醋酸钙不动杆菌合成吡咯喹啉醌发酵条件优化及干旱胁迫下对辣椒生长的影响

Acinetobacter calcoaceticus CDWB36: optimization of fermentation conditions for pyrroloquinoline quinone production and effect on growth of pepper under drought stress

摘要:

【目的】研究菌株CDWB36及其代谢产物吡咯喹啉醌(pyrroloquinoline quinone,PQQ)对辣椒抗旱促生的应用效果，为多功能菌剂的开发利用提供优良的菌种资源。【方法】结合形态学和16S rRNA基因对菌株CDWB36进行分类学鉴定；利用HPLC法和光谱法对PQQ进行检测；通过单因素试验探究菌株产PQQ的最佳发酵条件；通过盆栽试验，明确含PQQ的菌剂对干旱胁迫下辣椒生长、生理生化、土壤养分以及根际微生物群落结构的影响。【结果】菌株CDWB36经鉴定为醋酸钙不动杆菌，具备生产PQQ的能力。其最佳发酵条件为酵母粉10 g/L、混合氮源(硫酸铵:谷氨酸:酪氨酸=2:1:1)4 g/L、MgSO₄ 1.0 g/L、CaCl₂ 0.40 g/L、接种量0.5%、温度28℃和pH 6.5。优化后，在摇瓶水平上发酵7 d该菌株PQQ产量可达61.48 mg/L，较优化前提升3.3倍。盆栽试验结果表明，PQQ菌剂处理后，辣椒株高、茎粗、地上部和地下部鲜重较CK分别增加35.05%、8.22%、14.41%、51.70%，促生效果优于PQQ溶液。此外，PQQ菌剂显著提高叶片抗氧化酶活性和渗透调节物质含量，并显著增加土壤养分含量。同时，它明显改变了辣椒根际土壤中细菌和真菌的相对丰度，其中芽孢杆菌属、曲霉属和嗜热链球菌属相对丰度分别为CK的1.99倍、1.38倍和8.75倍。【结论】醋酸钙不动杆菌CDWB36具有合成PQQ的能力，适宜的发酵条件能够显著提高PQQ产量；该菌株发酵液对处于干旱胁迫下的辣椒生长具有显著的促进作用，其中PQQ是发挥这一作用的关键物质，在植物抗逆促生领域具有广阔的应用前景。

关键词:醋酸钙不动杆菌;吡咯喹啉醌;发酵优化;抗旱促生;根际微生物群落

Abstract:

[Objective] To explore the effects of strain CDWB36 and its metabolite pyrroloquinoline quinone (PQQ) on the drought resistance and growth of pepper, so as to provide efficient strain resources for the development and utilization of multifunctional microbial agents. [Methods] A strain CDWB36 was identified based on the morphological characteristics and the 16S rRNA gene-based phylogenetic tree. HPLC and spectroscopy were employed to detect PQQ. The fermentation conditions were optimized by single factor tests with PQQ production as the indicator. The effects of the PQQ-containing microbial agent on the growth, physio-biochemical characteristics, soil nutrients, and rhizosphere microbial community structure of pepper under drought stress were determined by pot experiments. [Results] Strain CDWB36 was identified as Acinetobacter calcoaceticus and it had the ability to produce PQQ. The optimum conditions of strain CDWB36 for producing PQQ were 10 g/L yeast powder, 4 g/L mixed nitrogen sources (ammonium sulfate:glutamic acid:tyrosine=2:1:1), 1.0 g/L MgSO₄, 0.40 g/L CaCl₂, 0.5% inoculum amount, 28 ℃, and pH 6.5. The PQQ production of the strain in shake flasks after 7 days of fermentation at the optimized conditions reached 61.48 mg/L, which increased by 3.3 times compared with that before optimization. Compared with CK, the PQQ-containing microbial agent increased the plant height, stem diameter, aboveground fresh weight, and belowground fresh weight of pepper by 35.05%, 8.22%, 14.41%, and 51.70%, respectively, demonstrating better plant growth-promoting effect than the PQQ solution. Moreover, the PQQ-containing microbial agent significantly improved the activities of antioxidant enzymes and the content of osmoregulatory substances (soluble sugar, soluble protein, and proline) in leaves, while increasing the soil nutrient content. The PQQ-containing microbial agent significantly changed the relative abundance of bacteria and fungi in the rhizosphere soil of pepper, increasing the relative abundance of Bacillus, Aspergillus, and Streptococcus by 1.99 times, 1.38 times, and 8.75 times, respectively, compared with CK. [Conclusion] A. calcoaceticus CDWB36 has the ability to produce PQQ. Optimizing the fermentation conditions can effectively enhance the PQQ production. The fermentation broth of CDWB36 significantly promotes pepper growth under drought stress, and PQQ is a key substance in the broth for promoting pepper growth. Therefore, the strain has broad application prospects in enhancing the stress resistance and promoting the growth of plants.

Key words:Acinetobacter calcoaceticus;pyrroloquinoline quinone;fermentation condition optimization;drought resisting and growth promoting;rhizosphere microbial community