Growth rate of Chlorella pyrenoidosa in vinasse medium and its evaluation as bioethanol

production’s feedstock

Megawati1*, Wahyudi Budi Sediawan2, Astrilia Damayanti1, Hanifah1, Fatkhulil Jannah Eva
Agustina1

1
Chemical Engineering Department, Engineering Faculty, Universitas Negeri Semarang

Building E1 2nd Floor, Kampus UNNES Sekaran, Gunungpati, Semarang, Indonesia, 50282

2
Chemical Engineering Department, Engineering Faculty, Universitas Gadjah Mada, Yogyakarta

*
megawati@mail.unnes.ac.id

Abstract
The purpose objective of this research is to study the growth of Chlorella pyrenoidosa in vinasse
waste,. The main focus is focusing on the effect of vinasse volume ratio and nutrition addition
towards the size of cell, optical density, and its growth rate of the cell. Growth rate parameter is
used on design calculation of photobioreactor in pilot and industry scales. Chlorella pyrenoidosa
was cultivated in fresh water and vinasse medium (20% and 30%) in mini ponds (3 L), ),
equipped with lighting using 3280 lumens lamp, aeration with air (0.05% CO2), and guillard
nutrition, and pH 8.. Acidity level was set at pH 8, while Rroom temperature and humidity are
relativelywere kept constant during the research. As the results, Ssize of Chlorella pyrenoidosa
cell are were increased if cultivated in vinasse medium and with given guillard addition every 2 2
two days,. Tthe size of cell is increasing,was increased from 3.0-3.6; 4.1-8.6; and 4.8-6.3 m,
each from cultivation result in fresh water, vinasse with nutrition per every 2 days, and without
nutrition per 2every two2 days, respectively. Carbohydrate composition in Chlorella
pyrenoidosa from cultivation result with vinasse is increasing sharply was sharply increased
compared to in fresh water, which areis from 11.00% into 47.47%. ThereforeIn conclusion,
Chlorella pyrenoidosa from cultivation result in vinasse waste is very potential as a feedstock for
bioethanol production. Chlorella pyrenoidosa’s protein and lipids are better to be extracted first
before converted into bioethanol. Specific growth of Chlorella pyrenoidosa in vinasse is was
also increased compared in to the one which use fresh water as medium, which are from 0.062
into 0.087/ day-1.

Keywords: bioethanol, Chlorella pyrenoidosa, fresh water, growth rate, nutrition, vinasse
Highlights
Chrorella pyrenoidosa grow very well in vinasse if given by additional nutrition (10 word)
The size of Chrorella pyrenoidosa cell that cultivated in vinasse are bigger (11 word)
Carbohydrate composition of Chrorella pyrenoidosa cell that cultivated in vinasse were
increased (12 kata)
Specific growth of Chrorella pyrenoidosa in vinasse is increasing if given by additional nutrition
(13 kata)
Chrorella pyrenoidosa that cultivated in vinasse is very prospecthighly potential to be converted
into bioethanol (14 kata)

Introduction
Chrorella pyrenoidosa is one of microalga species that lives in fresh water from which belongs
to Chlorophyta division and Chlorella genus that lives in fresh water. This microalga species can
grow well in climates with warm temperatures like the area of Java Island, therefore much
cultivation areas are developedis matched with climates in Java island, so it is much cultivated
(Hadiyanto et al., 2012). This Chlorella species can grow into 10,000 cells in 24 h and the . Llife
phase of Chlorella pyrenoidosa is between 11-15 h (Ma’rufatin, 2016). Chemical compounds
that can be found in Chlorella areis proteins, carbohydrates, amino acids, carotenoids, vitamins
and minerals (de Araújo, 2019; Pacheco et al., 2015). In the last decade, Chlorella has been
extensively explored to be used in many applications including pharmacy, food and animal
feedstock, natural dyes or, and cosmetics (Safi, 2014). Even Rrecently, Chlorella has been tried
to be utilized for biofuel production, which one of them arewhich is bioethanol (Carioca, 2010;
Chisti, 2008; Posten and Scaub; 2009, Yeh 2012). All this time, bioethanol is produced from
sugar-based feedstocks (sugar beets, sugarcane), starch-based feedstocks (corn, wheat, barley,
etc.), and lignocellulosic materials (bagasse, corn, rice straw, rice husk, switchgrass, and so on)
(Chen et al., 2013; Sambusiti et al., 2015; Singh et al., 2019). Material that based on sugar and
starch were still very expensive and competing over interest with food material and feed.
Meanwhile material based on lignocellulose is very cheap and easy to be handled. However, the
production cost into ethanol is still very expensive. Furthermore, the presence of lignin that hard
to be fermented into ethanol or other biodegradation method makes lignocellulosic material not
yet demanded to be developed in industrial scale. Therefore, more profitable feedstock is needed
to be developed. Microalgae could become one of the alternatives (Sambusiti et al., 2015).
Lignin contents in microalgae, especially Chlorella is very low, so it become easy to be
hydrolyzed and then fermented into bioethanol (Ho et al., 2012). Several species of
microalgae contain many carbohydrates (37-55%) (Brennan et al., 2010). In addition,
carbohydrates are often used in microalgae cell wall (cellulose, hemicellulose, and pectin) (Singh
et al., 2019).

Bioethanol from microalgae is often called as third generation bioethanol (Chen et al., 2013;
Jambo et al., 2016). Although there have been many attempts to develop it, However, ratio of
bioethanol production towards feedstock need of Chlorella pyrenoidosa is still small due to low
content . This is because the contentT of cellulose and hemicellulose are smallin microalgae and
microalgaeit’s cellular structure that is hard to be penetrated by enzymes (Rodrigues et al.,
2011). Lately, fFollowing the development of biodiesel, those kind of problem need to be
resolved and microalgae growth engineering needs to be done (Chen et al., 2013). The purpose is
to increase theing carbohydrate content in microalgae, so its’s conversion level to bioethanol will
beis increased. This engineering process is categorized into fourth-generation bioethanol
(Niphadkar et al., 2017).

One of engineering method of microalgae growth is using vinasse as mixture of it’s growth
medium (Pancha et al., 2019). Besides species of microalgae, carbohydrate content in microalgae
were also affected by cultivation condition (medium, CO2, nitrogen, temperature, pH, light
intensity, and photobioreactor types) (Pancha et al., 2015; Singh et al., 2019). It is done because
vVinasse is a ethanol waste product from molasses that contains many useful chemical
compounds, which arenamely ammonia and inorganic phosphate, and it is relatively low in
organic matter content (Marques et al., 2013). In addition, most of wastewater contains organics
carbon, nitrogen, phosphorus and other compounds, which makes them suitable for microalgae
cultivation (Pacheco et al., 2015).
According to Kendirlioglu (2017), photosynthesis process on Chlorella can’t do
photosynthesiscould not be transpired in the absence of without lights. Because of that, lights
from lamp or sunlight are very important as it used as the energy source for photosynthesis
process. In addition, microalgae can grow very well at lamp lighting condition 8000 lux
(Gunawan et al., 2018), aeration using CO2 with rate of 200 mL/min (Tang et al., 2011), pH range
of 5.7-8.1 (Tang et al., 2011) and temperature of 25-28 oC (Sachdeva et al., 2016). Fresh air
containing 0.03% of CO2 can be utilized for Chlorella aeration. Usage of air as substitute of pure
CO2 is also for efficiency of the process efficiency when applied on industrial scale. Microalgae
growth using fresh air for aeration has been done by Tang et al., (2011). The result showeds that
concentration of Chlorella pyredoinosa wasis high, in rate of 0.87 + 0.01 g/L. Meanwhile,
Chlorella growth using 50% v/v CO2 at rate of 200 L/h resulting on concentration of 0.87 + 0.01
g/L. That means aeration using air as CO2 supply couldcould be applied. Beside that, previous
researches showed that the concentration of CO2 aeration above 5% could be harmful to
microalgal cells and inhibit the microalgal growth (Tang et al., 2011).

Beside condition that needs to be kept, Chlorella also need nutritions on it’s growth. One of
nutrition that widely used is guillard solution. This solution is one of microalgae nutrition that
consist of NaNO3 75 g/L dH2O, NaH2PO4H2O 75 g/L dH2O, NaSi3.9H2O 75 g/L dH2O, trace
metal solution and vitamin solution (Guillard, 1975). According to Ong et al (2010), guillard
nutrition has been utilized for growth of Chlorella sp. microalgae. In summary, Tthis research
studies the growth of Chrorella pyrenoidosa in vinasse medium. Influence of vinasse volume
ratio and guillard addition as nutrition periodically toward cell sizes, cell composition and
growth rate areis also discussedstudied.

Materials and methods

Chlorella pyrenoidosa seeds were obtained from Ugoo planktonshop, Purworejo Regency,
Central Java. These seeds were grown in aqueous medium, at pH 8. and The seed was not too
thick and have a light green colorthe color of the seeds is light green and not too thick. When the
seeds were arrived at the Laboratory of Biomass, Chemical Engineering, Universitas Negeri
Semarang, the seeds were immediately aerated and lighted, so they couldan adapt to the
environment (Asunthkar et al., 2016). Aeration useds an AC/DC air pump (Amara, mini AC/DC
AA 6603) with an air velocity of 2 x 3 L/min and a pressure of 2 x 0.015 Mpa for two days. As
for Llighting, was using a lamp (Philips, 52 W) was used. Before being cultured, the seed
solution was analyzed for its optical density using UV-Vis spectrometry (Thermo Scientific,
Genesys 10UV) and for its shape and size of the cells using digital microscope (Camlab,
BA210).

Cultivation and harvesting of Chlorella pyrenoidosa microalgae in fresh water as medium

Cultivation of Chlorella pyrenoidosa was carried out in 2 two mini open ponds (dia. 8 x length
31 cm) that were made from transparent plastic. The Chlorella seed solution was mixed with
distilled water at a ratio of 1: 1 in the ponds and then guillard nutrition (2 mL) were added.
Before aerated, vinasse medium in the ponds were analyzed for its optical density using UV-Vis
Spectrophotometer. Air for CO2 supply is fed through a hose (dia. 5 mm) using an air pump. This
pump has 2 outputs with each speed of 3 L/min each. The lamp used as a light source has a
lumens of 3280 lumens. This lamp was placed outside the pond with a distance about 15 cm.
During growth, every day at 8:30 a.m. and 2:00 p.m., the temperature and light strength of each
pond was measured using an optical thermometer (Xueliee, GM 320) and luxmeter (Benetech,
GM 1010), and also samples (5 mL) were taken to observe its optical density using UV-Vis
spectrometry at a wavelength of 570 nm. Analysis was done through 5 times dilution for sample
that were obtained before 19 days. This cultivation was operated carried out for 14 days.

After 14 days, Chlorella pyrenoidosa cultivation results are ready to be harvested. Chlorella
pyrenoidosa solution was added by NaOH (Merck KGaA, 1.06498.1000) to turn off microalgae
(Kurnia et al., 2018) .followeod by Pprecipitation will occur and waited for 24 h. To separate the
Chlorella biomass with from the liquid, the solution iwas filtered using a chamois cloth with a
size of 60 μm and the microalgae was taken using an iron spatula. The filtered Chlorella
pyrenoidosa cells were washed with distilled water for three times to remove the other solid
components. The obtained Chlorella pyrenoidosa biomass obtained after filtration was then dried
using an oven (Memmert, UN 160 161 Liter) gradually. At the first step, Chlorella pyrenoidosa
were heated at a temperature of 30 oC for 12 h and then weighed. For the second step, Chlorella
pyrenoidosa were heated again for 10 min and weighed. If the weight was not constant, then it
was heated again for 10 min and the process were repeated until the constant weight were
obtained. A Piece of Chlorella pyrenoidosa that obtained were stored inside a sealed plastic bag.
The products of Chlorella pyrenoidosa were analyzed by their functional groups using Fourier
Transform Infrared-FTIR (Shimadzu Scientific Instruments IR-Prestige-21) at Universitas
Gadjah Mada and its lignocellulose composition were analyzed in CV Chem-mix Pratama,
Yogyakarta. FTIR analyzation method can be widely used to find out type and number of the
functional constituents in molecules (Wagner et al., 2010; Ozer et al., (2016).

Cultivation and harvesting of Chlorella pyrenoidosa in vinasse as medium

Vinasse used as cultivation medium of Chlorella pyrenoidosa were obtained from Madubaru
sugar factory, Yogyakarta. Before used, vinasse was analyzed for the number of COD. Also,
vinasse was pretreated to remove its impurities and inhibitor. Early stage of The pretreatment
was performed by filtering the vinasse twiceis two-times filterization using centrifuge
(Zentrifugen Rotofix, 32A) usingon 4000 rpm speed. After that, vinasse awere dilluted by
distillate water so that theto get volume ratio become 20 and 30% v/v volume ratio. The next
stage iwas sterilization, which iwas done by puttingcooking vinasse solution in an autoclave
(TOMY Autoclave, ES-315) at 121oC for 15 min and after its ’was cooled, the vinasse iwas
stored in freezer at 4 oC. Before being used as medium, optical density of vinasse was analyzed
using UV-Vis Spectrophotometer. Cultivation iwas done by the same method as using the fresh
water medium. Cultivation in vinasse was done in 2 condition, with addition of guillard nutrition
every 2 days and with nutrition addition only at early stage of cultivation.

Harvest method for cultivation using vinasse medium is almost same with using fresh water
medium. After 20 days, Chlorella pyrenoidosa from cultivation result awere ready to be
harvested. Cultivation solution was precipitated by centrifuge at 3400 rpm for 20 min. The
precipitate was taken and dried with same method for Chlorella pyrenoidosa that was
cultivatedion using fresh water medium.

Growth rate of Chlorella pyrenoidosa

Specific growth is calculated on exponential growth rate phase condition and expressed by Eq.
(1), in which μ is specific growth (A/h), X0 is initial optical density of the exponential phase (A),
and Xt is optical density at any time t of exponential phase (A), t is time (h).

ln ( X t ) −ln ( X 0)
μ= (1)
❑❑❑❑

Eq. (1) could be solved by doing linear regression between ln(Xt) as y-axis towards t as x-axis.
Slope value from regression result’s equation is the value of  (specific growth) and the
intercepts result is the value of ln(X0). Average error value is the ratio of X0 value from
regression result toward experimental data (Eq. 2).

X t −X 0
Error= | X0 |x 100 % (2)

Multiplication time or generation time (td) is the time needed by Chlorella pyrenoidosa to
multiply. For calculation of multiplication time (td), Eq (3) could be used.

0.693
t d= (3)
μ

Meanwhile, biomass productivity (P) are calculated based on Eq. (4), with Xf and Xi are final and
initial biomass concentration, respectively, and tt is the total time of cultivation.

( ❑❑ ❑❑ )
(4)
❑❑

Results and Discussion

Effect of vinasse volume ratio on size of Chlorella pyrenoidosa cell
The measurements result of Chlorella pyrenoidosa cells used in this study are presented in Fig.
1A and 1B. Chlorella pyrenoidosa has cells with size of 3.0-3.6 m. According to Hadiyanto et
al. (2012), the shape of Chlorella pyrenoidosa cells is round. Aand based on Takahashi (2018),
the size of Chlorella cell is around 4.1-4.8 m. Meanwhile, microalgale cells range in size from
5 to 50 m (Pacheco et al., 2015). That means, the size of Chlorella pyrenoidosa cell that used
on this research is smaller than Chlorella specie’s cell size in general. Condition of medium and
growth affect the cell size of microalgae (Domenighini and Giardano; Jebsen et al., 2012).
Meanwhile, Fig. 1C and 1D show the size of Chlorella pyrenoidosa cell from cultivation result
in vinasse medium with the addition of nutrients on a regular basis every 2 days. It appears that
number and size of Chlorella pyrenoidosa cells in vinasse medium are more and bigger than in
fresh water medium. The size of Chlorella pyrenoidosa cell are 4.1-4.4 and 4.4-8.6 m, for 20
and 30% vinasse volume ratio, respectively. The number of cells in a 30% vinasse volume ratio
is also more concentrated than the 20% one. In contrast, Fig. 1E and 1F show that without the
addition of nutrients on a regular basis every 2 days, the size of Chlorella pyrenoidosa cell in
30% vinasse (4.8-6.1 m) is smaller than 20% vinasse (5.1-6.3 m), for 20 and 30% vinasse
volume ratio, respectively. In addition, the number of cells in a 30% vinasse ratio are less than
20% one. This indicates that nutrition must be given periodically when Chlorella pyrenoidosa is
grown in a more concentrated vinasse medium.

(Figure 1)

Effect of vinasse as medium on growth of Chlorella pyrenoidosa

Chlorella pyrenoidosa growth cultivation in fresh water medium (Sample A and B) was carried
out in 2 ponds. The growth resultsprofile can be seen in Fig. 2A. Both ponds show almost similar
results. The growth of Chlorella pyrenoidosa in the beginning is very fast, then ramps briefly at
100 to 150 h. After that, its growth returned quickly. It means that Chlorella pyrenoidosa is a
type of microalgae that is easily adaptable (Pacheco et al., 2015). Optical density of Chlorella
pyrenoidosa in fresh water medium shows a range between 0.512 to 1.494 A. If assumed that
correlation between optical density (X) with microalgae biomass concentration (Y) is Y = 0.215
X -0.0239 (R2 = 0.9946) (Tang et al., 2011). Hence, the highest biomass concentrations of
Chlorella pyrenoidosa cultivated in fresh water medium on sample A and B are 0.29 and 0.30 g
of dry biomass per liter of cultivation medium volume, respectively. According to Gunawan et
al., (2018), the biomass concentration of Chlorella pyrenoidosa cultivated at the operation
condition of: light intensity 2000 lux/h, 84 h, CO2 rate 0.034%, and G15 Deptan nutrition (5%
NO3, 18% K2O, 3% MgO, 8% S, 0.35% Fe, 0.02% Mn, 0.02% Zn, and 0.015% Boron) couldan
achieve around 0.288 g/L. The growth of Chlorella pyrenoidosa in fresh water medium was also
studied by Asunthkar et al. (2016) with variations in lamp types as a source of energy. The
results show that the optical density range of Chlorella pyrenoidosa is between 0.3 to 0.65 A.
This density was achieved after 240 h and 1986 lux irradiation. In this study, the lamp used hads
3280 lumens and is installed at a distance of 0.15 m (resulting in an irradiation strength of 11600
lux). Therefore, the higher the irradiation power, the faster the growth of Chlorella pyrenoidosa
(Asunthkar et al., 2016).

Meanwhile, the growth of Chlorella pyrenoidosa in vinasse medium was carried out in 4
conditions, that is at: a volume ratio of 20 and 30% vinasse, without and with the addition of
guillard nutrition every 2 days. Vinasse thatThe used vinasse has a COD number of 33.3 mg/L
and pH of 2.53. The optical density measurement results are presented in Fig. 2B. This figure
shows that the constant periods Chlorella pyrenoidosa growth were found to be earlier and
longer, which is before 50 h, and up to 200 h. This indicates that Chlorella pyrenoidosa requires
earlier and longer adaptations in vinasse medium than in fresh water medium. After a constant
phase, Chlorella pyrenoidosa increases grows again, even sharply in 30% vinasse medium. The
optical density of Chlorella pyrenoidosa in vinasse medium is higher than in fresh water one. As
have been explained before, vinasse waste still contains many chemical compounds, which are
ammonia and organic compounds. These compounds can be used for supporting the growth of
Chlorella microalgae (Candido and Lombardi, 2016; Marques et al., 2013).

The optical densitiesy of Chlorella pyrenoidosa in vinasse medium with the addition of nutrients
per every 2 days are ranged from 2.728 to 9.385 and 0.854 to 16.944 A for the vinasse volume
ratio of 20 and 30%, respectively. Hence, Chlorella pyrenoidosa growth in 20% vinasse medium
is better than in fresh water one, and microalgae growth in 30% vinasse medium better than in
20% vinasse one.
Meanwhile, the optical density densities of Chlorella pyrenoidosa in vinasse medium without
addition of nutrients per every 2 days are ranged between 0.687 to 14.185 and 0.444 to 10.67 A
for the vinasse volume ratio of 20 and 30%, respectively (see Fig. 2C). Therefore, in 30%
vinasse volume ratio without nutrition addition per every 2 days, Chlorella pyrenoidosa can’t did
not grow well. It means that, if Chlorella pyrenoidosa wants to be grown in a more concentrated
vinasse medium, then regular nutrition addition must be done periodically. However, if nutrition
that given are too much, Chlorella pyrenoidosa will experience the nutrient stress of the
physiological status (Meng et al., 2014).

(Figure 2)

FTIR spectra of Chlorella pyrenoidosa cultivated in fresh water and vinasse as growth
medium
According to Meng et al. (2014), there are three main compound that can be found in Chlorella
pyrenoidosa, which are protein (28.1%), carbohydrate (16.3%), and lipid (11.5%). All three of
these compounds are analyzed using FTIR and the analyzation result was similar with the
traditional chemical methods. In this research, the result of FTIR of Chlorella pyrenoidosaCp
microalgae resulted from cultivation in fresh water can be seen in Fig. 3A and 3B. Identification
of Chlorella pyrenoidosa cells functional group is done by using Table 1 and affirmed by some
supporting information, which are.Identifikasi gugus fungsi sel Chlorella pyrenoidosa
menggunakan TabThe composition is presenteds on Table 1 dan dikuatkan oleh beberapa
informasi mendukung, yaitu Tt. There are three main regions that relate to macromolecular
pools, according to Giordano et al. (2001): the lipid band (around 1740 cm-1), the amide I and
amide II bands representing, proteins (around 1660 and around 1540 cm-1), and the carbohydrate
region (1200-900 cm-1). On this research, carbohydrates are observed in 1026 cm -1 with content
of 11%. Accordingly,Therefore, carbohydrate composition in this Chlorella pyrenoidosa is
smaller than previous research resultabout (16.3%. Carbohydrate composition is
small,Komposisi ini sangat kecil), so it needed to be improved if it will be converted into
bioethanol. One of the efforts to improve it, as has been done in this research, is using vinasse as
growth medium. In addition,

However, its protein composition is high (about 28.136%). Before being processed into
bioethanol, Chlorella pyrenoidosait will be more useful if the protein were extracted first.
Protein can be used as human nutrition, animal feed, and aquaculture nutrition. Protein extraction
in Chlorella can be done by enzymatic hydrolysis and chemical extraction. (Bleakley and
Hayes, 2017). Meanwhile, its lipid composition (about 31%) is more than in the previous
researches (11.5%) l. Likes protein extraction, lipid extraction also better to be done first,it is far
better if the lipid also extracted before Chlorella pyrenoidosa processed into bioethanol. Lipid
extraction can be done using non-polar solvent. Diethyl ether methanol solvent (1:1 ratio) was
the best solvent for Synechocytis sp. lipid extraction. Yields produced is about 24.3%
(Ashokhumar et al., 2019). Lipid that obtained could also be converted into biodiesel (Hadiyanto
et al., 2012). Waste from protein extraction result could be extracted to obtain its lipid, then are
processed further into biofuels (Adamakis et al., 2018). Step of biomass utilization into various
chemical products is often called as biorefinery, which are massly developed so that the process
become efficient and economic (Vanthoor-Koopmans et al., 2013). Microalgal biorefinery
concept were the main choice so that the conversion of microalgae into bioethanol is very
economical (Singh et al., 2019; Sambusiti et al., 2015). For instance, in an integrated process,
lipid could be extracted first. Furthermore, de-oiled biomass residues can be used for
biohydrogen production (Das, 2015).

(Figure 3)

(Table 1)

FTIR analyzation result for Chlorella pyrenoidosa from cultivation result on vinasse medium are
presented on Fig. 6C and D, for without nutrition and with nutrition, respectively-F and Table 2.
Carbohydrate composition are increasing slowly compared with to cultivation result in fresh
water. Carbohydrates were observed on band 1072 and 1087 dan 1049 cm-1 for Chlorella
pyrenoidosa that cultivated in 20% and 30% vinasse with nutrition and without per every 2 days,
respectively, respectively. If compared with the cultivation result in fresh water, hydrocarbon
content of cultivation result in 320% vinasse medium with nutrition is higher, which is 22.55%,
while in freshwater medium, hydrocarbon content is just 11%. This indicates that Chlorella
pyrenoidosa biomass will contain higher carbohydrates if cultivated in vinasse medium, even
with nutrition addition every 2 days. If compared with 30 % vinasse medium with nutrition, its
carbohydrate content are very high, which is 47.47%.

Nutrition gives very strong effect towards carbohydrates content of Chlorella pyrenoidosa that
cultivated in vinasse medium. Without nutrition, its carbohydrate contents are small (only
21.25%). Nutrition is very affecting FTIR result from algal cell has been delivered by
Domenighini and Giordano (2009) results reported that FTIR result from algal cell are greatly
affected by the nutrition given. That means, Chlorella pyrenoidosa that cultivated in vinasse will
produce biomass with high carbohydrates if given by nutrition every 2 days. If carbohydrate
content increases because Chlorella pyrenoidosa is cultivated in vinasse medium, protein are the
contrary, which is. It decreases from about 33% into about 10 %., mMeanwhile lipids are slightly
decreased, from 30% into 22%. However, lipid decreasement are not experienced by all
Chlorella pyrenoidosa. Protein content in Chlorella pyrenoidosa biomass is slightly increases on
20% vinasse medium with nutrition per every 2 days.

Kinetic growth of Chlorella pyrenoidosa

The natural logarithm of optical density of Chlorella pyrenoidosa plotted against time gives
curves (Fig. 4A-C), the slope of which is the specific growth rate. Values of specific growth rate
was reported in Table 3. Specific growth rate of Chlorella pyrenoidosa that cultivated in vinasse
medium (20%) with nutrition addition every 2 days (0,074 day-1) is faster than in freshwater
medium (0.062 day-1), as well as for 30 % vinasse with nutrition addition (0.087 day-1) the
growth of Chlorella pyrenoidosa become faster. On the contrary, for cultivation of Chlorella
pyrenoidosa in vinasse medium that not given nutrition every 2 days, but only given on early
cultivation, the growth of Chlorella pyrenoidosa become slower, which is 0.044 and 0.023 day-1,
for 20 dan 30% vinasse, respectively. It means, vinasse as medium could accelerate the growth
of Chlorella pyrenoidosa if given by nutrition regularly, and Chlorella pyrenoidosa if given
nutrition periodicallybila diberi nutrisi secara berkala, and the more concentrated vinasse that
used, the faster its growth. The shape form of six variation linierization line of Chlorella
pyrenoidosa is almost same, except for Chlorella pyrenoidosa from cultivation result on 30%
vinasse without nutrition per every 2 days. This was caused by lack of nutrition during Chlorella
pyrenoidosa growth. So, although Chlorella pyrenoidosa are cultivated in vinasse medium,
nutrition must be given continually. Nutrition will help microalgae to adapt with medium, other
than light (Jebsen et al., 2012). Values of specific growth of Chlorella pyrenoidosa in this
research is smaller than previous research. On this research, specific growth are ranged from
0.062 to 0.087 day-1. Meanwhile, on previous research, the value is 0.69 day -1, 8 times higher
(Tang et al., 2011). This research were usingused different nutrition, which are were (per litre): 1
mg Na2MG EDTA, 36 mg CaCl2.2H2O, 75 mg MgSO4.7H2O, 40 mg K2HPO4.3H2O, 2.86 mg
H3BO3, 1.81 mg MnCl2.4H2O, 0.222 mg ZnSO4.7H2O, 0.079 mg CuSO4.5H2O, 0.05 mg
CoCl2.6H2O, 0.391 mg NaMoO4.2H2O, and 1500 mg NaNO3. Guillard that used on this
research was cheap, so deeper economical analyzation is needed when it going to be utilized for
industrial scale (Guillard, 1975). Medium is indeed very influence toward the growth of
microalgae. In fact, there are a research that specifically investigate the differences of medium
(Bristol, Chu, Bold 3N, TAP(-), and BG-11) towards the specific growth rate of S. obliquus the
result are as follows 0.172; 0.170; 0.186; 0.179; and 0.175 day-1, respectively.

(Figure 4)

(Table 3)

Conclusion
The size of Chlorella pyrenoidosa cell was bigger when cultivated in vinasse medium and given
nutrition every 2 days compared with without nutrition and also than cultivated in fresh water,
which are 3.0-3.6 m (in fresh water medium), 4.1-8.6 m (in vinasse with nutrition every 2
days), 4.8-6.3 m (in vinasse without nutrition everyper 2 days). Specific growth of Chlorella
pyrenoidosa in vinasse medium with nutrition is faster (0.087 day-1), than without nutrition
(0.023 day-1), and in fresh water medium (0.062 day-1). Carbohydrate composition in Chlorella
pyrenoidosa that cultivated in vinasse medium which given by nutrition every 2 days is about
47.47%, higher than Chlorella pyrenoidosa that cultivated in vinasse medium without nutrition
given every 2 days (21.25%), and which cultivated in fresh water medium (11.00%). Therefore,
Chlorella pyrenoidosa that cultivated in vinasse medium must be given by nutrition in order to
increase the carbohydrates, so that it can becomes a prospect as feedstock raw materialof for
bioethanol production.
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