentrations in the examined cyanobacterial strains. The error bars show the standard deviation (n = 3).

Fig. 6. The effect of temperature on MWE concentrations in the examined cyanobacterial strains. The error bars show the standard deviation (n = 3).

which produced the most of SWE compounds under the given conditions. A negative interaction of this strain with all other treatment conditions was also observed, indicating a low production of SWE compounds under their influence. A positive interaction with UV irradiance was also observed in the S2 strain, while the positive interaction between UV irradiance and the

treated with the UV irradiance. Most of the aquatic strains show a strong negative interaction with this factor (red fields), indicating a very low production of SWE compounds under the given conditions. On the contrary, the highest positive interaction (green fields) was observed in two ‘strain by treatment’ combinations indicating a high positive interaction between the S1 strain and UV irradiance (L3 ± N), 7

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Fig. 7. The effect of different light source on MWE concentrations in the examined cyanobacterial strains. The error bars show the standard deviation (n = 3).

compound production. The +N medium gave the highest contribution to the interactions since most of them were achieved in the nitrogenrich environment. A high interaction rate was observed between three ‘strain to treatment’ combinations, indicating a strong impact of the high pH (P3), temperature (T3) and UV irradiation (L3) on the metabolic response of soil strains (S1-S4) (dark green fields). The red fields

soil S3 and S4 strains was observed only in the medium without added nitrogen (L3−N). The influence of selected treatment variables on the production of MWE compounds is presented on the heat-map graph (Fig. 9). A high variety of the ‘strain to treatment’ interactions was observed which demonstrated an important impact of these factors on the MWE

Fig. 8. The heat-map graphic display of the ‘strain to treatment’ interactions. Green fields mark their positive interaction (high SWE production) while the red fields mark their low interaction (low SWE production). The zero (white field) marks an absense of interaction. Abbreviations: P1 (pH 6.0); P2 (pH 7.5); P3 (pH 9.0); T1 (10–15 °C); T2 (20–25 °C); T3 (30–35 °C); L1 (white fluorescent light); L2 (natural daylight); L3 (UV irradiation); +N (growth medium with NaNO3); −N (growth medium with traces of nitrogen); W – aquatic strain, S – soil strain. 8

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Fig. 9. The heat-map graphic display of the ‘strain to treatment’ interactions. Green fields mark their positive interaction (high MWE production) while the red fields mark their low interaction (low MWE production). The zero (white field) marks an absense of interaction. Abbreviations: P1 (pH 6.0); P2 (pH 7.5); P3 (pH 9.0); T1 (10–15 °C); T2 (20–25 °C); T3 (30–35 °C); L1 (white fluorescent light); L2 (natural daylight); L3 (UV irradiation); +N (growth medium with NaNO3); −N (growth medium with traces of nitrogen); W – aquatic strain, S – soil strain.

compounds as biomarkers in paleoclimate and paleoenvironmental research. Correlating the abundance of SWE and MWE compounds with the various treatments through the double-centered interaction matrix analysis revealed the favorable (or unfavorable) growth conditions for the production of the SWE and MWE compounds. The production of SWE compounds varied considerably among the analysed cyanobacterial strains (Figs. 2–4 and 8). A high positive interaction between UV irradiance as a factor and the soil strains (NSCCC S1-NSCCC S4) was observed (Fig. 8), indicating that the given strains achieved the highest production of SWE compounds under that particular condition, i.e. UV irradiation and low nitrogen contents in the medium (treatment L3−N). This can be explained through the sunscreen role of scytonemin and scytonemin-like compounds as a first line of protection against harmful UV irradiation (Ehling-Schulz et al., 1997; Rastogi et al., 2014; Pathak et al., 2016). Also, an increase in scytonemin synthesis was observed in the nitrogen-depleted environment (Fleming and Castenholz, 2008). Most of the aquatic strains showed very low positive or negative, close to zero, interaction with the applied treatments, revealing the rather low production of SWE compounds under given conditions (Figs. 2–4 and 8). This finding confirms the photoprotective role of SWE compounds and demonstrates its narrow environment-specificity, a necessary biomarker trait. A few studies on scytonemin demonstrated its structural stability over different stress factors such as temperature, high UV irradiation and low/high pH values (Ehling-Schulz et al., 1997; Abed et al., 2008; Fulton et al., 2012; Rastogi and Incharoensakdi, 2014b). Scytonemin remained intact after two months of continuous exposure to UV irradiation (Fleming and Castenholz, 2007). It has been preserved in sediment structures of archaic origin (Leavitt et al., 1997; Verleyen et al., 2005; Fleming and Castenholz, 2007), while scytonemin-like pigments were found preserved in stromatolites from Proterozoic (Golubic and Hofmann, 1976). Scytonemin was also found in lake and marine sediments (Leavitt et al., 1997), in the layers of rocks exposed to high irradiation (Vítek et al., 2014), and in the surface layers of biocrusts (Garcia-Pichel and Castenholz, 1991; Svirčev et al., 2013) which all indicate their structural stability. Generally, scytonemin is often found in the upper layers of

define a negative interaction, characterized by a low production of the compound. 3.3. Presence of SWE compounds in loess sediment, BLCs samples and cyanobacterial cultures isolated from BLCs The presence of SWE compounds was analyzed in 6 loess samples, 4 BLCs and 14 cyanobacterial cultures isolated from BLC samples. The data presented in Fig. 10 are normalized to total weight. The presence of SWE compounds was observed in all loess samples (Fig. 10a), as well as in all BLC samples (Fig. 10b). The presence of SWE compounds was observed in all cyanobacterial cultures isolated from BLCs (Fig. 11). A much higher concentration of SWE compounds was detected in the NSCCC L24 strain (0.0932 Aλ × mg−1), followed by NSCCC L30 (0.0682 Aλ × mg−1) and NSCCC L29 (0.0669 Aλ × mg−1). All other strains had approximately the same concentration of SWE compounds (from 0.0421 to 0.0210 Aλ × mg−1), except that the lowest concentration was observed in NSCCC L25 (0.0138 Aλ × mg−1). 4. Discussion Presented research conducted on 15 aquatic and soil cyanobacterial strains gave us an insight into the ability of the strains to synthesize UV protective compounds under different physico-chemical conditions. SWE compounds were present in 8 out of 15 studied cyanobacterial strains, while the presence of MWE compounds was observed in all 15 strains. Due to methodological limitations in this study, the analyzed compounds could not be absolutely recognized as scytonemin and MAAs. However, the applied methods were previously used in many studies where the presence of scytonemin and MAAs was confirmed (Garcia-Pichel and Castenholz, 1992, 1993; Garcia-Pichel et al., 1993; Brenowitz and Castenholz, 1997; Dillon and Castenholz, 1999; Dillon et al., 2002; Fleming and Castenholz, 2007, 2008; Mushir et al., 2014; Vítek et al., 2014). Therefore, it is postulated that the presence of SWE and MWE compounds are closely related to the presence of scytonemin and MAAs. Our study hypothesizes the potential of SWE and MWE 9

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Fig. 10. Concentration of SWE compounds in: A – LOESS samples; B – BLC samples.

past. The impact of light intensity on cyanobacterial pigment distributions is highlighted by the presence of SWE compounds in the BLC samples. Additionally there have been numerous studies that have shown the effects of light on scytonemin concentrations (Ehling-Schulz

microbial mats (Garcia-Pichel and Castenholz, 1991; Proteau et al., 1993; Ehling-Schulz et al., 1997; Balskus et al., 2011), synthesized as a response to UV exposure (Garcia-Pichel and Castenholz, 1991), which could be used when evaluating the intensity of the UV radiation in the 10

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Fig. 11. Concentration of SWE compounds in cyanobacterial cultures isolated from BLC samples.

and also of Tolypothrix, Scytonema and Lyngbya. These species are commonly known as constituents of BLC (Pócs, 2009). For example, the filamentous type of BLC is mostly formed by cyanobacteria Scytonema, Tolypothrix and Lyngbya, containing a large quantity of scytonemin, which is effective in screening UV irradiation (Garcia-Pichel and Castenholz, 1991; Proteau et al., 1993; Castenholz and Garcia-Pichel, 2012). This type of BLC occurs primarily in desert, semidesert and woodland areas, as well as on calcareous rocks (Pócs, 2009). Furthermore, differences in overall irradiance and sunlight experienced over the growing season at each site could impose an influence on scytonemin production. Shields (2017) reported that the west bank of a lake received ~5.0% more sunlight hours than the east bank – Similar differences in sunlight exposure may have contributed to the variability observed in the present study. Lastly, the different concentrations of SWE detected in the samples might be attributed to the shading effect by other microbiota which reduced the need for scytonemin. Such an observation was made in the study of Uveges et al. (2018) where scytonemin was found at a very low concentration, or below the detection limit in several samples. The low concentrations of scytonemin were presumed to be due to the shading effect of charophyte algae that covered the microbialite surfaces. Scytonemin concentrations corresponded to the received UV irradiance level which indicates the potential of scytonemin to be used as a relative water depth/UV irradiance proxy in ancient microbialites (Uveges et al., 2018). Scytonemin was the most predominant pigment in all mats, except in one (Abed et al., 2018), and showed clear variation among the different sites (Abed et al., 2018). It was found that the concentration of scytonemin and its abundance relative to that of chlorophyll a decreased logarithmically with depth in desert cyanobacterial soil crusts (Bowker et al., 2002) and in water (Fleischmann, 1989; Morris et al., 1995; Moorhouse et al., 2018; Uveges et al., 2018) which is consistent with the function of scytonemin as a UV screening pigment. Pigment abundances in freshwater microbialites showed a strong dependence on water depth (Uveges et al., 2018). The concentrations of scytonemin in benthic microbial mat communities showed a distinct correlation with

et al., 1997; Garcia-Pichel and Castenholz, 1991; Schäfer et al., 2006; Kao et al., 2012; Uveges et al., 2018). Considering the stability of scytonemin, environmental variations through time might be reconstructed by tracking the occurrence and abundance of scytonemin over longer time scales. Since the SWE compounds were observed in this study in the loess sediment samples (Fig. 10a), cyanobacteria-dominated BLCs (Fig. 10b), and cyanobacterial strains isolated from BLCs (Fig. 11), these compounds may be considered as indicators of the UV-exposed land surfaces characterized by open patches of land, covered mostly by cyanobacteria-dominated biocrusts and very scarce, probably grassy vegetation. Considering that the content of SWE compounds depends on the intensity of UV irradiation, their content in BLC samples vary through time and its highest concentrations appear to be associated with intense UV irradiation and the length of exposure. The observed higher values of SWE compounds in BLC samples comparing to the values in analyzed strains are due to the significantly longer (maybe even years) exposure to the outer stress factors such as UV irradiation and desiccation, whereas the strains were stressed for a limited time in the laboratory. As shown in Fig. 10, the four BLC samples from Ruma loess section show different concentrations of SWE compounds, and our results show that also the production of SWE compounds varies in strains isolated from the same location. The variability seen in SWE concentrations from BLC samples is likely due to several factors. There are different types of crusts considering the morphological characteristics (Komáromy, 1976) and the functional characteristics (Belnap et al., 2001). Some specific morphological features reflect environmental conditions and survival strategies (Pócs, 2009). The stress which different crust types are exposed to (e.g. low temperatures, high levels of photosynthetically useful irradiation and UV irradiation, repeating freeze-thaw cycles, nutrient depletion) varies in intensity and quality (Pócs, 2009). Cyanobacteria have developed different strategies for their survival among harsh conditions. The cyanobacteria present in BLC (Table 2) were composed of Leptolyngbya, as the most dominant,


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2018); stable C and N isotopic analyses (Liu and Huang, 2005; Zech et al., 2011, 2013; Obreht et al., 2014; Marković et al., 2018); phytoliths (Liu et al., 1996; Lu et al., 1996; Kuklewicz et al., 2014); fossil pollen assemblages (Davis et al., 2003; Seppä et al., 2004; Rousseau et al., 2006); magnetic susceptibility and other mineral magnetic properties (Kukla and An, 1989; Maher et al., 1994; Porter, 2001; Singer and Verosub, 2007; Marković et al., 2018). The shortcomings of traditional proxies are becoming evident in some respects. For example, leaf wax cannot be used to reflect relative input of woody plants and grasses (Li et al., 2016). Land snails contain insufficient amounts of carbon in the small terrestrial gastropod shells for the relevant analyses (Pigati et al., 2010). Loess deposits are unreliable in preserving pollen (Sun et al., 1997; Carter, 2000). There are also inconsistencies in the magnitude of coeval magnetic susceptibility peaks of the Loess Plateau (Guo et al., 2001; Bloemendal and Liu, 2005) and magnetic properties may show the opposite relation to climate conditions in different regions (Begét et al., 1990). Further, n-alkanes tend to give insufficient information for environmental reconstruction (Marković et al., 2018). Due to certain shortcomings of these traditional biomarkers in the context of loess, it is beneficial to introduce a new approach into the analysis of loess. The application of a potential proxy based on pigment biomarkers can thus provide additional data about conditions where other organic compounds are limited or not well preserved. Cyanobacterial metabolites provide a good basis for development of novel biomarker proxies due to their widespread distribution and dominance in harsh environments. If cyanobacteria played a role in loess formation, many studies based on organic geochemistry biomarkers, such as n-alkanes, δ13C and δ15N isotopes and similar proxies used so far in Southeastern Europe (e.g. Schatz et al., 2011; Zech et al., 2013; Obreht et al., 2014, 2019; Schreuder et al., 2016), may need to be reconsidered for the contribution of cyanobacteria. Furthermore, cyanobacterial biomarkers could provide a new approach for the qualitative reconstruction of semi-arid to arid regions and explain the relationship between climate, sediment formation and ecological conditions. Each loess layer carries information on the prevailing climatic conditions at the time of the formation (Marković et al., 2008, 2015, 2018; Perić et al., 2019). The combination of different approaches of sedimentology, magnetism, geochemistry, geochronology and biomarkers allows a more complete analysis of climate change and paleoclimate reconstruction. BLC samples were sampled from different locations in the Ruma brickyard at different times of the year, even in different years. There is not enough data to correlate the description of the sampling location and the time of sampling with the observed concentrations of SWE compounds. The analyses of BLC samples should be taken as a proof of concept that SWE compounds are present in BLC samples. In future, an evaluation whether the SWE compounds have a systematic and consistent distribution in BLC has to be done. In order to constrain SWE compounds as a new proxy for UV irradiation for paleoclimate reconstruction, additional studies focusing on morphological variables of the selected crusts, quantification of chlorophyll a as a (cyanobacterial) biomass indicator vs. scytonemin, as well as further field and laboratory studies are required to establish a reliable proxy. The use of scytonemin/SWE compounds as a biomarker for regional/global UV conditions is expected to require that a statistically significant number of samples from different locations are analyzed. Extensive work is still required to explore the full ecological and paleoclimate importance of SWE and MWE compounds that show potent biomarker properties as new qualitative paleoclimate proxies. However, such studies require the use of e.g. HPLC-DAD (HighPerformance Liquid Chromatography with Diode-Array Detection) and LCMS/MS (Liquid chromatography-tandem mass spectrometry) to separate the target compounds from other organic molecules that are also found in modern and ancient sediments. These methods provide a more secure identification of scytonemin and MAAs (Airs et al., 2001; Squier et al.,

depth and intensity of UV light (Hodgson et al., 2004; Verleyen et al., 2005; Abed et al., 2008). In addition to UV exposure, there are other factors that influence scytonemin production. Increases in temperature, higher desiccation levels, oxidative and osmotic stress, and also nitrogen-limiting conditions have been shown to enhance scytonemin production in cyanobacteria (Bergmann and Welch, 1990; Peterson and Grimm, 1992; Peterson et al., 1994; Dillon et al., 2002; Fleming and Castenholz, 2007). However, neither the temperature nor salinity were thought to induce a strong response in scytonemin production by cyanobacteria (Dillon et al., 2002; Abed et al., 2008), and also the role of nitrogen fixation does not appear to be significant for scytonemin production (Shields, 2017). Also the results from our study showed that influence of different temperatures or pH values was small (Figs. 2 and 3). The conclusion is that the variation in UV irradiation is likely the strongest control on scytonemin production (Uveges et al., 2018). Thus, although the production of SWE compounds may be influenced, in some amount, by other environmental conditions, the results showed largely consistent dependencies across tested samples under UV light. As such, pigment quantification can be used to investigate the contributions of light regimes. MWE compounds were produced by all analyzed strains. A higher production of MWE compounds was mostly observed in soil strains (NSCCC S1–NSCCC S4) (Figs. 5–-7). Treatment conditions of 35 °C (+N), UV light (+N) and pH 9.0 (+N) were highlighted as the most favorable conditions for MWE compound production in the soil strains indicating an adaption for those conditions (Fig. 9). A higher production of MWE compounds was also observed in strains grown in the presence of nitrogen indicating its importance for the pigment production, which is also demonstrated for MAAs (Yadav et al., 2011). Contrary to SWE compounds, the factors which lead to stimulation of the synthesis of MWE compounds were more various, and included UV, temperatures, pH and nitrogen. MAAs are also general stress-induced compounds, and in nature, the production of MAAs is a result of several environmental factors, which is also confirmed in this study (Fig. 9). Demanding environmental conditions lead to a wider amplitude of metabolic responses, resulting in the production of specific substances, some of which may be good biomarker candidates in paleoreconstructions (Pantelić, 2017). A higher concentration of MWE compounds in a specific layer of loess sediment could provide information on a rise of local temperature or higher input of nitrogen, intensive UV exposure or a transition to more alkaline conditions. In combination with other proxies MWE compounds could contribute to the reconstruction of paleoclimatic conditions. Gröniger and Häder (2000) discussed the stability of MAAs against pH, different solvents, temperature and strong UV irradiation, even in combination of UV with heat (75 ± 2 °C) for up to 6 h (Sinha et al., 2000). The same was concluded in research by Rastogi and Incharoensakdi (2014a–c) on cyanobacteria belonging to the genera Gloeocapsa, Lyngbya and Arthrospira. The study of lake sediments by applying the cyanobacteria biomarker analysis is of special interest in reconstructing the evolution of an environment. Aspects of the paleoproductivity and environmental changes are inferred from the studies of sedimentary pigments (Danladi and Salihoglu, 2016; Kaiser et al., 2016) as well as diatom (Rybak and Dickman, 1988), cyanobacteria and protist communities (Savichtcheva et al., 2011; Domaizon et al., 2013; Deshpande et al., 2014; Hou et al., 2014). Cyanobacterial production of UV-protective compounds that are preserved over time provides a proxy for the reconstruction of past solar UV irradiation (Garcia-Pichel and Castenholz, 1991; Rozema et al., 2002; Verleyen et al., 2005; Fulton et al., 2012) as well as aridity (Fulton et al., 2012). Some of the traditional methods used as proxies in paleoclimatic reconstructions of loess are land snail assemblage (Marković et al., 2005, 2018); leaf-wax biomarkers (Xie et al., 2002; Zhang et al., 2006; Zech et al., 2009; Häggi et al., 2014; Marković et al., 12

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2002; Rastogi and Incharoensakdi, 2014b). Cyanobacterial biomarkers should not be viewed only as a climatic proxy but also as an additional parameter and a diagnostic tool for the modelling of changes in stress conditions, both on a regional and global scale. The combination of geochemical and cyanobacterial biomarker proxies is a useful approach that has the potential to assist in tracking the effects of changing climate. Results presented in this manuscript are important for the understanding of paleoclimate signals and the development of new proxies for loess and lacustrine (or even marine) sediments. Because pigments could be key biomarkers from which we can better understand past environmental conditions on Earth, as well as on other planets such as Mars (Wynn-Williams et al., 2002; Parnell et al., 2006; Varnali et al., 2009), the full potential of cyanobacterial pigments as biomarkers in paleoclimatic studies of loess should be evaluated through future paleoclimate reconstruction studies.

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Effect of environmental factors on the synthesis of scytonemin, a UV-screening pigment, in a cyanobacterium (Chroococcidiopsis sp.). Arch. Microbiol. 177, 322–331. Domaizon, I., Savichtcheva, O., Debroas, D., Arnaud, F., Villar, C., Pignol, C., Alric, B., Perga, M.E., 2013. DNA from lake sediments reveals the long-term dynamics and diversity of Synechococcus assemblages. Biogeosciences 10, 3817–3838. Dulić, T., Meriluoto, J., Palanački Malešević, T., Gajić, V., Važić, T., Tokodi, N., Obreht, I., Kostić, B., Kosijer, P., Khormali, F., Svirčev, Z., 2017. Cyanobacterial diversity and toxicity of biocrusts from the Caspian Lowland loess deposits, North Iran. Quatern. Int. 429 (Part B), 74–85. https://doi.org/10.1016/j.quaint.2016.02.046. Ehling-Schulz, M., Bilger, W., Scherer, S., 1997. UV-B-induced synthesis of photoprotective pigments and extracellular polysaccharides in the terrestrial cyanobacterium Nostoc commune. J. Bacteriol. 179 (6), 1940–1945. 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5. Conclusion Cyanobacterial crusts are very well developed on the loess surfaces, however, research on their metabolites as biomarkers is still a largely untouched area. The results reported in this paper describe the effects of pH, temperature and light source combined with the effects of nitrogen contents in the growth medium on the production of SWE and MWE compounds. The data were analyzed statistically by double-centered interaction matrix analysis. The application of this statistical tool together with a heat-map visualization revealed the increased production of certain secondary metabolites in certain environmental conditions. The analyzed cyanobacterial strains showed metabolic responses when treated with different stress factors. The production of SWE compounds (with the reference to scytonemin) was clearly increased by UV irradiation. The production of MWE compounds (with reference to MAAs) was increased by several stress factors including pH, temperature, nitrogen content and UV irradiation. The production of SWE and MWE compounds across examined stress conditions, and the presence of SWE compounds in BLCs and loess samples indicate the potential of these compounds to be regarded as biomarkers in geological research of loess sediments. Acknowledgments The authors would like to acknowledge the funding of the Ministry of Education, Science and Technological Development of the Serbian Government (project number: 176020), and the mobility support of the Erasmus + Programme of the European Union (agreement numbers: 2015-2-FI01-KA107-022151 and 2017-1-FI01-KA107-034440). Preparation of this publication was partly performed within the framework of the EUROWEB+ (European Research and Educational Collaboration with Western Balkans) scholarship program. The authors declare that there are no associations that could pose a conflict of interest in connection with this manuscript. References Abed, R.M.M., Kohls, K., Schoon, R., Scherf, A.-K., Schacht, M., Palinska, K.A., Al-Hassani, H., Hamza, W., Rullkötter, J., Golubic, S., 2008. Lipid biomarkers, pigments and cyanobacterial diversity of microbial mats across intertidal flats of the arid coast of the Arabian Gulf (Abu Dhabi, UAE). FEMS Microbiol. Ecol. 65 (3), 449–462. Abed, R.M.M., Palinska, K.A., Köster, J., 2018. Characterization of microbial mats from a Desert Wadi ecosystem in the Sultanate of Oman. Geomicrobiol J. 35 (7), 601–611. Airs, R.L., Atkinson, J.E., Keely, B.J., 2001. Development and application of a high resolution liquid chromatographic method for the analysis of complex pigment distributions. J. Chromatogr. A 917 (1–2), 167–177. Arai, T., Nishijima, M., Adachi, K., Sano, H., 1992. Isolation and structure of a UV absorbing substance from the marine bacterium Micrococcus sp. AK-334. Mar. Biotechnol. Instit. Tokyo 88–94. Balskus, E.P., Case, R.J., Walsh, C.T., 2011. The biosynthesis of cyanobacterial sunscreen scytonemin in intertidal microbial mat communities. FEMS Microbiol. Ecol. 77 (2), 322–332. https://doi.org/10.1111/j.1574-6941.2011.01113.x.


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