Algal Res.2016 Dec;20:7-15

Enhanced lipid selective extraction from Chlorella vulgaris without cell sacrifice.

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Current potential application of microalgae for biofuel production without cell sacrifice in aqueous-organic biphase system

Madiha Atta, Attaullah Bukhari, Ani Idris*

Institute of Bioproduct Development, Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering, Universiti Technologi Malaysia,81310,UTM, Johor Bahru, Johor, Malaysia 



To enhance the lipid content and reduce the energy intensive dewatering and drying steps our objective is to develop milking technique without killing C. vulgaris cells by using the biocompatible organic solvent in aqueous organic biphase system. This new method can milk the cells for very long time (30 days) for lipid production. Dodecane mediated milking of lipids in C.vulgaris has the potential to be more sustainable than other solvents because of (a) high oxygen vector capacity; (b) low molecular toxicity; (c) optimum solvent recovery; (d) maximum cell viability. Thus biocompatible organic solvents dodecane can milk lipids and recycle the C. vulgaris cells to produce more lipids without cell sacrifice. Milking technique would be feasible and advantageous 1) in reducing the cell harvesting and concentrating; 2) reducing the cell destruction with maximum viability for long periods; 3) purity and high lipid selectivity; 4) higher biomass and lipid content. This throughput can break the production cost barrier and enhance the yield in the industrialization of microalgae derived biofuel. Whole-cell biocatalysis in aqueous-oganic biphase system is used for the production of high-value secondary metabolites with a greater affinity to organic solvent, immiscible with the aqueous cell phase. This throughput can break the production cost barrier and enhance the yield in the industrialization of microalgae derived biofuel. Whole-cell biocatalysis in aqueous-oganic biphase system is used for the production of high-value secondary metabolites with a greater affinity to organic solvent, immiscible with the aqueous cell phase.



At present, the substitution of fossil fuels by biofuels or biofuel appears to be an effective strategy to meet not only the future world energy demands but also the requirement for reducing greenhouse gasses (GHG) and particularly carbon emissions originating mostly from fossil fuels combustion (Ullah et al., 2015). Therefore, the focus on bioenergy as an alternative to fossil energy has increased tremendously in recent times because of global warming problems.



Today mankind is suffering from some pressing challenges like hunger, lack of energy and the deterioration of the environment. The burning of fossil fuels further aggravates the problem of CO2 emission (29 Gt to 40.2 Gt in 2030) which leads to high contribution in global warming (Rossi et al., 2015). Thus increase in global energy demand along with escalating price and rapid depletion of fossil fuels and global warming issues rekindled the prevalence of renewable and sustainable energy sources.

Current potential feed stock for biofuels are energy containing molecules like sugars, oils and cellulose obtained from food crops (first generation biofuel; FGB), lignocellulose, residues of non-food crops like straw; bagasse, forest residues, waste cooking oil and animal fats are second generation biofuels (SGB) but they have some limitations (Aro, 2016; Chisti, 2007). Of course FGB and SGB have grasped more attention due to diminishing fossils resources; increasing world population, more demand of energy and materials, modern life style and environmental concerns. However these biofuels come from food based crops presents a problem when there is not enough food to feed everyone.

Thus food versus fuel debate has led to an extensive research to find better and sustainable alternative for biofuel. Among all the biofuel producing feedstocks, microalgae, the third generation biofuel (TGB) is a potentially superior alternative to food based crops due to its fast growing rate, high lipid accumulation (40 % of the dry weight in certain species) and cost efficient cultivation using saline water and grown on non-arable land (Ahmad et al., 2011; Widjaja et al., 2009)

However microalgae biofuels production has its own limitations and disadvantages. The major disadvantage is limited knowledge of the life cycle assessment (LCA) of microalgae biofuels with respect to sustainable energy source. Furthermore, there are many technological and investment issues such as high production cost, strain selection, cultivation related issues, harvesting issues, lipid extractions, biofuel production and high initial capital investment. For example; the production cost of microalgae biofuel is about 5 – 7 times higher than for lignocellulosic biomass (Vassilev and Vassileva, 2016).

Extraction of lipids from microalgae may be one of the least developed areas among the algal biofuel production processes (Demirbas, 2011b). Each algal cell has a rigid cell wall which makes oil extraction complicated. In order to reduce the production cost of microalgae biofuels, downstream processing which involves lipid extraction must be cost efficient. Several approaches are available to extract lipids from harvested algal biomass, including bead beating, osmotic shock, ultrasonic extraction, Supercritical fluid extraction, enzymatic extraction, solvent extraction and microwave heating (Hernández et al., 2014).

“No harvesting method has yet been identified as being efficient, reliable and at reasonable cost” (Montagne et al., 2013). Although consequential advances in microalgae biotechnology have taken place, still it is in infancy due to the inefficiencies in drying and extraction process. In microalgae, extraction of lipids or other metabolite is usually done by harvesting the biomass, cell disruption and then recovery of the final product from the culture broth. Drying and grinding are energy intensive steps and kill the microalgal cells that needs to be grown again for biomass production. In the extraction process, dewatering, drying and cell disruption are the steps that decreases the yield and have greater impact on overall production cost of microalgae biofuel. Cell disruption step alone requires 30% additional cost input on the total production cost of extraction process (Kim et al., 2013). Besides, huge amount of water is required for dewatering (i-e 3000 gallon water required/gallon biofuel) the algal culture. Most of the microalgae can grow to the biomass density of only a few grams per liter water, processes built upon dry biomass were unlikely to be economical from the point of energy inputs involved (Harun et al., 2011). Furthermore extraction and purification steps for microalgal lipids generate huge amounts of organic wastes.

Simultaneous extraction and production or in situ extraction is a new arising biotechnology recently introduced. Milking (simultaneous production and extraction from viable microalgal biomass) integrates current harvesting and post harvesting processes reduces the dewatering, drying and grinding steps. It is a kind of in-situ extraction process whereby after extracting the desired products, the algal cells are recycled back to the culture thus allowing to produce the desired products once again. In this way the same cells could be reused for second or third time (cycle) or more to produce the products without cultivating again. Milking is different from the classical extraction techniques in sense that milking technique employed the production of target molecules after a specific a number of extractions when recycled microalgae cultures were able to produce no more target product whereas freshly grown microalgae is needed for each extraction in classical extraction (Chaudry et al., 2017). This non-destructive extraction involves the microalgae to recycle back to the culture so that the live cells regenerate the lipids inside their body resulting in a sustainable system with high yields as compared to the destructive system. Normally between the extraction cycles, cultures are allowed to increase oxygen production, biomass and lipid content. It is an advance alternative method for the continuous and periodic removal of the target materials from the ‘live’ fermentation or cell culture without harvesting or killing the cells which can grow again for further biomass production. By comparing to the conventional methods, there are several advantages for lipid milking from microalgae, for instance, it is an eco-friendly solvent extraction technique to milk the lipid without killing the cells, and it occurs under aqueous-organic biphasic system utilizing biocompatible organic solvent, it does not require energy intensive dewatering and drying processes, it enhances the lipid productivity by recycling the algal cells (Chisti, 2007). Milking concept specifies that the extraction should not kill the cells. Consequently, milking does not require the constant need of culturing and re-growing the entire stock of algae, which has a typical timescale of a few hours to weeks. Lipid milking in microalgae can significantly reduce the downstream processes such as dewatering and drying steps thus reduces the cost of these steps (Vinayak et al., 2015).

To successfully milk particular product from living cells it is very important to consider factors as,: (i) viability of cells (ii) minimum molecular and phase toxicity (iii) biocompatible organic solvent (iv) solvent must have affinity for respective product (v) appropriate density gradient of solvent and aqueous phase (vi) enough solvent-cells contact time for extraction (vii) recovery and separation of the solvent from the final product (lipid) (viii) the location of product inside the cell (ix) mechanism of extraction. These parameters vary from type to type of cells, product to product and the same for solvents. All these are very supportive in order to enhance the yield so as to reduce the overall cost of fuel (Aro, 2016).

The concept of milking was first applied about 2000 years ago to higher plants such as the rubber plant (Ciesielski, 1999), maple syrup in 19th century (Svanberg et al., 2012), recently halophilic bacteria (Sauer and Galinski, 1998) and now microalgae. Microalgae synthesize secondary metabolites that are used in nutraceuticals, pharmaceuticals, and cosmeceuticals. These compounds are called high-value molecules (HVM) as they are quite expensive. For example the price of astaxanthin obtained from marine microalgae would be $14,000 US kg-1 in 2018 (Badr et al., 2014). By applying milking technique, these compounds are directly extracted from the culture broth thus cut down the energy intensive dewatering and drying steps. When applied to low value compounds such as biofuels, milking is also a potential technique to enhance the concept of recycling the microalgae cells for lipid extraction to cost effective production of fuel (Hejazi et al., 2004).

Currently milking has been applied for valuable products from bacteria, microalgae and diatoms by using several methods such as osmotic shock, pulsed electric field, spontaneous oozing and utilizing biocompatible organic solvents (Vinayak et al., 2015). Sauer and Galinski (1998) extracted ectoines from halophilic bacterium Halomonas elongate (Sauer and Galinski, 1998). When transferred to low salinity medium, 15% (w/v) of NaCl, the cells of Halomonas elongata rapidly milk their ectoines to achieve their equilibrium. The cells then regenerate their ectoines when placed into the medium containing higher salt concentration. By repeating the same procedure up to 9 cycles (1 cycle over a period of 1 day) they can produce 155 mg of ectoine per cycle per gram cell dry weight of ‘bacterial milking’. Nagata et al. applied the same procedure of bacterial milking on Brevibacterium sp. and found that over a repeated cycle of 7 days, this bacterium was able to produce 2.4g/L of ectoines at the salt stress of 1.5 to 2.4 M NaCl (Nagata et al., 2008).

Pulsed electric field is another method of milking. In this method, short electric pulses were given to the cell which ultimately releases its components from the cell. The punctured cell membrane heals later on. This method is applied on yeast, cyano- bacteria, and microalgae. In this method cell viability depends on length, intensity, frequency and repetition of electric pulses (Coustets et al., 2013; Ganeva et al., 2003; Sheng et al., 2011).

Secretion or spontaneous oozing is the accumulation of a specific set of products by an organism into its surrounding environment. Some bacteria, cyanobacteria and green algae secrete lipids into external environment. For example Botryococcus braunii secretes its oil into the outer matrix. Vinayak et al. (2015) reported that a diatom strain Diadesmis confervaceae oozes lipid droplets into the culture medium. This is a natural and very slow process.

An alternative and efficient milking approach is to use biocompatible organic solvents. In this type of process, the cells were directly exposed to the solvents that extract the desire products from the cell into culture medium. Frenz et al. (1989) reported the milking of Botryococcus braunii utilizing biocompatible organic solvents for the extraction of long chain (C30-C37) hydrocarbons. The cells remained alive after repeated exposure to hexane. Later Hejazi et al. (2002) conducted a series of studies on Dunaliella salina for the milking of β-carotene. They concluded that biocompatible organic solvents with log p > 6 such as n-dodecane are most suitable for extracting β carotene from the live culture of D. salina.

(Hejazi et al., 2004). MALA et al. (2010) extracted carotenoids by milking from Spirulina platensis utilizing propanol as a biocompatible organic solvent in aqueous-organic biphase system with the maximum cell viability of 90%. In our previous study we used dodecane and tetradecane for the lipid extraction from C. vulgaris without cell sacrifice. Results showed that dodecane was the biocompatible solvent for C. vulgaris with the maximum retention of cell viability (Atta et al 2016). Figure 1 describes the cell viability of C.vulgaris under dodecane (a) and tetradecane (b).




Fig 1. Microscopic image (magnification at 100x) of cell viability of C.vulgaris under (a) dodecane (b) tetradecane. (Biological microscope Olympus CX31, Japan).


Moheimani et al (2013, 2014) non-destructively and repeatedly harvested the external oil (hydrocarbons) from Botryococcus braunii CCAP 807/2, and BOT-22 using heptane as an extracting solvent. In both the studies the productivity of milking cultures were higher than that of the conventional cultures. (Badr et al., 2014) concluded that among the biocompatible organic solvents: decane, dodecane tetradecane, and hexadecane and ethyl oleate; decane has the highest extraction ability of extracellular β-carotene from Dunaliella bardawil by applying milking technique.

Researchers investigating the use of the milking technique utilizing biocompatible organic solvents to extract high value molecules (HVM) for example, nutraceuticals and to increase the yield (Greenwell et al., 2009; Hejazi et al., 2004). Milking might be cost effective if it is applied to low value molecules such as lipids for the production of biofuels. (Rashid et al., 2014; Zhu et al., 2008). Recently milking had been induced for the successful extraction of lipids from Nannochloropsis sp and Botryococcus braunii by employing the biocompatible organic solvents. The maximum percentage of lipids obtained in those studies were 415.5 mg/ L and 117.3 mg/L under the influence of 10% v/v of tetradecane (log p 7.60) and hexadecane (log p 8.80) respectively in a single cycle (Zhang et al., 2011).

Lipid producing microalgae generally accumulate the lipids inside the cells called lipid bodies, lipid droplets or oloesomes. Biogenesis of lipid droplets occurs in the ER (endoplasmic reticulum), a major location for neutral lipid synthesis, as a nascent droplet from the cytosolic leaflet of the membrane. The enzymes that catalyse this synthesis are integral membrane proteins, which ultimately encounter the substrates for TG (triglyceride) synthesis, such as diacylglycerol and fatty acyl CoA, at the cytosolic surface of the ER. Intercellular lipases hydrolyse the neutral lipids yielding fatty acids. During photosynthesis in chloroplast, microalgae utilize CO2 in the presence of light in photosystem I (PSI) and photosystem II (PSII) and produces large amount of energy storage molecules; adenosine triphosphate (ATP), dinucleotide phosphate-oxidase (NADPH) and ribulose-1, 5- biphosphate (RuBP). In oil containing seed plants, acetyl CoA involve cytosolic glycolysis to phosphoenolpyruvate (PEP), then it enters to plastid where it is converted to pyruvate and then to acetyl CoA (Hu et al., 2008). The malonyl CoA is then transferred to the acylcarrier protein (ACP), where it undergoes series of condensation reaction for fatty acid biosynthesis by condensing enzyme, 3-ketoacyl ACP synthase III (KAS III). Chain elongation in fatty acid happens by the thioesterase (TE) which selectively hydrolyzes the thiol ester bonds of acyl-ACPs to release free fatty acids. Further conversion of acylCoA to diacylglyceride (DAG) and triacylglycerol (TAG) occurs in endoplasmic reticulum and then releases as a lipid droplets in cytosol. TAG is stored in the form of energy in microalgae and observed in their stationary growth phase. Genetics of the algal species and environmental conditions are responsible for the composition of this fatty acid i-e TAG (Abe et al., 2014; Miranda et al., 2015). Ribulose-1, 5- biphosphate carboxylase /oxygenase (Rubisco) then covert ribulose-1, 5- biphosphate (RuBP) into glyceraldehyde-3-phosphate (G3P), a primary precursor for triacylglycerol (TAG) synthesis. This G3P converts acetyl CoA to malonyl CoA, catalyzed by acetyl CoA carboxylase (ACCase) on one hand and starch catalysed by ADP-glucose pyrophosphorylase (AGPase), on the other hand.

On the basis of the result of the efficient extraction of lipid, milking experiment was performed for very long time (long term milking for 30 days) under 20% dodecane concentration from C.vulgaris cells. Dodecane could milk lipids under continuous recirculation in the aqueous-organic biphase system. This is an important step in the development of the lipid selective milking for biofuel production from C.vulgaris.

The important aspect of using aqueous-organic biphase system is simultaneous production and extraction of the target compound with selectivity and high product recovery. Results obtained from long period of milking (long term milking) shows that lipid content was higher (2595.3±70.23mg/L) in aqueous-organic biphase system as compared to that extracted without milking (2132±31.11mg/L). This shows that increase in extraction time results in increase in the lipid content in aqueous-organic phase in milking process under continuous recirculation of organic solvent.


Figure 2. Lipid content of long term milking culture and control culture (without solvent).


Figure 3 Shows the FAME components of C.vulgaris obtained from milking and that of the control culture. Results show that dominant fatty acid in both the milking culture and control culture were palmitic (39.11±), (30.15±7.03) and stearic acid (31.2±3.31), (22.43±1.12) respectively. However the percentages of these fatty acids were higher in milking culture as compared to those obtained in control culture. Linoleic acid (C18:2n6c) which is also a prominent fatty acid methyl ester of C.vulgaris shows its higher concentration in control culture whereas in milking culture its concentration is very low. Composition of Fatty acid methyl ester of long term milking experiment shows that milking could produce appropriate biofuel from C.vulgaris.


Figure 3. Gas chromatographic analysis of FAME components of milked lipids and control culture without solvent.


Application of two phase bioreactors in microalgal biotechnology has been reported previously. Frenz et al. extracted hydrophobic hydrocarbons from the microalga, Botryococcus braunii, by exposing the biomass (cells) for a short time to hexane in biphase system (Frenz et al., 1989). Application of aqueous-organic bioreactors in extraction of bioproducts from fermentation and living cells has revealed auspicious outcomes. Leon et al. (1998) reported it would be possible to reach selective extraction of product from the microalgae cells and it can improve productivity and yield.

The overall yield of lipid extraction in the two-phase system can be increased considerably. For successful application of these two-phase system several factors have to be considered. Obviously, not only amassing of bioproducts inside the cells is required but also rather intra-cellular compound ought to be excreted by the cells in order to simplify product recovery (Buitelaar et al., 1991). Therefore design of a two-phase-system in which simultaneous production and extraction is possible, is suggested as the initial step. In the development of biphase reactors, segregation of cells and its bioproducts, activity and stability of cells are largely involved technically and economically practicable processes (Zijlstra et al., 1998). Application of aqueous-organic biphase system is a useful technique for improvement of yield and productivity of fermentations. Fermentative extraction of lipids in-situ can break the barrier of higher cost of biofuel production and in future can prove most promising extraction technique. Moreover, this continuous production or recycling of the algal cultures overcome the low productivity and high cost in producing high-value molecules (HVM) such as fucoxanthin, beta-carotene, and astaxanthin.



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