J Appl Phycol (2009) 21:239–246 DOI 10.1007/s10811-008-9355-z
Effects of organic carbon sources on growth, photosynthesis, and respiration of Phaeodactylum tricornutum Xiaojuan Liu & Shunshan Duan & Aifen Li & Ning Xu & Zhuoping Cai & Zhangxi Hu
Received: 9 January 2008 / Revised and accepted: 20 May 2008 / Published online: 6 September 2008 # Springer Science + Business Media B.V. 2008
Abstract The growth, photosynthesis, and respiration of the marine diatom Phaeodactylum tricornutum were examined under photoautotrophic and mixotrophic conditions. 100 mM glycerol, acetate, and glucose significantly increased specific growth rate, and mixotrophic growth achieved higher biomass concentrations. Under mixotrophic conditions, respiration rate (Rd) and light compensation irradiance (Ic) were significantly higher, but net maximum photosynthetic O2 evolution rate (Pm) and saturation irradiance (Ik) were depressed. Organic carbon sources decreased the cell photosynthetic pigment content and chlorophyll a to c ratio, but with a higher carotenoid to chlorophyll a ratio. Ratios of variable to maximum chlorophyll fluorescence (Fv/Fm) and 77 K fluorescence spectra of mixotrophic cells indicated a reduced photochemical efficiency of photosystem II. The results were accompanied by lower electron transport rate. Therefore, organic carbon sources reduced the photosynthesis efficiency, and the enhancement of biomass of P. tricornutum implied that organic carbon sources had more pronounced effects on respiration than on photosynthesis. Keywords Mixotrophic . Organic carbon sources . Photosynthesis . PS II . Respiration
X. Liu : S. Duan (*) : A. Li : N. Xu : Z. Cai : Z. Hu Institute of Hydrobiology, Jinan University, Shipai, Guangzhou 510632, China e-mail:
[email protected] X. Liu College of Food Science, South China Agricultural University, Guangzhou 510632, China
Introduction Although most microalgae are photoautotrophs, some microalgae can use organic carbon substances as the sources of energy and carbon for cell growth. Mixotrophy is growth in which organic carbon is assimilated in the light simultaneously with CO2 fixation. Much work has been done on mixotrophic growth of the green algae Chlamydomonas reinhardtii (Heifetz et al. 2000; Chen and Johns 1996), Chlorella (Endo et al. 1977; Lalucat et al. 1984; Ip and Chen 2005), and Haematococcus pluvialis (Kobayashi et al. 1992; Kang et al. 2005; Jeon et al. 2006). Certain cyanobacteria such as Synechococcus (Vernotte et al. 1992; Kang et al. 2004), Spirulina platensis (Marquez et al. 1993; Chen and Zhang 1997), and Anabaena variabilis (Valiente et al. 1992; Mannan and Pakrasi 1993) are known to grow mixotrophically when supplied with appropriate carbon sources. Some diatoms such as Phaeodactylum tricornutum (Cooksey 1974; García et al. 2000, 2005), Navicula saprophila (Kitano et al. 1997), and Nitzschia (Kitano et al. 1997; Wen and Chen 2000, 2002) are able to grow mixotrophically. These microalgae can use different organic carbon sources, such as glucose, acetate, and glycerol. Although P. tricornutum has been regarded as a typical diatom in many studies, it does not possess the heavily silicified cell wall characteristic of many Bacillariophyta. Depending on the growth form the wall is either unsilicified (except for the girdle bands) or has a single small silicified valve (Borowitzka and Volcani 1978). This makes P. tricornutum an organism particularly useful for the physiological study of diatoms because its cell walls are easily ruptured in a French press, after which preparation of cellular organelles is facilitated (Milner et al. 1950). Moreover, P. tricornutum has been studied intensively as a potential source of polyunsaturated fatty acids (PUFAs), mainly
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eicosapentaenoic acid (EPA). Cultivation is mainly under photoautotrophic conditions, and this mode of growth results in a relatively low growth rate and biomass concentration. Culturing P. tricornutum in mixotrophic conditions could potentially yield a higher biomass concentration (García et al. 2005). Mixotrophy is growth in which CO2 and organic carbon are assimilated simultaneously and, hence, both respiratory and photosynthetic metabolism have to operate concurrently (Ogawa and Aiba 1981; Marquez et al. 1993). For some microalgae, photosynthesis and the oxidative phosphorylation of organic carbon substances seem to function independently. The growth rate in mixotrophic conditions is approximately the same as the sum of the growth rate in the photoautotrophic and heterotrophic cultures, such as Chlorella regularis (Endo et al. 1977), C. vulgaris (Ogawa and Aiba 1981), and S. platensis (Marquez et al. 1993). On the other hand, organic carbon metabolism may exert an opposing influence on photosynthesis. Glucose can reduce the apparent affinity for CO2 in CO2 fixation in the species such as Chlorella sp.VJ79 (Lalucat et al. 1984), and C. vulgaris UAM101 (Martinez and Orus 1991). Glucose can also depress photosynthetic O2 evolution, such as Aphanocapsa 6714 (Der-Vartanian et al. 1981), and Galdieria sulphuraria (Oesterhelt et al. 2007). In the study of Steinmüller and Zetsche (1984), glucose was shown to have a strong inhibitory effect on the synthesis of the Calvin cycle enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and the light gathering proteins phycocyanin (PC) and allophycocyanin (APC) in Cyanidium caldariam, mainly by modulation of levels of translatable messenger RNA for these proteins. Oesterhelt et al. (2007) also showed that glucose could reduce photochemical efficiency of photosystem II (PS II) and levels of the PS II reaction centre protein D1. Many studies of photosynthesis have been carried out with acetategrown C. reinhardtii cells. Growth of cells under increasing concentrations of acetate culture reduced the photosynthetic CO2 fixation and net O2 evolution, without effects on respiration and PS II efficiency (Heifetz et al. 2000). Acetate can also reduce carbonic anhydrase (CA) activity and expression of cah-1 encoding CA (Fett and Coleman 1994). Kindle (1987) and Goldschmidt-Clermont (1986) showed that acetate can inhibit the light-harvesting chlorophyll a/bbinding gene cab11-1 mRNA abundance and expression of rbcS encoding RuBPCase. In the unicellular green alga Chlorogonium elongatum, acetate inhibited the synthesis of RuBPCase and its mRNAs (Steinbiß and Zetsche 1986). Moreover, acetate also repressed the activities of rbcL and rbcScah-1 encoding Rubisco, and psbA encoding protein D1 (Kroymann et al. 1995). Some other studies also suggested that glycerol assimilation by Pyrenomonas salina resulted in a reduction of photosynthetic components associated with light-harvesting. The cell phycoerythrin content, phycoery-
J Appl Phycol (2009) 21:239–246
thrin to chlorophyll ratio, degree of thylakoid packing, number of thylakoids•cell−1, and PS II particle size were also reduced (Lewitus et al. 1991). In Cyanothece sp., glycerol addition in the light produced small differences in the pigment content and ultrastructure (Schneegurt et al. 1997). However, there are also some exceptions. Glucose could enhance the net photosynthesis rate in Synechococcus sp. PCC7002 (Kang et al. 2004) and the PS II photochemical efficiency Ф II in Synechocystis sp. PCC 6803 (Wang et al. 2000). This might because the glucose promoted the donation of electrons to the plastoquinone pool from the respiratory substance, and the transforming of energy was promoted by photosynthetic system, which provided the energy needed by anabolism of cells caused by the glucose added to the medium (Wang et al. 2000). There is little information available about the photosynthetic activity of diatom under mixotrophic growth. Previous work and our early experiments have shown that P. tricornutum is capable of mixotrophic growth at the expense of glycerol, acetate or glucose (García et al. 2000, 2005). So we chose glycerol, acetate and glucose as the organic carbon sources. In this paper, the photosynthetic response of P. tricornutum to organic carbon sources was studied. The rates of growth, respiration, photosynthetic O2 evolution, the content of pigment, 77 K fluorescence spectra, chlorophyll fluorescence, and electron transport rate were examined with the photoautotrophic and mixotrophic strains.
Materials and methods Phaeodactylum tricornutum was obtained from the Institute of Hydrobiology, Jinan University (Guangzhou, China). Batch cultures (100 mL volume) were grown in f/2 medium (Guillard and Ryther 1962) in 250-mL flasks without aeration under aseptic conditions. Cultures were illuminated with white fluorescence light (50 μmol photons m−2 s−1) on a 12:12 h light:dark cycle at 20°C. The pH of the medium was 7.4, and the salinity was 30‰. For mixotrophic treatments, the media were supplied with different organic carbon sources. Glycerol, acetate, and glucose were separately sterilized by filtration through 0.22-μm pore membranes. The final concentration of each organic carbon source was 100 mM carbon. Cultures used for inocula were acclimated to the organic medium by repeated transfer of cells in the mid-exponential growth phase into fresh medium. After 3 months of repeated transfers, the cells were acclimated to the organic medium, and there were no significant differences in the growth rate of several sequential cultures. After acclimation to the organic medium, exponentially growing populations were inoculated into fresh media to start the experiment. The culture medium was supplemented with 50 µg.mL−1 kanamycin sulfate to help minimize bacterial
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Table 1 Growth parameters of Phaeodactylum tricornutum under photoautotrophic and mixotrophic conditions
Specific growth rate (day−1) Maximal biomass (mg L−1)
Growth conditions Photoautotrophy
Mixotrophy with glycerol
Mixotrophy with acetate
Mixotrophy with glucose
0.11±0.02 460±3
0.18±0.04 713±11
0.15±0.03 587±8
0.14±0.03 555±10
contamination during repeated transfers under mixotrophic growth conditions. Cultures were tested for bacterial contamination throughout the experiment by inoculating tubes containing f/2 medium plus 1.5% yeast extract with 1 mL of the culture and examing these tubes for bacterial growth. All parameters were performed in triplicate. Cell counts were determined using CASY-TT SchärfeSystem (ALIT, Germany). Dry weight was measured after centrifugation at 10,000 g for 10 min and drying at 60°C to constant weight. Chlorophyll a and c concentrations were determined according to the method of Jeffrey and Humphrey (1975). Carotenoid concentration was determined according to Wellburn (1994). Photosynthetic O2 evolution by intact cells was measured using a Clark-type O2 electrode (Hansatech, UK). Cells were harvested by centrifugation and resuspended in fresh medium to the same final concentrations. Dark respiration rate was measured with the same cell suspension before each measurement of net photosynthetic rate. The net maximum photosynthetic rate (Pm), respiration rate (Rd), and initial slope (α) at limiting photo flux densities were determined by fitting a three-parameter model (Henley 1993): P ¼ Pm tanhða I=Pm Þ þ Rd , where P is the net photosynthesis rate, and I the light level. Ik, saturation irradiance, was calculated as ðPm þ Rd Þ=a, and Ic, compensation irradiance, as Rd /a. Absorption spectra were recorded by a UV-2450 dualwavelength/beam recording spectrophotometer at room temperature (Shimadzu, Japan). The cell pigments were extracted with acetone and adjusted to the same chlorophyll absorbance near 660 nm. The scanning speed was 200 nm min−1, and the scanning scope was 400 to 750 nm with a 2-nm slit width. Fluorescence emission spectra at 77 K were recorded by an F-4500 fluorescence spectrophotometer (Hitachi, Japan). The cells were adjusted to the same chlorophyll absorbance near 680 nm. The scanning speed was 240 nm min−1, and the scanning scope was 650 to 750 nm with a 2-nm slit width. Chl a fluorescence was excited at 495 nm. The maximum photochemical efficiency of PS II was measured using a XE-PAM fluorometer (Walz, Germany). Fv =Fm ¼ ðFm F0 Þ=Fm , where F0 and Fm are the minimal and maximal fluorescence yields of a dark-adapted sample, with all PS II reaction centers fully open or closed, respectively. Fv is the variable fluorescence. Prior to fluorescence measurements, cells were dark-adapted for 15 min.
The intensity of measuring light and actinic light were 0.05 μmol photons m−2 s−1 and 200 μmol photons m−2 s−1, respectively, and saturation pulse was 1,200 μmol photons m−2 s−1. Means were compared using a t-test with a significance level of 0.05.
Results Effects of organic carbon sources on the growth Effects of organic carbon sources (glycerol, acetate, or glucose) on the growth of P. tricornutum are shown in Table 1. Organic carbon sources can significantly increase specific growth rate (p
