Li YANG*, Hiroto MAEDA, Takeshi YOSHIKAWA, Gui-qin ZHOU

1. College of Environment, Hohai University, Nanjing 210098, P. R. China

2. College of Environment, Nanjing University of Technology, Nanjing 210009, P. R. China

3. Laboratory of Microbiology, Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, Japan

Algicidal effect of bacterial isolates of Pedobacter sp. against cyanobacterium Microcystis aeruginosa

Li YANG*1,2, Hiroto MAEDA3, Takeshi YOSHIKAWA3, Gui-qin ZHOU2

1. College of Environment, Hohai University, Nanjing 210098, P. R. China

2. College of Environment, Nanjing University of Technology, Nanjing 210009, P. R. China

3. Laboratory of Microbiology, Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, Japan

The aim of this study was to isolate algicidal bacteria so as to control harmful cyanobacterium Microcystis aeruginosa (M. aeruginosa) blooms using biological methods. Nine bacterial strains were isolated to inhibit the growth of M. aeruginosa, among which the MaI11-5 bacterial strain exhibited remarkable algicidal activity against M. aeruginosa cells during the test. Based on the 16S rDNA analysis, the isolated MaI11-5 was identified as Pedobacter sp. through morphology and homology research. The results of cocultivation of the cyanobacteria with MaI11-5 algicidal isolates showed obvious algicidal activity against cyanobacterial cells. The algicidal effect of MaI11-5 exceeded 50% after two days, exceeded 70% after four days, and reached 80% after seven days. The observation results with a scanning electron microscope showed that the cyanobacterial cells aggregated and produced mucous-like substances when cocultivated with the algicidal bacteria. The results indicated that the MaI11-5 bacterial strain may possess a novel function for controlling harmful blooms and further studies will provide new insights into its role in water environment.

algicidal bacteria; Pedobacter sp.; algicidal effect; Microcystis aeruginosa

1 Introduction

As a result of eutrophication of lakes and reservoirs in recent years, cyanobacterial blooms have become a common problem and attracted extensive attention worldwide (Dai et al. 2008). Frequent outbreaks of cyanobacterial blooms have posed great threats to human health because of the deteriorated freshwater quality and serious economic damage to aquaculture. Furthermore, because of the widespread distribution of cyanobacteria in natural lakes and rivers, the possibility of water blooms of toxic strains in recreational water is considerable. Microcystis aeruginosa (M. aeruginosa), one of the most common toxic strains, always produces microcystins, which are harmful to aquatic organisms and humans (Jacquet et al. 2004; Ernst et al. 2006; Palikova et al. 2007) and are identified as a tumor-promotingcompound (Falconer 1999; Oberholster et al. 2004; Zurawell et al. 2005). Therefore, the development of some useful techniques to predict and eliminate the impacts of cyanobacterial blooms is urgently required. Many approaches and techniques have been used to control blooms (Oberholster et al. 2004; Sengco and Anderson 2004; Choi et al. 2005), among which biological control has been seen as an economical and environment-friendly solution due to the low treatment cost and no secondary pollution (Sigee et al. 1999; Manage et al. 2001; Mayali and Doucette 2002).

Since the first report on the successfully isolated algicidal bacterium, many researchers have focused on laboratory studies of algicidal bacteria against marine (Imai et al. 1995; Kim et al. 2009) and freshwater (Kim et al. 2008; Ren et al. 2010) algae, and thought of algicidal bacteria as the most promising solution due to their advantages of fast reproduction, high efficiency, and host specificity (Mu et al. 2007). In this study, we totally isolated nine algicidal bacterial stains, among which a bacterial strain of Pedobacter sp., MaI11-5, exhibited distinctive cyanobacterial growth inhibition on M. aeruginosa. Although a lot of reports have demonstrated the algicidal effects of some algicidal bacteria against marine and freshwater algae, using Pedobacter sp. as algicidal bacteria against cyanobacterium M. aeruginosa has been rarely reported. The aims of this study were to study the algicidal effect of MaI11-5 against M. aeruginosa cells and to investigate the algicidal process during cocultivation of the bacteria with cyanobacterial cells with a scanning electron microscope (SEM).

2 Materials and methods

2.1 Algal culture

M. aeruginosa NIES-843, which was isolated from the samples collected in Kasumigaura Lake in Ibaraki, Japan during a cyanobacterial bloom in August 1997, was supplied by the Microbial Culture Collection at the National Institute for Environmental Studies (NIES), Japan. The axenic cyanobacteria culture was incubated with aeration in an MA medium (Ichimura 1979) at 25℃ under a 12L:12D light-dark cycle with a light intensity of 40 μmol/(m2·s). The data of optical density absorption at 680 nm (Abs680nm) were monitored with a microplate reader (Tecan Infinite M200, Switzerland) and showed the cell densities of the NIES-843 cyanobacterial samples.

2.2 Isolation and screening of algicidal bacteria

Algicidal bacteria were isolated from activated sludge samples collected in a sewage treatment plant in Japan in February 2011. The water samples were serially diluted (ten-fold dilutions) and 0.1 mL aliquots of each dilution were spread onto nutrient broth (NB) agar plates (which contained 1% peptone, 0.5% beef extract, 0.2% NaCl, and 1.5% agar and had a pH value of 7.0), followed by incubation for three days at 23℃ . Individual bacterial colonies with distinct morphology were chosen and inoculated onto fresh NB agar plates forpurification and screening. To screen out algicidal bacteria, the bacterial isolates were transferred separately into each well of a 96-well microplate, in which each well contained 0.1 mL of M. aeruginosa cyanobacterial culture, followed by cocultivation at 23℃ for ten days. The cocultures in the 96-well microplate were observed day by day until the color of cultures in some wells changed from green to transparent, and then the bacterial isolates in these wells were selected as algicidal bacterial candidates. All the candidates repeated the cocultivation procedure three times to confirm their inhibitory effects on cyanobacterial growth.

2.3 Characterization and identification of bacterial strains

Morphological characters of the isolated algicidal bacterial strains were observed both on NB agar plates and under a light microscope after Gram staining. After being cultivated in the NB liquid medium, bacterial cells were collected by centrifugation (at a velocity of 6 000g for 10 min at 20 )℃ from bacterial cultures, and then subjected to sequential DNA extraction by the DNA extraction kit ISOIL (Nippon Gene, Japan). The 16S ribosomal RNA genes (16S rDNA) were amplified by polymerase chain reaction (PCR) using a forward primer PrSSUF.1 (5’-agagtttgatcctggctcag-3’) and a reverse primer EbITS23S (5’-gggttbccccattcgganatc-3’) in a 50 μL reaction volume, which contained 10× Ex Taq Buffer (Takara Bio Inc., Japan), dNTPs (100 μmol each), forward and reverse primers (0.5 μmol each), 0.025 units/μL Ex Taq DNA polymerase (Hot-Start Version, Takara Bio Inc., Japan), and 1 μL of bacterial DNA. The PCR thermal cycling procedure consisted of an initial denaturation at 94?C for 1 min, followed by 25 cycles of denaturation at 94?C for 30 s, a primer annealing at 56?C or 59?C for 30 s, an extension at 72?C for 1.5 min, and a final elongation at 72?C for 7 min. The PCR products were checked using 1% agarose gel electrophoresis, and then recovered with the MinElute Gel Extraction Kit (Qiagen, Germany). Nucleotide sequences of PCR products were determined with the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., U.S.A.), and the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems Inc., U.S.A.). Inner primers, PrSSUF.3 (5’-tgccagcagccgcggta-3’), PrSSUF.5 (5’-ttaagtcccgcaacgagcg-3’), and PrSSUR.2 (5’-cgctcgttgcgggacttaa-3’), were used for the sequencing reaction. The 16S rDNA sequences of the bacterial strains were aligned with their relatives belonging to the same genera, and phylogenetic trees were constructed using the maximum likelihood method (Felsenstein 1981) with a program megablast (Zhang et al. 2000). The sequences of the bacterial 16S rDNA fragments were submitted to the DNA Data Bank of Japan (DDBJ), and the accession numbers of all bacterial stains were obtained.

2.4 Algicidal effect of isolated bacteria against M. aeruginosa

Algicidal isolates were inoculated into 20 mL of NB medium and incubated with rotary shaking (with a frequency of 100 revolutions per minute (rpm)) at 23℃ for 48 h, and then the bacterial cultures were concentrated by centrifugation (6 000g for 10 min at 25℃), followed by two washes with an MA medium and suspension again in the MA medium to obtainbacterial cell suspensions. The absorbance at 600 nm (Abs600nm) of bacterial suspension was adjusted at 3.0 after diluting the culture with the MA medium. The bacterial suspensions, whose volumes were 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL, were added in a number of separate batches into the wells of a 24-well microplate, in which 0.5 mL of M. aeruginosa culture had been dispensed in each well. Of the six samples, the 0 mL bacterial suspension was regarded as a control sample and others as treatment samples. After adjustment of the volume of each well up to 1.5 mL with the MA medium, the cyanobacterial cells were cocultivated with the algicidal isolates at 25?C under a 12L:12D light-dark cycle with a light intensity of 40 μmol/(m2·s). During a ten-day cocultivation period, 100 μL of every coculture were transferred into a 96-well microplate every few days and the cyanobacterial cell densities of the cocultures were monitored with the microplate reader. The growth of algae was estimated by Abs680nmand the algal cell densities were monitored every two days in the first four days, and then every three days in the next six days. The algicidal effect (E) of the bacterial isolates was calculated by Eq. (1):

wheretT andtC are the values of Abs680nmin the presence or absence of the algicidal bacteria after the incubation time (t) of the treatment sample and control sample, respectively, and0T and0C are the initial values oftT andtC, respectively. In order to observe the algicidal process with SEM (Hitachi High-Tech TM-1000, Japan), after the cocultures were sampled during the cocultivation period, the SEM samples were pretreated as described in the following procedures: the cyanobacterial cells and algicidal bacteria in the 100 μL coculture were trapped onto 0.2 μm membranes (Whatman, U.K.) by filtering, immediately followed by the air-drying of the membranes and sputter-coating of them with gold.

3 Results and discussion

3.1 Isolation and screening of algicidal bacteria

In total, 96 bacterial strains were isolated from the samples and nine of the 96 strains exhibited cyanobacterial growth inhibition on M. aeruginosa NIES-843 cells. Among these nine bacterial isolates, the MaI11-5 strain showed the most distinctive algicidal effect against the cyanobacterium NIES-843, so it was chosen and subjected to further experiments.

3.2 Characterization and identification of algicidal bacterial strain

The MaI11-5 bacterial strain appeared gray-white and showed spreading and growing colonies on an NB agar plate. It was characterized as a Gram-negative bacterium, and its morphology was observed with SEM and showed a rod shape with cell sizes of 1.5 to 2.5 μm in length and 0.5 μm in width. After the PCR amplification of 16S rDNA, the electrophoresis profile of the PCR product of the MaI11-5 bacterial strain was displayed, and the fragment size was between 1375 and 1584 bp. Through comparison with available sequences in the GenBank sequence database, the 16S rDNA sequences of all algicidal strains were aligned (Table 1). Themegablast analysis revealed that the MaI11-5 strain was affiliated with the genus Pedobacter, and the most homologous species was Pedobacter steynii (P. steynii, accession number: AM491372), whose sequence identity was 99% (Table 1). MaI11-5 clustered with P. steynii in the phylogenetic tree (Fig. 1), supporting their close relationship. The sequences of other algicidal bacterial strains were identified as the same procedure as MaI11-5 and the analysis results are listed in Table 1. All of the algicidal strains were registered in the DDBJ database under the accession numbers of AB666450 (MaI11-1), AB666451 (MaI11-2), AB666452 (MaI11-3), AB666453 (MaI11-4), AB666454 (MaI11-5), AB666455 (MaI11-7), AB666456 (MaI11-8), AB666457 (MaI11-9), and AB666458 (MaI11-10).

Table 1Sequences producing significant alignments of algicidal strains

Fig. 1Phylogenetic tree of 16S rDNA of MaI11-5 strain

3.3 Algicidal effect of MaI11-5 strain against M. aeruginosa

During the cocultivation of the MaI11-5 algicidal bacterial strain and cyanobacterial cells, the Abs680nmvalues of the cocultures were regularly monitored and algicidal effects were calculated using Eq. (1). As shown in Fig. 2, all of the additions of algicidal bacterial suspensions from 0.1 to 0.5 mL induced a gradual increase of algicidal effects in thecocultivation test, showing that MaI11-5 exhibited remarkable algicidal activity against the cyanobacterial cells. Furthermore, the algicidal effect of the additions from 0.2 to 0.5 mL exceeded 50% after two days, exceeded 70% after four days, and reached 80% after seven days. Although the addition of 0.1 mL was not so sufficient to suppress the cyanobacterial growth as completely as other additions of bacterial suspension, its algicidal effect exceeded 40% after two days, reached 65% after four days, and exceeded 70% after seven days. The results indicated that a very small additional amount of MaI11-5 cells can successfully inhibit the growth of M. aeruginosa cells in the natural freshwater environment.

Fig. 2Algicidal effect of different additional amounts of MaI11-5 bacterial strain on growth of M. aeruginosa

During the cocultivation of the MaI11-5 bacterial strain with M. aeruginosa NIES-843, samples were transferred from cocultures and subjected to SEM observation every two or three days. As shown in the SEM image, after three days of cocultivation, the cyanobacterial cells (a in Fig. 3(a)) aggregated and their surfaces were rough and irregularly shaped. The SEM image in Fig. 3(b), which is the magnification profile of b in Fig. 3(a), showed that the MaI11-5 isolates did not directly attach to the surface of cyanobacterial cells, and there were large quantities of mucous excretions around the algal cells. While in the SEM image of M. aeruginosa cells in axenic culture (Fig. 4), there were no mucous-like substances and the appearance of the cyanobacterial cells displayed a round shape and smooth surface and were dispersed individually. Based on these SEM observations, it can be speculated that the defense mode of mucous-like excretion release behavior of the cyanobacterial cells may be stimulated bythe algicidal bacterial attack and contribute to their aggregation. Such defense mode of cyanobacteria can also be stimulated with some other factors, such as desiccation (Potts 1999), protistan grazing (Pajdak-Stos et al. 2001), and UV irradiation (Pattanaik et al. 2007). Besides, it was reported that the Sphingobacterial isolates were not dominant among all the classes in natural water samples, but the genus Pedobacter was the majority of the class Sphingobacteria and some results also showed that the Pedobacter bacterial strain can inhibit the cyanobacterial growth to a certain extent (Berg et al. 2009).

Fig. 3SEM images of MaI11-5 algicidal bacterial strain with M. aeruginosa in coculture

Fig. 4SEM image of M. aeruginosa cells in axenic culture (×5000)

4 Conclusions

Nine algicidal bacterial strains were isolated from the activated sludge samples collected from a sewage treatment plant in Japan. One of the nine bacterial strains, the MaI11-5 bacterial strain, was identified by the biological morphology analysis, sequence analysis of 16S rDNA, and phylogenetic analysis. The results indicated that MaI11-5 belonged to Pedobacter sp. The cocultivation test results of the MaI11-5 isolates with M. aeruginosa displayed obvious algicidal activity against cyanobacterial cells. SEM observations were also carried out and revealed that the MaI11-5 algicidal bacteria attacked cyanobacterial cells indirectly and some extracellular compounds with a mucous-like appearance were released by cyanobacterial cells out of self-defense. Previous reports on the algicidal mechanism of Pedobacter sp. have rarely been found, so further studies on the MaI11-5 bacterial strain will be carried out to discover more novel functions for controlling harmful blooms.

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This work was supported by the Basic Research Program of Jiangsu Province (Grant No. BK2012828), the grant of Greater Nagoya Project in Environmental Science, and the Open Laboratory Project of Nanjing University of Technology (Grant No. 2012-2013-138).

*Corresponding author (e-mail: njut.yl@njut.edu.cn)

Received Oct.13, 2011; accepted Jul. 8, 2012