cell disruption for microalgae biorefineries

Biotechnology Advances xxx (2015) xxx–xxx

JBA-06890; No of Pages 18

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Research review paper

Cell disruption for microalgae biorefineries

E. Günerken a,b,⁎, E. D'Hondt a, M.H.M. Eppink b, L. Garcia-Gonzalez a, K. Elst a, R.H. Wijffels b,c

a VITO NV, Boeretang 200, 2400 Mol, Belgiumb Wageningen University, Bioprocess Engineering, AlgaePARC, P.O. Box 16, 6700 AA Wageningen, Netherlandsc University of Nordland, Faculty of Biosciences and Aquaculture, N-8049 Bodø, Norway

⁎ Corresponding author at: Flemish Institute for TechnoE-mail addresses: [email protected] (E. Günerk

(L. Garcia-Gonzalez), [email protected] (K. Elst), rene.wij

http://dx.doi.org/10.1016/j.biotechadv.2015.01.0080734-9750/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Günerken E, et alj.biotechadv.2015.01.008

a b s t r a c t
a r t i c l e i n f o
Article history:Received 4 September 2014Received in revised form 6 January 2015Accepted 27 January 2015Available online xxxx

Keywords:Cell disruptionBead millingHigh pressure homogenizationHigh speed homogenizationUltrasonicationMicrowave treatmentPulsed electric field treatmentNon-mechanical cell disruptionMicroalgaeBiorefinery

Microalgae are a potential source for various valuable chemicals for commercial applications ranging fromnutraceuticals to fuels. Objective in a biorefinery is to utilize biomass ingredients efficiently similarly to pe-troleum refineries in which oil is fractionated in fuels and a variety of products with higher value. Down-stream processes in microalgae biorefineries consist of different steps whereof cell disruption is the mostcrucial part. To maintain the functionality of algae biochemicals during cell disruption while obtaininghigh disruption yields is an important challenge. Despite this need, studies onmild disruption of microalgaecells are limited. This review article focuses on the evaluation of conventional and emerging cell disruptiontechnologies, and a comparison thereof with respect to their potential for the future microalgaebiorefineries. The discussed techniques are bead milling, high pressure homogenization, high speed ho-mogenization, ultrasonication, microwave treatment, pulsed electric field treatment, non-mechanical celldisruption and some emerging technologies.

© 2015 Elsevier Inc. All rights reserved.

Contents

General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Cell disruption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Mechanical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Bead milling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0High pressure homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0High speed homogenization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Ultrasonication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Microwave treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Pulsed electric field treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Non-mechanical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Enzymatic cell lysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Chemical cell disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

New developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Mechanism of cell disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Effect on product quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Specific energy consumption (kWh/kg dry biomass) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Practical scalability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Gaps in data comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Future needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

logical Research (VITO), Boeretang 200, VITO MAT Building, 2400 Mol, Belgium. Tel.: +32 485 202413.en), [email protected] (E. D'Hondt), [email protected] (M.H.M. Eppink), [email protected]@wur.nl (R.H. Wijffels).

, Cell disruption for microalgae biorefineries, Biotechnol Adv (2015), http://dx.doi.org/10.1016/

http://dx.doi.org/10.1016/j.biotechadv.2015.01.008

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2 E. Günerken et al. / Biotechnology Advances xxx (2015) xxx–xxx

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

General introduction

Microalgae are considered as an industrially interesting source forthe sustainable production of numerous products because of signifi-cantly higher growth rates, photosynthetic efficiencies and processoptimization possibilities compared to conventional terrestrial plantsand are already used in many commercial applications including food,animal feed, cosmetics, pollution abatement, and therapeutics(Brennan and Owende, 2010; Butler, 2006; Chisti, 2007; de Stefanoet al., 2011; Harun et al., 2011; Hazar and Aydin, 2010; Huang et al.,2010; Li et al., 2008; Liu et al., 2008; Posten and Walter, 2012a,b;Rodolfi et al., 2009; Solovchenco et al., 2008; Spolaore et al., 2006;Thamsiriroj and Murphy, 2009; Tornabene et al., 1983; Usui andIkenouchi, 1997). Moreover, microalgae can be cultured in marginalareas in brackish or saline water resulting in a lower water and landfootprint (Tsukahara and Sawayama, 2005). Despite these advan-tages, microalgae also have their limitations. One of the major eco-nomic bottlenecks cited in the literature due to the high energydemand is downstream processing. The biorefinery concept, analo-gous to petroleum refineries, aims to fractionate biomass into fuelsand multiple added-value co-products simultaneously by focusingon downstream processes (Chisti, 2007; Clark et al., 2009; DOE,2010; IEA Bioenergy, 2009; Sánchez Mirón et al., 2003; SchmidStraiger, 2009). The main issue in designing a biorefinery is optimiz-ing the balance between products and energy to obtain a maximumfinancial profit (Anastas and Zimmerman, 2003). Products cannot berecovered effectively from microalgae using methods designed forproduct extrusion from crops such as soybeans since the microalgaemorphology is different from land crops. Microalgae cells are small,covered with a relatively thick cell wall and products are usually lo-cated in globules or bound to cell membranes, making extraction ofintracellular products challenging. Additionally, the cell wall structureof microalgae is complex and poorly understood (Gerken et al., 2013;

Fig. 1. Classification of the c

Please cite this article as: Günerken E, et al, Cell disruption for microalgj.biotechadv.2015.01.008

Scholz et al., 2014) and is known to have an important effect on thedisruption efficiency. However, there are no broad studies investigat-ing the relation between cell wall composition disruption efficiencyand energy consumption. Thus, inter- and intra-species variationsand variations observed from different cultivation conditions makepredictions or extrapolations very difficult. Some microalgal cells arevery easy to break so a mild or more energy efficient disruption tech-nique can be chosen. However, calculating a universal energy con-sumption value for a given cell disruption method and thereforemaking a direct comparison of different techniques is impossible.

Despite these challenges, efficient cell disruption is an essential pre-treatment step tomaximize product recovery frommicroalgae biomass.A feasible energy-efficient cell disruptionmethod should be establishedto ensure a low operating cost, high product recovery, and high qualityof the extracted products.

This review article focuses on the evaluation of the fundamentals,physics, and case studies of conventional cell disruption techniques, al-ready in use for microalgae, as well as emerging mild disruption tech-nologies. All techniques are evaluated and compared with respect tothe potential for the future microalgae biorefineries.

Cell disruption

A variety of disruptionmethods is currently available for cell disrup-tion. In general, these techniques are divided into two main groupsbased on the working mechanism of microalgal cellular disintegration,i.e., (i) mechanical and (ii) non-mechanical methods (Fig. 1)(Agerkvist and Enfors, 1990; Chen et al., 2009; Chisti and Moo-Young,1986; Lee et al., 1998, 2010; Middelberg, 1995; Mutanda et al., 2011).In this section, microalgae cell disruption methods and related casestudies will be discussed, divided into mechanical and non-mechanical methods, and evaluated in terms of suitability for mildmicroalgae biorefinery. An overview of the parameters affecting the

ell disruption methods.

ae biorefineries, Biotechnol Adv (2015), http://dx.doi.org/10.1016/



3E. Günerken et al. / Biotechnology Advances xxx (2015) xxx–xxx

disruption yield for the studied cell disruption processes is provided inTable 1, the summary of the case studies in Tables 2–8, and the compar-ison of cell disruption methods in Table 9.

Mechanical methods

Destruction of the cell wall in a non-specific manner is usuallyachieved by mechanical forces such as solid-shear forces (e.g., beadmill, high speed homogenization), liquid-shear forces (e.g., high pres-sure homogenization, microfluidization), energy transfer through

Table 1Process parameters of the cell disruption methods.

Disruption method Mechanism of cell disruption

Bead milling Mechanical compaction andshear stress

High pressure homogenization Cavitation and shear stress

High speed homogenization Cavitation and shear stress

Ultrasonication Cavitation and free radicalformation

Microwave treatment Temperature increase,molecular energy increase

Pulsed electric field treatment Proliferation due to electricity

Enzymatic lysis Enzyme substrate interaction

Chemical treatment Chemical substrate interaction


waves (e.g., ultrasonication, microwave), currents (e.g., pulsed electricfield) or heat (e.g., thermolysis, autoclaving).

Bead millingHigh disruption efficiency in single-pass operations, high through-

put, high biomass loading, good temperature control, commerciallyavailable equipment, easy scale up procedures, and low labor intensityare the primary factors that make bead milling an interesting cell dis-ruption method with high potential for industrial implementation(Bierau et al., 1999; Jahanshahi et al., 2002). The most common design

Process parameters References

Agitation disk design, speedBead filling, size, materialDry weightFeed rateGrowth phase and conditionsMicroalgae typeTimeCooling

Engler (1985),Kula and Schütte (1987)

Cycle numberDry weightFlow rateGrowth phase and conditionsHomogenizator designMicroalgae typePressure

Schütte et al. (1983),Kula and Schütte (1987)

Blade design, speedDry weightGrowth phase and conditionsMicroalgae typeTime

Shirgaonkar et al. (1998),Kumar and Pandit (1999)

Cycle number and timeDry weightGrowth phase and conditionsMicroalgae typePower of ultrasound

Chandler et al. (2001),Taylor et al. (2001),Onyeche et al. (2002),Fykse et al. (2003),Borthwick et al. (2005),Gogate and Pandit (2008)

AgitationDry weightGrowth phase and conditionsMicroalgae typePower of microwaveTime

Pan et al. (2002),Terigar et al. (2010),Balasubramanian et al. (2011)

Conductivity (electrolyte concentration)CurrentDry weightGrowth phase and conditionsMicroalgae typeOscillationTime

Brown et al. (1992),Muraji et al. (1993),Ganeva et al. (1995),Qin et al. (1995),Ho and Mittal (1996),Muraji et al. (1998),Muraji et al., 1999;Ade Omowaye et al. (2003)

AgitationDry weightEnzyme concentrationEnzyme typeGrowth phase and conditionsMicroalgae typeOxygen levelType and amount of bufferTemperaturePressureTime

Andrews and Asenjo (1987),Harrison (1991)

AgitationChemical concentrationChemical typeDry weightGrowth phase and conditionsMicroalgae typeTemperatureTime

Middelberg (1995),Mendes Pinto et al. (2001),Harun and Danquah (2011b),Harun et al. (2011),Halim et al. (2012b),Miranda et al. (2012)




Table 2Summary and comparison of case studies on bead milling.

Micro-algae Product Conditions Scale Outcome Analyses Reference

Bead millingScenedesmus quadricauda (fresh) Disrupted

biomassBallotini beads, 33% beadfilling, 2800 rpm agitator speed,5 min, 5% DCW

1 l grindingchamber,

55% cell disintegration Cell count Hedenskog et al. (1969)Scenedesmus quadricauda(spray dried)

87% cell disintegration

Scenedesmus quadricauda(fresh)

0.35–0.5 mm beads, 50% beadfilling, 1450 rpm agitator speed,40 l/h flow rate, 5% DCW

5 liter grindingchamber

90% cell disintegration

Chlorella sp. Disruptedbiomass

7.5 kW, 0.5 mm ZrO2 beads,70% beads filling, 15.8% DCW,62 kg/h feed rate, 90 min

1.5 l grindingchamber

98.5% cell disintegrationof Chlorella

Cell count,dry weight

Doucha and Lívanský (2008)

3.3 kW, 0.42–0.58 mmglass beads, 82% beads filling,10.7% DCW, 3 kg/h feed rate


99.9% cell disintegrationof Chlorella and 90.2% celldisintegration of bacteria

25 kW, 0.6–0.8 mm ZrO2 beads,85% beads filling, 12.4% DCW,35 kg/h Feed rate


85.29% cell disintegrationof Chlorella and 81.2% celldisintegration of bacteria

3 kW, 0.3–0.4 mm glass beads,85% beads filling, 6.9% DCW,10 kg/h Feed rate, 3000 rpmagitator speed, 2 cycles

0.6 l grindingchamber,

98–99% cell disintegrationof Chlorella and 99.5% celldisintegration of bacteria

Tetraselmis sp. Protein 3.3–4 kW, 0.3–0.4–0.6 mmceramic beads, 65% bead filling,12% DCW, 1.5 l/min flow rate,30 min


21% of proteins transferredto algae juice after treatment

Total protein Schwenzfeier et al. (2011)


for this system is shown in Fig. 2. The shaftmay carry agitators of varieddesign (concentric or eccentric disks or rings) that export kinetic energyto small steel, glass or ceramic beads in the chamber resulting in multi-ple collisions (Chisti and Moo-Young, 1986). It is hypothesized that thesuspended cells are disrupted in the bead collision zones by compactionor shear forces (Bunge et al., 1992; Melendres et al., 1992) with energytransfer from the beads to the cells (MacNeill et al., 1985).

Based on the results of the case studies, summarized in Table 2, it isconcluded that increasing the bead diameter has a positive effect whenthe beads are smaller than 0.5 mm and has a negative effect above0.5 mm (Doucha and Lívanský, 2008). Additionally, high density beads(e.g., zirconium) are more effective in media with high viscosity whilelow density beads (e.g., glass) are preferred in low viscous media(Schütte and Kula, 1990; Doucha and Lívanský, 2008; Hedenskog et al.,1969). Increasing the treatment time, agitator tip speed (peripheral veloc-ity 5–10 m·s−1), number of cycles and bead filling up to 85% of grindingchamber volume have a positive effect on the disruption process (Douchaand Lívanský, 2008; Hedenskog et al., 1969; Postma et al., 2014). Increas-ing dry cell weight (DCW; 0.5–8% w/w) and biomass flow rate (kg DCW/h) negatively affect the cell disruption efficiency. However, increasingthese parameters positively affect the cost of the cell disintegrationprocess by reducing the specific energy consumption (Doucha andLívanský, 2008; Postma et al., 2014). The effect of biomass flow rate onspecific energy consumption shown in Fig. 6. The biomass flow rate isgiven as DCW influent (kg/h) and the specific energy consumption(kWh/kg) is calculated based on total energy consumed (kWh) to disruptper kg (in dry basis) of microalgae biomass. Doucha and Lívanský (2008)recorded, for an increasing retention time from 1.3 to 2.3min, an increaseof 70% in biomass disruption efficiency and a decrease of 44% in the spe-cific energy consumption. Oppositely, at significantly larger retentiontimes (16 and 28 min), the specific energy consumption increased with32% because of a lower throughput. The energy consumption of singlepass bead milling operation of Chlorella sp. by using a Netzsch, LabstarLS1 recorded as 0.85 kWh/kg dry weight by Doucha and Lívanský(2008). Similarly, Postma et al. (2014) calculated the energy consumptionfor disrupting Chlorella sp. by a semi continiously operated Dyno-Mill Re-search Lab as 0.81 kWh/kg dry weight. The recorded values are just 13%-14% of the caloric value of microalgae biomass calculated by Weyer et al.(2010) (6.083 kWh/kg). In practice, however, the specific energy con-sumption highly depends on DCW concentration/load, the species andthe growth conditions of biomass.


Despite many positive characteristics, the high energy demand ofbead milling make it less favorable for microalgae biorefineries. The inef-ficient energy transfer from the rotating shaft to the individual cells(Schütte and Kula, 1990) and energy conversion into heat (Doucha andLívanský, 2008) require intensive, energy demanding cooling to allowthe recovery of functional fragile products (e.g., RuBisCO). Additionally,the formation of veryfine cell debris and non-selective distribution of bio-chemicals over the soluble and solid phase (Günerken et al., 2013) resultin increased downstream processing costs. Although protein extractabili-ty and digestibility are increased after treatment (Hedenskog et al., 1969;Schwenzfeier et al., 2011) and the method is effective against microbialand fungal infestations present in the microalgae culture (Doucha andLívanský, 2008;Hedenskog et al., 1969), it is not an ideal disruptionmeth-od for mild microalgae biorefineries.

High pressure homogenizationHigh pressure homogenizers (HPHs) are especially suitable for

emulsification processes. Various valve-seat configurations are availablefor HPHs to optimize the disruption efficiency (Masucci, 1985; Pandolfe,1993). The cell suspension flows radially across a valve, strikes an im-pact ring, exits the valve and flows either to a second valve or to a dis-charge (Fig. 3). Cell disruption is thus achieved through high-pressureimpact (shear forces) of the accelerated fluid jet on the stationaryvalve surface as well as hydrodynamic cavitation from the pressuredrop induced shear stress (Chisti and Moo-Young, 1986; Halim et al.,2012a,b). Cavitation is defined as a 3-step phenomenon taking placein short time intervals (micro to milliseconds) that starts with the for-mation of bubbles, followed by growth and ends with the collapse ofmicrobubbles. This causes the release of large amounts of energy intoa very small volume. Very high energy densities (energy released perunit volume) are obtained locally which leads to cell disruption(Petrier et al., 1998). An overview of the case studies is given inTable 3 and discussed below.

The literature on HPH shows that a high working pressure follow-ed by the cycle number have the most positive effect on cell disrup-tion efficiency. Lower DCW concentrations and culture stress levels(N-depletion) were significant but to a minor extent and the nozzlediameter was determined as not effective (Halim et al., 2012a,b).The specific energy consumption of HPH is highly dependent onDCW concentration, algae species and the growth conditions of bio-mass. In different studies, the specific energy consumption varies




Table 3Summary and comparison of case studies on high pressure homogenization.


High pressure homogenizationNannochloropsissalina

Anaerobic digestionand biogas fromtreated biomass

100 bar, 2 passes,0.875% DCW

35 ml 32.6% increase in biogasproduction in comparisonwith untreated biomass

Biogas production Schwede et al. (2011)

Chlorococcum sp. Disrupted biomass 850 bar, 0.85%DCW, 4 passes

200 ml Over 90% cell disintegration,83% of colony diameterreduction after first pass

Intact cell count,average colonydiameter measurement

Halim et al. (2012b)

146.94 kWh/kg drybiomass energy consumption

Energy calculations byusing the data fromHalim et al. (2012b)

Lee et al. (2012)

Nannochloropsisoculata

Disrupted biomass,lipid

2760 bars, 0.1% (wet w/w)approx. 0.023–0.035% DCWcell concentration, 4 passes,nitrogen depleted culture

15 ml 67% cell disintegration,8.5 times more oil extractionthan undisrupted algae

Intact cell count,total lipid

Samarasinghe et al. (2012a)

Nannochloropsisoculata

Disrupted biomass 2100 bar, 0.15% (wet w/w)approx. 0.015–0.023% DCW,cell concentration, 100 μmNozzle, 3 passes

15 ml ≈100% cell disintegration Intact cell count Samarasinghe et al. (2012b)

Nannochloropsis sp. Protein 1500 bar, 1% DCW cellconcentration, 6 passes,nitrogen depleted culture

250 ml ≈91% Protein extraction Bradford proteinanalysis

Grimi et al. (2014)


from 0.25 kWh/kg (1% DCW, N-depleted) to 147 kWh/kg (0.85% DCW,no stress) (Grimi et al., 2014; Halim et al., 2012b; Lee et al., 2012). Thelowest recorded specific energy consumption is approximately 4.1% ofthe microalgae biomass' caloric value (6.083 kWh/kg).

Although HPH is, together with bead milling, the most preferredmethod for the industrial scale cell disruption of microalgae, there aresome disadvantages. The main drawback of using HPH in the mildmicroalgae biorefinery is the use of low dry cell weight concentrations(0.01–0.85% w/w). This increases the energy demand of downstreamprocessing and water footprint due to isolation of products from dilutestreams. Also the non-selective intracellular compound release, difficul-ties to break hard cell walls and the generation of very fine cell debrisare among main problems of HPH. Finally, the reduced digestibility ofproteins after treatment (Janczyk et al., 2005; Komaki et al., 1998) canindicate that HPH is not a mild technique and thus not suitable for theisolation of fragile functional compounds.

High speed homogenizationA high-speed homogenizer (HSH) is a stirring device at high rpm

and usually consists of a stator–rotor assembly, preferably made of

Table 4Summary and comparison of case studies on high speed homogenization.

Micro-algae Product Conditions Scale Outcome

High speed homogenizationNannochloropsis sp. Lipid 10,000 rpm for 1 min,

%6 DCW≈16 ml Wet extra

high speereached 7extractio

Nannochloropsis sp. Lipid 12,000 rpm, 1:50(g/ml) biomass:solvent,2% DCW

50 ml %38 ± 2extractio

Phaeodactylumtricornutum

Antioxidant 14,000 rpm, 30 s,1:1 (v/v) EtOH(or MetOH)/water solvent,approx. 0.12% DCW

5 ml EtOH: ≈3ascorbic aactivityMetOH: ≈ascorbic aactivity

Pavlova lutheri 14,000 rpm, 30 s,1:1 (v/v) EtOH(or MetOH)/water solvent,approx. 0.36% DCW

EtOH: ≈2ascorbic aactivityMetOH: ≈ascorbic aactivity


stainless steel, with a variety in designs of stators and rotors. The effec-tive cell disruption mechanisms are hydrodynamic cavitation, generat-ed by stirring at high rpm, and shear forces at the solid–liquidinterphase. When the impeller tip speed reaches a critical value(8500 rpm), hydrodynamic cavitation occurs due to a local pressure de-creases nearly down to the vapor pressure of the liquid (Kumar andPandit, 1999; Shirgaonkar et al., 1998). Subsequently, as the liquidmoves away from the impeller, the liquid pressure restores proportionalto the decrease in velocity and the distance from impeller tip and causesthe collapse of the cavities (Gogate, 2011). An overview of HSH casestudies is given in Table 4 and the main characteristics for the mildmicroalgae biorefinery are discussed below.

High speed homogenization is themost simple, very effective, butaggressive cell disruption method. Advantages are short contacttimes and the potential to process suspensions with relatively highdry cell weight concentration (2–6% w/w) thus reducing the waterfootprint and downstream process costs. Additionally, with HSHincreased extraction yields of different biochemicals were observed(Balasubramanian et al., 2013; González-Delgado and Kafarov,2012; Guedes et al., 2013; Khoo et al., 2011; Wang and Wang,

Analyses Reference

ction yield withd homogenization5.8–78% of dryn yield

Total lipid analysis Wang and Wang (2011)

(w/w) lipidn

Total lipid analysis Balasubramanian et al. (2013)

0 mg equivalentcid/l antioxidant

22.5 mg equivalentcid/l antioxidant

Total intracellularantioxidant determination(ascorbic acid equivalent)

Guedes et al. (2013)

2.5 mg equivalentcid/l antioxidant

20 mg equivalentcid/l antioxidant





2011). Unfortunately, the lowest energy consumption is 156.4% ofthe microalgae biomass' caloric value and protein denaturation dueto shear induced local and bulk temperature increase make thismethod less favorable for mild microalgae biorefinery.

UltrasonicationDuring an ultrasonic treatment, the energy of high frequency acous-

tic waves initiates a cavitation process and a propagating shock waveforms jet streams in the surrounding medium causing cell disruptionby high shear forces (Chisti and Moo-Young, 1986; Mendes Pintoet al., 2001). Numerous designs for ultrasonic systems (Fig. 4) are avail-able for different purposes such as micro/nano emulsion production,cell disruption and product extraction. For bacterial cell disruption, ul-trasonic disrupters operating at 20, 40 kHz and 1 MHz are proposed(Chandler et al., 2001; Fykse et al., 2003; Taylor et al., 2001), but nowa-days only large scale 18, 20, 24, and 30 kHz ultrasonication devices arein use due to energy consumption concerns. In literature the specific en-ergy consumption ranges from efficient disruption with 0.06 kWh/kg(Hielscher, 2011) over inefficient disruption with 36.67 kWh/kg

Table 5Summary and comparison of case studies on ultrasonication.

Micro-algae Product Conditions Scale

UltrasonicationStichococcus sp. Chlorophyll a 70 W, 90 s 3 cycles

with 5 min breaks3 ml

Chlorella sp.Scenedesmusdimorphus

Lipid 100 W, 2 min, 2 cycles 15 ml

Chlorellaprotothecoides

Botryococcus sp. Lipid 10 kHz, 5 min,0.5% DCW

100 ml

Chlorellavulgaris

Scenedesmus sp.

Nannochloropsissalina

Anaerobicdigestionand Biogas fromtreatedBiomass

200 W, 45 s,30 kHz

Analyticavolume ngiven

Chlorellavulgaris

Lipid 600 W, 30 s 34cycles with 5second breaks

Laborator(N50 ml),volume ngiven

Chlorella sp. Lipid 50 kHz, 15 min,0.5% DCW

100 ml

Nostoc sp.

Tolypothrix sp.

Chlorococcumsp.

Disruptedbiomass

130 W, 5 min,5 cycles, 0.85%DCW

200 ml

Scenedesmusobliquus

Fermentablesugars

200 W, 30 s 5 cycleswith 10 min breaks,approx. 7–10% DWC

5 ml

SynechocystisPCC 6803

Lipid 2 kW, 3 min, 52 °Coutflow temperature,approx. 0.2% DCW

Analyticavolume ngiven

2 kW, 30 s 15 cycles with30 s breaks, 26 °C outflowtemperature, approx.0.2% DCW


(Halim et al., 2012b) to efficient disruption with 100 kWh/kg (Leeet al., 2010). The lowest specific energy demand found in literaturewas provided by a device manufacturer and the only shared parameterof the process was the 15% DCW concentration of microalgae feedstock.To reduce the amount of energy needed for cell disruption, ultrasonic vi-bration is frequently combined with chemical cell disruption methods(Nanu et al., 2011; Prabakaran and Ravindran, 2011; Priego Capoteand de Castro, 2007; Sheng et al., 2012; Sherrit et al., 1999; Thoe et al.,1998). In literature the direct effect of ultrasonication on solubilizationand conversion of biochemicals is also studied. The positive effect onsoluble chemical oxygen demand, and nutritional value, the insignifi-cant effect of lipid solubilization and conversion to fermentable sugarsand negative effect on monodigestion determined by different studies(Janczyk et al., 2005; Lee et al., 2010; Miranda et al., 2012; Schwedeet al., 2011; Sheng et al., 2012). An overview of case studies is given inTable 5.

Several forces are behind the mechanism of ultrasonic cell disrup-tion. Ultrasonic vibrations from the emitting tip result in acoustic cav-itation that can disrupt cells as discussed in the High pressure

Outcome Analyses Reference

Local heat caused degradationof chlorophyll a

Chlorophyll a Schumann et al.(2005)

Lipid recovery 21 wt.%No considerable difference incomparison with methods

Total lipids, dryweight

Shen et al. (2009)

Lipid recovery 10.7 wt.%Considerable difference incomparison with methodsLipid recovery 8.8 wt.%Considerable difference incomparison with methods

Total lipid, fattyacid composition

Lee et al. (2010)

Lipid recovery 8 wt.%No considerable difference incomparison with methodsLipid recovery 9 wt.%No considerable difference incomparison with methods

l,ot

21% decrease in biogas productionin comparison with untreatedbiomass

Biogas production Schwede et al.(2011)

y

ot

5.11 fold more extraction thanuntreated cells

Total lipid Zheng et al.(2011)

2.625 fold more extraction thanuntreated cells

Total lipid Prabakaran andRavindran (2011)

2.57 fold more extraction thanuntreated cells3.625 fold more extraction thanuntreated cellsNearly no cell disruption, ≈70%of colony diameter reductionafter 3rd cycle

Intact cell count,average colonydiameter

Halim et al.(2012b)

36.67 kWh/kg dry biomassenergy consumption

Energy calculationsby using the datafrom Halim et al.(2012b)

Lee et al. (2012)

Complex sugars were convertedto fermentable sugars, yield:0.025 equal gram of glucose/grambiomass

Total sugars,monosaccharides

Miranda et al.(2012)

l,ot

27.8% (w/w) Lipid release, SCODincrease as much as 29.8% of totalCOD of biomass

Total lipid, SCODanalysis

Sheng et al. (2012)

14.77% (w/w) lipid release, SCODincrease as much as 6.7% of totalCOD of biomass





homogenization section, but cavitation also results in thermolysis ofwater around the bubbles forming highly reactive free radicals(H•, HO•, and HOO•) (Riesz et al., 1985) that react with the substancesin water. Bubble implosion and fragmentation during acoustic cavita-tion produce micro-regions of extreme conditions with estimatedtemperatures as high as 5000 °C and pressures up to 100 MPa. Duringtreatment, the sample temperature can increase significantly with 50to 90 °C (Borthwick et al., 2005; Chandler et al., 2001; Taylor et al.,2001; Zhang et al., 2007) and destroy proteins and other intracellularmetabolites (Borthwick et al., 2005; Gogate and Pandit, 2008; Sartoryand Grobbelaar, 1984; Schumann et al., 2005; Suslick, 1990). Accord-ing to Gogate and Pandit (2008) the mechanical mechanismsresulting from the intense turbulence associated with liquid circula-tion currents (Luche, 1999; Mason and Lorimer, 1988; Mason andLorimer, 2002) are more effective on the ultrasonic cell disruptionyield than the chemical changes such as the formation of free radicals.The formation of free radicals, however, is themain cause according toZhang andHua (2000) and Zhang et al. (2007). Themajor drawback ofultrasonication of microalgae biomass is the relatively low cell disrup-tion efficiency for some microalgae species and the local and overallheat production. Temperature control during treatment can improveproduct quality, however, the effectiveness of cell disruption de-creases significantly (Sheng et al., 2012). The possibility of combiningultrasonication with different solvent systems or other disruptionmethods to increase the efficiency and decrease the energy demand,remains interesting for the mild microalgae biorefinery concept.

Microwave treatmentMicrowave treatment at 2450MHz is known as the optimal value for

heating, drying and cell disruption (Vasavada, 1986). When a suspen-sion is exposed to microwaves, the microwaves interact selectivelywith the dielectric or polar molecules (e.g., water) and cause localheating as a result of frictional forces from inter- and intramolecularmovements (Amarni and Kadi, 2010). The free water concentration incells contributes to the microwave efficiency for cell disruption. Waterexposed to microwaves reaches the boiling point fast resulting in ex-pansion within the cell and an increase in the internal pressure(Chuanbin et al., 1998). The local heat and pressure combined with

Table 6Summary and comparison of case studies on microwave treatment.

Micro-algae Product Conditions Scale

Microwave treatmentChlorella sp. Lipid 2450 MHz, 100 °C,

5 min, 0.5% DCW100 ml

Nostoc sp.

Tolypothrix sp.

Chlorellavulgaris

Lipid 2450 MHz, 100 °C,5 min

Laboratory (N250 ml),Volume not given

SynechocystisPCC 6803

Lipid 1.4 kW, 57 °C, 1 min Analytical, Volumenot given

1.4 kW, 26 °C, 30 streatment 30 s pause

Scenedesmusobliquus

Lipid 1.2 kW, 2450 MHz, 7.6%DCW, 95 °C, 30 min

Laboratory (N50 ml),Volume not given

Botryococcus sp. Lipid 100 °C, 2450 MHz,5 min, 0.5% DCW

100 mlChlorella vulgarisScenedesmus sp.Dunaliella tertiolecta Pigments 56 °C, 50 W, 5 min,

0.16% DCW in acetone30 ml

Cylindrothecaclosterium


the microwave induced damage to the cell membrane/wall, facilitatesthe recovery of intracellular metabolites (Choi et al., 2006; Rosenbergand Bogl, 1987). To distinguish the effect of microwaves from micro-wave induced temperature increase, the yield of microwave treatmentcompared to a regular heat treatment at the same temperature and37.5–44.4% of total yield determined as related to microwaves(Balasubramanian et al., 2011). However, since only a fraction of thewater is held inside the cells, the majority of the radiation energy isabsorbed by the surrounding medium and lost as heat (Lee et al.,2012) causing protein aggregation and denaturation (Woo et al., 2000).

As shown in Table 6, the variations in species and the DCW (0.16–7.6%) concentrations make a direct comparison of the specific energyconsumption impossible. The potential of using high DCW concentra-tions compared to some other techniques is beneficial for the specificenergy consumption. However, since the disruptive effect is mainlybased on the absorption of microwave energy by water molecules andsubsequently the formation of heat and radicals (Amarni and Kadi,2010; Chuanbin et al., 1998), it can be derived that the effect of micro-wave treatment is higher on diluted suspensions in comparison withconcentrated suspensions. Advantages of microwave treatment are ef-fectiveness, even for robustness, and easy scaled-up (Balasubramanianet al., 2011) because of the simplicity of the technique (Lee et al.,2010). The temperature increase is more homogeneous compared toconventional heating, thus heat related denaturation occurs less readily(Pasquet et al., 2011). Depending on the microalgae species microwavetreatment is even more efficient than both ultrasonication and beadmilling (Prabakaran and Ravindran, 2011; Zheng et al., 2011). Addition-ally, disruption can be combined with selective extraction (microwaveassisted extraction, MAE) which is superior to ultrasonication and mi-crowave heating in terms of speed, efficiency and protection againstthermal denaturation (Balasubramanian et al., 2011; Pasquet et al.,2011).

Even though microwave assisted (extraction) processes have thepotential to increase the extraction yield and decrease the amount ofsolvent, there are also numerous problems. The technique is limited topolar solvents and not suitable for volatile target compounds (Zhenget al., 2011). The formation of free radicals, temperature increase andchemical conversion could interfere with the recuperation of fragile

Outcome Analyses Reference

2.25 fold more extractionthan untreated cells

Total lipid Prabakaran andRavindran (2011)

2.21 fold more extractionthan untreated cells5.33 fold more extractionthan untreated cells3.875 fold more extractionthan untreated cells

Total lipid Zheng et al. (2011)

1.13 fold more extraction thanuntreated cells, SCOD increaseas much as 14.5% of total CODof biomass

Total lipid, SCODanalysis

Sheng et al. (2012)

1.05 fold more extraction thanuntreated cells, SCOD increaseas much as 4.4% of total COD ofbiomass77% of recoverable oil(1.64 fold of only heating method)extracted

Total lipid,lipidcomposition

Balasubramanianet al. (2011)

28.6% (w/w) Lipid extraction Total lipid Lee et al. (2010)10% (w/w) Lipid extraction10.4% (w/w) Lipid extraction≈4.5 μg/l Chlorophyll-a,≈1.4 Chlorophyll-b,≈1.3 β,β-carotene extraction

Pigmentanalysis

Pasquet et al. (2011)

≈4.9 μg/l Chlorophyll-a,≈3.8 fucoxanthin extraction





functional compounds making microwave treatment less favorable formild microalgae biorefinery as a cell disruption method.

Pulsed electric field treatmentPulsed electricfield (PEF) or high intensity electricfield pulse (HELP)

uses an external electric field to induce a critical electrical potentialacross the cell membrane/wall. Cell disruption by PEF is caused by elec-tromechanical compression and electric field-induced tension inducingpore formation in the membrane/wall (electroporation) (BarbosaCánovas et al., 1999; Ho and Mittal, 1996; Tsong, 1990; Weaver andChizmadzhev, 1996; Zimmermann et al., 1985). The size and numberof the pores is directly related to the electric field strength and pulses.It has been demonstrated that pore formation can be reversible or irre-versible (Rols et al., 1990; Tsong, 1990; Weaver et al., 1988). Reversiblecell membrane/wall damage occurs if the total area of induced pores issmall in comparison to the total surface area of the membrane/wall.On the other hand, if the ratio of total pore area to total membrane/wall area exceeds a certain limit as a result of a process at relativelyhigher field strength, the membrane/wall is no longer able to repair it-self and is irreversibly damaged.

PEF does not only destroy the cell wall, but also affects themoleculesinside the cells. Though temperature increase is not the mechanism ofcell disruption, the increase in bulk temperature during treatmentleads to a reduced nutritional value and protein digestibility (Janczyket al., 2005), the decomposition of fragile compounds (Sheng et al.,2011) and an increased extraction of lipids (Eing et al., 2013; Shenget al., 2012; Zbinden et al., 2013) and proteins (Coustets et al., 2013).The specific energydemand, calculatedwith literature data, strongly de-pends on the concentration of the suspension and ranges from 0.42kWh/kg for 10% DCW (Eing et al., 2013) to 239 kWh/kg for 0.03%DCW (Sheng et al., 2011, 2012). An overview of the case studies isgiven in Table 7.

Pulsed electric field can be scaled-up easily and combined with dif-ferent biomass treatment methods. However, the solution, which willbe treated, must be free of ions, i.e., electrically non-conductive, thuslimiting the use of this cell disruption method in mild microalgaebiorefineries. PEF treatment of marine microalgae would require pre-washing and deionization to increase the electrical resistance of themedium surrounding the cells. Additionally, the energy consumptionand cell disruption yield vary dramatically related to the medium com-position. For example, the increased conductivity associated with therelease of compounds from disrupted microalgal cells causes local tem-perature increases and subsequently a decrease in cell disruption

Table 7Summary and comparison of case studies on pulsed electric field.

Micro-algae Product Conditions Scale O

Pulsed electric fieldSynechocystis PCC6803

Lipid 59.67–239 kWh/kg,36–54 °C outflowtemperature, 0.03% DCW

Analytical, volumenot given

DRl

Synechocystis PCC6803

Lipid 120 kWh/kg, 46 °C outflowtemperature, 0.037% DCW

Analytical, volumenot given

Ec

120 kWh/kg, 36 °C outflowtemperature, 0.037% DCW

1u

Nannochloropsissalina

Protein 15.44–30.89 kWh/kg, 37 °Coutflow temperature,0.0545–0.109% DCW

1.08 ml 4tu

Chlorella vulgaris 2.3 kWh/kg, 37 °C outflowtemperature, 0.73% DCW

Auxenochlorellaprotothecoides

Lipid 0.42–0.63 kWh/kg, 10% DCW 2.112 ml Ow

Ankistrodesmusfalcatus

Lipid 5.8 kWh/kg, 0.19% DCW 4 ml Oe


efficiency. The decrease in disruption efficiency due to the release of in-tercellular compounds makes this technique less suitable for the mildmicroalgae biorefinery.

Non-mechanical methods

Non-mechanical methods often involve cell lysis with chemicalagents, enzymes or osmotic shock (Agerkvist and Enfors, 1990; Chistiand Moo-Young, 1986; Lee et al., 1998; Lee et al., 2010; Middelberg,1995; Mutanda et al., 2011). These methods are perceived as more be-nign than mechanical processes since cells are often only perforated orpermeabilized rather than being shredded. For example, chemical andenzymatic methods rely on selective interaction with the cell wall ormembrane components that modifies the cell boundary layer andallows products to leach (Middelberg, 1995; Vogels and Kula, 1992).An overview of case studies for non-mechanical methods is given inTable 8.

Enzymatic cell lysisEnzymatic lysis is an excessively studied cell disruption method due

to its biological specificity, mild operating conditions, low energy re-quirements, low capital investment, and the prevention of aggressivephysical conditions such as high shear stress (Andrews and Asenjo,1987; Harrison, 1991). Since the discovery of lysozyme, many re-searchers have contributed to the understanding of the mechanismsand other basic aspects of lytic enzymes (Salazar and Asenjo, 2007).Glycosidases, glucanases, peptidases and lipases are the main enzymeclasses that have been investigated for cell lysis of differentmicroorgan-isms. During lysis, enzymes bind to specific molecules in the cell mem-brane/wall to hydrolyze the bonds resulting in cell membrane/walldegradation (McKenzie and White, 1991).

Enzymatic treatments on microalgae have been tested in functionof lipid extraction (Zheng et al., 2011) and the conversion of biomassinto biogas through the hydrolysis of polysaccharides, i.e., hemicellu-lose and saccharides of cell wall, and subsequent fermentation (Choiet al., 2010; Fu et al., 2010; Harun and Danquah, 2011a). The main pa-rameter influencing the disruption yield in enzymatic processes is thetype of enzyme (Zheng et al., 2011) because of the specificy of themechanism. The type of enzyme also largerly determines processcosts and therefore enzyme immobilization (Fu et al., 2010) couldbe a solution to allow the implemention of large scale processes. Incontrast to the specificy, other high value products can be convertedresulting in the loss of valuable end products such as astaxanthin

utcome Analyses Reference

W loss % 1.37–% 9.54,educed solvent need foripid extraction

Cell viability,Total lipids,Lipid composition

Sheng et al. (2011)

xtraction similar to untreatedells, SCOD increase 4.9%

Total lipid,SCOD analysis

Sheng et al. (2012)

.09 fold more extraction thanntreated cells, SCOD 1.4%fold more extraction with waterhan methanol extraction ofntreated cells

Bradford total protein,SDS-PAGE

Coustets et al. (2013)

ver 3 fold more extractionith ethanol

Water soluble drydontents,Carbohydrate,Lipids

Eing et al. (2013)

ver 2 fold more extraction withthyl acetate-methanol

Microscopicinvestigation,Total lipids,FAME analysis

Zbinden et al. (2013)




Table 8Summary and comparison of case studies on enzymatic lysis and chemical treatment.


Enzymatic treatmentChlamydomonasreinhardtiiUTEX 90

Dextrin Thermostableα-amylase0.005%, 90 °C, 30 min

Laboratory,volume notgiven

25.21 g/l dextrin Dextrin Choi et al. (2010)

Haematococcuspluvialis

Astaxanthin 0.1% protease K and0.5% driselase, 1 h, pH5.8, 30 °C

Laboratory,volume notgiven


Astaxanthin, totalcarotenoids

Mendes Pinto et al.(2001)

Chlorellavulgaris

Lipid Cellulase, 10 h, pH 4.8,55 °C, 5 mg/l enzyme

Analytical,volume notgiven


Total lipid Zheng et al. (2011)

Lysozyme, 10 h, 55 °C,5 mg/l enzyme


Snailase, 2 h, 37 °C,5 mg/l enzyme


Chlorellapyrenoidosa

Carbohydratesfromcellulose,lipids

Cellulase, 24 h, pH 4.6,50 °C, 140 mg/m2

enzyme,2% DCW

15 ml 62% cellulose hydrolysis, 75%increaset in lipid extraction

Total carbohydrates,reducing sugar,immobilized enzymecontent, FAME analysis

Fu et al. (2010)

Chemical treatmentHaematococcuspluvialis

Astaxanthin 0.1 M HCl,15–30 min

Laboratory,volume not given


Astaxanthin, totalcarotenoids

Mendes Pinto et al.(2001)

0.1 M NaOH,15–30 min


1.8–2.2 fold more extractionthan untreated cells

Chlorococcumhumicola

Fermentablesugars

0.56 M (v/v) H2SO4,160 °C, 15 min


Complex sugars were convertedto fermentable sugars, 0.52 gethanolfermentation from treatedmicroalgaebiomass

Carbohydrates, ethanol Harun andDanquah(2011b)

Chlorococcuminfusionum

Fermentablesugars

0.3 M NaOH, 120 °C,60 min, 5% DCW

Laboratory,100 ml

Complex sugars were converted tofermentable sugars, 0.26 g ethanolfermentation for per gram treatedmicroalgae biomass

Ethanol, glucose, cell size Harun et al. (2011)

Chlorococcum sp. Fermentablesugars

1.51 M H2SO4, 160 °C,45 min, 0.85% DCW

Laboratory,volumenot given

Proteins and pigments weredestroyed.Complex sugars were converted tofermentable sugars

Intact cell count, averagecolony diameter

Halim et al.(2012b)

Scenedesmusobliquus

Fermentablesugars

1 M H2SO4, 120 °C,30 min, 10% DCW

Laboratory,5 ml

Complex sugars were converted tofermentable sugars, yield: 0.286equal g of glucose/g biomass

Total sugars,monosaccharides

Miranda et al.(2012)


due to oxidation (Mendes Pinto et al., 2001). Working in anaerobicconditions would require process adaptations, but could be a solutionto prevent product degradation. Because of a strong dependancy be-tween the concentrations of the reagents, i.e., reactive bonds in thebiomass and the activity and amount of enzymes, finding a good bal-ance between the amount of biomass and (immobilized) enzymes isneeded for an economically interesting process. Increasing DCW con-centration up to 1% enhances the cell disruption efficiency (Harun andDanquah, 2011a), but further increase between 2 and 6% results in adecreased efficiency (Fu et al., 2010; Harun and Danquah, 2011a). De-pending on the type of enzyme, other parameters like temperature,pH, salt concentration and the biomass lipid content can cause signif-icant changes in the disruption efficiency (Fu et al., 2010; Harun andDanquah, 2011a).

Generally, an enzymatic treatment is gentle, has a high selectivityand scale-up is relatively easy. Compared to microwave andultrasonication, an enzymatic treatment can even result in a betterlipid extraction yield (Zheng et al., 2011). However, somedrawbacks af-fecting the efficiency of a disruption process, are long process times,thus a low production capacity compared to mechanical or chemicaldisruption, and product inhibition (Harun and Danquah, 2011a).Today the main limitations of using enzymes in the biorefinery aretheir high cost and the fact that not many enzymes are suitable foralgae disruption. Enzyme immobilization could lower the neededamount of enzymes and additionally reduce the downstream processcosts since separation of the enzymes from the products would beavoided. Since the effectiveness of the same enzyme differs for different


microalgae and reaction times are generally long, the technique couldpotentially be improved by combining specific (immobilized) enzymeswith other mechanical techniques.

Chemical cell disruptionCell disruption can be caused by a large variety of chemical com-

pounds such as antibiotics, chelating agents, chaotropes, detergents,solvents, hypochlorites, acids and alkali. The selectivity, suitability andefficiency of these compounds are dependent on the cell wall composi-tion of the microorganism (Middelberg, 1995). As all of the chemicalsubstances disrupt the cells differently, there are several mechanismsof chemical cell disruption. Antibiotics usually inhibit the productionof cell membrane components, while chelating agents bind the cationsthat cross-bridge adjacent cell membrane molecules and chaotropesmake the surrounding medium less hydrophilic. Detergents form mi-celles together with membrane molecules while solvents dissolve orperforate cell membrane/wall. Bases saponify the membrane lipidswhile acids lead to poration of the cell membrane/wall (Halim et al.,2012b; Harun and Danquah, 2011b; Harun et al., 2011; Mendes Pintoet al., 2001; Middelberg, 1995; Miranda et al., 2012).

Chemicals such as surfactants and oxidizing chemicals (e.g., surfac-tant, ozone, chlorine, UV-B) are already used to inhibit eutrophication.There are several studies on cell disruption of microalgae with theseagents (Cheng et al., 2010; Ebenezer et al., 2012; Glembin et al., 2013;Huang and Kim, 2013; Hung and Liu, 2006; Miao and Tao, 2009; Pavlićet al., 2005; Ulloa et al., 2012), however, the product quality is highly af-fected by either oxidation (Phe et al., 2005; Saby et al., 1997; Virto et al.,





2005) or disruption agent contamination. Some emerging technologiesrelated to these chemicals, such as pressure-assisted ozonation (PAO) oradvanced oxidation processes (AOP) and peroxone treatment, exist(Huang et al., 2014; Nguyen et al., 2013), however, they are notdiscussed in detail in this review because they are not considered asmild for use in the future biorefinery because of their effect on productquality.

Solvent induced cell disruption. The use of solvents in literature on themicroalgae biorefinery is mainly focused on the extraction of specificbiochemicals, e.g., astaxanthin and c-phycocyanin. Some researchhowever combines extraction with disruption (Benavides andRito-Palomares, 2006; Benavides et al., 2008; Cisneros et al., 2004;Kang and Sim, 2007, 2008) or with cultivation in aqueous two phasesystems (Hejazi and Wijffels, 2004; Hejazi et al., 2004; Jin Young et al.,2004; Kleinegris et al., 2011; León, 2003; Mojaat et al., 2008).Kleinegris et al. (2011) showed that cell death was the mechanism ofthis extraction process which can affect the cell growth rate inrecultivation of treated batches (Jin Young et al., 2004). Additionally, di-rect contact of biomass with the organic phase led to aggregation andthe high concentration of cell fragments resulted in a difficult phase sep-aration and contamination in recultivation (Jin Young et al., 2004).

The data of solvent induced disruption is not displayed in Table 8due to the lack of specific cell disruption data. Despite this, it can be con-cluded that cell disruption with aqueous two-phase systems has poten-tial for the biorefinery as a relatively mild and selective extraction/celldisruption step. However, there are still some problems concerning ef-ficiency, toxicity and economic feasibility, and knowledge on the celldisruption characteristics is incomplete.

Acid & alkali treatment. Acid treatment has been applied to variousmicroalgae biomasses. Acid treatments at high temperatures (≈160°C) generally lead to a higher degree of cell disruption than the sametreatments at lower temperatures (≈120 °C) (Halim et al., 2012b;Harun and Danquah, 2011b; Mendes Pinto et al., 2001; Miranda et al.,2012). Harun et al. (2011) and Mendes Pinto et al. (2001) showedthat the average particle size in alkali-treated samples is decreased.Many small sized cell fragments were formed and microscopic studiesrevealed a less distinct cell wall structure indicating an effective cell dis-ruption. However, high temperatures (120 °C) are needed and alkali in-duced protein denaturation (Molina-Grima et al., 2003) making thistechnique less favorable for mild microalgae biorefinery.

For the recovery of unstable or fragile molecules, mild temperaturesand relatively low concentrations of chemicals are preferred. If thechemical treatment could be combined with other techniques for celldisruption, the resulting mild process would be more suitable for themicroalgae biorefinery. However, the effect of acid and alkali on cellconstitutes, such as denaturation effect of alkali on proteins and degra-dation of pigments by acid, are the problems that should be overcomebefore applying acid and alkali treatment in mild microalgaebiorefineries.

New developments

New developments as well as the new technologies in microalgaecell disruption are emerging rapidly including explosive decompres-sion, atomic force microscopy, laser treatment, microfluidizer, pulsedarc, high frequency focused ultrasonication and cationic polymer coatedmembranes. Studies related to the aforementioned methods are de-scribed below.

Dierkes et al. (2012) studied explosive decompression with CO2,propane or butane of a 18.11% DCW suspension of Haematococcuspluvialis for simultaneous cell disruption and lipid and astaxanthinextraction. As a result, 72.3–92.6% of astaxanthin and 80–100% oflipid was extracted. Because of the high DCW concentration in the in-fluent thus lowering the energy consumption per kg of disrupted


biomass, high extraction yields due to the effectiveness of the tech-nique, mild temperature and the use of gaseous products instead ofliquid, often aggressive chemicals, explosive decompression is apromising technique for the mild microalgae biorefinery. McMillanet al. (2013) used laser technologies to disrupt 30 μl of cell suspen-sions and showed that the method in analytical scale results inrapid cell disruption in comparison with other cell disruption tech-niques. The disruption is induced by high temperature and highshear and because this technique is not scalable, laser treatment inthis form is not a potential disruption method for mild microalgaebiorefinery.

A microfluidizer is a high shear fluid processors that functions as ahigh pressure homogenizer in which large pressure differences accel-erate cell suspension. The difference with the high pressure homoge-nizers is the generation of hydrodynamic impact by branching theflow and reconnecting the branches instead of using impact walls.This reduces cavitation making the method more mild than high pres-sure homogenization (Microfluidics Corp., 2012). The cells aredisrupted by shear and by explosive decompression of the gasentrapped in the sample liquid. Overall, the microfluidizer needs ahigher pressure thus more energy to disrupt the cells in comparisonto explosive decompression. Because of the possibility of treatinghigh DCW concentration samples, no solvent usage, mild temperatureand the potential for up-scaling, microfluidization is a promising tech-nique for mild microalgae biorefinery.

Boussetta et al. (2013) studied pulsed arch technology (i.e., pulsedelectric discharge) to disrupt grape seeds. Although this techniquewas not studied onmicroalgae, it is included as an emerging technology.By using high amplitude electricity discharges for short time courses (inμs level), big cavities are produced causing extreme pressure and tem-perature differences aswell as extreme shear stress. The energy require-ment was calculated as only 1/47 of the energy needed to disrupt grapeseeds with PEF. Although this technology is one of the most aggressivecell disruption technologies studied, it has the potential to be modifiedto a milder cell disruption method, since it has low sample-electriccharge interaction time and temperature/shear effects could be reducedby modifying the vessel design.

Wang et al. (2014) compared high frequency focused ultrasonication(3.2MHz, 40W)with the conventional ultrasonication (20 kHz, 100W)with Scenedesmus dimorphus (UTEX 417) and Nannochloropsis oculata(UTEX 2164) as model microalgae. The data showed that for the samecell disruption efficiency, high frequency focused ultrasonication had ahigher energy efficiency. Additionally, both techniques can be used inseries to acquire a better cell disruption efficiency.

Yoo et al. (2014) used a very gentle method wherein a membranewith a coated cationic polymer disturbs the local electrostatic equilibri-um of the amphiphilic microalgae cell membrane caused by direct con-tact with the tertiary-amine cations. The microalgal culture was simplyshaken together with the membrane resulting in the bursting of thecells.

Comparison

The methods of cell disruption were discussed individually permethod in the Cell disruption section. To evaluate the differences, acomparison of specific parameters is discussed in the following para-graphs. Themethods are compared qualitatively based on theirworkingmechanism, efficiency, product quality, process parameters, energy de-mand, and scalability. Themain characteristicswith a focus on industrialapplicability are summarized in Table 9.

Mechanism of cell disruption

When the methods are evaluated based on the mechanism of celldisruption, it is clear that the best known mechanical cell disruptiontechniques such as bead milling and high speed homogenization are




Table 9Comparison of cell disruption methods in terms of key aspects.

Disruption method Mildness Selective product recovery Optimum DCW concentration Energy consumption Practical scalability Repeatability

Bead milling Yes/no No Concentrated High/medium Yes HighHigh pressure homogenization Yes/no No Diluted/concentrated High/medium Yes HighHigh speed homogenizer No No Diluted High/medium Yes High/mediumUltrasound Yes/no No Diluted Medium/low Yes/no MediumMicrowave Yes/no No Diluted High/medium Yes/no MediumEnzymatic lysis Yes Yes Diluted Low Yes HighChemical treatment Yes/no Yes Diluted/concentrated Medium/low Yes HighPulsed electric field Yes/no No Very diluted/diluted High/medium/low Yes/no Medium


using solid–liquid interfacial shear forces which have a high cell disrup-tion efficiency. The cell disruption during bead milling, high pressurehomogenization and high speed homogenization is explained by fluidand solid–liquid interfacial shear forces while energy transfer throughwaves or currents causes the effects in ultrasound, pulsed electric field(PEF) and microwave treatments. Other techniques apply energybeams directly such as laser or pulsed arc induced cell disruption.Some energy transfer types such as microwave, laser, PEF or heatallow longer transfer distances in comparison with techniques basedon cavitation, such as bead milling, high pressure homogenization,high speed homogenization, ultrasonic treatment and pulsed arc. Non-mechanical methods are based on chemical interactions of organic(e.g., enzymes, organic solvents) or inorganic (alkaline, acid treatment)molecules with the molecules of the cell membrane/wall with or with-out thermal energy transfer.

Effect on product quality

During cell disruption, the aforementioned forces cause phenomenasuch as cavitation, temperature and pressure changes,molecular energyvariations, production of free radicals, solid shear, interfacial shear and/or hydrodynamic shear as mentioned in the Cell disruption section.These phenomena can occur individually or together and can affectthe final product quality through degradation of the algal constituentsand/or the formation of impurities. In cavitational methods, all the phe-nomena occur together and cause extreme temperature, pressure vari-ation, high shear rate and formation of free radicals, which highly effectproduct quality and extraction efficiency. Thomas and Geer (2010)reviewed the effect of shear on proteins and concluded that

Fig. 2. BeadAvailable from: AGT-Mining [In


hydrodynamic shear alone is not causing proteins denaturation, but in-terfacial shear stress (air–liquid or solid–liquid)was identified as the ef-fective and predominant mechanism for disruption and proteindenaturation. Thus cell disruption methods that depend on interfacialshear stress such as HSH, HPH and US can cause damage to the proteins(Thomas and Geer, 2010). Additionally, cavitation duringHPH, HSH andultrasonication can lead to the formation of free radicals causing oxida-tion which highly effects product quality (Luche, 1999; Mason andLorimer, 1988; Mason and Lorimer, 2002; Riesz et al., 1985; Zhangand Hua, 2000; Zhang et al., 2007). Product quality, however, is mainlymeasured indirectly via for example digestibility (Komaki et al. (1998)for HPH and Janczyk et al. (2005) for PEF).

In contrast to single cavitation source methods such as HSH, HPHand ultrasonication, cavity formation is more uniform in bead millsresulting in more homogeneous cell disruption. During cell disruptionwith energy waves, the molecular energy and temperature rise. For ex-ample, PEF causes energy variation in adherent cell membrane/wallmolecules, resulting in electroporation of the cell membrane/wall andmicrowave treatment increases the energy of intra- and extracellularwater molecules resulting in cell disruption through the expansion ofintracellular water. Molecular energy variations may cause radical for-mation and unwanted reactions such as oxidation which might reduceproduct quality. Chemical and enzymatic cell disruption may form un-wanted side products that can be present in the end product togetherwith the reagents requiring specific downstream processing steps. Theeffect of cell disruption on product quality is also related to the mor-phology and biochemical composition of microalgae. Komaki et al.(1998) investigated the effect of cell disruption on digestibility with 3different strains of Chlorella vulgaris and showed that the digestibility

mill.ternet], 2014 http://www.agt.cl/mining/doc/6_dmq_mill.pdf.


http://www.agt.cl/mining/doc/6_dmq_mill.pdf



Fig. 3. High pressure homogenizator (Kumar et al., 2013).


of one strain remained unchangedwhile for the two others a significantdecrease was observed. Overall, the effect of cell disruption on productquality can be a direct indicator of the mildness of the cell disruptionprocess. Next to product quality, the selectivity of a disruption processhas an impact on product quality and thedownstreamprocess steps. Se-lectivity in the context of cell disruption is referred to as targeting spe-cific cell compounds. With respect to algae cell disruption, onlychemical treatment and enzymatic lysis are described as selective inthe state of the art.

Specific energy consumption (kWh/kg dry biomass)

The evaluation of literature data on energy consumption and cost ef-fectiveness of the different cell disruption techniques is economicallyrelevant and more straightforward and accurate than comparing celldisruption efficiencies or the increase of biomass utilization (e.g., extrac-tion efficiency, digestibility, mono-digestibility). To allow comparisonbetween the different techniques, in this review the specific energy

Fig. 4. Different designs oAvailable from: Apollo Ultrasonics [


consumption (kWh/kg) was calculated as total energy consumed(kWh) to disrupt 1 kg of dry microalgae biomass (= consumedenergy / (treated biomass ∗ cell disruption yield)). However, the com-parison is only valid if similar conditions are being used (microalgaestrain, stage of growth, fermentation system, DCW concentration etc.).Therefore, the comparison is more indicative than quantitative.

For the non-mechanical methods, energy consumption is changingproportionally to treatment time, temperature and stirring. The settingsof these parameters, and therefore the energy consumption, are relatedto the type of disruption agent, cell properties, morphology, fermenta-tion conditions (e.g., open pond, photobioreactor, temperature, medi-um) and stage of growth. Generally for mechanical methods, theenergy consumption and cost effectiveness are influenced by processparameters, DCW concentration, the scale, type of microalgae, fermen-tation conditions (e.g., open pond or photobioreactor) and stage ofgrowth, whereof the dry cell weight concentration has a strong influ-ence. To allow comparison between continuous and batch mechanicalprocesses, the biomass treatment rate, kg of dry biomass disrupted (ef-fluent) or treated (influent) per hour (kg/h) together with cell disrup-tion yield, can be used which depend on dry cell weight concentrationof the processed mixture (kg/L), flow rate (L/h) or batch volume andtreatment time (h) as important parameters for industrial cell disrup-tion. As shown in Tables 2–8, DCW concentrations between 0.015 and15.8%, flow rates (bead milling) between 1.5 and 282.25 L/h and treat-ment times from milliseconds to hours were used in different cell dis-ruption methods. PEF and ultrasonic treatment need relatively shorttreatment times (milliseconds to minutes), but use more diluted DCW(PEF = 0.03–0.2%; ultrasonic treatment = 0.2–0.85% DCW) whileothers, i.e., enzymatic lysis and bead milling, need treatment times ofminutes to days with higher DCW concentrations (enzymatic lysis =1–2%; bead milling = 5–15% DCW). Additionally, the disruption effi-ciencies for bead mill, high pressure homogenizer and high speed ho-mogenizer are positively affected by an increasing dry cell weightuntil a certain level (15–25%; see Bead milling, High pressurehomogenization and High speed homogenization sections and refer-ences therein), while other methods such as microwave treatment(see the Microwave treatment section) are negatively affected. Aggres-sive mechanical techniques, such as bead mill, high pressure homoge-nizer and high speed homogenizer, consume per method nearly thesame amount of energy to process a unit of volume, independent

f ultrasonic systems.Internet], 2014 http://www.apolloultrasonics.co.uk/sonifier.html.


http://www.apolloultrasonics.co.uk/sonifier.html



Fig. 5. The effect of DCW concentration on specific energy consumption of pulsed electricfields. Both the horizontal and vertical axis are in a logarithmic scale.Data from Sheng et al. (2011), Coustets et al. (2013) and Eing et al. (2013).


whether the feed is diluted or concentrated. Hence, for these methods,processing higher DCW concentrations per unit of time is more cost ef-fective. For example, Doucha and Lívanský (2008) reported that the en-ergy consumption of bead milling can be reduced from 10.3 to 0.86kWh/kg by changing the process parameters.

Several authors studied the energy consumption of cell disruptiontechniques. An overview of the data is given in Table 10. The literaturediscussed below will show that energy consumption is highly relatedto both process and design parameters. This makes a universal compar-ison based on all studies and articles not possible since the data does notshow any trends. Several authors compared different methods at lowDCW concentrations, i.e., ultrasonication (Lee et al., 2012) and HPH(Halim et al., 2012b; Lee et al., 2012), beadmilling andmicrowave treat-ment (Lee et al., 2010). According to these studies, high pressure ho-mogenization has the highest energy consumption, followed bymicrowave treatment and ultrasonication. McMillan et al. (2013) stud-ied the disruption of Nannochloropsis oculata cells without dewateringand compared microwave treatment, water bath, laser treatment andhigh speed homogenization. They concluded that microwave treatmentis the most energy consuming method, followed by water bath, lasertreatment, and high speed homogenization. Boer et al. (2012) useddata from GEA (2011), Doucha and Lívanský (2008), Hielscher (2011)and Diversified Technologies (2010) and calculated the energy con-sumption of different commercial cell disruption methods at higherDCW concentrations. They concluded that bead milling consumes themost energy followed by high pressure homogenization, PEF and ultra-sound. It must be noted that the data from the company websites(Diversified Technologies, 2010; GEA, 2011; Hielscher, 2011) is not ver-ified by literature data. Balasubramanian et al. (2011) used comparableDCW concentrations as Boer et al. (2012) and obtained an energy con-sumption of 10 times the value of HPH for solvent assisted microwavetreatment.

In an attempt to evaluate trends in specific energy consumption(kWh/kg), some literature data on PEF and bead milling were plottedin Figs. 5 and 6 respectively. For continuousmethods, the biomass treat-ment rate (kg/h), i.e., biomass disrupted per unit of time, has a strong ef-fect on the specific energy consumption. Unfortunately, this parametercould not be used for PEF due to lack of literature data on disruption ef-ficiencies. However, PEF data was plotted in a logarithmic scale and acorrelation was found with DCW concentration (Fig. 5). Since thesedata points originate from different types of microalgae, it appearsthat the energy consumption for PEF is less dependent on the type ofmicroalgae. On the other hand, the specific energy consumption forultrasonication showed no correlation with DCW concentration andwas therefore not shown in Fig. 5. Apparently, the type of microalgaeand growth conditions have a more profound effect on the energy effi-ciency of this disruption process. As shown in Fig. 6, the biomass treat-ment rate correlates well to the specific energy consumption ofdifferent laboratory/pilot scale bead milling equipment. More specific,

Table 10Comparison of cell disruption methods in terms of specific energy consumption.

Microalgae DCW (%) Cell disruptionmethod

Chlorococcum sp. 0.85% HPHUltrasonication

Scenedesmus sp. 7.5% Solvent assisted microwave treatmentBotryococcus sp., Chlorella sp.,Scenedesmus sp.

0.5% Microwave treatment

Nannochloropsis oculata 0.14% Microwave treatmentWater BathLaser treatmentHSH

Chlorella sp. 3.5% Bead milling15% HPH

Isocrysis sp. 25% PEFSpecies not given 15% Ultrasound


two correlation curves are found that correspond to the installedpower (kW) of the specific equipment used. Therefore, the energy con-sumption for bead milling is highly related to a design parameter, i.e.,installed power (kW), and the biomass treatment rate (kg/h). However,it should be noted that each equipment has its own optimal feed flowrate/biomass flow rate and DCW limitations since higher feed flowrate and DCW lead to a lower cell disruption efficiency. For example, a3-fold increase of the flow rate of a 12.5% DCW feed to Fryma KorumaMS 18 led to a 42% decrease in cell disruption yield. For Dyno-MillKDL-Pilot-A, on the other hand, a 12-fold flow rate increase led to a10% the cell disruption yield decrease. Two DCW concentrations weretested for Netzsch, Labstar LS1. A 2-fold increase in biomass treatmentrate resulted in a 22.9% decrease in cell disruption efficiency when theDCW concentration was 14.17%. For a 6.94% DCW microalgae suspen-sion, a 3-fold increase in biomass treatment rate resulted in a decreaseof only 3.7% in cell disruption yield.

A recent study on the disruption of Tetraselmis suecica throughatomic force microscopy by Lee et al. (2013) measured an energy con-sumption of 0.000187 kWh/kg on analytical scale. These experimentswere performed tomeasure the actual energy needed for single cell dis-ruption. The large difference between the energy need to disrupt cellsdirectly on analytical and preparative scale demonstrates the influenceof energy transfer. The energy consumption of non-mechanicalmethods is therefore relatively low compared to mechanical methodssince disruption is not directly related to a mechanical or physical ener-gy transfer. Asmentioned before, comparing the energy consumption ofcell disruption methods can be ambiguous. Nevertheless, differentmethods for one microalgae or one method for several microalgae are

Specific energy consumption(kWh/kg disrupted biomass)

Reference

146.94 Lee et al. (2012), Halim et al. (2012b)36.67 Lee et al. (2012)2.67 Balasubramanian et al. (2011)

116.67 Lee et al. (2010)

17.26 McMillan et al. (2013)4.673.70.125

10 Boer et al. (2012), Doucha and Lívanský (2008)0.25 Boer et al. (2012), GEA, (2011)0.07 Boer et al. (2012), Hielscher (2011)0.06 Boer et al. (2012), Diversified Technologies (2010)




Fig. 6. The effect of biomass flow rate on specific energy consumption of bead milling with different equipment with similar grinding chamber volume and different dry cell weight con-centrations (Doucha and Lívanský, 2008).


comparable. Additionally, numerous LCA studies onmicroalgae produc-tion highlight cultivation and dewatering/drying before extraction asmajor energy consumers within the overall process (e.g., Razon andTan, 2011; Xu et al., 2011; Soratana et al., 2014). Generally, energyand labor are the most predominant costs, thus cost effectiveness of acell disruption method does not only depend on the energy consump-tion of the cell disruption process itself but also is the resultant of theoverall energy demand, supplementary chemicals, labor, and other op-erational and capital expenditures. For example, an energy efficient celldisruptionmethod that needs an extremely skilled person to be operat-ed may not be cost effective because of the high labor costs.

Practical scalability

Practical scalability of cell disruption methods is related to manycharacteristics, but cost effectiveness and product quality are the mostimportant for implementation on large scale. Bead milling, high-pressure homogenization, high speed, and ultrasonication treatmentsare the most frequently used mechanical methods on laboratory-scalefor microalgal cell disruption. For industrial-scale applications, beadmilling, HPH and HSH are considered as the most feasible methods(Chisti and Moo-Young, 1986; Harrison et al., 2003). Nowadays, otherindustrial scale cell disruption equipment is available on the marketsuch as ultrasonication with 100 m3/h (Hielscher, 2011) or PEF unitswith 10 m3/h (Diversified Technologies, 2010) capacities.

Gaps in data comparison

Tobuild up a knowledge database to allow the selection of a suitable,scalable cell disruption method for mild microalgae biorefineries, com-parable data is needed. As concluded from the comparisons, all the pa-rameters (e.g., microalgae type, growth conditions, process conditions)influence cell disruption efficiency and energy consumption. Consider-ing the diversified data available in literature and from providers, it iscurrently not possible to select the best universal disruption techniquefor the future mild microalgae biorefinery. For example, LCA studiesthat calculate Capital Expenditures (CAPEX) and Operational Expendi-tures (OPEX), usually rely on literature data and tend to choose organ-isms or process types they consider to be similar to their studied system.


In the following, several examples emphasize the importance or ef-fect of these parameters on overall LCA calculations. Several authorsused energy consumption data from literature wherein other types ofmicroalgae were used while the effect of microalgae was proven to besevere (see the Specific energy consumption (kWh/kg dry biomass)section) (Razon and Tan, 2011; Xu et al., 2011; Yuan et al., 2014). Simi-larly, several authors used energy data from theGEA companywebpage(0.25 kWh/kg on dry basis)which claims that energy consumption is in-dependent from the type of algae used (Adesanya et al., 2014; Handleret al., 2012; Stephenson et al., 2010; Yanfen et al., 2012). Other studies,however, already proved a significantly higher energy consumption(146.94 kWh/kg on dry basis) with the same technique (Halim et al.,2012a,b; Lee et al., 2012). Sometimes, the reference for the energy con-sumption is not mentioned and thus not traceable (Mata et al., 2014) ordata on the technique is not found in the reference (Soratana et al.,2014). Based on these studies, it can be stated the LCA studies in thisfield are not straightforward.

Future needs

To develop a large scale energy-efficient cell disruption method formicroalgae biorefineries, a considerable amount of research is still need-ed. New methods should consider overall cost effectiveness includingpre-processing and downstreamprocessing, controllability, energy con-sumption at least below the caloric value of algae (≈21 kJ/g), mildness,adaptability and extractability/recoverability of products as key aspects.To achieve an economically feasible mild microalgae biorefinery, it isalso necessary to fully utilize the biomass and to minimize total processcost by applying process intensification approaches.

Within process intensification approaches for cell disruption, it isimportant to design deviceswith improved process control and reducedspecific energy demand (kWh/kg; see the Bead milling section). Thisimprovement could be achieved by focusing to equipment designs gen-erating specific pulses (e.g., electric, steam, ultrasonic). Alternatively,the combination of conventional techniques, e.g., ultrasonication withchemical/enzymatic pre-treatment, could reduce the energy demandof mechanical disruptionmethods. To reduce the amount of unit opera-tions, two phase aqueous systems could be promising since disruptionand functional product separation can be achieved in one step. Explo-sive decompression also has the potential to provide cell disruption





and hydrophilic/hydrophobic compounds separation in one step aswellas simultaneous reagent separation. This method has been excessivelystudied for bacteria and yeast, but was until recent generally ineffectiveonmicroalgae cells. Process intensification for explosive decompressionin a continuous operating mode should be studied in more detail. An-other aspect of process intensification is to design the cell disruptionprocess to empower the subsequent downstream process. An exampleis the emerging method of cationic polymer coated membrane treat-ment since the reagent is already separated from the products throughimmobilization. In summary, methods combining cell disruption andproduct separation in one unit operation should be developed with im-proved process control, reduced specific energy demand and zero dis-ruption agent contamination.

A considerable amount of research is still needed for somepromisingemerging methods, such as cationic polymer coated membrane treat-ment and explosive decompression, to achieve conventional cell disrup-tion method efficiencies. To support future research and to be able tocompare cell disruption efficiencies, a generic method for data collec-tion should be used systematically to produce comparable data. Thedata in literature should allow the calculation of at least specific energyconsumption for disrupting a unit of microalgae biomass (see the Beadmilling section). This could result in a higher level of understandingwhichwould accelerate the development of newmildmethods. This re-search area could also benefit from a better understanding in the rela-tionship between cell wall characteristics and disruption efficiencies.Additionally, the cell wall characteristics are influenced by themicroalgae type, growth conditions, growth phase and the existenceof stress factors which could be studiesmore thoroughly. To summarizethe latter, a structured methodology is needed to build up a knowledgedatabase to allow discrimination in the selection of a suitable full scaleinstallation.

Conclusions

Industrial microalgae biorefineries are not yet feasible due to highoperational costs. Cell disruption, because of the high energy demandand effect on the efficiency of subsequent steps, is the most crucialstep in biorefinery. In this review several methods have been describedfor microalgae cell disruption. Themain aspects of an industrially inter-esting microalgae cell disruption method include energy efficiency,mildness, selectivity, controllability and universality. Recent studies in-dicate that mechanical methods are optimal for industrial scale cell dis-ruption, but have high specific energy consumption. Non-mechanicalmethodsmight affect product quality to a minor extent, have lower en-ergy needs, might obtain a higher selectivity and may provide uniformcell disruption. However, treatment time is longer and at large scale rel-atively large treatment vessels are required, the demand for chemicalsis costly, a waste stream is generated and side products can be formeddue to the increased temperatures. Additionally, the process itself ismore difficult to control compared to mechanical methods. As a result,energy demanding mechanical methods such as high-pressure homog-enizers or beadmills are generally preferred for large-scale applications.However, these conventional mechanical methods require energy in-tensive cooling for the isolation of fragile compounds. On the otherhand, the efficiency of mild cell disruption methods dramaticallychanges from one microalgae species to another related to their cellmembrane/wall composition and morphology. Among the new devel-opments on microalgae cell disruption, laser and pulsed electric arctechnologies are not suitable for scale-up, microfluidizer is aggressivewith high shear and ultrasonication is known as not universal andstrongly dependent on type of microalgae and cultivation conditions.Other emerging technologies such as cationic polymer coated mem-brane treatment and explosive decompression are promising, but a con-siderable amount of research is needed to obtain acceptable disruptionefficiencies.


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