解磷菌株黑曲霉PSFM发酵条件优化研究

Optimization of fermentation condition for phosphate-solubilizing fungi (Aspergillus niger)

【目的】利用液体培养法探究不同碳源、氮源、无机盐和微量元素浓度以及发酵时间、温度、初始pH、转速、接种量等条件对解磷菌解磷特性的影响,获得解磷菌的最佳发酵培养基和培养条件。【方法】采用单因素及正交试验法,对一株分离自土壤的解磷真菌黑曲霉(Aspergillus niger)PSFM菌株的基础发酵培养基和发酵条件(发酵时间0～10d、温度20～40℃、初始pH值3.0～11.0、接种量1%～11%、转速120～300rpm)进行优化,测定其发酵液吸光度值,以解磷量的高低作为评价发酵培养基及发酵条件优劣的指标。【结果】分别在无机磷和有机磷液体培养基、简单培养基(SP)、NBRIY、NBRIP、NBRIYP培养基中培养6d后,以无机磷液体培养基的PSFM菌株解磷能力最高,解磷量为526.42mg/L。在12种不同碳源培养基中,PSFM均能正常生长,其解磷能力由大到小依次为:木糖>葡萄糖>乳糖>蔗糖>麦芽糖>半乳糖>山梨糖>甘露糖>果糖>可溶性淀粉>鼠李糖>甘油。以硝酸钠、尿素、酵母浸膏、氯化铵、硝酸镁、硝酸铵、色氨酸、乙酸铵、牛肉浸膏、胰-蛋白胨、甘氨酸、酪氨酸、丙氨酸和精氨酸为氮源时,硫酸铵为唯一氮源的解磷效果最好,菌株PSFM发酵液中有效磷含量最高(990.31mg/L)。菌株PSFM的解磷能力均随着最佳培养基中NaCl、KCl、MgSO4·7H2O、FeSO4·7H2O和MnSO4·7H2O浓度的增加呈先提高后降低趋势,当其浓度分别为0.03%、0.01%、0.03%、0.001%、0.001%时,菌株PSFM的解磷能力均最强。在不同发酵条件下,以发酵6d、温度28℃、初始pH6.0、5%接种量、转速300rpm的PSFM解磷能力最强,解磷量分别为599.24、528.23、603.69、530.57和731.48mg/L。【结论】最适合PSFM菌株的基础发酵培养基为无机磷液体培养基,碳源为1.0%木糖,氮源为0.10%硫酸铵,最佳无机盐配方为:NaCl0.10g/L、KCl0.70g/L、MgSO·47H2O 0.70g/L、FeSO4·7H2O 0.01g/L、MnSO·47H2O 0.01g/L;最佳培养条件为:温度28℃,初始pH6.0,转速180rpm,接种量5%,发酵时间为6d。

【Objective】The present experiment was aimed to study the effects of different ingredients and cultural conditions on phosphate solubilization capacity of Aspergillus niger strain PSFM （isolated from soil） for obtaining best fermentation conditions. 【Method】The treatments included different media （inorganic and organic P, SP, NBRIY, NBRIP and NBRIYP）, C （xylose, glucose, lactose, sugar, maltose, galactose, sorbose, mannose, fructose, soluble starch, rhamnose, glycerin） and N （NaNO3, urea, yeast extract, NH4Cl, Mg（NO3）2, （NH4）2SO4, tryptophan, ammonium acetate, beef extract, pancreas-petone, glycine, tyrosine, alanine and arginine） sources, micro- and macronutrients （NaCl, KCl, MgSO4·7H2O, FeSO4·7H2O and MnSO4·7H2O）, fermentation time （0-10 days）, temperature （20-40℃）, initial pH （3.0-11.0）, inoculation quantity （1-11%）, and rotation speed of incubator shaker （120-300 rpm）. The orthogonal tests were performed to optimizethe fermentation conditions of phosphate-solubilizing fungi. The absorbance of fermentation liquid was determined to calculate the solubilized phosphate content. 【Result】The solubilized phosphate content in inorganic liquid medium was highest （526.42 mg/L） at 6 days after culturing in different media compared to the other mediums. Out of 12 carbon sources, the best growth of PSFM strain was recorded in xylose followed by glucose, while it was least in medium containing rhamnose and glycerin. Out of all the N sources, （NH4）2SO4 showed significantly higher solubilized phosphate content （990.31 mg/L） compared to the other N sources. With the increase of NaCl, KCl, MgSO4·7H2O, FeSO4·7H2O and MnSO4· 7H2O concentration in inorganic phosphorus medium, the phosphate solubilization capacity of PSFM strain increased initially, followed by a decrease, and it reached the highest at their 0.03, 0.01, 0.03, 0.001 and 0.001% concentration, respectively. The phosphate solubilization content of PSFM strain was recorded highest as 599.24, 528.23, 603.69, 530.57 and 731.48 mg/L in 6 days of fermentation, 28℃ temperature, 6.0 of initial pH, 5% inoculation quantity and 300 rpm rotation speed, respectively. 【Conclusion】The results revealed that inorganic phosphorus medium containing 1.0% xylose （carbon source）, 0.10% （NH4）2SO4 （nitrogen source）, 0.1, 0.7, 0.7, 0.01, and 0.01 g/L concentrations of inorganic salts, viz., NaCl, KCl, MgSO4·7H2O, FeSO4·7H2O and MnSO4·7H2O, respectively, was the best when 5% Aspergillus niger strain PSFM was cultured for 6 days at 28℃ temperature, initial pH 6.0 and 300 rpm rotation speed.