The Study Of Metabolic Mechanism On Insoluble Phosphate Solubilization Process By Penicillium Oxalicum

Phosphorus(P),one of the key maximum nurtrients,is dispensable for animal and human growth.The content of P in soil is abundant accounting for 0.1%-0.15,but most of which exist as insoluble P.The level of bioavailable P is very low,which only accounts for 10-20 mg/L.How to transform the insoluble P to bioavailable P efficiently and sustainably become heat point in recent researches.Phosphate solubilizing microorganism(PSM),which can increase level of bioavailable P,play crucial roles in soil P cycling.Many studies have investigated the mechanisms of PSM,among them the ability of PSM associated with organic acids is generally accepted.However,most of the studies are focusing on the types of organic acids and the effect evaluation of organic acids.It is unknowledgeable about the specific mechanisms and regulations on P solubilization genes and intracellular metabolic pathways,which hamper the utilization of PSM on soil P cycles and agricultural applications.In this study we screened high effective P solubilizing fungus(PSF),then the solubilizing performance of tricalcium phosphate(TP)and iron phosphate(IP)was test and optimized the experimental factors during culture.Also we showed the qualitative and quantitative analysis for types and patterns of organic acids during P solubilization performance.The response models of two key genes which maybe related to release gluconic acid were also studied.Finally,according to metabolimics analysis,the figure about association networks of P solubilization by PSF was drawn.The result are as followed:An insoluble P solubilizing fungus named PSF-4 was isolated from soil at abandoned factory based on medium that TP and IP were the P source.The maximal concentration of P in TP and IP treatments was 2270.17 mg/L,216.81 mg/L,respectively.Cluster of ITS r DNA sequence of PSF-4 showing that microbe belong to Penicillium oxalicum.After optimization of experimental factors,spores concentration-10~7/m L,temperature-28oC and rotation speed-150 rpm are considered as optimal experimental conditions.The qualitative and quantitative analysis for change of organic acids on suspensions by high performance liquid chromatography(HPLC)was conducted,suggesting the more diverse types of organic acids detected in TP treatment,such as oxalic,malic,formic acid,etc.and the major component of acids are monocarboxylic and dicarboxylic.On the other hand,compared to TP treatment,the higher concentration of acids was detected in IP treatment and the major types of acids are citric and gluconic acid.Both of two treatments reached the maximal concentration of acids on day 5,where the concentration of IP about 4-fold higher than that in TP and major component acids are dicarboxylic and tricarboxylic.Also,the concentration of suspension increases with the proportion of dicarboxylic and tricarboxylic increase within 7 days.Before addition of PSF-4,no significant changes of P solubilization performance were found on all treatments with the maximal concentration of each organic acid on day 5.The addition of H~+caused TP solubilization thoroughly but only a slight increase in IP solubilization performance,indicating H~+acted as different roles with PSF-4 response of P source.The expression patterns of pyrroloquinolinequinone-C(pqq C)gene and glucose dehydrogenase(gcd)gene,which are key associated with P solubilization,were studied under the condition of insoluble P and soluble P(CK).It indicated that the transcription of pqq C gene in IP was overexpressed than other treatments and the expression in IP was 1.96-fold than TP treatment by the method of Real-time quantitative PCR(q-PCR).And the expression of gcd gene in TP,IP and CK was 2.4×10~6,2.1×10~6,1.5×10~6,respectively.In additional,synthesis and resolving of gluconic acid was consistent with the changing trend of two gene's expression,suggesting pqq C gene involved in generating gluconic acid.But,the highest expression of gcd found in TP can be interpreted by gcd gene not only involved in synthesis but also resolved gluconic acid into ATP for metabolic activity.73 types of significant difference metabolites were detected in TP,IP and CK treatments by analysis of non-targeted metabonomics,which containing phosphate,organic acids,sugars,nucleosides,etc.It was found saccharose resolved into mannose and glucose in intracellular and the level of glucose phosphate and fructose phosphate was lower than other treatments.In addition,the activation of TCA cycle and glycerolipid metabolism detected up-regulation,As a result,organic acids such as intracellular gluconic acid,oxalic acid and citric acid were synthesized and released into the medium to achieve the degradation of insoluble P.Glyoxylate pathway was detected down-regulation,and the metabolism related to cell multiplication and cytoderm growth such as amino acid,nucleoside,fatty acid metabolisms were up-regulation,suggesting the intracellular energy metabolisms and substance synthesis were affected by addition of insoluble P,which could multiply biomass and stimulate synthesis metabolism likes protein,cytodern and so on in response of metallic ions.Finally,we verified the metallic ions and insoluble P to affect P solubilization performance and microbial growth of PSF-4 through secretion diversities of organic acids and intracellular metabolisms.