Vulgaris UTEX Research Articles

Experimental determination of radiative characteristics of carotenoid-producing microalgae

Currently, the demand for carotenoid pigments of natural origin has increased due to their diverse application in the industry. One of the natural sources of these compounds are microalgae, which are photosynthetic organisms of high biotechnological value. The production of carotenoid pigments from microalgal cultures are in the scope of sustainability but faces the challenge of optimizing the design and operation of new technologies; biomass production is first maximized and subsequently subjected to stress to induce the synthesis and accumulation of this type of pigments. In this context, the design and scaling of photobioreactors (FBRs) for the cultivation of microalgae is crucial for this type of process. The main objective of the present research work is focused on the application of a new experimental methodology to estimate the radiative characteristics of a microalgae quickly and independently in suspension. Strain Chlorella vulgaris UTEX 2714 (shows high growth rates and accumulate carotenoids such as canthaxanthin and astaxanthin) was used as sample. Extinction coefficient (β), albedo (ω) and asymmetry parameter (g) of the phase function (Φ) were estimated. These parameters define the solution of the radiative transfer equation (RTE) and they are fundamental to characterize the effect of microalgae on radiative transport within any FBR. Both β, and ωwere estimated at different concentrations of microalgae in suspension and were independent of the biomass concentration under the experimental conditions. This study suggests that Φ of Chlorella vulgaris UTEX 2714 can be described using the Henyey-Greenstein model. The radiative characterization of the microalgae was achieved, which is essential for the description of the radiative transport within a FBR and the development of better kinetic models for the scaling of this type of bioprocess.

Examining the impact of carbon dioxide levels and modulation of resulting hydrogen peroxide in Chlorella vulgaris

The eukaryotic green alga Chlorella vulgaris UTEX 395 was cultured under carbon dioxide (CO2) concentrations ranging from 0.04% to 15% in order to examine the effect of CO2 on algal growth, biomass composition and reactive oxygen species (ROS) accumulation in the culture medium. Supplying 5% CO2 yielded the highest biomass growth rate (μ = 0.35 day−1) compared with 0.04% (μ = 0.15 day−1) and 15% (μ = 0.19 day−1) CO2 conditions. Experimental evidence showed that increasing CO2 levels from 0.04% to 2% and above did not alter overall protein content significantly but did enhance C16:1 and C18:1 monounsaturated fatty acid (MUFA) composition by 3.5 and 2 fold, respectively, reducing C18:3 polyunsaturated fatty acid (PUFA) levels. Interestingly, bubbling 5% and 15% CO2 increased one type of ROS, H2O2 levels, in sterile medium by 1.8 to 2 μM while growing C. vulgaris substantially lowered these H2O2 levels. The ability to lower H2O2 levels, which was reduced for non-viable algal cells, was also observed with C. protothecoides UTEX 29 and C. sorokiniana UTEX 1230. In order to understand the impact of H2O2 directly, 10 μM and 25 μM H2O2 were added daily to 0.04% CO2-bubbled C. vulgaris cultures. Periodic H2O2 addition did not affect the growth of C. vulgaris or change its biomass composition. These findings demonstrate C. vulgaris can thrive at elevated concentrations of CO2 and also showed the capacity of microalgae to reduce the ROS level, specifically H2O2, present in a CO2 bubbling environment.

The establishment of new protein expression system using N starvation inducible promoters in Chlorella

Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.

Biomass and lipid production from Chlorella vulgaris UTEX 26 cultivated in 2 m3 raceway ponds under semicontinuous mode during the spring season

A Chlorella vulgaris UTEX 26 semicontinuous culture was implemented in 2000 L raceways with M medium during spring season at greenhouse conditions. Areal biomass productivities between 20 and 26 g m−2 d−1 were reached on the third day. The maximal areal lipid productivity obtained was 6.1 g m−2 d−1 and an increment in the saturated fatty acids (SFA) proportion (C14–C18) was favored in comparison with the fatty acids obtained with M medium in photobioreactors of 1 L and photoperiod light:darkness 12:12 h. After the eighth day of the culture or biomass concentrations above 0.25 g L−1, the microalgal cultures were prone to contamination by ciliates and amoebae, due to the sugars excreted by C. vulgaris UTEX 26. The periodical addition of NH4HCO3 to the microalgal culture maintained the ammonium concentration between 25 and 50 mg L−1, which contributed to diminish the contamination risks by protozoa.

Investigation of simultaneous lutein and lipid extraction from wet microalgae using Nile Red as solvatochromic shift probe

Microalgae have been proposed as an alternative lutein source due to their high productivity, reliability, and versatility. In this study lutein and lipid extraction from wet Chlorella vulgaris UTEX 265 was investigated. The lutein production was monitored throughout the microalgal growth phase and several extraction parameters such as the sample size, drying method, and cell disruption method were investigated. The performance of solvents on lutein extraction was compared using Nile Red as a solvatochromic polarity probe. The simultaneous lutein and lipid extraction was also studied for different polarities using an ethanol-hexane binary solvent at the optimal solvent compositions suitable for lutein extraction. Among the solvents investigated, 3:1 (v/v) ethanol/hexane was recognized as the optimal solvent for lutein and lipid co-extraction, which contributed to a 13.03 mg g−1 lutein and 101.8 mg g−1 FAME yield. The saponifiable lipids content (86.9% w/w) was higher than conventional extraction methods. Based on our results, wet extraction approach exhibits good potential, while the bead-beater is the most suitable technique for cell disruption and lutein extraction.

Assessment of Chlorella vulgaris and indigenous microalgae biomass with treated wastewater as growth culture medium

The aim of the present work was to evaluate the feasibility of microalgae cultivation using secondary treated domestic wastewater. Two Chlorella vulgaris strains (CICESE and UTEX) and an indigenous consortium, were cultivated on treated wastewater enriched with and without the fertilizer Bayfolan®. Biomass production for C. vulgaris UTEX, CICESE and the indigenous consortium grown in treated wastewater was 1.167±0.057, 1.575±0.434 and 1.125±0.250g/L, with a total lipid content of 25.70±1.24, 23.35±3.01and 20.54±1.23% dw, respectively. The fatty acids profiles were mainly composed of C16 and C18. Regardless of the media used, in all three strains unsaturated fatty acids were the main FAME (fatty acids methyl esters) accumulated in a range of 45–62%. An enrichment of treated wastewater with Bayfolan® significantly increased the production of biomass along with an increase in pigments and proteins of ten and threefold, respectively.

Investigation of Chlorella vulgaris UTEX 265 Cultivation under Light and Low Temperature Stressed Conditions for Lutein Production in Flasks and the Coiled Tree Photo-Bioreactor (CTPBR).

Lutein has an increasing share in the pharmaceutical and nutraceutical market due to its benefits to eye health. Microalgae may be a potential source for lutein production while the expense limits the commercialization. In this study, a coiled tubular tree photobioreactor (CTPBR) design was investigated for cultivating the cold tolerant microalgae Chlorella vulgaris UTEX 265 under various conditions for lutein production. The influence and interaction of light irradiance strength, lighting cycle, and temperature on microalgae and lutein production efficiency at low temperature range were also studied in flasks via response surface method (RSM). The results demonstrated that 14h day-light, 120μmol photons m-2s-1, and 10°C was the optimal condition for algae growth and lutein production at low temperature experimental ranges. C. vulgaris UTEX 265 showed good potential to produce lutein in cold weather, and the optimum lutein production was contrary to the specific lutein content but corresponds to the trend of optimum growth. Additionally, fast growth (μ=1.50day-1) and good lutein recovery (11.98mgg-1day-1) in CTPBR were also achieved at the low irradiance stress condition and the low temperature photo-inhibition conditions.

Genome-Scale Metabolic Model for the Green Alga Chlorella vulgaris UTEX 395 Accurately Predicts Phenotypes under Autotrophic, Heterotrophic, and Mixotrophic Growth Conditions.

The green microalga Chlorella vulgaris has been widely recognized as a promising candidate for biofuel production due to its ability to store high lipid content and its natural metabolic versatility. Compartmentalized genome-scale metabolic models constructed from genome sequences enable quantitative insight into the transport and metabolism of compounds within a target organism. These metabolic models have long been utilized to generate optimized design strategies for an improved production process. Here, we describe the reconstruction, validation, and application of a genome-scale metabolic model for C. vulgaris UTEX 395, iCZ843. The reconstruction represents the most comprehensive model for any eukaryotic photosynthetic organism to date, based on the genome size and number of genes in the reconstruction. The highly curated model accurately predicts phenotypes under photoautotrophic, heterotrophic, and mixotrophic conditions. The model was validated against experimental data and lays the foundation for model-driven strain design and medium alteration to improve yield. Calculated flux distributions under different trophic conditions show that a number of key pathways are affected by nitrogen starvation conditions, including central carbon metabolism and amino acid, nucleotide, and pigment biosynthetic pathways. Furthermore, model prediction of growth rates under various medium compositions and subsequent experimental validation showed an increased growth rate with the addition of tryptophan and methionine.

A novel culture medium designed for the simultaneous enhancement of biomass and lipid production by Chlorella vulgaris UTEX 26

A novel culture medium to enhance the biomass and lipid production simultaneously by Chlorella vulgaris UTEX 26 was designed in three stages of optimization. Initially, a culture medium was inferred applying the response surface method to adjust six factors [NaNO3, NH4HCO3, MgSO4·7H2O, KH2PO4, K2HPO4 and (NH4)2HPO4], which were selected on the basement of BBM (Bold’s Basal Medium) and HAMGM (Highly Assimilable Minimal Growth Medium) culture media. Afterwards, the nitrogen source compound was optimized to reduce both, ammonium and nitrate concentrations. As result of the optimization process, the proposed culture medium improved 40% the biomass (0.73gL−1) compared with the BBM medium and 85% the lipid concentration (281mgL−1), with respect to HAMGM medium. Some culture media components concentrations were reduced up to 50%. Gas chromatography analysis revealed that C16:0, C18:0, C18:1, C18:2 and C18:3 were the major fatty acids produced by C. vulgaris UTEX 26.

Evaluation of a Two-Phase Extraction System of Carbohydrates and Proteins from Chlorella vulgaris Utex 1803

Evaluation of a Two-Phase Extraction System of Carbohydrates and Proteins from Chlorella vulgaris Utex 1803

Synergistic interaction between metal ions in the sea salts and the extracellular polymeric substances for efficient microalgal harvesting

Artificial sea salts were found to cause microalgal flocculation and was so rather effectively in the presence of extracellular polymeric substances (EPS). EPS-producing species Ettlia sp. YC001 formed flocs at relatively low salt concentration: at 3.5g/L, it reached up to 90% of the flocculation within 2h. EPS non-producer Chlorella vulgaris UTEX 265, on the other hand, was less responsive to the salts even at 7g/L and it had only 42% of floc formation. This phenomenon of salt-mediated flocculation appeared to be brought about mainly by calcium ion, as it has had greater affinity to the EPS than any other ions including the divalent magnesium ion. This sea salt-based coagulation may serve as one environmental-friendly alternative to commonly used chemical flocculants, which also potentially possess an economic merit.

Comparison of biomass and lipid production under ambient carbon dioxide vigorous aeration and 3% carbon dioxide condition among the lead candidate Chlorella strains screened by various photobioreactor scales

Chlorella species from the UTEX collection, classified by rDNA-based phylogenetic analysis, were screened based on biomass and lipid production in different scales and modes of culture. The lead candidate strains of C. sorokiniana UTEX 1230 and C. vulgaris UTEX 395 and 259 were compared between conditions of vigorous aeration with filtered atmospheric air and 3% CO2 shake-flask cultivation. The biomass of UTEX 1230 produced 2 times higher at 652mgL−1 dry weight under both ambient CO2 vigorous aeration and 3% CO2 conditions, while UTEX 395 and 259 under 3% CO2 increased to 3 times higher at 863mgL−1 dry weight than ambient CO2 vigorous aeration. The triacylglycerol contents of UTEX 395 and 259 increased more than 30 times to 30% dry weight with 3% CO2, indicating that additional CO2 is essential for both biomass and lipid accumulation in UTEX 395 and 259.

Chlorella sorokiniana (formerly C. vulgaris) UTEX 2714, a non-thermotolerant microalga useful for biotechnological applications and as a reference strain

Molecular analyses employing sequencing of the complete ribosomal RNA cistron (18S rDNA, ITS1, 5.8S rDNA, ITS2, and 28S rDNA) and transcriptome analysis of the RuBisCO gene (rbcL) were done on Chlorella vulgaris UTEX 2714. The constructed phylogenetic trees showed that C. vulgaris UTEX 2714 is Chlorella sorokiniana. Growth analysis and production of chlorophyll a over a range of increasing cultivation temperatures (27–40 °C) showed that this strain is far less thermotolerant in comparison to a common C. sorokiniana strain. A change in the taxonomic designation of strain UTEX 2714 is proposed.

Biological system development for GraviSat: A new platform for studying photosynthesis and microalgae in space

Microalgae have great potential to be used as part of a regenerative life support system and to facilitate in-situ resource utilization (ISRU) on long-duration human space missions. Little is currently known, however, about microalgal responses to the space environment over long (months) or even short (hours to days) time scales. We describe here the development of biological support subsystems for a prototype “3U” (i.e., three conjoined 10-cm cubes) nanosatellite, called GraviSat, designed to experimentally elucidate the effects of space microgravity and the radiation environment on microalgae and other microorganisms. The GraviSat project comprises the co-development of biological handling-and-support technologies with implementation of integrated measurement hardware for photosynthetic efficiency and physiological activity in support of long-duration (3–12 months) space missions. It supports sample replication in a fully autonomous system that will grow and analyze microalgal cultures in 120 μL wells around the circumference of a microfluidic polymer disc; the cultures will be launched while in stasis, then grown in orbit. The disc spins at different rotational velocities to generate a range of artificial gravity levels in space, from microgravity to multiples of Earth gravity. Development of the biological support technologies for GraviSat comprised the screening of more than twenty microalgal strains for various physical, metabolic and biochemical attributes that support prolonged growth in a microfluidic disc, as well as the capacity for reversible metabolic stasis. Hardware development included that necessary to facilitate accurate and precise measurements of physical parameters by optical methods (pulse amplitude modulated fluorometry) and electrochemical sensors (ion-sensitive microelectrodes). Nearly all microalgal strains were biocompatible with nanosatellite materials; however, microalgal growth was rapidly inhibited (∼1 week) within sealed microwells that did not include dissolved bicarbonate due to CO2 starvation. Additionally, oxygen production by some microalgae resulted in bubble formation within the wells, which interfered with sensor measurements. Our research achieved prolonged growth periods (>10 months) without excess oxygen production using two microalgal strains, Chlorella vulgaris UTEX 29 and Dunaliella bardawil 30 861, by lowering light intensities (2–10 μmol photons m−2 s−1) and temperature (4–12 °C). Although the experiments described here were performed to develop the GraviSat platform, the results of this study should be useful for the incorporation of microalgae in other satellite payloads with low-volume microfluidic systems.

Comparative Analyses of Three Chlorella Species in Response to Light and Sugar Reveal Distinctive Lipid Accumulation Patterns in the Microalga C. sorokiniana

While photosynthetic microalgae, such as Chlorella, serve as feedstocks for nutritional oils and biofuels, heterotrophic cultivation can augment growth rates, support high cell densities, and increase triacylglycerol (TAG) lipid content. However, these species differ significantly in their photoautotrophic and heterotrophic characteristics. In this study, the phylogeny of thirty Chlorella strains was determined in order to inform bioprospecting efforts and detailed physiological assessment of three species. The growth kinetics and lipid biochemistry of C. protothecoides UTEX 411, C. vulgaris UTEX 265, and C. sorokiniana UTEX 1230 were quantified during photoautotrophy in Bold's basal medium (BBM) and heterotrophy in BBM supplemented with glucose (10 g L−1). Heterotrophic growth rates of UTEX 411, 265, and 1230 were found to be 1.5-, 3.7-, and 5-fold higher than their respective autotrophic rates. With a rapid nine-hour heterotrophic doubling time, Chlorella sorokiniana UTEX 1230 maximally accumulated 39% total lipids by dry weight during heterotrophy compared to 18% autotrophically. Furthermore, the discrete fatty acid composition of each strain was examined in order to elucidate lipid accumulation patterns under the two trophic conditions. In both modes of growth, UTEX 411 and 265 produced 18∶1 as the principal fatty acid while UTEX 1230 exhibited a 2.5-fold enrichment in 18∶2 relative to 18∶1. Although the total lipid content was highest in UTEX 411 during heterotrophy, UTEX 1230 demonstrated a two-fold increase in its heterotrophic TAG fraction at a rate of 28.9 mg L−1 d−1 to reach 22% of the biomass, corresponding to as much as 90% of its total lipids. Interestingly, UTEX 1230 growth was restricted during mixotrophy and its TAG production rate was suppressed to 18.2 mg L−1 d−1. This constraint on carbon flow raises intriguing questions about the impact of sugar and light on the metabolic regulation of microalgal lipid biosynthesis.

Discovery of small molecule hyperlipid accumulating triggers of algae Chlamydomonas reinhardtii identified via in vivo phenotype based screen (597.1)

A large scale in vivo high throughput screen (HTS) was performed to identify small compounds that stimulate lipid production and accumulation using the model organism Chlamydomonas reinhardtii. The HTS employed a 384‐well microplate format in which cells were allowed to grow in the presence of the compounds at 10 μM final concentration for 72 hours. Accumulation of intracellular lipids was assessed using the lipophilic dye Nile Red and growth was monitored at OD600. The screening library included 43,736 compounds (ChemBridge, Corp). A total of 367 active compounds were identified that stimulated lipid accumulation to > 2.5‐fold and did not affect cell viability to give a hit rate of 0.8%. Primary hits were “cherry picked” and the sub‐set of compounds were retested using an 8‐point dose response assay (0.25‐30 µM). Hits were further assessed visually using a Nikon Ti‐inverted microscope (60X) to reconfirm lipid droplet accumulation was induced.. The final set of hits included 306 compounds. These were further compared for structural similarities using Tibco Spotfire Lead Discovery package and resulted in three main structural clusters: compounds with a piperazine, nitrobenzene, and/or adamantine group. The impact of the compounds on cellular metabolism was also characterization by quantifying Chl A, B, starch, protein and toxicity analysis. Currently, we are examining compound effectiveness in additional algal species such as Chlorella sorokiniana UTEX 1230, Tetrachlorella alterans UTEX 2453, C. protothecoides UTEX 29, C. vulgaris UTEX 395, Nannochloropsis sp.Grant Funding Source: Supported by EPSCoR

Enhancing lipid productivity of Chlorella vulgaris using oxidative stress by TiO2 nanoparticles

Ability to increase the lipid production in microalgae is one of the heavily sought-after ideas to improve the economic feasibility of microalgae-derived transportation fuels for commercial applications. We used the oxidative stress by TiO2 nanoparticles, a well-known photocatalyst, to induce lipid production in microalgae. Chlorella vulgaris UTEX 265 was cultivated under various concentrations of TiO2 ranging from 0.1 to 5 g/L under UV-A illumination. Maximum specific growth rate was affected in responding to TiO2 concentrations. In the presence of UV-A, chlorophyll concentration was decreased at the highest concentration of TiO2 (5 g/L TiO2) by oxidative stress. The fatty acid ethyl ester (FAME) composition analysis suggested that oxidative stress causes the accumulation and decomposition of lipids. The highest FAME productivity was 18.2 g/L/d under low concentrations of TiO2 (0.1 g/L) and a short induction time (two days). The controlled condition of TiO2/UV-A inducing oxidative stress (0.1 g/L TiO2 and two days induction) could be used to increase the lipid productivity of C. vulgaris UTEX 265. Our results show the possibility of modulating the lipid induction process through oxidative stress with TiO2/UV-A.

Improvement of lipid productivity on chlorella vulgaris using waste glycerol and sodium acetate

Although microalgae have great potential as a raw material for biodiesel production it is necessary to increase both biomass and lipids productivity. One way to achieve this goal is the implementation of mixotrophic cultures and the regulation of carbon/nitrogen ratio. The present work aims to improve the productivity of biomass and lipids in Chlorella vulgaris UTEX 1803 using waste glycerol from biodiesel production (1, 5 and 10% v/v) and sodium acetate (5, 10 and 20 mM) as carbon sources together with modification of the initial concentration of nitrogen (1.02; 1.47 and 2.94mM de NaNO3). All experiments were performed at 23±1ºC, with light: dark cycles of 12:12 h during 5 days.
 In biomass production was achieved a significant increase (80% higher that autothrophic cultures without modification). The best percentages of lipids exceeded control culture up to 2.18. Lipid productivities were also found 2.83 and 3.5 times greater than control.
 Results show the possibility of increasing the production of biomass and lipids by applying the carbon/nitrogen ratio using as a carbon source waste glycerol of the biodiesel industry that opens up great possibilities for the re-use of this residue thus increasing the sustainability of the process in general, Also has been proved that carbon/nitrogen ratio using sodium acetate is an interesting alternative due to his low cost carbon/nitrogen ratio using sodium acetate is an interesting alternative.

Improvement of lab-scale production of microalgal carbohydrates for biofuel production

This work studied the improvement of biomass and carbohydrate (glucose and xylose) lab–scale productivity in Chlorella vulgaris UTEX 1803 through the use of the carbon/nitrogen ratio. In order to do so, mixotrophic cultures were made by the modification of initial concentration of CH3COONa (5, 10 and 20 mM) and NaNO3 (0.97, 1.94 and 2.94 mM). All treatments were maintained at 23 ± 1ºC, with light/dark cycles of 12h : 12h for 5 days.It was found that in addition to the carbon/nitrogen ratio, time also influences the concentration of biomass and carbohydrates. The treatment containing 10 mM acetate: 1.94 mM nitrate, reached a concentration of 0.79 g/L of biomass, 76.9 μg/mL of xylose and 73.7 μg/mL of glucose in the fifth day. However, the treatmentcontaining 20 mM acetate: 0.97 mM nitrate produced 1.04 g/L of biomass, 78.9 μg/mL of xylose and 77.2 μg/mL of glucose in the third day, while in the same day the treatment containing 0 mM acetate: 2.94 mM nitrate, produced 0.55 g/L of biomass, 40.2 μg/mL of xylose and 31.3 μg/mL of glucose.The use of carbon/nitrogen ratios improved biomass productivity (from 0.55 to 1.04 g/L) as well as xylose (from 40.2 to 78.9 μg/mL) and glucose (from 31.3 to 77.2 μg/mL) concentration, representing an improvement of up to two times the production of both biomass and carbohydrates in only 3 days of culture.

Increased pigment and lipid content, lipid variety, and cell and population size of the microalgae Chlorella spp. when co-immobilized in alginate beads with the microalgae-growth-promoting bacterium Azospirillum brasilense.

Three strains of the freshwater microalgae used for wastewater treatment, Chlorella vulgaris and Chlorella sorokiniana co-immobilized separately in alginate beads with the microalgae-growth-promoting bacterium Azospirillum brasilense Cd, resulted in significant changes in microalgal-population size, cell size, cell cytology, pigment, lipid content, and the variety of fatty acids produced in comparison with microalgae immobilized in alginate without the bacterium. Cells of C. vulgaris UTEX 2714 did not change in size, but the population size within the beads significantly increased. On the other hand, C. vulgaris UTEX 395 cells grew 62% larger, but their numbers did not increase. The population of C. sorokiniana UTEX 1602 increased, but not their cell size. The content of pigments chlorophyll a and b, lutein, and violoaxanthin increased in all microalgal species. The lipid content also significantly increased in all three strains, and the number of different fatty acids in the microalgae increased from four to eight. This study indicates that the microalgae-growth-promoting bacterium induced significant changes in the metabolism of the microalgae.