Search

Dichloromethane (DCM; CH2Cl2) is a toxic groundwater pollutant that also has a detrimental effect on atmospheric ozone levels. As a dense non-aqueous phase liquid, DCM migrates vertically through groundwater to low redox zones, yet information on anaerobic microbial DCM transformation remains scarce due to a lack of cultured organisms. We report here the characterisation of DCMF, the dominant organism in an anaerobic enrichment culture (DFE) capable of fermenting DCM to the environmentally benign product acetate. Stable carbon isotope experiments demonstrated that the organism assimilated carbon from DCM and bicarbonate via the Wood–Ljungdahl pathway. DCMF is the first anaerobic DCM-degrading population also shown to metabolise non-chlorinated substrates. It appears to be a methylotroph utilising the Wood–Ljungdahl pathway for metabolism of methyl groups from methanol, choline, and glycine betaine. The flux of these substrates from subsurface environments may either directly (DCM, methanol) or indirectly (choline, glycine betaine) affect the climate. Community profiling and cultivation of cohabiting taxa in culture DFE without DCMF suggest that DCMF is the sole organism in this culture responsible for substrate metabolism, while the cohabitants persist via necromass recycling. Genomic and physiological evidence support placement of DCMF in a novel genus within the Peptococcaceae family, ‘Candidatus Formimonas warabiya’.

Gas-liquid flow in annular geometries is the one of the most frequently encountered flow conditions in petroleum industry, either during drilling operations if aerated fluids are used, or production stages, if the produced fluid is under bubble point pressure. With the increase in the interest in horizontal / extended reach wells, understanding the flow behavior of gas-liquid mixtures in horizontal wells is essential for better pressure control downhole. Although two-phase fluid flow is studied intensively for circular pipes, there exists a lack of information about aerated fluid flow behavior inside annular geometries, both theoretically and experimentally. Existing two-phase fluid flow models available in the literature developed for circular pipes are performing poorly for annular geometries. Using hydraulic diameter definitions or effective diameter terms simply give inaccurate results for both flow pattern estimations and friction pressure loss determination. This study aims to identify the flow patterns of gasified fluids, and to determine frictional pressure losses for two phase flow through horizontal eccentric annular geometry. In order to develop the liquid holdup, Digital Image Processing Techniques have been used. Friction pressure losses are determined by applying two different methods; i) Modifying Lockhart-Martinelli parameter, and ii) Modifying Beggs and Brill's method, originally developed for circular pipes. Experiments have been conducted at Middle East Technical University (METU) Multiphase Flow Loop using air-water mixtures with various in-situ flow velocities. A digital camera is used for recording each test dynamically for the identification of flow patterns and the measurement of liquid holdup. Friction pressure losses are recorded during each test. The comparison of modified models with experimental data indicates that liquid holdup and friction pressure losses can be estimated with a reasonable accuracy. The information obtained from this s, ASME, Fluids Engn Div

Bacteria capable of dechlorinating the toxic environmental contaminant dichloromethane (DCM, CH2Cl2) are of great interest for potential bioremediation applications. A novel, strictly anaerobic, DCM-fermenting bacterium, ‘‘DCMF’’, was enriched from organochlorine-contaminated groundwater near Botany Bay, Australia. The enrichment culture was maintained in minimal, mineral salt medium amended with dichloromethane as the sole energy source. PacBio whole genome SMRTTM sequencing of DCMF allowed de novo, gap-free assembly despite the presence of cohabiting organisms in the culture. Illumina sequencing reads were utilised to correct minor indels. The single, circularised 6.44 Mb chromosome was annotated with the IMG pipeline and contains 5,773 predicted protein-coding genes. Based on 16S rRNA gene and predicted proteome phylogeny, the organism appears to be a novel member of the Peptococcaceae family. The DCMF genome is large in comparison to known DCM-fermenting bacteria. It includes an abundance of methyltransferases, which may provide clues to the basis of its DCM metabolism, as well as potential to metabolise additional methylated substrates such as quaternary amines. Full annotation has been provided in a custom genome browser and search tool, in addition to multiple sequence alignments and phylogenetic trees for every predicted protein, http://www.slimsuite.unsw.edu.au/research/dcmf/.

Drilling with aerated muds is becoming more often used in underbalanced drilling operations. One of the major challenges that has to be faced in such operations is the estimation of the physical behavior of aerated fluids inside the annulus. In this study, experiments have been conducted at METU Multiphase Flow Loop using air-water mixtures with various in-situ flow velocities of 0-120 and 0-10 ft/s, respectively. This study aims to develop a model to estimate the frictional pressure losses for two-phase flow through horizontal eccentric annular geometry. In order to estimate the frictional pressure losses, three different methods were developed: (i) definition of new friction factors by using experimental data; (ii) modification of Lockhart-Martinelli pressure loss correction factor; and (iii) modification of Beggs and Brill model by changing the equation constants. The comparison of the developed models with experimental data has shown that frictional pressure losses can be estimated with a reasonable accuracy.

Flow patterns, liquid holdup, and frictional pressure gradient are three importance parameters to study the multiphase drilling fluid behavior. Although two-phase fluid flow is studied in detail for pipes, there exists a lack of information about aerated fluid flow behavior inside a wellbore. This study aims to identify the flow patterns of gasified fluids flowing inside a horizontal annulus, and to develop a method for measurement of liquid holdup by using the image processing techniques. Experiments have been conducted at Middle East Technical University (METU) Multiphase Flow Loop using air-water mixtures with various in-situ flow velocities. A digital high-speed camera is used for recording each test dynamically for the identification of flow patterns and the measurement of liquid holdup.

Drilling with aerated muds is becoming more often used in underbalanced drilling operations. One of the major challenges that has to be faced in such operations is the estimation of the physical behavior of aerated fluids inside the annulus. In this study, experiments have been conducted at METU Multiphase Flow Loop using air-water mixtures with various in-situ flow velocities of 0-120 and 0-10 ft/s, respectively. This study aims to develop a model to estimate the frictional pressure losses for two-phase flow through horizontal eccentric annular geometry. In order to estimate the frictional pressure losses, three different methods were developed: (i) definition of new friction factors by using experimental data; (ii) modification of Lockhart-Martinelli pressure loss correction factor; and (iii) modification of Beggs and Brill model by changing the equation constants. The comparison of the developed models with experimental data has shown that frictional pressure losses can be estimated with a reasonable accuracy.

Flow patterns, liquid holdup, and frictional pressure gradient are three importance parameters to study the multiphase drilling fluid behavior. Although two-phase fluid flow is studied in detail for pipes, there exists a lack of information about aerated fluid flow behavior inside a wellbore. This study aims to identify the flow patterns of gasified fluids flowing inside a horizontal annulus, and to develop a method for measurement of liquid holdup by using the image processing techniques. Experiments have been conducted at Middle East Technical University (METU) Multiphase Flow Loop using air-water mixtures with various in-situ flow velocities. A digital high-speed camera is used for recording each test dynamically for the identification of flow patterns and the measurement of liquid holdup.

Dehalobacter sp. strain TeCB1 was isolated from groundwater near Sydney, Australia, that is polluted with a range of organochlorines. The isolated strain is able to grow by reductive dechlorination of 1,2,4,5-tetrachlorobenzene to 1,3- and 1,4-dichlorobenzene with 1,2,4-trichlorobenzene being the intermediate daughter product. Transient production of 1,2-dichlorobenzene was detected with subsequent conversion to monochlorobenzene. The dehalogenation capability of strain TeCB1 to respire 23 alternative organochlorines was examined and shown to be limited to the use of 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene. Growth on 1,2,4-trichlorobenzene resulted in the production of predominantly 1,3- and 1,4-dichlorobenzene. The inability of strain TeCB1 to grow on 1,2-dichlorobenzene indicated that the production of monochlorobenzene during growth on 1,2,4,5-tetarchlorobezene was cometabolic. The annotated genome of strain TeCB1 contained only one detectable 16S rRNA gene copy and genes for 23 full-length and one truncated Reductive Dehalogenase (RDase) homologs, five unique to strain TeCB1. Identification and functional characterization of the 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene RDase (TcbA) was achieved using native-PAGE coupled with liquid chromatography tandem mass spectrometry. Interestingly, TcbA showed higher amino acid identity with tetrachloroethene reductases PceA (95% identity) from Dehalobacter restrictus PER-K23 and Desulfitobacterium hafniense Y51 than with the only other chlorinated benzene reductase [i.e., CbrA (30% identity)] functionally characterized to date.

© 2016 Jugder, Ertan, Bohl, Lee, Marquis and Manefield.Organohalides are recalcitrant pollutants that have been responsible for substantial contamination of soils and groundwater. Organohalide-respiring bacteria (ORB) provide a potential solution to remediate contaminated sites, through their ability to use organohalides as terminal electron acceptors to yield energy for growth (i.e., organohalide respiration). Ideally, this process results in non- or lesser-halogenated compounds that are mostly less toxic to the environment or more easily degraded. At the heart of these processes are reductive dehalogenases (RDases), which are membrane bound enzymes coupled with other components that facilitate dehalogenation of organohalides to generate cellular energy. This review focuses on RDases, concentrating on those which have been purified (partially or wholly) and functionally characterized. Further, the paper reviews the major bacteria involved in organohalide breakdown and the evidence for microbial evolution of RDases. Finally, the capacity for using ORB in a bioremediation and bioaugmentation capacity are discussed.

Dehalobacter sp. strain UNSWDHB can dechlorinate up to 4 mM trichloromethane at a rate of 0.1 mM per day to dichloromethane and 1,1,2-trichloroethane (1 mM, 0.1 mM per day) with the unprecedented product profile of 1,2-dichloroethane and vinyl chloride. 1,1,1-trichloroethane and 1,1-dichloroethane were slowly utilized by strain UNSWDHB and were not completely removed, with minimum threshold concentrations of 0.12 mM and 0.07 mM respectively under growth conditions. Enzyme kinetic experiments confirmed strong substrate affinity for trichloromethane and 1,1,2-trichloroethane (Km = 30 and 62 µM respectively) and poor substrate affinity for 1,1,1-trichloroethane and 1,1-dichloroethane (Km = 238 and 837 µM respectively). Comparison of enzyme kinetic and growth data with other trichloromethane respiring organisms (Dehalobacter sp. strain CF and Desulfitobacterium sp. strain PR) suggests an adaptation of strain UNSWDHB to trichloromethane. The trichloromethane RDase (TmrA) expressed by strain UNSWDHB was identified by BN-PAGE and functionally characterized. Amino acid comparison of homologous RDases from all three organisms revealed only six significant amino acid substitutions/deletions, which are likely to be crucial for substrate specificity. Furthermore, strain UNSWDHB was shown to grow without exogenous supply of cobalamin confirming genomic-based predictions of a fully functional cobalamin synthetic pathway.

Organohalide respiring bacteria (ORB) are capable of utilising organohalides as electron acceptors for the generation of cellular energy and consequently play an important role in the turnover of natural and anthropogenically-derived organohalides. In this study, the response of a Dehalobacter sp. strain UNSWDHB to the addition of trichloromethane (TCM) after a 50 h period of its absence (suffocation) was evaluated from a transcriptomic and proteomic perspective. The up-regulation of TCM reductive dehalogenase genes (tmrABC) and their gene products (TmrABC) was confirmed at both transcriptional and proteomic levels. Other findings include the upregulation of various hydrogenases (membrane-associated Ni-Fe hydrogenase complexes and soluble Fe-Fe hydrogenases), formate dehydrogenases, complex I and a pyrophosphate-energized proton pump. The elevated expression of enzymes associated with carbon metabolism, including complete Wood Ljungdahl pathway, during TCM respiration raises interesting questions on possible fates of intracellular formate and its potential role in the physiology of this bacterium. Overall, the findings presented here provide a broader view on the bioenergetics and general physiology of Dehalobacter UNSWDHB cells actively respiring with TCM.