The D. vulgaris
model used here was based on genome annotation (Heidelberg et al, 2004
) and the Microbes online database (http://www.microbesonline.org/
). As the focus of our study is on fundamental reactions of carbon and energy metabolism in the two organisms, the complete model was simplified to include only central metabolism and amino-acid biosynthesis pathways. All other biosynthetic reactions were condensed into single equations for each major biomass component: DNA, RNA, protein, carbohydrate (assumed to be glycogen), phospholipid, peptidoglycan, and lipopolysaccharide. A reaction was then written to combine these components in the proper ratio for biomass synthesis. We assumed that biomass composition of D. vulgaris
is the same as in E. coli
(Pramanik and Keasling, 1997
). Using this composition, 1 g DCW of biomass is equal to 43.4 mmol of carbon atoms. This value was used to calculate biomass yield on a carbon atom basis. The resulting model contained 86 reactions and 75 intracellular metabolites (Appendix A
A model of M. maripaludis
central metabolism was constructed on the basis of putative gene functions from the genome annotation (Hendrickson et al, 2004
), supplemented by current physiological knowledge of the organism. Three pathways were included for nitrogen assimilation: NH4+
uptake from the medium, N2
fixation, and utilization of externally supplied alanine. Carbon uptake was allowed to occur from CO2
via the CO dehydrogenase/acetyl-CoA synthase pathway, or from acetate via acetyl-CoA synthetase. Formate could also be oxidized to CO2
. Although there are gaps in some of the amino-acid biosynthesis pathways (Hendrickson et al, 2004
), the complete pathways were included because this organism is not auxotrophic for any amino acids. As with the D. vulgaris
model, all other biosynthetic pathways were condensed. Because there have been no reports of detailed biomass composition measurements in Archaea, in constructing the model it was assumed that DNA, RNA, phospholipids, and protein combine in the same proportion as in E. coli
(Pramanik and Keasling, 1997
). The methanogen does not have an outer membrane. Instead it possesses an S layer; therefore, peptidoglycan and lipopolysaccharide were not included. Although this biomass composition is a crude estimate, it has been demonstrated that changing macromolecular composition does not have a large effect on central carbon or energy metabolism (Edwards and Palsson, 2000b
). The final M. maripaludis
model contained 82 reactions and 72 intracellular metabolites (Appendix B
For the simulation of syntrophic growth, the two metabolisms were treated as separate compartments within the same model, similar to the way by which different organelles are treated in eukaryotic cell models (Famili et al, 2003
; Forster et al, 2003
). A third ‘compartment' was added to represent the culture medium, through which certain metabolites could be transferred between the two organisms. Transport fluxes were added for metabolites that could be taken up by or secreted from each organism. Finally, exchange fluxes were added to represent accumulation or depletion of metabolites from the bulk medium. These exchange fluxes were either set equal to zero (indicating no net accumulation), set to the experimental measurement, or allowed to be flexible. The particular conditions for each simulation are described in the Results section. The final co-culture model contained 170 reactions and 147 metabolites. Note that the same compounds in different compartments are treated as separate metabolites and are connected by transport fluxes.
Flux-balance analysis involves writing a mass balance on each metabolite in the network, applying a steady-state assumption (Savinell and Palsson, 1992
; Vallino and Stephanopoulos, 1993
), and solving the resulting system of equations. As there are more unknown reaction rates (metabolic fluxes) than metabolites, the system of equations is underdetermined and thus does not have a unique solution. A common method to obtain a unique flux distribution in such a case is to define an objective function, such as biomass production, and use linear programming to find the optimal solution with respect to this objective function (Varma and Palsson, 1994
; Pramanik and Keasling, 1997
). Simulations were performed using the Fluxanalyzer program (Stelling et al, 2002
; Klamt et al, 2003
), which operates within the Matlab environment (The Mathworks, Natick, MA). The objective considered, unless stated otherwise, was maximization of the following function: 1.0 × D. vulgaris
biomass + 0.1 × M. maripaludis
biomass. This resulted in the maximization of D. vulgaris
growth as the primary objective, with M. maripaludis
as a secondary objective. There are possibly multiple solutions maximizing D. vulgaris
growth, and this function assures that the solution that also maximizes M. maripaludis
growth is chosen.
Construction and characterization of syntrophic interaction
Strains and culture conditions D. vulgaris
Hildenborough (NCIMB 8303) was obtained from J Wall (Columbia, MO). Wild-type Methanococus maripaludis
S2 (Whitman et al, 1986
) and a M. maripaludis
mutant strain MM 709 lacking both annotated formate dehydrogenases (Wood et al, 2003
), were used in this study.
S2 was routinely cultured in mineral McC medium amended with 10 mM acetate (Jones et al, 1983
) at 37°C in tubes or bottles with ~80% headspace consisting of 80% H2
at 30 psi and constant shaking at 200 r.p.m.
Although M. maripaludis and D. vulgaris should theoretically be able to grow syntrophically in the absence of sulfate, they are typically cultivated under different concentrations of sodium and magnesium. Before establishing a syntrophic culture, it was necessary to find concentrations for these two ions that were permissible for both organisms. To develop a medium suitable for syntrophic growth with D. vulgaris, the effect of sodium and magnesium concentration on M. maripaludis was investigated by modifying the sodium and magnesium chloride concentrations in McC as follows: 17 mM NaCl and 2 mM MgCl2, 50 mM NaCl and 4 mM MgCl2, 120 mM NaCl and 5.9 mM MgCl2. The methanogen demonstrated comparable growth rate at all ion concentrations tested. Growth of D. vulgaris was not affected at magnesium sulfate concentrations as high as 40 g/l, but sodium chloride slowed growth at concentrations as low as 2 g/l (the extra amount added to the media). On the basis of these results, we have modified McC media to accommodate syntrophic growth. This B3 medium (see the composition below) without sulfate added was used for most experiments with co-cultures.
D. vulgaris was grown on B3m medium (pH 7.2) at 37°C in an 80% N2:20% CO2 headspace that composed ~60% of the culture vessel. B3m medium containing (per liter) 0.25 g NaCl, 5.5 g MgCl2·6H2O, 0.1 g CaCl2·2H2O, 1.0 g NH4Cl, 0.1 g KCl, 1.4 g Na2SO4, 1.04 g Na2 PIPES, 25 mM NaHCO3, 5.75 mM K2HPO4, 0.001 g resazurine, 0.078 g Na2S·9H2O, 1 ml of Thauer's vitamins (containing per liter 0.02 g biotin, 0.02 g folic acid, 0.1 g pyridoxine HCl, 0.05 g thiamine HCl, 0.05 g riboflavin, 0.05 g nicotinic acid, 0.05 g DL pantothenic acid, 0.05 g p-aminobenzoic acid, 0.01 g vitamin B12), 1 ml of trace minerals (contained per liter 1.0 g FeCl2·4H2O, 0.5 g MnCl2·4H2O, 0.3 g CoCl2·4H2O, 0.2 g ZnCl2, 0.05 g Na2MoO4·4H2O, 0.02 g H3BO3, 0.1 g NiSO4·6H2O, 0.002 g CuCl2·2H2O, 0.006 g Na2SeO3·5H2O, 0.008 g Na2 WO4·2H2O), and 30 mM lactate. A modification of B3m medium (B3n) that did not contain PIPES was used in some experiments as indicated.
D. vulgaris and M. maripaludis were cultured syntrophically at atmospheric pressure in B3m or B3n medium without Na2SO4 at 30°C without shaking in an 80% N2:20% CO2 headspace that composed ~30% of the culture vessel (25 ml Balsch tube or 250 ml bottles). As necessary, pressure in the headspace was monitored using a needle pressure gauge. Syntrophic associations were initiated by mixing equal volumes of stationary phase D. vulgaris and M. maripaludis cultures, removing excess culture medium by centrifugation and resuspending in B3n medium lacking sodium sulfate. Samples of established syntrophic associations and D. vulgaris and M. maripaludis samples were stored at −80°C in 10% glycerol.
Microbial contamination was monitored by microscopic observation and by plating co-cultures on TSA (Difco) and R2A (Difco) agar and incubating plates at 30°C in aerobic and anoxic conditions.
Experimental determination of growth and metabolic activity
To characterize growth of the syntrophic association between D. vulgaris and M. maripaludis, four 240 ml bottles containing 177 ml B3n media and an 80% N2:20% CO2 headspace were inoculated with 10 ml of a stationary phase culture of the syntrophic association. This inoculum had been previously grown in B3m medium in batch culture by serial transfer through eight transfers at a 20-fold dilution rate. Thus, before the start of our experiment, both species had approximately 35 generations to acclimate to the syntrophic growth conditions. Immediately after inoculation and at several time intervals for the next 176 h, samples were taken from each bottle for measurement of total biomass, ratio of cell types, 16S rRNA ratio quantification, and lactate, acetate, methane, carbon dioxide, and hydrogen concentration. Pressure was measured at each time interval. The methods used for these assays are described below.
To characterize growth of M. maripaludis in monoculture, 0.5 ml of an overnight culture of M. maripaludis was inoculated into three 240 ml bottles with an 80% H2:20% CO2 headspace at 30 psi pressure. Each bottle contained 60 ml of B3n media amended with 10 mM Na acetate, 2 g yeast extract per liter, and higher concentrations of NaCl (4 g/l), cysteine (3 mM), and sodium sulfide (0.5 g/l). At several time intervals for 25 h, pressure of the headspace was measured and samples were taken for measurement of biomass, lactate, acetate, methane, carbon dioxide, and hydrogen.
The concentration of organic acids (lactate, pyruvate, acetate, formate, and fumarate) and inorganic ions (sulfate, phosphate) in culture fluids were determined using a Dionex 500 system equipped with an AS11HC column. In some cases, the concentration of organic acids was also measured on an HPLC equipped with an HPX 78 (Bio-Rad) column. Hydrogen concentrations were determined with a RGD2 Reduction Gas Detector (Trace Analytical) with 60/80 MOLE SIEVE 5A column (72 × 1/8 inch) with N2 as carrier gas. The concentration of methane and carbon dioxide was measured on a GC equipped with a TCD and 80/100 HAYESEP Q column (72 × 1/8 inch) with helium as carrier gas.
The concentrations of ethanol and glycerol in the syntrophic culture supernatant were measured enzymatically. Ethanol was measured using alcohol dehydrogenase (Sigma A3263) and aldehyde dehydrogenase (Sigma A6338), and glycerol using the enzyme glycerol dehydrogenase, both using the protocols suggested by Sigma.
Biomass, protein, and RNA measurements
For protein measurements, cell pellets were resuspended in 250 mM NaOH and frozen at −20°C. Protein was measured using Bradford reagent (Pierce) according to the manufacturer's recommendations. It was assumed that the composition of D. vulgaris and M. maripaludis cells was 60% protein in all culture conditions tested. Thus, the experimentally determined protein concentration was multiplied by 2 to estimate total biomass in most cases. Alternatively, the biomass of the syntrophic culture was estimated in some cases from the optical density using a conversion factor of 1 OD600=0.385 g/l dry weight. This conversion factor was determined by drying biomass of a syntrophic culture on filters overnight at 105°C.
At each time interval in syntrophic growth experiments, a biomass sample was frozen at −80°C. RNA was extracted from these samples using the Master Pure kit (Epicentre Biotechnology) according to the manufacturer's directions. The RNA in these samples was subsequently separated and analyzed using capillary electrophoresis in a Bioanalyzer (Agilent) according to the manufacturer's instructions. The 16S rRNA fragments of D. vulgaris and M. maripaludis migrate at slightly different rates and produce separate peaks in this electrophoresis method. The ratio of the peak areas of the two species was calculated to determine the 16S rRNA ratio.
Microscopic observation of syntrophic association
During cultivation, samples of the syntrophic association were viewed under the light microscope, Olympus BH2, to check for purity. During growth characterization studies, 100 μl of the culture was immediately fixed in a solution composed of 225 μl phosphate-buffered saline and 75 μl of 37% formaldehyde, and stored at 4°C for at least 24 h. These samples were subsequently stained for 24 h by addition of 50 μl of a 500 μg/ml solution of dichlorotriazinylaminofluorescein, a fluorescent protein stain. Diluted samples were filtered onto a black nitrocellulose membrane with 0.22 μm pore size, mounted onto a microscope slide and viewed under an epifluorescence microscope LSM5 PASCAL (Zeiss). The ratio of D. vulgaris-shaped cells (vibriod) and M. maripaludis-shaped cells (cocci) was counted on 10 randomly selected fields for each slide.