Halophilic and halotolerant microorganisms represent promising sources of salt-tolerant enzymes that could be used in various biotechnological processes where high salt concentrations would otherwise inhibit enzymatic transformations. Considering the current need for more efficient biocatalysts, the present study aimed to explore the microbial diversity of five under- or uninvestigated salty lakes in Romania for novel sources of hydrolytic enzymes. Bacteria, archaea and fungi were obtained by culture-based approaches and screened for the production of six hydrolases (protease, lipase, amylase, cellulase, xylanase and pectinase) using agar plate-based assays. Moreover, the phylogeny of bacterial and archaeal isolates was studied through molecular methods. From a total of 244 microbial isolates, 182 (74.6%) were represented by bacteria, 22 (9%) by archaea, and 40 (16.4%) by fungi. While most bacteria synthesized protease and lipase, the most frequent hydrolase produced by fungi was pectinase. The archaeal isolates had limited hydrolytic activity, being able to produce only amylase and cellulase. Among the taxonomically identified isolates, the best hydrolytic activities were observed in halotolerant bacteria belonging to the genus Bacillus and in extremely halophilic archaea of the genera Haloterrigena and Halostagnicola. Therefore, the present study highlights that the investigated lakes harbor various promising species of microorganisms able to produce industrially valuable enzymes.

Due to the presence of efflorescences and improper microclimate conditions for conservation, pink-pigmented areas were reported in two historic monuments in Northern and Central part of Romania. The aims of the present study were to find the nature of pink pigments observed on the pictorial layer, original and infilling mortar, to investigate the presence of carotenoids both on mural paintings and in the isolated halophilic bacterial strains and to preliminary characterize and identify the producing strains. Their role in the aesthetical biodeterioration of historic monuments was also pointed out. Obtained Raman spectra of the pink pigments extracted both from the isolated bacterial cultures (molecularly identified as mostly related to Halobacillus hunanensis and Halobacillus naozhouensis) and from the mural painting samples contain diagnostic bands of carotenoids. These results were confirmed by FTIR spectroscopy. The strong Raman signal of bacterial carotenoids detected on mural painting indicated their potential use as biomarker molecules in the evaluation of contamination and state of conservation of mural paintings and lithic monuments. Our results contribute to opening a new direction in cultural heritage restoration to assess the conservation status on the basis of interdisciplinary research, starting with spectroscopic methods (Raman, FTIR) and confirmed by microbiological analysis.

Salt-tolerant enzymes produced by halophilic and halotolerant microorganisms have been proposed to be used in various applications that involve high saline conditions. Considering their biotechnological significance and the current need for more efficient producers of such catalysts, the present study aimed to evaluate the extracellular proteolytic, esterolytic, cellulolytic and xylanolytic activities of some halotolerant strains, and to characterize their functional parameters. A total of 21 bacterial and fungal strains belonging to the genera Bacillus, Virgibacillus, Salinivibrio, Salinicoccus, Psychrobacter, Nocardiopsis, Penicillium, Aspergillus, and Emericellopsis were assayed by quantitative methods. Among them, the members of the Bacillus genus exhibited the highest catalytic activities. The exoenzymes produced by three selected Bacillus strains were active over wide ranges of salinity, temperature and pH. Proteases were active at 20–80 °C, pH 6–10, and 0–1 M NaCl, while esterases showed good catalytic activities at 20–80 °C, pH 7.5–10, and 0–4 M NaCl. Cellulases and xylanases were active at 20–80 °C, pH 5–10, and 0–5 M NaCl. Due to such properties, these hydrolases could be used in a newly proposed application, namely to clean aged consolidants and organic deposits accumulated over time from the surfaces of salt-loaded wall paintings.

Considering that most industrial processes are carried out under harsh physicochemical conditions, which would inactivate enzymes from commonly isolated mesophilic organisms, current studies are geared toward the identification of extremophilic microorganisms producing enzymes resistant to extreme salt concentrations, temperature and pH. Among the extremophiles, halophilic microorganisms are an important source of salt-tolerant enzymes that can be used in varying biotechnological applications. In this context, the aim of the present work was to isolate and identify halophiles producing hydrolases from the Atacama Desert, one of the harshest environments on Earth. Isolates were recovered from halite samples and screened for the presence of seven different hydrolase activities (amylase, caseinase, gelatinase, lipase, pectinase, cellulase and inulinase) using agar plate-based assays. From a total of 23 halophilic bacterial isolates, most showed lipolytic (19 strains) and pectinolytic (11 strains) activities. The molecular identification of eight selected isolates showed a strong similarity to members of the Halomonas and Idiomarina genera. Therefore, the present study represents a preliminary, but essential, step to identify novel biological sources of extremozymes in an environment once thought to be devoid of life.

One of the current research objectives is the development of new films for the conservation of glass heritage objects. The value of historical glass objects is given by the technology and raw materials used in production as well as their transparency and color. Their colors are correlated with oxide composition rich in transitional metals, which decrease resistance of corrosive agents from the atmosphere. In this paper, SiO2-ZnO gels have been designed to protect historical glass objects. The sol–gel method used to obtain gels is a powerful tool for functionalizing different materials. An important functionalization is the antibacterial activity. By applying a gel, the coated material is able to decrease the growth of bacteria. After deposition, some gels must be strengthened by heat treatment. The effect of ZnO content (10 mol% and 20 mol%) on the properties of the studied gels was investigated by Differential scanning calorimetry (DSC), Fourier transform infrared (FTIR), X-ray diffraction (XRD), Scanning electron microscopy (SEM), and antibacterial tests. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and the halotolerant bacterium, Virgibacillus halodenitrificans, isolated from a salt crystal from Unirea mine, Slănic Prahova, Romania, were used. The gel Gel 2 (SiO2-ZnO (20 mol%)) showed the best properties.

Cellulases are a group of enzymes that are used in many biotechnological processes. Since most of the enzymes synthesised by mesophilic microorganisms are unstable in industrial environments, it is necessary to direct research towards extremophile cellulolytic microorganisms because the enzymes synthesised by them are stable and active even in harsh physicochemical conditions. In the present investigation, our aim was to isolate and identify some microbial cellulolytic strains from a hypersaline lake located in Romania and to determine their optimal growth conditions. Of a total of 25 microbial strains isolated, only one extreme halotolerant bacterial strain was able to produce an endoglucanase. Based on molecular identification, we identified this cellulolytic strain as a species of Bacillus genus, most closely related to Bacillus zhangzhouensis. Optimal growth conditions were found to be at 15˚C, pH 7.5 and 2 M NaCl. Endoglucanase activity of this bacterial strain is influenced by both salinity and temperature. The most significant endoglucanase activity was detected in the presence of 3 M NaCl, after 72 h of incubation at 15˚C. In this situation, the amount of glucose released from a volume of 0.5 mL of 2% (w/v) carboxymethyl cellulose substrate is equivalent to 2.05 mg. In conclusion, this study represents the first preliminary characterization of a B. zhangzhouensis strain that has the ability to degrade cellulose and that demonstrates tolerance to high salt concentrations.

This study highlights the decomposing role through the hydrolytic activities of fungi isolated from natural environments represented by brackish and hypersaline lakes in Romania. Novel strains belonging to the Penicillium, Aspergillus, and Emericellopsis genera were isolated and screened for the ability to produce extracellular hydrolytic enzymes, i.e., proteases, lipases, amylases, cellulases, xylanases, and pectinases. According to salt requirements, they were classified as moderate halophilic and halotolerant strains. Agar plate-based assays with Tween 80, slide cultures with organic deposits, and quantitative evaluation allowed the selection of Aspergillus sp. BSL 2-2, Penicillium sp. BSL 3-2, and Emericellopsis sp. MM2 as potentially good decomposers of organic matter not only in lakes but also on deposits covering the mural paintings. Experiments performed on painted experimental models revealed that only Penicillium sp. BSL 3-2 decomposed Paraloid B72, transparent dispersion of casein, beeswax, sunflower oil, and soot. Moreover, using microscopic, spectroscopic, and imaging methods, it was proved the efficiency of Penicillium sp. BSL 3-2 for decomposition of organic deposits artificially applied on frescoes fragments.

Marine microorganisms have evolved a large variety of metabolites and biochemical processes, providing great opportunities for biotechnologies. In the search for new hydrolytic enzymes and antimicrobial compounds with enhanced characteristics, the current study explored the diversity of cultured and uncultured marine bacteria in Black Sea water from two locations along the Romanian coastline. Microbial cell density in the investigated samples varied between 65 and 12.7 × 103 CFU·mL−1. The total bacterial community identified by Illumina sequencing of 16S rRNA gene comprised 185 genera belonging to 46 classes, mainly Gammaproteobacteria, Alphaproteobacteria, Flavobacteriia, and 24 phyla. The 66 bacterial strains isolated on seawater-based culture media belonged to 33 genera and showed variable growth temperatures, growth rates, and salt tolerance. A great fraction of these strains, including Pseudoalteromonas and Flavobacterium species, produced extracellular proteases, lipases, and carbohydrases, while two strains belonging to the genera Aquimarina and Streptomyces exhibited antimicrobial activity against human pathogenic bacteria. This study led to a broader view on the diversity of microbial communities in the Black Sea, and provided new marine strains with hydrolytic and antimicrobial capabilities that may be exploited in industrial and pharmaceutical applications.