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Algal cell rupture using high pressure


homogenization as a prelude to oil extraction
ARTICLE in RENEWABLE ENERGY DECEMBER 2012
Impact Factor: 3.36 DOI: 10.1016/j.renene.2012.04.039

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Renewable Energy 48 (2012) 300e308

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journal homepage: www.elsevier.com/locate/renene

Technical note

Algal cell rupture using high pressure homogenization as a prelude to oil


extraction
Nalin Samarasinghe, Sandun Fernando*, Ronald Lacey, William Brock Faulkner
Department of Biological and Agricultural Engineering, Scoates Hall, 2117 TAMU, Texas A&M University, College Station, TX 77843, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 11 June 2011
Accepted 20 April 2012
Available online 12 June 2012

Research associated with extraction of lipids from algae and the use of algal biomass for transportation
fuel production is still in its infancy. Measurement techniques associated with algal systems are still not
well established and, as a result, pitfalls exist. This paper discusses several measurement and characterization techniques that were attempted while developing an algal oil extraction system and their
effectiveness. Measurements were made primarily to quantify the degree of algal cell breakage using
high pressure homogenization e as a prelude to solvent extraction.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Algae
Homogenization
Particle sizing
Cell counting
Cell breakage

1. Introduction
The potential use of micro-algal biomass and lipids as substrates
for fuel production has attracted widespread attention in recent
times [1e3]. However, extraction of lipids from algal cells is
a challenge because of the presence of rigid cell walls surrounding
the algal cells, difculty in handling cells that are only a few
microns in size, and the ubiquitous moisture that interferes with
extraction solvents.
To be effective for algae, an extraction system should: 1)
encompass a method to rupture individual cell walls and 2)
accommodate a high moisture environment. Cost effectively
rupturing cell walls of algae, which are only a few microns in
diameter and in a slurry-phase, is a signicant engineering challenge. Although mechanical cell breakage via extrusion-like
systems is a possibility, the presence of large amounts of water
hinders development of adequate shear to promote cell wall
rupture. The presence of lipids in exterior membranes of algal cells
further increases shear force requirements to generate adequate
shear. Therefore, a pragmatic cell wall breakage method should
accommodate materials that are in a high moisture slurry-phase
environment.
Our experiments suggest that harvested algae, depending on the
harvesting method, could contain as high as 99% (w/w) moisture
(1% solids) (wet basis). The algal solids concentration in a
* Corresponding author.
E-mail address: sfernando@tamu.edu (S. Fernando).
0960-1481/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.renene.2012.04.039

non-concentrated efuent directly from an algal pond or a bioreactor


would contain only 0.1% (w/w) (or 1 g/L) solids. Algae slurry harvested
via occulation and coagulation prior to dewatering contain
approximately 1% solids (w/w) (or 10 g/L). Moisture could be reduced
to approximately 85e90% w/w with centrifugation or 70% if occulation and sedimentation is used [4]. However, further removal of
moisture can only be obtained by drying. Consequently, the moisture
removal costs prior to processing algae can be substantial [5].
To avoid high drying costs, effective lipid extraction techniques
for algae should tolerate high moisture environments. One of the
most signicant drawbacks of conventional solvent extraction
techniques is the interference of polar water with the migration of
non-polar lipophilic solvents into the lipid-containing cellular
matrix-hindering efcient transfer of oil in to the solvent phase. As
a result, using conventional hydrophobic oil extraction solvents like
hexane for algal oil extraction is estimated to cost six-fold more
than similar processes in oil seed industry [5]. The amount of water
present in algae increases the amount of extractant needed for the
process [6e10]. Consequently, laboratory-scale extractions use
a hydrophilic-hydrophobic solvent combination (such as methanol/
chloroform) that makes the solvent-system amphiphilic [6].
However, it is unlikely that such a system would be commercially
feasible because of high downstream solvent separation costs.
In recent years, various methods of cell disruption techniques
such as autoclaving, bead beating, microwaving, osmotic shock and
sonication for disrupting cells of various micro-algal species has
been investigated by several authors [11,12]. Some of these
methods have shown to yield almost three times higher lipid yields,

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308

without cell disruption, compared to traditional Bligh and Dyer


method. However most of these methods cannot be scaled up for
industrial level and the costs for separating methanol from aqueous
phase is difcult.
Supercritical CO2 has been suggested for extracting lipids [13].
However, the need of high pressures (in the neighborhood of
300 bars) makes the process energy intensive. Also it is important
to note that supercritical liquid extraction also requires a pretreatment to ensure high lipid yields e which adds to the processing
costs [13].
Enzymatic hydrolysis [14] of algal cell wall has been suggested
with the goal of weakening the cell wall to assist lipid extraction.
Studies have shown high lipid extraction efciencies, as much as
84%. However sophisticated process and cost of enzymes makes
such processes yet impractical in indusial scale.
These challenges led us to consider development of a solvent
extraction technique that is tolerant to high moisture and that is
scalable. The rst step in oil extraction is rupturing the algal cell
walls to facilitate solvent migration deep in to the lipid-containing
algal cellular matrix. It was decided to use high pressure homogenization to rupture cell walls. Selection of homogenization was
inspired by favorable recommendations made by previous studies
and the relative ease of scalability. However, during this initial
design and development phase, signicant challenges that are
unique to algal processing were identied. The objective of this
paper is to introduce several measurement techniques and characterization methods attempted, discuss associated challenges
when using these techniques and make recommendations on the
suitability of these methods in algae-based process development.
1.1. Homogenization of algae and particle size detection
The rationale for homogenization is several-fold, the most
evident being the ability of a homogenizer to handle slurry-phase
materials in a continuous stream. It was hypothesized that the
sudden pressure differential between the nozzle and the outside
environment combined with shear forces generated when the high
velocity uid stream passes through the nozzle would induce
rupturing of the cell walls. Similar cell lysing operations have been
performed on yeast and Escherichia Coli cells [15e18]. Also, the
increase of temperature during homogenization was speculated to
assist in softening of the cell walls that in turn would help in the
rupturing process. An attractive advantage of high pressure
homogenization is the relative ease of scalability. For example,
there are industrial scale systems that have throughput capacities
over 50,000 L/h at moderate pressure (<68.948 MPa) and up to
5750 L/h at high pressure (up to 310.264 MPa).
The high pressure homogenizer used for this study was
purchased from NanoDeBEE, BEE International, South Easton,
Massachusetts (Fig. 1). In this homogenizer, the pressure applied on
a sample could be varied between 10,000 (68.948 MPa) to
45,000 PSI (310.264 MPa). The average throughput of the equipment was 50 ml/min. For this study, three nozzles with orice
diameters of 100, 130, and 190 mm were used. A reciprocating
piston driven by pressurized hydraulic uid was used to force the
material through the nozzle creating a high velocity jet. This liquid
jet was then forced through a homogenization cell where high
intra-material shear forces were generated. These shear forces are
dependent on the pressure applied, viscosity of the uid, and the
nozzle size used. In case of algae cell breakage, the resultant
temperature (25e70  C) and the number of passes (1e6 passes)
through the nozzle were also considered.
An initial screening study was designed towards detecting
whether homogenization had an effect on algal cell wall rupture.
For this experiment, we used Nannochloris oculata (UTEX 1998)

301

Fig. 1. High-pressure homogenizer.

which have a particle size of approximately 1e2 mm per cell and


was grown in Bold-modied Basel media. During the inception of
the studies, Malvern Mastersizer 2000 (Malvern Instruments Ltd.,
Westborough, MA) with a Hydro SM attachment was used for
quantitative particle sizing assessment while optical and confocal
laser scanning microscopy was used for qualitative assessment.
Algal samples harvested using coagulation and centrifugation
were used. The Malvern Mastersizer utilizes Mie theory laser
diffraction [19,20] and can detect particle sizes ranging 0.1 and
200 mm. Particles were dispersed in methanol before injection into
the Malvern. A particle refractive index of 1.46 (based on cellulose)
and a dispersant refractive index of 1.36 were used. Particle size
distributions of the algal samples were measured before and after
homogenization at various pressures and number of passes
(Table 1).
Fig. 2 depicts the particle size distribution of algal broth after
homogenization under varying conditions. The variables are named
according to the labeling described in Table 1. The variable named
Algae-Raw represents the control (non-homogenized) sample.
A bimodal distribution was observed for homogenized algae.
One peak was observed at 50 mm for the control algae, and 2 peaks
were observed for homogenized algae around 38 and 58 mm. We
conjecture that the peak at 50 mm represents algal ocks formed by
the harvesting mechanism used (occulation). However, some of
those ocks may break in to smaller ones during homogenization,
yielding a bimodal distribution. In order to explain this observation,
a series of microscopic images of samples prior to and after
homogenization were analyzed. A representative selection of two
such images is depicted in Fig. 3. While some algal cells stayed
segregated in the medium after homogenization, others tended to
agglomerate forming clusters. Also, larger cells were observed
scattered throughout the sample, which we suspect to be
a contaminant (see Fig. 3 left).
Although a reduction of the average particle size was expected
as a result of homogenization, we did not observe any signicant,
uniform shift of the particle size distribution(s). Regardless of the
intensity variation of the variables, a signicant correlation

Table 1
Parameter variables used during the initial study.
Sample ID

Nozzle

Z5:25:2:1R
Z5:40:1:1R
Z5:40:2:1R
Z5:25:1:1R

Z5-130
Z5-130
Z5-130
Z5-130

Pressure

mm
mm
mm
mm

25,000
40,000
40,000
25,000

PSI
PSI
PSI
PSI

Number of passes
(172.369
(275.790
(275.790
(172.369

MPa)
MPa)
MPa)
MPa)

Initial algae concentration was approximately 35 g/L solids.

2
1
2
1

302

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308

Fig. 2. Particle size distributions of algal samples (from 10 to 100 mm) after various
homogenization treatments. (Note that, 25 PSI  103 172.369 MPa and
40 PSI  103 310.264 MPa).

between any of the variables and particle size distribution was not
observed. However, particle size distribution data did not match
observations made with the microscopic images. The images suggested that most of the algal cells were almost entirely ruptured
after homogenization (see Fig. 3 right). Hypothesized explanations
for this observation include:
1. The cells, though ruptured, retain their size and shape (analogous to a partially ruptured but intact rubber ball) while some
agglomerate into ocks.
2. Since larger particles (in this case non disrupted cells and cell
agglomerates) scatter a signicantly larger amounts of light as
compared to smaller particles (in this case cell debris) at the
same concentration, cell debris may get shielded from
appearing in the particle size distribution data [21].
In order to rule out the rst possibility above, a series of
experiments were carried out with a particle sizer with a lower
minimum detection limit (Delsa Nano eC, Beckman Coulter, Opa
Locka, Florida). If the rst hypothesis is correct, particle size should
increase or remain the same after homogenization. The detection
range of this particle seizer was 0.6 nme10 mm. A standard operating procedure was developed to measure the particle size of algae
without using a special solvent. The following parameters for water

were used in measurements: refractive index of 1.33; viscosity of


0.89 cP; and dielectric constant of 78.3.
A sample particle size distribution of N. oculata containing
medium in its native state (non-homogenized) and after homogenizing four times through a Z8 Nozzle (195 mm diameter) at
10,000 PSI (68.948 MPa) is shown in Fig. 4. Note that the samples
were taken directly from the bioreactor, without using any harvesting mechanism like coagulation and/or centrifugation. Here, it
was observed that the peak particle size was around 2 mm e unlike
50 mm in the previous study. Also, average particle size decreased
after homogenization. These observations conrm our suspicion
that the larger average particle size spawned by the earlier experiment was due to algal ocks (which likely resulted from harvesting
procedures used). Although we were able to obtain precise particle
size readings for native algae, experiments with addition of standardized particles of 0.1 mm (much smaller than that of algal cells)
indicated that the particle sizer was not able to detect the smaller
particles when these co-exist with larger particles. It is hypothesized that the larger particles shield the smaller particles from
detection (conrming our initial suspicion). This suggests that even
if some fraction of algal cells is broken, the fraction of broken cells
could escape undetected as the particle sizing equipment generate
size distribution(s) as fractions (or percentages) of only detectable
material (for example, if 50% of cells with initial diameter D were
ruptured and all the broken cell material was shielded since those
were small, the particle sizer would still predict that 100% cells are
of diameter D after cell rupture). Thus the size distribution results
before and after cell breakage may not give accurate insights to
make effective, quantitative comparisons on how much cells were
broken. As a result, we decided to abandon using particle sizers for
detecting algal cell rupturing.
1.1.1. Cell breakage quantication via hemocytometry
Subsequent to the rst round of experiments, the suitability of
using cell counting via hemocytometry to quantify the degree of
algal cell disruption after homogenization was evaluated. A
hemocytometer is a slide with a gridded chamber that attaches
onto an optical microscope. The gridded chamber accepts a known
volume of liquid sample. The number of microscopic bodies (such
as algae) can then be counted via observation under the microscope. Since the volume of the sample is known, it is possible to
calculate the cell density with reasonable accuracy.
Initial algal cell counting experiments were conducted using
a reusable hemocytometer with 100 mm chamber depth. However,
this option was not considered to be feasible due to difculties in
cell counting resulting from overlapping cells in the 100 mm deep

Fig. 3. Live algae (left) and a homogenized algal sample (right).

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308

303

Fig. 5. Algal photobioreactor.

Table 2
List of variables used for the screening study.

Fig. 4. Particle size distribution of a raw N. oculata algal sample.

chamber. Alternatively, a disposable version of cell counters with


shallower chambers (C-Chip DHC-S01 semen counting chambers
from Incyto, Chungnam-do, Korea) was used. These are Neubauer
improved counting chambers with 10 ml volumes and designed for
accommodating smaller-sized cells.
For imaging the cells in the counting chamber, a Zeiss Axiphot
optical microscope was used with 20 object resolution. A digital,
black and white camera was used for capturing sections of the
counting chamber separately as the entire counting grid was not
able to be captured in one image using the required magnication.
A freeware with image processing capabilities (ImageJ 1.42q from
National Institute of Health, USA) was used for counting cells in the
images [22,23]. This method is widely adopted to detect degree of
cell disruption in the medical/biological community [24,25]. The
degree of cell breakage was characterized as a percentage of the
total number of intact cells observed per unit volume. Double
counting of cells in multiple images was avoided by cropping
images along the gridline.
For growing algae used in this experiment, Bold-modied Basel
[26] fresh water concentrated solution (Sigma Aldrich, St. Louis,
Missouri) was used as the nutrient medium. This was selected because
other collaborating research groups were successfully using it for
growing N. oculata. Ammonium chloride and potassium phosphate
was used as the nitrogen and phosphorus sources. Although using CO2
enriched air stream is desired, there are reports where successful
algae culturing was done using air without CO2 enrichment. In this
study, 12-h day and night illumination cycle was simulated using 2,
15 W orescent bulbs (Fig. 5). To increase lipid concentration, algal
cultures were subjected to nitrogen starvation for two weeks. Samples
which were subjected to this treatment are named as stressed algae
in this paper. Samples which were not grown in nutrient depleted
medium are referred as non-stressed algae.
2. Screening study for algal breakage using high pressure
homogenization
Parameters listed in Table 2 were identied as possible variables
that can affect the degree of cell rupture during homogenization.
A two-level fractional factorial design (252) was adopted to

Factor

Variable High

Extent of stress
Concentration
No of Passes
Pressure
Nozzle size

A
B
C
D
E

Low

Stressed
Non Stressed
100 g/L
1 g/L
1
4
25,000 PSI (172.369 MPa) 10,000 PSI (68.948 MPa)
100 mm (Z4)
195 mm (Z8)

investigate the preliminary effects of these factors. In the experimental design, the generator terms D AB and E AC were used to
construct the alias structure in the statistical analytical software.
The software generates aliases as shown in the Table 3. Any pattern
observed in a term should be expected from its aliases terms as
well [27].
After processing the samples, the number of remaining intact
cells was counted using the aforementioned hemocytometry
technique.
The difference of the intact cell count before and after homogenization directly correlates to the extent of cell disruption. A
collection of microscopic images with the cells in the gridded
counting chambers are depicted in Fig. 6. Intact cells, with
increasing pressure treatment, disappear in the solution matrix
because ruptured cell particles are smaller than the resolution
selected to image the intact cells.
In order to capture the degree of cell breakage, the intact cell
fraction after homogenization was calculated according to the
following formula:

Cell fraction

Cell density in sample after treatment


Cell density in sample before treatment

Results were analyzed using Design Expert software (Stat-Ease,


Inc., Minneapolis, Minnesota). Results of analysis of variance
(ANOVA) are depicted in Table 4. The analysis revealed that nozzle
Table 3
Aliases for the screening study.
Term

Aliases

A-stress
B-concentration
C-no of passes
D-pressure
E-nozzle
BC
BE

BD, CE
AD
AE
AB
AC
DE
CD

304

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308

Fig. 6. Counting chambers loaded with algal broth with (a) no processing, (b) 1 pass through the 195 mm homoginising nozel at 10 PSI  103(68.948 MPa) (c) 2 pass through the
195 mm homoginising nozel at 10 PSI  103(68.948 MPa).

size did not signicantly affect the degree of cell breakage however,
note that the interaction term BC, concentration e nozzle size, is
signicant since the concentration term is signicant. This observation is further illustrated in the Fig. 7 in the form of a Pareto
analysis. This analysis depicts the value of the square root of the
remaining cell fraction after subjecting to considered variables and
their interactions. According to this analysis, the degree of stress,
number of passes and pressure are inversely related to intact cell

fraction and concentration (Note: the negative effects means that


the increase in the factor value decreases remaining cell fraction).
The two interaction terms, i.e., cell concentration & number of
passes and concentration & nozzle size, are directly related to the
intact cell fraction (Note: positive effects means that the increase
in the factor value increases the remaining cell fraction).
Plots shown in Fig. 8 demonstrate the variation of intact cell
fraction with the variation of selected variables. A square root

Table 4
Analysis of variance (ANOVA) table [partial sum of squares e type III].
Source

Sum of squares

df

Mean square

F value

p-value Prob > F

Model
A-stress
B-concentration
C-no of pass
D-pressure
E-nozzle
BC
BE
Pure error
Cor total

2.996
0.058
0.510
0.362
0.974
0.003
0.725
0.364
0.012
3.008

7
1
1
1
1
1
1
1
16
23

0.428
0.058
0.510
0.362
0.974
0.003
0.725
0.364
0.001

561.116
76.478
668.904
474.067
1276.643
3.302
950.783
477.638

<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0880
<0.0001
<0.0001

Fig. 7. Pareto analysis of the effects of various variables on degree of cell breakage.

Signicant

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308

305

Fig. 8. Variation of cell fraction with two selected factors, when other factors are xed at given value. (Pressure in PSI and nozzle size in mm) (a) pressure 10000.00 PSI
(68.948 MPa), stress 0.50, nozzle size 147.50 mm; (b) pressure 25,000 PSI (172.364 MPa), stress 0.50, nozzle size 147.50 mm; (c) concentration 1.00, no of passes 2.50,
nozzle 147.50 mm; (d) concentration 10.00, no of passes 2.50, nozzle 147.50 mm; (e) no of passes 1.00, stress 0.50, pressure 17,500 PSI (120.658 MPa) (f) no of
passes 4.00, stress 0.50, pressure 17,500 PSI (120.658 MPa).

306

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308

Table 5
List of variables for full-factorial-design.
Nozzle size

Pressure

100 mm (Z4)
130 mm (Z5)
195 mm (Z8)

10
20
30
40

PSI
PSI
PSI
PSI






Number of passes
103 (68.948 MPa)
103(137.895 MPa)
103(206.843 MPa)
103(275.790 MPa)

1e6 (six levels)

transformation was performed on the intact cell fraction to obtain


an approximately normal distribution (tested by plotting the BoxCox plot). Individual plots depict the variation of cell fraction in
relation to selected two factors while the other factors were kept
xed. According to the plots, it is evident that lower concentrations
and higher number of passes (Fig. 8(a) and (b)) resulted in higher
degrees of cell breakage. Also note that at lower concentrations,
nozzle size had an inversely proportional relationship with cell
fraction while at higher algal concentrations the relationship was
positively correlated.
To further elucidate the effect of homogenization conditions on
the degree of cell breakage, a full factorial experiment was performed using selected variables. Algal concentration and the extent
of stress were xed by using non stressed algal broth having

0.675 A optical density (0.15 mg/ml) for all experimental units.


These variables were xed at given values since at the onset of the
study, access to stressed or concentrated algal samples were
limited. The effect of variation of these values will be investigated in
a later study. However, the homogenizer nozzle size was varied,
primarily to conrm the ndings of the previous screening study
(252 two-level fractional factorial design). The list of variables for
this full-factorial-design is shown in Table 5.
Subsequent to above treatments, each sample was placed in
Neubauer improved counting chambers and the number of cells
remaining intact were counted as described earlier.
Fig. 9 clearly depicts that the increase of pressure and the
number of passes reduces the visible cell count, which correlates to
increased cell disruption. This is conrmed from the optical
microscopic images as depicted in Fig. 3 as well. Even in this
experiment, nozzle size was determined to be not signicant conrming the observations from previous screening experiments.
It was observed that the cell count increased after rst and
second runs through the homogenizer at 10,000 PSI (68.948 MPa).
This can be attributed to breakage of cell coagulations into individual cells.
Nozzle size did not affect the degree of cell rupture, which
implies that the impact of shear exerted upon the cell walls from

Fig. 9. Fraction of cells remaining intact at different pressures and number of passes using different nozzle diameters. (a) 195 mm nozzle (b) 130 mm nozzle (c) 100 mm nozzle (d)
change in cell fraction with nozzle size and pressure after 2 passes. (Please note that the pressure is given in PSI  103).

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308

307

imperative that there is an optimum level of cell disruption


required to cause adequate exposure of lipid bodies while not
causing downstream separation issues.
3. Conclusions

Fig. 10. Desirability of pressure and nozzle size to minimize cell fraction after 4th pass.

the nozzle walls was minimal. Intuitively, smaller nozzles should


impart more resistance to forward movement of the cell mass
rendering more shear onto the algal cell walls. Apparently, this is
not the case and the pressure differential alone has a signicant
effect on rupturing the cell walls. This is a signicant nding since it
shows the possibility of using a larger nozzle size without
compromising the cell lyses, given that the pressure differential
across the nozzle is maintained. Increasing the nozzle size is
desirable for the processes because a larger nozzle is less likely to
clog during real-life operation.
Fig. 10 depicts the desirability of pressure and nozzle size to
minimize the fraction of intact cells after the 4th pass. In this case
desirability predictions are useful to select the most desired
combination of nozzle size and pressure that result in maximum
cell breakage. Therefore, it is possible to conclude that higher
pressures are desirable to obtain higher degrees of cell breakage.
One of the basic questions consistently raised is the energy
requirements of high pressure homogenization. Work done by the
piston can be calculated by multiplying operating pressure (P) and
the volume (V) of the algal broth processed. Accordingly, the
theoretical energy requirement for processing 1 m3 of algal slurry
in 10,000 PSI (68.95 MPa) pressure in a single pass is 69 MJ in this
unit. This should be compared with the energy available in algal
broth to evaluate energy efciency of the process. If it is assumed
that the specic energy of dried algae is 20 MJ/kg, solids concentration (wt) of algal slurry should be at least 0.345% to get a net
energy output from homogenization process. Since efciency losses
should be accounted for (and possibly multiple passes may be
required to obtain adequate cell rupture), it is obvious that the
homogenizer should be able to handle slurries of much higher
solids concentration. In fact, during preliminary studies we have
processed slurries up to 10% solids (directly from centrifugation) in
the homogenizer without any difculty. These preliminary calculations project the feasibility of using homogenization for
commercial scale algae processing. However a more detailed
energy analysis is necessary after deciding the optimal conditions
(e.g. number of passes and pressure) that maximize oil yields.
Another point to ponder is the fate of non-lipid algal debris after
homogenization. Although increased cell rupture maximizes
solvent accessibility to lipids, this also increases the number of
smaller-sized non-lipid particles suspending in the matrix.
Preliminary studies with solvent extraction have indicated that
heavily homogenized algal slurry tends to misciblize the lipophilic
extraction solvent in water making a hard-to-separate single-phase
solution. This may be as a result of compounds from cellular matrix
acting as amphiphiles making emulsions. Accordingly, it is

Particle sizing was attempted as a quantitative method to


evaluate the extent of breakage of N. oculata algal cells. Issues with
formation of algal ocks and shielding effects limit the use of
particle sizing to detect the degree of cell breakage. However, cell
counting using a Neubauer improved cell counter proved to
successfully discern the degree of cell breakage quantitatively.
High pressure homogenization proved to be an effective technique to rupture N. oculata cell walls. An initial partial factorial
design showed that the outlet diameter of the nozzle does not
signicantly affect the degree of cell wall lyses. The most signicant
parameters were the pressure differential across the nozzle and the
number of passes through the homogenizer. The concentration of
the algal culture and the level of stress of the culture were also
signicant but to a lesser degree.
Acknowledgments
This research was funded by the National Alliance of Advance
Biofuels and Bioproducts (NAABB) via Department of Energy. The
authors acknowledge the staff of Microscopy and Imaging Center
(especially Dr. Stanislav Vitha for assisting us with the acquisition of
optical microscopic images) and Material Characterization Facility
(especially Dr. Amanda Young for her assistance in obtaining
spectrouorometric data) at Texas A&M University.
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