Some species of micro-algae have been cultured intensively such as Spirulina sp

Part I

LITERATURE STUDY ON PROSPECT DEVELOPMENT OF MICRO-ALGAE IN WEST NUSA TENGGARA1.1 Introduction

In a very basic knowledge, micro-algae (phytoplankton) are well known as a foundation of food chains (or webs) in aquatic ecosystems. Without phytoplankton, there will be no living things can survive in the earth planet. Phytoplankton form the foundation on which the very survival of aquatic food webs depends (Hader et al., 1995). Harder et al. (1995), also reported that depletion of ozone layer of 16% could result in a 5% loss in phytoplankton, which equals a loss of about 7 million tons of fish per year.

Researches on investigation of micro-algal based production for alternative useful natural substances or environmental friendly natural sources for oils and food are developed rapidly. In the early 1980s, algal biotechnology has focused on commercial production of high value substances such as carotenoids, lipids, fatty acid, and pharmaceuticals (Knox et al., 1994, p. 751). The other most possible sources of environment friendly oils and foods (food suplements) are algae, mainly micro-algae (phytoplankton). No less than around 300 fresh and sea waters species of micro-lagae, mostly green algae and diatoms have been researched for finding alternative natural sources of oils and foods or for finding species that can grow under extremes temperature, salinity and pH (Chaugule, 2009,). Panji and Suhariyanto (2003), reported, that Spirulina platensis contain about 58% protein on dry matter basis. Large amounts of carotenoid (,-carotene) can be well accumulated Dunaliella salina grown at high silinity (10 times the salinity of sea water) with high light intensity (Knox et al., 1994, p.751). Algae is a darling of biofuel backers because it requires little to no maintenance while growing and doesnt compete with land for agriculture (Anonimous, 2009c).

Some other basic potential needs of human can be possiblely taken from micro-lagae. Development of algae cultivation as an energy source of an economically and eco-friendly alternative biofuels (Chaugule, 2009) is only possible to be done by intensive culturing of algae. Furthermore, the autors mention that, (1) micro-algae is, 8 to 25 times productive than palm and 40 to 120 times than rapeseed, the highest potential energy yield temperate vegetable oil crop; (2) The total oil content in algae can be up to 70% of their dry weight; (3) Micro-algae are capable of producing more than 30 times the amount of oil (per year per unit area of land) when compared to oil seed crops (Chaugule, 2009). It was also reported that 30 grams of algae per square meter per day can be possible produced, which would yield 4,000 gallons of biodiesel fuel per acre annually (Anonimous, 2009a). Production rates of over 100,000 gallons per acre per year considered to be fully achievable using closed photo bio-reactor systems (Anonimous, 2009b).

Tropical waters could be provide much more species of fresh and sea water algae that potential for sources of oils dan foods. The most possible way to take these benefits from algae is cultivation of unialgal species. Study and investigation of some potential tropical species of micro-alga are urgently to be done for fulfil of some fundamental economic needs (mainly foods and biofuels).1.2 Micro-algae and Potency To Be Cultured

1.2.1 The Important of Micro-algaeSome species of micro-algae have been cultured intensively such as Spirulina sp. for feeding of fish (Suhariyanto, 1992, personal comunication), Chlorella sp. (Prihantini, 1993) and Scenedesmus sp., and Micrastrias sp. for food suplement (Prihantini, 1992, personal comunication), Nannoclhoropsis oculata for feeding of larvae and zooplankton in laboratory treatment (Suharni, 2006), and Chaetoceros gracilis for feeding of pearl larvae in laboratory (Sony, 2003, personal comunication). Spirulina has been well recognized and used worldwide as a tradisional source of protein in food industry (Vonshak, 2009). Spirulina has also been cultured for foods (Hi-liena, :Linagreen, Linavina, and Spilin) resources and traded comercially (Kabinawa, 1993). Richmond (1986) reported that Spirulina can be used as food for malnutrision teraphy, and for inhibiting and curing cancer. Spirulina platensis has been also cultured for fish feed (Panji and Suhariyanto, 2003). Chlorella has been applied as protein and vitamin suplement resources (Tampubolon and Silalahi, 1993). Chlorella based Products are Sun-Chlorella, Sun-Siberian, Sun-Ukogi, and wakasa (Kabinawa, 1993). Chlorella contents a number of important minerals such as : vitamin, enzyme, saturated and unsaturated patty acids, carbohydrate, and pigment. All these minerals are essential for natural feed of fishes, molusca, and for source of food (Steenblock, 1987). Scenedesmus has also been used as food in Japan (Tampubolon and Silalahi, 1993). Scenedesmus provides economic comercial value to be converted as food suplement, since this alga contents higher protein compared to other cultivated crops (Richmond, 1986). Scenedesmus can survive in polution waters (Trainor, 1984), so this micro-alga has been widely used for wastes treatment. Some researches on the population growth of Scenedesmus in liquid westes were done by Richmond (1986), Lavoie and de la Noe (1986), and Agustini and Susilaningsih (1997). Chlorella and Scenedesmus were studied as sources of protein (Knox et al., 1994, p.751).

Some high value economical products producing algae such as Spirulina, Scenedesmus, and Microcystis, including Anabaena, Nostoc, and Oscillatoria were also prominent recorded abundently in the waters of Lombok island (Japa, unpublised data). On the other hand, some of these algae (Microcystis, Anabaena, Nostoc, and Oscillatoria) were also reported as toxin producing algae. Japa and Hallegraeff (2005), fully identified five species of sea water pennate diatom genus Pseudo-nitzschia, Pseudo-nitzschia froudulenta, P. granii, P. pungens, P. pseudodelicatissima, and P. turgidula in coastal waters of Lombok island. Two of these diatom (P. pungens and P. pseudodelicatissima) have reported responsible on neurotoxin production in Argetinan waters (Ferrario et al., 1999).

1.2.2 Cultivated Species of Micro-algae

In some very small scale of laboratory unialgal cultures were conducted for small researches such as : Scenedesmus sp. (Yoosy, 2000), Chlorella spp. (Johari, 1999), Chlorella (Prihantini, 1993), Chlorella pyrenoidosa (Atria, 1994, and Hasanah, 1997). The average peak (22.812 x 106 sel/ml) population density of Chlorella spp. was reported to occure in the 20% concentration of topu liquid wastes treatment (Johari, 1999). Pure uniselular stock cultures of Chlorella spp. in this research were developed in Beneck medium. Meanwhile, in the media made of coconut water, Chlorella pyrenoldosa reached an average peak (41,687,500 sel/ml) population in 0.5% treatment (Hasanah, 1997). Scenedesmus dimorphus - A unicellular algae in the class Chlorophyceae is one of the preferred species for oil yield for biodiesel HYPERLINK "http://www.oilgae.com/ref/glos/biodiesel.html" \o "Click here to see Biodiesel, Definition, Glossary, Details"

(Anonimous, 2009b). Nannochloropsis oculata is also a pavorite marine species cultured in the division of fisheries of the Indonesian Sciences Agency, Pemenang Lombok Barat. Nannochloris oculata is also called Nannochloropsis salina, and Spirulina sp., are currently being studied for their suitability as a mass-oil producing crop (Anonimous, 2009b). Spirulina sp. is also cultured for oils production. Spirulina platensis grown on media containing latex serum content carotenoid (-carotene) in high amount (Panji and Suhariyanto, 2001). Panji et al. (2005) reported, Spirulina platensis culturing on serum latex medium illuminated with 20 W TL lamp at room temperature produced a mximum biomass of superpxide dismutase (SOD). The culture results showed that Spirulina platensis grows successfully in diluted human urine, and yields maximal biomass at urine dilution ratios of 140~240 (Feng and Wu, 2006). Botryococcus sp. reported to have capability in producing biodiesel higher than palm or other introduced natural sources such as safflower, sunflower, palm, soybean, coconut, and castor oils plant (Kabinawa, 2009).

1.2.3 Mass Cultivation of Micro-algae

Dunaliella bardawil (= Dunaliella salina) has been intensively cultured for natural sources of , -carotene, glyserol and protein meal in Australia, Israel, and United States (Borowitzka and Borowitzka, 1995, in Clayton and King, 1995). The world largest open culturing systems fo Dunaliella salina are conducted at Hutt Lagoon in Western Australia and Whyalla in South Australia (Knox et al., 1994, p.751). Large scale system (100 m2 surface area and 7 m3 volume) of Spirulina culture has been operated in France (Chaumont et al., 1988 in Clayton and King, 1995). In Indonesia, Spirulina platensis has also been cultured in commercial scale (2,400 m2 ponds) for food suplement (Panji et al., 2004), fish feed (Suharyanto, 2003, Personal Comunication; Panji dan Suhariyanto 2001; 2003); and for multifunctional foods (Panji et al., 2005).

1.2.4 Miro-alga of Lombok and Sumbawa Islands

1.2.4.1 Fresh Water Micro-algae Species

Inventarisation of fresh water micro-algae species from around Lombok and Sumbawa island was done by Jumadil (2004), Latifah (2005), Japa (2007).

Species of fresh water micro-algae recorded abundantly in the waters of fish pond, Lingsar West Lombok were Fragillaria sp., Aktinastrum hantzehi, and Oscillatoria sp. (Latifah, 2005). Fresh water micro-algae such as Spirulina, Scenedesmus, Nostoc, Anabaena, Phacus, Oscillatoria, Dinobrion, and Spyrogira were recorded abundantly in the early rice planting season in paddy fields (Japa, 2007, and Japa unpublished data). Some these micro-algae were reported to have capability in producing high economic value products including environmental friendly biofuels (Kabinawa, 1993; Richmond, 1986; Tampubolon and Silalahi, 1993; and Anonimous, 2009b).1.2.4.2 Sea Water Miro-algae Species

Sea water micro-algae from Indonesia, mainly Lombok and Sumbawa islands never been researhced, except the diatom community of Sumatera and Java sea waters done by Allen and Cupp in 1935 (Allen and Cupp, 1935), and Hustett in 1948 for the community of Sumatera, Java, and Bali (Hustett, 1948). The community of sea water micro-algae of around coastal waters of both Lombok and Sumbawa islands was reported by Japa et al. (1997), Japa (2000), Feranita (2002), Japa and Suripto (2003), Sumarni (2003), Japa and Karnan (2004), Japa et al. (2004), Astuti (2004), Japa and halleagraeff (2005), Japa and Santoso (2005), Harisanty et al. (2005), . Sources of both fresh and sea waters samples collected in around Lombok and Sumbawa island are presented in Table 1.

Japa and Karnan (2004) reported total of 25 families containing 129 species of fully identified of phytoplankton from coastal waters of Mataram. The most abondance genus of this phytoplankton community is Chaetoceros. Nineteen families (14 families of Bacillariophyceae class and 5 families of Dinophyceae class) containing 71 species of phytoplankton were fully identifyed from Lembar harbur waters (Japa and Santoso, 2005). The family containing most number of members is Chaetoceraceae. Diatom genus Chaeotceros was also reported to abondant in Lembar houbur waters (Japa, 2000), in the coastal waters of Kerta Sari West Sumbawa (Sumarni, 2003), in the fish sanctuary of gili Ranggo Serewe bay east Lombok (Japa et al., 2004). Maidiati (2004) identified 39 taxa of Dinophyta in the coastal waters of Sekotong, West Lombok. Taxa of class Bacillariophyceae and Dinophyceae were recorded 160 and 29, respectively in Ampenan bay (Aisyah, 2004). Sari (2004), identified 111 taxa of Bacillariophyceae in Sekotong bay waters. In Alas Strait, Rosayanti (2005), identified 140 species of diatom class Bacillariophyceae, and genus Chaetoceros was the most abundant taxa. The most dominant species was Asterionella japonica and Chaetoceros mitra. Dominant taxa of micro-alga recorded in some different phytoplankton water samples is given in Table 1.

Table 1. Proportion Number of Taxa in Different Water Sample SourcesNo.Sample Colectors and Dates of ColectionLocalityDominant Taxa

1. Japa, Suripto, and Zulkifli, August 1997Asin Lake, Meno island Thalassiothrix

2. Japa, July November 2000Lembar Bay, West Lombok Chaetoceros

3. Feranita, January February, 2001 Kayangan Horbour, East Lombok Chaetoceros

4. Sulistiawati, July Ausgust, 2002 Batujai Dam, Central Lombok Microcystis

5. Maliana, Feberuary 2003 Amor-Amor Bay, West Lombok Skeletonema

6. Japa and Suripto, 2003 Coastal Water Sambelia, East Lombok Chaetoceros

7. Sumarni, April 2003 Kerta Sari, West Sumbawa Chaetoceros

8. Japa and Karnan, June 2004 Coastal Waters of Ampenan Asterionella

9. Japa and Suripto, August 2004 Coastal Waters of Mataram Chaetoceros

11. Sari, March May 2003 Coastal Waters of Sekotong, South West Lombok Asterionella

12. Japa dan Santoso, October 2005 Lembar Horbour, South Mest Lombok Chaetoceros

13. Latifah, May 2005 Fish Pond, Lingsar West Lombok Scenedesmus

14. Japa, July 2003 Saleh Bay, Bima Chaetoceros

15. Susilayati, November 2006 Lingsar, West Lombok Aktinastrum

16. Oktapiani, July August, 2007 Pengga Dam, South Central Lombok Peridinium

18. Japa, Desember 2007 Batujai Dam, Central Lombok Microcystis

17. Husnaini August September, 2007 Grupuk Bay Kuta, South Central Lombok Rhizosolenia

18. Japa, June 2007 Dam of Muncan Central Lombok Nostoc

19. Japa, February 2009 Pengga Dam, South Central Lombok Nostoc

20. Japa, May 2009 Paddy Fields Central Lombok Spirulina

21. Japa, Juni 2009 Asin Lake, Meno island, North West Lombok Pleurosigma

1.3 Media for Micro-algae CultivationSuch media commonly used for unimicro-algae culturing are :1.3.1 Beneck Medium

Composition : in 1000 ml distil water added with 200 mg KH2PO4, 100 mgr MgSO4.7H2O, 500 mgr NaNO3, and 5 mgr FeCl3.6H2O (Atria, 1994).

1.3.2 Allen Medium

Composition : 99,9 ml of distil water added with 132 mgr (NH4)SO4 , 27,2 mgr K2HPO4 , 24,6 mgr MgSO4.7H2O, 7,4 mgr CaCl2.2H2O and 0,01 ml Allen Metals, pH 2,5 (Anonimous, 1997).

Composition of Allen Metals : 100 ml of distil water added with 30,16 mgr Fe-EDTA, 1,79 mgr MnCl2.4H2O, 2,86 mgr H3BO3, 0,22 mgr ZnSO4.7H2O, 0,079 mgr CuSO4.5H2O, 0.13 mgr (NH4)6MoO24.4H2O, and 0.023 mgr NH4VO3.

1.3.3 HUT Medium

Composition : 100 ml of distil water added with 2 mgr K2HPO4 , 2,5 mgr MgSO4.7H2O, 40 mgr Sodium acetate, 4 mgr Potassium citrate, 60 mgr Polypeptone, 40 mgr Yeast extract, 0,05 (gr vitamin B12, and 0,04 mgr Thiamine HCl, pH 6,4 (Anonimous, 1997).

1.3.4 MKM Medium

Composition : 50 ml of distil water added with 50 ml sea water, 1,5 gr agar, 250 (gr Fe-citrate, 2 mgr MgSO4.7H2O, 2,5 mgr K2HPO4 , and 75 mgr KNO3 (Anonimous, 1997).

1.3.5 Larutan A5 Medium

Composition : 100 ml of distil water added with 286 mgr H3BO3, 250 mgr MnSO4.7H2O, 22,2 mgr ZnSO4.7H2O, 7,9 mgr CuSO4.5H2O, and 2,1 mgr Na2MoO4.2H2O (Anonimous, 1997).

1.4 Materials and Method

1.4.1 Method

This project is going to be done in 3 steps. Two braodly steps are 2nd and 3th. In the first step, the project activities are focused on study literatures and mapping of natural micro-algae sources in Lombok island. Culturing of unimicro-algae in small or laboratory scale is going to be started in the 2nd step. During this step, trial and error in cultivation unimicro-lagae is the main activity. Culturing of some different species on some different natural basis media will also be done in this step. The main target of Lombok local fresh water strains of micro-algae to be cultured are Botryococcus sp., Closterium sp., and Scenedesmus sp.. The fastest growing strains are then culturred in larger scale in the 3th step. Larger scale of culturing and extraction or obtaining of commercial products of micro-algae such as bio-fuels are also going to done in final step of this project. Larger scale culturing will be conducted in both outdoor and indoor (laboratory) treatments.

1.4.2 Materials

Artificial media used for culturing are Beneck medium (Table 1), Bristol's modified medium (Table 2), and modified CHU 13 medium. (Table 3). Natural basis media will prepared by using liquid weste of tofu industry and cocconut waters. Both the tofu liquid westes and cocconut waters are present abundance in Lombok island, mainly in the West Lombok.

1.4.3 Micro-algal Culture Harvesting SystemsA Cost-effective harvesting major methods for micro-algal culturing are filtration, sedimentation, flotation and flocculation (Borowitzka and Borowitzka, 1995, in Clayton and King, 1995). Filter cloths or micro-screens of 50 to 100 m pore size is useful for filtering Spirulina (Richmond, 1988, in Clayton and King, 1995). Filtration is the main method of harvesting applied in this project.1.5. BudgetNo.Activity and MaterialsVol.UnitVol.UnitPrice ($USA)

1.Visit Study

a.LIPI Pemenang North Lombok1time40

b.Biotech. of Forestry and LIPI Cibinong, Bogor2Person1time200400

Sub-total440

2.Searching refferences1pack.9898

3.Transport for Field investigation and sampling

a.Mataram - North Lombok1Time4040

b.Mataram - Central Lombok1Time5050

c.Mataram - East Lombok1Time7070

Sub-total160

4.Small scale of threatments (preliminary screening)

a.Meterials: Bristol's Modified Medium2pack.4.01198.0238

b.Equipments:

1).Cultur continer (Jar)2unit3.16.2

2).TL 20 Watt1unit8.58.5

3).Room Preparations1month4040

4).Thermometer2unit5.110.2

5).Spectrophotometer2month1122

6).Autoclave2month1020

7).24 hours timer swich2unit40.881.6

8).Silicon tube1.7m24.341.31

9).Flex slang1.7m11.519.55

10).Micropipette2unit18.236.4

11).Sedgwick-rafter2unit12.424.8

12).Filter cartridge 20, 5, 0,5 um1set150150

13).CO2 regulator thank1set9898

14).Sample bottle2unit612

15).Glass aparattus1pack.4242

16).Fresh water2month714

17).Aquades100Liter0.1616

Sub-total650.5838

5.Personil

a.Junior Consultance 5 days a week2people2month100400

b.Senior Consultance 2 days a week1person2month200400

c.Enumerator (Laborant)2people2month50200

Sub-total1000

6.Institutional Fee (Faculty and University)2month250500

TOTAL COST FOR 1St STEP2498.584

Part II

RESEARCH PLAN (2nd phase)SAMPLING OF FRESH WATER MICRO-ALGAE IN WEST NUSA TENGGARA2.1 Scope Research

2.1.1 Survey Location

2.1.2 Sampling and Identification

2.1.3 Small Scale Culturing

2.1.4 Harvesting

2.1.5 Analyzing for biomass production2.2 Materials and Methods

2.2.1 Equipments

a. Aerators

b. Plankton netc. Pyrex Bottles or Jard. Sample Bottles

e. Spectrophotometer2.2.2 Materials

2.2.2.1. Chemicals

Chemical materials needed are presented in Table 1, 2 and 3 below.

a. Beneck Medium

Table 1. Composition of Beneck Medium: in 1000 ml distil water added with :No.SubstancesWeighVolum Prepared (mg/L)

1.KH2PO4200 mg

2.MgSO4.7H2O100 mgr

3.NaNO3500 mgr

4.FeCl3.6H2O5 mgr

b. Bristol's Modified Medium

Table 2. Composition of six stock solutions, each with one of the following salts dissolved in 400 milliliters of water:

No.SubstancesWeigh (gr)Volum Prepared (ml)

1.Sodium nitrate (NaNO3)10400

2.Calcium chloride (CaCl2 2H2O)1400

3.Magnesium sulfate (MgSO4 7H2O)3400

4.Potassium phosphate dibasic (K2HPO4)3400

5.Potassium phosphate monobasic (KH2PO4)7400

6.Sodium chloride (NaCl)1400

Note: Take 10 mL of each stock solution and add to 900 mL of distilled water. Next add one drop of 1% ferric chloride solution, 40 mL of Pringsheim's soil-water extract and autoclave. This will give you a total volume of one liter of medium. c. CHU 13 Medium

Table 3. Composition of modified CHU 13 medium

No.SubstancesVolum (mg/L)Volum Prepared (mg/L)

1. KNO3 400400,000

2. K2HPO4 8080,000

3. MgSO4 heptahydrate 200200,000

4. CaCl2 dihydrate 107107,000

5. Ferric Citrate 2020,000

6. Citric acid 100100,000

7. CoCl2 dihydrate 107107,000

8. H3BO3 5.725,720

9. MnCl2 tetrahydrate 3.673,670

10. ZnSO4 heptahydrate 0.44440

11. CuSO4 pentahydrate 0.16160

12. Na2MoO4 0.08484

13. 1 drop of 0.072 N H2SO4

Note: Bring volume up to 1000 mL in de-ionized water, pH to 7.5 then autoclave

2.2.3 MethodsCulturing of unimicroalgae in small or laboratory scale is going to be started in the 2nd step. During this step, trial and error in cultivation unimicro-lagae is the main activity. Culturing of some different species on some different either artificial and natural basis media will be done in this step.The microalgae will be cultured are focused on large green microalgae species. The main target of Lombok local fresh water strains of microalgae to be cultured are Botryococcus sp., Closterium sp., Micrastreas sp., and Scenedesmus sp..2.3 Budget

No.MaterialsVol.UnitPrices

($USA)Need

(gr)Cost($USA)

1.Bristol's Modified Medium

a.10 g sodium nitrate (NaNO3)250g22.8201.824

-1 g calcium chloride (CaCl2 2H2O)100g33.220.664

-3 g magnesium sulfate (MgSO4 7H2O)100ml40.362.418

-3 g potassium phosphate dibasic 1000ml78.5120.942

-7 g potassium phosphate monobasic (KH2PO4)1000ml23.8140.3332

-1 g sodium chloride (NaCl)100ml2620.52

b.1% ferric Chloride100g19.781.576

Sub Total For 2 L Solution8.2772

Sub Total For 1000 L Solution4138.6

c.Pringsheim's Soil Water

d.Calcium carbonate (CaCO3).500g54.4202.176

e.Fresh water1months5315

f.Aquades1l0.121000120

g.Soil sample1g0.08100075

Sub Total4,350.776

2.Aparattus

a.Plankton net1unit85185

b.Silicon tube7.6m24.376243

c.Flex slang7.6m11.576115

d.Basin1m31502300

e.TL 20 Watt1unit16464

f.24 hours timer swich1unit74.82149.6

g.Micropipette1unit20.2240.4

h.Sedgwick-rafter (Wards) 50x20x10mm1set12.4224.8

i.Rafractometer1month8324

j.Hanna Instruments1month12336

k.Spectrophotometer1month24372

l.lWater Thermometer 1unit13113

m.Air Thermometer1unit11111

n.Regulator CO2 gas1month30390

o.Flex slang7.6m11.576115

Sub-total1,382.8

3.Room design preparation

a.Air condition1Unit5050

b.Electricity1set2020

c.Fresh water supply1set1515

d.UV TL 20 watt Lamps10unit10100

e.Aerator sets10unit10100

f.Long table3unit2575

Sub-total360

4.Transportation

a.Mataram East Lombok 141040

b.Mataram Central Lombok141040

c.Mataram West Lombok141040

d.Mataram North Lombok141040

e.Local transportations1pack.100100

Sub-total260

3.Personil

a.Senior Consultant 2 days a week1person3months200600

b.Junior Consultant 5 days a week2people3months100600

c.Enumerator (Laborant)2people3months50300

Sub-total1,500

4.Institutional (Faculty and Uniersity)3months250750750

Total Cost for 2nd Step8,828.351

Part III

UPGRADED AND LARGER SCALE CULTURING OF TARGET FRESH WATER MICRO-ALGAE (3th phase)3.1 Scope Research

3.1.1 Larger Scale Culturing

3.1.2 Harvesting

3.1.3 Extraction and Bioactive Substances Screening3.2 Materials and Methods

3.2.1 Equipments

Aerators

Spectrophotometer

Sample Bottles

Pyrex BottlesBasin

3.2.2 Materials

3.2.2.1. Chemicals

3.2.3 Methods

Larger scale of culturing and extraction or obtaining of commercial products of micro-algae such as bio-fuels are going to done in final step of this project. Larger scale culturing will be conducted in both outdoor and indoor (laboratory) treatments.Micro-algal culture harvesting systems, a cost-effective harvesting major methods for micro-algal culturing are filtration, sedimentation, flotation and flocculation (Borowitzka and Borowitzka, 1995, in Clayton and King, 1995). Filter cloths or micro-screens of 50 to 100 m pore size is useful for filtering Spirulina (Richmond, 1988, in Clayton and King, 1995).3.3 BudgetNo.MaterialsVol.PriceNeedCoast $ USA

1.Materials

a.Aquades1L30000.12360

b.Fresh water1month5210

c.Sample bottles1unit1.2500600

d.Filter paper1rol20240

Sub-total1,010

2.2. Aparattus

a.Cloth filter1month9218

b.Autoclave1month6212

c.Buckets1month762152

d.Electricity1month552110

e.Spectophotometer1month11111

f.Sedgwick-rafter (Wards) 50x20x10mm1set12.4224.8

Sub-total327.8

3.Local Transportation1package100

4.Personil

a.Senior Consultance 2 days a week1person2months200400

b.Junior Consultance 5 days a week2people2months100400

c.Enumerator (Laborant)1people2months50100

Sub-total900

5.Institutional (Faculty and Uniersity)2months250500500

TOTAL Budget of 3th phase2,837.8

References

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Allen, W.E., and Cupp, 1935, Plankton Diatom of the Java Sea, Annales du jardin Botanique de Buitenzorg, 44 : 101-174.

Anonimous, 1997, Nies-Collection List Of Strains 5th Eddition, Microalgae and Protozoa, Microbiol Culture Collection National Institude for Environmental Studies, Environment Agency Japan.Anonimous, 2009a, What is Algae Oil?, http : // www. algaeproductionsystems.com / equipment. html, 25 Mei 2009, 10.06 wita

Anonimous, 2009b, Algal Oil Yields, http://www.oilgae.com/algae/oil/yield/yield.html, 25 Mei 2009, 10.17 witaAnonimous, 2009c, U.K. algae biofuel co. BioMara launches, http : // cleantech.com / news / 4338 / uk-algae-biofuel-co-biomara-launche, 25 Mei 2009, 10.35.

Atria, M., 1994, Pengaruh Beberapa Konsentrasi Urea Terhadap Pertumbuhan Chlorella pyrenoidosa Chick dalam Medium Beneck Modifikasi, Skripsi, Program S1 Jurusan Biologi, Fakultas MIPA Universitas Indonesia, Depok.

Borowitzka, M.A., and L.J. Borowitzka, 1995, Algal Biotechnology, In, M.N. Clayton and R.J. King (Editors), Biology of Marine Plants, Longman, Australia

Chaugule, B.B., 2009, Algae : An oil Crop of Future, http://www.svlele.com/algae.htm, 25 Mei 2009, 09.53 WITA.

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Materials and Budged 1st Step : LITERATURE SEARCH

1. Visit Study toVol.UntPrice$ USA

a. LIPI Pemenang North Lombok 1time404040

b. PT Bumi Blanting East Lombok1time505050

2. Searching refferences 1package989898

3. Transport for Field investigation and sampling

a. Mataram - North Lombok1time4040

b. Mataram - Central Lombok1time5050

c. Mataram - East Lombok1time7070

Sub-total 160

4. Small scale of threatments

(preliminary screening)

Meterials: Bristol's Modified Medium2package4.01198.0238

Equipments:

a. Cultur continer2unit3.16.2

b. TL 20 Watt1unit8.58.5

c. Room Preparations1month4040

d. Thermometer2unit5.110.2

e. Spectrophotometer2month1122

f. Autoclave2month1020

g. 24 hours timer swich2unit40.881.6

h. Silicon tube1.7m 24.341.31

i. Flex slang1.7m11.519.55

j. Micropipette2unit18.236.4

k. Sedgwick-rafter2unit12.424.8

l. Filter cartridge 20, 5, 0,5 um1set150150

m. CO2 regulator thank1set9898

n. Sample bottle2unit612

o. Glass aparattus1package4242

p. Fresh water2month714

q. Aquades100l0.1616

Sub-total650.5838

5. Personil

a. Junior Consultance 5 days a week2people2months100400

b. Senior Consultance 2 days a week1person2months200400

c. Enumerator (Laborant)2people2months50200

Sub-total1000

6. Institutional Fee (Faculty and University)2months250500500

Total Cost for 1st Step 2498.584

2nd Step: BIOREACTOR AND MEASUREMENTS

1. Materialspreciseneed Cost$ USA

a. Bristol's Modified Medium

-10 g sodium nitrate (NaNO3)250g22.8201.824

-1 g calcium chloride (CaCl2 2H2O)100g33.220.664

-3 g magnesium sulfate (MgSO4 7H2O)100ml40.362.418

- 3 g potassium phosphate dibasic 1000ml78.5120.942

(K2HPO4)

- 7 g potassium phosphate monobasic 1000ml23.8140.3332

(KH2PO4)

- 1 g sodium chloride (NaCl)100ml2620.52

b.1% ferric Chloride100g19.781.576

Subtotal for 2L soltion8.2772

Subtotal for 1000L soltion 4138.6

c. Pringsheim's Soil Water

d. Calcium carbonate (CaCO3).500g54.4202.1762.176

e. Fresh water1months531515

f. Aquades1l0.121000120120

g. Soil sample1g0.0810007575

Sub-total4350.776

2. Aparattus

a. Plankton net1unit85185

b. Silicon tube7.6m24.376243

c. Flex slang7.6m11.576115

d. Basin1m31502300

e. TL 20 Watt1unit16464

f. 24 hours timer swich1unit74.82149.6

g. Micropipette1unit20.2240.4

h. Sedgwick-rafter (Wards) 50x20x10mm1set12.4224.8

i. Rafractometer1month8324

j. Hanna Instruments1month12336

k. Spectrophotometer1month24372

l. Water Thermometer 1unit13113

m. Air Thermometer1unit11111

o. Regulator CO2 gas1month30390

p. Flex slang7.6m11.576115

Sub-total1382.8

3. Personil

a. Junior Consultance 5 days a week2people3months100600

b. Senior Consultance 2 days a week1person3months200600

c. Enumerator (Laborant)2people3months50300

Sub-total1500

4. Institutional (Faculty and Uniersity)3months250750750

Total Cost for 2nd Step7983.576

3th Step: ALGAE PROCESSING

preciseneedCost$ USA

1. Materials

a. Aquades1L30000.12360

b. Fresh water1month5210

c. Sample bottles1unit1.2500600

d. Filter paper1rol20240

Sub-total1010

2. Aparattus

a. Cloth filter1month9218

b. Autoclave1month6212

c. Buckets1month762152

d. Electricity1month552110

e. Spectophotometer1month11111

f. Sedgwick-rafter (Wards) 50x20x10mm1set12.4224.8

g. Sub-total327.8

3. Personil

a. Junior Consultance 5 days a week2people2months100400

b. Senior Consultance 2 days a week1person2months200400

c. Enumerator (Laborant)1people2months50100

Sub-total900

4. Institutional (Faculty and Uniersity)2months250500500

TOTAL2737.8

Summary of Research Cost

1st Step:LITERATURE SEARCH ( 2 months )2498.584

2nd Step:BIOREACTOR AND MEASUREMENTS ( 3 months )7983.576

3th Step:ALGAE PROCESSING (2 months)2737.8

Total Cost for 3th Step ( 7 months )13219.96