Transcriptome and Metabolome Profiling of a Novel Isolate Chlorella sorokiniana G32 (Chlorophyta) Displaying Enhanced Starch Accumulation at High Growth Rate Under Mixotrophic Condition

Chlorella sorokiniana is one of the most productive microalgal species with a high potential for the production of biofuels and other high value-added molecules. Many studies have focused on its capability of mixotrophic growth using reduced organic carbon and growth pattern shift between autotrophic and mixotrophic conditions. In this study, we investigated growth patterns of a novel isolate, C. sorokiniana G32, under mixotrophic growth conditions supplemented with a low level (1.25 g L–1) and a high level (5 g L–1) of glucose. Physiological, transcriptomic (i.e., RNA-seq), and metabolomic (i.e., LC-MS/MS) methods were used. We showed that peak growth based on OD680nm absorbance is ∼4-fold higher with high glucose vs. low glucose supplementation. Photosynthetic efficiency (Fv/Fm) in G32 mixotrophic cultures with high or low glucose supplementation remains identical to that of G32 phototrophic growth. We also found that the conversion rate between absorbance-based cell density and cell dry weight with high glucose supplementation was lower than with low glucose. This suggests that more cell biomass is produced under high glucose treatment than with low glucose. The result was confirmed via sucrose density gradient centrifugation. It is likely that accumulation of high concentration of starch may account for this effect. Transcriptomic analysis of G32 cultures (i.e., via RNA-seq) in response to reciprocal change of glucose levels reveals that expression of a subset of differentially expressed genes (DEGs) is correlated with the amount of glucose supplementation. These DEGs are designated as glucose-specific responsive (GSR) genes. GSR genes are enriched for a number of energy metabolic pathways. Together with metabolomics data (i.e., LC-MS/MS), we show that under high-level supplementation, glucose is preferentially oxidized through an oxidative pentose phosphate pathway. Collectively, our results indicate the mechanism of regulation of glucose assimilation and energy metabolism in G32 under mixotrophic conditions with different levels of glucose supplementation revealed by transcriptomic and metabolomic analyses. We propose that C. sorokiniana G32 has the potential for the production of high value-added molecules.

Effect of Anaerobic Digestate on the Fatty Acid Profile and Biodiesel Properties of Chlorella sorokiniana Grown Heterotrophically

The growth kinetics and the lipid and protein content of the microalgal species Chlorella sorokiniana (CS) grown heterotrophically in growth media containing glycerol and increasing amounts of anaerobic digestate (AD) equal to 0%, 15%, 30%, and 50% was studied. The effect of the AD on the fatty acid (FA) distribution of the bio-oil extracted from the CS, as well as on the fatty acid methyl ester (FAME) properties such as the saponification number (SN), the iodine value (IV), the cetane number (CN), and the higher heating value (HHV) was also estimated. The percentage of AD in the growth medium affects the rate of carbon uptake. The maximum carbon uptake rate occurs at about 30% AD. Protein and lipid content ranged from 32.3–38.4% and 18.1–23.1%, respectively. Fatty acid distribution ranged from C10 to C26. In all AD percentages the predominant fatty acids were the medium chain FA C16 to C18 constituting up to about 89% of the total FA at 0% AD and 15% AD and up to about 54% of the total FA at 30% AD and 50% AD. With respect to unsaturation, monounsaturated FA (MUFA) were predominant, up to 56%, while significant percentages, up to about 38%, of saturated FA (SFA) were also produced. The SN, IV, CN, and HHV ranged from 198.5–208.3 mg KOH/g FA, 74.5–93.1 g I/100 g FAME, 52.7–56.1, and 39.7–40.0 MJ/kg, respectively. The results showed that with increasing AD percentage, the CN values tend to increase, while decrease in IV leads to biofuel with better ignition quality.

Harvesting of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium using different techniques

ABSTRACT: The harvesting process is a current challenge for the commercial production of microalgae because the biomass is diluted in the culture medium. Several methods have been proposed to harvest microalgae cells, but there is not a consensus about the optimum method for such application. Herein, the methods based on sedimentation, flocculation, and centrifugation were evaluated on the recovery of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium. C. sorokiniana BR001 was cultivated using a low-nitrogen medium to trigger the accumulation of neutral lipids and neutral carbohydrates. The biomass of C. sorokiniana BR001 cultivated in a low-nitrogen medium showed a total lipid content of 1.9 times higher (23.8 ± 4.5%) when compared to the biomass produced in a high-nitrogen medium (12.3 ± 1.2%). In addition, the biomass of the BR001 strain cultivated in a low-nitrogen medium showed a high content of neutral carbohydrates (52.1 ± 1.5%). The natural sedimentation-based process was evaluated using a sedimentation column, and it was concluded that C. sorokiniana BR001 is a non-flocculent strain. Therefore, it was evaluated the effect of different concentrations of ferric sulfate (0.005 to 1 g L-1) or aluminum sulfate (0.025 to 0.83 g L-1) on the flocculation process of C. sorokiniana BR001, but high doses of flocculant agents were required for an efficient harvest of biomass. It was evaluated the centrifugation at low speed (300 to 3,000 g) as well, and it was possible to conclude that this process was the most adequate to harvest the non-flocculent strain C. sorokiniana BR001.

Aislamiento y caracterización molecular de cepas fitoplanctónicas obtenidas en Engabao, Guayas-Ecuador.

El interés industrial en microorganismos fitoplanctónicos como microalgas y cianobacterias se debe al elevadopotencial biotecnológico que poseen en sintetizar compuestos bioactivos, a la facilidad de adaptarse adiferentes fuentes de nutrientes, condiciones extremas de supervivencia y a la capacidad bioabsorbente demetales pesados, principalmente en aguas residuales. El objetivo del presente estudio consistió en aislar ycaracterizar fenotípica y genotípicamente microorganismos fitoplanctónicos de piscinas de estabilización deEngabao, Guayas-Ecuador, mediante el uso de diferentes medios de cultivos y de los marcadores molecularesITS, ITS2, LSU y 16S RNAr. Los resultados del cultivo celular durante siete días mostraron que la microalgaChls1 con medio BG11 pH 7.8 alcanzó 25.4 x 106cel mL-1, la cianobacteria Syn1 fue de 340.6 x 106cel mL-1,mientras que para Nod1 se empleó análisis de clorofila a alcanzando al séptimo día 6.33 μg mL-1. Los estudiosmoleculares se realizaron empleando un protocolo modificado de extracción y purificación de ADNg obteniendouna óptima concentración y calidad del ADN para las tres cepas: Chls1 146 ng µL-1, Syn1 326 ng µL-1 y Nod1158.8 ng µL-1. El uso de análisis polifásicos en base a caracteres morfológicos y ADNg de las cepas, se amplificópor PCR y se secuenció; las cepas identificadas fenotípica y genotípicamente mediante los primers ITS2, ITS yLSU corresponde a la microalga Chlorella sorokiniana con 99,48%, 99,07% y 99,77% de similitud de identidad,mientras que los aislados cianobacterianos Synechococcus sp. y Nodosilinea sp. con el análisis de la región 16SRNAr presenta porcentajes de identidad de 97,47% y 99,83%; a pesar de no emplear cebadores específicos paraestos microorganismos, el uso de varias regiones, aumenta la confiablidad en la identificación taxonómica yfilogenética; estas especies poseen alto potencial biotecnológico y de gran impacto económico.

Effect of Different Cultivation Modes (Photoautotrophic, Mixotrophic, and Heterotrophic) on the Growth of Chlorella sp. and Biocompositions

In the past, biomass production using microalgae culture was dependent on inorganic carbon sources as microalgae are photosynthetic organisms. However, microalgae utilize both organic and inorganic carbon sources, such as glucose. Glucose is an excellent source of organic carbon that enhances biomass yield and the content of useful substances in microalgae. In this study, photoautotrophic, mixotrophic, and heterotrophic cultivation conditions were applied to three well-known strains of Chlorella (KNUA104, KNUA114, and KNUA122) to assess biomass productivity, and compositional changes (lipid, protein, and pigment) were evaluated in BG11 media under photoautotrophic, mixotrophic, and heterotrophic conditions utilizing different initial concentrations of glucose (5, 10, 15, 20, and 25 g L−1). Compared to the photoautotrophic condition (biomass yield: KNUA104, 0.35 ± 0.04 g/L/d; KNUA114, 0.40 ± 0.08 g/L/d; KNUA122, 0.38 ± 0.05 g/L/d) glucose was absent, and the biomass yield improved in the mixotrophic (glucose: 20 g L−1; biomass yield: KNUA104, 2.99 ± 0.10 g/L/d; KNUA114, 5.18 ± 0.81 g/L/d; KNUA122, 5.07 ± 0.22 g/L/d) and heterotrophic conditions (glucose: 20 g L−1; biomass yield: KNUA104, 1.72 ± 0.26 g/L/d; KNUA114, 4.26 ± 0.27 g/L/d; KNUA122, 4.32 ± 0.32 g/L/d). All strains under mixotrophic and heterotrophic conditions were optimally cultured when 15–20 g L−1 initial glucose was provided. Although bioresourse productivity improved under both mixotrophic and heterotrophic conditions where mixotrophic conditions were found to be optimal as the yields of lipid and pigment were also enhanced. Protein content was less affected by the presence of light or the concentration of glucose. Under mixotrophic conditions, the highest lipid content (glucose: 15 g L−1; lipid content: 68.80 ± 0.54%) was obtained with Chlorella vulgaris KNUA104, and enhanced pigment productivity of Chlorella sorokiniana KNUA114 and KNUA122 (additional pigment yield obtained with 15 g L−1 glucose: KNUA 114, 0.33 ± 0.01 g L−1; KNUA122, 0.21 ± 0.01 g L−1). Also, saturated fatty acid (SFA) content was enhanced in all strains (SFA: KNUA104, 29.76 ± 1.31%; KNUA114, 37.01 ± 0.98%; KNUA122, 33.37 ± 0.17%) under mixotrophic conditions. These results suggest that mixotrophic cultivation of Chlorella vulgaris and Chlorella sorokiniana could improve biomass yield and the raw material quality of biomass.

Use of Microalgae Biomass for Fortification of Food Products from Grain

This article describes the use of Chlorella sorokiniana biomass additives in pasta recipes to enrich the product with biologically active phytonutrients, as well as to achieve the desired color range without the use of synthetic dyes. Samples of dry biomass were obtained by the cultivation of microalgae C. sorokiniana (strain), its quality indicators and nutritional value were determined for use as a food additive. A method of using dry biomass of microalgae C. sorokiniana as a phytoadditive to replace 5% of flour mixture for effective enrichment of pasta with biologically active phytonutrients was proposed. The choice of the optimal amount of addition of microalgae biomass was proved since it turned out that the replacement of flour should be no more than 5% due to the distinct fish flavor of the final product. The present study was conducted to evaluate the effect of adding dry biomass of Chlorella microalgae on total protein, lipid, chlorophyll, and carotenoid content. Substitution of 5% of pasta flour led to an increase in the content of proteins and lipids to 15.7 ± 0.50% and 4.1 ± 0.06%, respectively. Meanwhile, the addition of microalgae Chlorella to pasta has helped to increase the content of polyunsaturated fatty acids, chlorophyll, and carotenoids which are necessary for the prevention of foodborne diseases. The aim of this study is to develop pasta recipe with additives of microalgae biomass C. sorokiniana and study their quality indicators.