Nitrifying of total viable bacterial population, prior to

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.

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 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