Yeasts and heavy metals:
some aspects of physiology and theoretical biotechnology.
Supervisor - Prof. Alexander Rapoport
Institute of Microbiology & Biotechnology,
University of Latvia, Riga
The thesis extends to 78 pages including 23 figures and 11 tables. The list of references contains 126 sources. The thesis consists of overall Introduction, Summaries in Latvian, English and Russian, 3 chapters, joint list of references and Appendix.
Increased knowledge about ecotoxicological effects and further legislative demands for reductions of toxic compounds in industrial emissions require an adequate research and development activities. In the area of wastewater treatment heavy metals represent an important class of contaminants.
The possibility of application of fungi and yeasts for heavy metal sorption from aqueous solutions was pointed out recently by many authors and shown as comparable to sorption of heavy metals on commercial ion exchange resins, activated carbon and metal oxides. However, the detailed mechanism remains not fully understood.
Fungal biomass can be used for this purpose in an intact state or after its specific treatment. Thus, the use of dehydrated biomass was shown at the Laboratory of Cell Biology as a promising tool to sorb the precious metals. Dehydration and rehydration of yeasts are complicated processes, which result in marked shifts in the metabolism, chemical composition and structure cells. A wide spectrum of physiological characteristics for yeast cells, subjected to the dehydration-rehydration procedure, including the state of anabiosis, - extensively was studied in the Institute of Microbiology and Biotechnology, University of Latvia for many years and the number of generalizations, related to this phenomenon, is well-documented. This theoretical background could provide the starting point for a new line of investigations related to the yeast sorption activity and yeast response to the metal stress.
Until now the mechanisms of metal sorption by dehydrated yeasts have been relatively unstudied. A wide spectrum of approaches was demonstrated in publications, i.e. yeast treatment with acid, alkali, detergents, cell immobilization in column, autoclaving etc. At the same time the main methodological tool for estimation of sorption activity is the Langmuir sorption isotherm. Although the sorption process occurred in microbial cell is extremely complicated, therefore it can not be explained on the basis of physical and chemical sorption processes alone due to the following reasons. First, detoxification process in the cell, i.e. metabolism-dependent binding of metal, can include the metal transformation into chemically inert form and further deposition. Second, along with the first rapid stage of adsorption process there is a slow long-term metal absorption within the cell, which demonstrate the metal- and organism-specificity.
In respect to the complicity of the biosorption mechanisms, some limitations and assumptions were done in the scope of our research.
- The real waste waters have many non-identified components which can influence the sorption process. Therefore, we have chosen the "model" for investigation which includes a limited range of factors influencing the interrelations "yeast : heavy metal". On the one hand, for this model the culture of Candida utilis was taken as a biosorbent, on the other hand, chromium (VI) (K2Cr2O7 as a source of chromium) - as a heavy metal. Among factors which can influence metal sorption, the various physiological state of C. utilis as well the content of sorption media were studied.
- Chromium sorption by yeasts in the presence of another heavy metal was designated as a "dual-metal-situation". In turn, chromium sorption without other metals - as a "single-metal-situation". In a dual-metal-situation the cation (Me2+) and anion (Cr2O72-) were presented. The studied metals belong to the different groups, according to the Pearson' classification of metals declared their softness or hardness and therefore prediction of their adsorption behavior.
- As biosorption of metal includes both, adsorption and absorption, the join term "sorption" was used in this study.
- The term "incubation" was used in experiments with non-growing cells incubated in aqueous solution with chromium. The term "cultivation" was used in experiments with growing cells in the molasses medium.
- The concentration of metal was chosen in respect to the task. Thus, the maximum sorption activity was determined at the chromium concentration 100 mg l-1, whereas an influence of various factors in dynamics was studied at lower metal concentration, i.e. 10 mg l-1.
- The period of cell incubation with metal was chosen taking into account an activity of process in dynamics. In our experiments the Cr(VI) sorption by C. utilis was shown even in 24; 48; an 72 h of incubation. The duration longer of 72 h was not reflected in these thesis, because i) of particular loss of viability in yeast population, cell lysis, which lead to an increase of intracellular compounds in the sorption media which can interfere with viable cells in sorption kinetics, as well to provoke infection; ii) of inefficiency from the biotechnological point of view.
- The Langmuir sorption isotherm is foreseen for the study of sorption kinetics in the equilibrium metal concentration, i.e. the amount of adsorbed and desorbed metal is equal. During yeast incubation with chromium the equilibrium in this context has not been achieved, therefore we used the likely Langmuir isotherm for various periods of incubation (from 20 min to 72 h), using the term "residual metal concentration", but not "equilibrium metal concentration".
The aim of this work was to investigate the interaction between yeast biomass and heavy metals in order to provide the necessary background for developing an effective technology of waste water treatment.
The main tasks of this work were:
- To estimate and compare Cr(VI) sorption by intact and dehydrated C. utilis cells from chromate solution.
- To reveal the main factors which could influence the sorption kinetics.
- To compare Cr(VI) uptake by non-growing and growing culture of C. utilis in single metal situation and in dual metal situation (in the presence of two-valent cation).
- To study the toxic effect of Cr(VI), Cu(II), Cd(II), Zn(II), Pb(II) to the non-growing (intact and dehydrated) and growing C. utilis, using an inhibitory effect and metal uptake by cells as test criterion.
Investigations were carried out in the Institute of Microbiology & Biotechnology, University of Latvia, Laboratory of Cell Biology, since 1994. The cell electrophoretic mobility and metal localization were studied in collaboration with Dr.B.Arrio and Dr.M.Zinovieva (Institute of Biochemistry, University of Paris XI). Investigations on the dual-metal systems, as well as the chromate ions stability were carried out in collaboration with Dr.D.Millers and Dr.L.Grigorjeva (Institute of Solid State Physics, University of Latvia)
Hexavalent chromium in the form of [Cr2O7]2- and [CrO4]2- anions, generally acknowledged as Cr(VI), is a hazardous environmental contaminant. Various industrial processes generally produce Cr(VI), e.g. chrome plating, manufacture of pigments, leather tanning, wood processing, etc. Heavy metals removal from wastewaters can be realized using physico-chemical methods, as well as biosorption. During last years the potential of fungi and yeasts in heavy metal recovery from aqueous solutions is under intensive investigation. Yeast is a low cost product which in some cases is received in large quantities as a by-product. However, the detailed mechanism of this process remains not fully understood.
The aim of this work was to investigate the interaction of yeast cells with heavy metals, in particular Cr(VI), under various conditions, with a special reference to the physiological state of intact and dehydrated-rehydrated yeasts and physico-chemical properties of sorption media.
Investigations were carried out (since 1994) in the Institute of Microbiology & Biotechnology, University of Latvia, Laboratory of Cell Biology, as well as in collaboration with the Institute of Solid State Physics, University of Latvia and the University of Paris (Sud).
The results of this work demonstrated that Cr(VI) sorption by dehydrated C. utilis cells was much more active at the beginning of incubation as compared with intact cells, whereas the maximum Cr(VI) uptake by intact and dehydrated biomass was found to be similar and achieved of about 6-7 mg Cr(VI) per g dry weight. Yeast dehydration resulted in some changes of the cell structure, which could make the mechanism of the [Cr(VI):cell] interrelation different, than that of intact cells. In this respect the sorption process by intact and dehydrated cells was studied in details in order to reveal the main factors that could influence the sorption kinetics. It was shown that [Cr2O7]2- was converted into [CrO4]2- during incubation of dehydrated biomass with K2Cr2O7. The considerably enhanced Cr(VI) sorption activity by dehydrated cells in the buffered media (pH 8.0) additionally confirmed the difference in sorption mechanisms of intact and dehydrated C. utilis cells.
The changes of cell surface charge upon sorption process were studied. Electrophoretic mobility (EPM) of intact and dehydrated C. utilis was increased after incubation with Cr(VI). The values of EPM correlated with Cr(VI) sorption activity of tested samples. Observed differences of the electronegativity among the cells within one population indicated to the heterogenity of biomass in respect to the cell sorption activity.
Cr(VI) sorption was compared with Cu2+, Cd2+, Zn2+ and Pb2+ sorption by intact and dehydrated C. utilis cells. It was observed, that uptake of chromate anions occurred much more slowly than uptake of cations. Uptake of tested metals by 1 g dw C. utilis was as following: 7 mg (135 *M) Cr, 23 mg (362 *M) Cu, 39 mg (188 *M) Pb, 19 mg (170 *M) Cd, 28 mg (428 *M) Zn. Combination of Me2+ and chromate anions gave a synergistic effect for Cr(VI) sorption, except Cd and Zn, which inhibited Cr(VI) sorption by dehydrated cells.
Additional experiments were done in order to compare sorption activity of non-growing and growing cells. Culture C. utilis, which grew in the presence of Cr(VI), resulted in the same specific Cr(VI) uptake by cells, as compared to the resting cells, i.e. 7 mg Cr g dw-1. Some Cr(VI) reduction into trivalent form was detected during growth. In spite of the total growth inhibition of C. utilis at Cr(VI) concentrations up to 500 mg l-1 in the medium, cell invertases activity was demonstrated.
A toxic effect of Cr(VI), Cu, Cd, Zn, Pb to the intact and dehydrated cells, as well as growing culture was investigated. For the growing culture a toxic effect was studied, using an inhibition of growth and metal uptake by cells as test criteria. Uptake of lead by growing cells was the highest among tested metals without any detectable changes in the growth activity. The growth of C. utilis with tested metals was accompanied by morphological changes, which were specific for each metal.
Summarized knowledges will improve the application methods of heavy metal sorption using yeast biomass. Obtained results were published in 7 scientific papers and 8 abstracts and presented in 8 international conferences and congress on biotechnology.