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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 (

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.

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.

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.

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.

© 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.

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.

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.

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.

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.

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.

Pulsed Electric Field (PEF) pre-treatment, applied on fresh microalgae Auxenochlorella protothecoides, induces spontaneous release of a substantial water fraction and enables subsequent lipid extraction using ethanol-hexane blends. In this study, fresh microalgae suspensions were treated with PEF and incubated under inert conditions. Incubation promotes the release of ions and carbohydrates and increases the yields of subsequent lipid extraction thus enabling a considerable reduction of PEF-treatment energy. With a 20 h incubation period at 25 °C, almost total lipid extraction is achieved with a specific PEF-treatment energy of only 0.25 MJ/kgDW. Incubation on ice remains beneficial but less efficient than at 25 °C. Additionally, incubating microalgae cells in suspension at 100 gDW/L or in a dense paste, was almost equally efficient. Correlation between the different results suggests that spontaneous release of ions and carbohydrates facilitates more successful lipid extraction. A direct causality between the two phenomena remains to be demonstrated.

Efficient extraction of valuable products from microalgae and utilization of the residual biomass for biofuel production appear to bring economic benefits for the microalgae biorefineries. The pulsed electric field (PEF) method has been proposed as a promising pre-treatment for microalgae wet extraction. For conversion of microalgae biomass and residues, hydrothermal liquefaction (HTL) is considered a suitable method. Here, a combination of PEF-assisted valuables extraction of microalgae and HTL of the residual biomass is studied for the first time. The microalgae Auxenochlorella protothecoides, Chlorella vulgaris, and Scenedesmus almeriensis were cultivated, harvested, treated by PEF, and then subjected to lipid extraction, protein extraction, or extraction of amino acids after enzymatic protein hydrolysis. The residual biomass obtained from PEF-assisted valuables extraction was subjected to HTL in micro autoclaves at a temperature of 350 °C and pressure of 25 MPa for 15 min holding time. Product yields and analytical results obtained by elemental content measurement, 1H-nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectroscopy, and gel permeation chromatography show that PEF alone has no significant direct influence on microalgae HTL. The harsh HTL conditions decisively determine the product yields and biocrude quality. However, PEF enhances the lipid extraction yield from 4 wt% to 33 wt%. Consequently, the biocrude yield decreases from 58 wt% to 43.2 wt%. Moreover, PEF boosts the protein extraction yield from almost zero to 41.6 wt% of the total protein, resulting in an increased biocrude yield by about 2 wt%. Finally, PEF accelerates the formation of amino acids by enzymatic hydrolysis, improving the extraction efficiency up to 150% in the first 60 min of the extraction. The extracted residue promises to produce a biocrude yield higher by 6 wt% and a better biocrude quality with lower nitrogen content. An overall mass balance of PEF-assisted valuables extraction and HTL is presented.

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.

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.

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.