Hans-Peter E. Kohler

Occurrence and fate of antibiotics as trace contaminants in wastewaters, sewage sludges, and surface waters

Environmental analytical studies show that trace concentrations of antibacterial agents (antibiotics) occur in hospital and municipal wastewaters and in the aquatic environment. Fluoroquinolones and macrolides, two important human-use antibiotic classes, were studied in detail. The results are discussed regarding input sources and behavior in wastewater treatment and rivers. The fluoroquinolones ciprofloxacin and norfloxacin are substantially eliminated in wastewater treatment (80-90%) by sorption transfer to sewage sludge. In digested sludges the fluoroquinolones occur at mg/kg levels. Ciprofloxacin and norfloxacin are further removed in the Glatt river by 66 and 48%, respectively. The most abundant macrolide clarithromycin was detected at 57 to 330 ng/l concentrations in treated wastewater effluents. Different compositions of the macrolides (clarithromycin and erythromycin-H 2 O) determined in treated effluents of three wastewater treatment plants can be explained by distinct consumption patterns, in one case due to an international airport located in the catchment area. Residual levels of clarithromycin in the Glatt river were up to 75 ng/l with no apparent removal in the river. These results provide important information on environmental exposures, which can be incorporated into environmental risk assessments of the particular chemicals.

Biochemistry of Microbial Degradation of Hexachlorocyclohexane and Prospects for Bioremediation

SUMMARY Lindane, the γ-isomer of hexachlorocyclohexane (HCH), is a potent insecticide. Purified lindane or unpurified mixtures of this and α-, β-, and δ-isomers of HCH were widely used as commercial insecticides in the last half of the 20th century. Large dumps of unused HCH isomers now constitute a major hazard because of their long residence times in soil and high nontarget toxicities. The major pathway for the aerobic degradation of HCH isomers in soil is the Lin pathway, and variants of this pathway will degrade all four of the HCH isomers although only slowly. Sequence differences in the primary LinA and LinB enzymes in the pathway play a key role in determining their ability to degrade the different isomers. LinA is a dehydrochlorinase, but little is known of its biochemistry. LinB is a hydrolytic dechlorinase that has been heterologously expressed and crystallized, and there is some understanding of the sequence-structure-function relationships underlying its substrate specificity and kinetics, although there are also some significant anomalies. The kinetics of some LinB variants are reported to be slow even for their preferred isomers. It is important to develop a better understanding of the biochemistries of the LinA and LinB variants and to use that knowledge to build better variants, because field trials of some bioremediation strategies based on the Lin pathway have yielded promising results but would not yet achieve economic levels of remediation.

High-Throughput Identification of Microbial Transformation Products of Organic Micropollutants

During wastewater treatment, many organic micropollutants undergo microbially mediated reactions resulting in the formation of transformation products (TPs). Little is known on the reaction pathways that govern these transformations or on the occurrence of microbial TPs in surface waters. Large sets of biotransformation data for organic micropollutants would be useful for assessing the exposure potential of these TPs and for enabling the development of structure-based biotransformation prediction tools. The objective of this work was to develop an efficient procedure to allow for high-throughput elucidation of TP structures for a broad and diverse set of xenobiotics undergoing microbially mediated transformation reactions. Six pharmaceuticals and six pesticides were spiked individually into batch reactors seeded with activated sludge. Samples from the reactors were separated with HPLC and analyzed by linear ion trap-orbitrap mass spectrometry. Candidate TPs were preliminarily identified with an innovative post-acquisition data processing method based on target and non-target screenings of the full-scan MS data. Structures were proposed following interpretation of MS spectra and MS/MS fragments. Previously unreported microbial TPs were identified for the pharmaceuticals bezafibrate, diazepam, levetiracetam, oseltamivir, and valsartan. A variety of previously reported and unreported TPs were identified for the pesticides. The results showed that the complementary use of the target and non-target screening methods allowed for a more comprehensive interpretation of the TPs generated than either would have provided individually.

Chirality of pollutants—effects on metabolism and fate

Abstract In most cases, enantiomers of chiral compounds behave differently in biochemical processes. Therefore, the effects and the environmental fate of the enantiomers of chiral pollutants need to be investigated separately. In this review, the different fates of the enantiomers of chiral phenoxyalkanoic acid herbicides, acetamides, organochlorines, and linear alkylbenzenesulfonates are discussed. The focus lies on biological degradation, which may be enantioselective, in contrast to non-biotic conversions. The data show that it is difficult to predict which enantiomer may be enriched and that accumulation of an enantiomer is dependent on the environmental system, the species, and the organ. Racemization and enantiomerization processes occur and make interpretation of the data even more complex. Enantioselective degradation implies that the enzymes involved in the conversion of such compounds are able to differentiate between the enantiomers. “Enzyme pairs” have evolved which exhibit almost identical overall folding. Only subtle differences in their active site determine their enantioselectivities. At the other extreme, there are examples of non-homologous “enzyme pairs” that have developed through convergent evolution to enantioselectively turn over the enantiomers of a chiral compound. For a better understanding of enantioselective reactions, more detailed studies of enzymes involved in enantioselective degradation need to be performed.

Is biological treatment a viable alternative for micropollutant removal in drinking water treatment processes

In western societies, clean and safe drinking water is often taken for granted, but there are threats to drinking water resources that should not be underestimated. Contamination of drinking water sources by anthropogenic chemicals is one threat that is particularly widespread in industrialized nations. Recently, a significant amount of attention has been given to the occurrence of micropollutants in the urban water cycle. Micropollutants are bioactive and/or persistent chemicals originating from diverse sources that are frequently detected in water resources in the pg/L to μg/L range. The aim of this review is to critically evaluate the viability of biological treatment processes as a means to remove micropollutants from drinking water resources. We first place the micropollutant problem in context by providing a comprehensive summary of the reported occurrence of micropollutants in raw water used directly for drinking water production and in finished drinking water. We then present a critical discussion on conventional and advanced drinking water treatment processes and their contribution to micropollutant removal. Finally, we propose biological treatment and bioaugmentation as a potential targeted, cost-effective, and sustainable alternative to existing processes while critically examining the technical limitations and scientific challenges that need to be addressed prior to implementation. This review will serve as a valuable source of data and literature for water utilities, water researchers, policy makers, and environmental consultants. Meanwhile this review will open the door to meaningful discussion on the feasibility and application of biological treatment and bioaugmentation in drinking water treatment processes to protect the public from exposure to micropollutants.

Differential Degradation of Nonylphenol Isomers by Sphingomonas xenophaga Bayram

ABSTRACT Sphingomonas xenophaga Bayram, isolated from the activated sludge of a municipal wastewater treatment plant, was able to utilize 4-(1-ethyl-1,4-dimethylpentyl)phenol, one of the main isomers of technical nonylphenol mixtures, as a sole carbon and energy source. The isolate degraded 1 mg of 4-(1-ethyl-1,4-dimethylpentyl)phenol/ml in minimal medium within 1 week. Growth experiments with five nonylphenol isomers showed that the three isomers with quaternary benzylic carbon atoms [(1,1,2,4-tetramethylpentyl)phenol, 4-(1-ethyl-1,4-dimethylpentyl)phenol, and 4-(1,1-dimethylheptyl)phenol] served as growth substrates, whereas the isomers containing one or two hydrogen atoms in the benzylic position [4-(1-methyloctyl)phenol and 4-n-nonylphenol] did not. However, when the isomers were incubated as a mixture, all were degraded to a certain degree. Differential degradation was clearly evident, as isomers with more highly branched alkyl side chains were degraded much faster than the others. Furthermore, the C9 alcohols 2,3,5-trimethylhexan-2-ol, 3,6-dimethylheptan-3-ol, and 2-methyloctan-2-ol, derived from the three nonylphenol isomers with quaternary benzylic carbon atoms, were detected in the culture fluid by gas chromatography-mass spectrometry, but no analogous metabolites could be found originating from 4-(1-methyloctyl)phenol and 4-n-nonylphenol. We propose that 4-(1-methyloctyl)phenol and 4-n-nonylphenol were cometabolically transformed in the growth experiments with the mixture but that, unlike the other isomers, they did not participate in the reactions leading to the detachment of the alkyl moiety. This hypothesis was corroborated by the observed accumulation in the culture fluid of an as yet unidentified metabolite derived from 4-(1-methyloctyl)phenol.

Mass flows of endocrine disruptors in the Glatt River during varying weather conditions.

This study focused on the occurrence and behaviour in wastewater and surface waters of several phenolic endocrine disrupting compounds (EDCs) including parabens, alkylphenolic compounds, phenylphenol (PhP) and bisphenol A (BPA). Analytical procedures using solid-phase-extraction and LC-MS/MS techniques were applied to samples of influents and effluents of wastewater treatment plants (WWTPs) discharging into the Glatt River (Switzerland) as well as to river water samples. A mass flow analysis provided insight into the main sources and the fate of these contaminants during different weather conditions. Concentrations in influents were in the low microg/L range for most analytes. Removal of parabens in the WWTPs was mostly above 99%. Nonylphenol polyethoxylates (A(9)PEO) removal amounted to 98%, but in some cases nonylphenoxy acetic acid (A(9)PEC) or nonylphenols (NP) were formed. In effluents, concentrations were highest for the A(9)PEC, A(9)PEO and NP. Concentrations in river water were in the high ng/L range for alkylphenolic compounds and in the low ng/L range for BPA, PhP and the parabens. During the sampling period, in which several rain events occurred, both water flows and mass flows varied strongly. Mass flows in WWTP effluents and in the river increased with increasing water flows for most compounds indicating that higher water flows do not lead necessarily to a proportional dilution of the pollutants. Throughout the low water flow period, mass flows predicted from the known inputs were similar to the actual mass flows at the end of the river for most analytes. For none of the EDCs, significant in-stream removal could be observed. In the periods with high water flows, mass flows in the river were much higher than can be explained by the initially defined sources. Discharge of untreated wastewater influent into the river was assessed as an additional source. Adding this source improved the mass balance for some, but not all of the analytes.

A novel metabolic pathway for degradation of 4-nonylphenol environmental contaminants by Sphingomonas xenophaga bayram : ipso-hydroxylation and intramolecular rearrangement

Several nonylphenol isomers with α-quaternary carbon atoms serve as growth substrates for Sphingomonas xenophaga Bayram, whereas isomers containing hydrogen atoms at the α-carbon do not (Gabriel, F. L. P., Giger, W., Guenther, K., and Kohler, H.-P. E. (2005) Appl. Environ. Microbiol. 71, 1123–1129). Three metabolites of 4-(1-methyloctyl)-phenol were isolated in mg quantities from cultures of strain Bayram supplemented with the growth substrate isomer 4-(1-ethyl-1,4-dimethyl-pentyl)-phenol. They were unequivocally identified as 4-hydroxy-4-(1-methyl-octyl)-cyclohexa-2,5-dienone, 4-hydroxy-4-(1-methyl-octyl)-cyclohex-2-enone, and 2-(1-methyl-octyl)-benzene-1,4-diol by high pressure liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, two metabolites originating from 4-n-nonylphenol were identified as 4-hydroxy-4-nonyl-cyclohexa-2,5-dienone and 4-hydroxy-4-nonyl-cyclohex-2-enone by high pressure liquid chromatography-mass spectrometry. We conclude that nonylphenols were initially hydroxylated at the ipso-position forming 4-alkyl-4-hydroxy-cyclohexa-2,5-dienones. Dienones originating from growth substrate nonylphenol isomers underwent a rearrangement that involved a 1,2-C,O shift of the alkyl moiety as a cation to the oxygen atom of the geminal hydroxy group yielding 4-alkoxyphenols, from which the alkyl moieties can be easily detached as alcohols by known mechanisms. Dienones originating from nongrowth substrates did not undergo such a rearrangement because the missing alkyl substituents at the α-carbon atom prevented stabilization of the putative α-carbocation. Instead they accumulated and subsequently underwent side reactions, such as 1,2-C,C shifts and dihydrogenations. The ipso-hydroxylation and the proposed 1,2-C,O shift constitute key steps in a novel pathway that enables bacteria to detach α-branched alkyl moieties of alkylphenols for utilization of the aromatic part as a carbon and energy source.

Biotransformation of Selected Iodinated X-ray Contrast Media and Characterization of Microbial Transformation Pathways

Iodinated X-ray contrast media (ICM) are commonly detected in the aquatic environment at concentrations up to the low microgram per liter range. In this study, the biotransformation of selected ICM (diatrizoate, iohexol, iomeprol, and iopamidol) in aerobic soil-water and river sediment-water batch systems was investigated. In addition, microbial transformation pathways were proposed. Diatrizoate, an ionic ICM, was not biotransformed, while three nonionic ICM were transformed into several biotransformation products (TPs) at neutral pH. Iohexol and iomeprol were biotransformed to eleven TPs and fifteen TPs, respectively, while eight TPs were detected for iopamidol. Since seven of the TPs detected during biotransformation had not been previously identified, mass fragmentation experiments were completed to elucidate the chemical structures. Oxidation of primary alcoholic moieties, cleavage of the N-C bonds (i.e., deacetylation and removal of hydroxylated propanoic acids), and decarboxylation are potential reactions that can explain the formation of the identified TPs. Iohexol and iomeprol had similar biotransformation rates, while iopamidol was biotransformed slower and to a lesser extent. A LC tandem MS method confirmed the presence of ICM TPs in aqueous environmental samples. Fifteen of the ICM TPs were even detected in drinking water with concentrations up to 120 ng/L.

An integrated process for the production of toxic catechols from toxic phenols based on a designer biocatalyst

We describe the biocatalytic production of 3-phenylcatechol from 2-phenylphenol with the whole cell biocatalyst Escherichia coli JM101 (pHBP461). The recombinant produces 2-hydroxybiphenyl 3-monooxygenase, an enzyme from Pseudomonas azelaica HBP1. This enzyme introduces a hydroxyl-group at the C3-position of a variety of 2-substituted phenols, such as 2-phenylphenol. This permits the biocatalytic production of 3-substituted catechols, which are difficult to synthesize chemically. Both 2-phenylphenol and 3-phenylcatechol are highly toxic to E. coli. The toxic effects of 2-phenylphenol were minimized by feeding this substrate to the reactor at a rate slightly below the maximum biooxidation rate. As a result, the substrate concentration in the reactor remained below toxic levels during the bioconversion. The toxic product formed was removed by continuous adsorption on the solid resin Amberlite XAD-4. To this end the reaction mixture, containing the biocatalyst, was pumped continuously through an external loop with a fluidized bed of the resin. This resin efficiently and quantitatively adsorbed both 3-phenylcatechol and the remaining trace amounts of 2-phenylphenol. Consequently, the concentrations of these compounds were kept at subtoxic levels (below 100 mg L-1) and gram amounts of 3-phenylcatechol were produced with space-time yields of up to 0.39 g L-1 h-1. The product was recovered from the resin by acidic methanol elution and purified by recrystallization from n-hexane resulting in overall yields exceeding 59%. The optimized system served as a surprisingly simple and efficient integrated process, that allows the bioconversion of toxic substrates to toxic products with whole cell biocatalysts.