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In microalgal biodieselproduction, the extraction of lipids and dewatering of the biomass is anenergy-intensive process. The extraction of biofuels mainly contained twomethods; cell disruption methods and total lipid extraction methods. 1.6.1 Cell disruption methods for lipidextractionCelldisruption is a method in which biomolecules within the cells are released andisolated from rest of the material, so that they can be analyzed, experimentedand studied upon. The cell disruption methods are divided into threecategories: Biological methods, chemical methods and mechanical methods. Thebiological methods involve utilization of enzyme for polysaccharide and/orprotein degradation.

The chemical methods comprise involvement of chemical treatmentsand osmotic shock. There are different kind of biological methods including theuse of microwave, bead beating, ultrasonication, high pressure homogenizationand electroporation. 1.6.1.1 Mechanical methodsMicroalgalstrains contain rigid cell wall which prevent release of intracellular products(extractin of oil) or breaking them require excess of energy. The greatestadvantage of this method is that, it is universally applied to biomassregardless of its species and has lower risk of degradation and degeneration ofthe target products. Harrison, (1991) provided mechanical disruption methodssuch as bead beating, HPH and grinding using pestle or mortar, but there arefew methods which were applicable on wet biomass such as ultrasonication.

   1.6.1.2 Bead beatingBeadbeating also known as ball mill and bead mill, is a simple method of celldisruption which open the cell wall of algal cell by shaking in a closedcontainer filled with small beads and target cells (Fig 3).

The small beads of0.1-6 mm are made up of glass,ceramic or steel.  The cell walls ofalgal cells are disrupted by friction and collision with the beads. Beadbeating method can disrupt cell within few minutes and is a common method ofextracting DNA from biological material (Robe et al. 2003). Compared with othermethods such as HPH, ultrasnication and homogenization, bead beating methodshowed maximum extraction rate from wet pellets of Botryococcus braunii UTEX 572. In fact, 28.

6% (dry weight basis) oflipid was isolated using mixture of methanol and chloroform (1:2v/v) which is1.96 fold higher than that of with the control (without any cell disruptiontreatment) (Lee et al. 1998).

  However,other studies comparison of bead beating with other cell disruption methodsshowed that, it is not an efficient approach for algal cell disruption (Chenget al. 2010; prabakaran and Ravindran, 2011; Sheng et al. 2012; Zheng et al.2011). There are various factors which affect the efficiency of bead beatingmethod that are, the shaking rate, the bead size, the shape of container, theamount and types of bead used.  Thesefactors will not only influence cell disruption but also effect the consumptionof energy.

The disadvantage of this method is that it is hard to scale-up andrequires an extensive cooling system to prevent the thermal degradation of thetarget products but this method is advantageous because of the simplicity ofthe equipment and the rapidness of the treatment.  1.6.1.3 MicrowaveThe microwave is an electromagnetic wave, having300MHz-300GHz frequency which is higher than that of radio waves and lower thanthat of infrared waves.  Only small rangeof microwave frequency (2450 MHz) is used in microwave ovens and this frequencyis sufficient for cell disruption because it can rotate the dipole of –OH bondsin alcohol and water. The use of microwave radiation is advantageous due to itsquick penetration into biomass and cause cell disruption.

The microwavesrapidly heat the biomass and affect the weak hydrogen bonds in the cell envelopes.  For example, the extraction efficiency ofsupercritical carbon dioxide from lyophilized chlorella vulgaris was improved upto 2.6 fold after 6 min of 800 Wmicrowave 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 showedthat water bath was not significantly preferred over the use of microwaveradiation.

Due to the rapid heating and pretreatment at 95°C, two-foldimprovement in lipid extraction was achieved as compared to pretreatment at 80°C when the slurry wasextracted with hexane through liquid: liquid extraction. In another study, 5g/Ldiluted biomass of Scenedesmus sp., C. vulgaris and Botryococcus was treated with bead beating, ultrasonication,microwave, autoclaving and osmotic shock and then subjected to equivalentvolume of chloroform and methanol (1:1 v/v) for 5 min.

The results of thisstudy demonstrated that, the microwave method is an efficient method because ofhigher extraction (2 to 4 fold higher) yield as compare to control (withoutcell disruption technique). Prabakaran and Ravindran, (2011) conducted similarstudy on Tolypothrix sp., Chlorella sp. and Nostoc sp and found that microwave and ultrasonication exhibitedbest performance. Microwave has various disadvantages along with advantageslike the requirement of vast cooling system, degradation of thermally labileproducts and consumption of tremendous amount of electricity when applied onlarge-scale. 1.

6.1.4 UltrasonicationThe ultrasonication method is a well known method of celldisruption which utilizes the ‘Cavitation effect’ caused by ultrasound in aliquid. Ultrasonic treatment (18 kHz– 1 MHz) can disintegrate fibrous, cellulosic materials into fine particles andbreak the wall of cell structure (Saranya et al.

2014). When liquid is radiatedby ultrasound, small “vacant regions” known as microbubbles are formed byacoustic waves. If ultrasound is applied in a sufficient amount, it willcompress the microbubbles to their minimum radii and implode, thereby producingheat light (sonoluminescence), shockwaves and free radicals, which can damagethe cell envelops of microorganisms (Miller et al. 1996 and Miller et al. 2002).The ultrasonication process is affected by temperature and viscosity of theliquid medium.

Mainly lower temperature is favorable for effective sonication,so the liquid medium should be cool down continuously because the temperatureincreases rapidly due to heat dissipation (Jiang et al. 2006).  The cavitation effect of ultrasonication ismore intense at low frequency (18-40 kHz) than that of high frequency (400-800kHz) (Cravotto et al. 2008).  Yoo et al.(2012) applied ultrasound of lower frequency (40 kHz) to Scenedesmus obliquus YSW15 biomass for 60 min, to increasefermentation yield.

The yield was increased after 15 min of the pretreatmentand damaged caused by ultrasound was observed after 60 min through atomic forcemicroscopy (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 disruptioncapacity but it is not applicable on pilot scale due to extensive requirementof ultrasonic power and cooling system or the cavitation effect occurs in smallregions near ultrasonic probes (Fig 4).

      1.6.1.5High pressure homogenizationHigh pressure homogenization was invented byCharles Stacy French, also known as French press. The HPH is a positivedisplacement pump which forces cell suspension through a valve, beforeimpacting the stream at high velocity on an impact ring. HPH utilizes hydraulicshear force for cell disruption which is generated when the slurry under highpressure is sprayed through a narrow tube.

This approach has mainly been usedfor sterilization and for extraction of the internal substances ofmicroorganism. This method has various advantages like having low risk fthermal degradation, low heat formation, low cooling cost, no dead volume inthe reactor, and easy to scale up. Various investigation compared HPH withother methods and shows that HPH exhibited the highest cell disruptionefficiency. Sheng et al. (2012) evaluate the cell disruption efficiency bymeasuring the requirement of soluble chemical oxygen demand forSynechocystis PCC 6803 biomass (20.6 g/L) and found that HPH at 2,600psi 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 andmeasuring the colony diameters. The results showed that HPH can destroy 70% ofcells at 500-800 bar pressure but the efficiency can be increased with higherpressure and cell concentration. Along with various advantages, HPH hasdisadvantages like it requires long treatment time and consume large amount ofenergy. Therefore, some modification is required to improve HPH apparatus whichwill short the treatment time and reduce the energy consumption.     1.6.1.

6ElectroporationThe electroporation is amicrobiological technique which involves short burst of high voltage to asample placed between two electrodes (Fig 5). This method has been used toisolate or insert DNA into the cells. When a high intensity electric field isapplied, it will create electrical potential across the cell membrane whichleads to rapid electrical breakdown and local structural changes in the cellmembrane or in cell wall (Joshi and Schoenbach, 2000). Local structural changesdramatically increase the permeability of the membrane, results in rupturedmicrobial cells. Sometimes, these changes can be overcome by a healing processwhen the electric field is removed. However, a strong range of electric fieldhas been applied which cause damage in the cell envelops beyond their healingability and can induce permanent cell disruption.

  The cell disruption efficiency of pulseelectric field (PEF) compared with heat treatment on Synechocystis PCC6803 suspension (0.3 g/L) (Sheng et al. 2011). A small number of cells wereruptured and stained with SYTOX green when the culture was treated with heat,whereas almost every cell treated with PEF was ruptured and stained with SYTOXgreen, shows efficiency of PEF. Now a day’s electroporation receiving attentionfrom industries too like OriginOil, developed tubular and tabular equipmentwhich use electric field for cell lyses (Eckelberry et al. 2010).

A patent hasbeen filed by NLP which involves the electrolysis of microalgae for biodieselproduction (Zheng et al. 2011). The electroporation is a promising methodbecause of its simplicity, provides financial and environmental benefits,consume lower amount of energy because it works for nano-to micro-second.    1.6.2Chemical method1.6.2.

1Chemical treatmentsInaddition to mechanical and biological methods, various chemicals for celldisruption exist such as plymyxin, lysine polymers, protamine, polycationicpeptides and cationic detergents (Vaara, 1992). These chemicals will increasepermeability of cells. The cells will rupture, if the permeability exceedscertain limit. The cell envelope is hydrolyzed by acid and alkali treatment.The protein layer of cell envelope can also be hydrolyzed by heating on highertemperature.

Miranda et al. (2012) increases ethanol fermentation yield to95.6% through dry S.obliquus with 2Nsulfuric acid treatment as compared to control cells subjected to harshquantitative acid hydrolysis with 76% sulfuric acid.

The wet biomass of Chlorella sp and Scenedesmus sp were treated with acids and alkalis for step-wiseextraction. 1M sulfuric acid and 5M sodium hydroxide was required for cellenvelopes treatment at 90°C for 30 min. After this,0.5M sulfuric acid was added to dissolve chlorophyll and to precipitate thefree fatty acids (extracted out with hexane), involves 60% of the total lipidrecovery (Sathish and Sims, 2012).  It isan interesting investigation because it helps to separate out lipids from thatof chlorophylls, which is a by-product of conventional lipid extraction. Saradaet al. (2006) extracted out astaxanthin (antioxidant supplemented with higheconomic value) from H.

pluvialis.Among various chemicals, the best cell disruption chemical reagents includeshydrochloric acid (HCl), dimethyl sulfoxide (DMSO), acetone and organic acids,which led to the recovery of 94% of the total astaxanthin from the cellbody.  The performance of 4N HCl was muchhigher than that of 19% methanol and 67% DMSO.

Despite of high cell disruptionefficiency, it has various disadvantages like corrosive nature ofalkalis and acids, higher cost at pilot scale; chemicals affect the content ofcells when used incorrectly and have various health and safety risks. 1.6.2.2 Osmotic shocksSuddenincrease or decrease of salt concentration in the liquid medium disturbs thebalance of osmotic pressure between the interior and the exterior of the cellsis known as osmotic shock. There are two osmotic stresses that can damagecells: hypo-osmotic stress and hyper-osmotic stress. Hypo-osmotic stress occurswhen salt concentration is lower in the medium and water flows into the cellsto balance the osmotic pressure.

The cells swell or burst if the stress is toohigh. In contrast, hyper-osmotic stress occurs when the salt concentration ishigher in the exterior and fluids inside the cells diffuse outwards causingshrinkage or damage to the cell envelopes. Hypo-osmotic shock is a normalprocedure used for the extraction of substances from microorganisms.

Buthypo-osmotic shock requires a large amount of water which makes it unrealisticat 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-liquidextraction from wild type and cell wall-less mutant strains of Chlamydomonas reinhardtii.

Osmotic shockis an inexpensive and simple approach but its performance is not efficient andrequire tremendous amount of water with high salinity.  1.6.

3 Biological methodAnotherstrategy to achieve cell lysis is to use digestive enzyme which will degradethe cell envelope. Different strains or cell types have different cell wall andcell membrane, thus the use of enzyme will depend upon the microbes present.Geciova et al. (2002) and Harrison, (1991) used phages for cell envelopedegradation but most of the investigations utilize enzymes for cell disruptionbecause enzymes are commercially available and the most easily controlbiological material.

The enzymatic method have various advantages over othermethods like mild reaction conditions and the high selectivity (degrade aspecific chemical linkage) whereas mechanical methods destroy almost everyparticle existing in the solution and chemical methods sometimes induceside-reactions of the target products. Braun and Aach, 1975 degraded theenvelope of Chlorella microalgae (hasvery resistant sporopollenin layers) after 90 hours of incubation with mixtureof enzymes (cellulose, hemicellulase and pectinase) and found that 80% of thecells were converted into osmotic labile state cells without rigid cell walls.The enzymatic method is effective only when enzymes are chosen carefully. Themajor pitfall of this method is the cost of the enzymes. There are two methodswith whom we can reduce the cost i.e. immobilization of the enzymes and thecombination of this process with other methods (Fig 6).

 Table4. 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 methods1.6.4.

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

  1.6.4.2 Bligh and dyer methodTheBligh and Dyer method is a widely practiced method for lipid extactions,wherein proteins are precipitated in the interface of two liquid phases (Blighand Dyer, 1959). The lipids were isolated from homogenized cells by using 1:2(v/v) chloroform/methanol. The lipids were separated out from chloroform layerand processed by various procedures.

The Bligh and Dyer method is very similarto that of Folch method but mainly differs in solvent/solvent andsolvent/tissue ratios. This method is stillwidely used for pilot scale extraction processes and for the estimation oflipids. Many modifications have been adopted by researchers i.e. addition of 1MNaCl to prevent binding of acidic lipids to denatured lipids, 0.2M phosphoricacid 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 oflipidsMatyashet al. (2008) suggested a recent and rigorous method which is a modification ofthe Folch/Bligh and dyer method. Methyl-tert- butyl ether (MTBE) was used as asolvent for the recovery of almost all major classes of lipids.

This methodprovides an accurate lipidome profile. The extraction of lipids was easybecause of the formation of a low density, lipid containing organic upperphase. The general procedure for lipid extraction is addition of 1.5 ml ofmethanol and 5 ml of MTBE in 200 ml of sample, followed by 1hr incubation atroom temperature. After the addition of water (1.

25 ml), the mixture wasallowed to incubate for 10 minutes at room temperature. The upper organic phase(having lipids) was separated out after centrifugation and vacuum dried todrain off the excess solvent. The extracted lipids can be directly used forfurther study or were dissolved in 200ml of chloroform/methanol/water(60/30/4.5 v/v/v) for storage.

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