Algal Research 3 (2014) 61–65

Contents lists available at ScienceDirect

Algal Research
journal homepage: www.elsevier.com/locate/algal

Short communication

Aqueous extraction of proteins from microalgae: Effect of different cell

disruption methods
Carl Safi a,b,⁎, Alina Violeta Ursu c, Céline Laroche c, Bachar Zebib a,b, Othmane Merah a,b,
Pierre-Yves Pontalier a,b, Carlos Vaca-Garcia a,b,d
a
Université de Toulouse, INP-ENSIACET, LCA (Laboratoire de Chimie Agro-industrielle), F-31030 Toulouse, France
b
INRA, UMR 1010 CAI, F-31030 Toulouse, France
c
Université Blaise Pascal, Polytech, Institut Pascal, UMR CNRS 6022, 24, Avenue des Landais 63174 Aubière, France
d
King Abdulaziz University, Jeddah, Saudi Arabia

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

Article history: The microalgal structure has been investigated to evaluate the release of proteins in aqueous media from five
Received 18 July 2013 microalgae after conducting different cell disruption techniques: manual grinding, ultrasonication, alkaline treat-
Received in revised form 3 December 2013 ment, and high-pressure treatment. After conducting cell disruption, the protein concentration in water was de-
Accepted 15 December 2013
termined for all the microalgae and the results are discussed within the context of their cell wall structure. It was
Available online 3 January 2014
found that the aqueous media containing most protein concentration followed the order: high-pressure cell
Keywords:
disruption N chemical treatment N ultrasonication N manual grinding. Fragile cell-walled microalgae were
Manual grinding mostly attacked according to the following order: Haematococcus pluvialis b Nannochloropsis oculata b Chlorella
High-pressure homogenization vulgaris b Porphyridium cruentum ≤ Arthrospira platensis.
Ultrasonication © 2013 Elsevier B.V. All rights reserved.
Chemical treatment
Cell wall structure

1. Introduction to recover different components. The efficiency of cell disruption was

usually evaluated by extracting a single component especially lipids be-
Microalgae were first exploited for their capacity to accumulate pro- fore and after applying the treatment or by microscopic observation. To
teins and, through time, interest in this biomass took a new course espe- our knowledge, studies of microalgal proteins have been focused on:
cially during the last two decades with increasing demand for evaluating the nitrogen to protein conversion factor [14–18]; finding
sustainable energy. This biomass proved to be an important source of the best method to analyze proteins and differentiate between soluble
lipids suitable for biodiesel production. Hence, many of the studies and non-soluble proteins [19]; and analyzing the behavior of proteins
were concentrated on lipid extraction for fuel purposes, neglecting the at the air/water interface [20].
potential of microalgae to produce proteins and other high-value com- Therefore, the present study focuses on evaluating the effect of dif-
ponents [1]. However, until now all studies and estimates confirmed ferent cell disruption techniques on protein extractability in water of
that costs of production of biodiesel from microalgae remain high [2,3] five different microalgae having different cell wall macrostructures.
and far from being competitive with fossil fuel. Researchers are there- Namely, the Cyanobacterium Arthrospira platensis, which has a relative-
fore turning towards valuing other components present in the ly fragile cell wall, composed mainly of murein and no cellulose [21,22].
microalgae such as proteins, pigments, dyes, sugars, etc. The Chlorophycean Chlorella vulgaris and the Eustigmatophyceae
Extracting the totality of a specific component from microalgae is Nannochloropsis oculata, which have a cell wall mainly composed of cel-
often prevented by the intrinsic rigidity of its cell wall. To overcome lulose and hemicelluloses [23]. Another Chlorophycean Haematococcus
this barrier, an initial operation unit of cell disruption is required to per- pluvialis has a thick trilaminar cell wall composed of cellulose and spo-
mit complete access to the internal components and facilitate the ex- ropollenin [12,24,25]. The composition of its cell wall, similar to that
traction process. Hence, many cell disruption techniques have been of spores, makes this microalga less permeable and extremely resistant
tested to break the cell wall of microalgae such as bead milling [4,5], to mechanical treatments [26]. Finally, the Rodophythe Porphyridium
ultrasonication [6–8], microwave radiation [9], enzymatic treatment cruentum, which lacks a true cell wall, but instead is encapsulated by a
[10,11], cell homogenizer [12] and high-pressure cell disruption [13] layer of sulfurized polysaccharides [27–32].
In addition, the microalgae selected in this study have a cytoplasm
⁎ Corresponding author at: Université de Toulouse, INP-ENSIACET, LCA (Laboratoire de
containing soluble proteins, and they all have a chloroplast except for
Chimie Agro-industrielle), F-31030 Toulouse, France. Tel.: +33 6 50 45 29 65. A. platensis, which instead has thylakoids bundles circling the peripheral
E-mail address: carl.safi@ensiacet.fr (C. Safi). part of the cytoplasm with their associated structures, the phycobilisomes

2211-9264/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.algal.2013.12.004
62 C. Safi et al. / Algal Research 3 (2014) 61–65

(containing the phycobiliproteins) present on the surface of the thyla- 2.4.3. Ultrasonication
koids like in the chloroplast of P. cruentum [21]. Furthermore, the chloro- This treatment was carried out using a VC-750HV (20 kHz, 13 mm
plast also contains soluble proteins and a central pyrenoid, which is a non- probe) ultrasonic processor on 0.5 g of dry cells dispersed in 25 mL dis-
membrane, bound organelle composed of RuBisCO. tilled water. Total treatment time was 30 min in cycles of 5 s of
In this study, proteins released in the aqueous media were evaluated ultrasonication and 15 s of resting time in order to prevent overheating
and discussed considering the cell wall macrostructure of each the sample.
microalga along with the effect of each cell disruption technique used.
2.4.4. Manual grinding
2. Materials and methods Dry microalgae were manually ground using a mortar for 5 min, and
then 0.5 g was dispersed in 25 mL distilled water for 2 h. Samples were
2.1. Microalgae taken for protein analysis.

The microalgae selected are the Cyanobacteria Arthrospira platensis

2.4.5. Chemical treatment
(strain PCC 8005), two different Chlorophyceaen Chlorella vulgaris
Mother solutions were prepared with approximately 500 mL of dis-
(strain SAG 211–19), and Haematococcus pluvialis (unknown strain),
tilled water and 2 N NaOH was added to adjust the solution to pH 12 for
one Rhodophyta Porphyridium cruentum (strain UTEX 161), and the
maximum protein solubility. A sample of 0.5 g of freeze-dried biomass
Eustigmatophyceae Nannochloropsis oculata (unknown strain).
was added to 25 mL of mother solution. The mixture was then stirred
Each microalga was cultivated in a different culture medium.
for 2 h at 40 °C. The separation of the supernatant from the pellet was
Hemerick medium was used for P. cruentum, Sueoka medium for
conducted by centrifugation at 10,000 g for 10 min at 20 °C. The super-
C. vulgaris, Basal medium for H. pluvialis, Conway medium for N. oculata
natant was then adjusted to pH 3 with 0.1 M HCl in order to precipitate
and Zarrouk medium for A. platensis. All strains were grown in batch
the proteins. The protein isolate was collected after centrifugation at
mode in a 10 L indoor tubular air-lift photo-bioreactor (PBR at 25 °C
10,000 g for 10 min at 20 °C and the pellet was neutralized with
[33] inoculated from a prior culture in a flat panel air-lift PBR (1 L). Cul-
0.01 M NaOH [20]. Samples were taken for protein analysis.
ture mixing was achieved by sterile air injection from the bottom of the
PBR. The pH and temperature were recorded by a pH/temperature
probe (Mettler Toledo SG 3253 sensor), and monitored by the acquisi- 2.5. Lowry method
tion software LabVIEW. The pH was regulated at 7.5 with CO2 bubbling.
Microalgae were harvested during the exponential growth phase and After every disruption treatment, the liquid/solid separation was
concentrated by centrifugation, and then supplied as frozen paste conducted by centrifugation at 10,000 g for 10 min at room tempera-
from Alpha Biotech (Asserac, France). The biomass concentration of ture and the supernatant was analyzed by the Lowry method. [34]
the paste was 20–24% dry weight. A calibration curve was prepared using bovine standard albumin at a
concentration range of 0 to 1500 μg mL−1. In order to measure the pro-
2.2. Reagents tein content, 0.2 mL of each standard or samples containing the crude
protein extract was withdrawn and then 1 mL of modified Lowry re-
The Lowry kit (a prepared mixture of Lowry reagent, BSA standards agent was added to each sample. Each sample was then vortexed and
and 2 N Folin-Ciocalteu reagents) was from Thermo Scientific. NaOH incubated for 10 min. After incubation, 100 μL of Folin-Ciocalteu Re-
granules and 37% HCl were purchased from Sigma Aldrich and used as agent (1 N) was added and again vortexed and incubated for 30 min.
received. The blue color solution was then measured at 750 nm with a UV-1800
Shimadzu spectrophotometer.

2.3. Microalgae pre-treatment

2.6. Elemental analysis
2.3.1. Freeze-drying
The frozen paste of crude microalga (about 70 g) was directly intro- Total nitrogen was evaluated by using a Perkin Elmer 2400 series II
duced to a Fisher Bioblock Scientific Alpha 2–4 LD Plus device (Illkirch, elemental analyzer. Microalgal samples (2 mg) were placed in tin cap-
France). The pressure was reduced to 0.010 bar and the temperature sules and heated at 925 °C, using pure oxygen as the combustion gas
was further decreased to − 80 °C and freeze-drying was conducted and pure helium as the carrier gas, and the nitrogen concentration
under vacuum for 48 h. Dry biomass was stored under anhydrous con- was evaluated. For all the previous analyses, three experiments were
ditions. Before any disruption treatment, the cells were vigorously conducted separately with all the microalgae.
rehydrated in distilled water to ensure good homogeneity of the
sample. 2.7. Confocal laser scanning microscopy

2.4. Microalgae treatments Cells were observed with an SP2-AOBS confocal laser-scanning mi-
croscope from Leica microsystems (Nanterre-France). The fluoro-
2.4.1. Control chrome calcofluor white that binds to the cell wall was added to the
Cells (0.5 g) were dispersed for 2 h in 25 mL distilled water and the samples. When excited at 488 nm, the cells are identified as light blue
supernatant was recovered by centrifugation at 10,000 g for 10 min at colored.
20 °C for protein analysis. This treatment was considered as a blank to
compare with the other extraction treatments.
2.8. Statistical analysis

2.4.2. High-pressure cell disruptor Three experiments were conducted separately on all microalgae and
A TS Haiva series, 2.2-kW, disrupter from Constant Systems Limited their protein extract. Statistical analyses were carried out on Microsoft
(Northants, UK), was applied, in two passes at a pressure of 2700 bar, to Excel 2011 and Statgraphics Sigma Express. ANOVA test was carried
a biomass sample at a concentration of 2% dry weight (0.5 g of dry cells out and measurements of three replicates for each sample were repro-
dispersed in 25 mL distilled water). ducible for ±5% of the respective mean values.
 C. Safi et al. / Algal Research 3 (2014) 61–65 63

Table 1 disruption. On the other hand, for C. vulgaris, N. oculata and H. pluvialis,
Protein and nitrogen content for each microalga based on three replicates for three only a majority of cells were completely disrupted, while a few cells
experiments ± SD (n = 9).
maintained their globular form.
Microalga Total nitrogen (%.dw-1) Total proteins (% dw−1)

H. pluvialis 8.27 ± 0.08 51.7 ± 0.43 4. Discussion

N. oculata 7.41 ± 0.40 46.5 ± 2.14
C. vulgaris 7.81 ± 0.18 49.6 ± 1.04 The goal of the present study was to highlight the release of protein
A. platensis 8.53 ± 0.20 53.5 ± 1.10
into aqueous media after the application of different cell disruption
P.cruentum 9.04 ± 0.70 57.3 ± 3.84
techniques. The results do not only rely on the mechanical rigidity of
the cell wall of each microalga but also on its chemical characteristics.
Indeed, having a deep understanding of the macrostructure is necessary
3. Results in order to evaluate the release of components after any treatment was
conducted on the cells. This approach has been considered in a study
The total protein in crude microalgae was determined by obtaining conducted by Jubeau et al. in order to selectively extract intracellular
total nitrogen through elemental analysis and converting it into protein components such as proteins and phycoerythrin after cell disruption
percentage using the conversion factor found for each crude microalga of P. cruentum [13]. Moreover, the freeze-drying process that conserves
in the study conducted by Safi et al. [18]. In all cases, the total protein the samples well makes the protein extraction harder for some species
content was high, ranging from 46 to 57% dw (Table 1). [18]. In addition, after freeze-drying the cells are more aggregated,
The fraction of soluble proteins released in water after each cell dis- which lowers the contact surface with the extracting solvent, and also
ruption technique is presented in Fig. 1. The fraction of soluble protein could affect the integrity of the cell wall in fragile species [35,36].
in the total protein present in the microalgae was also evaluated and Osmosis is the net movement of solvent (water) molecules through
all these results are given in Table 2. a partially permeable membrane into a region of higher solute concen-
In this work, four cell disruption techniques are compared, along tration. Water usually travels through the membrane, the vacuole, the
with a control in distilled water, in order to evaluate protein exiting chloroplast, and the mitochondria by diffusing across the phospholipid
by diffusion through the membranes and walls into water media. The bilayer via water channels (aquaporins), which are proteins embedded
recovery yield in the control ranges from 6.5% dw with H. pluvialis to in the cell membrane that regulate the flow of water. Hence, the water
25% dw with P. cruentum. The latter alga is considered as fragile and only treatment was not considered as a cell disruption technique, but it
the former as resistant to disruption. Among the tested techniques, was carried out in this study as a reference control for the other tech-
high-pressure cell disruption was the best technique for all the niques. Surprisingly, the dispersion of microalgae in water released up
microalgae, with a recovery yield of 41% to 90% dw. Moreover, the lowest to 19–25% of soluble proteins per dry weight (Table 2) from A. platensis
protein concentration for all microalgae was obtained in the water con- and P. cruentum, coloring the water light blue for the former and light red
trol and through manual grinding, especially for rigid cell walled for the latter. This indicates that water penetrated the cell walls of both
microalgae. A relative difference was noticed in the concentration of microalgae but also succeeded in penetrating the intra-thylakoids space
protein released between the microalgae with fragile and rigid cell of A. platensis and permeated the chloroplast of P. cruentum to slightly dis-
walls. P. cruentum released the most compared to A. platensis. After solve the phycobilisomes present on the thylakoid membranes. On the
ultrasonication a minor increase in protein concentration was notice- contrary, the osmosis phenomenon was not strongly effective for the
able for the green microalgae, especially for C. vulgaris, and a more im- green microalgae, which are known to have rigid cell walls that resist
portant increase was observed for the A. platensis and P. cruentum. water permeating the structure and, thus, releasing only 6–10% proteins
Furthermore, the chemical treatment showed a significant increase of (Table 2).
protein released (N. oculata and C. vulgaris statistically released the Taking into account the standard deviation of three samples consid-
same protein concentration) (Fig. 1). ered for the green microalgae (C. vulgaris, N. oculata and H. pluvialis), all
In order to better interpret these results, microscopic observation the values of released proteins after water treatment and manual grind-
was carried out. The laser scanning confocal microscopic images pre- ing shown in Fig. 1 are statistically equivalent, indicating again the resis-
sented in Fig. 2 showed that in the cases of P. cruentum and A. platensis tance of their cell walls after manual grinding. This was not the case for
a total disruption of the cell wall occurred after high pressure cell the A. platensis and P. cruentum with stronger coloration of water

60

50

40
Protein %. dw

Blank
Manual grinding
30 Ultrasonication
Chemical

20 High-pressure homogenization

10

H. pluvialis N. oculata C. vulgaris A. platensis P. cruentum

Fig. 1. Soluble protein concentration released in water after each cell disruption technique (±SD for three replicates in three experiments (n = 9)).
64 C. Safi et al. / Algal Research 3 (2014) 61–65

Table 2
Concentration of water-soluble protein from total protein released in the aqueous phase
after cell disruption.

Protein yield (%)

Microalga Blank Manual Ultrasonication Chemical High-pressure

grinding homogenization

H. pluvialis 6.5 ± 0.2 7.4 ± 0.1 8.5 ± 0.0 15.8 ± 0.1 41.0 ± 3.7
N. oculata 8.1 ± 0.1 9.7 ± 0.0 13.5 ± 0.1 31.1 ± 2.0 52.3 ± 0.6
C. vulgaris 9.7 ± 0.5 9.0 ± 0.1 18.1 ± 0.0 33.2 ± 0.0 52.8 ± 0.6
A. platensis 19.0 ± 0.1 35.0 ± 1.2 47.1 ± 0.9 53.4 ± 0.2 78.0 ± 2.8
P. cruentum 24.8 ± 0.3 49.5 ± 0.7 67.0 ± 0.9 73.5 ± 1.2 90.0 ± 2.4

Based on three replicates for three experiments ± SD (n = 9).

Protein yield was calculated according to the following equation: proportion of hydrosoluble
protein in total protein = (PLowry / Nea × NTP) × 100 (%).
Nea: total nitrogen in initial biomass (% dw) obtained by elemental analysis.
NTP: nitrogen-to-protein conversion factor from Safi et al. [18].
PLowry: water-soluble protein.

associated with increase in protein concentration, indicating that the in-

ternal structure of both microalgae is being further altered, and simulta-
neously facilitating the penetration of water to dissolve more proteins.
Ultrasonication produces cavitation in cells and facilitates cell dis-
ruption; it did not cause any change for H. pluvialis, but showed a
minor effect on the cell wall of N. oculata and C. vulgaris by possibly
making it difficult for water to extract cytoplasmic proteins without al-
tering the structure of their chloroplast. The concentration of soluble
proteins and coloration kept increasing for the fragile cell-walled
microalgae by releasing 47–68% dw.
Chemical treatment was a key process that showed increases in pro-
tein concentration compared to the other treatments. P. cruentum lacks
a well-defined cell wall. Since protein solubility is dependent on pH, cell
wall characteristics and chemical composition [11], the high pH easily
solubilized proteins without any resistance from its pseudo-cell wall.
But in the case of the green microalgae, the sodium hydroxide is able
to perform a process similar to mercerization, by penetrating the micro-
crystalline structure of the cellulosic cell walls of the green microalgae
[18]. The alkaline solution can also easily dissolve the hemicelluloses at-
tached to cellulose as it has been demonstrated during the refining of
lignocellulosic substrates (straw, bran, and wood). In addition, it indi-
cates that this treatment gave more access to the cytoplasmic proteins,
and recovered the same concentration of proteins from N. oculata and
C. vulgaris (Fig. 1). However, the sporopollenin contained in the most
rigid cell wall (H. pluvialis) is known to be extremely resistant to chem-
ical agents [26], which explains the low recovery of soluble proteins.
A. platensis has a cell wall rich in amino sugars cross-linked with
oligopeptide chains. The former are labile in alkaline conditions by
deamidation of the N-acetylglucosamine and the latter are soluble in al-
kaline conditions. Therefore, the cell wall becomes permeable allowing
the alkaline extraction of proteins [18]. Hence, all these results demon-
strate that the chemical action acts in synergy with the mechanical
characteristics of the cell wall.
High-pressure cell disruption was the most efficient technique for all
microalgae; the concentration of proteins was statistically the same for
the green microalgae, with evidence that the majority of the cells were
broken while some of them remained intact (Fig. 2). The chloroplast of
these species was also partially damaged, as it is revealed by the color-
ation in light green (chlorophyll) of the aqueous extract. Indeed, chloro-
phyll is a hydrophobic pigment; its presence in the aqueous phase
indicates the formation of micellar structures and it points to a possible
Fig. 2. Confocal laser scanning microscopy of five microalgae before (right) and after (left)
alteration of the chloroplast. The other indication is that some cell debris high-pressure cell disintegration. (A and B) C. vulgaris, (C and D) A. platensis, (E and F)
containing the green pigment were extremely reduced in size and did P. cruentum, (G and H) H. pluvialis, (I and J) N. oculata.
not precipitate in the pellet after centrifugation at 10000 g, leading to
a greenish color of the supernatant as it occurred in previous work [7].
Hence, after two passes, water had access to cytoplasmic proteins and hand, as expected according to their fragile cell wall (Table 2),
partially infiltrated the chloroplast. Therefore, this method released al- A. platensis and P. cruentum did not show much resistance, and the sol-
most half of the proteins present inside the rigid cell-walled microalgae uble protein concentration of total protein was 78% dw for the former
(Table 2), indicating again the resistance of their cell wall. On the other and 90% dw for the latter (Table 2). Moreover, an important coloration
 C. Safi et al. / Algal Research 3 (2014) 61–65 65

of the aqueous extract for both microalgae was observed with a pellet [14] J.A. Gerde, T. Wang, L. Yao, S. Jung, L.A. Johnson, B. Lamsal, Optimizing protein isola-
tion from defatted and non-defatted Nannochloropsis microalgae biomass, Algal Res.
having lost its red coloration for P. cruentum. This was also supported 2 (2013) 145–153.
by microscopic observation, showing that their structure was completely [15] C.V. Lopez, C. Garcia Mdel, F.G. Fernandez, C.S. Bustos, Y. Chisti, J.M. Sevilla, Protein
altered (Fig. 2). measurements of microalgal and cyanobacterial biomass, Bioresour. Technol. 101
(2010) 7587–7591.
The same order of rigidity was obtained in another study [18] that [16] S.O. Lourenço, E. Barbarino, P.L. Lavín, U.M. Lanfer Marquez, E. Aidar, Distribution of
took into account the values of the nitrogen to protein conversion fac- intracellular nitrogen in marine microalgae: calculation of new nitrogen-to-protein
tors before and after protein extraction and then attributed them to conversion factors, Eur. J. Phycol. 39 (2004) 17–32.
[17] S.O. Lourenço, E. Barbarino, U.M.L. Marquez, E. Aidar, Distribution of intracellular ni-
the rigidity of the cell walls. This result shows that in order to compare trogen in marine microalgae: basis for the calculation of specific nitrogen-to-protein
the efficiency of cell disruption technology, it is more accurate to use conversion factors, J. Phycol. 34 (1998) 798–811.
fragile cell algae such as P. cruentum. [18] C. Safi, M. Charton, O. Pignolet, F. Silvestre, C. Vaca-Garcia, P.-Y. Pontalier, Influence
of microalgae cell wall characteristics on protein extractability and determination of
The present study shows additional insight into the understanding
nitrogen-to-protein conversion factors, J. Appl. Phycol. (2012) 1–7.
of the recovery of proteins after different cell disruptions. Hence, [19] E. Barbarino, S.O. Lourenço, An evaluation of methods for extraction and quantification
among all the techniques used the high-pressure cell disruption was of protein from marine macro- and microalgae, J. Appl. Phycol. 17 (2005) 447–460.
the most efficient but not sufficient to recover more than 50% of the pro- [20] I.S. Chronakis, A.N. Galatanu, T. Nylander, B. Lindman, The behaviour of protein
preparations from blue-green algae (Spirulina platensis strain Pacifica) at the
teins for the green microalgae, indicating that more passes are required air/water interface, Colloids Surf. A Physicochem. Eng. Asp. 173 (2000) 181–192.
to completely disrupt their macrostructure, and thus more energy input [21] R.E. Lee, Phycology, 4th ed. New York, Cambridge University Press, Cambridge, En-
will be necessary. gland, 2008.
[22] H.K. Lu, C.C. Hsieh, J.J. Hsu, Y.K. Yang, H.N. Chou, Preventive effects of Spirulina
platensis on skeletal muscle damage under exercise-induced oxidative stress, Eur.
J. Appl. Physiol. 98 (2006) 220–226.
References [23] M.F. Payne, R.J. Rippingale, Evaluation of diets for culture of the calanoid copepod
Gladioferens imparipes, Aquaculture 187 (2000) 85–96.
[1] P.M. Foley, E.S. Beach, J.B. Zimmerman, Algae as a source of renewable chemicals: [24] C. Aflalo, Y. Meshulam, A. Zarka, S. Boussiba, On the relative efficiency of two- vs.
opportunities and challenges, Green Chem. 13 (2011) 1399–1405. one-stage production of astaxanthin by the green alga Haematococcus pluvialis,
[2] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (2007) 294–306. Biotechnol. Bioeng. 98 (2007) 300–305.
[3] L. Rodolfi, G. Chini Zittelli, N. Bassi, G. Padovani, N. Biondi, G. Bonini, et al., [25] A. Montsant, A. Zarka, S. Boussiba, Presence of a nonhydrolyzable biopolymer in the
Microalgae for oil: strain selection, induction of lipid synthesis and outdoor cell wall of vegetative cells and astaxanthin-rich cysts of Haematococcus pluvialis
mass cultivation in a low-cost photobioreactor, Biotechnol. Bioeng. 102 (2009) (Chlorophyceae), Mar. Biotechnol. (New York, NY) 3 (2001) 515–521.
100–112. [26] C. Hagen, S. Siegmund, W. Braune, Ultrastructural and chemical changes in the cell
[4] J. Doucha, K. Livansky, Influence of processing parameters on disintegration of wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore for-
Chlorella cells in various types of homogenizers, Appl. Microbiol. Biotechnol. mation, Eur. J. Phycol. 37 (2002) 217–226.
81 (2008) 431–440. [27] M. Adda, J.C. Merchuk, S. Arad, Effect of nitrate on growth and production of
[5] J.Y. Lee, C. Yoo, S.Y. Jun, C.Y. Ahn, H.M. Oh, Comparison of several methods for effec- cell-wall polysaccharide by the unicellular red alga Porphyridium, Biomass 10
tive lipid extraction from microalgae, Bioresour. Technol. 101 (Suppl. 1) (2010) (1986) 131–140.
S75–S77. [28] S. Arad, M. Adda, E. Cohen, The potential of production of sulfated polysaccharides
[6] T. Furuki, S. Maeda, S. Imajo, T. Hiroi, T. Amaya, T. Hirokawa, et al., Rapid and selec- from Porphyridium, Plant Soil 89 (1985) 117–127.
tive extraction of phycocyanin from Spirulina platensis with ultrasonic cell disrup- [29] S.M. Arad, O.D. Friedman, A. Rotem, Effect of nitrogen on polysaccharide production
tion, J. Appl. Phycol. 15 (2003) 319–324. in a Porphyridium sp. Appl. Environ. Microbiol. 54 (1988) 2411–2414.
[7] J.A. Gerde, M. Montalbo-Lomboy, L. Yao, D. Grewell, T. Wang, Evaluation of microalgae [30] S. Geresh, S. Arad, The extracellular polysaccharides of the red microalgae: chemis-
cell disruption by ultrasonic treatment, Bioresour. Technol. 125 (2012) 175–181. try and rheology, Bioresour. Technol. 38 (1991) 195–201.
[8] L. Gouveia, A.C. Oliveira, Microalgae as a raw material for biofuels production, J. Ind. [31] S. Geresh, A. Mamontov, J. Weinstein, Sulfation of extracellular polysaccharides of
Microbiol. Biotechnol. 36 (2009) 269–274. red microalgae: preparation, characterization and properties, J. Biochem. Biophys.
[9] H. Zheng, J. Yin, Z. Gao, H. Huang, X. Ji, C. Dou, Disruption of Chlorella vulgaris cells for Methods 50 (2002) 179–187.
the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, [32] T.M. Sobczuk, F.G. Camacho, E.M. Grima, Y. Chisti, Effects of agitation on the
bead milling, enzymatic lysis, and microwaves, Appl. Biochem. Biotechnol. 164 microalgae Phaeodactylum tricornutum and Porphyridium cruentum, Bioprocess
(2011) 1215–1224. Biosyst. Eng. 28 (2006) 243–250.
[10] J. Fleurence, The enzymatic degradation of algal cell walls: a useful approach for im- [33] K. Loubiere, J. Pruvost, F. Aloui, J. Legrand, Investigations in an external-loop airlift
proving protein accessibility? J. Appl. Phycol. 11 (1999) 313–314. photobioreactor with annular light chambers and swirling flow, Chem. Eng. Res.
[11] Y.W. Sari, M.E. Bruins, J.P.M. Sanders, Enzyme assisted protein extraction from rape- Des. 89 (2011) 164–171.
seed, soybean, and microalgae meals, Ind. Crop. Prod. 43 (2013) 78–83. [34] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the
[12] M.M. Mendes-Pinto, M.F.J. Raposo, J. Bowen, A.J. Young, R. Morais, Evaluation of dif- Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.
ferent cell disruption processes on encysted cells of Haematococcus pluvialis: effects [35] B. Cordero, D. Voltolina, Viability of mass algal cultures preserved by freezing and
on astaxanthin recovery and implications for bio-availability, J. Appl. Phycol. 13 freeze-drying, Aquac. Eng. 16 (1997) 205–211.
(2001) 19–24. [36] N. Rossignol, L. Vandanjon, P. Jaouen, F. Quéméneur, Membrane technology for
[13] S. Jubeau, L. Marchal, J. Pruvost, P. Jaouen, J. Legrand, J. Fleurence, High pressure dis- the continuous separation microalgae/culture medium: compared perfor-
ruption: a two-step treatment for selective extraction of intracellular components mances of cross-flow microfiltration and ultrafiltration, Aquac. Eng. 20 (1999)
from the microalga Porphyridium cruentum, J. Appl. Phycol. 25 (2012) 983–989. 191–208.