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Towards Zero Discharge of Wastewater from Floating Raceway Production Ponds Milestone No.. daniel.willett@dpi.qld.gov.au EXECUTIVE SUMMARY A major problem with intensified pond-based aq

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

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AUSTRALIA COMPONENT

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Towards Zero Discharge of Wastewater from Floating Raceway

Production Ponds (Milestone No 5)

D.J Willett1, C Morrison1, M.J Burke1, L Dutney1, and T Hoang2

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Department of Primary Industries and Fisheries, Bribie Island Aquaculture Research Centre, Bribie Island,

Queensland, Australia

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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 daniel.willett@dpi.qld.gov.au

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

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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 NOx) 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 floc treatment was the most promising technology to progress towards zero water discharge

bio-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

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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

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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

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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

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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)

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

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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)

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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

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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

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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

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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):

Cadd = Nww x ([C/N]mic/E)

Where:

Cadd is the amount of C required

Nww 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:

Cadd = Nww 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 Nww = 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

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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 8th Ed Software

Experiment 2

This trial tested the efficacy of shifting a plankton-dominated wastewater stream to a 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)

Bio-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

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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 8th Ed Software

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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

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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

0.0 1.0 2.0 3.0 4.0 5.0

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

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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

6 8 10 12 14 16 18 20 22

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 OC) 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

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0.0 1.0 2.0 3.0 4.0 5.0

0.0 0.5 1.0 1.5

0 20 40 60 80

0 20 40 60 80 100 120 140

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

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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

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

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)

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