Introduction used as the substrata for immobilization

IntroductionAmmoniatoxicity is one of the major problems in zero water exchange intensive shrimpculture system.

Optimum shrimp growth demands less than 0.1 mg/L unionizedammonia (NH3) in culture ponds (1.33 to 1.

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53 mg/L TAN at pH 8 and 28?– 30?C ) (Shan and Obbard 2001; Lin and Chen 2001, 2003). Its higherconcentration increases pH and reduces dissolved oxygen in blood, causes stressresulting in reduced feeding and disposition to diseases (Wickins 1976; Schuleret al. 2010).   Themost widely used methods for addressing ammonia toxicity in aquaculture is theestablishment of biological nitrification using biological filters, biofilmreactors and nitrifiying bacteria as such. Natural colonization of nitrifyingbacteria takes relatively longer time ( 4 to 8 weeks), and in intensive culturesystems naturally occurring nitrification may not be sufficient as it is beyondthe carrying capacity of the system, which  is sensitive to physical and chemical changessuch as salinity and/or temperature  (Maloneand Pfeiffer 2006; Emparanza 2009; Kuhn et al. 2010). In this context,bioaugmentation gains importance in bioremediation of TAN in intensive culturesystems.

In this process immobilization of nitrifiers prior to application hasturned out to be an attractive proposition considering its effectiveapplication. Immobilization technology has been used extensively in commercialbioreactor fermentations (Chen et al.2000; Nakano et al.

2004; Zala et al. 2004; Li and Logan 2004). Nitrosomonas europaea(ATCC 19718) immobilized  on biofiltershave been successfully applied to the removal of NH3  (Chung andHuang 1998), for the treatment of mixtures ofH2S and NH3 with a two-stage biofilter (Chung et al. 2007) andin a biotrickling filter packed with polyurethane foam (Ramirez et al. 2009). Several naturalmaterials (agar, agarose, collagen, alginates and chitosan, microbialcellulose) and synthetic polymer materials (polyacrylamide, polyurethane,polyethylene glycol and polyvinyl alcohol) have been used as the substrata forimmobilization (Jianlong et al. 1998;Fang et al.

2004; Rezaee et al. 2008;Boonpauk et al. 2011; Peirong and Wei2011). National Centre for AquaticAnimal Health (NCAAH) developed an economically viable and user friendlytechnology of bioaugmenting nitrification based on NBC (Achuthan et al.

2006) immobilized on wood powder asbiodegradable carrier, which would not leave any residue on degradation (Manju et al. 2009). Woodpowder from the plant species Ailanthus altissima was used for immobilization by adsorption. Subsequentlythe present work was undertaken for mass production of NBC immobilized on woodpowder and validation of the efficacy of the product for removal of TAN fromshrimp culture systems.Materials and Methods Nitrifying Bacterial Consortium (NBC)The Ammoniaoxidizing bacterial consortium for penaeid shrimp culture system (AMOPCU) (Achuthanet al. 2006) was generated in the nitrifyingbacterial consortia production unit (NBCPU) (Kumar et al. 2009) maintained atNCAAH.

 Wood powder Processing ofthe carrier materialWoodchips of the plant species Ailanthusaltissima were collected from local timber industry. They were dried,crushed and sieved to get particle size 300-500 µm. As lignin in wood powdermight interfere with the process of immobilization, it was delignified.Fordelignification, the method proposed by Wood and Saddler (1988) was employed asfollows: 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?Cfor 3 – 5 h on magnetic stirrer, with hourly correction of pH to 11.5 as perrequirement.

The suspension was filtered and the insoluble residue collected,washed, till pH dropped to neutrality. Delignified wood residue was dried at100?C before storage. Presence of lignin residue was checked bytreatment with hot sodium sulphite. To 0.5 g delignifiedmaterial 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 forimmobilization Thedevice was designed based on the requirements for obtaining maximum biomasswith in the shortest duration possible, and fabricated with locally availablematerials. The 50 L capacity cylindrical immobilization device with conicaltapering bottom was fitted with a stirrer assembly and an air diffuser at thebottom (0-500 rpm) (Fig. 1). Seawater (15 g/L salinity)(40 L) was chlorinated using sodium hypochlorite to attain 200 mg/L chlorine andafter twelve hours de-chlorinated using 15 g sodium thiosulphate. The vesselwas aerated for 2 days through a cartridge filter (0.2 µm) and plated out on toZoBell’s agar prepared in aged seawater (15 g/L) to record the presence oftotal viable bacterial population, prior to inoculating with the nitrifiers. Asnutrients, 10 mg / L NH4+-N and 2 mg/ L PO4- P (as NH4Cland KH2PO4) were added, pH adjusted to 7.5 using Na2CO3.

An aliquot of 4 L inoculum having 105cells/mL was introduced.  A quantity of800 g crushed, sieved and delignified wood powder from Ailanthus altissima (Pongalayam) as the substratum was added to theimmobilization tank and the aeration set at 6 L/min with an ambient temperature of 27°C. Samples wereanalysed daily for pH, TAN, NO2-N and NO3-N. The pH wasmaintained using aqueous 10% sodium carbonate. As the consumption of NH3+- N progressed; it was supplemented with aliquots of fresh substrate at an exponentialrate. The process was continued with daily monitoring of NH4+- N consumption and NO2-N and NO3-N production untilthe culture attained stationary phase Quantificationof immobilized nitrifying bacterial biomass            Quantificationof immobilized nitrifying bacterial biomass was accomplished following ATPbioluminescent method (Ukuku et al. 2005). ATP Extraction An aliquot of1 g (wet weight) sample was dropped in to test tube containing 5 mL boilingTris buffer (pH 7.

75, 0.1 M). The content was boiled for  further 60 s, subsequent to which the tubewas cooled and after centrifugation (1000 g) the supernatant used for ATP  estimation.ATP estimationStandardization             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 to1/10 of the primary standard. Placed a portion of the solution in a 1 cm quartscuvette 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 100ng 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 270µL 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/ mLBSA, 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 wayto draw calibration curve. Determination of nitrification potential of immobilizedNBC on wood powder            Thenitrifying potential of immobilized nitrifiers on wood powder was determined asfollows: The sample was filtered using tea filter, dried over blotting paperand maintained in a  desiccator undervaccum, without vaccum and also spread on a polythene sheet, all at roomtemperature (RT) (28±1? C). After drying, the content (1 g) was transferredto 100 mL Watson’s medium (1965) (composed of sea water (salinity 15 g/ L) withNH4+ – N (10 mg/ L), PO4- -P (2 mg/ L) and pH 8.0) and maintained on a shaker (Remi, India) at 100 rpm andthe activity was determined by measuring TAN consumption and NO2 -N/NO3 – N production. Determination of shelflife of immobilized NBC           Thenitrification potential of immobilized nitrifires after storage was determined asfollows: The sample was filtered using tea filter, dried over blotting paperand 1 g was aseptically transferred to polyethylene bags, sealed and maintainedin a box at RT.

Once in seven days 1 g each was transferred to 100 mL Watson’smedium (1965) and maintained on shaker at 100 rpm and the activity determinedby measuring TAN consumption and NO2-–N /NO3–N production.Determinationof 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 Lseawater based Watson’s medium (1965) in triplicates in conical flasks withaeration at the rate 1L per minute. Nitrification over a period of three dayswas monitored and measured in terms of TAN consumption and NO2–N/NO3–N production.Evaluationof nitrifying potency of immobilized NBC a)     In a simulated low stocking densityshrimp culture system.            The experimental design consisted of six tanks each holding 24 L 15 g/ L salinityseawater maintained under aeration at a rate of 2 L/ min.

Each set consisted ofcontrol and test tanks in triplicate. The experiment was conducted having 6shrimps/m2 with an average weight of 15 g ( P.monodon) maintained without water exchange and fed with commercialpelleted feed (CP Feed, Chennai, India) at a rate of 4% of body weight. After 8days when TAN loadings were 5 mg/ L, a quantity of 3 g immobilized NBC was appliedper tank. Water quality parameters such as, TAN, NO2–N,NO3–N, alkalinity and pH were monitored daily. b)     In a simulated high stockingdensity shrimp culture system.            The experiment was conducted in 100 L capacity fiber glass tanks. Theexperimental design consisted of six tanks, three each for control and testwith 15 g/ L seawater.

The shrimps were stocked at the rate of 24/m2with an average body weight of 8 – 10 g maintained without water exchange andfed with commercial pelleted feed (CP Feed, Chennai, India) at a rate of 4% ofthe body weight with a frequency of twice a day. After one week when the TANlevel became 10 mg/ L, 12 g immobilized NBC was added to the test tanks. Waterquality parameters such as, TAN, NO2- – N, NO3–N, alkalinity and pH were monitored daily.

ResultsImmobilization of NBC on wood powder            The substrate consumptionand product formation during mass immobilization of NBC meant for 15 g/ Lsalinity culture system is illustrated in Fig. 2. The system which started with  10mg/ L residual NH4 + – N consumed 583.6  mg/ L  NH4+- N over a period of75 days  with a total correspondingoutput of 415.6 mg/ L NO3– N. Growth curveshowed that there was  progressive buildup of NO2– N until seven days and subsequently itrapidly 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 toform NO3- -N. Determination of immobilized nitrifying bacterial biomassbased on ATP bioluminescent method            The immobilized nitrifying bacterial biomass  estimated at stationary phase of theculture  was  4.

24 x 107 CFU g/L. This resultwas based on the relationship 1.61 log CFU g/ L = 3.18 log fg/ L ATP (Ukuku etal.

2005).Determinationof nitrifying potential of immobilized NBC on wood powder            The immobilized NBC was dried under different conditions, such asdesiccation under vacuum and with out vacuum, and by spreading on polythenesheet all at room temperature (RT).  The results showed that TAN removal took placewithin a day in the experimental system inoculated with immobilized NBC. In thesystem 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 systeminoculated with NBC dried in dessiccator with out vacuum the removal andproduction were 7.09 and 3.7 mg/ L/day respectively. However, in thesystem inoculated with NBC dried by spreading at room temperature, TAN removalwas 8.

9 and NO2- – N production was 4.18 mg/ L/day. Onday 2nd in the systems inoculated with NBC dried in desiccator  with out vacuum and dried at room temperatureno residual TAN was detected, however, the one inoculated with vacuumdesiccated NBC the TAN removal was negligible. Following a similar trend,highest NO2- – N production was recorded in the systemsinoculated 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 hwith respect to the immobilized NBC stored for three weeks. The immobilizedNBC, 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 weekstook 96 h for the same (Table. 2).  On considering the rate of ammoniaconsumption by the immobilized consortia stored over a period, the one, whichwas stored for a week, could consume NH4+ – N up to 110 mg/ L with in 288 h.

Meanwhile, the samplesstored for prolonged period were showing reduced rate of consumption during thesame period.  Determinationof the quantity of immobilized nitrifiers required for treating unit volume ofwaterTo accomplish the above, varyingquantities of immobilized NBC on wood powder were incubated in 1L seawaterhaving 15 g/ L salinity. Initially TAN was maintained at 10 mg/ L. Nitrificationwas monitored in terms of per day TAN removal and NO2– Nproduction.

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 bacterialconsortium  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 was0.8 mg/ L.

The NO2– N production in the same systems was0.03, 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 apositive correlation (0.895). Evaluationof nitrifying potency of immobilized nitrifying bacterial consortia in a systemwith low stocking density.            TAN removal was obvious from point of addition of NBC to the system ninedays after its initiation (Fig.3). Aftertwo days, the entire TAN (4.

99 mg/ L) was removed in the test tanks where averagelowering of 2.75 mg/ L/ day of TAN was observed. In the corresponding control alowering 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 correspondingdecrease of NO2– N (Fig. 4).

A negative correlation was observed between TAN and NO3- N in thetests (r -0.59) indicating effective nitrification, whereas in the controltanks 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). Evaluationof nitrification potency of immobilized NBC in systems with high stockingdensity.            In the system with high stocking density, the entire(9.98 mg/ L) quantity of total ammonia nitrogen in the test tanks could be removedwithin five days (Fig.6). Averagelowering of 2.002 mg/ L/ day TAN was observed in the test tanks, whereas in thecorresponding 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), whichdeclined corresponding to the decline of TAN concentration (Fig. 7). MeanNO3- – N level increased from 0.1 to 6.39 mg/ L inthe test tanks, and remained at 0.005 to 1.89 mg/ L in the controlsystems.

NO3- – N levels were significantly higher (P< 0.05) in the treated systems compared to the control systems through outthe experimental period. TAN removal and drop in alkalinity were positivelycorrelated (r 0.769) Fig.8.Discussion            Intensivezero water exchange shrimp grow outs are specialised and highly dynamicaquauclture 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 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. 

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