# Presentation: 060301

## GTL       Microbial stress response and survival        DOESyntrophic associations of Desulfovibrio spp. and a hydrogenotrophic methanogen: Simple consortia for studying microbial stress response under environmental defensible growth conditions 

**GTL**       ***Microbial stress response and survival***        **DOE**Syntrophic associations of *Desulfovibrio *spp. and a hydrogenotrophic methanogen: Simple consortia for studying microbial stress response under environmental defensible growth conditions

**Abstract**

**Experimental design**

**Introduction**

**Sergey Stolyar, Beto Zuniga, Jonathan A. Miller,** **David Stahl** University of Washington, Seattle WA 98195

**Results**

*UW*

**Perturbations**
**Stressors**
**Different **
**species **
**and mutants**

**Bioreactors,**
**Controlled conditions**

**Data acquisition**

**Gas analysis: GC**

**Metabolites **
**and inorganic ions:**
**IC, HPLC **

**Cells for intracellular **
**metabolite analysis**

**Proteins and mRNA for**
**Proteomics and **
**microarrays**

- Figure 5.

- Figure 4.

- Figure 6.

- Figure 3.

**A**

**B**

-             Prior to establishing co-cultures of  *D. vulgaris* or *Desolfovibrio* sp. PT2  with the methanogen (*M. marapaludis*)* *we developed a common basal medium.  *M. maripaludis* is a marine isolate routinely grown at  22g/L of NaCl and 6.5g/L of MgSO4 (McC medium).  Thus, our initial studies evaluated growth of all organisms at varying salt concentrations, 2-20 g/L NaNO3 or NaCl, and 10-100g/L magnesium sulfate.  Both *Desulfovibrio* species tolerated 100g/L magnesium sulfate, with some reduction in growth rate at 40 g/L.  Growth of both strains was reduced at either 10g/L of NaCl or NaNO3, suggesting that growth rate is influenced primarily by sodium.  PT2 demonstrated somewhat greater salt tolerance than *D. vulgaris* (Fig. 3).  Similar characterization of *M*. *maripaludis* revealed that this putative marine organism had a relatively low sodium requirement .  M.maripaludis grew well in medium amended with as low as 17mM (1g/L) of NaCl and 2mM (0.2 g/L) of Mg SO4. Thus, our initial studies used a modified McC (McCm) medium, adjusted to 2g/L NaCl.

-       Pure cultures and co cultures were grown in 250 ml sealed flasks on McCm supplemented with three substrate formulations: 1)  ‘full’ medium (50 mM lactate, 50 mM acetate, 25 mM sulfate plus hydrogen and carbon dioxide in the head space), 2)  lactate only medium (50 mM), and 3) a sulfate-limited medium (50 mM lactate plus 4 mM sulfate).  The limiting sulfate medium was designed to enable establishment of a greater population of the SRB during initial development of the co-culture.  Cultures were monitored for optical density (OD600), CO2, H2, CH4, and SO4=, and duplicate samples were taken for nucleic acid analyses.  Greatest methane production and modest cell growth was observed using the limiting sulfate medium.  In contrast, very low methane production and cell growth was observed in the 50 mM lactate single cultures (Fig. 4, 5, and 6).

- Figure 7.

-       In preparation for studies in continuous co-culture, the FairMenTec Bioreactor has been installed in our lab and tested with three batch runs of *Desulfovibrio sp.* PT2 in LS medium supplemented with 20 mM of lactate and 28 mM of sulfate at 300C.  This strain demonstrated a very long lag and a tendency aggregate or attach when the system was sparged with N2.   Good growth in suspension was observed in the absence of sparging.  We are now evaluating sparging with higher purity N2 to determine if aggregation/attachment was a response to a contaminating gas (e.g., O2). (Fig 2.)
-         A fixed-bed bioreactor (3mm glass bead support substratum) was designed and fabricated for developing more complex biofilm communities derived from field site inocula.  The main bioreactor has a total volume of approx. 500 ml and a void volume of 225 ml.. The system has recalculating loop with flow rate up to 3L per hour and  an influent feed of 1 L per 24 hours (Fig 1 and 7) and equipped with two probes for pH and RedOx potential control.

_**References**_
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- Traore AS, Gaudin C, Hatchikian CE, Le Gall J, Belaich JP. Energetics of growth of a defined mixed culture of Desulfovibrio vulgaris and Methanosarcina barkeri: maintenance energy coefficient of the sulfate-reducing organism in the absence and presence of its partner. J Bacteriol. 1983 Sep;155(3):1260-4.

-           Although long thought to be relatively restricted in catabolic range, organisms having the capacity to respire sulfate are now recognized to be remarkably versatile.  Processes they mediate include the degradation of aromatics compounds once thought to be refractory to anaerobic degradation, including benzene (Aeckersberg *et al*. 1991; Deweerd *et al*. 1991;  Lovley *et al*. 1995; Londry *et al.* 1999), inorganic fermentation of sulfur compounds (Bak, 1987), the reduction of a variety of metals including radionuclides, and a limited capacity to respire oxygen ( Lovley and Phillips 1992; Cypionka 2000).  Their habitat range includes freshwater, marine and hypersaline aquatic systems, cold oceanic sediments, the deep subsurface, hydrothermal vents, and hotsprings.  Notably, their environmental distribution is not restricted by low sulfate-availability or the presence of oxygen.  For example, some SRB (e.g., *Desulfovibrio* species) have the capacity to growsyntrophically with hydrogenotrophic methanogens in the absence of sulfate.  Representatives of both groups are found together in deep sediments, granitic rocks and deep groundwater (Fry et al., 1997; Stroes-Gascoyne et al., 1997, Motamedi& Pedersen, 1998).
-       A key charge of the Applied and Environmental Core is to develop defensible environmental simulators for studies of stress response under conditions that approximate key biotic and abiotic conditions of the field.  We elected to initially simulate environmental conditions that allow for growth of SRB in the absence of sulfate. This can be achieved by growing the SRB in syntrophic co-culture with a methanogen.  Although the basic physiology of this interaction, based on interspecies hydrogen transfer, has been studied for years (Bryant et al., 1977, McInerney & Bryant, 1981; see for review Shink
- 1996), there is little understanding of genetic regulation, possible modification of electron transport systems, or how interacting partners respond to stress.  For example, we earlier observed that genes encoding sulfite reductase (dsrAB) continue to be expressed in co-culture of Desulfovibrio sp. PT2 and methanogen (Brusseau 1998). Also, several gene clusters coding putative hydrogenases and formate dehydrogenases have been found in genomic sequence of D. vulgaris Hildenborough.
-      The development of a *D. vulgaris* - *M. maripaludis* co-culture will provide a genetically tractable and environmentally relevant model system.  Genome sequences are available for both organisms and both are amenable to genetic manipulation.  Initial studies suggest that D. *vulgaris*  strain PT2 is better adapted to syntrophic growth.  This strain, isolated from low-sulfate reactor system, should provide insight into possible variation in physiology and stress response of closely related *Desulfovibrio* species.

- Figure 1.

- Figure 2.

-          A key objective of the Applied and Environmental Core is to develop defensible environmental simulators for studies of bacterial stress response and survival.  We elected to initially simulate conditions that allow sulfate reducing bacteria (SRB) to grow without sulfate respiration, achieved by syntrophic co-culture with a hydrogenotrophic methanogen.  To evaluate this model, we examined the co-culture of two different *Desulfovibrio* species (*Desulfovibrio vulgaris *Hildenborough* *and *Desulfovibrio sp*. PT2) with a hydrogenotrophic methanogen (*Methanococcus maripaludis*).
-         Since *M. maripaludis* is a marine organism, growth of this methanogen and the desulfovibrio were first evaluated at varying salt concentrations (2-20 g/L NaNO3 or NaCl, and 10-100g/L MgSO4).  Both *Desulfovibrio* species demonstrated a modest reduction in growth at 40 g/L MgSO4 but tolerated concentrations as high as 100g/L.  Growth of both strains was reduced at either 10g/L NaCl or NaNO3, suggesting that growth rate was influenced primarily by sodium.  PT2 demonstrated somewhat greater salt tolerance than *D. vulgaris*.  Based on these observations a modified basal growth medium (McCm) of intermediate salt concentration (2 g/L NaCl, 5.5 MgCl2 g/L) was developed that supported good growth of all three organisms.
-        Stable batch co-cultures of each desulfovibrio paired with *M. maripaludis* were established on 50 mM lactate in the absence of sulfate.  Co-culture growth was verified by methane production and microscopy and has resulted in modest cell growth as measured by spectrophotometry.  No appreciable growth was observed in 50 mM lactate single cultures.
-        Additionally, a set of bioreactors for both continuous co-culture and biofilm studies has been designed and installed in our laboratory.

**   ****Effect of NaCl and MgCl on Growth of *****D. vulgaris***** and *****Desulfovibrio PT2***

**Fixed-bed bioreactor system**