Cristina Becerra-Castro

Wastewater reuse in irrigation: A microbiological perspective on implications in soil fertility and human and environmental health

The reuse of treated wastewater, in particular for irrigation, is an increasingly common practice, encouraged by governments and official entities worldwide. Irrigation with wastewater may have implications at two different levels: alter the physicochemical and microbiological properties of the soil and/or introduce and contribute to the accumulation of chemical and biological contaminants in soil. The first may affect soil productivity and fertility; the second may pose serious risks to the human and environmental health. The sustainable wastewater reuse in agriculture should prevent both types of effects, requiring a holistic and integrated risk assessment. In this article we critically review possible effects of irrigation with treated wastewater, with special emphasis on soil microbiota. The maintenance of a rich and diversified autochthonous soil microbiota and the use of treated wastewater with minimal levels of potential soil contaminants are proposed as sine qua non conditions to achieve a sustainable wastewater reuse for irrigation.

Pseudometallophytes colonising Pb/Zn mine tailings: A description of the plant–microorganism–rhizosphere soil system and isolation of metal-tolerant bacteria

The plant-microorganism-soil system of three pseudometallophytes (Betula celtiberica, Cytisus scoparius and Festuca rubra) growing in a Pb/Zn mine was characterised. Plant metal accumulation, soil metal fractions (rhizosphere and non-vegetated) and bacterial densities were determined. Total Cd, Pb and Zn in non-vegetated soils was up to 50, 3000 and 20,000 mg kg(-1) dry weight, respectively. The residual fraction dominated non-vegetated soils, whereas plant-available fractions became important in rhizosphere soils. All plant species effectively excluded metals from the shoot. F. rubra presented a shoot:root transport factor of ≤0.2 and this population could be useful in future phytostabilisation trials. Culturable bacterial densities and diversity were low (predominantly Actinobacteria). Rhizosphere soils hosted higher total and metal-tolerant bacterial densities. Seventy-four metal-tolerant rhizobacteria were isolated, and characterised genotypically (BOX-PCR, 16S rDNA) and phenotypically [Cd/Zn tolerance, biosurfactant production and plant growth promoting (PGP) traits]. Several isolates resisted high concentrations of Cd and Zn, and only a few presented PGP traits. Fourteen isolates were evaluated for promoting plant growth of two species (Salix viminalis and Festuca pratensis). Thirteen inoculants enhanced growth of F. pratensis, while only three enhanced growth of S. viminalis. Growth enhancement could not always be related to isolate PGP traits. In conclusion, some isolates show potential application in phytostabilisation or phytoextraction techniques.

Endophytic and rhizoplane bacteria associated with Cytisus striatus growing on hexachlorocyclohexane-contaminated soil: isolation and characterisation

Inoculation of plants with their associated microorganisms is a promising strategy for improving phytoremediation of organic contaminants. Isolation and characterisation of these organisms from plants growing in contaminated sites will permit the identification of candidate strains for re-inoculation studies. The diversity of culturable endophytic and rhizoplane bacteria found in association with Cytisus striatus plants growing at a hexachlorocyclohexane (HCH)-contaminated site was studied. A total of 97 strains of endophytic bacteria were isolated from the root, stem and leaf tissues, and 49 from the rhizoplane. They were further characterised genotypically (BOX-PCR, 16S rDNA sequencing, presence of linA and linB genes) and phenotypically (trace metal tolerance, capacity to produce biosurfactants and plant growth promoting (PGP) traits). Proteobacteria and Actinobacteria dominated the isolate collection, and taxonomic diversity was strongly tissue-specific. The linA and linB genes were not detected in the isolate collection. The majority of isolates had at least one of the PGP traits tested, whereas biosurfactant-producing strains were less frequent. Resistance to more than one trace metal was generally restricted to endophytes isolated from shoot tissues. The PGP characteristics found in an important number of the bacterial isolates obtained in this study could be particularly useful for exploiting the phytoremediation potential of C. striatus.

Rhizobacterial inoculants can improve nickel phytoextraction by the hyperaccumulator Alyssum pintodasilvae

AimRhizobacteria can influence plant growth and metal accumulation. The aim of this study was to evaluate the effect of rhizobacterial inoculants on the Ni phytoextraction efficiency of the Ni-hyperaccumulator Alyssum pintodasilvae.MethodIn a preliminary screening 15 metal-tolerant bacterial strains were tested for their plant growth promoting (PGP) capacity or effect on Ni bioaccumulation. Strains were selected for their Ni tolerance, plant growth promoting traits and Ni solubilizing capacity. In a re-inoculation experiment five of the previously screened bacterial isolates were used to inoculate A. pintodasilvae in two contrasting Ni-rich soils (a serpentine (SP) soil and a sewage sludge-affected agricultural (LF) soil).ResultsPlant growth was greater in serpentine soil (where it grows naturally) than in the LF soil, probably due to Cd phytotoxicity. Rhizobacterial inoculants influenced plant growth and Ni uptake and accumulation, but the effect of the strains was dependent upon soil type. The increase in plant biomass and/or Ni accumulation significantly promoted shoot Ni removal.ConclusionOne strain (Arthrobacter nicotinovorans SA40) was able to promote plant growth and phytoextraction of Ni in both soil types and could be a useful candidate for future field-based trials.

Ozonation and UV254 nm radiation for the removal of microorganisms and antibiotic resistance genes from urban wastewater

Conventional wastewater treatment has a limited capacity to reduce antibiotic resistant bacteria and genes (ARB&ARG). Tertiary treatment processes are promising solutions, although the transitory inactivation of bacteria may select ARB&ARG. This study aimed at assessing the potential of ozonation and UV254nm radiation to inactivate cultivable fungal and bacterial populations, and the selected genes 16S rRNA (common to all bacteria), intI1 (common in Gram-negative bacteria) and the ARG vanA, blaTEM, sul1 and qnrS. The abundance of the different microbiological parameters per volume of wastewater was reduced by ∼2 log units for cultivable fungi and 16S rRNA and intI1 genes, by∼3-4 log units, for total heterotrophs, enterobacteria and enterococci, and to values close or below the limits of quantification for ARG, for both processes, after a contact time of 30min. Yet, most of the cultivable populations, the 16S rRNA and intI1 genes as well as the ARG, except qnrS after ozonation, reached pre-treatment levels after 3days storage, suggesting a transitory rather than permanent microbial inactivation. Noticeably, normalization per 16S rRNA gene evidenced an increase of the ARG and intI1 prevalence, mainly after UV254nm treatment. The results suggest that these tertiary treatments may be selecting for ARB&ARG populations.

Rhizosphere Microbial Densities and Trace Metal Tolerance of the Nickel Hyperaccumulator Alyssum Serpyllifolium Subsp. Lusitanicum

In this study we determine culturable microbial densities (total heterotrophs, ammonifiers, amylolytics and cellulolytics) and bacterial resistance to Co, Cr, and Ni in bulk and rhizosphere soils of three populations of the Ni-hyperaccumulator Alyssum serpyllifolium subsp. lusitanicum and the excluder Dactylis glomerata from ultramafic sites (two populations in Northeast (NE) Portugal (Samil (S), Morais (M)) and one population in Northwest (NW) Spain (Melide (L)). The relationship between bioavailable metal concentrations (H2O-soluble) and microbial densities were analysed. Significant differences in microbial densities and metal-resistance were observed between the two species and their three populations. The hyperaccumulator showed higher microbial densities (except cellulolytics) and a greater rhizosphere effect, but this was only observed in S and M populations. These populations of A. serpyllifolium also showed selective enrichment of Ni-tolerant bacteria at the rhizosphere where Ni solubility was enhanced (densities of Ni-resistant bacteria were positively correlated with H2O-soluble Ni). These rhizobacteria could solubilise Ni in the soil and potentially improve phytoextraction strategies.

Bacterially Induced Weathering of Ultramafic Rock and Its Implications for Phytoextraction

ABSTRACT The bioavailability of metals in soil is often cited as a limiting factor of phytoextraction (or phytomining). Bacterial metabolites, such as organic acids, siderophores, or biosurfactants, have been shown to mobilize metals, and their use to improve metal extraction has been proposed. In this study, the weathering capacities of, and Ni mobilization by, bacterial strains were evaluated. Minimal medium containing ground ultramafic rock was inoculated with either of two Arthrobacter strains: LA44 (indole acetic acid [IAA] producer) or SBA82 (siderophore producer, PO4 solubilizer, and IAA producer). Trace elements and organic compounds were determined in aliquots taken at different time intervals after inoculation. Trace metal fractionation was carried out on the remaining rock at the end of the experiment. The results suggest that the strains act upon different mineral phases. LA44 is a more efficient Ni mobilizer, apparently solubilizing Ni associated with Mn oxides, and this appeared to be related to oxalate production. SBA82 also leads to release of Ni and Mn, albeit to a much lower extent. In this case, the concurrent mobilization of Fe and Si indicates preferential weathering of Fe oxides and serpentine minerals, possibly related to the siderophore production capacity of the strain. The same bacterial strains were tested in a soil-plant system: the Ni hyperaccumulator Alyssum serpyllifolium subsp. malacitanum was grown in ultramafic soil in a rhizobox system and inoculated with each bacterial strain. At harvest, biomass production and shoot Ni concentrations were higher in plants from inoculated pots than from noninoculated pots. Ni yield was significantly enhanced in plants inoculated with LA44. These results suggest that Ni-mobilizing inoculants could be useful for improving Ni uptake by hyperaccumulator plants.

Nickel Solubilizing Capacity and Characterization of Rhizobacteria Isolated from Hyperaccumulating and Non-Hyperaccumulating Subspecies of Alyssum Serpyllifolium

Bacterial strains were isolated from the rhizosphere of three populations of the Ni-hyperaccumulator Alyssum serpyllifolium subsp. lusitanicum (A. pintodasilvae; M, S, and L), one population of Ni-hyperaccumulator A. serpyllifolium subsp. malacitanum (A. malacitanum; SB), and one population of the non-hyperaccumulator A. serpyllifolium subsp. serpyllifolium (A. serpyllifolium; SN). Isolates were characterized genotypically by BOX-PCR genomic DNA fingerprinting and comparative sequence analysis of partial 16S rRNA gene, and phenotypically by their Ni tolerance (0–10 mM), presence of plant growth promoting traits (indoleacetic acid (IAA)-, siderophore-, or organic acid-production, and phosphate solubilization) or capacity to produce biosurfactants. Among the collection of rhizobacteria, 84 strains were selected (according to their BOX-PCR profiles and phenotypic characteristics) to assess their ability to modify Ni extractability from Ni-rich (serpentine) soils. Metabolites produced by 13 of the isolates mobilized soil Ni (originating from the rhizosphere of both Ni-hyperaccumulators and non-hyperaccumulator). In contrast, Ni extraction using culture medium filtrates which had supported the growth of 29 strains was significantly reduced. The remaining strains had no effect on Ni mobility. Bacterial induced Ni mobilization was not related to Ni resistance or the phenotypic traits tested. Isolates with potential use in phytoremediation techniques will be further studied in a plant-microorganism-soil system.

Assessment of copper and zinc salts as selectors of antibiotic resistance in Gram-negative bacteria.

Some metals are nowadays considered environmental pollutants. Although some, like Cu and Zn, are essential for microorganisms, at high concentrations they can be toxic or exert selective pressures on bacteria. This study aimed to assess the potential of Cu or Zn as selectors of specific bacterial populations thriving in wastewater. Populations of Escherichia coli recovered on metal-free and metal-supplemented culture medium were compared based on antibiotic resistance phenotype and other traits. In addition, the bacterial groups enriched after successive transfers in metal-supplemented culture medium were identified. At a concentration of 1mM, Zn produced a stronger inhibitory effect than Cu on the culturability of Enterobacteriaceae. It was suggested that Zn selected populations with increased resistance prevalence to sulfamethoxazole or ciprofloxacin. In non-selective culture media, Zn or Cu selected for mono-species populations of ubiquitous Betaproteobacteria and Flavobacteriia, such as Ralstonia pickettii or Elizabethkingia anophelis, yielding multidrug resistance profiles including resistance against carbapenems and third generation cephalosporins, confirming the potential of Cu or Zn as selectors of antibiotic resistant bacteria.

Proteobacteria become predominant during regrowth after water disinfection

Disinfection processes aim at reducing the number of viable cells through the generation of damages in different cellular structures and molecules. Since disinfection involves unspecific mechanisms, some microbial populations may be selected due to resilience to treatment and/or to high post-treatment fitness. In this study, the bacterial community composition of secondarily treated urban wastewater and of surface water collected in the intake area of a drinking water treatment plant was compared before and 3-days after disinfection with ultraviolet radiation, ozonation or photocatalytic ozonation. The aim was to assess the dynamics of the bacterial communities during regrowth after disinfection. In all the freshly collected samples, Proteobacteria and Bacteroidetes were the predominant phyla (40-50% and 20-30% of the reads, respectively). Surface water differed from wastewater mainly in the relative abundance of Actinobacteria (17% and <5% of the reads, respectively). After 3-days storage at light and room temperature, disinfected samples presented a shift of Gammaproteobacteria (from 8 to 10% to 33-65% of the reads) and Betaproteobacteria (from 14 to 20% to 31-37% of the reads), irrespective of the type of water and disinfection process used. Genera such as Pseudomonas, Acinetobacter or Rheinheimera presented a selective advantage after water disinfection. These variations were not observed in the non-disinfected controls. Given the ubiquity and genome plasticity of these bacteria, the results obtained suggest that disinfection processes may have implications on the microbiological quality of the disinfected water.