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* Corresponding author: Srinivas, RDepartment of Zoology, College of Science and Technology, Andhra University, Visakhapatnam-530 003

ISSN: 0976-3031

RESEARCH ARTICLEIMPACT OF PROBIOTICS ON CULTURE PONDS OF PENAEID SHRIMPS, AT EAST COAST OF

ANDHRA PRADESH, INDIA

*1Srinivas, R., 1Manjulatha, C and 2Ramesh Babu, K

1Department of Zoology, College of Science and Technology, Andhra University, Visakhapatnam-530 0032Department of Marine Living Resources, Andhra University

ARTICLE INFO ABSTRACT

The current study was aimed to focus on the probiotics effect on shrimp culture ponds. The

study evaluates the probiotics effects on three ponds one control pond and two experimental

ponds during two consecutive years 2009 to 2010. The TVC count in water and sediments inPond A and Pond B in summer and winter for the year 2009 was recorded as 2.33 10 0.16, 2.08 10 0.12, 2.68 10 0.15, 2.43 10 0.23, 2.33 10 0.30, 1.75 10 0.16, 2.77 10 0.31, 2.02 10 0.15.and the TVC count in water and sediments in Pond

A and Pond B in summer and winter for the year 2010 was recorded as 2.85 10 0.19,0.35 10 0.53, 3.65 10 0.21, 1.32 10 0.12 ,0.29 10 0.47, 0.27 10 0.44,2.67 10 0.35, 0.19 10 0.15 at 120 days of culture period. The maximum TVC countwas noticed in culture pond was 94 10 0.30, 26.5 10 0.19 during summer 2009 and

2010 respectively. The statistical analysis of the present study revealed that there was asignificant difference in Vibrio count was observed in between control and experimental

ponds.

INTRODUCTION

In aquaculture environment the bottom gets deteriorated by leftover feed due to excess feeding, fecal matter and dead algae.Anaerobic conditions set in due to oxygen depletion and in

turn causes increase in the levels of carbon dioxide anddecrease in p

Hlevels. The management of the culture pond

without causing stress to shrimp is the right approach for

successful crop. The major functions of the probiotics are todegrade organic waste in pond bottom, reduced accumulated

bottom sludge, and accelerate the removal of hydrogensulphide, ammonia, nitrites in pond water and suspended solidsreduction.

Microbial diseases occur at all stages of shrimp aquaculture

and are responsible for considerable economic losses(Leightner, 1996; Moriarty, 1998; Sung et al., 2001).Probiotics are scientifically blended concentration of selected,adopted and cultured bacterial formulations plus enzymes and

special buffers fermented which cereal and mineral substratefor use in shrimp farming (Ravichandran and ShaickJalaluddin, 1999). The use of probiotics in aquaculture mayproduce various beneficial effects has been proven beyonddoubt (Balcazar et al., 2006; Kesarcodi-Watson et al., 2008).

The research on use of probiotics in aquatic animals isincreasing with the demand for environment friendlyaquaculture (Gatesoupe, 1999). Improved water quality has

especially been associated with probiotics it has been reportedthat use ofBacillus sp. improved water quality, survival andgrowth rates and the health status of the juvenile Penaeusmonodon and reduced the pathogenic Vibriosis (Dalmin et al.,

2001). Use of commercial probiotics in Penaeus vannamei

ponds can reduce concentrations of nitrogen and phosphorusand increase the shrimp yields (Wang et al., 2005). Recently,

Nimrat et al., (2011) developed an effectively novel probioticfor rearing of L. vannamei through microencapsulated orfreeze-dried forms of microbial probiotics: bacteria, yeast and

microalgae. The current study has taken up by application ofprobiotics in the shrimp culture pond ecosystem in

consideration of frequency and dosage of probiotics on pondwater and sediments in one control and two experimentalponds both in summer and winter seasons during two

consecutive years 2009 to 2010.

MATERIALS AND METHODS

The present study was carried out in coastal area of AndhraPradesh, India. The study area Amplam is located 18 20Nlatitude and 84 10E longitude in Srikakulam district wherethe control pond and experimental pond A & pond B were

located. The studies were conducted on the effect of the soiland water probiotics in the culture ponds for the analysis of thetotal Vibrio count as a comparative data between the controlpond and experimental pond A & pond B. The required waterand sediment samples for analysis from the control ponds and

experimental culture ponds of the farms were collected in airtight plastic bottles and these water samples were given tonearby laboratories for bacterial analysis. The experiments

were performed for two consecutive years 2007 & 2008 inboth summer and winter seasons for four crops in the controland experimental ponds. The total Vibrio count analysis in thelaboratories were done in the ZMA (Zobell Marine Agar) and

Available Online at http://www.recentscientific.com

International Journal

of Recent Scientific

ResearchInternational Journal of Recent Scientific ResearchVol. 4, Issue, 7, pp.1050 1055, July, 2013

Article History:

Received 16th, June, 2013

Received in revised form 28th, June, 2013

Accepted 17th, July, 2013

Published online 30th July, 2013

Copy Right, IJRSR, 2013, Academic Journals. All rights reserved.

Key words:Probiotics, Vibrio, Penaeus monodon, Sediment.

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International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 1050 - 1055, July, 2013

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TCBS (Thiosulphate Citrate Bilesucrose Agar) medium understrict and hygienic microbiological laboratory conditions at

M/s. Lotus Ganges Laboratories, Visakhapatnam. The waterparameters of salinity, pH, temperature, observed andmaintained during the study period of two years in both

summer and winter crops of both the seasons from the cultureponds. The water parameters were observed at the study pondsby electronic meters. The salinity readings were taken by

ATAGO refractometer, the temperature was recorded by theThermometer, and the p

Hreadings were taken by Hanna p

HPen

- Scan-2.

Statistical analysis of the data

The statistical package used for interpreting the available datais microcal-origin pro 8.0 Version for evaluation of the total

Vibrio count (TVC) by taking the values from the average datadrawn during study period. The results were evaluatedstatistically to observe the decrease of total Vibrio count withapplication of probiotics in the experimental ponds andwithout probiotics in the control ponds. Significant reduction

of the TVC in the pond water and sediment was observed.

RESULTS

The present experiment was carried out on the frequency anddose of application of the soil & water probiotics and the

effect on the pond water & sediment by recording the resultsof one control and two experimental ponds with respect to thetotal Vibrio count (TVC) observed periodically before

stocking of post larvae and at every 30days interval in the cropun till the harvest. In the present study at Amplam during theculture period in the summer crop of year 2009, the physico-chemical parameters such as salinity, p

H, and temperature of

the study ponds ranged between 16-22 %, 7.9-8.5, and 31-

33C respectively. Total Vibrio count (TVC) of the pond waternoticed as 2.3310 0.16 cfu ml

-1and 2.0810 0.12 cfu

ml-1 in the experimental ponds A & B respectively at 120 daysof culture. In the control pond, the TVC is recorded as

1.5710 0.40 cfu ml-1

and 2.4010 0.13 cfu mg-1

in thepond water and sediment respectively at 90 days of culture(DOC).

The shrimp P.monodon in this control pond was harvested at22.5 g on 107

thday with the incidence of white spot disease.

At the same time the TVC in the in the sediment of the

experimental ponds A & B, were recorded as 2.6810 0.15cfu mg

-1and 2.4310 0.23 cfu mg

-1respectively at 120 days

of culture. These experimental ponds A & B were harvestednormally at 28.0 g & 29.0 g on 125th day respectively. (Table

1, Fig 1).

The physico-chemical parameters in the winter crop during the

year 2009 such as salinity, pH, and temperature of the study

ponds ranged between 15-20 , 7.5-8.6, and 25-28Crespectively. The TVC results recorded as 3.3710 0.80 cfuml

-1, 2.3310 0.30 cfu ml

-1and 1.7510 0.16 cfu ml

-1in

water of control, experimental pond A & pond B at 90 days &120 days of culture respectively .

Where as in the sediment the total Vibrio count is noticed as

3.6810 0.20 cfu mg-1

, 2.7710 0.31 cfu mg-1

and2.0210 0.15 cfu mg-1 in control, experimental pond A &

pond B respectively at 90 & 120 days of culture.

Fig 1 Total Vibrio Count of culture ponds at Amplam

during summer 2009

Fig 2 Total Vibrio Count of culture ponds at Amplam

during winter 2009

Fig 3Total Vibrio Count of culture ponds at Amplam during summer2010

Fig 4 Total Vibrio Count of culture ponds at Amplam during

winter 2010

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Table 1 Total Vibrio Count of culture ponds at Amplam during summer 2009 Control pond

S.No Days of culture Salinity ppt pH Temp C TVC Water cfu/ml TVC Sediment cfu/mg

1 Before stocking 19 7.9 31 NIL NIL

2 30 20 8.3 32 94 10 0.30 33.7 10 0.16

3 60 20 8.3 32 0.23 10 0.32 0.20 10 0.19

4 90 19 8.5 32 1.57 10 0.40 2.40 10 0.13

5 120 - - - - -Control pond harvested due to white spot disease at 22.5 g on 107 th day

Pond A

S.No DOC Salinity ppt pH Temp C TVC Water cfu/ml TVC Sediment cfu/mg

1 BEFORE 19 7.9 31 NIL NIL

2 30 DOC 21 8.2 32 0.15 10 0.26 0.12 10 0.19

3 60 DOC 20 8.3 32 0.23 10 0.32 0.20 10 0.19

4 90 DOC 19 8.5 32 1.57 10 0.40 2.40 10 0.13

5 120 DOC 16 8.4 31 2.33 10 0.16 2.68 10 0.15

Experimental pond A harvested normally at 28 g on 125th day

Pond B



2 30 DOC 21 8.2 32 NIL 0.10 10 0.35

3 60 DOC 20 8.3 32 0.18 10 0.13 0.15 10 0.244 90 DOC 20 8.4 31 0.34 10 0.14 2.38 10 0.19

5 120 DOC 17 8.5 32 2.08 10 0.12 2.43 10 0.23Experimental pond B harvested normally at 29 g on 125 th day

Table 2 Total Vibrio Count of culture ponds at Amplam during winter 2009 Control Pond



2 30 DOC 18 7.9 26 0.20 10 0.48 2.82 10 0.75

3 60 DOC 19 8.2 25 2.35 10 0.30 2.95 10 0.39

4 90 DOC 19 8.6 25 3.37 10 0.80 3.68 10 0.20

5 120 DOC - - - - -Control pond harvested due to white spot disease at 16 g on 94th day

Pond A

S.No DOC Salinity ppt pH Temp C TVC Water cfu/ml TVC Sediment cfu/mg1 BEFORE 15 7.9 28 NIL NIL

2 30 DOC 17 8.2 27 0.11 10 0.10 NIL

3 60 DOC 18 8.1 27 0.31 10 0.52 0.27 10 0.29

4 90 DOC 19 8.4 25 1.78 10 0.38 2.45 10 0.27

5 120 DOC 20 8.3 25 2.33 10 0.30 2.77 10 0.31Experimental pond A harvested normally at 26 g on 124 th day

Pond B

S.N0 DOC Salinity Ppt pH Temp C TVC Water cfu/ml TVC Sediment cfu/mg


2 30 DOC 17 7.9 27 0.10 10 0.13 NIL

3 60 DOC 18 8.2 26 0.22 10 0.15 0.19 10 0.22

4 90 DOC 19 7.9 26 1.03 10 0.45 1.00 10 0.15

5 120 DOC 20 8.1 25 1.75 10 0.16 2.02 10 0.15Experimental pond B harvested normally at 27 g on 125 th day

Table 3 Total Vibrio Count of culture ponds at Amplam during summer 2010 Control Pond

S.No DOC Salinity Ppt pH Temp C TVC Water cfu/ml TVC Sediment cfu/mg


2 30 DOC 23 8.5 31 0.28 10 0.41 0.39 10 0.43

3 60 DOC 25 8.7 32 1.55 10 0.19 2.55 10 0.37

4 90 DOC 29 8.3 32 17.8 10 0.46 2.95 10 0.16

5 120 DOC 31 8.5 28 53.3 10 0.77 70.8 10 1.11Control pond harvested normally at 31.5 g on 137th day

Pond A

S.No DOC Salinity Ppt pH Temp C TVC Water cfu/ml TVC Sediment cfu/mg


2 30 DOC 22 8.5 30 0.16 10 0.19 0.27 10 0.46

3 60 DOC 26 8.7 31 NIL NIL

4 90 DOC 28 8.8 31 0.29 10 0.61 0.30 10 0.365 120 DOC 30 8.7 29 2.85 10 0.19 3.65 10 0.21

Experimental pond A harvested normally at 35 g on 139 th day

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The shrimp P.monodon cultured in the control pond isharvested at 16.0 g on 91st day with white spot disease and the

experimental ponds A & B were harvested normally at 26.0 g& 27.0 g on 124

thday & 125

thday respectively. (Table 2, Fig

2). The salinity, pH, and temperature of the study ponds were

recorded between 19-31 , 8.2-8.8, and 28-32C respectivelyin the summer crop of year 2010. The total Vibrio colonies inthe pond water were noticed as 53.310 0.77 cfu ml -1,2.8510 0.19 cfu ml

-1and 0.3510 0.53 cfu ml

-1in

control, experimental pond A & pond B respectively. In thesediment of the ponds the TVC is recorded as 70.810 1.11

cfu mg-1

, 3.6510 0.21 cfu mg-1

and 1.3210 0.12 cfumg

-1in control, experimental pond A & pond B respectively.

All the above TVC readings were recorded at 120 days of

culture period. Here in this crop the shrimp P.monodon in thecontrol pond, experimental pond A & pond B were harvestednormally at 31.5 g, 35.0 g, and 36.5 g on 137 th day, 139th day

& 141st

day respectively. (Table 3, Fig 3). In the winter crop ofthe year 2010, The parameters like salinity, pH, andtemperature of the study ponds ranged between 14-18 , 7.4-

8.9, and 25-28C respectively. The total Vibrio count in thepond water was recorded as 26.510 0.19 cfu ml

-1, 0.2910

0.47 cfu ml-1 and 0.2710 0.44 cfu ml-1 in control,

experimental pond A & pond B respectively in the sedimentthe readings were noticed as 5.1210 0.33 cfu mg

-1,

2.6710 0.35 cfu mg-1

and 0.1910 0.15 cfu mg-1

respectively in control,experimental pond A & pond B. The TVC values were

observed at 60 days in control pond and at 120 days inexperimental pond A & pond B. The control pond is harvestedon 64th day at 7.0 g which is harvested due to the cross

contamination by white spot disease carried from theneighboring farm. In this crop of shrimp P.monodon theexperimental pond A & pond B were harvested normally at30.5 g & 32.0 g on 129

thday & 130

thday respectively. (Table

4, Fig 4).

DISCUSSION

In present study great focus was done on the frequency and

dosage of application of the soil & water probiotics and theeffect on the pond water and sediment. The results of thepresent study have shown that positive benefits of probiotics towater and bottom soil quality. Boyd et al., (1994) found thatthe accumulated sediments, in shrimp ponds in Thailand werelargely composed of mineral soil eroded from the pond banks.Smith 1996 found that 70-80% of the accumulated sediments

in prawn ponds were a mixture of quartz, kaolinite and mica-illite mineral 5-10%, amorphous iron and aluminum & siliconoxides, 5-10% organic matter and volatile compounds and

elevated levels of trace elements. Smith (1996) did not find arelationship between sediment accumulation and factors suchas organic inputs, pond management or productivity. Hopkins

et al., (1994) working in plastic lined ponds, suggested that thesource of the accumulated sediments in a pond are uneatenfood faces, decaying plankton, air borne debris, eroded soil

Pond B



2 30 DOC 22 8.4 30 NIL 0.16 10 0.19

3 60 DOC 26 8.6 31 NIL NIL

4 90 DOC 28 8.8 32 NIL NIL

5 120 DOC 30 8.7 28 0.35 10 0.53 1.32 10 0.12Experimental pond B harvested normally at 36.5 g on 141 st day

Table 4 Total Vibrio Count of culture ponds at Amplam during winter 2010 Control Pond



2 30 DOC 17 8.6 27 8.97 10 0.38 2.65 10 0.19

3 60 DOC 18 8.9 26 26.5 10 0.19 5.12 10 0.33

4 90 DOC - - - - -

5 120 DOC - - - - -Control pond harvested due to white spot disease at 7 g on 64 th day

Pond A



2 30 DOC 15 8.5 27 0.14 10 0.22 NIL

3 60 DOC 16 8.4 26 NIL NIL4 90 DOC 17 8.3 26 0.21 10 0.25 2.17 10 0.50

5 120 DOC 17 8.5 26 0.29 10 0.47 2.67 10 0.35

Experimental pond A harvested normally at 30.5 g on 129 th day

Pond B



2 30 DOC 16 8.2 27 NIL NIL

3 60 DOC 16 8.5 26 NIL NIL

4 90 DOC 17 8.4 26 0.12 10 0.14 0.17 10 0.14

5 120 DOC 16 8.6 25 0.27 10 0.44 0.19 10 0.15Experimental pond B harvested normally at 32 g on 130 th da

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and microorganisms. Smith (1993) found that the accumulatedsediments in prawn ponds contained large amounts of silica

and that diatoms were significant part of the sediments.Accumulated sediments in shrimp ponds are highly reduced(Guo, 1986) enriched in organic matter (Boyd and Tucker

1992; Martin et al., 1998). Ram et al., (1982) found that thedensity of aerobic and anaerobic bacteria in the pond bottomsoil is 2 to 4 orders of magnitude higher than the density of

these groups in the water column. Moriarty (1996) found thatthe bacterial count in the sediments of shrimp ponds fertilizedwith chicken manure was 4x1013 bacteria m-2 which was 2 to 3

orders of magnitude higher than in the water column. Burford

et al., (1998) reported a bacteria count of 15.5x109

cellsg-1

inthe centre of shrimp pond where sludge accumulated, and

8.1x109

cellsg-1

at the periphery of the pond.

The transience of aquatic microbes may legitimate theextension of the probiotic concept to living microbialpreparations use to treat aquaculture ponds. Moriarty (1998)

proposed to extend the definition of probiotics to microbialwater additives. However these extensions would make too

vague definition of Tannock (1997). In 1991 Porubcanreported on two attempts at bacterial treatments to improvewater quality and production yield ofPenaeus monodon. Forthis Porubcan made floating biofilters, pre-inoculated with

nitrifying bacteria decreased the amounts of ammonia andnitrite in the rearing water. This treatment increase shrimpsurvival. The introduction ofBacillus spp., in proximity to

pond aerators reduced chemical oxygen demand (COD) andincreased shrimp harvest (Porubcan, 1991).

In the present study by application of probiotics the twoexperimental ponds showed positive results in the shrimp

production when compared to the control ponds. The watersamples of all the ponds were analyzed periodically for total

Vibrio colonies and the findings were revealed and the total

Vibrio colonies were increased in control ponds of bothseasons in the two years of study period. There is significantreduction in the total Vibrio colonies in the experimental ponds

in both pond water and pond sediment. It is evident from theobtained results from the present study that the reduction of thetotal Vibrio colonies with increased dosage of the pondprobiotic and also by increase the frequency of applicationfrom 15 days duration to 7 days duration. The positive bacteriain the probiotics reduced the proliferation of the pathogenic

bacteria in all the ponds where the application of probiotics isfollowed. At the same time due to the probiotic application thetoxic gases of the pond bottom were reduced and the oxidation

of the organic matter is observed in the culture ponds.

CONCLUSION

The results of the current study suggested that the decreasedTVC count was recorded when the probiotics were used for thecurrent experimentation for two consecutive years of the study

both in summer and winter seasons. The significant differencein TVC values were noticed between control and experimentalponds.

Acknowledgements

The author express thank to the Head, Department of Zoology,

Andhra University for carried out this research work

References

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