Recycling of Nutrients from Trash Fish Wastewater for Microalgae Production As Health

Recycling of Nutrients from Trash Fish Wastewater for Microalgae Production as Health and Pharmaceutical Products and Renewable Energy

Abstract

Trash fish feeding of cage fish can result in marine pollution. Whole and chopped trash fish can leach pollutants such as ammonia, phosphate and protein into surrounding waters. Reduction of pollution can be achieved by recycling the wastewater generated from trash fish feeding for cultivation of microalgae. Apart from larviculture, microalgae are potent candidates for the production of health and pharmaceutical products. Two microalage, Chlorella saccharophila and

Nannochloropsis sp. have the potential to produce high amounts of polyunsaturated fatty acids

called ω-3. 31.8 mg of EPA (Eicosapentaenoic acid, C20:5n-3) can be obtained from 1 g dry weight of Nannochloropsis sp. Furthermore, high oil content ranging from 10.7 to 13.6% for

Chlorella saccharophila and 9.3% for Nannochloropsis sp. TFA (Total fatty acids/cell dry weight) can be extracted from them respectively. An alternative biofuel derived from microalgae is feasible due to the fact that there is no competition for terrestrial occupation.

Introduction

Trash fish are low-priced fish that are typically used as feed for cultured fish, ducks, dogs and cats. Types of trash fish include hair-tail, gizzard-shad, scad, golden sardine, mackerel, grunt and other species. Usually it is a mixture of two or more species. Most marine fish farmers use trash fish to feed their cage fish. When a bucket of fresh trash fish stands for a certain period, some red leachate is observed on the top. The leachate is regarded as wastewater. Thus, most fish farmers will wash the trash fish with water before fish feeding and then discard both the leachate and the wastewater. Alternatively, fresh trash fish may not be supplied fresh daily and would be kept in refrigeration. Frozen trash fish will be defrosted in water before feeding. As a result, wastewater will also be generated from the defrosting process. Adult cage fish are usually fed a whole trash fish while juvenile fish would be fed trash fish that are first chopped into smaller pieces. We refer to the wastewater generated from all the aforesaid procedures as trash fish wastewater.

The fact that fish farming causes water pollution has been well documented (Olsen et al. 2008;

Wu 1995; Foy and Rosell 1991; Lam 1990), but the details of sources of pollution were not fully identified and no recycling alternative for the pollutants was proposed. Working over 30 years in aquatic environment and wastewater treatment in the market revealed that most of fish farmers have given up their business in Hong Kong. As a matter of fact, the marine environment has become more polluted than before despite a reduction on fish harvested by fishing vessels. Now we consume the aforesaid trash fish due to the fact we have to suffer from this man-made pollution. Untreated industrial effluents, petroleum spills and other leakage containing heavy metals (Gillan et al. 2005; Khansari et al. 2005) and toxicants (Aas et al. 2000; Payne and

Penrose 1975) are the main sources of pollution for acute and chronic aquatic poisoning. Some studies (Arup 1989; Wu and Lee 1989) have recommended pellet feed to replace trash fish for fish farming as nutrient leakage is less with pellet feed. Aside from the disadvantages of applying pellet feed to substitute trash fish, marine pollution continues to be a problem even though fish farmers have been utilizing pellet feed for many years. It is worth noting that trash fish are collected from the ocean while pellet feed is formulated from terrestrial plants. Feeding fish pellet feed is the equivalent of dumping pollutants from the terrestrial environment into the marine ecosystem. Trash fish, however, are a part of the marine ecosystem, their consumption by cage fish ensures the food supply-chain remains within the ocean environment. Accordingly, the nutrient balance remains the same provided that the cage fish are not harvested for human consumption. The main point is that a fish culture zone should be established where there is a high rate of water exchange area but not in a bay. Then, the nutrient balance can be maintained.

This explains why cage cultures have been run in open waters in various countries (Maldonado et al. 2005; Feng et al. 2004).

Several studies (Arup 1989; Yoonaisil and Hertrampf 2006; Hata et al. 1988; Hamada and

Kumagai 1988) pointed out that trash fish is rich in nutrients (Illustration 1-Table 1). This raises the question of whether there is an application for trash fish wastewater? It may be applied as an organic fertilizer to grow algae, vegetables, horticultural products and for other plantation means.

However, we are interested in aquatic applications and so, this study will concentrate on this field. The study was designed qualitatively and quantitatively to investigate the kinds of nutrients and the amounts leached out when trash fish was soaked in water (similar to fish feeding). Did the amounts of nutrients increase when the trash fish was soaked for a longer period? Did the same amount of nutrients leach out from the trash fish for both whole and chopped forms?

About three-quarters of the earth’s surface is covered by ocean; the mass culture of algae taken in the sea pose no resource competition as the counterpart – for terrestrial occupation. They are not only potent candidates to recycle pollutants generated by man, but they also produce valuable products for our consumption and other applications. Apart from the traditional utilization of microalage for larviculture (i.e. for rotifer, copepod and artemia), they have also been found in health, pharmaceutical and biofuel applications.

WHO (1998) has published an issue to emphasize the importance of natural carotenoids to our health. Similarly, the essence of chlorophyll and carotenoids of algae has recently been found for health and pharmaceutical applications. Microalgae appeared in different colours such as green, red, brown, yellow, blue and so on only when they grow to a high cell density. Natural pigments like β-Carotene (Van Rooyen et al. 2008) and chlorophyll (Sarkar et al. 1994) keep animals in healthy condition while accessory pigments such as zeaxanthin, lutein, astaxanthin, etc. are effective for natural colour development in animals like fish, bird and cattle (Borowitzka 1988;

Cohen 1986). Recently, accessory pigments were identified as natural antioxidants (Rodriguez-

Garcia and Guil-Guerrero 2008) and act as free radicals scavengers (Wang et al. 2010) to protect our body cells from ‘bleaching’. Nishino et al. (2008) reported that many carotenoids extracted from microalage have been demonstrated to be effective for the inhibition of various human cancer cells for the control of carcinogenesis such as: α-carotene for liver and colon; lutein for lung and skin; zeaxanthin for skin, lung and liver; fucoxanthin for skin, duodenum, colon and the liver. Phycobiliproteins such as phycoerythrin from red algae and phycocyanin from blue-green algae have been extracted for the applications of medical diagnosis (Phycofluors) (Glazer and

Stryer 1984).

Furthermore, algal polysaccharide produced by Phaeodactylum tricornutum and Chlorella stigmatophora has been identified to be effective for anti-inflammatory and immunomodulatory activities (Guzman et al. 2003). Sulphated exopolysaccharide of a brown alga called fucoidan has been produced by various companies (Kamerycah 2010; Seaherb 2010) in the market as various health and pharmaceutical products to treat different types of cancers (Maruyama et al.

2006). Spirulina and Chlorella health products have been on the market for many years.

Two genus microalgae, namely Chlorella and Nannochloropsis, were used to recycle trash fish wastewater for this study. A brief introduction is provided here. Green microalgae (Chlorophyte) including Chlorella and others are rich in long chain polyunsaturated fatty acids (ω-3/6), pigments like chlorophyll and carotenoids such as carotene and lutein (and violaxanthin, antheraxanthin, zeaxanthin and others) are also present. It also produces multi-vitamins, 18 amino acids and contains several minerals (Yaeyama 2010). The products were used as dietary, health and pharmaceutical supplement and were found nontoxic for consumption (Day 2009). Chlorella extract has been identified to have antioxidant properties (Rodriguez-Garcia and Guil-

Guerrero 2008; Wang et al. 2010) and could aid in the suppression of cancer cell (Rodriguez-

Garcia and Guil-Guerrero 2008; Tanaka et al. 2001; Hasegawa et al. 2002) as well as the stimulation of antibodies (Kralovec et al. 2007). Lutein of Chlorella was reported to have antiglycative activity in diabetic complications (Sun et al. 2010).

Yellow-green micro-algae (Eustigmatophyte) include Nannochloropsis, Monodus and others are

rich in EPA while AA (Arachidonic acid, C20:4n-6) and GLA (γ-Linolenic acid, C18:3n-6) are also present (Nichols and Appleby 1969). Nannochloropsis produces multi-vitamins (Brown et al.

1999; Durmaz 2007); several minerals and most essential amino acids (Rebolloso-Fuentes et al.

2001). Its total lipids accumulation is about 18.4% of dry weight (Rebolloso-Fuentes et al. 2001).

Salvesen et al. (2000) identified that the antibacterial substances were abundant in slow-growing cultures of N. oculata and others. In addition to chlorophyll, violaxanthin and vaucheriaxanthin are the two major pigments for this group (Gladu et al. 1995; Gentile and Blanch 2001).

Violaxanthin, antheraxanthin and zeaxanthin are the major pigments of C.ellipsoidea, which can have an antiproliferative effects on human colon cancer (Cha et al. 2008). There was no toxicity found for consumption of Nannochloropsis biomass (Andrés et al. 1992).

In addition to the aforesaid useful products, microalgae also contain a valuable amount of oil that can be extracted as polyunsaturated oils and biofuels. Recently, apart from health products, ω-3 polyunsaturated fatty acids have been tested in many pharmaceutical applications, especially for EPA. For the purpose of this study, an evaluation of the fatty acids profile for the two microalgae cultivated from the wastewater of trash fish has been undertaken.

Biofuel from microalgae is another important benefit because it is sustainable and renewable, unlike the fossil-based fuels. Use of microalgae provides us with one more solution for producing clean energy. The oil content of each microalga depends on its culture conditions and the type of species (Demirbas and Demirbas 2010; Lv et al. 2010). Microalga like Chlorella species is studied mostly for its application as a biodiesel. The heating value may approach that of the terrestrial plant seed (Illustration 2-Table 2). Thus, it is a potent candidate for providing a third generation biofuel alternative in the future. Since trash fish is harvested from the sea, we prefer the input effort to reduce pollution arisen from trash fish feeding to the introduction of terrestrial pellet feed into the ocean. We believe that this pollution may be reduced by recycling it into useful products that we have mentioned in the preceding paragraphs or even other alternatives. Accordingly, the recycling of trash fish wastewater for microalgal culture has been attempted here.

So the main objectives of this study were as follows: first, to introduce various valuable products produced by microalgae; second, to characterize the nutrient profile of trash fish wastewater; third, to compare pollution levels from whole and chopped form of trash fish; fourth, to test if trash fish wastewater can support algal growth; fifth, to evaluate the fatty acid profile and its amount for the two microalgae; and sixth, to demonstrate the recycling of pollutants into health and pharmaceutical products and a renewable energy based on oil content respectively.

Methods

Overview of the study

This experiment was designed to evaluate the impact on pollution from nutrients leached from both whole and chopped form as practised by fish farmers. The nutrients analysed were ammonia, nitrate, nitrite, orthophosphate and protein; each analysis was done according to individual standard chemical methods. The reasons why these particular nutrients were selected for analysis are as follows: (a) unionized ammonia molecules are toxic to aquatic animals (Merck

1987; Phillips 1985; Trussell 1972); (b) ammonium is selectively chosen as a nitrogen source by

Chlorella (Schuler et al. 1952; Syrett and Fowden 1952) and other microalgae (Krom et al. 1989) for assimilation; (c) nitrite is toxic to aquatic animals (Merck 1987; Chen and Chin 1988;

Almendras 1987); (d) phytoplankton needs soluble phosphate for growth and the reactive phosphate is an approximate measure of it (Phillips 1985); and (e) researchers (Yoonaisil and

Hertrampf 2006; Hamada and Kumagai 1988; Foscarini 1988) have shown that trash fish are rich in nitrogen, phosphate, proteins as well as other nutrients so that they may support algal growth.

Thus, it is worth evaluating the potential of recycling organic pollutants into health, pharmaceutical products and biofuel.

Trash fish wastewater analysis and the amount of nutrients in whole versus chopped trash fish Polyethylene pails with size of 0.28 m in diameter, 0.5 m height and total volume of 30 L were used to compare the leaching rate of nutrients from both whole and chopped trash fish. A tap was installed near the bottom of each pail to collect the wastewaters at different time intervals. The experiment was designed to be similar to the real situation when fish farmers feed their cage fish.

For routine feeding a large amount of trash fish is dumped into the cages at one time; since the fish cannot eat them all at once, some trash fish settles at the bottom of the cage or passes through the net. During this settling, a certain amount of nutrients will leach out, from the trash fish. So it was used to estimate the amount of nutrients which leached out from trash fish over different time intervals to see if it was time related. The same amount (1200 g) of trash fish (both whole and chopped) was dropped and soaked in the same volume of seawater (30 L/pail) for different time intervals. Samples (three replicates for both treatments) were collected after 5 min and then at 15 min intervals for 1 h to determine whether nutrient leaching varied with time. It was also designed to estimate the degree of pollution resulting from the two methods and to see if they were significantly different. The two methods were checked using the t test if the calculated value was different from the tabulated value at the 95 % confidence level (Zar 1984).

Analysis of nutrients from the trash fish wastewater

Overview of the analysis procedures

The samples were analysed on the same day; but if same day analysis was not possible, samples were stored in deep freeze and analysed within a few days. Samples were centrifuged at 7000 rpm for 15 min and the composition of the supernatant was analysed. Ammonia, nitrite, nitrate, phosphate and protein were tested for using a Philips Pye Unicam, PU 8600 UV/VIS spectrophotometer and a 1 cm cell. All standards and samples were analysed in three replicates against each individual reagent blank. A reagent blank was composed of deionized water and the reagents required for each nutrient. Each time a set of standards must be prepared as close as to the concentration of the samples as possible.

Total ammonia

The total ammonia includes the toxic unionized ammonia molecules and the ammonium ions.

The following method was based on Strickland and Parsons (1972) and Adams (1991). Ammonia reacts with phenol in the presence of the oxidizing agent sodium hypochlorite in alkaline solution to give indophenol blue. After adding all the reagents to samples and standards, the tubes are covered with paraffin film, keep out of direct light and allowed to develop in a dark place. The absorbance is then read from a spectrophotometer at 640 nm against a reagent blank after 2 h and the colour is good for 24 h. This method is suitable for both seawater and freshwater samples.

Nitrite

Nitrite reacts with sulphanilamide in acid solution (~ pH 2.0 ) to form a diazonium compound which then reacts with N-(1-naphthyl)-ethylenediamine dihydrochloride (NED) to form a reddish purple azo compound. The following method is based on Phillips (1985), is not affected by salinity and is suitable for both freshwater and seawater. If the concentration of the sample is too high, dilution is required, otherwise the reddish purple will turn into yellow. After adding all the reagents to the samples and standards, they are left for 10 min and then the absorbance read from a spectrophotometer against a reagent blank at 540 nm. The reddish purple colour is stable for 2 h.

Nitrate

A hydrazine-reduction method suggested by Bower and Hansen (1980) for the determination of nitrate in seawater was introduced here. Nitrate was reduced to nitrite in 2 h by hydrazine sulphate in the presence of copper catalyst and buffered with cyclohexyl-aminopropane sulfonic acid and sodium hydroxide (pH~10) at room temperature. After 2 h reduction, the excess hydrazine was destroyed by acetone to stop the reduction. When the nitrate has been reduced to nitrite this was determined as in the method described above . If seawater samples were measured, at least a 50 times dilution with deionized water must be done. The absorbance is read from a spectrophotometer against a reagent blank at 540 nm.

Reactive (Ortho) phosphate

Phosphates that respond to colour colorimetric tests without preliminary hydrolysis or digestion by oxidation of samples are termed reactive phosphorus. It is an approximate measure of soluble phosphorus available for phytoplankton growth. The following method was based on Adams

(1991).

Phosphate reacts with molybdate to form molybdo-phosphoric acid in acid solution which can be reduced to the intensely coloured molybdenum blue complex. After adding all the reagents to the standards and samples, 20 min (<2 h) should be allowed to develop the blue colour and then the absorbance is read at 880 nm against a reagent blank.

Protein

Since proteins vary greatly in their amino acid compositions and hence exhibit different properties, so different assay methods have been introduced. Lowry et al. (1951) introduced the most common techniques although the method was modified later by Peterson (1979).

Phosphomolybdic-tungstic (a mixed acid) is the active constituent in Folin-Ciocalteu's phenol reagent. Copper ions in alkaline solution facilitate electron transfer to the amino acids (tyrosine and tryptophan), thus resulting in a reduction of the mixed acid by loss of oxygen atoms from the tungstate/molybdate. As a result, several reduced species are produced, giving a blue colour. The following method was based on Harrison and Thomas (1988) which was modified from Lowry.

Standard solutions using albumin are prepared for each measurement. After adding all the reagents to the standards and samples, they are left for 30 min to allow both colour development and the precipitate to settle down. The absorbance is read from a spectrophotometer at 750 nm against a reagent blank.

Counting of microalgal cells

We have stock cultures of Chlorella saccharophila and Nannochloropsis sp. Their cells were counted using hemocytometers (namely Neubauer Improved) as suggested by Schoen (1988).

Application of trash fish wastewater for microalgal cultures

The wastewater was collected from 5 kg trash fish soaked in 2 L of seawater for 20 min. Four sets of culture flasks of equal volume (2 L each) were set up, two for C. saccarophila called cultures (a) and (b); one for Nannochloropsis sp.; and the other as a control. There were totally eight 2-L culture flasks. Each set of the culture flasks was conducted in duplicate. All samples from each of the duplicates were analysed in triplicates and their means were used to plot figures and draw tables. Two sets of culture flasks, namely (a) and (b) with different salinity were tested for C. saccarophila. All air ducts, glass tubings, flasks, culture media and nutrients were autoclaved for 20 min at 126 °C under 103 kPa pressure. 100 mL of the autoclaved trash fish wastewater were added to each flask except the control. 2 mL of pure culture of C. saccarophila

(~ 1x102 cells/mL) were inoculated into cultures (a) and (b) and the control. Similarly, 2 mL of pure culture of Nannochloropsis sp (~ 1x102 cells/mL) were inoculated into the culture flask for

Nannochloropsis sp. They were cultured under the same ambient temperature near window subjected to about 12 h: 12 h (day: night) sunlight. Air was supplied via a central air-blower. The growth of the microalage was studied by counting the number of cells using a hemocytometer for each week. Ambient temperature was read by a Bibby digital pen thermometer. pH value of the cultures was measured by Cole-Parmer pH meter, model 5985 with two-buffer-calibration (i.e. pH 4 and 7). The salinity of the cultures was checked by a temperature compensated American

Optical (AO) hand-held refractometer.

Analysis of fatty acid profile from the microalgal cultures

When the two cultures (a) and (b) of C. saccarophila turned deep green and the flask of

Nannochloropsis sp. turned to deep yellow-green (~ 50-60 d), they were harvested and centrifuged to have freeze dry cells for extraction. Cell dry weight content was estimated by drying the cells at 80 ℃ in a vacuum oven until a constant weight was obtained. Dry biomass

(~100 g) of each alga was added to a mixture of 500 mL methanol and 25 mL acetyl chloride and

500 mL methanol. The slurry was put into a pressure vessel and held in ultrasonic bath for 15 min. The pressure vessel was warmed in a boiling bath for 30 min at a maximum value of 3.5 atm. Then, the vessel was cooled to an ambient level in a water bath. The vessel was washed with 500 mL hexane and added to the biomass slurry for filtration with a Buchner funnel. The liquid phase was separated after 15 min. The top hexane layer was obtained and concentrated with rotary evaporator under nitrogen gas. The concentrated extract (ester) was applied to an HP

6890 capillary gas chromatographer (Hewlett-Packard, Palo Alto, CA) equipped with a FID

(Flame–ionization detector) and a Supelco (Bellefonte, PA) Omegawa 250 capillary column

(30x0.25 mm). The carrier gas was nitrogen. The column was initially set at 170 ℃ and was finally brought to 225 ℃ at 1 ℃ /min. The FID was kept at 270 ℃. The fatty acid methyl esters were identified by referring to the standards purchased from Sigma Chemical Co. The quantities

of fatty acids were enumerated from the peak areas on the chromatogram applying C17:0

(Heptadecanoic acid) as the internal standard.

Results and Discussion

Nutrients leached from whole and chopped trash fish Illustration 3-Fig. 1 shows that the trash fish wastewater contained the required nutrients. It was composed of two fundamental nutrients, i.e. nitrogen and phosphorus. However, there was no nitrate or nitrite in the wastewater. It was rich in ammonia which was chosen selectively by most phytoplankton (Krom et al. 1989) for its nitrogen assimilation. It was also rich in orthophosphate which is indispensable for algal growth (Phillips 1985). Apart from these nutrients for growth, protein was also found in significant amounts. However, the nutrients like ammonia, reactive phosphate and protein from the chopped form were higher than those from the whole form. It was also noted that the concentration for each of these three nutrients collected for both forms increased as they were soaked for longer periods.

Statistical analysis revealed that the amounts of the three nutrients (i.e. ammonia, phosphate and protein) collected from whole trash fish wastewater were significantly less than those of the chopped form. Since the calculated value (7.669) from the t test for ammonia was greater than the tabulated value (2.776), the ammonia leached from the two forms were significantly different

(p<0.05). Similarly the reactive phosphate and protein leached from the two forms were also significantly different (p<0.05) because their calculated values (4.486 and 7.367) from the t test were greater than the tabulated values (2.776 and 2.776) at p <0.05 respectively.

Chemical analysis of trash fish wastewater demonstrated that it was rich in ammonia, phosphate and protein. This result concurred with the conclusions of Yoonaisil and Hertrampf (2006). Other investigators (Hamada and Kumagai 1988; Foscarini 1988) also showed that trash fish is rich in nitrogen, phosphate and proteins as well as other nutrients. For the mass production of marine

Chlorella, Watanabe (1982) and Rothbard (1975) showed that both ammonia and phosphate were required. Fortunately, the wastewater contained both of them. Therefore, it was expected that it was suitable for microalgal culture and this study had demonstrated this to be true.

Fish farmers usually feed their adult fish with whole trash fish while the trash fish was chopped into pieces or made into a paste for their fish fingerlings. The experiment proved that if trash fish is chopped into pieces, more nutrients leached into open waters causing marine pollution. It is recommended, therefore, that cage fish be fed with whole trash fish rather than chopped fish.

The experiment also demonstrated that if the trash fish was soaked in water for a longer period, more nutrients leached into the open waters. Accordingly, it is recommended that fish farmers feed their cage fish with a few trash fish at a time rather than by dumping a great deal into the cages at the same time. Thus, trash fish would be devoured quickly, which would reduce the likelihood of trash fish settling in or at the bottom of cages. This should result in reduced leaching, which would mean reduced marine pollution.

It has been well documented (Olsen et al. 2008; Foy and Rosell 1991) that fish farming is a source of marine pollution. This study has explained how one of the pollution sources derives from trash fish feeding.

Growth of the microalgae enriched with trash fish wastewater

The number of cells of C. saccarophila and Nannochloropsis sp. can be counted using a haemocytometer (Improved Neubauer) under high power of 250x. The cell size of the former was approximately 10 μm while that the latter was as small as 5-8 μm.

From Illustration 4-Fig. 2, it is clear that trash fish wastewater is suitable for application as an organic fertilizer for the two C. saccarophila cultures, namely (a) and (b) and Nannochloropsis sp. The maximum number was 7x107, 8 x107 and 9x107cells/mL for cultures (a) and (b) of C. saccarophila and Nannochloropsis sp. respectively. The control remained clear and no vial cells were found from the beginning to the end of this study. The exponential phase for both cultures

(a) and (b) of C. saccarophila was very similar and extended for about 65 d to give their maximum cell numbers; Nannochloropsis sp. took a longer time of about 77 d. After about 35 d, the Chlorella cultures turned light green while the Nannochloropsis culture appeared a weaker green on day 42. The former cultures turn into a deep green after about 50 d while the latter turned into greenish yellow after about 63 d. The growth rate for C. vulgaris was 0.99/d (Illman et al. 2000) while that of Nannochloropsis gaditana was 0.56/d (Gentile and Blanch, 2001). This explains why the former grew faster than the latter. Cultures (a) and (b) remained deep green for about 25 to 30 d. Then cultures (a) and (b) of C. saccarophila became pale green and the cell counts dropped that wrinkled and decayed cells were found in the dead phase. Similarly,

Nannochloropsis took a longer time than culture (a) to evolve to its dead phase. Therefore, trash fish wastewater can promote the growth of Chlorella and Nannochloropsis.

During the entire culture period, the pH dropped very little (8.7-7.2) for Chlorella cultures. The salinity range for culture (a) was 18-22 ‰ while that of culture (b) was 13-17 ‰. The pH only dropped slightly from 8.7-7.6 for Nannochloropsis culture while its salinity was in the range of

18-25 ‰. On the other hand, the control remained colourless because there was no increase in cell numbers and the pH range showed an even smaller variation (8.7-8.3). The algal cultures maintained a constant pH range even when the cell number was increasing. The pH range may not have shifted very much in trash fish wastewater because of the presence of a high protein content in the culture media. Holum (1985) had shown previously that protein may be used as a pH buffer.

When ammonium is present with other nitrogen compounds (i.e. nitrate), it is selectively chosen for Chlorella assimilation (Schuler et al. 1952; Kaplan et al. 1986). Arup (1989) (Illustration 1-

Table 1) and this study (Illustration 3-Fig.1) reveals that trash fish wastewater is rich in ammonia so it promotes algal growth.

Fatty acids profile of the microalgae

Illustration 5-Table 3 shows C. saccarophila contains high amount of long carbon chain unsaturated fatty acids such as cis-lenoleic acid called LA (18:2n-6: 14.4-32.6% of total fatty acids) and α-linolenic acid called ALA (18:3n-3: 13.0-19.6% of total fatty acids).

Nannochloropsis sp. can synthesize high portion of EPA (20:5n-3: 34.2% of total fatty acids) and a small amount of ARA (20:4n-6: 4.1% of total fatty acids), LA (1.8 %) and ALA (0.3 %). These essential fatty acids are called ω-3/6. Other sources include fruits, vegetables, etc. except EPA.

EPA is primarily produced by certain microalgae and other microbes such as bacteria. Fresh water algae cannot synthesize it except Monodus (a yellow-green fresh water microalga). Only a small amount of DHA (22:6n-3: 0.1-0.5 % of total fatty acid) was found in both algal species. As a result, C. saccarophila is not a potent microagla used to produce EPA and DHA because only

0.1-0.5 mg of them can be obtained from 1 g dry weight of the alga. However, Nannochloropsis sp. is a potential candidate selected to produce EPA because 31.8 mg of it may be extracted from

1 g of its dry weight. Yellow-green micro-algae (Eustigmatophyte) include Nannochloropsis,

Monodus and others are rich in EPA while AA and GLA are also present (Nichols and Appleby

1969). This study agreed with former studies that it is rich in EPA and the presence of AA though there was no GLA found. The variation may be arisen from the difference in culture conditions and species.

Fish oil is rich in ω-3 (especially DHA and EPA), but fish itself cannot synthesize it so it must be obtained from the food chain instead. Fish liver oil has been utilized as a health additive for many years. It has been reported that Greenland Eskimos (EPA level in blood higher than other countries) seldom suffer form coronary heart disease because they consume diet that mainly comprises oil-rich fish (Kromhout et al. 1985). Recently, apart from health products, ω-3 polyunsaturated fatty acids have been tested in many pharmaceutical applications, especially for

EPA. They can regulate normal cardiac function and reduce blood pressure, decrease cholesterol level in serum, anti-agglomerate for blood cells, platelets and blood clot in blood vessel, promote immunity to prevent type 2 diabetes, hypertension, skin and kidney disorder and cancer, inhibit certain enzymes to protect prostate, stimulate anti-inflammation for arthritis and development of eye and brain (Kamal-Eldin and Yanishlieva 2002). ω-3 polyunsaturated fatty acids have been added to many health products such as Usana. Triomega (extracted from cod liver oil), produced by Seven Seas Ltd.(UK), is concentrated in EPA and DHA enriched with vitamins (i.e. A, D and

E); it can provide a synergistic effect for the relief of arthritis pain and maintenance of healthy joints for humans due to its anti-inflammation function (Curtis et al. 2000). EPA was also tested in hospital where it was observed to increase the body weight of cancer patients in preparation for further medical treatment (Wigmore et al. 2000).

Oil content and calorific value of the microalgae

Illustration 5-Table 3 also reveals that C. saccarophila gave 10.7-13.6% of total fatty acids of its cell dry weight enriched with the trash fish wastewater. Similarly, Nannochloropsis sp. accumulated 9.3% of total fatty acids of its cell dry weight. However, C. saccarophila gave a higher value than that of Nannochloropsis sp. The variation in total lipid content of C. saccarophila may arise from salinity difference between the two cultures (a) and (b). As mentioned in the aforesaid section that culture (a) was cultured in a higher salinity concentration range of 18-22 ‰ while that of culture (b) with the range of 13-17 ‰. As a result, the lower salinity level of culture (b) may promote its total lipid content to 13.6% of total fatty acids.

Lipid and fatty acids are components of all cells, where they serve as a source of metabolites and energy as well as membrane constituents. Lipids generally contain fatty acids esterified to glycerol, sugar or bases. It is generally recognized that the lipid content for most microalgae is about 10% of dry weight under normal autotrophic culture conditions (Cohen 1986). Some microalagal species can produce a high oil content under specific conditions. Oh et al. (2010) demonstrated that Chlorella minutissima gave maximum biomass of 8.3 g-dry wt./L and lipid content of 23.2% (w/w) subjected to warm seawater (>30 °C). Singh et al. (2010) reported that some efficient lipid producer algae contain more than 30% of their cell weight as lipids. Gao et al. (2010) can grow Chlorella protothecoides heterotrophically with glucose as the carbon source and accumulate high proportion of lipids. They also found that the lipid yield was 35.7% higher than that using glucose when sweet sorghum juice used as the carbon source.

The variation in biomass and oil content of algae depends on environmental factors such as temperature influence (Oh et al. 2010; Converti et al. 2009; Boussiba et al. 1987), level of irradiance (Lv et al. 2010), salinity content (Renaud and Parry 1994), strength of aeration

(Krienitz and Wirth 2006), CO2 mass transfer (Lv et al. 2010), concentration of nitrogen in culture medium (Lv et al. 2010; Converti et al. 2009), algal species (Converti et al. 2009) and others (Gao et al. 2010; Ong et al. 2010). The lipid content of Chlorella can be improved by optimum culture conditions (Lv et al. 2010). Converti et al. (2009) demonstrated that the lipid content of Chlorella and Nannochloropsis was influenced by culture temperature and nitrogen level in medium. The lipid content was double when the temperature was increased from 20 to

25 ℃ for Nannochloropsis. It was further supported by Rocha et al. (2003) that the optimal temperature for Nannochloropsis was 25±5 ℃. However, the lipid content of Chlorella was reduced when temperature was higher than 30 ℃. The lipid content can also be increased by reducing nitrogen content of the culture media (compared with optimal) for both microalgae. Lv

et al. (2010) showed that when the cultivation conditions were controlled at 1.0 mM KNO3, 1.0%

−2 −1 CO2 and 60 μmol photons m s at 25 °C, the highest lipid productivity of Chlorella vulgaris obtained was 40 mg/L/d. These studies indicated that the lipid content of Chlorella,

Nannochloropsis and other algae can be increased by the adjustment of various factors at their optimal range. Further study is required to optimize fatty acids and lipid production of the microalgae used to recycle the trash fish wastewater.

Several studies (Illustration 2-Table 2) pointed that most Chlorella species gave a calorific value range of 6692-9536 Cal/g while a Nannochloropsis species also gave a calorific value range of 5879-7815 Cal/g (Pan et al. 2010). The heating value of the microalgae approaches that of plant seed oil (Singh et al. 2010; Sams 1998). Therefore, from a sustainability view point, this renewable bio-oil derived from the aquatic environment could be used as an alterative biofuel.

Conclusions

Marine pollution has had a disastrous impact on aquatic life and has reduced valuable resources derived from the ocean. The commercial activity of cage fish farming is one of sources of marine pollution. The recycling of wastes generated from this activity into potentially valuable products must be considered. The production of algae derived from trash fish wastewater is feasible and environmentally friendly. Furthermore, valuable products come from the natural sea provide more alternatives in addition to the terrestrial to us. These products include health and pharmaceutical products and bio-oils, which are sustainable and renewable.

Acknowledgements

I deeply appreciate the support and participation of those fish farmers and my colleague for taking samples and setting up the equipment and tools at site, workshop and laboratory. I would also like to express my sincere thanks to the testing laboratories for GC analysis.

References

Aas E, Baussant, T, Balk L, Liewenborg B, Andersen OK (2000) PAH metabolites in bile,

cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil

exposure: a laboratory experiment with Atlantic cod. Aqu Toxicol 51(2):241-258

Adams VD (1991) Water and wastewater examination manual, 2nd printing. Lewis

Almendras JME (1987) Acute nitrite toxicity and methemoglobinemia in juvenile milkfish

(Chanos chanos Forsskal). Aquaculture 61:33-40

Andrés M, Raúl C, Luis L, Mariane L (1992) Evaluation of marine microalga Nannochloropsis

sp. as a potential dietary supplement. Chemical, nutritional and short term toxicological

evaluation in rats. Nutr Res 12(10):1273-1284 Arup O (1989) Assessment of the environmental impact of marine fish culture in Hong Kong.

Final report submitted to the Environmental Protection Department, Hong Kong

Government

Borowitzka MA (1988) Vitamins and fine chemicals from micro-algae. In: Borowitzka MA,

Borowitzka LJ (eds) Micro-algal biotechnology, pp 153-196

Boussiba S, Vonshak A, Cohen Z, Avissar Y, Richmond A (1987) Lipid and biomass production

by the halotolerant microalga Nannochloropsis salina. Biomass 12(1):37-47

Bower CE, Hansen TH (1980) A simplified hydrazine-reduction method for determinating high

concentrations of nitrate in recirculated seawater. Aquaculture 21:281-286

Brown MR, Mular M, Miller I, Farmer C, Trenerry C (1999) The vitamin content of microalage

used in aquaculture. J Appl Phycol 11:247-255

Cha KH, Koo SY, Lee DU (2008) Antiproliferation effects of carotenoids extracted from

C.ellipsoidea and C.vulgaris on human colon cancer cells. Agri Food Chem 56:10521-

Chen JC, Chin TS (1988) Acute toxicity of nitrite to tiger prawn, Penaeus monodon larvae.

Aquaculture 69:253-262

Cohen Z (1986) Products from microalgae. In: Richmond A (ed) Handbook of microalgal mass

culture. CRC Press, pp 421-454

Converti A, Casazza AA, Ortiz EY, Perego P, Del Borghi M (2009) Effect of temperature and

nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem Eng Process: Process Intens 48(6):1146-

Curtis CL, Hughes CE, Flannery CR, Little CB, Harwood JL, Caterson B (2000) N-3 fatty acids

specifically modulate catabolic factors involved in articular cartilage degradation. J Biol

Chem 275(2):721-724

Day AG, Brinkmann D, Franklin S, Espina K, Rudenko G, Roberts A, Howse KS (2009) Safety

evaluation of a high-lipid algal biomass from Chlorella protothecoides. Regul Toxicol

Pharmacol 55(2):166-180

Demirbas A, Demirbas MF (2010) Importance of algae oil as a source of biodiesel. En Con

Manage. doi:10.1016/j.enconman.2010.06.055 [accessed 12.07.2010]

Durmaz Y (2007) Vitamin E (α-tocopherol) production by the marine microalgae

Nannochloropsis oculata (Eustigmatophyceae) in nitrogen limitation. Aquaculture 272 (1-

4):717-722

Feng YY, Hou LC, Ping NX, Ling TD, Kyo CI (2004) Development of mariculture and its

impacts in Chinese coastal waters. Rev Fish Biol Fish 14:1-10

Foscarini R (1988) Intensive farming procedure for red seabream (Pagrus major) in Japan.

Aquaculture 72:191-246

Foy RH, Rosell R (1991) Loadings of nitrogen and phosphorus from a Northern Ireland fish

farm. Aquaculture 96:17-30 Fulke AB, Mudliar SN, Yadav R, Shekh A, Srinivasan N, Ramanan R, Krishnamurthi K, Devi

SS, Chakrabarti T (2010) Bio-mitigation of CO2, calcite formation and simultaneous

biodiesel precursors production using Chlorella sp. Bioresour Technol 101(21):8473-8476

Gao CF, Zhai Y, Ding Y, Wu QY (2010) Application of sweet sorghum for biodiesel production

by heterotrophic microalga Chlorella protothecoides. Appl En 87(3):756-761

Gentile MP, Blanch HW (2001) Physiology and xanthophylls cycle. Biotechnol Bioeng 75:1-12

Gillan DC, Danis B, Pernet P, Joly G, Dubois P (2005) Structure of sediment- associated

microbial communities along a heavy-metal contamination gradient in the marine

environment. Appl Environ Microbiol 71(2):679–906

Gladu PK, Patterson GW, Wikfors GH, Smith BC (1995) Sterol, fatty acid, and pigment

characteristics of UTEX2341, a marine estigmatophyte identified previously as Chlorella

minutissima (Chlorophyceae). J Phycol 31(5):774-777

Glazer AN, Stryer L (1984) Emerging techniques. Trends Biochem Sci 9:423-427

Guzman S, Gato A, Lamela M, Freire-Garabal M, Calleja JM (2003) Anti-inflammatory and

immunomodulatory activities of polysaccharide from Chlorella stigamatophora and

Phaeodactylum tricornutum. Phytother Res 17:665-670

Hamada M, Kumagai H (1988) Chemical composition of Sardine scale. Nip Sui Gak

54(11):1987-1992

Harrison PJ, Thomas TE (1988) Biomass measurements: protein determination. In: Lobban CS,

Chapman DJ, Kremer BP (eds) Experimental phycology. Cambridge University Press, pp 27-34

Hasegawa T, Matsuguchi T, Noda K., Tanaka K, Kumamoto S, Shoyama Y, Yoshikai Y (2002)

Toll-like receptor 2 is at least partly involved in the antitumor activity of glycoprotein from

Chlorella vulgaris. Int Immunopharmacol 2(4):579-589

Hata M, Sato Y, Yamaguchi T (1988) The chemical and amino acid compositions in tissues of

cultured and wild Coho Salmon, Oncorhynchus kisutch. Nip Sui Gak 54(8):1365-1370

Holum JR (1985) Introduction to organic and biological chemistry. CRC Press, pp 340-342

Illman AM, Scragg AH, Shales SW (2000) Increase in Chlorella strains calorific values when

grown in low nitrogen medium. Enzy Microb Technol 27(8):631-635

Kamal-Eldin A, Yanishlieva NV (2002) N-3 fatty acids for human nutrition: stability

considerations. Eur J Lip Sci Technol 104:825-836

Kamerycah Inc (2010) Fucoidan Umi No Shizuku. Dba Kanefuku America. Available at,

[accessed 17.08.2010]

Kaplan D, Richmond AE, Dubinsky Z, Aaronson S (1986) Algal nutrition. In: Richmond A (ed)

Handbook of microalgal mass culture. CRC Press, pp 147-198

Khansari FE, Ghazi-Khansari M, Abdollahi M (2005) Heavy metals content of canned tuna fish.

Food Chem 93:293–296

Kralovec JA, Metera KL, Kumar JR, Watson LV, Girouard GS, Guan Y, Carr RI, Barrow CJ,

Ewart HS (2007) Immunostimulatory principles from Chlorella pyrenoidosa-part 1:

isolation and biological assessment in vitro. Phytomedicine 14(1):57-64 Krienitz L, Wirth M (2006) The high content of polyunsaturated fatty acids in Nannochloropsis

limnetica (Eustigmatophyceae) and its implication for food web interactions, freshwater

aquaculture and biotechnology. Limnol-Ecol Manage Inland Waters 36(3):204-210

Krom MD, Erez J, Porter CB, Ellner S (1989) Phytoplankton nutrient uptake dynamics in

Earthen marine fishponds under Winter and Summer conditions. Aquaculture 76:237-253

Kromhout D, Bosschieter EB, Coulander CL (1985) The inverse relation between fish

consumption and 20-year mortality from coronary heart disease. New Eng J Med

312(19):1205-1209

Lam CWY (1990) Pollution effects on marine fish culture in Hong Kong. Asian Mar Biol 7:1-7

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin phenol

reagent. J Biol Chem 193:265-275

Lv JM, Cheng LH, Xu XH, Zhang L, Chen HL (2010) Enhanced lipid production of Chlorella

vulgaris by adjustment of cultivation conditions. Bioresour Technol 101(17):6797-6804

Maldonado M, Carmona MC, Echeverria Y, Riesgo A (2005) The environmental impact of

Mediterranean cage fish farms at semi-exposed locations: does it need a re-assessment?

Helgoland Mar Res 59:121-135

Maruyama H, Tamauchi H, Lizuka M, Nakano T (2006) The role of NK cells in antitumor

activity of dietary fucoidan from Undaria pinnatifida sporophylls (Mekabu). Planta Media

72:1415-1417

Merck (1987) Ammonium determination. In: Rapid test handbook. Merck, p 301 Nichols BW, Appleby RS (1969) The distribution and biosynthesis of arachidonic acid in algae.

Phytochemistry 8:423-427

Nishino H, Murakoshi M, Tokuda H, Stomi Y (2008) Cancer prevention by carotenoids. Arc

Biochem Biophys. doi:10.1016/j.abb.2008.09.011 [accessed 15.09.2008]

Oh SH, Kwon MC, Choi WY, Seo YC, Kim GB, Kang DH, Lee SY, Lee HY (2010) Long-term

outdoor cultivation by perfusing spent medium for biodiesel production from Chlorella

minutissima. J Biosci Bioeng 110(2):194-200

Olsen LM, Holmer M, Olsen Y (2008) Final report-perspectives of nutrient emission from fish

aquaculture in coastal waters: literature review with evaluated state of knowledge. Fishery

and Aquaculture Industry Research Fund, Norway

Ong SC, Kao CY, Chiu SY, Tsai MT, Lin CS (2010) Characterization of the thermal-tolerant

mutants of Chlorella sp. with high growth rate and application in outdoor photobioreactor

cultivation. Bioresour Technol 101(8):2880-2883

Pan P, Hu CW, Yang WY, Li YS, Dong LL, Zhu LF, Tong DM, Qing RW, Fan Y (2010) The

direct pyrolysis and catalytic pyrolysis of Nannochloropsis sp. residue for renewable bio-

oils. Bioresour Technol 101(12):4593-4599

Payne JF, Penrose WR (1975) Induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase in

fish by petroleum. Bull Environ Contam Toxicol 14(1): 112-116

Peterson GL (1979) Review of the Folin phenol protein quantitation method of Lowry,

Rosebrough, Farr and Randall. Analy Biochem 100:201-220 Phillips MJ (1985) Analysis of nutrients. In: Stirling HP (ed) Chemical and biological methods of

water analysis for aquaculturists. University of Stirling

Rebolloso-Fuentes MM, Navarvo-Perez A, Garcia-Camacho F, Ramos-Miras JJ, Guil-Guerrero

JL (2001) Biomass nutrient profiles of the microalga Nannochloropsis. J Agri Food Chem

49:2966-2972

Renaud SM, Parry DL (1994) Microalgae for use in tropical aquaculture II: effect of salinity on

growth, gross chemical composition and fatty acid composition of three species of marine

microalgae. J Appl Phycol 6:347-356

Rocha JMS, Garcia JEC, Henriques MHF (2003) Growth aspects of the marine microalga,

Nannochloropsis gaditana. Biomol Eng 20(4-6):237-242

Rodriguez-Garcia I, Guil-Guerrero JL (2008) Evaluation of the antioxidant activity of three

microalgal species for use as dietary supplements and in the preservation of foods. Food

Chem 108(3):1023-1026

Ross AB, Biller P, Kubacki ML, Li H, Lea-Langton A, Jones JM (2010) Hydrothermal

processing of microalgae using alkali and organic acids. Fuel 89(9):2234-2243

Rothbard S (1975) Control of Euplotes sp. by formalin in growth tanks of Chlorella sp. used as

growth medium for the rotifer, Brachionus plicatilis which serves as feed for hatchings.

Bamidgeh 27(4):100-109

Salvesen I, Reitan KI, Skjermo J, Oie G (2000) Microbial environments in marine larviculture:

impacts of algal growth rate on bacterial load in six microalgae. Aquaculture 8:275-287 Sams T (1998) Exhaust components of biofuels under real world engine conditions. In: Martini

N, Schell J (eds) Proceedings of the symposium of plant oils as fuels: present state of

science and future developments. Springer, pp 64-75

Sarkar D, Sharma A, Talukder G (1994) Chlorophyll and chlorophyllin as modifiers of genotoxic

effects. Mutat Res 318(3):239-247

Schoen S (1988) Cell counting. In: Lobban CS, Chapman DJ, Kremer BP (eds) Experimental

phycology. Cambridge University Press, pp 16-22

Schuler JF, Diller VM, Kersten HJ (1952) Preferential assimilation of ammonium ion by

Chlorella vulgaris. Plant Physiol 27:299-303

Scragg AH, Illman AM, Carden A, Shales SW (2002) Growth of microalgae with increased

calorific values in a tubular bioreactor. Biom Bioen 23(1):67-73

Seaherb (2010) Fucoidan. Available at, [accessed 17.08.2010]

Singh A, Nigam PS, Murphy JD (2010) Mechanism and challenges in commercialisation of algal

biofuels. Bioresour Technol 2010. doi:10.1016/j.biortech.2010.06.057 [accessed

05.07.2010]

Strickland JDH, Parsons TR (1972) A practical handbook of seawater analysis, 2nd ed. Bulletin

No. 167, Fisheries Research Board of Canada

Sun Z, Peng XF, Liu J, Fan KW, Wang MF, Chen F (2010) Inhibitory effects of microalgal

extracts on the formation of advanced glycation endproducts (AGEs). Food Chem

120(1):261-267 Syrett PJ, Fowden L (1952) The assimilation of ammonia by nitrogen-starved cells of Chlorella

vulgaris. III. The effect of addition of glucose on products of assimilation. Physiol Plant

5:558-566

Tanaka K, Shoyama Y, Yamada A, Noda K, Konishi F, Nomoto K (2001) Immunopotentiating

effects of a glycoprotein from Chlorella vulgaris strain CK and its characteristics. Stud Nat

Prod Chem 25(6):429-458

Trussell RP (1972) The percent unionized ammonia in aqueous ammonia solutions at different

pH levels and temperature. J Fish Res Board Can 29:1505-1507 van Rooyen J, Esterhuyse AJ, Engelbrecht AM, du Toit EF (2008) Health benefits of a natural

carotenoid rich oil: a proposed mechanism of protection against ischaemia/reperfusion

injury. Asia Pacif J Clin Nutr 17(S1):316-319

Wang HM, Pan JL, Chen CY, Chiu CC, Yang MH, Chang HW, Chang JS (2010) Identification of

anti-lung cancer extract from Chlorella vulgaris C-C by antioxidant property using

supercritical carbon dioxide extraction. Process Biochem.

doi:10.1016/j.procbio.2010.05.023 [accessed 28.05.2010]

Watanabe T (1982) The production of food organisms with particular emphasis rotifers. In:

FAO/UNDP Training course on seabass spawning and larval rearing held at the National

Institute of Coastal Aquaculture (NICA), Sonhkila, Thailand, pp 26-28

WHO (World Health Organization) (1998) Carotenoids, vol. 2. IARC Handbook of cancer

prevention. International Agency for Research on Cancer Wigmore SJ, Barber MD, Ross JA, Tisdale MJ, Fearon CH (2000) Effect of oral

Eicosapentaenoic acid on weight loss in patients with pancreatic cancer. Nutr Can

36(2):177-184

Wu RSS, Lee JHW (1989) Grow-out mariculture techniques in tropical waters: a case study of

problems and solutions in Hong Kong. Adv Trop Aqu 9:729-736

Wu RSS (1995) The environmental impact of marine fish culture: towards a sustainable future.

Mar Poll Bull 31(4-12):159-166

Yaeyama (2010) The nutritious superstar-green alga foodstuffs: Chlorella (Midori-no-sachi).

Available at, http://www.yaeyama.com [accessed 17.08.2010]

Yoonaisil T, Hertrampf JW (2006) An effect of nucleotides in the Asian seabass: a uniform and

faster growth of fish fed chopped trash fish with nucleotides was demonstrated in this trail

with Asian seabass in Thailand. Aqua Culture Asia Pacific Magazine, November/December,

Zar JH (1984) Biostatistical analysis, 2nd ed. Prentice-Hall

Illustrations Illustration 1

Table 1 Chemical composition of trash fish from various sources.

Parameter Nutrients of trash Increase pollutants of Composition of trash Sardine scale Integument of wild fish (mg/g dry wt) chopped fish (mg/L) fish as fed basis (%) (g/100g scale) Coho Salmon (%)

a N-NO2 0.0314 0.0046 - - - b g N-NO3 0.253 ND - - - c N-NH3 1.547 0.1355 - - - TONd 10.3 3.295 - - - TNe 12.1 3.4377 - 6.24 18.9 f P-PO4 2.548 0.9307 1.21 21.08 (g/100g ash) - Protein - - 11.2 36.25 - Reference Arup 1989 Arup 1989 Yoonaisil and Hamada and Hata et al. 1988 Hertrampf 2006 Kumagai 1988 a Nitrite. b Nitrate. c + Total ammonia (NH4 + Unionized ammonia molecules). d Total organic nitrogen. e Total nitrogen. f Reactive phosphate. g Not detected. Illustration 2

Table 2 Some comparative calorific values for various types of biofuels.

Source Chlorella Nannochloropsis Rapeseed oil

C. vulgaris C. sp. C. vulgaris C. emersonii.

Calorific value 7983-9536 6931 6692 6931 5879-7815 8843 (Cal/g) Reference Ross et al. Fulke et al. Scragg et al. Illman et al. Pan et al. Sams 2010 2010 2002 2000 2010 1998 Illustration 3

Fig. 1. Comparison of whole and chopped trash fish leachate with time.

) Protein (Chopped) L

/ Ammonia (Whole) g 140 Nitrite (Whole) m ( Nitrate (Whole) s t Phosphate (Whole) n e

i 120 Protein (Whole) r t Ammonia (Chopped) u n

Nitrite (Chopped) f

o 100 Nitrate (Chopped)

n Phosphate (Chopped) o i

t Protein (Chopped) a r

t 80 n e

c Protein (Whole) n

o 60 C

40 Phosphate (Chopped)

Phosphate (Whole) 20 Ammonia (Chopped)

Ammonia (Whole)

0 Nitrate and Nitrite 5 15 30 45 60

Time taken (min)

Illustration 4 Fig. 2. Growth curves of cultures (a) and (b) of Chlorella saccharophila and Nannochloropsis sp. enriched with trash fish wastewater.

Chlorella saccharophila culture (a) 900 Chlorella saccharophila culture (b) Nannochloropsis Control

L 800 m

700 5 0 1

l 500 e c

400 r e

b 300 m u

0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 Number of days

Illustration 5 Table 3 Fatty acid (%) profile for cultures (a) and (b) of Chlorella saccharophila and Nanochloropsis sp.

Fatty Chlorella saccharophila Nanochloropsis sp. acid (a) (b)

12:0 0.1±0.06a traceb 0.4±0.04a 13:0 0.4±0.24 0.8±0.10a traceb 14:0 0.3±0.07 0.3±0.04 4.0±0.24 14:1 0.6±0.11 1.0±0.17 0.0±0.00 15:0 0.3±0.06 0.2±0.04 0.2±0.03 15.1 0.0±0.00 0.2±0.04 0.0±0.00 16:0 0.0±0.00 16.6±0.36 12.3±0.14 Unknown 5.5±0.99 7.1±0.17 26.4±0.41 16:1 2.3±0.13 3.9±0.17 6.5±0.31 16:2 7.3±0.09 5.2±0.18 2.1±0.13 17:0 0.6±0.04 0.2±0.02 0.5±0.05 17:1 4.8±0.32 10.8±0.29 0.9±0.09 18:0 1.7±0.67 2.4±0.47 1.1±0.31 18:1n9c 6.0±1.75 16.8±0.19 4.1±0.27 18:2n-6(LA) 32.6±3.27 14.4±0.39 1.8±0.07 18:3n-3(ALA) 13.0±0.99 19.6±0.51 0.3±0.07 18:4 0.2±0.09 trace 0.0±0.00 20:0 0.1±0.04 trace 0.0±0.00 20:2 0.2±0.21 0.0±0.00 0.3±0.05 20:3 0.0±0.0 0.0±0.00 0.5±0.07 20:4n-6(ARA) 0.0±0.0 0.0±0.00 4.1±0.05 20:5n-3(EPA) 0.1±0.17 0.2±0.02 34.2±0.50 22:5n-3(DPA) 0.0±0.0 0.0±0.00 0.0±0.00 22:6n-3(DHA) 0.5±0.11 0.1±0.03 0.3±0.07 TFAc 10.7±0.68 13.6±0.76 9.3±0.05 EPA (mg/g) 0.1±0.02 0.2±0.04 31.8±0.31 DHA (mg/g) 0.5±0.10 0.2±0.05 0.3±0.07 a Data expressed as mean ± SD of triplications. b % of fatty acid is equal to or less than 0.1% of total fatty acids. c Total fatty acid content (%) = (TFA/cell dry weight) x 100%.