Chlorophyll Extraction Research Articles

Extraction of chlorophyll a from Tetradesmus obliquus—a method upgrade

Nowadays, the use of algae is prevalent for both industrial and agricultural purposes. The determination of chlorophyll (Chl) content is a commonly used method for estimating the phytoplankton abundance in different water bodies or biomass density of algal cultures. The aim of the present work is to optimise the efficiency of the Chl extraction from the green alga Tetradesmus obliquus using methanol as extracting solvent. The extraction efficiency was estimated by measuring the Chl a concentration of the extracts using fluorescence spectroscopy. To increase the extraction yield, glass fibre filters with algal cells on top were treated with 10% (v/v) formalin prior to the extraction. We found that this pretreatment significantly enhanced the extraction yield of Chl without its chemical decomposition. We also found that the optimal cell concentration for Chl determination ranged from 1.44 × 104 to 3.60 × 105 cells/mL and the extraction efficiency was lower when the cell density of the culture was out of this range. These results highlight the importance of the optimization of the pigment extraction for the studied algal species.

Extraction of Phycocyanin and Chlorophyll from Spirulina by “Green Methods”

Phycocyanin is a pigment–protein complex from the group of phycobiliproteins obtained from Spirulina (Arthrospira platensis), with possibilities for various applications in food and pharmaceutical technologies. It is a natural colorant for food and cosmetic products. This study aimed to investigate the effect of ultrasonic and microwave extraction conditions on antioxidant activity (AOA), chlorophyll content, and the content and purity index of phycocyanin in aqueous and alcoholic extracts of Spirulina (Arthrospira platensis). For this purpose, ultrasonic extraction with water or ethanol was performed at 20 °C, 30 °C, and 40 °C for 1, 2, and 3 h at an ultrasonic frequency of 36 kHz, 40 kHz, and 45 kHz. Microwave water extraction was performed for 60 s, 120 s, and 180 s. For each of the obtained samples, three parallel measurements of antioxidant activity were made by DPPH and FRAP methods, and chlorophyll content and phycocyanin yield and purity index were determined spectrophotometrically. Ultrasonic extraction resulted in a higher yield and purity index of phycocyanin compared to microwave extraction. The highest yield of 14.88 mg g−1 with a purity index of 1.60 was achieved at a temperature of 40 °C for one hour and an ultrasonic wave frequency of 40 kHz. A relatively low yield of 4.21 mg g−1, but with a purity index of 2.67, was obtained at a temperature of 30 °C, a time of two hours, and an ultrasonic frequency of 40 kHz. Chlorophyll b content at 20 °C, for two hours and ultrasonic frequency 40 kHz was 1.400 mg g−1. The study proposes ultrasonic extraction as a green method to obtain phycocyanin of varying purity index that may be used for food, cosmetic, or biomedical purposes.

A genome-wide association study identifies genes associated with cuticular wax metabolism in maize.

The plant cuticle is essential in plant defense against biotic and abiotic stresses. To systematically elucidate the genetic architecture of maize (Zea mays L.) cuticular wax metabolism, 2 cuticular wax-related traits, the chlorophyll extraction rate (CER) and water loss rate (WLR) of 389 maize inbred lines, were investigated and a genome-wide association study (GWAS) was performed using 1.25 million single nucleotide polymorphisms (SNPs). In total, 57 nonredundant quantitative trait loci (QTL) explaining 5.57% to 15.07% of the phenotypic variation for each QTL were identified. These QTLs contained 183 genes, among which 21 strong candidates were identified based on functional annotations and previous publications. Remarkably, 3 candidate genes that express differentially during cuticle development encode β-ketoacyl-CoA synthase (KCS). While ZmKCS19 was known to be involved in cuticle wax metabolism, ZmKCS12 and ZmKCS3 functions were not reported. The association between ZmKCS12 and WLR was confirmed by resequencing 106 inbred lines, and the variation of WLR was significant between different haplotypes of ZmKCS12. In this study, the loss-of-function mutant of ZmKCS12 exhibited wrinkled leaf morphology, altered wax crystal morphology, and decreased C32 wax monomer levels, causing an increased WLR and sensitivity to drought. These results confirm that ZmKCS12 plays a vital role in maize C32 wax monomer synthesis and is critical for drought tolerance. In sum, through GWAS of 2 cuticular wax-associated traits, this study reveals comprehensively the genetic architecture in maize cuticular wax metabolism and provides a valuable reference for the genetic improvement of stress tolerance in maize.

Green synthesis of a magnesium single-atom catalyst from Spinacia oleracea chlorophyll extracts for sustainable electrocatalytic nitrate reduction to ammonia

Magnesium single atoms derived from natural leaves for sustainable ammonia production.

Extraction and Spectrophotometric Determination of Chlorophyll Content and Carotenoids from Cocos nucifera L. Leaf using Various Solvents in Saurashtra Region

Chlorophyll and carotenoids are significant photosynthetic pigments present in higher plants, algae and cyanobacteria. Plants contain three major pigments called chlorophyll a, chlorophyll b, and chlorophyll c. Chlorophyll participates in the photosynthesis process, which transforms light energy into chemical energy. Species differences in chlorophyll pigment concentration exist. Orange, yellow and vivid red colors in vegetables and fruits are due to carotenoids. The health of plants is significantly influenced by these two pigments. In addition to having excellent antioxidant qualities, chlorophylls and carotenoids show therapeutic effects on oxidative and inflammatory disorders. They also possess anti-cancer effects. Chlorophyll and carotenoids support healthy blood coagulation and help in protecting skin, hormonal balancing, and deodorization. The major objective of this study is to identify plant with a high content of carotenoids and chlorophyll since both of these bioactive compounds have several applications in herbal medicine. In the current work, carotenoids and chlorophyll were extracted from three varieties of Cocos nucifera L. – Dwarf, DT and tall of Veraval coastof Saurashtra region, Gujarat using DMSO, methanol, diethyl ether and 80% acetone. The amounts of chlorophyll and carotenoids were measured using a spectrophotometer. Maximum extraction of chlorophyll and carotenoids was found in methanol and DMSO from the leaves of the dwarf variety of C. nucifera L.

MdDEWAX decreases plant drought resistance by regulating wax biosynthesis

Apple epidermal wax protects plants from environmental stresses, determines fruit gloss and improves postharvest storage quality. However, the molecular mechanisms underlying the biosynthesis and regulation of apple epidermal waxes are not fully understood. In this study, we isolated a MdDEWAX gene from apple, which localized in the nucleus, expressed mainly in apple fruit, and induced by drought. We transformed the MdDEWAX gene into Arabidopsis, and found that heterologous expression of MdDEWAX reduced the accumulation of cuticular waxes in leaves and stems, increased epidermal permeability, the rate of water loss, and the rate of chlorophyll extraction of leaves and stems, altered the sensitivity to ABA, and reduced drought tolerance. Meanwhile, overexpression or silencing of the gene in the epidermis of apple fruits decreased or increased wax content, respectively. This study provides candidate genes for breeding apple cultivars and rootstocks with better drought tolerance.

Kinetics of pulsed electric fields assisted pigment extraction from Chlorella pyrenoidosa

Kinetics οf chlorophyll extraction from the microalga Chlorella pyrenoidosa, assisted by Pulsed Electric Fields (PEF), were studied. Treated (4.6 kV/cm, 0–179.2 kJ/kg) and untreated biomass was suspended in ethanolic solutions (75–95% v/v) and incubated at 30–60 °C for up to 6 h. Higher PEF treatment specific energy, ethanolic content of extraction solvent and extraction temperature led to higher recovery of pigments and antioxidants (chlorophyll and lutein, respectively). Chlorophyll characteristic extraction time depended only on extraction temperature (2.16 h at 30 °C vs. 0.61 h at 40 °C). A treatment of 179.2 kJ/kg before extraction with 95% ethanol at 60 °C for 4 h led to maximum recovery of both chlorophyll and lutein (18.2 and 1.17 mg/g dry biomass, respectively). Lutein was extracted more selectively at 30 °C which led to extracts with higher concentration in antioxidants (28.83 mg Trolox/g dry extract from treated samples with 179.2 kJ/kg and a four-hour extraction with 95% ethanol). Industrial relevanceChlorophylls and carotenoids from C. pyrenoidosa have garnered significant interest by the food industry for application as natural colorants and strong antioxidants in food products. Low energy requirements, short treatment times and selectivity establish Pulsed Electric Fields as a suitable technology to improve the extraction of these ingredients. An overall mathematical model of chlorophyll extraction, incorporating all process parameters, is a useful industrial tool for reliable predictions and the estimation of extraction efficiency under different process conditions.

Optimal chlorophyll extraction conditions and postharvest stability in Moringa (M. Oleifera) leaves

This study aimed to assess the optimal conditions for the extraction of chlorophyll and the stability of chlorophyll with postharvest storage period in Moringa Oleifera leaves harvested in 3 different years for its preservation and lifespan. For this purpose, chlorophylls a (Chla) and b (Chlb) were extracted from the leaves using acetone, methanol, N, N-dimethylformamide (DMF), and the ‘green’ solvent (ethanol). In addition, the chlorophylls were extracted under various conditions, including temperatures (4, 25, and 45 °C), and times (10, 30, and 60 min) from dry leaves that were harvested in different years (2020, 2021, and 2022). The results showed that the Chla content extracted exceeded that of Chlb in the four solvents in all temperatures and extraction times, except for acetone and ethanol extracts under 45 °C at 30 and 60-min extraction times in samples harvested in 2022. An increase in extraction time and temperature resulted in higher chlorophyll content. Overall chlorophyll content decreased with the increasing postharvest storage period, particularly in methanol and ethanol extracts. The Chlorophyll Stability Index showed that chlorophyll is stable in moringa, such that the chlorophyll content obtained in a 2-year postharvest storage period samples was also found to be high. In general, the chlorophyll obtained from this study was found to be compatible with what is used in the industrial market. This suggests that the chlorophyll from moringa is stable and can be considered a major source of chlorophyll.

Chlorophyll Sensitization of TiO2: A Mini-Review

Recent studies have shown that chlorophyll sensitization can improve the performance of semiconductors like TiO2 in photocatalytic reactions and light-harvesting technologies, such as solar cells. Faced with the search for renewable energy sources and sustainable technologies, the application of this natural pigment has been gaining prominence. The present work addresses some of the main possibilities of chlorophyll-TiO2 combination, presenting the most relevant aspects affecting chlorophyll extraction and TiO2 sensitization.

New green biorefineries from cyanobacterial-microalgal consortia: Production of chlorophyll-rich extracts for the cosmetic industry and sustainable biogas

Microalgae are promising and sustainable candidates for developing biorefineries to obtain valuable bioproducts and bioenergy. However, key challenges, such as low biomass production, high-cost harvesting, and non-efficient extractions, are restricting its large-scale production. Symbiotic relationships between microalgae and cyanobacteria can simultaneously mitigate these technical and economic restrictions. This research aims to develop sustainable and cost-effective biorefineries from two microalgae-cyanobacteria consortia to produce valuable ingredients for the cosmetic field (chlorophylls) and bioenergy (biogas). Solvent screening and cell disruption experiments were carried out to optimise the chlorophyll extraction protocols. Green solvents were chosen for both consortia. The mildness method (vortexing) was enough to achieve the maximum extraction level of chlorophylls (4.8 ± 0.2 mg/g) using 96% ethanol from a consortium composed of Chlorella vulgaris, Tetraselmis sp., and Kamptonema sp. The use of bead-beating was even more effective using water in the case of the consortium of C. vulgaris and Arthrospira platensis (13.5 ± 1.1 mg/g). High-potential antioxidant chlorophyll extracts were obtained for the cosmetic sector. As ethanol traces were found in the residual biomass of the C. vulgaris, Tetraselmis sp., and Kamptonema sp. consortium, the highest cumulative biomethane production (472 ± 32 mLCH4/gVS) was achieved with this residual biomass, the corresponding value for the initial consortium being significantly lower (239 ± 32 mLCH4/gVS). The study concludes through the overall mass balances of the best biorefineries that it is possible to recover up to 100% of both consortia weight as cosmetic ingredients, biogas, and fertilisers or cultivation media.

Deep eutectic solvent dispersive liquid–liquid microextraction methods for the analysis of chlorophyll natural colorant (E140) via microwave assisted sample preparation

In the current research paper, extraction and analysis of chlorophyll, one of the bioactive compounds that is usually utilized as natural pigments (E140) and obtained from many leafy green plants and seaweeds, have been carried out via deep eutectic solvent-based dispersive liquid–liquid phase microextraction (DES-UA-DLLME) from plants and food pruducts. The DES-UA-DLLME method was developed by utilizing deep eutectic solvent (DES) which was prepared by heating process with a 1:1 ratio of choline chloride and urea (ChCl: U). Analytical parameters such as pH, DES volume, sample volume, centrifugation and mixing time, DES type, matrix effect, etc. were optimized to ensure the extraction efficiency of chlorophyll. The limit of detection (LOD), limit of quantification (LOQ), intra-day and inter-day relative standart devision (RSD) were found as 0.007 µg/mL, 0.023 µg/mL, 1.3 %, and 1.9 %, respectively. The accuracy of the method was tested with water sample addition/recovery studies. The DES-UA-DLLME method has been applied to food, water, and plant samples. The food products and plants were prepared with the help of ultrasonic and microwave extraction for spectrophotometric analysis of chlorophyll. According to literature survey, the DES-UA-DLLME method and the MWAE method combination was used to the first time for extraction of chlorophyll (E140) from plants.

Changing of Chlorophyll Contents in Caulerpa lentillifera After Five-Day Harvest

Caulerpa lentillifera is a marine macroalgae widely consumed in Asia Pacific, including Thailand. Quality and freshness are the main criteria for consumers. Chlorophyll is an important fundamental pigmentation and acts as green in C. lentillifera. It can be variable according to light and growth, and then it can indicate the quality of C. lentillifera products. This research aimed to observe changing chlorophyll contents in C. lentillifera after harvest during five days from two different months- May and June and two different ponds-ponds 1 and 2 by chlorophyll extraction and measurements of chlorophyll fluorescence. The result showed that the contents of total chlorophyll and chlorophyll A differed between months, but the pattern did not significantly change even in different months and ponds within five days after harvest.

Effect of blanching, freezing and microwave-assisted pretreatment methods on chlorophyll extraction from Sauropus androgynus leaves

Sauropus androgynus is grown commonly in Vietnam, which has a high content of bioactive substances including chlorophyll, polyphenols, and antioxidant activity. Sauropus androgynus is used as food or a colorant for traditional Vietnamese cakes. Green pigments are employed in various industrial sectors, such as food, drinks, soap, and drug. Chlorophyll can be replaced synthetic green pigments that might be unhealthy. In this study, the effect of chlorophyll extraction efficiency and perceptional green color by extraction condition combinined with physical pretreatment methods was investigated using Sauropus androgynus leave as a model. Fresh raw materials were pre-treated by blanching, freezing and microwave treatment at 55w/g for 3 min and then extracted with 40% (v/v) ethanol solvent. The best chlorophyll recovery efficiency (CRE) from Sauropus androgynus by heat pretreatment methods with and without microwave assistance was 32.035% and 25.784% of chlorophyll content in material; or 1.081 mg/g d. w and 0.862 mg/g d. w of fresh leaves, respectively. The chlorophyll extract is then spray-dried to form a water-dispersible powder. Chlorophyll extracted with microwave-assisted pretreatment having high CRE that has potential applications in food production, but green color is not much more favorable than the non-microwave assisted sample.

Leaf chlorophyll contents in some European pear cultivars grafted on different rootstocks and its relation with growth and yield

Aim of study: To investigate the effect of different combinations of pear rootstocks and cultivars on the contents of leaf photosynthetic pigments and their relation with some growth and yield characteristics. Area of study: Ondokuz Mayis University, Samsun, Türkiye, in two years, 2021 and 2022. Material and methods: The pear cultivars ‘Santa Maria’, ‘Williams’, and ‘Deveci’ were grafted on eight rootstocks: two quince clonal rootstocks (BA29 and QA), five pear clonal rootstocks (FOX9, FOX11, OH×F333, OH×F87, FAROLD40) and Pyrus communis L. seedlings. Growth and yield attributes were calculated and chlorophyll and carotenoid contents were determined by the spectrophotometric method, using 99.8% methanol solvent for chlorophyll (chl) extraction. Main results: Chl a/b in the case of rootstocks, chl a and total carotenoids in the cultivars were found statistically significant; the ratio chl a/b ranged 1.71-2.30 in the case of rootstocks; in the case of cultivars chl a ranged from 17.77 (cv. ‘Santa Maria’) to 19.88 (cv. ‘Deveci’) μg mL-1, and carotenoids ranged 0.21-0.95 μg mL-1. Under the main impact of cultivars, rootstocks and their combinations, a significant difference was seen in the growth and yield attributes. Research highlights: A negative correlation coefficient was observed between photosynthetic pigments and morphological characteristics; however, the correlation coefficient was positive for canopy volume and annual shoot growth. Canopy management, especially with the use of rootstocks and cultivars that result in weaker growth, is helpful for improving chlorophyll content and yield performances.

Correlation of the SPAD index with absolute leaf pigments in irrigated lettuce culture in the Northwest Paraná, Brazil

Photosynthesis is the most important biochemical process that takes place in plants and chlorophyll is the main pigment involved in it. Current destructive methods for determining chlorophyll content are accurate, but time-consuming and relatively expensive. The SPAD (Soil Plant Analysis Development) is a portable chlorophyll meter that provides an instant and nondestructive reading of the green color of leaves. As an important source of vitamins and antioxidants such as vitamins C, carotenoids and fiber content, lettuce (Lactuca sativa L.) plays a crucial role in human nutrition, being one of the most popular staple plant foods among the population. Considering the possibility of management based on the characteristics of photosynthetic pigments, the study aimed to relate the SPAD index values with the content of leaf pigments in lettuce. The study was conducted in the Northwest region of Paraná, Brazil. The experiment was carried out in a completely randomized design with 48 leaf samples being determined the SPAD index and the content of leaf pigments. The determination of the SPAD index was performed with the SPAD-502 equipment (Minota®) with three readings per sample. For the extraction of leaf pigments, chlorophyll (a; b), total chlorophyll (a + b) and carotenoids, acetone was used as an extractor and the reading was performed in a spectrophotometer. The SPAD index was correlated with the levels of chlorophyll a (0.93), chlorophyll b (0.89), total chlorophyll (0.93) and carotenoids (0.95) in lettuce leaves. Through the SPAD, the chlorophyll content in lettuce leaves can be determined in a representative way.

Ultrasound-Assisted Extraction of Chlorophyll from Pea Pod Waste: Optimization, Kinetics, and Stability Study

Ultrasound-Assisted Extraction of Chlorophyll from Pea Pod Waste: Optimization, Kinetics, and Stability Study

Simulation and Economic Analysis of the Biotechnological Potential of Biomass Production from a Microalgal Consortium

The biomass of microalgae and the compounds that can be obtained from their processing are of great interest for various economic sectors. Chlorophyll from green microalgae has biotechnological applications of great potential in different industrial areas such as food, animal feed, pharmaceuticals, cosmetics, and agriculture. In this paper, the experimental, technical and economic performance of biomass production from a microalgal consortium (Scenedesmus sp., Chlorella sp., Schroderia sp., Spirulina sp., Pediastrum sp., and Chlamydomonas sp.) was investigated in three cultivation systems (phototrophic, heterotrophic and mixotrophic) in combination with the extraction of chlorophyll (a and b) on a large scale using simulation; 1 ha was established as the area for cultivation. In the laboratory-scale experimental stage, biomass and chlorophyll concentrations were determined for 12 days. In the simulation stage, two retention times in the photobioreactor were considered, which generated six case studies for the culture stage. Subsequently, a simulation proposal for the chlorophyll extraction process was evaluated. The highest microalgae biomass concentration was 2.06 g/L in heterotrophic culture, followed by mixotrophic (1.98 g/L). Phototrophic and mixotrophic cultures showed the highest chlorophyll concentrations of 20.5 µg/mL and 13.5 µg/mL, respectively. The simulation shows that higher biomass and chlorophyll production is attained when using the mixotrophic culture with 72 h of retention that we considered to evaluate chlorophyll production (a and b). The operating cost of the entire process is very high; the cultivation stage has the highest operating cost (78%), mainly due to the high energy consumption of the photobioreactors.

Application of a Fluorescence-Based Instrument Prototype for Chlorophyll Measurements and Its Utility in an Herbicide Algal Ecotoxicity Assay

Project Aquafluosense was designed to develop prototypes for a modular fluorescence-based instrumental setup for in situ measurement of major water quality parameters. A fluorometer was developed for algal density estimation based on the fluorescent excitation of chlorophyll. The appropriate type of sample holder microplate was determined, along with the need for dark acclimation, prior to the measurements during the instrument’s development. Model species of green (Raphidocelis subcapitata) and blue-green alga (Microcystis aeruginosa) were applied in forms of pure monocultures and their mixtures, and improved analytical limits of detection were achieved (3.70 × 103 cell/mL and 1.13 × 105 for R. subcapitata and M. aeruginosa, respectively). The fluorescence-based determination of algal density was validated by conventional methods, such as cell counting in a Bürker chamber, optical density measurement, and chlorophyll extraction with ethanol. The signals obtained by the fluorometer correlated well with the conventional methods. Pearson r coefficients (applied where the correlation was linear) were ≥0.988 and Spearman ρ coefficients (applied where the correlation was not linear) were >0.976, indicating a strong and positive correlation. The applicability of the developed fluorometer was demonstrated in a growth inhibition ecotoxicity assay on R. subcapitata using the herbicide active ingredient isoxaflutole. During the assay, light intensity (continuous, 104.9 ± 14.9 µE/m2/s), temperature (22 ± 2 °C), pH of algal media (pH = 6–7 for Zehnder and Allen media, as well), and intensity of stirring (continuous, 100 rpm) were controlled. The results indicated that the FluoroMeter Module is applicable for screening algal toxicity: the observed ratio of fluorescence decrease determined by fluorescence induction provided significantly lower toxicity values (EC50: 0.015 ± 0.001 µg/mL) compared to values determined by the optical density (EC50: 0.034 ± 0.004 µg/mL) and chlorophyll a content (EC50: 0.033 ± 0.000 µg/mL).

3D bioprinting of mineralizing cyanobacteria as novel approach for the fabrication of living building materials.

Living building materials (LBM) are gaining interest in the field of sustainable alternative construction materials to reduce the significant impact of the construction industry on global CO2 emissions. This study investigated the process of three-dimensional bioprinting to create LBM incorporating the cyanobacterium Synechococcus sp. strain PCC 7002, which is capable of producing calcium carbonate (CaCO3) as a biocement. Rheology and printability of biomaterial inks based on alginate-methylcellulose hydrogels containing up to 50 wt% sea sand were examined. PCC 7002 was incorporated into the bioinks and cell viability and growth was characterized by fluorescence microscopy and chlorophyll extraction after the printing process. Biomineralization was induced in liquid culture and in the bioprinted LBM and observed by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and through mechanical characterization. Cell viability in the bioprinted scaffolds was confirmed over 14 days of cultivation, demonstrating that the cells were able to withstand shear stress and pressure during the extrusion process and remain viable in the immobilized state. CaCO3 mineralization of PCC 7002 was observed in both liquid culture and bioprinted LBM. In comparison to cell-free scaffolds, LBM containing live cyanobacteria had a higher compressive strength. Therefore, bioprinted LBM containing photosynthetically active, mineralizing microorganisms could be proved to be beneficial for designing environmentally friendly construction materials.

Chlorophylls as Natural Bioactive Compounds Existing in Food By-Products: A Critical Review.

Chlorophylls are a group of naturally occurring pigments that are responsible for the green color in plants. This pigment group could have numerous health benefits due to its high antioxidant activity, including anti-inflammatory, anti-cancer, and anti-obesity properties. Many food by-products contain a high level of chlorophyll content. These by-products are discarded and considered environmental pollutants if not used as a source of bioactive compounds. The recovery of chlorophylls from food by-products is an interesting approach for increasing the sustainability of food production. This paper provides insight into the properties of chlorophylls and the effect of different treatments on their stability, and then reviews the latest research on the extraction of chlorophylls from a sustainable perspective.