Ana I. Peláez

Metaproteogenomic insights beyond bacterial response to naphthalene exposure and bio-stimulation.

Microbial metabolism in aromatic-contaminated environments has important ecological implications, and obtaining a complete understanding of this process remains a relevant goal. To understand the roles of biodiversity and aromatic-mediated genetic and metabolic rearrangements, we conducted ‘OMIC’ investigations in an anthropogenically influenced and polyaromatic hydrocarbon (PAH)-contaminated soil with (Nbs) or without (N) bio-stimulation with calcium ammonia nitrate, NH4NO3 and KH2PO4 and the commercial surfactant Iveysol, plus two naphthalene-enriched communities derived from both soils (CN2 and CN1, respectively). Using a metagenomic approach, a total of 52, 53, 14 and 12 distinct species (according to operational phylogenetic units (OPU) in our work equivalent to taxonomic species) were identified in the N, Nbs, CN1 and CN2 communities, respectively. Approximately 10 out of 95 distinct species and 238 out of 3293 clusters of orthologous groups (COGs) protein families identified were clearly stimulated under the assayed conditions, whereas only two species and 1465 COGs conformed to the common set in all of the mesocosms. Results indicated distinct biodegradation capabilities for the utilisation of potential growth-supporting aromatics, which results in bio-stimulated communities being extremely fit to naphthalene utilisation and non-stimulated communities exhibiting a greater metabolic window than previously predicted. On the basis of comparing protein expression profiles and metagenome data sets, inter-alia interactions among members were hypothesised. The utilisation of curated databases is discussed and used for first time to reconstruct ‘presumptive’ degradation networks for complex microbial communities.

Microbial diversity and metabolic networks in acid mine drainage habitats.

Acid mine drainage (AMD) emplacements are low-complexity natural systems. Low-pH conditions appear to be the main factor underlying the limited diversity of the microbial populations thriving in these environments, although temperature, ionic composition, total organic carbon, and dissolved oxygen are also considered to significantly influence their microbial life. This natural reduction in diversity driven by extreme conditions was reflected in several studies on the microbial populations inhabiting the various micro-environments present in such ecosystems. Early studies based on the physiology of the autochthonous microbiota and the growing success of omics-based methodologies have enabled a better understanding of microbial ecology and function in low-pH mine outflows; however, complementary omics-derived data should be included to completely describe their microbial ecology. Furthermore, recent updates on the distribution of eukaryotes and archaea recovered through sterile filtering (herein referred to as filterable fraction) in these environments demand their inclusion in the microbial characterization of AMD systems. In this review, we present a complete overview of the bacterial, archaeal (including filterable fraction), and eukaryotic diversity in these ecosystems, and include a thorough depiction of the metabolism and element cycling in AMD habitats. We also review different metabolic network structures at the organismal level, which is necessary to disentangle the role of each member of the AMD communities described thus far.

Biodegradation of Oil Tank Bottom Sludge using Microbial Consortia

We present a rationale for the selection of a microbial consortia specifically adapted to degrade toxic components of oil refinery tank bottom sludge (OTBS). Sources such as polluted soils, petrochemical waste, sludge from refinery-wastewater plants, and others were used to obtain a collection of eight microorganisms, which were individually tested and characterized to analyze their degradative capabilities on different hydrocarbon families. After initial experiments using mixtures of these strains, we developed a consortium consisting of four microorganisms (three bacteria and one yeast) selected in the basis of their cometabolic effects, emulsification properties, colonization of oil components, and degradative capabilities. Although the specific contribution each of the former parameters makes is not clearly understood, the activity of the four-member consortium had a strong impact not only on linear alkane degradation (100%), but also on the degradation of cycloalkanes (85%), branched alkanes (44%), and aromatic and sulphur–aromatic compounds (31–55%). The effectiveness of this consortium was significantly superior to that obtained by individual strains, commercial inocula or an undefined mixture of culturable and non-culturable microorganisms obtained from OTBS-polluted soil. However, results were similar when another consortium of four microorganisms, previously isolated in the same OTBS-polluted soil, was assayed.

Microbial stratification in low pH oxic and suboxic macroscopic growths along an acid mine drainage.

Macroscopic growths at geographically separated acid mine drainages (AMDs) exhibit distinct populations. Yet, local heterogeneities are poorly understood. To gain novel mechanistic insights into this, we used OMICs tools to profile microbial populations coexisting in a single pyrite gallery AMD (pH ∼2) in three distinct compartments: two from a stratified streamer (uppermost oxic and lowermost anoxic sediment-attached strata) and one from a submerged anoxic non-stratified mat biofilm. The communities colonising pyrite and those in the mature formations appear to be populated by the greatest diversity of bacteria and archaea (including ‘ARMAN’ (archaeal Richmond Mine acidophilic nano-organisms)-related), as compared with the known AMD, with ∼44.9% unclassified sequences. We propose that the thick polymeric matrix may provide a safety shield against the prevailing extreme condition and also a massive carbon source, enabling non-typical acidophiles to develop more easily. Only 1 of 39 species were shared, suggesting a high metabolic heterogeneity in local microenvironments, defined by the O2 concentration, spatial location and biofilm architecture. The suboxic mats, compositionally most similar to each other, are more diverse and active for S, CO2, CH4, fatty acid and lipopolysaccharide metabolism. The oxic stratum of the streamer, displaying a higher diversity of the so-called ‘ARMAN’-related Euryarchaeota, shows a higher expression level of proteins involved in signal transduction, cell growth and N, H2, Fe, aromatic amino acids, sphingolipid and peptidoglycan metabolism. Our study is the first to highlight profound taxonomic and functional shifts in single AMD formations, as well as new microbial species and the importance of H2 in acidic suboxic macroscopic growths.

Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters.

Herein, we applied a community genomic approach using a naphthalene‐enriched community (CN1) to isolate a versatile esterase (CN1E1) from the α/β‐hydrolase family. The protein shares low‐to‐medium identity (≤ 57%) with known esterase/lipase‐like proteins. The enzyme is most active at 25–30°C and pH 8.5; it retains approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold‐adapted enzyme. CN1E1 has a distinct substrate preference compared with other α/β‐hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200–21 000 units g−1 protein at 40°C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio‐selectivity (1.0–55 000 units g−1 protein), including (±)‐menthyl‐acetate, (±)‐neomenthyl acetate, (±)‐pantolactone, (±)‐methyl‐mandelate, (±)‐methyl‐lactate and (±)‐glycidyl 4‐nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad‐spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo‐assembly (i.e. through ester bonds) of multi‐aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available.

Bioreactor treatment of municipal solid waste landfill leachates: Characterization of organic fractions

Quantitative and qualitative changes in organic matter were studied at different stages of treatment in a bioreactor designed to process leachates from a municipal solid waste landfill. The particulate matter (PM) and macromolecular fractions of the dissolved organic matter with solubility properties comparable to humic (acid-insoluble) and fulvic (acid-soluble) acid fractions (AI, AS, respectively) from the incoming black liquid, the bioreactor content, and the final processed effluent were isolated, quantified, and characterized by visible and infrared (IR) spectroscopies. The macromolecular signature either aliphatic (glycopeptides, carbohydrates) or aromatic (coinciding with infrared patterns of lignin, tannins etc.) enabled us to characterize the different organic fractions during the course of microbial transformation. The results reveal significant changes in the nitrogen speciation patterns within the different organic fractions isolated from the wastewater. The final increase in the relative proportions of nitrogen in the least aromatic AS fraction during microbial transformation could be related to protein formation inside the bioreactor. After biological treatment and ultrafiltration, the amount of organic matter was reduced by approximately 70%, whereas aromaticity increased in all fractions, indicating preferential elimination of aliphatic wastewater compounds. Most of the remaining fractions at the end of the process consisted of a yellow residue rich in low molecular weight AS fractions.

A fatal case of Nocardia otitidiscaviarum pulmonary infection and brain abscess: taxonomic characterization by molecular techniques

We report on a rare case of pulmonary Nocardiosis and brain abscess caused by Nocardia otitidiscaviarum in an elderly woman with chronic obstructive pulmonary disease. Taxonomic identification involved phenotypic testing, restriction fragment length polymorphism (RFLP), and complete 16S rRNA gene sequencing.

Actinobacteria Cyclophilins: Phylogenetic Relationships and Description of New Class- and Order-Specific Paralogues

Cyclophilins are folding helper enzymes belonging to the class of peptidyl-prolyl cis-trans isomerases (PPIases; EC 5.2.1.8) that catalyze the cis-trans isomerization of peptidyl-prolyl bonds in proteins. They are ubiquitous proteins present in almost all living organisms analyzed to date, with extremely rare exceptions. Few cyclophilins have been described in Actinobacteria, except for three reported in the genus Streptomyces and another one in Mycobacterium tuberculosis. In this study, we performed a complete phylogenetic analysis of all Actinobacteria cyclophilins available in sequence databases and new Streptomyces cyclophilin genes sequenced in our laboratory. Phylogenetic analyses of cyclophilins recovered six highly supported groups of paralogy. Streptomyces appears as the bacteria having the highest cyclophilin diversity, harboring proteins from four groups. The first group was named “A” and is made up of highly conserved cytosolic proteins of approximately 18 kDa present in all Actinobacteria. The second group, “B,” includes cytosolic proteins widely distributed throughout the genus Streptomyces and closely related to eukaryotic cyclophilins. The third group, “M” cyclophilins, consists of high molecular mass cyclophilins (∼30 kDa) that contain putative membrane binding domains and would constitute the only membrane cyclophilins described to date in bacteria. The fourth group, named “C” cyclophilins, is made up of proteins of approximately 18 kDa that are orthologous to Gram-negative proteobacteria cyclophilins. Ancestral character reconstruction under parsimony was used to identify shared-derived (and likely functionally important) amino acid residues of each paralogue. Southern and Western blot experiments were performed to determine the taxonomic distribution of the different cyclophilins in Actinobacteria.

Use of Endophytic and Rhizosphere Bacteria To Improve Phytoremediation of Arsenic-Contaminated Industrial Soils by Autochthonous Betula celtiberica

ABSTRACT The aim of this study was to investigate the potential of indigenous arsenic-tolerant bacteria to enhance arsenic phytoremediation by the autochthonous pseudometallophyte Betula celtiberica. The first goal was to perform an initial analysis of the entire rhizosphere and endophytic bacterial communities of the above-named accumulator plant, including the cultivable bacterial species. B. celtibericas microbiome was dominated by taxa related to Flavobacteriales, Burkholderiales, and Pseudomonadales, especially the Pseudomonas and Flavobacterium genera. A total of 54 cultivable rhizobacteria and 41 root endophytes, mainly affiliated with the phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria, were isolated and characterized with respect to several potentially useful features for metal plant accumulation, such as the ability to promote plant growth, metal chelation, and/or mitigation of heavy-metal stress. Seven bacterial isolates were further selected and tested for in vitro accumulation of arsenic in plants; four of them were finally assayed in field-scale bioaugmentation experiments. The exposure to arsenic in vitro caused an increase in the total nonprotein thiol compound content in roots, suggesting a detoxification mechanism through phytochelatin complexation. In the contaminated field, the siderophore and indole-3-acetic acid producers of the endophytic bacterial consortium enhanced arsenic accumulation in the leaves and roots of Betula celtiberica, whereas the rhizosphere isolate Ensifer adhaerens strain 91R mainly promoted plant growth. Field experimentation showed that additional factors, such as soil arsenic content and pH, influenced arsenic uptake in the plant, attesting to the relevance of field conditions in the success of phytoextraction strategies. IMPORTANCE Microorganisms and plants have developed several ways of dealing with arsenic, allowing them to resist and metabolize this metalloid. These properties form the basis of phytoremediation treatments and the understanding that the interactions of plants with soil bacteria are crucial for the optimization of arsenic uptake. To address this in our work, we initially performed a microbiome analysis of the autochthonous Betula celtiberica plants growing in arsenic-contaminated soils, including endosphere and rhizosphere bacterial communities. We then proceeded to isolate and characterize the cultivable bacteria that were potentially better suited to enhance phytoextraction efficiency. Eventually, we went to the field application stage. Our results corroborated the idea that recovery of pseudometallophyte-associated bacteria adapted to a large historically contaminated site and their use in bioaugmentation technologies are affordable experimental approaches and potentially very useful for implementing effective phytoremediation strategies with plants and their indigenous bacteria.

Bacterial, Archaeal, and Eukaryotic Diversity across Distinct Microhabitats in an Acid Mine Drainage

Acid mine drainages are characterized by their low pH and the presence of dissolved toxic metallic species. Microorganisms survive in different microhabitats within the ecosystem, namely water, sediments, and biofilms. In this report, we surveyed the microbial diversity within all domains of life in the different microhabitats at Los Rueldos abandoned mercury underground mine (NW Spain), and predicted bacterial function based on community composition. Sediment samples contained higher proportions of soil bacteria (AD3, Acidobacteria), as well as Crenarchaeota and Methanomassiliicoccaceae archaea. Oxic and hypoxic biofilm samples were enriched in bacterial iron oxidizers from the genus Leptospirillum, order Acidithiobacillales, class Betaproteobacteria, and archaea from the class Thermoplasmata. Water samples were enriched in Cyanobacteria and Thermoplasmata archaea at a 3–98% of the sunlight influence, whilst Betaproteobacteria, Thermoplasmata archaea, and Micrarchaea dominated in acid water collected in total darkness. Stalactites hanging from the Fe-rich mine ceiling were dominated by the neutrophilic iron oxidizer Gallionella and other lineages that were absent in the rest of the microhabitats (e.g., Chlorobi, Chloroflexi). Eukaryotes were detected in biofilms and open-air water samples, and belonged mainly to clades SAR (Alveolata and Stramenopiles), and Opisthokonta (Fungi). Oxic and hypoxic biofilms displayed higher proportions of ciliates (Gonostomum, Oxytricha), whereas water samples were enriched in fungi (Paramicrosporidium and unknown microbial Helotiales). Predicted function through bacterial community composition suggested adaptive evolutive convergence of function in heterogeneous communities. Our study showcases a broad description of the microbial diversity across different microhabitats in the same environment and expands the knowledge on the diversity of microbial eukaryotes in AMD habitats.