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Algalcellruptureusinghighpressurehomogenizationasapreludetooilextraction

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Renewable Energy 48 (2012) 300e308

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Technical note

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

Nalin Samarasinghe, Sandun Fernando*, Ronald Lacey, William Brock FaulknerDepartment 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

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

Keywords:AlgaeHomogenizationParticle sizingCell countingCell breakage

* Corresponding author.E-mail address: sfernando@tamu.edu (S. Fernando

0960-1481/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.renene.2012.04.039

a b s t r a c t

Research associated with extraction of lipids from algae and the use of algal biomass for transportationfuel production is still in its infancy. Measurement techniques associated with algal systems are still notwell established and, as a result, pitfalls exist. This paper discusses several measurement and charac-terization techniques that were attempted while developing an algal oil extraction system and theireffectiveness. Measurements were made primarily to quantify the degree of algal cell breakage usinghigh pressure homogenization e as a prelude to solvent extraction.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The potential use of micro-algal biomass and lipids as substratesfor fuel production has attracted widespread attention in recenttimes [1e3]. However, extraction of lipids from algal cells isa challenge because of the presence of rigid cell walls surroundingthe algal cells, difficulty in handling cells that are only a fewmicrons in size, and the ubiquitous moisture that interferes withextraction 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 effectivelyrupturing cell walls of algae, which are only a few microns indiameter and in a slurry-phase, is a significant engineering chal-lenge. Although mechanical cell breakage via extrusion-likesystems is a possibility, the presence of large amounts of waterhinders development of adequate shear to promote cell wallrupture. The presence of lipids in exterior membranes of algal cellsfurther increases shear force requirements to generate adequateshear. Therefore, a pragmatic cell wall breakage method shouldaccommodate materials that are in a high moisture slurry-phaseenvironment.

Our experiments suggest that harvested algae, depending on theharvesting method, could contain as high as 99% (w/w) moisture(1% solids) (wet basis). The algal solids concentration in a

).

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non-concentrated effluent directly froman algal pond or a bioreactorwouldcontainonly0.1%(w/w) (or1g/L) solids.Algae slurryharvestedvia flocculation and coagulation prior to dewatering containapproximately 1% solids (w/w) (or 10 g/L).Moisture could be reducedto approximately 85e90% w/w with centrifugation or 70% if floccu-lation and sedimentation is used [4]. However, further removal ofmoisture can only be obtained by drying. Consequently, themoistureremoval costs prior to processing algae can be substantial [5].

To avoid high drying costs, effective lipid extraction techniquesfor algae should tolerate high moisture environments. One of themost significant drawbacks of conventional solvent extractiontechniques is the interference of polar water with the migration ofnon-polar lipophilic solvents into the lipid-containing cellularmatrix-hindering efficient transfer of oil in to the solvent phase. Asa result, using conventional hydrophobic oil extraction solvents likehexane for algal oil extraction is estimated to cost six-fold morethan similar processes in oil seed industry [5]. The amount of waterpresent in algae increases the amount of extractant needed for theprocess [6e10]. Consequently, laboratory-scale extractions usea hydrophilic-hydrophobic solvent combination (such asmethanol/chloroform) that makes the solvent-system amphiphilic [6].However, it is unlikely that such a system would be commerciallyfeasible because of high downstream solvent separation costs.

In recent years, various methods of cell disruption techniquessuch as autoclaving, bead beating, microwaving, osmotic shock andsonication for disrupting cells of various micro-algal species hasbeen investigated by several authors [11,12]. Some of thesemethods have shown to yield almost three times higher lipid yields,

Fig. 1. High-pressure homogenizer.

Table 1Parameter variables used during the initial study.

Sample ID Nozzle Pressure Number of passes

Z5:25:2:1R Z5-130 mm 25,000 PSI (172.369 MPa) 2Z5:40:1:1R Z5-130 mm 40,000 PSI (275.790 MPa) 1Z5:40:2:1R Z5-130 mm 40,000 PSI (275.790 MPa) 2Z5:25:1:1R Z5-130 mm 25,000 PSI (172.369 MPa) 1

Initial algae concentration was approximately 35 g/L solids.

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308 301

without cell disruption, compared to traditional Bligh and Dyermethod. However most of these methods cannot be scaled up forindustrial level and the costs for separatingmethanol from aqueousphase is difficult.

Supercritical CO2 has been suggested for extracting lipids [13].However, the need of high pressures (in the neighborhood of300 bars) makes the process energy intensive. Also it is importantto note that supercritical liquid extraction also requires a pretreat-ment to ensure high lipid yields e which adds to the processingcosts [13].

Enzymatic hydrolysis [14] of algal cell wall has been suggestedwith the goal of weakening the cell wall to assist lipid extraction.Studies have shown high lipid extraction efficiencies, as much as84%. However sophisticated process and cost of enzymes makessuch processes yet impractical in indusial scale.

These challenges led us to consider development of a solventextraction technique that is tolerant to high moisture and that isscalable. The first step in oil extraction is rupturing the algal cellwalls to facilitate solvent migration deep in to the lipid-containingalgal cellular matrix. It was decided to use high pressure homog-enization to rupture cell walls. Selection of homogenization wasinspired by favorable recommendations made by previous studiesand the relative ease of scalability. However, during this initialdesign and development phase, significant challenges that areunique to algal processing were identified. The objective of thispaper is to introduce several measurement techniques and char-acterization methods attempted, discuss associated challengeswhen using these techniques and make recommendations on thesuitability 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 mostevident being the ability of a homogenizer to handle slurry-phasematerials in a continuous stream. It was hypothesized that thesudden pressure differential between the nozzle and the outsideenvironment combined with shear forces generated when the highvelocity fluid stream passes through the nozzle would inducerupturing of the cell walls. Similar cell lysing operations have beenperformed on yeast and Escherichia Coli cells [15e18]. Also, theincrease of temperature during homogenization was speculated toassist in softening of the cell walls that in turn would help in therupturing process. An attractive advantage of high pressurehomogenization is the relative ease of scalability. For example,there are industrial scale systems that have throughput capacitiesover 50,000 L/h at moderate pressure (<68.948 MPa) and up to5750 L/h at high pressure (up to 310.264 MPa).

The high pressure homogenizer used for this study waspurchased from NanoDeBEE, BEE International, South Easton,Massachusetts (Fig. 1). In this homogenizer, the pressure applied ona sample could be varied between 10,000 (68.948 MPa) to45,000 PSI (310.264 MPa). The average throughput of the equip-ment was 50 ml/min. For this study, three nozzles with orificediameters of 100, 130, and 190 mm were used. A reciprocatingpiston driven by pressurized hydraulic fluid was used to force thematerial through the nozzle creating a high velocity jet. This liquidjet was then forced through a homogenization cell where highintra-material shear forces were generated. These shear forces aredependent on the pressure applied, viscosity of the fluid, and thenozzle size used. In case of algae cell breakage, the resultanttemperature (25e70 �C) and the number of passes (1e6 passes)through the nozzle were also considered.

An initial screening study was designed towards detectingwhether homogenization had an effect on algal cell wall rupture.For this experiment, we used Nannochloris oculata (UTEX 1998)

which have a particle size of approximately 1e2 mm per cell andwas grown in Bold-modified Basel media. During the inception ofthe studies, Malvern Mastersizer 2000 (Malvern Instruments Ltd.,Westborough, MA) with a Hydro SM attachment was used forquantitative particle sizing assessment while optical and confocallaser scanning microscopy was used for qualitative assessment.

Algal samples harvested using coagulation and centrifugationwere used. The Malvern Mastersizer utilizes Mie theory laserdiffraction [19,20] and can detect particle sizes ranging 0.1 and200 mm. Particles were dispersed in methanol before injection intothe Malvern. A particle refractive index of 1.46 (based on cellulose)and a dispersant refractive index of 1.36 were used. Particle sizedistributions of the algal samples were measured before and afterhomogenization at various pressures and number of passes(Table 1).

Fig. 2 depicts the particle size distribution of algal broth afterhomogenization under varying conditions. The variables are namedaccording 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 peakswere observed for homogenized algae around 38 and 58 mm. Weconjecture that the peak at 50 mm represents algal flocks formed bythe harvesting mechanism used (flocculation). However, some ofthose flocks 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 afterhomogenization were analyzed. A representative selection of twosuch images is depicted in Fig. 3. While some algal cells stayedsegregated in the medium after homogenization, others tended toagglomerate forming clusters. Also, larger cells were observedscattered throughout the sample, which we suspect to bea contaminant (see Fig. 3 left).

Although a reduction of the average particle size was expectedas a result of homogenization, we did not observe any significant,uniform shift of the particle size distribution(s). Regardless of theintensity variation of the variables, a significant correlation

Fig. 2. Particle size distributions of algal samples (from 10 to 100 mm) after varioushomogenization treatments. (Note that, 25 PSI � 103 ¼ 172.369 MPa and40 PSI � 103 ¼ 310.264 MPa).

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308302

between any of the variables and particle size distribution was notobserved. However, particle size distribution data did not matchobservations made with the microscopic images. The images sug-gested that most of the algal cells were almost entirely rupturedafter homogenization (see Fig. 3 right). Hypothesized explanationsfor this observation include:

1. The cells, though ruptured, retain their size and shape (analo-gous to a partially ruptured but intact rubber ball) while someagglomerate into flocks.

2. Since larger particles (in this case non disrupted cells and cellagglomerates) scatter a significantly larger amounts of light ascompared to smaller particles (in this case cell debris) at thesame concentration, cell debris may get shielded fromappearing in the particle size distribution data [21].

In order to rule out the first possibility above, a series ofexperiments were carried out with a particle sizer with a lowerminimum detection limit (Delsa� Nano eC, Beckman Coulter, OpaLocka, Florida). If the first hypothesis is correct, particle size shouldincrease or remain the same after homogenization. The detectionrange of this particle seizer was 0.6 nme10 mm. A standard oper-ating procedurewas developed to measure the particle size of algaewithout using a special solvent. The following parameters for water

Fig. 3. Live algae (left) and a hom

were used in measurements: refractive index of 1.33; viscosity of0.89 cP; and dielectric constant of 78.3.

A sample particle size distribution of N. oculata containingmedium in its native state (non-homogenized) and after homoge-nizing four times through a Z8 Nozzle (195 mm diameter) at10,000 PSI (68.948 MPa) is shown in Fig. 4. Note that the sampleswere taken directly from the bioreactor, without using any har-vesting mechanism like coagulation and/or centrifugation. Here, itwas observed that the peak particle size was around 2 mm e unlike50 mm in the previous study. Also, average particle size decreasedafter homogenization. These observations confirm our suspicionthat the larger average particle size spawned by the earlier exper-iment was due to algal flocks (which likely resulted fromharvestingprocedures used). Although we were able to obtain precise particlesize readings for native algae, experiments with addition of stan-dardized particles of 0.1 mm (much smaller than that of algal cells)indicated that the particle sizer was not able to detect the smallerparticles when these co-exist with larger particles. It is hypothe-sized that the larger particles shield the smaller particles fromdetection (confirming our initial suspicion). This suggests that evenif some fraction of algal cells is broken, the fraction of broken cellscould escape undetected as the particle sizing equipment generatesize distribution(s) as fractions (or percentages) of only detectablematerial (for example, if 50% of cells with initial diameter D wereruptured and all the broken cell material was shielded since thosewere small, the particle sizer would still predict that 100% cells areof diameter D after cell rupture). Thus the size distribution resultsbefore and after cell breakage may not give accurate insights tomake effective, quantitative comparisons on how much cells werebroken. As a result, we decided to abandon using particle sizers fordetecting algal cell rupturing.

1.1.1. Cell breakage quantification via hemocytometrySubsequent to the first round of experiments, the suitability of

using cell counting via hemocytometry to quantify the degree ofalgal cell disruption after homogenization was evaluated. Ahemocytometer is a slide with a gridded chamber that attachesonto an optical microscope. The gridded chamber accepts a knownvolume of liquid sample. The number of microscopic bodies (suchas algae) can then be counted via observation under the micro-scope. Since the volume of the sample is known, it is possible tocalculate the cell density with reasonable accuracy.

Initial algal cell counting experiments were conducted usinga reusable hemocytometer with 100 mm chamber depth. However,this option was not considered to be feasible due to difficulties incell counting resulting from overlapping cells in the 100 mm deep

ogenized algal sample (right).

Fig. 5. Algal photobioreactor.

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

Table 2List of variables used for the screening study.

Factor Variable High Low

Extent of stress A Stressed Non StressedConcentration B 100 g/L 1 g/LNo of Passes C 1 4Pressure D 25,000 PSI (172.369 MPa) 10,000 PSI (68.948 MPa)Nozzle size E 195 mm (Z8) 100 mm (Z4)

Table 3Aliases for the screening study.

Term Aliases

A-stress BD, CEB-concentration ADC-no of passes AED-pressure ABE-nozzle ACBC DEBE CD

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308 303

chamber. Alternatively, a disposable version of cell counters withshallower chambers (C-Chip DHC-S01 semen counting chambersfrom Incyto, Chungnam-do, Korea) was used. These are Neubauerimproved counting chambers with 10 ml volumes and designed foraccommodating smaller-sized cells.

For imaging the cells in the counting chamber, a Zeiss Axiphotoptical microscope was used with 20� object resolution. A digital,black and white camera was used for capturing sections of thecounting chamber separately as the entire counting grid was notable to be captured in one image using the required magnification.A freeware with image processing capabilities (“ImageJ 1.42q” fromNational Institute of Health, USA) was used for counting cells in theimages [22,23]. This method is widely adopted to detect degree ofcell disruption in the medical/biological community [24,25]. Thedegree of cell breakage was characterized as a percentage of thetotal number of intact cells observed per unit volume. Doublecounting of cells in multiple images was avoided by croppingimages along the gridline.

For growing algae used in this experiment, Bold-modified Basel[26] fresh water concentrated solution (Sigma Aldrich, St. Louis,Missouri)wasusedas thenutrientmedium. Thiswasselectedbecauseother collaborating research groups were successfully using it forgrowing N. oculata. Ammonium chloride and potassium phosphatewasusedas thenitrogenandphosphorus sources.AlthoughusingCO2enriched air stream is desired, there are reports where successfulalgae culturing was done using air without CO2 enrichment. In thisstudy, 12-h day and night illumination cycle was simulated using 2,15 W florescent bulbs (Fig. 5). To increase lipid concentration, algalcultureswere subjected tonitrogen starvation for twoweeks. Sampleswhichwere subjected to this treatment are named as “stressed” algaein this paper. Samples which were not grown in nutrient depletedmedium are referred as “non-stressed” algae.

2. Screening study for algal breakage using high pressurehomogenization

Parameters listed in Table 2 were identified as possible variablesthat can affect the degree of cell rupture during homogenization.A two-level fractional factorial design (25�2) was adopted to

investigate the preliminary effects of these factors. In the experi-mental design, the generator terms D¼AB and E¼ ACwere used toconstruct the alias structure in the statistical analytical software.The software generates aliases as shown in the Table 3. Any patternobserved in a term should be expected from its aliases terms aswell [27].

After processing the samples, the number of remaining intactcells was counted using the aforementioned hemocytometrytechnique.

The difference of the intact cell count before and after homog-enization directly correlates to the extent of cell disruption. Acollection of microscopic images with the cells in the griddedcounting chambers are depicted in Fig. 6. Intact cells, withincreasing pressure treatment, disappear in the solution matrixbecause ruptured cell particles are smaller than the resolutionselected to image the intact cells.

In order to capture the degree of cell breakage, the intact “cellfraction” after homogenization was calculated according to thefollowing formula:

Cell fraction ¼ Cell density in sample after treatmentCell 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

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 the195 mm homoginising nozel at 10 PSI � 103(68.948 MPa).

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308304

size did not significantly affect the degree of cell breakage however,note that the interaction term BC, concentration e nozzle size, issignificant since the concentration term is significant. This obser-vation is further illustrated in the Fig. 7 in the form of a Paretoanalysis. This analysis depicts the value of the square root of theremaining cell fraction after subjecting to considered variables andtheir interactions. According to this analysis, the degree of stress,number of passes and pressure are inversely related to “intact cell

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

Source Sum of squares df Mean squa

Model 2.996 7 0.428A-stress 0.058 1 0.058B-concentration 0.510 1 0.510C-no of pass 0.362 1 0.362D-pressure 0.974 1 0.974E-nozzle 0.003 1 0.003BC 0.725 1 0.725BE 0.364 1 0.364Pure error 0.012 16 0.001Cor total 3.008 23

Fig. 7. Pareto analysis of the effects of vario

fraction” and concentration (Note: the negative effects means thatthe increase in the factor value decreases remaining cell fraction).The two interaction terms, i.e., cell concentration & number ofpasses and concentration & nozzle size, are directly related to the“intact cell fraction” (Note: positive effects means that the increasein the factor value increases the remaining cell fraction).

Plots shown in Fig. 8 demonstrate the variation of intact cellfraction with the variation of selected variables. A square root

re F value p-value Prob > F

561.116 <0.0001 Significant76.478 <0.0001

668.904 <0.0001474.067 <0.0001

1276.643 <0.00013.302 0.0880

950.783 <0.0001477.638 <0.0001

us variables on degree of cell breakage.

Fig. 8. Variation of cell fraction with two selected factors, when other factors are fixed at given value. (Pressure in PSI and nozzle size in mm) (a) pressure ¼ 10000.00 PSI(68.948 MPa), stress ¼ 0.50, nozzle size ¼ 147.50 mm; (b) pressure ¼ 25,000 PSI (172.364 MPa), stress ¼ 0.50, nozzle size ¼ 147.50 mm; (c) concentration ¼ 1.00, no of passes ¼ 2.50,nozzle ¼ 147.50 mm; (d) concentration ¼ 10.00, no of passes ¼ 2.50, nozzle ¼ 147.50 mm; (e) no of passes ¼ 1.00, stress ¼ 0.50, pressure ¼ 17,500 PSI (120.658 MPa) (f) no ofpasses ¼ 4.00, stress ¼ 0.50, pressure ¼ 17,500 PSI (120.658 MPa).

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308 305

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

Nozzle size Pressure Number of passes

100 mm (Z4) 10 PSI � 103 (68.948 MPa) 1e6 (six levels)130 mm (Z5) 20 PSI � 103(137.895 MPa)195 mm (Z8) 30 PSI � 103(206.843 MPa)

40 PSI � 103(275.790 MPa)

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308306

transformation was performed on the intact cell fraction to obtainan approximately normal distribution (tested by plotting the Box-Cox plot). Individual plots depict the variation of cell fraction inrelation to selected two factors while the other factors were keptfixed. According to the plots, it is evident that lower concentrationsand higher number of passes (Fig. 8(a) and (b)) resulted in higherdegrees of cell breakage. Also note that at lower concentrations,nozzle size had an inversely proportional relationship with cellfraction while at higher algal concentrations the relationship waspositively correlated.

To further elucidate the effect of homogenization conditions onthe degree of cell breakage, a full factorial experiment was per-formed using selected variables. Algal concentration and the extentof stress were fixed by using non stressed algal broth having

Fig. 9. Fraction of cells remaining intact at different pressures and number of passes usingchange in cell fraction with nozzle size and pressure after 2 passes. (Please note that the p

0.675 A optical density (0.15 mg/ml) for all experimental units.These variables were fixed at given values since at the onset of thestudy, access to stressed or concentrated algal samples werelimited. The effect of variation of these values will be investigated ina later study. However, the homogenizer nozzle size was varied,primarily to confirm the findings of the previous screening study(25�2 two-level fractional factorial design). The list of variables forthis full-factorial-design is shown in Table 5.

Subsequent to above treatments, each sample was placed inNeubauer improved counting chambers and the number of cellsremaining intact were counted as described earlier.

Fig. 9 clearly depicts that the increase of pressure and thenumber of passes reduces the visible cell count, which correlates toincreased cell disruption. This is confirmed from the opticalmicroscopic images as depicted in Fig. 3 as well. Even in thisexperiment, nozzle size was determined to be not significant -confirming the observations from previous screening experiments.

It was observed that the cell count increased after first andsecond runs through the homogenizer at 10,000 PSI (68.948 MPa).This can be attributed to breakage of cell coagulations into indi-vidual cells.

Nozzle size did not affect the degree of cell rupture, whichimplies that the impact of shear exerted upon the cell walls from

different nozzle diameters. (a) 195 mm nozzle (b) 130 mm nozzle (c) 100 mm nozzle (d)ressure is given in PSI � 103).

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

N. Samarasinghe et al. / Renewable Energy 48 (2012) 300e308 307

the nozzle walls was minimal. Intuitively, smaller nozzles shouldimpart more resistance to forward movement of the cell massrendering more shear onto the algal cell walls. Apparently, this isnot the case and the pressure differential alone has a significanteffect on rupturing the cell walls. This is a significant finding since itshows the possibility of using a larger nozzle size withoutcompromising the cell lyses, given that the pressure differentialacross the nozzle is maintained. Increasing the nozzle size isdesirable for the processes because a larger nozzle is less likely toclog during real-life operation.

Fig. 10 depicts the “desirability” of pressure and nozzle size tominimize the fraction of intact cells after the 4th pass. In this casedesirability predictions are useful to select the most desiredcombination of nozzle size and pressure that result in maximumcell breakage. Therefore, it is possible to conclude that higherpressures are desirable to obtain higher degrees of cell breakage.

One of the basic questions consistently raised is the energyrequirements of high pressure homogenization. Work done by thepiston can be calculated by multiplying operating pressure (P) andthe volume (V) of the algal broth processed. Accordingly, thetheoretical energy requirement for processing 1 m3 of algal slurryin 10,000 PSI (68.95 MPa) pressure in a single pass is 69 MJ in thisunit. This should be compared with the energy available in algalbroth to evaluate energy efficiency of the process. If it is assumedthat the specific energy of dried algae is 20 MJ/kg, solids concen-tration (wt) of algal slurry should be at least 0.345% to get a netenergy output fromhomogenization process. Since efficiency lossesshould be accounted for (and possibly multiple passes may berequired to obtain adequate cell rupture), it is obvious that thehomogenizer should be able to handle slurries of much highersolids concentration. In fact, during preliminary studies we haveprocessed slurries up to 10% solids (directly from centrifugation) inthe homogenizer without any difficulty. These preliminary calcu-lations project the feasibility of using homogenization forcommercial scale algae processing. However a more detailedenergy 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 afterhomogenization. Although increased cell rupture maximizessolvent accessibility to lipids, this also increases the number ofsmaller-sized non-lipid particles suspending in the matrix.Preliminary studies with solvent extraction have indicated thatheavily homogenized algal slurry tends to misciblize the lipophilicextraction solvent inwater making a hard-to-separate single-phasesolution. This may be as a result of compounds from cellular matrixacting as amphiphiles making emulsions. Accordingly, it is

imperative that there is an optimum level of cell disruptionrequired to cause adequate exposure of lipid bodies while notcausing downstream separation issues.

3. Conclusions

Particle sizing was attempted as a quantitative method toevaluate the extent of breakage of N. oculata algal cells. Issues withformation of algal flocks and shielding effects limit the use ofparticle sizing to detect the degree of cell breakage. However, cellcounting using a Neubauer improved cell counter proved tosuccessfully discern the degree of cell breakage quantitatively.

High pressure homogenization proved to be an effective tech-nique to rupture N. oculata cell walls. An initial partial factorialdesign showed that the outlet diameter of the nozzle does notsignificantly affect the degree of cell wall lyses. The most significantparameters were the pressure differential across the nozzle and thenumber of passes through the homogenizer. The concentration ofthe algal culture and the level of stress of the culture were alsosignificant but to a lesser degree.

Acknowledgments

This research was funded by the National Alliance of AdvanceBiofuels and Bioproducts (NAABB) via Department of Energy. Theauthors acknowledge the staff of Microscopy and Imaging Center(especially Dr. Stanislav Vitha for assisting uswith the acquisition ofoptical microscopic images) and Material Characterization Facility(especially Dr. Amanda Young for her assistance in obtainingspectrofluorometric data) at Texas A&M University.

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