The use of fossil fuels is unsustainable due to limited supply and also due to large emissions of Carbon dioxide due to the effect of global warming. Biofuel is a viable option but can, as produced today; only provide a limited amount of fuels needed. Biofuels are presently derived from terrestrial plants, which require large amounts of arable land. Biofuels from microalgae on the other hand do not necessarily require arable land and can theoretically replace fossil fuels absolutely. Biofuels from microalgae could use industry waste water as growth medium particularly paper industry waste water is an interesting potential provider due to its high nitrogen and phosphorus in waste water. In this research work marine microalgae Nannochloropsis Salina was cultivated using f/2 medium using modified air lift photo-bioreactor along with the paper industry effluent waste water, The doubling time calculated from optical density attained at 48 hrs the cell count almost doubled during this period. Since the marine species is sensitive to pH we need to maintain the pH at 7 below 7indicated the decreased biomass levels in culture. The lipid extraction was studied using solvent methods. The functional compounds in lipids FAME were studied using GC-MS analysis, the Nannochloropsis salina showed qualities of growing in fresh water and brackish water apart from the marine water which is a desirable characteristic for algal phycoremediation

1. http://www.iaeme.com/IJMET/index.asp 1471 editor@iaeme.com
International Journal of Mechanical Engineering and Technology (IJMET)
Volume 10, Issue 01, January 2019, pp. 1471–1477, Article ID: IJMET_10_01_149
Available online at http://www.iaeme.com/ijmet/issues.asp?JType=IJMET&VType=10&IType=1
ISSN Print: 0976-6340 and ISSN Online: 0976-6359
© IAEME Publication Scopus Indexed
BIOFUEL PRODUCTION FROM MARINE
MICROALGAE Nannochloropsis Salina USING
PAPER MILL EFFLUENTS
P. Gurumoorthy*
Department of Biotechnology and Chemical Engineering,
Arulmigu Meenakshi Amman College of Engineering, Tamilnadu, India
A. Saravanan
Department of Chemical Engineering, Hindustan Institute of Technology and Science,
Padur, Chennai, Tamilnadu, India
*Corresponding Author: biogp77@gmail.com
ABSTRACT
The use of fossil fuels is unsustainable due to limited supply and also due to large
emissions of Carbon dioxide due to the effect of global warming. Biofuel is a viable
option but can, as produced today; only provide a limited amount of fuels needed.
Biofuels are presently derived from terrestrial plants, which require large amounts of
arable land. Biofuels from microalgae on the other hand do not necessarily require
arable land and can theoretically replace fossil fuels absolutely. Biofuels from
microalgae could use industry waste water as growth medium particularly paper
industry waste water is an interesting potential provider due to its high nitrogen and
phosphorus in waste water. In this research work marine microalgae Nannochloropsis
Salina was cultivated using f/2 medium using modified air lift photo-bioreactor along
with the paper industry effluent waste water, The doubling time calculated from
optical density attained at 48 hrs the cell count almost doubled during this period.
Since the marine species is sensitive to pH we need to maintain the pH at 7 below
7indicated the decreased biomass levels in culture. The lipid extraction was studied
using solvent methods. The functional compounds in lipids FAME were studied using
GC-MS analysis, the Nannochloropsis salina showed qualities of growing in fresh
water and brackish water apart from the marine water which is a desirable
characteristic for algal phycoremediation
Key words: f/2 medium, Nannochloropsis salina, GC-MS, microalgae, FAME.
Cite this Article: P. Gurumoorthy, A. Saravanan, Biofuel Production from Marine
Microalgae Nannochloropsis Salina Using Paper Mill Effluents, International Journal
of Mechanical Engineering and Technology 10(1), 2019, pp. 1471–1477.
http://www.iaeme.com/IJMET/issues.asp?JType=IJMET&VType=10&IType=1

2. P. Gurumoorthy, A. Saravanan
http://www.iaeme.com/IJMET/index.asp 1472 editor@iaeme.com
1. INTRODUCTION
The exploitation of fossil fuels is unsustainable due to the insufficient availability and the
consequential widespread green-house gas emissions Chisti (2008); Ekendahl, et al. (2010).
The production of renewable biofuels such as ethanol and biodiesel are today chiefly
produced from terrestrial plants such as sugar cane, rapeseed, and palm that require enormous
amounts of arable land John, et al. (2011); Chisti (2008); Griffiths and Harrison (2009); Ma
and Hanna (1999); Xu, Miao and Wu (2006). The use of arable land for the manufacturing of
biofuels is contentious since this competes with food production Chisti (2008); Griffiths and
Harrison (2009).
Microalgae on the other hand, can be cultivated without the need of arable land and are
also in general much more efficient than the terrestrial plants in capturing solar energy
Malcata (2011). According to Li et al (2008a) switchgrass, which is the best growing
terrestrial plant, can acclimatize less than 0.5% of the total solar energy received in a
characteristic midlatitude location whereas microalgae may adapt up to 10% Li, et al. (2008a).
The doubling time and water requirements are also factors that significantly favour
microalgae. The distinctive doubling time for algae is approximately 24 hours Chisti (2008);
Malcata (2011). According to Kliphuis et al (2010) microalgae requires around 1.5 liters of
water to produce 1 liter of biodiesel compared to 10 000 liters for land crops Kliphuis, et al.(
2010). Chisti (2008) has approximated that the USA would need to use over 60% of its
agricultural cropping land to be able to cover its need for biodiesel using palm. This can be
compared to approximately 3% for a microalga with 30% oil content (Chisti 2008).
Microalgae requires huge amounts of CO2, it takes approximately 1.8 ton CO2 to generate
1 ton of microalgal biomass Chisti (2008). To be able to attain the high cell densities that are
required to make algal cultivation profitable CO2 needs to be supplied in concentrations
higher than in the air. To supplementary improve the prospects of microalgae cultivations
industrial flue gas and waste water could be used. Several types of waste waters include
phosphorous, nitrogen and trace metals which are essential for microalgal development, and
the exploit of this would diminish mutually costs and eutrophication effects Chisti (2008);
Kim (2013); Huang, et al. (2010); Kong (2009); Patil, et al. (2008); Gurumoorthy (2016,).
With soaring oil prices and increased environmental awareness microalgae seems very
promising. The thriving application of algal biomass for biofuel manufacturing will basically
depend on the development and harvesting of algal biomass at cheaper costs. The current
study is an effort to decrease the waste water pollution and produce the algal biomass and
biofuels from nutrient rich paper industry effluents.
Nannochloropsis is a genus with non-flagellate small cells, 2-4 μm in diameter Tomaselli
(2007). Like the other known species of Eustigmatophyceae the cells are green coccoid that
are either single, in pairs or in colonies Barsanti and Gualtieri (2005). Nannochloropsis has a
cell wall of polysaccharides and do not accumulate starch Tomaselli (2007). It has received a
great deal of interest as a source of polyunsaturated fatty acids, since it can accumulate large
amounts of eicosapentaenoic acid. Nannochloropsis is commonly cultivated in fish hatcheries
as feed for rotifers and to enhance growth in larvae tanks Tredici (2009). The optimum pH for
growth for N. salina is 9, but it can grow in the range of pH 5-10.5 (SERI Microalgal
Technology Research group 1986). The temperature range for growth lies between 17-32°C
with optimum at around 28°C (SERI Microalgal Technology Research group 1986).
2. COLLECTION OF MICROALGAE AND PAPER INDUSTRY
EFFLUENTS
Marine microalgae Nannochloropsis salina culture samples were delivered from Central
marine fisheries research institute, Chennai, Tamilnadu. The paper industry waste effluents

3. Biofuel Production from Marine Microalgae Nannochloropsis Salina Using Paper Mill Effluents
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where collected in and around Karur district, Tamil nadu. The industrial effluents were
collected in aseptic bottles and transported to the laboratory
3. BIOMASSPRODUCTION USING SEMICONTINUOUS AIRLIFT
PHOTOBIOREACTOR
The algal cultures were maintained in air conditioned room and 100 ml Nannochloropsis
salina samples were inoculated in 1000 ml conical flask containing sea water was enriched
with paper industry waste water effluents and f/2 medium and the seawater pH is maintained
at 7 after inoculation, a compressor is attached to the flask the air from the compressor will be
utilized for aeration and mixing process for light source 19v led bulb was fitted near the
photo-bioreactor for lightening purpose in the night since the algae grows in dark for
photosynthesis we fit the 19v led bulb so the algae can utilize the light source in night for
biomass production Figure 1. The pH is maintained correctly to increase the biomass when
there is decrease in pH leads to less biomass production. Periodically the algal growth was
examined in terms of the increase in the optical density at 550 nm. The results showed algal
growth increased and attained exponential phase in 72 hours and the f/2 medium was used
sparingly since the waste effluents contain more nitrogen and phosphorous source which is
suitable for algal biomass production. The results showed that the algal growth continued till
the 30th
day and then it started declining due to environmental conditions and decrease in pH
levels.
Figure 1 Airlift photo-bioreactor used for algal biomass production
4. ANALYSIS OF ALGAL BIOMASS
Algal biomass was measured by taking the dry weight of algae at different intervals. From
100 ml of algal broth biomass was extracted at day 5, 10, 15, 20 and 25. The measured algal
biomass of the entire sample is shown in Table1. These values are the mean of three
determinations ± Standard deviation. There was a progressive increase in growth of the algal
sample. By the end of 25 days Nannochloropsis salina showed high dry biomass i.e. 5.3±0.26
(g/100ml±SD). The dry biomass is further used for lipid extraction

4. P. Gurumoorthy, A. Saravanan
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Table 1 Algal biomass measurements
SAMPLE DAYS DRY WEIGHT MEASUREMENTS
(g/100ml±SD)
Nannochloropsis
salina
5th
0.66±0.00
10th
1.45±0.30
15th
2.49±0.07
20th
4.50±0.20
25th
5.3±0.26
The data obtained in Table 1 were statistically analyzed using Analysis of variance two
way classification to determine whether there is significant difference between the species and
there is significant effect of different times
5. ALGAL LIPID EXTRACTION
To harvest the algal dry mass cultures were filtered using Whatman No. 41 filter paper and
the biomass was dried in hot air oven for 24 hours at 80o
C Bagchi et al., (2015). Two hundred
grams of the dry algal mass Figure 2 was wrapped in Watman’s filter paper, sealed and kept
in the middle chamber of the soxhlet extractor to run extraction. The round-bottomed flask of
the soxhlet was filled with 200ml ethanol and the soxhlet was placed on a hot plate at a
temperature of 70o
C Redfern et al., (2014) and the Soxhlet apparatus was kept running for 36
hours for ten cycles to complete the lipid extraction process and the lipid content obtained
from 200 grams of algal biomass is 38ml lipid content
Figure 2 Dry algal biomass inside Soxhlet apparatus
6. TRANSESTERIFICATION
Biodiesel molecules are mixture of fatty acids methyl esters (FAMEs) produced from
transesterification reaction between triglycerides esters (vegetable oil or animal fat) and
alcohol (methanol) in presence of alkalis that acts as a catalyst. In a transestrification process,
each mole of triglyceride is converted into a mole of fatty acids methyl esters (FAMEs) using
three moles of methanol. The algal lipids are with 2 grams of NaOH and 110 ml of methanol
and the mixture was stirred continuously for 20 min on a magnetic stirrer and kept in a
incubator for 58 hrs at 60 rpm to separate the biodiesel and sedimented layers the uppermost
layer that is the biodiesel was separated carefully and was measured the algal biodiesel
obtained from transestrification is 20ml. Biodiesel was washed using 5% water a number of
times until a clear solution is obtained. Biodiesel was stored at room temperature for 12 hours.

5. Biofuel Production from Marine Microalgae Nannochloropsis Salina Using Paper Mill Effluents
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Final volume of biodiesel was measured and pH was checked Sander and Murthy, 2010. Use
of methanol resulted in soap formation because the small methyl group surrounds the fatty
acids, while the free fatty acids form long chains Stavarache et al., (2007).
7. GC-MS ANALYSIS OF FAME
GCMS was employed to study the Algal FAME using JEOL GC MATE II data system was
used the time range was 60 to 600 ionizations and it generated three different peaks the figure
3 Shows the GCMS peak values and their respective retention time Gurumoorthy et al (2016),
Table 2 Shows the composition of fatty acid methyl esters in microalgae Nannochloropsis
salina
Figure 3 Shows the GC-MS peak values for Fatty Acid Methyl Esters
Table 2 Shows the composition of fatty acid methyl esters in microalgae Nannochloropsis salina
Peak No Retention
Time (min)
Description of the ester Chemical
formula
Scan Ions
1 8.16 Tetradecanoic acid, methyl
ester ( Myristic acid cetane
number C14:0)
C14H28O2 1376 2171
2 8.76 Octadecanoic acid, methyl
ester (Oleic acid cetane
number C18:1)
C14H28O2 1492 2016
3 9.18 Oxiranedodeconoic acid, 3
octyl cis
C22H42O3 1573 1899
4 9.65 Tetradecanoic acid, methyl
ester ( Myristic acid cetane
number C14:0)
C14H28O2 1623 2181
5 9.94 Pentadecanoic acid, 14
methyl, methyl ester
C15H30O2 1718 1330
6 10.26 1,2 benzendicarboxylic
acid, bis(2methylpropyl)
ester
C8H6O4 1780 1543
7 10.5 9 Hexadecanoic acid (
Palmitic acid Cetane
number C16:0)
C16H32O2 1880 2061
8 11.12 10 –octadecenoic acid,
methyl ester (Oleic acid
cetane number C18:1)
C19H38O2 1946 1148

6. P. Gurumoorthy, A. Saravanan
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8. CONCLUSIONS
The main goal of this study was to determine whether marine microalgae are able to grow in
waste water from paper industry effluents. The results indicate that they can thrive on these
environments. The GC-MS fatty acid profile of Nannochloropsis salina displayed high
percentage of C18:1 Oleic acid which has been suggested as a compound for enrichment in
biodiesel and the presence C14:0 Myristic acid which is the chief component present in
biodiesel and C16:0 Palmitic acid which is also a major biodiesel component present in this
fatty acid profile which eventually proves that the Nannochloropsis salina species is an apt
able candidate for biodiesel production and also it is useful for phycoremediation this study
reveals that this marine algae can be well utilized for treating paper industry waste water and
as well as for the production of biodiesel.
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