Enhanced production of microalgal lipids using a heterotrophic marine microalga Thraustochytrium sp. BM2

Enhanced production of microalgal lipids using a heterotrophic marine microalga Thraustochytrium sp. BM2

Biochemical Engineering Journal 154 (2020) 107429 Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.el...

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Biochemical Engineering Journal 154 (2020) 107429

Contents lists available at ScienceDirect

Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej

Regular article

Enhanced production of microalgal lipids using a heterotrophic marine microalga Thraustochytrium sp. BM2

T

Chun-Yen Chena,**, Meng-Hsiu Leeb, Cheng-Di Dongc, Yoong Kit Leongd, Jo-Shu Changb,d,e,* a

University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan c Department of Marine Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung, Taiwan d Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung, Taiwan e Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan b

H I GH L IG H T S

sp. BM2 can utilize glycerol as a carbon source. • Thraustochytrid steep liquor was used as the inexpensive nitrogen source. • Corn high lipid content of over 70% has been achieved. • Very sea salt and 0.15 g/L inoculum size was optimal. • 2% • Maximal lipid productivity of 1.052 g/L/d was achieved.

A R T I C LE I N FO

A B S T R A C T

Keywords: Heterotrophic cultivation Thrasutochytrium sp. Microalgal lipids Glycerol Corn steep liquor

In this study, the potential of Thraustochytrium sp. BM2 for cost-effective lipid production was investigated. Lipid content and productivity of BM2 reached 76% and 37 mg/L/h when corn steep liquor (12.5 g/L) and glycerol (10 g/L) was used as nitrogen and carbon source respectively. Supplementing an optimal amount of sea salt (2% w/v) further enhanced the lipid content to 79%. The inoculum size and age were also evaluated for their effect on lipid accumulation. A 48-h pre-culture at 0.15 g/L loading further improved the lipid productivity to 43.86 mg/L/h (or 1.052 g/L/d), which is a 340% increase when compared with the control test. Thus, Thraustochytrium sp. BM2 strain could serve as a low-cost and high-lipid-yield heterotrophic lipid producer using glycerol (a by-product of biodiesel manufacturing process) as carbon source and an inexpensive nitrogen source (i.e., corn steep liquor).

1. Introduction Due to the fast depletion of petroleum fuels and the accompanying environmental issues with their continuous use, biofuels are being widely studied recently and are regarded as one of promising solutions to the energy dilemma we encounter today. Biodiesel, biologically derived diesel oil, is commonly added in automobile engines as an alternative fuel to substitute petrochemical diesel. Currently, biodiesel is mainly manufactured from plant oil (e.g., soy bean oil) or animal fat (e.g., beef tallow). However, biodiesel from these sources can only partly fulfil the existing demand for transport fuels [1]. Oil-rich microalgae, also dubbed as oleaginous algae are being considered as a



feedstock for biodiesel production because of their higher growth rate compared to oil-producing plants such as jatropha and rapeseed and their remarkable lipid accumulating potential [2]. However, the commercial use of microalgae for biodiesel faces many challenges owing to low productivity and high cost related to the photosynthetic efficiency of algal strain, photobioreactor design, harvesting method and extraction techniques [3]. Algal oil from the low-priced biomass was estimated to cost $2.80/L without accounting for the transesterification process, and the current price is still four times higher than petro-diesel price [4]. In 2010, the US navy and Dynamic Fuels formed a joint venture for algal oil production, and the cost of the algal biodiesel was US$ 27 per gallon [5]. Normalization of baseline assumption form

Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan. Corresponding author. E-mail addresses: [email protected] (C.-Y. Chen), [email protected] (J.-S. Chang).

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https://doi.org/10.1016/j.bej.2019.107429 Received 27 August 2019; Received in revised form 25 October 2019; Accepted 5 November 2019 Available online 06 November 2019 1369-703X/ © 2019 Published by Elsevier B.V.

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sequentially in the flask experiments including the carbon source types (glycerol, glucose, fructose and sucrose each 10 g/L), low-cost nitrogen sources (such as corn steep liquor (CSL), mono-sodium glutamate (MSG), whey powder each 10 g/L), different CSL concentrations (5.0, 7.5, 10.0, 12.5, 15.0, 17.5 g/L) and sea salt concentration (0, 1, 2, 3, 4% w/v). The inoculum condition including different seed culture time (48,72 h) and inoculum size (0.05 g/L, 0.10 g/L, 0.15 g/L, 0.20 g/L) were also tested.

various sectors indicated that the cost of algal diesel could be between 3.5 US$ per kg [6]. A cost analysis revealed that using open systems like raceways, wastewater as a nutrient source and using monochromatic light as a light source for algal cultivation could decrease the microalgae biomass production costs from 2.71 to 0.73 $/kg [7]. Life cycle analysis of biodiesel production from microalgae indicated that capital and operating costs should be reduced by at least 50% to make this venture profitable compared to petrodiesel [8]. Cost reduction could be accomplished by inexpensive sources of water, CO2 and fertilizers [9] as well as by improving the biomass and lipid productivities of microalgae [10]. Thus, cost reduction in case of microalgal biomass production and efficient use of the abundant by-product (biodiesel derived glycerol) are the main challenges for microalgal biodiesel. Microalgal strain selection, cultivation conditions, harvesting and processing technologies should be considered for the sake of improving the efficiency of biodiesel production [10]. Glycerol is synthesized as the main by-product during transesterification process of biodiesel production. However, impurities present in the crude by-product make it hard to use directly for commercial purpose and purification is expensive [11]. However, that crude glycerol could be used as a carbon source for microalgae like Thraustochytrids [12,13]. Thraustochytrids are heterotrophic protists, which uses organic carbon as their carbon and energy source in dark conditions [14]. Heterotrophic cultivation is highly advantageous due to the possibilities of high cell density fermentations in conventional bacterial fermenters with associated increase in biomass and lipid productivities [15]. Although Thraustochytrids now are commonly used for polyunsaturated fatty acid (PUFAs) production, they have been developed as a promising material for biodiesel production such as with Schizochytrium sp. S056 [16] and Aurantiochytrium limacinum SR21 (ATCC MYA-1381) [17]. Due to the high palmitic acid (C16:0) content which is an essential property to improve oxidative and thermal stability of biodiesel, Thraustochytrid lipid-derived biodiesel meets the American Society for Testing and Materials (ASTM) standard requirements for commercial use [18]. The main aim of this study was to optimize lipid production from an indigenous Thraustochytrid strain utilizing low cost resources like glycerol. The culture parameters like carbon source, nitrogen source and concentration and inoculum conditions were evaluated and optimized for maximal lipid content and productivity.

2.3. Determination of microalgal biomass concentration A 5 mL of cell suspension was collected from the bioreactor, and then the sample was centrifuged at 6000 rpm for 2 min to remove the culture medium. Then, the sample was washed twice with 5 mL of deionized water and transferred to an aluminum pan which is preweighed. The aluminum pan was placed in an infrared moisture determination balance (FD-720, KETT, Japan) at 100℃ until all the water was withdrawn. Then, the pan was weighed again with the dried biomass. The difference in the weight of the pan with and without the dry biomass is the weight of the biomass and the biomass concentration in g/L is then calculated accordingly. 2.4. Determination of biomass productivity, biomass yield and specific growth rate The biomass productivity (mg/L/h), biomass yield (g biomass/g carbon source) and specific growth rate (μ) were calculated according to the following equation: Biomass productivity (mg/L/h)

=

ΔX X − Xinitial = t Δt t Biomass yield (g / g carbon source)

=

ΔX Xt = ΔSt St − Sinitial Specific growth rate (h−1)

dlnX ⎞ =⎛ ⎝ dt ⎠max where X is biomass concentration (g/L), ΔX (mg/L) is the variation of biomass concentration, ΔSt is the variation of carbon source (g/L) and Δt is the cultured time (h).

2. Materials and methods 2.1. Microalgae strains and seed culture preparations

2.5. Determination of the carbon source concentration in culture medium Thraustochytrium sp. BM2 was isolated from full-strength water located in Beiman, Tainan. The isolate was identified as Thraustochytrium sp. based on the morphology appearance observed by microscope and 23S rDNA sequence analysis [19]. The strain is a member of the phylum Labyrinthulomycota of the kingdom Chromista, and the genus Thraustochytrium. The strain was maintained on DG agar plates containing sea salt 30 g/L, glucose 4 g/L, peptone 1 g/L, yeast extract 2 g/L and agar 30 g/L. Stocks were stored at −80 °C in glycerol. Seed culture was cultivated in GYP medium consisting sea salt 30 g/L, glucose 10 g/L, peptone 5 g/L and yeast extract 5 g/L. Standard inoculum condition was 0.05 g/L with 72 h culture.

The culture broth was collected from the bioreactor and centrifuged at 10,000 rpm for 2 min, and the biomass was separated. Then, the supernatant was filtered with 0.22 μm pore size filter to remove the impurities and diluted appropriately with deionized water. After that, the initial and residual carbon source concentration in the medium were determined by high performance liquid chromatography (HPLC) equipped with refractive index detector (2414, Waters, USA), ICSep ICE-COREGEL 87H3 column (Transgenomic, USA), pump (2695, Waters) and column oven (Temperature control moduleⅡ, Waters). The injection volume of sample was 20 μL, and the HPLC column temperature was set at 70℃. The mobile phase was 0.008 N H2SO4 at flow rate of 0.4 mL/min.

2.2. Microalgae cultivation conditions Erlenmeyer flasks (500 mL, glass) were used to conduct the experiments and the working volume was 250 mL with 175 rpm agitation speed at 25 °C. Standard medium contained 10 g/L glucose, 5 g/L peptone, 5 g/L yeast extract and 30 g/L sea salt. The main aim of this study was to evaluate sustainable or cheaper carbon sources and nutrients to grow Thraustochytrium sp. BM2 to reduce the cost of algaebased lipid production. Several medium compositions were examined

2.6. Determination of the initial total nitrogen concentration in the culture medium Total nitrogen analysis was determined using the total nitrogen reagent commercial kit from SUNTEX®. A 0.5 mL of the liquid sample and 0.5 mL deionized water were mixed. The reaction tubes were then heated at 100 °C and the reagents were added sequentially. Finally, 2

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total nitrogen concentration (ppm) of each sample was automatically measured by AQUALYTIC® Photometer AL400. 2.7. Determination of total lipid content of the microalgal lipids Lipid analysis was done as fatty acid methyl esters (FAMEs) through the direct transesterification according to method described in the work of Lepage and Roy [20]. The cell pellets were harvested by centrifugation (6000 rpm, 3 min) and then lyophilized. A 40 mg of lyophilized cells were mixed with 8 mL of 0.5 N KOH in methanol and boiled for 15 min for saponification. Then, 8 mL of 1 N HCl in methanol and 10 mL of 14% (v/v) BF3/CH3OH were added into the mixture and boiled again. Saturated NaCl solution was added to prevent emulsification and 4 mL hexane was added for solvent extraction. The FAMEs were finally collected from the upper hexane layer after centrifuging (6000 rpm, 5 min). The resulting FAMEs were analyzed by gas chromatography (Master GC, DANI, Italy), equipped with a fused silica capillary column (100 m long and 0.25 mm in internal diameter). Helium was used as the carrier gas with a flow rate of 1.0 mL/min. The temperature of injector and detector was set at 260 and 280 °C, respectively. The oven temperature was programmed from 140 to 240 °C with an increase rate of 4 °C/min, and maintained at 240 °C for 20 min. The total lipid content was quantified by the relative peak area of each kind of fatty acid to the internal standard (C19:0). The acquired concentration (mg/L) was transformed to the lipid content of biomass (mg/g) by dividing the lyophilized cell weight (mg) and multiplying the diluted volume (L). 2.8. Determination of lipid production, maximum lipid productivity and lipid yield The lipid production (mg/L), maximum lipid productivity (mg/L/h) and lipid yield (g /g glycerol) were presented from the following equation: Lipid production (mg/L) Fig. 1. Effect of carbon source (with a fixed concentration of 10 g/L) on (a) maximum biomass production and productivity, (b) biomass yield and specific growth rate, and (c) maximum lipid content and productivity of Thraustochytrium sp. BM2. (N.A, not available).

= Lipid contentt (%) × Xt Maximum lipid productivity (mg/L/h)

Lipid contentt (%) × Xt ⎞ =⎛ t ⎠max ⎝

source for growth and metabolism. Fructose was also not an effective carbon source due to poor biomass growth and high residual sugar. Glycerol was effective as a sole carbon source for BM2 with biomass production, productivity, yield, and specific growth rate of 4.04 g/L, 73.75 mg/L/h, 0.41 g/g substrate and 0.33 h−1 respectively and the results obtained were similar to that of glucose. Glycerol has been successfully used as a carbon source for Thraustochytrids for the production of omega-3 fatty acids and carotenoids [22–24]. Thraustochytrium sp. AMCQS5-5 achieved the highest biomass concentration of 8.32 g/L with 40 g/L glycerol as the carbon source [23]. The biomass growth obtained in this study using 10 g/L glycerol is comparable or better than other reported studies. Fructose as a carbon source showed the worst cell growth performance for Thraustochytrium sp. BM2, though the lipid content and productivity were close to that obtained with glucose. Lipid content and productivity of Thraustochytrium sp. BM2 grown on different carbon sources are shown in Fig. 1(c). A maximum lipid content of 30% was obtained with glycerol and the productivity was approximately 18 mg/L/h. The lipid content and maximum lipid productivity with glycerol as a carbon source was 1.5 times higher than that of glucose and fructose. However, Chi and coworkers reported that there was no significant difference in lipid content and productivity while using glucose or glycerol in cultivation of Schizochytrium limacinum SR21 [25]. Different species and strains differ in their metabolic potential and there are still many unknown pathways in

Lipid yield (g / g carbon substrate)

=

Lipid contentt (%) × Xt ΔCt

where Lipid contentt and Xt are the lipid content (% biomass) and biomass concentration (g/L) at time t. ΔCt is the variation of carbon substrate (g/L). 3. Results and discussion 3.1. Effect of different carbon sources on microalgal lipid production Thraustochytrids are known to use numerous carbon sources and glucose is the most common one for cultivation. Other substrates like glycerol, monosaccharides, disaccharides and polysaccharides (starch) have also been used. Different carbon sources could result in distinct growth condition and product profile [21]. Therefore, the effect of different carbon sources on the growth of Thraustochytrium sp. BM2 was investigated in this section. Four different carbon sources - glucose, glycerol, fructose and sucrose were tested and the concentration of each carbon source was maintained at 10 g/L in the culture medium. Biomass growth and lipid content is shown in Fig. 1. As it can be seen from the figure, Thraustochytrium sp. BM2 is incapable of utilizing sucrose as a sole carbon 3

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peptone. On the contrary, monosodium glutamate and whey powder were not preferred by Thraustochytrium sp. BM2 as poor biomass growth and poor glycerol utilization was shown. However, as shown in Fig. 2 (c), lipid content and lipid productivity with corn steep liquor reached 66% and 31.08 mg/L/h, respectively. The lipid content and productivity were three times and two times higher, respectively compared to yeast extract and peptone group. Schizochytrium limacinum SR21 also attained the highest fatty acid content and DHA yield of 37% and 1.7 g/L, respectively while using 10 g/L corn steep liquor as the nitrogen source [27]. According to Jakobsen and coworkers, total lipid content would increase while using low level of nitrogen in Thraustochytrids cultivation, which mean nitrogen starvation would improve lipid accumulation [28]. Total nitrogen concentration of corn steep liquor was 19.5 g/L, which was six times lesser than yeast extract and peptone (123.5 g/L) for the same weight as used in the experiments. Due to the low nitrogen content and high C/N ratio, lipid accumulation in Thraustochytrium sp. BM2 was higher. Another appealing feature is that the cost of the 1/6th of peptone plus yeast extract was still higher than that of full strength corn steep liquor. As for monosodium glutamate and whey powder, the lipid content was higher than that of corn steep liquor, but lipid productivity was very low due to poor cell growth. Therefore, considering the lipid productivity and cost, corn steep liquor was chosen as the optimal nitrogen source in the following tests. 3.3. Effect of corn steep liquor (CSL) concentration on microalgal lipid production Different concentrations of nitrogen source would have obvious influence on either biomass or lipid content in Thraustochytrids. The carbon/nitrogen (C/N) ratio, manipulating the switch between protein and lipid synthesis, was usually applied to investigate the integrated effect of carbon and nitrogen on lipid accumulation [29]. Many studies reported that high C/N ratio in culture medium would boost lipid accumulation in Thraustochytrids [28,30]. Therefore, the effect of corn steep liquor concentration (5, 7.5, 10, 12.5, 15, 17.5 g/L) with fixed glycerol concentration (10 g/L) on biomass and lipid production was investigated in this section to find out the optimal C/N ratio and the results are shown in Fig. 3. Corn steep liquor concentration under 10 g/L and above 15 g/L, especially 5 g/L, showed lower biomass production than other groups as shown in Fig. 3 (a). It might indicate that very low and very high nitrogen concentrations could severely affect growth and biomass accumulation of BM2. As for lipid content and lipid productivity (illustrated in Fig. 3 (c)), the results revealed that lipid content decreased with increasing corn steep liquor concentration, which was similar to other reported studies. A remarkably high lipid content of 76% by weight and lipid productivity of 37 mg/L/h were obtained with 12.5 g/ L of corn steep liquor concentration. This indicated that at optimal nitrogen source concentration, biomass and lipid content increased significantly. Hence, corn steep liquor concentration of 12.5 g/L was selected to be the optimal for lipid production by Thraustochytrium sp. BM2.

Fig. 2. Effect of nitrogen source (with a fixed concentration of 10 g/L) on (a) maximum biomass production and productivity, (b) biomass yield and specific growth rate, and (c) maximum lipid content and productivity of Thraustochytrium sp. BM2. (YEP, yeast extract and peptone; CSL, corn steep liquor; MSG, monosodium glutamate; WHEY, whey powder).

Thraustochytrid metabolism. In summary, Thraustochytrium sp. BM2 is capable of utilizing glycerol as a sole carbon source efficiently and it could replace glucose in the following experiments. 3.2. Effect of different nitrogen sources on microalgal lipids production Different nitrogen source in medium would affect microalgal growth condition and lipid content in the cells. Inorganic nitrogen sources and organic nitrogen source were both utilized by different kinds of Thraustochytrids. Chen and his coworkers showed that inorganic nitrogen sources like NaNO3, (NH4)2SO4 and organic nitrogen sources like urea, monosodium glutamate, corn steep liquor, peptone, yeast extract, tryptone were utilized by Aurantiochytrium sp. BR-MP4A1 [26]. Therefore, in order to replace yeast extract and peptone in the culture medium for Thraustochytrium sp. BM2, three kinds of low cost nitrogen sources (corn steep liquor, monosodium glutamate, whey powder at 10 g/L) were evaluated for their efficiency as a nitrogen source. As shown in Fig. 2 (a) and (b), using yeast extract and peptone as nitrogen source resulted in the best performance regarding biomass growth. Highest maximum biomass production (4.24 g/L), maximum biomass productivity (72.7 g/L/h), biomass yield (0.41 g/g glycerol) and specific growth rate (0.30 h−1) was observed with yeast extract and

3.4. Effect of sea salt concentration on microalgal lipids production Members of Thraustochytrids are usually marine and are adapted to the variation of salinity in sea water. Presence of sodium ion is indispensable for their osmotic pressure manipulation, metabolism, and ATP production, which may directly influence diverse physiological and biochemical mechanisms related to cell growth and lipid production [31]. Although Thraustochytrids can tolerate a wide range of salinity from as low as 1% to full-strength seawater, but high concentration of chloride ion is corrosive to the conventional bioreactors [32]. Therefore, different sea salt concentration (0, 1, 2, 3, 4% w/v) were investigated in this section to evaluate their effect on lipid production by 4

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Fig. 3. Effect of corn steep liquor concentration on (a) maximum biomass production and productivity, (b) biomass yield and specific growth rate, and (c) maximum lipid content and productivity of Thraustochytrium sp. BM2.

Fig. 4. Effect of sea salt concentration on (a) maximum biomass production and productivity, (b) biomass yield and specific growth rate, and (c) maximum lipid content and productivity of Thraustochytrium sp. BM2. (N.A, not available).

Thraustochytrium sp. BM2 and the results are summarized in Fig. 4. As seen in Fig. 4 (a) and (b), Thraustochytrium sp. BM2 could not grow in the absence of sea salt, which meant it is incapable of growth in fresh water. Biomass increased till 2% sea salt, and there was no significant improvement above 2% sea salt concentration. The highest biomass production, biomass productivity, biomass yield and specific growth rate were obtained at 2% sea salt concentration and were 3.89 g/L, 57.50 g/L/h, 0.36 g/g glycerol and 0.19 h−1, respectively. Lipid content and productivity with different salinity is illustrated in Fig. 4 (c). Results indicated the maximal lipid content of the biomass varied from about 70–79% at all sea salt concentrations used, with the highest at 79% with 2% sea salt concentration. However, the lipid productivity at 2 and 3% sea salt concentration (37.35 and 36.54 mg/L/ h) was relatively higher than 1 and 4% (21.93 and 27.17 mg/L/h). Previous literature has reported that sodium serves as the major contributor in osmotic adjustment followed by chloride [32]. In addition to osmotic adjustment, sodium is essential in the uptake of phosphate and magnesium into the cell [33]. The result indicates that 2% sea salt concentration has provided enough sodium and chloride ion for cell osmotic adjustment as well as cell metabolism in Thraustochytrium sp. BM2 growth cycle. Furthermore, 2% sea salt concentration is sufficient to induce intracellular lipid accumulation of the microalgae. Studies have shown that other Thraustochytrid strains require very high sea salt concentrations in the range of 25–30%. Schizochytrium mangrovei required a high sea salt concentration in the range of 15–22.5%

for maximal lipid accumulation, which was in the range of 16–33.2% [34]. Schizochytrium mangrovei strain Sk-02 did not show any significant difference in lipid accumulation in the sea salt concentration range of 15-25 g/L (1.5–2.5%) [35]. So, the requirement and response of Thraustochytrids to salinity are highly strain specific, and it is beneficial that BM2 required very low sea salt concentration at 2% resulting in the highest lipid content of 79% with a lipid productivity of 37.35 mg/L/d. Regarding cost and corrosion effect on fermenter, 2% w/ v sea salt was chosen as the optimal salinity for Thraustochytrium sp. BM2 cultivation. 3.5. Effect of inoculum conditions on microalgal lipids production Inoculum conditions had been rarely studied and regarded as a factor in microbial growth kinetics, but it is evident that inoculum conditions could influence the outcome of a fermentation. Recent studies have indicated the significance of inoculum size on the capability of a microbial population to initiate growth [36]. An increase in inoculum size to 4.16% increased lipid and DHA accumulation in Thraustochytrium sp. ATCC 26,185. Increase in inoculum size not only promotes rapid biomass growth and higher lipid accumulation, it shortens the fermentation time as well [35]. Since very few studies evaluated the effect of inoculum condition on Thraustochytrids cultivation, different inoculum age (48 h and 72 h) and inoculum size (0.05, 0.10, 0.15, 0.20 g/L) were examined in this section to find out the 5

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6

[40] [41] [42] [43] This study

[39]

52 171 1400 1140 – 336 1683 13 38 43.3 57 50 ± 2 – 79 Thraustochytrium striatum Thraustochytrium sp. CR01 Thraustochytrium sp. T01 Thraustochytrium sp. ATCC 26185 Thraustochytrium sp. BM2

0.4 0.45 3.23 2 – – 2.13 Glucose, yeast extract, batch, flask cultures Glucose, yeast extract, fed batch, flask cultures Maltose, yeast extract, batch, flask cultures Corn syrup, yeast extract, batch, flask cultures Glucose, soya peptone, yeast extract, batch, flask cultures Glucose, yeast extract, batch, bioreactor culture Glycerol, corn steep liquor, batch, flask cultures Thraustochytrium striatum

Lipid content (% of DCW) Biomass productivity (g/L/d)

optimal lipid productivity with Thraustochytrium sp. BM2. As shown in Fig. 5, age of pre-culture did not significantly influence biomass production or lipid productivity when the inoculum size was 0.05 g/L and the biomass concentration and lipid productivity were 3.8 g/L and 34 mg/L/h, respectively. However, maximum biomass productivity and specific growth rate with 48 h pre-culture (62.92 g/L and 0.18 h−1) was higher than 72 h pre- culture (55.50 g/L and 0.16 h−1). Hong et al showed that exponential phase or early stationary phase cells promoted higher biomass accumulation in Aurantiochytrium sp. KRS101 [37]. Hence, 48 h-aged seed culture (most probably in the late exponential phase/early stationary phase) was selected. As mentioned previously, inoculum size is another important factor affecting biomass and lipid production. As shown in Fig. 5, maximum biomass production, maximum biomass productivity, and maximum lipid productivity increased with increase in inoculum size. A high lipid content of 70% was attained irrespective of the inoculum size and age. However, lipid productivity increased with increase in inoculum size by accompanied biomass growth until 0.15 g/L inoculum and then levelled off. Highest maximum biomass productivity and lipid productivity were 88.75 and 43.86 mg/L/h respectively with inoculum size 0.15 g/L. Rosa et al. [38] showed that a pre-culture grown in low C/N medium and transferred to the fermenter at 10% v/v highly improved DHA yield from Aurantiochytrium limacinum SR21, suggesting that higher inoculum size can positively influence lipid accumulation. Therefore, combining results with inoculum age and size, 48 h-aged pre-culture

Cultivation conditions

Fig. 5. Effect of inoculum conditions on (a) maximum biomass production and productivity, (b) biomass yield and specific growth rate, and (c) maximum lipid content and productivity of Thraustochytrium sp. BM2.

Microalgae species

Table 1 Comparison of heterotrophic lipid production performance of Thraustochytrium sp. BM2 obtained from this study with others from related literature.

Lipid productivity (mg/L/d)

References

C.-Y. Chen, et al.

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and 0.15 g/L initial biomass concentration were selected for microalgal lipid production with Thraustochytrium sp. BM2. When compared with the related reports regarding the performance of microalgal lipid production with heterotrophic microalgal cultivation, it can be seen that Thraustochytrium sp. BM2 has the highest total lipid content (up to 79%) among the literature (Table 1), along with a very high lipid productivity of 43.86 mg/L/h (or 1.052 g/L/d). Hence, we present a new Thraustochytrium sp. BM2 strain with high lipid production potential and a suitable candidate for sustainable biodiesel production using waste glycerol as a carbon source. Further studies regarding the use of industry derived waste crude glycerol and scale up in fermenters are in progress.

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