Ozonolytic degradation of Tetracycline Antibiotic ...

© by PSP Volume 25 – No. 8/ 2016, pages 2928-2934

Fresenius Environmental Bulletin

OZONOLYTIC DEGRADATION OF TETRACYCLINE ANTIBIOTIC: EFFECT OF PH Osman Gulnaz1, Gokhan Sezer2 1

Faculty of Education, Department of Science and Technology Education, Cukurova University, 01330, Adana, TURKEY 2 Faculty of Art and Science, Department of Biology, Osmaniye Korkut Ata University, 80000, Osmaniye, TURKEY

Chlortetracycline, oxytetracycline and tetracycline, which are a kind of tetracycline antibiotics, are broad spectrum antibiotics and act as protein synthesis inhibitor on bacteria. Tetracycline antibiotics have been widely used against to both Gram (+) and Gram (-) microorganisms. In recent years, broad-spectrum antibiotics have been used against to gram-positive and gramnegative pathogens, but the microbial resistance against to antibiotics occurs after the first use of these agents for the treatment of infectious diseases. In natural systems, presence of antibiotics leads to the development of multi-resistant strains of bacteria [1, 4-5]. Antibiotics are low biodegradable substances due to their biocidal activity and they cannot be completely eliminated in conventional wastewater treatment plant. For this reason, removal of antibiotics from wastewater is very important environmental problem [5-9]. A number of physicochemical methods have been used to remove toxic substances from wastewaters such as chemical precipitation, photocatalytic or oxidative degradation [10-13]. Among these techniques advanced oxidation processes (AOPs) are the most efficient methods. AOPs have already been successfully applied for the treatment of industrial effluents containing toxic or recalcitrant (difficult to biodegradation) organic pollutants. Ozone is well known oxidation agent in the AOPs and has been applied to many fields in water and wastewater treatment [14-15]. Many studies have been conducted on the removal of antibiotics using AOPs process, but in previous studies, any observation has not been stated about antibacterial activity of tetracycline and degradation products after AOPs. In this ozonolytic degradation of tetracycline and removal of antimicrobial activity of degradation products were evaluated. Effect of initial pH and initial antibiotic concentrations were determined on ozonolytic degradation of tetracycline.

ABSTRACT In this study, ozonolytic degradation of protein synthesis inhibitor antibiotic tetracycline was evaluated. Effect of pH on ozonolytic degradation was studied at pH 3, 7, and 11, and the most efficient pH was determined to be 3. The results showed that more than 50% of the tetracycline was degraded within 10 min and it was completely removed after 40 min in all initial antibiotic concentrations. The degradation kinetic was well described by a pseudo first-order kinetic model. The first-order rate constant decreases with increasing of the initial antibiotic concentrations. The kinetic rate constants were determined between 0.128 and 0.091 min-1. The total organic carbon (TOC) removal was monitored during ozonolytic antibiotic degradation (120 min). The percentage TOC removal after 120 min ozone treatment of tetracycline was determined to be 33, 28 and 28% at pH 3, 7 and 11, respectively. Disc diffusion technique was performed for antibacterial susceptibility testing. Inhibition zone diameter was decreased with ozone treatment. Inhibition zone was not determined for Escherichia coli K-12 (ATCC) after 10 min and for Bacillus subtilis (NRRL B-354) after 15 min ozone treatment at pH 3, 7 and 11. The inhibition zone of tetracycline on the Staphylococcus epidermidis (NRRL B-4268) was removed after 15 min ozone treatment at pH 7 and 11, however, after 20 min ozone treatment it was removed at pH 3. KEYWORDS: Antibiotic, Tetracycline-HCl, Ozonation, Antimicrobial activity; Kinetic model.

INTRODUCTION Several hundred different antibiotics have been used as human and veterinary medicine to treat or prevent microbial infections [1]. The annually 100.000–200.000 tons antibiotic has been used worldwide, for this reason antibiotics their metabolized products have been detected in various environment such as groundwater, surface water and sediment samples in the range of nano gram to microgram concentrations [2-3].

MATERIALS AND METHODS Ozonation experiments. Commercially available tetracycline-HCl antibiotic was obtained from a local pharmacy, Adana-Turkey. Ozone gas 2928



was produced from pure oxygen (99.9%) by an Ozo-1VTT model ozone generator (Ozomax, Canada). Ozonation experiments were performed 1200 ml closed cylindrical glass reactor containing 800 mL tetracycline solution. Ozone gas was diffused into the tetracycline solution at the bottom of the reactor. The effect of initial pH on ozonolytic degradation was determined at pH 3, 7 and 11, using an antibiotic concentration of 400 mgL-1. The effect of initial concentrations of tetracycline on ozonolytic degradation was determined at 100, 200, 400, 600, 800 and 1000 mgL-1 at pH 3.0. The pH was adjusted by adding 0.1 N HCl or 0.1 N NaOH and all experiments were performed at ambient temperature, 20°C±0.5. Tetracycline concentrations were determined with HPLC analysis (Thermo spectra system p1000, UV 2000, AS 3000). Hypersil C18 column was used (250 mm long, 4.6 mm i.d. and 5µm particle size) and isocratic elution was carried out and acetonitrile/ water/ methanol (20/60/20) at a flow rate of 1 ml min-1 (at 356 nm). The TOC removal was determined using the Hach Lange total organic carbon test kit (DR 2800). All measurements were made at least in duplicate or triplicate.

shown that the degradation of tetracycline at pH 3 was very fast and more than 50% tetracycline was removed at first 5 min ozone treatment and 98% of tetracycline was removed at 20 min. Ozone and hydroxyl radicals have effect on degradation of tetracycline at the neutral and alkali conditions. 400 350

-1

Ct(mgL )

300

pH 3 pH 7 pH 11

250 200 150 100 50 0 0

10

20

30

40

time (min)

FIGURE 1 Effect of pH on ozonolytic tetracycline degradation However, acidic conditions of ozone do not decompose to hydroxyl radicals and thus direct ozonolytic effects are shown in acidic pH conditions. The removal percentages of tetracycline after 40 min. were 98.7, 98.3 and 97.6% at pH 3, 7 and 11, respectively. Depending on pH, ozone reacts with organic compounds in two different ways. One of reaction way is direct attack at acidic pH and another way is free radical attack at basic pH [18]. Ozone directly attacks the conjugated double bonds in the dye [19]. Electrophilic attack by molecular ozone is very selective against nucleophilic carbons, multiply bonded components such as carbon–carbon and nitrogen–nitrogen. At higher pH, during the ozonolytic reaction, the ozone can also react with hydroxyl radicals and ozone decomposes to the hydroxyl radical. Hydroxyl radical is a very powerful (E0 = 2.80 V) and nonselective oxidant. Moreover, radical oxidation is more efficient and faster than the direct oxidation. The direct oxidation efficiency by ozone depends on the pollutants and the solution of pH [20]. This was also seen in our experiments, where ozone efficiency was observed in the order of pH 3 > pH 7 > pH 11. The initial pH values were decreased with increasing reaction time from 3 to 2.2, 7 to 3.4 and 11 to 6.3 within 40 min due to formation of organic acids [21-22].

Bacterial strains and antibacterial susceptibility testing. In this study, disc diffusion technique was used for antibacterial susceptibility testing [17]. Bacillus subtilis (NRRL B-354), Escherichia coli K-12 (ATCC), Staphylococcus epidermidis (NRRL B-4268) were selected as standard organisms. Bacterial strains were cultured on Lauria-Bertani (LB) agar plates and incubated for 24 h at 37 °C. After incubation, bacterial concentration was adjusted to McFarland standard no 0.5 and inoculated on the Muller–Hinton agar plates. After swabbing the bacteria, antibiotic free paper test disks (Oxoid) were put on to agar plates. 10 µL antibiotic and/or oxidation products inoculated paper disk and all plates were incubated for 24 h at 37 °C. The pH of the samples was adjusted to 7 by the addition of H2SO4 or NaOH before disk diffusion test. The antimicrobial activity was evaluated by measuring the zone diameter (mm). All tests were made at least in duplicate or triplicate.

RESULTS AND DISCUSSION The Effect of pH on degradation of Tetracycline. The effect of initial pH on ozonolytic degradation tetracycline was given in Fig.1. It was

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FIGURE 2 The UV-Vis spectra of tetracycline at pH 3 during ozon degradation (t is time; min) The UV-Vis spectra were given in Fig 2 at pH 3 and 400 mgL-1 initial tetracycline concentrations. The UV spectrum of tetracycline in Fig 2 shows that tetracycline has two characteristic peaks at 275 and 360 nm. During the ozonolytic degradation experiments, this two characteristic peak decreased with increased ozone contacting time of tetracycline.

Degradation of tetracycline kinetic was analyzed by using pseudo first order kinetic equation. Pseudo First order rate constants were calculated with the slope of the plot of  Ln (C t / C 0 ) versus time t.



d [C ]  k[C ] dt

(1)

1000 900

0

800 -1

600 500 400 300

-2

ln(Ct/C0)

-1

Ct(mgL )

-1

C0 (mgL ) 100 200 400 600 800 1000

700

-1

-3

-4

200 100

-5

0 0

5

10

15

20

25

30

35

C0 (mgL ) 100 200 400 600 800 1000

40

-6

time (min)

10

20

30

40

time (min)

FIGURE 3 Degradation of various initial concentrations of tetracycline at pH 3 (t is time min).

FIGURE 4 Linear fit of pseudo first order kinetic at pH 3

Kinetic. Effect of initial tetracycline concentrations on ozonolytic degradation was shown in Fig 3. Degradation time of tetracycline was decreased with increasing of initial tetracycline concentrations. At the beginning of the ozonation process, the degradation of the tetracycline solution rapidly was decreased moreover more than 50% of the tetracycline was degrade within 5 min at the diluted tetracycline concentrations. The tetracycline was not detected by HPLC analysis after 40 min ozone treatment.

Plots of linear form of the pseudo first order kinetic model were given in Fig. 4. The degradation kinetics of tetracycline can be described with pseudo first-order reaction model under the experimental conditions [23]. Pseudo first order rate constants of tetracycline at pH 3 were given in Table 1.

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test method. Bacillus subtilis (NRRL B-354), Escherichia coli K-12 (ATCC), Staphylococcus

TABLE 1 Pseudo first order kinetic constant at pH 3 Tetracycline (mgL-1)

K (min-1)

r

100

0,1289

0,989

100

200

0,1308

0,996

95

400

0,128

0,994

600

0,1101

0,991

800

0,1041

0,995

1000

0,091

0,994

TOC removal (%)

105

The first-order rate constant decreases with increasing of the initial antibiotic concentrations. The higher initial tetracycline concentrations of ozone react with much many molecules at lower concentration. Hence, much more ozone requires at higher substrate concentrations. The first-order regression coefficient of straight line was determined above 0.991. Finally, it can be deduced that pseudo-first-order model fit with degradation kinetic at the given test condition.

90 85 80 75

pH 3 pH 7 pH 11

70 65 0

20

40

60

80

100

120

time (min)

FIGURE 5 Total organic carbon removal various pH

TOC removal. It is well known that ozone readily react with double bonds and aromatic rings compounds presenting in their structures [24]. It can be seen that the possible reaction centers are more susceptible to ozone electrophilic attack. The TOCt/ TOC0 ratio is helpful to assess the mineralization of target substances. TOC removal was carried out with 1gL-1 initial concentration of tetracycline, at pH 3, 7 and 11. The TOC removal of the tetracycline solution was monitored at regular intervals of time (0-120 min) and results was given in Fig. 5. According to Fig. 5, TOC was decreased much more slowly due to fact that many intermediates organic compounds such as aldehydes, phenols, etc. occured after ozonoliytic reaction of tetracycline. 15 ozonolytic degradation products of tetracycline were determined by using Liquid chromatography–triple quadrupole mass spectrometry at pH 2.2 [21]. The mineralization of these intermediates organic compounds are very slowly, for this reason TOC removal cannot be completely determined under the experimental conditions. The TOC removal was determined at 120 min ozone treatment to be 33, 28 and 28% at pH 3, 7 and 11, respectively. Similar results were given by Khan et al., [21] as 15% TOC removal after 30 min ozone treatment of tetracycline antibiotic at pH 2.2

epidermidis (NRRL B-4268) were used as test organisms. The 1000mgL-1 tetracycline was ozonated 3, 5, 10, 15, 20 and 40 min at pH 3, 7 and 11 to determine antibiotic inhibition zone. The disk diffusion tests results were given in Fig. 6 and Table 2. The amount of swelling from the edge of each disk and the inhibition zone diameter in the agar plate were given in millimeter (mm). The test was repeated at least three times, for each sample. The inhibition zone results of tetracycline at three different pH conditions were given in Fig. 3. Decreasing of inhibition zone of parent compound on petri plaque clearly is shown. The inhibition zone diameter was decreased with ozone treatment. Inhibition zone was not determined for Escherichia coli K-12 (ATCC) after 10 min ozone treatment at pH 3, 7 and 11. Similar results were obtained for Bacillus subtilis (NRRL B-354), after 15 min ozone treatment parent compounds of inhibition zone were disappeared all pH conditions. Testing of tetracycline and by products for Staphylococcus epidermidis (NRRL B-4268) at pH 7 and 11 inhibition zones were disappeared at 15 min ozone treatment, however, the inhibition zone was removed after 20 min ozone treatment at pH 3. Antibiotics cannot be removed by conventional wastewater treatment processes. Advanced oxidation processes (AOPs) are efficient environmental friendly methods for removal of antibiotics from wastewater [25]. Tetracycline acts as antibiotics inhibition of protein synthesis. In this study, during the ozonation processes of tetracycline were degraded and changed structure of the main molecules.

Antibacterial activity. To obtain detailed information on microbial test of ozonolytic degradation of tetracycline and degradation by products were determined by using disk diffusion

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TABLE 2 Zone inhibition test results at pH 3,7 and 11 Time (min)

Sample no

0 3 5 10 15 20 40

1 2 3 4 5 6 7

0 3 5 10 15 20 40

1 2 3 4 5 6 7

0 3 5 10 15 20 40

1 2 3 4 5 6 7

B.subtilis E. coli S. epidermidis (NRRL B-354) (ATCC), (NRRL B-4268) Inhibition zone (mm) at pH 3 21 16 20 19 15 17 18 12 16 17 10 14 14 -11 --8 ---Inhibition zone (mm) at pH 7 22 17 20 21 16 18 18 14 17 16 11 14 14 -11 ------Inhibition zone (mm) at pH 11 21 17 20 20 14 18 18 12 16 16 10 15 13 -13 -------

FIGURE 6 Antibacterial susceptibility of tetracycline and oxidation by products

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Therefore, degraded molecules cannot be inhibited protein synthesis since it cannot be bound of ribosome sub units.

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CONCLUSIONS In summary, ozone has been a method successfully used for removal of tetracycline from aqueous solution. During the ozonation processes more than 50% of the tetracycline was degraded within 10 min and it was completely removed after 40 min. The degradation kinetic was well described by a pseudo first-order kinetic model. The firstorder rate constant was decreased with increasing of the initial tetracycline concentrations. The kinetic rate constants were determined between 0.128 and 0.091 min-1. The TOC removal is helpful to assess the mineralization of tetracycline and percentage TOC removal was determined through 120 min ozone treatment to be 33, 28 and 28% at pH 3, 7 and 11, respectively. Ozonolytic degradation experiments shows that ozonolytic degradation is an effective method for removal of antibacterial activity of tetracycline for test organisms E. coli K-12 and B. subtilis (NRRL B-354) and S. epidermidis (NRRL B-4268).

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[19] Peng, R.Y., Fan, H.J., (2005). Ozonalytic kinetic order of dye decoloration in aqueous solution. Dyes and Pigments. 67, 153-159. [20] Robinson, T., McMullan, G., Merchant, R., Nigam, P., (2001). Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresource Technol. 77, 247–255. [21] Khan, H.M., Bae, H., Jung J.Y., (2010). Tetracycline degradation by ozonation in the aqueous phase: Proposed degradation intermediates and pathway. Journal of Hazardous Materials, 181, 659–665. [22] Rosal, R., Rodriguez, A.P., Melon, J.A., Mezcua, M., Hernando, M.D., Leton, P., Garcia-Calvo, E., Aguera, A., Fernandez-Alba, A.R., (2008). Removal of pharmaceuticals and kinetics of mineralization by O3/H2O2 in a biotreated municipal wastewater. Water Res., 42, 3719-3728. [23] Kim, I.H., Tanaka, H., Iwasaki, T., Takubo, T., Morioka, T., Kato, Y., (2008). Classification of the degradability of 30 pharmaceuticals in

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Received: Accepted:

18.09.2015 12.01.2016

CORRESPONDING AUTHOR Osman Gulnaz, Faculty of Education, Department of Science and Technology Education, Cukurova University, 01330, Adana, TURKEY E-mail: [email protected]

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