Influence of Carbon Source on Biological Nitrogen Removal by Immobilised Bacteria

Wilawan Khanitchaidecha, Tatsuo Sumino, Futaba Kazama

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DOI: 10.4236/jwarp.2010.26059                

Abstract

Acetate, ethanol, and hydrolysed rice were used as external carbon sources in an ammonium removal process employing immobilised bacteria. The influence of the carbon source on the occurrence of free cells and total nitrogen removal efficiency was examined at C/N ratios of 1.5 (low), 2.5 (medium), and 3.5 (high). At the low C/N ratio, no free cells were found in the reactors and the use of acetate as the carbon source resulted in the highest total nitrogen removal efficiency, followed by ethanol and hydrolysed rice. The occurrence of free cells in reactors fed with acetate and ethanol led to a negligible increase in the total nitrogen removal efficiency with increasing C/N ratio. The results suggest that acetate is the most appropriate carbon source for nitrogen removal and that the number of free cells should be minimized to achieve the highest efficiency during long-term operation.

Keywords

Nitrogen Removal, Nitrification and Denitrification, Carbon Sources

1. Introduction

During the past few decades, biological ammoniumnitrogen (NH₄-N) removal has emerged as one of the most interesting methods for water and wastewater treatment. The system consists of a nitrification process (NH₄-N ® NO₃-N (nitrate-nitrogen)) followed by denitrification to produce non-toxic nitrogen gas (NO₃-N ® N₂). In general, nitrification is an aerobic process and denitrification is an anaerobic process. A source of organic carbon is an important component of the denitrification process. Several sources of carbon have been used including acetate [1-3], methanol [2,4], ethanol [1,3], glucose [3], peptone [3], glycerol [5], and lactic acid [5]. Additionally, solid waste-derived carbon sources such as molasses [6], corncobs [7], and excess sludge [8] have also been used. Table 1 provides a comparison of the nitrogen removal efficiency obtained using various carbon sources. Of these, acetate and ethanol are attractive and versatile substrates due to biodegradability, low consumption (mg-C/mg-N), and low toxicity. Despite the effort invested in exploring biological nitrogen removal, there are few reports on the influence of the carbon source on N removal efficiency, particularly acetate vs. ethanol [9,10], and no research on the occurrence of free cells when using various carbon sources. Free cells are commonly found in immobilised sludge systems, and result in ineffective NH₄-N removal. The present work examines the influence of external carbon sources including acetate, ethanol, and hydrolysed rice (representing a solid waste source) on nitrogen removal. The use of hydrolysed rice in NH₄-N removal provides a potential means of waste reduction.

2. Materials and Methods

2.1. Solution Preparation

Simulated wastewater influent solution containing 40 mg/L of NH₄-N was prepared by mixing 0.19 g/L of (NH₄)₂SO₄, 0.48 g/L of NaHCO₃, 0.05 g/L of KCl, 0.11 g/L of CaCl₂·2H₂O, 0.1 g/L of MgSO₄·7H₂O, and 0.02 g/L of Na₂HPO₄·12H₂O. The carbon concentration of acetate and ethanol feed solutions was fixed at 7.1 g/L.

Hydrolysed rice was prepared by adding 35 g of rice and 18 mL of 6 M HCl to 1L of tap water. The solution was heated to 90ºC for 24 hours. The resulting carbon concentration of the hydrolysed rice solution was 6.5-7.0 g/L of total organic carbon (TOC) and ~0.1 g/L of volatile organic carbon (VOC).

Table 1. Nitrogen removal efficiency and C/N ratio requirement observed using various carbon sources.

2.2. Polyethylene Glycol (PEG) Pellet Preparation

Sludge was collected from the first sedimentation tank of a drinking water plant in Kofu, Japan. A 34.5 mL portion of 10% concentrated sludge was mixed with 10.5 mL of a solution containing PEG pre-polymer and promoter, and 5 mL of potassium persulfate (K₂S₂O₈) was added to initiate polymerization. The polymerized gel was cut into 3 mm cubes referred to as PEG pellets.

2.3. Reactor Set-up

A 3L acrylic reactor (12.5 × 16 × 20 cm) was separated into two identical chambers (the aeration and non-aeration chambers) by an acrylic plate perforated by a number of small holes (Figure 1). The aeration chamber contained 8 g of support material (polyester textile, Networking of Engineering and Textile Processing (NET) Company, Japan) and 0.05 L of 10% concentrated sludge. The bulk dissolved oxygen (DO) concentration was maintained at 5-6 mg/L, and the NH₄-N solution (influent) was continuously supplied at a rate of 0.2 L/h. The non-aeration chamber contained 0.6 L of PEG pellets, and the bulk DO concentration ranged from 4-5 mg/L. In order to investigate the effect of carbon source at various C/N ratios, the carbon source solution was fed to the non-aeration chamber for 10 min every 4 hours at a rate of ~42 mL/h (for the 1.5 C/N ratio), ~70 mL/h (for the 2.5 C/N ratio), or ~98 mL/h (for the 3.5 C/N ratio). The hydraulic retention time of the reactors was approximately 12 hours.

2.4. Analytical Methods

The influent and effluent were analysed for NH₄-N, NO₂-N, and NO₃-N concentrations using standard methods [12]. The total suspended solids (TSS) and VOC

Figure 1. Schematic diagram of reactor design and operation.

contents were measured at irregular intervals in accordance with standard methods [12]. The organic carbon concentration was analysed using an organic carbon analyser (Shimadzu TOC-5050A). The temperature, pH, and DO concentration of the reactor were measured onsite.

3. Results and Discussion

In the aeration chamber of all reactors, the complete liquid recirculation produced by continuous aeration resulted in attachment of the sludge bacteria to the supporting material. The conditions in this chamber included a high DO concentration and continuous influent NH₄-N feed, resulting in nitrification being the main process in this chamber. Cultivation of nitrifying bacteria was indicated by a change in colour of the attached bacteria from dark to light brown. Nitrification was not the rate-limiting step in the process, since the zero concentration of NH₄-N in the effluent indicated complete nitrification (Figures 2(a-d)).

The non-aeration chamber directly communicated with the aeration chamber through the perforated divider, and the water containing high concentrations of NO₃-N and DO was immediately introduced to the non-aeration chamber. The DO concentration in the non-aeration chamber was 4-5 mg/L. Although this concentration is somewhat higher than usual for denitrification, the limited oxygen diffusion into the pellet and oxygen consumption by bacteria near the pellet surface led to formation of an anaerobic zone in the pellet core where denitrification could occur. The NO₃-N and added carbon (i.e. acetate, ethanol) diffused into the core as explained by Jun et al. [13]. The denitrifying bacteria were concentrated in the pellet core, while the region near the surface predominantly contained competing aerobic heterotrophic bacteria. In the present work, the denitrification process was the rate-limiting step of NH₄-N removal and the efficiency of the denitrification process depended on the growth rate of competitive bacteria, the carbon consumption, and the carbon source. The effect of carbon

Figure 2. Nitrogen time course for (a) acetate-fed reactor; (b) ethanol-fed reactor; (c) hydrolysed rice-fed reactor, and (d) zero carbon feed reactor.

source on denitrification and growth of competing bacteria was examined during long-term operation (60 days).

Figure 3 depicts the total N (NH₄-N + NO₂-N + NO₃-N) removal efficiency for reactors fed with various carbon sources. It is not helpful to compare the efficiency be-

Conflicts of Interest

The authors declare no conflicts of interest.

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