Zeev Ronen

Anaerobic-Aerobic Process for Microbial Degradation of Tetrabromobisphenol A

ABSTRACT Tetrabromobisphenol A (TBBPA) is a flame retardant that is used as an additive during manufacturing of plastic polymers and electronic circuit boards. Little is known about the fate of this compound in the environment. In the current study we investigated biodegradation of TBBPA, as well as 2,4,6-tribromophenol (TBP), in slurry of anaerobic sediment from a wet ephemeral desert stream bed contaminated with chemical industry waste. Anaerobic incubation of the sediment with TBBPA and peptone-tryptone-glucose-yeast extract medium resulted in a 80% decrease in the TBBPA concentration and accumulation of a single metabolite. This metabolite was identified by gas chromatography-mass spectrometry (GC-MS) as nonbrominated bisphenol A (BPA). On the other hand, TBP was reductively dehalogenated to phenol, which was further metabolized under anaerobic conditions. BPA persisted in the anaerobic slurry but was degraded aerobically. A gram-negative bacterium (strain WH1) was isolated from the contaminated soil, and under aerobic conditions this organism could use BPA as a sole carbon and energy source. During degradation of BPA two metabolites were detected in the culture medium, and these metabolites were identified by GC-MS and high-performance liquid chromatography as 4-hydroxybenzoic acid and 4-hydroxyacetophenone. Both of those compounds were utilized by WH1 as carbon and energy sources. Our findings demonstrate that it may be possible to use a sequential anaerobic-aerobic process to completely degrade TBBPA in contaminated soils.

Isolation from Agricultural Soil and Characterization of a Sphingomonas sp. Able To Mineralize the Phenylurea Herbicide Isoproturon

ABSTRACT A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the α-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.

Field observation of flow in a fracture intersecting unsaturated chalk

Flow through a natural fracture crossing unsaturated chalk in an arid region was investigated in a field experiment using a specially designed experimental setup. The setup allowed complete control of the flow domain inlet and outlet. Water flux into and out of the fracture was measured in small segments of the fracture openings, and flow trajectories were identified using seven fluorobenzoic acid tracers. A 5 day percolation experiment on a 5.3 m long fracture showed significant spatial and temporal variability of the flow regime. Flow through fracture openings did not reach a steady state either in individual segments or across the entire flow domain, although the boundary conditions were kept relatively steady for the entire duration of the experiment. Flow trajectories within the fracture plane varied over time; however, most of the flow was confined to small sections of the fracture. Over 70% of the flux was transmitted through <20% of the studied fracture openings. Observations from the tracer tests suggest that flow paths can coexist near each other without water mixing, probably because the fracture fill generates unconnected flow paths across the main fracture void.

Growth in Coculture Stimulates Metabolism of the Phenylurea Herbicide Isoproturon by Sphingomonas sp. Strain SRS2

ABSTRACT Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the β-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of 14C-labeled isoproturon to 14CO2 and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.

Effect of organic and inorganic nitrogenous compounds on RDX degradation and cytochrome P-450 expression in Rhodococcus strain YH1.

We hypothesized that biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)—a widely used explosive contaminating soil and groundwater—by Rhodococcus strain YH1 is controlled by the presence of external nitrogen sources. This strain is capable of degrading RDX while using it as sole nitrogen source under aerobic conditions. Both inorganic and organic nitrogen sources were found to have a profound impact on RDX-biodegradation activity. This effect was tested in growing and resting cells of strain YH1. Nitrate and nitrite delayed the onset of RDX degradation by strain YH1, while ammonium inhibited it almost completely. In addition, 2,4,6-trinitrotoluene (TNT) inhibited RDX degradation and growth of strain YH1. On the other hand, tetrahydrophthalamide did not influence biodegradation or growth. Growth on RDX induced the expression of a cytochrome P-450 enzyme that is suggested to be involved in the first step in the aerobic pathway of RDX degradation, as identified by SDS-PAGE analysis. Ammonium and nitrite strongly repressed cytochrome P-450 expression. Our findings suggest that effective RDX bioremediation by strain YH1 requires the design of a treatment scheme that includes initial removal of ammonium, nitrite, nitrate and TNT before RDX degradation can take place.

Sequential biodegradation of TNT, RDX and HMX in a mixture

We describe TNTs inhibition of RDX and HMX anaerobic degradation in contaminated soil containing indigenous microbial populations. Biodegradation of RDX or HMX alone was markedly faster than their degradation in a mixture with TNT, implying biodegradation inhibition by the latter. The delay caused by the presence of TNT continued even after its disappearance and was linked to the presence of its intermediate, tetranitroazoxytoluene. PCR-DGGE analysis of cultures derived from the soil indicated a clear reduction in microbial biomass and diversity with increasing TNT concentration. At high-TNT concentrations (30 and 90 mg/L), only a single band, related to Clostridium nitrophenolicum, was observed after 3 days of incubation. We propose that the mechanism of TNT inhibition involves a cytotoxic effect on the RDX- and HMX-degrading microbial population. TNT inhibition in the top active soil can therefore initiate rapid transport of RDX and HMX to the less active subsurface and groundwater.

The influence of antiscalants on biofouling of RO membranes in seawater desalination.

Antiscalants are surface active polyelectrolyte compounds commonly used in reverse osmosis (RO) desalination processes to avoid membrane scaling. In spite of the significant roles of antiscalants in preventing membrane scaling, they are prone to enhance biofilm growth on RO membranes by either altering membrane surface properties or by serving as nutritional source for microorganisms. In this study, the contribution of antiscalants to membrane biofouling in seawater desalination was investigated. The effects of two commonly used antiscalants, polyphosphonate- and polyacrylate-based, were tested. The effects of RO membrane (DOW-Filmtec SW30 HRLE-400) exposure to antiscalants on its physico-chemical properties were studied, including the consequent effects on initial deposition and growth of the sessile microorganisms on the RO membrane surface. The effects of antiscalants on membrane physico-chemical properties were investigated by filtration of seawater supplemented with the antiscalants through flat-sheet RO membrane and changes in surface zeta potential and hydrophobicity were delineated. Adsorption of antiscalants to polyamide surfaces simulating RO membranes polyamide layer and their effects on the consequent bacterial adhesion was tested using a quartz crystal microbalance with dissipation monitoring technology (QCM-D) and direct fluorescent microscopy. A significant increase in biofilm formation rate on RO membranes surface was observed in the presence of both types of antiscalants. Polyacrylate-based antiscalant was shown to enhance initial cell attachment as observed with the QCM-D and a parallel plate flow cell, due to rendering the polyamide surface more hydrophobic. Polyphosphonate-based antiscalants also increased biofilm formation rate, most likely by serving as an additional source of phosphorous to the seawater microbial population. A thicker biofilm layer was formed on the RO membrane when the polyacrylate-based antiscalant was used. Following these results, a wise selection of antiscalants for scaling control should take into account their contribution to membrane biofouling propensity.

Metabolism of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a contaminated vadose zone.

The aim of this study was to explore biodegradation potential of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a deep contaminated unsaturated zone over Israels coastal aquifer. While anaerobic biodegradation potential was observed throughout the profile down to the water table at a depth of 45 m, aerobic biodegradation was limited to the surface of the unsaturated zone. Traces of nitroso-RDX intermediates were detected in the soil samples, indicating possible in situ activity. Polymerase chain reaction and denaturing gradient gel electrophoresis analysis revealed that the microbial population in the soil consisted of protobacteria, but no known RDX degraders were detected. However, a 16S rRNA gene sequence most similar to Sphingomonas sp. was detected at all depths. Biodegradation rates were faster in the surface (0 and 1m) versus deeper soil samples (22 and 45 m) and were not affected under anaerobic conditions by the presence of nitrate, indicating a concurrent reduction of both compounds. RDX half-life in the surface soil was mostly dependent on carbon content and to lesser extent on soil moisture. Biomineralization of RDX to CO(2) was confirmed by incubating surface soil with (14)C-labeled RDX. An aerobic RDX-degrading bacterium, identified as Gordonia sp., was isolated from the soil: it degraded RDX aerobically and produced 4-nitro-2,4-diazabutanal. This study, the first to explore RDX biodegradation in the deep vadoze zone, indicates biodegradation potential throughout the profile, which is likely to support natural attenuation.

Soil nitrifying enrichments as biofilter starters in intensive recirculating saline water aquaculture

Abstract Intensive recirculating aquaculture relies on biofilters to sustain satisfactory water quality in the ponds. Establishment of new biofilters in aquaculture ponds without a start-up culture requires a long period of time and may therefore cause significant losses and environmental harm due to discharge of nitrogen-rich effluents. A laboratory scale setup (7-l aquaria with shrimp and fish) demonstrated that an external start-up nitrifying enrichment culture performed similarly to the natural bacterial population of an established pond biofilter, and superior to the performance of similar biofilters without a start-up culture (control). Ammonia concentration in the control treatment increased daily and reached 18 mg l −1 during a 14-day experiment, whereas in the treated aquaria, it averaged less than 2 mg l −1 . Fish growth and survival were similar in the treated aquaria (average growth of 0.45 g/14 days, and 95% survival) and significantly higher than in the control (average growth of 0.0 g/14 days, and 80% survival). The source for the enrichment cultures was soil samples collected from the region where the farm is situated. This approach may lead to the development of bacterial amendments (probiotic products) that can be used as start-up cultures for new operations or damaged filters, and potentially enhance nitrification in established filters. As the cultures are collected from soils, it is unlikely that they will be contaminated with fish disease-causing agents. This will improve water quality and consequently aquatic animal production.

Activity and survival of tribromophenol-degrading bacteria in a contaminated desert soil

A strain of bromophenol degrading bacteria was isolated from a contaminated desert soil. The isolate identified as Achromobacter piechaudii and designated as strain TBPZ was able to metabolize both 2,4,6-tribromophenol and chlorophenols. The degradation of halophenols resulted in the stechiometric release of bromide or chloride. Growth and degradation of bromophenol were enhanced in the presence of yeast extract. To follow the survival of an introduced bacteria in the contaminated soil, TBPZ was transformed with a plasmid carrying a gene for kanamycin resistance and the lux CDABE operon from the luminescent bacteria Vibrio fischeri under the control of a constitutive promoter producing strain TBPZ-N61. The activity of the transformed bacteria was not affected by the insertion of the plasmid. Specific detection of the introduced isolate in the contaminated soil samples was achieved by selection on kanamycin. Survival of the introduced bacteria, TBPZ-N61, in the contaminated soil was influenced by soil moisture. Biodegradation of TBP occurred only in soil with at least 25% water content. Addition of yeast extract increased the survival and the activity of the introduced bacteria. The current study demonstrated that the limiting factors controlling pollutant degradation in a contaminated desert soil are water content, nutrient availability and the bioaugmentation of an appropriate microbial population.