Integrated Wastewater Treatment and Aquaculture Production

A report for the Rural Industries Research and Development Corporation by Martin S Kumar and Michael Sierp

May 2003

RIRDC Publication No 03/026 RIRDC Project No SAR-16A

 2003 Rural Industries Research and Development Corporation. All rights reserved.

ISBN 0642 58594 6 ISSN 1440-6845 Integrated Wastewater Treatment and Aquaculture Production Publication No. 03/026 Project No. SAR-16A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Dr Martin S Kumar SARDI Aquatic Sciences Centre, PO Box 120 Henley Beach South Australia 5022 Phone: (08) 82002400 Fax: (08) 82002481 E-mail: kumar.martin@saugov.sa.gov.au

RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: E-mail: Website: 02 6272 4539 02 6272 5877 rirdc@rirdc.gov.au. http://www.rirdc.gov.au

Published in May 2003 Printed on environmentally friendly paper by Canprint

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Foreword
Water is crucial to Australias natural and economic wealth, and as demand for water increases, the efficient use of wastewater gains importance. The degradation of aquatic environments is also of concern in Australia. Overuse of water resources, coupled with inadequate management, has resulted in significant resource degradation. Better management of water resources is necessary, along with improved human interaction with the water cycle. The trend is towards the management of the total water cycle, so that all water is managed to include the efficient reuse of wastewater. Opportunities need to be found to minimise wastewater and to re-use agricultural effluent for production, and then to return cleaner effluent to the environment. This project deals with these important issues faced by intensive farming industries. It provides base line information for the development of a model for integrated treatment of wastewater piggery effluent and aquaculture. The report is a new addition to RIRDCs diverse range of over 900 research publications and
forms part of our Future Agricultural Systems R&D program and the Resilient Agricultural Systems sub-program, which aims to foster the development of agri-industry systems that have sufficient diversity, flexibility and robustness to be resilient and respond to challenges and opportunities. This project was funded from RIRDC Core Funds which are provided by the Federal Government. Most of our publications are available for viewing, downloading or purchasing on-line through our website: Downloads at www.rirdc.gov.au/reports Purchases at www.rirdc.gov.au/eshop

Simon Hearn Managing Director Rural Industries Research and Development Corporation

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Acknowledgements
The project was funded by the Rural Industries Research and Development Corporation and this is gratefully acknowledged. Appreciation is extended to Mr Rob Lewis, Executive Director, SARDI and Dr Anthony Cheshire, Chief Scientist, SARDI Aquatic Sciences for their encouragement, provision of facilities and support for undertaking this project. Thanks to Mr Steven Clarke for the constructive comments and editing the report. We are grateful to Mr Terry Tierney, Principal and Mr Dean Cresswell, Vice Principal, Urrbrae Agricultural High School for their kind assistance and support throughout the investigation. We are indebted to Dr Yongshun Xiao for assisting in experimental design and data analysis. Special thanks to Mr Thai Thanh Binh and Mr Mai Van Ha for their input and technical assistance. Finally we extend appreciation to two of our colleagues Mr Brett hall and Mr Tony Olson for their excellent technical assistance.

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Table of Contents
Foreword............................................................................................................................................................... iii Acknowledgements............................................................................................................................................... iv Table of Contents .................................................................................................................................................. v Executive Summary ............................................................................................................................................. vi 1 Introduction........................................................................................................................................................ 1 1.1 Rationale....................................................................................................................................................... 1 1.2 Industry background ..................................................................................................................................... 1 2 Objective ............................................................................................................................................................. 3 3 Literature review................................................................................................................................................ 4 3.1 A successful concept..................................................................................................................................... 4 3.2 Live stock aquaculture integrated systems ................................................................................................... 4 3.3 Wastefed aquaculture.................................................................................................................................... 5 3.4 Health Issues and Waste-Fed Aquaculture ................................................................................................... 7 4 Research strategies and methodology .............................................................................................................. 9 4.1 Experimental plan....................................................................................................................................... 10 4.2 The Urrbrae integrated wastewater treatment system................................................................................. 12 4.3 Analytical methods ..................................................................................................................................... 12 4.4 Experimental protocol and analysis ............................................................................................................ 13 4.5 Experiment on effects of mussel culture, duckweed and fish on nutrient uptake. ...................................... 15 5 Results ............................................................................................................................................................... 16 5.1 Optimum nutrient concentration for maximum nutrient depletion. ............................................................ 16 0.0001 ................................................................................................................................................................... 19 (k)5.2 Seasonal influence on biological wastewater treatment. ........................................................................ 23 5.2 Seasonal influence on biological wastewater treatment.............................................................................. 24 5.3 Impact of zooplankton on nutrient depletion .............................................................................................. 31 5.4 Tolerance of fish to various nutrient concentrations................................................................................... 37 5.5 Impact of duckweed, mussels, Rainbowfish and their combination on nutrient depletion ......................... 39 6 Discussion.......................................................................................................................................................... 47 7 Conclusion and recommendation ................................................................................................................... 55 8 Future strategy ................................................................................................................................................. 56 8 Future strategy ................................................................................................................................................. 57 8.1 Research strategies and methodology......................................................................................................... 57 9 References......................................................................................................................................................... 59

Executive Summary
Intensive animal systems housing pigs, poultry, turkeys, dairy, feed lot cattle and horses have caused an animal waste disposal problem in parts of Australia. The efficient recycling and reuse of both nutrients and water are likely to become essential requirements for these and other intensive animal husbandry systems in the future, particularly given the focus on ecologically sustainable development A proactive approach from the livestock industry in dealing with its environmental issues and long term sustainability has wide benefits including improvement in farm income, rural employment and sustainability of the livestock industries. The management of all wastes from a number of industries will also be a platform for non fossil fuel energy production. This project deals with the disposal of piggery effluent. The pig industry is facing many challenges, with the major one being profitability. Factors affecting profitability range from the price received for the product to the high cost of inputs and increasing regulation on effluent and other waste disposal. This regulation has become increasingly stringent in Europe and Australia is now experiencing increasing government regulation on piggery wastes. An example of this is the introduction of load-based licensing in NSW where the producer will be charged for pollutants released into the environment. The main aim of the project is to obtain baseline information for the development of integrated wastewater treatment model and to provide direct input to the Urrbrae integrated treatment system for optimising its efficiency. The experiments conducted in this project produced the following key information: The optimum total nitrogen level to produce maximum algal biomass is about 100mg/l. However, the nutrient depletion rate was greater in higher concentrations. A retention time of eight days in summer and 14 days in winter will reduce 70-85% of the ammonia and about 50-60% of the soluble phosphorous content from the digested wastewater. The retention time in algal ponds during winter can be reduced by diluting the wastewater. The digested wastewater did not contain any total and faecal coliform. The properly digested wastewater appears to be safe from pathogens such as coliform. Both silver perch and rainbowfish can be cultured in wastewater with the latter species more tolerant to high nutrient levels. The duckweed and mussels along with fish could be used as an important plants and animals for nutrient removal purpose. Duckweed is highly efficient in removing ammonia from the wastewater.

Recommendations The current expensive aerobic digester could be replaced with simple anaerobic type that can produce biogas. The present system cannot be recommended to farmers as it is expensive and complicated to operate. It also needs to be modified to ensure manual operation if any breakdown in the electronic system happens. The high rate algal pond with heavy hydraulic paddle wheel can be replaced with an extra two duckweed ponds. This will increase the efficiency of ammonia removal and reduce the energy cost. Most importantly the system is easier to operate.

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The dilution of wastewater from the digester needs to be adjusted to a concentration closer to 100mg/l nitrogen. This will optimise the efficiency of the algal pond. The flow rate in the algal and duckweed ponds needs to be adjusted to retain water for the minimum period required during winter and summer. This will enable proper biological treatment of water. The quality of the water going into the fishpond will need to be maintained. Mussels could be used in the fishpond with silver perch, rainbowfish and aquatic plants. The aquatic plants and mussels enhance the nutrient removal in the fishpond. There is a need to undertake research to determine the optimum density and combination of organisms to maximise the wastewater treatment. Since most of the results were compiled from the microcosm and mesocosm experiments, further pond level experiments are required to refine the results. The experiments conducted in this project were carried out in still water. It is important to repeat the treatments in the flow-through system to determine the levels more precisely Further research is necessary to establish a commercial scale demonstration system. The information from this pilot project should also be used to establish a best practice commercial scale integrated farming system model to treat intensive livestock industry waste (in particular piggery waste utilisation).

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1 Introduction
1.1 Rationale
Intensive livestock production faces the problem of disposal of organic waste. Guidelines used in the Netherlands control the amount of effluent that can be disposed by limiting the application rate of nitrogen and phosphorus. The objective of these guidelines is to limit eutrophication of surface water and prevent groundwater contamination. The environmental impacts of intensive animal farming are also of concern in Australia where a variety of state government agencies have introduced guidelines for the disposal of animal waste. Intensive piggeries, poultry and cattle feedlots produce considerable effluent with a high pollution potential from nutrients such as nitrogen, phosphorus and carbon and odour. These industries are likely to face increasing regulation. The efficient recycling and reuse of both nutrients and water is also likely to become a requirements to achieve ecologically sustainable development The pig industry faces many challenges including the price received for the product, the high cost of inputs and regulations on waste disposal. An example is the introduction of load-based licensing in NSW where the producer is charged for pollutants that are released into the environment (Henman and Smits, 2000).

1.2 Industry background

1.2.1 Pig industry

In September 1997the Australian pig industry was made up of 3,252 different herds comprising 301,854 sows. Of this total, South Australia had 665 herds with 47,846 sows. In 1995-96 the South Australian pig industry produced approximately 957,000 market pigs annually, representing about $100 million in farm gate value and a further $110 million in value adding within the processing, distribution and retailing sectors. National per capita pork consumption is about 18.8kg and rising. The Pig Research and Development Corporation (PRDC) has allocated $1.5 million over the next five years to develop management options that ensure that the environment is not adversely effected by pig meat production (Johnson, 2000). Lenis and Jongblood (1994) state that when all dietary factors are taken into consideration then 60% of the nitrogen and 80% of the phosphorus fed to pigs is excreted into the effluent. Studies carried out by McGahan and Sliedregt (2000) also indicated that the majority of the nutrients are excreted with nitrogen excretion ranging from 56.1 to 63.4%, phosphorous excretion from 62.5 to 72% and potassium excretion from 77.6% to 82%. Odour is another major problem for the pig industry. The ability to expand existing piggeries or develop new sites is usually determined by the availability of adequate separation distances. Regulatory authorities are increasingly requiring odour disperser modelling to determine the required separations. 1.2.2 Poultry Industry In 1994/95 the Australian egg industry produced some 200 million eggs with a farmgate value of approximately $320 million (Mitchell and Derksema, 1997). This industry must also deal with the disposal of solid wastes including cage manure, dead birds and hatchery waste. Unless they are quickly and carefully treated, the waste generates odours and methane, attracts flies, encourages disease, lowers working conditions,
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affects production and releases pollutants. The proximity of many intensive poultry enterprises the rural-urban fringe makes also this industry particularly vulnerable to complaints from neighbours. There are approximately 9.2 million layer hens in Australia (Mc Master, 1995). Each bird produces approximately 145g of faecal matter per day with a moisture content of 80% making a national total of 1335 metric tonnes per day. Manure moisture content usually decreases to an average of 50% by the time of disposal and therefore daily production of solid waste is about 535 tonne/day (Mitchell and Derksema 1997). Poultry manure is a potential pollutant because it comprises high organic content, high nutrient composition (both nitrogen and phosphorous) and high pathogen content. However, it also has great potential as a resource if managed correctly. 1.2.3 Dairy Industry The dairy industry is a major rural industry and ranks third behind the wheat and beef industries in Australia. The gross value of dairy production at farm-gate prices in 1998/99 was around $3 billion. The awareness of environmental issues within the dairy industry has increased greatly over the past five to six years. (Australian Industry Dairy part 1). In 1990 the Environment Protection Authority of Victoria showed that dairy farms were contributing to the decline in water quality in the Kiewa River. A collaborative approach was taken to address the situation, with the formation of the North East Victoria Milking Shed Waste Management Group. According to Crocos (1992) a 120-cow dairy produces approximately 2.9 ML of wastewater per year. The waste produced by a single dairy cow is equivalent to about ten humans. The emphasis in dairy pollution control in Australia is on controlling contamination of surface waters (Wrigley, 1994). In the United States, Europe and United Kingdom, additional emphasis is placed on pollution of ground water, excessive nutrient concentration of soil and control of odours. Pollution of ground water is also a problem in specific parts of Australia with highly permeable soils. 1.2.4 Environmental considerations Wastewater treatment usually involves additional cost. However, if the treatment itself produces income, prevents pollution and complies with environmental standards, it increases the profitability and enhances the sustainability of the industry. Waste can become a resource (Kumar et al 2000). For example, wastewater can be used for aquaculture to produce both fish and aquatic plants and there are a number of models that can be adapted to suit individual situations and resources. The incorporation of aquaculture into wastewater treatment to reclaim nutrients and release clean effluent has proved successful in many parts of the world. Purification levels have reached those attained by the best alternative treatment methods. There are successful enterprises in Germany, England, China, Thailand and India (Ryther 1990; Edwards 1990).

2 Objective
This project sets out to design and develop an integrated wastewater treatment model for the integration of aquaculture with intensive livestock production for practical application on farms situations in South Australia or similar conditions. The specific objectives were: To understand the nutrient load and composition of digested pig effluent. To determine optimum nutrient concentration for biological treatment using algal culture for maximum nutrient depletion. To evaluate the seasonal influence in biological wastewater treatment. To understand algal and nutrient dynamics. To understand animal and plant species for integrated wastewater treatment and aquaculture production.

3 Literature review
3.1 A successful concept
Wastewater is not necessarily a pollutant but a nutrient resource that can be recycled through integrating farming practices. Traditional practices of recycling effluent through agriculture, horticulture and aquaculture have been in used in several countries (Gopakumar et al. 2000). Well-known examples are the sewage-fed fish culture in Munich, Germany and the bheries in Calcutta, India. . Aquaculture is common in many developing countries and has been adapted as a standardised technology for treatment of wastewater. A wealth of technical, economical and social data were presented in the Proceedings of International Seminar on Wastewater Reclamation and Reuse for Aquaculture (Edwards and Pullin, 1990. Several countries including China, Germany, Hungary, India, Israel, Peru and Thailand contributed research and development information to the proceedings. It is clear that excellent potential exists to expand sewage fed aquaculture. Properly designed and managed sewage-fed fishponds offer a low cost wastewater treatment and use opportunity. Papers published in the proceedings clearly demonstrate that five to seven tonnes/ha/year fish production is achievable in tropical climates. The South Australian Research Development Institute (SARDI) conducted a national workshop on integrated wastewater treatment aquaculture production (Kumar 2000). This workshop included participants from India, Vietnam, United States, Samoa and Malaysia. . Researchers from Vietnam and India presented papers on sewage treatment aquaculture within their respective countries but there were concerns regarding environmental impact and health issues in the development of integrated farming practices and its produce in Australia The quantity of wastewater generated has been increasing with population growth. Apart from domestic sewage, a number of industrial effluents and solid wastes are generated in such quantities that treatment has become difficult. Gopakumar et al (2000) described different processes of wastewater treatment including conventional activated sludge and trickling filter methods, oxidation/waste stabilization ponding, aerated lagoons and variations of anaerobic treatment systems. A more recently developed process known as the Up-flow Anaerobic Sludge Blanket (UASB) is also available. (Pearson et al., 1987; Sarikaya and Saatci, 1987; Curtis et al., 1992; Mill et al., 1992; Schlegel, 1993; Oron, 1994; Hammad, 1996; Pearson et al., 1996). Many of the systems described use energy-based treatment processes and only a few of them lead to any resource recovery through root zone treatment, the incorporation of wetland systems and the use of aquatic macrophytes and aquaculture. Duckweeds grow relatively well on sewage water and effectively help in reduction of BOD5, suspended solids and algae (Culley and Epps, 1973; Ozimek, 1983; Oron, 1994). The treatment of domestic sewage through aquaculture is based on taking advantage of macrophytes to trap nutrients together with the traditional use of wastewater in fish farming.

3.2 Live stock aquaculture integrated systems

Integrated fish andlivestock farming is an excellent innovation for recycling of organic wastes and optimum production of high-quality protein at low cost. Cattle manure has been used extensively in India as the major source of organic manure in carp polyculture (Lakshmanan et al., 1971; Singh et al., 1973). Manure is normally applied at
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5000-10,000 kg/ha/yr, in low productive ponds but can be used as high as 25 tonnes/ha/yr (Lakshmanan et al., 1971). According to Gopakumar et al (2000) a cow weighing about 450kg excretes 12000kg of dung and 8000 litres of urine per year. The biological oxygen demand (BOD) of cattle manure is lower than that of other livestock manure because it is already partially decomposed by rumen microorganisms. Studies in India showed that two or three head of cattle can provide sufficient manure to fertilise a one-hectare pond. This form of farming results in the production of both milk and fish, which increases revenue and reduces working expenditure. Addition of biogas slurry results in better growth of plankton and higher carp production than raw cow dung alone (Kalyani and Shetty, 1987). The use of biogas technology also gives a much higher nutrient recovery than the conventional manure application (Chawla, 1986; Gaur et al., 1990). Pigs have a high efficiency of food conversion and are often capable of reproducing and sustaining themselves by scavenging on farm wastes and kitchen refuge (Gopakumar et al 2000). Pig excreta has certain advantages over cow, horse, sheep and goat excreta for fish culture because pigs have a limited ability to consume roughage and the excreta contains lesser amounts of cellulose, hemicellulose and lignin that resist decomposition and blanket the soil at the bottom of the pond. The waste produced by 20-30 pigs is equivalent to one tonne of ammonium sulphate applied to the soil. Fish-pig integration is practiced in China, Taiwan, Vietnam, Thailand, Hong Kong, Malaysia, Hungary and some other European countries (Buch et sl., 1979; Tan and Huat, 1980; Janesirisak, 1980; Cruz and Shehadeh, 1980; Woynarovich, 1980; Chen and Yen, 1980). According to Gopakumar et al (2000) several exotic breeds of pigs have been introduced to India to augment pork production. The size of the pen is based on the number of pigs to be reared, with floor space of three to four m2 for every pig weighing 70-90 kg. When pigs are fed a concentrate containing 15% organic matter, 0.6% nitrogen, 0.5% phosphorous and 0.4% potash their dung is as an excellent fertiliser for enhancing the biological productivity of ponds (Gopakumar et al 2000). Some of the dung is also consumed by the fish. According to Jhingaran (1991), each fully grown pig voids between 500-600 kg dung in a year and the excreta released by 30-40 pigs is adequate to fertilize one hectare of water resulting in a yield of six to seven tonnes of fish/ha/yr. Stocking fingerlings of six carp species at a combined density of 8500/ha, Sharma and Das (1988) recorded production levels of 6792kg/ha/ in one year. Sharma and Olah (1986) recorded production rates of 18.4 kg/ha/day by recycling the pig manure in a fish polyculture pond, without addition of supplementary feeding and inorganic fertilization. Fish production of 4000-7000kg/ha/year without addition of supplementary feeding and inorganic fertilisation has been achieved additional to 4000-5000 kg live weight of pig meat (Gopakumar et al 2000).

3.3 Wastefed aquaculture

Wastefed aquaculture dates back more than a century in Germany. Perin (1990) recorded over ninety installations across the country ranging from small single ponds, receiving wastewater from only few dozen people up to large systems such as at Munich with 233ha designed to treat the wastewater from 500,000 people and to produce a gross fish yield of 100-150 tonnes per year. The main fish species used are common carp (Cyrpinus carpio) and tench (Tinca tinca). The German system was designed to operate
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in temperate latitudes with low stocking densities and fish yields are low compared to those gained in tropical climates. The first experimental sewage-fed fish culture in Hungary was at Fonyod on a water surface area of 211 ha (Ponyi et al. 1973, 1974). A five-year research program followed by a commercial operation and monitoring program was implemented (1979-1983). Technical guidelines for domestic sewage-fed fishponds were developed and approved by government in 1982 (Olah 1990). The sewage-fed fishpond technology in Hungary can receive, process, utilise and purify domestic sewage and produce 12 to 20kg/ha/day. It applies basic principles of complete grazing pressure on both planktonic and benthic communities by implementing polyculture methods using silver carp and common carp, which are able to use both the planktonic and benthic food resources. The results indicate that the nutrients such as ammonia have been reduced from 48-50mg/l to just 0.3-0.5 mg/l, total nitrogen from 50-55mg/l to 2-3mg/l, and total phosphorus reduced from 10-12mg/l to 0.70-1mg/l. At the same time the oxygen level was increased significantly to 8.3mg/l. In China fish culture using human excreta is regarded as a traditional form of agriculture (Zhang 1990). In 1985 the total area of waste-fed aquaculture in China was 8000 ha, with a total fish production of 30,000t/year. This method of production was reviewed in the 1970s and continued to receive approval. Most of the waste-fed aquaculture in China is located near the cities and contributes fish products to city markets. According to Chen et al. (1982) the maximum production of phytoplankton and zooplankton standing crops maintaining 6-8mg/l of total nitrogen or equivalent of 3.54.8mg/l of ammonia in wastewater for fishponds may be permissible. Silver carp (Hypophthalmichthys molitrix) and common carp and bighead carp are the main species stocked along with common carp and Crucian carp (Carassius auratus). The stocking density is usually 15000/ha of 20-30g fingerlings. Fish yields from waste-fed ponds were generally two to four times higher than those from ordinary fish farms. The production ranged from 1.5-11t/ha/year. The efficient use of food resources coupled with year round growing conditions allows high production. The big problem in waste-fed aquaculture in China is that the industrial toxic waste is sometimes mixed with municipal sewage. Wastewater has been used in aquaculture and agriculture near Hanoi for many decades. Tuan (1990) described the three systems involving fish culture that are used: fish culture alone fish-rice rotation, and fish-rice-vegetable rotation. Average net yield from fish culture is about 2.1tonnes/ha/year. Yield has been increased from four to seven tonnes/ha/yr) by controlling the sewage flow and thereby adjusting the nitrogen/phosphorus ratio along with the organic load. Tilapia (Oreochromis mossambicus and O.niloticus), silver carp and common carp are the main species used. Luu and Kumar (2000) described the status of sewage-fed aquaculture systems in Vietnam. Fish culture is a common practice in medium size towns and cities where the sewage output is relatively high and the catchment area is suitable for aquaculture, Sewage is available all year in larger cities and the intermediate catchment areas are invariably used for aquaculture. It is also widely practiced in most of the sewage lakes.
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In Hanoi, there is a daily discharge of 320,000m3 of sewage that is gravity-fed to flood plains of Thanh Tri district where it is used and treated in agricultural-aquacultural systems. Other lakes like Truc Bach and West Lake and Bay Mau Lake are simple catchment areas for domestic sewage and are used for aquaculture. The sewage lakes or ponds are usually stocked with fingerlings of silver carp, grass carp, Indian Rohu and Mrigal carp, tilapia and common carp. In the intermediate catchment lakes, where the water level is manageable, and less exchangeable, algal blooms develop quickly sometimes resulting in sudden planktonic collapse and dissolved oxygen depletion to critically low limits. The ratio of species such as silver carp and tilapia that feed on phytoplankton, algae and detritus can be increased to 5060% in these lakes. On the other hand, in sewage lakes and ponds where water is periodically pumped to balance the nitrogen content, the fish species thriving predominantly on detritus, zooplankton and zoobenthos can be stocked at a higher ratio. Stocking density depends on the quality of sewage. A stocking density of up to four fingerlings/m2 within the size range of 30 to70g is used in India. With control of the stocking density, fish productivity of these ponds/lakes reaches to 5-7 t /ha/year without other inputs of feed, fertilisers and chemicals. The use of aquaculture for wastewater treatment has been evaluated in India with positive results through a five-year research program at the Central Institute of Freshwater Aquaculture, Bhubaneswar. An Aquaculture Sewage Treatment Plant (ASTP) comprising duckweed and fish culture was developed in India. The ASTP comprises a set of duckweed ponds where algae and duckweed is used to remove nutrients and reduce BOD and COD levels, complemented by fishponds holding ponds for marketing. (CIFA 1998). The system can receive primary-treated sewage after the removal of solids. The intake BOD levels for the ASTP are in the range of 100-150 mg/l and consequently it may be necessary to incorporate an anaerobic unit where the organic load and BOD levels are very high. Duckweed culture, before the fishponds, aids in the removal of excessive nutrient concentration and residues. The waste contains BOD5 levels of about 100 mg/l after treatment in the system with a total retention period of five days, the final effluent BOD5 is brought down to 18 - 22 mg/l, meeting the required Indian standards for discharge into natural waters. The fishponds are stocked with five carp species; catla (Catla catla), rohu (Labeo rohita), mrigal (Cirrhinus migrila), silver carp and common carp. The treated effluent from the duckweed culture is released into the fishponds through control valves and fish are harvested when they attain marketable size after eight to12 months of stocking. The production levels recorded from the fish ponds are in the range of three to four t/ha/year.

3.4 Health Issues and Waste-Fed Aquaculture

Fishponds serve as facultative ponds for sewage treatment and the system also provides for oxygen input from the photosynthesising algae and macrophytes (CIFA1998). The macrophytes also serve as nutrient pumps, reducing the eutrophication effects that the sewage is likely to cause in the natural waters. Ponding reduces the bacterial loads by two to three log units and bacteriophage loads by three to four log units even at a sewage loading of 100 kg COD/ha/d. Faecal coliform concentrations are reduced by four log units within 24 hours of retention in the ponds with no evidence of an increase in the concentration of excreted micro organisms in pond water.

Studies in India showed that about one million litres per day of domestic sewage could be treated over an area of one hectare by using water hyacinth which also reduces the BOD and COD by 89 and 71% along with removal of 89 % nitrogen and 50 % of phosphorus.. Public health concerns have been raised about the suitability for consumption of fish and shellfish from wastefed systems because of the microbial load of the produce, the possibilities that it harbours human pathogens, accumulation of pesticide residues and heavy metals. Consequently, wastefed aquaculture systems are being modified with the incorporation of plant cultivation prior to application of wastewaters to the fishponds, followed by necessary depuration measures. In Munich, the government laboratories regularly monitor the waste-fed aquaculture fish. The published information indicates that all values are below critical levels required by the German Federal Bureau of Health. Human pathogens have never been found in fish flesh. Values of heavy metals and aromatic hydrocarbons in fish are said to comply with government standards (Prein 1990). Counts of bacteria, including coliforms reveal that properly functioning wastewater and fish culture installations have high reduction values of over 99% compared to the diluted wastewater. A survey conducted in the fish farming area of Wuxi, Jiansu Province, China on animal manure found that the manured pond water and fish body surface mucus of Chinese carp did not reveal any human intestinal pathogenic bacteria. When the fish from the manured pond were de-scaled and rinsed the amount of coliform bacteria was reduced by 100-1000 times to almost the same amount in the fish from the non-manured pond. Studies also found that after proper washing, fish cultured in the manured pond is hygienic as human food (Jieyi et al 1994). In a full-scale demonstration study in Suez, Egypt, about 400M3/day of raw sewage was treated using a multi-compartment stabilisation pond system. The effluent was used for rearing Tilapia and Grey Mullet. The fish were subjected to an extensive monitoring program. Bacteriological examination revealed that in all samples the fish mussels were free of bacterial contaminants. The study concluded that fish reared in the treated effluent at the Suez experimental station is suitable for human consumption (Easa et al 1995)

4 Research strategies and methodology

In nutrient and water dynamics studies, the scale of a system can affect the outcomes. Lessons learnt from small-scale experiments where high levels of replication are possible may not be applicable to a large scale where experiments are less tractable. This research project is designed to understand fish, zooplankton, phytoplankton and nutrients in three levels of experiments: microcosm, mesocosm and ponds. Microcosms have become an important tool for such investigations since together with appropriately scaled field studies; there is benefit through replicability. The results obtained from microcosm experiments will be taken into consideration when designing the mesocosm experiment. Similarly, the pond experiment will be structured based on the results of both micro and mesocosm experiments. Outline of research strategy
Aquarium Small Tank Ponds (Commercial scale trial and implementation)

RIRDC SRA 16 Project proposed

Regional demonstration site (temperate conditions)

Experimental integrated wastewater treatment system

Urrbrae Wastewater Treatment and I t t d Biosystems

Algal Pond

Livestock

Digester

Macro & Micro Algae

Organic Waste

Fish Pond

Fish & Aquatic Plants

Wetland

Horticulture/ Agriculture

4.1 Experimental plan

The approach includes standardise chemical and biological analytical methods and experiments to achieve the objectives in an orderly manner. Two pilot scale experiments were completed to standardise the analytical methods. The microcosm experiments were designed to determine optimum concentration for biological treatment through microalgal production to obtain maximum nutrient depletion after characterising the nutrient quality in terms of composition of nitrogen (ammonia and nitrate) and phosphorous, The experiments were then focussed to understand the effect of zooplankton on nutrient depletion and to determine the nutrient tolerance of the fish species for aquaculture. A combination of aquatic plants, freshwater mussels and fish were used in a trial to determine the appropriate combination of these species to improve water quality through nutrient depletion.

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Focus of the experiments and expected outcomes

Nutrient characterisation

Optimum nutrient concentration level for biological treatment (maximum nutrient depletion and algal production)
Nutrient quality and composition

Wastewater dilution factor

Zooplankton influence on nutrient depletion

Optimum wastewater retention time and zooplankton species tolerance and influence on nutrient depletion

Fish species tolerance to nutrient level

Fish species suitability for wastewater treatment aquaculture production

Suitable aquatic plants and animal combination required for integrated wastewater treatment and . aquaculture production

Fish, molluscs and aquatic plant influence in nutrient

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The piggery effluent used for this experiment was collected from Urrbrae Agriculture High School (UAHS), South Australia. This small piggery unit has 12 sows, 70 growers and 15 weaners. The piggery, which is mainly used for teaching purposes, has been producing about 8600 litres of liquid and 930 litres of solid waste per week. A prototype model of integrated wastewater treatment system is in place near the piggery to treat the effluent and to undertake research and development to optimise the system to develop a proper treatment model. All the experiments conducted in this project are for optimising efficiency of this system.

4.2 The Urrbrae integrated wastewater treatment system

The Urrbrae integrated wastewater treatment system comprises an aerobic reactor, two high rate algal ponds, two ponds for fish production and constructed wetlands. Animal waste is collected in a reception pit from both the piggery and dairy and is fed semi-continuously to an aerobic reactor. Air is supplied via a compressor and membrane diffuser to the 28 m3 aerobic reactor where the natural microbial population degrades the organic material to carbon dioxide, water and microbial biomass. Control of the rate of aeration determines the form of nitrogen present in the final effluent. The aerobic process significantly reduces the organic content (BOD & COD) of the slurry, transforms nitrogen and produces an effluent with an inoffensive odour. However, the effluent still contains significant quantities of nitrogen and phosphorus. The slurry is separated into solid and liquid phases following aerobic treatment. The solid phase is composted and the liquid phase is diluted using water from on-site wetlands and then passes into two high-rate algal ponds. These are shallow channels where the water is mixed slowly using a paddlewheel. Prolific algal growth occurs accompanied by nutrient removal via biotic and abiotic processes. The algae produced in the high rate algal ponds contain approximately 50% crude protein. Treated effluent from the high rate algal ponds is fed into two aquaculture ponds for the production of fish using the algae and zooplankton as a source of nutrients and energy. The final effluent water from the fishpond is returned to the wetlands.

4.3 Analytical methods

Nutrients (nitrite, nitrate, ammonia, total nitrogen, phosphorous, soluble reactive phosphorous), algal biomass estimation, dissolved oxygen, pH, salinity and temperature were analysed according to APHA (1985). Universal Pocket Meter MultiLine P4 probes were also used to measure nitrate and ammonia. P4 probes were also used for measuring pH, temperature conductivity and salinity. Statistical analysis was performed by use of the generalised linear model of the form.

Nutrient / A lg ae(i, day) = (i ) .day -

day

(i,day)

Day is the number of days from the start of the experiment

is intercept (i)is the effect of treatment i, and characterise the effects of day, and (i,day) is the
multiplicate error term(assumed to be log normally distributed).

, and are parameters to be estimated

12

4.4 Experimental protocol and analysis

4.4. 1 Experiment to determine optimum nitrogen concentration for nutrient depletion through algal culture.

This experiment was performed in eight-litre capacity aquariums from the 29th of March 2000 for 15 days. The digested wastewater was collected from the anaerobic digester and measured for total Kjeldhal nitrogen and total phosphorus. Five treatment dilutions with three replicates of each were used. The five treatment were 0% (no waste water added), 25%(25% wastewater+ 75% tap water), 50%(50% waste water + 50% tap water), 75%(75% waste water+ 25% tap water) and 100% (100% waste water. All treatments were inoculated with of pond water to enhance the growth of algae. The inoculum water was sampled from the Urrbrae biosystem fishpond, which was sieved to remove zooplankton. A stock solution for each treatment was prepared and divided equally between the eight- litre aquariums. The aquaria were placed outdoors under ambient conditions and water quality parameters and algal biomass samples were taken daily. Sampling and measurements were performed at 12:00 midday. Water quality parameters tested included temperature, pH, dissolved oxygen, salinity and specific conductivity. Total alkalinity, ammonia-N, nitrite-N, nitrate-N and soluble reactive P was analysed. Light intensity was measured by using a Licor RS 232 sensor logger. The nutrient levels were statistically compared for each treatment and with the time (days) and total biomass (micro algae) production to determine optimum concentration for wastewater treatment. Nutrient reduction rate was estimated = 1-Final value/initial value X 100.
4.4.2 Experiment on seasonal influence in nutrient uptake by micro algae with temperature, day length and light intensity.

An experiment to determine the seasonal influence of temperature, day length and light intensity on nutrient depletion and water quality in digested piggery wastewater by algae and also to indicate coliforms level of wastewater was undertaken. It was carried out from the 17th of July 2000 over 14 days using the aquariums in two environment control rooms and under outdoor ambient conditions. The initial sample of piggery wastewater was tested for total Kjeldahl-N, nitrate-N, nitrite-N, ammonia-N, total P, soluble reactive P, BOD, alkalinity, total coliforms and faecal coliforms. Wastewater was diluted to the pre-determined optimum concentration of 100mg/l nitrogen based on the results obtained in the previous experiment described above under 4.4.1. Three replicates were used for each treatment. The three treatments were: Winter (average day length / temperature / light intensity). Summer (average day length / temperature / light intensity). Ambient (day length / temperature / light intensity) used for this experiment. Average winter and summer temperatures were based on local weather data and set to increase to 15oC for winter and 25oC for the summer. Halogen grow-lights were used for artificial light. Day lengths used were indicative of local seasons with the winter room set to a 12/12 hr light/dark cycle and the summer at a 14/10 light/dark cycle. Samples were taken and tested for water quality and micro algal biomass every 2 days over this period. The inoculum water was sampled from the Urrbrae biosystem fishpond and sieved to remove zooplankton. A stock solution for each treatment was then prepared whereby the individual aquariums received their eight litres of solution. Samples were then taken every two days
13

and tested for nitrate-N, nitrite-N, ammonia-N, total phosphorous, soluble reactive phosphorus. Samples were also tested every two days for temperature, pH, dissolved oxygen, conductivity, salinity and BOD. Faecal coliform testing was also performed on the stock solutions initially and at the end of the experiment. Statistical analysis was carried out: To determine the best treatment in terms nutrient reduction and algal production. To determine the influence of temperature on nutrient depletion: To compare time as a factor during nutrient depletion.
4.4.3 Experiment on zooplankton interference on nutrient depletion by algae.

An experiment was conducted to determine the impact of zooplankton on water quality and nutrient depletion by algae in digested piggery wastewater. A treatment was also included to test the effect of Daphnia carinata on water quality and nutrient depletion by algae in digested piggery wastewater. The trial was conducted from the 25th of September 2000 for 14 days under ambient conditions. Three treatments were applied with three replicates conducted in aquaria at the pre-determined optimum wastewater concentration of 100mg.l-1N (based on experiment 4.4.1) and an inoculum of pond water for the growth of algae. The three treatments were: Wastewater with alga and zooplankton inoculation including an extra inoculation of specifically Daphnia carinata. Wastewater with alga and zooplankton inoculation. Wastewater with alga inoculation (zooplankton filtered out).

The initial sample of piggery wastewater was tested for total Kjeldahl-N, nitrate-N, nitrite-N, ammonia-N, total P, soluble reactive P and alkalinity. The inoculum water was sampled from the Urrbrae biosystem fishpond. The Daphnia inoculation was grown separately on site. A stock solution for each treatment was then prepared whereby the individual aquariums received an equal amount of inoculum. Samples were taken at noon every two days for algae and water quality. Water quality parameters tested included nitrate-N, ammonia-N, and soluble reactive P. Samples were also tested every two days for temperature, pH, dissolved oxygen, conductivity and salinity.
4.4.4 Experiment on fish tolerance to various levels of nutrient

An experiment was conducted to test the tolerance of juvenile Silver perch (Bidyanus bidyanus) and Murray River rainbowfish (Melanotaenia fluviatilis) to various wastewater concentrations. It was conducted from the 25th of September 2000 for seven days under ambient conditions. Five treatments with concentrations of 0, 1, 2, 20 and 100 mg.l-1 wastewater received equal amount of inoculum of pond water for the growth of algae. The experiment was carried out with three replicates of each treatment and four fish were introduced in each replicate. Fish mortalities were checked at increasing intervals starting from three hours, six hours, nine hours 12 hours, 18 hours, 24 and then on every 24 hours. The inoculum water was sampled from the Urrbrae biosystem fishpond. Water quality parameters temperature, pH and dissolved oxygen were measured. Mortality rate of the fish species with time was compared to determine the tolerance level of each species.

14

4.5 Experiment on effects of mussel culture, duckweed and fish on nutrient uptake.
A trial was conducted to determine the appropriate species composition involving freshwater mussels, rainbowfish and duckweed on nutrient uptake and water quality. It was conducted from the 8th of December 2000 over 15 days under ambient conditions in 12 fibreglass tanks at Urrbrae High School. Each tank held a concentration of 5 mg.l-1 total nitrogen wastewater and received an inoculum of pond water for the growth of algae and 10 rainbowfish. Four treatments were applied with three replicates including mussels (Velesumio ambiguus), duckweed (Lemnaceae lemna), mussels and duckweed and a control with no mussels or duckweed. Mussels were introduced as eight mature individuals per tank and duckweed at 50g per tank. The inoculum water was sampled from the Urrbrae biosystem fishpond. Fish and mussel mortalities were recorded as well as growth of duckweed. Samples were taken at noon every three days for algae and water quality. Water quality parameters tested included nitrate-N; ammonia-N and soluble reactive P. Samples were also tested every two days for temperature, pH, dissolved oxygen, conductivity, and salinity. The results were statistically analysed to determine the best treatment in terms of nutrient depletion.

15

5 Results
5.1 Optimum nutrient concentration for maximum nutrient depletion.
The total nitrogen content in the digested wastewater varied between 175 nitrogen to 350mg/l during the normal functioning of the unit provided there was a regular output to the high rate algal ponds. However, wastewater containing over 500mg/l nitrogen was collected when waste was retained in the digester over 10 days. The tank was designed to function on a seven-day retention period. The nitrogen and phosphorous ratio varied between 4.4: 1 to 8.8 :1. The high levels of nitrogen concentration allowed various levels of dilutions for undertaking this experiment. The treatment system was restricted for teaching the principles of wastewater treatment and to undertake research and development for developing an efficient treatment unit. The unit aerobic digester and the hydraulic system in the high rate algal ponds are being constantly reviewed, as these systems are prototypes. This experiment was conducted during March 2000 under ambient conditions of a typical South Australian summer. The temperature ranged between 20 to 35oC. The day length was longer and the treatment received maximum light during the experimental period. The nutrient factor considered for dilution for various treatments for this experiment was total nitrogen. The dissolved oxygen content remained steady throughout the trials. The pH levels of the treatments gradually increased from slightly acidic (6.5) to alkaline condition (7.5) towards the end of the experiment. A slight increase in salinity and alkalinity was noticed during the trial for all treatments.

Table 1 Showing the changes in the nutrient level after 15 days of experiment Initial Mean Reducti % Mean Reduction % Nitrog nitrate on rate/ reduction ammonia rate/ reduct en reduction day reduction day ion content mg/l mg/l mg/l mg/ mg/l 0 0. 0 0. 0 0 0. 50 10.483 0.699 52.76 36.5966 2.440 79.59 100 13.247 0.883 39.90 39.98 2.666 66.71 150 14.067 0.938 31.45 41.07 2.738 57.25 200 24.900 1.66 41.85 51.9 3.46 60.00 An interesting result was the reduction rate and variation of ammonia and nitrate content over time. Table 1 shows the final change in the level of ammonia, nitrate and phosphorus. The reduction rate was higher in higher concentration treatment for nitrate and ammonia. However, the percentage reduction (50, 100, and 150 mg/l nitrogen) and the reduction rate was more or less the same. The treatments of 25% and 50% (equal to 50mg/l and 100mg/l Nitrogen) produced nutrient depletion more rapidly than other treatments. Nutrient reduction was rapid over the first five days of with approximately 75% of the depletion occurring within this period. Maximum nutrient depletion occurred in all treatments, after 13 to 14 days. The nutrient changes and other water quality measurements are presented in fig: 1.a-i.

16

The corresponding algal biomass estimation indicated that the treatment 50% (100mg/l) produced the highest algal biomass of any treatment. It was closely followed by the 25% and 75% wastewater treatments. Four main groups of algae were found in the wastewater namely, Chlorella, Scenedesmus, Ankistrodesmus and filamentous.

Table 2 Showing the results of the analysis of linear model. R-Square Nitrite Nitrate Ammonia S.R. Phosphorous Total Algal Biomass 0.463732 0.993611 0.980071 0.977526 0.883648 C.V. 50.38778 4.226434 7.668615 7.813659 19.90949 Root MSE 0.16532 0.11657 0.21890 0.24499 0.64432 Log Mean 0.32809 2.75809 2.85453 3.13546 3.23624

T tests were carried out to determine the significance for the variables.

Table 3 Least square means of log nitrate content Treat 0 25 50 75 100 LSMean 0.0000000 0 2.8668465 8 3.3562032 3 3.6865446 3 3.8808476 1 Std Err LSMean 0.0173770 5 0.0173770 5 0.0173770 5 0.0173770 5 0.0173770 5 Pr > | T | HO:LSMe an = 0 1.0000 0.0001 0.0001 0.0001 0.0001 N 45 45 45 45 45 Bon Grouping E D C B A

Statistical analysis based on the generalised linear model indicated that the goodness of fit for nitrite, as a dependent variable was poor. (R-Square=0.463732). The other important nutrients considered for the analysis including nitrate (0.993611) ammonia (0.980071), and total algal biomass (0.883648) exhibited a good relationship. The details of the analysis are presented in table 2. Table 4 indicates the log mean square, F and probability values.

17

Table 4 Indicating mean square, F and probability values.

Nitrite Source Treatment LDAY DAY Log Nitrate

DF 4 1 1

Type III SS 4.76298978 0.03564556 0.14520094

Mean Square F Value 1.19074745 0.03564556 0.14520094 43.57 1.30 5.31

Pr > F 0.0001 0.2547 0.0221

Treatment 4 Log DAY 1 DAY 1 Log Ammonia

454.465824 0.579501 2.340504

113.616456 0.579501 2.340504

8361.35 42.65 172.24

0.0001 0.0001 0.0001

Treatment 4 492.202507 LDAY 1 0.003231 DAY 1 2.357855 Log Total Algal Biomass Treatment LDAY DAY 4 1 1 121.952707 0.955420 9.588139

123.050627 0.003231 2.357855

2567.92 0.07 49.21

0.0001 0.7954 0.0001

30.488177 0.955420 9.588139

73.44 2.30 23.10

0.0001 0.1327 0.0001

The nitrate levels were not significantly different for all the treatments except in the control. In the case of nitrate, ammonia, and total algal production, the mean levels were significantly different from each other. The depletion rate of nutrient was found to be higher as the nitrogen concentration in the wastewater was higher. The value of depletion per day was closer (but different) in the case of 25%, (=50mg/lN) 50%, (=100mg/lN) and 75%(=150mg/lN) of wastewater treatment. The ammonia reduction rate in these treatments ranged from 2.4mg/l/day to 2.7 mg/l/day. Similarly the nitrogen reduction rate ranged between 0.7mg/l/day to 0.94mg/l/day. However, the treatment containing 200mg/l the reduction rate of ammonia and nitrate were 3.46mg/l/day and 1.66mg/l/day respectively. The total algal production was significantly different for various treatments and the results are shown in table 6. According to the statistical model estimation of the optimum algal production can be obtained from 34% of wastewater concentration, equivalent to 68mg/l nitrogen level in the wastewater. However, the depletion rate and algal production in 50% (100mg/l) wastewater treatment recorded the highest algal biomass (352.34mg/l) at the end of the experiment (on day 14). A decreasing trend in the algal biomass was noticed in the concentration higher than 100mg/l. The algal production in both 50% and 25% concentrations were not significantly different. T test comparison on algal biomass production is presented in table 6. Means with the same letter are not significantly different. Both 75% and 100% wastewater treatments were classified into one category. 25 and 50% treatments were classified into another category and the control classified in a separate category.

18

Table 5 Log least square means log ammonia content

Treatment % wastewater 0 25 50 75 100

LSMean 0.00000000 2.93200197 3.43136456 3.83163674 4.07764666

Std Err LSMean 0.326321 2 0.326321 2 0.326321 2 0.326321 2 0.326321 2

Pr > | T | HO:LSMean = 0 1.0000 0.0001 0.0001 0.0001 0.0001

N 45 45 45 45 45

Bon Grouping E D C B A

Table 6 Least square means of log total algal biomass Treat 0 25 50 75 100 LSMean 0.96193915 4.11500110 4.42152027 3.14711173 2.88584918 Std Err LSMean 0.16964200 0.14072699 0.14072699 0.14072699 0.14072699 Pr > | T | HO:LSMean =0 0.0001 0.0001 0.0001 0.0001 0.0001 N 45 45 45 45 45 Bon Grou ping C A A B B

pH
8.5 8 7.5 pH 7 6.5 6 1 2 3 4 5 0% 6 7 8 9 75% 10 11 12 100% 13 14 15 day 25%

Fig 1(a) Showing the pH variation (optimum nutrient concentration experiment).

19

Salinity
4 3.5 concenttration (0/00) 3 2.5 2 1.5 1 0.5 0 -0.5 1 2 3 4 5 6 7 day 8 9 10 11 12 13 14 15

(b)
Conductivity
8000 7000 Conductivity (uS/cm) 6000 5000 4000 3000 2000 1000 0 -1000 1 2 3 4 5 6 7 day 8 9 10 11 12 13 14 15

(c)
Temperature
40 35 Temp (C) 30 25 20 15 1 2 3 4 5 6 7 8 day 9 10 11 12 13 14 15

0%

25%

50%

75%

100%

(d)
Fig 1(b,c,&d) Showing the salinity, conductivity and temperature(optimum nutrient concentration experiment).

20

Nitrite
1.4 1.2 concentration (mg/l) 1 0.8 0.6 0.4 0.2 0 -0.2 1 2 3 4 5 6 7 8 day 9 10 11 12 13 14 15

(e)
Nitrate
70 60 concentration (mg/l) 50 40 30 20 10 0 1 2 3 4 5 6 7 8 day 9 10 11 12 13 14 15

(f)
Ammonia
100 90 concentration (mg/l) 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 day 9 10 11 12 13 14 15

(g)
0%

25%

50%

75%

100%

Fig 1(e,f,&g) Showing the nitrite, nitrate and ammonia variation (optimum nutrient concentration experiment).

21

Alkalinity
700 600 500 units 400 300 200 100 0 1 2 3 4 5 6 7 8 day 9 10 11 12 13 14 15

(h)
Dissolved Oxygen
12 concentration (mg/l) 10 8 6 4 2 0 1 2 3 4 5 6 7 8 day 9 10 11 12 13 14 15

0%

25%

50%

75%

100%

(i)
Fig 1(h,&i) Showing the alkalinity and dissolved oxygen(optimum nutrient concentration experiment).

22

40 30 concentration (mg/l) 20 10 0 -10 2 4

Chlorella by treatment

8 day

10

12

14

(j)
Scenedesmus by treatment
concentration (mg/l) 200 150 100 50 0 -50 2 4 6 8 day 10 12 14

Ankistrodesmus by treatment
200 concentration (mg/l) 150 100 50 0 -50 2 4 6 8 day 10 12 14

(k)
Totals by treatment
500 concentration (mg/l) 400 300 200 100 0 -100 2 4 6 8 day 10 12 14

(l)
0% 25%

50%

75%

100%

Fig 1(j,k&l) Showing algal biomass variations (optimum nutrient concentration experiment).

23

5.2 Seasonal influence on biological wastewater treatment.

South Australian temperate climatic conditions brought dramatic changes in the biological activity. Temperatures reached extreme levels during summer (forties oC) and reduced to very low levels up to 2oC in winter. Temperature plays a vital role in the breaking down of organic materials and has a major influence on the metabolic activities of organisms. Therefore biological treatment will be impacted by the climatic changes during the year. Considering these factors, it was important to collect basic information on the seasonal impact on algal production and nutrient depletion. This experiment is focussed on determining the depletion rate of various nutrients and algal production under the two major seasons such as winter and summer. The details of the results are presented in figure 2(a-l). As expected summer conditions enhanced the nutrient depletion rate and algal production. As the experiment was conducted in winter, the results were more or less similar in ambient and winter controlled conditions. The initial and final analysis for faecal and total coliform indicated that the digested wastewater has negligible pathogenic bacteria levels. The dissolved oxygen levels rose sharply to supersaturated levels particularly in summer conditions. During the experimental period the biological oxygen demand (BOD) within the aquariums dropped from high to low levels. The ammonia-N concentration, the largest nitrogen budget of the nitrogenous compounds was reduced most effectively under summer conditions. In summer the final level of ammonia (after 14 days) is 5 times lower than that in winter and ambient conditions. About 80% of the ammonia content was depleted in the first eight days (from 95mg/l to 20.5mg/l) of the experiment. In winter conditions, the depletion rate was comparatively slower and it took twice as much time to reach equivalent levels in summer. It was observed that in summer it took 8 days to deplete down to 20.5mg/l from the initial level of 95mg/l. This level of depletion was achieved in winter within about 14 days. The trend of nitrate depletion was similar in the case of winter and ambient conditions. In summer conditions, the nitrate levels were very low compared to ambient and winter and depleted rapidly during the first 8 days of the experiments whereas in ambient and winter controlled conditions the nitrate levels rose slightly up to 6 days and then dropped rapidly. In all the treatments the soluble reactive phosphorous reduced steadily during the 14 days of experiment. It is interesting to note that the ambient treatment conditions were similar to the summer conditions in terms of the trend in phosphorus depletion for the experiment duration.

Table 7 results of linear model analysis.

Nutrient Nitrate Ammonia S.R. Phosphorous Total Algal Biomass

R-Square 0.586410 0.754143 0.735091 0.941814

C.V. 56.19330 13.70299 6.149272 12.35724

Root MSE 0.34003 0.53006 0.05825 0.40031

Log Mean 0.60512 3.86817 0.94728 3.23950

24

The details of algal production are given in figure 2 (j,k&l). The algae Chlorella, Ankistrodesmus, Scenedesmus, and Filamentous type were the main species observed in all treatments. The level of algal production was found to be similar in ambient and winter conditions. At the end of the experiment an average of 80.7 and 91.7mg/l of algal biomass was recorded for ambient and winter conditions respectively. At the same time under summer conditions the algal production was rapid and reached an average of 304mg/l after 14 days. Generalised linear model analysis results are presented in table 7. The R-square value for all nutrient level indicates that the goodness fit of the model is good. The probability, F and Mean Square values are provided in table 8.

Table 8 Showing mean square, F and probability of winter, summer and ambient treatments

Log Nitrate Source

DF

Type III SS 11.7090947 3.0377965 2.1636253 21.5044520 2.0135527 7.6003703 0.10077776 0.01300280 0.08267077 11.7090947 3.0377965 2.1636253

TREAT 2 LDAY 1 DAY 1 Log Ammonia TREAT 2 LDAY 1 DAY 1 Log S.R.Phosphorous TREAT 2 LDAY 1 DAY 1 Log Total Algal Biomass TREAT 2 LDAY 1 DAY 1

Mean Square 5.8545474 3.0377965 2.1636253 10.7522260 2.0135527 7.6003703 0.05038888 0.01300280 0.08267077 5.8545474 3.0377965 2.1636253

F Value 36.53 18.96 13.50 38.27 7.17 27.05 14.85 3.83 24.36 36.53 18.96 13.50

Pr > F 0.0001 0.0001 0.0005 0.0001 0.0096 0.0001 0.0001 0.0551 0.0001 0.0001 0.0001 0.0005

T tests were carried out to compare winter, ambient and summer conditions. Treatments were grouped and differentiated by letters. Means with the same letter are not significantly different. Treatment comparisons clearly indicated that the winter and ambient conditions performed more or less similarly and they are not significantly different for nitrogen compound (nitrite and ammonia) and algal biomass production. However, phosphorus depletion in summer and ambient conditions were classified in the same group (Table 9 -11).

25

Table-9 Nitrate least square means Treatment AMBIENT SUMMER CER WINTER CER LSMean 0.80014348 0.16821481 0.84698919 Std Err LSMean 0.07420162 0.07420162 0.07420162 Pr > | T | HO:LSM ean = 0 0.0001 0.0271 0.0001 N 21 21 21 Bon Grouping A B A

Table-10 Ammonia least square means

Treat AMBIENT SUMMER CER WINTER CER

LSMean 4.16478847 3.05201176 4.38771282

Std Err LSMean 0.11566753 0.11566753 0.11566753

Pr > | T | HO:LSM ean = 0 0.0001 0.0001 0.0001

N 21 21 21

Bon Grouping A B A

Table-11 S.R.Phosphorous least square means

Treat AMBIENT SUMMER CER WINTER CER

LSMean 0.92410501 0.91418165 1.00355042

Std Err LSMean 0.01271136 0.01271136 0.01271136

Pr > | T | HO:LSM ean = 0 0.0001 0.0001 0.0001

N 21 21 21

Bon Grouping B B A

26

Nitrate
4 3.5 3 2.5 units 2 1.5 1 0.5 0 2 4 6 8 day 10 12 14

(a)
Ammonia
120 100 80 units 60 40 20 0 -20 2 4 6 8 day 10 12 14

(b)
SR Phosphorous
3

2.5 units 2 1.5 2 4 6 8 day 10 12 14

(c)

ambient

Summer CER

Winter CER

Fig 2(a,b,c) Showing variations in nitrate, ammonia and SR phosphorous

27

D.Oxygen
20 15 10 5 0 -5 2 4 6 8 day 10 12 14

(d)
Salinity
0.25 0.2 0.15 units 0.1 0.05 0 -0.05 2 4 6 8 day 10 12 14

(e)
Conductivity
900 800 700 units 600 500 400 300 200 2 4 6 8 day 10 12 14

(f)
ambient Summer CER Winter CER

Fig 2 (d,e&f) Showing variation in oxygen, salinity and conductivity

units

28

Temperature (C)
32 30 28 26 units 24 22 20 18 16 14 2 4 6 8 day 10 12 14

(g)
pH
12 11 10 pH 9 8 7 2 4 6 8 day 10 12 14

(h)
Total green algae
400 350 300 biomass mg/l 250 200 150 100 50 0 -50 2 4 6 8 day ambient Summer CER Winter CER 10 12 14

(i)
Fig 2(g,h&i) Showing variations in temperature, pH and total algal biomass

29

Ambient conditions
100 80 biomass mg/l 60 40 20 0 -20 2 4 6 8 day 10 12 14

(j)

Summer conditions
400 350 300 biomass mg/l 250 200 150 100 50 0 -50 2 4 6 8 day 10 12 14

(k)
Winter condition
120 100 biomass mg/l 80 60 40 20 0 -20 2 4 6 8 day Chlorella mg/l Filamentous mg/l Scenedesmus mg/l Total Green algae mg/l Ankistrodesmus mg/l 10 12 14

(l)
Fig 2(j,k&l) Showing alga species variation in different seasons

30

5.3 Impact of zooplankton on nutrient depletion

An experiment was conducted to determine whether a mixture of naturally occurring zooplankton and the addition of an efficient algal consumer such as Daphinia made any impact on the nutrient depletion rate and to understand the tolerance of zooplankton at the level of nutrient concentration optimum to algal culture. Under microcosm conditions, wastewater containing approximately 100mg/l nitrogen concentration appeared to be optimum for algal production. There was negligible amount of zooplankton present on the second day. Zooplankton including Daphinia did not survive in 100mg/l nitrogen concentration. Treatment 1 (inoculated with algae, zooplankton and Daphina carinata), treatment 2 (inoculated with algae and zooplankton), and treatment 3 (inoculated with only micro-algae) did not show any difference in nutrient depletion. The experiment was conducted at an average temperature of 24oC. Similar to the previous experiments the pH level turned alkaline. The dissolved oxygen level increased to super saturation level after 10 days and subsequently dropped to about 18mg/l towards the end of the experiment. The details of the result are shown in figure-3 (a-i). The nitrate level decreased steadily and reached a minimum within 10 days of the experiment. Although there is a variation in the level of nitrate change between treatments, it is not statistically significant. The ammonia content in wastewater decreased rapidly up to 12 days and reached minimum level (app.100 to 4mg/l) towards the end of the experiment in treatments two and three. However, in treatment one the ammonia depletion was comparatively slower; the ammonia content recorded after 14 days was 21.6mg/l. The soluble phosphorus level was found to increase during the initial eight days and subsequently decreased to the minimum level towards the end of the experiment. The introduction of a general inoculum including zooplankton does not have a major effect on nutrient depletion. The introduction of Daphnia may have had an initial effect on the rate of ammonia depletion by algae. Daphnia is the only zooplankton species able to impact upon the proliferation of algae due to a high grazing rate and has a small effect under the conditions of this experiment. Hence Daphnia should be avoided for the most effective nutrient depletion. Statistical analysis using generalised linear model has been presented in table 12. The goodness of fit for soluble reactive phosphorous is poor (R-Square =0.252517). The relationship in all other nutrient level and total algal biomass is good.
Table 12 showing the results of the analysis of linear model.

Nutrient Nitrate Ammonia S.R. Phosphorous Total Algal Biomass

R-Square 0.684009 0.533126 0.252517 0.769109

C.V. 826.2432 25.77667 -192.6214 33.31609

Root MSE 0.23321 0.94799 0.61221 1.02592

Log Mean 0.02823 3.67772 -0.31783 3.07935

31

Table 13 Showing mean square, F and probability values

Log Nitrate Source

DF

Type III SS 1.25144616 4.63516892 1.7044138 49.6066701 1.57110504 4.75976244 3.035962 172.262002

Mean Square 0.62572308 4.63516892 0.8522069 49.6066701 0.78555252 4.75976244 1.517981 172.262002

TREAT 2 LDT 1 Log Ammonia TREAT 2 LDT 1 Log S.R.Phosphorous TREAT 2 LDT 1 Log Total Algal Biomass TREAT 2 LDT 1

F Value 11.50 85.22 0.95 55.20 2.10 12.70 1.44 163.67

Pr > F 0.0001 0.0001 0.3943 0.0001 0.1336 0.0008 0.2461 0.0001

T test was carried out to compare the significance of treatments for both nutrients and algal biomass levels. Means with the same letter are not significantly different. In general these treatments were not significantly different from each other.
Table-14 Nitrate least square means

Treat 1 2 3

LSMean 0.23845862 -0.0367158 -0.1170654

Std Err LSMean 0.05496902 0.05496902 0.05496902

Pr > | T | HO:LSMe an = 0 0.0001 0.5072 0.0381

N 18 18 18

Bon Grouping A B B

Table-15 Ammonia least square means

Treat 1 2 3

LSMean 3.91372982 3.485080.22 3.63435009

Std Err LSMean 0.22344438 0.22344438 0.22344438

Pr > | T | HO:LSMe an = 0 0.0001 0.0001 0.0001

N 18 18 18

Bon Grouping A A A

Table-16 S.R. Phosphorous least square means

Treat 1 2 3

LSMean - 0.2339942 -0.5556339 -0.1638673

Std Err LSMean 0.14429982 0.14429982 0.14429982

32

Pr > | T | HO:LSMe an = 0 0.1112 0.0003 0.2615

N 18 18 18

Bon Grouping A A A

Table 17 Total algal biomass least square means

Treat 1 2 3

LSMean 2.95941931 3.41051005 2.86812924

Std Err LSMean 0.24181164 0.24181164 0.24181164

Pr > | T | HO:LSMe an = 0 0.0001 0.0001 0.0001

N 18 18 18

Bon Grouping A A A

The nitrate level in the control treatment is significantly different from treatment two and three. Treatments two and three are not significantly different with regard to the variation of the nitrate levels. However, ammonia, phosphorous and total algal biomass levels were not significantly different between treatments.

33

Nitrate

3.5 concentration (mg/l) 3 2.5 2 1.5 1 0.5 0 0 2 4 day 6 8 10 12

(a)

Ammonia

160 140 120 100 80 60 40 20 0 -20 0 2 4 day 6 8 10 12

(b)
SR Phosphorus

concentration (mg/l)

2 concentration (mg/l) 1.5 1 0.5 0 -0.5 0 2 4 6 day 8 10 12

(c)
algae & zooplankton & Daphnia algae & zooplankton algae

Fig 3a,b&c Showing variation in nitrite, ammonia and SR Phosphorus

34

Tem perature

34 32 30 28 26 24 22 20 18 16 0 2 4 6 day 8 10 12

Temperature (C)

(d)
pH

11 10.5 10 9.5 pH 9 8.5 8 7.5 7 0 2 4 day 6 8 10 12

(e)
35 concentration (mg/l) 30 25 20 15 10 5 0 -5 0 2 4

D.Oxygen

6 day

10

12

(f)

algae & zooplankton & Daphnia

algae & zooplankton

algae

Fig 3(d,e&f) Showing the variations in temperature, pH and dissolved oxygen

35

Treatment: algae & zooplanton & Daphnia

250 200 concentration (mg/l) 150 100 50 0 -50 0 2 4 6 day 8 10 12

(g)
350 300 concentration (mg/l) 250 200 150 100 50 0 -50 0 2

Treatment: algae & zooplankton

6 day

10

12

(h)
Treatment: algae

250 concentration (mg/l) 200 150 100 50 0 -50 0 2 4 6 day 8 10 12

(i)
chlorella s cenedes m us ankistrdesm us total green algae

Fig 3(g,h&i) Showing the variation in algal species biomass

36

5.4 Tolerance of fish to various nutrient concentrations.

Wastewater with a 100mg/l nitrogen level was lethal to both species silver perch and rainbowfish. Similarly, the 20mg/l treatment was also not suitable for these fish with mortality occurring within 24 hours. Both juvenile silver perch and rainbowfish died within the first time increment in the 100 mg/l N treatment. Silver perch mortalities appeared to be more prevalent than rainbowfish in the other treatments. There was no mortality in 2mg/l and the control treatments. Details of the experimental results are provided in the figure 4(a&b). The optimum maximum sublethal total Kjeldahl-N concentration for the survival of these fish lies between two and 20 mg/l N. In all treatments the dissolved oxygen levels increased rapidly towards super-saturated toxic levels towards the 7th day. Rainbowfish had a higher survival rate in extreme conditions than silver perch. Although dissolved oxygen supersaturation is present in levels toxic to fish at day seven, it should be recognised that the biosystem is in fact a flow-through system and as such could be manipulated to alleviate this occurrence. The results of the analysis of the parameter estimates are given in the table 18.
Table 18. Analysis of parameter estimates

Parameter INTERCEPT FISH rainbows FISH silver perch TREATMENT 0 TREATMENT 1 TREATMENT 2 TREATMENT 20 TREATMENT 100 TEMPERATURE DT SCALE

DF 1 1 0 1 1 1 1 0 1 1 0

Estimate 4.2061 -0.4107 0.0000 NR NR -2.8233 -0.6783 0.0000 -0.2008 -0.1690 1.0000

Std Err 1.3508 0.3712 0.0000 NR NR 0.6672 0.3942 0.0000 0.0623 0.0313 0.0000

ChiSquare Pr>Chi 9.6958 1.2240 . NR NR 17.9039 2.9608 . 10.3865 29.2401 . 0.0018 0.2686 . NR NR 0.0001 0.0853 . 0.0013 0.0001 .

37

Rainbowfish mortality
5

3 Mortality (No)

-1 3 6 12 18 30 54 78 102

hrs

Silver Perch mortality

5

3 Mortality (No)

-1 3 6 12 18 hrs 0 1 2 20 100 30 54 78 102

Fig 4 (a&b) Showing Rainbowfish and Silver perch mortality.

38

5.5 Impact of duckweed, mussels, Rainbowfish and their combination on nutrient depletion
An experiment was conducted to provide base line information on the influence and suitability of aquatic plants and animals in wastewater treatment. . Mussels are filter feeding animals that mainly bottom feeders. Rainbowfish is a planktivorus species that moves within the water column. Duckweed is a surface-growing aquatic plant. All three orgnisms were used in this experiment to evaluate their suitability for wastewater treatment. The temperature averaged 25.7oC throughout the experiment and varied between 20.75oC to 29.6oC. The pH and oxygen levels remained steady except for a slight increase during day six and day nine. No significant change in salinity and nitrate levels were detected throughout the experiment. The graphs (figure 5.a - i) indicate little effect on the reduction of nutrients between treatments. The duckweed treatment reduced the ammonia nitrogen faster than the other treatments. Mussels did not affect the water quality at this concentration. The ammonia levels steadily decreased up to day nine and then remained steady until the end of the experiments. Phosphorus levels fluctuated in the control and duckweed treatments. However, a gradual decline was noticed in the mussel and duckweed and mussel treatments. The algal biomass decreased except for the control where until day nine when a steady increase in the biomass was recorded. The algal biomass deceased gradually and reached minimum levels (0.94mg/l) towards the end of the experiment. The results of algal studies are presented in fig 5(j i) Statistical analysis using generalised linear model was carried out to determine the goodness fit of the treatments. The results are provided in the table 19
Table 19 Results of the analysis of linear model

Nutrient Nitrate Ammonia S.R. Phosphorous Total Algal Biomass

R-Square 0.228407 0.306264 0.430474 0.491446

C.V. -13.82086 132.3915 -54.53048 309.8303

Root MSE 0.24704 0.52693 1.17454 1.80223

Log Mean -1.78746 0.39801 -2.15391 0.58168

This analysis clearly indicates that the goodness fit is relatively poor except for total algal biomass. The log mean square, F and probability values are given in table-20. The oxygen level remained above 5mg/l except in the control treatment where it averaged 4.8 mg/l. The duckweed treatment effectively depleted ammonia as approximately 70% of the initial content was removed by day nine. However, the data recorded on days 12 and 15 showed that ammonia content gradually increased again. A T test was used to compare the significance of treatment for both nutrients and total algal biomass. The ammonia depletion rate was significantly different between treatments (table-21-24). However, the nitrate level did not differ significantly between treatments. The SR phosphorus content was significantly higher in the duckweed treatment. However, the level of SR phosphorus was similar in mussels and mussels and duckweed treatments. It appears that there may be some relationship with mussels and phosphorus depletion. The control treatment maintained a maximum level of algae (average 23.9mg/l) and the mussel treatment was the least averaging 3.2mg/l.
39

Table 20 Showing mean square, F and probability values of duckweed and mussel experiment.

Log Nitrate Source DF TREAT 3 LDAY 1 DAY 1 Log Ammonia TREAT 3 LDAY 1 DAY 1 Log S.R.Phosphorous TREAT 3 LDAY 1 DAY 1 Log Total Algal Biomass TREAT 3 LDAY 1 DAY 1

Type III SS 0.26385579 0.06014645 0.00285589 2.14594047 2.82065583 1.79455663 28.2329664 18.3623620 20.6744724 60.3410890 3.1083504 16.4458396

Mean Square 0.08795193 0.06014645 0.00285589 0.71531349 2.82065583 1.79455663 9.4109888 18.3623620 20.6744724 20.1136963 3.1083504 16.4458396

F Value 1.44 0.99 0.05 2.58 10.16 6.46 6.82 13.31 14.99 6.19 0.96 5.06

Pr > F 0.2410 0.3253 0.8296 0.0632 0.0024 0.0139 0.0008 0.0007 0.0004 0.0011 0.3324 0.0286

Table -21 Nitrate :Least Square Means

Treat CONTROL DUCKWEED MUSSELS MUSSELS + DUCKWEED

LSMean -1.79623121 -1.86614632 -1.68250395 -1.80495693

Std Err LSMean 0.06378604 0.06378604 0.06378604 0.06378604

Pr > | T | HO:LSMean =0 0.0001 0.0001 0.0001 0.0001

N 15 15 15 15

Bon Grouping A A A A

40

Table-22 Ammonia: Least Square Means

Treat CONTROL DUCKWEED MUSSELS MUSSELS + DUCKWEED

LSMean 0.30859198 0.16927369 0.43032165 0.68384697

Std Err LSMean 0.13605259 0.13605259 0.13605259 0.13605259

Pr > | T | HO:LSM ean = 0 0.0273 0.2188 0.0026 0.0001

N 15 15 15 15

Bon Grouping A B A A

Table-23. S.R. Phosphorous : Least Square Means

Treatment CONTROL DUCKWEED MUSSELS MUSSELS + DUCKWEED

LSMean -2.64345033 -0.81393371 -2.57219164 -2.61887754

Std Err LSMean 0.32631725 0.34234585 0.35750165 0.36542057

Pr > | T | HO:LSMean =0 0.0001 0.0222 0.0001 0.0001

N 15 15 15 15

Bon Grouping A B A A

Table-24. Total Algal Biomass: Least Square Means

Treat CONTROL DUCKWEED MUSSELS MUSSELS + DUCKWEED

LSMean 2.04478057 0.63950239 -0.77894866 0.40994791

Std Err LSMean 0.46538661 0.46538661 0.46538661 0.48219449

Pr > | T | HO:LSMean =0 0.0001 0.1752 0.1001 0.3991

N 15 15 15 15

Bon Grouping A A B A

The fish and mussel mortalities were monitored during this experiment. In general, the mortalities observed were relatively low, with just over one fish out of 10 fish used for the experiment. While maximum mortality was noticed in mussel and the mussel andduckweed treatment (16.7%), the lowest mortality was observed in the control (6% fish). The mortality of mussel was lower than that for fish. The mortality rate of fish in the mussel and mussel andduckweed treatments averaged about 4% and 8% respectively.

41

Temperature

32 30 28
Temp (C)

26 24 22 20 18 0 3 6
day

12

15

(a)

pH 11 10 pH 9 8 7 0 3 6 day control duckweed mussels mussels+duckweed 9 12 15

(b)
Fig 5 (a & b) Showing temperature and pH of various treatments.

42

Salinity 0.25 concentration (0/00 0.2 0.15 0.1 0.05 0 0 3 6 day 9 12 15

(c)
Conductivity
900 Conductivity (uS/cm) 880 860 840 820 800 780 760 740 0 3 6 day 9 12 15

(d)
D.Oxygen 9 concentration (mg/l 8 7 6 5 4 3 2 0 3 6 day 9 12 15

(e)

control

duckweed

mussels

mussels+duckweed

Fig 5 (c, d & e) Showing salinity, conductivity and dissolved oxygen.

43

Nitrates 0.25 concentration (mg/l 0.2 0.15 0.1 0.05 0 0 3 6 day 9 12 15

(f)
Ammonia
4.5 4 concentration (mg/l) 3.5 3 2.5 2 1.5 1 0.5 0 0 3 6 day 9 12 15

(g)
SR Phosphorous 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -0.1 -0.2 0 3 6 day control duckweed mussels mussels+duckweed 9 12 15

(h)
Fig 5 (f, g & h) Showing variation in nitrate, ammonia and SR phosphorus in different treatments 44

concentration (mg/l

Control
60 concentration (mg/l) 50 40 30 20 10 0 -10 3 6 9 day 12 15

(i)
Duckweed
60 concentration (mg/l) 50 40 30 20 10 0 -10 3 6 9 day 12 15

(j)
Mussels
12 10 8 6 4 2 0 -2 3 6 9 day 12 15

concentration (mg/l)

chlorella micractinium

scenedesmus closterium

ankistrdesmus totals

(k)

Fig 5 (i, j & k) Showing variation in different algal species for control, duckweed and mussel treatments.

45

Mussels & duckweed

60 50 concentration (mg/l) 40 30 20 10 0 -10 3 6 9 day 12 15

chlorella micractinium

scenedesmus closterium

ankistrdesmus totals

(l)
Fig 5 (l) Showing variation in different algal species for mussel and duckweed treatment

Total gree algae

70 60 concentration (mg/l) 50 40 30 20 10 0 -10 3 6 9 day 12 15

control

duckweed

mussels

mussels+duckweed

(m)

Fig 5 (m) Showing variation in total algae for various treatments

46

6 Discussion
One of the major objectives of this project is to show how to minimise water use and then treat any wastewater for recycling. The Urrbrae system requires clean water for diluting the wastewater coming out of the digester. It is important to determine the optimum dilution factor to minimise the clean water usage and maximise biological treatment. The first experiment focussed on determining the optimum nitrogen dilution level for maximum nitrogen depletion through biological treatment using algal production. The components of nitrogen such as nitrite, nitrate and ammonia along with total algal biomass were considered. This provided a direct input in the form of baseline information required to optimise the performance of the high rate algal pond that receives the diluted wastewater. It is also important to determine the optimum retention time in the high rate algal pond before wastewater from this discharged into aquaculture ponds downstream. The relationship between various nitrogenous components and their use by the total algal biomass has been the subject of a number of studies. The different forms of nitrogen can be separated into organic and inorganic, as well as particulate and dissolved components. Particulate organic nitrogen is found in living biomass and detritus, while soluble organic nitrogenous materials are released into the water from excretion, secretion, and decomposition processes (Keeney, 1973). Soluble inorganic nitrogen is represented primarily by four different molecules: nitrate (NO3-), nitrite (NO2), ammonia (NH3 /NH4 +), and nitrogen gas (N2 ). The three primary nitrogen molecules for algal growth are ammonia, nitrate, and nitrogen gas (Fogg, 1975; Bold and Wynne, 1978). Ammonia is the preferred form for plant growth because the incorporation of nitrate requires additional metabolic energy and enzymatic activity (Goldman and Horne, 1983). Both algae and bacteria incorporate ammonia very rapidly (Sugiyama and Kawai, 1979). In addition, some algae can metabolise various forms of dissolved organic nitrogen, as well as remove ammonium ions adsorbed to suspended particulate matter (Grobbelaar, 1983). The incorporation of nitrogen gas into algal biomass occurs through a process known as nitrogen fixation. The rate of algal biomass production clearly indicated that the optimum concentration of total nitrogen is between 50-100mg/l in the water. Further dilution studies will more precisely indicate the nutrient level suited to a commercial scale operation. It was clear from the experiment that up to 80% of ammonia and nitrate will be removed within the first eight days of treatment at a temperature ranging between 22 to 29oC. Oxygen levels in the water and the minimum levels of ammonia and nitrate mainly control the nitrite levels. Transformations between different forms of nitrogen in ponds are influenced by pond environmental conditions (Keeney, 1973). For example, ammonia is the principal nitrogenous by-product of organic decomposition and experiences different fates depending on where in the pond the ammonia is produced. In waters containing dissolved oxygen, ammonia not incorporated by algae can be oxidised (i.e., add oxygen) through microbial processes. The oxidation of ammonia first to nitrite and then to nitrate is called nitrification (NH 4 + NO 2 - NO 3 -). In this two-step process, the microbial transformation of ammonia to nitrite is much slower than the subsequent microbial transformation of nitrite to nitrate (Cavari, 1977; Goldman and Horne, 1983). This has two implications; first, algal uptake of ammonia can be relatively fast, and competition for ammonia between algae and bacteria is predominantly in favour of the algal community; second, the relatively rapid oxidation of nitrite to nitrate means that very little nitrite accumulates in toxic waters. Throughout the
47

experiment high levels of dissolved oxygen were maintained as the trial was done in aquarium (microcosm). Nitrite levels were minimum throughout the experiment. Therefore, it was decided to focus on the key compounds such as ammonia, nitrate and soluble phosphorous depletion rate along with algal biomass production.
Table -25 Showing a comparison of algal production by treatment (wastewater %)

Table 26 Comparison on significant difference in total algal production General Linear Models Procedure Alpha= 0.05 Confidence= 0.95 df= 92 MSE= 0.415148 Critical Value of T= 2.87631 Total log algal biomass Comparisons significant at the 0.05 level are indicated by '***'. Treatment Simultaneou Difference Simultaneous Comparison s Lower Between Upper Confidence Means Confidence Limit Limit 50 - 25 -0.2654 0.3065 0.8784 50 - 75 0.7025 1.2744 1.8463 *** 50 - 100 0.9637 1.5357 2.1076 *** 50 - 0 2.0476 2.6741 3.3006 *** 25 - 50 25 - 75 25 - 100 25 - 0 75 - 50 75 - 25 75 - 100 75 - 0 100 - 50 100 - 25 100 - 75 100 - 0 0 - 50 0 - 25 0 - 75 0 - 100 -0.8784 0.3960 0.6572 1.7410 -1.8463 -1.5398 -0.3107 0.7731 -2.1076 -1.8011 -0.8332 0.5119 -3.3006 -2.9941 -2.0262 -1.7649 -0.3065 0.9679 1.2292 2.3676 -1.2744 -0.9679 0.2613 1.3997 -1.5357 -1.2292 -0.2613 1.1384 -2.6741 -2.3676 -1.3997 -1.1384 0.2654 1.5398 1.8011 2.9941 -0.7025 -0.3960 0.8332 2.0262 -0.9637 -0.6572 0.3107 1.7649 -2.0476 -1.7410 -0.7731 -0.5119

*** *** *** *** *** *** *** *** *** *** *** *** ***

The three main types of algae recorded were Chlorella, Scenedesmus and Ankistrodesmus sp. Algal production was rapid up to 12 days after the start of experiments but continued to increase at a slower rate from the 12th to 14th day. Analysis of the algal production rate (table-25) indicated that 25 and 50% wastewater dilution treatments were not significantly different. Similarly, no significant difference in algal production rate was observed between the 75 and 100% dilutions of wasterwater treatments. However, maximum mean value of algal production was recorded for 50% (100mg/l N) wastewater treatment followed by 25% wastewater treatment. The trend in algal production in comparison with nitrogen concentration clearly indicated that further increase (>100mg/l) in nutrient concentration affected the rate of algal production.

48

The factors required for algal growth include: Primary nutrients (C, N, P) as well as hydrogen and oxygen. Minor and micronutrients. Sufficient light energy in the water column. Suitable water temperatures for growth. Algae will continue to grow as long as all of the above requirements are met. The rate of production depends highly on the quantity and quality/suitability of the factors listed above. When one or more of the stated requirements are not available for growth, then algal productivity is said to be limited by that condition. Limitation of algal growth is best described by Liebigs Law of the Minimum, which says that algal productivity will be limited by the element present in least supply relative to algal requirements (OBrien, 1974; Goldman and Horne, 1983). Changes in temperature and light are the two main causes for algal production to vary significantly between seasons. Since the experiment on the seasonal impact on algal production was conducted during winter, the ambient treatments and controlled winter treatment were more or less the same. However, variations were observed in ambient conditions particularly with temperature and light intensity. The winter controlled treatment and ambient conditions produced similar results making the results obtained in this experiment highly relevant for implementation. The nutrient depletion and algal production rate was found to be twice as fast in summer, than in winter because of the greater biological activity This provides important information with regard to wastewater retention time. According to this microcosm experiment, a retention time of six or seven days in summer is similar to that in the winter period of 12-14 days. This provides two important guidelines for wastewater treatment in South Australian conditions: Under Urrbrae wastewater treatment model, a retention period for summer being six or seven days and in winter 12-14 days in algal ponds without the hydraulic paddle wheel system (just aeration or shallow ponds with flow-through system) will reduce 90% of the ammonia level if the total nitrogen concentration is 100mg/l. The retention time can be kept the same for winter and summer provided the dilution of wastewater in winter is increased to twice that in summer. The other option is to decrease the flow rate of wastewater from the algal pond in winter in order to increase the retention time Large-scale culture of microalgae on manure from livestock operations is both a wastewater treatment process and a high-yielding source of protein feed for livestock and aquaculture. A major problem is the control of grazing zooplankton, particularly rotifers and cladocerans (Oswald, 1980). These organisms can severely reduce algal cultures in as little as two days, attaining densities in excess of 105/l and reducing the algal population by as much as 90%. Such grazing in mass cultures presents operational and economic problems for combined wastewater treatment-protein production operations in terms of effluent quality degradation and loss of biomass. A number of methods have been investigated for zooplankton control but with limited success. Early attempts at control through heating, centrifugation or the application of chemicals were ineffective or detrimental to algal growth (Burlew, 1953). Tests of a wide variety of synthetic toxicants have not identified any commercially available compounds that are both economical and free of toxic residues (Tamiya, 1956; Loosanoff et al., 1957; Kiser et al., 1963; Lee, 1979). Reducing dissolved oxygen concentration through high organic loading was found to be ineffective because many zooplankton are able to survive extended periods of anoxia through physiological tolerance (Schluter and Groeneweg, 1981) or by
49

aggregation at the aerobic surface layers (Lee, 1979; Schluter, 1979). The direct physical removal of zooplankton () was investigated by Benemann et al (1978) who found that larger forms (primarily cladocerans) were easily removed at reasonable flow rates using a 350micron m screen. However, smaller screen sizes down to 150 micro m employed for rotifer removal were subject to clogging while still passing some rotifers and most eggs. A more effective control measure is pH adjustment. O'Brien and DeNoyelles (1972) reported that pH values of 10.5 to 11 were fatal to zooplankton in a naturally eutrophic pond. Lee (1979) was able to rid wastewater algal cultures of cladocerans by raising the pH to 9.5. Since mass algal cultures on wastewater medium generally contain significant concentrations of ammonia, the observed toxic effects of pH on zooplankton may be due to either an increased hydroxide ion concentration or increased free ammonia levels accompanying elevated pH. If free ammonia is the controlling variable then the effects of pH alone may not be lethal and explains why algal cultures often exceed a pH of 10 without noticeably affecting zooplankton populations (Benemann et al., 1978; Lincoln and Hall, 1980). Mortality at high pH may also be due to indirect effects, or may indicate the lack of adaptation of a population, as suggested by the work of Schluter and Groeneweg (1981) who found the rotifer Brachionus rubens to be intolerant of pH values exceeding 9.5 even at low ammonia concentrations. The experiments on zooplankton interference clearly were designed to determine the tolerance of zooplanktons at 100mg/l total nitrogen concentration. The naturally occurring zooplankton from pond water and cultured Daphnia were tested to find if they could survive under experimental conditions. The results of treatment one with algae, zooplankton and cultured Daphnia , treatment two with algae and zooplankton, and treatment three algae alone were not significantly different from each other. This is mainly due the mortality of zooplankton during early stages of the experiment. No zooplankton were recorded after negligible amounts were found on the initial day. The pH level rose to 10.8 under experimental conditions particularly after day three of the experiment. The elimination of zooplankton may have been the result of elevated pH levels coupled with high ammonia concentration. According to Lincoln et al 1983 the infestation of rotifers (Brachionus rubens) and cladocerans (Diaphanosoma Brachyurum) in a 0.1 ha high-rate algal pond were eliminated by temporarily raising the un-ionized (free) ammonia concentration to approximately 20 mg/l (as N) by addition of ammonium hydroxide solution. The 24-h LD100 for Brachionus was found to be 17 mg/l free ammonia-N and that for Diaphanosoma just under 20 mg/l. Ammonium hydroxide treatments did not inhibit microalgal populations but rather promoted rapid recovery of algal densities to pre-infestation levels. Manipulation of free ammonia concentrations provides an economical and effective means of zooplankton control in mass algal cultures. This experiment on zooplankton interference clearly indicated that there was no impact on nutrient depletion and algal production at a concentration of 100mg/l total nitrogen in wastewater. The 100mg/l total nitrogen concentration of effluent may be safe from zooplankton interference for biological (microalgal culture) treatment of wastewater under the given Urrbrae conditions. One of the major challenges faced during this research was to find an appropriate fish species for aquaculture. After careful review and commercial consideration, the ornamental fish, Rainbowfish and the more marketable silver perch were tested for their tolerance to higher levels of nutrients. The optimum concentration for algal production was 100mg/l total
50

nitrogen concentration so this was selected as the baseline along with a broad range of other concentrations, (20mg/l and 2mg/l and 0mg/l) to test the tolerance of both fish. The South Australian Research and Development Institutes (SARDI) collaborative studies with Research Institute for Aquaculture (RIA), Vietnam indicated that up to 2mg/l under highland Vietnam condition were optimum for fish production. The 2mg/l N treatment was included to determine how the fish responded in South Australian conditions. From the experiment the following points were apparent: Fish behavior appeared to be normal in 2mg/l N concentration of wastewater There was 50% of fish mortality in both species () during the initial 12 hours of treatment in 20mg/l N concentration. Safer concentration levels in South Australian conditions were approximately 2-20mg/l N Rainbowfish were more tolerant to higher nutrient levels than silver perch. Further studies are required to determine the precise level of nutrient concentration for fish culture under South Australian conditions while treating the wastewater. Optimum nutrient levels required for fish culture may not necessarily be suitable for optimum biological treatment of wastewater. When aquaculture is used as a tool for wastewater treatment the nutrient concentration levels need to be regulated in consideration of factors such as the tolerance level of the species of fish to achieve optimum efficiency in fish production as well as wastewater treatment.

51

Table -26 Comparisons on significant difference in phosphorous levels General Linear Models, Bonferroni (Dunn) T tests for variable: Log Soluble phosphorous Alpha= 0.05 Confidence= 0.95 df= 41 MSE= 1.37954 Critical Value of T= 2.77235 Comparisons significant at the 0.05 level are indicated by '***'. Treatment Simultaneous Difference Simultaneous Comparison Lower Between Upper Confidence Means Confidence Limit Limit DUCKWEED 0.0862 1.4454 2.8047 *** MUSSELS + DUCKWEED DUCKWEED 0.2028 1.5620 2.9212 *** MUSSELS DUCKWEED 0.3351 1.6386 2.9421 *** CONTROL MUSSELS + DUCKWEED DUCKWEED MUSSELS + DUCKWEED MUSSELS MUSSELS + DUCKWEED CONTROL MUSSELS DUCKWEED MUSSELS MUSSELS + DUCKWEED MUSSELS CONTROL CONTROL DUCKWEED CONTROL MUSSELS + DUCKWEED CONTROL MUSSELS -2.8047 -1.4454 -0.0862 ***

-1.2719

0.1165

1.5050

-1.1409

0.1931

1.5271

-2.9212 -1.5050

-1.5620 -0.1165

-0.2028 1.2719

***

-1.2574

0.0766

1.4106

-2.9421 -1.5271

-1.6386 -0.1931

-0.3351 1.1409

***

-1.4106

-0.0766

1.2574

A safer 5mg/l N concentration was selected for trial in the next experiment using rainbowfish, which showed a greater tolerance. A filter feeding bottom dwelling freshwater mussel and duckweed were also included to use the complete water column. Fish was the common factor in all treatments. The treatment included (a) duckweed, (b) mussels and (c) duckweed and mussels. Among the nutrients measured, ammonia and nitrate levels were not significantly different between treatments. However in the case of soluble phosphorus depletion, the duckweed treatment was found to be significantly different from other
52

treatments. Maximum ammonia reduction occurred in the duckweed treatment while soluble phosphorous reduction was greater in all other treatments. Trials using different densities of mussels and duckweed needs together with varying fish densities are required to obtain more precise information. Table- 27 Comparisons of treatments for significant difference in algal biomass General Linear Model:. Alpha= 0.05 Confidence 0.95 df= 53 MSE= 3.248031 Critical Value of T= 2.74091 Bonferroni (Dunn) T tests for variable: Log total algal biomass Comparisons significant at the 0.05 level are indicated by '***'. Treatment Simultaneou Difference Simultaneous Comparison s Lower Between Upper Confidence Means Confidence Limit Limit CONTROL -0.3985 1.4053 3.2090 DUCKWEED CONTROL -0.3529 1.4828 3.3184 MUSSELS + DUCKWEED CONTROL 1.0200 2.8237 4.6275 *** MUSSELS DUCKWEED CONTROL DUCKWEED MUSSELS + DUCKWEED DUCKWEED MUSSELS MUSSELS + DUCKWEED CONTROL MUSSELS + DUCKWEED DUCKWEED MUSSELS + DUCKWEED MUSSELS MUSSELS CONTROL MUSSELS DUCKWEED MUSSELS MUSSELS + DUCKWEED -3.2090 -1.7582 -1.4053 0.0775 0.3985 1.9132

-0.3853

1.4185

3.2222

-3.3184

-1.4828

0.3529

-1.9132

-0.0775

1.7582

-0.4947

1.3409

3.1766

-4.6275 -3.2222 -3.1766

-2.8237 -1.4185 -1.3409

-1.0200 0.3853 0.4947

***

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The mean total algal biomass level was significantly different in the controls as compared with the mussel treatment. The details of the comparison between treatments are provided in table-27. The results in this experiment showed a relationship between nutrient depletion and duckweed, mussel and microalgae. However, further studies are required at mesocosm and pond level to provide appropriate advice on the suitable combination of plant and animal species required in integrated wastewater treatment models.

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7 Conclusion and recommendation

This project provides baseline information for the development of an integrated wastewater treatment model and to provide direct input to the Urrbrae integrated treatment system for optimising its efficiency. The experiments conducted in this project produced the following information: a) b) The optimum total nitrogen level to produce maximum algal biomass is about 100mg/l. However, the nutrient depletion rate was higher at higher concentrations. A retention time of eight days in summer and 14 days in the winter will reduce 7085% of the ammonia and about 50-60% of the soluble phosphorous content from the digested wastewater. However, by diluting the wastewater further during the winter season the retention time in the algal pond could be reduced. The digested wastewater did not contain any total and faecal coliform. The results indicate that properly digested wastewater is safe from pathogens such as coliform. Both silver perch and rainbowfish can be cultured in wastewater. However, rainbowfish are more tolerant to high nutrient levels. The duckweed and mussels along with fish can be used for wastewater treatment. The duckweed was found to be highly efficient in removing ammonia from wastewater.

c) d) e)

A number of recommendations can be provided on the Urrbrae treatment system. These include:

The aerobic digester is expensive and should be replaced with simple anaerobic type that can produce biogass. The present system cannot be recommended to farmers as it is both expensive and complicated. It also needs to be modified in order to enable manual operation if any breakdown occurs in the electronic system. The high rate algal pond with hydraulic paddle wheel can be replaced by two extra duckweed ponds. The duckweed plants will increase the efficiency of ammonia removal and reduce the energy cost. Moreover, the system will be more reliable. The dilution of wastewater coming out of the digester needs to be adjusted to a concentration of about100mg/l nitrogen. This will optimise the efficiency of the algal pond. The flow rates of the algal and duckweed ponds need to be adjusted to retain water for the minimum period required during winter and summer. This will enable proper biological treatment of water. The quality of water going into the fishpond will need to be maintained. Along with fish, aquatic plants and mussels could be used in the fishpond. The aquatic plants and mussels will enhance the nutrient removal in the fishpond. There is a dire need to undertake research to determine the optimum density and combination of organisms to maximise the wastewater treatment. Since most of the results were compiled from microcosm and mesocosm experiments, pond level experiments have to be done in order to fine tune the information provided. The experiments conducted in this project were conducted in still water. It is important to repeat the best treatments in a flow through system to precisely find the treatment levels.

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Recommended Model for Urrbrae Wastewater Treatment Unit

Flow adjustments For retention time Digester 7days

Expected Nutrient depletion

Dilution tank

100mg/l N

Algal pond

10 days

<10mg/l N

Duckweed pond

Fish pond

Fish pond

< 0.1mgN 10 days

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8 Future strategy
This study addressed the optimum concentration for biological treatment of wastewater treatment; optimum water retention time; optimum nutrient concentration for fish culture; suitable species composition and winter summer influence on biological wast water treatment. A wider objective is to establish a commercial scale integrated wastewater treatment system using piggery waste to: Develop water and nutrient reuse technology applicable to commercial live stock industries Optimise bio-energy production performance in terms of cost and efficiency Optimise aquaculture (aquatic plants and animals) production in terms of cost and efficiency Examine the potential for optimal usage for horticulture production in the integrated treatment system Develop a closed farming system for minimising organic pollution Study the quality of aquaculture product and water to monitor pathogens Establish a method of salt removal from wastewater Develop extension documentation for use by industry and farmers.

8.1 Research strategies and methodology

A strategy is required to use the information from these small-scale projects and focus on the livestock industry waste (in particular piggery waste) to establish a best practice commercial scale integrated farming system. The research and development is planned for three years and in three phases. First stage: Setting up infrastructure facility: A demonstration facility at a site such as Roseworthy Campus or similar infrastructure facility that has a commercial pig and poultry unit, dairy and horse enterprise and considerable land available to develop an integrated biosystem incorporating aquaculture, agroforestry, pasture/crop rotations and horticulture is required. The facility will include an anaerobic digester and associated systems to treat piggery waste and feed treated waste into aquaculture ponds linked with horticulture and a salt removal system. Digester biosolids, water quality and pathogens will be analysed. Results from SAR 16 projects will be used to initially set up the infrastructure and biosystem operation. Second stage: Establish commercial scale operation: Research will focus on adoption and improvement of digester performance and to optimise wastewater treatment, aquaculture and horticulture production. This adaptive research will be performed with the support of tank experiments to test both aquaculture and horticulture product for pathogens and to develop depuration methods. Research will also be undertaken to remove salt from wastewater using clays and zeolites and an anion exchanger. Removal of salt will be an option depending on the level of salt in the water, which is an issue in some parts of Australia. Third stage: Establish and demonstrate commercial scale operation:

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This phase will involve demonstrating a commercial operation based on the trials conducted in the second year and refining the techniques and undertake a cost benefit analysis. Workshops involving key stakeholders including industry representatives, farmers, policy persons and staff from teaching and research institutions will be conducted and training and extension materials will be prepared. The establishment of this commercial biosystem will serve as the Australian model for pig and other intensive Industries and give farmers best practice systems for adoption. Information will be disseminated to Australian farmers through networks and publications and the demonstration of the commercial model to encourage adoption. The recommendations of the studies will also be made available to the policy makers in Australia in the renewable energy resource sector.

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