Bioresource Technology 164 (2014) 86–92
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On-line modeling intracellular carbon and energy metabolism of Nannochloropsis sp. in nitrogen-repletion and nitrogen-limitation cultures Dongmei Zhang a,b, Fei Yan a,b,1, Zhongliang Sun a,b, Qinghua Zhang a, Shengzhang Xue a, Wei Cong a,⇑ a b
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China University of Chinese Academy of Sciences, Beijing 100049, China
h i g h l i g h t s On-line monitoring and calculation of carbon and energy metabolism of microalgae. Only 30–60% of carbon fixed in Calvin cycle was used for biomass material. Protein, lipids, carbohydrates, and nucleic acids contents were calculated on-line. The energy for biomass formation and maintenance were quantitatively analyzed. Light respiration rate decreased with the intracellular nitrogen reduction.
a r t i c l e
i n f o
Article history: Received 27 February 2014 Received in revised form 22 April 2014 Accepted 24 April 2014 Available online 4 May 2014 Keywords: Microalgae Carbon metabolism Energy metabolism On-line monitor Biomass composition
a b s t r a c t In this study, a photobioreactor cultivation system and a calculation method for on-line monitoring of carbon and energy metabolism of microalgae were developed using Nannochloropsis sp. in nitrogenrepletion and nitrogen-limitation cultures. Only 30–60% of carbon fixed in Calvin cycle was used for biomass and the rest was lost in light respiration. The net fixed carbon was assumed to be incorporated into protein, lipids, carbohydrates, and nucleic acids, whose contents calculated on-line fitted well with the experimental measurements. Intracellular ATPs were quantitatively divided for biomass production and cell maintenance, and the result is in accordance with known reports. Due to light limitation induced by high cell concentration in batch cultures, the proportion of CO2 loss in light respiration and the proportion of energy for maintenance rapidly increased in culturing process. Nitrogen starvation reduced the light respiration, thus decreasing CO2 loss and maintenance energy, but no effect on ATP requirement for cell growth. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae have recently received specific attention as the most potential biofuel feedstock due to their unique characteristics such as rapid growth rate, high lipid content and no requirement of high quality agriculture land (Chisti, 2007; Scott et al., 2010). In addition, microalgae can utilize CO2 in flue gases as carbon source, which can mitigate the increasing CO2 emission and help reduce the green house effect (Wang et al., 2008). The growth rate and composition of microalgae vary greatly under different environmental conditions. There have been known ⇑ Corresponding author. Tel.: +86 10 8262 7060; fax: +86 10 8262 7074. 1
E-mail address: [email protected] (W. Cong). This author should be considered as co-first author.
http://dx.doi.org/10.1016/j.biortech.2014.04.083 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
reports on the effects of cultural conditions on cell growth rate and lipid productivity (Breuer et al., 2013; Lv et al., 2010), whereas little information is available on the dynamic change of intracellular carbon and energy flux of algae according to growth conditions. Photoautotrophic organisms utilize CO2 and light energy to synthesize biomolecules. In eukaryotic microalgal cells, photosynthesis and respiration are two important processes that coexist in light. CO2 is fixed through photosynthesis, but a fraction of carbon is lost by respiration in the form of CO2 evolution. The respiration should not only be regarded as carbon loss because the precursor and the energy generated during respiration are necessary for the formation of high-value biomass components such as proteins and lipids (Wilhelm and Jakob, 2011). The biomass composition usually varies with environmental conditions, and light respiration rate may change according to types of components synthesized. Therefore,
D. Zhang et al. / Bioresource Technology 164 (2014) 86–92
light respiration rate should be an important factor in carbon metabolism. However, it is usually considered to have a negative impact on growth, and is assumed to remain constant in classical macroscopic modeling approaches (Takache et al., 2012). The conventional measurements of cell composition require the disruption of the cell and can only be carried out off-line, thus being invasive and time-consuming (Carvalho et al., 2009; Kliphuis et al., 2012). Although several models have been developed for microalgal growth and lipid accumulation (Mairet et al., 2011; Packer et al., 2011), the methods for calculating biomass concentration and contents of main components on-line are not reported. On energy metabolism, some theoretical estimations of the energy in the form of ATP required for biomass production are solely based on the main biomass composition. This method gives a value lower than that experimental measurements. Additional energy is required for the assembly of biopolymers into growing biomass defined as ‘‘growth-associated maintenance’’ and cell maintenance called ‘‘non-growth-associated maintenance’’ (Baart et al., 2008). Kliphuis et al. (2012) quantified the energy parameters for biomass formation and maintenance of Chlamydomonas reinhardtii by performing chemostat experiments at different growth rates. Monitoring and analysis of intracellular carbon and energy flux could provide further insight on intracellular metabolic processes. To the best of our knowledge, there has been no known report on on-line calculations of carbon and energy allocation in culture of microalgae. In this study, a photobioreactor cultivation system and a calculation method were established to monitor process parameters and acquire information on carbon and energy flux on-line. For oleaginous microalgae, light intensity and nitrogen stress are the most widely studied approaches to regulate cell growth rate and lipid production. (Breuer et al., 2012; Liu et al., 2012; Pal et al., 2011). In this study, Nannochloropsis sp., a promising marine alga for biodiesel production (Quinn et al., 2012), was used as a model strain to investigate the change of carbon and energy flux in nitrogenrepletion (NR) and nitrogen-limitation (NL) batch cultures in a self-built photobioreactor culture system.
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AAA 4.6 150 mm; Agilent, USA). After extraction of lyophilized algal powder with modified Bligh and Dyer method, lipid content was determined gravimetrically, and the relative fatty acid composition was determined by GC analysis using capillary column (VF-5ht 30 m 0.25 mm; Agilent, USA) and flame ionization detection (Liang et al., 2013). Nucleic acids were not measured, DNA and RNA contents were set at 0.35% and 2.20%, respectively, according to the literature (Rebolloso-Fuentes et al., 2001). Nitrate content in the medium was measured with an ion chromatograph (Metrohm, 761 Compact IC, CH), and intracellular nitrogen quota was determined with an elemental analyzer (Vario EL III CHN, GER). 2.3. Photobioreactor culture system Batch cultures were carried out in a cylindrical air-lift glass photobioreactor (U0.165 m 1.0 m, working volume of 18 L). Continuous illumination was provided by 16 fluorescent lamps (tubes) arrayed symmetrically around the bioreactor, providing the incident light intensity of 120 lE/(m2 s). The pH, dissolved oxygen (DO), and dissolved carbon dioxide (DCO2) of the medium were monitored by autoclavable pH electrode, DO electrode, and DCO2 electrode, respectively (Mettler-Toledo, CH). The pressure in the photobioreactor was measured by a pressure transducer and maintained at 10 ± 1 kPa above ambient pressure. Aeration was supplied with air filtrated through a 0.22-lm gas filter at the flow rate of 1.0 L/min, monitored by an air mass flow controller (Brooks, USA). CO2 was intermittently injected into the bioreactor to control pH at 7.8 ± 0.2, monitored by a CO2 mass flow controller (Brooks, USA). The off-gas was channeled to the CO2/O2 analysis unit, comprising a dehumidifier, a pressure regulator, and a gas mass spectrometry (Sunny Hengping, CHN). The CO2/O2 analysis unit was designed with multiple channels. Part of the air in the inlet was channeled to the CO2/O2 analysis unit for cross-calibration. The software developed by our lab was used to collect and store the data on-line in a local computer. 2.4. Light respiration rate measurement
2. Methods 2.1. Microorganism and culture medium The Nannochloropsis sp. used in this study was obtained from Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences. The algae were cultivated in sterilized modified artificial f/2-Si medium (Guillard and Ryther, 1962). NaNO3 was used as the sole nitrogen source. In NR culture, NaNO3 was added to the medium according to the biomass production every day, with 0.8 g/L of initial concentration. In NL culture, initial NaNO3 concentration was set at 0.375 g/L.
The light respiration rate was determined by measuring the rate of post-illumination O2 uptake (Kliphuis et al., 2011). The photosynthesis and respiration rates were measured by a Clark-type electrode (Hansatech Oxylab, UK), referring to the method described by Langner et al. (2009). A computer controlled the program of increasing light intensity from 50 to 500 lE/m2 s (each light irradiance with 3-min duration) alternating with subsequent dark phase of 3 min. Both oxygen increase rate and decrease rate were recorded, representing net oxygen evolution rates (PN) and respiration oxygen consumption rate, respectively. This respiration rate represents the oxygen consumption during the light period, denoted as RL. Gross photosynthetic oxygen production (PG) was derived from PN corrected by corresponding RL.
2.2. Analytical methods 2.5. PQ monitoring on-line and carbon flux analysis The biomass concentration was determined by gravimetric method. An aliquot of culture was sampled and centrifuged (5000 rpm, 8 min), washed twice with distilled water to remove adhering inorganic salts, lyophilized for more than 48 h, and weighed. Each experimental measurement was performed in triplicate and averaged. Carbohydrates content was measured according to the phenol/ concentrated sulfuric acid method, and glucose was used as a standard (Dubois et al., 1956). Total protein content was assayed by the Lowry method, and bovine serum albumin was used as a standard (Lowry et al., 1951). The relative amino acid composition was determined by HPLC using amino acid analysis column (Eclipse
For photoautotrophic organisms, CO2 and inorganic nitrogen + NO 3 , NO2 and NH4 are used as carbon source and nitrogen source, respectively. PQ (Photosynthetic Quotient) was determined from the ratio between O2 evolution rate and CO2 uptake rate (Eriksen et al., 2007; Kroon and Thoms, 2006). Bubbled CO2 into the phtotobioreactor was utilized by cells for biomass (CX) or dissolved in medium (Caq), or escaped with off-gas (CO). The carbon balance in the photobioreactor could be described as follows:
C in ¼ C X þ C aq þ C O
ð1Þ
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where, CO2 in the inlet (Cin) and outlet (CO) were monitored on-line. To calculate the carbon utilized for biomass (CX), the change of dissolved inorganic carbon in the medium (Caq) had to be determined. First of all, the conception of ‘‘C supply cycle’’ was proposed: when pH was above the high set-point, a constant flow of CO2 was added until pH decreased to below the low set-point. The carbon was absorbed by the cells or escaped from the medium gradually, resulting in the increase of pH. When pH reached the high set-point again, another CO2 injection began. This process was called one ‘‘C supply cycle’’. The calculation of carbon balance was carried out in a ‘‘C supply cycle’’ period. In this process, taking into account the carbonate chemical equilibrium, CO2 injection induced a series of reactions in the medium as shown in Appendix A. Caq at the end of ‘‘C supply cycle’’ should be back to the beginning value. However, in general, Caq increased with extracellular nitrate utilized, because 1 molecule H+ was consumed for each molecule NO 3 absorbed by cells and an equimolar CO2 was added to regenerate these H+ (Eriksen et al., 2007). In NR culture, the absorption of nitrate was assumed to be proportional to CO2 absorption according to the intracellular element composition. In NL culture, this proportion was taken as a Michaelis–Menten function of the external nitrogen concentration (Mairet et al., 2011):
mN ¼
mm NðtÞ NðtÞ þ K s
ð2Þ
where, mN is N absorption rate (g/mol CO2), Ks is the half-saturation constant for the substrate (g/L), and mm is the maximum uptake rate (g/mol CO2). In addition, intracellular nitrogen quota qn (g/gdcw) could be calculated based on extracellular N concentration and biomass production:
X 0 qn0 þ ðN0 NðtÞÞ qn ðtÞ ¼ XðtÞ
ð3Þ
where, X(t) and X0 are the biomass concentration (g/L) at time t and at the beginning; qn0 is the initial intracellular nitrogen quota. O2 evolution rate could be calculated based on Eq. (4):
O2in ¼ O2X þ O2aq þ O2O
ð4Þ
where, the left side of the equation represents the flow of O2 in the air entered into the photobioreactor (O2in ). The right side of the equation is the sum of O2 produced by the cells (O2X ), dissolved oxygen in the medium (O2aq ), and the flow of O2 in the off-gas (O20 ). O2 in the inlet (O2in ) and outlet (O20 ) were monitored on-line through gas mass spectrometry, and O2aq was monitored using DO electrode. Therefore, PQ was obtained with following equation:
PQ ¼ O2X =C X
ð5Þ
Based on PQ, RL and intracellular main metabolic reactions (Appendix B), the intracellular carbon flux was analyzed. Net evolved molar amount of O2 was set as PQ per net molecule CO2 fixed, and the simultaneous molar amount of O2 consumption by light respiration was set as r. The gross O2 evolved in light reaction of photosynthesis in chloroplast was the sum of net O2 evolved and O2 consumed in light respiration, which was (PQ + r) molecules. The light reactions can be illustrated by the classical Z-scheme of linear electron transport chain. Based on the photosynthesis equation, 8 molecules photons were required to split 2 molecules H2O and release 1 molecule O2, and 1 molecule CO2 was fixed in Calvin cycle in dark reactions, accordingly (Cornet et al., 1998). The additional ATP generation in cyclic pathway in light reactions was usually small (Kliphuis et al., 2012). To simplify the calculation, the cyclic pathway was not included in our model. Therefore, the ratio between CO2 uptake and O2 evolved was set as 1:1 in photosynthesis. Thus, the gross molar amount of CO2 uptake is
(PQ + r), among which only 1 molecule carbon is ultimately used to synthesize cell materials and the rest carbon are expelled out of the cell. This part of carbon flux in photoautotrophic microalgae should not only be regarded as loss. Instead, the biosynthesis of more reduced components such as lipids and proteins from the sugar C skeletons needs additional reductants and ATP, which is inevitably associated with decarboxylation reactions. The reductants, such as NADPH, NADH, and FADH2, provide reducing power for more reduced components synthesis and generate energy by entering the electron transport chain (ETC) (Wilhelm and Jakob, 2011). When r represents the molar amount of O2 consumption in ETC, the molar amount of CO2 released in this process is r. The reductants (NADH/NADPH) generated based on the rest carbon stock (PQ-1) are directly incorporated into the synthesis more reduced components. Therefore, the gross fixed carbon in Calvin cycle is: (1) converted into biomass building blocks (CB); (2) decarboxylated to generate NADH/NADPH for reduction reaction to synthesize cellular components, causing CO2 loss (CR); (3) decarboxylated to generate NADH/FADH2, which is oxidized to produce additional energy, causing O2 consumption and CO2 loss (CE). These carbon metabolism reactions are intertwined to each other. Here, to demonstrate the pattern of carbon flux, three categories are manually constructed. The proportion of each of these three parts is calculated as follows:
CB ¼
1 PQ þ r
ð6Þ
CR ¼
PQ 1 PQ þ r
ð7Þ
CE ¼
r PQ þ r
ð8Þ
The partition of the net fixed 1 unit of carbon to the main components can be reflected by the change of PQ, e.g., the increased carbon flux into more reduced products (such as proteins) results in the increase of PQ (Kroon and Thoms, 2006). Since the biomass is mainly composed of proteins, carbohydrates, lipids and nucleic acids, the PQ of the whole cell can be regarded as the molecular weighted sum of the respective PQ of these main components. For de novo cell materials in a period of time:
PQ ¼
4 X ni PQ i
ð9Þ
il 4 X ni ¼ 1
ð10Þ
i¼l
where, i = 1, 2, 3, 4 represents the macromolecules: proteins, carbohydrates, lipids, nucleic acids, respectively; ni is the molar proportion; PQi is the PQ of the macromolecule i, whose calculation process can be found in Appendix C. The mole weight of the de novo synthesis part is the molecular weighted sum of the respective mole weight:
M¼
4 X ni M i
ð11Þ
i¼l
where, Mi is the molecular weight of the macromolecules i (g/mol). Therefore, the increased biomass, DX (g/L) and weight of macromolecule i, Dwi (g/L) are obtained by:
DX ¼ C X M=0:97
ð12Þ
Dwi ¼ C x ni Mi
ð13Þ
where, CX is the net CO2 incorporated in biomass (mol/L), which could be monitored on-line. 0.97 represents that only the five major
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elements (C, H, O, N, and P) are considered based on the fact that these elements form 97% of cell biomass. The rest was considered as ash, which was mainly comprised of sulfur and other inorganic minerals, though they are also necessary for cell growth (Chen et al., 2011; Kroon and Thoms, 2006). According to Geider et al. (1998), synthesis of proteins and nucleic acids are proportional to the nitrogen assimilation.
PðtÞ ¼ aðNðtÞ N0 Þ þ P0
ð14Þ
NuðtÞ ¼ bðNðtÞ N0 Þ þ Nu0
ð15Þ
where, P(t), Nu(t) and N(t) are the protein concentration (g/L), nucleic acids concentration (g/L), and extracellular nitrogen content (g/L) in the medium at time t, respectively; P, Nu, and N0 are the respective initial values of P(t), Nu(t), and N(t). a and b represent protein synthesis and nucleic acids synthesis coefficients, respectively. The combination of Eqs. (9)–(15) can be used to monitor the changes of biomass concentration and the contents of proteins, carbohydrates, lipids and nucleic acids on-line in the culture process. 2.6. Energy flux analysis The amount of energy in the form of ATP is difficult to determine and it varies with different microorganisms and growth conditions. The energy requirement of cell can be divided into the energy used for biomass formation and that used for maintenance (Pirt, 1965). The energy for biomass formation refers to the energy for synthesis of biopolymers and for growth-associated maintenance (GAM) which is the energy consumed in assembling biopolymers into biomass. The energy for maintenance is called ‘‘non growth-associated maintenance’’ (NGAM) (Kliphuis et al., 2012; Taymaz-Nikerel et al., 2010). The main energy metabolism can be determined by intracellular carbon flux analysis and quantitative calculation of reductants and ATP. Light is taken up in the light reaction of photosynthesis with expelling of O2 and formation of NADPH and ATP. CO2 is fixed in the form of GAP (Glyceraldehyde-3-phosphate) in Calvin cycle driven by the reducing power and energy from the light reaction. A fixed stoichiometry ratio of 2 molecules NADPH and 3 molecules ATP generated per molecule O2 produced during linear electron transport in chloroplast was hypothesized, which exactly fits the requirements of Calvin cycle (Kliphuis et al., 2012). A fraction of the GAP is moved to the cytosol, where it enters the glycolysis, tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) to synthesize precursors of macromolecules such as lipids and proteins. The additional reductants and ATP for biosynthesis of lipids and proteins from the sugar C skeletons may be obtained from photosynthesis or from light respiration, whose proportion is unclear due to the little knowledge of the microalgal metabolic pathways (Johnson and Alric, 2013). As assumed above, CO2 fixation in Calvin cycle consumed all NADPH and ATP generated in light reaction. Therefore, the additional energy for more reduced component synthesis came only from light respiration for a simplification. According to Appendix B, in glycolysis and TCA cycle, 1 molecule GAP is oxidized and completely decomposed, generating 5 molecules NADH, 1 molecule FADH2, 3 molecules CO2 and 3 molecules ATP. In the electron transport chain, the relationship between ATP synthesis and reductant consumption was assumed to be as follows: 1 molecule NADH yields 2.5 molecules ATP with 0.5 molecules O2 consumption and 1 molecule FADH2 yields 1.5 molecules ATP with 0.5 molecules O2 consumption. Therefore, when 1 molecule GAP is completely oxidized and decomposed, it will consume 3 molecules O2 and generate 3 molecules CO2 and 17 molecules ATP, among which 14 molecules ATP come from
89
oxidative phosphorylation and 3 molecules ATP come from substrate phosphorylation. It was assumed that NADPH was only generated in PPP, and 2 molecules NADPH were produced per molecule CO2 released. NADPH and part of NADH were directly involved in the reduction reaction for biomass production. Based on these assumptions, the quantitative analysis of intracellular ATP could be performed. 3. Results and discussion 3.1. Biomass concentration prediction for NR and NL cultures The initial cell concentrations were set as 0.24 g/L in both NR and NL cultures. In NL culture, the extracellular nitrate was depleted at day 4. However, the intracellular nitrogen pool could support further cell growth (Li et al., 2008). The cell growth rate of Nannochloropsis sp. in NL cultures kept the same as that in NR culture until day 6, and then slowed down. Due to the limited culture time, the cell growth was not completely halted at the end of NL culture. The calculated results fitted well with the experimental measurements in both NR and NL cultures. Generally, the relative deviations between the experimental measurements and calculated values were less than 8%. Additional, in NR culture, the absorption rate of nitrate was proportional to CO2 absorption, which was set as 0.125 mol N/mol CO2, according to the intracellular element composition. In NL culture, Michaelis–Menten equation for the nitrate absorption was fitted based on the nitrate concentrations in medium measured by ion chromatograph. The maximal uptake rate (vm) and half-saturation constant (Ks) were 1.802 g N/mol CO2 and 0.0082 g N/L, respectively. 3.2. Carbon flux analysis To understand the carbon metabolism of algal cells, the light respiration was investigated, which occurs simultaneously with photosynthesis in eukaryotic microalgal growth. The change of light respiration rate (RL) with the gross phostosynthesis rate (PG) of Nannochloropsis sp. is shown in Fig. 1. Light respiration was enhanced with the increase of photosynthesis rate, which was consistent with a previous report showing that higher gross photosynthesis rate led to higher respiratory activity for fast growth rate (Kliphuis et al., 2011). The relationship between RL and PG was also investigated under different intracellular nitrogen quota (qn). Fig. 1 shows that RL decreased significantly with nitrogen starvation. The slopes of RL to PG are almost constant, irrespectively of qn. However,
Fig. 1. Relationship between RL and PG of Nannochloropsis sp. for different qn.
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Fig. 2. Time course of the photosynthesis rate, the respiration rate and the qn of Nannochloropsis sp. [(—) PG; (- - -) RL; () calculated qn, (h) measured qn; black for NR culture; red for NL culture]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the intercepts, denoted by Rm, change significantly with different qn. There is a linear relationship between Rm and qn (Rm = 0.0833 qn – 0.0002, R2 = 0.99). As depicted in Fig. 2, in NR and NL cultures, through O2 evolution rate and qn monitored and calculated on-line, PG and RL were obtained. The gross photosynthesis rate (PG) decreased rapidly with culture time in both NR and NL cultures, mainly due to light limitation. Early in the two cultures, the values of PG were almost the same. From day 4, PG of NL culture became lower due to nitrogen depletion. The decrease of PG did not mean the decrease of growth rate because the biomass concentration in NL culture was the same as in NR culture in day 4 and day 5. Because in NL culture, RL decreased with nitrogen depletion, thus the decreasing CO2 loss in respiration relieved the decreasing CO2 fixation in the weakening photosynthesis. In addition, nitrogen starvation may decrease the degree of reduction of de novo biomass, suggesting that the same O2 evolution may result in more CO2 fixation, and thereby compensate for the low photosynthesis rate compared with NR condition (Jakob et al., 2007). Therefore, the decrease of cell growth
Fig. 3. PQ, carbon flux and component contents of Nannochloropsis sp. in NR and NL cultures [h, and for measured data of protein content, lipid content, and carbohydrate content, respectively, and the lines in corresponding colors for calculated results; green line for calculated nucleic acids content; a for NR culture; b for NL culture]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
D. Zhang et al. / Bioresource Technology 164 (2014) 86–92
rate was hysteretic compared with the decrease of photosynthesis rate under nitrogen starvation. As shown in Fig. 2, the respiration activity decreased with the decrease in photosynthesis, but mainly depended on intracellular nitrogen quota (qn). The initial value of qn was set as 0.08 g/gdcw, and the calculated results of qn fitted well with the measurements. The relative deviations between experimental measurements and calculated values were less than 5%. The time courses of PQ and CB, CR, CE calculated on the analysis of carbon flux in NR and NL cultures are shown in Fig. 3. CB decreased and CO2 loss in respiration increased with culture time in both cultures because of the decrease of photosynthesis rate in limited light. It can be seen that CB was only 30–60% of the total carbon fixed in Calvin cycle, and the rest was expelled out through decarboxylation reactions in light respiration. In ND culture, nitrogen depletion reduced the respiration rate. Therefore, CB of NL culture did not decrease as rapidly as CB of NR culture. The carbon in the CR part was responsible for supplying reductants for reduced component synthesis. CR/CB represented the reductants requirement per molecule biomass production. Nitrogen depletion caused protein synthesis arrest, whereas lipid and carbohydrate synthesis may be induced, thereby reducing the degree of biomass reduction. In Fig. 3, it is shown that PQ is lower in late period of NL culture, and the corresponding CR/CB of NL culture is lower than that of NR culture. The carbon fixed for biomass materials was used to produce proteins, lipids, carbohydrates and nucleic acids, whose contents were reflected by PQ. PQ of NL culture started to decrease from day 3, indicating that the intracellular carbon pathway changed, because extracellular nitrogen source was insufficient to support protein and nucleic acid synthesis. The proportion of carbon flux to lipids and carbohydrates increased. PQs of lipids and carbohydrates were 1.383 and 1.0, respectively, according to Appendix C. The value of PQ was approximately 1.35 after nitrogen depletion, suggesting that lipid synthesis was predominant in carbon partition. For calculation of protein and nucleic acid contents, their synthesis coefficients were set as 5.512 g/g N and 0.290 g/g N, respectively. The contents of major intracellular components are shown in Fig. 3a3 and b3. The calculation results fitted the experimental measurement very well. The absolute deviations between experimental measurements and calculated values of contents of protein, lipids, and carbohydrates were less than 0.04 g/g in general. Eriksen et al. reported that PQ depended on the composition of the produced biomass and the type of nitrogen source, independent on other environmental conditions (Eriksen et al., 2007). The models in this study could successfully describe the dynamic change of microalgae growth in batch cultures. 3.3. Energy flux analysis Besides the energy required for organic materials synthesis, additional energy is required for growth-associated maintenance (GAM) and non-growth-associated maintenance (NGAM) (Baart et al., 2008; Kliphuis et al., 2012). The energy parameters for biomass formation and maintenance of Nannochloropsis sp. in NR and ND cultures were analyzed, and the results are presented in Fig. 4. The energy in the form of ATP required per molecule C-biomass production increased rapidly with culture time, because of the increasing maintenance energy. The ATP demand for maintenance was calculated based on the relationship between RL and PG (Fig. 1). The intercept represents the respiration consumption with no cell growth, i.e., the maintenance requirement. Based on the reactions in Appendix B, for 1 unit of O2 consumption in respiration, 1 unit of carbon (GAP) degraded to CO2, and yielded 17/3 units of ATP. The ATP requirement for maintenance was determined to be approximately 2.2 mmol/gh in NR culture, and 2.1–0.98 mmol/gh in NL culture, which decreased with nitrogen
91
Fig. 4. Intracellular ATP consumption per mole C-biomass production of Nannochloropsis sp. in NR and NL cultures [a for NR culture; b for NL culture].
starvation. Kliphuis et al. worked out that the ATP required for the maintenance of C. reinhardtii was 2.85 mmol/gh (Kliphuis et al., 2012), similar to the value obtained in our study. In late culture period, high cell concentration and low specific cell growth rate resulted in a low fraction of energy for growth. The maintenance could account for more than 60% of total energy demand. Therefore, cultivating microalgae at high specific growth rate was important to attain high biomass yield on energy. Wilhelm and Jakob (2011) presented that the theoretical ATP requirement on the base of biosynthetic pathways for the main cell components were 3.67 units of ATP for carbohydrates, 6.33 units of ATP for lipids, and 5.33 units of ATP for proteins on 1 unit of carbon. As shown in Fig. 4, our experimental results for ATP requirement of biomacromolecules synthesis (growth minus GAM) was around 5 molecules ATP per molecule C-biomass production, which were in the range of 3.67–6.33. In NL culture, protein synthesis stopped and the accumulations of carbohydrates and lipids were accelerated after the nitrogen depletion. Lipids can store more ATP than other macromolecules. Therefore, in nitrogen starvation, lipids accumulate to prevent the photo-oxidative damage of the cell by incorporating the excess of energy over growth demand (Breuer et al.,
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2013). However, as shown in Fig. 4, the ATP demand for biomacromolecules did not vary significantly, indicating that the cell capacity for energy storage did not increase in nitrogen starvation condition, mainly because the increase in carbohydrate synthesis occurred along with lipid accumulation, lowering the energy fixation. The energy for NGAM in NR and NL cultures appeared to be constant in the culture process. 4. Conclusion Carbon and energy metabolism of Nannochloropsis sp. in NR and NL cultures were analyzed on-line. Carbon fixed in Calvin cycle was quantitatively divided into three parts: for biomass, for reductants and for energy. The main biomass components contents were calculated on-line, fitted well with the measurements. The energy parameters for biomass production and cell maintenance were also obtained. Therefore, the photobioreactor culture system and calculation method presented here could be used to gain some dynamic information on microalgae metabolism. Acknowledgements This study was supported by Twelfth-five-year National Key Task program (2011BAD14B02), 863 Program (2013AA065801) and Natural Science Foundation of China (21376246). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.04. 083. References Baart, G.J.E., Willemsen, M., Khatami, E., de Haan, A., Zomer, B., Beuvery, E.C., Tramper, J., Martens, D.E., 2008. Modeling Neisseria meningitidis B metabolism at different specific growth rates. Biotechnol. Bioeng. 101 (5), 1022–1035. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2013. Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus. Bioresour. Technol. 143, 1–9. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2012. The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour. Technol. 124, 217–226. Carvalho, A.P., Monteiro, C.M., Malcata, F.X., 2009. Simultaneous effect of irradiance and temperature on biochemical composition of the microalga Pavlova lutheri. J. Appl. Phycol. 21 (5), 543–552. Chen, M., Tang, H., Ma, H., Holland, T.C., Ng, K.Y.S., Salley, S.O., 2011. Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta. Bioresour. Technol. 102 (2), 1649–1655. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Cornet, J.F., Dussap, C.G., Gros, J.B., 1998. Kinetics and energetics of photosynthetic micro-organisms in photobioreactors. In: Scheper, T. (Ed.), Advances in Biochemical Engineering Biotechnology. Springer, Heidelberg, pp. 153–222. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28 (3), 350–356. Eriksen, N.T., Riisgard, F.K., Gunther, W.S., Iversen, J.J.L., 2007. On-line estimation of O2 production, CO2 uptake, and growth kinetics of microalgal cultures in a gastight photobioreactor. J. Appl. Phycol. 19 (2), 161–174.
Geider, R.J., MacIntyre, H.L., Kana, T.M., 1998. A dynamic regulatory model of phytoplanktonic acclimation to light, nutrients, and temperature. Limnol. Oceanogr. 43 (4), 679–694. Guillard, R.R., Ryther, J.H., 1962. Studies of marine planktonic diatoms.1. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8 (2), 229–239. Jakob, T., Wagner, H., Stehfest, K., Wilhelm, C., 2007. A complete energy balance from photons to new biomass reveals a light- and nutrient-dependent variability in the metabolic costs of carbon assimilation. J. Exp. Bot. 58 (8), 2101–2112. Johnson, X., Alric, J., 2013. Central carbon metabolism and electron transport in Chlamydomonas reinhardtii: metabolic constraints for carbon partitioning between oil and starch. Eukaryotic Cell 12 (6), 776–793. Kliphuis, A.M.J., Janssen, M., van den End, E.J., Martens, D.E., Wijffels, R.H., 2011. Light respiration in Chlorella sorokiniana. J. Appl. Phycol. 23 (6), 935–947. Kliphuis, A.M.J., Klok, A.J., Martens, D.E., Lamers, P.P., Janssen, M., Wijffels, R.H., 2012. Metabolic modeling of Chlamydomonas reinhardtii: energy requirements for photoautotrophic growth and maintenance. J. Appl. Phycol. 24 (2), 253–266. Kroon, B.M.A., Thoms, S., 2006. From electron to biomass: a mechanistic model to describe phytoplankton photosynthesis and steady-state growth rates. J. Phycol. 42 (3), 593–609. Langner, U., Jakob, T., Stehfest, K., Wilhelm, C., 2009. An energy balance from absorbed photons to new biomass for Chlamydomonas reinhardtii and Chlamydomonas acidophila under neutral and extremely acidic growth conditions. Plant Cell Environ. 32 (3), 250–258. Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C.Q., 2008. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biotechnol. 81 (4), 629–636. Liang, K., Zhang, Q., Gu, M., Cong, W., 2013. Effect of phosphorus on lipid accumulation in freshwater microalga Chlorella sp.. J. Appl. Phycol. 25 (1), 311–318. Liu, J., Yuan, C., Hu, G., Li, F., 2012. Effects of light intensity on the growth and lipid accumulation of microalga Scenedesmus sp. 11-1 under nitrogen limitation. Appl. Biochem. Biotechnol. 166 (8), 2127–2137. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1), 265–275. Lv, J.M., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L., 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101 (17), 6797–6804. Mairet, F., Bernard, O., Masci, P., Lacour, T., Sciandra, A., 2011. Modelling neutral lipid production by the microalga Isochrysis aff. galbana under nitrogen limitation. Bioresour. Technol. 102 (1), 142–149. Packer, A., Li, Y., Andersen, T., Hu, Q., Kuang, Y., Sommerfeld, M., 2011. Growth and neutral lipid synthesis in green microalgae: a mathematical model. Bioresour. Technol. 102 (1), 111–117. Pal, D., Khozin-Goldberg, I., Cohen, Z., Boussiba, S., 2011. The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp.. Appl. Microbiol. Biotechnol. 90 (4), 1429–1441. Pirt, S.J., 1965. The maintenance energy of bacteria in growing cultures. Proc. R. Soc. Lond. B 163 (991), 224–230. Quinn, J.C., Yates, T., Douglas, N., Weyer, K., Butler, J., Bradley, T.H., Lammers, P.J., 2012. Nannochloropsis production metrics in a scalable outdoor photobioreactor for commercial applications. Bioresour. Technol. 117, 164–171. Rebolloso-Fuentes, M.M., Navarro-Perez, A., Garcia-Camacho, F., Ramos-Miras, J.J., Guil-Guerrero, J.L., 2001. Biomass nutrient profiles of the microalga Nannochloropsis. J. Agric. Food Chem. 49 (6), 2966–2972. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A.G., 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21 (3), 277–286. Takache, H., Pruvost, J., Cornet, J.-F., 2012. Kinetic modeling of the photosynthetic growth of Chlamydomonas reinhardtii in a photobioreactor. Biotechnol. Prog. 28 (3), 681–692. Taymaz-Nikerel, H., Borujeni, A.E., Verheijen, P.J.T., Heijnen, J.J., van Gulik, W.M., 2010. Genome-derived minimal metabolic models for Escherichia coil MG1655 with estimated in vivo respiratory ATP stoichiometry. Biotechnol. Bioeng. 107 (2), 369–381. Wang, B., Li, Y., Wu, N., Lan, C.Q., 2008. CO(2) bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Wilhelm, C., Jakob, T., 2011. From photons to biomass and biofuels: evaluation of different strategies for the improvement of algal biotechnology based on comparative energy balances. Appl. Microbiol. Biotechnol. 92 (5), 909–919.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
On-line modeling intracellular carbon and energy metabolism of Nannochloropsis sp. in nitrogen-repletion and nitrogen-limitation cultures Dongmei Zhang a,b, Fei Yan a,b,1, Zhongliang Sun a,b, Qinghua Zhang a, Shengzhang Xue a, Wei Cong a,⇑ a b
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China University of Chinese Academy of Sciences, Beijing 100049, China
h i g h l i g h t s On-line monitoring and calculation of carbon and energy metabolism of microalgae. Only 30–60% of carbon fixed in Calvin cycle was used for biomass material. Protein, lipids, carbohydrates, and nucleic acids contents were calculated on-line. The energy for biomass formation and maintenance were quantitatively analyzed. Light respiration rate decreased with the intracellular nitrogen reduction.
a r t i c l e
i n f o
Article history: Received 27 February 2014 Received in revised form 22 April 2014 Accepted 24 April 2014 Available online 4 May 2014 Keywords: Microalgae Carbon metabolism Energy metabolism On-line monitor Biomass composition
a b s t r a c t In this study, a photobioreactor cultivation system and a calculation method for on-line monitoring of carbon and energy metabolism of microalgae were developed using Nannochloropsis sp. in nitrogenrepletion and nitrogen-limitation cultures. Only 30–60% of carbon fixed in Calvin cycle was used for biomass and the rest was lost in light respiration. The net fixed carbon was assumed to be incorporated into protein, lipids, carbohydrates, and nucleic acids, whose contents calculated on-line fitted well with the experimental measurements. Intracellular ATPs were quantitatively divided for biomass production and cell maintenance, and the result is in accordance with known reports. Due to light limitation induced by high cell concentration in batch cultures, the proportion of CO2 loss in light respiration and the proportion of energy for maintenance rapidly increased in culturing process. Nitrogen starvation reduced the light respiration, thus decreasing CO2 loss and maintenance energy, but no effect on ATP requirement for cell growth. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae have recently received specific attention as the most potential biofuel feedstock due to their unique characteristics such as rapid growth rate, high lipid content and no requirement of high quality agriculture land (Chisti, 2007; Scott et al., 2010). In addition, microalgae can utilize CO2 in flue gases as carbon source, which can mitigate the increasing CO2 emission and help reduce the green house effect (Wang et al., 2008). The growth rate and composition of microalgae vary greatly under different environmental conditions. There have been known ⇑ Corresponding author. Tel.: +86 10 8262 7060; fax: +86 10 8262 7074. 1
E-mail address: [email protected] (W. Cong). This author should be considered as co-first author.
http://dx.doi.org/10.1016/j.biortech.2014.04.083 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
reports on the effects of cultural conditions on cell growth rate and lipid productivity (Breuer et al., 2013; Lv et al., 2010), whereas little information is available on the dynamic change of intracellular carbon and energy flux of algae according to growth conditions. Photoautotrophic organisms utilize CO2 and light energy to synthesize biomolecules. In eukaryotic microalgal cells, photosynthesis and respiration are two important processes that coexist in light. CO2 is fixed through photosynthesis, but a fraction of carbon is lost by respiration in the form of CO2 evolution. The respiration should not only be regarded as carbon loss because the precursor and the energy generated during respiration are necessary for the formation of high-value biomass components such as proteins and lipids (Wilhelm and Jakob, 2011). The biomass composition usually varies with environmental conditions, and light respiration rate may change according to types of components synthesized. Therefore,
D. Zhang et al. / Bioresource Technology 164 (2014) 86–92
light respiration rate should be an important factor in carbon metabolism. However, it is usually considered to have a negative impact on growth, and is assumed to remain constant in classical macroscopic modeling approaches (Takache et al., 2012). The conventional measurements of cell composition require the disruption of the cell and can only be carried out off-line, thus being invasive and time-consuming (Carvalho et al., 2009; Kliphuis et al., 2012). Although several models have been developed for microalgal growth and lipid accumulation (Mairet et al., 2011; Packer et al., 2011), the methods for calculating biomass concentration and contents of main components on-line are not reported. On energy metabolism, some theoretical estimations of the energy in the form of ATP required for biomass production are solely based on the main biomass composition. This method gives a value lower than that experimental measurements. Additional energy is required for the assembly of biopolymers into growing biomass defined as ‘‘growth-associated maintenance’’ and cell maintenance called ‘‘non-growth-associated maintenance’’ (Baart et al., 2008). Kliphuis et al. (2012) quantified the energy parameters for biomass formation and maintenance of Chlamydomonas reinhardtii by performing chemostat experiments at different growth rates. Monitoring and analysis of intracellular carbon and energy flux could provide further insight on intracellular metabolic processes. To the best of our knowledge, there has been no known report on on-line calculations of carbon and energy allocation in culture of microalgae. In this study, a photobioreactor cultivation system and a calculation method were established to monitor process parameters and acquire information on carbon and energy flux on-line. For oleaginous microalgae, light intensity and nitrogen stress are the most widely studied approaches to regulate cell growth rate and lipid production. (Breuer et al., 2012; Liu et al., 2012; Pal et al., 2011). In this study, Nannochloropsis sp., a promising marine alga for biodiesel production (Quinn et al., 2012), was used as a model strain to investigate the change of carbon and energy flux in nitrogenrepletion (NR) and nitrogen-limitation (NL) batch cultures in a self-built photobioreactor culture system.
87
AAA 4.6 150 mm; Agilent, USA). After extraction of lyophilized algal powder with modified Bligh and Dyer method, lipid content was determined gravimetrically, and the relative fatty acid composition was determined by GC analysis using capillary column (VF-5ht 30 m 0.25 mm; Agilent, USA) and flame ionization detection (Liang et al., 2013). Nucleic acids were not measured, DNA and RNA contents were set at 0.35% and 2.20%, respectively, according to the literature (Rebolloso-Fuentes et al., 2001). Nitrate content in the medium was measured with an ion chromatograph (Metrohm, 761 Compact IC, CH), and intracellular nitrogen quota was determined with an elemental analyzer (Vario EL III CHN, GER). 2.3. Photobioreactor culture system Batch cultures were carried out in a cylindrical air-lift glass photobioreactor (U0.165 m 1.0 m, working volume of 18 L). Continuous illumination was provided by 16 fluorescent lamps (tubes) arrayed symmetrically around the bioreactor, providing the incident light intensity of 120 lE/(m2 s). The pH, dissolved oxygen (DO), and dissolved carbon dioxide (DCO2) of the medium were monitored by autoclavable pH electrode, DO electrode, and DCO2 electrode, respectively (Mettler-Toledo, CH). The pressure in the photobioreactor was measured by a pressure transducer and maintained at 10 ± 1 kPa above ambient pressure. Aeration was supplied with air filtrated through a 0.22-lm gas filter at the flow rate of 1.0 L/min, monitored by an air mass flow controller (Brooks, USA). CO2 was intermittently injected into the bioreactor to control pH at 7.8 ± 0.2, monitored by a CO2 mass flow controller (Brooks, USA). The off-gas was channeled to the CO2/O2 analysis unit, comprising a dehumidifier, a pressure regulator, and a gas mass spectrometry (Sunny Hengping, CHN). The CO2/O2 analysis unit was designed with multiple channels. Part of the air in the inlet was channeled to the CO2/O2 analysis unit for cross-calibration. The software developed by our lab was used to collect and store the data on-line in a local computer. 2.4. Light respiration rate measurement
2. Methods 2.1. Microorganism and culture medium The Nannochloropsis sp. used in this study was obtained from Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences. The algae were cultivated in sterilized modified artificial f/2-Si medium (Guillard and Ryther, 1962). NaNO3 was used as the sole nitrogen source. In NR culture, NaNO3 was added to the medium according to the biomass production every day, with 0.8 g/L of initial concentration. In NL culture, initial NaNO3 concentration was set at 0.375 g/L.
The light respiration rate was determined by measuring the rate of post-illumination O2 uptake (Kliphuis et al., 2011). The photosynthesis and respiration rates were measured by a Clark-type electrode (Hansatech Oxylab, UK), referring to the method described by Langner et al. (2009). A computer controlled the program of increasing light intensity from 50 to 500 lE/m2 s (each light irradiance with 3-min duration) alternating with subsequent dark phase of 3 min. Both oxygen increase rate and decrease rate were recorded, representing net oxygen evolution rates (PN) and respiration oxygen consumption rate, respectively. This respiration rate represents the oxygen consumption during the light period, denoted as RL. Gross photosynthetic oxygen production (PG) was derived from PN corrected by corresponding RL.
2.2. Analytical methods 2.5. PQ monitoring on-line and carbon flux analysis The biomass concentration was determined by gravimetric method. An aliquot of culture was sampled and centrifuged (5000 rpm, 8 min), washed twice with distilled water to remove adhering inorganic salts, lyophilized for more than 48 h, and weighed. Each experimental measurement was performed in triplicate and averaged. Carbohydrates content was measured according to the phenol/ concentrated sulfuric acid method, and glucose was used as a standard (Dubois et al., 1956). Total protein content was assayed by the Lowry method, and bovine serum albumin was used as a standard (Lowry et al., 1951). The relative amino acid composition was determined by HPLC using amino acid analysis column (Eclipse
For photoautotrophic organisms, CO2 and inorganic nitrogen + NO 3 , NO2 and NH4 are used as carbon source and nitrogen source, respectively. PQ (Photosynthetic Quotient) was determined from the ratio between O2 evolution rate and CO2 uptake rate (Eriksen et al., 2007; Kroon and Thoms, 2006). Bubbled CO2 into the phtotobioreactor was utilized by cells for biomass (CX) or dissolved in medium (Caq), or escaped with off-gas (CO). The carbon balance in the photobioreactor could be described as follows:
C in ¼ C X þ C aq þ C O
ð1Þ
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where, CO2 in the inlet (Cin) and outlet (CO) were monitored on-line. To calculate the carbon utilized for biomass (CX), the change of dissolved inorganic carbon in the medium (Caq) had to be determined. First of all, the conception of ‘‘C supply cycle’’ was proposed: when pH was above the high set-point, a constant flow of CO2 was added until pH decreased to below the low set-point. The carbon was absorbed by the cells or escaped from the medium gradually, resulting in the increase of pH. When pH reached the high set-point again, another CO2 injection began. This process was called one ‘‘C supply cycle’’. The calculation of carbon balance was carried out in a ‘‘C supply cycle’’ period. In this process, taking into account the carbonate chemical equilibrium, CO2 injection induced a series of reactions in the medium as shown in Appendix A. Caq at the end of ‘‘C supply cycle’’ should be back to the beginning value. However, in general, Caq increased with extracellular nitrate utilized, because 1 molecule H+ was consumed for each molecule NO 3 absorbed by cells and an equimolar CO2 was added to regenerate these H+ (Eriksen et al., 2007). In NR culture, the absorption of nitrate was assumed to be proportional to CO2 absorption according to the intracellular element composition. In NL culture, this proportion was taken as a Michaelis–Menten function of the external nitrogen concentration (Mairet et al., 2011):
mN ¼
mm NðtÞ NðtÞ þ K s
ð2Þ
where, mN is N absorption rate (g/mol CO2), Ks is the half-saturation constant for the substrate (g/L), and mm is the maximum uptake rate (g/mol CO2). In addition, intracellular nitrogen quota qn (g/gdcw) could be calculated based on extracellular N concentration and biomass production:
X 0 qn0 þ ðN0 NðtÞÞ qn ðtÞ ¼ XðtÞ
ð3Þ
where, X(t) and X0 are the biomass concentration (g/L) at time t and at the beginning; qn0 is the initial intracellular nitrogen quota. O2 evolution rate could be calculated based on Eq. (4):
O2in ¼ O2X þ O2aq þ O2O
ð4Þ
where, the left side of the equation represents the flow of O2 in the air entered into the photobioreactor (O2in ). The right side of the equation is the sum of O2 produced by the cells (O2X ), dissolved oxygen in the medium (O2aq ), and the flow of O2 in the off-gas (O20 ). O2 in the inlet (O2in ) and outlet (O20 ) were monitored on-line through gas mass spectrometry, and O2aq was monitored using DO electrode. Therefore, PQ was obtained with following equation:
PQ ¼ O2X =C X
ð5Þ
Based on PQ, RL and intracellular main metabolic reactions (Appendix B), the intracellular carbon flux was analyzed. Net evolved molar amount of O2 was set as PQ per net molecule CO2 fixed, and the simultaneous molar amount of O2 consumption by light respiration was set as r. The gross O2 evolved in light reaction of photosynthesis in chloroplast was the sum of net O2 evolved and O2 consumed in light respiration, which was (PQ + r) molecules. The light reactions can be illustrated by the classical Z-scheme of linear electron transport chain. Based on the photosynthesis equation, 8 molecules photons were required to split 2 molecules H2O and release 1 molecule O2, and 1 molecule CO2 was fixed in Calvin cycle in dark reactions, accordingly (Cornet et al., 1998). The additional ATP generation in cyclic pathway in light reactions was usually small (Kliphuis et al., 2012). To simplify the calculation, the cyclic pathway was not included in our model. Therefore, the ratio between CO2 uptake and O2 evolved was set as 1:1 in photosynthesis. Thus, the gross molar amount of CO2 uptake is
(PQ + r), among which only 1 molecule carbon is ultimately used to synthesize cell materials and the rest carbon are expelled out of the cell. This part of carbon flux in photoautotrophic microalgae should not only be regarded as loss. Instead, the biosynthesis of more reduced components such as lipids and proteins from the sugar C skeletons needs additional reductants and ATP, which is inevitably associated with decarboxylation reactions. The reductants, such as NADPH, NADH, and FADH2, provide reducing power for more reduced components synthesis and generate energy by entering the electron transport chain (ETC) (Wilhelm and Jakob, 2011). When r represents the molar amount of O2 consumption in ETC, the molar amount of CO2 released in this process is r. The reductants (NADH/NADPH) generated based on the rest carbon stock (PQ-1) are directly incorporated into the synthesis more reduced components. Therefore, the gross fixed carbon in Calvin cycle is: (1) converted into biomass building blocks (CB); (2) decarboxylated to generate NADH/NADPH for reduction reaction to synthesize cellular components, causing CO2 loss (CR); (3) decarboxylated to generate NADH/FADH2, which is oxidized to produce additional energy, causing O2 consumption and CO2 loss (CE). These carbon metabolism reactions are intertwined to each other. Here, to demonstrate the pattern of carbon flux, three categories are manually constructed. The proportion of each of these three parts is calculated as follows:
CB ¼
1 PQ þ r
ð6Þ
CR ¼
PQ 1 PQ þ r
ð7Þ
CE ¼
r PQ þ r
ð8Þ
The partition of the net fixed 1 unit of carbon to the main components can be reflected by the change of PQ, e.g., the increased carbon flux into more reduced products (such as proteins) results in the increase of PQ (Kroon and Thoms, 2006). Since the biomass is mainly composed of proteins, carbohydrates, lipids and nucleic acids, the PQ of the whole cell can be regarded as the molecular weighted sum of the respective PQ of these main components. For de novo cell materials in a period of time:
PQ ¼
4 X ni PQ i
ð9Þ
il 4 X ni ¼ 1
ð10Þ
i¼l
where, i = 1, 2, 3, 4 represents the macromolecules: proteins, carbohydrates, lipids, nucleic acids, respectively; ni is the molar proportion; PQi is the PQ of the macromolecule i, whose calculation process can be found in Appendix C. The mole weight of the de novo synthesis part is the molecular weighted sum of the respective mole weight:
M¼
4 X ni M i
ð11Þ
i¼l
where, Mi is the molecular weight of the macromolecules i (g/mol). Therefore, the increased biomass, DX (g/L) and weight of macromolecule i, Dwi (g/L) are obtained by:
DX ¼ C X M=0:97
ð12Þ
Dwi ¼ C x ni Mi
ð13Þ
where, CX is the net CO2 incorporated in biomass (mol/L), which could be monitored on-line. 0.97 represents that only the five major
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elements (C, H, O, N, and P) are considered based on the fact that these elements form 97% of cell biomass. The rest was considered as ash, which was mainly comprised of sulfur and other inorganic minerals, though they are also necessary for cell growth (Chen et al., 2011; Kroon and Thoms, 2006). According to Geider et al. (1998), synthesis of proteins and nucleic acids are proportional to the nitrogen assimilation.
PðtÞ ¼ aðNðtÞ N0 Þ þ P0
ð14Þ
NuðtÞ ¼ bðNðtÞ N0 Þ þ Nu0
ð15Þ
where, P(t), Nu(t) and N(t) are the protein concentration (g/L), nucleic acids concentration (g/L), and extracellular nitrogen content (g/L) in the medium at time t, respectively; P, Nu, and N0 are the respective initial values of P(t), Nu(t), and N(t). a and b represent protein synthesis and nucleic acids synthesis coefficients, respectively. The combination of Eqs. (9)–(15) can be used to monitor the changes of biomass concentration and the contents of proteins, carbohydrates, lipids and nucleic acids on-line in the culture process. 2.6. Energy flux analysis The amount of energy in the form of ATP is difficult to determine and it varies with different microorganisms and growth conditions. The energy requirement of cell can be divided into the energy used for biomass formation and that used for maintenance (Pirt, 1965). The energy for biomass formation refers to the energy for synthesis of biopolymers and for growth-associated maintenance (GAM) which is the energy consumed in assembling biopolymers into biomass. The energy for maintenance is called ‘‘non growth-associated maintenance’’ (NGAM) (Kliphuis et al., 2012; Taymaz-Nikerel et al., 2010). The main energy metabolism can be determined by intracellular carbon flux analysis and quantitative calculation of reductants and ATP. Light is taken up in the light reaction of photosynthesis with expelling of O2 and formation of NADPH and ATP. CO2 is fixed in the form of GAP (Glyceraldehyde-3-phosphate) in Calvin cycle driven by the reducing power and energy from the light reaction. A fixed stoichiometry ratio of 2 molecules NADPH and 3 molecules ATP generated per molecule O2 produced during linear electron transport in chloroplast was hypothesized, which exactly fits the requirements of Calvin cycle (Kliphuis et al., 2012). A fraction of the GAP is moved to the cytosol, where it enters the glycolysis, tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) to synthesize precursors of macromolecules such as lipids and proteins. The additional reductants and ATP for biosynthesis of lipids and proteins from the sugar C skeletons may be obtained from photosynthesis or from light respiration, whose proportion is unclear due to the little knowledge of the microalgal metabolic pathways (Johnson and Alric, 2013). As assumed above, CO2 fixation in Calvin cycle consumed all NADPH and ATP generated in light reaction. Therefore, the additional energy for more reduced component synthesis came only from light respiration for a simplification. According to Appendix B, in glycolysis and TCA cycle, 1 molecule GAP is oxidized and completely decomposed, generating 5 molecules NADH, 1 molecule FADH2, 3 molecules CO2 and 3 molecules ATP. In the electron transport chain, the relationship between ATP synthesis and reductant consumption was assumed to be as follows: 1 molecule NADH yields 2.5 molecules ATP with 0.5 molecules O2 consumption and 1 molecule FADH2 yields 1.5 molecules ATP with 0.5 molecules O2 consumption. Therefore, when 1 molecule GAP is completely oxidized and decomposed, it will consume 3 molecules O2 and generate 3 molecules CO2 and 17 molecules ATP, among which 14 molecules ATP come from
89
oxidative phosphorylation and 3 molecules ATP come from substrate phosphorylation. It was assumed that NADPH was only generated in PPP, and 2 molecules NADPH were produced per molecule CO2 released. NADPH and part of NADH were directly involved in the reduction reaction for biomass production. Based on these assumptions, the quantitative analysis of intracellular ATP could be performed. 3. Results and discussion 3.1. Biomass concentration prediction for NR and NL cultures The initial cell concentrations were set as 0.24 g/L in both NR and NL cultures. In NL culture, the extracellular nitrate was depleted at day 4. However, the intracellular nitrogen pool could support further cell growth (Li et al., 2008). The cell growth rate of Nannochloropsis sp. in NL cultures kept the same as that in NR culture until day 6, and then slowed down. Due to the limited culture time, the cell growth was not completely halted at the end of NL culture. The calculated results fitted well with the experimental measurements in both NR and NL cultures. Generally, the relative deviations between the experimental measurements and calculated values were less than 8%. Additional, in NR culture, the absorption rate of nitrate was proportional to CO2 absorption, which was set as 0.125 mol N/mol CO2, according to the intracellular element composition. In NL culture, Michaelis–Menten equation for the nitrate absorption was fitted based on the nitrate concentrations in medium measured by ion chromatograph. The maximal uptake rate (vm) and half-saturation constant (Ks) were 1.802 g N/mol CO2 and 0.0082 g N/L, respectively. 3.2. Carbon flux analysis To understand the carbon metabolism of algal cells, the light respiration was investigated, which occurs simultaneously with photosynthesis in eukaryotic microalgal growth. The change of light respiration rate (RL) with the gross phostosynthesis rate (PG) of Nannochloropsis sp. is shown in Fig. 1. Light respiration was enhanced with the increase of photosynthesis rate, which was consistent with a previous report showing that higher gross photosynthesis rate led to higher respiratory activity for fast growth rate (Kliphuis et al., 2011). The relationship between RL and PG was also investigated under different intracellular nitrogen quota (qn). Fig. 1 shows that RL decreased significantly with nitrogen starvation. The slopes of RL to PG are almost constant, irrespectively of qn. However,
Fig. 1. Relationship between RL and PG of Nannochloropsis sp. for different qn.
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Fig. 2. Time course of the photosynthesis rate, the respiration rate and the qn of Nannochloropsis sp. [(—) PG; (- - -) RL; () calculated qn, (h) measured qn; black for NR culture; red for NL culture]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the intercepts, denoted by Rm, change significantly with different qn. There is a linear relationship between Rm and qn (Rm = 0.0833 qn – 0.0002, R2 = 0.99). As depicted in Fig. 2, in NR and NL cultures, through O2 evolution rate and qn monitored and calculated on-line, PG and RL were obtained. The gross photosynthesis rate (PG) decreased rapidly with culture time in both NR and NL cultures, mainly due to light limitation. Early in the two cultures, the values of PG were almost the same. From day 4, PG of NL culture became lower due to nitrogen depletion. The decrease of PG did not mean the decrease of growth rate because the biomass concentration in NL culture was the same as in NR culture in day 4 and day 5. Because in NL culture, RL decreased with nitrogen depletion, thus the decreasing CO2 loss in respiration relieved the decreasing CO2 fixation in the weakening photosynthesis. In addition, nitrogen starvation may decrease the degree of reduction of de novo biomass, suggesting that the same O2 evolution may result in more CO2 fixation, and thereby compensate for the low photosynthesis rate compared with NR condition (Jakob et al., 2007). Therefore, the decrease of cell growth
Fig. 3. PQ, carbon flux and component contents of Nannochloropsis sp. in NR and NL cultures [h, and for measured data of protein content, lipid content, and carbohydrate content, respectively, and the lines in corresponding colors for calculated results; green line for calculated nucleic acids content; a for NR culture; b for NL culture]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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rate was hysteretic compared with the decrease of photosynthesis rate under nitrogen starvation. As shown in Fig. 2, the respiration activity decreased with the decrease in photosynthesis, but mainly depended on intracellular nitrogen quota (qn). The initial value of qn was set as 0.08 g/gdcw, and the calculated results of qn fitted well with the measurements. The relative deviations between experimental measurements and calculated values were less than 5%. The time courses of PQ and CB, CR, CE calculated on the analysis of carbon flux in NR and NL cultures are shown in Fig. 3. CB decreased and CO2 loss in respiration increased with culture time in both cultures because of the decrease of photosynthesis rate in limited light. It can be seen that CB was only 30–60% of the total carbon fixed in Calvin cycle, and the rest was expelled out through decarboxylation reactions in light respiration. In ND culture, nitrogen depletion reduced the respiration rate. Therefore, CB of NL culture did not decrease as rapidly as CB of NR culture. The carbon in the CR part was responsible for supplying reductants for reduced component synthesis. CR/CB represented the reductants requirement per molecule biomass production. Nitrogen depletion caused protein synthesis arrest, whereas lipid and carbohydrate synthesis may be induced, thereby reducing the degree of biomass reduction. In Fig. 3, it is shown that PQ is lower in late period of NL culture, and the corresponding CR/CB of NL culture is lower than that of NR culture. The carbon fixed for biomass materials was used to produce proteins, lipids, carbohydrates and nucleic acids, whose contents were reflected by PQ. PQ of NL culture started to decrease from day 3, indicating that the intracellular carbon pathway changed, because extracellular nitrogen source was insufficient to support protein and nucleic acid synthesis. The proportion of carbon flux to lipids and carbohydrates increased. PQs of lipids and carbohydrates were 1.383 and 1.0, respectively, according to Appendix C. The value of PQ was approximately 1.35 after nitrogen depletion, suggesting that lipid synthesis was predominant in carbon partition. For calculation of protein and nucleic acid contents, their synthesis coefficients were set as 5.512 g/g N and 0.290 g/g N, respectively. The contents of major intracellular components are shown in Fig. 3a3 and b3. The calculation results fitted the experimental measurement very well. The absolute deviations between experimental measurements and calculated values of contents of protein, lipids, and carbohydrates were less than 0.04 g/g in general. Eriksen et al. reported that PQ depended on the composition of the produced biomass and the type of nitrogen source, independent on other environmental conditions (Eriksen et al., 2007). The models in this study could successfully describe the dynamic change of microalgae growth in batch cultures. 3.3. Energy flux analysis Besides the energy required for organic materials synthesis, additional energy is required for growth-associated maintenance (GAM) and non-growth-associated maintenance (NGAM) (Baart et al., 2008; Kliphuis et al., 2012). The energy parameters for biomass formation and maintenance of Nannochloropsis sp. in NR and ND cultures were analyzed, and the results are presented in Fig. 4. The energy in the form of ATP required per molecule C-biomass production increased rapidly with culture time, because of the increasing maintenance energy. The ATP demand for maintenance was calculated based on the relationship between RL and PG (Fig. 1). The intercept represents the respiration consumption with no cell growth, i.e., the maintenance requirement. Based on the reactions in Appendix B, for 1 unit of O2 consumption in respiration, 1 unit of carbon (GAP) degraded to CO2, and yielded 17/3 units of ATP. The ATP requirement for maintenance was determined to be approximately 2.2 mmol/gh in NR culture, and 2.1–0.98 mmol/gh in NL culture, which decreased with nitrogen
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Fig. 4. Intracellular ATP consumption per mole C-biomass production of Nannochloropsis sp. in NR and NL cultures [a for NR culture; b for NL culture].
starvation. Kliphuis et al. worked out that the ATP required for the maintenance of C. reinhardtii was 2.85 mmol/gh (Kliphuis et al., 2012), similar to the value obtained in our study. In late culture period, high cell concentration and low specific cell growth rate resulted in a low fraction of energy for growth. The maintenance could account for more than 60% of total energy demand. Therefore, cultivating microalgae at high specific growth rate was important to attain high biomass yield on energy. Wilhelm and Jakob (2011) presented that the theoretical ATP requirement on the base of biosynthetic pathways for the main cell components were 3.67 units of ATP for carbohydrates, 6.33 units of ATP for lipids, and 5.33 units of ATP for proteins on 1 unit of carbon. As shown in Fig. 4, our experimental results for ATP requirement of biomacromolecules synthesis (growth minus GAM) was around 5 molecules ATP per molecule C-biomass production, which were in the range of 3.67–6.33. In NL culture, protein synthesis stopped and the accumulations of carbohydrates and lipids were accelerated after the nitrogen depletion. Lipids can store more ATP than other macromolecules. Therefore, in nitrogen starvation, lipids accumulate to prevent the photo-oxidative damage of the cell by incorporating the excess of energy over growth demand (Breuer et al.,
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2013). However, as shown in Fig. 4, the ATP demand for biomacromolecules did not vary significantly, indicating that the cell capacity for energy storage did not increase in nitrogen starvation condition, mainly because the increase in carbohydrate synthesis occurred along with lipid accumulation, lowering the energy fixation. The energy for NGAM in NR and NL cultures appeared to be constant in the culture process. 4. Conclusion Carbon and energy metabolism of Nannochloropsis sp. in NR and NL cultures were analyzed on-line. Carbon fixed in Calvin cycle was quantitatively divided into three parts: for biomass, for reductants and for energy. The main biomass components contents were calculated on-line, fitted well with the measurements. The energy parameters for biomass production and cell maintenance were also obtained. Therefore, the photobioreactor culture system and calculation method presented here could be used to gain some dynamic information on microalgae metabolism. Acknowledgements This study was supported by Twelfth-five-year National Key Task program (2011BAD14B02), 863 Program (2013AA065801) and Natural Science Foundation of China (21376246). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.04. 083. References Baart, G.J.E., Willemsen, M., Khatami, E., de Haan, A., Zomer, B., Beuvery, E.C., Tramper, J., Martens, D.E., 2008. Modeling Neisseria meningitidis B metabolism at different specific growth rates. Biotechnol. Bioeng. 101 (5), 1022–1035. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2013. Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus. Bioresour. Technol. 143, 1–9. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2012. The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour. Technol. 124, 217–226. Carvalho, A.P., Monteiro, C.M., Malcata, F.X., 2009. Simultaneous effect of irradiance and temperature on biochemical composition of the microalga Pavlova lutheri. J. Appl. Phycol. 21 (5), 543–552. Chen, M., Tang, H., Ma, H., Holland, T.C., Ng, K.Y.S., Salley, S.O., 2011. Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta. Bioresour. Technol. 102 (2), 1649–1655. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Cornet, J.F., Dussap, C.G., Gros, J.B., 1998. Kinetics and energetics of photosynthetic micro-organisms in photobioreactors. In: Scheper, T. (Ed.), Advances in Biochemical Engineering Biotechnology. Springer, Heidelberg, pp. 153–222. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28 (3), 350–356. Eriksen, N.T., Riisgard, F.K., Gunther, W.S., Iversen, J.J.L., 2007. On-line estimation of O2 production, CO2 uptake, and growth kinetics of microalgal cultures in a gastight photobioreactor. J. Appl. Phycol. 19 (2), 161–174.
Geider, R.J., MacIntyre, H.L., Kana, T.M., 1998. A dynamic regulatory model of phytoplanktonic acclimation to light, nutrients, and temperature. Limnol. Oceanogr. 43 (4), 679–694. Guillard, R.R., Ryther, J.H., 1962. Studies of marine planktonic diatoms.1. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8 (2), 229–239. Jakob, T., Wagner, H., Stehfest, K., Wilhelm, C., 2007. A complete energy balance from photons to new biomass reveals a light- and nutrient-dependent variability in the metabolic costs of carbon assimilation. J. Exp. Bot. 58 (8), 2101–2112. Johnson, X., Alric, J., 2013. Central carbon metabolism and electron transport in Chlamydomonas reinhardtii: metabolic constraints for carbon partitioning between oil and starch. Eukaryotic Cell 12 (6), 776–793. Kliphuis, A.M.J., Janssen, M., van den End, E.J., Martens, D.E., Wijffels, R.H., 2011. Light respiration in Chlorella sorokiniana. J. Appl. Phycol. 23 (6), 935–947. Kliphuis, A.M.J., Klok, A.J., Martens, D.E., Lamers, P.P., Janssen, M., Wijffels, R.H., 2012. Metabolic modeling of Chlamydomonas reinhardtii: energy requirements for photoautotrophic growth and maintenance. J. Appl. Phycol. 24 (2), 253–266. Kroon, B.M.A., Thoms, S., 2006. From electron to biomass: a mechanistic model to describe phytoplankton photosynthesis and steady-state growth rates. J. Phycol. 42 (3), 593–609. Langner, U., Jakob, T., Stehfest, K., Wilhelm, C., 2009. An energy balance from absorbed photons to new biomass for Chlamydomonas reinhardtii and Chlamydomonas acidophila under neutral and extremely acidic growth conditions. Plant Cell Environ. 32 (3), 250–258. Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C.Q., 2008. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biotechnol. 81 (4), 629–636. Liang, K., Zhang, Q., Gu, M., Cong, W., 2013. Effect of phosphorus on lipid accumulation in freshwater microalga Chlorella sp.. J. Appl. Phycol. 25 (1), 311–318. Liu, J., Yuan, C., Hu, G., Li, F., 2012. Effects of light intensity on the growth and lipid accumulation of microalga Scenedesmus sp. 11-1 under nitrogen limitation. Appl. Biochem. Biotechnol. 166 (8), 2127–2137. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1), 265–275. Lv, J.M., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L., 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101 (17), 6797–6804. Mairet, F., Bernard, O., Masci, P., Lacour, T., Sciandra, A., 2011. Modelling neutral lipid production by the microalga Isochrysis aff. galbana under nitrogen limitation. Bioresour. Technol. 102 (1), 142–149. Packer, A., Li, Y., Andersen, T., Hu, Q., Kuang, Y., Sommerfeld, M., 2011. Growth and neutral lipid synthesis in green microalgae: a mathematical model. Bioresour. Technol. 102 (1), 111–117. Pal, D., Khozin-Goldberg, I., Cohen, Z., Boussiba, S., 2011. The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp.. Appl. Microbiol. Biotechnol. 90 (4), 1429–1441. Pirt, S.J., 1965. The maintenance energy of bacteria in growing cultures. Proc. R. Soc. Lond. B 163 (991), 224–230. Quinn, J.C., Yates, T., Douglas, N., Weyer, K., Butler, J., Bradley, T.H., Lammers, P.J., 2012. Nannochloropsis production metrics in a scalable outdoor photobioreactor for commercial applications. Bioresour. Technol. 117, 164–171. Rebolloso-Fuentes, M.M., Navarro-Perez, A., Garcia-Camacho, F., Ramos-Miras, J.J., Guil-Guerrero, J.L., 2001. Biomass nutrient profiles of the microalga Nannochloropsis. J. Agric. Food Chem. 49 (6), 2966–2972. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A.G., 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21 (3), 277–286. Takache, H., Pruvost, J., Cornet, J.-F., 2012. Kinetic modeling of the photosynthetic growth of Chlamydomonas reinhardtii in a photobioreactor. Biotechnol. Prog. 28 (3), 681–692. Taymaz-Nikerel, H., Borujeni, A.E., Verheijen, P.J.T., Heijnen, J.J., van Gulik, W.M., 2010. Genome-derived minimal metabolic models for Escherichia coil MG1655 with estimated in vivo respiratory ATP stoichiometry. Biotechnol. Bioeng. 107 (2), 369–381. Wang, B., Li, Y., Wu, N., Lan, C.Q., 2008. CO(2) bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Wilhelm, C., Jakob, T., 2011. From photons to biomass and biofuels: evaluation of different strategies for the improvement of algal biotechnology based on comparative energy balances. Appl. Microbiol. Biotechnol. 92 (5), 909–919.
