# Presentation: 482183

## Applied Environmental Microbiology Core

**Applied Environmental Microbiology Core**

- Terry C. Hazen (LBNL, Core Team Leader)
- Matthew Fields (ORNL)
- Hoi-Ying Holman (LBNL)
- Martin Keller (Diversa Inc.)
- David Stahl (University of Washington)
- Dorothea Thompson (ORNL)
- Judy Wall (University of Missouri)
- Jizhong Zhou (ORNL)

- (Consultant)

## AEMC Mission

- AEMC is the source of environmental data and samples that determine the stressors that will be studied, provides the environments for growing the organisms to be tested, simulates stressed environments, and verifies the conceptual models to determine how these stress regulatory pathways control the biogeochemistry of contaminated sites

## Main Goals

- Develop criteria for monitoring the integrity (health) and altering the trajectory of an environmental biological system (process control).
- More complete understanding of the diversity and environmental context of stress response.

## Specific Aims

- 1. Survey and map DOE sites contaminated by metals and radionuclides using chemical and molecular/ microbiological parameters to determine major microbial populations and potential stressors for *Desulfovibrio vulgaris*, *Geobacter metallireducens*, and *Shewanella oneidensis* MR1.
- 2. Determine the rank priority of these stressors in terms of their ability to affect metal/radionuclide bioreduction by either direct or indirect processes, and to establish the normal active range of the stressor in metal/radionuclide contaminated environments.
- 3. Determine the incidence and activity of the three target bacteria, and closely related relatives, in the test metal/radionuclide contaminated environments and collect isolates for analysis by the Functional Genomics Core for comparison using 16S RNA profiling.

## Specific Aims, cont.

- 4. Create defensible environmental simulators that can replicate key features of field site chemical and biological structure to mimic stress conditions for single populations and later for microbial communities (chemostats to soil columns from 10 μm to 1 m size systems).
- 5. Provide large quantities of cells in various stress states for the Functional Genomics Core’s physiological monitoring facility, and molecular interaction studies.
- 6. Provide environmental simulators for testing stressor effects on mutants, large insert clones, expression analysis, etc., for elucidating critical parts of the stress regulation pathway.
- 7. Develop testable conceptual models of stress regulatory pathways based on results of the Computational Core that could predict natural attenuation and suggest biostimulatory strategies for immobilization of metals and radionuclides at DOE contaminated sites.

## Specific Aims, cont.

- 8. Test conceptual models of stress regulatory pathways and effects on contaminate site biogeochemistry using competent soil columns with different levels of complexity over the active range of the stressors
- 9. Validate conceptual models using field tests at contaminated sites that utilize specific functional gene arrays developed from the stress regulatory pathways.
- 10. Alter field conditions or test along gradients to verify stress regulatory model efficiency for predicting natural attenuation or suggesting biostimulatory strategies for immobilization of metals and radionuclides.

## Slide 7

## Research Elements

- chemical and molecular/microbiological mapping of field sites to identify major microbial populations and potential system stressors
- development of reactor systems that replicate key features of field site chemical and biological structure
- testing of different stressors using appropriate controlled reactor systems in combination with a general measure of physiological stress (rRNA synthesis)
- development of simplified reactor systems containing the target organism, mutant variants, and native populations that are closely related to the target organism
- field validation of conceptual models using chemical and molecular/microbiological probes and assays based on the stress regulatory pathways developed during the overall project.

## Experimental Core Facilities

- Environmental Molecular Microbiology Facility (ORNL, LBNL, U. Wash., Diversa Inc.)
- Environmental Simulation and Culture Facility (LBNL, U. Wash., Diversa Inc.)

## AEMC Milestones

_**Oak Ridge National Laboratory **_*(Note: Expression profiling will be done in part with environmental simulators, these milestones are shared with the Functional Genomics Core)*
**Year 1 (July - September, 2002):** Begin survey and mapping of ORNL sites for stressors and target organisms.  Obtain isolates from site for culturing.  Initial expression profiling experiments with *S. oneidensis*
**Year 2 (October, 2002 – September 2003):** Continue survey and mapping of ORNL site.  Expression profiling experiments with *S. oneidensis*; Initial expression profiling experiments with *D.vulgaris* and *G. metallireducens*
**Year 3 (October, 2003 – September 2004):**  Expression profiling experiments with *S. oneidensis*; Expression profiling experiments with regulatory mutants of *S. oneidensis*; Expression profiling experiments with *D.vulgaris* and *G. metallireducens*

## AEMC Milestones, cont.

_**University of Washington**_
**Year 1 (July - September, 2002): **Establish high-speed sorting capability
**Year 2 (October, 2002 – September 2003):** Characterize microbiological and chemical structure of field sites;** **Recovery of specific population by high-speed sorting
**Year 3 (October, 2003 – September 2004):** Development of DNA microarray probe set; Bioreactor set-up and operation

## AEMC Milestones, cont.

_**University of Missouri at Columbia**_* Note: Environmental simulator, chemostat and field testing will also involve Dr. Wall, so her milestones are also in the Functional Genomics Core where the bulk of her tasks are associated.*
**Year 1 (July - September, 2002):**  Develop conjugation system for *D. vulgaris*.  Begin development of DNA microarrays for *D. vulgaris*.
**Year 2 (October, 2002 – September 2003):**  Disrupt the restriction-modification system in *D. vulgaris*.  Continued development of DNA microarrays for *D. vulgaris*.
**Year 3 (October, 2003 – September 2004):**  Develop a system that will allow transformation of *D. vulgaris *with dsDNA and double recombination.  DNA microarray analysis of wild-type *D. vulgaris* subjected to various stresses.

## AEMC Milestones, cont.

_**Lawrence Berkeley National Laboratory**_
**Year 1 (July - September, 2002): **Begin survey and mapping of ORNL sites for stressors and target organisms.  Obtain isolates from site for culturing.  Set up environmental simulator facility with PLFA and scanning laser confocal microscopy.  Do preliminary studies to determine protocols for target bacteria for SFTIR spectra and unique signatures.  Begin development of environmental chambers.  Provide field data to Computational Core.
**Year 2 (October, 2002 – September 2003):** Continue survey and mapping of ORNL site and begin survey and mapping at LLNL site. Obtain isolates from LLNL site for culturing.  Establish rank priority of stressors at the ORNL site.  Continue development of environmental chambers.  Analyze SFTIR and PLFA signatures for stressed and unstressed bacteria.  Initiate stressor studies in chemostats and soil columns for expression analysis, PLFA, SFTIR, etc.  Begin cell production for Functional Genomics Core. Provide field and lab data to Computational Core.
**Year 3 (October, 2003 – September 2004):** Continue survey and mapping of LLNL for stressors and target organisms. Continue development of environmental chambers.  Continue stressor studies in chemostats and soil columns for expression analysis, PLFA, SFTIR, etc.  Analyze SFTIR and PLFA signatures for stressed and unstressed bacteria.  Continue cell production for Functional Genomics Core. Provide field and lab data to Computational Core.

## AEMC Milestones, cont.

**Diversa, Inc.**
- Year 1 (July - September, 2002):  Begin work on large insert cloning.
- Year 2 (October, 2002 – September 2003):  Begin protein profiling of wild-type *S. oneidensis*.  Begin work on BioPanning.
- Year 3 (October, 2003 – September 2004):  Complete protein profiling of of wild-type *S. oneidensis*.  Continued work on BioPanning.

## Highlights

## Highlights

## Highlights

- University of Washington has established syntrophic growth in reactors with *D. vulgaris* and *Methanococcus maripaludis*, a hydrogenotrophic methanogen so that *D. vulgaris* can be grown in the absence of sulfate.

## Highlights

## Highlights

- UW, LBNL, and UM have purchased and set up specialized fermentors for growing *D. vulgaris*.  Media are being tested and protocols developed for growth curves and QA/QC.

## Highlights

**Diversa** has extracted small insert DNA from FRC samples and amplified them with the Phi29 polymerase.  The amplified DNA was examined in pulsed field gel and shown to have sized up to ~50 kb.

## Highlights

- As part of the development of the design of the chemostats, lipid signatures of *D. vulgaris* were also compared during different growth conditions.  Cells were grown in a container on a stir plate and stirred at a moderate speed.  Glass slides were suspended to provide a vertical surface for cell growth.  After 7 days, the culture was divided into three fractions:  planktonic, attached, and flocculated.  Shifts were seen in the type of lipids depending on the growth condition, especially in the fraction of unsaturated lipids and branched (iso) lipids.

## Highlights

**Diversa** has shown via diversity indexing of the amplified large fragment gDNA from sample FB609, that they can successfully amplify undetectable amounts of gDNA of multiple organisms from FRC samples.

## Highlights

- Lipid signatures of oxygen stressed cells (1) inoculation of aerated No-Fe Baars medium followed by growth in the anaerobic box, (2) show that new lipids were formed in the oxygenated-medium growth.

## Oxygen Stress Biomass

- Parameters for Biomass production
- 10% inoculum of primary culture
- 30°C incubation
- Shake at 100 rpm
- BAARs modified medium (-Fe)
- Sparge oxygen as air at a rate of 57ml/min for a final concentration of 8 ppm oxygen in un-inoculated medium
- H2S purge issue

## Oxygen Stress Biomass

_**BAARs modified medium ATCC# 1249**_
- MgSO4  2g/L
- Sodium citrate 5g/L
- CaSO4 1g/L
- NH4Cl 1g/L
- K2HPO4 0.5g/L
- Sodium lactate 3.5g/L
- Yeast extract 1g/L
- pH 7.5

_**Lactate Sulfate Medium**_
- Na2SO4   7g/L
- MgSO4   2g/L
- NH4Cl  1.1g/L
- CaCl  .9g/L
- K2HPO4  .4g/L
- HEPES  6g/L
- Trace Minerals 12.5ml
- Thauers Vitamins 10ml
- Sodium Lactate  6.7g/L
- Yeast Extract  1g/L
- pH 7.2

**FeS**

**Floc**

## Oxygen Stress Biomass

## Oxygen Stress Biomass

## Oxygen Stress Biomass

- QA/QC
- Colony morphology - Purity streak
- Cell morphology - Microscopy
- Phenotypic characterization - PLFA
- DNA characterization - RFLP

## Oxygen Stress Biomass

- Within 1 month Biomass will be produced for shipping to ORNL, Diversa, UCB, Sandia?     0 time and 5 hours?!!!!
- Second experiment in 2 months?
- Times?
- Growth curves and comparative oxygen tests on Geobacter and Shewanella?

## Other Tasks

- Complete Data Summary of FRC
- Stress Review on metal reducers?
- FairManTech Production Protocols
- Attached Production Protocols
- LLNL Field samples?

## Tree of Life