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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.