文章摘要
张亚珍,向媛羚,张庆明,康茜琳,张恒星,张伟,韩颖.外源刺激剂强化芽孢杆菌高效修复Cr(Ⅵ)污染水体[J].农业环境科学学报,2024,43(5):1100-1113.
外源刺激剂强化芽孢杆菌高效修复Cr(Ⅵ)污染水体
Exogenous stimulants enhance the efficacy of remediation of Cr(Ⅵ)-contaminated water by Bacillus sp.
投稿时间:2023-11-23  
DOI:10.11654/jaes.2023-0992
中文关键词: Cr(Ⅵ)污染  非包埋型刺激剂  包埋型刺激剂  芽孢杆菌  Cr(Ⅵ)还原效率
英文关键词: Cr(Ⅵ) pollution  non-embedded stimulant  embedded stimulant  Bacillus sp.  Cr(Ⅵ) reduction efficiency
基金项目:国家科技重大专项(2019YFC1805900);四川省自然科学基金项目(2022NSFSC1223)
作者单位E-mail
张亚珍 西南科技大学生命科学与工程学院, 四川 绵阳 621010  
向媛羚 西南科技大学生命科学与工程学院, 四川 绵阳 621010  
张庆明 西南科技大学应用技术学院, 四川 绵阳 621010  
康茜琳 西南科技大学生命科学与工程学院, 四川 绵阳 621010  
张恒星 西南科技大学生命科学与工程学院, 四川 绵阳 621010  
张伟 西南科技大学分析测试中心, 四川 绵阳 621010  
韩颖 西南科技大学生命科学与工程学院, 四川 绵阳 621010 595749991@qq.com 
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中文摘要:
      目前,利用微生物修复六价铬[Cr(Ⅵ)]污染的土壤和水体已经在实验室及场地大量应用,但由于土著微生物的竞争及场地的寡营养条件,导致外源铬(Cr)高效还原微生物定殖困难。鉴于此,本研究从成本低廉且无二次污染的氮源型/碳源型/矿物型/离子型的微生物刺激剂筛选出发,探讨了不同刺激剂添加方式(非包埋型和包埋型)对抗铬芽孢杆菌(Bacillus sp.)高效还原Cr(Ⅵ)的影响,并采用扫描电子显微镜-能谱(SEM-EDS)对芽孢杆菌表面进行表征。结果表明,甘蔗粉(碳源型)、豆粕粉(氮源型)、钙离子[Ca(Ⅱ),离子型]、蒙脱石(矿物型)这四种刺激剂的添加显著增加了芽孢杆菌对Cr(Ⅵ)的还原效率,进一步利用单因素多水平试验确定了它们的最佳投加量分别为10 g∙L-1、10 g∙L-1、10 mmol∙L-1、8 g∙L-1,在第8天时与裸菌相比,分别添加这4种刺激剂的芽孢杆菌对Cr(Ⅵ)的还原率分别提高了17.48、15.56、11.70、18.84个百分点。在单因素试验的基础上,挑选2个效果最佳的刺激剂甘蔗粉和蒙脱石,利用响应曲面法建立了混合型刺激剂(甘蔗粉+蒙脱石)与菌的最佳投加量,结果显示甘蔗粉投加量为8 g∙L-1、蒙脱石投加量为6 g∙L-1、菌剂投加量为0.3%(V/V)时,培养8 d后,与裸菌相比,添加混合刺激剂的芽孢杆菌对Cr(Ⅵ)还原率增加了32.53个百分点,显著高于单一非混合刺激组。考虑到刺激剂直接加入环境亦会被土著微生物利用,导致对外源铬还原微生物刺激作用的长效性差,本研究还探究了包埋型缓释刺激剂对芽孢杆菌还原Cr(Ⅵ)的长效刺激作用,结果发现,在包埋型刺激剂的辅助下,芽孢杆菌对Cr(Ⅵ)的还原能力随着时间延长而不断增加,其Cr(Ⅵ)还原率分别从86.08%(第2天)增加到97.09%(第28天),添加了非包埋型混合刺激剂的菌从86.92%(第2天)下降到75.93%(第28天)。SEM观察修复28 d的菌,发现未加任何刺激剂的裸菌出现严重皱缩,部分断裂,形态结构不完整;添加非包埋混合刺激剂的菌出现轻微皱缩,点状断裂,形态结构较为完整;而添加包埋型刺激剂的菌光滑,形态结构完整,表面有绒毛状产物,EDS显示绒毛状产物O和Cr含量相对于其他元素较多,包埋型刺激剂提升了菌细胞表面参与的铬固定,降低了铬对细菌细胞的毒性。研究表明,混合刺激剂还原效果优于单一非混合刺激组,确定最佳投加量分别为甘蔗粉(8 g∙L-1)、蒙脱石(6 g∙L-1)和菌剂(0.3%)。利用非包埋型刺激剂有利于短期内快速提升菌对Cr(Ⅵ)还原,而包埋型刺激剂使菌还原Cr(Ⅵ)具有长效性并有效地降低了Cr(Ⅵ)毒性。
英文摘要:
      Microbial remediation of hexavalent chromium Cr(Ⅵ) contamination in soil and water has been extensively studied to date in both laboratory and field settings. Challenges arise due to competition among indigenous microorganisms and the nutrient-deficient conditions of the sites, adversely affecting the colonization of exogenous chromium(Cr)-reducing microbes. To address this issue, this study embarked on screening low-cost and non-secondary pollution nitrogen/carbon/mineral/ion-based microbial stimulants. We investigated the impact of various stimulant application methods(non-embedded and embedded) on the efficacy of reduction of Cr(Ⅵ) by Bacillus sp. and we characterized the Bacillus sp. surface using scanning electron microscopy-energy dispersive spectroscopy(SEM-EDS). The results indicate that separate addition of sugarcane powder(carbon source), soybean meal(nitrogen source), calcium ion Ca(Ⅱ)(ion type), and montmorillonite(mineral type) significantly increased the efficiency of Bacillus sp. to reduce Cr(Ⅵ). Further single-factor multi-level experiments determined their optimal dosages as 10 g∙L-1, 10 g∙L-1, 10 mmol∙L-1, and 8 g∙L-1, respectively. On the 8th day, compared to the bare bacteria, the addition of these four stimulants increased the reduction of Cr(Ⅵ) by 17.48, 15.56, 11.70, and 18.84 percent points, respectively. Building on the single-factor experiments, sugarcane powder and montmorillonite, the two most effective stimulants, were chosen for a response surface methodology to establish the optimal dosage of a combined stimulant(sugarcane powder + montmorillonite) with the bacteria. The results revealed an optimal dosage of 8 g∙L-1 for sugarcane powder, 6 g∙L-1 for montmorillonite, and 0.3%(V/V) for the bacterial agent. After 8 days of cultivation, compared to the bare bacteria, the addition of the combined stimulant increased the reduction of Cr(Ⅵ) by 32.53 percent points, which was significantly more than the reduction by the single stimulant groups. Considering that direct addition of stimulants into the environment can be utilized by indigenous microorganisms, resulting in poor long-term effectiveness for stimulating exogenous microbial chromium reduction, this study further explored the long-term stimulatory effect of embedded slow-release stimulants on the reduction of Cr(Ⅵ) by Bacillus sp. We found that with the assistance of embedded stimulants, the ability of Bacillus sp. to reduce Cr(Ⅵ) increased continuously over time, with Cr(Ⅵ) reduction levels rising from 86.08%(two days) to 97.09%(28 days). In contrast, for bacteria treated with non-embedded combined stimulants, the reduction levels decreased from 86.92%(two days) to 75.93%(28 days). SEM observations of bacteria after 28 days of remediation showed significant shrinkage, partial rupture, and incomplete morphological structure in bare bacteria without any stimulant. Bacteria treated with non-embedded combined stimulants exhibited slight shrinkage, punctate ruptures, and relatively complete morphological structures. In contrast, bacteria treated with embedded stimulants appeared smooth, with intact morphological structures and a fuzzy surface. EDS analysis revealed a higher content of O and Cr in the fuzzy structures compared with other elements, indicating that the embedded stimulants enhanced the fixation of Cr on bacterial cell surfaces, thus reducing the toxicity of Cr toward bacterial cells. In summary, this study shows that the reduction effect of mixed stimulant is better than that of single unmixed stimulant groups, and the optimal dosages are 8 g∙L-1 for sugarcane powder, 6 g∙L-1 for montmorillonite, and 0.3% for bactericide. The use of non-embedded stimulants is conducive to rapid improvement of the reduction of Cr(Ⅵ) by bacteria in the short term, while embedded stimulants provide bacteria with a more long-term capacity to reduce Cr(Ⅵ) and thus effectively reduce the toxicity of Cr(Ⅵ).
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