Performance of Zero Water Discharge (ZWD) System with Nitrifying Bacteria and Microalgae Chaetoceros calcitrans Components in Super Intensive White Shrimp (Litopenaeus vannamei) Culture

This research was aimed to study the performance of Zero Water Discharge (ZWD) by using nitrifying bacteria and microalgae Chaetoceros calcitrans in 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 NH 4+ (0.07–0.69 mg/L), NO 2- (0–3.2 mg/L), NO 3- (1.04–4.29 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 NO 2- 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%), specific growth rate (7.7 ± 0.11%) and feed conversion ratio (1.27 ± 0.29) was obtained in ZWD, while in control the figures were significantly different: total weight (160.48 ± 6.62 g), mean body weight (5.45 ± 0.28 g), survival rate (27.22 ± 2.09%), specific 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


Introduction
Crustaceans are sources of high quality protein and its production contributes significantly to the gross national product of Indonesian fishery sector. National production of crustaceans in Indonesia reached up to 639,589 tonnes in 2013 with L. vannamei contributed about 60% of 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 for a year) [2], with penaeid shrimp as the dominating crustacean commodity, standing at approximately 80% of the total shrimp export [3].
Because of its significant contribution to the Indonesia's economic growth, the increment of shrimp production has become a national policy and recently the production is targeted to reach around 700,000 tonnes. Although the production tends to increase annually, shrimp farm productivity in Indonesia is still a big question mark, since this increment could probably caused by the extensification of shrimp land farming. In Indonesia and other countries (Vietnam, India, Ecuador, China, Malaysia, Philippines) most shrimp farming 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 of dissolved N-inorganic compound NH4 + (ammonium) [6]. To overcome this unfavorable condition, farmers generally conduct a frequent water renewal. Unfortunately, due to biosecurity and environmental reasons, water renewal is not considered practically. Furthermore, water renewal method is also known 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). To this point, the results are still unpredictable in terms of production stability. Recently, the use of Zero Water Discharge by using nitrifying bacteria and Chlorella has been applied on cultivation of giant freshwater prawn (Macrobrachium rosenbergii) [8]. The application of zero water discharge was reported to help increase survival rate by 10-20%. Based on this promising result, the development of zero water discharge system as an alternative zoo technique in super intensive white shrimp culture needs to be evaluated further.
Zero water discharge provides an improvement for batch system and a modification of green water technology, especially in optimizing the nutrient cycle (inorganic and particulate nitrogen). The ammonium, nitrite, and nitrate level can be maintained at acceptable level by adding nitrifying bacteria and microalgae Chaetoceros calcitrans. These microorganisms have a positive effect on increasing water quality. Nitrifying bacteria can convert ammonium to nitrite (Nitrosomonas)

Abstract
This research was aimed to study the performance of Zero Water Discharge (ZWD) by using nitrifying bacteria and microalgae Chaetoceros calcitrans in 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 NH 4 + (0.07-0.69 mg/L), NO 2 -(0-3.2 mg/L), NO 3 -(1.04-4.29 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 NO 2 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%), specific growth rate (7.7 ± 0.11%) and feed conversion ratio (1.27 ± 0.29) was obtained in ZWD, while in control the figures were significantly different: total weight (160.48 ± 6.62 g), mean body weight (5.45 ± 0.28 g), survival rate (27.22 ± 2.09%), specific 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 calcitrans Components in Super
Intensive White Shrimp (Litopenaeus vannamei) Culture and nitrite to nitrate (Nitrobacter) [9], while biomass of microalgae has an important role in nitrate reduction and as food supplement for shrimps [10].

Materials and Method
Activation and cultivation of nitrifying bacteria C. calcitrans Culture of nitrifying bacteria was obtained from the Laboratory of Microbiology SITH ITB. Nitrifying bacteria was activated in liquid medium of Winogradsky and scaled up to a volume of 100 L as expressed in Figure 1 [8].
Culture of C. calcitrans that were used in this study was obtained from the Laboratory of Aquatic Ecosystem Analysis SITH ITB. C. calcitrans was activated in Guillard medium and scaled up to a volume of 40 L as illustrated in Figure 2 [11].

Acclimatization of white shrimp post larvae
White shrimps PL-10 used in this study were obtained from PT. Suri Tani Pemuka Indramayu, West Java and acclimatized at room temperature (25 ± 1°C) at the Laboratory of Aquatic Ecosystem Analysis, School of Life Sciences and Technology, Institut Teknologi Bandung (SITH ITB), Indonesia to reach the PL-15 stadia.

Conditioning of zero water discharge system
The study was conducted in cylindrical fiber tank filled with 300 L sterilized and filtered seawater at 30 ppt salinity. As illustrated in Figure 3, ZWD system consists of four compartments: (1) microbial component: nitrifying bacteria and microalgae C. calcitrans which play a role in nitrogen cycle of 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 of diatom C. calcitrans and nitrifying bacteria, (4) filtered and sterilized culture water as shrimp growth media, (5) feeding tray to control amount of feed delivered daily, and (6) CaCO 3 gravel that is used as substrate for nitrifying bacteria colonization and as pH buffering agent.
Before starting the culture, the ZWD system was conditioned by inoculating 10 L of nitrifying bacteria (at 10 6 CFU/ml) and 12 L of C. calcitrans (at 10 6 cells/ml) into the ZWD systems and total volume is 300 L. The ammonium breakdown capacity of nitrifying bacteria was conditioned by adding 3 g of NH 4 Cl (≈ 10 mg/L of ammonium) to the system. The conditioning was performed until the NH 4 + and NO 2 level decreased to approximately 0 mg/L.

Treatment implementation
There were two treatments done in triplicate for this research: conventional culture (without any addition of nitrifying bacteria and C. calcitrans and 80% of the water volume was renewed once in four weeks) as the control, and ZWD system (with the addition of nitrifying bacteria and C. calcitrans). In order to compensate water losses due to siphoning and evaporation, every two weeks six liters (2% of culture volume) new seawater was added into the culture system. After the conditioning, PL-15 shrimp was stocked to the conventional and ZWD culture with stocking density of 120 ind/0.3m 3 which refers to the super intensive stocking density (≈400 ind/m 3 ) [12]. Every two weeks, 5 L of nitrifying bacteria and 12 L of microalgae C. calcitrans were added at a density of ± 10 6 CFU/ml and ± 10 6 cells/ml, respectively, into the ZWD system. The experiment was conducted for 90 days culture period.

Feeding management
The amount of feed was adjusted weekly based on measurement of mean body weights, estimation of survival rate, and feeding rate ( 1). The formula for feeding calculation is : where : SD is stocking density (individual/tank), MBW is mean body weight (gr), SR is Survival rate (%), FR is feeding rate (%) Feed was placed on feeding tray and monitored frequently to   provide information of daily delivered feed accuracy ( Table 1). The size and condition of shrimp can be checked and their consumption rate estimated based on the left-over feed in the tray. The feeding scheme was modified from Tacon [13]. Feed were delivered five times a day at 08:00 am, 11:00 am, 14:00 pm, 17:00 pm and 20:00 pm.

Biological and Microbiological Parameter Measurement
Biological parameters: Total weight, mean body weight (MBW), specific growth rate (SGR), survival rate (SR) and feed conversion ratio (FCR) were measured during culture period. The survival rate of the shrimp was calculated by using the following equation [8]: where: SR is survival rate , N o is initial shrimp number, N t is final shrimp number, t is culture period (day).
Shrimp specific growth rate was calculated by using the following equation: where: SGR is specific growth rate, W 1 is initial body weight (g) at time T 1 (day), and W 2 is final body weight (g) at time T 2 (day) Microbiological parameters were measured by total bacterial count or total plate count method [15]. Total bacteria was counted every week by plating water sample on nutrient agar (NA) medium, while total Vibrio sp. count was conducted every four weeks using thiosulphate citrate bile salt agar (TCBS) medium [16].

Conditioning of zero water discharge system
During conditioning period, it was observed that nitrifying bacteria was able to convert 10 mg/L of ammonium to 0 mg/L in 5 days (average of breakdown capacity was 2 mg/L per day) as shown in Figure 4. The conversion of ammonium to nitrite can be seen from the increment of nitrite concentration which reached 3 mg/L on day 5 and remained steady until day 15 of culture period (Figure 4). By day 17, the nitrite level had plummeted to nearly 0 mg/L. As a final product of nitrification, nitrate had been accumulated, in which the level was around 60 mg/L by the end of culture period (day 17).
Conditioning process becomes a crucial step prior to shrimp stocking and culture. This step enables ZWD system to convert toxic NH 4 + and NO 2 into less harmful substances NO 3 -. From the result, it can be seen that ammonium oxidizing bacteria was activated on day 5 of conditioning period. However, longer conditioning period of 17 days was taken to activate nitrite oxidizing bacteria. For nitrite oxidizing bacteria, longer activation period is due to slower growth or doubling time of nitrite oxidizing bacteria compared to ammonium oxidizing bacteria [9]. As the second step of nitrification process, NO 2 oxidation takes place after NH 4 + oxidation reaction. This consecutive reaction also contributes to slower activation process for nitrite oxidizing bacteria since the reaction depends heavily on NO 2 availability of the first reaction [9].
Even though the nitrification process was able to be achieved during 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 better activation process of nitrite oxidizing bacteria in stock culture during maintenance, prior to being inoculated into ZWD system.

Physical and chemical parameters measurement
The range of ammonium, nitrite and nitrate concentrations documented in conventional and ZWD rearing technique are presented in Table 2. Ammonium, nitrite and nitrate concentrations were still in tolerance levels for white shrimp in both rearing strategies [17][18][19].
The trend for ammonium, nitrite and nitrate concentration during 90 days of culture period is presented in Figure 5. It is noticeable that the ammonium, nitrite and nitrate level tended to slightly increase. In ZWD system, ammonium, nitrite and nitrate levels ranged between 0.07-0.69 mg/L, 0-3.15 mg/L, 1.04-42.9 mg/L, respectively, while in conventional (batch) system, it ranged between 0.20-0.59 mg/L, 0-3.2 mg/L, 1.38-14.17 mg/L, respectively. Based on these levels, all dissolved N-inorganic levels measured during 90 days culture period were not significantly different in both rearing systems (p>0.05). In spite of this condition, the breakdown capacity of ZWD system on NH 4 + and NO 2 levels were higher compared to conventional (batch) system. This is because a higher amount of feed (≈ 1178.28 g) was delivered to ZWD system; around 44% higher than that of conventional system (≈ 656.15 g). This contributed to a higher organic matter accumulation and

Ammonium
Nitrite Nitrate  therefore had an effect on higher ammonium (NH 4 + ) accumulation in the system [20]. Based on [21] estimation, it was assumed that the amount of feed given to shrimp in ZWD for 90 days could produce a total NH 4 + of 55.20 mg/L in shrimp culture which is equivalent to 0.61 mg/L per day. In contrast, total ammonia produced by shrimp for 90 days in conventional system was 30.73 mg/L, which is equivalent to 0.34 mg/L per day (total of 656.15 g feed delivery). From this estimation, it can be stated that the ammonium breakdown capacity with average ammonium conversion rate of 2 mg/L/day was higher in ZWD system than that of conventional culture system.
Better ammonium and nitrite conversion rate were determined by measuring higher nitrate accumulation after 90 days culture period (Figure 5c). At the end of culture period, nitrate level of 42.9 mg/L at ZWD system was significantly higher (p<0.05) compared to conventional system (14.17 mg/L). This level of final nitrification product indicates that ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) performs well in the system. They contributed in maintaining a stable and low toxic NH 4 + and NO 2 level in culture water, despite the feed delivery was approximately 180% higher in ZWD system.
Besides shrimp productivity, the accumulation of NH 4 + and NO 2 levels in ZWD should be taken as a serious consideration, since the presence of these toxic substances increases the amount of feed needed for better growth and to avoid shrimp's system failure. Based on this fact, an adequate system conditioning process (Figure 3) becomes a critical step prior to system operation. This conditioning will provide us with certainty on ZWD performance, NH 4 + and NO 2 breakdown capacity in particular.
Relatively low NH 4 + and NO 2 levels also probably had an effect on marine diatom C. calcitrans inclusion of ZWD system. C. calcitrans can uptake both NO 3 and NH 4 + as N source during assimilation process, thus, the level of NH 4 + and NO 2 can be maintained at an acceptable level. Keeping the ammonium, nitrite and nitrate in a low and stable level is important for shrimp cultivation. An excessive amount of ammonium might reduce growth, increase oxygen consumption and ammonia-N excretion, alter concentration of hemolymph protein and free amino acid levels, and even cause mortality [22], whereas a high level of nitrite could induce methemoglobin formation; which might result in hypoxia and cyanosis [23] and later contribute to retardation of shrimp growth and death [24].
Predicted microbial loop during 90 days culture period can be described as in Figure 6. In general, nutrient cycle processes that are involved in ZWD systems include nitrogen and carbon cycle. It can be seen that excessive feed input will not only contribute to shrimp growth, but also to the accumulation of organic matter in water, together with shrimp excreted metabolism [25]. The organic matter will then be utilized by heterotrophic bacteria and converted into NH 4 + through ammonification process. Furthermore, NH 4 + ions will be oxidized to form toxic substance NO 2 by chemolithotroph ammonium oxidizing bacteria and NO 2 will be converted into less toxic substance NO 3 by chemolithotroph nitrite oxidizing bacteria, respectively [26]. Finally, NO 3 as one of macronutrients will be utilized through assimilation process by photoautotrophic organism, microalgae C. calcitrans, which serves as shrimp live feed and plays a role in increasing O 2 level in water [27][28]. Microalgae biomass also contributes to organic matter increment in water, together with chemolithotrophic and heterotrophic bacteria.
Among three microbial components that have a role in ZWD microbial loop, only two (photoautotrophic and chemolithotrophic bacteria) were applied in this ZWD system, while the heterotrophic component still has not been used yet. However, for further development of the ZWD system, application of functional heterotrophic component will be taken as crucial step. Hopefully, the use of the three components altogether could help stabilizing and lowering the microbial diversity, as well as enhancing the ammonification, nitrification, and assimilation processes in culture water.
Appropriate culture ZWD condition will create a better environment and growth for the shrimp, even though it might contribute to the decrement of dissolved oxygen budget due to different shrimp survival (90.82% compared to 27.22%) and higher organic input. However, in this system, the DO level decrement was less than 1 mg/L (7.42 ± 0.52 mg/L in conventional and 6.81 ± 0.5 mg/L in ZWD) after 90 days culture period. Despite there was no significant effect of DO level decrement, this result should be taken as a serious consideration in further application and development of ZWD system, especially if the culture process is going to be conducted for a duration of more than 3 months (4 months of normal culture period), in which the shrimp density and the accumulation of organic matter might  reduce the oxygen budget of the system. From the result, it seemed that the dissolved oxygen budget could be covered by oxygen production of microalgae and constant aeration in ZWD system [32].

Microbiological parameter measurement
Total bacteria counted in conventional and ZWD for 12 weeks culture period is given in Figure 7.
Based on the results, total bacteria count during the trial tended to increase in both rearing system. In ZWD, maximum number of Total Bacterial Count and total Vibrio sp. were 1 × 10 10 CFU/ml and 4.8 × 10 2 CFU/ml, respectively, while in conventional system the figures stood at 6.7 × 10 10 CFU/ml and 1.05 × 10 2 CFU/ml, respectively Table  3. There were no significant differences on Total Bacterial Count and total Vibrio sp. obtained during culture period between ZWD and conventional culture system (p>0.05).
It is noticeable that excessive organic matter accumulation in ZWD system did not significantly increase the number of bacteria, despite it has been widely known that the availability of higher organic content could promote the growth of heterotrophic bacteria [33]. The presence of nitrifying bacteria and C. calcitrans was well documented in controlling the growth of opportunistic bacteria and indigent Vibrio sp. in shrimp culture. Nitrifying bacteria can compete with other bacteria in terms of space and oxygen [34] and marine diatom secrete fatty acids and esters that can act as antibacterial compounds [35]. It is not surprising that the total Vibrio sp. in ZWD system was below pathogenicity level of 10 6 CFU/ml [36], even when shrimp culture in ZWD contained higher shrimp density and higher input of feed.
A low total number of bacteria in conventional system are suspected due to regular dilution by water renewal (80 % every four weeks) and also due to less organic matter load of feed and biological wastes (less shrimp density) accumulated during the culture period.

Biological parameter measurement
Shrimp culture productivity was observed based on mean body weight (MBW), total weight, survival rate (SR), specific growth rate (SGR) and feed conversion ratio (FCR) measurement Table 4.
In ZWD system, better shrimp culture performance was obtained after 90 days culture period. Measurements of biological parameters, including mean body weight (MBW) of 8.24 ± 0.84 g, total weight of 923.38 ± 42.15 g, Survival Rate (SR) of 90.82 ± 2.5%, and specific growth rate (SGR) of 7.7 ± 0.11%, were significantly higher in ZWD than those of conventional culture system (MBW: 5.45 ± 0.28 g, Total Weight: 160.48 ± 6.62 g, SR: 27.22 ± 2.09%, and SGR 7.24 ± 0.05). The FCR in ZWD (1.27) were significantly lower than that of conventional culture system (4.10) (p<0.05). It is of note that the value of two critical culture parameters, SR and FCR, were still in tolerance range of shrimp culture, in which the SR and FCR are expected to be between 51 to 91% and 1.5 to 2.6, respectively [13]. Better culture performance obtained in ZWD culture system was due to better and stable water quality maintained during culture period, even though more feed amount was delivered to support shrimp growth at higher shrimp density.
Beside the advantages of nitrifying bacteria application on water quality (NH 4 + and NO 2 -), the use of microbial component, marine diatom C. calcitrans, has also contributed in creating a favorable condition by providing shading effect and alternative feed for shrimp [37]. Both of these factors have a significant role in reducing cannibalism due to less water visibility (minimize predation during moulting) and as a supplement diet for shrimp.   Besides the information on physico-chemical, microbiological and biological parameters, a cost estimation of shrimp production using both systems also become an important step that needs to be conducted before further application of this new culture approach in aquaculture industry. Estimation was calculated in assumption to produce 100 kg shrimp during 90 days culture period, based on shrimp culture performance (total weight, FCR, MBW, SR) obtained from this research, seed cost, feed cost and water consumption as shown in Table 5.
The calculation was made based on biological parameters obtained during 90 days culture period. Better shrimp growth and survival implied in better and lower FCR in ZWD which resulted in profit by 2,375,240 IDR. Based on this analysis, the use of zero water discharge system for super intensive shrimp rearing technology can improve the profit of the culture compared to the conventional batch system with the same initial shrimp density. This economic advantage was due to better culture performance on SR, MBW, and FCR measured in ZWD system. During culture period, the use of ZWD system also consumed less water volume (52.1 m 3 ) compared to conventional batch system (78 m 3 ) to produce 100 kg of shrimps as shown in Table 4. This condition shows that the use of ZWD system will not only contribute in sustaining a more cost effective system, but also in minimizing water quality disturbance and reducing wastewater release into the environment.

Conclusion
Based on this research, Zero Water Discharge system can be used as an alternative for shrimp culture since ZWD was able to enhance white shrimp culture performance (water quality, SR, growth and FCR).