rRNA and poly-beta-hydroxybutyrate dynamics in bioreactors subjected to feast and famine cycles

Abstract

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-beta-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage.

FIG. 1.

Concentrations of acetate (mmol C/liter) (○), PHB (mmol C/liter) (▾), ammonium (mmol/liter) (▪), and biomass (mmol C/liter) () (left axis) and oxygen uptake rates (mmol/liter · h) (⋄) (right axis) during a pulse experiment. (a) PHB(+) in a semibatch reactor. (b) PHB(−) in a semibatch reactor. (c) PHB(+) in a chemostat reactor. (d) PHB(−) in a chemostat reactor. Note that the left y axes are broken between 12 and 21 mM.

FIG. 2.

Optical density at 420 nm for semibatch reactors (a) and chemostat reactors (b) and specific OUR (c) calculated from the dissolved oxygen concentration for the semibatch reactors. •, PHB(+) cultures; ▾, PHB(−) cultures.

FIG. 3.

Specific growth rate during the feast phase as a function of the level of RNA in the biomass. ○ and •, PHB(+) cultures; ▿ and ▾, PHB(−) cultures; solid symbols, semibatch reactor; open symbols, chemostat reactor.

FIG. 4.

Degradation of PHB in the PHB(+) cultures during the famine phase for the semibatch reactor (▪) and the chemostat reactor (•). The lines indicate the fit obtained with equation 5.

FIG. 5.

Major axis regression to determine the parameters of the metabolic model using equations 14, 15, and 16. The terms on the left of the equations were plotted on the y axis, while the multipliers of δ (terms on the right of the equations) were plotted on the x axis. For the regression line: δ = 2.88 ± 0.17 mol ATP (mol NADH)⁻¹, m_ATP = 0.100 ± 0.042 mol ATP (mol C · h)⁻¹), R² = 0.98. ○ and •, PHB(+) cultures; ▿ and ▾, PHB(−) cultures; solid symbols, semibatch reactor; open symbols, chemostat reactor.