# Nitrate Removal from Wastewater through Biological Denitrification with OGA 24 in a Batch Reactor

^{1}

^{2}

^{*}

## Abstract

**:**

## 1. Introduction

_{3}

**removal abilities [9,10,11,12,13]. Depending on their characteristics, different bacteria are employed in different waste treatment facilities, with a preference towards those microorganisms capable of combined heterotrophic nitrification and aerobic denitrification. However, other characteristics are often desirable, for example bacteria with a marked resistance to high salinity, are generally employed in the treatment of polluted seawater [14] and strains isolated from critically polluted environments are used for the treatment of special industrial wastes, such as tannery wastewater [15].**

^{−}## 2. Materials and Methods (or Experimental)

#### 2.1. Bacteria and Media

_{3}, analytical grade, Carlo Erba) as electron acceptors. To obtain rapid population expansion, the strain was grown aerobically in rich Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract and 0.5% NaCl). For both growth conditions, the bacterium was incubated at 37 °C. For assays of nitrate respiration, frozen (−80 °C) cultures in 20% glycerol were revived by incubating 24 h in LB medium at 37 °C with constant shaking at 150 rpm. Cells were collected by centrifugation at 2.500 × g at room temperature, washed twice with water saline solution (0.9% NaCl) and pre-incubated in VG medium with 10 mM NO

_{3}

^{−}and 10mM acetate at concentration corresponding to the optical density at λ = 600 nm (OD

_{600}) of 0.02 A.U. (OD

_{600}= 1 A.U. ~1 × 10

^{9}individuals/mL). The growth was carried out in 100 mL flasks sealed with rubber stoppers to ensure anaerobic conditions at 37 °C for 24 h without shaking. After pre-incubation cells were harvested and inoculated at similar concentration in fresh VG medium for bioreactor assays.

#### 2.2. Experimental Set Up

_{3}

^{−}]

_{0}= 350 mg/L (total volume V

_{0}= 250 mL).

_{tot}= 400 mL) and equipped with several probes: dissolved oxygen (DO, Vernier Software and Technology, Beaverton, OR, USA), pH (Vernier Software and Technology) and Nitrate Ion Selective Electrode (NO

_{3}

^{− }ISE, Vernier Software and Technology). All the probes were connected to a LabQuest2 data logger (Vernier Software and Technology) for data acquisition and storage.

_{2}] ~0 mg/L (~20 min). The bioreactor was placed in an air thermostat at a temperature of 30 ± 1 °C and stirred continuously at 200 rpm. Phosphate salts in the VG medium acted as buffer keeping the pH around 7 during the whole course of the experiments. All the experiments were performed in triplicate and the data reported represent their average values.

_{3}

^{−}ISE was calibrated by using 1 mg/L and 1000 mg/L standard solutions of KNO

_{3}(Carlo Erba, Analytical grade) and a series of intermediate solutions obtained by dilution. We checked the correct functioning of our experimental apparatus by performing a simple semi-batch experiment. We infused in a glass reactor containing a known volume of nitrate solution (V

_{0}= 250 mL, [NO

_{3}

^{−}]

_{0}= 10 mg/L) a second nitrate solution ([NO

_{3}

^{−}]

_{in}= 13.6 g/L) by a syringe pump (NE-300, New Era Pump Systems) at a constant flow rate (F

_{in}= 2.11 μL/min). We then monitored over time the concentration of nitrate ions in solution ([NO

_{3}

^{−}](t)) by using our calibrated NO

_{3}

^{−}ISE and we compared the experimental values with the theoretical curve Equation (1), which describes the nitrates evolution in time. Here the nitrate ions do not undergo any reaction and the only variation is due to the input of the known amount of nitrates and to the dilution effect. The comparison between the experimental and the theoretical data reported in Figure 2 reveals a very good response of the NO

_{3}

^{−}ISE. The same results were obtained in the presence of a VG medium having the same composition as that used in the bioreactor.

_{3}

^{−}] determined with the two methods was found to be lower than 2%.

**Figure 2.**(

**a**) Sketch of the Semi-batch reactor used for the experimental setup tests; (

**b**) Comparison between the experimental data (black squares) and the theoretical curve (black line) of [NO

_{3}

^{−}](t) in a semi-batch reactor where V

_{0}= 250 mL, [NO

_{3}

^{−}]

_{0}= 10 mg/L, [NO

_{3}

^{−}]

_{in}= 13.6 g/L and F

_{in}= 2.11 μL/min.

## 3. Results and Discussion

^{7}individuals/mL), Azospira sp. OGA 24 was then inoculated in the batch bioreactor in the presence of the VG medium, acetate as the organic substrate (electron donor) and [NO

_{3}

^{−}] = 350 mg/L (electron acceptor). The bioreactor was previously sparged with argon in order to obtain anaerobic conditions, so that the bacteria could use nitrates as the only final electron acceptors for their respiratory functions. Thanks to the ad hoc experimental setup and to the analytical methods employed, the kinetics of the nitrates consumption and of the bacterial population growth could be recorded simultaneously and with a continuous sampling rate. Figure 3 shows a typical time series of the nitrate consumption operated by OGA 24 (black trace, right axis) and the corresponding growth curve of the bacteria (red trace, left axis). The whole dynamics could be divided into three main regions for both the curves. The first region represents a rather long (0–~1800 min) acclimation period (A in the figure) where bacteria adapt to the anaerobic condition and to the high concentration of nitrates and their population slightly increases (OD 0.02→~0.3 A.U.); correspondingly the concentration of nitrates slowly decreases from 350 mg/L to ~320 mg/L. The quite long acclimation period could certainly be reduced by starting experiments with a larger population of bacteria, i.e., by increasing the duration of the cells culture. The acclimation period is followed by the exponential region (B in the figure, ~1800–~2500 min) where nitrates are rapidly depleted until their concentration reaches a value of about 0 mg/L. The rapid consumption of the electron acceptor species corresponds to a fast increase of the bacteria population (OD ~0.3→~1.2 A.U.). Finally, a plateau region (C in the figure) indicates that the system reached a steady state where nitrates are completely consumed and the number of bacteria is stationary in time (~1.2 × 10

^{9}individuals/mL).

**Figure 3.**Experimental curves of nitrates reduction operated by the azospira OGA 24. Black squares represent nitrates concentration over time, measured by means of the NO

_{3}

^{−}ISE; red squares show the increase of the solution optical density over time, i.e., the growth of the bacteria population. Three different regions can be identified:

**A**: acclimation zone;

**B**: exponential consumption of nitrates (increase of the bacterial population);

**C**: steady state.

_{3}

^{−}] time series of Figure 3, where the rate of consumption of the electron acceptor is maximum. In order to characterize the kinetics of the denitrification process, data from different experiments where fitted both to a linear equation (blue dashed line, fitting interval 2000–2400 min) having the form

^{−1}·min

^{−1}) is the kinetic constant of the zero-order kinetics, and to an exponential equation (red line, fitting interval 2000–2800 min) having the form

^{−1}) is the kinetic constant of the first-order kinetics. Data where fitted by means of a Levenberg–Marquardt [24] nonlinear regression algorithm, which yielded a value of ${k}_{d}^{0}$ = 0.63 ± 0.03 mg L

^{−1}min

^{−1}and ${k}_{d}^{1}$ = 4 ± 0.5 × 10

^{−3}min

^{−1}. For both the fitting procedures the value of the R-squared index was higher than 0.99, meaning that both the mechanisms proposed for the denitrification process were plausible.

**Figure 4.**(

**a**) Exponential fitting (red line, Equation (3)) and linear fitting (blue dashed line, Equation (2)) of the [NO

_{3}

^{−}] consumption during the exponential period; (

**b**) Fitting of the bacterial growth curve by means of the logistic Equation (4). The inset shows the bacterial growth curve when the bacterial population is considered constant during the acclimation period.

_{0}is the relative bacterial population size, A = ln(N

_{∞}/N

_{0}) is the maximum value reached, λ is the lag time, i.e., the time of the acclimation period (region A in Figure 3) and μ

_{m}is the maximum specific growth rate [25]. The slight increase of the population size during the acclimation period is amplified in the logarithmic scale and it makes difficult a good fitting to the Equation (4), as showed in the main graphic of Figure 4b. The values of the fitting parameters were found to be A = 4.14 ± 0.04 (which roughly corresponds to a population increase of 60 times, i.e., 10 times larger than the value obtained from OD measurements), μ

_{m}= 1.6 ± 0.04 × 10

^{−3}min

^{−1}and an unrealistic lag time λ = 28 ± 10 min, with R-squared = 0.95. In order to obtain a better fitting and a more realistic value for the lag time, the population size was considered as constant during the acclimation period (inset in Figure 4). In this case, the fitting procedure yielded A = 3.83 ± 0.04 (which roughly corresponds to a population increase of 50 times in accordance with the OD measurements), μ

_{m}= 9.9 ± 0.4 × 10

^{−3}min

^{−1}and λ = 1689 ± 12 min, with R-squared = 0.99. The specific growth rate differs by almost one order of magnitude in the two cases and it is difficult at this stage to obtain a proper characterization of the growth dynamics. However, the value of ${k}_{d}^{1}$ was found to be in between these two extremes, therefore the value of μ

_{m}can be reliable in both cases.

_{in}, equals to the outgoing denitrified water, F

_{out}, i.e., the volume of the reactor, V(t), is in stationary state: F

_{in}= F

_{out}= F (m

^{3}/d), V(t) = V

_{0}(m

^{3}). Moreover, the bacterial population is constant and in the exponential growth stage.

_{3}

^{−}]

_{in}is the concentration of nitrates in the wastewater, [NO

_{3}

^{−}]

_{0}is the initial concentration of nitrates at time t = 0, R

_{d}represents the kinetics of the denitrification reactions and [NO

_{3}

^{−}](t) is the concentration of nitrates in time, which corresponds to the concentration of nitrates in the outgoing wastewater. When we consider the zero-order kinetics for the nitrates consumption, the system is described by a differential equation having the form

_{3}

^{−}] ≡ [NO

_{3}

^{−}](t). Being both F and V

_{0}constant, k

_{0}= F/V

_{0}(d

^{−1}) can be defined as the reciprocal of the mean residence time of a molecule in the reactor and the integral form of the Equation (5) yields the variation in time of nitrates

_{3}

^{−}]

_{ss}, i.e., the concentration in the outgoing wastewater when the system is in continuous working regime, can be found by Equating (5) to zero or by calculating the limit of Equation (6) for t → ∞ and obtain

^{−1}·d

^{−1}) and the values for k

_{0}and [NO

_{3}

^{−}]

_{in}of a real wastewater treatment plant with a potential of < 100,000 A.E. (F = 210 m

^{3}/d, V

_{0}= 93 m

^{3}, k

_{0}= 2.25 d

^{−1}) and [NO

_{3}

^{−}]

_{in}= 450 mg/L to find that [NO

_{3}

^{−}]

_{ss}~47 mg/L which corresponds to a reduction of nitrate of about 90%. This value meets the requests of Italian legislation that requires a minimum reduction of nitrate of 70% for this kind of treatment plant. The process was also modeled considering a first-order kinetics and using ${k}_{d}^{1}$ in the place of ${k}_{d}^{0}$ , in this case

**Figure 5.**Scheme of the anoxic denitrification process in a Continuously Stirred Tank Reactor (CSTR).

## 4. Conclusions

_{3}

**], the stage with the maximum rate of nitrates consumption can be described by both a zero- and a first-order kinetics. The main motivation of the present work was to assess the effectiveness of OGA 24 as denitrifying agent; therefore a better characterization of the kinetic mechanism will be done in future works.**

^{−}## Acknowledgments

## Author Contributions

## Conflicts of Interest

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**MDPI and ACS Style**

Rossi, F.; Motta, O.; Matrella, S.; Proto, A.; Vigliotta, G.
Nitrate Removal from Wastewater through Biological Denitrification with OGA 24 in a Batch Reactor. *Water* **2015**, *7*, 51-62.
https://doi.org/10.3390/w7010051

**AMA Style**

Rossi F, Motta O, Matrella S, Proto A, Vigliotta G.
Nitrate Removal from Wastewater through Biological Denitrification with OGA 24 in a Batch Reactor. *Water*. 2015; 7(1):51-62.
https://doi.org/10.3390/w7010051

**Chicago/Turabian Style**

Rossi, Federico, Oriana Motta, Simona Matrella, Antonio Proto, and Giovanni Vigliotta.
2015. "Nitrate Removal from Wastewater through Biological Denitrification with OGA 24 in a Batch Reactor" *Water* 7, no. 1: 51-62.
https://doi.org/10.3390/w7010051