MODELLING OF MICROBIAL METABOLISM STOICHIOMETRY:

APPLICATION IN BIOLEACHING PROCESSES

Artin Hatzikioseyian, Marios Tsezos

National Technical University of Athens (NTUA), School of Mining and Metallurgical Engineering,

Laboratory of Environmental Science and Engineering, Heroon Polytechniou 9, Zografou 15780,

Athens, Greece. Tel: +30 210 772 2172, +30 210 772 2271. Fax: +30 210 772 2173.

artin@metal.ntua.gr, tsezos@metal.ntua.gr

ABSTRACT

The present paper reviews an approach to theoretical evaluation of stoichiometric reactions thatsupport microbial growth as applied to environmental problems, with special emphasis on bioleachingof sulfide minerals. Our approach is based on the bioenergetic concepts developed by McCarty,according to which microbial redox reactions can be formulated by combining three half̀reactions: theelectron donor reaction, the electron acceptor reaction and the biomass synthesis reaction. In this studyonly chemolithotrophic autotrophs are considered, as this is the case of interest in bioleachingapplications.

Keywords: Microbial Stoichiometry, Bioleaching, Bioenergetics, Thermodynamics.

INTRODUCTION

The stoichiometry of a chemical reaction provides basic information about the nature and the quantitiesof chemical species consumed and produced. In the case of microbiological reactions that supportmicrobial growth, this information deals with the carbon and energy sources consumed, the terminalelectron acceptor utilized, other metabolic products formed, as well as the quantity of the biomassproduced. Such information is useful and necessary for the design of any biotechnological process.In principle an equation of microbial metabolism can be written in the following simplified schematic

form:

(1)

This generalized equation can be formulated for any combination of carbon source, energy source

and terminal electron acceptor following the approach presented by McCarty, [1-5, 7-9]. The

application of this methodology in the case of bioleaching of sulfide minerals is presented below.

STOICHIOMETRY IN MICROBIAL METABOLISM

When microorganisms utilize a chemical molecule (organic or inorganic) as electron donor, a portion

of the electrons (fe0) from the donor is transferred to the terminal electron acceptor and a portion of the

free energy released in this reaction is used for the production of new biomass. For these two processes

the following equation should apply:

(2)

The fraction fs, that is used for net cell synthesis may be lower than the maximum fraction fs0, which

is denoted with the superscript 0, when maintenance requirements are neglected. Correspondingly, fe is

higher than the value of fe0.

For the formulation of any microbial metabolic reaction, three half reactions should be selected and

combined:

(a) The cell synthesis reaction (Rc),

(b) The electron donor reaction (Rd), and

(c) The electron acceptor reaction (Ra)

According to the methodology followed and for reasons of uniformity, all the half reactions are

written on the basis of one-electron change (e- eq), with the electrons present on the left hand side of the

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Proceedings of the 16th International Biohydrometallurgy Symposium 25 – 29 September 2005Editors: STL Harrison, DE Rawlings and J Petersen Cape Town, South AfricaISBN: 1-920051-17-1 Produced by Compress www.compress.co.za

equation, regardless of whether the reaction involves oxidation or reduction. The overall metabolic

reaction (R) is the sum of the half reactions according to the equation:

(3)

The negative sign of an electron donor reaction means that this reaction should be reversed beforeadding it to the others.The most important mineral decomposing microorganisms in bioleaching applications are the iron and

sulfide oxidizing chemolithotrophs. These microorganisms grow autotrophically by fixing atmospheric

CO2. Chemolithotrophs may obtain their energy by oxidizing either ferrous iron (Fe2+) or reduced formsof inorganic sulfur compounds (some use both) as an electron donor. Other chemolithotrophs obtain theirenergy from oxidizing some other oxidizable inorganic molecules. Oxygen is used as the terminal electron

acceptor, although many of the sulfur oxidizing microorganisms are able to use ferric iron in place ofoxygen as electron acceptor alternatively. This latter ability is important in non-aerated heap reactors inwhich oxygenmight not penetrate to the bottomof the heap.Typically, oxygen is introducedby aerationof

an iron – and/or sulfur containing mineral suspension in water or, in the case of heap leaching, byirrigation. Because sulfuric acid is produced during the oxidation of the inorganic sulfur, thesemicroorganisms grow in low-pH environment. Most mineral bio-oxidation processes take place at a pHbetween 1.4 and 1.6, at which ferric iron is soluble. The modest nutritional requirements of these

microorganisms are provided by small quantities of inorganic fertilizer, which can be added to ensure thatnitrogen, phosphorous, potassium and other trace elements are available [6].In mineral biooxidation processes that operate at 408C or less, the most important microorganisms

are believed to be a consortium of Gram-negative bacteria. These include the iron- and sulfur-oxidizingAcidithiobacillus ferrooxidans (previously known as Thiobacillus ferrooxidans), the sulfur oxidizingAcidithiobacillus thiooxidans (previously known as Thiobacillus thiooxidans) and Acidithiobacillus caldus

(previously known as Thiobacillus caldus) and the iron-oxidizing Leptospirillum ferrooxidans andLeptospirillum ferriphilum which become established in bioleaching processes at temperatures rangingfrom 30 to 458C. So mesophilic biooxidation of ferrous iron has been extensively studied [6].

Fewer studies have been reported on microorganisms that dominate bioleaching consortia attemperatures of 508C or above. However these are believed to include Acidithiobacillus caldus, someLeptospirillum spp. bacteria belonging to some Gram-positive genera Sulfobacillus and Acidimicrobiumand frequently members of the archaea genus Ferroplasma, [6].

Recently an increased interest in the application of high temperature processes (65 to 808C) utilizingthermophilic archaea such as Sulfolobus acidocaldarius, Sulfolobus metallicus, Acidianus brierleyi andMetallosphaera sedula, has been shown, [6].

Table 1 presents the most important electron donor half reactions involved in bioleachingapplications. Reaction presents the oxidation of ferrous to ferric ion. As can be seen, the reaction isindependent of [H+] concentration, so pH changes do not affect the free energy of the reaction.

Nevertheless, the reaction usually takes place at pH values below 1.5 in the bulk phase at which bothFe2+ and Fe3+ remain soluble. Reactions -, present the oxidation of different forms of inorganic sulfurto sulfate in bioleaching. In these reactions sulfur at different oxidation stages (-2, 0, +2, +4 in the

form of sulfides, elemental sulfur, thiosulfate and sulfite respectively), is oxidized to sulfate, in whichsulfur has an oxidation state of +6. All these reactions are pH dependent, meaning that [H+]concentration affects significantly the value of the free energy of reaction. It should be noted that all thereactions in Table 1, are written as electron-consuming half reactions, with the electrons appearing on

the left hand side of the equation, although these reactions operate in the reverse direction because oftheir electron donor function in bioleaching.Table 2, presents the most important electron acceptor reactions in bioleaching. They typically

involve oxidation of sulfide minerals under aerobic conditions with oxygen as terminal electronacceptor, as in reaction (9). Alternatively, under oxygen limited conditions, ferric iron might act asterminal electron acceptor as shown in reaction (10).

Finally, Table 3 presents the synthetic reactions involved in formation of cell biomass, utilizing twoalternative nitrogen sources: ammonium (reaction (11)) or nitrate (reaction (12) ). The formula weightC5H7O2N, represents the new cell material and is very commonly used in representing the microbialbiomass in environmental biotechnology, [7-9].

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Table

1.Themost

importantelectrondonorreactionsforbioleachingapplications.

5

Table

2.Themost

importantelectronacceptorreactionsforbioleachingapplications.

Table

3.Cellsynthesisreactionswithammonium

ornitrate

asnitrogen

source.

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The free energy released in each of the half reactions can be calculated from the free energy of

formation of the reactants and the products. Such values can be found in the literature, [7, 9]. Inbiochemistry the free energy change of a reaction is calculated at 258C, assuming 1M concentration forall aqueous species and 1 atm partial pressure for gases. Because the optimum pH at which most

bacteria grow is around neutral (most are neutrophiles) the pH of the growth medium is fixed at 7.0 i.e.[H+]=10-7. In addition, intracellular pH is generally assumed to be circumneutral, regardless ofwhether an acidophile, a neutrophile or an alkaliphile is considered. This free energy is given with the

notation DG0 and takes into account the neutral pH of the micro and macro environment of the cells.However, in most bioleaching, processes bacteria grow at very acid pH (e.g. pH 1.5 to 2). This acidity isthe result of H+ production from the oxidation of sulfur to sulfate. In these cases it is much moreappropriate to calculate the standard free energy for each half reaction without any pH correction, (i.e.

[H+]=1M is assumed in the bulk phase), although still the intracellular pH of the cells remaincircumneutral. This free energy is given with the notation DG0 , and is presented in the second column ofthe tables. Thus, for biochemical reactions taking place inside the cell membrane or in neutral pH

values, DG0 is the most appropriate selection, whereas for reactions taking place in the bulk phase ofbioleaching the corrected value of DG0 can be selected instead.For any reaction given in the generalized form, (where A1 . . . Ak are the reactants and Am . . . An are

the products of the reaction with u1. . . uk, um . . . un the stoichiometric coefficient of the reactants andproducts respectively):

(13)

the correction for the nonstandard conditions can be calculated by the equation

(14)

where, DG0 is the free energy of the reaction under standard conditions (temperature 258C,concentration of all species 1M and partial pressure of gases 1atm), T the absolute temperature in Kand R the ideal gas constant 8.314 J/mol K.By using equation we are able to recalculate the changes of the free energy of reactions at different pH

values as shown in Tables 1-3. For pH adjustment we have recalculated the free energy of reaction at[H+]=1M, (pH values 0). As can be seen the [H+] concentration changes the values of the reaction freeenergy significantly and should not be neglected. In contrast, temperature does not significantly affect

the free energy of reaction when pH remains constant. As an example, in the range from 258C to 808C,the free energy of reaction is changed from 16.41 KJ/e-eq to the value of 14.54 KJ/e-eq when the pH is1.5, (calculations not shown). Therefore, free energy corrections are important when the concentrations

of electron donor or electron acceptor are very low (much lower than 1M) or when pH valuessignificantly deviate from pH 7. These conditions are common in bioleaching processes.

ESTIMATION OF THE FRACTION fs0

The fraction of the electrons that are used in cell synthesis fs0, can be estimated from the cell yield, if

adequate experimental data are available.

Typical maximum values for fs0 are given in Table 4, and are appropriate for young, rapidly growing

bacterial cells, [8]. In general reactions between electron donors and acceptors that yield more energyallow for the shunting of more energy to biosynthetic reactions resulting in higher values of fs

0. Thus,the heterotrophic metabolism of carbohydrates, proteins, fatty acids etc. under aerobic conditions has

an indicative fs0 value in the range of 0.60-0.70, whereas autotrophic biomass formed from CO2 and

inorganic nitrogen with the aid of inorganic electron donors, exhibit fs0 values between 0.04-0.20. For

old or slowly growing cultures, values of fs would be less than fs because a larger portion of the energy is

used for cell maintenance than for cell synthesis. The value of fs also depends upon the characteristics ofthe bacterial species, the energy transfer efficiencies and the environmental conditions of the growingcells.

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Table 4. Typical values for fs0 for bacterial reactions, [8].

Alternatively, a theoretical value of fs0 can be estimated from a thermodynamic analysis after

identifying the half reactions for the electron donor, electron acceptor and for cell synthesis, [9]. Thisapproach has been used for the background of this paper in estimating the effect of pH and temperatureon the value of fs

0.

OVERALL REACTION STOICHIOMETRY FOR BIOLEACHING APPLICATIONS

Table 5, presents the effect of pH and temperature on the fs0 values for the cases of Fe(II) and sulfide

oxidation reactions. The values have been estimated by applying the thermodynamic approachpresented by Rittmann, [9], (the details of the calculations are not shown here).It is interesting to note the low fs

0 for Fe(II) oxidation at pH 7, which is increased significantly when

the pH of the bulk phase is lowered to 1.5. Typical calculated maximum values of fs0 for Fe(II)

oxidation range from 0.1 to 0.15 and are lower than values observed when different forms of sulfur arethe electron donors, (see Table 4). This is due to the low energy yield of the redox reaction when Fe(II) is

used as electron donor. With regard to the effect of temperature when the pH remains constant, the cellyield is decreased slightly as the temperature is increased.In the same table, the effect of pH and temperature on the value of fs

0 for sulfur oxidizing bacteria ispresented for the case of S2- oxidation. Typical maximum values of fs

0 range from 0.21 to 0.26 and are

higher than the values observed for Fe(II) oxidation. It is important to notice that in the case of sulfideoxidation, changing the [H+] concentration from 10-7 M to 10-1.5 M, increases the value of fs

0 by 1.2times from (0.213 to 0.250) whereas the corresponding change for the case of Fe(II) oxidation is 9.4

times from (0.012 to 0.113). Increase of temperature decreases slightly the fraction of the electrons thatare used for cell synthesis, as can be seen form the values calculated.

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Table 5. The effect of pH and temperature on the value of fs0 for iron and sulfide oxidizing bacteria.

Tables 6 and 7 overleaf present the overall reaction stoichiometry for Fe(II) and S2- oxidation at 40C

and pH 1.5. The values of fs0 used, are these presented in Table 5.

Calculations of overall Fe(II) oxidation indicate that the maximum amount of biomass produced is

113*0.0056/56=11.3 mg cells/g Fe oxidized, the maximum amount of CO2 fixed is estimated to be0.0278*44/56= 21.8 mg CO2/g Fe oxidized, whereas the minimum oxygen consumption is estimated tobe 0.2223*32/56=127 mg O2/ g Fe oxidized.

Similar calculations for the overall reaction for S2- oxidation show that the maximum amount ofbiomass produced is 113*0.0125/0.125*32=353 mg cells/g S2- oxidized, the maximum amount of CO2

fixed is estimated to be 0.0623*44/0.125*32= 685 mg CO2/g S2- oxidized, whereas the minimum oxygen

consumption is estimated to be 0.1878*32/0.125*32=1502 mg O2/ g S2- oxidized.

CONCLUSIONS

A reliable and relatively simple methodology for developing a quantitative model for the overallmetabolic reaction for bioleaching applications has been presented. By using the value of fs

0 themaximum production of biomass and CO2 fixation can be estimated. Likewise, the minimum oxygenrequirement for the oxidation of a given amount of Fe(II) or S2- can be calculated. In practice, as fs is

lower than fs0, because of cell maintenance energy requirements and other electron transfer

inefficiencies in the electrons transfer chain, these values should be adjusted accordingly by applying theappropriate value of fs .

REFERENCES

[1] McCarty P.L., Thermodynamics of Biological Synthesis and Growth, In: Baers J. Editor.

Advances in Water Pollution Research: Proceedings of the 2nd International Conference on WaterPollution Research. Oxford, England: Pergamon Press, Inc., (1965), 169-199.

[2] McCarty P.L., Energetics and bacterial growth, The Fifth Rudolf Research Conference, Rutgers.

The State University, 1969, New Bruswick, NJ.[3] McCarty P.L., Energetics and bacterial growth, In: Faust S.D., Hunter J.V., (ed.), Organic

compounds in aquatic environments. New York: (1971), Marcel Dekker.

[4] McCarty P.L., Energetics of organic matter degradation, In: Mitchell R. (ed.). Water PollutionMicrobiology. New York, 1972, Wiley Interscience.

[5] McCarty P.L., Stoichiometry of biological reactions, Progress in Water Technology, 7, (1975),

157-172.[6] Rawlings D.E., Dew D., and du Plessis C., Biomineralization of metal-containing ores and

concentrates, TRENDS in Biotechnology, 21, 1, (2003), 38-44.[7] Grady C.P.L., Lim H.C. Biological Wastewater Treatment Theory and Applications, Marcel

Dekker Inc. New York and Basel, 1980.[8] Sawyer C.N., McCarty P.L., Parkin G.F., Chemistry for Environmental Engineering and Science,

Fifth Edition, McGraw-Hill International Edition, 2003.

[9] Rittmann B.E., McCarty P.L., Environmental Biotechnology: Principles and Applications,McGraw-Hill, 2001.

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Table

6.TheoverallreactionofFe(II)oxidationat408C

andpH

1.5

Table

7.TheoverallreactionofS2-oxidationat408C

andpH

1.5

10