A diesel fuel-contaminated aquifer in Menziken, Canton of Aargau, Switzerland, was in situ bioremediated from 1989 to 1994 by adding O 2 , , , and through an infiltration well. After a remediation time of 3.5 years, aquifer material from Ϫ 3Ϫ ϩ NO PO NH 3 4 4the contaminated zone was excavated and found to contain Ͼ10 6 hydrocarbon-degrading microorganisms/g and 1.15 Ϯ 0.15 mg/g weathered diesel fuel comprizing mainly isoprenoid alkanes and an unresolved complex mixture (UCM) of unknown components. Samples of this material were incubated for up to 470 days in aerobic and anaerobic microcosms. The microbial activity was determined by measuring the production of inorganic carbon and the consumption of O 2 and . The degradation of the weathered diesel fuel Ϫ NO 3 was quantified by infrared spectroscopy and by capillary gas chromatography. In aerobic microcosms, all isoprenoid alkanes and most of the UCM were biodegraded as long as a nitrogen source was present. The O 2 consumption could be stimulated by adding KH 2 PO 4 and by elevating the temperature to 22ЊC. In anaerobic microcosms with KNO 3 , was consumed, inorganic carbon was produced,Ϫ NO 3 and the isoprenoid alkanes and the UCM were partially metabolized. In some selected microcosms, the consumption rate was Ϫ NO 3 stimulated by adding external substrates such as toluene, o-xylene, m-xylene, p-xylene, n-alkanes, or fatty acids. Mineralization of toluene, naphthalene, and hexadecane to CO 2 under denitrifying conditions was confirmed by using [ 14 C]-labelled substrates. Keywords-BioremediationHydrocarbons Biodegradation Aerobic AnaerobicEnviron. Toxicol. Chem. 15, 1996 T.P.-A. Bregnard et al.

A diesel fuel-contaminated aquifer was bioremediated in situ by the injection of oxidants (O 2 and NO 3 ؊) and nutrients in order to stimulate microbial activity. After 3.5 years of remediation, an aquifer sample was excavated and the material was used (i) to isolate bacterial strains able to grow on selected hydrocarbons under denitrifying conditions and (ii) to construct a laboratory aquifer column in order to simulate the aerobic and denitrifying remediation processes. Five bacterial strains isolated from the aquifer sample were able to grow on toluene (strains T 2 to T 4 , T 6 , and T 10), and nine bacterial strains grew on toluene and m-xylene (strains M 3 to M 7 and M 9 to M 12). Strains T 2 to T 4 , T 6 , and T 10 were cocci, and strains M 3 to M 7 and M 9 to M 12 were rods. The morphological and physiological differences were also reflected in small sequence variabilities in domain III of the 23S rRNA and in the 16S rRNA. Comparative sequence analyses of the 16S rRNA of one isolate (T 3 and M 3) of each group revealed a close phylogenetic relationship for both groups of isolates to organisms of the genus Azoarcus. Two 16S rRNA-targeted oligonucleotide probes (Azo644 and Azo1251) targeting the experimental isolates, bacteria of the Azoarcus tolulyticus group, and Azoarcus evansii were used to investigate the significance of hydrocarbon-degrading Azoarcus spp. in the laboratory aquifer column. The number of bacteria in the column determined after DAPI (4,6-diamidino-2-phenylindole) staining was 5.8 ؋ 10 8 to 1.1 ؋ 10 9 cells g of aquifer material ؊1. About 1% (in the anaerobic zone of the column) to 2% (in the aerobic zone of the column) of these bacteria were detectable by using a combination of probes Azo644 and Azo1251, demonstrating that hydrocarbon-degrading Azoarcus spp. are significant members of the indigenous microbiota. More than 90% of the total number of bacteria were detectable by using probes targeting higher phylogenetic groups. Approximately 80% of these bacteria belonged to the ␤ subdivision of the class Proteobacteria (␤-Proteobacteria), and 10 to 16% belonged to the ␥-Proteobacteria. Bacteria of the ␣-Proteobacteria were present in high numbers (10%) only in the aerobic zone of the column. Diesel fuel-contaminated soils and aquifers can be partially remediated by pumping hydrocarbons occurring in free phase back to the soil surface or by stripping the subsurface with air (7). Residual hydrocarbons, however, are often trapped in cracks and pores of the subsurface, and they may be removed by in situ bioremediation. This technique is usually based on the infiltration of water supplemented with oxidants (e.g., O 2 and NO 3 Ϫ) and/or nutrients (e.g., NH 4 ϩ and PO 4 3Ϫ) to stimulate the catabolic activity of microorganisms in the subsurface and thereby the biodegradation of the hydrocarbons (18, 23-25, 32). An in situ bioremediation process was applied in a diesel fuel-contaminated aquifer in Menziken, Switzerland (23). Groundwater supplemented with O 2 (329 M) and NO 3

In 2016, the United Nations declared the need for urgent action to combat the global threat of antimicrobial resistance (AMR). In support of this effort, the pharmaceutical industry has committed to measures aimed at improving the stewardship of antibiotics both within and outside the clinic. Notably, a group of companies collaborated to specifically address concerns related to antibiotic residues being discharged from manufacturing sites. In addition to developing a framework of minimum environmental expectations for antibiotic manufacturers, science‐based receiving water targets were established for antibiotics discharged from manufacturing operations. This paper summarizes the holistic approach taken to derive these targets and includes previously unpublished, company‐generated, environmental toxicity data.

Microcosm studies were conducted under nitrate-reducing conditions with diesel fuel-contaminated aquifer material from a site treated by in situ bioremediation. In the microcosms, the consumption of nitrate and the production of inorganic carbon were strongly stimulated by the addition of the isoprenoid alkane pristane (2,6,10,14-tetramethylpentadecane). Within 102 days enrichment cultures degraded more than 90% of the pristane supplied as coatings on reticulated sinter glass rings. The study demonstrates that pristane can no longer be regarded as recalcitrant under anaerobic conditions.

Microbial cultures enriched from a diesel fuel-contaminated aquifer were able to grow on p-xylene under denitrifying conditions. The oxidation of p-xylene to CO 2 was coupled to the reduction of NO 3 ؊. The enrichment cultures also grew on toluene and m-xylene, but they did not degrade benzene, ethylbenzene, and o-xylene.

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A microbial culture enriched from a diesel fuel-contaminated aquifer was able to grow on 1,3,5-trimethylbenzene (1,3,5-TMB) and 1,2,4-TMB under N(inf2)O-reducing conditions, but it did not degrade 1,2,3-TMB. The oxidation of 1,3,5-TMB to CO(inf2) was coupled to the production of biomass and the reduction of N(inf2)O. N(inf2)O was used to avoid toxic effects caused by NO(inf2)(sup-) accumulation during growth with NO(inf3)(sup-) as the electron acceptor. In addition to 1,3,5-TMB and 1,2,4-TMB, the culture degraded toluene, m-xylene, p-xylene, 3-ethyltoluene, and 4-ethyltoluene.