ARTICLE The Microalga Parachlorella kessleriA Novel Highly Efcient Lipid Producer

ibyl,2 Kater ina Bis ova ,1 Shigeyuki Kawano,3 Vladislav Cepa k,2 Xiuling Li,1 Pavel Pr 1 1 1 1 m Zachleder, Ma ria C , Irena Bra nyikova , Milada V z tova kova Vile mly n, Laboratory of Cell Cycle of Algae, Institute of Microbiology, AS CR, Opatovicky ebon , Czech Republic; telephone: 420-384-340-480; fax: 420-384-340-415; 379 81 Tr e-mail: zachleder@gmail.com 2 ebon , Czech Republic Institute of Botany, AS CR, Algological Centre, Tr 3 Department of Integrated Sciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba, Japan
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ABSTRACT: The alga Parachlorella kessleri, strain CCALA 255, grown under optimal conditions, is characterized by storage of energy in the form of starch rather than lipids. If grown in the complete medium, the cultures grew rapidly, producing large amounts of biomass in a relatively short time. The cells, however, contained negligible lipid reserves (110% of DW). Treatments inducing hyperproduction of storage lipids in P. kessleri biomass were described. The cultures were grown in the absence or vefold decreased concentration of either nitrogen or phosphorus or sulfur. Limitation by all elements using vefold or 10-fold diluted mineral medium was also tested. Limitation with any macroelement (nitrogen, sulfur, or phosphorus) led to an increase in the amount of lipids; nitrogen limitation was the most effective. Diluted nutrient media (5- or 10-fold) were identied as the best method to stimulate lipid overproduction (60% of DW). The strategy for lipid overproduction consists of the fast growth of P. kessleri culture grown in the complete medium to produce sufcient biomass (DW more than 10 g/L) followed by the dilution of nutrient medium to stop growth and cell division by limitation of all elements, leading to induction of lipid production and accumulation up to 60% DW. Cultivation conditions necessary for maximizing lipid content in P. kessleri biomass generated in a scale-up solar open thin-layer photobioreactor were described. Biotechnol. Bioeng. 2013;110: 97107. 2012 Wiley Periodicals, Inc.
Correspondence to: V. Zachleder Contract grant sponsor: Ministry of Education, Youth and Sports of the Czech Republic Contract grant number: OE09025; LH12145 Contract grant sponsor: Grant Agency of the Czech Republic Contract grant number: P503/10/1270; P501/10/P258 Contract grant sponsor: CREST of Japan Science and Technology Agency Contract grant sponsor: Technology Agency of the Czech Republic Contract grant number: TE 01020080 Contract grant sponsor: Academy of Sciences of the Czech Republic Contract grant number: RVO 61388971 Received 2 April 2012; Revision received 4 june 2012, Accepted 22 june 2012 Accepted manuscript online 5 July 2012; Article rst published online 18 July 2012 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.24595/abstract) DOI 10.1002/bit.24595

KEYWORDS: carbon dioxide; light intensity; limitation by elements; lipid hyperproduction; Parachlorella kessleri; thin-layer photobioreactor

Introduction
Considering natural resource depletion and climate change, lipid-based algal biofuels are an alternative to conventional fossil fuels, due to the high productivity of algal biomass vansky , 2009; Rodol et al., (Chisti, 2007; Doucha and L 2009; Singh et al., 2011b) and the ability of algae to recycle CO2 originating from ue gas (Benemann, 1997; Brown, 1996; Douskova et al., 2009). Algae are considered as the only alternative to current biofuel crops such as corn and soybean, as they do not require arable land (Chisti, 2007; Hu et al., 2008; Singh et al., 2011a). Increased interest has been focused on microalgae not only due to their high growth rate and high photosynthetic efciency, but particularly due to the possibility of controlling their metabolism to produce relatively high contents of nyikova et al., energy-rich compounds, either starch (Bra 2011) and/or lipids (Chen et al., 2011; Deng et al., 2009; Lee, 2011). Under favorable growth conditions, algae synthesize fatty acids, principally as substrates for esterication into glycerol-based polar lipids (e.g. glycolipids and phospholipids), the main constituent of membranes. However, under unfavorable environmental or stress conditions, many algae alter their lipid biosynthetic pathways towards the synthesis and accumulation of neutral lipids (2050% cell dry weight), mainly in the form of triacylglycerols (Hu et al., 2008), that are typically stored as cytoplasmic lipid bodies (Goodson et al., 2011; Yu et al., 2007). Neutral lipids can be readily converted to biodiesel or other fuel types through both

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existing and emerging lipid rening processes (Biller and Ross, 2011; Francisco et al., 2010; Halim et al., 2011; Patil et al., 2011). However, oleaginous microalgal strains that have been used to date often showed much lower growth rates than many non-oleaginous species (Hu et al., 2008; Sheehan et al., 1998), leading to unacceptably expensive biofuel production. Previous studies have shown that some microalgae (e.g. selected strains of Chlorella) can be directed to accumulate high levels of starch as a storage compound; this may be used nyikova et al., for industrial production of bioethanol (Bra 2011). Several approaches like inhibitory treatments by cycloheximide or uorodeoxyuridine, or limitation of macroelements, such as phosphorus (Ballin et al., 1988), etl k et al., 1988), or nitrogen (Zachleder et al., sulfur (S 1988), were used to stimulate overproduction of starch in nyikova et al., 2011). microalgae (Bra Although the mechanisms of induction of lipid production can be different from starch, there are several common approaches inducing both starch and lipid overproduction. It is widely believed that the lipid content could be increased by nitrogen or phosphate limitation (Mutlu et al., 2011; Reitan et al., 1994; Tornabene et al., 1983). Currently, nitrogen limitation is the most frequently used treatment to enhance lipid production in microalgae (Rodol et al., 2009). It was also shown that the algal strains appropriate for overproduction of starch are not usually suitable for nyikova et al., overproduction of lipids and vice versa (Bra 2011; Li et al., 2010a,c). Many algae, particularly green algae (Chlorophyta), use starch as the primary carbon and energy storage compound and in Chlamydomonas reinhardtii under nitrogen deciency (N) conditions, its content can reach up to 45% of cell dry weight (Li et al., 2010a) or higher (60% of DW) in some Chlorella strains if induced by sulfur deciency or inhibited nyikova et al., 2011). Some algae may by cycloheximide (Bra accumulate similar amounts of starch and lipids under stress conditions (Li et al., 2011; Ramazanov and Ramazanov, 2006), whereas others only transiently accumulate starch (Collen et al., 2004). Substantial differences in both biomass and lipid productivities as well as in nal content of neutral lipids can be found among various Chlorella strains. Recently, a highly productive strain, Chlorella vulgaris ibyl et al., 2012) was identied and CCALA 256 (Pr described. Another promising strain, Parachlorella kessleri CCALA 255, characterized by high biomass and lipid ibyl et al., 2012), is being presented in detail productivity (Pr in this study. In the present study, the controlled production of oil reserves was tested under conditions of the absence or limitation of some macroelements (nitrogen, sulfur, and phosphorus) as well as limitation of all elements in mineral media by dilution. To conrm the laboratory ndings for potential industrial use, lipid production was tested in algal cultures grown in an outdoor scale-up thin layer photobioreactor.

Materials and Methods

Microorganism and Culturing The green microalga P. kessleri (Krienitz et al., 2004), strain CCALA 255, was provided by the Culture Collection ebon , Czech of Autotrophic Organisms (CCALA) in Tr Republic (http://www.butbn.cas.cz/ccala/index.php). In the collection, the strain has been maintained on agar slants under irradiance of about 23 mmol/(m2 s), 12/12 h (light/ dark) regime and at a temperature of 12158C. Laboratory cultivation units consisted of glass cylinders (inner diameter 36 mm, height 500 mm), which were placed in a thermostatic bath (308C) and continuously illuminated by a panel dimmable uorescent lamps (OSRAM DULUX L55W/950 Daylight, Italy) allowing adjustment of the incident light intensity from 16 to 780 mmol/(m2 s). The cylinders were aerated using a mixture of air and CO2 (2%, v/v). The volume of the algal suspension in each cylinder was 300 mL, and each cylinder was supplied with gas at a ow rate of 15 L/h. For the mineral medium used for experimental cultures, and a description of both laboratory and scale up outdoor nyikova et al. (2011). cultivation units see Bra

Nutrient Limitation P. kessleri cultures were grown in complete medium to attain the required cell concentration for any given experiment (usually between 0.3 and 0.75 g of DW/L). The original mineral medium was removed from the cell suspension by centrifugation and the cells were re-suspended in fresh mineral medium (control) or in one of the nutrient-limited nyikova et al., 2011). media (for more details, see Bra

Biomass Determination For dry weight determination, biomass was separated from the medium by centrifugation of 2 mL of the cell suspension in pre-weighed microtubes at 3,000g for 5 min; the sediment was dried at 1058C for 12 h and weighed on an analytical nyikova Sartorius 1601 MPB balance (as described in Bra et al., 2011). Cell volume and concentration were measured using a Beckman Coulter Multisizer III (Coulter Corporation, Miami, FL) by diluting 1050 mL of xed (0.2% glutaraldehyde) cell suspension into 10 mL of 0.9% NaCl (w/v) electrolyte solution.

Measurement of Light Intensity A quantum/radiometer-photometer (LI-COR, Inc.) was used. In the culture unit, dimmable uorescent tubes were used for adjustment of irradiance. The mean light intensity (I) was calculated according to the LambertBeer equation: I (Ii It)/ln(Ii/It), where Ii is the incident light intensity

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measured at the surface of the culture vessel and It is the transmitted light intensity measured at the rear side of culture vessel.

Nile Red Fluorescence Determination of Lipids The algal suspension was xed with glutaraldehyde to a nal concentration of 0.25% (v/v) and loaded into wells (100 mL per well) of a 96-well plate. Four microlitre of Nile Red solution (were added to wells, uorescence intensity was measured using a 96-well plate luminometer (Tecan innite 200, Switzerland) with the following lters: excitation 485 nm (bandwidth 20 nm), emission 595 nm (bandwidth 10 nm). Glyceryl trioleate (Sigma, T7140) was used as a lipid standard to obtain a calibration curve. Fluorescence intensity was compared to the total lipid content determined gravimetrically to quantify the relative uorescence intensity values.

Determination of Chlorophyll Content The algal suspension (10 mL) was centrifuged at 4,000g for 3 min and the sediment was collected. Phosphate buffer, 7.7 pH (1 mL), a pinch of MgCO3, and Zircon beads (500 mL, size 0.7) were added to the sediment, which was then disintegrated by vortexing (Vortex Genie 2, Scientic Industries, Inc., Bohemia, NY) for 10 min. Acetone (4 mL, 100%) was added, mixed well, and centrifuged at 4,000g for 3 min. The supernatant was drained into a calibrated test tube using an exhauster/air pump, closed with a stopper and left standing in a dark-block. Another 4 mL of acetone (80%) were added to the sediment, mixed well, and centrifuged at 4,000g for 3 min. Using an exhauster/air pump, the supernatant was drained off to the same calibrated test tube used in the preceding step and topped up with 80% acetone up to 10 mL. Absorbances at 750, 664, 647, 470, 450 nm were measured in a 1 cm path length cuvette using the spectrophotometer (Shimadzu UV-1800 S). Calculation of chlorophyll content was based on absorbances at different wavelengths and was carried out according to equations published previously (MacKinney, 1941).

Fatty Acids in Lipids This analysis was carried out in the Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology, ASCR ezanka et al. (2010). in Prague as described by R

Results
Effect of Nutrient Limitation When cultures of P. kessleri were grown in the complete medium under optimal growth conditions (308C, 2% CO2, incident light intensity of 780 mmol/(m2 s)), they rapidly grew, and their biomass reached approx. 8.0 g of DW/L in 4 days (Fig. 1). However, accumulation of lipids was very slow. No lipid bodies were found in cells until at least 2 days of culture, and only small lipid bodies were observed in some cells of 5-day culture (Fig. 2). The relative content of lipids in the cells of 4-day culture was not more than 10% of DW (Fig. 3D). Starch content reached a maximum of approx. 15% in 1-day, thereafter gradually decreased to 10% during 4 days of culture period (Fig. 3D). When nitrogen, sulfur, or phosphorus was eliminated from the complete medium, the growth rate severely decreased, especially in the case of nitrogen free medium (Fig. 1A). Their biomass of 4-day culture was <2.0 g of DW/ L. In contrast, the elimination of any of the above elements caused an accumulation of lipids. Elimination of nitrogen induced a very fast lipid accumulation (Fig. 2). Small lipid bodies were observed in the cells of 1-day culture, and lipid body number increased in the course of culture. The lipid bodies fused together, and the most of the intracellular space was occupied by large lipid bodies in 5 days. The elimination of phosphorus also induced a lipid accumulation, and the cells of 5-day culture accumulated lipid at nearly the same extent as under nitrogen elimination. Although the elimination of sulfur induced a lipid accumulation, the quantity of accumulated lipids was less than under elimination of nitrogen or phosphorus. Sulfur elimination had another undesirable effect. The

Starch Analysis A modication of the method of (McCready et al., 1950) was nyikova et al., 2011). used as described previously (Bra

Visualization of Lipids Using Nile Red Fluorescence Intracellular lipid droplets were stained using the neutral lipidspecic dye, Nile Red (9-diethylamino-5H-benzo(a)phenoxazine-5-one), following the protocol described earlier (Eltgroth et al., 2005) with slight modications. Briey, 1 mL of the cell suspension was xed with glutaraldehyde at a nal concentration of 0.25% (v/v) and stained with 4 mL of Nile Red (Sigma, N3013) stock solution (0.5 mg/mL of acetone) that was stored in the dark at 48C. Samples were observed after 5 min using an epiuorescence OLYMPUS BX 51 microscope equipped with the lter combination U-MNU2 (360370 nm excitation and >420 nm emission). Photomicrographs were taken with a digital camera DP72, and processed using Adobe Photoshop 7.

Lipid Analyses Gravimetric Lipid Determination Analyses have been carried out as described previously ibyl et al., 2012). (Pr

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Effect of CO2 Limitation The optimal growth of the cultures required aeration with 2% CO2. The limitation of CO2 by aeration with air caused the decrease in growth (Fig. 3). Decrease in growth by CO2 limitation occurred also under a condition of nitrogen limitation to 20% of the complete medium. However, no difference in growth was observed between 2% CO2 and air when the cultures were grown in the medium diluted to 10% of the complete medium. No signicant differences in the time course of starch content were observed between cultures grown with 2% CO2 and air, regardless of nutrient conditions. In contrast, the rates of the accumulation of lipids were decreased by CO2 limitation under conditions of nutrient limitation. Fluorescence microscopic observation also demonstrated the decrease in lipid accumulation by CO2 limitation (Fig. 4). The intracellular space of the cells grown with air-aeration was less occupied by lipid bodies than that of the cells grown with 2% CO2-aeration. The total chlorophyll content was also affected by CO2 limitation. As for the cultures grown in the complete medium, the total chlorophyll content reached a maximum in 3 days and slowly decreased thereafter under the condition of sufcient CO2 supply (Fig. 5). However, under the condition of CO2 limitation, the total chlorophyll content reached a maximum in 1-day, and soon decreased even though the cultures still continued to increase in biomass (Fig. 3A). As for the cultures grown under conditions of nutrient limitation, the total chlorophyll content decreased from the start of culture, and the decrease rates were made faster by CO2 limitation.

Figure 1. Variation in biomass concentration (DW g/L) in cultures of Parachlorella kessleri. Cultures were grown in complete mineral medium (1 medium) (A and B), or in various element limiting growth media as follows: nitrogen (N), phosphorus (P), sulfur (S) free media, or 10-fold diluted complete mineral medium (0.1 medium) (A), vefold lower content of either nitrogen (0.2 N), phosphorus (0.2 P), or sulfur (0.2 S) media, or vefold diluted complete mineral medium (0.2 medium) (B). The cultures were cultivated in a laboratory photobioreactor under continuous illumination of an incident light intensity of 780 mmol/(m2 s) and at the same initial biomass concentrations (0.3 g/L).

Effect of Mean Irradiance on Lipid Yield The cultures started to be grown with the mean irradiances at 125, 160, 183, 216, and 292 mmol/(m2 s) by the adjustment of the initial biomass at 4.0, 2.0, 1.0, 0.5, and 0.25 g DW/L. The cultures of an initial biomass below 2.0 g/L started to accumulate lipids 2 days after inoculation, and continued for 23 days (Fig. 6). The relative lipid content reached approx. 50%. However, the cultures of an initial biomass of 2.0 and 4.0 g/L delayed to start accumulation of lipids for 1-day, and their relative lipid contents and lipid productivities were substantially lower (Table I), indicating that the mean irradiance was insufcient to lipid production. The sufcient production of lipids was carried out with the mean irradiances beyond 125 mmol/(m2 s) at which the cultures of an initial biomass of 1.0 g/L reached the maximum of relative lipid content.

number of dead cells (blue cells in Fig. 2) rapidly increased with cultivation time. For the avoidance of the undesirable effects of lack of the above elements, nitrogen, sulfur, or phosphorus was decreased to 20% of the complete medium instead of the elimination of them. The cultures slowly grew, and the biomass in 4 days reached 2.43 g of DW/L (Fig. 1). The decrease in nitrogen caused an accumulation of lipids, and the relative content of lipids reached 45% in 4 days (Fig. 3E). On the other hand, changes in starch content were not effected by the decrease in nitrogen except for the slight lowering of the content. An incubation in a medium diluted to 20% or 10% of the complete medium was also reduced the growth (Fig. 1). The most effective accumulation of lipids was achieved by the cultures grown in the medium diluted to 10%, with the relative content of 55% (Fig. 3F).

Fatty Acid Composition The fatty acid composition of the cultures grown under different conditions was analyzed. Although the lipids were mainly composed of fatty acids of C16 and C18 under all conditions tested, the ratio of saturated and unsaturated

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Figure 2. Time course of lipids accumulation during batch cultivation of P. kessleri. Lipid bodies were stained using Nile Red (yellow); autouorescence of chloroplasts is seen in red. Cultures were grown in complete mineral medium (1 medium), or in either nitrogen (N), phosphorus (P) or sulfur (S) free media. The cultivation time for all variants is indicated in the left column. The same initial biomass concentration (0.3 g/L) was used for all cultures and no detectable lipid bodies were seen in cells at the beginning (0 h; not shown in gure). Scale bar 10 mm.

fatty acids altered by culture conditions (Table II). When cultures were grown in the complete medium under the optimal growth condition, the ratio of saturated, and unsaturated fatty acids was 0.917. The lipids of the cultures grown under nutrient limitation were rich in unsaturated fatty acids. The ratio of saturated and unsaturated fatty acids decreased to 0.55 under the best condition for lipid accumulation, that is, 10-fold diluted medium.

Large Scale Industrial Thin-Layer Bioreactor The cultures were incubated in the complete medium put in a large-scale thin-layer bioreactor to simulate the industrial production of lipid-rich biomass. Unfortunately climatic conditions during cultivation were unfavorable, low light

intensities, and temperatures, so that the cultures grew at about half the rate of the optimal growth conditions in the laboratory (Fig. 7). Starch content decreased to 2% in the rst 4 days, and thereafter increased gradually. It took another 8 days to reach approx. 15%, corresponding to the values in the laboratory. On the 14th day from the beginning of the experiment, the 10-fold diluted medium was added to the reactor. An accumulation of lipids occurred 4 days after the addition, and the lipid content reached a maximum in next 3 days. It was slightly delayed compared to laboratory conditions, probably resulting from insufcient light intensity. The measurements of mean light intensity showed that the cultures in the thin-layer bioreactor efciently absorbed light, resulting in that the biomass in the outdoor thin-layer bioreactor reached almost 2 times higher (14 g/L) than in the

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Figure 3. Changes in biomass concentration (DW in g/L) (AC) and relative starch and lipid content (% of DW) (DF) in asynchronous cultures of P. kessleri aerated by air (empty symbols) or by a mixture of air and CO2 (2%) (lled symbols). The cultures were grown in a laboratory photobioreactor under continuous illumination of an incident light intensity of 780 mmol/(m2 s) either in a complete mineral medium (1 medium) or in a medium of vefold lower nitrogen content (0.2 N), or in 10-fold diluted medium (0.1 medium). The initial biomass concentration (0.75 g/L) was identical for all variants.

laboratory bioreactor (8 g/L) even though the incident light intensity of outdoor conditions was lower. The lipid content of the outdoor cultures reached 25%, comparable to the laboratory cultures of an initial biomass of 2.0 g/L (Fig. 6).

Discussion
Algae producing starch as a primary energy storage compound usually have a relatively low lipid content. The starch reserves can be increased markedly by macroelement limitation in some algal species or strains such as etl k et al., Scenedesmus quadricauda (Ballin et al., 1988; S 1988; Zachleder et al., 1988), C. vulgaris strain CCALA 924 nyikova et al., 2011) or Tetraselmis subcordiformis (Yao (Bra

et al., 2012). A high starch content (about 4045% of DW) was also found under nitrogen-decient conditions in C. reinhardtii (Li et al., 2010b,c). Among all the macroelements tested, sulfur limitation promoted the highest accumulation of starch and the longest maintenance time of starch-enriched cells. Sulfur limitation was also successfully tested under eld conditions using the pilot-scale nyikova et al., outdoor thin-layer solar photobioreactor (Bra 2011). However, in contrast to the strains above, a decrease in macroelement concentration by depletion or omission of nitrogen, phosphorus or sulfur from mineral medium (Figs. 2, 3, and 5) induced the hyper-accumulation (to about 60% of DW) of reserve lipids instead of starch. This stress-induced high lipid production could, however, differ

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Figure 4. Time course of lipid accumulation during batch cultivation of P. kessleri in cultures aerated either by air or by a mixture of air and CO2 (2%). Lipid bodies were stained using Nile Red (yellow); autouorescence of chloroplasts is seen in red. Cultures grew either in a medium containing vefold less nitrogen (0.2 N) or in vefold diluted medium (0.2 medium). The cultivation time for all variants is indicated in the left column. The same initial biomass concentration (0.3 g/L) was used for all cultures and no detectable lipid bodies were seen in cells at the beginning (0 h; not shown in gure). Scale bar 10 mm.

substantially amongst various Chlorella and Parachlorella ibyl et al., 2012). Nutrient limitation-induced strains (Pr increased lipid production has been described previously many times but hyper-accumulation to values of about 60% of DW has no often been achieved and in all cases, it was attained at a much lower biomass density (Illman et al., ibyl et al., 2012; Rodol et al., 2009). 2000; Pr Nutrient limitation with all tested macroelements had a positive impact on lipid hyper-accumulation. Previously, it was found that natural depletion of elements during growth of C. vulgaris was the most effective way to increase lipid productivity (Stephenson et al., 2010). This approach also effectively induced lipid accumulation, leading to very ibyl et al., 2012). high lipid productivity (0.325 g/L/day) (Pr The problem is that exhaustion of elements to a level enabling induction of lipid production is attained at so

high a biomass concentration that the mean light intensity per cell is too low to supply sufcient energy for lipid production. So, in order to use this very easy and economically advantageous procedure, it is necessary to grow cell cultures in an appropriately diluted medium. Depletion of nutrients in such a medium is attained at a lower biomass concentration and thus at a higher mean light intensity (see Fig. 6) which is needed for successful lipid production. Using this treatment, biomass accumulation ceased (and oil production was triggered) at values between 2 and 3.70 g/L of DW depending on the type of nutrient limitation (Fig. 1B). The commercial price of carbon dioxide, as a source of carbon for cellular metabolism in algal cultures, represents more than 40% of the biomass price (Kadam, 1997). Utilization of waste CO2 from fuel gases of various sources

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Changes in total chlorophyll content in P. kessleri cultures aerated either by air (empty symbols) or by a mixture of air and CO2 (2%) (lled symbols). The cultures were grown in a laboratory photobioreactor under continuous illumination of an incident light intensity of 780 mmol/(m2 s) either in complete mineral medium (1 medium) or in a medium containing vefold less nitrogen (0.2 N), or in 10-fold diluted medium (0.1 medium). The initial biomass concentration (0.75 g/L) was identical for all.

Figure 5.

(incinerators, power stations, lime, biogas stations, etc.) proved to be feasible (Douskova et al., 2009). In practice, however, it is sometimes difcult to locate bioreactors close to waste CO2 sources. However, a two-step procedure for lipid production can be applied because the expensive components for algal growth (CO2 and fertilizers) are required only in the rst step, for biomass accumulation. The second step, induction of lipid accumulation, can be performed at a low CO2 concentration in air (Fig. 3E and F), due to slowed growth under nutrient limitation (Fig. 2); the input of CO2 from air is sufcient. For the large-scale production of lipid-enriched biomass, a relatively high input of light energy per algal cell must be assured. Under conditions of the present experiments, the minimum mean light intensity for production of reserve lipids was about 150 mmol/(m2 s). This limit can differ in various strains but to keep irradiation of cultures above this limit is one of the most important requirements for lipid production. In a thin layer algal suspension (7 mm) grown in outdoor cultures, mean light intensity sufcient for lipid production can be attained at very high biomass concentrations (13 g/L) and relatively low incident light intensity (Fig. 7). In addition to the present ndings, the convenience of a thin vansky , 2006, 2009) for biofuel layer scale-up (Doucha and L production was veried, both for starch overproduction nyikova as a feedstock for bioethanol production (Bra et al., 2011) and for overproduction of lipids for possible ibyl et al., 2012). production of biodiesel (Pr Starch is a major storage carbohydrate in many algae and higher plants, and its biosynthesis shares common precursors with lipid biosynthesis. Previous studies have indicated interactions between the two pathways, although

Figure 6. Changes in biomass concentration (DW in g/L) (A) and in relative lipid content (% of DW) (B) in cultures of P. kessleri grown at different mean light intensities (C) as affected by various initial biomass concentrations (DW 4, 2, 1, 0.5, and 0.25 g/L, curve 1, 2, 3, 4, 5 respectively) (A). The cultures were grown in a laboratory photobioreactor at 308C at a constant incident light intensity of 780 mmol/(m2 s) in 10-fold diluted complete mineral medium. Numerals on curves indicate the same variants illustrated in panels (AC).

the regulation of carbon partitioning between starch and lipid biosynthetic pathways is still not well understood (Rawsthorne, 2002; Weselake et al., 2009). Under nutrient limitation, some algal species or strains can inhibit the starch biosynthetic pathway, redirecting the

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Table I.

Biomass, lipid content, and productivity at different mean light intensities. Lipids Productivity Abs. (g/L) 1.2 1.1 1.6 1 1 Rel. % (DW) 15 25 44 45 51 Biomass (g/L/d) 0.80 0.42 0.40 0.30 0.35 Lipid (g/L/d) 0.34 0.30 0.60 0.50 0.58 Mean light intensity from start to end mmol/(m2 s) 12569 160104 183125 216148 292160

Biomass from start to end (g/L) 47.8 24.75 13.4 0.51.75 0.251.5

Total (g/L) 3.8 2.75 2.4 1.25 1.25

photosynthetic carbon ux toward lipid biosynthesis, resulting in lipid hyper-accumulation as an alternate carbon and energy reserve for cells under stress (Li et al., 2011; Wang et al., 2009). After 2 days of nitrogen starvation, lipid bodies increased twofold in the C. reinhardtii starch-less mutant compared to the wild type. The present ndings in P. kessleri conrmed that this species, similarly to some other algae, accumulated lipids under conditions disrupting starch accumulation. Our ndings also suggest that the cessation of starch biosynthesis, as well as the slow decrease in starch content, was not related to lipid biosynthesis in nutrient-limited cells, because a similar decrease in starch content also occurred in cultures grown in the complete medium with no or low accumulation of storage lipids. In the green microalga Pseudochlorococcum sp., starch content decreased after nitrogen depletion, while neutral lipids rapidly increased to 52.1% of cell dry weight (Li et al., 2011). However, Li et al. (2011) found, in contrast to the results of Wang et al. (2009) and the present ndings, that partial inhibition of the enzymes of starch biosynthesis and degradation resulted in a decrease in neutral lipid content, indicating that conversion of starch to neutral lipid may have contributed to the overall neutral lipid accumulation (Li et al., 2011). These contradictory results indicate that different species of algae may have evolved distinct strategies to redirect carbon ux from carbohydrate pathways towards lipid biosynthesis. Some algae may accumulate similar amounts of starch and lipid under stress conditions such as nitrogen limitation (Li et al., 2011; Ramazanov and Ramazanov, 2006), whereas others only transiently accumulate starch (Collen et al., 2004).

To verify laboratory ndings, experimentation using large-scale production units is the only way to prove real outcomes of any proposed approach. Unfortunately, to date almost no large scale production studies giving relevant data on the production of lipid-enriched algal biomass have been carried out. There are only two exceptions; the study on Nannochloropsis (Rodol et al., 2009) and the work on ibyl et al., 2012). Though using Chlorella vulgaris (Pr different production systems, both studies demonstrated high maximal lipid productivity (over 0.250 g/L/day and up to 0.326 g/L/day, respectively), and total lipid content (60% and over 30% DW, respectively), albeit at relatively low biomass concentrations (up to 1.5 g DW/L and up to 6 g DW/L, respectively). In the present experiments with P. kessleri grown in a scale-up thin layer outdoor PBR, if transferred into 10-fold

Table II. Fatty acid composition of Parachlorella kessleri algal biomass in cultures grown in the complete medium and nutrient-limited cultures. 1 medium (%) 32.4 14.3 8.4 42.5 0.917 0.2 N (%) 24.9 11.2 18.5 42.0 0.597 0.2 medium 0.1 medium (%) (%) 28.8 12.1 16.8 40.0 0.874 25.7 8.7 10.1 52.4 0.550

Fatty acid Palmitic Stearic Oleic Linoleic Saturated/ unsaturated

Scheme 16:0 18:0 18:1 18:2

Only FAs of an amount higher than 1% of the total FA content are shown.

Changes in biomass concentration in g/L (DW), cell volume in mm3 (V) and cell number in 107/mL (N) (A), and relative content of both starch and lipid in % of DW) (B) in cultures of P. kessleri grown in a scale-up thin-layer photobioreactor in complete mineral medium for 14 days followed by addition of a 10-fold diluted complete mineral medium. Variations in mean light intensity in mmol/(m2 s) are given in the panel (B).

Figure 7.

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diluted complete mineral medium, the lipid content attained a maximum of 25% DW after 7 days, in a biomass of 14 g DW/L, corresponding to a lipid productivity of 0.500 g/L/day. These achievements make this alga a viable option for industrial lipid production. However, bottlenecks with low mean sunlight intensity must be prevented. It can be attained by a combination of two conditions: Firstly an area with sufcient sunlight intensity for the maximum number of sunny days per year and secondly a thin layer photobioreactor, where turbulent ows of a thin layer of algae (<1 cm) increases the utilization of light energy. This type of PBR provides further benets; a high algal density reduces the risk of contamination by other species and decreases the nancial input for harvesting the biomass by centrifugation.

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