Bioresource Technology 102 (2011) 7388–7392

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Nutrient removal of effluent from quail farm through cultivation of Wolffia arrhiza
T. Suppadit ⇑
The Graduate School of Social and Environmental Development, National Institute of Development Administration, Bangkapi, Bangkok 10240, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this work was to study the nutrient removal using the Wolffia arrhiza during the treat-
Received 19 April 2011 ment of laying quails farm effluent. The relationship between W. arrhiza biomass and treatment time, the
Received in revised form 20 May 2011 change in water qualities, and nitrogen-balance (N-balance) were evaluated. The results showed that a
Accepted 22 May 2011
biomass of 12.0 g of W. arrhiza per liter of effluent and a treatment period of 30 days were found to pro-
Available online 30 May 2011
vide the best conditions for W. arrhiza’s growth and the quality of the treated effluent in terms of biolog-
ical oxygen demand, suspended solids, total phosphorus, nitrate, total ammonia nitrogen and total
Keywords:
Kjeldahl nitrogen. The pH and salinity were similar for each level of biomass. The W. arrhiza biomasses
Aquatic plant
Biomass
of 4.00–12.0 g/l of effluent were suitable for W. arrhiza survival over time. Since W. arrhiza can fix N in
Quail domestication the atmosphere, it can grow very well in effluent containing a low level of N.
Wastewater Ó 2011 Elsevier Ltd. All rights reserved.
Water purification

1. Introduction
The Japanese quail (Coturnix japonica) is an excellent bird for
commercial domestication because of its rapid growth, high rate
of laying, fecundity and environmental resistance when compared
to chickens (Iwamoto et al., 2008). In recent years, laying quail
domestication in Thailand has developed rapidly (Suppadit
et al., 2009). The total census of laying quails in 2009 was about
2.60 million and often concentrated within a small area especially
in the central zone of country (e.g. Angthong, Ayudhya, Lopburi,
Phichit, Saraburi, Singburi and Suphanburi) (Department of Live-
stock Development, 2010). This zone accounts for approximately
84.0% of the total laying quail production (Suppadit, 2010).
Although the large scale intensive laying quail farms satisfy the
demands of people for egg product, however, they results in large
volumes of wastewater in small region, which takes serious pol-
lution to the environment nearby (Suppadit et al., 2005). Because
at least 50.0–150 m3 of wastewater, whose chemical oxygen de-
mand content is 5000–10,000 mg/l and biological oxygen demand
(BOD5) content is 2500–5500 mg/l, are poured into the environ-
ment in an intensive farm every day (Suppadit, 2010). Based on
investigation, most of the intensive quail farms are lacking effi-
cient wastewater treatment and integrated utilization facilities
(Suppadit, 2010). Generally, wastewater is released into effluent
⇑ Tel.: +66 2 727 3334; fax: +66 2 374 4413.
E-mail address: tawatc.s@nida.ac.th
ponds on the farm where aquatic plants are found in abundance
(Suppadit et al., 2005). Optimum controls of wastewater require
knowledge of nutrient removal (Bolan et al., 2009; Chen et al.,
2010). It is possible that the aquatic plant could absorb organic
substances from wastewater, allowing farmers to recycle the
water (Suppadit et al., 2008; Agunbiade et al., 2009). A water-
recirculatory system is a way to lower the cost of water in animal
domestication (Bolan et al., 2004).
For all these reasons, the idea of studying the nutrient removal
of effluent from laying quail farms by using Wolffia arrhiza has
emerged. W. arrhiza is a small circular floating weeds about
1.00 mm in length that lives in tropical and subtropical water
sources (Fujita et al., 1999). In Thailand, W. arrhiza grows quickly
and absorbs large amounts of nutrient (Suppadit et al., 2008). In
addition, W. arrhiza provides high nutrition, especially protein
(6.80–45.0%) (Faskin, 1999), so it can be used to make animal
feed, which Wolffia meal was successfully tested as protein
replacement for soybean meal in diets of laying hens (Chanti-
ratikul et al., 2010b) and meat Japanese quails (Chantiratikul et
al., 2010a). From these characteristics, W. arrhiza may be applica-
ble to the removal of nutrients from effluent or polluted water
like that in natural water sources, while also serving as a pro-
ducer of protein. Although the effluent purification capabilities
of duckweeds, including spirodela and Lemna spp. (Sutton and Or-
ens, 1977; Matsumoto, 1981; Hammouda et al., 1995), have been
widely studied, the applicability of W. arrhiza to water purifica-
tion has received little attention. For this reason, this paper has
studied the biomass of W. arrhiza and evaluated its nutrient re-
moval capability and nitrogen balance (N-balance) in effluent
after cultivation W. arrhiza.

0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.05.061
 T. Suppadit / Bioresource Technology 102 (2011) 7388–7392
2. Methods
The quality of the effluent, the biomass of the W. arrhiza, and
the N-balance of the effluent and the W. arrhiza were assessed in
a 5  3 factorial arrangement with 4 replications in a completely
randomized design, with the first factor being a W. arrhiza bio-
masses of 0, 4.00, 8.00, 12.0, and 16.0 g/l of effluent, and the second
factor being the experimental time period of 10, 20, and 30 days.
The data were subjected to an analysis of variance and compari-
sons among means were investigated using Duncan’s New Multi-
ple Range Test of the Statistical Analysis System, version 6.12
(SAS Institute, 1996).
The effluent ponds of laying quail farm were located at the Am-
phon Farm, Inburi district, Singburi province. A closed zero-water
exchange system was used for the ponds in the farm area. The
experiment was carried out from September 2010 to January
2011. Effluent totaling 1100 l was randomly collected in the ponds
using grab sampling method (Ohio Department of Health, 2009).
The water quality was analyzed at the Thailand Institute of Scien-
tific and Technological Research, Pathum Thani province prior to
the start of the experiment. W. arrhiza was obtained from local
markets located at Muang district, Sukhothai province. It was
washed, planted in clean water for 7 days, sampled randomly,
and analyzed for total Kjeldahl nitrogen (TKN) according to AOAC
official method, 979.09 (AOAC, 1990) prior to start of the
experiment.
The effluent was placed into 20 black plastic containers (50.0 l/
container). Varying weights of W. arrhiza (0, 200, 400, 600, and
800 g) were placed in the containers for each treatment (4 replica-
tions) and cultured in an experimental house for 30 days.
Effluent from each treatment was sampled at 10.00 a.m. at the
start of the experiment and after 10, 20 and 30 days. The effluent
temperature during the trial was 21.5–30.0 °C. Cheng and Stomp
(2009) reported that optimum temperature for maximum growth
of W. arrhiza was between 17.5–30.0 °C. Water quality was ana-
lyzed for BOD5 (5210 B), suspended solids (SS; 2540 D), pH
(4500-H+ B), salinity (2520 B), total phosphorus (TP; 4500 PE), ni-
trate (NO3 ; 4500-NO3 E), total ammonia nitrogen (TAN; 4500-NH3
G), and TKN (4500-Norg B) using the standard methods for the
examination of water and wastewater (APHA, AWWA and WEF,
1998). In each replication, effluent was kept in 1.00 l polyethylene
bottles, cooled in an icebox, and sent to the laboratory. At the end
of the experiment, W. arrhiza in each replication was filtered using
a net for about 5 min. Then these biomasses were weighed after
the effluent was sampled and analyzed for TKN using the proce-
dures of AOAC official method, 979.09 (AOAC, 1990).
Investigation was made on the N-balance taking into account
the total N in the system containing the TKN value in the effluent
and the W. arrhiza (before and after the experiment), TKN losses
due to volatilization, transformation and collected water samples,
and N fixation from the atmosphere by the W. arrhiza.
3. Results and discussion
3.1. Effluent quality
3.1.1. BOD5
The values of BOD5 at the starting biomasses of the W. arrhiza
when different treatment times were applied were different
(P < 0.05) (Fig. 1a). The W. arrhiza biomass of 12.0 g/l and the treat-
ment time of 30 days gave the highest BOD5 removal; the residual
BOD5 was 28.0 mg/l. The BOD5 of the effluent decreased over time
in the 0–12.0 g/l treatments. This might be because the W. arrhiza
adsorbed organic substances from the effluent for its growth
resulting in a decreasing demand for the digestion of organic sub-
7389
stances (Chaiyakum and Predalumburt, 1994; Suppadit et al.,
2008). However, at the W. arrhiza biomass level of 16.0 g/l, the
BOD5 had dropped by 29.4% after 10 days and then increased at
days 20 and 30 to be 19.1% and 8.82% less than the day 0 value
for this treatment, respectively. At this level, the W. arrhiza died,
and most likely led to the high level of the organic substances in
the water.
3.1.2. SS
The levels of SS in the various W. arrhiza biomass treatments
were different when different treatment times were applied
(P < 0.05) (Fig. 1b). The highest reduction in SS was obtained when
a W. arrhiza biomass of 12.0 g/l and a treatment time of 30 days
were employed. This resulted in an SS of 43.0 mg/l from an initial
SS of 105 mg/l. Naturally, even without the W. arrhiza, SS was
decreasing over time by gravity sedimentation (Bolan et al.,
2004); however, with the presence of the plant, SS decreased at a
faster rate. The SS in the form of organic matter might be decom-
posed by microorganisms (Bunsirichai, 2004) and turned into
nutrients for the W. arrhiza (Suppadit et al., 2008). Similar to the
findings for the BOD5 levels, in the W. arrhiza treatment of
16.0 g/l, the SS increased; the reasons for this were the same as
for BOD5.
3.1.3. pH
There was no significant difference (P > 0.05) between the vari-
ous W. arrhiza biomass treatments and the length of treatment
time (Fig. 1c). It was also found that in experimental units with
4.00–16.0 g of W. arrhiza, the W. arrhiza was still adapting to its
environment and its growth may have been in a lag phase; there-
fore, its photosynthesis was incomplete and some of W. arrhiza
died, resulting in an accumulation of CO2, NO3 , SO24 , PO24 and
TAN (Jiwyam, 1997). Although TAN caused an increase in pH (Pan-
yakapo, 2002), other compounds suspended in the treated effluent
caused it to decrease. As a consequence for the final pH, the treated
effluent was in a slightly basic state.
3.1.4. Salinity
The salinities of the treated effluent were between 5.15 and
5.26 ppt (Fig. 1d). There was no significant difference (P > 0.05) be-
tween the various W. arrhiza biomass treatments and the length of
treatment times. Khondker et al. (1993) reported the maximum
biomass of W. arrhiza from effluent within a salinity range of
3.00–6.00 ppt. The maximum salinity was 5.26 ppt when a W. arrh-
iza biomass of 12.0 or 16.0 g/l and the treatment time of 30 days
were used. This was likely partly due to water evaporation and
some water being used for the growth of the W. arrhiza.
3.1.5. Total phosphorus
A comparison between various starting biomasses of W. arrhiza
and various treatment times showed differences in the TP of each
biomass level and treatment time (P < 0.05) (Fig. 1e). The lowest
reduction in TP was from an initial TP level of 0.950 mg/l dropping
to 0.830 mg/l when the W. arrhiza biomass of 16.0 g/l and the
treatment time of 30 days were applied, whereas the highest
reduction of TP left only 0.0600 mg/l of TP remaining when a W.
arrhiza biomass treatment of 12.0 g/l and the treatment time of
30 days were used. This result was similar to that of BOD5. This
might be because the W. arrhiza used phosphorus in the form of
orthophosphate for its growth (Chowwanklang, 1991; Pansawad,
2002; Suppadit et al., 2008). Khondker et al. (1993) reported that
orthophosphate concentration had significant positive correlation
with the biomass of W. arrhiza.
7390 T. Suppadit / Bioresource Technology 102 (2011) 7388–7392

a 80.0
b 120.0
*****
*****
70.0
100.0
60.0
0 g/l 0 g/l
80.0
BOD5 (mg/L)

50.0

SS (mg/L)
4.00 g/l 4.00 g/l

40.0 8.00 g/l 60.0 8.00 g/l

12.0 g/l 12.0 g/l
30.0
16.0 g/l 40.0 16.0 g/l
20.0
20.0
10.0

0.0 0.0
0 10 20 30 0 10 20 30
Days after experiment Days after experiment

8.30 5.30
c d
8.20
8.10 5.25
8.00
0 g/l 0 g/l

Salinity (ppt)
7.90
4.00 g/l 5.20 4.00 g/l
7.80
pH

8.00 g/l 8.00 g/l

7.70
12.0 g/l 5.15 12.0 g/l
7.60
16.0 g/l 16.0 g/l
7.50
7.40 5.10

7.30
7.20 5.05
0 10 20 30
Days after experiment Days after experiment

1.200 0.900
e f 0.800 *****
1.000 *****
0.700
0 g/l 0 g/l
Nitrate (mg/L)

0.800 0.600
TP (mg/L)

4.00 g/l 4.00 g/l

0.500
0.600 8.00 g/l 8.00 g/l
0.400
12.0 g/l 12.0 g/l
0.400 16.0 g/l 0.300 16.0 g/l
0.200
0.200
0.100

0.000 0.000
0 10 20 30 0 10 20 30
Days after experiment Days after experiment

7.00 35.0
g h *****
6.00 30.0

5.00 0 g/l
25.0 0 g/l
TKN (mg/L)
TAN (mg/L)

4.00 4.00 g/l 20.0 4.00 g/l

8.00 g/l 8.00 g/l
3.00 12.0 g/l 15.0 12.0 g/l
16.0 g/l 16.0 g/l
2.00 10.0

1.00 5.0

0.00 0.0
0 10 20 30 0 10 20 30
Days after experiment Days after experiment

Fig. 1. BOD5 (a), SS (b), pH (c), Salinity (d), TP (e), NO3 (f), TAN (g) and TKN (h) at different W. arrhiza biomasses and treatment periods. ⁄ indicates P < 0.05 vs other treatment
periods.

3.1.6. Nitrate-NO3
When different W. arrhiza biomasses and treatment times were
used, the NO3 levels were also different (P < 0.05) (Fig. 1f). After
the experiments were completed, the remaining NO3 levels ranged
from 0.0550 to 0.430 mg/l, down from the initial NO3 value of
0.760 mg/l. The highest reduction of NO3 was found for the W.
arrhiza biomass treatment of 12.0 g/l and the treatment time of
30 days, leaving a remaining NO3 level of 0.0550 mg/l, whereas
the lowest reduction of NO3 was found for the W. arrhiza biomass
treatment of 0 g/l and the treatment time of 10 days, leaving a
 T. Suppadit / Bioresource Technology 102 (2011) 7388–7392 7391

4.00 tended to increase as the treatment time increased by up to

An increase of watermeal biomass (g)

5.82 mg/l. This might be because of the death of some W. arrhiza

3.00 plants.

2.00 *
* 4.00 g/l
1.00 * *
0.00
10 20
-1.00
-2.00
-3.00
Days after experiment
Fig. 2. W. arrhiza’s biomass at different starting biomasses and treatment periods. ⁄
indicates P < 0.05 vs other treatment periods.
remaining NO3 level of 0.430 mg/l. This might be because the W.
arrhiza adsorbed NO3 for its growth (Pansawad, 2002; Tuntulweat
and Tuntulweat, 2004; Suppadit et al., 2008). However, for the
treatments with the W. arrhiza biomass of 16.0 g/l, the NO3 value
increased because the W. arrhiza biomass was too dense and some
of the plants died. The W. arrhiza was decomposed by bacteria,
which in turn resulted in nitrification, adding NO3 to the effluent
(Ong et al., 2010).
3.1.7. Total ammonia nitrogen-TAN
When different starting biomasses of W. arrhiza and treatment
times were applied, the concentrations of TAN were different
(P < 0.05) (Fig. 1g). When the W. arrhiza biomass treatment of
12.0 g/l and the treatment time of 30 days were used, the lowest
TAN, 0.180 mg/l, was achieved. From the initial concentration of
TAN, which was 2.30 mg/l, the TAN tended to decrease as the bio-
mass and the treatment time increased. This might be because the
higher the biomass, the higher the adsorption of TAN from the
water (Tuntulweat and Tuntulweat, 2004; Suppadit et al., 2008).
However, when the W. arrhiza biomass of 16.0 g/l was used, TAN
3.1.8. TKN
8.00 g/l When different starting biomasses of W. arrhiza and treatment
12.0 g/l times were applied, the TKN levels in the effluent were also differ-
16.0 g/l ent (P < 0.05) (Fig. 1h). The lowest level of TKN contamination in
30
the effluent was 2.80 mg/l and was obtained from the biomass
treatment of 12.0 g and the treatment time of 30 days. This might
be because the W. arrhiza used N in the form of TAN and NO3 for its
growth (Mahakhan, 2004; Suppadit et al., 2008), and this resulted
in TKN levels of 2.80–28.2 mg/l. The initial value of TKN was
30.2 mg/l. In contrast, when the W. arrhiza biomass of 16.0 g/l
and the treatment times of 20 and 30 days were used, the level
of TKN increased, since there was an excess of W. arrhiza that led
to its decomposition, transforming it into N in the effluent. The rea-
son for this was similar to the phenomenon for TAN and NO3
levels.
3.2. Biomass
Significant differences (P < 0.05) in W. arrhiza biomass levels a
rose as the treatment period lengthened. The initial biomass level
of 4.00 g/l and the treatment time of 30 days yielded the highest
increase in biomass, about 3.22 g, whereas the initial biomass level
of 16.0 g/l and the treatment time of 30 days showed the greatest
decrease in biomass, about 1.88 g (Fig. 2). This might be because
there was a high density of biomass. It was found that the lower
the initial amount of biomass, the higher the survival of W. arrhiza
and the higher the initial amount of biomass, the lower the survival
of W. arrhiza. This is similar to the trend that was reported by Sup-
padit et al. (2008) and Cheng and Stomp (2009). The initial amount
of biomass and optimize production continuous harvesting of W.
arrhiza biomass were necessary and important the removal of
nutrient in effluent. Therefore, daily harvesting of the incremental
growth of the W. arrhiza in container averaging approximately
0.100 g/l of effluent was recommended (Skillicorn et al., 1993). Be-

5.04 mg 0.968 mg
30.2 mg 4 30.2 mg 4
1 1
0 mg 520 mg
5 5
0 mg 0 g/l 288 mg 4 g/l
2 2
23.6 mg 23.6 mg
0 mg 6 227 mg 6
3 a 1.58 mg 3 b 0.832 mg
7 7

0.660 mg 0.622 mg
30.2 mg 4 30.2 mg 4
1 1
752 mg 953 mg
5 5
576 mg 8 g/l 864 mg 12 g/l
2 2
23.6 mg 23.6 mg
171 mg 6 83.7 mg 6
3 c 0.740 mg 3 d 0.678 mg
7 7

164 mg KN in effluent KN in effluent

4 1 4
30.2 mg (before experiment) (after experiment)
1
1,017 mg 2 KN in W. arrhiza KN in W. arrhiza
5
5 (before experiment) (after experiment)
1,152 mg 16 g/l
2 N fixation from KN loss i n volatili zation
23.6 mg 3 6
6 atmosphere and transformation
23.6 mg
3 e 1.24 mg 7
KN loss in collected
7 water samples

Fig. 3. N-balance at 0 (a), 4.00 (b), 8.00 (c), 12.0 (d) and 16.0 (e) g biomasses of W. arrhiza.
7392 T. Suppadit / Bioresource Technology 102 (2011) 7388–7392
cause of W. arrhiza accumulation led to obstruction of light on the
water and decreased in dissolved oxygen (Fujita et al., 1999).
3.3. N-balance
The total N in the system was composed of the total N in the
effluent before the experiment, which was equal for all experimen-
tal units, and the total N in the W. arrhiza before the experiment,
depending on the initial amount of W. arrhiza biomass – the great-
er the amount of W. arrhiza biomass, the higher the level of total N
(Fig. 3). During the experiment, total N was lost when some efflu-
ent was removed for analysis. Also, the total N was lost due to the
ammonia volatilization and/or transformation of N (Bolan et al.,
2004), the pathways of N removal in the effluent was biological
denitrification, leading to the emission of nitrous oxide and dini-
trogen gas (Groffman and Crawford, 2003). However, there was
an increase in the total N from the N2-fixation process of atmo-
spheric N by cyanobacteria living in the W. arrhiza fronds (Iqbal,
1999), as a result of the increases in the total N mentioned, the re-
sults showed an increase in the total N released from the system,
which was accumulated in the biomass. The results also showed
that the total N in the W. arrhiza at the end of the experiment in-
creased as the biomass increased whereas the total N in the treated
effluent decreased as the biomass increased except for the biomass
level of 16.0 g/l. In this case, the total N in the treated effluent in-
creased when the biomass treatment of 16.0 g/l was used because
some of the plants died due to an excessive density of biomass and
released N into effluent.
4. Conclusions
The biomass level of 12.0 g W. arrhiza/l effluent and the treat-
ment time of 30 days showed the best performance in water qual-
ity improvement in this experiment. The growth of the W. arrhiza
declined when a biomass concentration of 16.0 g/l was employed,
because of excessive W. arrhiza. N could be fixed from atmosphere
by cyanobacteria living in W. arrhiza fronds. Thus, W. arrhiza can
grow very well in effluent containing a low level of N. The utiliza-
tion of the W. arrhiza for treating the laying quail farm effluent is
practical and feasible when optimal conditions are met and a suit-
able biomass concentration and time period is utilized.
Acknowledgements
The author would like to acknowledge the Thailand Institute of
Scientific and Technological Research for supporting laboratory
and the Amphon farm for providing necessary facilities.
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