optimization of medium components for high biomass and lipid production of tryblionella hungarica...

Egypt. J. Exp. Biol. (Bot.), 11(1): 41 – 50 (2015) © The Egyptian Society of Experimental Biology

ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://my.ejmanger.com/ejeb/

R E S E A R C H A R T I C L E

Em an I . Abdel -Aal Jelan Mofeed

OPTIMIZAT ION OF MEDIUM COMPONENTS FOR HIGH BIOMASS AND LIPID PRODUCTION OF THE FRESHWATER DIATOM TRYBLIONELLA HUNGARICA NIOF-DM-017 BY USING PLACKETT-BURMAN DESIGN

ABSTRACT: Biofuel productions from microalgae are considered as potential alternative fuel sources of fossil fuels. The ideal candidate species for biodiesel production need both high biomass and lipid production. In this study, the Plackett-Burman design was used to evaluate the relative importance of ten nutritional independent components to biomass and lipid production in Tryblionella hungarica NIOF-DM-017. These components were Ca(NO3)2.4H2O, K2HPO4.3H2O, MgSO4.7H2O, Na2SiO3.9H2O, Na2CO3, FeCl3.6H2O, Na2.EDTA.2H2O, glucose, NaCl and pH, where twelve different runs of media were conducted. The biomass and lipid content of the control Navicula medium were 0.133 gL-1 and 21.4%, after five days of growth. Most of components found to contribute largely for the biomass and lipids production. The medium run 8 exhibit lowest biomass and lipids of 0.032 gL-1 and 11.6%, while runs 11 and 12 recorded the highest biomass (0.274 and 0.268 gL-1, respectively). Slight to significant decreases were recorded in the lipid content in different experimental runs. The ligand nature of EDTA found to inhibit the growth of the diatom in the runs especially of high EDTA concentration (runs 3, 5, 9, and 10). The results of the main effects of the studied components were used to prepare two verification medium to evaluate the accuracy of the applied Plackett-Burman design. Form the maximum biomass verification medium, the biomass production increased by 85.7% and lipids by 42.9%, when compared to the control medium. Meanwhile, the prepared verification medium for maximum lipids resulted in increase in biomass by 30.8% and lipid content with 61.2% after five days of experimental period. The lipid profile of T. hungarica cultured on Navicula medium and the two verification media was mainly composed of fatty acids and hydrocarbons. The fatty acids comprise about 53%, 64%, and 59.4% of the total lipids of the test diatom cultured in control medium, verification medium for high biomass and verification medium for high lipids, respectively. In general, non-considerable changes were recorded in fatty acids profile between control and verification media. The FAMEs of T. hungarica were dominated by methyl palmiteate (C16: 0), methyl palmitoleate (C16: 1) and methyl linoleate myristate (C18: 2), which are suitable for biodiesel production.

KEY WORDS: Biomass, Lipids, Plackett-Burman, Tryblionella hungarica

CORRESPONDENCE: Em an I . Abdel -Aal National Institute of Oceanography and Fisheries (NIOF), Cairo, Egypt E-mail: emanibrahim2002@gmail .com

Jelan Mofeed

Department of Aquatic Environment, Faculty of Fish Resources, Suez University, Suez, Egypt ARTICLE CODE: 05.02.15

INTRODUCTION: The world energy consumption predicted

to increase by 54% between 2001 and 2025. In order to meet future needs for energy shortage, considerable focus is being directed towards the development of sustainable al ternat ive energy sources (EIA, 1998). Feasible alternative renewable and environmental ly friendly sources of fuels are needed to replace pet roleum-based fossil fuels (Vasudevan and Briggs, 2008). Biofuels from microalgae is now consider as a potential alternative to fossil fuels (Chisti, 2008; Hu et al., 2008; Fields et al. , 2014) because of their advantages of higher photosynthetic efficiency, higher biomass production, high levels of oils and starch for biodiesel and bioethanol production (Griff iths and Harrison, 2009). Diatoms and certain green microalgae have attracted attention for the use in biofuel product ion because they have between 20-80% oil by weight of dry mass (Bajhaiya et al., 2010; Fields et al. , 2014). Diatoms are the primary consti tuent of the marine plankton community, typically representing more than 70% of the total plankton, and are est imated to contribute up

Egypt. J. Exp. Biol. (Bot.), 11(1): 41 – 50 (2015)


42

to 40% of the total oceanic primary production (Sumper and Brunner, 2008). Diatoms produce lipids (e.g., tri acylglycerols: TAGs) and carbohydrates as reserve food material during the vegetative period of growth (Becker, 2004). Some algae were reported to produce up to 60% of their cel lular mass as TAGs. These TAGs can be easily converted into biodiesel (Sheehan et a l., 1998). The chemical conversion of the oil to i ts corresponding biodiesel (fatty acid alkyl esters) is called transesteri fication (Demirbas, 2009). Biodiesel i s better than diesel fuel in terms of sulphur content, f lash point, aromatic content and biodegradabili ty (Bala, 2006).

Media composition and growth conditions influence the culture growth and thus the biomass and lipid content. A number of factors are known to influence the biomass and lipid production of microalgae, such as deficiency of nitrogen (Richardson et al., 1969; Illman et al., 2000) and silicon (Lynn et al. , 2000), phosphate limitation (Reitan et al., 1994), high salinity (Rao et al. , 2007), light intensity (Kojima and Zhang, 1999) and iron content of the medium (Liu et al., 2008). In order to make the biofuel production from microalgae more economic, it is very necessary to use native strains that can be easily cultured and have high lipid production and also adapt for the climates (Chisti, 2007; Jiang et al., 2014). Biomass and lipid content of microalgae can be increased with properly optimized media composition. Conventional optimization may a time-consuming and laborious process involving large number of experiments. The Plackett-Burman design (PBD) (Plackett and Burman, 1946) provides an efficient way of sorting out a large number of variables and ident ifying the most important ones. Numerous reports have proved the applicability of PBD in the optimization of media components for various culture activities (Yu et al., 1997; Li et al., 2007; Abedin and Taha, 2008). Diatom species, for example Nitzschia species characterized by having a simple nutritional requirements, fast growth rates and high lipid production (Chagoya et al. , 2014; Jiang et al., 2014). The present study aims at testing the PBD for screening the key factors affecting the biomass and lipid production of the native strain of the diatom Tryblionella hungarica and to assess its feasibility for biodiesel production.

MATERIAL AND METHODS: Algal strain and culture medium:

The tested alga Tryblionella hungar ica used in the present study (Fig. 1) was isolated from El-Salam Canal, Egypt (31° 45` & 34° 05` E and 30° 42` & 31° 24` N). Pure culture of T. hungarica (Grunow) Frenguelli 1942 (Basionym: Nitzschia hungarica Grunow 1862 (NIOF DM-017) was obtained by repeated streaking and plating at pH 7.5 ± 1 using standard isolation and culturing techniques

(Stein, 1973) in modified Navicula medium (Starr, 1978). The composition of Navicula medium (gL- 1) is 0.1 g Ca(NO 3)2.4H2O, 0.14 g K2HPO4.3H2O, 0.025 g MgSO4.7H2O, 0.1 g NaSiO3.9H2O, 0.02 g NaCO 3, 1.0 ml iron stock solution (one liter iron solution contains 5.0 g FeCl3.6H2O and 30 g Na2.EDTA.2H2O), and 1.0 ml trace elements solution (one li ter trace element solution contains 2.8 g H3BO3, 0.9 g MnCl 2.4H2O, 0.125 g ZnCl2, 0.08 g CuSO4.5H2O, 0.9 g Na2MoO4.2H2O, and 0.014 g CoCl2.6H2O). The diatom was purifi ed by the method described in Van Der Werff (1955) and identif ied under microscopic examination by the according to Schoeman and Archibald (1976) and Kociolek (2011).

Growth characteristics of the test alga: The tested alga T. hungarica NIOF-DM-

017 was cultured in 250 ml Erlenmeyer flasks containing 90 ml Navicula nutrient medium, inoculated by four days old culture to obtain initial concentration of 5000 cell /ml. Three replicate f lasks were prepared, the culture flasks were incubated at 25 ± 2oC under continuous light intensity of 51.35 ± 2.7 µmol photons m -2 s- 1 for eight days. Every two days, a sample was taken and the algal growth was est imated by direct cell count using the standard haemocytometer technique (APHA, 2005). The division time (Tg), the t ime required for cells to divide (in days) was calculated using the fol lowing equations (Gui llard, 1973): The division time:

(Tg) = 0.6931 / µ; µ = ln X2 - ln X1 / (t2 – t1) Where, µ = growth rate, X 1 = cel l count at time t1, X2 = cel l count at time t2.

Plackett-Burman design: The Plackett-Burman design for ten

variables: nine nutritional independent variables, one physiological factor (pH) and one dummy variable were used to evaluate

Fig. 1. Tryblionella hungarica (Grunow) Frenguelli; a: the tested isolate T. hungarica NIOF-DM-017, b: after Kociolek (2011).

Abdel-Aal & Mofeed, Optimization of Medium Components for High Biomass and Lipid Production of Tryblionella Hungarica …..


43

thei r relative importance for biomass and l ipid production in the diatom T. hungarica . These factors were Ca(NO3)2.4H2O, K2HPO4.3H2O, MgSO4.7H2O, Na2SiO3.9H2O, Na2CO3, FeCl3.6H2O, Na2.EDTA.2H2O, Glucose, NaCl and pH. Each factor was tested at three levels of concentrations (low "-1", medium "0" and high "+1") shown in table 1. Twelve dif ferent medium runs shown in table 2 were performed in triplicates. The experimental design was prepared by the software Design-Expert® Version 9 (Stat-Ease, Inc. USA). The experiments were carried out in 500 ml Erlenmeyer flasks each containing 200 ml of each medium run and control cul ture in Navicula medium was also run parallel. To avoid the precipitation of the medium component, the stock solutions of the medium components were steril ized separately and the medium runs were prepared under asepti c condition. The flasks were inoculated with three days fresh cultures to obtain initial concentration of 5000 cel l/ml. The culture flasks were incubated at 25 ± 2 ºC under

continuous l ight intensi ty 51.35 (± 2.7) µmol photons m -2 s -1 for five days. The whole experiment was repeated two times to make sure of the results and the mean results of both experiments were recorded. Table 1. Plackett-Burman experimental design with low and

high levels of selected factors

No. Variables Unit Level -1 (Low)

Level - 0 (Navicula

cont.medium)

Level +1 (High)

1 Ca(NO3)2.4H2O gL-1 0.05 0.1 0.3 2 K2HPO4.3H2O gL-1 0.07 0.14 0.42 3 MgSO4.7H2O gL-1 0.0125 0.025 0.075 4 Na2SiO3.9H2O gL-1 0.05 0.1 0.3 5 Na2CO3 gL-1 0.01 0.02 0.06 6 FeCl3.6H2O gL-1 0.0025 0.005 0.015 7 Na2.EDTA.2H2O gL-1 0.015 0.03 0.09 8 pH 6.5 7.5 8.5 9 Glucose gL-1 1.5 0 4.5 10 NaCl gL-1 0.33 0 0.99 11 Dummy - - -

Table 2. Mean biomass and lipid production in Tryblionella hungarica after applying Plackett-Burman design with 12 runs of medium.

Variables

Run

s

Ca(

NO

3)2.

4H

2O

K2H

PO

4.3H

2O

MgS

O4.

7H2

O

Na 2

SiO

3.9H

2O

Na 2

CO

3

FeC

l 3.6H

2O

Na.

ED

TA.2

H2O

pH

Glu

cose

N

aCl

salin

ity

Dum

my

Mea

n bi

omas

s pr

oduc

tion

(g.L

-1)

% in

crea

se (+

) or

dec

reas

e (-)

Sign

ifica

nce

leve

l

Mea

n lip

id

prod

uctio

n (%

)

% in

crea

se (+

) or

dec

reas

e (-)

Sign

ifica

nce

leve

l

0 (control medium) 0 0 0 0 0 0 0 0 0 0 0 0.133 21.4 1 1 -1 -1 -1 1 -1 1 1 -1 1 1 0.0497 -62.6 *** 19.5 -9.6 NS 2 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 0.0867 -34.8 *** 20.0 -7.07 NS 3 -1 1 -1 1 1 -1 1 1 1 -1 -1 - - 4 1 -1 1 1 -1 1 1 1 -1 -1 -1 0.142 6.76 NS 18.0 -17.2 NS 5 1 1 1 -1 -1 -1 1 -1 1 1 -1 - - 6 1 1 -1 -1 -1 1 -1 1 1 -1 1 0.145 9.023 NS 17.69 -18.7 * 7 -1 -1 1 -1 1 1 -1 1 1 1 -1 0.142 6.767 NS 17.2 -21.2 * 8 -1 1 1 1 -1 -1 -1 1 -1 1 1 0.032 -75.9 *** 11.6 -49.5 *** 9 -1 -1 -1 1 -1 1 1 -1 1 1 1 - - 10 -1 1 1 -1 1 1 1 -1 -1 -1 1 - - 11 1 -1 1 1 1 -1 -1 -1 1 -1 1 0.274 106 *** 19.5 -9.6 NS 12 1 1 -1 1 1 1 -1 -1 -1 1 -1 0.268 101.5 *** 19.3 -10.6 NS

(-): No growth of the alga on these runs; NS: Non-significant. (*): Significant decrease at P ≤ 0.05; (***): Very highly significant increase or decrease at P ≤ 0.001.

Determination of algal biomass: The dry weight biomass was determined

gravimetrically according to Samorì et al. (2010). The cultures were centrifuged at 10,000 x g, washed twice in sterile saline (0.85% NaCl), centrifuged, the biomass transferred to aluminum crucibles previously dried at 60°C for 24 hours and weighed. The algal biomass was dried at 60°C unt il constant weight. The dried biomass was expressed as gL-1. Determination of lipid content:

The total l ipids were extracted using the modified method of Bligh and Dyer (1959). The

dried algal biomass was suspended in 4 ml methanol and 3 ml chloroform and incubated for 24 h at 25°C. After 24 h, the mixture was agitated in a vortex for 2 min, 3 ml of chloroform was added, again the mixture was agitated in a vortex for 1 min, then transferred to a clean previously dried and weighed glass vial . The previous ext raction method was repeated again unt il the extract become almost clear. The extraction mixture was added to that in the glass vial and the solvents evaporated to dryness in a water bath at 70oC. The weight of the glass vial was



44

again recorded and lipid contents were expressed as % dry weight. Data analysis:

The data obtained from the different runs were analysed by the equations of Fossi et al. (2005). The main effect of each variable was determined with the fol lowing equation:

E x i = (ΣHx i - ΣLx i) / N Where Exi i s the variable main effect, Hxi

and Lxi are the concentrat ion of the variable at high level and low level of the same variable, and N is the number of trials divided by 2. A main effect with a posi tive sign indicates that the high concentration of this variable i s nearer to optimum and a negati ve sign indicates that the low concentration of thi s variable is nearer to optimum. The variance of effect of each variable was calculated as follows: Vx i = (ΣHx i - ΣLx i)2 / N.

The experimental error was calculated as follows: R = ΣV xd

2 / n Where Vxd i s mean square of dummy

variables and n is number of dummy variables. The factors showing larger effects were determined using F-test: F = Vx i / R

Where, R is the experimental error (mean square for error), Vxd is the mean square of dummy variables; Vxi is the mean square of variable and n is number of dummy variables. Verification experiments:

In order to evaluate the accuracy of the applied Plackett-Burman design, the predicted optimum levels of the independent variables, for high biomass and/or lipid production, were used to prepare two verification media, one for maximum biomass product ion (A) and another one for maximum lipid product ion (B). The prepared media were inoculated with three days fresh cultures to obtain initial concentration of 5000 cel l/ml. The culture flasks were incubated at 25 ± 2oC under continuous light intensi ty of 51.35 (± 2.7) µmol photons m -2 s- 1 for seven days. The biomass and l ipid content were determined at the day five and seven of the incubation period. The verif ication experiment was carried out in triplicates. Estimation of lipid profiles:

The chemical composition of the lipids extracted from the algal biomass cultured in the two verification media for five days were estimated by Gas chromatography and mass spectroscopy (GC-MS) and compared with that of the control medium. The transesterification method of McDonough et a l. (1999) was used to convert fatty acids to fatty acids methyl esters (FAME). The FAME profi les were determined using an Agilent mass spectrometric detector, with a direct capill ary interface and fused si lica capi llary column PAS- 5 ms (30 mm x 0.25 um film thickness). Samples were injected under the following conditions: Hel ium was used as

carrier gas at approximately 1 ml/min., pulsed spli tless mode. The solvent delay was 3 min. and the inject ion size was 1.0 µL. The mass spectrophotometric detector was operated in electron impact ionization mode ioning energy of 70 e.v. scanning from m/z 50 to 500. The ion source temperature was 230ºC and the quadruple temperature was 150ºC. The electron multiplier voltage (EM voltage) was maintained 1250v above auto tune. The instrument was manually tuned using perfluorotributyl amine (PFTBA). The GC temperature program was started at 60ºC then elevated to 280ºC at rate of 8ºC /min, and 10 min. hold at 280ºC the detector and injector temperature were set at 280ºC and 250ºC, respectively. Wiley 7n and NIST 05 mass spectral data base was used in the identification of the separated peaks.

RESULTS: A glance on figure 2 revealed that

Tryblionella hungarica had a characteristic fast growth, where it completes its growth cycle within less than one week. The calculated division time was about 0.77 d - 1 (= about 18 h). This means that the tested diatom isolate could be doubled in number every 18 h. It i s worth mentioning that, the purpose of using Plackett-Burman design was to screening and select the important nutritional factors affecting biomass and l ipid content of the diatom T. hungarica NIOF DM-017. The biomass and lipid production of the run 0 (the control Navicula medium) was 0.133 gL- 1 and 21.4% (Table 2). For the other experimental runs, a very high significant (P ≤ 0.001) increase in biomass production was recorded in the runs no. 11 (0.274 gL - 1) and 12 (0.268 gL -1). Meanwhile, the experimental runs 4, 6, and 7 attained almost non-significant (P ≤ 0.05) increase i n biomass production. The biomass in these runs was fluctuated between 0.142 and 0.145 gL - 1, whi le the experimental run 8 exhibited a very poor growth of 0.032 gL - 1 (Table 2). The tested diatom was not able to grow in the medium runs 3, 5, 9, and 10.

0 2 4 6 8 1010000

100000

1000000

1E7

Alg

al c

ell c

ount

(ml-1

)

Tim

e (d

ays)

Cell count Division time

Incubation time (Days)Division time

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Fig. 2. Growth curve and division time of T. hungarica NIOF-DM-017.



45

Concerning l ipid production, non-significant differences (P ≤ 0.05) in l ipid content were recorded between the experimental runs no. 1, 2, 4, 11, and 12, where the l ipid content fluctuated between 18.0 and 20% (Table 2). However, the experimental runs 6 and 7 showed a significant decrease (P ≤ 0.05) in lipid production. The minimum lipid production with very high significant decrease (P ≤ 0.001) was recorded within the experimental run 8 “11.6%” (Table 2).

Regarding the contribution of the tested variables to biomass and li pid production, some variables showed positi ve, whereas main effects showed negative effects on biomass and/or l ipid production (Table. 3). As it was i llustrated in table 3, the experimental error found to be 0.0005 in case of algal biomass and the effect of the dummy factor was - 0.023, which indicates successful experimental work. The posi tive main effect of the variables; Ca(NO3)2.4H2O, MgSO4.7H2O, NaSiO3.9H2O, NaCO3 and FeCl3.6H2O indicate that the maximum biomass production of tested diatom T. hungarica require higher concentrations from these components, than the concentrations used in the Plackett-Burman design (Table 3). In contrast, the negati ve main effect of K2HPO4.3H2O, Na2EDTA.2H2O, pH, NaCl and glucose, indicate that the tested diatom required concentrations lower than that used in the design. Among the screened variables, Ca(NO3)2.4H 2O, NaCO3, FeCl 3.6H2O and pH found to had posi tive main effects on lipid production; while K2HPO4.3H2O, MgSO4.7H2O, NaSiO3.9H2O, Na2EDTA.2H2O, NaCl and glucose had negative effects (Table 3). The high F value of most of the tested medium components revealed that these components contribute largely for the biomass and lipids production (Table 3). The contribution of MgSO4.7H2O and glucose to biomass production and FeCl3.6H2O to l ipid production were very low (Table 3). Table 3. Influence of medium variables on biomass and lipid

content of T. hungarica NIOF-DM-017. Main effect F value Error = R

Variables Biomass Lipids Biomass Lipids Biomass Lipids

Ca(NO3)2.4H2O 0.103 7.53 120.3 317.7 K2HPO4.3H2O -0.042 - 7.60 19.59 323.7 MgSO4.7H2O 0.007 - 1.69 0.519 16.16 Na2SiO3.9H2O 0.048 - 0.99 26.97 5.582 Na2CO3 0.055 1.37 33.93 10.49

FeCl3.6H2O 0.043 0.26 20.42 0.393 Na.EDTA.2H2O -0.126 - 11.3 180.1 715.1 pH -0.018 4.19 4.387 98.72 Glucose -0.003 - 5.67 0.095 180.0 NaCl -0.026 - 1.27 7.667 8.96 Dummy -0.023 - 1.04 6.0 6.0

0.0005 1.07

In order to evaluate the accuracy of the applied Plackett-Burman design, a verification media for maximum biomass and l ipid

product ion were applied to compare between the predicted optimum levels of independent variables. Form the veri fication medium (A), it was found that the biomass product ion, after five days of growth, increased to 0.247 gL - 1 with 85.7% increase when compared to the production at control medium (0.133 gL- 1) and lipid content increased to 30.6% with 42.9% increase when compared to its production at control medium about 21.5% (Fig. 3a). Meanwhile, the verification medium (B) results in increase in biomass with 30.8% and l ipid content with 61.2% when compared to its production at control medium (Fig. 3b). . A noticeable decl ine in biomass and l ipid content at day seven of the incubat ion period was evident in the two verifi cation medium (Fig. 3). From al l the previous results, the composition of the predicted optimized medium for high biomass production suggested as follows (g/l ): Ca(NO3)2.4H2O (0.3), K2HPO4.3H2O (0.07), MgSO4.7H2O (0.075), NaSiO3.9H2O (0.3), Na2CO3 (0.06), FeCl3.6H2O (0.015), Na.EDTA.2H2O (0.015), NaCl (0.33), glucose (1.5) and pH (6.5), while the predicted opt imized medium for high l ipid production is as follows (gL -1): Ca(NO3)2.4H2O (0.3), K2HPO4.3H2O (0.07), MgSO4.7H2O (0.0125), NaSiO3.9H2O (0.05), Na2CO3 (0.06), FeCl3.6H2O (0.015), Na.EDTA.2H2O (0.015), NaCl (0.33), glucose (1.5) and pH (8.5).

0.000.020.040.060.080.100.120.140.160.180.200.220.240.260.280.30

Bio

mas

s pr

oduc

tion

(g. L

-1)

Biomass Lipids a

0246810121416182022242628303234363840

Day 7

Verification medium A

Lipi

d co

nten

t (%

)Control medium Day 5

a

0.000.020.040.060.080.100.120.140.160.180.200.220.240.260.280.30

Bio

mas

s pr

oduc

tion

(g. L

-1)

Biomass Lipids b

0246810121416182022242628303234363840

Day 7Verification medium B

Li

pid

cont

ent (

%)

Control medium Day 5

b Fig. 3 (a & b). Effect of verification media (A) and (B) on

biomass and lipid content of T. hungarica NIOF-DM-017.



46

The l ipid composition of T. hungarica cultured on Navicula medium and the veri fication media was mainly composed of fatty acids and hydrocarbons (Table 4). The fatty acids comprise about 53% of the total lipids of the tested diatom cultured in control medium, out of them, 21.5% saturated fatty acids and 31.8% unsaturated fatty acids (Table 4). A slight increase in the percentage of fatty acids in T. hungarica l ipids cul tured on the verificat ion media was recorded. For the test alga cultured in verification medium for high biomass, the fatty acids was about 64% of the total lipids of the test diatom, out

of them, 26.3% saturated fatty acids and 37.5% unsaturated fatty acids, whi le in the verif ication medium for high lipids, the fatty acids was about 59.4% of the total l ipids of the test diatom, out of them, 22.4% saturated fatty acids and 37% unsaturated fatty acids (Table 4). In general, non-considerable changes were recorded in fatty acids profil e between the control and verificat ion media. The FAMEs were dominated by methyl palmiteate (C16: 0), methyl palmitoleate (C16:1) and methyl li noleate myristate (C18: 2) (Table 4).

Table 4. Lipid composition at day 5 of Tryblionella hungarica NIOF-DM-017 cultured in control Navicula medium and two verification media

Lipid composition Structure M. wt. Control medium

Verification medium for biomass

Verification medium for lipids

Fatty acids: 16: 1 C17H32O2 268 10.14 8.56 7.56 16: 0 C17H34O2 270 18.69 21.49 17.96 18: 1 C18H34O2 282 5.76 n.d* 4.04 18: 2 C19H34O2 294 3.81 8.19 10.59 20: 5 C21H32O2 316 6.19 7.68 6.62 21: 1 C22H42O2 338 5.95 13.14 8.14 29: 0 C30H60O2 480 2.78 4.85 4.47 Hydrocarbons: Dodecane C12H26 170 n.d n.d 0.67 Hexadecane C16H34 226 1.43 n.d 2.22 Heptadecane C17H36 240 1.52 n.d 12.2 Octadecane C18H38 254 n.d 2.113 3.45 Nonadecene C19H38 266 1.91 n.d 6.08 Eicosane C20H42 282 9.62 8.505 0.67 Heneicosane C21H44 296 n.d 0.722 n.d Tricosane C23H48 324 n.d 1.598 1.68 Tetracosane C24H50 338 8.62 3.454 6.73 Pentacosane C25H52 352 3.43 7.732 2.46 Hexacosane C26H54 366 9.52 n.d n.d Hexatriacontane C36H74 506 6.43 5.825 2.39 Saturated fatty acids: 21.47 26.34 22.43 Monounsaturated fatty acids 21.85 21.7 19.74 Polyunsaturated fatty acids 10 15.87 17.21 Total fatty acids: 53.32 63.91 59.38 Total unsaturated fatty acids: 31.85 37.57 36.95 Total hydrocarbons 42.48 29.949 38.55

*n.d = not detectable

DISCUSSION: Feasible and sustainable production of

biodiesel from microalgae has become in a focus of immense and intense global concern. Many studies highlighted the importance of biomass of certain microalgae, including diatoms, as reliable feedstock for biodiesel production (Sheehan et al., 1998; Chisti, 2008; Mata et al., 2010). Although it became evident that microalgae represent a renewable resources of biodiesel that maintains the potential to completely displace fossil diesel, yet the commercial production is still far away from reality (Chisti, 2007). One of the major objectives of the present study was to evaluate the potentiality of the diatom T. hungarica as reliable feedstock of biodiesel. Diatoms can grow extremely rapidly compared with other oil crops; they can double their biomass within 5 h to 24 h (Ramachandra et al., 2009). The growth characteristics of the tested diatom are in agree with the growth rates of the diatoms studied by

Gilstad and Sakshaug (1990) who recorded that the division time of some studied diatoms fluctuated between 0.41 d -1 (Amphiprora sp.) to 0.60 d- 1 (Thalassiosira antarctica and T. bulbosa). Many studies revealed that microalgae tend to grow and build up biomass under optimal growth conditions, but this is not necessary accompanied with production of high lipid contents (Chisti, 2007; Sharma et al., 2012; Jiang et al., 2014). This may explain the very high significant (P ≤ 0.001) increase in biomass production in media runs 11 and 12, in which 106% and 101.5% increase were recorded (Table 2) without any considerable changes in lipid contents. The growth inhibition of the tested diatom in the medium runs 3, 5, 9, and 10 could be attributed to the high Na2.EDTA.2H2O concentration in these runs of media, which may explained by the following suggestion: The ligand nature of EDTA could be results in decreasing the availabil ity of some



47

elements, especially iron, to the algal cell when EDTA concentration is high (Gerringa et al., 2000; Matz et al. , 2004). Also, the high calculated F values of 180.1 and 715.1 (Table 3) which indicate that EDTA had great effect on biomass and lipid production. Further more, the negative main effect (- 0.126 and - 11.3) of Na2.EDTA.2H2O indicates that this medium component is required, by the tested alga, in concentrations lower than the concentration used in these runs.

Media composition and growth conditions influence the growth and thus the biomass and lipid content. Many studies reported that the limitation of some nutrients (e.g., N, Si, C, P, iron and salinity), osmotic stress, radiation, pH, temperature and heavy metals affect the biomass and lipid accumulation of algae (Taguchi et al., 1987; Hu, 2004; Griffiths and Harrison, 2009; Gardner et al., 2011; Ruangsomboon et al., 2012). The effect of N, P, Mg, Si, iron and pH concentrations on the tested diatom are evident. Hu (2004) reported that an increase in salinity may result in slightly increase in total lipid content of certain microalgae, but the excess salinity has a negative effect on growth of originally fresh water microalgae. These results agreed with the results obtained in this study, in which the tested diatom needs low concentrations of NaCl for both biomass and lipid production.

Many previous studies indicate that glucose is the most common organic carbon source that widely used in mixotrophic algal cultures and the highest lipid productivities of Chlorella, Nannochloropsis and Scenedesmus species were attained when their mixotrophic cultures were supplemented with glucose as an organic carbon source (Marquez et al., 1995; Liang et al., 2009; Zhao et al., 2012). In this context, many investigators (Cheirsilp and Torpee, 2012; Wang et al. , 2012; Zhao et al., 2012) reported that biomass and lipid production of mixotrophic cultures of many green microalgae are attained at relatively lower glucose concentrations of 1.25 - 1.5 g glucose l -

1. These results are in accordance with our results, which indicated that the tested diatom T. hungarica recoded to need 1.5 g glucose l -1

for high biomass and lipid production. Chisti (2007) estimated that an algal species needs 30% lipid per dry weight in order to be a possible candidate for biofuel production. Diatoms on average have 37.8% lipid per dry weight when grown under nutrient deplete conditions (Hu et al., 2008). Therefore, based on lipid percentage alone, the two prepared verification media for maximum biomass and lipids achieves lipid content greater than 30%. These results could make T. hungarica a possible candidate for biodiesel production.

Concerning lipid profile, microalgae reported to synthesize fatty acids suitable for conversion into biodiesel. These fatty acids

include medium-chain (C10–C14), long-chain (C16–18) and very-long-chain (C20), which may be saturated or unsaturated (Hu et al., 2008). In general, saturated and mono-unsaturated fatty acids are predominant in most algal species (Borowitzka, 1988). For bacillariophyceae, the major fatty acids are C16: 0, C16: 1 and the polyunsaturated fatty acids (PUFAs), especially (C20: 5) and (C22: 6) (Basova, 2005). The chemical composition of the lipids extracted from T. hungarica cultured in the control Navicula medium and the two veri fication medium seems to an extent similar fatty acids profile reported by Basova (2005), where C16: 0, C16: 1 were the dominant fatty acids and comprise about 54, 47, and 43% of the total fatty acids of T. hungarica cultured in the control Navicula medium, verification medium for high biomass and verification medium for high lipids, respectively.

Besides the high saturated and monounsaturated fatty acid content in T. hungarica, there is an abundance of eicosapentaenoic acid, methyl ester (20: 5) (polyunsaturated fatty acids), where it represent diatoms 6.19, 7.68, and 6.62% of lipids of T. hungarica cultured in the control Navicula medium, verification medium for high biomass and verification medium for high lipids. In this context, Renaud et al. (1994) and Chen et al. (2007) reported diatoms as a potential of eicosapentaenoic acid, for example Nitzschia laev is. In general, both TAGs and long chain fatty acids comprise ideal feedstock of biodiesel when transesterified to the corresponding methyl esters (Laurens et al. , 2012). The high hydrocarbon content in T. hungarica l ipids indicated that it is possible feedstock for hydrocarbon fuels (e.g. Jet fuel). The biosynthesize of hydrocarbons by diatoms was reported by many scientists (Belt et al., 2001 & 2006; Sinninghe Damste´ et al., 2004). Studies of Largeau et al. (1980) and Templier et al. (1984) indicated that palmitic acid and oleic acid are significant precursors of hydrocarbons.

CONCLUSIONS: Screening the nutrient factors affecting

the biomass and lipid content of the diatom Tryblionella hungarica NIOF-DM-017 by using the Plackett-Burman experimental design revealed that most of the variables contributed largely to biomass and/or lipid production in this diatom. The effect of the different medium runs on biomass and lipid production was evident. The verification experiments revealed the accuracy of the applied Plackett-Burman design. The biomass was increased by 85.7% and lipids by 42.9% compared to the production at control medium. Tryblionella hungarica NIOF-DM-017 show to be very rich in C16 and C18 fatty acids and hydrocarbons, thus it is a suitable feedstock for biodiesel and hydrocarbon fuel production.



48

REFERENCES: Abedin RMA, Taha HM. 2008. Antibacterial and

antifungal act ivity of cyanobacteria and green microalgae. Evaluat ion of media components by Plackett-Burman design f or ant imicrobial activity of Spirulina platensis. Global J. Biotechnol. Biochem. 3(1): 22-31.

APHA. 2005. Standard methods for the examinat ion of water and wastewater. 21st ed. American public health association, 800 I Street, NW , W ashington, DC 20001- 3710.

Bajhaiya AK, Mandotra SK, Suseela MR, Toppo K, Ranade S. 2010. Algal Biodiesel: the next generation biofuel for India. Asian J. Exp. Biol. Sci., 1(4): 728-739.

Bala BK. 2006. Studies on biodiesel from transformation of vegetable oils f or diesel engines. Energy Educ. Sci. Technol., 15: 1-45.

Basova MM. 2005. Fatty ac id composit ion of lipids in microalgae. Int. J. Algae, 7: 33–57.

Becker W. 2004. Microalgae in human and animal nutrit ion. In: “Handbook of microalgal culture: biotechnology and applied phycology. (Richmond A. Ed.)”. Blackwell Sc ience, Oxf ord, pp. 312–351

Belt ST, Allard WG, Masse´ G, Robert JM, Rowland SJ. 2001. Structural characterisat ion of C30 highly branched isoprenoid alkenes (rhizenes) in the marine diatom Rhizosolenia setigera. Tetrahedron Lett., 42(32): 5583–5585.

Belt ST, Massé G, Rowland SJ, Rohmer M. 2006. Highly branched isoprenoid alcohols and epoxides in the diatom Haslea ostrearia Simonsen. Org. Geochem., 37(2): 133–145.

Bligh EG, Dyer W J. 1959. A rapid method of total l ipid extraction and purif ication. Can. J. Biochem. Physiol., 37(8): 911–917.

Borowitzka MA. 1988. Vitamins and f ine chemicals. In: “Micro-algal Biotechnology. (Borowitzka MA, Borowitzka LJ. eds.)”. Cambridge Univers ity Press: Cambridge, pp. 153-196.

Chagoya J, Brown J, Gomez M, Zhang J, Jiang Y, Laverty K, Brown L, Quigg A, Burow M. 2014. Media optimization and lipid formation of two native diatoms for cultivat ion in the Southwest Texas desert. J. Appl. Phycol., 26(5): 2075-2085.

Cheirsilp B, Torpee S. 2012. Enhanced growth and lipid production of microalgae under mixotrophic culture condit ion: Effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour. Technol., 110: 510–516.

Chen GQ, Jiang Y, Chen F. 2007. Fatty acid and lipid class composition of the eicosapentaenoic acid-producing microalga Nitzschia laevis. Food Chem., 104(4): 1580–1585.

Chist i Y. 2007. Biodiesel from microalgae. Biotechnol. Adv. 25(3): 294-306.

Chist i Y. 2008. Biodiesel from microalgae beats bioethanol. Trend. Biotechnol., 26(3): 126-131.

Demirbas A. 2009. Production of biodiesel from algae oils. Energy Source A, 31(2): 163-168.

EIA. 1998. Annual energy out look 1999, with projections to 2020, in DOE/EIA-0383. Energy Information Administration, Department of Energy, W ashington, DC, USA.

Fields M, Hise A, Lohman E, Bell T, Gardner R,

Corredor L, Moll K, Peyton B, Characklis G, Gerlach R. 2014. Sources and resources: Importance of nutr ients, resource allocation, and ecology in microalgal cult ivation for lipid accumulation. Appl. Microbiol. Biotechnol., 98(11): 4805–4816.

Foss i B T, Tavea F, Ndjouenkeu R. 2005. Production and part ial characterizat ion of a thermostable amylase from ascomycetes yeast strain isolated form starchy soil. Afr. J. Biotechnol., 4(1): 14-18.

Gardner R, Peters P, Peyton B, Cooksey KE. 2011. Medium pH and nitrate concentration effects on accumulation of tr iacylglycerol in two members of the chlorophyta. J. Appl. Phycol., 23(6): 1005–1016.

Gerringa LJA, Baar HJW, Timmermans KR. 2000. A comparison of iron limitation of phytoplankton in natural oceanic waters and laboratory media conditioned with EDTA. Mar. Chem. 68: 335–346.

Gilstad M, Sakshaug E. 1990. Growth rates of ten diatom species from the Barents Sea at different irradiances and day Lengths. Mar. Ecol. Prog. Ser., 64: 169-173.

Griff iths MJ, Harr ison STL. 2009. Lipid productivity as a key characterist ic for choosing algal species for biodiesel production. J. Appl. Phycol., 21(5): 493–507.

Guil lard RRL. 1973. D ivis ion rates. Handbook of Phycological Methods. Cambridge University Press, Cambridge, pp. 320.

Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A. 2008. Microalgal triacylglycerols as feedstock for biofuel production: Perspect ives and advances. Plant J., 54(4): 621-639.

Hu Q. 2004. Environmental effects on cell composit ion. In: “Handbook of microalgal culture: biotechnology and applied phycology. (Richmond A. Ed.)”. Blackwell, Victor ia, pp. 83–93.

Il lman AM, Scragg AH, Shales SW . 2000. Increase in Chlorella strains calor if ic values when grown in low nitrogen medium. Enzyme Microb. Tech., 27(8): 631-635.

Jiang Y, Laverty K S, Brown J, Nunez M, Brown L, Chagoya J, Burow M, Quigg A. 2014. Effects of f luctuating temperature and silicate supply on the growth, biochemical composit ion and lipid accumulat ion of Nitzschia sp. Bioresour. Technol., 154: 336–344.

Kociolek P. 2011. Tryblionella calida. In Diatoms of the United States. Retr ieved May 24, 2015, from http://westerndiatoms. colorado.edu/ taxa/species/tryblionella_calida.

Kojima E, Zhang K. 1999. Growth and hydrocarbon production of microalga Botryococcus braunii in Bubble column photobioreactors. J. Biosc ienc. Bioeng., 87(6): 811-815.

Largeau C, Casadevall E, Berkaloff C, Dhamelincourt P. 1980. Sites of accumulation and composit ion of hydrocarbons in Botryococcus braunii. Phytochemistry, 19(6): 1043–1051.

Laurens L, ML, Quinn M, W ychen VS, Templeton WD, Wolfrum JE. 2012. Accurate and reliable



49

quantif ication of total microalgal fuel potential as fatty ac id methyl esters by in situ transesterif ication. Anal. Bioanal. Chem., 403(1): 167–178.

Li Y, Liu Z, Cui F, Liu Z. 2007. Application of Plackett-Burman experimental des ign and Doehlert design to evaluate nutr it ional requirements for xylanase product ion by Alternaria mali ND-16. J. Appl. Microbiol. Biotechnol., 77(2): 285-291.

Liang YN, Sarkany N, Cui Y. 2009. Biomass and lipid productivit ies of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnol. Lett., 31(7): 1043–1049.

Liu ZY, Wang GC, Zhou BC. 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresourc. Technol., 99(11): 4717-4722.

Lynn SG, Kilham SS, Kreeger DA, Interlandi SJ. 2000. Effect of nutr ient availability on the biochemical and elemental stoichiometry in freshwater diatom Stephanodiscus minutulus (Bacillar iophyceae). J. Phycol., 36(3): 510-522.

Marquez FJ, Nishio N, Nagai S, Sasaki K. 1995. Enhancement of biomass and pigment production during growth of Spirulina platensis in mixotrophic culture. J. Chem. Technol. Biot., 62(2): 159–164.

Mata TM, Mart ins AA, Caetano NS. 2010. Microalgae for biodiesel production and other applications, Renew. Sustain. Energ. Rev., 14(1): 217-232.

Matz CJ, Christensen MR, Bone AD, Gress CD, Widenmaier SB, Weger HG. 2004. Only iron-l imited cells of the cyanobacter ium Anabaena f los-aquae inhibit growth of the green alga Chlamydomonas reinhardtii. Can. J. Bot., 82(4): 436–442.

McDonough LP, Fogelman D, Shin JS, Brunner AM, Lein HD. 1999. Salmonella enterica Serotype Dublin Infection: an Emerging Infectious Disease for the Northeastern United States. J. Clin. Microbiol., 37(8): 2418-2427.

Plackett RL, Burman JP. 1946. The design of optimum mult ifactor ial experiments. Biometr ika, 33(4): 305-325.

Ramachandra TV, Mahapatra DM, Karthick B. 2009. Milking diatoms for sustainable energy: biochemical engineering versus gasoline-secreting diatom solar panels. Ind. Eng. Chem. Res., 48(19): 8769–8788.

Rao RA, Dayananda C, Sarada R, Shamala TR, Ravishankar, GA. 2007. Effect of salinity on growth of green alga Botryococcus braunii and its constituents. Bioresour. Technol., 98(3): 560–564.

Reitan KI, Rainuzzo JR, Olsen Y. 1994. Ef fect of nutrient limitat ion of fatty ac id and lipid content of marine microalgae. J. Phycol., 30: 972-979

Renaud SM, Parry DL, Thinh LV. 1994. Microalgae for use in tropical aquaculture I: Gross chemical composit ion and fatty acid composit ion of twelve species of microalgae f rom the Northern Terr itory, Australia. J. Appl. Phycol., 6(3): 337–345.

Richardson B, Orcutt DM, Schwertner HA, Martinez CL, W ickline HE. 1969. Ef fects of nitrogen

limitat ion on the growth and composit ion of unicellular algae in continuous culture. Appl. Microbiol., 18(2): 245-250.

Ruangsomboon S. 2012. Ef fect of light, nutrient, cultivation t ime and salinity on l ipid production of newly isolated strain of the green microalga, Botryococcus braunii KMITL 2. Bioresource Technol., 109: 261–265.

Samorì C, Torr i C, Samorì G, Fabbri D, Gallett i P, Guerr ini F, Pistocchi R, Tagliavini E. 2010. Extract ion of hydrocarbons from microalga Botryococcus braunii with switchable solvents. Bioresource Technol., 101(9): 3274 -3279.

Schoeman FR, Archibald REM. 1976. The diatom flora of southern Africa. Pretoria: C.S.I.R. Special Report WAT 50.

Sharma KK, Schuhmann H, Schenk PM. 2012. High lipid induction in microalgae for biodiesel production. Energies, 5(5): 1532-1553.

Sheehan J, Dunahay T, Benemann J., Roessler P. 1998. A look back at the U.S. Department of Energy’s Aquatic Species Program: biodiesel from algae. Golden, Colorado, pp. 325.

Sinninghe Damsté JS, Muyzer G, Abbas B, Rampen SW, Massé G, Allard W G, Belt ST, Robert JM, Rowland SJ, Moldowan JM, Barbant i SM, Fago FJ, Denisevich P, DahlJ, Trindade LAF, Schouten S. 2004. The r ise of the rhizosolenid diatoms. Science, 304(5670): 584–587.

Starr RC. 1978. The Culture Collection of Algae at The University of Texas at Austin. J. Phycol., 14(suppl.): 47-100.

Stein JR. 1973. Hand book of phycological methods, culture methods and growth measurements. Cambridge at the University Press, pp. 417.

Sumper M, Brunner E. 2008. Silica biomineralisation in diatoms: the model organism Thalass ios ira pseudonana. Chembiochem, 9(8): 1187–1194.

Taguchi S, Hirata JA, Laws EA. 1987. Silicate defic iency and lipid synthesis of marine diatoms. J. Phycol., 23(Supll. S2): 260-267.

Templier J, Largeau C, Casadevall E. 1984. Mechanism of non-isoprenoid hydrocarbon biosynthesis in Botryococcus braunii. Phytochemistry, 23: 1017–1028.

Van Der W erff A. 1955. A new method of concentrating and cleaning diatoms and other organisms. Int. Ver. Theor. Angew. Limnol. Verh. 12: 276–277.

Vasudevan PT, Br iggs M. 2008. Biodiesel production--current state of the art and challenges. J. Ind. Microbiol. Biotech., 35(5): 421-430.

W ang H, Fu R, Pei G. 2012. A study on lipid production of the mixotrophic microalgae Phaeodacty lum tr icornutum on various carbon sources. Afr. J. Microbiol. Res., 6(5): 1041-1047.

Yu X, Hallett SG, Sheppard J, Watson AK. 1997. Application of Plackett-Burman experimental design to evaluate nutr itional requirements for the product ion of Colletor ichum coccodes spores. Appl. Microbiol. Biotechnol., 47(3): 301-305.

Zhao G, Yu J, Jiang F, Zhang X, Tan T. 2012. The effect of different trophic modes on l ipid accumulation of Scenedesmus quadricauda. Bioresource Technol., 114: 466–471.



50

دراسة العناصر الغذائیة التي تؤثر على إنتاج الكتلة الحیوية والدھون في طحلب المیاه العذبة

Tryblionella hungarica NFDM-017بإستخدام تصمیم بالكیت بیرمان

**و جیالن مفید* إيمان ابراھیم عبدالعال المعھد القومي لعلوم البحار والمصايد *

ثروة السمكیة، جامعة السويس، السويس، مصرقسم البیئة المائیة، كلیة ال**

وإستخدمت النتائج السابقة في تحضیر %). 21.4(الدھون ) A(وسطین غذائین إحدھما لإلنتاج الكتلي األقصي

Aوجد أن الوسط ). B(واألخري لإلنتاج األعلي من الدھون ٪ والدھون 85.7إنتاج الكتلة الحیوية بنسبة أدي الي زيادة

ادي الي زيادة في الكتلة B ، والوسط ٪42.9بنسبة ٪، بالمقارنة 61.2٪ والمحتوى الدھني 30.8الحیوية بنسبة

للدھون GC/MS وبإستخدام تحالیل. مع الكنترولالمستخلصة من الكتلة الحیوية المزروعة علي األوساط

أساسا من والكنترول وجد أنھا تتكون A ،Bالغذائیة ربونات، وقد مثلت األحماض األحماض الدھنیة والھیدروك

٪ من إجمالي الدھون، 59.4٪ و 64٪، 53الدھنیة حوالي بشكل عام و سجلت االحماض الدھنیة . على التوالي

C16:0 ،C16:1 و C18:2 في دھون األوساط أعلي نسب .الثالثة، مما يجعلھا مصدر مناسب إلنتاج الوقود الحیوي

:المحكمون ان قسم النبات، علوم طنطا محمد األنور حسین عثم.د.أ زينب خلیل قسم النبات، علوم القاھرة .د.أ

يمثل اإلنتاج اإلقتصادى المستدام للكتلة الحیوية في للطحالب الدقیقة العمود الفقري إلنتاج الوقود الحیوي

لتقییم بالكیت بیرمانستخدام تصمیم إھذه الدراسة تم عشرة عناصر غذائیة إلنتاج الكتلة األھمیة النسبیة ل

Tryblionella hungaricaالحیوية والدھون في الدياتوم NIOF-DM-017 . ،ھذه العناصر ھي كربونات الكالسیوم

فوسفات البوتاسیوم، كربونات الصوديوم، كبريتات ، EDTAالماغنسیوم، سلیكات الصوديوم، كلوريد الحديديك،

تم حیث . رجة الحموضةالجلوكوز، كلوريد الصوديوم، ود إثني عشر وسط غذائي، تختلف فیما بینھا في تحضیر

وجد أن معظم العناصر لھا . تركیزات العناصر موضع الدراسةتأثیر علي إنتاج الكتلة الحیوية ومحتوي الدھون في

و أبرزت النتائج المتحصل علیھا ان . الطحلب موضع الدراسةسجلت ) لتر/ جرام0.032(اقل إنتاجیة للكتلة الحیوية

0.274( بینما سجلت أعلي انتاجیة 8في الوسط رقم كما لوحظ إنعدام نمو الطحلب . 11≠ في الوسط ) لتر/جرام

والتي تحتوي علي تركیزات ) 10 و 9، 5، 3(في األوساط وقد سجلت كل االوساط الغذائیة إنخفاض . EDTAعالیة من

الغذائي طفیف في محتوى الدھون مقارنة بالوسط من الجدير بالذكر، أن إنتاجیة الطحلب علي ). كنترول(

ومحتوي لتر/ جرام0.133الوسط الغذائي الكنترول كانت

optimization of medium components for high biomass and lipid production of tryblionella hungarica...

Documents