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In microalgal biodiesel
production, the extraction of lipids and dewatering of the biomass is an
energy-intensive process. The extraction of biofuels mainly contained two
methods; cell disruption methods and total lipid extraction methods.

 

1.6.1 Cell disruption methods for lipid
extraction

Cell
disruption is a method in which biomolecules within the cells are released and
isolated from rest of the material, so that they can be analyzed, experimented
and studied upon. The cell disruption methods are divided into three
categories: Biological methods, chemical methods and mechanical methods. The
biological methods involve utilization of enzyme for polysaccharide and/or
protein degradation. The chemical methods comprise involvement of chemical treatments
and osmotic shock. There are different kind of biological methods including the
use of microwave, bead beating, ultrasonication, high pressure homogenization
and electroporation.

 

1.6.1.1 Mechanical methods

Microalgal
strains contain rigid cell wall which prevent release of intracellular products
(extractin of oil) or breaking them require excess of energy. The greatest
advantage of this method is that, it is universally applied to biomass
regardless of its species and has lower risk of degradation and degeneration of
the target products. Harrison, (1991) provided mechanical disruption methods
such as bead beating, HPH and grinding using pestle or mortar, but there are
few methods which were applicable on wet biomass such as ultrasonication. 

 

1.6.1.2 Bead beating

Bead
beating also known as ball mill and bead mill, is a simple method of cell
disruption which open the cell wall of algal cell by shaking in a closed
container filled with small beads and target cells (Fig 3). The small beads of
0.1-6 mm are made up of glass,
ceramic or steel.  The cell walls of
algal cells are disrupted by friction and collision with the beads. Bead
beating method can disrupt cell within few minutes and is a common method of
extracting DNA from biological material (Robe et al. 2003). Compared with other
methods such as HPH, ultrasnication and homogenization, bead beating method
showed maximum extraction rate from wet pellets of Botryococcus braunii UTEX 572. In fact, 28.6% (dry weight basis) of
lipid was isolated using mixture of methanol and chloroform (1:2v/v) which is
1.96 fold higher than that of with the control (without any cell disruption
treatment) (Lee et al. 1998).  However,
other studies comparison of bead beating with other cell disruption methods
showed that, it is not an efficient approach for algal cell disruption (Cheng
et al. 2010; prabakaran and Ravindran, 2011; Sheng et al. 2012; Zheng et al.
2011). There are various factors which affect the efficiency of bead beating
method that are, the shaking rate, the bead size, the shape of container, the
amount and types of bead used.  These
factors will not only influence cell disruption but also effect the consumption
of energy. The disadvantage of this method is that it is hard to scale-up and
requires an extensive cooling system to prevent the thermal degradation of the
target products but this method is advantageous because of the simplicity of
the equipment and the rapidness of the treatment.

 

 

1.6.1.3 Microwave

The microwave is an electromagnetic wave, having
300MHz-300GHz frequency which is higher than that of radio waves and lower than
that of infrared waves.  Only small range
of microwave frequency (2450 MHz) is used in microwave ovens and this frequency
is sufficient for cell disruption because it can rotate the dipole of –OH bonds
in alcohol and water. The use of microwave radiation is advantageous due to its
quick penetration into biomass and cause cell disruption. The microwaves
rapidly heat the biomass and affect the weak hydrogen bonds in the cell envelopes.  For example, the extraction efficiency of
supercritical carbon dioxide from lyophilized chlorella vulgaris was improved upto 2.6 fold after 6 min of 800 W
microwave radiation exposures (Dejoye et al. 2011). Balasubramanian et al.
(2011) compared the heating effects of microwave radiation and water bath on a Scenedesmus obliquus at two temperatures
(80 and 95 °C). The results showed
that water bath was not significantly preferred over the use of microwave
radiation. Due to the rapid heating and pretreatment at 95°C, two-fold
improvement in lipid extraction was achieved as compared to pretreatment at 80°C when the slurry was
extracted with hexane through liquid: liquid extraction. In another study, 5g/L
diluted biomass of Scenedesmus sp., C. vulgaris and Botryococcus was treated with bead beating, ultrasonication,
microwave, autoclaving and osmotic shock and then subjected to equivalent
volume of chloroform and methanol (1:1 v/v) for 5 min. The results of this
study demonstrated that, the microwave method is an efficient method because of
higher extraction (2 to 4 fold higher) yield as compare to control (without
cell disruption technique). Prabakaran and Ravindran, (2011) conducted similar
study on Tolypothrix sp., Chlorella sp. and Nostoc sp and found that microwave and ultrasonication exhibited
best performance. Microwave has various disadvantages along with advantages
like the requirement of vast cooling system, degradation of thermally labile
products and consumption of tremendous amount of electricity when applied on
large-scale.

 

1.6.1.4 Ultrasonication

The ultrasonication method is a well known method of cell
disruption which utilizes the ‘Cavitation effect’ caused by ultrasound in a
liquid. Ultrasonic treatment (18 kHz– 1 MHz) can disintegrate fibrous, cellulosic materials into fine particles and
break the wall of cell structure (Saranya et al. 2014). When liquid is radiated
by ultrasound, small “vacant regions” known as microbubbles are formed by
acoustic waves. If ultrasound is applied in a sufficient amount, it will
compress the microbubbles to their minimum radii and implode, thereby producing
heat light (sonoluminescence), shockwaves and free radicals, which can damage
the cell envelops of microorganisms (Miller et al. 1996 and Miller et al. 2002).
The ultrasonication process is affected by temperature and viscosity of the
liquid medium. Mainly lower temperature is favorable for effective sonication,
so the liquid medium should be cool down continuously because the temperature
increases rapidly due to heat dissipation (Jiang et al. 2006).  The cavitation effect of ultrasonication is
more intense at low frequency (18-40 kHz) than that of high frequency (400-800
kHz) (Cravotto et al. 2008).  Yoo et al.
(2012) applied ultrasound of lower frequency (40 kHz) to Scenedesmus obliquus YSW15 biomass for 60 min, to increase
fermentation yield. The yield was increased after 15 min of the pretreatment
and damaged caused by ultrasound was observed after 60 min through atomic force
microscopy (AFM) and energy-filtering transmission electron microscopy
(EF-TEM). The biogas fermentation yield of diluted Scenedesmus sp (4g/L) was also increased by ultrasonication
(Gonzalez- Fernandez et al. 2012). This method has strong cell disruption
capacity but it is not applicable on pilot scale due to extensive requirement
of ultrasonic power and cooling system or the cavitation effect occurs in small
regions near ultrasonic probes (Fig 4).    

 

 

1.6.1.5
High pressure homogenization

High pressure homogenization was invented by
Charles Stacy French, also known as French press. The HPH is a positive
displacement pump which forces cell suspension through a valve, before
impacting the stream at high velocity on an impact ring. HPH utilizes hydraulic
shear force for cell disruption which is generated when the slurry under high
pressure is sprayed through a narrow tube. This approach has mainly been used
for sterilization and for extraction of the internal substances of
microorganism. This method has various advantages like having low risk f
thermal degradation, low heat formation, low cooling cost, no dead volume in
the reactor, and easy to scale up. Various investigation compared HPH with
other methods and shows that HPH exhibited the highest cell disruption
efficiency. Sheng et al. (2012) evaluate the cell disruption efficiency by
measuring the requirement of soluble chemical oxygen demand for
Synechocystis PCC 6803 biomass (20.6 g/L) and found that HPH at 2,600
psi was the best cell disruption method. Halim et al. (2012) compared efficiency of HPH with bead beating,
ultrasonication and sulfuric acid in wet biomass of Chlorococcum sp. through cell counting and
measuring the colony diameters. The results showed that HPH can destroy 70% of
cells at 500-800 bar pressure but the efficiency can be increased with higher
pressure and cell concentration. Along with various advantages, HPH has
disadvantages like it requires long treatment time and consume large amount of
energy. Therefore, some modification is required to improve HPH apparatus which
will short the treatment time and reduce the energy consumption.   

 

1.6.1.6
Electroporation

The electroporation is a
microbiological technique which involves short burst of high voltage to a
sample placed between two electrodes (Fig 5). This method has been used to
isolate or insert DNA into the cells. When a high intensity electric field is
applied, it will create electrical potential across the cell membrane which
leads to rapid electrical breakdown and local structural changes in the cell
membrane or in cell wall (Joshi and Schoenbach, 2000). Local structural changes
dramatically increase the permeability of the membrane, results in ruptured
microbial cells. Sometimes, these changes can be overcome by a healing process
when the electric field is removed. However, a strong range of electric field
has been applied which cause damage in the cell envelops beyond their healing
ability and can induce permanent cell disruption.  The cell disruption efficiency of pulse
electric field (PEF) compared with heat treatment on Synechocystis PCC
6803 suspension (0.3 g/L) (Sheng et al. 2011). A small number of cells were
ruptured and stained with SYTOX green when the culture was treated with heat,
whereas almost every cell treated with PEF was ruptured and stained with SYTOX
green, shows efficiency of PEF. Now a day’s electroporation receiving attention
from industries too like OriginOil, developed tubular and tabular equipment
which use electric field for cell lyses (Eckelberry et al. 2010). A patent has
been filed by NLP which involves the electrolysis of microalgae for biodiesel
production (Zheng et al. 2011). The electroporation is a promising method
because of its simplicity, provides financial and environmental benefits,
consume lower amount of energy because it works for nano-to micro-second. 

 

 

1.6.2
Chemical method

1.6.2.1
Chemical treatments

In
addition to mechanical and biological methods, various chemicals for cell
disruption exist such as plymyxin, lysine polymers, protamine, polycationic
peptides and cationic detergents (Vaara, 1992). These chemicals will increase
permeability of cells. The cells will rupture, if the permeability exceeds
certain limit. The cell envelope is hydrolyzed by acid and alkali treatment.
The protein layer of cell envelope can also be hydrolyzed by heating on higher
temperature. Miranda et al. (2012) increases ethanol fermentation yield to
95.6% through dry S.obliquus with 2N
sulfuric acid treatment as compared to control cells subjected to harsh
quantitative acid hydrolysis with 76% sulfuric acid. The wet biomass of Chlorella sp and Scenedesmus sp were treated with acids and alkalis for step-wise
extraction. 1M sulfuric acid and 5M sodium hydroxide was required for cell
envelopes treatment at 90°C for 30 min. After this,
0.5M sulfuric acid was added to dissolve chlorophyll and to precipitate the
free fatty acids (extracted out with hexane), involves 60% of the total lipid
recovery (Sathish and Sims, 2012).  It is
an interesting investigation because it helps to separate out lipids from that
of chlorophylls, which is a by-product of conventional lipid extraction. Sarada
et al. (2006) extracted out astaxanthin (antioxidant supplemented with high
economic value) from H. pluvialis.
Among various chemicals, the best cell disruption chemical reagents includes
hydrochloric acid (HCl), dimethyl sulfoxide (DMSO), acetone and organic acids,
which led to the recovery of 94% of the total astaxanthin from the cell
body.  The performance of 4N HCl was much
higher than that of 19% methanol and 67% DMSO. Despite of high cell disruption
efficiency, it has various disadvantages like corrosive nature of
alkalis and acids, higher cost at pilot scale; chemicals affect the content of
cells when used incorrectly and have various health and safety risks.

 

1.6.2.2 Osmotic shocks

Sudden
increase or decrease of salt concentration in the liquid medium disturbs the
balance of osmotic pressure between the interior and the exterior of the cells
is known as osmotic shock. There are two osmotic stresses that can damage
cells: hypo-osmotic stress and hyper-osmotic stress. Hypo-osmotic stress occurs
when salt concentration is lower in the medium and water flows into the cells
to balance the osmotic pressure. The cells swell or burst if the stress is too
high. In contrast, hyper-osmotic stress occurs when the salt concentration is
higher in the exterior and fluids inside the cells diffuse outwards causing
shrinkage or damage to the cell envelopes. Hypo-osmotic shock is a normal
procedure used for the extraction of substances from microorganisms. But
hypo-osmotic shock requires a large amount of water which makes it unrealistic
at the industrial scale (Cheng et al. 2010, Prabakaran and Ravindran, 2011).
Yoo et al. (2012) showed that the hyper-osmotic shock using sodium chloride
(NaCl) and sorbitol increased the yield of lipid through liquid-liquid
extraction from wild type and cell wall-less mutant strains of Chlamydomonas reinhardtii. Osmotic shock
is an inexpensive and simple approach but its performance is not efficient and
require tremendous amount of water with high salinity.

 

1.6.3 Biological method

Another
strategy to achieve cell lysis is to use digestive enzyme which will degrade
the cell envelope. Different strains or cell types have different cell wall and
cell membrane, thus the use of enzyme will depend upon the microbes present.
Geciova et al. (2002) and Harrison, (1991) used phages for cell envelope
degradation but most of the investigations utilize enzymes for cell disruption
because enzymes are commercially available and the most easily control
biological material. The enzymatic method have various advantages over other
methods like mild reaction conditions and the high selectivity (degrade a
specific chemical linkage) whereas mechanical methods destroy almost every
particle existing in the solution and chemical methods sometimes induce
side-reactions of the target products. Braun and Aach, 1975 degraded the
envelope of Chlorella microalgae (has
very resistant sporopollenin layers) after 90 hours of incubation with mixture
of enzymes (cellulose, hemicellulase and pectinase) and found that 80% of the
cells were converted into osmotic labile state cells without rigid cell walls.
The enzymatic method is effective only when enzymes are chosen carefully. The
major pitfall of this method is the cost of the enzymes. There are two methods
with whom we can reduce the cost i.e. immobilization of the enzymes and the
combination of this process with other methods (Fig 6).

 

Table
4. Different cell disruption methods used for different types of organisms

Sr. No

Cell Disruption method used

Efficient method

Organism Used

Lipid Content (%)

References

1

Bead beating
Microwaves
Sonication
Osmotic shock

Microwaves
 

Scenedesmus sp.
Chlorella
vulgaris
Botryococcus sp.

11.5%
11%
28.6%

Lee et al.
2010

2

Sonication
Osmotic Shock
Microwave
Autoclave
Bead beating

Sonication

Tolypothrix sp.
Nostoc sp.
Chlorella sp.

14%
18.2%
20.1%

Prabakaran et
al. 2011

3

Grinding
Sonication
Bead Beating
Enzymatic
lysis
Microwaves

Grinding

Chlorella
vulgaris

29%

Zheng et al.
2011

4

Grinding
Bead Vortexing
Osmotic Shock
Water bath
Sonication
Shake mill

Osmotic shock

Thraustochytrium sp. AMCQS5-5
Schizochytrium sp. S31

29.1%
 
48.7%

Byreddy et al.
2015

 

1.6.4 Total lipid extraction methods

1.6.4.1 Folch method

The
selective lipids have been extracted from a complex mixture of organic
compounds by using various organic solvents (in combination or individually).
The Folch method (Folch et al. 1957) involves the extraction of lipids from
endogenous cells using chloroform-methanol in 2:1 v/v. The homogenized cells
were mixed in equal quantity with one- fourth volume of saline solution and
mixed well. The resulting mixture was separated out in two layers and upper
phase contained lipids. This method is still used with some modification for
the estimation of algal lipids spectrophotometrically. The Folch method is a
rapid and easy method to process large number of samples but it is less
sensitive as compare to other latest procedures.

 

 

1.6.4.2 Bligh and dyer method

The
Bligh and Dyer method is a widely practiced method for lipid extactions,
wherein proteins are precipitated in the interface of two liquid phases (Bligh
and Dyer, 1959). The lipids were isolated from homogenized cells by using 1:2
(v/v) chloroform/methanol. The lipids were separated out from chloroform layer
and processed by various procedures. The Bligh and Dyer method is very similar
to that of Folch method but mainly differs in solvent/solvent and
solvent/tissue ratios. This method is still
widely used for pilot scale extraction processes and for the estimation of
lipids. Many modifications have been adopted by researchers i.e. addition of 1M
NaCl to prevent binding of acidic lipids to denatured lipids, 0.2M phosphoric
acid and HCl to improves lipid recovery (Hajra, 1974; Jensen et al. 2008)
addition of 0.5% acetic acid increased the recovery of acid phosphor-lipids
(Weerheim et al. 2002).

 

1.6.4.3 Extraction of all classes of
lipids

Matyash
et al. (2008) suggested a recent and rigorous method which is a modification of
the Folch/Bligh and dyer method. Methyl-tert- butyl ether (MTBE) was used as a
solvent for the recovery of almost all major classes of lipids. This method
provides an accurate lipidome profile. The extraction of lipids was easy
because of the formation of a low density, lipid containing organic upper
phase. The general procedure for lipid extraction is addition of 1.5 ml of
methanol and 5 ml of MTBE in 200 ml of sample, followed by 1hr incubation at
room temperature. After the addition of water (1.25 ml), the mixture was
allowed to incubate for 10 minutes at room temperature. The upper organic phase
(having lipids) was separated out after centrifugation and vacuum dried to
drain off the excess solvent. The extracted lipids can be directly used for
further study or were dissolved in 200ml of chloroform/methanol/water
(60/30/4.5 v/v/v) for storage.

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