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External sources of organic carbon are often added to biological processes to facilitate denitrification and nitrogen removal by providing energy and carbon to heterotrophic bacteria. External carbon is needed especially in post-denitrification systems where wastewater carbon is not
normally available. One of the factors in selecting an appropriate substrate source is that denitrification rates differ depending on the type of carbon added. The overall objectives of this research were two-fold: (1) to compare denitrification rates for organisms using different external
carbon sources; and (2) to investigate acclimation of biomass to the various external carbon sources. Three lab-scale sequencing batch reactors (SBRs) were employed in the study, each with a different carbon substrate – methanol, ethanol or acetate. The units were seeded with sludge
from a step feed biological nutrient removal (BNR) system that did not use any external carbon. The reactors were operated at 20°C and a solids retention time (SRT) of approximately 10 days with a cycle time of 6 hours. Each SBR cycle commenced with influent feeding (tap water spiked with
nitrate), followed by a 2.25 hour aeration period. Aeration was then stopped and a 0.25 hour period ensured that dissolved oxygen (DO) decreased to zero before adding the carbon substrate. After a further 2 hour anoxic period, the SBRs were aerated for 0.25 hours before settling and decanting.
The operating mode was selected to mimic the exposure of denitrifying biomass to alternating aerobic-anoxic conditions typically encountered in practice. Monitored parameters included pH, DO, temperature, nitrate, nitrite, chemical oxygen demand (COD), mixed liquor suspended solids (MLSS),
and mixed liquor volatile suspended solids (MLVSS). All parameters were measured according to Standard Methods (1998). Soluble nitrate, nitrite and COD were tested using the Hach DR4000 Spectrophotometer. During the first 2.25 hours (aerated) nitrate concentration remained essentially constant
at a level determined by the influent concentration. Over 0.25 hours after aeration stopped (DO stripping) there was a small decrease in nitrate due to endogenous denitrification. After the carbon substrate was added at 2.5 hours, the nitrate showed a linear decrease over the remainder of
the unaerated phase. Steady state was attained in the SBRs after approximately 40 days (4 SRTs). The in-situ specific denitrification rate (SDNR) was calculated at intervals of a few days from start up from the slope of the nitrate profile after substrate addition. The steady state in-situ
SDNR values for methanol, ethanol and acetate were 24, 41 and 53 mgNO3-N/gVSS/hr, respectively. Once the systems had stabilized (after 3 to 4 SRTs) a series of ex-situ SDNR tests were performed using mixed liquor from each SBR with each of the three substrates (3 ×
3 tests). A total of nine ex-situ SDNR experiments were performed using a matrix of the three different acclimated biomass and the three different substrates. As expected, each substrate produced the highest SDNR rate with biomass acclimated to that specific substrate. It is interesting to
note that addition of acetate or ethanol to methanol-acclimated biomass exhibited very low SDNRs of 2.9 and 2.7 mgNO3-N/gVSS/hr, respectively. Use of ethanol with acetate acclimated biomass produced a high SDNR value of 27.2 mgNO3-N/gVSS/hr. High F/M
tests are also being conducted to assess the maximum specific growth rates (μMAX) of organisms using the different substrates (Dold et al., 2005). The SBR profiles will be simulated using the BioWin process simulator to confirm the μMAX estimates obtained from the
High F/M tests. Information from this study will be significant for systems practising external carbon addition for denitrification. The results to date demonstrate that biomass acclimated to acetate or ethanol exhibit high denitrification rates per unit VSS produced on the substrate (53
and 41 mgNO3-N/gVSS/hr) compared to the rate in the methanol-fed system (24 mgNO3-N/gVSS/hr). The results of the ex-situ tests are of particular interest for plants with methanol addition considering simultaneous addition of other substrates (at say
low operating temperatures in an effort to improve denitrification performance). The methanol-acclimated sludge exhibits very little denitrification with addition of acetate or ethanol.
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