The continuing reports of plastic pollution in various ecosystems highlight the threat posed by the ever-increasing consumption of synthetic polymers. Therefore, Pseudomonas capeferrum TDA1, a strain ...recently isolated from a plastic dump site, was examined further regarding its ability to degrade polyurethane (PU) compounds. The previously reported degradation pathway for 2,4-toluene diamine, a precursor and degradation intermediate of PU, could be confirmed by RNA-seq in this organism. In addition, different cell fractions of cells grown on a PU oligomer were tested for extracellular hydrolytic activity using a standard assay. Strikingly, purified outer membrane vesicles (OMV) of P. capeferrum TDA1 grown on a PU oligomer showed higher esterase activity than cell pellets. Hydrolases in the OMV fraction possibly involved in extracellular PU degradation were identified by mass spectrometry. On this basis, we propose a model for extracellular degradation of polyester-based PUs by P. capeferrum TDA1 involving the role of OMVs in synthetic polymer degradation.
Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation ...Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.
In this study, we suggest a clogging maintenance strategy and design alteration for constructed wetlands. Such a system could benefit rural communities who rely solely on constructed wetland systems ...for provision of household and agricultural water and for whom regular system shut-down for maintenance is essentially not viable. A newly established, pilot-scale horizontal subsurface flow constructed wetland was investigated by means of step-change tracer experiments. Sampling was carried out at multiple points down the length of the wetland and at three depths for each location. The mean residence time data were used to generate velocity profiles. This methodology enabled the development of a three-dimensional hydraulic model to identify dead zones and regions of short-circuiting within the system. Biomimetic principles were then incorporated to propose improvements to conventional constructed wetland design, which would potentially allow for better clogging management and continuous operation. In particular, modularizing the regions of the wetland most prone to clogging (those containing dead space) would accommodate isolation, removal and replacement of such sections, while still allowing treatment of wastewater in adjacent sections.
In this study, biomimetic principles were incorporated into a kinetic study of a pilot-scale, horizontal subsurface-flow constructed wetland (6.0 m × 1.0 m × 0.5 m) in Leipzig, Germany. The bed ...contained glacial gravel (4-8 mm) planted with Phragmites australis. Construction was completed in October 2013 and experiments commenced in August 2015. During establishment, the system was fed with only municipal tap water (165 L.d-1). The Phragmites root system had penetrated to the bottom of the wetland within 18 months. To break into the constructed wetland ‘black box’, the system was divided into a three-dimensional grid of sample ports. Initially, the wetland was physicochemically characterized (prior to addition of nutrients from an external source) in order to quantify the natural, baseline state. Thereafter, an impulse-response tracer test was conducted, using a fluorometer, for continual measurement of uranine concentration. 100% tracer recovery was achieved. The RTD was multi-modal - indicating by-pass flow - and showed long tailing due to mixing, diffusive effects and dead zones. Kinetic performance was investigated via monitoring total organic carbon and total nitrogen degradation, with a continual feed of artificial domestic wastewater (110 mg.L-1 COD). 93% reduction in TOC and TN was achieved for 5 weeks (11 November - 08 December 2015), despite high inflow loading (69.9 g.m-3.d-1 TOC; 28.1 g.m-3.d-1 TN) and colder temperatures. There was a general decline in reaction rate and rate constant from late October to early December. The average rates of TOC and TN removal were 65.08 +- 2.16 g.m-3.d-1 and 26.22 +- 0.68 g.m-3.d-1, respectively (Tanks-In-Series model). These results are the first set in a series. Continual observation and repetition of these experiments into long-term operation will deepen understanding of the internal development and performance of constructed wetlands, as is in line with the biomimetic approach, and provide the basis of a framework for improved wetland design.
Understanding the flow behaviour and accounting for time in constructed wetlands is necessary for the modelling and design of these systems. The addition of a tracer into the feed of a horizontal ...subsurface flow wetland allows the researcher to understand the flow of the fluid through the system, especially if the mass of the tracer is conserved (i.e. the mass of tracer injected is equal to the mass of tracer leaving the system). Conservative tracers, however, may pose a problem when it comes to the disposal of the effluent of the system if they are hazardous to the environment. In this study, the use of benzoate as a non-conservative degradable hydraulic tracer was investigated. The response curve of such a tracer is distorted due to its degradation within the wetland and hence the mass leaving the system is not equal to the mass injected. As a result, the typical hydraulic performance parameters obtained from tracer-response curves cannot be accurately calculated. In this paper, a curve-shift technique was developed by using a benzoate step-change curve as an input and using mathematical techniques to transform it into a conservative tracer-response curve. This was done through establishing a mathematical relationship between the retardation and hydrodynamic dispersion of benzoate and a known conservative tracer, uranine. This methodology was tested by conducting a dual tracer study using both benzoate and uranine and comparing the shifted benzoate response curve with the uranine response curve. Hydraulic parameters including mean residence time, effective volume ratio and hydraulic efficiency for each of these tracers were also compared.
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•Flow behaviour and hydraulics of constructed wetlands is still not fully understood.•Hydraulic modelling of constructed wetlands requires conservative tracers.•Use of a non-conservative tracer is investigated.•Methodology relating conservative and non-conservative tracers is proposed.
Here, we report the draft genome sequence of
sp. 15-1. This strain was isolated from a planted fixed-bed reactor based on its ability to degrade toluene under anaerobic conditions. The genome ...assembly consists of 5.4 Mb in 28 contigs and 5,095 coding sequences containing the genes involved in anaerobic toluene degradation.
Pseudomonas putida mt-2 harbors the TOL plasmid (pWWO), which contains the genes encoding the enzymes necessary to degrade toluene aerobically. The xyl genes are clustered in the upper operon and ...encode the enzymes of the upper pathway that degrade toluene to benzoate, while the genes encoding the enzymes of the lower pathway (meta-cleavage pathway) that are necessary for the conversion of benzoate to tricarboxylic acid cycle intermediates, are encoded in a separate operon. In this study, the effects of oxygen availability and oscillation on the expression of catabolic genes for enzymes involved in toluene degradation were studied by using P. putida mt-2 as model bacterium. Quantitative reverse transcription-PCR was used to detect and quantify the expression of the catabolic genes xylM (a key gene of the upper pathway) and xylE (a key gene of the lower pathway) in cultures of P. putida mt-2 that were grown with toluene as a carbon source. Toluene degradation was shown to have a direct dependency on oxygen concentration, where gene expression of xylM and xylE decreased due to oxygen depletion during degradation. Under oscillating oxygen concentrations, P. putida mt-2 induced or downregulated xylM and xylE genes according to the O₂ availability in the media. During anoxic periods, P. putida mt-2 decreased the expression of xylM and xylE genes, while the expression of both xylM and xylE genes was immediately increased after oxygen became available again in the medium. These results suggest that oxygen is not only necessary as a cosubstrate for enzyme activity during the degradation of toluene but also that oxygen modulates the expression of the catabolic genes encoded by the TOL plasmid.
Constructed wetlands are important ecosystems with respect to nitrogen cycling. Here we studied the activity and abundance of nitrogen transforming bacteria as well as the spatial distribution of ...nitrification, anaerobic ammonium oxidation (anammox), and denitrification processes in a horizontal subsurface-flow constructed wetland. The functional genes of the nitrogen cycle were evenly distributed in a linear way along the flow path with prevalence at the superficial points. The same trend was observed for the nitrification and denitrification turnover rates using isotope labeling techniques. It was also shown that only short-term incubations should be used to measure denitrification turnover rates. Significant nitrate consumption under aerobic conditions diminishes nitrification rates and should therefore be taken into account when estimating nitrification turnover rates. This nitrate consumption was due to aerobic denitrification, the rate of which was comparable to that for anaerobic denitrification. Consequently, denitrification should not be considered as an exclusively anaerobic process. Phylogenetic analysis of hydrazine synthase (hzsA) gene clones indicated the presence of Brocadia and Kuenenia anammox species in the constructed wetland. Although anammox bacteria were detected by molecular methods, anammox activity could not be measured and hence this process appears to be of low importance in nitrogen transformations in these freshwater ecosystems.
•The activity and abundance of nitrogen transforming bacteria were quantified.•Significant nitrate consumption under aerobic conditions was observed.•This nitrate consumption was due to aerobic denitrification.•Only short-term incubations should be used to measure denitrification turnover rates.•Anammox appears to be of low importance in nitrogen transformations in these ecosystems.
This paper describes the hydraulic performance of a start-up, pilot-scale, horizontal sub-surface flow constructed wetland (CW), located outdoors at the Helmholtz UFZ, Leipzig. This paper aims to ...investigate the impact of the method of hydraulic calculation in a pilot-scale system. Impulse-response tracer tests were conducted at multiple depths and locations throughout the system and the uranine concentration was measured using a fluorometer. In addition, the volumetric flow rate was closely monitored and climatic data was gathered to support the hydraulic results. Werner and Kadlec's modified residence time distribution (RTD) theory (originally developed for systems with large flow rate and volume fluctuations) was applied and the results compared to those obtained using classic RTD theory.
Progressive uranine dispersion, broadening of the RTD base, a change in peak shape and extended tailing were observed with increasing distance. All of these factors indicated deviation from plug flow and mixing effects with low-to-moderate dead volume. As this was a non-steady flow system, application of modified RTD theory ensured that the first moments of the normalized breakthrough curves and RTD functions were always unity. The Student's t-test (95% confidence) showed that the outlet RTDs calculated assuming steady-flow were significantly different, but those determined using the modified theory were closely comparable.
In general, a decrease in flow rate from inlet to outlet was observed and fluctuations in the outflow were linked to climatic conditions. August was characterized by the highest temperatures, high global radiation and high rates of evapotranspiration. Low or no outflow was recorded in conjunction with high evapotranspiration. The lowest temperatures, low global radiation, low evapotranspiration and high humidity were recorded in October, as well as the second highest rainfall (82 mm) after June (115 mm). Surges in outflow were observed with rain events.
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•Impulse-response tracer tests in a start-up, non-steady flow, pilot-scale HSSF CW•Fluctuations in outflow linked to vegetation and climatic conditions.•Comparison of results of classic RTD theory with Werner & Kadlec's modified method•Significant difference between outlet RTDs calculated using standard RTD theory.•Variable flow method should be used preferentially as outlet RTDs are comparable.
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