POME

1. Galley Proofs, RJC-5845/2020
ISSN: 0974-1496 | e-ISSN: 0976-0083 | CODEN: RJCABP
http://www.rasayanjournal.com
http://www.rasayanjournal.co.in
Rasayan J.Chem.,13(3),XXXX-XXXX(2020)
http://dx.doi.org/10.31788/RJC.2020.1335845
THE INFLUENCE OF PALM OIL MILL EFFLUENT (POME)
PRE-TREATMENT AS SCENEDESMUS DIMORPHUS
MICROALGAE CULTIVATION MEDIUM FOR BIODIESEL
FEEDSTOCKS
Zulkarnain Chaidir1*
, Ajie Syaputra1
, Elida Mardiah1
, Hilfi Pardi2
1Biochemistry Laboratory, Faculty of Mathematics and Natural Sciences Andalas University
Faculty of Mathematics and Natural Sciences, Andalas University, The Limau Manis Campus,
Padang 25163 Indonesia
2Department of Chemistry Education, Faculty of Teacher Training and Education Raja Ali Haji
Maritime University, Senggarang, Tanjungpinang, 29100 Indonesia
Email:hilfipardi@gmail.com
____________________________________________________________________________________
ABSTRACT
Palm Oil Mill Effluent (POME) is pollutant from the processing of fresh palm bunches into crude palm oil. The
POME still contains nutrients that can be utilized for microalgae growth. The purpose of this research was to
explore the influence of pre-treated POME using H2SO4 or HNO3 on its nutrition. The pre-treated POME was
applied on cultivation medium of Scenedesmus dimorphus microalgae. POME was pre-treatedusing H2SO4 or
HNO3with concentrate variance: 5%, 10% dan 30% (v/v). The pre-treated POME was added into Bold Basal
Medium (BBM) which was the cultivation medium. The Scenedesmus dimorphus was cultivated on medium that
containsa mix of BBM and POME. Harvesting was executed in the stationary phase. Harvested Scenedesmus
dimorphus was dried, and then its lipid was extracted by using Bligh & Dyer method. The transesterification was
performed to the lipid, and the result was examined using Gas Chromatography- Mass Spectra (GC-MS). This
research found the adding Bio (v/v). GC-MS analysis shows fatty acid content in extracted lipid was saturated fatty
acid and monounsaturated fatty acids which were palmitic acid, stearic acid, and 9-octadecenoic acid. Those fatty
acids that come from Scenedesmus dimorphus have the potential to become biodiesel feedstock.
Keywords: POME, Pre-treatment, Biodiesel, Scenedesmus Dimorphus, GC-MS.
© RASĀYAN. All rights reserved
INTRODUCTION
Currently, the majority energy needs are fulfilled by utilizing the fossil fuel. The extensive use of fossil
fuel without providing the alternative energy source will causes the extinction of fossil fuel. Moreover,
the combustion of fossil fuel causes unexpected gas emission such as CO2. Those gas emissions are not
friendly to environment, it can cause the global warming and greenhouse effect. On account of that, the
needs of alternative energy source which friendly to environment is inevitable. Currently, biodiesel is one
the alternative energy source that expected to substitute the fossil fuel [1-3]
.
Biodiesel is a renewable and environment friendly energy source, those characteristics draw the global
attention to succeed it as the alternative of fossil fuel. Biodiesel has identic physical and chemical
characteristic with fossil fuel, for that reason the biodiesel can be used by conventional engine. Moreover,
the advantage of biodiesel is the environment friendly emission from the combustion. These days,

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THE INFLUENCE OF PALMOIL MILL EFFLUENT Zulkarnain Chaidir et al.
biodiesel is already in the 3rd generation which produced from microorganism lipid. Microalgae is one of
the microorganisms that can produce lipid for biodiesel [4-6]
.
Microalgae become the promising of biodiesel feedstock because of the fast growth and high lipid
production. Additionally, microalgae do not require vast land to grow and not become foods source. The
growth of microalgae needs medium with high nutrition. Bold Basal Medium (BBM) is the one of the
microalgae growths medium, but the BBM costs a lot. Hence, it needs to explore the potential to lower
the BBM cost. On the other hand, industrial liquid waste contains the nutrition that needed by microalgae.
Previous researches explore the liquid waste from the processing of Fresh Palm Bunches (FPB) into
Crude Palm Oil (CPO) as nutrition for microalgae. Cheah et al (2018) cultivated Chlorella Sorokiniana
CY-1 microalgae using POME as growth medium. The result of Cheah et al research was 30 % (v/v)
POME produce 11.21% lipid content [7-10]
.
Indonesia is a country that produce huge volume of palm oil, it causes the palm oil industry grows
positively every year. Along with the growth of palm oil industry, the volume of POME which is the
liquid waste that have negative impact on environment also increases. POME requires further processing
to avoid environment polluting. H2SO4 or HNO3 can hydrolysis POME and the result can be utilized for
microalgae cultivation [10-12]
.
Scenedesmus Dimorphus is the better microalga in lipid production compared to Spiruline sp, or
Chlorella Sorokiniana CY-1. This research focused POME hydrolysis using H2SO4 or HNO3 to substitute
BBM as medium for microalgae cultivation. This research explored the variance of Hydrolysis addition
on cultivation medium of Scenedesmus Dimorphus. This research analysed the lipid content of harvested
Scenedesmus Dimorphus biomass.
EXPERIMENTAL
Tools
This research utilized following tools: cup glasses, centrifuge, autoclave, analytic scale (Metler),
Spectrophotometer UV-Vis (Genesys 20), light microscope (Dynatech), Vortex Genie Pulse, Gas
Chromatography- Mass Spectra (Shimazu), water bath shaker.
Materials
This research used following materials: POME, Bold Basal Medium (BBM), isolated Scenedesmus
Dimorphus microalgae, aquadest, H2SO4, HNO3, Methanol, Hexane, Chloroform, and NaOH.
Samples Collection and POME Pre-treament
POME was collected from PT. IncasiRaya,Pesisir Selatan, West Sumatera. POME was pre-treated using
H2SO4 1M or HNO3 1M. One glass cup of POME was poured and stirred with H2SO4, and another glass
cup of POME was poured and stirred with HNO3. The addition of H2SO4 and HNO3 was continued until
the POME reach pH = 1. After that, they were filtered, and the filtrates were sterilized using autoclave
with temperate set at 121°C for 15 minutes.
CreatingMediumforScenedesmusDimorphusMicroalgaeCultivation
Cultivation medium was created using variation of concentrate from POME pre-treatment. The variation
is 5%, 10%, and 10% (v/v). The medium was mixed with sterile BBM until the volume is 100 mL. NaOH
1M was ass added into medium until pH level was 7-8. Afterwards, cultivation medium was sterilized
with temperature at 121°C for 20 minutes.
Cultivation,IdentificationandDeterminingScenedesmusDimorphusMicroalgaeGrowth
Curve
Isolated Scenedesmus Dimorphus microalgae were collected from Biochemical Laboratories of
Universitas Andalas. They were cultivated on the medium with aeration by aerator. The lamp light was

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THE INFLUENCE OF PALMOIL MILL EFFLUENT Zulkarnain Chaidir et al.
set to 21 watts with 12 hours light/12 hours dark in the cultivation period. Pureness of Scenedesmus
Dimorphus microalgae was identified using light microscope at 400x zoom. Growth curve was
determined by checking time in day measurement versus absorbance level at wavelength 680 nm (OD680).
Harvesting Scenedesmus Dimorphus Microalgae
Scenedesmus Dimorphus Microalgae were harvested at final exponential phase or at starting stationary
phase. Harvesting was done by using centrifuge with speed at 3000 rpm for 15 minutes. Biomass was
taken and the transferred to petri dish. They ware aerated until dry.
ExtractionandFatty AcidAnalysis
Lipid extraction was performed using Bligh & Dyer method that modified by Cavonius[13]
. Twenty
milligrams of dry biomass were watered using 80 µL aquadest for 1 hour. Afterwards, addition of
methanol:chloroform (2:1) 300 µL was performed and it was vortexed for 20 seconds. After that it was
centrifuged at 2500 rpm for 5 minutes until chloroform, aquadest, and biomass phase formed. The
chloroform part was picked and transfer to vial bottle, they were put in oven with temperature at 50°C for
30 minutes. Microalgae lipids was calculated with following formula [13]
.
The transesterification was executed for each highest extracted lipid from every pre-treatment. The
transesterification used methanol and H2SO4. After that, incubation performed with temperature at 90°C
for 120 minutes. Next, Hexane and aquadest were added and vortexed 15. Hexane part was picked for
fatty acid methyl ester analysis by using Gas Chromatography-Mass Spectra (GC-MS).
RESULTS AND DISCUSSION
The InfluenceofPOME Pre-treatment and ConcentrationonMicroalgaeGrowth
The purpose of POME pre-treatment was to hydrolysis the organic compound inside POME. The result
from hydrolysis of organic compound and acid can become additional nutrition for BBM which utilized
as Scenedesmus Dimorphus cultivation medium. Variance of POME pre-treatment and concentration
caused different growth phase of Scenedesmus Dimorphus microalgae cell. The growth of Scenedesmus
Dimorphus microalgae cell was measured based the absorbance at Optical Density 680 nm (OD680).
Growth curve variance based on the variance of POME H2SO4 pre-treatment concentration is shown on
Figure 1.

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THE INFLUENCE OF PALMOIL MILL EFFLUENT Zulkarnain Chaidir et al.
0 5 10 15 20 25
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Absorbance
Time (day)
Control
5% POME +
BBM
10% POME +
BBM
30% POME +
BBM
Fig. 1: Growth Curve of Scenedesmus Dimorphus with Variation
POME Pre-Treatment H2SO4 concentration
This research proved that POME pre-treatment with H2SO4 increased nutrition in cultivation medium. The
value of optical density (OD680) is high, for that reason it can be concluded there was an increment on
number of microalgae cell. POME pre-treatment with H2SO4 converted lignocellulose compound into
cellulose and glucose. Cellulose and glucose were more convenient to be used as addition nutrition for
Scenedesmus Dimorphus growth. It showed that 10% POME concentration resulted the number of
nutrition for Scenedesmus Dimorphus growth more optimal compared to another POME concentration
variance. Light penetration was also better on the medium with 10 % POME. The light penetration helped
Scenedesmus Dimorphus growth. Light is an energy source for cell metabolism.
-2 0 2 4 6 8 10 12 14 16 18 20
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 Control
5% POME +
BBM
10% POME +
BBM
30% POME +
BBM
Absorbance
Time (day)
Fig. 2: Growth Curve of Scenedesmus Dimorphus with Variation POME
Pre-Treatment H2SO4 concentration
POME pre-treatment with HNO3 was faster in lignocellulose hydrolysis, it apparent in change medium
colour that become brighter than H2SO4 pre-treatment. Figure 2 shows that 10% POME concentration

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THE INFLUENCE OF PALMOIL MILL EFFLUENT Zulkarnain Chaidir et al.
have higher absorbance value compare to 5% POME and 10% POME. Cultivation medium with 10%
POME is the optimum medium for Scenedesmus Dimorphus growth. All cell growth influencing factor
such as nutrition and light were fulfilled, therefore Scenedesmus Dimorphus adapted faster on cultivation
medium with 10% POME.
TheInfluenceofPOME Pre-treatment and ConcentrationonLipidContent
Harvesting ScenedesmusDimorphus was performed at final exponential phase or starting stationary phase
of the growth. All obtained biomass was dried, and its lipid was extracted subsequently. Lipid extraction
was using Bligh Dreyer method. The highest lipid content was obtained when using 10% POME. All
nutrition for lipid creation in cell were supplied when using 10% POME and by light intensity adjustment.
Figure 3 shows the lipid content of Scenedesmus Dimorphus microalgae for each POME pre-treatment.
This research results showed that the highest increment of total lipid content was obtained when use 10%
POME instead of 5% POME or 30% POME. POME pre-treatment can increase the nutrition content in
cultivation medium. One of the nutrition was carbon as the result of lignocellulose hydrolysis to become
cellulose and glucose. The higher carbon in nutrition made lipid biosynthesis increases [14-18]
.
The higher POME concentration generated more carbon in cultivation medium nutrition. This
condition made cell more convenient to absorb nutrition from cultivation medium. Moreover, cell would
reduce the lipid biosynthesis process and produced less lipid because the cultivation medium had
provided all required nutrition
Control Nitric acid Sulphuric acid
0
5
10
15
20
25
Control
5% POME
10% POME
30% POME
Total
Lipid
(%)
Pre-treatment
Fig. 3: Total Lipid Microalgae Scenedesmus Dimorphus in Various
POME Pre-Tretament.
Fatty AcidMethyl EsterAnalysis
This research extracted fatty acid methyl ester content from the Scenedesmus Dimorphus
microalgae with the highest lipid content. The extraction result was analysed by using GC-MS.
The analysis result showed the extracted fatty acid content could be utilized as biodiesel
feedstock.

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Table 1. FAMEs Profiles
Methyl Ester Fatty Acid
% Area
Control
H2SO4
Pre-treatment
HNO3
Pre-treatment
Methyl Hexadecenoic Palmitic Acid (C16:0) 24.38 1.01 10.65
Methyl Octadecenoic Stearic Acid (C18:0) - 13.32 -
Methyl 9-octadecenoic Oleic Acid (C18:1) 39.29 22.91 17.64
Tabel 1 shows the fatty acid in Scenedesmus Dimorphus microalgae lipid which were Palmitic
Acid (C16:0), Stearic Acid (C18:0), and Oleic Acid (C18:1). Those fatty acid were saturated and
monounsaturated. The composition of fatty has influence on biodiesel characteristics such as
Oxidation Stability, cetane number, melting point, and viscosity. One of biodiesel quality is the
content of polyunsaturated fatty acids and saturated fatty acid. The number of double bonds have
effect on biodiesel oxidation stability.
9.
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