Báo cáo nghiên cứu khoa học " Towards Zero Discharge of Wastewater from Floating Raceway Production Ponds (Milestone No. 5) " ppt

Ministry of Agriculture & Rural Development



PROGRESS REPORT
Intensive in-pond floating raceway production of marine finfish (CARD VIE 062/04)



MILESTONE REPORT NO.5
Development of a zero-discharged system












Report Author: Michael Burke, Tung Hoang & Daniel Willet

December 2007
1













AUSTRALIA COMPONENT
2

Towards Zero Discharge of Wastewater from Floating Raceway
Production Ponds (Milestone No. 5)


D.J. Willett
1
, C. Morrison
1
, M.J. Burke
1
, L. Dutney

1
, and T. Hoang
2
1
Department of Primary Industries and Fisheries, Bribie Island Aquaculture Research Centre, Bribie Island,
Queensland, Australia.
2
Nha Trang University, International Centre for Research and Training, NHATRANG City, Vietnam

Correspondence: Daniel Willett, Bribie Island Aquaculture Research Centre, PO Box 2066 Bribie Island,
Queensland, 4507 Australia.



EXECUTIVE SUMMARY
A major problem with intensified pond-based aquaculture production systems has been
managing water quality and discharge quotas due to the accumulation of waste nutrients.
This is exacerbated in the current CARD project which demonstrated the very high
production capability of in-pond raceways in excess of 35 ton/ha of combined mulloway
and whiting. While the current operation managed water quality through exchanging water
(approximately 10% per day on average – see MS No.4), it is recognised that with water
conservation issues and environmental nutrient discharge impacts, flushing pond water to
waste is a less desirable solution. One of the original goals of this project was to
investigate strategies that limited water discharge to show that raceway production of fish
could be sustainable. This report summarises details of water remediation strategies
investigated to progress towards zero water discharge.
Waste sumps were installed into the raceways as a proposed means for collecting and
concentrating uneaten feed and faeces, thereby reducing nutrients entering the ponds. A
trial tested the effectiveness of these solids traps by comparing Total Solids, TN and TP
collected in the sump with those flowing out of the raceway through the end screens.

Results showed that the waste sumps are generally not effective at concentrating solids for
periodic removal. This was primarily due to flow dynamics within the raceways causing
eddies to form that keep solids from going down into the collector. In addition, fish within
the raceways continually stir up and resuspend particulate waste, allowing it to be expelled
into the pond. However, the sumps may be useful as a discharge point in a remediation
system which recirculates pond water via an external treatment pond.
3

An original objective of the project was to investigate the culture of the red marine
macrophyte Harpoon Weed (Asparagopsis armata) as a nutrient sink. While much
previous research at BIARC has looked to develop seaweed biofilters for pond-based
aquaculture, the culture of A. armata was novel and offered advantages over commonly
used green seaweed species, according to new literature. Several attempts to collect seed
stock and culture the specific tetrasporophyte phase of this species however proved
problematic and the seaweed failed to thrive and eventually died. Specific factors
responsible are discussed. Concurrent research at BIARC is developing technologies that
overcome many of the common impediments to seaweed culture and these are discussed in
light of future work evaluating A. armata as a biofilter.
Recent international research has demonstrated the successful use of bacterial-based
processes (termed Bio-floc treatment) for water quality management in pond-based
aquaculture. The concept involves manipulating substrate Carbon:Nitrogen ratios to
promote heterotrophic nutrient assimilation. A series of experiments were conducted to
determine whether bio-floc treatment may be incorporated effectively as part of the
raceway production system, specifically as an external component of a recirculating
system.
The trial defined a Carbon dose rate that achieved almost complete elimination of toxic N
species (TAN and NO
x
) from raceway effluent within 12 hours and prolonged the period
prior to remineralisation. A successful shift from a phytoplankton-dominated waste stream

to a bio-floc community was also achieved by applying this carbon dose in a replicated
continuous-flow treatment system. The bio-floc community was characterised by lower,
stable pH (8.0-8.2) and DO (6.9-8.8) levels, increased biomass and a decreased proportion
of phytoplankton present. This demonstrated that effluent treated in an external bio-floc
pond would be suitable for recirculation, and a schematic of a proposed integrated
production system is presented.
Of the wastewater remediation strategies investigated in this project, it is evident that bio-
floc treatment was the most promising technology to progress towards zero water
discharge.

INTRODUCTION
A major goal of this CARD project was to develop a pond-based fish production system
that is both sustainable and profitable, designed to increase production and improve stock
4

management efficiencies and ultimately make better use of existing unprofitable
aquaculture pond infrastructure in Australia and Vietnam. The development of low-cost in-
pond Floating Raceways (FRs) in this project has demonstrated an innovative approach to
larval rearing, juvenile nursery and fish growout. As reported in Milestone No.4, the FR
system within a pond a Bribie Island Aquaculture Research Centre demonstrated
production capability in excess of 35 ton/ha of combined mulloway and whiting.

An inherent problem of any pond-based production system is the accumulation of residual
organic matter (uneaten feed, faeces) and toxic inorganic nitrogen (specifically ammonia).
Even the best practices cannot avoid this since it has been shown that fish and shrimp only
assimilate on average about 25% of ingested food – the rest being excreted into the water
column predominately as ammonia (Boyd & Tucker 1998; (Funge-Smith and Briggs 1998;
Hargreaves 1998). This feeds phytoplankton blooms which are at best only a partial
nutrient sink in ponds stocked at densities above 5 ton/ha (Avnimelech 2003; Brune et al.
2003). Dense phytoplankton blooms can cause lethal DO and pH fluctuations and their

overgrowth can lead to bloom crashes and subsequent release of ammonia (Krom et al.
1989; Boyd 1995; Boyd 2002; Ebeling et al. 2006). Water exchange is usually required to
alleviate this problem and maintain suitable pond water quality; however with water
conservation issues and environmental nutrient discharge impacts, flushing pond water to
waste is becoming a less desirable solution.

Clearly, production of fish in the order of 35 ton/ha as demonstrated in this project cannot
be maintained without a means to remediate or exchange water. The current project
managed water quality using secchi depth as gauge of appropriate conditions and by
exchanging water (approximately 10% per day on average – see MS No.4). One of the
original goals of this project was to investigate strategies that limited water discharge. A
number of strategies were proposed, including the culture of Harpoon Weed (Asparagopsis
armata) as a nutrient sink; partitioning ponds to into ‘fish culture’ and ‘remediation’ zones;
and manipulating Carbon:Nitrogen ratios to promote bacterial nutrient processing. This
report will summarise details of water remediation strategies investigated, with particular
emphasis on partitioned bacterial nutrient processing as it became evident that this was the
most promising technology to progress towards zero water discharge.

Strategy 1: Raceway sump to trap solids
5


Background: Reducing direct nutrient input into production ponds reduces pressure on
biological remediation processes. The regular removal of uneaten feed and faeces directly
from raceways before it is allowed to enter the pond will prevent further nutrient release
and mineralisation from this waste source over the production period. The amounts of
these settleable solids within floating raceways will vary depending on feeding rates and
efficiencies. In turn, the ability to harvest these solids depends on flow dynamics within the
raceways and the design of the solids trap. A preliminary experiment was designed to
gauge the effectiveness of a solids trap built into the raceways as a means for reducing

nutrients entering the ponds.

Methods: Plastic stormwater drain sumps were inserted into the tail end floor of each
raceway as a solids trap (Fig 1). These sumps were connected via a flexible hose to a pump
on a timer which periodically (twice daily) pumped collected waste to a holding tank for
evaluation of nutrient content. On monthly occasions between February and October 2006,
water leaving the raceways through the end screen was also sampled and nutrient data was
compared with that from the sump waste to determine differences. Water quality analyses
evaluated Total Solids (TS), Total Nitrogen (TN) and Total Phosphorous (TP), and were
determined using validated laboratory protocols based on standard methods (American
Public Health Association 1989) and nutrient analysis equipment at BIARC.


6


Figure 1. Design and configuration of the solids trap inserted within the nursery raceways.
A plastic grate cover (not shown) prevented fish from entering the sump.

Results & Discussion: Nutrient analyses showed some small differences in concentration
between water pumped from the sump and water leaving the raceways through the end
screen (Table 1.) The greatest difference was with TS, where the sump captured on average
16% more solids than water discharged from the pond. Differences in TN and TP between
sump and raceway screen were smaller but still showed a marginally greater average
nutrient removal via the sump. This data cannot be statistically validated however because
monthly data from the raceway was from a single water sample (due to budgetary
constraints) whereby no measure of error rate can be determined. Regardless, the sump
was designed to trap and concentrate solids into a thick sludge that could be periodically
removed from the pond. It was clear that only a slightly more concentrated effluent was
captured by the sumps and their role in preventing nutrients entering the pond from the

raceways was limited. This suggests that the waste sumps are not effective at collecting
solids for periodic removal. However, they may be useful as a discharge point in a
remediation system which recirculates pond water via an external treatment pond. It is an
advantage, in this instance, to discharge the most concentrated effluent as possible into the
7

treatment pond, and this was employed in subsequent bio-floc remediation trials (see
below).

Similar waste removal systems were employed by Koo et al. (1995) in in-pond raceways
developed for channel catfish, and likewise their waste removal system showed poor
performance. The primary problem was due to inefficient settling of waste in the solids
collectors. A known difficulty with raceways is that when solids reach the end of the tank,
the hydraulic forces do not efficiently concentrate the solids around the drain. Water
reflected off the end wall generates turbulence, causing eddies to form that may keep solids
from going down into the collector (Van Wyk, 1999). In addition, fish within the raceways
continually stir up and resuspend particulate waste, allowing it to be expelled into the
pond.


Table 1. Differences in water collected from the solids trap and water leaving the raceway
through the end screen, over seven months (n=7).
Constituent Mean concentration
in water expelled
from raceway (mg/L)
Mean concentration
in water from sump
(mg/L)
Total Solids 15.4 18.35
Total Nitrogen 2.07 2.33

Total Phosphorous 0.78 0.83


Strategy 2: Evaluation of Harpoon Weed
Summary: The concept of using seaweeds as biofilters for removing waste nutrients from
fish and shrimp aquaculture operation is well known, with a seminal review by Neori et al
(2004) describing the state of the art of this technology. Presently, the most commonly
proposed and researched biofilters are green seaweeds from the genus Ulva and the red
seaweed Gracilaria. Yet, in practice most seaweed-based remediation systems have proven
not to be economically viable, mainly due to the low value of the produced seaweed and
the high labour and area requirements for its cultivation. Other physical impediments to the
culture of seaweeds in effluent from aquaculture ponds include their susceptibility to
epiphytism (Friedlander et al., 1987), infestation by grazers such as amphipods, and
8

competition for available nutrients with phytoplankton (Palmer 2005). These difficulties
are compounded by the accumulation of effluent particulate matter on the seaweed’s
surfaces. The result therefore in practice, is that growth rate of the seaweeds (and their
corresponding value as a nutrient sink) is very often limited and nutrient removal
efficiencies are below optimum rates achieved in scaled trials under more favourable
conditions (Palmer 2005; previous BIARC research).

The present CARD project proposed to investigate the performance of the red seaweed
Asparagopsis armata (also known as Harpoon Weed) as a sink for waste nutrients
generated in raceway production system. This species was selected on the basis of new
work by Schuenhoff & Mata (2004) which suggested that it had considerably greater
market value than other seaweeds due to its high concentration of halogenated organic
metabolites. Once extracted, these halogenated compounds are used for antifouling and in
the cosmetic industry as fungicides. Schuenhoff & Mata (2004) suggest that these
compounds are also responsible for limiting epibiota and epiphytes in culture – an

advantage over other cultured seaweeds. In addition, its reported removal rate of ammonia
is superior to that of Ulva species and it is also a native species to Australia (Fig 2).



Figure 2. Harpoon weed (Asparagopsis armata) growing on rocks in Moreton Bay, S.E.
Qld. Photo by Marine Botany Group, University of Qld (2003)


9

A proposal was drafted to collect harpoon weed from Moreton Bay as a seed stock to trial
its growth rate and nutrient uptake under effluent conditions generated in the raceway pond
at BIARC. In particular, it is the tetrasporophyte phase of the plant that is reported useful
for biofiltration. Several collecting expeditions were mounted in conjunction with marine
botanists from the University of Qld. Only a small amount of harpoon weed in its
tetrasporophyte phase was located. It was transferred to a production unit at BIARC and
supplied with pond effluent in order to cultivate larger quantities for use in a replicated
bioremediation trial. Unfortunately, the harpoon weed failed to thrive and eventually died
preventing the trial being conducted. It is uncertain whether seasonal or effluent-specific
factors were responsible. Given the previous considerable work conducted at BIARC
evaluating seaweed biofilters and the difficulty in locating, collecting and culturing this
specific macrophyte, plans for further trials were terminated for the current project. Future
work in evaluating this species as a biofilter, however, is planned as part of ongoing
BIARC wastewater remediation studies.

Based on current research at BIARC on seaweed biofilters, to effectively incorporate
seaweeds into a bioremediation system for pond-based aquaculture it appears that pre-
treatment of the effluent would be necessary so that competing plankton levels, fouling
organisms and suspended materials are reduced, and so that nutrients are converted into

forms available for direct plant uptake. Current work at BIARC, outside of the CARD
project, is assessing the role of polychaete-aided sand filtration as one such pre-treatment
option (Palmer 2007).

Strategy 3: Bacterial nutrient processing
Background: There is now recognition that promoting a swing from autotrophic
(phytoplankton-based) to heterotrophic (bacterial-based) processing of residual pond
nutrients has many advantages for water remediation. Sewage effluent treatment has long
employed bacterial digestion of organic matter in activated sludge systems (Arundel 1995)
and more recent studies have shown that suspended growth systems, where heterotrophic-
dominated processes regulate water quality, have great application for limited-water-
exchange shrimp and tilapia production (Avnimelech 1999; Burford, et al. 2003; Erler et
al. 2005). In aquaculture, these heterotrophic-dominated growth systems are generally
termed Bio-floc systems.

10

The challenge is to determine the best configuration for incorporating biofloc treatment as
part of the raceway production system. Two approaches are possible: in-pond biofloc
treatment or external biofloc treatment as part of a recirculating system.

Most studies on using bio-floc water remediation for aquaculture have advocated floc
formation within the culture pond as a supplementary source of dietary protein
(Avnimelech 1999; McIntosh et al. 2001; Erler et al. 2005) in addition to controlling water
quality. While increased feed utilisation is ideal, the excessive turbidity and high oxygen
demand created by bio-flocs may have a negative effect on fish cultured within floating
raceways. The high DO demands of the floc colony in addition to those of the cultured
species means that cultured stock are even more vulnerable in the event of any aeration
failure, especially in intensive production systems such as floating raceways. High
suspended solids levels can foul the gills of cultured animals and lead to bacterial,

protozoan and fungal infections (Boyd 1994). In addition, not all cultured species will
access or target the additional protein source provided by the bacterial flocs – especially
higher order species (non filter feeders).

Alternatively, establishing a bio-floc zone as a component of a treatment system external to
the culture pond (i.e. post-production) is a new approach for this technology and may be
more suited to FR production for the reasons detailed above. Waste nutrients potentially
could be captured within bio-flocs, which in turn are periodically harvested from the water
in isolation from the cultured stock. Significantly cleaner supernatant could then be
returned to the culture pond. While sedimentation ponds are routinely used in Australia to
treat post-production wastewater, local studies have shown they are generally ineffective at
reducing Total Nitrogen, mostly due to remineralisation and inadvertent discharge of the
dominating phytoplankton (Preston et al. 2000; Palmer 2005). Directly harvesting
phytoplankton is difficult and generally cost prohibitive to farmers, so a need exists for a
new approach to enhance the performance of post-production treatment ponds.

For a Bio-floc Pond (BFP) to effectively operate as a post-production wastewater
remediation system there must be mechanisms for converting phytoplankton-dominated
wastewater into a bio-floc community which packages nutrients into the more harvestable
‘floc’ form. A key mechanism for promoting heterotrophic assimilation of waste nutrients
is through the manipulation of substrate carbon:nitrogen (C:N) balance. Heterotrophic
11

bacteria utilise organic carbon as an energy source, which is required in conjunction with
nitrogen to synthesize protein for new cell material (Avnimelech 1999). For the bacteria to
metabolise available nitrogen efficiently into the floc, carbon must not be limiting.
Therefore, maintaining an appropriate C:N ratio by adding carbonaceous material is
necessary. Theoretical carbon requirements can be calculated based on the C:N ratio of
bacterial biomass, bacterial carbon assimilation efficiency and the bio-available N levels in
the pond water (Hargreaves 2006).

While a quantitative rationale for estimating C additions was described by (Avnimelech
1999), his equation was based on total ammonia nitrogen (TAN) residue. A complication is
that TAN is not the only form of nitrogen available to heterotrophic bacteria. Dissolved
organic nitrogen (DON) in particular, but also nitrite and nitrate can constitute a varying
but substantial portion of bio-available N in aquaculture wastewater (Preston et al. 2000)
and bacteria may scavenge these in addition or in preference to ammonia (Jorgensen et al.
1994). Therefore, C additions based solely on TAN level may be under-dosing.

Calculating real-time (i.e. on-the-day) bio-available N levels is difficult (particularly for
DON which requires laboratory digestion and analysis) whereas daily in-the-field testing
of TAN is standard practice, so we acknowledge the validity of Avnimelech’s (1999)
suggestion to use TAN as a convenient reference to gauge C requirements. The objective
of this study was to refine C dosing requirements based on real-time TAN readings for
more complete nutrient assimilation in discharged wastewater. A further objective was to
assess the ability to convert plankton-dominated wastewater into a bio-floc community
using these established C dose rates, within pilot-scale external treatment ponds.

Methods: A series of experiments were carried out at BIARC during 2006. The wastewater
source was the discharge from the sumps of the FRs containing the mulloway and whiting.
Molasses (37.5% C) was the carbohydrate source used to adjust substrate C:N ratios in
both experiments because it contains simple sugars, negligible nitrogen, is readily available
and relatively inexpensive.

Experiment 1
This trial investigated the effect of molasses addition at two application rates on
wastewater nutrient levels over a 48 hour period. Nine 3L tanks were filled with common
12

wastewater and supplied with continuous aeration to ensure thorough mixing. The

experiment was conducted in the dark to prevent photosynthesis. Three treatments in
triplicate were tested: Control, Molasses 1 and Molasses 2.

Molasses doses were based on the following equation (adapted from Avnimelech 1999):
C
add
= N
ww
x ([C/N]
mic
/E)

Where:
C
add
is the amount of C required
N
ww
is the bio-available N in wastewater
[C/N]
mic
is the C:N ratio of bacterial biomass [typically about 5 (Moriarty 1997;
Hargreaves 2005)]
E is the bacterial C assimilation efficiency [assumed to be 0.4 (Avnimelech 1999)]

Therefore:
C
add
= N
ww

x 12.5

According to this equation, 12.5 g C is needed to convert 1 g bio-available N into bacterial
biomass. Given that molasses is 37.5% C, 33.3 g of molasses is needed to convert 1 g bio-
available N.

A stock solution of molasses was prepared (100 g molasses L
-1
= 37.5 g C L
-1
) to aid
addition to the experimental tanks. Molasses 1 treatment was a single molasses dose based
on N
ww
= the real-time TAN level measured in the wastewater immediately prior to filling
experimental tanks. 'Molasses 2' treatment was based on double the amount of Molasses 1
to account for the extra ‘unmeasured’ bio-available N present. No molasses was added to
the Control treatment.

After molasses addition, two 50mL water samples (one filtered [0.45um] & one unfiltered)
were taken from each tank at regular intervals (0, 3, 6, 12, 24, 48 hrs). Nutrient
concentrations in the water samples were measured including Total Nitrogen [TN], Total
Phosphorus [TP], Total Ammonium Nitrogen [TAN], Nitrate/Nitrite [NOx], and Dissolved
Inorganic ortho-Phosphate [DIP]), Dissolved Organic Nitrogen [DON] and Dissolved
Organic Phosphorus [DOP]. Measurements were conducted using validated laboratory
13

protocols based on standard methods (American Public Health Association 1989) on a
Flow Injection analyser at BIARC. Data was statistically analysed using Arepmeasures
with treatment and time as parameters on Genstat 8

th
Ed Software.

Experiment 2
This trial tested the efficacy of shifting a plankton-dominated wastewater stream to a Bio-
floc community, using previously established C dose rates in a pilot-scale treatment
system. Wastewater was distributed into four concrete raceways (each 8.6m x 2.7m x
0.8m; Volume: 19,000L). Two raceways were established as replicate Bio-floc Ponds
(BFPs) and the remaining two as replicate Passive Settlement Ponds (PSP) (see Figure 3).
A two-day effluent retention time was tested. This is equivalent to a water exchange rate of
20% of production pond water per day into a treatment system that occupies 30% of farm
pond area (as this is typical of many Australian aquaculture farms using ponds), and
represents the most challenging, realistic demand a treatment system is likely to
experience. Flow of effluent through the treatment raceways was continuous to enable
more accurate monitoring.





Figure 3. Simulated post-production treatment ponds in the remediation trial showing
Bio-floc Pond (BFP) on left and Passive Settlement Pond (PSP) on right.

14

To simulate real conditions in the Passive Settlement Pond (PSP), there was no additional
aeration or stirring provided and wastewater discharged from the surface through a
standpipe. The Bio-floc Pond (BFP) used vigorous aeration with diffusers to ensure
thorough mixing and to restrict anaerobic zones within the raceway (Fig 3). Organic
carbon was added proportional to influent ammonia level as required to maintain

prescribed C:N ratios (as determined in Experiment 1), and averaged 200 ml of Molasses
every 2 days.

Weekly monitoring involved assessing untreated (influent) and treated discharged water
quality. A YSI multiprobe meter measured the Standard parameters (pH, temperature,
salinity, dissolved oxygen [DO]) during the experiment. Methods for determining nutrient
concentrations, total suspended solids [TSS], and Chlorophyll A [Chl-a] were as
described for Experiment 1.

Measurements assessed differences between bio-floc treatment and standard
phytoplankton-dominated PSP treatment. In addition, differences between the (untreated)
influent and post-treatment water were measured to assess the efficiency within each
treatment system. Changes in water quality parameters were statistically analysed using
Arepmeasures with treatment type and time as parameters on Genstat 8
th
Ed Software.

Results:

Experiment 1
Results for each constituent tested are described in detail in the paragraphs below and
displayed graphically in Figure 4.

Nitrogen
TAN levels in the un-dosed Control treatment increased significantly (p>0.01) during the
trial period. In contrast, at just three hours after a single addition of C, TAN levels in the
two molasses treatments had fallen by over 35% and were significantly (P>0.01) lower
than the control. By six hours TAN removal remained consistent between the two molasses
treatments with over 65% of TAN removed from the water. However, beyond six hours
TAN in the lower dose (Molasses 1) treatment began to rise again, suggesting the

exhaustion of available C supplies before complete ammonia assimilation occurred. The
15

higher dose (Molasses 2) continued to decrease significantly (p>0.01) so that after 12
hours, ammonia was virtually eliminated (96% removal). TAN levels began to increase
significantly (p>0.01) again after 24 hours in Molasses 1 and after 48 hours in Molasses 2,
presumably due to degradation of senescing phytoplankton not accounted for

Initially (3-6 hrs) the un-dosed Control treatment experienced a significant (p>0.01) release
of DON before maintaining the elevated level for the duration of the experiment. In
contrast, the addition of C provided a subdued and delayed (6-12hr) release of DON.
However 24 hours after C addition DON was significantly (p<0.01) reduced by 30% with
the lower C dose treatment (Molasses 1) and 85% with the higher dose (Molasses 2). The
DON levels returned to similar levels at the conclusion of the experiment 48 hrs after C
addition, suggesting an exhaustion of the available C

The TN levels were not significantly influenced (p>0.05) by C addition for the
experimental period. This Suggests the C addition can significantly influence the nutrient
processes without impacting the nutrient budget.

NOx levels were tested however the levels were negligible or below detectable levels
throughout the experimental period. High C:N ratios typically inhibit nitrification and
nitrifying bacteria are often out-competed by heterotrophic bacteria.

Phosphorus
The DIP levels followed the same trends as the TAN levels. The un-dosed Control
treatment increased significantly (p>0.01) during the trial period. Again, 6 hours after the
addition of C, DIP levels remained consistent between the two molasses treatments (with
50% of DIP removed), but after 12 hours the lower dose (Molasses 1) commenced rising
while the higher dose (Molasses 2) continued to decrease significantly (p>0.01) to almost

completely eliminating DIP (93% removal). DIP levels also began to rise significantly
after 24 hours in Molasses 1 and 48 hours in Molasses 2 as seen in the TAN levels.

DOP levels were significantly (p>0.05) lower in the Control samples but the level of C
dose did not significantly (p<0.05) effect the response.

16

Similarly to TN levels, C addition did not significantly effect (p<0.05) TP levels during the
experimental period. Again suggesting the C addition can significantly influence the
nutrient processes without affecting the nutrient budget.

0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 6 12 18 24 30 36 42 48
HOURS
TAN mg/L
Control
Molasses 1
Molasses 2

0.0
0.1
0.2
0.3

0.4
0.5
0 6 12 18 24 30 36 42 48
Hours
DIP mg/L
Control
Molasses 1
Molasses 2


0
2
4
6
8
10
0 6 12 18 24 30 36 42 48
HOURS
DON mg/L
Control
Molasses 1
Molasses 2

0.0
0.5
1.0
1.5
0 6 12 18 24 30 36 42 48
Hours
DOP mg/L

Molasses 2
Molasses 1
Control


0
4
8
12
16
20
0 6 12 18 24
Hours
TN mg/L
Control
Molasses 1
Molasses 2
0.0
1.0
2.0
3.0
4.0
5.0
0 6 12 18 24
Hours
TP mg/L
Control
Molasses 1
Molasses 2

Figure 4: Nutrient levels over the experimental period in controls and at two molasses
doses.

Experiment 2
Standard Parameters
Phytoplankton-dominated PSP treatment systems are characterised by the high pH (<8.5)
and DO (<8 mg/L) levels measured during the trial (See Figure 5). In the BFP treatment
both the DO & pH levels were significantly (p>0.05) lower compared to the PSP which
suggests a successful shift away from a phytoplankton dominated community (Funge-
Smith and Briggs 1998). Significant (p>0.05) fluctuations within the PSP (pH 8.14-9.08;
17

DO 9.74-19.16) treatment demonstrated the dangerous bloom/crash cycling typical in this
type of community (Hargreaves 2006). While the BFP (pH 8.00-8.17; DO 6.86-8.80)
system maintained consistent levels during the experimental period.







7.0
7.5
8.0
8.5
9.0
9.5
123456789101112
Week

pH
PSP
BFP
6
8
10
12
14
16
18
20
22
123456789101112
Week
DO mg/L
PSP
BFP
Figure 5: Water Quality measurements for pH and Dissolved Oxygen (DO)

Temperature and Salinity remained within biological limits for both systems. As expected,
the temperature was similar in both systems (15.3 – 21.0
O
C) on most occasions. Salinity
showed significant (p>0.01) fluctuations over time for both treatments due to rain events.
The salinity of the BFP was significantly(p<0.01) lower than PSP on a number of
occasions probably due to the more effective mixing of rain water which can float on top
of still seawater in the PSP.

Nutrient Analyses
In general, both treatments significantly (p<0.05) lowered the dissolved nutrients levels

present in the untreated water. The inorganic nitrogen (TAN and NOx) was effectively
eliminated from the untreated water by the BFP treatment. The BFP treatment preformed
significantly better than the PSP treatment for NOx (p<0.01) and DIP levels (p<0.01).
Importantly, this suggests a more efficient removal of the toxic components of wastewater
occurs in the BFP treatment (See Figure 6).

TN &TP levels in the BFP treatment were significantly (p<0.01) higher than levels present
in PSP. The BFP treatment also significantly (p<0.01) increased the TN levels from the
untreated water (influent). In contrast, the PSP significantly reduced the TN levels of the
Untreated water suggesting PSPs are more efficient at overall nutrient removal at this
stage. The high levels of TN & TP suggest efficient processing and assimilation of
nutrients to biomass.

18








0.0
0.5
1.0
1.5
123456789101112
WEEK
g/LNOx m

UNTREATED
PSP
BFP
0.0
0.2
0.4
0.6
0.8
123456789101112
WEEK
TAN mg/L
UNTREATED

PSP
BFP
0.0
0.1
0.2
0.3
0.4
0.5
123456789101112
WEEK
DIP mg/L
UNTREATED
PSP
BFP
0.0
1.0
2.0
3.0
4.0
5.0
123456789101112
WEEK
TN mg/L
BFP
UNTREATED
PSP
0.0
0.5
1.0
1.5

123456789101112
WEEK
TP mg/L
BFP
UNTREATED
PSP
0
20
40
60
80
123456789101112
WEEK
TSS mg/L
BFP
UNTREATED
PSP
0.0
0.5
1.0
1.5
2.0
123456789101112
WEEK
DON mg/L
BFP
PSP
UNTREATED
0
20

40
60
80
100
120
140
123456789101112
WEEK
ChlA ug/L
BFP
UNTREATED
PSP
Figure 6: Nutrient levels during the experimental period in untreated influent and from
bio-floc ponds and passive settlement ponds.

Two characteristics of the BFP system explain the elevated nutrients levels. Firstly the
BFP suspends and digests the organic matter (nutrients) within the water column.
Secondly, the formation of bio-flocs (with the efficient digestion of nutrients) means that
nutrients can become concentrated within water column of the BFP thus providing the
elevated TN & TP levels. As there were significantly (p<0.05) higher DON levels detected
19

in the BFP treatment than in the Untreated water, N may be accumulating in a refractory
DON form as suggested by other researchers such as (Erler et al. 2005). In contrast, within
the PSP system organic material (nutrients) settles out of the water column, but later
reminerialises causing the elevated levels normally seen in PSPs later in the season
(Preston et al. 2000). Improved containment of the bio-floc (separation from water
column) will dramatically increase the efficiency of the BFP treatment and is discussed
later. Further research into whether DON accumulates will also assist to address this issue.

TSS, another indicator of water column biomass, confirmed the trend that the BFP
treatment significantly (p<0.01) increased biomass (TSS levels) present compared to both
Untreated and PSP samples. Figure 6 displays results for all nutrients.

Interestingly, Chlorophyll A (ChlA) levels in the BFP treatment were significantly
(p<0.05) higher than ChlA levels present in Untreated samples on most occasions and was
significantly higher than the PSP treatment during the final three weeks (See Figure 6). A
heterotrophic community in a BFP treatment might be expected to have less photosynthetic
material (ChlA) than the phytoplankton dominated communities present in the untreated
water or PSP system. However, others have observed that C addition did not affect ChlA
levels in production system (Avnimelech 2001; Erler et al. 2005; Hari et al. 2006). The
higher ChlA levels in the BFP treatment can be explained by the retention of
phytoplankton within the floc material and thus within the system (i.e. concentrating the
phytoplankton). Hargreaves (2006) described suspended organic material in BFPs as
primarily made up of senescing algal cells colonised by bacteria. It is therefore, more
appropriate to look at the proportion of phytoplankton within the whole community
structure. Although the ChlA levels are higher in the BFP system, the community structure
has a lower proportion of phytoplankton than the PSP (See Figure 7).

Phytoplankton biomass can be estimated from the ChlA levels using the relationship: 1 mg
ChlA = 200mg dry weight (Pagand et al. 2000). Estimates of the contribution by
phytoplankton to the TSS levels recorded for each system were calculated. The graphs
below demonstrate the difference in community structure achieved by the applied
treatment. The PSP community was dominated by phytoplankton (57%) with a low
percentage (43%) of other particulates (including bacteria, and zooplankton etc.). In
20

contrast, the BFP community had a relatively low percentage of phytoplankton (41%) and
was dominated by other particulates (59%) presumably bacterial biomass.

PSP
0%
20%
40%
60%
80%
100%
123456789
Week
g/LTSS m
Other
Phytoplankton
BFP
0%
20%
40%
60%
80%
100%
123456789
Week
TSS mg/L

Other
Phytplankton
Figure 7: Proportion of phytoplankton present during the experimental period

Discussion: Increasing the C dose in BFPs to 30g C L
-1
achieves almost complete
elimination of dissolved nutrients within 12 hours and extends the period before a
significant remineralisation or release of these dissolved nutrients occurs. This suggests
that with higher C dosing, treatment systems require only 12 hours retention time to
process available dissolved nutrients and exceeding 24 hours will complicate the system
with remineralisation and reduce efficiency. The data also suggests that carbon plays a part
in the processing of DON, however the data was inconclusive and further work in this area
is required.

The subsequent experiment included the application of C at this higher dose rate to
demonstrate the effect on a phytoplankton-dominated waste-stream in a continuous flow
pilot-scale treatment system. By applying the higher C dose and BFP principles to
phytoplankton-dominated influent we demonstrated a clear shift to a bio-floc community.
A Bio-floc community can be characterised by the following criteria:
o Low levels of photosynthesis occurring indicated by lower and more stable pH
levels due to the release of carbon dioxide into the water column and lower DO
levels due to uptake of available oxygen (Hargreaves 2006).
o High nutrient levels (Burford, Thompson et al. 2003)
o High levels of organic matter (which can be measured by TN & TP) and low levels
of dissolved nutrients due to assimilation (Avnimelech 2003; Ebeling, et al. 2006).
21

o A high level of water column suspended material and a low proportion of
phytoplankton present in the community biomass (Burford et al. 2003).

The shift to a bio-floc community was indicated by the differences in the standard
parameters of DO and pH, which were lower in the predominantly ‘heterotrophic’ BFP
system compared to the primarily ‘photosynthetic’ PSP. Both systems maintained all
standard parameters within biological and EPA limits throughout the trial period. The
effect of adding a carbon source to lower pH has been previously discussed in many papers
(Pote et al. 1990; Avnimelech 2003). Our work confirms these findings and also achieved
consistency in DO and pH levels by adding molasses to the BFP system. It is well
accepted that the key to water quality management for production systems is stability
(DPI&F 2006) and this study shows the BFP system is successful in providing both
acceptable water quality and stability.

Both photosynthetic and Bio-floc communities assimilate dissolved inorganic nutrients and
the significant reduction in each of the dissolved inorganic nutrients is evidence that
assimilation occurred in both treatment systems trialled in this experiment. However the
BFP system did perform better in reducing the potentially toxic nitrogen species TAN and
NOx. Toxicity of un-ionised Ammonia is dependant on high pH, and temperature
(Hargreaves 1998). Therefore, low TAN levels in conjunction with the lower pH levels,
greatly reduces the risk of toxic un-ionised ammonia in BFP systems. Nitrite is also a
potentially toxic form of nitrogen and may accumulate due to incomplete nitrification
processes (Hargreaves 1998). The effective reduction of NOx (Nitrate+Nitrite) to low
levels compared to the Untreated water, suggests that assimilation rather than nitrification
is occurring in the BFP treatment. Assimilation reduces the presence of both Nitrate and
Nitrite and prevents nitrification, which can result in the accumulation of the toxic nitrite
intermediate.

This study demonstrated the potential of bio-floc treatment as an external component in a
recirculating production system. There is no need to discharge wastewater to the
environment so long as the toxic components of the water can be removed. As such, higher
TN and TP levels in a production system are not a concern to fish health while there is

limited TAN and NO
2
, and while DO levels can be maintained. This trial demonstrates that
those conditions can be achieved with bio-floc treatment. High TSS can be detrimental to
22

fish health as discussed earlier so the preferred production model would be an external
biofloc treatment as part of a recirculating system. For most effective performance, a
means to separate or exclude bio-flocs from the supernatant would permit the return of
treated water back to the production pond without a high BOD or TSS load. Schneider et
al. (2007) also reported a similar conclusion when trying to apply a bacteria reactor to clear
Recirculating Aquaculture System wastewater. Such a bio-floc exclusion device needs
further research but may be in the form of a mechanical particle filter such as a screen or
drum filter. Figure 8 shows a schematic representation of the proposed recirculating
system, which offers scope to grow and additional crop of prawns (or similar detritivore)
within the bio-floc pond, which graze on the nutrient-rich bio-flocs and have the added
benefit of helping to keep flocs in suspension.

Supernatant
returned to
production pond
Floc
excluder

Figure 8. Schematic representation of the proposed recirculating system, with external bio-
floc pond for water treatment.

Conclusion
Of the wastewater remediation strategies investigated in this project, it is evident that bio-
floc treatment, particularly as a component of an integrated recirculating production

system, is the most promising technology to progress towards zero water discharge.

Acknowledgements
This milestone report forms part of the Project ‘Intensive In-Pond Raceway Production of
Marine Finfish’ CARD VIE 062/04 funded by CARD (Collaboration for Agriculture
Research and Development) program through the Ministry of Agriculture and Rural
Aeration – F7 or
similar for O
2

delivery and
particle
suspension
Production Pond Bio-floc
Pond
New water
input for
evaporation
losses
Drain for periodic
sludge removal
Floating
raceways
Banana prawns
stocked at low
densities and unfed
– graze on flocs/
keep flocs
suspended
Paddlewheel

Organic-rich wastewater
removed from raceways
to Bio-floc Pond
23

Development of Vietnam. The research team would like to thank the Queensland
Department of Primary Industries and Fisheries, in particular Adrian Collins, Ben Russell
and Blair Chilton for their efforts in establishing the project. We also thank our
Vietnamese research colleagues ably led by Dr Tung Hoang (Director, International Centre
for Research & Training, Nha Trang University) for their valuable help and support
throughout this project.

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