Uranium mining effluents: What about the re-use of mining wastes to improve the bioproduction of industrially relevant bioactive compounds?

The shift towards a circular economy, where waste generation is minimized through waste re-use and the development of valorization strategies, is crucial for the establishment of a low carbon, sustainable, and resource-efficient economy. However, there is a lack of strategies for re-using and valorizing specific types of waste, particularly those containing naturally occurring radioactive materials (NORM), despite the prevalence of industrial activities that produce such waste due to their chemical and radiological hazards. Living organisms, including fungi, are valuable sources of bioactive compounds with various industrial applications. In this study, we assessed the growth and metabolic profile changes of three white rot fungi species in response to low concentrations of a uranium mine effluent containing NORM and metals to explore their potential for producing biotechnologically relevant bioactive compounds. The growth rate was assessed in three different culture media, with and without the uranium mine effluent (1% V/V)), and the metabolic profile was analyzed using FTIR-ATR spectroscopy. Results suggested an improvement in growth rates in media containing the uranium mine effluent, although not statistically significant. T. versicolor showed promise in terms of bioactive compound production. The production of droplets during growth experiments and significant metabolic changes, associated with the production of bioactive compounds like laccase, melanin, and oxalic acid, were observed in T. versicolor grown in mYEPDA with the uranium mine effluent. These findings present new research opportunities for utilizing waste to enhance the biotechnological production of industrially relevant bioactive compounds and promote the development of circular economy strategies for re-using and valorizing NORM-containing waste.

Inter-species bystander effect: Eisenia fetida and Enchytraeus albidus exposed to uranium and cadmium.

The bystander effect is commonly defined as the observation of effects in nonirradiated cells and tissues when the later are in contact with irradiated cells/ tissues. More recently the occurrence of bystander effect between organisms of the same species has been also demonstrated. Nevertheless, there is limited knowledge about this effect between soil dwelling organisms from different taxonomic groups, as well as in response to stressors other than ionizing radiation. Moreover, data reporting this phenomenon for soil invertebrates are scarce. The results herein presented contribute for the understanding of the impacts of cadmium and uranium in the DNA integrity of two terrestrial oligochaetes species (Eisenia fetida and Enchytraeus albidus). The evaluation was based on the quantification of the effects in the DNA integrity of the coelomocytes using the alkaline comet assay technique. This work reports the existence of bystander signaling from terrestrial earthworms to enchytraeids and from enchytraeids to earthworms when the organisms were exposed to Cd. These results reinforce that the bystander effect seems to be related with the genotoxic activity of stressors, and not exclusive of radiotoxic contaminants. Further, the bystander effect occurs between different species and under real environmental conditions, even in complex matrices, as the soil.

Metals and low dose IR: Molecular effects of combined exposures using HepG2 cells as a biological model.

Uranium mining sites produce residues rich in metals and radionuclides, that may contaminate all environmental matrices, exposing human and non-human biota to low doses of ionizing radiation (LDIR) and to the chemical toxicity of several metals. To date, experimental and radio-epidemiological studies do not provide conclusive evidence of LDIR induced cancer. However, co-exposures (LDIR plus other contaminants), may increase the risks. To determine the potential for genotoxic effects in human cells induced by the exposure to LDIR plus metals, HEPG2 cells were exposed to different concentrations of a uranium mine effluent for 96 h. DNA damage was evaluated using the comet assay and changes in the expression of tumor suppressor and oncogenes were determined using qPCR. Results show that effluent concentrations higher than 5%, induce significant DNA damage. Also, a significant under-expression of ATM and TP53 genes and a significant overexpression of GADD45a gene was observed. Results show that the exposure to complex mixtures cannot be disregarded, as effects were detected at very low doses. This study highlights the need for further studies to clarify the risks of exposure to LDIR along with other stressors, to fully review the IR exposure risk limits established for human and non-human biota.

Shiga toxin-producing Escherichia coli in wild ungulates.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens that live in the gastrointestinal tract of wildlife and cattle without causing disease. In humans, their colonization and infection lead to life-threatening disease. We investigated the occurrence of STEC in wild ungulates (wild boar, red deer and roe deer) inhabiting areas differently impacted by anthropogenic activities. STEC were detected in 9% (n = 6) of the samples and were recovered from the three species: 1 of wild boar, 4 of red deer and 1 of roe deer. All the isolates (n = 7) were non-O157 STEC encoding stx1 (n = 2; 29%) and/or stx2 genes (n = 6; 86%). O27:H30 was the most frequent serotype identified, followed by O146:H21 and O146:H28. Two STEC were O-untypable: ONT:H28 and ONT:H52. The phylo-groups identified were B1 (n = 3), E (n = 3) and F (n = 1). All the isolates recovered were susceptible to the different classes of antibiotics tested, although resistance genes were found in two strains. Apart from stx, all STEC encode many virulence factors (VF), particularly adhesins and/or other toxins. A strain with 13 VF collected from roe deer has a high enterohemorrhagic risk due to the presence of intimin, hemolysin and protease effectors genes. Enterohemorrhagic E. coli (EHEC) are implicated in the major cases of human infection and mortality, highlighting the zoonotic potential of wildlife-associated STEC. Wild ungulates are reservoirs of STEC potentially pathogenic to humans. Therefore, following the One Health concept, it is crucial to establish worldwide local monitoring programs that will benefit human, animal and environmental health.

RIBE at an inter-organismic level: A study on genotoxic effects in Daphnia magna exposed to waterborne uranium and a uranium mine effluent.

The induction of RIBE (Radiation Induced Bystander Effect) is a non-target effect of low radiation doses that has already been verified at an inter-organismic level in fish and small mammals. Although the theoretical impact in the field of environmental risk assessment (ERA) is possible, there is a gap of knowledge regarding this phenomenon in invertebrate groups and following environmentally relevant exposures. To understand if RIBE should be considered for ERA of radionuclide-rich wastewaters, we exposed Daphnia magna (<24 h and 5d old) to a 2% diluted uranium mine effluent for 48 h, and to a matching dose of waterborne uranium (55.3 µg L-1). Then the exposed organisms were placed (24 and 48 h) in a clean medium together with non-exposed neonates. The DNA damage observed for the non-exposed organisms was statistically significant after the 24 h cohabitation for both uranium (neonates p = 0.002; 5 d-old daphnids p = <0.001) and uranium mine effluent exposure (only for neonates p = 0.042). After 48 h cohabitation significant results were obtained only for uranium exposure (neonates p = 0.017; 5 d-old daphnids p = 0.013). Although there may be some variability associated to age and exposure duration, the significant DNA damage detected in non-exposed organisms clearly reveals the occurrence of RIBE in D. magna. The data obtained and here presented are a valuable contribution for the discussion about the relevance of RIBE for environmental risk assessment.

Uranium mining wastes: The use of the Fish Embryo Acute Toxicity Test (FET) test to evaluate toxicity and risk of environmental discharge.

Active and abandoned uranium mining sites often create environmentally problematic situations, since they cause the contamination of all environmental matrices (air, soil and water) with stable metals and radionuclides. Due to their cytotoxic, genotoxic and teratogenic properties, the exposure to these contaminants may cause several harmful effects in living organisms. The Fish Embryo Acute Toxicity Test (FET) test was employed to evaluate the genotoxic and teratogenic potential of mine liquid effluents and sludge elutriates from a deactivated uranium mine. The aims were: a) to determine the risk of discharge of such wastes in the environment; b) the effectiveness of the chemical treatment applied to the uranium mine water, which is a standard procedure generally applied to liquid effluents from uranium mines and mills, to reduce its toxicological potential; c) the suitability of the FET test for the evaluation the toxicity of such wastes and the added value of including the evaluation of genotoxicity. Results showed that through the FET test it was possible to determine that both elutriates and effluents are genotoxic and also that the mine effluent is teratogenic at low concentrations. Additionally, liquid effluents and sludge elutriates affect other parameters namely, growth and hatching and that water pH alone played an important role in the hatching process. The inclusion of genotoxicity evaluation in the FET test was crucial to prevent the underestimation of the risks posed by some of the tested effluents/elutriates. Finally, it was possible to conclude that care should be taken when using benchmark values calculated for specific stressors to evaluate the risk posed by uranium mining wastes to freshwater ecosystems, due to their chemical complexity.

The lanthipeptides of Bacillus methylotrophicus and their association with genomic islands.

Bacillus methylotrophicus strains are known for their potential as plant-growth promoters and as microbial pesticides that effectively control plant diseases caused by bacteria and fungi. Over the past few years, a wide diversity of their secondary metabolites has been extensively characterized. Among these are the RiPPs lanthipeptides, which are an important and growing group of notable compounds. The increasing interest in B. methylotrophicus species, accompanied by the development of high throughput sequencing techniques, has resulted in a substantial number of full genomes being available. Here, an in silico analysis was performed on these genomes in order to survey the presence of lanthipeptide biosynthetic clusters. It was found that the pan genome of B. methylotrophicus only encoded the biosynthesis of mersacidin and amylolysin, which are lanthipeptides with antibacterial activity. However, the amylolysin gene cluster identified was comprised of more genetic elements than those previously described, and it had certain features of two-peptide lantibiotics. Additionally, it was also established that the association of lanthipeptides with genomic islands (GIs) was not confined to mersacidin. This was also found for the amylolysin cluster as well as other class I and class II lanthipeptides, supporting the idea that their production is probably related to functional adaptation.

Bioengineering of lanthipeptides in Escherichia coli: assessing the specificity of lichenicidin and haloduracin biosynthetic machinery.

The lichenicidin and haloduracin biosynthetic machinery specificity was investigated in vivo in Escherichia coli. Unlike previous reports using different hosts, it was found that the biosynthetic machineries of lichenicidin and haloduracin are highly specific to their dedicated peptide precursors. Likewise, the substitution of lichenicidin structural genes by chimeras of lichenicidin leader sequences and haloduracin core peptides did not yield mature haloduracin peptides. Despite these restrictions, it was found that the bifunctional enzyme HalT was able to process and export lichenicidin peptides. These findings corroborate the promiscuity of LanT enzymes reported for other lantibiotics, such as nukacin ISK-1 and lacticin 481.

Biomonitoring a human population inhabiting nearby a deactivated uranium mine.

Environmental exposure to uranium and its daughter radionuclides, has been linked to several negative effects such as those related with important physiological processes, like hematopoiesis, and may also be associated with genotoxicity effects. Herein, genotoxic effects, immunotoxicity, trace elements and C reactive protein (CRP) analyses, were performed in peripheral blood samples collected from individuals of a population living near a deactivated uranium mine. C reactive protein analysis was performed to exclude candidates with active inflammatory processes from further evaluations. DNA damage and immunotoxicity (immunophenotyping and immune cell counts) were evaluated by comet assay and flow cytometry, respectively. Significant DNA damage was observed in the peripheral blood samples from volunteers living in the Cunha Baixa village. A significant decrease of NK and T lymphocytes counts were observed in the individuals from the Cunha Baixa village, when compared with individuals from the reference site. Uranium and manganese levels were significantly higher in the Cunha Baixa village inhabitants. On the other hand, zinc levels were significantly lower in those individuals when compared with the volunteers from the control village. Results suggest that inhabitants from Cunha Baixa have a higher risk of suffering from serious diseases such as cancer, since high DNA damages were observed in peripheral blood leukocytes and also decreased levels of NK and T cells, which play an essential role in the defense against tumor growth.

A novel complex class 1 integron found in a Klebsiella pneumoniae isolate from Portugal.

Klebsiella pneumoniae Kp1 carrying a novel complex class 1 integron was isolated from an inanimate surface of a female ward sanitary facility in the Hospital Infante D. Pedro, Aveiro, central Portugal. The integron consists of two variable regions (VRs); VR1 was previously described in Escherichia coli and Vibrio cholerae, and VR2 contains an In37-like structure and is located downstream of an ISCR1 element. The integron was found on a plasmid of 225 kb. The qnrB10 gene, although present, is not associated with the complex class 1 integron.

Tn5090-like class 1 integron carrying bla(VIM-2) in a Pseudomonas putida strain from Portugal.

Three Pseudomonas putida strains containing bla(VIM-2) were isolated from an inanimate surface of a female ward sanitary facility in the Hospital Infante D. Pedro, Aveiro. A novel class 1 integron was found in strain Pp2 (aacA4/bla(VIM-2)/aac6'-IIc disrupted by an insertion sequence IS1382), and strain Pp1 was found to carry a class 1 integron (aacA7/bla(VIM-2)/aacC1/aacA4), which is described for the first time in this species. Strain PF1 carries a class 1 integron associated with a Tn5090-like transposon, constituting the first finding of this type of arrangement in a strain from Portugal. This association highlights further dissemination of bla(VIM-2) in environmental hospital isolates.

Genetic, biochemical, and individual responses of the teleost fish Carassius auratus to uranium.

Carassius auratus were exposed for 96 h to different concentrations of uranyl nitrate (corresponding to 0, 100, 450, and 2,025 microg U L(-1)) and killed after different postexposure periods (0, 48, and 96 h) to assess uranium bioaccumulation, peroxisome proliferation (catalase [CAT]), lipid peroxidation (thiobarbituric acid reactive substances [TBARS]), and DNA integrity in erythrocytes (comet assay). In addition, feeding behaviour was recorded as a general response to toxicant exposure. Results provided evidence of uranium bioaccumulation in muscle of C. auratus after exposure to the highest concentrations (450 and 2,025 microg U L(-1)). This tissue was able to depurate uranium to control levels 96 h after exposure ceased. However, no perturbations in feeding behaviour or cell damage were observed in the tested organisms, except for the apparent irreversible inhibition of CAT activity immediately after exposure in the highest concentration tested. Data on DNA integrity (comets) showed that waterborne uranium exposure was able to induce genotoxicity in C. auratus erythrocytes because fish exposed to all concentrations exhibited higher DNA damage than controls 96 h after exposure. No DNA damage repair was apparent throughout the postexposure period, which was contrary to a recovery scenario. This experiment provides evidence of uranium's ability to induce physiologic impairment and genotoxicity in freshwater fish at environmentally relevant concentrations.

In99, an In100-related integron, its occurrence and prevalence in clinical Pseudomonas aeruginosa strains from a central region of Portugal.

In99, a possible ancestor of In100, is a class 1 integron associated with carbenicillinase (blaPSE) and aminoglycoside resistance genes [aac(6')-Ib and aadA2]. In99 was present in 8 of 81 clinical isolates of Pseudomonas aeruginosa from unrelated patients collected in different years. The strains fell into two clonal groups and exhibited resistance to beta-lactams and aminoglycosides.

Genetic characterization of a new thermotolerant Bacillus licheniformis strain.

A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector.