Effectiveness of plant residues for biological nitrate reduction in freshwater systems

A laboratory study was conducted to determine the effectiveness of plant residues as carbon sources and physical support for denitrifying bacteria in freshwater systems using a batch method. Main effects of plant residue types with different biochemical qualities and particle sizes were particularly examined also the interacting effect of those factors during the process. A preliminary experiment was conducted for one week to test whether the batch culture system would be satisfactory or not for the reduction of nitrate levels in a freshwater system. Nitrate was added into the system with different concentrations: 0, 100, and 200 mg/L KN0₃. Glucose was used for the preliminary experiment instead of plant residues, and added with different concentrations: 0, 50, 100, and 150 g/L. Phosphate (as K₂RP0₄) at 3 mg/L was added for the bacterial growth. Dionised water was used to fill up the system representing freshwater system. After being autoclaved for 20 minutes at 6.8 kg (15 lb) of pressure, all reactors except the controls were inoculated with 5-g sediment of Liffey Stream in Lincoln Village. The reactors were kept inside an incubator at 25°C for one week. The samples were taken at the beginning and end of the experiment, which were then analysed for nitrate-N, nitrite-N, and ammonium-N concentrations using the colorimetric method. The results showed that a significant reduction of nitrate-N occured using the batch culture system, whereas insignificant ammonium-N concentrations produced with high glucose and low nitrate concentrations. No nitrite-N concentration was detected in all samples. These results confirmed that the batch system was satisfactory for detecting nitrate reduction in water systems. After the preliminary experiment, a main laboratory experiment using the same batch culture system was conducted using two types of plant residue: straws of oat (Avena sativa) and pea (Pisum sativum L.) representing plant residues with high and low C/N ratios respectively. These plant residues were taken freshly from the field after harvest. Three different particle sizes: very fine (50.5 mm), medium (0.5 - 1 cm), and field size (≤ 5 cm) of each kind of the plant residues were used. Each plant residue was added in the amount of 50 g/L. Nitrate (KN0₃) was added at 200 mg/L, while phosphate (K₂RP0₄)) was added at 3 mg/L. After being filled up with dionised water and autoclaved for 20 minutes at 6.8 kg (15 lb) of pressure, 5-g sediment of Liffey stream in Lincoln Village were inoculated into each reactor except the controls, which received all the chemicals but no plant residues. All reactors were kept inside an incubator at 25°C. The main experiment was conducted for 8 weeks. The samples of incubated solution were taken at the beginning of the main experiment and then once a week for 8 weeks. The samples were analysed for nitrate-N, nitrite-N, and ammonium-N concentrations using the colorimetric method, the pH values using the electrometric method, and the number of denitrifying bacteria using the Most-Probable-Number method. Average daily temperatures of the incubator were recorded at the same time daily over the whole period of the main experiment. Results were statistically analysed using Repeated Measures ANOVA to examine the effects of particle size, plant residue types and the incubation time as well as the interacting effect between those factors on the nitrate reduction process. The results obtained showed that each of the two plant residues studied was effective as the physical support and carbon source for the nitrate reducing bacteria during the batch experiment. Particle size and plant residue type interacting with the incubation time significantly affected nitrate reduction process. Oat straws showed significantly rapid and higher nitrate reduction than pea straws (P < 0.001). Larger particle sizes of both plant residues caused significantly higher nitrate reduction than smaller particle sizes, especially the particle size of ≥ 5 cm. On the other hand, no significant variation of the nitrite concentrations occurred suggesting there might be several other factors influence the production of nitrite in the system. Only plant residue types independent of the incubation time significantly affected ammonium production (P < 0.001). As the present study involved only two types of plant residues, further studies, both laboratory and field experiments, should be undertaken to ascertain the effects of plant residue qualities on the process of nitrate reduction in the freshwater systems.