Microalga Neochloris Oleoabundans Research Articles

Combined enzymatic and mechanical cell disruption and lipid extraction of green alga Neochloris oleoabundans.

Microalgal biodiesel is one of the most promising renewable fuels. The wet technique for lipids extraction has advantages over the dry method, such as energy-saving and shorter procedure. The cell disruption is a key factor in wet oil extraction to facilitate the intracellular oil release. Ultrasonication, high-pressure homogenization, enzymatic hydrolysis and the combination of enzymatic hydrolysis with high-pressure homogenization and ultrasonication were employed in this study to disrupt the cells of the microalga Neochloris oleoabundans. The cell disruption degree was investigated. The cell morphology before and after disruption was assessed with scanning and transmission electron microscopy. The energy requirements and the operation cost for wet cell disruption were also estimated. The highest disruption degree, up to 95.41%, assessed by accounting method was achieved by the combination of enzymatic hydrolysis and high-pressure homogenization. A lipid recovery of 92.6% was also obtained by the combined process. The combined process was found to be more efficient and economical compared with the individual process.

Diacylglycerol acyltransferase type 2 cDNA from the oleaginous microalga Neochloris oleoabundans: cloning and functional characterization

Currently, biodiesel production from microalgae is technically, but not yet economically, feasible. Increasing microalgal triacylglycerol (TAG) content via genetic engineering could be a potential approach to improve the efficiency of biodiesel production. In all organisms studied so far, diacylglycerol acyltransferase (DGAT) is the primary enzyme of TAG biosynthesis. In this study, we cloned the cDNA encoding DGAT type 2 (NeoDGAT2) from the oleaginous microalga Neochloris oleoabundans. The open reading frame of NeoDGAT2 cDNA was 978 bp encoding a protein of 325 amino acids. Although N. oleoabundans has been classified in the class Chlorophyceae, NeoDGAT2 was closely related to Treboxiophycean DGAT2 as it shared 80 % amino acid identity with CvDGAT2-2 of Chlorella variabilis, but distantly related to Chlorophycean DGAT2 as it shared only 34 % identity with CrDGAT2A (DGTT4) of Chlamydomonas reinhardtii. As suggested by hydropathy analysis, NeoDGAT2 might contain 4 to 5 transmembrane domains. We tested whether NeoDGAT2 cDNA encoded a protein with DGAT activity by functional complementation assay in yeast Saccharomyces cerevisiae mutant H1246. NeoDGAT2 was able to compensate the endogenous DGAT2 activity of mutant H1246 and restore the lipid body formation and TAG synthesis. The successful cloning of cDNA encoding a protein with DGAT activity provides a candidate for genetic manipulation in microalgae to increase TAG content for biodiesel production.

Potential application of algicidal bacteria for improved lipid recovery with specific algae

The utility of specific strains of natural algicidal bacteria isolated from shallow wetland sediments was evaluated against several strains of algae with potential immediate or future commercial value. Two strains of bacteria, Pseudomonas pseudoalcaligenes AD6 and Aeromonas hydrophila AD9, were identified and demonstrated to have algicidal activity against the microalgae Neochloris oleoabundans and Dunaliella tertiolecta. These bacteria were further evaluated for the potential to improve lipid extraction using a mild solvent extraction approach. Aeromonas hydrophila AD9 showed a nearly 12-fold increase in lipid extraction with D.tertiolecta, while both bacteria showed a sixfold improvement in lipid extraction with N.oleoabundans.

Growth, morphology and photosynthetic responses of Neochloris oleoabundans during cultivation in a mixotrophic brackish medium and subsequent starvation

The green microalga Neochloris oleoabundans is able to grow in both low and high salinity media and is largely studied for its capability to accumulate lipids under starvation. Moreover, N. oleoabundans is a mixotrophic alga, and then organic carbon addition can promote its growth. This research aims to study the morpho-physiological aspects, with a particular attention on the photosynthetic response, both during mixotrophic growth and starvation in brackish media, more sustainable than freshwater cultivation. In the first step, the alga was cultivated mixotrophically in a brackish medium added with an apple waste product; in the second one, cells were starved also to verify lipid induction. Results indicate that growth is highly promoted during the first week of mixotrophic cultivation, while photosynthetic pigments and lipids are over-produced during the following three weeks of cultivation. In parallel, in mixotrophic cultures the maximum PSII quantum yield was enhanced during the exponential phase of growth. Interesting changes affected the mixotrophic cultures with respect to the partitioning of absorbed light energy. Starvation of both 7-day-grown mixotrophic and autotrophic cultures caused growth inhibition, pigments and photosynthesis downshifting, and concomitantly promoted evident lipid synthesis.

The synchronized cell cycle of Neochloris oleoabundans and its influence on biomass composition under constant light conditions

The effect of cell cycle stage on biomass composition of the green microalgae Neochloris oleoabundans was investigated. N. oleoabundans was grown under constant light conditions in a flat panel photobioreactor operated as a turbidostat. Even though light conditions were constant, a synchronized cell division was observed with the cells dividing by multiple fission during the natural night. Presumably, the circadian clock was responsible for ‘gating’ cell division to this specific time frame. Oscillations in starch, protein and pigment content were observed during the cell cycle. These oscillations could be solely contributed to the cell cycle stage of the synchronized culture, since all experimental conditions were kept constant. A maximum in starch, protein and fatty acid content was obtained just before cell division. Biomass yield was also greatly influenced by the cell cycle and declined to a minimum during cell division. These findings highlight that knowledge of the cell cycle is of importance in microalgae process optimization.

A model for customising biomass composition in continuous microalgae production

A kinetic model is presented that describes functional biomass, starch and storage lipid (TAG) synthesis in the microalga Neochloris oleoabundans as a function of nitrogen and light supply rates to a nitrogen-limited turbidostat cultivation system. The model is based on the measured electron distribution in N. oleoabundans, which showed that starch is the primary storage component, whereas TAG was only produced after an excess of electrons was generated, when growth was limited by nitrogen supply. A fixed 8.6% of the excess electrons ended up in TAG, suggesting close metabolic interactions between nitrogen assimilation and TAG accumulation, such as a shared electron pool. The proposed model shows that by manipulating the cultivation conditions in a light or nitrogen limited turbidostat, algal biomass composition can be customised and the volumetric productivities and yields of the major biomass constituents can be changed on demand.

Biomass and lipid productivity of Neochloris oleoabundans under alkaline–saline conditions

We explored the effect of pH and nitrogen supply on the biomass and fatty acid productivities of the microalga Neochloris oleoabundans cultivated in saline medium under continuous light. During light-limited and nitrogen-replete conditions, the maximal volumetric biomass productivity was 0.98gDWL−1d−1 at pH8.2 and 0.47gDWL−1d−1 at pH10. Apparently microalgal growth costs more energy at high pH, resulting in lower biomass productivities. With the fatty acid content being 2-fold higher at pH10 (8% versus 15% w/w), the maximal volumetric fatty acid productivity was 75.2mgFAL−1d−1 at pH8.2 and 66.6mgFAL−1d−1 at pH10. When high pH was combined with nitrogen depletion, fatty acids accumulated up to 29% (w/w), leading to the highest fatty acid productivity observed in this study (112.4mgFAL−1d−1). These findings stimulate further development of a two-stage process for optimal overall lipid productivity.

Optimization of clean extraction methods to isolate carotenoids from the microalga Neochloris oleoabundans and subsequent chemical characterization using liquid chromatography tandem mass spectrometry

A novel experimental design was used to optimize the extraction of carotenoids from Neochloris oleoabundans using pressurized liquid extraction with food-grade solvents such as ethanol and limonene. Experimental factors, including the extraction temperature and the solvent composition, were optimized using a three-level factorial design. The response variables extraction yield and total amount of carotenoids were assessed. The statistical analysis of the results provided mathematical models to predict the behavior of the responses as a function of the factors involved in the process. The optimum conditions predicted by the model developed in this study were 112 °C as the extraction temperature and 100% ethanol as the extraction solvent. Chemical characterization of the extracts obtained was performed by means of high-performance liquid chromatography-tandem mass spectrometry. The results obtained demonstrated that, under certain growth conditions (photoautotrophically cultured in a medium supplemented with 0.3 g L(-1) KNO3), N. oleoabundans accumulated significant total amounts of the carotenoids (from 57.4 to 120.2 mg carotenoids per gram of extract depending on the extraction conditions), mainly lutein, cantaxanthin, zeaxanthin, and astaxanthin monoesters and diesters.

Transcriptomic analysis of the oleaginous microalga Neochloris oleoabundans reveals metabolic insights into triacylglyceride accumulation

BackgroundThe lack of sequenced genomes for oleaginous microalgae limits our understanding of the mechanisms these organisms utilize to become enriched in triglycerides. Here we report the de novo transcriptome assembly and quantitative gene expression analysis of the oleaginous microalga Neochloris oleoabundans, with a focus on the complex interaction of pathways associated with the production of the triacylglycerol (TAG) biofuel precursor.ResultsAfter growth under nitrogen replete and nitrogen limiting conditions, we quantified the cellular content of major biomolecules including total lipids, triacylglycerides, starch, protein, and chlorophyll. Transcribed genes were sequenced, the transcriptome was assembled de novo, and the expression of major functional categories, relevant pathways, and important genes was quantified through the mapping of reads to the transcriptome. Over 87 million, 77 base pair high quality reads were produced on the Illumina HiSeq sequencing platform. Metabolite measurements supported by genes and pathway expression results indicated that under the nitrogen-limiting condition, carbon is partitioned toward triglyceride production, which increased fivefold over the nitrogen-replete control. In addition to the observed overexpression of the fatty acid synthesis pathway, TAG production during nitrogen limitation was bolstered by repression of the β-oxidation pathway, up-regulation of genes encoding for the pyruvate dehydrogenase complex which funnels acetyl-CoA to lipid biosynthesis, activation of the pentose phosphate pathway to supply reducing equivalents to inorganic nitrogen assimilation and fatty acid biosynthesis, and the up-regulation of lipases—presumably to reconstruct cell membranes in order to supply additional fatty acids for TAG biosynthesis.ConclusionsOur quantitative transcriptome study reveals a broad overview of how nitrogen stress results in excess TAG production in N. oleoabundans, and provides a variety of genetic engineering targets and strategies for focused efforts to improve the production rate and cellular content of biofuel precursors in oleaginous microalgae.

Neochloris oleoabundans grown in enriched natural seawater for biodiesel feedstock: Evaluation of its growth and biochemical composition

The freshwater microalga Neochloris oleoabundans was used to study algal lipid production in enriched natural seawater, in order to assess its suitability as biodiesel feedstock. Optimal and nitrogen-stress (N-stress) conditions were analyzed. Under optimal conditions, the strain’s growth rate was 0.73 div day−1 and the biomass concentration was 1.5gL−1, while it had a maximum lipid yield under N-stress conditions (lipid content: 26% of dry weigh and lipid productivity: 56mgL−1 day−1). Lipid accumulation was mainly due to a significant increase of triacylglycerol content. Neutral lipids were characterized by a dominance of monounsaturated fatty acids and displayed a fatty acid profile that is suitable for biodiesel. This work offers an interesting alternative for sustainable microalgal oil synthesis for biodiesel production without using freshwater resources. However, further studies are necessary in order to optimize the lipid productivities required for commercial biodiesel production.

Growth of oil accumulating microalga Neochloris oleoabundans under alkaline–saline conditions

The effect of elevated pH and salt concentration on the growth of the freshwater microalga Neochloris oleoabundans was investigated. A study was conducted in 24-well plates on the design of a growth medium and subsequently applied in a photobioreactor. An artificial seawater medium with reduced Ca2+ and PO43- could prevent mineral precipitation at high pH levels. Growth was characterized in this new medium at pH8.1 and at pH10.0, with 420mM of total salts. Specific growth rates of 0.08h−1 at pH8.1 and 0.04h−1 at pH10.0 were obtained under controlled turbidostat cultivation. The effect of nitrogen starvation on lipid accumulation was also investigated. Fatty acids content increased not only with nitrogen limitation but also with a pH increase (up to 35% in the dry biomass). Fluorescence microscopy gave visual proof that N. oleoabundans accumulates oil bodies when growing in saline conditions at high pH.

Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor

The fresh water microalga Neochloris oleoabundans was investigated for its ability to accumulate lipids and especially triacylglycerols (TAG). A systematic study was conducted, from the determination of the growth medium to its characterization in an airlift photobioreactor. Without nutrient limitation, a maximal biomass areal productivity of 16.5 g m −2 day −1 was found. Effects of nitrogen starvation to induce lipids accumulation was next investigated. Due to initial N. oleoabundans total lipids high content (23% of dry weight), highest productivity was obtained without mineral limitation with a maximal total lipids productivity of 3.8 g m −2 day −1. Regarding TAG, an almost similar productivity was found whatever the protocol was: continuous production without mineral limitation (0.5 g m −2 day −1) or batch production with either sudden or progressive nitrogen deprivation (0.7 g m −2 day −1). The decrease in growth rate reduces the benefit of the important lipids and TAG accumulation as obtained in nitrogen starvation (37% and 18% of dry weight, respectively).

Effect of a salt‐osmotic upshock on the edaphic microalga Neochloris oleoabundans

Abstract. The edaphic microalga Neochloris oleoabundans (isolated from desert soil) was subjected to a salt‐osmotic shock from 0 to 0·6 kmol m−3 NaCl. The effect of the osmotic upshock on the cell composition was determined. The cell dry weight and the lipid, glycerol and soluble amino acids contents remained unchanged during 5d of osmotic upshock. The protein content increased after 2d of initial osmotic shock, and it appears to be a long‐term haloadaptation process of the cells. The most important short‐term effects of salt osmotic upshock were a decrease in polysaccharide content and an increase in the soluble carbohydrate content of Neochloris oleoabundans cells. Within the first 4h after the initial shock, there was a transfer of carbon units from polysaccharides to sucrose which was independent of photosynthesis. The increase of intracellular concentration of sucrose contributed to cell osmoregulation.