Antimicrofouling properties of chosen marine plants: An eco-friendly approach to marine microfoulers

Original Research Paper

Antimicrofouling properties of chosen marine plants: An eco-friendlyapproach to restrain marine microfoulers

S. Prakash a,1,n, N.K. Ahila b, V. Sri Ramkumar b,d, J. Ravindran c, E. Kannapiran b,1,nn

a Research Institute, SRM University, Kattankulathur 603203, Kancheepuram District, Tamil Nadu, Indiab Department of Zoology, DDE, Alagappa University, Karaikudi 600003, Tamil Nadu, Indiac CSIR-National Institute of Oceanography, Regional Centre, Dr Salim Ali Road, PB. no. 1913, Kochi 682018, Indiad Department of Environmental Biotechnology, School of Environmental Sciences, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India

a r t i c l e i n f o

Article history:Received 11 September 2014Received in revised form7 November 2014Accepted 11 November 2014

Keywords:Marine biofilm bacteriaSeaweedsSeagrassesAntimicrofoulingCytotoxicityAntifouling

a b s t r a c t

Biofouling is a panic issue in the marine environment where the major perpetrator is the biofilm formingmicrobial cells like bacterial groups. Hence, the present study was focused to study the diversity anddensity of marine biofilm forming bacteria on different experimental panels immersed in Palk Bayregion. The results are inferred that the density and distribution of biofilm forming bacterial groups weresignificantly (Po0.05) varied, whereas the Pseudomonas spp. (15.78–22.22%) had maximum distributionin the immersed all the test panels. The current antifouling paints create toxic effects on non-targetorganisms. Of late, natural products were replaced with current toxic antifouling problems. In thepresent study, the four marine plants viz. two seaweeds (Sarconema furcellatum, Sargassum wightii)species and two seagrasses (Syringodium isoetifolium, Cymodocea serrulata) species were selected toscreen their antimicrofouling activity. From this, the crude acetone extract of S. furcellatum exhibited thegood antimicrofouling activity over the other marine plant extracts against test microfoulers; anti-bacterial (770.16 to 1370.26 mm) with least concentration of MIC and MBC values (12.5–25 mg/ml and25–50 mg/ml), antimicroalgal (50–300 mg/ml) and Artemia cytotoxicity (LC50 133.88 mg/ml; Po0.001)and anticrustacen activity was significantly (Po0.05) increased mortality with increasing test concen-trations of crude extracts. Also, phytochemical studies of the four marine plants revealed the presence ofchemical constituents such as flavanoids, alkaloids, phenols and sugars. Further studies on thepurification and identification of active compounds from S. furcellatum might help to characterize thenature of eco-friendly antifouling compounds filed study.

& 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The undesirable formation of biotic deposits on artificial or naturalsurface systems immersed in seawater is called biofouling, and this iscurrently one of the most imperative tribulations in the offshoremarine establishment (Callow, 1986; Gerhart et al., 1988). Chief foulingcommunities which play a significant role in the fouling cycle areaquatic bacteria, unicellular microalgae such as diatoms and cyano-bacteria (Qian et al., 2007). These ubiquitous microfouling membersattach to the immersed surface by complex biochemical glue calledExtracellular Polysaccharide Substances (EPS) (Salta et al., 2013).

Microfouling plays a critical role in the settlement of macrofoulerslike macroalgae larvae of barnacle, mollusks, bryozoans, polychaetes,tunicates, coelenterates etc. The formation and attachment of microand macrofouling communities are influenced by environmentalfactors and surface nature of immersed substrata (Immanuel et al.,2005).

Biofouling creates problems such as surface alternation, speedreduction and increase in fuel consumption of ships, corrosion,weight increase and distortion of the initial configuration of sub-merged man-made structures (Schultz, 2007), damage the aquacul-ture equipments and cause disease in fish and shell fishescommunities (Fitridge et al., 2012). All these problems lead to a hugeeconomic loss. As a result, commercial antifoulants came in tomarket to manage the problems of fouling. But, they are found toaffect the nontarget aquatic organisms (Konstantinou and Albanis,2004; Zhou et al., 2006) and it has also been recognized as the globalenvironmental problem since they cause marine pollution. For

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http://dx.doi.org/10.1016/j.bcab.2014.11.0021878-8181/& 2014 Elsevier Ltd. All rights reserved.

n Corresponding author.nn Corresponding author.E-mail addresses: [email protected] (S. Prakash),

[email protected] (E. Kannapiran).1 These authors contributed equally.

Please cite this article as: Prakash, S., et al., Antimicrofouling properties of chosen marine plants: An eco-friendly approach to restrainmarine microfoulers. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.11.002i

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instance, the copper based antifoulant, tributyltin (TBT) have beenbanned by IMO (International Maritime Organisation) (Yebra et al.,2004).

Hence, there is a need for the development of effective and novelenvironmentally compatible antifouling compounds from the nat-ural resources to control fouling cycle. The natural antifouling com-pounds can be one of the best replacement options for the mostsuccessful antifouling technology currently available (Raveendranand Limna Mol, 2009). Recently, the marine natural products (suchas secondary metabolites) found to exhibit strong antifoulingproperties which have been separated and identified from numberof marine organisms including seaweeds (Manilal et al., 2010;Goecke et al., 2012; Ramirez et al., 2012), seagrasses (Xu et al.,2005; Mayavu et al., 2009; Prabhakaran et al., 2012; Iyapparaj et al.,2013, 2014), sponges, ascidians, bryozoans, and gorgonians (Clare,1996) from different coastal regions. Information's available onbiofouling cycling and biological properties of marine plants fromThondi coastal region is very scanty. Therefore, the present inves-tigation was made to understand the dynamics of biofilm formingbacterial communities on different immersed substrata and toscreen antimicrofouling properties of marine plants.

2. Material and methods

2.1. Biofilm samples collection

The present investigation was carried out in Thondi coastal water,southeast coast of Tamil Nadu, India (Latitude: 91 440N and Long-itude: 791 000E). Three different experimental panels such as PVC(15�6 cm2), wood (Artocarpus hirsutus) (15�6 cm2) and titanium(15�3 cm2) were immersed to a depth of one meter below thesurface water, using wooden raft in Thondi coast for a period of fourdays. To enumerate biofilm forming bacteria, the biofilm slimesamples from each test panels were collected up to 96 h with a timeinterval of 24 h using sterile cotton swabs and transported to thelaboratory (Wahl et al., 1994). Physico-chemical parameters such astemperature, salinity and pH of the water samples were alsomeasured in the field itself with the help of a thermometer,refractometer (Agato, Japan) and pH pen (pHep, Henna instrumentPvt. Ltd., Portugal) respectively. The level of dissolved oxygen, nitrite,nitrate, inorganic phosphate, total phosphorus, and ammonia in thewater sample collected from the study area was estimated as per theprocedure given by Grasshoff et al. (1983) and APHA (1985).

2.2. Isolation and identification of biofilm forming bacterial strains

The biofilm slime samples collected from all the experimentalpanels were serially diluted up to 105 using filter sterilized seawaterand 100 ml of each diluents was spread on individual Zobell MarineAgar (2216E) plates and incubated at 37 1C for 48 h. The bacterialcolonies developed on the plates were counted and their populationdensity was expressed as CFU/ml. The morphologically distinctbiofilm bacterial colonies were purified and identified up to genuslevel by Bergey's Manual of Determinative Bacteriology (Holt et al.,1996). The pure individual bacterial colonies were maintained onslants for further study.

2.3. Generic level confirmation on selected biofilm film bacterialstrains

The predominant biofilm bacterial strains were identified andconfirmed up to genus level using Probabilistic Identification ofBacteria (PIB) software package (Bryant, 1995), an implementationof Bayes' theorem byWest et al. (1986). An identification score, as theWilcox probability (P) was calculated for identification thresholds of

P40.99 for all the isolated biofilm bacterial strains. As a result, 10biofilm film forming bacterial strains such as Pseudomonas sp.,(P40.987), Vibrio sp., (P40.993), Proteus sp., (P40.978), Salmonellasp., (P40.998), Serratia sp., (P40.986), Escherichia sp., (P40.994),Morganella sp., (P40.988), Staphylococcus sp., (P40.997), Bacillus sp.,(P40.999) and Micrococcus sp., (P40.995) were identified.

2.4. Antimicrofouling activities of marine plants

The young and healthy seaweeds (Sarconema furcellatum, Sargas-sum wightii and seagrass species (Syringodium isoetifolium, Cymodo-cea serrulata) were collected from the present study area. Thesamples were identified by referring to the keys methodology asdescribed by Umamaheswara Rao (1987) and Kuo and den Hartog(2001). The collected samples were thoroughly washed with runningtap water to remove associated debris and shade dried for 3–5 days.The dried samples were powdered, weighed and subjected to coldpercolation by soaking them individually in two different organicsolvents i.e. acetone and diethyl ether (HPLC grade) at roomtemperature for a week. Then the crude extracts were obtained byconcentrating them under vacuum at 37 1C and stored for furtherstudies.

2.4.1. Antibacterial properties of chosen marine plantsThe antibacterial activity of the crude extracts obtained from

marine plants species was tested against biofilm bacterial strainsusing an agar well diffusion method (Perez et al., 1990). Each wellwas loaded with 100 μl of DMSO containing 500 μg/ml of crudeextract and incubated for 24 h at 37 1C. 100 μl of DMSO withoutextract was maintained as negative control. The zone of inhibitionwas measured from the edge of the well to the clear zone inmillimeter (mm). Based on the results, all the acetone extractswere chosen for further research.

2.4.2. Bacteriostaic (MIC) and bactericidal concentration (MBC)of crude extract of marine plants

The Minimum Inhibitory Concentration (MIC) and MinimumBactericidal Concentration (MBC) were screened by the macro-tube dilution method (Trampuz et al., 2007). Each strains of biofilmforming bacteria (0.01 ml) with the cell density of 2�108 cell/ml(Amsterdam, 1996) were added to series of tubes containing 0.5 ml ofvarying concentrations of crude acetone extract of marine plants(12.5, 25, 50, 75, 100, 150, 200, 250 and 300 mg/ml). Then, the totalvolume was made up to 1ml by sterile Zobell marine broth andincubated at 37 1C for 24 h in thermostat shaker. The tubes wereexamined for microbial growth by turbidity observation. To determinethe MIC and MBC, a loopful of inoculum was streaked onto Zobellmarine agar plates and incubated at 37 1C for 24 h. The concentrationwhich inhibits the bacterial growth was recorded as MIC value and atwhich there is no visible bacterial growth on ZMA plates as MBC.

2.4.3. Antimicroalgal activityThe crude acetone extract of marine plants was tested against

fouling microalgal strains by following the method of Thabard et al.(2009). The two brown marine microalgae namely Chaetoceros sp.,Pavlora sp., and four green marine microalgae Nannochloropsis sp.,Dunaliella sp., Chorella sp., and Tetraselmis sp. were obtained frommarine algal culture unit, CMFRI (Central Marine Fisheries ResearchInstitute), Tuticorin, Tamil Nadu, India. The individual seed culturewas maintained in Conway medium at 20 1C in 12 h light periodsfor 7 days. Different concentrations (25–300 mg/ml) of crudeextracts in the carrier solvent of acetone: methanol (1:3) werecoated in the 96 well flat bottom microplates (Polystyrene). Eachmicroalgal strain of 100 ml volume with a initial density of 1�105

cells/ml was loaded in the extract coated plates and incubated at

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20 1C under 12 h light periods for five days. The least concentrationof crude extract which inhibits algal growth was recorded as MICand the concentration which completely the ceased the algalgrowth as Minimal Algicidal Concentration (MAC).

2.4.4. Anticrustacean assayAnticrustacean assay is one of the simplest screening techniques to

study the cytotoxicity of bioactive compounds (He et al., 2001) andoften the crustacean, Artemia is used as the model organism. Antic-rustacean activity was followed by the modified method described bySri Ramkumar et al. (2014). In brief, the cysts of brine shrimp (Artemiasalina) were hatched in a conical vessel (1 L) filled with filteredseawater under constant aeration for 24–48 h. Ten active larvae(I instar) were collected from brighter portion of the hatching chamberusing capillary glass tube and placed in a test tube containing 10ml ofbrine solution with varying concentrations (12.5, 25, 50, 75, 100, 125,150, 175 and 200 μg/ml) of crude extract. Seawater without extract waskept as negative control and maintained at room temperature for 24 hunder light. After 24 h of exposure, the number of larvae surviving ineach test concentrationwas counted and the LC50 values were analyzedby probit analysis and the percentage of larval mortality were calcu-lated. All the antimicrofouling assays were carried out in triplicates.

2.5. Phytochemical analysis on crude extract of marine plants

Primary chemical constituent's present in the crude acetoneextract of marine plants were determined by following the methoddescribed by Harborne (1973).

2.6. Statistical analysis

All data were expressed as mean7SD. The analysis of variance(one way-ANOVA) was performed for the mean value of biofilmbacterial density on different experimental panels at different timeintervals, which was compared by post-hoc multiple range test usingTukey's – HSD (Po0.05). Pearson's correlation analysis was per-formed between biofilm bacterial density on different panels andphysico-chemical parameters of the source water sample. Thepercentage mortality of A. salina larvae in the difference test conc-entrations and negative control was compared by Dunnett's test withvaried significant levels (Po0.0001; Po0.001; Po0.05). The statis-tical analysis was carried out by using SPSS 16.0 (SPSS Inc. USA).Probit analysis of toxicity data was materialized with EPA probitanalysis software (Version1.5), USA.

Fig. 1. Biofilm bacterial density (CFU/ml�103) on different immersed substrata exposed in Thondi coastal water. Each value is a mean7SD of three replicates; bars withdifferent letters are statistically significant from each other (Po0.05) subsequent post-hoc multiple comparison with Tukeys-HSD test.

Table 1Percentage composition of biofilm bacterial strains on different immersed experimental panels (12–96 h).

Biofilm bacterial strains Experimental panels Total isolates (%)

Wooden PVC Titanium

Grams negativePseudomonas spp. 6 (15.78) 4 (22.22) 8 (19.04) 18 (18.36) 86 (87.75)Vibrio spp. 7 (18.4) 3 (16.66) 7 (16.66) 17 (17.34)Proteus spp. 0 (0.0) 0 (0.00) 6 (14.28) 6 (6.12)Salmonella spp. 6 (15.78) 0 (0.00) 7 (16.66) 13 (13.26)Serratia spp. 6 (15.78) 4 (22.22) 4 (9.52) 14 (14.28)Escherichia spp. 6 (15.78) 3 (16.66) 3 (7.14) 12 (12.24)Morganella spp. 2 (5.3) 0 (0.00) 4 (9.52) 6 (6.12)

Grams positiveStaphylococcus spp. 2 (5.3) 1 (5.55) 1 (2.38) 4 (4.08) 12 (12.24)Bacillus spp. 1 (2.6) 1 (5.55) 1 (2.38) 3 (3.06)Micrococcus spp. 2 (5.3) 2 (11.11) 1 (2.38) 5 (5.10)Total isolates 38 (38.77) 18 (18.36) 42 (42.85) 98 (99.9)

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3. Results and discussion

To survive in diverse and fluctuating physical and chemical con-ditions of the marine environment, microbes have evolved mechan-isms of attaching to immersed surfaces and biofouling formingcommunities including biofilms structures. Therefore, the fluctuationof microfouling community was undoubtedly influenced by changingenvironmental conditions (Thiyagarajan et al., 2003). The physico-chemical parameters recorded in the present study is depicted inTable S1. The surface water temperature, salinity, pH and dissolvedoxygen varied from 30 to 32 1C, 34 to 36 ppt, 8.2 to 8.6, and 6.45 to6.54 ml/L, respectively. Other parameters like nitrite (2.76–2.82 mM),nitrate (5.68–5.73 mM) and reactive silicate (17.5–18.4 mM) weremoderately fluctuated during the study period. The inorganic phos-phate (5.15–5.4 mM), total phosphorus (17.04–17.08 mM) and ammo-nia (0.034–0.038 mM) also showed meager variations.

In conformity with the present study, Fletcher and Marshall(1982) reported that the pattern of bacterial colonization onsubmerged substrata was influenced by the mixture of physicaland chemical characteristics of the substrata as well as sourcewater. The Pearson's correlation and coefficient analysis revealedthat the biofilm forming bacterial density on wood and PVC panelswere positively correlated with temperature (0.198; 0.242), pH(0.831; 0.852), nitrite (0.419; 0.441), inorganic phosphate (0.419;0.441), ammonia (0.412; 0.448) and negatively correlated withother parameters. But, the bacterial density on the titanium panelshowed a positive correlation with pH (0.361), silicate (0.334) andinorganic phosphate (0.227) and negative correlation was noticedwith other parameters (Table S2).

In the present study, the biofilm bacterial density (CFU/ml) ondifferent experimental panels were in the following order: titanium(56–302�103 CFU/ml)4wood (15–167�103 CFU/ml)4PVC (23–136�103 CFU/ml) during different time intervals (12–96 h) (Fig. 1).Supportively, Immanuel et al. (2005) reported that the TVC of biofilmbacterial density on different experimental panels were in the order ofstainless steel4wood4carbon steel4FRP panels after 24–72 h of

Table

2Antiba

cterialactivity

ofmarineplants

extractag

ainst

biofi

lmform

ingba

cteria.

Nam

eofth

emarineplants

Solven

ts%

ofex

trac

tion

Antibac

terial

activity

(zoneofinhibition–mm

a )

Pseudo

mon

assp

.Vibriosp

.Pro

teussp

.Sa

lmon

ella

sp.

Serratia

sp.

Esch

erichia

sp.

Mor

ganella

sp.

Stap

hyloc

occu

ssp

.Bac

illussp

.Micro

coccussp

.

S.furcellatum

A2.43

107

0.46

117

0.25

97

0.29

77

0.22

97

0.22

107

0.21

77

0.16

137

0.29

87

0.16

87

0.24

D1.22

97

0.22

127

0.16

07

0.00

107

0.25

07

0.00

137

0.29

07

0.00

137

0.16

07

0.00

07

0.00

S.wightii

A2.23

37

0.12

97

0.26

57

0.17

47

0.21

87

0.21

87

0.22

87

0.24

107

0.24

57

0.33

97

0.22

D1.45

07

0.00

117

0.17

07

0.00

107

0.22

07

0.00

97

0.17

07

0.00

127

0.24

07

0.00

07

0.00

S.isoe

tifoliu

m(R)

A1.95

27

0.08

07

0.00

07

0.00

07

0.00

37

0.12

07

0.00

07

0.00

47

0.09

07

0.00

07

0.00

D1.11

47

0.17

07

0.00

07

0.00

07

0.00

37

0.12

07

0.00

07

0.00

37

0.00

07

0.00

27

0.12

S.isoe

tifoliu

m(L)

A2.48

37

0.21

67

0.29

57

0.16

67

0.24

57

0.21

67

0.22

07

0.00

87

0.21

47

0.08

87

0.16

D1.24

47

0.19

07

0.00

07

0.00

07

0.00

07

0.00

07

0.00

07

0.00

77

0.21

07

0.00

57

0.25

C.serrulata(R)

A2.93

37

0.12

07

0.00

37

0.16

07

0.00

47

0.2

07

0.00

37

0.12

27

0.17

07

0.00

07

0.00

D1.62

37

0.12

07

0.00

07

0.00

07

0.00

07

0.00

07

0.00

07

0.00

27

0.16

07

0.00

07

0.00

C.serrulata(L)

A2.06

77

0.16

67

0.16

47

0.21

37

0.12

07

0.00

87

0.17

07

0.00

77

0.21

97

0.24

87

0.22

D1.74

27

0.08

57

0.21

07

0.00

47

0.17

07

0.00

67

0.16

07

0.00

07

0.00

07

0.00

47

0.12

aEa

chva

lueis

mea

n7

SDthreereplic

ates;A:aceton

e;DE:

diethyl

ether;R:root

sample;L:

leaf

sample;%of

extraction

areex

pressed

thedry

weigh

tof

thesample

obtained

200g�1of

alga

ean

dseag

rass

individual

species.

Fig. 2. (a) Bacteriostaic (MIC) and (b) Bactericidal (MBC) concentration of crudeacetone extract of marine plants against biofilm bacterial strains.






immersion at Kanyakumari coast, India. Palanichamy et al. (2002) havealso studied the biofilm bacterial load on various substrata immersedin Tuticorin port seawater and found that the pattern of fouling was inthe order of Brass4Stainless Steel (SS)4PVC4Copper. The aboveobservations primarily reflect the influence of different substrata onthe adherence of bacteria.

In the marine environment, 90% of bacteria are Gram negativeand it is better adapted to survive in the marine environment (Daset al., 2006). In the present study, a total of 98 morphologicaldistinct biofilm bacterial strains were isolated from different imm-ersed substrata at different time interval (12–96 h) and identifiedbased on biochemical characterization. The Gram negative bacterialgroups i.e. Pseudomonas, Vibrio, Proteus, Salmonella, Serratia, Escher-ichia and Morganella were found to be of higher percentages(87.75%) than Gram positive groups (12.24%) which include Staphy-lococcus, Bacillus and Micrococcus. The distribution of biofilm form-ing bacterial population on different experimental panels was in theorder of titanium (42.85%)4wood (38.77%)4PVC (18.36%)(Table 1). In all the experimental panels, Pseudomonas spp. wasthe predominant genera and it varied from 15.78% to 22.22%,followed by Serratia spp., (9.52–22.22%) and Vibrio spp., (16.66–18.4%). The Escherichia was found to be maximum (16.66%) on PVCpanel and minimal on wood. The genera Proteus, Salmonella and

Morganella occurred only on titanium panel and not in other panels(Table 1). Many researchers have explored similar bacterial diversityfrom different experimental panels (Palanichamy et al., 2002;D’souza and Bhosle (2003b); Immanuel et al., 2005).

Currently, marine natural products (MNPs) are used as apromising biopotent and ecofriendly antifoulers in maritime andpharmaceutical industries. A broad range of secondary metabolitesfrom marine plants such as seaweed, seagrass demonstrate a widespectrum of bioactivity (Paul, 1992) and it have been proved as achemical defense mechanism against fouling organisms (Xu et al.,2005; Bazes et al., 2009; Plouguerne et al., 2010 a,b; Thabard et al.,2011; Prabhakaran et al., 2012; Ramirez et al., 2012). In the presentstudy, crude acetone extract obtained from S. furcellatum showed100% growth inhibitory activity against the tested biofilm bacterialstrains. The maximum zone of inhibition (1370.29 mm) wasrecorded against Staphylococcus sp., whereas, minimum (770.16 mm) was against Salmonella sp., and Morganella sp. However,crude diethyl ether extract of the same seaweed species showedless inhibitory activity against five out of 10 biofilm bacterial strainsand its zone of inhibition ranged between 970.22 and 1370.16 mm. Next to, the crude acetone extract of S. wightii registeredmoderate inhibitory activity. The acetone extracts from root and leafsamples of S. isoetifolium and C. serrulata registered lesser inhibitory

Table 3Antimicroalgal properties of marine plants extract against different microalgae.

Microalgal strains Types of microalgae Antimicroalgal activity (lg/ml)

S. furcellatum S. wightii S. isoetifolium (L) C. serrulata (L)

Chaetoceros sp. Brown microalgal strains 50 200 50 250Pavlora sp. 150 250 200 200Nannochloropsis sp. Green microalgal strains 100 200 200 300Dunaliella sp. 200 250 50 150Chorella sp. 50 100 50 150Tetraselmis sp. 50 200 150 200

Fig. 3. Anticrustacean activity and percentage mortality of marine crustacean, A. salina larvae exposed to different concentration of crude extract of marine plants.Mean7SD value indicates a significant percentage mortality of A. salina larvae at different test concentration compared with the control (one-way ANOVA, Dunnett test,(nPo0.05; nnPo0.0001).






activity than seaweed extracts (Table 2). But, the previous study byIyapparaj et al. (2014) evidenced that the methanolic (high polar)extract of marine angiosperm S. isoetifolium and C. serrulata hadexcellent antifouling potential, when compared to our results(acetone4diethyl ether). This may be due to variant solvents fromhigh to low polarity.

However, crude acetone extract of seagrass leaves showed mod-erate inhibitory activity than the root samples and it ranged from 3 to9 mm (C. serrulata) and 3 to 8 mm (S. isoetifolium) (Table 2). Similarly,the seagrass species such as Zostera marina (eel grass), C. serrulata andS. isoetifolium also have potent antifouling metabolites against foulingorganisms including biofilm bacteria, fungi, diatoms, protozoan,larvae of invertebrates, spores of macroalgae and mussels withoutany toxicity levels (Clare, 1996; Mayavu et al., 2009; Iyapparaj et al.,2013, 2014). The results showed that the acetone extract of marineplants exhibited very good antimicrofouling activity than othersolvents expect in methanolic extracts of seagrasses species.

The potent crude acetone extract of S. furcellatum exhibitedbacteriostatic activity against Pseudomonas sp., Vibrio sp., Escherichiasp., Staphylococcus sp., at a minimum concentration of 12.5 mg/ml,whereas the other biofilm bacterial strains were inhibited at 25–50 mg/ml concentration. Followed by, S. wightii (25–100 mg/ml), S.isotefolium leaf (12.5–100 mg/ml) and C. serulata leaf (12.5–200 mg/ml) (Fig. 2). The varying MBC of crude extract of S. furcellatum

(25–75 mg/ml), S. wightii (50–150 mg/ml), S. isoteifolium (50–200 mg/ml) and C. serulata (50–250 mg/ml) exhibited the bactericidal activityagainst biofilm bacterial strains (Fig. 2).

The toxic and ecofriendly antifouling compounds could be effec-tive against most fouling organisms, whereas it poorly inhibits themicroalgal slime like diatoms and cyanobacterial strains (Molino andWetherbee, 2008). Antimicroalgal activity of acetone extract ofmarine plants (Table 3) showed good antimicroalgal activity againstthe test microalgal strains after 5 days. Among them, S. furcellatumand S. isoetifolium exhibited excellent antimicroalgal activity againstChaetoceros sp., Chorella sp., Tetraselmis sp., and Dunaliella sp. at aleast concentration of 50 mg/ml. Whereas, the S. wightii and C.serrulata extracts showed moderate activity against Chlorella sp.,and Dunaliella sp. at a least concentration of 100 and 150 mg/ml. Themicroalgal strains such as Pavlora sp. and Nanochloropsis sp., werealso inhibited at a concentration of 300 mg/ml of crude acetoneextract of marine plants. Similarly, a quite few authors have alsoreported the antimicroalgal activity of different solvent extracts ofSargassum against diatoms, cyanobacteria, and microalgal strains(Bazes et al., 2009; Plouguerne et al., 2010b; Cho, 2013). Recently,Iyapparaj et al. (2014) had reported that the antimicroalgal activity ofcrude methanolic extracts from seagrasses species (C. serrulata and S.isoetifolium) had strong antimicroalgal activity at a least concentra-tion (10.0 and 1.0 mg/ml) against marine fouling microalgal strains.

Table 4Cytotoxicity effects of crude acetone extract of marine plants against the larvae of Artemia salina.

Concentrations No oflarvaeexposed

Crude acetone extract of marine plants


No ofresponse

LC50 (LCL–UCL)

r2No ofresponse

LC50 (LCL–UCL)

r2No ofresponse

LC50 (LCL–UCL)

r2No ofresponse

LC50 (LCL–UCL)

r2

Control 10 0 LC50

133.88 mg/ml(112.39–162.69)

0.876n 0 LC50

111.38 mg/ml(95.10–126.74)

0.959nn 0 LC50

141.54 mg/ml(118.05–178.89)

0.860n 0 LC50

115.94 mg/ml(95.65–138.19)

0.973nn

12.5 10 0 0 0 025 10 0 0 0 050 10 1 0 1 175 10 1 2 1 2100 10 2 3 2 3125 10 3 6 3 5150 10 5 8 4 6175 10 7 9 6 8200 10 10 10 10 10

L: leaf sample; LCL: lower confidence limit; UCL: upper confidence limit; r2: regression coefficient.n Po0.001.

nn Po0.0001.

Table 5Primary phytochemical constituents of crude acetone extract of selected marine plants.

Phytochemical constituents Marine plants


Alkaloids þ � � �Phenolics þ þ þ þSaponins � � � �Flavonoids þ þ þ þCarboxylic acid � � þ �Coumarins � þ � �Quinones � � � �Proteins þ � þ þSteroids � þ � �Tannins � þ � �Sugars þ � þ þGlycosides � � � �Terpenoids � � � �Resins � � � �

�: absent; þ: present.






Toxicity against A. salina is an indication of potential against marinefouling organisms, more specifically crustaceous foulers such asbarnacles (Persoone and Castritsi-Catharios, 1989). The cytotoxic effectof crude extracts from marine halophytic plants such as seaweed,seagrass and mangroves has displayed different levels of lethal toxicityconcentrations (Bragadeeswaran et al., 2011; Saeidnia et al., 2012). Inthe present study also, the tested crude extract of marine plantsshowed significant mortality of A. salina larvae at 200 mg/ml concen-tration. It has been noted that an increase or decrease in theconcentration of crude extracts reflected on the mortality of A. salinalarvae (Fig. 3). The anticrustacean activity of marine plant extractsshowed significant (Po0.05) mortality of test organisms at differenttest concentrations. The significance of lethal concentration (LC50)were as follows: S. furcellatum (133.88 mg/ml; r2: 0.876; Po0.001), S.wightii (111.38 mg/ml; r2: 0.959; Po0.0001), S. isoteifolium (141.54 mg/ml; r2: 0.860; Po0.001) and C. serrulata (115.94 mg/ml; r2: 0.973;Po0.0001) (Table 4).

The marine plants are rich in pharmaceutically imperative second-ary metabolites such as alkaloids, flavonoids, glycosides, phenolics,tannins, saponins, and steroids (Eluvakkal et al., 2010). In the presentstudy also, the phytochemical constituents of crude acetone extract ofmarine plants revealed the presence of variety of primary chemicalconstituents including phenolics and flavonoids and also other che-mical constituents such as alkaloides, carboxylic acid, coumarine,proteins, steriods and sugars (Table 5). Similarly, several authors havealso reported the phytochemical constituents from marine plants vizseaweed and seagrass (Vergeer et al., 1995; Clare, 1996; Jeeva et al.,2012). Clare (1996) reported the presence of rich amount of phenolicderivatives in the marine plants such as Sargassum spp., and Z. marina,which also inhibited the fouling organisms such as bacteria, micro-algae and cyprids larvae of barnacles at a least concentration withouttoxic effects. It has been reported that the primary chemical constitu-ents such as phenolics groups found in the marine plant extracts haveinhibited the marine microfoulers.

4. Conclusion

In general, present results clearly noticed that the antimicor-fouling potential of chosen marine plants against microfoulingorganisms and thus substantiate the non/less toxic nature of theextracts. Understanding the new metabolites from the marinesource will be a valuable topic of further investigation on purifica-tion and identification of active compounds was tested against thecommon Indian macro-fouler likes mussel (Perna indica), mollusc(Patella vulgata) and cybrid larvae of barnacles and field evaluationis being aimed at eco-friendly antifouling paint development.

Conflicts of interest

All authors have no potential conflict of interest.

Acknowledgment

The authors are thankful to the authorities of School of MarineScience, Alagappa University, Karaikudi Tamil Nadu, India, forproviding facilities to carry out this work.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bcab.2014.11.002.

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Antimicrofouling properties of chosen marine plants: An eco-friendly approach to marine microfoulers