A new paradigm for producing astaxanthin from the unicellular green alga Haematococcus pluvialis.

The unicellular green alga Haematococcus pluvialis has been exploited as a cell factory to produce the high-value antioxidant astaxanthin for over two decades, due to its superior ability to synthesize astaxanthin under adverse culture conditions. However, slow vegetative growth under favorable culture conditions and cell deterioration or death under stress conditions (e.g., high light, nitrogen starvation) has limited the astaxanthin production. In this study, a new paradigm that integrated heterotrophic cultivation, acclimation of heterotrophically grown cells to specific light/nutrient regimes, followed by induction of astaxanthin accumulation under photoautotrophic conditions was developed. First, the environmental conditions such as pH, carbon source, nitrogen regime, and light intensity, were optimized to induce astaxanthin accumulation in the dark-grown cells. Although moderate astaxanthin content (e.g., 1% of dry weight) and astaxanthin productivity (2.5 mg L(-1) day(-1) ) were obtained under the optimized conditions, a considerable number of cells died off when subjected to stress for astaxanthin induction. To minimize the susceptibility of dark-grown cells to light stress, the algal cells were acclimated, prior to light induction of astaxanthin biosynthesis, under moderate illumination in the presence of nitrogen. Introduction of this strategy significantly reduced the cell mortality rate under high-light and resulted in increased cellular astaxanthin content and astaxanthin productivity. The productivity of astaxanthin was further improved to 10.5 mg L(-1) day(-1) by implementation of such a strategy in a bubbling column photobioreactor. Biochemical and physiological analyses suggested that rebuilding of photosynthetic apparatus including D1 protein and PsbO, and recovery of PSII activities, are essential for acclimation of dark-grown cells under photo-induction conditions. Biotechnol. Bioeng. 2016;113: 2088-2099. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Biofertilizer and biostimulant properties of the microalga Acutodesmus dimorphus.

Microalgae represent a potential sustainable alternative for the enhancement and protection of agricultural crops. Cellular extracts and dry biomass of the green alga Acutodesmus dimorphus were applied as a seed primer, foliar spray, and biofertilizer, to evaluate seed germination, plant growth, and fruit production in Roma tomato plants. A. dimorphus culture, culture growth medium, and different concentrations (0, 1, 5, 10, 25, 50, 75, and 100 %) of aqueous cell extracts in distilled water were used as seed primers to determine effects on germination. Seeds treated with A. dimorphus culture and with extract concentrations higher than 50 % (0.75 g mL-1) triggered faster seed germination-2 days earlier than the control group. The aqueous extracts were also applied as foliar fertilizers at various concentrations (0, 10, 25, 50, 75, and 100 %) on tomato plants. Extract foliar application at 50 % (3.75 g mL-1) concentration resulted in increased plant height and greater numbers of flowers and branches per plant. Two dry biomass treatments (50 and 100 g) were applied 22 days prior to seedling transplant and at the time of transplant to assess whether the timing of the biofertilizer application influenced the effectiveness of the biofertilizer. Biofertilizer treatments applied 22 days prior to seedling transplant enhanced plant growth, including greater numbers of branches and flowers, compared to the control group and the biofertilizer treatments applied at the time of transplant. The A. dimorphus culture, cellular extract, and dry biomass applied as a biostimulant, foliar spray, and biofertilizer, respectively, were able to trigger faster germination and enhance plant growth and floral production in Roma tomato plants.

Vernalophrys algivore gen. nov., sp. nov. (Rhizaria: Cercozoa: Vampyrellida), a New Algal Predator Isolated from Outdoor Mass Culture of Scenedesmus dimorphus.

Microbial contamination is the main cause of loss of biomass yield in microalgal cultures, especially under outdoor environmental conditions. Little is known about the identities of microbial contaminants in outdoor mass algal cultures. In this study, a new genus and species of vampyrellid amoeba, Vernalophrys algivore, is described from cultures of Scenedesmus dimorphus in open raceway ponds and outdoor flat-panel photobioreactors. This vampyrellid amoeba was a significant grazer of Scenedesmus and was frequently associated with a very rapid decline in algal numbers. We report on the morphology, subcellular structure, feeding behavior, molecular phylogeny, and life cycle. The new amoeba resembles Leptophrys in the shape of trophozoites and pseudopodia and in the mechanism of feeding (mainly by engulfment). It possesses two distinctive regions in helix E10_1 (nucleotides 117 to 119, CAA) and E23_1 (nucleotides 522 and 523, AG) of the 18S rRNA gene. It did not form a monophyletic group with Leptophrys in molecular phylogenetic trees. We establish a new genus, Vernalophrys, with the type species Vernalophrys algivore. The occurrence, impact of the amoeba on mass culture of S. dimorphus, and means to reduce vampyrellid amoeba contamination in Scenedesmus cultures are addressed. The information obtained from this study will be useful for developing an early warning system and control measures for preventing or treating this contaminant in microalgal mass cultures.

Molecular mechanisms of the coordination between astaxanthin and fatty acid biosynthesis in Haematococcus pluvialis (Chlorophyceae).

Astaxanthin, a red ketocarotenoid with strong antioxidant activity and high commercial value, possesses important physiological functions in astaxanthin-producing microalgae. The green microalga Haematococcus pluvialis accumulates up to 4% fatty acid-esterified astaxanthin (by dry weight), and is used as a model species for exploring astaxanthin biosynthesis in unicellular photosynthetic organisms. Although coordination of astaxanthin and fatty acid biosynthesis in a stoichiometric fashion was observed in H. pluvialis, the interaction mechanism is unclear. Here we dissected the molecular mechanism underlying coordination between the two pathways in H. pluvialis. Our results eliminated possible coordination of this inter-dependence at the transcriptional level, and showed that this interaction was feedback-coordinated at the metabolite level. In vivo and in vitro experiments indicated that astaxanthin esterification drove the formation and accumulation of astaxanthin. We further showed that both free astaxanthin biosynthesis and esterification occurred in the endoplasmic reticulum, and that certain diacylglycerol acyltransferases may be the candidate enzymes catalyzing astaxanthin esterification. A model of astaxanthin biosynthesis in H. pluvialis was subsequently proposed. These findings provide further insights into astaxanthin biosynthesis in H. pluvialis.

Cellular capacities for high-light acclimation and changing lipid profiles across life cycle stages of the green alga Haematococcus pluvialis.

The unicellular microalga Haematococcus pluvialis has emerged as a promising biomass feedstock for the ketocarotenoid astaxanthin and neutral lipid triacylglycerol. Motile flagellates, resting palmella cells, and cysts are the major life cycle stages of H. pluvialis. Fast-growing motile cells are usually used to induce astaxanthin and triacylglycerol biosynthesis under stress conditions (high light or nutrient starvation); however, productivity of biomass and bioproducts are compromised due to the susceptibility of motile cells to stress. This study revealed that the Photosystem II (PSII) reaction center D1 protein, the manganese-stabilizing protein PsbO, and several major membrane glycerolipids (particularly for chloroplast membrane lipids monogalactosyldiacylglycerol and phosphatidylglycerol), decreased dramatically in motile cells under high light (HL). In contrast, palmella cells, which are transformed from motile cells after an extended period of time under favorable growth conditions, have developed multiple protective mechanisms--including reduction in chloroplast membrane lipids content, downplay of linear photosynthetic electron transport, and activating nonphotochemical quenching mechanisms--while accumulating triacylglycerol. Consequently, the membrane lipids and PSII proteins (D1 and PsbO) remained relatively stable in palmella cells subjected to HL. Introducing palmella instead of motile cells to stress conditions may greatly increase astaxanthin and lipid production in H. pluvialis culture.

Critical evaluation and modeling of algal harvesting using dissolved air flotation.

In this study, Chlorella zofingiensis harvesting by dissolved air flotation (DAF) was critically evaluated with regard to algal concentration, culture conditions, type and dosage of coagulants, and recycle ratio. Harvesting efficiency increased with coagulant dosage and leveled off at 81%, 86%, 91%, and 87% when chitosan, Al(3+) , Fe(3+) , and cetyl trimethylammonium bromide (CTAB) were used at dosages of 70, 180, 250, and 500 mg g(-1) , respectively. The DAF efficiency-coagulant dosage relationship changed with algal culture conditions. Evaluation of the influence of the initial algal concentration and recycle ratio revealed that, under conditions typical for algal harvesting, it is possible that the number of bubbles is insufficient. A DAF algal harvesting model was developed to explain this observation by introducing mass-based floc size distributions and a bubble limitation into the white water blanket model. The model revealed the importance of coagulation to increase floc-bubble collision and attachment, and the preferential interaction of bubbles with larger flocs, which limited the availability of bubbles to the smaller sized flocs. The harvesting efficiencies predicted by the model agree reasonably with experimental data obtained at different Al(3+) dosages, algal concentrations, and recycle ratios. Based on this modeling, critical parameters for efficient algal harvesting were identified.

Assessment of key biological and engineering design parameters for production of Chlorella zofingiensis (Chlorophyceae) in outdoor photobioreactors.

For the design of a large field of vertical flat plate photobioreactors (PBRs), the effect of four design parameters-initial biomass concentration, optical path length, spacing, and orientation of PBRs-on the biochemical composition and productivity of Chlorella zofingiensis was investigated. A two-stage batch process was assumed in which inoculum is generated under nitrogen-sufficient conditions, followed by accumulation of lipids and carbohydrates in nitrogen-deplete conditions. For nitrogen-deplete conditions, productivity was the most sensitive to initial biomass concentration, as it affects the light availability to individual cells in the culture. An initial areal cell concentration of 50 g m(-2) inoculated into 3.8-cm optical path PBR resulted in the maximum production of lipids (2.42 ± 0.02 g m(-2) day(-1)) and carbohydrates (3.23 ± 0.21 g m(-2) day(-1)). Productivity was less sensitive to optical path length. Optical path lengths of 4.8 and 8.4 cm resulted in similar areal productivities (biomass, carbohydrate, and lipid) that were 20 % higher than a 2.4-cm optical path length. Under nitrogen-sufficient conditions, biomass productivity was 48 % higher in PBRs facing north-south during the winter compared to east-west, but orientation had little influence on biomass productivity during the spring and summer despite large differences in insolation. An optimal spacing could not be determined based on growth alone because a tradeoff was observed in which volumetric and PBR productivity increased as space between PBRs increased, but land productivity decreased.

Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production.

Isochrysis is a genus of marine unicellular microalgae that produces docosahexaenoic acid (DHA, C22:6), a very long chain polyunsaturated fatty acid (PUFA) of significant health and nutritional value. Mass cultivation of Isochrysis for DHA production for human consumption has not been established due to disappointing low DHA productivity obtained from commonly used Isochrysis strains. In this study, 19 natural Isochrysis strains were screened for DHA yields and the results showed that the cellular DHA content ranged from 6.8 to 17.0 % of total fatty acids with the highest DHA content occurring in the exponential growth phase. Isochrysis galbana #153180 exhibited the greatest DHA production potential and was selected for further investigation. The effects of different light intensities, forms, and concentrations of nitrogen, phosphorus, and salinity on growth and DHA production of I. galbana #153180 were studied in a bubble column photobioreactor (PBR). Under favorable culture conditions, I. galbana #153180 contained DHA up to 17.5 % of total fatty acids or 1.7 % of cell dry weight. I. galbana #153180 was further tested in outdoor flat-plate PBRs varying in light path length, starting cell density (SCD), and culture mode (batch versus semicontinuous). When optimized, record high biomass and DHA productivity of I. galbana #153180 of 0.72 g L(-1) day(-1) and 13.6 mg L(-1) day(-1), or 26.4 g m(-2) day(-1) and 547.7 mg m(-2) day(-1), respectively, were obtained, suggesting that I. galbana #153180 may be a desirable strain for commercial production of DHA.

A flexible culture process for production of the green microalga Scenedesmus dimorphus rich in protein, carbohydrate or lipid.

Microalgae have the ability to undergo programmatic changes in photosynthetic carbon partitioning and thus cellular biochemical composition, particularly in the relative amounts of crude protein, lipids, and carbohydrate, in response to changes in environmental and culture conditions. In this study, a novel strategy that employs a single microalgal strain Scenedesmus dimorphus grown in a single cultivation platform to produce protein-, carbohydrate- or lipid-rich biomass, as so desired, was introduced. With the combined manipulation of nitrogen availability and light intensity and cell inoculation density, it was successfully demonstrated that highest yields for protein and carbohydrate were 0.2 and 0.7 g L(-1) d(-1), respectively, which could be obtained in early stages of cultivation, while the highest yield for lipid, 0.17 g L(-1) d(-1), occured in a late stage of cultivation.

Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii.

Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production.

Influence of growth phase on harvesting of Chlorella zofingiensis by dissolved air flotation.

The effects of changes in cellular characteristics and dissolved organic matter (DOM) on dissolved air flotation (DAF) harvesting of Chlorella zofingiensis at the different growth phases were studied. Harvesting efficiency increased with Al(3+) dosage and reached more than 90%, regardless of growth phases. In the absence of DOM, the ratio of Al(3+) dosage to surface functional group concentration determined the harvesting efficiency. DOM in the culture medium competed with algal cell surface functional groups for Al(3+), and more Al(3+) was required for cultures with DOM than for DOM-free cultures to achieve the same harvesting efficiency. As the culture aged, the increase of Al(3+) dosage due to increased DOM was less than the decrease of Al(3+) dosage associated with reduced cell surface functional groups, resulting in overall reduced demand for Al(3+). The interdependency of Al(3+) dosage and harvesting efficiency on concentrations of cell surface functional groups and DOM was successfully modeled.

SUSCEPTIBILITY AND PROTECTIVE MECHANISMS OF MOTILE AND NON MOTILE CELLS OF HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) TO PHOTOOXIDATIVE STRESS(1).

The life cycle of the unicellular green alga Haematococcus pluvialis consists of motile and nonmotile stages under typical growing conditions. In this study, we observed that motile cells were more susceptible than nonmotile cells to high light, resulting in a decrease in population density and photo-bleaching. Using two Haematococcus strains, CCAP 34/12 (a motile cell dominated strain) and SAG 34/1b (a nonmotile cell dominated strain), as model systems we investigated the cause of cell death and the protective mechanisms of the cells that survived high light. The death of motile cells under high light was attributed to the generation of excess reactive oxygen species (ROS), which caused severe damage to the photosynthetic components and the membrane system. Motile cells were able to dissipate excess light energy by nonphotochemical quenching and to relax ROS production by a partially up-regulated scavenging enzyme system. However, these strategies were not sufficient to protect the motile cells from high light stress. In contrast, nonmotile cells were able to cope with and survive under high light by (i) relaxing the over-reduced photosynthetic electron transport chain (PETC), thereby effectively utilizing PETC-generated NADPH to produce storage starch, neutral lipid, and astaxanthin, and thus preventing formation of excess ROS; (ii) down-regulating the linear electron transport by decreasing the level of cytochrome f; and (iii) consuming excess electrons produced by PSII via a significantly enhanced plastid terminal oxidase pathway.

Growth and neutral lipid synthesis in green microalgae: a mathematical model.

Many green microalgae significantly increased their cellular neutral lipid content when cultured in nitrogen limited or high light conditions. Due to their lipid production potential, these algae have been suggested as promising feedstocks for biofuel production. However, no models for algal lipid synthesis with respect to nutrient and light have been developed to predict lipid production and to help improve the production process. A mathematical model is derived describing the growth dynamics and neutral lipid production of green microalgae grown in batch cultures. The model assumed that as the nitrogen was depleted, photosynthesis became uncoupled from growth, resulting in the synthesis and accumulation of neutral lipids. Simulation results were compared with experimental data for the green microalgae Pseudochlorococcum sp. For growth media with low nitrogen concentration, the model agreed closely with the data; however, with high nitrogen concentration the model overestimated the biomass. It is likely that additional limiting factors besides nitrogen could be responsible for this discrepancy.

Microwave-assisted nile red method for in vivo quantification of neutral lipids in microalgae.

In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.

Life-cycle analysis on biodiesel production from microalgae: water footprint and nutrients balance.

This research examines the life-cycle water and nutrients usage of microalgae-based biodiesel production. The influence of water types, operation with and without recycling, algal species, geographic distributions are analyzed. The results confirm the competitiveness of microalgae-based biofuels and highlight the necessity of recycling harvested water and using sea/wastewater as water source. To generate 1 kg biodiesel, 3726 kg water, 0.33 kg nitrogen, and 0.71 kg phosphate are required if freshwater used without recycling. Recycling harvest water reduces the water and nutrients usage by 84% and 55%. Using sea/wastewater decreases 90% water requirement and eliminates the need of all the nutrients except phosphate. The variation in microalgae species and geographic distribution are analyzed to reflect microalgae biofuel development in the US. The impacts of current federal and state renewable energy programs are also discussed to suggest suitable microalgae biofuel implementation pathways and identify potential bottlenecks.

Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under nitrogen-limited conditions.

Photosynthetic carbon partitioning into starch and neutral lipid was investigated in the oleaginous green microalga Pseudochlorococcum sp. When grown under low light and nitrogen-replete conditions, the algal cells possessed a basal level of starch. When grown under high light and nitrogen-limited conditions, starch synthesis was transiently up-regulated. After nitrogen depletion, starch content decreased while neutral lipid rapidly increased to 52.1% of cell dry weight, with a maximum neutral lipid productivity of 0.35 g L(-1)D(-1). These results suggest that Pseudochlorococcum used starch as a primary carbon and energy storage product. As nitrogen was depleted for an extended period of time, cells shift the carbon partitioning into neutral lipid as a secondary storage product. Partial inhibition of starch synthesis and degradation enzymes resulted in a decrease in neutral lipid content, indicating that conversion of starch to neutral lipid may contribute to overall neutral lipid accumulation. Biotechnological application of Pseudochlorococcum sp. as a production strain for biofuel was assessed.

Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae).

The green alga Haematococcus pluvialis produces large amounts of the pink carotenoid astaxanthin under high photon flux density (PFD) and other oxidative stress conditions. However, the regulation and physiological role of carotenogenesis leading to astaxanthin formation is not well understood. Comparative transcriptional expression of five carotenoid genes along with growth and pigment composition as a function of PFD was studied using a wild-type and an astaxanthin-overproduction mutant of H. pluvialis NIES144. The results indicate that astaxanthin biosynthesis was mainly under transcriptional control of the gene encoding carotenoid hydroxylase, and to a lesser extent, the genes encoding isopentenyl isomerase and phytoene desaturase, and to the least extent, the genes encoding phytoene synthase and carotenoid oxygenase. The expression of a plastid terminal oxidase (PTOX) gene ptox2 underwent transient up-regulation under elevated PFDs, suggesting that PTOX may be functionally coupled with phytoene desaturase through the plastoquinone pool and may play a role in reducing redox-potential-dependent and oxygen-concentration-dependent formation of reactive oxygen species in the chloroplast. Over-expression of both the carotenogenic and PTOX genes confers to the astaxanthin-overproduction mutant more effective photoprotective capability than that of the wild type under photooxidative stress.

Inhibition of starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtii.

Starch and neutral lipids are two major carbon storage compounds in many microalgae and plants. Lipids are more energy rich and have often been used as food and fuel feedstocks. Genetic engineering of the lipid biosynthesis pathway to overproduce lipid has achieved only limited success. We hypothesize that through blocking the competing pathway to produce starch, overproduction of neutral lipid may be achieved. This hypothesis was tested using the green microalga Chlamydomonas reinhardtii and its low starch and starchless mutants. We discovered that a dramatic increase in neutral lipid content and the neutral lipid/total lipid ratio occurred among the mutants under high light and nitrogen starvation. BAFJ5, one of the mutants defective in the small subunit of ADP-glucose pyrophosphorylase, accumulated neutral and total lipid of up to 32.6% and 46.4% of dry weight (DW) or 8- and 3.5-fold higher, respectively, than the wild-type. These results confirmed the feasibility of increasing lipid production through redirecting photosynthetically assimilated carbon away from starch synthesis to neutral lipid synthesis. However, some growth impairment was observed in the low starch and starchless mutants, possibly due to altered energy partitioning in PSII, with more excitation energy dissipated as heat and less to photochemical conversion. This study demonstrated that biomass and lipid production by the selected mutants can be improved by physiological manipulation.

Harvesting algal biomass for biofuels using ultrafiltration membranes.

The objective of this paper is to develop efficient technologies for harvesting of algal biomass using membrane filtration. Foulants were characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. Anti-fouling strategies were established, such as using air-assisted backwash with air scouring, and optimizing operational conditions. A model was also developed to predict the flux decline and final concentration based on a resistance-in-series analysis and a cake development calculation. The results showed that the buildup of the algal cake layer and adsorption of algogenic organic matter (AOM) (mainly protein, polysaccharides or polysaccharide-like substances) on the membrane caused membrane fouling. The cake layer buildup was removed by conducting an air-assisted backwash every 15 min. The adsorbed AOM could be removed by soaking the membrane in 400mg/L NaClO for 1h. In our experiment the algal suspension was concentrated 150 times, to give a final cell concentration of 154.85g/L. The harvesting efficiency and average flux were 46.01 g/(m(2)h) and 45.50 L/(m(2)h), respectively. No algae were found in the permeate, which had an average turbidity of 0.018 Nephelometric Turbidity Units (NTU). The flux decline predicted by the model under different conditions was consistent with the experimental results.

Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol.

Many microalgae and plants have the ability to synthesize large amounts of triacylglycerol (TAG) that can be used to produce biofuels. Presently, TAG-based biofuel production is limited by the feedstock supply. Metabolic engineering of lipid synthesis pathways to overproduce TAGs in oleaginous microalgae and oil crop plants has achieved only modest success. We demonstrate that inactivation of ADP-glucose pyrophosphorylase in a Chlamydomonas starchless mutant led to a 10-fold increase in TAG, suggesting that shunting of photosynthetic carbon partitioning from starch to TAG synthesis may represent a more effective strategy than direct manipulation of the lipid synthesis pathway to overproduce TAG.