Nitrate removal from groundwater using an activated sludge as a source of bacteria

Life on Earth relies on the existence of water (Saleh & Gupta 2012; Saravanan et al. 2014). Water pollution by organic and inorganic compounds is a matter of great public concern (Gupta et al. 2002; Gupta & Saleh 2013; Jerroumi et al. 2020). Recent anthropogenic activities are greatly increasing the amount of nitrogen cycling between the living world and the soil, and the water and the atmosphere (Galloway et al. 2003). The main sources of nitrate pollution are agricultural activities (especially fertilization), wastewater, leakage from landfills, and nitrogen solubility in the atmosphere (Abdel-Aziz et al. 2019). The nitrate contamination of groundwater has become an environmental concern and a public health problem. In fact, nitrate is a highly mobile form of nitrogen in the soil, making it a dangerous pollutant in water, and can cause adverse effects on human health, including methemoglobinemia in infants (Chen et al. 2017; Zhai et al. 2017). For those reasons, the World Health Organization and Morocco stipulated that the maximum nitrate concentration in drinking water is 50 mg NO₃⁻/L (⁠⁠) (WHO 2011; NM03.7.001).

In fact, bacterial cells take longer to grow and adapt to the new environment (Shimp & Pfaender 1987). In the same way, Dey & Mukherjee (2010) and Cozma et al. (2012) tested the biodegradation of phenol in a batch reactor. They found that the duration of adaptation phase increased with the substrate concentration. Indeed, the evolution of the adaptation phase was mentioned by some authors (Lekhlif et al. 2015b). They treated a synthetic petrochemical wastewater containing benzoic acid in a laboratory scale using a sequential batch reactor (SBR). They tested concentrations of synthetic solutions of 50, 100, 150, and 200 mg/L of benzoic acid, which were inoculated by an activated sludge collected from the municipal sewage treatment plant and aerated for acclimatization in synthetic solution of 200 mg/L of benzoic acid.

This paper is dedicated to the investigation of the nitrate removal efficiency of nitrate-rich groundwater as a function of several parameters such as the C/N/P ratio required for bacterial multiplication, the bacteria adaptation, and the aeration period.

In this study, we treated the nitrate-rich groundwater with heterotrophic denitrification (HD) using activated sludge as a source of bacteria and glycerol as a source of carbon. In addition, a comparison of process performance using continuous and batch aeration is provided.

The nitrate-contaminated synthetic water was prepared from sodium nitrate, phosphorus, and glycerol in a Chemical Oxygen Demand (COD)/N/P ratio of 50/4/1. Pure glycerol was used as an organic substrate. The salts of sodium nitrate and sodium phosphate were used as the source of nitrogen and the source of phosphorus, respectively. The seeding of the reactor was carried out using bacteria extracted from the activated sludge. The composition of the synthetic water for 5 L of distilled water is presented in Table 1.

The pH was determined using a consort C6010 pH meter, whose probe also measures the temperature. Turbidity was measured using a turbidimeter type HACH 2100P. The nitrate and nitrite concentrations were measured using an ion chromatograph device of the type ΩMetrohm 881 Compact IC pro according to the standard NF EN ISO 10304-1. A Metrosep A Supp 5-250/4.0 column, composed of an anion exchange resin, was used for anion separation. The mobile phase consisted of a mixed solution of 3.2 mM Na₂CO₃ and 1 mM NaHCO₃ with a flow rate of 0.7 mL/min. The peaks of the different ions analyzed were identified and quantified by MagIC Net software, version 2.3.

In this study, four tests were performed:

• The first test studied the effect of the COD/N/P ratio on nitrate removal efficiency. For this test, three ratios were used: 100/4/1, 50/4/1, and 25/4/1, respectively.

• The second test studied the effect of adaptation on nitrate removal efficiency for synthetic waters with an initial concentration of nitrate (100 mg-nitrate/L), which was prepared according to the ratio of 50/4/1.

• The third test studied the effect of aeration on nitrate removal efficiency by comparing batch and continuous aeration using synthetic water containing 100 mg/L of nitrate, which was prepared according to the 50/4/1 ratio.

• The fourth test consisted of nitrate removal by avoiding the accumulation of nitrites, which are produced by the reduction of nitrates by changing the aeration cycle.

In each of these tests, the first synthetic solution was added to the submerged aerobic fixed-film reactor, which was filled with a clean packing. After the total nitrate removal or the stability of concentration in the case of the first adaptations, the bioreactor was evacuated and drained out for 15 min. Furthermore, the second synthetic solution was added without washing the bioreactor. This procedure was used for all the tests cited above. Samples were taken and filtered through syringe filters with a porosity of 0.45 μm to determine nitrate and nitrite concentrations. All the experiments were repeated three times and average values were reported.

To investigate the effect of COD/N/P ratio on nitrate removal, we varied the COD/N/P ratio to reveal its effect on the nitrate removal efficiency for the first adaptation phase of denitrifying bacteria. In this study, we used three ratios: 100/4/1, 50/4/1, and 25/4/1.

For the other two ratios (COD/N/P = 50/4/1, 25/4/1), limiting conditions after 12 h were noticed. In fact, the removal rates were 60.6 and 27.19%, respectively. The COD/N/P ratio is a limiting factor influencing the HD rate, since organic carbon is essential for the survival of heterotrophic bacteria. Indeed, a high C/N ratio could accelerate the growth of heterotrophic denitrifying bacteria in the biofilm and thus favor the total denitrification rate (Zhou et al. 2007; Warneke et al. 2011).

In this figure, an increase in pH is observed for the different tests. This is probably due to the process of anoxic HD that takes place within the biomass attached as a biofilm to the packing supports. This results in the degradation of biofilm proteins (release of hydroxide ions). Indeed, many authors (Vives Fàbregas 2004; Akın & Ugurlu 2005) have observed this phenomenon. This behavior may also be due to the release of carbon dioxide. Lekhlif et al. (2015a) observed the same evolution in tests carried out on glycerol. Other authors focused on the causes of the increase in pH. The detachment of the biofilm can trigger a phenomenon of degradation of proteins in the biomass. Et-taleb et al. (2014) showed that the increase in pH can be explained by the consumption of nitrogen compounds during reactor aeration. They showed identical pH behavior after almost 2 h of aeration in an SBR system.

The third adaptation test, however, shows a slight increase in turbidity. This could be the result of the endogenous respiration process that occurs in the biofilm after the organic substrate has been almost exhausted (Zhao et al. 2015; Kherbeche et al. 2017), causing the biofilm to slough off the packing material.

Figure 5 shows that nitrites are produced during the aerobic treatment process. These nitrites are probably formed in areas where aeration is inaccessible when oxygen diffusion becomes limiting, especially in the deep layers of the biofilm; the transformation of nitrates into nitrites takes place in an anoxic environment. The nitrite production increases as the biofilm matures and becomes more compact. Their concentration then decreases slightly over time.

Many researchers noted that this denitrification occurs in an aerobic environment (Robertson et al. 1988; Ahn 2006; Joshi et al. 2014). Cheikh et al. (2013) studied the novel water denitrification technique based on immobilized bacterial biomass using various plastic wastes as supports in packed columns. They showed that the denitrifying bacteria, obtained from activated sludge present in a local municipal wastewater treatment plant, were capable of treating the water at an inlet nitrate concentration of 600 mg/L with an elimination rate close to 100%. Many researchers used activated sludge, collected from wastewater treatment plants, as a source of denitrifying bacteria (Zhao et al. 2012; Maintinguer et al. 2013).

As for nitrates, we noted a decrease in their concentration, which improved as the bacteria became adapted. Nitrate removal or reduction results from the heterotrophic degradation process, in which bacteria consume nitrogen as a trace element, i.e., nitrogen assimilation into the biomass. It also occurs through the process of anoxic denitrification within the biofilm, as demonstrated by the denitrification process (reduction of nitrates to nitrites) outlined above. Many authors have observed this behavior of nitrates in the first stage of the denitrification reaction. De Filippis et al. (2013) observed that during the denitrification of highly loaded wastewater in an SBR reactor, nitrates were reduced to nitrite ions and then totally reduced to nitrogen gas. Aouati et al. (2017) conducted a biological denitrification study on synthetic waters using a mixed culture taken from a treatment plant. They chose sodium succinate, the buffer mixture of acetic acid, and sodium acetate and date flour as the carbon source. They showed that nitrate degradation using all three carbon sources is complete and does not stop at the nitrite stage after 80 h of treatment. The occurrence of nitrite is probably related to the removal of nitrate according to the denitratation reaction that takes place within the biofilm in the absence of oxygen.

Figure 5 also shows that the nitrite concentration ultimately stabilizes for all three tests. It reaches values of 0.86, 5.79, and 38.85 mg-nitrite/L for the first, second, and third adaptation, respectively. This stabilization can probably be explained by the absence of nitrite-reducing enzymes (nitrite reductase).

To remove accumulated nitrite, we changed the aeration mode. Subsequently, we noticed a decrease in the concentration of nitrite until it disappeared completely. The complete elimination of the produced nitrites was obtained after 12 h of treatment under discontinuous aeration (aeration period = 1 h). This elimination can be explained by the production of enzymes (nitrite reductase) capable of transforming nitrites into nitrogen gas.

The tests carried out in this study on the heterotrophic biological denitrification reaction under alternative aeration, using activated sludge as a source of bacteria, of synthetic water contaminated by nitrates. The results obtained draw the following conclusions:

In all the tests, the pH increased due to the anoxic HD process that takes place in the biomass attached as a biofilm to the plastic packing supports. Turbidity varied initially for the first addition and after each replenishment of the bioreactor with the synthetic solution. It was affected by the solid suspension of activated sludge used to inoculate the synthetic solution as well as by loose biofilm particles that were not drained. Nitrates are removed over time, especially when the bacteria are adapted. Nitrate removal could be explained either by a heterotrophic assimilation process, in which the bacteria consume nitrogen as a trace element or by an anoxic process that settles within the biofilm when the diffusion of oxygen becomes limiting. Furthermore, the accumulation of nitrites can be explained by the absence of the enzyme nitrite reductase. The complete disappearance of nitrites is mainly due to the change in the aeration mode. The efficiency of the HD reaction increased to 99.42% after 8 h of treatment with the total disappearance of nitrite after 12 h of treatment under discontinuous aeration (aeration period = 1 h).

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.