Short Communication
Characterization of the thermal-tolerant mutants of Chlorella sp. with high
growth rate and application in outdoor photobioreactor cultivation
Seow-Chin Ong, Chien-Ya Kao, Sheng-Yi Chiu, Ming-Ta Tsai, Chih-Sheng Lin
*Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan
a r t i c l e
i n f o
Article history: Received 17 June 2009
Received in revised form 6 October 2009 Accepted 7 October 2009
Available online 7 November 2009 Keywords: Carbon dioxide Chlorella sp. Growth Microalgae Thermal-tolerant mutant
a b s t r a c t
In this study, two thermal-tolerant mutants of Chlorella sp. MT-7 and MT-15, were isolated. In indoor cul-tivation, specific growth rate (l, d1) of the mutants were 1.4 to 1.8-fold at 25 °C and 3.3 to 6.7-fold at 40 °C higher than those of wild type. The carbon dioxide fixation rate of both microalgal mutants was also significantly higher than that of wild type. In outdoor closed cultivation, where the temperature of cul-ture broth was 41 ± 1 °C, the lof mutant strain MT-15 was 0.238 d1during an 8-day cultivation. Whereas, the growth of wild type was inhibited in the outdoor cultivation. Our results show that the iso-lated microalgal strains are adaptable to be applied in outdoor cultivation in subtropical zones.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Global warming, which has been a concern in world-wide, is due to the increasing carbon dioxide (CO2) level in atmosphere.
The global atmospheric concentration of CO2has increased from
a pre-industrial value of about 280–379 ppm in 2005. Microalgae have very efficient photosynthesis, grow faster than other plants and are able to convert CO2to biomass efficiently. In recent years,
biological CO2 fixation using microalgal photosynthesis has
emerged as a potential option because of its effectiveness and eco-nomical in CO2reduction. Among the microalgae which were
stud-ied in CO2fixation, Chlorella sp. has been used in many studies and
shows high CO2fixation rate (Cheng et al., 2006; Chiu et al., 2009b; de Morais and Costa, 2007).
During outdoor cultivation with solar as the light source, bio-mass productivity is strongly affected by environmental factors such as irradiation and temperature (Ugwu et al., 2007). The tem-perature of microalgal culture broth in photobioreactors can in-crease to about 40 °C by irradiation of sunlight in subtropical zones. The microalgal growth would be highly inhibited at such high temperature if the cultivation is not provided with cooling system. Thermal-tolerant species could grow well under high tem-perature and would significantly reduce the cooling costs (Ono and Cuello, 2007).
Many thermal-tolerant microalgal strains have been isolated from hot springs (Hsueh et al., 2007; de Bashan et al., 2008). How-ever, it is time consuming to purify the cultures from other micro-organisms. In the present study, two thermal-tolerant mutant strains of Chlorella sp. were isolated by mutagenic chemical treat-ment. The growth pattern, CO2fixation rate and lipid content of the
microalgae were determined for characterizations of the isolated Chlorella sp. mutant strains.
2. Methods
The microalga (wild type) Chlorella sp. was obtained from Taiwan Fisheries Research Institute, Tung-Kang, Taiwan. The microalga was cultured in artificial sea water in each batch culture with the medium which has the composition (per liter) of 750 mg NaNO3, 44.11 mg NaH2PO4H2O, 43.6 mg Na2EDTA, 31.6 mg
FeCl36H2O and micronutrients (trace elemental solution)
includ-ing 1.8 mg MnCl24H2O, 0.1 mg CoCl26H2O, 0.1 mg CuSO45H2O,
0.23 mg ZnSO47H2O, 0.06 mg Na2MoO4, 1 mg vitamin B1, 5
l
gvitamin B12and 5
l
g biotin (Chiu et al., 2009b).The wild type cells were mutagenized following the method de-scribed by Chaturvedi et al. (Chaturvedi and Fujita, 2006) with some modifications. About 1 107cells of Chlorella sp. were treated with
100 mM ethyl methane sulfonate (EMS) for 1 h, and each approxi-mate 1 103cells were plated on agar plates and were incubated
at 40 °C. The bigger colonies were selected and cultivated in indoor vertical bubble column photobioreactor (cultured volume is 4 L;
Chiu et al., 2009b) subsequently. The growth rate of the mutant 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.10.007
*Corresponding author. Address: Department of Biological Science and Technol-ogy, National Chiao Tung University, No.75 Po-Ai Street, Hsinchu 30068, Taiwan. Tel.: +886 3 5131338; fax: +886 3 5729288.
E-mail address:lincs@mail.nctu.edu.tw(C.-S. Lin).
Bioresource Technology 101 (2010) 2880–2883
Contents lists available atScienceDirect
Bioresource Technology
strains were compared with the wild type at the cultivation temper-atures of 25, 30, 35 and 40 °C. All the cultures were aerated with 5% (v/v) CO2continuously at 0.25 vvm (volume gas per volume media
per min) and supplied with light intensity of 300
l
mol m2s1.The microalgal growth based on the biomass concentration (g L1)
was determined by spectrophotometric method (Chiu et al., 2008). The specific growth rate (
l
, d1) was measured during theexponen-tial growth of microalgae (Ono and Cuello, 2007). For the analysis of lipid content, microalgal lipid was determined according to the method reported byChiu et al. (2009a).
The growth experiment of semicontinuous cultivation was per-formed when the biomass concentration in the batch cultures reached to about 1 g L1(OD
682= 5). In the semicontinous
cultiva-tion, half of the culture broth was replaced and 0.5-fold of medium was added each day.
The CO2fixation rate of the microalgal cultures was determined
when the cultures were grown at 40 °C by the semicontinuous cultivation with an influent of 5% CO2 at 0.25 and 0.5 vvm.
The CO2 concentration in airstreams, i.e., CO2(g), was measured
using a Guardian Plus Infrared CO2Monitor D-500 with a
sensitiv-ity of 0.05% of CO2(g) (Edinburgh Instruments, Livingston, UK).
The CO2 fixation rate (mg min1) was determined as, RCO2¼
½ðP0þ
q
ghÞyCO2in P0yCO2outFairMwCO2=8:314 TVculturein which, yCO2inpresents the amount of CO2 influent (%), yCO2out presents the
amount of CO2effluent (%), P0is atmospheric pressure (Pa),
q
isdensity of liquid (kg m3), h is vertical distance of culture medium
(m), Fairis gas flow rate (L min1), MwCO2is molecular weight of CO2
(g mol1), T is absolute temperature (K), and V
culture is volume of
culture medium (L) (Cheng et al., 2006).
The mutant strain which was the most tolerant to the high tem-perature of 40 °C was cultivated in a large scale of outdoor closed and vertical bubble column photobioreactor (cultured volume is
40 L). The experiment was carried out over a period of 8-day dur-ing the summer of 2008. The cultures were supplied with 5% (v/v) CO2and 0.25 vvm aeration rate. The photosynthetic photon flux at
the photobioreactor location during the day time was averagely 1500
l
mol m2s1. A nighttime light supply of 300l
mol m2s1was also established.
A Student’s t-test was used to evaluate differences between groups of discrete variables. A value of p < 0.05 was considered sta-tistically significant.
3. Results and discussion
Fig. 1demonstrates that the
l
of Chlorella sp. mutant MT-7 and MT-15 in indoor cultivation were 1.4- and 1.8-fold at 25 °C and 3.3- and 6.7-fold at 40 °C cultivation compared with those of the wild type during an 8-day cultivation, respectively. The wild type cultures did grow poor at 35 and 40 °C. The mutant strains showed the maximuml
at 30 °C cultivation and moderate growth at 35 and 40°C. The optimal temperature for most microalgal specieswas in a range of 22–28°C. This was confirmed that 25 °C was
the optimal temperature for growth of the wild type Chlorella sp. in this study. Our results indicate that the microalgal mutants MT-7 and MT-15 are thermal-tolerant and have high growth po-tential. It is identified that the growth and thermal-tolerant poten-tial of the mutated microalgal strains selected in the present study are comparable to those of microalgal strains isolated from the nat-ure (de Bashan et al., 2008; Ono and Cuello, 2007).
Table 1demonstrates the lipid contents of wild type, MT-7 and MT-15 cultivated at 25, 30, 35 and 40 °C. The average lipid content of both mutant strains was lower than wild type across all of the cul-tivation temperatures. The lipid content of wild type in stationary phase of cultivation was significantly higher (p < 0.05) than that in exponential phase at 25, 30 and 35 °C. But, there was no effect of the growth phase on the lipid accumulation in both mutant strains when the cultivation temperatures were ranging from 25 to 40 °C, except MT-15 cultivated at 25 °C. Overall, the mutant strains were not able to accumulate lipid efficiently compared to the wild type. However, the mutant strains isolated in this study grew faster than the wild type strain and were tolerant at higher temperature. There-fore, when the microalgal cells cultured at 35 and 40 °C, but not at 25 and 30 °C, the daily lipid productivities of the mutant strains at high-er temphigh-erature (35 and 40 °C) whigh-ere greathigh-er than the wild type.
In the semicontinuous cultivation at 25 °C, the growth of mu-tant strains was faster than that of wild type. The growth of MT-7, MT-15 and wild type was maintained consistently at biomass concentration from 0.8 to 1.6 g L1, 0.9 to 1.8 g L1 and 0.45 to
0.90 g L1, respectively, each day during an 8-day cultivation
(Fig. 2a). In the semicontinuous cultivation at 40 °C, the wild type culture was grown from biomass concentration about 0.4 g L1on
the first day of semicontinuous cultivation. During an 8-day semi-continuous culture, the biomass concentration of wild type culture gradually decreased. However, MT-7 and MT-15 strain grew from
0 0.2 0.4 0.6 0.8 1.0 25 30 35 40 Wild Type M7 M15
µ
(day
-1)
Temperature (
°C)
Fig. 1. Specific growth rate (l, d1
) of Chlorella sp. wild type and mutant strain MT-7 as well as MT-15 at cultivation temperature of 25, 30, 35 and 40 °C.
Table 1
Effect of growth phase and temperature on lipid content (%) of wild type, mutant strain MT-7 and MT-15.
Strain 25°C 30°C 35°C 40°C
Exponentiala
Stationaryb
Exponential Stationary Exponential Stationary Exponential Stationary Wild type 12.3 ± 0.5 22.5 ± 0.9* 12.5 ± 0.7 17.3 ± 1.7* 14.7 ± 1.7 19.4 ± 2.3* 14.1 ± 0.6 16.2 ± 0.6
MT-7 12.8 ± 1.5 11.5 ± 0.9 10.3 ± 1.7 9.9 ± 1.0 12.6 ± 0.6 12.9 ± 2.5 12.4 ± 0.8 12.0 ± 0.7
MT-15 9.2 ± 1.1 13.8 ± 0.6* 10.4 ± 0.6 11.5 ± 0.7 11.7 ± 0.7 9.6 ± 0.7 11.8 ± 0.5 8.8 ± 0.5*
a
Sample was collected during the exponential phase after 3 days of cultivation.
b
Sample was collected during the stationary phase after 13 days of cultivation.
*There were significant differences (p < 0.05) in the lipid content of the culture collected at stationary phase compared to the exponential phase for each strain at each
cultivation temperature. Each data indicates the mean ± SD, which was measured from three independent cultures.
0.4 to 0.9 g L1 on the first day, and the growth was stable and
maintaining from 0.6 to 1.2 g L1from the third day onward, after
which half of the culture broth was replaced with fresh medium daily (Fig. 2b). In short, the mutant strains, but not the wild type strain, were able to maintain growth potential at an environmental temperature of 40 °C.
The CO2fixation rate of the microalgae cultured in a
semicon-tinuous cultivation was determined when the microalgal strains
were grown to the biomass concentration of 1, 2 or 3 g L1 at
40 °C (Table 2). The aerated CO2concentration was 5% and the
aer-ation rate was set as 0.25 and 0.5 vvm. Overall, the results show that there was significant higher of CO2fixation rate at 0.25 vvm
compared to 0.5 vvm CO2aeration rate in all the cultures. In
addi-tion, significant higher of CO2fixation rate was found at higher
bio-mass concentration compared to lower biobio-mass concentration (i.e., 3 > 2 > 1 g L1) in all the cultures.
The fixation rate of CO2increased when the biomass
concentra-tion of cultures increases. This is the reason that more CO2 was
captured by the microalgae to maintain the growth at a higher bio-mass concentration. High density cultures result in higher viscosity which subsequently increase gas retention time for CO2absorption
and therefore enhance the CO2 removal efficiency (Chiu et al., 2008). Significant higher of CO2fixation rate was also found when
the aeration rate decreases in the result of the increasing of CO2
absorption from bubbling gas. This is caused by the increase of sur-face area per unit gas volume of the bubble which will also en-hance the CO2removal efficiency (Chiu et al., 2009b).
Chlorella sp. MT-15 was selected and applied for the cultivation in an outdoor closed photobioreactor as it was the faster-growing strain cultivated at 40 °C. The microalgal culture used sunlight for photosynthesis in the day, and artificial light with intensity of 300
l
mol m2s1was supplied to the surface of thephotobioreac-tor at night. During the experimental period of 8 days, the average air temperature was 29 ± 2 °C, while the average culture broth temperature was 41 ± 1 °C. The wild type culture did not seem
0 0.4 0.8 1.2 1.6 2.0 Wild type MT-7 MT-15 0 0.4 0.8 1.2 1.6 0 1 2 3 4 5 6 7 8
Biomass concentration (g L
-1)
Cultivation time (days)
(a)
25
°C
(b)
40
°C
Fig. 2. Growth profiles of Chlorella sp. mutant strain MT-7 and MT-15 compared to wild type in the semicontinuous cultivation with 5% CO2aeration at (a) 25 °C and (b) 40 °C.
Table 2
The CO2fixation rate of wild type, mutant strain MT-7 and MT-15 for the cultivation
at 40 °C with 5% CO2aeration. Aeration rate (vvm)a Biomass concentration (g L1)
CO2fixation rate (RCO2, mg min 1) Wild type MT-7 MT-15 0.25 1 11.80 ± 0.10b 12.45 ± 0.47 10.92 ± 0.23 2 NDc 14.43 ± 0.16 14.86 ± 0.20 3 ND 16.89 ± 0.19 17.72 ± 0.19 0.5 1 15.52 ± 0.11 18.24 ± 1.03 17.13 ± 0.24 2 ND 19.43 ± 0.76 21.38 ± 0.77 3 ND 21.14 ± 0.70 25.65 ± 1.03
aThe aeration rates of CO
2at 0.25 vvm and 0.5 vvm are equal to 89.43 mg min1
and 178.86 mg min1CO
2, respectively.
b Each data indicates the mean ± SD, which was measured from three
indepen-dent cultures.
C
ND, not detected. The wild type cultures would not reach to the biomass con-centration of 2 g L1
and 3 g L1
at the cultivated condition. Therefore, the RCO2
could not be detected.
growing; however, MT-15 grew well with
l
= 0.238 d1. Nighttimegrowth delays were not observed because the cultures were sup-plied with artificial light. The maximum volumetric productivity obtained from MT-15 cultivated up to 40 °C in batch culture in this study, was 0.35 g L1d1. During the summer in subtropical zones,
study of outdoor microalgal cultivation was mostly supplied with cooling system by circulating thermostatic water through the pho-tobioreactors (Chini Zittelli et al., 2006). However, in this study, the cultures of mutant strain could grow well at the environmental temperature reaching 40 °C without any operation to lower the temperature.
4. Conclusion
The mutant strains Chlorella sp. MT-7 and MT-15 isolated in this study are thermal-tolerant, grow fast with a high density (i.e., high biomass concentration) at temperature of 40 °C, and could capture CO2 with a significantly high efficiency compared to their wild
type. In addition, MT-7 and especially MT-15 have the potential to be applied at outdoor cultivation in subtropical region without cooling system, thereby reducing the cost of outdoor cultivation.
Acknowledgements
This work was supported by the grants from the National Sci-ence Council (NSC) and ‘‘Aim for the Top University Plan” of the National Chiao Tung University and Ministry of Education, Taiwan.
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