Bioremediation of environments contaminated with mercury. Present and perspectives.

Mercury is a highly toxic heavy metal whose emission sources can be both natural and the result of anthropic activity. Its polluting action on soils, and its ability to spread through the atmosphere and aquatic environments, constitutes a threat to human and environmental health; both for its bioaccumulation capacity and for biomagnification through the trophic chain. For this reason, there is a growing scientific and social interest in the reduction of this heavy metal in ecosystems. Bioremediation based on the use of microorganisms and/or plants is postulated as a sustainable alternative to traditional physicochemical methods. The main strategies used for this purpose (individually or in combination) are the volatilization of the contaminant, biosorption, phytoextraction and phytoremediation. All these tools are based on taking advantage of the natural and evolutionary capacity that different organisms have developed to adapt to the presence of various pollutants in the environment. Based on the consulted bibliography, these bioremediation methodologies focus on the use of microorganisms (freely or associated with plants) have been successfully applied in different ecosystems, postulating themselves as a respectful alternative for the future for the recovery of degraded environments. For these reasons there is a growing interest in the scientific community to design and use new techniques in a "One Health" context, which allow interpreting the positive impact of bioremediation. In this sense, the universalization of Omics techniques has allowed to abound in the knowledge of new bacterial taxa, and their biotechnological application. This study pretends to cover the present knowledge about mercury bioremediation techniques. In the same way, some new techniques and perspectives are presented in order to expand the frontiers of future research.

Reduced Antibiotic Resistance in the Rhizosphere of Lupinus albus in Mercury-Contaminated Soil Mediated by the Addition of PGPB.

The emergence of antibiotic resistance (AR) poses a threat to the "One Health" approach. Likewise, mercury (Hg) pollution is a serious environmental and public health problem. Its ability to biomagnify through trophic levels induces numerous pathologies in humans. As well, it is known that Hg-resistance genes and AR genes are co-selected. The use of plant-growth-promoting bacteria (PGPB) can improve plant adaptation, decontamination of toxic compounds and control of AR dispersal. The cenoantibiogram, a technique that allows estimating the minimum inhibitory concentration (MIC) of a microbial community, has been postulated as a tool to effectively evaluate the evolution of a soil. The present study uses the metagenomics of 16S rRNA gene amplicons to understand the distribution of the microbial soil community prior to bacterial inoculation, and the cenoantibiogram technique to evaluate the ability of four PGPB and their consortia to minimize antibiotic resistance in the rhizosphere of Lupinus albus var. Orden Dorado grown in Hg-contaminated soils. Results showed that the addition of A1 strain (Brevibacterium frigoritolerans) and its consortia with A2, B1 and B2 strains reduced the edaphic community´s MIC against cephalosporins, ertapenem and tigecycline. The metagenomic study revealed that the high MIC of non-inoculated soils could be explained by the bacteria which belong to the detected taxa,. showing a high prevalence of Proteobacteria, Cyanobacteria and Actinobacteria.

Oxidative stress protection and growth promotion activity of Pseudomonas mercuritolerans sp. nov., in forage plants under mercury abiotic stress conditions.

SAICEUPSMT strain was isolated from soils in the mining district of Almadén (Ciudad Real, Spain), subjected to a high concentration of mercury. Using the plant model of lupinus, the strain was inoculated into the rhizosphere of the plant in a soil characterized by a high concentration of mercury (1,710 ppm) from an abandoned dump in the mining district of Almadén (Ciudad Real, Spain). As a control, a soil with a minimum natural concentration of mercury, from a surrounding area, was used. Under greenhouse conditions, the effect that the inoculum of the SAICEUPSMT strain had on the antioxidant capacity of the plant was studied, through the quantification of the enzymatic activity catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), and glutathione reductase (GR). Likewise, the capacity of the plant to bioaccumulate mercury in the presence of the inoculum was studied, as well as the effect on the biometric parameters total weight (g), shoot weight (g), root weight (g), shoot length (cm), root length (cm), total number of leaves (N), and total number of secondary roots (No). Finally, in view of the results, the SAICEUPSMT strain was identified from the phenotypic and genotypic point of view (housekeeping genes and complete genome sequencing). The inoculum with the SAICEUPSMT strain in the presence of mercury produced a significant reduction in the enzymatic response to oxidative stress (CAT, APX, and SOD). It can be considered that the strain exerts a phytoprotective effect on the plant. This led to a significant increase in the biometric parameters total plant weight, root weight and the number of leaves under mercury stress, compared to the control without abiotic stress. When analyzing the mercury content of the plant with and without bacterial inoculum, it was found that the incorporation of the SAICEUPSMT strain significantly reduced the uptake of mercury by the plant, while favoring its development in terms of biomass. Given the positive impact of the SAICEUPSMT strain on the integral development of the plant, it was identified, proving to be a Gram negative bacillus, in vitro producer of siderophores, auxins and molecules that inhibit stress precursors. The most represented fatty acids were C16:0 (33.29%), characteristic aggregate 3 (22.80%) comprising C16:1 ω7c and C16: 1ω6c, characteristic aggregate 8 (13.66%) comprising C18:1 ω7c, and C18: 1 cycle ω6c and C 17:0 (11.42%). From the genotypic point of view, the initial identification of the strain based on the 16S rRNA gene sequence classified it as Pseudomonas iranensis. However, genome-wide analysis showed that average nucleotide identity (ANI, 95.47%), DNA-DNA in silico hybridization (dDDH, 61.9%), average amino acid identity (AAI, 97.13%), TETRA (0.99%) and intergenic distance (0.04) values were below the established thresholds for differentiation. The results of the genomic analysis together with the differences in the phenotypic characteristics and the phylogenetic and chemotaxonomic analysis support the proposal of the SAICEUPSMT strain as the type strain of a new species for which the name Pseudomonas mercuritolerans sp. is proposed. No virulence genes or transmissible resistance mechanisms have been identified, which reveals its safety for agronomic uses, under mercury stress conditions.

Effect of Plant Growth-Promoting Bacteria on Biometrical Parameters and Antioxidant Enzymatic Activities of Lupinus albus var. Orden Dorado Under Mercury Stress.

Heavy metal contamination of soils is a large-scale environmental problem. It leads to significant disqualification of the territory, in addition to being a source of the potential risk to human health. The exposure of plants to mercury (Hg) generates responses in its growth and their oxidative metabolism. The impact of increasing concentrations of Hg on the development of Lupinus albus var. Orden Dorado seedlings has been studied, as well as the plant's response to the maximum concentration of Hg that allows its development (16 µg ml-1). The result shows that only the inoculum with plant growth promoting bacteria (PGPB) allows the biometric development of the seedling (root length, weight, and number of secondary roots) and prevents the toxic effects of the heavy metal from aborting the seedlings. Specifically, treatments with strains 11, 20 (Bacillus toyonensis), 48 (not determined), and 76 (Pseudomonas syringae) are interesting candidates for further PGPB-assisted phytoremediation trials as they promote root biomass development, through their PGPB activities. The plant antioxidant response has been analyzed by quantifying the catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) enzyme activity in the root, under 16 µg ml-1 of HgCl2 and different PGPB treatments. Results show that, although Hg stress generally induces enzyme activity, strains 31 and 69I (Pseudomonas corrugata) and 18 and 43 (Bacillus toyonensis) can keep SOD and APX levels close to those found in control without Hg (p < 0.01). Strain 18 also shows a significant reduction of GR to control levels without Hg. The present work demonstrates the benefit of PGPB treatments in situations of high Hg stress. These findings may be a good starting point to justify the role of PGPB naturally isolated from bulk soil and the rhizosphere of plants subjected to high Hg pressure in plant tolerance to such abiotic stress conditions. More studies will be needed to discover the molecular mechanisms behind the phytoprotective role of the strains with the best results, to understand the complex plant-microorganism relationships and to find effective and lasting symbioses useful in bioremediation processes.

Valorization as a biofertilizer of an agricultural residue leachate: Metagenomic characterization and growth promotion test by PGPB in the forage plant Medicago sativa (alfalfa).

The abuse of chemical fertilizers in intensive agriculture has turned out in the contamination of ground and the soil on which they are applied. Likewise, the generation, storage, and destruction of plant residues from the agri-food industry poses a threat to the environment and human health. The current situation of growing demand for food implies the urgent need to find sustainable alternatives to chemical fertilizers and the management of agricultural waste. Valorization of this plant residue to produce natural biofertilizers using microbiological treatments is presented as a sustainable alternative. The microbial activity allows the transformation into simple molecules that are easily absorbed by plants, as well as the stimulation of plant growth. This double direct and indirect action induced significant increases against the variables of germination, viability, and biomass (dry weight). To guarantee biosafety, it is necessary to use new bio-technological tools, such as metagenomics, which allow the taxonomic analysis of microbial communities, detecting the absence of pathogens. In the present paper, a physicochemical and metagenomic characterization of a fertilizer obtained from agricultural plant waste valorization is carried out. Likewise, fertigation treatments were tested to which the Plant Growth Promoting Bacteria (PGPB) Pseudomonas agronomica and Bacillus pretiosus were added, both independently and in consortium. Metagenomic analysis has identified taxa belonging to the kingdoms Bacteria and Archaea; 10 phyla, 25 families, 32 genera and 34 species, none of them previously described as pathogenic. A 1/512 dilution of the fertilizer increased the germination rate of Medicago sativa (alfalfa) by 16% at 144 h, compared to the treatment without fertilizer. Both the fertilizer and the addition of PGPB in a double direct and indirect action induced significant increases against the variables of germination, viability, and biomass (dry weight). Therefore, the use of an agricultural residue is proposed, which after the addition of two new species is transformed into a biofertilizer that significantly induces plant growth in Mendicago sativa plants.

Plant growth promotion of the forage plant Lupinus albus Var. Orden Dorado using Pseudomonas agronomica sp. nov. and Bacillus pretiosus sp. nov. added over a valorized agricultural biowaste.

Introduction: The overexploitation of natural ecosystems and the evolution of climate change currently force us to design new strategies for more sustainable agronomic uses. The recovery of plant residues, as an alternative to agrochemicals, can help alleviate these problems, for example, through its use for the synthesis of biofertilizers. In this work, the effect of the organic fertilizer matrix ORGAON® from the valorization of horticultural waste is tested, to which two strains of bacteria (and their consortium) are added (SAICEU11T identified as Bacillus pretiosus and SAICEU22T identified as Pseudomonas agronomica), selected for their demonstrated ability to promote plant growth (PGPB), on the lupine forage plant (Lupinus albus). Methods: For the synthesis of the biofertilizer, both strains were added to the ORGAON® organic matrix separately, until reaching a final optical density (OD) of 0.5 McFarland in each case in the irrigation matrix. As a control, sterile ORGAON® (ORGAON®st) was used, also supplemented with the PGPB strains and a chemical fertilizer widely used in agronomy (Chem-F). With these treatments, a 6-week experiment was started under controlled laboratory conditions and on agricultural substrate, to recreate field conditions as accurately as possible. All the tests were carried out with 9 repetitions and 3 replicates of each treatment. After harvest, the improvements on the following biometric variables were studied for each treatment: total weight (Weight_T, g), shoot weight (Weight_S, g), root weight (Weight_R, g), number of leaves (Leaves, No.), shoot length (Length_S), root length (Length_R) and number of secondary roots (Roots, No.). Likewise, the identification of the tested strains and their description as new species was carried out. For this, they were studied from the phenotypic point of view (Transmission electron microscopy (TEM), metabolic profile, PGP activities, fatty acid profile and Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)) and genotypic (sequencing of the main housekeeping genes and sequencing of the whole genome, genomic characteristics (dDDH and ANI) and phylogenetic analysis). Results and discussion: After the statistical analysis of the results, it is shown that the individual addition of both strains on the ORGAON® and ORGAON®st organic matrix improve certain biometric variables. In the case of the SAICEU11T (Bacillus pretiosus) strain, the variables root weight (Weight_R, g), total weight (Weight_T, g) and length of the plant, and number of secondary roots (Roots, No.) significantly improve, while in the case of the strain SAICEU22T (Pseudmonas agronomica), a significant improvement of root length (Length_R) and number of secondary roots (Roots, No.) is demonstrated. On the other hand, the genotaxonomic analysis showed that both species have not been described to date. The identification based on the main housekeeping genes, show that for the Bacillus strain (SAICEU11T) the sequence similarity of the 16S rRNA was 100%, gyrB 92.69%, rpoB 97.70% and rpoD 94.67%. For the Pseudomonas strain (SAICEU22T) the results were 100% for 16S rRNA, 98.43% for rpoD and 96.94% for gyrB. However, in both cases, the dDDH and ANI values, as well as the phylogenetic analysis, show that both species are below the species threshold, which would support the hypothesis that both are new species, in line with the chemotaxonomic results obtained by MALDI-TOF spectrometry and fatty acid profile. To verify the biosafety in their handling and release into the natural environment, we have ruled out the presence of genes that encode virulence factors or resistance to antibiotics, concluding that they are suitable for use in the field to improve the yield of crop plants. Type strains are SAICEU11T (= DSM 114702T = CECT30674T) for Bacillus pretiosus and SAICEU22T (= DSM 114959T = CECT30673T) for Pseudomonas agronomicae.

Automated detection of archaeological mounds using machine-learning classification of multisensor and multitemporal satellite data.

This paper presents an innovative multisensor, multitemporal machine-learning approach using remote sensing big data for the detection of archaeological mounds in Cholistan (Pakistan). The Cholistan Desert presents one of the largest concentrations of Indus Civilization sites (from ca 3300 to 1500 BC). Cholistan has figured prominently in theories about changes in water availability, the rise and decline of the Indus Civilization, and the transformation of fertile monsoonal alluvial plains into an extremely arid margin. This paper implements a multisensor, multitemporal machine-learning approach for the remote detection of archaeological mounds. A classifier algorithm that employs a large-scale collection of synthetic-aperture radar and multispectral images has been implemented in Google Earth Engine, resulting in an accurate probability map for mound-like signatures across an area that covers ca 36,000 km2 The results show that the area presents many more archaeological mounds than previously recorded, extending south and east into the desert, which has major implications for understanding the archaeological significance of the region. The detection of small (<5 ha) to large mounds (>30 ha) suggests that there were continuous shifts in settlement location. These shifts are likely to reflect responses to a dynamic and changing hydrological network and the influence of the progressive northward advance of the desert in a long-term process that culminated in the abandonment of much of the settled area during the Late Harappan period.

Who's better at spotting? A comparison between aerial photography and observer-based methods to monitor floating marine litter and marine mega-fauna.

Pollution by marine litter is raising major concerns due to its potential impact on marine biodiversity and, above all, on endangered mega-fauna species, such as cetaceans and sea turtles. The density and distribution of marine litter and mega-fauna have been traditionally monitored through observer-based methods, yet the advent of new technologies has introduced aerial photography as an alternative monitoring method. However, to integrate results produced by different monitoring techniques and consider the photographic method a viable alternative, this 'new' methodology must be validated. This study aims to compare observations obtained from the concurrent application of observer-based and photographic methods during aerial surveys. To do so, a Partenavia P-68 aircraft equipped with an RGB sensor was used to monitor the waters off the Spanish Mediterranean coast along 12 transects (941 km). Over 10000 images were collected and checked manually by a photo-interpreter to detect potential targets, which were classified as floating marine macro-litter, mega-fauna and seabirds. The two methods allowed the detection of items from the three categories and proved equally effective for the detection of cetaceans, sea turtles and large fish on the sea surface. However, the photographic method was more effective for floating litter detection and the observer-based method was more effective for seabird detection. These results provide the first validation of the use of aerial photography to monitor floating litter and mega-fauna over the marine surface.

Multivariate factorial analysis to design a robust batch leaching test to assess the volcanic ash geochemical hazard.

A method to obtain robust information on short term leaching behaviour of volcanic ashes has been developed independently on the sample age. A mixed factorial design (MFD) was employed as a multivariate strategy for the evaluation of the effects of selected control factors and their interactions (amount of sample (A), contact time (B), and liquid to solid ratio or L/S (C)) on the leaching process of selected metals (Na, K, Mg, Ca, Si, Al, V, Mn, Fe, and Co) and anions (Cl(-) and SO(4)(2-)). Box plots of the data acquired were used to evaluate the reproducibility achieved at different experimental conditions. Both the amount of sample (A) and leaching time (B) had a significant effect on the element stripping whereas the L/S ratio influenced only few elements. The lowest dispersion values have been observed when 1.0 g was leached with an L/S ratio equal to 10, shaking during 4 h. The entire method is completed within few hours, and it is simple, feasible and reliable in laboratory conditions.