Process Biochemistry 40 (2005) 1565–1572 www.elsevier.com/locate/procbio Chromium(III) and (VI) tolerance and bioaccumulation in yeast: a survey of cellular chromium content in selected strains of representative genera H. Ksheminskaa, D. Fedorovycha, L. Babyaka, D. Yanovycha, P. Kaszyckib, H. Koloczekb,* a Institute of Cell Biology, National Academy of Science of Ukraine, Dragomanov Street 14/16, 79005 Lviv, Ukraine b ´ University of Agriculture, Biochemistry Department, Al. 29 Listopada 54, 31-425 Krakow, Poland Received 10 September 2003; accepted 16 May 2004Abstract Fifty-one wild type, naturally occurring yeast strains belonging to various systematic groups were screened for chromium(III) and (VI)uptake at growth-inhibitory concentrations of the metal. Yeast cells were supplemented with Cr at the moment of inoculation with 0.03 mgd.w. biomass/ml and then cultivated for 3 days in optimal growth media. The tolerance to Cr varied depending on the strain tested and the yeastcultures proved to be generally more sensitive to Cr(VI) (concentration range: 0.1–0.5 mM) than to Cr(III) (0.25–5 mM). The levels of cellularCr content ranged from 0.29 to 11.10 mg/g d.w. and 0.21–3.3 mg/g d.w. for Cr(III) and Cr(VI), respectively. Distribution diagrams of the cell-accumulated Cr were constructed for the tested strain population, and the general uptake tendency of middle-range amounts of Cr(III), andlow-range levels of Cr(VI) was revealed. The cell-accumulated Cr levels were similar at identical, non-toxic concentrations of either Cr formsupplemented to the medium. Electron microscopic images proved that cytoplasm and cellular organelles were the ultimate targets foraccumulation of both valences of the metal. The extreme cases of the strains revealing either the lowest or the highest Cr tolerance and uptakecapabilities are discussed in terms of possible bioremediation mechanisms. The applicability of the strains in both environmental andnutritional practice was also considered.# 2004 Elsevier Ltd. All rights reserved.Keywords: Chromium bioremediation; Cr(VI) and Cr(III) toxicity; Yeast survey1. Introduction oxidation states of this element, ranging from 2À to 6+, the most common and stable are Cr(VI) and Cr(III). Both forms Chromium uptake and bioremediation by yeast are gain- are excessively released into the environment; for exampleing much attention since these eukaryotic microorganisms Cr(III) prevails in efﬂuents from tanneries and pigment-have proved to be useful in biotechnological practice. Yeast producing plants whereas the sources of Cr(VI) are, amonghas been applied in the management of Cr-containing waste others: metallurgy, mining, fossil fuel combustion, woodas well as in nutritional supplementation of this trace metal. preservation and cooling installation efﬂuents .Environmental risk caused by Cr contamination is due to a Cr(VI) compounds are known to be extremely toxic tovariety of industrial applications of chromium, which lead living organisms, causing allergies, eczema, irritations, andﬁnally to heavy pollution of soils, ground and surface waters, respiratory track disorders [2,4]; they are also stronglyand the atmosphere . The chemistry of environmentally- mutagenic and cancerogenic [5,6]. This toxic action isreleased Cr compounds is very complex [2,3]. Among many due to the negatively charged hexavalent Cr ion complexes which can easily cross cellular membranes by means of * Corresponding author. Tel.: +48 12 413 38 74; fax: +48 12 413 38 74. sulfate ionic channels , and then undergo immediate E-mail address: firstname.lastname@example.org (H. Koloczek). reduction reactions leading to formation of various reactive0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.procbio.2004.05.012
1566 H. Ksheminska et al. / Process Biochemistry 40 (2005) 1565–1572intermediates [8–10]. These intermediates are themselves of Sciences, Lviv, Ukraine. Strain descriptions are givenharmful to cell organelles, proteins and nucleic acids according to the nomenclature used by appropriate source[5,11,12]. collections. Cr(III) has also been shown to negatively affect cellular Yeast cultures were grown in optimal (rich) liquid media,structures [5,7,13]; however, its toxicity observed in vivo is at a pH of 5.0, containing (per litre): 20 g glucose, 3 gmuch lower as compared to Cr(VI). This fact can be (NH4)2SO4, 0.5 g KH2PO4, 0.2 g MgSO4Á7H2O, 0.2 gaccounted for by the presence of positively charged com- CaCI2Á6H2O, 2 g yeast extract, 2 g peptone, and 2 mg biotin.plexes that are the predominant form of trivalent Cr, which The source of iron (0.2 mg/l) was provided by Mohr salts.are much less soluble and less suitable for transport inside Cells were inoculated at low biomass densities (approxi-cells. Still, the organically-bound Cr(III) derivatives might mately 0.03 mg d.w./ml). Yeast cells were grown in 100 mlalso be transported across cell membranes by some as yet Erlenmeyer ﬂasks on a gyro-shaker (200 rpm) at 30 8C forunknown mechanism, as pointed out by Raspor et al.  three days. Biomass was checked turbidimetrically, as opti-and Srivastava et al. . cal density at 540 nm (OD540). Yeast dry mass, expressed in On the other hand, trivalent Cr has been found to be an mg per ml of the cell suspension, was calculated based on theessential trace element involved in protein structure stabi- appropriate calibration curves as OD540 Â Dilution/1.9.lisation and lipid and glucose metabolism [15,16]. Dietary Viability studies were made by cell surface plating on PetriCr requirements for humans have been determined as 25– dishes and incubating for 48 h in optimal media containing50 mg per day and the most convenient natural source of the 2% bacto-agar.metal seems to be the non-toxic and stable organically-bound Cr present in chromium-enriched biomass. 2.2. Incubation with Cr(VI) and Cr(III) As a useful means for bioremediation of environmentalchromium contamination, yeasts were used to treat Cr- Sterile chromium salt stock solutions (100 mM), Cr(VI)containing efﬂuents in order to remove toxic compounds — K2Cr2O7, and Cr(III) — Cr2(SO4)3Á16H2O were addedfrom waters and soils [17–23]. They were also found to be directly to the cultivation media at concentrations given invery suitable organisms capable of conducting a bioprocess Section 3. Cr(III) was soluble at a cultivation media pH ofaimed at obtaining chromium-enriched biomass used for 5.0. This acidity prevented trivalent chromium from formingbalanced nutrition of mammals and humans [13,24]. In complexes and olation ( and our observations). For eachparticular, yeasts which are found effective in accumulation strain tested, the level of Cr(III) and Cr(VI) supplemented toof aggressive Cr compounds and able to bioconvert them the medium was growth-inhibitory, i.e. it led to a 40–60%into stable, non-toxic and bioavailable forms might be inhibition of yeast culture density growth at the cultivationemployed for successful environmental control. conditions described above. In order to determine the appro- The aim of this study is to screen a variety of yeast genera priate Cr concentrations to be applied, preliminary chro-in terms of Cr(III) and Cr(VI) uptake capacity using cell mium-sensitivity tests were performed for all of the strains,cultures cultivated at optimal conditions, in the presence of as typically presented in Fig. 1. Cell viability checks provedgrowth-inhibitory chromium concentrations. that in all ofcases incubation with sublethal Cr concentra- tions had no impact on cell survival. Determination of the Cr level in yeast cells was performed in samples containing2. Materials and methods approximately 50–100 mg d.w. of yeast. Cells were washed three times with distilled water. Specimens were wet-miner-2.1. Yeast strains, growth conditions and viability studies alised by gently heating in a nitric acid/hydrogen peroxide solution. The total chromium content was determined by Fifty-one yeast strains that were used in the study are listed means of atomic absorption spectroscopy (AAS) using ain Table 1. The strains represent various systematic groups Varian spectrometer model Spectr AA-20B. In several cases,such as Saccharomyces, Zygosaccharomyces, Pichia, Can- Cr determinations were conﬁrmed by the technique ofdida, Debaryomyces, Schwanniomyces, Cryptococcus, Kluy- spectroﬂuorimetric measurement of the ﬂuorescent probeveromyces, Hansenula and several others. They were PTQA (2-(a-pyridyl)thioquinaldinamide)  with a Hita-obtained from the yeast strain collections: American Type chi model F-4500 spectroﬂuorimeter. The analyses of cel-Culture Collection (ATCC), Halle University Collection, lular Cr accumulation were performed for at least threeGermany (H), Collection of the Institute of Biochemistry independent experimental runs.and Physiology of Microorganisms, Pushchino, Russia(IBPhM), National Collection of Yeast Cultures, UK 2.3. Electron microscopy(NCYC), Russian General Collection of Microorganisms(Vsierossijskaja Kollekcija Mikroorganizmov, VKM), and Microscopic images were obtained with a TESLA elec-Centraal Bureau voor Schimmelcultures, Baarn, Holland tron microscope, model BS 500. The specimens of 24 h cell(CBS). The other strains were obtained from the laboratory cultures were ﬁxed with glutaraldehyde and were not furthercollection of the Institute of Cell Biology, National Academy contrasted in order to avoid generating imaging artefacts.
1568 H. Ksheminska et al. / Process Biochemistry 40 (2005) 1565–1572 d.w., respectively). On the other hand, in chromium sensitive C. parapsilosis CBS 88 and C.macedoniensis Y-1507, rela- tively low levels of cellular chromium were determined (0.70 and 0.51 mg/g d.w., respectively). The highest amounts of Cr(III) taken up by the cultures were obtained for C.curvata Y-39 (11.10 mg/g d.w.) and Candida sp. Y-454 (7.53 mg/g d.w.), and both strains were found to be resistant to trivalent Cr (5 and 4 mM, respectively). For the case of Cr(VI), all of the sensitive strains (below 0.2 mM Cr(VI)) showed weak chromate uptake (<0.6 mg/g d.w.). The tolerant strain Pichia guilliermondii Y-1256 (grown at 0.5 mM Cr(VI)) revealed unusually low Cr con- tent (0.28 mg/g d.w.). Among the mostly efﬁcient accumu- lators were Kluyveromyces bulgaricus Y-1494 and K. lactis Y-1527, which revealed the levels of 3.05 and 3.30 mg/g d.w. determined after growth in the presence of 0.4 and 0.3 mM Cr(VI), respectively. When the uptake tendency of both Cr valences is con-Fig. 1. Growth inhibition of P. guilliermondii ATCC 201911 culturestreated with chromium(VI) at concentrations 0.125 mM (a), 0.25 mM sidered, there appears to be no correlation for most of the(b), 0.5 mM (c), 0.75 mM (d), and 1 mM (e). (D) growth kinetics in the strains. In this respect, however, K. lactis Y-1527 stands out,absence of Cr, at optimal medium conditions. as an example of a strain capable of accumulating high levels of both Cr(III) and Cr(VI) (see Table 1). At the same time, such strains as Pichia pastoris NCYC 175 (cellular Cryeast tested (see Fig. 1 for an example). For the case of content: 0.29 mg Cr(III)/g d.w. and 0.32 mg Cr(VI)/gCr(III), the concentration ranged from 0.25–5 mM, and for d.w.) and Hansenula polymorpha NCYC 2309 (0.66 mgCr(VI), from 0.1–0.5 mM. The screening of the yeast cell Cr(III)/g d.w. and 0.40 mg Cr(VI)/g d.w. revealed rather asensitivity to chromium(III) and chromium(VI) and of the poor uptake of both Cr forms.uptake of the metal after a 3-day incubation is given in Table The distribution characteristics of the strains tested in1. Cellular levels of Cr accumulated by the yeast cultures Table 1, based on the cell accumulation data, are presentedshowed some dependence on the initial concentration of the in Figs. 2 and 3. In Fig. 2, the strains have been groupedmetal in the medium; however, various strains revealed a according to the cellular chromium content of Cr(III) (Fig.wide range of uptake capabilities. In particular, several 2A) and Cr(VI) (Fig. 2B), as indicated in the ﬁgure legend.strains are listed which exhibited unusual characteristics Fig. 3 presents the distribution map where the coordinates ofregarding chromium tolerance and/or uptake. For the case of each strain are expressed as the levels of cell-accumulatedCr(III), Kluyveromyces thermotolerans VKM Y-894, Can- Cr(III) and Cr(VI).dida membranaefaciens Y-918 and Y-1510 were found to be Electron microphotographs obtained for Pichia guillier-chromium sensitive (50% growth-inhibition at 0.5 mM con- mondii ATCC 201911 after 24 h exposure to inhibitorycentration), yet, they tended to accumulate relatively high chromium concentrations are shown in Fig. 4. The cells,cellular levels of the metal (3.95, 3.33, and 1.91 mg Cr/g grown to a density of 1 mg d.w./ml, were incubated withFig. 2. Frequency distribution of strains as tested in Table 1. based on the level of cell-accumulated chromium. Each bar represents the number of strains whichaccumulated Cr within the concentration ranges (in mg of Cr per g dry weight): panel A — Cr(III): 0–0.5 (a), 0.51–1.0 (b), 1.1–1.5 (c), 1.6–2.0 (d), 2.1–2.5 (e),2.6–3.0 (f), 3.1–3.5 (g), 3.6–4.0 (h), and >4.0 (i); panel B — Cr(VI): 0–0.25 (a), 0.26–0.50 (b), 0.51–0.75 (c), 0.76–1.0 (d), 1.1–1.25 (e), 1.26–1.5 (f), 1.51–1.75(g), 1.76–2.0 (h), and >2.0 (i).
H. Ksheminska et al. / Process Biochemistry 40 (2005) 1565–1572 1569 are based on the cell culture physiological response to chromium. Therefore, the concentrations of both Cr(III) and Cr(VI) used to treat yeast cultures varied several fold. In order to compare cellular uptake of both chromium forms, in several cases identical levels of Cr(III) and Cr(VI) were applied. The results obtained for selected strains are pre- sented in Fig. 5. Note that out of the three strains tested, only P. pastoris NCYC 175 grown in the presence of 0.5 mM Cr(III) and Cr(VI) accumulated the hexavalent form more efﬁciently than the trivalent form. In the other cases (P. angusta NCYC 2311, H. polymorpha NCYC 2309) similar levels of cellular chromium were obtained. 4. Discussion Yeasts are a very diverse group of eukaryotic microor- ganisms [26,27] and the studies of their interaction withFig. 3. Yeast strain distribution diagram based on the levels of cell-accumulated Cr(III) and (VI), as given in Table 1. The insert reveals the chromium and other heavy metals may reveal differentpositions of two additional strains with exceptionally high levels of cellular resistance and bioremediation strategies. The aim of thisCr(III): Candida sp. IBPhM Y-454 (7.53 mg/g d.w.) and C. curvata VKM Y- study was to compare a number of yeasts of various genera39 (11.10 mg/g d.w.). in terms of their sensitivity to, and the uptake potential for Cr(III) and Cr(VI), as revealed by living biomass cultivated at optimal conditions in liquid rich growth media. Our data4 mM Cr(III) (Fig. 4B) and 1 mM Cr(VI) (Fig. 4C). The indicate profound differences within the group of the yeastexample of a control image (absence of chromium in the strains studied, which is in agreement with other observa-medium) is shown in Fig. 4A, where typical round-shaped tions  where the diversity was found not only amongcells can be seen with poorly visible cellular structures. It different genera but even between the strains belonging toshould be stressed that the images were obtained after the same taxonomic group. The results of this paper also givespecimen ﬁxation with glutaraldehyde only, and that no evidence against the view of some authors that metal uptakecontrasting techniques, involving any heavy metal, were depends mainly on surface binding mechanisms rather thanapplied, which could have interfered with the chromium- on metabolic activity .rich regions and thus could have generated possible imaging Experimental work presented here provides, for the ﬁrstartefacts. The photographs in sections B and C reveal the time, a systematic survey on yeast of representative genera.effect of chromium on cellular structures as well as the In a complementary study, similar screening involvingpossible intracellular targets for the bioremediated metal. various yeast genera was made by Batic et al. ; however,Signiﬁcant changes in cellular interior contrast and density the work focused on Cr(III) toxicity rather than Cr accu-could be observed after interaction with both Cr(III) and mulation, and, unlike our tests done in liquid media, theCr(VI) compounds indicating the intracellular deposition of experiments were performed using the technique of surfacechromium. In the case of Cr(III) presence (Fig. 4B), the plating on solid media. The studies on chromium tolerancecellular organelles were much more structured relative to the and interaction with yeast, as published by other authors,control, and it was typical to observe dark entities (grains), were usually limited to the strains of single, selected sys-located preferentially in the vacuole, which seem to be the tematic groups. So far, the most extensively examined yeastsend-effects of Cr(III) accumulation. The trivalent form of the were Saccharomyces sp., Schwanniomyces occidentalis (seemetal, however, did not affect the general appearance and  and the references therein) and the environmentalshape of the cells. isolates of Candida sp. and Rhodotoruloides sp. [7,17,30]. The addition of Cr(VI) (Fig. 4C) to the culture medium More recently, bioremediation of chromium was studiedresulted in the formation of similar structures in the cell with Candida intermedia , C. utilis  as well as withinterior and the appearance of the dark grains found in wild-type and mutant strains of Schizosaccharomycesregions which could be identiﬁed as vacuole. However, pombe , and Pichia guilliermondii .these grains were much sharper and denser in relation to The uptake of chromium strongly depends on a variety ofCr(III) supplementation. Hexavalent chromium was also factors, with the metal valence, concentration and the naturefound to affect yeast cell morphology, causing irregular of the chemical complex of Cr itself being the most impor-shape, as exempliﬁed by the left-hand image of Fig. 4C. tant. The cellular content of this metal has also been proved As indicated earlier, the accumulation data presented in to be dependent upon the cultivation mode [13,24], mediaTable 1 were obtained at growth-inhibitory conditions that composition and availability of energy source metabolites
1570 H. Ksheminska et al. / Process Biochemistry 40 (2005) 1565–1572Fig. 4. Electron microscopic images of P. guilliermondii ATCC 201911 cultivated at control conditions (A) and incubated for 24 h with chromium: 4 mMCr(III) (B) (Cr cellular level; 0.83 mg/g d.w.), and 1 mM Cr(VI) (C) (Cr cellular level; 0.53 mg/g d.w.). Bars: 1 mm.
H. Ksheminska et al. / Process Biochemistry 40 (2005) 1565–1572 1571 of the strains (n = 27) were found to exhibit relatively low uptake capabilities (0.25–0.5 mg/g d.w.). Based on the data presented in this paper it is not likely that the problem of Cr toxicity is closely and exclusively related to Cr overaccumulation, as suggested by several authors [7,13,17,22,36]. Among the yeasts examined, sev- eral strains can be found which were tolerant to Cr and were still able to accumulate high levels of the metal (see Section 3 and Table 1). At the same time, the results of this study may enable strains to be chosen for further bioremediation researchFig. 5. Cellular chromium uptake obtained after treatment with Cr(III) which should include characterisation of Cr(III) and Cr(VI)(light bars) and Cr(VI) (dark bars) at identical concentrations of the metal transport and/or biosorption, metabolism of Cr complexes(given in brackets) in 3-day cultures of: (1) P. angusta (0.5 mM), (2) P. and intermediates inside cells, detoxiﬁcation pathways aspastoris (0.5 mM), (3) H. polymorpha (1 mM) grown after inoculation with well as bioconversion into biologically available forms.approximately 0.03 mg cells/ml. Strains having unusual characteristics such as hypersensi- tivity, hyperaccumulation, elevated tolerance or low perme-, yeast growth phase and biomass initial density [13,32] ability towards Cr compounds could be selected andas well as on the presence of certain modulators such as ions subjected to detailed studies.(e.g. sulfate, phosphate, ferrous), chelators, riboﬂavin, etc. Regarding possible environmental applications, it should([33,34], and Fedorovych et al., unpublished). be pointed out that both Cr(III) and Cr(VI) forms are All the yeasts employed in this study were wild-type identiﬁed in industrial efﬂuents and waste and that bothstrains previously isolated from natural sources. This work valences of the metal can be detrimental to living cellswas focused on the living yeast biomass only in order to provided they reach intracellular regions. The main ﬁndingreveal physiological conditions affecting the bioaccumula- of this work is that yeast cells, under certain conditions, cantion process. That is why the concentration of Cr supple- accumulate Cr(III) with an efﬁciency comparable to Cr(VI)mented to the culture cultivation media was based on a 40– (cf. Fig. 5). This observation indicates either the existence of60% growth inhibition criterion. Such sub-lethal Cr levels independent transport mechanism(s), as was considered byhad no effect on cell survival but rather induced a stress Raspor et al. , or, at the very least, the permeability of theresponse as observed by a prolonged lag growth phase and cell membrane to trivalent chromium species. Althoughthe hampering of the yeasts’ proliferation potential (not Cr(III) was found to be relatively less harmful to yeast,shown). In this respect the dark, grained structures found which is in agreement with other studies [13,22,29], its toxicinside cells as seen in electron microscopy photographs (Fig. effect was still apparent at higher concentrations. If these4) are the stabilised ﬁnal products of chromium metabolism observations are true for other eukaryotes, there would be aby viable yeast. However, in order to explain the detailed strong environmental demand to remove Cr(III) contami-nature of the metal complexes and to describe the intracel- nants, too, since these species could not be regarded as ‘‘lesslular chromium distribution, further studies, involving toxic’’ or not-accumulated. So far, the general strategy for Crmicrospeciation analyses, are required. Based on the micro- (and other heavy metal) pollution control has relied uponscopic images, it is possible that the deposition target of the dissimilatory metal reduction [10,37,38], that is the uptakebioremediated Cr species are cellular organelles, preferen- of toxic and permeable Cr(VI) species by microorganismstially vacuole, rather than cell walls and plasma membranes. and plants and then subsequent bioremediation and conver-The latter cellular structures seem not to change their sion into less toxic trivalent forms. This work may thuscontrast when the yeast is exposed to either Cr(III) or Cr(VI). imply that the environmental risk caused by Cr(III) con-Still, Nevertheless the contribution of physical biosorption tamination is at least underevaluated.mechanisms to the total uptake of chromium might be Finally, the study shows that yeasts are a very hetero-signiﬁcant and should not be neglected [13,35]. geneous group in terms of chromium tolerance and accu- The chromium sensitivity thresholds and accumulation mulation. It appears that they have evolved a variety oflevels determined for Cr(III) did not correlate with those of mechanisms for metal uptake and metabolism. For thatCr(VI) for the distributions of strains examined (Table 1 and reason, the research which would involve laboratory trans-Figs. 2 and 3). The majority of the strains (total n = 51) were port-defective mutants as well as mutants tolerant or sensi-sensitive to a concentration of 1–2 mM Cr(III) (n = 24) and tive to Cr is of high scientiﬁc interest in terms of helping to0.3 mM Cr(VI) (n = 22). In the case of Cr(III) the total elucidate the mechanisms of uptake. Such mutants are beingnumber of strains revealed a Gaussian-like distribution over subjected to separate studies and the results will be pub-the cellular Cr content, i.e. most of the strains tended to lished in future papers. The complexity of bioremediationaccumulate Cr within the middle-range of 0.5–3.0 mg/g d.w. processes in yeast is still a challenging problem with respect(n = 34) (Fig. 2). However, in the case of Cr(VI) the majority to environmental pollution control and nutritional practice.
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