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Microbial electrosynthesis (MES) for bioelectro carbon dioxide (CO2) recycling is an interesting and sustainable opportunity to exploit off gases from industrial facilities and convert them into valuable energy sources. In the present study, a two-step process based on coupling a bioelectrochemical system (BES) and heterotrophic microalgae Auxenochlorella protothecoides is proposed to convert carbon dioxide into a biodiesel compatible oil. The MES effluent was further processed in a heterotrophic microalgae batch reactor, where the acetate previously synthesized from CO2 was converted into bio-oil in a subsequent, extraction-free step. Two MES reactors were operated in batch mode at an applied cathodic potential of 0.8 V vs. SHE (standard hydrogen electrode) for 95 days. The system reached a concentration of up to 13 g L 1 of acetate (at a maximum production rate of 0.29 g L 1 d 1). Microbial community analysis revealed the presence of Clostridium spp. in both reactors. In a second stage, the effluent from the biocathode was transferred to microalgae reactors containing A. protothecoides to assess oil production. The bio-oil content was up to 22% w/w (dry weight), sufficient to further explore the feasibility of microalgae-to-oil recovery in the future. According to our estimations, 7.59 kg CO2 can be converted into 1 kg acetate, which can be used to grow heterotrophically 1.11 kg dry algae; an overall balance of 0.03 kg bio-oil produced per kg CO2 captured was assessed. The oil obtained can be further processed to produce a biodiesel compatible with EU requirements for biofuels This research was funded by the Spanish Ministry of Science through the grant RTI2018-098360-B-I00 and the Agency for Business Competitiveness of the Government of Catalonia (ACCIO) through the DigesTake Project (COMRDI16-1-0061). E. ´ P.-V. is grateful for the Research Training grant from the University of Girona (IFUdG2018/52). S. P. is a Serra Hunter ´ Fellow (UdG-AG-575) and acknowledges the funding from the ICREA Acad`emia award. LEQUiA and Ecoaqua have been recognized as consolidated research groups by the Catalan Government with codes 2017-SGR-1552 and 2017SGR-548, respectively

An investigation on which pH levels would affect the growth of Auxenochlorella protothecoides.

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.

Hydrothermal liquefaction of Auxenochlorella pyrenoidosa (AuP) and Arthrospira platensis (ArP) at 350 °C for 1 h produced algal biocrudes (BCs), BC(AuP) and BC(ArP), with yields of 41.82 and 36.60 wt.%, respectively. These two algal BCs were cut into five distillate fractions (DFs) of 25–100 °C (DF0), 101–200 °C (DF1), 201–300 °C (DF2), 301–400 °C (DF3), and ≥401 °C (DR) using atmospheric distillation under N2 atmosphere. The total yields of DF1, DF2, and DF3 from either BC(AuP) or BC(ArP) are at least 60 wt.%. All the DFs, from either AuP or ArP, showed different yields and elemental and molecular compositions. Next, the DF1, DF2, and DF3 DFs were each blended with used engine oil (UEO) at a mass ratio of 1:1 and treated at 400 °C for 4 h with an additional 0.1 kgPt/C/kgfeed under 6 MPa H2. The presence of UEO could dilute the DF, avoid solvent extraction of the product oil, favor desulfurization of the upgraded oil, and be directly recovered as a major part of the product oil. Catalytic hydrotreatment of the DF and UEO blends led to a higher upgraded oil yield (>79 wt.%) and lower coke (

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.

This study aimed to optimize the growth conditions of the microalgae Auxenochlorella protothecoides to maximize the biomass and lipid content. The pH values and acetate concentrations of a tris-acetate-phosphate medium were varied for a photoheterotrophically grown culture of A. protothecoides. Approximately 23% of the microalgal biomass consisted of saturated fatty acids (SFAs). The highest lipid content (47.8%) was achieved with a medium containing 17 mM acetate at pH 6.2, and a medium containing 50 mM acetate at pH 5.8 resulted in the highest SFA content (43.7%). Surface response methodology was used to analyze the effects of two independent variables, pH (5.8–7.2) and acetate (0–35 mM), on lipid and biomass content. Four quadratic models, using the Box-Wilson central composite design, were proposed to correlate the optimal pH and acetate concentrations that would achieve the maximum biomass and lipid content. The study demonstrated that medium containing 24.4 mM acetate at pH 5.96 resulted in the highest lipid content (47.8%), and resulted in the highest biomass (1537 mg/L). Microalgae growth under these optimized conditions can enhance biomass and lipid productivity and can provide suitable conditions for high-quality biodiesel.

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.

The bio-flocculation ability of UTEX 2341 was studied for the purpose of improving microalgae harvesting efficiency to cut the high cost of biofuel production. The algae cells of UTEX 2341 cultured under heterotrophic and municipal wastewater conditions were found to have better self-flocculation ability, with flocculation rates of 92% and 85% at 2 h, respectively. Moreover, the flocculation rates of 16 freeze-dried microalgae powder samples cultured under different stress conditions were 0~72% with an algae powder dosage of 35 mg L−1. The flocculation efficiency of DIM, DCd1, DT28, and L6S was stable under different pH of 3~9 and temperatures of 15~50 °C. For samples of IM, LCd0.6, LMn2, and LZn2, the flocculation efficiency decreased or increased respectively with increased pH or temperatures. Though the flocculation properties of the eight samples showed wide differences, their flocculant compositions were almost the same with unknown components occupying large proportions. More studies needed to be further carried out to reveal the flocculation mechanisms and analyze the flocculation abilities in practical application, which would be conducive to future large-scale application of the bio-flocculation method and also cost reduction.

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.

Background Both APETALA2/Ethylene Responsive Factor (AP2/ERF) superfamily and R2R3-MYB family were from one of the largest diverse families of transcription factors (TFs) in plants, and played important roles in plant development and responses to various stresses. However, no systematic analysis of these TFs had been conducted in the green algae A. protothecoides heretofore. Temperature was a critical factor affecting growth and lipid metabolism of A. protothecoides. It also remained largely unknown whether these TFs would respond to temperature stress and be involved in controlling lipid metabolism process. Results Hereby, a total of six AP2 TFs, six ERF TFs and six R2R3-MYB TFs were identified and their expression profiles were also analyzed under low-temperature (LT) and high-temperature (HT) stresses. Meanwhile, differential adjustments of lipid pathways were triggered, with enhanced triacylglycerol accumulation. A co-expression network was built between these 18 TFs and 32 lipid-metabolism-related genes, suggesting intrinsic associations between TFs and the regulatory mechanism of lipid metabolism. Conclusions This study represented an important first step towards identifying functions and roles of AP2 superfamily and R2R3-MYB family in lipid adjustments and response to temperature stress. These findings would facilitate the biotechnological development in microalgae-based biofuel production and the better understanding of photosynthetic organisms’ adaptive mechanism to temperature stress.

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.

Background The yield of microalgae biomass is the key to affect the accumulation of fatty acids. A few microalgae can assimilate organic carbon to improve biomass yield. In mixotrophic cultivation, microalgae can use organic carbon source and light energy simultaneously. The preference of the main energy source by microalgae determines the biomass yield. Auxenochlorella protothecoides is an oleaginous mixotrophic microalga that can efficiently assimilate glucose and accumulate a large amount of biomass and fatty acids. The current study focused on the effect of light on the growth and glucose assimilation of A. protothecoides. Results In this study, we found that the uptake and metabolism of glucose in A. protothecoides could be inhibited by light, resulting in a reduction of biomass growth and lipid accumulation. We employed comparative proteomics to study the influence of light on the regulation of glucose assimilation in A. protothecoides. Proteomics revealed that proteins involving in gene translation and photosynthesis system were up-regulated in the light, such as ribulose-phosphate 3-epimerase and phosphoribulokinase. Calvin cycle-related proteins were also up-regulated, suggesting that light may inhibit glucose metabolism by enhancing the production of glyceraldehyde-3-phosphate (G3P) in the Calvin cycle. In addition, the redox homeostasis-related proteins such as thioredoxin reductase were up-regulated in the light, indicating that light may regulate glucose uptake by changing the redox balance. Moreover, the increase of NADH levels and redox potential of the medium under illumination might inhibit the activity of the glucose transport system and subsequently reduce glucose uptake. Conclusions A theoretical model of how glucose assimilation in A. protothecoides is negatively influenced by light was proposed, which will facilitate further studies on the complex mechanisms underlying the transition from autotrophy to heterotrophy for improving biomass accumulation.

Light (wavelength, intensity, and light/dark cycle) have been considered as one of the most important parameters for microalgae cultivation. In this paper, the effect of medium frequency intermittent light on Auxenochlorella pyrenoidosa (formerly Chlorella pyrenoidosa) cultivation was investigated. Three parameters of intermittent light, light intensity, light/dark ratio, and light/dark cycle were employed and the influence of these parameters on the productivity of Auxenochlorella pyrenoidosa was studied. The biomass yield and growth rates were mainly affected by the light fraction and cycle time. Light with 220 &mu, E m&minus, 2 s&minus, 1 light intensity was determined as the optimal light intensity for biomass production. At the light intensity of 420 &mu, 1, the results indicated that the intermittent light improved the biomass production with larger light/dark ratio compared with the continuous light. At a lower mean light intensity over time, the intermittent light should be more suitable for biomass growth and the decrease in the light/dark ratio (L/D) will lead to a higher biomass productivity. The light/dark cycle time has little influence on the biomass yield.

Heterotrophic cultures are the most effective approach to overcome low growth rate challenge in the most commercial microalgae. However, the mechanism through which heterotrophic condition regulates algae metabolism are not completely clear. Alternative Splicing (AS) is a common posttranscriptional process by which transcriptome and proteome plasticity increases at different environmental conditions. To identify and characterize of AS events in Auxenochlorella protothecoides microalga grown in autotrophic and heterotrophic, RNA-Seq data were analysed. We found that AS increased with the transition from autotrophic to heterotrophic condition. 705 and 660 differentially expressed (DEG) and spliced (DAS) genes were identified for A.protothecoides was transferred from autotrophic to heterotrophic condition, respectively. Moreover, there was slight coverage between DEG and DAS genes. Furthermore, functional analysis showed that the DAS genes are most frequently related to ion binding and stimulus response. The results also indicated that prevalence of Intron retention is associated with down-regulation of the genes involved in carotenoid biosynthesis. This study provides valuable insights into transcriptional and posttranscriptional plasticity of microalgae during growth mode change.

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.

Algae hold great potential as a source of renewable fuel due to their ability to produce refinery-compatible diesel and jet fuel precursors. Significant effort has been made to maximize productivity under photoautotrophic growth conditions; however, little progress has been made to discover and understand reduced carbon assimilation pathways or enzymatic degradation of complex carbon substrates in algae. We purport that utilization of plant-based carbon substrates in addition to photosynthesis (mixotrophic growth) for biochemical assimilation into biomass, biofuels, and bioproducts, can increase cultivation productivity and improve the economic viability of algal-derived biofuels. Herein we report that a freshwater production strain of microalgae, Auxenochlorella protothecoides UTEX 25, is capable of directly degrading and utilizing non-food plant substrates, such as switchgrass, for cell growth. Glycome profiling of plant substrates before and after addition to A. protothecoides cultures demonstrates the utilization of xyloglucans. Genomic, proteomic and transcriptomic analyses revealed the identity of many enzymes that are hypothesized to be involved in complex carbohydrate degradation, including several family 5 and 9 glycosyl hydrolases. This work paves the way for future designer engineering of plant‑carbon utilization to further improve productivity of algal production strains.

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.

There is significant potential for employing algae in tertiary wastewater treatment, however, little is known about the contribution of algae-bacteria synergy toward treatment performance. This study demonstrates potential synergy in the treatment of three winery wastewater samples. Two strains of green algae, Auxenochlorella protothecoides and Chlorella sorokiniana were tested and each removed > 90% of nitrogen, > 50% of phosphate, and 100% of acetic acid in the wastewater. Both algae strains grew significantly faster on wastewaters compared to growth on minimal media. Organic carbon in the wastewater apparently played a limited role in algal growth enhancement. When cultured on sterile-filtered wastewater, A. protothecoides increased soluble COD loadings in two of the three wastewaters and C. sorokiniana secreted an insoluble film. Culturing algae with the native wastewater microbial community negated the secretion of algal photosynthate, allowing for simultaneous reductions in COD and nutrient concentrations. Both algae species stimulated bacterial growth in a strain-specific way, suggesting unique responses to algal photosynthate. Cofactor auxotrophy for thiamine, cobalamin, and biotin is widespread among algae and these cofactors are typically obtained from bacteria. Sequencing the wastewater microbial community revealed bacteria capable of synthesizing all three cofactors while liquid chromatography with mass spectrometry (LCMS) and bio-assays revealed the presence of thiamine metabolites in the wastewaters. These cofactors likely increased algal growth rates, particularly for A. protothecoides, which cannot synthesize thiamine de-novo but can salvage it from degradation products. Collectively, these results demonstrate that bacteria and algae provided synergistic growth benefits, potentially contributing to higher levels of wastewater treatment than either organism type alone.

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.

This work intends to promote the growth of microalgae biomass with simultaneous remediation of an agro-industrial wastewater. Winery wastewater (WW) was used as growth media for the cyanobacteria Arthrospira maxima and the green microalgae Scenedesmus obliquus, Auxenochlorella protothecoides and Chlorella vulgaris, under mixotrophic and heterotrophic conditions. The latter species stands out under mixotrophic conditions, with removals of TOC and TN above 90%. Biomass production and pollutant removal were influenced by the initial WW concentration. Maximum removal values within 8 days of incubation were 92, 91, 49 and 40% for COD, TN, polyphenols and P-PO4, respectively, and 147.5 mg L−1 d−1 of biomass productivity. C. vulgaris biomass showed higher carotenoid content (maximum of 8.7 mg/g) when grown in WW, compared to autotrophic conditions (6.5 mg/g), making the bioremediation process more viable with the production of valuable by-products such as pigments. As the pollutant load removed by the microalgae does not allow reach the legal limits of release treated waters in natural water courses, a tertiary treatment process was applied. A post-treatment by photocatalysis in a UV LEDs photoreactor with TiO2-supported in Raschig rings was proposed for the removal of COD and polyphenols from a high loaded WW. The heterogeneous photocatalytic process was efficient in removing 80% of total polyphenols and 40% of COD, allowing the release of the treated water in superficial water courses since complies with the legal limits (COD below 150 mg L−1).

The compositions of three microalgal strains that have potential as biofuel feedstocks were evaluated to investigate how their biomass could be best utilized in a portfolio of products in an algal biofuel biorefinery. These strains, Chlorella vulgaris, Chlorella sorokiniana and Auxenochlorella (Chlorella) protothecoides, can grow both heterotrophically and phototrophically. An underlying principle of this project was that a continuous and maximally productive growth system will be required for commercial-scale production of algal biomass. Alternatively, a two-stage, mixed trophic state production process could be utilized to achieve this goal. For this reason, our research focused on these strains grown phototrophically to produce biomass and grown heterotrophically with nitrogen stressing to induce lipid production. The compositions reported in this work should be reflective of what is possible in continuous phototrophic or heterotrophic systems or in a two-stage mixed trophic system that are en...

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.

Pulsed Electric Fields, a known technique for permeabilization of cell membranes, can considerably foster intracellular component extraction from microalgae. However, it is currently uncertain in what way, apart from the cell membrane, the cell wall is affected during pulsation. In this study, fresh Auxenochlorella protothecoides and Chlorella vulgaris were subjected to treatment with pulsed electric fields and energy input of 1.5 MJ/kgDryWeight. Subsequently the biomass was fed into a High Pressure Homogenizer for 5 passes at 1500 bar. The percentage of intact cells after each pass was determined through cell counting and compared with Control biomass that underwent the same homogenization. No major difference on the disruption degree of pulsed and control samples was observed, indicating that the resistance to mechanical stress of the cell, a function of the cell wall, is not affected by pulsed electric fields. Scanning Electron Microscopy observation also showed no superficial or structural cell alteration after pulsation.

Microalgae are a source of potentially healthy and sustainable nutrients. However, the bioaccessibility of these nutrients remains uncertain. In this study, we analyzed the biomass composition of five commercial Chlorella and Auxenochlorella strains, and Chlorella vulgaris heterotrophically cultivated in our laboratory. Protein accounted for 65% ± 3% (w w-1) dry matter (DM) in all biomasses, except for the lab-grown C. vulgaris that contained 20% (w w-1) DM protein. The lipid content was comparable and ranged between 7%–10% (w w-1) DM. Most of the biomasses had a ω6-polyunsaturated fatty acids (PUFAs)/ω3-PUFAs ratio < 4, as recommended by nutritional experts. A recently published harmonized protocol for in vitro digestion was used to evaluate lipid and protein bioaccessibility. Protein bioaccessibility ranged between 60%–74% for commercial Chlorella and Auxenochlorella biomasses and was 43% for the lab-grown C. vulgaris. Lipid bioaccessibility was < 7% in commercial biomasses and 19% in the lab-grown C. vulgaris. Taken together, the results show that microalgae are promising sources of bioaccessible protein. The limited lipid bioaccessibility indicates the need for alternative upstream and downstream production strategies., Frontiers in Nutrition, 7, ISSN:2296-861X

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.

The objective of this work was to test the impact of anaerobic digester microorganisms on algal growth, composition, and nutrient removal from digestate. Culture studies were carried out to determine the impacts of the microbial community on treatment of poultry litter anaerobic digestate by two strains of green algae: Auxenochlorella protothecoides and Chlorella sorokiniana. The results showed that the community doubled the growth of A. protothecoides but had no impact on C. sorokiniana growth. A similar result was observed for nutrient removal where the microbial community increased the capacity of A. protothecoides to remove ammonium and phosphate. The impact of the microbial community on biomass composition was minimal for both algae types.

Wastewater treatment is an energy-intensive process and a net emitter of greenhouse gas emissions. A large fraction of these emissions is due to intensive aeration of aerobic bacteria to facilitate break-down of organic compounds. Algae can generate dissolved oxygen at levels in excess of saturation, and therefore hold the potential to partially displace or complement mechanical aeration in wastewater treatment processes. The objective of this study was to develop an internally consistent experimental and modeling approach to test the hypothesis that algal photosynthetic aeration can speed the removal of organic constituents by bacteria. This framework was developed using a simplified wastewater treatment process consisting of a model bacteria (Escherichia coli), a model algae (Auxenochlorella protothecoides), and a single carbon source that was consumable by bacteria only. This system was then tested both with and without the presence of algae. A MATLAB model that considered mass transfer and biological kinetics was used to estimate the production and consumption of O2 and CO2 by algae and bacteria. The results indicated that the presence of algae led to 18-66% faster removal of COD by bacteria, and that roughly one-third of biochemical oxygen demand was offset by algal photosynthetic aeration.

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.

Algal growth is often inhibited in full-strength anaerobic digestate. The objective of this study was to investigate conditions under which digestate pretreatment using bacteria is effective in promoting algal growth, nutrient removal, and favorable changes in algal biomass composition. Batch culture experiments were carried out using low- and high-strength municipal sludge anaerobic digestate, two algae strains of varying sensitivity to digestate inhibitors, short and long pretreatment periods, and axenic vs. non-axenic algal cultures. Pretreatment of digestate increased algal growth up to 40%, N assimilation (up to 29%), and P removal (340%) by Chlorella sorokiniana (resilient algae) when grown on high-strength digestate. Pretreatment did not increase algal growth or nutrient assimilation when Chlorella sorokiniana was combined with low-strength digestate. The more sensitive strain, Auxenochlorella protothecoides, benefitted from pretreatment on both low- and high-strength digestate, even preventing complete cell death in the latter. Pretreatment increased starch content but not lipid content of algae.

Culturing algae with bacteria has been explored as a means of promoting algal growth and altering algal biochemical composition. Plant growth promoting bacteria (PGPB) are of particular interest given their ability to produce phytohormones that are bioactive in microalgae. However, many non-plant growth promoting bacteria also promote algal growth. The objective of this study was to test the benefits of a model PGPB versus non-PGPB in a side-by-side study, evaluating improvement in algal growth rates and impacts on biomass composition. Three green microalgae strains were tested with three strains of bacteria: Azospirillum brasilense (a model PGPB), Escherichia coli (a non-PGPB), and Bacillus megaterium (a potential PGPB). A. brasilense produces the auxin phytohormone indole-3-acetic acid. A. brasilense promoted growth in two of the three algae strains by 39–69% but did not promote growth in Chlorella sorokiniana (strain UTEX 2805). E. coli promoted growth in all three algae strains by 16–64%. That the non-PGPB, E. coli, performed as well as the PGPB, A. brasilense, suggests the importance of symbiotic mechanisms that are ubiquitous among many bacteria. One such mechanism, cell-to-cell O2-CO2 exchange, may have been important given significant consumption of algal photosynthate by bacteria. B-vitamin exchange is another widespread mechanism of algal-bacterial symbiosis. The riboflavin metabolite, lumichrome, was detected in low concentrations in co-cultures of A. brasilense (0.4–0.6 ng/mL) and E. coli (5.5–13 ng/mL). These concentrations of lumichrome were found to provide small but statistically significant growth benefits for C. sorokiniana UTEX 2805 and Auxenochlorella protothecoides.

Pulsed electric field (PEF) treatment of microalgae has been demonstrated to exhibit processing advantages compared to conventional processing. Besides low specific energy requirements, PEF treatment can separate microalgae biomass into aqueous and oleaginous fractions. To minimize processing energy demand, the treatment of pre-concentrated microalgae suspensions is advantageous. For suspensions with a biomass density of approximately 80 g of dry weight per liter, microalgae precipitation at the electrodes of cross-linear treatment chambers was observed. This phenomenon is examined using the microalgae species Chlorella vulgaris and Auxenochlorella protothecoides. An interrelation between pH and zeta potential on the one hand and the amount of precipitated microalgae on the electrodes on the other hand is emerging. Impact of electric pulse parameters and pH was investigated and suggested that precipitation is caused by electrophoretic forces. The results indicate that shorter pulses can be used to reduce the unwanted precipitation. Industrial relevance The manuscript describes an undesirable effect occurring while performing pulsed electric field (PEF) treatment on microalgae. We have indeed observed that microalgae tend to precipitate on the surface of the treatment electrodes during PEF processing. This precipitation of microalgae results in the distortion of the flow through the treatment chamber and in the modification of the electric field distribution. At an industrial scale, this will affect the process efficiency and therefore increase processing costs. In the worst case, the affected electroporation reactors might be blocked and require additional maintenance, generating extra costs. We demonstrate that the precipitation is induced by the interaction between the electric field and the surface-charged algae. Driving parameters for the precipitation effect were identified and strategies to avoid it are proposed in the manuscript.

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.

Oxidative stress caused by chemicals brings about mass mortalities in aquatic organisms and in this context, antioxidants and heat shock protein 70 (Hsp70) stimulating compounds represent potential alternatives to alleviate this problem during aquaculture. The antioxidant capacity and efficacies of five Malaysian indigenous microalgae, namely Auxenochlorella pyrenoidosa, Chlorella vulgaris, Messastrum gracile, Desmodesmus subspicatus and Parachlorella kessleri to regulate Hsp70 expression in the brine shrimp Artemia, an important live food species, were examined. A. pyrenoidosa contained the highest total phenol and flavonoid content (48.3 mg GAE/g extract and 34.7 mg QE/g) across all the microalgae examined in this study, whereas the lowest occurred with C. vulgaris (24.4 mg GAE/g extract and 14.0 mg QE/g). The ferric reducing antioxidant power of A. pyrenoidosa, C. vulgaris, M. gracile, D. subspicatus and P. kessleri respectively stood at 0.78, 0.62, 1.01, 0.77 and 0.68 ferrous equivalents (mM), with trolox equivalent antioxidant capacity assay revealed that A. pyrenoidosa possessed the highest scavenging activity at 1 mg/mL concentration. Interestingly, application of 1.0–2.0 × 106 cells/mL C. vulgaris, M. gracile, D. subspicatus, and P. kessleri as feed enhanced Hsp70 accumulation in Artemia, indicating that these microalgae consist stimulating compound(s) which potentially promote the synthesis of the 70-kDa stress protein in this crustacean species. Taken together, results from this study revealed that microalgae possess various antioxidant properties and coupled with their abilities to induce Hsp70, they are potential oxidative stress control alternatives in Artemia and perhaps other aquatic organisms used in aquaculture.

Real-time reverse quantitative transcription PCR (RT-qPCR) is a commonly used method for gene expression analysis. However, the accuracy of RT-qPCR analysis greatly depends on the stability of the reference genes selected for data normalization. Unfortunately, there have been no systematic evaluation studies on reference genes in the unicellular green alga Auxenochlorella protothecoides. Ten genes (ACT, COX6B, CYB5R4, EF1α, GAPDH, rbcL, RPL19, RPL32, RPS10 and TUA) were chosen as candidate reference genes. The expression stability of each reference gene was determined by applying geNorm, NormFinder, BestKeeper and comprehensive ranking based on geomean ranking values. In order to validate the suitability and reliability of the candidate reference genes selected for data normalization, two key functional genes related to lipid metabolism were also selected as target genes. The results revealed that RPL32 was the most stably expressed and suitable reference genes under all experimental conditions of the analyses; CYB5R4 was also a suitable reference gene under continuous heterotrophic culture condition, and RPL19 was top-ranked and stably expressed under both low and high temperature stress. These results provided a list of suitable reference genes for accurate normalization of heterotrophic A. protothecoides time-course samples under different temperature stress in qPCR experiments. This study will be of very great reference value to assess the stability of candidate reference genes in other heterotrophically grown algae.

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.

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

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.