Chapter 3

RESULTS AND DISCUSSION

3.1 Experimental Results of Chlorococcalean Algae Biodiesel

This chapter explores the results obtained for method describes in Chapter 2, such as a database of Algae found in State, Algae Collection Results, and Algae Growth Data of Selected Microalgae, Biodiesel Characterization by FTIR and GCMS Results and Properties of Prepared Biodiesel Fuel.

3.1.1 A database of Algae found in Maharashtra State

Biodiversity is the variety of plant and animal in particular ecosystem. The human induced activities cause serious threats to the biodiversity, which ultimately leads to environmental degradation. The knowledge about biodiversity of water reservoir along with its present conservation status and maintenance of its natural properties will help in sustainable utilization of human mankind. The aim of present investigation to prepare database of algae phytoplankton found in Maharashtra State which can be exploited for making of biodiesel.

Maharashtra occupies the western and central part of the country and has a long coastline stretching nearly 720 kilometer along the Arabian Sea [1]. The is the more prominent physical feature of Maharashtra which is separated from the Kokan coastline by Ghats. The state is surrounded by Gujarat to the North West, to the north, Chattisgarh to the east, to the south east, to the south and Goa to the south west. The main rivers of the state are Krishna, Bhima, Godavari, Tapi-Purna and Wardha-Wainganga [1][2]. The Maharashtra has typical monsoon climate, with hot, rainy and cold weather seasons. The winter in January and February is followed by summer between March and May and the monsoon season between June and September. The summer is extreme with March, April and May as the hottest months. The temperature varies between 22 ˚C and 39 ˚C during this season. Rainfall starts normally in the first week of June while winter starts in September. The region to region seasonal variation found in Maharashtra. The flora of Maharashtra is heterogeneous in composition. The most of the forest area have low annual rainfall (50-70 cm), a mean annual temperature of 25- 27˚C and low humidity. Some of the forest areas are converted into wildlife reserves which help to preserving the biodiversity of Maharashtra state [3].

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The study of algae found in Maharashtra was done according to their administrative divisions. Maharashtra consists of six administrative divisions which are further divided into 36 districts. The administrative divisions and districts are as follows: i. Amrawati : Amarawati, Akola, Buldhana, Washim, Yewatmal. ii. : Ahemdnagar, Dhule, Jalgaon, Nandurbar, Nashik. iii. Aurangabad : Aurangabad, Beed, , Jalana, Latur, , Osmnabad, Parbhani. iv. Pune : Kolhapur, Pune, Sangli, Satara, Solapur. v. : Bhandara, Chandrapur, Gadchiroli, Gondia, Nagpur, Wardha. vi. Kokan : Mumbai, Mumbai-upnagar, Ratnagiri, Raigad, Sindhudurga, Thane.

Table 3.1: The Maharashtra States administrative divisions and districts, Algae Source Reservoir and reference used

Sr. No. Name Of District Algae Source Reservoir Reference No. Division Amarawati 1 Amrawati Agriculture Soil, Nal- 4, 5 Damyanti Reservoir 2 Buldhana Lonar Lake 6 3 Washim Fresh water Reservoir 7 4 Yawatmal Fresh Water Dam 8 Division: Nashik 5 Ahemdnagar Bhandardara Dam, 9, 10,11, 12 6 Dhule Amarawati Dam, MIDC 13, 14, 15 7 Jalgaon MIDC, Hartala Lake 13, 14 8 Nandurbar Ordinary Drinking water 13 9 Nashik Gangapur Dam 16

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Division: Aurangabad 10 Aurangabad Air born algae, Ajanta 17, 18 water fall 11 Beed Jalgaon Nala of Ashti 19, 20 12 Hingoli Siddheshwar Dam 21 13 Jalana Jayakwadi project 22, 23 14 Latur Manjara River 24 15 Nanded Vishnupuri Reservoir, 25,26 Kundrala Dam 16 Osmanabad Dhanegaon 27,28 17 Parbhani Masoli reservoir 29 Division: Pune 18 Kolhapur Lake 30 19 Pune Different places 31, 12 20 Sangli Bharatnagar Lake, 32, 33 21 Satara Dhakani 34 22 Solapur Urban Lakes 114 Division: Nagpur 23 Chandrapur Tadoba Lake 125 24 Gadchiroli Bothali (Mendha) Lake 131 25 Gondia Chulband dam 35 26 Nagpur Lake 36 27 Wardha Mahakali Water 37, 38, 39 Reservoir Division: Kokan 28 Mumbai Aarey Lake 40 29 Ratnagiri Fresh water 41 30 Raigad Vishrale, Krishnale, 42 Dewale Lake 31 Sindhudurga Malwan 43 32 Thane Aarey Lake 40

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Table 3.2: Algae separated according to its Class found in water reservoir

Sr. Name Of the

No. District

iophyceae

Cyanophyceae Chlorophyceae Bacillariophyceae Xanthophyceae Euglenophyceae Zygnematophycea Flagillar Myxophyceae Dinophyceae Desmidiceae Chrysophyceae Charophyceae Ulvophyceae Trebouxiphyceae Division: Amarawati

1 Amrawati + + + + + ------

2 Buldhana + ------

3 Washim ------

4 Yawatmal + + ------

Division: Nashik

5 Ahemdnagar + + + + + + ------

6 Dhule + + + - + ------

7 Jalgaon + + + - + ------

8 Nandurbar - - + ------

9 Nashik + + ------+ - - - - -

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Division: Aurangabad

10 Aurangabad + + + ------

11 Beed + ------

12 Hingoli + + + - + - - - - + + + + -

13 Jalana + ------

14 Latur + + + - + ------

15 Nanded + + + - + ------

16 Osmanabad + + + - + ------

17 Parbhani + + + - + + ------

Division: Pune

18 Kolhapur + + + + + + ------

19 Pune + + + ------

20 Sangli + + + - - + + ------

21 Satara + + + - + + - + ------

22 Solapur + + + - + + ------

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Division: Nagpur

23 Bhandara

24 Chandrapur - + + - + ------

25 Gadchiroli ------

26 Gondia + ------

27 Nagpur + + + + + ------

28 Wardha + + + + + ------

Division: Kokan

29 Mumbai + + + ------

30 Ratnagiri + ------

31 Raigad - + + ------+

32 Sindhudurga + - + - - + ------

33 Thane + + + ------

Note: “+” sign indicate = Present, “-” sign indicate = absent.

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The algae found in Maharashtra State were in large extent due to its availability of water reservoir like river, lake and dam. Algae are very large and diverse group of simple, autotrophic organisms and ranging from unicellular to multicellular. The classification of algae on the basis of pigment, external form, chromatophore shape, reserve food products, cell wall, nucleus, chromosome, type of reproduction and ecological data. Basically algae are two type`s macro and micro respectively. The algae database was studied are shown in Table 3.1 and 3. 2.

i. Algae found in Amrawati Division of Maharashtra State

The Amrawati division consists of five districts, Amrawati, Akola, Buldgana, Washim and Yawatmal. Fule U.W. et al. (2012) reported that the plankton diversity in Nal-Damayanti (Simbhora) reservoir in Taluka Morshi, District, Amrawati. The evaluation result was consisting of four groups of algae. The algae phytoplankton possesses 23 species; Chlorophyceae was dominant by counting 11 species followed by Cynophyceae by 5 species, Bacillariophyceae by 4 species and Euglenophyceae by 2 species. Cherian K.J. et al. (2012) was observed algae in crop soil field of Orange, Jowar, Tuar, Soyabean, Cotton, Ground and vegetable field. In all 49 algal species identified from this field out of which 25 belonged of Chlorophyceae, 1 belonged to Xanthophyceae, 6 belonged to Euglenophyceae and 17 belonged to Bacillariophyceae. The growth of algae promoted due to presence of high organic compound in soil crop field. Algae help to prevent the erosion of soil and increase fertility of soil. The greater biodiversity of algae in pond and soil crop field was observed in Amrawati.

Deshmukh D.V. et al. (2014) was isolated four genera of Cynophyceae from alkaline Lake Lonar, situated in the . Mukund Dhore et al. (2012) reported, the macrophytes found in fresh water bodies of . The macrophytes of family Hydrocharitaceae, Characeae, Hydrocharitaceae, Najadaceae, Aponogetonaceae, Ceratophyllaeae, Hydrocharitaeae, Typhaceae, Hydrocharitaceae and Plantaginaceae were observed.

Joshi P.P. (2012) was evaluated algal species from Yawatmal district. The algae species were observed from family Hydrochartaceae, Potamogetonaceae, Convolvulaceae, Hydrocharitaceae, Polygonaceae, Najadaceae, Cyperaceae, Cladophoraceae, Typhaceae, Characeae, Lemnaceae, Nostocaceae, Nymphacaceae,

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Paniceae, Graminaea, Chlorophyceae and Salviniaceae. The potential algal flora was found in Amrawati Division of Maharashtra State.

ii. Algae found in Nashik Division of Maharashtra State

The Nashik division consists of five districts, Ahemdnagar, Dhule, Jalgaon, Nandurbar and Nashik. Dhamak R. M. et al. (2013) reported phytoplankton from Bhanardara Dam, Ahemdnagar district. The variation in the group of algae was seen in which 5 species of Cyanophceae, 22 species of Chlorophyceae, 14 species of Bacillariophceae has been observed. Pingle S.D. et al. (2009) reported algae found in fresh water reservoir in belonging class Cyanophyceae, Chlorophyceae, Bacillariophyceae, Euglenophyceae, Xanthophyceae and Dinophyceae. Nerpagar P.B. et al. (2011) investigated algae from Dhule and Jalgaon District. The 36 algal taxa were reported, 9 belonging from Cyanophyceae, 8 belonging from Chlorophyceae, 11 belonging from Bacillariophyceae and 6 belonging from Euglenophyceae. The class Bacillariophyceae is dominant group observed than the other group.

Mahajan K.D. (2012) reported, algal biodiversity of North Maharashtra region in which diatoms an important group found in aquatic ecosystem. The forty two diatoms taxa were reported. Thakur H.A. et al. (2008) has investigated algae found in fresh water reservoir Gangapur Dam of . The 25 number of algal taxa were found which comprises 5 genera and 17 species of Cynophyceae, 3 genera and 5 species of Chlorophyceae, 2 genera and 3 species of Charophyceae. The member of class Cynophyceae are relatively dominant algae was observed in Gangapur Dam in Nashik district. iii. Algae found in Aurangabad Division of Maharashtra State

The Aurangabad division consists of eight districts, Aurangabad, Beed, Hingoli, Jalana, Latur, Nanded, Osmnabad and Parbhani. Jadhav M. et al. (2010) reported the diversity of Airborn algae In the Atmosphere of Aurangabad district. The 49 airborn algal taxa were found, in which 29 genera belonging Cynophyceae, Chlorophyceae and Bacillariophyceae. The Cynophyceae members dominated airborn algal flora.

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Taekar S. M. (2014) reported the Chlorococcales algae found in Jalgao Nala of Ashti Taluka in . The total 9 genera and 22 species of Chlorococcales were recorded.

Shaikh P.R. et al. (2012) reported the survey of algae from Siddheshwar reservoir of . The 24 algae genera were found In which 7 belonging from Chlorophyceae, 6 belonging from Bacillariophyceae, 4 belonging from Zygnematophyceae, 2 belonging from Cynophyceae, 2 belonging from Flagillariophyceae, 1 belonging from Ulvophyceae, 1 belonging from Trebouxiphyceae and 1 belonging from Euglenophyceae were recorded.

Andhale S.B. et al. (2009) studied the algae found in from Jalana District. The 5 genera and 32 species of Cynophyceae recorded. Khanpure N.N. et al. (2013) investigated the algae found in Manjara River from Lature District. The algae comprises of 12 species of Chlorophyceae, 9 species of Cynophyceae, 13 species of Basillariophyceae and 4 species of Euglenophyceae. Among the phytoplankton of Manjara River the Chlorophyceae was dominant group observed.

Pawale R.G. (2014) recorded the algae found in Vishnupuri Reservoir . During the survey of phytoplankton study the seasonal variation occurred. The phytoplankton observed in the reservoir 9 species of Chlorophyceae, 7 species of Cynophyceae, 11 species of Basillariophyceae and 6 species of Euglenophyceae recorded. Lokhande M.V. et al. (2009) studied Phytoplankton diversity of of Dhanegaon Reservior, Dhanegaon and Osmanabad Distict. Due to seasonal variation the phytoplankton count was different in monsoon, winter and summer. The algae species of Chlorophyceae, Cynophyceae, Basillariophyceae and Euglenophycea were recorded.

Kadam S. U. et al. (2010) investigated Phytoplankton Biodiversity of Masooli Reservoir, . The algae identified are members of Chlorophyceae, Cynophyceae, Basillariophyceae, Euglenophycea, Dinophyceae, Filamentous algae, Oedogonium species, Cladophora, Glomerata and Spirogyra.

iv. Algae found in Pune Division of Maharashtra State

The Pune division consists of five districts, Kolhapur, Pune, Sangli, Satara and Solapur. Leela J. Bhosale et al. (2010) investigated Phytoplankton in The Lakes in

92 and around Kolhapur City. The number of phytoplankton and filamentous algae were found which belonging from Chlorophyceae, Cynophyceae, Basillariophyceae, Euglenophycea, Dinophyceae, Xanthophyceae and Chrysophyceae.

Patil V. S. et al. (2014) explored the algae from Pune District. The 29 species have been recorded which comprises 31 genera and 29 species of Cynophyceae, 7 genera of Chlorophyceae and 3 genera of Bacillariophyceae. Sarawade A.B et al. (2014) reported Plankton Diversity in Krishna River, Sangli. The 53 species were found with the group of Cynophyceae, Bacillariophyceae, Chlorophyceae, Hydrophyceae and Desmidiceae. The Chlorophyceae was dominant group with 22 species recorded. Leela J. Bhosale et al. (2010) studied the algae found in lake from Sangli District. The seasonal variation was found and the Euglenophyceae, Chlorophyceae were dominant group reported. Jagtap M.N.et al. (2012) isolated algae from Urban Lakes of Solapur City. The phytoplankton of class Cynophyceae, Bacillariophyceae, Chlorophyceae, Euglenophycea and Dinophyceae were reported. v. Algae found in Nagpur Division of Maharashtra State

The Nagpur division consists of six districts, Bhandara, Chandrapur, Gadchiroli, Gondia, Nagpur and Wardha. Telkhade P.M. (2009) investigated algae from Tadoba Andhari Tiger Reserve (TATR) of . The study of phytoplanktonic results the 35 species belonging from Chlorophyceae, Bacillariophyceae, Euglenophycea and Myxophyceae. The Euglenophycea and Bacillariophyceae were found dominant. Tijare R. (2013) explored algae from Bothali (Mendha) Lake, Gadchiroli District. The lake is old Malgujari Talav type. The seasonal variation in population of algae was observed. The 16 genera were recorded of which 6 belongs to Chlorophyceae, 4 to Basillariophyceae, 3 to Cynophyceae and 1 to Euglenophyceae.

Sahare P.C. et al. (2012) investigated algae diversity of Chulband Dam from . The Chulband dam shows variety of fresh water algae of class Cynophyceae. The 32 forms of Cynophyceae were found in different season. Kumari P. et al. (2008) assess the algae from water Lakes in Nagpur City. During the study a total 50 algae species were recorded. Out of these, 10 species belonged to Cynophyceae, 20 to Chlorophyceae, 13 to Bacillariophyceae, 4 to Euglenophyceae and 1 to Cryptophyceae, Xanthophyceae and Pyrrhophyceae. Dalal I.P.et al. (2012)

93 reported Fresh Water Algae of Mahakali Water Reservior of . This study results 37 algae belonging from Chlorophyceae, 24 of Bacillariophyceae, 1of species Xanthophyceae, 1 of Euglenophyceae and 9 species of Cynophyceae were identified. vii. Algae found in Kokan Division of Maharashtra State

The Kokan division consists of six districts, Mumbai, Mumbai-upnagar, Ratnagiri, Raigad, Sindhudurga and Thane. Atikah Yusuf Moosa et al. (2013) evaluated algae from Aarey Lake which comprises the Thane and Mumbai District. The algae species of Bacillariophyceae was occurring dominant in lake. Vaidya S. et al. studied the algae from Ratnagiri District. This study results the fresh water Chlorococcles dominant algae. Shashikala R.S.Prajapati et al. explore the algae from Vishrale, Krishnale and Dewale Lake from Raigad District. In all the three lakes 16 genera of algae were recorded. Out of which 7 genera belong to the Chlorophyceae, 4 genera of Bacillariophyceae and 5 genera of Myxophyceae. Among all algae Chlorophyceae was dominant. Parulekar, A.H. (1981) explores the fauna of Malvan from Kokan District. The seasonal variation was observed in population of algae. The Bacillariophyceae, Dinophyceae and Cynophyceae were reported.

Maharashtra is the third largest state by area in . Most of the area in Maharashtra is covered by forest. The climate of state is favorable to fulfill the growth conditions of plant. Maharashtra State shows the huge biodiversity not only terrestrial but also for aquatic environment. Maharashtra State possesses the fresh water reservoir except Lonar Lake. Lonar Lake is alkaline in nature. The algae are found with high potential which comprises various divisions of algae. The Chlorophyceae and Cyanophyceae were dominant algae found in most of water reservoirs. Algae are focused as modern biomass for biodiesel production, due to its huge availability, productivity of lipid, easy screening, harvesting and drying. Hence, algae can be exploited for making of biodiesel.

3.1.2 Algae Collection Results

The algae have been collected aseptically from site of Godawari River and Nizarneshwar Dam, Ahemdnagar (M.S.) India. It was preserved in 4% formalin solution and Lugol`s solution as described in method 3.1.3. The macroalgae and

94 microalgae sample identification results that, the 79 species were observed in collected sample which belonging from class Cyanophyceae (20), Chlorophyceae (44), Bacillariophyceae (9), Euglenophyceae (2), Xanthophyceae (2) and Dinophyceae (2). The obtained results of algae collection were match with [54] Pingle et al., (2009) was studied algae and water reservoir assessment from Ahmednagar District. The observed algae species shown in Table 3.3 to 3.8

Table 3.3: Cynophycean member Algal species

Sr. No. Algae Species Name 1 Lingbya majar Menegh. Ex. Gimont 2 Lingbya majuscula Harvey ex. Gomont 3 Nostoc linkia sp. 4 Nostoc punctiforme (Kuetz.) hariot 5 Nostoc haetai Dixit. 6 Nostoc spongiformere Ag.ex.Born. et Flah. 7 Anabaena constricta (Szafer) Geither. 8 Nodularia spumigena sp. 9 Chlorococcus minor Kuetz. Naegeli 10 Gomphospaeria aponia var. delicatula virieux 11 Gleotrichia ghosei sp. 12 Oscillatoria Subbrevis sp. 13 Oscillatoria tenuis Ag.ex.Gomont. 14 Oscillatoria mougeotii Kurtz. 15 Oscillatoria agardhii Gomont 16 Microcystis flow-aquae (Wittr.) Kirchner 17 Microcystis protocystis Crow 18 Microcysis aeruginosa Kuetz 19 Chroococcus turgidus (Kuetz.) Naeg. 20 Chroococcus giganteus west.W.

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Table 3.4: Chlorophycean member Algal species

Sr. No. Algae Species Name 1 Cosmarium viridis (Chorda) Josh 2 Cosmarium contractum Kirchn 3 Euastrum dubium Nageli 4 Micrasterias zeilanica sp. 5 Scenedesmus bijugatus (Turpin) Kuetzing. 6 Scenedesmus acuminatus (Lagerheim) Chodat. 7 Scenedesmus quadricauda sp. 8 Scenedesmus bijugatus var. alternans (Reinsch) Hansgirg. 9 Scenedesmus dimorphus (Turpin) Kuetzing. 10 Scenedesmus indicus sp. 11 Scenedesmus acuminatus (Lagerheim) Chodat. 12 Micractinium pusillum sp. 13 Spirigyra mirabilis (Hass.) Kuetzing 14 Zygema sterile transeau in Transeau. 15 Closterium cynthica De. Not 16 Pandorina morum (muell.) Bory 17 Sphaerocystis schroeteri Chodat. 18 Dictyospharium erhenbergianum Naegeli 19 Eudorina elegans Ehrenberg. 20 Volvox globator Linn. 21 Chlorella vulgaris Beyerink 22 Oocystis elliptica W. West. 23 Ankisrodesmus spiralis (Turner) Lemm. 24 Ankistrodesmus falcatus (Corda) Ralfs. 25 Kircheneriella lunaris (Kirchner) Moebius. 26 Tetraedron regulare Kuetzing 27 Tetrodron sp. 28 Tetradron gracile (Reinsch) Hangsirg. 29 Golenkinia radiate (Chod.) Wille.

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30 Hydrodictyon reticulatum (L.) Lagerheim 31 Pediastrum simplex (Meyen) Lemm. 32 Pediastrum simplex var. duodenarium (Bailery) Rabenhorst 33 Pediastum ovatum (Her.) A. Braun. 34 Pediastum duplex var. subgranulatum Raciboski. 35 Pediastrum boranum (Turp.) Meneghini. 36 Pediastrum duplex var.reticulatum Lagerheim. 37 Chamydocapa ampla (Kuetz.) Fott. 38 Ulothrix cylindricum Prescott. 39 Chlamydocapa elegans (Roth.) C.A.Agarth. 40 Rhizoclonium crassipellitum West & West. 41 Oedogenium latiusculum Tiff. 42 Oedognium sp. 43 Coleastrum cambrcum Archer. 44 Coleastrum microporium Naegeli

Table 3.5: Bacillariophyceae member Algal Species

Sr. Algae Species Name No. 1 Fragilaria intermedia Grun 2 Fragilaria ungeriana sp. 3 Synedra tabulate (Ag) Kutz 4 Navicula cupsidata Kutz 5 Pinnularia acrosphaeria (breb) W. Smith 6 Cymbella gracilis (Rabh) Celve 7 Melosira granulata (Her.) Ralfs 8 Melosira islandica O. Muell. 9 Cyclotella meneginiana Kuetzing

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Table 3.6: Euglenophyceae member Algal Species

Sr. Algae Species Name No. 1 Euglena gracilis Kleps 2 Phacus curvicauda Swirenao

Table 3.7: Xanthophycea emember Algal Species

Sr. Algae Species Name No. 1 Botryococcus braunii kuetzing 2 Vaucharia terrestris (Vauch.) De Candolle

Table 3.8: Dinophyceae member Algal Species

Sr. Algae Species Name No. 1 Ceratium Hirundinella (O.F.Muell) Dujardin 2 Glenodinium quadridens (Stein) Schiller

3.1.3 Algae Growth Data of Selected Microalgae

The growth rate analysis behavior of microalgae strain was assessed in four different media i.e. Chu 10 modified, Bold Basal media, Chu 13 Modified, BG11 media. The growth rate of the Chlorella vulgaris, Chlorococcum humicola, Ankistrodesmus convolutes, Botryococcus braunii, Clamydomaonas pertusa and Scenedesmus dimorphus microalgae strain in culture media was determining by using a UV-visible spectrophotometer at 680 nm [55]. The maximum growth rate of microalgae was studied by plotting graph of day versus absorbance which is shown in Figure 3.1, 3.3, 3.5, 3.7, 3.9 and 3.11 and Figure 3.2, 3.4, 3.6, 3.8, 3.10 and 3.12 shows the response of microalgae growth for different growth media for 21 days. The growth study of the Chlorococcalean Microalgae species was done by using UV- visible Spectrophotometer and measuring optical density at 680 nm. The results were

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tabulated in Table 3.9. Biomass productivity and Lipid productivity for Microalgae for selected Media (mg/l) was tabulated in Table 3.10. The cell density or concentration of microalgae was directly proportional to the absorbance of medium. The strain culture with green color variation was observed during growth rate study because of lag, exponential and stationary phase of algae. The lag phase was initial phase of multiplication of algae cell, the exponential is the growth phase where multiplication of algae cell took place while in stationary phase the multiplication of cells was usually minimum and absorbance remain constant.

Table 3.9: Growth study of the Chlorococcalean Microalgae species by measuring optical density by using UV- visible Spectrophotometer at 680 nm (Nitumani et al. 2011)

Day Name of Microalgae Cv Ch Ac Bb Cp Sd

1 0.10 0.12 0.08 0.13 0.10 0.14 2 0.14 0.15 0.12 0.15 0.13 0.18 3 0.16 0.19 0.17 0.19 0.15 0.22 4 0.18 0.23 0.21 0.21 0.19 0.25 5 0.20 0.25 0.25 0.25 0.22 0.29 6 0.22 0.27 0.29 0.28 0.26 0.33 7 0.25 0.28 0.32 0.30 0.29 0.38 8 0.28 0.30 0.35 0.33 0.31 0.41 9 0.32 0.32 0.39 0.34 0.34 0.46 10 0.34 0.38 0.43 0.36 0.36 0.49 11 0.37 0.41 0.46 0.38 0.39 0.53 12 0.39 0.47 0.51 0.42 0.44 0.56 13 0.43 0.49 0.59 0.46 0.48 0.58 14 0.46 0.50 0.62 0.49 0.53 0.61 15 0.49 0.51 0.69 0.52 0.56 0.65 16 0.52 0.52 0.69 0.58 0.59 0.67

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17 0.55 0.53 0.69 0.63 0.63 0.70 18 0.59 0.55 0.69 0.67 0.67 0.72 19 0.62 0.56 0.69 0.67 0.71 0.72 20 0.62 0.57 0.69 0.67 0.71 0.72 21 0.62 0.57 0.69 0.67 0.71 0.72

Figure 3.1: Growth study of Chlorella vulgaris at 680 nm

Figure 3.2: Response of Chlorella vulgaris Microalgae for Different Growth Media

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Figure 3.3: Growth study of Chlorococcum humicola at 680 nm

Figure 3.4: Response of Chlorococcum humicola Microalgae for Different Growth Media

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Figure 3.5: Growth study of Ankistrodesmus convolutes at 680 nm

Figure 3.6: Response of Ankistrodesmus convolutes Microalgae for Different Growth Media

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Figure 3.7: Growth study of Botryococcus braunii at 680 nm

Figure 3.8: Response of Botryococcus braunii Microalgae for Different Growth Media

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Figure 3.9: Growth study of Clamydomaonas pertusa at 680 nm

Figure 3.10: Response of Clamydomaonas pertusa Microalgae for Different Growth Media

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Figure 3.11: Growth study of Scenedesmus dimorphus at 680 nm

Figure 3.12: Response of Scenedesmus dimorphus Microalgae for Different Growth Media

It was observed that the Chlorella vulgaris species (Figure 3.2) showed maximum dry biomass weight for Chu 10 modified (786.2 mgl-1) than Chu 13 modified (691.3 mgl-1), BBM (638.7 mgl-1) and minimum in BG 11 (526 mgl-1). The growth curve of Chlorella vulgaris for Chu 10 (mod.) media (Figure 3.1), the lag phase on 1st day, exponential phase occurs on 18th day and stationary phase observed maximum growth at 19th and 20th day. The biomass productivity of Chlorella vulgaris

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was observed 0.629 gl-1d-1 and lipid extracted from biomass was 0.273 gl-1d-1, the 43.40% lipid content was obtained.

The similar results in growth pattern were found by Kuei-Ling Yeh et al., (2011) [56], they reported the growth rate of Chlorella vulgaris in Modified Bristol’s medium. The lipid productivity was 0.63 gl-1d-1 for MBL and 3.7 gl-1d-1 for Modified Bristol’s medium. The variation in lipid content observed was due to nutrients in growth medium of culture. The factors like CO2 concentration and light intensity play important role in both productivity of mass culture and lipid productivity. According to Blanchemain and Grizeau, (1996) [62]; cell growth of Chlorella vulgaris was depend on light intensity. The lipid content 44-47% was achieved under 50 and 100 µmol photons m-2s-1 whereas 35-40% was obtained under 20-400 µmol photons m-2s-1 hence our result for Chlorella vulgaris in Chu 10 modified medium was similar to referred literature.

The Chlorococcum humicola (Figure 3.4) showed maximum dry biomass weight for BBM (219.5 mgl-1), for Chu 10 modified (203.7mgl-1) and for Chu 13 modified (174.6 mgl-1) and minimum in BG 11(135.2 mgl-1). The remarkable growth rate was observed in BBM (Figure 3.3), the lag phase on first 3 days, the exponential growth phase was seen on 20th day and stationary phase was observed on 21st day. The biomass productivity of Chlorococcum humicola in BBM was observed 0.094 gl- 1d-1 and lipid extracted from biomass was 0.029 gl-1d-1, the 30.85% lipid content was obtained.

Durga M.M. et al., (2013) [57], has reported that the growth in exponential phase of Chlorococcum humicola in artificial wastewater enhanced during incubation which was matched with the present experimental data of Chlorococcum humicola. The similar observation was reported by Sudarat C. et al., (2012) [58], the biomass -1 -1 productivity of Chlorococcum humicola in 0.033 gl d achieved in 3NBBM.

The Ankistrodesmus convolutes species showed (Figure 3.6) maximum dry biomass weight for BBM (347.5 mgl-1), for Chu 10 modified (176.5mgl-1) and for Chu 13 modified (134.6 mgl-1) and minimum in BG 11(129.3 mgl-1). The Ankistrodesmus convolutes species (Figure 3.5) showed that the positive growth rate for BBM with exponential phase till 15th day while stationary phase observed maximum growth rate on 16th day onwards. The biomass productivity was observed

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0.047 gl-1d-1 and lipid extracted from biomass was 0.018 gl-1d-1, the 38.29% lipid content was obtained. Nitumani Kalita et al., (2011) [57] reported the growth study of Ankistrodesmus sp., the stationary phase similar to observed results of Ankistrodesmus convolutes sp.

The Botryococcus braunii species showed (Figure 3.8) maximum dry biomass weight for Chu 13 modified (978.2 mgl-1), for Chu 10 modified (629.3 mgl-1), BBM (527.8 mgl-1) and minimum in BG 11 (398.4 mgl-1). It showed better growth result (Figure 3.7) in Chu 13 (mod.) medium, the growth phase observed up to the 18th day and stationary phase observed from 19th day. The biomass productivity of Botryococcus braunii in Chu 13 modified was observed 0.692 gl-1d-1 and lipid extracted from biomass was 0.147 gl-1d-1, the 21.24% lipid content was obtained. Dayananda C. et al., (2007) [59] reported similar observation,

The Clamydomaonas pertusa showed (Figure 3.10) maximum dry biomass weight for Chu 13 modified (750.4 mgl-1), for BBM (520.6 mgl-1), BG 11 (362.5 mgl- 1) and minimum in Chu 10 modified (286.5 mgl-1). The Clamydomaonas pertusa (Figure 3.9) showed the better growth results in Chu 13 (mod.) media, the growth phase observed up to the 19th day and stationary phase observed from 20th day. The biomass productivity of Clamydomaonas pertusa in Chu 13 modified was observed 0.059 gl-1d-1 and lipid extracted from biomass was 0.019 gl-1d-1, the 32.20% lipid content was obtained.

The species Scenedesmus dimorphus showed (Figure 3.12) maximum dry biomass weight for BG 11(245.5 mgl-1), for BBM (167.8 mgl-1), for Chu 13 modified (159.7 mgl-1) and minimum in Chu 10 modified (120.2 mgl-1). The Scenedesmus dimorphus (Figure 3.11) showed the better growth results in BG11 medium, the growth phase observed up to the 18th day and stationary phase observed from 19th day, maximum growth of strain. The stationary phase of all microalgae strain showed the linear growth pattern and the variation in exponential phase was seen. The biomass productivity of Scenedesmus dimorphus in BG11 was observed 0.095 gl-1d-1 and lipid extracted from biomass was 0.016 gl-1d-1, the 16.84% lipid content was obtained. Reda A.I. Abou-Shanab et al., (2011) [60] reported similar results in BBM.

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Table 3.10: Biomass productivity and Lipid productivity of Microalgae of selected Media Sr. Name of Microalgae Biomass Lipid No. productivity Productivity (gl-1d-1) (gl-1d-1)

1 Chlorella vulgaris 0.629 0.273 2 Chlorococcum humicola 0.094 0.029 3 Ankistrodesmus convolutes 0.047 0.018 4 Botryococcus braunii 0.692 0.147 5 Clamydomaonas pertusa 0.059 0.019 6 Scenedesmus dimorphus 0.095 0.016

Figure 3.13: Biomass Productivity of Microalgae

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Figure 3.14: Lipid Productivity of Microalgae

Figure 3.15: Lipid Content of Microalgae

However, the growth study of microalgae results that, the biomass productivity of Chlorella vulgaris was 0.629 gl-1d-1, lipid extracted from biomass was 0.273 gl-1d-1 and the 43.40% lipid content. The biomass productivity of Chlorococcum humicola in BBM was 0.094 gl-1d-1 and lipid extracted from biomass was 0.029 gl-1d- 1, the 30.85% lipid content. For the Ankistrodesmus convolutes in BBM, biomass productivity was 0.047 gl-1d-1 and lipid extracted from biomass was 0.018 gl-1d-1, the 38.29% lipid content. The biomass productivity of Botryococcus braunii in Chu 13

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modified was 0.692 gl-1d-1 and lipid extracted from biomass was 0.147 gl-1d-1, the 21.24% lipid content. The biomass productivity of Clamydomaonas pertusa in Chu 13 modified was 0.059 gl-1d-1, lipid extracted from biomass was 0.019 gl-1d-1and 32.20% lipid content. While the biomass productivity of Scenedesmus dimorphus in BG11 was 0.095 gl-1d-1 and lipid extracted from biomass was 0.016 gl-1d-1, the 16.84% lipid content was observed (Figure 3.13, 3.13 & 3.14)

The results obtained for the biomass productivity and lipid productivity in different growth media are shown in Table 3.11. On the basis of standard data, the medium was selected for the cultivation of mass algae.

Table 3.11: Growth Media used for selected microalgae during mass Cultivation

Sr. No. Name of Microalgae Name of Media 1 Chlorella vulgaris Chu 10 modified 2 Chlorococcum humicola Bold Basal Media 3 Ankistrodesmus Bold Basal Media convolutes 4 Botryococcus braunii Chu 13 modified 5 Clamydomaonas pertusa Chu 13 modified 6 Scenedesmus dimorphus BG11

3.1.4 Acid Value /Free Fatty Acid (FFA) of Algae Oil

The acid value of macroalgae and microalgae oil was evaluated by method 3.1.8. The acid value (AV) is the weight of KOH in mg needed to neutralize the acids present in 1g of fat and is a measure of the free fatty acids (FFA) present in the fat or oil. FFA is half amount of acid value of oil or fat. The obtained results of fatty acid value (mg KOH/g) for algae oil was shown in Table 3.12. The acid value for Chlorococcalean Macro-algae Oil was AV (2.24) and FFA (1.12); for Chlorella vulgaris Oil was AV (1.75) and FFA (0.87); for Chlorococcum humicola, AV (2.16) and FFA (1.08); for Ankistrodesmus convolutes, AV (2.08) and (1.08); for Botryococcus braunii, AV (1.95) and FFA (0.97); for Clamydomaonas pertusa, AV

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(2.25) and FFA (1.12) and Scenedesmus dimorphus AV (1.93) and FFA (0.86) were observed.

The Acid value and Free Fatty Acid value of algae Biodiesel was shown in Table 3.13. The acid value for Chlorococcalean Macro-algae Oil was (0.22) and FFA (0.11); for Chlorella vulgaris Oil was AV (0.16) and FFA (0.08); for Chlorococcum humicola, AV (0.14) and FFA (0.07); for Ankistrodesmus convolutes, AV (0.17) and (0.08); for Botryococcus braunii, AV (0.12) and FFA (0.06); for Clamydomaonas pertusa, AV (0.19) and FFA (0.09) and Scenedesmus dimorphus AV (0.13) and FFA (0.06) were observed.

Table 3.12: Acid values and free fatty acid values of Algae Oils

Sr. No. Algae Oil Acid value FFA Value 1 Chlorococcalean Macroalgae 2.24 1.12 2 Chlorella vulgaris 1.75 0.87 3 Chlorococcum humicola 2.16 1.08 4 Ankistrodesmus convolutes 2.08 1.04 5 Botryococcus braunii 1.95 0.97 6 Clamydomaonas pertusa 2.25 1.12 7 Scenedesmus dimorphus 1.93 0.86

Table 3.13: Acid values and free fatty acid values of Algae Biodiesel

Sr. No. Algae Biodiesel Acid value FFA Value 1 Chlorococcalean Macroalgae 0.22 0.11 2 Chlorella vulgaris 0.16 0.08 3 Chlorococcum humicola 0.14 0.07 4 Ankistrodesmus convolutes 0.17 0.08 5 Botryococcus braunii 0.12 0.06 6 Clamydomaonas pertusa 0.19 0.09 7 Scenedesmus dimorphus 0.13 0.06

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3.2 Characterization of Chlorococcalean Algae Biodiesel

(Acid, Base Transesterification Reaction)

3.2.1 Characteristics of Macroalgae Biodiesel

3.2.1.1 FTIR Data of Macroalgae Oil & Acid, Base Catalyzed

Biodiesel

Figure 3.16: FTIR spectrum of Chlorococcalean Macroalgae Oil

[AL 1]

Figure 3.17: FTIR spectrum of Chlorococcalean Macroalgae Biodiesel

[ALE1 (ac) TR]

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Figure 3.18: FTIR spectrum of Chlorococcalean Macroalgae Biodiesel

[ALE1 (bc) TR]

The FTIR Spectroscopy is the method to determine the functional groups present in obtained product. The obtained results of FTIR spectrum of oil and biodiesel evaluated with help of standard references [63] [64]. The biodiesel is mixture of fatty acid methyl esters. Don Pavia (2009), reported[63] the characteristic functional group frequency of hydroxyl group (O-H) occurs at 3400-2400 cm-1, for ester group carbonyl (C=O) frequency occurs at 1750-1735 cm-1, for alkyl carbon oxygen bond stretch (C-O) occurs at 1300-1000 cm-1 and long chain carbon more than four the frequency occurs at 700-800 cm-1.

The FTIR spectrum of Macroalgae oil (Figure 3.16) shows the υ (O-H) -1 3 stretching of hydroxyl group of fatty acids at 3381.03 cm , υ as (sp C-H) stretching of hydrocarbon at 2920.78 cm-1, υ (C=O) stretching of carbonyl group from fatty -1 acids at 1710.49 cm , methylene (CH2) groups have a characteristic bending -1 absorption at 1461.43 cm . The methyl δ s (CH3) group have characteristic bending -1 absorption is 1376.68 cm , the υ (C-O) stretching of alkyl carbon and oxygen from -1 fatty acids is at 1162.30 cm and δ r (CH2) bending (rocking) motion associated with

four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 722.41 cm-1.

The FTIR spectrum of Macroalgae Biodiesel [ALE1 (ac) TR] (Figure 3.17) 3 -1 shows υ as (sp C-H) stretching of hydrocarbon at 2921.78 cm , υ (C=O) stretching -1 of carbonyl group from fatty acids methyl ester at 1741.25 cm , methylene (CH2) -1 groups have a characteristic bending absorption at 1460.92 cm . The methyl δ s (CH3) -1 group have characteristic bending absorption is 1373.13 cm , the υ (C-O) stretching

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-1 of alkyl carbon and oxygen from fatty acids is at 1169.35 cm and δ r (CH2) bending

(rocking) motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 722.86 cm-1.

The FTIR spectrum of Macroalgae Biodiesel [ALE1 (bc) TR] (Figure 3.18) 3 -1 shows υ as (sp C-H) stretching of hydrocarbon at 2920.00 cm , υ (C=O) stretching -1 of carbonyl group from fatty acids methyl ester at 1740.77 cm , methylene (CH2) -1 groups have a characteristic bending absorption at 1461.47 cm . The methyl δ s (CH3) -1 group have characteristic bending absorption is 1371.93 cm , the υ (C-O) stretching -1 of alkyl carbon and oxygen from fatty acids is at 1170.83 cm and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 723.57 cm-1.

The given FTIR data shows the characteristic values of functional groups for oil and its biodiesel of algae. The nature of spectrum or peak intensity in oil and its biodiesel are different. The characteristic carbonyl frequency for oil was 1710.49 cm-1 while ester carbonyl group frequency 1741.25 cm-1 and 1740.77 cm-1 for biodiesel hence, it shows that complete conversion of oil into biodiesel takes place.

3.2.1.2 Gas Chromatography Data of Acid Catalyzed Biodiesel

Figure 3.19: The qualitative peaks of Gas Chromatography spectrum of Chlorococcalean Macroalgae Biodiesel

[ALE1 (ac) TR]

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Table 3.14: Chlorococcalean Macroalgae Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE1 (ac) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW Tetradecanoic acid methyl

1 22.765 13.59 ester C15H30O2 242 Hexadecanoic acid methyl

2 25.42 24.92 ester C17H34O2 270 9-Octadecenoic acid methyl

3 27.513 12.5 ester C19H36O2 296 Octadecanoic acid methyl

4 27.765 4.99 ester C19H38O2 298

3.2.1.3 Mass Spectroscopy Data of Acid Catalyzed Biodiesel

Mass Spectrum i: Tetradecanoic acid, methyl ester

Mass Spectrum ii: Hexadecanoic acid, methyl ester

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Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: Octadecanoic acid, methyl ester

Figure 3.20: Mass Spectra of Fatty Acid Methyl Esters of Macroalgae Biodiesel

3.2.1.4 Molecular Structures of Fatty Acid Methyl Esters of Acid

Catalyzed Biodiesel

O

O

i. Tetradecanoic acid methyl ester (C15H30O2)

O

O

ii. Hexadecanoic acid methyl ester (C17H34O2)

O

O

iii. 9-Octadecenoic acid methyl ester (C19H36O2)

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O

O

iv. Octadecanoic acid methyl ester (C19H38O2)

Figure 3.21: Molecular Structures of Fatty Acid Methyl Ester of Macroalgae Biodiesel

3.2.1.5 Gas Chromatography Data of Base Catalyzed Biodiesel

Figure 3.22: The qualitative peaks of Gas Chromatography spectrum of Chlorococcalean Macroalgae Biodiesel [ALE1 (bc) TR]

Table 3.15: Chlorococcalean Macroalgae Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formula and their Molecular Weights [ALE1 (bc) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 22.789 7.16 Tetradecanoic acid methyl ester C15H30O2 242

2 25.462 12.84 Hexadecanoic acid methyl ester C17H34O2 270

3 27.54 6.48 9-Octadecenoic acid, methyl ester C19H36O2 296

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3.2.1.6 Mass Spectroscopy Data of Base Catalyzed Biodiesel

Mass Spectrum i: Tetradecanoic acid, methyl ester

Mass Spectrum ii: Hexadecanoic acid, methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Figure 3.23: Mass Spectra of Fatty Acid Methyl Esters Macroalgae Biodiesel

3.2.1.7 Molecular Structures of Fatty Acid Methyl Esters of Base Catalyzed Biodiesel

O

O

i. Tetradecanoic acid methyl ester (C15H30O2)

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O

O

ii. Hexadecanoic acid methyl ester (C17H34O2)

O

O

iii. 9-Octadecenoic acid, methyl ester (C19H36O2)

Figure 3.24: Molecular Structures of Fatty Acid Methyl Esters of Macroalgae Biodiesel

Gas chromatography Mass Spectroscopy is used to separate and identify the chemical components present in the biodiesel. Gas chromatographic spectrum shows number of constituents present in analyzed sample and mass spectrum provide information about molecular structure. The obtained results were evaluated and confirmed by using the standard MS library NIST. The acid catalyzed Chlorococcalean Macroalgae Biodiesel [ALE1 (ac) TR] (Figure 3.19) shows Gas chromatographic spectrum with the various peak obtained at different retention times. In acid catalyzed Biodiesel of Macroalgae, four types of esters obtained at retention times (min) at 22.765, 24.92, 27.513 and 27.765. Mass spectrum fatty acid methyl esters of energy liquid fuel shown in Figure 3.20 respectively. The three components of biodiesel at retention times 22.765, 24.92 and 27.765 shows the base peak at m/z 74.05 (Figure 3.20: i, ii, iv), it is characteristic peak occurs due to McLafferty rearrangement [66]; the one component at 27.513 shows base peak at m/z 55 (Figure 3.20: iii). Table 3.14 shows the peak obtained with various retention times, percentage areas, name of the obtained compound and molecular formula. Figure 3.21 shows the molecular structure of FAMEs of obtained biodiesel product.

The Macroalgae Biodiesel contains higher content of Hexadecanoic acid methyl ester (24.92 %), Tetradecanoic acid methyl ester (13.59 %), 9-Octadecenoic acid methyl ester (12.5 %) and Octadecanoic acid methyl ester (4.99 %). The

119

biodiesel is mixture of saturated and unsaturated fatty acid methyl ester. The Macroalgae Biodiesel contain 43.5% saturated FAME and unsaturated 12.5 FAME.

The obtained FAME was ranging between C15 to C19.

The base catalyzed Chlorococcalean Macroalgae Biodiesel [ALE1 (bc) TR] (Figure 3.22) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In base catalyzed Biodiesel of Macroalgae, three types of major esters obtained at retention times (min) at 22.789, 25.462 and 27.54. Mass spectrum fatty acid methyl ester of energy liquid fuel shown in Figure 3.24 respectively. The two components of biodiesel at retention time 22.789 and 25.462 shows the base peak at m/z 74.05 (Figure 3.23: i, ii), this characteristic peak occurs due to McLafferty rearrangement [66]. The one component at 27.54 shows base peak at m/z 55 (Figure 3.23: iii). Table 3.15 shows the peak obtained with various retention times, percentage area, name of the obtained compound and molecular formula. Figure 3.24 shows the molecular structure of FAMEs of obtained biodiesel product.

The Macroalgae Biodiesel contains higher content of Hexadecanoic acid methyl ester (12.84 %), Tetradecanoic acid methyl ester (7.16 %) and 9-Octadecenoic acid methyl ester (6.48 %). The Macroalgae Biodiesel contain 43.5% saturated FAME

and unsaturated 12.5 FAME. The obtained FAMEs were ranging between C15 to C19.

The obtained results were compared with Jatropha biodiesel as standard. Sanjaykumar D. et al. (2012), reported [67] that the Jatropha biodiesel contain saturated and unsaturated FAMEs at ranging between C13 to C19. Pokharkar et al. (2012) had [68] investigated the undi biodiesel, the observed FAMEs range at C8 to C23. Brain J. Krohn et al. (2010), reported [69] that the macroalgae biodiesel possesses the FAME in

the range C12 to C18. Thus, the acid and base catalyzed biodiesel of macroalgae shows the variation in composition of FAMEs.

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3.2.2 Characteristics of Microalgae: Chlorella vulgaris Biodiesel

3.2.2.1 FTIR Data of Microalgae Oil & Acid, Base Catalyzed Biodiesel

Figure 3.25: FTIR spectrum of Chlorella vulgaris Oil

[AL2]

Figure 3.26: FTIR spectrum of Chlorella vulgaris Biodiesel [ALE2 (ac) TR]

Figure 3.27: FTIR spectrum of Chlorella vulgaris Biodiesel [ALE2 (bc) TR]

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The FTIR spectrum of Chlorella vulgaris oil (Figure 3.25) shows the υ (O-H) -1 3 stretching of hydroxyl group of fatty acids at 3366.89 cm , υ as (sp C-H) stretching of hydrocarbon at 2924.12 cm-1, υ (C=O) stretching of carbonyl group from fatty -1 acids at 1743.29 cm , methylene (CH2) groups have a characteristic bending -1 absorption at 1461.06 cm . The methyl δ s (CH3) groups have characteristic bending -1 absorption at 1375.68 cm , the υ (C-O) stretching of alkyl carbon and oxygen from -1 fatty acids is at 1158.32 cm and δ r (CH2) bending (rocking) motion associated with

four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 720.15 cm-1. The FTIR spectrum of Chlorella vulgaris Biodiesel [ALE2 (ac) TR] (Figure 3 -1 3.26) shows υ as (sp C-H) stretching of hydrocarbon at 2922.78 cm , υ (C=O) -1 stretching of carbonyl group from fatty acids methyl ester at 1743.61cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1460.21 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption is 1372.48 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1160.01 cm

and δ r (CH2) bending (rocking) motion associated with four or more methylene -1 (CH2) groups in an open chain of fatty acid occurs at about 721.21 cm .

The FTIR spectrum of Chlorella vulgaris Biodiesel [ALE2 (bc) TR] (Figure 3 -1 3.27) shows υ as (sp C-H) stretching of hydrocarbon at 2922.06 cm , υ (C=O) -1 stretching of carbonyl group from fatty acids methyl ester at 1743.61 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1458.65 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption is 1369.59 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1158.10 cm

and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 720.51 cm-1.

The FTIR analysis shows that in the Chlorella vulgaris algal oil and biodiesel, the –OH group of acid is appeared in oil while it not observed in spectrum of algal biodiesel, the appearance of peaks in algal oil and its biodiesel was different.

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3.2.2.2 Gas Chromatography Data of Acid Catalyzed Biodiesel

Figure 3.28: The qualitative peaks of Gas Chromatography spectrum of Chlorella vulgaris microalgae Biodiesel [ALE2 (ac) TR]

Table 3.16: Chlorella vulgaris Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE2 (ac) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 25.378 27.95 Hexadecanoic acid methyl ester C17H34O2 270 27.444 28.27 9,12-Octadecadienoic acid (Z,Z)-,

2 methyl ester C19H34O2 294

3 27.508 37.22 9-Octadecenoic acid methyl ester C19H36O2 296

4 27.757 6.56 Octadecanoic acid, methyl ester C19H38O2 298

3.2.2.3 Mass Spectroscopy Data of Acid Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

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Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: Octadecanoic acid, methyl ester

Figure 3.29: Mass Spectra of Fatty Acid Methyl Esters [ALE2 (ac) TR]

3.2.2.4 Molecular Structures of Fatty Acid Methyl Esters of Acid

Catalyzed Biodiesel

O

O

i. Hexadecanoic acid methyl ester (C17H34O2)

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O

O

ii. 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (C19H34O2)

O

O

iii. 9-Octadecenoic acid methyl ester (C19H36O2)

O

O

iv. Octadecanoic acid, methyl ester (C19H38O2)

Figure 3.30: Molecular Structures of Fatty Acid Methyl Esters

[ALE2 (ac) TR]

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3.2.2.5 Gas Chromatography Data of Base Catalyzed Biodiesel

Figure 3.31: The qualitative peaks of Gas Chromatography spectrum of Chlorella vulgaris microalgae Biodiesel [ALE2 (bc) TR]

Table 3.17: Chlorella vulgaris Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE2 (bc) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 25.378 15.33 Hexadecanoic acid, methyl ester C17H34O2 270 2 27.445 14.66 9,12-Octadecadienoic acid (Z,Z)-,

methyl ester C19H34O2 294

3 27.508 20.37 9-Octadecenoic acid, methyl ester C19H36O2 296

4 27.759 2.78 Octadecanoic acid, methyl ester C19H38O2 298

3.2.2.6 Mass Spectroscopy Data of Base Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

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Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: Octadecanoic acid, methyl ester

Figure 3.32: Mass Spectra of Fatty Acid Methyl Esters [ALE2 (bc) TR]

3.2.2.7 Molecular Structures of Fatty Acid Methyl Esters of Base Catalyzed Biodiesel

O

O

i. Hexadecanoic acid, methyl ester (C17H34O2)

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O

O

ii. 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (C19H34O2)

O

O

iii. 9-Octadecenoic acid, methyl ester (C19H36O2)

O

O

iv. Octadecanoic acid, methyl ester (C19H38O2)

Figure 3.33: Molecular Structures of Fatty Acid Methyl Esters [ALE2 (bc) TR]

The acid catalyzed Chlorococcalean microalgae Chlorella vulgaris Biodiesel [ALE2 (ac) TR] (Figure 3.28) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In acid catalyzed Biodiesel of Chlorella vulgaris four types of esters are obtained at retention times (min) at 25.378, 27.444, 27.508 and 27.757. Mass spectra of fatty acid methyl esters of energy liquid fuel are shown in Figure 3.29. The two components of biodiesel at retention times 25.378 and 27.757 shows the base peak at m/z 74.05 (Figure 3.29: i, iv), the characteristic peak occurs due to McLafferty rearrangement [66]. The one component at 27.444 shows base peak at m/z 81.5 (Figure 3.29: ii) and at 27.508 shows the base peak at m/z 55 (Figure 3.29: iii). Table 3.16 shows the peak obtained with various retention timess, percentage areas, name of the obtained compounds, molecular formulae their molecular weights. Figure 3.29 shows the molecular structure of FAMEs of obtained biodiesel product.

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The acid catalyzed Chlorococcalean microalgae Chlorella vulgaris Biodiesel contains higher content of 9-Octadecenoic acid methyl ester (37.22 %), 9,12- Octadecadienoic acid (Z,Z)-, methyl ester (28.27 %), Hexadecanoic acid methyl ester (27.378 %) and Octadecanoic acid methyl ester (6.56 %). The biodiesel is a mixture of saturated and unsaturated fatty acid methyl esters. The Chlorella vulgaris Biodiesel contain 34.51% saturated FAMEs and unsaturated 65.49% FAMEs. The obtained

FAMEs were ranging between C17 to C19.

The base catalyzed Chlorococcalean microalgae Chlorella vulgaris Biodiesel [ALE2 (bc) TR] (Figure 3.31) shows Gas chromatographic spectra with the various peaks obtained at different retention times. In acid catalyzed Biodiesel of Chlorella vulgaris four types of esters are obtained at retention times (min) at 25.378, 27.445, 27.508 and 27.759. Mass spectra fatty acid methyl esters of energy liquid fuel shown in Figure 3.32. The two components of biodiesel at retention times 25.378 and 27.759 show the base peak at m/z 74.05 (Figure 3.32: i, iv), the characteristic peak occurs due to McLafferty rearrangement [66]. The one component at 27.445 show base peak at m/z 81.5 (Figure 3.32: ii) and at 27.508 shows the base peak at m/z 55 (Figure 3.32: iii). Table 3.16 shows the peak obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.33 shows the molecular structure of FAMEs of obtained biodiesel product.

The base catalyzed Chlorococcalean microalgae, Chlorella vulgaris Biodiesel contains higher content of 9-Octadecenoic acid methyl ester (20.37 %), 9,12- Octadecadienoic acid (Z,Z)-, methyl ester (14.66 %), Hexadecanoic acid methyl ester (15.33 %) and Octadecanoic acid methyl ester (2.78 %). The biodiesel is a mixture of saturated and unsaturated fatty acid methyl esters. The Chlorella vulgaris Biodiesel contains 18.11 % saturated FAMEs and unsaturated 35.03 % FAMEs. The obtained [65] FAMEs were ranging between C17 to C19. The similar results were made by Prafulla D Patil et al. (2010).

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3.2.3 Characteristics of Microalgae: Chlorococcum humicola Biodiesel 3.2.3.1 FTIR Data of Microalgae Oil & Acid, Base Catalyzed Biodiesel

Figure 3.34: FTIR spectrum of Chlorococcum humicola Oil [AL 3]

Figure 3.35: FTIR spectrum of Chlorococcum humicola Biodiesel [ALE3 (ac) TR]

Figure 3.36: FTIR spectrum of Chlorococcum humicola Biodiesel [ALE3 (bc) TR] The FTIR spectrum of Chlorococcum humicola oil (Figure 3.34) shows the υ -1 3 (O-H) stretching of hydroxyl group of fatty acids at 3373.64 cm , υ as (sp C-H)

130

stretching of hydrocarbon at 2923.54 cm-1, υ (C=O) stretching of carbonyl group -1 from fatty acids at 1742.53 cm , methylene (CH2) groups have a characteristic -1 bending absorption at 1461.49 cm . The methyl δ s (CH3) group have characteristic -1 bending absorption at 1375. 43 cm , the υ (C-O) stretching of alkyl carbon and -1 oxygen from fatty acids are at 1156.14 cm and δ r (CH2) bending (rocking) motion

associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 721.82 cm-1. The FTIR spectrum of Chlorococcum humicola Biodiesel [ALE3 (ac) TR] 3 -1 (Figure 3.35) shows υ as (sp C-H) stretching of hydrocarbon at 2922.12 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1742.47 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1460.72 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1370.72 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1157.97 cm

and δ r (CH2) bending (rocking) motion associated with four or more methylene -1 (CH2) groups in an open chain of fatty acid occurs at about 723.37 cm .

The FTIR spectrum of Chlorococcum humicola Biodiesel [ALE3 (bc) TR] 3 -1 (Figure 3.36) shows υ as (sp C-H) stretching of hydrocarbon at 2924.00 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1745.66 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1459.96 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1375.90 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1164.49 cm

and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 725.94 cm-1.

The FTIR analysis shows that the Chlorococcum humicola algal oil and biodiesel, the –OH group of acid is disappeared, which is not observed in spectrum of algal biodiesel, the appearance of peak in algal oil and its biodiesel was different.

131

3.2.3.2 Gas Chromatography Data of Acid Catalyzed Biodiesel

Figure 3.37: The qualitative peaks of Gas Chromatography spectrum of Chlorococcum humicola microalgae Biodiesel [ALE3 (ac) TR]

Table 3.18: Chlorococcum humicola Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE3 (ac) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 12.911 8.95 Octanoic acid, methyl ester C9H18O2 158

2 16.599 8.35 Decanoic acid, methyl ester C11H22O2 186

3 19.901 38.23 Dodecanoic acid, methyl ester C13H26O2 214

4 22.77 20.28 Tetradecanoic acid methyl ester C15H30O2 242

5 25.374 9.87 Hexadecanoic acid, methyl ester C17H34O2 270

6 27.491 7.74 9-Octadecenoic acid methyl ester C19H36O2 296

3.2.3.3 Mass Spectroscopy Data of Acid Catalyzed Biodiesel

Mass Spectrum i: Octanoic acid, methyl ester

132

Mass Spectrum ii: Decanoic acid, methyl ester

Mass Spectrum iii: Dodecanoic acid, methyl ester

Mass Spectrum iv: Tetradecanoic acid, methyl ester

Mass Spectrum v: Hexadecanoic acid, methyl ester

133

Mass Spectrum vi: 9-Octadecenoic acid methyl ester

Figure 3.38: Mass Spectra of Fatty Acid Methyl Esters [ALE3 (ac) TR]

3.2.3.4 Molecular Structures of Fatty Acid Methyl Esters of Acid

Catalyzed Biodiesel

O

O

i. Octanoic acid, methyl ester (C9H18O2)

O

O

ii. Decanoic acid, methyl ester (C11H22O2)

O

O

iii. Dodecanoic acid, methyl ester (C13H26O2)

O

O

iv. Tetradecanoic acid methyl ester (C15H30O2)

134

O

O

v. Hexadecanoic acid, methyl ester (C17H34O2)

O

O

vi. 9-Octadecenoic acid methyl ester (C19H36O2)

Figure 3.39: Molecular Structures of Fatty Acid Methyl Esters

[ALE3 (ac) TR]

3.2.3.5 Gas Chromatography Data of Base Catalyzed Biodiesel

Figure 3.40: The qualitative peaks of Gas Chromatography spectrum of Chlorococcum humicola microalgae Biodiesel

[ALE3 (bc) TR]

135

Table 3.19: Chlorococcum humicola Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE3 (bc) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 12.93 8.55 Octanoic acid, methyl ester C9H18O2 158

2 16.617 8.00 Decanoic acid, methyl ester C11H22O2 186

3 19.948 33.93 Dodecanoic acid, methyl ester C13H26O2 214

4 22.808 20.58 Tetradecanoic acid, methyl ester C15H30O2 242

5 25.403 10.97 Hexadecanoic acid, methyl ester C17H34O2 270 6 27.443 3.3 9,12-Octadecadienoic acid (Z, Z),

methyl ester C19H34O2 294

7 27.518 8.02 9-Octadecenoic acid, methyl ester C19H36O2 296

8 27.774 4.98 Octadecanoic acid, methyl ester C19H38O2 298

3.2.3.6 Mass Spectroscopy Data of Base Catalyzed Biodiesel

Mass Spectrum i: Octanoic acid, methyl ester

Mass Spectrum ii: Decanoic acid, methyl ester

136

Mass Spectrum iii: Dodecanoic acid, methyl ester

Mass Spectrum iv: Tetradecanoic acid, methyl ester

Mass Spectrum v: Hexadecanoic acid, methyl ester

Mass Spectrum vi: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

137

Mass Spectrum vii: 9-Octadecenoic acid methyl ester

Mass Spectrum viii: Octadecanoic acid, methyl ester

Figure 3.41: Mass Spectra of Fatty Acid Methyl Esters

[ALE3 (bc) TR]

3.2.3.7 Molecular Structures of Fatty Acid Methyl Esters of Base

Catalyzed Biodiesel

O

O

i. Octanoic acid, methyl ester (C9H18O2)

O

O

ii. Decanoic acid, methyl ester (C11H22O2)

138

O

O

iii. Dodecanoic acid, methyl ester (C13H26O2)

O

O

iv. Tetradecanoic acid, methyl ester (C15H30O2)

O

O

v.Hexadecanoic acid, methyl ester (C17H34O2)

O

O

vi. 9,12-Octadecadienoic acid (Z, Z)-,methyl ester (C19H34O2)

O

O

vii. 9-Octadecenoic acid, methyl ester (C19H36O2)

139

O

O

viii. Octadecanoic acid, methyl ester (C19H38O2)

Figure 3.42: Molecular Structures of Fatty Acid Methyl Esters

[ALE3 (bc) TR]

The acid catalyzed Chlorococcalean microalgae Chlorococcum humicola Biodiesel [ALE3 (ac) TR] (Figure 3.37) shows Gas chromatographic spectrum with various peaks obtained at different retention times. In acid catalyzed Biodiesel of Chlorococcum humicola six types of ester obtained at retention times (min) at 12.911, 16.599, 19.901, 22.77, 25.374 and 27.491. Mass spectrum of fatty acid methyl esters of energy liquid fuel shown in Figure 3.38 The five components of biodiesel at retention times 12.911, 16.599, 19.901, 22.77 and 25.374 shows the base peak at m/z 74.05 (Figure 3.38: i-v), the characteristic peak occurs due to McLafferty rearrangement [66]. The one component at 27.491 shows base peak at m/z 55 (Figure 3.38: vi). Table 3.18 shows the peak obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.39 shows the molecular structures of FAMEs of obtained biodiesel product.

The acid catalyzed Chlorococcalean microalgae Chlorococcum humicola Biodiesel contains higher content of Dodecanoic acid, methyl ester (38.23%), Tetradecanoic acid methyl ester (20.28%), Hexadecanoic acid methyl ester (9.87 %), Octadecanoic acid methyl ester (8.95 %), Decanoic acid, methyl ester (8.35%) and 9- Octadecenoic acid methyl ester (7.74%). The Chlorococcum humicola Biodiesel contains 85.68 % saturated FAMEs and unsaturated 7.74 % FAMEs. The obtained

FAMEs were ranging between C9 to C19.

The base catalyzed Chlorococcalean microalgae Chlorococcum humicola Biodiesel [ALE3 (bc) TR] (Figure 3.40) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In base catalyzed Biodiesel of Chlorococcum humicola eight types of esters were obtained at retention times (min) at

140

12.93, 16.617, 19.948, 22.808, 25.403, 25.403, 27.443, 27.518 and 27.774. Mass spectrum of fatty acid methyl esters of energy liquid fuel are shown in Figure 3.41. The six components of biodiesel at retention times 12.93, 16.617, 19.948, 22.808, 25.403, 25.403 and 27.774, shows the base peak at m/z 74.05 (Figure 3.41: i-v, viii), its characteristic peak occurs due to McLafferty rearrangement [66]. The one component at 27.443 shows base peak at m/z 81.5 (Figure 3.41: vi) and at 25.403 shows the base peak at m/z 55 (Figure 3.41: vii). Table 3.19 shows the peak obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.42 shows the molecular structures of FAMEs of obtained biodiesel product.

The base catalyzed Chlorococcalean microalgae Chlorococcum humicola Biodiesel contains higher content of Dodecanoic acid, methyl ester (33.93 %), Tetradecanoic acid, methyl ester (20.58%), Hexadecanoic acid, methyl ester (10.97 %), Octanoic acid, methyl ester (8.55 %), 9-Octadecenoic acid, methyl ester (8.02 %), Decanoic acid, methyl ester (8 %) and Octadecanoic acid, methyl ester (4.98 %). The biodiesel is a mixture of saturated and unsaturated fatty acid methyl esters. The Chlorococcum humicola Biodiesel contains 87.01 % saturated FAMEs and unsaturated 11.32 % FAMEs. The obtained FAMEs were ranging between C9 to C19.

The similar results were reported by Sudarat Chaichalerm [58].

141

3.2.4 Characteristics of Microalgae: Ankistrodesmus convolutes Biodiesel 3.2.4.1 FTIR Data of Microalgae Oil & Acid, Base Catalyzed Biodiesel

Figure 3.43: FTIR spectrum of Ankistrodesmus convolutes Oil [AL4]

Figure 3.44: FTIR spectrum of Ankistrodesmus convolutes Biodiesel [ALE4 (ac) TR]

Figure 3.45: FTIR spectrum of Ankistrodesmus convolutes Biodiesel [ALE4 (bc) TR]

142

The FTIR spectrum of Ankistrodesmus convolutes oil [AL4] (Figure 3.43) -1 shows the υ (O-H) stretching of hydroxyl group of fatty acids at 3381.22 cm , υ as (sp3 C-H) stretching of hydrocarbon at 2923.09 cm-1, υ (C=O) stretching of carbonyl -1 group from fatty acids at 1741.08 cm , methylene (CH2) groups have a characteristic -1 bending absorption at 1460.68 cm . The methyl δ s (CH3) group have characteristic -1 bending absorption at 1376.12 cm , the υ (C-O) stretching of alkyl carbon and -1 oxygen from fatty acids are at 1160.02 cm and δ r (CH2) bending (rocking) motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 721.24 cm-1. The FTIR spectrum of Ankistrodesmus convolutes Biodiesel [ALE4 (ac) TR] 3 -1 (Figure 3.44) shows υ as (sp C-H) stretching of hydrocarbon at 2922.09 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1742.88 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1458.14 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1366.02 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1163.02 cm

and δ r (CH2) bending (rocking) motion associated with four or more methylene -1 (CH2) groups in an open chain of fatty acid occurs at about 721.57 cm .

The FTIR spectrum of Ankistrodesmus convolutes Biodiesel [ALE4 (bc) TR] 3 -1 (Figure 3.45) shows υ as (sp C-H) stretching of hydrocarbon at 2922.75 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1743.21 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1457.85 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1366.21 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1163.15 cm

and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 721.34 cm-1.

The FT-IR analysis shows that in the Ankistrodesmus convolutes algal oil and biodiesel, the –OH group of acid is disappeared and not observed in spectrum of algal biodiesel, the appearance of peaks in algal oil and its biodiesel were different.

143

3.2.4.2 Gas Chromatography Data of Acid Catalyzed Biodiesel

Figure 3.46: The qualitative peaks of Gas Chromatography spectrum of Ankistrodesmus convolute microalgae Biodiesel [ALE4 (ac) TR]

Table 3.20: Ankistrodesmus convolutes Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weight [ALE4 (ac) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 25.413 19.43 Hexadecanoic acid methyl ester C17H34O2 270 2 27.477 23.53 9,12-Octadecadienoic acid (Z,Z)-,

methyl ester C19H34O2 294

3 27.586 34 9-Octadecenoic acid methyl ester C19H36O2 296

4 27.816 19.47 Octadecanoic acid, methyl ester C19H38O2 298

5 30 3.57 Methyl 18-methylnonadecanoate C21H42O2 326

144

3.2.4.3 Mass Spectroscopy Data of Acid Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: Octadecanoic acid, methyl ester

145

Mass Spectrum v: 11-Eicosenoic acid, methyl ester

Figure 3.47: Mass Spectra of Fatty Acid Methyl Esters

[ALE4 (ac) TR]

3.2.4.4 Molecular Structures of Fatty Acid Methyl Esters of Acid

Catalyzed Biodiesel

O

O

i. Hexadecanoic acid methyl ester (C17H34O2)

O

O

ii. 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (C19H34O2)

O

O

iii. 9-Octadecenoic acid methyl ester (C19H36O2)

146

O

O

iv. Octadecanoic acid, methyl ester (C19H38O2)

O

O

v . Methyl 18-methylnonadecanoate (C21H42O2)

Figure 3.48: Molecular Structure of Fatty Acid Methyl Esters

[ALE4 (ac) TR]

3.2.4.5 Gas Chromatography Data of Base Catalyzed Biodiesel

Figure 3.49: The qualitative peaks of Gas Chromatography spectrum of Ankistrodesmus convolute microalgae Biodiesel [ALE4 (bc) TR]

147

Table 3.21: Ankistrodesmus convolutes Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE4 (bc) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 25.389 15.36 Hexadecanoic acid, methyl ester C17H34O2 270 2 27.461 18.59 9,12-Octadecadienoic acid (Z, Z)

methyl ester C19H34O2 294

3 27.539 30.55 9-Octadecenoic acid methyl ester C19H36O2 296

4 27.783 14.11 Octadecanoic acid, methyl ester C19H38O2 298

5 30.005 2.05 Methyl 18-methylnonadecanoate C21H42O2 326

6 32.986 12.93 Methyl 20-methyl-heneicosanoate C23H46O2 354

3.2.4.6 Mass Spectroscopy Data of Base Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

148

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: Octadecanoic acid, methyl ester

Mass Spectrum v: 11-Eicosenoic acid, methyl ester

Mass Spectrum vi: Methyl 20-methyl-heneicosanoate

Figure 3.50: Mass Spectra of Fatty Acid Methyl Esters

[ALE4 (bc) TR]

149

3.2.4.7 Molecular Structures of Fatty Acid Methyl Esters of Base

Catalyzed Biodiesel

O

O

i. Hexadecanoic acid, methyl ester (C17H34O2)

O

O

ii. 9,12-Octadecadienoic acid (Z, Z) methyl ester (C19H34O2)

O

O

iii. 9-Octadecenoic acid methyl ester (C19H36O2)

O

O

iv. Octadecanoic acid, methyl ester (C19H38O2)

O

O

v. Methyl 18-methylnonadecanoate (C21H42O2)

150

O

O

vi. Methyl 20-methyl-heneicosanoate (C23H46O2)

Figure 3.51: Molecular Structures of Fatty Acid Methyl Esters [ALE4 (bc) TR]

The acid catalyzed Chlorococcalean microalgae Ankistrodesmus convolutes Biodiesel [ALE4 (ac) TR] (Figure 3.46) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In acid catalyzed Biodiesel of Ankistrodesmus convolutes five types of esters are obtained at retention times (min) at 25.41, 27.477, 27.586, 27.816 and 30. Mass spectrum of fatty acid methyl esters of energy liquid fuel shown in Figure 3.47 The three components of biodiesel at retention times 25.41, 27.816 and 30 shows the base peak at m/z 74.05 (Figure 3.47: i, iv, v), the characteristic peak occurs due to McLafferty rearrangement [66]. The component at 27.477 shows base peak at m/z 81.5 (Figure 3.47: ii) and the one components at 27.586 shows base peak at m/z 55 (Figure 3.47: iii). Table 3.20 shows the peak obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.48 shows the molecular structures of FAMEs of obtained biodiesel product. The acid catalyzed Chlorococcalean microalgae Ankistrodesmus convolutes Biodiesel contains higher content of 9-Octadecenoic acid methyl ester (34 %), 9,12- Octadecadienoic acid (Z,Z), methyl ester (25.53 %), Octadecanoic acid, methyl ester (19.47 %), Hexadecanoic acid methyl ester (19.43%) and Methyl 18- methylnonadecanoate (3.57 %). The Ankistrodesmus convolutes Biodiesel contain 42.47 % saturated FAMEs and unsaturated 57.53 % FAMEs. The obtained FAMEs were ranging between C17 to C21.

The base catalyzed Chlorococcalean microalgae of Ankistrodesmus convolutes Biodiesel [ALE4 (bc) TR] (Figure 3.49) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In base catalyzed Biodiesel of Ankistrodesmus convolutes six types of esters are obtained at retention times (min) at 25.389, 27.461, 27.539, 27.783, 30.005 and 32.986. Mass

151

spectrum of fatty acid methyl esters of energy liquid fuel is shown in Figure 3.50. The six components of biodiesel at retention time’s 25.389, 27.783, 30.005 and 32.986 shows the base peak is at m/z 74.05 (Figure 3.50: i, iv, v, vi), this characteristic peak occurs due to McLafferty rearrangement [66]. The component at 27.461 shows base peaks at m/z 81.5 (Figure 3.50: ii) and at 27.539 shows the base peak at m/z 55 (Figure 3.50: iii). Table 3.21 shows the peaks obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.51 shows the molecular structure of FAMEs of obtained biodiesel product.

The base catalyzed Chlorococcalean microalgae Ankistrodesmus convolutes Biodiesel contains higher content of 9-Octadecenoic acid methyl ester (27.539 %); 9, 12-Octadecadienoic acid (Z, Z) methyl ester (18.59 %), Hexadecanoic acid, methyl ester (15.36 %), Octadecanoic acid, methyl ester (14.11 %), Methyl 20-methyl- heneicosanoate (12.93 %) and Methyl 18-methylnonadecanoate (2.05 %). The biodiesel is a mixture of saturated and unsaturated fatty acid methyl esters. The Ankistrodesmus convolutes Biodiesel contain 46.5 % saturated FAMEs and unsaturated 49.14 % FAMEs. The obtained FAMEs were ranging between C17 to C23.

152

3.2.5 Characteristics of Microalgae: Botryococcus braunii Biodiesel

3.2.5.1 FTIR Data of Microalgae Oil & Acid, Base Catalyzed Biodiesel

Figure 3.52: FTIR spectrum of Botryococcus braunii Oil

[AL 5]

Figure 3.53: FTIR spectrum of Botryococcus braunii Biodiesel

[ALE5 (ac) TR]

Figure 3.54: FTIR spectrum of Botryococcus braunii Biodiesel [ALE5 (bc) TR]

153

The FTIR spectrum of Botryococcus braunii oil [AL5] (Figure 3.52) shows -1 3 the υ (O-H) stretching of hydroxyl group of fatty acids at 3405.10 cm , υ as (sp C- H) stretching of hydrocarbon at 2923.00 cm-1, υ (C=O) stretching of carbonyl group -1 from fatty acids at 1742.30 cm , methylene (CH2) groups have a characteristic -1 bending absorption at 1459.98 cm . The methyl δ s (CH3) group have characteristic -1 bending absorption at 1373.34 cm , the υ (C-O) stretching of alkyl carbon and -1 oxygen from fatty acids is at 1160.06 cm and δ r (CH2) bending (rocking) motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 721.11 cm-1. The FTIR spectrum of Botryococcus braunii Biodiesel [ALE5 (ac) TR] 3 -1 (Figure 3.53) shows υ as (sp C-H) stretching of hydrocarbon at 2921.50 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1742.93 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1460.00 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1368.54 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1160.81 cm

and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 721.02 cm-1.

The FTIR spectrum of Botryococcus braunii Biodiesel [ALE5 (bc) TR] 3 -1 (Figure 3.54) shows υ as (sp C-H) stretching of hydrocarbon at 2922.24 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1743.24 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1459.94 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1369.40 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1162.21 cm

and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 721.81cm-1.

The FTIR analysis shows that the Botryococcus braunii algal oil and biodiesel, the –OH group of acid is disappeared and which not observed in spectrum of algal biodiesel, the appearance of peak in algal oil and its biodiesel were different.

154

3.2.5.2 Gas Chromatography Data of Acid Catalyzed Biodiesel

Figure 3.55: The qualitative peaks of Gas Chromatography spectrum of Botryococcus braunii microalgae Biodiesel

[ALE5 (ac) TR]

Table 3.22: Botryococcus braunii Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE5 (ac) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 25.386 10.66 Hexadecanoic acid, methyl ester C17H34O2 270 2 27.464 15.26 9,12-Octadecadienoic acid (Z, Z)

methyl ester C19H34O2 294

3 27.563 30.81 9-Octadecenoic acid, methyl ester C19H36O2 296

4 27.775 6.16 Octadecanoic acid, methyl ester C19H38O2 298

5 29.727 2.02 11-Eicosenoic acid, methyl ester C21H40O2 324

6 30.002 2.23 Methyl 18-methylnonadecanoate C21H42O2 326

7 32.903 5.03 Methyl 20-methyl-heneicosanoate C23H46O2 354

8 36.603 1.5 Tetracosanoic acid, methyl ester C25H50O2 382

9 39.535 26.34 Dodecanoic acid, 1,2,3- C39H74O6 638 propanetriyl ester

155

3.2.5.3 Mass Spectroscopy Data of Acid Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: 11-Eicosenoic acid, methyl ester

156

Mass Spectrum v: Methyl 18-methylnonadecanoate

Mass Spectrum vi: Methyl 20-methyl-heneicosanoate

Mass Spectrum vii: Tetradecanoic acid, methyl ester

Mass Spectrum viii: Dodecanoic acid, 1, 2, 3-propanetriyl ester

Figure 3.56: Mass Spectra of Fatty Acid Methyl Esters [ALE5 (ac) TR]

157

3.2.5.4 Molecular Structures of Fatty Acid Methyl Esters of Acid

Catalyzed Biodiesel

O

O

i. Hexadecanoic acid, methyl ester (C17H34O2)

O

O

ii. 9,12-Octadecadienoic acid (Z, Z) methyl ester (C19H34O2)

O

O

iii. 9-Octadecenoic acid, methyl ester (C19H36O2)

O

O

iv. Octadecanoic acid, methyl ester (C19H38O2)

O

O

v. 11-Eicosenoic acid, methyl ester (C21H40O2)

158

O

O

vi. Methyl 18-methylnonadecanoate (C21H42O2)

O

O

vii. Methyl 20-methyl-heneicosanoate (C23H46O2)

O

O

viii. Tetracosanoic acid, methyl ester (C25H50O2)

O

O O O

O O

ix. Dodecanoic acid, 1,2,3-propanetriyl ester (C39H74O6)

Figure 3.57: Molecular Structures of Fatty Acid Methyl Esters [ALE5 (ac) TR]

159

3.2.5.5 Gas Chromatography Data of Base Catalyzed Biodiesel

Figure 3.58: The qualitative peaks of Gas Chromatography spectrum of Botryococcus braunii microalgae Biodiesel [ALE5 (bc) TR]

Table 3.23: Botryococcus braunii Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE5 (bc) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 25.408 13.16 Hexadecanoic acid methyl ester C17H34O2 270 2 27.47 20.4 9,12-Octadecadienoic acid (Z,Z)-,

methyl ester C19H34O2 294

3 27.612 32.4 9-Octadecenoic acid methyl ester C19H36O2 296

4 27.799 8.54 Octadecanoic acid, methyl ester C19H38O2 298

5 29.736 2.74 11-Eicosenoic acid, methyl ester C21H40O2 324

6 30.01 3.21 Methyl 18-methylnonadecanoate C21H42O2 326

7 32.924 6.88 Methyl 20-methyl-heneicosanoate C23H46O2 354

8 36.615 2.85 Tetracosanoic acid, methyl ester C25H50O2 382

160

3.2.5.6 Mass Spectroscopy Data of Base Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: 11-Eicosenoic acid, methyl ester

161

Mass Spectrum v: Methyl 18-methylnonadecanoate

Mass Spectrum vi: Methyl 20-methyl-heneicosanoate

Mass Spectrum vii: Tetradecanoic acid, methyl ester

Figure 3.59: Mass Spectra of Fatty Acid Methyl Esters [ALE5 (bc) TR]

3.2.5.7 Molecular Structures of Fatty Acid Methyl Esters of Base

Catalyzed Biodiesel

O

O

i. Hexadecanoic acid methyl ester (C17H34O2)

162

O

O

ii. 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (C19H34O2)

O

O

iii. 9-Octadecenoic acid methyl ester (C19H36O2)

O

O

iv. Octadecanoic acid, methyl ester (C19H38O2)

O

O

v. 11-Eicosenoic acid, methyl ester (C21H40O2)

O

O

vi. Methyl 18-methylnonadecanoate (C21H42O2)

O

O

vii. Methyl 20-methyl-heneicosanoate (C23H46O2)

163

O

O

viii. Tetracosanoic acid, methyl ester (C25H50O2)

Figure 3.60: Molecular Structures of Fatty Acid Methyl Esters [ALE5 (bc) TR]

The acid catalyzed Chlorococcalean microalgae Botryococcus braunii Biodiesel [ALE5 (ac) TR] (Figure 3.55) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In acid catalyzed Biodiesel of Botryococcus braunii nine types of ester are obtained at retention times (min) at 25.386, 27.464, 27.563, 27.775, 29.727, 30.002, 32.903, 36.603 and 39.535. Mass spectrum of fatty acid methyl esters of energy liquid fuel is shown in Figure 3.56. The five components of biodiesel at retention times 25.386, 27.775, 30.002, 32.903 and 36.603, show the base peak at m/z 74.05 (Figure 3.56: i, iv, vi, vii), this characteristic peak occurs due to McLafferty rearrangement [66]. The two components at 27.464 and 39.535 show base peak at m/z 81.5 (Figure 3.56: ii, ix) and the two components at 27.563 and 29.727 show base peak at m/z 55 (Figure 3.56: iii, v). Table 3.22 shows the peak obtained with various retention times, percentage areas, names of the obtained compounds, molecular formulae and their molecular weights. Figure 3.57 shows the molecular structures of FAMEs of obtained biodiesel product. The acid catalyzed Chlorococcalean microalgae Botryococcus braunii Biodiesel contains higher content of 9-Octadecenoic acid methyl ester (30.81 %), Dodecanoic acid, 1,2,3-propanetriyl ester (26.34 %), 9,12-Octadecadienoic acid (Z, Z) methyl ester (15.26%), Hexadecanoic acid, methyl ester (10.66%), Octadecanoic acid, methyl ester (6.16 %), Methyl 20-methyl-heneicosanoate (5.03 %), Methyl 18- methylnonadecanoate (2.23 %), 11-Eicosenoic acid, methyl ester (2.02) and Tetracosanoic acid, methyl ester (1.5 %). The Botryococcus braunii Biodiesel contain 51.92 % saturated FAMEs and unsaturated 48.09 % FAMEs. The obtained FAMEs were ranging between C17 to C25.

164

The base catalyzed Chlorococcalean microalgae of Botryococcus braunii Biodiesel [ALE5 (bc) TR] (Figure 3.58) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In base catalyzed Biodiesel of Botryococcus braunii eight types of esters obtained at retention times (min) at 25.408, 27.47, 27.612, 27.799, 29.736, 30.01, 32.924 and 36.615. Mass spectrum of fatty acid methyl esters are shown in Figure 3.59. The six components of biodiesel at retention times 25.408, 27.799, 30.01, 32.924 and 36.615 shows the base peak at m/z 74.05 (Figure 3.59: i, iv, vi, vii, viii), this characteristic peak occurs due to McLafferty rearrangement [66]; the one component at 27.47 shows base peak at m/z 81.5 (Figure 3.59: ii) and at 27.612 and 29.736 shows the base peak at m/z 55 (Figure 3.59: iii, v). Table 3.23 shows the peaks obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.60 shows the molecular structure of FAMEs of obtained biodiesel product.

The base catalyzed Chlorococcalean microalgae Botryococcus braunii Biodiesel contains higher content of 9-Octadecenoic acid methyl ester (32.4 %); 9,12- Octadecadienoic acid (Z, Z) methyl ester (20.4 %), Hexadecanoic acid, methyl ester (13.16 %), Octadecanoic acid, methyl ester (8.54 %), Methyl 20-methyl- heneicosanoate (6.88 %) and Methyl 18-methylnonadecanoate (3.21 %), Tetracosanoic acid, methyl ester (2.85 %) and 11-Eicosenoic acid, methyl ester (2.74 %). The biodiesel is a mixture of saturated and unsaturated fatty acid methyl esters. The Botryococcus braunii Biodiesel contain 34.64 % saturated FAMEs and

unsaturated 55.54 % FAMEs. The obtained FAMEs were ranging between C17 to [59] C25.Similar observation were made by Dayananda C. (2007) .

165

3.2.6 Characteristics of Microalgae: Clamydomaonas pertusa Biodiesel 3.2.6.1 FTIR Data of Microalgae Oil & Acid, Base Catalyzed Biodiesel

Figure 3.61: FTIR spectrum of Clamydomaonas pertusa Oil [AL 6]

Figure 3.62: FTIR spectrum of Clamydomaonas pertusa Biodiesel [ALE6 (ac) TR]

Figure 3.63: FTIR spectrum of Clamydomaonas pertusa Biodiesel [ALE6 (bc) TR]

The FTIR spectrum of Clamydomaonas pertusa oil [AL6] (Figure 3.61) shows -1 3 the υ (O-H) stretching of hydroxyl group of fatty acids at 3409.79 cm , υ as (sp C-

166

H) stretching of hydrocarbon at 2922.59 cm-1, υ (C=O) stretching of carbonyl group -1 from fatty acids at 1743.30 cm , methylene (CH2) groups have a characteristic -1 bending absorption at 1460.01 cm . The methyl δ s (CH3) group have characteristic -1 bending absorption at 1374.17 cm , the υ (C-O) stretching of alkyl carbon and -1 oxygen from fatty acids is at 1159.47 cm and δ r (CH2) bending (rocking) motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 719.52 cm-1. The FTIR spectrum of Clamydomaonas pertusa Biodiesel [ALE6 (ac) TR] 3 -1 (Figure 3.62) shows υ as (sp C-H) stretching of hydrocarbon at 2922.85 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1744.06 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1459.19 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1369.28 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1161.68 cm

and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 720.90 cm-1.

The FTIR spectrum of Clamydomaonas pertusa Biodiesel [ALE6 (bc) TR] 3 -1 (Figure 3.63) shows υ as (sp C-H) stretching of hydrocarbon at 2922.72 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1743.98 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1460.31 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1373.14 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1159.74 cm

and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 720.25cm-1.

The FT-IR analysis shows that in the Clamydomaonas pertusa algal oil and biodiesel, the –OH group of acid is appeared in algal oil spectrum while it not observed in spectrum of algal biodiesel; the appearance of peaks in algal oil and its biodiesel were different.

167

3.2.6.2 Gas Chromatography Data of Acid Catalyzed Biodiesel

Figure 3.64: The qualitative peaks of Gas Chromatography spectrum of Clamydomaonas pertusa microalgae Biodiesel

[ALE6 (ac) TR]

Table 3.24: Clamydomaonas pertusa Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE6 (ac) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW 1 25.365 4.89 Hexadecanoic acid, methyl

ester C17H34O2 270 2 27.454 14.33 9,12-Octadecadienoic acid

(Z,Z)-, methyl ester C19H34O2 294 3 27.527 14.17 9-Octadecenoic acid methyl

ester C19H36O2 296

4 27.757 2.92 Octadecanoic acid, methyl ester C19H38O2 298 5 29.735 7.4 11-Eicosenoic acid, methyl

ester C21H40O2 324

6 29.996 1.63 18-methyl nonadecanoic acid C21H42O2 methyl ester 326 7 32.6 39.08 13-Docosenoic acid, methyl

ester C23H44O2 352

168

3.2.6.3 Mass Spectroscopy Data of Acid Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: Octadecanoic acid, methyl ester

169

Mass Spectrum v: 11-Eicosenoic acid, methyl ester

Mass Spectrum vi: 18-methyl nonadecanoic acid methyl ester

Mass Spectrum vii: 13-Docosenoic acid, methyl ester

Figure 3.65: Mass Spectra of Fatty Acid Methyl Esters

[ALE6 (ac) TR]

3.2.6.4 Molecular Structures of Fatty Acid Methyl Esters of Acid Catalyzed Biodiesel

O

O

i. Hexadecanoic acid, methyl ester (C17H34O2)

170

O

O

ii. 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (C19H34O2)

O

O

iii. 9-Octadecenoic acid methyl ester (C19H36O2)

O

O

iv. Octadecanoic acid, methyl ester (C19H38O2)

O

O

v.11-Eicosenoic acid, methyl ester (C21H40O2)

O

O

vi. 18-methyl nonadecanoic acid methyl ester (C21H42O2)

171

O

O

vi. 13-Docosenoic acid, methyl ester (C23H44O2)

Figure 3.66: Molecular Structures of Fatty Acid Methyl Esters of Biodiesel

3.2.6.5 Gas Chromatography Data of Base Catalyzed Biodiesel

Figure 3.67: The qualitative peaks of Gas Chromatography spectrum of Clamydomaonas pertusa microalgae Biodiesel [ALE6 (bc) TR]

Table 3.25: Clamydomaonas pertusa Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weight [ALE6 (bc) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 25.368 7.01 Hexadecanoic acid, methyl ester C17H34O2 270 2 27.44 13.46 9,12-Octadecadienoic acid (Z, Z)-

,methyl ester C19H34O2 294

3 27.501 21.54 9-Octadecenoic acid methyl ester C19H36O2 296

4 29.733 10.4 11-Eicosenoic acid, methyl ester C21H40O2 324

5 32.546 47.59 13-Docosenoic acid, methyl ester C23H44O2 352

172

3.2.6.6 Mass Spectroscopy Data of Base Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: 11-Eicosenoic acid, methyl ester

173

Mass Spectrum v: 13-Docosenoic acid, methyl ester

Figure 3.68: Mass Spectra of Fatty Acid Methyl Esters [ALE6 (bc) TR]

3.2.6.7 Molecular Structures of Fatty Acid Methyl Esters of Base

Catalyzed Biodiesel

O

O

i. Hexadecanoic acid, methyl ester (C17H34O2)

O

O

ii. 9,12-Octadecadienoic acid (Z, Z)-,methyl ester (C19H34O2)

O

O

iii. 9-Octadecenoic acid methyl ester (C19H36O2)

O

O

iv. 11-Eicosenoic acid, methyl ester (C21H40O2)

174

O

O

v. 13-Docosenoic acid, methyl ester (C23H44O2)

Figure 3.69: Molecular Structures of Fatty Acid Methyl Esters [ALE6 (bc) TR]

The acid catalyzed Chlorococcalean microalgae Clamydomaonas pertusa Biodiesel [ALE6 (ac) TR] (Figure 3.64) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In acid catalyzed Biodiesel of Clamydomaonas pertusa seven types of esters were obtained at retention times (min) at 25.365, 27.454, 27.527, 27.757, 29.735, 29.996 and 32.6. Mass spectrum of fatty acid methyl esters of energy liquid fuel is shown in Figure 3.65. The three components of biodiesel at retention times 25.36, 27.757 and 32.6 show the base peak at m/z 74.05 (Figure 3.65: i, iv, vi). This characteristic peak occurs due to McLafferty rearrangement [66]. The one component at 27.454 shows base peak at m/z 81.5 (Figure 3.65: ii) and the three components at 27.527, 29.735 and 29.996 shows base peak at m/z 55 (Figure 3.65: iii, v, vii). Table 3.24 shows the peaks obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.66 shows the molecular structure of FAMEs of obtained biodiesel product. The acid catalyzed Chlorococcalean microalgae Clamydomaonas pertusa Biodiesel contains higher content of 13-Docosenoic acid, methyl ester (39.08 %), 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (14.33 %), 9-Octadecenoic acid methyl ester (14.17 %), 11-Eicosenoic acid, methyl ester (7.4 %), Hexadecanoic acid, methyl ester (4.89%), Octadecanoic acid, methyl ester (2.92 %) and Methyl 18- methylnonadecanoate (1.63 %). The Clamydomaonas pertusa Biodiesel contains 9.44 % saturated FAMEs and unsaturated 74.98 % FAMEs. The obtained FAMEs were ranging between C17 to C23.

The base catalyzed Chlorococcalean microalgae of Clamydomaonas pertusa Biodiesel [ALE6 (bc) TR] (Figure 3.67) shows Gas chromatographic spectrum with

175 the various peaks obtained at different retention times. In base catalyzed Biodiesel of Clamydomaonas pertusa five types of esters are obtained at retention times (min) at 25.368, 27.44, 27.501, 29.733 and 32.546. Mass spectrum of fatty acid methyl esters is shown in Figure 3.68. The one component of biodiesel at retention time 25.368 shows the base peak at m/z 74.05 (Figure 3.68: i), this characteristic peaks occurs due to McLafferty rearrangement [66]. The one component at 27.44 show the base peak at m/z 81.5 (Figure 3.68: ii) and at 27.501, 29.733 and 32.546 shows the base peaks at m/z 55 (Figure 3.68: iii, iv, v). Table 3.25 shows the peak obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.69 shows the molecular structures of FAMEs of obtained biodiesel product.

The base catalyzed Chlorococcalean microalgae Clamydomaonas pertusa Biodiesel contains higher content of 13-Docosenoic acid, methyl ester (47.59%), 9- Octadecenoic acid methyl ester (21.54%), 9,12-Octadecadienoic acid (Z, Z) methyl ester (13.46 %), Hexadecanoic acid, methyl ester (7.01 %) and 11-Eicosenoic acid, methyl ester (10.4% ). The biodiesel is a mixture of saturated and unsaturated fatty acid methyl esters. The Clamydomaonas pertusa Biodiesel contain 7.01 % saturated FAMEs and unsaturated 92.99 % FAMEs. The obtained FAMEs were ranging between C17 to C23.

176

3.2.7 Characteristics of Microalgae: Scenedesmus dimorphus Biodiesel

3.2.7.1 FTIR Data of Microalgae Oil & Acid, Base Catalyzed Biodiesel

Figure 3.70: FTIR spectrum of Scenedesmus dimorphus Oil [AL7]

Figure 3.71: FTIR spectrum of Scenedesmus dimorphus Biodiesel [ALE7 (ac) TR]

Figure 3.72: FTIR spectrum of Scenedesmus dimorphus Biodiesel [ALE7 (bc) TR] The FTIR spectrum of Scenedesmus dimorphus oil [AL7] (Figure 3.70) shows -1 3 the υ (O-H) stretching of hydroxyl group of fatty acids at 3372.12 cm , υ as (sp C-

177

H) stretching of hydrocarbon at 2924.68 cm-1, υ (C=O) stretching of carbonyl group -1 from fatty acids at 1772.19 cm , methylene (CH2) groups have a characteristic -1 bending absorption at 1460.36 cm . The methyl δ s (CH3) group have characteristic -1 bending absorption at 1376.87 cm , the υ (C-O) stretching of alkyl carbon and -1 oxygen from fatty acids is at 1160.70 cm and δ r (CH2) bending (rocking) motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 720.79 cm-1. The FTIR spectrum of Scenedesmus dimorphus Biodiesel [ALE7 (ac) TR] 3 -1 (Figure 3.71) shows υ as (sp C-H) stretching of hydrocarbon at 2921.91 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1742.82 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1460.67 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1370.36 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1161.16 cm

and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 721.21 cm-1.

The FTIR spectrum of Scenedesmus dimorphus Biodiesel [ALE7 (bc) TR] 3 -1 (Figure 3.72) shows υ as (sp C-H) stretching of hydrocarbon at 2921.90 cm , υ -1 (C=O) stretching of carbonyl group from fatty acids methyl ester at 1743.19 cm , -1 methylene (CH2) groups have a characteristic bending absorption at 1453.55 cm . -1 The methyl δ s (CH3) group have characteristic bending absorption at 1351.86 cm , -1 the υ (C-O) stretching of alkyl carbon and oxygen from fatty acids is at 1177.21 cm

and δ r (CH2) bending motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 722.39 cm-1.

The FTIR analysis shows that in the Scenedesmus dimorphus algal oil and biodiesel, the –OH group of acid is disappeared and not observed in spectrum of algal biodiesel, the appearance of peak in algal oil and its biodiesel were different.

178

3.2.7.2 Gas Chromatography Data of Acid Catalyzed Biodiesel

Figure 3.73: The qualitative peaks of Gas Chromatography spectrum of Scenedesmus dimorphus microalgae Biodiesel [ALE7 (ac) TR]

Table 3.26: Scenedesmus dimorphus Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE7 (ac) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 25.325 15.87 Hexadecanoic acid, methyl ester C17H34O2 270 2 27.473 34.62 9,12-Octadecadienoic acid (Z,Z)-,

methyl ester C19H34O2 294

3 27.546 41.43 9-Octadecenoic acid methyl ester C19H36O2 296

4 29.766 8.09 11-Eicosenoic acid, methyl ester C21H40O2 324

179

3.2.7.3 Mass Spectroscopy Data of Acid Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z), methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum vi: 11-Eicosenoic acid, methyl ester

Figure 3.74: Mass Spectra of Fatty Acid Methyl Esters [ALE7 (ac) TR]

180

3.2.7.4 Molecular Structures of Fatty Acid Methyl Esters of Acid

Catalyzed Biodiesel

O

O

i. Hexadecanoic acid, methyl ester (C17H34O2)

O

O

ii. 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (C19H34O2)

O

O

iii. 9-Octadecenoic acid methyl ester (C19H36O2)

O

O

iv. 11-Eicosenoic acid, methyl ester (C21H40O2)

Figure 3.75: Molecular Structures of Fatty Acid Methyl Esters [ALE7 (ac) TR]

181

3.2.7.5 Gas Chromatography Data of Base Catalyzed Biodiesel

Figure 3.76: The qualitative peaks of Gas Chromatography spectrum of Scenedesmus dimorphus microalgae Biodiesel [ALE7 (bc) TR]

Table 3.27: Scenedesmus dimorphus Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE7 (bc) TR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 25.376 17.56 Hexadecanoic acid, methyl ester C17H34O2 270 2 27.448 21.83 9,12-Octadecadienoic acid (Z, Z)-,

methyl ester C19H34O2 294

3 27.519 32.64 9-Octadecenoic acid, methyl ester C19H36O2 296

4 27.769 15.48 Octadecanoic acid, methyl ester C19H38O2 298

5 30 1.89 Methyl 18-methylnonadecanoate C21H42O2 326

3.2.7.6 Mass Spectroscopy Data of Base Catalyzed Biodiesel

Mass Spectrum i: Hexadecanoic acid, methyl ester

182

Mass Spectrum ii: 9,12-Octadecadienoic acid (Z, Z)-,methyl ester

Mass Spectrum iii: 9-Octadecenoic acid methyl ester

Mass Spectrum iv: Octadecanoic acid, methyl ester

Mass Spectrum v: Methyl 18-methylnonadecanoate

Figure 3.77: Mass Spectra of Fatty Acid Methyl Esters [ALE7 (bc) TR]

183

3.2.7.7 Molecular Structures of Fatty Acid Methyl Esters of Base

Catalyzed Biodiesel

O

O

i. Hexadecanoic acid, methyl ester (C17H34O2)

O

O

ii. 9,12-Octadecadienoic acid (Z, Z)-, methyl ester (C19H34O2

O

O

iii. 9-Octadecenoic acid, methyl ester (C19H36O2)

O

O

iv. Octadecanoic acid, methyl ester (C19H38O2)

O

O

v. Methyl 18-methylnonadecanoate (C21H42O2)

Figure 3.78: Molecular Structures of Fatty Acid Methyl Esters [ALE7 (bc) TR]

184

The acid catalyzed Chlorococcalean microalgae Scenedesmus dimorphus Biodiesel [ALE7 (ac) TR] (Figure 3.73) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In acid catalyzed Biodiesel of Scenedesmus dimorphus four types of esters were obtained at retention times (min) at 25.325, 27.473, 27.546 and 29.766. Mass spectrum of fatty acid methyl esters is shown in Figure 3.74. The one component of biodiesel at retention time 25.325 shows the base peak at m/z 74.05 (Figure 3.74: i). This characteristic peak occurs due to McLafferty rearrangement [66]. The one component at 27.473 shows base peak at m/z 81.5 (Figure 3.74: ii) and the two components at 27.546 and 29.766 show base peak at m/z 55 (Figure 3.74: iii, iv). Table 3.26 shows the peaks obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.75 shows the molecular structures of FAMEs of obtained biodiesel product.

The acid catalyzed Chlorococcalean microalgae Scenedesmus dimorphus Biodiesel contains higher content of 9-Octadecenoic acid methyl ester (41.43 %); 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (34.62 %), Hexadecanoic acid, methyl ester (15.87%), 11-Eicosenoic acid, methyl ester (8.09 %). The Scenedesmus dimorphus Biodiesel contain 15.87 % saturated FAMEs and unsaturated 84.14 %

FAMEs. The obtained FAMEs were ranging between C17 to C21.

The base catalyzed Chlorococcalean microalgae of Scenedesmus dimorphus Biodiesel [ALE7 (bc) TR] (Figure 3.76) shows Gas chromatographic spectrum with the various peaks obtained at different retention time. In base catalyzed Biodiesel of Scenedesmus dimorphus five types of esters were obtained at retention times (min) at 25.376, 21.83, 32.64, 15.48 and 1.89. Mass spectrum of fatty acid methyl esters shown in Figure 3.76 The three components of biodiesel at retention times 25.376, 15.48 and 1.89 show the base peak at m/z 74.05 (Figure 3.77: i, iv, v). This characteristic peak occurs due to McLafferty rearrangement [66]; the one component at 21.83 shows base peak at m/z 81.5 (Figure 3.77: ii) and at 32.64 shows the base peak at m/z 55 (Figure 3.77: iii) and their molecular weights. Figure 3.78 shows the molecular structures of FAMEs of obtained biodiesel product.

The base catalyzed Chlorococcalean microalgae Scenedesmus dimorphus Biodiesel contains higher content of 9-Octadecenoic acid methyl ester (32.64 %),

185

9,12-Octadecadienoic acid (Z, Z) methyl ester (21.83 %), Hexadecanoic acid, methyl ester (17.56 %), Octadecanoic acid, methyl ester (15.48 %) and Methyl 18- methylnonadecanoate (1.89 %). The biodiesel is a mixture of saturated and unsaturated fatty acid methyl esters. The Scenedesmus dimorphus Biodiesel contains 34.93 % saturated FAMEs and unsaturated 54.47 % FAMEs. The obtained FAMEs [60] were ranging between C17 to C21. Reda A.I. Abou-Shanab et al. (2011), reported similar results.

186

3.3 Chlorococcalean Algae Biodiesel Characterization

(In-situ Transesterification Reaction)

3.3.1 Characteristics of Macroalgae Biodiesel

3.3.1.1 FTIR Data of Macroalgae Oil & Biodiesel

Figure 3.79: FTIR spectrum of Chlorococcalean Macroalgae powder

Figure 3.80: FTIR spectrum of Chlorococcalean Macroalgae Biodiesel

[ALE1 InsTR]

The FTIR spectrum of Macroalgae powder [AL1 InsTR] (Figure 3.79) shows -1 3 the υ (O-H) stretching of hydroxyl group of fatty acids at 3402.20 cm , υ as (sp C- H) stretching of hydrocarbon at 2972.10 cm-1, υ (C=O) stretching of carbonyl group -1 from fatty acids at 1741.75 cm , methylene (CH2) groups have a characteristic -1 bending absorption at 1458.00 cm . The υ (C-O) stretching of alkyl carbon and -1 oxygen from fatty acids is at 1206.43 cm and δ r (CH2) bending (rocking) motion

associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 711.68 cm-1.

187

The FTIR spectrum of Macroalgae Biodiesel [ALE1 InsTR] (Figure 3.80) 3 -1 shows υ as (sp C-H) stretching of hydrocarbon at 2925.81 cm , υ (C=O) stretching -1 of carbonyl group from fatty acids methyl ester at 1743.60 cm , methylene (CH2) -1 groups have a characteristic bending absorption at 1461.94 cm . The methyl δ s (CH3) -1 group have characteristic bending absorption at 1373.22 cm , the υ (C-O) stretching -1 of alkyl carbon and oxygen from fatty acids are at 1170.71 cm and δ r (CH2)

bending (rocking) motion associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 725.19 cm-1.

The given FTIR data shows the characteristic values of functional groups for oil and its biodiesel of algae. The nature of spectrum or peak intensity in oil and its biodiesel are different. The characteristic carbonyl frequency for oil and biodiesel are differed.

188

3.3.1.2 Gas Chromatography Data of Biodiesel

Figure 3.81: The qualitative peaks of Gas Chromatography spectrum of Chlorococcalean Macroalgae Biodiesel [ALE1 InsTR]

Table 3.28: Chlorococcalean Macroalgae Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE1 InsTR]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 12.49 1.01 Dodecanoic acid, methyl ester C13H26O2 214

2 14.95 4.94 Tetradecanoic acid, Methyl ester C15H30O2 242 9-Hexadecenoic acid, methyl ester

3 17.07 4.23 (Z) C17H32O2 268

4 17.19 29.15 Hexadecanoic acid, methyl ester C17H34O2 270 9,12-Octadecadienoic acid (Z,Z)

5 18.92 8.12 methyl ester C19H34O2 294

6 18.98 18.63 9-Octadecenoic acid(Z),methyl ester C19H36O2 296

7 19.2 4.04 Octadecanoic acid, methyl ester C19H38O2 298

189

3.3.1.3 Mass Spectroscopy Data of Biodiesel

Mass Spectrum i: Decanoic acid, methyl ester

Mass Spectrum ii: Tetradecanoic acid, methyl ester

Mass Spectrum iii: Hexadecanoic acid, methyl ester

Mass Spectrum iv: 9-Hexadecenoic acid, methyl ester (Z)

190

Mass Spectrum v: 9,12-Octadecadienoic acid (Z, Z), methyl ester

Mass Spectrum vi: 9-Octadecenoic acid methyl ester

Mass Spectrum vii: Octanoic acid, methyl ester

Figure 3.82: Mass Spectra of Fatty Acid Methyl Esters [ALE1 InsTR]

3.3.1.4 Molecular Structures of Fatty Acid Methyl Esters of

Macroalgae Biodiesel

O

O

i. Dodecanoic acid, methyl ester (C13H26O2)

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O

O

ii. Tetradecanoic acid, Methyl ester (C15H30O2)

O

O

iii. 9-Hexadecenoic acid, methyl ester (Z) (C17H32O2)

O

O

iv. Hexadecanoic acid, methyl ester (C17H34O2)

O

O

v.9,12-Octadecadienoic acid (Z,Z) methyl ester (C19H34O2)

O

O

vi. 9-Octadecenoic acid(Z) methyl ester (C19H36O2)

O

O

v. Octadecanoic acid, methyl ester (C19H38O2) Figure 3.83: Molecular Structures of Fatty Acid Methyl Esters [ALE1 InsTR]

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The Chlorococcalean Macroalgae Biodiesel [ALE1 InsTR] (Figure 3.81) shows Gas chromatographic spectrum with the various peaks obtained at different retention times. In macroalgae biodiesel seven types of esters are obtained at retention times (min) at 12.49, 14.95, 17.07, 17.19, 18.92, 18.98 and 19.2. Mass spectrum of fatty acid methyl esters is shown in Figure 3.82 respectively. The four components of biodiesel at retention times 12.49, 14.95, 17.07, 17.19, 18.92 and 19.2 shows the base peak at m/z 74.05 (Figure 3.82: i, ii, iv, vii). This characteristic peak occurs due to McLafferty rearrangement [66]. The one component at 18.92 shows base peak at m/z 81.5 (Figure 3.82: v) and the two components at 17.07, 17.19 show base peak at m/z 55 (Figure 3.82: iii, vi). Table 3.28 shows the peaks obtained with various retention times, percentage area, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.83 shows the molecular structures of FAMEs of obtained biodiesel product.

The acid catalyzed Chlorococcalean Macroalgae Biodiesel contains higher content of Hexadecanoic acid, methyl ester (29.15 %), 9-Octadecenoic acid(Z) methyl ester (18.63 %), 9,12-Octadecadienoic acid (Z,Z) methyl ester (8.12 %), 9- Hexadecenoic acid, methyl ester (4.23 %), Tetradecanoic acid, Methyl ester (4.94 %), Octadecanoic acid, methyl ester (4.04 %) and Dodecanoic acid, methyl ester (1.01 %). The Macroalgae Biodiesel contains 39.01 % saturated FAMEs and unsaturated 30.98

% FAMEs. The obtained FAMEs were ranging between C13 to C19.

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3.3.2 Characteristics of Microalgae: Chlorella vulgaris Biodiesel

3.3.2.1 FTIR Data of Microalgae Oil & Biodiesel

Figure 3.84: FTIR spectrum of Chlorella vulgaris powder

Figure 3.85: FTIR spectrum of Chlorella vulgaris Biodiesel

[ALE2 InsTR]

The spectrum Chlorella vulgaris algae powder [AL2 InsTR] (Figure 3.84) -1 shows the υ (O-H) stretching of hydroxyl group of fatty acids at 3296.48 cm , υ as (sp3 C-H) stretching of hydrocarbon at 2921.06 cm-1, υ (C=O) stretching of carbonyl -1 group from fatty acids at 1745.46 cm , methylene (CH2) groups have a characteristic -1 bending absorption at 1458.00 cm . The υ (C-O) stretching of alkyl carbon and -1 oxygen from fatty acids is at 1139.50 cm and δ r (CH2) bending (rocking) motion

associated with four or more methylene (CH2) groups in an open chain of fatty acid occurs at about 722.02 cm-1.

The FTIR spectrum of Chlorella vulgaris Biodiesel [ALE2 InsTR] (Figure 3 -1 3.85) shows the υ as (sp C-H) stretching at 3026.10 cm , υ (C=O) stretching of ester

194

-1 -1 at 1743.06 cm , δ s (CH2) bending of methylene group at 1460.01cm , υ (C-O) -1 stretching of alkyl carbon and oxygen from fatty acid methyl ester at 1029.92 cm , δ r -1 (CH2) bending of methylene group was observed at 729.04 cm .

The FTIR analysis is carried out for both Chlorella vulgaris algae powder and its biodiesel, the hydroxyl group (–OH) of fatty acid is disappear which not observed in FTIR spectrum of obtained biodiesel product. The appearance of peaks in algal oil and its biodiesel are different.

3.3.2.2 Gas Chromatography Data of Microalgae Chlorella vulgaris Biodiesel

Figure 3.86: The qualitative peaks of Gas Chromatography spectrum of Chlorella vulgaris Biodiesel [ALE2 (In-situ transesterification)]

Table 3.29: Chlorella vulgaris Biodiesel Components with Retention Times, % Areas, Name of the Compounds, Molecular Formulae and their Molecular Weights [ALE2 (In-situ transesterification)]

Molecular Peak R.Time Area% Name of the Compound Formula MW

1 8.914 0.26 Octanoic acid, methyl ester C9H18O2 158

2 10.58 0.45 Decanoic acid, methyl ester C11H22O2 186 5,8-Octadecadienoic acid, methyl

3 11.94 1.59 ester C19H34O2 294 Pentadecanoic acid, 14-methyl-,

4 12.11 23.75 methyl ester C17H34O2 270

5 13.33 54.95 11-Octadecenoic acid, methyl ester C19H36O2 296

6 13.47 4.59 Octadecanoic acid, methyl ester C19H38O2 298

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3.3.2.3 Mass Spectroscopy Data of Biodiesel

Mass Spectrum i: Octanoic acid, methyl ester

Mass Spectrum ii: Decanoic acid, methyl ester

Mass Spectrum iii: 5,8-Octadecadienoic acid, methyl ester

Mass Spectrum iv: Pentadecanoic acid, 14-methyl-, methyl ester

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Mass Spectrum v: 11-Octadecenoic acid, methyl ester

Mass Spectrum vi: Octadecanoic acid, methyl ester

Figure 3.87: Mass Spectra of Fatty Acid Methyl Esters [ALE2 InsTR]

3.3.2.4 Molecular Structures of Fatty Acid Methyl Esters of Microalgae

Biodiesel

O

O

i. Octanoic acid, methyl ester (C9H18O2)

O

O

ii. Decanoic acid, methyl ester (C11H22O2)

O

O

197

iii. 5,8-Octadecadienoic acid, methyl ester (C19H34O2)

O

O

iv. Pentadecanoic acid, 14-methyl-, methyl ester (C17H34O2)

O

O

v.11-Octadecenoic acid, methyl ester (C19H36O2)

O

O

vi. Octadecanoic acid, methyl ester (C19H38O2)

Figure 3.88: Molecular Structures of Fatty Acid Methyl Esters of Biodiesel

The Chlorococcalean Chlorella vulgaris Biodiesel [ALE2 InsTR]

(Figure 3.86) shows Gas chromatographic spectrum with the various peak obtained at different retention times. In Chlorella vulgaris six types of esters are obtained at retention times (min) at 8.914, 10.58, 11.94, 12.11, 13.33 and 13.47. Mass spectrum of fatty acid methyl esters are shown in Figure 3.82. The four components of biodiesel at retention times 8.914, 10.58, 12.11, and 13.47 shows the base peak at m/z 74.05 (Figure 3.87: i, ii, iv, vi). This characteristic peak occurs due to McLafferty rearrangement [66]; the one component at 11.94 shows base peak at m/z 81.5 (Figure 3.87: iii) and the one component at 13.33 shows base peak at m/z 55 (Figure 3.87: v). Table 3.29 shows the peak obtained with various retention times, percentage areas, name of the obtained compounds, molecular formulae and their molecular weights. Figure 3.88 shows the molecular structures of FAMEs of obtained biodiesel product.

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The acid catalyzed Chlorococcalean Chlorella vulgaris Biodiesel contains higher content of 11-Octadecenoic acid, methyl ester (54.95 %), Pentadecanoic acid, 14-methyl-, methyl ester (23.75 %), Octadecanoic acid, methyl ester (4.59 %); 5,8- Octadecadienoic acid, methyl ester (1.59 %), Decanoic acid, methyl ester (0.45%) and Octanoic acid, methyl ester (0.26%). The Chlorella vulgaris Biodiesel contains 29.05 % saturated FAMEs and unsaturated 56.54 % FAMEs. The obtained FAMEs were ranging between C9 to C19.

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3.4 Calculation of Cetane Number of Biodiesel The calculation of Cetane value of biodiesel by using method explain in Chapter second 2.3.2. The following results were obtained.

Table 3.30: Cetane Number of Biodiesel

Algae Biodiesel Cetane Value of Biodiesel Macroalgae. 117.16 Chlorella v. 74.28

Chlorococcum h. 67.60

Ankistrodesmus c. 62.72

Botryococcus b. 69.11

Clamydomonas p. 69.84

Scenedesmus d. 58.84

Macroalgae (InsTR) 77.168

Chlorella v. (InsTR) 67.726

Jatopha 54.00

The obtained results are compared with ASTM standard. The ASTM method D613 shows minimum limit of Cetane number is 47. Our results clearly indicate that the Cetane value of prepared biodiesel fuel match with ASTM standard [69].

The FTIR and GCMS data of acid, base catalyzed transesterified biodiesel and in-situ transesterified product of both macroalgae and microalgae species biodiesel is compared with standard and references. Here Jatropha biodiesel was taken as standard. Sanjaykumar N.D. (2013) reported [67] that the Jatropha biodiesel is one of the permissive alternative energy sources. The jatropha plant possesses variety of genus over 170 in number in the tropical and subtropical regions of world. It has yield per hector of more than four times that of soyabean. The seeds of jatropha contain 20% saturated fatty acid and 80% unsaturated acid, and they yield 25-40% oil by weight. Due to perennial life cycle, jatropha is produce widely. Seed yield under cultivation can range 1500-2000 kg -1 hectare, yields 540-680 liters -1 oil [75]. The oil

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conversion into biodiesel gives mixture of saturated and unsaturated fatty acid methyl esters. The FTIR spectroscopy shows the functional groups of product (FAMEs). The fatty acid methyl esters gives the carbonyl vibrational bond frequency at 1741.60 cm ˉ1,carbon oxygen alkoxy bond of ester is at 1461.94 cm ˉ 1 while finger print region shows the long chain methylene carbon hydrogen bond frequency at 729.04 cm ˉ 1 & 694.33 cm ˉ 1.

GCMS analysis shows that the Jatropha biodiesel contains 92.68 % unasurated

FAMEs and 7.32 % saturated FAMEs. It consists of FAMEs in range C13-C19.

The obtained results of algae biodiesel were compared with the Jatropha biodiesel. The FTIR analysis results of macroalgae biodiesel were similar to Standard reference. The characteristic carbonyl frequency of ester carbonyl group frequency occurs at 1741.25 cm-1 and 1740.77 cm-1 for macroalge biodiesel similar to Jatropha biodiesel [67] at 1741.60 cm-1. The macroalgae biodiesel consist of major FAMEs C15:0, C17:0, C24:0, C19:1, C19:0. Macroalgae biodiesel contain 20-43.5% saturated FAMEs and 48-30.98% Unsaturated FAMEs. The similar results were obtained [65] by Prafulla D. Patil et al. (2010). Pokharkar R. D. (2012) had prepared [68] and characterised biodiesel of undi seed. The conclusion of study shows that undi biodiesel contain [69] FAMEs ranging between C9 to C24. Brain J. Krohn et al. (2010), reported that the

macroalgae biodiesel possesses the FAME in the range C12 to C18; C12:0, C14:0, C16:0, C18:1, 2, 3, C18:0. Thus, the acid and base catalyzed biodiesel of macroalge shows the variation in composition of FAMEs. The FTIR spectrum of Microalgae biodiesel of Chlorella vulgaris shows strong absorption peak of carbonyl group at 1743.61 cm-1. The GCMS of acid catalyzed biodiesel shows mixture of FAMEs ranging from C9 to C19. Chlorella vulgaris biodiesel contain 18.11-34.51 % saturated FAMEs and 35.03-65.49 % unsaturated FAMEs. It consist of major FAMEs were C9:0, C11:0, C19:1, C17:1. The results match with the standard. Similar results were obtained by Reda A.I. Abou- [60] Shanab (2010) , they obtained major range of FAMEs between C16-C18.

The FTIR spectrum of Microalgae biodiesel of Chlorococcum humicola shows strong absorption carbonyl frequency peak at 1742.47 cm-1. The GCMS of acid

catalyzed biodiesel shows mixture of FAMEs ranging from C9 to C19. It consists of major FAMEs C9:0, C11:0, C13:0, C15:0, C17:0, C19:1. Chlorococcum humicola

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biodiesel contain 85.68-87.01 % saturated FAMEs and 7.74-11.32 % Unsaturated FAMEs. These results are similar to the reference standard. Sudarat Chaichalerm et al. (2012), reported [58] that the Chlorococcum humicola biodiesel consist of saturated and unsaturated fatty acid 32-45%, which comprises FAMEs range C12-C18. Its FAMEs consist of C12:0, C14:0, C16:0, C17:0, C18:0, C18:1, 2, 3. Our results are similar to reference data.

The FTIR spectrum of Microalgae biodiesel of Ankistrodesmus convolutes shows strong absorption peak of carbonyl group at 1742.88 cm-1. The GCMS of acid

catalyzed biodiesel shows mixture of FAMEs ranging from C17 to C23. It consist major FAMEs C17:0, C19:2, C19:1, C21:0, C23:0. Ankistrodesmus convolutes biodiesel contain 42.47-46.5 % saturated FAMEs and 49.14-57.53 % unsaturated FAMEs. These results match with the standard results.

The FTIR spectrum of Microalgae biodiesel of Botryococcus braunii shows strong absorption carbonyl frequency peak at 1742.93 cm-1. The GCMS of acid

catalyzed biodiesel shows mixture of FAMEs ranging between C17 to C25. It consist of C17:0, C19:0, C19:1,2, C21:0, C21:1,2, C23:0, C25:0. Botryococcus braunii biodiesel contain 34.64-51.92 % saturated FAMEs and 48.09-55.54 % Unsaturated FAMEs. The similar results were obtained by Dayananda C. et al. (2007) [59]. He has reported that FAMEs range between C21-C33. Thus our results match with references.

The FTIR spectrum of Microalgae biodiesel of Clamydomaonas pertusa shows strong absorption carbonyl frequency peak at 1744.06 cm-1. The GCMS of acid

catalyzed biodiesel shows mixture of FAMEs ranging between C17 to C23. The FAMEs were C17:0, C19:2; C21:0, C23:0. Clamydomaonas pertusa Biodiesel contain 7.01-9.44 % saturated FAMEs and 74.98-92.99 % Unsaturated FAMEs. Thus our results are similar to standard jatropha biodiesel results. The FTIR spectrum of Microalgae biodiesel of Scenedesmus dimorphus shows strong absorption carbonyl frequency peak at 1742.82 cm-1. The GCMS of acid

catalyzed biodiesel shows mixture of FAMEs ranging from C17 to C21. Scenedesmus dimorphus Biodiesel contain 15.87-34.93 % saturated FAMEs and 54.47-84.14 % unsaturated FAMEs. The obtained results are similar to standard reference used. Reda A.I. Abou-Shanab et al. (2011) [60] reported that Scenedesmus sp. consist of major FAMEs C16:0 and C18:1.

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The acid value, saponification value, iodine value and cetane number are calculated. The obtained results are compared with ASTM standard. The ASTM method D613 shows [69] minimum limit of Cetane number 47. Pokharkar et al., (2009) reported that the biodiesel of Sesbania sesban meets the Cetane value at 69.73 which is closer to ASTM standards [76]. Our results clearly indicate that the Cetane values of prepared biodiesel fuels match with ASTM standard. The Macroalgae biodiesel show CN value 117.16, for Chlorella sp. CN to be 74.28, for Chlorococcum sp. CN to be 67.60, Ankistrodesmus sp. CN to be 62.72, Botryococcus sp. CN to be 69.11, Clamydomonas sp.CN to be 69.11, Scenedesmus sp. CN to be 58.84, Macroalgae (InsTR) CN to be 77.168, Chlorella sp. (InsTR) is 67.726 and for Jatropha is 54.00, for different fatty acid methyl ester Hence, our results clearly indicate that the obtained data was match with standard value of biodiesel. Result shows that liquid fuel is various FAME compositions of saturated and unsaturated fatty acid methyl ester. The macroalgae and microalgae Chlorella vulgaris, Chlorococcum humicola, Ankistrodesmus convolutes, Botryococcus braunii, Clamydomaonas pertusa and Scenedesmus dimorphus sp. give saturated FAMEs and higher unsaturated FAMEs. The Jatropha biodiesel consist of 20% saturated FAMEs and 80% unsaturated FAMEs. The quality of fuel is directly depending upon the fatty acid composition of biofuels. Ignition quality is improved with increase in Cetane number and Cetane number of fatty acid increases with increase in chain length. The methyl esters of oleic acid, palmitate acid, methyl stearate has good ignition property [74]. Thus, results of this study indicate that the macroalgae and microalgae species are valuable for production of biodiesel.

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