Produced water exposure alters bacterial response to biocides.

Microbial activity during the holding and reuse of wastewater from hydraulic fracturing operations, termed produced water, may lead to issues with corrosion, sulfide release, and fouling. Biocides are applied to control biological activity, often with limited efficacy, which is typically attributed to chemical interactions with the produced water. However, it is unknown whether there is a biologically driven mechanism to biocide tolerance in produced water. Here, we demonstrate that produced water exposure results in an enhanced tolerance against the typically used biocide glutaraldehyde and increased susceptibility to the oxidative biocide hypochlorite in a native and a model bacteria and that this altered resistance is due to the salinity of the produced water. In addition, we elucidate the genetic response of the model organism Pseudomonas fluorescens to produced water exposure to provide a mechanistic interpretation of the altered biocide resistance. The RNA-seq data demonstrated the induction of genes involved in osmotic stress, energy production and conversion, membrane integrity, and protein transport following produced water exposure, which facilitates bacterial survival and alters biocide tolerance. Efforts to fundamentally understand biocide resistance mechanisms, which enable the optimization of biocide application, hold significant implications for greening of the fracturing process through encouraging produced water recycling. Specifically, these results suggest the necessity of optimizing biocide application at the level of individual shale plays, rather than historical experience, based upon produced water characteristics and salinity.

Type 2 quorum sensing monitoring, inhibition and biofilm formation in marine microrganisms.

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 µM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 µM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that QSI compounds must be selected in the specific system in which they are to function, and they cannot easily be transferred from one QS system to another.

Anti-biofilm performance of three natural products against initial bacterial attachment.

Marine bacteria contribute significantly towards the fouling consortium, both directly (modern foul release coatings fail to prevent "slime" attachment) and indirectly (biofilms often excrete chemical cues that attract macrofouling settlement). This study assessed the natural product anti-biofilm performance of an extract of the seaweed, Chondrus crispus, and two isolated compounds from terrestrial sources, (+)-usnic acid and juglone, against two marine biofilm forming bacteria, Cobetia marina and Marinobacter hydrocarbonoclasticus. Bioassays were developed using quantitative imaging and fluorescent labelling to test the natural products over a range of concentrations against initial bacterial attachment. All natural products affected bacterial attachment; however, juglone demonstrated the best anti-biofilm performance against both bacterial species at a concentration range between 5-20 ppm. In addition, for the first time, a dose-dependent inhibition (hormetic) response was observed for natural products against marine biofilm forming bacteria.

Substrate-dependent modulation of the enzymatic catalytic activity: reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed.

Purification and characterization of maltooligosaccharide-forming α-amylase from moderately halophilic Marinobacter sp. EMB8.

Maltooligosaccharides especially maltotriose and maltotetraose producing amylases are highly desirable for application in bread making and other food industries. A maltotriose and maltotetraose producing amylase from moderately halophilic Marinobacter sp. EMB8 is described. Under optimized culture conditions, 48.0 IU/mL amylase was obtained. The enzyme was purified to homogeneity by ultrafiltration, DEAE cellulose and Sephadex G-75 column chromatography with 52% yield and 76-fold purification. It was a monomeric protein of 72 kDa. The amylase had many novel features viz. stability up to 20% NaCl, 80 °C temperature, pH 6.0-11.0 and in wide range of organic solvents at high concentrations. The enzyme efficiently hydrolyzed starch into maltooligosaccharides rich in maltotriose and maltotetraose. These novel properties make the Marinobacter sp. amylase a potentially useful enzyme.

[Identification and characterization of a novel hydrocarbon-degrading Marinobacter sp. PY97S].

OBJECTIVE: To identify and characterize a hydrocarbon-degrading bacterium isolated from the sediment of the Yellow Sea. METHODS: We used 16S rRNA gene sequences based phylogenetic analysis, physiological and biochemical characterization, DNA G + C content assaying, determination of cellular fatty acids, testing of carbon sources and respiratory lipoquinone and experiment of DNA-DNA relatedness. Its capability of degrading aliphatic hydrocarbons in ONR7a media supplemented with nine n-alkanes, separately, as sole source of carbon and energy was further determined. RESULTS: The Gram-negative isolate PY97S was a member of the genus Marinobacter, catalase-and oxidase-positive, and with Q-9 as its predominant respiratory lipoquinone. The similarity between its 16S rRNA gene and that of its most closely related type strain in GenBank Marinobacter koreensis DD-M3(T) was 96.93%, and their level of DNA relatedness was 46.7%. The appropriate temperature for its growth ranged from 15 degrees C to 35 degrees C with the optimum of 30 degrees C, the appropriate initial acidity from pH 6.0 to 9.5 with the optimum of pH 7.0, and the appropriate salinity (NaCl) from 0% to 10% with the optimum of 0%. It metabolized many carbohydrates and organic acids and was sensitive to diverse antibiotics including ampicillin and piperacillin. The G + C content of its genomic DNA was 48.2 mol%. The major fatty acids were 2-methyl C15:0 (29.97%), C16: 1omega7c (27.22%), C12:0 (22.22%) and C16: 1omega9c (5.73%). CONCLUSION: The isolate PY97S was identified as a petroleum hydrocarbon-degrading novel species of genus Marinobacter, holding the potential of being applied in the bioremediation of oil spill.

The potential of nano-structured silicon oxide type coatings deposited by PACVD for control of aquatic biofouling.

SiO(x)-like coatings were deposited on glass slides from a hexamethylsiloxane precursor by plasma-assisted CVD (PACVD). Surface energies (23.1-45.7 mJ m(-1)) were correlated with the degree of surface oxidation and hydrocarbon contents. Tapping mode AFM revealed a range of surface topologies with Ra values 1.55-3.16 nm and RMS roughness 1.96-4.11 nm. Settlement of spores of the green alga Ulva was significantly less, and detachment under shear significantly more on the lowest surface energy coatings. Removal of young plants (sporelings) of Ulva under shear was positively correlated with reducing the surface energy of the coatings. The most hydrophobic coatings also showed good performance against a freshwater bacterium, Pseudomonas fluorescens, significantly reducing initial attachment and biofilm formation, and reducing the adhesion strength of attached bacterial cells under shear. Taken together the results indicate potential for further investigation of these coatings for applications such as heat exchangers and optical instruments.