Mechanisms Of PH Value,Clay Minerals,and Heavy Metals Affecting The Phosphate Solubilization Of Aspergillus Niger NJDL-12 Strain

Phosphorus（P）is one of the essential nutrient elements for plant growth.There is often no shortage of total phosphorus in the soil,but the concentration of available P for plant is very low.In agriculture,excessive application of phosphate fertilizer is often used to provide the required P to plants.Due to the adsorption and fixation of metal cations and clay minerals in the soil,most of the P applied to the soil is stored in the form of invalid state,resulting in insufficient concentration of available P in the soil and extremely low utilization rate of P.At the same time,long-term excessive application of chemical fertilizers will also cause soil compaction,acidification,non-point source pollution,and water pollution,which will affect human health.Therefore,improving the supply capacity of available P in the soil is of great significance for maintaining agricultural production and improving the agricultural ecological environment.P biogeochemical cycle is affected by a variety of soil factors,such as pH value,minerals,temperature,humidity,texture,and heavy metal cations.In this paper,three factors of pH value,clay minerals,and heavy metal cations were studied.pH value affects the absorption of nutrients by microorganisms and the activity of enzymes in the metabolic process by causing changes in the cell membrane charge,which in turn affects the growth and metabolic activities of microorganisms.Clay minerals are the active centers of the soil system and can affect the growth,reproduction,and behavior of microorganisms through adsorption.It is difficult to independently study the pH value or clay minerals in the soil system,so for the pH value and clay minerals,this study mainly analyzes their effects on the release and migration of P by constructing an ideal environment（solution system）.In addition,P in the soil is easy to combine with heavy metals to form phosphates,resulting in the superimposed effect of available P deficiency and heavy metal pollution.For heavy metal cations,the mechanism of heavy metal stress on the chemical cycle of P was analyzed in combination with the solution system and field experiments in soils containing high concentrations of lead（Pb）.As the main driver of the soil P cycle,phosphate-solubilizing microorganisms（PSMs）can not only secrete organic acids,inorganic acids,and protons to dissolve inorganic P sources,but also can secrete enzymes（phosphatase and phytase）to mineralize organic P sources.Therefore,PSMs have a great influence on the activation and turnover of insoluble phosphate in the soil.Aspergillus niger（A.niger）is a phosphate-solubilizing fungus（PSF）with high-efficiency phosphate-solubilizing ability that has been widely studied.Based on this,this article took A.niger NJDL-12 strain as the main research object,combined with laboratory culture experiment and field experiment,systematically discussed the effects of pH value,clay mineral types and different heavy metal cations on phosphate dissolution and their mechanism.The main research contents and results are as follows:1.Study on the mechanism of typical environmental factors affecting phosphate solubilization by A.nigerIn this study,two typical factors,pH value and clay minerals,were used for experiments.The most common organophosphorus compound（phytic acid calcium,OP）in the soil was selected as the P source.The growth and metabolic activities（oxalic acid concentration and phytase activity）of A.niger were investigated by accurately regulating the pH value of the environment.We studied the changing trend of dissolved OP by A.niger through initial adjustment of pH value（EI treatment）and continuous adjustment to keep pH value stable（EC treatment）.In the EI treatments,A.niger secreted organic acids to reduce the pH value of the environment during the growth process and can still survive in a strong acidic environment（pH=1.5）.When pH=2.5-4.5,the biomass of A.niger in EI treatment was higher than that in other treatments,but the concentration of P in the solution was lower than that in other treatments.A.niger could secrete more oxalic acid and phytase when the pH was 3.5-6.5,and the phytase concentration（0.056μmol/min/m L）was the highest at pH=6.5.For EC treatment,A.niger could also survive at pH=1.5.When pH=2.5,the biomass was the highest,but the P concentration and phytase activity in the solution were the lowest.The concentration of oxalic acid and phytase activity were higher when the pH=3.5-6.5,and the phytase concentration（0.075μmol/min/m L）was the highest at pH=5.5.By adding three soil typical clay minerals（kaolinite（Kao）,palygorskite（Pal）,and montmorillonite（Mon））,the influence of clay minerals to growth activity of A.niger and the adsorption of phosphate and the dissolution of phosphate minerals（hydroxylapatite,HAp）by A.niger were analyzed,and then the influence of typical clay minerals on the mechanism of P dissolution by A.niger was studied.The results showed that Kao and Pal were wrapped by hyphae and formed large particles,while Mon particles were easily dispersed around hyphae,which strongly stimulated the activity of A.niger(up to 30.1 mg C L^-1 medium L^-1 for 36 h,higher than other treatments)and secreted more organic acids.All these factors promoted the dissolution of HAp and the release of P.However,due to the strong adsorption capacity of Mon to organic acids and mineral particles,it will eventually lead to the reduction of available P in the environment in the short term（five days）.This study is of great significance for the future discussion of the effects of pH and clay minerals in the soil environment on the dissolution of insoluble phosphates by A.niger.2.Study on the effect of heavy metal cations on the phosphate solubilization mechanism of A.nigerThree types of heavy metals（lead（Pb）,cadmium（Cd）,copper（Cu））with different concentrations were incubated with A.niger to investigate heavy metals influenced on fungal phosphate solubilization mechanism in aqueous solution.Fungal biomass,P utilization rate,metabolic activity（oxalic acid,phosphatase activity,and respiration activity）,morphology,the characterization of the differentially expressed genes,and heavy metal cation concentration were analyzed.The results showed that the toxicity order of the three heavy metal cations to A.niger was:Cd²⁺>Cu²⁺>Pb²⁺.Low concentration of Pb²⁺（100 mg/L）could stimulate the activity of A.niger.High concentration of Pb²⁺（1000 mg/L）could inhibit the biomass,phosphorus utilization,and metabolic activity of A.niger.Cd²⁺（50 mg/L）and Cu²⁺（300 mg/L）significantly inhibited the biomass and oxalic acid secretion of A.niger,and this inhibition was more evident with the increase of Cd²⁺and Cu²⁺concentrations.TEM results showed that A.niger mainly formed a new barrier to resist high concentration of Pb²⁺（1000 mg/L）.Cd²⁺and Cu²⁺affected the morphology of A.niger,and then had a toxic effect on A.niger.Moreover,RNA-seq results showed that under Pb²⁺stress,the gene encodingα-1,3-glucan synthase in the cell wall was up-regulated,suggesting that Pb²⁺could be toxic to A.niger by changing the structure of the cell wall.However,the genes encoding AAA ATPase protein and efflux pump antibiotic resistance protein were significantly down-regulated under Cd²⁺and Cu²⁺stress,indicating that the metabolism of A.niger was inhibited by Cd²⁺and Cu²⁺stress,and the toxic heavy metal cations could not be excreted in time.3.Study on the mechanism of phosphate solubilization by A.niger in Pb-contaminated soilIn this chapter,a two-year fungal resistance experiment was conducted by adding a high concentration of Pb²⁺（about 3000 mg/L）to the soil to evaluate the change of phosphate-dissolving ability of A.niger under Pb²⁺stress.The differences in the dissolution of two typical phosphates,fluorapatite（FAp）and bioapatite（BAp）,were also compared.The results showed that A.niger could colonize stably in the soil and resist the high concentration of Pb²⁺stress by analyzing the topsoil fungal community and morphology.A.niger secreted organic acids to dissolve BAp and FAp during the growth process,which significantly increased the water-soluble P concentration in surface soil（from 1.2-3.1mg/kg to 4.9-12.6 mg/kg）.A.niger had a better effect on the dissolution of P in BAp.The phosphate released by A.niger dissolving apatite can combine with Pb²⁺to form Pb-oxalate,but only in the treatment of FAp addition,the formation of pyromorphite was found,which all increased the fixation of Pb²⁺in soil and enhanced the resistance of A.niger to the high concentration of Pb²⁺.This study provides a theoretical basis for further research on the biological activation and dynamic changes of P in complex soil systems.