利用光合细菌进行微生物修复：一种降低辛硫磷在养殖水中积累的低成本方法

Microbial remediation with photosynthetic bacteria: a low-cost approach to reducing phoxim accumulation in aquaculture water

摘要:

[背景] 中国是农业生产大国，渔林农牧占比庞大。有机农药无论在畜牧业还是水产养殖业都有广泛的应用。有机磷农药（Organophosphorus Pesticide，OP）是应用最广泛的有机农药，具有低毒和不易残留的优点。OP在水体中大量积累可通过生物富集作用间接影响人体健康，由此产生的生殖毒性不容忽视。光合细菌作为环境友好型水体有益菌，部分菌种具有降解有机农药的功能。[目的] 自上海海洋大学明湖中分离纯化得到一株光合细菌（编号SPZ）。探究其对辛硫磷的耐受程度及降解效果，为养殖水体中有机磷农药的生物降解提供目的菌株。[方法] 利用16S rRNA基因序列分析方法对目标菌株进行种属鉴定；利用紫外分光光度法测定分离菌株SPZ和标准菌株ST在不同接种量下的OD₆₆₀并测定实验周期内光合细菌在不同浓度辛硫磷中OD₆₆₀的变化趋势，以示辛硫磷对光合细菌的毒性作用；利用高效液相色谱法（High Performance Liquid Chromatography，HPLC）测定菌株对水体辛硫磷的降解能力；通过HPLC测定加热致死菌与活菌对水体辛硫磷的降解能力，确定菌株对辛硫磷的降解方式。[结果] 16S rRNA基因序列分析表明菌株SPZ与红假单胞菌属相似度高达99%以上，与沼泽红假单胞菌ATCC 17001菌株聚为一支，置信度为100%；菌株SPZ与菌株ST的适宜接种量为10%；菌株SPZ可耐受100 mg/L的辛硫磷，当浓度超过该值后，辛硫磷对光合细菌生长产生显著的抑制作用；当光合细菌进入生长稳定期后添加辛硫磷，1 d时可相对降解辛硫磷（12.97%-26.69%，20.0 mg/L；24.25%-32.85%，2.0 mg/L；16.66%-34.59%，0.2 mg/L），3 d时可相对降解辛硫磷（46.63%-53.95%，20.0 mg/L；24.78%-30.34%，2.0 mg/L；31.92%-39.25%，0.2 mg/L），5 d时可相对降解辛硫磷（93.65%-97.72%，20.0 mg/L；67.69%-74.41%，2.0 mg/L；10.34%-24.27%，0.2 mg/L）。[结论] 菌株SPZ作为一种常见光合细菌，能够有效去除水体中的辛硫磷农药，具有生物修复功能，在水产养殖和有机磷农药污染水处理中有广阔的前景。

Abstract:

[Background] China is a major agricultural producer, with a large proportion of fishery, forestry, agriculture, and animal husbandry. Organic pesticides are widely used in animal husbandry and aquaculture, particularly the organophosphorus pesticides (OPs) which feature low toxicity and little residue. OP indirectly affects human health through biological enrichment, and the resulting reproductive toxicity cannot be ignored. Photosynthetic bacteria are environmentally friendly beneficial bacteria in water, some of which can degrade organic pesticides. [Objective] We isolated a photosynthetic strain (No. SPZ) from the Minghu Lake of Shanghai Ocean University and tested its tolerance to and degradation of phoxim, aiming at provide a strain for the removal of OPs in aquaculture water. [Methods] The strains used in this study were identified by 16S rRNA gene sequencing. The OD₆₆₀ of the isolated strain SPZ and the standard strain ST at different inoculum sizes was measured by UV spectrophotometry to determine the optimal inoculum size. The toxicity of phoxim to photosynthetic bacteria was determined based on the changing trends of OD₆₆₀ of the bacteria exposed to phoxim at different concentration levels. The phoxim-degrading ability of SPZ and ST was determined by high performance liquid chromatography (HPLC), and that of live and dead (from heating) SPZ was also detected (HPLC) to clarify the degradation mechanism. [Results] The 16S rRNA gene sequence analysis demonstrated that SPZ showed 99% similarity to Rhodopseudomonas and clustered with Rhodopseudomonas palustris ATCC 17001 (confidence: 100%). The optimal inoculation volume fractions of SPZ and ST were 10%. SPZ can tolerate 100 mg/L phoxim, and phoxim significantly inhibited the growth of the photosynthetic bacteria at the concentration > 100 mg/L. Phoxim was added to photosynthetic bacteria at logarithmic growth phase and the relative degradation of phoxim was 12.97%-26.69% (at 20.0 mg/L), 24.25%-32.85% (at 2.0 mg/L), and 16.66%-34.59% (at 0.2 mg/L) after 1 day, 46.63%-53.95% (at 20.0 mg/L), 24.78%-30.34% (at 2.0 mg/L), and 31.92%-39.25% (at 0.2 mg/L) after 3 days, and 93.65%-97.72% (at 20.0 mg/L), 67.69%-74.41% (at 2.0 mg/L), and 10.34%-24.27% (at 0.2 mg/L) after 5 days. [Conclusion] The common photosynthetic SPZ can effectively remove the phoxim in water. It has a broad application prospect in aquaculture and OPs-contaminated wastewater treatment.