2. ZWD principle

annual growth rate of 8.8% [2]. Global aquaculture production has reached 73.8 million tons in 2014 with an estimated value of USD160.2 billion. It shared about 44.14% of total fishery production. In the next decade (2025), FAO predicted that aquaculture sector would share 52% of the total fishery production [1]. Along with the prediction, Indonesia has a great potential to develop the aquaculture sector. Indonesia is one of main producers of both capture and aquaculture fishery commodities because it is supported by its geographical condition. Indonesia is an archipelagic country that has great potential in fisheries sector. It consisted of 17,500 islands and located between two big oceans, Pacific and Indian Ocean. Moreover, Indonesia is a country crossed by equator line and ranked as world's 4th longest coastline, which indicates a high diversity of aquatic organisms, including marine biota [3]. So, there are many fishery commodities grown in Indonesia. Currently, Indonesia ranks as the second top both capture and aquaculture producers after People's Republic of China, contributing 6.48 and 14.36 million tons, respectively, to worldwide production [1]. One of main commodities is crustaceans that produced both capture and aquaculture practices. In fact, most of productions were obtained from aquaculture. In 2014, shrimp capture production only contributed about 30% of the total shrimp production or approximately 273,133 tons [4]. Shrimp commodities

rank as the top by annual total aquaculture production from aquatic animal.

up to Rp 275.2 billions [7].

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Most of shrimp production is dominated by white shrimp (Litopenaeus vannamei) which is also exported to several countries in the world, such as United States of America, Japan, People's Republic of China, United Kingdom, Malaysia, etc. [5]. Trend of white shrimp production has increased significantly with an average growth of 22.46%. This increment production was due to ease of cultivation practice, in case of availability of seed, cultivation period, and more resistance to environmental changes. Another species, giant freshwater prawn has the opportunity to become a main commodity due to high economic value. In 2013, prawn production reached approximately 3.171 tons, which has been cultivated in several site, such as West Kalimantan, Bali, West Java, and East Java [6]. Although it is still small in number compared to white shrimp, production volume continued to rise in recent years. Ministry of Maritime Affairs and Fisheries Republic of Indonesia seriously began promoting the cultivation of prawn, started in 2015, they have allocated a national capital budget for prawn production

However, a high production scale does not ensure sustainability of shrimp aquaculture industry, because currently most shrimp farms use conventional culture practices, such as batch or flow-through system. It is true that conventional shrimp rearing strategies are still widely applied and profitable due to its simplicity and acceptable production cost, but since the cultivation relies on natural environment with less control to water quality and disease or predation, this condition leads to unpredictable culture performances [8]. Furthermore, the accumulation of harmful substances in culture water from uneaten feed and excretion (e.g., ammonium and nitrite) is very likely exceeds the tolerance limits, causing a decrement of culture survival rate and thus affecting overall shrimp productivity in conventional culture system [9]. Besides, the system is considered as not environmentally friendly, because untreated effluent water can pollute the surrounding aquatic environment [8]. In term of space requirement, the system occupies a large production area and requires close distance to coastal area to ensure seawater access. These circumstances contribute to impractical shrimp farming Water body is habitat for all aquatic animals, including shrimp and prawn. Consequently, the key for success cultivation is to keep the habitat favorable for shrimp to grow. So that, it is crucial to maintain water quality in tolerance range for shrimp growth. Water quality includes physical, chemical, and biological parameters particularly temperature, dissolved oxygen, and toxic nitrogen substance concentrations [15]. Temperature and dissolved oxygen parameters can be manipulated by physical treatment such as using aerator and water heater, while toxic nitrogen substances have dealt with biological treatment system usually utilizing microbial-based treatment.

Toxic nitrogen substances produced from excretion activity of shrimp and their feed residue, such as ammonium and nitrite, disturb metabolic balance of the shrimps, making them more prone to disease that causes several disadvantages, including reduced body weight, increased mortality, and eventually decrease production yield [16–18]. As this has become one major problem in aquaculture, ammonium and nitrite removal management is a major concern in ZWD system. In natural aquatic ecosystems, microorganism present in water body maintains a balance concentration of each nitrogen compounds. As ammonium and nitrite concentration in intensive aquaculture systems build up much faster than in natural ecosystems, we cannot rely on naturally occurring microorganisms in the ponds. Their low population size cannot cope with the rate of ammonium accumulation, and therefore, addition of microorganism is needed.

This system uses the principle of microbial loops adapted from natural ecosystems. Toxic nitrogen substances present in ammonium and nitrite form can be converted into nitrate which is less toxic substance through consecutive nitrification microbial process. ZWD system aims to improve water quality through recycling chemical waste [19]. While conventional system (e.g. flow-through) requires a continuous new water supply to avoid waste accumulation in the culture, ZWD recycles ammonium, nitrite, and nitrate using microorganism consortia, and therefore, it reduces water usage significantly. Ammonium, nitrite, and nitrate level can be maintained using addition of heterotrophic bacteria, nitrifying bacteria, and microalgae, regularly [13].

#### 2.1. State of the art

Based on the principle explained earlier, the most crucial thing is the selection of microbial components that have functions in maintaining water quality and are harmless to the animals being cultivated. In addition, selected microbes may act as probiotics such as to counteract pathogenic attacks from Vibrio sp. in shrimp farming [19]. Since this system refers to nutrient cycles in aquatic habitat, the selected microbes should have a role in the alteration of toxin substances into harmless substance produced in the cultivation system. The system emphasizes nitrogen nutrient cycle because nitrogen toxin is very dangerous if it accumulates excessively.

producer that also provides oxygen that can raise the DO level, and their biomass may be

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Because the ZWD system relies on microbial components added to the system, different animal cultures will have different microbial components added, and the system must be favorable for microbial components to live. For a simple example, we have to consider about native microbial habitat; marine microbes will be suitable for marine animal farming and freshwater microbes for freshwater animal farming. Figure 1 is an example of ZWD system in the cultivation of white shrimp, so the microbial components used are marine heterotrophic bacteria, marine AOB, marine NOB, and marine microalgae

2.2. Distinctive characteristics in ZWD system compared to other microbial-based systems As the aquaculture industry grows rapidly in the world, it encourages research to create technology that leads to the sustainability in aquaculture industry. One of main research areas is the utilization of microbes that are now widely used in aquaculture industry. It can play a role as a food source such as microalgae for the larval phase [23, 24], maintain water quality such as using ammonium as a source of nitrogen for microbial metabolism [21, 25, 26], fight against disease such as immunostimulant that trigger antibodies or directly interact antagonistically with path-

The application of microbes in aquaculture is conducted into microbial-based closed systems, such as the ZWD system. The term of zero water discharge has many versions; it can be zero water exchange [14, 29–31], limited water discharge [32, 33], minimal discharge system [34], minimal effluent discharge [35], minimal exchange system [36], etc. All such systems have the same principle that is minimizing water use and re-recycling water used by involving the role of microbes. ZWD system is an improvement from batch system with an emphasis on microbial manipulation in rearing tanks. ZWD system can be interpreted as no water discharge during culture period, additional water that put into the system is to balance water level due to water losses caused siphoning and evaporation. It is approximately 2% of culture volume in

So far, the existing microbial form used may be in consortium, biofilm, periphyton, biofloc forms or has separated compartments such as biofilter in recirculation aquaculture system (RAS). In ZWD system, the form of microbial used is consortia that have been added regularly to the system during cultivation period. The purpose of additional microbial consortia regularly is to control microbial loop works in appropriate way. In addition, the presence of microbial control is to keep dominancy of selected microbes that play a role in predicted microbial loop. However, to maintain the availability of microbial cultures, the system must be equipped with separated microbial cultivation facilities. Consequently, there is control to maintain microbial culture from contamination and to keep the microbes in their optimum growth. Table 1 below is a summary of the characteristics of each

possible as food source for cultivated animals in the system.

(Chaetoceros calcitrans).

ogen [19, 27, 28].

every 6 weeks [13].

microbial-based closed system.

Figure 1 shown below is an example for the estimation of nutrient cycle and microbial loop that occur in the ZWD system [13]. The greatest accumulation of toxic compounds in cultivation is from animal feed and feces. These compounds are mostly organic matters, which can be degraded by heterotrophic bacteria into inorganic compounds. Inorganic compounds, such as ammonium and nitrite, which became the focus, have to be removed. The ammonium and nitrite should be converted into less harmful compounds such as nitrate by oxidation. Microbes that can do the oxidation process from inorganic compounds are litoautotrophic bacteria [20]. There are two stages of the oxidation processes: (1) the conversion from ammonium to nitrite and (2) nitrite to nitrate. Ammoniumoxidizing bacteria (AOB) convert ammonium to nitrite, for example, Nitrosomonas, and nitrite-oxidizing bacteria (NOB) convert nitrite to nitrate, such as Nitrobacter [21]. Even though nitrate is a harmless substance, the tolerance range in aquaculture system is no more than 200 ppm [22]. Therefore, it is necessary to search microbes that can utilize nitrate. Some microalgae can use nitrate as a source of nitrogen, so that addition of microalgae is important in this system. In addition, at the trophic level, microalgae act as

Figure 1. Schematic of nutrient cycle in ZWD system [13].

producer that also provides oxygen that can raise the DO level, and their biomass may be possible as food source for cultivated animals in the system.

2.1. State of the art

300 Biological Resources of Water

Figure 1. Schematic of nutrient cycle in ZWD system [13].

sively.

Based on the principle explained earlier, the most crucial thing is the selection of microbial components that have functions in maintaining water quality and are harmless to the animals being cultivated. In addition, selected microbes may act as probiotics such as to counteract pathogenic attacks from Vibrio sp. in shrimp farming [19]. Since this system refers to nutrient cycles in aquatic habitat, the selected microbes should have a role in the alteration of toxin substances into harmless substance produced in the cultivation system. The system emphasizes nitrogen nutrient cycle because nitrogen toxin is very dangerous if it accumulates exces-

Figure 1 shown below is an example for the estimation of nutrient cycle and microbial loop that occur in the ZWD system [13]. The greatest accumulation of toxic compounds in cultivation is from animal feed and feces. These compounds are mostly organic matters, which can be degraded by heterotrophic bacteria into inorganic compounds. Inorganic compounds, such as ammonium and nitrite, which became the focus, have to be removed. The ammonium and nitrite should be converted into less harmful compounds such as nitrate by oxidation. Microbes that can do the oxidation process from inorganic compounds are litoautotrophic bacteria [20]. There are two stages of the oxidation processes: (1) the conversion from ammonium to nitrite and (2) nitrite to nitrate. Ammoniumoxidizing bacteria (AOB) convert ammonium to nitrite, for example, Nitrosomonas, and nitrite-oxidizing bacteria (NOB) convert nitrite to nitrate, such as Nitrobacter [21]. Even though nitrate is a harmless substance, the tolerance range in aquaculture system is no more than 200 ppm [22]. Therefore, it is necessary to search microbes that can utilize nitrate. Some microalgae can use nitrate as a source of nitrogen, so that addition of microalgae is important in this system. In addition, at the trophic level, microalgae act as Because the ZWD system relies on microbial components added to the system, different animal cultures will have different microbial components added, and the system must be favorable for microbial components to live. For a simple example, we have to consider about native microbial habitat; marine microbes will be suitable for marine animal farming and freshwater microbes for freshwater animal farming. Figure 1 is an example of ZWD system in the cultivation of white shrimp, so the microbial components used are marine heterotrophic bacteria, marine AOB, marine NOB, and marine microalgae (Chaetoceros calcitrans).

#### 2.2. Distinctive characteristics in ZWD system compared to other microbial-based systems

As the aquaculture industry grows rapidly in the world, it encourages research to create technology that leads to the sustainability in aquaculture industry. One of main research areas is the utilization of microbes that are now widely used in aquaculture industry. It can play a role as a food source such as microalgae for the larval phase [23, 24], maintain water quality such as using ammonium as a source of nitrogen for microbial metabolism [21, 25, 26], fight against disease such as immunostimulant that trigger antibodies or directly interact antagonistically with pathogen [19, 27, 28].

The application of microbes in aquaculture is conducted into microbial-based closed systems, such as the ZWD system. The term of zero water discharge has many versions; it can be zero water exchange [14, 29–31], limited water discharge [32, 33], minimal discharge system [34], minimal effluent discharge [35], minimal exchange system [36], etc. All such systems have the same principle that is minimizing water use and re-recycling water used by involving the role of microbes. ZWD system is an improvement from batch system with an emphasis on microbial manipulation in rearing tanks. ZWD system can be interpreted as no water discharge during culture period, additional water that put into the system is to balance water level due to water losses caused siphoning and evaporation. It is approximately 2% of culture volume in every 6 weeks [13].

So far, the existing microbial form used may be in consortium, biofilm, periphyton, biofloc forms or has separated compartments such as biofilter in recirculation aquaculture system (RAS). In ZWD system, the form of microbial used is consortia that have been added regularly to the system during cultivation period. The purpose of additional microbial consortia regularly is to control microbial loop works in appropriate way. In addition, the presence of microbial control is to keep dominancy of selected microbes that play a role in predicted microbial loop. However, to maintain the availability of microbial cultures, the system must be equipped with separated microbial cultivation facilities. Consequently, there is control to maintain microbial culture from contamination and to keep the microbes in their optimum growth. Table 1 below is a summary of the characteristics of each microbial-based closed system.


main and supporting facilities of ZWD systems for crustacean cultivation, particularly white shrimp and giant freshwater prawn. Besides, the selection of microbial components that are suitable for shrimp and prawn culture and how to prepare the cultivation will be explained in

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As ZWD system is an improvement system of batch culture, the main facilities provided are similar to batch system. ZWD system installation can be constructed in a rectangular or circular culture tank that is equipped with several basic utilities commonly used in aquacul-

1. Culture tanks for nitrifying, heterotrophic bacteria, and microalgae culture. Separation is necessary for easier maintenance purpose. Proper maintenance is critical to keep optimum performance of microbial components. Tank sizes of nitrifying bacteria and microalgae are suggested to have minimal capacity about 20% from culture tanks, while tank size of heterotrophic bacteria is suggested to have minimal capacity about 2.5%

2. Aeration equipment including aerator, silicon hose, and air stone. The aerator provides continuous oxygen supply with airflow rate of 28 L/min [49]. Proper aeration is critical, not only to provide oxygen to shrimp for effective feed utilization and growth, but as importantly to oxidize liquid, solid, and gaseous waste in the system. Oxygen level in water must be maintained between 4 and 6 ppm [44]. However, 6 ppm is recommended to support optimum growth. With high inputs of feed, there is higher demand for oxygen by shrimps and by the microbial community in the

3. A net covering is used to avoid pollutant entry to culture pond, and it is more important to reduce water evaporation, which can affect salinity level significantly. In addition, covering reduces light penetration through the water column to suit intensity level for

6. CaCO3 and gravel, as a substrate for nitrifying bacteria attachment as well as a buffering

In addition, shelter is required for some crustacean such as prawn (Macrobrachium rosenbergii De Man). Prawn is much more aggressive than shrimp. There is a risk of cannibalistic behavior that emerges when prawns are cultured at high density, especially during their grow-out phase and molting period [11]. Without shelter, they do not have enough niche for each individual. Several shelters that have been proved in previous studies were textile vertical substrate [11] and cubical bamboo shelters [51]. These shelters have been proven to improve prawn culture productivity. Figure 2 below represents the components

this section.

3.1. Design construction and facility

from culture tanks.

water.

agent.

of ZWD system.

ture. Here is the list of facilities for ZWD system.

the microalgae population in water.

4. A thermometer to monitor daily culture temperature.

5. Feeding trays to administer and control sufficient daily feed amount.

Table 1. Characteristics of microbial-based system.

### 3. Preparation of ZWD system

Proper designed systems and good microbial management are important parts to optimize production efficiency in intensive cultivation using ZWD system. This section will describe the main and supporting facilities of ZWD systems for crustacean cultivation, particularly white shrimp and giant freshwater prawn. Besides, the selection of microbial components that are suitable for shrimp and prawn culture and how to prepare the cultivation will be explained in this section.

#### 3.1. Design construction and facility

As ZWD system is an improvement system of batch culture, the main facilities provided are similar to batch system. ZWD system installation can be constructed in a rectangular or circular culture tank that is equipped with several basic utilities commonly used in aquaculture. Here is the list of facilities for ZWD system.


3. Preparation of ZWD system

Table 1. Characteristics of microbial-based system.

Microbialbased. closed system

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Zero water discharge

– Low/no water discharge

Biofloc – Low/no water discharge

Periphyton – Low/no water discharge

Biofilm – Low water discharge

RAS – No water discharge

Green water technique

– Improved system from batch system – Emphasize in microbial manipulation

– Improved system from batch system

– Emphasize in C/N ratio in the system

– Improved system from batch system

– Improved system from batch system

– Microbial compartment is in biofilter – Biofilter has defined microbial consortia – Isolated and clear-water system

– Low water discharge – Use batch system

– No control of microbial consortia in biofilm – Biofilm can also be a food source for cultured animal

Defined biofilm – Biofilm production needs additional reactor and attachment substrate

– No control in microbe community in the system

– Main purpose is to provide natural food for cultured animal

– Nitrogen toxic compound removal by microbial loop system – Microbial consortia added regularly to the system – Microbial component is kept dominant in the system

– Need additional compartment for separated microbial cultivation

– Add carbon source to enhance heterotrophic bacteria consortium

– Need organic substrate i.e. bamboo to periphyton attachment

– 'waste' Nitrogen is converted to high concentration of total suspended solid (microbial biomass) that can act as highly protein feed for cultured animal – Consider well mixing and aeration to compensate BOD in the system

– Input organic matter i.e. manure and chemical fertilizers to trigger periphyton growth – Sometimes, needs additional carbon source to maintain C/N ration in the system – Periphyton acts as nitrogen toxic removal system and food source for cultured animal

– Nitrogen toxic compound removal was done by formed biofilm during culture period

– Defined microbial consortia in biofilm (predominantly nitrifying bacteria)

– Many treatment process involved including physical and chemical treatment

– Main purpose is biologically secured and hygiene aquaculture product – Investment cost and operational cost is higher than other systems

– Mostly autotrophic microalgae used as microbial component in the system – Utilized chemical fertilizer and organic waste to trigger phytoplankton grow

– Main purpose is to remove nitrogen toxic substance in system – Can be applied in the system or in external unit i.e. biofilter

Proper designed systems and good microbial management are important parts to optimize production efficiency in intensive cultivation using ZWD system. This section will describe the

Characteristics References

[13]

[26, 37, 38]

[39–41]

[18]

[42, 43]

[44–46]

[47, 48]


In addition, shelter is required for some crustacean such as prawn (Macrobrachium rosenbergii De Man). Prawn is much more aggressive than shrimp. There is a risk of cannibalistic behavior that emerges when prawns are cultured at high density, especially during their grow-out phase and molting period [11]. Without shelter, they do not have enough niche for each individual. Several shelters that have been proved in previous studies were textile vertical substrate [11] and cubical bamboo shelters [51]. These shelters have been proven to improve prawn culture productivity. Figure 2 below represents the components of ZWD system.

According to previous studies, the most common nitrifying bacteria that were stable during cultivation period were Nitrosomonas sp. and Nitrobacter sp. [13]. Nitrosomonas and Nitrobacter are Gram-negative and aerobe obligate that is used as final electron acceptor. The bacteria can grow and multiply as individual units or in the form of biofilms. Nitrosomonas reproduces with binary fission, while Nitrobacter reproduces with budding. Nitrosomonas and Nitrobacter have different generation time, Nitrosomonas is every 8 h, and Nitrobacter is every 12 h. After 72 h, the population size of Nitrosomonas will be eight times greater than the population size of the

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Several inhibition factors affect the nitrification process. Inhibitors may be short-term or longterm impact to their enzymatic activity. Some factors that can inhibit the rate of nitrification, there are alkaline pH, temperature, oxygen, salinity, organic and inorganic compounds, substrate for attachment and sunlight [53]. From mentioned factors, sunlight is an important factor to be taken into attention because sunlight can decrease the activity of bacteria

The cultivation of nitrifying bacteria was performed in Winogradsky medium and in strong aeration with no light conditions (covered with black plastic). At the beginning of cultivation, bacterial culture of nitrification is activated by adding 10 ppm of ammonium. Ammonium levels are measured daily until it reaches 0 ppm. Furthermore, the activity of nitrifying bacteria is enhanced by continuously increasing the ammonium level up to 50 ppm. Later, the culture was scaled up to 10 L and then up to 500 L. The culture substrate used was CaCO3 and gravel.

Besides nitrifying bacteria, microalgae are also an important component in ZWD system. Through their metabolism, microalgae take up nitrate obtained from final nitrification process by nitrifying bacteria as a nitrogen source [13]. Microalgae have the ability to conduct photosynthesis, which captures energy from light to synthesize organic carbon from inorganic carbon (CO2). It accumulates organic carbon in forms of starch or other carbohydrates. Along

Figure 3. (a) Schematic diagram of nitrifying bacteria reactor [11, 13]; and (b) real nitrifying bacteria culture.

Nitrosomonas and Nitrobacter in oxidizing ammonium and nitrite compounds [54].

Figure 3 below shows schematic reactor for nitrifying bacteria cultivation.

Nitrobacter [53].

3.2.2. Autotrophic microalgae

Figure 2. Basic facilities of ZWD system [13, 50].

#### 3.2. Microbial components

The microbial component of ZWD system can be understood through three key functional groups: nitrifying bacteria, autotropic microalgae, and heterotrophic bacteria. When managed correctly, a diverse healthy microbial community contributes directly and indirectly to shrimp nutrition and growth while processing excess nitrogen waste in the system. Once established, the community becomes stable, competitively excluding harmful opportunistic pathogen and therefore improving health and immune competence of shrimps. The key to maximize these benefits is in understanding and managing the microbial community in the system.

#### 3.2.1. Nitrifying bacteria

Nitrifying bacteria live in a wide variety of habitats, including soil, freshwater, seawater, rocks, and sediment. Nitrifying bacteria are widely used in aquaculture practice and usually in the form of ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). AOB derive energy through the process of catabolism of ammonium into nitrite; the bacteria included are genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio. While NOB oxidize nitrite to nitrate, the bacteria included are genera Nitrobacter, Nitrococcus, Nitrospira, and Nitrospina. The bacteria are classified into litoautotrophic bacteria because they use inorganic compounds as a source of energy and CO2 as a carbon source [52].

According to previous studies, the most common nitrifying bacteria that were stable during cultivation period were Nitrosomonas sp. and Nitrobacter sp. [13]. Nitrosomonas and Nitrobacter are Gram-negative and aerobe obligate that is used as final electron acceptor. The bacteria can grow and multiply as individual units or in the form of biofilms. Nitrosomonas reproduces with binary fission, while Nitrobacter reproduces with budding. Nitrosomonas and Nitrobacter have different generation time, Nitrosomonas is every 8 h, and Nitrobacter is every 12 h. After 72 h, the population size of Nitrosomonas will be eight times greater than the population size of the Nitrobacter [53].

Several inhibition factors affect the nitrification process. Inhibitors may be short-term or longterm impact to their enzymatic activity. Some factors that can inhibit the rate of nitrification, there are alkaline pH, temperature, oxygen, salinity, organic and inorganic compounds, substrate for attachment and sunlight [53]. From mentioned factors, sunlight is an important factor to be taken into attention because sunlight can decrease the activity of bacteria Nitrosomonas and Nitrobacter in oxidizing ammonium and nitrite compounds [54].

The cultivation of nitrifying bacteria was performed in Winogradsky medium and in strong aeration with no light conditions (covered with black plastic). At the beginning of cultivation, bacterial culture of nitrification is activated by adding 10 ppm of ammonium. Ammonium levels are measured daily until it reaches 0 ppm. Furthermore, the activity of nitrifying bacteria is enhanced by continuously increasing the ammonium level up to 50 ppm. Later, the culture was scaled up to 10 L and then up to 500 L. The culture substrate used was CaCO3 and gravel. Figure 3 below shows schematic reactor for nitrifying bacteria cultivation.

#### 3.2.2. Autotrophic microalgae

3.2. Microbial components

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Figure 2. Basic facilities of ZWD system [13, 50].

3.2.1. Nitrifying bacteria

The microbial component of ZWD system can be understood through three key functional groups: nitrifying bacteria, autotropic microalgae, and heterotrophic bacteria. When managed correctly, a diverse healthy microbial community contributes directly and indirectly to shrimp nutrition and growth while processing excess nitrogen waste in the system. Once established, the community becomes stable, competitively excluding harmful opportunistic pathogen and therefore improving health and immune competence of shrimps. The key to maximize these

Nitrifying bacteria live in a wide variety of habitats, including soil, freshwater, seawater, rocks, and sediment. Nitrifying bacteria are widely used in aquaculture practice and usually in the form of ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). AOB derive energy through the process of catabolism of ammonium into nitrite; the bacteria included are genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio. While NOB oxidize nitrite to nitrate, the bacteria included are genera Nitrobacter, Nitrococcus, Nitrospira, and Nitrospina. The bacteria are classified into litoautotrophic bacteria because they

benefits is in understanding and managing the microbial community in the system.

use inorganic compounds as a source of energy and CO2 as a carbon source [52].

Besides nitrifying bacteria, microalgae are also an important component in ZWD system. Through their metabolism, microalgae take up nitrate obtained from final nitrification process by nitrifying bacteria as a nitrogen source [13]. Microalgae have the ability to conduct photosynthesis, which captures energy from light to synthesize organic carbon from inorganic carbon (CO2). It accumulates organic carbon in forms of starch or other carbohydrates. Along

Figure 3. (a) Schematic diagram of nitrifying bacteria reactor [11, 13]; and (b) real nitrifying bacteria culture.

with other physiological processes, microalgae produce high-quality vitamin and minerals [58]. Moreover, microalga also has a good nutritional composition for aquaculture animals. Microalgae usually serve as a live feed at larval or early juvenile stage. Selected microalgae must have rapid growth rates, can cultivate to mass culture, and are stable growth to any environmental fluctuations.

Several species of microalgae that have been documented to success cultivation since 1997 are Isochrysis sp., Pavlova lutheri, C. calcitrans, Chaetoceros muelleri, Chaetoceros gracilis, Thalassiosira pseudonana, Skeletonema spp., Tetraselmis suecica, Navicula spp., Nitzschia spp. [55–59]. In practice, diatoms such as C. calcitrans, C. muelleri, or C. gracilis proved to increase shrimp productivity. Moreover, cell wall of these microalgae contains silicate, which is an important mineral for building exoskeleton of shrimps [60]. In addition, as microalgae cell density increases throughout their growth, it reduces light penetration to water body, so shrimp is not directly exposed to light (i.e., shading effects). Shading effect improves the production of shrimp, even though their exact mechanism of action remains unclear [61].

Microalgae begin to be cultivated from small scale (1 L) to large scale approximately 500 L. The cultured microalgae are diatoms (C. calcitrans, C. muelleri, and C. gracilis) for white shrimp and Chlorella sp. for prawn. For diatoms, medium used is f/2 medium [62] for stock culture up to 1 L, while medium used for Chlorella stock culture is Bold's Basal Medium [63]. Commercial media consisted of chemical fertilizer are used for large scale. Fertilizer must comprise a source of nitrogen, phosphate, silicate, and a small portion of the mineral. Examples of fertilizers used are NPK, Urea, ZA, mineral concentrates, etc. The cultivation uses batch system with condition parameter as follows: temperature is at interval 25–30C, light intensity 3000–5000 lux, pH between 7.0–8.5, and aeration rate 3 L/min. Initial density is 105 CFU/mL and is incubated for 7–10 days until density reaches approximately 106 CFU/mL. Figure 4 shows 1 L stock culture and 500 L scale-up culture for diatom C. calcitrans.

bacteria, species of heterotrophic bacteria varies among different psychochemical conditions. Most common predominant species are Bacillus megaterium and Bacillus flexus [66]. B. megaterium is an example of well-studied bacteria for aquaculture application. The bacteria secretes high amount of extracellular enzymes, such as protease, carbohydrolase, and lipase, that can increase feed intake and digestibility in shrimp [67]. Other species that are also beneficial are Shewanella algae and Halomonas aquamarina. These natural-occurred bacteria have

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Recent research progress proved that several heterotrophic bacteria genus (e.g. Bacillus sp. and Pseudomonas sp.) associated with microalgae through a mutual symbiosis. Although these heterotrophs have no direct role in controlling nitrogen cycle, their presence was proven to suppress growth of Vibrio spp. Mechanism of pathogenicity was done by quorum sensing [68]. This pathogen causes shrimp's mortality only when their population reaches a certain number of cells at least 106 CFU/mL [69]. Therefore, dominance of friendly heterotrophic bacteria can avoid pathogenic bacteria growth in the water, minimizing the risk of pathogen infection to the shrimp's gut.

Medium for culturing heterotrophic bacteria is nutrient broth medium for stock culture, while commercial medium is made from beef broth and ammonium chloride for large-scale cultivation. Heterotrophic bacteria were cultivated up to 15 L and then incubated for 24 h or until they reached the cell density of 107 CFU/mL. Figure 5 below shows heterotrophic bacteria B.

There are several steps in conditioning of ZWD system; they are (1) microbial maturation in culture animal tanks; (2) acclimatization and stocking of shrimp or prawn; (3) feeding man-

agement; and (4) microbial manipulation that will be described in detail below.

been studied and found to increase shrimp weight significantly [19].

Figure 5. (a) Culture stock and (b) large scale cultivation of Bacillus megaterium.

megaterium that are cultivated for culture stock and large-scale reactor.

4. Conditioning of ZWD system

#### 3.2.3. Heterotrophic bacteria

Heterotrophic bacteria can also uptake ammonium and nitrate as their nitrogen source [64]. The main advantage of adding particular heterotrophic bacteria is related to growth rate of heterotrophic bacteria that exhibit much faster than nitrifying bacteria [65]. Just like nitrifying

Figure 4. (a) Small scale and (b) large scale of Chaetoceros calcitrans in batch system.

Figure 5. (a) Culture stock and (b) large scale cultivation of Bacillus megaterium.

with other physiological processes, microalgae produce high-quality vitamin and minerals [58]. Moreover, microalga also has a good nutritional composition for aquaculture animals. Microalgae usually serve as a live feed at larval or early juvenile stage. Selected microalgae must have rapid growth rates, can cultivate to mass culture, and are stable growth to any

Several species of microalgae that have been documented to success cultivation since 1997 are Isochrysis sp., Pavlova lutheri, C. calcitrans, Chaetoceros muelleri, Chaetoceros gracilis, Thalassiosira pseudonana, Skeletonema spp., Tetraselmis suecica, Navicula spp., Nitzschia spp. [55–59]. In practice, diatoms such as C. calcitrans, C. muelleri, or C. gracilis proved to increase shrimp productivity. Moreover, cell wall of these microalgae contains silicate, which is an important mineral for building exoskeleton of shrimps [60]. In addition, as microalgae cell density increases throughout their growth, it reduces light penetration to water body, so shrimp is not directly exposed to light (i.e., shading effects). Shading effect improves the production of shrimp, even

Microalgae begin to be cultivated from small scale (1 L) to large scale approximately 500 L. The cultured microalgae are diatoms (C. calcitrans, C. muelleri, and C. gracilis) for white shrimp and Chlorella sp. for prawn. For diatoms, medium used is f/2 medium [62] for stock culture up to 1 L, while medium used for Chlorella stock culture is Bold's Basal Medium [63]. Commercial media consisted of chemical fertilizer are used for large scale. Fertilizer must comprise a source of nitrogen, phosphate, silicate, and a small portion of the mineral. Examples of fertilizers used are NPK, Urea, ZA, mineral concentrates, etc. The cultivation uses batch system with condition parameter as follows: temperature is at interval 25–30C, light intensity 3000–5000 lux, pH between 7.0–8.5, and aeration rate 3 L/min. Initial density is 105 CFU/mL and is incubated for 7–10 days until density reaches approximately 106 CFU/mL. Figure 4 shows 1 L stock culture

Heterotrophic bacteria can also uptake ammonium and nitrate as their nitrogen source [64]. The main advantage of adding particular heterotrophic bacteria is related to growth rate of heterotrophic bacteria that exhibit much faster than nitrifying bacteria [65]. Just like nitrifying

though their exact mechanism of action remains unclear [61].

and 500 L scale-up culture for diatom C. calcitrans.

Figure 4. (a) Small scale and (b) large scale of Chaetoceros calcitrans in batch system.

3.2.3. Heterotrophic bacteria

environmental fluctuations.

306 Biological Resources of Water

bacteria, species of heterotrophic bacteria varies among different psychochemical conditions. Most common predominant species are Bacillus megaterium and Bacillus flexus [66]. B. megaterium is an example of well-studied bacteria for aquaculture application. The bacteria secretes high amount of extracellular enzymes, such as protease, carbohydrolase, and lipase, that can increase feed intake and digestibility in shrimp [67]. Other species that are also beneficial are Shewanella algae and Halomonas aquamarina. These natural-occurred bacteria have been studied and found to increase shrimp weight significantly [19].

Recent research progress proved that several heterotrophic bacteria genus (e.g. Bacillus sp. and Pseudomonas sp.) associated with microalgae through a mutual symbiosis. Although these heterotrophs have no direct role in controlling nitrogen cycle, their presence was proven to suppress growth of Vibrio spp. Mechanism of pathogenicity was done by quorum sensing [68]. This pathogen causes shrimp's mortality only when their population reaches a certain number of cells at least 106 CFU/mL [69]. Therefore, dominance of friendly heterotrophic bacteria can avoid pathogenic bacteria growth in the water, minimizing the risk of pathogen infection to the shrimp's gut.

Medium for culturing heterotrophic bacteria is nutrient broth medium for stock culture, while commercial medium is made from beef broth and ammonium chloride for large-scale cultivation. Heterotrophic bacteria were cultivated up to 15 L and then incubated for 24 h or until they reached the cell density of 107 CFU/mL. Figure 5 below shows heterotrophic bacteria B. megaterium that are cultivated for culture stock and large-scale reactor.
