ELSEVIER

Aquacultural Engineering, Vol. 15, No. 1, pp. 67-80,1996

0 1995 Elsevier Science Limited
Printed in Great Britain. All rights reserved
0144~@09/96/$9.50
0144.8609(95)00020-8
Nitrification in Three-stage and Single-stage Floating
Bead Biofilters in a Laboratory-scale Recirculating
Aquaculture System
L. L. Hargrove, P. W. Westerman & T. M. Losordo
Biological and Agricultural Engineering Department, North Carolina State
University, Raleigh, NC 276957625, USA
(Received 13 August 1994; accepted 3 May 1995)
ABSTRACT
Previous research with biological filters has indicated that nitri~cation
eficiency may be increased by separating the media into multiple
stages. This study compared nitrification in two floating-bead biofilter
treatments: a three-stage biofilter vs a single-stage or solitary biofilter;
having similar total bead volumes for both treatments, and three
replicates of each treatment. The filters supported a maximum of 15.6
kg of hybrid tilapia (Oreochromis niloticus x Oreochromis aureus) in
a 366 liter recirculating system. Generally, the single-stage biofilter had
somewhat greater reduction of total ammonia nitrogen (TAN) than the
three-stage biofilter; especially when the fish feeding rate was increased
to 14.85 kg/day-(m3 of beads) to challenge the filters. The three-stage
treatment usually had difserences between stages regarding net reduction
of TAN (usually between 5 and 15% reduction of influent
concentration in each stage), but there were no consistent trends
between stages. Both treatments displayed less than optimal total
suspended solids (TSS) filtering capacity and inconsistent agitation
during daily flushing of filters, which may have led to variable biojloc
retention time and increased variability of TAN removal.
INTRODUCTION
The use of similar biofilters in sequence or series is not a widely
practiced technique in aquaculture. However, Miller and Libey (1985)
used a four-stage rotating biological contactor (RBC) to treat water
68 L. L. Hagrove, i? u! Westernan, T M, Losordo
from a fish culture tank and found that 80-90% of TAN removal
occurred in the first two stages. They did not report differences
between the first two stages. Also, Kaiser and Wheaton (1983)
suggested that for removing fish culture organics and ammonia, two
smaller volume filters in series may operate more efficiently than one
larger volume filter operating alone. They referred to the results of
Heukelekian et al. (1952) who reported that two high-rate trickling
filters operating in series were 2.5-3 times more efficient at removing
BOD (biochemical oxygen demand) than when operated in parallel.
Kaiser and Wheaton (1983) also referred to Srna and Baggaley
(1975), who reported that for two trickling filters in series, the first
would operate at a higher pH and support more Nitrosomonas and
the second would have a lower pH and be more favorable for
Nitrobacter.
A three-stage floating-bead biofilter was proposed for study in
anticipation that the competition between the bacteria populations
would be lessened. The supposition was that the greatest amount of
dissolved organics would be consumed in the first filter by the highly
prolific heterotrophic bacteria. The carbon dioxide produced by the
heterotrophs would, in turn, encourage more Nitrosomonas growth in
the second filter of the series. Nitrosomonas activity produces
hydrogen ions which cause a decrease in the pH and encourage
Nitrobacter growth in the third stage of the series (Srna and Baggaley,
1975; Srna, 1975). Owing to the separation of these essential bacteria
and the removal of suspended solids and BOD between stages, the
three-stage biofilter should have greater nitrification capacity than the
single-stage biofilter.
The bead filter is attractive because it displays higher solids capture
and lower water loss during cleaning than a conventional upflow sand
biofilter (Wimberly, 1990; Malone et al, 1993). The major objectives
of this study were to develop a laboratory-scale recirculating system
to culture fish and to compare the performance of a three-stage bead
filter to that of a single-stage bead filter with equal media volume.
METHODS AND MATERIALS
A laboratory-scale prototype system of six filters, three single-stage
and three series filters (each made up of three stages), was used to
support a maximum of 15-6 kg of tilapia in a rectangular rearing tank
with water volume averaging 348 liters. Fish stocking was done in
increments on days 1, 15, 26 and 49 with fish weighing 100-200 g.
Lab-scale recirculating aquaculture system 69
The fish were fed 36% protein floating catfish fingerling food twice a
day by hand for the greater part of the study. The crude protein, fat
and fiber contents were 36, 45 and 6%, respectively. Feed rate
initially was 1.48 kg/day-(m3 of beads) and was gradually increased (as
more fish were added) to 13.20 kg/day-(m3 of beads) by day 67. On
day 68, feed rate was increased to 14.85 kg/day-(m3 of beads) to
challenge the filters. This resulted in high TAN (45 mg/liter), so the
feed was decreased to 12.38 kg/day-(m3 of beads) by day 72.
The filter components were designed by Dr Ron Malone at
Louisiana State University, fabricated by Armant Aquaculture, Inc.,
at Vacherie, Louisiana, and assembled at North Carolina State
University.
The filters were constructed of clear PVC pipe with an inside
diameter of 16.19 cm. This PVC casing was fitted with conical plastic
inserts and two slotted plastic screens and flat plastic caps on the
ends to retain the beads (see Fig. 1). A white PVC pipe (3.33 cm
OD) in the center of the filter provided influent to the filter below
the bottom screen. The single-stage filter vessel height was O-762 m,
and the vessel height of each stage of the series filter was O-381 m.
The media within the filters was post-consumer (recycled)
polyethylene plastic beads with a specific surface area (SSA) of 983
m2/m3. Table 1 gives the specifications for the filters and media used.
The filters were cleaned by backflushing once a day. To backflush,
all filters were completely voided of water via the waste port near the
bottom of the filter. As water left the filter, air was drawn in through
the manometer on the top of the filter and entered the filter below
the beads resulting in air bubbling up through the bead bed, breaking
it up, and releasing solids and biofloc. Most of the beads were
expected to fall into the bottom chamber of the filter and the solids
would fall through slots in the bottom plastic screen. To be discussed
later, the backflushing did not consistently have good bubble action in
all filters. The backflush water was collected in a 114-liter barrel and
allowed to settle for about 15 min. After settling, 76-95 liters of
supernatant were removed with a wet-dry vacuum system. This water
was returned to the rearing tank; the remaining high-solids water was
discarded. Aerated dechlorinated water (usually about 35 liter) was
added daily to make up for the losses due to evaporation and wasting
(including the foam fractionator).
Dissolved oxygen and temperature were monitored with portable
meters on site. Air was added to the water in the rearing tank by a
network of silica air stones. A foam fractionator utilized two air lines
from the low-pressure air manifold. The foam fractionator was used
70 L. L. Hargrove, I ? W Westerman, I : M. Losordo
THREE-STAGE SERIES FILTER
SOLITARY FILTER
Fig. 1. Schematic of bead filter treatments: solitary and three-stage series filters.
to remove fine solids, reduce dissolved organics like protein, and
control carbon dioxide. Alkalinity was monitored on site with the
methyl orange titration method and was controlled using sodium
bicarbonate. On-site pH checks were done with a hand-held meter.
The filter network included 1.25 cm PVC ball valves in the water
line from which samples were taken. Sample bottles were rinsed at
least 3 times before the sample was taken. Samples were collected
Lab-scale recirculating aquaculture gstem 71
TABLE 1
Summary of Filter Specifications
Characteristic Series titer
(each stage)
Series filter
(all stages)
Solitary
filter
Depth of beads (cm) (with center pipe) 8.26 24.8 24.1
Volume of beadsa (m) 1.71 x 1o-3 5.13 x 1o3 4.97 x 1o-3
Weight of beads (g) 910 2732 2543
Active surface areab (m) 1.68 5.04 4.89
Passive surface area (m) 0.52 1.55 0.87
Total surface aread (m) 2.20 6.59 5.76
Cross-sectional area (CSA) of filters is =0*021 m *. bActive surface area (ASA): the
surface area of bead media. Passive Surface Area (PSA): the surface area of walls
of the tubing, pipe, filter casing, etc. dTotal surface area (TSA): the sum of ASA and
PSA.
before feeding and backflushing and transported to the Water
Quality/Waste Management Laboratory at North Carolina State
University and analyzed using Standards Methods (API-IA, 1989).
Performance of the filters was evaluated by measuring filter
reductions (in and out samples) of TAN (total ammonia nitrogen),
N02-N (nitrite-nitrogen), TSS (total suspended solids) and CBOD
(5-day carbonaceous biochemical oxygen demand). The N03-N
(nitrate-nitrogen) accumulation rates were also monitored. The
operational goals included maintaining the DO (dissolved oxygen)
above 5.0 mg/liter, TAN and N02-N below 0.5 mg/liter, and alkalinity
above 100 mg/liter as CaC03.
The beads were taken from two filters after sampling was
completed on day 88. Therefore, days 91, 93 and 95 are not included
in most plots. By removing the beads, the nitrifying capacity of the
passive surface areas (other than bead surface area) could be
assessed.
RESULTS AND DISCUSSION
General performance
System performance was monitored almost daily. Median values for
DO, temperature and pH were 6.8 mg/liter, 29.5C and 7.43.
Maintaining a constant flow rate common to all filters was difficult.
Flow was affected by accumulation of biofloc, uneaten feed and feces
72 L. L. Hargrove, I! W Westerrnan, T. M. Losordo
$3.5
23
J
8 2.5
B 2
ii
t
1.5
+ Solitary Average
+ Series Average
I L
9 16 32 42 44 51 67 70 72 74 77 79 61 64 66 66
Days After First Stocking of Fish
Fig. 2. Treatment mean flow rates as measured periodically.
1
SO.6
6
0
+ Rearing Tank
--t Solitary Average
- Series Average
1 7 16 21 28 32 37 44 51 58 65 70 74 79 84 88
Days
Fig. 3. TAN concentrations in rearing tank and treatment effluents (peak values of
45 occurred at all three locations).
on the pump screens, in fittings, and within the laboratory-scale
filters. The average flow rates for treatments measured periodically
are shown in Fig. 2. Despite regular cleaning of tubing and fittings,
the series flow rate was often lower than the solitary flow rate. In an
attempt to achieve equal flow rates for the two treatments, the head
tanks for the solitary filters were lowered on day 70 to obtain a flow
rate of about 2.5 liter/min. Then, to increase both flow rates to
35-4.0 liter/min, a section of pipe was added to raise the head tanks
on day 80. Within a few days, the flow rate had decreased to about 3
liter/min. Effect of flow rate on TAN reduction is discussed later.
Figure 3 shows the TAN concentrations measured during the study.
TAN concentrations were highest on days 70 and 72. The peak
concentration occurred on day 70 at 45 mg TAN/liter. This resulted
from an increase in feed rate on day 68. The feed rate was decreased
Lab-scale recirculating aquaculture system
1 7 16 21 20 32 37 44 51 58 65 70 74 79 84 88
Days
Fig. 4. NO*-N concentrations in tank and treatment effluents (day 72,
mg/liter, SE = 14.7 mg/liter; day 74, SO = 13.1 mg/liter, SE = 13.1
(SO = solitary, SE = series.)
73
so = 14.9
mgjliter).
on day 72. The filters reduced TAN to below 0.5 mg/liter by day 74.
Figure 4 shows the N02-N concentrations during the study.
Increasing the feed rate on day 68 resulted in a higher range of
concentrations (13-14.6 mg/liter) on days 72 and 74, but N02-N
returned to less than 1 mg/liter within 10 days.
TAN reductions
TAN concentration reductions were typically 20-40% on average for
each treatment, but varied from less than zero to nearly 60% (Fig. 5).
The production of TAN on day 30 was either an analytical error or
due to pockets of anaerobic mineralization of organic N to TAN in
the filter. Generally, the reductions were similar for the solitary and
series filters, except for a period after the feed rate was increased
when the series had lower TAN reduction.
The solitary filters tended to have a higher TAN mass reduction
rate than the series filters, particularly from day 70 to 79 (Fig. 6)
when the flow rates were around 2-2.5 liter/min for both treatments
(Fig. 2). When the flow rate was increased (day 80) the two
treatments operated at a more similar rate. Four variations of TAN
mass reduction rate (MRR) were also investigated: (1) MRR per
total surface area (MRR/TSA) (TSA is the sum of passive (non-bead)
surface area and active (bead) surface area); (2) MRR per TSA per
flow rate (liter/min); (3) MRR per active surface area (ASA) of
beads; and (4) MRR per ASA per liter/min. Results were very similar
for all measures. The mean TAN MRR/ASA for the solitary
treatment was nearly equal to that of the Wimberly (1990) study with
74 L. L. Hargrove, l? W Westennan, T. M. Losordo
Fig. 5. TAN concentration reduction (%) for treatments.
A- Sditary Average
-c Series Average
\I
t 1 I
E 1 16 32 42 44 51 67 70 72 74 77 79 81 84 06 88
Fig. 6. TAN mass reduction rate per active surface area.
laboratory-scale bead filters (Table 2). The series filter average TAN
reduction was less.
The TAN mass reduction rate data for days 9-88 were analyzed
statistically using the pooled t-test (Table 3). Average values for the
solitary and series filter replicates were used. The pooled t-test was
also conducted on the average values of MRR/TSA, MRRITSA-
LPM, MRR/ASA and MRR/ASA-LPM for TAN. The tests were
conducted with and without negative values on all days, on the period
before the feed rate was increased, and after the feed rate increase
on day 68. Almost all of the TAN measures that included all days
indicated that there was a significant difference between the two
treatment means (with the solitary means being greater). However,
the period prior to the feed rate challenge had no significant
difference between the means.
Lab-scale recirculating aquaculture system 75
TABLE 2
Comparison of Bead Studies Regarding TAN Reduction
Bead type SSA TAN reduction rateb
(m*/m) (mg TAN/m*-day)
Source
Plastic beads (3-4 mm diameter)
Post-consumer plastic beads
(3-4 diameter, 6-9 mm
long)
1231
983
82 Wimberly, 1990
81 (for solitary filter) This study
54 (for series filter )
SSA = specific surface area of beads. bAverages for one backlhrsh per day. Rate
is per active surface area (ASA).
TABLE 3
Summary of Statistics for Mass Reduction Ratea
MRRb MRR/TSA MRRfTSA-LPMd MRRIASA MRR/ASA-LPMd
SO(a)SE SO(a)SE SO(a)SE SO(a)SE O(a)SE
All days (days 9-88)
TAN 397( > )270 69( > )41 25.0( > )14.3 81.2( > )535 29.4( > )18.7
TANf 397( = )293 69( > )44 25+0( > )16.1 81.2( > )58.1 29.4( = )21.1
Days prior to feed rate increase (days 9-67)
TAN 299.5( = )258.9 52( = )39 16.3( = )14.9 61.2( = )51.3 19.2( = )19.5
Days after feed rate increase (days 70-80)
TAN 473( > )278 82( > )42 31.7( > )13.9 96.6( > )55-l 37.3( > )18.2
TANf 473( = )323 82( > )49 31.7( > )17.2 96.6( > )64-l 3.73( > )22.5
Abbreviations: ASA = active surface area; TSA = total surface area; LPM =
liters/min; MRR = mass reduction rate; SE = series filter mean; SO = solitary
filter mean. (a) represents the statistical association between the treatment means
with a level of significance of 0.05 for pooled t-test ( = implies no significant
difference; > implies significant difference). bUnits: mg/day. Units: mg/day-m*.
dUnits: mg/day-m*-(liter/min). Negative values included in analysis. No negative
numbers included in analysis.
Flow rate and concentration effects on TAN reduction
Regression analyses were conducted on flow rate vs TAN
concentration reduction (days 51-88) flow rate vs TAN mass
reduction (days 9-88) and influent TAN concentration vs TAN
concentration reduction (days 49-88) for the averages of the series
and solitary treatments. Only the flow rate vs TAN concentration
reduction had regression slope significantly different from zero (Figs
7 and 8). One negative value for TAN reduction in the series
76 L. L. Hargrove, I ? K Westernan, I : M. Losordo
0 I I
1.5 2 2.5 3 3.5 4 4.5
Solitary Flow Rate (Umin)
Fig. 7. Solitary flow rate vs TAN concentration reduction (regression equation:
y = -0.040~ + 0.234, and R2 =O-33).
0.14
0.12..
z
t 0.1
,E 0.06
z
s
d
0.06
* 0.04
$
0.02
0 I I I
1.5 2 2.5 3 3.5 4 4.5
Series Flow Rate (Llmin)
Fig. 8. Series flow rate vs TAN concentration reduction (regression equation:
y = 0*031x - 0.006, and R2 =O-22).
treatment, indicating TAN production in the filter, is omitted from
the evaluation. TAN concentration reduction in the solitary filter
decreased with increasing flow rate (Fig. 7) which would be expected
because residence time for TAN conversion in the filter is less for
higher flow rates. The opposite occurred for the series filter (Fig. 8)
with no obvious explanation. However, both regressions had low R2
values.
The TAN mass reduction per day would be expected to remain
fairly constant with varying flow rate, assuming reaction rates in
the filter are influenced more by microbial populations than by flow
rate effects on mass transfer in the biofilm. The TAN mass reduc-
tion ranges for the treatments were about 200-600 mg/day for the
solitary treatment and about 50-500 mg/day for the series treatment.
There was no significant correlation between TAN mass reduction
and flow rate.
Lab-scale recirculating aquaculture system 77
20
r ~STIEFP ??STZFBF ??ST3EFP I
Days
Fig. 9. Mean TAN concentration reduction (% of stage 1 influent) for each stage
of the series filter.
There was no significant relationship between influent
concentration and concentration change. The solitary filter generated
higher TAN reductions than the series filter. Similar influent
concentrations at lower flow rates should produce a higher TAN
reduction per pass because of longer residence time in the filter.
Since the series filter experienced lower flow rates than the solitary,
the lower level of TAN reduction for the series was not expected.
TAN reduction in stages
Starting with day 58, influent and effluent of each stage of the series
filters were analyzed to determine variability in TAN removal among
the stages. Until then, stage 1 and stage 2 effluents were not
measured. Also, before day 49 the rearing tank sample was used as
the influent sample for all treatments. Differences between
concentrations measured in the rearing tank and in the influents to
the six filter systems were generally small. Figure 9 displays the TAN
percent reduction for each stage based on stage 1 influent
concentration. Two values go off the scale for day 77; the actual
percent reductions for day 77 are -124, 9 and 112% for stages 1, 2
and 3, respectively. This indicates that stage 1 possibly had anaerobic
zones that produced TAN, while TAN reduction was extremely high
in stage 3. Days 67-74 show much variability, possibly resulting from
the shock brought on by the challenge of increased feed rate on day
68. For most of the study the total net reduction for the three-stage
series filters was at least 25%. There is no substantial pattern among
the stages. However, generally there are differences between the
L. L. Hatgrove, I ? U? Westerrnan, T. hf. Losordo
Days
Fig. 10. TAN concentration reduction (%) of no beads (NB) versus beads (B) for
solitary (SO) and series (SE) filters.
stages, which indicates that biological activity, pH or some other
factors are affecting TAN reduction in the various stages. Further
study with larger biofilters may be beneficial in determining what
factors are causing differences between stages.
Other factors affecting TAN removal
In general, the series filters showed as much as a 2 mg/liter DO drop
vs a 1 mg/liter drop in the solitary filters, particularly in the latter
portions of the study. The higher DO drop in the series filters
indicates more microbial activity in the series treatment, but the
lower TAN reductions suggests that the greater microbial activity was
mainly heterotrophic activity.
The beads of one solitary filter and one series filter were removed
on day 88 and stored in separate buckets. This was done to assess the
nitrification occurring on passive surface area (surfaces other than
media surface area). In Fig. 10, the two beadless treatments showed
essentially the same amount of concentration change in TAN, with
the exception of day 95. The series change for the beadless filters on
day 95 was - 18.7%. The results indicated that passive surfaces are
probably supporting between 10 and 30% of the TAN reduction. This
is important to consider when comparing various filter systems.
Variation in TAN removal may have been affected by inconsistent
agitation during flushing of the filters after the first week. Once the
biofloc began to build up on the beads, there was often inadequate
bubble action to agitate the beads, and consequently the beads
tended to stick together and not fall to the bottom section of the
Lab-scale recirculating aquaculture system 79
filter. The solitary filters and the first stage of the series filters were
the most difficult to backflush. However, substantial solids were
removed from all filters during daily flushing. It is possible that longer
biofloc residence time in the filter can increase TAN reduction rate.
Malone et al. (1993) has indicated that maximum nitrification in bead
filters occurs with a biofloc residence time of 2-3 days when influent
TAN concentration is 0.2-0.4 mg/liter, and with 4-7 days residence
time when influent TAN concentrations are less than 0.1 mg_/liter.
The laboratory-scale filters did not perform as well as expected in
the removal of CBOD and TSS. Generally, none of the filters
displayed outstanding CBOD removal over the 4-week period of
CBOD sampling. The average rearing tank CBOD (excluding the first
day) was 31 mg/liter. The average rearing tank TSS for all 4 days was
84 mg/liter. Yet none of the influent and effluent samples for the
filters showed consistent solids removal. Because the time of sampling
was just before flushing, the data may not reflect the overall CBOD
and TSS removal efficiency of the filters. More samples taken at
smaller time intervals between flushes or a composite method of
sampling with time between flushes would be needed to better
estimate average performance of the filters.
SUMMARY AND CONCLUSIONS
The average TAN mass reduction rate based on bead surface area
was 54 mg/m*-day for the series filter and 81 mg/m-day for the
solitary filter. However, there were no significant differences between
the treatments until the feed rate was increased on day 68 to
challenge the filters. The series filters recovered more slowly that the
solitary filters after the high feed rate challenge period began.
Backflush cleaning action was less than desired for both treatments,
and may have resulted in variability in TAN removal rates because of
variable accumulation of solids in the treatments.
The results with the laboratory-scale filters do not support the
hypothesis that a three-stage series bead biofilter would produce
greater nitrification. Although there were differences in TAN removal
between stages of the three-stage filter, the trends were not
consistent. In addition, economic factors would have to be included
when considering multi-stage filters, because multiple smaller units
would cost more than a single unit of the same volume.
80 L. L. Hargrove, I ? W Westerntan, I : M. Losordo
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Kaiser, G. E. & Wheaton, F. W. (1983). Nitrification filters for aquatic
culture systems: state of the art. J. World Maricult. Sot., 14, 302-24.
Malone, R. F., Chitta, B. S. & Drennan, D. G. (1993). Optimizing
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Miller, G. E. & Libey, G. S. (1985). Evaluation of three biological filters
suitable for aquacultural applications. J. World Maricult. Sot., 16, 158-68.
Srna, R. F. (1975). A modular nitrification filter design based on a study of
the kinetics of nitrification of marine bacteria. Proc. World Maricult. Sot.,
6, 463-78.
Srna, R. F. & Baggaley, A. (1975). Kinetic response of perturbed marine
nitrification systems. J. WPCe 47, 473-86.
Wimberly, D. M. (1990). Development and evaluation of a low-density
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M.S. Thesis, Louisiana State University, Baton Rouge, Louisiana.