Iron enriched yeast biomass – A promising mineral feed supplement

Bioresource Technology 98 (2007) 1622–1628

Iron enriched yeast biomass – A promising mineral feed supplement Maja Pan, Barbara Pinkur, Matevq Kuntarib, Peter Raspor

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University of Ljubljana, Biotechnical Faculty, Food Science and Technology Department, Chair of Biotechnology, Jamnikarjeva 101, 1111 Ljubljana, Slovenia Received 10 April 2006; received in revised form 8 June 2006; accepted 8 June 2006 Available online 28 August 2006

Abstract Yeast biomass enriched with iron could represent a new and safer solution for prevention from anaemia development. Such an iron source is less toxic and has better absorbability in organisms. The purpose of our research was the determination of the most suitable iron source in the cultivation medium for the yeast Saccharomyces cerevisiae, regarding good growth and iron accumulation in cells. Iron(III) citrate, iron(III) chloride, iron(III) nitrate and Fe–EDTA complex were used. The uptake of the chosen iron compound, Fe(III) citrate, by the yeasts Candida intermedia and Kluyveromyces marxianus was also investigated. DiVerent growth behaviour of the three yeast strains in the presence of Fe(III) citrate was observed. The highest amounts of accumulated iron in S. cerevisiae, C. intermedia and K. marxianus biomass were about 13, 20 and 34 mg Fe g¡1 dry wt., respectively. To optimise the accumulation of iron in K. marxianus and to characterise iron enriched yeast biomass, further experiments are needed. © 2006 Published by Elsevier Ltd. Keywords: Yeasts; Growth; Iron compounds; Accumulation; Feed supplement

1. Introduction Iron is the most abundant and the most versatile trace element in the body. It is an important component of molecules that undergoes redox reactions in cells – vital in life on one hand but also potentially toxic on the other, since reactive oxygen species may be generated, which are known to damage proteins, lipids and DNA (De Freitas and Meneghini, 2001; Eaton and Qian, 2002). Iron deWciency as well as iron overload has serious or even fatal consequences (Aisen et al., 2001). However, the iron-poor nutrition, impaired iron absorption from dietary sources, mostly due to interaction with food components, lowered bioavailability of iron in industrially manufactured food or disturbances in regulation of iron transport, metabolism and resorption lead to the development of iron deWciency anaemia in animals or humans (Hurrell, 2002; Lieu et al., 2001).

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0960-8524/$ - see front matter © 2006 Published by Elsevier Ltd. doi:10.1016/j.biortech.2006.06.002

Although iron deprivation is of limited practical signiWcance in farm livestock, anaemia can develop in young animals, especially piglets, causing illness, subnormal growth and higher mortality (Stehlik-Tomas et al., 2003; Underwood and Suttle, 1999). Iron deprivation can be treated by injection of ferrous salts (e.g. ferrous sulphate), iron-dextran or a dextrin or more recently polynuclear ferric hydroxide complexes. The other possibility is supplementation of iron by the oral route, using diVerent inorganic ferric or ferrous salts of iron or organic chelates, such as iron proteinate or amino acid iron chelate (Legssyer et al., 2003; Underwood and Suttle, 1999; Wienk et al., 1999). However, many inorganic preparations proved to have poor availability, some of them have also shown toxic side eVects, often attributable to reduction potential of such compounds and consecutively reduction of ferric iron by superoxide anion and participation in Fenton chemistry. On the other hand, there is no convincing evidence that provision of iron in chelated forms enhances bioavailability (Close, 1998; Emerit et al., 2001; Fox et al., 1997; Legssyer et al., 2003; Lund et al., 1999; Underwood and Suttle, 1999).

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Yeast biomass enriched with iron, when used as a feed supplement, could represent a new and safer solution for prevention from anaemia development by animals. Iron bound to organic carriers, such as diVerent macromolecules in yeast cells, proved to have better absorbability in the organism and it is less toxic. In addition, yeast biomass is a good source of proteins, essential amino acids and vitamins (Janzso et al., 1993). As for other organisms, iron is an essential nutrient for yeasts, too, although on the other hand, it can be potentially toxic to cells. That is why the uptake and utilization of iron in yeast cells is tightly regulated (Philpott et al., 2002; Protchenko et al., 2001). Transport mechanisms, iron localization and storage in yeast cells have been widely studied in Saccharomyces cerevisiae, since this yeast is a good model organism for clariWcation of iron metabolism in higher eukaryotes (De Freitas et al., 2003; Eide, 1998; Eide et al., 1992; Gakh et al., 2002; Gaudreau et al., 2001; Haas, 2003; Kim et al., 2003; Kucej and Foury, 2003; Lesuisse et al., 2003; Radisky and Kaplan, 1999; Schröder et al., 2003). The metal uptake process by microorganisms is complex and dependent on the chemistry of metal ions, speciWc surface properties of the organism, cell physiology and the physico-chemical inXuence of the environment (Goyal et al., 2003). Therefore, with the purpose to produce iron enriched yeast biomass, the Wrst step in our research was to search for the most suitable source of iron in the cultivation medium for the yeast S. cerevisiae, regarding good growth and iron accumulation in cells. Subsequently, we wanted to investigate the uptake of the chosen iron compound by yeast strains diVerent than S. cerevisiae. Yeasts, capable of using waste materials, would be especially interesting from the point of view of biomass production. A good example is Kluyveromyces marxianus, which can grow on dairy wastes using whey as a source of nutrients. 2. Methods 2.1. Microorganisms In our experiments we used yeasts Candida intermedia ZIM 156, K. marxianus ZIM 1867 and S. cerevisiae ZIM 2155 (S288C), obtained from the Collection of Industrial Microorganisms of Biotechnical Faculty, University of Ljubljana. Cultures were maintained and stored on agar plates at 28 °C. 2.2. Media Chemically deWned medium (CDM) reported by Pan et al. (2004) was used for inoculum preparation and for control cultures. Agar plates for storing and maintaining the viability of yeast culture at 28 °C were prepared by addition of 20 g l¡1 agar to the CDM.

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CDM was supplemented separately by the following iron compounds: iron(III) citrate monohydrate (C6H5FeO7 · H2O), iron(III) chloride hexahydrate (FeCl3 · 6H2O), iron (III) nitrate nonahydrate (Fe(NO3)3 · 9H2O) and Fe–EDTA complex (prepared from Fe(NO3)3 · 9H2O and EDTA– Na2 · 2H2O in molar ratio of 1:3, respectively). Iron compounds were added from stock solutions in appropriate volumes to give the initial concentrations of 0.1, 1, 10, 50, 100 mM Fe(III) citrate or 10, 50, 70, 100 M iron(III) chloride, iron(III) nitrate and Fe–EDTA in the medium. Initial concentrations of iron in the media were checked by Flame Atomic Absorption Spectrometry (FAAS), using VARIAN SPECTRA AA-110. A fairly good match between the amount of Fe added to the media and experimentally determined Fe in the media before yeast cultivations was obtained for all iron compounds, with maximal deviations ranging from 10% for FeCl3 · 6H2O and Fe(NO3)3 · 9H2O to 30% in the case of iron(III) citrate and Fe–EDTA. pH of the media was adjusted to 4.0 with 5 M NaOH. The attempts to use beside Fe(III) also Fe(II) compounds as iron source in yeast cultivation medium were unsuccessful, since iron(II) oxalate dihydrate (C2FeO4 · 2H2O) and iron(II) sulphate heptahydrate (FeSO4 · 7H2O) were not soluble under experimental conditions. As iron(III) citrate proved to be the most suitable iron source in the case of S. cerevisiae (see results), this compound was also used for cultivations of yeasts K. marxianus and C. intermedia that were performed in CDM containing Fe in concentration range already described above. 2.3. Culture conditions Inoculum for all cultivations was prepared by transferring 3-day-old culture from agar plate to 75 ml CDM (initial OD650 around 0.15) followed by cultivation in shaking Xasks (28 °C, 200 rpm) to the late exponential phase of growth, which was typical of each yeast strain. 200 ml CDM containing single iron compounds in proper concentrations was inoculated by 10 ml inoculum in the case of S. cerevisiae or 20 ml in the case of K. marxianus and C. intermedia. The Xasks were agitated on a rotary shaker (200 rpm) at 28 °C for 24 h. Three separate cultivations were performed for each concentration of each iron compound and for control cultures, where iron compounds were omitted. The growth of yeasts during cultivations was followed by measuring OD650 in few hour intervals and growth curves (OD650 vs. time) were designed. 2.4. Determination of total accumulated iron in yeast biomass After 24-h cultivation, two samples of the broth from each cultivation Xask were centrifuged (5 min, 4000 rpm) and the supernatant discarded. The pellet was washed three times with distilled water in order to remove loosely

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associated fraction of iron from yeast cell surface. Washed yeast biomass was dried at 105 °C to constant mass and digested with addition of 1 ml of 65% (v/v) HNO3 to 20 mg dry biomass followed by heating for 30 min at 140 °C. After cooling down, the samples were diluted with bidistilled water up to 5 ml and the content of iron was analysed by FAAS. The amount of total iron was calculated per gram of dry weight (mg Fe g¡1 dry wt.). 3. Results and discussion In order to determine the most suitable source of iron in yeast cultivation medium and its optimal concentration for yeast growth and accumulation of iron in the cells, cultivations of S. cerevisiae in media supplemented by Fe(III) citrate, FeCl3 · 6H2O, Fe(NO3)3 · 9H2O or Fe–EDTA were performed. In the case of FeCl3 · 6H2O and Fe(NO3)3 · 9H2O we could examine the eVect of iron concentration only by addition of maximum 100 M Fe to the medium, since above that concentration precipitation occurred. The reason for precipitation could be the appearance of iron hydroxides, their polymerisation or formation of poorly

soluble iron phosphate (ChipperWeld, 2003; Cotton et al., 1999). Due to similar diYculties with dissolving, the same concentration range was used for the Fe–EDTA complex. The most suitable iron compound in this respect proved to be Fe(III) citrate, as the concentration range in the medium could be raised up to 100 mM without formation of any precipitates. Fig. 1a–d represents growth curves (OD650 vs. time) for yeast S. cerevisiae obtained after 24-h cultivation in the presence of diVerent iron compounds in diVerent concentrations. It is noticeable that all tested iron compounds, irrespective of iron concentration, enhanced the yeast growth when compared to the control. This positive eVect can be seen in Fig. 1b–d, when comparing the Wnal OD650 (after 24 h) of control experiments with the Wnal OD650 for growth in the presence of FeCl3 · 6H2O, Fe(NO3)3 · 9H2O and Fe–EDTA, which are about 1.2 and 1.4, respectively. Higher OD650 were reXected in higher biomass concentrations in broth, where OD650 1.4 for S. cerevisiae corresponds to about 1 g l¡1 dry wt. Similar or even higher values of Wnal OD650, ranging from 1.3 to 1.6, were obtained in the case of Fe(III) citrate as a source of iron in the medium, with the exception of 100 mM iron concentration, where no

Fig. 1. Growth of yeast Saccharomyces cerevisiae in the presence of (a) Fe(III)–citrate, (b) Fe–EDTA, (c) FeCl3 · 6H3O, (d) Fe(NO3)3 · 9H2O (24 h, 28 °C, 200 rpm).

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Fig. 2. Growth of yeast Kluyveromyces marixianus in the presence of Fe(III)–citrate (24 h, 28 °C, 200 rpm).

essential diVerence comparing to the control could be observed (Fig. 1a). Such behaviour could be explained Wrst, by the addition of high portion of iron stock solution to the medium (around 25% v/v), when 100 mM iron concentration in CDM was being prepared, which led to dilution of the medium comprising consequently lower amounts of other nutrients or second, by toxic eVects of high concentration of Fe(III) citrate. Beside iron-induced toxicity, usually expressed as oxidative damage of cell constituents (Kruszewski, 2003), the anionic part of iron compound could play an important role, as well. While citrate as an intermediate is secreted from yeast cells to the environment and acts as an important transporter and donor of iron for cell surface reductases (Lesuisse and Labbe, 1994), its role inside yeast cells has not been completely clariWed. However, some authors (Chen et al., 2002) reported toxicity of citrate inside the cells, depending on concentrations of iron in citosol and in mitochondria. We cannot conclude from the results of our experiment, which part of Fe(III) citrate is responsible for inhibition of yeast growth. Literature data of similar studies (Stehlik-Tomas et al., 2003) demonstrate, that the highest amounts of biomass were obtained by cultivating the yeast S. cerevisiae in molasses media containing iron in concentration range from 10.7 to 14.3 mM Fe, using FeCl3 · 6H2O as an iron source. Based on the results for S. cerevisiae we chose Fe(III) citrate as the most suitable iron compound for further experiments with K. marxianus and C. intermedia. When cultivating yeasts K. marxianus in CDM containing Fe(III) citrate in concentration range of 0.1–100 mM, we observed a slightly diVerent growth behaviour comparing to S. cerevisiae. It is evident from Fig. 2 that OD650 at 0.1 mM and 1.0 mM Fe(III) citrate in the medium is lower than for control experiment, what was also reXected in lower biomass concentration in broth (results not shown). Higher concentrations of Fe(III) citrate (from 10 to 100 mM) did not sig-

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Fig. 3. Growth of yeast Candida intermedia in the presence of Fe(III)– citrate (24 h, 28 °C, 200 rpm).

niWcantly inXuence OD650 or biomass concentration, although a slight stimulation of growth could be observed, when compared to control. On the other hand, no used concentration of Fe(III) citrate (from 0.1 to 100 mM) caused growth enhancement of the yeast C. intermedia comparing to control (Fig. 3). DiVerent growth behaviour of three yeast strains we used for research in the presence of iron citrate is quite diYcult to explain, since there are almost no literature data regarding iron metabolism of yeast genera diVerent than Saccharomyces (see Section 1). Still, it is well known, that S. cerevisiae requires certain amounts of iron, which plays an important role in the reactions of citric acid cycle and in many parts of respiration chain (Stehlik-Tomas et al., 2003). According to Jones and Gadd (1990), the optimal iron concentration in media for cultivation of yeast S. cerevisiae would be between 0.1 and 9 M, while the medium we used for control cultivations (CDM) contained 35.8 M Fe added in the form of FeCl3 · 6H2O (Pan et al., 2004). The results of our research indicate that even higher iron concentrations (e.g. 100 M) positively inXuenced the growth of the yeast S. cerevisiae. Yeast cells are able to control iron levels by regulating iron intake and sequestration in response to nutritional iron conditions (De Freitas et al., 2003). Uptake of iron by yeasts from the media containing iron compounds in diVerent concentrations was followed by the determination of total accumulated iron in yeast cells after 24-h cultivations. As can be clearly seen from the results presented in Tables 1 and 2, the accumulation of iron in yeast biomass is dependent on iron source and its concentration in the environment on one hand and yeast strain on the other. Increasing the concentration of FeCl3 · 6H2O or Fe(NO3)3 · 9H2O in the medium up to 100 M (10, 50, 70, 100 M) caused also progressive increase of iron accumulation in biomass of the yeast S. cerevisiae, with the maximum amount about 2 mg Fe g¡1 dry wt. obtained at 100 M

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Table 1 Accumulation of iron in biomass (mg g¡1 dry wt.) of the yeast Saccharomyces cerevisiae after 24-h cultivation in the chemically deWned medium (CDM) containing iron compounds in diVerent concentrations Iron compound

Initial concentration of Fe in CDM (M)

FeCl3 · 6H2O Fe(NO3)3 · 9H2O Fe–EDTA a b

0a

10

50

70

100

0.081 § 0.019 0.090 § 0.003 0.072 § 0.018

0.449 § 0.028 0.573 § 0.056 0.152 § 0.015

1.371 § 0.249 1.407 § 0.055 0.095 § 0.007

1.649 § 0.032 1.640 § 0.070 –b

1.979 § 0.147 2.345 § 0.068 0.095 § 0.003

Control. Not determined.

Table 2 Accumulation of iron in biomass (mg g¡1 dry wt.) of the yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus and Candida intermedia after 24-h cultivation in the chemically deWned medium (CDM) containing Fe(III)–citrate in concentration range from 0 to 100 mM Yeast

S. cerevisiae K. marxianus C. intermedia a

Initial concentration of Fe in CDM (mM) 0.0a

0.1

1.0

10.0

50.0

100.0

0.031 § 0.003 0.025 § 0.005 0.110 § 0.035

2.125 § 0.055 0.613 § 0.024 0.495 § 0.033

13.931 § 0.073 20.990 § 0.822 8.916 § 0.664

2.290 § 0.171 33.576 § 1.480 20.400 § 3.347

1.084 § 0.058 33.016 § 1.616 20.245 § 1.053

0.547 § 0.037 33.601 § 1.805 12.781 § 1.702

Control.

Fe (Table 1). While no signiWcant diVerence in iron uptake could be seen for iron chloride and iron nitrate, the results proved to be quite diVerent when we added iron complexes to the medium (Fe–EDTA and Fe(III) citrate) (Tables 1 and 2). Supplementation of the medium with Fe–EDTA had almost no inXuence on iron accumulation in biomass of the yeast S. cerevisiae, when compared to control (Table 1). On the other hand, the experiments with Fe(III) citrate, which were performed using higher iron concentrations in the medium (up to 100 mM), gave interesting results (Table 2). The amount of accumulated iron increased up to 1 mM Fe in the medium, while above that concentration (10, 50, 100 mM) it dropped again. Yeast cells accumulated about 2 mg Fe g¡1 dry wt. at 0.1 mM and 10 mM iron concentrations, while the addition of 100 mM Fe inhibited iron uptake, which was only 0.5 mg Fe g¡1 dry wt. The highest amount of iron in S. cerevisiae cells, obtained at 1 mM Fe in the medium, was about 13 mg Fe g¡1 dry wt. Possible explanations for lower iron accumulation at higher concentrations in the medium could be found in some previous investigations. Lesuisse et al. (1987) found out that higher concentrations of Fe(III) in the medium caused decrease of negative potential at yeast cell surface due to saturation of negative groups near iron transporters, which otherwise participate in iron uptake. Consequently, the accumulation of iron would be inhibited, what is consistent with our results, too. On the other hand, Tuttle et al. (2003) discussed the possibility of iron eZux out of yeast cells at higher concentrations of iron in the cell interior. This could also be a reason for lower amounts of accumulated iron in S. cerevisiae at concentrations of Fe(III) citrate higher than 1 mM. The fact, that a type of Fe(III) complex aVects the intake of iron into yeast cells has already been established. It was partly attributed to diVerent aYnities of metalloreductases

at cell surface for single complexes (Lesuisse et al., 2001; Philpott et al., 2002). Namely, a great part of iron is accumulated in cell walls, either at non-speciWc sites or bound to proteins (Protchenko et al., 2001). Furthermore, some complexes are very stable in the medium, providing less free iron for binding to cell walls. Additional interpretation of our results regarding Fe(III)–EDTA as iron source in the medium could be found in ability of EDTA to chelate iron in the form of Fe(II). Fe(II), which originates from the reduction of Fe(III) (from Fe(III)–EDTA complex) with metalloreductases at the cell surface, is again bound to free EDTA and as Fe(II)–EDTA complex it is inaccessible to yeast cells (Lesuisse et al., 1987). Still, no data about the exact amounts of accumulated iron in yeast biomass when using diVerent iron complexes in the medium could be found in literature. Thus, when comparing the amounts of total accumulated iron in the cells of S. cerevisiae after cultivation in the presence of diVerent iron compounds at the same concentration (0.1 mM), with the exception of Fe–EDTA, no diVerence could be found, since the values are about 2 mg Fe g¡1 dry wt. (Tables 1 and 2). We could not examine the eVect of iron source on its uptake by yeasts at higher iron concentrations, since supplementation of the medium with amounts giving concentrations above 0.1 mM was only possible for iron citrate (explanation above). The results of iron accumulation by yeasts C. intermedia and K. marxianus after cultivation in the presence of Fe(III) citrate are presented in Table 2. The optimal iron concentration in the medium for uptake by both yeast strains was 10 mM, as the achieved amounts of accumulated iron at that concentration were 20 mg Fe g¡1 dry wt. and 34 mg Fe g¡1 dry wt. for C. intermedia and K. marxianus, respectively. The addition of iron citrate to the medium in higher concentrations (50 or 100 mM) did not enhance iron

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uptake, in the case of C. intermedia we obtained even lower amounts of accumulated iron (about 13 mg Fe g¡1 dry wt.). Comparing the accumulation of iron from the media containing Fe(III) citrate in biomass of the yeast strains belonging to three diVerent genera, S. cerevisiae, C. intermedia and K. marxianus, we could conclude that the uptake mechanisms diVer. Evidently, the enrichment eYciency of yeast biomass with iron is strongly dependent on a yeast strain. Since there are almost no existent data in the literature, one can only speculate ascribing those diVerences between yeast strains or genera to existence of various transport systems or diVerent cell walls composition. In our research, K. marxianus had the best potential in this regard, accumulating 34 mg Fe g¡1 dry wt. at 10 mM Fe(III) citrate in the medium, what proved to be a high amount, if compared to some data from corresponding literature. Stehlik-Tomas et al. (2003) obtained the highest amount of ac- cumulated iron in biomass of the yeast S. cerevisiae of 2.5 mg Fe g¡1 dry wt., where they used FeCl3 at concentrations ranging from 10 to 14 mM as an iron source and the culture conditions were semi-aerobic. Some previous studies on this subject were also performed in vitro. Goyal et al. (2003) exposed washed yeast biomass to diVerent iron concentrations and obtained maximum iron uptake of 16.9 mg Fe g¡1 dry wt. at 0.9 mM iron concentration in solution. As indicated before, S. cerevisiae is mostly used as a working organism in the studies on iron uptake by yeasts. Based on our Wndings it would be interesting to use K. marxianus instead of S. cerevisiae as a subject for further research, especially when this iron enriched yeast biomass is intended for a mineral supplement for animal feed. It is obvious from our results that further experiments are needed to optimise the accumulation of iron in K. marxianus and to characterise iron enriched yeast biomass, which could be also used in human nutrition, since these yeasts belong to the GRAS group. Acknowledgements Ministry of Education, Science and Sport and Ministry of Agriculture, Forestry and Food, Republic of Slovenia (Project No. V4-0756-0481-02) supported the study. The authors are thankful to Dr. Milabib and Dr. Kbanbar from the Department of Environmental Sciences at “Joqef Stefan” Institute in Ljubljana for performing AAS analyses. References Aisen, P., Enns, C., Wessling-Resnick, M., 2001. Chemistry and biology of eukaryotic iron metabolism. The International Journal of Biochemistry & Cell Biology 33, 940–959. Chen, O.S., Hemenway, S., Kaplan, J., 2002. Genetic analysis of iron citrate toxicity in yeast: implication for mammalian iron homeostasis. In: Proceedings of the National Academy of Sciences of the United States of America, vol. 99 (26), pp. 16922–16927. ChipperWeld, J.R., 2003. Iron. Properties and determination. In: Caballero, B., Trugo, C.L., Finglas, M.P. (Eds.), Encyclopedia of Food

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