Extent of intracellular storage in single and dual substrate systems under pulse feeding.

The study investigated the effect of acetate/starch mixture on the formation of storage biopolymers as compared with the storage mechanism in systems fed with these compounds as single substrates. Experiments involved two sequencing batch reactor sets operated at steady state, at sludge ages of 8 and 2 days, respectively. Each set included three different runs, one fed with acetate, the other with starch and the last one with the acetate/starch mixture. In single substrate systems with pulse feeding, starch was fully converted to glycogen, whereas 25 % of acetate was utilized for direct microbial growth at sludge age of 8 days, together with polyhydroxybutyric acid (PHB) storage. The lower sludge age slightly increased this fraction to 30 %. Feeding of acetate/starch mixture induced a significant increase in acetate utilization for direct microbial growth; acetate fraction converted to PHB dropped down to 58 and 50 % at sludge ages of 8 and 2 days respectively, while starch remained fully converted to glycogen for both operating conditions. Parallel microbiological analyses based on FISH methodology confirmed that the biomass acclimated to the substrate mixture sustained microbial fractions capable of performing both glycogen and PHB storage.

Aerobic metabolism of mixed carbon sources in sequencing batch reactor under pulse and continuous feeding.

The aerobic metabolism of a mixture of acetate and starch was studied with main emphasis on their interaction and the effect on their storage as PHB and glycogen, respectively. Pulse feeding strongly increased the storage of both substrates; however, the presence of starch decreased PHB storage whereas the presence of acetate did not affect glycogen storage. Indeed, 13C NMR isotopomer analysis suggested an increase of acetate utilization towards TCA cycle, due to an increased request of ATP production for glycogen biosynthesis regulated by ADP-GlcPPase. This in turn influenced the partition flux for pyruvate synthesis between TCA cataplerosis and glyoxylate shunt. The corresponding reduction of PHB synthesis was in agreement with the competition for HS-CoA between KGDH activity and acetyl-CoA for PHB synthesis pathway. As a practical consequence, bioprocesses for PHA production from volatile fatty acids could be negatively affected from other carbon sources, such as unfermented carbohydrates.

Effect of feeding regime and the sludge age on the fate of acetate and the microbial composition in sequencing batch reactor.

The study investigated the effects of the feeding regime on the substrate metabolism under aerobic conditions. Throughout the study, two parallel sequencing batch reactors were operated using either short-term (pulse) or long-term (almost continuous) feeding of acetate at two different sludge ages of two days and eight days. The microbial characterization studies showed that the feeding regime did not change the microbial composition as determined by the fluorescent in situ hybridization (FISH) analysis, however did strongly affect the substrate utilization mechanism. Additionally, the same microbial culture was able to utilize acetate with or without poly-ß-hydroxybutyrate (PHB) storage under pulse or continuous feeding, respectively. Conversely, the selected sludge ages induced significant changes in the microbial composition and floc structure, however, the well settling and bulking biomass selected at the sludge ages of eight days and two days, respectively, did not significantly affect the substrate utilization mechanism, where storage or growth prevailed as a result of either pulse or continuous feeding.

Respirometric evaluation and modelling of acetate utilization in sequencing batch reactor under pulse and continuous feeding.

The study investigated the effect of feeding regime and sludge age on acetate utilization. Parallel sequencing batch reactors (SBRs) were operated at steady-state with pulse and continuous feeding of acetate at sludge ages of 8 and 2 days. Acetate was always partially converted to poly-ß-hydroxybutyrate (PHB). The adopted model remained equally applicable to oxygen uptake rate and PHB profiles reflecting different feeding regimes and culture history. Sludge age was significant on the rate parameters of storage and direct growth (k(STO), µ(H1)), while the feeding regime affected half saturation coefficients (K(STO), K(S1)). Changing the sludge age from 8 days to 2 days reduced the k(STO) value from 8.0 day(-1) to 6.5 day(-1) and increased the corresponding µ(H1) value from 1.5 day(-1) to 2.5 day(-1), regardless of the feeding regime; conversely, changing from pulse to continuous feeding reduced K(STO) while increasing K(S) for the SBR operation at the same sludge age.

Effect of feeding and sludge age on acclimated bacterial community and fate of slowly biodegradable substrate.

The study investigated the effect of feeding regime and sludge age on starch utilization. For this purpose, parallel sequencing batch reactors were operated with pulse and continuous feeding of soluble starch at sludge ages of 8 and 2 days. Pulse feeding induced almost complete conversion of starch to glycogen, while storage was lowered and accompanied with direct growth under continuous feeding, regardless of sludge age. Low sludge age did not alter simultaneous storage and utilization for direct growth but it slightly favoured direct utilization due to faster growing biomass. Experimental results suggested adsorption of starch onto biomass as a preliminary removal mechanism prior to hydrolysis at sludge age of 8 days. Adsorption was not noticeable as substrate removal, glycogen generation and dissolved oxygen decrease were synchronous at sludge age of 2 days. Bacterial community always included fractions storing glycogen although sludge age only affected the relative magnitude of filamentous growth.

Effect of high nitrate concentration on PHB storage in sequencing batch reactor under anoxic conditions.

The study investigated effect of high influent nitrate concentration on poly-beta-hydroxybutyrate, (PHB), storage in a sequencing batch reactor, (SBR), under anoxic conditions. Acetate was fed as pulse during anoxic phase, sustained with external nitrate feeding. SBR operation involved three runs at steady state with COD/N ratios of 3.84, 2.93 and 1.54 gCOD/gN, where external nitrate concentrations gradually increased from 50 mg N/l to 114 mg N/l and 226 mg N/l, in 1st, 2nd and 3rd runs, respectively. In 1st run, acetate was fully converted into PHB with the storage yield value of 0.57-0.59 gCOD/gCOD, calculated both in terms of PHB formation and NO(X) utilization, confirming storage was the sole substrate utilization mechanism. In the following runs, PHB formation was reduced and the storage yield based on PHB dropped down to 0.40 and 0.33 gCOD/gCOD with increasing influent nitrate concentrations, indicating that higher portions of acetate were diverted to simultaneous direct growth. The observations suggested that nitrite accumulation detected at low COD/N ratios was responsible for inhibition of PHB storage.

Effect of feeding pattern on biochemical storage by activated sludge under anoxic conditions.

This study investigated the effect of feeding pattern on bacterial storage under anoxic conditions, emphasizing previous adaptation of biomass to a long term feeding condition, under steady-state operation. Storage was evaluated in a sequencing batch reactor (SBR) system operated in a sequence of anoxic/aerobic phases, fed with acetate as the sole carbon source during anoxic conditions. The experimental results indicated that biochemical storage of acetate as PHB occurred when substrate was fed as a pulse, while acetate was removed mostly through direct microbial growth under continuous feeding. For pulse feeding, the anoxic yield, Y(STOD) was calculated as 0.58mg COD (mgCOD)(-1) in two different ways, using mass balances on acetate utilization, PHB generation and nitrate consumption. This value was supported by parallel batch tests and similar results in the literature. Similarly, the rate coefficient for storage under anoxic conditions, k(STOD) was computed as 9.3day(-1) based on basic stoichiometry and model calibration of experimental data. Batch tests conducted with biomass adapted to different feeding patterns showed that substrate storage was insignificant when the feed was added continuously at low concentrations, even if the biomass was previously adapted to storage. Similarly biomass acclimated to continuous feeding could not store the excess substrate although fed instantly. For the operating conditions selected for the study, storage response was significant only with a microbial culture with metabolic activities previously adapted to storage and with short-term substrate feeding at high concentrations.