In situ†protein-SIP highlights Burkholderiaceae as key players degrading toluene by para ring hydroxylation in a constructed wetland model.

In constructed wetlands, organic pollutants are mainly degraded via microbial processes. Helophytes, plants that are commonly used in these systems, provide oxygen and root exudates to the rhizosphere, stimulating microbial degradation. While the treatment performance of constructed wetlands can be remarkable, a mechanistic understanding of microbial degradation processes in the rhizosphere is still limited. We investigated microbial toluene removal in a constructed wetland model system combining 16S rRNA gene sequencing, metaproteomics and (13) C-toluene in situ protein-based stable isotope probing (protein-SIP). The rhizospheric bacterial community was dominated by Burkholderiales and Rhizobiales, each contributing about 20% to total taxon abundance. Protein-SIP data revealed that the members of Burkholderiaceae, the proteins of which showed about 73% of (13) C-incorporation, were the main degraders of toluene in the planted system, while the members of Comamonadaceae were involved to a lesser extent in degradation (about 64% (13) C-incorporation). Among the Burkholderiaceae, one of the key players of toluene degradation could be assigned to Ralstonia pickettii. We observed that the main pathway of toluene degradation occurred via two subsequent monooxygenations of the aromatic ring. Our study provides a suitable approach to assess the key processes and microbes that are involved in the degradation of organic pollutants in complex rhizospheric ecosystems.

Aerobic Toluene Degraders in the Rhizosphere of a Constructed Wetland Model Show Diurnal Polyhydroxyalkanoate Metabolism.

UNLABELLED: Constructed wetlands (CWs) are successfully applied for the treatment of waters contaminated with aromatic compounds. In these systems, plants provide oxygen and root exudates to the rhizosphere and thereby stimulate microbial degradation processes. Root exudation of oxygen and organic compounds depends on photosynthetic activity and thus may show day-night fluctuations. While diurnal changes in CW effluent composition have been observed, information on respective fluctuations of bacterial activity are scarce. We investigated microbial processes in a CW model system treating toluene-contaminated water which showed diurnal oscillations of oxygen concentrations using metaproteomics. Quantitative real-time PCR was applied to assess diurnal expression patterns of genes involved in aerobic and anaerobic toluene degradation. We observed stable aerobic toluene turnover by Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis was upregulated in these bacteria during the day, suggesting that they additionally feed on organic root exudates while reutilizing the stored carbon compounds during the night via the glyoxylate cycle. Although mRNA copies encoding the anaerobic enzyme benzylsuccinate synthase (bssA) were relatively abundant and increased slightly at night, the corresponding protein could not be detected in the CW model system. Our study provides insights into diurnal patterns of microbial processes occurring in the rhizosphere of an aquatic ecosystem. IMPORTANCE: Constructed wetlands are a well-established and cost-efficient option for the bioremediation of contaminated waters. While it is commonly accepted knowledge that the function of CWs is determined by the interplay of plants and microorganisms, the detailed molecular processes are considered a black box. Here, we used a well-characterized CW model system treating toluene-contaminated water to investigate the microbial processes influenced by diurnal plant root exudation. Our results indicated stable aerobic toluene degradation by members of the Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis in these bacteria was higher during the day, suggesting that they additionally fed on organic root exudates and reutilized the stored carbon compounds during the night. Our study illuminates microbial processes occurring in the rhizosphere of an aquatic ecosystem.

Role of plants in nitrogen and sulfur transformations in floating hydroponic root mats: A comparison of two helophytes.

Knowledge about the roles helophytes play in constructed wetlands (CWs) is limited, especially regarding their provision of organic rhizodeposits. Here, transformations of inorganic nitrogen and sulfur were monitored in a CW variety, floating hydroponic root mat (FHRM), treating synthetic wastewater containing low concentration of organic carbon. Two helophytes, Phragmites australis and Juncus effusus, were compared in duplicates. Striking differences were found between the FHRM of the two helophytes. Whereas ammonium was removed in all FHRMs to below detection level, total nitrogen of 1.15 ± 0.4 g m(-2) d(-1) was removed completely only in P. australis systems. The mats with J. effusus displayed effective nitrification but incomplete denitrification as 77% of the removed ammonium-nitrogen accumulated as nitrate. Furthermore, the P. australis treatment units showed on average 3 times higher sulfate-S removal rates (1.1 ± 0.45 g m(-2) d(-1)) than the systems planted with J. effusus (0.37 ± 0.29 g m(-2) d(-1)). Since the influent organic carbon was below the stoichiometric requirement for the observed N and S transformation processes, helophytes' organic rhizodeposits apparently contributed to these transformations, while P. australis provided about 6 times higher bioavailable organic rhizodeposits than J. effusus.

Long-term starvation and subsequent recovery of nitrifiers in aerated submerged fixed-bed biofilm reactors.

The effectiveness of three operational strategies for maintaining nitrifiers in bench-scale, aerated, submerged fixed-bed biofilm reactors (SFBBRs) during long-term starvation at 20 degrees C were evaluated. The operational strategies were characterized by the resulting oxidation-reduction potential (ORP) in the SFBBRs. The activity rates of the nitrifiers were measured and the activity decay was expressed by half-life times. It was found that anoxic and alternating anoxic/aerobic conditions were the best ways to preserve ammonia-oxidizing bacteria (AOB) during long starvation periods and resulted in half-life times of up to 34 and 28 days, respectively. Extended anaerobic conditions caused the half-life for AOB to decrease to 21 days. In comparison, the activity decay of nitrite-oxidizing bacteria (NOB) tended to be slightly faster. The activity of AOB biofilms that were kept for 97 days under anoxic conditions could be completely recovered in less than one week, while over 4 weeks was needed for AOB kept under anaerobic conditions. NOB were more sensitive to starvation and required longer recovery periods than AOB. For complete recovery, NOB needed approximately 7 weeks, regardless of the starvation conditions applied. Using the fluorescence in situ hybridization (FISH) technique, Nitrospira was detected as the dominant NOB genus. Among the AOB, the terminal restriction fragment length polymorphism (TRFLP) technique showed that during starvation and recovery periods, the relative frequency of species shifted to Nitrosomonas europaea/eutropha, regardless of the starvation condition. The consequences of these findings for the operation of SFBBRs under low-load and starvation conditions are discussed.

Spatial characterization of microbial sulfur cycling in horizontal-flow constructed wetland models.

Constructed wetlands (CWs) are a cost-effective technology for wastewater treatment in which plant-microorganism relationships play a key role in transforming pollutants. However, there is little knowledge about the spatial organization of microbial metabolic processes in CWs. Here we show the structuring of microbial transformation of inorganic sulfur compounds (ISCs) in two horizontal subsurface-flow CW models fed with sulfate-rich artificial wastewater. One model was fully planted with Juncus effusus, while the other was planted only in the middle to investigate further the influence of the plant on ISC transformations. Chemical analyses revealed that sulfate reduction and re-oxidation of sulfide/sulfur occurred simultaneously along the flow paths, with net reduction at the beginning of the CWs, where organic carbon from the influent was still present, and predominant re-oxidation in the downstream sections. Porewater ISC concentrations hardly differed between the two CWs. However, analysis of the bacterial communities showed that sulfur cycling in the fully planted CW was much higher. Total bacterial abundances were about 50 times and 3-4 orders of magnitude higher in the rhizoplane than in porewater and on gravel, respectively, as quantified by qPCR determination of the 16S rRNA gene. Sequencing of 16S rRNA gene amplicons revealed that bacterial communities on the roots and in the porewater differed substantially, apparently a consequence of the fluxes of oxygen and exudates from the roots. Furthermore, we observed partitioning of ISC transforming bacteria into different niches of the CWs. The results of the chemical and microbial analyses collectively support that extensive sulfur cycling occurred in the rhizospheres of the CW models. The study is relevant to the treatment of sulfur-containing wastewater and the elucidation of microbial communities involved in biogeochemical activities to improve water quality.

Extracellular degradation of a polyurethane oligomer involving outer membrane vesicles and further insights on the degradation of 2,4-diaminotoluene in Pseudomonas capeferrum TDA1.

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.

A curve-shift technique for the use of non-conservative organic tracers in constructed wetlands.

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.

An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene.

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.

High stability and fast recovery of expression of the TOL plasmid-carried toluene catabolism genes of Pseudomonas putida mt-2 under conditions of oxygen limitation and oscillation.

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.

Increasing ibuprofen degradation in constructed wetlands by bioaugmentation with gravel containing biofilms of an ibuprofen-degrading Sphingobium yanoikuyae.

The aim of this study was to investigate the removal of ibuprofen in laboratory scale constructed wetlands. Four (planted and unplanted) laboratory-scale horizontal subsurface flow constructed wetlands were supplemented with ibuprofen in order to elucidate (i) the role of plants on ibuprofen removal and (ii) to evaluate the removal performance of a bioaugmented lab scale wetland. The planted systems showed higher ibuprofen removal efficiency than an unplanted one. The system planted with Juncus effusus was found to have a higher removal rate than the system planted with Phalaris arundinacea. The highest removal rate of ibuprofen was found after inoculation of gravel previously loaded with a newly isolated ibuprofen-degrading bacterium identified as Sphingobium yanoikuyae. This experiment showed that more than 80 days of CW community adaptation for ibuprofen treatment could be superseded by bioaugmentation with this bacterial isolate.

Toward Biorecycling: Isolation of a Soil Bacterium That Grows on a Polyurethane Oligomer and Monomer.

The fate of plastic waste and a sustainable use of synthetic polymers is one of the major challenges of the twenty first century. Waste valorization strategies can contribute to the solution of this problem. Besides chemical recycling, biological degradation could be a promising tool. Among the high diversity of synthetic polymers, polyurethanes are widely used as foams and insulation materials. In order to examine bacterial biodegradability of polyurethanes, a soil bacterium was isolated from a site rich in brittle plastic waste. The strain, identified as Pseudomonas sp. by 16S rRNA gene sequencing and membrane fatty acid profile, was able to grow on a PU-diol solution, a polyurethane oligomer, as the sole source of carbon and energy. In addition, the strain was able to use 2,4-diaminotoluene, a common precursor and putative degradation intermediate of polyurethanes, respectively, as sole source of energy, carbon, and nitrogen. Whole genome sequencing of the strain revealed the presence of numerus catabolic genes for aromatic compounds. Growth on potential intermediates of 2,4-diaminotoluene degradation, other aromatic growth substrates and a comparison with a protein data base of oxygenases present in the genome, led to the proposal of a degradation pathway.

Hydraulic study of a non-steady horizontal sub-surface flow constructed wetland during start-up.

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.

Anoxic nitrogen cycling in a hydrocarbon and ammonium contaminated aquifer.

Nitrogen fate and transport through contaminated groundwater systems, where N is both ubiquitous and commonly limits pollutant attenuation, must be re-evaluated given evidence for new potential microbial N pathways. We addressed this by measuring the isotopic composition of dissolved inorganic N (DIN = NH4+, NO2-, and NO3-) and N functional gene abundances (amoA, nirK, nirS, hszA) from 20 to 38 wells across an NH4+, hydrocarbon, and SO42- contaminated aquifer. In-situ N attenuation was confirmed on three sampling dates (0, +6, +12 months) by the decreased [DIN] (4300 - 40 µM) and increased δ15N-DIN (5‰-33‰) over the flow path. However, the assumption of negligible N attenuation within the plume was complicated by the presence of alternative electron acceptors (SO42-, Fe3+), both oxidizing and reducing functional genes, and N oxides within this anoxic zone. Active plume N cycling was corroborated using an NO2- dual isotope based model, which found the fastest (∼10 day) NO2- turnover within the N and electron donor rich central plume. Findings suggest that N cycling is not always O2 limited within chemically complex contaminated aquifers, though this cycling may recycle the N species rather than attenuate N.

Novel approach using substrate-mediated radiolabelling of RNA to link metabolic function with the structure of microbial communities.

A novel concept was developed applying radioisotope-labelled substrate incorporation into the biomass. The resulting radiolabelled RNA was used both as an indicator of activity and as a template for gaining structural and functional information about a substrate-utilizing microbial community. Sequences of PCR products are separated via cloning or using molecular fingerprinting techniques. Nucleic acids from predominant clones or the whole molecular fingerprinting pattern are transferred to a membrane and hybridized with the radiolabelled sample RNA. Scanning of the hybridized blots for radioactivity indicates the members involved in the utilization of the substrate. This novel 'random walk' approach using radioisotope probing was evaluated in a model community experiment.

Draft Genome Sequence of Magnetospirillum sp. Strain 15-1, a Denitrifying Toluene Degrader Isolated from a Planted Fixed-Bed Reactor.

Here, we report the draft genome sequence of Magnetospirillum 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.

Molecular characterization of dichloromethane-degrading Hyphomicrobium strains using 16S rDNA and DCM dehalogenase gene sequences.

A phylogenetic analysis of 6 strains of dichloromethane (DCM) utilizing bacteria was performed. Based on the almost complete 16S rDNA sequence determination, all strains clustered together and showed high sequence similarity to Hyphomicrobium denitrificans, except for the strain MC8b, which is only moderately related to them and probably represents a distinct species. The 16S rDNA-based phylogenetic tree was compared to the one obtained from the DNA sequence data of the dcmA gene coding DCM dehalogenase, the key enzyme of DCM utilization. The topology of the two trees is in good agreement and may suggest an ancient origin of DCM dehalogenase, but also raises questions about the original role of the enzyme.

Nitrogen transforming community in a horizontal subsurface-flow constructed wetland.

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.

Physiological evidence for the presence of a cis-trans isomerase of unsaturated fatty acids in Methylococcus capsulatus Bath to adapt to the presence of toxic organic compounds.

The physiology of the response in the methanotrophic bacterium Methylococcus capsulatus Bath towards thermal and solvent stress was studied. A systematic investigation of the toxic effects of organic compounds (chlorinated phenols and alkanols) on the growth of this bacterium was carried out. The sensitivity to the tested alkanols correlated with their chain length and hydrophobicity; methanol was shown to be an exception to which the cells showed a very high tolerance. This can be explained by the adaptation of these bacteria to growth on C1 compounds. On the other hand, M. capsulatus Bath was very sensitive towards the tested chlorinated phenols. The high toxic effect of phenolic compounds on methanotrophic bacteria might be explained by the occurrence of toxic reactive oxygen species. In addition, a physiological proof of the presence of cis-trans isomerization as a membrane-adaptive response mechanism in M. capsulatus was provided. This is the first report on physiological evidence for the presence of the unique postsynthetic membrane-adaptive response mechanism of the cis-trans isomerization of unsaturated fatty acids in a bacterium that does not belong to the genera Pseudomonas and Vibrio where this mechanism was already reported and described extensively.

Cells of Pseudomonas putida and Enterobacter sp. adapt to toxic organic compounds by increasing their size.

The phenol-degrading solvent-tolerant bacterium Pseudomonas putida P8 changed its cell shape when grown in the presence of aromatic compounds such as phenol and 4-chlorophenol. The sizes of cells that had been growing after addition of different concentrations of the toxic compounds were measured using a coulter counter that calculates the sizes of the rod-shaped bacteria to diameters of virtual spheres. The cells showed an increase in the diameter depending on the toxic effects of the applied concentrations of both solvents. The same effect was measured for an alkanol degrading bacterium, Enterobacter sp. VKGH12, in the presence of n-butanol. The reaction of the cells to different concentrations of n-butanol was examined by scanning electron microscopy. With this technique it could be shown that the size of the bacteria increased with increasing concentrations of n-butanol. These changes in cell size were dependent on the cellular activity and occurred only after addition of non-lethal concentrations. In the presence of lethal concentrations that completely inhibited cell growth, the cell sizes were similar to those of cells without intoxication. Taking into account the mathematical formula for spherical and cylindrical diameter and surface, respectively, the cells reacted to the presence of organic solvents by decreasing the ratio between surface and volume of the cells and therefore reducing their relative surfaces. As the cell surface and especially the cytoplasmic membrane are the major targets for the toxic effects of membrane-active compounds, this reduction of the relative surface represents an adaptive response to the presence of such compounds.