B I O L Ecología de los Microorganismos

Transcripción

1 B I O L Ecología de los Microorganismos Long-Term Chromium Bio-Immobilization at the Hanford 100H Site: Geochemical and Microbiological Response to Slow Release Electron Donor Terry C. Hazen Lawrence Berkeley National Laboratory The focus of these studies is to understand the coupled hydraulic, geochemical, and microbial conditions necessary to maximize Cr(VI) bioreduction and minimize Cr(III) reoxidation in groundwater. Here we present the application of slow release electron donor during a field-scale treatability study over an 18-month period. Samples were taken at intervals pre- and post-injection of a 13 C-labeled slow release polylactate compound (HRC) used to stimulate indigenous microbial populations to immobilize hexavalent chromium. Redox potential, ph, dissolved oxygen (DO), nitrate, chromium (VI), and sulfate concentrations in groundwater were monitored. Stable isotope enrichment in dissolved inorganic pools was followed and a fluorescent antibody used to visualize the presence of a sulfate reducer. Following HRC injection (27 days) reducing conditions (-130 mv) had established with a corresponding disappearance of DO and nitrate. Cr(VI) concentrations declined steadily over 6 weeks. Analysis of delta 13 C ratios in dissolved inorganic carbon confirmed microbial metabolism of the labeled HRC. Hydrogen sulfide production was first observed after about 20 days post-injection and this corresponded with the enrichment of a Desulfovibrio species identified using fluorescent antibodies. Bacterial densities have remained high (>10 7 cells/ml), Fe(II), and Cr(VI) concentrations in the monitoring and pumping wells have remained below up-gradient concentrations for the first 12 months. Lower parts of the aquifer have maintained sulfate reducing conditions and when pumping was applied to downgradient wells DO declined significantly in the upper parts of the aquifer. A number of sulfate reducers have been isolated from the deeper parts of the aquifer. HRC was still present more than a year after initial injection and was maintaining the overall Cr (VI) levels at non-detectable, suggesting that this may be a long-term bioimmobilization of Cr (VI). Geochemical analysis of groundwater coupled with stable isotope and microbial monitoring allowed for accurate tracking of microbial processes during this field treatability study. EXAMEN 1: Miércoles 20 de septiembre SEMINARIO: Jueves 21 de septiembre 10:40 am/b-280

2 Acceso a documentos de la clase! Prontuario.pdf Propuesta.pdf Forney.pdf Massol_01.pdf Massol_02.pdf

3 Dogma CENTRAL ph alto DNA Población 1 RNA Población I1 bajo alto T ( o C) Proteínas alto Nutrientes

4 Dogma CENTRAL ph alto DNA Población 1 RNA Población I1 bajo T ( o C) alto Proteínas alto Nutrientes COMPETITIVE EXCLUSION PRINCIPLE No two species can occupy precisely the same ecological niche If two species coexist in a stable environment, they do so by niche differentiation. Niche differentiation occurs through adaptive evolution. If there is no niche differentiation, then one species will eliminate or exclude the other.

5 ej. PATOGENOS/DEPREDADORES Sp. I Sp. I Sp. I Sp. I Interacciones MAYOR DIVERSIDAD Sp. I Sp. I Sp. II Sp. II Sp. I Sp. II RECURSOS

6 Effect of limiting substrate on growth

7 Survival under nutrientlimited conditions Growth of bacterial populations in the environment is typically limited by the availability of 1 or more nutrients Monod Growth Equation µ = µ max S K s + S µ = Growth rate µ max = Maximal growth rate S = Substrate concentration K s = Substrate concentration where growth rate is one-half maximum rate

8 µ and K S Relationships B For any D (= µ), the organism with the lowest K S will out-compete an organism with a higher K S. PANEL A. At all D, K SA < K SB and A is more competitive, i.e., µ A > µ b. PANEL B. At low [S] K SA < K SB and µ A > µ b organism A is more competitive; while at other [S] K SB > K SA and µ b > µ A organism B is more competitive.

9 4. Cierto (A) o Falso (B): Las bacterias sólo tienen un cromosoma circular, algunas también tienen plásmidos. Vibrio cholera tiene dos cromosomas, algunas bacterias tienen su cromosoma lineal.

10 5. Cierto (A) o Falso (B): Imágenes de Satélite (Remote Sensing) no son utilizadas para detectar brotes de Vibrio cholera en el planeta. BIOCOMPLEXITY Dr. Rita Colwell

11 Fitoplancton: A plant plankton; a rapid build-up in abundance of phytoplankton, usually in response to nutrient build-up, can result in a `bloom'; microscopic plant life that floats in the open ocean. México Peru Animal plankton; animals (mostly microscopic) which drift freely in the water column. Zooplancton: Copépodos

12 Approx. 7 días Microorganismos Alto contenido de Fe

13 Métodos de cultivo Por qué sólo recuperamos 0.1% o menos de la diversidad microbiana? Platos de Agar (Siglo 19) R. Koch, Walter Hasse & esposa & Juliius Petri

14 Cultivo de Microorganismos Fastidiosos 1. Agentes solidificantes Agar y agentes inhibidores, uso de gellan gum como agente solidificante aumneta el número de microorganismos cultivables por un factor de 10.

15 2. Factores de crecimiento desconocidos (ej. quorum sensing) Necesidad de factores de crecimiento o alguien que remueva ciertos desperdicios (Growth Factors Network). Cultivo con sobrenadante filtro-esterilizado de otros donde este organismo aun no cultivado está creciendo. 3. Sintrofismo (ej. H2 Transfer: sintrofomonas y metanogénicas) Interspecies requirements for growth of microbes in natural communities. Agitación puede ser fatal al dispersar agregados o disipar gradientes necesarios para crecimiento. 4. Concentración de nutrientes (ej. N & P en los océanos en escala de µm: DOC es 1000X menos en la naturaleza que en LB) 5. El tiempo de generación en la naturaleza es en escala de días, semanas, meses o años [no minutos como en cultivos domesticados] Paciencia: Se necesitan más estudiantes graduados y subgraduados...

16 Quien eres y qué haces? Cultivo y caracterización Análisis de DNA (ej. 16S rrna, Phylochips) Microscopía/RAMAN Cuán rápido trabajas? Process rate Enzyme activity RNA (no incubation necessary)

17 nitrogen fixation atmospheric deposition river input water column denitrification benthic denitrification Is the ocean losing NO 3? 15 burial benthic nitrogen fixation Codispoti 1995; Gruber and Sarmiento 1997; Middelburg et al all units in Tg N y -1

18 Denitrification Pathway NO 3 NO 2 NO N 2 O N 2 NO - 3 NO - 2 NO N 2 O N 2 Nitrate Reductase nargh Nitrite Reductase nirk or nirs Nitric Oxide Reductase norcb Nitrous Oxide Reductase nosz nirs nirk norb (A) norb (B) nosz

19 Lyzozyme Enzymatic Permeabilization In situ RT- PCR with unlabeled nucleotides In situ Hybridization (FISH) Cy3 as the Fluorescently Labeled Probe Protease K Cell Fixation 4% Paraformaldehyde Epifluorescence microscopy

20 1. Fijación (química con 4% paraformaldehido) 2. Permeabilizar las células 3. Transcriptasa al reverso 4. PCR 5. Hibridización 6. Lavado 7. Microscopía de fluoresencia

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22 Fase Exponencial Fase Estacionaria Fase Estacionaria Control Negativo Ps. G 179 (nirk)

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25 Co-Culture of P. denitrificans with a non-denitrifying rod in stationary phase.