b i o l 4 3 6 5 ecología de los microorganismosacademic.uprm.edu/~amassol/biol4365/massol_03.pdfb i...
TRANSCRIPT
B I O L 4 3 6 5Ecología de los Microorganismos
Long-Term Chromium Bio-Immobilization at the Hanford 100H Site: Geochemical andMicrobiological Response to Slow Release Electron Donor
Terry C. HazenLawrence Berkeley National Laboratory
The focus of these studies is to understand the coupled hydraulic, geochemical, andmicrobial conditions necessary to maximize Cr(VI) bioreduction and minimize Cr(III)reoxidation in groundwater. Here we present the application of slow release electron donorduring a field-scale treatability study over an 18-month period. Samples were taken atintervals pre- and post-injection of a
13C-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 sulfateconcentrations in groundwater were monitored. Stable isotope enrichment in dissolvedinorganic pools was followed and a fluorescent antibody used to visualize the presence ofa sulfate reducer. Following HRC injection (27 days) reducing conditions (-130 mV) hadestablished with a corresponding disappearance of DO and nitrate. Cr(VI) concentrationsdeclined steadily over 6 weeks. Analysis of delta
13C ratios in dissolved inorganic carbon
confirmed microbial metabolism of the labeled HRC. Hydrogen sulfide production was firstobserved after about 20 days post-injection and this corresponded with the enrichment of aDesulfovibrio species identified using fluorescent antibodies. Bacterial densities haveremained 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 pumpingwas applied to downgradient wells DO declined significantly in the upper parts of theaquifer. A number of sulfate reducers have been isolated from the deeper parts of theaquifer. HRC was still present more than a year after initial injection and was maintainingthe overall Cr (VI) levels at non-detectable, suggesting that this may be a long-termbioimmobilization of Cr (VI). Geochemical analysis of groundwater coupled with stableisotope and microbial monitoring allowed for accurate tracking of microbial processesduring this field treatability study.
EXAMEN 1: Miércoles 20 de septiembreSEMINARIO: Jueves 21 de septiembre 10:40 am/B-280
¡Acceso a documentos de la clase!
• http://academic.uprm.edu/~amassol/biol4365/Prontuario.pdf
• http://academic.uprm.edu/~amassol/biol4365/Propuesta.pdf
• http://academic.uprm.edu/~amassol/biol4365/Forney.pdf
• http://academic.uprm.edu/~amassol/biol4365/Massol_01.pdf
• http://academic.uprm.edu/~amassol/biol4365/Massol_02.pdf
Nutrientes
T (oC)
pH
bajo
alto
alto
alto
DNA
RNA
Proteínas
DogmaCENTRAL
Población 1
Población I1
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.
Nutrientes
T (oC)
pH
bajo
alto
alto
alto
DNA
RNA
Proteínas
DogmaCENTRAL
Población 1
Población I1
Sp. IISp. I
RECURSOS
Sp. II Sp. IISp. I Sp. I
InteraccionesMAYOR
DIVERSIDADSp. I Sp. I Sp. I Sp. I
ej. PATOGENOS/DEPREDADORES
Effect of limiting substrate on growth
Monod Growth Equation
µ = µmax
Survival under nutrient-limited conditions
S
Ks + S
µ = Growth rateµmax = Maximal growth rateS = Substrate concentrationKs = Substrate concentration where growth rate is one-half maximum rate
Growth of bacterial populations in the environment is typically limited by the availability of 1 or more nutrients
µ and KS Relationships
B
For any D (= µ), the organism with the lowest KS will out-compete an organism with a higher KS.
PANEL A. At all D, KSA < KSB and A is more competitive, i.e., µA > µb.
PANEL B. At low [S] KSA < KSB and µA > µb ∴ organism A is more competitive; while at other [S] KSB > KSA and µb > µA ∴ organism B is more competitive.
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.
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
Zooplancton: Copépodos
México
Peru
Animal plankton; animals (mostly microscopic) which drift freely in the water column.
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.
• Alto contenido de Fe• Microorganismos
Approx. 7 días
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
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.
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...
¿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)
95-285
benthicdenitrification
water columndenitrification
nitrogenfixation
60-250
110-200
benthic nitrogen fixation
burial
atmosphericdeposition
10-25
15
30-50
all units in Tg N y-1
25-75river input
Codispoti 1995; Gruber and Sarmiento 1997; Middelburg et al. 1996
Is the ocean losing NO3?
N2O
Nitrate Reductase
narGH
Nitrite ReductasenirK or nirS
Nitric Oxide Reductase
norCB
Nitrous Oxide Reductase
nosZ
NO3- NO2
- NO N2
nirSnirK
norB (A)norB (B)
nosZ
NO3 NO2 NO N2O N2
Denitrification Pathway
Lyzozyme
Protease K
In situ Hybridization
(FISH)
In situ RT-PCR with unlabeled
nucleotides
EnzymaticPermeabilization
Epifluorescence microscopy
Cell Fixation
4% Paraformaldehyde
Cy3 as the Fluorescently Labeled Probe
1. Fijación (química con 4% paraformaldehido)2. Permeabilizar las células3. Transcriptasa al reverso4. PCR5. Hibridización6. Lavado7. Microscopía de fluoresencia
Control NegativoPs. G 179 (nirK)
Fase Exponencial
Fase Estacionaria
Fase Estacionaria
Co-Culture of P. denitrificans with a non-denitrifying rod in stationary phase.