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.
1. 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 [1–6]. 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 [7–10].
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 [13–15].
Aminoglycosides are the commonly used broad-spectrum 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 [20–22]. 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 [29–31].
Kanamycin resistance (KanR) 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 [32–34]. 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.
2. Material and Methods2.1. 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±2μmol 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 (OD750) on every alternating day [41]. Additionally, all the experiments were repeated three times.
2.2. 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 μL of the cultured microalgae with approximately adjusted initial cell density (1 × 104 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.
2.3. 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 (F0) is measured by using modulated light of low intensity to avoid the reduction of the PSII primary electron acceptor (QA) [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 (F0) fluorescence level corresponds to the fluorescence measured shortly before the application of a saturation pulse. Maximum fluorescence level (Fm) 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].(1)Y=Yield=Fm-F0Fm=FvFm.Relative photosynthetic inhibition of the investigated samples with respect to the reference sample was calculated by the following formula:(2)Relative Photosynthetic Inhibition%=100Y2-Y1Y2.
2.4. Protein Isolation and Quantification
The tested antibiotics are believed to interfere with the protein synthesis machinery; hence, at the end of the experiment, the whole cell protein was isolated by total protein extraction kit (Invent Biotechnologies). The extracted protein was quantified by BCA protein quantification assay kit (Pierce Biotechnology), while bovine serum albumin (BSA) was used as a standard. The extracted protein was electrophoresed on SDS-PAGE with 30% acrylamide : bisacrylamide solution and dyed for 1 h with coomassie brilliant blue G-250 (sigma). The gels were destained overnight with destaining solution and documented.
3. 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 × 103 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 × 106 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 (OD750) (Figures 2(a), 2(b), 3(a), and 3(b)).
(a) Colony forming units of D. pulchellum on kan and tet agar plates. (b) Colony forming units of M. pusillum on kan and tet agar plates. X-axis represents concentrations of antibiotics and colony forming units (CFU) per mL−1 are shown along the y-axis. Values are means ± SE, n=3.
(a) Growth of D. pulchellum in AF6 medium supplemented with kan. (b) Growth of D. pulchellum in AF6 medium supplemented with tet. X-axis represents days and absorbance values at 750 nm (OD750) are shown along the y-axis. Values are means ± SE, n = 3.
(a) Growth of M. pusillum in AF6 medium supplemented with kan. (b) Growth of M. pusillum in AF6 medium supplemented with tet. X-axis represents days and absorbance values at 750 nm (OD750) are shown along the y-axis. Values are means ± SE, n=3.
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)).
(a) Modulated fluorescence yield of D. pulchellum treated with kan. X-axis represents days and fluorescence yield values are shown along the y-axis. (b) Relative photosynthetic inhibition of D. pulchellum treated with kan. X-axis represents days and inhibition percentages are shown along the y-axis. Values are means ± SE, n=3.
(a) Modulated fluorescence yield of D. pulchellum treated with tet. X-axis represents days and fluorescence yield values are shown along the y-axis. (b) Relative photosynthetic inhibition of D. pulchellum treated with tet. X-axis represents days and inhibition percentages are shown along the y-axis. Values are means ± SE, n = 3.
(a) Modulated fluorescence yield of M. pusillum treated with kan. X-axis represents days and fluorescence yield values are shown along the y-axis. (b) Relative photosynthetic inhibition of M. pusillum treated with kan. X-axis represents days and inhibition percentages are shown along the y-axis. Values are means ± SE, n = 3.
(a) Modulated fluorescence yield of M. pusillum treated with tet. X-axis represents days and fluorescence yield values are shown along the y-axis. (b) Relative photosynthetic inhibition of M. pusillum treated with tet. X-axis represents days and inhibition percentages are shown along the y-axis. Values are means ± SE, n = 3.
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).
Standard curve for Bovine Serum Albumin (BSA). X-axis indicates BSA concentrations and absorbance values at 562 nm are shown along the y-axis. Values are means ± SE, n = 3.
Protein quantities of D. pulchellum and M. pusillum treated with kan and tet. X-axis represents concentrations of antibiotics, kan and tet; y-axis represents protein concentrations in μg mL−1. Values are means ± SE, n=3.
4. 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 (OD750), 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 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.
5. 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.
Abbreviationskan:
Kanamycin
tet:
Tetracycline
Y1:
Fluorescence yield of the investigated sample
Y2:
Fluorescence yield of the reference
Y=Fv/Fm:
Quantum yield of photosystem II (PSII)
F0:
Minimum fluorescence value
Fm:
Maximum fluorescence value
Fv:
Variable fluorescence
SDS-PAGE:
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
BCA:
Bicinchoninic acid
BSA:
Bovine serum albumin.
Competing Interests
The authors declare that there is no conflict of interests regarding the publication of this article and regarding the funding received.
Acknowledgments
This work was supported by Dongseo University Frontier Project-2012; authors are thankful for their support.
HansonM. R.BogoradL.Effects of erythromycin on membrane-bound chloroplast ribosomes from wild-type Chlamydomonas reinhardi and erythromycin-resistant mutants1977479327928910.1016/0005-2787(77)90110-12-s2.0-0017660795KallqvistT.RomstadR.Effects of agricultural pesticides on planktonic algae and cyanobacteria: examples of interspecies sensitivity variations199413117131NyströmB.BjörnsäterB.BlanckH.Effects of sulfonylurea herbicides on non-target aquatic micro-organisms—growth inhibition of micro-algae and short-term inhibition of adenine and thymidine incorporation in periphyton communities199947192210.1016/s0166-445x(99)00007-72-s2.0-0345062227LuC. M.ChauC. W.ZhangJ. H.Acute toxicity of excess mercury on the photosynthetic performance of cyanobacterium, S. platensis—assessment by chlorophyll fluorescence analysis2000411-219119610.1016/s0045-6535(99)00411-72-s2.0-0034009902JuneauP.DewezD.MatsuiS.KimS.-G.PopovicR.Evaluation of different algal species sensitivity to mercury and metolachlor by PAM-fluorometry2001454-558959810.1016/s0045-6535(01)00034-02-s2.0-0034817298Peña-VázquezE.Pérez-CondeC.CostasE.Moreno-BondiM. C.Development of a microalgal PAM test method for Cu(II) in waters: comparison of using spectrofluorometry20101961059106510.1007/s10646-010-0487-y2-s2.0-79960558547Lopez-RodasV.AgreloM.CarrilloE.FerreroL. M.LarrauriA.Martín-OteroL.CostasE.Resistance of microalgae to modern water contaminants as the result of rare spontaneous mutations200136217919010.1017/S09670262010031092-s2.0-0034931479NashS. M. B.QuayleP. A.SchreiberU.MüllerJ. F.The selection of a model microalgal species as biomaterial for a novel aquatic phytotoxicity assay200572431532610.1016/j.aquatox.2005.02.0042-s2.0-17544377012HerloryO.BonzomJ.-M.GilbinR.Sensitivity evaluation of the green alga Chlamydomonas reinhardtii to uranium by pulse amplitude modulated (PAM) fluorometry2013140-14128829410.1016/j.aquatox.2013.06.0072-s2.0-84880373110Suresh KumarK.DahmsH.-U.LeeJ.-S.KimH. C.LeeW. C.ShinK.-H.Algal photosynthetic responses to toxic metals and herbicides assessed by chlorophyll a fluorescence20141041517110.1016/j.ecoenv.2014.01.0422-s2.0-84896031035NathanC.CarsO.Antibiotic resistance—problems, progress, and prospects2014371191761176310.1056/nejmp14080402-s2.0-84908577353BryskierA.2005Washington, DC, USAASM PressDoyleM. P.BustaF. F.CordsB. R.Antimicrobial resistance: implications for the food system2006537113710.1111/j.1541-4337.2006.00004.xSchlüterA.KrauseL.SzczepanowskiR.GoesmannA.PühlerA.Genetic diversity and composition of a plasmid metagenome from a wastewater treatment plant20081361-2657610.1016/j.jbiotec.2008.03.0172-s2.0-50149108551SzczepanowskiR.LinkeB.KrahnI.GartemannK.-H.GützkowT.EichlerW.PühlerA.SchlüterA.Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics200915572306231910.1099/mic.0.028233-02-s2.0-69949162470RechtM. I.FourmyD.BlanchardS. C.DahlquistK. D.PuglisiJ. D.RNA sequence determinants for aminoglycoside binding to an A-site rRNA model oligonucleotide1996262442143610.1006/jmbi.1996.05262-s2.0-1842388479RechtM. I.DouthwaiteS.PuglisiJ. D.Basis for prokaryotic specificity of action of aminoglycoside antibiotics199918113133313810.1093/emboj/18.11.31332-s2.0-0033152492SuzukiJ.KunimotoT.HoriM.Effects of kanamycin on protein synthesis: inhibition of elongation of peptide chains19702329910110.7164/antibiotics.23.992-s2.0-0014736610KestellD.LaiS.LiangG.WatersS.WladichukA.Effects of kanamycin and streptomycin on the macromolecular composition of streptomycin-sensitive and resistant Escherichia coli strains20022103108FoterM. J.PalmerC. M.MaloneyT. E.Antialgal properties of various antibiotics19533505508HarrassM. C.KindigA. C.TaubF. B.Responses of blue-green and green algae to streptomycin in unialgal and paired culture19856111110.1016/0166-445x(85)90015-32-s2.0-0021965971Halling-SørensenB.Algal toxicity of antibacterial agents used in intensive farming200040773173910.1016/s0045-6535(99)00445-22-s2.0-0033981490GallowayR. E.Selective conditions and isolation of mutants in salt-tolerant, lipid-producing microalgae199026475276010.1111/j.0022-3646.1990.00752.xKvíderováJ.HenleyW. J.The effect of ampicillin plus streptomycin on growth and photosynthesis of two halotolerant chlorophyte algae200517430130710.1007/s10811-005-7293-62-s2.0-23844459249GarrodL. P.LambertH. P.O'GradyF.19815thEdinburgh, UKChurchill Livingstone PressWHO2013Geneva, SwitzerlandWorld Health OrganizationPestkaS.The use of inhibitors in studies of protein synthesis19743026128210.1016/0076-6879(74)30030-42-s2.0-0016023673MisumiM.TanakaN.Mechanism of inhibition of translocation by kanamycin and viomycin: a comparative study with fusidic acid198092264765410.1016/0006-291x(80)90382-42-s2.0-0018817529SchnappingerD.HillenW.Tetracyclines: antibiotic action, uptake, and resistance mechanisms1996165635936910.1007/s0020300503392-s2.0-0030163126ChopraI.RobertsM.Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance200165223226010.1128/mmbr.65.2.232-260.20012-s2.0-0034973583ConnellS. R.TraczD. M.NierhausK. H.TaylorD. E.Ribosomal protection proteins and their mechanism of tetracycline resistance200347123675368110.1128/aac.47.12.3675-3681.20032-s2.0-0344443255VieraM. H.PerezO. A.BermanB.Incyclinide200732320921410.1358/dof.2007.032.03.10833082-s2.0-34250310117DaviesJ.DaviesD.Origins and evolution of antibiotic resistance201074341743310.1128/MMBR.00016-102-s2.0-77957980707RichardsC.PantanowitzL.DezubeB. J.Antimicrobial and non-antimicrobial tetracyclines in human cancer trials201163215115610.1016/j.phrs.2010.10.0082-s2.0-79251612333RyanM. E.UsmanA.RamamurthyN. S.GolubL. M.GreenwaldR. A.Excessive matrix metalloproteinase activity in diabetes: inhibitions by tetracycline analogues wit zinc reactivity20018330531610.2174/09298670133735982-s2.0-0035031111NečasJ.Responses of cell populations of three chlorococcal algae to the action of streptomycin1971135-633834810.1007/BF02930922SuJ. Q.YangX.ZhengT.HongH.An efficient method to obtain axenic cultures of Alexandrium tamarense—a PSP-producing dinoflagellate200769342543010.1016/j.mimet.2006.07.0052-s2.0-34247633477WilkensS. L.MaasE. W.Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments2012112234635210.1111/j.1365-2672.2011.05197.x2-s2.0-84855880102BashirK. M. I.KimM.StahlU.ChoM.Microalgae engineering toolbox: selectable and screenable markers201621222423510.1007/s12257-015-0386-4WatanabeM. M.20006thTsukuba, JapanNIESAlkhamisY.QinJ. G.Cultivation of Isochrysis galbana in phototrophic, heterotrophic, and mixotrophic conditions20132013998346510.1155/2013/9834652-s2.0-84893776674MarkhamJ. W.HagmeierE.Observations on the effects of germanium dioxide on the growth of macro-algae and diatoms198221212513010.2216/i0031-8884-21-2-125.1SchreiberU.Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorometer198691-226127210.1007/bf000297492-s2.0-0000347609GentyB.BriantaisJ.-M.BakerN. R.The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence19899901879210.1016/s0304-4165(89)80016-9