Yana Ilieva

The engineering, resource, and financial constraints in space and spacecraft so far have not allowed the incorporation of biological components into a closed-loop bioregenerative life support system (BLSS), despite decades of research. The expected increase in deep-space exploration and planetary bases with limited access to Earth-based resources necessitates the development of self-sustaining hybrid BLSS technology. The created physicochemical systems, together with photosynthetic organisms and bacteria, aim to revitalize the air, produce food, and recycle nutrients and water in mutually beneficial mini-ecosystems. While plants are best in the function of food production and bacteria in waste recycling, the incorporation of microalgae would add immense benefits in optimizing the life support system (LSS) and increasing the degree of closure. Microalgal photobioreactors (PBRs) could perform wastewater treatment (WWT), removing the nitrogen (N) and phosphorus (P) in the human-derived wastewater (WW), and couple it with converting carbon dioxide (CO₂) from the cabin to oxygen (O₂) and food production. As microalgal WWT on Earth is an emerging field with engineering hurdles, power, mass, volume, microgravity fluid dynamics, and other constraints have also prevented their operations in space. However, in space vehicles, there is no need for large upscaling of a laboratory prototype system, and the WW effluent is easier to predict, facilitating microalgal extraplanetary use in comparison to Earth treatment plants. These factors, combined with the qualities of microalgae such as surface-to-volume efficiency, fast growth rate, high yield, and tolerability to WW, etc., have led to many preliminary testbeds, prototypes, and ground demonstrations from space agencies, space centers, and academia, which show promising results. Microalgal participation in space WWT is beyond current operational practice; however, PBRs are on the space agenda, and the scientific community is elaborating the technologies that would allow their successful implementation.

Coronaviruses are worldwide-distributed RNA viruses with zoonotic potential and the ability to jump from one host species to another, including humans. Even after the COVID-19 pandemic, the search for new, biologically active substances with anti-coronavirus activity continues to be a critical milestone for human health protection. In the framework of a complex engineering strategy, we cultivated the microalgal species Scenedesmus acutus in two different innovative types of flat-plate photobioreactors (PBR1 and K1) for CO₂ utilization and biomass production with special features. Isolated extracts from the microalgal biomass of each one were compared for their anti-coronavirus potential. The design of both PBRs allows a hydrodynamic regime to achieve best fluid flow distribution in their sections, therefore providing the optimal so-called flashing light effect. Of course, this is achieved under well-controlled operational conditions. A strain of beta coronavirus 1 (BCoV, bovine coronavirus) replicated in MDBK cells was used as an in vitro model for the evaluation of the antiviral activity of both extracts. The cell viability, number of survived BCoV particles, and cytopathic effect were evaluated after pre-incubation of the virus with the extracts or direct treatment. The extracts’ samples exhibited evident antiviral activity—extract 1 (from PBR1) in concentrations ≥ 200 µg/mL and extract 2 (from K1) in concentrations ≥150 µg/mL. The ddPCR result revealed significant diminishment of the BCoV particles in samples treated with higher concentrations of the extracts. The phytochemical analysis for certain main groups of compounds (flavonoids, polyphenols, carotenoids, and lipids) showed some differences for both extracts, which could be a possible reason for the observed difference in the antiviral activity. In conclusion, the innovative PBRs are a good platform for studying microalgal growth kinetics by applying different stress conditions from hydrodynamics and mass transfer subsystems. Both extracts showed promising potential for the isolation of metabolites with antiviral activity against BCoV and could be an object for future pharmacological investigations.

Urban areas and suburbs are facing many environmental problems, one of which is the increasing pollution due to the accumulation of cellulosic waste. This article presents a laboratory study on the microbial biodegradation processes of paper as a cellulosic substrate under anaerobic and mesophilic cultivation conditions. A bacterial consortium with cellulose-degrading activity, as well as 4 individual strains originating from a methanogenic anaerobic bioreactor (BRA), was isolated, identified, and characterized. The results demonstrated that the consortium degraded 57.14% of the cellulose matrix within 20 days. Among the individual colonies, colonies 1 and 2 (identified as Clostridium tertium and Agromyces rhizospherae) exhibited lower activities (35.37% and 34.79%, respectively), while colony 3 (Clostridium paraputrificum) displayed the highest activity (83.74%). The mixture of all four colonies achieved lower degradation (21.22%). The performed metagenomic analysis of the microbial consortium revealed a wide variety of different bacterial genera, among which Clostridium, Bacteroides, and Ruminiclostridium dominate, and the species Bacteroides oleiciplenus, Clostridium butyricum, and Ruminiclostridium papyrosolvens. Scanning electron microscopy visualized the adhesion and morphological features of the degrading microbial population. Additional experiments on the development of a laboratory model for the anaerobic biodegradation of cellulose were carried out in BRAs by using different working volumes. A maximal level of cellulose decomposition was achieved in the BRA with a working volume of 1 L, reaching 71.0% cellulose decomposition on day 20. Long-term storage studies confirmed the survival and well-preserved activity of the consortium and individual isolates, demonstrating their potential for the development of bioconversion technologies.

Objectives: Research on the antimicrobial effect of Hypericum plant constituents is very rarely accompanied by studies of the cytotoxic effect on cell lines. In the current study, besides microbiological tests, an investigation of the cytotoxicity of Hypericum active ingredients on five non-tumorigenic cell lines, as well as research into the effect on other factors of host homeostasis, was performed. Methods: The main methods applied included an MTT assay, the broth microdilution method (BMD), real-time PCR, live cell imaging with Hoechst dye, Western blot, an enzyme-linked immunosorbent assay (ELISA), and skin irritation test on rabbits. Results: The mean inhibitory concentrations (IC₅₀) of six selected agents—previously phytochemically characterized extracts and compounds—ranged from 0.63 to 48 µg/mL. Due to their strong antimicrobial effect and favorable cytotoxic profile, the extract RochC from Hypericum rochelii and the compound olympiforin B from Hypericum olympicum were selected for subsequent studies at their previously determined minimum inhibitory concentrations (MICs) against Staphylococcus aureus—0.625 and 1 µg/mL, respectively. These doses were lower than their IC₅₀ values and the maximum tolerated concentrations (MTCs), according to ISO 10993-5, Annex C, for fibroblast cells, including a human gingival line. The MIC values of RochC and Olympiforin B against the cariogenic Streptococcus mutans were 6 and 3 µg/mL, respectively, values lower than the IC₅₀ values of the gingival cells. Olympiforin B inhibited the gene expression of the staphylococcal biofilm-related genes icaA and icaD, while RochC induced icaA and had a versatile effect on icaD. The MIC values for lactobacilli strains were higher than for S. aureus. The phytoconstituents did not cause cytopathic effects or apoptosis in CCL-1 fibroblasts at 2 × MIC. However, the agents at 1 × MIC significantly induced Atg5 and Atg7, proteins related to autophagy. Cytochrome P450 was not induced in liver cells, with the exception of a dose of 2 × MIC of RochC. The agents did not irritate rabbit skin in vivo at a dose of even 10 × MIC. Conclusions: The extract and compound have potential for further pharmacological development.

Background/Objectives: Hypericum cerastoides (Spach) N. Robson is a lesser-known species with potential pharmacological importance. This study aimed to profile phenolic compounds in its aerial parts and assess biological activities of isolated constituents, focusing on radical-scavenging, anti-α-glucosidase, and pro-lipase effects. Methods: Phenolic compounds from H. cerastoides aerial parts were dereplicated via UHPLC-HRMS/MS. The structures of isolated compounds were determined using spectroscopic methods (1D and 2D NMR, UV, and HRMS-ESI). Radical-scavenging was evaluated by DPPH and ABTS assays; anti-α-glucosidase and pro-lipase activities were measured by LC-MS. Results: UHPLC-HRMS profiling of a hydroalcoholic extract tentatively identified and quantified 39 phenolic compounds, mainly flavonoids and hydroxycinnamic acid derivatives. Furthermore, two new phenolic compounds, namely hypercerastoside A (HC4) and hypercerastoside B (HC6), together with three known compounds, coumaroylquinic acid (HC1), myricetin-3-O-glycoside (HC2), and myricetin-3-O-galactoside (HC3), as well as two artifacts, namely methyl ester of chlorogenic acid (HC5) and hypercerastoside C (HC7), were isolated from the ethylacetate extract of the aerial parts of title plant. Compounds HC2, HC3, and HC5 displayed the highest radical-scavenging activity. The anti-α-glucosidase test showed that compounds HC1 (IC₅₀ = 44 µM) and HC3 (IC₅₀ = 206 µM) possessed similar activity to acarbose (IC₅₀ = 206 µM). Myricetin glycosides HC2 and HC3 enhanced lipase activity fivefold at 200 µM. Conclusions: H. cerastoides is a promising source of bioactive phenolic compounds with significant radical-scavenging and enzyme-modulating activities. These preliminary findings support further exploration of its therapeutic potential, especially for oxidative stress-related disorders, type 2 diabetes, and cachexia.

Nanotechnology offers alternative approaches to the discovery of anticancer drugs. Hydrophobic bioactive components can be included in the cores of amphiphilic nanocarriers, which leads to the formation of a water-dispersible product with improved bioavailability, facilitated excretion, and reduced systemic toxicity in the treated organisms. This study was aimed at the formation of polymer nanocarriers, loaded with anticancer drug precursor podophylotoxin (PPT) or PPT-containing juniper leaf extracts, seeking to study their antineoplastic activity in A-431 epidermoid carcinoma cells and HaCaT normal keratinocytes. The amphiphilic, biodegradable, and biocompatible MPEG-b-PLA diblock copolymer was self-assembled in aqueous media into nanosized particles, whose physicochemical characteristics were studied by dynamic light scattering, transmission electron microscopy, and other methods. High encapsulation efficiency was determined for the PPT component-loaded micelles. DNA fragmentation, cell cycle arrest, nuclear condensation, membrane lipid order assessment, reactive oxygen species, and apoptosis induction by the loaded nanocarriers in A-431 or HaCaT cells were analyzed by the comet assay, FACS, Hoechst DNA staining, Laurdan generalized polarization, and other methods. As a result of various cellular processes induced by the PPT component-loaded nanoparticles, effector caspase-3 and caspase-7 activation showed selectivity towards tumor cells compared to the normal cells. The newly obtained PPT-containing nanoparticles have applications as potential drugs in the prospective nanomedicine.

Background: Nowadays, the microbial degradation of cellulose represents a new perspective for reducing cellulose waste from industry and households and at the same time obtaining energy sources. Methods: We isolated and enriched two aerobic (at 37 °C and 50 °C) and one anaerobic microbial consortium from an anaerobic bioreactor for biogas production by continuous subculturing on peptone cellulose solution (PCS) medium supplemented with 0.3% treated or untreated Whatman filter paper under static conditions. Samples were taken every 7 days until day 21 to determine the percentage of cellulose biodegradation. We determined the antimicrobial resistance of aerobic and anaerobic consortia and some single colonies by disc diffusion method, against 42 clinically applied antibiotics. PCR analyses were performed to search for the presence of eight genes for cellulolytic activity and nine genes for antibiotic resistance. By metagenomics analysis, the bacterial and fungal genus distributions in the studied populations were determined. Results: Aerobes cultured at 50 °C degraded cellulose to the greatest extent (47%), followed by anaerobes (24–38%) and aerobes (8%) cultured at 37 °C. The bacterial sequence analysis showed that the dominant phyla are Bacillota and Bacteroidetes and genera—Paraclostridium, Defluvitalea, Anaerobacillus, Acetivibrio, Lysinibacillus, Paenibacillus, Romboutsia, Terrisporobacter, Clostridium, Sporanaerobacter, Lentimicrobium, etc. in a different ratio depending on the cultivation conditions and the stage of the process. Some of these representatives are cellulolytic and hemicellulolytic microorganisms. We performed lyophilization and proved that it is suitable for long-term storage of the most active consortium, which degrades even after the 10th re-inoculation for a period of one year. We proved the presence of ssrA, ssrA BS and blaTEM genes. Conclusions: Our findings demonstrated the potential utility of the microbial consortium of anaerobes in the degradation of waste lignocellulose biomass.

Compared to other reptile groups in Europe, lizards have generally been neglected and understudied in terms of microbiota research. In this study, we aimed to isolate, identify and characterize the aerobic cloacal microflora of wild-dwelling lizard hosts. We examined a total of 86 individuals from five species belonging to three families: the European green lizard (Lacerta viridis), the common wall lizard (Podarcis muralis), the meadow lizard (Darevskia praticola) (Lacertidae), the European snake-eyed skink (Ablepharus kitaibelii) (Scincidae) and the European slow worm (Anguis fragilis) (Anguidae) which co-occur in a low-mountain region in Western Bulgaria. In general, a similar composition of the resident microbial communities in the cloaca was found, accompanied by variation in the relative abundance of some bacterial taxa between the lizard species. A variety of Gram-negative and Gram-positive bacteria was isolated from the cloacal samples. Some of these bacteria are also known as opportunistic pathogens, both for hosts and humans. The bacterial species Hafnia alvei, Pseudomonas aeruginosa, Klebsiella oxytoca and representatives of Enterobacter spp., Citrobacter spp. and Enterococcus spp. were among the most prevalent.

A complex evaluation of antimicrobial activities of microalgae, including those relevant to wastewater treatment (WWT), in light of the integrated biorefinery concept, is performed. An example of this concept is linking a commercial microalgal system to plants, factories, or farms that emit polluted wastewater (WW). The microalgae would not only metabolize the pollutants—such as nitrogen (N) and phosphorus (P)—from the WW, thus fueling their biomass, but they would exert an antibacterial effect against the pathogenic bacteria there. The biomass then could be harvested and used for biofertilizers, biofuels, and bioplastics and might possibly be utilized as animal feed, antimicrobial and other pharmaceutical agents. A large amount of the research on the antimicrobial activity and WWT potential focuses on the families Chlorellaceae and Scenedesmaceae, which are also some of the most commercially used strains of microalgae. For that reason, they are the species chosen for the current review. Furthermore, the increasing antimicrobial resistance necessitates the search for antibiotic alternatives, and the antibacterial and antifungal activity of Chlorellaceae and Scenedesmaceae is very promising. Microalgae are rich in antibacterial compounds like polyunsaturated fatty acids (PUFAs), polysaccharides, carotenoids, proteins, etc., and for that reason, their extracts possess antimicrobial effects. The in vitro antimicrobial activity of Chlorellaceae and Scenedesmaceae families has varied in a broad range from low to strong activity or no effect. Several strains have fulfilled the criteria for outstanding and high activity, especially C. vulgaris and other Chlorellaceae spp., with an effect equal to or better than the control antibiotics. There were several strains with minimum inhibitory concentrations (MIC) below 80 µg/mL and even 10 and 1.5 µg/mL; some species also had inhibition zones (IZ) over 30 mm, even as high as 48 mm. In vivo results are also promising but scarce, and all this warrants further in vivo and in situ studies—from animal models to clinical and environmental trials. Altogether, important data in the light of the circle economy, the urgent necessity to decrease CO₂ emissions to fight climate change, and to curb the harmful influence of future pandemics are presented. This review paves the way for further utilizing the total potential of a microalgal system.

With the increasing rate of the antimicrobial resistance phenomenon, natural products gain our attention as potential drug candidates. Apart from being used as nutraceuticals and for biotechnological purposes, microalgae and phytoplankton have well-recognized antimicrobial compounds and proved anti-infectious potential. In this review, we comprehensively outline the antimicrobial activity of one genus of cyanobacteria (Arthrospira, formerly Spirulina) and of eukaryotic microalgae (Dunaliella). Both, especially Arthrospira, are mostly used as nutraceuticals and as a source of antioxidants for health supplements, cancer therapy and cosmetics. Their diverse bioactive compounds provide other bioactivities and potential for various medical applications. Their antibacterial and antifungal activity vary in a broad range and are strain specific. There are strains of Arthrospira platensis with very potent activity and minimum inhibitory concentrations (MICs) as low as 2–15 µg/mL against bacterial fish pathogens including Bacillus and Vibrio spp. Arthrospira sp. has demonstrated an inhibition zone (IZ) of 50 mm against Staphylococcus aureus. Remarkable is the substantial amount of in vivo studies of Arthrospira showing it to be very promising for preventing vibriosis in shrimp and Helicobacter pylori infection and for wound healing. The innovative laser irradiation of the chlorophyll it releases can cause photodynamic destruction of bacteria. Dunaliella salina has exhibited MIC values lower than 300 µg/mL and an IZ value of 25.4 mm on different bacteria, while Dunaliella tertiolecta has demonstrated MIC values of 25 and 50 μg/mL against some Staphylococcus spp. These values fulfill the criteria for significant antimicrobial activity and sometimes are comparable or exceed the activity of the control antibiotics. The bioactive compounds which are responsible for that action are fatty acids including PUFAs, polysaccharides, glycosides, peptides, neophytadiene, etc. Cyanobacteria, such as Arthrospira, also particularly have antimicrobial flavonoids, terpenes, alkaloids, saponins, quinones and some unique-to-them compounds, such as phycobiliproteins, polyhydroxybutyrate, the peptide microcystin, etc. These metabolites can be optimized by using stress factors in a two-step process of fermentation in closed photobioreactors (PBRs).