Carbon metabolism and energy conversion of Synechococcussp. PCC 7942 under mixotrophic conditions: comparisonwith photoautotrophic condition

Riming Yan & Du Zhu & Zhibin Zhang & Qingui Zeng &

Ju Chu

Received: 29 October 2010 /Revised and accepted: 15 April 2011 /Published online: 13 May 2011# Springer Science+Business Media B.V. 2011

Abstract To investigate the carbon metabolism and energyconversion efficiency of the cyanobacterium Synechococ-cus sp. PCC 7942 under mixotrophic conditions, we studiedits growth characteristics in mixotrophic cultures withglucose and with acetate, respectively, and further discussedthe carbon metabolism and energy utilization based onmetabolic flux analysis. Results showed that both glucoseand acetate could enhance the growth of Synechococcus sp.PCC 7942. The metabolic flux through the glycolyticpathway, tricarboxylic acid cycle, and mitochondrial oxi-dative phosphorylation was affected by the two organicsubstrates. Additionally, the cellular composition was alsomodulated by glucose and acetate. Under mixotrophicconditions, glucose exerts more significant impact on thediminishment of photochemical efficiency. Although thecontribution of light energy was smaller, the cell yieldsbased on total energy in mixotrophic cultures were highercompared with that of photoautotrophic one. On the basis

of chlorophyll fluorescence analysis, the actual energyconversion efficiencies based on ATP synthesis in thephotoautotrophic, glucose-mixotrophic, and acetate-mixotrophic cultures were evaluated to be 4.59%, 5.86%,and 6.60%, respectively.

Keywords Synechococcus sp. PCC 7942 .Mixotrophiccultivation . Carbon metabolism . Energy conversion .

Metabolic flux analysis . Chlorophyll fluorescence analysis

Nomenclatureaij Stoichiometric coefficient of metabolite i in the

jth reaction (−)A m×n Matrix of stoichiometric coefficient (−)A0 Side area of flask receiving incident light (m2)Eab Light energy absorbed by the biomass unit

(kJ g−1 h−1)EP Actual light energy for photosynthesis (kJ

g−1 h−1)EC Chemical energy received from organic carbon

resources (kJ g−1 h−1)EC,ACE Chemical energy originated from acetate

(kJ g−1 h−1)EC,GLC Chemical energy originated from glucose

(kJ g−1 h−1)ET The total absorbed energy (kJ g−1 h−1)I0 Incident light intensity (μmol m−2s−1)Ka Effective absorption coefficient of light (m−1)r Vector of m-dimensional metabolite accumula-

tion rate (mmol g−1 h−1)rATP Relative rate of ATP synthesized (mmol g−1 h−1)rACE Relative rate of acetate consumption (mmol

g−1 h−1)rex Subvector of extracellular metabolite accumula-

tion rate (mmol g−1 h−1)

R. Yan :D. Zhu : Z. Zhang :Q. ZengKey Laboratory of Protection and Utilization of Subtropic PlantResources of Jiangxi Province, Jiangxi Normal University,Nanchang 330022, China

R. Yan : J. ChuState Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237, China

J. Chue-mail: juchu@ecust.edu.cn

D. Zhu (*)Key Laboratory for Research on Active Ingredients in NaturalMedicine of Jiangxi Province, Yichun University,Yichun 336000, Chinae-mail: zhudu12@163.com

J Appl Phycol (2012) 24:657–668DOI 10.1007/s10811-011-9683-2

rGLC Relative rate of glucose consumption (mmolg−1 h−1)

ri Accumulation rate of metabolite i (mmol g−1 h−1)rin Subvector of intracellular metabolite accumula-

tion rate (mmol g−1 h−1)R Reactor radius (m)v n-Dimensional flux vector (mmol g−1 h−1)vj Flux through reaction j (mmol g−1 h−1)X Cell concentration DCW (g L−1)YATP Yield of cell mass based on ATP generation

(g mol-ATP−1)YE Cell yield based on total light energy (g kJ−1)ΔGo

ACE Free energy change of acetate complete oxidation(kJ mol−1)

ΔGoATP Free energy change of ATP hydrolysis (kJ mol−1)

ΔGoGLC Free energy change of glucose complete oxida-

tion (kJ mol−1)μ Specific growth rate (h−1)8 Vector of measurement noise variance–

covariance (−)ΦPSII The efficiency of photosystem II photochemistry

(−)ΨATP The efficiency of energy conversion on ATP

synthesis (%)

Introduction

Synechococcus is one of the largest genera of theprokaryotic blue-green algae (cyanobacteria). It hasattracted extensive attention in recent years because ofthe potential application for industrial CO2 removal andproduction of many valuable metabolites. Synechococcussp. PCC 7942 (previously known as Anacystis nidulansR2) holds a special place in many research fields ofcyanobacteria such as acquisition of inorganic carbon(Tchernov et al. 2001), transport and regulation ofnitrogen compounds (Vazquez-Bermudez et al. 2002),response to iron deprivation (Sandstrom et al. 2002),adaptation to environmental variations (Sarcina et al.2001), etc. Synechococcus sp. PCC 7942, can be alsoreliably transformed by exogenously added DNA. As aphotoautotroph, the growth of Synechococcus sp. PCC7942 strictly depends on incident light. Its cultivation isstill difficult because the available light energy isinsufficient in low light, whereas photoinhibition occursin high light. Mixotrophic or heterotrophic cultivation areappropriate ways to solve this problem (Lee et al. 1987).However, Synechococcus sp. PCC 7942 has been regardedas an obligate photoautotroph (Kratz and Myers 1955;Zhang et al. 1998), and many studies have been done inphotoautotrophic cultures (e.g., Tsinoremas et al. 1994;Sauer et al. 2001; Bogos et al. 2010). Despite of that, there

have been studies which suggest that some obligatephotoautotrophic cyanobacteria can assimilate organiccarbon sources (Hoare et al. 1967; Pelroy et al. 1972;Pearce and Carr 1967; Khoja and Whitton 1971).Takahashi et al. (1998) initially found that, underillumination and nitrogen-starvation, a supplement ofacetate could drastically improve poly-3-hydroxybutyrate(PHB) productivity of Synechococcus sp. PCC 7942.Kang et al. (2004) also found that glucose could markedlyaccelerate growth and enhance the photosynthetic rate inmixotrophic culture. These studies indicate that Synechococcuscould grow mixotrophically. However, there are few papersdealing with the mechanism of growth promotion ofSynechococcus cells under mixotrophic conditions. Therefore,it is important to probe the growth characteristics and thecarbon flux distribution in Synechococcus sp. PCC 7942aiming to improve productivity.

In the light, mixotrophic cells of microalgae can utilizelight and organic substrates as energy sources and convert thelight energy into chemical energy through photosynthesis.Extensive studies have focused on light energy utilizationunder photoautotrophic, mixotrophic, and heterotrophic con-ditions (e.g., Aiba 1982; Molina et al. 1997; Miyake et al.1999; Benemann 2000; Yang et al. 2000). However, therehave been few studies dealing with the energetics and carbonmetabolism of Synechococcus sp. PCC 7942 under mixo-trophic conditions.

As a powerful tool for evaluating metabolic distribu-tion within cells, intracellular flux analysis has beenapplied to many microbial cultures for the purpose ofimprovement of production processes (Vallino andStephanopoulos 1993; Shi et al. 1997; Melzer et al.2007; Iwatani et al. 2008). Knowledge of intracellular fluxdistributions under different conditions makes it possibleto understand the fundamental metabolism in detail andgives useful information which can be applied to bioreac-tor design and control strategies of culture systems. Withrespect to evaluating energy utilization in microalgalcultures, metabolic flux analysis (MFA) was employed toinvestigate the light energy utilization of Chlorellapyrenosidosa (Yang et al. 2000), Synechocystis sp.PCC6803 (Yang et al. 2002), and Synechococcus sp.MA19 (Nishioka et al. 2002). These studies provided afundamental understanding of the energy conversion fromthe supplied energy to biomass formation. Since theenergy supplied by light cannot entirely be absorbed byphotosynthetic organisms, the energetic yield and conver-sion efficiency of microalgal cells in those studies wereunderestimated because they were calculated based ontotal supplied light energy instead of actual absorbed lightenergy. Therefore, it is appropriate to take the photochem-ical efficiency into account when calculating the energeticefficiency of photosynthetic microalgae cells.

658 J Appl Phycol (2012) 24:657–668

In the present study, Synechococcus sp. PCC 7942 wascultivated under photoautotrophic and mixotrophic condi-tions using glucose and acetate separately as carbonsources. MFA was applied to elucidate the metabolism ofcells grown on different carbon and energy sources. Basedon the results of flux distribution, the utilization propertiesand conversion efficiency of energy are discussed in termsof ATP formation.

As one of the most common and useful techniques inphotosynthesis research, the chlorophyll fluorescencesignals can provide rapid, real-time information onphotosynthesis and light utilization (Campbell et al.1998). In the present paper, the actual light energyconversion of Synechococcus sp. PCC 7942 was evaluatedon the basis of MFA and chlorophyll fluorescence analysis(CFA).

Materials and methods

The cyanobacterium Synechococcus sp. PCC 7942 wasobtained from the FACHB-Collection of the Institute ofHydrobiology, Chinese Academy of Sciences. Themineral culture medium used was BG11 basal mediumaccording to Zhu et al. (2003). Different concentrationsof glucose (from 2 to 10 g L−1) or acetate (from 0.5 to5 g L−1) were added to the culture medium for themixotrophic conditions. The cells were grown in a 5.0-Lbatch bioreactor (ALG-XL, Guoqiang BioengineeringEquipment Co., Ltd, Shanghai) with a working volumeof 3.0 L. The culture was illuminated by the parallelfluorescent lamps (Philips, 8 W) around the bioreactor,giving an incident light intensity of 47.3 μmol photonsm−2 s−1 on the surface of the medium, which representslight-limiting conditions. Air was introduced into thevessel at a flow rate of 1.5×10−3 m3h−1, and cellsuspension was agitated at 50 revolutions min−1 by amagnetic drive. Temperature was kept at 28°C, and themedium pH was controlled at 9–10.

For the determination of cell dry weight, duplicatesamples of the culture were washed with 0.5 M HCl, rinsedwith distilled water, and dried overnight at 105°C. Theamounts of glucose and acetate in the culture medium weredetermined with a glucose kit and gas chromatography,respectively. The chlorophyll and carotenoid concentrationswere measured upon pigment extraction in 100% methanolat 4°C overnight (Lichtenthaler 1987). Total lipids wereextracted with chloroform/methanol and weighed (Piorrecket al. 1984). The amount of protein in cell extracts wasdetermined by Folin phenol reagent using bovine serumalbumin as a standard. The total carbohydrate contentin the suspension of cell extracts was measured usingphenol sulfuric acid method (Dubois et al. 1956). A

CO2/O2 gas analyzer (Model PA200, Sichuan InstrumentCo. Ltd., Chongqing) was used to measure the CO2

uptake rate and the O2 evolution rate of cells. Incidentlight intensity, I0, was measured with a PAR sensor(Model FGH-1, Photoelectric Instrument Factory ofBeijing Normal University, China). The chlorophyllfluorescence parameter of the photochemical efficiency(ΦPSII) of photosystem II (PSII) was measured using anAquaPen (AP100, Photon Systems Instruments, CzechRepublic).

The light energy absorbed by the biomass unit or thespecific light energy absorption rate, Eab, was calculatedaccording to the following equation (Iehana 1990; Yang etal. 2000).

Eab ¼ 2I0pRX

1�Z p=2

0cos f exp �2RKaX cos f½ �df

!ð1Þ

where I0 is the incident light intensity, R indicates thereactor radius, and X represents the cell concentration. Theeffective absorption coefficient of light, Ka, was determinedfrom the data of the light transmittance against light pathlength measured at various cell concentrations.

Metabolic network

The central metabolic network of Synechococcus sp. PCC7942 cells grown under mixotrophic conditions is shownin Fig. 1. The main pathway of carbon metabolism inSynechococcus sp. PCC 7942 (Yan et al. 2009) includesglycolysis, pentose phosphate pathway, incomplete tricar-boxylic acid (TCA) cycle, and photophosphorylation, andthe glucose or acetate is utilized as organic carbonsources. In the glucose-mixotrophic culture, glucose ismetabolized through glycolysis, pentose phosphate (PP)pathway, and TCA cycle with CO2 release by respiration,whereas CO2 is fixed through photosynthesis (Calvincycle). As for acetate-mixotrophic condition, acetatekinase (AK) activates acetate in an ATP-dependentreaction to acetyl phosphate, and further formed acetyl-CoA by phosphotransacetylase (PTA; Pearce and Carr1967). Moreover, ATP as an energetic compound ismainly produced in three metabolic pathways—substratephosphorylation (direct ATP), oxidative phosphorylation,and photophosphorylation. In the first pathway, ATP isproduced from the metabolites reactions in all pathways.In the second, ATP is generated by using NADH or FADHvia the electron transport chain with O2 as a terminalelectron acceptor in mitochondria. As in the last pathway,on the other hand, ATP synthesis occurs in the course ofconverting light energy into chemical energy through thephotosynthetic light reactions in chloroplasts.

J Appl Phycol (2012) 24:657–668 659

Calculation of metabolic flux

A stoichiometric model combined with measurements ofsubstrate and extracellular products was applied to theestimation of intracellular metabolite fluxes. The metaboliteaccumulation rates can be expressed as follows.

ri ¼X

aijvj i¼ 1; 2;:::;m; and j¼ 1; 2;:::;nð Þ ð2Þ

where ri is the accumulation rate of a metabolite i, aij astoichiometric coefficient, and vj the flux through jthreaction. The metabolite i and reaction j involved in themetabolic network in Synechococcus sp. PCC 7942 cellsare specified in Appendices A and B, respectively. Basedon the mass balances of respective metabolites, thefollowing equation can be obtained:

Av ¼ r ð3Þ

G6P

Ru5P

F6P

X5P R5P

GAP

PGA

RuDP

PEP

AcCoA

CO2

CO2

CO2

CO2

PYR

OAAISOCIT

¦Á-KGCO2

E4P

S7P

v5 v11

v4

v3v7

v8v6

v1

v16v2

v17

v21

v20

v19

v9v10

ATPADP

NADP NADPH

ADPATP

v18 NADNADH

NADNADH

FUM

SUCCoA

NADHNAD

FADH2

FAD

ATP

ADP

v22

v23

NADH 2 ATP

0.5 O2

FADH2 ATP

0.5 O2

2NADPH

v26

v272H2O O2+H

2NADP 2NADPH

2ADP 2ATP

v28

ADPATP

ACE AcPv24 v25

ADPATP

Biomass

2NADP

GLC

v12ADP

ATP

Fig. 1 Central metabolic path-way in Synechococcus sp. PCC7942 cells under mixotrophicconditions

660 J Appl Phycol (2012) 24:657–668

where A is an m×n matrix of stoichiometric coefficients, van n-dimensional flux vector, and r an m-dimensionalmetabolite accumulation rate vector. The weighted least-square solution to Eq. 4, provided that A is of full rank, isobtained as follow:

ð4Þ

where 8 is the matrix of measurement noise variance–covariance concerning the vector r.

The elements of r were divided into two subvectors, rexand rin, which correspond to extracellular and intracellularcompounds, respectively. The rates of production orconsumption for extracellular compounds (elements of rex)were directly measured. The elements of rin correspondingintracellular metabolites were set to zero by steady-state forconcentration of intracellular metabolites. In the presentpaper, MFA was performed at 120 h because Synechococ-cus sp. PCC 7942 was grown in light-limited conditions,and the growth rate was approximately stable. The fluxdistribution under the photoautotrophic and mixotrophicconditions was calculated using MATLAB® version7.0.0.19920 (R14).

The elements of r for the present case are given inAppendix A, where the measured extracellular compoundsare cell mass, CO2, and O2. Detailed representation ofmetabolic reactions, which yield the values of stoichiomet-ric coefficients in matrix A, is shown in Appendix B.

Analysis of energy utilization

Under photoautotrophic and mixotrophic conditions, lightis the vital energy source for cell growth and metabolitesynthesis. The light energy absorbed by the light-harvestingantennae is dispatched into four competitive pathways—photochemical reactions, heat dissipation, fluorescenceemission, and energy transfer (Campbell et al. 1998). Onlya limited fraction of light energy is used for photochemistry.Therefore, it is appropriate to take the photochemicalefficiency of photosystem II, ΦPSII, into account whenevaluating the efficiency of light utilization. So, the actuallight energy for photosynthesis, EP, can be defined asfollows:

EP ¼ ΦPSIIEab ð5ÞTo evaluate the actual cell yield based on energy in the

mixotrophic culture, the total absorbed energy, ET, includeslight energy absorbed by the cell and chemical energyreceived from organic carbon resources:

ET ¼ EP þ EC ð6Þwhere EC is the chemical energy received from organicsubstrates. In the present paper, this energy originated from

glucose (EC,GLC) or acetate (EC,ACE), and calculated asfollows:

EC;GLC ¼ rGLC � �ΔGoGLC

� � ð7Þ

EC;ACE ¼ rHAC � �ΔGoACE

� � ð8Þwhere rGLC and rACE are related to the flux values ofglucose and acetate uptake, respectively. The values ofΔGo

GLC= −2,803 kJ mol−1 and ΔGoACE= −874.4 kJ mol−1

were adopted in the present study.The light and chemical energy captured by the cell are

converted into ATP for cell synthesis. Therefore, the cellyield on energy plays a vital role in investigating theefficiency of energy utilization. In the present paper, thecell yield on ATP generation and on total energy, YATP andYE, were defined as follows:

YATP ¼ m=rATP ð9Þ

YE ¼ mET

ð10Þ

where μ is the specific growth rate of Synechococcus sp.PCC 7942 and rATP is the ATP generation rate.

To further understand the energy conversion ofSynechococcus sp. PCC 7942, the efficiency of energyconversion on ATP formation, ΨATP , was introduced inthis study:

ΨATP ¼ rATP � �ΔGoATP

� �ET

� 100 ð11Þ

In Eq. 11, the value of ΔGoATP=−29.3 kJ mol−1 was

adopted in the present paper.

Results and discussion

Batch cultures under mixotrophic conditions

Batch cultures of Synechococcus sp. PCC 7942 wereconducted under different mixotrophic conditions in orderto collect data for the metabolic analysis. Mixotrophiccultures were performed at an irradiance of 47.3 μmolphotons m−2 s−1 and different concentrations of glucose oracetate. The effect of organic substrate concentrations onmixotrophic growth over 10 days is shown in Fig. 2. It wasfound that both 4.0 g L−1 glucose and 2.0 g L−1 acetateenhance the maximum cell growth. This result suggestedthat Synechococcus sp. PCC 7942 could grow in mixo-trophic culture in the presence of organic carbon at lowconcentration.

J Appl Phycol (2012) 24:657–668 661

v ¼ ATϕ�1A� ��1

ATϕ�1r

To further investigate the growth characteristics of Syne-chococcus sp. PCC 7942 in mixotrophic conditions, the cellswere grown separately in photoautotrophic and mixotrophiccultures at an irradiance of 47.3 μmol photons m−2 s−1. Asignificant improvement in the biomass concentration wasobserved exactly as expected. The growth rates of Synecho-coccus sp. PCC 7942 under mixotrophic conditions in thepresence of glucose or acetate were much higher comparedwith those from photoautotrophic growth. The addition,especially of acetate produced more significant effect ongrowth (Fig. 3), indicating the ability of Synechococcus sp.PCC 7942 to grow on organic carbon sources in mixotrophiccultures. When supplying light and organic carbon simulta-neously as energy sources, a much higher final cellconcentration was obtained.

Except for growth rate, the composition of mixotrophiccells was also different from that of photoautotrophic cellsat stationary phase. Carbohydrates increased in the presence

of glucose and higher lipid contents were attained in theculture with acetate. These results suggest that glucose andacetate play an important role in enhancing cell formationbecause they could serve as the precursors for carbohydrateand lipid synthesis, respectively. However, the mixotrophiccultures contained much less chlorophyll in comparison tothe photoautotrophic culture (Fig. 4). The decreasingchlorophyll in the mixotrophic cultures resulted from theinhibition by organic substrates (Stadnichuk et al. 1998).Furthermore, the decline of chlorophyll in the mixotrophiccultures reduced the dependence on light as the energyrequired for cell growth which now originated from bothlight and organic carbon. The decrease in chlorophyllcontent is also a way to relieve photoinhibition (Nakajimaand Ueda 1999).

Metabolic flux distribution in Synechococcus sp. PCC 7942

Based on the metabolic network model and the experimen-tal data, considerable metabolic information could beextracted from intracellular flux distributions. A compari-son of metabolic fluxes of Synechococcus sp. PCC 7942between photoautotrophic and mixotrophic cultures isshown in Fig. 5. The metabolic flux of carbon was dividedinto Calvin cycle and glycolytic pathway branches at thenode of phosphoglycerate acid, and more than 80% carbonmetabolism flowing into the former in the three cases. TheCalvin cycle predominated in the carbon flux distribution inboth photoautotrophic and mixotrophic cultures. An obvi-ous split was also found at the node of fructose-6-phosphate(F6P) in the PP pathway. The metabolic flux of formingglucose-6-phosphate (G6P) from F6P reverses in themixotrophic culture in the presence of glucose. Thisphenomenon may be explained by the excessive accumu-

Fig. 4 Main cellular composition of Synechococcus sp. PCC 7942cells under photoautotrophic and mixotrophic (with glucose or withacetate) conditions

Fig. 3 Comparison of Synechococcus sp. PCC 7942 growth underphotoautotrophic (black square) and mixotrophic (with glucose blackcircle; with acetate black triangle) conditions

Fig. 2 The effect of organic substrates concentration on the growth ofSynechococcus sp. PCC 7942 cell in mixotrophic cultures

662 J Appl Phycol (2012) 24:657–668

lation of G6P. It is widely known that the F6P is the onlypathway to form G6P for carbohydrate synthesis underphotoautotrophic conditions. However, the cell could takeup and assimilate glucose in the glucose-mixotrophicculture and then generate abundant G6P which is catalyzedby the hexokinase. Moreover, the abundant G6P drasticallyenhanced carbon flux through the glycolysis pathway and

produced a large amount of carbohydrates and ATP for cellsynthesis.

As the glyoxylate cycle does not exist in themetabolic network of Synechococcus sp. PCC 7942, theacetate is absorbed by cells and forms acetyl coenzyme A(AcCoA) in the presence of AK and PTA in the acetate-mixotrophic culture. The abundant AcCoA promotes the

G6P

Ru5P

F6P

X5P R5P

GAP

PGA

RuDP

PEP

AcCoA

CO2

CO2

CO2

CO2

PYR

OAAISOCIT

¦Á-KGCO2

E4P

S7PATPADP

NADP NADPH

ADPATP

NADNADH

NAD

NADH FUM

SUCCoA

NADHNAD

FADH2

FAD

NADH 2 ATP

0.5 O2

FADH2 ATP

0.5 O2

2NADPH

2H2O O2+H

2NADP 2NADPH

2ADP 2ATP

ADPATP

ACE AcP

ADPATP

Biomass

2NADP

GLC

ADP

ATP

100100100

176163177

77.764.677.7

6.1-0.45.9

2.63.02.5

100100100

32.031.731.7

65.465.365.8

32.732.733.0

32.732.632.8

22.534.620.1

15.123.49.9

5.98.67.7

10.717.34.0

00

14.3 2.73.64.7 0.25

0.190.22

546562

121102107

32.732.632.8

010.1

0

00

14.3

2.73.64.7

0.250.190.22

0.250.190.22

Fig. 5 Metabolic flux distribu-tion of Synechococcus sp. PCC7942 under photoautotrophicand mixotrophic conditions atculture time of 120 h. The fluxvalues are normalized by theflux for CO2 fixation (the fixa-tion rates were 1.46, 1.73, and1.80 mmol g−1 h−1 in photoau-totrophic, glucose-mixotrophic,and acetate-mixotrophic cul-tures, respectively), and theupper, middle, and lowernumerals are the flux valuescalculated from the photoauto-trophic, glucose-mixotrophic,and acetate-mixotrophic culture

J Appl Phycol (2012) 24:657–668 663

flux distribution through the TCA cycle and is partiallyresponsible for the carbon metabolism and cell synthesisunder mixotrophic conditions. On one hand, the superflu-ous AcCoA produces α-ketoglutarate (α-KG) via the TCAcycle for cell formation. On the other hand, it decreasesthe flux of AcCoA formation through glycolysis pathwayby feedback regulation, and the accumulated phospho-enolpyruvate and pyruvate flood to the outlet for cellsynthesis. Furthermore, as an important precursor, AcCoAcan enhance the productivity of lipids, which is consistentwith the results shown in Fig. 4. From the above analysis,it is suggested that as organic carbon sources, both glucoseand acetate are vital precursors for biomass formation inthe mixotrophic cultures. Since CO2 fixation correlateswith the Calvin cycle closely, the flux through the Calvincycle was constant regardless of the culture conditionswhen the metabolic flux was normalized by CO2 fixation.

Energy metabolism and conversion in the photoautotrophicand mixotrophic cultures based on MFA

Energy metabolism of Synechococcus sp. PCC 7942in the photoautotrophic and mixotrophic cultures

Table 1 shows the fluxes involved in the generation andutilization of ATP in the photoautotrophic, glucose-mixotrophic, and acetate-mixotrophic cultures based onthe data from the MFA. In the photoautotrophic culture,light is the sole energy source for cell growth. Undermixotrophic conditions, the energy is provided not only bylight but also by the organic carbon source taken up by thecells. Both of these are transformed into ATP and reducingpotential for various energy demands inside the cells. Theproduction of ATP is derived from three processes—photophosphorylation, substrate phosphorylation (directATP), and oxidative phosphorylation (Yang et al. 2000).Photophosphorylation was the predominant source forATP production regardless of the culture conditions.

About 60% of total ATP is derived from photophosphor-ylation in the mixotrophic cultures, which is less than thatin the photoautotrophic culture (87%). It is apparent that,in both mixotrophic cultures, a considerable fraction ofthe above 32% of the total ATP was formed fromoxidative phosphorylation, suggesting its non-negligiblerole in ATP production. Since the glucose and acetate aretaken up by the cell and promote the metabolic fluxthrough glycolysis and TCA cycle in the mixotrophiccultures, the activity of the respiratory chain is enhanced,and more ATP is formed from oxidative phosphorylationthan in the photoautotrophic culture. This result indicatesthat the organic substrates can also provide energy besidesbeing precursors for cell growth under mixotrophicconditions.

In terms of ATP consumption, the Calvin cycle is themain ATP sink, regardless of the trophic conditions,because it requires a large amount of ATP for CO2 fixation.The ATP demand for the assimilation of CO2 accounted forabout 81.5, 78.7, and 79.5% of the total in the photoauto-trophic, glucose-mixotrophic, and acetate-mixotrophic cul-tures, respectively. It was also found that the ATP demandfor cell synthesis in mixotrophy was larger than that ofautotrophy. This result is consistent with the cell growthdata in Fig. 3. Meanwhile, the ATP expenditure ofSynechococcus sp. PCC 7942 under mixotrophic conditionincludes not only the ATP demand for CO2 fixation, cellsynthesis, and maintenance, but also the energy requirementfor organic substrate uptake (Table 1).

As discussed above, light was the sole energy contrib-utor in the photoautotrophic culture, but both light andorganic substrates were energy sources for growth in themixotrophic cultures. In the three cases, the energyprovided by light and/or organic substrates was absorbedby the microalgal cells, harvested by the photosystems, andthen transformed into ATP for various energy demandsinside the cells. Thus, the conversion of total energy basedon ATP synthesis involves two energy forms, the energy

Photoautotrophic Mixotrophic

Glucose Acetate

ATP production

Photophosphorylation 4.15 3.37 4.13

Direct ATP 0.27 0.53 0.34

Oxidative phosphorylation 0.83 1.84 2.17

ATP consumption

Calvin cycle 3.59 4.35 5.34

Synthesis of cell mass 0.51 0.73 0.77

Glucose uptake – 0.17 –

Acetate uptake – – 0.28

Table 1 The generation andutilization of ATP in the photo-autotrophic and mixotrophiccultures of Synechococcus sp.PCC 7942 (mmolATP g−1 h−1)

664 J Appl Phycol (2012) 24:657–668

supplied by light and the energy derived from glucose/acetate. Figure 6 shows the energy contribution and thephotochemical efficiency in the photoautotrophic andmixotrophic cultures. The capture of light energy forphotosynthesis under photoautotrophic culture was about45% of the total absorbed light energy, higher than thatobserved under mixotrophic conditions (34% and 39%).The low energy availability in the mixotrophic culture wasthe result of the reduced photosynthetic pigments in thecells as shown in Fig. 4. Furthermore, the photosyntheticapparatus was disturbed due to the presence of organicsubstrate (Spring et al. 2009). Despite the inefficient lightenergy utilization in mixotrophy, light was still thepredominant contributor to growth. Light supplied about83 and 91% of the total energy in the mixotrophic culturewith glucose and acetate, respectively.

Estimation of energy conversion in the photoautotrophicand mixotrophic cultures

The energy economy of the algae cultures could beevaluated through the cell yield based on energy utilization.Although light is the predominant energy source in bothphotoautotrophic and mixotrophic cultures, only a smallportion of the absorbed light energy could be used forphotochemical reactions. Therefore, the photochemical

efficiency and the actual light energy for photosynthesisshould be taken into consideration when evaluating energyutilization. On the basis of MFA and the relative rate ofATP synthesized, the bioenergetic yields are shown inTable 2. It is apparent that the values of YE in bothmixotrophic cultures were much higher (7.52×10−3 and8.55×10−3 g kJ−1) than that in the photoautotrophic one(5.07×10−3 g kJ−1). Under mixotrophic conditions, theprocess of oxidative phosphorylation was prompted by thedegradation of organic substrates. Consequently, the bioen-ergetic yield through oxidative phosphorylation was higherthan that through the photophosphorylation, as the former ismore efficient. Not surprisingly, the energetic growth yieldin the photoautotrophic culture was lower due to theinefficient conversion of light energy into biomass asdiscussed above. Therefore, the difference in the wayenergy is supplied might result in the different bioenergeticyields. Since the two mixotrophic cultures were conductedwith rather low concentration of glucose or acetate, lightwas still the foremost energy source for biomass formationand ATP generation instead of the organic substrates.Accordingly, there was little difference in the values ofYATP between the mixotrophic and the photoautotrophiccultures, compared with the differences of YE.

To further investigate light energy utilization the effi-ciency of energy conversion was also studied. Table 2 alsoshows the efficiency of energy conversion based on ATPsynthesis. The values of ΨATP in the photoautotrophic,glucose-mixotrophic, and acetate-mixotrophic cultures were4.59, 5.86, and 6.60%, respectively. This indicates that theenergy conversion in mixotrophic conditions was moreefficient than that in photoautotrophic conditions. Asdiscussed above, the difference was due to the differentenergy sources supplied to the cultures. Apparently, thelight energy was more difficult to trap and convert to ATPby the cells, compared with the organic substrates. If theenergy conversion efficiency through photosynthetic elec-tron transport could be improved, a higher availability ofATP from the absorbed energy would be expected. It is alsonotable that the energy conversion efficiency recorded inthis paper was higher than that reported by Miyake and co-workers (Miyake et al. 1999; Yoon et al. 2006; Hata et al.2000). This difference might be caused by the different

Fig. 6 Energy absorption of Synechococcus sp. PCC 7942 in differentcultures. The photochemical efficiency of Photosystem II (whitecircle) and the percentage of energy contribution are also shown

Photoautotrophic Mixotrophic

Glucose Acetate

μ (h−1) 0.017±0.003 b 0.022±0.004 a 0.025±0.003 a

YE (10−3 g kJ−1) 5.07±0.11 c 7.52±0.15 b 8.55±0.23 a

YATP (g mol−1) 3.24±0.10 b 3.76±0.12 a 3.80±0.13 a

ΨATP (%) 4.59±0.23 c 5.86±0.22 b 6.60±0.27 a

Table 2 The energy utilizationof Synechococcus sp. PCC 7942in the photoautotrophic andmixotrophic cultures

Values in the same row followedby the same letter did not differsignificantly at P=0.05 (Tukey'stest)

J Appl Phycol (2012) 24:657–668 665

calculation methods of light energy utilization for micro-algae growth. The actual light energy for photosynthesisrather than the supplied energy or absorbed energy wasadopted in the present study. The energy supplied by lightcould not be absorbed and used for photochemistry entirelyby the algae. Therefore, the value of conversion efficiencywould be underestimated regardless of the light trappingefficiency and the photochemical efficiency as discussedabove. Actually, it has been suggested that the maximumthermodynamic efficiency of ATP formation from theactual absorbed energy was above 15% in a culture of C.pyrenosidosa (Yang et al. 2000).

From the above analysis, the investigation of lightconversion inefficiency of Synechococcus sp. PCC 7942 indifferent culture conditions should be a promising tool forimproving the photosynthetic productivity of microalgae.

Conclusion

Synechococcus sp. PCC 7942 has been regard as anobligate photoautotroph, although some studies found thatit was able to assimilate organic substrates in the presenceof light. In the present study, it was further proven thatSynechococcus sp. PCC 7942 was able to grow inmixotrophic cultures. Both glucose and acetate couldenhance cell growth, and the latter was more effective. Onthe basis of MFA, it was found that the metabolic fluxthrough the glycolytic pathway in mixotrophic cultures wasstimulated by glucose whereas it was depressed by acetate,while the flux through the tricarboxylic acid cycle increasedin both cases. Glucose was able to provide both chemicalenergy and precursors for cell formation, and acetate playsan important role in precursor supply for lipid synthesis aswell. Under mixotrophic conditions, both glucose andacetate reduced the photochemical efficiency, while glucosehas a more significant impact on the diminishment ofphotochemical efficiency. To compare the energy economyof Synechococcus sp. PCC 7942 in different cultures, theutilization and conversion efficiency of energy in photoau-totrophic and mixotrophic cultures were evaluated, basedon the MFA and CFA. The results revealed that the biomassyield on the total absorbed energy was the lowest in thephotoautotrophic cultivation and that the mixotrophiccultures displayed more efficient utilization of energy forbiomass productivity. These data also suggest that mixo-trophy is a potential way for high-density microalgaecultivation. These results may illuminate a new directionin that it is possible to cultivate in mixotrophic culture forthis supposedly obligate photoautotrophic alga. In addition,the analysis of the energy utilization efficiency in differentconditions has been shown to be very useful for providinginformation regarding carbon source utilization and energy

conversion and guidance to improve the algal cell cultureperformance.

Acknowledgements This work was supported by National NaturalScience Foundation (No. 20506009); Natural Science Foundation ofJiangxi province (No. 0530095); and The Education Department ofJiangxi province (No. GJJ08147). We thank the Key Lab of Protectionand Utilization of Subtropic Plant Resources for expert technicalassistance. Moreover, we also wish to express our thanks to theanonymous reviewers for constructive comments.

Appendix A. Metabolite for constructionof accumulation rate vector

1 AcCoA Acetyl coenzyme A

2 ACE Acetate

3 AcP Acetylphosphate

4 α-KG α-Ketoglutarate

5 ATP Adenosine 5’-triphosphate

6 CO2 Carbon dioxide

7 E4P Erythrose-4-phosphate

8 F6P Fructose-6-phosphate

9 FADH2 Flavin adenine dinucleotide, reduced form

10 FUM Fumarate

11 G3P Glyceraldehyde-3-phosphate

12 G6P Glucose-6-phosphate

13 GLC Glucose

14 ISOCIT Isocitrate

15 NADH Nicotinamide adenine dinucleotide, reduced form

16 NADPH Nicotinamide adenine dinucleotide phosphate,reduced form

17 O2 Oxygen

18 OAA Oxaloacetate

19 PEP Phosphoenolpyruvate

20 PGA Phosphoglycerate acid

21 PYR Pyruvate

22 R5P Ribose-5-phosphate

23 Ru5P Ribulose-5-phosphate

24 RuDP Ribulose-1,5-bisphosphate

25 S7P Sedoheptulose-7-phosphate

26 SUCCoA Succinyl coenzyme A

27 X5P Xylulose-5-phosphate

Appendix B. Biochemical reactions for flux estimation

Calvin cycle and pentose phosphate pathway

1. H2Oþ CO2þRuDP ) 2PGA

666 J Appl Phycol (2012) 24:657–668

2. PGA þ ATP þ NADPH þ H ) GAP þ ADP þNADPþ Pi

3. 2GAPþH2O ) F6Pþ Pi4. F6P , G6P5. G6Pþ 2NADPþH2O ) Ru5Pþ CO2þ2NADPH6. F6Pþ GAP ) X5Pþ E4P7. E4Pþ GAPþH2O ) S7Pþ Pi8. S7PþGAP ) R5Pþ X5P9. R5P ) Ru5P

10. X5P ) Ru5P11. Ru5PþATP ) RuDPþADP

Glycolytic pathway and incomplete tricarboxylic acidcycle

12. GLCþ ATP ) G6Pþ ADP13. G6P , F6P14. F6Pþ ATP ) 2GAPþ ADP15. GAPþ NADþ Piþ ADP , PGAþ ATPþ NADH16. PGA , PEPþH2O17. PEPþADP ) PYRþATP18. PYRþNADþCOA ) AcCoAþNADHþCO2

19. PEPþCO2 þH2O ) OAAþ Pi20. OAAþAcCoAþH2O , ISOCITþCoA21. ISOCITþNAD , aKGþNADHþCO2

22. OAAþNADH , FUMþNADþH2O23. FUMþ FADH2þATPþCoA , SUCCoAþADPþ

Piþ FAD

AcCoA formation

24. HACþATP ) AcPþADP25. AcPþCoA ) AcCoA

Oxidative phosphorylation (P/O=2)

26. NADHþ0:5O2þ2ADPþ2Piþ 2H)H2OþNADþ2ATP

27. FADH2 þ 0:5O2 þADPþ Piþ 2H ) H2Oþ FADþATP

Light reactions

2 8 . 2H2O þ 2NADP þ 2ADP þ 2Pi þ 0:125APF )2NADPHþ 2Hþ 2ATP þ O2

Synthesis of biomass

29. G6Pþ 2ATP ) CAR30: R5Pþ1:235ASPþ2:185GLNþ0:61GLYþ1:22FTHFþ

0:61CO2þ8:68ATPþ0:765NAD)RNAþ2:185GLUþ0:845FUMþ1:22THFþ8:68ADPþ8:68Piþ0:765NADH

31. R5Pþ1:22ASPþ 2:06GLNþ 0:5GLYþ1:22FTHFþ0:5CO2 þ NADPHþ 0:78NADþ8:22ATP) DNAþ0:72FUMþ2:06GLUþ1:22THFþ8:22ADPþ8:22Piþ0:78NADHþNADP

32. 0:07888ALA þ 0:05193AGR þ 0:02902ASN þ0:09701ASPþ 0CYSþ0:07362GLUþ0:02356GLNþ

0:0723GLY þ 0:01534HIS þ 0:05127ILE þ0:08151LEU þ 0:0631LYS þ 0:01578MET þ0:03812PHE þ 0:06573PRO þ 0:03155SER þ0:07493THR þ 0:01249TRP þ 0:03812TYR þ0:09202VAL þ 4ATP ) PROT

3 3 . GAP þ NADH þ 16AcCoA þ 26:65NADPH þ28H þ 14ATPþO2 ) DGþNADþ26:65NADPþ14ADPþ14Piþ16CoAþH2O

3 4 . 8SUCCoA þ 8Gly þ 10AcCoA þ 9ATP þ15NADPH þ Mg2þ þ MYTHF ) CHLO þ4NH3 þ 14CO2 þ 15NADP þ THF þ 9ADP

3 5 . 0:573PROTþ 0:231CARþ 0:142DGþ0:007DNAþ0:024RNAþ0:0086CHLO ) Biomass

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