Recycling of Nutrients from Trash Fish Wastewater for Microalgae

Article ID: WMC002027 ISSN 2046-1690

Recycling of Nutrients from Trash Fish Wastewaterfor Microalgae Production as Health andPharmaceutical Products and Renewable EnergyAuthor(s):Dr. Hing Chan

Corresponding Author:Dr. Hing Chan,Manager, Marine Resources Technologies, Fisherman's New Village, Tap Mun, N.T. - China

Submitting Author:Dr. Hing Chan,Manager, Marine Resources Technologies, Fisherman's New Village, Tap Mun, N.T. - China

Article ID: WMC002027

Article Type: Original Articles

Submitted on:19-Jul-2011, 01:05:37 AM GMT Published on: 19-Jul-2011, 07:30:30 PM GMT

Article URL: http://www.webmedcentral.com/article_view/2027

Subject Categories:MICROBIOLOGY

Keywords:Trash Fish wastewater, Microalgae, Health-Pharmaceutical Products, Biofuels

How to cite the article:Chan H . Recycling of Nutrients from Trash Fish Wastewater for Microalgae Productionas Health and Pharmaceutical Products and Renewable Energy . WebmedCentral MICROBIOLOGY2011;2(7):WMC002027

Source(s) of Funding:

No funding from other parties

Competing Interests:

No conflict of interest

Additional Files:

Manuscript

Text only

Illustrations 1-5

WebmedCentral > Original Articles of 19

http://www.webmedcentral.com/article_view/2027

http://www.webmedcentral.com//articlefiles/2e1dead26fb6bc5fec5b8724833e682c.doc

http://www.webmedcentral.com//articlefiles/e1a002762d6489683aaea971da43bdea.doc

http://www.webmedcentral.com//articlefiles/b47d1e68631cef961dbefd019a7cad05.doc

WMC002027 Downloaded from http://www.webmedcentral.com on 09-Aug-2011, 07:54:54 AM

Recycling of Nutrients from Trash Fish Wastewaterfor Microalgae Production as Health andPharmaceutical Products and Renewable Energy

Abstract

Trash fish feeding of cage fish can result in marinepollution. Whole and chopped trash fish can leachpollutants such as ammonia, phosphate and proteininto surrounding waters. Reduction of pollution can beachieved by recycling the wastewater generated fromtrash fish feeding for cultivation of microalgae. Apartfrom larviculture, microalgae are potent candidates forthe production of health and pharmaceutical products.Two microalage, Chlorella saccharophila andNannochloropsis sp. have the potential to producehigh amounts of polyunsaturated fatty acids called ω-3.31.8 mg of EPA (Eicosapentaenoic acid, C20:5n-3)can be obta ined f rom 1 g dry we ight o fNannochloropsis sp. Furthermore, high oil contentranging from 10.7 to 13.6% for Chlorella saccharophilaand 9.3% for Nannochloropsis sp. TFA (Total fattyacids/cell dry weight) can be extracted from themrespectively. An alternative biofuel derived frommicroalgae is feasible due to the fact that there is nocompetition for terrestrial occupation.

Introduction

Trash fish are low-priced fish that are typically used asfeed for cultured fish, ducks, dogs and cats. Types oftrash fish include hair-tail, gizzard-shad, scad, goldensardine, mackerel, grunt and other species. Usually itis a mixture of two or more species. Most marine fishfarmers use trash fish to feed their cage fish. When abucket of fresh trash fish stands for a certain period,some red leachate is observed on the top. Theleachate is regarded as wastewater. Thus, most fishfarmers will wash the trash fish with water before fishfeeding and then discard both the leachate and thewastewater. Alternatively, fresh trash fish may not besupplied fresh daily and would be kept in refrigeration.Frozen trash fish will be defrosted in water beforefeeding. As a result, wastewater will also be generatedfrom the defrosting process. Adult cage fish areusually fed a whole trash fish while juvenile fish would

be fed trash fish that are first chopped into smallerpieces. We refer to the wastewater generated from allthe aforesaid procedures as trash fish wastewater.

The fact that fish farming causes water pollution hasbeen well documented (Olsen et al. 2008; Wu 1995;Foy and Rosell 1991; Lam 1990), but the details ofsources of pollution were not fully identified and norecycling alternative for the pollutants was proposed.Working over 30 years in aquatic environment andwastewater treatment in the market revealed that mostof fish farmers have given up their business in HongKong. As a matter of fact, the marine environment hasbecome more polluted than before despite a reductionon fish harvested by fishing vessels. Now we consumethe aforesaid trash fish due to the fact we have tosuffer from this man-made pollution. Untreatedindustrial effluents, petroleum spills and other leakagecontaining heavy metals (Gillan et al. 2005; Khansariet al. 2005) and toxicants (Aas et al. 2000; Payne andPenrose 1975) are the main sources of pollution foracute and chronic aquatic poisoning. Some studies(Arup 1989; Wu and Lee 1989) have recommendedpellet feed to replace trash fish for fish farming asnutrient leakage is less with pellet feed. Aside from thedisadvantages of applying pellet feed to substitutetrash fish, marine pollution continues to be a problemeven though fish farmers have been utilizing pelletfeed for many years. It is worth noting that trash fishare collected from the ocean while pellet feed isformulated from terrestrial plants. Feeding fish pelletfeed is the equivalent of dumping pollutants from theterrestrial environment into the marine ecosystem.Trash fish, however, are a part of the marineecosystem, their consumption by cage fish ensuresthe food supply-chain remains within the oceanenvironment. Accordingly, the nutrient balanceremains the same provided that the cage fish are notharvested for human consumption. The main point isthat a fish culture zone should be established wherethere is a high rate of water exchange area but not in abay. Then, the nutrient balance can be maintained.This explains why cage cultures have been run inopen waters in various countries (Maldonado et al.2005; Feng et al. 2004).



Several studies (Arup 1989; Yoonaisil and Hertrampf2006; 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 ofwhether there is an application for trash fishwastewater? It may be applied as an organic fertilizerto grow algae, vegetables, horticultural products andfor other plantation means. However, we areinterested in aquatic applications and so, this study willconcentrate on this field. The study was designedqualitatively and quantitatively to investigate the kindsof nutrients and the amounts leached out when trashfish was soaked in water (similar to fish feeding). Didthe amounts of nutrients increase when the trash fishwas soaked for a longer period? Did the same amountof nutrients leach out from the trash fish for both wholeand chopped forms?

About three-quarters of the earth’s surface is coveredby ocean; the mass culture of algae taken in the seapose no resource competition as the counterpart – forterrestrial occupation. They are not only potentcandidates to recycle pollutants generated by man, butthey also produce valuable products for ourconsumption and other applications. Apart from thetraditional utilization of microalage for larviculture (i.e.for rotifer, copepod and artemia), they have also beenfound in health, pharmaceutical and biofuelapplications.

WHO (1998) has published an issue to emphasize theimportance of natural carotenoids to our health.Similarly, the essence of chlorophyll and carotenoidsof algae has recently been found for health andpharmaceutical applications. Microalgae appeared indifferent colours such as green, red, brown, yellow,blue and so on only when they grow to a high celldensity. Natural pigments like β-Carotene (VanRooyen et al. 2008) and chlorophyll (Sarkar et al. 1994)keep animals in healthy condition while accessorypigments such as zeaxanthin, lutein, astaxanthin, etc.are effective for natural colour development in animalslike fish, bird and cattle (Borowitzka 1988; Cohen1986). Recently, accessory pigments were identifiedas natural antioxidants (Rodriguez-Garcia andGuil-Guerrero 2008) and act as free radicalsscavengers (Wang et al. 2010) to protect our bodycells from ‘bleaching’. Nishino et al. (2008) reportedthat many carotenoids extracted from microalage havebeen demonstrated to be effective for the inhibition ofvarious human cancer cells for the control ofcarcinogenesis such as: α-carotene for liver and colon;lutein for lung and skin; zeaxanthin for skin, lung andliver; fucoxanthin for skin, duodenum, colon and theliver. Phycobiliproteins such as phycoerythrin from red

algae and phycocyanin from blue-green algae havebeen extracted for the applications of medicaldiagnosis (Phycofluors) (Glazer and Stryer 1984).

Furthermore, algal polysaccharide produced byPhaeodactylum tr icornutum and Chlorel lastigmatophora has been identified to be effective foranti-inflammatory and immunomodulatory activities(Guzman et al. 2003). Sulphated exopolysaccharide ofa brown alga called fucoidan has been produced byvarious companies (Kamerycah 2010; Seaherb 2010)in the market as various health and pharmaceuticalproducts to treat different types of cancers (Maruyamaet al. 2006). Spirulina and Chlorella health productshave been on the market for many years.

Two genus microalgae, namely Chlorella andNannochloropsis, were used to recycle trash fishwastewater for this study. A brief introduction isprovided here. Green microalgae (Chlorophyte)including Chlorella and others are rich in long chainpolyunsaturated fatty acids (ω-3/6), pigments likechlorophyll and carotenoids such as carotene andlutein (and violaxanthin, antheraxanthin, zeaxanthinand others) are also present. It also producesmulti-vitamins, 18 amino acids and contains severalminerals (Yaeyama 2010). The products were used asdietary, health and pharmaceutical supplement andwere found nontoxic for consumption (Day 2009).Chlorella extract has been identified to haveantioxidant properties (Rodriguez-Garcia andGuil-Guerrero 2008; Wang et al. 2010) and could aidin the suppression of cancer cell (Rodriguez-Garciaand Guil-Guerrero 2008; Tanaka et al. 2001;Hasegawa et al. 2002) as well as the stimulation ofantibodies (Kralovec et al. 2007). Lutein of Chlorellawas reported to have antiglycative activity in diabeticcomplications (Sun et al. 2010).

Yellow-green micro-algae (Eustigmatophyte) includeNannochloropsis, Monodus and others are rich in EPAwhile AA (Arachidonic acid, C20:4n-6) and GLA (γ-Linolenic acid, C18:3n-6) are also present (Nichols andAppleby 1969). Nannochloropsis producesmulti-vitamins (Brown et al. 1999; Durmaz 2007);several minerals and most essential amino acids(Rebolloso-Fuentes et al. 2001). Its total lipidsaccumulation is about 18.4% of dry weight(Rebolloso-Fuentes et al. 2001). Salvesen et al. (2000)identified that the antibacterial substances wereabundant in slow-growing cultures of N. oculata andothers. In addition to chlorophyll, violaxanthin andvaucheriaxanthin are the two major pigments for thisgroup (Gladu et al. 1995; Gentile and Blanch 2001).Violaxanthin, antheraxanthin and zeaxanthin are themajor pigments of C.ellipsoidea, which can have an



antiproliferative effects on human colon cancer (Cha etal. 2008). There was no toxicity found for consumptionof Nannochloropsis biomass (Andrés et al. 1992).

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

Biofuel from microalgae is another important benefitbecause it is sustainable and renewable, unlike thefossil-based fuels. Use of microalgae provides us withone more solution for producing clean energy. The oilcontent of each microalga depends on its cultureconditions and the type of species (Demirbas andDemirbas 2010; Lv et al. 2010). Microalga likeChlorella species is studied mostly for its applicationas a biodiesel. The heating value may approach thatof the terrestrial plant seed (Illustration 2-Table 2).Thus, it is a potent candidate for providing a thirdgeneration biofuel alternative in the future. Since trashfish is harvested from the sea, we prefer the inputeffort to reduce pollution arisen from trash fish feedingto the introduction of terrestrial pellet feed into theocean. We believe that this pollution may be reducedby recycling it into useful products that we havementioned in the preceding paragraphs or even otheralternatives. Accordingly, the recycling of trash fishwastewater for microalgal culture has been attemptedhere.

So the main objectives of this study were as follows:first, to introduce various valuable products producedby microalgae; second, to characterize the nutrientprofile of trash fish wastewater; third, to comparepollution levels from whole and chopped form of trashfish; fourth, to test if trash fish wastewater can supportalgal growth; fifth, to evaluate the fatty acid profile andits amount for the two microalgae; and sixth, todemonstrate the recycling of pollutants into health andpharmaceutical products and a renewable energybased on oil content respectively.

Methods

Overview of the study

This experiment was designed to evaluate the impacton pollution from nutrients leached from both wholeand chopped form as practised by fish farmers. Thenutrients analysed were ammonia, nitrate, nitrite,

orthophosphate and protein; each analysis was doneaccording to individual standard chemical methods.The reasons why these particular nutrients wereselected for analysis are as follows: (a) unionizedammonia molecules are toxic to aquatic animals(Merck 1987; Phillips 1985; Trussell 1972); (b)ammonium is selectively chosen as a nitrogen sourceby Chlorella (Schuler et al. 1952; Syrett and Fowden1952) and other microalgae (Krom et al. 1989) forassimilation; (c) nitrite is toxic to aquatic animals(Merck 1987; Chen and Chin 1988; Almendras 1987);(d) phytoplankton needs soluble phosphate for growthand the reactive phosphate is an approximatemeasure of it (Phillips 1985); and (e) researchers(Yoonaisil and Hertrampf 2006; Hamada and Kumagai1988; Foscarini 1988) have shown that trash fish arerich in nitrogen, phosphate, proteins as well as othernutrients so that they may support algal growth. Thus,it is worth evaluating the potential of recycling organicpollutants into health, pharmaceutical products andbiofuel.

Trash fish wastewater analysis and the amount ofnutrients in whole versus chopped trash fish

Polyethylene pails with size of 0.28 m in diameter, 0.5m height and total volume of 30 L were used tocompare the leaching rate of nutrients from both wholeand chopped trash fish. A tap was installed near thebottom of each pail to collect the wastewaters atdifferent time intervals. The experiment was designedto be similar to the real situation when fish farmersfeed their cage fish. For routine feeding a largeamount of trash fish is dumped into the cages at onetime; since the fish cannot eat them all at once, sometrash fish settles at the bottom of the cage or passesthrough the net. During this settling, a certain amountof nutrients will leach out, from the trash fish. So it wasused to estimate the amount of nutrients whichleached out from trash fish over different time intervalsto see if it was time related. The same amount (1200 g)of trash fish (both whole and chopped) was droppedand soaked in the same volume of seawater (30 L/pail)for different time intervals. Samples (three replicatesfor both treatments) were collected after 5 min andthen at 15 min intervals for 1 h to determine whethernutrient leaching varied with time. It was also designedto estimate the degree of pollution resulting from thetwo methods and to see if they were significantlydifferent. The two methods were checked using the ttest if the calculated value was different from thetabulated value at the 95 % confidence level (Zar1984).

Analysis of nutrients from the trash fishwastewater



Overview of the analysis procedures

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

Total ammonia

The total ammonia includes the toxic unionizedammonia molecules and the ammonium ions. Thefollowing method was based on Strickland andParsons (1972) and Adams (1991). Ammonia reactswith phenol in the presence of the oxidizing agentsodium hypochlorite in alkaline solution to giveindophenol blue. After adding all the reagents tosamples and standards, the tubes are covered withparaffin film, keep out of direct light and allowed todevelop in a dark place. The absorbance is then readfrom a spectrophotometer at 640 nm against a reagentblank after 2 h and the colour is good for 24 h. Thismethod is suitable for both seawater and freshwatersamples.

Nitrite

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

Nitrate

A hydrazine-reduction method suggested by Bowerand Hansen (1980) for the determination of nitrate inseawater was introduced here. Nitrate was reduced tonitrite in 2 h by hydrazine sulphate in the presence ofc o p p e r c a t a l y s t a n d b u f f e r e d w i t hcyclohexyl-aminopropane sulfonic acid and sodiumhydroxide (pH~10) at room temperature. After 2 h

reduction, the excess hydrazine was destroyed byacetone to stop the reduction. When the nitrate hasbeen reduced to nitrite this was determined as in themethod described above. If seawater samples weremeasured, at least a 50 times dilution with deionizedwater must be done. The absorbance is read from aspectrophotometer against a reagent blank at 540 nm.

Reactive (Ortho) phosphate

Phosphates that respond to colour colorimetric testswithout preliminary hydrolysis or digestion by oxidationof samples are termed reactive phosphorus. It is anapproximate measure of soluble phosphorus availablefor phytoplankton growth. The following method wasbased on Adams (1991).

Phosphate reacts with molybdate to formmolybdo-phosphoric acid in acid solution which can bereduced to the intensely coloured molybdenum bluecomplex. After adding all the reagents to the standardsand samples, 20 min (<2 h) should be allowed todevelop the blue colour and then the absorbance isread at 880 nm against a reagent blank.

Protein

Since proteins vary greatly in their amino acidcompositions and hence exhibit different properties, sodifferent assay methods have been introduced. Lowryet al. (1951) introduced the most common techniquesalthough the method was modified later by Peterson(1979). Phosphomolybdic-tungstic (a mixed acid) isthe active constituent in Folin-Ciocalteu's phenolreagent. Copper ions in alkaline solution facilitateelectron transfer to the amino acids (tyrosine andtryptophan), thus resulting in a reduction of the mixedac id by loss o f oxygen a toms f rom thetungstate/molybdate. As a result, several reducedspecies are produced, giving a blue colour. Thefollowing method was based on Harrison and Thomas(1988) which was modified from Lowry.

Standard solutions using albumin are prepared foreach measurement. After adding all the reagents tothe standards and samples, they are left for 30 min toallow both colour development and the precipitate tosettle down. The absorbance is read from aspectrophotometer at 750 nm against a reagent blank.

Counting of microalgal cells

We have stock cultures of Chlorella saccharophila andNannochloropsis sp. Their cells were counted usinghemocytometers (namely Neubauer Improved) assuggested by Schoen (1988).

Application of trash fish wastewater for microalgalcultures

The wastewater was collected from 5 kg trash fish



soaked in 2 L of seawater for 20 min. Four sets ofculture flasks of equal volume (2 L each) were set up,two for C. saccarophila called cultures (a) and (b); onefor Nannochloropsis sp.; and the other as a control.There were totally eight 2-L culture flasks. Each set ofthe culture flasks was conducted in duplicate. Allsamples from each of the duplicates were analysed intriplicates and their means were used to plot figuresand 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 20min at 126 °C under 103 kPa pressure. 100 mL of theautoclaved trash fish wastewater were added to eachflask except the control. 2 mL of pure culture of C.saccarophila (~ 1x102 cells/mL) were inoculated intocultures (a) and (b) and the control. Similarly, 2 mL ofpure culture of Nannochloropsis sp (~ 1x102 cells/mL)were inoculated into the cul ture f lask forNannochloropsis sp. They were cultured under thesame ambient temperature near window subjected toabout 12 h: 12 h (day: night) sunlight. Air was suppliedvia a central air-blower. The growth of the microalagewas studied by counting the number of cells using ahemocytometer for each week. Ambient temperaturewas read by a Bibby digital pen thermometer. pHvalue of the cultures was measured by Cole-ParmerpH meter, model 5985 with two-buffer-calibration (i.e.pH 4 and 7). The salinity of the cultures was checkedby a temperature compensated American Optical (AO)hand-held refractometer.

Analysis of fatty acid profile from the microalgalcultures

When the two cultures (a) and (b) of C. saccarophilaturned deep green and the flask of Nannochloropsissp. turned to deep yellow-green (~ 50-60 d), they wereharvested and centrifuged to have freeze dry cells forextraction. Cell dry weight content was estimated bydrying the cells at 80ºC in a vacuum oven until aconstant weight was obtained. Dry biomass (~100 g)of each alga was added to a mixture of 500 mLmethanol and 25 mL acetyl chloride and 500 mLmethanol. The slurry was put into a pressure vesseland held in ultrasonic bath for 15 min. The pressurevessel was warmed in a boiling bath for 30 min at amaximum value of 3.5 atm. Then, the vessel wascooled to an ambient level in a water bath. The vesselwas washed with 500 mL hexane and added to thebiomass slurry for filtration with a Buchner funnel. Theliquid phase was separated after 15 min. The tophexane layer was obtained and concentrated withrotary evaporator under nitrogen gas. Theconcentrated extract (ester) was applied to an HP

6890 capillary gas chromatographer (Hewlett-Packard,Palo Alto, CA) equipped with a FID (Flame–ionizationdetector) and a Supelco (Bellefonte, PA) Omegawa250 capillary column (30x0.25 mm). The carrier gaswas nitrogen. The column was initially set at 170ºCand was finally brought to 225ºC at 1ºC /min. The FIDwas kept at 270ºC. The fatty acid methyl esters wereidentified by referring to the standards purchased fromSigma Chemical Co. The quantities of fatty acids wereenumerated f rom the peak areas on thechromatogram applying C17:0 (Heptadecanoic acid) asthe internal standard.

Results and Discussion

Nutrients leached from whole and chopped trashfish

Illustration 3-Fig. 1 shows that the trash fishwastewater contained the required nutrients. It wascomposed of two fundamental nutrients, i.e. nitrogenand phosphorus. However, there was no nitrate ornitrite in the wastewater. It was rich in ammonia whichwas chosen selectively by most phytoplankton (Kromet al. 1989) for its nitrogen assimilation. It was alsorich in orthophosphate which is indispensable for algalgrowth (Phillips 1985). Apart from these nutrients forgrowth, protein was also found in significant amounts.However, the nutrients like ammonia, reactivephosphate and protein from the chopped form werehigher than those from the whole form. It was alsonoted that the concentration for each of these threenutrients collected for both forms increased as theywere soaked for longer periods.

Statistical analysis revealed that the amounts of thethree nutrients (i.e. ammonia, phosphate and protein)collected from whole trash fish wastewater weresignificantly less than those of the chopped form.Since the calculated value (7.669) from the t test forammonia was greater than the tabulated value(2.776), the ammonia leached from the two formswere significantly different (p<0.05). Similarly thereactive phosphate and protein leached from the twoforms 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 wastewaterdemonstrated that it was rich in ammonia, phosphateand protein. This result concurred with the conclusionsof Yoonaisil and Hertrampf (2006). Other investigators(Hamada and Kumagai 1988; Foscarini 1988) alsoshowed that trash fish is rich in nitrogen, phosphate



and proteins as well as other nutrients. For the massproduction of marine Chlorella, Watanabe (1982) andRothbard (1975) showed that both ammonia andphosphate were required. Fortunately, the wastewatercontained both of them. Therefore, it was expectedthat it was suitable for microalgal culture and this studyhad demonstrated this to be true.

Fish farmers usually feed their adult fish with wholetrash fish while the trash fish was chopped into piecesor made into a paste for their fish fingerlings. Theexperiment proved that if trash fish is chopped intopieces, more nutrients leached into open waterscausing marine pollution. It is recommended, therefore,that cage fish be fed with whole trash fish rather thanchopped fish.

The experiment also demonstrated that if the trash fishwas soaked in water for a longer period, morenutrients leached into the open waters. Accordingly, itis recommended that fish farmers feed their cage fishwith a few trash fish at a time rather than by dumpinga great deal into the cages at the same time. Thus,trash fish would be devoured quickly, which wouldreduce the likelihood of trash fish settling in or at thebottom of cages. This should result in reducedleaching, which would mean reduced marinepollution.

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

Growth of the microalgae enriched with trash fishwastewater

The number of cells of C. saccarophila andNannochloropsis sp. can be counted using ahaemocytometer (Improved Neubauer) under highpower of 250x. The cell size of the former wasapproximately 10 µm while that the latter was as smallas 5-8 µm.

From Illustration 4-Fig. 2, it is clear that trash fishwastewater is suitable for application as an organicfertilizer for the two C. saccarophila cultures, namely(a) and (b) and Nannochloropsis sp. The maximumnumber was 7x107, 8 x107 and 9x107cells/mL forcultures (a) and (b) of C. saccarophila andNannochloropsis sp. respectively. The controlremained clear and no vial cells were found from thebeginning to the end of this study. The exponentialphase for both cultures (a) and (b) of C. saccarophilawas very similar and extended for about 65 d to givetheir maximum cell numbers; Nannochloropsis sp.took a longer time of about 77 d. After about 35 d, theChlorella cultures turned light green while the

Nannochloropsis culture appeared a weaker green onday 42. The former cultures turn into a deep greenafter about 50 d while the latter turned into greenishyellow after about 63 d. The growth rate for C. vulgariswas 0.99/d (Illman et al. 2000) while that ofNannochloropsis gaditana was 0.56/d (Gentile andBlanch, 2001). This explains why the former grewfaster than the latter. Cultures (a) and (b) remaineddeep green for about 25 to 30 d. Then cultures (a) and(b) of C. saccarophila became pale green and the cellcounts dropped that wrinkled and decayed cells werefound in the dead phase. Similarly, Nannochloropsistook a longer time than culture (a) to evolve to its deadphase. Therefore, trash fish wastewater can promotethe growth of Chlorella and Nannochloropsis.

During the entire culture period, the pH dropped verylittle (8.7-7.2) for Chlorella cultures. The salinity rangefor culture (a) was 18-22 ‰ while that of culture (b)was 13-17 ‰. The pH only dropped slightly from8.7-7.6 for Nannochloropsis culture while its salinitywas in the range of 18-25 ‰. On the other hand, thecontrol remained colourless because there was noincrease in cell numbers and the pH range showed aneven smaller variation (8.7-8.3). The algal culturesmaintained a constant pH range even when the cellnumber was increasing. The pH range may not haveshifted very much in trash fish wastewater because ofthe presence of a high protein content in the culturemedia. Holum (1985) had shown previously thatprotein may be used as a pH buffer.

When ammonium is present with other nitrogencompounds (i.e. nitrate), it is selectively chosen forChlorella assimilation (Schuler et al. 1952; Kaplan et al.1986). Arup (1989) (Illustration 1-Table 1) and thisstudy (Illustration 3-Fig.1) reveals that trash fishwastewater is rich in ammonia so it promotes algalgrowth.

Fatty acids profile of the microalgae

Illustration 5-Table 3 shows C. saccarophila containshigh amount of long carbon chain unsaturated fattyacids such as cis-lenoleic acid called LA (18:2n-6:14.4-32.6% of total fatty acids) and α-linolenic acidcalled ALA (18:3n-3: 13.0-19.6% of total fatty acids).Nannochloropsis sp. can synthesize high portion ofEPA (20:5n-3: 34.2% of total fatty acids) and a smallamount of ARA (20:4n-6: 4.1% of total fatty acids), LA(1.8 %) and ALA (0.3 %). These essential fatty acidsare called ω-3/6. Other sources include fruits,vegetables, etc. except EPA. EPA is primarilyproduced by certain microalgae and other microbessuch as bacteria. Fresh water algae cannot synthesizeit except Monodus (a yellow-green fresh watermicroalga). Only a small amount of DHA (22:6n-3:



0.1-0.5 % of total fatty acid) was found in both algalspecies. As a result, C. saccarophila is not a potentmicroagla used to produce EPA and DHA becauseonly 0.1-0.5 mg of them can be obtained from 1 g dryweight of the alga. However, Nannochloropsis sp. is apotential candidate selected to produce EPA because31.8 mg of it may be extracted from 1 g of its dryweight. Yellow-green micro-algae (Eustigmatophyte)include Nannochloropsis, Monodus and others are richin EPA while AA and GLA are also present (Nicholsand Appleby 1969). This study agreed with formerstudies that it is rich in EPA and the presence of AAthough there was no GLA found. The variation may bearisen from the difference in culture conditions andspecies.

Fish oil is rich in ω-3 (especially DHA and EPA), butfish itself cannot synthesize it so it must be obtainedfrom the food chain instead. Fish liver oil has beenutilized as a health additive for many years. It hasbeen reported that Greenland Eskimos (EPA level inblood higher than other countries) seldom suffer formcoronary heart disease because they consume dietthat mainly comprises oil-rich fish (Kromhout et al.1985). Recently, apart from health products, ω-3polyunsaturated fatty acids have been tested in manypharmaceutical applications, especially for EPA. Theycan regulate normal cardiac function and reduce bloodpressure, decrease cholesterol level in serum,anti-agglomerate for blood cells, platelets and bloodclot in blood vessel, promote immunity to prevent type2 diabetes, hypertension, skin and kidney disorder andcancer, inhibit certain enzymes to protect prostate,stimulate anti-inflammation for arthritis anddevelopment of eye and brain (Kamal-Eldin andYanishlieva 2002). ω-3 polyunsaturated fatty acidshave been added to many health products such asUsana. Triomega (extracted from cod liver oil),produced by Seven Seas Ltd.(UK), is concentrated inEPA and DHA enriched with vitamins (i.e. A, D and E);it can provide a synergistic effect for the relief ofarthritis pain and maintenance of healthy joints forhumans due to its anti-inflammation function (Curtis etal. 2000). EPA was also tested in hospital where it wasobserved to increase the body weight of cancerpatients 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 itscell dry weight enriched with the trash fish wastewater.Similarly, Nannochloropsis sp. accumulated 9.3% oftotal fatty acids of its cell dry weight. However, C.saccarophila gave a higher value than that of

Nannochloropsis sp. The variation in total lipid contentof C. saccarophila may arise from salinity differencebetween the two cultures (a) and (b). As mentioned inthe aforesaid section that culture (a) was cultured in ahigher salinity concentration range of 18-22 ‰ whilethat of culture (b) with the range of 13-17 ‰. As aresult, the lower salinity level of culture (b) maypromote its total lipid content to 13.6% of total fattyacids.

Lipid and fatty acids are components of all cells, wherethey serve as a source of metabolites and energy aswell as membrane constituents. Lipids generallycontain fatty acids esterified to glycerol, sugar orbases. It is generally recognized that the lipid contentfor most microalgae is about 10% of dry weight undernormal autotrophic culture conditions (Cohen 1986).Some microalagal species can produce a high oilcontent under specific conditions. Oh et al. (2010)demonstrated that Chlorella minutissima gavemaximum biomass of 8.3 g-dry wt./L and lipid contentof 23.2% (w/w) subjected to warm seawater (>30 °C).Singh et al. (2010) reported that some efficient lipidproducer algae contain more than 30% of their cellweight as lipids. Gao et al. (2010) can grow Chlorellaprotothecoides heterotrophically with glucose as thecarbon source and accumulate high proportion oflipids. They also found that the lipid yield was 35.7%higher than that using glucose when sweet sorghumjuice used as the carbon source.

The variation in biomass and oil content of algaedepends on environmental factors such astemperature influence (Oh et al. 2010; Converti et al.2009; Boussiba et al. 1987), level of irradiance (Lv etal. 2010), salinity content (Renaud and Parry 1994),strength of aeration (Krienitz and Wirth 2006), CO2

mass transfer (Lv et al. 2010), concentration ofnitrogen in culture medium (Lv et al. 2010; Converti etal. 2009), algal species (Converti et al. 2009) andothers (Gao et al. 2010; Ong et al. 2010). The lipidcontent of Chlorella can be improved by optimumculture conditions (Lv et al. 2010). Converti et al.(2009) demonstrated that the lipid content of Chlorellaand Nannochloropsis was influenced by culturetemperature and nitrogen level in medium. The lipidcontent was double when the temperature wasincreased from 20 to 25ºC for Nannochloropsis. It wasfurther supported by Rocha et al. (2003) that theoptimal temperature for Nannochloropsis was 25±5ºC.However, the lipid content of Chlorella was reducedwhen temperature was higher than 30ºC. The lipidcontent can also be increased by reducing nitrogencontent 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 mMKNO3, 1.0% CO2 and 60 µmol photons m−2 s−1 at 25ºC,the highest lipid productivity of Chlorella vulgarisobtained was 40 mg/L/d. These studies indicated thatthe lipid content of Chlorella, Nannochloropsis andother algae can be increased by the adjustment ofvarious factors at their optimal range. Further study isrequired to optimize fatty acids and lipid production ofthe microalgae used to recycle the trash fishwastewater.

Several studies (Illustration 2-Table 2) pointed thatmost Chlorella species gave a calorific value range of6692-9536 Cal/g while a Nannochloropsis speciesalso gave a calorific value range of 5879-7815 Cal/g(Pan et al. 2010). The heating value of the microalgaeapproaches that of plant seed oil (Singh et al. 2010;Sams 1998). Therefore, from a sustainability viewpoint, this renewable bio-oil derived from the aquaticenvironment could be used as an alterative biofuel.

Conclusions

Marine pollution has had a disastrous impact onaquatic life and has reduced valuable resourcesderived from the ocean. The commercial activity ofcage fish farming is one of sources of marine pollution.The recycling of wastes generated from this activityinto potentially valuable products must be considered.The production of algae derived from trash fishwastewater is feasible and environmentally friendly.Furthermore, valuable products come from the naturalsea provide more alternatives in addition to theterrestrial to us. These products include health andpharmaceutical products and bio-oils, which aresustainable and renewable.

Acknowledgements

I deeply appreciate the support and participation ofthose fish farmers and my colleague for takingsamples and setting up the equipment and tools at site,workshop and laboratory. I would also like to expressmy sincere thanks to the testing laboratories for GCanalysis.

References

1.Aas E, Baussant, T, Balk L, Liewenborg B, AndersenOK (2000) PAH metabolites in bile, cytochromeP4501A and DNA adducts as environmental riskparameters for chronic oil exposure: a laboratory

experiment with Atlantic cod. Aqu Toxicol51(2):241-258 2.Adams VD (1991) Water and wastewaterexamination manual, 2nd printing. Lewis3.Almendras JME (1987) Acute nitrite toxicity andmethemoglobinemia in juvenile milkfish (Chanoschanos Forsskal). Aquaculture 61:33-404.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 inrats. Nutr Res 12(10):1273-1284 5.Arup O (1989) Assessment of the environmentalimpact of marine fish culture in Hong Kong. Finalreport submitted to the Environmental ProtectionDepartment, Hong Kong Government 6.Borowitzka MA (1988) Vitamins and fine chemicalsfrom micro-algae. In: Borowitzka MA, Borowitzka LJ(eds) Micro-algal biotechnology, pp 153-1967.Boussiba S, Vonshak A, Cohen Z, Avissar Y,Richmond A (1987) Lipid and biomass production bythe halotolerant microalga Nannochloropsis salina.Biomass 12(1):37-478.Bower CE, Hansen TH (1980) A simplifiedhydrazine-reduction method for determinating highconcentrations of nitrate in recirculated seawater.Aquaculture 21:281-286 9.Brown MR, Mular M, Miller I, Farmer C, Trenerry C(1999) The vitamin content of microalage used inaquaculture. J Appl Phycol 11:247-25510.Cha KH, Koo SY, Lee DU (2008) Antiproliferationeffects of carotenoids extracted from C.ellipsoidea andC.vulgaris on human colon cancer cells. Agri FoodChem 56:10521-1052611.Chen JC, Chin TS (1988) Acute toxicity of nitrite totiger prawn, Penaeus monodon larvae. Aquaculture69:253-26212.Cohen Z (1986) Products from microalgae. In:Richmond A (ed) Handbook of microalgal mass culture.CRC Press, pp 421-45413.Converti A, Casazza AA, Ortiz EY, Perego P, DelBorghi M (2009) Effect of temperature and nitrogenconcentration on the growth and lipid content ofNannochloropsis oculata and Chlorella vulgaris forbiodiesel production. Chem Eng Process: ProcessIntens 48(6):1146-115114.Curtis CL, Hughes CE, Flannery CR, Little CB,Harwood JL, Caterson B (2000) N-3 fatty acidsspecifically modulate catabolic factors involved inarticular cartilage degradation. J Biol Chem275(2):721-72415.Day AG, Brinkmann D, Franklin S, Espina K,Rudenko G, Roberts A, Howse KS (2009) Safetyevaluation of a high-lipid algal biomass from Chlorella



protothecoides. Regul Toxicol Pharmacol55(2):166-180 16.Demirbas A, Demirbas MF (2010) Importance ofalgae oil as a source of biodiesel. En Con Manage. doi:10.1016/j.enconman.2010.06.055 [accessed12.07.2010]17.Durmaz Y (2007) Vitamin E (α-tocopherol)production by the marine microalgae Nannochloropsisoculata (Eustigmatophyceae) in nitrogen limitation.Aquaculture 272 (1-4):717-72218.Feng YY, Hou LC, Ping NX, Ling TD, Kyo CI (2004)Development of mariculture and its impacts in Chinesecoastal waters. Rev Fish Biol Fish 14:1-1019.Foscarini R (1988) Intensive farming procedure forred seabream (Pagrus major) in Japan. Aquaculture72:191-24620.Foy RH, Rosell R (1991) Loadings of nitrogen andphosphorus from a Northern Ireland fish farm.Aquaculture 96:17-3021.Fulke AB, Mudliar SN, Yadav R, Shekh A,Srinivasan N, Ramanan R, Krishnamurthi K, Devi SS,Chakrabarti T (2010) Bio-mitigation of CO2, calciteformation and simultaneous biodiesel precursorsproduction using Chlorella sp. Bioresour Technol101(21):8473-8476 22.Gao CF, Zhai Y, Ding Y, Wu QY (2010) Applicationof sweet sorghum for biodiesel production byheterotrophic microalga Chlorella protothecoides. ApplEn 87(3):756-761 23.Gentile MP, Blanch HW (2001) Physiology andxanthophylls cycle. Biotechnol Bioeng 75:1-1224.Gillan DC, Danis B, Pernet P, Joly G, Dubois P(2005) Structure of sediment- associated microbialcommunities along a heavy-metal contaminationgradient in the marine environment. Appl EnvironMicrobiol 71(2):679–90625.Gladu PK, Patterson GW, Wikfors GH, Smith BC(1995) Sterol, fatty acid, and pigment characteristics ofUTEX2341, a marine estigmatophyte identifiedpreviously as Chlorella minutissima (Chlorophyceae).J Phycol 31(5):774-77726.Glazer AN, Stryer L (1984) Emerging techniques.Trends Biochem Sci 9:423-42727.Guzman S, Gato A, Lamela M, Freire-Garabal M,Cal le ja JM (2003) Ant i - inf lammatory andimmunomodulatory activities of polysaccharide fromChlorella stigamatophora and Phaeodactylumtricornutum. Phytother Res 17:665-67028.Hamada M, Kumagai H (1988) Chemicalcomposit ion of Sardine scale. Nip Sui Gak54(11):1987-199229.Harrison PJ, Thomas TE (1988) Biomassmeasurements: protein determination. In: Lobban CS,Chapman DJ, Kremer BP (eds) Experimental

phycology. Cambridge University Press, pp 27-3430.Hasegawa T, Matsuguchi T, Noda K., Tanaka K,Kumamoto S, Shoyama Y, Yoshikai Y (2002) Toll-likereceptor 2 is at least partly involved in the antitumoractivity of glycoprotein from Chlorella vulgaris. IntImmunopharmacol 2(4):579-589 31.Hata M, Sato Y, Yamaguchi T (1988) The chemicaland amino acid compositions in tissues of cultured andwild Coho Salmon, Oncorhynchus kisutch. Nip SuiGak 54(8):1365-137032.Holum JR (1985) Introduction to organic andbiological chemistry. CRC Press, pp 340-34233.Illman AM, Scragg AH, Shales SW (2000) Increasein Chlorella strains calorific values when grown in lownitrogen medium. Enzy Microb Technol 27(8):631-63534.Kamal-Eldin A, Yanishlieva NV (2002) N-3 fattyacids for human nutrition: stability considerations. EurJ Lip Sci Technol 104:825-83635.Kamerycah Inc (2010) Fucoidan Umi No Shizuku.Dba Kanefuku America. Available at, [accessed17.08.2010]36.Kaplan D, Richmond AE, Dubinsky Z, Aaronson S(1986) Algal nutrition. In: Richmond A (ed) Handbookof microalgal mass culture. CRC Press, pp 147-19837.Khansari FE, Ghazi-Khansari M, Abdollahi M (2005)Heavy metals content of canned tuna fish. Food Chem93:293–29638.Kralovec JA, Metera KL, Kumar JR, Watson LV,Girouard GS, Guan Y, Carr RI, Barrow CJ, Ewart HS(2007) Immunostimulatory principles from Chlorellapyrenoidosa-part 1: isolation and biologicalassessment in vitro. Phytomedicine 14(1):57-6439.Krienitz L, Wirth M (2006) The high content ofpolyunsaturated fatty acids in Nannochloropsislimnetica (Eustigmatophyceae) and its implication forfood web interactions, freshwater aquaculture andbiotechnology. Limnol-Ecol Manage Inland Waters36(3):204-210 40.Krom MD, Erez J, Porter CB, Ellner S (1989)Phytoplankton nutrient uptake dynamics in Earthenmarine fishponds under Winter and Summerconditions. Aquaculture 76:237-25341.Kromhout D, Bosschieter EB, Coulander CL (1985)The inverse relation between fish consumption and20-year mortality from coronary heart disease. NewEng J Med 312(19):1205-120942.Lam CWY (1990) Pollution effects on marine fishculture in Hong Kong. Asian Mar Biol 7:1-743.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ(1951) Protein measurement with Folin phenol reagent.J Biol Chem 193:265-27544.Lv JM, Cheng LH, Xu XH, Zhang L, Chen HL (2010)Enhanced lipid production of Chlorella vulgaris byadjustment of cultivation conditions. Bioresour Technol



101(17):6797-6804 45.Maldonado M, Carmona MC, Echeverria Y, RiesgoA (2005) The environmental impact of Mediterraneancage fish farms at semi-exposed locations: does itneed a re-assessment? Helgoland Mar Res59:121-13546.Maruyama H, Tamauchi H, Lizuka M, Nakano T(2006) The role of NK cells in antitumor activity ofdietary fucoidan from Undaria pinnatifida sporophylls(Mekabu). Planta Media 72:1415-141747.Merck (1987) Ammonium determination. In: Rapidtest handbook. Merck, p 30148.Nichols BW, Appleby RS (1969) The distributionand biosynthesis of arachidonic acid in algae.Phytochemistry 8:423-42749.Nishino H, Murakoshi M, Tokuda H, Stomi Y (2008)Cancer prevention by carotenoids. Arc BiochemBiophys. doi:10.1016/j.abb.2008.09.011 [accessed15.09.2008]50.Oh SH, Kwon MC, Choi WY, Seo YC, Kim GB,Kang DH, Lee SY, Lee HY (2010) Long-term outdoorcultivation by perfusing spent medium for biodieselproduction from Chlorella minutissima. J Biosci Bioeng110(2):194-20051.Olsen LM, Holmer M, Olsen Y (2008) Finalreport-perspectives of nutrient emission from fishaquaculture in coastal waters: literature review withevaluated state of knowledge. Fishery andAquaculture Industry Research Fund, Norway52.Ong SC, Kao CY, Chiu SY, Tsai MT, Lin CS (2010)Characterization of the thermal-tolerant mutants ofChlorella sp. with high growth rate and application inoutdoor photobioreactor cultivation. Bioresour Technol101(8):2880-2883 53.Pan P, Hu CW, Yang WY, Li YS, Dong LL, Zhu LF,Tong DM, Qing RW, Fan Y (2010) The direct pyrolysisand catalytic pyrolysis of Nannochloropsis sp. residuefor renewable bio-oi ls. Bioresour Technol101(12):4593-4599 54.Payne JF, Penrose WR (1975) Induction of arylhydrocarbon (benzo[a]pyrene) hydroxylase in fish bypetroleum. Bull Environ Contam Toxicol 14(1):112-11655.Peterson GL (1979) Review of the Folin phenolprotein quantitation method of Lowry, Rosebrough,Farr and Randall. Analy Biochem 100:201-22056.Phillips MJ (1985) Analysis of nutrients. In: StirlingHP (ed) Chemical and biological methods of wateranalysis for aquaculturists. University of Stirling57.Rebolloso-Fuentes MM, Navarvo-Perez A,Garcia-Camacho F, Ramos-Miras JJ, Guil-Guerrero JL(2001) Biomass nutrient profiles of the microalgaNannochloropsis. J Agri Food Chem 49:2966-297258.Renaud SM, Parry DL (1994) Microalgae for use in

tropical aquaculture II: effect of salinity on growth,gross chemical composition and fatty acid compositionof three species of marine microalgae. J Appl Phycol6:347-35659.Rocha JMS, Garcia JEC, Henriques MHF (2003)Growth aspects of the marine microalga,Nannoch lo rops is gad i tana. B iomol Eng20(4-6):237-242 60.Rodriguez-Garcia I, Guil-Guerrero JL (2008)Evaluation of the antioxidant activity of threemicroalgal species for use as dietary supplements andin the preservat ion of foods. Food Chem108(3):1023-1026 61.Ross AB, Biller P, Kubacki ML, Li H, Lea-Langton A,Jones JM (2010) Hydrothermal processing ofmicroalgae using alkali and organic acids. Fuel89(9):2234-224362.Rothbard S (1975) Control of Euplotes sp. byformalin in growth tanks of Chlorella sp. used asgrowth medium for the rotifer, Brachionus plicatiliswhich serves as feed for hatchings. Bamidgeh27(4):100-10963.Salvesen I, Reitan KI, Skjermo J, Oie G (2000)Microbial environments in marine larviculture: impactsof algal growth rate on bacterial load in six microalgae.Aquaculture 8:275-28764.Sams T (1998) Exhaust components of biofuelsunder real world engine conditions. In: Martini N,Schell J (eds) Proceedings of the symposium of plantoils as fuels: present state of science and futuredevelopments. Springer, pp 64-7565.Sarkar D, Sharma A, Talukder G (1994)Chlorophyll and chlorophyllin as modifiers of genotoxiceffects. Mutat Res 318(3):239-24766.Schoen S (1988) Cell counting. In: Lobban CS,Chapman DJ, Kremer BP (eds) Experimentalphycology. Cambridge University Press, pp 16-2267.Schuler JF, Diller VM, Kersten HJ (1952)Preferential assimilation of ammonium ion by Chlorellavulgaris. Plant Physiol 27:299-30368.Scragg AH, Illman AM, Carden A, Shales SW(2002) Growth of microalgae with increased calorificvalues in a tubular bioreactor. Biom Bioen 23(1):67-7369.Seaherb (2010) Fucoidan. Available at, [accessed17.08.2010]70.Singh A, Nigam PS, Murphy JD (2010) Mechanismand challenges in commercialisation of algal biofuels.B i o r e s o u r T e c h n o l 2 0 1 0 . doi:10.1016/j.biortech.2010.06.057 [accessed05.07.2010]71.Strickland JDH, Parsons TR (1972) A practicalhandbook of seawater analysis, 2nd ed. Bulletin No.167, Fisheries Research Board of Canada72.Sun Z, Peng XF, Liu J, Fan KW, Wang MF, Chen F



(2010) Inhibitory effects of microalgal extracts on theformation of advanced glycation endproducts (AGEs).Food Chem 120(1):261-26773.Syrett PJ, Fowden L (1952) The assimilation ofammonia by nitrogen-starved cells of Chlorellavulgaris. III. The effect of addition of glucose onproducts of assimilation. Physiol Plant 5:558-56674.Tanaka K, Shoyama Y, Yamada A, Noda K,Konishi F, Nomoto K (2001) Immunopotentiatingeffects of a glycoprotein from Chlorella vulgaris strainCK and its characteristics. Stud Nat Prod Chem25(6):429-458 75.Trussell RP (1972) The percent unionizedammonia in aqueous ammonia solutions at differentpH levels and temperature. J Fish Res Board Can29:1505-1507 76.Van Rooyen J, Esterhuyse AJ, Engelbrecht AM, duToit EF (2008) Health benefits of a natural carotenoidrich oil: a proposed mechanism of protection againstischaemia/reperfusion injury. Asia Pacif J Clin Nutr17(S1):316-319 77.Wang HM, Pan JL, Chen CY, Chiu CC, Yang MH,Chang HW, Chang JS (2010) Identification of anti-lungcancer extract from Chlorella vulgaris C-C byantioxidant property using supercritical carbon dioxidee x t r a c t i o n . P r o c e s s B i o c h e m .doi:10.1016/j.procbio.2010.05.023 [accessed28.05.2010]78.Watanabe T (1982) The production of foodorganisms with particular emphasis rotifers. In:FAO/UNDP Training course on seabass spawning andlarval rearing held at the National Institute of CoastalAquaculture (NICA), Sonhkila, Thailand, pp 26-2879.WHO (World Health Organization) (1998)Carotenoids, vol. 2. IARC Handbook of cancerprevention. International Agency for Research onCancer80.Wigmore SJ, Barber MD, Ross JA, Tisdale MJ,Fearon CH (2000) Effect of oral Eicosapentaenoic acidon weight loss in patients with pancreatic cancer. NutrCan 36(2):177-18481.Wu RSS, Lee JHW (1989) Grow-out mariculturetechniques in tropical waters: a case study ofproblems and solutions in Hong Kong. Adv Trop Aqu9:729-73682.Wu RSS (1995) The environmental impact ofmarine fish culture: towards a sustainable future. MarPoll Bull 31(4-12):159-166 83.Yaeyama (2010) The nutritious superstar-greenalga foodstuffs: Chlorella (Midori-no-sachi). Availableat, http://www.yaeyama.com [accessed 17.08.2010]84.Yoonaisil T, Hertrampf JW (2006) An effect ofnucleotides in the Asian seabass: a uniform and fastergrowth of fish fed chopped trash fish with nucleotides

was demonstrated in this trail with Asian seabass inThailand. Aqua Culture Asia Pacific Magazine,November/December, 20-2185.Zar JH (1984) Biostatistical analysis, 2nd ed.Prentice-Hall.



Parameter Nutrients oftrash fish (mg/gdry wt)

Increasepollutants ofchopped fish(mg/L)

Composition oftrash fish as fedbasis (%)

Sardine scale(g/100g scale)

Integument ofwild CohoSalmon (%)

N-NO2 a 0.0314 0.0046 - - -

N- NO3 b 0.253 NDg - - -

N-NH3 c 1.547 0.1355 - - -

TONd 10.3 3.295 - - -

TNe 12.1 3.4377 - 6.24 18.9

P-PO4f 2.548 0.9307 1.21 21.08 (g/100gash)

-

Protein - - 11.2 36.25 -Reference Arup 1989 Arup 1989 Yoonaisil and

H e r t r a m p f2006

Hamada andKumagai1988

Hata et al.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.

Illustrations

Illustration 1

Table 1 Chemical composition of trash fish from various sources.



Source Chlorella Nannochloropsis Rapeseed oil

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

Calorific value (Cal/g) 7983-9536 6931 6692 6931 5879-7815 8843

Reference Ross et al.2010 Fulke et al.2010 Scragg et al.2002 Illman et al. 2000 Pan et al. 2010 Sams 1998

Illustration 2

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



Illustration 3

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



Illustration 4

Fig. 2. Growth curves of cultures (a) and (b) of Chlorella saccharophila and Nannochloropsis sp. enriched with trash fishwastewater.



Fatty acid Chlorella saccharophila(a) (b)

Nanochloropsis sp.

12:0 0.1±0.06a trace b 0.4±0.04a13:0 0.4±0.24 0.8±0.10a traceb14:0 0.3±0.07 0.3±0.04 4.0±0.2414:1 0.6±0.11 1.0±0.17 0.0±0.0015:0 0.3±0.06 0.2±0.04 0.2±0.0315.1 0.0±0.00 0.2±0.04 0.0±0.0016:0 0.0±0.00 16.6±0.36 12.3±0.14Unknown 5.5±0.99 7.1±0.17 26.4±0.4116:1 2.3±0.13 3.9±0.17 6.5±0.3116:2 7.3±0.09 5.2±0.18 2.1±0.1317:0 0.6±0.04 0.2±0.02 0.5±0.0517:1 4.8±0.32 10.8±0.29 0.9±0.0918:0 1.7±0.67 2.4±0.47 1.1±0.3118:1n9c 6.0±1.75 16.8±0.19 4.1±0.2718:2n-6(LA) 32.6±3.27 14.4±0.39 1.8±0.0718:3n-3(ALA) 13.0±0.99 19.6±0.51 0.3±0.0718:4 0.2±0.09 trace 0.0±0.020:0 0.1±0.04 trace 0.0±0.020:2 0.2±0.21 0.0±0.0 0.3±0.05

Illustration 5

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



20:3 0.0±0.0 0.0±0.0 0.5±0.0720:4n-6(ARA) 0.0±0.0 0.0±0.0 4.1±0.0520:5n-3(EPA) 0.1±0.17 0.2±0.02 34.2±0.5022:5n-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±0.07TFAc 10.7±0.68 13.6±0.76 9.3±0.05EPA (mg/g) 0.1±0.02 0.2±0.04 31.8±0.31DHA (mg/g) 0.2±0.05

0.3±0.070.2±0.050.3±0.07

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%.



DisclaimerThis article has been downloaded from WebmedCentral. With our unique author driven post publication peerreview, contents posted on this web portal do not undergo any prepublication peer or editorial review. It iscompletely the responsibility of the authors to ensure not only scientific and ethical standards of the manuscriptbut also its grammatical accuracy. Authors must ensure that they obtain all the necessary permissions beforesubmitting any information that requires obtaining a consent or approval from a third party. Authors should alsoensure not to submit any information which they do not have the copyright of or of which they have transferredthe copyrights to a third party.

Contents on WebmedCentral are purely for biomedical researchers and scientists. They are not meant to cater tothe needs of an individual patient. The web portal or any content(s) therein is neither designed to support, norreplace, the relationship that exists between a patient/site visitor and his/her physician. Your use of theWebmedCentral site and its contents is entirely at your own risk. We do not take any responsibility for any harmthat you may suffer or inflict on a third person by following the contents of this website.


Recycling of Nutrients from Trash Fish Wastewater for Microalgae

Documents

Transcript of Recycling of Nutrients from Trash Fish Wastewater for Microalgae