Optimization of the medium for the growth of Nitrobacter winogradskyi by statistical method

Although Nitrobacter winogradskyi is an important chemoorganotrophic organism for the study of nitrite-oxidizing bacteria physiology as well as nitrification, until now, the mixotrophic medium for this organism growth has not been optimized, comprehensively. In this study, we aimed to improve the growth medium of N. winogradskyi using the one-factor-at-a-time (NaNO₂ , glycerol, pH) method. In addition, a further experimental design was carried out based on central composite design with response surface methodology. Different combinations of the three cultural parameters were fitted by multiple regression analysis to calculate the predicted response. Our results suggest that optimal culture condition for the growth of N. winogradskyi was a modified DSMZ 756a medium containing NaNO₂ (5·74 g l^-1 ) and glycerol (37·88 mmol l^-1 ), pH 7·83, a temperature of 28°C and agitation at 120 rev min^-1 . The results from a validation experiment (bacterial growth: OD₆₀₀ 1·0293) were close to the value predicted by the quadratic model (OD₆₀₀ 1·0994). In addition, we uncovered the potential mechanism at the cellular and ultrastructural levels. The results indicated that glycerol in the media enhanced the rate of cell division and cell growth by increasing the accumulation of polyphosphates and phosphorus, and high concentrations of NaNO₂ provided sufficient energy for growth and contributed to the generation of carboxysomes in cells for CO₂ fixation.

Due to the extremely slow growth rate and the low growth yield of ammonia-oxidizing bacteria and NOB (nitrite-oxidizing bacteria), nitrification is still the rate-limiting step of nitrogen cycle in the current research. Nitrobacter winogradskyi, an important NOB, participates in the second step of nitrification in water and soil. This study reported an optimized culture condition for N. winogradskyi, which increased the growth yield by 5·06 times than that in the basal medium and uncovered the potential mechanism. We expect our study will contribute to the research on water and soil nitrogen cycle. In addition, the optimized culture conditions have the potential to be suitable for the chemoorganotrophic growth of other nitrifiers.