Use of stable isotope probing to assess the fate of emerging contaminants degraded by white-rot fungus

The widespread of emerging contaminants in the environment and their potential impact on humans is a matter of concern. White-rot fungi are cosmopolitan organisms able to remove a wide range of pharmaceuticals and personal care products (PPCP) through cometabolism (i.e. laccases and peroxidases) or detoxification mechanisms (i.e. cytochrome P450 system). However, the use of PPCP as carbon source for these organisms is largely unexplored. Here, we used carbon stable isotope tracer experiments to assess the fate of anti-inflammatory diclofenac (DCF) and UV filter benzophenone-3 (BP3) during degradation by Trametes versicolor. The comparison between carbon isotopic composition of emitted carbon dioxide from 13C-labelled DCF ([acetophenyl ring-13C6]-DCF) and 13C-BP3 ([phenyl-13C6]-BP3) versus their 12C-homologue compounds showed mineralization of about 45% and 10% of the 13C contained in their respective molecules after 9 days of incubation. The carbon isotopic composition of the bulk biomass and the application of amino acid-stable isotope probing (SIP) allowed distinguishing between incorporation of 13C from BP3 into amino acids, which implies the use of this emerging contaminant as carbon source, and major intracellular accumulation of 13C from DCF without implying the transformation of its labelled phenyl ring into anabolic products. A mass balance of 13C in different compartments over time provided a comprehensive picture of the fate of DCF and BP3 across their different transformation processes. This is the first report assessing biodegradation of PPCP by SIP techniques and the use of emerging contaminants as carbon source for amino acid biosynthesis

Assessment of aerobic biodegradation of lower-chlorinated benzenes in contaminated groundwater using field-derived microcosms and compound-specific carbon isotope fractionation

Biodegradation of lower chlorinated benzenes (tri-, di- and monochlorobenzene) was assessed at a coastal aquifer contaminated with multiple chlorinated aromatic hydro- carbons. Field-derived microcosms, established with groundwater from the source zone and amended with a mixture of lower chlorinated benzenes, evidenced biodegradation of monochlorobenzene (MCB) and 1,4-dichlorobenzene (1,4-DCB) in aerobic microcosms, whereas the addition of lactate in anaerobic microcosms did not enhance anaerobic reduc- tive dechlorination. Aerobic microcosms established with groundwater from the plume consumed several doses of MCB and concomitantly degraded the three isomers of dichloroben- zene with no observable inhibitory effect. In the light of these results, we assessed the applicability of compound stable isotope analysis to monitor a potential aerobic remediation treatment of MCB and 1,4-DCB in this site. The carbon isotopic fractionation factors ( ε) obtained from field-derived microcosms were -0.7 ¿ ± 0.1 ¿ and -1.0 ¿ ± 0.2 ¿ for MCB and 1,4-DCB, respectively. For 1,4-DCB, the carbon isotope fractionation during aerobic biodegra- dation was reported for the first time. The weak carbon isotope fractionation values for the aerobic pathway would only allow tracing of in situ degradation in aquifer parts with high extent of biodegradation. However, based on the carbon isotope effects measured in this and previous studies, relatively high carbon isotope shifts (i.e., δ13 C > 4.0 ¿ ) of MCB or 1,4- DCB in contaminated groundwater would suggest that their biodegradation is controlled by anaerobic reductive dechlorination

Integrative isotopic and molecular approach for the diagnosis and implementation of an efficient in-situ enhanced biological reductive dechlorination of chlorinated ethenes

Based on the previously observed intrinsic bioremediation potential of a site originally contaminated with perchloroethene (PCE), field-derived lactate-amended microcosms were performed to test different lactate isomers and concentrations, and find clearer isotopic and molecular parameters proving the feasibility of an in-situ enhanced reductive dechlorination (ERD) from PCE-to-ethene (ETH). According to these laboratory results, which confirmed the presence of Dehalococcoides sp. and the vcrA gene, an in-situ ERD pilot test consisting of a single injection of lactate in a monitoring well was performed and monitored for 190 days. The parameters used to follow the performance of the ERD comprised the analysis of i) hydrochemistry, including redox potential (Eh), and the concentrations of redox sensitive species, chlorinated ethenes (CEs), lactate, and acetate; ii) stable isotope composition of carbon of CEs, and sulphur and oxygen of sulphate; and iii) 16S rRNA gene sequencing from groundwater samples. Thus, it was proved that the injection of lactate promoted sulphate-reducing conditions, with the subsequent decrease in Eh, which allowed for the full reductive dechlorination of PCE to ETH in the injection well. The biodegradation of CEs was also confirmed by the enrichment in 13C and carbon isotopic mass balances. The metagenomic results evidenced the shift in the composition of the microbial population towards the predominance of fermentative bacteria. Given the success of the in-situ pilot test, a full-scale ERD with lactate was then implemented at the site. After one year of treatment, PCE and trichloroethene were mostly depleted, whereas vinyl chloride (VC) and ETH were the predominant metabolites. Most importantly, the shift of the carbon isotopic mass balances towards more positive values confirmed the complete reductive dechlorination, including the VC-to-ETH reaction step. The combination of techniques used here provides complementary lines of evidence for the diagnosis of the intrinsic biodegradation potential of a polluted site, but also to monitor the progress, identify potential difficulties, and evaluate the success of ERD at the field scale

Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain)

The bioremediation potential of an aquifer contaminated with tetrachloroethene (PCE) was assessed by combining hydrogeochemical data of the site, microcosm studies, metabolites concentrations, compound specific-stable carbon isotope analysis and the identification of selected reductive dechlorination biomarker genes. The characterization of the site through 10 monitoring wells evidenced that leaked PCE was transformed to TCE and cis-DCE via hydrogenolysis. Carbon isotopic mass balance of chlorinated ethenes pointed to two distinct sources of contamination and discarded relevant alternate degradation pathways in the aquifer. Application of specific-genus primers targeting Dehalococcoides mccartyi species and the vinyl chloride-to-ethene reductive dehalogenase vcrA indicated the presence of autochthonous bacteria capable of the complete dechlorination of PCE. The observed cis-DCE stall was consistent with the aquifer geochemistry (positive redox potentials; presence of dissolved oxygen, nitrate, and sulphate; absence of ferrous iron), which was thermodynamically favourable to dechlorinate highly chlorinated ethenes but required lower redox potentials to evolve beyond cis-DCE to the innocuous end product ethene. Accordingly, the addition of lactate or a mixture of ethanol plus methanol as electron donor sources in parallel field-derived anoxic microcosms accelerated dechlorination of PCE and passed cis-DCE up to ethene, unlike the controls (without amendments, representative of field natural attenuation). Lactate fermentation produced acetate at near-stoichiometric amounts. The array of techniques used in this study provided complementary lines of evidence to suggest that enhanced anaerobic bioremediation using lactate as electron donor source is a feasible strategy to successfully decontaminate this site

Hydrogen isotope fractionation during biodegradation of 1,2-dichloroethane: potential for pathway identification using a multi-element (C, Cl and H) isotope approach

Even though multi-element isotope fractionation patterns provide crucial information to identify contaminant degradation pathways in the field, those involving hydrogen are still lacking for many halogenated groundwater contaminants and degradation pathways. This study investigates for the first time hydrogen isotope fractionation during both aerobic and anaerobic biodegradation of 1,2-dichloroethane (1,2-DCA) using five microbial cultures. Transformation-associated isotope fractionation values (ε_bulk^H) were: -115 ± 18¿ (aerobic C-H bond oxidation), -34 ± 4¿ and -38 ± 4¿ (aerobic C-Cl bond cleavage via hydrolytic dehalogenation), -57 ± 3¿ and -77 ± 9¿ (anaerobic C-Cl bond cleavage via reductive dihaloelimination). The dual element C-H isotope approach (ΛC-H = Δδ2H/Δδ13C ≈ ε_bulk^H/ε_bulk^C, where Δδ2H and Δδ13C are changes in isotope ratios during degradation) resulted in clearly different ΛC-H values: 28 ± 4 (oxidation), 0.7 ± 0.1 and 0.9 ± 0.1 (hydrolytic dehalogenation), 1.76 ± 0.05 and 3.5 ± 0.1 (dihaloelimination). This result highlights the potential of this approach to identify 1,2-DCA degradation pathways in the field. In addition, distinct trends were also observed in a multi (i.e., Δδ2H vs Δδ37Cl vs Δδ13C) isotope plot, which opens further possibilities for pathway identification in future field studies. This is crucial information to understand the mechanisms controlling natural attenuation of 1,2-DCA and to design appropriate strategies to enhance biodegradation

Distinct dual C-Cl isotope fractionation patterns during anaerobic biodegradation of 1,2-dichloroethane: potential to characterize microbial degradation in the field

This study investigates, for the first time, dual C-Cl isotope fractionation during anaerobic biodegradation of 1,2-dichloroethane (1,2-DCA) via dihaloelimination by Dehalococcoides and Dehalogenimonas-containing enrichment cultures. Isotopic fractionation of 1,2-DCA (εbulkC and εbulkCl) for Dehalococcoides (−33.0 ± 0.4¿ and −5.1 ± 0.1¿) and Dehalogenimonas-containing microcosms (−23 ± 2¿ and −12.0 ± 0.8¿) resulted in distinctly different dual element C-Cl isotope correlations (Λ = Δδ13C/Δδ37Cl ≈ εbulkC/εbulkCl), 6.8 ± 0.2 and 1.89 ± 0.02, respectively. Determined isotope effects and detected products suggest that the difference on the obtained Λ values for biodihaloelimination could be associated with a different mode of concerted bond cleavage rather than two different reaction pathways (i.e., stepwise vs concerted). Λ values of 1,2-DCA were, for the first time, determined in two field sites under reducing conditions (2.1 ± 0.1 and 2.2 ± 2.9). They were similar to the one obtained for the Dehalogenimonas-containing microcosms (1.89 ± 0.02) and very different from those reported for aerobic degradation pathways in a previous laboratory study (7.6 ± 0.1 and 0.78 ± 0.03). Thus, this study illustrates the potential of a dual isotope analysis to differentiate between aerobic and anaerobic biodegradation pathways of 1,2-DCA in the field and suggests that this approach might also be used to characterize dihaloelimination of 1,2-DCA by different bacteria, which needs to be confirmed in future studies

Evaluación fitoquímica y de actividad antimicrobiana de dos extractos de plantas amazónicas

Se hicieron análisis fitoquímico preliminar y antimicrobiano a los extractos etanólicos y las fracciones de éter de petróleo y acetato de etilo obtenidas por partición líquido-líquido, de las plantas amazónicas: Crepidospermum goudotianum (Burseraceae) e Irlbachia alata (Gentianaceae). Las dos plantas presentaron alcaloides, fenoles, cumarinas, saponinas, esteroides y/o triterpenoides. La fracción de éter de petróleo de C. goudotianum presentó actividad antifúngica frente a C. albicans

Evaluación fitoquímica y de actividad antimicrobiana de dos extractos de plantas amazónicas

Se hicieron análisis fitoquímico preliminar y antimicrobiano a los extractos etanólicos y las fracciones de éter de petróleo y acetato de etilo obtenidas por partición líquido-líquido, de las plantas amazónicas: Crepidospermum goudotianum (Burseraceae) e Irlbachia alata (Gentianaceae). Las dos plantas presentaron alcaloides, fenoles, cumarinas, saponinas, esteroides y/o triterpenoides. La fracción de éter de petróleo de C. goudotianum presentó actividad antifúngica frente a C. albicans

Bioremediation of emerging pollutants from sewage sludge by fungal bioaugmentation

Current wastewater treatment processes are not able to completely remove many organic pollutants. The increasing use of the sludge derived from wastewater treatment plants (WWTP) in agricultural lands therefore becomes a source for micropollutants to enter the environment. An eco-friendly biotechnological treatment which employs the white-rot fungus Trametes versicolor was assessed to remove several groups of pharmaceuticals at pre-existent concentrations from sewage sludge. First, two different strategies were applied in sterile conditions to demonstrate the colonization and degrading ability of the fungus: solidphase systems with dehydrated sludge and a lignocellulosic substrate, and slurry bioreactors with sludge from the outlet of an anaerobic digester, all from the same WWTP. Fungal colonization and activity were monitored with ergosterol content, laccase activity and a degradation test (ND24). The solid phase biopile treatment resulted in the complete removal of seven out of 14 detected pharmaceuticals, and between 42-80% for the remaining compounds. Meanwhile, the bioslurry reactor produced a complete elimination of eight out of 24 pharmaceutical agents detected, and 26-92% for the others. However, for the same compounds, in most of the cases the solid-phase treatment showed higher removal efficiency, plus better results in terms of reducing the toxicity of the sludge after the treatment. Next step consisted in applying T. versicolor in non-sterile sludge, which was assessed in solid-phase biopiles, considering the better fungal performance on these systems. Success in the fungal bioaugmentation was monitored by community analyses, which compared the bioaugmented (TVB) and non-bioaugmented (NB) systems. DGGE profiles revealed some inhibition caused by the fungus over bacterial community and also the predominance of T. versicolor in the TVB-systems up to 21-d (half-treatment), to later disappear by the end of the process and being replaced by other fungi. Results permitted to find the relationship of the fungal survival with the degradation of pharmaceuticals on time. Results include the identification of the most abundant bacterial/fungal taxons present in the sludge biopiles. After 42-d of treatment, removals over 50% for eight out of the nine therapeutic agents detected were obtained; only carbamazepine could not be removed at all, contrary to sterile conditions. Overall results suggest that mycoremediation is a potential strategy for the degradation of emerging pollutants from sludge.Spanish Ministries MMAMRM (project-010/PC08/3-04) and MICIN (project-CTQ2010-21776-C2-01). The Department of Chemical Engineering (UAB) is the Unit of Biochemical Engineering of the Xarxa de Referència en Biotecnologia de la Generalitat de Catalunya. Rodríguez-Rodríguez acknowledges UCR-CSIC collaboration

Continuous fungal treatment of non-sterile veterinary hospital effluent: pharmaceuticals removal and microbial community assessment

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well.Authors want to acknowledge the UAB veterinary hospital staff for their kind permission and help for the samplings. This work has been funded by the Spanish Ministry of Economy and Competitiveness and FEDER (projects CTM2013-48545-C2 and AIB2010PT-00169) and supported by the Generalitat de Catalunya (Consolidated Research Groups 2014-SGR-476 and 2014-SGR-291). The Department of Chemical Engineering of the Universitat Autonoma de Barcelona (UAB) is a member of the Xarxa de Referencia en Biotecnologia de la Generalitat de Catalunya. M. Badia-Fabregat and D. Lucas acknowledge the predoctoral grants from UAB and from the Spanish Ministry of Education, Culture and Sports (AP-2010-4926), respectively. The authors also thank the Portuguese Foundation for Science and Technology (FCT) Strategic Project PEst-OE/EQB/LA0023/2013, Project FCOMP-01-0124-FEDER-027462 co-funded by Operational Competitiveness Programme, FEDER, and Project "BioEnv-Biotechnology and Bioengineering for a sustainable world," REF. NORTE-07-0124-FEDER-000048, co-funded by Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER