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A lytic bacterium' s potential application in biofuel production through

directly lysing the diatom Phaeodactylum tricornutum cell

Article  in  Algal Research · September 2015

DOI: 10.1016/j.algal.2015.08.023

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1Q1 A lytic bacterium's potential application in biofuel production through

2 directly lysing the diatom Phaeodactylum tricornutum cell

F
3Q2 Zhangran Chen a,b,1, Bangzhou Zhang a,1, Jingyan Zhang a, Xueqian Lei a, Huajun Zhang a, Yi Li a, Luxi Yang a,
Wei Zheng a, Yun Tian a, Lisa A. Boughner c, Hong Xu a,⁎, Tianling Zheng a,b,⁎⁎

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4

5 a
State Key Laboratory of Marine Environmental Science and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystem, School of Life Sciences, Xiamen University,
6

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Xiamen 361005, China
7 b
ShenZhen Research Institute of Xiamen University, ShenZhen 518057, China
8 c
Center for Microbial Ecology, Michigan State University, East Lansing 48824, USA
9

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1 0 a r t i c l e i n f o a b s t r a c t

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11 Article history: The isolation of microorganisms able to break the cell wall of algal species would contribute to the release of 21
12 Received 5 September 2014 starch and lipid molecules for biofuel production. In this study, a special plaque-forming bacterium, forming 22
13 Received in revised form 6 July 2015 plaques of about 2 cm in diameter, was found to target the diatom Phaeodactylum tricornutum. To monitor the
D 23
14 Accepted 30 August 2015
dynamic effects of the bacterium on P. tricornutum, samples from both normal algal cultures and lysates were 24
15 Available online xxxx
analyzed by fluorescence microscopy and denaturing gradient gel electrophoresis. The DGGE results showed 25
26
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16 Keywords:
that there was no different band between normal algal cultures and lysate samples, suggesting the bacterium's
17 Phaeodactylum tricornutum low abundance. Several different culture mediums were tried, and the obtained bacteria from the lysates were 27
18 DGGE further investigated using the plaque-forming method. Finally, one isolated bacterium grown on culture medium 28
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19 Labrenzia containing P. tricornutum cells showed the special lytic activity. The results from sequence and phylogenetic 29
20 Biofuel analysis show that this bacterium belongs to the genus of Labrenzia in the family Rhodobacteraceae. This 30
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study demonstrates the isolation of a lytic bacterium that has a potential for biofuel production through 31
P. tricornutum ‘cracking’. 32
33 © 2015 Published by Elsevier B.V.
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37
35
34
36
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38 1. Introduction it could grow in salt water, is photosynthetically efficient, has a fast 53

growth rate, and could easily be grown in large scale applications with- 54
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39 Diatoms are among the most common types of phytoplankton [55] out utilizing significant agricultural resources [1,13,33,52,53,58]. 55
40 and are believed to be responsible for around one-fifth of the primary One of the most expensive steps in the conversion of biomass may 56
41
Q3 productivity on Earth (Bowler et al., 2008). An important representative be cell disruption. Researchers became interested in using bacteria to 57
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42 from the pennate diatoms, Phaeodactylum tricornutum has gained much lyse algal cells from observing the control of harmful algal blooms 58
43 attention from scientists for a number of reasons. For example, the (HABs) with bacteria [30,31,61,62], which may be superior to chemical 59
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44 genomic sequence of P. tricornutum supports the development of this and physical methods in view of cost, convenience, and large scale suit- 60
45
Q4 species as a model system for molecular-based studies ([45]; Bowler ability. Current literature describes algicidal bacteria with direct or indi- 61
46 et al., 2008). The ability of the marine microalga P. tricornutum to be rect algicidal (release of algicidal compounds) activity towards HAB 62
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47 immobilized on calcium alginate beads make it possible for in situ diatom species [15,28,37], but few report on bacteria showing indirect 63
48 toxicity testing of estuarine systems and other environments [38,44]. algicidal activity in connection to P. tricornutum [4,14]. P. tricornutum 64
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49 Also, driven by the increasing price of petroleum-based fuels and has come into focus due to its high lipid content (20–60% dry weight) 65
50 growing concerns about climate change, the potential for biodiesel under certain growth conditions [27]. Though possible to utilize bacterial 66
51 production from P. tricornutum is of great interest [16,32,46]. The infection as a means of releasing P. tricornutum's ‘stored lipids’, there are 67
52 microalgae P. tricornutum is an advantageous biofuel candidate because surprisingly few researches directly utilizing microorganisms to disrupt 68
microalgal cell walls for downstream biofuel production [5]. 69
Currently, there is no literature describing the relationships between 70
⁎ Corresponding author. P. tricornutum and plaque-forming microorganisms. Thus, one plaque- 71
⁎⁎ Corresponding author at: State Key Laboratory of Marine Environmental Science and forming microorganism isolated from the Xiamen Sea, attracted our 72
Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystem, School of
strong interest. The aims of the present study were: (i) to confirm 73
Life Sciences, Xiamen University, Xiamen 361005, China.
E-mail addresses: wshwzh@xmu.edu.cn (H. Xu), hxu@xmu.edu.cn (T. Zheng). the biological nature of this plaque-forming microorganism, (ii) to 74
1
These authors contributed equally to this work. observe the dynamic interactions of the algae and bacterium after 75

http://dx.doi.org/10.1016/j.algal.2015.08.023
2211-9264/© 2015 Published by Elsevier B.V.

Please cite this article as: Z. Chen, et al., A lytic bacterium's potential application in biofuel production through directly lysing the diatom
Phaeodactylum tricornutum cell, Algal Res. (2015), http://dx.doi.org/10.1016/j.algal.2015.08.023
 2 Z. Chen et al. / Algal Research xxx (2015) xxx–xxx

76 co-inoculation; and (iii) to determine the taxonomic identity of 10 min. The supernatant was discarded, and the pellet was resuspended 135
77 the plaque-forming bacteria KD531. The current investigation into in 5 mL of 90% ethanol, mixed thoroughly on a vortex mixer and allowed 136
78 a lytic bacterium that targets P. tricornutum, a promising candidate to rest overnight. Afterwards, the overnight settled pellet was removed 137
79 for biofuel production, will enrich our understanding for biological using centrifugation and then the chlorophyll a concentration was 138
80 disruption of microalgae. determined [24]. 139

81 2. Materials and methods 2.4. Antibiotic tests 140

82 2.1. Algal cultures and growth conditions In order to identify how antibiotics affect the lytic activity of the 141
microbe, confirming it to be either a protist or bacterium, cycloheximide 142
83 Cultures of experimental algae, P. tricornutum MEL106, were sup- and penicillin–streptomycin were introduced into the algae-lytic 143
84 plied by the State Key Laboratory of Marine Environmental Science microbe system. Cycloheximide (Sigma, Chemical Company) which 144
85 (Xiamen University). The cultures were incubated in sterile f/2 medium inhibits eukaryotic protein synthesis, was used to control protozoan 145
86 (75 mg NaNO3, 5 mg NaH2PO4•H2O, 4.36 mg Na2EDTA•2H2O, 3.15 mg growth and it has previously been used to remove protozoa without 146

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87 FeCl3•6H2O, 0.01 mg CoCl2•6H2O, 0.18 mg MnCl2•4H2O, 0.006 mg affecting the bacterial population in aerobic systems [20]. The initial 147
88 NaMoO4•2H2O, 0.1 mg thiamine•HCl, 0.5 μg vitamin B12, 0.5 μg biotin, concentration of cycloheximide (20 mg/mL), which could inhibit the 148

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89 in 1 L seawater) [19] prepared with 0.45 μm of filtered seawater at growth of nanoflagellates and reduce or eliminate predators was 149
90 20 ± 1 °C under a 12/12-h light–dark cycle of approximately 50 μmol adjusted to a final concentration of 0.2 mg/mL in the culture system 150
91 photons m−2s−1. outlined below [40]. While the penicillin–streptomycin solution 151

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(10,000 units penicillin/mL, 10 mg streptomycin/mL, in 0.9% NaCl; 152
92 2.2. Discovery of the special plaque-forming microorganism Sigma Corp., St. Louis, Missouri), which is known to prevent the growth 153
of bacteria, was added as follows: Treatment 1: 20 mL P. tricornutum 154

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93 Water samples (from 50 to 100 L) of an algal bloom were collected from culture + 2.2 mL PtV1; Treatment 2: 20 mL P. tricornutum 155
94 the Xiamen Sea in China, prefiltered through 5 μm then 0.45 μm-sized culture + 2 mL PtV1 + 200 μL cycloheximide; Treatment 3: 20 mL 156

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95 membranes (Millipore, Bedford, USA) to remove larger sized micro- P. tricornutum culture +2 mL PtV1 + 200 μL penicillin–streptomycin; 157
96 organisms, and further filtered through PVDF 0.22-μm-pore-size filters and Treatment 4: 20 mL P. tricornutum culture +2 mL PtV1 + 100 μL 158
97 (Millipore, USA). The filtrate were then concentrated into 1 to 2 L by ultra- penicillin–streptomycin + 100 μL cycloheximide. As for the control
D 159
98 filtration with P2B030A05 Biomax 30 k (Millipore, USA) mounted on a group, 2 mL of f/2 medium was inoculated into the system instead of 160
99 Pellicon standard system (1996). The concentrated filtrate was stored in the PtV1. All the control and treatment groups were set up in triplicate 161
100 the dark at 4 °C. In order to isolate the lytic microorganisms, 5 mL of the and grew with the conditions as described in Section 2.1 for about 162
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101 concentrated filtrate was inoculated into an exponentially growing 10 days. During this time, 200 μL of each sample was collected on 163
102 P. tricornutum culture (25 mL) and incubated at 20 °C using the same light- days 0, 1, 2, 4, 6, 8 and 10 to measure the fluorescence intensity by 164
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103 ing conditions mentioned in Section 2.1, while filtrate inactivated at 121 °C Spectra max M2 (Molecular Devices Corporation, USA) (excitation 165
104 for 30 min served as the control. The lytic microorganism was purified from wavelength = 440 nm; emission wavelength = 680 nm). 166
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105 the lysate through two extinction dilution cycles [49] and a double-agar
106 overlay plaque assay [26]. In brief, the dilution cycles involved the algal 2.5. The dynamics of bacteria and P. tricornutum cell 167
107 lysate undergoing a 10-fold dilution series in modified f/2 medium.
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108 Aliquots (1 mL) of each dilution step were added to a test tube containing To find the optimal time to easily isolate the lytic microbe of interest, 168
109 4 mL of exponentially growing P. tricornutum (completed in triplicates). the changing dynamics of algal and bacterial cell numbers were traced 169
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110 Then, the algal lysate in the most dilute of the first assay tubes was carried by using an epifluorescence microscope (Olympus BX41, Chiyoda-ku, 170
111 over to the second extinction dilution procedure. Lastly, the resultant lysate Tokyo, Japan) with a digital camera (Olympus DP50, Tokyo, Japan). 171
112 in the final endpoint dilution was used as a clonal lysate. The double-agar 2 mL of the newly-prepared lysate (or f/2 medium as a control) were 172
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113 overlay plaque assay was completed as follows. Serial dilutions of lysate added into 20 mL of exponentially growing P. tricornutum culture. 173
114 were prepared in f/2 medium. The tubes containing 4 mL of overlay Two subsamples (1 mL each) were collected over the 10 days and 174
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115 medium (0.7% agar in f/2) were placed in a boiling water bath to melt fixed by 2.5% glutardialdehyde (preparation in 0.1 M pH = 7.4 phos- 175
116 the agar, then kept in a 48–50 °C water bath before use. A fraction phate buffer), then stored at 4 °C before analysis. The fixed subsamples 176
117 (100 μL) of each final endpoint dilution was mixed with 1 mL of exponen- were filtered through 0.22-μm track-etched filter (Millipore, USA) to re- 177
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118 tially grown P. tricornutum, kept at room temperature for 5 min, transferred tain the algae and bacteria. One subsample was used for epifluorescence 178
119 to the warmed 4 mL overlay medium, mixed and poured onto the underlay microscopy, while the other for denaturing gradient gel electrophoresis 179
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120 plates (1.2% agar in f/2). Any piece of the agar within the plaque were (DGGE). The method for epifluorescence microscopy (SYBRGreen I as 180
121 picked off and resuspended in f/2 overnight. Thereafter, 100 μL of the sus- dye and phenylenediamines as antifading reagents) observation was 181
122 pension and 1 mL P. tricornutum together with 4 mL of the upper medium based on Lunau et al. [34]. The filter membrane with the collected 182
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123 were again poured onto the lower medium. Pure plaque of lytic micro- algal and bacterial cells was put on a glass slide and then observed at 183
124 organism was established by repeating the double-agar overlay plaque a magnification of 400 times under ultraviolet excitation light of fluores- 184
125 assay several times. cence microscopy. For each slide, cells in five representative visual fields 185
were counted and an average value was calculated to reflect the cell 186
126 2.3. The lytic microbe size numbers. 187

127 To check the size of the lytic microbe and to determine the alga-lytic 2.6. Amplification of 16S rDNA genes and denaturing gradient gel 188
128 pattern, the P. tricornutum lysate (hereafter called PtV1) that resulted electrophoresis (DGGE) 189
129 from the plaque was filtered through 8, 5, 3, 1, 0.45, and 0.22 μm
130 diameter-pore-sized filter membranes (Millipore, Bedford, USA). The In order to perform molecular investigations on the lytic micro- 190
131 supernatant (2 mL) was then added into the exponentially growing organism of interest, the other subsamples mentioned in Section 2.5 191
132 host P. tricornutum (20 mL) while cultures inoculated with unfiltered were for DGGE analysis. DNA was extracted from the filter membrane 192
133 lysate and sterile f/2 served as controls (all were completed in triplicate). samples as described previously [56]. Briefly, the filter membrane was 193
134 After 7 days, all the cultures were centrifuged at 5000 × g at 4 °C for washed repeatedly by the DNA extraction buffer (Tris 50 mM, EDTA 194

Please cite this article as: Z. Chen, et al., A lytic bacterium's potential application in biofuel production through directly lysing the diatom
Phaeodactylum tricornutum cell, Algal Res. (2015), http://dx.doi.org/10.1016/j.algal.2015.08.023
 Z. Chen et al. / Algal Research xxx (2015) xxx–xxx 3

195 50 mM, NaCl 100 mM, pH 8.0), the buffer (with washed off microbes) was 2.8. DNA extraction, PCR and phylogeny construction 255
196 then transferred to a 4.5 mL centrifuge tube, along with 2.5 mg ml−1
197 lysozyme (Lulong Biotech, Xiamen), and warmed at 37 °C for 1 h with in- Both the subsamples in the plaque and the lytic bacteria grown 256
198 termittent shaking. Followed by the addition of 1% SDS and 0.2 mg ml−1 on the PTF medium were collected for DNA extraction. Briefly, the exca- 257
199 protease K, the tubes were then heated at 65 °C for 2 h with shaking every vated plaques were soaked in SM buffer (50 mM Tris–HCl, 100 mM 258
200 15 min, then 5 M NaCl and 10% preheated CTAB/NaCl were added, mixed NaCl, 10 mM MgSO4, pH = 7.5) overnight, followed by centrifugation 259
201 and the tubes were heated at 65 °C for 15 min. Thereafter, the subsamples at 10,000 × g at 4 °C for 10 min and the supernatant was discarded. 260
202 in the tubes were processed by the isometric phenol/chloroform/ The genomic DNA of bacteria was extracted following the method of 261
203 isoamylol, chloroform/isoamylol, isopropanol and 70% ethanol and the Ausubel et al. [2]. The 16S rDNA gene was amplified using PCR with 262
204
Q5 obtained DNAs were dissolved in 100 μL TE buffer (Tris–HCl 10 mmol/L the primer pair P27F and P1492R [12]. Purification of the PCR product 263
205 (pH 8.0), EDTA 1 mmol/L). A highly variable region (V3) of the 16 s was carried out using a TIANquick Midi purification kit (TIANGEN, 264
206 rRNA gene was amplified using PCR primers. DGGE was used to separate China) and the purified PCR product was cloned into vector pMD19-T 265
207 mixtures of 167-bp fragments amplified from the V3 region of the 16S and sequenced. Sequences of related taxa were downloaded from the 266
208 rRNA gene. One universal primer 517R was based on the conserved GenBank database and the EzTaxon-e server (http://eztaxon-e. 267

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209 region (Escherichia coli positions at 517–534: 5′-ATTACCGCGGCTGC ezbiocloud.net/) [25]. A phylogenetic analysis was performed using 268
210 TGG-3′) and the other primer 341F complemented a region conserved MEGA version 4 [47] after multiple alignment of data using DNAMAN 269

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211 among members of the domain Bacteria (E. coli positions 341–358:5′- (version 5.1). Evolutionary distances and clustering were performed 270
212 CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGC. by using the neighbor-joining method [43]. The resulting tree topology 271
213 ACGGGGGGCCTACGGGAGGCAGCAG-3′) was incorporated into an was evaluated using a bootstrap analysis based on 1000 replicates. 272

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214 underlined GC clamp [35,59]. This primer combination amplified a
215 fragment suitable for subsequent DGGE analysis. The PCR system
(25 μL in total) for 16S rDNA V3:0.25 μL of each 341F/534R (10 μm),

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216 3. Results 273
217 0.4 μL dNTP (10 mM).
218 2.5 μL 10 × Buffer, 1.25 U Taq DNA polymerase (TaKaRa, Dalian) and 3.1. Purification of the plaque 274

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219 2 μL DNA template. The PCR thermocycler conditions were as follows:
220 initial denaturation 94 °C for 5 min, 10 cycles of denaturation 94 °C for The concentrated filtrate through 0.22-μm-pore-size filters (5 mL) 275
221 1 min, annealing 65 °C for 1 min with 1 °C decrease per cycle, extending was added to the exponentially growing P. tricornutum cultures
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222 72 °C for 1 min, followed by another 20 cycles of denaturation 94 °C for (20 mL) and the same volume of 121 °C inactivated supernatant served 277
223 1 min, annealing 55 °C for 1 min, extending 72 °C for 1 min and the final as the control. After the P. tricornutum was incubated with the filtrate for 278
224 extending 72 °C for 10 min. Small pieces of selected DGGE bands were 14 days, the color had changed from brown to transparent, an apparent 279
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225 excised from the gel using a sterile razor blade. The pieces of acrylamide aggregation had formed at the bottom. The lysate was added into the 280
226 containing the DNA bands were placed in a sterilized microfuge tube host algae again and the same algal death process was observed 281
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227 and 50 μL sterilized ddH2O was added. The samples were subjected to (Fig. 1A), suggesting that the lysate had a stable alga-lytic activity. 282
228 passive diffusion overnight at 4 °C. The supernatant was used as a tem- Furthermore, plaque-forming assays of the lysate were performed on 283
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229 plate for PCR amplification using the primers 341F without GC clamp algal plates, and all plates developed plaques 2–5 cm in diameter after 284
230 and 517R, with the reaction conditions as before. The PCR products 10 days expanding to the whole plate when given enough time 285
231 were sequenced (Life Technology, Shanghai) to see what kind of bacte- (Fig. 1B, C). By repeating this procedure several times, we determined 286
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232 ria existed in the phycosphere of P. tricornutum, the sequences were there is only one kind of plaque-forming microbe present in the plaques. 287
233 then aligned with closely related 16S rDNA sequences from GenBank.
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3.2. Size of the plaque-forming microorganism and its alga-lytic mode 288
234 2.7. Isolation and cultivation of lytic bacteria
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Our results from using the filtrates of different pore-sized filter 289
235 Lytic bacteria were isolated from both plaques and lysates of membranes to infect the P. tricornutum, revealed that the size of the 290
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236 P. tricornutum in which lytic bacteria were discovered. In order to obtain lytic microorganism was between 1 and 3 μm in length, as shown in 291
237 a pure colony of the plaque-forming bacterium, PTF culture medium Fig. 2. When compared with the control group, there was little differ- 292
238 (1.0% agar added into exponential phase P. tricornutum cultures) was ence in the content of chlorophyll a in the filtrate from pore sizes 0.22 293
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239 used. For bacterial isolation, the approximate block size of plaques and 1 μm, meaning that P. tricornutum could not be infected by the fil- 294
240 were excavated with a sterile razor blade, soaked in the liquid f/2 trate from membranes less than 1 μm. These results indicate that the 295
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241 medium overnight for passive diffusion release of the bacterium from microorganism adopted a direct, rather than indirectly lytic approach 296
242 agar, while the lysates were gradient diluted. Afterwards, the sub- since the 0.22 μm filtrate had no lytic activity. 297
243 samples from the lysate and agar suspension were streaked onto PTF
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244 plates and other conventional culture mediums, such as 2216E [7,8],
245 YMA [42], SSA [17]and Gause I medium [18]. The plates were cultured 3.3. Antibiotic tests 298
246 at 20 ± 1 °C for 7 days. Colonies that developed on this culture medium
247 were retested for lytic activity on P. tricornutum using the double-agar As seen in Fig. 3A, cycloheximide had an adverse effect on the 299
248 overlay plaque assay. Plaques caused by the bacterium would be the growth of P. tricornutum (red bar), with the relative fluorescence inten- 300
249 designated ‘special lytic microorganism’. After 15 days, only one bacterium sity values decreasing from 300 to 70. There was no significant differ- 301
250 grown in PTF, designated as KD531, formed plaques on the agar plate. ence observed between the groups with and without the addition of 302
251 Since KD531 grown in PTF could cause the formation of plaque while penicillin–streptomycin (Fig. 3A), indicating that when used at the des- 303
252 no plaque formed for KD531 grown in 2216E, KD531 colonies from both ignated concentrations, penicillin–streptomycin had no negative effects 304
253 culture mediums were selected for transmission electron microscopy to on the growth of P. tricornutum. However, in Fig. 3B, when comparing 305
254 compare whether there were any morphological differences. the fluorescence values between Treatment 1 (P. triconutum + PtV1) 306
and 3 (P. triconutum + PtV1 + antibiotics), the addition of penicillin– 307
streptomycin in Treatment 3 resulted in a gradual increase of algal 308
cells, which means that the lytic effects in PtV1 was due to a bacterium. 309

Please cite this article as: Z. Chen, et al., A lytic bacterium's potential application in biofuel production through directly lysing the diatom
Phaeodactylum tricornutum cell, Algal Res. (2015), http://dx.doi.org/10.1016/j.algal.2015.08.023
 4 Z. Chen et al. / Algal Research xxx (2015) xxx–xxx

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Fig. 1. P. tricornutum was co-cultured with lysate PtV1 both in liquid and agar plate medium. A. Growth of P. tricornutum incubated with PtV1 10 days post-infection. B. Plaque formation on
the P. tricornutum lawn plate (90 mm petri dish) after 10 days. Plaques 2 to 5 cm in diameter were seen. C. Plaque formation on the whole P. tricornutum lawn plate after 15 days.

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310 3.4. The dynamics of P. tricornutum + PtV1 during the infection process 3.5. DGGE analysis during the infection process 328

311 In order to evaluate the change in cell numbers during the infection
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There was a sharp variation in the bacterial numbers during a short 329
312 process and when the microorganism of interest demonstrated lytic ac- time (3rd–6th day) in the treatment group, and it was surprising to us 330
313 tivity, serial subsamples were collected over 10 days, and cell numbers what kind of microorganism could grow so rapidly. Hence, samples 331
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314 were determined under fluorescence microscopy. The samples were were collected throughout the entire algal infection experiments for 332
315 stained with SYBR Green, which can bind to intracellular dsDNA and DGGE analysis to examine for differential banding patterns. We expected 333
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316 fluoresce green under ultraviolet (UV) light. The algae naturally fluo- that if the lytic microbe of interest was abundant in the experimental 334
317 resce red under UV light due to chloroplast autofluorescence. Hence, system, there would be an intense V3 region band present in the treatment 335
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318 we counted the red signal as P. tricornutum and the green signal as the groups but not in the control groups. However, as shown in Fig. 5, there 336
319 lytic bacteria. As seen in Fig. 4, there is no significant change in the were almost no apparent differences between the control and treatment 337
320 bacterial abundance among the control groups while the bacterial groups. There were a total of eight different bands shared in all samples. 338
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321 counts in the treatment group increased from 2 × 107 to 9 × 107 during The sequence analysis from bands 1 to 8 showed them to be most closely 339
322 the 3rd to 4th days and then the bacterial numbers sharply decreased related to members of the Alphaproteobacteria (Band 1 (KT225549), 6 340
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323 from 9 × 107 to 4 × 107. As for P. tricornutum, in the absence of the (KT225554), at 99.0%; and 7 (KT225555), at 100%), Rhodobacteraceae 341
324 lytic bacteria, the algal cell numbers gradually increased from 1 × 106 (Bands 2 (KT225550), 4 (KT225552) and 5 (KT225553), at 99.0%), 342
325 on the 1st day to 3 × 106 on the 10th day which was just the time for Flavobacteriaceae (Band 3 (KT225551), at 93.0%), and Phaeodactylum 343
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326 the rapid growth of P. tricornutum cells. It was intriguing that the numbers tricornutum (Band 8 (KT225556), 100%). The results showed that 344
327 of bacteria changed so fast. bacteria from the above members existed both in with and without the 345
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lysates. However, we were still unable to obtain information concerning 346

the lytic microbe of interest. 347
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3.6. Identification of the plaque-forming bacteria 348

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It was difficult to get a pure culture of this lytic microbe when it had 349
such low abundance, we therefore tried a variety of different mediums, 350
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such as 2216E, YMA, SSA, Gause I medium, and PTF culture medium. Col- 351
onies that formed on these culture mediums were tested for ability to 352
form plaques on algal agar plates. Among them, one bacterium, KD531 353
was isolated by the PTF culture medium after 8 days and was able to con- 354
sistently lyse the algal cells, but it showed no activity when grown in 355
2216E (Fig. S1). The results from KD531 sequence (GenBank: KJ948638) 356
and phylogenetic analysis show that this bacterium (Culture collection 357
number: MCCC 1 K00274) belongs to the genus of Labrenzia in the family 358
Rhodobacteraceae (Fig. 6). Fig. 7 shows TEM images of the KD531 bacte- 359
rium grown in PTF medium. Organisms from the Rhodobacteraceae family 360
Fig. 2. Size of the microorganisms of interests inferred from filtering the lysate of P. tricornutum. are characteristically rich in poly-β-hydroxybutyrate (PHB). From the 361
The different pore-sized membranes, 0.22, 0.45, 1, 3, 5 and 8 μm were used, and “f/2” refers to
TEM image, it can be seen that the bacterium KD531 was rich in white 362
medium with no lysate and served as the control. P. tricornutum was inoculated with the
control and treatment groups for about 8 days; thereafter, the amount of chlorophyll α was cavitation materials which may be PHBs and there were more micro- 363
determined. All data are mean ± 1.SE (n = 3). tubule structures around the bacteria when grown in PTF medium. 364

Please cite this article as: Z. Chen, et al., A lytic bacterium's potential application in biofuel production through directly lysing the diatom
Phaeodactylum tricornutum cell, Algal Res. (2015), http://dx.doi.org/10.1016/j.algal.2015.08.023
 Z. Chen et al. / Algal Research xxx (2015) xxx–xxx 5

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Fig. 3. The effect of different antibiotic additions on A the growth of P. tricornutum and B the lytic activity of PtV1 during the 10 days.
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365 4. Discussion resources, algal cell wall disruption by microbes in a large scale ap- 372
plication would be quite advantageous [5]. In our study, we isolated 373
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366 4.1. Discovery of the plaque-forming microbes towards P. tricornutum a lytic and previously uncultured microorganism that could form 374
large plaques (5 cm) on the P. tricornutum algal plate. To further in- 375
367 P. tricornutum was selected as a model organism and as a potential vestigate what the plaque-forming microbe was in this study, namely 376
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368 source of biodiesel. Besides, this species exhibits a high amount of whether a protist, virus, or bacterium, as they have been reported as 377
369 additionally marketable polyunsaturated fatty acids [3] and the avail- being able to form plaques on the different algal plates [7,8,15,22,41, 378
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370 ability of its full genome, makes the organism popular for research in 51], we worked to gradually eliminate the possibility of it being virus 379
371 photobiotechnology [11]. In order to utilize the algal lipid and starch or protist. 380
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N
U

Fig. 4. The bacterial and algal abundance over 10 days were determined using fluorescence microscopy, stained with SYBR Green. The left Y axis reflects the bacterial cell counts and the
right Y axis reflects the algal abundance. Data are mean ± SD for three replicates.

Please cite this article as: Z. Chen, et al., A lytic bacterium's potential application in biofuel production through directly lysing the diatom
Phaeodactylum tricornutum cell, Algal Res. (2015), http://dx.doi.org/10.1016/j.algal.2015.08.023
 6 Z. Chen et al. / Algal Research xxx (2015) xxx–xxx

4.3. Efforts to trace the molecular information of the microbe during the 396
infection process 397

At first, it was hard for us to obtain the lytic bacteria in pure isolation 398
using the PtV1 system, which was a mixture of cracked microalgal cells, 399
released organic material, and the lytic bacteria among others, although 400
we tried different conventional culture mediums (2216E, YMA, SSA, 401
Gause I medium) and conditions. The obtained isolations showed no 402
lytic activity in the discussed plaque-forming assays. We then consid- 403
ered that the lytic bacterium was not dominant in quantity and needed 404
special nutritional requirements which were not met with the conven- 405
tional culture medium. To determine whether the low abundance of the 406
lytic bacterium obstructed our isolation efforts, we used the lysate 407
samples from the fourth day when the abundance of bacteria reached 408

F
the highest (Fig. 4) for the bacteria isolation and plaque-forming test. 409
However, the isolations did not form plaques, suggesting the outburst 410

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of bacteria numbers on the fourth day was not due to the large produc- 411
tion of lytic bacteria, but because of the release of other bacteria in the 412
Fig. 5. DGGE band analysis of control and treatment samples during the infection process. phycosphere of P. tricornutum. Despite this, we inferred that the num- 413

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DNA was extracted and analyzed from samples on day 0 to day 8 after infection.
bers of the lytic bacterium also increased during the lysis process, and 414
if the bacterial density did increase, we may be able to track the mole- 415
cular information of the lytic bacterium by comparing the samples in 416

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the control and treatment groups using DGGE. However, as depicted 417
381 4.2. The size and property of the plaque-forming microbes in Fig. 5, there were no differential DGGE bands between the treatment 418

P
samples and the controls, which was contrary to our initial hypothesis. 419
382 The use of different sized membrane filters showed that the size of The possible reason for this observation may be that the abundance of 420
383 the lytic microbe was 1–3 μm (Fig. 2), which excluded the possibility the microorganism was below the detection limit of DGGE: that is to 421
of it being a virus (normal virus size b0.22 μm) [39]. In addition, protists
384
D
say, it had no quantitative advantages, while it did lead to the death of 422
385 are reported to be 1–100 μm [10], so the microorganism of interest P. tricornutum and releasing large numbers of bacteria to the 423
386 might be either a protist or bacterium. However, according to Fig. 3, phycosphere of the algae. Consequently, the fluorescence microscopy 424
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387 penicillin–streptomycin inhibited the lytic activity of PtV1, yet the and DGGE results, suggested that though the lytic bacteria KD531 was 425
388 addition of penicillin–streptomycin (Treatment 3) allowed for algal not highly abundant in the algal phycosphere, it could influence the 426
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389 growth, unlike in Treatment 1 (no antibiotics), suggesting that the abundance of other bacteria by lysing the host algae. Through the 427
390 lytic microbe in PtV1 was most likely a bacterium. A previous study DGGE results here, bacterium from Phyllobacteriaceae, Rhodobacteraceae 428
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391 shows lytic bacteria that were isolated using a similar plaque assay and Flavobacterium dominated in the phycosphere of P. tricornutum. 429
392 that can form plaques of only about 7 mm in diameter [15]. It was Organisms belonging to the family Phyllobacteriaceae of the α- 430
393 intriguing that the plaques remained circular in shape throughout the proteobacteria are able to reduce nitrate to nitrite then to molecular 431
E

394 entire plaque assay, and given enough time unexpectedly extended to nitrogen [29], while the physiological properties of bacterial species 432
395 lysing all the algae present necessitating further investigation. affiliated with Rhodobacteraceae are highly diverse, including aerobic 433
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Fig. 6. 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 N50% are shown. Bar, 0.005 nucleotide substitution rate (Knuc) units.

Please cite this article as: Z. Chen, et al., A lytic bacterium's potential application in biofuel production through directly lysing the diatom
Phaeodactylum tricornutum cell, Algal Res. (2015), http://dx.doi.org/10.1016/j.algal.2015.08.023
 Z. Chen et al. / Algal Research xxx (2015) xxx–xxx 7

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Fig. 7. The bacterial morphology under TEM. (A, B) KD531 culture in PTF medium. The scale bars are 0.5 μm.

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434 anoxygenic photosynthesis, aerobic sulfite oxidation, organic sulfur focused on the discovery and isolation of a bacterium capable of lysing 479
435 compound degradation, lignin degradation, methylotrophy and anti- algae cells, it shows promise for the utilization of bacteria to directly dis- 480

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436 biotic production [23]. Members of the genus Flavobacterium are rupt microalgae in the production of biofuels. Future studies on axenic 481
437 found in a wide range of habitats such as soil, sediment, fresh water, P. tricornutum could focus on tracking the lipid content through the 482
438 seawater and micromats and display a variety of physiological charac- lytic cycle, potentially revealing the detailed lytic mechanism from the 483

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439 teristics [57]. Although there are many reports about their ecological aspects of algal morphology and physiology, protein, and key genes 484
440 function, no reports yet directly describe the relationship between related to cell growth and lipid synthesis. However, an additional 485
441 them and P. tricornutum, and whether they may play an important practical concern is the risk presented to a large scale algal cultivation
D 486
442 part in the algal growth. facility where undesired cross-contamination of a lytic bacterium 487
could be detrimental. There are many advantages to using this lytic 488
443 4.4. The special nutritional requirement and potential use of the bacterium, or the PtV1 lysate, to lyse the algal cells once sufficient 489
E
444 plaque-forming microbe biomass is cultivated. However, many industrial safe guards would 490
need to be implemented to avoid undesired infection of the algal 491
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445 As for the special nutritional requirements, our suspicion that the biomass by the lytic bacterium. 492
446 bacteria may have special metabolic requirements from outside of
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447 P. tricornutum and conventional culture medium was based on a report

5. Conclusions 493
448 about chlorellavorus bacterium whose growth occurs only in the pres-
449 ence of live Chlorella cells [9]. The lytic bacterium Labrenzia sp. KD531
E

The results from this study demonstrate the feasibility for using bac- 494
450 was finally isolated from both the plaques and lysates with PTF culture
terial infection to efficiently lyse P. tricornutum. Through DGGE analysis, 495
451 medium and it could form vivid plaques. Interestingly, the lytic bacteri-
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bacteria found in the phycosphere of P. tricornutum were mainly from 496

452 um showed no lytic activity when grown on eutrophic culture medium
the families Rhizobiales, Rhodobacteraceae and Flavobacteriaceae. Even 497
453 2216E but it did show lytic activity when grown in the f/2 medium plus
given such a complicated relationship between algae and bacteria, one 498
454 the algae. According to Fig. 7, KD531 grown in PTF (A) accumulated less
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bacterium isolated from the Xiamen Sea which was of low abundance 499
455 white inclusions compared to its growth in 2216E (B). While in oligotro-
in the system, was able to regulate the quantitative interaction of 500
456 phic PTF medium, there were many microtubule-like structures around
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other bacteria through lysing the algae. The bacterium of interest 501
457 the bacterium cell. A similar study to ours indicated that the Saprospira
could form ever-larger plaques, unlike viruses and other bacteria. Spe- 502
458 sp. strain SS98-5 cells became motile under low-nutrient condition and
cial medium with addition of algal cells allowed us to obtain pure colo- 503
459 then numerous microtubule-like fibril structures were found intracellu-
C

nies of this special lytic bacterium. The ability to utilize beneficial 504
460 larly [60], suggesting to us that the microtubule-like fibril structures
materials following algal cell wall degradation by this bacterium 505
461 might play important roles for the lytic bacterium to directly interact
would be an effective method to begin the production of bioethanol 506
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462 with the algal cell.

and other biofuel molecules from P. tricornutum. 507
463 Bacteria from the family Rhodobacteraceae have been reported to
Supplementary data to this article can be found online at http://dx. 508
464 have algicidal activity. However, studies on the relationship between
U

doi.org/10.1016/j.algal.2015.08.023. 509
465 species in the Rhodobacteraceae sp. and P. tricornutum are rare. Hence,
466 this work is the first report on species of the Rhodobacteraceae being
467 able to form plaques on diatom plates. In this study, we finally isolated Uncited references 510
Q6
468 the lytic bacterium from a complex system in which it was not the
469 dominant organism. The result of one bacterium showing distinct lytic [6,21,36,48,50,54] 511
470 effects in different mediums, suggests that the algal cell might supply
471 some essential nutrients to the lytic bacterium. In addition, the lytic
472 bacterium KD531 accumulated poly-β-hydroxybutyrate (PHB), which Acknowledgments 512
473 tends to be synthesized by various microorganisms under unbalanced
474 growth conditions, serving as internal energy and carbon reserve. This work was financially supported by the National Natural Science 513
475 Hence, on the one hand, P. tricornutum cells could be lysed by KD531 Foundation (41376119, 41476095, 40930847), the Science and Tech- 514
476 releasing stored lipids within the algae to the environment. On the nology Innovation Funds of Shenzhen (JCYJ20120615161239998), 515
477 other hand, KD531 could synthesize the PHB by taking up the nutritional Prof. I.J. Hodgkiss of the University of Hong Kong is thanked for help 516
478 materials released from the algae as substrate. Although this research with the English. 517

Please cite this article as: Z. Chen, et al., A lytic bacterium's potential application in biofuel production through directly lysing the diatom
Phaeodactylum tricornutum cell, Algal Res. (2015), http://dx.doi.org/10.1016/j.algal.2015.08.023
 8 Z. Chen et al. / Algal Research xxx (2015) xxx–xxx

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Please cite this article as: Z. Chen, et al., A lytic bacterium's potential application in biofuel production through directly lysing the diatom
Phaeodactylum tricornutum cell, Algal Res. (2015), http://dx.doi.org/10.1016/j.algal.2015.08.023
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