Intensive reported to be extremely low, due to slow

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.

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            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, 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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