Effect of Different Separation Techniques and Storage Temperatures on the Viability of Marine Microalgae, Chaetoceros calcitrans, during Storage
The aim of this study was to optimize and to propose the suitable separation method and storage conditions for specific species of microalgae. The performance of different separation methods for the recovery of cell biomass of marine microalgae, Chaetoceros calcitrans, from the culture broth was evaluated. The microalgae were cultivated using 10 L photobioreactor. The microalgae cell cultures were concentrated either by centrifugation, tangential flow filtration or flocculation and then stored at different temperatures (-20, 4 and 27°C) to investigate the optimum storage conditions for C. calcitrans prior to different downstream processing methods. High concentration of cell in slurry (4.88x107 cells mL-1) was obtained using centrifugation as compared to tangential flow filtration (4.14x107 cells mL-1), flocculation with chitosan (1.56x107 cells mL-1) and flocculation with Magnafloc®LT 25 (8.24x106 cells mL-1). Storage of C. calcitrans biomass at chilled temperature (4°C) directly after the harvesting using these four different separation methods resulted in extended shelf life (> 4 weeks). Frozen biomass (-20°C) fails to preserve the quality of C. calcitrans after they were revived in fresh medium. C. calcitrans flocculated with 0.5 mg L-1 Magnafloc®LT 25 was able to maintain the quality of the cells after storage at 27°C for more than 2 weeks. However, flocculation of cells with 20 mg L-1 chitosan, centrifugation at 8000 rpm for 10 min and tangential flow filtration process at transmembrane pressure of 20 psi failed to retain the quality of biomass after storage for 2 weeks at 27°C.
April 27, 2010; Accepted: June 15, 2010;
Published: September 24, 2010
The use of microalgae as conventional diet for many aquatic organisms is deal
with the need for live culture (Montaini et al.,
1995). Alternative diet such as preserved microalgae is important to replace
the demand of live microalgae which increase the production cost. Microalgae
can be harvested using different separation methods such as flocculation (Knuckey
et al., 2006; Heasman et al., 2001;
Bilanovic et al., 1988), centrifugation (Heasman
et al., 2001; McCausland et al., 1999)
and filtration (Heasman et al., 2001; McCausland
et al., 1999). The algae concentrates are susceptible to damage if
not stored under appropriate conditions (Heasman et al.,
2001). The main problem in the preservation and storage of microalgae concentrate
is related to their shelf-life, which depends on the species and storage conditions
(Ponis et al., 2008). Therefore, it is important
to maintain the shelf life of the microalgae by maintaining the cells viability
and consequently sustain their cell contents and chemical integrity.
Microalgal concentrates stored at low temperature is a promising alternative
to the fresh algae (McCausland et al., 1999;
Heasman et al., 2001). Tetraselmis suecica
suspension maintained at 4°C in darkness showed no significant difference
in cells viability after 50 days of storage while maintaining the fatty acid
profile over 90 days (Montaini et al., 1995).
However, storage at low temperature is not suitable for industrial applications
due to significant increase in cost and equipment used. The use of cryoprotectant
for improving the preservation of microalgae has also been studied (Heasman
et al., 2001; Molina-Grima et al., 1994).
However, this cryopreservation technique is too expensive to be used for microalgae
cells for subsequent use as aquaculture feeds.
Although, several separation methods have been proposed for effective harvesting
of microalgae from culture broth, the suitability of each method might be specific
to certain species. Some robust methods may not be suitable for delicate microalgae
cells such as Pavlova lutheri (Ponis et al.,
2008; Heasman et al., 2000) and Tahitian
Isochrysis (Heasman et al., 2000). In addition,
the viability and stability of the cells during storage may not only be influenced
by the storage conditions but also by the harvesting method. The present study
investigated the effect of storage temperature (-20, 4 and 30 °C) on the
viability and stability of the microalgae cells, C. calcitrans, harvested
using different separation methods (centrifugation, flocculation and membrane
filtration). The ability of the cell concentrates to revive after 4 weeks of
storage was also investigated. The information obtained may be used to optimize
and propose the suitable separation method and storage conditions for specific
species of microalgae.
MATERIALS AND METHODS
Microalgae and cultivation method: The microalgae, C. calcitrans, obtained from Aquatic Animal Health Unit, Faculty of Veterinary Medicine, Universiti Putra Malaysia in 2008 was used throughout this study. The microalgae was cultured in 10 L photobioreactor using Conway medium at 29 ppt salinity, with the addition of 0.02 g L-1 of silica. The temperature within the photobioreactor was regulated at 20±2°C by air-conditioning. Aeration to the culture was provided by air bubbling through the air sparger. The pH of the medium was maintained at 8±0.2 by sparging with a mixture of air and carbon dioxide (CO2) at a ratio of 97:3. Cultures were grown under illumination by white fluorescent light (4500-5000 Lux) for 12 h during day time and 12 h in the dark during night time. Cells were harvested at late logarithmatic growth phase (after 6 days of cultivation) and subjected to different separation methods for the separation of cells from the culture broth.
Methods for cell separation: The different separation methods (flocculation,
centrifugation and membrane filtrations) were used in this study. The optimized
flocculation method as reported in our previous study (Harith
et al., 2009) was used. Polyelectrolyte flocculant, Magnafloc®LT
25, was used for flocculation of C. calcitrans. The pH of the culture
was adjusted to pH 10.2 followed by the addition of 0.5 mg L-1 of
polyelectrolyte into 500 mL of the culture. Flocculation of C. calcitrans
was also conducted using chitosan as a flocculant. In this method, 20 mg L-1
of the polymer was added to 500 mL of culture and pH was adjusted to 8. In both
flocculation methods, the cultures were mixed vigorously (200 rpm) for 1 min,
followed with slow mixing (50 rpm) for 2 min. Flocculation was allowed under
gravity without any stirring and surface water was siphoned off at the end of
flocculation (4 h). The flocs were resuspended into single cells by adjusting
the pH to 7, which was achieved by the addition of hydrochloric acid (HCl).
For centrifugation method, bench top centrifuge (Appendorf, 5510R) was used. Centrifugation of the C. calcitrans culture was carried out in 50 mL centrifuge tube (Vivantis) with screw caps at 8000 rpm for 10 min. The temperature during centrifugation was maintained at 20°C. After the centrifugation, supernatant was discarded and the cell pellet was resuspended in 5 mL of the clear supernatant to obtain cell concentrate with a concentration of 10-fold higher than the harvested culture broth from the photobioreactor. This cell concentrate was then used for the storage study.
The laboratory scale Tangential Flow Filtration (TFF) system (Millipore, United State) was used for the separation of C. calcitrans by membrane filtration method. About 500 mL of C. calcitrans broth was filtered along the surface of a Pellicon XL device (Durapore PVDF, 0.65 μm, 50 cm2). The filtration was operated at transmembrane pressure of 20 psi. The filtration was conducted continuously until the final retentate volume of 70 mL was achieved to obtain a volumetric reduction factor (VRF) of 7.1.
Storage study and analytical procedures: The C. calcitrans cells concentrates obtained from the different separation methods were placed in 1.5 mL vial without the addition of cryoprotective agents. For storage studies, the cell concentrates were stored at different temperatures (-30, 4 and 27°C) in dark conditions. During storage, samples were withdrawn at time intervals (0, 1, 2, 3 and 4 weeks) for analysis.
The ability of the stored cells to revive was evaluated by inoculating 1.0 mL of the culture into 20 mL of fresh Conway medium. The culture was allowed to grow under continuous illumination (4500-5000 Lux) at 20°C and the growth of cells was monitored by measuring the optical density of cells at A750 nm. The culture was also examined under the microscope to evaluate whether the increase in cell optical density was purely due to growth of microalgae or bacterial contaminants.
The viability of the microalgae cells during storage was measured using staining
method. The culture sample (20 mL) was treated with 1 mL of 1% (w/v) stock solution
of Evans Blue and allowed to stand at room temperature for 30 min (Heasman
et al., 2001). The stained cells were examined under the light microscope
(Leica DMLB, Germany). The dead cells were stained blue due to the penetration
of the stain through the cell wall, whereas the viable cells remained unstained.
Cell number was counted using Haemacytometer and the percentage of viable cells
is calculated using Eq. 1:
The harvesting efficiency of C. calcitrans cells from the culture broth using different separation methods is shown in Table 1, where different separation methods produced different final cell concentrations. The highest cell concentration (4.88x107 cells mL-1) was obtained with centrifugation, followed by the separation using TFF (4.14x107 cells mL-1). Reduced final cell concentration was obtained with flocculation using either chitosan or Magnafloc® LT 25, where the final cell concentration obtained was 1.56x107 cells mL-1 and 8.24x106 cells mL-1, respectively. The highest harvesting efficiency (100%) was obtained in separation using TFF. The separation using centrifugation and flocculation with both Magnafloc® LT 25 and chitosan gave the same harvesting efficiency (> 95%).
The changes in viability of C. calcitrans harvested from the culture
broth using different separation methods during storage at different temperatures
are shown in Fig. 1a-d. A slight reduction
in cell viability with storage time was observed at all temperatures investigated.
However, the difference in cell viability with different storage temperatures
was not significantly different. The use of different separation methods also
did not show significant variation in cell viability during storage.
Table 2 shows the effect of separation methods and storage
temperatures on the ability of stored C. calcitrans to revive when inoculated
into fresh medium at 30°C. For all separation methods used in this study,
the cells stored at 4°C were able to be revived even after 4 weeks of storage.
The cells harvested by flocculation using Magnafloc®LT 25 revived
after 2 weeks of storage at 27°C. On the other hand, cells harvested using
TFF, centrifugation and flocculation with chitosan failed to be revived after
2 weeks storage at 24°C.
||Effect of different cell separation methods used to separate
C. calcitrans cells from the culture broth on the harvesting efficiency
and the final cell number in the concentrate. Respective cell density was
used in the preservation procedure
||Change in the viability of C. calcitrans cells, separated
using various method, during storage at different temperatures. (a) Flocculation
with 0.5 mg L-1 Magnafloc® LT 25 at pH 10.2 followed
by resuspension at pH 7, (b) Flocculation with 20 mg L-1 chitosan
at pH 8 followed by resuspension at pH 7, (c) Centrifugation at 8000 rpm;
10 min and (d) Tangential flow filtration at TMP = 20 psi
||Effect of different cell separation methods used to separate
C. calcitrans cells from the culture broth on the quality of the
cell concentrate during storage at different temperatures. The quality of
cells was evaluated by their ability to revive when inoculated into the
fresh medium and incubated at 30°C
|+: Able to revive with increase in growth, : Not able
to revive the growth after 5 days of cultivation
Frozen cell concentrates (-20°C) show very poor quality cultures and none
of the cells concentrates were able to revive after 2 weeks of storage regardless
of the separation methods used.
Results from this study clearly indicate that the storage temperature greatly
influenced the quality of the stored microalgae cultures to a greater extent
than the separation method. Since cell concentrate harvested using flocculation
with Magnafloc®LT 25 had lower cell density (8.24x106
cells mL-1) and can easily be revived after 4 week of storage at
4°C, it can be said that the cell density of the cell concentrate may also
play an important role in the storage of C. calcitrans cells to maintain
high cell viability and quality. This result is supported by
Cordero and Voltolina (1997) who reported that loss in viability of microalgae
was strongly correlated with cell concentration, where reduction in survival
rate was increased with increasing cell density. Therefore, high cell viability
and quality could be obtained by formulating the cell concentrate with lower
cell density prior to storage.
According to Heasman et al. (2001), optimum combinations
of harvesting and storage conditions had to be specifically tailored to individual
species of microalgae in order to maximize the effective shelf life of the cell
concentrates. Furthermore, the success in preservation methods depends on the
microalgae species and method of harvesting. The quality of the preserved cell
concentrate was normally evaluated as the starter to initiate the new culture
using fresh medium (Jaouen et al., 1999), which
is widely used as indirect method to confirm the viability and stability of
the cell concentrates. A major prerequisite to extend the shelf-life of the
microalgae cells is the maintenance of membrane integrity, cells contents and
chemical integrity (Heasman et al., 2001).
Results from the present study indicated that the storage of C. calcitrans
cells at 4°C yielded the highest quality of cells. Montaini
et al. (1995) and McCausland et al. (1999)
also claimed that preservation at low temperatures was the preferred method
to maintain high cell viability of microalgae species. However, reduced quality
and shelf life of several microalgae such as Skletonema costatum, P.
lutheri, Chaetoceros muelleri and T. Iso concentrates prepared
using super centrifuge prior to storage at 4.0±0.5°C was reported
by Heasman et al. (2000).
Storage at low temperature is the simplest way for maintaining the cells quality.
Reduced temperature slows both metabolic processes and changes including oxidative
denaturation of essential vitamins and highly unsaturated fatty acids (HUFAs),
autolysis and microbial degradation while maintaining the cells viability (Heasman
et al., 2001). Preservation of concentrated microalgae cell at low
temperature (chilled conditions) is a potential method of retarding microbial
degradation of stored concentrates since all microorganisms have definite minimum,
maximum and optimum growth temperature. However, storage at temperature below
0°C resulted to the formation of C. calcitrans cell concentrate in
the frozen form. Frozen cultures need to be thawed before use and this process
resulted in injury to the cell wall (Ben-Amotz and Gilboa,
1980). Excessive damage to the C. calcitrans cells during thawing
of the stored cells in the frozen form (stored at -20°C) is the possible
explanation for the failure of the cells to be revived even after a short storage
period, as observed in this study.
Results from this study demonstrated that the storage temperature showed a greater influence to the quality of C. calcitrans cells than the method of harvesting cells from the culture broth. For all separation methods used (flocculation (Magnafloc®LT 25, chitosan), centrifugation and TFF), the preferred storage temperature to maintain the quality of C. calcitrans cells was at chilled condition (4°C). For cell concentrate harvested by flocculation with Magnafloc®LT 25 followed by resuspension to pH 7 using hydrochloric acid, the quality of cells could be maintained up to 2 weeks of storage at 27°C. This may be related to low cell density of the cell concentrate as compared to those produced by other separation method. Frozen cultures (-20°C) were unable to revive in fresh medium regardless of the separation methods used.
The authors would like to acknowledge the Johor Satellite Biotechnology Project, Malaysia, for the funding support (grant number: BSP(J)BTK/004(4)) of this study.
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