Studies of biouptake and transformation of mercury by a typical unicellular diatom Phaeodactylum tricornutum

DENG GuiFu 1 ZHANG TianWen 1 YANG LiMin 1 WANG QiuQuan ) 0 1 0 State Key Laboratory of Marine Environmental Science, Xiamen University , Xiamen 361005, China 1 Department of Chemistry & the Key Laboratory of Analytical Science, College of Chemistry and Chemical Engineering, Xiamen University , Xiamen 361005, China Mercury (Hg) is a toxic heavy metal with its biogeochemical cycling in the ocean depending on the type and behavior of the oceanic microalgae. The present work aimed to evaluate bioaccumulation and transformation of Hg by Phaeodactylum tricornutum, a typical unicellular diatom, when exposed to the extremely high level of Hg in order to understand the possible mechanisms of acute stress response. P. tricornutum can accumulate Hg (its bioaccumulation factor is at 104 level), and the 96 h EC50 was estimated to be 145 g L-1. The amounts of surface-bound Hg being about 1.2 to 4.8 times higher than those of intracellular Hg under exposure to HgCl2 (from 20 to 120 g L-1 concentrations) suggested that the cell wall of P. tricornutum is an important fence towards Hg. After entering the P. tricornutum cell, Hg underwent transformation in its chemical form via interactions with high molecular weight sulfur-containing proteins (accounting for 68% of the intracellular Hg), and glutathione as well as the induced phytochelatins (PCs) (24% Hg) which alleviated the toxicity of HgCl2. In addition, the existence of organic ligands greatly influenced the uptake and transformation behavior of P. tricornutum towards HgCl2, especially in the case of cysteine (Cys), which increased the uptake of Hg, but alleviated the toxicity of Hg towards P. tricornutum due to the fact that Cys is an important precursor for the synthesis of PCs inside the cell. The uptake process of Hg by P. tricornutum was in agreement with the Freundlich isotherm, suggesting a typical heterogeneous sorption process. More importantly, we observed the conversion of HgCl2 into methylmercury inside the P. tricornutum cells and its release into the culture solution using HPLC/CVG-AFS and GC-MS, although the mechanism needs to be further investigated. - Microorganisms are able to accumulate heavy metals via both ways of surface-bound sorption and intracellular involvement [1]. The cell walls of microorganisms contain many different functional groups such as amine, carboxyl, hydroxyl, sulfates and phosphates, which interact with heavy metals. Microorganisms can also produce proteins and/or polypeptides such as metallothioneins and other cysteine-rich peptides which complex heavy metals and thus detoxify them in the cells [2]. On the other hand, many kinds of natural and anthropogenic ligands are always present in the aquatic environment, and heavy metals may form complexes with such existing ligands resulting in various chemical species, determining significantly the modes and amounts of the heavy metals to enter into the cells of microorganisms. In addition, some heavy metals may be transformed to methylated compounds by microorganisms [3]. All these accumulation and transformation processes affect the fate and transport of heavy metals in the environment. As one of the typical toxic heavy metals, mercury (Hg) pollution is a global problem because of its persistence, bioaccumulation and toxicity in the environment [4,5], and The Author(s) 2012. This article is published with open access at Springerlink.com therefore is a risk factor for the health of people [6,7]. Hg can take a myriad of pathways to enter the environment, but one of the main sources of Hg to most aquatic ecosystems is the atmospheric deposition of Hg through long-distance atmospheric transport from anthropogenic and natural sources. Generally, the concentration of total Hg is less than 0.12 g L1 in seawater [5], however, the Hg concentration is as high as 2.3 g L1 or even 260 g L1 in some seriously polluted areas [8,9]. Many marine organisms take part in and may play an important role in its biogeochemical cycling in the ocean. Phaeodactylum tricornutum is a unicellular diatom distributed widely in oceanic and fresh waters. It is at the bottom of aquatic food chains, and may be consumed by the next higher level. It represents a major entry point of toxic heavy metals to the organisms at higher trophic levels, leading to their accumulation and bioamplification in higher organisms along the food chains [10,11]. Additionally, because of its known structure and genome [12], it has frequently been used as a model diatom in studies of algal physiology and ecology as well as studies of heavy metal transportation and transformation in the oceanic ecosystem [13]. However, previous studies on P. tricornutum were mainly focused on the accumulation, transformation and toxicity of cadmium and copper, those concerning Hg are scarce [14]. The purpose of our present study was to investigate the bioaccumulation and transformation of Hg by P. tricornutum. The accumulation behavior and toxicity of various Hg species to P. tricornutum as well as the conversion of Hg species inside the cell were studied when P. tricornutum was exposed to the extremely high level of Hg species in order to understand the possible mechanisms of acute stress response and the role of microalgae during the biogeochemical cycling of Hg in the marine environment. Reagents and chemicals 3-N-morpholino-propane-sulfonic acid (MOPS) purchased from Sangon Biological Engineering Technology & Services Co. Ltd. (SBETS, Shanghai, China) was used to control the acidity of seawater culture solutions at pH 7.5. Chymotrypsin, -lactoglobulin, insulin, vitamin B12 and glutathione from SBETS were used as molecular weight standards to calibrate the molecular weight of Hg associated with proteins and peptides expressed in P. tricornutum using size-exclusion chromatography. HgCl2, humic acid (HA), tryptophan (Trp), methionine (Met), cysteine (Cys), nitrilotriacetic acid (NTA) and ethylenediamine tetraacetic acid disodium salt (EDTA), which were used as model ligands during Hg exposure experiments towards P. tricornutum, were obtained from Sinopharm Chemical Reagent Co. Ltd. (SCR, Shanghai China). Sodium borohydride (NaBH4), tributyl phosphine (TBP), acetonitrile (ACN), trifluoroacetic acid (TFA) as well as 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), used for the reduction and derivatization of phytochelatins (PCs) during the extraction, HPLC separation and determination of PCs, were purchased from SigmaAldrich (St. Louis, MO, USA); dichloromethane (CH2Cl2), methanol, mercaptoethanol (-ME), dithioerythreitol (DTE), ammonium acetate (CH3COONH4) and formic acid (HCOOH), used for selective methylmercury (MeHg) extraction and HPLC-AFS determination with photo-induced cold vapor generation (CVG) [1517], were purchased from Merck (Darmstadt, Germany); sodium tetraphenylborate (NaBPh4), also from SCR, was used to derivatize MeHg for GC-MS analysis; and ultrapure water (18 M) was prepared with a Milli-Q system (Mill (...truncated)