Comprehensive Analysis of Biomass from Chlorella sorokiniana Cultivated with Industrial Flue Gas as the Carbon Source.

Chlorella sorokiniana, isolated from a pond adjacent to a cement plant, was cultured using flue gas collected directly from kiln emissions using 20 L and 25000 L photobioreactors. Lipids, proteins, and polysaccharides were analyzed to understand their overall composition for potential applications. The lipid content ranged from 17.97% to 21.54% of the dry biomass, with carotenoid concentrations between 8.4 and 9.2 mg/g. Lutein accounted for 55% of the total carotenoids. LC/MS analysis led to the identification of 71 intact triacylglycerols, 8 lysophosphatidylcholines, 10 phosphatidylcholines, 9 monogalactosyldiacylglycerols, 12 digalactosyldiacylglycerols, and 1 sulfoquinovosyl diacylglycerol. Palmitic acid, oleic acid, linoleic acid, and α-linolenic acid were the main fatty acids. Polyunsaturated fatty acid covers ≥ 56% of total fatty acids. Protein isolates and polysaccharides were also extracted. Protein purity was determined to be ≥75% by amino acid analysis, with all essential amino acids present. Monomer analysis of polysaccharides suggested that they are composed of mainly D-(+)-mannose, D-(+)-galactose, and D-(+)-glucose. The results demonstrate that there is no adverse effect on the metabolite profile of C. sorokiniana biomass cultured using flue gas as the primary carbon source, revealing the possibility of utilizing such algal biomass in industrial applications such as animal feed, sources of cosmeceuticals, and as biofuel.

Characterization of Neutral Lipids of the Oleaginous Alga Micractinum inermum.

An oleaginous microalga Micractinum inermum isolated from Mariana Lake, AB, Canada was cultured in a 1000 L photobioreactor with an f/2 medium to study its lipid content and neutral lipid profile. Algal biomass was collected at the stationary phase contained a significant amount of lipids (44.2%), as determined by Folch's method. The lipid was fractionated into neutral lipid, glycolipid and phospholipid fractions. The neutral lipid constitutes almost 77.3% of the total lipid species and is mainly composed of triacylglycerols (TAGs) determined by a proton NMR study. UHPLC-HRMS analysis allows us for the first time to identify 81 TAGs in the neutral lipid fraction of M. inermum. The fatty acid acyl side chains were identified based on fragment ions observed in MSMS analysis. TAGs with fatty acid acyl chains 18:1/18:1/18:1, 18:1/18:1/16:0, 18:2/18:1/16:0, and 18:2/18:2/18:0 were the major ones among the identified TAGs. Fatty acid analysis further supports the fact that oleic acid was the major fatty acid present in the neutral lipid fraction of M. inermum constituting 41.7%, followed by linoleic acid at 21.5%, and palmitic acid at 21.2%. The saturated and monounsaturated fatty acids were 67.8% or higher in the lipid fraction. Long-chain fatty acids were only present in a minor quantity. The results clearly demonstrate that M. inermum is an excellent source for TAGs.

Photosynthetic conversion of carbon dioxide from cement production to microalgae biomass.

Production of microalgae is a potential technology for capturing and recycling carbon dioxide from cement kiln emissions. In this study, a process of selecting a suitable strain that would effectively utilize carbon dioxide and generate biomass was investigated. A down-selection screening method was applied to 28 strains isolated from the area surrounding a commercial cement plant. In laboratory-scale (1 L) continuous-mode chemostats, observed productivity was > 0.9 g L-1 d-1 for most strains studied. Chlorella sorokiniana (strain SMC-14M) appeared to be the most tolerant to cement kiln gas emissions in situ, delivered under control of a pH-stat system, and was down-selected to further investigate growth and biomass production at large-scale (1000 L) cultivation. Results demonstrated little variability in lipid, crude protein, and carbohydrate composition throughout growth between kiln-gas grown algal biomass and biomass produced with laboratory grade CO2. The growth rate at which the maximum quantity of CO2 from the emissions is recycled also produced the maximum amount of the targeted biomass components to increase commercial value of the biomass. An accumulation of some heavy metals throughout its growth demonstrates the necessity to monitor the biomass cultivated with industrial flue gases and to carefully consider the potential applications for this biomass; despite its other attractive nutritional properties. KEY POINTS: • Studied high biomass producing algal strains grown on CO2 from cement flue gas. • Chlorella sorokiniana SMC-14M grew well at large scale, in situ on cement flue gas. • Demonstrated the resulting commercial potential of the cultured algal biomass.

Transcriptome analysis of lipid metabolism in response to cerium stress in the oleaginous microalga Nannochloropsis oculata.

Nannochloropsis oculata can accumulate large amounts of lipids under rare earth element (REE) conditions. However, the lipid accumulation mechanism responsible for REE stress has not been elucidated. In this study, the effects of cerium (the most abundant REE) on the growth and lipid accumulation of N. oculata were investigated. The de novo transcriptome data of N. oculata under cerium conditions were subsequently collected and analyzed. The results showed that N. oculata exhibited good cerium-resistance ability, showed slightly decrease in biomass but significantly increase in lipid content (55.8 % dry cell weight) under 6.0 mg/L cerium condition. Meanwhile, about 83.4 % cerium was biological fixated. Through transcriptome analysis, we found that the inhibited photosynthesis and carbon fixation pathways coupled with the stress-sensitive expression of ribosome biogenesis genes acclimatized the cells to REE stress. The active glycolysis pathway accelerated carbon flux to pyruvate and acetyl-CoA, and the upregulation of glycerol kinase and phosphatidate cytidylyltransferase genes further induced lipid accumulation. In addition, cerium downregulated the acyl-CoA oxidase and triacylglycerol lipase genes, which inhibited the degradation of lipids. Therefore, different responses to cerium demonstrate how N. oculata cells adapt to REE stress, and this knowledge may be used to extend our understanding of triacylglycerol (TAG) and the synthesis of other important metabolites.

Microalgae as Sources of High-Quality Protein for Human Food and Protein Supplements.

As a result of population growth, an emerging middle-class, and a more health-conscious society concerned with overconsumption of fats and carbohydrates, dietary protein intake is on the rise. To address this rapid change in the food market, and the subsequent high demand for protein products, agriculture, aquaculture, and the food industry have been working actively in recent years to increase protein product output from both production and processing aspects. Dietary proteins derived from animal sources are of the highest quality, containing well-balanced profiles of essential amino acids that generally exceed those of other food sources. However, as a result of studies highlighting low production efficiency (e.g., feed to food conversion) and significant environmental impacts, together with the negative health impacts associated with the dietary intake of some animal products, especially red meats, the consumption of animal proteins has been remaining steady or even declining over the past few decades. To fill this gap, researchers and product development specialists at all levels have been working closely to discover new sources of protein, such as plant-based ingredients. In this regard, microalgae have been recognized as strategic crops, which, due to their vast biological diversity, have distinctive phenotypic traits and interactions with the environment in the production of biomass and protein, offering possibilities of production of large quantities of microalgal protein through manipulating growing systems and conditions and bioengineering technologies. Despite this, microalgae remain underexploited crops and research into their nutritional values and health benefits is in its infancy. In fact, only a small handful of microalgal species are being produced at a commercial scale for use as human food or protein supplements. This review is intended to provide an overview on microalgal protein content, its impact by environmental factors, its protein quality, and its associated evaluation methods. We also attempt to present the current challenges and future research directions, with a hope to enhance the research, product development, and commercialization, and ultimately meet the rapidly increasing market demand for high-quality protein products.

Screening of two freshwater green microalgae in pulp and paper mill wastewater effluents in Nova Scotia, Canada.

In recent years, the use of microalgae as feedstock for many marketable products, such as animal/aqua feeds, bioplastics and fertilizers, has gained renewed interest due to their fast growth potential coupled with relatively high lipid, carbohydrate and nutrient content. An algal biorefinery at an industrial site has the potential to sustainably and profitably convert carbon dioxide emissions into microalgal biomass and concomitantly reduce nitrogen and phosphorus from wastewaters. Industrial wastewaters are a potential alternative to traditional media used for large-scale microalgal cultivation. Pulp and paper mills are major consumers of water resources and discharge a huge amount of water to nearby lakes or rivers. This study investigated whether pulp and paper mill waste water is suitable for microalgal cultivation with the aim of achieving significant biomass production. Six different process waters from one Canadian pulp and paper mill were tested with two freshwater green microalgae. All of these waters were unable to support growth of microalgae due to inadequate nutrient concentrations, colour, turbidity and possible toxicity issues.

A Rat Study to Evaluate the Protein Quality of Three Green Microalgal Species and the Impact of Mechanical Cell Wall Disruption.

The present study was conducted to evaluate the protein quality of microalgae species Chlorella vulgaris (CV), Chlorella sorokiniana (CS), and Acutodesmus obliquus (AO) and assess the impact of mechanical cell wall disruption. Male Sprague-Dawley rats, around 156 g after adaptation, were placed in metabolic cages and fed experimental diets that were either protein-free or contained 10% protein solely from one of the undisrupted or disrupted CV, CS, and AO. After 3 days, feces were collected for a period of 5 days and analyzed together with diet samples for crude protein contents. Apparent protein digestibility, true protein digestibility, amino acid score, and protein digestibility-corrected amino acid score were calculated. In vitro protein digestibility was measured using the pepsin-pancreatin method and the in vitro protein digestibility-corrected amino acid score was calculated. The crude protein contents of CV, CS, and AO were 53.5, 50.2, and 40.3%, respectively. The amino acid score of the first limiting amino acid was 1.10, 1.27, and 0.86, true protein digestibility was 64.7, 59.3, and 37.9% and protein digestibility-corrected amino acid score was 0.63, 0.64, and 0.29, respectively, for CV, CS, and AO. Mechanical cell disruption significantly improved protein digestibility without a substantial impact on the amino acid profile and score, resulting in the increase of protein digestibility-corrected amino acid score to 0.77, 0.81, and 0.46, respectively, for disrupted CV, CS, and AO. There was a strong correlation between in vitro protein digestibility and apparent protein digestibility (r = 0.986), and also between in vitro protein digestibility-corrected amino acid score and in vivo protein digestibility-corrected amino acid score (r = 0.994). The results suggest that the CV and CS are acceptable sources of protein for humans and animals and quality can be markedly improved by mechanical cell wall disruption. Additionally, in vitro protein digestibility measured using the pepsin-pancreatin method may be used to screen protein product candidates, save animals, reduce cost, and accelerate product development.

Nitric Oxide Mediates Nitrite-Sensing and Acclimation and Triggers a Remodeling of Lipids.

Nitric oxide (NO) is an intermediate of the nitrogen cycle, an industrial pollutant, and a marker of climate change. NO also acts as a gaseous transmitter in a variety of biological processes. The impact of environmental NO needs to be addressed. In diatoms, a dominant phylum in phytoplankton, NO was reported to mediate programmed cell death in response to diatom-derived polyunsaturated aldehydes. Here, using the Phaeodactylum Pt1 strain, 2E,4E-decadienal supplied in the micromolar concentration range led to a nonspecific cell toxicity. We reexamined NO biosynthesis and response in Phaeodactylum NO inhibits cell growth and triggers triacylglycerol (TAG) accumulation. Feeding experiments indicate that NO is not produced from Arg but via conversion of nitrite by the nitrate reductase. Genome-wide transcriptional analysis shows that NO up-regulates the expression of the plastid nitrite reductase and genes involved in the subsequent incorporation of ammonium into amino acids, via both Gln synthesis and Orn-urea pathway. The phosphoenolpyruvate dehydrogenase complex is also up-regulated, leading to the production of acetyl-CoA, which can feed TAG accumulation upon exposure to NO. Transcriptional reprogramming leading to higher TAG content is balanced with a decrease of monogalactosyldiacylglycerol (MGDG) in the plastid via posttranslational inhibition of MGDG synthase enzymatic activity by NO. Intracellular and transient NO emission acts therefore at the basis of a nitrite-sensing and acclimating system, whereas a long exposure to NO can additionally induce a redirection of carbon to neutral lipids and a stress response.

Maximizing the productivity of the microalgae Scenedesmus AMDD cultivated in a continuous photobioreactor using an online flow rate control.

In this study, production of the microalga Scenedesmus AMDD in a 300 L continuous flow photobioreactor was maximized using an online flow (dilution rate) control algorithm. To enable online control, biomass concentration was estimated in real time by measuring chlorophyll-related culture fluorescence. A simple microalgae growth model was developed and used to solve the optimization problem aimed at maximizing the photobioreactor productivity. When optimally controlled, Scenedesmus AMDD culture demonstrated an average volumetric biomass productivity of 0.11 g L-1 d-1 over a 25 day cultivation period, equivalent to a 70 % performance improvement compared to the same photobioreactor operated as a turbidostat. The proposed approach for optimizing photobioreactor flow can be adapted to a broad range of microalgae cultivation systems.

Label-free hyperspectral nonlinear optical microscopy of the biofuel micro-algae Haematococcus Pluvialis.

We consider multi-modal four-wave mixing microscopies to be ideal tools for the in vivo study of carotenoid distributions within the important biofuel microalgae Haematococcus pluvialis. We show that hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy generates non-invasive, quantitative real-time concentrations maps of intracellular carotenoid distributions in live algae.

Switchable hydrophilicity solvents for lipid extraction from microalgae for biofuel production.

A switchable hydrophilicity solvent (SHS) was studied for its effectiveness at extracting lipids from freeze-dried samples of Botryococcus braunii microalgae. The SHS N,N-dimethylcyclohexylamine extracted up to 22 wt.% crude lipid relative to the freeze-dried cell weight. The solvent was removed from the extract with water saturated with carbon dioxide at atmospheric pressure and recovered from the water upon de-carbonation of the mixture. Liquid chromatography-mass spectrometry (LC-MS) showed that the extracted lipids contained high concentrations of long chain tri-, di- and mono-acylglycerols, no phospholipids, and only 4-8% of residual solvent. Unlike extractions with conventional organic solvents, this new method requires neither distillation nor the use of volatile, flammable or chlorinated organic solvents.

Suitability of Soxhlet extraction to quantify microalgal Fatty acids as determined by comparison with in situ transesterification.

To assess Soxhlet extraction as a method for quantifying fatty acids (FA) of microalgae, crude lipid, FA content from Soxhlet extracts and FA content from in situ transesterification (ISTE) were compared. In most cases, gravimetric lipid content was considerably greater (up to sevenfold) than the FA content of the crude lipid extract. FA content from Soxhlet lipid extraction and ISTE were similar in 12/18 samples, whereas in 6/18 samples, total FA content from Soxhlet extraction was less than the ISTE procedure. Re-extraction of residual biomass from Soxhlet extraction with ISTE liberated a quantity of FA equivalent to this discrepancy. Employing acid hydrolysis before Soxhlet extraction yielded FA content roughly equivalent to ISTE, indicating that acidic conditions of ISTE are responsible for this observed greater recovery of FA. While crude lipid derived from Soxhlet extraction was not a useful proxy for FA content for the species tested, it is effective in most strains at extracting total saponifiable lipid. Lipid class analysis showed the source of FA was primarily polar lipids in most samples (12/18 lipid extracts contained <5% TAG), even in cases where total FA content was high (>15%). This investigation confirms the usefulness of ISTE, reveals limitations of gravimetric methods for projecting biodiesel potential of microalgae, and reinforces the need for intelligent screening using both FA and lipid class analysis.

Triacylglycerol profiling of microalgae strains for biofuel feedstock by liquid chromatography-high-resolution mass spectrometry.

Biofuels from photosynthetic microalgae are quickly gaining interest as a viable carbon-neutral energy source. Typically, characterization of algal feedstock involves breaking down triacylglycerols (TAG) and other intact lipids, followed by derivatization of the fatty acids to fatty acid methyl esters prior to analysis by gas chromatography (GC). However, knowledge of the intact lipid profile could offer significant advantages for discovery stage biofuel research such as the selection of an algal strain or the optimization of growth and extraction conditions. Herein, lipid extracts from microalgae were directly analyzed by ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) using a benchtop Orbitrap mass spectrometer. Phospholipids, glycolipids, and TAGs were analyzed in the same chromatographic run, using a combination of accurate mass and diagnostic fragment ions for identification. Using this approach, greater than 100 unique TAGs were identified over the six algal strains studied and TAG profiles were obtained to assess their potential for biofuel applications. Under the growth conditions employed, Botryococcus braunii and Scenedesmus obliquus yielded the most comprehensive TAG profile with a high abundance of TAGs containing oleic acid.

Integration of microalgae cultivation with industrial waste remediation for biofuel and bioenergy production: opportunities and limitations.

There is currently a renewed interest in developing microalgae as a source of renewable energy and fuel. Microalgae hold great potential as a source of biomass for the production of energy and fungible liquid transportation fuels. However, the technologies required for large-scale cultivation, processing, and conversion of microalgal biomass to energy products are underdeveloped. Microalgae offer several advantages over traditional 'first-generation' biofuels crops like corn: these include superior biomass productivity, the ability to grow on poor-quality land unsuitable for agriculture, and the potential for sustainable growth by extracting macro- and micronutrients from wastewater and industrial flue-stack emissions. Integrating microalgal cultivation with municipal wastewater treatment and industrial CO(2) emissions from coal-fired power plants is a potential strategy to produce large quantities of biomass, and represents an opportunity to develop, test, and optimize the necessary technologies to make microalgal biofuels more cost-effective and efficient. However, many constraints on the eventual deployment of this technology must be taken into consideration and mitigating strategies developed before large scale microalgal cultivation can become a reality. As a strategy for CO(2) biomitigation from industrial point source emitters, microalgal cultivation can be limited by the availability of land, light, and other nutrients like N and P. Effective removal of N and P from municipal wastewater is limited by the processing capacity of available microalgal cultivation systems. Strategies to mitigate against the constraints are discussed.

Genomic imprinting in Drosophila has properties of both mammalian and insect imprinting.

Genomic imprinting is a process that marks DNA, causing a change in gene or chromosome behavior, depending on the sex of the transmitting parent. In mammals, most examples of genomic imprinting affect the transcription of individual or small clusters of genes whereas in insects, genomic imprinting tends to silence entire chromosomes. This has been interpreted as evidence of independent evolutionary origins for imprinting. To investigate how these types of imprinting are related, we performed a phenotypic, molecular, and cytological analysis of an imprinted chromosome in Drosophila melanogaster. Analysis of this chromosome reveals that the imprint results in transcriptional silencing. Yet, the domain of transcriptional silencing is very large, extending at least 1.2 Mb and encompassing over 100 genes, and is associated with decreased somatic polytenization of the entire chromosome. We propose that repression of somatic replication in polytenized cells, as a secondary response to the imprint, acts to extend the size of the imprinted domain to an entire chromosome. Thus, imprinting in Drosophila has properties of both typical mammalian and insect imprinting which suggests that genomic imprinting in Drosophila and mammals is not fundamentally different; imprinting is manifest as transcriptional silencing of a few genes or silencing of an entire chromosome depending on secondary processes such as differences in gene density and polytenization.

Beta-adrenergic stimulation enhances the heat-shock response in fish.

We have taken advantage of the unique properties of nucleated rainbow trout (Oncorhynchus mykiss) red blood cells (rbcs) to demonstrate that beta-adrenergic stimulation with the agonist, isoproterenol, significantly enhanced the heat-induced induction of heat-shock proteins (Hsps) in trout rbcs without affecting hsp expression on its own. Furthermore, this beta-adrenergic potentiation of hsp expression occurred only at physiologically relevant concentrations of adrenergic stimulation. In further experiments, we found that adrenaline increased 100-fold and noradrenaline increased 50-fold in trout after a 1-h heat shock at 25 degrees C, approximately 12 degrees C above acclimation temperature. This is the first time the adrenergic heat-shock response has been described for a temperate fish species. We conclude that beta-adrenergic stimulation enhances hsp expression in trout rbcs following heat stress, indicating physiological regulation of the cellular stress response in fish.

Expression and regulation of carbonic anhydrases in the marine diatom Thalassiosira pseudonana and in natural phytoplankton assemblages from Great Bay, New Jersey.

BLAST searches of expressed sequence tag libraries have revealed putative homologs of the archetypal diatom delta-carbonic anhydrase (CA) TWCA1 (for Thalassiosira weissflogii CA) in a broad range of eukaryotic phytoplankton including haptophytes, prasinophytes and dinoflagellates. Four putative homologs of TWCA1 are also reported and described from a search of the genomic sequence of Thalassiosira pseudonana and designated TWCA(Tp1-4). The delta-CA class is therefore more widely distributed in marine phytoplankton than previously thought. In particular, it is not restricted to the diatoms like the cadmium-containing enzyme, CDCA, seems to be (zeta-CA class). Zinc status strongly influences growth rate in marine diatoms. We observed a decrease in the specific growth rate of T. pseudonana from 2.1 to 1.3 day(-1) when the unchelated Zn concentration (Zn') was lowered from 20 to 5 pM. In the same cultures, we detected a drop in whole-cell CA activity of about 50% in the most Zn-limited cells that occurred simultaneously with a large downregulation of TWCA(Tp1), TWCA(Tp2) and CDCA(Tp) gene transcripts and protein. These three genes were also found to be strongly upregulated by low pCO(2) in a manner typical of many algal CA enzymes. We have also conducted field experiments in diatom-dominated natural phytoplankton assemblages sampled from Great Bay of the coast of New Jersey. We observed increased total CA activity in parallel with increased expression of homologous twca and cdca gene transcripts in water incubated at increasingly higher pH values. Immunoblot and transcript expression analyses demonstrated a clear upregulation of CDCA transcript and protein as incubation pH increased both in the lab and in the field indicating that this CA is expressed under a broad range of environmental conditions and not restricted to low pCO(2) and low Zn.

Expression and inhibition of the carboxylating and decarboxylating enzymes in the photosynthetic C4 pathway of marine diatoms.

There is evidence that the CO(2)-concentrating mechanism in the marine diatom Thalassiosira weissflogii operates as a type of single-cell C(4) photosynthesis. In quantitative-PCR assays, we observed 2- to 4-fold up-regulation of two phosphoenolpyruvate carboxylase (PEPC) gene transcripts in Thalassiosira pseudonana cells adapted to low pCO(2), but did not detect such regulation in Phaeodactylum tricornutum grown under similar conditions. Transcripts encoding phosphoenolpyruvate carboxykinase did not appear to be regulated by pCO(2) in either diatom. In T. pseudonana and T. weissflogii, net CO(2) fixation was blocked by 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (a specific inhibitor of PEPC), but was restored by about 50% and 80%, respectively, by addition of millimolar concentrations of KHCO(3). In T. pseudonana, T. weissflogii, and P. tricornutum, rates of net O(2) evolution were reduced by an average of 67%, 55%, and 62%, respectively, in the presence of 20 microm quercetin, also an inhibitor of PEPC. Quercetin promoted net CO(2) leakage from inhibited cells to levels in excess of the equilibrium CO(2) concentration, suggesting that a fraction of the HCO(3)(-) taken up is fated to leak back into the medium as CO(2) when PEPC activity is blocked. In parallel to these experiments, in vitro assays on crude extracts of T. pseudonana demonstrated mean inhibition of 65% of PEPC activity by quercetin. In the presence of 5 mm 3-mercaptopicolinic acid (3-MPA), a classic inhibitor of phosphoenolpyruvate carboxykinase, photosynthetic O(2) evolution was reduced by 90% in T. pseudonana. In T. weissflogii and P. tricornutum, 5 mm 3-MPA totally inhibited net CO(2) fixation and O(2) evolution. Neither quercetin nor 3-MPA had a significant inhibitory effect on photosynthetic O(2) evolution or CO(2) uptake in the marine chlorophyte isolates Chlamydomonas sp. or Dunaliella tertiolecta. Our evidence supports the idea that C(4)-based CO(2)-concentrating mechanisms are generally distributed in diatoms. This conclusion is discussed within the context of the evolutionary success of diatoms.

Inorganic carbon limitation and light control the expression of transcripts related to the CO2-concentrating mechanism in the cyanobacterium Synechocystis sp. strain PCC6803.

The cyanobacterium Synechocystis sp. strain PCC6803 possesses three modes of inorganic carbon (Ci) uptake that are inducible under Ci stress and that dramatically enhance the efficiency of the CO(2)-concentrating mechanism (CCM). The effects of Ci limitation on the mRNA transcript abundance of these inducible uptake systems and on the physiological expression of the CCM were investigated in detail in this cyanobacterium. Transcript abundance was assessed with semiquantitative and real-time reverse transcriptase-polymerase chain reaction techniques. Cells aerated with CO(2)-free air for 30 min in the light, but not in the dark, depleted the total [Ci] to near zero levels. Under these conditions, the full physiological expression of the CCM was apparent within 2 h. Transcripts for the three inducible Ci uptake systems, ndhF3, sbtA, and cmpA, showed near-maximal abundance at 15 min under Ci limitation. The transcriptional regulators, cmpR and ndhR, were more moderately expressed, whereas the rbcLXS and ccmK-N operons and ndhF4/ndhD4/chpX and ccaA genes were insensitive to the low-Ci treatment. The combined requirement of low Ci and light for the expression of several CCM-related transcripts was examined using real-time reverse transcriptase-polymerase chain reaction. CmpA, ndhF3, and sbtA were strongly expressed in the light, but not in the dark, under low-Ci conditions. We could find no evidence for induction of these or other CCM-related genes by a high-light treatment under high-CO(2) conditions. This provided evidence that high-light stress alone could not trigger the expression of CCM-related transcripts in Synechocystis sp. PCC6803. Potential signals triggering induction of the high-affinity state of the CCM are discussed.