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

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 in algal culture. We studied the effect of kanamycin and tetracycline on the growth and photosynthetic activity of two chlorophyte microalgae, Dictyosphaerium pulchellum and Micractinium pusillum. We measured CFU mL−1 on agar plates, optical density, fluorescence yields, and photosynthetic inhibition. Our results showed a significant effect of 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 the whole cell protein. A significant reduction in protein quantity was observed at concentrations more than 5 mg L−1, except M. pusillum, which showed only a slight reduction in protein quantity even at the maximum tested concentration of tet (30 mg L−1). 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.


Introduction
Microalgae are gaining importance in medical, pharmaceutical, and food industry. With the increasing applications of microalgae, it is mandatory to investigate growth conditions and potential growth inhibitors. Herbicides, antibiotics, and heavy metals are toxic to microalgae even at low concentrations [1][2][3][4][5][6]. Studying the survival and adoption of microalgae in the contaminated environment is not an insignificant question and to a certain extent, the microalgae could survive in contaminated environments [7][8][9][10].
In the past decade antibiotics use and resistance have been the focus of the world leading organizations, including the Center of Disease Control (CDC) and the World Health Organization (WHO). Alexander Fleming and Howard Walter Florey warned the world first time about the antibiotic resistance while receiving 1945 Nobel Prize for the discovery of penicillin [11]. Antibiotic resistance has been a productive research topic for scientists in the medical field [12]. Anthropogenic activities including use of antibiotics in agriculture, aquaculture, and waste disposal have been linked with the antibiotic resistance [13][14][15].
Aminoglycosides are the commonly used broadspectrum antibiotics, that is, streptomycin, kanamycin, and amikacin. Aminoglycosides are characterized as multifunctional hydrophilic carbohydrates with several amino and hydroxyl activities having higher affinities to the prokaryotic rRNA [16,17]. Suzuki et al. studied the effect of kanamycin on bacterial protein inhibition [18]. Kestell et al. reported the effect of kanamycin and streptomycin on the macromolecular composition of Escherichia coli strains [19]. The inhibitory effect of streptomycin had been reported to microalgae species at a concentration of 0.5 to 150 mg L −1 [20][21][22]. Galloway reported a halotolerant algae Amphora coffeaeformis resistance to streptomycin [23]. Kvíderová and Henley reported the effect of ampicillin and streptomycin on the growth and photosynthetic activity of halotolerant chlorophyte algae species [24]. However, a limited or no literature is available on the structural studies of aminoglycosides interaction with RNA sequences.
Kanamycin is a broad-spectrum aminoglycoside antibiotic, isolated from bacterium Streptomyces kanamyceticus [25]. It is considered an important medication needed in a basic health system and it has been listed in the WHO's list of Essential Medicines [26]. Kanamycin interacts with the 30S ribosomal subunit resulting in a significant amount of mistranslation and prevents translocation during protein synthesis [27,28], whereas tetracyclines bind to the 16S part of the 30S ribosomal subunit and prevent amino-acyl tRNA to attach at A-site of mRNA-ribosome complex, ultimately inhibiting protein synthesis as well as cell growth [29][30][31].
Kanamycin resistance (Kan R ) is mainly due to the cytoplasmic aminoglycoside phosphotransferase that inactivates kanamycin by covalent phosphorylation. On the other hand, tetracyclines are a group of broad-spectrum antibiotics, but their general application has been shortened because of the inception of antibiotic resistance [32][33][34]. Cells can become resistant to tetracyclines by one of the three mechanisms: enzymatic inactivation of tetracycline, efflux, and ribosomal protection [35].
Antibiotics tolerance of prokaryotic microorganisms has been described by leading scientists, but there are just a few reports available on the antibiotic tolerance study of eukaryotic microalgae [20,22,23,36]. No doubt, antibiotics are normally considered effective against prokaryotic microorganisms, but they are extensively used in microalgae culture screening [37,38], in aquaculture, and for screening of genetic transformants [39]; hence, there is a need to check the effects of the antibiotics against eukaryotic microalgae.
This work was planned to determine the activity of two important antibiotics, kanamycin sulfate and tetracycline hydrochloride, against the freshwater eukaryotic microalgae species, Dictyosphaerium pulchellum and Micractinium pusillum. Colony forming units, optical density, fluorescence yields, and photosynthetic inhibitions were measured. The antibiotics used in this study are believed to interact with the protein synthesis machinery; hence, the whole cell protein was also extracted and quantified.

Microalgae Cultivation and Treatment.
The eukaryotic freshwater microalgae species, Dictyosphaerium pulchellum and Micractinium pusillum, used in this study were obtained from the Korea Marine Microalgae Culture Center (KMMCC), Busan, South Korea. Stock cultures were stored on the modified AF6 agar slants [40]. The cultures were streak plated and purified by subculturing by at least 5-6 times before use. Both microalgae species were cultivated in 250 mL flasks with 150 mL, modified AF6 medium while incubating at 25 ± 2 ∘ C, 50 ± 2 mol photons m −2 s −1 and 50% humidity. Antibiotics, kanamycin sulfate (Amresco), and tetracycline hydrochloride (Bio101) with different concentrations ranging from 0 to 30 mg L −1 were used. Growth rates were calculated by measuring the absorbance at 750 nm (OD 750 ) on every alternating day [41]. Additionally, all the experiments were repeated three times.

Screening
Tests. The spread plate method according to Markham and Hagmeier [42], with slight modifications, was used to obtain colonies of the tested microalgae on agar plates. 200 L of the cultured microalgae with approximately adjusted initial cell density (1 × 10 4 cells mL −1 ) was spread plated on AF6-agar plates supplemented with different concentrations of kan and tet ranging from 0 to 30 mg L −1 . Plates were incubated under constant light intensities and the growth was observed for three weeks.

Modulated Fluorescence and Photosynthetic Inhibition
Measurement. Fluorescence yields of algae samples treated with different concentrations of kan and tet were measured by toxy-PAM dual channel yield analyzer (Heinz Walz GmbH, Effeltrich, Germany). The toxicity test is based on extremely sensitive measurement of the effective quantum yield (Y), of photosystem II (PSII), via assessment of chlorophyll fluorescence yield by following the saturation pulse method [43,44]. Fluorescence of the dark adopted algal samples ( 0 ) is measured by using modulated light of low intensity to avoid the reduction of the PSII primary electron acceptor ( ) [43]. In order to induce an equilibrium state for the photosynthetic electron transport, prior to measurement of fluorescence, algal cells were adapted to darkness for 20 min.
In the toxy-PAM blue light is used for excitation and fluorescence is assessed at a wavelength above 650 nm. The ( 0 ) fluorescence level corresponds to the fluorescence measured shortly before the application of a saturation pulse. Maximum fluorescence level ( ) corresponds to the maximal fluorescence measured during a saturation pulse. The effective PSII overall quantum yield of the photochemical energy conversion was calculated by the formula given by Genty et al. [44].
Relative photosynthetic inhibition of the investigated samples with respect to the reference sample was calculated by the following formula:

Results
During this study, antibiotic sensitivity of two freshwater eukaryotic microalgae was assessed. Microalgae species showed significant sensitivities to the tested antibiotics as indicated by their colony forming units, fluorescence yields, and protein concentrations. The CFU mL −1 of D. pulchellum reduced significantly with the increasing concentrations of kan and tet. The CFU mL −1 of 3.50 × 10 3 was observed with kan at a concentration of 30 mg L −1 , but at the same concentration of tet, no colony was observed (Figure 1(a)). There was a reduction in CFU of M. pusillum with increasing concentration of tet, but CFU of 1.09 × 10 6 was observed even at the maximum tested concentration (Figure 1(b)). A substantial decrease in CFU of M. pusillum was observed with increasing concentrations of kan. Similar results were achieved with growth measurement study at absorbance of 750 nm (OD 750 ) (Figures 2(a), 2(b), 3(a), and 3(b)).
The fluorescence yields and photosynthetic inhibition percentages of tested algal species against kan and tet showed significant variations. Initially, D. pulchellum showed a slight increase in fluorescence yield with kan and tet at concentrations of 5 and 10 mg L −1 but after the 3rd day of inoculation, a significant reduction in fluorescence yield was observed with all the tested concentrations as compared to the control (0 mg L −1 ) (Figures 4(a) and 5(a)). This species showed photosynthetic inhibition at all the tested concentrations after the 3rd day of culturing (Figures 4(b) and 5(b)). M. pusillum also showed variation in fluorescence yield and photosynthetic inhibition. When treated with kan, the concentrations, 20 and 30 mg L −1 , did not show fluorescence yield even at the 11th day of experiment, but when treated with the same concentration of tet it showed a slight variation in fluorescence yields (Figures 6(a) and 7(a)). A variable degree of photosynthetic inhibition percentages was achieved when treated with kan and tet (Figures 6(b) and 7(b)). Whole cell protein from both microalgae species was extracted and quantified by BCA protein quantification assay, while BSA was used as a control. A BSA standard curve was drawn with optical density values at 562 nm versus BSA concentrations (Figure 8). An increase in protein quantity was observed with kan and tet at a concentration of 5 mg L −1 , but a significant reduction in protein quantity was observed at the higher concentrations ( Figure 9). However, M. pusillum showed only a slight reduction in protein quantity even at the maximum tasted concentration of tet (30 mg L −1 ).

Discussion
The antibiotic sensitivity has been reported for different microorganisms, but there is a limited or no literature available on the antibiotic sensitivity characteristics of microalgae. During this study, antibiotic sensitivity characteristics of two freshwater eukaryotic microalgae species D. pulchellum and M. pusillum were evaluated against the two important protein synthesis inhibiting antibiotics, kanamycin sulfate and tetracycline hydrochloride. The sensitivity of D. pulchellum and M. pusillum to kanamycin and tetracycline was estimated by colony forming units on agar plates, variation in whole cell protein quantities, modulated fluorescence yields, and relative photosynthetic inhibition percentages. Microalgae species showed significant sensitivities against the tested antibiotics as indicated by their fluorescence kinetics and protein concentrations. D. pulchellum showed reduction in growth with both antibiotics; even a clear difference in the extracted protein quantities was observed. M. pusillum also showed reduction when tested against kan but showed only a slight reduction in growth on tet agar plates even at the highest tested concentration (30 mg L −1 ). Interestingly, when absorbance was tested at 750 nm (OD 750 ), this species also showed reduction in growth with increasing concentrations of tet; however, a minor growth was observed at the maximum tested concentration (30 mg L −1 ). This species may also show inhibition at higher concentrations of tet. Both the tested species showed significant reduction in growth at kan and tet concentrations higher than 10 mg L −1 . The minimum inhibitory concentration for D. pulchellum was recorded as 6 mg L −1 with kan and 8 mg L −1 with tet, whereas the minimum inhibitory concentration of kan against M. pusillum was recorded as 8 mg L −1 . To further confirm the effect of the tested antibiotics, whole cell protein from both microalgae species was extracted and quantified by BCA protein quantification assay while BSA was used as a standard. The results of isolated proteins were quite interesting; both the tested antibiotics showed increase in protein 6 BioMed Research International quantity with kan and tet at a concentration of 5 mg L −1 but a clear reduction in protein quantity was observed at higher concentrations. However, M. pusillum showed only a slight reduction in protein quantity even at the maximum tasted concentration of tet (30 mg L −1 ). Whether the tested antibiotics at low concentrations accelerated the growth or not cannot be concluded at this stage. Further study and biochemical analyses are required to support the findings. This basic study can further aid the microalgae genetic engineers in construction of molecular markers and in microalgae stock culture maintenance.

Conclusion
Kanamycin and tetracycline are routinely used for human and animals. The sensitivity of D. pulchellum and M. pusillum was studied to kanamycin and tetracycline through colony forming units on agar plates, variation in protein concentrations, quantum yields, and photosynthetic inhibition percentages. Both the tested species showed significant reduction in growth at kan and tet concentrations higher than 10 mg L −1 except M. pusillum which showed growth even at the maximum tested concentration of tetracycline (30 mg L −1 ). 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. Bicinchoninic acid BSA: Bovine serum albumin.