thealterationsofbiofilmformationandepscharacteristicsofa diatom...

Research ArticleThe Alterations of Biofilm Formation and EPS Characteristics of aDiatom by a Sponge-Associated Bacterium Psychrobacter sp

Xiaojian Zhou 12 Jie Meng1 Zhaowei Yu1 Li Miao 1 and Cuili Jin 12

1College of Environmental Science and Engineering Yangzhou University No 196 Huayang West Street Hanjiang DistrictYangzhou Jiangsu China2Marine Science and Technology Institute Yangzhou University No 196 Huayang West Street Hanjiang DistrictYangzhou Jiangsu China

Correspondence should be addressed to Xiaojian Zhou zhouxiaojianyzueducn and Cuili Jin cljinyzueducn

Received 3 November 2017 Revised 26 February 2018 Accepted 8 March 2018 Published 24 June 2018

Academic Editor Joaquim Ruiz

Copyright copy 2018 Xiaojian Zhou et al is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A sponge-associated bacterium which was identified as Psychrobacter sp in this study was found with high activity againstbiofilm formation of benthic diatoms including Amphora sp Nitzschia closterium Nitzschia frustulum and Stauroneis sp eactivity against diatom biofilm formation by the tested strain was confirmed mostly in the culture supernatant and could beextracted using organic solvents Treatment with its supernatant crude extract significantly reduced the cells of Stauroneis spforming biofilm and slightly increased the cells floating in the culturemedium which results in the ratio of biofilm cellfloating cellaltering from 0736 in control to 0414 in treatment Use of the supernatant crude extract led to increased production of ex-tracellular polymeric substances (EPSs) by diatom Stauroneis sp from 1666 to 4159 (gg cell dry weight) e increase in EPSproduction was mainly contributed by soluble EPS (SL-EPS) and followed by the EPS that was tightly bound to biofilm cells (BF-TB-EPS) In addition the supernatant crude extract caused significant changes in the monosaccharides composition of the EPS ofStauroneis sp Specifically glucuronic acid (Glc-A) and N-acetyl-D-glucosamine (Glc-NAc) in BF-TB-EPS were 55 fold de-creased and 1219 fold increased respectively Based on our findings we proposed that these changes in monosaccharidescomposition might lead to a decreased biofilm formation efficiency of diatom

1 Introduction

Biofouling of ship hulls by biofilms of algae and bacteria andby larger organisms for example barnacles and mussels(macrofouling) causes large economic losses worldwide [1]In addition to increased drag and decreased speed by 8ndash21of ships biofilms on ship hulls also contribute for inducingsubsequent macrofouling [2] Every surface that is immersedin seawater becomes rapidly covered with an unavoidablebiofilm However biofilm formation is a complex multistageprocess and not yet thoroughly investigated [3] e mi-crobial communities of biofilms are commonly dominatedby diatoms in light-penetrating marine habitats [4ndash6]Benthic diatoms which are capable of forming biofilm evenon the most fouling resistant surfaces play a key role in thebiofilm development [7] erefore interruption of diatom

biofilm formation is an essential and challenging step forresolving the biofouling problem [8 9]

Photoautotrophic biofilms are composed of cells that areembedded in extracellular polymeric substances (EPSs) [10]Marine benthic diatoms secrete large amounts of EPSs intothe surrounding environment as much as approximately30ndash60 of photoassimilated carbon [11 12] ese EPSs aremainly composed of carbohydrates and proteins and playvarious roles in the life of diatoms EPS can act as a diffusivebarrier against chemicals physical stress dehydration andpredator grazing EPS can also cause the retention of exo-enzymes or cellular metabolites and nutrient sequestrationfrom aquatic environments EPS also promotes sorption ofcompounds cell-to-cell communication and formation ofmicrocolonies [11 13 14] Besides EPS acts as a type of gluethat is primarily used by benthic diatoms for aggregation and

HindawiScientificaVolume 2018 Article ID 1892520 10 pageshttpsdoiorg10115520181892520

gripping to substrates and is also involved in the motilitysystem and substratum adhesion of diatoms [15ndash17] ere-fore EPS is proposed to be a key component for diatom cells toform biofilms on the substratum beneath water [16]

In marine environments sponges are commonlyfouling-free due to their chemical defenses against foulingorganisms [18 19] Many natural compounds were isolatedfrom sponges and found to be similar to those from mi-croorganisms Moreover some of them were verified beingmicrobially produced by sponge-associated microorganisms[20] Strong antimicrobial and antifouling activities in-cluding inhibition against the biofilm formation and larvalsettlement of typical fouling organisms such as Hydroideselegans or Amphibalanus amphitrite were found among themetabolites of sponge-associated bacteria [9 18 19 21ndash28]However few study on how the metabolites of active bac-terial strains affect diatom biofilm formation and whethertreatment with these metabolites provokes changes in di-atom EPS is reported Currently the active natural productsfrom sponge-associated bacteria against diatom biofilmformation have not been sufficiently investigated yet [29]

Our previous studies highlighted the strong activitiesagainst biofilm formation of several diatom species by crudeextracts from the whole culture of some sponge-associatedbacteria [29 30] Among the tested diatoms the benthicdiatom Stauroneis sp was used as a model organism in thisstudy because its gliding mechanism and monosaccharidecomposition of the EPS are very clear [31 32] In the presentstudy we found another active sponge-associated strain andwould like to clarify several questions (1) what portion ofcrude extracts from active bacterial culture is responsible forthe activity against diatom biofilm formation (2) whetherthe active strain inhibits diatom biofilm formation by al-tering cell distribution in biofilm and planktonic phaserather than by reducing total cell biomass in the wholeculture and (3) whether correlating responses in the EPSproduction or composition occur with the poor biofilmformation efficiency of diatoms when treated by the activestrain

2 Materials and Methods

21 Bacterial Culture Sponge-associated bacteria wereoriginally collected from San Juan Island WashingtonCollection isolation and purification of the strains wereperformed by Coastal Marine Laboratory in Hong KongUniversity of Science and Technology (HKUST) as describedpreviously [33] One strain of UST050418-708 was selectedfor this study since we found that it can be active againstdiatom biofilm formation [29]

A stock culture of strain UST050418-708 was stored inthe Marine Science and Technology Institute YangzhouUniversity e stock solution (1ml) was inoculated in10ml of the peptone-yeast extract medium (P-Y mediumcontaining 03 yeast extract and 05 peptone in artificialseawater (ASW)) and incubated for about two days to theexponential phase at 23degC with shaking at 120 rpm ebacterial culture in the exponential phase was then in-oculated in 500ml of the P-Y medium and incubated for

three days in the stationary phase under the same condi-tions and used for extract preparation [29]

22 Strain Identification e genomic DNA of strainUST050418-708 was extracted and its 16S rDNA was am-plified and sequenced by Sangon Biotech (Shanghai) CoLtd as described by Jin et al [29]e obtained sequence wassubmitted to GenBank e strain was identified by com-paring the submitted sequence with those available inGenBank databases Similar sequences were aligned usingmultiple sequence alignment program MEGA Gaps andpositions with ambiguities were excluded from the phylo-genetic analysis Phylogenetic analysis was performed usingthe neighbour-joining method described by Li et al [34]

23 Extract Preparation from Bacterial Culture e crudeextract was collected from the bacterial culture as describedby Jin et al [29] In detail the extraction solvent of ethylacetate (EA) acetone 95 5 (vv) was added with a ratio ofextraction solvent bacterial culture 1 1 (vv) and shakenvigorously for 1 h After standing and stratification theorganic phase was separated and dried on a rotary evapo-rator (37degC) to obtain the whole culture crude extract Forthe preparation of supernatant crude extract the bacterialcells were separated from the culture medium by centrifu-gation (5880timesg 20min 15degC) and the supernatant wasthen extracted with the same extraction solvent and pro-cedure as above Each crude extract was dissolved indimethylsulfoxide (DMSO) for subsequent bioassays

24 Diatom Culture Four benthic diatoms Amphora spNitzschia closterium Nitzschia frustulum and Stauroneissp were obtained from the Key Laboratory of MaricultureMinistry of Education Ocean University of China

e diatoms were cultured by the method of Jin et al[29] In detail ASW-based Guillardrsquos f2 culture medium[35] was used and the culture conditions of 23degC 100 μmolphotons mminus2middotsminus1 illumination and light dark 12 h 12 hcycle were employed Diatom films collected prior to assayswere suspended and washed with ASW twice adjusted toapproximately 1times 105 cellsmiddotmlminus1 in ASW and used in fol-lowing diatom biofilm formation assays

25 Diatom Biofilm Formation Assays Using Crude ExtractsDiatom biofilm formation assays for crude extracts wereperformed in 24-well polystyrene plates (353047 BectonDickinson Labware) following the method described by Jinet al [29] Algal suspension in ASW (1ml) and the crudeextract dissolved in DMSO (50 μl) were added to each wellon the plate to achieve the final concentration of 100 μgmiddotmlminus1for the crude extract in the well For the control wells 1ml ofthe algal suspension in ASW and 50 μl DMSOwere includedAfter 24 h incubation at the above diatom culture condi-tions the floating cells were removed by pipetting and thebiofilm cells were then counted using an inverted micro-scope (Nikon ECLIPSE TS 100) Triplicates were done foreach treatment and control e inhibition ratios (R) were

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expressed in terms of the percentages for each sampleR () 1minus (biofilm cell number of treatment)(mean bio-film cell number of controls) [29 36]

26 DiatomBiofilm Formation Assays Using Various Portionsof the Bacterial Culture To examine the possible trophiccompetition between the living bacterial cells and diatomsthe benthic diatom Stauroneis sp was used in this experi-ment Diatom Stauroneis sp under optical and electronmicroscopes is shown in Figure 1 Prior to the bioassays thedensities of the suspended Stauroneis sp were adjusted usinga hemocytometer to 1times 105 cellsmiddotmlminus1 e bacterial isolateswere collected from stationary-phase cultures after 72 hincubations in the P-Y medium at 23degC and 120 rpm e

collected bacterial culture was centrifuged (20min 5880timesgand 15degC) e supernatant was collected and used as theldquosupernatantrdquo for the bioassay e pellet was resuspendedin the same volume of the fresh P-Y medium and used asldquocellsrdquo for the bioassay

Assays were conducted in 24-well polystyrene plates(353047 Becton Dickinson Labware) using two volumeratios (bacteria diatom volume ratios of 3 7 and 5 5)When the ratio was 3 7 each well contained 07ml of algalsuspension in the f2 medium and 03ml of bacterial aliquotsof the supernatant or cells When the ratio was 5 5 themixture in each well was composed of 05ml of the algalsuspension and 05ml of bacterial aliquots e total volumeof each well for both ratios was 1ml and controls contained03 or 05ml of the sterilized P-Ymedium instead of bacterialaliquots Plates were incubated for 24 h at the same diatomculture conditions mentioned above e contents in thewells were washed by gently pipetting algal cells still at-tached to the bottom of the wells were counted and theinhibition ratios (R) were calculated following the methoddescribed above [29 36]

27 Treatment of Growing Diatom Using Bacterial Superna-tant Crude Extract e benthic diatom Stauroneis sp wasused in this experiment e diatom was cultured in 2-Lconical flasks Each flask contained 1500ml of freshly in-oculated Stauroneis sp with a cell density of 1times 105 cellsmiddotmlminus1in the sterilized f2 medium Bacterial supernatant crudeextract dissolved in DMSO was added to a final concentrationof 150 μgmiddotmlminus1 at a DMSO content of 05 (vv) e controlincluded 05 (vv) DMSO onlye triplicated flasks for bothtreatment and control were incubated for 39 days at 23degC with100 μmol photons mminus2middotsminus1 and a 12 h 12 h light dark cycle

28 EPS Preparation from Diatoms Grown in the Presence ofBacterial Supernatant Crude Extract e EPS fractionationprocedures for the suspension fraction (cells floating in themedia) and biofilm fraction (cells embedded in biofilmattached to the flask bottom) were performed as described byXu et al [37] After completing the incubation all flasks wereshaken on a shaker at 70 rpm for 10min e suspensionfraction was carefully decanted and collected after shakingen ASW was added to the flask and diatom cells in thebiofilm were carefully scratched down and suspended edetailed methods for the EPS fractionation cell densitydetection and cell dry weight measurement were as follows

e suspension fraction of diatom culture was countedfor cell density after shaking using a hemocytometer undermicroscopy and then centrifuged at 1707timesg for 20min toseparate diatom cells and the supernatant e supernatantof suspension fraction was collected to measure the solubleEPS (SL-EPS) that is the EPS fraction that could be re-moved by soft perturbation e pellets were resuspended inASW and heated at 40degC overnight followed by three washeswith ASW and centrifugation at 1707timesg for 20min eASW washes were collected to measure the EPS tightlybound to floating cells (F-TB-EPS) and the final pellets were

(a)

(b)

(c)

Figure 1 Scanning electronmicroscope (a) and optical microscope((b) for valve view and (c) for girdle view) images of diatomStauroneis sp

Scientifica 3

placed in a 100degC oven and heated to constant weight tomeasure the mass of floating cells

For the biofilm fraction of diatom culture cells weresuspended in the same volume of ASW as the originalculture by vigorous shaking and counted for cell densityusing a hemocytometer under microscopy After beingcentrifuged at 1707timesg for 20min the supernatant wascollected to measure the EPS loosely bound to the biofilmcells (BF-LB-EPS) And the pellets were treated in the sameway as that for F-TB-EPS and the supernatant was collectedto measure EPS that tightly bound to biofilm cells (BF-TB-EPS) e final pellets were heated at 100degC until constantweight to measure the weight of biofilm cells

All the EPS fraction samples were precipitated with 3-foldvolumes of ethanol e solution was left overnight in therefrigerator (4degC) [38] e final precipitate was collected bycentrifugation and washed three times with 2-fold volumes ofacetone and dichloromethane subsequently to obtain thecrude EPS e crude EPS was dried under a stream of ni-trogen gas weighed and stored at minus20degC After deproteinationusing the Sevag method and desalting using dialysis (35 kDa)the purified EPS was obtained by rotary evaporation andfreeze-drying for determination of monosaccharide compo-sitions [39]

29 Determination of Monosaccharide Compositions in EPSe monosaccharide compositions of EPSs were determinedusing high-performance liquid chromatography (HPLC) afterderivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP)[40] As standards 11 monosaccharides were used mannose(Man) glucuronic acid (Glc-A) N-acetyl-D-glucosamine(Glc-NAc) xylose (Xyl) galactose (Gal) arabinose (Ara)fucose (Fuc) glucose (Glc) galacturonic acid (Gal-A)rhamnose (Rha) and glucosamine hydrochloride (GlcN)e HPLC system (L2000 Hitachi Japan) was equipped witha diode array detector (DAD L-2455 Hitachi Japan) installedin tandem at the outlet of the column (LaChrom ODS C185 microm 46mmtimes 250mm Hitachi Japan) and mounted withan ODS precolumn e used solvents were 83 methanoland a 17 potassium dihydrogen phosphate-sodium hy-droxide buffer solution (01M pH 86) at a fixed flow rate of07mlmiddotminminus1 at 25degC A volume of 10 microl of the sample wasinjected into the column using an autosampler (L-2200Hitachi Japan) and the UV absorption at λ 245 nm wasdetectedeHPLC analyses were performed at least twice foreach sample

e data were analysed by the method of Yang et al [41]to determine the monosaccharide ratio of each sample ecorrection factors (f12) and molar ratios (R12) betweenevery two monosaccharides ((1) and (2)) were calculatedusing the following equations respectively

f12 A2m2( 1113857

A1m1( 1113857 (1)

R12 f12 lowastA1prime

A2prime1113888 1113889 (2)

where A1 and A2 and m1 and m2 are the peak area andweight for two component monosaccharides in the standardsolution respectively and A1prime and A2prime are the peak areas forthe component monosaccharide of the tested samples [41]

e content of one of the identifiedmonosaccharides (X)is set as 1 and the mole contents of other monosaccharideswere calculated based on f12 and R12 between X and each ofothers e mole percentage of each monosaccharide wascalculated as its mole content divided by the sum of the molecontents of all identified monosaccharides

For diatoms treated by the supernatant crude extract of theactive strain the amplitude of variation () for each mono-saccharide in an EPS fraction was calculated as the variation(mole percentages) between the treatment and control dividedby the mole percentage in the control

210 Statistical Analysis All calculations were performedwith at least triplicate samples Statistical analyses werecarried out using the IBM SPSS statistics 22 e differencesamong treatments in each experiment were compared usingthe independent t-test or one-way analyses of variance(ANOVA) followed by the LSD test with a threshold forsignificance of 001

3 Results

31 Identification of Active Strain Genomic DNA of strainUST050418-708 was extracted and the 16S rDNA was PCRamplified and sequencede nearly complete 16S rRNA genesequence of strain UST050418-708 (1431bp) was obtained andsubmitted to GenBank with an accession number (MF179520)Comparative analysis of the 16S rRNA gene sequence withsequences deposited in GenBank using BLASTshowed that thestrain belong to the genus Psychrobacter and has a very highsimilarity (100) with Psychrobacter glacincola (Figure 2)erefore the strain was identified as Psychrobacter sp basedon the 16S rDNA sequence

32 gte Activity against Diatom Biofilm Formation by CrudeExtract fromWhole Bacterial Culture A crude extract fromwhole culture of the tested strain was prepared and used inthe diatom attachment assays At concentration of 100μgmiddotmlminus1the crude extract from the whole culture had very high activitiesagainst diatom biofilm formation and inhibited four diatomspecies from attaching to the bottom of 24-well plates withinhibition ratios (R) higher than 90 (Figure 3) ForN closteriumand Stauroneis sp the inhibition ratios were over 98

33gte Activity against Diatom Biofilm Formation by VariousPortions of the Bacterial Culture Since the whole cultureextract of tested strain inhibited diatom biofilm formationwith very high efficiencies the supernatant and cells fromthe bacterial culture (without extraction) were used in di-atom biofilm formation assays Regardless of the used ratiosbetween bacteria and diatom being 3 7 or 5 5 both twofractions of the bacterial culture showed activities againstdiatom biofilm formation (Figure 4) Among all treatments

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the supernatant showed the highest activity against diatombiolm formation with R 95 when the ratio betweenbacteria and algae was 3 7 (Figure 4) When the ratio wasadjusted to 5 5 the supernatant fraction maintained highactivity with R 93 us the supernatant of the testedstrain possessed higher activity against diatom biolmformation than the cells Statistical analysis indicated thatthe supernatant showed signicantly higher activity thancells and that the change in the ratio (bacteria diatom)signicantly ashyected the activities of the cells rather than thesupernatant

34 Eect of the Bacterial Supernatant Crude Extract on theGrowth and Biolm Formation of Diatom Since the su-pernatant was the most eshyective portion of the bacterialculture a supernatant crude extract was prepared and usedto treat the growing diatom of Stauroneis sp e totalweight of dry cells and cell density for the whole culture oftreated diatom showed minor decrease from 0578 g to

0504 g (by percentage of 128) and from 9557 times105mlminus1to 8409 times105mlminus1 (by percentage of 120) respectively(Table 1) Importantly the dry cell weight in the biolmfraction signicantly decreased from 0245 g (control) to0147 g (treatment) with a decrease percentage of 400 Atthe same time the dry cell weight in the suspension fractionincreased slightly with 72 (from 0333 g to 0357 g)without a signicant dishyerence

In the cases of cell density the similar tendency wasobserved as that of the dry cell weight e cell density ofthe biolm (detected by resuspending) reduced by 370from 4091times 105mlminus1 in the control to 2577times105mlminus1 inthe treatment while the cell density in culture suspension

Stauroneis sp84

86

88

90

92

94

96

98

100

102

Inhi

bitio

n ra

tio (R

)

Amphora spNitzschiaclosterium

Nitzschiafrustulum

Figure 3 Activities against diatom biolm formation by the crudeextract from the whole culture of Psychrobacter sp e inhibitingratios (R) were calculated after 24 h incubations Triplicates weretested for each treatment and control and the means and standarddeviations are shown as closed columns and bars respectively

A

B

A

C

0

20

40

60

80

100

120

Inhi

bitio

n ra

tio (R

)

CellSupernatant

Bacteriadiatom = 37Bacteriadiatom = 55

Figure 4 Activities against diatom biolm formation by bacteriumPsychrobacter sp with dishyerent portions e numbers of 7 3 or5 5 indicate the volume ratios with which diatom Stauroneis sp insuspension was mixed with the supernatant or cells suspension inthe fresh culture medium e inhibiting ratios (R) were calculatedafter 24 h incubations Triplicates were tested for each treatmentand control and the means and standard deviations are shown ascolumns and bars respectively Signicance was tested for eachtreatment separately Samedishyerent letters above the bars indicatenoa statistical dishyerence in determination by one-way analyses ofvariance (ANOVA) followed by LSD test (Plt 001) respectively

Legionella longbeachae NSW150 (NC013861)Methylococcus capsulatus strain Bath (NC002977)

Thalassolituus oleivorans MIL-1 (NC020888)

Marinobacter aquaeolei VT8 (NC008740)Moraxella catarrhalis RH4 (NC014147)

UST050418-708 (MF179520)Psychrobacter glacincola strain ANT9253 (AY167308)100

100

54

39

001

Figure 2 Phylogenetic tree based on 16S rDNA of strain UST050418-708 e evolutionary history was inferred using the neighbour-joining method e numbers at the nodes indicate the bootstrap values based on neighbour-joining analyses of 1000 sample data sets etree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree eevolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of basesubstitutions per site Bar of 001 represents per nucleotide position e numbers in parentheses are accession number of sequencesEvolutionary analyses were conducted in the MEGA6 software package

Scientica 5

increased by 67 from 5466times105mlminus1 to 5833times105mlminus1without a significant difference e decreases in the sum ofcell dry weight and cell density in the treatment with bac-terial supernatant crude extract were contributed by thesignificant reduction in the biofilm fraction

In the whole culture of diatom 42 of diatom cells (interms of cell dry weight) formed the biofilm in the controlTreatment with supernatant crude extract from the testedstrain significantly decreased the percentage of biofilm cellsto 29 whereas the percentage of floating cells increasedfrom 58 to 71 Accordingly the ratio of biofilmcellsfloating cells decreased from 0736 to 0414 For datadescribed by cell density the consistent results occurredDue to the good correlation between dry cell weight andcell density the subsequent results were expressed by drycell weight only

35 Effect of the Bacterial Supernatant Crude Extract on EPSProduction of Growing Diatom e EPS of diatom Staur-oneis sp grown in the presence of the supernatant crudeextract of the tested strain were fractionated and measurede treatment of supernatant crude extract led to significantincrease in the EPS dry weight (Table 2) with minor re-duction in total biomass (Table 1) Among different EPSfractions the treatment led to a higher proportion of SL-EPS(from 7400 to 9129) in the EPS distribution Takingthe slight variance of the biomass into account the EPSproduction per diatom cell dry weight was 1666 (gg celldry weight) in the control and increased to 4159 (gg celldry weight) with treatment (249-fold) (Table 2) e su-pernatant crude extract led the EPS production per diatom

biomass to increase especially for SL-EPS (308-fold higherthan control) e production of BF-TB-EPS per biomassalso significantly increased to 271 (gg cell dry weight)which was 176-fold higher than that of 154 (gg cell dryweight) in the control

36 Effect of the Bacterial Supernatant Crude Extract on theMonosaccharide Compositions of Diatom EPS Fractionse EPS fractions of Stauroneis sp were hydrolysed andsubjected to HPLC analysis e results of the control andtreatment (growing in the presence of the supernatant crudeextract of tested strain) are shown in Table 3 and Figure 5respectively

As shown in Table 3 the EPS of the untreated Stauroneissp included nine monosaccharides of Man GlcN Rha Glc-AGlc-NAc Glc Gal Xyl and Fuc which were identified bycomparison to standards e soluble EPS fraction and otherfractions showed qualitatively similar monosaccharidescompositions e major monosaccharide in the SL-EPSfraction was Xyl with significant levels of Gal Man Glc-Aand GlcN and slight levels of Fuc Glc and Glc-NAc edominant monosaccharide in each fraction varied for bothF-TB-EPS and BF-TB-EPS Glc was the dominant mono-saccharide with mole percentages of 379 and 610 re-spectively Xyl was the most abundant monomer of SL-EPSwith a mole percentage of 370 while Man was mostabundant in BF-LB-EPS with a mole percentage of 232

As shown in Figure 5 treatment with the supernatantcrude extract from the tested strain led to altered levels of allmonosaccharides Among the four fractions treatment withthe supernatant crude extract caused the largest changes in

Table 1 Cell distributions in the floating phase and biofilm of diatom Stauroneis sp incubated in the presence of bacterial supernatant crudeextract

Cell distributionControl Treatment

Dry weight (g) Cell density (times105mlminus1) Percentage () Dry weight (g) Cell density (times105mlminus1) Percentage ()Biofilm 0245plusmn 0014 4091plusmn 0218 42 0147plusmn 0016lowastlowast 2577plusmn 0255lowastlowast 29Floating 0333plusmn 0013 5466plusmn 0199 58 0357plusmn 0021 5833plusmn 0326 71Sum 0578plusmn 0008 9557plusmn 0124 mdash 0504plusmn 0010lowastlowast 8409plusmn 0157lowastlowast mdashBiofilmfloating 0736plusmn 0066 0750plusmn 0063 mdash 0414plusmn 0070lowastlowast 0444plusmn 0068lowastlowast mdashCell weights and cell densities were independently measured at least three times and the means and standard deviations are shown Independent t-test wasused to compare the control and treatment Percentages were calculated based on the means of cell weights and indicated the cell distribution between biofilmand floating phases lowastlowastPlt 001

Table 2 EPS production per biomass of diatom Stauroneis sp incubated in the presence of bacterial supernatant crude extract

FractionControl Treatment

SL-EPS F-TB-EPS

BF-LB-EPS

BF-TB-EPS Total SL-EPS F-TB-

EPSBF-LB-EPS

BF-TB-EPS Total

EPS dry weight (g) 713plusmn001

033plusmn005

179plusmn001

038plusmn002

963plusmn003

1913plusmn001lowastlowast

039plusmn005


040plusmn002


Percentage () 7400 346 1862 392 100 9129 187 494 190 100EPS production (gg cell dryweight)

1233plusmn036

100plusmn013

734plusmn048

154plusmn004

1666plusmn050


110plusmn012

705plusmn004



Dry weights of fractions were independently measured at least three times and the means and standard deviations are shown Percentages were calculatedbased on themeans of dry weights EPS production was calculated as the EPS weight divided by cell dry weight andmeans and standard deviations are shownIndependent t-test was used to compare the control and treatment lowastlowastPlt 001

6 Scientifica

BF-TB-EPS and the smallest in SL-EPS In the ninemonomers detected only four monomers altered in thesame direction in all four EPS fractions Glc-A and Galalways decreased and Xyl and Fuc increased in all EPSfractions Glc-NAc exhibited the largest increase (1219) inBF-TB-EPS followed by Man (667) in F-TB-EPS echanges in other monomers were less than 200

4 Discussion

In our previous study several strains with remarkable ac-tivity against diatom biolm formation of Amphora spNitzschia closterium Sellaphora sp and Stauroneis sp werescreened from a sponge-associated bacterial bank [29] Inextension screening the UST050418-708 strain was foundand identied as Psychrobacter sp in this study (Figure 2) Itsactivity against diatom biolm formation was conrmed tobe higher than those of most strains in the previous study

[29] with an inhibition ratio of gt90 against all four testeddiatom species of Amphora sp Nitzschia closterium Nitz-schia frustulum and Stauroneis sp in Figure 3

In natural habitats microphytobenthic (MPB) biolmsare widespread and are mainly composed of diatoms andbacteria [5] Inside these biolms multiple interactions existbetween MPB and bacteria including trophic pathways andother potential interactions including competition for nu-trients and negative cellcell interactions [4] Understandingwhether trophic competition between bacteria and diatomsis important for activities against diatom biolm formationthe culture of tested strain was divided into cells and thesupernatant to investigate their eshyect on diatom biolmformation separately e results in Figure 4 show that thesupernatant was signicantly more eshyective than cells againstdiatom biolm formation and that the competition fornutrients did not signicantly contribute to the inhibitioneshyect of the tested strain against biolm formation of

0

ndash100ndash80ndash60ndash40ndash20

20406080

100120140160180200600800

100012001400

Am

plitu

de o

f var

iatio

n (

)

GlcN Rha Glc-A Glc-NAc Xyl FucMan Glc Gal

SL-EPSF-TB-EPS

BF-LB-EPSBF-TB-EPS

Figure 5 Variation amplitude for monosaccharides in EPS of diatom Stauroneis sp treated by supernatant crude extract of Psychrobactersp SL-EPS and F-TB-EPS were prepared from the suspension phase of the treated diatom Stauroneis sp culture and BF-LB-EPS and BF-TB-EPS were prepared from the biolm phase Based on the HPLC proles of samples and standardmonosaccharides the correction factors(f12) molar ratios (R12) and mole percentages were calculatede amplitude of variation () for each monosaccharide in an EPS fractionwas calculated as the variation (mole percentages) between the treatment and control divided by the mole percentage in the control

Table 3 Monosaccharide composition for EPS fractions prepared from untreated diatom Stauroneis sp

FractionsMonosaccharides (mol)

Man GlcN Rha Glc-A Glc-NAc Glc Gal Xyl FucSL-EPS 148 104 0 122 08 29 155 370 66F-TB-EPS 40 79 130 109 11 379 241 0 12BF-LB-EPS 232 161 0 24 117 136 139 130 61BF-TB-EPS 178 19 06 19 01 610 149 07 09Mole percentage (mol) of each monosaccharide was calculated on the basis of HPLC proles of each fractions and data represent the average of duplicateexperiments

Scientica 7

Stauroneis sp Our results were similar to many reports of thecell-free supernatant such as the supernatant of Pseudomonasfluorescens containing the quorum sensing signal affecting thegrowth biofilm development and spoilage potential ofShewanella baltica [42] the cell-free supernatant of a marinebacterium Pseudoalteromonas haloplanktis containing a signalmolecule that identifies as a long-chain fatty acid active againstStaphylococcus epidermidis [43 44] and the spent medium ofa coisolated bacteria inducing diatom Achnanthidium minu-tissimum capsule and biofilm formation [10] erefore wepropose that metabolites of the tested strain in the supernatantare responsible for the activity

e results in Table 1 indicate that the extract from theculture supernatant of the tested strain significantly reducesthe biomass of diatoms which formed biofilm in the cultureand did not change the floating biomass significantly estable biomass in the floating phase indicates no significantlethal effect of the crude extract e decreased biomass inthe biofilm phase proves that the extract made the cellsdifficult to form biofilm and to grow to high density etreatment did significantly alter the distribution of plank-tonic versus biofilm cells e significant changes in the celldistribution proved that the supernatant of the tested strainpossessed high activity against diatom biofilm formationrather than lethal effect [18 19]

We were interested in how the supernatant crude extractled to the changes in the EPS fractions Diatom cells in theMPB biofilms secrete a wide range of EPS which are majorcomponents of the biofilm matrix [4ndash6] ese EPS havebeen described as regulators of bacterial development[5 45] erefore the EPS is absolutely necessary for biofilmformation and plays important functions in the interactionsof MPB and bacteria Algal EPS production is consideredbeing regulated by environmental factors [11 12 46] eresponses in the EPS production are assumed to be an at-tempt of diatom to adapt to environmental changes [47ndash49]In the present study the treatment of supernatant crudeextract of the tested strain led diatom Stauroneis sp toproduce 2-fold more total EPS both in terms of total EPSweight and cell quota as shown in Table 2 e increase inEPS production indicates that the supernatant crude extractof the tested strain made diatoms difficult to form biofilmand that the treated diatom was struggling to completebiofilm formation by producing more total EPS

Besides the responses in the total EPS production of thetreated diatom being observed further investigation on theresponses of various fractions of EPS was also carried oute EPS of diatoms can be classified in two main fractionsone of which is colloidal EPS that are soluble in saline waterand excreted in the vicinity of cells and the other of which isthe bound EPS that is tightly attached to the algal cell wallBound EPS may be involved in the cell-cell communicationof the bacteria-diatom consortium in addition to havingadhering properties such communication is expected tocontribute to biofilm development and surface colonization[5] In a previous study the diatom Achnanthidium min-utissimum which normally does not form biofilm and inwhich the cells grow completely suspended was induced toform biofilm in the presence of a coisolated bacteria [10]e

experiments following the changes of different fractions ofthe diatom EPS found stable total amount with reducingdissolved and increasing insoluble EPS [10] In our in-vestigation of responses in various fractions of EPS (Table 2)the increase in EPS production (gg cell dry weight) wasmainly contributed by SL-EPS and BF-TB-EPS in thetreatment SL-EPS is produced by both biofilm cells andfloating cells in the culture BF-TB-EPS should be the keyfraction for biofilm formation [10] It appears that treateddiatom cells must produce more BF-TB-EPS to completebiofilm formation than untreated cells Moreover the in-creased production of BF-TB-EPS suggests that the treatedEPS exhibited lower efficiency to embed diatom cells ontothe substrate surface to form biofilm compared to those fromthe control diatom

To understand low efficiency of treated EPS in biofilmformation the monomeric composition of EPS was studiede treatment of supernatant crude extract of the testedstrain effected remarkable changes on the EPS monomericcompositions of the diatom Stauroneis sp As shown inFigure 5 the EPS of treated diatom contained less Glc-A andGal and more Xyl and Fuc in all of the EPS fractionscompared to the control e content of Glc-NAc increasedwith the largest amplitude of variation by 1219 in BF-TB-EPS ere are reports which proposed that surface-activepolysaccharides such as acidic sugars including uronicacids and sulfonic sugars were correlated with the co-agulation efficiency [50] It was also reported that more than90 of the EPS fraction being composed of different acidicpolysaccharides led to the strong adhesive nature of Am-phora sp [38] erefore the reduced content of acidicsugars such as Glc-A and increased content of alkalinesugars such as Glc-NAC in the EPS of diatom Stauroneis spmight be important for the low efficiency of treated EPS andthe activity of the supernatant crude extract from the testedstrain against diatom biofilm formation

e active strain was identified as Psychrobacter spa genus with many reported characteristics including coldand salt tolerance and a unique cellular fatty acid content[51 52] e activity against diatom biofilm formation ofPsychrobacter species is reported here for the first timeFurther studies to isolate the active metabolites produced bythis strain should lead to the discovery of new activecompounds against diatom biofilm formation

In conclusion a sponge-associated bacteria strainUST050418-708 which was identified as Psychrobactersp and found sharing very high 16S rDNA sequencesimilarities with Psychrobacter glacincola in this studypossesses remarkable activities against biofilm formationof different species of benthic diatoms e activity forthis strain was found in the culture supernatant e crudeextract of the supernatant altered cell distribution of diatomStauroneis sp such that fewer cells formed biofilms Impor-tantly the supernatant crude extract of the tested Psychro-bacter strain caused significant changes not only in theproductions of BF-TB-EPS and SL-EPS but also in mono-saccharide composition of the diatom Stauroneis sp especiallya decrease in Glc-A of all EPS fractions and an increase in Glc-NAC of BF-TB-EPS Metabolites of this strain are proposed as

8 Scientifica

a promising source for novel active compounds against diatombiofilm formation

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is study was supported by the National Natural ScienceFoundation of China (nos 41776156 41106113 and 41271521)the Key Project Chinese Ministry of Education (no 211065)the Natural Science Foundation of Jiangsu Province (noBK2010322) and Science and Technology Innovation Foun-dation of Yangzhou University (2016CXJ049) e authorsacknowledge Dr Likui Zhang for English corrections

References

[1] V P L Mol T V Raveendran and P S ParameswaranldquoAntifouling activity exhibited by secondary metabolites ofthe marine sponge Haliclona exigua (Kirkpatrick)rdquo In-ternational Biodeterioration and Biodegradation vol 63 no 1pp 67ndash72 2009

[2] M P Schultz ldquoEffect of coating roughness and biofouling onship resistance and poweringrdquo Biofouling vol 23 no 5pp 331ndash341 2007

[3] M Mejdandzic T Ivankovic M Pfannkuchen et al ldquoCol-onization of diatoms and bacteria on artificial substrates in thenortheastern coastal Adriatic Seardquo Acta Botanica Croaticavol 74 no 2 pp 407ndash422 2015

[4] H Agogue CMallet F OrvainM D Crignis F Mornet andC Dupuy ldquoBacterial dynamics in a microphytobenthic bio-film a tidal mesocosm approachrdquo Journal of Sea Researchvol 92 no 2 pp 36ndash45 2014

[5] F Orvain M D Crignis K Guizien S Lefebvre C Malletand E Takahashi ldquoTidal and seasonal effects on the short-term temporal patterns of bacteria microphytobenthos andexopolymers in natural intertidal biofilms (Brouage France)rdquoJournal of Sea Research vol 92 no 18 pp 6ndash18 2014

[6] G J C Underwood and D M Paterson ldquoe importance ofextracellular carbohydrate production by marine epipelicdiatomsrdquo Advances in Botanical Research vol 40 no 5pp 183ndash240 2003

[7] B Vanelslander C Paul J Grueneberg et al ldquoDaily bursts ofbiogenic cyanogen bromide (BrCN) control biofilm forma-tion around a marine benthic diatomrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 109 no 7 pp 2412ndash2417 2012

[8] S Cao J Wang D Li and D Chen ldquoEcological and socialmodeling for migration and adhesion pattern of a benthicdiatomrdquo Ecological Modelling vol 250 no 1753 pp 269ndash2782013

[9] V Kumar D Rao T omas S Kjelleberg and S EganldquoAntidiatom and antibacterial activity of epiphytic bacteriaisolated from Ulva lactuca in tropical watersrdquo World Journal ofMicrobiology and Biotechnology vol 27 no 7 pp 1543ndash15492010

[10] M Windler K Leinweber C R Bartulos B Philipp andP G Kroth ldquoBiofilm and capsule formation of the diatomAchnanthidium minutissimum are affected by a bacteriumrdquoJournal of Phycology vol 51 no 2 pp 343ndash355 2015

[11] G Pierre M Graber B A Rafiliposon et al ldquoBiochemicalcomposition and changes of extracellular polysaccharides(ECPS) produced during microphytobenthic biofilm devel-opment (Marennes-Oleron France)rdquo Microbial Ecologyvol 63 no 1 pp 157ndash169 2012

[12] G Pierre J M Zhao F Orvain C Dupuy G L Klein andM Graber ldquoSeasonal dynamics of extracellular polymericsubstances (EPS) in surface sediments of a diatom-dominatedintertidal mudflat (MarennesndashOleron France)rdquo Journal ofSea Research vol 92 pp 26ndash35 2014

[13] C M Bennke T R Neu B M Fuchs and R AmannldquoMapping glycoconjugate-mediated interactions of marineBacteroidetes with diatomsrdquo Systematic and Applied Micro-biology vol 36 no 6 pp 417ndash425 2013

[14] L Verneuil J Silvestre I Randrianjatovo C E Marcato-Romain E Girbal-Neuhauser and F Mouchet ldquoDoublewalled carbon nanotubes promote the overproduction ofextracellular protein-like polymers in Nitzschia palea anadhesive response for an adaptive issuerdquo Carbon vol 88pp 113ndash125 2015

[15] A W Decho ldquoMicrobial biofilms in intertidal systems anoverviewrdquo Continental Shelf Research vol 20 no 10-11pp 1257ndash1273 2000

[16] M J Higgins P Molino P Mulvaney and R Wetherbeeldquoe structure and nanomechanical properties of the adhesivemucilage that mediates diatom-substratum adhesion andmotilityrdquo Journal of Phycology vol 39 no 6 pp 1181ndash11932003

[17] B A Wustman M R Gretz and K D Hoagland ldquoExtra-cellular matrix assembly in diatoms (Bacillariophyceae) (Ι Amodel of adhesives based on chemical characterization andlocalization of polysaccharides from the marine diatomAchnanthes longipes and other diatoms)rdquo Plant Physiologyvol 113 no 4 pp 1059ndash1069 1997

[18] P Y Qian Y Xu and N Fusetani ldquoNatural products asantifouling compounds recent progress and future per-spectivesrdquo Biofouling vol 26 no 2 pp 223ndash234 2009

[19] P Y Qian Z R Li Y Xu Y X Li and N Fusetani ldquoMini-review marine natural products and their synthetic analogs asantifouling compounds 2009ndash2014rdquo Biofouling vol 31 no 1pp 101ndash122 2015

[20] M W Taylor R Radax D Steger and M Wagner ldquoSponge-associated microorganisms evolution ecology and bio-technological potentialrdquo Microbiology and Molecular BiologyReviews vol 71 no 2 pp 295ndash347 2007

[21] S Dash C L Jin O O Lee Y Xu and P Y Qian ldquoAntibacterialand antilarval-settlement potential and metabolite profiles ofnovel sponge-associated marine bacteriardquo Journal of IndustrialMicrobiology and Biotechnology vol 36 no 8 pp 1047ndash10562009

[22] S Dash Y Nogata X J Zhou et al ldquoPoly-ethers fromWinogradskyella poriferorum antifouling potential time-course study of production and natural abundancerdquo Bio-resource Technology vol 102 no 16 pp 7532ndash7537 2011

[23] S V Dobretsov and P Y Qian ldquoEffect of bacteria fromsurface of the green seaweedUlva reticulata on marine micro-and macrofoulingrdquo Biofouling vol 18 no 3 pp 276ndash2872002

[24] N Fusetani ldquoBiofouling and antifoulingrdquo Nature ProductsReports vol 21 no 1 pp 94ndash104 2004

[25] J Kennedy P Baker C Piper et al ldquoIsolation and analysis ofbacteria with antimicrobial activities from the marine spongeHaliclona simulans collected from Irish watersrdquo MarineBiotechnology vol 11 no 3 pp 384ndash396 2009

Scientifica 9

[26] O O Lee and P Y Qian ldquoe chemical control of bacterialepiosis and larval settlement of Hydroides elegans in the redspongeMycale adherensrdquo Biofouling vol 19 pp 171ndash180 2003

[27] O C S Santos P V M L Pontes J F M Santos G MuricyM Giambiagi-deMarval and M S Laport ldquoIsolation char-acterization and phylogeny of sponge-associated bacteria withantimicrobial activities from Brazilrdquo Research in Microbiol-ogy vol 161 no 7 pp 604ndash612 2010

[28] V iel and J F Imhoff ldquoPhylogenetic identification ofbacteria with antimicrobial activities isolated from Mediter-ranean spongesrdquo Biomolecular Engineering vol 20 no 4ndash6pp 421ndash423 2003

[29] C L Jin X Y Xin S Y Yu et al ldquoAntidiatom activity ofmarine bacteria associated with sponges from San Juan IslandWashingtonrdquo World Journal of Microbiology and Bio-technology vol 30 no 4 pp 1325ndash1334 2014

[30] X Y Xin G H Huang X J Zhou et al ldquoPotential antifoulingcompounds with antidiatom adhesion activities from thesponge-associated bacteria Bacillus pumilusrdquo Journal ofAdhesion Science and Technology vol 31 no 9 pp 1028ndash10432017

[31] J L Lind K Heimann E A Miller C van VlietN J Hoogenraad and R Wetherbee ldquoSubstratum adhesionand gliding in a diatom are mediated by extracellular pro-teoglycansrdquo Planta vol 203 no 2 pp 213ndash221 1997

[32] M J Mcconville R Wetherbee and A Bacic ldquoSubcellularlocation and composition of the wall and secreted extracel-lular sulphated polysaccharidesproteoglycans of the diatomStauroneis amphioxys Gregoryrdquo Protoplasma vol 206 no 1pp 188ndash200 1999

[33] O O Lee Y H Wong and P Y Qian ldquoInter- and in-traspecific variations of bacterial communities associated withmarine sponges from San Juan Island Washingtonrdquo Appliedand Environmental Microbiology vol 75 no 11 pp 3513ndash3521 2009

[34] H Li H Sun X Bai et al ldquoHC2 of Pseudomonas sp inducedenteritis in Hippocampus japonicasrdquo Aquaculture Researchvol 47 no 6 pp 2027ndash2030 2016

[35] R R L Guillard and J H Ryther ldquoStudies of marineplanktonic diatoms I Cyclotella nana Hustedt and Detonulaconfervacea Cleverdquo Canadian Journal of Microbiology vol 8no 2 pp 229ndash239 1962

[36] J Leflaive and L Ten-Hage ldquoImpairment of benthic diatomadhesion and photosynthetic activity by 2E4E-decadienalrdquoResearch in Microbiology vol 162 no 9 pp 982ndash989 2011

[37] H C Xu H Y Cai G H Yu and H L Jiang ldquoInsights intoextracellular polymeric substances of cyanobacteriumMicrocystis aeruginosa using fractionation procedure andparallel factor analysisrdquo Water Research vol 47 no 6pp 2005ndash2014 2013

[38] S J Zhang C Xu and P H Santschi ldquoChemical compositionand 234 (IV) binding of extracellular polymeric substances(EPS) produced by the marine diatom Amphora sprdquo MarineChemistry vol 112 no 1-2 pp 81ndash92 2008

[39] J R Liang X X Ai Y H Gao and C P Chen ldquoMALDI-TOFMS analysis of the extracellular polysaccharides released bythe diatom gtalassiosira pseudonanardquo Journal of AppliedPhycology vol 25 no 2 pp 477ndash484 2013

[40] H X Wang J Zhao D M Li et al ldquoComparison of poly-saccharides of Haliotis discus hannai and Volutharpaampullaceal perryi by PMP-HPLC-MSn analysis upon acidhydrolysisrdquo Carbohydrate Research vol 415 pp 48ndash53 2015

[41] X Yang Y Zhao Q Wang H Wang and Q MeildquoAnalysis of the monosaccharide components in Angelica

polysaccharides by high performance liquid chromatog-raphyrdquo Analytical Sciences vol 21 no 10 pp 1177ndash11802005

[42] A Zhao J Zhu X Ye Y Ge and J Li ldquoInhibition of biofilmdevelopment and spoilage potential of Shewanella baltica byquorum sensing signal in cell-free supernatant from Pseu-domonas fluorescensrdquo International Journal of Food Micro-biology vol 230 pp 73ndash80 2016

[43] A Casillo R Papa A Ricciardelli et al ldquoAnti-biofilm activityof a long-chain fatty aldehyde from Antarctic Pseudoalter-omonas haloplanktis TAC125 against Staphylococcus epi-dermidis biofilmrdquo Frontiers in Cellular and InfectionMicrobiology vol 23 no 7 p 46 2017

[44] E Parrilli R Papa S Carillo et al ldquoAnti-biofilm activity ofPseudoalteromonas haloplanktis tac125 against Staphylococcusepidermidis biofilm evidence of a signal molecule in-volvementrdquo International Journal of Immunopathology andPharmacology vol 28 no 1 pp 104ndash113 2015

[45] H V Lubarsky C Hubas M Chocholek et al ldquoe stabi-lisation potential of individual and mixed assemblages ofnatural bacteria and microalgaerdquo PLoS One vol 5 no 11article e13794 2010

[46] G Pletikapic V Zutic I Vinkovic Vrcek and V SvetlicicldquoAtomic force microscopy characterization of silver nano-particles interactions with marine diatom cells and extra-cellular polymeric substancerdquo Journal of MolecularRecognition vol 25 no 5 pp 309ndash317 2012

[47] X X Ai J R Liang Y H Gao et al ldquoMALDI-TOF MSanalysis of the extracellular polysaccharides released by thediatom gtalassiosira pseudonana under various nutrientconditionsrdquo Journal of Applied Phycology vol 27 no 2pp 673ndash684 2015

[48] S N Aslam C Tania D Nomas and G J C UnderwoodldquoProduction and characterization of the intra- and extracel-lular carbohydrates and polymeric substances (EPS) of threesea-ice diatom species and evidence for a cryoprotective rolefor EPSrdquo Journal of Phycology vol 48 no 6 pp 1494ndash15092012

[49] B Gugi C T Le C Burel P Lerouge W Helbert andM Bardor ldquoDiatom-specific oligosaccharide and poly-saccharide structures help to unravel biosynthetic capabilitiesin diatomsrdquoMarine Drugs vol 13 no 9 pp 5993ndash6018 2015

[50] J S Chow C Lee and A Engel ldquoe influence of extra-cellular polysaccharides growth rate and free coccoliths onthe coagulation efficiency of Emiliania huxleyirdquo MarineChemistry vol 175 pp 5ndash17 2015

[51] B M Barney B D Wahlen E Garner J Wei andL C Seefeldt ldquoDifferences in substrate specificities of fivebacterial wax ester synthasesrdquo Applied and EnvironmentalMicrobiology vol 78 no 16 pp 5734ndash5745 2012

[52] P S Chain J J Grzymski M A Ponder N IvanovaP W Bergholz and G D Bartolo ldquoe genome sequenceof Psychrobacter arcticus 273-4 a psychroactive Siberianpermafrost bacterium reveals mechanisms for adaptation tolow-temperature growthrdquo Applied and Environmental Mi-crobiology vol 76 no 7 pp 2304ndash2312 2010

10 Scientifica

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

gripping to substrates and is also involved in the motilitysystem and substratum adhesion of diatoms [15ndash17] ere-fore EPS is proposed to be a key component for diatom cells toform biofilms on the substratum beneath water [16]

In marine environments sponges are commonlyfouling-free due to their chemical defenses against foulingorganisms [18 19] Many natural compounds were isolatedfrom sponges and found to be similar to those from mi-croorganisms Moreover some of them were verified beingmicrobially produced by sponge-associated microorganisms[20] Strong antimicrobial and antifouling activities in-cluding inhibition against the biofilm formation and larvalsettlement of typical fouling organisms such as Hydroideselegans or Amphibalanus amphitrite were found among themetabolites of sponge-associated bacteria [9 18 19 21ndash28]However few study on how the metabolites of active bac-terial strains affect diatom biofilm formation and whethertreatment with these metabolites provokes changes in di-atom EPS is reported Currently the active natural productsfrom sponge-associated bacteria against diatom biofilmformation have not been sufficiently investigated yet [29]

Our previous studies highlighted the strong activitiesagainst biofilm formation of several diatom species by crudeextracts from the whole culture of some sponge-associatedbacteria [29 30] Among the tested diatoms the benthicdiatom Stauroneis sp was used as a model organism in thisstudy because its gliding mechanism and monosaccharidecomposition of the EPS are very clear [31 32] In the presentstudy we found another active sponge-associated strain andwould like to clarify several questions (1) what portion ofcrude extracts from active bacterial culture is responsible forthe activity against diatom biofilm formation (2) whetherthe active strain inhibits diatom biofilm formation by al-tering cell distribution in biofilm and planktonic phaserather than by reducing total cell biomass in the wholeculture and (3) whether correlating responses in the EPSproduction or composition occur with the poor biofilmformation efficiency of diatoms when treated by the activestrain

2 Materials and Methods

21 Bacterial Culture Sponge-associated bacteria wereoriginally collected from San Juan Island WashingtonCollection isolation and purification of the strains wereperformed by Coastal Marine Laboratory in Hong KongUniversity of Science and Technology (HKUST) as describedpreviously [33] One strain of UST050418-708 was selectedfor this study since we found that it can be active againstdiatom biofilm formation [29]

A stock culture of strain UST050418-708 was stored inthe Marine Science and Technology Institute YangzhouUniversity e stock solution (1ml) was inoculated in10ml of the peptone-yeast extract medium (P-Y mediumcontaining 03 yeast extract and 05 peptone in artificialseawater (ASW)) and incubated for about two days to theexponential phase at 23degC with shaking at 120 rpm ebacterial culture in the exponential phase was then in-oculated in 500ml of the P-Y medium and incubated for

three days in the stationary phase under the same condi-tions and used for extract preparation [29]

22 Strain Identification e genomic DNA of strainUST050418-708 was extracted and its 16S rDNA was am-plified and sequenced by Sangon Biotech (Shanghai) CoLtd as described by Jin et al [29]e obtained sequence wassubmitted to GenBank e strain was identified by com-paring the submitted sequence with those available inGenBank databases Similar sequences were aligned usingmultiple sequence alignment program MEGA Gaps andpositions with ambiguities were excluded from the phylo-genetic analysis Phylogenetic analysis was performed usingthe neighbour-joining method described by Li et al [34]

23 Extract Preparation from Bacterial Culture e crudeextract was collected from the bacterial culture as describedby Jin et al [29] In detail the extraction solvent of ethylacetate (EA) acetone 95 5 (vv) was added with a ratio ofextraction solvent bacterial culture 1 1 (vv) and shakenvigorously for 1 h After standing and stratification theorganic phase was separated and dried on a rotary evapo-rator (37degC) to obtain the whole culture crude extract Forthe preparation of supernatant crude extract the bacterialcells were separated from the culture medium by centrifu-gation (5880timesg 20min 15degC) and the supernatant wasthen extracted with the same extraction solvent and pro-cedure as above Each crude extract was dissolved indimethylsulfoxide (DMSO) for subsequent bioassays

24 Diatom Culture Four benthic diatoms Amphora spNitzschia closterium Nitzschia frustulum and Stauroneissp were obtained from the Key Laboratory of MaricultureMinistry of Education Ocean University of China

e diatoms were cultured by the method of Jin et al[29] In detail ASW-based Guillardrsquos f2 culture medium[35] was used and the culture conditions of 23degC 100 μmolphotons mminus2middotsminus1 illumination and light dark 12 h 12 hcycle were employed Diatom films collected prior to assayswere suspended and washed with ASW twice adjusted toapproximately 1times 105 cellsmiddotmlminus1 in ASW and used in fol-lowing diatom biofilm formation assays

25 Diatom Biofilm Formation Assays Using Crude ExtractsDiatom biofilm formation assays for crude extracts wereperformed in 24-well polystyrene plates (353047 BectonDickinson Labware) following the method described by Jinet al [29] Algal suspension in ASW (1ml) and the crudeextract dissolved in DMSO (50 μl) were added to each wellon the plate to achieve the final concentration of 100 μgmiddotmlminus1for the crude extract in the well For the control wells 1ml ofthe algal suspension in ASW and 50 μl DMSOwere includedAfter 24 h incubation at the above diatom culture condi-tions the floating cells were removed by pipetting and thebiofilm cells were then counted using an inverted micro-scope (Nikon ECLIPSE TS 100) Triplicates were done foreach treatment and control e inhibition ratios (R) were

2 Scientifica








(a)

(b)

(c)


Scientifica 3






f12 A2m2( 1113857

A1m1( 1113857 (1)


A2prime1113888 1113889 (2)





3 Results




4 Scientifica





Stauroneis sp84

86

88

90

92

94

96

98

100

102

Inhi

bitio

n ra

tio (R

)


Nitzschiafrustulum


A

B

A

C

0

20

40

60

80

100

120

Inhi

bitio

n ra

tio (R

)

CellSupernatant







100

54

39

001


Scientica 5













SL-EPS F-TB-EPS

BF-LB-EPS


EPSBF-LB-EPS

BF-TB-EPS Total


033plusmn005

179plusmn001

038plusmn002

963plusmn003


039plusmn005


040plusmn002



1233plusmn036

100plusmn013

734plusmn048

154plusmn004

1666plusmn050


110plusmn012

705plusmn004




6 Scientifica


4 Discussion




0


20406080

100120140160180200600800

100012001400

Am

plitu

de o

f var

iatio

n (

)


SL-EPSF-TB-EPS

BF-LB-EPSBF-TB-EPS





Scientica 7









8 Scientifica




Acknowledgments


References


























Scientifica 9





























10 Scientifica



Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









(a)

(b)

(c)


Scientifica 3






f12 A2m2( 1113857

A1m1( 1113857 (1)


A2prime1113888 1113889 (2)





3 Results




4 Scientifica





Stauroneis sp84

86

88

90

92

94

96

98

100

102

Inhi

bitio

n ra

tio (R

)


Nitzschiafrustulum


A

B

A

C

0

20

40

60

80

100

120

Inhi

bitio

n ra

tio (R

)

CellSupernatant







100

54

39

001


Scientica 5













SL-EPS F-TB-EPS

BF-LB-EPS


EPSBF-LB-EPS

BF-TB-EPS Total


033plusmn005

179plusmn001

038plusmn002

963plusmn003


039plusmn005


040plusmn002



1233plusmn036

100plusmn013

734plusmn048

154plusmn004

1666plusmn050


110plusmn012

705plusmn004




6 Scientifica


4 Discussion




0


20406080

100120140160180200600800

100012001400

Am

plitu

de o

f var

iatio

n (

)


SL-EPSF-TB-EPS

BF-LB-EPSBF-TB-EPS





Scientica 7









8 Scientifica




Acknowledgments


References


























Scientifica 9





























10 Scientifica



Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







f12 A2m2( 1113857

A1m1( 1113857 (1)


A2prime1113888 1113889 (2)





3 Results




4 Scientifica





Stauroneis sp84

86

88

90

92

94

96

98

100

102

Inhi

bitio

n ra

tio (R

)


Nitzschiafrustulum


A

B

A

C

0

20

40

60

80

100

120

Inhi

bitio

n ra

tio (R

)

CellSupernatant







100

54

39

001


Scientica 5













SL-EPS F-TB-EPS

BF-LB-EPS


EPSBF-LB-EPS

BF-TB-EPS Total


033plusmn005

179plusmn001

038plusmn002

963plusmn003


039plusmn005


040plusmn002



1233plusmn036

100plusmn013

734plusmn048

154plusmn004

1666plusmn050


110plusmn012

705plusmn004




6 Scientifica


4 Discussion




0


20406080

100120140160180200600800

100012001400

Am

plitu

de o

f var

iatio

n (

)


SL-EPSF-TB-EPS

BF-LB-EPSBF-TB-EPS





Scientica 7









8 Scientifica




Acknowledgments


References


























Scientifica 9





























10 Scientifica



Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






Stauroneis sp84

86

88

90

92

94

96

98

100

102

Inhi

bitio

n ra

tio (R

)


Nitzschiafrustulum


A

B

A

C

0

20

40

60

80

100

120

Inhi

bitio

n ra

tio (R

)

CellSupernatant







100

54

39

001


Scientica 5













SL-EPS F-TB-EPS

BF-LB-EPS


EPSBF-LB-EPS

BF-TB-EPS Total


033plusmn005

179plusmn001

038plusmn002

963plusmn003


039plusmn005


040plusmn002



1233plusmn036

100plusmn013

734plusmn048

154plusmn004

1666plusmn050


110plusmn012

705plusmn004




6 Scientifica


4 Discussion




0


20406080

100120140160180200600800

100012001400

Am

plitu

de o

f var

iatio

n (

)


SL-EPSF-TB-EPS

BF-LB-EPSBF-TB-EPS





Scientica 7









8 Scientifica




Acknowledgments


References


























Scientifica 9





























10 Scientifica



Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018














SL-EPS F-TB-EPS

BF-LB-EPS


EPSBF-LB-EPS

BF-TB-EPS Total


033plusmn005

179plusmn001

038plusmn002

963plusmn003


039plusmn005


040plusmn002



1233plusmn036

100plusmn013

734plusmn048

154plusmn004

1666plusmn050


110plusmn012

705plusmn004




6 Scientifica


4 Discussion




0


20406080

100120140160180200600800

100012001400

Am

plitu

de o

f var

iatio

n (

)


SL-EPSF-TB-EPS

BF-LB-EPSBF-TB-EPS





Scientica 7









8 Scientifica




Acknowledgments


References


























Scientifica 9





























10 Scientifica



Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



4 Discussion




0


20406080

100120140160180200600800

100012001400

Am

plitu

de o

f var

iatio

n (

)


SL-EPSF-TB-EPS

BF-LB-EPSBF-TB-EPS





Scientica 7









8 Scientifica




Acknowledgments


References


























Scientifica 9





























10 Scientifica



Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018










8 Scientifica




Acknowledgments


References


























Scientifica 9





























10 Scientifica



Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





Acknowledgments


References


























Scientifica 9





























10 Scientifica



Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






























10 Scientifica



Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


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