Appl Microbiol Biotechnol (2001) 57:791–798
DOI 10.1007/s00253-001-0835-1
O R I G I N A L PA P E R
H. Shan · J.P. Obbard
Ammonia removal from prawn aquaculture water using immobilized
nitrifying bacteria
Received: 10 April 2001 / Received revision: 31 August 2001 / Accepted: 7 September 2001 / Published online: 1 November 2001
© Springer-Verlag 2001
Abstract Intensive prawn aquaculture in tropical re-
gions is associated with high concentrations of total am-
moniacal nitrogen (TAN) as a result of high rates of
prawn excretion and feed loading. Excessive TAN can
adversely effect productivity and result in adverse im-
pacts on coastal waters. Cultures of indigenous nitrifying
bacteria were enriched from intensive prawn aquaculture
pond water using continuous and batch enrichment tech-
niques. Cultures were capable of TAN removal over a
wide range of initial TAN concentrations – up to
200 mg/l. Cultures were immobilized onto porous clay
pellets to enhance cell density and applied to culture me-
dium and TAN-augmented pond water under aerobic
conditions to determine TAN removal proficiency. Im-
mobilized cultures were able to achieve a high TAN re-
moval proficiency in pond water – even at a low density
of 0.1 pellet per liter. A concentration of less than 0.5 mg
TAN/l could be maintained under a fed-batch condition
of 3.2 mg TAN/l per day, after an initial 2-day lag phase.
A simplified and effective culture enrichment process
was developed for culture immobilization onto pellets
using TAN-augmented pond water. Overall, pellet immo-
bilization of indigenous nitrifying bacteria represents a
potentially effective TAN control system for prawn
aquaculture in low-cost, but intensive tropical prawn
farms.
Introduction
The rapid development of the shrimp aquaculture indus-
try in tropical countries has led to adverse environmental
impacts. Thailand is the world’s major exporter of
prawns, with a capacity in excess of 250,000 tonnes per
annum (FAO/NACA 1995; ASSC 1996; Department of
Fisheries 1996). Following Thailand’s success, other
H. Shan · J.P. Obbard (✉)
Department of Chemical and Environmental Engineering,
4 Engineering Drive 4, National University of Singapore, 129791
e-mail: chejpo@nus.edu.sg
Fax: +65-779-1936
countries in Asia including India, Myanmar, Cambodia,
Vietnam, Indonesia, China, the Philippines and Malaysia
have instituted their own programs to develop the prawn
farming industry. As a result, over 80% of the world’s
production of prawns is now located in Asia (Csavas
1994). To meet demand, up to 85% of prawn production
is based on intensive cultivation practices – typified by
ultra-high stocking densities and feed loadings (Shang et
al. 1998). Under such practices, as much as 40% of the
pond water may be exchanged every few days to remove
toxic waste metabolites (Deb 1998).
Discharge of nutrient-enriched wastewater and bot-
tom sediments from prawn ponds into adjacent coastal
waters has frequently resulted in eutrophication and oxy-
gen depletion with adverse consequences for marine
ecology and fisheries (Paez-osuna et al. 1998). Depend-
ing upon prawn stocking density, total pollution loading
during an aquaculture production cycle for total phos-
phorous, nitrogen and suspended solids can be as high as
321, 668 and 215,000 kg/ha per cycle, respectively
(Dierberg and Kiattisimkul 1996). Aquatic conditions in
the pond itself can be become toxic, as total ammoniacal
nitrogen (TAN) can accumulate rapidly as a result of the
high metabolic excretion rate of intensively fed prawn
stock (Hopkins et al. 1995). Optimum prawn growth re-
quires a concentration of less than 0.1 ppm unionized
ammonia, i.e. 1.33–1.53 mg/l TAN at pH 8.0 and tem-
perature of 28–30 °C in pond water. Effective manage-
ment of water quality in prawn ponds is therefore a criti-
cal pre-requisite not only for maximizing productivity,
but also for mitigating the adverse impacts of discharg-
ing prawn pond water and sediments.
A number of techniques have been developed in re-
cent years for the control of TAN concentrations in aqua-
culture systems. Submerged flow biofilters (Abeysinghe
et al. 1996), high-rate linear-path trickling nitrification
filters (Twarowska et al. 1997), bench-scale fluidized
bed bioreactors (Ng et al. 1996) and continuous bioreac-
tors using immobilized alginate beads (Kim et al. 2000)
have all been evaluated in pilot scale treatment systems
with varying degrees of success. Certainly, achieving
792
high rates of TAN removal under conditions of continu-
ous TAN production can be a major challenge. For ex-
ample, a secondary treatment system combined with ef-
fluent biofiltration tested at an experimental prawn farm
by Chin and Ong (1997) achieved only a 25% TAN re-
moval rate. Key constraints on operational efficiency in-
clude high capital costs, as well as technical problems
during establishment, start-up and maintenance of the
technology. Furthermore, the application of sophisticated
treatment technologies may be inappropriate for low-cost
prawn aquaculture systems, as commonly found in de-
veloping tropical countries.
Bioaugmentation involves the seeding of water-puri-
fying bacteria into aquaculture systems. Nitrification
takes place by two groups of autotrophic bacteria, Nitro-
somonas spp and Nitrobacter spp., both of which com-
prise slow-growing species. Both groups exist naturally
in aquaculture water, but are readily washed out by fre-
quent water exchange during the production cycle there-
by impairing their potential beneficial effect on water
quality. Commercial bioaugmentation products are avail-
able, but their efficacy is questionable as suppliers of
such products often overstate their potential (Young
1976). Reasons for the failure of inocula to function in
aquaculture as they do in axenic culture include: inade-
quate substrate concentration and cell density, inter-spe-
cific competition with indigenous microorganisms lead-
ing to growth inhibition, and an insufficient acclimatiza-
tion period to affect bioremediation (Stephenson and
Stephenson 1992). The challenge of maintaining a viable
culture of nitrifying bacteria at high cell density in the
active growth phase is a key factor in providing an effec-
tive in situ treatment for prawn aquaculture water.
The aim of this study was to combine the merits of
bioaugmentation together with cell immobilization for
the control of TAN concentrations in prawn aquaculture
water using enriched cultures of nitrifying bacteria. The
potential advantages of using such a technique include:
achievement of longer biomass retention time in pond
water, maintenance of a high microbial cell density, opti-
mization of microbial growth and metabolic rates, and
protection from inhibitory compounds (Travieso et al.
1995; Yang 1997). As such, for our experimental purpos-
es, a culture of indigenous nitrifying bacteria was en-
riched from a tropical commercial prawn farm and sub-
sequently immobilized on an inert, high-surface-area
Table 1 Enrichment culture
medium Component Constituent
Artificial sea water NaCl
KCl
MgSO4·7H2O
CaCl2
FeSO4·7H2O
Sediment extract Growth factor
Prawn food Heterotrophic nutrient (chemical oxygen demand)
Ammonium chloride Total ammoniacal nitrogen
Inorganic carbon NaHCO3
Phosphate K2HPO4
growth support medium. The use of an indigenous bio-
mass for nitrification has potential benefits with respect
to reduced inter-specific competition and acclimatization
period. Specifically, this report describes the use of an
immobilized culture of nitrifying bacteria to affect TAN
removal in both aqueous culture medium and actual
prawn aquaculture water under laboratory conditions.
Overall, our objective was the development of a novel
and economically viable technique for in situ TAN man-
agement in tropical prawn aquaculture water.
Materials and methods
Culture medium and inoculum
A surface sediment sample (0–5 cm depth) was taken from a com-
mercial prawn aquaculture pond in Singapore for the preparation
of a sediment extract for use in an enrichment culture medium.
The extract was used in culture medium in order to supply micro-
nutrients or other growth factors present in the pond water, but ab-
sent from culture medium. Culture medium was prepared using ar-
tificial seawater and other constituents, as specified in Table 1.
The sediment extract was prepared by mixing 100 g of sedi-
ment and 100 ml of sterile de-ionized water prior to autoclaving
(121 °C, 15 min) to kill the indigenous biomass. The sediment
mixture was subsequently settled prior to filtration of the superna-
tant through a Whatman glass microfiber filter 934-AH. Following
filtration, the extract was again autoclaved (121 °C, 15 min). A
concentration of 10 ml of sediment extract per liter of culture me-
dium was used and was taken from the same stock each time cul-
ture medium was prepared for experimentation. The initial TAN
concentrations used in culture medium (between 3.0 and 4.0 mg/l)
was selected based on measured TAN values prevailing in the
pond water under a normal aquaculture cycle (2.5–5.0 mg/l, un-
published data). In order to simulate actual pond water conditions,
a chemical oxygen demand (COD) similar to that of the pond wa-
ter (100–150 mg/l) was established in the culture medium by the
addition of dissolved prawn food, and the pH was adjusted to 8.0
using sodium bicarbonate. Finally, all components of the medium
were mixed, stirred for 2 h, pre-filtered, sterilized by 0.2-µm pore
filtration and kept at 4 °C prior to use. Inoculation was undertaken
by adding one part of unsterilized sediment to nine parts of culture
medium (w:v) for both closed (batch) and open (continuous) en-
richment cultures.
Open (continuous) system culture enrichment
A 1-l triple-necked flask with a 500-ml working volume of culture
medium was used as a continuous chemostat fermenter. The cul-
ture was incubated at 30 °C in darkness. Fresh sterile medium was
added continuously at a rate of 4 ml/h; the rate was based on
Concentration (per l)
23.40 g
0.75 g
7.00 g
0.5 g
0.001 g
10 ml
0.44 g
2.5–5.0 mg TAN
0.2 mg
0.1 mg

25–50% of the reported minimum µmax of nitrifying bacteria of
0.024 h–1 (Underhill 1990) to prevent the elution of slow-growing
nitrifying bacteria. TAN concentrations in the influent were in-
creased gradually over the 40-day enrichment period from 2.5 to
5.0 mg/l by 0.5 mg/l increments every 8 days. The culture was
continuously agitated with a magnetically coupled stirrer
(200 rpm) and aerated with moisturized compressed air at 2 l/min
via a sterilization filter. The medium was maintained at a constant
working volume using an overflow device and a sample of efflu-
ent was taken once every 3 days for analyses of pH and TAN, us-
ing a pH meter and HACH DR/2000 spectrophotometer, respec-
tively. Nitrate was also analyzed using the spectrophotometer from
day 30 onward. The continuous enrichment culture was proceeded
until the TAN concentration in the effluent was maintained at a
constant level close to zero.
Closed (batch) system culture enrichment
Batch enrichment was carried out in 250-ml sterile conical flasks
with a working volume of 100 ml of inoculated culture medium.
Incubation conditions were identical to those used for continuous
enrichment. TAN and pH were measured daily, as specified above,
but nitrate was not measured due to the limited culture volume.
Sub-culture into fresh sterile media at a 1:9 ratio (v:v) was carried
out when 90% of TAN in the growth medium was depleted. Sub-
culturing was terminated when a comparatively higher TAN re-
moval rate and shorter lag phase (i.e. on-set of TAN removal)
were achieved than in previous batch generations.
Determination of TAN tolerance of enriched culture
The tolerance of nitrifying bacteria (from batch and continuous
enrichments) of high concentrations of TAN was determined. All
treatment and incubation conditions were identical to those speci-
fied for batch enrichment, except initial TAN concentrations were
adjusted to 10, 50, 100 and 200 mg/l, respectively. The upper level
of 200 TAN mg/l was the same as that measured in the pore water
of bottom sediments taken from the commercial prawn pond at the
completion of a 16-week aquaculture cycle (unpublished data).
Immobilization of nitrifying cultures
Associations of nitrifying bacteria cultures derived from the batch
and continuous systems were immobilized onto sterile, buoyant,
porous clay pellets (diameter: 1 cm; weight: 1 g). The specific sur-
face area and pore size of the clay pellet were determined using a
Brunauer-Emmett-Teller analyzer and scanning electron microsco-
py, respectively. Two-liter suspensions of batch and continuously
enriched cultures were incubated at 30 °C in separate 5-l conical
flasks with an initial TAN concentration of 200 mg/l. The flasks
were kept in darkness and continuously aerated; the pH was main-
tained between 7.5 and 8.2. Sterile clay pellets (autoclaved at
121 °C, 15 min) were added to cultures when the log phase of
TAN removal was detected, i.e. between 30 and 50 mg/l TAN re-
moval per day. Culture immobilization onto pellets was then un-
dertaken at 6, 30, and 72 h for the batch-enriched culture to deter-
mine the optimal immobilization period, and 30 h for the continu-
ously enriched culture.
Culture-immobilized pellet testing
Once the optimal immobilization period had been established, cul-
ture-enriched pellets were tested for TAN removal proficiency im-
mediately following culture immobilization using conditions iden-
tical to those for the batch enrichment, except that rotation speed
was reduced to 120 rpm to avoid pellet attrition. A dosage of ei-
ther 1 or 4 pellets per 100 ml of growth medium was used, and
pellets were sub-cultured under identical conditions upon initial
793
TAN depletion to determine their continued TAN removal effica-
cy. The procedure was repeated for the continuously enriched cul-
ture-immobilized pellets (using pellets immobilized for 30 h) at a
dosage of 1 pellet per 100 ml of culture medium. Again, pellets
were sub-cultured as above.
Finally, water taken from the prawn aquaculture pond was test-
ed under laboratory conditions to determine the effectiveness of
culture-immobilized pellets at TAN removal. The initial TAN con-
centration in pond water was augmented by the addition of ammo-
nium chloride to give a TAN concentration in the range of
3–4 mg/l. Pellets were tested at the following dosages:
– 1 pellet per 100 ml of fresh unsterilized pond water. Upon
TAN depletion, the pellets were transferred to fresh pond water
to determine the continued efficacy of their TAN removal po-
tential. This was repeated twice.
– Four immobilized pellet-packs in 4 l of fresh unsterilized
prawn pond water. Each pellet-pack consisted of ten pellets en-
closed in nylon mesh and adjusted to neutral density. Follow-
ing initial TAN depletion, 3.2–4.2 mg/l of TAN was added dai-
ly to simulate continuous TAN release in the prawn aquacul-
ture system, and sodium bicarbonate was added to regulate the
pH. The experiment was continued until TAN depletion in a
control (i.e. pond water minus immobilized pellets) was ob-
served. Removal occurred as a result of growth of indigenous
nitrifying bacteria in the pond water itself. The experiment was
repeated twice more, but using a lower pellet density, i.e. 1 pel-
let-pack per 4 l of pond water, and finally a single pellet per 4 l
of pond water.
Simplified enrichment process
As TAN removal was found to occur in prawn pond water without
immobilized pellet addition, albeit with a lag phase of 6 days, a
simplified enrichment procedure was developed. This was accom-
plished by raising the initial TAN concentration of pre-settled
pond water to 200 mg/l, providing aeration and maintaining the
pH between 7.5 and 8.2. Sediment extract (Table 1) was supplied
to optimize conditions for the growth of nitrifying bacteria. Sterile
clay pellets were added to pond water when the TAN removal rate
was greater than 30 mg/l per day and the culture was subsequently
immobilized for 30 h. Culture-immobilized pellets were then
checked initially for TAN removal efficacy using unsterilized
pond water at a density of 1 pellet per 4 l of fresh pond water. This
was followed by further testing at a lower pellet dosage of 1 pellet
per 10 l of pond water. The test method was the same as above,
with a daily TAN addition rate of 3.2 mg/l to simulate continual
TAN production due to prawn excretion and feed loading under
field conditions.
Results
Culture enrichment
The relationship between the duration of culture (days)
for continuously enriched and TAN removal, as well as
corresponding nitrate generation in culture medium ef-
fluent is shown in Fig. 1. The relationship between batch
generation and the lag phase (days) for TAN removal for
batch-enriched culture, together with the highest daily
TAN removal rate (mg/l per day) measured in each batch
generation is given in Fig. 2.
794

Fig. 1 Relationship between duration of culture (days) for contin-

uously enriched culture and TAN removal as well as correspond-
ing nitrate generation

Fig. 2 Relationship between batch generation and lag phase for

TAN removal, and the highest TAN removal rate (mg/l per day)
for each batch generation

Fig. 3 Maximum TAN removal rate in culture medium with vary-

ing initial concentrations of TAN for both batch- and continuous-
ly-enriched cultures
Fig. 4a–c Pore-size characteristics of clay pellet used for immobi-
lization. a Surface of clay pellet, b inner edge of clay pellet, c in-
ner center of clay pellet
TAN tolerance of enriched cultures

Maximum TAN removal rates in culture medium with
various initial TAN concentrations for continuously en-
riched and batch-enriched cultures are given in Fig. 3.
Immobilization of enriched cultures
Electron microscopy images of the pore size characteris-
tics of the surface, inner edge and center of clay pellets
used for culture immobilization are shown in Fig. 4a–c.
TAN removal in culture medium using
culture-immobilized pellets
TAN removal rates in culture medium dosed with batch-
enriched pellets (4 pellets/100 ml) that had been immobi-
lized for different time periods (6, 30, and 72 h) are
shown in Fig. 5a–g, for primary and sub-cultures. Re-
sults for pellets that had been immobilized for 30 h with
continuously enriched cultures (1 pellet/100 ml) are
shown in Fig. 6a–c, for primary and sub-cultures. For
 795
Fig. 5a–f TAN removal in cul-
ture medium dosed with batch-
enriched pellets (4 pellets/
100 ml) immobilized for vari-
ous time periods. a, c, e Prima-
ry culture, b, d, f sub-culture;
a, b 6-h incubation; c, d 30-h
incubation; e, f 72-h incubation

Fig. 6a, b TAN removal in

culture medium dosed with
continuously-enriched pellets
(1 pellet/100 ml) immobilized
for 30 h. a Primary culture,
b sub-culture

Fig. 7 TAN removal in culture medium dosed with batch-enriched

pellets (1 pellet/100 ml) immobilized for 30 h
▲

Fig. 8a, b TAN removal in aquaculture pond water dosed with

pellets (1 pellet/100 ml) culture-immobilized for 30 h. a Primary
culture, b sub-culture
796
Fig. 9a–c TAN removal rate in aquaculture pond water with dif-
ferent dosages of culture-immobilized pellets. Arrows represents
TAN addition. a One pellet pack per liter pond water, b one pellet
pack per 4 l pond water, c one pellet per 4 l pond water
comparison, TAN removal rates for pellets (1 pel-
let/100 ml) that had been immobilized with batch-en-
riched culture for 30 h are shown in Fig. 7.
TAN removal in pond water using culture-immobilized
pellets
TAN removal rates in aquaculture pond water dosed with
one culture-immobilized pellet per 100 ml of water are
shown in Fig. 8a–c, for primary and sub-cultures. Simi-
larly, results using culture-immobilized pellets at dosag-
es of 1 pellet-pack per liter, 1 pellet-pack per 4 l, and
1 pellet per 4 l of pond water augmented with daily TAN
addition are shown in Fig. 9a–c, respectively.
Simplified enrichment process
TAN removal rates in aquaculture pond water dosed with
pellets immobilized using the simplified enrichment pro-
cess at 1 pellet per 4 l and 1 pellet per 10 l of pond water
Fig. 10a, b TAN removal rate in aquaculture pond water with dif-
ferent dosages of culture-immobilized pellets using simplified en-
richment process. Arrows represents TAN addition. a One pellet
per 4 l pond water, b one pellet per 10 l pond water
augmented with daily TAN addition are shown in Fig.
10 a and 10b, respectively.
Discussion
This study has resulted in the isolation of effective cul-
tures of indigenous bacteria that could be immobilized
onto porous clay pellets for the removal of TAN from
both culture medium and prawn pond water. Nitrifying
bacteria were purposefully enriched under heterotrophic
conditions so that cultures were representative and pre-
acclimatized to actual prawn aquaculture pond water
conditions. Although it is likely that heterotrophic bacte-
ria were present in the enriched culture associations,
TAN removal proficiency by nitrifying bacteria was
nonetheless potent. The ability to enrich and immobilize
indigenous culture associations of TAN-removing bacte-
ria from pond water is a positive development compared
to the practice of adding non-indigenous cultures of ni-
trifying bacteria in aquaculture practice. Bioaugmenta-
tion using non-indigenous cultures is often problematic
due to inadequate substrate concentration and cell densi-
ty, inter-specific competition, growth inhibition and
an insufficient acclimatization period (Young 1976;
Stephenson and Stephenson 1992). In contrast, cultures
enriched in this study were indigenous, pre-acclimatized
and easily immobilized for effective TAN removal.
Culture enrichment
For continuous culture enrichment, the TAN concentra-
tion in culture medium effluent decreased gradually and
was maintained close to zero from day 40 onward

(Fig. 1). This was achieved even though influent TAN
concentration increased by 0.5 mg/l on a regular basis
from 2.5 to 5.0 mg/l over the 40-day period. Depletion of
TAN corresponded with a measured nitrate concentration
in effluent of approximately 5.0 mg/l from day 40 on-
ward, indicating conversion of TAN to nitrate and the
completion of the enrichment process. For the batch cul-
ture enrichment, the lag phase for the initiation of TAN
removal was reduced to less than 2 days by the sixth
generation and this lag phase was maintained up to the
ninth generation (Fig. 2). The peak TAN removal rate
from the sixth generation onward was in excess of
2.0 mg/l per day and reached a maximum in the ninth
generation of 3.0 mg/l per day. Overall, results from the
batch enrichment process showed that five subcultures
were sufficient to achieve an effective association of ni-
trifying bacteria with a short lag phase and high TAN re-
moval function. While the number of sub-cultures re-
quired is specific to the pond water used for this study, it
serves as useful guidance for other aquaculture systems.
TAN tolerance of enriched cultures
For both cultures, the maximum TAN removal rates in-
creased with an increasing initial TAN concentration
(Fig. 3). Both cultures could exert a TAN removal func-
tion far in excess of TAN concentrations normally pres-
ent in prawn pond water as a result of prawn excretion
and food addition (2.5–5.0 mg/l). The highest TAN re-
moval rates occurred in culture medium with TAN con-
centrations equivalent to those found in sediment pore
water (i.e. up to 200 mg/l). This implies that nitrifying
cultures enriched from prawn pond water were exerting
TAN removal at only a fraction of their maximum capac-
ity and may be potent in controlling TAN accumulation
in pond water at high immobilized cell densities.
Immobilization of enriched cultures
The specific surface area of clay pellets used for culture
immobilization was in the range of 1.3–3.4 m2/g and the
pore size distribution of the pellet surface, inner edge
and inner center was 1–10 µm, 2–10 µm and 20–300 µm,
respectively (see Fig. 4a–c). For effective culture immo-
bilization, a pore diameter of between two and five times
the cell diameter is known to be optimal for cell immobi-
lization in aqueous systems (Murray and Van den Berg
1981; Wang and Wang 1988). As the cell size of
Nitrobacter and Nitrosomonas species are 0.5–0.8×
1.0–2.0 µm and 0.7–1.5×1.0–2.4 µm, respectively (Holt
et al. 1994), it was concluded that pellets were well suit-
ed to immobilize enriched nitrifying bacteria on the pel-
let surface and sub-surface.
797
TAN removal in culture medium using
culture-immobilized pellets
Batch-enriched cultures immobilized onto pellets for 6 h
removed in excess of 3.0 mg TAN/l per day during the
log phase of TAN removal, but required a lag phase of
2–4 days prior to the initiation of TAN removal in prima-
ry and sub-cultures (Fig. 5a, b). By contrast, 30- and
72-h immobilized pellets exerted TAN removal in cul-
ture medium immediately and achieved a removal rate of
approximately 4.0 mg/l per day. Removal proficiency
was maintained or improved in the sub-cultures for both
sets of pellets (Fig. 5c, d and Fig. 5e, f). A TAN removal
proficiency of approximately 3.5 mg/l per day was
achieved for continuously enriched cultures immobilized
on pellets for 30 h at a dosage of 1 pellet/100 ml of cul-
ture medium (Fig. 6a, b). The maximum TAN removal
rate for the batch-enriched culture for this pellet immobi-
lization period and density was approximately 3.75 mg/l
per day (Fig. 7). Consequently, batch-enriched cultures
immobilized on pellets for 30 h were used for measure-
ment of TAN removal in aquaculture pond water.
TAN removal in pond water using culture-immobilized
pellets
TAN removal rates during log phase for 30-h culture im-
mobilized pellets in aquaculture pond water at a density
of 1 pellet per 100 ml exceeded rates achieved in culture
media – reaching a maximum of 4.2–6.7 mg TAN/l per
day (Fig. 8a). TAN removal was initiated immediately in
sub-culture and the TAN removal rate was maintained
(Fig. 8b). There was no evidence that suspended solids
in pond water interfered with TAN removal efficiency of
the pellets as a result of biofouling. At lower pellet dos-
ages (down to 1 pellet per 4 l of pond water), TAN re-
moval was also initiated immediately and TAN concen-
trations could be maintained at 0.2 mg/l under fed-batch
culture conditions (Fig. 9a–c). Overall, it is apparent that
the culture was highly proficient at removing TAN when
TAN was continuously supplied at concentrations similar
to that produced in an aquaculture as a result of prawn
excretion and food addition to pond water.
Simplified enrichment process
It can be seen in Fig. 9a that TAN removal occurred in
unsterilized aquaculture pond water in the absence of
pellet addition after the sixth day – as a result of the
growth of indigenous nitrifying bacteria in the pond wa-
ter. Using the simplified enrichment process at an aug-
mented initial concentration of 200 mg TAN/l in pond
water, a TAN removal rate in excess of 30 mg/l per day
was achieved after 14 days of culture growth (unpub-
lished data). Subsequently, indigenous nitrifying bacteria
were immobilized onto pellets for 30 h. TAN removal in
the presence of culture-immobilized pellets using the
798
simplified enrichment process was found to be as effec-
tive as when pellets immobilized with enriched cultures
were used. At a low pellet dosage of 1 pellet per 4 l of
pond water, a lag phase of TAN removal was not appar-
ent (Fig. 10a). Even at the lowest pellet density tested,
i.e. 1 pellet per 10 l of pond water, after an initial 2-day
lag phase, the TAN concentration could be maintained at
0.5 mg/l under the fed-batch culture conditions of 3.2 mg
TAN/l per day (Fig. 10b). Thus, a potent culture of nitri-
fying bacteria can be readily immobilized onto clay pel-
lets from aquaculture pond water and this culture could
maintain low TAN concentrations under conditions of
continuous TAN enrichment.
Overall, it can be concluded that effective control of
TAN concentrations in prawn pond water under aerobic
laboratory conditions has significant and positive impli-
cations with respect to prawn productivity and removal
of TAN from prawn aquaculture water. Assuming a pel-
let density of 1 pellet per 10 l pond water, treatment of
1 ha of aquaculture pond, of 1-m depth would, theoreti-
cally, require 1×106 pellets – representing a pellet vol-
ume of approximately 2.5 m3. The maximum TAN re-
moval rate of continuous bioreactors using immobilized
alginate beads (Kim et al. 2000) was 52.0 g/m3 per day.
Based on a TAN removal rate per unit of media surface
area, submerged flow biofilters (Abeysinghe et al, 1996),
high-rate linear-path trickling nitrification filters
(Twarowska et al. 1997) and bench-scale fluidized bed
bioreactors using activated carbon granules as media (Ng
et al. 1996) achieved TAN removal efficiencies of 1.05,
0.33, and 0.33 g/m2 per day, respectively. To achieve a
TAN removal proficiency identical to that found in this
study (3.2 mg/l per day, or 32,000 g TAN/day for a 1 ha
aquaculture system of 1-m depth), the technology pro-
posed by Kim et al. (2000) would require a reactor vol-
ume of 615 m3. Similarly, filter media volumes needed
for the technologies proposed by Abeysinghe et al.
(1996), Twarowska et al. (1997) and Ng et al. (1996)
would approximate to 216, 233, and 156 m3, respective-
ly. Comparatively, therefore, the use of pellet-immobi-
lized nitrifying bacteria cultures represents a potent tech-
nology for the control of TAN in prawn aquaculture.
Furthermore, the indigenous nitrifying cultures can in-
duce immediate TAN removal without the constraints of
acclimatization and inter-specific competition associated
with the use of proprietary and non-indigenous cultures.
In conclusion, the use of high-surface-area clay pel-
lets as an immobilization medium for metabolically ac-
tive nitrifying bacteria in aerated prawn aquaculture sys-
tems represents a viable alternative for the treatment of
pond water in tropical regions in situ, where factors of
low-cost and ease of application are paramount. Further-
more, pellet recovery upon harvest drainage also has
cost-saving implications, as pellets could be readily re-
cycled for use in subsequent aquaculture. Further studies
are currently under way to evaluate this technology in
prawn aquaculture systems in the field.
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