Speleomycology of Air in Stopica Cave (Serbia).

Fungi can colonize organic matter present in subterranean sites and have a significant role as dwellers in different microniches of cave habitats. In order to analyze the content of airborne fungal propagules in different parts of "Stopica Cave," a touristic site in Serbia, air sampling was carried out in three seasons during 2020, prior to and during the onset of COVID-19 pandemic. Culturable mycobiota was identified using both microscopic techniques and ITS region/BenA gene barcoding, while multivariate analyses were employed to establish the link between fungal taxa and different environmental factors. The maximal measured fungal propagule concentrations were recorded during spring sampling which were based on fungal propagule concentration categories; the cave environment matches the category V. A total of 29 fungal isolates were identified, while Aspergillus, Cladosporium, Fusarium, Lecanicillium, Mucor, and Penicillium were the most diverse genera. According to the trophic mode, most of the isolated fungal species were pathotrophs (75.86%), but when regarding ecological guilds, the most dominant were undefined saprobes and animal pathogens (41.38% for each). Show caves are especially vulnerable to human impacts, and the fungal propagules' concentration within the caves could be good indices for the level of ecological disturbance.

Early Antioxidative Response to Desiccant-Stimulated Drought Stress in Field-Grown Traditional Wheat Varieties.

Extended drought affects the production and quality of wheat (Triticum aestivum L.), one of the world's most important food crops. Breeding for increased drought resistance is becoming increasingly important due to the rising demand for food production. Four old traditional Croatian wheat cultivars were used in the present study to examine the early antioxidant response of flag leaves to desiccant-stimulated drought stress and to identify drought-tolerant cultivars accordingly. The results indicate that the enzymatic antioxidant system plays the most significant role in the early response of adult wheat plants to drought stress and the removal of excessive H2O2, particularly GPOD and APX. Nada and Dubrava cultivars revealed the strongest activation of the enzymatic defense mechanism, which prevented H2O2 accumulation and lipid peroxidation. Additionally, the Nada cultivar also showed increased synthesis of proline and specific phenolic compounds, which both contribute to the increased stress tolerance. Among the cultivars investigated, cultivar Nada has the broadest genetic base, which may explain why it possesses the ability to activate both enzymatic and non-enzymatic defense mechanisms in an early response to drought stress. This suggests that old traditional wheat cultivars with broad genetic bases can be a valuable source of drought tolerance, which is especially important given the current climate change.

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.

Effect of the Addition of Hulless Barley Flour on the Quality of Short-Dough Cookies.

Short-dough cookies are one of the most popular cereal-based products in the world, but usually they are considered as foods with a low nutritional value. Therefore, the aim of this study was to investigate the possibility of replacing part of the wheat flour (WF) with hulless barley flour (HLBF), in order to improve the functional properties and nutritional value of the cookies. Cookies were prepared from composite flours in the ratios HLBF:WF 0:100, 25:75, 50:50, 75:25, and 100:0. The results show that as the HLBF content in the composite flour increases, the viscosity of the corresponding HLBF:WF slurries also increases, due to the high dietary fiber content (especially the high ß-glucan content), which is significantly higher when HLBF is included in the formulation. The addition of HLBF decreases the spread factor of the cookies, and increases their softness compared to the control cookies (100% WF), but these changes are not statistically significant up to 50% HLBF addition. The color of the HLBF:WF cookies is not significantly affected. A significant increase in total phenolic content (TPC) and increased antioxidant capacity (AOC) are observed in the HLBF:WF cookies. In addition, sensory evaluation confirms that WF can be replaced by up to 50% with HLBF without significant deterioration of the organoleptic properties of the cookies. It can be concluded that hulless barley serves as a promising raw material if the nutritional and functional properties of cereal-based products are to be improved.

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.

Phenolic Acid Profiles and Antioxidant Activity of Major Cereal Crops.

Phenolic acids (PAs) are a dominant group of phenolic compounds in cereals, existing mostly bound to compounds of cell wall. In this study, a total of 25 cereal grain samples, including wheat, winter and spring barley, corn, and popcorn, were evaluated for bound PAs and antioxidant activity in a two-year field trial. The PA contents, determined by HPLC, were significantly affected by cereal type. The mean total PA content was highest in popcorn and corn (3298 and 2213 µg/gdm, respectively), followed by winter and spring barley (991 and 908 µg/gdm, respectively) and wheat (604 µg/gdm). Ferulic acid was the most abundant, accounting from 62% to 83% of total PAs (in popcorn and winter and spring barley, respectively). Across cereals, p-coumaric (35-259 µg/gdm) and p-hidroxybenzoic (45-79 µg/gdm) were also dominant, while in corn and popcorn o-coumaric (71 and 89 µg/gdm, respectively) also occurred in higher content. The mean total phenol content ranged from 853 µg GAE/gdm (wheat) to 1403 µg GAE/gdm (winter barley) with DPPH scavenging activity from 14% to 67%, respectively. A significant influence of crop years on the ferulic acid and total PA content was found, while the variability of other PAs was dependent on the cereal type. The results indicated a high health benefit potential of selected cereals.

Distribution of ß-Glucan, Phenolic Acids, and Proteins as Functional Phytonutrients of Hull-Less Barley Grain.

Two hull-less barley varieties were roller-milled, and breaks (B) and reduction flours (C), shorts, and bran were collected. Shorts, which mainly originate from endosperm cells with a smaller amount of the outer layers, had the largest yield (48.87-51.54%). Ash (0.82-3.10%) and protein (9.95-14.8%) increased from flours toward shorts and bran, while starch decreased (82.31-48.69%). In contrast to clear distribution differences in protein content (bran > shorts > C > B), albumins/globulins content was lowest in bran (0.78-0.90 g/100 gdw), and their distribution between fractions was uneven and genotype dependent. Distribution of hordeins (6.69-10.49 g/100 gdw) was more distinct and generally decreased in order from bran > B > shorts > C. The proportion of nutritionally poor C-hordeins in total hordeins varied from 28.33% to 30.24%, without significant differences between fractions. The ß-glucan content varied from 0.80% to 7.49% with decreasing content in the order bran, shorts > C > B. Shorts and bran could be classified as moderate and high ß-glucan flour (5.70-7.22%). The total phenolic and antioxidant activities ranged from 0.91 to 2.21 mg GAE/gdw and 28.81-72.06%, respectively. Ferulic and sinapic acids determined by high-performance liquid chromatography (HPLC) were major contributors to the antioxidant activity (45.16-1026.91 ug/gdw and 18.93-206.52 ug/gdw), respectively. The yield and high content of phytonutrients make hull-less barley shorts suitable for the production of health-promoting food and food supplements.

Screening of Various Metabolites in Six Barley Varieties Grown under Natural Climatic Conditions (2016-2018).

Climatic changes influence considerably the distribution and occurrence of different secondary metabolites in cereals. The aim of this investigation was to assess the changes in metabolite prevalence observed in six different winter barley varieties over a statistically significant period of three years by linking agro-climatic conditions with metabolite concentrations in chosen samples. The results showed that temperatures and precipitation levels varied during the observed timeframe and that the multi-toxin concentrations followed the trend of changing climatic conditions depending on the variety. All quantified (fungal) metabolites showed significant variations throughout the years and, for some (tryptophol and the cyclic dipeptides cyclo(L-Pro-L-Tyr) and cyclo(L-Pro-L-Val)), an unexpected, but clear connection can be made with temperature changes and precipitation levels during the growing season.

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.

Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture.

Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.

Influence of carboxymethyl dextran and ferric citrate on the adhesion of CHO cells on microcarriers.

Due to the inherent risks of animal-derived raw materials, the biopharmaceutical industry has an increasing demand for serum-free and protein-free media for industrial cell culture bioprocesses. The absence of serum often changes the characteristics of mammalian cells, especially growth, productivity, and adherence properties. This study is mainly focused on the influence of media additives on cell adherence characteristics. An array of different carboxymethyl dextrans and different ferric citrate concentrations was tested with a number of CHO clones, using standard cell culture Roux-flasks and Cytoline 1 macroporous microcarriers. A prototype mixing system with controlled shear force input was developed as a screening system for adherence characteristics. The results of this evaluation revealed a negatively correlated dose-dependent influence on adhesion for ferric citrate. It was also found that certain carboxymethyl dextrans are capable of increasing the adherence on Roux-flasks and microcarriers.