# Presentation: 905694

## Growth of Desulfovibrio vulgaris in Sparged Systems Dominique Joyner, Sharon Borglin, Rick Huang, and Terry HazenVirtual Institute for Microbial Stress and Survival, Lawrence Berkeley National Laboratory

**Growth of *****Desulfovibrio vulgaris***** in Sparged Systems**** **Dominique Joyner,** **Sharon Borglin, Rick Huang, and Terry HazenVirtual Institute for Microbial Stress and Survival, Lawrence Berkeley National Laboratory

-  At 10% inoculum of log phase primary culture, experimental log phase occurs in the range of  3-13 h with a generation time of 1.5-3.0 h
-  The air sparged cultures showed no significant changes in any of the parameters over the same time periods; however, cells were viable
-  Differences in growth patterns of sparged and closed systems suggest that H2S may inhibit growth in the absence of Fe
-  OD, AODC, and protein assays gave comparable results for all experiments, though protein assays did not show as great a difference between sparged and non-sparged incubation
-  Measurable precipitate accumulates as a by-product of growth in both Fe (+) and Fe (-) medium.  It is not intact biomass as determined by DNA staining

- These data demonstrate the growth characteristics of *Desulfovibrio vulgaris* as defined by the conditions outlined for future oxygen stress experiments.  Anoxic sparging of the system results in an increase in cell growth, whereas oxic sparging limits cell growth as compared to the non-sparged control.
- Generation times for *Desulfovibrio* were calculated from each of the cell density assays performed.  These results correlate well with each other to yield similar cell densities.

- The data suggest survivability of *D. vulgaris* in the presence of oxygen.  The next step will be to demonstrate recovery and growth of an aerated culture as the oxygen concentration recedes.
- In further studies, dissolved oxygen in the medium will be measured with a fiber optic oxygen sensor to determine the concentration of oxygen in the system which inhibits growth of *D. vulgaris.*

- Since most contaminated subsurface environments are episodically exposed to oxygenated water via infiltration of snow melt and rain from the surface, and from recharge areas upgradient where oxygen has not been depleted, oxygen is a common stressor for anaerobes like *D. vulgaris* in the environment.  In addition, metal and radionuclide reduction by bacteria is dependent upon fairly stringent reducing (anaerobic) conditions which can be upset by any exposure to oxygen.  *D. vulgaris* is tolerant of oxygen via a number of metabolic mechanisms, negative aerotaxis, and via production of EPS.  The exact response sequence that *D. vulgaris* expresses and how oxygen affects that response are unknown.

- The overall objective of this first experiment is to document the array of changes that occur in cells exposed to air using the array of techniques being developed by the project.
- The objectives of this experiment are:
-  -Define baseline O2 stress changes in growth pattern that occur over 12h or
-      one generation time as measured by the techniques being developed
-  -Establish *D. vulgaris* baseline parameters for the techniques being tested
-  -Determine logistical constraints for sampling, shipping etc.
-  -Establish QA/QC verification of assay protocols
-  -Develop functional design criteria for future experiments

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

- 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

_**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

_**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

**FeS**

**Floc**

**Rationale**

**Objective**

**Parameters for Oxygen Stress Experiments**

**Validation of Isolate Purity**

**Results**

**Growth Medium**

**Conclusions and Future Work**