International Journal of Environment and Bioenergy, 2012, 4(3): 147-161
International Journal of Environment and Bioenergy
ISSN: 2165-8951
Florida, USA
Journal homepage: www.ModernScientificPress.com/Journals/IJEE.aspx
Article
Assessment for the Higher Production of Biodiesel from
Scenedesmus dimorphus Algal Species
Gulab Chand Shah *, Pushpendra Singh, Mahavir Yadav, Archana Tiwari
School of Biotechnology, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, Madhya Pradesh
(University of Technology of Madhya Pradesh), Airport Bypass Road, Gandhi Nagar, Bhopal-462 033
India
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: 07552678803; Fax: 0755-2742002.
Article history: Received 17 October 2012, Received in revised form 21 November 2012, Accepted 22
November 2012, Published 23 November 2012.
Abstract: Algae are the fastest-growing plants in the world, and this study demonstrates
the production of algal biodiesel from Scenedesmus dimorphus. Biodiesel is an alternative
fuel for conventional diesel, which is made from natural plant oils, animal fats, and waste
cooking oils. This paper discusses the economics of producing biodiesel fuel from algae
grown in open ponds. Microalgae have been identified as a potential biodiesel feedstock
due to their high lipid productivity and the process conditions are milder than those
required for pyrolysis and prevent the formation of by-products. Algae are very important
as a biomass source. Algae will some day be competitive as a source for biofuel. Algae can
be grown almost anywhere, even on sewage or salt water, and does not require fertile land
or food crops, and processing requires less energy than the algae provides. Algae can be a
replacement for oil based fuels, which is more effective and has no disadvantages. About
50% of their weight is oil. This lipid oil can be used to make biodiesel for cars, trucks, and
airplanes. Microalgae have much faster growth-rates than terrestrial crops.
Keywords: microalgae; biofuels; lipid; biomass; glycerol; transesterification.
1. Introduction
The search for sustainable and renewable fuels is becoming increasingly important as a direct
result of climate change and rising fossil-fuel prices. Commercial production of biodiesel or fatty acid
methyl ester (FAME) involves alkaline-catalyzed transesterification of triglycerides found in
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oleaginous food crops with methanol (Ronald et al., 2012). Biodiesel is produced from triglycerides
derived mainly from vegetable oils or animal fats. Recently, new oil production methods have been
investigated such as oil produced from algae and oleaginous yeasts indicating new sources of biodiesel
which, contrary to energy crops, do not conflict with the cultivation of land for food; therefore they can
offer alternatives to the food vs. fuels land use (Anestis et al., 2011). Petroleum-based fuels are
recognized as unsustainable energy source due to their depleting supplies and contribution to global
warming. Renewable biofuels are promising alternatives to petroleum-based fuels, among which
biodiesel has attracted the most attention in recent years (Liu et al., 2011). Competitive liquid biofuels
from various biomass materials by chemically and biochemically have been found promising methods
for near future. There are two global liquid transportation biofuels: bioethanol and biodiesel,
respectively (Fatih Demirbas, 2011).
Cost and environmental impact of conversion process
For a sustainable future of the planet, we must look into renewable energy sources which
implicitly include sustainable fuel sources. Based on the positive energy balance or life cycle analysis,
biodiesel is shown to be sustainable (Jidon and Naoko, 2010). We highlights the important aspects of
the biodiesel which will strengthen the prospect as the next generation green fuel. Four major areas are
discussed: (i) cost and environmental impact of conversion processes, (ii) efforts towards
environmentally benign and cleaner emissions, (iii) diversification of products derived from biodiesel
glycerol; and (iv) policy and government incentives (Pinzi et al., 2009).
Flow diagram of microalgal oil description
High acidic value of microalgae oil (MAO) makes them an inconvenient raw material for the
traditional biodiesel production. However, by means of a sequential acidic/basic transesterification,
coupled with a heat integration strategy, the opportunities of accepting microalgae oil as a biodiesel
precursor will increase (Sánchez et al., 2011).
Figure 1. Overview of biodiesel production.
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Conventional production methods
The direct use of vegetable oils as biodiesel is possible only by blending them with
conventional diesel fuel in a suitable ratio, but the direct usage of vegetable oils in diesel engines is not
technically possible because of their: (i) high viscosity, (ii) low stability against oxidation, and (iii) low
volatility (Robles Medina, 2009).
Sources of biodiesel and advantages of biodiesel as diesel fuel
There are more than 350 oil-bearing crops identified, among which only soybean, palm,
sunflower, safflower, cottonseed, rape seed and peanut oils are considered as potential alternative fuels
for diesel engines (Ayhan, 2007). The advantages of biodiesel as diesel fuel are liquid nature
portability, ready availability, renewability, higher combustion efficiency, lower sulfur and aromatic
content, higher cetane number and higher biodegradability (Ma and Hanna, 1999).
Availability and renewability of biodiesel
Biodiesel is the only alternative fuel so that low concentration biodiesel–diesel blends run on
conventional unmodified engines. It can be stored anywhere where petroleum diesel fuel is stored.
Biodiesel can be made from domestically produced, renewable oilseed crops such as soybean, rapeseed
and sunflower (Mittelbach and Remschmidt, 2004).
Lower emission by using biodiesel and disadvantages of biodiesel as diesel fuel
Combustion of biodiesel alone provides over a 90% reduction in total unburned hydrocarbons
(HC), and a 75 - 90% reduction in polycyclic aromatic hydrocarbons (PAHs) (Laforgia et al., 1994).
Major disadvantages of biodiesel are higher viscosity, lower energy content, higher cloud point and
pour point, higher nitrogen oxides emissions, lower engine speed and power , injector coking (Prakash
et al., 1998).
Biodiesel economy and algal strain and culture conditions
Economic advantages of biodiesel can be listed as follows: it reduces green house gas
emissions, it helps to reduce a country’s reliance on crude oil imports and supports agriculture by
providing a new labor and market opportunities for domestic crops (Palz et al., 2002). H. pluvialis
samples were collected from rainwater in Bahía Blanca Buenos Aires Province, Argentina. Unialgal
cultures were obtained by means of serial dilutions. Biflagellate cells were cultured in Bold’s Basal
Medium, containing 3.4 mM of sodium nitrate. The cells were kept at 24 οC (Stein, 1973).
Acceptability of microalgal biodiesel and improving economics of microalgal biodiesel
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Microalgal biodiesel will need to comply with existing standards in the United States, and the
relevant standard is the ASTM Biodiesel Standard D6751 (Knothe, 2006). Cost of producing
microalgal biodiesel can be reduced substantially by using a biorefinery based production strategy,
improving capabilities of microalgae through genetic engineering and advances in engineering of
photobioreactors (Sánchez et al., 2003).
Enhancing algal biology and biorefinery based production strategy
Genetic modification of microalgae has received little attention. Molecular level engineering
can be used to potentially: (1) increase photosynthetic efficiency to enable increased biomass yield on
light, (2) enhance biomass growth rate, (3) increase oil content in biomass, and (4) improve
temperature tolerance to reduce the expense of cooling (León-Bañares et al., 2004). Like a petroleum
refinery, a biorefinery uses every component of the biomass raw material to produce useable products.
Because all components of the biomass are used, the overall cost of producing any given product is
lowered (Mata-Alvarez et al., 2000).
2. Materials and Methods
2.1. Sample Collection and Media Preparation
Microalgal species were collected from the upper Lake, Bhopal, Madhya Pradesh, India. Lake
water used for media preparation came from upper lake and was stored in 20 L bottle in the dark to
prevent algal growth. The lake water was filtered twice before use through triple thickness whatman
filter paper followed by 0.45 µm whatman membrane filter. The filtered lake water was the stored at 4
o
C in container in the dark. Lake/sand mix soil was used for preparing soil extract. The soil extract was
prepared using the method by sifting dry soil once through a coarse sieve (1.00 mm) and twice through
a 500 µm sieve. One kilogram of the soil was then mixed into 2 L of deionized water. The mixture was
filtered through triple thickness Whatman No.1 filter paper, autoclaved for 20 min at 121 oC under 103
kPa and cooled overnight. It was then centrifuged at 1008 g for 5 min in the 50 mL polyethylene tubes
at room temperature. The supernatant was poured into 100 mL reagent bottles, and autoclaved for 20
min at 121 oC under 103.35 kPa. Once the bottles were cool, they were sealed with parafilm and stored
at 4 oC. (Table 1).
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Table 1. Composition of modified CHU 13 medium
S. No.
Composition
Stock Media 20X
( g/L)
Working media 1X
(mg/L)
1
KNO3
8
400
2
K2HPO4
1.6
80
3
MgSO4 7H2O
4
200
4
CaCl2
2.14
107
5
Ferric citrate
0.4
20
6
Citric acid
2
100
7
CoCl2 2H2O
2.14
107
8
H3BO3
114.4
5.72
9
MnCl2 4H2O
73.4
3.67
10
ZnSO4 7H2O
8.8
0.44
11
CuSO4 5H2O
3.2
0.16
12
NaMoO4
1.68
0.084
13
0.72N H2SO4
1 drop
2.2. Algal Culture and Measuring Algae Concentration through Spectrophotometry
Using the proper protocol insure that the algal culture is clean and not contaminated by
protozoan and bacteria. Stock culture of all species was maintained 100 mL culture media in 250 mL
conical flask and was subculture every 14 days. They were grown at constant temperature growth
room or cabinets under an irradiation provided by a combination of cool white and lights and a 14:10
day night period. Take out 1 mL of this medium and mix it with 9 mL of this fresh sterile liquid
medium, then have 100 microalgae in or 10 microalgae/mL, add 1 mL of this suspension to another 9
mL of fresh sterile liquid medium, each mL would now contain a single microalgae. This tube shows
any microbial growth, there is a very high probability that this growth has resulted from the
introduction of a single microorganism in the medium and represents the pure culture of those
microalgae. This pure culture of microalgae was used for optimization of growth condition and growth
media (Table 2). The scanning range is 200 - 760 nm, within the visible spectrum. Most culture peak at
670 - 705 nm because absorbance is directly proportional to concentration when only the algae
significantly absorb the appropriate wavelength, a protocol can be made by establishing a linear fit of
standard dilution. After the appropriate wave length is identified, concentrations of the starting sample
are measure and serial dilutions were prepared from that sample. Because the concentrations have been
measured and then calculated for the serial dilutions, the absorbance of each dilution is plotted against
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its concentration. Therefore, the linear curve fit is a plot of concentration vs. absorbance at the
appropriate wavelength.
Table 2. Shows the different media and condition for algal growth
S. No.
Sign
Media ingredients
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
CDW
CDT
CDR
CLW
CLT
CLR
SDW
SDT
SDR
SLW
SLT
SLR
MW
MT
MR
DW
DT
DR
LW
LT
LR
CHU Medium + Deionised Water
CHU Medium+ Deionised Water
CHU Medium + Deionised Water
CHU Medium + Lake water
CHU Medium + Lake water
CHU Medium + Lake water
Lake Soil + Deionised Water
Lake Soil + Deionised Water
Lake Soil + Deionised Water
Lake Soil + Lake water
Lake Soil + Lake water
Lake Soil + Lake water
CHU Medium + Lake Soil + Lake water (Mixed)
CHU Medium + Lake Soil + Lake water
CHU Medium + Lake Soil + Lake water
Deionised Water
Deionised Water
Deionised Water
Lake water
Lake water
Lake water
Place
Window
Tissue culture lab
Roof
Window
Tissue culture lab
Roof
Window
Tissue culture lab
Roof
Window
Tissue culture lab
Roof
Window
Tissue culture lab
Roof
Window
Tissue culture lab
Roof
Window
Tissue culture lab
Roof
2.3. Measurement of Dry Biomass and Scale up of Algal Culture
The total biomass as, total dry weight, was determined as follow: whatman (2.5 cm) filter was
washed in deionized water and dried in a 70 oC oven for 24 h and stored in a vacuum desiccator over
silica gel until needed. The filter was then weighed to 4 decimal places on an analytical balance. A 10
mL of algal culture was filtered until the filters were dried using a Millipore filtration apparatus. The
filter was then washed with 10 mL of 0.65 M ammonium formate solution to remove excess salt and
dried at 90 oC for 4 h and placed in vacuum desiccator over night. Filter was then weighed to four
decimal places. Dry weight was calculated as g/L after subtracting the filter weight from filter total
weight of sample. Sequencing algal culture vessels were used: (a) Innovate culture (test tube) house
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incoming culture of algal species. Replicate them for backups. Use them only in event an algae culture
become contaminated. (b) Stock cultures (125-mL flasks) are maintained aseptically and use to
inoculate starter and to restart stock cultures, and starter cultures (1000-mL or 2000-mL flasks ) are
used to inoculate carboys. (c) Carboys (5-gallons) are used to inoculate kalwalls, and Kalwalls (60-5
gallons) are used to large culture quantities of algae. (d) Open tanks are used to culture algae in very
large quantites. Selected stock culture of MT (CHU Medium +Lake soil +lake water) 250 mL flasks
and then inoculate into sterilized 1000 mL or 2000 mL flasks with CHU medium, lake soil and sterile
water media for starter algal culture production. Selected starter cultures (1 L/2 L flasks) and then
transfer into prepared 5-gallon carboy with CHU medium, lake soil and sterile lake water media for
large scale algal biomass production.
2.4. Growth Parameter and Harvest Algal Biomass
Growth parameters were: temperature 26 oC, pH 7.8, day/night period -14:10, aerationcontinuous, algal culture - 27.6 L. Algae can be harvested from 5-gallon carboy culture by the addition
of 0.2% ferric chloride in the algal culture and after the addition of the chemical flocculants algae
settled down in the bottom of the 5-gallon carboy. Settled biomass was removed out from the container,
and then dried in the oven for 2 d at 55 oC, and the dry algal biomass was used further oil extraction.
2.5. Lipid Determination and Oil Extraction
A 10 mL of algal culture was filtered using whatman 2.5 cm filters, rinsed with isotonic 0.65 M
ammonium format solution and stored at -20 oC for later lipid extraction. Before extraction the filters
were thawed at room temperature, then homogenized in a glass mortar with 5 mL of
methanol:chloroform:deionised water (2:1:0.8, v/v/v). The extracts were then transferred to 10 mL
glass stopper centrifuged tubes and centrifuged at 1008 g for 10 min at room temperature. The
supernatant was then carefully transferred to glass stopper graduted glass centrifuge tubes. The extract
was made up to 5.7 mL with fresh methanol: chloroform: deionised water and 1.5 mL of chloroform
was added, followed by 1.5 mL of deionised water and stirred. After partial phase separation had
occurred, the green chloroform bottom layer was transferred to dry, pre-weighed 4 mL glass vial over
KOH pellets overnight in vacuum desicators weighing. Total lipid content was then calculated as g/L.
The simplest method is mechanical crushing.
2.6. Determination of Fatty Acid Concentration
Prepare solvent mixture (95% ethanol/diethyl ether, 1/1, v/v), 0.1M KOH in ethanol, and 1%
phenolphthalein in 95% ethanol. Weigh 0.5 g of oil in conical flask and dissolve in at least 50 mL of
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solvent mixture. Titrate, with shaking, with the KOH solution (in a 25 mL burette in 0.1 mL) to the end
point of the indicator (5 drop of indicator), the pink color persisting for at least 10 s. The acid value is
calculated by the formula: 56.1  N  V/M, where V is the number of KOH solution used and N is
exact normality, M is the mass in g of sample.
2.7. Chemical-catalyzed Transesterification and Enzyme Catalyzed Transesterification
In a 150 mL Erlenmeyer flask with a magnetic stir bar, 0.25 g of anhydrous sodium hydroxide
was placed, and dissolved with 10 mL of methanol. The mixture was stirred until all of the sodium
hydroxide was dissolved. In a 250 mL beaker, 5 mL of algal oil was placed. The oil was heated to 60
o
C. Added 2 mL of the dissolved sodium hydroxide into the heated oil, immediate the mixture become
cloudy. Allow to stir for 30 min on high. Transfer content of beaker into a 250 mL separatory funnel.
Allow the mixture to separate for overnight. Transfer the glycerol (bottom layer) into a beaker from
biodiesel. Algal oil (0.25 g) and acyl accepter (methanol, ethanol, propanol-2, n-butanol) were taken in
the ratio of 1:4 in a screw-capped vail. To this mixture, 5 mL of enzyme preparation (crude) was added
and incubate at 40 oC with constant shaking at 200 rpm. Progress of the reaction was monitored by
removing aliquots (20 µL) at various time intervals (4, 8…24 h). The aliquots were appropriately
diluted (with hexane) and analysed by thin-layer chromatography.
2.8. Effect of Varied amount of Enzyme on Transesterification
Algal oil (0.25 g) and acyl acceptor (methanol, ethanol, propanol-2, and n-butanol) were taken
in the ratio of 1:4 in a screw-capped vial. The crude enzymes were prepared by adding of 20 mM
sodium phosphate buffer, pH 7.8 and take this crude lipase solution in varying amount viz, 1, 2, 3, 4,
and 5 mL. To the reaction mixture, these enzyme preparations were added and incubate at 40 oC with
constant shaking at 200 rpm for 24 h. The aliquots were appropriately diluted (with hexane) and
analysed by thin-layer chromatography.
2.9. Effect of Varied amount of Acyl Acceptor on Transesterification
Algal oil (0.25 g) and acyl acceptor (methanol, ethanol, propanol-2, and n-butanol) were taken
in the different ratio such as 1:1, 1:2, 1:3, 1:4, 1:5, inscew capped vail and in this mixture 5 mL of
prepared crude lipase enzyme preparation was added. The reaction mixture was incubated at 40 oC
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with constant shaking at 200 rpm for 24 h. The aliquots were appropriately diluted (with hexane) and
analysed by thin-layer chromatography.
2.10. Qualitative Analysis of Esters by Thin-layer Chromatography
The formation of methyl ester of algal oil in the reaction mixture was also analyzed by thinlayer chromatography with silca gel 60F254. The solvent system consisted of hexane/ethyl acetate
/acetic acid in the ratio of 90:10:1. The spot was detected in the UV light.
2.11. The Quantitive Analysis of Ester by Titration
Prepare solvent mixture 95% ethanol/diethyl ether (1/1, v/v), 0.1 M KOH in ethanol, and 1%
phenolphthalein in 95% ethanol. Total reaction mixture was placed in conical flask and dissolved in at
least 50 mL of the solvent mixture. Titrate, with shaking, with the KOH solution (in a 25 mL burette in
0.1 mL) to the end point of the indicator (5 drops of indicator) the pink colour persisting for at least 10
s. The acid value is calculated by the formula: 56.1  N  V/M, where V is the number of mL of KOH
solution used, N is exact normality, and M is the mass in g of the sample.
3. Results and Discussion
3.1. Optimization of Algal Culture
Although microalgae, especially Scenedesmus dimorphus is widely distributed in the upper
lake but these are not currently used for biotechnological applications. Microalgal culture is the same
as other microbial culture with the exception of the light requirement in autotrophic. For successful
microalgal culture, a suitable Scenedesmus dimorphus must be selected mainly based on general
physical, chemical and biological characteristics together with the growth optimization of selected
species on a CHU13 medium. High cell density mass production culture was unattainable due to a
number of physical, chemical and biological factors. These factors were summarized in Table 3. In this
study, microalgae were screened for high dry biomass production. Table 4 shows that all the different
conditions and media composition produced algal biomass with varying amount. The most one
productive was CHU medium + Lake soil + Lake water media used for microalgae culture incubate at
department roof followed by CHU medium + Lake soil + Lake water media at department window.
Therefore, CHU medium +Lake soil + Lake water for microalgae culture was incubated at tissue
culture lab. Temperature, light and nutrient media composition may represent the main limitation for
high biomass production rates for the outdoor and indoor cultivation of microalgae. The response of
selected species of microalgae such as Scenedesmus dimorphus to varying nutrient media composition
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is also an important consideration when considering the culture of these algae in closed cultivation
system in tissue culture laboratory. Although the effect of temperature, light, nutrient media
composition on the growth rate of laboratory microalgal culture was well, its effect in out door cultures
was not properly grown due to the high temperature in the day time period (Fig. 2). An out door (roof)
algal culture under goes a diuranal cycle which, in areas out of the tropics, may show a different of cell
component in summer seasons the maximum day temperature of over 41 oC, and temperature can be an
important limiting factor. When nutrient and temperature was not limiting the growth of microalgae,
light availability to the average cell becomes the dominant limiting factor, which has demonstrated a
decrease in the growth rate of microalgae, with an increase in self shading due to increased cell
density.
3.2. Biodiesel Production by Chemical-catalyzed and Lipase-catalyzed Transesterification
Biodiesel was prepared in the form of methyl ester of fatty acid from algae in oil using
chemical catalyzed tronsesterification reaction (Fig. 3). Fig. 4 shows the top layer in the separation
funnel is the biodiesel product and lower layer is glycerine. The lipase from Scenedesmus dimorphus
source (which ware prepared crude in laboratory) ware screened for transesterification. Fig. 5 shows
lipase from Scenedesmus dimorphus is capable of transesterification (Table 5) with methanol, ethanol
and propanol-2 showed by tubes 1, 2 and 3 respectively in which biodiesel (methanol ester, ethanol
ester and propanool-2ester) mix with algal oil in the upper layer and total reaction mixture in the lower
layer. The n-butanol has not transesterification into n-butanol ester which shows in TLC analysis (lane
4 in Fig. 7) but in tube 4 after lipase – catalyzed transestrification with algal oil observed a light green
coloured layer which might be algal oil. This suggested that when the number of carbon atom
increased in alcohol from 1 (methanol) to 4 (n-butanol), dissolving effect of substrate (algal oil) into
alcohol was increased. The lipase from Scenedesmus dimorphus source (which were prepared crude in
laboratory) ware screened for transesterification. Fig. 5 shows that lipase from Scenedesmus
dimorphus is capable of transestrification. With this enzyme, the yields of 44, 24, 22 and 0%
respectively for the methyl ester, ethyl ester, propanyl ester and butanyl ester could be obtained after
24 h (Table 6). The reaction could be qualitativety followed by TLC (Figs. 6 & 7) and quantitively by
titration (Fig. 5). The formation of biodiesel did increase beyond 24 h which is likely to be inactivation
of the lipase by substrate methanol. The inactivation of lipase during transesterification reactions has
also been observed by other.
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Table 3. Factor influencing algal growth
A biotic factors
Biotic factor
Operational factor
Light (quantity, quality)
Temperature
Nutrient requirement
O2 and CO2
Salinity
pH
Toxic chemicals
Pathogens( bacteria, fungi, viruses)
Competition by other algae
Shear produced by mixing
Dilution rate
Depth
Harvest frequency
Table 4. Algal dry biomass production at different condition and media
Media composition
CHU medium + Deionised water
CHU medium + Lake water
Lake soil+ Deionised water
Lake water + Lake soil
CHU medium + Lake water + Lake soil
Lake water
Deionised water
Algal dry biomass (g/L)
Department
Department
Department
Window
Roof
Culture lab
2.2
3.9
2.3
4.3
5.2
3.7
0.11
1.6
2.7
1.7
3.6
4.9
2.9
0.08
Figure 2. Optimization of algal culture.
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2
3.3
1.9
3.5
4.8
2.1
0.03
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Figure 3. Different steps for algal oil extraction.
Figure 4. The chemical catalyzed transesterificaation reaction (Upper layer layer is biodiesel and
lowerlayer is glycerine).
Figure 5. Graph shows the effect of incubation time on biodiesel production at 40 oC using 5 mL
Scenedesmus dimorphus.
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Figure 6. TLC analysis of reaction mixture during transesterification reaction.
Figure 7. TLC analysis of different ester.
4. Conclusions
As demonstrated above, microalgal biofuel is technically feasible. It is the only renewable
biofuel that can potentially completely relocate liquid fuel derived from gasoline. The financial side of
producing algal biofuel needs to recover significantly to be possible. Production of low-cost microalgal
biofuels requires primarily improvements to microalgal biology through genetic engineering. Use of
the biorefinery idea and advances in photo bioreactors engineering will further lower the cost of
fabrication. In view of their much greater yield than raceways, tubular photo bioreactors are likely to
be used in producing much of the algal biomass required for making biodiesel. Photobioreactors
provide a prohibited environment that can be modified to the specific stress of highly productive
microalgae to attain a consistently good annual give up of algal oil. Algal biodiesel creation can be
done through the use of chemical (acid, base). There is a recent attention in using powerless lipase or
immobilized whole cells as “green” alternatives to chemical catalysts to produce biofuels industrially.
However, the cost of the final enzymatic product remains a difficulty compared to the cheaper
substitute of using chemical catalysis. Using the apparatus of recombinant DNA technology, it is
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possible to increase the supply of appropriate lipases for biofuels fabrication. Protein engineering and
site-directed mutagenesis may be used to alter the enzyme-substrate specificity, stereo specificity, and
thermo stability, or to increase their catalytic efficiency, which will benefit biofuels construction, and
lower the cost of the generally process. Indeed, more research efforts should be focused in modifying
and cloning more lipases. Lipases can be immobilized to increase their prepared stability, making
reuse several times possible.
Acknowledgement
The authors thank to the all faculty members of Rajiv Gandhi Proudyogiki Vishwavidiyalya for
given support.
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