Journal of Environmental Biotechnology
Vol. 8, No. 2, 111–118, 2008

Original paper (regular paper)

Removal of Hexavalent Chromium in Vitro and from Contaminated Soils
by Chromate-Resistant Fungi from Chromium Deposits
TSUBASA FUKUDA, KADZUYO TSUTSUMI, YASUHIRO ISHINO, TAKAHIRO SATOU,AKANE OGAWA and HIROSHI MORITA*
Graduate School of Environmental Engineering, The University of Kitakyushu,
Hibikino, Wakamatsuku, Kitakyushu 808–0135, Japan* TEL: +81–93–695–3289 FAX: +81–93–695–3381* E-mail: morita01@hibikino.ne.jp(Received; 20 July, 2008/Accepted; 21 October, 2008)
Three chromate-resistant filamentous fungi, strains N2, N3 and N5, were selected from seven independent fungal isolates
indigenous to Cr(VI) contaminated soil based on their ability to decrease hexavalent chromium level in the growth medium.
Morphophysiological studies identified strain N2 as an Aspergillus sp. and strains N3 and N5 as Penicillium sp.. After
192 h growth in a medium containing 60 mg/L Cr(VI) and at near neutral pH, Aspergillus sp. N2 decreased the Cr(VI)concentration in the growth medium to a virtually undetected level, whereas both Penicillium sp. N3 and Penicillium sp. N5
decreased the Cr(VI) concentration by about 80%. However, acidic condition inhibited the Cr(VI) concentration reducing
ability of these strains. Interestingly, Cu(II), as a coexisting ion, enhanced the Cr(VI) reducing ability of Penicillium sp. N3
at strong acid pH condition. In both slurry-phase and water-phase bioremediation assays, all three strains decreased thelevels of Cr(VI) in polluted soil samples, suggesting that these chromate-resistant filamentous fungi might be useful in cleaning
up chromium contaminated sites.
Key words: Aspergillus sp., bioremediation, chromate-resistant fungi, Cr(VI) removal, Penicillium sp., polluted soils

1. Introduction
Chromium (Cr), a widespread environmental pollutant, isreleased from various industries including tanneries, metal
cleaning and processing, chromium plating, wood processing, and alloy formation3,30). In the developing as well as in
the underdeveloped countries, the industrial effluents are released directly or indirectly into the natural water resources,
mostly without proper treatment, posing a major threat tothe environment11). Among the different oxidative forms ofchromium, the hexavalent chromium (Cr(VI)) is the most
toxic and carcinogenic due to its high solubility in water,
rapid permeability through biological membranes, and subsequent interaction with the intracellular proteins and nucleic
acids13,21,26). Heavy metals in general cannot be biologicallytransformed into more or less toxic products and, hence,
persist indefinitely in the environment. Although presenceof Cr(VI) causes the chromate toxicity, further reduction
leads to the formation of stable, less soluble, and less toxic
Cr(III)4). Thus, reduction of potentially toxic Cr(VI) to lesstoxic Cr(III) is a useful process for remediation of Cr(VI)affected environments19).
Microbial viability is essential for biotransformation asthese reactions are enzyme mediated. Generally metal ionsare converted into insoluble form by specific enzyme medi-

ated reactions and are removed from the aqueous phase22).
There are reports of using live microbial systems for the
purpose of remediation of contaminated soils and waters7).
Higher fungi, yeast, bacteria, seaweed and materials derived
from plants (such as charcoal) are abundantly available innature and can be useful sources for low cost biosorbents6,29). The use of microbial cells as biosorbents for heavy
metals is a potential alternative to conventional methods
that are used to decontaminate liquid wastes. Several bacteria possess chromate reductase activity that can convert
Cr(VI) to a much less toxic and less soluble Cr(III), andthus the reduction of Cr(VI) by these enzymes offer affordable means for chromate bioremediation12). Detoxificationof Cr by naturally occurring microorganisms therefore provides a viable option to protect the environment from chromium toxicity. Moreover, continuous exposure of microbial
populations to heavy metal containing polluted environments selects resistant strains with higher level of tolerance
to heavy metals18).
Some microorganisms are able to interact with different
forms of Cr, making them attractive options for use in thefield of environmental biotechnology. In this respect, it isnoteworthy that the use of microbial biomass for the removalof Cr from the industrial wastewater and polluted water has
already been recognized6,24). Properties of some the micro-

FUKUDA et al.

112

organisms to both tolerate and reduce Cr(VI) enable theirapplication in biotechnological processes focusing on detoxification of hexavalent Cr. Chromate resistance has been
described in bacteria and fungi isolated from the Cr-contaminated environments. In the laboratory, chromate-resistantfungal strains were obtained by mutagenic induction6). Yeast
strains isolated from the Cr-contaminated environments
include those mostly from the genera Candida2,23) and to aminor extent from the genera Rhodosporidium23). In theseyeast strains, the general mechanism of chromate resistanceis limited to the increased ion uptake rather than thechemical reduction of the toxic specie2,23). However, otheryeasts such as Candida utilis20) and Candida maltosa25)
showed partial ability to reduce Cr(VI) and also the capability to accumulate Cr in the biomass. There are also several
recent reports examining the uptake and accumulation of
Cr(III) and (or) Cr(VI) by different yeasts5,14) and by filamentous fungi8,31).
In this work, we have isolated two chromate-resistantstrains of filamentous fungi indigenous to chromium deposit,
characterized their abilities to remove Cr(VI) from the contaminated soil.

2. Materials and Methods
2.1. Isolation of fungal strains and chromate resistance
test
Chromate-resistant filamentous fungi were isolated fromchromium deposits in Fukuoka, Japan. A sample suspension was prepared for each sample by adding about 1 g of
the fungi containing chromium deposit to 10 mL physiological saline solution. One hundred microliter of each sample
suspension was dropped into a culture plate, PG agar media
containing 500 mg/L Cr(VI) was poured on the top, andthe agar plates were incubated at 30°C for 1 week. The
microbial colonies (fungi) appearing on the culture plates
were isolated, purified and characterized based on theirmorphological structures such as color, texture, and diameter of the mycelia, and microscopic observation of thespore formation. Seven strains (N2, N3, N4, N5, N6, N7,
N8) were isolated from the PG solid medium containing
500 mg/L of Cr(VI), and further purified on culture plates.
Finally, the fungi were inoculated from the plates onto theagar slants and stored at 4°C until needed for further experiments. The PG agar medium consisted of 5 g bactopeptone, 2 g glucose, 15 g agar in 1.0 L deionized water.
The pH value of the medium was adjusted to 6.0 with 6 M
NaOH and 6 M HCl. Chromate-resistance test of the isolated strains was performed using PG liquid medium containing different concentrations of chromium oxide (0, 50,
100, 500 and 1000 mg/L) as Cr(VI).
2.2. Cr(VI) removal assay in liquid media
Each isolated strain was suspended in sterilized water andthe spore concentration was adjusted to approximately 1×
107 spores/mL. One milliliter of this spore suspension wasused for inoculating one hundred milliliter of a Cr(VI) containing liquid test medium in a 300 mL conical flask andincubated at 30°C for 8 days on a 60 rpm shaking incubator. The liquid test medium contained the same ingredientsas the PG agar medium described above except agar. After
autoclaving the medium at 120°C for 20 min, appropriate
amount of chromium oxide solution was aseptically added
to the medium to prepare Cr(VI) containing liquid test
medium of various final concentrations (25, 60, 100 and
200 mg/L).
The influence of pH on Cr(VI) removal was examined byvarying the pH of the reaction mixture from 3 to 6 andusing the initial Cr(VI) concentration of 60 mg/L.
Stock solutions of copper sulfate and zinc sulfate asZn(II) were prepared in distilled water, filter sterilized andappropriately diluted (50 mg/L final) in the PY liquid medium containing an initial Cr(VI) concentration of 60 mg/L
for determining the influence of Cu(II) or Zn(II) on Cr(VI)removal.
2.3. Cr(VI) removal assay in contaminated soils
Contaminated soil samples were collected from the chromium contaminated sites at Fukuoka, Japan. Soil samples
were manually collected and transported to the laboratory
in airtight polythene containers. The pH of the soil was 6.5.
Two types of laboratory-scale bioremediation assays werecarried out. In one type of assay (slurry-phase bioremediation assay), the mycelial biomass, obtained from a culturegrown for 3 days in liquid medium as described in the previous section, was transferred to 50 mL of fresh PG liquidmedium, followed by the addition of 10 g of contaminatedsoil. At this point, the DMW (Dry Mycelium Weight) of
each fungi was about 0.5 g/L. At various times during thecourse of incubation, aliquots were removed, centrifuged at4200 × g for 20 min and the supernatant was used for thedetermination of Cr(VI) concentration (see below).
In the second assay (water-phase bioremediation assay),
10 g of contaminated soil was washed by incubating with
90 mL of distilled water for 24 hours on a 100 rpm shaking
incubator. The pH value of the distilled water was adjusted
to 8.0 with 6 M NaOH and 6 M HCl. Mycelial biomass,
obtained from a culture grown for 3 days in liquid medium
as described in the previous section, was transferred to
10 mL of fresh PG liquid medium, followed by the additionof 90 mL of the wash supernatant. At this point, the DMWof each fungi was about 0.5 g/L. At various times during
the course of incubation, aliquots were removed, centrifuged at at 4200 × g for 20 min and the supernatant wasused for the determination of Cr(VI) concentration (see below).
2.4. Growth assessment of microorganisms
Dry mycelium weight (DMW) as a growth measure wasdetermined by drying the cells from the culture at 105°C for
24 h in an oven. DMW was defined as mycelium weight per
100 mL of liquid culture medium.

Removal of Cr(VI) by chromate-resistant fungi

113

2.5. Measurement of Cr (VI) and total Cr concentration
Concentrations of Cr(VI) were determined by the diphenylcarbazide method16), and total Cr was determined by inductively coupled plasma atomic emission spectrometry(ICP-AES, PerkinElmer Japan Co., Ltd.). Mycelia were
removed from the fermentation broth by filtration throughfilter paper and centrifugation at 4200×g for 20 min. The
supernatant fluid was filtered through a 0.45-μm-pore-size
membrane filter (Toyo Roshi Kaisha, Ltd.). Five mL ofculture supernatant was aseptically removed and transferred
to a glass test tube. The sample was acidified with 1 mL of
10% H2SO4. The sample was then treated with 1 mL of a
10 mg/mL diphenylcarbazide solution in acetone, and distilled water was added to a total volume of 50 mL. Cr(VI)reacts with diphenylcarbazide to form a bright pink color,the intensity of which is directly proportional to the Cr(VI)concentration. This solution was shaken for 30 s, and after
5 min the absorbance at 540 nm was read on a UV-vis
spectrophotometer (Amersham Pharmacia Biotech Co.,
Ltd.). In addition, this solution was analyzed by ICP-AES.
Cr(VI) concentrations were estimated from standard curves
generated with known amounts of Cr(VI) using potassium
chromate in the growth medium.

3. Results
Seven independent chromate-resistant filamentous fungi
were isolated from the chromium deposits, which are labeledhere as N2, N3, N4, N5, N6, N7 and N8. All seven strains
were tested for their ability to decrease the Cr(VI) concentration in the growth media at near neutral (pH 6.0) andstrong acidic (pH 3.0) conditions. As shown in Fig. 1, allseven fungal strains were able to efficiently decrease theCr(VI) concentration in the growth medium; however, there
were obvious differences in their efficiency of decreasing theCr(VI) concentration. In the case of strong acid pH condition (pH 3.0), the Cr(VI) concentration in the N2 culture
medium dropped from its initial concentration of 100 mg/L
to 30 mg/L (~70% reduction) after 192 h incubation. During the same time period and similar strong acidic pH condition, the strains N3, N5 and N7 decreased the Cr(VI)concentration in the growth media by 60%, and the strains
N4, N6 and N8 decreased the Cr(VI) concentration in thegrowth media by 35%, 50% and 45%, respectively. On theother hand, at the near neutral pH condition, the N5 andN6 strains decreased the Cr(VI) concentration by approximately 60%, the N2, N3 and N8 strains decreased theCr(VI) concentration by about 50%, and the strains N7 andN4 decreased the Cr(VI) concentration by about 40% and30%, respectively. The fungal isolate N2 was identified as
Aspergillus sp., whereas the isolates N3 and N5 were identified as Penicillium sp.15); however, we do not know theidentity of rest of the four isolated fungal strains. Thus, weused Aspergillus sp. N2, Penicillium sp. N3, and Penicillium sp. N5 for further investigation. All three strains (N2,

Fig. 1. Cr(VI) remaining (%) in the growth medium of cultures ofseven (N2, N3, N4, N5, N6, N7 and N8) chromate-resistantfungal strains isolated from the chromium deposits.
Each fungal strain was grown in the PG medium at 30°C for
192 h. The initial concentration of Cr(VI) in the medium was
100 mg/L. White bars depict percentage of Cr(VI) remaining
after 192 h growth in the medium at near neutral pH condition (pH 6.0). Black bars depict percentage of Cr(VI) remaining after 192 h growth in the medium at an acidic condition(pH 3.0).

N3 and N5) showed significant growth in the presence ofup to 25 mg/L Cr(VI), and were able to grow, albeit very
slowly, even in the presence of 1000 mg/L Cr(VI).
Next, we compared the abilities of the Aspergillus sp.
N2, Penicillium sp. N3, and Penicillium sp. N5 strains inlowering the concentration of Cr(VI) (initial concentration
60 mg/L) in the culture medium. Fig. 2A shows the results
when the medium pH was near neutral (pH 6.0). After
192 h of incubation in medium, Aspergillus sp. N2 exhibited a remarkable efficiency to diminish the Cr(VI) level inthe growth medium. The other two strains, N3 and N5,
were also able to decrease the Cr(VI) concentration in thegrowth medium by about 80%. In the Aspergillus sp. N2
culture, the yellow color due to the soluble Cr(VI) turned
into slightly turbid brownish color (result not shown). In
contrast, when grown in strongly acidic medium (pH 3.0),
these strains were able to decrease the Cr(VI) concentrationin the growth medium to a significantly lower extent than
the neutral pH condition (Fig. 2B). Whereas Aspergillus sp.
N2 and Penicillium sp. N3 decreased the Cr(VI) concentration in the medium under the strong acidic condition by
50%, Penicillium sp. N5 was able to reduce the Cr(VI)concentration by only 30%. However, the Cr(VI) concentration also decreased by about 30% in the medium of thecontrol culture without any inoculum.
Next, we cultured Aspergillus sp. N2, Penicillium sp. N3,
and Penicillium sp. N5 in liquid media containing different
concentrations (25–200 mg/L) of Cr(VI) and examined how
effectively they could remove chromate from the medium
after 192 h growth. Results of these experiments are shownin Fig. 3. As shown, the initial concentration of Cr(VI) in
the culture medium clearly affected the ability of thesestrains to decrease the Cr(VI) concentration in the medium
after 192 h of growth, and all three strains showed very

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FUKUDA et al.

Fig. 4. Effect of divalent cations on the Cr(VI) reducing propertiesof the isolated fungi. Each strain was grown in the PG medium at 30°C for 192 h. White bars, % reduction of Cr(VI) concentration when the strains were grown in the medium containing 60 mg/L Cr(VI). Gray bars, % reduction in the Cr(VI)concentration when the strains were grown in the medium
containing 60 mg/L Cr(VI) plus 50 mg/L Cu(II). Black bars,% reduction in the Cr(VI) concentration when the strains weregrown in the medium containing 60 mg/L Cr(VI) plus 50 mg/
L Zn(II).

Fig. 2. Time course of decrease in Cr(VI) concentration (mg/l) in
the growth medium of cultures initiated in PG medium at (A)near neutral pH 6.0 (B) acidic pH 3.0 supplemented with60 mg/L of Cr(VI).
Symbols represent: ̶●̶, blank; ̶△△̶, Aspergillus sp.
N2; ̶□□̶, Penicillium sp. N3; and ̶+̶, Penicillium sp.
N5.

Fig. 3. Percent decrease in Cr(VI) concentration in the growth medium containing different concentrations of Cr(VI).
Each fungal strain was grown in the PG medium at 30°C for
192 h. Symbols represent: ̶●̶, blank; ̶△△̶, Aspergillus
sp. N2; ̶□□̶, Penicillium sp. N3; and ̶+̶, Penicillium
sp. N5.

similar efficiency pattern in removing Cr(VI) from themedium. At lower initial Cr(VI) concentrations (25 mg/L
and 60 mg/L), Aspergillus sp. N2 completely removed theCr(VI) from the medium; in contrast, at initial Cr(VI) concentrations of 100 mg/L and 200 mg/L, N2 decreased theCr(VI) concentration in the media by 45% and 5%, respec-tively. Similarly, at initial Cr(VI) concentrations of 25 mg/L,
60 mg/L and 100 mg/L, Penicillium sp. N3 decreased theCr(VI) concentration in the growth medium by 75%, 75%
and 60%, respectively, but failed to decrease the Cr(VI)concentration when the initial concentration of Cr(VI) in
the medium was 200 mg/L. Likewise, at initial Cr(VI) concentrations of 25 mg/L, 60 mg/L, 100 mg/L and 200 mg/L,
Penicillium sp. N5 decreased the Cr(VI) concentration in
the growth medium by 85%, 85%, 60% and 20%, respectively. Thus, the higher initial Cr(VI) concentration was, thelower rate of Cr(VI) removal.
Microbial growth and Cr(VI) removal are influenced bymany environmental factors16), one of which is be the concentration of coexisting metal ions. It is known that copperand zinc commonly present in the Cr(VI) contaminatedwastewater or Cr(VI) contaminated soil33,34). Thus, we next
investigated the influence of the coexisting Cu(II) and Zn(II)ions on the Cr(VI) removing properties of Aspergillus sp.
N2, Penicillium sp. N3 and Penicillium sp. N5, and theresults are shown in Fig. 4. These experiments were performed using the culture medium containing 60 mg/L ofCr(VI) as the base growth medium to which either Cu(II) or
Zn(II) was added to a final concentration of 50 mg/L. As acoexisting ion, both Cu(II) and Zn(II) diminished the abilityof each strain in decreasing the Cr(VI) concentration in thegrowth medium, and the diminishing effect of Zn(II) was
higher than Cu(II). Diminishing effect of Cu(II) was, however, influenced by the initial pH of the growth medium.
Fig. 5A shows the time course of decrease in Cr(VI) concentration in the presence of Cu(II) at neutral pH. Cu(II)affected the abilities of Aspergillus sp. N2, Penicillium sp.
N3 and Penicillium sp. N5 in decreasing the Cr(VI) concentration. Although acidic condition inhibited the Cr(VI)concentration removing abilities of these strains, presenceof trace amount of Cu(II) under acid condition actuallyRemoval of Cr(VI) by chromate-resistant fungi

115

Table 1. DMW of isolated fungi that were grown in the presence of 60 mg/L of Cr(VI) at neutral pH (6.0)
Cr(VI) + Cu(II)Cr(VI)
StrainN2N3N5aDMW [g/L]
48 h

192 h

% Reduction of
Cr(VI)a

0.00
0.05
0.00

0.44
0.28
0.40

97.3
80.3
84.9

DMW [g/L]
48 h

192 h

% Reduction of
Cr(VI)a

0.00
0.07
0.00

0.01
0.37
0.34

26.8
68.5
55.4

Percentage reduction of Cr(VI) was evaluated from the Cr(VI) content remaining in the growth medium after 192 h growthFig. 5. Time course of decrease in Cr(VI) concentration in thegrowth media of cultures initiated in the PG medium supplemented with 60 mg/L of Cr(VI) and 50 mg/L of Cu(II) at (A)near neutral condition (pH 6.0) and (B) acidic condition(pH 3.0). Symbols represent: ̶●̶, blank; ̶△△̶, Aspergillus sp. N2; ̶□□̶, Penicillium sp. N3; and ̶+̶, Penicillium sp. N5.

enhanced the ability of Penicillium sp. N3, but not those ofAspergillus sp. N2 and Penicillium sp. N5, in decreasing
the Cr(VI) concentration in the growth medium (Fig. 5B).
Thus, when grown in the acidic medium (pH 3.0) containing 50 mg/L Cu(II), Penicillium sp. N3 decreased theCr(VI) concentration from its initial concentration of
60 mg/L to almost undetected level after 144 h incubation.
Table 1 summarizes the DMW of N2, N3 and N5 at 48
and 192 h of incubation in growth medium at neutral pH
containing different metal ions. In medium containing only
Cr(VI), the DMWs of Penicillium sp. N3 after 48 h and
192 h incubation were 0.05 g/L and 0.28 g/L, respectively.
Although Aspergillus sp. N2 and Penicillium sp. N5 did

not show any growth at 48 h incubation in the same medium, their DMWs after 192 h incubation (0.44 g/L and
0.40 g/L, respectively) suggested that they grew to the same
extent. Under this growth condition, Aspergillus sp. N2,
Penicillium sp. N3 and Penicillium sp. N5 decreased
Cr(VI) concentration by 97%, 80% and 85%, respectively.
The amount of Cr(VI) removed by the biomass of Aspergillus sp. N2, Penicillium sp. N3 and Penicillium sp. N5 was
111 mg/g, 143 mg/g, and 106 mg/g, respectively. In medium
containing both Cr(VI) and Cu(II), the DMW of both Penicillium sp. N3 and Penicillium sp. N5 were approximately
0.35 g/L after 192 h incubation, whereas Aspergillus sp.
N2 did not grow. Under this condition Aspergillus sp. N2,
Penicillium sp. N3 and Penicillium sp. N5 decreased
Cr(VI) concentration in the growth media by 23.9%, 68.5%
and 55.4%, respectively. Moreover, under this growth condition the amount of Cr(VI) removed by the biomass ofAspergillus sp. N2, Penicillium sp. N3 and Penicillium sp.
N5 was 145 mg/g, 603 mg/g and 46 mg/g, respectively.
To explore possible usefulness of Aspergillus sp. N2,
Penicillium sp. N3, and Penicillium sp. N5 for eliminating
Cr(VI) from the contaminated soil containing approximately
250 mg Cr(VI)/g soil, we performed two types of bioremediation assays. In the first type of bioremediation assay(slurry-phase assay), Cr(VI) was removed from the contaminated soil directly by each strain (Fig. 6). In this assay themycelial biomass, obtained from a 3-day culture in PG
medium that contained no added Cr(VI), was transferred to
the fresh PG liquid medium, followed by the addition of
non-sterilized contaminated soil and incubated further at
30°C. Cr(VI) concentration in the control culture medium(Blank) that did not contain any added fungi gradually increased up to 32 mg/L at 72 h post-incubation. However,
Cr(VI) concentration in the growth medium inoculated withany one of the three strains slowly increased until 24 h andthen remained stationary. Thus, after 72 h of incubation
with the Aspergillus sp. N2, Penicillium sp. N3, and Penicillium sp. N5 biomass, the Cr(VI) concentration of the soil
samples decreased by about 50%, 60% and 50%, respectively.
In the second type of bioremediation assay (water-phasebioremediation assay), Cr(VI) from the contaminated soil
was removed indirectly by Aspergillus sp. N2, Penicillium
sp. N3 and Penicillium sp. N5 (Fig. 7). For this purpose
the Cr(VI) was first leached out from the contaminated soil

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116

Fig. 6. Time course of changes in Cr(VI) levels in samples fromcontaminated soil using slurry phase bioremediation.
Symbols represent: ̶●̶, blank; ̶△△̶, Aspergillus sp.
N2; ̶□□̶, Penicillium sp. N3; and ̶+̶, Penicillium sp.
N5.

Fig. 7. Time course of changes in Cr(VI) levels in samples fromcontaminated soil using water phase bioremediation.
Symbols represent: ̶●̶, blank; ̶△△̶, Aspergillus sp.
N2; ̶□□̶, Penicillium sp. N3; and ̶+̶, Penicillium sp.
N5.

by shaking with distilled water at pH 8.0 for 24 h. We haveobserved that the pH of the water used for leaching determined the rate of chromate extraction from the contaminated water, the optimal pH being 8.0 (data not shown). At
this pH, 24 h shaking with water extracted out all chromium
present in the contaminated soil. The mycelial biomass,
obtained from a 3-day culture of one of the fungi in PG
medium that contained no added Cr(VI), was then mixed
with the chromium-leached water, and fresh PG liquidmedium (9 : 1), and the biomass mixture was incubatedfurther at 30°C. The pH of this biomass mixture was approximately 7.5, and the Cr(VI) concentration was approximately 25 mg/L. Fig. 7 shows the time dependent decreasein the Cr(VI) concentration by Aspergillus sp. N2, Penicillium sp. N3 and Penicillium N5. As shown, each strain wasable to decrease the Cr(VI) concentration significantly until
48 h (81% by Aspergillus sp. N2, 55% by Penicillium sp.
N3 and 71% by Penicillium sp. N5). Beyond 48 h incubation, the Cr(VI) concentration removing ability of eachstrain was slow. After 120 h incubation, Aspergillus sp. N2
decreased the Cr(VI) concentration in the mixture by almost 100%, whereas Penicillium sp. N3 and Penicillium
sp. N5 decreased the Cr(VI) concentration by 74% and85%, respectively. In the absence of any added fungal strain(control blank), the Cr(VI) concentration in the mixture did
not decrease. Then, total Cr concentration did not change
at each condition (data not shown).

lating chromate removing fungi and their use in the detoxification of chromate contaminated soil. The present study
describes the isolation of chromate-resistant Aspergillus sp.
(strain N2) and Penicillium sp. (strain N3 and strain N5)
strains, which can readily decrease the Cr(VI) concentrationin the growth media as well as from contaminated soil
samples. These three strains showed high degree of tolerance to chromate (1000 mg/L), which is greater than thepreviously reported chromate tolerance (25–200 mg/L) of
the Cr(VI)-resistant Aspergillus sp. Ed8 and Penicillium
sp. H13 strains1). Furthermore, the three fungi reported inthis study have the ability to grow and also have high activity
at low pH (3.0–6.0). In contrast, most reported microorganisms have the high ability to remove chromate at neutral
pH1,2,5,8,12–15).
Our other study suggested two mechanisms by whichchromate could be removed10). First, chromate could be reduced to a less toxic lower oxidation state by an enzymatic
reaction. Results described here showed Cr(VI) removal
ability at acidic pH was lower than at neutral pH. Wang etal.32) reported that reduction of chromate to a lower oxidation state by Enterobacter strain occurred between pH 6.5–
8.5 and the reduction was strongly inhibited at pH 5 andpH 9, while at pH 9.0 Achromobacter sp. completely reduced Cr(VI)16). However, since Cr(VI) reduction is enzymemediated, pH changes affects the enzyme ionization rate,changes the protein’s conformation and consequently affects
the enzyme activity. A coexisting ion could also affect the
enzyme activity. At neutral pH condition, the presence ofCu(II) affected the Cr(VI) removal abilities of these strains.
Cu(II) enhanced the Cr(VI) removing ability of Penicillium
sp. N3 at strong acid condition, causing a drop in the
Cr(VI) concentration in the culture medium from its initialconcentration of 60 mg/L to almost undetectable level after
144 h growth. This result is similar to that observed in
Bacillus sp. RE expressing a Cr(VI) reducing enzyme), where
the enzyme activity was enhanced by Cu(II) and Ni(II) andinhibited by Hg(II)9). Cr(VI) removal of Aspergillus sp. N2

4. Discussion
Metal-polluted environments pose serious health andecological risks. Metal containing industrial effluents constitute a major source of metallic pollution. Thus, researchers
are continuously searching for naturally occurring microbes
having better metallic pollutant transformation capabilities
over a wider range of microbial growth conditions. Becauseof the ever-increasing concerns about the chromate toxicity
in chromium deposits we have focused our attention in iso-

Removal of Cr(VI) by chromate-resistant fungi

and Penicillium sp. N3 was caused by enzymatic reaction
significantly10). Moreover, the addition of Cu(II) caused thegrowth of Aspergillus sp. N2 was inhibited drastically. The
DMW of Penicillium sp. N3 and Penicillium sp. N5 was
also influenced by the presence of Cu(II). Thus, the existence of metallic ion was important to grow fungi. Further
studies are necessary to extend our understanding of theeffects of the coexisting ions on the Cr(VI) reducing activityof the strains reported in this study.
Biosorption is the second mechanism by which chromate
concentration is reduced. The fungal cell wall can be regarded as a mosaic of different functional groups, which
could form coordination complexes with metals. Zafar etal.33) showed biosorption ability of Aspergillus strains isolated from wastewater treated soil. At 6 mM initial concentration of Cr (pH 4.5), Cr adsorption value of Aspergillus
sp. 2 (1.56 mg/g) exceeded the Cr adsorption value(1.20 mg/g) of the less tolerant counterpart Aspergillus sp.
1. Some Penicillium strains were also reported to adsorb
chromium33). For example, Penicillium purpurogenum havethe capacity of adsorbing 36.5 mg of Cr(VI) ions per g offungal biomass27). The chromium sorption by Aspergillus sp.
N2 and Penicillium sp. N3 was confirmed10). Under acidic
condition biosorption of chromium by these strains washigher than at neutral pH. The pH condition has a large
influence on chromium sorption of fungi. However, theinfluence of sorption on mycelia in the chromium removal
was little.
Aspergillus niger also has the ability to reduce and adsorb Cr(VI)28). When the initial concentration of Cr(VI) was
50 mg/L, Aspergillus niger mycelium removed 8.9 mg ofchromium/g dry weight of mycelium in 7 days. In the present study, Cr(VI) removed by the biomass of Aspergillus
sp. N2, Penicillium sp. N3 and Penicillium sp. N5 were
111 mg/g, 143 mg/g, and 106 mg/g, respectively. Thus, thechromium removal abilities of these strains are equal or
better than the other reported strains.
Aspergillus sp. N2, Penicillium sp. N3 and Penicillium
sp. N5 were able to decrease the initial Cr(VI) concentrations of the contaminated soils. While Cr(III) compounds
are very stable in soils, Cr(VI) is very unstable and is easily
mobilized in both acidic and alkaline soils34). Since Cr(VI)could be easily extracted from the soil into the aqueous
solution, we employed two types of bioremediation assays(slurry phase bioremediation and water phase bioremediation) to determine the effectiveness of the isolated strains inremoving Cr(VI) from the contaminated soils. In addition,
we adopted seed culture in these assays because inoculated
strains must grow preferentially. Our results clearly showedthat the fungal strains Aspergillus sp. N2, Penicillium sp.
N3 and Penicillium sp. N5 succeeded in reducing theCr(VI) concentration in non-sterilized soils. These results
suggest the potential applicability of Aspergillus sp. N2,
Penicillium sp. N3 and Penicillium sp. N5 for the remediation of Cr(VI) from the contaminated soil in the fields.

117

Acknowledgments
The authors thank Kaori Morita, Kazuhiro Noguchi,
Ying Wang, and Kana Morita for technical assistance.

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