H2O2 Pretreatment Research Articles

Liquefaction of pineapple peel: Pretreatment and process optimization

This study explored the optimization of pretreatment of pineapple peel for biogas generation. Pretreatments were carried out sulfuric acid and alkaline hydrogen peroxide prior to anaerobic digestion while the response surface methodology (RSM) was used for optimization of the pretreatment procedures. The physical, chemical, proximate and structural compositions of the peels were determined prior to and at the end of the pretreatment procedures. The dynamics of microorganisms in the reactors were also evaluated by rapid molecular methods while the Fourier Transform Infra-red (FTIR) spectroscopy was employed in the identification of the chemical changes as a result of pretreatments. The use of H2O2 pretreatment caused enormous lignin solubilization in the pineapple peel. In comparison, biogas production was 67% more in the alkaline pretreated pineapple peel than the biomass treated with acid and also 51% over the untreated samples. The total biogas volume produced from the acidic pretreated, alkaline pretreated, not sifted untreated and sifted untreated samples are 194.2 ± 3.0, 587.5 ± 5.2, 287.8 ± 2.1 and 245.4 ± 3.1 respectively. Thus, the alkaline pretreated experiment used lower retention time to achieve maximum gas production in this study. The use of alkaline H2O2 on lignocelluloses has remained unpopular prior to this study. However, its usage in this study yielded better result than all the conventional treatments in terms of lignin solubilization and improvement in methane yield. Economically, the use of H2O2 for pretreatment is adjudged feasible because the 1504 kWh t−1 TS thermal energy gain obtained from the biogas produced by the alkaline treated peel exceeded the 921 kWh t−1 TS used in the pretreatment. This gives a net thermal energy of 583 kWh t−1 TS. Whereas, the investment into acidic pretreatment of pineapple peel may not be economically justified because the total thermal energy gain of −200 kWh t−1 TS was far lower than the 1236 kWh t−1 TS thermal energy that was consumed during the pretreatment giving a net thermal energy of −1436 kWh t−1 TS. Therefore, the use of mild alkaline pretreatment is advocated in biogas generation from pineapple peel and also for biofertilizer production mostly in localities of mass production.

Comprehensive understanding of the non-productive adsorption of cellulolytic enzymes onto lignins isolated from furfural residues

Effective enzymatic hydrolysis of lignocellulosic materials to produce fermentable sugars requires close cooperation between cellulase components. However, lignin remaining in the pre-treated substrate plays an inhibitory role in the synergistic action of enzymes through irreversible adsorption. In this study, the effect of cellulolytic enzyme lignin (CEL) isolated from pre-treated furfural residues and dissolved lignins (DLs) precipitated from black liquor on cellulase and cellobiase adsorption behavior was investigated. Green liquor (GL) combined with H2O2 (GL-H2O2) or ethanol (GL-ethanol) pre-treatments markedly decreased the adsorption capacity of CELs. However, the DLs had the strongest binding ability, which was approximately two to three times higher than that of CELs. According to SDS–PAGE analyses, only CBHII and EGII in cellulase and enzymes of 245 kDa and 55 kDa molecular weight in cellobiase were easily bound onto CELs and DLs. Solid-state 13C NMR spectra and FT-IR analysis indicated that pre-treatments reduced the hydrophobic structure of aromatic rings, β-O-4, and carbonyl, and increased the number of hydrophilic carboxyl groups in CELs. For this reason, the hydrophobicity of CELs was decreased, thus reducing their adsorption capacity. Compared with GL-H2O2 pre-treatment, GL-ethanol pre-treatment had a stronger effect on decreasing enzyme adsorption of lignin due to its high hydrophilicity. Heavy damage to aromatic structures, low molecular weight, and the large number of aliphatic portions in DLs caused their strong affinity for enzymes, although their BET surface area and hydrophobicity were low.

Comparison of mild alkaline and oxidative pretreatment methods for biobutanol production from brewer’s spent grains

In this study, a comparative analysis of different pretreatment strategies on hydrolysis and fermentation of brewers’ spent grain for biobutanol production by C. beijerinckii is presented. Ozone (2.7% w/w, 30 min, 40–60% w/w humidity), sodium hydroxide (1–5 % w/w NaOH, 30 min, 120 ºC, 5–15 % w/w BSG) and hydrogen peroxide (5% w/w H2O2, pH 11.5, 60–180 min, 50 °C, 5–15 % w/w BSG) were the pretreatments analyzed.Ozonolysis was not very effective for either the degradation of lignin or the recovery of fermentable sugars in the enzymatic process under the tested operation conditions. Maximum butanol and ABE concentrations, 5.3± 0.2 g butanol/L and 6.8 ± 0.2 g ABE/L, were observed when low moisture content was used (40% w/w).The peroxide alkaline pretreatment was found to be the most effective for BSG, when it was conducted at 5% BSG for 60 min. Under these conditions, sugar enzymatic yields of 62.8% for glucose and 28.1% for xylose and arabinose, referred to untreated BSG, and product concentrations of 11.0± 0.2 g butanol/L and 13.7 ± 0.2 g ABE/L, were achieved. The alkaline pretreatment at 15% BSG with 1% w/w NaOH also provided high butanol and ABE concentrations (7.3 ± 0.1 g butanol/L and 8.9 ± 0.1 g ABE/L).Both the sodium hydroxide alkaline and peroxide alkaline methods were highly successful as pretreatments of BSG for ABE production, with overall yields of 44.4 g butanol/kg BSG and 54.0 g ABE/kg BSG for NaOH pretreatment (15% BSG, 1% w/w NaOH) and 45.1 g butanol/kg BSG and 56.1 g ABE/kg BSG for H2O2 pretreatment (5% BSG, 60 min).

Enhancement of neural stem cell survival, proliferation and differentiation by IGF-1 delivery in graphene oxide-incorporated PLGA electrospun nanofibrous mats.

The mammalian central nervous system has a limited ability for self-repair under injury conditions. The treatment of nerve injuries has been revolutionised with the development of tissue engineering techniques. However, the lack of bioactivity has severely restricted the application of biodegradable implants for neurogenesis. Therefore, surface modification of biomaterials is crucial to improve their bioactivity and promote endogenous repair mechanisms for nerve regeneration. Insulin-like growth factor 1 (IGF-1) is a growth factor for neuroprotection and neurogenesis. In this study, IGF-1 was successfully immobilised on graphene oxide (GO)-incorporated poly(lactic-co-glycolic acid) (PLGA) biodegradable electrospun nanofibres. For the in vitro investigation, neural stem cells (NSCs) were cultured on different nanofibres to observe various cellular activities. GO enhanced NSC survival under H2O2 pre-treatment and neuronal differentiation to some extent. More importantly, the immobilisation of IGF-1 onto the PLGA/GO nanofibres resulted in significantly increased NSC survival, proliferation, and differentiation. Findings from this study revealed that using PLGA/GO electrospun nanofibres to immobilise IGF-1 has excellent potential for the enhancement of the neuroprotective and neurogenic effects of nerve implants.

Enhancement of Methane Production from Banana Harvesting Residues: Optimization of Thermal–Alkaline Hydrogen Peroxide Pretreatment Process by Experimental Design

Increase of population has a significant effect on energy demand which results in depletion of fossil fuels, and this causes a search for alternative renewable energy sources. One of these alternatives is production of bioenergy and biofuel from renewable and non-food feedstocks such as lignocellulosic biomass and biowaste. However, lignocellulosic biomass needs pretreatment because of its complex structure. Effectiveness of thermal–alkaline H2O2 pretreatment process and determination of its optimum conditions using central composite design of RSM were evaluated for the enhancement of methane production from banana harvesting residues. Optimum process conditions for cost driven approach was determined as 50 °C reaction temperature, 2.73% H2O2 concentration and 6 h reaction time. As a result of pretreatment at optimum conditions, 40% increase on biochemical methane potential was obtained with 290 mLCH4/gVS methane production. SEM and FTIR results revealed surface disruption and lignin removal impacts of pretreatment, respectively. Thermal–alkaline H2O2 pretreatment was determined as an effective pretreatment process for banana harvesting residues.

Extraction and characterization of pepsin-solubilized collagen from snakehead (Channa argus) skin: Effects of hydrogen peroxide pretreatments and pepsin hydrolysis strategies

Abstract The effects of hydrogen peroxide (H2O2) pretreatments and pepsin hydrolysis strategies on the extraction of pepsin-solubilized collagen (PSC) from the skin of snakehead (Channa argus) were studied. The dependences of H2O2 bleaching on H2O2 concentrations (1%, 3%, and 6% (w/v)) and pH (6, 8, and 10) were examined, while the difference between the conventional and unconventional pepsin hydrolysis methods was compared. Results showed that the yield of snakehead skin PSC was highly dependent on the parameters of both H2O2 pretreatments and pepsin hydrolysis processes. The color of PSC was affected by pH more greatly than by H2O2 concentration. Compared with the conventional pepsin hydrolysis of fish skins, the use of pepsin after extraction of acid-solubilized collagen (ASC) could improve the color of PSC. Moreover, the electrophoretic study, infrared spectroscopy, and fibril formation measurement showed that the structural integrity of PSC was largely influenced by the pH of H2O2 pretreatments, suggesting that the H2O2 solution (3% (w/v), pH 10) was suitable for the bleaching of snakehead skins. Finally, the amino acid analysis, ultraviolet spectroscopy, and differential scanning calorimetry confirmed that the prepared collagen had high purity and thermal stability. The light-color collagen might be used as an alternative for mammalian collagens.

EFFECT OF COMBINATION OF LIQUID HOT WATER SYSTEM AND HYDROGEN PEROXIDE PRETREATMENT ON ENZYMATIC SACCHARIFICATION OF CORN COB

Alkaline hydrogen peroxide pretreatment is an effectively enhance the increasing enzymatic digestibility of lignocellulosic biomass for conversion to fuels and chemicals in the biorefinery processes. In this study, effects of H2O2 on monomeric sugar in the liquid fraction during hydrogen peroxide pretreatment and sugar after enzymatic hydrolysis from corncobs were studied under varying reaction conditions. The temperature (30-120oC) and H2O2 concentration (2.5-10%) efficiently promoted sugar yield in the piqued fraction and improved enzymatic hydrolysis of pretreated solids. The optimal condition for H2O2 pretreatment of corncob (H2O2 concentration of 5% using 60 oC for 2 h) increased hemicellulose solubilization into the aqueous phase, resulting into the maximized pentose yield of 61.88% (xylose + arabinose) in the aqueous phase. H2O2 pretreatment under the optimal conditions at 60oC for 2 h, leading to the enhance glucose yield from enzymatic hydrolysis of the pretreated biomass using 10 FPU/g CelluclastTM (85.66 %) and small amount of formation of inhibitory by-products. Combined with glucose in the aqueous phase, this resulted in the maxima 95.61% glucose recovery from the native corncob. This was related to changes in crystallinity and surface area of the pretreated biomass. Scanning electron microscopy (SEM) showed disruption of the intact biomass structure resulting increasing enzyme’s accessibility to the cellulose microfibers which showed higher crystallinity index compared to the native biomass as shown by X-ray diffraction with a marked increase in surface area as revealed by BET measurement. The results provided efficiency of H2O2 pretreatment on increasing sugar recovery and an efficient approach for its processing in biorefinery industry.

Hydrogen peroxide-induced salt tolerance in relation to antioxidant systems in pistachio seedlings

In this study, the effects of H2O2 pretreatment on growth and antioxidant system were investigated in pistachio rootstock Badami Rize Zarand during short term salt stress. The plants were subjected to NaCl stress (120 or 240 mM) for 7 days after pretreatment with 0, 1, 5 or 10 mM H2O2. Salinity stress significantly decreased seedling growth and ascorbic acid (ASA) and carotenoid (CAR) contents, whilst exogenous H2O2 application improved the growth and helped to reduce the percent reduction of total ASA and CAR contents under salt stress conditions. Ascorbate peroxidase (APX) and catalase (CAT) activities were strongly induced by salt stress after 48 h and reached the highest level at 168 h after spraying. H2O2-treated plants showed enzymatic activity higher than untreated plants, during the entire experimental period. Salinity treatments caused increase in plants GSH content with or without H2O2 pretreatment. GSH levels in the plants were previously treated with H2O2 were elevated even higher. Overall, better performance of pistachio seedlings under salt stress due to H2O2 pre-treatment might be due to improved better osmotic adjustment through coordination with CAT, and APX and enhanced non-enzymatic antioxidant accumulation under salt stress leading to a higher ability to withstand salt-induced effects.

Impact of Alkaline H₂O₂ Pretreatment on Methane Generation Potential of Greenhouse Crop Waste under Anaerobic Conditions.

This paper intended to explore the effect of alkaline H2O2 pretreatment on the biodegradability and the methane generation potential of greenhouse crop waste. A multi-variable experimental design was implemented. In this approach, initial solid content (3–7%), reaction time (6–24 h), H2O2 concentration (1–3%), and reaction temperature (50–100 °C) were varied in different combinations to determine the impact of alkaline H2O2 pretreatment. The results indicated that the alkaline H2O2 pretreatment induced a significant increase in the range of 200–800% in chemical oxygen demand (COD) leakage into the soluble phase, and boosted the methane generation potential from 174 mLCH4/g of volatile solid (VS) to a much higher bracket of 250–350 mLCH4/gVS. Similarly, the lignocellulosic structure of the material was broken down and hydrolyzed by H2O2 dosing, which increased the rate of volatile matter utilization from 31% to 50–70% depending on selected conditions. Alkaline H2O2 pretreatment was optimized to determine optimal conditions for the enhancement of methane generation assuming a cost-driven approach. Optimal alkaline H2O2 pretreatment conditions were found as a reaction temperature of 50 °C, 7% initial solid content, 1% H2O2 concentration, and a reaction time of six h. Under these conditions, the biochemical methane potential (BMP) test yielded as 309 mLCH4/gVS. The enhancement of methane production was calculated as 77.6% compared to raw greenhouse crop wastes.

Pretreatment with concurrent UV photocatalysis and alkaline H2O2 enhanced the enzymatic hydrolysis of sisal waste

This work studied a concurrent UV photocatalysis and alkaline H2O2 pretreatment (UHP) to enhance the subsequent enzymatic hydrolysis of sisal waste in comparison with alkaline H2O2 pretreatment (AHP). An optimal condition was identified for UHP at H2O2 charge 0.1 g/g dried sisal waste, pH 10.0, and UV radiation for 6 h. Under this condition, UHP led to a delignification rate of 76.6%, a conversion to reducing sugar at 71.2%, and a conversion to glucose at 91.6%, respectively. XRD, FT-IR and SEM analysis showed an increase in crystalline degree and significant changes in the structure of sisal during UHP. The current study implicates that UHP is more efficient than AHP in pretreating sisal waste, with reduced H2O2 charge, shortened pretreatment time, and enhanced enzymatic digestibility.

Two-stage anaerobic fermentation process for bio-hydrogen and bio-methane production from pre-treated organic wastes

In this study, the effect of pre-treatments including alkaline, acid and hydrogen peroxide on continuous hydrogen and methane production was investigated. Two different substrates as potatoes and bean wastes were used. Pre-treatment showed positive effect on bio-hydrogen and bio-methane production; higher bio-hydrogen and bio-methane production results using pre-treated samples than the control bioreactors (without pre-treatment), were recorded. In case of potatoes wastes, the hydrogen yield ranged between 126.4 and 252.7 mL-H2/g-TVS using pre-treated samples compared to 58.7 mL-H2/g-TVS observed in the reference test. Pre-treated bean wastes showed hydrogen yield of 93.0–152.1 mL-H2/g-TVS higher than 53.3 mL-H2/g-TVS measured in the control test. In the second stage, average methane yield results of 322.9–507.1 and 284.3–462.6 mL-CH4/g-TVS higher than 198.6 and 124.3 mL-CH4/g-TVS measured for potatoes and bean wastes control bioreactors, respectively. The best results were observed using H2O2 pre-treatment. The energy production efficiency was improved by combining H2 and CH4 bioreactors.

Polycyclic aromatic hydrocarbons affiliated with microplastics in surface waters of Bohai and Huanghai Seas, China

Microplastics (MPs) sized between 0.33 and 5 mm were collected using Manta trawls from ten surface seawater sites in Bohai and Huanghai Seas, China. A total of 1024 (Bohai Sea) and 132 (Huanghai Sea) microplastic pieces were classified, including polystyrene foams, polyethylene films and lines, and other plastic pellets, with concentrations of MPs ranging from 3 to 162 particles per 100 m3 (0.012–2.96 mg m−3). A pretreatment of MPs with 30% H2O2 in water did not significantly lower polycyclic aromatic hydrocarbon (PAH) concentrations on MPs compared to no H2O2 pretreatment. Measurements of PAHs carried on the collected MPs indicated that the concentrations of the sum of 16 PAHs were in the range of 3400−119,000 ng g−1. The sources of PAHs in Bohai and Huanghai Seas were highly similar, with petroleum and gasoline probably as the dominant sources. The present study shows the relative importance of MPs in regards to chemical transport in the marine environment. The combination of high concentrations of PAHs affiliated with MPs and the increasing magnitude of plastic pollution in the world's oceans demonstrates the considerable importance of MPs to the fate of PAHs in marine environments.

Enhanced Enzymatic Hydrolysis of Rice Straw Pretreated by Oxidants Assisted with Photocatalysis Technology.

This work evaluated the effectiveness of rice straw pretreatment using a TiO2/UV system in the presence of oxidants. The effects of TiO2 concentrations, pH and photocatalysis time were investigated. Inorganic oxidants including H2O2, K2S2O8, and KIO4 were added to further enhance the effect on enzymatic hydrolysis of rice straw. The TiO2/UV/ H2O2 pretreatment showed a higher amount of released reducing sugar (8.88 ± 0.10 mg/mL, compared to 5.47 ± 0.03 mg/mL in untreated sample). Composition analyses of rice straw after the TiO2/UV/H2O2 pretreatment showed partial lignin and hemicellulose removal. Moreover, structural features of untreated and pretreated rice straw were analyzed through FE-SEM, FT-IR, and XRD. This work suggests that H2O2 is an efficient addition for photocatalysis pretreatment of rice straw.

Particle size distribution in soils and marine sediments by laser diffraction using Malvern Mastersizer 2000—method uncertainty including the effect of hydrogen peroxide pretreatment

Purpose Methods for particle size distribution (PSD) determination by laser diffraction are not standardized and differ between disciplines and sectors. The effect of H2O2 pretreatment before a sonication treatment in laser diffraction analysis of soils and marine sediments was examined on soils with less than 1% C and some marine sediments.

Improved anaerobic digestion performance of Miscanthus floridulus by different pretreatment methods and preliminary economic analysis

During the anaerobic digestion (AD) of cellulosic substrate, an economical and environmental friendly pretreatment method should be selected to improve the AD performance. In this study, five pretreatment methods include four traditional pretreatment methods (alkali hydrogen peroxide pretreatment, NaOH pretreatment, HCl pretreatment and hot water pretreatment) and one new developed pretreatment (microaerobic pretreatment) method were selected to study the effects of pretreatment on the AD of Miscanthus floridulus. In addition, preliminary economic analysis of different pretreatment methods was also conducted. Results showed pretreatment could significantly improve the AD performance of Miscanthus. After alkali hydrogen peroxide pretreatment, microaerobic pretreatment, NaOH pretreatment, HCl pretreatment and hot water pretreatment the methane yields of Miscanthus were 41.1%, 25.5%, 18.7%, 15.7% and 10.2% higher than that of untreated sample. Though all these five pretreatment methods could improve the methane yield significantly, their action sites were different. H2O2 pretreatment and NaOH pretreatment were efficient in removal lignin from Miscanthus floridulus. Hemicellulose structure was also partly destroyed during H2O2 pretreatment and NaOH pretreatment. HCl pretreatment was efficient in destruction of hemicelluloses structure and partly destroyed the cellulose structure. Microaerobic pretreatment and hot water pretreatment were mainly acted in partly destruction of hemicelluloses and cellulose components. Preliminary economic analysis showed compared with un-pretreated group only microaerobic pretreatment was economically feasible. In addition, compared with other pretreatment methods, microaerobic pretreatment just needs limited amount of oxygen, which is more environmental friendly.

Hydrogen peroxide promotes the tolerance of soybeans to waterlogging

Abstract We investigated the role of pretreatment of seeds with hydrogen peroxide (H2O2) in promoting the tolerance of seedlings of Glycine max to waterlogging. Soybean seeds were pretreated with a solution of H2O2 70 mM or water for 24 h under aeration, and then were placed in plastic bags containing substrate. At 12 days after sowing, the seedlings were subjected to waterlogging for 32 days. There were three evaluations: 0, 16 days and 32 days of waterlogging. Biomass accumulation, root volume, stem diameter, gas exchanges, activity of antioxidant system and cell membrane damages were evaluated. The pretreatment of seeds with H2O2 generated seedlings with elevated activity of antioxidant enzymes, low H2O2 and O2−. content, and low cell membrane damages. Hydrogen peroxide pretreatment also resulted in increased content of photosynthetic pigments and net photosynthetic rate, high biomass accumulation, root volume and stem diameter. H2O2 pretreatment of soybeans seeds generated seedlings more tolerant to waterlogging. There is probably a stress memory involved in the process of induction of tolerance in soybeans mediated by hydrogen peroxide.

Degradation of p-Nitrophenol by Lignin and Cellulose Chars: H2O2-Mediated Reaction and Direct Reaction with the Char.

Chars and other black carbons are reactive toward certain compounds. Such reactivity has been attributed to reduction of O2 by persistent free radicals in the solid to H2O2, which then back-reacts with the solid to generate reactive oxygen species (ROS; especially HO•). We studied the decomposition of p-nitrophenol (PNP) by pure lignin and cellulose chars aged in moist air or a vacuum at room temperature for up to a month. In air, the chars chemisorbed oxygen, a portion of which was liberated as H2O2 when the char was submerged in water. The evolved H2O2 was simultaneously decomposed by the char. PNP reacted predominantly in the sorbed state and only reduction products (phenol, catechol) were identified. Aging the char in air sharply (within hours) reduced H2O2-producing capacity and free radical concentration, but more gradually reduced PNP decay rate over the month-long period. PNP decay was only modestly suppressed (12-30%) by H2O2 removal (catalase), and had little effect on the free radical signal (<6 radicals annihilated per 1000 PNP reacted). Contrasting with previous studies, the results show that direct reaction of PNP with char predominates over H2O2-dependent reactions, and the vast majority of direct-reacting sites are nonradical in character. Nonradical sites are also responsible in part for H2O2 decomposition; in fact, H2O2 pretreatment depleted PNP reactive sites. Lignin char was generally more reactive than cellulose char. The Fe impurity in lignin played no role. The results are relevant to the fate of pollutants in black carbon-rich environments and the use of carbons in remediation.

Tomato photorespiratory glycolate-oxidase-derived H2 O2 production contributes to basal defence against Pseudomonas syringae.

Despite being essential for C3 plants, photorespiration is believed to cause a significant crop yield loss even under future climates. However, how photorespiration affects plant basal defence still remains largely unknown. Here, we studied the involvement of photorespiration in tomato-Pseudomonas syringae pv. tomato DC3000 interaction focusing on three photorespiratory genes. Inoculation with P.syringae increased photorespiration rate (Pr) and expression of glycolate oxidase (GOX2), serine glyoxylate aminotransferase (SGT) and serine hydroxyl methyltransferase (SHMT1); however, inhibition of photorespiration by isonicotinic acid hydrazide decreased tomato basal defence against P.syringae. Furthermore, silencing of GOX2, SGT or SHMT1 genes in tomato decreased Pr but increased susceptibility to P.syringae, whereas transient overexpression of GOX2, SGT or SHMT1 in tobacco increased basal defence. Further study revealed that salicylic acid (SA) signalling is involved in GOX2-mediated, SGT-mediated and SHMT1-mediated defence. Moreover, H2 O2 pretreatment remarkably alleviated the GOX2 silencing-induced depression in basal defence and SA signalling, whereas it had no effect on that of SGT-silenced and SHMT1-silenced plants. Taken together, these results suggest that H2 O2 is critical for GOX2-modulated but not SGT-modulated or SHMT1-modulated SA signalling and subsequent basal defence against P.syringae. This work deepens the understanding of photorespiration-involved defence responses to bacterial attack in plants.

Hydrogen Peroxide-Induced Steviol Glycosides Accumulation and Enhancement of Antioxidant Activities in Leaf Tissues of Stevia rebaudiana Bertoni

Hydrogen peroxide (H2O2) functions as an abiotic stress elicitor for plants. In this study, the effect of oxidative stress caused by H2O2 on steviol glycosides (SGs) production and antioxidant activities of an in vitro grown antidiabetic plant, Stevia rebaudiana, has been investigated. Direct shoot organogenesis was performed, and after 4 weeks of culture, the shoots of S. rebaudiana were transferred to MS medium containing H2O2 (10 or 20 mM) and incubated in growth room for 6 h. It resulted in increased SGs production, such as rebaudioside A (Reb-A) and stevioside (ST), and enhancement of total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power and DPPH free radical scavenging activity when compared with control. The results clearly showed highest amount of SGs produced in MS medium containing 10 mM H2O2 [producing 2.97% Reb-A and 2.32% ST (w/w)], followed by the medium employing 20 mM H2O2 and control group. It is also evident that H2O2 pre-treatment caused significant increase in non-enzymatic antioxidants that play defensive role against an oxidative stress induced by H2O2. The described protocol has a great potential to be utilized for Reb-A production on commercial scale.

The wheat NB-LRR gene TaRCR1 is required for host defence response to the necrotrophic fungal pathogen Rhizoctonia cerealis.

SummaryThe necrotrophic fungus Rhizoctonia cerealis is the major pathogen causing sharp eyespot disease in wheat (Triticum aestivum). Nucleotide‐binding leucine‐rich repeat (NB‐LRR) proteins often mediate plant disease resistance to biotrophic pathogens. Little is known about the role of NB‐LRR genes involved in wheat response to R. cerealis. In this study, a wheat NB‐LRR gene, named TaRCR1, was identified in response to R. cerealis infection using Artificial Neural Network analysis based on comparative transcriptomics and its defence role was characterized. The transcriptional level of TaRCR1 was enhanced after R. cerealis inoculation and associated with the resistance level of wheat. TaRCR1 was located on wheat chromosome 3BS and encoded an NB‐LRR protein that was consisting of a coiled‐coil domain, an NB‐ARC domain and 13 imperfect leucine‐rich repeats. TaRCR1 was localized in both the cytoplasm and the nucleus. Silencing of TaRCR1 impaired wheat resistance to R. cerealis, whereas TaRCR1 overexpression significantly increased the resistance in transgenic wheat. TaRCR1 regulated certain reactive oxygen species (ROS)‐scavenging and production, and defence‐related genes, and peroxidase activity. Furthermore, H2O2 pretreatment for 12‐h elevated expression levels of TaRCR1 and the above defence‐related genes, whereas treatment with a peroxidase inhibitor for 12 h reduced the resistance of TaRCR1‐overexpressing transgenic plants and expression levels of these defence‐related genes. Taken together, TaRCR1 positively contributes to defence response to R. cerealis through maintaining ROS homoeostasis and regulating the expression of defence‐related genes.