生物炭固定解磷菌剂的材料制备及其解磷机制

Preparation and phosphorus-solubilizing mechanism exploration of a biochar-immobilized phosphorus-solubilizing bacterial agent

摘要:

目的 西南地区土壤酸化问题日益严重，该问题导致不稳定磷库转变为闭蓄态磷库，土壤磷有效性降低，进而影响作物产量并造成磷肥资源浪费。本研究以生物炭为载体，选用一株兼具溶解有机磷和无机磷功能的菌株作为固定化菌种，对其制备条件进行优化。在此基础上，评估生物炭固定化解磷菌剂的稳定性及其对难溶性磷的溶解效果，并探究其解磷机理。方法 以植物根际土壤为研究材料，利用选择性培养基分离筛选解磷菌。基于钼锑抗比色法量化细菌解磷能力，通过生理生化试验和分子生物学分析完成细菌分类鉴定。采用吸附法制备固定化菌剂，并利用单因素试验法确定生物炭固定化菌剂的最佳制备条件。通过傅里叶变换红外光谱、扫描电子显微镜等对其进行表征分析，进一步运用HPLC和荧光法分别对有机酸代谢谱和磷酸酶活性进行定量分析。结果 分离获得的固定化菌株为克雷伯氏菌(Klebsiella sp.)，其对卵磷脂和磷酸三钙的溶解量分别为236.5 mg/L和200.3 mg/L。基因组分析发现该菌株含有27个与有机磷和无机磷溶解转运相关的基因。单因子优化试验表明，在生物炭添加量为30.0 mg/mL、N107接菌量为6.0%、固定温度为30.0 ℃、固定时间为12.0 h的条件下所制备的固定化菌剂对卵磷脂和磷酸三钙的溶磷量相较于游离解磷菌分别提高了24.0%和22.5%。固定解磷菌的生物炭含有更多的含氧官能团，其总比表面积和外表面积较原始生物炭分别提高61.9%和165.1%。解析固定化菌剂的解磷机理，发现尽管其分泌的有机酸种类未发生明显影响变化，但是酒石酸、柠檬酸及总酸含量发生显著变化，且有效提高了培养液中酸性和碱性磷酸酶活性。结构方程模型显示，pH值与磷酸酶活性和有机酸含量密切相关，固定化菌剂通过提高磷酸酶活性和有机酸含量促进难溶性磷的活化。结论 固定化解磷菌剂的制备为难溶性磷活化提供了良好的生物修复材料，并为开发基于微生物组的绿色修复策略提供了创新视角。

Abstract:

Objective As soil acidification in southwestern China becomes increasingly severe, the labile phosphorus pool is transformed into a non-labile phosphorus pool, which reduces the availability of soil phosphorus, affecting crop yield and wasting phosphate fertilizer resources. In this study, we prepared a biochar-immobilized phosphorus-solubilizing bacterial agent with biochar as the carrier and a strain capable solubilizing both organic phosphorus and inorganic phosphorus as the immobilized strain and then optimized the preparation conditions. Furthermore, this bacterial agent was evaluated in terms of the stability and the solubilizing effects on insoluble phosphorus.Methods Selective media were used for the isolation of phosphorus-solubilizing bacteria from plant rhizosphere soil. The molybdenum-antimony colorimetric method was employed to quantify the ability of bacteria to solubilize phosphorus. The bacterial strain was identified through physiological and biochemical tests and molecular biological analysis. The immobilized bacterial agent was prepared by the adsorption method, and the preparation conditions were optimized by single factor experiments. The prepared agent was characterized by Fourier transform infrared spectrometry and scanning electron microscopy. Furthermore, the metabolic spectrum of organic acids and phosphatase activity were qualitatively and quantitatively tested by HPLC and the fluorescence method, respectively.Results The strain Klebsiella sp. was isolated for immobilization, and its abilities to solubilize lecithin and tricalcium phosphate were 236.5 mg/L and 200.3 mg/L, respectively. Genome analysis showed that the strain N107 carried 27 genes related to organic and inorganic phosphorus solubilization. The optimized preparation conditions were biochar addition of 30.0 mg/mL, N107 inoculation amount of 6.0%, immobilization temperature of 30.0 ℃, and immobilization time of 12.0 h. The bacterial agent prepared under the optimal conditions increased the phosphorus-solubilizing capacity for lecithin and tricalcium phosphate by 24.0% and 22.5%, respectively, compared with the free bacterial strain. The biochar-immobilized phosphorus-solubilizing bacterial agent contained more oxygen-containing functional groups, compared with the original biochar, its total specific surface area and external surface area increased by 61.9% and 165.1%, respectively. The mechanism of phosphorus solubilization by the immobilized bacterial agent was preliminarily analyzed. The results showed that the levels of tartaric acid, citric acid, and total acids changed significantly and the activities of acid and alkaline phosphatases in the culture medium were effectively improved, although the types of organic acids secreted by the agent had no obvious changes. The structural equation model showed that pH value was closely related to phosphatase activity and organic acid content, and the immobilized bacterial agent can promote the activation of insoluble phosphorus by increasing phosphatase activity and organic acid content.Conclusion The immobilized phosphorus-solubilizing bacterial agent prepared in this study provides a good bioremediation material for the activation of insoluble phosphorus. This study provides an innovative perspective for developing green remediation strategies based on microbiomes.