A Comparative Analysis of the ‘Green’ Techniques Applied for Polyphenols Extraction from Bioresources

From all the valuable biomass extractives, polyphenols are a widespread group of secondary metabolites found in all plants, representing the most desirable phytochemicals due to their potential to be used as additives in food industry, cosmetics, medicine, and others fields. At present, there is an increased interest to recover them from plant of spontaneous flora, cultivated plant, and wastes resulted in agricultural and food industry. That is why many efforts have been made to provide a highly sensitive, efficiently, and eco‐friendly methods, for the extraction of polyphenols, according to the green chemistry and sustainable development concepts. Many extraction procedures are known with advantages and disadvantages. From these reasons, the aim of this article is to provide a comparative analysis regarding technical and economical aspects related to the most innovative extraction techniques studied in the last time: microwave‐assisted extraction (MAE), supercritical fluid extraction (SFE), and ultrasound‐assisted extraction (UAE).     

Microwave assisted extraction of ursolic acid and oleanolic acid from Ocimum sanctum

Present study deals with the microwave assisted extraction (MAE) of ursolic acid (UA) and oleanolic acid (OA) from Ocimum sanctum leaves. UA and OA have been reported to possess significant medicinal properties. Various experimental parameters such as selection of solvent, solvent composition, irradiation time, microwave power, solid to solvent ratio, preleaching time and number of cycles were investigated to optimize the extraction process. Under optimum conditions of irradiation time (3 min), microwave power (272 W), solid to solvent ratio (1:30), preleaching time (10 min), maximum UA and OA has been extracted in one extraction cycle with ethanol: water (80:20) as a solvent. Maximum 86.76 and 89.64% of UA and OA was extracted under above mentioned optimized experimental conditions. MAE was also compared with the batch and ultrasound assisted extraction (UAE) method. As compared to batch and UAE, higher extraction yield of these important phytochemicals have been obtained through MAE in only 3 min.     

A comparative exploration of the phytochemical profiles and bio-pharmaceutical potential of Helichrysum stoechas subsp. barrelieri extracts obtained via five extraction techniques

We endeavoured to probe into and compare the possible effect(s) of different extraction techniques (accelerated solvent extraction (ASE), microwave-assisted extraction (MAE), ultrasonication-assisted extraction (UAE), maceration, and Soxhlet extraction (SE)) on the bioactivity (antioxidant and enzyme inhibitory activities) of the aerial parts of Helichrysum stoechas subsp. barrelieri (Ten.) Nyman. Total phenolic and flavonoid contents of the extracts obtained by different extraction methods followed the order of ASE > MAE > UAE > maceration > SE. Extract obtained by ASE was the most potent radical scavenger (219.92 and 313.12 mg Trolox equivalent [TE]/g, against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), respectively) and reducing agent (927.39 and 662.87 mg TE/g, for cupric reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP), respectively). Helichrysum stoechas extract obtained by UAE (18.67 mg ethylenediaminetetraacetic equivalent [EDTAE]/g) was the most active metal chelator and inhibitor of acetylcholinesterase (4.23 mg galantamine equivalent [GALAE]/g) and butyrylcholinesterase (6.05 mg GALAE/g) cholinesterase. Extract from maceration (183.32 mg kojic acid equivalent [KAE]/g) was most active against tyrosinase while ASE extract (1.66 mmol acarbose equivalent [ACAE]/g) effectively inhibited α-glucosidase. In conclusion, data amassed herein tend to advocate for the use of non-conventional extraction techniques, namely ASE and UAE, for the extraction of bioactive secondary metabolites from H. stoechas aerial parts.     

Analysis of fatty acids in A. szechenyianum Gay. by microwave‐assisted extraction and gas chromatography‐mass spectrometry

Introduction – Aconitum szechenyianum Gay. is a traditional Chinese medicinal herb with the detumescent and styptic effects and antitumor activity. There have been only a few researches on its chemical components, but no detailed report has appeared on its fatty acids. Objective – To develop a simple and effective method for the extraction of fatty acids from A. zechenyianum Gay. and then to investigate the fatty acid components. Methodology – Microwave‐assisted extraction (MAE) was optimized with response surface methodology, and the fatty acid compositions of extract were determined by GC–MS with previous derivatisation to fatty acid methyl esters (FAMEs). The results were compared with that obtained by classical Soxhlet extraction (SE). Results – Compared with SE, MAE showed significantly higher fatty acid yields, shorter extraction time, and lower energy and solvent consumption. The major fatty acids in A. szechenyianum Gay. are linoleic acid, palmitic acid, linolenic acid, oleic acid and stearic acid, and the unsaturated fatty acids occupy 66.4% of the total fatty acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Extraction of chlorophylls a and b from phytoplankton using standard extraction techniques *

SUMMARY. Pigments extracted in methanol, acetone and ethanol from three cultures of green algae and one blue-green alga revealed different extraction efficiencies depending on the species, the extraction solvent used and the extraction time. Chromatographic identification and quantitative measurements of chlorophylls a and b were made from six green algae. When extraction of pigments was incomplete, chlorophyll-b was extracted faster than chlorophyll-a. This effect was more pronounced for acetone extractions, whereas methanol extractions gave the stable ratios of chlorophyll b/a after about 6–10 h. When green algae are frequent, a 6–10 h methanol extraction, without any extra manipulations, is sufficient to ensure reliable ratios of chlorophyll b/a and extraction of the major proportion of the chlorophylls without risk of induced destruction of the chlorophylls.     

SOME FACTORS IN THE COMPETITION OR ANTAGONISM AMONG BACTERIA, ALGAE, AND AQUATIC WEEDS

Field observations of changes in the populations of aquatic weeds and phytoplankton have confirmed that aquatic weeds have antagonistic activity toward phytoplankton. Nutritional studies in the laboratory indicate that cultures of the aquatic weeds, Myriophyllum sp., Ceratophyllum sp., and duckweed (Lemma minor L.); liquid cultures of barley (Hordeum vulgare L., Dickson variety); and cultures of the filamentous green algae, Cladophora sp. and Pithophora oedogonium (Mont.) Withrock, will remain relatively free of epiphytes or competing phytoplankton if the cultures are nitrogen-limited. Field observations of Cladophora sp. have confirmed that the growth of epiphytes on the Cladophora is related to conditions of surplus available nitrogen compounds. It is proposed that this antagonistic activity may be due to a “nitrogen sink” effect in which the aquatic weeds or filamentous green algae prevent the growth of contaminating algae by competition for the limited nitrogen compounds available. However, the presence of bacteria-sized organisms which have selective toxicity to certain algae indicates that perhaps multiple factors exist. Discussed are the ecological implications of associations of certain algae with bacteria that have selective toxicities for other species of algae under certain environmental conditions such as nitrogen-limited growth.     

Application of microwave‐assisted extraction coupled with high‐speed counter‐current chromatography for separation and purification of Dehydrocavidine from Corydalis saxicola Bunting

Introduction – Dehydrocavidine is a major component of Corydalis saxicola Bunting with sedative, analgesic, anticonvulsive and antibacterial activities. Conventional methods have disadvantages in extracting, separating and purifying dehydrocavidine from C. saxicola. Hence, an efficient method should be established. Objective – To develop a suitable preparative method in order to isolate dehydrocavidine from a complex C. saxicola extract by preparative HSCCC. Methodology – The methanol extract of C. saxicola was prepared by optimised microwave‐assisted extraction (MAE). The analytical HSCCC was used for the exploration of suitable solvent systems and the preparative HSCCC was used for larger scale separation and purification. Dehydrocavidine was analysed by high‐performance liquid chromatography (HPLC) and further identified by ESI‐MS and 1H NMR. Results – The optimised MAE experimental conditions were as follows: extraction temperature, 60°C; ratio of liquid to solid, 20; extraction time, 15 min; and microwave power, 700 W. In less than 4 h, 42.1 mg of dehydrocavidine (98.9% purity) was obtained from 900 mg crude extract in a one‐step separation, using a two‐phase solvent system composed of chloroform–methanol–0.3 m hydrochloric acid (4 : 0.5 : 2, v/v/v). Conclusion – Microwave‐assisted extraction coupled with high‐speed counter‐current chromatography is a powerful tool for extraction, separation and purification of dehydrocavidine from C. saxicola. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of response surface methodology to optimise ultrasonic-assisted extraction of four chromones in Radix Saposhnikoviae

Introduction – Radix Saposhnikoviae is one of the most famous Chinese herbal medicines with many pharmacological activities towards inflammatory symptoms and antioxidation. Chromones are considered as one of the effective components. It is important to find a reasonable method to extract the chromones in S. divaricata. Objective – To develop an ultrasonic‐assisted extraction (UAE) to extract chromones in Radix Saposhnikoviae and to optimise extraction conditions. Methodology – Four chromones (prim‐O‐glucosylcimifugin, cimifugin, 5‐O‐methylvisammioside and sec‐O‐glucosylhamaudol) were extracted by the UAE method combined with response surface methodology (RSM). Box–Behnken design (BBD) was applied to evaluate the effects of three independent variables (ethanol concentration, extraction time and extraction temperature) on the chromones yield of Radix Saposhnikoviae. Results – Correlation analysis of the mathematical‐regression model indicated that a quadratic polynomial model could be employed to optimise the extraction of chromones by UAE method. The optimal conditions to obtain the highest chromones yield of Radix Saposhnikoviae were a solvent of 75% ethanol, an extraction time of 48 min and an extraction temperature of 67°C. Conclusion – Under these optimal conditions, the experimental values agreed closely with the predicted values. The analysis of variance indicated a high goodness of model fit and the success of RSM method for optimising chromones extraction in Radix Saposhnikoviae. Copyright © 2011 John Wiley & Sons, Ltd.     

Activated chemical defenses suppress herbivory on freshwater red algae

The rapid life cycles of freshwater algae are hypothesized to suppress selection for chemical defenses against herbivores, but this notion remains untested. Investigations of chemical defenses are rare for freshwater macrophytes and absent for freshwater red algae. We used crayfish to assess the palatability of five freshwater red algae relative to a palatable green alga and a chemically defended aquatic moss. We then assessed the roles of structural, nutritional, and chemical traits in reducing palatability. Both native and non-native crayfish preferred the green alga Cladophora glomerata to four of the five red algae. Batrachospermum helminthosum, Kumanoa holtonii, and Tuomeya americana employed activated chemical defenses that suppressed feeding by 30–60 % following damage to algal tissues. Paralemanea annulata was defended by its cartilaginous structure, while Boldia erythrosiphon was palatable. Activated defenses are thought to reduce ecological costs by expressing potent defenses only when actually needed; thus, activation might be favored in freshwater red algae whose short-lived gametophytes must grow and reproduce rapidly over a brief growing season. The frequency of activated chemical defenses found here (three of five species) is 3–20× higher than for surveys of marine algae or aquatic vascular plants. If typical for freshwater red algae, this suggests that (1) their chemical defenses may go undetected if chemical activation is not considered and (2) herbivory has been an important selective force in the evolution of freshwater Rhodophyta. Investigations of defenses in freshwater rhodophytes contribute to among-system comparisons and provide insights into the generality of plant–herbivore interactions and their evolution.     

Indolacetamid—ein Intermediat der Indolessigsäurebildung aus Indolacetonitril bei der Alge Furcellaria

Summary The red algae Furcellaria fastigiata, Nemalion multifidum, and the green alga Cladophora rupestris were able to hydrolyse IAN to IAA. The conversion of IAN to IAA by Furcellaria and probably by Cladophora included indoleacetamide as an intermediate.When the brown algae Fucus vesiculosus, Pylaiella litoralis, and Halidrys siliquosa were incubated with IAN, the only products formed were indolecarboxaldehyde and indolecarboxylic acid.     

Microwave‐assisted extraction of total bioactive saponin fraction from Gymnema sylvestre with reference to gymnemagenin: a potential biomarker

Objective – To develop a fast and ecofriendly microwave assisted extraction (MAE) technique for the effective and exhaustive extraction of gymnemagenin as an indicative biomarker for the quality control of Gymnema sylvestre. Methodology – Several extraction parameters such as microwave power, extraction time, solvent composition, pre‐leaching time, loading ratio and extraction cycle were studied for the determination of the optimum extraction condition. Scanning electron micrographs were obtained to elucidate the mechanism of extraction Results – The final optimum extraction conditions as obtained from the study were: 40% microwave power, 6 min irradiation time, 85% v/v methanol as the extraction solvent, 15 min pre‐leaching time and 25 : 1 (mL/g) as the solvent‐to‐material loading ratio. The proposed extraction technique produced a maximum yield of 4.3% w/w gymnemagenin in 6 min which was 1.3, 2.5 and 1.95 times more efficient than 6 h of heat reflux, 24 h of maceration and stirring extraction, respectively. A synergistic heat and mass transfer theory was also proposed to support the extraction mechanism Conclusion – Comparison with conventional extraction methods revealed that MAE could save considerable amounts of time and energy, whilst the reduction of volume of organic solvent consumed provides an ecofriendly feature. Copyright © 2009 John Wiley & Sons, Ltd.     

Extraction of astaxanthin from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier

Conventional solvent extraction methods cannot attain high‐quality antioxidant extracts from microalgae and also require solvent recovery and posttreatment. In this study, we utilized environmental friendly supercritical carbon dioxide fluid extraction (SFE‐CO₂) techniques to obtain pigment (i.e. astaxanthin) from Haematococcus pluvialis. The effects of key operating parameters on the extraction efficiency of astaxanthin were investigated, giving an optimal condition of H. pluvialis weight, 6.5 g; CO₂‐flow rate, 6.0 NL/min; extraction time, 20 min; extraction pressure, 4500 psi; volume of ethanol modifier added, 9.23 mL/g; extraction temperature, 50°C; modifier composition, 99.5%. Under these optimum conditions, the astaxanthin yield was 73.9% (10.92 mg/g dry H. pluvialis powder) after eight cycle of extraction cycles. The saponification index (C_S/C₀, representing the ratio of astaxanthin concentration after and before the saponification procedures) of the extract could be increased from 1 to 12.78 by saponification with 3.5 M NaOH.     

Optimisation of a microwave-assisted method for extracting withaferin A from Withania somnifera Dunal. using central composite design

Introduction – Recently, there have been growing attention on the modification and optimisation of new extraction and quantification methods, caused by the lack of environmentally friendly methodologies for the extraction of phytochemicals from complex matrices. In the case of pharmaceutical compounds, not only the extraction procedure but also the analysis method should be efficient, precise, fast and easy. Objectives – The essential pharmaceutical characteristics and trace concentration of withanolides led us to modify and optimise the previously reported extraction and quantification procedure for withaferin A (WA) as a candidate for withanolides. Matrial and methods – The WA from the air‐dried aerial part of Withania somnifera Dunal. was extracted using a microwave‐assisted extraction (MAE) technique. Four variables affecting the extraction procedure were optimised using the central composite design approach. The method of high‐performance thin‐layer chromatography assay was validated and applied for the quantification of each experiment. Results – The optimum values of factors were: extraction time (150 s), extraction temperature (68°C) and 17 mL of methanol : water in the ratio 25 : 75 as extracting solvent. The solvent system consisted of ethyl acetate : toluene : formic acid : 2‐propanol (7.0 : 2.0 : 0.5 : 0.5, v/v/v/v), and densitometric scanning at 220 nm was applied for the analysis. The dynamic linear range, LOD, LOQ and recovery with the inter‐day, and intra‐day RSDs of the developed method indicated the validity of the method. Conclusion – A pressurised MAE method for extracting WA from the plant's aerial part was optimised using factorial‐based design. The net effect of time, temperature, solvent volume and its ratio suggests that the yield of WA increases until each factor reaches its optimum value, and decreases with further increase in temperature or solvent ratio. Copyright © 2010 John Wiley & Sons, Ltd.     

Heavy metal uptake capacities by the common freshwater green alga Cladophora fracta

Macroalgae have received much attention for heavy metal removal in treatment of domestic wastewater. In this report, the uptake capacity of a common freshwater green alga, Cladophora fracta, for heavy metal ions (copper, zinc, cadmium, and mercury) was evaluated. The equilibrium adsorption capacities were 2.388?mg Cu²⁺, 1.623?mg Zn²⁺, 0.240?mg Cd²⁺, and 0.228?mg Hg²⁺ per gram of living algae at 18°C and pH?5.0. The removal efficiency for Cu²⁺, Zn²⁺, Cd²⁺, and Hg²⁺ were 99, 85, 97, and 98%, respectively. Greater removal efficiency was achieved when the concentrations of metal ions were at very low level. The results indicated that living algae are suitable for removal and recovery of heavy metal ions from aqueous solutions and can be a potential tool to treat industrial wastewater.     

Molecular phylogeny of the Cladophoraceae (Cladophorales,Ulvophyceae), with the resurrection of Acrocladus Nägeli and Willeella Børgesen,and the description of Lurbica gen. nov. and Pseudorhizoclonium gen. nov.

The taxonomy of the Cladophoraceae, a large family of filamentous green algae, has been problematic for a long time due to morphological simplicity, parallel evolution, phenotypic plasticity, and unknown distribution ranges. Partial large subunit (LSU) rDNA sequences were generated for 362 isolates, and the analyses of a concatenated dataset consisting of unique LSU and small subunit (SSU) rDNA sequences of 95 specimens greatly clarified the phylogeny of the Cladophoraceae. The phylogenetic reconstructions showed that the three currently accepted genera Chaetomorpha, Cladophora, and Rhizoclonium are polyphyletic. The backbone of the phylogeny is robust and the relationships of the main lineages were inferred with high support, only the phylogenetic position of both Chaetomorpha melagonium and Cladophora rupestris could not be inferred unambiguously. There have been at least three independent switches between branched and unbranched morphologies within the Cladophoraceae. Freshwater environments have been colonized twice independently, namely by the freshwater Cladophora species as well as by several lineages of the Rhizoclonium riparium clade. In an effort to establish monophyletic genera, the genera Acrocladus and Willeella are resurrected and two new genera are described: Pseudorhizoclonium and Lurbica.     

PHYLOGENETIC RELATIONSHIPS WITHIN CLADOPHORALES (ULVOPHYCEAE,CHLOROPHYTA) INFERRED FROM 18S rRNA GENE SEQUENCES,WITH SPECIAL REFERENCE TO AEGAGROPILA LINNAEI 1

The phylogenetic position of a freshwater green alga, Aegagropila linnaei (Cladophorales, Ulvophyceae), was investigated using nuclear 18S rRNA gene sequences. This alga has usually been called Cladophora aegagropila (L.) Rabenhorst or Cladophora sauteri (Nees ex Kütz.) Kütz. Based on morphology, it was formerly classified into the section Aegagropila or into the subgenus Aegagropila, together with several marine species of the genus Cladophora. This classification is not supported by the present phylogenetic analyses in which two very distinct Cladophorales clades are recognized. Aegagropila linnaei groups together in a well‐supported clade with Cladophora sp., Pithophora sp., Chaetomorpha okamurae, Arnoldiella conchophila, Wittrockiella lyallii, and Cladophora conchopheria. Aegagropila linnaei and its closely related species share some ultrastructural and biochemical characteristics, like pyrenoid structure, carotenoid composition, and cell wall composition. Freshwater species, included in the analysis, were located in two distantly related lineages, indicating that adaptation from a marine to a freshwater habitat has happened at least twice independently in the Cladophorales.     

Simultaneous Measurement of Fluoroquinolones in Eggs by a Combination of Supercritical Fluid Extraction and High Pressure Liquid Chromatography

Simultaneous detection of the fluoroquinolone antibiotics ciprofloxacin, enrofloxacin, ofloxacin, and norfloxacin in eggs by a combination of supercritical fluid extraction (SFE) and high pressure liquid chromatography (HPLC) was studied. Lipid matrices that have been considered to result in poor extraction and isolation of fluoroquinolones in eggs were removed first by SFE with supercritical CO₂ alone, and then the fluoroquinolones were extracted by SFE with supercritical CO₂ containing 20% (v/v) methanol for HPLC analysis. A time-course study of the extraction of lipid matrices of eggs suggested that the SFE method successfully removed the matrices within 20 min. When the fluoroquinolones added to control eggs were extracted by SFE, the extraction efficiency was similar to that by the solvent extraction method, giving the recovery percentages from 83 to 96% in a 40 min-extraction time. The fluoroquinolones extracted from eggs by SFE were analyzed simultaneously by HPLC equipped with a fluorescence detector with detection sensitivity at about 10 ppb for the detection limit. The standard calibration profiles of fluoroquinolones showed linear responses to HPLC, showing more than 0.995 for the mean r ² value. This is the first report of the simultaneous measurement of fluoroquinolones in eggs by a combination of SFE and HPLC. Using the SFE method allowed us to avoid extensive sample preparation such as solvent extraction and chromatographic cleanup that are basically required in extraction of fluoroquinolones.     

Oil production by six microalgae: impact of flocculants and drying on oil recovery from the biomass

Oil production in batch photoautotrophic cultures of the following microalgae is reported: the freshwater microalgae Chlorella vulgaris, Choricystis minor, and Neochloris sp.; the marine microalgae Nannochloropsis salina and Cylindrotheca fusiformis; and C. vulgaris grown in a full-strength seawater medium. In all cases, the solvent extraction of lipids from freeze-dried biomass is compared with extraction from the fresh biomass paste. For all algae, the oils could be extracted equally effectively from freeze-dried samples and the paste samples (67–88 % moisture by weight). Moisture content determinations of the biomass using the freeze-drying method and the high-temperature oven drying were found to be equivalent for all algae. The biomass recovered by flocculation with metal salts (aluminum sulfate, ferric chloride) followed by centrifugation had a certain amount of the flocculant irreversibly bound to it. Washing failed to remove the adsorbed flocculants. For all algae, the adsorbed flocculants did not interfere with oil recovery by solvent extraction. The solvent system of chloroform–methanol–water proved highly effective for quantitative extraction of the lipids from all algae.     

Microwave-assisted extraction of glycyrrhizic acid from licorice root

In the present study, a microwave-assisted extraction (MAE) technique has been developed for the extraction of glycyrrhizic acid (GA) from licorice root. Various experimental conditions, such as extraction time, different ethanol and ammonia concentration, liquid/solid ratios, pre-leaching time before MAE and material size for the MAE procedure were investigated to optimize the efficiency of the extraction. Under appropriate MAE conditions, such as extraction times of 4-5min, ethanol concentrations of 50-60% (v/v), ammonia concentrations of 1-2% (v/v) and liquid/solid ratios of 10:1(ml/g), the recovery of GA from licorice root with MAE was equivalent with conventional extraction methods. Those methods include extraction at room temperature (ERT), the traditional Soxhlet extraction, heat reflux extraction and ultrasonic extraction. Due to the considerable savings in time and solvent, MAE was more effective than the conventional methods. This novel method is suitable for fast extraction of GA from licorice root.     

Photosynthetic responses to temperature in tropical lotic macroalgae

A comparative analysis of the photosynthetic responses to temperature (10–30°C) was carried out under short‐term laboratory conditions by chlorophyll fluorescence and oxygen (0₂) evolution. Ten lotic macroalgal species from southeastern Brazil (20°11–20°48′S, 49°18–49°41′W) were tested, including Bacillariophyta, Chlorophyta, Cyanophyta, Rhodophyta and Xanthophyta. Temperature had significant effects on electron transport rate (ETR) only for three species (Terpsinoe musica, Bacillariophyta; Cladophora glomerata, Chlorophyta; and C. coeruleus, Rhodophyta), with highest values at 25–30°C, whereas the remaining species had no significant responses. It also had similar effects on non‐photochemical quenching and ETR. Differences in net photosynthesisldark respiration ratios at distinct temperatures were found, with an increasing trend of respiration with higher temperatures. This implies in a decreasing balance between net primary production and temperature, representing more critical conditions toward higher temperatures for most species. In contrast, high net photosynthesis and photosynthesisldark respiration ratios at high and wide ranges of temperature were found in three species of green algae, suggesting that these algae can be important primary producers in lotic ecosystems, particularly in tropical regions. Optimal photosynthetic rates were observed under similar environmental temperatures for five species (two rhodophytes, two chlorophytes and one diatom) considering both techniques, suggesting acclimation to their respective ambient temperatures. C. coeruleus was the only species with peaks of ETR and 0₂ evolution under similar field‐measured temperatures. All species kept values of ETR and net photosynthesis close to the optimum under a broad range of temperatures. Increased non‐photochemical quenching, as a measure of thermal dissipation of excess energy, toward higher temperatures was observed in some species, as well as positive correlation of non‐photochemical quenching with ETR, and were interpreted as two mechanisms of adaptation of the photosynthetic apparatus to temperature changes. Different optimal temperatures were found for individual species by each technique, generally under lower temperatures by 0₂ evolution, indicating dependence on distinct factors: increases in temperature generally induced higher ETR due to increased enzymatic activity, whereas increments of enzymatic activity were compensated by increased respiration and photorespiration leading to decreases in net photosynthesis.