ISSN 1063455X, Journal of Water Chemistry and Technology, 2016, Vol. 38, No. 1, pp. 51–55.

© Allerton Press, Inc., 2016.

Original Russian Text © A.M. Marchenko, G.N. Pshinko, V.Ya. Demchenko, V.V. Goncharuk, 2016, published in Khimiya i Tekhnologiya Vody, 2013, Vol. 38, No. 1, pp. 88–
96.

BIOLOGICAL METHODS
OF WATER TREATMENT

Bioleaching of Heavy Metals from Wastewater Sludge by Ferrous

Iron Oxidizing Bacteria
A. M. Marchenko*, G. N. Pshinko, V. Ya. Demchenko, and V. V. Goncharuk
Dumanskii Institute of Colloid Chemistry and the Chemistry of Water,
National Academy of Sciences of Ukraine, Kiev
*email: marchenkoolm@gmail.com
Received August 6, 2014

Abstract—Experiments on bacterial leaching of heavy metals from aerobically stabilized surplus activated
sludge at the Bortnichi aeration plant in Kiev have been conducted with addition of FeSO4 ⋅ 7H2O as a
stimulant of ironoxidizing bacteria. The mechanism of heavy metal leaching involving the ferrous iron
oxidation by bacteria with subsequent formation of Fe(OH)3 was confirmed. This process is accompanied
by the reduction of pH level of wastewater sludge that results in leaching of heavy metals.
DOI: 10.3103/S1063455X16010094
Keywords: bacterial leaching, ferrous iron, wastewater sludge, heavy metals, thionic ironoxidizing bacte
ria.

INTRODUCTION
Investigations of bacterial leaching (bioleaching) of heavy metals (HM) from wastewater sludge (WWS) [1,
2] are based on using bacteria Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans taking part in
bioleaching of metals from ores [3–5]. The purpose of the specified papers is to remove HM from the solid
phase of WWS for ensuring its safe use as agricultural fertilizers. The behavior of thionic acidophilic bacteria
was discussed earlier [6]. The role of bacteria A. ferrooxidans in the biospheric cycle of iron [7–9] that is
present in WWS at sufficiently high concentrations [1, 10] makes it possible to suggest a search for the reagent
less technique of HM leaching from WWS by using these bacteria. The action of the latter consists in oxidizing
Fe(II) to Fe(III), while the result of HM leaching from WWS is determined by the replacement of HM at
adsorption centers with H+ [1, 2, 11] and dissolution of HM salt forms with reduction of WWS pH level [1,
2]. An additional energy source representing iron in the form of Fe(II) sulfate or pyrite ore was used in paper
[12] for intensifying the growth of bacteria A. ferrooxidans.
There exist two viewpoints on the mechanism of bioleaching of HM from WWS using A. Ferrooxidans with
addition of Fe(II) compounds. According to the first point of view [13–17] this mechanism consists in bacte
ria oxidizing the sulphur of metal sulfides present in the sludge to metal sulfates: bacteria oxidize Fe(II) to
Fe(III) with subsequent chemical recovery of Fe(III) by metal sulfides resulting in formation of elemental sul
phur [18]. These bacteria oxidize it to sulfuric acid that transfers HM into the liquid phase of WWS. Such
mechanism of HM leaching is based on the concept of S2– oxidation by bacteria [18] and the results of deter
mination of metal forms in WWS by the sequential extraction procedures[19] indicating the presence of high
concentrations of metal sulfides in WWS. According to the second point of view [11, 14, 20–22] the most rel
evant factor in HM bioleaching from WWS is the oxidation of Fe(II) to Fe(III) that results in the rise of system
Eh and also due to high concentrations of Fe(III) in the transfer of the latter into hydroxide forms, which leads
to decrease of system pH and HM leaching. Such mechanism of HM leaching is based on formation of insol
uble Fe(III) forms, namely, jarosite [11, 12, 20, 22], shvermannite [23] and amorphous hydroxides [4, 11, 12,
20, 22, 24], while bacteria A. ferrooxidans are cultivated by using artificial media and WWS.
The purpose of this study is to investigate the process of HM bioleaching from WWS aimed at creating the
elective conditions for thionic bacteria oxidizing the ferrous iron. To this end, we considered an example of
the treatment of aerobically stabilized surplus activated sludge (ASS) from the Bortnichi aeration plant in
Kiev. Hence, the experiments on HM bioleaching were conducted using an inoculum of ironoxidizing bac
teria from WWS with addition of FeSO4 ⋅ 7H2O; we also investigated the growth of bacteria on solid media
with silica gel.

51
52 MARCHENKO et al.

EXPERIMENTAL
ASS in experiments was used as described in papers [6, 10]. The analysis of elements in its liquid phase and
investigation of bacteria were conducted by the methods proposed in paper [6].
Inoculum of ironoxidizing bacteria was obtained in the same way as in paper [2]: elective conditions were
created for bacteria oxidizing the ferrous iron by adding 1 vol % of FeSO4 ⋅ 7H2O to the sludge. Flasks with
such sludge were thermostated at 28°C on a suspended shaker until pH value was reduced from 7 to 2.5. For
an inoculum we used ASS treated with 1 vol % of Fe(II) sulfate as is described below.
In conducting four sets of tests on bioleaching of HM from ASS with addition of FeSO4 ⋅ 7H2O (Table 1)
the sludge was placed into round flatbottom flasks having volume 0.5 dm3 and then 1 or 0.5 vol % of Fe(II)
sulfate AR (taking into account that this reagent is in inoculum) and 10 vol % of inoculum. The flasks were
closed with cottongauze corks and thermostated at 28°C on a suspended shaker.

Table 1. Schemes of experimentation on bioleaching of heavy metals from ASS

Set of tests
Description of experimentation
I* II* III* IV** Control*
Volume of ASS, cm3 180 225 225 200 200
Weight of FeSO4⋅7H2O, g 1.125 2.25 2.25 2 –
Volume of inoculum, cm3 20 25 25 – –
ASS is an inoculum III – –
Number of tests 4 6 2 2 3
Amount of repetition of each test 2 2 1 1 1
Note: *Distilled water was added every day taking into account the water losses caused by evaporation; ** Flasks were frozen and
defreezed on a daily basis.

Values of pH and Eh were measured on a daily basis. Reinoculations and measurements of concentrations
of elements in ASS liquid phase were performed after pH reached the level of about 2.5 that was observed by
the third day of experiment of set II. For the two last tests of set II the measurements of metal concentrations
in the liquid phase were conducted during the entire process. In tests of set III for reducing the bacteria growth
flasks with the matter were put into a fridge, then into a freezer compartment, and after a few hours the samples
were defreezed. The “freezing–defreezing” procedure was performed every day. In tests of set IV, Fe(II) sulfate
in the amount of 1 or 0.5 vol % was added to ASS, but inoculum was not added. Samples of ASS without
FeSO4⋅7H2O and inoculum were used as control. The leaching process was assessed on the basis of pH vari
ation and the leaching efficiency, i.e., the part of HM in ASS liquid phase with respect to the HM total con
tent.

RESULTS AND DISCUSSION

Tests on Bioleaching of HM from ASS

Test data of set I (Fig. 1a) indicates that 0.5 vol % of Fe(II) sulfate is insufficient for overcoming the buffer
capacity of sludge. In tests of set II (see Fig. 1b) the largest difference as compared to the other sets consists in
the drop of pH during the first day (24 hours) of the process to the level of about ~3.5, while in the remaining
cases this pH level is achieved as a later time, if ever. Such drop of pH is determined by the introduction of
inoculum with high acidity and by dissolving FeSO4⋅7H2O. The test data of set II is presented below.
The humidity of treated ASS amounted to 98.3 ± 0.25%, i.e., it decreased from 98.6 ± 0.3% for the initial
ASS, while the ash content increased from 27.4 ± 0.7% to 37 ± 1%. Thus, the increased ash content completely
specifies the rise of humidity. The ash of treated sludge acquired the brown color indicating the presence of
ferric oxide (Fe2O3) to which Fe(OH)3 decomposed if heated to temperature >500°C [25] (ash content was
determined by burning at 600°C [26]. The tests of set II involved an addition of Fe(II) in the amount of
0.036 mol/dm3 to ASS; precipitation of the total added iron in the form Fe(OH)3 should have exceeded the
content of solids by 0.39% (the observed increase amounted to 0.3 ± 0.25%), while the rise of ash content
should have reached 37% that just had place. Since the concentration of total sulphur in the initial ASS is suf

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 BIOLEACHING OF HEAVY METALS 53

ficiently low (0.0031 mol/dm3), we can suggest that the main mechanism of HM bioleaching from WWS using
ironoxidizing bacteria consists in bacterial oxidation of Fe(II) to Fe(III) and precipitation of the latter in the
form of Fe(OH)3 with an increased acidity of WWS.
pH Eh, mV pH
Eh, mV
6 pH3 600
Eh1 EhII
pH2
600

6 EhIII
5
300 EhIV
pHK 300
pH1
EhK

4
4
pHIII 0
0
Eh3 pHІV

Eh2 pHII
3 2 –300
0 3 6 0 3 6 9
days days
(a) (b)
Fig. 1. Variation of ASS pH and Eh in tests of sets I (a) and II–IV (b) on bioleaching of heavy metals. Each index specifies
either the test number of a set (Arabic numerals) or the set number (Roman numerals). The control tests are designated by
index K.

For all metals the process efficiency with addition of inoculum (Table 2) is higher than without it. For V,
Cr, Ga, As, and Pb the efficiency is lower than for bioleaching using thionic acidophilic bacteria with addition
of elemental sulphur; for Zn, Rb, Cd, Sr, and Ba the efficiency is similar, while for Mn, Co, Ni, Cu, and U
the efficiency is higher [6]. The HM concentrations in liquid phase (Figs. 2 and 3) significantly increase during
the first 24 hours of the process that corresponds to the decrease of sludge pH to the level of about ~3.5.

Table 2. The efficiency of toxic component bioleaching (%) in tests of sets II and IV
Set of tests Set of tests
Metal Control Metal Control
II IV II IV
V 1.5 0.6 0.62 As 3 2.3 14.4
Cr 2 0.2 0.27 Rb 26 25.4 22.7
Mn – 62.2 13.9 Sr 42 34.4 16.2
Co – 46.1 28.3 Cd 79 34.9 1.7
Ni 80 32.4 6.7 Ba 2.5 2.2 0.7
Cu 30 15.0 6.4 Pb 0.7 0.3 2.3
Zn 94 19.4 3.2 U 6 1.7 2.0

Tests on Cultivation of Bacteria

The initial ASS inoculation of the Leathen medium (pHinit 3.5) led to formation of hydroxides after five
days (pHfin 2.5, Eh fin = 580 mV after 12 days). In the case of the enrichment culture inoculation of Leathen
solid medium we observed the growth of bacteria under investigation in the form of orange or brown spots that

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54 MARCHENKO et al.

sometimes projected above the gel surface and were always present in the thickness of medium [4]. In case of
the colony inoculation of Leathen liquid medium with addition of 1.25 vol % of NaCl we also observed the
formation of hydroxides [27]. The inoculation of Waksman’s solid medium with thiosulfate did not produce
any growth of bacteria. In case of the colony inoculation of glucose medium the bacterial growth also was not
observed. This led to a conclusion that bacteria were chemolithoautotrophs. The Gram stain procedure made
it possible to register (1000×) gramnegative rods (< 2 μm long and up to 1 μm wide).
3 3
С, mol/dm С, mol/dm
10–3 10–4 Cutot
Zntot

CuII
–4
Nitot
10 Mntot ZnII –5
10
NiII CuK
MnII

ZnK
–5
10 MnK –6
10 NiK
ZnIII

–6 MnIII
10
–7 NiIII
10

–7
10
0 2 4 6 days 0 2 4 6 days
(a) (b)
Fig. 2. Variation of the concentration of zinc and manganese (a), copper and nickel (b) in the ASS liquid phase in tests of sets
II and III on leaching of heavy metals. Each index specifies the test number in a set. The control tests are designated by
index K.
3 3
С, mol/dm С, mol/dm
10
–5
Pbtot 10
–4 Crtot

Cdtot –5
10
–6
10
CdII CrII
Cotot
–6
PbK 10
CoII
–7
10 PbII
–7 CrK
CdK 10
CoK
PbIII
–8
10 CoIII
–8
10
CrIII

–9
–9
10
10 0 2 4 6 days 0 2 4 6 days
(a) (b)
Fig. 3. Variation of the concentration of lead and cadmium (a), chromium and cobalt (b) in the ASS liquid phase in tests of
sets II and III on leaching of heavy metals. Indices are the same as in Fig. 2.

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 BIOLEACHING OF HEAVY METALS 55

CONCLUSIONS
It has been shown that leaching of heavy metals from wastewater sludge leads to bacterial oxidation of fer
rous iron with subsequent formation of Fe(OH)3. This is accompanied by WWS pH decrease that results in
leaching of heavy metals.

REFERENCES
1. Babel, S. and del Mundo Dacera, D., Waste Manag., 2006, vol. 26, pp. 988–1004.
2. Pathak, A., Dastidar, M.G., and Sreekrishnan, T.R., J. Environ. Manag., 2009, vol. 90, pp. 2343–2353.
3. Krebs, W., Brombacher, C., Bosshard, P.P., Bachofen, R., and Brandl, H., FEMS Microbiol. Rev., 1997, vol. 20,
pp. 605–617.
4. Leathen, W.W., Kinsel, N.A., and Braley, S.A., J. Bacteriol., 1956, vol. 72, pp. 700–704.
5. Dworkin, M., ch. Ed., The Prokaryotes. Proteobacteria: Alpha and Beta Subclasses, 3rd ed., in 7 vol., vol. 5, Springer,
2006.
6. Marchenko, A.M., Pshinko, G.N., Demchenko, V.Ya., and Goncharuk, V.V., Khimiya i Tekhnologiya Vody, 2015,
vol. 37, no. 6, pp. 564–573.
7. Ingledew, W.J., Biochim. et Biophys. Acta, 1982, vol. 683, pp. 89–117.
8. Ehrlich, H.L. and Newman, D.K., Geomicrobiology, 5th ed., New York: CRC Press, 2009.
9. Lu, S., Gischkat, S., Reiche, M., Akob, D.M., Hallberg, K.B., and Kusel, K., AEM, 2010, vol. 76, no. 24, pp. 8174–
8183.
10. Marchenko, A.M., Pshinko, G.N., and Demchenko, V.Ya., Khimiya i Tekhnologiya Vody, 2015, vol. 37, no. 4,
pp. 375–383.
11. Xiang, L., Chan, L.C., and Wong, J.W., Chemosphere, 2000, vol. 41, pp. 283–287.
12. Wong, J.W., Xiang, L., Gu, X.Y., and Zhou, L.X., Ibid., 2004, vol. 55, pp. 101–107.
13. Tyagi, R.D., Couillard, D., and Tran, F., Environ. Pollut., 1988, vol. 50, pp. 295–316.
14. Couillard, D. and Chartier, M., J. Biotechnol., 1991, vol. 20, pp. 163–180.
15. Chan, L.C., Gu, X.Y., and Wong, J.W., Adv. Environ. Res., 2003, vol. 7, pp. 603–607.
16. Gu, X.Y. and Wong, J.W., Chemosphere, 2007, vol. 69, pp. 311–318.
17. Peng, G., Tian, G., Liu, J., Bao, Q., and Zang, L., Desalination, 2011, vol. 271, pp. 100–104.
18. Adkh’ya, S., Alpert, K.A., Bukkel’, V., et al., Sovremennaya mikrobiologiya. Prokarioty (Modern Microbiology.
Prokaryotes), Lengeler, I., Drevs, G., and Shlegelyayu, Eds., in 2 vol., vol. 1, Moscow: Mir, 2009.
19. Stover, R.C., Sommers, L.E., and Silvera, D.J., J. Water Pollut. Control Fed., 1976, vol. 48, no. 9, pp. 2165–2175.
20. Blais, J.F., Tyagi, R.D., and Auclair, J.C., Water Res., 1993, vol. 27, no. 1, pp. 111–120.
21. Tyagi, R.D., Blais, J.F., and Auclair, J.C., Environ. Pollut., 1993, vol. 82, pp. 9–12.
22. Pathak, A., Dastidar, M.G., and Sreekrishnan, T.R., J. Hazard. Materials, 2009, vol. 171, pp. 273–278.
23. Liao, Y., Zhou, L., Bai, S., Liang, J., and Wang, S., Appl. Geochem., 2009, vol. 24, pp. 1739–1746.
24. Andreyuk, E.I. and Kozlova, I.A., Litotrofnye bakterii i mikrobiologicheskaya korroziya (Lithotrophic Bacteria and
Microbiological Corrosion), Kiev: Nauk. Dumka, 1977.
25. Nikol’skii, B.P., Spravochnik khimika (Reference Chemist), in 6 vol., 3rd ed., vol. 2, Leningrad: Khimiya, 1971.
26. Tereshchuk, A.I., Issledovanie i pererabotka osadkov stochnykh vod (Investigation and Processing of Wastewater
Sludge), Lvov: Vyshcha shk., 1988.
27. Holt, J., Krieg, N., Snit, P., Stakey, J., and Williams, S., Eds., Opredelitel’ bakterii Berdzhi (Bergey’s Manual of Deter
minative Bacteriology), in 2 vol., vol. 2, Moscow: Mir, 1997.

Translated by A. Zheldak

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