Metabolic network modelling of nitrification and denitrification under cyanogenic conditions

Abstract

Simultaneous nitrification and aerobic denitrification (SNaD) is a preferred method for single stage total nitrogen (TN) removal, which was recently proposed to improve wastewater treatment plant design. However, SNaD processes are prone to inhibition by toxicant loading with free cyanide (CN-) possessing the highest inhibitory effect on such processes, rendering these processes ineffective. Despite the best efforts of regulators to limit toxicant disposal into municipal wastewater sewage systems (MWSSs), free cyanide (CN-) still enters MWSSs through various pathways; hence, it has been suggested that CN- resistant or tolerant microorganisms be utilized for processes such as SNaD. To mitigate toxicant loading, organisms in SNaD have been observed to adopt a multiphase growth strategy to sequentially degrade CN- during primary growth and subsequently degrade TN during the secondary growth phase. However, CN- degrading microorganisms are not widely used for SNaD in MWSSs due to the inadequate application of suitable microorganisms (Chromobacterium violaceum, Pseudomonas aeruginosa, Thiobacillus denitrificans, Rhodospirillum palustris, Klebsiella pneumoniae, and Alcaligenes faecalis) commonly used in single-stage SNaD. The use of CN- degrading or resistant microorganisms for SNaD is a cost-effective method compared to the use of other methods of CN- removal prior to TN removal, as they involve multi-stage systems (as currently observed in MWSSs). The use of CN- degrading microorganisms, particularly when used as a consortium, presents a promising and sustainable resolution to mitigate inhibitory effects of CN- in SNaD. However, SNaD is known to be completely inhibited by CN- thus it is imperative to also study some thermodynamic parameters of SNaD under high CN- conditions to see the feasibility of the process. The Gibbs free energy is significant to understand the feasibility of SNaD, it is also vital to study Gibbs free energy to determine whether or not the biological reaction is plausible. The relationship between the rate of nitrification and Gibbs free energy was also investigated. The attained results showed that up to 37.55 mg CN-/L did not have an effect on SNaD. The consortia degraded CN- and achieved SNaD, with degradation efficiency of 92.9 and 97.7% while the degradation rate of 0.0234 and 0.139 mg/L/hr for ammonium-nitrogen (NH4-N) and CN- respectively. Moreover, all the free Gibbs energy was describing the individual processes were found to be negative, with the lowest Gibbs free energy being -756.4 and -1830.9 Kcal/mol for nitritation and nitratation in the first 48 h of the biological, reaction respectively. Additionally, a linear relationship between the rate of NH4-N and nitrite-nitrogen (NO2-N) degradation with their respective Gibbs free energy was observed. Linear model was also used to predict the relationship between NH4-N, NO2-N degradation and Gibbs free energy. These results obtained showed a good correlation between the models and the experimental data with correlation efficiency being 0.94 and 0.93 for nitritation, and nitratation, respectively. From the results found it can be deduced that SNaD is plausible under high cyanide conditions when cyanide degrading or tolerant microorganisms are employed. This can be a sustainable solution to SNaD inhibition by CN- compounds during wastewater treatment. Furthermore, a single strain was purified from the consortium and identified as Acinetobacter courvalinii. This bacterial strain was found to be able to perform sequential CN- degradation, and SNaD; an ability associated with multiphase growth strategy of the microorganism when provided with multiple nitrogenous sources, i.e. CN- and TN. The effect of CN- on nitrification and aerobic denitrification including enzyme expression, activity and protein functionality of Acinetobacter courvalinii was investigated. It was found that CN- concentration of up to 5.8 mg CN-/L did not affect the growth of Acinetobacter courvalinii. In cultures whereby the A. courvalinii isolate was used, degradation rates of CN- and NH4-N were found to be 2.2 mg CN-/L/h and 0.40 mg NH4-N/L/h, respectively. Moreover, the effect of CN- on NH4-N, nitrate-nitrogen (NO3-N) and NO2-N oxidizing enzymes was investigated, with findings indicating CN- did not affect the expression and activity of ammonia monooxygenase (AMO), but affected the activity of nitrate reductase (NaR) and nitrite reductase (NiR). Nevertheless, a slow decrease in NO2-N was observed after the addition of CN- thus confirming the activity of NaR and the activation of the denitrification pathway by the CN-. Moreover, five models’ (Monod, Moser, Rate law, Haldane, and Andrew’s model) ability to predict SNaD under CN- conditions, indicated that only Rate law, Haldane and Andrew’s models, were suited to predict both SNaD and CN- degradation. Due to low degradation rates of NH4-N and CN-, optimization of SNaD was essential. Therefore, response surface methodology was used to optimize the SNaD under CN- conditions. The physiological parameters that were considered for optimization were temperature and pH; with the result showing that the optimum for pH and temperature was 6.5 and 36.5oC respectively, with NH4-N and CN- degradation efficiency of 50 and 80.2%, respectively. Furthermore, the degradation kinetics of NH4-N and CN- were also studied under the optimum conditions in batch culture reactors, and the results showed that up to 70.6% and 97.3% of NH4-N and CN- were simultaneously degraded with degradation rates of 0.66 and 0.41 mg/L/h, respectively. The predictive ability of RSM was further compared with cybernetic models, and cybernetic models were found to better predict SNaD under CN- conditions. These results exhibited a promising solution in the management of inhibition effected of CN- towards SNaD at an industrial scale.