Neutral Lipid Content and Biomass Production in Skeletonema marinoi (Bacillariophyceae) Culture in Response to Nitrate Limitation

Neutral Lipid Content and Biomass Productionin Skeletonema marinoi (Bacillariophyceae) Culturein Response to Nitrate Limitation

Elena Bertozzini & Luca Galluzzi & Fabio Ricci &Antonella Penna & Mauro Magnani

Received: 11 January 2013 /Accepted: 6 May 2013 /Published online: 29 May 2013# Springer Science+Business Media New York 2013

Abstract Microalgae are one of the most promising biodiesel feedstocks due to theirefficiency in CO2 fixation and high neutral lipid productivity. Nutrient–stress conditions,including nitrogen starvation, enhance neutral lipid content, but at the same time lead to areduction of biomass. To maximize lipid production in the diatom Skeletonema marinoi, weinvestigated two different nitrogen starvation approaches. In the first experimental approach,inocula were effectuated in modified f/2 media with decreasing nitrogen concentration,while in the second experiment, nitrate concentration was gradually reduced through acollection/resuspension system in which the culture was periodically collected andresuspended in culture medium with a lower nitrate concentration. In the first approach,the neutral lipid accumulation was accompanied by a strong biomass reduction, as wasexpected, whereas the second experiment generated cultures with significantly higherneutral lipid content without affecting biomass production. The total proteins and totalcarbohydrates, which were also quantified in both experiments, suggest that in S. marinoi,neutral lipid accumulation during nutrient starvation did not derive from a new carbonpartition of accumulated carbohydrates.

Keywords Microalgae . Biofuel . Nitrogen depletion . Neutral lipid content . Growth strategy

Appl Biochem Biotechnol (2013) 170:1624–1636DOI 10.1007/s12010-013-0290-3

Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0290-3)contains supplementary material, which is available to authorized users.

E. Bertozzini (*) : L. GalluzziDepartment of Biomolecular Sciences, Biotechnology Section, University of Urbino,via Campanella 1, 61032 Fano, PU, Italye-mail: [email protected]

F. Ricci :A. PennaDepartment of Biomolecular Sciences, Environmental Biology Section, University of Urbino,viale Trieste, 296, 61032 Pesaro, PU, Italy

M. MagnaniDepartment of Biomolecular Sciences, University of Urbino,via Saffi 2, 61029 Urbino, PU, Italy

http://dx.doi.org/10.1007/s12010-013-0290-3

Introduction

Microalgae are highly adaptable unicellular photosynthetic organisms widely represented inall existing earth ecosystems. Due to their high replication rate and suitability for large-scalecultivation, microalgae are currently used in many fields, including the pharmaceutical,cosmetic, and food industries [1, 2]. Since the 1970s, the potential of microalgae as asubstrate for biodiesel production has also been the focus of research [3]. Biodiesel isformed by the fatty acid methyl esthers derived from a transesterification reaction betweentriglycerides and methanol. The chemical and physical qualities of biodiesel are closelyrelated to the properties of its parent oil [4]. Of all the substrates being considered forbiodiesel production, microalgae are the most promising [5–7]. In fact, microalgae have thepotential to produce more oil per acre than any other biodiesel feedstock [8] because they useCO2, water, and sunlight more efficiently than other plants [7]. Moreover, the highlyefficient CO2 consumption of intensive cultivation of microalgae could mitigate the green-house effect, reducing CO2 emission contextually to biodiesel production [9]. Sincemicroalgae can also grow in severe climatic conditions and non-arable land, competitionbetween intensive microalgal cultivation and traditional agriculture would be limited [9].Furthermore, some microalgal species can utilize organic and inorganic N and P fromwastewater [10], reducing the need for freshwater for irrigation. Finally, after oil production,remaining biomass could be used as a fertilizer or to produce bioethanol [11].

Nevertheless, an important parameter that must be taken into account in fuel evaluation is thelife cycle assessment (LCA) defined by the ISO 14040.2 standard. This parameter assesses theenvironmental implications and potential impact of a product from “cradle-to-grave.” Based onthe LCA, the undoubted potential of biodiesel production from microalgae is still a long wayfrom being realized [12]. In particular, the feasibility of microalgal species as a biodieselfeedstock depends on the optimization of biomass and neutral lipid productivity [13]. A carefuloptimization of culture conditions can induce microalgae to produce 60 % of their cellular massin neutral lipids [14]. Significant evidence of increased neutral lipid content in microalgae underculture stress conditions has been described in literature [14, 15], but this increase was alwaysaccompanied by a reduction of biomass productivity. Creating culture conditions that allowmaximal growth and high lipid productivity at the same time represents a significant challenge[16]. Specifically, it has been shown that nitrogen limitation strongly influences lipid metabo-lism in many algae [13, 17], but such limitations have adverse effects on biomass production.

Another interesting aspect related to lipid accumulation during nutrient starvation is theinteraction between the synthesis of lipid derivatives and carbohydrates. In fact, a morecomplete understanding of the regulatory networks that control the partition of carbonbetween lipids and other storage products will be very useful in the metabolic engineeringof algal cells to enhance neutral lipid content [18].

Conflicting data have been reported in literature: in some case, the neutral lipid produc-tion during N starvation has been shown to derive primarily from a de novo synthesis [19];in other cases, for example in diatoms, a conversion of carbon from carbohydrates into lipidshas been shown during silicon deficiency [20].

In the present study, the effects of different nitrogen depletion strategies were investigatedusing the diatom Skeletonema marinoi, very common in the NW Adriatic Sea, as a model[21]. Diatoms are particularly rich in triglycerides [22–24] and are one of the most promisinggroups for biodiesel production [8].

In order to identify the optimal culture conditions to elevate biomass productivity withhigh neutral lipid content, and to investigate the metabolic reassessment of this diatomduring nitrogen starvation, two different approaches were tested. In the first approach, S.

Appl Biochem Biotechnol (2013) 170:1624–1636 1625

marinoi was simply inoculated at different decreasing nitrate concentrations, while in thesecond experiment, nitrogen depletion was gradually introduced.

The results obtained in this work showed that accurate nitrogen depletion strategies canprovide reproducible and high neutral lipid content and biomass in S. marinoi cultures andsuggested that neutral lipid accumulation did not derive from a new carbon partition ofaccumulated carbohydrates in this diatom.

Materials and Methods

Microalgal Culture

Stock cultures of S. marinoi CBA4 were grown in f/2 medium [25, 26] at 21±1 °C, with adark/light cycle of 12:12 h (photosynthetic active radiation=1.5 W/m2). The inocula wereeffectuated at a final concentration of 25,000 cells/ml in a total volume of 600 ml. All culturemedia were prepared using Artificial Seawater Provasoli–McLachlan [25]

Experimental Approach 1: Microalgae Cultivation at Different Nitrogen Concentrations

In the first experimental approach, starting from the standard f/2 composition [25, 26], fourdifferent nitrate concentrations were tested (Table 1). Samples were collected from theinoculum on days 8, 20, and 15 for the evaluation of biomass, neutral lipid, total carbohy-drate, total protein, and N-NO3 concentrations.

Experimental Approach 2: Microalgae Adaptation at Low Nitrogen Concentrations

In the second experimental approach, cultures were centrifuged at 2,000×g for 5 min every 3or 4 days in order to remove the culture medium and resuspended in f/2 medium with halvednitrate concentration, maintaining the same cell concentration. For this experiment, twodifferent control cultures were considered: The first control was a S. marinoi CBA4 culturethat was collected every 3 or 4 days (as the sample culture), but resuspended in standard f/2medium (control), while a second control was a standard S. marinoi CBA4 culture inocu-lated and maintained in standard f/2 medium for the same time period as the other cultureswithout manipulation (standard culture). Starting from day 7, at each dilution, samples forneutral lipid, total protein, and total carbohydrate determination were collected. Biomassanalysis was also performed on day 3 to verify that a successful inoculum occurred.

Fluorometric Determination of Neutral Lipid Content

At each time point, three culture aliquots of 1.90 ml were collected and centrifuged at3,500×g for 20 min. The analysis of neutral lipid content was performed on the pellets using

Table 1 Nitrate concentration ofthe different modified f/2 culturemedia used in the “ExperimentalApproach 1: Microalgae Cultivationat Different Nitrogen Concentra-tions.” Other components were ac-cordingly with Guillard and Ryther[26] and Guillard [25]

Sample Final nitrate concentration (μM)

f/2 883.00 [25, 26]

1/2 NO3 441.50

1/4 NO3 220.75

No NO3 Not detectable

1626 Appl Biochem Biotechnol (2013) 170:1624–1636

the fluorescent stain Nile red in association with the standard addition method as previouslydescribed [27]. The spectrofluorometric determinations were performed using aspectrofluorophotometer RF-5301PC (Shimadzu, Japan).

Biomass Determination

Biomass values, expressed as cell concentration, were determined on three replicates of 1 mlculture through spectrophotometric analysis (spectrophotometer UV-2401 PC, Shimadzu,Japan), after identification of a linear correlation between optical density (OD) at 678 nmand cell concentration [28]. This correlation was determined by plotting the OD values ofdifferent aliquots of S. marinoi cultures with the corresponding cell concentrations obtainedusing a hemocytometer. The correlation was tested in a range from 1×107 to 1.5×109 cells/l(R2=0.991; Supplementary information Fig. 1). Both hemocytometer analysis and ODanalysis were performed in triplicate. For spectrophotometric analysis, filtered culture mediawere used as a blank.

In order to express biomass content as milligrams per liter, cell concentrations weremultiplied by 50 pg, the dry weight of the S. marinoi cell [29].

Total Protein Determination

At each time point, culture aliquots (5–10 ml) were collected on Isopore filter membranes(Millipore®) or centrifuged for 20 min at 2,200×g at 15 °C and maintained at −20 °C untilthe day of analysis.

Each filter/pellet was washed/resuspended in 1 ml of Milli-Q water, and protein analysiswas performed on 100 μl of sample according to the Peterson method [30]. Serial dilution ofBSA was used for standardization.

Total Carbohydrate Determination

At each time point, culture aliquots (5–10 ml) were collected on Isopore filter membranes(Millipore®) or centrifuged for 20 min at 2,200×g at 15 °C and maintained at −20 °C untilthe day of analysis.

Each filter/pellet was washed/resuspended in 4 ml of 0.5 M H2SO4 and incubated for 4 hat 100 °C for complex carbohydrate reduction. Reduced samples were centrifuged for 5 minat 2,000×g, and total carbohydrate analysis was performed on 1 ml of surnatant according tothe DuBois protocol [31]. Serial dilution of glucose was used for standard curve.

N-NO3 determination

Chemical analysis of dissolved inorganic nitrate (N-NO3) in culture supernatants wasperformed using the method of Parsons [32].

Statistics

Each culture condition was analyzed on three biological replicates. Reported values arealways the means ± standard deviation of three values. The statistical significance of theresults was tested with the Student–Newman–Keuls multiple comparative test (GraphPadInStat 3.06 version).


Results and Discussion

Microalgae Cultivation at Different Nitrogen Concentrations

In this experimental approach, four parallel inocula were performed in four different culturemedia with decreasing nitrate concentrations (Table 1). N-NO3 determination confirmed thereduced nitrogen concentration in N-depleted samples (Fig. 1).

The reduction of nitrate concentration in culture media induced a significant decrease inbiomass production (p<0.05 or p<0.001) (Fig. 2a). Specifically, when no nitrate was addedto the medium, the biomass reduction ranged from 63 % (day 8) to 89 % (day 15) comparedto the f/2 medium. Under the other conditions, in which nitrate concentration was below f/2levels, the biomass reduction was not as marked (between 11 and 18 %) but equallysignificant at days 8 and 15 (p<0.01 and p<0.05).

The fluorometric quantification of neutral lipid content showed that when nitrate con-centration is reduced to 1/2 and 1/4, no significant differences were recorded either in termsof milligrams per liter or as percentage of dry weight (Figs. 2b and 3a). Specifically, nitrogenanalysis showed that the neutral lipid accumulation was mostly affected by nitrate concen-tration below 40 μM (Fig. 1). In the case of total absence of nitrate in culture medium, theobserved negative effects on biomass also caused a very significant reduction in total neutrallipid concentration (p<0.001).

Nevertheless, although the neutral lipid content of the culture with no NO3, expressed asmilligrams per liter of culture, is always very significantly lower than that found in the otherculture conditions, the percentage of neutral lipid content per cell, in absence of NO3, is verysignificantly higher (p<0.001) than in the other culture conditions, specifically from 30 to64 % higher (Fig. 3a).

These data confirm that stress conditions enhance neutral lipid content but, as side effect,a decrease in biomass production can be rescountered [16, 33, 34].

Regarding carbohydrate content, all cultures showed an increase in carbohydrate contentfrom day 8 to day 15; however, the culture with no NO3 showed a significantly higher valuethan the other cultures (Fig. 3b) throughout the experiment. Moreover, on day 15, the 1/4NO3 culture also reached a higher carbohydrate content per cell than the f/2 and 1/2 NO3

Fig. 1 Nitrogen (N-NO3) concentrations on days 8, 10, and 15 from the inoculum on the supernatants pool ofS. marinoi CBA4 cultures maintained under different culture conditions (see “Materials and Methods”).Reported values were obtained from a pool of supernatants from three parallel inocula


cultures (p<0.05 and p<0.01, respectively). Many algae, in particular Chlorophyta, accu-mulate carbohydrate under nitrogen-deficient conditions [35] or, more generally, underconditions of stress. Regarding diatoms, starch accumulation was recorded in silicon-deficient conditions [20]. The results obtained in this experiment suggest that in the diatomS. marinoi, low levels of nitrate in culture media induce carbohydrate accumulation.

The total protein content per cell in the culture with no NO3 was from 70 to 80 % lowerthan that found under the other condition (Fig. 3c) suggesting a strong decrease in cellmetabolism.

Fig. 2 Biomass (a) and neutral lipid content (b) on days 8, 10, and 15 from the inoculum in S. marinoi CBA4cultures grown with different nitrate concentrations. Represented values are means ± standard deviation ofthree replicates. *p<0.05, significantly different from f/2; **p<0.01, very significantly different from f/2;***p<0.001, extremely significantly different from f/2

Fig. 3 Neutral lipid (a), total carbohydrate (b), and total protein (c) contents per cell on days 8, 10, and 15from the inoculum in S. marinoi CBA4 cultures grown with different nitrate concentrations, expressed aspercentage of dry weight. Represented values are means ± standard deviation of three replicates. *p<0.05,significantly different from f/2; **p<0.01, very significantly different from f/2; ***p<0.001, extremelysignificantly different from f/2

b



Microalgae Adaptation at Low Nitrogen Concentrations

In the second experimental approach, the S. marinoi culture was gradually adapted to lownitrogen concentration (NS culture), while in the control culture, nitrate concentration wasalways maintained at f/2 level. Finally, a standard culture was grown without manipulationfor the same time period as an additional control (see “Materials and Methods”).

N-NO3 analyses on the pools of supernatants under each of the different conditions wereperformed at each time point (Fig. 4). The nitrogen concentration in NS culture decreased, asexpected, at each dilution. In control culture, the N-NO3 concentration maintained very highlevels with a slight decrease in response to microalgae nitrogen consumption. In fact, in thelatest phases of the experiment, when biomass in control culture decreased (Fig. 5a), the N-NO3 concentration slightly increased.

Regarding the standard culture, where the S. marinoi cells were grown without anymanipulation, a gradual decrease in nitrate concentrations was observed. It is noteworthythat the nitrate concentration in the standard culture was never as low as it was in the NSculture.

The biomass trend in the control culture differed from the trends in the other cultures.Specifically, the collection/resuspension procedure without a decrease in nitrate concentra-tion significantly enhanced biomass production in S. marinoi. Furthermore, biomass valuesin the control culture were always significantly higher than those found in the NS andstandard cultures.

The reduction of nitrate concentrations significantly affected biomass in the NS culturefrom day 14 to the end of experiment (Fig. 5a). At this time point, the NS culture entered astationary growth phase where biomass values remained constant with no significantvariation until day 31.

Comparing biomass production in the NS and standard cultures, it can be observed thatwhile the NS culture entered a stationary phase, with no significant differences in biomassfrom day 17 to day 31, the standard culture entered a senescence phase. Specifically, from

Fig. 4 Nitrogen (N-NO3) concentrations of supernatant pool of S. marinoi CBA4 cultures maintained underdifferent culture conditions (see “Materials and Methods”). Reported values were obtained from a pool ofsupernatants from three parallel inocula


day 21 to day 31, the total biomass in the standard culture was reduced three times, and, inthe last phases of the experiment, the NS culture showed significantly higher biomass valuesthan the standard culture. This was probably due to the periodic addition of nutrients duringthe substitution of culture medium. The neutral lipid contents of the three different culturesare shown in Fig. 5b.

At day 21, the standard culture had the highest neutral lipid content, with significantlyhigher values than the control and NS cultures. In fact, it has been shown that even simpleculture aging is correlated to neutral lipid accumulation [36]. Nevertheless, from day 24 to

Fig. 5 Biomass (a) and neutral lipid content (b) in S. marinoi CBA4 cultures maintained in a gradual nitrogendepletion regimen (NS culture). The control was maintained under the same conditions as the NS culture butwithout nitrogen depletion. The standard culture was maintained in standard f/2 medium without manipula-tions for the same time period (see “Materials and Methods”). Represented values are means ± standarddeviation of three biological replicates. *p<0.05; **p<0.01; ***p<0.001



the end of the experiment, the neutral lipid content of the NS culture reached significantlyhigher values than the standard culture and control.

The percentage of neutral lipids compared to the dry weight of the NS culture wassignificantly higher than the control from day 21 (Fig. 6a). At this time, the increase wasonly related to the decrease in nitrate in the culture media, whereas the increase in neutrallipid content in the standard culture could only be attributed to culture aging (includingnutrient consumption). In fact, nitrate concentration was much higher in this culture than inthe NS culture (Fig. 4).

From day 21 until the end of the experiment, there was a sharp increase in neutral lipidcontent in the NS culture (Fig. 6a), due to the combined effect of culture aging and nitrogenstarvation [37].

This growth strategy induced a 200 % increase in neutral lipid content per cell in NSculture in respect to standard culture, allowing to obtain a percentage of neutral lipid per cellfrom the 17 % to the 38 % of dry weight (Fig. 6a).

This increase in neutral lipid content per cell allowed a 220 % increase in neutral lipidconcentration in NS culture, expressed as milligrams per liter, than the standard culture (Fig. 5b).

Carbohydrate contents showed a very similar trend compared to neutral lipid contents(Fig. 6b). Moreover, in the case of carbohydrates, the NS and standard cultures showed asignificantly higher carbohydrate content per cell than the control confirming, as observed inthe first experimental approach, that nitrogen starvation induces carbohydrate accumulationin S. marinoi.

This simultaneous increase in carbohydrate and neutral lipid contents per cell suggests ade novo synthesis of triglycerides under nitrogen starvation and not a new carbon partition ofaccumulated carbohydrates as observed in other strains [17, 34].

An opposite trend was observed regarding protein content (Fig. 6c). In this case, thepercentage of total protein compared to the dry weight of the control culture was alwayssignificantly higher than in the other cultures. These results confirm that the control culturehad a very active metabolism, since the periodic introduction of nutrients maintained thecells in a phase of active reproduction that was reflected in higher biomass values (Fig. 5a).

Conclusions

When grown in nitrogen-depleted conditions (Experimental Approach 1: MicroalgaeCultivation at Different Nitrogen Concentrations), S. marinoi significantly increased itsneutral lipid content per cell only in the total absence of nitrate in the culture media;however, under such conditions, biomass production was strongly inficiated and the neutrallipid content of the culture, expressed as milligrams per liter, was negatively affected.

The second growth strategy tested in this investigation consisted in the periodic collection ofthe S. marinoi culture and its subsequent resuspension in a culture medium in which nitrateconcentration gradually decreased. Using this strategy, biomass was not drastically affected,and, particularly in the last phases of the experiment (from day 24 to day 31), it is likely that theadditional effect of cell aging further increased the neutral lipid content, and triglyceride

Fig. 6 Neutral lipid (a), total carbohydrate (b), and total protein (c) contents per cell in S. marinoi CBA4cultures, expressed as percentage of dry weight, maintained in a gradual nitrogen depletion regimen (NSculture). The control was maintained under the same conditions as the NS culture but without nitrogendepletion. The standard culture was maintained in standard f/2 medium without manipulations for the sametime period (see “Materials and Methods”). Represented values are means ± standard deviation of threebiological replicates. *p<0.05; **p<0.01; ***p<0.001

R


concentration in the NS culture reached the highest level. In conclusion, the second growthstrategy appears to show that the gradual reduction of nitrate concentration in culture media,through the periodic collection and resuspension of the culture, is a good strategy to induceneutral lipid accumulation in the diatom S. marinoi without affecting biomass.

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Neutral Lipid Content and Biomass Production in Skeletonema marinoi (Bacillariophyceae) Culture in Response to Nitrate Limitation