摘　要：气单胞菌（Aeromonas spp.）是一类能感染多种水生动物的高致病性病原菌，氨氮和亚硝酸盐胁迫可增强其感染风险，采用益生菌进行水质调节和生物防治，因其无毒无残留、不引起抗药性而备受推崇。本研究以鱼类病原气单胞菌为指示菌，筛选拮抗芽孢杆菌（Bacillus spp.），并检测了所筛选菌株对氨氮和亚硝酸盐超标水样的降解效果；采用形态观察、生理生化鉴定以及分子生物学方法对其进行种属鉴定；采用皮肤敏感与浸泡攻毒试验检测其生物安全性；采用单因素与正交试验方法优化其产孢培养基与发酵条件；采用鱼类病原菌攻毒保护试验和池塘原位调节水质试验评估其在水产养殖上的应用潜能。本研究筛选到一株对气单胞菌有明显抑制效果的芽孢杆菌D34，种属鉴定为贝莱斯芽孢杆菌（Bacillus velezensis），能有效降解不同来源池水氨氮和亚硝酸盐；D34菌对哺乳动物的皮肤无感染作用，对草鱼鱼苗安全；其优化的产孢培养基与发酵条件为酵母浸粉5.0 g/L、生黄豆粉15.0 g/L、氯化钠10.0 g/L、硫酸镁8.0 g/L，发酵时间48 h、温度30℃、接种量5%、转速220 r/min，产孢率与芽孢数分别达到96.5%和2.03×109 CFU/mL；D34菌剂对夏花草鱼抗维氏气单胞菌（Aeromonas veronii）感染具有明显的保护效果，对亚硝酸盐严重超标的池塘的水质有很好的原位调节作用，24 h亚硝酸盐降解率达91.7%。研究表明：贝莱斯芽孢杆菌D34兼具生物防治与水质调节潜能，且生物安全性良好，可为绿色环保的水产微生态制剂研发提供新的微生物资源。
关键词：贝莱斯芽孢杆菌；病原气单胞菌；生物防治；水质调节；益生菌
中图分类号：S942.3          　　　　文献标志码：A       　　　　 DOI：10.3969/j.issn.1007-7146.2025.02.009

Aquaculture and Product Processing in Dongting Lake Area, Hunan Provincial Key Laboratory for Molecular Immunity Technology of Aquatic Animal Diseases, Hunan Engineering Research Center of Aquatic Organism Resources and Environmental Ecology, School of Life and Environmental Sciences, Hunan University of Arts and Science, Changde 415000, China; 2. School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, China; 3. Hengyang Medical School, University of South China, Hengyang 421009, China)
Abstract: Aeromonas spp. are highly pathogenic bacteria that can infect various aquatic animals, their infection risk can be increased under ammonia nitrogen and nitrite stress. The use of probiotics for water quality regulation and biocontrol is highly recommended for its non-toxic, residue free, and non-drug resistant properties. The antagonistic Bacillus spp. was screened using fish pathogenic Aeromonas spp. as indicator and their degradation effect on the water samples with excessive ammonia nitrogen and nitrite was tested. Morphological observation, physiological and biochemical identification, and molecular biology were used to identify the species. The skin sensitivity test and the immersion attack and infection method were used to detect its biosafety. The single factor and orthogonal experimental methods were used to optimize the sporulation medium and fermentation conditions. In addition, the fish pathogen toxicity protection test and the in-situ pond water quality regulation test were also used to evaluate the potential of the strain in aquaculture. In this study, a strain of Bacillus D34 with obvious inhibitory effect on Aeromonas spp. was identified as Bacillus velezensis, which could effectively degrade ammonia nitrogen and nitrite from different sources of pond water. Moreover, D34 had no infectious effect on the skin of mammals and was safe for the grass carp fry, its optimized sporulation medium and fermentation conditions were as follows: yeast maceration powder 5.0 g/L, raw soybean meal 15.0 g/L, sodium chloride 10.0 g/L, magnesium sulfate 8.0 g/L, fermentation time 48 h, temperature 30℃, inoculum amount 5%, rotational speed 220 r/min, under these conditions, its spore production rate and spore quantity reached 96.5% and 2.03×109 CFU/mL respectively. D34 microbial agent had significant protective effect against Aeromonas veronii infection on the summer fry of grass carp and good in-situ regulation effect on the pond water quality with serious nitrite overload, the nitrite degradation rate reached 91.7% in 24 h. Our results showed that Bacillus velezensis D34 has the potential for both biocontrol and water quality regulation as well as good biosafety, providing new microbial resources for the research and development of green and environmentally friendly aquatic microecological agents.
Key words: Bacillus velezensis; pathogenic Aeromonas spp.; biocontrol; water quality regulation; probiotics
（Acta Laser Biology Sinica, 2025, 34(2): 165-175）