Heterotrophic Cultivation of Microalgae for Production of Biodiesel

2 Recent Patents on Biotechnology 2011, Vol. 5, No. 2 Mohamed et al.

toautotrophic cultivation is not required for diesel engine applications [14]. Mixotrophic microalgae may exhibit a dual photoautotrophic and phagotrophic mode of nutrition [15]. However, certain observations on the mixotrophic al-gae suggested that phagotrophic growth reaches lower repro-ductive rates and incurs higher metabolic costs as compared to heterotrophic microalgae. Furthermore, its capacity for purely photoautotrophic growth is low when weighed against the obligately phototroph [16].

The advantages of using heterotrophic microalgae culti-vation method as compared to outdoor pond and photobiore-actor (PBR) system have been outlined by several research-ers. These include very high cell density, increased lipid con-tent, high specific growth rate and the ability to maintain axenic mono-culture to produce valuable higher purity me-tabolites [17-20]. The list of algal strains that are able to sus-tain heterotrophic growth has been compiled [21]. As such, identification of all strains in nature possessing such trait might not be the core theme for protection purposes, or as a matter of fact could really be classified as patentable subject matters. Intellectual property (IP) rights in algal oil indus-tries are by far dominated by works on PBR design and mi-croalgae pond layout as well as optimization processes [22]. Information from the international patent depositories reveal that the numbers of protections bestowed on technologies incorporating heterotrophic strains is smaller than technolo-gies related to photoautotrophic systems. This could be at-tributed to the limited number of commercially valuable strains that readily undergo heterotrophic growth [20] cou-pled by the skepticism towards heterotrophic model due to the elimination of the primary thermodynamics advantage of having biodiesel produced from the cell’s ability to harness sunlight and CO2 fixation. More patent applications are pre-dicted in the near future since most energy companies are still at the early start-up stage prioritizing on R&D works. This review describes and discusses some of the technolo-gies disclosed for IP protection with focus on the relevant bioprocess engineering, medium formulation, cultivation strategies and molecular improvement of microalgae strains for biodiesel production through heterotrophic cultivation of microalgae.

2. HISTORICAL PERSPECTIVE OF PRIOR ARTS

The historical perspective of prior arts related to het-erotrophic cultivation of microalgae is summarized in Table 1. The interest in heterotrophic system in the late 1950s [23] was initiated by the restricted success of outdoor mono-cultures towards two microalgae genera; cyanobacteria Spi-rulina spp and Dunaliella spp. Spirulina spp are able to tol-erate high pH and bicarbonate concentration and widely used for health supplement. On the other hand, Dunaliella spp are able to grow in extremely high salinities and widely used as source of -carotene [24]. Early development of synthetic medium to grow microalgae in the dark has been described in the US Patent filed in 1958 [25]. This patent is applicable for microalgae from the genus Chlorococcum and Chlorella, preferably of the species C. vulgaris and C. pyrenoidosa. The invention proposed growth medium consists of carbo-hydrate and proteinaceous materials, which emphasize on the use of urea (0.2% to 1.2% wv

-1) to enhance carotenoids pro-

duction. Carbohydrate sources could be any assimilable

starch or sugar with a ratio of 1: 0.4-0.8 to water soluble pro-tein. A variation of the medium employed for the production using a 378 L deep tank bioreactor was published in 1964 [26]. The latter version was specific on the critical range of ammonium or inorganic nitrates (i.e., between 0.05 to 0.6% wv

-1) to be used for effective cultivation. The data from this

patent indicates that the optimum growth (20 gL-1

of dried biomass) and carotenoids yield (70 to 85 mgL

-1 xanthophyll)

could be obtained from 2 to 11 days of cultivation. This give a substantial increase in cell density and carotenoid produc-tivity as compared to photoautotrophic algae cells, in which, the concentration rarely exceed 1-2 gL

-1.

Inventions pertaining to microalgae lipids began with the realization that the beneficial long chain polyunsaturated fatty acids (PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are not entirely synthe-sized de novo in oceanic fishes but acquired through ingest-ing zooplanktons that fed on photosynthetic microalgae [27]. Martek Biosciences was heavily involved in algal lipid-based technologies since the early 1980’s and holds the highest numbers of patent applications or awarded (127) for the development of viable heterotrophic processes for omega-3 fatty acid production. Among others, the identifica-tion of Nitzschia alba as high EPA-producing strain from the University of Texas (UTEX) culture collection [28, 29]. The cultivation of N. alba in bioreactor is claimed to yield 45 to 50 gL

-1 cells in 64 h. In this case, it produces about 50% ww

-

1 of triacylglycerol (TAG) with 4-5% contained EPA. Alter-

native process with regards to EPA-rich marine eukaryotes of the order Thraustochytriales has been described by Barclay of Omegatech [30]. The screening on 150 microal-gae isolates had led to the discovery of high omega-3 lipid producer, Schizochytrium sp. 31. The isolate exhibits fatty acid yield of about 10 times higher than the previous known ATCC strains and grow well under low salinities. Thus, it has the ability to remove the threat of corrosion as well as problems associated with the disposal of saline water. A final cell concentration of 21.7 gL

-1 obtained from the 48 h culti-

vation of Schizochytrium sp. 31 in a bioreactor would pro-vide 40% ww

-1 of fatty acids, in which, 25.6% representing

EPA. Additionally, inventions specific to the production of DHA from dinoflagellates Crypthecodinium cohnii were disclosed by Kyle et al. [31,32]. The strain has potential for industrial applications since DHA is the only PUFA present in sufficient quantities. The heterotrophic cultivation of C. cohnii starts with 10-20 gL

-1 glucose and 6-12 gL

-1 micronu-

trient-rich yeast extract in strength natural or artificial sea water and lasts for 72-120 h. About 80-150 gL

-1 glucose and

25-50 gL-1

yeast extract need to be continuously added to prevent nutrient starvation. Final cell concentration could e achieved from 20 to 40 gL

-1 having

15-30% ww

-1 of lipid, in

which, 20-35% consists of DHA in TAG form.

The United States policies on developing alternative en-ergy through the passage of the National Energy Act and the Public Utilities Regulatory Policies Act 1978 were in re-sponse to the energy crisis started by the 1978 oil embargo [33]. The legislation had set off the seminal works on algae as alternative to fossil fuels through a DOE-supported “Aquatic Species Program” under the purview of National Renewable Energy Laboratory (NREL). The USD25 million program that spanned for 18 years was one of the most

Heterotrophic Microalgae for Biodiesel Production Recent Patents on Biotechnology 2011, Vol. 5, No. 2 3

Table 1. Historical Perspective of Prior Arts in Patents Related to Heterotrophic Cultivation of Microalgae

Publication

Number Inventor and Year (Assignee) Microalgae Strains Brief Description of the Invention

US2949700 Kathrein (1960)

(Grain Processing Corporation)

Chlorococcum and Chlorella

(preferably C. vulgaricus

and C. pyrenoidosa)

Early known patent on method to cultivate microalgae in dark

condition. Medium consists of carbon and nitrogen sources, em-

phasizing on the use of urea for the production of -carotene and

xanthophylls

US3142135 Kathrein (1964)

(Grain Processing Corporation)

Chlorococcum and Chlorella

(preferably C. vulgaricus

and C. pyrenoidosa)

Dark cultivation of microalgae in large scale deep tank bioreactor

US5130242 Barclay (1992)

(Omegatech Inc.) Schizochytrium sp. 31

Method to cultivate heterotrophic microalgae specifically for high

concentration of lipid. In this case, the microbial products com-

prise of omega-3 highly unsaturated fatty acids

US5244921

US5567732

Kyle and Gladue (1993; 1996)

(Martek Biosciences Corp) Nitzschia alba

Production of eicosapentaenoic acid (EPA)-containing oil in

stirred tank reactor using medium containing glucose

US5407957

US5711983

Kyle et al. (1995; 1998)

(Martek Biosciences Corp) Crypthercodinium cohnii

Production of docosahexaenoic acid (DHA)-containing oil by

C. cohnii, which is an obligate heterotroph algae

US5578472

Ueda et al. (1996)

(Mitsubishi Jukogyo Kabushiki

Kaisha)

Chlamydomonas reinhardtii Method to produce bioethanol fuel from microalgae

CN1418946A Wu et al. (2003;2005)

(Tsinghua University) Chlorella sp.

Semi-aseptic method to culture heterotrophic Chlorella for bio-

diesel production

CN1446882A Formulation of medium for heterotrophic biodiesel production

through enzymatic hydrolysis of low grade starch

CN 1446883A Method for preparing biodiesel by using fast pyrolysis of tiny

algae powder

CN 1699516A

Preparation of biodiesel which comprises of charging a predeter-

mined amount of methanol into algal lipid in the presence of acid

catalyst and heating to a predetermined temperature

comprehensive research efforts to date on biofuel derived from microalgae. The discovery that many species produce as much as 60% ww

-1 of cellular oil droplets under certain

growth conditions, and that it could be manipulated as source of energy became the main driver of the program. Early em-phasis was on hydrogen production but the focus changed to biodiesel in the early 1980s. More than 3,000 strains have been collected from the western, northwestern and south-eastern regions of the continental U.S. and Hawaii, and screened for their oil-producing capacity. The key attention was on harnessing highly efficient photosynthetic strains that could tolerate variations in salinity, pH, temperature, and also for their ability to produce neutral lipids when grown in outdoor pond system. The collection was narrowed to the 300 most promising strains, primarily of green algae (Chlo-rophyceae) and diatoms (Bacillariophyceae) [34].

The open cultivation systems for microalgae encountered various problematic steps. Open ponds occasionally froze during the winter, which implies more productive scenarios would require warmer climates or the incorporation of waste heat to maintain the productivity. In term of growth, though forced starvation of nitrogen sources would lead to accumu-lation of lipid, nutrient deficiency actually reduces the over-

all rate of oil production in the culture due to concomitant decrease in cell growth rate. Low biomass concentration in pond systems would also cause the dewatering and harvest-ing steps to be energy-intensive and high cost. The initiative wavered throughout 1980’s and was abandoned by 1996. The closed-out report at that time had estimated the cost of algal oil processing at around USD40-70 per barrel. At the same time crude oil was traded at only USD25 per barrel, which make algal oil economically not viable [11]. Never-theless, studies on algal characterization, physiology, biochemistry, molecular genetic and process development for mass cultivation have progressed very well. After the closure of NREL program, only very few private corporations continued with research on bioenergy from algae and applica-tions for IP protection had been reduced significantly.

Interestingly, early development and patent filing en-deavor on the heterotrophic system specific to biofuel pro-duction could be found in the Asian region rather than Europe or the United States, where the follow-up technolo-gies to NREL program were more preoccupied with the pho-toautotrophic system. The process of making bioethanol from microalgae using starch accumulated inside Chlamy-domonas reinhardtii UTEX2247 as a starting material has


been claimed by Ueda et al. [35]. In the patented process, C. reinhardtii cells are first grown in a bioreactor with favor-able conditions for high cell growth. The culture broth is subsequently concentrated to achieve 10-20% solid content and undergo fermentation under dark and anaerobic condi-tions with pH regulated from 6 to 9. Alcoholization of starch ensues and ethanol is collected when concentration reached 5,000 to 50,000 ppm. The residual microalgae-cake already stripped of ethanol is further subjected to methanogenesis. The resultant methane gas can be burned for energy recovery and generated CO2 for carbon uptake by the following batch of microalgae cultivation. Hence, the design represents an interesting close loop concept with very nearly zero dis-charge level.

Research and development of biodiesel from heterotro-phic Chlorella have been actively pursued by the researchers from Tsinghua University. Most of their earlier IP disclo-sures at the start of 2003 were submitted to the China’s State Intellectual Property Office (SIPO). Initial approach to het-erotrophic cultivation was made by adopting two-steps semi-aseptic strategy [36]. The first stage requires the preparation of 400 mL inoculum culture in a defined medium containing 10 gL

-1 glucose to enhance cell growth, where log phase

could be reached after about 20-24 h of cultivation. The in-oculum is later transferred in non-sterile conditions into 3 L seed flask with agitation and aeration at flow rate ranging from 60 to 100 Lh

-1. Subsequent transfer to 10 L or 20 L

production bioreactor requires medium containing glucose and glycine at concentration ranging from 50 to 90 gL

-1 and

0.5 to 0.9 gL-1

, respectively. The patent describes that the color of microalgae cells change from green to a shiny light-yellow during the course of trophic conversion. The inven-tion also claimed that the cultivation cost could be greatly reduced while high-purity Chlorella cells could be main-tained.

The method for the preparation of cultivation medium for Chlorella based on enzymatic hydrolysis of low grade starch has also been patented [37]. The method requires the combi-natorial action of two enzymes (amylase and glucoamylase) for starch hydrolysis in the presence of extract from Asper-gillus biomass or soil extract to obtain an aqueous solution of glucose. Substantially high lipid content in algae cells was claimed for cultivation of Chlorella with this patented me-dium formulation. Harvested Chlorella cells are dried into powderized form with particulate size of an average diameter of 0.6 mm, where biodiesel could be extracted by fast pyro-lysis [38]. The powderized Chlorella biomass will pass through a pyrolysis system via nitrogen gas at flow rate rang-ing from 0.1 to 0.4 m

3 h

-1. The unit is maintained at tempera-

ture ranging from 300 to 600oC with heating rate in the range

from 400 to 1000oC s

-1. During the process the algae powder

is fed at the feeding rate ranging from 1 to 10 g min-1

. Pyro-lysis produces a stream of hydrocarbon gases and residual tar. Biodiesel is formed as a result of these gases undergoing condensation. The oil is claimed to have low viscosity, high flowability and calorific value ranging from 40 to 50 MJ kg

-

1, which is about 33% higher than the biodiesel obtained

from photoautotrophic system.

Biodiesel from Chlorella is also produced through trans-esterification in a treatment steps parallel to the process that

uses vegetable oil. A related patent discloses a procedure, in which, the lipid extracted from microalgae biomass is mixed with predetermined amount of methyl alcohol [39]. The variables of the process include molar ratio of methanol to algal oil (from 30:1 to 56:1), temperature (30

oC to 60

oC) and

time (5 to 7 h). The transesterification process shall be oper-ated in the presence of pure acid catalyst at 100% of the weight of raw oil and the reaction mixture shall be agitated continuously at 160 rpm. It is claimed that the process could produce high quality biodiesel with the yield of about 68% of the total lipid. The biodiesel has the density ranging from 0.864 to 0.875 kg L

-1 and the viscosity of 5.2 x 10

-4 Pa.s.

3. RECENT INNOVATIONS IN HETEROTROPHIC ALGAL OIL PRODUCTION

3.1. Microalgae Strains

Lipid-rich and fast growing microalgal strains must be employed to make sure that algae-biofuel process is industri-ally and economically viable. For microalgae strains to be effective in heterotrophic culture, it should (i) have the fac-ulty of cell division and active metabolisms in total darkness, (ii) able to grow in easy-to-sterile organic substrates, (iii) adapt very fast to environmental changes, and (iv) have the capacity to resist hydrodynamic stresses during bioreactor operation [20]. Microalgae oil-producing strains to be con-sidered should preferably possess lipid content of at least 20% by weight of its cell. Wild types microalgae accumulate lipid to a practical maximum of 40 to 60% of its cell weight while bio-engineered strains may reach as high as 80%. In-dustrially important oil-producing microalgae generally con-sist of phylum bacillariophyta (diatoms), chlorophyta (green algae), cyanophyta (blue-green algae), chrysophyta (golden-brown algae) and several strains from phylum haptophyta. Specific non-limiting examples of heterotrophically capable bacillariophytes genera suitable for biofuel production in-clude Amphipleura, Amphora, Chaetoceros, Cyclotella, Cymbella, Fragilaria, Hantzschia, Navicula, Nitzschia, Phaeodactylum, and Thalassiosira. Chlorophytes microalgae would consist of Ankistrodesmus, Botryococcus, Chlamydo-monas, Chlorella, Chlorococcum, Dunaliella, Monoraphid-ium, Neochloris, Oocycstis, Scenedesmus and Tetraselmis. Cyanophytes may include Oscillatoria and Synechococcus. A specific example of chrysophytes includes Boekelovia and as for haptophytes, non-limiting examples include Isochrysis and Pleurochrysis [40].

The various process technologies employing the het-erotrophic-capable microalgae species that have been pat-ented or pending protection to the respective inventors is listed in Table 2. This list of microalgae is mostly exemplar to demonstrate the workings of the specified invention and hence, definite success of any heterotrophic cultivation should not be restricted to exclusive application of the listed strains. Chlorella, particularly C. protothecoides, apparently has received the most attention by many innovators for pos-sible large scale biodiesel production. C. protothecoides are non-motile and spherical unicellular chlorophyte, which able to asexually reproduce rapidly during cultivation to reach a final cell densities of about 10-fold higher than other com-mon microalgae. Lipid content in heterotrophic C. pro-tothecoides cell (55% of its cell weight) is four-fold higher


Table 2. Recent Technologies Employing Heterotrophic Microalgae to Produce Biofuel

Publication Number Microalgae Strain(s) as Stipulated in the

Embodiment of Invention

Brief Description of Disclosed

Technology

Inventor(s)

(Assignee) and Year

US7,905,930 Chlorella spp., Dunaliella spp. Two stage process for producing oil from

microalgae

Oyler et al.

(Genifuel Corp.) - 2011

WO2010/046115A2 Nannochloropsis spp. Integrated concurrent process of pho-

toautotrophic and heterotrophic algae

Bellussi et al.

(ENI S.P.A) - 2010

WO2010/042842A2 Chlorella protothecoides EVG42050 Co-cultivation of algae with lignocellu-

losic-degrading microorganism strains

De Crecy

(Evolugate LLC) - 2010

US2011/0045564A1

Chlorella vulgaris, Chlorella sp. D101, Euglena

gracilis, Euglena sp. D405, Spirulina maxi-

ma,Spirulina sp. D11

Enhancement of algae bioproducts

through the use of symbiotic diazotroph-

attenuated stress co-cultivation

Dhamwichukorn – 2011

US2010/0317073A1 Chlorella protothecoides Genetic manipulation of C. protothecoides

to increase lipid yield

Sayre and Pereira (Ohio

State University Research

Foundation) - 2010

US2010/0279354A1 Chlorella protothecoides UTEX 25, Dunaliella salina Process flow of algae adaptation towards

agriculture waste to produce biofuels

De Crecy

(Evolugate LLC) -2010

US2010/0267122A1

Botryococcus braunii UTEX 572, Chlamydomonas

globosa, Chlorella minutissima, Chlorella pro-

tothecoides UTEX 25, Chlorella saccharophila

UTEX 2469, Chlorella vulgaris UTEX 2714, Cris-

cosphaera carterae UTEX LB1014, Dunaliella terti-

olecta UTEX LB999, Nannochloropsis oculata

UTEX LB1998, Scedenesmus bijuga, Spirulina plat-

ensis UTEX LB1926, Spirulina maxima UTEX

LB2342, Tetraselmis suecica UTEX LB2286, Tet-

raselmis chuii UTEX LB232, Phaeodactylum tricor-

nutum UTEX 646, Pleurochrysis carterae CCMP 647

Mixed microalgae cultivation in a waste-

water having high proportion of carpet

mill effluents

Chinnasamy et al.

- 2010

US2010/0151539A1

Prototheca moriformis UTEX 1435, Prototheca

krugani UTEX 329, Prototheca stagnora UTEX

1442, Prototheca zopfii UTEX 1438, Chlorella

luteoviridis SAG2214

Incorporation of sucrose utilization genes

and lipid modification genes into obligate

heterotroph for production of medium

chain fatty acids

Franklin et al.

(Solazyme Inc.) – 2010

US2009/0298159A1

EP2292782A1 Chlorella protothecoides

Two stage sequence of photoautotrophic

to heterotrophic cultivation

Wu and Xiong

(Tsinghua University) –

2011

US2009/0211150A1

CN 101230364A Chlorella protothecoides sp 0710

Process flow of screening, cultivation with

carbohydrate feed and oil extraction

Wu and Xiong

(Tsinghua University) -

2008

US2009/0209014A1 Schizochytrium limacinum SR21 High density cultivation on crude glycerol Chi et al.- 2009

US2009/0148928A1 Chlorella spp. Methods of shifting the algae trophism

from photoautotrophic to heterotrophic

Hackworth and Charlsberg

Jr - 2009

EP1780283A1 Phaeodactylum tricornutum Trophic conversion of obligate pho-

toautotroph via metabolic engineering

Apt and Allnutt

(Martek Biosciences Corp)

- 2007

than the lipid content in photoautotrophic cell (14%) [41]. Unequivocal claim of improvement with regard to final mi-croalgae cell density or lipid accumulation is evident in each of the disclosed invention. Nevertheless, the majority in the list embodies a two-trophic operation rather than a single “true” heterotrophic protocol. Moreover, the transition from photoautotrophic to heterotrophic conditions is mostly de-

scribed as a straight-forward two stages process. In practice, it might be fatal to certain microalgae strains when a sudden illumination stress of 24 h light (24L) to total darkness (24D) is imposed even to microalgae cultivated in enriched me-dium. Recently, it was revealed that Tetraselmis suecica could not survived in immediate dark cultivation. However, T. suecica cell would undergo gradual physiological changes


to accommodate heterotrophic conditions through thinning of cell walls, decreased in chlorophyll content and alteration in lipid, carbohydrate and protein profile when subjected to sequential photoperiod adaptation (12L:12D, 8L:16D, 4L:20D and 24D) over a period of long cultivation time [42].

3.2. Mono - Culture Process Development

A concept of heterotrophic shift by Hackworth and Charlberg Jr. is aimed at taking the advantage of both trophic conditions in altering the proportion and macromolecular characteristics of lipids in photosynthetic algae [43]. The gist of invention is at best, describes generalized steps on grow-ing large amount of Chlorella cells presumably in the most energy efficient and cost-effective manner. The method re-quires a plurality of modular photoautotrophic-supporting chambers such as open ponds, tubular or flat-panel PBRs or polybags arranged to form an integrated microalgae cultiva-tion system. Ideally, photoautotrophic system should be lo-cated adjacent to the exhaust gas emission points of either industrial processing, methane and coal electric power gen-eration or decarbonization plants of natural gas or other fuel gases. Cells grown to high density will be channeled to a dark cultivation system such as stirred tank bioreactor or closed ponds. The heterotrophic growth is instigated by add-ing sugars with concentration equivalent to 5% glucose. Lipid proceeds to maturation phase in heterotrophic cultiva-tion environment resulting in an increased yield and a final biodiesel profile close to petroleum diesel (Table 3). The inventors’ way of justifying the effectiveness of trophic shift as opposed to a strict photoautotrophic (AT) or heterotrophic (HT) cultivation is explained in terms of useful energy (Euse-

ful). Euseful is broadly defined as the amount of “desired end-product” gained and measured in an arbitrary unit (AU). It was reported that Euseful (AT HT) was higher than Euseful (HT HT). Appended charts in patent document vaguely states that an unknown basal medium with X g of sugar will fuel the cell growth of up to 150 AU, with 27% of the total AU will be converted to Euseful in (AT HT) shift-ing as compared to 15% Euseful in (HT HT) scenario. In this particular invention, Euseful refers to the amount of lipid produced in the culture, which can be expressed either as percentage assayed by the number and size of lipid globules viewed under a microscope, or quantified in percentage of desired fraction compared to the total fatty acid using mass spectrometry.

A more workable scheme from Genifuel Corporation based on the two-trophic system was recently issued a full patent status [40]. The invention relates to the process flow of using intermediate heterotroph to convert microalgae biomass and/or lignocellulosic materials into biodiesel or bioethanol Fig. (1). In the first stage, photosynthetic and dia-zotrophic (nitrogen fixing) blue-green cyanobacteria is culti-vated in greenhouse environments to produce large quantity of cell mass, which in turn, served as nitrogen source to the more prolific oil-producing microalgae. Non-limiting exam-ples of such strains include Chlorella sp and Dunaliella sp. This process flow can also introduce impurities to the het-erotrophic strains in the form of carried over protozoans grazing on cyanobacteria. An interesting but cautionary pro-vision in this design lists the use of viruses to invade and rupture foreign algal strains. Such viruses are readily avail-

able in algal ecosystem and specific to a single type of algae. Particular examples include Paramecium Bursaria Chlorella virus (PBCV-1) attacking certain types of Chlorella and cya-nophages SM-1, P-60 and AS-1 specific to the blue green microalgae, Synechococcus.

Sugars for cellular build-up are also sourced from de-

polymerization and saccharification of carbonaceous cellulo-ses or starches. Cellulosome, an array of cellulase enzyme complex, is use in this invention. This enzyme complex is employed to hydrolyze lignocellulosic matrixes together

with glycoproteinases, which proved to be more effective on the cell wall of feed algae. Typical process executions lead-ing to an oil recovery and biodiesel end-product requires rupturing the matured algal cell bodies with expeller or

press, lipid extraction using solvent, hydrogenation and transesterification of the extracted lipid. The manufacturing line also produces bioethanol to a lesser extent; in which the surplus microalgae cells or those cultivated in separate line

will be channeled to a hydrolization reactor for conversion into sugars. The description of fermentation sub-system is nonetheless conventional in its use of yeast to produce etha-nol, ethyl acetate and CO2.

The benefits of China, US and European Patent applica-

tions on two variations of C. protothecoides cultivation pro-tocols for biodiesel production; a two-stage heterotrophic bioreactors (HT HT) [44,45] as well as conditions permit-ting photoautotrophic to heterotrophic transition (AT HT)

[46,47] have been claimed by Wu and co-workers. Both methods used C. protothecoides sp. UTEX 0710 and claimed to reach an impressive final cell density of 108 gL

-1 with

high oil content (40-61% ww-1

). The initial seed culture

bioreactor in the process flow corresponds to the major dif-ference between the two protocols. Sunlight and CO2 fixa-tion in photoautotrophic (AT HT) operation were substi-tuted with medium containing glucose (preferably 5-30 gL

-1)

and yeast extract (preferably 1-10 gL-1

) in a strict heterotro-phic (HT HT) unit operation. The basal medium compo-nents consists of phosphate (KH2PO4 and K2HPO4), magne-sium and ferric sulphate, glycine, vitamin B1 and an assort-

ment of A5 trace mineral constituents. Axenic culture is maintained by the addition of antibiotic chloramphenicol (0.002-0.2 gL

-1) or monoflouroacetate (0.1-100 mM). The

seed culture bioreactor is operated at temperature ranging

from 20 to 45oC and mixing is created by impeller agitated at

speed ranging from 50 to 300 rpm.

The working volume of secondary production bioreactor is ranging from 5 L to 11,000 L. To reduce the cost of me-dium, glucose can be substituted with cheaper carbon

sources such as sucrose, fructose, corn starch hydrolysate, cassava starch hydrolysate, wheat starch hydrolysate, broomcorn juice or wastewater containing sugars from food and beverage industry. The optimal parameters to obtain

high-cell density cultivation are proposed as follows; inocu-lum size of 20-30% vv

-1, working temperature of 20-45

oC,

pH of 6.0 to 8.0, air flow rate of 100 – 200 Lh-1

, and the dis-solved oxygen in the culture is maintained at above 20%

saturation. During the cultivation, glucose (5 to 18 gL-1

) and yeast extract (0.5 to 1.0 gL

-1h

-1) are fed continuously after

about 128 h and the cultivation is extended to 213 h.


Table 3. Comparisons of Physical Characteristics between Algal-Oil and Petroleum–based Diesel [36]

Physical Property Biodiesel from Photoautotrophic

Chlorella

Biodiesel from Heterotrophic

Chlorella Diesel from Crude Petroleum

Fuel Density 1.06 kg L-1 0.86 to 0.92 kg L-1 0.75 to 1.0 kg L-1

Dynamic Viscosity 0.10 Pa.s 0.02 Pa.s 2-1000 Pa.s

Heating Value 30 MJ kg-1 41 MJ kg-1 42 MJ kg-1

Oxygen Content High Low Low

Fig. (1). Process flow diagram of two-stage photoautotrophic-heterotrophic biodiesel production [33]

Dual trophism strategy through an integrated process scheme has been invented by Bellussi et al. [48]. This inven-tion, published under the World Intellectual Property Orga-nization (WIPO), emphasized on concurrent cultivations of both photoautotrophic and heterotrophic systems equipped with recycling facility Fig. (2) in place of aforementioned sequential transfer from solar irradiation to dark condition. The process scheme was envisaged to consume smaller quantity of water and cultivation area as compared to pho-toautotrophic culture for biodiesel production. Microalgae of the genus Nannochloropsis, capable to grow in light and dark regime, has been used as a model system. This inven-tion relates to the cultivation of at least one phototroph and heterotroph strain or mix culture in the ponds or closed bioreactors. At the end of the cultivation, the algae cells are harvested either by means of sedimentation, decanting, or

flocculation to obtain concentrated algae cell suspension (18-25% by weight). The excess water is recirculated back to the cultivation units whereas the concentrated suspension is sub-jected to ‘hydrothermal treatment’ to extract the oil and aqueous carbohydrates/proteins solution. ‘Hydrothermal treatment’ refers to pressurized heating (150-330

oC, 0.5-18

MPa, 0.5 to 2 h) that causes cell breakage and separation of oil phase. The process also allows for some portion of poly-saccharides and proteins to be partially converted to algal oil with the rest hydrolyzed into glucoside or water-soluble pro-teins. Consequently, the oil is recovered for subsequent bio-fuel processing whereas the residual aqueous suspension rich in hydrosoluble nutrients is recycled back, possibly after further cooling to assimilate with the following batch of mi-croalgae in heterotrophic section. The gaseous by-products of hydrothermal treatment are equal to 10-25% of dried bio-


mass, which essentially made up of 80-90% CO2 by volume and C1 to C3 hydrocarbon gases in the remaining. These would be ideal for supplementing carbon for the growth of phototroph or upgraded in its hydrocarbon component as fuel gas. The invention claimed that the integrated two trophic process lines require 385 hectares of land and 12,000,000 ton yr

-1 of water to obtain specific productivity of 20,000 ton yr

-1

of algal oil. This only requires half of the occupied area and volume of water utilized by the single mode photoautotro-phic ponds.

Fig. (2). Integrated photoautotrophic-heterotrophic process flow for

biodiesel production from Nannochloropsis sp. [41]

A true heterotrophic cultivation scheme has been dis-closed by Chi et al. [49], where the concept of decoupling

the cell division, cell size enlargement, and fatty acid profil-ing by controlling optimal conditions in separate bioreactors is employed. The main objectives of the invention are to use the non omega-3 lipids as feed components in biodiesel and

simultaneously to enhance the DHA production as valuable side-product of Schizochytrium sp. microalgae for nutraceu-tical purposes. The invented process involved three distinct phases; i) proliferation of Schizochytrium limacinum SR 21

cells to raise the cell density, ii) improvement of lipid con-tent in the microalgae cells, and iii) intensification of lipid

concentration and enhancement of DHA production Fig. (3).

The entire process is intended as a segment to a biodiesel refinery giving solution to the glut of crude glycerol pro-duced from the hydrolysis of TAG feedstock. Crude glycerol differs from purified counterpart in a sense that, as a major

byproduct of biodiesel refinery, it contains roughly 70-80% of glycerol mixed with free fatty acids anions, mono and diglycerides fragments, alcohol and salts. Successful utiliza-tion of treated crude glycerol as substrate for S. limacinum is

viewed as integral to increase the viability of biodiesel in-dustry. True economic analyses shows that the current forms of biofuels are somewhat expensive on an equivalent-energy basis than fossil fuels in the absence of government subsidies

[10]. The first phase focuses on shunting the majority of cel-lular activity towards rapid reproduction of cell bodies as opposed to simultaneous cell division and growth. The metabolic flux during this stage is strong, since biosynthesis

of nucleic acids, enzymes and essential materials for cellular division requires large amounts of nitrogen and amino acids. Oxygen is consumed rapidly to oxidize crude glycerol into low molecular weight carbon and energy. Higher culture

temperature is preferred to maintain optimal enzyme activity. In the second phase, lipid starts to accumulate under the conditions of low temperature and nitrogen deficient in tan-dem with low oxygen consumption. Thus, manipulation of

the types and concentrations of nutrient, dissolved oxygen, and temperature are required to enhance the production be-tween the two stages.

In the first bioreactor, S. limacinum is cultivated using crude glycerol as substrate. The favorable conditions for

optimal cells multiplications are as follows: sufficient min-eral salts, high temperature (23-33

oC), low carbon concentra-

tion (10-40 gL-1

), high nitrogen concentration (1.0-1.5 gL-1

) and high dissolved oxygen (20% to 50% saturation). A final

cell density, ranging from 6 x 108 to 1 x 10

9 cells mL

-1 can

be obtained in cultivation via fed-batch or continuous mode for 24 h. The second bioreactor operates in fed-batch mode, where the cultivation conditions are switched to: 30

oC, dis-

solved oxygen (3% to 5% saturation), carbon concentration is increased from 30 to 50 gL

-1, nitrogen concentration is

kept at 0.5-1.0 gL-1

. The cultivation continues until the point of deceleration in the exponential growth phase. Cells start to

gain weight and accumulated lipids in the second bioreactor, where the DHA content in S. limacinum cell increases up to 13-17%. The DHA content is further increased in a third “polishing” bioreactor operated at a much lower temperature

(20-25oC), very low dissolved oxygen (0-0.5% saturation)

and a total shut-off in nitrogen supply. The outcome ex-pected from the three stage process is typically a five days operation that allows for a high algal cell density (150 gL

-1)

and high productivity (1.2 gL-1

h). The process also claimed that total lipid, PUFA, and DHA yields of 50%, 25% and 20% ww

-1 can also be obtained, respectively. The overall

process has opted for an in situ solvent extraction and trans-

esterification through drying process and followed by treat-ment using alcohol and base. Since this will eventually lead to a volatile form of fatty acid methyl esters (FAMES), the invention can benefit from the widely employed distillation

process in a typical refinery to fractionate the non omega-3 fatty acids from PUFA or DHA methyl esters.


3.3. Co-Cultivation Approach

The current approach of turning industrial, municipal or agricultural wastes containing indigestible fibrous materials into biodiesel is proving to be the antithesis to axenic culti-vation. Most of the disclosed processes are designed around the enzymatic hydrolysis to liberate sugars, subsequent car-bon catabolism, and fatty acid synthesis which take place during the assemblage of lignocellulose-degrading microor-ganisms (i.e., extracellular cellulase producers) and microal-gae. One such method adhering to the principle of symbiotic co-existence was filed by De Crecy for the US Patent and WIPO protections [50,51]. The main invention relates to the inoculation of a culture medium containing at least a compo-nent of cellulose, hemicellulose or lignin with a single mi-croorganism and a microalgal strain. The fermentation proc-ess will be first subjected to an aerobic-heterotrophic condi-tion, followed by anaerobic-photoautotrophic or anaerobic-heterotrophic condition.

The inclusion of pre-treatment step for plant-based feeds seems inevitable in today’s high yield bioconversion process. Pre-treatment such as comminution reduces the coarse cellu-lytic particulates, which in turn, increases the surface area for the reaction and made handling easier. Steam or acid explo-sion, and hydrothermalysis will further alter the macro/ mi-croscopic size and structure as well as submicroscopic chemical composition of the cellulosic materials. Under the first aerobic-heterotrophic conditions, the cellulases enzyme complex secreted by the microorganism employed in the process will hydrolyze carbohydrate fractions into monomer sugars. For a variety of embodiments, the subsequence step can proceed in two possible anaerobic conditions: either het-erotrophically or photoautotrophically. Under anaerobic-heterotrophic condition, the microorganisms employed se-crete cellulase enzymes to degrade the lignocellulosic mate-rials to fermentable sugars for the conversion to alcohols. On the other hand, alcoholic by-products and CO2 are produced by the microorganism in anaerobic-phototrophic condition, which are subsequently used by the phototrophs in the mix-ture. In both cases, C. protothecoides metabolize fermentable sugars from microbial hydrolysis to release CO2 for cell pro-liferation and fatty acids synthesis. The probable consortium

of archae, bacterium, yeast, or fungi, and the necessary mi-croalgae having to co-exist, adapting to alternating tro-phisms, and subcultured in heterogeneity of carbonaceous materials for a sufficient period of time is categorized as evolutionary modified organism (EMO). The EMO is pro-jected to demonstrate greater capacity in consuming cellu-losic feedstocks as compared to the unmodified wild-types and having high tolerant to the toxic by-products of pre-treatment procedure, especially furfural and acetate that can inhibit biofuel production.

In contrary to the idea of using mixed microalgae popula-tion in metabolizing numerous lignocellulosic residues, the method of using a plurality of microalgae strains solely to treat wastewater effluent from carpet industry has been in-vented [52]. Carpet mill wastewaters have the inherent dis-advantages of containing inorganic chemicals and dye color-ants imparting stresses and reduced illumination for growth of photoautotrophic microalgae. The particular disclosure had initially isolated and identified a “primary consortium” of 15 native algae strains from locations subjected to prolong exposure to wastewater from carpet industry. Out of these, Chlamydomonas globosa, Chlorella minutissima and Scenedesmus bijuga were found to be the beneficial strains for exploitation in biofuel production. Co-cultivation was extended to include marine microalgae of the genus Botryo-coccus, Chlorella, Criscosphaera, Dunaliella, Spirulina, Tetraselmis and Pleurochrysis from the UTEX collection. Treated and untreated effluent supported the growth of ma-rine microalgae, especially B. braunii, C. saccharophila, D. tertiolecta and P. carterae without any salt amendments. This result suggests that these marine microalgae have unique osmotic adjustment and regulation mechanisms to tolerate hypo-osmotic stress conditions. A combination of 85-90% wastewater from carpet industry with 10-15% mu-nicipal sewage accommodates good growth conditions to the consortium with 96% of nutrient removal could be accom-plished. Projection from the time-scale analysis indicates that the process has great potential for scale-up. It is claimed that 11.4 to 29.3 tons ha

-1 yr

-1 of biomass and 2100 to 4060 L ha

-1

yr-1

of algal oil can be obtained from the co-cultures of na-tive microbial isolates together with the selected marine strains. This patent claimed that the lipid content in microal-

Fig. (3). Sequential bioreactor units for decoupling the cell division, cell growth, and specific fatty acid production [42]


gal cells is ranged from 9.5% to 18% of their weight and only 65% of the algal oil can be converted into biodiesel.

Recently, a novel concept of ‘diazotroph-attenuated ni-trogen stress co-cultivation’ (DANSC) for biodiesel produc-tion has been filed for patent [53]. The application high-lighted on the minimal needs for exogenous nutrients by espousing co-cultivation technique of at least one algal spe-cies, an aerobic bacteria and a diazotroph as the requisite microorganism in the culture. Under continuous symbiotic conditions, the consortium is envisioned to be self-sustained by utilizing oxygen produced from a phototroph, CO2 from the respiration of bacterial species or the heterotroph, and drawing significant proportion of organic macronutrients from endogenous decomposed microalgae and bacterial cells. The inclusion of diazotrophic species is designed to provide sustainable environment for algal growth. In addi-tion, the advantages of nitrogen stress responses for inducing algal bioproducts synthesis is also preserved. This method greatly enhances the percentage of saturated and mono-unsaturated fatty acids relative to PUFA in triacylglycerol.

The inventive step comprises the inoculation and main-taining of algae: aerobic bacteria: diazotroph population at appropriate ratio throughout the cultivation process. The suitability of such cultivation is proposed for a broad spec-trum of microalgae phylum, covering strains from Chloro-phyta, Euglenophyta, Baccillariophyta, Microspora and Xanthophyta. The possible aerobic bacteria to be used in the process include Gamma proteobacteria, Actinobacteria, Ba-cilli, Beta proteobacteria and Alpha proteobacteria. Almost all bacterial taxonomic groups of diazotrophic microorgan-ism can be employed. However, strains from algae-like pro-tist or cyanophyta will be more appropriate since they al-ready possess both photosynthetic and nitrogen fixing capa-bility. The patent claimed that substantially high concentra-tion of algal culture (10-50%) can be obtained on continuous basis either in open or closed systems. Enhancement of lipid production relative to a non-symbiotic growth of respective microalgae cells up to 30-50% is also claimed. Total lipid in the form of TAG constituted to 30-80% and the percentage of saturated and mono-unsaturated fatty acids relative to PUFAs ranging from 10% to 30% ww

-1 could be obtained.

3.4. Strain Improvement

The need to expand on the existing pool of strains having alternate trophism can be resolved through genetic manipula-tion. Most phototrophs are unable to metabolize external organic compounds because of two reasons; i) lack of effi-cient uptake system of essential substrate (i.e., sugars) to permeate across the cell membrane or, ii) the existence of a ‘metabolic block’ to convert cellular respiration into ATP due to biosynthetic or degradative lesion in central metabo-lism [17]. The first claim of successful trophic conversion of an obligate photoautotrophic diatom Phaeodactylum tricor-nutum Bohlin was filed at European Patent Office in a 2007 [54]. In this invention, the Hup1 gene from Chlorella kessleri and Glut1 gene from human erythrocyte encoding glucose transporter protein were successfully inserted into P. tricornutum transformation vector pPha-T1 via biolistic pro-cedures. Transformation of P. tricornutum microalgae ex-pressing Glut1 protein turned the cells into facultative het-

erotroph, which capable to use glucose as the sole carbon source. Although the green algae, Volvox [55] and Cylindro-theca [56], transfected with Hup1 gene have the ability to catabolize glucose nonetheless they have no ability to grow in the dark. The invention provides comparative data indicat-ing equal growth rate and cell density ( 2 x 10

7 cells mL

-1)

between untransformed and recombinant P. tricornutum grown under strict photoautotrophic condition. The cultiva-tion of P. tricornutum transformant with Glut1 gene in basal medium supplemented with glucose (5-10 gL

-1) gave 5 times

higher cell density than the untransformed cells under nor-mal illumination. Strikingly, the principal claim of P. tricor-nutum transformant with Glut1 gene functionality within the diatom membrane region becomes apparent when the rate of growth and final cell density (5 x 10

8 cells mL

-1) in het-

erotrophic cultivation is about 10 to 20 fold higher than those obtained by wild type microalgae.

Selective inhibition and over-expression of one or more targeted metabolic genes in C. protothecoides through mo-lecular approach have been invented by Sayre and Pereira [57]. The inventive steps are unique in the sense that gene stacking technology is employed to collectively increase the photosynthetic light-usage efficiency of C. protothecoides prior to transfer into heterotrophic condition. Simultane-ously, lipid accumulation in transgenic cells is also im-proved. The photosynthetic light-usage efficiency is achieved by eliminating the cells’ chlorophyll a/b-binding LHC (light harvesting antennae) complex of thylakoid mem-branes. It is believed that over 90% of the energy absorbed by the LHC chlorophyll (Chl) is ineffectively re-radiated as fluorescence or lost as heat by non-photochemical quench-ing. Elimination of LHC complex through mutagenesis of the Chlorophyll a oxygenase gene (Cao) and LHCII-b gene shown to block Chlorophyll b synthesis and prevent accumu-lation of LHC complexes which bind Chl b. In the absence of LHC complex, light is absorbed by the proximal antennae Chls. The innovation allows for deeper light penetration when self-shading occurred at greater culture density and more efficient utilization of available photons absorbed by the Chl. Microalgae lacking in LHC complex or Chl b exhib-ited 10-fold higher rates of oxygen evolution per unit chlo-rophyll when compared to the wild-type cells.

Four genes are targeted for over-expression to increase lipid accumulation in the cells. Genes encoding acetyl CoA carboxylase (ACCase) and diacylglycerol acyl transferase (DGAT) in combination with other two genes encoding pro-tein, caleosin and olesin are inserted into Chlorella transfor-mation vector. ACCase catalyzes the first committed step of fatty acid synthesis through the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA while (DGAT) cata-lyzes the triacylglycerol synthesis from diacylglycerol. Pro-tein, caleosin and olesin are components of lipid storage vesicle. Enhanced lipid accumulation is correlated with ele-vated caleosin/olesin expression in C. protothecoides when cells are grown under nitrogen-limiting condition. It is only logical that their next measure would involve reducing starch synthesis which accounts for the other major storage form of reduced carbon in cells. Carbon sequestration to starch is reduced by suppressing the ADP glucopyrophosporylase (AGPase) involves in rate-limiting step of starch formation. This is done through introducing an expression cassette with


sequence encoding small inhibitory ribonucleic acid (siRNA) blocking AGPase expression. The AGPase RNAi element can be stacked together with ACCase, DGAT and caleosin/ olesin encoding genes to enhance the lipid yield. Starchless Chlorella is claimed to have higher growth rate and lipid content as compared to the wild-type cells by about 22% and 50%, respectively. Subsequent heterotrophic growth of C. protothecoides in the presence of glucose is associated with the loss of photosynthetic capacity. In the embodiment, it is claimed that C. protothecoides is able to use both, glycerol and glucose, as carbon source for heterotrophic growth. The final cell concentration of 1.7 gL

-1 and 1.9 gL

-1 is obtained in

cultivation using 1.8 gL-1

glycerol and 5.0 gL-1

glucose, re-spectively. Glucose-supported growth gave higher lipid ac-cumulation in the cells than the cultivation in glycerol. Lipid yield in the cells cultivated in glucose and glycerol is 52 and 15 times higher than the yield obtained in cultivation using control medium, respectively.

Modification of obligate heterotrophic, Prototheca, through genetic manipulation has been claimed by Franklin and co-workers [58]. The improved strain has higher effi-ciency in sucrose uptake as compared to the native cells. Lipids from Prototheca have shorter hydrocarbon chain length and higher degree of saturation than that synthesized by other microalgae. Greater saturation in fatty acids is pre-ferred for the production of biodiesel or high-end jet fuel. In addition, Prototheca lipids are free from chlorophyll and other carotenoids which may reduce the downstream cost of scrubbing biofuel for contaminants. The specific invention provides Prototheca with exogenous “sucrose utilization gene” encoding sucrose transporter, sucrose invertase, and hexokinases (e.g., glucokinases and fructokinases). Expres-sion of the first two enzymes allows Prototheca cell to util-ize sucrose in the culture and hydrolyze it into glucose and fructose. Fructokinase will be optionally expressed in the event where endogenous hexokinase activity is insufficient for maximum phosphorylation of fructose. Prototheca spe-cies integrated with yeast invertase gene (yInv) is found to successfully transit the heterologous invertase enzyme out-side of cells. The extracellular secretion will enable the Pro-totheca cells to grow on low-value agricultural by-products (e.g. molasses from sugarcane processing, forage sorghum and beet pulp) or cellulosic biomass (e.g., corn stover, rice hulls or stalk). The use of low cost raw materials is essential to ensure that the proposed biodiesel production method is economically viable.

This invention also describes the re-engineering of lipid pathway in Prototheca cells to alter the proportions and/or the properties of lipid molecules. It is claimed that the lipid content in genetically-modified Prototheca cells can be in-creased up to 70% of its dry cell weight. Excess lipid pro-duction in the transformant Prototheca cells is achieved via exogenous genes that direct the up-regulation and down-regulation of one or more key enzymes controlling the branch points in fatty acid biosynthetic pathway. Franklin et al. chose the amplification of pyruvate dehydrogenase, which increases the conversion of pyruvate to acetyl-CoA and targeted the ACCase and glycerol-3-phosphate-acyltransferase involved in fatty acid synthesis for over-expression. Fatty acid production is also enhanced through

up-regulating the acyl carrier protein (ACP), which binds to the growing acyl chains during fatty acid synthesis.

The recombinant Prototheca cells had also been modified to contain exogenous genes encoding “lipid modification enzymes” that have hydrolysis activity towards substrate with specific number of carbon atoms. The invention con-templates the expression of the main transgene encoding the fatty acyl-ACP thioesterases groups that engaged in the cleaving of C8, C10, C12 or C14 carbon chains from the ACP, and optionally includes one or more relevant modifica-tion enzymes from the fatty acyl-CoA/ aldehyde reductases, fatty acyl-CoA/ reductases, fatty aldehyde reductases and fatty aldehyde decarbonylases groups. The invention also includes an exogenous gene encoding stearoyl-ACP desatu-rase to provide modification with respect to lipid saturation. Upregulation of the gene can further increase the proportion of monounsaturates suitable for conversion to biodiesel. The disclosed invention claimed a C8-C14 lipid yield from 10 to 30% of the total lipids in Prototheca cell, with the proportion of C8, C10, C12, and C14 to be at least 0.3%, 2%, 2% and 4% of the total lipid, respectively. Such controlled profile is possible through successful targeting and expression of het-erologous fatty acyl-ACP thioesterases from Cuphea hook-eriana (preference for C8-C10 cleavage), Umbellularia cali-fornica (preference for C12 cleavage) and Cinnamomum camphorum (preference for C14 cleavage) within the Pro-totheca species.

By selecting and transfecting the desired combination of exogenous genes to be expressed, the invention has the ac-tual choice of tailoring the types of desired lipid-based end products generated by the Prototheca cells, which may then be extracted from the aqueous suspension. A fatty acid sev-ered from its ACP linkage by thioesterase usually forms a fatty acyl-CoA molecule through further enzymatic process-ing. If chosen to be expressed, the fatty acyl-CoA/ aldehyde reductase will catalyze the acyl-CoA molecule to an alcohol. Similarly, the fatty acyl-CoA/ reductase when present will catalyze the reduction of acyl-CoA molecule to an aldehyde form. In a different embodiment of invention in which the fatty acyl-CoA/ reductase is present together with a fatty aldehyde reductase, this third exogenous enzyme is respon-sible in further catalyzing the reduction of the aforemen-tioned aldehyde to an alcohol. If alkane or alkene is preferred over alcohol as the primary end-product of aldehyde reduc-tion in Prototheca cultivation, the transformant microalgae should instead inserted with an exogenous gene encoding a fatty aldehyde decarbonylase.

4. CURRENT AND FUTURE DEVELOPMENT

Current outlook on the application of heterotrophic mi-croalgae for biodiesel manufacturing points to a small per-centage of growth as shown by the small number of in-volvement by the active biodiesel producers. Nonetheless, advancement by certain proponent towards making biodiesel from heterotrophic microalgae at commercially viable cost proved to be encouraging. Solazyme, which ranked at 6 in the Hottest 50 Companies in Bioenergy for 2008-2009, runs a strictly dark algal biodiesel refinery operation. The tech-nology has commercial potential with the success story of delivering a contract for 84,000 L of biodiesel to the US


military under a short period of time. The present challenges on this technology are to reduce further the operating cost and to select the suitable organic substrates. Crop plants as primary feedstocks are subjected to seasonal variations and consumer competition, which might not provide a stable supply throughout the year. Carbon-rich cellulosic waste streams hold the greatest scale-up prospects that directly avoid adverse impacts on food and feed market. However, reaping the benefit of seemingly low-cost resources comes with a caveat. There will be an inevitable increase in the en-ergy expenses and cost by adding unrefined substrates which is incur through several levels of pre-treatment and hydroly-sis steps to convert them into feedstock. Cheaper processes to generate sugars from the deconstruction of lignocelluloses are still a sought-after technological advancement. The threat of contamination and competition with other microorganisms as well as the inhibition of growth by excess organic sub-strate are the problems that may arise by the application of full heterotrophic cultivation method. On the strain im-provement side, the algal isolation from unique local sources may help to ensure versatility and robustness of the strains to be employed in mass cultivation for biodiesel production. Innovations through evolutionary adaptation or genetic trans-formation can further overcome the cost barrier. Foreseeable non-limiting strategies include developing strains that can auto-flocculate, able to produce specific hydrocarbon chain length in low quality aqueous substrate, or variable salinities, or have weaker cell walls that break easily under low pres-sures or low heat treatment. The use of strains that are capa-ble to secrete oil directly into the culture is also highly pre-ferred. Ultimately, these algal characteristics will help to increase the efficiency of future dewatering process, down-stream lipid extraction, recovery and refinement into high quality biodiesel.

ACKNOWLEDGEMENT

The authors gratefully acknowledge the funding from Malaysian Department of Higher Education through the dis-bursement of Fundamental Research Grant Scheme (grant no. 02-10-07-297FR). The support of Malaysian Ministry of Higher Education and Universiti Putra Malaysia in providing a PhD scholarship to Mohd Shamzi Mohamed is also greatly appreciated.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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Heterotrophic Cultivation of Microalgae for Production of Biodiesel