Fish farm effluent as a nutrient source for algae

FUNDING: NRF-TWAS African Renaissance PhD Fellowship Programme (grant no. 99798); NEEDS Assessment Scholarship, Federal University of Technology Owerri One of the challenges of microalgae biotechnology is the cost of growth media nutrients, with microalgae consuming enormous quantities of fertilisers, more than other oil crops. The traditional use of synthetic fertilisers in mass cultivation of microalgae is associated with rising prices of crude oil and competition from traditional agriculture. The fact that fish farm wastewater (FFW) nutrients are released in the form preferred by microalgae (NH3 for nitrogen and PO4 -3 for phosphate), and the ability of microalgae to use nitrogen from different sources, can be exploited by using fish farm effluent rich in nutrients (nitrogen and phosphorus) in the cultivation of cheaper microalgae biomass for production of biodiesel. The cultivation of algae biomass in FFW will also serve as wastewater treatment. We reviewed the benefits and potential of fish effluent in algae cultivation for the production of biodiesel. Microalgae can utilise nutrients in FFW for different applications desirable for the production of biomass, including the accumulation of lipids, and produce a fuel with desirable properties. Also, treating wastewater and reducing demand for fresh water are advantageous. The high lipid content and comparable biodiesel properties of Chlorella sorokiniana and Scenedesmus obliquus make both species viable for FFW cultivation for biodiesel production.


Introduction
The ability of microalgae to adapt in a diverse environment is reflected in the patterns of lipids produced as well as their ability to synthesise various unusual compounds. 1 The kinetics of microalgae growth, lipid productivities, and the amount of biomass vary with the algal strain, culture, and physiological conditions. 2 Some species of microalgae, such as Dunaliella Salina, Chlamydomonas reinhardtii, Chlorella, and Botryococcus braunii can contain more than 60% lipid by dry cell weight. 3 However, microalgal species with high lipid accumulation (50-70% of dry cell weight) generally have a slow growth rate. 4 It is possible to find examples of microalgae that are fast growing and have a high lipid accumulation, e.g. Nannochloropsis oculata and Chlorella vulgaris ( Table 1), and that have been used for biodiesel production. 5 Increasing lipid production is possible through the cessation of cell division under environmental stress conditions. This switches from the synthesis of carbon dioxide (CO 2 ) to lipid production as energy storage and thereby increases the lipid content to 20-50% dry cell weight of mostly triacylglycerol. 2,[6][7][8][9] Environmental stress conditions that can lead to lipid production include: • low nitrogen concentration 10,11 ; • low temperature 12 ; • high light intensity 13 ; and • high ion concentrations 14 .
Microalgae have a higher growth rate when compared to land-based plants [19][20][21] and can be harvested every few days 22 . Microalgae require less land than other oil crops 21,23,24 and can be grown on marginal lands not required for food cultivation 25,26 . Microalgae feedstock has a high lipid production advantage with 15-300 times more oil than plant-based biomass 27,28 (Table 2). Microalgae can make use of nutrients, especially nitrogen and phosphorus, from different sources of waste, including concentrated animal feed operations, industrial and municipal wastewater, and agricultural run-off. 7,29 This offers cost-savings from the purchase of exogenous nutrients such as sodium nitrate and potassium phosphate 30 , reduces the use of fresh water 30,31 and provides the additional bioremediation benefits of wastewater treatment 7,29,32 . The production of microalgae biomass offers real opportunities for solving issues of CO 2 sequestration 32 , and at the same time, generates economic value through the conversion of CO 2 into energy and chemical products 33 , utilising about 1.83 kg of CO 2 for the production of 1 kg of microalgae biomass 18 .

Microalgae growth requirements
The use of wastewater to efficiently grow microalgae is dependent on different variables, including the concentration of essential nutrients (such as nitrogen, phosphorus, organic carbon), temperature, pH of the medium, availability of light, CO 2 , and oxygen. 37

Nutrients
Microalgae require nutrients for growth, particularly carbon (in the form of CO 2 ), nitrogen and phosphorus 18,38 (Table 3). To provide these nutrients, different recipes for algae culture media exist ( Table 4). The concentrations of nitrogen and phosphorus in the algae growth medium are considered fundamental factors affecting algae growth kinetics directly and are closely related to lipid accumulation and nutrient removal. 6 The main mechanism for nutrient removal by microalgae is by uptake into microalgae cells 6,39 , while the rate of nutrient removal is directly affected by the microalgae population growth rate 6 .
The usual carbon source for microalgae photosynthetic culture is CO 2 , supplied either continuously or intermittently, from industrial exhaust gases, atmospheric CO 2 , or chemically fixed CO 2 in the form of soluble carbonates, e.g. NaCO 3 and NaHCO 3 . 30,40 The efficiency at which microalgae cells use carbon through photosynthesis is directly proportional to the microalgal biomass production rate. 41 The pH change in microalgae cultures is predominantly from the consumption of CO 2 , while changes due to degradation of metabolites excreted or from the uptake of other nutrients are minimal. 42 Increasing the concentration of CO 2 can result in higher production of biomass and a decrease in pH which can cause harm to the microalgae physiology. 43 The next most important element required for the nutrition of microalgae is nitrogen. 44 Nitrogen is directly involved with primary metabolism as it constitutes protein and nucleic acids. 40,45 The nitrogen content of the biomass can vary from 1% to above 10% (even within the same species) and depends on the type and availability of the nitrogen source. 46 Microalgae cultivation utilises a higher amount of chemical fertilisers (N-fertiliser), about 8-16 tons N/H, than other oil-bearing terrestrial plants. 47 The use of nutrients from wastewater, especially agricultural sources rich in inorganic pollutants (nitrogen and phosphorus), can be one alternative to traditional chemical fertiliser sources. 47 Ammonia is the preferred form of nitrogen for micro-organisms. 46 Nutrients released from aquaculture are most suitable for the cultivation of algae as nitrogen is released as NH 3 and phosphorus as PO 4 -3 . 48 On the other hand, microalgae species with a fast growth rate prefer the primary source of nitrogen in the form of ammonia over nitrate 49 , although they can grow well with different sources of nitrogen 46,47,50 .
Assimilation of either NH 4 + or NO 3 is related to the pH of the growth medium. The pH of the growth medium could drop during active algal growth when ammonia is used as the only nitrogen source. This is due to the release of H + ions. On the other hand, pH increases when nitrate is used as the only nitrogen source in the growth medium. At high pH, nitrate could be lost due to volatilisation. However, it is important to ensure an adequate supply of this important nutrient to achieve a high growth rate. Culture media are formulated to supply nutrients in excess to avoid nutrients becoming a limiting factor, except in specific applications. 46 Another important nutrient for microalgae growth is phosphorus, even though it forms less than 1% by mass. 46 According to Kumar et al. 40 , phosphorus is the third most important nutrient for microalgae growth and is required in significantly excess supply because not all compounds of phosphorus are bioavailable, especially those combined with metal ions 18,46,51 . Microalgae store excess phosphorus in phosphate bodies, which they can use when phosphorus becomes limiting. The ratio of N:P in the growth medium is important, both in determining the growth potential and maintaining the dominance of cultured species in the culture. 46 In Mostert and Grobbelaar's 52 study, nitrogen was supplied at concentrations between 25 mg/L and 5000 mg/L for Scenedesmus sp., Chlorella sp. and Monoraphidium, with a suggested optimal nitrogen concentration for maximum productivity of between 2 mg/L and 619 mg/L and a variation on phosphorus of between 0.98 mg/L and 179 mg/L. Studies with Chlorella vulgaris at different ammonia concentrations obtained algae growth at all concentrations of algae. Low algae growth was obtained at very high ammonia concentrations (above 750 mg/L) and very low ammonia concentrations (below 10 mg/L) while maximum cell density was obtained at nitrogen concentrations between 20 mg/L and 250 mg/L, with no difference in specific growth rates. The growth rate in the different ammonia media studied was comparable to growth in commercial Bristol medium in which nitrate was the nitrogen source. 53 Adapted from Grobbelaar 46

Light
Light is an important requirement in microalgae growth, and should be delivered optimally to all microalgae cells within the culture. The highest photosynthetic efficiencies are realised at low light, as high light intensities not only cause inefficient use of absorbed light energy but also cause biochemical damage to photosynthetic machinery (photoinhibition), as well as a reduction in dry weight. 3 Generally, the light intensity requirement of microalgae cultivation is lower than the light intensity needed for higher plants. 40 Microalgae photosynthesis and productivity is equal to the efficiency of light conversion when the only limiting factor is light. 55,56 Generally, specific growth increases with an increase in irradiance to a maximum point beyond which inhibition may occur due to any further increase. 55

Temperature
One of the major factors controlling cellular, physiological, and morphological responses of microalgae is temperature. Generally, an increase in temperature increases the rate of metabolism, while a decrease in temperature decreases the growth of algae. Environmental parameters such as light intensity affect optimal temperature, with 20-25 °C reported as optimal for some species, and highest cell density occurring at 23 °C. 40

Fish farm wastewater
Globally, aquaculture has been one of the food production sectors with rapid development and production growth, significant investment, and technical innovation. 57 The main pollutants of concern in fish farm wastewater (FFW) are particulate and dissolved nutrients (nitrogen and phosphorus), and specific inorganic and organic compounds. 58 The volume of waste discharged from aquaculture depends on the feeding regime, stocking density, and feeding rate, as these three factors determine the quantity of feed used. 59

Nitrogen
Transformations of nitrogen are key biochemical processes in aquaculture systems, with protein as the major form of nitrogen in the fish feed. 45 In every ton of fish produced, approximately 132.5 kg nitrogen and 25.0 kg phosphorus are released to the environment. 59 Fish feed consumed is converted partially into fish biomass, egested as faeces or excreted through the gills as un-ionised ammonia, a major product of protein metabolism. 45,60,61 Most ammonia produced in fish occurs in the liver and is voided through the epithelial surface and renal routes. Production of ammonia also occurs in the kidney, intestine, and muscle due to the presence of the amino acid deamination enzyme in the tissues. 62 Ammonia in fresh water is from excretion via passive NH 3 diffusion across the branchial epithelium. Next to the gill, this NH 3 subsequently gets trapped as NH 4 + in an acidic boundary layer, which maintains the partial pressure gradient of blood-to-gill water NH 3 . 63 Urea is produced through argininolysis or hepatic uricolysis and is excreted through the gills, kidneys, skin or faeces.
Nitrogen loading in fish farms can be generally grouped into three sources 59,61 : 1. feed wasted due to poor management and farm practice; 2. poor feed quality, leading to poor feed stability and rapid dissolution of fish feed in water; and Large amounts of nitrogen in FFW are dissolved, with only 7-30% occurring in the form of particulates. 64 Nitrogen in aquaculture is predominantly excreted as ammonia 65 and only about 20-40% of total nitrogen is excreted as urea 62 . Ammonia nitrogen build-up is the second most limiting factor to an increased level of production in intensive aquaculture after dissolved oxygen. 66 Even at very low concentrations, ammonia -especially un-ionised (NH 3 ) ammonia -is toxic to fish, with maximum concentrations below 0.0125 mg/L seen as acceptable. 67 In flow-through aquaculture systems, most of the total nitrogen in the system is produced as ammonia while recirculating systems with biofilters produce mostly nitrates. 68 As the most reactive nitrogenous species, the pelagic microbial community quickly take ups ammonia and produces other nitrogenous species such as nitrate. 69 The rate of ammonia reaction in water is rapid, having a half-life of fewer than 50 ms for interconversion of NH 4 + to NH 3 . 70 However, temperature, pH, and salinity of the water affect the relative proportion of the two forms of ammonia. 70 In natural water, ammonia exists as a component of pH and temperature-dependent equilibrium. Aqueous ammonia, an ionised form of ammonium (NH 4 + ), is favoured within equilibrium pH (6.5 to 8.0), while a high pH >9 favours un-ionised form of ammonia (NH 3 ). 71

Phosphorus
Phosphorus is a limiting nutrient in a freshwater ecosystem 72,73 and is excreted through urine in fishes 74 . Excretion of phosphorus, usually 60-86% of dietary phosphorus, is related to the source of origin, which different species use in different ways. 65 Water quality can be influenced by phosphorus from aquaculture 75 , as elevated levels of phosphorus cause premature eutrophication 73 . Soluble phosphorus is not produced when feed with low phosphorus levels is consumed. 76 The particulate total phosphorus and particulate total nitrogen fractions of effluent from a salmonid farm range from 30% to 84% and 7% to 32%, respectively. 64,77,78 Phosphorus is usually not lost in an aquatic environment but remains conserved in a series of fractions as a result of dissolution, adsorption, and precipitation. 79 This changes the form of phosphorus availability from dissolved orthophosphates to phosphorus attached to the suspended load. 80 This makes phosphorus a useful indicator of the environmental impact of fish effluent. 81 Modern agriculture relies on non-renewable phosphate from rocks for phosphorus supply, which is estimated to run out in 50-100 years with the estimated increase in phosphorus use. 82 This makes it essential to recycle phosphorus in wastewater sources, manure, and even within production processes of biofuels to eliminate direct competition for phosphorus between algae cultivation and conventional agriculture. 83

Impact of aquaculture discharge to the environment
Aquaculture's impact on the environment depends on feed type, stocking density, species, culture method, and farm practices. 84 The concentration or total amount of effluents released and the capacity of the environment to assimilate the particular constituent also affects the impact of aquaculture on the environment. 58 Nitrogen and phosphorus as major constituents of fish loading can affect the environment as a whole as well as the rearing of the fish. 65 The introduction of organic and inorganic materials through feed for fishes has significantly impacted the nutrient and organic matter loading in coastal waters.
Rapidly growing intensive aquaculture systems would lead to various adverse effects on the environment. These effects might include: • increased release of nutrients, which leads to eutrophication of coastal waters [85][86][87][88] ; • shortage of drinking water resources as a result of release of toxic chemicals, including ammonia (NH 3 ) and nitrite (NO 2 ) from aquaculture, especially in intensive systems of fish culture 89 ; • reduction of wild-fish supplies which can affect the ecosystem through large input of wild-fish feed used in feeding carnivorous species, and also habitat modification for some aquaculture systems 90 ; • competition for land and disturbance of wild ecosystems from escaped farmed fish 91 ; • pollution from drug residues used in the prevention and treatment of diseases in aquaculture can lead to a change in biodiversity 92 ; and • environmental concerns from the use of chemicals (including antifoulants, vitamins) and the introduction of new genetic strains and pathogens. Cleaning of fouled cages can also add to the organic loading of the water. 84 Microbial nitrification and denitrification are reactions common in aquaculture systems, which lead to the release of nitrous oxide (N 2 O), a major greenhouse gas with 310 times more global warming potential than CO 2 over a lifespan of 100 years (Figure 1). Nitrous oxide destroys the ozone and has a lifespan of 114 years. It is estimated that aquaculture N 2 O emissions will contribute roughly 5.

Benefits of utilising microalgae in FFW nutrient recovery
The use of algae for nutrient removal, especially nitrogen and phosphorus, has been demonstrated and has numerous advantages. These advantages include: • low operating costs 30,54,89 by saving money for the purchase of exogenous nutrients such as potassium phosphorus and sodium nitrate; • saving of freshwater resources 89 ; • a suitable growth material with high tolerance 89 ; • pollutant conversion and effluent conversion to clean water 93 ; • extra income when economic important species are used 87 ; • increased productivity by eliminating pollutant nutrients 84 ; and • recycling nitrogen and phosphorus trapped in algae biomass as fertiliser avoids problems of sludge handling and oxygenated effluent discharge into the receiving water body. 54 The use of algae for nutrient removal is not environmentally dangerous as it follows the principles of the natural ecosystem and also does not lead to secondary pollutants as long as the biomass produced is reused. 94 Furthermore, the process is attractive for the treatment of secondary sludge as it has no carbon requirement for nitrogen and phosphorus removal. 54 Moreover, the use of wastewater from agricultural, industrial, and municipal activities can provide a sustainable and cost-effective means of cultivating algae for biofuels. 37 An alternative to synthetic fertiliser and eliminating the traditional use of synthetic fertilisers in the mass cultivation of algae is beneficial because of the rising prices of crude oil. 95 The use of residual nutrient and nutrient recycling can overcome the high cost of algae biomass production -a major drawback in algae biotechnology for biodiesel production. 96 Cultivation of microalgae also benefits the fish farmer by savings associated with the treatment of aquaculture wastewater before discharge, reducing demand for fresh water, and supplying algae biomass fish feed for the cultivation of fish. 97

Microalgae cultivation in FFW
Recently, studies using FFW have been carried out for different purposes. [97][98][99][100][101][102][103][104][105][106] Most of the studies 98-100,102,105 focused on the growth rate of algae in aquaculture wastewater, the rate of nutrient removal, the effect of aquaculture wastewater on algae composition, enhancing microalgae harvesting through bioflocculation by co-cultivation of microalgae with fungus and feed production. A few studies 97,101,103,104 determined the lipid content of the microalgae grown in FFW while fewer studies 97 went further to determine the fatty acid composition of the lipid accumulated. Enwereuzoh et al. 106 determined the quality of biodiesel from the FAME obtained from microalgae cultivated in FFW. However, most of the studies reviewed characterised the FFW used, determined biomass yield, and nutrient removal.
The characteristics of the FFW (Table 5) specific growth rate, biomass yield, biomass productivity, and lipid content ( Table 6) are provided. All the studies utilising FFW for microalgae cultivation agree that FFW has sufficient nutrients to support microalgae cultivation. The concentration of nutrients in FFW were 0.48-433 mg/L for ammonia, 0.13-157 mg/L for nitrate, 0.14-28 mg/L for nitrite and 0.42-16.9 mg/L for phosphorus. These ranges are lower than concentrations obtained in standard growth media. For instance, the higher range of 157 mg/L obtained in FFW is only about 10% of the concentration of nitrate in both BG11 and Modified Allen's media and 62.8% in Bold's Basal standard media ( Table 3). The lower biomass yield and productivity obtained in FFW when compared to the yield obtained in standard growth media have been attributed to the lower concentrations of nutrients in FFW. 100 In this review, the highest biomass yield of 2.96 g/L and biomass productivity of 160.96 mg/L/d were obtained in Ankistrodesmus falcatus -cultivated in FFW with 5.32 mg/L ammonia, 40.67 mg/L nitrate and 8.82 mg/L phosphorus -are lower than the biomass yield and productivity obtained in the same species cultivated in standard growth media. 104 Biomass yield and productivity in the same study increased with increased supplementation of nutrients. These findings also confirm that nutrients in FFW support the growth of microalgae but are not sufficient for comparable biomass yield and productivity obtained with standard growth media.

Conclusion
With an increasing world population and increased dependence on aquaculture for fish supplies, fish farm effluents are expected to grow. These effluents could provide nutrients for microalgae cultivation.
Several studies have shown that the cultivation of microalgae in aquaculture wastewater is suitable for microalgae growth and biomass productivity coupled with efficient nutrient removal. The replacement of inorganic fertilisers with nutrient-rich fish farm effluent would eliminate the cost of purchasing fertiliser. This should lead to cheaper cultivation of microalgae biomass production for biodiesel production. When high costs -one of the major setbacks of algae biotechnology -are eliminated, the potential of microalgae biodiesel will be enhanced. Fish farm effluent nutrient recycling for microalgae cultivation for biodiesel production will at the same time eliminate numerous negative environmental effects associated with nutrient-rich effluent discharge to the environment, while also reducing the volume of water used.