13 lsa - muthulakshmi.pdf

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Life Science Archives (LSA)

ISSN: 2454-1354

Volume – 1; Issue - 2; Year – 2015; Page: 84 - 89

© 2015 Published by JPS Scientific Publications Ltd. All rights reserved

Research Article

BIODIESEL ENERGY PRODUCTION FROM MICROALGAE - Spirogyra sp.,

Oscillatoria foreani AND Chlorella pyrenoidosa

R. Muthulakshmi* and K. Meenatchisundaram

PG Department of Microbiology, A.V.C College (Autonomous), Mayiladuthurai, Tamil Nadu, India.

Abstract

Algae are an economical choice for biodiesel production, because of its availability and low cost.

Microalgae were a good source for biodiesel production due to its lipid content in addition to its easy growth.

Hence, in the present study, fresh water microalgae viz., Spirogyra sp., Oscillatoria foreani and Chlorella

pyrenoidosa were chosen. The total lipid content was estimated in Spirogyra sp., Oscillatoria foreani and

Chlorella pyrenoidosa. Highest lipid content was observed in Oscillatoria foreani than other two species.

The dry weight percent were found to be higher in Oscillatoria foreani. After transesterification process the

extraction of oil and biomass observed were to be more in Oscillatoria foreani compared with other two

species. The pH varies between the two algal species and it is higher in Oscillatoria foreani. The present

investigation thus revealed higher oil extraction in Oscillatoria foreani. Microalgae Oscillatoria foreani were

found to be more beneficial than Spirogyra sp. and Chlorella pyrenoidosa commercially and economically

for production of biodiesel. The study also concludes that microalgae are a best source of biodiesel.

Article History Received : 18.05.2015

Revised : 29.03.2015

Accepted : 02.04.2015

Key words: Spirogyra sp., Oscillatoria foreani, Chlorella pyrenoidosa and Microalgae

1. Introduction

Biodiesel is a nontoxic and biodegradable

fuel that is obtained from renewable sources.

Biodiesel can be prepared from waste cooking oil

such as palm, soyabeen, canola, rice, bran,

sunflower, coconut, cornoil, fish oil, chicken fat

and algae, which would partly decrease the

dependency on petroleum based fuels. Microalgae

are sunlight-driven cell factories that convert

carbon dioxide to potential biofuels, foods, feeds

and high-value bioactives (Metting and Pyne,

1986). In addition, these photosynthetic

microorganisms are useful in bioremediation

applications and as nitrogen fixing biofertilizers.

This article focuses on microalgae as a potential

* Corresponding author: R. Muthulakshmi

Tel.: +91-7373784146

E-mail: [email protected]

source of biodiesel. Microalgae can provide

several different types of renewable biofuels.

These include methane produced by anaerobic

digestion of the algal biomass (Spolaore et al.,

2006); biodiesel derived from microalgal oil

(Roessler et al., 1994).

Biodiesel is produced currently from plant

and animal oils, but not from microalgae. This is

likely to change as several companies are

attempting to commercialize microalgal biodiesel.

Biodiesel is a proven fuel. Technology for

producing and using biodiesel has been known for

more than 50 years. Bioenergy is one of the most

important components to mitigate greenhouse gas

emissions and substitute of fossil fuels. The need

of energy is increasing continuously, because of

increases in industrialization and population. The

R. Muthulakshmi/Life Science Archives (LSA), Volume – 1, Issue – 2, Page – 84 to 89, 2015 85


basic sources of this energy are petroleum, natural

gas, and coal, hydro and nuclear. The major

disadvantage of using petroleum based fuels is

atmospheric pollution created by the use of

petroleum diesel. Petroleum diesel combustion is a

major source of greenhouse gas (GHG). Apart

from these emissions, petroleum diesel is also

major source of other air contaminants including

NOx, SOx, CO, particulate matter and volatile

organic compounds. The world is entering a

period of declining non-renewable energy

resources, popularly known as “Peak oil” with

energy demand is increasing the world’s oil

production is expected to decline in between one

and ten decades (Crokes, 2006). As a result of this

impending energy crisis, both governments and

private industry are examining alternative source

of energy. Other non-renewable sources of energy

exist, such as coal and uranium: however these

sources are limited and will also inevitably decline

in availability. Microalgae have been suggested as

very good candidates for fuel production because

of their advantages of higher photosynthetic

efficiency, higher biomass production and faster

growth compared to other energy crops. In this

study, Biodiesel is produced from microalgae such

as Spirogyra sp., Oscillatoria foreani and

Chlorella pyrenoidosa.

2. Materials and Methods

2.1. Sample collection

The microalgae sample viz., Spirogyra sp.,

Oscillatoria foreani and Chlorella yrenoidosa and

were collected from fresh water source at

Kalyanasozhapuram Village, Nagapattinam Dt.

2.2. Microscopic observation

The Collected algal species were

confirmed based on color cultural and microscopic

observation (Desikachary, 1959).

2.3. Mass cultivation of algal sample

The collected microalgal sample Spirogyra

sp., Oscillatoria foreani and Chlorella

pyrenoidosa were cultivated using BG-11 medium

in the laboratory (Aneja, 2002). BG-11 medium

was prepared and 50 ml were taken at each conical

flask and autoclaved at 121 °C for 30 minutes. The

medium were allowed to cool. The collected

samples were inoculated at aseptic condition. The

conical flasks were kept in an alternate light and

dark regime of 2 weeks at 25 ºC. After incubation

the algal colonies were appeared. After 20 days

the algal mat were collected and used for further

studies.

2.3. Assay of total lipids

Total lipid content was estimated in the

Spirogyra sp. and Oscillatoria foreani by the

method of Cox and Pearson (1962). One gm of

algal sample was dissolved in 250 ml of conical

flask. Few drops of phenolphthalein were added

and titrated against 0.1 N Potassium hydroxide.

Shake constantly until a pink colour which persists

for fifteen seconds is obtained.

Calculation:

Acid value (mg KOH/g) = 𝑻𝒊𝒕𝒓𝒂𝒕𝒆 𝒗𝒂𝒍𝒖𝒆

×𝑵𝒐𝒓𝒎𝒂𝒍𝒊𝒕𝒚𝒐𝒇𝑲𝑶𝑯

×𝟓𝟔.𝟏/𝑾𝒆𝒊𝒈𝒉𝒕𝒐𝒇𝒕𝒉𝒆𝒔𝒂𝒎𝒑𝒍𝒆 (𝒈)

2.4. Oil extraction

Algae were ground with motor and pestle

as much as possible. The ground algae were dried

for 20 min at 80°C in incubator for releasing

water. Hexane and ether solution (20 and 20 mL)

were mixed with the dried ground algae to extract

oil. Then the mixture was kept for 24 hrs for

settling (Sharif Hossain et al., 2008). The dried

ground algal sample were calculated using the

formula

𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 % = 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡/𝐹𝑟𝑒𝑠ℎ 𝑤𝑒𝑖𝑔ℎ𝑡 × 100

2.5. Biomass collection

The biomass of microalgae such as

Spirogyra sp., Oscillatoria foreani and Chlorella

pyrenoidosa were collected by filtration. Biomass

percentage was calculated using the formula.

𝐵𝑖𝑜𝑚𝑎𝑠𝑠 % = 𝐶𝑒𝑙𝑙𝑏𝑖𝑜𝑚𝑎𝑠𝑠/𝐷𝑟𝑦𝑤𝑒𝑖𝑔ℎ𝑡 × 100

2.6. Evaporation

The extracted oil was evaporated in

vacuum to release hexane and ether solutions

using rotary evaporator.



2.7. Mixing of catalyst and methanol

0.25 g NaOH was mixed with 24 ml

methanol and stirred properly for 20 min.

2.8. Biodiesel production

The mixture of catalyst and methanol was

poured into the algal oil in a conical flask. The

following reaction and steps were followed

National biodiesel Board.

2.9. Transesterification

The reaction process is called

transesterification. The conical flask containing

solution was shaken for 3 h by electric shaker at

300rpm.

2.10. Settling

After shaking the solution was kept for 16

h to settle the biodiesel and sediment layers

clearly.

2.11. Separation of biodiesel

The biodiesel was separated from

sedimentation by flask separator carefully.

Quantity sediment (glycerine, pigments, etc.) was

measured. The percentage of biodiesel were

calculated by the formula.

𝐵𝑖𝑜𝑑𝑖𝑒𝑠𝑒𝑙 % =𝑜𝑖𝑙𝑤𝑒𝑖𝑔ℎ𝑡/𝐹𝑟𝑒𝑠ℎ𝑤𝑒𝑖𝑔ℎ𝑡× 100

2.12. Washing

Biodiesel was washed by 5% water until it

was become clean.

2.13. Storage

Production of Biodiesel was measured by

using measuring cylinder; pH was measured and

stored for analysis.

3. Results

In this study the microalgae such as


pyrenoidosa were used for the production of

biodiesel. The investigated results were presented

in this chapter.

3.1. Microscopic observation

The collected three micro algal species

were screened by microscopically.

3.2. Mass culturing of Algal sample

The sample such as Spirogyra sp.,

Oscillatoria foreani and Chlorella pyrenoidosa

were cultivated in sterilized BG-11 medium

separately. After two weeks greenish algal

biomass were observed in the flask, cultivated

algal species were grown on the BG-11 agar

medium for further studies.

3.3. Estimation of total lipid

The total lipid content was estimated in


pyrenoidosa. In this study, maximum lipid level

was noted in Oscillatoria foreani compared than

other two species. The results were presented in

Table- 1.

3.4. Measurement of fresh weight and Dry

weight of the sample

The fresh weight of the microalgae viz.,


pyrenoidosa were noted. The dry weight was

noted after drying the sample in the incubator for

20 min at 80°C for releasing water content in the

sample. The percentage of dry weight was greater

in Oscillatoria foreani and Chlorella pyrenoidosa

than Spirogyra sp. species (Table - 2).

3.5. Measurement of oil extraction

The oil extraction of microalgae viz.,


pyrenoidosa were noted after the

transesterification process. The obtained

percentage of oil content was seemed to be higher

in Oscillatoria foreani. This represents that the

biodiesel (Methyl ester) production was found to

be maximum in Oscillatoria foreani (Table - 3).

3.6. Assay of total biomass

The biomass obtained after oil extraction

were seem to be higher in microalgae Oscillatoria

foreani when compared with other two species

(Table-4).

3.7. Determination of pH

There is a significant difference between

pH of the two microalgae viz., Spirogyra sp.,

Oscillatoria foreani and Chlorella pyrenoidosa

were noted (Table - 5). Overall, the sediment



(Glycerine, pigment and other element) were seen

to be higher in Oscillatoria foreani and Chlorella

pyrenoidosa than the Spirogyra sp.

4. Discussion

In the present study, microalgae viz.,


pyrenoidosa were analyzed for their biodiesel

production and compared with previous

theoretical and biostatistical reports. The results

revealed microalgae Oscillatoria foreani

comparatively show effective production of

biodiesel than other two species. Van Gerpen

(2005) reported that the biodiesel is a primary

advantages, that it is one of the most renewable

fuels and also non-toxic and biodegradable shay

(1993) reported that today, biodiesel has come to

mean a very specific chemical modification of

natural oils. The use of vegetable oils as

alternative fuels has been around for 100 years

when the inventor of the diesel engine Rudolph

Diesel first tested Peanut oil in his compression

ignition engine. One of the biggest advantages of

biodiesel compared to many other alternative

transportation fuels is that it can be used in the

existing diesel engines without modification, and

can be blended in at any ratio with petroleum

diesel.

Barnwal and Sharma (2005) found that

biodiesel performs as well as petroleum diesel,

while reducing emissions of particulate matter,

carbon monoxide (Co), Hydrocarbons and oxides

of sulphur (Sox). Other environmental benefits of

biodiesel include the fact that it is highly

biodegrable and appear to reduce emissions of air

toxins and carcinogens (relative to petroleum

diesel). The production of biofuels from algae

does not reduce atmospheric carbon-di-oxide,

because any Co2 taken out of the atmosphere by

the algal is returned when the biofuels are burned.

They do however eliminate the introduction of

new Co2 by displacing fossil hydrocarbon fuels.

(Murdoch University, Western Australia). Many

researchers prove the Algae are a good source of

biodiesel production than the commercial food

crops.

Hall et al. (1991) reported that biomass has

been focused on as an alternative energy source.

Since it is a renewable resource and it fixes Co2 in

the atmosphere through photosynthesis. If biomass

is grown in a sustained way, its combustion has no

impact on the Co2 fixed by photosynthesis. Shay

(1993) reported that among biomass, algae (Macro

and microalgae) usually have higher

photosynthetic efficiency than other biomass.

Shay (1993) suggested that algae were one of the

best sources of biodiesel. In fact algae are the

highest yielding feed stock for biodiesel. It can

produce up to 250 times the amount of oil per acre

as soyabeans.

Ladygina and Dehyukhina (2006) stated

that the national renewable energy laboratory

(NREL) has categorized approximately 300 sp of

microalgae that could be potential fuel sources. In

this study we selected microalgae viz., Spirogyra

sp., Oscillatoria foreani and Chlorella

pyrenoidosa for the production of biodiesel.

Spolaore et al. (2006) evaluated that microalgae

can provide several different types of renewable

biofuels. Many researchers reported the biodiesel

was derived from microalgal oil. Sheehan et al.

(1998) reported that the average lipid content of

the green algae, blue green algae ranges the

production of biodiesel. Canakci and van Gerpen

(2001) reported that Chlorella vulgaris known for

shorter chain fatty acids which are ideally suited

for biodiesel production. In my present research

work there is an increase in lipid content in

Oscillatoria foreani and than the blue green algae

Spirogyra sp. and finally this leads to the

increased production of biodiesel in the

microalgae Oscillatoria foreani.

Chisti (2007) suggested that microalgae

are capable of producing 30 times the amount of

oil per unit area of land, compared to terrestrial oil

seed crops. Metting (1996) oil content of

microalgae are usually between 20-50% (Dry

weight) while some strains can reach as high as

80%. Chisti (2007) found that oil level of 20-50%

is common in microalgae. Miao and Wu (2006)

reported that Algal biodiesel production was

typically performed by the extraction of algal oil

followed by transesterification. Hu et al. (2008)

reported that algae produced more oil in stressed

or unfavourable condition in comparison to

optimal growth condition. There are other ways to



make biodiesel, including direct use of blending,

microemulsions and pyrolysis (Ma and Hanna,

1999), but transesterification is the most common

method since the biodiesel product can be used

directly or in blends (Miao and Wu, 2006). In my

work the obtained biodiesel from algal extract

were also undergone the transesterification

process. Fukuda et al. (2001) suggested that

transesterification was catalysed by acids, alkalis

and lipase enzymes. Alkali catalysed

transesterification in about 4000 times faster than

the acid catalyzed reaction. Transesterification is a

typical process for the commercial production of

biodiesel. The use of organic solvents can increase

this extraction level to 99% but there is an

increased cost in processing to achieve this

(Metzger and Largeau, 2005). Using

transesterification allows for a single step process

that extracts and the algal oils and reacts then with

methanol result in biodiesel.

Belarbi et al. (2000) suggested that

production of methyl esters, or biodiesel, from

micro algal oil has been demonstrated although

the product was intended for pharmaceutical use.

Oil productivity, that is the mass of oil produced

per unit volume of the micro algal broth per day,

depends on the algal growth rate and the oil

content of the biomass. Microalgae with high oil

productivities are desired for producing biodiesel.

Yusuf (2007) explained about biofuel from

microalgae. He mentioned the biodiesel derived

from the oil crop is a potential renewable and

carbon neutral alternative to petroleum fuels.

Table – 1: Total lipid content of Test organisms

Table – 2: Measurement of Fresh weight and

Dry weight of the test organisms

Table – 3: Measurement of oil extraction

Table – 4: Assay of Total Biomass

S.

No.

Test

Organisms

Total Biomass

(After

Extraction)

% of

Biomass

1 Spirogyra sp. 3.5 ± 0.14 71.42

2 Oscillatoria

foreani

3.8 ± 0.16 73.07

3 Chlorella

vulgaris

3.7 ± 0.12 70.06

Table – 5: Determination of pH from Algal

Biodiesel

S.

No

Test Organisms pH Value

Biodiesel

1 Spirogyra sp. 6 ± 0.30

2 Oscillatoria foreani 7 ± 0.12

3 Chlorella vulgaris 7 ± 0.21

S.

No

Test

Organisms

Oil

extraction (g)

% of oil

extraction

1 Spirogyra sp. 2.8 ± 0.41 10.7

2 Oscillatoria

foreani

4.3 ± 0.20 17.9

3 Chlorella

vulgaris

3.6 ± 0.30 16.4

S.No Test organisms Total lipid

content (%)

1 Spirogyra sp.

14

2 Oscillatoria foreani

29

3 Chlorella vulgaris 18

S.

No.

Test

organisms

Fresh

weight

(g)

Dry

weight

(g)

Dry

weight

(%)

1 Spirogyra sp. 20 ±

0.95

12.5 ±

0.14

37.5

2 Oscillatoria

foreani

20 ±

1.13

9.5 ±

0.58

52.5

3 Chlorella

vulgaris

20 ±

1.12

10.5 ±

0.64

47.5



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