Levy Jacqueline L, Stauber Jenny L, Wakelin Steven A, Jolley Dianne F
Centre for Environmental Contaminants Research, CSIRO Land and Water, Private Mailbag 7, Lucas Heights, NSW 2234, Australia.
Chemosphere. 2009 Mar;74(9):1266-74. doi: 10.1016/j.chemosphere.2008.10.049. Epub 2008 Dec 19.
Although single-species laboratory toxicity tests with microalgae are sensitive and highly reproducible, they lack environmental realism. Interactions between algae and their associated bacteria, either in the plankton or in biofilms, may alter algal sensitivity to contaminants, which are not mimicked in laboratory toxicity tests. This study investigated the effects of simple algal-bacterial relationships on the sensitivity of laboratory-cultured algae to copper using 72-h algal growth-rate inhibition bioassays. Four species of microalgae were used, two isolates of each; a strain of algae with no microscopically visible and no culturable bacteria present (operationally defined as axenic) and a non-axenic strain. The four algae used were the marine diatom Nitzschia closterium, the freshwater green alga Pseudokirchneriella subcapitata and two tropical Chlorella spp. Under control conditions (no copper), N. closterium and P. subcapitata grew better in the presence of the bacterial community. Sensitivity to copper (assessed as the concentration to inhibit the growth rate by 50% after 72-h (IC50)) was not significantly different for the axenic and non-axenic strains of N. closterium, P. subcapitata or for Chlorella sp. (PNG isolate). At pH 5.7, the axenic Chlorella sp. (NT isolate) had a 72-h IC50 of 46mugCuL(-1), while in the presence of bacteria the IC50 increased (i.e., sensitivity decreased) to 208mugCuL(-1). However, when the bacterial status of both the operationally defined axenic and non-axenic cultures of N. closterium and Chlorella sp. (NT isolate) was investigated using polymerase chain reaction (PCR) amplification of 16S rRNA followed by DNA fingerprinting using denaturing gradient gel electrophoresis (DGGE), it was found that bacteria were actually present in all the algal cultures, i.e. the axenic cultures were not truly bacteria-free. Based on sequence information, the bacteria present were nearly all identified as alphaproteobacteria, and a number of isolates had high similarity to bacteria previously identified as symbionts or species endophytically associated with marine organisms. The "axenic" cultures contained less bacterial phylotypes than the non-axenic cultures, and based on band-intensity, also contained less bacterial DNA. This supported the findings of few differences in copper sensitivity between strains, and suggests that standard microalgal toxicity tests probably inadvertently use non-axenic cultures in metal assessment.
尽管使用微藻进行的单物种实验室毒性测试灵敏且高度可重复，但它们缺乏环境真实性。藻类与其相关细菌在浮游生物或生物膜中的相互作用，可能会改变藻类对污染物的敏感性，而实验室毒性测试并未模拟这些相互作用。本研究使用72小时藻类生长速率抑制生物测定法，调查了简单的藻菌关系对实验室培养藻类对铜敏感性的影响。使用了四种微藻，每种有两个分离株；一种在显微镜下看不到且无法培养出细菌的藻类菌株（实际定义为无菌）和一种非无菌菌株。所使用的四种藻类为海洋硅藻菱形藻、淡水绿藻斜生栅藻以及两种热带小球藻属。在对照条件下（无铜），菱形藻和斜生栅藻在有细菌群落存在时生长得更好。菱形藻、斜生栅藻或小球藻属（巴布亚新几内亚分离株）的无菌和非无菌菌株对铜的敏感性（评估为72小时后抑制生长速率50%的浓度（IC50））没有显著差异。在pH值为5.7时，无菌小球藻属（NT分离株）的72小时IC50为46μg Cu L⁻¹，而在有细菌存在时，IC50增加（即敏感性降低）至208μg Cu L⁻¹。然而，当使用16S rRNA的聚合酶链反应（PCR）扩增，随后使用变性梯度凝胶电泳（DGGE）进行DNA指纹分析，来研究菱形藻和小球藻属（NT分离株）在实际定义的无菌和非无菌培养物中的细菌状况时，发现所有藻类培养物中实际上都存在细菌，即无菌培养物并非真正无细菌。根据序列信息，所存在的细菌几乎都被鉴定为α变形菌，并且一些分离株与先前鉴定为共生体或与海洋生物内生相关的细菌具有高度相似性。“无菌”培养物中所含的细菌系统型比非无菌培养物少，并且根据条带强度，所含的细菌DNA也较少。这支持了菌株之间铜敏感性差异不大的研究结果，并表明标准的微藻毒性测试在金属评估中可能无意中使用了非无菌培养物。
