Biogeochemical evolution of a marine shore tailings deposit during bioremediation

Summary : Mining activities produce enormous amounts of waste material known as tailings which are composed of fine to medium size particles. These tailings often contain sulfides, which oxidation can lead to acid and metal contamination of water; therefore they need to be remediated. In this work a tailings bioremediation approach was investigated by an interdisciplinary study including geochemistry, mineralogy and microbiology. The aim of the work was to study the effect of the implementation of wetland above oxidizing tailings on the hydrogeology and the biogeochemical element cycles, and to assess the system evolution over time. To reach these goals, biogeochemical processes occurring in a marine shore tailings deposit were investigated. The studied tailings deposit is located at the Bahìa de Ite, Pacific Ocean, southern Peru, where between 1940 and 1996 the tailings were discharged from the two porphyry copper mines Cuajone and Toquepala. After the end of deposition, a remediation approach was initiated in 1997 with a wetland implementation above the oxidizing tailings. Around 90% of the tailings deposits (total 16 km2) were thus remediated, except the central delta area and some areas close to the shoreline. The multi-stable isotope study showed that the tailings were saturated with fresh water in spite of the marine setting, due to the high hydraulic gradient resulting from the wetland implementation. Submarine groundwater discharge (SGD) was the major source of SO4 2-, C1-, Na+, Fe2+, and Mn2+ input into the tailings at the original shelf-seawater interface. The geochemical study (aquatic geochemistry and X-Ray diffraction (XRD) and sequential extractions from the solid fraction) showed that iron and sulfur oxidation were the main processes in the non-remediated tailings, which showed a top a low-pH oxidation zone with strong accumulation of efflorescent salts at the surface due to capillary upward transport of heavy metals (Fe, Cu, Zn, Mn, Cd, Co, and Ni) in the arid climate. The study showed also that the implementation of the wetland resulted in very low concentrations of heavy metals in solution (mainly under the detection limit) due to the near neutral pH and more reducing conditions (100-150 mV). The heavy metals, which were taken from solution, precipitated as hydroxides and sulfides or were bound to organic matter. The bacterial community composition analysis by Terminal Restriction Fragment Length Polymorphism (T-RFLP) and cloning and sequencing of 16S rRNA genes combined with a detailed statistical analysis revealed a high correlation between the bacterial distribution and the geochemical variables. Acidophilic autotrophic oxidizing bacteria were dominating the oxidizing tailings, whereas neutrophilic and heterotrophic reducing bacteria were driving the biogeochemical processes in the remediated tailings below the wetland. At the subsurface of the remediated tailings, an iron cycling was highlighted with oxidation and reduction processes due to micro-aerophilic niches provided by the plant rhizosphere in this overall reducing environment. The in situ bioremediation experiment showed that the main parameter to take into account for the effectiveness was the water table and chemistry which controls the system. The constructed remediation cells were more efficient and rapid in metal removal when saturation conditions were available. This study showed that the bioremediation by wetland implementation could be an effective and rapid treatment for some sulfidic mine tailings deposits. However, the water saturation of the tailings has to be managed on a long-term basis in order to guarantee stability. Résumé : L'activité minière produit d'énormes quantités de déchets géologiques connus sous le nom de « tailings » composées de particules de taille fine à moyenne. Ces déchets contiennent souvent des sulfures dont l'oxydation conduit à la formation d'effluents acides contaminés en métaux, d'où la nécessité d'effectuer une remédiation des sites de stockage concernés. Le but de ce travail est dans un premier temps d'étudier l'effet de la bio-remédiation d'un dépôt de tailings oxydés sur l'hydrogéologie du système et les cycles biogéochimiques des éléments et en second lieu, d'évaluer l'évolution du processus de remédiation dans le temps. Le site étudié dans ce travail est situé dans la Bahía de Ite, au sud du Pérou, au bord de l'Océan Pacifique. Les déchets miniers en question sont déposés dans un environnement marin. De 1940 à 1996, les déchets de deux mines de porphyre cuprifère - Cuajone et Toquepala - ont été acheminés sur le site via la rivière Locumba. En 1997, une première remédiation a été initiée avec la construction d'une zone humide sur les tailings. Depuis, environ 90% de la surface du dépôt (16 km2) a été traité, les parties restantes étant la zone centrale du delta du Locumba et certaines zones proches de la plage. Malgré la proximité de l'océan, les études isotopiques menées dans le cadre de ce travail ont montré que les tailings étaient saturés en eau douce. Cette saturation est due à la pression hydraulique résultant de la mise en place des zones humides. Un écoulement d'eau souterrain sous-marin a été à détecté à l'interface entre les résidus et l'ancien fond marin. En raison de la géologie locale, il constitue une source d'entrée de SO4 2-, Cl-, Na+, FeZ+, et Mn2+ dans le système. L'analyse de la géochimie aquatique, la Diffraction aux Rayons X (XRD) et l'extraction séquentielle ont montré que l'oxydation du fer et .des sulfures est le principal processus se produisant dans les déchets non remédiés. Ceci a entraîné le développement d'une zone d'oxydation à pH bas induisant une forte accumulation des sels efflorescents, conséquence de la migration capillaire des métaux lourds (Fe, Cu, Zn, Mn, Cd, Co et Ni) de la solution vers la surface dans ce climat aride. Cette étude a montré également que la construction de la zone humide a eu comme résultats une précipitation des métaux dans des phases minérales en raison du pH neutre et des conditions réductrices (100-150mV). Les métaux lourds ont précipité sous la forme d'hydroxydes et de sulfures ou sont adsorbés à la matière organique. L'analyse de la composition de la communauté bactérienne à l'aide la technique T-RFLP (Terminal Restriction Fragment Length Polymorphism) et par le clonage/séquençage des gènes de l'ARNr 16S a été combinée à une statistique détaillée. Cette dernière a révélé une forte corrélation entre la distribution de bactéries spécifiques et la géochimie : Les bactéries autotrophes acidophiles dominent dans les déchets oxydés non remédiés, tandis que des bactéries hétérotrophes neutrophiles ont mené les processus microbiens dans les déchets remédiés sous la zone humide. Sous la surface de la zone humide, nos analyses ont également mis en évidence un cycle du fer par des processus d'oxydoréduction rendus possibles par la présence de niches micro-aérées par la rhizosphère dans cet environnement réducteur. L'expérience de bio-remédiation in situ a montré que les paramètres clés qui contrôlent l'efficacité du traitement sont le niveau de la nappe aquifère et la chimie de l'eau. Les cellules de remédiation se sont montrées plus efficaces et plus rapides lorsque le système a pu être saturé en eau. Finalement, cette étude a montré que la bio-remédiation de déchets miniers par la construction de zones humides est un moyen de traitement efficace, rapide et peu coûteux. Cependant, la saturation en eau du système doit être gérée sur le long terme afin de garantir la stabilité de l'ensemble du système.

Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil.

Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databases. The specificity of primers was tested on a variety of frc-containing and frc-lacking bacteria. The frc-primers were then used to develop PCR-DGGE and real-time SybrGreen PCR assays in soils containing various amounts of oxalate. Some PCR products from pure cultures and from soil samples were cloned and sequenced. Data were used to generate a phylogenetic tree showing that environmental PCR products belonged to the target physiological group. The extent of diversity visualised on DGGE pattern was higher for soil samples containing carbonate resulting from oxalate catabolism. Moreover, the amount of frc gene copies in the investigated soils was detected in the range of 1.64x10(7) to 1.75x10(8)/g of dry soil under oxalogenic tree (representing 0.5 to 1.2% of total 16S rRNA gene copies), whereas the number of frc gene copies in the reference soil was 6.4x10(6) (or 0.2% of 16S rRNA gene copies). This indicates that oxalotrophic bacteria are numerous and widespread in soils and that a relationship exists between the presence of the oxalogenic trees Milicia excelsa and Afzelia africana and the relative abundance of oxalotrophic guilds in the total bacterial communities. This is obviously related to the accomplishment of the oxalate-carbonate pathway, which explains the alkalinization and calcium carbonate accumulation occurring below these trees in an otherwise acidic soil. The molecular tools developed in this study will allow in-depth understanding of the functional implication of these bacteria on carbonate accumulation as a way of atmospheric CO(2) sequestration.

Development of a new chlamydiales-specific real-time PCR and its application to respiratory clinical samples.

Originally composed of the single family Chlamydiaceae, the Chlamydiales order has extended considerably over the last several decades. Chlamydia-related bacteria were added and classified into six different families and family-level lineages: the Criblamydiaceae, Parachlamydiaceae, Piscichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, and Waddliaceae. While several members of the Chlamydiaceae family are known pathogens, recent studies showed diverse associations of Chlamydia-related bacteria with human and animal infections. Some of these latter bacteria might be of medical importance since, given their ability to replicate in free-living amoebae, they may also replicate efficiently in other phagocytic cells, including cells of the innate immune system. Thus, a new Chlamydiales-specific real-time PCR targeting the conserved 16S rRNA gene was developed. This new molecular tool can detect at least five DNA copies and show very high specificity without cross-amplification from other bacterial clade DNA. The new PCR was validated with 128 clinical samples positive or negative for Chlamydia trachomatis or C. pneumoniae. Of 65 positive samples, 61 (93.8%) were found to be positive with the new PCR. The four discordant samples, retested with the original test, were determined to be negative or below detection limits. Then, the new PCR was applied to 422 nasopharyngeal swabs taken from children with or without pneumonia; a total of 48 (11.4%) samples were determined to be positive, and 45 of these were successfully sequenced. The majority of the sequences corresponded to Chlamydia-related bacteria and especially to members of the Parachlamydiaceae family.

Bloodstream and endovascular infections due to Abiotrophia defectiva and Granulicatella species.

BACKGROUND: Abiotrophia and Granulicatella species, previously referred to as nutritionally variant streptococci (NVS), are significant causative agents of endocarditis and bacteraemia. In this study, we reviewed the clinical manifestations of infections due to A. defectiva and Granulicatella species that occurred at our institution between 1998 and 2004. METHODS: The analysis included all strains of NVS that were isolated from blood cultures or vascular graft specimens. All strains were identified by 16S rRNA sequence analysis. Patients' medical charts were reviewed for each case of infection. RESULTS: Eleven strains of NVS were isolated during the 6-year period. Identification of the strains by 16S rRNA showed 2 genogroups: Abiotrophia defectiva (3) and Granulicatella adiacens (6) or "para-adiacens" (2). The three A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas 7 of 8 Granulicatella spp. strains were isolated from immunosuppressed patients, mainly febrile neutropenic patients. We report the first case of "G. para-adiacens" bacteraemia in the setting of febrile neutropenia. CONCLUSION: We propose that Granulicatella spp. be considered as a possible agent of bacteraemia in neutropenic patients.

The dermatophyte species Arthroderma benhamiae: intraspecies variability and mating behaviour.

Arthroderma benhamiae is a zoophilic dermatophyte belonging to the Trichophyton mentagrophytes species complex. Here, a population of A. benhamiae wild strains from the same geographical area (Switzerland) was studied by comparing their morphology, assessing their molecular variability using internal transcribed spacer (ITS) and 28S rRNA gene sequencing, and evaluating their interfertility. Sequencing of the ITS region and of part of the 28S rRNA gene revealed the existence of two infraspecific groups with markedly different colony phenotypes: white (group I) and yellow (group II), respectively. For all strains, the results of mating type identification by PCR, using HMG (high-mobility group) and α-box genes in the mating type locus as targets, were in total accordance with the results of mating type identification by strain confrontation experiments. White-phenotype strains were of mating type + (mt+) or mating type - (mt-), whilst yellow-phenotype strains were all mt-. White and yellow strains were found to produce fertile cleistothecia after mating with A. benhamiae reference tester strains, which belonged to a third group intermediate between groups I and II. However, no interfertility was observed between yellow strains and white strains of mt+. A significant result was that white strains of mt- were able to mate and produce fertile cleistothecia with the white A. benhamiae strain CBS 112371 (mt+), the genome of which has recently been sequenced and annotated. This finding should offer new tools for investigating the biology and genetics of dermatophytes using wild-type strains.

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

BACKGROUND: The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. RESULTS: We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. CONCLUSIONS: In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.

Estado reproductivo de la anchoveta peruana engraulis ringens durante los veranos 1996 y 1997

Mediante el análisis histológico de gónadas se muestra la variabilidad del estado reproductivo de la anchoveta peruana en distintas latitudes. En el año 1996 las áreas en las que se observó mayor actividad reproductiva estuvieron entre los grados 4°-14°S y 16° -18°S. El área 14°-16° se caracterizó por el predominio del estado de reposo sexual. En el año 1997 las áreas en las que hubo mayor actividad reproductiva fueron 4°-6°S y 8°-12°S; el estado de reposo sexual fue el predominante. Se observó un mayor porcentaje de hembras en desove en 1996 que en 1997, probablemente debido a que el verano de 1996 fue frío. Las hembras mayores de 14 cm fueron reproductivamente más activas que las de 12 a 14 cm.

Los Tintinidos (Protozoa: Ciliata) del mar peruano y sus relaciones con el fenómeno El Niño

Se ha estudiado la composición, abundancia y distribución de las especies de Tintinida (Protozoa: Ciliata) en relación con las condiciones ambientales, entre 1982 y 1985. Se han identificado 54 especies pertenecientes a 30 géneros y 12 familias. Se utilizaron 901 muestras colectadas con red estándar de fitoplancton en 15 cruceros realizados por el Instituto del Mar del Perú, entre la primavera de 1982 y el verano de 1985, a lo largo del litoral peruano, entre Puerto Pizarro (04' S) e llo (18°S). Además se estudiaron 216 muestras de agua obtenidas con botellas Niskin en 7 estaciones distribuidas a lo largo del perfil Callao (12° S) y a profundidades de 0, 10, 25 y 50 m. Las mayores densidades se presentaron en el verano de 1985, por las especies: Eutintinnus tubulosus, Hellicostomella longa y H. subulata, época considerada como normal de acuerdo a las condiciones oceanográficas. La composición por especies cambia con la distancia de la costa. Dentro de las 30 mn predominaron H. longa y H. subulata; y en la región oceánica: Eutintinnus similis, Oadayiella ganymedes y X ystonella treforti, especies de aguas subtropicales superficia­les. En la distribución vertical, las mayores concentraciones se encontraron entre los O y 25 m de profundidad, muy cerca de la costa.

Relationships of Scincid Lizards (Mabuya spp; Reptilia: Scincidae) from the Cape Verde Islands Based on Mitochondrial and Nuclear DNA Sequences

Partial DNA sequences from two mitochondrial (mt) and one nuclear gene (cytochrome b, 12S rRNA, and C-mos) were used to estimate the phylogenetic relationships among the six extant species of skinks endemic to the Cape Verde Archipelago. The species form a monophyletic unit, indicating a single colonization of the islands, probably from West Africa. Mabuya vaillanti and M. delalandii are sister taxa, as indicated by morphological characters. Mabuya fogoensis and M. stangeri are closely related, but the former is probably paraphyletic. Mabuya spinalis and M. salensis are also probably paraphyletic. Within species, samples from separate islands always form monophyletic groups. Some colonization events can be hypothesized, which are in line with the age of the islands. C-mos variation is concordant with the topology derived from mtDNA.

Estado reproductivo, fecundidad parcial y frecuencia de desove de la anchoveta peruana a inicios de otoño 1998. Crucero BIC Humboldt 9803-05 de Tumbes a Tacna

Durante la ejecución de este crucero, efectuado entre el 20 de marzo y el 7 de mayo de 1998, se estudió el estado reproductivo de la anchoveta, a través del análisis histológico, por grados latitudinales, registrándose anchoveta desovante en el área comprendida entre 8°S y 13°S. Desde el 14°S al 18°S la especie se encontró en un periodo de reposo reproductivo con valores de ovarios desovantes (con folículos post-ovulatorios y ovocitos hidratados) comparativamente menores a los registrados en la zona norte-centro. Los resultados de la muestra fueron ponderados a las capturas efectuadas por grados latitudinales. Los individuos jóvenes (11,5 a 14,0 cm) resultan ser la fracción de adultos más importante de la población, sobre todo en la zona sur. La fecundidad parcial estimada fue de 6.668 ovocitos por bache de desove, con una desviación estándar de 212, y el peso promedio de los ovarios en estado de hidratación fue de 1,25 g, lo cual resulta ser extremadamente bajo en comparación a lo observado a lo observado en años anteriores.

Estado reproductivo de la anchoveta peruana, Engraulis ringens; la sardina, Sardinops sagax sagax y la caballa, Scomber japonicus peruanus durante el crucero BIC Humboldt 9808-09

Estudio del estado reproductivo de la anchoveta, Engraulis ringens; la sardina Sardinops sagax sagax y la caballa Scomber japonicus peruanus, a través del análisis histológico, por grados latitudinales. Los resultados de la muestra fueron ponderados a las capturas por grados latitudinales. La anchoveta, distribuida básicamente entre los grados 14°S y 18°S y la sardina, entre los grados 9°S y 12°S, se la encontró desovando intensamente, mientras que a la caballa se la encontró, fundamentalmente, en una etapa de maduración. La fecundidad de la anchoveta fue estimada en 14.137 ovocitos por bache de desove por hembra.

Informe preliminar del crucero Unanue 7108

Presenta información recogida en el crucero oceanográfico 7108 realizado a bordo del B.A.P. Unanue, el cual tuvo como propósito realizar observaciones sobre los aspectos físicos, químico y de la fertidilidad de las aguas costeras, a fin de conocer las condiciones actuales del ambiente marino en el período invernal de 1971. La duración fue de 16 días (del 16 de agosto al 02 de setiembre de 1971), con paradas intermedias en el Callao, Los Órganos y Paita y con un recorrido total de 3012 millas náuticas que cubrió el área que comprende entre las latitudes 4°S - 18°S hasta una distancia máxima de 150 millas de la costa peruana.

Condición reproductiva de la anchoveta peruana y la vinciguerria en la primavera 2002

El estudio se ejecutó entre los 4 y 18°S, durante el crucero de Evaluación de Recursos Pelágicos 0209-11. Se realizaron 82 lances positivos para anchoveta y 36 para vinciguerria. La anchoveta, Engraulis ringens, tuvo un rango de longitud total (LT) de 10,5 a 17,0 cm. La fracción desovante (FD) >20,0% en la zona sur (15 a 17°S), con los valores más altos dentro de las 20 mn.

Climatología de la salinidad superficial del mar frente a la costa peruana 1960-2008

Análisis de la información recopilada por el Instituto del Mar del Perú (IMARPE), durante el período 1960 - 2008, en el área del Pacífico sudeste comprendida entre los 3 y 18°S y desde la costa hasta los 86°W, con datos filtrados y procesados. Se obtuvo promedios por cuadrados Marsden de un grado; se elaboraron tablas y mapas patrón mensuales, estacionales y anuales, una serie de tiempo de medias mensuales para la banda costera dentro de las 60 mn, referida a tres periodos: a) 1960-1975, b) 1976-1992 y c) 1993-2008, además de una serie de promedios mensuales por cuadrados Marsden de un grado de la salinidad superficial del mar (SSM), para las zonas norte (Tumbes y Paita), central (Chimbote, Callao y Pisco), y sur (San Juan e Ilo). La SSM frente al Perú presentó promedios mensuales entre 29,47 y 35,94 ups, que varían de acuerdo a la estación del año, situación geográfica, topografía, factores meteorológicos, descargas de los ríos, etc. Los promedios mensuales patrón (multianuales) tuvieron rango de 32,92 a 35,62 ups; en verano (enero, febrero y marzo) 33,30 a 35,62 ups y en invierno (julio, agosto y setiembre) 33,96 a 35,44 ups; se registraron valores de transición en otoño (abril, mayo, junio) y primavera (octubre, noviembre y diciembre). La salinidad <34,00 ups corresponde a masas de agua de la región tropical, principalmente del golfo de Panamá (alta precipitación), en la zona costera al norte de 4°30'S. Aguas con salinidades de 34,00 - 34,80 ups se extienden por el lado costero hasta 6°S en verano, y se repliegan en invierno. Alta salinidad superficial corresponde a las aguas de la región subtropical (alta evaporación), que se presentan principalmente al sur de los 6°S con valores de 35,1 hasta 35,60 ups. Estas aguas tienen mayor aproximación a la costa durante el verano austral. En toda el área de estudio, el rango de la fluctuación anual de los promedios patrón de la SSM fue de 32,92 a 35,62 ups (± 2,70 ups en el año); con variación mensual de -0,1 a 0,1 ups al sur de 6°S; y de -0,3 a 0,3 ups al norte de 6°S, la mayor variación se da en la zona norte (Tumbes y Paita). La serie de tiempo mensual (1960 - 2008) en la franja costera (0 - 60 mn), entre los 3°S y 18°S, mostró importantes fluctuaciones decadales, destacando salinidad relativamente baja en los años 60 y después de El Niño (EN) 1997 - 98 hasta la actualidad.

Estudios oceanográficos durante el crucero pelágico 0302-04

El crucero de evaluación hidroacústica de recursos pelágicos 0302-04 se realizó a bordo de dos barcos: el BIC Olaya (26 febrero al 6 abril, Salaverry – Ilo) y el BIC SNP2, del 4 al 29 de marzo (Puerto Pizarro – Salaverry), para estudiar la relación entre los recursos pelágicos y las condiciones ambientales. En términos generales las condiciones térmicas fueron: (a) temperaturas frías de Paita a Huacho; (b) temperaturas normales de Puerto Pizarro a Paita y de Huacho a Mollendo; (c) temperaturas cálidas al sur de Mollendo. Este comportamiento térmico estuvo asociado a la presencia de aguas ecuatoriales superficiales (AES) al norte de Punta Gobernador (5°30’S), aguas costeras frías (ACF) con una amplia distribución (80 mn al norte de Atico, 16°S), aguas subtropicales superficiales (ASS) ubicadas por fuera de las 60 mn con fuertes proyecciones hacia la costa, frente a Chimbote (25 mn, 9°S), Pisco (10 mn, 14°S) y Morro Sama (20 mn, 18°S); (d) observando la topografía de la isoterma de 15 °C, se deduce que la zona de convergencia entre los flujos del norte y sur se localizó entre Pimentel (7°S) y Salaverry (8°30’S)