Performance of Zero Water Discharge Zwd System With Nitrifying Bacteria and Microalgae Chaetoceros Calcitrans Components in Super Intensive White Shrimp Litopenaeus Vannamei Culture

7/26/2019 Performance of Zero Water Discharge Zwd System With Nitrifying Bacteria and Microalgae Chaetoceros Calcitrans

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Open Access

Volume 6 Issue 9 1000359J Aquac Res Development

ISSN: 2155-9546 JARD, an open access journal

Research Article Open Access

Suantika et al., J Aquac Res Development 2015, 6:9

http://dx.doi.org/10.4172/2155-9546.1000359

Research Article Open Access

AquacultureResearch & DevelopmentISSN: 2155-9546J

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Keywords: Zero Water Discharge system; nitriying bacteria;Chaetoceros calcitrans; Litopenaeus vannamei; Super intensive

IntroductionCrustaceans are sources o high quality protein and its production

contributes significantly to the gross national product o Indonesian

fishery sector. National production o crustaceans in Indonesia reached

up to 639,589 tonnes in 2013 with L. vannamei contributed about 60%

o the total production (386,314 tonnes) [1]. From January to June

2014, Indonesia shrimp export reached up to 13,000 tons per month (

almost 156,000 or a year) [2], with penaeid shrimp as the dominating

crustacean commodity, standing at approximately 80% o the total

shrimp export [3].

Because o its significant contribution to the Indonesias economic

growth, the increment o shrimp production has become a national

policy and recently the production is targeted to reach around 700,000tonnes. Although the production tends to increase annually, shrimp

arm productivity in Indonesia is still a big question mark, since this

increment could probably caused by the extensification o shrimp land

arming. In Indonesia and other countries (Vietnam, India, Ecuador,

China, Malaysia, Philippines) most shrimp arming are still cultured

traditionally, using external outdoor earthen pond, with less attention

paid on diseases and water quality control [4]. It is not surprising

that the system cannot maintain an adequate water quality and might

lead to disease outbreak. Later, this condition might contribute to

unpredictable shrimp yield production [5].

One major water quality parameter that contributes to these

conditions is the accumulation o dissolved N-inorganic compound

NH4+ (ammonium) [6]. o overcome this unavorable condition,armers generally conduct a requent water renewal. Unortunately,

due to biosecurity and environmental reasons, water renewal is not

considered practically. Furthermore, water renewal method is alsoknown to cause unstable water quality [7].

Many efforts had been conducted to develop and improve a new

approach in shrimp rearing strategy (e.g.: probiotic, biofloc, RAS). o

this point, the results are still unpredictable in terms o production

stability. Recently, the use o Zero Water Discharge by using nitriying

bacteria and Chlorella has been applied on cultivation o giant

reshwater prawn (Macrobrachium rosenbergii) [8]. Te application o

zero water discharge was reported to help increase survival rate by 10-

20%. Based on this promising result, the development o zero water

discharge system as an alternative zoo technique in super intensive

white shrimp culture needs to be evaluated urther.

Zero water discharge provides an improvement or batch system

and a modification o green water technology, especially in optimizing

the nutrient cycle (inorganic and particulate nitrogen). Te ammonium,

nitrite, and nitrate level can be maintained at acceptable level by

adding nitriying bacteria and microalgae Chaetoceros calcitrans. Tesemicroorganisms have a positive effect on increasing water quality.

Nitriying bacteria can convert ammonium to nitrite (Nitrosomonas)

*Corresponding author: Suantika G, Microbial Biotechnology Research Group,

School of Life Sciences and Technology, Institut Teknologi Bandung, Jalan Ganesha

10, Bandung 40132, Indonesia, Tel: +62-22-251-1575; E-mail: [email protected]

ReceivedMay 15, 2015; AcceptedJune 06, 2015; PublishedAugust 15, 2015

Citation: Suantika G, Lumbantoruan G, Muhammad H, Azizah FFN, Aditiawati

P (2015) Performance of Zero Water Discharge (ZWD) System with Nitrifying

Bacteria and Microalgae Chaetoceros calcitransComponents in Super Intensive

White Shrimp (Litopenaeus vannamei) Culture. J Aquac Res Development 6: 359.

doi:10.4172/2155-9546.1000359

Copyright: 2015 Suantika G et al. This is an open-access article distributedunder the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the

original author and source are credited.

Abstract

This research was aimed to study the performance of Zero Water Discharge (ZWD) by using nitrifying bacteria

and microalgae Chaetoceros calcitransin super intensive white shrimp (Litopenaeus vannamei) culture. The study

consists of three consecutive steps: (1) activating and cultivating of nitrifying bacteria and microalgae C. calcitrans,

(2) conditioning of zero water discharge system, and (3) using ZWD during shrimp culture along with control (a

conventional system in which the water was renewed every four weeks and without the addition of nitrifying bacteria

and C. calcitrans). Based on water quality parameters, low and stable NH4+ (0.070.69 mg/L), NO2-(03.2 mg/L),NO

3-(1.044.29 mg/L) were obtained in both systems during culture period. Higher feed amount of 1178.28 g in

ZWD system compared to the conventional one contributed to a same level of NH 4+and NO2-level during 90 days

culture period. At the end of the period, higher culture performance in terms of total weight (923.38 42.15 g), mean

body weight (8.24 0.84 g), survival rate (90.82 2.5%), specic growth rate (7.7 0.11%) and feed conversion

ratio (1.27 0.29) was obtained in ZWD, while in control the gures were signicantly different: total weight (160.48

6.62 g), mean body weight (5.45 0.28 g), survival rate (27.22 2.09%), specic growth rate (7.24 0.05%), and

feed conversion ratio (4.10 0.66). Based on this research, Zero Water Discharge system was able to maintain a

stable and an acceptable water quality for shrimp culture. Furthermore, it resulted in better shrimp growth, higher

survival rate, as well as lower FCR in high shrimp density.

Performance of Zero Water Discharge (ZWD) System with Nitrifying

Bacteria and Microalgae Chaetoceros calcitransComponents in SuperIntensive White Shrimp (Litopenaeus vannamei) CultureSuantika G*, Lumbantoruan G, Muhammad H, Azizah FFN and Aditiawati P

Microbial Biotechnology Research Group, School of Life Sciences and Technology, Institute Technology Bandung (ITB), Jalan Ganesha No. 10 Bandung 40132,

Indonesia
http://dx.doi.org/10.4172/2155-9546.1000359http://dx.doi.org/10.4172/2155-9546.1000359http://dx.doi.org/10.4172/2155-9546.1000359http://dx.doi.org/10.4172/2155-9546.1000359


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Citation: Suantika G, Lumbantoruan G, Muhammad H, Azizah FFN, Aditiawati P (2015) Performance of Zero Water Discharge (ZWD) System with

Nitrifying Bacteria and Microalgae Chaetoceros calcitransComponents in Super Intensive White Shrimp (Litopenaeus vannamei) Culture.

J Aquac Res Development 6: 359. doi:10.4172/2155-9546.1000359

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and nitrite to nitrate (Nitrobacter) [9], while biomass o microalgaehas an important role in nitrate reduction and as ood supplement orshrimps [10].

Materials and Method

Activation and cultivation of nitrifying bacteriaC. calcitrans

Culture o nitriying bacteria was obtained rom the Laboratoryo Microbiology SIH IB. Nitriying bacteria was activated in liquidmedium o Winogradsky and scaled up to a volume o 100 L as

expressed in Figure 1 [8].

Culture o C. calcitransthat were used in this study was obtainedrom the Laboratory o Aquatic Ecosystem Analysis SIH IB. C.calcitranswas activated in Guillard medium and scaled up to a volumeo 40 L as il lustrated in Figure 2 [11].

Acclimatization of white shrimp post larvaeWhite shrimps PL-10 used in this study were obtained rom P.

Suri ani Pemuka Indramayu, West Java and acclimatized at roomtemperature (25 1C) at the Laboratory o Aquatic EcosystemAnalysis, School o Lie Sciences and echnology, Institut eknologi

Bandung (SIH IB), Indonesia to reach the PL-15 stadia.

Conditioning of zero water discharge system

Te study was conducted in cylindrical fiber tank filled with 300L sterilized and filtered seawater at 30 ppt salinity. As illustrated inFigure 3, ZWD system consists o our compartments: (1) microbial

component: nitriying bacteria and microalgae C. calcitranswhich playa role in nitrogen cycle o the culture, (2) aeration line to provide and

maintain dissolved oxygen level and allow culture homogenization,

(3) paranet cover to reduce light illumination and enable the system to

promote better growth o diatom C. calcitransand nitriying bacteria,(4) filtered and sterilized culture water as shrimp growth media, (5)eeding tray to control amount o eed delivered daily, and (6) CaCO

3

gravel that is used as substrate or nitriying bacteria colonization andas pH buffering agent.

Beore starting the culture, the ZWD system was conditioned by

inoculating 10 L o nitriying bacteria (at 106CFU/ml) and 12 L o C.

calcitrans (at 106cells/ml) into the ZWD systems and total volume is300 L. Te ammonium breakdown capacity o nitriying bacteria was

conditioned by adding 3 g o NH4Cl ( 10 mg/L o ammonium) to thesystem. Te conditioning was perormed until the NH

4+and NO

2-level

decreased to approximately 0 mg/L.

Treatment implementation

Tere were two treatments done in triplicate or this research:

conventional culture (without any addition o nitriying bacteria and

C. calcitransand 80% o the water volume was renewed once in our

weeks) as the control, and ZWD system (with the addition o nitriying

bacteria and C. calcitrans). In order to compensate water losses due to

siphoning and evaporation, every two weeks six liters (2% o culture

volume) new seawater was added into the culture system. Afer the

conditioning, PL-15 shrimp was stocked to the conventional and ZWD

culture with stocking density o 120 ind/0.3m3

which reers to the superintensive stocking density (400 ind/m3) [12]. Every two weeks, 5 L

o nitriying bacteria and 12 L o microalgae C. calcitrans were added

at a density o 106 CFU/ml and 106cells/ml, respectively, into

the ZWD system. Te experiment was conducted or 90 days culture

period.

Feeding management

Te amount o eed was adjusted weekly based on measurement omean body weights, estimation o survival rate, and eeding rate (

1). Te ormula or eeding calculation is :

eed (gr) =SD MBW FR SR

where : SD is stocking density (individual/tank), MBW is meanbody weight (gr), SR is Survival rate (%), FR is eeding rate (%)

Feed was placed on eeding tray and monitored requently to

Figure 1:The bioreactor used for upscalling the nitrifying bacteria.

Figure 2:Scheme of batch system of C. calcitrans.

Figure 3:Schematic overview of ZWD system for white shrimp culture (L.

vannamei).
http://dx.doi.org/10.4172/2155-9546.1000359http://dx.doi.org/10.4172/2155-9546.1000359


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provide inormation o daily delivered eed accuracy (able 1). Te sizeand condition o shrimp can be checked and their consumption rateestimated based on the lef-over eed in the tray. Te eeding schemewas modified rom acon [13]. Feed were delivered five times a day at08:00 am, 11:00 am, 14:00 pm, 17:00 pm and 20:00 pm.

Physical and chemical water parameters measurement

Physical parameters:DO and temperature were measured everydayusing a digital meter Hach 40 qd, and pH level using a pH meterEutech Instruments. Chemical parameters: ammonium, nitrite, nitrateand orthophosphate were measured twice a week by using Nessler,Diazotation, Nitrate HCl, and Stannous Chloride methods, respectively[14].

Biological and Microbiological Parameter Measurement

Biological parameters: otal weight, mean body weight (MBW),specific growth rate (SGR), survival rate (SR) and eed conversion ratio(FCR) were measured during culture period. Te survival rate o theshrimp was calculated by using the ollowing equation [8]:

SR=Nt/N

o100%

where: SR is survival rate , Nois initial shrimp number, Ntis finalshrimp number, t is culture period (day).

Shrimp specific growth rate was calculated by using the ollowingequation:

SGR (%/day)=[ln (W2/W

1)/(

2-

1)100]

where: SGR is specific growth rate, W1is initial body weight (g) at

time 1(day), and W

2is final body weight (g) at time

2(day)

Microbiological parameters were measured by total bacterial countor total plate count method [15]. otal bacteria was counted every weekby plating water sample on nutrient agar (NA) medium, while totalVibriosp. count was conducted every our weeks using thiosulphate

citrate bile salt agar (CBS) medium [16].

Results and Discussion

Conditioning of zero water discharge system

During conditioning period, it was observed that nitriyingbacteria was able to convert 10 mg/L o ammonium to 0 mg/L in 5days (average o breakdown capacity was 2 mg/L per day) as shownin Figure 4. Te conversion o ammonium to nitrite can be seen romthe increment o nitrite concentration which reached 3 mg/L on day 5and remained steady until day 15 o culture period (Figure 4). By day17, the nitrite level had plummeted to nearly 0 mg/L. As a final producto nitrification, nitrate had been accumulated, in which the level wasaround 60 mg/L by the end o culture period (day 17).

Conditioning process becomes a crucial step prior to shrimpstocking and culture. Tis step enables ZWD system to convert toxicNH

4+and NO

2-into less harmul substances NO

3-. From the result, it

can be seen that ammonium oxidizing bacteria was activated on day 5o conditioning period. However, longer conditioning period o 17 dayswas taken to activate nitrite oxidizing bacteria. For nitrite oxidizing

bacteria, longer activation period is due to slower growth or doublingtime o nitrite oxidizing bacteria compared to ammonium oxidizingbacteria [9]. As the second step o nitrification process, NO

2- oxidation

takes place afer NH4

+ oxidation reaction. Tis consecutive reactionalso contributes to slower activation process or nitrite oxidizingbacteria since the reaction depends heavily on NO

2-availability o the

first reaction [9].

Even though the nitrification process was able to be achievedduring conditioning period, a long conditioning period (17 days)should be considered as a constraint in ZWD system because it will

require a longer culture period to run one shrimp production cycle.One alternative solution to overcome this disadvantage is a betteractivation process o nitrite oxidizing bacteria in stock culture during

maintenance, prior to being inoculated into ZWD system.

Physical and chemical parameters measurement

Te range o ammonium, nitrite and nitrate concentrationsdocumented in conventional and ZWD rearing technique are presented

in able 2. Ammonium, nitrite and nitrate concentrations were still intolerance levels or white shrimp in both rearing strategies [17-19].

Te trend or ammonium, nitrite and nitrate concentration during90 days o culture period is presented in Figure 5. It is noticeable that

the ammonium, nitrite and nitrate level tended to slightly increase. InZWD system, ammonium, nitrite and nitrate levels ranged between0.070.69 mg/L, 03.15 mg/L, 1.0442.9 mg/L, respectively, while in

conventional (batch) system, it ranged between 0.200.59 mg/L, 03.2

mg/L, 1.3814.17 mg/L, respectively. Based on these levels, all dissolvedN-inorganic levels measured during 90 days culture period were not

significantly different in both rearing systems (p>0.05). In spite o thiscondition, the breakdown capacity o ZWD system on NH

4+and NO

2-

levels were higher compared to conventional (batch) system. Tis isbecause a higher amount o eed ( 1178.28 g) was delivered to ZWDsystem; around 44% higher than that o conventional system ( 656.15

g). Tis contributed to a higher organic matter accumulation and

Mean Body

Weight (gr)Feeding Rate (%) Survival Rate (%)

Feeding Tray

Monitoring

Intervals (hours)


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thereore had an effect on higher ammonium (NH4

+) accumulationin the system [20]. Based on [21] estimation, it was assumed that theamount o eed given to shrimp in ZWD or 90 days could produce atotal NH

4+o 55.20 mg/L in shrimp culture which is equivalent to 0.61

mg/L per day. In contrast, total ammonia produced by shrimp or 90days in conventional system was 30.73 mg/L, which is equivalent to 0.34mg/L per day (total o 656.15 g eed delivery). From this estimation, itcan be stated that the ammonium breakdown capacity with averageammonium conversion rate o 2 mg/L/day was higher in ZWD system

than that o conventional culture system.

Better ammonium and nitrite conversion rate were determinedby measuring higher nitrate accumulation afer 90 days cultureperiod (Figure 5c). At the end o culture period, nitrate level o 42.9mg/L at ZWD system was significantly higher (p


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reduce the oxygen budget o the system. From the result, it seemed thatthe dissolved oxygen budget could be covered by oxygen production omicroalgae and constant aeration in ZWD system [32].

Microbiological parameter measurement

otal bacteria counted in conventional and ZWD or 12 weeksculture period is given in Figure 7.

Based on the results, total bacteria count during the trial tended toincrease in both rearing system. In ZWD, maximum number o otalBacterial Count and total Vibriosp. were 1 1010CFU/ml and 4.8 102 CFU/ml, respectively, while in conventional system the figuresstood at 6.7 1010CFU/ml and 1.05 102CFU/ml, respectively able3. Tere were no significant differences on otal Bacterial Count andtotal Vibrio sp. obtained during culture period between ZWD andconventional culture system (p>0.05).

It is noticeable that excessive organic matter accumulation inZWD system did not significantly increase the number o bacteria,

despite it has been widely known that the availability o higher organiccontent could promote the growth o heterotrophic bacteria [33]. Tepresence o nitriying bacteria and C. calcitranswas well documented

in controlling the growth o opportunistic bacteria and indigent Vibriosp. in shrimp culture. Nitriying bacteria can compete with otherbacteria in terms o space and oxygen [34] and marine diatom secrete

atty acids and esters that can act as antibacterial compounds [35]. Itis not surprising that the total Vibrio sp. in ZWD system was belowpathogenicity level o 106CFU/ml [36], even when shrimp culture in

ZWD contained higher shrimp density and higher input o eed.

A low total number o bacteria in conventional system are suspected

due to regular dilution by water renewal (80 % every our weeks) andalso due to less organic matter load o eed and biological wastes (lessshrimp density) accumulated during the culture period.

Biological parameter measurement

Shrimp culture productivity was observed based on mean bodyweight (MBW), total weight, survival rate (SR), specific growth rate

(SGR) and eed conversion ratio (FCR) measurement able 4.

In ZWD system, better shrimp culture perormance was obtainedafer 90 days culture period. Measurements o biological parameters,including mean body weight (MBW) o 8.24 0.84 g, total weighto 923.38 42.15 g, Survival Rate (SR) o 90.82 2.5%, and specificgrowth rate (SGR) o 7.7 0.11%, were significantly higher in ZWDthan those o conventional culture system (MBW: 5.45 0.28 g, otalWeight: 160.48 6.62 g, SR: 27.22 2.09%, and SGR 7.24 0.05). TeFCR in ZWD (1.27) were significantly lower than that o conventionalculture system (4.10) (p


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Besides the inormation on physico-chemical, microbiologicaland biological parameters, a cost estimation o shrimp production

using both systems also become an important step that needs to be

conducted beore urther application o this new culture approachin aquaculture industry. Estimation was calculated in assumption to

produce 100 kg shrimp during 90 days culture period, based on shrimpculture perormance (total weight, FCR, MBW, SR) obtained rom thisresearch, seed cost, eed cost and water consumption as shown in able 5.

Te calculation was made based on biological parameters obtainedduring 90 days culture period. Better shrimp growth and survivalimplied in better and lower FCR in ZWD which resulted in profit by

2,375,240 IDR. Based on this analysis, the use o zero water dischargesystem or super intensive shrimp rearing technology can improvethe profit o the culture compared to the conventional batch systemwith the same initial shrimp density. Tis economic advantage was

due to better culture perormance on SR, MBW, and FCR measured

in ZWD system. During culture period, the use o ZWD system alsoconsumed less water volume (52.1 m3) compared to conventional batchsystem (78 m3) to produce 100 kg o shrimps as shown in able 4. Tiscondition shows that the use o ZWD system will not only contribute

in sustaining a more cost effective system, but also in minimizingwater quality disturbance and reducing wastewater release into theenvironment.

Conclusion

Based on this research, Zero Water Discharge system can be used asan alternative or shrimp culture since ZWD was able to enhance whiteshrimp culture perormance (water quality, SR, growth and FCR).

Acknowledgement

We would like to thank Mr. Tirsa and PT . SuriTani PemukaunitIndramayu-

Japfa Group for the shrimp larvae used in this research.

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Citation: Suantika G, Lumbantoruan G, Muhammad H, Azizah FFN,

Aditiawati P (2015) SPerformance of Zero Water Discharge (ZWD) System

with Nitrifying Bacteria and Microalgae Chaetoceros calcitransComponents inSuper Intensive White Shrimp (Litopenaeus vannamei) Culture. J Aquac Res

Development 6: 359. doi:10.4172/2155-9546.1000359
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Documents

Performance of Zero Water Discharge Zwd System With Nitrifying Bacteria and Microalgae Chaetoceros Calcitrans Components in Super Intensive White Shrimp Litopenaeus Vannamei Culture