Tgp chi Cong nghi Sinh hgc 12(2): 387-392, 2014
E F F E C T S O F C U L T U R E C O N D I T I O N S O N T H E G R O W T H O F H E T E R O T R O P H I C B A C T E R I A S T R A I N S S l A N D S 2
Pham Thi Bich Dao', Le Thi Hue', Tran Dang Khoa^ Nguyen Thi Anh Dao', Nguyen Thi Kim Q u y \ Nguyen Thi Hoai Ha^
'institute of Microbiology and Biotechnology, Vietnam National University. Hanoi
^University of Engineering and Technology, Vietnam National University, Hanoi Received: 27 April 2014
Accepted: 19 June 2014 SUMMARY
Hoan Kiem lake, Hanoi is the habitat of many rare species of microalgae with endenucily and especially diversity of micraalgae. Microcystis aeruginosa m Hoan Kiem lake may form hamifiil blooms and secrete the microcystin toxm that can damage animals in the same aquanum Microcystin is cyclic heptapedtides that affect liver cells and lead to hypeiphosphorylation of proteins and breakdown of cytoskeletal filaments, liver haemorrtiage and death. Biode^adaiion is the one of the safe and nature treatments for removing microcystins fixim water Tins process involves indigenous bacteria and complex communities of microorganisms in lake water, Biodegradation can reduce both intracellular toxins within cyanobactenal cells and extracellular toxin released into waterbodies. Therefore, the inveshgahon for a biodegradation solution for this toxin by domestic bacteria is of necessity. In this study, effects of culture condihons on the ^ w t h of heterotrophic bacteria which biodegrade microcystin were examined. Two strains of heterotrophic bacteria were isolated fiom the w^er bodies of Hoan Kiem lake, called Sl and S2 and subjected to apolyphasic taxonomic study.
Both strains Sl, S2 were Gram-negative bacteria, mohle and rod-shaped Sphingomonas-\A.e bacterial strains, the surface was relatively smooth and the size ranges fi'om 0.31 to 0 5nm >= 0.92 to I 2jim viewed with a scanning electron microscope (SEM ^10 000). Phylogenetic analysis based on 16S rDNA sequence showed that strains Sl,S2 belonged to the genus Sphingomonas. Levels of 16S rDNA sequence similanty between stram Sl, S2 and the type strains of Sphingomonas qjecies were in the range of 97-99%. Optimal growth conditions of both strains were pH 7-8 and temperature 28-30°C
Key words. !6S rDNA, heterotrophic bacteria, Hoan Kiem lake. Sphingomonas
INTRODUCTION water management has been performed, water eutrophication and cyanobactenal blooms were not The genus Sphingomonas was proposed by significantly controlled.
Yabuuchi el al., (1990) with five sphingosine-
containing species and two genomospecies, and The utilization of natural microorganisms to subsequently the number of Sphingomonas species has degrade microcystin (MC) was considered as interested been increased considerably by reclassifications of bioremediation pathways for water contaminated with some species belonging to other genera and microcystm because of its cost-effective, efficiently descriptions of novel species (Busse et a!., 2005; Lee et microcystins removal, and nature protection (Edwards a/.,2001;Takeuchie(o/., 2001; Yabuuchi era/., 1999). el al., 2009). The aim of this study was to identify Meanwhile, it was proposed that Sphingomonas species Sphingomonas strains isolated from cyanobacterial should be assigned to three new genera, Sphingobium, blooms water in Hoan Kiem lake and culture conditions Novosphingobium and Sphmgopyxis, in addition to the for their growth to treat microcystin-contammated genus Sphingomonas sensu stricto. The genus water in Hoan Kiem lake.
Sphingomonas comprises of at least 36 species with validly published names (Takeuchi e(a/., 2001). Due to the increasingly sharp water eutrophication in recent years, cyanobactenal blooms occur frequently in Hoan Kiem lake, Hanoi. In 2011, sharp water eutrophication
even reached 4/5 of the total area of the lake. Although Heterotrophic bacteria were isolated from the water MATERIALS AND METHODS
Strain isolation and characterization
Pham Thi Bich Dao el al.
sample collected in Hoan Kiem lake by an enrichment culture approach using NA medium containing lOpg/l microcystin-LR. The cultures were incubated at pH 7 and 28''C for 16-24h in the liquid medium. The obtained colonies were streaked onto a NA agar plate.
Heterotrophic bacteria were selected as pigment producing bacterial sirams that formed a clear zone around the colomes on agar plate. Then, the colomes were cuIUired m LB medium containing lOpg/I microcystin-LR.
Phylogenetic 16S rDNA analysis
Total DNA of the strains were extracted by Saito &
Miura method with modification The extracted DNAs were used to be template tor amplification of 16S rDNA by primers 27F (5'- AGAGTTTGATCCCTGGCTCAG-3') and 1525R (5'- AAAGGAGGTGATCCAGCC-3') The amplification products were directly sequenced on an ABI PRISM model 310 Genetic Analyzer (Applied Biosystems, Foster, CA, USA). Multiple sequence alignments were performed with CLUSTAL X (version 1 83). Gaps and ambiguous bases were eliminated from calculation A phylogenetic tree was constructed by the neighbor- joimng method with MEGA 5 (Tamura el al, 2007) The confidence values of individual branches in the phylogenetic tree were determined by using the bootstrap analysis based on 1000 replications.
Effect of period cultivation, pH and temperature on the growth of strains
Two strains of Sphingomonas were cultivated in LB medium at 28°C on a rotary shaker (200rpm) for 72h and the growth of the strains were controlled by ODeoonm after each 6h. To select the optimal temperature and pH for the strains growth, the heterotrophic bacteria were incubated with different temperatures at 28, 30, 37, 40, 45, and/or 50°C and pH ranging from 6 to 10.
RESULTS AND DISCUSSION Isolation and characterization of Sl and S2
Two bacterial strains were cultured in LB medium (microcystin-LR was added at concentrations of lOpg/I as the major carbon and nitrogen sources) incubated at 28°C. After 3-5 days of incubation, colonies of S l , S2 had round shape, white, smooth, convex, glossy, about 1 -2mm in size.
Both of them were Gram-negative bacteria, rod- shaped cells, relatively smooth and the size range from 0.31 to 0.5pm x 0.92 to 1.2^lm (Fig. 1 A, 1B).
The existence of two indigenous bacteria strains SI, S2 using microcystin for their growth showed great potential in successflilly applying biodegradation to treat cyanobacterial toxin microcystin.
9^rf ^ •
A
Figure 1 . A: SEM image of the strain S I («10 000), B. SEM image of the strain S2 ("10 000).
Phylogenetic analysis of 16S rDNA
Extraction and punfication of DNA from bacteria were important steps because the quality of the DNA will affect the efficiency of the subsequent
experiments. The extracted DNA of bacterial strains 51 and S2 were used as templates for amplification of 16S rDNA by PCR using specific primers 27F and 1525R. PCR products of the heterotrophic strains S I , 52 were read by both terminals. The comparison of 388
Tgp chi Cong nghe Smh hgc 12(2): 387-392, 2014 16S rDNA sequences of heterotrophic bacteria S l , S2 with those of prokaryotic microorganisms published in GenBank showed that Sl and S2 strains closely related to the strains of the genus Sphingomonas.
The gene sequences of Sl (1263 bp in length) was similar 99% with species Sphingomonas sp.
AB681542, 9 8 % with species Sphingomonas sp., AF361178. The gene sequences of S2 (1217bp in length) was similar 9 8 % with that of species Sphingomonas sp,, EU814953, 97% with species Sphingomonas sp., JN857975. According to some studies date reported, strains Sphingomonas sp., AB681542, Sphingomonas sp., AF361178, Sphingomonas sp., EU814953, Sphingomonas sp., JN857975 were able to biodegrade polycyclic aromatic hydrocarbons. Evolutionary distances were
calculated to establish the database consisting Sphingomonas sequences and some other related taxa. Phylogenetic tree was drawn by neighbor- Joining method Knor measure = 0.005 nucleotide sequences. Bootstrap values (expressed as percentages of 1000 replicates) of 50% were shown at branch points. Phylogenetic tree (Fig 2) showed close relationship between heterotrophic bacteria S l and S2 with the order of nucleotides of strains in the genus Sphingomonas in the gene databank.
Thus, based on phenotypic, phylogenetic and genetic data, heterotrophic bactena S l , S2 isolated from Hoan Kiem lake were classified into Sphingomonas genus and named Sphingomonas sp., Sl and Sphingomonas sp., S2. Classification of Sphingomonas sp., Sl and Sphingomonas sp., S2 as follows:
Phylum- Proteobacteria Class: Alphaproteobacteria
Order; Sphingomonadales Family: Sphingomonadaceae
Genus: Sphingomonas
Figure 2, Neighbor-joining phylogenetic tree based on the 16S rDNA sequence indicated the position of strain S I , S2 in relation to other related species
Pham Thi Bich Dao et ai.
Suitable period for the isolated Sphingomonas growth
Growth curves (Fig. 3A, 3B) showed the best biomass taken in the logarithmic phase, corresponding to a period of 9 to 30 or 36h after starting incubation. The ODeoonm reached 2.036 and 1.993 respectively for Sphingomonas sp., Sl, S2.
The time period of 24h were selected for cultivation of both heterotrophic bacteria Sl and S2.
Optimal temperature for the strains' growth To study the effects of temperature on the growth of the heterotrophic bacteria Sphingomonas sp., SI, S2 were incubated at 28, 30, 37, 40, 45 and 50°C. In Sphingomonas sp., Sl, enzyme activity and biomass increased steadily at temperatures of 28 to
37°C and highest at 30°C, the ODsoonm reached 2.001. When temperatures rised, biomass and enzyme activity has decreased sharply, at 50°C ODjoonm was only 0.735 and enzyme activity reduced to 49.481% compared to that at 30°C (Fig. 4A).
Sphingomonas sp., S2 biomass reached the highest at 30°C, but the enzyme activity was found the highest at 28°C and continuously decreased as the temperature increased (Fig. 4B). Higher temperatures might affect the stability of the enzyme so the enzyme activity also reduced. Thus, the ability of enzyme biosynthesis of two species Sphingomonas sp., Sl and S2 was almost parallel to the growth of cells. The temperature for growth and ability of enzyme activity of two strains was 28- 30°C, which was similar with environmental water temperature.
6 \1 M 14 10 36 A2 *S 5* 60 66 ll Time (hours)
6 12 18 J* 30 36 41 4fl 54 60 66 12 Time (hours)
Figures. A. Effect of time on growth of Sphingomonas sp , S I , B' Effect oftime on growth of Sp/j/ngomonassp.. S2.
Figure 4. A: Effect of temperature on growth of Sphingomonas s p , S I ; B Effect of temperature on growth of Sphingomonas s p , S2.
Tgp chi Cdng nghe Sinh hgc 12(2): 387-392, 2014
Effect of p H
A dynamic change in the pH of aquatic environments from neutral to alkaline is associated with the occurrence of the blooms of cyanobactena in water bodies. However, all currently known MC-degrading bacteria have been isolated under neutral pH conditions. Hence, the mechanism which accounts for the rapid degradation of MC in aquatic environments remains unclear. After 36h of incubation at pH ranges from 6-8, both Sphingomonas sp., S l and S2 grew quite well and enzyme activity was also very high (Fig. 5A, 5B). It indicated the proper pH range for grovrth of both species was relativity large. In this range, at pH =7 the enzyme acrivity and biomass of Sphingomonas
sp., Sl were at the highest. Also at this pH, Sphingomonas sp., S2 presented the highest enzyme activity, but it achieved optimal biomass at p H 8 .
The grov/th of the Sphingomonas sp., S l , S2 was inhibited significantly under alkaline conditions with an initial pH=10. However, the optimum pH for the growth of the Sphingomonas sp., S l , S2 species was determined to be from 7 to 8, therefore, the Sphingomonas sp., S l , S2 species should be described as alkali tolerant. The pH of most water bodies changed dynamically during the occurrence of a water bloom and becomes highly alkaline. Hence, it was important to examine the effect of pH on the degradation activity of MC-degrading bacteria.
CZIEnzyme activity -fc-Ol cziEnzyme activity -•-OD600
Figure 5. A. Effect of pH on growth of Sp/imgomo/jassp , S I ; B Effect of pH on growth of Sp/i/'ngomonas sp , S2.
CONCLUSION REFERENCES
Two strains of heterotrophic bacteria S l , S2 were isolated from blooms in Hoan Kiem lake. They have round, white, smooth, convex, and glossy colonies of about l-2mm in size. Both strains S l , S2 was Gram-negative bacteria, rod-shaped cells, the surface was relatively smooth and the size range from 0.31 to 0.5^m >; 0.92 to 1.2pm. Based on the morphological characteristics and the 16S rDNA sequences comparison, these strains belonged to genus Sphingomonas. Culture duration for S I , S2 was about 30-36h. The optimum temperature for the growth was 28-30''C at pH 7-8.
Acknowledgements: We would like to thank officially the Hanoi Department of Science and Technology. Vietnam (grant OlC-09/01-2012-2) for the financial support to this study
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(Yurkov et al. 1997) comb nov, and emendahon of the Microbiol Immunol 34:99-\}9.
ANH HUOfNG CUA DIEU KIEN NUOI CAY DEN SINH TRlTCTNG CUA HAI CHIJNG VI KHUAN Dl DUOfNG Sl VA S2
Pham Thi Bich D i o ' , Le Thj Hue', Tr3n Dang Khoa^ Nguyin Thi Anh Dao', Nguyin Thj Kim Quy', Nguyen Thi Hoai Ha'"'
Vien Vi sinh vgt vd Cdng nghe sinh hgc, Dgi hgc Quoc gia Hd Ngi Truang Dgi hgc Cong nghe, Dgi hoc Quoc gia Hd Ngi
TOM TAT
Ho Hoan Kiem la moi tnictng s6ng cua cac lo^i vi tao quy hilm voi tinh d^c hihi va da dang vh thanh phin loai Microcystis aeruginosa tron^ ho Hoan Kiem c6 the hinh thanh cac dang no hoa dpc hgi gay anh huong den cac sinh vat thuy sinh trong ho. Microcystm 1^ d$c t6 vong heptapedtide gay anh huong len cac te hao gan va CO thS dan den sy phosphoryl hoa cac protein, pha v& cac soi cytoskeletal, xuat huyet gan va til vong. Phan hiiy sinh hpc la mpt trong nh&ng phuong phap xir ly an toan va tu nhien trong viec loai bo microcystin tir nuoc.
Qua trinh nay lien quan toi su co mat ciia vi khudn v^ cac lo^i vi sinh vat trong thiiy vyc. Sy phan hiiy sinh hoc lam giam cac dOc to ngi bao va ngoai bao giai phong vao moi truang nuoc. Vi the, viec tim kilm m$t phuang phap phan hiiy sinh hoc doc to microcystm boi cac vi khuan ban dja la rat can thiet. Trong nghien ciiu nay, cac anh huong ciia dieu kien nuoi cSy cua cac vi khuan dl dudng co kha nang phan hiiy microcystin da duoc xem xet. Hai chung vi khuan di duong dugc phan l|[p t6 miu nuoc ho Hoan Kiem, goi la Sl va S2 Ca hai chung diu la VI khudn Gram am, te bao hmh que, be mat tuong dfii nhin va co kich thuoc trong khoang 0.31 din 0.5pm X 0.92 den 1.2|im quan sat du6i kinh hien vi dien hi quet (SEM xJOOOO). Phan tich giai trinh tu 16S rDNA cho thay ca hai chiing Sl va S2 thupc chi Sphingomonas. Miic do tuong ddng ciia tririi ty 16S rDNA giua Sl, S2 vk Sphingomonas la 97-99%. Dieu ki§n nuoi ciy thich hgp diryc xac dinh v6i pH nim trong khoang tCr 7-8 va nhi^t dp tu 28-30°C
/•«• khoa: 16S rDNA, ho Hodn Kiem, vi khudn di du&ng, Sphingomonas
'Authorfor correspondence: Tel: +84-966688631; E-mail: nguyenhoaiha&.yahoo.com
