f Sulfonamide antibiotics have a wide application range in human and veterinary medicine. Because they tend to persist in the environment, they pose potential problems with regard to the propagation of antibiotic resistance. Here, we identified metabolites formed during the degradation of sulfamethoxazole and other sulfonamides in Microbacterium sp. strain BR1. Our experiments showed that the degradation proceeded along an unusual pathway initiated by ipso-hydroxylation with subsequent fragmentation of the parent compound. The NADH-dependent hydroxylation of the carbon atom attached to the sulfonyl group resulted in the release of sulfite, 3-amino-5-methylisoxazole, and benzoquinone-imine. The latter was concomitantly transformed to 4-aminophenol. Sulfadiazine, sulfamethizole, sulfamethazine, sulfadimethoxine, 4-amino-N-phenylbenzenesulfonamide, and N-(4-aminophenyl)sulfonylcarbamic acid methyl ester (asulam) were transformed accordingly. Therefore, ipso-hydroxylation with subsequent fragmentation must be considered the underlying mechanism; this could also occur in the same or in a similar way in other studies, where biotransformation of sulfonamides bearing an amino group in the para-position to the sulfonyl substituent was observed to yield products corresponding to the stable metabolites observed by us. Sulfonamides are widely used as antibiotics, antidiabetics, diuretics, antivirals, and anticancer agents (1-4), and thus, large amounts of these compounds enter the environment every year (5, 6). Contamination with sulfonamides poses environmental concern due to the potential development and dissemination of antibiotic resistances (7). Despite their ubiquity, their microbial metabolism and ultimate fate in the environment thereof are poorly understood.Several studies showed that sulfamethoxazole (SMX) (Fig. 1a), an important representative of sulfonamide compounds, undergoes partial degradation in wastewater treatment plants under aerobic and anaerobic conditions (8-11). We recently demonstrated that Microbacterium sp. strain BR1, a Gram-positive bacterium isolated from a membrane bioreactor treating effluent contaminated by several pharmaceuticals, was capable of mineralizing the 14 C-labeled aniline moiety of SMX when the latter was supplied as the sole carbon source (12). This was the first unambiguous indication that sulfonamides are subject to growth-linked metabolism in microorganisms.To our knowledge, Hartig (13) was the first to identify the aminated heteroaromatic side moieties of the sulfonamides SMX and sulfadimethoxine as stable metabolites after biodegradation with activated sludge. This result was recently confirmed by two groups that were able to isolate Microbacterium strains with the ability to degrade the sulfonamides sulfamethazine (SMZ) (14) and sulfadiazine (SDZ) (15). Additionally, both groups identified the aminated heteroaromatic side moieties of the sulfonamide as a stable metabolite after the degradation of the parent compound. Although these stable metabolites were identified, the i...
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.
In the present study, microbial toluene degradation in controlled constructed wetland model systems, planted fixed-bed reactors (PFRs), was queried with DNA-based methods in combination with stable isotope fractionation analysis and characterization of toluene-degrading microbial isolates. Two PFR replicates were operated with toluene as the sole external carbon and electron source for 2 years. The bulk redox conditions in these systems were hypoxic to anoxic. The autochthonous bacterial communities, as analyzed by Illumina sequencing of 16S rRNA gene amplicons, were mainly comprised of the families Xanthomonadaceae, Comamonadaceae, and Burkholderiaceae, plus Rhodospirillaceae in one of the PFR replicates. DNA microarray analyses of the catabolic potentials for aromatic compound degradation suggested the presence of the ring monooxygenation pathway in both systems, as well as the anaerobic toluene pathway in the PFR replicate with a high abundance of Rhodospirillaceae. The presence of catabolic genes encoding the ring monooxygenation pathway was verified by quantitative PCR analysis, utilizing the obtained toluene-degrading isolates as references. Stable isotope fractionation analysis showed low-level of carbon fractionation and only minimal hydrogen fractionation in both PFRs, which matches the fractionation signatures of monooxygenation and dioxygenation. In combination with the results of the DNA-based analyses, this suggests that toluene degradation occurs predominantly via ring monooxygenation in the PFRs. Biology-based remediation technologies (bioremediation) for the treatment of groundwater and soils polluted with organic compounds have been receiving high interest due to their low cost, high efficiency, and relative operational simplicity (1). Rhizoremediation is one such effective bioremediation approach, where the transformation of contaminants to innocuous products may be enhanced by plant-microbe interactions occurring in the rhizosphere (2-4). The plants provide the rhizospheric microbial community with root exudates such as carbohydrates, amino acids/amines, and organic acids (5) and thus promote the microbes' establishment and proliferation. Rhizoremediation is particularly effective in constructed wetlands. These treatment systems have been applied for the removal of even high loads of contaminants present in inflow waters (6-8). In addition to the input of labile organic carbon, the helophytes used in these systems have the capacity of channeling significant amounts of oxygen from the atmosphere through a specific tissues, the aerenchyma, into their roots, and thereby foster aerobic microbial activities in the rhizosphere (9).In order to enhance the performance of constructed wetlands through engineering optimizations of the systems, it is deemed necessary to understand the functionality of the relevant microbial community present in the rhizosphere, and some efforts have been pursued in this direction (7, 10). However, due to the presence of steep chemical gradients and variable environmental ...
Multidimensional compound-specific stable isotope analysis (CSIA) was applied in combination with RNA-based molecular tools to characterize methyl tertiary (tert-) butyl ether (MTBE) degradation mechanisms occurring in biofilms in an aerated treatment pond used for remediation of MTBE-contaminated groundwater. The main pathway for MTBE oxidation was elucidated by linking the low-level stable isotope fractionation (mean carbon isotopic enrichment factor [ C ] of ؊0.37‰ ؎ 0.05‰ and no significant hydrogen isotopic enrichment factor [ H ]) observed in microcosm experiments to expression of the ethB gene encoding a cytochrome P450 monooxygenase able to catalyze the oxidation of MTBE in biofilm samples both from the microcosms and directly from the ponds. 16S rRNA-specific primers revealed the presence of a sequence 100% identical to that of Methylibium petroleiphilum PM1, a well-characterized MTBE degrader. However, neither expression of the mdpA genes encoding the alkane hydroxylase-like enzyme responsible for MTBE oxidation in this strain nor the related MTBE isotope fractionation pattern produced by PM1 could be detected, suggesting that this enzyme was not active in this system. Additionally, observed low inverse fractionation of carbon ( C of ؉0.11‰ ؎ 0.03‰) and low fractionation of hydrogen ( H of ؊5‰ ؎ 1‰) in laboratory experiments simulating MTBE stripping from an open surface water body suggest that the application of CSIA in field investigations to detect biodegradation may lead to false-negative results when volatilization effects coincide with the activity of low-fractionating enzymes. As shown in this study, complementary examination of expression of specific catabolic genes can be used as additional direct evidence for microbial degradation activity and may overcome this problem.
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