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BNR 101: White Paper wateronline.com ■ Water Online The Magazine 20 preferentially consumed by ordinary denitrifying organisms, which reduce nitrate/nitrite to N 2 gas. Thus, PAO lose the competition for the carbon source, resulting in EBPR deterio- ration (Cho and Molof, 2004). Other authors (van Niel et al, 1998; Saito et al, 2004) have hypothesized that nitrate, nitrite, or other denitrification intermediates can cause inhibition of PAO, which ends up with a loss of EBPR activity. Piloting A Cure In a recent research project, we studied the loss of activity of EBPR observed in municipal WWTPs due to nitrate recycle with the objective of understanding this failure and provid- ing solutions to minimize its detrimental effect. A pilot plant (see Figure 1 on the previous page) was initially operated with A 2 /O configuration to obtain simultaneous removal of COD/N/P. After four months of working under stable con- ditions, a steady state with high nutrient removal capacity was achieved with a 70 percent of PAO (quantified by FISH [fluorescence in situ hybridization] microbial identification). The plant was moved to a modified Ludzack-Ettinger (MLE) anoxic/aerobic configuration to study the effect of the presence of nitrate in the anaerobic phase by placing both internal and external recycles to the anaerobic reactor, thus maximizing the nitrate load to the theoretically anaerobic reactor. The pilot plant was first fed with wastewater con- taining a high percentage of VFA (75 percent of 400 mg COD/L). The internal recycle was progressively increased up to an internal recycle ratio of 10, resulting in a high nitrate load to the anaerobic reactor and nitrate concentration in the anaerobic reactor higher than 7 mg NO 3 - - N/L. Even under these unfavorable operational conditions, the system was able to maintain a high P-removal efficiency above 85 percent. These results showed that carbon source was prefer- entially consumed in the EBPR process rather than ordinary heterotrophic denitrification, which is in disagreement with literature to date (Henze et al, 2008). In the next step of this study, the inlet carbon source was reduced to 200 mg COD/L, testing two types of substrate: VFA and sucrose. For the wastewater with high VFA content, the reduction in COD did not affect the P-removal process despite the nitrate concentration in the effluent increasing. Conversely, when the main substrate was a more complex carbon source (sucrose), most of the COD was used for heterotrophic deni- trification, which resulted in a drastic decrease of P-removal capacity. Thus, the major role of the nature of carbon source in EBPR deterioration by the nitrate presence in the anaerobic reactor was demonstrated (Guerrero et al., 2011). The results can be explained considering the processes usually assigned to denitrifying microorganisms. Under anaer- obic conditions, denitrifiers ferment complex carbon sources to produce VFA which are used by the PAO. The presence of nitrate prevents fermentation of complex carbon sources to VFA since this carbon source is directly denitrified, which is energetically more favorable. The experimental results show that nitrate input to the anaerobic reactor is not per se the direct cause of the loss of EBPR activity in urban WWTPs, but it is a key element that reduces VFA production — certainly having a negative effect. Therefore, WWTPs would be able to maintain biological P-removal despite the introduction of nitrate in the anaerobic reactor, provided that the presence of VFA is guaranteed. These results also allow rejecting the hypothesis of a possible inhibitory effect of nitrate or denitri- fication intermediates as suggested in the literature, since the pilot plant was able to maintain EBPR activity despite the high amount of recycled nitrate to the anaerobic reactor. Potential Solutions The results demonstrate that the presence of VFA ensures EBPR operation process despite the presence of nitrate in the anaerobic reactor. Thus, VFA addition as an external carbon source would also minimize the competition between PAO and denitrifiers in the scenario of low organic load wastewaters. However, the high cost associated with this addition makes it a difficult alternative to apply. One possible solution is the use of anaerobic fermentation of primary sludge (Moser-Engeler et al, 1998; Chanona et al, 2006) to generate a VFA-rich effluent. Another alternative is the addition of other low-cost external carbon sources to improve EBPR for COD-deficient wastewaters. We obtained promising results using the byproduct generated in biodiesel production (i.e., crude glycerol). The addition of this byproduct provides a carbon source for the denitrification of nitrate recycled and allows the in situ generation of VFA in the anaerobic reactor. The utilization of this carbon source requires the development of a proper microbial community able to degrade/ferment complex substrates as glycerol to produce VFA. A direct replacement strategy of the carbon source was successfully applied for glycerol, starting from the original microbial community developed in the pilot WWTP. EBPR was maintained using glycerol as the sole carbon source, with a proper selection of the fraction of anaerobic, anoxic, and aerobic phases. The anaerobic phase has to be long enough to facilitate fermentation of complex substrate to VFA and consumption of VFA by PAO. In addition, the aero- bic phase should allow net P uptake. Regarding the initial distribution of the biomass, the initial microbial consortium requires the presence of a fraction of organisms capable of fermenting the complex substrate to VFA, but these microorganisms are usually present in activated sludge of any WWTP. Another important opera- tional condition that needs to be considered is avoiding the Figure 2: Different strategies to apply EBPR were studied at this WWTP in Manresa, Spain.