Modification Of Fulvic Acid-like Substances In Spent Cooking Liquor And Its Mechanism To Enhance Salt Tolerance On Rice

Fulvic acid has been widely used as an important biostimulant to improve salt tolerance of rice,but the non-renewable property severely limited its large-scale application in agriculture.Spent cooking liquor,generated from the pulping process of pulp and paper industries,is the main water pollution source in China.Treatments as to reduce the organic matter load in spent cooking liquor representing of lignin only lead to a waste of resource not utilization.Previous research found that the organic matter in spent cooking liquor exhibited similar characteristic functional groups with that in natural fulvic acid.However,the bioactivity of the organic matter and its effect on salt tolerance of rice are not yet fully explored.The study employed the spent cooking liquor from ammonium sulfite pulping process as materials for the extraction of fulvic acid-like substance?PFA?,and compared the differences in chemical structure and bioactivity on salt tolerance of rice with the benchmark fulvic acid extracted from leonardite?LFA?;Fractionating PFA into different size fractions by ultrafiltration to elucidate the structure-bioactivity relationship of PFA;Using H₂O₂ as an oxidant and TiO₂ as a catalyst to chemical modify PFA and identify the optimal reaction condition for higher bioactivity;For lower molecular weight fulvic acid-like substances?LPFA,MW<5kDa?,laccase modification was conducted to study its effect on the content of active functional groups and the bioactivity of LPFA under the condition of different substrate concentrations,reaction time and reaction temperature;For high molecular weight fulvic acid-like substance?HPFA,MW>10kDa?,catalytic oxidation combined with laccase modification was employed to explore its possibility for improved bioactivity of HPFA;Transcriptome sequencing and real-time PCR?qRT-PCR?were used to explore the mechanism of PFA on salt tolerance of rice;Field experiments were conducted to verified the effects of PFA and controlled release urea on crop yield,nitrogen use efficiency and economic benefits in a rice-wheat rotation system.The main findings are listed below?1?Comparison on chemical structure and bioactivity of LFA and PFACompared with LFA,PFA contained more C,H,N,S elements and less O elements.PFA has a relative higher molecular weight,with more carbohydrates and lignin-derived aromatic structures but less carboxyl groups.Both LFA and PFA promoted the germination of rice seeds under salt stress and improved the growth of rice seedlings with increased the antioxidant enzyme activities on salt tolerance.But the efficiency concentrations on rice growth are significantly different.?2?Molecular composition of size-fractionated PFA and its relationship with bioactivity.PFA were fractioned into five classes including<1,1-3,3-5,10-50 and>50kDa.After fractionation,aromaticity and carboxyl content increased with increasing molecular weight,but protein/amino proportion decreased.Under salt stress,rice seedling growth was promoted by PFA with low molecular weight?<5kDa?,but inhibited by fraction with high molecular weight?>10kDa?.Principal component analysis suggested that promoted growth was more related with chemical structure?aliphatic structure,carboxyl group,and phenolic hydroxyl group?than with molecular weight.?3?Activation of H₂O₂/TiO₂ catalytic oxidation on chemical structure and bioactivity of PFAThe activation of H₂O₂/TiO₂ reduced the aromaticity of PFA-Os and increase its carboxyl group content.With increased reaction condition,the yield of PFA gradually decreased and the color became lighter.Among PFA-Os with varying degrees of oxidation,1:2 vol ratio of paper mill effluent and 30%H₂O₂ for 20min digestion at 90°C stands out to be the optimal catalytic oxidation conditions.Under this condition,the production got a higher bioactivity on salt tolerance of rice seedling.?4?Laccase modification on chemical structure and bioactivity of low molecular weight fulvic acid-like substanceFor the<5kDa molecular weight fulvic acid-like substance,laccase modification decreased the molecular weight and increased the content of functional groups like carboxyl and phenolic hydroxyl groups in the way of oxidation and demethylation.In addition,laccase modification reduced the aromaticity and the relative abundance of lignin,but increased the relative abundance of lipids,tannins,protein,and amino acids.Due to the increased content of the active-promoting functional group,the bioactivity of the modified LPFA had also been further improved.?5?H₂O₂/TiO₂ catalytic oxidation combined with laccase modification on chemical structure and bioactivity of HPFAH₂O₂/TiO₂ catalyzes oxidation degraded the lignin structure and increased content of carboxyl functional group.On this basis,laccase modification further increased phenolic hydroxyl functional group formed by demethylation.Through the combination of catalytic oxidation and laccase modification,the molecular weight of HPFA decreased and the content of functional groups increased.HPFA,which originally inhibited rice growth,was transformed into an active substance that promoted rice growth under salt stress.?6?Regulation mechanism of PFA on rice salt toleranceUnder salt stress,PFA alleviated ion toxicity by reducing the content of Na⁺.Transcriptome sequencing results showed that PFA influenced the expression genes relating to oxidoreductase activity,nutrient absorption,transmembrane transportion and cation transportion.qRT-RCR results further indicated that the gene of OsHKT6,OsHKT8 and OsCBL8 can be the potential key regulation gene.?7?Effect of PFA combined with controlled-release urea on yield,nitrogen utilization rate and economic benefits in rice-wheat rotation systemBased on the reasonable nitrogen supply of controlled release urea,the application of PFA enhanced the nitrogen absorption of crops and increased the supply strength of soil phosphate fertilizer.The synergistic application of controlled-release urea with PFA greatly improved rice and wheat yields,nitrogen utilization rate and economic benefits,thereby achieving efficient utilization of nutrient.