Heterotrophic nitri ﬁ cation and aerobic denitri ﬁ cation using pure-culture bacteria for wastewater treatment

Due to the high water demand and unsustainable water resource, wastewater reclamation and wastewater treatment prior to discharge have become current important issues. Various treatment technologies, such as biological processes, have been improved as alternatives. In this study, the biological nitrogen removal system using pure-culture Bacillus licheniformis was developed and used as an internal treatment unit in an aquarium to improve the ef ﬂ uent quality for water reuse. The ef ﬁ ciencies for NH 4 -N and total nitrogen (TN) removal and the quality of treated water veri ﬁ ed the occurrence of heterotrophic nitri ﬁ cation and aerobic denitri ﬁ cation; the nitri ﬁ cation rate was 0.84 mg/L-h and the denitri ﬁ cation rate was 0.62 mg/L-h. The maximal NH 4 -N and TN removal ef ﬁ ciencies were approximately 73% at the in ﬂ uent NH 4 -N of 30 mg/L. However, the other competitive heterotroph of Pseudomonas sp. was observed, which resulted in dramatically decreasing ef ﬁ ciencies and an enlarged ratio of carbon consumption and nitrogen removal. Although the overall performance of the B. licheniformis system was lower than the system using mixed-culture nitrifying and heterotrophic denitrifying microorganisms, the advantages of the B. licheniformis system were ease of operation and the fact that it is a land-limited treatment system. The research is ongoing to enhance performance and maintain excellent ef ﬁ ciency in a long-term operation.


INTRODUCTION
Over the past decade, the demand for fresh water has drastically increased with rapid growth in population, global climate change and growing scarcity of surface water and groundwater resources. The lack of fresh water has accelerated efforts to improve the current treatment technology for reclamation of wastewater, such as domestic wastewater.
The concept of water reclamation is to remove a high pollutant concentration in the wastewater to an acceptable level, and then reuse the water for other purposes, such as agricultural irrigation, surface water restoration, groundwater recharge and industrial manufacturing processes (Maryam & Buyukgungor ). One of the most important pollutants is ammonium-nitrogen (NH 4 -N) which is commonly found in many wastewater sources including household, industry, landfill and aquaculture. In the meanwhile, the discharge of NH 4 -N wastewater to the environment causes a poor quality of water resource and consequently affects water pollution from eutrophication.
The nitrogen contamination including NH 4 -N and its oxidized forms of nitrate (NO 3 -N) and nitrite (NO 2 -N) cannot be removed by common filtration, and the consumption of such contaminated water has negative impacts to human health, in particularly infants and pregnant women. The maximal level of nitrogen in safe drinking water has been set at 1.5 mg/L as NH 4 -N, 1.0 mg/L as NO 2 -N and 11.3 mg/L as NO 3 -N (World Health Organization (WHO) ). Similarly, the standard limitation of nitrogen in the reclaimable water is established and dependent on the type of water reuse, for example 45 mg/L as total nitrogen (TN) for restricted irrigation (i.e., orchards, industrial crops and fodder) and 30 mg/L as TN for unrestricted irrigation (i.e., public parks and urban uses) (Massachusetts Institute of Technology (MIT) ).

Various advanced technologies have been proposed for
water reclamation and reuse, especially for nitrogen removal; membrane filtration and biological processes are examples (Shrimali & Singh ). Due to the limit of using membranes, in terms of scaling and treatment capacity, the application of membrane filtration for wastewater treatment is inappropriate in some areas. On the other hand, the biological nitrogen removal process includes an aerobic condition in which nitrification occurs by autotroph  and an anoxic condition to allow denitrification by heterotroph (NO 3 -N to nitrogen gas conversion). The nitrification and denitrification processes are difficult to jointly operate in a single reactor, due to the low growth rate of autotrophic nitrifying microorganisms and the different environments for achieving nitrifying and denitrifying activity. Recently, the simultaneous nitrification and denitrification (SND) system was developed to achieve nitrogen removal using either immobilized or suspended sludge.
The system required a low dissolved oxygen (DO) of about 0.3-0.8 mg/L during the treatment, and the coexistence of nitrifying and denitrifying microorganisms was found (Pochana & Keller ; Peng & Qi ). To enhance the nitrification and denitrification rates, the SND system was operated via an intermittent air supply for providing the aerobic and anoxic conditions. The rates of nitrification and denitrification reached 18 and 6 mg/L-h, respectively (Khanitchaidecha et al. ). The significant factor for success in SND was an abundance of organic carbon. This is because complete denitrification cannot occur when the organic carbon is deficient. In the theoretical denitrification equation, 1.08 mole of organic carbon (i.e., methanol) is required to remove a mole of NO 3 -N (Tchobanoglous & Burton ).
However, in the practical operation of nitrification and denitrification, the ratio of organic carbon and nitrogen contents was reported in the range of 7.5-11.1 (Zhao et al. ). Of the above microorganisms, the Bacillus species is advantageous over the others and is the key in the wastewater treatment system. This is because Bacillus can consist of aerobes and facultative anaerobes which live in a wide range of habitats, thus a large volume of Bacillus in the treatment system is easily isolated from the environment. In addition, Bacillus is nontoxic and tolerant to temperature, pH and salt conditions. The objective of this study was to determine the ability of Bacillus licheniformis (B. licheniformis) to remove NH 4 -N, based on its heterotrophic nitrification and aerobic denitrification. The system containing B. licheniformis carriers was started up by step-wise increasing the initial NH 4 -N from 10 to 30 mg/L, and then continuously operated at the high NH 4 -N concentration of 40 mg/L. The organic carbon was maintained at the C/N ratio of 3.5. The efficiencies of NH 4 -N removal and TN removal, including NH 4 -N, NO 3 -N and NO 2 -N, were used to indicate the system performance. Both values were compared to the system using cultivated sludge from a wastewater treatment plant. The purpose of this study was to demonstrate the potential of the heterotrophic nitrification and aerobic denitrification process to enable the reuse of wastewater as an alternative water resource for human activity. The outcome of this study can reduce the demand on natural water resources. The nitrogen wastewater (influent) was prepared with the following chemicals (per 1 L); 0.15 g of NH 4 Cl, 0.48 g of NaHCO 3 , 0.02 g of KH 2 PO 4 and 0.48 g of CH 3 COONa.

MATERIALS AND METHODS
The influent was continuously fed to the reactor with a flow rate of 3 L/d. Air was continuously supplied to maintain a high DO of ∼5 mg/L. During the start-up, the NH 4 -N concentration was step-wise increased from 10 to 30 mg/L for bacteria adaptation, as summarized in Table 1. Further, another reactor (mixed-culture reactor) was also set up in accordance with the above procedure. However, the mixed-culture nitrifying-denitrifying sludge was attached on the porous materials, instead of B. licheniformis. The mixed-culture sludge was taken from a successful nitrification and heterotrophic denitrification system which was operated for over 3 months. The mixed-culture reactor was operated under continuous aeration (similarly to the B. licheniformis reactor) and intermittent aeration for the best performance.

Water quality analysis
The nitrogen removal ability of B. licheniformis was measured by two parameters: NH 4 -N removal efficiency and TN removal efficiency. The reduction of NH 4 -N concentration between the influent and effluent was only relevant for the NH 4 -N removal (2)

Microbial analysis
The pure culture of dominant bacteria was isolated according to the following procedure: (i) randomly take biomass samples from the porous materials; (ii) isolate on the nutrient broth by spread plate and streak plant techniques and keep at 37 C for 24-48 hours; and (iii) mix the pure culture solution with glycerol 30% and direct to a private company for further molecular analysis.

RESULTS AND DISCUSSION
The   The results are shown in Figure 3(a) and 3(b), the NH 4 -N and NO 3 -N were linearly reduced with the rates of 0.84 and 0.62 mg/L-h, during 24 hours. The NH 4 -N consumption rate was higher than the NO 3 -N consumption rate because B. licheniformis was familiarized with NH 4 -N feeding during start-up.
In this study, the NH 4 -N consumption represented the nitrification ability and the NO 3 -N consumption under aerobic conditions referred to the denitrification ability. Therefore, the nitrification and denitrification rates of B. licheniformis were lower than the autotrophic nitrification and heterotrophic denitrification rates, which were 4.23 and 4.15 mg/L-h, respectively, as reported in a previous study (Zeng et al. ).
The influent NH 4 -N was increased to the highest concentration of 40 mg/L during day 16-30, as shown in Therefore, some NH 4 -N at the higher concentration was untreated and then contaminated the effluent. In this study,     Figure 2(a)). On the other hand, the NH 4 -N removal efficiency reached 100% in the mixed-culture reactor, however there was almost zero TN removal. The oxidized NH 4 -N mainly remained in NO 3 -N form, as illustrated in Figure 5(a) and 5(b). Since the heterotrophic denitrification required an anoxic condition to transfer the NO 3 -N to gaseous nitrogen, the high DO of ∼5 mg/L during the treatment prevented the denitrification from occurring. Later, the condition of the mixed-culture reactor was changed to the intermittent air supply: 2 hours aeration and 2 hours non-aeration in a cycle. From Figure 5(a), the effluent NO 3 -N concentration was sharply reduced to 7.1 mg/L and some NO 2 -N of 9.6 mg/L was found. The TN removal efficiency was immediately increased to 51.6% and continued to 72.2% at the maximum. However, the efficiency of NH 4 -N removal slightly dropped to approximately 78.2% during the intermittent aeration. Since the NH 4 -N reduction referred to nitrification and the NO 3 -N reduction referred to denitrification, the ability for nitrification was greater than that for denitrification in the mixedculture reactor. From the DO measurement, the aerobic DO concentration of 5 mg/L decreased to around 1.5-2.0 mg/L in the non-aeration period. Therefore, the denitri- However, the latter reactor required specific conditions including the aeration period for nitrification and the in the B. licheniformis reactor indicates that the nitrogen removal system via heterotrophic nitrification and aerobic denitrification needs to be improved before using as the treatment system for wastewater and reclaimable water.