REVIEW
Enzymatic Cell Disruption of Microalgae Biomass
in Biorefinery Processes
Marie Demuez,1 Ahmed Mahdy,1,2 Elia Tom
as-Pej

o,1 Cristina Gonz
alez-Fern
andez,1
1,3
Mercedes Ballesteros
1
IMDEA Energy Institute, Biotechnological Processes for Energy Production Unit,
Av. Ram
on de la Sagra 3 28935, M
ostoles, Spain; telephone: þ34 917371126; fax: þ34
917371140; e-mail: marie.demuez@imdea.org
2
Department of Agricultural Microbiology, Faculty of Agriculture, Zagazig University,
44511, Zagazig, Egypt
3
CIEMAT, Renewable Energy Division, Biofuels Unit, Av. Complutense 40 28040,
Madrid, Spain
ABSTRACT: When employing biotechnological processes for the
procurement of biofuels and bio-products from microalgae, one of
the most critical steps affecting economy and yields is the “cell
disruption” stage. Currently, enzymatic cell disruption has delivered
effective and cost competitive results when compared to mechanical
and chemical cell disruption methods. However, the introduction of
enzymes implies additional associated cost within the overall
process. In order to reduce this cost, autolysis of microalgae is
proposed as alternative enzymatic cell disruption method. This
review aims to provide the state of the art of enzymatic cell
disruption treatments employed in biorefinery processes and
highlights the use of endopeptidases. During the enzymatic
processes of microalgae life cycle, some lytic enzymes involved in
cell division and programmed cell death have been proven useful in
performing cell lysis. In this context, the role of endopeptidases is
emphasized. Mirroring these natural events, an alternative cell
disruption approach is proposed and described with the potential to
induce the autolysis process using intrinsic cell enzymes.
Integrating induced autolysis within biofuel production processes
offers a promising approach to reduce overall global costs and
energetic input associated with those of current cell disruption
methods. A number of options for further inquiry are also
discussed.
Biotechnol. Bioeng. 2015;112: 1955–1966.
ß 2015 Wiley Periodicals, Inc.
KEYWORDS: microalgae; autolysis; biofuels; enzymatic
hydrolysis; proteases
Correspondence to: M. Demuez
Contract grant sponsor: Spanish Ministry of Economy and Competitiveness
Grant numbers: WW-ALGAS; ENE2013-45416-R
Received 17 March 2015; Revision received 5 May 2015; Accepted 6 May 2015
Accepted manuscript online 14 May 2015;
Article first published online 14 July 2015 in Wiley Online Library
(http://onlinelibrary.wiley.com/doi/10.1002/bit.25644/abstract).
DOI 10.1002/bit.25644
ß 2015 Wiley Periodicals, Inc.
Introduction
Increasing global demand for transport and bio-chemicals coupled
with the necessity to reduce greenhouse gas emissions, are driving
increasing interest in renewable alternatives to conventional fossil
fuels. Potentially valuable and game-changing alternatives include
those derived from microalgae. Microalgae are a diverse group of
unicellular photosynthetic eukaryote organisms that have received a
considerable attention in recent decades as a platform for biofuels
and bio-chemicals production. Some algae species are capable
of rapid growth rate (ex: Chlorella vulgaris, Scenedesmus sp.),
surviving in saline or brackish water (ex: Dunaliella sp.), and are
crucial for global carbon fixation (Field et al., 1998). Some others
present numerous advantageous properties for biofuels and bio-
chemicals production including naturally accumulating high
carbohydrate content (ex: Scenedesmus sp.; Porphyridium
cruentum) (Gonz
alez-Fern
andez and Ballesteros, 2012), high oil
content (ex: Botryococcus braunii; Nannochloropsis sp.) (Chisti,
2007), or producing valuable chemicals such as astanxanthin (ex:
Haematococcus sp.) (Lorenz and Cysewski, 2000). Moreover,
cultivation under environmental stress conditions such as nutrient
depletion has proven to be an efficient strategy to force
accumulation of carbohydrates or lipids (Gonz
alez-Fern
andez
and Ballesteros 2012; Sharma et al., 2012). Increasingly, microalgae
cells have the potential for metabolic or genetic engineering to
improve traits that increase production of biofuels and valuable co-
products in biorefinery processes (Potvin and Zhang, 2010;
Radakovits et al., 2010,2011; Rasala et al., 2014). While high-value
products from microalgae biomass have already been commercial-
ized, biofuels production processes are still not economically viable
to date (Parmar et al., 2011). In the case of value-added products,
the final price of the product can counterbalance the production
cost. In contrast, the overall process costs of biofuels production
from microalgae biomass must remain as low as possible for cost-
competitiveness with current conventional fuel production.
Consequently, the development of new technologies or strategies
Biotechnology and Bioengineering, Vol. 112, No. 10, October, 2015 1955
is required to lower the global cost of biofuel production processes. of biorefinery process that integrates the production of bioethanol
Several studies have shown that the cell disruption step of and biogas from microalgae biomass is presented in Figure 1.
microalgae processing represents a large proportion of the overall Microalgal cultivation is followed by the harvesting step where
process cost (Brennan and Owende, 2010; Molina Grima et al., entire cells are concentrated and recovered as a paste of cells.
2003). Current cell disruption technologies can be grouped into four Subsequently, a cell disruption step usually follows to break cell wall
categories: mechanical (bead-beating, milling, ultrasonication, and facilitate the release of intracellular compounds. Microalgae cell
high-pressure homogenization, and spray-drying), thermal (micro- debris containing carbohydrates in the form of cell walls and starch
wave, autoclaving, and freezing), chemical (organic solvents, can be further processed through a saccharification and
osmotic shock, and acid- alkali reactions) and biological processes fermentation to produce bioethanol. Then fermentation residues
(microbial degradation, enzymatic reactions) (G€unerken et al., are processed under anaerobic digestion to generate biogas.
2015). The review of G€unerken et al. (2015) focuses on the The advantages of enzymatic methods for microalgae cell
comparison of conventional and non-conventional emerging disruption are the mild reaction conditions, the absence of
techniques of microalgae cell disruption. Their potential application inhibiting by-products and the high selectivity of the reactions. An
for microalgae cell disruption in biorefinery is evaluated in terms of enzyme can selectively degrade a specific chemical linkage, whereas
disruption efficiency, product quality, process parameters, scal- mechanical methods destroy almost every particle existing in the
ability and specific energy consumption (G€unerken et al., 2015). solution, and chemical methods usually induce side-reactions on
The authors highlighted that specific energy consumption highly target products and consequently generating product damage.
depends on microalga species, growth conditions of biomass and Thanks to the low temperature and neutral pH reaction conditions,
cell dry weight loading. Mechanical methods display the highest enzymatic processes avoid the device corrosion that may occur
specific energy consumption (Boer et al., 2012; G€unerken et al., during thermochemical processes; thereby lowering equipment
2015). For non-mechanical methods, energy consumption is mostly costs. Therefore, enzymatic cell wall disruption methodology is
influenced by treatment time, temperature and stirring. Hence, new considered as more environmentally benign and less energy-
promising non-mechanical and mild technologies such as pulse
electric field (PEF) or biological cell disruption are emerging.
The biological processes include the degradation of microalgae
biomass using external addition of purified enzymes as single or
mixture of enzymes (Choi et al., 2010; Mahdy et al., 2014), or
external addition of an enzymatic extract from another micro-
organism (Ciudad et al., 2014; Ho et al., 2013). Biological cell
disruption can also be performed by microbial cell to cell
interaction in co-culture or infection, that induce the secretion of
lytic enzymes or active molecules causing algae death (Chen et al
2013; Cheng et al., 2013; Matsumoto et al., 2003; Mu~noz et al.,
2014).
This paper proposes a new enzymatic alternative for microalgae
cell wall disruption, namely, microalgae autolysis. The development
of a controlled or induced autolysis process offers a promising
approach to overcome traditional energy-consuming, corrosive or
polluting methods for microalgae cell wall disruption. Such an
approach represents a breakthrough towards a cost-efficient
industrial bioprocessing for producing biofuels from microalgae
biomass, a development which is now potentially within reach.
This review aims to provide an overview of hydrolytic enzymes
that have been demonstrated to be effective in microalgae
disruption and to improve biofuels production processes. Since
cell autolysis occurs naturally at certain moments of microalgae life
cycle such as cell division and programmed cell death (PCD),
microalgal hydrolytic enzymes involved in these natural events are
described. Furthermore, autolysis induction methods are reported
to perform autolysis. Taking advantage of the natural way of
microalgae to lyse, the potential of autolysis approach as an
integrated method for biofuel production process is also explored.

Enzymatic Treatment As Cell Disruption Method

Figure 1. General scheme of a biorefinery process from microalgae biomass.
For the production of bioethanol and biogas, an efficient cell
disruption is mandatory. In the interests of clarity, a general scheme

1956 Biotechnology and Bioengineering, Vol. 112, No. 10, October, 2015
consuming than mechanical and thermal pretreatments and
chemical catalytic hydrolysis options.
Some existing studies have determined the feasibility of
enzymatic processes to disrupt microalgae cell-wall (Gerken
et al., 2013; Harun and Danquah 2011; Rodrigues and Bon,
2011). Compared with mechanical or chemical cell disruption
methods, enzymatic methods have obtained very competitive
results in terms of disruption efficiency and biofuel production (Ho
et al., 2013; Kim et al., 2014; Lee et al., 2013). The specificity of the
chosen enzymes plays an important role in the efficiency of
microbial cell degradation, leading to the frequent application of
enzyme mixtures. Most investigations dealing with enzymatic cell
disruption utilize commercial enzymes, due to their high
concentration and in-depth characterization. The main drawback
of this method is the high cost of enzymatic cocktails. For that
reason, this review aims at proposing other alternatives.
Cell Wall-Degrading Enzymes Employed for
Microalgae Cell Disrurption in Biofuels
Production Processes
Microalgae cell walls consist of inner and outer cell wall layers. The
composition of the outer cell wall usually contains specific matrix
polysaccharides such pectin, chitin agar or alginates or the aliphatic
polymer algaenan (Scholz et al., 2014). The inner cell wall is usually
composed of microfibrillar cellulose and other materials such as
hemicellulose and glycoproteins (Domozych et al., 2012). The
composition, proportion and complexity of microalgae cell wall
structure will directly depend on phylogeny, development stage, cell
type and season. For example, the Chlamydomonas reinhardtii cell
wall is arranged in six distinct layers and contains intricated
cellulose-pectin complexes with others made of hydroxyproline-
rich glycoproteins (Imam et al., 1985). Besides, vegetative cells of
Scenedesmus, some species of Chlorella, Nannochloropsis sp., cysts
of Chlamydomonas, Haematococcus and Polytomella, and
Botryococcus colonies have been shown to contain the aliphatic
polymer algaenan in the cell wall (Scholz et al., 2014; Zych et al.,
2009). The diversity of composition and polymer arrangement of
microalgae cell wall ensures its mechanical strength, shape and
rigidity as necessary characteristics for cell protection against
infection or predators. Consequently, an effective microalgae cell
wall disruption method must be optimized for the targeted cell wall
to lyse.
A number of studies have reported the efficiency of enzymatic
cell wall disruption by adding externally single or cocktails of cell
wall-degrading enzymes. Several of them have already applied this
method to produce biofuels from hydrolyzed biomass (Choi et al.,
2010; Ho et al., 2013; Lee et al., 2013; Mahdy et al., 2014; Kim et al.,
2014). Table I summarizes the experimental conditions and results
of relevant studies employing enzymatic hydrolysis to disrupt
microalgal biomass.
Effect of Carbohydrases on Cell Wall Disruption
In the case of microalgae biomass containing carbohydrates,
enzymatic treatment can be designed to couple both cell wall
disruption and polysaccharides hydrolysis. It can be considered as a
saccharification step as it hydrolyzes both cell wall and storage
carbohydrates. This saccharification involves the use of cellulases,
amylases, and amyloglucosidases to convert polymeric carbohy-
drates into simple sugars. For cellulose-containing microalgae,
endo-b-(1,4)-D-glucanases are required to attack the amorphous
cellulose and split cellulosic chains. The exo-b-(1,4)-D-glucanase
cleaves the small fragments of cellulose into small oligosaccharides
such as cellobiose and cellodextrin. Finally, b-glucosidase enzymes
degrade cello-oligosaccharides into glucose. To degrade starch,
endo-amylases are first used to target the internal a-(1,4)-
glycosidic bond of starch to produce dextrin, and amyloglucosi-
dases are further needed to hydrolyze dextrin into glucose and
oligosaccharides such as maltose.
The studies of Harun and Danquah (2011), Fu et al. (2010), and
Lee et al. (2011) reported the use of cellulases to hydrolyze
microalgal biomass at different consistencies of 1, 2, and 5% (w/w
dry weight (DW)), respectively. They obtained saccharification yields
of 64.2, 58, and 47.3% based on the total amount of carbohydrates
contained in the biomass, which resulted in insufficient yields
compared to diluted-acid pretreatment. There is a direct correlation
between glucose yield and biomass consistency. A lower consistency
of microalgae biomass resulted in a higher sugar yield due to the
lower viscosity of the media. Additionally enzyme dosages need to
be optimized to reach a maximum of cell degradation using a
minimum of enzymes. In this context, Fu et al. (2010) attempted to
reduce the global cost of the process by immobilizing cellulases.
Although, immobilized enzymes could efficiently degrade the cell
envelopes of C. pyrenoidosa (62% after 72 h), the enzyme activity
was significantly reduced when the enzyme was recycled. After
recycling the enzyme five times, the relative hydrolysis yield
decreased to 40%, indicating that the recyclability of the
immobilized enzymes is of concern.
Rodrigues and Bon (2011) evaluated the addition of amylase,
together with cellulase and xylanase enzymes to improve the
hydrolysis yield of two Chlorella sp. biomass. These two Chlorella
sp. biomass were hydrolyzed at 10% (w/wDW) consistency, reaching
a saccharification yield of 67.5% and 53.2%. A significant
improvement on that achieved by Lee et al. (2011) whose hydrolysis
was made on lower biomass consistency (5% w/wDW). The
treatment of the microalgal cell wall with an appropriate
combination of several hydrolytic enzymes, rather than with single
hydrolytic enzyme, seems to be required to generate an efficient cell
disruption.
Lee et al. (2013) used Viscozyme L or amyloglucosidase (AMG
300L) to disrupt 5% (w/wDW) residual biomass of Dunaliella
tertiolecta after lipid extraction. Viscozyme L is a commercial
cocktail containing carbohydrases including b-glucanases, cellu-
lases, hemicellulases, xylanases, and arabinases activities. They
achieved 80.9% and 71.3% (w/w) hydrolysis yield using AMG 300L
and Viscozyme L, respectively. This is significantly higher than
hydrolysis obtained using 0.5 M HCl acid pretreatment. No synergic
effect was observed using the mixture 1:1 of AMG 300L and
Viscozyme L. As the major component of residual biomass was
starch, the saccharification efficiency of AMG 300L was better than
that of other enzymes. This finding illustrates that the degrading
Demuez et al.: Enzymatic Cell Disruption of Microalgae Biomass 1957
Biotechnology and Bioengineering
 Table I. Summary of studies utilizing enzymatic hydrolysis for microalgae cell wall disruption.

1958
Carbohydrate solubilization

Algae biomass Hydrolysis process

Hydrolysis conditions (temperature,

Consistency dry weight basis Carbohydrate content (%) pH, Saccharification yield
Species (%) (w/w) Enzymes incubation time) (%)* References

Chlorococum humicola 1 32.5 Cellulase 40 C, pH 4.8, 24 h 64.2 Harun and Danquah,
(2011)
Chlorella homosphaera Chlorella 10 NA (33) Cellulase, Xylanase, Amylase 50 C, pH 4.8, 24 h 24.5a–67.5b, 19.3a–53.2b Rodrigues and Bon,
zofingiensis (dry and ground) (2011)
Chlorella vulgaris NA NA Achromopeptidase, Cellulase, 30 C, 3 h 88 osmotically-labile Honjoh et al. (2003)
Chitosanase, Gluczyme,
Uskizyme
Chlorella sorokiniana, Nannochloropsis NA 18.2, 11.2, 14.5 Cellulases (Celluclast 1.5L, 55 C, pH 4.5, 72 h; 64a, 100a, 38.8a Hern
andez et al. (2015)
gaditana, Scenedesmus almeriensis Novozyme 188), 90 C, pH 6, 6 h;
(combined with diluted H2SO4) Amylases (Liquozyme 60 C, pH 4.5, 72 h
SC, DS Spirizyme Fuel)

Biotechnology and Bioengineering, Vol. 112, No. 10, October, 2015

Chlamydomonas reinhardtii 5 59.7 Amylase (Termamyl 120L, with 90 C, 30 min; 94 Choi et al. (2010)
protease activity), 55 C, pH 4.5;
Amyloglucosidase 30 min
(AMG300L)
Chlorella vulgaris (milled and 5 NA (25) Celluclast 1.5L Novo188 45 C, pH 4.6, 1 h 13a–47.3b Lee et al. (2011)
autoclaved)
Dunaliella tertiolecta 5 51.9 Amyloglucosidase (AMG300L) 55 C, pH 5.5, 2h 80.9, 71.3b Lee et al. (2013)
Viscozyme L
Chlorella vulgaris (bead-beating 10 22.4 Aspergillus aculeatus extract of 50 C, pH 4.8, 72 h 79 Kim et al. (2014)
pretreated) pectinase
Chlorella vulgaris 2 51 Pseudomonas sp. CL3 extract 45 C, 48 h 46.1a–90.4b Ho et al. (2013)
(cellulases amylase
activities)
Biogas production

Algae biomass Hydrolysis process

Hydrolysis conditions Increment in organic CH4 production

Consistency dry Carbohydrate (temperature, pH, matter solubilization increment fold
Species weight basis (%) content (%) (w/w) Enzymes incubation time) (increased sCOD) (increased % of biodegradability) References

Scenedesmus obliquus, 2 NA Cellulase, Endogalacturonase 50 C, 24 h 14.4x, 4x 14x, 10.4x Ometto et al. (2014)
Chlorella sorokiniana (DepolTM 40L), Esterase, Protease (LipomodTM 957)
Chlorella vulgaris, 1.6 18.6, 22.6 Viscozyme L, Alcalase 2.5L pH 5.5, pH 8; 50 C, 5 h 4.8x, 2.8x 1.4–1.5x (51%), 1.1x (14%) Mahdy et al. (2014)
Chlamydomonas
reinhardtii
Botryococcus braunii 0.45g dry wt NA Enzymatic extract of Anthracophyllum discolor Room T C 24 h NA 1.6x (62%) Ciudad et al. (2014)
 Lipid extraction

Algae biomass Hydrolysis process

Lipid extraction
Carbohydrate Hydrolysis conditions (%) (fold of
Consistency dry weight basis content (%) (temperature, Hydrolysis yield improved
Species (%) (w/w) Enzymes pH, incubation time) (%)* extraction) References

Chlorella vulgaris, 18 NA Snailase, Trypsin 37 C, 12 h; pH 5.8; pH 8 NA 49.8; 46.8; 11.7 Liang et al. (2012)
Scenedesmus
dimorphus,
Nannochloropsis
sp. (sonicated)
Chlorella vulgaris 1 NA Enzymatic extract of Flammeovirga p H8, 72 h NA 21.5 (2x) Chen et al. (2013)
yaeyamensis, (amylases cellulases
activities)
Chlorella vulgaris 1 9.5 Celluclast 1.5L, Novo188 50 C, pH 4.8, 72 h 85.3  10 (1.29x) Cho et al., (2013)

Chlorella vulgaris 10 25.8 Cellulase, Neutrase-Alcalase 55 C, pH 4.8, 10 h 60 C; 17.5 a–61.6b NA Zheng et al.
pH, 6 24 h (2012)
Chlorella pyrenoidosa 2 33 Immobilized cellulase 50 C, pH 4.6; 24 h, 72 h 58; 62 56 Fu et al. (2010)
Nannochloropsis oculta NA NA Viscozyme L, Proteinase K 37 C; 2 h 20 NA Horst et al. (2012)

NA, means not available.

*
Free sugars released relative to total initial carbohydrates content in biomass.
a
Value expressed in grams of glucose or reducing sugar per 100 g of dry biomass.
b
Value calculated from the given hydrolysis yield in g/100 g of dry biomass, dividing it by total carbohydrates content in biomass*1.1.

Biotechnology and Bioengineering

Demuez et al.: Enzymatic Cell Disruption of Microalgae Biomass
1959
enzymes must be chosen carefully according to each type of
biomass.
Effect of Carbohydrases and Proteases on Cell Wall
Disruption
To complement the action of carbohydrases enzymes, the use of
endopeptidases or proteases in enzymatic pretreatment for biofuels
production has gained particular interest as it strongly improves cell
lysis and biomass degradation (Horst et al., 2012; Mahdy et al.,
2014; Zheng et al., 2012). The combined action of glucanase and
protease at optimum dosage and hydrolysis conditions intensifies
the extraction efficiency.
Choi et al. (2010) hydrolyzed 5% (w/wDW) C. reinhardtii biomass
using the sequential action of two enzymatic cocktail, namely
Termamyl 120L (containing a-amylases and proteases activities)
and AMG 300L. The resulting sugar conversion yielded 94%. The
authors argued that improvement in hydrolysis yield was due to the
protease activities also present in the enzymatic cocktail Termamyl
120L, making possible the degradation of glycoproteins within cell
wall of C. reinhardtii. Enzymatic hydrolysis could disrupt the cell
wall structure of the biomass, releasing starch out and making it
accessible to starch-degrading enzymes. This study was the first to
report the effectiveness of proteases together with carbohydrase
enzymes to disrupt microalgae cells.
Being inspired by DNA extraction protocols using cell wall-
degrading enzymes, bacterial and yeast cell wall-degrading
enzymes including lysozyme, achromopeptidase, chitinase, chito-
sanase, lyticase, and pectinase, have also been tested (Gerken et al.,
2013; Honjoh et al., 2003).
Honjoh et al. (2003) prepared C. vulgaris protoplasts using a
mixture of cellulase, glucoamylase (Gluczyme), achromopeptidase,
chitosanase, and b-glucuronidase (Uskizyme). Furthermore,
Gerken et al. (2013) showed that Chlorella strains cell walls
were degraded by enzymes like chitinases, lysozymes and pectinase
whereas cellulase and amylase had no effect.
Lysozyme and achromopeptidase are collectively called pepti-
doglycan hydrolases. They catalyze the lysis of peptidoglycan
bacterial cell wall. Lysozyme is a glycoside hydrolase that splits the
polysaccharide chains at b-(1–4)-glycosidic links between two
units of N-acetyl-muramic acid. Achromopeptidase is a lysyl-
endopeptidase that splits polypeptide bridges that connect adjacent
glycan polymers. Amidase is the lytic enzyme responsible for the
cleavage of the junction between polysaccharides and peptides
(Salazar and Asenjo, 2007). Similarly, yeast cell wall-degrading
enzymes include b-(1–3)-glucanases, b-(1–6)-glucanases, man-
nanases, chitinases and proteases. Proteases act initially on the
yeast surface, allowing the exposition of the glucan layer to the
glucanases hydrolytic action (Salazar and Asenjo, 2007). In order to
mimic the natural lytic enzymes systems of bacteria and yeast, a
combined action of protease and glucanase is necessary to open a
sufficiently large hole in the cell wall to extrude its content.
Horst et al. (2012) evaluated cell wall disruption efficiency of one
marine eustimagtophyte (Nannochloropsis oculata) and two marine
diatoms (Phaeodactylum tricornutum and Thalassiosira pseudo-
nana) using Viscozyme, Driselase, proteinase K, crude papain, and
bromelain. Driselase is a mixture of laminarinase, pectinase,
1960 Biotechnology and Bioengineering, Vol. 112, No. 10, October, 2015
xylanase, and cellulase enzymes, so it belongs to the carbohydrase
as Viscozyme. Papain, bromelain, and proteinase K can be classified
as endopeptidases. Papain and bromelain are cysteine proteases
while proteinase K is an alkaline serine protease, both exhibit broad
specificity of cleavage site. Since cell walls were very different, the
results were very specific to each algal species. The best results, in
terms of cell breakage of N. oculata biomass, were obtained using
Viscozyme or proteinase K. The most efficient lysis of P. tricornutum
and T. pseudonana biomass were achieved with crude papain and
Driselase, respectively. Therefore, the best cell breakage was
achieved using mixtures of enzymes rather than single enzymes.
Lipid extraction efficiency using crude papain treated biomass and
heptane: IPA was similar to mechanically cell disrupted biomass
and chloroform:methanol (Horst et al., 2012).
Mahdy et al. (2014) studied the effect of Viscozyme L and a
serine endopeptidase (Alcalase 2.5L) on the solubilization of C.
reinhardtii and C. vulgaris biomass in order to enhance methane
production. The addition of Viscozyme supported the solubiliza-
tion of 86% and 96% of total carbohydrates of C. vulgaris and C.
reinhardtii biomass, respectively, while the combination of both
enzymes did not increase markedly the yield. The addition of
Alcalase allowed solubilizing 97% of total proteins of both
microalgal biomass. This enzymatic treatment renders almost all
carbohydrates and proteins available in soluble phase, making
them easily digestible for biogas production. In the case of C.
vulgaris biomass, the methane yield achieved by Viscozyme- and
Alcalase-treated biomass was improved by 14% and 51%,
respectively, compared to the methane yield obtained by the
raw material. Combination of both enzymes exhibited similar
results to Alcalase alone which corresponds to almost 85%
biodegradability.
Addition of protease is especially suitable for biogas production
as it takes advantage of both proteic and polysaccharidic
components of the biomass, increasing soluble organic matter
significantly (Mahdy et al., 2014; Ometto et al., 2014). High methane
yield obtained after enzymatic hydrolysis is the direct consequence
of efficient cell wall degradation which enables the removal of
components that resist bacterial degradation and limit the methane
production yield. Additionally, depending on algal species, this
efficient cell wall degradation enables the release of a higher
quantity of energetically valuable components such as sugars,
proteins and lipids.
In case of lipid extraction from microalgal biomass, the
utilization of cell wall-degrading enzymes such as a mixture of
carbohydrases and proteases focuses on making fractionation much
easier, which markedly raises lipid recovery (Chen et al., 2013;
Horst et al., 2012; Liang et al., 2012).
The combination of enzymatic treatment with other mechanical
or thermal methods such as milling, microwave or autoclave, in
softer conditions appears less energy consuming, and mitigates the
generation of side-products or toxics that could alter the end-
product. By combining sequential diluted sulphuric acid and
enzymatic treatments, Hern
andez et al. (2015) reached a complete
carbohydrate hydrolysis of Nannochloropsis gaditana biomass.
These aforementioned studies emphasized the potential of
enzymatic treatment in a biofuel refinery as it decreased the global
energy input in the process. The key obstacle for its widespread
plant-scale application is the cost of purified enzyme production or
commercial enzymatic cocktails.
Alternatives to the Expensive Enzymatic
Cocktails
Two prominent alternatives to reduce the cost of an enzymatic
process can be highlighted, namely, the production of these
enzymes by another microorganism (on-site production), and the
expression of these enzymes directly by the microalgae to degrade.
Use of Microbial Enzymatic Extract
By employing an enzymatic extract of a microbe, a blend of
enzymes is recovered to perform enzymatic hydrolysis of microalgal
biomass. Ho et al. (2013), Kim et al. (2014), and Ciudad et al. (2014)
used this method, and proved its efficiency in maximizing
hydrolysis yield. The enzymatic mixtures were extracted from
bacteria or fungi, gathering a complex composition of activities
together. Aspergillus aculeatus extracellular enzymes had abundant
pectinases activities together with cellulases and xylanases
activities, while enzymatic extract of the cellulose-hydrolyzing
bacterium Pseudomonas sp. CL3 contained a mixture of cellulases
and amylases activities (Ho et al., 2013; Kim et al., 2014). Both
enzymatic extracts were able to hydrolyze 2% and 10% (w/wDW) of
C. vulgaris biomass with the improved saccharification yield of
90.4% and 79%, respectively.
Chen et al. (2013) compared the lysis effect of a bacterial
enzymatic extract to the one generated by purified cellulase or
amylase enzymes on C. vulgaris biomass. Maximum sugar
releases were obtained on biomass treated with cellulase alone
and enzymatic extract, even though, sugar release rate was
markedly higher with cellulase alone. This suggested that
cellulase was the key enzyme of C. vulgaris cell wall disruption.
To get a reasonable rate of cell wall breakdown, enough cellulase
activity was required.
Microalgae Autolysis
This mechanism, known as autolysis or self-digestion, consists in
the destruction of tissues or cells of an organism by endogenous
enzymes and it is initiated by the release of digestive enzymes from
secretory vesicles. The microbial cell is being disrupted through the
action of its own enzymes. Microalgae autolysis is a natural event
that occurs during microalgae life cycle where cell wall-degrading
enzymes are acting. Few lytic enzymes involved in cell division and
programmed cell death of microalgae are described in the next
section (4.1). The advantage of microalgae self-digestion is that cell
wall-degrading enzymes produced are going to be strain-specific, so
especially suitable for the degradation the particular cell-wall
composition. In the context of an industrial bioprocess, an inductor
is required to activate the proper cell digestion and release all
intracellular material into broth culture. Currently, the only
induction of microalgae cell lysis reported so far, have been
investigated in the context of the programed cell death of algae
bloom and red tides by employing environmental stress conditions
(Affenzeller et al., 2009; Segovia and Berges, 2009; Vardi et al., 1999;
Zuppini, 2007). It is developed in the section 4.2.
The application of the performance of internal enzymes through
an autolysis process is a promising alternative. In the biofuels
production process, the autolysis method is proposed to replace the
conventional energy-consuming disruption method and remove the
cost associated with enzyme production.
While autolysis exploits endogenous lytic enzymes, the micro-
algal induced lysis is another enzymatic cell disruption approach in
which bacterial, viral or bacteriophage cell wall-degrading enzymes
are employed. Algicidal bacteria and virus have been described in
the context of terminations of harmfull algal blooms. Co-cultures of
algicidal bacterial or virus with microalgae to induce microalgae cell
disruption have already been reported, giving the opportunity of
applying this method for biofuels production (Chen et al., 2013;
Cheng et al., 2013; Matsumoto et al., 2003). Moreover, there are few
examples of genetically engineered inducible lytic system that prove
its feasibility. These systems have been achieved for the disruption
of cyanobacteria biomass Synechocystis sp. Cyanobacterial cell
disruption was triggered by the expression of the bacteriophage
holin- endolysin system under control of inducible promoter
activated by nickel (Liu and Curtiss 2009) or by green-light
illumination (Miyake et al., 2014). The development of a genetically
engineered inducible lysis system for microalgae is experiencing
limitations in that these microorganisms are eukaryotic and
genomic sequences of most microalgae are not yet available. Thus,
genetic engineering of microalgae is not yet developed to the same
extent as for prokaryotic organisms. Nevertheless, results illustrate
that the potential of microalgae cannot be neglected and new
molecular biology tools are constantly emerging in the last decade
(Potvin and Zhang 2010; Radakovits et al., 2010, 2011;
Rasala et al., 2014). Further inquiry into the inducible system
for microalgae cell disruption is necessary.
Cell Wall-Degrading Enzymes Involved
Microalgae Life Cycle
Microalgal Cell Division—Autolysins
Cell autolysis occurs naturally during particular events and sites of
the cell wall. Depending on cell type and environment stress, these
lysis events occur both during asexual and sexual cycle.
When asexual cell division takes place, vegetative cells undergo
fission or spore formation by mitosis to form 4–8 zoospores similar
to the parent (1n). Under starvation conditions or environmental
stress, vegetative cells differentiate into gametes (gametogenesis) of
the two mating types mtþ and mt. Through an adhesion process,
gametes of opposite mating types are attached to each other and
fuse to form a zygote (2n). The zygote proceeds to elaborate new
more dense and thick cell walls that ensure cell survival under
adverse environmental conditions (Goodenough et al., 2007).
Afterwards, the zygote undergoes meiosis to form four zoospores
(1n). During these events of fusion or fission, specific digestives
enzymes known as autolysins, are produced at restricted sites of the
cell wall to allow cell fusion, or the release of the daughter cells by
fission.
Demuez et al.: Enzymatic Cell Disruption of Microalgae Biomass 1961
Biotechnology and Bioengineering
 Autolysins are proteases capable of degrading cell walls in Gametolysins
localized sites and are characterized to be strain-specific and life
Gametolysins are 62 kDa zinc metalloproteases that have the
cycle stage-specific. Their presence has been reported in
capacity of selectively degrading HRGPs of the gamete walls during
Chlamydomonas reinhardtii (Adair and Apt, 1990; Buchanan
mating but has no activity on b-casein, hemoglobin, or other
et al., 1989; Matsuda et al., 1985), and Volvox carteri alga (Shimizu
proteins (Kinoshita et al., 1992; Matsuda et al., 1985). As a
et al., 2002). These digestive enzymes specifically target the
consequence, gametolysins are also known to be involved in
extracellular matrix protein of the microalgae cell wall known as
cleavage of cell surface receptors, cell-cell adhesion proteins,
hydroxyproline-rich glycoprotein (HRGPs) (Jaenicke et al., 1987).
chemotactic molecules interactions and release of apoptotic ligands
Whereas cell wall composition and rigidity of these volvocales vary
(Sternlicht and Werb, 2001). During the synthesis of theses
according to species or life cycle stage, HRGPs-like proteins are
enzymes, the inactive pre-enzyme is stored in the periplasm and
always present. HRGPs share highly conserved repeating amino
translocated over the cell membrane via a signal peptide. Later, the
acids motifs: X-(Hyp)3 and Ser-(Hyp)4, where hydroxyproline and
signal peptide is cut to allow the pro-enzyme to cross the membrane
serine act as specific sites for O-linked glycosylation (Woessner
and be released into the culture medium by signal induction. This
et al., 1994). The degree of glycosylation between HRPGs
signal induction is the flagellar agglutination between opposite
determines substrate specificity. This cross-linkage leads to the
mating gamete. Then, another protease, p-lysinase, which has the
stabilization of polyproline-II conformation that promotes cell wall
characteristics of a serine protease, converts the inactive pro-
rigidity. After mating and zygote formation, algae zygote up-
enzyme into an active extracellular metalloprotease (Snell et al.,
regulate expression of HRGPs-like proline-rich polypeptides within
1989). Many proteins representatives of the same family, as
15 min of cell wall autolysis, and a new cell wall matrix is assembled
metallopeptidase M11 and containing Pfam domain PF05548 have
within 3–4 h (Adair and Apt 1990; Buchanan et al., 1989). HRGPs
already been described: Matrix-metalloproteases (MMPs), LSG2
have been suggested to have the major role in defense mechanism
and Volvox metalloproteinases (VMPs) (Fukada et al., 2006;
against microbes, agglutination and adhesion by binding to cell
Shimizu et al., 2002). Using MEROPS and Pfam databases matrix-
surface glycoproteins (Woessner et al., 1994).
metalloproteases from M11 family are highly represented in
Depending on the cell wall to be degraded, autolysins can be
Chlorophyta genomes. Interestingly, the comparison of genes
divided into two different groups. The specific digestive
occurrence among the sequenced genomes revealed that metal-
proteases involved in vegetative cell wall degradation are
lopeptidases M11 are only represented in C. reinhardtii, C. variabilis
sporangins, which are also reported as vegetative lysin enzyme,
and V. carteri, without following any phylum specificity (Fig. S1).
v-lysin or VLE, and that to digest the gamete cell walls are
The induced secretion of those autolysins or endopeptidases may
known as gametolysins (also referred as gamete lytic enzyme,
be an interesting tool for algae cell autolysis and release of the
g-lysin or GLE) (Matsuda et al., 1985, 1994, 1995; Jaenicke et al.,
targeted component. Induction of VLE has the advantage to get the
1987). Both of these enzymes are endopeptidases, which mean
algae cell wall digested in a strain-specific and in a cell stage-specific
that they cleave peptide bonds of nonterminal amino acids
way, whereas the action of GLE is site-specific and strain-specific
within the protein.
(Jaenicke et al., 1987). Actually, C. reinhardtii autolysins (GLE) are
widely used in molecular biology. Transformations were achieved in
Sporangins wild-type microalgae with very low efficiencies. For this reason, the
removal of microalgae cell wall is currently performed to facilitate
Sporangins are calcium-dependent glycoproteases of 37–40 kDa,
the transformation procedure and reach higher transformation
characteristics of subtilisin-like serine endopeptidases from
efficiency. A protocol for cell wall removal for transformation
peptidase family S8 and containing the Pfam PF00082. These
procedure has been developed, and the detailed protocol for the
glycoproteases can cleave the peptide bonds on the carboxyl side of
production and purification of these autolysins is described in
a lysine or arginine residue (Matsuda et al., 1995; Siezen and
Buchanan et al. (1989).
Leunissen, 1997). The highly conserved catalytic triad Asp-His-Ser
A deeper understanding of the induction and control of the
is responsible for this activity. As in C. reinhardtii, Kubo et al. (2009)
autolytic system in microalgae could help to determine which lytic
showed that the green multicellular algae V. carteri encloses a
enzymes play a major role in this process. Furthermore, more insights
homologous hatching enzyme VheA of 125 kDa that presents 50%
about structure recognition of the enzyme are necessary to know the
identity with C. reinhardtii sporangin. During asexual division,
exact protein domain interaction with the specific feature of the cell
sporangial autolysins are synthetized and accumulated in the cell
wall HRPGs for a given algae strain. Structure-function relationship
wall of the flagella thanks to its transmembrane attachment. Later,
studies of the enzyme would be suitable for this purpose. The
sporangins are released into culture medium, concurrently with
identification of the key effects that trigger the secretion of those
cell wall digestion in order to release the vegetative cells
enzymes and promote the autolysis process is also crucial.
(Kubo et al., 2009). A computational analysis of the sequenced
Chlorophyta genomes, searching for the occurrence of cell wall-
degrading related genes, was carried out using MEROPS peptidase
Lytic Enzymes and Autolysis Inducers Involved in
database (Rawlings et al., 2014) and Pfam protein families database
Programed Cell Death (PCD)
(Finn et al., 2014). It revealed that subtilisin-type serine peptidases
from S8 family, containing Pfam PF00082, are well represented in Biological lysis events take place in algae cells during programed
chlorophyte (Fig. S1). cell death by self- or environmental induction. PCD is an

1962 Biotechnology and Bioengineering, Vol. 112, No. 10, October, 2015
irreversible and genetically controlled form of cell suicide. PCD
mechanism is essential for the proper cell development and is
programed and regulated in case of senescence or injured cells that
have engendered mitosis errors during cell division. PCD can be
mediated by different signal pathways; it can be caspase-dependent
(apoptosis) which involves cleavage or/and phosphorylation
cascades, or caspase-independent where autophagy or vacuolar-
like degradation proceeds. Apoptosis is an active cell mechanism. It
refers to specific morphological changes that occur during
genetically controlled cell death and initiated by a variety of
environment stimuli (Bidle and Falkowski, 2004; Franklin et al.,
2006). Although there is very few information in the literature about
microalgae PCD mechanisms, these morphological changes have
been well characterized in higher plants and animals. They include
cell shrinkage, chromatin condensation and DNA fragmentation,
blebbing and formation of apoptotic bodies, and phagocytosis.
Related biochemical changes encompass DNA degradation, de novo
protein expression, the activation of caspases proteases and
production of secondary messengers such as calcium ions and
reactive oxygen species (ROS) (Gordeeva et al., 2004). In animals,
stressed cells are specifically targeted and removed by phagocytosis
without releasing the cellular content. These characteristics are used
to discriminate between cell suicide and accidental cell death,
related as necrosis, which is a passive cell mechanism leading to the
release of cellular content and in an inflammatory response
(Bidle and Falkowski 2004; Franklin et al., 2006).
Caspases are known as cysteinyl-aspartate-specific proteases and
belong to C14 cysteine endopeptidase MEROPS family. Caspases are
recognized as key enzymes in apoptosis mechanisms in virtually all
organisms including metazoans, plants, fungi, protists, bacteria
and animals. However, only the caspases orthologues, referred as
metacaspases (meta signifies “after”) and caspases-like enzymes,
have been found in chlorophytes and phytoplankton. Moreover,
multiple sequence alignments and the comparison tridimensional
structures of caspases and metacaspases revealed large variations
leading to marked functional differences (Choi and Berges, 2013).
Currently, only few investigations are available on PCD or
apoptosis mechanisms in planktonic photosynthetic
microorganisms (Bidle and Falkowski, 2004), and chlorophytes
(Franklin et al., 2006). Hence, few insights are reported about which
kind of PCD process is occurring in microalgae (Choi and Berges,
2013; Gordeeva et al., 2004; Zuppini et al., 2010). However, inquiry
has focused effort on studying the induction of algal cell death in
some species of chlorophytes and phytoplankton, using environ-
mental stresses such as nitrogen limitations (Berges and Falkowski,
1998), UVradiation (Moharikar et al., 2006), intense light, darkness
(Segovia and Berges, 2009), CO2 limitation and oxidative stress
(Vardi et al., 1999), heat stress (Zuppini et al., 2007) and salt stress
(Affenzeller et al., 2009). Table II summarizes the relevant studies
inducing microalgae cell death under abiotic stress and reporting
protease activities during microalgae lysis.
In most of these studies, observed cell destruction mechanism
was analogous to apoptosis mechanism. It was defined as a form
of autocatalytic cell suicide in which an endogenous biochemical
pathway leads to apoptotic-like morphological changes, and
ultimately, cellular dissolution. Moreover a significant production
of ROS species and an increase of caspase-3-like proteases activity
were detected. The latter was observed in the unicellular
chlorophytes C. reinhardtii, Chlorella saccharophila, and
Dunaliella tertiolecta while using environmental stresses
including heat and darkness (Moharikar et al., 2006; Segovia
and Berges 2009; Zuppini et al., 2007). Noticeably, Segovia and
Berges, (2009) reported that the apoptosis-like phenotype of
D. tertiolecta PCD induced by darkness also comprehend the loss
of cell membrane integrity. A distinct PCD mechanism was
observed in Micrasterias denticulata when induced by high
salinity. The degradation of organelles occurred by autophagy,
indicating the involvement of caspase-independent PCD mech-
anism (Affenzeller et al., 2009). Moreover, Jimenez et al. (2009)
studied the PCD mechanism of Dunaliella viridis when induced
by different kind of environmental stresses, and demonstrated
that different cell death phenotypes could be induced. Senescence,
heat shock, UV irradiation, hyperosmosis and nitrogen starvation
stresses resulted in apoptosis- like, necrotic-like, aponecrotic-like,
paraptotic-like, autophagic/vacuolar-like cell death phenotypes.
The latter points out that PCD mechanism of unicellular

Table II. Summary of studies inducing microalgae cell death under abiotic stress.

Microalgae species Cell death inducer Cell death phenotype References

Green algae
Chlamydomonas reinhardtii
Dunaliella tertiolecta
Dunaliella tertiolecta
Dunaliella viridis
Chlorella saccharophila
Micrasterias denticulata
Dinoflagellate
Peridinium gatunense
Prorocentrum donghaiense
Amphidinium carterae
UV irradiation Caspase-3 homologue and protein of 28 kDa Moharikar et al. (2006)
Darkness and oxidative Apoptosis ROS, Caspase-like activities, loss Segovia and Berges, (2009)
stress of membrane integrity
Darkness Apoptosis Large increase of protease activity Berges and Falkowski, (1998)
Environmental stresses Apoptosis- Necrosis- Aponecrosis- Paraptosis Jimenez et al. (2009)
Autophagy Caspase-like DEVDase
Heat-stress High salinity Apoptosis ROS, Caspase 3-like activity Zuppini et al. (2007) Zuppini et al. (2010)
Salt stress Autophagy Affenzeller et al. (2009)
CO2 limitation and oxidative Apoptosis Cysteine proteases Vardi et al. (1999)
stress
Senescence Caspase 85aa Yang et al. (2008)
Senescence and darkness Paraptosis Leucine aminopeptidase protease Franklin and Berges, (2004)
activity

Demuez et al.: Enzymatic Cell Disruption of Microalgae Biomass 1963

Biotechnology and Bioengineering
eukaryotic microalgae is not following a general scheme and
depends on multiple factors.
The concept of PCD in microalgae involves a broad range of
important yet poorly understood phenomena in the microalgae cell life.
A better understanding of microalgae PCD mechanisms and the study
of PCD signal transduction pathways together with the characterization
of proteic activities that are effectively involved in microalgae cell
degradation, would allow researchers to manipulate cultures and have
a better control of induced cell autolysis. Furthermore, components
that make up the PCD signal transduction should be prime candidates
for the development of novel inductors of autolysis.
Integration of the Autolysis Method in a
Biorefinery Process
In a biorefinery process applied to the production of bioethanol and
biogas, the autolysis method is proposed to disrupt microalgae cell
wall. Figure 2 illustrates a schematic view of the proposed
biorefinery process applying microalgal autolysis disruption. The
induction of cell lysis is performed at a specific desired cell density
and culture stage. Once cells are disrupted and lysed, a
concentration step is required but it would be more beneficial to
not separate microalgae from the culture fluid and exploits lysed
microalgae in a continuous way, as wet route process. The genomic
analysis of occurring cell wall-degrading enzymes in Chlorophyta,
using MEROPS peptidase and Pfam protein families databases
showed that microalgal cell-wall degrading enzymes contains both
carbohydrase and protease enzymes (Fig. S1). Therefore,
Figure 2. Proposed integrated process of biofuels production from microalgae
biomass, involving autolysis as cell disruption.
1964 Biotechnology and Bioengineering, Vol. 112, No. 10, October, 2015
microalgae autolysis has the advantageous effect of cell wall
disruption together with degradation of carbohydrate polymers that
are present into cell wall and as intracellular storage. Resulting
biomass containing soluble sugars, proteins and remaining cells
debris, is processed directly without enzymatic treatment, as
feeding for fermentation to produce bioethanol. Sequentially,
fermentation residues would pass through an anaerobic digestion
for methane production. As proteins content is solubilized, an
improvement in methane production yield would be expected.
Conclusions
With the increasing interest in using algae for producing biofuels,
new cost-effective cell disruption procedures have to be developed
to overcome problems associated with traditional expensive
pretreatment methods. In this sense, the development of an in
situ controllable autolysis of microalgae is a promising
alternative. Induced autolysis of microalgae must be implemented
with strain-specific enzymes that digest the glycoproteins and
polysaccharidic matrix that constitute the algae cell wall. For this
reason, endogenous hydrolytic enzymes involved in cell wall
degradation mechanisms can be used for promoting autolysis to
microalgae. Controlled cell wall degradation would be induced at
demand for the expression of lytic enzymes involved in cell
autolysis during cell life cycle or PCD. The integration of induced
autolysis approach in an industrial bioprocess will depend on
process characteristics and on the nature of final product. While
the application of a microalgae autolysis may not be the optimal
solution in all settings, it offers a promising approach to
effectively disrupt the cell wall, and consequently, the cost
competitiveness of biofuels production.
Better understanding of the dynamics and mechanisms of
natural occurring lytic enzymes involved in microalgae autolysis
will give new opportunities for the development of innovative cost-
effective disruption methods. The use of induced autolysis of
microalgae is an encouraging development which can contribute to
an important breakthrough in the cost-efficient industrial
bioprocessing for producing biofuels and other high value products
from microalgae biomass.
The authors want to thank the Spanish Ministry of Economy and
Competitiveness for financial support to this project (WW-ALGAS,
ENE2013-45416-R). Ahmed Mahdy appreciates his Ph.D scholarship funded
by the Ministry of Higher Education of Egypt. Elia Tom
as-Pej
o acknowledges
the People Programme (Marie Curie Actions) of the European Union’s
Seventh Framework Programme (FP7/2007-2013) under REA grant
agreement n 291803.
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