See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/324390074

Depuration kinetics and bioaugumentation studies on ammonia using

pseudomonas putida for aquaculture systems

Article  in  Asian Journal of Microbiology, Biotechnology and Environmental Sciences · January 2018

CITATIONS READS

0 53

1 author:

Radhakrishnan Preetha
SRM Institute of Science and Technology
52 PUBLICATIONS   504 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

probiotics from indian fermented foods View project

probiotics from Indian Fermented Foods View project

All content following this page was uploaded by Radhakrishnan Preetha on 08 June 2018.

The user has requested enhancement of the downloaded file.

Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 20, No. (1) : 2018 : 247-251
© Global Science Publications
ISSN-0972-3005

DEPURATION KINETICS AND BIOAUGUMENTATION STUDIES

ON AMMONIA USING PSEUDOMONAS PUTIDA FOR
AQUACULTURE SYSTEMS

HEERTHANA V.R. AND PREETHA R.*

1
Department of Food and Process Engineering, SRM University Kaatankulathur, Chennai 603 203, India

(Recieved 26 May, 2017; accepted 14 July, 2017)

Key words : Depuration, Bioaugumentation, Ammonia, Elutriation, Bioremediation

Abstract- The present depuration study explain bio cleansing activity of ammonia at lab level using
immobilized P. putida cells with bentonite clay. The bentonite clay has wide scope for water treatment
with healing properties. The bioaugumentation study was conducted to analyze the biodegradation
ability of P.putida cells immobilized in bentoniteclay. The immobilization of the cells was carried out by
adhesion technique using bentonite clay. The bentonite clay was separated from quartz and barium
using elutriation technique using stokes law and other minerals present was characterized using
scanning electric microscopy (SEM) and energy dispersive spectroscopy (EDS). The mineral studies
conducted by elutriation technique using EDS shows the availability of molecular oxygen as 114.44% and
carbon (17.58%) which ensured the microhabitat for the growth of P. putida cells. The study determined
the effect of surface characteristics on the growth of bacteria depending on complex integrity between
the physical, chemical and biological factors in the clay. Other micronutrients availability such as Fe
(0.42%), Al (0.58%), and Si (0.84%) render the growth of bacteria and titanium present (0.11%) per weight
helps in flocculation of other contaminants present in aquaculture systems. Advanced water treatment
with bio filtration system facilitates aquaculture bioremediation to prevent quality and safety of fish and
shellfish.

INTRODUCTION in removing other contaminants such as naturally

An overall measure of the concentration of
biodegradable substance in environment is known
as depuration. In aerobic biodegradation, the most
energy comes from oxidation of carbon present at
innate reduced state. Pseudomonas putida that have
the ability to utilize pollutants such as alcohols,
fuels and solvents as well as natural organic
detritus as a source of energy(Hartig et al., 1999).
Similarly there are microorganism which are
ammonia-eaters and commonly known as
diazotrophs (anaerobic). Microorganisms can be
configured to help aquaculture environment to
reduce ammonia level that prevents brown blood
disease in fish and shellfish (Jungst et al., 1991).In
modern commercialpractice, bentonite clay
wasreported as tool for removing viral contami-
nants such as nor virus and hepatitis A. It was
found to be consistently effective, or was ineffective,
occurring marine vibrio’s (e.g. Vibrio parahaemolyticus
and Vibrio vulnificus), marine bio toxins. For
microbial contaminants, the risk gets enhanced by
the fact that those shellfish and fish are often eaten
raw or relatively lightly cooked. The risk may be
reduced further by appropriate treatment
following harvest. Over time, feeding and excreta
released produces nitrogen combining with
hydronium ions to form ionized (toxic) and
unionized ammonia. To evaluate the biodegra-
dation with respect ammonia disrupting the
balance Using first order kinetic model of
aquaculture system can be studied. Immobilization
of bacteria in clay and silicate components such as
montomorilline clay, improves color of the scales
and skeletal system of the aquatic animals (Carson
et al., 2009). Further Improves immunity, including
disease & parasite resistance in fish and shellfish.
Increased egg production with fewer deformities,

*Corresponding author’s email- preetha.r@ktr.srmuniv.ac.in

248 HEERTHANA AND PREETHA
enhanced growth rates with stronger with larger
fins and improved resistance to splitting (Schram
and Roques, 2010). Hence increases lifespan of
aquaculture organisms. Bentonite clay can be added
as a mineral supplement for preventative healing
and for anti-parasite benefits. It also balances the
aquaculture pond’s pH and assists in toxin removal
(including aflatoxin). The scope of the study was to
perform toxicokinetic studies to estimate ammonia
oxidation using P.putida ammonia oxidizing species
by bioaugumentation and immobilization studies
with bentonite clay.
MATERIALS AND METHODS
Selection of bacteria and Growth condition
Pseudomonas putida strain (MTCC NO: 10458) was
obtained from microbial type culture collection
and gene bank (MTCC) Chandigarh Punjab. The
pure culture obtained from the MTCC was further
plated on sing streak plate techniquewith growth
medium 3 requirements. The aerobic condition was
maintained in incubator for 48 hours at 30 oC.the
optical density of the cells grown in nutrient media
is analyzed using spectrophotometer at 610 nm for
the utilization of fresh cultures for immobilization
studies the colony forming unit is measured using
electronic colony counter. The cells are centrifuged
at 5000 rpm and the supernatant was decanted and
pelleted cells were used for immobilization using
bentonite clay.
Bentonite clay preparation and characterization
studies
Bentonite clay was obtained from Astraa
chemicals private LTD with food grade properties.
The bentonite clay is further pretreated by
allowing water into the mixture and allows the
sediments and inorganic contaminants to settle
down using elutriation technique using stoke law.
The clay was treated with acetic acid with 99.8%
purity to bring down the pH to uniform of 7pH for
facilitating the growth of the organism. The
mechanical procedure was by attrition, as follows:
in a 100 mL beaker were weighed 100 g of in nature
clay. In a graduated cylinder, 200 mL of distilled
water were added and homogenized at the 100
rpm in a homogenizing blender. All elutriation
system were connected in series and the water flow
was adjusted with the aid of a needle properly
calibrated, obtaining a flow rate of approximately
(28.0 ±1.2) g/min. The elutriation was monitored.
(Stokes et al., 1851) After separation, at room
temperature, the fractions collected were placed for
drying with the help of fan for three days. After
drying, samples were ground in a mortar and
pestle, and sieved in a sieve of number 100 mesh
(150 μm aperture) for characterization analysis.
Characterization methods of bentonite clay
Scanning electron microscopy and energy
dispersive spectroscopy (EDS)
Characterization of bentonite for the immobili-
zation study was done using scanning electron
microscopy along with EDS using scanning TEM
and E Beam lithography system infrastructure
utilized from SRM nanotechnology research Centre.
The technique of scanning electron microscopy
(SEM) allows to evaluate the morphology of the clay
particles16.the SEM analysis was conducted in low
vacuum at the magnification of 6000x, 12000x and
24000x. Composition of the bentonite was
analyzed using Energy Dispersive Spectroscopy
(EDS). Prior to the SEM-EDS measurements
performed by drying the mixture of bentonite
using the hot air oven at 110?C for 4 hours, then
smoothed with 150μm aperture size.
Immobilization of Pseudomonas putida in
bentonite clay
The bentonite clay dried and UV treated inside the
laminar hood to ensure nil pathogenic micro-
organism present in the bentonite clay. An empty
Petri-dish (diameter 60 mm) is weighed (including
the lid) and filtered suspension cell material is put
into the Petri-dish (Dunn et al., 2002) The Petri-dish
with the cell material is placed into an oven at 40 °C
for three days and viability of the cells was
checked by standard plate count method and the
plating is done periodically for six days (Wenting
Ma and Donghai Peng, 2017).
Depuration kinetics study on Pseudomonas putida
The five random samples obtained from fish ponds
were selected as the media and incubated with
fresh culture obtained calorimetrically at 610nm
with the value of 0.16 and incubated for 120 hrs and
after that water quality analysis was done as per
(APHA, 21th edition, 2005). Initial concentration of
ammonia before incubation of fresh cultures and
final concentration after incubation compared over
time with formula in refereeing to first order
depuration kinetics. Using the formula C = Co e-kt
 Depuration Kinetics and Bioaugumentation Studies on Ammonia Using Pseudomonas putida for 249

Where C is the concentration at any time t, Co is
the initial concentration, k is the first order rate
constant (depuration-essentially analogous to
decay) and t is the time.
RESULTS AND DISCUSSION
Bentonite clay characterization
Some of these grains with atypical geometry are
highlighted. On the other hand the fraction of the
material density obtained was 11.1mm the
elutriated sample was recorded in image focusing
5μm, 10 μm and 20 μm Fig. 1 denotes that the
elutriation is an efficient and promising method for
the selection, separation and purification of clay
materials, as it succeeded in its goal.
The bentonite has following composition in high
proportion required for the adhesion of cells to
grow the cells inside the bentonite by adhesion
technique. Fig. 2 denotes the effect on surface
characteristics of the clay. Other micronutrients
availability such as Fe (0.42%), Al (0.58%), and Si
(0.84%) render the growth of bacteria and titanium
present (0.11%) per weight helps in flocculation of
other contaminants present in aquaculture
systems and proves to fulfill advanced water treat-
ment with bio filtration system. The mineral
studies conducted by elutriation technique using
EDS shows the availability of molecular oxygen as
114.44% and carbon (17.58%) which ensured the
microhabitat for the growth of P. putida cells (Table
1).
Immobilization of Pseudomonas putida results
After 48 hour’s incubation the cells got attached to
the bentonite clay in the aerobic chamber and
started to produce daughter cells and surface of the

a) Low vacuum at 12000x with 10μm b) Low vacuum at 6000x with 20μm imaging

c) Low vacuum at 24000x with 5 μm

Fig. 1 Morphology of bentonite clay structure after elutriation Technique
250 HEERTHANA AND PREETHA

Table 1. Composition of bentonite clay used for immobilization study analyzed using energy dispersive
spectroscopy

El AN S e rie s u n n .C [ n o rm .C A t o m .C E r ro r ( 1
w t .% ] [ w t. % ] [ at. % ] Sigm a) [
w t. % ]
O 8 K - S e ri e s 69.39 4 8 .2 5 50.41 114.44
C 6 K - S e ri e s 38.56 2 6 .8 2 37.32 17.58
Si 14 K - S e ri e s 15.18 1 0 .5 6 6.28 0.84
Fe 26 K - S e ri e s 9.28 6 .4 5 1.93 0.42
Al 13 K - S e ri e s 8.22 5 .7 2 3.54 0.58
Ba 56 K - S e ri e s 2.12 1 .4 7 0.18 0.25
Cl 17 K - S e ri e s 0.88 0 .6 1 0.29 0.11
Ti 22 K - S e ri e s 0.17 0 .1 2 0.04 0.11
To tal 143.80 1 0 0 .0 0 99.99

Table 2. Plate count (log CFU.mL-1) of Pseudomonas putida initially was (1000 ± 10 mg/L, pH 11) and 10000± 10
(MTCC no: 10458) calculated during immobi-lization of mg/L, pH 11.5 was prepared by aqueous ammonia
whole cells using dynamic adhesion growth with with distilled water. At room temperature (about
bentonite clay and with nutrient medium at 30°C
30 °C), the whole cells of fresh culture of dry cell
Time With broth Petriplates weight 0.5μg was suspended. The sample was
(hours) (log CFU.mL-1) (log CFU.mL-1) tested every day till 30 days to analyze the changes
in pH values, concentrations of ammonia nitrogen,
3 8.67±0.16 4.84±0.03
48 8.56±0.46 4.06±0.67 nitrite, nitrate, and total nitrogen (TN). pH value
96 9.90±0.52 4.64±0.54 was measured by pH meter. Final concentrations of
144 9.94±0.42 4.62±0.59 ammonia after 96 hours are measured to 10.7mg/L,
192 9.64±0.09 4.51±0.43 which is 98% of the initial ammonia concentration.
240 9.36±0.03 5.68±1.07 At 10,000mg/L of initial ammonia concentration
99.7% of ammonia removed. After 5 days incu-
bation with immobilized P.putida water quality was
found to be in the acceptable limit (Table 3). The
study proves that the depuration kinetics study
with Pseudomonas putida can remove ammonia
effectively from the aquaculture pond and reduce
the toxicity to least level. Hence the tolerance to
ammonia toxicity at toxic pH shows the
adaptability of the organism to toxic environment.

CONCLUSION

The major problem arise due to increase in

Fig. 2 EDS graph with the presence of elements
in its valance shell
flask is viewed in microscope to check the viability
of the cells and cell count is given in Table 2.
Depuration kinetics study results on Pseudomonas
putida
Ammonia-containing aquaculture pond water
ammonia level is depletion of oxygen levels and
which is turn lead to the mortality of fish. The
sources of oxygen in aquaculture ponds;
photosynthesis process carried out by aquatic
plants, wavy wind and atmospheric air circulating
on the pond. Normal concentration of DO should
be 5 mg/L which is recommended for survival of
aquaculture species. Sensitivity to low levels of
dissolved oxygen leads to distressed environment,
especially when DO falls to 2-4 mg/L. At a
concentrations less than 2 mg/L fishes and shellfish
 Depuration Kinetics and Bioaugumentation Studies on Ammonia Using Pseudomonas putida for
Table 3. the chemical oxygen calculated along with other internal effects influencing COD in aquaculture ponds
Parameter Pond 1 Pond 2
pH 7.15±0.03a 6.10±0.01b
Temperature ( oC) 30.25±0.06b 30.15±0.06b
Salinity (ppt) 0.13±0.07e 0.43±0.06e
Dissolved Oxygen 5.15±0.07a 4.72±0.08b
(mg/L)
Chemical Oxygen 5.20±0.06a 7.42±0.15d
Demand (mg/L)
Results are represented as mean±SD mean is in column with superscript are not significantly different (p>0.05)
die due to oxygen depletion. Increases in oxygen
consumption can be caused by an over-abundance
of phytoplankton in the ecosystem, stratification
and increased organic waste entering the water.
The study focused on the new advanced study of
combining two effective treatments such as the
usage of bentonite clay of food grade (waste water
treatment) and Pseudomonas putida (biological
treatment) combined to form effective bioremedi-
ation tool. Also a biodegradation tool in aqua-
culture to eradicate ammonia from aquaculture
environment which is harmful to aquaculture
organism. The bentonite clay not only helps in
purifying the water but also helps in fighting the
viral attack and absorbs other pathogenic micro-
organism. The immobilized P.putida which has the
capability of high ammonia scavenging correlating
with the experimental data. The EDS and SEM
analysis proves that the morphology and surface
characteristics of the bentonite clay satisfies the
microhabitat to grow and multiply further on the
contact of surface of aquaculture ponds. The
activity of P.putida helps in balancing ammonia and
since the bentonite clay is of food grade it does not
offer any toxic effect to fish and shellfish at
aquaculture forum. Hence, wide scope for rendering
quality assurance and food safety at minimum cost
and energy utilization. Since the organism can
grow in aerobic condition and can with stand light,
pH, and salinity concentration. The wide usage of
this combined technology will be helpful for the
society to offer assistance to aquaculture sector to
increase the yield and empower blue revolution one
step forward with better productivity.
ACKNOWLEDGEMENTS
We thank Head Department of Chemical
Engineering and Head Department of Physics and
Nanotechnology, SRM University Kaatankulathur
View publication stats
251
Pond 3 Pond 4 Pond 5
7.00±0.02c 6.32±0.01d 6.72±0.02e
7.00±0.06a 28.54±0.06e 31.43±0.06a
1.00±0.11b 1.23±0.03b 2.97±0.21a
3.18±0.06d 3.24±0.03d 3.87±0.13a
10.11±0.72c 16.10±0.11b 17.30±0.54a
Chennai, for providing facilities for analysis.
REFERENCES
APHA 2005. Standard Methods for the Examination of Water
and Waste Water, 21st edn. American Public Health
Association, Washington, DC.
Carson, J.K. and Campbell, 2009. Minerals in soil select
distinct bacterial communities in their micro-
habitats. FEMS Microbiol. Ecol. 67: 381-388DOI:
10.1111/j.1574-6941.2008.00645.x
Dunne, W.M. 2002. Bacterial adhesion: seen any good
biofilms lately. Clin. Microbiol. Rev. 15 (2) : 155-166,
doi: 10.1128/CMR.
Hartig, E., Schiek, U., Vollack, K.U. and Zumft, W.G.
1999. Nitrate & nitrite control of respiratory nitrate
reduction in denitrifying Pseudomonas stutzeri by a
two-component regulatory system homologous to
NarXL of Escherichia coli. J Bacteriol. 181 (12) : 3658-
65. PMID: 10368138
Jungst, A., Braun, C. and Zumft, W.G. 1991. Close link-
age in Pseudomonas stutzeri of the structural genes
for respiratory nitrite reductase and nitrous oxide
reductase, and other essential genes for denitrifi-
cation. Mol Gen Genet. 225 (2) : 241-248.
Sir George Gabriel Stokes and Sir J. Larmor, 1851.On
the effect of the internal friction of fluids on the
motion of pendulums. Mathematical and Physical
Papers. 3 : 1880-1905.
Schram, E., Roques, J.A.C., Abbink, W., Spanings, T., de
Vries, P., Bierman, S., de Vis, H. van and Flik, G.
2010. The impact of elevated water ammonia con-
centration on physiology, growth and feed intake
of African catfish (Clarias gariepinus). Aquaculture.
306 (1-4) : 108-115.
Tomasso, J.R. 2012. Environmental nitrite and aqua-
culture: a perspective. Aquaculture International. 20
(6) : 1107-1116.
Wenting, Ma, Donghai Peng, Sharon, L. Walker, Bin
Cao, Chun-Hui Gao, Qiaoyun Huang and Peng
Cai, 2017. Bacillus subtilis biofilm development in
the presence of soil clay minerals and iron
oxidesnpj. Biofilms and Microbiomes. 3 : 3 -4.