INTRODUCTION
Microalgae produce many useful and beneficial compounds for the sustainability
of the ecosystem. Microalgae biomass is widely used as human food and aquaculture
feed. Microalgae biotechnology is increasingly recognized as one of the emerging
fields in the agriculture and aquaculture industries. In addition, microalgae
culture could also potentially provide a way of reducing the amount of accumulated
carbon dioxide in the atmosphere (Demirbas, 2011a;
Demirbas, 2010; Suali and Sarbatly,
2012). Recently, there has been a concerted interest in using microalgae
as an innovative and environmentally friendly source for production of oils
for renewable energy and pharmaceutical purposes (Demirbas,
2011b; Ilavarasi et al., 2011). Due to their
capability to produce structurally complex compounds, microalgae could potentially
serve as an important natural source of bioactive molecules (Ming
et al., 2012).
Mass cultivation of microalgae required the utilization of photobioreactors
for completely-controlled cultivation and large scale production. Until today,
a variety of photobioreactors have been proposed and developed for mass microalgae
cultivation (Richmond, 2008). However, these are either
too complex or too costly to be applied in large-scale production. Hence, the
utilization of photobioreactors are considered as capital-intensive approaches
due to the high development cost and they would only justified when a fine and
valuable chemical is to be produced (Brennan and Owende,
2010; De La Noue and De Pauw, 1988). However, the
rapidly growing aquaculture industry had created a growing demand for microalgae
as live feed source for larval feeding (Richmond, 2008).
Hence, the challenge faced worldwide is to reduce the construction costs of
photobioreactor systems to make them more economically competitive and viable
for adaptation for the mass production. There are several factors contributing
to the low productivity of microalgae. Some of them are photo-inhibition in
the intensely illuminated outer zones of photobioreactor, inefficient conversion
of the available light into the cell biomass, build-up of excess inhibitory
oxygen, depletion of biomass due to respiration in the photobioreactor’s
dark zones and inadequate mixing of CO2 and nutrients (Degen
et al., 2001; Sato et al., 2010).
Burlew (1953) stated that via the intermittent exposure,
photoautotrophic cells such as microalgae will have an improved utilization
of strong illumination. This approach could be adapted to increase the conversion
of light energy into biomass by allowing the microalgae cell to be repeatedly
exposed to the condition of strong light and darkness. This type of energy conversion
enhancement is known as the light-dark cycle or FLE (Kok,
1956; Terry, 1986).
Several designs of photobioreactors have been reported to attempt the application
of FLE for microalgae productivity enhancement (Degen et
al., 2001; Sato et al., 2010; Xu
et al., 2002; Yoshimoto et al., 2005).
However, a regular mixing pattern has not generally been implemented. Due to
that, this study had focus on a new photobioreactor that enhances productivity
by effectively utilizing the flashing-light effect and regular mixing channel.
This study emphasized on the engineering of a simple, low-cost, yet efficient
airlift photobioreactor. Cylinder body will be used to provide wide illumination
surface area and a draft tube to induce a regular light cycling and flow of
microalgae. Commonly, there are two basic approaches for exposing the cells
to intermittent illumination: the first method entails the use of a light source
or a system which provides illumination intermittently (Richmond,
2008). The application of flashing light source may only be acceptable for
low microalgae cell densities, in which mutual shading is essentially absent,
thus it is being useless for mass cultures and whenever high productivity is
expected. The second probability is the only practical by using a continuous
light source whether in the lab or outdoors and has the cells move at a high
frequency, in and out of the illuminated volume. The second approach was selected
to be utilized in this study.
This study was performed based on the objectives to determine the effect of
FLE on the growth performance of Chlorella sp. and the optimum aeration
flow rate to induce FLE for the maximum growth yield of Chlorella sp.
FLE system was design to provide two types of illumination areas: the dark region
and illuminated region. The illuminated cells (at the illuminated region), which
are replaced by dark cells, were shifted to the dark volume while these former
dark cells are in their turn, illuminated. In this manner, more microalgae cells
such as in dense cultures are exposed to light flashes per unit of time. Strong
light which is higher by an order of magnitude than saturating light, is in
effect diluted by being available in smaller doses to more microalgae cells
along a given time span, thus being used more efficiently as compared to the
light utilization of microalgae cells illuminated continuously in low density
or poorly stirred cultures.
MATERIALS AND METHODS
Cultivation of Chlorella sp.: Chlorella sp. was obtained
from stock culture collection in Live Feed Culture Laboratory of Institute of
Tropical Aquaculture, UMT. Chlorella sp. was obtained in the form 10
mL monoculture which then up-scaled and maintained in 1000 mL bottle. Conway
medium based on Tompkins et al. (1995) were used
as growth nutrient were added into 10 mL of inoculum and it was kept under illumination
in an air-conditioned room at temperature of 22±2°C. Cell count was
performed using a Haemocytometer technique coupled with light absorption at
678 nm via Shimadzu UV1800 Dual-Beam Spectrophotometer.
Specification of the airlift photobioreactor: The photobioreactor is
consists of three major parts: outer tube, draft tube and air duct. The outer
tube will provide an illumination surface area of 8.67x10-2 m2.
A concentric draft tube also called riser was located coaxially within the outer
tube. Four opening holes with the total area of 6x10-4 m2
were constructed in order to allow liquid circulate through the riser and down-comer.
By joining the circular cover, an air duct was located coaxially inside the
draft tube. The photobioreactor has working volume of 4x10-3 m3;
outer tube internal diameter 0.13 m; outer tube height 0.34 m; draft tube internal
diameter 9.2x10-1 m; draft tube height 0.37 m; air duct internal
diameter 5x10-3 m.
Design of the bioreactor was based on the study of (Xu
et al., 2002). Modification was done by introducing opaque draft
tube as replacement to transparent tube in order to create FLE (Fig.
1). In this study, three illumination conditions were introduced: intermittence,
continuous illumination and unlighted condition. Each illumination variables
was performed in triplicates. Both microalgae growth performance and aeration
flow rate of each replicates were recorded and analysed. Bright culture condition
(positive control) was established by removing the draft tube from the PBR system.
Dark culture condition (negative control) established with the same manner by
wrapping aluminium foils on the overall external structure of the PBR system.
|
| Fig. 1: |
Schematic diagram of the operating condition of the PBR system
under FLE condition (left), continuously illuminated condition (middle)
and unlighted condition (right). For FLE-PBR, culture will move in designated
speed alternatively to the dark region and the illuminated area during its
operation. Continuously illuminated PBR had no opaque draught tube which
exposed microalgae culture to illumination whereas unlighted condition PBR
had its external cylinder body coated with opaque black paint |
Aeration flow rate variables: Aeration flow rate was regulated by varying
the flow rate: low, intermediate and high. Low flow rate was established by
observing the lowest possible aeration flow rate required to allow a complete
liquid circulation from inside draught tube to the illumination area. On the
contrary, high flow rate was introduced by completely opening the aeration valve
allowing as maximum aeration as possible into the PBR system. Intermediate flow
rate was done by regulating the aeration at mid-point valve opening between
the low and high flow rate specified.
Volumetric flow rate measurement based on Recktenwald (2006)
was used to quantify each specified aeration flow rate. Ten replicates were
carried out for each flow rate before inoculation of microalgae performed. Regular
monitor of the aeration flow rate was performed to ensure uniform flow rate
throughout the culture period. As shown in Fig. 2, the low,
intermediate and high flow rates was predetermined at 16.94, 33.14 and 49.28
mL sec-1, respectively prior to inoculation of microalgae biomass
into each photobioreactor.
Liquid circulation velocity: A tracer method was employed for the measurement
of liquid circulation velocity in the airlift reactor (Chisti,
1989). A small amount of concentrated H2SO4 was added
to the reactor to adjust pH to about 3 and the reactor was bubbled with air
(superficial gas velocity in the riser, UGR ~ 40 mm sec-1)
for about 2 h.
|
| Fig. 2: |
Calculated aeration flow rate and its respective light exposure
period at different flow rates |
When the liquid showed no buffering over the pH range (pH = 4) of the measurements
and 200 mL of 8 M H2SO4 was poured instantaneously into
the top of the down-comer.
The linear liquid velocity (VLd) in the down-comer was determined
by dividing the vertical distance (0.3 m) between the tracer peaks of the two
pH electrodes with the corresponding time taken according to Miron
et al. (1999). VLd is represented the superficial velocity
(ULr) in the riser based in the continuity relationship by Chisti
(1989): ULrAr = VLrAr (1-εr)
= (VLdAd (1 – εd) = ULdAd,
where VLr is the linear liquid velocity in the down-comer, Ar
and Ad are cross-sectional area of the riser and the down-comer,
respectively. The mean circulation time (tc) in the airlift reactor
was defined by the sum of the residence time in the riser (tr) and
in the down-comer (td) and computed using analytical relationships
(Chisti, 1989): tc = tr+td
= Lr/ULr+Ld/ULd, where Lr
and Ld are the length of the riser and the down-comer, respectively.
As shown in Fig. 2, light exposure period per liquid circulation
regarding three predetermined aeration period is 3.99 sec for low, 1.71 sec
for intermediate and 1.1 sec for high aeration flow rate.
Microalgae growth parameter: Sampling of biomass was performed daily
until microalgae reached their death phase for about 12-14 days. Aliquot of
2 mL was taken and placed on haemocytometer for cell density determination under
light microscope. Observation was performed using National Optical (tm) microscope
coupled with MOTIC DMC-300 VGA camera for digital visualization. Absorbance
of biomass was determined at 678 nm as reference on calculated cell density.
A determination of cell density was computed using Microsoft Office ExcelTM
in order to form growth curve.
Numerical and graphical methods were employed to determine the significance
between both variables, i.e., flow rate and illumination condition. Maximum
cell production and net cell production throughout 14-days cultivation period
was performed using area under curve analysis by integrating the polynomial
equation of the growth curve. One-way ANOVA with 95% confidence interval was
utilized for comparison on the growth performance using Minitab 16TM.
RESULTS
Batch trial and establishment of photo-inhibition condition: Microalgae
cell density was recorded to form mean growth evolution curve and the data was
taken from six replications performed under photo-inhibition condition. Figure
3 shows growth evolution curve depicting a normal growth phase compared
to growth evolution under photo-inhibition condition. Cell growth under photo-inhibition
had suppressed growth, which was lower order exponential growth phase as compared
to cell growth under normal illumination. Illumination was evaluated for the
capacity to induce photo-inhibition in the microalgae population. Photo-inhibition
determination was done to ensure supplied illumination was at least at an order
higher than the light saturation value. Illumination capable of inducing photo-inhibition
was important in order to simulate the effect of FLE (Burlew,
1953; Kok, 1956; Richmond, 2008;
Terry, 1986; Yoshimoto et al.,
2005).
|
| Fig. 3: |
Microalgae growth evolutions under saturating illumination
as compared to normal illumination throughout 14 days treatment period |
Culturing condition under normal illumination which was light below saturation
point was performed in order to compare microalgae growth evolution to the illumination
and growth under photo-inhibition.
Microalgae growth performance under designated FLE-Photobioreactor:
Effects of FLE were measured at three different flow rate conditions; low, intermediate
and high flow rate. As shown in Fig. 4, mean growth evolution
of FLE, continuously illuminated culture condition and unlighted culture condition
throughout 14 days period were plotted. At low flow rate, continuously illuminated
culture condition and FLE growth performance in terms of maximum cell density,
maximum cell production and net cell production was not significantly different
from each other. FLE had lower order of growth at exponential phase and short
sustain at the stationary phase compared to bright culture condition. In this
condition, growth performance of microalgae at continuous illumination culture
condition was better than FLE culture condition. Low aeration flow rate caused
low liquid circulation velocity. At low liquid circulation velocity, alternating
frequency of light flash would also become lower. However, lower frequency of
light flash inversely proportional to light exposure period. At low flow rate,
light exposure period recorded was 3.99±0.037 sec. But, higher light
exposure period per liquid circulation contributed to higher dark period since
each cycle divided into two region only-light exposure and dark period.
At intermediate flow rate, differences between continuous illumination and
FLE culture condition could be observed at exponential and stationary phase
of microalgae growth. FLE had higher order of exponential growth and higher
maximal cell density reading as compared to continuous illumination culture
condition.
|
| Fig. 4: |
Microalgae growth evolutions at three illumination conditions-FLE,
bright condition and dark condition at low flow rate (top), intermediate
flow rate (middle) and high flow rate (bottom) throughout 14 days treatment
period |
No clear observable differences recorded at the death phase of both culture
conditions. Under intermediate flow rate, mean light exposure period recorded
was 2.02±0.19 sec. Microalgae culture was alternating between the illuminated
and dark condition at higher velocity thus reducing the dark period. This would
contribute to lower respiration and triacylglycerol metabolism for cell division.
However, the reduction would be at the right balance-not too low and not too
high-that metabolized triacylglycerol in the dark has been compensated with
its synthesis at the illuminated area.
Lastly, for the high flow rate, no clear differences observed on the pattern
of growth evolution between FLE and bright culture condition throughout the
culturing period. At high flow rate, microalgae culture was subjected to high
Reynolds which is the degree of vigorous mixing allowing sufficient contact
between microalgae cells and nutrient molecules inside the media. High flow
rate would also contribute to higher liquid circulation velocity which caused
the culture to pass through illuminated area in short time. Recorded light exposure
period for this flow rate was 1.1±0.22 sec.
Maximum cell density, cell production and treatment period between culture
conditions: Maximum cell density is defined as the given concentration of
microalgae biomass at certain time whereas maximum cell production is the cumulative
number of microalgae biomass concentration from inoculation to the day when
maximum cell production is achieved. In addition, net cell production is the
total cumulative number of microalgae biomass concentration from inoculation
to the 14 days period. As shown in Table 1, FLE had the highest
maximum cell density as compared to other culture conditions. However, FLE and
continuously illuminated culture at both the intermediate and high flow rate
still not significantly different at 95% confidence interval. In the contrary,
maximum cell production for FLE was significantly higher as compared to other
illumination conditions.
DISCUSSION
The study for batch and establishment of photo-inhibition focused on the growth
rate of cells and conducted in two conditions; photo-inhibition and normal illumination.
Under the condition of photo-inhibition, microalgae growth especially at the
exponential phase or log phase was suppressed as compared to the growth under
normal illumination. Under normal lighting, the supplied amount of illumination
was either at the same level or lesser than those could be consumed by the photon
reaction centre. According to Degen et al. (2001),
Chlorella sp. has the ability to absorb more photon than what it required
and this protect the cell from photo-damage. Richmond (2008)
supported that the species of plants would exhibit the same growth phase, whether
they are from lower or higher type, under optimum range of illumination, nutrition
and temperature and ambient water condition. Velikova et
al. (2000) showed that photo-inhibition has been related to PS II part
of photosynthetic electron transport chain. Strong illumination could at some
level contributed to the decrease in photosynthetic activity. Once absorbed
light energy reached reaction centre which exceeds its consumption, the photosynthetic
apparatus can be injured (Demmig-Adams and Adams, 1996;
Ruban et al., 1994). Under condition of normal
illumination, all of the growth phases were clearly depicted on the plotted
growth evolution curve.
Microalgae growth performance under designated FLE was measured at three different
flow rate conditions. For the low flow rate study, Degen
et al. (2001) reported consumption of biomass through respiration
happen in the dark zone of bioreactor. This is supported by Sukenik
et al. (1989) stating that dark condition plays role in photosynthesis
and growth since triacylglycerol is metabolized rapidly only in the dark.
| Table 1: |
Microalgae growth parameters at different flow rates and illumination
conditions. Statistically different means within each category with 95%
confidence interval is grouped in subsets represented with superscript |
 |
Long period of darkness inside FLE culture condition under this particular
flow rate would contribute to the loss of microalgae biomass thus suppressing
its growth especially at the exponential phase. As compared to intermediate
and high flow rate, both FLE and continuous illumination culture condition at
low flow rate had very low maximal cell density recorded at the stationary phase.
So, it could be presumed that supplied flow rate was too slow to supply optimal
mixing in addition to the long dark photoperiod. According to Richmond
(2008), sufficient mixing rate, quantify by the Reynolds number, at certain
minimum level is required in order to allow optimum cell growth. Flow rate that
was too slow would also not sufficient to prevent the formation of biofilm which
could be observed at almost PBR of this particular flow rate.
Under intermediated flow rate, growth performance under FLE culture condition
was observed to be better as compared to bright culture condition. Maximal cell
density yield and exponential growth order of FLE was observed to be higher
than the bright culture condition. Illumination used for the culturing condition
was considered strong light since it contributed to the photo-inhibition of
batch culture test. In order to utilize strong light efficiently for growth,
intermittent manner of illumination is required (Kok, 1956;
Sato et al., 2010; Sukenik
et al. 1989). Allowing culture media to flow between dark and illuminated
area would contribute to the effect of light dilution and also provide adequate
region for microalgae cell division. Lastly, under high flow rate condition,
shorter light exposure period leads to higher alternating frequency. At this
degree, the alternating frequency was very high until it reached the level where
dark photoperiod almost negligible approaching the characteristics of bright
culture condition. Thus, this explained why the difference between FLE culture
condition and bright culture condition was insignificant.
Taking FLE-PBR as constant variable in term of physical aspects in bioreactor-mixing
pattern, volume, growth media and illumination area-the effect between varieties
of flow rate introduced could be observed. One-way ANOVA was selected to test
the effect of different flow rate on the growth of microalgae inside the FLE-PBR
system. Significance within replicates between different flow rates had been
omitted to take into consideration the possibility of natural variation. This
comparison would depict the best aeration flow rate within and limited to the
engineered PBR system itself.
CONCLUSION
Effects of flashing light on the growth of Chlorella sp. in a bioreactor
system was elucidated successfully in this study. Intermediate flow rate of
25.07 mL sec-1 has been determined as the most optimum flow rate
as compared to the other three flow rates. Low flow rate was insufficient to
induce optimum Reynolds number for mixing and agitation of available nutrient
and microalgae biomass. Increase of flow rate did not necessarily contribute
to the increase in microalgae growth since it started to approach the characteristics
of continuous illumination at high level of liquid circulation velocity. Maximal
cell density of 3.113x106 cells mL-1 was recorded at the
intermediate flow rate which is higher than both low and high aeration flow
rate.
ACKNOWLEDGMENT
The authors would like to thank the laboratory staff of Institute of Tropical
Aquaculture at Universiti Malaysia Terengganu for their assistance during this
study. This project was financially funded by the Institute of Tropical Aquaculture’s
Master in Science Course work Structure Programme.
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