Evaluation of Organic Carbon from Anaerobic Sequencing Batch Reactor Effluent as a Carbon Source for Denitrification

The discharge of nitrate-rich effluent has adverse effect on the receiving environment and the public health of the polluted water users. The nitrates are eliminated in a denitrification step that requires reducing power in form of organic carbon. The objective of this study was to evaluate the potential of utilizing organic carbon in effluent from the anaerobic SBR as a carbon source for denitrification. Reactors were operated for one year using meat processing wastewater. Anaerobically treated abattoir wastewater equivalent to 5, 10 and 15% of aerobic SBR hydraulic volume were added to three separate reactors. A 12 h operating cycle consisted of the following periods: (a) filling, 0.30 h; (b) settling, 11 h and (d) decanting, 0.30 h for the anoxic reactor. A comparison between different carbon loads was performed based on biological carbon, nitrogen and phosphorus removal. Sufficient denitrification was achieved with 10% (aerobic SBR hydraulic volume) of anaerobically-treated abattoir wastewater. TCOD, BOD5, TKN, N02N, NO3N, PO4, TS, EC and temperature and turbidity were reduced by 78, 70, 91, 100, 98, 62, 39, 65, 71, 5 and 39% respectively, with effluent mean concentrations of 80 ± 5 mg/L, 54 ± 12 mg/L, 35 ± 4, 00 ± 0, 2 ± 1, 18 ± 1, 254 ± 12, 1.64 ± 0.01, 22.04 ± 0.02 and 738 ± 9 FAU. Organic carbon in effluent from the anaerobic SBR can be used as a carbon source for anoxic denitrification. However, the denitrification rate is affected by the organic carbon load used. Except TKN and o-PO43mg/L, all other parameters in the denitrified effluent met discharge standards.


Introduction
The discharge of nitrate-rich effluent has adverse effect on the receiving environment and the public health of the polluted water users [1,2]. Such effects manifest as toxicity to aquatic biota, eutrophication [3,4] and public health complications such as thyroid hypertrophy, methemoglobinemia, hypertension and cancer [5]. The World Health Organization has set a limit of 10 and 100 mg/L NO 3 for human and animal consumption, respectively. Wastewater above these limits requires treatment [6][7][8].
Nitrates are biologically removed via denitrification, anoxic reduction of NO 3 -→ NO 2 -→ NO → N 2 O → N 2 by heterotrophic bacteria such as Pseudonomas stutzeri, Alcaligenes faecalis and Ochrobactrum anthropi [9,10]. Activated sludge based sequencing batch reactor (SBR) allows the removal of carbon, nitrogen and phosphorus using a single reactor [11][12][13]. The key to efficient biological pollutant removal is transitioning between anaerobic, aerobic, and anoxic phases. Doing so promotes transformation of the carbon, nitrogen and phosphorus by triggering utilization of different electron acceptors and donors [14].
However, simultaneous phosphorus and nitrogen removal is not always successful [13,15]. Polyphosphate-accumulating organisms (PAO) and denitrifying bacteria are both heterotrophic [9,10], and thus able to take up organic carbon under anaerobic conditions and store it for growth once a suitable electron acceptor is available [14,[16][17][18]. In combined denitrification and EBPR systems, organic carbon availability is usually the limiting parameter [19,20], denitrifers and PAOs directly compete for the available organic carbon [11,21]. Both processes are inhibited by this competition [13,22]. Insufficient denitrification is improved by addition of an external organic carbon such as such as glucose, ethanol, acetate, methanol, aspartate, formic acid [18,23], molasses, sulfite waste liquor, whey and distillery stillage [24]. However, these solutions are costly, require an adaptation period, lead to excessive sludge production and are not always efficient [13]. Hence efficient and cost-effective solutions are needed.
The use of wastewater as an internal carbon source is a promising alternative [10]. However, denitrification is affected by the type and dose of the organic carbon source used, duration of each phase and time cycle. The choice of hydraulic retention times and sludge retention time is dependent on this optimization [25,26]. The objective of this study was to evaluate the potential of utilizing anaerobically treated meat processing wastewater as a carbon source for denitrification. A comparison between different carbon loads was performed based on biological carbon, nitrogen and phosphorus removal.

Model reactors
Three glass reactors (Figure 1), each with a total volume of 25 L and a working liquid volume of 20 L were set up at Makerere University Biochemistry Research Lab. These reactors were fed with nitrified effluent. To prepare the nitrified liquor, meat processing wastewater from City Abattoir, Kampala-Uganda was treated sequentially in anaerobic and aerobic SBR using a standard procedure as described by Mutua et al. [19]. Then, anaerobically treated abattoir wastewater equivalent to 5, 10 and 15% of aerobic SBR hydraulic volume were added to three separate reactors (the properties of the anaerobically treated wastewater are shown in Table 1.

Parameter
Outflow conc.   4 3-, Turbidity and TS are expressed in mg/L; Turbidity, EC, and temperature are expressed in (FAU), (ms/cm) and (•C), respectively. An 12 hour operating cycle consisted of the following periods: (a) filling, 0.30 h; (b) settling, 11 h and (d) decanting, 0.30 h for the anoxic reactors. At the end of each cycle, 10 litres of the supernatant was decanted, followed by feeding of an equal amount of wastewater. The system operated at a nominal Sludge Retention Time (SRT) of 5 days. The organic loading was 12.8 kg COD/m 3 /day, during the study period. Steady-state conditions were obtained after 3 months.

Statistical analysis
One-way analysis of variance (ANOVA) was used to compare treatment means. The results were expressed as mean ± SEM. The differences were considered significant, when P<0.05. Table 2 show the denitrification efficiencies obtained when 5, 10 and 15% organic carbon load were used.  removal efficiency significantly differed between 10% and 15% carbon load (p=0.000) ( Figure 2, Table 2).

Figures 2 to 7 and
The BOD removal efficiency did not significantly differ between 5% and each of 10% and 15% carbon load. However, there was significant difference in BOD removal efficiency between 10 and 15% carbon load (p=0.007) ( Figure 3, Table 2).
The TKN removal efficiency significantly differed between 5% and each of 10% (p=0.000) and 15% (p=0.000) organic carbon load and the TKN removal efficiency significantly differed between 10% and 15% organic carbon load (p=0.000) (   The NH4 -H removal efficiency did not significantly differ between 5% and each of 10%. However, there was significant difference in NH4 -H removal efficiency between 15% each of 5 and 10% carbon load (p=0.007) ( Table 2).
The TS removal efficiency did not significantly differ between 5% and 10% (p>0.05) carbon load. However, there was significant difference in TS removal efficiency between 15% and each of 5% and 10% organic carbon load (p<0.05) ( Table 2). The pH did not significantly differ between 5% and 15% (p>0.05) organic carbon load. However, there was significant difference in pH between 10% and each of 5% and 15% organic carbon load (p<0.05) ( Table 2).
The EC did not significantly differ between 5% and 15% (p>0.05) organic carbon load. However, there was significant difference in EC between 10% and each of 5% and 15% organic carbon load (p<0.05) ( Table 2). Temperature did not significantly differ between 10% and 15% (p>0.05) organic carbon load. However, there was significant difference in temperature between 5% and each of 10% and 15% organic carbon load (p<0.05) ( Table 2).
Denitrification is anoxic reduction of the NO 3 -→ NO 2 -→ NO → N 2 O → N 2 by heterotrophic bacteria such as Pseudonomas stutzeri, Alcaligenes faecalis and Ochrobactrum anthropi [9,10]. Most denitrifiers are facultative heterotrophic bacteria that use organic carbon as energy source and nitrite-nitrates as electron acceptors [15]. While most waters contain a reducing power in the form of organic substrate, it is difficult to preserve the reducing power required for denitrification, due to the necessary preceding aerobic oxidation step [24,26]. Consequently, sufficient organic carbon source must be provided for proper denitrification [18].
Limited carbon is known to cause repression of denitrifying enzymes [11] as shown when 5% organic carbon (C:N ratio 1.68) is added (Figures 2 and 3). The current interpretation of this phenomenon is that nitrate entering the anoxic phase is used as an electron acceptor in the growth of non-poly heterotrophs [14]. This reduces the amount of the substrate available for sequestration by the poly organisms and hence reduces the amount of phosphorous removal that can be achieved [15]. Moreover, phosphate removal efficiency is affected by competition for the organic substrate between denitrifiers and PAOs [13,19] leading to high phosphorus concentration in the effluent (Figure 7, Table 2). 10% organic carbon load had a C:N ratio of 1.89 mg COD.L -1 N-TKN which achieved complete nitrite removal (Figure 4). Negligible amounts of nitrates (2 ± 1 mg/L) remained within the system ( Figure 5, Table 2). This is consistent with research findings by Obaja et al. [25] and Rahman et al. [24] that complete denitrification is obtained when the C/N ratio is ≥ 1.7. According to Fabregas [27], the high DO concentration (14.40 mg/L -1 ) at the beginning of anoxic period could have lowered the level of biodegradability of the wastewater and hence complete nitrate removal was not achieved. Carbon decrease observed was as a result of both assimilative and dissimilative carbon utilization by denitrifying and other bacteria [20,24]. The COD concentration measured at the end of the cycle was from the fraction of slowly biodegradable substrate contained in the abattoir wastewater [15]. Denitrification resulted in a rise in alkalinity of the system [13,28,29], with corresponding increase in pH ( Table 2).
The rate of phosphate removal increases (Figures 6 and 7) after most of the denitrification had taken place because denitrifers have high affinity for organic carbon than PAOs [11]. The phosphorus uptake under anoxic conditions is attributable to the activity of denitrifying phosphorus accumulating organisms (DNPAO), capable of accumulating high amounts of polyphosphates [20]. Towards the end of anoxic phase, orthophosphorus had a slight increase with no corresponding total phosphorus increase. This observation can be attributed to anoxic orthophosphorus release by PAOs and total phosphorus absorption by sediments [22].
The use of 15% organic carbon load decreases C:N ratio (1.59). This system overload inhibits both denitrifiers and PAOs activity [13,15], decreasing nitrates and ortho-phosphorus removal efficiencies. The initial high NO 2-and NO 3-removal efficiencies were likely due to dilution factor. Figure 7: Comparison of PO 4 3removal efficiency in a denitrification step using 5, 10 and 15% organic carbon dose in a lab-scale SBR treating wastewater, (n=6).

Conclusion
Organic carbon in effluent from the anaerobic SBR can be used as a carbon source for anoxic denitrification and phosphorus removal. However, the denitrification rate is affected by the organic carbon load used. The best denitrification and phosphorus removal efficiencies are achieved with 10% (of the anoxic SBR operational volume) of the anaerobic effluent. Except o-PO 4 3--P (8 ± 1 mg/L) and TKN (35 ± 4 mg/L) all other parameters (BOD, TCOD, SCOD, TP, TSS, NH 4+ -N and turbidity) in the denitrified effluent met permissible discharge standards when 10% (of anoxic bioreactor) organic load was used.