Microalgae and their metabolites have great potential in areas such as food and feed manufacturing; the cosmetics and medicine industries; and bioenergy development(Skulberg,2000; Griesbeck et al., 2006; Rosenberg et al., 2008; Amit et al., 2010; Subashchandrabose et al., 2011). At present,photoautotrophic culture is widely adopted for most microalgae. Unfortunately this is heavily infl uenced by multiple environmental factors including light defi ciency; temperature fl uctuation; and microalgal,bacterial and protozoan contamination,all of which result in low and unstable productivity(Zhang et al., 1999; Lee,2001; Carvalho et al., 2006; Chisti,2007). Early studies have documented that many microalgal species are able to grow by metabolizing organic carbon(Chojnacka and Marquez-Rocha, 2004; Chen and Chen, 2006; Sloth et al., 2006; Bumbak et al., 2011; Perez-Garcia et al., 2011),making the heterotrophic culture of microalgae feasible. Such a mode of culture can avoid the limitations of phototrophic culture and lead to a high growth rate and high biomass yield(Miao and Wu, 2006; Sloth et al., 2006; Brennan and Owende, 2010; Bumbak et al., 2011). Potential valuable applications include the production of biofuels and food-grade microalgae material. Axenic microalgal strains are important in this mode of culture.

Many methods have been adopted for purifying microalgal strains and obtaining an axenic culture.These include plate streaking( Wigglesworth-Cooksey et al., 2001),lysozyme and sodium dodecyl sulfate treatment(Su et al., 2007),repeated centrifugation and rinsing(Bolch and Blackburn, 1996),sonication(Azma et al., 2010) and single cell isolation(Choi et al., 2008). The removal of bacteria from microalgae with antibiotics is widely undertaken(Spencer,1952; Cottrell and Suttle, 1993; Bolch and Blackburn, 1996; Lin et al., 2000; Vázquez-Martínez and Rodriguez, 2004; Bruckner and Kroth, 2009).

We found that the majority of the existing methods are species specifi c. In this study,we developed a method of batch-purifying microalgae species with antibiotics to obtain an axenic culture, and have successfully purifi ed 10 strains of microalgae in this way.

Pure cultures of microalgae(Table 1)were cultivated in f/2 medium(Guillard and Ryther, 1962)at 25±1°C and salinity 30 and under 70 μmol photons/(m ² ·s)white fl uorescent light following a cycle of 12 h light and 12 h dark. The solid f/2 medium contained 1.2% agar. All the experimental operations were carried out at a clean bench(SW-CJ-2D,Jinghua^®,Suzhou,China). Liquid culturing was carried out in a light growth chamber(GXZ-80A,Jiangnan^®,Ningbo,China) and solid culturing was carried out in a constant temperature incubator(DHP- 9052,Yiheng^®,Shanghai,China).

Table 1 Axenic strains used in this study

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Based on previous reports(Table 2),fi ve antibiotics(ampicillin,gentamycin sulfate,kanamycin,neomycin and streptomycin)were selected to remove the bacteria. Each microalga was inoculated in the stationary phase into fresh f/2 medium at a ratio of 1/10(volume/volume) and cultured for about 7 days. The mixture of fi ve antibiotics(each antibiotic at the same fi nal concentration of 200,600 and 1 000 mg/L for treatments A,B and C,respectively)was then added to the microalgal culture and cultivated for 3 days. After the antibiotics treatment,the following procedures were carried out:(1)1 mL of microalga was mixed with 2216E solid medium(ZoBell,1941)(containing 1.5% agar) and cultured at 25°C for 15 days. A preliminary evaluation of the eff ects of bacteria removal of the diff erent treatments was reached by counting the number of the bacterial colonies;(2)5 mL of microalga was transferred to 45 mL antibiotics-free f/2 medium and cultured for 5 days;(3)next,100 μL of the microalga was spread on f/2 solid medium and cultured under 70 μmol photons/(m ² ·s)irradiation with a light-dark regime of 12 h: 12 h. Algal colony formation and the eff ect of bacteria removal were examined to determine the optimal concentration of antibiotics for bacteria removal.

Table2 Antibiotics used for microalga purifi cation in early studies

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A microalgal colony of each original culture was transferred to a 100-mL fl ask containing 50 mL fresh f/2 medium and cultured for about 10–15 days to a cell density of 3.0×106 cells/mL(five strains of Nannochloropsis sp.); 0.25×106 cells/mL(two strains of Tetraselmis sp.); 0.2×106 cells/mL(two strains of Cylindrotheca sp.); and 1.0×106 cells/mL(one strain of Amphikrikos sp.). The combination of antibiotics at the optimal concentration was then added to the microalgae. During the following 3 days,the microalgae were blown thoroughly by a pipettor,90 times a day. After antibiotics treatment,5 mL of each microalgal culture was transferred to 45 mL of antibiotics-free f/2 medium and cultured for 5 days. Then,100 μL of each microalga was spread on solid f/2 medium containing fi ve antibiotics at 50 mg/L. The procedure is outlined in Fig. 1.

Fig. 1 Key steps in obtaining a batch axenic microalgal culture

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A single colony was transferred to and cultivated in fresh f/2 liquid medium for 5 days. Two methods were used to judge whether a microalga was axenic: the cells were stained with SYBR Green I(Solarbio^®,China)using the Bruckner and Kroth(2009)method and observed under a fl uorescence microscope(IX51,Olympus^®,Japan). Next,2-mL of microalga was transferred to 35 mL of H1 liquid medium and incubated for 5 days at 25°C,shaking at 200 r/min in a constant temperature shaker(HZQ-Q,HDL^®,Harbin,China). Finally,1-mL of microalga was mixed with semi-solid H1 solid medium and cultured at 25°C for 15 days. H1 medium was prepared by adding 5 g peptone and 1 g yeast extract to 1 L of f/2 liquid medium. H1 solid medium was prepared by adding 1.2%(w/w)agar to H1 liquid medium.

When microalgae treated with antibiotics in combination at three concentrations were transferred to antibiotics-free f/2 medium,the color of their cultures gradually deepened,except for Amphikrikos sp. treated with antibiotics at 600 and 1 000 mg/L(the color of both of them faded away gradually). In contrast to the fl ourishing growth of cells transferred to antibioticsfree media,all strains remaining in the medium containing antibiotics at concentrations of 600 and 1 000 mg/L bleached in approximately 10 days,a similar phenomenon to that observed by Bruckner and Kroth(2009). Obviously,when microalgae were treated with antibiotics at concentrations of 600 and 1 000 mg/L for more than 10 days,their growth was drastically inhibited. However,the algal cells treated with antibiotics at these two concentrations for 3 days could resume their growth quickly when they were transferred to antibiotics-free media.

When transferred to antibiotics-free medium C . closterium and T . chuii formed colonies after about 10days. However, Amphikrikos sp. and Nannochloropsis sp. took longer to form microalgal colonies, and this time-lag was antibiotics concentration-dependent(the higher the concentration,the greater the time needed). For example,Amphikrikos sp. treated by antibiotics at 200 mg/L and 600 mg/L formed colonies in about 20 and 30 days,respectively; however,no algal colony was formed when it was treated with antibiotics at 1 000 mg/L(Table 3).

Table 3 Performance of microalgae treated with antibiotics

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When the concentration of antibiotics increased from 200 mg/L to 600 mg/L,the number of bacterial colonies plummeted signifi cantly. However,antibiotics concentrations at 1 000 mg/L did not signifi cantly increase the effi ciency of bacteria removal(Table 3).

Taking all factors into account,the concentration of 600 mg/L was optimal for microalgal purifi cation.

Although bacteria were dramatically inhibited by antibiotics at 600 mg/L,a few bacterial colonies formed. To scavenge bacteria completely,the microalgae were repeatedly blown with a sterilized pipettor to separate the bacteria from the algal cells during antibiotics treatment. As shown in Fig. 2,bacteria were not observed under microscopy. Furthermore,no bacterial colony was found on H1 plates(Table 3). These observations indicated that the microalgal species(strains)had been purifi ed successfully.

Fig. 2 Microalgal cells treated with antibiotics at 600 mg/L with intensive blowing, stained with 0.3% SYBR Green I and observed under fl uorescence microscopy Bacteria and microalgal nuclei are green, while chloroplasts are red because of the autofl uorescence of chlorophyll. a and b: No1 before and after purifi cation; c and d: Cc1 before and after purifi cation; e and f: Am1 before and after purifi cation; g and h: Tc1 before and after purifi cation. White arrows point to bacteria in Figs.a, c, e and g and to microalgal nuclei in Figs.b, d, f and g. The staining time diff ers according to species, varying between 20 and 40 min. Scale bar=10 μm.

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In this study,we verifi ed that microalgae treated with fi ve antibiotics in combination at a concentration of 600 mg/L for 3 days could resume their growth quickly when transferred to antibiotics-free medium,establishing an eff ective method of batch removal of bacteria from microalgae.

Many microalgal species secrete polysaccharides and form gelatinous “sheaths”,which may wrap around bacteria and prevent their exposure to antibiotics( Subashchandrabose et al., 2011). As a result,a few bacteria may survive antibiotics treatment even when the concentration of antibiotics is high. We have tentatively isolated the bacteria surviving antibiotics treatment in 2216E medium containing fi ve antibiotics at concentrations of 200,600 and 1 000 mg/L. We found that the growth of these bacteria was completely inhibited,implying that the bacteria were sensitive to the antibiotics. Their survival may be due to their aggregation,which insulated them from the antibiotics. In this study,we intensively blew the microalgal culture with a sterilized pipettor to break up any bacterial aggregation,enhancing the removal of bacteria by antibiotics.

Two methods were adopted to verify that the microalgae were axenic: spreading on bacterial H1 plates, and staining with SYBR Green I. Both methods were easy to operate and highly eff ective. We have tentatively used the 16S rRNA gene to verify the purity of C . closterium and T . chuii ; however,all of the amplifi cation b and s were from chloroplasts. As a result,slight contaminations may be overlooked. The cost of sequencing clones was expensive.

The bacterial communities associated with diff erent microalgal strains may vary greatly,thus multiple kinds and amounts of antibiotics are essential for an eff ective purifying protocol. However,antibiotics may not only remove bacteria,but also inhibit the growth of microalgae. To avoid such inhibition,a rigorous procedure to determine the sensitivity of microalgae to each kind of antibiotic is always conducted in early methods. Obviously,such a strategy is not appropriate for batch purifi cation. Microalgal batch purifi cation should be easy so that it can be used routinely. The powerful recovery ability of microalgae provides us with an opportunity to solve this problem.

To the best of our knowledge,antibiotics treatment has often been used eff ectively to remove bacterial contamination from microalgal cultures(Underwood et al., 2004; Liu and Duan, 2006; Huang et al., 2007; Lin,2000 ; Li et al., 2009; Katoh et al., 2012). However,methods to batch-purify microalgae are limited, and the method we developed in this study may help to fi ll this methodological gap.

Microalgae treated with multiple antibiotics at high concentrations can resume their growth quickly when transferred to antibiotics-free medium. This observation enriches our underst and ing of microalgae and underlies our purifying protocol which is featured by easy operation,time and labor saving. Consequently,this method can contribute to discover heterotrophic and mixotrophic species,study the microalgal-microalgal or microalgal-bacterial interactions, and so on.