World J Microbiol Biotechnol (2014) 30:1325–1334 DOI 10.1007/s11274-013-1557-0
ORIGINAL PAPER
Antidiatom activity of marine bacteria associated with sponges from San Juan Island, Washington Cuili Jin • Xiaying Xin • Siyu Yu • Jingjing Qiu Li Miao • Ke Feng • Xiaojian Zhou
•
Received: 3 September 2013 / Accepted: 9 November 2013 / Published online: 15 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Crude extracts of 52 marine bacteria associated with sponges, which were collected from the sea near San Juan Island, Washington, USA, were screened using diatom attachment assays against Amphora sp., Nitzschia closterium, Sellaphora sp. and Stauroneis sp. to investigate their antidiatom activities. Among these samples, five expressed strong anti-adhesion effects on all four tested diatoms. There was no negative effect observed from those five active samples on the growth of Amphora sp. Those five active samples were prepared from respective isolates, which all belonged to the genus Bacillus based on 16S rRNA gene sequencing analysis. The results of present study indicate that Bacillus may play important roles for sponges’ chemical defence against biofouling of diatoms and that the metabolites of Bacillus may be a potential source of natural antifouling compounds. Keywords Sponges Bacteria Bacillus Antidiatom activity Growth
Introduction Attachment and growth of unwanted living organisms on man-made surfaces is generally referred to as biofouling
C. Jin X. Xin S. Yu J. Qiu L. Miao K. Feng X. Zhou (&) College of Environmental Science and Engineering, Yangzhou University, 196# of Huayang West Street, Hanjiang District, Yangzhou, Jiangsu, China e-mail: [email protected] C. Jin L. Miao X. Zhou Marine Science and Technology Institute, Yangzhou University, 196# of Huayang West Street, Hanjiang District, Yangzhou, Jiangsu, China
(Fusetani 2004; Kristensen et al. 2008). Marine biofouling is an extensive phenomenon resulting in large costs to engineered structures in ocean environments, such as ship hulls and offshore platforms, due to increased manpower, fuel and materials consumption, and dry-docking time (Limna Mol et al. 2009). To date, the antifouling strategies in use are mainly based on antifouling paints containing toxic chemicals. Among these toxic compounds, tributyltin (TBT) is not only the most effective antifouling agent but also one of the most detrimental biocides ever introduced to marine environments because it is not readily degraded in the natural marine environments and acts on both target and non-target organisms. This property has led the International Maritime Organisation (IMO) to prohibit its application to ships, effective from 17 September 2008 (Qian et al. 2010). Since that prohibition, researchers have made great strides in finding new environmentally friendly chemicals as substitutes for TBT (Fusetani 2004). Many sessile macroalgae and animals are free from marine fouling, as they release chemicals influencing the settlement, growth, and survival of fouling organisms. This is known as chemical defence against biofouling (Qian et al. 2010). Many macroalgae and animals themselves lack both chemical and non-chemical defences and are believed to rely on the secondary metabolites produced by their associated bacteria (Mizobuchi et al. 1996; Kon-ya et al. 1995). Among those sessile organisms, marine sponges, as a significant component of benthic communities throughout the world, have been the focus of recent studies aiming for the isolation of natural antifouling compounds (Qian et al. 2010; Fusetani 2004). Sponges form close associations with a wide variety and a large number of microorganisms. Many natural compounds isolated from sponges have been reported to be
123
1326
World J Microbiol Biotechnol (2014) 30:1325–1334
Table 1 Phylogenic status of bacterial isolates from sponges collected from San Juan Island, Washington Sample number
Strain designation
Next relative by GenBank alignment
Sequence homology (%)
Accession no.
1
UST050418-092
Bacillus pumilus SAFR-032
100
NC009848.1
2
UST050418-097
Bacillus pumilus SAFR-032
99
NC009848.1
3
UST050418-099
Clostridium beijerinckii NCIMB 8052
98
NC009617.1
4
UST050418-100
Bacillus pumilus SAFR-032
99
NC009848.1
5
UST050418-101
Bacillus megaterium DSM319
100
NC014303.1
6
UST050418-172
Psychrobacter arcticus 273-4
98
NC007204.1
7
UST050418-178
Staphylococcus haenolyticus JCSC1435
94
NC007168.1
8
UST050418-192
Pseudoalteromonas sp. SM9913
99
NC014803.1
9
UST050418-202
Bacillus sp.1NLA3E
86
NC021171.1
10
UST050418-205
Bacillus sp.1NLA3E
92
NC021171.1
11 12
UST050418-206 UST050418-279
Bacillus sp.1NLA3E Pseudomonas aeruginosa PAO1
88 95
NC021171.1 NC002516.2
13
UST050418-281
Bacillus pumilus SAFR-032
99
NC009848.1
14
UST050418-291
Bacillus pumilus SAFR-032
99
NC009848.1
15
UST050418-292
Pseudomonas syringae pv syringae B728a
99
NC007005.1
16
UST050418-303
Psychrobacter cryohalolentis K5
97
NC007969.1
17
UST050418-315
Bacillus pumilus SAFR-032
99
NC009848.1
18
UST050418-319
Paenibacillus terrae HPL-003
95
NC016641.1
19
UST050418-323
Bacillus pumilus SAFR-032
100
NC009848.1
20
UST050418-326
Bacillus pumilus SAFR-032
98
NC009848.1
21
UST050418-328
Bacillus pumilus SAFR-032
99
NC009848.1
22
UST050418-329
Bacillus pumilus SAFR-032
98
NC009848.1
23
UST050418-331
Bacillus pumilus SAFR-032
87
NC009848.1
24
UST050418-333
Bacillus pumilus SAFR-032
99
NC009848.1
25
UST050418-337
Microbacterium testaceum StLB037
88
NC015125.1
26
UST050418-368
Cellulophaga lytica DSM 7489
98
NC015167.1
27 28
UST050418-380 UST050418-390
Bacillus pumilus SAFR-032 Cellulophaga lytica DSM 7489
99 98
NC009848.1 NC015167.1
29
UST050418-392
Cellulophaga lytica DSM 7489
99
NC015167.1
30
UST050418-393
Zobellia galactanivorans
98
NC015844.1 NC018877.1
31
UST050418-416
Bacillus thuringiensis Bt407
99
32
UST050418-421
Geobacillus sp. WCH70
96
NC012793.1
33
UST050418-480
Psychrobacter arcticus 273-4
99
NC007204.1
34
UST050418-555
Bacillus thuringiensis Bt407
100
NC018877.1
35
UST050418-566
Pseudovibrio sp. FO-BEG1
98
NC016642.1 NC018877.1
36
UST050418-573
Bacillus thuringiensis Bt407
99
37
UST050418-579
Pseudoalteromonas sp. SM9913
97
NC014803.1
38
UST050418-580
Bacillus weihenstephanensis KBAB4
99
NC010184.1
39
UST050418-581
Bacillus thuringiensis Bt407
96
NC018877.1
40
UST050418-584
Pseudoalteromonas sp. SM9913
99
NC014803.1
41
UST050418-585
Pseudoalteromonas sp. SM9913
97
NC014803.1
42 43
UST050418-649 UST050418-681
Bacillus megaterium DSM319 Bacillus pumilus SAFR-032
98 98
NC014303.1 NC009848.1
44
UST050418-683
Bacillus pumilus SAFR-032
99
NC009848.1
45
UST050418-700
Paenibacillus sp. Y412MC10
86
NC013406.1
46
UST050418-703
Bacillus thuringiensis Bt407
97
NC018877.1
47
UST050418-714
Lysinibacillus fsaphaericus C3-41
93
NC010382.1
48
UST050418-724
Exiguobacterium sibiricum 255-15
98
NC010556.1
123
World J Microbiol Biotechnol (2014) 30:1325–1334
1327
Table 1 continued Sample number
Strain designation
Next relative by GenBank alignment
Sequence homology (%)
Accession no.
49
UST050418-735
Arthrobacter phenathrenivorans Sphe3
99
NC015145.1
50
UST050418-753
Bacillus weihenstephanensis KBAB4
99
NC010184.1
51
UST050418-760
Bacillus sp. 1NLA3E
91
NC021171.1
52
UST050418-763
Paenibacillus terrae HPL-003
94
NC016641.1
similar to microbial products or even verified as microbially produced (Taylor et al. 2007). In addition, certain metabolites of bacteria isolated from marine sponges display antimicrobial activities (Thiel and Imhoff 2003; Anand et al. 2006; Kennedy et al. 2009; Santos et al. 2010), and there are also many impressive reports on the antifouling activity of sponge-associated bacteria that have been published in past decades (Qian et al. 2010; Fusetani 2004). The organic extracts of Winogradskyella poriferorum, isolated from sponges, have been reported to be capable of effectively inhibiting the larval settlement of two typical fouling invertebrates of Hydroides elegans and Amphibalanus amphitrite, as well as the biofilm formation of two bacterial species (Dash et al. 2009). Lee and Qian (2003) found that the bacteria associated with marine sponges were able to suppress larval settlement of the tubeworm H. elegans. From a bacterium separated from marine sponge, Kon-ya et al. (1995) isolated ubiquinone-8, which inhibited larval settlement of barnacle larvae. Polyethers from a sponge-associated bacterium displayed inhibitory effects on both biofilm formation of marine bacteria and larval settlement of macrofoulers (Dash et al. 2011). As a result, sponge-associated bacteria have been recognised as a good choice to search for new antifouling compounds not only because sponges are free from biofouling but also because of such advantages as short growing time, easy control in the production of target substances, and especially vast production capacity (Fusetani 2004; Taylor et al. 2007; Qian et al. 2010). Biofouling can be divided into macro-fouling and micro-fouling based on fouling organisms, with the former including macro-algae and marine invertebrates such as barnacles, and the latter mainly referring to biofilm caused by bacteria and diatoms (Kristensen et al. 2008). Virtually, all surfaces of aquatic habitats are covered with a biofilm, whether formed by bacteria or/and diatoms. Biofilms play a crucial role in the attachment and subsequent growth of macro-organisms, which further leads to the formation of a complex biofouling community (Hoagland et al. 1993). Diatoms are always the major component in these biofilms as long as light can reach down to these habitats (Aumeier and Menzel 2012; Leflaive and Ten-Hage 2011a, b). Therefore, the use of antidiatom is an essential and challenging step to avoid the subsequent process of macro-
fouling. However, possibly due to the inherent, technical challenges in performing antidiatom assays (Kumar et al. 2010), reports of natural products from sponge-associated bacteria with antidiatom activity are rare in comparison with tremendous progress in the fields of antibacterial and anti-larval activity (Dobretsov and Qian 2002; Kumar et al. 2010). The objective of the present study was to assess the effect of sponge-associated bacteria on adhesion by potent biofilm-forming diatoms, including Amphora sp., Nitzschia closterium, Sellaphora sp., and Stauroneis sp. In total, 52 isolates of sponge-associated bacteria were screened for their antidiatom activities against four species of fouling diatoms. The EC50 of the five isolates with high antidiatom activities were tested against Amphora sp. and N. closterium. In addition, the isolates that expressed inhibitive or inductive activity against diatom attachment are discussed on the genus level.
Materials and methods Tested sponge-associated bacteria Bacteria associated with 39 sponge specimens collected from San Juan Island, Washington, USA, were isolated and purified. The collection, isolation and purification process were performed by Prof. Pei-Yuan Qian’s group from the Hong Kong University of Science and Technology (HKUST), and the detailed methods were described by Lee et al. (2009). Out of these samples, 52 isolates were selected randomly to screen using diatom attachment assays. Among the tested 52 isolates, UST050418-333 and UST050418-683 were identified based on comparative sequence analysis of their 16S rDNA genes by Sangon Biotech (Shanghai) Co., Ltd. in our preliminary study. The 16S rRNA gene sequencing of the other 50 bacterial isolates were performed by Prof. Pei-Yuan Qian’s group in HKUST. Using the 16S rRNA gene sequences, the taxonomic hierarchy of the isolates was determined using a Bayesian classifier available online through the Ribosomal Database Project II (Kumar et al. 2010). These strains were identified as belonging to five major groups of bacteria: alpha and gamma Proteobacteria, Actinobacteria,
123
1328
World J Microbiol Biotechnol (2014) 30:1325–1334
A
160 150 140 130 120 110
R (%)
100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10111213141516171819 202122 23 24 252627 28293031 32 33343536 37383940 41424344 4546 47484950 51 52
160
B
150 140 130 120 110
R (%)
100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10111213141516171819 2021 22 23 24 252627 28293031 323334 353637383940 414243444546 474849 5051 52 160
C
150 140 130 120 110
R (%)
100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10111213 1415161718192021222324252627282930313233343536373839 40414243444546474849505152
Sample
123
World J Microbiol Biotechnol (2014) 30:1325–1334
1329
The crude extracts were prepared from 52 isolates of sponge-associated bacteria using the method described by Dash et al. (2009). First, 1 ml of the bacteria stocks in 50 % glycerol were individually inoculated into 10 ml of peptone–yeast extract medium (P-Y medium, containing 0.3 % of yeast extract and 0.5 % of peptone in artificial sea water (ASW) and incubated at 28 °C until reaching the exponential phase (60 h). Then, the individual bacterial culture was transfer to 800 ml of P-Y medium and incubated until the stationary phase at 28 °C and 120 rpm for 84 h. Finally, each bacterial culture was collected. Ethyl acetate (EA) with 5 % (v/v) acetone was added to each bacteria culture with a ratio of 1:1 (v/v) and was shaken vigorously for 1 h for complete extraction. The organic phase containing the bacterial metabolites was separated from the aqueous phase and dried on rotary evaporator (37 °C) to obtain the crude extract. Each crude extract was weighed and re-dissolved with dimethylsulfoxide (DMSO) for bioassays.
chamber at 23 °C under controlled illumination (100 lmol photons m2/s1 provided by cool-white fluorescent lamps with a 12 h:12 h light: dark cycle). Prior to the assays, diatom films were collected from the culture flask, suspended in ASW, washed with ASW by centrifugation twice, re-suspended in ASW, and adjusted to suitable density by addition of ASW. The diatom attachment assays were run in 24-well plates, each well of which contained 1 ml of algal suspension in ASW with addition of crude extract dissolved in DMSO. The final concentration of the crude extract in the well was 100 lg/ml, and the DMSO content was 0.5 % (v/v). The control received 1 ml of algal suspension in ASW with 0.5 % (v/v) DMSO. Algal concentrations in the ASW were adjusted using a blood counting chamber with comparable biomass: 5 9 105 cells/ml for N. closterium, 1 9 105 cells/ml for the others. Plates were incubated for 24 h at 23 °C with 100 lmol photons m2/s1 illumination and a 12 h:12 h light: dark cycle. Then, the well contents were gently shaken by pipetting, and non-adherent cells were removed. The remaining cells, still fixed to the bottom of the wells, were counted using an inverted microscope (Nikon ECLIPSE TS 100). At least triplicates were tested for each treatment. The results were expressed as the ratios (R) in terms of percentages for each sample: R(%) = (adherent cell number of treatment)/(mean adherent cell number of controls) (Leflaive and Ten-Hage 2011a). The effects of samples were classified according to the method described by Ma et al. (2009) with slight modification. Crude extracts with R C 110 % were classified as ‘inductive’. Those with R B 10 % were classified as ‘inhibitive’, and the remaining with 10 % \ R\110 % were classified as ‘neutral’ (Ma et al. 2009). When calculating the EC50 of active isolates, the inhibition percentage was equal to (100 %-R).
Diatom attachment assays
Diatom growth assays
Four benthic diatoms were used in the diatom attachment assays. The diatoms Amphora sp., N. closterium, Sellaphora sp., and Stauroneis sp. were obtained from the Key Laboratory of Mariculture, Ministry of Education, Ocean University of China. Among of them, the genus Amphora is pre-eminent amongst the fouling diatoms and has a cosmopolitan distribution and an ability to grow in a wide range of conditions (Daniel et al. 1980). N. closterium is a common marine benthic diatom in the intertidal zone and often used in antidiatom assays (Li et al. 2011). Sellaphora sp. and Stauroneis sp. were isolated from natural biofilms and identified by scanning electron microscopy (SEM). The diatoms were maintained in 250 ml Erlenmeyer flasks in ASW-based Guillard’s f/2 culture medium (Guillard and Ryther 1962) in a temperature-controlled
This experiment was designed to determine the effect of crude extracts on Amphora sp. growth. The test was run in triplicate on 96-well microplates, each well of which contained 200 ll of exponentially growing cells of Amphora sp. at a concentration of 1 9 105 cells/ml in ASW. Then, the tested crude extracts, with a final concentration of 100 lg/ml, were added into the wells, before the plates were incubated under the same conditions as those in attachment assays. Algal growth was determined by measurements of optical density (OD, at 690 nm, by 318 Microplate reader, Shanghai Sanco, China) at the initial time and the 3rd day (Leflaive and Ten-Hage 2011b). The growth rate of Amphora sp. was calculated as l = (lnN2 - lnN1)/(t2 - t1), where N1 and N2 were OD690, at times t1 and t2, respectively (Silkina et al. 2012).
b Fig. 1 Antidiatom effect of 52 bacterial samples against N. closte-
rium, Stauroneis sp. and Sellaphora sp. The concentration of the crude extracts in all treatments was 100 lg/ml. R was calculated as (adherent cell number of treatment) divided by (mean adherent cell number of controls) and is shown as a percentage. Data plotted are the means ? SD of at least three replicates. a–c N. closterium, Stauroneis sp. and Sellaphora sp., respectively. Crude extracts with an R higher than 110 % were classified as ‘inductive’, R lower than 10 % ‘inhibitive’, and otherwise, R was classified as ‘neutral’. The control for diatom attachment (not shown in figure) is representative of no inhibition (i.e., 100 % attachment)
Firmicutes and Bacteroides. Detailed information is listed in Table 1. Crude extracts preparation of bacteria
123
1330
Data analysis The growth rate of Amphora sp. was analysed with oneway ANOVA followed by the LSR (Least significant range) test. In all cases, the thresholds for significance were 1 % and 5 %.
World J Microbiol Biotechnol (2014) 30:1325–1334
found to inhibit the attachment of at least one of the tested three diatom species. Importantly, there were 5 samples that inhibited the attachment of all tested three diatoms simultaneously. These samples were No. 4, 17, 24, 27 and 44. Antidiatom activities screening using Amphora sp.
Antidiatom activities screening using N. closterium, Stauroneis sp. and Sellaphora sp. As shown in Fig. 1a, in total, 52 crude extracts of spongeassociated bacteria were subjected to an antidiatom assay against N. closterium. For tested samples, a concentration of 100 lg/ml of all crude extracts greatly impaired the adhesion of N. closterium compared with the controls. In total, 11 samples (21 % of the all tested samples), including No. 2, 4, 13, 17, 24, 26, 27, 28, 29, 35 and 44, expressed strong antidiatom activity and inhibited N. closterium attachment with an R lower than 10 %. Different than in the assays using N. closterium, crude extracts expressed two types of effects against Stauroneis sp. in the assays. For Stauroneis sp., a total of 9 crude extracts, including No. 2, 4, 17, 19, 22, 24, 27, 35 and 44 (17 % of tested samples) displayed strong attachment inhibition effects. At the same time, 8 samples (No. 8, 9, 12, 18, 20, 31, 37 and 48) induced diatom adhesion with an R higher than 110 %. For example, sample No. 20 promoted diatom attachment at level higher than in the controls at the concentration of 100 lg/ml with an R as high as 130 % (Fig. 1b). Similar to Stauroneis sp., crude extracts of spongeassociated bacteria displayed both inductive and inhibitive effects on the diatom adhesion of Sellaphora sp. For the tested samples, crude extracts at a concentration of 100 lg/ml greatly inhibited attachment of Stauroneis sp. compared with those in the controls. Among the samples, 6 samples (12 % of tested samples, including No. 4, 5, 17, 24, 27 and 44) inhibited diatom attachments with an R lower than 10 %. In addition, 7 samples (No. 7, 14, 16, 34, 40, 41 and 46) strongly promoted Sellaphora sp. adhesion with an R higher than 110 % (Fig. 1c). There were eight and seven samples that displayed inductive effects on Stauroneis sp. and Sellaphora sp., respectively. These 15 samples promoted diatom attachment with an R higher than 110 %. However, none of these inductive samples was effective in promoting the attachment of both Stauroneis sp. and Sellaphora sp. In addition, there were 11, 9 and 6 samples found that displayed inhibiting effects against N. closterium, Stauroneis sp., and Sellaphora sp., respectively. In total, 14 samples were
123
To verify their broad inhibitive activities, the above 5 effective samples, which were active against all tested three diatoms, were tested against one of the most often tested fouling diatom species, Amphora sp. As shown in Fig. 2, all five samples were effective against Amphora sp. at a concentration of 100 lg/ml. All these five samples inhibited diatom adhesion with an R lower than 5 %. The highest R for these five samples was only approximately 4 %, and the most active sample with the lowest R was 1 % for sample No. 4. Therefore, these five samples displayed a strong ability to inhibit the attachment of not only N. closterium, Stauroneis sp. and Sellaphora sp. but also the most often tested fouling diatom species, Amphora sp. Antidiatom effects of 5 active samples against N. closterium and Amphora sp. Crude extracts with descending concentrations of the 5 active samples were further tested against two representative diatoms, N. closterium and Amphora sp. The extracts were tested at concentrations ranging from 100 to 0.39 lg/ml using diatom attachment assays performed in 24-well plates for 24 h as described previously. The antidiatom activities weakened with reduced concentration. The dramatic weakening in the activities were observed in a relatively
10 9 8 7 6
R (%)
Results
5 4 3 2 1 0
4
17
24
27
44
Sample
Fig. 2 Antidiatom effect of 5 active bacterial samples against Amphora sp. The diatom Amphora sp. was employed in this antidiatom bioassay. The concentration of the crude extracts in all treatments was 100 lg/ml. R was calculated as (adherent cell number of treatment) divided by (mean adherent cell number of controls) and was as a percentage. The data plotted are the means ? SD of at least three replicates. The control for diatom attachment (not shown in figure) is representative of no inhibition (i.e., 100 % attachment)
World J Microbiol Biotechnol (2014) 30:1325–1334 No.4 EC50=3.22 µg/ml
100
No.4 EC50=15.40 µg/ml
80
R (%)
R (%)
1331
60 40 20 0
50.00
25.00
12.50
6.25
3.13
1.56
100 80 60 40 20 0
50.00
25.00
12.50
6.25
100 80 60 40 20 0
R (%)
R (%)
No.17 EC50=5.62 µg/ml
50.00
25.00
12.50
6.25
3.13
80
20 6.25
1.56
0.78
0.39
3.13 No.24 EC50=12.53 µg/ml
20
No.27 EC50=3.88 µg/ml
80
100.00
50.00
25.00
12.25
60 40 20
6.25 No.27 EC50=19.32 µg/ml
100
R (%)
R (%)
6.25
40 12.50
100
80 60 40
50.00
25.00
12.50
6.25
3.13
20
1.56
0 No.44 EC50=8.39 µg/ml
R (%)
R (%)
12.50
60
0
100 80 60 40 20 0
25.00
80
R (%)
\
40
0
50.00
100
60
0
No.17 EC50=21.19 µg/ml
No.24 EC50=1.47 µg/ml
100
R (%)
1.56
100 80 60 40 20 0
3.13
100.00
50.00
25.00
12.50
6.25
Concentration (µg/ml)
Fig. 3 Antidiatom effect of 5 active bacterial samples against N. closterium. The diatom N. closterium was employed in this antidiatom bioassay. R was calculated as (adherent cell number of treatment) divided by (mean adherent cell number of controls) and is shown as a percentage. The data plotted are the means ? SD of at least three replicates. EC50 values were calculated based on the inhibitory rate, which was 100 %-R(%) and are shown in the right-top of each sub-figure labelled with the respective sample number
narrow concentration ranges for each sample and are described in Fig. 3 for N. closterium and Fig. 4 for Amphora sp. In Fig. 3, the R became higher than 50 % when the concentration was lower than 6.25, 6.25, 1.56, 6.25, and 12.50 lg/ml for samples No. 4, 17, 24, 27, and 44, respectively. As a result, the EC50 values were calculated for each sample, and the lowest value was 1.47 lg/ml for sample No. 24, whereas the highest value was 8.39 lg/ml for sample No. 44. The EC50 values for the other samples, No. 4, 17 and 27, were 3.22, 5.62 and 3.88 lg/ml,
100 80 60 40 20 0
50.00
25.00
12.50
6.30
3.13 No.44 EC50=21.07 µg/ml
100.00
50.00
25.00
12.50
6.25
Concentration (µg/ml)
Fig. 4 Antidiatom effect of 5 active bacterial samples against Amphora sp. The diatom Amphora sp. was employed in this antidiatom bioassay. R was calculated as (adherent cell number of treatment) divided by (mean adherent cell number of controls) and is shown as a percentage. The data plotted are the means ? SD of at least three replicates. The EC50 values were calculated based on the inhibitory rate, which was 100 %- R(%) and were shown on the righttop of each sub-figure labelled with the respective sample number
respectively. In Fig. 4, the R became higher than those in Fig. 3 for N. closterium at the same concentration for each sample. As the result, all of the EC50 against Amphora sp. were much higher than those for N. closterium and were calculated as 15.40, 21.19, 12.53, 19.32, and 21.07 lg/ml for samples No. 4, 17, 24, 27 and 44, respectively. Among these samples, sample No. 4 was the most effective at inhibiting adhesion of Amphora sp.
123
1332
World J Microbiol Biotechnol (2014) 30:1325–1334
0.25 **
**
0.2
**
** *
µ
0.15 0.1 0.05 0
4
17
24
27
44
Control
Sample
Fig. 5 Effect on diatom growth by 5 active bacterial samples against Amphora sp. The diatom Amphora sp. was employed in this growth bioassay. The concentration of the crude extracts in all treatments was 100 lg/ml. The results are expressed as the growth ratio (l). The data plotted are the means ? SD of at least three replicates. Data that are significantly different according to the LSR test (one-way ANOVA) are indicated by an asterisk above the bars. Single and double asterisks indicate statistical significance at the 0.05 and \0.01 levels, respectively
Effects on diatom growth by five active samples against Amphora sp. To test whether the antidiatom activity of five active samples is based on inhibiting diatom growth, algal growth assays were conducted against Amphora sp. No negative effect on algal growth was detected. The growth rate of Amphora sp. treated with the five samples was significantly higher than that of the control. An LSR test revealed that 4 samples (all but No. 44) resulted in the algal growth rate being significantly higher than the control (P \ 0.01). The growth rate of sample No. 44 was also higher than the control (P \ 0.05) (Fig. 5). All these 5 active samples prepared from respective isolates strongly inhibited diatom attachment without negative effects on diatom growth. These 5 active isolates were identified as belonging to the Bacillus genus based on 16S rRNA gene sequencing.
Discussion The antidiatom effects of the crude extracts from the sponge-associated bacterial isolates were examined in this study. The results in Fig. 1 verified that many of the metabolites of sponge-associated bacteria affect diatom settlement. Sponges are free from biofouling because of their chemical defence, which is closely related to their associated bacteria (Thomas and Jo¨rn 2009; Kennedy et al. 2009). The large community of microorganisms can compose up to 50 % volume of the sponge tissue, the density of which exceeds that of bacteria in seawater by two–three orders of magnitude (Wang 2006; Lee et al. 2009). Sponge-
123
associated bacteria have been screened for new antifouling natural products for many years, and some positive results have been found (Fusetani 2004). These studies also have verified that it is possible to utilise sponge-associated bacteria as a promising resource for the isolation of effective antifouling compounds (Qian et al. 2010). Most studies focus on the anti-microbiological and anti-larval settlement activities (Fusetani 2004). Diatoms are important first settlers on new surfaces, and prevention of diatom biofouling is a crucial and difficult step in antifouling research (Hoagland et al. 1993; Silkina et al. 2012). However, information concerning antidiatom activity by sponge-associated bacteria remains insufficient. Similar to the results obtained by Dobretsov and Qian (2004) and Kumar et al. (2010), both inductive and inhibitive effects of sponge-associated bacteria on diatom adhesion were found in this study, though there were differences in regards to specific diatom species. The inductive and inhibitive bacteria co-exist in sponges. These findings indicate that the integrated effect for a specific diatom is based on the relative abundance of bacterial species and their active products. The sponges benefit from both inductive- and inhibitive-associated bacteria because of their selective attraction or exclusion against specific diatoms. The inhibitive bacteria are most likely responsible for the anti-fouling functions. The inductive bacteria in sponges have been reported to play other important roles for the hosts, such as nitrogen-fixing bacteria providing the host organism with additional amounts of nitrogen or furnishing the host organisms with fixed carbon or nitrogen (Dobretsov and Qian 2004). In addition, the existence of different functional bacteria makes it possible to adjust the integrated functions according to varied conditions. Undoubtedly, such flexibility is advantageous for the host in the unsteady marine environment. The effects of a sample prepared from each isolate on different tested diatom species varied. For example, total of 21, 17, and 12 % of the tested samples were active against the attachment of N. closterium, Stauroneis sp. and Sellaphora sp., respectively. Some isolates even displayed an opposite effect against different tested diatoms. An extreme example was isolate 7, which displayed a neutral effect with an R of 46 % against N. closterium but relative inductive effects against Stauroneis sp. and Sellaphora sp. with R values of 109 and 147 %, respectively. This phenomenon is concordance with the properties of marine diatom settlement and adhesion varying between species (Li et al. 2011). Marine diatoms often attach to solid surfaces via adhesive extracellular polymeric substances (EPS) (Hoagland et al. 1993; de Brouwer et al. 2006; Becker 1996). EPS can undergo conformational changes to adapt to solid surfaces with different physicochemical properties to optimise the
World J Microbiol Biotechnol (2014) 30:1325–1334
adhesion process. These changes grant marine diatoms the ability to adhere to all types of solid surfaces, which consequently produces greater for antifouling techniques (Li et al. 2011; Silkina et al. 2012). In addition, most pennate diatoms possess a raphe, which correlates closely with the ability of the diatom to adhere to an inert substratum and to glide whilst maintaining adhesion. It is well documented that diatom cell gliding requires adhesion to the substratum at the raphe, and strands of EPS have been observed projecting from the raphe (Lind et al. 1997). Though the detailed relationship between the raphe and adhesion strength is not clear, it would be beneficial to screen multiple species of diatoms with different raphe types to determine effective antifouling substances that work against many species of diatoms. One reason is that the target diatom species respond with different sensitivity to a certain tested substance. The other reason is that antifouling applications are aimed at multiple species (Silkina et al. 2012). Thus far, target species in antidiatom studies have mostly been chosen from algal banks or natural biofilms mainly based on availability and maintenance (Kumar et al. 2010). The raphe systems vary for the diatoms used in the present report. The raphe branches of Sellaphora sp. have a groove on each side, and the raphe is straight with expanded external central raphe ends (Mann et al. 2009). The raphe system of N. closterium is traversed by a series of fibulae, which join directly to the valve face. The raphe of Stauroneis sp. is straight, whereas the central area is a ‘‘bow tie’’-shaped stauros composed of a non-striated, thickened central nodule that usually extends to the valve margin. The raphe of Amphora sp. is eccentric, lying close to the ventral margin, filiform, and dorsally arched (Daniel et al. 1980; Hilaluddin et al. 2011). The results from antidiatom assays with multiple species targets, especially those with different raphe types, would useful for solving diatom fouling problems in the future. Biofouling of marine structures, especially ship hulls, are characterised by two critical events: the irreversible adhesion of biofouling species and the proliferation of the microorganisms in biofilm (Kristensen et al. 2008). Accordingly, traditional antifouling paints rely on cytotoxic effects and new environmentally safe alternatives aim to interfere with the adhesion and/or growth of the fouling organisms (Kristensen et al. 2008; Leflaive and Ten-Hage 2011b). In the present study, all active tested isolates possessed anti-adhesion activity without anti-growth effect, which makes it possible to identify some active chemicals from these isolates that merely inhibit diatom without exerting any negative influence on normal algal growth. In this study, bacterial isolates were members of 15 genera that were affiliated with the Firmicutes (69 %), alpha and gamma Proteobacteria (17 and 2 %, respectively), Bacteroides (8 %), and the Actinobacteria (4 %). This
1333
bacterial diversity is greater than bacteria isolated from Antarctic sponges and Mediterranean sponges, as the former lack of Firmicutes and the latter lack Bacteroidetes (Mangano et al. 2009; Hentschel et al. 2001). In total, 80 % of the isolates belonged to genera Bacillus, Pseudoalteromonas, Psychrobacter, Cellulophaga and Paenibacillus. In total, seven genera expressed inductive effects, including Pseudoalteromonas, Bacillus, Staphylococcus, Pseudomonas, Psychrobacter, Paenibacillus and Exiguobacterium. In contrast, the genus Bacillus outstandingly and significantly inhibited attachment of diatoms. Marine Bacillus species are often isolated from sediments and invertebrates. The species of this genus are known to generate spores under adverse conditions, such as low nutrient availability, irradiation and chemical disinfectants (Pabel et al. 2003). Metabolites produced by members of the genus Bacillus are known to have various biological activities, such as antimicrobial, antifungal, and generally cytotoxic properties (MuschollSilberhorn et al. 2007; Ortega-Morales et al. 2008). The epibiotic bacteria of Bacillus isolated from different host organisms have been demonstrated to be able to inhibit the formation of bacteria biofilm, the settlement of barnacle B. amphitrite larvae and algal spores of Ulva lactuca (Burgess et al., 2003). It has been reported that an isolate of Bacillus genus from U. lactuca had antidiatom activity against the pennate diatom Cylindrotheca fusiformis (Kumar et al. 2010). In the present study, members of the Bacillus genus isolated from sponges significantly suppressed multispecies diatom attachment. The results provide solid evidence that Bacillus species are a good source for the isolation of potentially new antifouling agents. In conclusion, this study demonstrated that five isolates of sponge-associated bacteria, belonging to the Bacillus genus, could suppress the attachment of Amphora sp., N. closterium, Sellaphora sp., and Stauroneis sp. Future work will be directed toward determining the chemical properties and the action mode of their metabolites. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 41076097, 41006097, 41106113, and 41271521), Key Project Chinese Ministry of Education (No. 211065), Natural Science Foundation of Jiangsu Province (No. BK2010322), Natural Science Foundation for College and University of Jiangsu Province (No. 11KJB170011), and SRF for ROCS, SEM for Xiaojian Zhou.
References Anand TP, Bhat AW, Shouche YS, Roy U, Siddharth J, Sarma SP (2006) Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India. Microbiol Res 161:252–262 Aumeier C, Menzel D (2012) Secretion in the diatoms. In: Vivanco JM, Baluska F (eds) Secretions and exudates in biological systems. Springer, Heidelberg, pp 221–250
123
1334 Becker K (1996) Exopolysaccharide production and attachment strength of bacteria and diatoms on substrates with different surface tensions. Microb Ecol 32:23–33 Burgess JG, Boyd KG, Armstrong E, Jiang Z, Yan L, Berggren M, May U, Pisacane T, Granmo A, Adams DR (2003) The development of a marine natural product-based antifouling paint. Biofouling 19:197–205 Daniel GF, Chamberlain AHL, Jones EBG (1980) Ultrastructural observations on the marine fouling diatom Amphora. Helgolinder Meeresunters 34:123–149 Dash S, Jin CL, Lee OO, Xu Y, Qian PY (2009) Antibacterial and antilarval-settlement potential and metabolite profiles of novel sponge-associated marine bacteria. J Ind Microbiol Biotechnol 36:1047–1056 Dash S, Nogata Y, Zhou XJ, Zhang YF, Xu Y, Guo XR, Zhang XX, Qian PY (2011) Poly-ethers from Winogradskyella poriferorum: antifouling potential, time-course study of production and natural abundance. Bioresour Technol 102:7532–7537 de Brouwer JFC, Cooksey KE, Wigglesworth-Cooksey B, Staal MJ, Stal LJ, Avci R (2006) Time of flight-secondary ion mass spectrometry on isolated extracellular fractions and intact biofilms of three species of benthic diatoms. J Appl Microbiol 65:562–572 Dobretsov SV, Qian PY (2002) Effect of bacteria from surface of the green seaweed Ulva reticulata on marine micro- and macrofouling. Biofouling 18:276–287 Dobretsov S, Qian PY (2004) The role of epibotic bacteria from the surface of the soft coral Dendronephthya sp. in the inhibition of larval settlement. J Exp Mar Biol Ecol 299:35–50 Fusetani N (2004) Biofouling and antifouling. Nat Prod Rep 21:94–104 Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can J Microbiol 8:229–239 Hentschel U, Schmid M, Wagner M, Fieseler L, Gernert C, Hacker J (2001) Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola. FEMS Microbiol Ecol 35:305–312 Hilaluddin F, Leaw CP, Lim PT (2011) Morphological observation of common pennate diatoms (Bacillariophyceae) from Sarawak estuarine waters. Annu Microsc 11:12–23 Hoagland KD, Rosowski JR, Gretz MR, Roemer SC (1993) Diatom extracellular polymeric substances: function, fine structure, chemistry, and physiology. J Phycol 29:537–566 Kennedy J, Baker P, Piper C, Cotter PD, Walsh M, Mooij MJ, Bourke MB, Rea MC, O’Connor PM, Ross RP, Hill C, O’Gara F, Marchesi JR, Dobson ADW (2009) Isolation and analysis of bacteria with antimicrobial activities from the marine sponge Haliclona simulans collected from Irish waters. Mar Biotechnol 11:384–396 Kon-ya K, Shimidzu N, Otaki N, Yokoyama A, Adachi K, Miki W (1995) Inhibitory effect of bacterial ubiquinones on the settling of barnacle, Balanus Amphitrite. Experientia 51:153–155 Kristensen JB, Meyer RL, Laursen BS, Shipovskov S, Besenbacher F, Poulsen CH (2008) Antifouling enzymes and the biochemistry of marine settlement. Biotechnol Adv 26:471–481 Kumar V, Rao D, Thomas T, Kjelleberg S, Egan S (2010) Antidiatom and antibacterial activity of epiphytic bacteria isolated from Ulva lactuca in tropical waters. World J Microbiol Biotechnol 27(7):1543–1549 Lee OO, Qian PY (2003) The chemical control of bacterial epiosis and larval settlement of Hydroides elegans in the red sponge Mycale adherens. Biofouling 19(Suppl):171–180 Lee OO, Wong YH, Qian PY (2009) Inter- and intraspecific variations of bacterial communities associated with marine sponges from
123
World J Microbiol Biotechnol (2014) 30:1325–1334 San Juan Island, Washington. Appl Environ Microbiol 75:3513–3521 Leflaive J, Ten-Hage L (2011a) Impairment of benthic diatom adhesion and photosynthetic activity by 2E, 4E-decadienal. Res Microbiol 162:982–989 Leflaive J, Ten-Hage L (2011b) Effects of 2E, 4E-decadienal on motility and aggregation of diatoms and on biofilm formation. Microb Ecol 61:363–373 Li Y, Liu CM, Yang JY, Gao YH, Li XS, Que GH, Lu JR (2011) Anti-biofouling properties of amphiphilic phosphorylcholine polymer films. Colloid Surf B Biointerfaces 85:125–130 Limna Mol VP, Raveendran TV, Parameswaran PS (2009) Antifouling activity exhibited by secondary metabolites of the marine sponge, Haliclona exigua (Kirkpatrick). Int Biodeter Biodegra 63:67–72 Lind JL, Heimann K, Miller EA, van Vliet C, Hoogenraad NJ, Wetherbee R (1997) Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans. Planta 203:213–221 Ma YX, Liu PL, Yu SB, Li DT, Cao SM (2009) Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates. Acta Ecol Sin 29:222–226 Mangano S, Michaud L, Caruso C, Brilli M, Bruni V, Fani R, Lo Giudice A (2009) Antagonistic interactions between psychrotrophic cultivable bacteria isolated from Antarctic sponges: a preliminary analysis. Res Microbiol 160:27–37 Mann DG, Evans KM, Chepurnov VA, Nagai S (2009) Morphology and formal description of Sellaphora bisexualis sp. nov. (Bacillariophyta). Fottea 9(2):199–209 Mizobuchi S, Abachi K, Miki W (1996) Antifouling polyhydroxysterols isolated from a Palauan octocoral of Sinularia sp. Fish Sci 62(1):98–100 Muscholl-Silberhorn A, Thiel V, Imhoff JF (2007) Abundance and bioactivity of cultured sponge-associated bacteria from the Mediterranean. Sea Microbial Ecol 55:94–106 Ortega-Morales B, Chan-Bacab M, Miranda-Tello E, Fardeau M, Carrero J, Stein T (2008) Antifouling activity of sessile bacilli derived from marine surfaces. J Ind Microbiol Biotechnol 35:9–15 Pabel CT, Vater J, Wilde C, Franke P, Hofemeister J, Adler B, Bringmann G, Hacker J, Hentschel U (2003) Antimicrobial activities and matrix-assisted laser desorption/ionization mass spectrometry of Bacillus isolates from the marine sponge Aplysina aerophoba. Mar Biotechnol 5:424–434 Qian PY, Xu Y, Fusetani N (2010) Natural products as antifouling compounds: recent progress and future perspectives. Biofouling 26(2):223–234 Santos OCS, Pontes PVML, Santos JFM, Muricy G, GiambiagideMarval M, Laport MS (2010) Isolation, characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil. Res Microbiol 161:604–612 Silkina A, Bazes A, Mouget JL, Bourgougnon N (2012) Comparative efficiency of macroalgal extracts and booster biocides as antifouling agents to control growth of three diatom species. Mar Pollut Bull 64:2039–2046 Taylor MW, Radax R, Steger D, Wagner M (2007) Sponge-associated microorganisms: evolution, ecology, and biotechnological potential. Microbiol Mol Boil R 71:295–347 Thiel V, Imhoff JF (2003) Phylogenetic identification of bacteria with antimicrobial activities isolated from Mediterranean sponges. Biomol Eng 20:421–423 Thomas H, Jo¨rn P (2009) Polyketide synthases of bacterial symbionts in sponges -evolution-based applications in natural products research. Phytochemistry 70:1841–1849 Wang G (2006) Diversity and biotechnological potential of the sponge-associated microbial consortia. J Ind Microbiol Biotechnol 33:545–551
ORIGINAL PAPER
Antidiatom activity of marine bacteria associated with sponges from San Juan Island, Washington Cuili Jin • Xiaying Xin • Siyu Yu • Jingjing Qiu Li Miao • Ke Feng • Xiaojian Zhou
•
Received: 3 September 2013 / Accepted: 9 November 2013 / Published online: 15 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Crude extracts of 52 marine bacteria associated with sponges, which were collected from the sea near San Juan Island, Washington, USA, were screened using diatom attachment assays against Amphora sp., Nitzschia closterium, Sellaphora sp. and Stauroneis sp. to investigate their antidiatom activities. Among these samples, five expressed strong anti-adhesion effects on all four tested diatoms. There was no negative effect observed from those five active samples on the growth of Amphora sp. Those five active samples were prepared from respective isolates, which all belonged to the genus Bacillus based on 16S rRNA gene sequencing analysis. The results of present study indicate that Bacillus may play important roles for sponges’ chemical defence against biofouling of diatoms and that the metabolites of Bacillus may be a potential source of natural antifouling compounds. Keywords Sponges Bacteria Bacillus Antidiatom activity Growth
Introduction Attachment and growth of unwanted living organisms on man-made surfaces is generally referred to as biofouling
C. Jin X. Xin S. Yu J. Qiu L. Miao K. Feng X. Zhou (&) College of Environmental Science and Engineering, Yangzhou University, 196# of Huayang West Street, Hanjiang District, Yangzhou, Jiangsu, China e-mail: [email protected] C. Jin L. Miao X. Zhou Marine Science and Technology Institute, Yangzhou University, 196# of Huayang West Street, Hanjiang District, Yangzhou, Jiangsu, China
(Fusetani 2004; Kristensen et al. 2008). Marine biofouling is an extensive phenomenon resulting in large costs to engineered structures in ocean environments, such as ship hulls and offshore platforms, due to increased manpower, fuel and materials consumption, and dry-docking time (Limna Mol et al. 2009). To date, the antifouling strategies in use are mainly based on antifouling paints containing toxic chemicals. Among these toxic compounds, tributyltin (TBT) is not only the most effective antifouling agent but also one of the most detrimental biocides ever introduced to marine environments because it is not readily degraded in the natural marine environments and acts on both target and non-target organisms. This property has led the International Maritime Organisation (IMO) to prohibit its application to ships, effective from 17 September 2008 (Qian et al. 2010). Since that prohibition, researchers have made great strides in finding new environmentally friendly chemicals as substitutes for TBT (Fusetani 2004). Many sessile macroalgae and animals are free from marine fouling, as they release chemicals influencing the settlement, growth, and survival of fouling organisms. This is known as chemical defence against biofouling (Qian et al. 2010). Many macroalgae and animals themselves lack both chemical and non-chemical defences and are believed to rely on the secondary metabolites produced by their associated bacteria (Mizobuchi et al. 1996; Kon-ya et al. 1995). Among those sessile organisms, marine sponges, as a significant component of benthic communities throughout the world, have been the focus of recent studies aiming for the isolation of natural antifouling compounds (Qian et al. 2010; Fusetani 2004). Sponges form close associations with a wide variety and a large number of microorganisms. Many natural compounds isolated from sponges have been reported to be
123
1326
World J Microbiol Biotechnol (2014) 30:1325–1334
Table 1 Phylogenic status of bacterial isolates from sponges collected from San Juan Island, Washington Sample number
Strain designation
Next relative by GenBank alignment
Sequence homology (%)
Accession no.
1
UST050418-092
Bacillus pumilus SAFR-032
100
NC009848.1
2
UST050418-097
Bacillus pumilus SAFR-032
99
NC009848.1
3
UST050418-099
Clostridium beijerinckii NCIMB 8052
98
NC009617.1
4
UST050418-100
Bacillus pumilus SAFR-032
99
NC009848.1
5
UST050418-101
Bacillus megaterium DSM319
100
NC014303.1
6
UST050418-172
Psychrobacter arcticus 273-4
98
NC007204.1
7
UST050418-178
Staphylococcus haenolyticus JCSC1435
94
NC007168.1
8
UST050418-192
Pseudoalteromonas sp. SM9913
99
NC014803.1
9
UST050418-202
Bacillus sp.1NLA3E
86
NC021171.1
10
UST050418-205
Bacillus sp.1NLA3E
92
NC021171.1
11 12
UST050418-206 UST050418-279
Bacillus sp.1NLA3E Pseudomonas aeruginosa PAO1
88 95
NC021171.1 NC002516.2
13
UST050418-281
Bacillus pumilus SAFR-032
99
NC009848.1
14
UST050418-291
Bacillus pumilus SAFR-032
99
NC009848.1
15
UST050418-292
Pseudomonas syringae pv syringae B728a
99
NC007005.1
16
UST050418-303
Psychrobacter cryohalolentis K5
97
NC007969.1
17
UST050418-315
Bacillus pumilus SAFR-032
99
NC009848.1
18
UST050418-319
Paenibacillus terrae HPL-003
95
NC016641.1
19
UST050418-323
Bacillus pumilus SAFR-032
100
NC009848.1
20
UST050418-326
Bacillus pumilus SAFR-032
98
NC009848.1
21
UST050418-328
Bacillus pumilus SAFR-032
99
NC009848.1
22
UST050418-329
Bacillus pumilus SAFR-032
98
NC009848.1
23
UST050418-331
Bacillus pumilus SAFR-032
87
NC009848.1
24
UST050418-333
Bacillus pumilus SAFR-032
99
NC009848.1
25
UST050418-337
Microbacterium testaceum StLB037
88
NC015125.1
26
UST050418-368
Cellulophaga lytica DSM 7489
98
NC015167.1
27 28
UST050418-380 UST050418-390
Bacillus pumilus SAFR-032 Cellulophaga lytica DSM 7489
99 98
NC009848.1 NC015167.1
29
UST050418-392
Cellulophaga lytica DSM 7489
99
NC015167.1
30
UST050418-393
Zobellia galactanivorans
98
NC015844.1 NC018877.1
31
UST050418-416
Bacillus thuringiensis Bt407
99
32
UST050418-421
Geobacillus sp. WCH70
96
NC012793.1
33
UST050418-480
Psychrobacter arcticus 273-4
99
NC007204.1
34
UST050418-555
Bacillus thuringiensis Bt407
100
NC018877.1
35
UST050418-566
Pseudovibrio sp. FO-BEG1
98
NC016642.1 NC018877.1
36
UST050418-573
Bacillus thuringiensis Bt407
99
37
UST050418-579
Pseudoalteromonas sp. SM9913
97
NC014803.1
38
UST050418-580
Bacillus weihenstephanensis KBAB4
99
NC010184.1
39
UST050418-581
Bacillus thuringiensis Bt407
96
NC018877.1
40
UST050418-584
Pseudoalteromonas sp. SM9913
99
NC014803.1
41
UST050418-585
Pseudoalteromonas sp. SM9913
97
NC014803.1
42 43
UST050418-649 UST050418-681
Bacillus megaterium DSM319 Bacillus pumilus SAFR-032
98 98
NC014303.1 NC009848.1
44
UST050418-683
Bacillus pumilus SAFR-032
99
NC009848.1
45
UST050418-700
Paenibacillus sp. Y412MC10
86
NC013406.1
46
UST050418-703
Bacillus thuringiensis Bt407
97
NC018877.1
47
UST050418-714
Lysinibacillus fsaphaericus C3-41
93
NC010382.1
48
UST050418-724
Exiguobacterium sibiricum 255-15
98
NC010556.1
123
World J Microbiol Biotechnol (2014) 30:1325–1334
1327
Table 1 continued Sample number
Strain designation
Next relative by GenBank alignment
Sequence homology (%)
Accession no.
49
UST050418-735
Arthrobacter phenathrenivorans Sphe3
99
NC015145.1
50
UST050418-753
Bacillus weihenstephanensis KBAB4
99
NC010184.1
51
UST050418-760
Bacillus sp. 1NLA3E
91
NC021171.1
52
UST050418-763
Paenibacillus terrae HPL-003
94
NC016641.1
similar to microbial products or even verified as microbially produced (Taylor et al. 2007). In addition, certain metabolites of bacteria isolated from marine sponges display antimicrobial activities (Thiel and Imhoff 2003; Anand et al. 2006; Kennedy et al. 2009; Santos et al. 2010), and there are also many impressive reports on the antifouling activity of sponge-associated bacteria that have been published in past decades (Qian et al. 2010; Fusetani 2004). The organic extracts of Winogradskyella poriferorum, isolated from sponges, have been reported to be capable of effectively inhibiting the larval settlement of two typical fouling invertebrates of Hydroides elegans and Amphibalanus amphitrite, as well as the biofilm formation of two bacterial species (Dash et al. 2009). Lee and Qian (2003) found that the bacteria associated with marine sponges were able to suppress larval settlement of the tubeworm H. elegans. From a bacterium separated from marine sponge, Kon-ya et al. (1995) isolated ubiquinone-8, which inhibited larval settlement of barnacle larvae. Polyethers from a sponge-associated bacterium displayed inhibitory effects on both biofilm formation of marine bacteria and larval settlement of macrofoulers (Dash et al. 2011). As a result, sponge-associated bacteria have been recognised as a good choice to search for new antifouling compounds not only because sponges are free from biofouling but also because of such advantages as short growing time, easy control in the production of target substances, and especially vast production capacity (Fusetani 2004; Taylor et al. 2007; Qian et al. 2010). Biofouling can be divided into macro-fouling and micro-fouling based on fouling organisms, with the former including macro-algae and marine invertebrates such as barnacles, and the latter mainly referring to biofilm caused by bacteria and diatoms (Kristensen et al. 2008). Virtually, all surfaces of aquatic habitats are covered with a biofilm, whether formed by bacteria or/and diatoms. Biofilms play a crucial role in the attachment and subsequent growth of macro-organisms, which further leads to the formation of a complex biofouling community (Hoagland et al. 1993). Diatoms are always the major component in these biofilms as long as light can reach down to these habitats (Aumeier and Menzel 2012; Leflaive and Ten-Hage 2011a, b). Therefore, the use of antidiatom is an essential and challenging step to avoid the subsequent process of macro-
fouling. However, possibly due to the inherent, technical challenges in performing antidiatom assays (Kumar et al. 2010), reports of natural products from sponge-associated bacteria with antidiatom activity are rare in comparison with tremendous progress in the fields of antibacterial and anti-larval activity (Dobretsov and Qian 2002; Kumar et al. 2010). The objective of the present study was to assess the effect of sponge-associated bacteria on adhesion by potent biofilm-forming diatoms, including Amphora sp., Nitzschia closterium, Sellaphora sp., and Stauroneis sp. In total, 52 isolates of sponge-associated bacteria were screened for their antidiatom activities against four species of fouling diatoms. The EC50 of the five isolates with high antidiatom activities were tested against Amphora sp. and N. closterium. In addition, the isolates that expressed inhibitive or inductive activity against diatom attachment are discussed on the genus level.
Materials and methods Tested sponge-associated bacteria Bacteria associated with 39 sponge specimens collected from San Juan Island, Washington, USA, were isolated and purified. The collection, isolation and purification process were performed by Prof. Pei-Yuan Qian’s group from the Hong Kong University of Science and Technology (HKUST), and the detailed methods were described by Lee et al. (2009). Out of these samples, 52 isolates were selected randomly to screen using diatom attachment assays. Among the tested 52 isolates, UST050418-333 and UST050418-683 were identified based on comparative sequence analysis of their 16S rDNA genes by Sangon Biotech (Shanghai) Co., Ltd. in our preliminary study. The 16S rRNA gene sequencing of the other 50 bacterial isolates were performed by Prof. Pei-Yuan Qian’s group in HKUST. Using the 16S rRNA gene sequences, the taxonomic hierarchy of the isolates was determined using a Bayesian classifier available online through the Ribosomal Database Project II (Kumar et al. 2010). These strains were identified as belonging to five major groups of bacteria: alpha and gamma Proteobacteria, Actinobacteria,
123
1328
World J Microbiol Biotechnol (2014) 30:1325–1334
A
160 150 140 130 120 110
R (%)
100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10111213141516171819 202122 23 24 252627 28293031 32 33343536 37383940 41424344 4546 47484950 51 52
160
B
150 140 130 120 110
R (%)
100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10111213141516171819 2021 22 23 24 252627 28293031 323334 353637383940 414243444546 474849 5051 52 160
C
150 140 130 120 110
R (%)
100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10111213 1415161718192021222324252627282930313233343536373839 40414243444546474849505152
Sample
123
World J Microbiol Biotechnol (2014) 30:1325–1334
1329
The crude extracts were prepared from 52 isolates of sponge-associated bacteria using the method described by Dash et al. (2009). First, 1 ml of the bacteria stocks in 50 % glycerol were individually inoculated into 10 ml of peptone–yeast extract medium (P-Y medium, containing 0.3 % of yeast extract and 0.5 % of peptone in artificial sea water (ASW) and incubated at 28 °C until reaching the exponential phase (60 h). Then, the individual bacterial culture was transfer to 800 ml of P-Y medium and incubated until the stationary phase at 28 °C and 120 rpm for 84 h. Finally, each bacterial culture was collected. Ethyl acetate (EA) with 5 % (v/v) acetone was added to each bacteria culture with a ratio of 1:1 (v/v) and was shaken vigorously for 1 h for complete extraction. The organic phase containing the bacterial metabolites was separated from the aqueous phase and dried on rotary evaporator (37 °C) to obtain the crude extract. Each crude extract was weighed and re-dissolved with dimethylsulfoxide (DMSO) for bioassays.
chamber at 23 °C under controlled illumination (100 lmol photons m2/s1 provided by cool-white fluorescent lamps with a 12 h:12 h light: dark cycle). Prior to the assays, diatom films were collected from the culture flask, suspended in ASW, washed with ASW by centrifugation twice, re-suspended in ASW, and adjusted to suitable density by addition of ASW. The diatom attachment assays were run in 24-well plates, each well of which contained 1 ml of algal suspension in ASW with addition of crude extract dissolved in DMSO. The final concentration of the crude extract in the well was 100 lg/ml, and the DMSO content was 0.5 % (v/v). The control received 1 ml of algal suspension in ASW with 0.5 % (v/v) DMSO. Algal concentrations in the ASW were adjusted using a blood counting chamber with comparable biomass: 5 9 105 cells/ml for N. closterium, 1 9 105 cells/ml for the others. Plates were incubated for 24 h at 23 °C with 100 lmol photons m2/s1 illumination and a 12 h:12 h light: dark cycle. Then, the well contents were gently shaken by pipetting, and non-adherent cells were removed. The remaining cells, still fixed to the bottom of the wells, were counted using an inverted microscope (Nikon ECLIPSE TS 100). At least triplicates were tested for each treatment. The results were expressed as the ratios (R) in terms of percentages for each sample: R(%) = (adherent cell number of treatment)/(mean adherent cell number of controls) (Leflaive and Ten-Hage 2011a). The effects of samples were classified according to the method described by Ma et al. (2009) with slight modification. Crude extracts with R C 110 % were classified as ‘inductive’. Those with R B 10 % were classified as ‘inhibitive’, and the remaining with 10 % \ R\110 % were classified as ‘neutral’ (Ma et al. 2009). When calculating the EC50 of active isolates, the inhibition percentage was equal to (100 %-R).
Diatom attachment assays
Diatom growth assays
Four benthic diatoms were used in the diatom attachment assays. The diatoms Amphora sp., N. closterium, Sellaphora sp., and Stauroneis sp. were obtained from the Key Laboratory of Mariculture, Ministry of Education, Ocean University of China. Among of them, the genus Amphora is pre-eminent amongst the fouling diatoms and has a cosmopolitan distribution and an ability to grow in a wide range of conditions (Daniel et al. 1980). N. closterium is a common marine benthic diatom in the intertidal zone and often used in antidiatom assays (Li et al. 2011). Sellaphora sp. and Stauroneis sp. were isolated from natural biofilms and identified by scanning electron microscopy (SEM). The diatoms were maintained in 250 ml Erlenmeyer flasks in ASW-based Guillard’s f/2 culture medium (Guillard and Ryther 1962) in a temperature-controlled
This experiment was designed to determine the effect of crude extracts on Amphora sp. growth. The test was run in triplicate on 96-well microplates, each well of which contained 200 ll of exponentially growing cells of Amphora sp. at a concentration of 1 9 105 cells/ml in ASW. Then, the tested crude extracts, with a final concentration of 100 lg/ml, were added into the wells, before the plates were incubated under the same conditions as those in attachment assays. Algal growth was determined by measurements of optical density (OD, at 690 nm, by 318 Microplate reader, Shanghai Sanco, China) at the initial time and the 3rd day (Leflaive and Ten-Hage 2011b). The growth rate of Amphora sp. was calculated as l = (lnN2 - lnN1)/(t2 - t1), where N1 and N2 were OD690, at times t1 and t2, respectively (Silkina et al. 2012).
b Fig. 1 Antidiatom effect of 52 bacterial samples against N. closte-
rium, Stauroneis sp. and Sellaphora sp. The concentration of the crude extracts in all treatments was 100 lg/ml. R was calculated as (adherent cell number of treatment) divided by (mean adherent cell number of controls) and is shown as a percentage. Data plotted are the means ? SD of at least three replicates. a–c N. closterium, Stauroneis sp. and Sellaphora sp., respectively. Crude extracts with an R higher than 110 % were classified as ‘inductive’, R lower than 10 % ‘inhibitive’, and otherwise, R was classified as ‘neutral’. The control for diatom attachment (not shown in figure) is representative of no inhibition (i.e., 100 % attachment)
Firmicutes and Bacteroides. Detailed information is listed in Table 1. Crude extracts preparation of bacteria
123
1330
Data analysis The growth rate of Amphora sp. was analysed with oneway ANOVA followed by the LSR (Least significant range) test. In all cases, the thresholds for significance were 1 % and 5 %.
World J Microbiol Biotechnol (2014) 30:1325–1334
found to inhibit the attachment of at least one of the tested three diatom species. Importantly, there were 5 samples that inhibited the attachment of all tested three diatoms simultaneously. These samples were No. 4, 17, 24, 27 and 44. Antidiatom activities screening using Amphora sp.
Antidiatom activities screening using N. closterium, Stauroneis sp. and Sellaphora sp. As shown in Fig. 1a, in total, 52 crude extracts of spongeassociated bacteria were subjected to an antidiatom assay against N. closterium. For tested samples, a concentration of 100 lg/ml of all crude extracts greatly impaired the adhesion of N. closterium compared with the controls. In total, 11 samples (21 % of the all tested samples), including No. 2, 4, 13, 17, 24, 26, 27, 28, 29, 35 and 44, expressed strong antidiatom activity and inhibited N. closterium attachment with an R lower than 10 %. Different than in the assays using N. closterium, crude extracts expressed two types of effects against Stauroneis sp. in the assays. For Stauroneis sp., a total of 9 crude extracts, including No. 2, 4, 17, 19, 22, 24, 27, 35 and 44 (17 % of tested samples) displayed strong attachment inhibition effects. At the same time, 8 samples (No. 8, 9, 12, 18, 20, 31, 37 and 48) induced diatom adhesion with an R higher than 110 %. For example, sample No. 20 promoted diatom attachment at level higher than in the controls at the concentration of 100 lg/ml with an R as high as 130 % (Fig. 1b). Similar to Stauroneis sp., crude extracts of spongeassociated bacteria displayed both inductive and inhibitive effects on the diatom adhesion of Sellaphora sp. For the tested samples, crude extracts at a concentration of 100 lg/ml greatly inhibited attachment of Stauroneis sp. compared with those in the controls. Among the samples, 6 samples (12 % of tested samples, including No. 4, 5, 17, 24, 27 and 44) inhibited diatom attachments with an R lower than 10 %. In addition, 7 samples (No. 7, 14, 16, 34, 40, 41 and 46) strongly promoted Sellaphora sp. adhesion with an R higher than 110 % (Fig. 1c). There were eight and seven samples that displayed inductive effects on Stauroneis sp. and Sellaphora sp., respectively. These 15 samples promoted diatom attachment with an R higher than 110 %. However, none of these inductive samples was effective in promoting the attachment of both Stauroneis sp. and Sellaphora sp. In addition, there were 11, 9 and 6 samples found that displayed inhibiting effects against N. closterium, Stauroneis sp., and Sellaphora sp., respectively. In total, 14 samples were
123
To verify their broad inhibitive activities, the above 5 effective samples, which were active against all tested three diatoms, were tested against one of the most often tested fouling diatom species, Amphora sp. As shown in Fig. 2, all five samples were effective against Amphora sp. at a concentration of 100 lg/ml. All these five samples inhibited diatom adhesion with an R lower than 5 %. The highest R for these five samples was only approximately 4 %, and the most active sample with the lowest R was 1 % for sample No. 4. Therefore, these five samples displayed a strong ability to inhibit the attachment of not only N. closterium, Stauroneis sp. and Sellaphora sp. but also the most often tested fouling diatom species, Amphora sp. Antidiatom effects of 5 active samples against N. closterium and Amphora sp. Crude extracts with descending concentrations of the 5 active samples were further tested against two representative diatoms, N. closterium and Amphora sp. The extracts were tested at concentrations ranging from 100 to 0.39 lg/ml using diatom attachment assays performed in 24-well plates for 24 h as described previously. The antidiatom activities weakened with reduced concentration. The dramatic weakening in the activities were observed in a relatively
10 9 8 7 6
R (%)
Results
5 4 3 2 1 0
4
17
24
27
44
Sample
Fig. 2 Antidiatom effect of 5 active bacterial samples against Amphora sp. The diatom Amphora sp. was employed in this antidiatom bioassay. The concentration of the crude extracts in all treatments was 100 lg/ml. R was calculated as (adherent cell number of treatment) divided by (mean adherent cell number of controls) and was as a percentage. The data plotted are the means ? SD of at least three replicates. The control for diatom attachment (not shown in figure) is representative of no inhibition (i.e., 100 % attachment)
World J Microbiol Biotechnol (2014) 30:1325–1334 No.4 EC50=3.22 µg/ml
100
No.4 EC50=15.40 µg/ml
80
R (%)
R (%)
1331
60 40 20 0
50.00
25.00
12.50
6.25
3.13
1.56
100 80 60 40 20 0
50.00
25.00
12.50
6.25
100 80 60 40 20 0
R (%)
R (%)
No.17 EC50=5.62 µg/ml
50.00
25.00
12.50
6.25
3.13
80
20 6.25
1.56
0.78
0.39
3.13 No.24 EC50=12.53 µg/ml
20
No.27 EC50=3.88 µg/ml
80
100.00
50.00
25.00
12.25
60 40 20
6.25 No.27 EC50=19.32 µg/ml
100
R (%)
R (%)
6.25
40 12.50
100
80 60 40
50.00
25.00
12.50
6.25
3.13
20
1.56
0 No.44 EC50=8.39 µg/ml
R (%)
R (%)
12.50
60
0
100 80 60 40 20 0
25.00
80
R (%)
\
40
0
50.00
100
60
0
No.17 EC50=21.19 µg/ml
No.24 EC50=1.47 µg/ml
100
R (%)
1.56
100 80 60 40 20 0
3.13
100.00
50.00
25.00
12.50
6.25
Concentration (µg/ml)
Fig. 3 Antidiatom effect of 5 active bacterial samples against N. closterium. The diatom N. closterium was employed in this antidiatom bioassay. R was calculated as (adherent cell number of treatment) divided by (mean adherent cell number of controls) and is shown as a percentage. The data plotted are the means ? SD of at least three replicates. EC50 values were calculated based on the inhibitory rate, which was 100 %-R(%) and are shown in the right-top of each sub-figure labelled with the respective sample number
narrow concentration ranges for each sample and are described in Fig. 3 for N. closterium and Fig. 4 for Amphora sp. In Fig. 3, the R became higher than 50 % when the concentration was lower than 6.25, 6.25, 1.56, 6.25, and 12.50 lg/ml for samples No. 4, 17, 24, 27, and 44, respectively. As a result, the EC50 values were calculated for each sample, and the lowest value was 1.47 lg/ml for sample No. 24, whereas the highest value was 8.39 lg/ml for sample No. 44. The EC50 values for the other samples, No. 4, 17 and 27, were 3.22, 5.62 and 3.88 lg/ml,
100 80 60 40 20 0
50.00
25.00
12.50
6.30
3.13 No.44 EC50=21.07 µg/ml
100.00
50.00
25.00
12.50
6.25
Concentration (µg/ml)
Fig. 4 Antidiatom effect of 5 active bacterial samples against Amphora sp. The diatom Amphora sp. was employed in this antidiatom bioassay. R was calculated as (adherent cell number of treatment) divided by (mean adherent cell number of controls) and is shown as a percentage. The data plotted are the means ? SD of at least three replicates. The EC50 values were calculated based on the inhibitory rate, which was 100 %- R(%) and were shown on the righttop of each sub-figure labelled with the respective sample number
respectively. In Fig. 4, the R became higher than those in Fig. 3 for N. closterium at the same concentration for each sample. As the result, all of the EC50 against Amphora sp. were much higher than those for N. closterium and were calculated as 15.40, 21.19, 12.53, 19.32, and 21.07 lg/ml for samples No. 4, 17, 24, 27 and 44, respectively. Among these samples, sample No. 4 was the most effective at inhibiting adhesion of Amphora sp.
123
1332
World J Microbiol Biotechnol (2014) 30:1325–1334
0.25 **
**
0.2
**
** *
µ
0.15 0.1 0.05 0
4
17
24
27
44
Control
Sample
Fig. 5 Effect on diatom growth by 5 active bacterial samples against Amphora sp. The diatom Amphora sp. was employed in this growth bioassay. The concentration of the crude extracts in all treatments was 100 lg/ml. The results are expressed as the growth ratio (l). The data plotted are the means ? SD of at least three replicates. Data that are significantly different according to the LSR test (one-way ANOVA) are indicated by an asterisk above the bars. Single and double asterisks indicate statistical significance at the 0.05 and \0.01 levels, respectively
Effects on diatom growth by five active samples against Amphora sp. To test whether the antidiatom activity of five active samples is based on inhibiting diatom growth, algal growth assays were conducted against Amphora sp. No negative effect on algal growth was detected. The growth rate of Amphora sp. treated with the five samples was significantly higher than that of the control. An LSR test revealed that 4 samples (all but No. 44) resulted in the algal growth rate being significantly higher than the control (P \ 0.01). The growth rate of sample No. 44 was also higher than the control (P \ 0.05) (Fig. 5). All these 5 active samples prepared from respective isolates strongly inhibited diatom attachment without negative effects on diatom growth. These 5 active isolates were identified as belonging to the Bacillus genus based on 16S rRNA gene sequencing.
Discussion The antidiatom effects of the crude extracts from the sponge-associated bacterial isolates were examined in this study. The results in Fig. 1 verified that many of the metabolites of sponge-associated bacteria affect diatom settlement. Sponges are free from biofouling because of their chemical defence, which is closely related to their associated bacteria (Thomas and Jo¨rn 2009; Kennedy et al. 2009). The large community of microorganisms can compose up to 50 % volume of the sponge tissue, the density of which exceeds that of bacteria in seawater by two–three orders of magnitude (Wang 2006; Lee et al. 2009). Sponge-
123
associated bacteria have been screened for new antifouling natural products for many years, and some positive results have been found (Fusetani 2004). These studies also have verified that it is possible to utilise sponge-associated bacteria as a promising resource for the isolation of effective antifouling compounds (Qian et al. 2010). Most studies focus on the anti-microbiological and anti-larval settlement activities (Fusetani 2004). Diatoms are important first settlers on new surfaces, and prevention of diatom biofouling is a crucial and difficult step in antifouling research (Hoagland et al. 1993; Silkina et al. 2012). However, information concerning antidiatom activity by sponge-associated bacteria remains insufficient. Similar to the results obtained by Dobretsov and Qian (2004) and Kumar et al. (2010), both inductive and inhibitive effects of sponge-associated bacteria on diatom adhesion were found in this study, though there were differences in regards to specific diatom species. The inductive and inhibitive bacteria co-exist in sponges. These findings indicate that the integrated effect for a specific diatom is based on the relative abundance of bacterial species and their active products. The sponges benefit from both inductive- and inhibitive-associated bacteria because of their selective attraction or exclusion against specific diatoms. The inhibitive bacteria are most likely responsible for the anti-fouling functions. The inductive bacteria in sponges have been reported to play other important roles for the hosts, such as nitrogen-fixing bacteria providing the host organism with additional amounts of nitrogen or furnishing the host organisms with fixed carbon or nitrogen (Dobretsov and Qian 2004). In addition, the existence of different functional bacteria makes it possible to adjust the integrated functions according to varied conditions. Undoubtedly, such flexibility is advantageous for the host in the unsteady marine environment. The effects of a sample prepared from each isolate on different tested diatom species varied. For example, total of 21, 17, and 12 % of the tested samples were active against the attachment of N. closterium, Stauroneis sp. and Sellaphora sp., respectively. Some isolates even displayed an opposite effect against different tested diatoms. An extreme example was isolate 7, which displayed a neutral effect with an R of 46 % against N. closterium but relative inductive effects against Stauroneis sp. and Sellaphora sp. with R values of 109 and 147 %, respectively. This phenomenon is concordance with the properties of marine diatom settlement and adhesion varying between species (Li et al. 2011). Marine diatoms often attach to solid surfaces via adhesive extracellular polymeric substances (EPS) (Hoagland et al. 1993; de Brouwer et al. 2006; Becker 1996). EPS can undergo conformational changes to adapt to solid surfaces with different physicochemical properties to optimise the
World J Microbiol Biotechnol (2014) 30:1325–1334
adhesion process. These changes grant marine diatoms the ability to adhere to all types of solid surfaces, which consequently produces greater for antifouling techniques (Li et al. 2011; Silkina et al. 2012). In addition, most pennate diatoms possess a raphe, which correlates closely with the ability of the diatom to adhere to an inert substratum and to glide whilst maintaining adhesion. It is well documented that diatom cell gliding requires adhesion to the substratum at the raphe, and strands of EPS have been observed projecting from the raphe (Lind et al. 1997). Though the detailed relationship between the raphe and adhesion strength is not clear, it would be beneficial to screen multiple species of diatoms with different raphe types to determine effective antifouling substances that work against many species of diatoms. One reason is that the target diatom species respond with different sensitivity to a certain tested substance. The other reason is that antifouling applications are aimed at multiple species (Silkina et al. 2012). Thus far, target species in antidiatom studies have mostly been chosen from algal banks or natural biofilms mainly based on availability and maintenance (Kumar et al. 2010). The raphe systems vary for the diatoms used in the present report. The raphe branches of Sellaphora sp. have a groove on each side, and the raphe is straight with expanded external central raphe ends (Mann et al. 2009). The raphe system of N. closterium is traversed by a series of fibulae, which join directly to the valve face. The raphe of Stauroneis sp. is straight, whereas the central area is a ‘‘bow tie’’-shaped stauros composed of a non-striated, thickened central nodule that usually extends to the valve margin. The raphe of Amphora sp. is eccentric, lying close to the ventral margin, filiform, and dorsally arched (Daniel et al. 1980; Hilaluddin et al. 2011). The results from antidiatom assays with multiple species targets, especially those with different raphe types, would useful for solving diatom fouling problems in the future. Biofouling of marine structures, especially ship hulls, are characterised by two critical events: the irreversible adhesion of biofouling species and the proliferation of the microorganisms in biofilm (Kristensen et al. 2008). Accordingly, traditional antifouling paints rely on cytotoxic effects and new environmentally safe alternatives aim to interfere with the adhesion and/or growth of the fouling organisms (Kristensen et al. 2008; Leflaive and Ten-Hage 2011b). In the present study, all active tested isolates possessed anti-adhesion activity without anti-growth effect, which makes it possible to identify some active chemicals from these isolates that merely inhibit diatom without exerting any negative influence on normal algal growth. In this study, bacterial isolates were members of 15 genera that were affiliated with the Firmicutes (69 %), alpha and gamma Proteobacteria (17 and 2 %, respectively), Bacteroides (8 %), and the Actinobacteria (4 %). This
1333
bacterial diversity is greater than bacteria isolated from Antarctic sponges and Mediterranean sponges, as the former lack of Firmicutes and the latter lack Bacteroidetes (Mangano et al. 2009; Hentschel et al. 2001). In total, 80 % of the isolates belonged to genera Bacillus, Pseudoalteromonas, Psychrobacter, Cellulophaga and Paenibacillus. In total, seven genera expressed inductive effects, including Pseudoalteromonas, Bacillus, Staphylococcus, Pseudomonas, Psychrobacter, Paenibacillus and Exiguobacterium. In contrast, the genus Bacillus outstandingly and significantly inhibited attachment of diatoms. Marine Bacillus species are often isolated from sediments and invertebrates. The species of this genus are known to generate spores under adverse conditions, such as low nutrient availability, irradiation and chemical disinfectants (Pabel et al. 2003). Metabolites produced by members of the genus Bacillus are known to have various biological activities, such as antimicrobial, antifungal, and generally cytotoxic properties (MuschollSilberhorn et al. 2007; Ortega-Morales et al. 2008). The epibiotic bacteria of Bacillus isolated from different host organisms have been demonstrated to be able to inhibit the formation of bacteria biofilm, the settlement of barnacle B. amphitrite larvae and algal spores of Ulva lactuca (Burgess et al., 2003). It has been reported that an isolate of Bacillus genus from U. lactuca had antidiatom activity against the pennate diatom Cylindrotheca fusiformis (Kumar et al. 2010). In the present study, members of the Bacillus genus isolated from sponges significantly suppressed multispecies diatom attachment. The results provide solid evidence that Bacillus species are a good source for the isolation of potentially new antifouling agents. In conclusion, this study demonstrated that five isolates of sponge-associated bacteria, belonging to the Bacillus genus, could suppress the attachment of Amphora sp., N. closterium, Sellaphora sp., and Stauroneis sp. Future work will be directed toward determining the chemical properties and the action mode of their metabolites. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 41076097, 41006097, 41106113, and 41271521), Key Project Chinese Ministry of Education (No. 211065), Natural Science Foundation of Jiangsu Province (No. BK2010322), Natural Science Foundation for College and University of Jiangsu Province (No. 11KJB170011), and SRF for ROCS, SEM for Xiaojian Zhou.
References Anand TP, Bhat AW, Shouche YS, Roy U, Siddharth J, Sarma SP (2006) Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India. Microbiol Res 161:252–262 Aumeier C, Menzel D (2012) Secretion in the diatoms. In: Vivanco JM, Baluska F (eds) Secretions and exudates in biological systems. Springer, Heidelberg, pp 221–250
123
1334 Becker K (1996) Exopolysaccharide production and attachment strength of bacteria and diatoms on substrates with different surface tensions. Microb Ecol 32:23–33 Burgess JG, Boyd KG, Armstrong E, Jiang Z, Yan L, Berggren M, May U, Pisacane T, Granmo A, Adams DR (2003) The development of a marine natural product-based antifouling paint. Biofouling 19:197–205 Daniel GF, Chamberlain AHL, Jones EBG (1980) Ultrastructural observations on the marine fouling diatom Amphora. Helgolinder Meeresunters 34:123–149 Dash S, Jin CL, Lee OO, Xu Y, Qian PY (2009) Antibacterial and antilarval-settlement potential and metabolite profiles of novel sponge-associated marine bacteria. J Ind Microbiol Biotechnol 36:1047–1056 Dash S, Nogata Y, Zhou XJ, Zhang YF, Xu Y, Guo XR, Zhang XX, Qian PY (2011) Poly-ethers from Winogradskyella poriferorum: antifouling potential, time-course study of production and natural abundance. Bioresour Technol 102:7532–7537 de Brouwer JFC, Cooksey KE, Wigglesworth-Cooksey B, Staal MJ, Stal LJ, Avci R (2006) Time of flight-secondary ion mass spectrometry on isolated extracellular fractions and intact biofilms of three species of benthic diatoms. J Appl Microbiol 65:562–572 Dobretsov SV, Qian PY (2002) Effect of bacteria from surface of the green seaweed Ulva reticulata on marine micro- and macrofouling. Biofouling 18:276–287 Dobretsov S, Qian PY (2004) The role of epibotic bacteria from the surface of the soft coral Dendronephthya sp. in the inhibition of larval settlement. J Exp Mar Biol Ecol 299:35–50 Fusetani N (2004) Biofouling and antifouling. Nat Prod Rep 21:94–104 Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can J Microbiol 8:229–239 Hentschel U, Schmid M, Wagner M, Fieseler L, Gernert C, Hacker J (2001) Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola. FEMS Microbiol Ecol 35:305–312 Hilaluddin F, Leaw CP, Lim PT (2011) Morphological observation of common pennate diatoms (Bacillariophyceae) from Sarawak estuarine waters. Annu Microsc 11:12–23 Hoagland KD, Rosowski JR, Gretz MR, Roemer SC (1993) Diatom extracellular polymeric substances: function, fine structure, chemistry, and physiology. J Phycol 29:537–566 Kennedy J, Baker P, Piper C, Cotter PD, Walsh M, Mooij MJ, Bourke MB, Rea MC, O’Connor PM, Ross RP, Hill C, O’Gara F, Marchesi JR, Dobson ADW (2009) Isolation and analysis of bacteria with antimicrobial activities from the marine sponge Haliclona simulans collected from Irish waters. Mar Biotechnol 11:384–396 Kon-ya K, Shimidzu N, Otaki N, Yokoyama A, Adachi K, Miki W (1995) Inhibitory effect of bacterial ubiquinones on the settling of barnacle, Balanus Amphitrite. Experientia 51:153–155 Kristensen JB, Meyer RL, Laursen BS, Shipovskov S, Besenbacher F, Poulsen CH (2008) Antifouling enzymes and the biochemistry of marine settlement. Biotechnol Adv 26:471–481 Kumar V, Rao D, Thomas T, Kjelleberg S, Egan S (2010) Antidiatom and antibacterial activity of epiphytic bacteria isolated from Ulva lactuca in tropical waters. World J Microbiol Biotechnol 27(7):1543–1549 Lee OO, Qian PY (2003) The chemical control of bacterial epiosis and larval settlement of Hydroides elegans in the red sponge Mycale adherens. Biofouling 19(Suppl):171–180 Lee OO, Wong YH, Qian PY (2009) Inter- and intraspecific variations of bacterial communities associated with marine sponges from
123
World J Microbiol Biotechnol (2014) 30:1325–1334 San Juan Island, Washington. Appl Environ Microbiol 75:3513–3521 Leflaive J, Ten-Hage L (2011a) Impairment of benthic diatom adhesion and photosynthetic activity by 2E, 4E-decadienal. Res Microbiol 162:982–989 Leflaive J, Ten-Hage L (2011b) Effects of 2E, 4E-decadienal on motility and aggregation of diatoms and on biofilm formation. Microb Ecol 61:363–373 Li Y, Liu CM, Yang JY, Gao YH, Li XS, Que GH, Lu JR (2011) Anti-biofouling properties of amphiphilic phosphorylcholine polymer films. Colloid Surf B Biointerfaces 85:125–130 Limna Mol VP, Raveendran TV, Parameswaran PS (2009) Antifouling activity exhibited by secondary metabolites of the marine sponge, Haliclona exigua (Kirkpatrick). Int Biodeter Biodegra 63:67–72 Lind JL, Heimann K, Miller EA, van Vliet C, Hoogenraad NJ, Wetherbee R (1997) Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans. Planta 203:213–221 Ma YX, Liu PL, Yu SB, Li DT, Cao SM (2009) Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates. Acta Ecol Sin 29:222–226 Mangano S, Michaud L, Caruso C, Brilli M, Bruni V, Fani R, Lo Giudice A (2009) Antagonistic interactions between psychrotrophic cultivable bacteria isolated from Antarctic sponges: a preliminary analysis. Res Microbiol 160:27–37 Mann DG, Evans KM, Chepurnov VA, Nagai S (2009) Morphology and formal description of Sellaphora bisexualis sp. nov. (Bacillariophyta). Fottea 9(2):199–209 Mizobuchi S, Abachi K, Miki W (1996) Antifouling polyhydroxysterols isolated from a Palauan octocoral of Sinularia sp. Fish Sci 62(1):98–100 Muscholl-Silberhorn A, Thiel V, Imhoff JF (2007) Abundance and bioactivity of cultured sponge-associated bacteria from the Mediterranean. Sea Microbial Ecol 55:94–106 Ortega-Morales B, Chan-Bacab M, Miranda-Tello E, Fardeau M, Carrero J, Stein T (2008) Antifouling activity of sessile bacilli derived from marine surfaces. J Ind Microbiol Biotechnol 35:9–15 Pabel CT, Vater J, Wilde C, Franke P, Hofemeister J, Adler B, Bringmann G, Hacker J, Hentschel U (2003) Antimicrobial activities and matrix-assisted laser desorption/ionization mass spectrometry of Bacillus isolates from the marine sponge Aplysina aerophoba. Mar Biotechnol 5:424–434 Qian PY, Xu Y, Fusetani N (2010) Natural products as antifouling compounds: recent progress and future perspectives. Biofouling 26(2):223–234 Santos OCS, Pontes PVML, Santos JFM, Muricy G, GiambiagideMarval M, Laport MS (2010) Isolation, characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil. Res Microbiol 161:604–612 Silkina A, Bazes A, Mouget JL, Bourgougnon N (2012) Comparative efficiency of macroalgal extracts and booster biocides as antifouling agents to control growth of three diatom species. Mar Pollut Bull 64:2039–2046 Taylor MW, Radax R, Steger D, Wagner M (2007) Sponge-associated microorganisms: evolution, ecology, and biotechnological potential. Microbiol Mol Boil R 71:295–347 Thiel V, Imhoff JF (2003) Phylogenetic identification of bacteria with antimicrobial activities isolated from Mediterranean sponges. Biomol Eng 20:421–423 Thomas H, Jo¨rn P (2009) Polyketide synthases of bacterial symbionts in sponges -evolution-based applications in natural products research. Phytochemistry 70:1841–1849 Wang G (2006) Diversity and biotechnological potential of the sponge-associated microbial consortia. J Ind Microbiol Biotechnol 33:545–551
