Piracetam (2-oxo-1-pyrrolidine acetamide) is a popular cognitive enhancer, which has recently been detected in waste and drinking water. Nootropic drugs are designed to affect human metabolism and act on the nervous system, but their environmental effects have yet to be the subject of detailed studies. In this report, we present the efficient biodegradation of the cognitive enhancer, piracetam. Two bacterial strains capable of using this compound as the sole carbon source were isolated and later identified as Ochrobactrum anthropi strain MW6 and Ochrobactrum intermedium strain MW7. The compound's mineralization and the cleavage of the heterocyclic ring were shown in the experiments with 14C-labeled piracetam. This is also the first report of a pharmaceutical's degradation by the Ochrobactrum genus. This study presents model microorganisms that can be used in further investigation of piracetam's degradation pathways as well as enzymes and genes involved in the process.

The wide application of sulfonamide (SA) antibiotics in human and veterinary medicine contributes to the accumulation of these antibiotics in the environment and the corresponding onset of antibiotic resistance among bacteria. Microbacterium sp. BR1 is capable of mineralizing sulfamethoxazole and other SAs via a novel mechanism. The genetic basis of SA elimination by BR1 remains unknown. Development of an efficient plasmid transfer protocol for Microbacterium sp. BR1 is highly desirable, as it would open the door to genetic analysis and manipulation of its genome. Here we report that intergeneric Escherichia coli–Microbacterium spp. BR1 conjugation is an efficient way to introduce various plasmids into BR1. The generated transconjugants were stable in the presence of antibiotics and the plasmids showed no signs of rearrangements. Nevertheless, the plasmids were rapidly lost in the absence of selection. We also show that the cumate-inducible beta-glucuronidase reporter gene functions in BR1 and is strictly regulated. Our results set the working ground for further genetic manipulations of BR1, such as the overexpression of sulfonamide degradation genes or the selection of strong microbacterial promoters.

The consumption of nootropic drugs has increased tremendously in the last decade, though the studies on their environmental fate are still scarce. Nootropics are bioactive compounds designed to alter human's physiology therefore the adverse effects towards wildlife can be expected. In order to understand their environmental impact, the knowledge on their transformation pathways is necessary. Methylphenidate belongs to the most prescribed neuro-enhancers and is among the most favored smart drugs used in non-medical situations. It is metabolized in human liver and excreted as ritalinic acid. Here, we showed for the first time that ritalinic acid can be biodegraded and used as a sole carbon and nitrogen source by various microbial strains originating from different environmental samples. Five axenic strains were isolated and identified as: Arthrobacter sp. strain MW1, MW2 and MW3, Phycicoccus sp. strain MW4 and Nocardioides sp. strain MW5. Our research provides the first insight into the metabolism of ritalinic acid and suggests that it may differ depending on the strain and growth conditions, especially on availability of nitrogen. The isolates obtained in this study can serve as model organisms in further studies on the catabolism of ritalinic acid and methylphenidate but potentially also other compounds with similar structures. Our findings have important implication for the sewage epidemiology. We demonstrated that ritalinic acid is subject to quick and efficient biodegradation thus its use as a stable biomarker should be reconsidered.

Dechlorination patterns of three tetrachlorobenzene isomers, 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-TeCB, were studied in anoxic microcosms derived from contaminated harbor sludge. The removal of doubly, singly, and un-flanked chlorine atoms was noted in 1,2,3,4- and 1,2,3,5-TeCB fed microcosms, whereas only singly flanked chlorine was removed in 1,2,4,5-TeCB microcosms. The thermodynamically more favorable reactions were selectively followed by the enriched cultures with di- and/or mono-chlorobenzene as the main end products of the reductive dechlorination of all three isomers. Based on quantitative PCR analysis targeting 16S rRNA genes of known organohalide-respiring bacteria, the growth of Dehalococcoides was found to be associated with the reductive dechlorination of all three isomers, while growth of Dehalobacter, another known TeCB dechlorinator, was only observed in one 1,2,3,5-TeCB enriched microcosm among biological triplicates. Numbers of Desulfitobacterium and Geobacter as facultative dechlorinators were rather stable suggesting that they were not (directly) involved in the observed TeCB dechlorination. Bacterial community profiling suggested bacteria belonging to the phylum Bacteroidetes and the order Clostridiales as well as sulfate-reducing members of the class Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to fastidious dechlorinators. Our results provide a better understanding of thermodynamically preferred TeCB dechlorinating pathways in harbor environments and microbial guilds enriched and active in anoxic TeCB dechlorinating microcosms.

The aim of this study was to evaluate the ability of bacterial cultures isolated from cattle, poultry or pig faeces and manure to produce rhamnolipids, as well as to investigate the influence of interspecies communication on possible quantitative differences in the production of rhamnolipid congeners. Initial screening methods (oil spreading, drop collapse, haemolytic activity and emulsification activity) showed that approximately 36% of the 51 isolated cultures exhibited the ability to produce biosurfactants. Subsequent studies using a selected culturable mixed culture (which included Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli) revealed that only P. aeruginosa was able to produce this biosurfactant. HPLC–MS analysis showed that the surface active compounds were rhamnolipids. Further comparative studies confirmed that the total yield of rhamnolipids was notably higher in the bioreactor inoculated with the selected mixed culture (940.58 ± 1.10 mg/L) compared to the bioreactor inoculated with the axenic strain of P. aeruginosa (108.47 ± 0.41 mg/L). Twelve rhamnolipid congeners were identified during cultivation of the selected mixed culture, whereas six congeners were detected during cultivation of the sole axenic strain of P. aeruginosa. Furthermore, increased production of rhamnolipids was observed when the concentration of autoinducer molecules (AI-2) responsible for interspecies signaling increased, suggesting the influence of quorum-sensing communication on biosynthesis efficiency. This observation may be of importance for large-scale production of this biosurfactant, as it opens new possible solutions based on the use of mixed cultures or external addition of stimulating autoinducers.

Carbon isotope fractionation of sulfamethoxazole (SMX) during biodegradation by Microbacterium sp. strain BR1 (ipso-hydroxylation) and upon direct photolysis was investigated. Carbon isotope signatures (δ(13)C) of SMX were measured by LC-IRMS (liquid chromatography coupled to isotope ratio mass spectrometry). A new LC-IRMS method for the SMX metabolite, 3-amino-5-methylisoxazole (3A5MI), was established. Carbon isotope enrichment factors for SMX (ε(C)) were -0.6 ± 0.1‰ for biodegradation and -2.0 ± 0.1‰ and -3.0 ± 0.2‰ for direct photolysis, at pH 7.4 and pH 5, respectively. The corresponding apparent kinetic isotope effects (AKIE) for ipso-hydroxylation were 1.006 ± 0.001; these fall in the same range as AKIE in previously studied hydroxylation reactions. The differences in SMX and 3A5MI fractionation upon biotic and abiotic degradation suggest that compound specific stable isotope analysis (CSIA) is a suitable method to distinguish SMX reaction pathways. In addition, the study revealed that the extent of isotope fractionation during SMX photolytic cleavage is pH-dependent.

Anaerobic reductive dechlorination of hexachlorobenzene (HCB) and three isomers of tetrachlorobenzene (TeCB) (1,2,3,4-, 1,2,3,5- and 1,2,4,5-TeCB) was investigated in microcosms containing chloroaromatic contaminated river sediment. All chlorobenzenes were dechlorinated to dichlorobenzene (DCB) or monochlorobenzene. From the sediment, a methanogenic sediment-free culture was obtained which dechlorinated HCB, pentachlorobenzene, three TeCB isomers, three trichlorobenzene (TCB) isomers (1,2,3-, 1,2,4- and 1,3,5-TCB) and 1,2-DCB. Dechlorination involved multiple pathways including the removal of doubly flanked, singly flanked and isolated chlorine substituents. 454-pyrosequencing of partial bacterial 16S rRNA genes amplified from selected chlorobenzene dechlorinating sediment-free enrichment cultures revealed the presence of a variety of bacterial species, including Dehalobacter and Dehalococcoides mccartyi, that were previously documented as organohalide respiring bacteria. A genus with apparent close relationship to Desulfitobacterium that also has been associated with organohalide respiration, composed the major fraction of the operational taxonomic units (OTUs). Another major OTU was linked with Sedimentibacter sp., a genus that was previously identified in strict co-cultures of consortia reductively dehalogenating chlorinated compounds. Our data point towards the existence of multiple interactions within highly chlorinated benzene dechlorinating communities.

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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.

An enrichment culture dominated by one type of Dehalococcoides sp. (83% of clones) was characterised. This culture, originally derived from contaminated groundwater from the area of Bitterfeld-Wolfen (Saxony-Anhalt, Germany), dehalogenates chlorinated ethenes to ethene. Further, the culture also dehalogenated vinyl bromide (VB) and 1,2-dichloroethane (DCA) to ethene, 1,2,3,4- and 1,2,3,5-tetrachlorobenzene (TeCB), penta- and hexachlorobenzene (PeCB and HCB) to trichlorobenzenes (TCB), lindane to monochlorobenzene (MCB) and pentachlorophenol (PCP) to 2,3,4,6-tetrachlorophenol (TeCP). Growth was proven by quantitative PCR for all active cultures, except for those with TeCB, lindane and PCP. The growth yields obtained ranged from (2.9 ± 0.7) × 10(7) cells μmol(-1) Br(-) released on VB to (34.8 ± 5.4) × 10(7) cells μmol(-1) Cl(-) released on VC. Genes coding for nine putative reductive dehalogenases, the enzymes that mediate the respiratory process of dehalogenation, were identified. Phylogenetic analysis revealed eight reductive dehalogenases with similar sequences in other Dehalococcoides strains and one unique sequence.

The impact of four electron acceptors on hydrocarbon-induced methanogenesis was studied. Methanogenesis from residual hydrocarbons may enhance the exploitation of oil reservoirs and may improve bioremediation. The conditions to drive the rate-limiting first hydrocarbon-oxidizing steps for the conversion of hydrocarbons into methanogenic substrates are crucial. Thus, the electron acceptors ferrihydrite, manganese dioxide, nitrate or sulfate were added to sediment microcosms acquired from two brackish water locations. Hexadecane, ethylbenzene or 1-(13)C-naphthalene were used as model hydrocarbons. Methane was released most rapidly from incubations amended with ferrihydrite and hexadecane. Ferrihydrite enhanced only hexadecane-dependent methanogenesis. The rates of methanogenesis were negatively affected by sulfate and nitrate at concentrations of more than 5 and 1 mM, respectively. Metal-reducing Geobacteraceae and potential sulfate reducers as well as Methanosarcina were present in situ and in vitro. Ferrihydrite addition triggered the growth of Methanosarcina-related methanogens. Additionally, methane was removed concomitantly by anaerobic methanotrophy. ANME-1 and -2 methyl coenzyme M reductase genes were detected, indicating anaerobic methanotrophy as an accompanying process [Correction added 16 December after online publication: 'methyl coenzyme A' changed to 'methyl coenzyme M' in this sentence]. The experiments presented here demonstrate the feasibility of enhancing methanogenic alkane degradation by ferrihydrite or sulfate addition in different geological settings.

“Food, Agriculture and Fisheries and Biotechnology” is one of 10 thematic areas in the Cooperation programme of the European Union's 7th Framework Programme for Research, Technological Development and Demonstration Activities (FP7). With a budget of nearly €2 billion for the period 2007–2013, its objective is to foster the development of a European Knowledge-Based Bio-Economy (KBBE) by bringing together science, industry and other stakeholders that produce, manage or otherwise exploit biological resources. Biotechnology plays an important role in addressing social, environmental and economic challenges and it is recognised as a key enabling technology in the transition to a green, low carbon and resource-efficient economy. Biotechnologies for non-health applications have received a considerable attention in FP7 and to date 61 projects on industrial, marine, plant, environmental and emerging biotechnologies have been supported with a contribution of €262.8 million from the European Commission (EC). This article presents an outlook of the research, technological development and demonstration activities in biotechnology currently supported in FP7 within the Cooperation programme, including a brief overview of the policy context.

A Dehalococcoides-dominated culture coupling reductive dechlorination of tetrachloroethene (PCE) to ethene to growth was enriched from a European field site for the first time. Microcosms were set up using groundwater from a chlorinated ethene-contaminated anaerobic aquifer in Bitterfeld (Germany). Active, lactate-amended microcosms capable of PCE dechlorination to ethene without the accumulation of intermediates were used for further enrichment. After three transfers on lactate as an electron donor and PCE as an electron acceptor, the enrichment was transferred to parallel cultures with one of the chlorinated ethenes as an electron acceptor and acetate and hydrogen as the carbon and energy source, respectively. After three more transfers, a highly purified culture was derived that was capable of dechlorinating PCE with hydrogen and acetate as the electron donor and carbon source, respectively. PCR, followed by denaturing gradient gel electrophoresis, cloning and sequencing revealed that this culture was dominated by a Dehalococcoides sp. belonging to the Pinellas group. Investigation of substrate specificity in the parallel cultures suggested the presence of a novel Dehalococcoides that can couple all dechlorination steps, from PCE to ethene, to energy conservation. Quantitative real-time PCR confirmed growth with PCE, cis-dichloroethene, 1,1-dichloroethene or vinyl chloride as electron acceptors. The culture was designated BTF08 due to its origin in Bitterfeld.

Carbon isotope fractionation was observed during dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) by a mixed culture containing Dehalococcoides ethenogenes strain 195. Fractionation was examined when 1,2,3,4-TeCDD was added as the only chlorinated compound and when 1,2,3,4-TeCDD was added with a known growth substrate, tetrachloroethene (PCE). The 1,2,3,4-TeCDD was dechlorinated to 1,2,4-trichlorodibenzo-p-dioxin (1,2,4-TrCDD) which was enriched in 13C relative to 1,2,3,4-TeCDD with isotope separation factors, εC, of 1.3±0.2‰ and 1.7±0.4‰ (average±95% confidence interval (CI)) in cultures with and without PCE, respectively. The 1,2,4-TrCDD was further dechlorinated to 1,3-dichlorodibenzo-p-dioxin (1,3-DCDD) which was depleted in 13C relative to 1,2,4-TrCDD with εC of −2.4±0.4‰ and −2.9±0.8‰ (average±95% CI) in cultures with and without PCE, respectively. This demonstrates carbon isotope fractionation during sequential reductive dechlorination of PCDDs, where isotope fractionation during dechlorination of the intermediate was substantial and a 13C depleted lightly chlorinated PCDD congener was ultimately formed during dechlorination of more highly chlorinated PCDD congeners. Despite reproducible, statistically significant differences between isotope compositions of the parent, 1,2,3,4-TeCDD and daughter, 1,2,4-TrCDD and 1,3-DCDD congeners in triplicate bottles of both treatments, fractionation factors for 1,2,3,4-TeCDD could not be determined for all replicates by regression analysis of the plot of the Rayleigh equation. It is possible that dissolution of 1,2,3,4-TeCDD imposed a kinetic limitation on dechlorination, thus masking isotope fractionation during its dechlorination.