Removal of nitrate from groundwater by eggshell biowaste

The importance of removing pollutants, especially hazardous cationic heavy metals and anions, is increasing as a result of growing water demands in semi-arid areas all over the world. Pollution is, therefore, one of the most serious problems affecting our reality and expresses its seriousness in more than one area in the aspects of community life. The problem of pollution will exacerbate over time and years and will lead to an increasing demand for qualified water due to the increase in population. Consequently, there will be an increase in pressure on the quantity and quality of water abstracted (Rezaee et al. 2008). One of the most serious issues of water contamination is the presence of the nitrate anion. As a natural source, nitrate presents at moderate concentrations resulting from the degradation of organic nitrogen-containing compounds. Their presence in nature is mostly in rocks, organic matter, and soil. The presence of decaying organic matter deep in the earth is a polluting source of nitrates in nature as well (Hagerty & Taylor 2012).

The main cause of high levels of nitrate is the intensive use of fertilizers and pesticides as well as the filtration of wastewater into the aquifer. Many factors can affect nitrate groundwater contamination levels, including fertilizer utilization rate, chemicals introduced in the manufacture of explosives and unloading them as waste materials and also from source cultivation of leguminous crops acting on the stabilization of nitrogen in the atmosphere which raise nitrate concentration (Afkhami 2003). For the purpose of prevention, the World Health Organization (WHO) has established a maximum nitrate concentration in water of 50 mg/L (WHO 2016), while the US Environmental Protection Agency (EPA) has set a maximum permissible nitrate concentration of 10 mg/L (Buss et al. 2005).

Nitrate is currently one of the most important water pollution problems in many countries around the world including India, the UK, Saudi Arabia, North America, Australia, Changshu, Morocco, China, and Iran (Mohseni-Bandpi et al. 2013). It was recently observed that the Gaza Strip was exposed to high nitrate levels ranging from 40 mg/L to more than 300 mg/L. This indicates the danger of nitrates as exceeding international standards according to the World Health Organization (WHO 2016). People with health problems suffer from the risk of this ion. It has been observed that children less than six months of age, as well as the fetus, are most susceptible to this ion when the nitrate concentration is higher than 50 mg/L. Often nitrate leads to death in infants younger than six months of age. This is produced when the nitrate is converted to nitrite in the body of the fetus where the nitrite interferes with the energy-carrying oxygen in the blood, where the symptoms are shortness of breath and change of skin colour to blue (WHO 2017). Many studies have been conducted to treat the nitrate ion to provide an appropriate solution for these ions using inexpensive economical methods. Many physical and chemical techniques have been reported such as ion exchange (Song et al. 2012), reverse osmosis (Ali & El-Aassar 2018), catalytic reduction (Teng et al. 2018), electrodialysis (Kikhavani et al. 2014) and electrochemistry (Safari et al. 2015). These techniques are highly undesirable because of poor selectivity, high operating and maintenance costs and high salt generation as waste after treatment. In such a context, nitrate ions should therefore be disposed of or treated in safer ways (Apshankar & Goel 2020). New systems, such as biological denitrification removal systems, have been used as an example of anoxic microbial processes, which have been used as a way of dealing with the risks of this ion. This operation when performed can convert nitrate into nitrogen in four enzymatic steps via the following intermediates: nitrite (NO₂⁻), nitrogen oxide (NO) and dinitrogen oxide (N₂O) (Torrentó et al. 2010; Kong et al. 2016). These techniques require high economic costs. Therefore, attention has been paid to the use of biowastes as materials in the removal process. The purpose of this use is to reduce economic cost and avoid adverse side-effects (Intharapat et al. 2013).

One of the biowastes used in the removal process was egg husks. Several studies have been conducted on this aspect of treatment (Zulfikar & Setiyanto 2013; El-Kemary et al. 2018). Eggshells are available in more than one place, especially in open areas without being treated in advance as useless (Tsai et al. 2008; Nasrollahzadeh et al. 2016). One of the interesting characteristics of eggshells indicated is that they contains a high number of pores and have an attractive ability that can be used in an excellent manner. Eggshells are estimated to contain 7,000–17,000 pores (Stadelman et al. 2017). The chemical composition (%w/w) of eggshell has been reported as follows: calcium carbonate (94%), magnesium carbonate (1%), calcium phosphate (1%) and organic matter (4%) (Stadelman 2000; Tangboriboon et al. 2018). Eggshells have the ability to absorb some heavy metals as well as organic compounds and have given more effective results in removals. Eggshells can be used as feed for cattle and as organic fertilizer in agriculture (Pramanpol & Nitayapat 2006). Eggshells are used to remove nitrates from artificial groundwater contaminated with nitrates. Eggshells have a high clearance capacity in the decontamination of synthetic water from nitrate (Xu et al. 2016). A biochar composite with nano-zerovalent iron and eggshell powder composite exhibited higher nitrate removal efficiency even in the presence of the highest concentration (100 mg L⁻¹) of coexisting chloride ions (Ahmad et al. 2018).

The previous study of Xu et al. (2016) was based on the construction of a complex eggshell system for nitrate removal using external sulfur-based autotrophic denitrification, while in the new system presented in this work no sulfur-based nutrients were added. In the present study, the effects of various parameters were studied and are discussed in detail.

Eggshells were collected fresh from the distribution points of the local egg production factories. After collection, the eggshells with their membranes were washed by distilled water several times as preliminary cleaning from inside and outside to remove suspended contaminants. The eggshells then were air-dried for 12 hours at room temperature (RT) and were preserved to be dried at different temperatures (20–80 °C) for 24 hours. The eggshells were then ground using pestle and mortar to suitable particle sizes. The ground eggshells were sieved to different particle sizes using standard numbered sieves. Three types of particles with different size ranges were collected (coarse = 2.8 mm–710 μm, medium = 710–90 μm and fine <90 μm). The eggshell powders were stored in airtight containers for further use. No other chemical treatments were used before the adsorption experiments. The dried eggshells were used for incubation at variable temperatures (15–45 °C) using an incubator at different time intervals before being used as an adsorbent for nitrate removal.

Stock nitrate solutions (NO⁻_3aq) (1,000–1,500 mg/L) were prepared using distilled water from pure potassium nitrate in volumetric flasks and shielded from direct sunlight by covering with aluminium foil and stored in a refrigerator. Dilute solutions of the desired concentrations (50–1,500 mg/L) were prepared by dilution using distilled water when required.

Different pH values (3.5–8) were controlled using acetic acid (0.1 M)/sodium acetate (0.1 M) solution mixture.

Preliminary experiments were conducted to determine the percentage of nitrate removal from aqueous solution by eggshell biosorbent at different concentrations (50, 100, 250, 500, 1,000 and 1,500 ppm NO₃⁻) under room temperature (RT) using 10 g of the adsorbent powder of particle size (90–710 μm) which was dried at 80 °C. Each nitrate solution (100 mL) was mixed with the eggshell substrate for six days. Determination of nitrate removed was conducted at different time intervals and tested at λ_max = 220 nm using UV.

The effect of incubation temperature of eggshell adsorbent and adsorbate solution was examined at different temperatures. The biosorbent eggshell (10 g) of particle size (90–710 μm) was used to investigate the effect of the incubation temperature of both the adsorbent and adsorbate at various duration times. The adsorbate solution was controlled at pH 7–7.5 using acetate solution.

The effect of drying temperature of eggshell adsorbent was examined at different values (20, 37, 45 and 70 °C). The adsorbent eggshell (10 g) of particle size (710–90 μm) was mixed with the adsorbate solution (100 mL, 100 ppm) and was incubated at 29 °C (∼RT) at different time intervals.

The percentage of nitrate removal from aqueous solution by eggshell biosorbent was examined using 10 g of the ground eggshell at different particle sizes (coarse = 2.8 mm–710 μm, medium = 710–90 μm and fine <90 μm). The pH was controlled at 7.0–7.5 and the drying and incubation temperatures of both the biosorbent and adsorbate solution were controlled at 45 °C and 29^o C respectively. The adsorbate (nitrate solution) (100 mL, 100 ppm) was mixed with the eggshell biosorbent and the amount of nitrate removed was determined.

The effect of adsorbent dose on the % efficiency of nitrate removal was studied. Different doses of eggshell biosorbent material were used (5, 10 and 15 g) of particle size (710–90 μm). The nitrate solution (100 mL) was mixed with the eggshell biosorbent for four days. Determination of nitrate absorbed was conducted at different time intervals.

Different pH values were used to examine the effect of pH on % nitrate removal by eggshell bio-absorbent. The various pH values were controlled using acetic acid–acetate solutions at pH 3.5–9.0. The nitrate solution (100 mL, 100 ppm) was mixed with the eggshell bio-adsorbent and the amount of nitrate removed was tested.

After several developments and changing factors to reach the best removal of nitrate from a synthetic nitrate solution, real groundwater samples were tested for nitrate from various locations in the Gaza Strip at the optimum conditions (biosorbent dose: 10 g, pH: 6.0–7.0, drying temperature: 45 °C, particle size: 90–710 μm, incubation adsorbent temperature: 20 °C and incubation adsorbate temperature: 37 °C). A volume of 100 mL of the groundwater was mixed with 10 g of the eggshell biosorbent and the optimum factors were controlled. After 48 hours the amount of nitrate removed was determined. The test was repeated three times.

The recovery procedure of the eggshell biosorbent after its use for nitrate removal was conducted by testing the eggshell material in repeated experiments. Tests were carried out either after washing of the previously used material or without washing. A volume of 100 mL of the water of NO₃⁻ of 100 ppm initial concentration was mixed with 10 g of the recovered eggshell biosorbent at the optimum conditions. Each experiment was carried out six times at 12- and 24-hour intervals.

A polyethylene column (30 cm long, 5 cm diameter) was washed sequentially with 0.1 M nitric acid, distilled water, and acetic acid. It was then dried with hot air and packed with a bed (80.0 g, particle size of 90–710 μm) of the eggshell biosorbent. The packed material was activated by elution with 0.1 M acetate solution for controlling the adsorbent material at pH 6–7. A nitrate solution of (500 mL, 100 mg/L) was added to the column and incubated at 37 °C for 24 hours in the incubator. The solution then was eluted from the column at different flow rates under gravity. The eggshell biosorbent column removal efficiency of nitrate was quantified.

In order to identify the mechanism of denitrification by the eggshell biosorbent, during optimization of the parameters, two control samples were conducted. The samples contained 100 mL of 100 ppm nitrate and 10 g eggshell biosorbent, and were incubated for eight hours and closed tightly with aluminum foil in order to prevent attacks by various types of bacteria, especially denitrification bacteria. The samples were then preserved for three days and tested for nitrate.

In order to detect if any nitrate converted to nitrite during the removal of nitrate by the eggshell biosorbent, a nitrite test was conducted by the sulphanilamide spectrophotometric standard method (Michalski & Kurzyca 2006). The reagent for nitrite analysis was prepared (Figure 1). Phosphoric acid (50 mL, 85%) was dissolved in 200 mL distilled water followed by 5 g of sulfanilamide, then 0.5 g of N-(1-naphthyl) ethylenediamine was added to the mixture with stirring. The solution mixture was then completed to 500 mL by distilled water.

Because of ion instability, the samples of nitrite were analyzed immediately after collection. A sample solution (1 mL) was withdrawn using a micropipette and then 1 mL of nitrite reagent was mixed for one minute until a pink colour appeared. All samples were determined at λ_max = 543 nm by applying a calibration curve.

Investigating the effect of contact time required to reach equilibrium is essential for designing batch adsorption experiments. As shown in Figure 2, it is observed that the percentage of nitrate removed decreases as the initial concentration of nitrate solution increases for all the tested concentrations and becomes constant after five days contact time, when saturation is achieved and the sorption process attains equilibrium. The rapid adsorption rate after the second day of contact time could be explained by the growth of the available number of denitrification bacteria. It has been reported that some bacteria such as aerobic mesophilic bacteria, enterobacteria, enterococci and staphylococci could live on the eggshell adsorbent surface (Miretzky et al. 2008). Among these bacteria, aerobic mesophilic bacteria have a high affinity for consuming nitrate because most denitrifying bacteria grow in an aerobic environment (Miretzky et al. 2008).

The decrease in % nitrate removal by increasing concentration (Figure 2) can be explained by the absorption of an adequate amount of nitrate from the adsorbate (nitrate solution) according to the available number of adsorption pores or active sites within the fixed amount of eggshell adsorbent dose, so only a small amount of nitrate that is available for removal in these preliminary conditions was achieved, especially at the high concentration of nitrate. In other words, the substrate becomes saturated at a certain concentration of adsorbate (Murugan & Subramanian 2006). The sorption is also normally controlled by the diffusion process from the bulk to the surface, which is related to nitrate concentration (Bhaumik et al. 2012). With the exception of the 1,000 and 1,500 ppm initial concentrations, the increasing concentration gradient acts as an increasing driving force to overcome all mass-transfer resistances of the nitrate between the aqueous and solid phase, leading to an increasing equilibrium sorption until sorbent saturation is achieved. A similar trend has been reported for chromium and fluoride removal by geo-materials (Bhatti et al. 2010) and activated charcoal (Murugan & Subramanian 2006).

According to various studies, the denitrification bacteria (Torrentó et al. 2010) which grow onto the eggshell could be of two types: autotrophic and heterotrophic. Autotrophic denitrifying bacteria utilize carbon dioxide or bicarbonates as a carbon source to produce cell biomass (Sharma & Sobti 2012), but this type of bacteria is rarely present in the present system as no reduced species are present that could be utilized by this type of bacteria for energy production such as hydrogen or other forms of reduced sulfur materials (e.g., S⁰, S²⁻, S₂O₃²⁻, S₄O₂²⁻, or SO₃²⁻). In addition, the process is very slow by these bacteria, which need a long contact time, and hence it is hard to apply (Zhang et al. 2009). Denitrification by heterotrophic bacteria is the dissimilative reduction of nitrate (NO₃⁻) to nitrogen gas (N₂), through the production of nitrite (NO₂⁻) and gaseous nitric oxide (NO) and nitrous oxide (N₂O) intermediates in four enzymatic steps (Torrentó et al. 2010). This process is performed by heterotrophic bacteria under anoxic conditions and uses nitrate as a terminal electron acceptor in the presence of a carbon and energy source (Madigan et al. 1997). An electron donor is required as a carbon and energy source to fuel the denitrification process (Phillips 1997). The organic membrane which is accompanied by the calcite eggshell is considered a source of carbon for the heterotrophic bacteria. The acetate that is added to control the pH of the medium is another source of carbon that enhances the activity of the heterotrophic bacteria.

Table 1 summarizes the maximum removal capacity at each initial concentration of nitrate in mg/g of the eggshell biosorbent on day 5. It is shown from Table 1 that the maximum amount of nitrate absorbed by the eggshell material is 2.4 mg/g eggshell in the case of 500 ppm initial concentration of nitrate. This amount decreases to 1.7 mg/g and 0.3 mg/g in the case of 1,000 ppm and 1,500 ppm initial concentrations of nitrate respectively. This could be attributed to clogging and deterioration of the eggshell active sites by increasing nitrate concentration, so the mass transfer of nitrate decreases dramatically. More optimization of conditions to activate the eggshell for efficient removal at various concentrations was investigated in the following sections.

Temperature is considered an important factor for the biological treatment process, as it affects the metabolic activities of the microbial population, and the settling characteristics of biological solids (Guo et al. 2013).

It is shown from Table 2 that the best efficiency for removing nitrate from the aqueous solutions was nearly on the third day when the eggshell biosorbents were incubated at 15–20 °C and the adsorbate solution incubated at 29 °C. This could be attributed to the good temperature conditions for denitrification bacteria growth. This is in agreement with many researchers (Xu et al. 2016). No nitrate removal is observed at temperatures ≥70 °C, which is due to the inhibition or dying of bacteria.

The denitrification process with this eggshell biosorbent system process would be an attractive system owing to the potential to reduce nitrate completely at moderate temperatures that are close to the real groundwater temperature.

The drying temperature of the eggshell is an important factor since it influences the metabolic activities of the microbial population (Guo et al. 2013).

Biological reaction rates in a suspended growth increase with rising temperature until an optimal temperature, above which enzymatic proteins denature and the rates decrease (Zhang et al. 2009). It is shown from Table 3 that the removal of nitrate increases with time up to the third day for all the samples at the different drying temperatures. The relation between % removal of nitrate versus drying temperature on the third day is given in Figure 3. It is shown from Figure 3 that the drying temperature increases the amount of nitrate removed up to 45 °C, which is the optimal temperature, then decreasing slightly at 70 °C, whereas a slight inhibition occurs at low temperatures. At moderate temperatures of 30–45 °C, removal of nitrate is more efficient than at low or high temperatures, which is attributed to the preservation of denitrification bacteria on the eggshell surface (Pramanpol & Nitayapat 2006).

The particle size is an important parameter owing to its effect on % removal efficiency and on the amount of nitrate adsorbed per unit weight of biosorbent. The results in Table 4 reveal that the amount of nitrate removed increases with time for the three ranges of particle sizes and reaches the maximum after three days, when saturation takes place. It is observed also, the removal % of nitrate of the coarse and fine materials is 92% and 89%, respectively, whereas medium material shows higher efficiency (94%).

Although fine eggshell material possesses the largest surface area, it experiences some particle solvation in aqueous solution at high duration time. Tsai et al. (2008) attributed the reduction in adsorption ability associated with decrease in membrane particle size to the use of excess time in the milling process, which can cause a structural change due to a reduction of crystallinity and affect adsorption ability. The material of medium particle size (710–90 μm) was selected for further experiments due to its stability and acceptable potential for nitrate removal.

In order to explain the effect of drying temperature of the eggshell in parallel with the effect of particle size on the % removal of nitrate, a detailed investigation was conducted and the results are shown in Table 5. It is obvious that there is a significant effect of particle size and drying temperature on the % of nitrate removal. The optimum conditions were found for the medium-particle-size material (710–90 μm) as discussed previously and the drying of the eggshell at 45 °C. The moderate drying temperature range (37–45 °C) that gave the highest % removal of nitrate is attributed to its availability for the growth of bacteria and their reproduction (Yusuf et al. 2016) in addition to preservation of vital process in denitrification and preventing its death (Pramanpol & Nitayapat 2006). On the other hand, as the drying temperature increases, the % removal decreases, and it was found that at temperatures higher than 80 °C no significant nitrate removal was detected. The high temperature affects the active sites and biochemical functional structure. The enzyme (destructive), on the other hand, plays a role at the level of DNA, as it denatures the hydrogen bond between the two strands of the double helix of the DNA (Rouzina & Bloomfield 2001).

It is observed from Figure 4 that in the first three days the % of nitrate removed increases with increasing of absorbent dose. Such a trend is mostly attributed to an increase in the sorptive surface area and the availability of more active adsorption sites with a higher amount of adsorbent (Miretzky et al. 2008; Kumar et al. 2010). After four days equilibrium is attained and the % of nitrate removal is nearly the same with a slight increase for the larger dose (98% for 15 g dose) comparing with both the smaller doses (93% for both 5 and 10 g doses) which is attributed to the availability of the low doses to absorb more nitrate over a long time.

The results in Figure 5 illustrate that the amount of nitrate removed increases with increasing pH value and reaches the maximum at pH 6.0–7.5. This result is in agreement with the study of Xu et al. (2016), who reported that eggshell can maintain a neutral condition in a range of pH 7.05–7.74 in sulfur-based autotrophic denitrification with eggshell for a nitrate-contaminated synthetic groundwater treatment process, and remove 97% of nitrate in synthetic groundwater at this pH range. Metcalf & Eddy (1991) reported that the optimum pH for denitrification typically lies within the range of 7 to 8, depending on the microbial community. Other researchers also observed a stable pH range (pH 6.9–7.5) in autotrophic denitrification systems (Van der Hoek et al. 1992; Guo et al. 2013).

The principal component of the shell is CaCO₃ in the form of the mineral calcite.

In aqueous solution, the carbonate species derived from calcite are H₂CO₃, HCO₃^−, and CO₃²⁻, the proportions of which are determined by the pH of the resulting solution. The pH of mixtures of eggshell and solutions of the adsorbate all eventually attained a pH >7. From this finding it is concluded that sufficient amounts of carbonates are solubilized from the shells to a faintly alkaline pH (Pramanpol & Nitayapat 2006) and that this explains the tendency of the raw eggshell biosorbent to remove nitrate. The effect of pH can be explained by the ion-exchange mechanism of adsorption in which the important role is played by carbonate groups on the eggshells that have cation-exchange properties. At lower pH values, eggshell tends to adsorb protons. It is probable that at lower pH nitrates are converted to nitric acid and attack the eggshell surface and so some Ca²⁺ ions are formed and thus deactivate the eggshell surface (Zulfikar & Setiyanto 2013; Xu et al. 2016).

In conclusion, a neutral or slightly basic medium enhances the adsorption of nitrate and degradation by denitrification bacteria.

It is observed also that maximum absorption was achieved nearly after three days, which emphasizes the improved efficiency of the acetate solution at pH 6–7.5 where 91.5% of nitrate was removed. This is in agreement with other studies reported for the denitrification processes. Therefore, a range of 7.05–7.74 maintained by eggshell is desirable for denitrifying bacteria to remove nitrate (Xu et al. 2016). The pH range 6–7 was used for further investigations in this study. The proposed mechanism of nitrate removal is shown in Figure 6, which is in agreement with that proposed by Xu et al. (2016). The final products produced as a result of the denitrification process are nitrogen (N₂) and nitrous oxide (N₂O) gases that get away from the substrate. This process enhances a sustainable activation of the denitrification of the bacteria (Xu et al. 2016). This was confirmed by observation of gas bubbles and use of control samples in addition to obtaining no N-nitrite and ammonia compounds. The results of this study have superior advantages compared with that of Xu et al. (2016), who construct a complex system as they studied the effect of different sulfur/eggshell (w/w) ratios to evaluate the feasibility of the sulfur-based autotrophic denitrification with eggshell (SADE) process for nitrate removal. They added cultivated microorganisms from anaerobic sludge in a liquid nutrient medium for 60 days. Their system used a nutrient medium consisting of Na₂S₂O₃·5H₂O, NaHCO₃, KNO₃, KH₂PO₄, NH₄Cl, MgSO₄·7H₂O, FeSO₄·7H₂O and CaCl₂·2H₂O. The enriched biomass was maintained under anaerobic conditions at 30 °C and the nutrient medium was replaced every two days, with further equilibration.

The improvement of the efficiency of the biosorbent eggshell for removing nitrate from aqueous solutions from the previous results can be summarized in the following steps:

• 1

  - Biosorbent dose: 10 g

• 2

  - pH control: 6.0–7.0

• 3

  - Time: 3 days

• 4

  - Drying temperature: 45 °C

• 5

  - Particle size: 90–710 μm

• 6

  - Incubation adsorbent temperature: 20 °C

• 7

  - Incubation adsorbate temperature: 37 °C

Control of the previous parameters was applied to different initial concentrations of the adsorbate (50, 100, 250, 500, 1,000 and 1,500 ppm) in order to examine their effect on the removal of nitrate. The determination of the nitrate absorbed was conducted at different time intervals for three days. The results are shown in Figure 7.

From Figure 7, it is observed that the % of nitrate removed decreases with the increasing of the initial concentration of nitrate and becomes constant after three days for all tested concentrations, when saturation is achieved. Table 6 summarizes the maximum amount of nitrate removed at each initial concentration of nitrate in mg/g of the eggshell bio-absorbent. It is shown from Table 6 that the maximum amount of nitrate absorbed by the eggshell material was 8.25 mg/g eggshell in the case of 1,500 ppm initial concentration of nitrate. Comparing these results with those shown in Table 1 it is clear that the amount of nitrate removal improved nearly for all concentrations from 1:1 (the minimum) for 100 ppm initial concentration to 27.5:1 (the maximum) for 1,500 ppm. This improvement is attributed to the high activation of the surface of the eggshell substrate by the optimization system, which includes pH value, particle size, drying temperature and incubation temperature.

The used eggshell adsorbate was re-tested for nitrate removal at the optimum conditions with or without reconditioning. Six cycles of measurements using previously tested eggshell adsorbate were conducted with and without washing by water and acetate solution. The cycles were repeated every 24 hours and tested at two time intervals (12 and 24 hrs) to check the recovery activity of the adsorbate. The results are given in Table 7. It is observed from Table 7 that the eggshell biosorbent preserved its activity toward the removal of nitrate from the water samples. The results show that the removal efficiency is 79–92% in the case of washed samples while the removal efficiency is 89–93% in the case of unwashed samples. This is attributed to the presence of denitrification bacteria that sustained its activity on the eggshell adsorbate.

Different real samples from various locations of groundwater in the Gaza Strip were tested for nitrate removal by the eggshell biosorbent at the optimized conditions. The nitrate was analyzed before and after applying the eggshell by the batch process and the results are given in Table 8. The original nitrate concentration range for all the tested samples was (86–223 ppm). It is observed that the efficiency of nitrate removal was in the range (77.6–93%), where it is not only dependent on nitrate concentration but also on the water matrix. The differences of % nitrate removal are obviously due to the variation in the water matrix at each location, which contain various anionic and cationic species as seen from the values of electrical conductivity (EC) in Table 8, which affect the removal efficiency. These species could compete with the nitrate ions to be adsorbed into the eggshell adsorbent, even when there is a high selectivity of the eggshell biosorbent toward the nitrate ions at the optimized conditions. It seems that two factors affect the percentage of nitrate removal: the concentration of nitrate and the concentration of competing ions present as observed from the value of EC of the examined water samples. It is obvious that at high EC values, the % removal of nitrate decreases slightly as the high concentration of ionic species affects the diffusion of nitrate through the porous eggshell adsorbate.

Applying the eggshell adsorbate as a stationary phase for nitrate removal from the water samples was investigated. The eluted solution was examined for nitrate removal as a function of flow rate, and the results are given in Table 9. It is obvious that at a fast flow rate (≥15 mL/min) the removal efficiency is only around 53–59%, but as the flow rate decreases the removal efficiency increases and reaches 90%, which is attributed to more contact between the active sites and the nitrate ions. The optimum flow rate was at ≤2 mL/min.

In order to identify the mechanism of denitrification by the eggshell biosorbent, during optimization of the parameters, two control samples containing eggshell biosorbent without exposure to other optimized parameters were conducted. The results showed no nitrate removal. This result emphasized that the main factor for the removal of nitrate is the bacterial activity and no other chemical factors contributed significantly to nitrate degradation. The porosity of the eggshell biosorbent activates the adsorption of the nitrate ions where they become available for the denitrification bacteria. The denitrification bacteria can grow with the suitable parameters that were investigated in this study.

In order to investigate the kind of transformation of nitrate after adsorption, experimental analysis of nitrite and ammonia was conducted after the disappearance of the nitrate. The results showed that neither nitrite nor ammonia was detected. This interesting result suggests a strong probability that nitrate was degraded to volatile nitrogen and nitrogen oxide by the denitrification bacteria (Torrentó et al. 2010). Further studies are recommended in order to identify the types of microorganisms and the mechanism of the denitrification process.

Nitrate in synthetic and real groundwater was effectively reduced by the eggshell biosorbent process with a nitrate removal rate of up to 95%. The results showed that the removal efficiency of nitrate was related to particle size and the best particle size was at (710–90 μm). Experimental data showed that nitrate adsorption increased by increasing the pH value of the solution and the optimum pH was at (6–7.5). The results showed that the equilibrium time for nitrate removal was after 24 h. Nitrate removal was temperature-dependent and the best results were found when eggshell adsorbent and adsorbate were incubated at 37 °C and in the case of eggshell drying before use as biosorbent at 45 °C. The removal efficiency increases with increasing eggshell dose up to saturation. The removal efficiency also increases with increasing nitrate initial concentration up to saturation. For the recovery of the eggshell biosorbent, the results showed that the removal efficiency is 79–92% in the case of washed samples while the removal efficiency is 89–93% in the case of unwashed samples. The results showed the efficient removal by eggshells in the process of treatment of nitrate ions in real samples was in the range (77–93%).

The authors would like to thank the Islamic University-Gaza and the Universiti Malaysia Terengganu, Malaysia, for technical and experimental guidance and support.

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