HAYATI Journal of Biosciences 24 (2017) 149e155

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HAYATI Journal of Biosciences

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hayati-journal-of-biosciences

The Effects of Audible Sound for Enhancing the Growth Rate of

Microalgae Haematococcus pluvialis in Vegetative Stage
Marcelinus Christwardana,1,2* H. Hadiyanto1,3,4**
1
Center of Biomass and Renewable Energy (C-BIORE), Department of Chemical Engineering, Diponegoro University, Jl Prof. H. Soedarto, SH, Tembalang,
Semarang 50275, Indonesia.
2
Graduate School of Energy and Environment, Department of New Energy Engineering, Seoul National University of Science and Technology, 232
Gongneung-ro, Nowon-gu, Seoul 01811, Republic of Korea.
3
Department of Chemical Engineering, Diponegoro University, Jl Prof. H. Soedarto, SH, Tembalang, Semarang 50275, Indonesia.
4
Master Program of Environmental Science, School of Postgraduate Studies, Diponegoro University, Jl Imam Bardjo, SH No 3, Semarang 50241, Indonesia.

a r t i c l e i n f o a b s t r a c t

Article history: Physico-stimulant like audible sound is one of the new promising methods for enhancing microalgae
Received 28 April 2017 growth rate. Here, microalgae Haematococcus pluvialis was cultivated with the addition of audible sound
Received in revised form with titles “Blues for Elle” and “Far and Wide.” The objective of this research was to evaluate the effect of
22 August 2017
audible sound to the growth and productivity of microalgae. The experiment has been conducted by
Accepted 23 August 2017
Available online 14 September 2017
exposing the audible sound for 8 h in 22 days to microalgae cultivation. The result showed that
microalgae H. pluvialis treated by the music “Blues for Elle” shows the highest growth rate (0.03 per day),
and 58% higher than the one without audible sound. The average number of cells in stationary phase is
KEYWORDS:
audible sound,
0.76
104 cells/mL culture and the productivity is 3.467
102 cells/mL/day. The pH of microalgae
biomass productivity, medium slightly decreases because of proton production during photosynthesis process. The kinetic rate
growth rate, constant (kapp) is 0.078 per day, reaction half-life (t1/2) is 8.89 days, and catalytic surface (Ksurf) is
Haematococcus pluvialis 1.66
105/day/cm2. In conclusion, this audible sound is very useful to stimulate microalgae growth rate,
especially H. pluvialis.
Copyright © 2017 Institut Pertanian Bogor. Production and hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction feedstock. Some microalgae can accumulate fatty acid which can be
extracted to be bio-oil (Nur and Hadiyanto, 2015). In addition,
Microalgae are unicellular microorganisms, which can be used microalgae have been used for wastewater treatment because they
as feedstock for producing biofuel or other value-added products can capture carbon and nutrition from wastewater and also remove
(Choksi et al., 2015; Hu et al., 2008; Shah et al., 2016) such as CO2 from flue gas (Christenson and Sims, 2011; Olaizola, 2003).
docosahexaenoic and eicosapentaenoic (Grima et al., 2003), omega- That is why microalgae are very useful for food, energy, and
3 (Barclay et al., 1994), and protein (Hadiyanto et al., 2012). Most of wastewater treatment area.
them contains protein, lipid, inorganic elements, polysaccharides, One of microalgae is Haematococcus pluvialis. These microalgae
and also pigments such as chlorophyll, xanthophyll, zeaxanthin, have four cycles of life; vegetative cell growth, encystment, matu-
canthaxanthin, astaxanthin, and b-carotene. ration, and germination (Kobayashi et al., 1997), but generally
In food or pharmaceutical area, microalgae pigments, vitamin, consist only of vegetative (green stage) and maturation (red stage)
and functional bioactive compound are collected as functional food (Park et al., 2014). H. pluvialis has a valuable pigment which can be
or drugs because most of them has antioxidant properties, which is used as an antioxidant, called astaxanthin (3,30 -dihydroxy-b-caro-
needed by living organisms, especially human and animals tene-4,40 -dione) (Lorenz and Cysewski, 2000; Ranga Rao et al.,
(Hadiyanto et al., 2013). Microalgae also have been used as biofuel 2010). For human body, astaxanthin is 54 times more powerful
than b-carotene, 65 times more powerful than vitamin C, 100 times
more effective than tocopherol (Borowitzka, 2013; Koller et al.,
* Corresponding author. rez-Lopez et al., 2014), and good for eye
2014; Miki, 1991; Pe
** Corresponding author.
E-mail addresses: marcelinus@seoultech.ac.kr (M. Christwardana), hadiyanto@
health, central nervous system, immune system, anti-aging, and
live.undip.ac.id (H. Hadiyanto). fertility. Astaxanthin is not only produced by H. pluvialis, but also by
Peer review under responsibility of Institut Pertanian Bogor. yeast, salmon, trout, krill, shrimp, crayfish, and crustaceans

http://dx.doi.org/10.1016/j.hjb.2017.08.009
1978-3019/Copyright © 2017 Institut Pertanian Bogor. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
150 M. Christwardana, H. Hadiyanto

(Higuera-Ciapara et al., 2006; Ranga Rao et al., 2014). Astaxanthin is

resulted from the secretion process of H. pluvialis during maturation
stage, whereas chlorophyll was still produced in vegetative stage.
In previous research, effect of physico-stimulation such as
addition of audible sound in plant growth has been performed by
several researchers (Cai et al., 2014; Creath and Schwartz, 2004; Gu
et al., 2013; Hassanien et al., 2014; Hou and Mooneyham, 1999; Hou
et al., 2009). For microalgae experiment, Jiang et al. (2012) con-
ducted the audible sound effect experiment on chlorella and Cai
et al. (2016) conducted on Picochlorum oklahomensis. But, none
exploring the type of sound which was used and only focusing on
the frequency of sound.
Audible sound is very important in this experiment. Larsen and
Gilbert (2013) have been arranged the music called “microbial
bebop” from microbial deoxyribose nucleic acid (DNA) genes
sequence. That music consists of “Blues for Elle,” “Bloom,” “Far and
Wide,” and “Fifty Degrees North, Four Degrees West,” where each
music has different function if it is used for microbe. Other exper-
iment about arranging notation based on live creature's DNA genes
sequences had been performed by Sousa et al. (2005). Figure 1. Schematic of Haematococcus pluvialis cultivation set-up.
In this experiment, we used specific songs called “Blues for Elle”
10 AM to 6 PM. The power of sound was controlled by controller in
and “Far and Wide” as audible sound because that music was
music player. For control variable, culture with no addition of
created to stimulate photosynthesis, nutrient consumption, and
audible sound was set with the same parameter of temperature,
temperature resistance for microbe or microalgae. On the other
light, aeration rate, and pH.
hand, “Bloom” and “Fifty Degrees North, Four Degrees West” were
not used as audible sound because they were created especially for
cyanobacteria and pseudomonas, which is avoided in H. pluvialis 2.3. Biomass measurement
growth (Larsen and Gilbert, 2013). Microalgae growth rate would Most studies said that the maximum absorbance for microalgae
be determined by measuring optical density (OD) and manual cell was observed at 680 nm as the maximum peak of chlorophyll to
counting method using hemocytometer. estimate biomass concentration. Eq. 1 was followed to determine
microalgae growth rate. The OD was monitored using bench
spectrophotometer UV-1800 (Angstrom Advanced, Massachusetts,
2. Materials and Methods USA). Around 20 mL of culture was collected into the sample bottle
every day during growth period and used for growth rate and pH
2.1. Materials measurement. The pH of culture itself was checked by using pH
H. pluvialis strain (UTEX #2505) was used in this experiment meter Fisher Scientific S90525 (Fisher Scientific, Shanghai, China).
and was cultured in 1 L flask with the addition of optimal
bregas et al., 2000), which OD2  OD1
haematococcus medium (OHM) (Fa m¼ (1)
consists of (in g/L) KNO3 0.41, Na2HPO4 0.03, MgSO4.7H2O 0.246, t2  t1
CaCl2.2H2O 0.11, (in mg/L) Fe(III)citrate.H2O 2.62, CoCl2.6H2O 0.011,
where, m is microalgae growth rate, OD is at 680 nm, and t is time
CuSO4.5H2O 0.012, Cr2O3 0.075, MnCl2.4H2O 0.98, Na2MoO4.2H2O
(day).
0.12, SeO2 0.005, and (in mg/L) biotin 25, thiamine 17.5, and B12 15
The second method to measure growth rate was using hemo-
(in g/L), then placed in a chamber. After 3 days or if the cell con-
cytometer. In this method, 1 mL of culture was injected to hemo-
centration was 0.4
104 cells/mL of culture, that culture was
cytometer. H. pluvialis was observed by using microscope BA310
transferred into acrylic photobioreactor which contains 60 L
(Motic, Xiamen, China), total cells number in eight areas was
distilled water (include OHM). The temperature of environment
counted manually and then divided by eight. Then the resulted
was set to 25 C and checked every day using Infrared Thermometer
number was multiplied by 104. Eq. 2 was used to
NUB8380H (Nubee, Guangdong, China). Some cool white LED
measure microalgae growth rate.
lamps were placed beside photobioreactor which resulted in
photosynthetic photon flux of 20.3 mmol/m2/s. Culture was left for N2  N1
22 days with dark:light cycle was 12:12, and OHM would be m¼ (2)
t2  t1
injected into the culture every 2 days. Aeration with the rate of
100 mL/min was fed into the photobioreactor without any addi- where, m is microalgae growth rate, N is cells number (cells/mL),
tional CO2. and t is time (day).

2.2. Audible sound treatment

2.4. Kinetic model measurement
Previously, Cai et al. (2015) reported that 2000 Hz is the major
Profile of pseudoefirst-order kinetics of H. pluvialis biomass
frequency component in most audible sound in nature. Based on
during 22 days of cultivation following Eq. 3 (Rokhina et al., 2010).
that, music or audio with power 60 dB was set as audible sound and
average frequency of the audible sound was measured by using c0
Audacity software (Audacity Team, New York, USA). Audible sound ln ¼ kapp
t (3)
c
was played from music player HCB-811 (Hyundai, Seoul, South
Korea) and placed between the two photobioreactors as shown in where, C0 is the initial number of H. pluvialis cells, C is the final
Figure 1 (approximately 20 cm). The audible sound was continu- number of H. pluvialis cells, kapp is apparent rate constant (per day),
ously generated for microalgae growth, and played 8 h per day from and t is time (day).
 Audible sound effect on microalgae H. pluvialis growth 151

The slope of a linear plot of ln (C0/C) versus time gives the 3.2. Morphology of H. pluvialis
apparent increasing rate constant. Also, there is a correlation be- Morphology of H. pluvialis before and after the cultivation time
tween the reaction rate constant and the reaction half-life in the was investigated using digital microscope, and they are shown in
pseudoefirst-order reactions that can be expressed as following Eq. Figure 3. From that figure, the color of H. pluvialis in 0 day was light
4 (Rokhina et al, 2010). green, which represented the chlorophyll inside the cells
(Figure 3A). Another thing is young H. pluvialis did not have layered
ln 2 cell wall, which made that cell easy to burst. In after green stage
t1 ¼ (4)
2 kapp cultivation, the layered cell wall has been made and the cell wall of
H. pluvialis become thick. That thick layered cell wall was used to
where, t1 is half-life reaction and kapp is apparent rate constant (per protect microalgae from burst by environment during astaxanthin
2
day). accumulation (Montsant et al., 2001). At this time, the color of
The novel catalytic parameter (Ksurf) was reported by Sakkas H. pluvialis was reddish green, where red color represented the
et al. (2004), which describes the participation of the surface area starting accumulation of astaxanthin (Figure 3B). It means, that
in the process. Ksurf is derived by the partition of the apparent ki- culture was ready to transfer to the next stage (maturation stage).
netic rate, estimated for the studied process with the value of the
surface area of the catalyst. As audible sound achieves the micro-
3.3. Correlation between OD and number of cells
algae by propagating through bioreactor wall, we can say that the
It is meaningful to measure the accurate microalgae cell. For
surface area of the bioreactor wall is the surface area of physico-
that, we correlated between OD and manual cell counting as a
stimulant following Eq. 5.
method to determine the number of microalgae cell in large scale
kapp application. Figure 4 shows the correlation between OD at 680 nm
Ksurf ¼ (5) and manual cells counting using hemocytometer with the value is
S
y ¼ 4.858
or 1 OD ¼ 4.858
104 cells/mL culture.
where, Ksurf is catalytic surface, kapp is apparent rate constant (per
day), and S is surface area of the catalyst (m2/g) 3.4. Effect of different frequency on the growth rate of H.
pluvialis
It is very important to determine the growth rate of H. pluvialis.
3. Results For doing that, we tried to measure the growth rate by using the
first method e correlation between OD and cultivation time. From
3.1. Characterization of audible sound Figure 5, the following two things can be noticed: (1) the culture
It is important to characterize the audible sound before used as which was treated by “Blues for Elle” had the highest growth rate
physico-stimulant for enhancing the growth rate of H. pluvialis. For than other variable with the value 0.03 per day (growth rate values
that, frequency at 60 dB and octave analysis of background sound for treatment by “Far and Wide” and control were 0.015 and 0.011
from different audible sound “Blues for Elle” and “Far and Wide” per day, respectively) and (2) the OD680 of H. pluvialis which was
were measured. For doing that, we measured the average fre- treated by “Blues for Elle” had average value of 0.158 or 52% higher
quency, frequency level pattern, and octave analysis of background than control, whereas H. pluvialis which was treated by “Far and
sound of two kinds of audible sound. From the result as shown in Wide” only increase up to 26%.
Figure 2A, “Far and Wide” had octave analysis of background sound
more stable than “Blues for Elle,” although both was set at sound 3.5. Effect of different frequency on biomass productivity of
level 60 dB. While the pattern of frequency level from “Far and H. pluvialis
Wide” and “Blues for Elle” is shown in Figure 2B. From that figure, The other important thing to be considered is H. pluvialis
intensity of frequency level from “Far and Wide” is also more stable biomass productivity measurement. There are some factors which
than “Blues for Elle,” compatible with phenomena in Figure 2A, can affect the productivity, such as lag phase time, growth rate, and
where the average frequency of “Far and Wide” and “Blues for Elle” average OD at stationary phase as shown in Table 1. From Table 1,
audible sound are 0.24 and 0.28 kHz, respectively. we can see that the lag phase time of control (no additional sound)

Figure 2. (A) Octave analysis of background sound and (B) level of frequency intensity from “Far and Wide” and “Blues for Elle” audible sound.
152 M. Christwardana, H. Hadiyanto

Figure 3. Microscopic images of Haematococcus pluvialis at (A) day 0 and (B) day 22.

Table 1. Lag phase time, growth rate, average maximal OD, and biomass produc-
tivity of Haematococcus pluvialis

Audible sound Lag Growth Average Biomass

time rate max OD productivity
(day) (per day) (
102 cells/mL/day)

Control (no music) 5 0.011 0.104 2.297

Blues for Elle 3 0.030 0.158 3.467
Far and Wide 5 0.015 0.125 2.760

OD ¼ optical density.

Average maximal OD for control, treated by “Blues for Elle” and

“Far and Wide” are 0.104, 0.158, and 0.125 or 5.052, 7.627, and
6.073
103 cells/mL. Maximal achieved OD was obtained from
average OD at stationary phase from day 12 to day 22. The highest
average maximal OD was culture which was treated by “Blues for
Elle,” compatible with the result in Figure 5. Productivity was ob-
tained by following Eq. 6.
Figure 4. Calibration curve between absorbance (OD680) and microalgae cells number.

X
P¼ (6)
t

where,

 P is productivity (cells/mL/day)
 X is average cells number at stationary phase (cells/mL)
 t is total cultivation time (day).

Regarding microalgae productivity, from that equation, the

highest productivity was culture which was treated by “Blues for
Elle” is 3.467
102 cells/mL/day with total cultivation time was 22
days. Productivity of control (without audible sound) and culture
which was treated by “Far and Wide” are 2.297 and 2.76
102 cells/
mL/day, respectively. We can say that addition of “Blues for Elle”
audible sound is very effective to increase productivity up to
50.94%, which is higher compared with “Far and Wide” as only
increased up to 20.16%.

Figure 5. Growth of Haematococcus pluvialis in different types of audible sound based 3.6. pH Phenomena during H. pluvialis cultivation
on their optical density. It is important to know the pH behavior related with audible
sound treatment in H. pluvialis. From Figure 6, initially, pH was set
and culture which was treated by “Far and Wide” are 5 days from in 6.5e6.8, then it gradually decreased. The decreasing pH during
day 7 to day 12 with growth rate 0.011 and 0.015 per day, respec- cultivation was due to releasing proton into medium during
tively, whereas culture which was treated by the music “Blues for photosynthesis process. Because this cultivation is heterotrophic
Elle” has lag phase time only 3 days from day 9 to day 12 with activity, illumination, aeration, and nutrient would affect the
growth rate 0.03 per day. microalgae growth rate.
 Audible sound effect on microalgae H. pluvialis growth 153

“Blues for Elle” has kapp value 0.078 per day or 1.63 times higher than
control (0.048 per day), whereas H. pluvialis which was treated with
“Far and Wide” had kapp value 0.061 per day or 1.27 times higher
than control. H. pluvialis which was treated with “Blues for Elle” also
has t1/2 value 8.89 day, 38.43% lower than control (the t1/2 value of
control and treated by “Far and Wide” are 14.44 and 11.36 days,
respectively). Whereas, Ksurf value of H. pluvialis which was treated
with “Blues for Elle” was 1.66
105/day/cm2, 1.63 times higher
than control (Ksurf value of H. pluvialis which was treated with “Far
and Wide” and control are 1.29 and 1.02
105/day/cm2,
respectively.

4. Discussion

In microbial especially microalgae, there are usually five stages

or phases in their growth. That phases consist of lag or induction
phase, log or exponential phase, declining relative growth phase,
stationary phase, and death phase. Dry biomass, manual cell
counting, and OD are usually used to determine the number of
Figure 6. pH of Haematococcus pluvialis culture during the cultivation of vegetative biomass in the culture. It is difficult to measure the accurate
phase. number of microalgae cells although using cell counting method by
hemocytometer. For that, OD is mostly used to represent the
number of cells inside culture. According to Banerjee et al. (1993),
the most accurate indirect method for microalgae cells counting
measurement was dry biomass, but its accuracy would decrease for
large scale application (Pirt, 1975). By correlating the OD at 680 nm
and cells number of microalgae, the accurate amount of biomass
can be achieved.
Notation of “Blues for Elle” which was mapped to temperature,
nutrient reactivity, and chlorophyll activity (photosynthetic) give
the big impact in H. pluvialis photosynthesis and growth because it
can increase the microalgae metabolism. In this case, “Far and
Wide” only affect the photosynthetic activity and temperature.
Beside that, the unstable frequency intensity level gives the impact
in growing and cell dividing processes. The genre of “Blues for Elles”
which has the unstable rhythm also plays an important role on this.
In this experiment, frequency of edible sound is lower than other
references, for instance, the experiment conducted by Jiang et al.
(2011) and Jiang et al. (2012) used audible sound with frequency
0.4 kHz to grow Chlorella pyrenoidosa.
The “Blues for Elle” audible sound decreases thermodynamic
Figure 7. Pseudoefirst-order of increasing biomass kinetic model.
phase transition, which illustrates the enhancement of the fluidity
of the cell wall and membrane of H. pluvialis than “Far and Wide”
Audible sound like “Blues for Elle” and “Far and Wide” had (Hassanien et al. 2014). It means, it will enhance the H. pluvialis cells
capability to stimulate photosynthesis process. During photosyn- to grow and divide faster and easily. Generally, in plant, cell division
thesis, microalgae took nutrient source from medium and released cycle consists of four phases: G1-phase, S-phase, G2-phase, and M-
higher proton. It made the decreasing pH of H. pluvialis, which was phase (Depamphilis, 2003). Sound waves increase the capacity of
treated by “Blues for Elle” and “Far and Wide” faster than control. indule-3-acetic acid metabolism and inhibit the abscisic
acid metabolism during cell division process (Lovelli et al. 2012;
3.7. Kinetic study of audible sound as physico-stimulant Zhang et al. 2006), which cause the H. pluvialis is difficult to stress.
In previous section, we observed the strong dependence be- In the microalgae cycle, longer lag phase time is meaningless if
tween the growth rate and the frequency of audible sound. An in- the growth rate is low. Capability of the music “Blues for Elle” can
crease in the growth rate of H. pluvialis with an increase in the stimulate the growth of microalgae by affecting amino acid and
biomass weight or absorbance was also reported. DNA sequences, relevant with our reference (Takahashi and Miller,
The Pseudo-first order kinetic model, apparent reaction rate 2007).
constants (kapp) and correlation coefficients (R) are shown in Figure 7 The electric potential from audible sound frequency made
and Table 2. From Table 2, H. pluvialis which was treated with H. pluvialis cell membranes could be changed by enhancing plas-
malemma and Adrenoleukodystrophy mRNA exists in the outer-
most part of cells which consists of membrane lipid and protein
Table 2. Relating kinetic parameters of increasing biomass of Haematococcus
pluvialis
(Sun and Xi, 1999; Jia et al. 2003). As the effect, lipid accumulation
inside H. pluvialis also occurred faster. The audible sound frequency
Audible sound kapp (per day) t1/2 (day) Ksurf (
105/day/cm2) R2
also improves the cell membrane deformability which would
Control (no music) 0.048 14.44 1.02 0.96 change under external force (Wang et al., 2001). It made that cells
Blues for Elle 0.078 8.89 1.66 0.87 have stronger resistance to the new environment and other in-
Far and Wide 0.061 11.36 1.29 0.87
hibitors such as protozoa or fungus.
154 M. Christwardana, H. Hadiyanto

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