Intensive zero water exchange shrimp grow outs arespecialised and highly dynamic aquauclture production systems wherebioremediation 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 decomposingorganic matter generated from phyto and zooplankton.
Unionized ammonia (NH3)is the toxic species and its percentagedepends upon the variation of pH and tempreature; at high pH and tempreaturethe NH3 concentrations shoots up. In nature nitrifyingbacteria bring forth the oxidation of ammonia to nitrite and to comapartivelyinnocuous nitrate, the process termed nitrification. In biological ammonia removal systems, nitrifyingactivity of suspended bacteria has been reported to be extremely low, due toslow growth rate (Bower and Turner 1981; Furukwa et al. 1993). With out theaddition of nitrifiers as start up culture, 2-3 months are needed to establishnitrification in marine systems (Manthe and Malone 1987) and 2-3 weeks in freshwater (Masser et al. 1999). There is an agreement, among researchers andbetween laboratory research and commercial application, on the fact that saltwater systems need much longer start up period. Under such situations,immobilization techniques have been found useful to overcome the delay in theinitiation of nitrification (Sung Koo et al.
2000). For such applications,nitrifying bacteria have to be generated in large quantity, and an importantconsideration of which is cost – effectiveness. The medium optimized here hasbeen seawater based and required only addition of the substrate NH4+- N as NH4Cl and Ca CO3 to maintain optimum pH. Thedesign consisted of 50 L conical tapering fermentation tank made of fiberreinforced plastic. An electrically operated stirrer/agitator is used toaccomplish agitation and mix up of the carrier material and nitrifying bacteriato maintain them in suspension. The fermentation tank has been made opaque andplaced well protected from sunlight, as the visible and UV light rays arelethal to nitrifying organisms (Johnstone and Jone 1988; Diab and Shilo 1988).
The carrier material, wood powder, used forimmobilization was locally available and inexpensive. Initially 50 L seawaterwas chlorinated with 300 mg/ L sodium hypochlorite and subsequently aerated toremove chlorine and supplemented with sodium thiosulphate to ensure its totalremoval. The carrier material was sterilized by autoclaving at 15 lbs for 15min. NBC was drawn from nitrifying bacterial production unit (Kumar et al. 2009).
Active 4 L NBC (105cells/mL) and 800 g wood powder were introduced in to 46 L seawater basedmedium in the device for immobilization of NBC and maintained in suspension. Whenpopulation of nitrifying bacteria gets established under steady stateconditions residual nitrite shall be too low to be detected with progressivebuilding up of nitrate according to the observations made by Achuthan et al. (2006) during the enrichment of nitrifyingbacterial cultures form shrimp ponds, and by Kumar et al. (2009) during their mass production. It has also been establishedthat nitrite oxidation to nitrate is more rapid than the preceeding step (Stenseland Barnad 1992). This was proved to be true in the present study over andagain as nitrite turned out to be below detectable level after seven to ninedays of initiation of immobilization. The time period required for immobilizationof NBC was determined by inoculating immobilizedNBC into fresh medium and analyzing the TAN removal rates which were0.27,0.
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 theprocessing of immobilized NBC after harvest with out loss of its nitrifyingpotency was developed. Two methods could be evolved, one was drying the woodpowder immobilized with nitrifiers by spreading at room temperature and the otherwas drying in a desiccator with out vaccum. NBC processed by both the methodsexhibited significant TAN removal compared to the one processed under vaccum (P< 0.05).
Reduction in the nitrifying activity of immobilized NBC processedin a vacuum desiccator might be due to the excessive loss of moisture contentfrom the preparation. Theshelf life of bacterial products happens to be a major issue in all commercialapplications. During this experiment 1 g each immobilized NBC was stored insealed polythene bags at room temperature for a period of one week to twelve weeks, and thestorage of nitrifiers over a period of three months under ambient conditionsdid not affect the nitrifying potency. Evaluationof immobilized NBC in the low and high stocking culture systems showed aremarkable reduction in the TAN concentration in the tests. The TANconcentration in the test tanks of low stocking density was 4.99 mg/ L whenimmobilised NBC was applied, and within two days, it could be fully removed. Meanwhilein the high stocking density culture system, 9.
98 mg/ L TAN could be totally removedwithin five days. NO2– N also showed depletion afterslight increase initially in both the cases demonstrating effective functioningof the two stage nitrification. Meanwhile NO3- –N stood between4 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 ofcertain level of nitrite accumulation for activating nitrite oxidizers in theconsortium until steady state equilibrium was reached (Sharmaand Ahler 1977; Smith et al. 1997; Vadivelu et al. 2007).TAN removal and drop inalkalinity showed a positive correlation in these systems. The conversion of NH4+- N to NO2– Nconsumed alkalinity in the form of Ca CO3 supplemented.
Alkalinityin the form of bicarbonate and carbonate become one of the carbon sources apartfrom carbon dioxide for nitrifying bacteria (Chen et al. 2006). Alkalinity isnormally consumed at approximately 7.
14 g/L/ N oxidized duringnitrification (Villaverde et al. 1997; Timmonas et al. 2002). At the end of theexperiment of the high stocking density system, the percentage survival ofshrimp in the test was 83.
3 ± 8.9% and in the control 45.5 ± 9.9%. Nitrate level was significantly higherin the tests compared to controls where it was found not getting built up demonstratingincomplete nitrification (Sandu et al. 2002).Overall, it was concluded that theeffective control of TAN in shrimp culture systems could be achieved throughthe application of immobilized NBC.
Novelty of this work lies on the fact thatthe proposed system is ideal for the removal of toxic NH4+- N in a high stocking density zerowater exchange shrimp culture system. The immobilization system is easy to befabricated and the wood powder can bemade available at ease as the plant (Ailanthus altissima ) is cultivatedfor soft timber widely, and is economically viable and degradable.