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Prototheca spp. are achlorophyllous algae, ubiquitous in nature. An increasing number of human and animal cases of Prototheca infection (protothecosis) are reported, and antifungal azoles, which inhibit sterol 14α-demethylase (CYP51/ERG11) involved in ergosterol biosynthesis, have empirically been used for the treatment of protothecosis. Although Prototheca, like fungi, has ergosterol in the cell membrane, efficacy of the antifungal azoles in the treatment of protothecosis is controversial. For investigating the interaction of azole drugs with Prototheca CYP51/ERG11, the CYP51/ERG11 genomic genes of four strains of P. wickerhamii and one strain each of P. cutis and P. miyajii were isolated and characterized in this study. Compared with the CYP51/ERG11 gene of chlorophyllous Auxenochlorella Protothecoides, it is possible that ProtothecaCYP51/ERG11 gene, whose exon-intron structure appeared to be species-specific, lost introns associated with the loss of photosynthetic activity. Analysis of the deduced amino acid sequences revealed that Prototheca CYP51/ERG11 and fungal CYP51/ERG11 are phylogenetically distant from each other although their overall structures are similar. Our basic in silico studies predicted that antifungal azoles could bind to the catalytic pocket of Prototheca CYP51/ERG11. It was also suggested that amino acid residues away from the catalytic pocket might affect the drug susceptibility. The results of this study may provide useful insights into the phylogenetic taxonomy of Prototheca spp. in relationship to the CYP51/ERG11 structure and development of novel therapeutic drugs for the treatment of protothecosis. Lay Summary Cases of infection by microalgae of Prototheca species are increasing. However, effective treatment has not been established yet. In this study, gene and structure of Prototheca’s CYP51/ERG11, an enzyme which might serve as a target for therapeutic drugs, were characterized for the first time.

In this study, iron sources and their optimum concentrations for high-quality biodiesel production were determined on Auxenochlorella protothecoides microalgae. Three iron compounds—ferrous sulfate, ferric ethylenediaminetetraacetic acid (EDTA), and ferric chloride—were assessed regarding concentration, specific growth rate, lipid, and lipid quality in relation to fatty acid methyl ester (FAME) composition of microalgal biomass. Auxenochlorella protothecoides demonstrated high resistivity to high concentrations of iron species, still showing an ability to grow in the presence of iron concentrations as high as 21.60 mM. The highest saturated fatty acid (SFA), at 78.5% of FAME, was observed at 1.15 mM ferric chloride, and the highest biomass generation was observed at 1.08 mM ferrous sulfate. In addition, biodiesel and diesel fuel qualities were satisfactory with microalgae cultivated at 0.2 and 14.4 mM ferrous sulfate, 7.19 mM ferric EDTA, and 0.07–21.58 mM ferric chloride concentrations. The former may be more practical, since the use of this iron compound gives the desired result in a broader range. Overall, it can be inferred that high-quality lipid and biodiesel production can be obtained by changing the concentrations and sources of iron compounds.

The vital function of mitochondrial alternative oxidase (AOX) pathway in optimizing photosynthesis during plant de-etiolation has been well recognized. However, whether and how AOX impacts the chloroplast biogenesis in algal cells remains unclear. In the present study, the role of AOX in regulating the reassembly of chloroplast in algal cells was investigated by treating Auxenochlorella protothecoides with salicylhydroxamic acid (SHAM), the specific inhibitor to AOX, in the heterotrophy to autotrophy transition process. Several lines of evidences including delayed chlorophyll accumulation, lagged reorganization of chloroplast structure, altered PSI/PSII stoichiometry, and declined photosynthetic activities in SHAM treated cells indicated that the impairment in AOX activity dramatically hindered the development of functioning chloroplast in algal cells. Besides, the cellular ROS levels and activities of antioxidant enzymes were increased by SHAM treatment, and the perturbation on the balance of NAD+/NADH and NADP+/NADPH ratios was also observed in A. protothecoides lacking AOX activity, indicating that AOX was essential in promoting ROS scavenging and keeping the redox homeostasis for algal chloroplast development during greening. Overall, our study revealed the essentiality of mitochondrial AOX pathway in sustaining algal photosynthetic performance and provided novel insights into the physiological roles of AOX on the biogenesis of photosynthetic organelle in algae.

Biolubricants refer to eco-friendly, biodegradable, and non-toxic lubricants. Their applications are still limited compared to mineral oils; however, their sustainable credentials are making them increasingly attractive. Vegetable oils are frequently used for this purpose. However, vegetable oils have issues of low lipid productivity, dependence on climatic conditions, and need for agricultural land. Microbial oils represent a more sustainable alternative. To ensure their widespread applicability, the suitability of microbial oils from a physicochemical point of view needs to be determined first. In this study, oils obtained from various oleagenic microbes—such as microalgae, thraustochytrids, and yeasts—were characterized in terms of their fatty acid profile, viscosity, friction coefficient, wear, and thermal stability. Oleaginous microalgal strains (Auxenochlorella protothecoides and Chlorella sorokiniana), thraustochytrids strains (Aurantiochytrium limacinum SR21 and Aurantiochytrium sp. T66), and yeast strains (Rhodosporidium toruloides and Cryptococcus curvatus) synthesized 64.5%, 35.15%, 47.89%, 47.93%, 56.42%, and 52.66% of lipid content, respectively. Oils from oleaginous microalgae (A. protothecoides and C. sorokiniana) and yeasts (R. toruloides and C. curvatus) possess excellent physicochemical and tribological qualities due to high amount of monounsaturated fatty acids (oleic acid C18:1 content, 56.38%, 58.82%, 46.67%, 38.81%) than those from oleaginous thraustochytrids (A. limacinum SR21 and Aurantiochytrium sp. T66; 0.96%, 0.08%, respectively) supporting their use as renewable and biodegradable alternatives to traditional mineral oil-based lubricants. Oil obtained from microalgae showed a lower friction coefficient than oils obtained from yeasts and thraustochytrids.

This study examined the effects of monochromatic illumination (blue, red, green and yellow) employing light-emitting diodes (LEDs), trophic conditions (photoautotrophic and mixotrophic), and nitrogen availability (high and low peptone concentration) on the growth and biochemical composition of Auxenochlorella protothecoides. The results revealed that mixotrophic conditions did not favor A. protothecoides, giving lower growth rates compared to heterotrophy (dark conditions). However, mixotrophy gave significantly higher growth rates compared to photoautotrophy. The best light wavelengths for mixotrophic cultivation were that of white and red. In all cases investigated in this study, high peptone concentration (4 g/L) resulted in decreased growth rates. Regarding the biochemical composition of A. protothecoides, the strongest effect, irrespective of trophic conditions, was caused by nitrogen availability (peptone concentration). Specifically, at nitrogen replete conditions (4 g/L peptone), biomass was rich in proteins (32–67%), whereas under deplete conditions (0.5 g/L peptone), A. protothecoides accumulated mainly carbohydrates (up to 56%). Mixotrophic conditions generally favored higher carbohydrate content, whereas photoautotrophic conditions favored higher protein content. The different illumination spectra did not have any clear effect on the biochemical composition (metabolites content), except that, in all trophic conditions, the use of the green spectrum resulted in higher chlorophyll b content. Chlorophyll a fluorescence studies revealed that the trophic conditions and the high peptone concentrations impacted the photosystem II (PSII) performance, and also affected plastoquinone re-oxidation kinetics and the heterogeneity of the PSII reaction centers.

Microalgae can accumulate substantial amounts of triacylglycerol, the primary feedstock for biodiesel production, under different stress conditions. In this study, exposure to alkaline pH (pH 10) using carbonate-bicarbonate buffer, nitrogen starvation, and both were applied to induce triacylglycerol accumulation in Auxenochlorella protothecoides KP7. In addition to triacylglycerol accumulation, growth, biochemical composition, and fatty acid profiles were examined in detail and compared. Our results illustrated that the combination of nitrogen starvation and alkaline pH led to significant increases in triacylglycerol and starch contents and drastic decreases in chlorophyll and protein contents compared with their control levels. Triacylglycerol levels, the triacylglycerol/total lipid ratio, and lipid productivities were significantly increased after 7 days of cultivation under the combined stress compared with the effects of nitrogen starvation alone. After prolonged cultivation under the combined stress, triacylglycerol content reached up to 25.01% of the dry cell weight and constituted up to 81.25% of total lipids. Under the combined stress, the major fatty acids in the total lipid of A. protothecoides KP7 were C18:1 (57.5%), C18:2 (19.5%), C16:0 (12.5%), and C18:3 (5.6%), and the percentages of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids represented 16.3, 58.4, and 25.1% of the total fatty acids, respectively. Furthermore, the combined stress was associated with the lowest iodine value (97.64 g I-2 (100 g)(-1)), highest oxidative stability (7.29 h), and highest cetane number (55.26) among the examined conditions. The use of nitrogen-free medium containing carbonate-bicarbonate buffer for cultivation appears promising for enhancing triacylglycerol production in A. protothecoides.

Under ultra-high cadmium (Cd) stress, large amounts of glutathione are produced in Auxenochlorella protothecoides UTEX 2341, and the lipid content increases significantly. Glutathione is the best reductant that can effectively remove Cd, but the relationship between lipid accumulation and the cellular response to Cd stress has not been ascertained. Integrating analyses of the transcriptomes and lipidomes, the mechanism of lipid accumulation to Cd tolerance were studied from the perspectives of metabolism, transcriptional regulation and protein glutathionylation. Under Cd stress, basic metabolic pathways, such as purine metabolism, translation and pre-mRNA splicing process, were inhibited, while the lipid accumulation pathway was significantly activated. Further analysis revealed that the transcription factors (TFs) and genes related to lipid accumulation were also activated. Analysis of the TF interaction sites showed that ABI5, MYB_rel and NF-YB could further regulate the expression of diacylglycerol acyltransferase through glutathionylation/deglutathionylation, which led to increase of the triacylglycerol (TAG) content. Lipidomes analysis showed that TAG could help maintain lipid homeostasis by adjusting its saturation/unsaturation levels. This study for the first time indicated that glutathione could activate TAG synthesis in microalga A. protothecoides, leading to TAG accumulation and glutathione accumulation under Cd stress. Therefore, the accumulation of TAG and glutathione can confer resistance to high Cd stress. This study provided insights into a new operation mode of TAG accumulation under heavy metal stress.

Herein, we report a new process for intensified hydro-upgrading of a biocrude produced from the hydrothermal processing of Auxenochlorella pyrenoidosa (AuP) in tetralin at a mass ratio of 1:1 with the addition of 10 wt.% Pt/C and 8 MPa H2 at different temperatures (300-480 °C) and times (1-8 h). Under the selected reaction conditions, the gaseous products were replaced with fresh H2 at regular time intervals; this process was then continued until the total reaction time was over. Increasing the number of H2 replacement times decrease the C, N, O, and S contents and increased the H content. The treated bio-oil mainly consisted of aromatic, saturated and unsaturated hydrocarbons. Overall, H2 replacement removed some of the denitrogenation, desulfurization, and deoxygenation products and avoided secondary reactions of gaseous products with the bio-oil, thus promoting the hydrogenation reaction and N, O, and S removal from the treated bio-oil.

The influence of pH and substrate concentration on the mixotrophic growth of five microalgae species in presence of acetate or butyrate was evaluated. Acutodesmus obliquus, Auxenochlorella protothecoides, two strains of Chlamydomonas reinhardtii, and Chlorella sorokiniana were cultivated at pH from 5 to 10. Acetate was efficiently assimilated by all strains while butyrate uptake was greatly strain dependent. Growth rates at pH values above 8 were reduced while values below 5 or 6 inhibited growth on acetate and butyrate respectively. The influence of acetic and butyric acid concentration was tested. It was demonstrated that the main factor affecting microalgae growth is the concentration of undissociated acid, which can be controlled by pH adjustment. The strains exhibited inhibition threshold concentrations of 71–207 mg·L−1 for acetic acid and of 13–25 mg·L−1 for butyric acid. These findings are crucial for proper control of processes coupling bacterial fermentation and microalgae cultures.

Temperature is one of the critical environmental factors that influence microalgal growth, lipid content and fatty acid (FA) composition. However, the molecular mechanism underlying regulations of FA metabolism under low and high temperature stress in oleaginous microalgae remains unclear. In this study, integrated analyses of transcriptome, proteome and fatty acid profilings were performed for the first time in Auxenochlorella protothecoides UTEX 2341. Under low and high temperature (LT and HT) stress, a total of 5565 and 4757 genes, and 1311 and 728 proteins were differentially expressed respectively. 65 actively expressed genes and 61 proteins involved in FA metabolism were identified. A strong positive correlation between the genes' transcript and protein levels existed in FA metabolism (r = 0.80, p-value

© 2018 Elsevier B.V. Auxenochlorella protothecoides is a known thiamine auxotroph but our past work has shown that it can synthesize thiamine if provided with the precursor molecule 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Partial thiamine auxotrophy is common in microalgae with important ramifications for global phytoplankton productivity as well as engineering applications of algae. While thiamine deficiency can greatly depress algae growth and lipid content, the detailed metabolic impacts of thiamine deficiency are not well understood. We used metabolomics to study the response to thiamine-limited and replete conditions in mixotrophic A. protothecoides. We also investigated the impacts of exogenous HMP addition and the use of spent medium from another green algae, C. sorokiniana, as a source of thiamine metabolites. This is the first study, to our knowledge, that addresses metabolic impacts of thiamine deficiency and alleviation in green microalgae. Thiamine deficient cultures exhibited accumulation of pyruvate and α-ketoglutarate, indicating bottlenecks at the pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) complexes. Both PDH and OGDH require thiamine pyrophosphate (TPP) as a cofactor. Transketolase also requires TPP but we only observed build-up of ribose-5-phosphate when glucose was supplied as a substrate. As expected, thiamine and HMP addition could alleviate these metabolic bottlenecks while greatly increasing algal growth, neutral lipid and starch content. Spent medium from C. sorokiniana only appeared to partially alleviate thiamine deficiency and resulted in build-up of isocitrate and glycolate, metabolites that appeared relatively unaffected by the presence or absence of thiamine. Interestingly, longer culture time of C. sorokiniana when preparing the spent medium led to much higher availability of thiamine metabolites. Thus, under the right conditions, it may be possible to co-culture mutually beneficial algae species and/or recycle spent cultivation medium to overcome auxotrophy in algae.

Enzymatic hydrolysis and decoloration are indispensable processes for the production of biological peptides from microalgae materials. Herein, this study aimed to investigate the effects of enzymatic hydrolysis and activated carbon decoloration on antioxidative activities and peptidomic patterns of microalgae protein hydrolysates based on a peptidomic approach. Protein hydrolysates with different hydrolysis time (2–16 h) showed significantly different antioxidative activities based on DPPH and ABTS assays. Regarding the peptidomic pattern, principal component analysis (PCA) models revealed that hydrolysates were classified into discriminable clusters depending on the hydrolysis and decoloration processes. Protein hydrolysates contained many aromatic amino acid containing peptides, such as GDSLYPG, SADPETF, FPALN, FPGDY, and LTDWV, contributing to the classification of PCA models. Activated carbon decoloration at the concentration of 5 mg/mL significantly increased the color lightness by 38.20%, reduced antioxidative activities by 29.78% and 33.59% based on DPPH and ABTS methods, and reduced relative abundances of peptides, especially aromatic amino acid containing peptides. Fifteen selected peptides showed strong ABTS radical scavenging abilities and ten of these peptides were correlated with the antioxidative properties of hydrolysates. The monitoring of dynamic production processes at a molecular scale would contribute to the production and quality control of bioactive peptides from Auxenochlorella pyrenoidosa.

Culturing microalgae using dairy-wastes offers the opportunity of producing valuable biomass for different industrial applications. The capability of four Chlorella species and a recombinant Chlamydomonas reinhardtii strain to mixotrophically grow in wastewaters from an Italian dairy factory was investigated. A robust algal growth could be efficiently sustained in these wastes, despite the abundance of d -Lactose (~4% w/v), that could not be metabolized by any microalgal species. Non axenic cocultivation of microalgae together with microbial communities from the dairy wastes resulted in a marked decrease of their pollution load, thus reducing the necessity of expensive treatments before their discharge in the municipal sewage system. Microalgae cultivated using dairy-wastes were characterized by a lipid content ranging from 12% to 21% (w/w), with Auxenochlorella protothecoides reaching the highest lipid productivity (~0.16 g/L/d) whereas the transplastomic C. reinhardtii strain expressing a thermostable β-glucosidase reached a recombinant enzyme productivity of 0.18 mg/L/d.

In recent years, there has been growing interest in the biomass of unicellular algae as a source of valuable metabolites. The main limitations in the commercial application of microbial biomass are associated with the costs of production thereof. Maize silage is one of the main substrates used in biogas plants in Europe. The effects of sterilized agricultural liquid digestate (LD) from methane fermentation of maize silage on the growth rates, macro and micronutrient removal efficiency, lipid content, and fatty acid profile in Auxenochlorella protothecoides were investigated. The results indicate that A. prothecoides can proliferate and accumulate lipids with simultaneous reduction of nutrients in the 1:20 diluted liquid digestate. The rate of nitrogen and phosphorus removal from the liquid digestate was 79.45% and 78.4%, respectively. Cells growing in diluted liquid digestate exhibited the maximum lipid content, i.e., 44.65%. The fatty acid profile of A. prothecoides shows a decrease in the content of linolenic acid by 20.87% and an increase in oleic acid by 32.16% in the LD, compared with the control. The liquid digestate changed the content of monounsaturated fatty acids and polyunsaturated fatty acids. The cells of A. protothecoides growing in the liquid digestate were characterized by lower PUFA content and higher MUFA levels.

Human completely rely on food and nutrition supply to absorb enough lutein, which is an important antioxidant beneficial to health of eyes and heart. In plants, lycopene epsilon-cyclase (LCYE) is the key enzyme in lutein synthesis pathway, where lycopene is converted into δ-carotene, α-carotene and lutein. In this study, for the first time, we successfully cloned CvLCYE, the evolutionarily conserved lycopene cyclase in the genome of Chlorella vulgaris that is a green alga rich in lutein. Homology analysis showed that the amino acid sequence of CvLCYE protein is factually high identical with that of other aquatic green algae including Chlorella variabilis, Auxenochlorella protothecoides strain CS-41, Chromochloris zofingiensis and Chlamydomonas reinhardtii. Meanwhile, the UPGMA tree analysis of CvLCYE protein sequence showed the consistent results. Bioinformatic analysis of CvLCYE protein revealed a typical lycopene cyclase domain (Pfam05834) between the 64th–475th amino acid. The 3D structure prediction of CvLCYE protein displayed a structure of beta strand-alpha helix-beta strand, which is conserved in all known lycopene epsilon-cyclases. Subcellular localization tests demonstrated that mature CvLCYE-GFP fused protein localized to cytoplasm in Arabidopsis protoplasts. Importantly, targeted HPLC analysis on lycopene content are performed with Escherichia coli strain expressing CvLCYE, which displayed faded pink of consumption of lycopene, in comparison to strain expressing empty vector, which maintained pink because of accumulation of lycopene. HPLC data strongly suggested the capability of CvLCYE to catalyze lycopene. Furthermore, enzymatic activity of CvLCYE was also clearly verified in Chlamydomonas reinhardtii that is the model organism for algae research. Interestingly, we found that the lutein content in transgenic C. reinhardtii expressing CvLCYE increased by about 2.3 times than that of wild type strain. All these results provide insight into the positive role played by CvLCYE in lutein synthesis and lay a promising foundation for the molecular breeding of lutein metabolic pathway in Chlorella vulgaris and other algae.

Algae have gained attention for production of fuels and chemicals, and treatment of wastewater. The high cost of algae cultivation, however, has limited industry adoption for these applications. Developing methods to increase algal growth rates and lipid content has emerged as an important strategy toward reducing production costs, and significant research effort has been exerted in this area. We have reported previously that co-culturing the green alga, Auxenochlorella protothecoides, with Escherichia coli under mixotrophic conditions led to 2–6 fold increases in algal growth, doubling of neutral lipid content, and elevated nutrient removal rates compared to axenic growth, indicative of a symbiotic relationship. In the present work, we reveal that symbiosis stems largely from E. coli's provision of thiamine derivatives and degradation products to A. protothecoides. LCMS showed that residual cell-free medium obtained from axenic E. coli culture contained roughly 1.15 nM thiamine pyrophosphate and 4.0–9.1 nM of the thiamine precursor and degradation product, 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). These compounds were found to promote the growth, lipid content, and glucose uptake of A. protothecoides, while dramatically improving substrate utilization efficiency. Due to widespread cofactor auxotrophy among algae, the co-culture results presented here likely extend to a large number of microbial community systems. We show that algal-algal symbiosis based on cofactor exchange is also possible, opening a new frontier in algae cultivation management. These findings highlight the potential of engineered microbial communities for improved algal biofuel production and wastewater treatment.

Plant growth-promoting bacteria, such as Azospirillum brasilense, have the potential to significantly increase algal growth rates through a variety of mechanisms including the production of indole-3-acetic acid, an auxin hormone. A. brasilense promotion of growth in Chlorella sorokiniana is well-established for co-cultures suspended in alginate bead structures, however, its impacts on other types of green algae grown in suspended cultures is not well-understood. The objective of this research was to determine the impact of A. brasilense and indole-3-acetic acid on growth promotion and energy storage product accumulation in suspended cultures of C. sorokiniana and Auxenochlorella protothecoides. Suspended cultures were grown in lab-scale photobioreactors under the following conditions: algae grown on chemical medium (control), in co-culture with A. brasilense, chemical medium supplemented with exogenous indole-3-acetic acid, and algae grown on spent medium from A. brasilense. The results showed that co-cultures and exogenous indole-3-acetic acid stimulated growth in both algae types but the effect was stronger in C. sorokiniana. These same treatments also suppressed neutral lipids (particularly triacylglycerol) and starch during exponential growth of C. sorokiniana. Indole-3-acetic acid and co-cultures suppressed starch in A. protothecoides. Spent medium resulted in slight growth promotion in C. sorokiniana but significant growth suppression in A. protothecoides. It also led to significantly different compositional changes compared to using live A. brasilense, indicating that bioactive constituents in A. brasilense secretions are transient or that physical cell attachment is important for ensuring adequate mass transfer of these constituents. Overall, the findings suggest that indole-3-acetic acid and live A. brasilense mobilize cellular energy resources for growth.

Cultivating algae in anaerobic digestate has the potential to produce valuable bio-products with economic and environmental benefits. Growing algae in wastewater will immobilize the nutrients as well as produce biomass suitable for biofuel and animal feed production. However, culturing algae on high-strength anaerobic digestates faces technical challenges. One of the major challenges is the presence of inhibitory compounds in digestate that suppress algal growth. Volatile fatty acids (VFA) are commonly found in the effluent of anaerobic processes. VFA concentrations can exceed 1 g/L in digesters operating under unstable or overloaded conditions. VFAs present in digestate can significantly harm downstream algal growth, potentially leading to culture crashes. We tested the inhibitory effects of two model VFAs (propionic acid and butyric acid) on two different strains of green microalgae. We observed that the VFAs had a strong inhibitory effect on algal growth with EC50 concentrations of ~400 mg/L. Therefore, removing VFAs from the digestate would be beneficial for algae cultivation. Here, we report a pretreatment process for the effluent that uses a titanium oxide-based nanomaterial designed to oxidize VFA and other organic molecules through photocatalysis. Two model green algae, Chlorella sorokiniana and Auxenochlorella protothecoides were cultivated in the presence of VFAs (originally 1g/L for both propionic acid and butyric acid) with and without nanomaterial treatment. With the nanomaterial treatment, we observed a 20-30% reduction in VFA concentrations and X% faster algal growth. Further research is needed to understand more inhibitory compounds in digestate and to develop nanomaterials with more efficient removal capability.

The ultra-high Cd polluted environment is a special habitat in nature. Analysis of the biological adaptation and resistance mechanism of Auxenochlorella protothecoides UTEX234 to ultra-high Cd stress would offer some inspiring understanding on Cd detoxification mechanism and help discovering highly active bioremediation agents. In this study, integrated analyses of the transcriptome, multi-physiological and biochemical data and fatty acid profilings of UTEX2341 were performed for the first time. It was found that exogenous Ca ions could alleviate Cd stress. Manganese-dependent superoxide dismutase and peroxidase also participated in intracellular detoxification. And non-enzymatic antioxidants rather than one specific enzymatic antioxidant were suggested to be used as “core antioxidants”, which witnessed better performance in Cd detoxification. In addition, Cd stress improved sixteen alkane value and biofuel yield and quality.

In order to enhance lutein accumulation and to explain the reasons for the difference in lutein accumulation under photoautotrophic and heterotrophic conditions, different culture modes and the associated transcriptome profiles were investigated in Auxenochlorella protothecoides. The heterotrophic-photoautotrophic transition culture mode was investigated for lutein accumulation, changing from organic carbon to increase biomass in dark fermentation to irradiation under nitrogen rich conditions. This strategy increased the lutein content 10 times along with chloroplast regeneration and little biomass loss in 48 h. The highest lutein productivity and production in the heterotrophic-photoautotrophic transition culture reached 12.36 mg/L/day and 34.13 mg/L respectively within seven days. Furthermore, compared to the photoautotrophic conditions, most genes involved in lutein biosynthesis and photosystem generation were down-regulated during heterotrophic growth. By contrast, two &beta, ring hydroxylases were transiently upregulated, while violaxanthin de-epoxidase and zeaxanthin epoxidase were mostly downregulated, which explained the extremely low lutein content of heterotrophic cells. Nevertheless, the lutein proportion in total carotenoids reached nearly 100%. This study is the first to our knowledge to report on a comparative transcriptome analysis of lutein biosynthesis, and it provides a promising strategy to boost lutein production in A. protothecoides.

A novel highly selective lutein-producing marine microalga was isolated and identified using high throughput screening (HTS). Phylogenetic analysis based on 18S rRNA gene sequence revealed it belongs to a microalgae class (Trebouxiophyceae), and is closely related to Auxenochlorella spp. The novel strain was designated as Auxenochlorella sp. strain LEU27. Based on HPLC-DAD and HPLC-MS analyses, strain LEU27 produced a total of 1203.9 ± 98 μg g−1 dry cells of carotenoids, including a remarkable amount (996.6 ± 98 μg g−1 dry cells) of pure lutein under heterotrophic growth conditions. This is the first report that describes a natural, highly selective lutein-producing marine microalga isolated by HTS approach.

Algae were investigated in the past as a potential source of biofuel and other useful chemical derivatives. Magnetic separation of algae by iron oxide nanoparticle binding to cells has been proposed by others for dewatering of cellular mass prior to lipid extraction. We have investigated feasibility of magnetic separation based on the presence of natural iron stores in the cell, such as the ferritin in Auxenochlorella protothecoides (A. p.) strains. The A. p. cell constructs were tested for inserted genes and for increased intracellular iron concentration by inductively coupled plasma atomic absorption (ICP-AA). They were grown in Sueoka's modified high salt media with added vitamin B1 and increasing concentration of soluble iron compound (FeCl3 EDTA, from 1× to 8× compared to baseline). The cell magnetic separation conditions were tested using a thin rectangular flow channel pressed against interpolar gaps of a permanent magnet forming a separation system of a well-defined fluid flow and magnetic fringing field geometry (up to 2.2 T and 1,000 T/m) dubbed “magnetic deposition microscopy”, or MDM. The presence of magnetic cells in suspension was detected by formation of characteristic deposition bands at the edges of the magnet interpolar gaps, amenable to optical scanning and microscopic examination. The results demonstrated increasing cellular Fe uptake with increasing Fe concentration in the culture media in wild type strain and in selected genetically-modified constructs, leading to magnetic separation without magnetic particle binding. The throughput in this study is not sufficient for an economical scale harvest.

Microalgae are a sustainable energy source because of their lipid storage capacity. However, the cultivation of microalgae requires optimization to produce a high biomass and lipid content. Stressing the cell is a common approach that is applied to increase the lipid content of the algae. Within this context, sodium chloride (NaCl) stress and magnesium (Mg2+) limitations were applied individually and mutually to understand their potential effects on biomass, lipid quantity, and the fatty acid methyl esters (FAME) profile of Auxenochlorella protothecoides. Saturated fatty acid (SFA) contents as high as 43.9% were obtained at 0 mg L−1 Mg2+ with 5 g L−1 NaCl. The highest linoleic acid content (35.2%) was obtained at 0.3 mg L−1 Mg2+ with 2.5 g L−1 NaCl. However, the highest biomass was achieved at 18.5 mg L−1 Mg2+ with 5.0 g L−1 NaCl according to surface response methodology. These optimization data could be useful for producing a feasible and sustainable biodiesel production strategy with high biomass and lipid productivity.

Specific growth rates (SGR) of freshwater algae species (Chlorella vulgaris, Auxenochlorella protothecoides, and Chlorella sorokiniana) and the marine species Nannochloropsis oculata on various nitrogen sources (ammonium carbonate, ammonium chloride, sodium nitrate, and urea) could be determined by in vivo chlorophyll-a autofluorescence. These preferences could be determined before large pH changes occurred in the media, with no significant difference (P > 0.05) between buffered and non-buffered media. In all algal species, acclimatization was observed with no significant difference (P > 0.05) between SGRs of the second and third cultivations. ANOVA of SGRs in the acclimatized second and third cultivations revealed preferences for nitrogen sources among most of the algae; C. vulgaris preferred sodium nitrate over other nitrogen sources, A. protothecoides adapted to urea after no growth in the first cultivation, and the SGRs of N. oculata showed an aversion for sodium nitrate over other nitrogen sources (P < 0.05).

The only algae which are able to inflict disease on humans and other mammals through active invasion and spread within the host tissues belong to either of two genera: Chlorella and Prototheca. Whereas Chlorella infections are extremely rare, with only two human cases reported in the literature, protothecosis is an emerging disease of humans and domestic animals, especially dairy cows. The genus Prototheca, erected by Kruger in 1894, has undergone several significant revisions, as more phenotypic, chemotaxonomic, and molecular data have become available. Due to this, a large number of Prototheca strains have been accumulated in public culture collections, over the years, where they still exist under outdated or invalid infraspecific or species names. In this study, the partial cytb gene was used as a marker to revise the taxonomy and nomenclature of a set of Prototheca strains, preserved in major algae culture repositories worldwide. Within the genus, two main lineages were observed, with a dominance of typically dairy cattle-associated (i.e. P. ciferrii, formerly P. zopfii gen. 1, the here validated P. blaschkeae, and one newly erected species, namely P. bovis, formerly P. zopfii gen. 2) and human-associated (i.e. P. wickerhamii, P. cutis, P. miyajii) species, respectively. In the former lineage, three newly described species were allocated, namely P. cookei sp. nov., P. cerasi sp. nov., and P. pringsheimii sp. nov., and the lecto- and epitypified P. zopfii species. The second, or so-called P. wickerhamii lineage, incorporated a newly proposed species of P. xanthoriae sp. nov. These protothecans were shown as the closest relatives of the photosynthetic genera, Chlorella and Auxenochlorella. The environmental species P. ulmea was synonymized with the lecto- and epitypified P. moriformis species. For circumscription and differentiation of Prototheca spp., the use of phenotypic characters, and morphology in particular, is of limited value and should rather be auxiliary to molecular marker-based approaches. As demonstrated in our previous study and corroborated in the present one, the cytb gene provides higher resolution than the conventional rDNA markers, and currently represents the most efficient barcode for the Prototheca algae.

This study evaluates the effect of different concentrations of glucose supplementation on growth, lipid accumulation, and the fatty acid profile in the Auxenochlorella protothecoides. Addition of glucose promoted the growth rate and decreased the chlorophyll content. Compared with photoautotrophic cells, an increase in the lipid content was observed in mixotrophic cells. The glucose addition induced changes in the fatty acid profile. Higher content of saturated fatty acids was found in the case of cells growing in the glucose-free medium. Oleic acid was the predominant component in mixotrophic cells supplemented with 5gL(-1) glucose, while linoleic acids dominated in cultures supplemented with both 1 and 3gL(-1) glucose. The use of glucose was associated with decreased levels of linolenic acid and PUFA. The changes in the fatty acid profile in mixotrophic cells are favourable for biodiesel production.

Using wastewater to grow algae is probably the most promising route to reduce production costs associated with nutrients and water. In this study, a newly isolated facultative heterotrophic freshwater microalgae strain, Auxenochlorella protothecoides UMN280, was examined for algal growth, wastewater nutrient removal efficiency, and lipid accumulation in batch and semi-continuous cultivation with various hydraulic retention time using concentrated municipal wastewater (CMW) as cultivation media. The results of the 6 day batch cultivation showed that the maximal removal efficiencies for total nitrogen, total phosphorus, chemical oxygen demand (COD) and total organic carbon (TOC) were over 59%, 81%, 88% and 96%, respectively, with high growth rate (0.490 d −1 ), high biomass productivity (269 mg L −1 d −1 ) and high lipid productivity (78 mg L −1 d −1 ). Further fatty acid methyl ester (FAME) analysis showed that the microalgal lipids were mainly composed of C16/C18 fatty acids (accounting for over 94% of total fatty acid), which are suitable for high-quality biodiesel production. The system could be scaled up from 100 mL flasks to 25 L BIOCOIL reactors, and semi-continuously operated at hydraulic retention time of 3 days with a net biomass productivity of 1.51 g L −1 d −1 of dried algae.

Microalgae are promising feedstocks for biodiesel, where the high proportion of monounsaturated fatty acid such as oleic acid (C18:1) is preferred. To regulate fatty acid desaturation in microalgae, the relationship among nitrate concentration, fatty acid composition and the expression levels of desaturase genes was explored. Dynamic variations of fatty acid profiles suggested nitrate could induce desaturation of C18 fatty acids. The content of C18:1 inAuxenochlorella pyrenoidosawas 30.88% at 0 g l−1nitrate concentration compared with 0.48% at 1.5 g l−1. The expressions of relative delta-9, 12 and 15 fatty acid desaturase genes (Δ9, Δ12 and Δ15FADs) were further investigated. The 330% upregulated expression of Δ9FAD in logarithmic phase at 0 g l−1resulted in C18:1 accumulation. Moreover, nitrate replenishment caused a sharp reduction of C18:1 from 34.79% to 0.22% and downregulation of Δ9FAD expression to 1% of the nitrate absence level, indicating the pivotal role of Δ9FAD in C18:1 accumulation. Finally, overexpression of Δ9FAD inEscherichia coliandSaccharomyces cerevisiaeresulted in an increase of C18:1, confirming its ability of desaturating C18:0. The results could provide a new approach and scientific guidance for the improvement of biodiesel quality and industrialization of high-valued chemicals by means of metabolic engineering.

Despite the capacity to accumulate ~70% w/w of lipids, commercially produced unicellular green alga A. protothecoides may become compromised due to the high cost of phosphate fertilizers. To address this limitation A. protothecoides was selected for adaptation to conditions of 100× and 5× lower phosphate and peptone, respectively, compared to 'wild-type media'. The A. protothecoides showed initial signs of adaptation by 45-50 days, and steady state growth at ~100 days. The low phosphate (P)-adapted strain produced up to ~30% greater biomass, while total lipids (~10% w/w) remained about the same, compared to the wild-type strain. Metabolomic analyses indicated that the low P-adapted produced 3.3-fold more saturated palmitic acid (16:0) and 2.2-fold less linolenic acid (18:3), compared to the wild-type strain, resulting in an ~11% increase in caloric value, from 19.5kJ/g for the wild-type strain to 21.6kJ/g for the low P-adapted strain, due to the amounts and composition of certain saturated fatty acids, compared to the wild type strain. Biochemical changes in A. protothecoides adapted to lower phosphate conditions were assessed by comparative RNA-Seq analysis, which yielded 27,279 transcripts. Among them, 2,667 and 15 genes were significantly down- and up-regulated, at >999-fold and >3-fold (adjusted p-value

Taxonomy and phylogeny of twenty two microalgal isolates were examined using both universal and newly designed molecular primers. Among the isolates, Scenedesmus bijugus, Coelastrella sp., Auxenochlorella protothecoides, and Chlorella sp. were particularly promising in terms of producing lipids as measured by fatty acid methyl esters (FAME) analysis and significant concentration of carotenoids. A comparative experiment showed that S. bijugus and Chlorella sp. were the most promising candidates (L(-)(1)d(-)(1), with biomass) 174.77±6.75, 169.81±5.22mg, lipids 40.14±3.31, 39.72±3.89mg, lutein 0.47, 0.36mg, and astaxanthin 0.27, 0.18mg respectively. The fatty acids produced by these microalgal isolates were mainly palmitic, stearic, oleic, linoleic, and linolenic acid. The freshwater microalgal isolate S. bijugus be the most suitable isolate for producing biodiesel and carotenoids, due to high productivity of biomass, lipids, metabolites, and its suitable fatty acid profile.

Autophagy is a cellular degradation process that recycles cytoplasmic components in eukaryotes. Although intensively studied in yeast, plants, and mammals, autophagy in microalgae is not well understood. Auxenochlorella protothecoides is a green microalga that has the ability to grow either autotrophically when under light or heterotrophically when in media containing glucose. The two growth modes are inter-convertible and transition between them is accompanied by drastic changes in morphology and cellular composition; however, the mechanisms underlying these changes are unknown. In this study, we identified autophagy-related genes and characterized their roles in the degradation of lipid droplets during the heterotrophy-to-autotrophy (HA) transition in A. protothecoides. Most of the proteins constituting the eukaryotic “core machinery” were conserved in A. protothecoides. Two proteins, Atg4 and Atg8, were further investigated. A. protothecoides ATG4 was cloned from a cDNA library and expressed within yeast, and was able to functionally restore the autophagy pathway in atg4Δ yeast during nitrogen starvation. Furthermore, Atg8, which displayed high sequence identity with its yeast homolog, was able to conjugate to phosphatidylethanolamine (PE) in vitro and was recruited to the phagophore assembly site in yeast. We also identified a C-terminal glycine residue, G118, that was the cleavage site for Atg4. Finally, we used confocal and transmission electron microscopy to reveal that autophagic-like vacuoles were detectable in algal cells during the HA transition. Our data suggested that the lipid droplets in heterotrophic cells were engulfed directly by the autophagic-like vacuole instead of via autophagosomes.

The present study introduced an integrated method for the production of biodiesel from microalgal oil. Heterotrophic growth of Chlorella protothecoides resulted in the accumulation of high lipid content (55%) in cells. Large amount of microalgal oil was efficiently extracted from these heterotrophic cells by using n-hexane. Biodiesel comparable to conventional diesel was obtained from heterotrophic microalgal oil by acidic transesterification. The best process combination was 100% catalyst quantity (based on oil weight) with 56:1 molar ratio of methanol to oil at temperature of 30 degrees C, which reduced product specific gravity from an initial value of 0.912 to a final value of 0.8637 in about 4h of reaction time. The results suggested that the new process, which combined bioengineering and transesterification, was a feasible and effective method for the production of high quality biodiesel from microalgal oil.

Microalgae of the Chlorella clade are extensively investigated as an environmentally friendly source of renewable biofuels and high-value nutrients. In addition, essentially unprocessed Chlorella serves as wholesome food additive. A recent study on 80 commercial Chlorella preparations revealed an unexpected variety of protein-linked N-glycan patterns with unprecedented structural features, such as the occurrence of arabinose. Two groups of products exhibited a characteristic major N-glycan isobaric to the Man2GlcNAc2XylFuc N-glycan known from pineapple stem bromelain, but tandem mass spectrometry (MS/MS) analysis pointed at two types of N-glycan different from the bromelain structure, as well as from each other. Here we report the exact structures of these two novel N-glycan structures, elucidated by nuclear magnetic resonance spectroscopy and MS/MS, as well as on their phylogenetic context. Despite their humble size, these two N-glycans exhibited a very different design with structural features unrelated to those recently described for other Chlorella-clade strains. The major glycans of this study presented several novel structural features such as substitution by arabinose or xylose of the internal N-acetylglucosamine, as well as methylated sugars. ITS1-5.8S-ITS2 rDNA barcode analyses revealed that the xylose-containing structure derived from a product primarily comprising Scenedesmus species, and the arabinose-containing glycan type related to Chlorella species (SAG211-34 and FACHB-31) and to Auxenochlorella. This is another example where characteristic N-glycan structures distinguish phylogenetically different groups of microalgae.

The aim of this work was to establish a process for the heterotrophic growth of green microalgae using forest biomass hydrolysates. To provide a carbon source for the growth of the green microalgae, two forest biomasses (Norway spruce and silver birch) were pretreated with a hybrid organosolv-steam explosion method, resulting in inhibitor-free pretreated solids with a high cellulose content of 77.9% w/w (birch) and 72% w/w (spruce). Pretreated solids were hydrolyzed using commercial cellulolytic enzymes to produce hydrolysate for the culture of algae. The heterotrophic growth of A. protothecoides was assessed using synthetic medium with glucose as carbon source, where the effect of sugar concentration and the carbon-to-nitrogen ratio were optimized, resulting in accumulation of lipids at 5.42 ± 0.32 g/L (64.52 ± 0.53% lipid content) after 5 days of culture on glucose at 20 g/L. The use of birch and spruce hydrolysates was favorable for the growth and lipid accumulation of the algae, resulting in lipid production of 5.65 ± 0.21 g/L (66 ± 0.33% lipid content) and 5.28 ± 0.17 g/L (63.08 ± 0.71% lipid content) when grown on birch and spruce, respectively, after only 120 h of cultivation. To the best of our knowledge, this is the first report of using organosolv pretreated wood biomass hydrolysates for the growth and lipid production of microalgae in the literature. The pretreatment process used in this study provided high saccharification of biomass without the presence of inhibitors. Moreover, the lipid profile of this microalga showed similar contents to vegetable oils which improve the biodiesel properties.