Physiology of syntrophic propionate oxidizing bacteria

Under methanogenic conditions, with protons and carbon dioxide as intermediate and ultimate electron acceptors, complex organic material is degraded in several steps to methane and carbon dioxide. About 15% of the total carbon compounds are degraded via propionate as an intermediate. Propionate is oxidized to acetate, carbon dioxide and hydrogen. For thermodynamical reasons, this reaction can only proceed if the partial pressure of hydrogen is kept very low. Hydrogenotrophic organisms, e.g. methane bacteria or sulphate reducing bacteria, are syntrophic partner in this process of interspecies hydrogen transfer. Recently, however, it was hypothesized that also formate could be an important compound via which the electrons are transferred to the partner organism.The aim of this study was to obtain better fundamental understanding of biochemical and physiological aspects of obligate syntrophic propionate oxidizing bacteria. The presence of an obligate partner organism makes such studies very difficult. In this thesis different methods were used to overcome these difficulties: 1) techniques which do not require pure cultures, e.g. the use of specifically labelled compounds, 2) growth of the acetogen in pure culture by either using artificial electron acceptors or metabolic intermediates, and 3) determining the acetogen specific enzymes by subtracting the activities measured for the pure culture of the electron scavenging partner organism from the activities found in a defined biculture.Using invivo high-resolution 13C-NMR, evidence was found for the involvement of the succinate pathway in propionate oxidation by a methanogenic coculture (Chapter 1). The addition of [3-13C]-labelled propionate clearly showed succinate as an intermediate, and the ultimate breakdown product acetate was labelled equally in the C-1 and C-2 positions. In addition, denovo synthesis of propionate from propionate was observed. The 13C-label randomized completely between the C-3 and C-2 of propionate. Apparently propionate and succinate were interconverted at a high rate. These results were in accordance with the data published by others.The interconversion of propionate and succinate offered the possibility to study the role of carboxylation reactions in propionate metabolism in some anaerobic bacteria (Chapter 3). This was done in a very easy way by the inclusion of [3- 13C] propionate and H 13C0 3 -, which gave insight into the process of randomization and the types of (de)carboxylating enzymes involved. Both the propionate oxidizer in a methanogenic coculture and Syntrophobacterwolinii were shown to degrade propionate via the. succinate pathway involving a transcarboxylase.Chapter 4 deals with a two-liquid-phase electron removal system including the artificial, water soluble redox mediator propylviologen sulphonate (PVS). The organic phase dibutylphtalate was used as reservoir for the electron acceptor 2-anilino-1,4-naphtoquinone. In the abiotic two-liquid-phase system, electrons were transfered from the medium into the organic phase. The indicator organism Acidaminobacterhydrogenoformans oxidized glutamate to acetate without evolution of hydrogen (or formate). However, results indicated that the hydrogen partial pressure obtained by this method, was not low enough to clearly influence the metabolism of the bacterium. Besides possible toxicity problems, the relatively low midpoint redox potential of PVS (-390 mV) may have been the problem for efficient electron transfer at the required hydrogen partial pressure of 10-5 atm. to cause a shift in electron flow during glutamate oxidation. In a syntrophic propionate oxidizing coculture the electron scavenging methane bacteria could not be replaced by the artificial electron acceptor. PVS was tested both as redox mediator in the two-liquid-phase system, and as terminal electron acceptor.The metabolic intermediates pyruvate and fumarate were tested for growth in pure culture of the propionate oxidizing organism in a methanogenic coculture (Chapter 5). A propionate fermentation was performed with pyruvate as the substrate. 13C-NMR showed the involvement of the succinate pathway in the formation of propionate. The isolated organism, however, did not oxidize propionate in coculture with hydrogenotrophic methanogens. Moreover, a sulphate reducer appeared to be present in the original coculture. A syntrophic sulphidogenic propionate oxidizing coculture was obtained by repeated transfer of the coculture in medium with propionate and sulphate. To test whether the (slow growing) obligate syntrophic acetogen can be grown on other substrates than propionate, could not be tested because of the contaminating organisms.Chapter 6 is the first report on enzyme measurements in syntrophic propionate oxidation. As Syntrophobacterwolinii grows in a defined biculture with a Desulfovibrio species, it was possible to use cell-free extracts of a pure culture of the latter organism as a blanc. Most enzymes involved in the succinate pathway, including the key enzyme propionyl-CoA:oxaloacetate transcarboxylase, were demonstrated in S.wolinii . This confirms the results found by 13C-NMR (Chapter 3).Further, S.wolinii appeared to have a lower growth yield than Desulfobulbuspropionicus . This difference is explained in terms of energy conservation mechanisms. Comparison of growth rates of three syntrophic propionate oxidizing cocultures showed that hydrogenotrophic sulphate reducers are more efficient than methanogens during interspecies hydrogen transfer. The more negative Gibbs free energy change under sulphidogenic conditions compared to methanogenic conditions, is thought to contribute to this phenomenon.The final chapter (7) deals with the use of 13C-NMR in a complex biological system. Propionate degradation was followed in mesophilic methanogenic granular sludge at 55°C. Because of the non-steady conditions, transient intermediary products accumulated in the medium. The addition of fumarate as secondary substrate stimulated propionate conversion. Propionate and succinate appeared to be direct precursors of each other during propionate metabolism. Selective labelling of one of the substrates offered the possibility to study turnovers ofdifferent compounds. Moreover, interrelated biochemical processes could, in this way, be investigated in a relatively easy way.