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Use of <i>Streptomyces fradiae</i> and <i>Bacillus megaterium</i> as probiotics in the experimental culture of tiger shrimp <i>Penaeus monodon</i> (Crustacea, Penaeidae)

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AACL BI OFLUX

Aqu a cu lt u r e , Aqu a r iu m , Con se r va t ion & Le gisla t ion

I n t e r n a t ion a l Jou r n a l of t h e Bioflu x Socie t y

Use of

St r e pt om y ce s fr a dia e

a n d

Ba cillu s

m e ga t e r ium

a s pr obiot ics in t h e e x pe r im e n t a l

cu lt u re of t ige r sh r im p

Pe n a e u s m on odon

( Cr u st a ce a , Pe n a e ida e )

1

Sheikh Aft abuddin,

2

M. Abul Kashem ,

1

M. Abdul Kader,

1

M. Nurul Azim Sikder, and

3

M. Abdul Hak im

1

I nst it ut e of Marine Sciences and Fisheries, Univ ersit y of Chit t agong, Bangladesh; 2 Prim e Shrim p Hat chery Lim it ed, Kolat oly , Cox’s Bazar, Bangladesh; 3Depart m ent of Microbiology, Univ ersit y of Chit t agong, Bangladesh. Corresponding aut hor: S. Aft abuddin,

em ail: aft abim sf@y ahoo. com

Abst r a ct. Shr im p hat cheries oft en beset wit h diseases, m ainly t he bact er ial infect ion and ant ibiot ics ar e widely used for pr event ion of disease. Pr esent ly, beneficial bact er ia ( pr obiot ics) ar e used t o pr event diseases inst ead of ant ibiot ics and incr easing t he pr oduct ion. I n t he pr esent st udy, t he t wo new m icr obial st r ains Bacillus m egat er ium and St r ept om yces fr adiae isolat ed fr om m angr ove sedim ent s wer e applied ( exper im ent al cult ur e) for t he post lar val r ear ing of Penaeus m onodon which is com par ed wit h cont r ol cult ur e t anks ( wit hout pr obiot ics) . The wat er qualit y condit ion such as t em per at ur e ( 27- 29o

C) , salinit y ( 26- 28‰ ) and dissolved oxygen ( 4. 7- 5. 0 m g L- 1

) of bot h cont r ol and exper im ent al cult ur e wer e m or e or less sim ilar . Concent r at ion of am m onia and pH wer e significan t ly differ ent ( p< 0. 05) bet ween t he cont r ol and exper im ent al cult ur e dur ing t he st udy per iod. The feed assim ilat ion efficiency is higher ( above 8 0% ) in exper im ent al cult ur e t ank when com par ed t o cont r ol t ank ( 74. 76% ) . The gr owt h r at e was higher - 1. 70 and 1. 67 in S. fr adiae, 1. 66 and 1. 63 in B. m egat er ium - t hr ough feed and wat er , r espect ively, while in cont r ol t ank it was 1. 4. The FCR values wer e 2. 06 an d 2. 12 in S. fr adiae t r eat ed t anks t hr ough feed and wat er , r espect ively, while 2. 51 and 2. 55 wer e obser ved in B. m egat erium t r eat ed t hr ough feed and wat er r espect ively. The FCR value was found higher ( 4. 02) in t he cont r ol t ank. The aver age t ot al het er ot r ophic bact er ia ( THB) and t ot al pr esum pt ive Vibr io bact er ia bot h in cult ur e wat er and post lar vae wer e lower dur ing exper im ent al cult ur e in com par ed t o cont r ol cult ur e. The pr esent st udy indicat es t hat t he pr obiot ic t r eat m ent using t wo new m icr obial st r ains such as B. m egat erium and S. fr adiae would help in bet t er aquacult ur e pr oduct ion.

Ke y W or ds: Penaeus m onodon, post lar vae, St r ept om yces fr adiae, Bacillus m egat er ium, pr obiot ics.

I n t r odu ct ion. Shrim p farm s are growing t hroughout t he w orld t o m eet t he dem and for shrim p w hich is one of t he m ost popular t y pes of seafood in t he w orld and Bangladesh is no ex cept ion. The t iger shrim p, Penaeus m onodon is one of t he largest shrim p am ong penaeids and it is m ost suit able for aquacult ure in Bangladesh ( Aft abuddin et al 2005) . Shrim p cult iv at ion and ex port in Bangladesh have undergone rapid ex pansion ov er t he last t w o decades. During 2008- 2009 fiscal y ear, ex port of shrim p and fish product s w as 117. 31 m illion Lbs earning 454. 53 m illion US dollar. Shrim p alone cont ribut ed about 78% of t ot al ex port earning from fishery product ( BFFEA 2009) . Wit h t he rapid ex pansion of shrim p grow out farm , t he hat chery indust ry has progressed rapidly during t he last t en y ears. There are 50 shrim p hat cheries ( at v arious st ages of operat ion and m anagem ent ) in Bangladesh and t hey produced nearly 5 billion post larv ae in 2004 ( Aft abuddin & Kader 2006) w hich w as sufficient t o supply t he ent ire shrim p cult ure indust ry in Bangladesh. How ev er, m any of t hese hat cheries had t o st op product ion in t he last t hree y ears due t o inv asion of different pat hogens in t heir sy st em ( Aft abuddin et al 2008) .

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and significant infect ious pat hogens ( Light ner 1996; Moriart y 1997; Vaseeharan et al 2005; Aft abuddin et al 2008) . Bact erial diseases are t he m aj or problem affect ing t he shrim p hat cheries and m ost of t he m ass m ort alit ies report ed in shrim p (P. m onodon) hat cheries are associat ed wit h lum inous bact eria ( Lavilla- Pit ogo et al 1990; Jirav anichpaisal et al 1994; Karunasagar et al 1994) . Vibrio spp. are by far t he m aj or bact erial pat hogens and can cause sev ere m ort alit ies, part icularly in hat cheries. Vibriosis is caused by a num ber of Vibrio species including: V. harv ey i, V. v ulnificus, V. parahaem oly t icus, V. alginoly t icus, V. penaeicida ( Brock & Light ner 1990; I shim aru et al 1995) . Different ant ibiot ics i. e. Ox y t et racy cline, Chloram phenicol, Ery t hrom y cin, Furazolidone, Neom ycin sulphat e, Kenam y cin sulphat e, Ciprocin and ot hers are used in P. m onodon shrim p hat cheries in Bangladesh ( Aft abuddin & Kader 2006) . Prolonged ex posure of a bact erial populat ion t o a giv en ant ibiot ic can result in developm ent of pat hogens t hat are resist ant t o t hose ant ibiot ics. This resist ance can occur eit her by m ut at ion wit hin t he t arget bact erial populat ion or by inadv ert ent select ion of a m inor percent age of t he original populat ion t hat is innat ely resist ant ( Cham berlain 1988) . On t he ot her hand, t he use of beneficial bact eria ( probiot ics) t o displace pat hogens by com pet it iv e processes is being used in t he anim al indust ry as a bet t er rem edy t han adm inist ering ant ibiot ics and is now gaining accept ance for t he cont rol of pat hogens in aquacult ure ( Hav enaar et al 1992) . The probiot ics are liv e m icrobial feed supplem ent s, used for env ironm ent - friendly aquacult ure. The probiot ics are usually applied t hrough w at er or feed, t hey act beneficially in im proving t he w at er qualit y, increasing grow t h of t he shrim p and m aking t he anim als act ive and healt hy ( Gat esoupe 1999) . The probiot ics includes Vibrios, Pseudom onades, lact ic acid bact eria, bacilli and y east s. Am ong t hem

Bacillus spp. and y east s are com m only st udied probiot ics ( Cherif et al 2001; Vaseeharan & Ram asam y 2003; Dahan et al 2003; Duc et al 2004; Soundarapandian & Babu 2010) . Marine environm ent cont ains a wide range of dist inct m icroorganism s t hat are not present in t he t errest rial environm ent ( Ram esh & Mat hiv anan 2009) . Many prom ising bioact iv e com pounds including ant im icrobial, ant it um our, im m unosuppressiv e agent s and enzy m es are being discov ered from m arine act inom y cet es ( Tanak a & Om ura 1990) . Marine m angrov e ecosy st em s are a great source for isolat ion of new m icrobes t hat are pot ent ially rich t o produce t he im port ant bioact ive m et abolit es ( Mangam uri et al 2012) . The environm ent of t he m angrov e ecosy st em is saline, and highly rich in organic m at t er due t o it s v arious m icrobial enzy m at ic and m et abolic act ivit ies ( Kizhekk edat hu & Paruk ut t y am m a 2005) . Approx im at ely 70% of t housands of nat urally occurring ant ibiot ics hav e been isolat ed from m arine act inom y cet es ( Takizaw a et al 1993) and t he m aj orit ies w ere isolat ed from t he genus St rept om y ces ( Zheng et al 2000; At t a & Ahm ad 2009; Reddy et al 2011) . Marine act inom y cet es, especially St rept om y ces, hav e been considered in ant ibiot ic product ion for chem ical fact ories ( Ok azaki & Ok am i 1972; Ellaiah & Reddy 1987) . How ev er, in addit ion, lit t le inform at ion w as found on t he applicat ion of

St rept om y ces as probiot ics in aquacult ure ( Das et al 2006) .

On t he ot her hand, Bacillus m egat erium is a gram posit iv e, spore producing bact eria. I t is found in div erse environm ent s from rice paddies t o dried food, seaw at er, sedim ent s, fish, norm al flora, and ev en in bee honey ( Vary 1994) . Tax onom ically, B. m egat erium is posit ioned int o t he B. subt ilis group of Bacilli ( Priest 1993; Vary 1994) .

I n cont rast t o ot her bacilli st rains B. m egat erium has an adv ant age, t hat no alk aline prot ease is present ( Vary et al 2007) . This fact enables ex cellent product ion and secret ion of foreign prot eins wit hout degradat ion ( Meinhardt et al 1989; Ry gus & Hillen 1991) . I n com pare t o gram negat iv e organism s lik e Escherichia coli, it does not produce endot ox ins associat ed wit h t he out er m em brane (Vary et al 2007) . These feat ures m ak e

B. m egat erium w ell applicable in food and ev en in pharm aceut ical indust ry . B. m egat erium has econom ic im port ance because of it s com m ercially im port ant enzy m es such as penicillin am idase and st eroid hy drolases ( Vary et al 2007) .

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bact erial st rains hav e led t o t he use of probiot ic feed addit iv es in t he aquacult ure indust ry.

Therefore, t he present st udy w as undert ak en t o ex am ine t he effect s of B. m egat erium

and S. fradiae as probiot ics w hen t reat ed t hrough feed and cult ure m edium of P. m onodon. Wat er qualit y param et ers of cult ured environm ent , net grow t h efficiency , assim ilat ion, food conv ersion rat io ( FCR) , grow t h rat e, t ot al het erot rophic bact eria ( THB) and t ot al Vibrio bact eria w ere also st udied, wit h a view t o dev elop a pot ent m icrobial supplem ent t o ensure bet t er aquacult ure product ion m aint aining t he environm ent friendly.

M a t e r ial an d Me t h od. I n t he present st udy, st rains of B. m egat erium and S. fradiae

w ere isolat ed from m angrov e sedim ent s of Chok aria m angrov e area, Cox ’s Bazar, Bangladesh ( Figure 1) . This st udy w as carried out from January 2011 t o Decem ber 2011 in Prim e Shrim p Hat chery Lim it ed, Cox ’s Bazar, where adequat e research facilit ies for t he st udy of hat chery operat ion and m anagem ent w ere readily av ailable and Shrim p & Fish Disease Diagnosis Laborat ory , I nst it ut e of Marine Sciences and Fisheries, Univ ersit y of Chit t agong, Bangladesh.

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I sola t ion a n d iden t ifica t ion of Ba cillus m e ga t e r iu m. The sedim ent sam ples w ere collect ed by insert ing a grab sam pler at 1- 2 m et er dept h. The sam pler w as st erilized wit h 70% alcohol before sam pling. The sam ple w as t hen t ransferred t o a st erilized poly et hylene bag. Sedim ent w as dried ov ernight in a lam inar flow hood and w hen clum ping occurred, w as grinded wit h m ort ar and pest le. Ten gram of sedim ent sam ple w as suspended in 100 m l dist illed w at er in a 200 m l conical flask and serial dilut ions w ere m ade. From t he serial dilut ions 100 µl of soil suspension at different dilut ions were draw n and poured ont o t he Blood Agar m edium and incubat ed at 35 t o 37oC for 24- 48 hours ( HPA 2007) . Bact erial cells from t y pical colonies w ere observ ed wit h a light m icroscope ( 400X) and purified on nut rient agar m edium . Colonies w ere w hit e, round sm oot h and shiny . During t he m icroscopic ex am inat ion all t he isolat es w ere found gram posit iv e rods. The isolat ed colonies w ere ident ified as B. m egat erium using st andard m orphological, phy siological and biochem ical plat e and t ube t est s ( Holt et al 1994) . All st rains w ere st ored in Luria- Bert ani ( LB) brot h cult ures w it h st erile gly cerol ( 15% v⁄v ) .

I sola t ion a n d iden t ifica t ion of St r e pt om yce s fr a dia e. The collect ed sedim ent sam ples w ere air- dried asept ically . All t he m arine sedim ent sam ples w ere subj ect ed t o pre- heat t reat m ent prior t o serial dilut ion. Pre- heat t reat m ent w as perform ed by incubat ing t he sedim ent sam ples in a w at er bat h at 50o C for 60 m inut es ( Takizaw a et al 1993) . Ten gram s of sedim ent sam ples w ere suspended in 95 m l of st erile aged seaw at er ( 7 day old seaw at er) and t hese suspensions w ere considered as 10- 1 dilut ion. St arch casein agar ( SCA) m edium w as used for isolat ion of St rept om y ces spp. Serial dilut ions w ere done and ov erlaid on t he surface of SCA. The m edium consist ed of 10 g soluble st arch, 0.3g casein, 2g KNO3, 2g KH2PO4, 0. 05g MgSO4. 7H2O, 2g NaCl, 0. 02g CaCO3, 0. 01g FeSO4. 7H2O, 20g Agar and nat ural aged sea w at er 1000m l. The final pH of t he m edium w as adj ust ed t o 7.0 ± 2 w it h 0. 1 N NaOH before st erilizat ion ( Ram esh & Mat hiv anan 2009) . The m edium w as supplem ent ed wit h 50 m g L- 1 and 20 m g L- 1 of t et racy cline and ny st at in respect iv ely as ant ibact erial and ant ifungal agent s t o inhibit t he bact erial and fungal cont am inat ion ( Reddy et al 2011) . The plat es w ere incubat ed at room t em perat ure 28± 2oC for one w eek .

Pu r ifica t ion. The appearance and grow t h of m arine act inom y cet es w ere observ ed ev ery day on SCA plat es. Again, t he colonies hav e been observ ed using a light m icroscope for t heir filam ent ous nat ure, spores, widt h of hyphae and spiral sporophores. I ndividual colonies w ere pick ed up, and subcult ured on SCA and on I nt ernat ional St rept om yces Proj ect m edium 2 ( I SP2) i.e. Yeast ext ract - m alt ex t ract agar ( Pridham et al 1956) by repeat ed st reaking t o ascert ain t heir purit y . The m edium consist ed of Bact o Yeast Ex t ract ( Difco, USA) 4g, Bact o Malt Ext ract ( Difco, USA) 10g, Bact o Dex t rose ( Difco, USA) 4g, dist illed w at er 1 lit er, Bact o agar 20g and adj ust ed pH 7. 3. The pure cult ures of m arine act inom y cet es w ere sub- cult ured in SCA slant s, incubat ed at room t em perat ure for 5–7 day s t o achiev e good sporulat ion, and t hen preserv ed in 20% gly cerol vials at - 80oC ( William s & Cross 1971) . The inocula used in all t he ex perim ent s w ere sev en- day - old cult ures, unless ot herwise st at ed.

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Ex pe r im e n ta l se t up. Healt hy and act iv e post larv ae ( PLs2 0 -2 2) w ere acclim at ized t o t he laborat ory condit ions for 5 day s. One hundred PL w ere used for each t reat m ent t ank of 100 lit ers capacit y. The t ank w as filled wit h 90 lit ers of filt ered seaw at er and w as aerat ed using air- st ones. Salinit y w as m aint ained bet w een 22 and 25 ppt . The ex perim ent al anim als w ere fed wit h form ulat ed feed ( Brine Shrim p flakes, Higashim aru 3, Singapore) at 10% body w eight of t he PL.

The ex perim ent w as done in t w o w ay s using t he cult ures of B. m egat erium and S. fradiae. One w ay w as t hat t he cult ures w it h a densit y of 109 cells per m l and t ot al 10m l w ere inoculat ed int o t he t ank having t he 90 lit ers of seaw at er considering t hat t his sm all am ount of cult ure m edia w ould not be affect ed t he ex perim ent . Anot her w ay w as t hat t he cult ures w ere m ix ed t horoughly wit h feed at 109 cells per gram of t he feed. The t reat ed feed w as giv en t w o t im es a day at 7. 00 a. m . and 7. 00 p. m . All t he t reat m ent s ( w at er and feed) w ere done at an int erv al of ev ery 5 day s of t he t ot al 60 day s of cult ure. I n cont rol t ank s, no m icrobial t reat m ent eit her t hrough w at er or t hrough feed w as m ade. Wat er ex change w as m ade for 35% of t he cult ure w at er ev ery m orning. Ten t ank s w ere used ( fiv e for Bacillus spp. and fiv e for St rept om y ces spp. ) for conduct ing t he ex perim ent . ANOVA t est w as done t o see t he t reat m ent effect s of t he new probiot ics used.

W a t e r pa r a m e te r s an a lysis. The w at er qualit y param et ers of t he probiot ics (B. m egat erium and S. fradiae) t reat ed and cont rol t ank s w ere regularly m onit ored. Wat er qualit y param et ers such as salinit y , t em perat ure, pH, dissolv ed ox y gen, and am m onia w ere est im at ed daily in t he m orning hours. The w at er salinit y w as m easured by using a hand refrect om et er ( ATAGO, Japan) . The pH of t he w at er w as m easured by using elect ronic pen pH m et er m anufact ured by Hanna I nst rum ent al Com pany ( Singapore) . Wat er t em perat ure w as m easured by using a digit al t herm om et er ( DT801, China) . Dissolv ed oxy gen ( m g L- 1) , and am m onia ( m g L- 1) w ere m easured by HANNA inst rum ent s product s, Singapore, Model no. HI 3810 and HI 3826, respect iv ely.

M icr obial e va lu a t ion. For enum erat ion of t ot al het erot rophic bact eria ( THB) in w at er and post larv ae t he pour plat e m et hod w as used wit h st erile Zobell’s m arine agar m edium ( Hi- m edia, Mum bai, I ndia) . For presum pt ive Vibrio count TCBS and TSA m edium ( Ox oid, England) w ere used. This w ork w as carried out for 10 day s int erv al i. e. 10t h, 20t h, 30t h, 40t h, 50t h and 60t h day s of cult ure period.

An a lysis of sh r im p gr ow t h. The shrim p grow t h in t erm s of lengt h and w eight w as m easured aft er 60 day s of cult ure. Tot al lengt h w as m easured w it h t he help of a plast ic graduat ed scale. Fresh w eight w as t aken by w eighing t he liv e shrim ps on an elect ronic balance. From t he dat a collect ed, t he food consum pt ion, m ean product ion w eight , assim ilat ion, m et abolism , growt h rat e, relat ive grow t h rat e, digest ibilit y, gross conv ersion efficiency , net conv ersion efficiency, food conv ersion rat io ( FCR) and food- coefficient w ere calculat ed by Crisp ( 1971) , Qasim & East erson ( 1974) and East erson ( 1987) . Before feeding and w at er ex change, unut ilized feed and faecal m at t er w ere collect ed separat ely by siphoning out t he w at er using a 2 cm ( dia) plast ic hosepipe t o det erm ine t he feed assim ilat ion rat e. The collect ed unut ilized feed and faecal m at t er w ere placed separat ely on a pre- w eight ed filt er paper ( WHATMANTM No. 1) , cleaned wit h dist illed w at er, dried in an ov en at 60oC for 24- h and t hen w eight ed.

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t reat ed wit h food and from 59. 83 x 103 t o 39. 22x 106 cfu m l- 1 and 6. 1± 1. 1x 102 t o 1. 99± 0. 35x 104 cfu g- 1 in w at er and post larv ae respect ively w hile t reat ed wit h w at er. I n t he S. fradiae t reat ed t ank ’s post larv ae, t he THB ranged from 3. 48± 1. 24x 103 t o 3. 45± 0. 82x 104 in feed ex perim ent and from 8. 8± 2. 3x 102 t o 6. 1± 1. 2x 103 cfu g- 1 in w at er ex perim ent . The THB v alues are m uch different bet w een t he cont rol and t reat ed t ank s ( Figure 2) . The observ ed t ot al Vibrio bact eria w ere lower in S. fradiae and B. m egat erium

t reat ed t ank com pared t o cont rol t ank ( Figure 3) and t he v alues w ere significant ly different ( p< 0. 05) . The average THB and Vibrio bact eria in w at er and post larv ae are show n in Table 2.

Table 1 Different phy siological and biochem ical charact erist ics of B. m egat erium and S. fradiae

Test s B. m egat erium ( n= 10) S. fradiae ( n= 10)

Gram st aining + +

Mot ilit y + / - -

Cat alase + +

Reduct ion of nit rat e - ( d) ND

Liquefact ion of Gelat in + +

St arch hy droly sis + -

Cit rat e t est + +

Egg y olk react ion - ND

Anaerobic ut ilizat ion of glucose - ND

Voges prosk auer react ion - -

Grow t h at 45oC - +

Grow t h at NaCl 7% ( w / v ) - +

Melanin Pigm ent on Pept one y east ex t ract –iron agar m edium ( I SP- 6)

ND +

Ty rosin decom posed ( I SP- 7) + / - +

Ly sozy m e- resist ant - -

Acid produced from

Manint ol + -

Glucose + +

Acet ylglucosam ine + ND

Arbut in + ND

D- fruct ose + + / -

D- t rehalose + + / -

Ribose + ND

Rham ose ND +

Carbon ut ilizat ion D- glucose L- arabinose

Sucrose I nosit ol Rham nose

+ ND ND ND ND

+ + - - +

“ + ” = 90- 10 0% st r ains posit ive; “ - ” = 90- 100 % st r ains ar e negat ive; ND = Not done; “ + / - ” = 50- 50% st r ains ar e posit ive; “ –( d) ” = m ost st r ains ar e negat ive.

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t he cont rol t ank . This finding coincide wit h t he w ork s of Moriart y ( 1998) , Prabhu et al ( 1999) , Dalm in et al ( 2001) and Das et al ( 2006) .

Figure 2. Tot al het erot rophic bact eria ( cfu m l- 1) observed in cont rol and t reat ed t anks wat er.

Figure 3. No. of Vibr io bact eria cfu m l- 1 ( 102) in t reat ed and cont rol t anks wat er.

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rat e and surviv al rat e ( Soundarapandian & Babu 2010) . The av erage w at er qualit y param et ers of ex perim ent al and cont rol t anks w ere show n in Table 2.

Table 2 Wat er qualit y param et ers and t ot al het erot rophic bact eria ( THB) and Vibrio ( TVC) count s

in P. m onodon cult ure t ank s t reat ed wit h S. fradiae and B. m egat erium

Average value Variables

Cont rol

B. m egat erium t reat ed wit h

feed

S. fradiae t reat ed wit h

feed

B. m egat erium t reat ed wit h

wat er

S. fradiae t reat ed wit h

wat er

Tem perat ure (oC) 29 27. 6 27. 2 27. 4 27

Salin it y ( ppt ) 28 27 27 26 26

Dissolv ed oxy gen

( m g L- 1) 4. 8 4. 7 5. 0 4. 8 4. 9

pH 6. 8 7. 5 7. 7 7. 6 7. 5

Am m onia ( NH3 –

N) ( m g L- 1) 0. 45 0. 025 0. 018 0. 022 0. 020

THB ( cfu m l- 1)

in wat er 10. 33± 0. 5x10

4

21.54± 1.2x 104 22.98± 0.8x 104 19. 56± 0. 6x104

22.52± 1.1x 104

THB ( cfu g- 1)

in post larvae 2. 3± 0. 3x10

3

11. 2± 1. 5x1 03

18. 5± 0. 9x1 03

4. 14± 0. 7x 103

6. 42± 0. 9x1 03

Vibr ios ( cfu m l- 1)

in wat er 469. 66 ± 1. 2x1 0

2

63.33± 0.90x 102

60.66± 1.0x 102 72.83± 0.72 x 102 62.33± 1.3x 102

Vibr ios ( cfu m l- 1)

in post larvae 233.11 ± 2. 3x1 0

1

10.12± 0.5x 101 9. 36± 0. 3x1 01

13.38± 0.45x 101 14.12± 0.65x 101

NB: Values ar e av er age of 60 day s of analysis for t em per at ur e, salinit y , DO and pH; THB ( t ot al het er ot r ophic bact eria) and Vibr ios ar e av er ages of 6 day s of analy sis ( 10t h, 20t h, 30t h, 40th, 50t h and 60th day s of cult ur e t im e) .

The opt im um range of t em perat ure for t he black t iger shrim p larv al rearing is bet ween 28 t o 32oC ( Kannupandi et al 2002) . The t em perat ure v aried from 27 t o 29oC in bot h cont rol and ex perim ent al t ank s during t he st udy period. There w ere no m ark ed differences in t em perat ure bet ween cont rol and ex perim ent al t ank s of t he present st udy .

P. m onodon can t olerat e a w ide range of salinit y . The range of salinit y wit h w hich t he norm al grow t h of P. m onodon can be m aint ained is bet w een 15 and 30 ppt ( Chen 1976; Chen 1985) . I n P. m onodon, t he opt im um salinit y range for grow t h m ay change during t heir life hist ory wit h 10 ppt for early post larv ae ( Valencia 1976) , 15- 20 ppt for lat e post larv ae ( Chen 1976) and 15- 25 ppt for j uv enile ( Chen 1984) . I n t he present st udy , t he salinit y ranged bet w een 26 and 28 ppt on all t he t ank s. There were slight differences bet w een cont rol and t reat ed t ank s (S. fradiae and B. m egat erium) . Salinit y w as slight ly higher in S. fradiae and B. m egat erium t reat ed t ank s m ix ed wit h feed t han m ixed in w at er. For a shrim p hat chery , t he recom m ended salinit y range is 28- 35 ppt ( Kannupandi et al 2002) .

The av erage DO lev el ranged bet w een 4. 7 and 5. 0 m g L- 1 in t he ex perim ent al and cont rol t ank s during t he st udy and t here w as no significant difference ( p> 0. 05) not iced bet w een t he cont rol and t reat ed t ank s. Liao & Murai ( 1986) report ed t hat t he respirat ion rat e of P. m onodon rem ained const ant at dissolv ed ox y gen ( DO) concent rat ions abov e 3. 0 t o 4. 0 m l L- 1 at salinit ies 4- 45 ppt and t em perat ures of 20- 30oC.

The av erage pH w as observ ed from 6. 8 t o 7. 7 in bot h cont rol and t reat ed t anks. There w as a significant difference ( p< 0. 05) bet w een t he cont rol and t reat ed t ank s. Law ( 1988) report ed t hat P. m onodon could t olerat e pH dow n t o 6. 0 t o 6. 5. High m ort alit y w as observ ed at pH below 6. 0. I n t he present st udy t he pH lev el w as less in t he cont rol t ank ( 6. 8) and considerably high in ex perim ent al t ank ( 7. 7) . The result s point ed t hat probiot ics (B. m egat erium and S. fradiae) present in t he ex perim ent al t ank s were helpful in m aint aining t he pH at desired lev el.

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diffuse readily across cell m em brane ( From m & Gillet t e 1968; Em erson et al 1975) . The react ion of NH3 depends on pH, t em perat ure and t o a lesser ext ent on salinit y ( Bow er & Bidw ell 1978) . As pH or t em perat ure rises, NH3 increases relat iv e t o NH4+ , and t he t ox icit y of am m onia t o anim als increases ( Chien 1992) . The av erage am m onia lev el ranged bet w een 0. 018 t o 0. 45 m g L- 1 in t he ex perim ent al and cont rol t ank s during t he st udy . There w as a significant difference ( p< 0. 05) bet w een t he cont rol and t reat ed t ank s. Chien ( 1992) report ed t hat t he safe lev els of am m onia for v arious st ages of P. m onodon

are 0. 15, 0. 1 and 0. 08 m g L- 1 for PL3 0 - 50, j uv enile and adolescent respect iv ely .

Sh r im p gr ow t h. The result s obt ained from t he ex perim ent are present ed in Table 3a. Grow t h ( in t erm s of w et w eight gain) w as significant ly higher ( p< 0. 05) in t he t reat ed t han in t he cont rol t ank s. The percent of increm ent w as 100. 10 and 98. 09 in B. m egat erium t reat ed t hrough feed and w at er respect iv ely . The percent increm ent w as 102. 33 and 100. 44 in S. fradiae t reat ed t hrough feed and w at er respect iv ely w hile in t he cont rol group it w as 89. 32 percent ( Table 3b) .

Table 3a Weight gain of P. m onodon post larv aeaft er 60 day s cult ure t reat ed wit h new probiot ics

S. fradiae and B. m egat erium ( m ixed wit h feed and w at er)

Treat m ent

In it ia l w e ig h t w 1 F in a l w e ig h t w 2 W e ig h e d m e a n s (W ) = w 1 + w 2 /2 F e e d c o n s u m e d ( C ) = fe e d g iv e n -u n u ti li z e d fe e d F a e c a l o u tp u t (F ) A s s im il a ti o n ( A ) = F e e d c o n s u m e d fa e c a l m a tt e r (C -F ) P ro d u c ti o n G ro w th (P ) = ( w 2 -w 1 ) M e ta b o li s m ( R ) = C -( P + F )

Cont rol t ank 0. 34 2. 32 1. 33 5. 35 1. 35 4. 00 1. 98 2. 02

S. fradiae

( m ix ed wit h feed) 0. 34 4. 28 2. 31 4. 78 0. 75 4. 03 3. 94 0. 09

S. fradiae

( m ix ed wit h w at er ) 0. 35 3. 90 2. 12 4. 50 0. 85 3. 65 3. 55 0. 10

B. m egat erium

( m ix ed wit h feed) 0. 33 3. 60 1. 96 4. 92 0. 90 4. 02 3. 27 0. 75

B. m egat erium

( m ix ed wit h w at er) 0. 35 3. 44 1. 89 4. 85 0. 88 3. 97 3. 09 0. 88

The relat ive grow t h rat e in shrim p w as higher in t reat ed t ank s t han in cont rol. The best relat iv e grow t h rat es 1. 70 and 1. 67 w ere observ ed in t he t ank inoculat ed wit h S. fradiae

respect iv ely t hrough feed and w at er. The grow t h rat es w ere 1. 66 and 1. 63 respect ively in t he B. m egat erium t reat ed t hrough feed and wat er. How ev er, t he low est growt h rat e 1. 48 w as recorded in t he cont rol t ank ( Table 3b) .

The shrim p show ed high assim ilat ion efficiency ( digest ibilit y ) of 84. 30 and 81. 85% in S. fradiae and B. m egat erium t reat ed t hrough feed and w at er respect iv ely. Assim ilat ion efficiency w as also observ ed at 81. 70 and 81. 11% in B. m egat erium and S. fradiae t reat ed t hrough feed and w at er respect iv ely ( Table 3b) . Qasim & East erson ( 1974) observ ed t he assim ilat ion efficiency ranged from 89 t o 97% in t he shrim p,

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Table 3b FCR and relat iv e grow t h of P. m onodon post larv ae aft er 60 day s cult ure t reat ed wit h

new probiot ics S. fradiae and B. m egat erium ( m ixed w it h feed and w at er)

Treat m ent

A s s im il a ti o n e ff ic ie n c y o r d ig e s ti b il it y = A /C ( % ) G ro s s g ro w th ( c o n v e rs io n ) e ff ic ie n c y ( k1 ) = P /C ( % ) N e t g ro w th ( c o n v e rs io n ) e ff ic ie n c y ( k2 ) = P /A ( % ) F o o d C o n v e rs io n R a ti o = T o ta l fo o d c o n s u m e d /T o ta l li v e w e ig h t in c re a s e d F o o d c o -e ff ic ie n t C /P R e la ti v e G ro w th R a te = P /W x 1 G ro w th R a te = F w - Iw /W x 6 0

Cont rol t ank 74. 76 37. 00 49. 50 4. 02 2. 70 1. 49 89. 32

S. fradiae

( m ix ed wit h feed) 84. 30 82. 42 97. 76 2. 06 1. 21 1. 70 102. 33

S. fradiae

( m ix ed wit h w at er ) 81. 11 78. 88 97. 26 2. 12 1. 26 1. 67 100. 44

B. m egat erium

( m ix ed wit h feed) 81. 70 66. 46 81. 34 2. 51 1. 50 1. 66 100. 10

B. m egat erium

( m ix ed wit h w at er) 81. 85 63. 71 77. 83 2. 55 1. 56 1. 63 98. 09

The gross grow t h ( conv ersion) efficiency in t he shrim ps w as 82. 42 and 78. 88% t hrough feed and w at er respect iv ely in S. fradiae t reat ed t ank . Gross grow t h ( conv ersion) efficiency were 66. 46 and 63. 71% in B. m egat erium t reat ed t hrough feed and w at er respect iv ely ( Table 3b) . The gross conv ersion efficiency w as found v ery high in S. fradiae

t reat ed t anks in com parison t o t he cont rol and B. m egat erium t reat ed t ank s. I n crust aceans, t he report ed gross grow t h ( conv ersion) efficiency w as 4- 68% ( Qasim & East erson 1974) .

The highest v alue of net conv ersion efficiency in shrim ps w as recorded as 97. 76 and 97. 26% in S. fradiae t reat ed t hrough feed and w at er respect iv ely . The net conv ersat ion efficiency w as 81. 34 and 77. 83 in B. m egat erium t reat ed t hrough feed and w at er respect iv ely. Net conv ersion efficiency in t he cont rol t ank w as 49. 50 ( Table 3b) . Qasim & East erson ( 1974) report ed t hat net conv ersion efficiency w as 24 t o 71% in crust aceans. The m axim um food coefficient v alue w as 2. 70 recorded in anim als of t he cont rol t ank , follow ed by t he v alues of 1. 56 and 1. 50 in B. m egat erium and 1. 26 and 1. 21 in S. fradiae t reat ed t ank s t hrough feed and w at er, respect iv ely .

The food conv ersion rat io ( FCR) w as low er in t reat ed t ank s t han in cont rol t ank . The FCR v alues w ere 2. 06 and 2. 12 in S. fradiae t reat ed t ank t hrough feed and w at er respect iv ely w hile 2. 51 and 2. 55 w ere observ ed in B. m egat erium t reat ed t hrough feed and w at er, respect iv ely. The FCR v alue is higher 4. 02 in t he cont rol t ank .

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t he t iger shrim p pond im prov es w at er qualit y grow t h and product ions of P. m onodon. The beneficial effect is at t ribut ed t o t he efficiency of bact eria in ant agonism s t o pat hogen, gut colonizat ion w it h possible adhesion t o int est inal m ucus ( Ev ely n 1996) and t o increase resist ance of t he host t o pat hogens.

The act inom y cet es i. e. St ept om y ces sp. are w ell k now n for t heir product ion of ant ibiot ics, grow t h- prom ot ing subst ances. Therefore, it is probably t hat t he S. fradiae

used in t he present st udy has im prov ed t he grow t h of shrim ps t hrough grow t h prom ot ing subst ances produced and in m aking t he shrim ps healt hy by reducing t he load of pat hogenic vibrios in t he cult ure m edium t hrough ant ibiot ics form ed. I t is t herefore, necessary t o charact erize t he grow t h prom ot ing subst ances and ant ibiot ics produced by t he act inom y cet es t o dev elop t hem as m icrobial product ion for bet t er aquacult ure pract ices.

Moriart y ( 1998) has not ed an increase of praw n surv iv al in t he pond w at er inoculat ed wit h bacillus species. This is at t ribut ed t o t he w at er qualit y im prov ed by t he bact erial inoculat ions because of degradat ion of organic m at t er. This t reat m ent also has decreased t he pat hogenic lum ionous v ibrio species and ot her bact eria ( Maeda 1994) . The ant agonist ic act ivit ies of Bacillus species m ay be m ediat ed by m any inhibit ory subst ances, such as organic acids, hy drogen perox ide ( Ringø & Gat esoupe 1998) . The st rains of bacillus are also k now n t o com pet e for nut rient s and for space for pat hogenic bact eria. These m echanism s of bacillus m ay even prev ent t he em ergence of resist ant st rains of pat hogenic bact eria, a w ell- k now n risk of ant ibiot ics t reat m ent s ( Moriart y 1998) .

Con clusion. The t w o m icrobial species nam ely B. m egat erium and S. fradiae hav e been prov ed t o hav e pot ent ial for t heir use as probiot ics in shrim p aquacult ure t o im prov e w at er qualit y, balance m icrobial populat ion and t o reduce pat hogenic bact eria. This pract ice is a new approach, deserving m ore at t ent ion. There is a great scope for applicat ion of m arine act inom y cet es as probiot ics for a glorious fut ure of aquacult ure.

Ack n ow le dge m e n t. The aut hors are t hank ful t o Professor Dr. Md. Abul Kalam Azad, Dean, Facult y of Science, Univ ersit y of Chit t agong and Professor Dr. Md. Ashraful Azam Khan, Direct or, I nst it ut e of Marine Sciences and Fisheries, Univ ersit y of Chit t agong, Bangladesh for prov iding t he fund t hrough special allocat ion no. 350/ P&D/ 7- 28/ 2012 ( Part - 3) .

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Received: 27 Januar y 2 013. Accept ed: 07 Febr uar y 2 013. Published online: 04 Mar ch 201 3. Aut hor s:

Sheikh Aft abuddin, I nst it ut e of Mar ine Sciences and Fisher ies, Univer sit y of Chit t agong, Chit t agong- 433 1, Bangladesh, e- m ail: aft abim sf@yahoo. com

Abul Kashem , Pr im e Shr im p Hat cher y Lim it ed, Hat cher y Zone, Kolat oly, Cox’s Bazar , Bangladesh, e- m ail: m d. kashem @gm ail. com

Abdul Kader ,I nst it ut e of Mar ine Sciences and Fisher ies, Chit t agong Univer sit y, Chit t agong- 4331, Bangladesh, e- m ail: m akader im s@yahoo. com

Nur ul Azim Sikder , I nst it ut e of Mar ine Sciences and Fisher ies, Chit t agong Univer sit y, Chit t agong- 4331, Bangladesh, e- m ail: sikder im s@gm ail.com

Abdul Hakim , Depar t m ent of Micr obiology, Univer sit y of Chit t agong, Chit t agong- 433 1, Bangladesh, e- m ail: hakim cu@yahoo. com

This is an open- access ar t icle dist r ibut ed under t he t er m s of t he Cr eat ive Com m ons At t r ibut ion License, which per m it s unr est r ict ed use, dist r ibut ion and r epr oduct ion in any m edium , pr ovided t he or iginal aut hor and sour ce ar e cr edit ed.

How t o cit e t his ar t icle:

Imagem

Figure 1.  Sedim ent  sam pling area of t he present  inv est igat ion.
Table 1  Different  phy siological and biochem ical charact erist ics of B.  m egat erium  and  S
Figure 2. Tot al het erot rophic bact eria ( cfu m l - 1 )  observed in cont rol and t reat ed t anks wat er
Table 2   Wat er qualit y  param et ers and t ot al het erot rophic bact eria ( THB)  and Vibrio ( TVC)  count s
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