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 a b s t r a c t

Article history: Experiments were conducted to find out the molecular features, infection process of a special alga pla-
Received 8 June 2014 que-forming microorganism and its potential influence on the biomass of Chlorella vulgaris during the
Received in revised form 3 July 2014 infection process. Direct contact between the algal cell and the bacterium may be the primary steps
Accepted 5 July 2014
needed for the bacterium to lyse the alga. Addition of C. vulgaris cells into f/2 medium allowed us obtain
Available online 15 July 2014
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
Keywords:
the bacterium could influence both the dry weight and lipid content of C. vulgaris. This study demon-
Chlorella vulgaris
Biomass
strated that direct cell wall disruption of C. vulgaris by the bacterium would be a potentially effective
Labrenzia method to utilize the biomass of microalgae.
Direct contact Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Up to now, many articles have already discussed the possibility

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 pro-
duction 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 appli-
cation (Cheng et al., 2013).
⇑ Corresponding author. Address: School of Life Sciences, Xiamen University,
Xiamen 361002, China. Tel.: +86 (592) 2183217/2184528.
E-mail address: wshwzh@xmu.edu.cn (T. Zheng).
1
These authors contributed equally to this work.
of utilizing various microorganisms (viruses and bacterial) to dis-
rupt 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-degrad-
ing 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 com-
pound 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, partic-
ularly 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.

http://dx.doi.org/10.1016/j.biortech.2014.07.021
0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
 Z. Chen et al. / Bioresource Technology 169 (2014) 784–788
In this study, a plaque-forming microorganism on Chlorella vul-
garis plates was isolated. Unlike other plaque-forming microorgan-
isms 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 deter-
mine 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 transmis-
sion 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 NaH2PO4
H2O, 4.36 mg Na2EDTA
2H2O,
3.15 mg FeCl3
6H2O, 0.01 mg CoCl2
6H2O, 0.18 mg MnCl2
4H2O,
0.006 mg NaMoO4
2H2O, 0.1 mg thiamine
HCl, 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 immedi-
ately 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 bot-
tom. The responsible pathogen in the lysate was tested and puri-
fied 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 pla-
ques 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 occur-
rence of plaque, the morphology and diameter of the targeted pla-
ques were determined at various time intervals.
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
785
subsample was applied to a film-coated copper grid and phospho-
tungstic acid, or ammonium molybdate (Sigma) solutions were
then dropped onto the grid. The stained samples were examined
in a JEM2100HC (Japan) transmission electron microscope acceler-
ated at 100 kV.
2.5. Cultivation of the plaque-forming bacterium
In order to obtain a pure colony of the plaque-forming bacte-
rium, 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 isola-
tion, 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 for-
mation 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 concen-
trated 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 exponen-
tial 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
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. Reg-
ularly, after 15 days, the plaque sizes increased to an average of
1.3 cm.
786 Z. Chen et al. / Bioresource Technology 169 (2014) 784–788
A single plaque agar block was excavated and prepared for the
new formation of plaque. The morphology and diameter of one tar-
geted 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 thereaf-
ter until 2.0 cm. The average size varied from 1.0 to 1.5 cm in diam-
eter. 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 diam-
eter. 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 bacte-
ria in the plaque were large. In the middle of the plaque, they gath-
ered 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, sin-
gle bacterial could be witnessed in contact with the algal cell
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.
(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 bacte-
ria 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 plaque-
formation 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
 Z. Chen et al. / Bioresource Technology 169 (2014) 784–788
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 bacteriolog-
ical culture media (Coder and Starr, 1978), the presence of nutri-
ents from the Chlorella cells promoted bacterial growth. In spite
of this, the research between Rhodobacteraceae sp. and biofuel mic-
roalgae are few, let alone the direct algicidal patterns by Rhodob-
acteraceae 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. Phy-
logenetic analysis comparison of strain KD531 with other Rhodob-
acteraceae 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 physio-
logical 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). How-
ever, there are no literatures about plaque formation on the algae
plate by Rhodobacteraceae. Further, the relationship between Rho-
dobacteraceae 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, Chlo-
rella 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
787
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 spe-
cial 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 Sci-
ence 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.
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