Ammonia Kuhn et al. 2010). In this

Ammonia
toxicity is one of the major problems in zero water exchange intensive shrimp
culture system. Optimum shrimp growth demands less than 0.1 mg/L unionized
ammonia (NH3) in culture ponds (1.33 to 1.53 mg/L TAN at pH 8 and 28?
– 30?C ) (Shan and Obbard 2001; Lin and Chen 2001, 2003). Its higher
concentration increases pH and reduces dissolved oxygen in blood, causes stress
resulting in reduced feeding and disposition to diseases (Wickins 1976; Schuler
et al. 2010).   

The
most widely used methods for addressing ammonia toxicity in aquaculture is the
establishment of biological nitrification using biological filters, biofilm
reactors and nitrifiying bacteria as such. Natural colonization of nitrifying
bacteria takes relatively longer time ( 4 to 8 weeks), and in intensive culture
systems naturally occurring nitrification may not be sufficient as it is beyond
the carrying capacity of the system, which  is sensitive to physical and chemical changes
such as salinity and/or temperature  (Malone
and Pfeiffer 2006; Emparanza 2009; Kuhn et al. 2010).

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In this context,
bioaugmentation gains importance in bioremediation of TAN in intensive culture
systems. In this process immobilization of nitrifiers prior to application has
turned out to be an attractive proposition considering its effective
application. Immobilization technology has been used extensively in commercial
bioreactor fermentations (Chen et al.
2000; Nakano et al. 2004; Zala et al. 2004; Li and Logan 2004). Nitrosomonas europaea
(ATCC 19718) immobilized  on biofilters
have been successfully applied to the removal of NH3  (Chung and
Huang 1998), for the treatment of mixtures of
H2S and NH3 with a two-stage biofilter (Chung et al. 2007) and
in a biotrickling filter packed with polyurethane foam (Ramirez et al. 2009). Several natural
materials (agar, agarose, collagen, alginates and chitosan, microbial
cellulose) and synthetic polymer materials (polyacrylamide, polyurethane,
polyethylene glycol and polyvinyl alcohol) have been used as the substrata for
immobilization (Jianlong et al. 1998;
Fang et al. 2004; Rezaee et al. 2008;
Boonpauk et al. 2011; Peirong and Wei
2011).

National Centre for Aquatic
Animal Health (NCAAH) developed an economically viable and user friendly
technology of bioaugmenting nitrification based on NBC (Achuthan et al. 2006) immobilized on wood powder as
biodegradable carrier, which would not leave any residue on degradation (Manju et al. 2009). Wood
powder from the plant species Ailanthus altissima was used for immobilization by adsorption. Subsequently
the present work was undertaken for mass production of NBC immobilized on wood
powder and validation of the efficacy of the product for removal of TAN from
shrimp culture systems.

Materials and Methods

Nitrifying Bacterial Consortium (NBC)

The Ammonia
oxidizing bacterial consortium for penaeid shrimp culture system (AMOPCU) (Achuthan
et al. 2006) was generated in the nitrifying
bacterial consortia production unit (NBCPU) (Kumar et al. 2009) maintained at
NCAAH.

 Wood powder

 Processing of
the carrier material

Wood
chips of the plant species Ailanthus
altissima were collected from local timber industry. They were dried,
crushed and sieved to get particle size 300-500 µm. As lignin in wood powder
might interfere with the process of immobilization, it was delignified.

For
delignification, the method proposed by Wood and Saddler (1988) was employed as
follows: 1 g crushed wood powder was immersed in 50 mL tap water containing 1%
V/V H2O2, with 0.1 N NaOH,
pH of the suspension adjusted to 11.5. The suspension was stirred gently at 25?C
for 3 – 5 h on magnetic stirrer, with hourly correction of pH to 11.5 as per
requirement. The suspension was filtered and the insoluble residue collected,
washed, till pH dropped to neutrality. Delignified wood residue was dried at
100?C before storage. Presence of lignin residue was checked by
treatment with hot sodium sulphite. To 0.5 g delignified
material aliquots of 5 mL aqueous hot sodium sulfite (5g/500 mL) were added.
Release of magentha coloured liquor indicated delignification.

Immobilization of nitrifying bacterial consortium (NBC)
on wood powder

 Device for
immobilization

The
device was designed based on the requirements for obtaining maximum biomass
with in the shortest duration possible, and fabricated with locally available
materials. The 50 L capacity cylindrical immobilization device with conical
tapering bottom was fitted with a stirrer assembly and an air diffuser at the
bottom (0-500 rpm) (Fig. 1). Seawater (15 g/L salinity)
(40 L) was chlorinated using sodium hypochlorite to attain 200 mg/L chlorine and
after twelve hours de-chlorinated using 15 g sodium thiosulphate. The vessel
was aerated for 2 days through a cartridge filter (0.2 µm) and plated out on to
ZoBell’s agar prepared in aged seawater (15 g/L) to record the presence of
total viable bacterial population, prior to inoculating with the nitrifiers. As
nutrients, 10 mg / L NH4+-
N and 2 mg/ L PO4- P (as NH4Cl
and KH2PO4) were added, pH adjusted to 7.5 using Na2CO3.
An aliquot of 4 L inoculum having 105cells/
mL was introduced.  A quantity of
800 g crushed, sieved and delignified wood powder from Ailanthus altissima (Pongalayam) as the substratum was added to the
immobilization tank and the aeration set at 6 L/
min with an ambient temperature of 27°C. Samples were
analysed daily for pH, TAN, NO2-N and NO3-N. The pH was
maintained using aqueous 10% sodium carbonate. As the consumption of NH3+
– N progressed; it was supplemented with aliquots of fresh substrate at an exponential
rate. The process was continued with daily monitoring of NH4+
– N consumption and NO2-N and NO3-N production until
the culture attained stationary phase

Quantification
of immobilized nitrifying bacterial biomass

            Quantification
of immobilized nitrifying bacterial biomass was accomplished following ATP
bioluminescent method (Ukuku et al. 2005).

ATP Extraction

 An aliquot of
1 g (wet weight) sample was dropped in to test tube containing 5 mL boiling
Tris buffer (pH 7.75, 0.1 M). The content was boiled for  further 60 s, subsequent to which the tube
was cooled and after centrifugation (1000 g) the supernatant used for ATP  estimation.

ATP estimation

Standardization

            A primary ATP standard was prepared by dissolving 10 mg high purity ATP
(sodium salt) dissolved in 10 mL distilled water. The solution was diluted to
1/10 of the primary standard. Placed a portion of the solution in a 1 cm quarts
cuvette and measured the absorbance at 259 nm.  
Concentration of ATP was calculated using the equation A = Elc, where A
= absorbance at 259 nm; E = ATP molar extinction coefficient (15.4 x 103);  l = path length of cuvette (1 cm) and c =
concentration of ATP in moles/ L. Working standards of 10, 30, 50, 70 and 100
ng ATP per µL were prepared in 0.02 M tris buffer (pH 7.74).

            An aliquot of 30 µL sample was added to an optical sensing cell and 270
µL Luciferase – Luciferin reagent (39 µg/ mL Luciferase, 78 µg/ mL Luciferin,
1.1 mmol/ L EDTA 2 Na, 11 mmol/ L magnesium acetate tetrahydrate, 1.1 mg/ mL
BSA, 0.6 mmol/L DTT, and 25 mmol/ L Tris – acetate (pH 7.8) was added subsequently.
Luminescent intensity was measured using Luminometer (Turner Bio systems, USA).  ATP standards were also analyzed the same way
to draw calibration curve.

Determination of nitrification potential of immobilized
NBC on wood powder

            The
nitrifying potential of immobilized nitrifiers on wood powder was determined as
follows: The sample was filtered using tea filter, dried over blotting paper
and maintained in a  desiccator under
vaccum, without vaccum and also spread on a polythene sheet, all at room
temperature (RT) (28±1? C). After drying, the content (1 g) was transferred
to 100 mL Watson’s medium (1965) (composed of sea water (salinity 15 g/ L) with
NH4+ – N (10 mg/ L), PO4- –
P (2 mg/ L) and pH 8.0) and maintained on a shaker (Remi, India) at 100 rpm and
the activity was determined by measuring TAN consumption and NO2 –
N/NO3 – N production.

Determination of shelf
life of immobilized NBC

          The
nitrification potential of immobilized nitrifires after storage was determined as
follows: The sample was filtered using tea filter, dried over blotting paper
and 1 g was aseptically transferred to polyethylene bags, sealed and maintained
in a box at RT. Once in seven days 1 g each was transferred to 100 mL Watson’s
medium (1965) and maintained on shaker at 100 rpm and the activity determined
by measuring TAN consumption and NO2-–N /NO3–N
 production.

Determination
of the quantity of immobilized NBC required for treating unit volume of water

            Activated immobilized nitrifiers of 0.1 to 1 g were administrated to 1 L
seawater based Watson’s medium (1965) in triplicates in conical flasks with
aeration at the rate 1L per minute. Nitrification over a period of three days
was monitored and measured in terms of TAN consumption and NO2–N/
NO3–N production.

Evaluation
of nitrifying potency of immobilized NBC

a)     
In a simulated low stocking density
shrimp culture system.

            The experimental design consisted of six tanks each holding 24 L 15 g/ L salinity
seawater maintained under aeration at a rate of 2 L/ min. Each set consisted of
control and test tanks in triplicate. The experiment was conducted having 6
shrimps/m2 with an average weight of 15 g ( P.monodon) maintained without water exchange and fed with commercial
pelleted feed (CP Feed, Chennai, India) at a rate of 4% of body weight. After 8
days when TAN loadings were 5 mg/ L, a quantity of 3 g immobilized NBC was applied
per tank. Water quality parameters such as, TAN, NO2–N,
NO3–N, alkalinity and pH were monitored daily.

b)     
In a simulated high stocking
density shrimp culture system.

            The experiment was conducted in 100 L capacity fiber glass tanks. The
experimental design consisted of six tanks, three each for control and test
with 15 g/ L seawater. The shrimps were stocked at the rate of 24/m2
with an average body weight of 8 – 10 g maintained without water exchange and
fed with commercial pelleted feed (CP Feed, Chennai, India) at a rate of 4% of
the body weight with a frequency of twice a day. After one week when the TAN
level became 10 mg/ L, 12 g immobilized NBC was added to the test tanks. Water
quality parameters such as, TAN, NO2- – N, NO3–
N, alkalinity and pH were monitored daily.

Results

Immobilization of NBC on wood powder

            The substrate consumption
and product formation during mass immobilization of NBC meant for 15 g/ L
salinity culture system is illustrated in Fig. 2. The system which started with  10
mg/ L residual NH4 + – N consumed 583.6  mg/ L  NH4+- N over a period of
75 days  with a total corresponding
output of 415.6 mg/ L NO3– N. Growth curve
showed that there was  progressive build
up of NO2– N until seven days and subsequently it
rapidly declined and NO3– N began to accumulate. After that,
no residual nitrite could be detected and oxidation of NH4+
– N and NO2- -N was found to take place simultaneously to
form NO3- -N.

Determination of immobilized nitrifying bacterial biomass
based on ATP bioluminescent method

            The immobilized nitrifying bacterial biomass  estimated at stationary phase of the
culture  was  4.24 x 107 CFU g/L. This result
was based on the relationship 1.61 log CFU g/ L = 3.18 log fg/ L ATP (Ukuku et
al. 2005).

Determination
of nitrifying potential of immobilized NBC on wood powder

            The immobilized NBC was dried under different conditions, such as
desiccation under vacuum and with out vacuum, and by spreading on polythene
sheet all at room temperature (RT).

 The results showed that TAN removal took place
within a day in the experimental system inoculated with immobilized NBC. In the
system inoculated with NBC dried in vacuum desiccator the TAN removal and NO2-
– N production were 1.07 and 0.25 mg/ L/ day respectively. In the system
inoculated with NBC dried in dessiccator with out vacuum the removal and
production were 7.09 and 3.7 mg/ L/day respectively. However, in the
system inoculated with NBC dried by spreading at room temperature, TAN removal
was 8.9 and NO2- – N production was 4.18 mg/ L/day. On
day 2nd in the systems inoculated with NBC dried in desiccator  with out vacuum and dried at room temperature
no residual TAN was detected, however, the one inoculated with vacuum
desiccated NBC the TAN removal was negligible. Following a similar trend,
highest NO2- – N production was recorded in the systems
inoculated with NBC dried by spreading at room temperature (Table. 1).

Determination of shelf life of immobilized NBC

                             In this experiment,
total NH3+ – N removal (10 mg/ L) took place within 48 h
with respect to the immobilized NBC stored for three weeks. The immobilized
NBC, which were stored for 8 weeks, could consume NH4+ –
N (10 mg/ L) within 72 h, and others having a storage period up to 12 weeks
took 96 h for the same (Table. 2). 

On considering the rate of ammonia
consumption by the immobilized consortia stored over a period, the one, which
was stored for a week, could consume NH4+ – N up to 110 mg/ L with in 288 h. Meanwhile, the samples
stored for prolonged period were showing reduced rate of consumption during the
same period. 

Determination
of the quantity of immobilized nitrifiers required for treating unit volume of
water

To accomplish the above, varying
quantities of immobilized NBC on wood powder were incubated in 1L seawater
having 15 g/ L salinity. Initially TAN was maintained at 10 mg/ L. Nitrification
was monitored in terms of per day TAN removal and NO2– N
production. On day 1, the TAN removal rates in the systems  inoculated with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1 g wood powder immobilized with nitrifying bacterial
consortium  were 1.05, 3.98, 4.11, 4.2, 4.2,
4.3, 4.4, 4.62, 4.39, 4.98 mg/ L  respectively, whereas in the control it was
0.8 mg/ L. The NO2– N production in the same systems was
0.03, 0.98, 1.83, 2.08, 2.34, 2.01, 2.38, 2.18, 2.09, 2.99 respectively,
whereas in the control it was 0.098 mg/ L (Table.
3). TAN removal per day and quantity of immobilized NBC showed a
positive correlation (0.895).

Evaluation
of nitrifying potency of immobilized nitrifying bacterial consortia in a system
with low stocking density.

            TAN removal was obvious from point of addition of NBC to the system nine
days after its initiation (Fig.3). After
two days, the entire TAN (4.99 mg/ L) was removed in the test tanks where average
lowering of 2.75 mg/ L/ day of TAN was observed. In the corresponding control a
lowering of 0.76 mg/ L/ day, three fold lower than the test tanks, was seen.  On the second day, the NO2–
N production in the test tank was 2.26 mg/ L and an increased out put of 3.9 mg/
L/ day on the third day. On subsequent days it declined, and by the time NO3-
– N production had commenced (on the 4th day onwards) with a corresponding
decrease of NO2– N (Fig. 4).
A negative correlation was observed between TAN and NO3- N in the
tests (r -0.59) indicating effective nitrification, whereas in the control
tanks elevated levels of NH4+ – N and NO2– N could be recorded.
TAN removal and drop in alkalinity (Fig.5)
in the test tanks were positively correlated (r = 0.838).

Evaluation
of nitrification potency of immobilized NBC in systems with high stocking
density.

            In the system with high stocking density, the entire
(9.98 mg/ L) quantity of total ammonia nitrogen in the test tanks could be removed
within five days (Fig.6). Average
lowering of 2.002 mg/ L/ day TAN was observed in the test tanks, whereas in the
corresponding control tanks it was 0.58 mg/ L/ day. In the tests NO2-
–N registered maximum value on the 14th day (3.35 mg/ L), which
declined corresponding to the decline of TAN concentration (Fig. 7). Mean
NO3- – N level increased from 0.1 to 6.39 mg/ L in
the test tanks, and remained at 0.005 to 1.89 mg/ L in the control
systems. NO3- – N levels were significantly higher (P
< 0.05) in the treated systems compared to the control systems through out the experimental period. TAN removal and drop in alkalinity were positively correlated (r 0.769) Fig.8. Discussion             Intensive zero water exchange shrimp grow outs are specialised and highly dynamic aquauclture production systems where bioremediation of NH4+ - N and NO2-- N are the vital processes for  successful culmination of the culture. NH4+ - N originates from  animal excreta, uneaten feed and decomposing organic matter generated from phyto and zooplankton. Unionized ammonia (NH3) is the  toxic species and its percentage depends upon the variation of pH and tempreature; at high pH and tempreature the NH3 concentrations shoots up. In nature nitrifying bacteria bring forth the oxidation of ammonia to nitrite and to comapartively innocuous nitrate, the process termed  nitrification. In biological ammonia removal systems, nitrifying activity of suspended bacteria has been reported to be extremely low, due to slow growth rate (Bower and Turner 1981; Furukwa et al. 1993). With out the addition of nitrifiers as start up culture, 2-3 months are needed to establish nitrification in marine systems (Manthe and Malone 1987) and 2-3 weeks in fresh water (Masser et al. 1999). There is an agreement, among researchers and between laboratory research and commercial application, on the fact that salt water systems need much longer start up period. Under such situations, immobilization techniques have been found useful to overcome the delay in the initiation of nitrification (Sung Koo et al. 2000). For such applications, nitrifying bacteria have to be generated in large quantity, and an important consideration of which is cost – effectiveness. The medium optimized here has been seawater based and required only addition of the substrate NH4+ - N as NH4Cl and Ca CO3 to maintain optimum pH. The design consisted of 50 L conical tapering fermentation tank made of fiber reinforced plastic. An electrically operated stirrer/agitator is used to accomplish agitation and mix up of the carrier material and nitrifying bacteria to maintain them in suspension. The fermentation tank has been made opaque and placed well protected from sunlight, as the visible and UV light rays are lethal to nitrifying organisms (Johnstone and Jone 1988; Diab and Shilo 1988). The carrier material, wood powder, used for immobilization was locally available and inexpensive. Initially 50 L seawater was chlorinated with 300 mg/ L sodium hypochlorite and subsequently aerated to remove chlorine and supplemented with sodium thiosulphate to ensure its total removal. The carrier material was sterilized by autoclaving at 15 lbs for 15 min. NBC was drawn from nitrifying bacterial production unit (Kumar et al. 2009).             Active 4 L NBC (105 cells/mL) and 800 g wood powder were introduced in to 46 L seawater based medium in the device for immobilization of NBC and maintained in suspension. When population of nitrifying bacteria gets established under steady state conditions residual nitrite shall be too low to be detected with progressive building up of nitrate according to the observations made by Achuthan et al. (2006) during the enrichment of nitrifying bacterial cultures form shrimp ponds, and by Kumar et al. (2009) during their mass production. It has also been established that nitrite oxidation to nitrate is more rapid than the preceeding step (Stensel and Barnad 1992). This was proved to be true in the present study over and again as nitrite turned out to be below detectable level after seven to nine days of initiation of immobilization.             The time period required for immobilization of NBC was determined by inoculating  immobilized NBC into fresh medium and analyzing the TAN removal rates which were 0.27,0.29.0.35 and 0.35 g/ m2/ day on the 7th, 8th, 9th and 10th day respectively (Manju et al. 2009).             A simple technique for the processing of immobilized NBC after harvest with out loss of its nitrifying potency was developed. Two methods could be evolved, one was drying the wood powder immobilized with nitrifiers by spreading at room temperature and the other was drying in a desiccator with out vaccum. NBC processed by both the methods exhibited significant TAN removal compared to the one processed under vaccum (P < 0.05). Reduction in the nitrifying activity of immobilized NBC processed in a vacuum desiccator might be due to the excessive loss of moisture content from the preparation.              The shelf life of bacterial products happens to be a major issue in all commercial applications. During this experiment 1 g each immobilized NBC was stored in sealed polythene bags at room temperature for  a period of one week to twelve weeks, and the storage of nitrifiers over a period of three months under ambient conditions did not affect the nitrifying potency.             Evaluation of immobilized NBC in the low and high stocking culture systems showed a remarkable reduction in the TAN concentration in the tests. The TAN concentration in the test tanks of low stocking density was 4.99 mg/ L when immobilised NBC was applied, and within two days, it could be fully removed. Meanwhile in the high stocking density culture system, 9.98 mg/ L TAN could be totally removed within five days. NO2-- N also showed depletion after slight increase initially in both the cases demonstrating effective functioning of the two stage nitrification. Meanwhile NO3- –N stood between 4 to 6 mg/ L. In these systems, the TAN oxidation was established within a day, but the NO2-- N oxidation took 4 days. The delay in nitrite oxidation could have been due to the requirement of certain level of nitrite accumulation for activating nitrite oxidizers in the consortium until steady state equilibrium was reached (Sharma and Ahler 1977; Smith et al. 1997; Vadivelu et al. 2007). TAN removal and drop in alkalinity showed a positive correlation in these systems. The conversion of NH4+ - N to NO2-- N consumed alkalinity in the form of Ca CO3 supplemented. Alkalinity in the form of bicarbonate and carbonate become one of the carbon sources apart from carbon dioxide for nitrifying bacteria (Chen et al. 2006). Alkalinity is normally consumed at approximately 7.14 g/L/ N oxidized during nitrification (Villaverde et al. 1997; Timmonas et al. 2002). At the end of the experiment of the high stocking density system, the percentage survival of shrimp in the test was 83.3 ± 8.9% and in the control 45.5 ± 9.9%. Nitrate level was significantly higher in the tests compared to controls where it was found not getting built up demonstrating incomplete nitrification (Sandu et al. 2002). Overall, it was concluded that the effective control of TAN in shrimp culture systems could be achieved through the application of immobilized NBC. Novelty of this work lies on the fact that the proposed system is ideal for the removal of toxic NH4+ - N in a high stocking density zero water exchange shrimp culture system. The immobilization system is easy to be fabricated and the wood powder  can be made available at ease as the plant ( Ailanthus altissima ) is cultivated for soft timber widely, and is economically viable and degradable.  

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