Two stage cultivation of a Nannochloropsis mutant for biodiesel feedstock

J Appl Phycol DOI 10.1007/s10811-014-0490-4 Two-stage cultivation of a Nannochloropsis mutant for biodiesel feedstock Yen Thi Thai Doan & Jeffrey Philip Obbard Received: 26 May 2014 / Revised and accepted: December 2014 # Springer Science+Business Media Dordrecht 2014 Abstract Optimization of mass microalgae cultures is required for the efficient production of biodiesel feedstock in terms of total fatty acid (TFA) content and a conducive fatty acid profile A mutant strain of Nannochloropsis sp (MT-I5), as modified via random mutagenesis and flow cytometric cell sorting, was investigated in both a single- and two-stage cultivation using 250 L laboratory raceway ponds Culture was based on photoautotrophic biomass production (stage 1) followed by a switch to photomixotrophic growth induced by adding sodium acetate (2 mM) (stage 2) The biomass yield of the mutant in two-stage cultivation was maintained at a level similar to that of the one-stage photoautotrophic culture, but TFA content was increased by 2.3-fold The fatty acid profile of MT-I5 also had an increased level of desirable saturated fatty acids (SFA) for use as a biodiesel feedstock, i.e from 43 to 48 % of TFA, as well as a decreased level of less desirable polyunsaturated fatty acids (PUFA), i.e from 22 to % of TFA The two-stage cultivation process is of interest for the mass culture of microalgae for biofuel feedstocks, as biomass productivity can be maximized during the first stage of culture until N-starvation is achieved, followed by the enhanced synthesis of SFA in the second stage of culture by adding sodium acetate as a fixed-carbon source Y T T Doan School of Environmental Science and Technology, Hanoi University of Sciences and Technology, Hanoi, Vietnam J P Obbard Environmental Studies Centre, Qatar University, PO Box 2713, Doha, Qatar Y T T Doan (*) Dai Co Viet, Hanoi, Vietnam e-mail: yenhanhnguyet@yahoo.com Keywords Nannochloropsis Eustigmatophyceae Raceway pond Photomixotrophic Two-stage cultivation Biodiesel Introduction Biodiesel production from microalgae lipids is increasingly regarded as a more sustainable alternative to conventional biodiesel feedstocks that are derived from terrestrial bioenergy crops (Borowitzka and Moheimani 2013; Demirbas 2011; Williams and Laurens 2010) However, to render microalgae-based fuels feasible from a technical and economic perspective, effective procedures for increasing biomass productivity as well as improving intracellular fatty acid profile of the selected strain are an imperative Microalgae are usually cultivated in photobioreactors and/ or open raceway ponds The key advantage of open pond systems relative to photobioreactors is their lower capital and operating costs (Sheehan et al 1998) In the US Aquatic Species Program, conducted from 1978 to 1996, raceway ponds were lauded as the optimal design for biomass production and were used for both experimentation and technoeconomic modelling for potential commercialization of the technology (Sheehan et al 1998) After preliminary screening of cell growth rates, fatty acid profile and physiological characteristics, lipid productivity of the selected microalgae strain is the key parameter for biodiesel feedstock production (Griffiths and Harrison 2009) Moreover, the selected microalgae strain must be produced in large quantities to yield sufficient intracellular lipids for generation of biodiesel feedstock Nutrient concentrations, culture depth and the rate of mixing in raceway ponds are all key variables that need to be optimized for maximum biomass and lipid productivity (Schenk et al 2008) Many studies have been conducted to optimize these parameters for the growth of a multitude of J Appl Phycol microalgae species (Chiaramonti et al 2013; Moazami et al 2012) Certain microalgae can grow mixotrophically in the presence of a supplemented organic carbon source (e.g glycerol, acetate, glucose, etc.) in the growth medium, and this generally leads to higher productivities than autotrophic growth alone (Yang et al 2000) Moreover, photomixotrophic cultivation has been reported to lead to a higher cellular fatty acid content (Feng et al 2005; Hu and Gao 2003; Oh et al 2010) Glucose is the widely utilized carbon source in laboratory studies, but is prohibitively expensive for use in mass cultivation Acetic acid or acetate derived from anaerobic fermentation processes can be substituted for glucose as a lower cost carbon alternative (Bhatnagar et al 2011) As the enhancement of overall lipid yield in large-scale microalgae biomass production is the primary goal for biofuel feedstock production, the use of improved strains of microalgae is often a prerequisite N-starvation stress is known to enhance algal lipid content, but biomass productivity reduces as cell division decreases, thereby resulting in little or no net productivity gains (Pruvost et al 2009; Solovchenko et al 2008; Tomabene et al 1983) Adding an appropriate organic carbon source to the culture medium to induce photomixotrophic growth, at the appropriate cell growth phase, may prove an effective method to increase overall lipid yield Although photomixotrophic growth has been shown to enhance intracellular lipid accumulation, bacteria may simultaneously contaminate and overgrow a photomixotrophic culture However, when microalgae predominate in the culture medium, it is less likely that bacteria will overwhelm the culture—at least in a short-term batch production mode For biodiesel feedstock, it is not only the biomass productivity that is of primary importance but also the lipid yield or actual biodiesel value as measured via fatty acid methyl ester (FAME) productivity (Griffiths and Harrison 2009; Rai et al 2013) The fatty acid profile of the microalgae strain used has a direct influence on final biodiesel properties For biodiesel derived from Nannochloropsis oils, the fatty acid oxidative stability is a major issue as Nannochloropsis spp have a high content of polyunsaturated fatty acids (PUFA) such as oleates (C18:1), linoleates (C18:2) and eicosapentaenoate (C20:5, EPA) which are highly susceptible to oxidation (Knothe 2013) The fatty acid profiles are also affected by cell culture conditions including temperature, light intensity, growth medium and the stage of cell growth (Converti et al 2009; Knothe 2013) This study aimed to induce photomixotrophic growth of a mutant strain of Nannochloropsis sp (MT-I5) in a secondary stage of growth upon N-starvation to determine the effect on cell growth and intracellular lipid accumulation The fatty acid profile of a mutant strain of Nannochloropsis sp (MT-I5) was investigated using a two-stage cultivation in the presence of an organic carbon source (i.e sodium acetate) in batch mode Kinetic cell growth and total fatty acid (TFA) under singlestage (i.e photoautotrophic growth only (pond 1)) and twostage (i.e photoautotrophic growth followed by photomixotrophic growth (pond 2)) were compared The effect of sodium acetate on cellular fatty acid composition of MT-I5 was also evaluated Materials and methods Mutant strain of Nannochloropsis and culture conditions A mutant strain of Nannochloropsis, MT-I5, with enhanced intracellular fatty acid content, was used for this study MT-I5 was derived from a locally isolated marine strain of N a n n o c h l o ro p s i s s p u s i n g t h e m u t a g e n e t h y l methanesulfonate (EMS) to induce random mutagenesis and then subsequently isolated via flow cytometric cell sorting, as reported in our previous study (Doan and Obbard 2012) MT-I5 was cultivated in 250 L laboratory raceway using 0.2 μm filtered nutrient-enriched seawater, i.e f/2 Guillard medium (Guillard 1975) Culture medium was maintained at a depth of 15 cm in the pond to allow light penetration to the base of the culture, and water loss via evaporation was regularly compensated to maintain volume The culture was initiated using a 15 % (v/v) mid-log-phase inoculum (equivalent to an initial concentration of 38 mg.L−1) Pond cultures were maintained at 28±1 °C and illuminated with fluorescent light at an irradiation of 60 μmol photons m2.s−1 over a 12:12 h dark/light period Irradiance was measured using a LI-COR Photometer LI-250A Two-stage cultivation in laboratory raceway ponds MT-I5 was cultured photoautotrophically throughout the entire cultivation period of 27 days (pond 1) In a second pond (pond 2), MT-I5 was cultivated in two distinct stages: i.e stage 1, photoautotrophically over the first 11 days (as for pond 1), then for further 16 days, i.e stage 2, where sodium acetate (NaAc at mM) was added to the medium The pH of the culture medium in ponds and was measured thrice daily MT-I5 biomass (dry weight) and TFA profile and content for both ponds were determined throughout the cultivation period The nitrate concentration of the culture medium was measured spectrophotometrically using a Shimadzu UV-1601 spectrophotometer (wavelength of 220 nm) according to the method described by Collos et al (1999) A nitrate standard curve was established based on sodium nitrate standards at concentrations ranging from to 750 μM Determination of biomass concentration Triplicates of 100 mL of algae suspension were taken every 2–3 days from each culture pond and passed through a Whatman glass microfiber filter paper (GF/F, diameter 4.7 cm) and then washed in 0.5 M of ammonium formate (twice) to remove salts Filter Biomass-Pond NO3-Pond NO3-Pond 800 700 200 600 500 150 400 100 300 200 50 100 0 12 16 20 24 28 Total FAME content (% dry wt.) 250 Biomass-Pond Nitrate concentration (µM) Biomass concentration (mg.L-1) J Appl Phycol 35 Pond 30 Pond 25 20 15 10 5 Cultivation time (days) 11 14 17 20 23 26 Cultivation time (days) Fig Growth curves and nitrate reduction of culture medium in a single-stage cultivation (pond 1) and two-stage cultivation (pond 2) The arrow indicates addition of NaAc into pond on day 11 Each data indicates the mean of triplicates+SD Fig Fatty acid content of cultures with single-stage cultivation (pond 1) and two-stage cultivation (pond 2) The arrow indicates addition of NaAc into pond Data are expressed as means+SD (n=3) papers containing algae biomass were dried in an oven at 80 °C to dryness and then weighed to determine biomass dry weight Results Biomass production in raceway ponds Determination of fatty acid profile and FAME content One hundred microlitres of algal suspension of MT-I5 was centrifuged at 5000 rpm for 10 and then washed in 0.5 M ammonium formate to produce biomass pellets Pellets were then lyophilized in a freeze drier (Christ Alpha 2-4, Germany) to dryness to analyse for TFA content and the fatty acid methyl ester (FAME) profile which was used as a proxy measurement of intracellular fatty acid composition FAME content was measured via a one-step transesterification of cellular fatty acids using a 14 % boron trifluoride-methanol mix (Sigma-Aldrich, Singapore) at 100 °C for 120 FAMEs were then separated in n-hexane and analysed using a gas chromatography-mass spectrometer (GC-QP2010, Shimadzu, Japan) fitted with a DB-5MS capillary column 30 m in length, 0.25 mm in film thickness and an internal diameter of 0.25 μm Helium was used as the carrier gas The detector and injection temperature were set at 280 °C The temperature programme of the GC column was as follows: 50 °C held for min; increased at a rate of 10 °C.min−1 to 150 °C and held for min; at 185 °C held for min; and at 300 °C held for The mutant strain of Nannochloropsis sp (MT-I5) was generated from our previous study (Doan and Obbard 2012) and had an enhanced lipid content relative to its wild type (i.e 51 vs 34 % of dry weight (DW)) Culture of MT-I5 in the laboratory 250 L raceway ponds showed that cell concentrations dropped slightly during the first days (see Fig 1), after which an exponential growth was observed This extended lag phase (i.e days) was most likely due to dilution of the inoculum at the start of experiment The biomass concentration in both ponds increased 4-fold after 11 days of cultivation NaAc (2 mM) was added to pond as a fixed-carbon source on day 11 (start of stage 2, see the arrow in Fig 1) The biomass concentration in pond 2, upon culture termination on day 27, was 171 mg.L−1 only slightly less than that in pond (i.e 191 mg.L−1), as shown in Table Biomass productivity for both cultures in the first 11 days was 11–12 mg.L−1.day−1 and this subsequently reduced by half from day 12 to day 27 most likely due to nitrogen deprivation Adding organic carbon in pond induced a pH reduction from a range of pH 8.5– 9.3 to 7.2–8.2, while the pH in pond maintained within the range of pH 9.0–9.4 Table Biomass concentration and productivities of mutant MT-I5 Nannochloropsis under single-stage (photoautotrophic mode) and two-stage cultivation (photoauto- and photomixotrophic mode) Cultivation mode Pond 1—single phase (photoautotrophic) Pond 2—biphasic (photoauto- and photomixotrophic) *Biomass concentration determined at day 27 Biomass concentrationa (mg.L−1) 191 171 Biomass productivity (mg.L−1.day−1) Day 6–11 Day 11–27 12 11 5.7 42.8±2.6 35.4±2.6 21.9±0.7 8.5±1.2 44.8±0.1 42.3±0.4 12.9±4.0 19.9±0.9 36.9±0.0 32.2±0.0 30.9±0.1 7.4±1 42.3±2.0 39.7±3.1 18.0±5.0 5.3±0.5 36.8±2.7 28.7±1.0 34.5±1.8 4.0±0.2 33.8±1.8 29.0±2.0 37.3±0.2 3.7±0.8 39.5±2.3 28.7±4.3 31.8±7.5 3.9±0.5 47.6±2.4 31.6±3.2 20.8±1.2 2.2±0.1 57.1±3.8 20.1±8.1 22.8±4.8 2.5±0.3 56.5±0.4 26.6±1.2 16.8±3.2 1.7±1.0 56.6±0.5 22.8±0.9 20.6±3.0 1.8±0.4 SFA (% DW) MUFA (% DW) PUFA (% DW) FAME productivity (mg.L−1.day−1) P-1 P-1 P-2 P-1 P-2 P-1 P-2 P-1 P-2 20 13 11 Days Fatty acid composition of MT-I5 in single-stage (P-1: pond 1) and two-stage growth (P-2: pond 2) Data are expressed as means+SD (n=3) Table The final total fatty acid content, measured as FAME, in MTI5 from ponds and was 12 vs 30.6 % of cell dry weight (DW), respectively, at day 27 Figure shows a markedly elevated FAME content in the NaAc-supplemented culture (pond 2) after adding NaAc on day 11, while an increase of FAME in under single-stage MT-I5 cultivation (pond 1) was not apparent, even though nitrate concentrations were almost identical in both ponds The PUFA content, as a sum of C20:5n3, C20:4n6 and C18:2, of the MT-I5 in pond increased from 20.6 to 34.5 % (w/w) of TFA for the first 13 days of culture, while in pond from 16.8 to 37.3 % (w/w) for the first 11 days Then, the PUFA content in MT-I5 in pond decreased to 21.9 % at the day 27, while the PUFA concentration in MT-I5 in pond reduced significantly from day 11 to day 27, i.e % of TFA (see Table 2) This shows that NaAc addition to MT-I5 not only altered the fatty acid quantity but also altered its fatty acid composition Additionally, this PUFA concentration in both ponds at day 11 (end of stage 1) was significantly higher than that observed in flask cultures, i.e 32–37 vs 14.6 % (data from flask cultures given in (Doan and Obbard 2012)) In this experiment, the addition of NaAc in N-limited cultures of MT-I5 was tested with respect to induced fatty acid accumulation The FAME content of NaAcsupplemented culture (i.e in pond 2) was in the range of 21–28 % DW The levels measured were lower than those reported by Liang et al (2009) who showed that a culture of Chlorella vulgaris supplemented with % (v/v) NaAc had a cellular TFA content of 31–34 % of DW Table shows that the C16:0 and C16:1 fatty acids were predominant in both MT-I5 cultures in ponds and However, the NaAc-amended culture in pond had a reduced content of eicosapentaenoic acid (EPA, 20:5n3) as 4.6 % of TFA In contrast, the percentage of C20:5n3 in the photoautotrophic MT-I5 culture, i.e pond 1, was 4-fold higher than that in the NaAc-amended culture Furthermore, the composition of the increased FAME content in the NaAcsupplemented culture was significantly higher to that of the photoautotrophic MT-I5 culture, i.e 30.6 vs 12.0 % DW Overall, the relatively high proportion of C14:0, C16:0 and C16:1 and lowered percentage of long chain C20:5n3 fatty acids show that growth of MT-I5 under photomixotrophic culture following N-limitation is more favourable for P-2 Fatty acid content and composition P-1 27 P-2 Nitrate concentrations in ponds and decreased between day and day 21 as biomass growth increased (see Fig 1) The N-concentration decreased from 750±10 μM nitrate at day to 100–200 μM nitrate on day 11, i.e the day NaAc was added to pond 2, and then to about 18±5 μM nitrate on day 21 for both ponds (see Fig 1) 47.6±0.4 45.3±0.6 7.1±1.0 19.4±1.3 J Appl Phycol J Appl Phycol Table Fatty acid composition (% of TFA) and total FAME content (% of DW) of NaAc-amended cultures and unamended cultures at the day 27 Data are expressed as means+SD (n=3) Pond Pond C14:0 C16:1 C16:0 C18:2 C18:1 C18:0 C20:4n6 C20:5n3 FAME (% DW) 5.3±0.2 6.8±0.1 29.4±0.5 33.0±0.4 35.1±1.7 39.3±0.8 1.6±0.6 0.7±0.1 6.0±1.8 12.3±0.3 2.3±0.8 1.5±0.4 4.1±0.7 1.9±0.2 16.2±1.6 4.6±0.8 12.0±2.9 30.6±0.7 biodiesel feedstock production than unamended photoautotrophic cultures Discussion Biomass production MT-I5 growth was tested in single-stage control (pond 1) and two-stage cultivation (pond 2) over 27 days to determine biomass and TFA production Pond was not supplemented with sodium acetate as a fixed organic carbon source and served as the photoautotrophic control In contrast, sodium acetate (2 mM) was added to pond after 11 days of culture The photomixotrophic growth of microalgae is often typified by the overwhelming predominance of algal cells, such that bacteria are unlikely to compete in such conditions—at least in short-term batch production mode (in this case 16 days) This cultivation practice is suited to large-scale microalgae cultivation, where two-stage growth production is practised, where the second stage commences when cell density is increased by 4–5 times, and cell growth is in exponential phase Figure shows that the growth curves of MT-I5 in ponds and were similar in stage 2, implying that NaAc supports fatty acid synthesis rather than biomass production The slight reduction in biomass production in pond vs pond is likely a result of a reduction in pH of the culture medium (i.e from 9.5 to 7.2–8.2) Nlimitation inevitably results in reduced biomass productivity, but usually triggers lipid accumulation However, if N-limitation is not severe, then cell division may proceed based on the utilization of intracellular Nreserves (Li et al 2008) In this study, N-limitation was likely induced following by the addition of sodium acetate as the fixed organic carbon into pond A correlation of the biomass growth, nitrogen depletion and fatty acid content, as shown in Figs and 2, indicates that addition of sodium acetate in photomixotrophic growth of MT-I5 strain increased total fatty acid content by 2.3-fold Fatty acid content and composition The decline in relative PUFA levels under photomixotrophic growth of MT-I5 in pond supports previous studies that reported reductions in EPA and PUFA content of NaAc-enrichment mixotrophic cultures aerated with CO2 in one-stage cultivation (Hu and Gao 2003; Rai et al 2013) Addition of NaAc has also been previously reported to have a significant effect on lipid accumulation during early stationary phase for Chlorella sorokiniana or Chlorella pyrenoidosa (Qiao and Wang 2009; Rai et al 2013) In our study, NaAc was added on day 11, following a 4-fold increase in MT-I5 cell density under photoautotrophic growth The MT-I5 culture in pond was then switched to photomixotrophic growth upon N-limitation of the culture This technique, i.e maximizing biomass in the first stage of culture, could avoid bacteria contamination until N-starvation is reached, followed by addition of an organic carbon source in a second stage of culture to enhance cellular lipid accumulation, and is beneficial to scale up production in larger cultivation systems Addition of sodium acetate, as a fixed-carbon source in stage 2, resulted in an increase of the C/N ratio as well as pH of the culture medium and induced the synthesis of fatty acids Biomass production continued in the presence of NaAc and does not decline under N-limitation, as reported earlier by Rodolfi et al (2009) Rattanapoltee and Kaewkannetra (2014) reported increased lipid content and fatty acid synthesis after adding sugarcane bagasse in a two-stage Scenedesmus acutus cultivation However, in contrast with this study, the PUFA content from the bagasse-supplemented cultures was increased (mostly C18:2 and C18:3) A two-stage culture process has been previously utilized in mass algae culture (Chi et al 2009; Huntley and Redalje 2007), but without the use of fixed-carbon sources Acetate has been applied as a supplemental carbon source to microalgae cultures in several other studies in single-stage cultivation only (Hu and Gao 2003; Pyle and Wen 2008) Our study has shown that addition of NaAc, upon N-limitation, results in an overall enhancement of cell growth in the first-stage of culture, as well as an increase in cellular TFA content in the second-stage of culture relative to an unamended photoautotrophic culture In two-stage cultivation, cell division is promoted in stage under a photoautotrophic growth condition, and TFA accumulation and a favourable fatty acid profile are promoted in stage under a photomixotrophic growth condition for the mutant MT-I5 strain of Nannochloropsis sp J Appl Phycol Conclusions The mutant strain MT-I5 of Nannochloropsis sp has an ability to grow photomixotrophically in pilot-scale 250 L raceway ponds in the presence of sodium acetate (at mM) as fixed-carbon source Addition of NaAc induced lipid biosynthesis substantially without reducing biomass production Moreover, the addition of NaAc in a two-stage cultivation process reduced the relative percentage of longer chain PUFA present Overall, this study shows that an improved phenotype of Nannochloropsis sp subjected to a twostage 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economics Energy Environ Sci 3:554–590 Yang C, Hua Q, Shimizu K (2000) Energetics and carbon metabolism during growth of microalgal cells under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions Biochem Eng J 6:87–102 ... growth and intracellular lipid accumulation The fatty acid profile of a mutant strain of Nannochloropsis sp (MT-I5) was investigated using a two- stage cultivation in the presence of an organic carbon... single -stage cultivation (pond 1) and two- stage cultivation (pond 2) The arrow indicates addition of NaAc into pond on day 11 Each data indicates the mean of triplicates+SD Fig Fatty acid content of. .. compared The effect of sodium acetate on cellular fatty acid composition of MT-I5 was also evaluated Materials and methods Mutant strain of Nannochloropsis and culture conditions A mutant strain