Large-scale Culture of a Tropical Marine Microalga Chaetoceros calcitrans (Paulsen) Takano 1968 at Different Temperatures Using Annular Photobioreactors
Outdoor mass culture of microalgae in the tropical area is important to minimize its production cost. This study evaluates the growth of Chaetoceros calcitrans in 120 L annular photobioreactors at indoor temperature (Treatment I, 25±2°C) and outdoor tropical ambient temperature, (Treatment II, 30±6°C). Each treatment was done in duplicates. For both treatments, C. calcitrans was first grown in starter columns of 10 L capacity for a period of 7 days at 25±2°C. After 7 days, the 9 L culture was transferred to the annular photobioreactors and subsequently brought to a final volume of 100 L by adding 20 L fresh medium every 5 days. There was no significant difference (p>0.05) in the dry weight of microalgae grown in natural light and those grown indoor. The results suggest that C. calcitrans can be grown in outdoor conditions, hence, saving time and microalgae production cost for the larviculture industry.
Microalgae form a very important component of the live feed production system
in aquaculture hatcheries due to their high content of valuable biochemical
products such as antioxidants and polyunsaturated fatty acids (Natrah
et al., 2007; Goh et al., 2009, 2010).
Essential live feed organisms such as rotifers and copepods require microalgae
not only as feed items, but also for enrichment of their nutritive values (Farhadian
et al., 2008, 2009; Khatoon
et al., 2009; Banerjee et al., 2011;
Khatoon et al., 2012). Due to their important role
in providing a feed resource in aquaculture production, simple large scale microalgae
production is necessary for larviculture industry. In the past, most hatcheries
in Asia have been using plastic bags, tanks and open ponds for their algae culture.
Our preliminary studies indicate that the production of the microalgae in plastics
bags is about ten times lower than that propagated in photobioreactors. Efficient
production of microalgae in photobioreactors are now necessary not only due
to rapid development of aquaculture industry but also for other uses such as
production of biofuel (Dayananda et al., 2007;
Chisti and Yan, 2011; Demirbas,
2011), foods, pharmaceutical and nutraceutical products (Ghasemi
et al., 2007; Abd El-Baky et al., 2008).
With more than 90% (62,423,187 tonnes) of worlds aquaculture producers
located in Asia (FAO, 2010), a tremendous amount of food
is needed to sustain the growing demand of the aquaculture industry. Mass production
of microalgae economically without compromising the purity is urgently needed.
Most open ponds or tank grown microalgae are at a disadvantage with lower productivity
and prone to contaminations (Chisti, 2007; Chen
et al., 2009). In addition, outdoor temperatures can be a limiting
factor to the growth of microalgae (Mata et al.,
2010). Temperature also affects the growth rate of microalgae (Sriharan
et al., 1991; Chen et al., 2012; Fukao
et al., 2012). Another study conducted by Cho
et al. (2007) suggested that temperature has a more significant effect
on the maximum density of Nannochloris oculata as compared to salinity.
Weiss et al. (1985) reported that although the
optimal growth for Tetraselmis suecica was found to be at 25-27°C,
cultures were able to tolerate a wider range of temperature (15-32°C), when
nutrient supply was abundant. On the other hand, cell metabolism such as nitrate
metabolism in Thalassiosira pseudonana decreased when the alga was cultured
at suboptimal temperature (Berges et al., 2002).
Under hatchery conditions, environmental factors such as temperature and light
can undergo drastic fluctuations which may affect the quality of the microalgae
(Veron et al., 1996; Ak et
al., 2008). Thus, culture optimization of microalgae species by growing
them under ambient temperature is one important strategy for successful and
economical larviculture activity. This study was designed to elucidate the capacity
of an important live feed species, Chaetoceros calcitrans in 120 L capacity
annular photobioreactors under ambient conditions compared to the indoor temperature
under which it is normally cultured.
MATERIALS AND METHODS
Microalgae culture: Pure isolate of C. calcitrans (UPMC-A0010)
was obtained from Aquatic Animal Health Unit (AAHU), Faculty of Veterinary Medicine,
Universiti Putra Malaysia. Natural seawater (30 ppt) filtered through membrane
filter paper of 0.45 μm pore size (Sartorius, Germany) was used for preparation
of f-medium (Guillard and Ryther, 1962) to grow stock
and primary cultures of C. calcitrans. The pH level was adjusted to
7.8-8.2 before autoclaving at 121°C for 15 min.
Experimental design: Four annular photobioreactors (Series F and M-TI
50/40, Zittelli et al., 2006) previously disinfected
with sodium hypochlorite and rinsed using filtered tap water through 0.1 μm
filter bags, were used in the experiment. Two bioreactors were placed in a temperature
(25±2°C) controlled room (Treatment I) while another two were placed
under ambient temperature of 30±6°C outdoor under shade (Treatment
II). Each bioreactor was inoculated with C. calcitrans at the initial
cell concentrations of approximately 4.37x106 cells mL-1
(Treatment I) and 4.36x106 cells mL-1 (Treatment II) in
20 L culture media, using inocula prepared in starter columns. Thereafter, the
cultures were diluted with 20 L of fresh medium every 5 days (5th, 10th, 15th,
20th day) until a final volume of 100 L was reached. The cultures were illuminated
using two cool daylight fluorescent lamps (cool daylight lamps, OSRAM L58W/865,
Germany) set at 12L:12D cycle throughout the experimental period.
Inoculum: Prior to the start of the experiment, four starter columns (10 L capacity) were inoculated with C. calcitrans starting from 1.5 L and were scaled up to 3, 6 and 9 L every 2 days for 7 days. All cultures in the starter columns were grown at 25±2°C and were supplied with air filtered through 0.45 μm air filter (Sartorius, Germany). Industrial pure carbon dioxide was provided to the cultures to maintain the pH at 7.8-8.2 in the culture vessels. The cultures were illuminated using cool daylight fluorescent lamps (cool daylight lamps, OSRAM L58W/865, Germany) set at 12L:12D cycle.
Analysis of growth parameters: Samples were collected daily in the morning
(0830-0900) for dry weight measurements. Dry weight of the biomass was determined
by filtering a known volume of the cells through a precombusted Whatman GF/C
filter paper (105°C, 4 h, cooled and weighed to constant weight) and washed
with 0.5 M ammonium formate. The filter paper with biomass was then dried at
105°C for 4 h, cooled and weighed until constant dry weight (modified from
Coutteau, 1996). Specific growth rate, μ was estimated
using the following formula:
where, W0 is the dry weight at the beginning of the selected time interval (days); Wt is dry weight at the end of the selected time interval (days) and tt-t0 is the time interval (days) for the incubation time.
Statistical analysis: Collected data were analyzed using independent sample t-test. Significant differences were determined at 0.05 level of probability. Statistical analysis was done using SPSS 17.0 for Windows (SPSS Inc. Chicago. IL, USA).
This study illustrated that a diatom, Chaetoceros calcitrans can be grown at outdoor ambient temperature using photobioreactors, indicating that this species could tolerate the daily fluctuations of the local temperature. In fact, cultures grown in outdoor ambient temperature were not significantly different (p>0.05) from those grown indoor.
The average room temperature in Treatment I was 25±2°C while in
Treatment II the average room temperature was 30±6°C. The pH ranges
for Treatment I and II were 8.6-9.8 and 8.6-9.9, respectively (Table
1). Specific growth rates for Treatment I (0.06/day) and II (0.05/day) were
not significantly different (p>0.05).
||Growth of Chaetoceros calcitrans in 120 L annular photobioreactor
measured in terms of dry weight (g L-1), bars represent a 20
L dilution with medium (Mean±SE, n = 2)
||Ranges of pH of culture, means of culture room temperature
and specific growth rates of Chaetoceros calcitrans during the culture
|Values (Mean±SE) in a row with the same superscript
are not significantly different at p>0.05
|| Means of dry weight in Treatments I and II
|Value (Mean±SE, n = 2) in each column with same superscript
are not significantly different at p>0.05
Upon the first dilution, there was a sharp reduction in biomass in terms of
dry weight. Dry weight of C. calcitrans decreased after each dilution
(5th, 10th and 15th day) except for the dilution on day 20. The dilution at
day 5 reduced the algal concentration to lower than that of the initial concentration
(Fig. 1). However, the cultures were able to adapt to this
dilution and increased in cell concentration from day 7 onwards.
Generally, the outdoor cultures showed a better growth at the beginning of the study but after day 14, biomass production of indoor cultures surpassed the outdoor cultures. However, a similar trend of biomass production was observed in both indoor and outdoor cultures (Fig. 1). There was no significant difference (p>0.05) between the average dry weight measurements of Treatment I (0.29±0.02 g L-1) and Treatment II (0.28±0.01 g L-1) (Table 2). Maximum biomass achieved were (0.49 g L-1) and (0.39 g L-1) for indoor and outdoor cultures, respectively.
In the present study of C. calcitrans large scale culture, there were
no significant differences in the growth rates in Treatment I and Treatment
II, indicating that the microalgae could quickly adapt to the new temperature
without prior acclimatization (Fig. 1). Chaetoceros calcitrans
was originally grown indoor (25±2°C) and our results showed that
C. calcitrans was able to tolerate the high ambient temperature range
of 24-36°C. Renaud et al. (2002) demonstrated
that Chaetoceros sp. grew best at 30°C but also showed that tolerance
to high temperatures of 33 and 35°C with a moderate growth rate of 0.53/day.
This finding was further supported by Araujo and Garcia
(2005) and Banerjee et al. (2011) who reported
that C. calcitrans grow best at temperature ranging from 25-36°C.
The continuous growth of C. calcitrans after every dilution is an indication
that it was able to adapt to the sudden changes in environmental conditions
(Fig. 1). During the experiment, three growth phases were
observed. Lag phase of 7 days was observed while exponential phase was observed
thereafter. The cultures progressed into stationary phase after day 10 as the
growth rate was reduced. The sharp decrease after the first culture medium addition
could be due to the dilution while the cultures were still in adaptation stage
and the cell density was still low. Nevertheless, cultures in both treatments
managed to adapt to their respective culture environments. Probably, the addition
of fresh culture medium to the bioreactor provided adequate nutrients to the
microalgae cells (Fig. 1).
In the natural environment, the effect of temperature is not only shown physically
by changes in growth but also at cell metabolism level such as photosynthesis,
carbon and nitrogen uptake (Anning et al., 2001;
Berges et al., 2002; Araujo
and Garcia, 2005). Anning et al. (2001) demonstrated
that light harvesting pigments increased in abundance with increasing temperature
and suggested that at low temperatures, carbon fixation mediated by photosynthetic
enzymes is affected. Meanwhile, nitrogen uptake in Thalassiosira pseudonana
was shown to decrease with increasing temperatures suggesting that nitrate metabolism
in this diatom is mediated through enzymatic activities (Berges
et al., 2002). In fact, other factors could also modulate the effects
of temperature on microalgae growth. For instance, Chinnasamy
et al. (2009) reported that high temperature (50°C) reduced the
growth of Chlorella vulgaris cultures when 6% of CO2 was provided
to the cultures and those grown at ambient CO2 levels showed no growth.
Addition of carbon dioxide is also reported to alter the biochemical composition
of microalgae through a higher production of protein Araujo
and Garcia (2005) while extending the exponential phase of algal culture
(Fabregas et al., 2001).
In the present study, there was no significant difference (p>0.05) in the
biomass production of cultures maintained at temperature 25±2°C (treatment
I) and ambient temperature of 30±6°C (treatment II). Average specific
growth rate was 0.06/day and 0.05/day for treatment I and II, respectively.
McGinnis et al. (1997) demonstrated that Chaetoceros
muelleri achieved optimum growth rate at 30°C with 4.0 doublings/day
as compared to those cultured at 20°C while Banerjee
et al. (2011) also reported that C. calcitrans grew better
in outdoor conditions as compared to laboratory cultures in 1 L culture medium.
However, cultures in the previous studies were not grown in a large volume as
in the present study where 120 L photobioreactors were used. Apart from that,
a previous study by Banerjee et al. (2011) also
pointed out that the cultures were acclimatized to the study conditions, whereas
in the present study, inoculums were grown indoor before the study period. Thus,
further studies on the acclimatization of microalgae to outdoor conditions and
biomass production in annular photobioreactors should be carried out to elucidate
the effects of temperature on microalgae.
Culturing microalgae in outdoor environment can achieve higher yields, leading
to a reduction of operating cost compared to cultures cultivated indoors (Lopez-Elias
et al., 2005). In the present study, biomass production in terms
of dry weight in treatment II at the end of the study (25 days, 0.35 g L-1)
was similar to the biomass achieved during the first 10 days of the experiment
(0.36 g L-1). It can be suggested that microalgae cultured in treatment
II conditions can be harvested earlier. The biomass obtained in the present
study was lower than the previous study in 1 L (Banerjee
et al., 2011), probably due to the fact that the 20 L dilution was
done every 5th day.
Chaetoceros calcitrans can be mass cultivated in the outdoor tropical ambient temperatures. This study suggested that cultivation of C. calcitrans under ambient temperature conditions using photobioreactors were able to produce higher biomass in a shorter period of time. In fact, there was no significant difference between the outdoor and indoor mass culture (p>0.05). Further studies are needed to establish standard protocol for large scale production of microalgae in photobioreactors.
We would like to thank Dr. Hazel Monica Matias-Peralta for isolating the C. calcitrans used in this study. We would also like to thank Dr. Sanjoy Banerjee, Dr. Helena Khatoon and Mr. Perumal Kuppan for their technical assistance. This study was funded by Johor Satellite Biotechnology Project grant No. BSP(J)/BTK001(4).
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