The Effect of Kanamycin and Tetracycline on Growth and Photosynthetic Activity of Two Chlorophyte Algae

Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 5656304, 8 pages http://dx.doi.org/10.1155/2016/5656304 Research Article The Effect of Kanamycin and Tetracycline on Growth and Photosynthetic Activity of Two Chlorophyte Algae Khawaja Muhammad Imran Bashir and Man-Gi Cho Department of Biotechnology, Division of Energy and Bioengineering, Dongseo University, Busan, Republic of Korea Correspondence should be addressed to Man-Gi Cho; [email protected] Received 20 April 2016; Revised 22 July 2016; Accepted 26 July 2016 Academic Editor: Jinsong Ren Copyright © 2016 K. M. I. Bashir and M.-G. Cho. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Antibiotics are routinely used in microalgae culture screening, stock culture maintenance, and genetic transformation. By studying the effect of antibiotics on microalgae growth, we can estimate the least value to inhibit growth of undesired pathogens inalgal culture. We studied the effect of kanamycin and tetracycline on the growth and photosynthetic activity of two chlorophyte −1 microalgae, Dictyosphaerium pulchellum and Micractinium pusillum. We measured CFU mL on agar plates, optical density, fluorescence yields, and photosynthetic inhibition. Our results showed a significant effectof kan and tet on the tested microalgae species except tet, which showed a minor effect on M. pusillum. Both antibiotics are believed to interact with the protein synthesis machinery; hence, the inhibitory effect of the tested antibiotics was further confirmed by isolation and quantification of thewhole −1 cell protein. A significant reduction in protein quantity was observed at concentrations more than 5mgL , except M. pusillum, −1 which showed only a slight reduction in protein quantity even at the maximum tested concentration of tet (30 mg L ). This study can further aid in aquaculture industry, for the maintenance of the microalgae stock cultures and it can also help the microalgae genetic engineers in the construction of molecular markers. 1. Introduction aquaculture, and waste disposal have been linked with the antibiotic resistance [13–15]. Microalgae are gaining importance in medical, pharmaceu- Aminoglycosides are the commonly used broad- tical, and food industry. With the increasing applications of spectrum antibiotics, that is, streptomycin, kanamycin, and microalgae, it is mandatory to investigate growth conditions amikacin. Aminoglycosides are characterized as multifunc- and potential growth inhibitors. Herbicides, antibiotics, and tional hydrophilic carbohydrates with several amino heavy metals are toxic to microalgae even at low concentra- and hydroxyl activities having higher affinities to the tions [1–6]. Studying the survival and adoption of microalgae prokaryotic rRNA [16, 17]. Suzuki et al. studied the effect of in the contaminated environment is not an insignificant kanamycin on bacterial protein inhibition [18]. Kestell et al. question and to a certain extent, the microalgae could survive reported the effect of kanamycin and streptomycin on the in contaminated environments [7–10]. macromolecular composition of Escherichia coli strains [19]. The inhibitory effect of streptomycin had been reported to In the past decade antibiotics use and resistance have −1 been the focus of the world leading organizations, including microalgae species at a concentration of 0.5 to 150 mg L the Center of Disease Control (CDC) and the World Health [20–22]. Galloway reported a halotolerant algae Amphora Organization (WHO). Alexander Fleming and Howard Wal- coffeaeformis resistance to streptomycin [23]. Kv´ıderovaand´ ter Florey warned the world first time about the antibiotic Henley reported the effect of ampicillin and streptomycin resistance while receiving 1945 Nobel Prize for the discovery on the growth and photosynthetic activity of halotolerant of penicillin [11]. Antibiotic resistance has been a productive chlorophyte algae species [24]. However, a limited or research topic for scientists in the medical field [12]. Anthro- no literature is available on the structural studies of pogenic activities including use of antibiotics in agriculture, aminoglycosides interaction with RNA sequences. 2 BioMed Research International Kanamycin is a broad-spectrum aminoglycoside antibi- 2.2. Screening Tests. The spread plate method according to otic, isolated from bacterium Streptomyces kanamyceticus Markham and Hagmeier [42], with slight modifications, was [25]. It is considered an important medication needed in used to obtain colonies of the tested microalgae on agar a basic health system and it has been listed in the WHO’s plates. 200 L of the cultured microalgae with approximately 4 −1 list of Essential Medicines [26]. Kanamycin interacts with adjusted initial cell density (1 × 10 cells mL )wasspread the 30S ribosomal subunit resulting in a significant amount plated on AF6-agar plates supplemented with different con- −1 of mistranslation and prevents translocation during protein centrations of kan and tet ranging from 0 to 30 mg L . synthesis [27, 28], whereas tetracyclines bind to the 16S part Plates were incubated under constant light intensities and the of the 30S ribosomal subunit and prevent amino-acyl tRNA growth was observed for three weeks. to attach at A-site of mRNA-ribosome complex, ultimately inhibiting protein synthesis as well as cell growth [29–31]. Kanamycin resistance (KanR)ismainlyduetothecyto- 2.3. Modulated Fluorescence and Photosynthetic Inhibition plasmic aminoglycoside phosphotransferase that inactivates Measurement. Fluorescence yields of algae samples treated kanamycin by covalent phosphorylation. On the other hand, with different concentrations of kan and tet were measured by tetracyclines are a group of broad-spectrum antibiotics, but toxy-PAM dual channel yield analyzer (Heinz Walz GmbH, their general application has been shortened because of the Effeltrich, Germany). The toxicity test is based on extremely inception of antibiotic resistance [32–34]. Cells can become sensitive measurement of the effective quantum yield (Y), resistant to tetracyclines by one of the three mechanisms: of photosystem II (PSII), via assessment of chlorophyll fluorescence yield by following the saturation pulse method [43, enzymatic inactivation of tetracycline, efflux, and ribosomal protection [35]. 44]. Fluorescence of the dark adopted algal samples ( 0)is measured by using modulated light of low intensity to avoid Antibiotics tolerance of prokaryotic microorganisms has been described by leading scientists, but there are just a the reduction of the PSII primary electron acceptor ( )[43]. few reports available on the antibiotic tolerance study of In order to induce an equilibrium state for the photosynthetic eukaryotic microalgae [20, 22, 23, 36]. No doubt, antibi- electron transport, prior to measurement of fluorescence, otics are normally considered effective against prokaryotic algal cells were adapted to darkness for 20 min. microorganisms, but they are extensively used in microalgae In the toxy-PAM blue light is used for excitation and fluorescence is assessed at a wavelength above 650 nm. culture screening [37, 38], in aquaculture, and for screening of genetic transformants [39]; hence, there is a need to check The ( 0) fluorescence level corresponds to the fluorescence measured shortly before the application of a saturation pulse. the effects of the antibiotics against eukaryotic microalgae. This work was planned to determine the activity of two Maximum fluorescence level ( ) corresponds to the max- important antibiotics, kanamycin sulfate and tetracycline imal fluorescence measured during a saturation pulse. The hydrochloride, against the freshwater eukaryotic microal- effective PSII overall quantum yield of the photochemical gae species, Dictyosphaerium pulchellum and Micractinium energy conversion was calculated by the formula given by pusillum. Colony forming units, optical density, fluorescence Genty et al. [44]. yields, and photosynthetic inhibitions were measured. The antibioticsusedinthisstudyarebelievedtointeractwiththe ( −) = = 0 = V . protein synthesis machinery; hence, the whole cell protein Yield (1) was also extracted and quantified. Relative photosynthetic inhibition of the investigated samples with respect to the reference sample was calculated by the 2. Material and Methods following formula: 2.1. Microalgae Cultivation and Treatment. The eukary- 100 ( −) otic freshwater microalgae species, Dictyosphaerium pulchel- = 2 1 . Relative Photosynthetic Inhibition% (2) lum and Micractinium pusillum, used in this study were 2 obtained from the Korea Marine Microalgae Culture Center (KMMCC), Busan, South Korea. Stock cultures were stored 2.4. Protein Isolation and Quantification. The tested antibi- on the modified AF6 agar slants [40]. The cultures were streak otics are believed to interfere with the protein synthesis plated and purified by subculturing by at least 5-6 times machinery; hence, at the end of the experiment, the whole before use. Both microalgae species were cultivated in 250 mL cell protein was isolated by total protein extraction kit (Invent flasks with 150 mL, modified AF6 medium while incubating ∘ −2 −1 Biotechnologies). The extracted protein was quantified by at 25 ± 2 C, 50 ± 2 mol photons m s and 50% humidity. BCA protein quantification assay kit (Pierce

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