Microcystis aeruginosa grown in different defined media leads to different cultivable heterotrophic bacteria composition that could influence cyanobacterial morphological characteristics and growth properties

Cyanobacterial blooms involving Microcystis spp. often pose severe problems to the environment and general community due to their persistent presence in eutrophic water bodies and potential to form blooms. Bacterial associations are known to alter microenvironment of Microcystis and potentially influence their development. This study aimed to study cultivable heterotrophic bacteria composition that developed symbiotically with Microcystis aeruginosa naturally as well as those cultured under defined media and their possible effects on the morphology and growth properties of the cyanobacterium. M. aeruginosa (UPMC-A0051) was isolated during a bloom from Putrajaya Lake, Malaysia and characterized as a non microcystin-producing cyanobacterium using PCR and chromatographic methods. Associated heterotrophic bacteria were then isolated and identified from the culture media as well as the lake where the cyanobacterium was originally isolated. A total of 16 bacterial species were isolated from the lake and none of them were similar to the bacteria associated with M. aeruginosa cultured in artificial media. Cultivable heterotrophic bacteria composition associated with M. aeruginosa were also distinct in different culture media, despite the same inoculum. These bacteria were classified under Actinobacteria, α.-Proteobacteria and β-Proteobacteria. Under different bacterial associations, M. aeruginosa cultivated in defined media showed different colony morphology and growth properties. The present study demonstrated that distinct bacterial composition observed in different culture media could be responsible for dissimilar cyanobacterium morphology and growth rate, particularly on the clustering pattern. In the axenic culture, the growth of M. aeruginosa was significantly reduced indicating the influence of associated bacteria on the development of cyanobacterial colonies.


140
Thermal Cycler (Biometra, Jena, Germany). Prior to sequencing, the PCR products were gel-141 purified using gel/ PCR DNA fragments extraction kit (Geneaid, New Taipei City, Taiwan). 142 Sequencing of these purified products was performed in bi-direction with both forward and 143 reverse primers by Apical Scientific Laboratories Sdn Bhd (Selangor, Malaysia) using ABI 144 3770 sequencer (Applied Biosystems, Foster City, USA). The microcystin primers, tox4f (forward primer) and tox4r (reverse primer), adopted from 149 Kurmayer et al. [14] that target the mcyB A1 domain were used in this study. The previously 150 extracted Microcystis sp. DNA was utilized as the PCR template. A total volume of 25 µL 151 PCR reaction was prepared as above. The PCR thermal cycling protocols were an initial 152 denaturation at 94°C for 10 min, followed by 35 cycles at 94°C for 30 s (denaturation), at 153 50°C for 30 s (annealing), 72°C for 1 min (extension) and final extension at 72°C for 7 min 154 with the use of T-Personal Thermal Cycler (Biometra, Jena, Germany). As for direct 155 sequencing, the amplified mcyB products were first gel-purified using gel/PCR DNA 156 fragments extraction kit (Geneaid, New Taipei, Taiwan) as per manufacturer's instructions 8 MS/MS) 164 Dense growing Microcystis cultures from two media were centrifuged at 5000×g for 15 min 165 and pellets were lyophilized and stored at -20°C prior to analysis. Extractions of microcystins 166 were performed by sonicating 0.1 g lyophilized Microcystis cells in 70% methanol (w/v) for 167 20 minutes. Three similar extractions were conducted and pooled together and then, the 168 residue was evaporated at 60°C. Methanol solution (10%) was used to redissolve the residue. 169 The extract was fractionated using a preconditioned ODS solid phase extraction (SPE) 170 cartridge (Merck, New Jersey, USA).

171
A step gradient of 30% to 100% methanol in water (v/v) was used to elute the extract.  All treatments were conducted in triplicates. The culture was gently agitated to ensure the 193 culture uniformity before each growth measurement. Axenic cultures of Microcystis sp. were 194 prepared using the procedures described previously [16][17]. Axenic condition was examined 195 using epifluorescence microscope techniques [16][17].  was centrifuged at 6000×g for 10 min. The supernatant was streaked on six different agar 207 plates i.e. Muller Hilton agar, eosin methylene blue (EMB) agar, brain-heart infusion agar, 208 210 hand, the pellet was resuspended in 1 mL TE buffer (Tris 10 mM; EDTA 1 mM, pH 8.0).

211
After that, 10 mL distilled water was added and sonicated three times for 3 min each time 212 with intervals of 3 min. The solution was inoculated onto the six different agar plates as 213 mentioned previously and incubated at 25°C for five days. Bacteria from the environment 214 (the same date and location of the Microcystis sp. isolation) were also isolated using the 215 streaking approach on same types of agars mentioned previously.   differences were accepted as significant when P < 0.05.   (Fig 2). The phycocyanin operon-based phylogeny is demonstrated to be distinct from that 306 derived 16S rRNA gene, reflecting the fact that even organisms with 99% of similarity in the 307 ribosomal gene may belong to different groups in terms of their pigmentation physiology.  . In this study, any compounds with a fragmentation pattern 331 characteristic of microcystin standards was not detected within the range (Fig 4). Therefore, 332 this implies the isolated M. aeruginosa (UPMC-A0051) in the present study did not belong to 333 a microcystin-producing strain.  Most bacteria were found to grow on Muller Hilton, brain-heart infusion and EMB media.  (Table 3A).

362
Both of them were found present in the medium as free-moving and attached bacteria.  (Table 3A). This is in line with 370 Shen et al. [4] who reported that some Microcystis species harbour heterotrophic bacteria 371 within their large mucilaginous colonies that might be responsible for the aggregation of 372 cyanobacterial cells.   Microcystis aeruginosa (UPMC-A0051) grown in BG11 medium was found to multiply and 528 formed colonies faster than that cultivated in BBM medium (Fig 6).  It is interesting to note that the space between solitary cells within the clathrate of M. 548 aeruginosa grown in BBM media was found to be more intense that those grown in BG11, 549 and this space increased with the growing period (Fig 6). Microscystis aeruginosa in BG11 550 culture displayed a firmly bound colony, indicating smaller spaces in between cells in a 551 colony, similar to the M. aeruginosa collected from the environment (Fig 1). This space was colonies cultured in BG11 medium were more compact compared to those in BBM medium.

587
As compared to their axenic counterpart, M. aeruginosa grown in association with the 588 bacteria showed significantly higher biomass production (p < 0.05). This study illustrated that