Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR.

The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, blaTEM, blaSHV, and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs' biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments.

Structure of fungal communities in sequencing batch reactors operated at different salinities for the selection of triacylglyceride-producers from a fish-canning lipid-rich waste stream.

Oleaginous fungi natively accumulate large amounts of triacylglycerides (TAG), widely used as precursors for sustainable biodiesel production. However, little attention has been paid to the diversity and roles of fungal mixed microbial cultures (MMCs) in sequencing batch reactors (SBR). In this study, a lipid-rich stream produced in the fish-canning industry was used as a substrate in two laboratory-scale SBRs operated under the feast/famine (F/F) regime to enrich microorganisms with high TAG-storage ability, under two different concentrations of NaCl (SBR-N: 0.5 g/L; SBR-S: 10 g/L). The size of the fungal community in the enriched activated sludge (EAS) was analyzed using 18S rRNA-based qPCR, and the fungal community structure was determined by Illumina sequencing. The different selective pressures (feeding strategy and control of pH) implemented in the enrichment SBRs throughout operation increased the abundance of total fungi. In general, there was an enrichment of genera previously identified as TAG-accumulating fungi (Apiotrichum, Candida, Cutaneotrichosporon, Geotrichum, Haglerozyma, Metarhizium, Mortierella, Saccharomycopsis, and Yarrowia) in both SBRs. However, the observed increase of their relative abundances throughout operation was not significantly linked to a higher TAG accumulation.

Dynamics of PHA-Accumulating Bacterial Communities Fed with Lipid-Rich Liquid Effluents from Fish-Canning Industries.

The biosynthesis of polyhydroxyalkanoates (PHAs) from industrial wastes by mixed microbial cultures (MMCs) enriched in PHA-accumulating bacteria is a promising technology to replace petroleum-based plastics. However, the populations' dynamics in the PHA-accumulating MMCs are not well known. Therefore, the main objective of this study was to address the shifts in the size and structure of the bacterial communities in two lab-scale sequencing batch reactors (SBRs) fed with fish-canning effluents and operated under non-saline (SBR-N, 0.5 g NaCl/L) or saline (SBR-S, 10 g NaCl/L) conditions, by using a combination of quantitative PCR and Illumina sequencing of bacterial 16S rRNA genes. A double growth limitation (DGL) strategy, in which nitrogen availability was limited and uncoupled to carbon addition, strongly modulated the relative abundances of the PHA-accumulating bacteria, leading to an increase in the accumulation of PHAs, independently of the saline conditions (average 9.04 wt% and 11.69 wt%, maximum yields 22.03 wt% and 26.33% SBR-N and SBR-S, respectively). On the other hand, no correlations were found among the PHAs accumulation yields and the absolute abundances of total Bacteria, which decreased through time in the SBR-N and did not present statistical differences in the SBR-S. Acinetobacter, Calothrix, Dyella, Flavobacterium, Novosphingobium, Qipengyuania, and Tsukamurella were key PHA-accumulating genera in both SBRs under the DGL strategy, which was revealed as a successful tool to obtain a PHA-enriched MMC using fish-canning effluents.

Salinity is the major driver of the global eukaryotic community structure in fish-canning wastewater treatment plants.

Fish-canning wastewater is characterized frequently by a high content of salt (NaCl), making its treatment particularly difficult; however, the knowledge of the effect of NaCl on eukaryotic communities is very limited. In the present study, the global diversity of eukaryotes in activated sludges (AS) from 4 different wastewater treatment plants (WWTPs) treating fish-canning effluents varying in salinity (0.47, 1.36, 1.72 and 12.76 g NaCl/L) was determined by sequencing partial 18S rRNA genes using Illumina MiSeq. A greater diversity than previously reported was observed in the AS community, which comprised 37 and 330 phylum-like and genera-like groups, respectively. In this sense, the more abundant genus-like groups (average relative abundance (RA) > 5%) were Adineta (6.80%), Lecane (16.80%), Dictyostelium (7.36%), Unclassified_Fungi7 (6.94%), Procryptobia (5.13) and Oocystis (5.07%). The eukaryotic communities shared a common core of 25 phylum-like clades (95% of total sequences); therefore, a narrow selection of the eukaryotic populations was found, despite the differences in the abiotic characteristics of fish-canning effluents and reactor operational conditions inflicted. The differences in NaCl concentration were the main factor that influenced the structure of the eukaryotic community, modulating the RAs of the different phylum-like clades of the common core. Higher levels of salt increased the RAs of Ascomycota, Chlorophyta, Choanoflagellata, Cryptophyta, Mollusca, Nematoda, Other Protists and Unclassified Fungi. Among the different eukaryotic genera here found, the RA of Oocystis (Chlorophyta) was intimately correlated to increasing NaCl concentrations and it is proposed as a bioindicator of the global eukaryotic community of fish-canning WWTPs.

Biodegradation of methyl and butylparaben by bacterial strains isolated from amended and non-amended agricultural soil. Identification, behavior and enzyme activities of microorganisms.

The aim of the present study was to investigate the kinetics of methylparaben (MPB) and butylparaben (BPB) removal, two emerging pollutants with possible endocrine disrupting effects, from agricultural soil with and without amendment with compost from sewage sludge used as biostimulant. Compound removal is explained by a first-order kinetic model with half-life times of 6.5/6.7 days and 11.4/8.2 days, in presence/absence of compost, for MPB and BPB respectively. % R2 for the fitted model were higher than 96% in all cases. Additionally, isolation of bacteria capable to grow using MPB or BPB as carbon source was also carry out. Laboratory tests demonstrated the ability of these bacteria to biodegrade MPB and BPB from culture media in more than 95% in some cases. These strains showed high ability to biodegrade the compounds. Ten isolates, most of them related to Gram positive bacteria of the genus Bacillus, were identified by 16S rRNA gene sequencing. The study of the enzymatic activities of the isolates revealed both esterase (C4) and esterase-lipase activities.

Performance and microbial community structure of an aerobic granular sludge system at different phenolic acid concentrations.

The present work aims to use aerobic granular sludge technology for the treatment of wastewater containing high organic matter loads and a mixture of phenolic compounds normally present in olive washing water. The physicochemical performance of five bioreactors treating different concentrations of mixture of phenolic acid was monitored to observe the response of the systems. The bioreactors that operated at 50, 100 and 300 mg L-1 did not show relevant changes in terms of performance and granules properties, showing high ratio of phenolic compound removal ratio. However, the bioreactors operated with high phenolic compound concentrations showed low rates of organic matter, nitrogen and phenolic acid removal. In the same way, high concentrations of phenolic compounds determined the disintegration of the granular biomass. Next-generation sequencing studies showed a stable community structure in the bioreactors operating with 50, 100 and 300 mg L-1 of phenolic acids, with the genera Lampropedia and Arenimonas, family Xanthobacteraceae and Fungi Pezizomycotina as the dominant phylotypes. Conversely, the reactors operated at 500 and 600 mg L-1 of phenolic substances promoted the proliferation of Oligohymenophorea ciliates. Thus, this study suggests that aerobic granular sludge technology could be useful for the treatment of wastewaters such as olive washing water.

Physiologic and metabolic characterization of a new marine isolate (BM39) of Pantoea sp. producing high levels of exopolysaccharide.

BACKGROUND: Marine environments are the widest fonts of biodiversity representing a resource of both unexploited or unknown microorganisms and new substances having potential applications. Among microbial products, exopolysaccharides (EPS) have many physiological functions and practical applications. Since EPS production by many bacteria is too scarce for practical use and only few species are known for their high levels of production, the search of new high EPS producers is of paramount importance. Many marine bacteria, that produce EPS to cope with strong environmental stress, could be potentially exploited at the industrial level. RESULTS: A novel bacterium, strain BM39, previously isolated from sediments collected in the Tyrrhenian Sea, was selected for its production of very high levels of EPS. BM39 was affiliated to Pantoea sp. (Enterobacteriaceae) by 16S rRNA gene sequencing and biochemical tests. According to the phylogenetic tree, this strain, being quite far from the closest known Pantoea species (96% identity with P. agglomerans and P. ananatis) could belong to a new species. EPS production was fast (maximum of ca. 21 g/L in 24 h on glucose medium) and mainly obtained during the exponential growth. Preliminary characterization, carried out by thin layer and gel filtration chromatography, showed that the EPS, being a glucose homopolymer with MW of ca. 830 kDa, appeared to be different from those of other bacteria of same genus. The bacterium showed a typical slightly halophilic behavior growing optimally at NaCl 40 ‰ (growing range 0-100 ‰). Flow cytometry studies indicated that good cell survival was maintained for 24 h at 120 ‰. Survival decreased dramatically with the increase of salinity being only 1 h at 280 ‰. The biochemical characterization, carried out with the Biolog system, showed that MB39 had a rather limited metabolic capacity. Its ability, rather lower than that of P. agglomerans, was almost only confined to the metabolization of simple sugars and their derivatives. Few alcohols, organic acids and nitrogen compounds were partially used too. CONCLUSIONS: Strain BM39, probably belonging to a new species, due to its remarkable EPS production, comparable to those of known industrial bacterial producers, could be suggested as a new microorganism for industrial applications.

Bacterial community structure and enzyme activities in a membrane bioreactor (MBR) using pure oxygen as an aeration source.

A pilot-scale membrane bioreactor was used to treat urban wastewater using pure oxygen instead of air as a source of aeration, to study its influence on bacterial diversity and levels of enzyme activities (acid and alkaline phosphatases, glucosidase, protease, and esterase) in the sludge. The experimental work was developed in two stages influenced by seasonal temperature. Operational parameters (temperature, pH, BOD5, COD, total and volatile suspended solids) were daily monitored, and enzyme activities measured twice a week. Redundancy analysis (RDA) was used to reveal relationships between the level of enzyme activities and the variation of operational parameters, demonstrating a significant effect of temperature and volatile suspended solids. Bacterial diversity was analyzed by temperature-gradient gel electrophoresis of PCR-amplified partial 16S rRNA genes. Significant differences in community structure were observed between both stages. Sequence analysis revealed that the prevalent Bacteria populations were evolutively close to Alphaproteobacteria (44%), Betaproteobacteria (25%) and Firmicutes (17%).

Biological treatment of two-phase olive mill wastewater (TPOMW, alpeorujo): polyhydroxyalkanoates (PHAs) production by Azotobacter strains.

Azotobacter chroococcum H23 (CECT 4435), Azotobacter vinelandii UWD, and Azotobacter vinelandii (ATCC 12837), members of the family Pseudomonadaceae, were used to evaluate their capacity to grow and accumulate polyhydroxyalkanoates (PHAs) using two-phase olive mill wastewater (TPOMW, alpeorujo) diluted at different concentrations as the sole carbon source. The PHAs amounts (g/l) increased clearly when the TPOMW samples were previously digested under anaerobic conditions. The MNR analysis demonstrated that the bacterial strains formed only homopolymers containing beta-hydroxybutyrate, either when grown in diluted TPOMW medium or diluted anaerobically digested TPOMW medium. COD values of the diluted anaerobically digested waste were measured before and after the aerobic PHA-storing phase, and a clear reduction (72%) was recorded after 72 h of incubation. The results obtained in this study suggest the perspectives for using these bacterial strains to produce PHAs from TPOMW, and in parallel, contribute efficiently to the bioremediation of this waste. This fact seems essential if bioplastics are to become competitive products.

Metabolic characterization of a strain (BM90) of Delftia tsuruhatensis showing highly diversified capacity to degrade low molecular weight phenols.

A novel bacterium, strain BM90, previously isolated from Tyrrhenian Sea, was metabolically characterized testing its ability to use 95 different carbon sources by the Biolog system. The bacterium showed a broad capacity to use fatty-, organic- and amino-acids; on the contrary, its ability to use carbohydrates was extremely scarce. Strain BM90 was identified and affiliated to Delftia tsuruhatensis by molecular techniques based on 16S rRNA gene sequencing. D. tsuruhatensis BM90, cultivated in shaken cultures, was able to grow on various phenolic compounds and to remove them from its cultural broth. The phenols used, chosen for their presence in industrial or agro-industrial effluents, were grouped on the base of their chemical characteristics. These included benzoic acid derivatives, cinnamic acid derivatives, phenolic aldehyde derivatives, acetic acid derivatives and other phenolic compounds such as catechol and p-hydroxyphenylpropionic acid. When all the compounds (24) were gathered in the same medium (total concentration: 500 mg/l), BM90 caused the complete depletion of 18 phenols and the partial removal of two others. Only four phenolic compounds were not removed. Flow cytometry studies were carried out to understand the physiological state of BM90 cells in presence of the above phenols in various conditions. At the concentrations tested, a certain toxic effect was exerted only by the four compounds that were not metabolized by the bacterium.

Production of chitinolytic enzymes by a strain (BM17) of Paenibacillus pabuli isolated from crab shells samples collected in the east sector of central Tyrrhenian Sea.

Nineteen bacterial isolates were grown in shaken cultures in media containing chitin as carbon source and different additional nitrogen sources such as yeast nitrogen base (YNB), yeast extract (YE), corn steep liquor (CSL) and ammonium sulfate. Strain BM17 showed the highest activity (200 U/l) in medium containing Chitin (1%) and YNB (0.5%). Molecular analysis of the 16S rRNA gene showed that strain BM17 belongs to the species Paenibacillus pabuli (99.72% homology). The enzyme activity started after 12-24 h; exponential enzyme production was recorded from the 24th h and lasted till the 96th h of incubation when activity peaked to decrease thereafter. Medium optimisation was carried out by Response Surface Methodology (RSM) considering the effects of chitin, corn steep liquor and yeast extract. BM17 chitinolytic activity was induced by chitin but the increase of its concentration did not have significant effects on the enzyme activity. By contrast, the nitrogen source, particularly YE, strongly affected the enzyme production.