Ferrous Iron Oxidation MBR JMPark

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Continuous biological ferrous iron oxidation in a

submerged membrane bioreactor

D. Park, D.S. Lee and J.M. Park

Department of Chemical Engineering, School of Environmental Science and Engineering, AdvancedEnvironmental Biotechnology Research Center, POSTECH, San 31, Hyoja-dong, Nam-gu, Pohang,

Kyungbuk 790-784, Republic of Korea (E-mail: [email protected]; [email protected];

[email protected])

Abstract Microbial oxidation of ferrous iron may be available alternative method of producing ferric iron,

which is a reagent used for removal of H2S from biogas. In this study, a submerged membrane bioreactor

(MBR) system was employed to oxidize ferrous iron to ferric iron. In the submerged MBR system, we could

keep high concentration of iron-oxidizing bacteria and high oxidation rate of ferrous iron. There was

membrane fouling caused by chemical precipitates such as K-jarosite and ferric phosphate. However, a

strong acidity (pH 1.75) of solution and low ferrous iron concentration (below 3000 mg/l) significantly

reduced the fouling of membrane module during the bioreactor operation. A fouled membrane module could

be easily regenerated with a 1 M of sulfuric acid solution. In conclusion, the submerged MBR could be used

for high-density culture of iron-oxidizing bacteria and for continuous ferrous iron oxidation. As far as our

knowledge concerns, this is the first study on the application of a submerged MBR to high acidic conditions

(below pH 2).

Keywords H2S removal; iron-oxidizing bacteria; jarosite; submerged membrane bioreactor

Introduction

Biological oxidation of ferrous iron is a well-researched area in the bioleaching and treat-

ment of acid mine drainage (Jensen and Webb, 1995a; Nemati et al., 1998). A variety of

iron-oxidizing bacteria have been isolated from acidic mine drainage or places where an

ore body is naturally exposed to water and the atmosphere. Most of them have been

characterized as Acidithiobacillus ferrooxidans and Leptospirillium ferroxidans. Ferrous

iron is oxidized according to the following reaction:

2FeSO4 H2SO4 0:5O2Iron oxidizingbacteria! Fe2SO43 H2O 1

The oxidizing property of ferric iron produced in this reaction makes it an useful reagent

in the removal of H2S from sour gases (Jensen and Webb, 1995b; Pagella and De Faveri,

2000). In stage 1, a solution of ferric sulfate contacts sour gas in an absorber. The sol-

ution absorbs hydrogen sulfide and oxidizes it to elemental sulfur while the ferric sulfate

is reduced to ferrous sulfate:

H2S Fe2SO43 ! S # 2FeSO4 H2SO4 2

In stage 2, elemental sulfur is recovered by solid-liquid separation. The ferrous sulfate

solution is sent to a microbial oxidation tank in stage 3, where iron-oxidizing bacteria

reoxidize ferrous iron to ferric iron according to Equation 1. The oxidized solution is

then recycled to the absorber in stage 1 to repeat the cycle.

Of the two reactions involved in the chemobiological process for the removal of H2S,

the biological oxidation of ferrous iron to ferric iron is the rate-limiting step ( Jensen and

Webb, 1995a). Over the years, a number of studies have been aimed at improving the

rate of ferrous iron oxidation by iron-oxidizing bacteria. Several experimental systems

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with batch and continuous flow modes of operation have been carried out, and various

reactor configurations were designed, trying to obtain better results (Table 1). Among

them, an immobilized bioreactor system showed most efficient performence of ferrous

iron oxidation. However there are still some problems such as cells wash-out at high

liquid flow rate and detachment of cell from support media by air flow. In this study, a

submerged MBR system was employed to keep high concentration of cells and high oxi-

dation rate of ferrous iron. Effects of influent pH, ferrous iron concentration and dilution

rate on the performance of the submerged MBR were investigated. As far as our knowl-

edge concerns, this is the first study on the application of a submerged MBR to biological

ferrous iron oxidation.

Material and methods

Organism, medium and growth conditions

The culture used in this study was screened from the activated sludge of a domestic

wastewater treatment plant (Pohang, Korea). It was a complex mixture of autotrophic

bacterial strains capable of oxidizing ferrous iron to ferric iron, largely consisting of

Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans (Park et al., 2005). These

iron-oxidizing bacteria were grown in a FeSO4-based 9K medium. The compositon of the

medium is as follows: FeSO47H2O, 5.0 g/l; (NH4)2SO4, 3.0g/l; K2HPO4, 0.5 g/l;

MgSO47H2O, 0.5 g/l; KCl, 0.1 g/l; Ca(NO3)2, 0.01 g/l. The initial pH was adjusted to 2.0

using 5 M H2SO4 solution.

Submerged membrane bioreactor system

The membrane module was an ultra-micro hollow fiber (Sterapore-L, Mitsubishi Rayon)

made of polyethylene. The main part of the bioreactor was a glass column with a diam-

eter of 5 cm and a height of 51 cm (Figure 1). The membrane module was fully sub-

merged into the medium in the column. The bottom part incorporated inlets for the fresh

medium and air, the top part incorporated outlets for the effluent and air. The working

volume of the submerged MBR system was 500 ml and air flow rate was 2 litres per min-

ute. Bioreactor was operated at room temperature (20 258C) under various conditions as

shown in Table 2. Samples were taken from the effluents and analyzed for pH, total iron

and ferrous iron concentrations. Also, suspended solids (SS) and protein concentrations in

the bioreactor were analyzed during the bioreactor operation.

Analytical methods

A colorimetric method was used to measure the ferrous iron and total iron concentrations

in the effluent (Karamanev et al., 2002). The red-orange colored complex, formed from

1,10-phenanthroline and ferrous iron in acidic solution, was spectrophotometrically

Table 1 Comparison of ferrous iron oxidation rate in various reactor configurations

Reference Reactor configuration Support media Oxidation rate

(g/lh)

Braddocket al., 1984 Chemostat Free cells 0.12Nikolov et al., 2002 Rotating biological contactor Polyvinyl chloride disks 1.64Karamanev and Nikolov, 1988 Fluidized bed Expanded polystyrene 1.68Grishin and Tuovinen, 1988 Packed bed Glass bead 8.1

Mazuelos et al., 2000 Packed bed Siliceous stone particles 11.25Lancy and Tuovinen, 1984 Packed bed Calcium alginate 0.52Nikolov et al., 1988 Packed bed Polyvinyl chloride turnings 1.8Mesa et al., 2002 Packed bed Polyurethane foam 4

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analyzed at 510 nm (SPECTRONICO GENESYSTM 5, Spectronic Ins.). The yellow

colored complex, formed from 5-sulfosalicylic acid and total iron in basic solution, was

spectrophotometrically analyzed at 425 nm. Since most of SS in the bioreactor system

were ferric precipitates such as jarosites and ferric phosphate, SS concentration did not

reflect the concentration of iron-oxidizing bacteria in the bioreactor. Thus to measure the

iron-oxidizing bacteria, the total protein concentration in the bioreactor was analyzed by

a modified Ramsays method (Mesa et al., 2000).

SEM/EDS analysis

Scanning electron microscopy (SEM)/energy dispersive X-ray spectroscopy (EDS) anal-

ysis was used to observe and analyze the surface of membrane module before and after

run of bioreactor. At the end of each run of bioreactor, membrane module was washed

with deionized-distilled water several times and cut into approximately 0.5 cm sized

pieces. Cut modules were freeze-dried to maintain the structure of membrane module and

stored in a desiccator before SEM/EDS analysis. Dried modules were gold-coated and

analyzed with a scanning electron microscope equipped with energy dispersive X-ray

spectrometer (SEM 515, Philips).

Results and discussion

Effect of influent pH

Under the same condition of ferrous iron concentration of 1000 mg/l and liquid flow rate

of 300ml/h, the sumberged MBR was operated at various influent pHs such as 1.50,

1.75, 2.00 and 2.50. In the case of influent pH 1.50, ferrous iron concentration in theeffluent increased to 868 mg/l and protein concentration did not changed in the bioreactor

(Figure 2). These results imply that iron-oxidizing bacteria could not oxidize ferrous iron

to ferric iron and not use obtained electrons for cell growth at pH 1.50. While iron-oxi-

dizing bacteria could completly oxidize the ferrous iron to ferric iron and grew up to 416

and 478 mg-protein/l in 270 hours at both influent pH 1.75 and 2.00 (Figures 3 and 4).

Figure 1 Schematic diagram of the submerged MBR

Table 2 Operational conditions of the submerged MBR

pH Fe(II) (mg/l) Flow rate (ml/h)

Run 1 1.50 1000 300Run 2 1.75 1000 300Run 3 2.00 1000 300

Run 4 2.50 1000 300Run 5 1.75 10000 300Run 6 1.75 3000 300Run 7 1.75 3000 300 6000

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Total iron concentrations in the effluent were the same to those in the influent and SS

concentrations decreased slightly due to the sampling and attachment of SS on the wall

of the bioreactor. These results imply that there was no formation of ferric precipitates.

The pHs in the effluents of both Run 2 and 3 did not changed. The submerged MBR

could be operated without any problem such as a membrane fouling for 270 hours. In the

case of Run 4, ferrous iron concentration decreased slowly in the effluent but protein con-

centration increased slowly in the bioreactor. The total iron concentration in the effluent

was about 840 mg/l and SS concentration increased to 26.5 g/l, which indicate the for-

mation of ferric precipitates in the bioreactor. However, the operation of Run 4 ( Figure 5)

was stopped due to the fouling of membrane module. The membrane fouling might be

resulted from the deposition or adsorption of SS including ferric precipitates on the sur-

face of the membrane or within the pore. It has been known that A. ferroxidans can grow

in the pH range of 1.52.5 (Nemati et al., 1998). However, above pH 2 various ferric

precipitates can be formed (Jensen and Webb, 1995a) and may result in the fouling of

membrane module. Therefore, pH 1.75 is the optimal condition for both cell growth and

prevention of membrane fouling.

Effect of ferrous iron concentration

Under the same condition of influent pH 1.75, the bioreactor was operated with the

increase of ferrous iron concentration. Figures 6 and 7 show the effects of ferrous iron

concentrations. For Run 5, ferrous iron concentration in the effluent decreased to

Figure 2 Run 1 (influent pH 1.50, 1000 mg/l of Fe(II))


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1111 mg/l and protein concentration incresed from 20 to 88 mg/l (Figure 6). These results

imply that iron-oxidizing bacteria could oxidize ferrous iron to ferric iron under high con-

centration of ferrous iron. However, total iron concentration in the effluent decreased to

5790 mg/l and SS concentration increased to 48 g/l, which indicate the formation of ferric

precipitates in the bioreactor. The membrane module was fouled in 58 hours after all.

Generally, the ferrous iron oxidation by iron-oxidizing bacteria is a proton consuming

reaction, but ferric precipitation reactions produce protons (Jensen and Webb, 1995a).

The increase in the pH value from 1.75 to 2.09 in the effluent resulted from these various

reactions. For Run 6, iron-oxidizing bacteria could completely oxidize ferrous iron of

3000 mg/l and grew up to 702 mg of protein per litre in 142 hours (Figure 7). In the case

of Run 2, the protein concentration in the bioreactor was 264 mg/l at 148 hours (Figure 3).

The total iron concentration in the effluent was nearly the same to that in the influent and

SS concentration increased a little. The pH in the effluent increaed to 1.98, but this value

was lower than that in Run 5. The bioreactor could be operated without any membrane

fouling problem under the operational condition of Run 6.

Effect of dilution rate

Under the optimum condition, the submerged MBR was operated for 263 hours. Initially,

the concentration of ferrous iron in the effluent decreased and then reached at a steady

state in 70 hours (Figure 7). Then the liquid flow rate was increased stepwise until the

bioreactor showed the oxidation efficiency of ferrous iron above 50%; i.e. dilution rate



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increased from 0.6 to 12 h21

(Figures 8 and 9). The required time to achieve steady state

conditions varied depending on the liquid flow rate, but normally it was one day. As

dilution rate increased, the oxidation rate of ferrous iron increased, but oxidation effi-

ciency decreased. Below dilution rate of 5.4h21

, the oxidation efficiency was above

90%. However, the oxidation efficiency was about 60% at dilution rate of 12 h21. On the

contrary the oxidation rate of ferrous iron increased from 1.8 to 21.7 g/lh, which was

higher than that of other reactors that have been reported ( Table 1). In the aspect of cell

growth, iron-oxidizing bacteria could grow up to 2.4 g-protein/l (Figure 8), which corre-

sponds to 5.5 1010

cells/l (Mesa et al., 2000). It has been known that the maximumspecific grow rate of A. ferrooxidans is 0.060.25 h

21(Nemati et al. 1998). Thus dilution

rate above 0.25 h21

may result in the cells wash out due to the high liquid flow rate

(Braddock et al., 1984; Nikolov and Karamanev, 1992). However, the wash out of iron-

oxidizing bacteria did not occurred at even dilution rate of 12 h21

in our bioreactor sys-

tem. It is the main advantage of the submerged MBR system that cells can be remained

within the bioreactor even at high liquid flow rate (Park et al. 2004). Therefore, the sub-

merged MBR system could be successfully used for high-density culture of iron-oxidizing

bacteria and high oxidation rate of ferrous iron.

Fouling and regeneration of membrane module

Although the submerged MBR system could be used for high-density culture of iron-

oxidizing bacteria and high oxidation rate of ferrous iron, membrane fouling was major


Figure 6 Run 5 (influent pH 1.75, 10,000mg/l of Fe(II))

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constraints to the application of the submerged MBR. To characterize the cause of mem-

brane fouling, the surface of the membrane module before and after experimental runs

was observed by SEM (Figure 10). The surface of original module was very clean and

has a lot of micro-pore. However, the surface of module after each run was coated with

particles and the degree of adhesion depended on both influent pH and ferrous iron con-

centration. The degree of membrane fouling increased with increasing influent pH and

ferrous iron concentration. For further more information about particles causing mem-

brane fouling, the surface chemistry of module was analysed by EDS (Table 3). Since the

original module is made of polyethylene (Z[CH2ZCH2]nZ), carbon was a major element

presented on the module surface. Oxygen was detected as contaminant of the module. At

ferrous iron concentration of 1000 mg/l, micro-pores of the module after Run 2 did not

nearly get blocked with particles (Figure 10(b)) and a small amount of iron was detected

on the module surface (Table 3). While micro-pores of the module after Run 4 were com-

pletely blocked with a solid overlayer (,0.3mm thick) and various particles containing

microorganism which might be A. ferrooxidans (rod shaped, 0.50.6mm wide by

1.02.0mm long, with rounded ends, occurring singly or in pairs) as shown in

Figure 10(c). EDS analysis showed that iron, phosphorus and sulfur existed on the mod-

ule surface and the amount of oxygen increased largely. These results imply that ferric

phosphate (FePO4) and other ferric precipitates were formed during Run 4. At the

influent pH 1.75, the degree of the module fouling increased with incresing ferrous iron

Figure 9 Oxidation rate according to dilution rate


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concentration (Figure 10 (b,d,e)). Particulary, micro-pores of the module after Run 5

were completely blocked by 0.11mm-sized granules which were different in shape to

particles presented on the module surface after Run 4, any microorganism-shaped par-

ticles was not observed in SEM images (Figure 10(e)). Surface chemistry of the module

after Run 5 was oxygen . carbon . iron . sulfur . potassium. But phosphorus was

not detected (Table 3).

In general, ferric iron has an extremely low solubility above pH 2.5. However, jarosite

with the general formula MFe3(SO4)2(OH)6 can be formed chemically around pH 2 as

follows:

3Fe3 M 2HSO24 6H2O!MFe3SO42OH6 8H 3

where M K, Na, NH4 or H3O

(Jensen and Webb, 1995a). Jarosite precipitation is

dependent on the pH, ionic composition and concentration of the medium. In an acidic

ferric sulfate medium containing various cations and anions, a number of other ferric iron

species may be present. Apart from ferric hydrolysis products and several jarosites, ferric

Figure 10 SEM images of surface of membrane modules

Table 3 Atomic percentages of element covered on surface of membrane modules before and after run

C O Fe P S K

Original module before run 96.6 3.4 N.D. N.D. N.D. N.D.

Module after Run 2 (pH 1.75, 1 000 m g/l of Fe(II)) 93.9 5.9 0.2 N.D. N.D. N.D.Module after Run 4 (pH 2.50, 1 000 m g/l of Fe(II)) 29.7 51. 6 11.1 5 .6 2.0 N.D.Module after Run 5 (pH 1.75, 1 0,000 m g/l of Fe(II)) 23.8 58. 8 9.9 N.D. 5.4 2.1

N.D. Not Detected.

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complexes with sulphate and ferric precipitates of phosphates and oxyhydroxides may

occur. Jones et al. (2003) reported that an uniform solid ferric phosphate overlayer

(,0.2mm thick) was produced on the arsenopyrite surface during iron oxidation by

A. ferrooxidans. Based on element contents, composition of ferric precipitates could be

analyzed as follows: for Run 4-ferric phosphate 50%, K-jarosite 0%, others 50%; for Run

5-ferric phosphate 0%, K-jarosite 64%, others 36%.

Meantime, in order to regenerate the fouled membrane module it was contacted with a

1 M of sulfuric acid solution for 10 minutes. As can be seen in Figure 10(f), the membrane

module after regeneration was very clean and no precipitates did exist. Thus the 1 M of sul-

furic acid solution was chosen as a regeneration agent of the fouled membrane module.

As mentioned above, membrane module was fouled during the run of the submerged

MBR in the FeSO4-based 9K medium. The degree of membrane fouling increased with

increasing influent pH and ferrous iron concentration. In consideration of both cell growth

and membrane fouling, the best condition for the submerged MBR system was deter-

mined as follows; influent pH 1.75 and ferrous iron concentration of 3000 mg/l. As an

alternative method preventing ferric precipitates causing membrane fouling, we tested a

new medium reported most recently (Kim et al., 2002). However, these tries could not

prevent the membrane fouling caused by chemical precipitates. Thus, there should be

further studies about new medium reducing the formation of ferric precipitates.

Conclusions

H2S is an undesirable contaminant of various gas streams and the broad variations in the

chemical and physical nature of the sour gases are mirrored by the numerous physico-

chemical processes available at present for treating gas. Since there are no universally

adopted methods, the prospects for adoption of microbiologically based processes are

very promising. Particularly promising is the application of chemoautotrophic bacteria of

the genus Acidithiobacillus, which has already shown encouraging results (Jensen and

Webb, 1995b).

Iron-oxidizing bacteria can oxidize ferrous iron to ferric iron, which is a reagent used

for removal of H2S from biogas. In this chemobiological process, the biological oxidation

of ferrous iron to ferric iron is the rate-limiting step. In this study, the submerged MBR

system was employed to enhance oxidation rate of ferrous iron by iron-oxidizing bacteria.

In the submerged MBR system, the effects of influent pH, ferrous iron concentration and

dilution rate were examined. Iron-oxidizing bacteria showed optimum growth rate at pH

2 and grew fast with increases in ferrous iron concentration and dilution rate. However,

there was membrane fouling caused by chemical precipitates depending on both influent

pH and ferrous iron concentration. Around pH 2, various jarosites including K-jarosite

were formed and caused the membrane fouling. On the contrary, ferric phosphate was a

main precipitate causing serious membrane fouling above pH 2.5. The degree of mem-

brane fouling could be reduced with decreasing influent pH and ferrous iron concen-

tration. In consideration of both cell growth and membrane fouling, the best condition for

the submerged MBR system was determined as influent pH 1.75 and ferrous iron concen-

tration of 3000 mg/l. Under this condition, iron-oxidizing bacteria could grow up to 2.4 g-

protein/l. For 12 h21

of dilution rate, the oxidation efficiency of ferrous iron was 60%

and the oxidation rate of ferrous iron was 21.7 g/lh, which was higher than that of other

reactors that have been reported so far. Meantime, the fouled membrane module could be

easily regenerated with a 1 M of sulfuric acid solution. However, there is still a need for

optimising regeneration conditions.

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The submerged MBR system could be used for high-density culture of iron-oxidizing

bacteria and continuous ferrous iron oxidation. As far as our knowledge concerns, this is

the first study on the application of a submerged MBR to high acidic conditions.

Acknowledgements

The work was financially supported by the Korea Science and Engineering Foundation

through the Advanced Environmental Biotechnology Research Center at Pohang Univer-

sity of Science and Technology.

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