Bacteriobiota of the Cave Church of Sts. Peter and Paul in Serbia-Culturable and Non-Culturable Communities' Assessment in the Bioconservation Potential of a Peculiar Fresco Painting.

The principal aim of this study was to determine bacterial diversity within the Cave Church of Sts. Peter and Paul, via culturable and non-culturable approaches, and elucidate the antifungal potential of autochthonous antagonistic bacterial isolates against biodeteriogenic fungi. Furthermore, whole-genome sequencing of selected bacterial antagonists and the analysis of genes included in the synthesis of secondary metabolites were performed. With the highest RA values, determined in metabarcoding analysis, phyla Actinobacteriota (12.08-54.00%) and Proteobacteria (25.34-44.97%) dominated most of the samples. A total of 44 different species, out of 96 obtained isolates, were determined as part of the culturable bacteriobiota, with the predominance of species from the genus Bacillus. Bacillus simplex was the only isolated species simultaneously present in all investigated substrata within the church. The best antagonistic activity against 10 biodeteriogenic fungi was documented for Streptomyces anulatus, followed by Bacillus altitudinis, Chryseobacterium viscerum, and Streptomyces sp. with their highest PGI% values ranging of from 55.9% to 80.9%. These promising results indicate that characterized bacteria are excellent candidates for developing biocontrol strategies for suppressing deteriogenic fungi responsible for the deterioration of investigated fresco painting. Finally, isolate 11-11MM, characterized as Streptomyces sp., represents a new species for science prompting the need for further study.

The Discovery of the Rare Chara baueri (Charales, Charophyceae) in Serbia.

Chara baueri is one of the rarest charophytes worldwide. It had been considered extinct in Europe for more than a century, from the 1870s to 2006, when it was rediscovered in Germany. The current distribution of this species is limited to a few localities in Europe (Germany, Poland and Russia), and one locality in Asia (Kazakhstan). We present a new finding of Chara baueri, to be a significant contribution to the species ecology and biogeography, and helping to review and update the current scarce knowledge. Chara baueri was discovered in Serbia and monitored for two vegetative seasons in 2018 and 2019, along with the associated macrophyte vegetation and water quality parameters. The morphology and ecology data of the species are presented comparatively with the literature data and the biogeography is critically reviewed. The population in Serbia is the first verified record of Chara baueri in southern Europe. Considering the recent findings and the knowledge accumulated in these records, Chara baueri was very possibly never extinct at all, but overlooked in Europe for the entire 20th century. We suggest that waterfowl migrating from the northern parts of Europe should be considered as the important spreading agent of Chara baueri in southern regions.

Biofilms in caves: easy method for the assessment of dominant phototrophic groups/taxa in situ.

Domination of certain aerophytic phototrophic group or specific taxon in biofilms is connected with biofilm features recognised in situ. Well-developed, gelatinous, olive to dark-green biofilms are composed mostly of coccoid cyanobacterial forms. The same features, characterised biofilms dominated by one coccoid taxon, except the latter were vividly coloured. Gloeobacter caused the appearance of purple, Gloeocapsa representatives yellow and Chroococcidiopsis black biofilm. The brown to the dark colour of heterocytous biofilms was mainly caused by Nostoc. Simple trichal Cyanobacteria were occasionally present in biofilm, except in one blue-coloured sample. According to the principal component analysis (PCA), well-developed and gelatinous biofilms were correlated with Cyanobacteria, while scanning electron microscopy (SEM) revealed richness of extracellular polymeric substances (EPS) in such biofilms. Biofilm with calcified cyanobacterium (Geitleria cf. calcarea) was also found. Chlorophyta-abundant biofilms (many rich in Desmococcus), thinner than cyanobacterial, were predominantly green and occasionally yellow and blue. Many were dry when observed in situ (confirmed with PCA), with few being moistened (i.e. Klebsormidium-dominant). Diatom biofilms were usually developed on sediment, mosses or near seeping water (demonstrated by PCA) and were also thinner than cyanobacterial ones. Compared to cyanobacterial biofilms, SEM showed less developed EPS in those rich in diatoms and green algae, where microorganisms are more exposed to the environment. The study demonstrates an easy method for biofilm assessment based on visual characterisation and provides encouragement for more frequent biofilm investigation in caves that can be important from an ecological, biological, biotechnological point of view and which assessment can have an irreplaceable role in potential monitoring and protection.

ATP bioluminescence method: tool for rapid screening of organic and microbial contaminants on deteriorated mural paintings.

The extent of the microbial contamination of the seventeenth-century wall paintings in the nave of the old Church of the Holy Ascension (Veliki Krcimir, Serbia) was evaluated via newly implemented ATP bioluminescence method, and traditional cultivation-based method, utilising commercially available dip slides. To assess the validity of ATP, as a biomarker for rapid detection of mural surface contamination, obtained zones of cleanliness values, in range from 1.0 to 5.3, were compared to documented total microbial counts, ranging between seven and 247 CFU/cm2. Small coefficients of determination, 0.0106-0.0385, suggest poor correlation between microbial counts and surface ATP levels; however, zones of cleanliness values are of great help in determining the high points of contamination, aka 'hotspots', which should be given special attention during sampling and investigation using other methods. In addition, various aspects of the possible implementation of the ATP bioluminescence method in an integrated system of wall painting conservation are discussed.

Cyanobacterial effects in Lake Ludos, Serbia - Is preservation of a degraded aquatic ecosystem justified?

Cyanobacteria are present in many aquatic ecosystems in Serbia. Lake Ludos, a wetland area of international significance and an important habitat for waterbirds, has become the subject of intense research interest because of practically continuous blooming of cyanobacteria. Analyses of water samples indicated a deterioration of ecological condition and water quality, and the presence of toxin-producing cyanobacteria (the most abundant Limnothrix redekei, Pseudanabaena limnetica, Planktothrix agardhii and Microcystis spp.). Furthermore, microcystins were detected in plants and animals from the lake: in macrophyte rhizomes (Phragmites communis, Typha latifolia and Nymphaea elegans), and in the muscle, intestines, kidneys, gonads and gills of fish (Carassius gibelio). Moreover, histopathological deleterious effects (liver, kidney, gills and intestines) and DNA damage (liver and gills) were observed in fish. A potential treatment for the reduction of cyanobacterial populations employing hydrogen peroxide was tested during this study. The treatment was not effective in laboratory tests although further in-lake trials are needed to make final conclusions about the applicability of the method. Based on our observations of the cyanobacterial populations and cyanotoxins in the water, as well as other aquatic organisms and, a survey of historical data on Lake Ludos, it can be concluded that the lake is continuously in a poor ecological state. Conservation of the lake in order to protect the waterbirds (without urgent control of eutrophication) actually endangers them and the rest of the biota in this wetland habitat, and possibly other ecosystems. Thus, urgent measures for restoration are required, so that the preservation of this Ramsar site would be meaningful.

Cyanobacteria and cyanotoxins in fishponds and their effects on fish tissue.

Cyanobacteria can produce toxic metabolites known as cyanotoxins. Common and frequently investigated cyanotoxins include microcystins (MCs), nodularin (NOD) and saxitoxins (STXs). During the summer of 2011 extensive cyanobacterial growth was found in several fishponds in Serbia. Sampling of the water and fish (common carp, Cyprinus carpio) was performed. Water samples from 13 fishponds were found to contain saxitoxin, microcystin, and/or nodularin. LC-MS/MS showed that MC-RR was present in samples of fish muscle tissue. Histopathological analyses of fish grown in fishponds with cyanotoxin production showed histopathological damage to liver, kidney, gills, intestines and muscle tissues. This study is among the first so far to report severe hyperplasia of intestinal epithelium and severe degeneration of muscle tissue of fish after cyanobacterial exposure. These findings emphasize the importance of cyanobacterial and cyanotoxin monitoring in fishponds in order to recognize cyanotoxins and their potential effects on fish used for human consumption and, further, on human health.