Boris A. Kolvenbach

Biodegradation of sulfamethoxazole and other sulfonamides by Achromobacter denitrificans PR1.

This study aimed to isolate and characterize a microbial culture able to degrade sulfonamides. Sulfamethoxazole (SMX)-degrading microorganisms were enriched from activated sludge and wastewater. The resultant mixed culture was composed of four bacterial strains, out of which only Achromobacter denitrificans PR1 could degrade SMX. This sulfonamide was used as sole source of carbon, nitrogen and energy with stoichiometric accumulation of 3-amino-5-methylisoxazole. Strain PR1 was able to remove SMX at a rate of 73.6 ± 9.6 μmol SMX/gcell dryweighth. This rate more than doubled when a supplement of amino acids or the other members of the mixed culture were added. Besides SMX, strain PR1 was able to degrade other sulfonamides with anti-microbial activity. Other environmental Achromobacter spp. could not degrade SMX, suggesting that this property is not broadly distributed in members of this genus. Further studies are needed to shed additional light on the genetics and enzymology of this process.

Isolation of Bacterial Strains Capable of Sulfamethoxazole Mineralization from an Acclimated Membrane Bioreactor

ABSTRACT In this study, we isolated five strains capable of degrading 14C-labeled sulfamethoxazole to 14CO2 from a membrane bioreactor acclimatized to sulfamethoxazole, carbamazepine, and diclofenac. Of these strains, two belonged to the phylum Actinobacteria, while three were members of the Proteobacteria.

Shedding Light on Selenium Biomineralization: Proteins Associated with Bionanominerals

ABSTRACT Selenium-reducing microorganisms produce elemental selenium nanoparticles with particular physicochemical properties due to an associated organic fraction. This study identified high-affinity proteins associated with such bionanominerals and with nonbiogenic elemental selenium. Proteins with an anticipated functional role in selenium reduction, such as a metalloid reductase, were found to be associated with nanoparticles formed by one selenium respirer, Sulfurospirillum barnesii.

ipso-Hydroxylation and subsequent fragmentation — a novel microbial strategy to eliminate sulfonamide antibiotics

ABSTRACT 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.

Degradation and Metabolism of Tetrabromobisphenol A (TBBPA) in Submerged Soil and Soil−Plant Systems

Contamination by tetrabromobisphenol A (TBBPA), the most widely used brominated flame retardant, is a matter of environmental concern. Here, we investigated the fate and metabolites of (14)C-TBBPA in a submerged soil with an anoxic-oxic interface and planted or not with rice (Oryza sativa) and reed (Phragmites australis) seedlings. In unplanted soil, TBBPA dissipation (half-life 20.8 days) was accompanied by mineralization (11.5% of initial TBBPA) and the substantial formation (60.8%) of bound residues. Twelve metabolites (10 in unplanted soil and 7 in planted soil) were formed via four interconnected pathways: oxidative skeletal cleavage, O-methylation, type II ipso-substitution, and reductive debromination. The presence of the seedlings strongly reduced (14)C-TBBPA mineralization and bound-residue formation and stimulated debromination and O-methylation. Considerable radioactivity accumulated in rice (21.3%) and reed (33.1%) seedlings, mainly on or in the roots. While TBBPA dissipation was hardly affected by the rice seedlings, it was strongly enhanced by the reed seedlings, greatly reducing the half-life (11.4 days) and increasing monomethyl TBBPA formation (11.3%). The impact of the interconnected aerobic and anaerobic transformation of TBBPA and wetland plants on the profile and dynamics of the metabolites should be considered in phytoremediation strategies and environmental risk assessments of TBBPA in submerged soils.

Fate and metabolism of tetrabromobisphenol A in soil slurries without and with the amendment with the alkylphenol degrading bacterium Sphingomonas sp. strain TTNP3.

Transformation of ring-(14)C-labelled tetrabromobisphenol-A (TBBPA) was studied in an oxic soil slurry with and without amendment with Sphingomonas sp. strain TTNP3, a bacterium degrading bisphenol-A. TBBPA degradation was accompanied by mineralization and formation of metabolites and bound-residues. The biotransformation was stimulated in the slurry bio-augmented with strain TTNP3, via a mechanism of metabolic compensation, although this strain did not grow on TBBPA. In the absence and presence of strain TTNP3, six and nine metabolites, respectively, were identified. The initial O-methylation metabolite (TBBPA-monomethyl ether) and hydroxytribromobisphenol-A were detected only when strain TTNP3 was present. Four primary metabolic pathways of TBBPA in the slurries are proposed: oxidative skeletal rearrangements, O-methylation, ipso-substitution, and reductive debromination. Our study provides for the first time the information about the complex metabolism of TBBPA in oxic soil and suggests that type II ipso-substitution could play a significant role in the fate of alkylphenol derivatives in the environment.

The degradation of alkylphenols by Sphingomonas sp. strain TTNP3 – a review on seven years of research

Over the past seven years, we have been working with Sphingomonas sp. strain TTNP3, a bacterium capable of growing on numerous alkylphenolic compounds as a source of carbon and energy. We succeeded in elucidating an unusual pathway involving an attack at the quaternary alpha-carbon atom of the substrate, a position previously thought to be highly resistant to biodegradation. Combining analytical and bioanalytical methods, a good understanding of the reaction mechanisms, the enzymes catalysing them and the organization of the genes encoding them could be gained. First studies on the use of Sphingomonas sp. strain TTNP3 in wastewater treatment have been performed revealing promising results.

Identification of the flavin monooxygenase responsible for ipso substitution of alkyl and alkoxyphenols in Sphingomonas sp. TTNP3 and Sphingobium xenophagum Bayram

We previously showed that opdA from Sphingomonas sp. PWE1 encodes a putative flavin monooxygenase capable of transforming octylphenol (OP) via type II ipso substitution. Here, we demonstrate that an opdA homolog is responsible for OP and related alkyl/alkoxyphenol degradation in the nonylphenol degrader Sphingomonas sp. TTNP3. PCR and Southern blot analyses revealed that TTNP3 contained an opdA homolog, while a TTNP3 derivative unable to grow on nonylphenol (TTNP3d) did not. OpdA expression was confirmed in wild-type TTNP3 via two dimensional gel electrophoresis. Activity was restored to TTNP3d following complementation with opdA. Sequence analysis of an opdA homolog from another nonylphenol degrader, Sphingobium xenophagum Bayram, revealed that the predicted protein sequences from PWE1 and Bayram were identical, but differed from TTNP3 by four amino acids. In order to assess differences, we heterologously expressed the two unique opdA homologs and compared their effect on the disappearance of five alkyl/alkoxyphenol substrates and subsequent appearance of hydroquinone. For all substrates, except OP, the levels of substrate disappearance and hydroquinone appearance were significantly lower in cultures expressing odpATTNP3 than those expressing opdAPWE1/Bayram. These differences in substrate specificity were consistent with an in silico model which predicted that two of the amino acid differences between odpATTNP3 and opdAPWE1/Bayram lay in a putative substrate binding pocket. While these strains are known to use the same type II ipso substitution mechanism for alkylphenol degradation, this work provides the first preliminary evidence that opdA homologs also encode the type I ipso substitution activity responsible for the degradation of alkoxyphenols.

Fate of Tetrabromobisphenol A (TBBPA) and Formation of Ester- and Ether-Linked Bound Residues in an Oxic Sandy Soil

Bound-residue formation is a major dissipation process of most organic xenobiotics in soil. However, both the formation and nature of bound residues of tetrabromobisphenol A (TBBPA) in soil are unclear. Using a 14C-tracer, we studied the fate of TBBPA in an oxic soil during 143 days of incubation. TBBPA dissipated with a half-life of 14.7 days; at the end of incubation, 19.6% mineralized and 66.5% formed bound residues. Eight extractable metabolites were detected, including TBBPA methyl ethers, single-ring bromophenols, and their methyl ethers. Bound residues (mostly bound to humin) rapidly formed during the first 35 days. The amount of those humin-bound residues then quickly decreased, whereas total bound residues decreased slowly. By contrast, residues bound to humic acids and fulvic acids increased continuously until a plateau was reached. Ester- and ether-linked residues accounted for 9.6-27.0% of total bound residues during the incubation, with ester linkages being predominant. Residues bound via ester linkages consisted of TBBPA, TBBPA monomethyl ether, and an unknown polar compound. Our results indicated that bound-residue formation is the major pathway of TBBPA dissipation in oxic soil and provide first insights into the chemical structure of the reversibly ester-linked bound residues of TBBPA and its metabolites.

Enhanced Transformation of Tetrabromobisphenol A by Nitrifiers in Nitrifying Activated Sludge

The fate of the most commonly used brominated flame retardant, tetrabromobisphenol A (TBBPA), in wastewater treatment plants is obscure. Using a (14)C-tracer, we studied TBBPA transformation in nitrifying activated sludge (NAS). During the 31-day incubation, TBBPA transformation (half-life 10.3 days) was accompanied by mineralization (17% of initial TBBPA). Twelve metabolites, including those with single benzene ring, O-methyl TBBPA ether, and nitro compounds, were identified. When allylthiourea was added to the sludge to completely inhibit nitrification, TBBPA transformation was significantly reduced (half-life 28.9 days), formation of the polar and single-ring metabolites stopped, but O-methylation was not significantly affected. Abiotic experiments confirmed the generation of mono- and dinitro-brominated forms of bisphenol A in NAS by the abiotic nitration of TBBPA by nitrite, a product of ammonia-oxidizing microorganisms (AOMs). Three biotic (type II ipso-substitution, oxidative skeletal cleavage, and O-methylation) and one abiotic (nitro-debromination) pathways were proposed for TBBPA transformation in NAS. Apart from O-methylation, AOMs were involved in three other pathways. Our results are the first to provide information about the complex metabolism of TBBPA in NAS, and they are consistent with a determining role for nitrifiers in TBBPA degradation by initiating its cleavage into single-ring metabolites that are substrates for the growth of heterotrophic bacteria.