Pressurized extraction of unsaturated fatty acids and carotenoids from wet Chlorella vulgaris and Phaeodactylum tricornutum biomass using subcritical liquids

The objective of this study was to investigate the extraction of lipids, for example, mono‐ and polyunsaturated fatty acids (PUFA) as well as carotenoids, from wet microalgae biomass using pressurized subcritical extraction solvents, which meet the requirements of food and feed applications. To demonstrate the effect of the solvent and temperature on the lipid yield, we chose two microalgae species, viz. Chlorella vulgaris and Phaeodactylum tricornutum, differing in their biochemical composition fundamentally. In case of P. tricornutum, ethanol showed the highest fatty acid yield of 85.9% w/w. In addition to eicosapentaenoic acid (EPA), the ethanolic extracts contained exceptional amounts of fucoxanthin (up to 26.1 mg/g d. w.), which can be beneficial to protect unsaturated fatty acids from oxidation processes and in terms of human nutrition. For C. vulgaris, a fatty acid yield of 76.5% w/w was achieved from wet biomass using ethyl acetate at 150°C. In general, an increase in the extraction temperature up to 150°C was found to be important in terms of fatty acid yield when extracting wet microalgae biomass. The results suggest that it is possible to efficiently extract both fatty acids and carotenoids from wet microalgae by selecting suitable solvents and thus circumvent energy‐intensive drying of the biomass.     

Process optimization for extraction of carotenoids from shrimp waste with vegetable oils

Shrimp waste is an important source of natural carotenoid. Studies were carried out to determine the extraction yield of shrimp waste carotenoids in different vegetable oils. Highest yield was obtained by extraction using refined sunflower oil compared to groundnut oil, gingelly oil, mustard oil, soy oil, coconut oil and rice bran oil. The extraction yield of carotenoids in sunflower oil was significantly influenced by level of oil to waste (p < 0.05), time (p < 0.01) and temperature (p < 0.001) of heating waste with oil before centrifugation to separate pigmented oil. A regression equation was derived for carotenoid yield as a function of time of heating, temperature of heating and oil level to waste. The optimized conditions for extraction of shrimp waste carotenoids in sunflower oil were determined to be oil level to waste of 2, temperature of 70 degrees C and heating time of 150 min.     

Optimization and economic evaluation of ultrasound extraction of lutein from Chlorella vulgaris

The main carotenoid in Chlorella vulgaris is lutein. The ultrasound alone or together with enzymatic pretreatment for the extraction of lutein from C. vulgaris was optimized using response surface methodology (RSM) to improve the extraction process. The optimal ultrasound extraction condition was: ultrasound frequency, 35 kHz; ultrasound intensity, 56.58 W/cm²; extraction temperature, 37.7°C; extraction time, 5 h; and ratio of solvent to solid, 31 mL/g, where the lutein recovery was 3.16 ± 0.03 mg/g wet C. vulgaris. The optimal enzymatic pretreatment was: reaction time, 2 h; enzyme concentration, 1.23% (v/w); pH, 4.5, and temperature 50°C. The optimal ultrasound extraction with enzymatic pretreatment was: ultrasound frequency, 35 kHz; ultrasound intensity, 56.58 W/cm²; extraction temperature, 37.7°C; extraction time, 162 min; and ratio of solvent to solid, 35.6 mL/g wet C. vulgaris, where the extraction yield of lutein was 3.36 ± 0.10 mg/g wet C. vulgaris. This was much higher than for ultrasound treatment alone. The surface areas of microalga cells treated by ultrasound with/without enzymatic pretreatment increased significantly, which might contribute to the increase in lutein yield. There were no significant differences in structure, color, and antioxidant activity of lutein between the ultrasound and conventional methods. The highest cost of the crude and lutein was obtained by the ultrasound with enzymatic pretreatment due to the complex process and liquid waste in the enzymatic pretreatment process, but the ultrasound treatment alone was the lowest. Therefore, ultrasound extraction is the most economical method for the extraction of microalgal lutein.     

Effect of biomass pre-treatment and solvent extraction on beta-carotene and lycopene recovery from Blakeslea trispora cells

The production of carotenoids from Blakeslea trispora cells in a synthetic medium has been reported, with the main products being beta-carotene, lycopene, and gamma-carotene. The effect of biomass pretreatment and solvent extraction on their selective recovery is reported here. Eight solvents of class II and III of the International Conference of Harmonization: ethanol, methanol, acetone, 2-propanol, pentane, hexane, ethyl acetate, and ethyl ether, and HPLC analysis were used for the evaluation of their selectivities towards the three main carotenoids with regard to different biomass pre-treatment. The average C(max) values (maximum concentration of caronoids in a specific solvent) were estimated to 16 mg/L with the five out of eight solvents investigated, whereas methanol, pentane, and hexane gave lower values of 10, 11, and 9 mg/L, respectively. The highest carotenoid yield was obtained in the case of wet biomass, where 44-56% is recovered with one solvent and three extractions and the rest is recovered only after subsequent treatment with acetone; thus, four extractions of 2.5 h are needed. Two extractions of 54 min are enough to recover carotenoids from dehydrated biomass, with the disadvantage of a high degree of degradation. Our results showed that, for maximum carotenoid recovery, ethyl ether, 2-propanol, and ethanol could be successfully used with biomass without prior treatment, whereas fractions enriched in beta-carotene or lycopene can be obtained by extraction with the proper solvent, thus avoiding degradation due to time-consuming processes.     

Effect of Biomass Pre-Treatment and Solvent Extraction on β-Carotene and Lycopene Recovery from Blakeslea trispora Cells

Abstract

The production of carotenoids from Blakeslea trispora cells in a synthetic medium has been reported, with the main products being β-carotene, lycopene, and γ-carotene. The effect of biomass pretreatment and solvent extraction on their selective recovery is reported here. Eight solvents of class II and III of the International Conference of Harmonization: ethanol, methanol, acetone, 2-propanol, pentane, hexane, ethyl acetate, and ethyl ether, and HPLC analysis were used for the evaluation of their selectivities towards the three main carotenoids with regard to different biomass pre-treatment. The average C_max values (maximum concentration of caronoids in a specific solvent) were estimated to 16 mg/L with the five out of eight solvents investigated, whereas methanol, pentane, and hexane gave lower values of 10, 11, and 9 mg/L, respectively. The highest carotenoid yield was obtained in the case of wet biomass, where 44–56% is recovered with one solvent and three extractions and the rest is recovered only after subsequent treatment with acetone; thus, four extractions of 2.5 h are needed. Two extractions of 54 min are enough to recover carotenoids from dehydrated biomass, with the disadvantage of a high degree of degradation. Our results showed that, for maximum carotenoid recovery, ethyl ether, 2-propanol, and ethanol could be successfully used with biomass without prior treatment, whereas fractions enriched in β-carotene or lycopene can be obtained by extraction with the proper solvent, thus avoiding degradation due to time-consuming processes.     

Isolation of chlorophylls and carotenoids from freshwater algae using different extraction methods

Usually marine algae are an excellent source of pigments for different commercial sectors. Freshwater macroalgae can be exploited as a good source of biologically active compounds provided an appropriate extraction method is developed. The efficiency of four methods, like microwave‐assisted (MAE), ultrasound‐assisted extraction (UAE), supercritical fluid extraction (SFE) with ethanol as a co‐solvent, as well as conventional Soxhlet extraction were studied in the same conditions (time, solvent and temperature) for the recovery of chlorophylls and carotenoids from three freshwater green algae species: Cladophora glomerata, Cladophora rivularis and Ulva flexuosa. UV‐Vis spectrophotometry was used to determine chlorophyll a, chlorophyll b and total carotenoid content in obtained extracts. The results of this study showed that the advantages of novel extraction techniques (MAE and UAE) include higher yield and, in consequence, lower costs compared to traditional solvent extraction techniques. These methods were much more efficient in freshwater green algae pigment recovery than the classic Soxhlet extraction as well as SFE.     

硅藻金色奥杜藻色素的HPLC分析与超临界CO2萃取研究

以硅藻金色奥杜藻(Odontella aurita)为实验材料,利用高效液相色谱法分析了其色素组成与含量,采取超临界CO2萃取技术研究了从干藻粉内提取岩藻黄素的条件。结果表明,该藻主要含有岩藻黄素、硅甲藻黄素、β-胡萝卜素、硅藻黄素等类胡萝卜素以及叶绿素a和叶绿素c1,其中岩藻黄素为该藻含量最高的类胡萝卜素。色素的萃取率与压强、温度、夹带剂含量以及萃取时间呈正相关,夹带剂含量对萃取率影响最大,CO2流速的影响最小;与有机溶剂法相比,超临界CO2萃取岩藻黄素效率略低,而更利于岩藻黄素的选择性萃取及分离提纯;岩藻黄素的SFE-CO2适宜条件为压强400 bar、温度50℃、CO2流速0.2 L/min、夹带剂比例10%、萃取时间2～3 h。     

Antioxidant potential of microalgae in relation to their phenolic and carotenoid content

In the past decades, food scientists have been searching for natural alternatives to replace synthetic antioxidants. In order to evaluate the potential of microalgae as new source of safe antioxidants, 32 microalgal biomass samples were screened for their antioxidant capacity using three antioxidant assays, and both total phenolic content and carotenoid content were measured. Microalgae were extracted using a one-step extraction with ethanol/water, and alternatively, a three-step fractionation procedure using successively hexane, ethyl acetate, and water. Antioxidant activity of the extracts varied strongly between species and further depended on growth conditions and the solvent used for extraction. It was found that industrially cultivated samples of Tetraselmis suecica, Botryococcus braunii, Neochloris oleoabundans, Isochrysis sp., Chlorella vulgaris, and Phaeodactylum tricornutum possessed the highest antioxidant capacities in this study and thus could be a potential new source of natural antioxidants. The results from the different types of extracts clearly indicated that next to the well-studied carotenoids, phenolic compounds also contribute significantly to the antioxidant capacity of microalgae.     

Subcritical Water Hydrolysis of Microalgal Biomass for Protein and Pyrolytic Bio-oil Recovery

Microalgae are a promising source of protein and biofuels. This study involved the extraction of soluble proteins from raw microalgae using subcritical water hydrolysis followed by pyrolysis of the resulting spent microalgal biomass for bio-oil production. The extraction process produced solubilized protein in amounts up to 10 wt% of the dry biomass. The effects of hydrolysis temperature (150–220 °C), process time (90–180 min), and initial pH (2–12) on the chemical compositions and reactivity of the spent biomass as biofuel intermediates were investigated. It was found that when the temperature and time increased, the protein and carbohydrate fractions of the spent biomass were reduced, while their lipid fraction increased. A low initial pH led to lower protein content in the spent biomass. Compared with the raw microalgae, the spent biomass gave a higher yield of pyrolytic bio-oil that contained much less of the N-containing compounds and higher amounts of long-chain fatty acids (C₁₆) and C₁₄–C₂₀ long-chain hydrocarbons. In addition, enhanced energy recovery and a reduction in the energy consumption of the pyrolysis process were the other benefits acquired from the protein extraction. Therefore, subcritical water hydrolysis was considered to be an effective process to recover solubilized proteins, enhance the properties of the spent biomass, improve the energy balance of the subsequent pyrolysis process, and raise the quality of the bio-oil.     

Efficient and environmentally friendly method for carotenoid extraction from Paracoccus carotinifaciens utilizing naturally occurring Z-isomerization-accelerating catalysts

This study aimed to improve the efficiency of Paracoccus carotinifaciens-derived carotenoid (astaxanthin, adonirubin, adonixanthin) extraction using environmentally friendly Z-isomerization-accelerating catalysts. Adding naturally occurring catalysts such as isothiocyanates and polysulfides to the extraction solvent significantly improved the efficiency of carotenoid extraction, likely because of enhanced solubility of carotenoid Z-isomers compared with all-E-isomers. Indeed, addition of the catalysts markedly increased the content of carotenoid Z-isomers in the resulting extract. The use of a catalyst allyl isothiocyanate, which is abundantly included in Brassicaceae plant family, at high extraction temperature and long extraction time led to increased carotenoid recovery and Z-isomer content. These findings will enhance the efficiency of organic solvent-based extraction of carotenoids from carotenoid-rich sources. Numerous studies have reported that the Z-isomers of carotenoids exhibit greater bioavailability and antioxidant capacity than the all-E-isomers. Hence, the method proposed here utilizing Z-isomerization-accelerating catalysts could enhance both the extraction efficiency and beneficial health effects of carotenoids.     

Pigment Production by Cultured Florets of Chrysanthemum morifolium

Individual florets (4–5 mm long) of a purple cultivar(Fandango) of the horticultural chrysanthemum (Chrysanthemummorifolium Ramat) were taken from flower buds just prior toopening and cultured in a sterile liquid medium (containinginorganic salts and sucrose) at 15 °C under a 12-h day.For the first 14 days increase in wet weight was exponential.Anthocyanin appeared on the third day and was then synthesizedrapidly. Chlorophyll and carotenoid were present initially:carotenoid levels rose quickly while chlorophyll remained almostconstant. Highest pigment content and most growth were foundwhen the florets were grown on 3 per cent sucrose. However,the highest anthocyanin concentration was found with 4 per centsucrose, the highest carotenoid concentration with 0.6 per centsucrose. No anthocyanin was produced when the florets were grownat 6 or 30 °C; maximum yield was at 15 °C. Most carotenoidwas formed at 30 °C and most chlorophyll was found at 20–5°C. All florets from 1 to 7 mm long could be cultured. Theseresults are discussed in relation to flower colour and pigmentformation in vivo.     

In Situ Enzymatic Conversion of <Emphasis Type="Italic">Nannochloropsis oceanica</Emphasis> IMET1 Biomass into Fatty Acid Methyl Esters

Conventionally, production of methyl ester fuels from microalgae occurs through an energy-intensive two-step chemical extraction and transesterification process. To improve the energy efficiency, we performed in situ enzymatic conversion of whole algae biomass from an oleaginous heterokont microalga Nannochloropsis oceanica IMET1 with the immobilized lipase from Candida antarctica. The fatty acid methyl ester yield reached 107.7% for dry Nannochloropsis biomass at biomass to t-butanol to methanol weight ratio of 1:2:0.5 and a reaction time of 12 h at 25 °C, representing the first report of efficient whole algae biomass conversion into fatty acid methyl esters at room temperature. Different forms of algal biomass including wet Nannochloropsis biomass were tested. The maximum yield of wet biomass was 81.5%. Enzyme activity remained higher than 95% after 55 days of treatment (equal to 110 cycles of reaction) under the conditions optimized for dry algae biomass conversion. The low reaction temperature, high enzyme stability, and high yield from this study indicate in situ enzymatic conversion of dry algae biomass may potentially be used as an energy-efficient method for algal methyl ester fuel production while allowing co-product recovery.     

Production of Dunaliella salina biomass rich in 9-cis-beta-carotene and lutein in a closed tubular photobioreactor

Performance of Dunaliella salina cultures outdoors in a closed tubular photobioreactor has been assessed. Optimization of conditions involved verification of the effect of several determining factors on the yield of both biomass and carotenoids. Maximal biomass productivity (over 2g (dry weight) m(-2) d(-1) or 80 gm(-3) d(-1)) was achieved at 38 cm s(-1), flow rate; 2 x 10(9) cells l(-1), initial population density; 25 degrees C, temperature; semi-continuous regime, keeping a cell density interval between 2 x 10(9) and over 4 x 10(9) cells l(-1). Coverage of the tubular loop with a sunshade screen to avoid light-induced damage of cells was essential to maintain growth performance. The cellular beta-carotene level increased significantly during the light period, as also did that of lutein. The rise in the beta-carotene level could be accounted by the 9-cis-isomer, with all-trans-beta-carotene remaining steady during the light period. By sunset, the ratio between 9-cis- and all-trans-isomers of beta-carotene amounted to 1.5, with over 60% of total beta-carotene corresponding to the 9-cis-isomer. Removal of sunshade enhanced carotenoid accumulation by cells to reach up to 10% of dry biomass. Cultivation of Dunaliella in closed tubular photobioreactor, thus represents a suitable approach for the production of a high-quality microalgal biomass enriched in the valuable 9-cis-isomer of beta-carotene and lutein.     

Oil extraction from microalgae for biodiesel production

This study examines the performance of supercritical carbon dioxide (SCCO₂) extraction and hexane extraction of lipids from marine Chlorococcum sp. for lab-scale biodiesel production. Even though the strain of Chlorococcum sp. used in this study had a low maximum lipid yield (7.1 wt% to dry biomass), the extracted lipid displayed a suitable fatty acid profile for biodiesel [C18:1 (∼63 wt%), C16:0 (∼19 wt%), C18:2 (∼4 wt%), C16:1 (∼4 wt%), and C18:0 (∼3 wt%)]. For SCCO₂ extraction, decreasing temperature and increasing pressure resulted in increased lipid yields. The mass transfer coefficient (k) for lipid extraction under supercritical conditions was found to increase with fluid dielectric constant as well as fluid density. For hexane extraction, continuous operation with a Soxhlet apparatus and inclusion of isopropanol as a co-solvent enhanced lipid yields. Hexane extraction from either dried microalgal powder or wet microalgal paste obtained comparable lipid yields.     

Zero-waste biorefinery of oleaginous microalgae as promising sources of biofuels and biochemicals through direct transesterification and acid hydrolysis

Oleaginous microalgae are considered as promising sources of biofuels and biochemicals due to their high lipid content and other high-value components such as pigments, carbohydrate and protein. This study aimed to develop an efficient biorefinery process for utilizing all of the components in oleaginous microalgae. Acetone extraction was used to recover microalgal pigments prior to processes for the other products. Microalgal lipids were converted into biodiesel (fatty acid methyl ester, FAME) through a conventional two-step process of lipid extraction followed by transesterification, and alternatively a one-step direct transesterification. The comparable FAME yields from both methods indicate the effectiveness of direct transesterification. The operating parameters for direct transesterification were optimized through response surface methodology (RSM). The maximum FAME yield of 256 g/kg-biomass was achieved when using chloroform:methanol as co-solvents for extracting and reacting reagents at 1.35:1 volumetric ratio, 70 °C reaction temperature, and 120 min reaction time. The carbohydrate content in lipid-free microalgal biomass residues (LMBRs) was subsequently acid hydrolyzed into sugars under optimized conditions from RSM. The maximum sugar yield obtained was 44.8 g/kg-LMBRs and the protein residues were recovered after hydrolysis. This biorefinery process may contribute greatly to zero-waste industrialization of microalgae based biofuels and biochemicals.     

Ultrasonication assistance increases the efficiency of isoflavones extraction from kudzu (Pueraria lobata Ohwi) roots waste

This study assesses the use of ultrasonication to improve the extraction process of classical solvent extraction methods for extracting isoflavones from the kudzu roots waste. The kudzu roots waste was produced after squeezing fresh kudzu roots to make juice. The effects of extraction time, extraction temperature, ultrasonic power, and ethanol concentration in ethanol/water mixtures were investigated. The extraction yield was found to increase with extraction time and temperature. The application of ultrasonication-assisted extraction (UAE) increased the extraction yield of water/ethanol mixture (20:80) at 25°C 3 fold. A maximum amount (7.28 g) of isoflavone was obtained from 100 g of dried kudzu roots waste by UAE with water/ethanol mixture (20:80) for 6 h at 80°C. Combining the use of ultrasonication with conventional vacuum evaporation method also reduced the concentration time for extracts from 45 to 24 min.     

An efficient method for the sequential production of lipid and carotenoids from the Chlorella Growth Factor-extracted biomass of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>

Efficient methodology for simultaneous extraction of multiple bioactive compounds from microalgae still remains a major challenge. The present study provides a method for the sequential production of three major products: Chlorella Growth Factor (CGF, a nucleotide-peptide complex enriched with vitamins, minerals, and carbohydrates), lipid, and carotenoids from Chlorella vulgaris biomass in an economically feasible manner. After protein-rich CGF was extracted, the spent biomass was found to contain 12% lipid and 3% carotenoids when extracted individually, compared to that of the un-utilized (fresh) biomass (lipid, 14%; carotenoids, 4%). When extracted simultaneously using conventional methods, the yield of lipid from “CGF and carotenoids-extracted biomass,” and carotenoids from “CGF and lipid-extracted biomass” were significantly reduced (50%). However, simultaneous extraction using different solvent mixtures such as hexane:methanol:water and pentane:methanol:water mixture-augmented lipid yield by 38.5% and carotenoids by 14%, and additionally retained chlorophyll and its derivatives. Column chromatographic approach yielded sequential production of lipid (18%), lutein (9%) with better yields as well as without chlorophyll interference. Different geometric isomers of lutein all-E-(trans)-(3R,3′R,6′R)-β,ε-carotene-3,3′diol, 9Z(cis)-(3R,3′R,6′R)-β,ε-carotene-3,3′diol, and 13Z(cis)-(3R,3′R,6′R)-β,ε-carotene-3,3′diol were purified by HPLC and elucidated by CD, UV, NMR, FT-IR, and Mass spectra. In conclusion, the study provides an efficient and economically viable methodology for sequential production of lipid and lutein along with its geometrical isomers without chlorophyll influence and yield loss from the protein-rich CGF-extracted spent biomass of marine microalga, Chlorella vulgaris.     

Effect of light and temperature on the cyanobacterium <Emphasis Type="Italic">Arthronema africanum</Emphasis> - a prospective phycobiliprotein-producing strain

The effect of light intensity (50–300 μmol photons m⁻² s⁻¹) and temperature (15–50°C) on chlorophyll a, carotenoid and phycobiliprotein content in Arthronema africanum biomass was studied. Maximum growth rate was measured at 300 μmol photons m⁻² s⁻¹ and 36°C after 96 h of cultivation. The chlorophyll a content increased along with the increase in light intensity and temperature and reached 2.4% of dry weight at 150 μmol photons m⁻² s⁻¹ and 36°C, but it decreased at higher temperatures. The level of carotenoids did not change significantly under temperature changes at illumination of 50 and 100 μmol photons m⁻² s⁻¹. Carotenoids were about 1% of the dry weight at higher light intensities: 150 and 300 μmol photons m⁻² s⁻¹. Arthronema africanum contained C-phycocyanin and allophycocyanin but no phycoerythrin. The total phycobiliprotein content was extremely high, more than 30% of the dry algal biomass, thus the cyanobacterium could be deemed an alternative producer of C-phycocyanin. A highest total of phycobiliproteins was reached at light intensity of 150 μmol photons m⁻² s⁻¹ and temperature of 36°C, C-phycocyanin and allophycocyanin amounting, respectively, to 23% and 12% of the dry algal biomass. Extremely low (<15°C) and high temperatures (>47°C) decreased phycobiliprotein content regardless of light intensity.     

Carotenoid Formation by Staphylococcus aureus

The carotenoid pigments of Staphylococcus aureus U-71 were identified as phytoene; zeta-carotene; delta-carotene; phytofluenol; a phytofluenol-like carotenoid, rubixanthin; and three rubixanthin-like carotenoids after extraction, saponification, chromatographic separation, and determination of their absorption spectra. There was no evidence of carotenoid esters or glycoside ethers in the extract before saponification. During the aerobic growth cycle the total carotenoids increased from 45 to 1,000 nmoles per g (dry weight), with the greatest increases in the polar, hydroxylated carotenoids. During the anaerobic growth cycle, the total carotenoids increased from 20 nmoles per g (dry weight) to 80 nmoles per g (dry weight), and only traces of the polar carotenoids were formed. Light had no effect on carotenoid synthesis. About 0.14% of the mevalonate-2-(14)C added to the culture was incorporated into the carotenoids during each bacterial doubling. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The incorporation and turnover of (14)C indicated the carotenes were sequentially desaturated and hydroxylated to form the polar carotenoids.     

Aphid (Hemiptera: Aphididae) resistance in wheat near-isogenic lines

Plant and aphid biomass, photosynthetic pigment (chlorophylls a and b and carotenoids) concentrations, and chlorophyll a/b and chlorophyll/carotenoid ratios were quantified in aphid-infested 'Tugela' near-isogenic lines (Tugela, Tugela-Dn1, Tugela-Dn2, and Tugela-Dn5). The objectives were to quantify changes of photosynthetic pigments (chlorophylls a and b, and carotenoids) caused by aphid feeding and assess resistance of wheat isolines through aphid and plant biomass analysis. Biomass of bird cherry-oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae)-infested plants was lower than Russian wheat aphid, Diuraphis noxia (Mordvilko) (Hemiptera: Aphididae),- infested plants. When infested by D. noxia, all lines showed increased biomass over time, except Tugela where biomass decreased on day 12. No difference in plant biomass was detected among R. padi-infested and uninfested wheat lines. Biomass of D. noxia from Tugela (D. noxia-susceptible) was significantly higher than from plants with Diuraphis noxia-resistant Dn genes. Diuraphis noxia biomass from Tugela-Dn1 and Dn2 lines was not different from each other, but they were lower than from Tugela-Dn5. In contrast, there was no difference in R. padi biomass among wheat lines. Concentrations of chlorophylls a and b and carotenoids were significantly lower in D. noxia-infested plants compared with R. padi-infested and uninfested plants. When infested by D. noxia, chlorophyll a and b concentrations were not different among wheat lines on day 3, but they were lower in Tugela and Tugela-Dn1 than in Tugela-Dn2 and -Dn5 plants on days 6 and 12. However, no difference was detected in chlorophyll a/b or chlorophyll/carotenoid ratio among Tugela lines. The study demonstrated that Dn genes in the Tugela isolines conferred resistance to D. noxia but not to R. padi. Tugela-Dn1 was antibiotic, Tugela-Dn2 was tolerant and antibiotic, and Tugela-Dn5 was moderately antibiotic.