3
Iron-Reducing Organisms
A mixed culture of metal-reducing organisms was collected
from a fen in Houghton County, Michigan, where natural
iron reduction was observed, as shown in Figure 3.
In the natural environment, red-colored iron hydrox-
ide precipitate formation in anaerobic bogs indicates the
presence of metal-reducing organisms. These metal-reduc-
ing organisms naturally reduce iron, and the reduced iron
ions form iron hydroxide precipitate when in contact with
the air. These organisms were used for the iron leaching
experiments as their metal leaching activity was previously
observed in preliminary experiments [21]. Organisms were
initially cultured in preliminary iron-leaching experiments.
Following this primary cultivation, the organisms were
transferred and used as inoculants in subsequent iron leach-
ing experiments to assess their leaching efficiency.
Nutrient Media
Typha latifolia was used as a source of nutrient media for
the growth and survival of iron-reducing organisms. T. lati-
folia was fermented using fermentative organisms collected
from the same source as iron-reducing organisms to pro-
duce an organic-rich liquid. An additional 5 g/L of sucrose
was added to the organic-rich liquid, which fermented to
organic acids and lowered the pH to ≈5. High-performance
liquid chromatography was used to analyze the organic
compounds in the liquid, as shown in Table 2.
Iron Bioleaching Flask Experiments
All of the flask bioleaching experiments were conducted
in triplicates. They were conducted in a 250 ml Buchner
flask with 200 g of iron ore tailings from either source A or
source B, nutrient media, and iron-reducing organisms. The
experimental setup for iron leaching is shown in Figure 4.
The sidearm of the iron leaching flask was connected
to a 100 ml Erlenmeyer flask. The small flask contained
distilled water, preventing the backflow of oxygen to the
leaching flask to maintain an anaerobic leaching environ-
ment. Fresh nutrient media was supplied to the leaching
flask at a rate of 300 ml per week (5 increments of 60 ml),
providing the organisms with sufficient energy for growth
and survival. Overflow solution from the leaching flask was
collected to analyze pH and dissolved iron concentration.
The pH of the solution obtained from the leaching
flask was measured using a Thermo Scientific ™ pH meter.
Iron concentration in the overflow solution was analyzed
using the spectrophotometric method, which is based on
the formation of a strongly orange-colored complex by
Fe2+ ions with 1,10 phenanthroline indicator [22]. X-ray
fluorescence and X-ray diffraction techniques were used to
study the composition of the iron ore tailing samples after
and before the leaching experiment.
RESULTS AND DISCUSSION
Dissolved iron concentrations for bioleaching experiments
on the Source A and Source B tailings for 368 days are
shown in Figure 5 and Figure 6. In a few days, the dissolved
iron concentration reached above 600 mg/L as iron-reduc-
ing organisms had already adapted to the iron-leaching
environment. As experiments progressed, the iron concen-
tration reached above 1000 mg/L on day 86. A maximum
concentration of 1177 mg/L was observed in iron bioleach-
ing on tailings from source A. pH was observed in the range
of 3.95 to 5.36, and a total of 49% of the iron present was
dissolved over 368 days.
Iron leaching activity on the source B tailings contain-
ing hematite was low in the beginning. As microbial activity
increased, concentrations above 500 mg/L were observed,
reaching a maximum of 619 mg/L on day 58th. As experi-
ments progressed, a drop in dissolved iron concentrations
was observed, which was due to the decrease in organic
compounds in T. latifolia media. pH was slightly increased
during this period.
Fresh T. latifolia plants from the swamp were used for
fermentation to produce more organic compounds and
were added after day 225. After getting fresh T. latifolia,
Figure 3. Iron-bearing seepage from an anaerobic bog in
Houghton County, Michigan [20]
Table 2. Composition of organic-rich liquid by fermentation
of T. latifolia
Organic compounds Concentration (mg/L)
Glucose 390
Acetic acid 205
Fructose 146
Citric acid 29
Lactic acid 25
Formic acid 23
Iron-Reducing Organisms
A mixed culture of metal-reducing organisms was collected
from a fen in Houghton County, Michigan, where natural
iron reduction was observed, as shown in Figure 3.
In the natural environment, red-colored iron hydrox-
ide precipitate formation in anaerobic bogs indicates the
presence of metal-reducing organisms. These metal-reduc-
ing organisms naturally reduce iron, and the reduced iron
ions form iron hydroxide precipitate when in contact with
the air. These organisms were used for the iron leaching
experiments as their metal leaching activity was previously
observed in preliminary experiments [21]. Organisms were
initially cultured in preliminary iron-leaching experiments.
Following this primary cultivation, the organisms were
transferred and used as inoculants in subsequent iron leach-
ing experiments to assess their leaching efficiency.
Nutrient Media
Typha latifolia was used as a source of nutrient media for
the growth and survival of iron-reducing organisms. T. lati-
folia was fermented using fermentative organisms collected
from the same source as iron-reducing organisms to pro-
duce an organic-rich liquid. An additional 5 g/L of sucrose
was added to the organic-rich liquid, which fermented to
organic acids and lowered the pH to ≈5. High-performance
liquid chromatography was used to analyze the organic
compounds in the liquid, as shown in Table 2.
Iron Bioleaching Flask Experiments
All of the flask bioleaching experiments were conducted
in triplicates. They were conducted in a 250 ml Buchner
flask with 200 g of iron ore tailings from either source A or
source B, nutrient media, and iron-reducing organisms. The
experimental setup for iron leaching is shown in Figure 4.
The sidearm of the iron leaching flask was connected
to a 100 ml Erlenmeyer flask. The small flask contained
distilled water, preventing the backflow of oxygen to the
leaching flask to maintain an anaerobic leaching environ-
ment. Fresh nutrient media was supplied to the leaching
flask at a rate of 300 ml per week (5 increments of 60 ml),
providing the organisms with sufficient energy for growth
and survival. Overflow solution from the leaching flask was
collected to analyze pH and dissolved iron concentration.
The pH of the solution obtained from the leaching
flask was measured using a Thermo Scientific ™ pH meter.
Iron concentration in the overflow solution was analyzed
using the spectrophotometric method, which is based on
the formation of a strongly orange-colored complex by
Fe2+ ions with 1,10 phenanthroline indicator [22]. X-ray
fluorescence and X-ray diffraction techniques were used to
study the composition of the iron ore tailing samples after
and before the leaching experiment.
RESULTS AND DISCUSSION
Dissolved iron concentrations for bioleaching experiments
on the Source A and Source B tailings for 368 days are
shown in Figure 5 and Figure 6. In a few days, the dissolved
iron concentration reached above 600 mg/L as iron-reduc-
ing organisms had already adapted to the iron-leaching
environment. As experiments progressed, the iron concen-
tration reached above 1000 mg/L on day 86. A maximum
concentration of 1177 mg/L was observed in iron bioleach-
ing on tailings from source A. pH was observed in the range
of 3.95 to 5.36, and a total of 49% of the iron present was
dissolved over 368 days.
Iron leaching activity on the source B tailings contain-
ing hematite was low in the beginning. As microbial activity
increased, concentrations above 500 mg/L were observed,
reaching a maximum of 619 mg/L on day 58th. As experi-
ments progressed, a drop in dissolved iron concentrations
was observed, which was due to the decrease in organic
compounds in T. latifolia media. pH was slightly increased
during this period.
Fresh T. latifolia plants from the swamp were used for
fermentation to produce more organic compounds and
were added after day 225. After getting fresh T. latifolia,
Figure 3. Iron-bearing seepage from an anaerobic bog in
Houghton County, Michigan [20]
Table 2. Composition of organic-rich liquid by fermentation
of T. latifolia
Organic compounds Concentration (mg/L)
Glucose 390
Acetic acid 205
Fructose 146
Citric acid 29
Lactic acid 25
Formic acid 23