Metagenomics approach to identify lignocellulose-degrading enzymes in the gut microbiota of the Chinese bamboo rat cecum

BACKGROUND: Lignocellulose is considered a renewable organic material, but the industrial production of biofuel from lignocellulose is challenging because of the lack of highly active hydrolytic enzymes. The guts of herbivores contain many symbiotic microorganisms that have evolved to hydrolyze plant lignocellulose. Chinese bamboo rats mainly consume high-fiber foods, indicating that some members of the intestinal tract microbiota digest lignocellulose, providing these rats with the energy required for growth. RESULTS: Here, we used metagenomics to analyze the diversity and functions of the gut microbiota in Chinese bamboo rats. We identified abundant populations of lignocellulose-degrading bacteria, whose main functions involved carbohydrate, amino acid, and nucleic acid metabolism. We also found 587 carbohydrate-active enzyme genes belonging to different families, including 7 carbohydrate esterase families and 21 glycoside hydrolase families. The glycoside hydrolase 3, glycoside hydrolase 1, glycoside hydrolase 43, carbohydrate esterase 4, carbohydrate esterase 1, and carbohydrate esterase 3 families demonstrated outstanding performance. CONCLUSIONS: The microbes and enzymes identified in our study expand the existing arsenal of proficient degraders and enzymes for lignocellulosic biofuel production. This study also describes a powerful approach for targeting gut microbes and enzymes in numerous industries.

Progress in detoxification of inhibitors generated during lignocellulose pretreatment / 生物工程学报

Lignocellulose can be hydrolyzed by cellulase into fermentable sugars to produce hydrogen, ethanol, butanol and other biofuels with added value. Pretreatment is a critical step in biomass conversion, but also generates inhibitors with negative impacts on subsequent enzymatic hydrolysis and fermentation. Hence, pretreatment and detoxification methods are the basis of efficient biomass conversion. Commonly used pretreatment methods of lignocellulose are chemical and physic-chemical processes. Here, we introduce different inhibitors and their inhibitory mechanisms, and summarize various detoxification methods. Moreover, we propose research directions for detoxification of inhibitors generated during lignocellulose pretreatment.

Progress in the production of lignocellulolytic enzyme systems using Penicillium species / 生物工程学报

The efficient production of lignocellulolytic enzyme systems is an important support for large-scale biorefinery of plant biomass. On-site production of lignocellulolytic enzymes could increase the economic benefits of the process by lowering the cost of enzyme usage. Penicillium species are commonly found lignocellulose-degrading fungi in nature, and have been used for industrial production of cellulase preparations due to their abilities to secrete complete and well-balanced lignocellulolytic enzyme systems. Here, we introduce the reported Penicillium species for cellulase production, summarize the characteristics of their enzymes, and describe the strategies of strain engineering for improving the production and performance of lignocellulolytic enzymes. We also review the progress in fermentation process optimization regarding the on-site production of lignocellulolytic enzymes using Penicillium species, and suggest prospect of future work from the perspective of building a "sugar platform" for the biorefinery of lignocellulosic biomass.

Biodegradation of furan aldehydes in lignocellulose hydrolysates / 生物工程学报

Lignocellulose is the most abundant renewable organic carbon resource on earth. However, due to its complex structure, it must undergo a series of pretreatment processes before it can be efficiently utilized by microorganisms. The pretreatment process inevitably generates typical inhibitors such as furan aldehydes that seriously hinder the growth of microorganisms and the subsequent fermentation process. It is an important research field for bio-refining to recognize and clarify the furan aldehydes metabolic pathway of microorganisms and further develop microbial strains with strong tolerance and transformation ability towards these inhibitors. This article reviews the sources of furan aldehyde inhibitors, the inhibition mechanism of furan aldehydes on microorganisms, the furan aldehydes degradation pathways in microorganisms, and particularly focuses on the research progress of using biotechnological strategies to degrade furan aldehyde inhibitors. The main technical methods include traditional adaptive evolution engineering and metabolic engineering, and the emerging microbial co-cultivation systems as well as functional materials assisted microorganisms to remove furan aldehydes.

Single cell oil production by a novel yeast Trichosporon mycotoxinivorans for complete and ecofriendly valorization of paddy straw

Background: Oleaginous yeasts can be grown on different carbon sources, including lignocellulosic hydrolysate containing a mixture of glucose and xylose. However, not all yeast strains can utilize both the sugars for lipogenesis. Therefore, in this study, efforts were made to isolate dual sugar-utilizing oleaginous yeasts from different sources. Results: A total of eleven isolates were obtained, which were screened for their ability to utilize various carbohydrates for lipogenesis. One promising yeast isolate Trichosporon mycotoxinivorans S2 was selected based on its capability to use a mixture of glucose and xylose and produce 44.86 ± 4.03% lipids, as well as its tolerance to fermentation inhibitors. In order to identify an inexpensive source of sugars, nondetoxified paddy straw hydrolysate (saccharified with cellulase), supplemented with 0.05% yeast extract, 0.18% peptone, and 0.04% MgSO4 was used for growth of the yeast, resulting in a yield of 5.17 g L−1 lipids with conversion productivity of 0.06 g L−1 h−1 . Optimization of the levels of yeast extract, peptone, and MgSO4 for maximizing lipid production using Box­Behnken design led to an increase in lipid yield by 41.59%. FAME analysis of single cell oil revealed oleic acid (30.84%), palmitic acid (18.28%), and stearic acid (17.64%) as the major fatty acids. Conclusion: The fatty acid profile illustrates the potential of T. mycotoxinivorans S2 to produce single cell oil as a feedstock for biodiesel. Therefore, the present study also indicated the potential of selected yeast to develop a zero-waste process for the complete valorization of paddy straw hydrolysate without detoxification

Advances in the synthesis of biobutanol by consolidated bioprocessing from lignocellulose / 生物工程学报

Consolidated bioprocessing (CBP) is a multi-step process in a bioreactor, which completes hydrolase production, enzymatic hydrolysis, and microbial fermentation. It is considered to be the most promising process for the production of second-generation biofuels because of its simple steps and low cost. Due to the complexity of lignocellulose degradation and the butanol synthesis pathway, few wild microorganisms can directly utilize lignocellulose to synthesize butanol. With the development of synthetic biology, single-bacterium directly synthesizes butanol using lignocellulose by introducing a butanol synthesis pathway in the cellulolytic Clostridium. However, there are still some problems such as heavy metabolic load of single bacterium and low butanol yield. Co-culture can relieve the metabolic burden of single bacterium through the division of labor in different strains and can further improve the efficiency of butanol synthesis. This review analyzes the recent research progress in the synthesis of biobutanol using lignocellulose by consolidated bioprocessing from both the single-bacterium strategy and co-culture strategy, to provide a reference for the research of butanol and other biofuels.

Improvement of ethanol production from birch and spruce pretreated with 1-H-3-methylmorpholinium chloride

Background: Pretreatment is the critically important step for the production of ethanol from lignocelluloses. In this study, hardwood birch (Betula pendula) and softwood spruce (Norway spruce) woods were pretreated with a newly synthesized morpholinium ionic liquid, 1-H-3-methylmorpholinium chloride ([HMMorph][Cl]), followed by enzymatic hydrolysis and fermentation to ethanol. Results: [HMMorph][Cl] was synthesized using inexpensive raw materials, i.e., hydrochloric acid and N-methyl morpholine, following a simple process. The influence of pretreatment time (2, 3, 5, and 8 h) and temperature (120 and 140°C) in terms of hydrolysis efficiency was investigated. Glucose yields from enzymatic hydrolysis were improved from 13.7% to 45.7% and 12.9% to 51.8% after pretreatment of birch and spruce woods, respectively, under optimum pretreatment conditions (i.e., at 140°C for 3 h) as compared to those from pristine woods. Moreover, the yields of ethanol production from birch and spruce were increased to 34.8% and 44.2%, respectively, while the yields were negligible for untreated woods. Conclusions: This study demonstrated the ability of [HMMorph][Cl] as an inexpensive agent to pretreat both softwood and hardwood.

Multi-enzyme complex of white rot fungi in saccharification of lignocellulosic material

ABSTRACT The multi-enzyme complex (crude extract) of white rot fungi Pleurotus ostreatus, Pleurotus eryngii, Trametes versicolor, Pycnosporus sanguineus and Phanerochaete chrysosporium were characterized, evaluated in the hydrolysis of pretreated pulps of sorghum straw and compared efficiency with commercial enzyme. Most fungi complexes had better hydrolysis rates compared with purified commercial enzyme.

Evaluation of biogas and syngas as energy vectors for heat and power generation using lignocellulosic biomass as raw material

The use of nonrenewable energy sources to provide the worldwide energy needs has caused different problems such as global warming, water pollution, and smog production. In this sense, lignocellulosic biomass has been postulated as a renewable energy source able to produce energy carriers that can cover this energy demand. Biogas and syngas are two energy vectors that have been suggested to generate heat and power through their use in cogeneration systems. Therefore, the aim of this review is to develop a comparison between these energy vectors considering their main features based on literature reports. In addition, a techno-economic and energy assessment of the heat and power generation using these vectors as energy sources is performed. If lignocellulosic biomass is used as raw material, biogas is more commonly used for cogeneration purposes than syngas. However, syngas from biomass gasification has a great potential to be employed as a chemical platform in the production of value-added products. Moreover, the investment costs to generate heat and power from lignocellulosic materials using the anaerobic digestion technology are higher than those using the gasification technology. As a conclusion, it was evidenced that upgraded biogas has a higher potential to produce heat and power than syngas. Nevertheless, the implementation of both energy vectors into the energy market is important to cover the increasing worldwide energy demand.

Production of thermostable ß-glucosidase and CMCase by Penicillium sp: LMI01 isolated from the Amazon region

Background: Cellulolytic enzymes of microbial origin have great industrial importance because of their wide application in various industrial sectors. Fungi are considered the most efficient producers of these enzymes. Bioprospecting survey to identify fungal sources of biomass-hydrolyzing enzymes from a high-diversity environment is an important approach to discover interesting strains for bioprocess uses. In this study, we evaluated the production of endoglucanase (CMCase) and ß-glucosidase, enzymes from the lignocellulolytic complex, produced by a native fungus. Penicillium sp. LMI01 was isolated from decaying plant material in the Amazon region, and its performance was compared with that of the standard isolate Trichoderma reesei QM9414 under submerged fermentation conditions. Results: The effectiveness of LMI01 was similar to that of QM9414 in volumetric enzyme activity (U/mL); however, the specific enzyme activity (U/mg) of the former was higher, corresponding to 24.170 U/mg of CMCase and 1.345 U/mg of ß-glucosidase. The enzymes produced by LMI01 had the following physicochemical properties: CMCase activity was optimal at pH 4.2 and the ß-glucosidase activity was optimal at pH 6.0. Both CMCase and ß-glucosidase had an optimum temperature at 60°C and were thermostable between 50 and 60°C. The electrophoretic profile of the proteins secreted by LMI01 indicated that this isolate produced at least two enzymes with CMCase activity, with approximate molecular masses of 50 and 35 kDa, and ß-glucosidases with molecular masses between 70 and 100 kDa. Conclusions: The effectiveness and characteristics of these enzymes indicate that LMI01 can be an alternative for the hydrolysis of lignocellulosic materials and should be tested in commercial formulations.

Isolation of fungi from dung of wild herbivores for application in bioethanol production

ABSTRACT Producing biofuels such as ethanol from non-food plant material has the potential to meet transportation fuel requirements in many African countries without impacting directly on food security. The current shortcomings in biomass processing are inefficient fermentation of plant sugars, such as xylose, especially at high temperatures, lack of fermenting microbes that are able to resist inhibitors associated with pre-treated plant material and lack of effective lignocellulolytic enzymes for complete hydrolysis of plant polysaccharides. Due to the presence of residual partially degraded lignocellulose in the gut, the dung of herbivores can be considered as a natural source of pre-treated lignocellulose. A total of 101 fungi were isolated (36 yeast and 65 mould isolates). Six yeast isolates produced ethanol during growth on xylose while three were able to grow at 42 °C. This is a desirable growth temperature as it is closer to that which is used during the cellulose hydrolysis process. From the yeast isolates, six isolates were able to tolerate 2 g/L acetic acid and one tolerated 2 g/L furfural in the growth media. These inhibitors are normally generated during the pre-treatment step. When grown on pre-treated thatch grass, Aspergillus species were dominant in secretion of endo-glucanase, xylanase and mannanase.

Cellulase activity and dissolved organic carbon release from lignocellulose macrophyte-derived in four trophic conditions

Abstract Considering the importance of lignocellulose macrophyte-derived for the energy flux in aquatic ecosystems and the nutrient concentrations as a function of force which influences the decomposition process, this study aims to relate the enzymatic activity and lignocellulose hydrolysis in different trophic statuses. Water samples and two macrophyte species were collected from the littoral zone of a subtropical Brazilian Reservoir. A lignocellulosic matrix was obtained using aqueous extraction of dried plant material (≈40 °C). Incubations for decomposition of the lignocellulosic matrix were prepared using lignocelluloses, inoculums and filtered water simulating different trophic statuses with the same N:P ratio. The particulate organic carbon and dissolved organic carbon (POC and DOC, respectively) were quantified, the cellulase enzymatic activity was measured by releasing reducing sugars and immobilized carbon was analyzed by filtration. During the cellulose degradation indicated by the cellulase activity, the dissolved organic carbon daily rate and enzyme activity increased. It was related to a fast hydrolysable fraction of cellulose that contributed to short-term carbon immobilization (ca. 10 days). After approximately 20 days, the dissolved organic carbon and enzyme activity were inversely correlated suggesting that the respiration of microorganisms was responsible for carbon mineralization. Cellulose was an important resource in low nutrient conditions (oligotrophic). However, the detritus quality played a major role in the lignocelluloses degradation (i.e., enzyme activity) and carbon release.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Fermentative hydrogen production from agroindustrial lignocellulosic substrates

To achieve economically competitive biological hydrogen production, it is crucial to consider inexpensive materials such as lignocellulosic substrate residues derived from agroindustrial activities. It is possible to use (1) lignocellulosic materials without any type of pretreatment, (2) lignocellulosic materials after a pretreatment step, and (3) lignocellulosic materials hydrolysates originating from a pretreatment step followed by enzymatic hydrolysis. According to the current literature data on fermentative H2 production presented in this review, thermophilic conditions produce H2 in yields approximately 75% higher than those obtained in mesophilic conditions using untreated lignocellulosic substrates. The average H2 production from pretreated material is 3.17 ± 1.79 mmol of H2/g of substrate, which is approximately 50% higher compared with the average yield achieved using untreated materials (2.17 ± 1.84 mmol of H2/g of substrate). Biological pretreatment affords the highest average yield 4.54 ± 1.78 mmol of H2/g of substrate compared with the acid and basic pretreatment - average yields of 2.94 ± 1.85 and 2.41 ± 1.52 mmol of H2/g of substrate, respectively. The average H2 yield from hydrolysates, obtained from a pretreatment step and enzymatic hydrolysis (3.78 ± 1.92 mmol of H2/g), was lower compared with the yield of substrates pretreated by biological methods only, demonstrating that it is important to avoid the formation of inhibitors generated by chemical pretreatments. Based on this review, exploring other microorganisms and optimizing the pretreatment and hydrolysis conditions can make the use of lignocellulosic substrates a sustainable way to produce H2.

Potential of giant reed (Arundo donax L. ) for second generation ethanol production

Background The production of second generation ethanol from lignocellulosic biomasses that have not had their potential fully explored as feedstock is of great importance. Arundo donax is one these biomasses. It is a promising grassy plant to be used as a renewable resource for the production of fuels and chemicals, because of its fast growth rate, ability to grow in different soil types and climatic conditions. The present study evaluated its use as feedstock for the production of second generation ethanol. Results Initially its chemical characterization was carried out, and a protocol for fractioning the biomass through diluted acid pretreatment followed by alkaline pretreatment was developed, providing a solid fraction which was undergone to enzymatic hydrolysis reaching 42 g/L of glucose, obtained in 30 h of enzymatic hydrolysis. This partially delignified material was subjected to a simultaneous saccharification and fermentation (SSF) process, resulting in an ethanol concentration of 39 g/L at 70 h. Conclusions The fermentability of the pretreated biomass was performed successfully through the conception of simultaneous saccharification and fermentation resulting in approximately 75 L of ethanol per ton of cellulose.

Poly R decolorization and APPL production by Streptomyces violaceoruber and Streptomyces spiroverticillatus

Two mesophilic streptomycetes (S. violaceoruber and S. spiroverticillatus) were selected to study their Poly R-478 decolorization ability and lignocellulose solubilizing activity. Both strains were able to degrade Poly R-478 dye and ferulic acid during growth on a minimal salts medium. The Poly R-478 decolorizing activities of both strains were induced by adding different carbon sources to the culture media. S. violaceoruber could decolorize 63% of Poly R-478 after 24 h. Both strains could solubilize straw and produce acid-precipitable polymeric lignin (APPL) with different efficiency. From the major extracellular enzymes recovery of both strains on rice and wheat straw, we can predicate that the biodegradation process was partial indicating a possible utilization in biological delignification.

Response of ligninolytic macrofungi to the herbicide atrazine: dose-response bioassays / Respuesta de macrohongos ligninolíticos al herbicida atrazina: bioensayos dosis-respuesta

The effect of atrazine concentrations on mycelial growth and ligninolytic enzyme activities of eight native ligninolytic macrofungi isolated in Veracruz, México, were evaluated in a semi-solid culture medium. Inhibition of mycelial growth and growth rates were significantly affected (p = 0.05) by atrazine concentrations (468, 937, 1875, and 3750 mg/l). In accordance with the median effective concentration (EC50), Pleurotus sp. strain 1 proved to be the most tolerant isolate to atrazine (EC50 = 2281.0 mg/l), although its enzyme activity was not the highest. Pycnoporus sanguineus strain 2, Daedalea elegans and Trametes maxima showed high laccase activity (62.7, 31.9, 29.3 U mg/protein, respectively) without atrazine (control); however, this activity significantly increased (p < 0.05) (to 191.1, 83.5 and 120.6 U mg/protein, respectively) owing to the effect of atrazine (937 mg/l) in the culture medium. Pleurotus sp. strain 2 and Cymatoderma elegans significantly increased (p < 0.05) their manganese peroxidase (MnP) activities under atrazine stress at 468 mg/l. The isolates with high EC50 (Pleurotus sp. strain 1) and high enzymatic activity (P. sanguineus strain 2 and T. maxima) could be considered for future studies on atrazine mycodegradation. Furthermore, this study confirms that atrazine can increase laccase and MnP activities in ligninolytic macrofungi.

Se evaluó el efecto de diferentes concentraciones de atrazina sobre el crecimiento micelial y la actividad enzimática de ocho macrohongos ligninolíticos aislados en Veracruz, México. La inhibición del crecimiento micelial y la tasa de crecimiento diaria fueron significativamente (p < 0,05) afectadas por todas las dosis de atrazina (468, 937, 1875 y 3750 mg/l) adicionadas al medio de cultivo. De acuerdo con la concentración efectiva media (CE50), Pleurotus sp. cepa 1 fue el aislamiento más tolerante a la atrazina (CE50 = 2281 mg/l), aunque sus actividades enzimáticas no fueron altas. Pycnoporus sanguineus cepa 2, Daedalea elegans y Trametes maxima mostraron actividades altas de lacasa (62,7, 31,9 y 29,3 U mg/proteína, respectivamente) en ausencia de atrazina (control); estas actividades se incrementaron (p < 0,05) significativamente (191,1, 83,5 y 120,6 U mg/proteína, respectivamente) en presencia de atrazina (937 mg/l) en el medio de cultivo. Pleurotus sp. cepa 2 y Cymatoderma elegans incrementaron significativamente (p < 0,05) sus actividades de manganeso peroxidasa (MnP) bajo la concentración de 468 mg/l de atrazina. Los aislamientos con alta CE50 (Pleurotus sp. cepa 1) y alta actividad enzimática (P. sanguineus cepa 2 y T. maxima) podrían ser considerados para futuros estudios en la micodegradación de atrazina. Además, el presente estudio confirma que la atrazina puede incrementar las actividades lacasa y MnP en macrohongos ligninolíticos.

Evaluation of polymeric adsorbent resins for efficient detoxification of liquor generated during acid pretreatment of lignocellulosic biomass.

Production of fuel ethanol from lignocellulosic biomass conventionally includes biomass pretreatment, hydrolysis, and fermentation. The liquor generated during dilute acid pretreatment of biomass contains considerable quantities of pentose sugars as well as various degradation products of sugars and lignin, like furfural, hydroxymethyl furfural (HMF), organic acids, aldehydes and others, which are known to be inhibitory for microbial growth. This pentose rich liquor is a potent resource which can be used to produce alcohol or other value added metabolites by microbial fermentation. However, the presence of these inhibitory compounds is a major hindrance and their removal is essential for efficient utilization of this byproduct stream. In the present work, the polymeric adsorbent resins, XAD-4, XAD-7 and XAD-16 were evaluated for their ability to adsorb fermentation inhibitors like furfural and HMF from the acid pretreated liquor. These resins could remove 55-75% of furfural and 100% of HMF and more than 90% sugar remained un-adsorbed in the pretreated liquor. Desorption of furfural from stationary phase was evaluated by using ethanol and hot water. The results suggest that these polymeric resins may be used for detoxification of acid pretreatment liquor with selective removal of sugar degradation products without affecting the sugar content in the solution.

Influence of novel lignocellulosic residues in a biobed biopurification system on the degradation of pesticides applied in repeatedly high doses

Background: The biobed is a simple biopurification system used to prevent the point-source pesticide contamination that occurs at farm level. The typical composition of the biomixture used in this system is soil, peat and straw in volumetric proportions of 1:1:2. The principal component is straw due to its positive effects on biological activity and thus pesticide degradation. However, access to straw can be limited in some regions, so it must be replaced by other more readily available lignocellulosic residues. Results: Therefore, two alternate lignocellulosic materials (barley husks and pine sawdust) were evaluated as partial substitutes for straw. The degradation of a repeatedly applied mixture of six pesticides by these alternates was assessed. The microbial respiration and fluorescein diacetate (FDA) hydrolysis activity were also assessed. The results showed that the highest degradation efficiency was found in mixtures containing straw and barley husks. Each biomixtures tested achieved a high degradation (50 to 90%) of all the pesticides used except iprodione. Repeated applications of pesticides resulted in a slowing of the degradation rate of all pesticide types in all biomixtures. FDA activity and microbial respiration were higher in the biomixtures containing barley husks and straw compared to the mixture with pine sawdust, a result consistent with the pesticide degradations observed. Conclusions: This paper demonstrates that the straw in the traditional biomixture can be partially replaced by other lignocellulosic materials to efficiently degrade a mixture of pesticides, even when the pesticides are added in successive applications and high concentrations.

Evaluation of changes in lipid peroxidation, ROS production, surface structures, secondary metabolites and yield of linseed (Linum usitatissimum L.) under individual and combined stress of ultraviolet-B and ozone using open top chambers.

The individual and interactive effects of supplemental UV-B (sUV-B) (ambient + 7.2 kJ m-2 d-1) and elevated O3 (ambient + 10 ppb) were evaluated under field conditions using open top chambers on two cultivars, Padmini and T-397 of linseed (Linum usitatissimum L.). Mean monthly surface level of O3 concentrations varied from 27.7 ppb to 59.0 ppb during the experimental period. Both UV-B and O3 induced the production of ROS (H2O2 and O2.-), resulting in significant damage of membranes due to lipid peroxidation and electrolyte leakage. Synthesis of secondary metabolites (flavonoids, anthocyanin, lignin and wax) was also enhanced in all the treatments, whereas biomass and yield were reduced. Alterations in frequency of stomata and wax distribution were also observed through scanning electron microscopy (SEM). Cultivar Padmini was found to be more sensitive because of higher damage of membrane vis-a-vis reduction in biomass and seed yield. However, concentrations of flavonoids, anthocyanin, lignin and wax were higher in T-397, suggesting its relative resistance against applied stress. Combined exposure of sUV-B and O3 was less harmful, as compared to their individual treatment. Among the three treatments, O3 was found to be more detrimental for overall growth and sUV-B for economic yield.