水环境中重金属如汞(Hg)、铜(Cu)、锌(Zn)、镉(Cd)等污染严重,其中汞在生物体内可转变为毒性更强的甲基汞、乙基汞等有机汞,对健康的危害大[1]。在水生食物网中,汞可显著影响藻类生长增殖[2],探讨微藻对汞胁迫的生理响应机制具有理论和实践意义[3-5]。有研究表明,藻细胞不断由胞内释放藻源性有机物(algal organic matter, AOM)到胞外,AOM是水体有机碳、氮及磷的重要来源,且极易与汞发生相互作用,将其吸附在藻细胞表面,进而影响汞在水环境中的形态分布、流动、毒性以及藻类的生长繁殖和发育[6-7];已经确认藻细胞对汞毒性的防御机制之一是AOM与汞的结合。

藻类生长发育繁殖的过程中会大量产生AOM,其部分包裹在细胞外围或者直接分泌释放至周围环境中,形成胞外有机质(extracellular organic matter, EOM),还有部分作为藻细胞基质的主要部分直接贮存在细胞内部,即胞内有机质(intracellular organic matter, IOM)[8]。AOM成分复杂,主要由蛋白质、糖类、腐殖酸等多种生物大分子组成,还存在藻毒素和嗅味物质等有毒有害物质,是一类具有丰富共轭体系的化合物,且具有光吸收能力[9]。因此,可以采用原子吸收光谱、荧光光谱、高效液相色谱等技术评估AOM与重金属之间的相互作用,紫外可见吸收光谱法、荧光光谱法分别用于分析AOM中分子共轭体系及其荧光特征,且因操作简单便捷及破坏性低被广泛运用[10-12]。二维相关光谱(2D-COS)可以用来解决重叠峰问题,揭示AOM结构随外界扰动的变化序列,可用来探索金属离子与AOM中特定分子基团的结合特性[13-14]。但用光谱学技术研究藻类在重金属胁迫下AOM中胞内外有机质变化特性的相关报道较少。

不同藻类对汞的耐受性和响应机制不同,大多数藻类对Hg较为敏感,0.002 mg·L-1 Hg2+对蛋白核小球藻和斜生栅藻有明显的毒性效应[15];>0.025 mg·L-1的Hg2+显著抑制锥状斯式藻的生长[16];超过0.1 mg·L-1的Hg2+会明显抑制中肋骨条藻的生长状态甚至出现死亡[17];0.14 mg·L-1的Hg2+使四尾栅藻的生长受到显著抑制[18]。但雪衣藻(Chlamydomonas nivalis)分布在类似南极、北极的低温、强紫外辐射、贫营养等多重极端环境中,是研究极端环境下藻类抗逆性的理想材料[19-20],且通过预实验发现雪衣藻能够在较高浓度Hg2+下生长。为寻找藻类细胞中耐受汞胁迫的内在机制,需要选择耐受极端环境的优良藻株为对象,为此,本文选用雪衣藻为材料,以生理活性和AOM的释放特性为切入点,基于叶绿素荧光、紫外可见光谱、三维荧光光谱及同步荧光光谱等方法,探究雪衣藻在汞胁迫下响应与调节机制,以期认识汞对藻细胞的毒性效应。

1 材料与方法(Materials and methods)

1.1 材料及培养条件

雪衣藻(Chlamydomonas nivalis)由中国科学院水生生物研究所黄开耀研究员惠赠,选用Bold 3N培养基(121 ℃灭菌20 min),置于温度(10±0.5) ℃、光强35 μmol photons·m-2·s-1、光暗比为12 h∶12 h的光照培养箱中培养,每天定时3次摇匀。离心收集生长于对数期的雪衣藻,初始接种密度OD680=0.3,接种于含不同浓度Hg2+的培养基中。结合已有重金属对藻类耐受性研究及预实验结果,设置处理组Hg2+浓度为0.005、0.05、0.5、1、2、5 mg·L-1,以0 mg·L-1作为对照组,且每组设3个平行,置于光照培养箱中培养96 h。

1.2 比生长速率(μ)和叶绿素a(Chl.a)含量的测定

细胞密度和680 nm处吸光度有很好的线性关系,其生长状况用紫外可见分光光度计(UV-1780,Shimadzu,日本)测定680 nm处的吸光度,表示为OD680[21],根据藻细胞生长情况计算比生长速率,公式如下:

μ=

式中:N0和N1分别表示起始时间t0和取样时间t1的测定值。

在96 h取1 mL混匀藻液于离心管中,经处理后进行紫外可见光扫描,测定在666、653 nm处的吸光度,具体步骤参考文献[22]。Chl.a的计算公式如下:

Chl.a=15.65×A666-7.34×A653

1.3 叶绿素荧光参数的测定

取Hg2+胁迫96 h混匀藻液2 mL置于测量瓶中,暗适应20 min后用Aqua-Pen-100荧光计(Photo Systems Instruments, Czech Republic)测定叶绿素荧光参数:最大光化学效率(Fv/Fm)[23]。测定过程中注意避光。

1.4 溶解性有机碳(dissolved organic carbon, DOC)释放速率的测定

DOC一般能通过孔径为0.45 μm滤膜,包括未被过滤的有机物中的碳含量,代表水体中溶解有机物质的总和[24]。分别在0、96 h时,取20 mL混匀藻液于离心管中,使用孔径0.45 μm滤膜进行抽滤,使用TOC测定仪(MultiN/C 3100,Analytik Jena AG,德国)测定藻液DOC含量。分别再取20 mL混匀藻液用烘干的GF/C滤膜进行抽滤,置于45 ℃烘箱中至恒重后称重,滤膜前后差值即为藻的干质量。DOC释放速率(RDOC,μmol·g-1·h-1)指单位质量雪衣藻(干质量)在单位时间内引起的藻液DOC含量的变化,RDOC的计算公式为[25]:

RDOC=

式中:Ct为试验结束时藻液的DOC的浓度(mg·L-1);C0为空白对照组DOC的浓度(mg·L-1);V为所取的藻液体积(L);WD为一定体积藻液的干质量(kg);MC为碳的相对分子质量;t为试验处理时间(h)。

1.5 AOM的提取与测定

提取方法[26-27]:雪衣藻胞外有机质采用离心法提取,取混匀的藻液在3 500 r·min-1转速下离心10 min,用0.45 μm混合纤维滤膜对上清液进行过滤,得到雪衣藻EOM。胞内有机质采用反复冻融法进行提取,将提取完EOM的离心管内的藻细胞沉淀物用超纯水进行清洗,混合均匀后置于离心机中进行离心,如此重复此过程清洗3次,以去除残留的EOM。用超纯水定容至原体积,之后放置在-80 ℃冰箱中冷冻12 h,45 ℃恒温水浴锅中融化8 h,采用冻融法重复3次。然后再对含藻水样3 500 r·min-1转速下离心10 min,用0.45 μm混合纤维滤膜对上清液进行过滤,得到雪衣藻IOM。按照上述方法提取汞胁迫96 h后雪衣藻胞内外有机质水溶液用于测定。

1.5.1 DOC含量的测定

使用TOC测定仪(MultiN/C 3100,Analytik Jena AG,德国)测定雪衣藻胞内外DOC含量[28]。

1.5.2 紫外可见光谱的测定

将提取到的胞内外有机物水溶液放于1 cm石英比色皿中,使用紫外可见分光光度计(UV-1780,Shimadzu,日本)测定200～800 nm处的紫外可见光谱。光谱中的不同位置的峰代表不同类型的物质,峰的高低可以表示物质的相对含量[29]。溶解性有机质中的官能团可通过特定波长的紫外吸收率来表征,254 nm处的吸光度(A254)代表腐殖质分子的不饱和程度,包括具有碳碳双键的不饱和化合物[30]。

1.5.3 三维荧光光谱的测定

荧光光谱法已经广泛用于对水环境中溶解性有机物的定性鉴定和定量分析,激发-发射矩阵(EEM)涵盖了其组成和官能团结构的大量信息,因此可以用来测定AOM中存在的蛋白质、腐殖酸等有机物[31]。取96 h时提取的1 mL雪衣藻胞内外有机物水溶液,使用荧光光度计(日立F-2700,日本)测定3D-EEM光谱。测试条件如下:激发波长(Ex)范围为220～550 nm,步距5 nm;发射波长(Em)范围为220～550 nm,步距5 nm,激发和发射狭缝宽度均为10 nm。

1.5.4 同步荧光光谱的测定

取96 h时提取的1 mL雪衣藻胞内外有机物水溶液,使用荧光光度计(FL-5600,Perkin-Elmer,美国)测定同步荧光光谱,扫描范围为220～550 nm,激发和发射的狭缝宽度均为5 nm,扫描速度为2 400 nm·min-1,增量为1 nm,Δλ的恒定偏移量为55 nm。光谱中各峰对应物质为[32]:类蛋白质峰(220～300 nm)含有类酪氨酸(220～250 nm)和类色氨酸(250～300 nm);类腐殖酸峰(300～500 nm)含有富里酸(300～380 nm)和腐殖酸(380～500 nm)物质。

1.6 统计分析方法

每组处理设置3个平行,数据统计分析利用SPSS 22.0 (IBM, USA)进行Kruskal-Wallis单因素ANOVA分析及成对比较,相关曲线和图形用软件Origin 2021进行拟合绘制。组间不同小写字母表示差异显著,P<0.05。使用MATLABR 2018b和drEEM工具箱对3D-EEM光谱进行PARAFAC分析,得到相关组分及最大荧光强度(Fmax)。使用Origin 2021软件对同步荧光光谱进行2D-COS分析,绘制同步-异步谱图,通过Noda法则[33]判断同步和异步互相相关峰的符号,进而探究物质结合的先后顺序。

2 结果(Results)

2.1 比生长速率和Chl.a含量

雪衣藻的比生长速率和Chl.a含量如图1所示。与对照组相比,0.005、0.05 mg·L-1的Hg2+低浓度处理组的比生长速率无显著变化,Chl.a含量显著下降(P<0.05);0.5、1、2、5 mg·L-1的处理组比生长速率均显著降低(P<0.05),Chl.a含量显著下降(P<0.01)且2、5 mg·L-1高浓度处理组的比生长速率为负值,雪衣藻的生长受到严重抑制。

2.2 叶绿素荧光参数Fv/Fm

雪衣藻Fv/Fm的变化如图2所示,雪衣藻Fv/Fm随Hg2+浓度的增高而降低,低于2 mg·L-1处理组的Fv/Fm在0.6以上,藻细胞可以维持正常活性,2、5 mg·L-1处理组的Fv/Fm降到0.1以下,活性受到显著抑制(P<0.01)。

2.3 AOM中DOC含量及释放速率

由图3可知,正常情况下,雪衣藻胞内DOC含量高于胞外,但在Hg2+的胁迫下,胞内外有机质含量发生显著变化。随着Hg2+浓度的增高,EOM中DOC含量升高,IOM中DOC含量降低。不同浓度Hg2+对雪衣藻的DOC释放速率有显著影响,DOC释放速率随Hg2+浓度的增加而升高。

2.4 AOM紫外光谱图及参数特征分析

如图4所示,低于2 mg·L-1处理组中,EOM紫外光谱图中峰的位置在300 nm左右,IOM在250～275 nm之间,且2、5 mg·L-1高浓度处理组中峰均不明显。与对照组相比,0.005 mg·L-1最低浓度处理组EOM峰值降低、IOM峰值升高,其他处理组中EOM光谱峰值随着Hg2+浓度的增加而升高,IOM峰值则降低。雪衣藻胞内A254大于胞外,且与对照组相比,随着Hg2+浓度的增加,EOM A254升高,IOM A254降低,胞内外有机质种类和不饱和程度发生改变。

2.5 AOM三维荧光图谱特征分析

图5展示雪衣藻AOM荧光组分的EEM光谱图及激发/发射载荷,表1中列出各荧光组分的最大λex/λem及物质描述。雪衣藻EOM中组分1(C-1)和组分3(C-3)均属于类蛋白质荧光组分,组分2(C-2)属于类腐殖酸荧光组分,该组分中包含A峰和C峰荧光物质;IOM中C-1、C-2均属于类蛋白质荧光组分,C-3中A峰属于类腐殖酸荧光组分。

由图6可知,不同浓度的Hg2+处理96 h后,与对照组相比,胞外类蛋白质和类腐殖酸含量上升,其中类色氨酸含量显著增加;胞内类蛋白质含量在0.005、0.05 mg·L-1处理组中增加,在大于0.05 mg·L-1处理组中下降,类腐殖酸在小于1 mg·L-1的处理组中含量降低,2、5 mg·L-1处理组中含量增加。结果表明,随着Hg2+含量的增高,雪衣藻类蛋白质组分胞外含量增高,胞内先升高后降低;类腐殖酸组分胞外含量增高,胞内先降低后升高。

2.6 AOM同步荧光光谱特征分析

如图7所示,EOM中含有275 nm处的类色氨酸和少量350～425 nm处的富里酸和腐殖酸物质峰;IOM中的物质峰含有225 nm处的类酪氨酸、275 nm处的类色氨酸,以及少量类腐殖酸物质峰,且随着Hg2+浓度的升高,荧光强度发生改变。类色氨酸含量在胞外升高,胞内降低;胞内类酪氨酸含量在低浓度(<1 mg·L-1)中升高,高浓度中降低。同步图和异步图中各交叉峰符号的二维荧光相关结果如表2所示,Hg2+处理96 h后,在胞内外有机质的同步和异步图中选取4个较为明显的峰,通过对比各交叉峰的符号,可知Hg2+与EOM结合亲和力的顺序为375 nm >233 nm>275 nm>285 nm,与IOM结合亲和力的顺序为285 nm>275 nm>225 nm>375 nm,说明EOM结合敏感性为富里酸>类蛋白质,IOM为类蛋白质>富里酸。

3 讨论(Discussion)

重金属对藻类的生长繁殖、形态结构等具有重要影响,能够通过影响光合作用直接影响藻类的生长状态和分布[36-38]。在实验室培养的单一藻类体系中,Chl.a含量与藻密度显著线性相关,因此Chl.a含量也是表征藻类生物量变化的有效指标[39]。不同浓度Hg2+对雪衣藻的耐受实验结果表明,处理96 h后,雪衣藻在1 mg·L-1 Hg2+浓度下能基本维持生长和细胞活性,低浓度(0.005、0.05 mg·L-1)对藻类影响较小,但随Hg2+浓度的增加,生长速率降低,Chl.a含量下降,Fv/Fm降低,这与Hg2+对栅藻进行短期胁迫后的生长响应相似[18]。Hg2+能影响藻细胞的生物量和生长活性,不同藻类受Hg2+胁迫的影响程度与其细胞大小、细胞内可与金属结合的化合物类型和含量、离子作用位点、金属区室化等解毒机制的差异性有关[40-41]。在同一浓度Hg2+胁迫下的毒性效应越小,藻细胞的致死率越低,说明耐受性越强,雪衣藻能耐受1 mg·L-1的Hg2+,耐受性较强。有研究表明,Hg2+取代了来自叶绿素中心的镁离子,并作为镁离子的非荧光活性替代品,且通过与参与叶绿素生物合成酶的功能巯基基团相互作用,损坏叶绿体的结构,降低色素含量,影响光吸收和光合作用,同时降低了光合活性和二氧化碳的固定效率,进一步影响藻细胞生长和生物量生产[42-43]。

藻类对重金属胁迫较为敏感但有一定的耐受性,其中一个原因是胞内外存在多糖、蛋白质和脂质等有机质,有机络合金属的形成可以降低金属对藻类的毒性[44]。有研究表明,在Cd2+的毒性作用下,AOM中蛋白质、脂质等有机质的含量受到显著影响,这与藻细胞的解毒机制有关[45]。DOM中有效官能团的类型和密度决定了藻类对Cu2+毒性效应和生物吸附率的影响[46]。因此,本文从AOM变化特性为切入点,探究藻细胞与汞之间的相互作用。在Hg2+胁迫下,雪衣藻AOM含量和种类都发生显著变化,随Hg2+浓度的增加,DOC释放速率升高,EOM中DOC含量升高,IOM中降低,与Cu2+与Cd2+胁迫链状亚历山大藻(Alexandrium catenella)时DOC释放速率变化一致[47],这一趋势可能是细胞通过产生更多的DOC来缓解毒性,维持正常生长代谢。2、5 mg·L-1高浓度处理组藻细胞已丧失细胞活性,推测是藻细胞死亡后释放出大量的有机质。采用紫外可见光谱和荧光光谱法可进一步探究雪衣藻AOM结构和组成变化。

紫外光谱及参数变化结果中,0.005 mg·L-1处理组EOM光谱峰降低、IOM升高可能是由于重金属刺激藻细胞产生有机物,进而结合重金属形成复合有机物以缓解毒性。Hg2+含量较低时与胞外有机质结合就可以达到解毒效果,只有较少部分进入细胞内,但随着Hg2+浓度的增高,胞内有机质可能需要释放到胞外结合重金属,并依靠新陈代谢将Hg2+通过细胞膜转移入细胞内结合有机质,进一步缓解毒性[48]。三维荧光光谱结果显示,在Hg2+的作用下,雪衣藻AOM中类蛋白质中的酪氨酸、色氨酸以及类腐殖酸物质发生变化,这与Hg2+与其他胞外聚合物结合的物质相似[49]。当藻细胞受到轻微毒性时,会释放类腐殖酸物质,同时细胞内产生类蛋白质结合重金属,在一定范围内重金属浓度越高,类蛋白质相对含量越高,但当重金属含量超过某一阈值,会损伤甚至破坏藻细胞结构,类蛋白质的合成受影响且大量结合重金属离子,含量降低。2、5 mg·L-1处理组中胞外类蛋白质和类腐殖酸含量升高,可能是因为胁迫下死亡藻细胞的破碎释放出大量的荧光物质以及细菌对非荧光物质的降解,这与衰老或死亡的藻细胞破碎后类蛋白质和类腐殖酸的荧光强度变化相似[50]。

有研究表明,细胞为缓解汞离子毒性,会合成结合蛋白或者多肽,从而把部分汞排到细胞外,减少细胞内汞离子的含量[51]。有机分子的动态释放取决于金属的性质和浓度,藻细胞的AOM与不同金属离子的结合亲和力和结合位点也存在差异,如Cu2+与Cd2+胁迫Alexandrium catenella时各荧光组分变化不同[47],莱茵衣藻响应Cd2+与Pb2+胁迫的作用位点不同[52]。根据同步荧光光谱结果所示,雪衣藻IOM 225 nm波段在低于1 mg·L-1的处理组中荧光强度增强,是由于Hg2+与无机或其他有机组分的相互作用导致胞内类酪氨酸物质发生变化。275 nm处类色氨酸波段荧光强度降低可能是因为IOM通过各种络合作用与Hg2+形成复合物,且部分释放到胞外与Hg2+结合,防止其大量进入细胞内。同时,AOM各组分与金属离子结合顺序也存在差异,通过2D-COS来解释AOM响应外部扰动的结构变化顺序,进一步探索金属离子与AOM中特定分子基团的强度变化和结合性质[53],结果显示雪衣藻胞外有机质的结合顺序为富里酸>类蛋白质,胞内则相反。随着汞离子浓度的增加,雪衣藻AOM组成和特性发生变化,EOM和IOM之间存在显著差异,这对进一步理解有机质与重金属结合性能,探索AOM在缓解Hg2+毒性方面的作用提供更深入的见解。

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Responses of Physiological Activities and Algal Organic Matter of Chlamydomonas nivalis to Mercury Stress

Ai Xiaohan1,2, Tu Xiaojie1, Xu Pingping1,2, Xia Yixue1,2, Bi Yonghong1,*

1. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China 2. University of Chinese Academy of Sciences, Beijing 100049, China

Abstract：Mercury (Hg) can significantly affect algal growth and proliferation, but the response characteristics of algal cells to mercury stress is not clear. In this study, Chlamydomonas nivalis were selected and exposed to different Hg2+ concentrations for 96 h. Results showed that: algal cell’s tolerance concentration was 1 mg·L-1 Hg2+; algal biomass and growth state were significantly affected by Hg2+ stress. With the increase of concentration of Hg2+, the dissolved organic carbon release rate increased, extracellular proteins-like and humic acids increased, intracellular proteins-like first increased and then decreased, and the sensitivity of extracellular organic matter to Hg2+ binding was in the order of fulvic acid >protein-like, variation of intracellular organic matter sensitivity was the opposite. The growth, intra- and extracellular organic matter content and structural composition were significantly affected by mercury stress, the dynamic change of algal organic matter was the cellular response to mercury stress, which can enhance its tolerance ability and alleviate the toxicity of mercury. The results contributed to understanding the algal cell’s detoxification mechanism to mercury stress.

Keywords：mercury; Chlamydomonas nivalis; toxic effects; algal organic matter

基金项目：国家重点研发计划课题(2020YFA0907402)

第一作者：艾晓寒(2000—),女,硕士研究生,研究方向为藻类生理生态学,E-mail: aixh@ihb.ac.cn

* 通信作者(Corresponding author), E-mail: biyh@ihb.ac.cn

DOI：10.7524/AJE.1673-5897.20230412001

艾晓寒, 涂晓杰, 许萍萍, 等. 汞胁迫下雪衣藻的生理活性及其藻源性有机物分析[J]. 生态毒理学报,2023, 18(6): 245-256

Ai X H, Tu X J, Xu P P, et al. Responses of physiological activities and algal organic matter of Chlamydomonas nivalis to mercury stress [J]. Asian Journal of Ecotoxicology, 2023, 18(6): 245-256 (in Chinese)

收稿日期：2023-04-12

录用日期：2023-06-22

文章编号：1673-5897(2023)6-245-12

中图分类号：X171.5

文献标识码：A

Received 12 April 2023

accepted 22 June 2023

通信作者简介：毕永红(1974—),男,博士,研究员,主要研究方向为水域生态学。