Hydrogenase does not confer significant benefits to Azotobacter vinelandii growing diazotrophically under conditions of glucose limitation

Abstract

The presumed beneficial effect of hydrogenase on growth of diazotrophic bacteria was reinvestigated with carbon-limited chemostat cultures of the hydrogenase-deficient mutant hoxKG of Azotobacter vinelandii and its parent. The results revealed that hydrogen recycling was too low to benefit the cellular energy metabolism or activities of nitrogenase and respiration.Reduction of dinitrogen to ammonia by nitrogenase is accompanied by the production of hydrogen (17). Since organisms which express an uptake hydrogenase are able to recycle hydrogen, it has been proposed that recycling of hydrogen benefits diazotrophic growth (5, 19). Yates and colleagues showed that hydrogenase-negative mutants of Azotobacter chroococcum were outcompeted by the wild type, when growing diazotrophically in sucrose-limited mixed populations (1,20). These results have become widely accepted as evidence for the beneficial effect of hydrogen recycling on carbon-limited growth of diazotrophs (2, 19). However, results obtained with mixed populations may be questioned because it is very likely that the wild type utilized hydrogen produced not only by itself but also by the mutant. In fact, the present communication shows that the amount of hydrogen produced by glucose-limited hydrogenase-positive Azotobacter vinelandii is too low to benefit diazotrophic growth.Glucose-limited cultures of mutant hoxKG of A. vinelandii and its parent strain (15) were grown with 15 mM glucose in a pH-controlled chemostat (6, 13). Dissolved oxygen concentrations (DOC) of 135 and 11.3 M were measured and kept constant as described previously (13). In order to prevent washing out of the culture, the dilution rate (D) was not increased above 0.25 h Ϫ1 (6, 13). Since in carbon-limited chemostat cultures the ratio of protein to biomass is constant (6), steady-state biomass levels of the cultures were estimated on the basis of protein (10). At both DOC, the mutant and its parent exhibited the same dependence of protein formation on D (Fig. 1). Both strains consumed all of the carbon and energy source, glucose. Consequently, yield coefficients (Y, grams of biomass formed per mole of substrate consumed) were identical as well. The maintenance coefficients and the theoretical maximum Y were extrapolated from plots of 1/Y versus 1/D (12) (Fig. 2). The theoretical maximum Y of both strains were 20 g/mol, and maintenance processes required 1.0 and 16 mmol of glucose per g of protein and h at 11.3 and 135 M DOC, respectively. The identical maintenance coefficient and Y values may be considered sufficient to infer lack of any significant differences in the efficiency of the energy metabolism of both strains. Nevertheless, in the following, we examined the suggestion that hydrogenase benefits diazotrophs by giving rise to extra ATP formation or to protection of nitrogenase against oxygen (1,20).Adenine nucleotide levels were determined according to published methods (9,11,18). The different parameters of the energy status of both strains as compiled in Table 1 do not reveal any ...

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations

“…In diazotrophic organisms and particularly in aerobic azotobacters, the high energy requirement of nitrogenase contributes significantly to the high maintenance activities known for these organisms (16,29). It was reported before that increases in the dissolved oxygen concentration were accompanied by increases in the maintenance requirements of diazotrophic cultures of A. vinelandii and decreases in the Y values (14,17). Importantly, however, it was also reported that the true growth yield (Y G ) remained constant in cultures grown at different dissolved oxygen concentrations and carbon sources.…”

“…Since diazotrophic cultures can be considered energy limited and since the concentration of the energy source in the feed (S R ) was constant, it follows that the observed D-dependent increases in the steady-state levels of both S (ATP) and X were due to an increase in the efficiency of energy utilization (Y ATP ) for biomass formation, i.e., dinitrogen fixation. Augmentation of Y may be explained by the fact that, upon increasing the substrate supply with increasing D, the relative influence of maintenance functions on Y decreases (17,22). In diazotrophic cultures of A. vinelandii, the unusually high maintenance activities with glucose and other carbon sources vary largely in direct proportion to the oxygen concentration (14,17).…”

“…Augmentation of Y may be explained by the fact that, upon increasing the substrate supply with increasing D, the relative influence of maintenance functions on Y decreases (17,22). In diazotrophic cultures of A. vinelandii, the unusually high maintenance activities with glucose and other carbon sources vary largely in direct proportion to the oxygen concentration (14,17). Therefore, when increasing D at the higher oxygen concentrations, the relative influence of maintenance activities on Y does not decrease in A. vinelandii to a negligibly low level as it does in other bacteria (22).…”

“…A. vinelandii wild-type strain OP (ATCC 13705) as well as the mutant strains MK5 (12,16) and hoxKG (17) were grown diazotrophically in an oxygen-and pH-controlled chemostat. Dissolved oxygen concentrations were measured and kept constant as described previously (25).…”

“…In situ respiratory activities were estimated on the basis of the gas flow rate through the culture and the difference between oxygen concentrations at the gas inlet and outlet. Oxygen concentrations were determined by gas chromatography (17).…”