DOI: 10.3897/biorisk.22.117668

Authors: Stephany Toschkova, Sevginar Ibryamova, Darina Ch. Bachvarova, Teodora Koynova, Elitca Stanachkova, Radoslav Ivanov, Nikolay Natchev, Tsveteslava Ignatova-Ivanova

Abstract: One of the main problems of the world's oceans, reported by many scientific studies, is the microplastic pollution. Within the Black Sea, one of the main sources of pollution is the same, which is caused by the diverse anthropogenic activities. The present study demonstrated detailed microplastics contamination of in five fish species important for the commercial fishing (Garfish, Мullet, Knout goby, Pontic shad and Mediterranean horse mackerel). They were collected from the Sozopol area on the Bulgarian Black Sea coast. Within each microplastic morphological group, three size classes were recognised: 100–200 µm, 25–100 µm and ≤ 25 µm. Microplastics were found in all studied tissues of the fish, but in varying proportions of pellets, fibres and fragments. Pellets were most frequently isolated, followed by irregularly-shaped fragments and fibres were the least numerous. The bulk of insulated plastics are made of polyethylene (PE) and polyethylene terephthalate (PET). Our results pointed out serious pollution with plastic particles in the Bulgarian Black Sea aquatory, which, in the future, may seriously affect the health of the fish population and also human health.

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DOI: 10.3897/biorisk.21.111253

Authors: Sevginar F. Ibryamova, Stephany Toschkova, Darina Ch. Bachvarova, Elitca Stanachkova, Radoslav I. Ivanov, Nikolay D. Natchev, Tsveteslava V. Ignatova-Ivanova

Abstract: In the present work, we investigated the dynamics of the microbial communities in the wedge clam Donax trunculus (Linnaeus, 1758) from the Bulgarian coastal waters of the Black Sea. The samples were collected in the period of January 2020 until December 2022 from Arkutino, Ahtopol, Obzor and Tsarevo. The BIOLOG system was used for microbiological determination. In our investigation were isolated the following microorganisms: Enterococcus cancerogenus, Enterococcus hirae, Escherichia vulneris, Citrobacter farmeri, Acinetobacter gyllenbergii, Enterococcus hirae, Escherichia vulneris, Enterobacter cloacae, Escherichia hermannii, Pseudomonas mendocina, Pseudomonas fulva, Pseudomonas alcaligenes, Pseudomonas putida, Acinetobacter johnsonii, Acinetobacter gyllenbergii, Enterococcus hirae, Escherichia vulneris, Enterococcus gallinarum, Citrobacter sedakii, Pseudomonas putida, Streptococcus lugdunensis, Enterococcus casseliflavus, Vibrio cincinnatiensis, Vibrio alginolyticus, Vibrio parahaemolyticus, Enterococcus hirae, Streptococcus aureus, Staphylococcus lugdunensis and Enterococcus casseliflavus. During the winter period, we detected the presence of Pseudomonas sp. – P. alcaligenes, P. putida, and A. gyllenbergii. In the autumn months we isolated C. sedakii, C. farmeri, A. gyllenbergii, A. johnsonii, P. fulva and E. casseliflavus. In the spring, E. cancerogenus, E. hirae and Pseudomonas mendocina were found. During the summer, the highest biodiversity of microorganisms - E. hirae, E. vulneris, E. cloacae, E. gallinarum, P. putida, V. cincinnatiensis, V. alginolyticus, V. parahaemolyticus, S. aureus, E. hermannii and S. lugdunensis were registered. Although our three-year research showed that some species are permanent and others are transient, we tend to accept the conclusion that there is only a transient microbiota in mussels and it changes depending on environmental conditions or is a result of pollution of the Black Sea.

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DOI: 10.3897/biorisk.21.111983

Authors: Aleksandrs Petjukevičs, Inta Umbraško, Natalja Škute

Abstract: This study describes an express method for identifying microorganisms: Pseudomonas aeruginosa by standard Raman spectroscopy, without surface-enhanced Raman spectroscopy (SERS). The short-wavelength 514 nm Ar-Ion laser was used for P. aeruginosa spectral identification in the Raman shift range from 3200 cm−1 to 200 cm−1. The research results showed a high analytical and diagnostic sensitivity of the technology to the express identification of P. aeruginosa and can be used as one of the reliable methods. The proven technology is promising for further research of other microorganisms due to several significant advantages of the method. It does not require long-term cultivation of bacteria and special sample preparation, additional expensive reagents or consumables.

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DOI: 10.3897/biorisk.20.97581

Authors: Gloria Georgieva, Trayana Nedeva, Marina Badalova, Veronika Deleva, Valentin Savov

Abstract: Plant Growth Promoting Rhizobacteria (PGPR) represent a microbial community that exerts growth-promoting capabilities in plants by various mechanisms. Among the PGPR genera, Pseudomonas spp. deserves special attention. It is due to its characteristic traits, like the production of phytohormones and siderophores, solubilization of minerals and phosphates, and plant protection from biotic and abiotic stress. These PGPR properties depend on the microorganism and its plant counterpart. The use of microbial strains as bioinoculants must consider the physiological and economic aspects of the process, and the plant growth stimulating effect has to be checked and proved. This study aimed to explore the PGP capacity of Pseudomonas putida 1046 strain in a model plant system of the economically important corn culture (Zea mays). The effect of the strain’s metabolic status on the plant germination capacity was evaluated. Bacterial cultures, grown 16 h and 48 h, were explored for the treatment of the corn seeds at three experimental concentrations: 0.1, 0.2, and 0.4%, and monitoring of their germination capacity through the growth indicators length of the radicle, length of the coleoptile, and the number of lateral roots. The data obtained outline the positive effect of Pseudomonas putida 1046 on the germination capacity of corn when applied at 0.2% concentration. The in vitro treatment of the model plants with 0.2% suspension resulted in a 22.87%–28.33% increase in the length of the radicle, a 35.96%–49.56% increase in the length of the coleoptile, and a 5.41–16.67% increase in the number of the lateral roots. High values of the vigour index (2125 for 16 h and 2721 for 48 h culture) were also registered. The strain’s ability to produce siderophores of hydroximate type and exhibit phosphate solubilizing activity is proved. The optimal treatment parameters of the corn seeds comprise the application of 0.2% suspension of 16 h grown Pseudomonas putida 1046 strain for five days.

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DOI: 10.3897/biorisk.20.97795

Authors: Michaella Petkova, Nadezhda Nankova, Viktoriya Kancheva, Silvena Boteva, Anelia Kenarova, Galina Radeva

Abstract: This study presents the distribution of bacterial and fungal abundances in long-term copper (Cu) contaminated soils in Topolnitsa-Pirdop valley – a highly industrialized zone with a number of mines and processing plants for copper and other non-ferrous metals. The bacterial (16S rRNA gene copies) and fungal (ITS rRNA gene copies) were estimated using quantitative PCR technique in five topsoils, differently Cu contaminated (ranging from 28.05 to 198.9 mg kg-1). Bacterial abundance varied in a range of 1.68 × 1011 to 3.24 × 101116S rRNA genes, whereas fungi amounted from 1.95 × 108 to 6.71 × 108 ITS rRNA genes. Fungal and bacterial abundances were significantly (fungi) and insignificantly (bacteria) influenced by Cu contamination. The fungal/bacterial ratio related negatively with soil Cu, which shifted microbial communities’ structure towards bacterial dominance. Since the ratio between bacteria and fungi are vital in explaining many soil functions, the calculated changes in this ratio indicated deterioration in soil quality, being of primary importance for plant production.

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DOI: 10.3897/biorisk.20.104660

Authors: Kalina Danova

Abstract:

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DOI: 10.3897/biorisk.18.80357

Authors: Sevginar Ibryamova, Stephany Toschkova, Borislava Pavlova, Elitca Stanachkova, Radoslav Ivanov, Nikolay Natchev, Nesho Chipev, Tsveteslava Ignatova-Ivanova

Abstract: This paper presents a study on the microbial status of different fish species and their habitats in the Bulgarian Black Sea area. The samples were collected in the period of January 2021 until March 2021. The fish species we used in this study were Black Sea turbot (Scophthalmus maximus), round goby (Neogobius melanostomus), shore rockling (Gaidropsarus mediterraneus) and European anchovy (Engraulis encrasicolus). The BIOLOG system was used for microbiological determination. From the different fish species, different species of microorganisms were isolated (using selective nutrient media). From the torbut, we isolated species Enterococcus villorum with 24 × 103 cells in 1 ml, Moraxella nonliquefaciens with 70 × 103 cells in 1 ml and Pseudomonas synxantha with 123 × 103 cells. Pseudomonas putida was isolated from the round goby with 20 × 103 cells in 1 ml. The species Streptococcus entericus with 123 × 103 cells in 1 ml was isolated from the shore rockling. Pseudomonas fulva with 60 × 103 cells in 1 ml was isolated from the European anchovy. A total of 223 × 103 cells in 1 ml of Pseudomonas agarici were isolated from Trachinus draco. Pseudomonas tolaasii with 145 × 103 cells in 1 ml were isolated from Merlangius merlangus. A different species of bacteria of the genus Pseudomonas was found for each of the investigated species of Black Sea fish. Apparently, the species Pseudomonas is characteristic of marine Teleostei and is important for the life and metabolism of these vertebrates. These microorganisms probably are resident species and developed not as result of pollution or environmental change.

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DOI: 10.3897/biorisk.17.77484

Authors: Desislava Mantovska, Detelina Petrova, Lybomira Yocheva, Zhenya Yordanova

Abstract: Stachys thracica Davidov – Thracian woundwort is a Balkan endemic plant included in The Red Data Book of Bulgaria with conservational status “rare”. The plants from genus Stachys have a long history of use to treat various diseases, inflammatory conditions, coughs, ulcers, genital tumors, and infected wounds. Due to its limited distribution the information on the biological activity and chemical composition of S. thracica is rather scarce. The aim of the present research is the comparative determination of the antimicrobial activity of methanolic extracts obtained from in situ wild, in vitro cultivated and ex vitro adapted S. thracica plants. The in vitro shoot culture of the Thracian woundwort was maintained in hormone-free MS medium under controlled environmental conditions. The methanolic extracts from in situ, in vitro cultivated and ex vitro adapted S. thracica plants were active mainly against Gram-negative bacteria. All three extracts showed equal activity against Acinetobacter calcoaceticus. The establishment of in vitro shoot culture and its subsequent adaptation in ex vitro conditions was an appropriate alternative approach for the ex situ conservation of S. thracica as well as for the study of its biological activity.

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DOI: 10.3897/biorisk.17.77097

Authors: Sevginar Ibryamova, Borislava Pavlova, Elitca Stanachkova, Seniha Salim, Aysel Lyatif, Dimitar Dimitrov, Darina Bachvarova, Nesho Chipev, Nikolay Natchev, Tsveteslava Ignatova-Ivanova

Abstract: The main goal of the present study was to investigate the impact of the state of the environment on the microbiota of the wedge clam Donax trunculus (Linnaeus, 1758) from the region of Arkutino (Bulgarian Black Sea aquatory). The species Enterococcus hirae was isolated during the summer (from May to August). The species P. mendocina prefers the warmer months and the species P. alcaligenes the colder ones. The temperature followed a course of decrease during the period September 2020 to January 2021, followed by a slow increase from February 2021. Comparing May 2020 with May 2021, it became evident that in 2021 the temperature was 1.5 °C lower. We can say that the number of the species P. alcaligenes was twice as high in May 2021. It is likely that this species preferred lower optimum temperatures and constant other parameters. For the species Enterococcus hirae such dependence was not observed - the number remained constant in May, but with increasing temperature the number of microorganisms decreased during the summer months. The species seemed to preferably develop at pH 7.78. The species A. gyllenbergii preferably grows at temperatures between 20.3–25.7 °C and the optimal temperature was 25.7 °C. For C. farmeri the optimum conditions were temperature 26.2 °C and pH 7.3. The species E. vulneris was probably related not only to the increase in water temperature, but also to the anthropogenic factor, as it was found only in July.

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DOI: 10.3897/biorisk.17.77463

Authors: Mihaela Belouhova, Dobromira Yaneva, Yana Topalova

Abstract: To stimulate plant development in phytoremediation or in the cultivation of non-food crops in potentially contaminated soils, a biotechnologically created product could be applied. The aim of this study was to explore the possibility of creation of biofertiliser, based on activated sludge combined with bacterial strain with detoxifying and plant growth promoting properties. The presented study is focused on the effect of phenol in the following concentrations: 5 mg/l, 100 mg/l, 250 mg/l, 500 mg/l and 1000 mg/l on the metabolic activity of Brevibacillus laterosporus BT271. The gradual increased concentration of phenol was used to study the metabolic activity of mineralised activated sludge and B. laterosporus BT271. The CTC/DAPI staining showed high activity of the bacteria even at the highest concentration. The greatest amount of biomass was accumulated at 5 mg/l phenol (4.44 × 107 cells/ml). At this toxicant concentration, a total dehydrogenase activity of 5.72 × 10-4 μg H+/ml*min was found. Studies of the metabolic activity of microorganisms in experiments involving a combination of mineralised activated sludge, B. laterosporus BT271 and phenol at three concentrations (5 mg/l, 250 mg/l and 1000 mg/l) showed the highest value for dehydrogenase activity in the variant with average phenolic concentration (up to 6.39 × 10-6 μg H+/ml*min. The results proved the detoxification potential of B. laterosporus BT271 when different concentrations of phenol were present. The combination of a mineralised activated sludge and selected highly active biodegrading B. laterosporus BT271 showed valuable properties of detoxification and metabolic activity and keep these potentials up to 1000mg/l phenol.

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DOI: 10.3897/biorisk.17.77306

Authors: Lubov Sharapova, Nadezhda Yurina

Abstract: Proteins similar to Hli (high light inducible) proteins of cyanobacteria are present in all photosynthetic eukaryotes and are necessary for survival in various stressful conditions, although their exact function is not fully understood. In current study, the recombinant stress-induced protein HliA of cyanobacterium Synechocystis was isolated and characterised for the first time. The synthetic gene of HliA protein was created and cloned into plasmid for expression of recombinant protein with Hisx6-tag at the C-terminus in bacteria. Recombinant HliA protein of Synechocystis was isolated by metal-affinity chromatography. The HliA protein was reconstituted with chlorophyll a and carotenoids. Using circular dichroism spectroscopy, it was shown that chlorophyll a and carotenoids interact in vitro with the HliA protein. The binding of pigments to the HliA protein favours the protective function of this protein. Apparently, Hli proteins are involved in the coordinated delivery of pigments for the biogenesis of photosynthetic complexes, thereby reducing the risk of accumulation of phototoxic free chlorophyll molecules. Current results are important for understanding the processes of photoprotection in either cyanobacteria or algae and higher plants.

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DOI: 10.3897/biorisk.17.77381

Authors: Veronika Yordanova, Yovana Todorova, Mihaela Belouhova, Lyubomir Kenderov, Valentina Lyubomirova, Yana Topalova

Abstract: The upper Iskar sub-catchment is one of Bulgaria’s most important economic and socially significant water sources because of its role in supplying Sofia with drinking water. Among the critical factors that carry potential high-risk levels for water quality in this hydrosystem are the discharge from the Samokov Wastewater Treatment Plant (WWTP), diffuse pollution from agriculture, and the percolation of untreated sewage from the small villages. In this study, we assessed the effect of treated wastewater effluent on water quality, and on the ecological state and microbial communities in the river sector of Samokov’s WWTP discharge area. The assessment was based on the complex use of chemical and microbiological indicators and biological quality elements. The concentrations of organics, nutrients and microcomponents were determined with results confirming the expected increase for parameters associated with the discharge of urban wastewater. The ecological state, according to macrozoobenthos indicators, was “good” throughout the river sector but local deterioration was registered in a proximal location downstream of the WWTP outfall. The analysis of stream water and bed sediment microbial communities by a fluorescent technique showed the high metabolic activity and intensive transformation processes in addition to high abundance registered with standard cultivation methods. The importance of the studied sub-catchment for the functioning of the urban water cycle, and for the quality of Sofia’s drinking water, underlines the need to extend an existing monitoring program with a more detailed assessment of the environmental impact of wastewater discharge.

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DOI: 10.3897/biorisk.17.76939

Authors: Michaella Petkova, Anelia Kenarova, Silvena Boteva, Stela Georgieva, Christo Chanev, Galina Radeva

Abstract: The non-target effect of the fungicide QuadrisR on the bacterial community from grassland loamy sand (LS) and cropland clay loam (CL) soils with unknown history of fungicide usage was investigated. QuadrisR was applied to soil mesocosms at 0.0 mg kg-1 (Az0), 2.90 mg kg-1 (Az1), 14.65 mg kg-1 (Az2) and 35.0 mg kg-1 (Az3) calculated towards the active ingredient azoxystrobin (Az). Response of bacterial communities to QuadrisR was investigated during a 120-day incubation experiment, evaluating the shifts in bacterial catabolic profiles by the community-level physiological profiling (CLPP) technique and Biolog EcoPlatesTM method. QuadrisR decreased the overall catabolic activity (AWCD) of soil bacterial communities and the rate of decrease was independent of soil type and fungicide concentration. Fungicide affected negatively the utilisation of amines and positively that of amino acids in both soil types, whereas the effects on other carbon guilds (carbohydrates, carboxylic acids and polymers) corresponded closely to the respective soil type and fungicide concentration. Results indicated the presence of non-target effects of QuadrisR on bacterial functioning; hence, it is important to address the fungicide side-effects on soil health.

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DOI: 10.3897/biorisk.17.77458

Authors: Radina Nikolova, Michaella Petkova, Nikolai Dinev, Anelia Kenarova, Silvena Boteva, Dimitar Berov, Galina Radeva

Abstract: In the present study, the correlation between bacterial abundance and soil physicochemical properties along the heavy metal contamination gradient in the area of non-ferrous metal processing plant was assessed. Our results showed that bacterial abundance (number of heterotrophic bacteria and number of 16S rRNA gene copies) decreased with 45–56% (CFU) and 54–87% (16S rRNA gene) along the Zn, Pb and Cd contamination gradient. The total bacterial abundance (16S rRNA gene) increased exponentially in contrast to the abundance of heterotrophic bacteria. The reduction of bacterial abundance in heavily contaminated soil indicated that the soil properties (soil pH, total organic carbon, inorganic ions, soil texture) could modify the effects of heavy metals and the response of microorganisms to that stress in long-term contaminated soils.

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DOI: 10.3897/biorisk.15.58455

Authors: Jessica Borbolla-Vazquez, Paul Ugalde-Silva, José León-Borges, Job Alí Díaz-Hernández

Abstract: The large increase in population in Cancun, Mexico has increased domestic, agricultural and industrial activities, resulting in inadequate solid and liquid waste management that can affect underground aquifers. One of the factors which affects water quality is coliform bacteria. The present study focused on determining the presence of total and faecal coliforms in ten urban cenotes in Cancun. Sampling was carried out in the dry and rainy seasons of 2018. The Most Probable Number (MPN) technique was used to determine the concentration of coliform bacteria. The results from the analyses indicate that the ten cenotes are contaminated with total and faecal coliforms. Additionally, the concentration of coliforms increases during the rainy season. We conclude that all the cenotes are contaminated with faecal coliforms and suggest that more studies are necessary to determine the origin of this contamination and the impact on the ecosystem.

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DOI: 10.3897/biorisk.12.12156

Authors: Sreenivas Reddy Bathula, A. Rakhimol

Abstract: This report recapitulates the diverse aspects of biological safety. Biological laboratory is a space that facilitates the handling and storage of microorganisms, their components or their derivatives. Laboratories that handle dangerous pathogens have to act in a responsible manner to manage the safety and security threats posed by these pathogens. This necessity was foreground in the December 2008 World at risk report, which specifically demanded bioscience laboratories that handle dangerous pathogens to implement a unified laboratory biorisk management framework to enhance their safety and security. The report also discusses the guidelines of biosafety regulations provided by World Health Organization (WHO) that are necessary to adequately and sustainably manage these biorisks and helps in better understanding of risk governance approaches for laboratories that handle dangerous pathogens to achieve the ultimate goal of minimizing or preventing the occurrence and consequences of human error within the laboratory environment: the biorisk management approach, composed of biosafety, laboratory biosecurity and ethical responsibility. It preferably provides an agreement between authorities, the public, and the scientific community establishing trust and societal safety and security, while enabling the continued progress of science. Biorisk management approach demonstrates that biorisks in all their potential forms are appropriately addressed, managed and minimized. Thus, biorisk management has become an important aspect of the development and sustainability of biological activities.

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DOI: 10.3897/biorisk.3.29

Authors: Aharon Oren, Danny Ionescu, Muna Hindiyeh, Hanan Malkawi

Abstract: The freshwater thermal springs of Zerka Ma'in, located in Jordan in the mountains of Moab east of the Dead Sea, are densely inhabited by cyanobacteria up to the highest temperature of 63°C. We have investigated the cyanobacterial diversity of these springs and their outflow channels by microscopic examination, culture-dependent and culture-independent phylogenetic analysis, and by physiological studies of selected isolates of special interest. Both unicellular and filamentous types of cyanobacteria are present, and we identified morphological types such as Thermosynechococcus, Chroogloeocystis, Fischerella (Mastigocladus), Scytonema (occurring as large masses at lower temperatures), and others. Although morphologically similar cyanobacteria have been identified in hot springs world-wide, the Zerka Ma’in strains were phylogenetically distinct based on 16S rRNA gene sequence analysis. Considerable diversity was detected also in the gene sequences of nifH (nitrogenase reductase), encoding one of the key enzymes involved in nitrogen fixation. Nitrogen fixation in a Mastigocladus isolate obtained from the springs was investigated in further depth. The heterocystous strain could fix nitrogen (as assayed by acetylene reduction) at tem peratures up to 53°C.

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