Bioresource Technology 169 (2014) 784–788
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Influence of plaque-forming bacterium, Rhodobacteraceae sp. on the growth of Chlorella vulgaris Zhangran Chen a,b,1, Jingyan Zhang a,b,1, Xueqian Lei a, Bangzhou Zhang a, Guanjing Cai a, Huajun Zhang a, Yi Li a, Wei Zheng a,b, Yun Tian a, Hong Xu a, Tianling Zheng a,b,⇑ a State Key Laboratory for Marine Environmental Sciences and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystem, School of Life Sciences, Xiamen University, Xiamen 361005, China b ShenZhen Research Institute of Xiamen University, ShenZhen 518057, China
h i g h l i g h t s Bacterial infection is a feasible approach to disrupt microalgal cell walls. Biomass and lipid content can be affected during the process. Special plaques on C. vulgaris unlike viruses can be caused by the bacterium. KD531 in oligotrophic conditions showed algicidal activity while not 2216E.
a r t i c l e
i n f o
Article history: Received 8 June 2014 Received in revised form 3 July 2014 Accepted 5 July 2014 Available online 15 July 2014 Keywords: Chlorella vulgaris Biomass Labrenzia Direct contact
a b s t r a c t Experiments were conducted to find out the molecular features, infection process of a special alga plaque-forming microorganism and its potential influence on the biomass of Chlorella vulgaris during the infection process. Direct contact between the algal cell and the bacterium may be the primary steps needed for the bacterium to lyse the alga. Addition of C. vulgaris cells into f/2 medium allowed us obtain the object bacterium. The 16S rRNA gene sequence comparisons results showed that the plaque-forming bacterium kept the closest relationship with Labrenzia aggregata IAM 12614T at 98.90%. The existence of the bacterium could influence both the dry weight and lipid content of C. vulgaris. This study demonstrated that direct cell wall disruption of C. vulgaris by the bacterium would be a potentially effective method to utilize the biomass of microalgae. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Driven by the increasing price of petroleum-based fuels and growing concern about global climate change, biofuels have aroused public attention. Microalgae, such as Chlorella, have been proposed as a potential feedstock for biofuel and biomaterial production because they are able to accumulate considerable amounts of starch and lipid as the products of photosynthesis (Brennan and Owende, 2010). However, the cost of waste treatment and energy consumption associated with the chemical or physical methods to disrupt algal cells, which is a very significant step (Halim et al., 2012) for biofuel production stands in the way of large-scale application (Cheng et al., 2013). ⇑ Corresponding author. Address: School of Life Sciences, Xiamen University, Xiamen 361002, China. Tel.: +86 (592) 2183217/2184528. E-mail address: [email protected] (T. Zheng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biortech.2014.07.021 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Up to now, many articles have already discussed the possibility of utilizing various microorganisms (viruses and bacterial) to disrupt the cell walls of microalgae. For example, it may be possible to utilize viral infection as a biological treatment to disrupt cell walls and make the stored starch more accessible to starch-degrading enzymes (Cheng et al., 2013). A significant amount of literature has reported that bacterium could either lyse the algal cell directly or indirectly through releasing chemical materials. Li et al. report that Mangrovimonas yunxiaonensis LY01 release a chemical compound which causes the death of Alexandrium tamarense at the molecular, physiological and biochemical level (Li et al., 2014). However, the isolation of directly algicidal bacteria is rare, particularly of those that could have direct contact with the cell walls of the biofuel-producing microalga. Utilizing bacteria to break the cell wall of this algal species would contribute to the release of the starch and lipid for biofuel production, and hence, it would be significant to isolate any algicidal bacteria which could lyse the cell wall of microalgae.
Z. Chen et al. / Bioresource Technology 169 (2014) 784–788
In this study, a plaque-forming microorganism on Chlorella vulgaris plates was isolated. Unlike other plaque-forming microorganisms targeting Chlorella, during which the plaque diameter almost remained the same, the plaque diameter in our study gradually increased to about 2 cm. The aims of this study were (1) to determine the taxonomic identity of the microorganism involved, (2) to record the formation process of the plaque, (3) to document the contact mode between alga and microorganism through transmission electron microscopy, and (4) to roughly understand how the microorganism affect the biomass of C. vulgaris during the algicidal process. 2. Methods 2.1. Algal cultures and growth conditions An axenic clonal strain of C. vulgaris was cultured in f/2 medium (75 mg NaNO3, 5 mg NaH2PO4H2O, 4.36 mg Na2EDTA2H2O, 3.15 mg FeCl36H2O, 0.01 mg CoCl26H2O, 0.18 mg MnCl24H2O, 0.006 mg NaMoO42H2O, 0.1 mg thiamineHCl, 0.5 lg vitamin B12, 0.5 lg biotin, in 1 L seawater) (Guillard, 1975) prepared with 0.45 lm of filtered seawater) at 20 ± 1 °C under a 12/12-h light– dark cycle of approximately 50 lmol of photons m 2s 1. 2.2. Sample collection Water samples (0.5 m below the surface) in an algal bloom were collected from Xiamen sea of China. The samples were immediately filtered through a 3-lm-pore-size filter, to remove larger eukaryotic microorganisms. Then the treated samples were stored at 4 °C for further analysis, which involved 5 mL of the supernatant being inoculated into an exponentially growing C. vulgaris culture (20 mL) and incubated at 20 °C using the same lighting conditions as mentioned above. Algal cultures inoculated with supernatant inactivated at 121 °C served as controls. 2.3. Isolation of plaque-forming microorganisms and formation process of the plaque After the C. vulgaris cultures were inoculated with the samples for about 18 days, the color of the lysate changed from green to transparent and an apparent aggregation of cells formed at the bottom. The responsible pathogen in the lysate was tested and purified via the plaque assay procedure (Acosta et al., 2014). This double-agar overlay plaque assay briefly involved 15 mL of 1.2% f/2 agar medium (underlay) being dispensed onto 90 mm petri plates and 0.7% f/2 agar medium (overlay). Samples of 100 lL of lysate were mixed with 1 mL of C. vulgaris cells in the exponential phase, transferred to tubes with 4 mL warmed overlay medium, mixed and then poured onto the underlay plate. The single plaques formed were picked up and resuspended in f/2 overnight. Pure plaques of algicidal microorganisms were established by repeating the method several times. All these plates were incubated at 20 °C using the same lighting conditions for 25 days as mentioned above and plates added with f/2 served as the control. After the occurrence of plaque, the morphology and diameter of the targeted plaques were determined at various time intervals.
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subsample was applied to a film-coated copper grid and phosphotungstic acid, or ammonium molybdate (Sigma) solutions were then dropped onto the grid. The stained samples were examined in a JEM2100HC (Japan) transmission electron microscope accelerated at 100 kV. 2.5. Cultivation of the plaque-forming bacterium In order to obtain a pure colony of the plaque-forming bacterium, 50 mL exponential phase C. vulgaris culture were centrifuged at 5000g at 4 °C for 10 min and the supernatant was discarded while the algae precipitated were separately added into 100 mL f/2 (agar 1.0%), designated as CVF culture medium. For the isolation, a small-sized block was excavated and treaked onto the CVF plates. Also, another plaque block was soaked in the f/2 overnight and then the plate smearing method was used. The plates were cultured in 20 ± 1 °C for 7 day. The obtained colony were further cultured in lipid CVF and 2216E for several days for the plaque formation test by plaque assay procedure as described above. After 15 days, one bacterium, later designated as KD531, grown in CVF formed the plaque on the agar plate. As KD531 grown in CVF could cause the formation of plaque while no plaque formed for KD531 grown in 2216E, KD531 colonies in both culture medium were selected for transmission electron microscopy to see whether there were any difference. 2.6. DNA extraction, polymerase chain reaction (PCR) and phylogeny construction The genomic DNA of bacterium was extracted according to the method of Ausubel et al. (2002) and the 16S rRNA gene was amplified using PCR with the primer pair P27F and P1492R. The purified PCR product was cloned into vector pMD19-T and sequenced. Sequences of related taxa were downloaded from the GenBank database and the EzTaxon-e server (http:// eztaxon-e.ezbiocloud.net/) (Kim et al., 2012). Phylogenetic analysis was performed using MEGA version 4 (Tamura et al., 2007) after multiple alignment of data using DNAMAN (version 5.1). Evolutionary distances and clustering were performed using the neighbor-joining method (Saitou and Nei, 1987). 2.7. The algicidal effect of KD531 on the biomass of C. vulgaris In order to understand how KD531 affected the biomass of C. vulgaris during the algicidal process, the algal cells were concentrated at 6000g for 10 min and dried and measured for the dry weight determination, then the total lipid was extracted using a mixture of chloroform and methanol (Lee et al., 2010). As for the sample preparation, addition of 10% f/2 medium into the exponential growth phase algae served as the control while addition of C. vulgaris lysate caused by the plaque in which KD531 grew served as the treatment. The subsamples were collected every other day for about 14 days. 3. Results and discussion 3.1. Plaque formation and the plaque formation process
2.4. Transmission electron microscopy observation in the plaque In order to understand the infection process of the bacterium, agar blocks prepared from the boundary, middle and out of plaque zones of plates were suspended in f/2 liquid medium for 15 min. The subsamples were examined with a negative stain as previously described (Kaplun-Frischoff and Touitou, 1997). Briefly, a drop of
C. vulgaris cells and the C. vulgaris lysate which contained the plaque-forming microorganisms were mixed and poured onto the lower medium. The plates were cultured in the light condition as described above for more than 3 days when plaques formed. Regularly, after 15 days, the plaque sizes increased to an average of 1.3 cm.
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A single plaque agar block was excavated and prepared for the new formation of plaque. The morphology and diameter of one targeted plaque that first occurred in the plate was recorded during the following 25 days. A small round-shaped plaque about 0.3 cm in diameter occurred on the 3rd day and then the diameter increased to 0.5 cm in 6 days, and surprisingly, went up sharply to 1.0 cm on the 7th day. A gradual increase was observed thereafter until 2.0 cm. The average size varied from 1.0 to 1.5 cm in diameter. It was intriguing that some small white colonies appeared on the plaque ring in the 20th day and became more obvious on the 25th day, and this white colony was collected for further analysis. A pathogen which formed 2 mm sized plaques on C. vulgaris was previously reported (Chen et al., 2014). Previous study showed that, the Chlorella viruses were isolated by plaque assay and they could form plaques about 1 to 4 mm in diameter. For example, Hoshina described the basic diameter of the plaque caused by the viruses were 3 to 4 mm in their study (Imamura et al., 2010) and there are no other reports concerning plaques of 2.0 cm diameter. It is just this interesting phenomenon aroused our strong attention.
3.2. Plaques under transmission electron microscopy For the TEM observation, plaques were collected on the 18th day when the diameter reached the maximum achieved by the end of the sampling time. As seen in Fig. S1, the numbers of bacteria in the plaque were large. In the middle of the plaque, they gathered together and lysed the algae (Fig. S1B), in other fields, many of them contacted the surface of the C. vulgaris cell (Fig. S1C) and might have been on the way to lyse the algae. At the boundary, single bacterial could be witnessed in contact with the algal cell
(Fig. S1D, E, F) and the cell shell separated from the contact site seen at a larger magnification (Fig. S1G). With regard to the zone from outside the plaque, no direct contact between algal cells and bacteria was observed. There existed a distance for the bacteria to move toward the algae (Fig. S1H). Based on the results, the bacterium (1.0 2.0 lm) directly contacted with the algal cell wall, then the algal cell shell divided into half at the contact site and the cell finally was lysed. However, whether after contact with the algal cell, the bacterium released an enzyme to degrade the cell wall was not sure.
3.3. TEM of the bacterial colony After culture for about 5 days in the CVF culture medium, a small colony designated as KD531, was isolated. It is intriguing that, although the bacterium grows fastest in lipid 2216E, it still showed no algicidal activity. The TEM image of the bacterium grown in both CVF and 2216E showed that it had a size of 0.7–0.9 2.0–3.2 lm subpolar flagellum (Fig. S1 I and J). During the plaque formation tests, addition of C. vulgaris cell into the oligotrophic medium f/2 maintained the growth and plaqueformation activity of KD531 while no such phenomenon occurred for KD531 grown in 2216E. Actually, the lysing process of Chaetoceros ceratosporum by strain SS98-5 on a double layer agar plate shows a similar result to ours (Furusawa et al., 2003). In their study, they find that the microtubule-like structures observed in these bacterial cells are related to their gliding motility. Later, Yoshikawa note that SS98-5 are motile by gliding under low-nutrient conditions (Yoshikawa et al., 2008). Combining our results with this literature, the gliding motility of algicidal bacteria may play an important part in the plaque-forming process as
Fig. 1. Neighbor-joining tree showing the phylogenetic positions of strain KD531 and representatives of some other related taxa, based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. Only bootstrap values >50% are shown. Bar, 0.005 nucleotide substitution rate (Knuc) units.
Z. Chen et al. / Bioresource Technology 169 (2014) 784–788
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Fig. 2. The dry weight and total lipid content change of C. vulgaris caused by the algicidal plaque-forming bacteria KD531 for 14 days.
KD531 also had more microtubule-like structures for colony grown in CVF according to the TEM images. Unlike chlorellavorus bacterium, whose growth occurs only in the presence of live Chlorella cells and not on various bacteriological culture media (Coder and Starr, 1978), the presence of nutrients from the Chlorella cells promoted bacterial growth. In spite of this, the research between Rhodobacteraceae sp. and biofuel microalgae are few, let alone the direct algicidal patterns by Rhodobacteraceae sp. 3.4. Phylogenetic analysis of the strain 16S rRNA gene sequence comparisons revealed that strain KD531 was 98.90% similar to Labrenzia aggregata IAM 12614T. Phylogenetic analysis comparison of strain KD531 with other Rhodobacteraceae reference strains suggested that strain KD531 was near to the Labrenzia aggregata IAM 12614T (Fig. 1). Bacteria from the family Rhodobacteraceae have been reported to have the algicidal activity. The abundance and diverse physiological characteristics within this group suggest that they play important roles in marine ecosystems, such as the degradation of aromatic compounds (Buchan et al., 2005) and the biogeochemical cycles of carbon and sulfur (Wagner-Döbler and Biebl, 2006). However, there are no literatures about plaque formation on the algae plate by Rhodobacteraceae. Further, the relationship between Rhodobacteraceae sp. and C. vulgaris is rare, hence, this would be the first study about Rhodobacteraceae sp. which could form plaques on the microalgae plate. 3.5. The algicidal effect of KD531 on the biomass of C. vulgaris Fig. 2 shows the dynamic change of algal biomass including dry weight and total lipid content. When exposed to the bacterial lysate for a continuous time, the dry weight of algal cells fluctuated at about 0.16 g/L in the 12th day, and then decreased to 0.13 g/L finally while in the control, the value slightly increased. Regarding to the total lipid content, no significant change can be seen before 6 days, while the lipid content decreased gradually thereafter. A stable gradual increase occurred during the whole process in the control group into which f/2 was added (Fig. 2). In this study, Chlorella vulgaris grew for about 6 days normally until the bacterial numbers are sufficiently high to induce significant cell lysis and later the bacterial numbers reached a critical density where they outnumbered the Chlorella cells, causing a steady decrease in algal
numbers. Hence, disrupt of the algae cell wall by the bacteria would, make it convenient for us to utilize the lipid and starch in algae. 4. Conclusion The results from this study showed the feasibility of utilization of bacterial infection to degrade the cell wall of C. vulgaris. The special bacterium could formed ever-larger plaque, which was unlike virus and other bacterium. It not only cause the crack of algal cell but also influence the lipid accumulation. Utilization of beneficial materials after algal cell wall degradation by the bacterium would be a potentially effective method to the subsequent production of bioethanol and other biofuel molecules. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (41376119, 40930847), the Public Science and Technology Research Funds for Projects on the Ocean (201305016), and the Science and Technology Innovation Funds of Shenzhen (JCYJ20120615161239998) and the Program for Changjiang Scholars and Innovative Research Team in University (41121091). Prof. I. J. Hodgkiss of the University of Hong Kong is thanked for help with the English. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.07. 021. References Acosta, E.G., Piccini, L.E., Talarico, L.B., Castilla, V., Damonte, E.B., 2014. Changes in antiviral susceptibility to entry inhibitors and endocytic uptake of dengue-2 virus serially passaged in Vero or C6/36 cells. Virus Res. 184, 39–43. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., Struhl, K., 2002. Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology. John Wiley & Sons Inc., Hoboken, NJ. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sustain. Energy Rev. 14 (2), 557–577. Buchan, A., González, J.M., Moran, M.A., 2005. Overview of the marine Roseobacter lineage. Appl. Environ. Microbiol. 71 (10), 5665–5677.
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Chen, Z., Lei, X., Zhang, B., Yang, L., Zhang, H., Zhang, J., Li, Y., Zheng, W., Tian, Y., Liu, J., Zheng, T., 2014. First report of Pseudobodo sp., a new pathogen for a potential energy-producing algae: Chlorella vulgaris cultures. PLoS ONE 9 (3), e89571. Cheng, Y.-S., Zheng, Y., Labavitch, J.M., VanderGheynst, J.S., 2013. Virus infection of Chlorella variabilis and enzymatic saccharification of algal biomass for bioethanol production. Bioresour. Technol. 137, 326–331. Coder, D.M., Starr, M.P., 1978. Antagonistic association of the chlorellavorus bacterium (‘‘Bdellovibrio’’ chlorellavorus) with Chlorella vulgaris. Curr. Microbiol. 1 (1), 59–64. Furusawa, G., Yoshikawa, T., Yasuda, A., Sakata, T., 2003. Algicidal activity and gliding motility of Saprospira sp. SS98-5. Can. J. Microbiol. 49 (2), 92–100. Guillard, R.R., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Culture of Marine Invertebrate Animals. Plenum, pp. 29–60. Halim, R., Harun, R., Danquah, M.K., Webley, P.A., 2012. Microalgal cell disruption for biofuel development. Appl. Energy 91 (1), 116–121. Imamura, N., Hoshina, R., Shimizu, M., Makino, Y., Haruyama, Y., Ueda, S., Kato, Y., Kasahara, M., Ono, B., 2010. Isolation and characterization of a virus (CvVBW1) that infects symbiotic algae of Paramecium bursaria in Lake Biwa, Japan. Virol. J. 7. Kaplun-Frischoff, Y., Touitou, E., 1997. Testosterone skin permeation enhancement by menthol through formation of eutectic with drug and interaction with skin lipids. J. Pharm. Sci. 86 (12), 1394–1399.
Kim, O.-S., Cho, Y.-J., Lee, K., Yoon, S.-H., Kim, M., Na, H., Park, S.-C., Jeon, Y.S., Lee, J.H., Yi, H., 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62 (Pt 3), 716–721. Lee, J.-Y., Yoo, C., Jun, S.-Y., Ahn, C.-Y., Oh, H.-M., 2010. Comparison of several methods for effective lipid extraction from microalgae. Bioresour. Technol. 101 (1), S75–S77. Li, Y., Zhu, H., Guan, C., Zhang, H., Guo, J., Chen, Z., Cai, G., Lei, X., Zheng, W., Tian, Y., Zheng, T., 2014. Towards molecular, physiological, and biochemical understanding of photosynthetic inhibition and oxidative stress in the toxic Alexandrium tamarense induced by a marine bacterium. Appl. Microbiol. Biotechnol. 98, 4637–4652. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4 (4), 406–425. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24 (8), 1596– 1599. Wagner-Döbler, I., Biebl, H., 2006. Environmental biology of the marine Roseobacter lineage. Annu. Rev. Microbiol. 60, 255–280. Yoshikawa, T., Nakahara, M., Tabata, A., Kokumai, S., Furusawa, G., Sakata, T., 2008. Characterization and expression of Saprospira cytoplasmic fibril protein (SCFP) gene from algicidal Saprospira spp. strains. Fish. Sci. 74 (5), 1109–1117.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Influence of plaque-forming bacterium, Rhodobacteraceae sp. on the growth of Chlorella vulgaris Zhangran Chen a,b,1, Jingyan Zhang a,b,1, Xueqian Lei a, Bangzhou Zhang a, Guanjing Cai a, Huajun Zhang a, Yi Li a, Wei Zheng a,b, Yun Tian a, Hong Xu a, Tianling Zheng a,b,⇑ a State Key Laboratory for Marine Environmental Sciences and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystem, School of Life Sciences, Xiamen University, Xiamen 361005, China b ShenZhen Research Institute of Xiamen University, ShenZhen 518057, China
h i g h l i g h t s Bacterial infection is a feasible approach to disrupt microalgal cell walls. Biomass and lipid content can be affected during the process. Special plaques on C. vulgaris unlike viruses can be caused by the bacterium. KD531 in oligotrophic conditions showed algicidal activity while not 2216E.
a r t i c l e
i n f o
Article history: Received 8 June 2014 Received in revised form 3 July 2014 Accepted 5 July 2014 Available online 15 July 2014 Keywords: Chlorella vulgaris Biomass Labrenzia Direct contact
a b s t r a c t Experiments were conducted to find out the molecular features, infection process of a special alga plaque-forming microorganism and its potential influence on the biomass of Chlorella vulgaris during the infection process. Direct contact between the algal cell and the bacterium may be the primary steps needed for the bacterium to lyse the alga. Addition of C. vulgaris cells into f/2 medium allowed us obtain the object bacterium. The 16S rRNA gene sequence comparisons results showed that the plaque-forming bacterium kept the closest relationship with Labrenzia aggregata IAM 12614T at 98.90%. The existence of the bacterium could influence both the dry weight and lipid content of C. vulgaris. This study demonstrated that direct cell wall disruption of C. vulgaris by the bacterium would be a potentially effective method to utilize the biomass of microalgae. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Driven by the increasing price of petroleum-based fuels and growing concern about global climate change, biofuels have aroused public attention. Microalgae, such as Chlorella, have been proposed as a potential feedstock for biofuel and biomaterial production because they are able to accumulate considerable amounts of starch and lipid as the products of photosynthesis (Brennan and Owende, 2010). However, the cost of waste treatment and energy consumption associated with the chemical or physical methods to disrupt algal cells, which is a very significant step (Halim et al., 2012) for biofuel production stands in the way of large-scale application (Cheng et al., 2013). ⇑ Corresponding author. Address: School of Life Sciences, Xiamen University, Xiamen 361002, China. Tel.: +86 (592) 2183217/2184528. E-mail address: [email protected] (T. Zheng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biortech.2014.07.021 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Up to now, many articles have already discussed the possibility of utilizing various microorganisms (viruses and bacterial) to disrupt the cell walls of microalgae. For example, it may be possible to utilize viral infection as a biological treatment to disrupt cell walls and make the stored starch more accessible to starch-degrading enzymes (Cheng et al., 2013). A significant amount of literature has reported that bacterium could either lyse the algal cell directly or indirectly through releasing chemical materials. Li et al. report that Mangrovimonas yunxiaonensis LY01 release a chemical compound which causes the death of Alexandrium tamarense at the molecular, physiological and biochemical level (Li et al., 2014). However, the isolation of directly algicidal bacteria is rare, particularly of those that could have direct contact with the cell walls of the biofuel-producing microalga. Utilizing bacteria to break the cell wall of this algal species would contribute to the release of the starch and lipid for biofuel production, and hence, it would be significant to isolate any algicidal bacteria which could lyse the cell wall of microalgae.
Z. Chen et al. / Bioresource Technology 169 (2014) 784–788
In this study, a plaque-forming microorganism on Chlorella vulgaris plates was isolated. Unlike other plaque-forming microorganisms targeting Chlorella, during which the plaque diameter almost remained the same, the plaque diameter in our study gradually increased to about 2 cm. The aims of this study were (1) to determine the taxonomic identity of the microorganism involved, (2) to record the formation process of the plaque, (3) to document the contact mode between alga and microorganism through transmission electron microscopy, and (4) to roughly understand how the microorganism affect the biomass of C. vulgaris during the algicidal process. 2. Methods 2.1. Algal cultures and growth conditions An axenic clonal strain of C. vulgaris was cultured in f/2 medium (75 mg NaNO3, 5 mg NaH2PO4H2O, 4.36 mg Na2EDTA2H2O, 3.15 mg FeCl36H2O, 0.01 mg CoCl26H2O, 0.18 mg MnCl24H2O, 0.006 mg NaMoO42H2O, 0.1 mg thiamineHCl, 0.5 lg vitamin B12, 0.5 lg biotin, in 1 L seawater) (Guillard, 1975) prepared with 0.45 lm of filtered seawater) at 20 ± 1 °C under a 12/12-h light– dark cycle of approximately 50 lmol of photons m 2s 1. 2.2. Sample collection Water samples (0.5 m below the surface) in an algal bloom were collected from Xiamen sea of China. The samples were immediately filtered through a 3-lm-pore-size filter, to remove larger eukaryotic microorganisms. Then the treated samples were stored at 4 °C for further analysis, which involved 5 mL of the supernatant being inoculated into an exponentially growing C. vulgaris culture (20 mL) and incubated at 20 °C using the same lighting conditions as mentioned above. Algal cultures inoculated with supernatant inactivated at 121 °C served as controls. 2.3. Isolation of plaque-forming microorganisms and formation process of the plaque After the C. vulgaris cultures were inoculated with the samples for about 18 days, the color of the lysate changed from green to transparent and an apparent aggregation of cells formed at the bottom. The responsible pathogen in the lysate was tested and purified via the plaque assay procedure (Acosta et al., 2014). This double-agar overlay plaque assay briefly involved 15 mL of 1.2% f/2 agar medium (underlay) being dispensed onto 90 mm petri plates and 0.7% f/2 agar medium (overlay). Samples of 100 lL of lysate were mixed with 1 mL of C. vulgaris cells in the exponential phase, transferred to tubes with 4 mL warmed overlay medium, mixed and then poured onto the underlay plate. The single plaques formed were picked up and resuspended in f/2 overnight. Pure plaques of algicidal microorganisms were established by repeating the method several times. All these plates were incubated at 20 °C using the same lighting conditions for 25 days as mentioned above and plates added with f/2 served as the control. After the occurrence of plaque, the morphology and diameter of the targeted plaques were determined at various time intervals.
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subsample was applied to a film-coated copper grid and phosphotungstic acid, or ammonium molybdate (Sigma) solutions were then dropped onto the grid. The stained samples were examined in a JEM2100HC (Japan) transmission electron microscope accelerated at 100 kV. 2.5. Cultivation of the plaque-forming bacterium In order to obtain a pure colony of the plaque-forming bacterium, 50 mL exponential phase C. vulgaris culture were centrifuged at 5000g at 4 °C for 10 min and the supernatant was discarded while the algae precipitated were separately added into 100 mL f/2 (agar 1.0%), designated as CVF culture medium. For the isolation, a small-sized block was excavated and treaked onto the CVF plates. Also, another plaque block was soaked in the f/2 overnight and then the plate smearing method was used. The plates were cultured in 20 ± 1 °C for 7 day. The obtained colony were further cultured in lipid CVF and 2216E for several days for the plaque formation test by plaque assay procedure as described above. After 15 days, one bacterium, later designated as KD531, grown in CVF formed the plaque on the agar plate. As KD531 grown in CVF could cause the formation of plaque while no plaque formed for KD531 grown in 2216E, KD531 colonies in both culture medium were selected for transmission electron microscopy to see whether there were any difference. 2.6. DNA extraction, polymerase chain reaction (PCR) and phylogeny construction The genomic DNA of bacterium was extracted according to the method of Ausubel et al. (2002) and the 16S rRNA gene was amplified using PCR with the primer pair P27F and P1492R. The purified PCR product was cloned into vector pMD19-T and sequenced. Sequences of related taxa were downloaded from the GenBank database and the EzTaxon-e server (http:// eztaxon-e.ezbiocloud.net/) (Kim et al., 2012). Phylogenetic analysis was performed using MEGA version 4 (Tamura et al., 2007) after multiple alignment of data using DNAMAN (version 5.1). Evolutionary distances and clustering were performed using the neighbor-joining method (Saitou and Nei, 1987). 2.7. The algicidal effect of KD531 on the biomass of C. vulgaris In order to understand how KD531 affected the biomass of C. vulgaris during the algicidal process, the algal cells were concentrated at 6000g for 10 min and dried and measured for the dry weight determination, then the total lipid was extracted using a mixture of chloroform and methanol (Lee et al., 2010). As for the sample preparation, addition of 10% f/2 medium into the exponential growth phase algae served as the control while addition of C. vulgaris lysate caused by the plaque in which KD531 grew served as the treatment. The subsamples were collected every other day for about 14 days. 3. Results and discussion 3.1. Plaque formation and the plaque formation process
2.4. Transmission electron microscopy observation in the plaque In order to understand the infection process of the bacterium, agar blocks prepared from the boundary, middle and out of plaque zones of plates were suspended in f/2 liquid medium for 15 min. The subsamples were examined with a negative stain as previously described (Kaplun-Frischoff and Touitou, 1997). Briefly, a drop of
C. vulgaris cells and the C. vulgaris lysate which contained the plaque-forming microorganisms were mixed and poured onto the lower medium. The plates were cultured in the light condition as described above for more than 3 days when plaques formed. Regularly, after 15 days, the plaque sizes increased to an average of 1.3 cm.
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A single plaque agar block was excavated and prepared for the new formation of plaque. The morphology and diameter of one targeted plaque that first occurred in the plate was recorded during the following 25 days. A small round-shaped plaque about 0.3 cm in diameter occurred on the 3rd day and then the diameter increased to 0.5 cm in 6 days, and surprisingly, went up sharply to 1.0 cm on the 7th day. A gradual increase was observed thereafter until 2.0 cm. The average size varied from 1.0 to 1.5 cm in diameter. It was intriguing that some small white colonies appeared on the plaque ring in the 20th day and became more obvious on the 25th day, and this white colony was collected for further analysis. A pathogen which formed 2 mm sized plaques on C. vulgaris was previously reported (Chen et al., 2014). Previous study showed that, the Chlorella viruses were isolated by plaque assay and they could form plaques about 1 to 4 mm in diameter. For example, Hoshina described the basic diameter of the plaque caused by the viruses were 3 to 4 mm in their study (Imamura et al., 2010) and there are no other reports concerning plaques of 2.0 cm diameter. It is just this interesting phenomenon aroused our strong attention.
3.2. Plaques under transmission electron microscopy For the TEM observation, plaques were collected on the 18th day when the diameter reached the maximum achieved by the end of the sampling time. As seen in Fig. S1, the numbers of bacteria in the plaque were large. In the middle of the plaque, they gathered together and lysed the algae (Fig. S1B), in other fields, many of them contacted the surface of the C. vulgaris cell (Fig. S1C) and might have been on the way to lyse the algae. At the boundary, single bacterial could be witnessed in contact with the algal cell
(Fig. S1D, E, F) and the cell shell separated from the contact site seen at a larger magnification (Fig. S1G). With regard to the zone from outside the plaque, no direct contact between algal cells and bacteria was observed. There existed a distance for the bacteria to move toward the algae (Fig. S1H). Based on the results, the bacterium (1.0 2.0 lm) directly contacted with the algal cell wall, then the algal cell shell divided into half at the contact site and the cell finally was lysed. However, whether after contact with the algal cell, the bacterium released an enzyme to degrade the cell wall was not sure.
3.3. TEM of the bacterial colony After culture for about 5 days in the CVF culture medium, a small colony designated as KD531, was isolated. It is intriguing that, although the bacterium grows fastest in lipid 2216E, it still showed no algicidal activity. The TEM image of the bacterium grown in both CVF and 2216E showed that it had a size of 0.7–0.9 2.0–3.2 lm subpolar flagellum (Fig. S1 I and J). During the plaque formation tests, addition of C. vulgaris cell into the oligotrophic medium f/2 maintained the growth and plaqueformation activity of KD531 while no such phenomenon occurred for KD531 grown in 2216E. Actually, the lysing process of Chaetoceros ceratosporum by strain SS98-5 on a double layer agar plate shows a similar result to ours (Furusawa et al., 2003). In their study, they find that the microtubule-like structures observed in these bacterial cells are related to their gliding motility. Later, Yoshikawa note that SS98-5 are motile by gliding under low-nutrient conditions (Yoshikawa et al., 2008). Combining our results with this literature, the gliding motility of algicidal bacteria may play an important part in the plaque-forming process as
Fig. 1. Neighbor-joining tree showing the phylogenetic positions of strain KD531 and representatives of some other related taxa, based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. Only bootstrap values >50% are shown. Bar, 0.005 nucleotide substitution rate (Knuc) units.
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Fig. 2. The dry weight and total lipid content change of C. vulgaris caused by the algicidal plaque-forming bacteria KD531 for 14 days.
KD531 also had more microtubule-like structures for colony grown in CVF according to the TEM images. Unlike chlorellavorus bacterium, whose growth occurs only in the presence of live Chlorella cells and not on various bacteriological culture media (Coder and Starr, 1978), the presence of nutrients from the Chlorella cells promoted bacterial growth. In spite of this, the research between Rhodobacteraceae sp. and biofuel microalgae are few, let alone the direct algicidal patterns by Rhodobacteraceae sp. 3.4. Phylogenetic analysis of the strain 16S rRNA gene sequence comparisons revealed that strain KD531 was 98.90% similar to Labrenzia aggregata IAM 12614T. Phylogenetic analysis comparison of strain KD531 with other Rhodobacteraceae reference strains suggested that strain KD531 was near to the Labrenzia aggregata IAM 12614T (Fig. 1). Bacteria from the family Rhodobacteraceae have been reported to have the algicidal activity. The abundance and diverse physiological characteristics within this group suggest that they play important roles in marine ecosystems, such as the degradation of aromatic compounds (Buchan et al., 2005) and the biogeochemical cycles of carbon and sulfur (Wagner-Döbler and Biebl, 2006). However, there are no literatures about plaque formation on the algae plate by Rhodobacteraceae. Further, the relationship between Rhodobacteraceae sp. and C. vulgaris is rare, hence, this would be the first study about Rhodobacteraceae sp. which could form plaques on the microalgae plate. 3.5. The algicidal effect of KD531 on the biomass of C. vulgaris Fig. 2 shows the dynamic change of algal biomass including dry weight and total lipid content. When exposed to the bacterial lysate for a continuous time, the dry weight of algal cells fluctuated at about 0.16 g/L in the 12th day, and then decreased to 0.13 g/L finally while in the control, the value slightly increased. Regarding to the total lipid content, no significant change can be seen before 6 days, while the lipid content decreased gradually thereafter. A stable gradual increase occurred during the whole process in the control group into which f/2 was added (Fig. 2). In this study, Chlorella vulgaris grew for about 6 days normally until the bacterial numbers are sufficiently high to induce significant cell lysis and later the bacterial numbers reached a critical density where they outnumbered the Chlorella cells, causing a steady decrease in algal
numbers. Hence, disrupt of the algae cell wall by the bacteria would, make it convenient for us to utilize the lipid and starch in algae. 4. Conclusion The results from this study showed the feasibility of utilization of bacterial infection to degrade the cell wall of C. vulgaris. The special bacterium could formed ever-larger plaque, which was unlike virus and other bacterium. It not only cause the crack of algal cell but also influence the lipid accumulation. Utilization of beneficial materials after algal cell wall degradation by the bacterium would be a potentially effective method to the subsequent production of bioethanol and other biofuel molecules. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (41376119, 40930847), the Public Science and Technology Research Funds for Projects on the Ocean (201305016), and the Science and Technology Innovation Funds of Shenzhen (JCYJ20120615161239998) and the Program for Changjiang Scholars and Innovative Research Team in University (41121091). Prof. I. J. Hodgkiss of the University of Hong Kong is thanked for help with the English. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.07. 021. References Acosta, E.G., Piccini, L.E., Talarico, L.B., Castilla, V., Damonte, E.B., 2014. Changes in antiviral susceptibility to entry inhibitors and endocytic uptake of dengue-2 virus serially passaged in Vero or C6/36 cells. Virus Res. 184, 39–43. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., Struhl, K., 2002. Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology. John Wiley & Sons Inc., Hoboken, NJ. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sustain. Energy Rev. 14 (2), 557–577. Buchan, A., González, J.M., Moran, M.A., 2005. Overview of the marine Roseobacter lineage. Appl. Environ. Microbiol. 71 (10), 5665–5677.
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