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.