Operación de reactores biológicos para el tratamiento del agua

Traballo fin de mestrado (UDC.CIE). Biotecnoloxía avanzada. Curso 2011/201

Valorization of bioethanol by-products to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentationS.G., A.T. and G.E. thank their grants (BES-2017-081677, FJC2019-041664-I and RYC2018-024846-I, respectively) funded by MCIN/AEI/ 10.13039/501100011033, and by “ERDF A way of making Europe” and “ESF Investing in your future”. Authors would like to thank the use of USC Mass Spectrometry and Proteomics facilities and Bioetanol Galicia S.A. for the supply of the substrates used in the fermentations. The authors belong to the Galician Competitive Research Groups (GRC)_ ED431C-2021/37. The program is co-funded by FEDER (UE)S

Use of dual element isotope analysis and microcosm studies to determine the origin and potential anaerobic biodegradation of dichloromethane in two multi-contaminated aquifers

Many aquifers around the world are impacted by toxic chlorinated methanes derived from industrial processes due to accidental spills. Frequently, these contaminants co-occur with chlorinated ethenes and/or chlorinated benzenes in groundwater, forming complex mixtures that become very difficult to remediate. In this study, a multi-method approach was used to provide lines of evidence of natural attenuation processes and potential setbacks in the implementation of bioremediation strategies in multi-contaminated aquifers. First, this study determined i) the carbon and chlorine isotopic compositions (δ¹³C, δ³⁷Cl) of several commercial pure phase chlorinated compounds, and ii) the chlorine isotopic fractionation (εCl = −5.2 ± 0.6‰) and the dual CCl isotope correlation (ΛC/Cl = 5.9 ± 0.3) during dichloromethane (DCM) degradation by a Dehalobacterium-containing culture. Such data provide valuable information for practitioners to support the interpretation of stable isotope analyses derived from polluted sites. Second, the bioremediation potential of two industrial sites contaminated with a mixture of organic pollutants (mainly DCM, chloroform (CF), trichloroethene (TCE), and mono-chlorobenzene (MCB)) was evaluated. Hydrochemistry, dual (CCl) isotope analyses, laboratory microcosms, and microbiological data were used to investigate the origin, fate and biodegradation potential of chlorinated methanes. At Site 1, δ¹³C and δ³⁷Cl compositions from field samples were consistent with laboratory microcosms, which showed complete degradation of CF, DCM and TCE, while MCB remained. Identification of Dehalobacter sp. in CF-enriched microcosms further supported the biodegradation capability of the aquifer to remediate chlorinated methanes. At Site 2, hydrochemistry and δ¹³C and δ³⁷Cl compositions from field samples suggested little DCM, CF and TCE transformation; however, laboratory microcosms evidenced that their degradation was severely inhibited, probably by co-contamination. A dual CCl isotopic assessment using results from this study and reference values from the literature allowed to determine the extent of degradation and elucidated the origin of chlorinated methanes

Enrichment and characterization of anaerobic bacteria degrading organohalide compounds

La freqüent contaminació d’aigua subterrània per compostos organohalogenats és un greu problema degut als riscs humans i ecològics que se’n deriven. La bioremediació és un tècnica sostenible que permet superar algunes de les limitacions que presenten els tractaments fisicoquímics. En aquest estudi ens proposem obtenir i caracteritzar cultius que contenen bacteris anaerobis capaços de degradar compostos organohalogenats ambientalment perillosos i que es puguin aplicar per a la bioremediació d’aqüífers in situ. En treballs previs realitzats al nostre laboratori es va obtenir un cultiu enriquit que contenia un bacteri dehalorespirador del gènere Dehalogenimonas a partir de sediments de la desembocadura del riu Besòs (Barcelona) que degrada alcans amb halògens situats en carbons adjacents. En aquesta tesis, s’ha identificat la dehalogenasa reductora (RDasa) d’aquesta Dehalogenimonas implicada en la conversió de dibromur d’etilè (EDB) al compost innocu etilè combinant tècniques de proteòmica basades en gels d’electroforesis, tests enzimàtics i nano-cromatografia líquida acoblada a espectrometria de masses (nLC-MS/MS). Aquesta RDasa es va designar com a EdbA. EdbA és la primera RDasa identificada entre les espècies d’aquest gènere bacterià que catalitza una reacció de debromació. A més, és la primera RDasa que s’ha demostrat funcional i que no té cap subunitat B de fixació a la membrana citoplasmàtica codificada de forma adjacent en el seu genoma. Addicionalment, s’ha detectat un enzim ortolog a l’enzim responsable de la degradació de 1,2-diclorpropà a propé (DcpA) com a única RDasa en cultius que transformen 1,2,3-triclorpropà a clorur d’alil mitjançant la combinació de tècniques d’ultracentrifugació, gels d’electroforesis i nLC-MS/MS. Aquesta DcpA es va detectar en la fracció de la membrana tal i com predeien les eines bioinformàtiques emprades. El mecanisme pel qual aquestes dues RDases identificades es fixen a les membranes és encara desconegut. En aquesta treball s’ha obtingut un segon consorci bacterià estable provinent de llots d’una planta de tractament d’aigües residuals industrials i aplicant estratègies de d’enriquiment del cultiu i tècniques de dilució fins a l’extinció. Aquest cultiu fermenta diclorometà (DCM) i dibromometà (DBM) en acetat i format. S’ha demostrat que el bacteri responsable de la fermentació d’aquests dihalometans és un Dehalobacterium i s’ha procedit al seu aïllament. Tanmateix, les interaccions sinèrgiques entre les espècies del consorci han impedit el seu aïllament. Mitjançant la selecció de colònies en cultius semi sòlids, canvis en la composició del medi i l’ús de antibiòtics, s’ha assolit un cultiu on l’abundància de Dehalobacterium és del 67%. L´acompanyen bacteris dels gèneres Acetobacterium i Desulfovibrio, tal i com revelen els anàlisis de genoteques. El fraccionament dels isòtops de carboni durant la fermentació de DCM per aquest cultiu s’ha determinat mitjançant l’anàlisi d’isòtops estables de compostos específics (CSIA). El valor obtingut de -27 ± 2‰ difereix del prèviament publicat per una soca de Dehalobacter (-15.5 ± 1.5‰) que també fermentava DCM. Aquests valors són significativament diferent dels obtinguts per bacteris metilotròfics degradadors de DCM (que varien de -45 a -61‰) i podria permetre la distinció entre vies de degradació de DCM en treballs de bioremediació in situ. Finalment, s’ha demostrat que la presència de co-contaminants que es detecten freqüentment amb DCM, tals com tricloroetilè (TCE), 1,2-dicloroetà (1,2-DCA), cis-dicloroetilè (cis-DCE), 1,1,2-tricloroetà (1,1,2-TCA), àcid perfluorooctanoic (PFOA) i 3,4-dicloroanilina (3,4-DCA) no provoca una inhibició significativa en la degradació de DCM pel cultiu amb Dehalobacterium a les concentracions testades. La concentració de cloroform de 100 mg/L provoca una total inhibició. De manera similar, la presència de 200 mg/L d’àcid perfluorooctanosulfonic (PFOS) i ≥ 25 mg/L de diuron provoquen una inhibició severa, impedint la degradació completa de DCM. Tanmateix, l’activitat degradadora de DCM es recupera quan els cultius inhibits es transfereixen a medi fresc sense co-contaminants.La frecuente contaminación de las aguas subterráneas por compuestos organohalogenados es un grave problema ambiental debido a los riesgos ecológicos y para la salud humana de ella derivados. La bioremediación es una tecnología sostenible que evita algunos inconvenientes que presentan los tratamientos físico-químicos. En este estudio nos proponemos obtener y caracterizar cultivos que contengan bacterias anaerobias que degraden compuestos organohalogenados ambientalmente peligrosos con potencial para la bioremediación in situ de aguas subterráneas. En trabajos previos de nuestro grupo de investigación, se obtuvo un cultivo enriquecido en bacterias del género Dehalogenimonas procedente de sedimentos del estuario del río Besós (Barcelona) que degrada alcanos con halógenos situados en carbonos adyacentes. En esta tesis se ha identificado la dehalogenasa reductora (RDasa) de esta cepa de Dehalogenimonas implicada en la conversión del dibromuro de etileno (EDB) al compuesto inocuo eteno combinando técnicas de proteómica basadas en geles de electroforesis, ensayos enzimáticos y nano-cromatografía líquida de alta resolución (nLC-MS/MS). Esta RDasa es designada EdbA, y constituye la primera RDasa identificada en este género bacteriano que cataliza una reacción de debromación. Además, es también la primera RDasa en ser demostrada funcional sin una subunidad B de anclaje a la membrana codificada de forma adyacente en el genoma. Adicionalmente, se ha detectado una única RDasa en cultivos que transforman 1,2,3-tricloropropano a cloruro de alilo combinando técnicas de ultracentrifugación, geles de electroforesis y nLC-MS/MS. Esta enzima ortóloga a DcpA, la responsable de la degradación de 1,2-dicloropropano a propeno, ha sido detectada en la fracción proteica de membrana, lo cual concuerta con las predicciones realizadas mediante herramientas bioinformáticas. El mecanismo por el cual EdbA y esta DcpA se anclan a la membrana citoplasmática es desconocido, atribuyéndose a proteínas todavía no descritas. En este trabajo se ha obtenido un segundo consorcio bacteriano estable a partir de lodos de una planta de tratamiento de aguas residuales industriales aplicando técnicas de cultivo de enriquecimiento y dilución por extinción. Este cultivo fermenta diclorometano (DCM) y dibromometano (DBM) a acetato y formato. Se ha demostrado que la bacteria responsable de la fermentación pertenece al género Dehalobacterium, y se ha procedido a su aislamiento. Sin embargo, las interacciones sinérgicas existentes entre las especies del consorcio han impedido obtener un cultivo puro. Seleccionando colonias en medio de cultivo semisólido, aplicando antibióticos y cambios en la composición del medio, se ha obtenido una abundancia relativa de Dehalobacterium del 67%. Le acompañan bacterias de los géneros Acetobacterium y Desulfovibrio, tal y como se detectó mediante análisis de genotecas. El fraccionamiento isotópico del carbono durante la fermentación del DCM por este cultivo fue determinado mediante análisis de isótopos estables de compuestos específicos (CSIA). El valor obtenido, -27 ± 2‰, difiere del publicado previamente para una cepa de Dehalobacter que también fermenta el DCM (-15.5 ± 1.5‰). Estos valores son significativamente diferentes de los obtenidos con bacterias metilotróficas degradadoras de DCM (-45 a -61‰), y podrían permitir diferenciar vías de degradación de DCM en trabajos de bioremediación in situ. Finalmente, se ha demostrado que la presencia de co-contaminantes que se detectan frecuentemente con el DCM, como el tricloroetileno (TCE), 1,2-dicloroetano (1,2-DCA), cis-dicloroetileno (cis-DCE), 1,1,2-tricloroetano (1,1,2-TCA), ácido perfluorooctanoico (PFOA) y 3,4-dicloroanilina (3,4-DCA) no provocan una inhibición significativa en la degradación de DCM por parte del cultivo de Dehalobacterium, a las concentraciones estudiadas. Una concentración de cloroformo de 100 mg/L provoca una inhibición total. De manera similar, 200 mg/L de sulfonato de perfluoroctano (PFOS), y ≥ 25 mg/L de diuron provocan una inhibición severa, impidiendo la degradación completa del DCM. Sin embargo, la actividad degradadora de DCM se recupera cuando los cultivos inhibidos se transfieren a medio libre de co-contaminantes.The widespread groundwater contamination by organohalide compounds is of a major concern due to the human and ecological risks derived from it. Bioremediation is a sustainable technology that overcomes some limitations of the physical-chemical remediation techniques on these water bodies. In this study, we aimed to obtain and characterize cultures containing anaerobic bacteria capable of degrading organohalide compounds of environmental concern with potential for in situ groundwater bioremediation. In previous work carried out in our laboratory a highly enriched culture containing organohalide-respiring bacteria from the genus Dehalogenimonas degrading vicinally halogenated alkanes was obtained from sediments of the river Besós estuary (Barcelona). In this thesis, the reductive dehalogenase (RDase) from this Dehalogenimonas strain responsible for the catalysis of ethylene dibromide (EDB) to the innocuous ethene was identified combining gel-based proteomic techniques, specific enzymatic tests and nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS). This RDase is therefore designated as EdbA, for ethylene dibromide RDase subunit A. EdbA is the first RDase identified for debrominating catalytic activity among species of this genus. Moreover, it is the first RDase shown to be functional for respiration without an adjacent membrane-anchoring subunit B encoded on the genome. Additionally, combining ultracentrifugation, gel electrophoresis and nLC-MS/MS, an orthologous enzyme of the dichloropropane-to-propene RDase (DcpA) was the only RDase detected in 1,2,3-trichloropropane-to-allyl chloride dehalogenating cultures. This DcpA was detected in the membrane fraction of the crude protein extract, in accordance to its predicted subcellular localization by bioinformatics tools and it is also not co-localised with an rdhB gene. The membrane-anchoring mechanisms of these RDases remains not known and may rely in yet-unidentified proteins. A second stable bacterial consortium was obtained in the present work from slurry samples of an industrial wastewater treatment plant with a combination of enrichment culture strategies and the dilution-to-extinction technique. This culture was demonstrated to ferment dichloromethane (DCM) and dibromomethane (DBM) into acetate and formate. The Dehalobacterium sp. present in this culture was shown to be the responsible for the dihalomethanes fermentation, and the isolation of this strain was attempted. However, the synergic interactions existing among the different accompanying species present in the bacterial consortia impeded the isolation. Despite a pure culture was not achieved via picking up colonies from semisolid agar cultures, changes in the medium composition, and the application of selected antibiotics, a final relative abundance of Dehalobacterium sp. of 67 % was attained. As determined by clone library analysis, bacteria from the genera Acetobacterium and Desulfovibrio remained present in the culture. The carbon isotope fractionation during DCM fermentation by this culture was determined by compound-specific stable isotope analysis (CSIA). The value obtained was -27 ± 2‰ and differs from the previously published value of -15.5 ± 1.5‰ of a Dehalobacter sp. performing also DCM fermentation. These values are yet significantly different from those reported for facultative methylotrophic bacteria degrading DCM (ranging from -45 to -61‰), and this would allow for further differentiation of these degradation pathways during in situ bioremediation works. Finally, the potential inhibitory effect of selected frequent groundwater co-contaminants over DCM degradation by the Dehalobacterium-containing culture was assessed for further in situ bioremediation applications. Trichloroethylene (TCE), 1,2-dichloroethane (1,2-DCA), cis-dichloroethylene (cis-DCE), 1,1,2-trichloroethane (1,1,2-TCA), perfluorooctanoic acid (PFOA), and 3,4-dichloroaniline (3,4-DCA) did not show significant inhibitory effects at the concentrations tested. Differently, a total inhibition was caused with a chloroform concentration of 100 mg/L. Also, the presence of 200 mg/L of perfluorooctanesulfonic acid (PFOS), as well as concentrations higher than 25 mg/L of the pesticide diuron caused a severe inhibitory effect, preventing the full depletion of DCM. Nevertheless, DCM degrading activity was recovered when inhibited cultures were transferred to co-contaminant free medium

Enrichment and characterization of anaerobic bacteria degrading organohalide compounds

La freqüent contaminació d’aigua subterrània per compostos organohalogenats és un greu problema degut als riscs humans i ecològics que se’n deriven. La bioremediació és un tècnica sostenible que permet superar algunes de les limitacions que presenten els tractaments fisicoquímics. En aquest estudi ens proposem obtenir i caracteritzar cultius que contenen bacteris anaerobis capaços de degradar compostos organohalogenats ambientalment perillosos i que es puguin aplicar per a la bioremediació d’aqüífers in situ. En treballs previs realitzats al nostre laboratori es va obtenir un cultiu enriquit que contenia un bacteri dehalorespirador del gènere Dehalogenimonas a partir de sediments de la desembocadura del riu Besòs (Barcelona) que degrada alcans amb halògens situats en carbons adjacents. En aquesta tesis, s’ha identificat la dehalogenasa reductora (RDasa) d’aquesta Dehalogenimonas implicada en la conversió de dibromur d’etilè (EDB) al compost innocu etilè combinant tècniques de proteòmica basades en gels d’electroforesis, tests enzimàtics i nano-cromatografia líquida acoblada a espectrometria de masses (nLC-MS/MS). Aquesta RDasa es va designar com a EdbA. EdbA és la primera RDasa identificada entre les espècies d’aquest gènere bacterià que catalitza una reacció de debromació. A més, és la primera RDasa que s’ha demostrat funcional i que no té cap subunitat B de fixació a la membrana citoplasmàtica codificada de forma adjacent en el seu genoma. Addicionalment, s’ha detectat un enzim ortolog a l’enzim responsable de la degradació de 1,2-diclorpropà a propé (DcpA) com a única RDasa en cultius que transformen 1,2,3-triclorpropà a clorur d’alil mitjançant la combinació de tècniques d’ultracentrifugació, gels d’electroforesis i nLC-MS/MS. Aquesta DcpA es va detectar en la fracció de la membrana tal i com predeien les eines bioinformàtiques emprades. El mecanisme pel qual aquestes dues RDases identificades es fixen a les membranes és encara desconegut. En aquesta treball s’ha obtingut un segon consorci bacterià estable provinent de llots d’una planta de tractament d’aigües residuals industrials i aplicant estratègies de d’enriquiment del cultiu i tècniques de dilució fins a l’extinció. Aquest cultiu fermenta diclorometà (DCM) i dibromometà (DBM) en acetat i format. S’ha demostrat que el bacteri responsable de la fermentació d’aquests dihalometans és un Dehalobacterium i s’ha procedit al seu aïllament. Tanmateix, les interaccions sinèrgiques entre les espècies del consorci han impedit el seu aïllament. Mitjançant la selecció de colònies en cultius semi sòlids, canvis en la composició del medi i l’ús de antibiòtics, s’ha assolit un cultiu on l’abundància de Dehalobacterium és del 67%. L´acompanyen bacteris dels gèneres Acetobacterium i Desulfovibrio, tal i com revelen els anàlisis de genoteques. El fraccionament dels isòtops de carboni durant la fermentació de DCM per aquest cultiu s’ha determinat mitjançant l’anàlisi d’isòtops estables de compostos específics (CSIA). El valor obtingut de -27 ± 2‰ difereix del prèviament publicat per una soca de Dehalobacter (-15.5 ± 1.5‰) que també fermentava DCM. Aquests valors són significativament diferent dels obtinguts per bacteris metilotròfics degradadors de DCM (que varien de -45 a -61‰) i podria permetre la distinció entre vies de degradació de DCM en treballs de bioremediació in situ. Finalment, s’ha demostrat que la presència de co-contaminants que es detecten freqüentment amb DCM, tals com tricloroetilè (TCE), 1,2-dicloroetà (1,2-DCA), cis-dicloroetilè (cis-DCE), 1,1,2-tricloroetà (1,1,2-TCA), àcid perfluorooctanoic (PFOA) i 3,4-dicloroanilina (3,4-DCA) no provoca una inhibició significativa en la degradació de DCM pel cultiu amb Dehalobacterium a les concentracions testades. La concentració de cloroform de 100 mg/L provoca una total inhibició. De manera similar, la presència de 200 mg/L d’àcid perfluorooctanosulfonic (PFOS) i ≥ 25 mg/L de diuron provoquen una inhibició severa, impedint la degradació completa de DCM. Tanmateix, l’activitat degradadora de DCM es recupera quan els cultius inhibits es transfereixen a medi fresc sense co-contaminants.La frecuente contaminación de las aguas subterráneas por compuestos organohalogenados es un grave problema ambiental debido a los riesgos ecológicos y para la salud humana de ella derivados. La bioremediación es una tecnología sostenible que evita algunos inconvenientes que presentan los tratamientos físico-químicos. En este estudio nos proponemos obtener y caracterizar cultivos que contengan bacterias anaerobias que degraden compuestos organohalogenados ambientalmente peligrosos con potencial para la bioremediación in situ de aguas subterráneas. En trabajos previos de nuestro grupo de investigación, se obtuvo un cultivo enriquecido en bacterias del género Dehalogenimonas procedente de sedimentos del estuario del río Besós (Barcelona) que degrada alcanos con halógenos situados en carbonos adyacentes. En esta tesis se ha identificado la dehalogenasa reductora (RDasa) de esta cepa de Dehalogenimonas implicada en la conversión del dibromuro de etileno (EDB) al compuesto inocuo eteno combinando técnicas de proteómica basadas en geles de electroforesis, ensayos enzimáticos y nano-cromatografía líquida de alta resolución (nLC-MS/MS). Esta RDasa es designada EdbA, y constituye la primera RDasa identificada en este género bacteriano que cataliza una reacción de debromación. Además, es también la primera RDasa en ser demostrada funcional sin una subunidad B de anclaje a la membrana codificada de forma adyacente en el genoma. Adicionalmente, se ha detectado una única RDasa en cultivos que transforman 1,2,3-tricloropropano a cloruro de alilo combinando técnicas de ultracentrifugación, geles de electroforesis y nLC-MS/MS. Esta enzima ortóloga a DcpA, la responsable de la degradación de 1,2-dicloropropano a propeno, ha sido detectada en la fracción proteica de membrana, lo cual concuerta con las predicciones realizadas mediante herramientas bioinformáticas. El mecanismo por el cual EdbA y esta DcpA se anclan a la membrana citoplasmática es desconocido, atribuyéndose a proteínas todavía no descritas. En este trabajo se ha obtenido un segundo consorcio bacteriano estable a partir de lodos de una planta de tratamiento de aguas residuales industriales aplicando técnicas de cultivo de enriquecimiento y dilución por extinción. Este cultivo fermenta diclorometano (DCM) y dibromometano (DBM) a acetato y formato. Se ha demostrado que la bacteria responsable de la fermentación pertenece al género Dehalobacterium, y se ha procedido a su aislamiento. Sin embargo, las interacciones sinérgicas existentes entre las especies del consorcio han impedido obtener un cultivo puro. Seleccionando colonias en medio de cultivo semisólido, aplicando antibióticos y cambios en la composición del medio, se ha obtenido una abundancia relativa de Dehalobacterium del 67%. Le acompañan bacterias de los géneros Acetobacterium y Desulfovibrio, tal y como se detectó mediante análisis de genotecas. El fraccionamiento isotópico del carbono durante la fermentación del DCM por este cultivo fue determinado mediante análisis de isótopos estables de compuestos específicos (CSIA). El valor obtenido, -27 ± 2‰, difiere del publicado previamente para una cepa de Dehalobacter que también fermenta el DCM (-15.5 ± 1.5‰). Estos valores son significativamente diferentes de los obtenidos con bacterias metilotróficas degradadoras de DCM (-45 a -61‰), y podrían permitir diferenciar vías de degradación de DCM en trabajos de bioremediación in situ. Finalmente, se ha demostrado que la presencia de co-contaminantes que se detectan frecuentemente con el DCM, como el tricloroetileno (TCE), 1,2-dicloroetano (1,2-DCA), cis-dicloroetileno (cis-DCE), 1,1,2-tricloroetano (1,1,2-TCA), ácido perfluorooctanoico (PFOA) y 3,4-dicloroanilina (3,4-DCA) no provocan una inhibición significativa en la degradación de DCM por parte del cultivo de Dehalobacterium, a las concentraciones estudiadas. Una concentración de cloroformo de 100 mg/L provoca una inhibición total. De manera similar, 200 mg/L de sulfonato de perfluoroctano (PFOS), y ≥ 25 mg/L de diuron provocan una inhibición severa, impidiendo la degradación completa del DCM. Sin embargo, la actividad degradadora de DCM se recupera cuando los cultivos inhibidos se transfieren a medio libre de co-contaminantes.The widespread groundwater contamination by organohalide compounds is of a major concern due to the human and ecological risks derived from it. Bioremediation is a sustainable technology that overcomes some limitations of the physical-chemical remediation techniques on these water bodies. In this study, we aimed to obtain and characterize cultures containing anaerobic bacteria capable of degrading organohalide compounds of environmental concern with potential for in situ groundwater bioremediation. In previous work carried out in our laboratory a highly enriched culture containing organohalide-respiring bacteria from the genus Dehalogenimonas degrading vicinally halogenated alkanes was obtained from sediments of the river Besós estuary (Barcelona). In this thesis, the reductive dehalogenase (RDase) from this Dehalogenimonas strain responsible for the catalysis of ethylene dibromide (EDB) to the innocuous ethene was identified combining gel-based proteomic techniques, specific enzymatic tests and nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS). This RDase is therefore designated as EdbA, for ethylene dibromide RDase subunit A. EdbA is the first RDase identified for debrominating catalytic activity among species of this genus. Moreover, it is the first RDase shown to be functional for respiration without an adjacent membrane-anchoring subunit B encoded on the genome. Additionally, combining ultracentrifugation, gel electrophoresis and nLC-MS/MS, an orthologous enzyme of the dichloropropane-to-propene RDase (DcpA) was the only RDase detected in 1,2,3-trichloropropane-to-allyl chloride dehalogenating cultures. This DcpA was detected in the membrane fraction of the crude protein extract, in accordance to its predicted subcellular localization by bioinformatics tools and it is also not co-localised with an rdhB gene. The membrane-anchoring mechanisms of these RDases remains not known and may rely in yet-unidentified proteins. A second stable bacterial consortium was obtained in the present work from slurry samples of an industrial wastewater treatment plant with a combination of enrichment culture strategies and the dilution-to-extinction technique. This culture was demonstrated to ferment dichloromethane (DCM) and dibromomethane (DBM) into acetate and formate. The Dehalobacterium sp. present in this culture was shown to be the responsible for the dihalomethanes fermentation, and the isolation of this strain was attempted. However, the synergic interactions existing among the different accompanying species present in the bacterial consortia impeded the isolation. Despite a pure culture was not achieved via picking up colonies from semisolid agar cultures, changes in the medium composition, and the application of selected antibiotics, a final relative abundance of Dehalobacterium sp. of 67 % was attained. As determined by clone library analysis, bacteria from the genera Acetobacterium and Desulfovibrio remained present in the culture. The carbon isotope fractionation during DCM fermentation by this culture was determined by compound-specific stable isotope analysis (CSIA). The value obtained was -27 ± 2‰ and differs from the previously published value of -15.5 ± 1.5‰ of a Dehalobacter sp. performing also DCM fermentation. These values are yet significantly different from those reported for facultative methylotrophic bacteria degrading DCM (ranging from -45 to -61‰), and this would allow for further differentiation of these degradation pathways during in situ bioremediation works. Finally, the potential inhibitory effect of selected frequent groundwater co-contaminants over DCM degradation by the Dehalobacterium-containing culture was assessed for further in situ bioremediation applications. Trichloroethylene (TCE), 1,2-dichloroethane (1,2-DCA), cis-dichloroethylene (cis-DCE), 1,1,2-trichloroethane (1,1,2-TCA), perfluorooctanoic acid (PFOA), and 3,4-dichloroaniline (3,4-DCA) did not show significant inhibitory effects at the concentrations tested. Differently, a total inhibition was caused with a chloroform concentration of 100 mg/L. Also, the presence of 200 mg/L of perfluorooctanesulfonic acid (PFOS), as well as concentrations higher than 25 mg/L of the pesticide diuron caused a severe inhibitory effect, preventing the full depletion of DCM. Nevertheless, DCM degrading activity was recovered when inhibited cultures were transferred to co-contaminant free medium

Enrichment and characterization of anaerobic bacteria degrading organohalide compounds /

Bibliografia.La freqüent contaminació d'aigua subterrània per compostos organohalogenats és un greu problema degut als riscs humans i ecològics que se'n deriven. La bioremediació és un tècnica sostenible que permet superar algunes de les limitacions que presenten els tractaments fisicoquímics. En aquest estudi ens proposem obtenir i caracteritzar cultius que contenen bacteris anaerobis capaços de degradar compostos organohalogenats ambientalment perillosos i que es puguin aplicar per a la bioremediació d'aqüífers in situ. En treballs previs realitzats al nostre laboratori es va obtenir un cultiu enriquit que contenia un bacteri dehalorespirador del gènere Dehalogenimonas a partir de sediments de la desembocadura del riu Besòs (Barcelona) que degrada alcans amb halògens situats en carbons adjacents. En aquesta tesis, s'ha identificat la dehalogenasa reductora (RDasa) d'aquesta Dehalogenimonas implicada en la conversió de dibromur d'etilè (EDB) al compost innocu etilè combinant tècniques de proteòmica basades en gels d'electroforesis, tests enzimàtics i nano-cromatografia líquida acoblada a espectrometria de masses (nLC-MS/MS). Aquesta RDasa es va designar com a EdbA. EdbA és la primera RDasa identificada entre les espècies d'aquest gènere bacterià que catalitza una reacció de debromació. A més, és la primera RDasa que s'ha demostrat funcional i que no té cap subunitat B de fixació a la membrana citoplasmàtica codificada de forma adjacent en el seu genoma. Addicionalment, s'ha detectat un enzim ortolog a l'enzim responsable de la degradació de 1,2-diclorpropà a propé (DcpA) com a única RDasa en cultius que transformen 1,2,3-triclorpropà a clorur d'alil mitjançant la combinació de tècniques d'ultracentrifugació, gels d'electroforesis i nLC-MS/MS. Aquesta DcpA es va detectar en la fracció de la membrana tal i com predeien les eines bioinformàtiques emprades. El mecanisme pel qual aquestes dues RDases identificades es fixen a les membranes és encara desconegut. En aquesta treball s'ha obtingut un segon consorci bacterià estable provinent de llots d'una planta de tractament d'aigües residuals industrials i aplicant estratègies de d'enriquiment del cultiu i tècniques de dilució fins a l'extinció. Aquest cultiu fermenta diclorometà (DCM) i dibromometà (DBM) en acetat i format. S'ha demostrat que el bacteri responsable de la fermentació d'aquests dihalometans és un Dehalobacterium i s'ha procedit al seu aïllament. Tanmateix, les interaccions sinèrgiques entre les espècies del consorci han impedit el seu aïllament. Mitjançant la selecció de colònies en cultius semi sòlids, canvis en la composició del medi i l'ús de antibiòtics, s'ha assolit un cultiu on l'abundància de Dehalobacterium és del 67%. Ĺacompanyen bacteris dels gèneres Acetobacterium i Desulfovibrio, tal i com revelen els anàlisis de genoteques. El fraccionament dels isòtops de carboni durant la fermentació de DCM per aquest cultiu s'ha determinat mitjançant l'anàlisi d'isòtops estables de compostos específics (CSIA). El valor obtingut de -27 ± 2‰ difereix del prèviament publicat per una soca de Dehalobacter (-15.5 ± 1.5‰) que també fermentava DCM. Aquests valors són significativament diferent dels obtinguts per bacteris metilotròfics degradadors de DCM (que varien de -45 a -61‰) i podria permetre la distinció entre vies de degradació de DCM en treballs de bioremediació in situ. Finalment, s'ha demostrat que la presència de co-contaminants que es detecten freqüentment amb DCM, tals com tricloroetilè (TCE), 1,2-dicloroetà (1,2-DCA), cis-dicloroetilè (cis-DCE), 1,1,2-tricloroetà (1,1,2-TCA), àcid perfluorooctanoic (PFOA) i 3,4-dicloroanilina (3,4-DCA) no provoca una inhibició significativa en la degradació de DCM pel cultiu amb Dehalobacterium a les concentracions testades. La concentració de cloroform de 100 mg/L provoca una total inhibició. De manera similar, la presència de 200 mg/L d'àcid perfluorooctanosulfonic (PFOS) i ≥ 25 mg/L de diuron provoquen una inhibició severa, impedint la degradació completa de DCM. Tanmateix, l'activitat degradadora de DCM es recupera quan els cultius inhibits es transfereixen a medi fresc sense co-contaminants.La frecuente contaminación de las aguas subterráneas por compuestos organohalogenados es un grave problema ambiental debido a los riesgos ecológicos y para la salud humana de ella derivados. La bioremediación es una tecnología sostenible que evita algunos inconvenientes que presentan los tratamientos físico-químicos. En este estudio nos proponemos obtener y caracterizar cultivos que contengan bacterias anaerobias que degraden compuestos organohalogenados ambientalmente peligrosos con potencial para la bioremediación in situ de aguas subterráneas. En trabajos previos de nuestro grupo de investigación, se obtuvo un cultivo enriquecido en bacterias del género Dehalogenimonas procedente de sedimentos del estuario del río Besós (Barcelona) que degrada alcanos con halógenos situados en carbonos adyacentes. En esta tesis se ha identificado la dehalogenasa reductora (RDasa) de esta cepa de Dehalogenimonas implicada en la conversión del dibromuro de etileno (EDB) al compuesto inocuo eteno combinando técnicas de proteómica basadas en geles de electroforesis, ensayos enzimáticos y nano-cromatografía líquida de alta resolución (nLC-MS/MS). Esta RDasa es designada EdbA, y constituye la primera RDasa identificada en este género bacteriano que cataliza una reacción de debromación. Además, es también la primera RDasa en ser demostrada funcional sin una subunidad B de anclaje a la membrana codificada de forma adyacente en el genoma. Adicionalmente, se ha detectado una única RDasa en cultivos que transforman 1,2,3-tricloropropano a cloruro de alilo combinando técnicas de ultracentrifugación, geles de electroforesis y nLC-MS/MS. Esta enzima ortóloga a DcpA, la responsable de la degradación de 1,2-dicloropropano a propeno, ha sido detectada en la fracción proteica de membrana, lo cual concuerta con las predicciones realizadas mediante herramientas bioinformáticas. El mecanismo por el cual EdbA y esta DcpA se anclan a la membrana citoplasmática es desconocido, atribuyéndose a proteínas todavía no descritas. En este trabajo se ha obtenido un segundo consorcio bacteriano estable a partir de lodos de una planta de tratamiento de aguas residuales industriales aplicando técnicas de cultivo de enriquecimiento y dilución por extinción. Este cultivo fermenta diclorometano (DCM) y dibromometano (DBM) a acetato y formato. Se ha demostrado que la bacteria responsable de la fermentación pertenece al género Dehalobacterium, y se ha procedido a su aislamiento. Sin embargo, las interacciones sinérgicas existentes entre las especies del consorcio han impedido obtener un cultivo puro. Seleccionando colonias en medio de cultivo semisólido, aplicando antibióticos y cambios en la composición del medio, se ha obtenido una abundancia relativa de Dehalobacterium del 67%. Le acompañan bacterias de los géneros Acetobacterium y Desulfovibrio, tal y como se detectó mediante análisis de genotecas. El fraccionamiento isotópico del carbono durante la fermentación del DCM por este cultivo fue determinado mediante análisis de isótopos estables de compuestos específicos (CSIA). El valor obtenido, -27 ± 2‰, difiere del publicado previamente para una cepa de Dehalobacter que también fermenta el DCM (-15.5 ± 1.5‰). Estos valores son significativamente diferentes de los obtenidos con bacterias metilotróficas degradadoras de DCM (-45 a -61‰), y podrían permitir diferenciar vías de degradación de DCM en trabajos de bioremediación in situ. Finalmente, se ha demostrado que la presencia de co-contaminantes que se detectan frecuentemente con el DCM, como el tricloroetileno (TCE), 1,2-dicloroetano (1,2-DCA), cis-dicloroetileno (cis-DCE), 1,1,2-tricloroetano (1,1,2-TCA), ácido perfluorooctanoico (PFOA) y 3,4-dicloroanilina (3,4-DCA) no provocan una inhibición significativa en la degradación de DCM por parte del cultivo de Dehalobacterium, a las concentraciones estudiadas. Una concentración de cloroformo de 100 mg/L provoca una inhibición total. De manera similar, 200 mg/L de sulfonato de perfluoroctano (PFOS), y ≥ 25 mg/L de diuron provocan una inhibición severa, impidiendo la degradación completa del DCM. Sin embargo, la actividad degradadora de DCM se recupera cuando los cultivos inhibidos se transfieren a medio libre de co-contaminantes.The widespread groundwater contamination by organohalide compounds is of a major concern due to the human and ecological risks derived from it. Bioremediation is a sustainable technology that overcomes some limitations of the physical-chemical remediation techniques on these water bodies. In this study, we aimed to obtain and characterize cultures containing anaerobic bacteria capable of degrading organohalide compounds of environmental concern with potential for in situ groundwater bioremediation. In previous work carried out in our laboratory a highly enriched culture containing organohalide-respiring bacteria from the genus Dehalogenimonas degrading vicinally halogenated alkanes was obtained from sediments of the river Besós estuary (Barcelona). In this thesis, the reductive dehalogenase (RDase) from this Dehalogenimonas strain responsible for the catalysis of ethylene dibromide (EDB) to the innocuous ethene was identified combining gel-based proteomic techniques, specific enzymatic tests and nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS). This RDase is therefore designated as EdbA, for ethylene dibromide RDase subunit A. EdbA is the first RDase identified for debrominating catalytic activity among species of this genus. Moreover, it is the first RDase shown to be functional for respiration without an adjacent membrane-anchoring subunit B encoded on the genome. Additionally, combining ultracentrifugation, gel electrophoresis and nLC-MS/MS, an orthologous enzyme of the dichloropropane-to-propene RDase (DcpA) was the only RDase detected in 1,2,3-trichloropropane-to-allyl chloride dehalogenating cultures. This DcpA was detected in the membrane fraction of the crude protein extract, in accordance to its predicted subcellular localization by bioinformatics tools and it is also not co-localised with an rdhB gene. The membrane-anchoring mechanisms of these RDases remains not known and may rely in yet-unidentified proteins. A second stable bacterial consortium was obtained in the present work from slurry samples of an industrial wastewater treatment plant with a combination of enrichment culture strategies and the dilution-to-extinction technique. This culture was demonstrated to ferment dichloromethane (DCM) and dibromomethane (DBM) into acetate and formate. The Dehalobacterium sp. present in this culture was shown to be the responsible for the dihalomethanes fermentation, and the isolation of this strain was attempted. However, the synergic interactions existing among the different accompanying species present in the bacterial consortia impeded the isolation. Despite a pure culture was not achieved via picking up colonies from semisolid agar cultures, changes in the medium composition, and the application of selected antibiotics, a final relative abundance of Dehalobacterium sp. of 67 % was attained. As determined by clone library analysis, bacteria from the genera Acetobacterium and Desulfovibrio remained present in the culture. The carbon isotope fractionation during DCM fermentation by this culture was determined by compound-specific stable isotope analysis (CSIA). The value obtained was -27 ± 2‰ and differs from the previously published value of -15.5 ± 1.5‰ of a Dehalobacter sp. performing also DCM fermentation. These values are yet significantly different from those reported for facultative methylotrophic bacteria degrading DCM (ranging from -45 to -61‰), and this would allow for further differentiation of these degradation pathways during in situ bioremediation works. Finally, the potential inhibitory effect of selected frequent groundwater co-contaminants over DCM degradation by the Dehalobacterium-containing culture was assessed for further in situ bioremediation applications. Trichloroethylene (TCE), 1,2-dichloroethane (1,2-DCA), cis-dichloroethylene (cis-DCE), 1,1,2-trichloroethane (1,1,2-TCA), perfluorooctanoic acid (PFOA), and 3,4-dichloroaniline (3,4-DCA) did not show significant inhibitory effects at the concentrations tested. Differently, a total inhibition was caused with a chloroform concentration of 100 mg/L. Also, the presence of 200 mg/L of perfluorooctanesulfonic acid (PFOS), as well as concentrations higher than 25 mg/L of the pesticide diuron caused a severe inhibitory effect, preventing the full depletion of DCM. Nevertheless, DCM degrading activity was recovered when inhibited cultures were transferred to co-contaminant free medium

Genome Sequence, Proteome Profile, and Identification of a Multiprotein Reductive Dehalogenase Complex in Dehalogenimonas alkenigignens Strain BRE15M

Bacteria of the genus Dehalogenimonas respire with vicinally halogenated alkanes via dihaloelimination. We aimed to describe involved proteins and their supermolecular organization. Metagenomic sequencing of a Dehalogenimonas -containing culture resulted in a 1.65 Mbp draft genome of Dehalogenimonas alkenigignens strain BRE15M. It contained 31 full-length reductive dehalogenase homologous genes (rdhA), but only eight had cognate rdhB gene coding for membrane-anchoring proteins. Shotgun proteomics of cells grown with 1,2-dichloropropane as an electron acceptor identified 1152 proteins representing more than 60% of the total proteome. Ten RdhA proteins were detected, including a DcpA ortholog, which was the strongest expressed RdhA. Blue native gel electrophoresis (BNE) demonstrating maximum activity was localized in a protein complex of 146-242 kDa. Protein mass spectrometry revealed the presence of DcpA, its membrane-anchoring protein DcpB, two hydrogen uptake hydrogenase subunits (HupL and HupS), an iron-sulfur protein (HupX), and subunits of a redox protein with a molybdopterin-binding motif (OmeA and OmeB) in the complex. BNE after protein solubilization with different detergent concentrations revealed no evidence for an interaction between the putative respiratory electron input module (HupLS) and the OmeA/OmeB/HupX module. All detected RdhAs comigrated with the organohalide respiration complex. Based on genomic and proteomic analysis, we propose quinone-independent respiration in Dehalogenimonas

Bundling the removal of emerging contaminants with the production of ligninolytic enzymes from residual streams

Enzymes offer interesting features as biological catalysts for industry: high specificity, activity under mild conditions, accessibility, and environmental friendliness. Being able to produce enzymes in large quantities and having them available in a stable and reusable form reduces the production costs of any enzyme-based process. Agricultural residues have recently demonstrated their potential as substrates to produce ligninolytic enzymes by different white rot fungi. In this study, the biotechnological production of a manganese peroxidase (MnP) by Irpex lacteus was conducted through solid-state fermentation (SSF) with wheat straw as substrate and submerged fermentation (SmF) employing wheat straw extract (WSE). The obtained enzyme cocktail also showed manganese-independent activity (MiP), related to the presence of a short MnP and a dye-decolorizing peroxidase (DyP) which was confirmed by shotgun proteomic analyses. In view of the enhanced production of ligninolytic enzymes in SmF, different parameters such as WSE concentration and nitrogen source were evaluated. The highest enzyme titers were obtained with a medium formulated with glucose and peptone (339 U/L MnP and 15 U/L MiP). The scale-up to a 30 L reactor achieved similar activities, demonstrating the feasibility of enzyme production from the residual substrate at different production scales. Degradation of five emerging pollutants was performed to demonstrate the high oxidative capacity of the enzyme. Complete removal of hormones and bisphenol A was achieved in less than 1 h, whereas almost 30% degradation of carbamazepine was achieved in 24 h, which is a significant improvement compared to previous enzymatic treatments of this compoundOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was funded by the Spanish Ministry of Science and Innovation: MODENA Project (CTQ2016-79461-R) and the WooBAdh project (PCI2018-092866, ERA-CoBioTech program)S

Interspecies interaction and effect of co-contaminants in an anaerobic dichloromethane-degrading culture

An anaerobic stable mixed culture dominated by bacteria belonging to the genera Dehalobacterium, Acetobacterium, Desulfovibrio, and Wolinella was used as a model to study the microbial interactions during DCM degradation. Physiological studies indicated that DCM was degraded in this mixed culture at least in a three-step process: i) fermentation of DCM to acetate and formate, ii) formate oxidation to CO₂ and H₂, and iii) H₂/CO₂ reductive acetogenesis. The 16S rRNA gene sequencing of cultures enriched with formate or H₂ showed that Desulfovibrio was the dominant population followed by Acetobacterium, but sequences representing Dehalobacterium were only present in cultures amended with DCM. Nuclear magnetic resonance analyses confirmed that acetate produced from ¹³C-labelled DCM was marked at the methyl ([2-¹³C]acetate), carboxyl ([1-¹³C]acetate), and both ([1,2-¹³C]acetate) positions, which is in accordance to acetate formed by both direct DCM fermentation and H₂/CO₂ acetogenesis. The inhibitory effect of ten different co-contaminants frequently detected in groundwaters on DCM degradation was also investigated. Complete inhibition of DCM degradation was observed when chloroform, perfluorooctanesulfonic acid, and diuron were added at 838, 400, and 107 μM, respectively. However, the inhibited cultures recovered the DCM degradation capability when transferred to fresh medium without co-contaminants. Findings derived from this work are of significant relevance to provide a better understanding of the synergistic interactions among bacteria to accomplish DCM degradation as well as to predict the effect of co-contaminants during anaerobic DCM bioremediation in groundwater

Interspecies interaction and effect of co-contaminants in an anaerobic dichloromethane-degrading culture

An anaerobic stable mixed culture dominated by bacteria belonging to the genera Dehalobacterium, Acetobacterium, Desulfovibrio, and Wolinella was used as a model to study the microbial interactions during DCM degradation. Physiological studies indicated that DCM was degraded in this mixed culture at least in a three-step process: i) fermentation of DCM to acetate and formate, ii) formate oxidation to CO₂ and H₂, and iii) H₂/CO₂ reductive acetogenesis. The 16S rRNA gene sequencing of cultures enriched with formate or H₂ showed that Desulfovibrio was the dominant population followed by Acetobacterium, but sequences representing Dehalobacterium were only present in cultures amended with DCM. Nuclear magnetic resonance analyses confirmed that acetate produced from ¹³C-labelled DCM was marked at the methyl ([2-¹³C]acetate), carboxyl ([1-¹³C]acetate), and both ([1,2-¹³C]acetate) positions, which is in accordance to acetate formed by both direct DCM fermentation and H₂/CO₂ acetogenesis. The inhibitory effect of ten different co-contaminants frequently detected in groundwaters on DCM degradation was also investigated. Complete inhibition of DCM degradation was observed when chloroform, perfluorooctanesulfonic acid, and diuron were added at 838, 400, and 107 μM, respectively. However, the inhibited cultures recovered the DCM degradation capability when transferred to fresh medium without co-contaminants. Findings derived from this work are of significant relevance to provide a better understanding of the synergistic interactions among bacteria to accomplish DCM degradation as well as to predict the effect of co-contaminants during anaerobic DCM bioremediation in groundwater