Reutilization of immobilized fungus Rhizopus sp. LG04 to reduce

Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Reutilization of immobilized fungus Rhizopus sp. LG04 to
reduce toxic chromate
H. Liu1*, L. Guo1*, S. Liao2 and G. Wang1
1 State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
2 College of Basic Sciences, Huazhong Agricultural University, Wuhan, China
Keywords
biobeads, chromate-reducing fungus,
immobilization, repeated chromate reduction,
Rhizopus.
Correspondence
Gejiao Wang, State Key Laboratory of
Agricultural Microbiology, College of Life
Science and Technology, Huazhong
Agricultural University, Wuhan 430070,
China. E-mails: [email protected];
[email protected]
*Joint first authors.
2011 ⁄ 2128: received 16 December 2011,
revised 16 January 2012 and accepted 19 January 2012
doi:10.1111/j.1365-2672.2012.05257.x
Abstract
Aims: Most of the researches investigating immobilized fungi in chromate
[Cr(VI)] bioremediation have used dead cells to adsorb Cr(VI). Therefore, the
aim was to identify a Cr(VI)-reducing fungus with the ability of reducing the
toxic Cr(VI) into the much less toxic Cr(III) and to apply the immobilized living fungus in continual reduction of Cr(VI).
Methods and Results: Cr(VI) reduction occurred using both free fungi and
immobilized living Rhizopus sp. LG04. The Cr(VI) bioreduction by the free
fungi was achieved mainly by bioreduction coupled with a small amount of
biosorption on the cell surfaces. LG04 spores immobilized with 3% polyvinyl
alcohol and 3% sodium alginate produced the most stable and efficient biobeads. When the LG04 biobeads were washed and transferred into fresh medium
containing 42 mg l)1 of Cr(VI), the biobeads could be reused to reduce Cr(VI)
for more than 30 cycles during an 82-day operation period. Interestingly, as
the cycles increased, the time required for complete reduction stabilized at
approximately 2Æ5 days, which was faster than that obtained using the free
fungi (4Æ5 days). The pH value of the solution decreased from 6Æ60 ± 0Æ10 to
3Æ85 ± 0Æ15 after each reduction cycle, which may be because the metabolic
products of the fungus changed the environmental pH or because there was an
accumulation of the organo-Cr(III) complex.
Conclusions: The results indicate that using the immobilized living fungus for
the removal of Cr(VI) has the advantages in being stable, long-term treatment,
easy to re-use and less biomass leakage.
Significance and Impact of the Study: To our knowledge, this study reports
the first successful use of immobilized living Rhizopus for the repeated reduction of Cr(VI).
Introduction
The widespread use of chromium (Cr) in different industrial activities, including electroplating, metallurgy, leather
tanning, paint pigmentation and wood preservation, has
produced large quantities of Cr-contaminated wastes. The
discharge of these Cr-contaminated wastes into the environment in solid and liquid forms could result in serious
environmental pollution and human health problems,
even at low concentrations (Kotas and Stasicka 2000; Nickens et al. 2010). Cr usually exists in the highly toxic, sol-
uble Cr(VI) form and less toxic, less soluble Cr(III) form
(Codd et al. 2001). The Cr(III) form generated by
microbe is thought to primarily form undissolved inorganic matter, such as Cr(OH)3 or Cr2O3 precipitates.
However, recent studies suggest a more complicated and
diverse fate of Cr(III), that is, Cr(VI) reduction in the
presence of cellular organic metabolites forms not only
insoluble but also soluble organo-Cr(III) end-products
(Puzon et al. 2005; Dogan et al. 2011). The reduction of
the hazardous Cr(VI) to Cr(III) is an efficient approach
to remove Cr(VI) from water and soil systems (He et al.
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Chromate reduction using immobilized Rhizopus
H. Liu et al.
2010). The conventional physical ⁄ chemical technologies
used in Cr(VI)-contaminated wastewater treatment are
expensive or require large amounts of chemical reagents
that may become secondary environmental pollutants
(Komori et al. 1990). Cr(VI) removal through the reduction and ⁄ or adsorption by micro-organisms has received
increasing attention as an alternative to the conventional
technology in recent years because it is cost-effective and
environmental friendly (Dursun et al. 2003; Zouboulis
et al. 2004).
Most of the Cr(VI) bioremediation studies were performed using free and immobilized bacteria, but showed
major problems with reusability and leakage (White et al.
1995). Research investigating Cr(VI) reduction by fungi is
still limited. The aerobic reduction of Cr(VI) has been
reported for many free bacteria and only a few fungi,
including Bacillus (Camargo et al. 2003; He et al. 2010),
Lysinibacillus (He et al. 2011), Intrasporangium (Yang
et al. 2009), Pseudomonas (Park et al. 2000; Ganguli and
Tripathi 2002), Escherichia coli (Bae et al. 2005), Microbacterium (Pattanapipitpaisal et al. 2001), Shewanella
(Myers et al. 2000; Vaimajala et al. 2002), Aspergillus
(Coreno-Alonso et al. 2009) and Trichoderma (MoralesBarrera and Cristiani-Urbina 2008). Techniques for the
immobilization of the microbes have been attracting
worldwide attention because immobilization has the
advantages of increased stability, the potential for reuse,
easier solid–liquid separation and minimal clogging in
continuous systems (Poopal and Laxman 2008). Several
support materials have been reported for bio-immobilization, such as natural materials [e.g. sodium alginate (SA),
agar and carrageenan] and synthetic matrices [e.g. polyvinyl alcohol (PVA), polyethylene glycol and polyacrylamide (PA)]. Natural gel matrices have the disadvantages
of abrasion and biodegradation (Leenen et al. 1996),
whereas synthetic gels have better mechanical properties
and are less biodegradable. The choice of the immobilization matrix is a key factor for the successful bioremediation of heavy metal pollution by microbial cells.
Different techniques for the immobilization of bacterial
cells during Cr(VI) reduction have been reported, including the following: Intrasporangium sp. Q5-1 entrapped in
PVA–SA beads (Yang et al. 2009), Cr(VI) reduction in
continuous culturing columns by immobilized cells of
Microbacterium liquefaciens MP30 (Pattanapipitpaisal
et al. 2001) and Bacillus sp. ES 29 (Camargo et al. 2004),
Serratia marcescens attached on activated carbon as a biofilm (Bruijn and Mondaca 2000) and Pseudomonas
embedded in agar–agar films on a cellulose acetate membrane (Konovalova et al. 2003). However, the low reusability, biomass leakage and low biomass of the
immobilized bacteria have limited their use in Cr(VI)
bioremediation compared with that of immobilized living
652
fungi. Most research investigating immobilized fungi
in Cr(VI) bioremediation has used dead cells to adsorb
Cr(VI) and ⁄ or Cr(III) ions, which could not remediate
Cr(VI) completely (Bai and Abraham 2003; Li et al.
2008). To our knowledge, only one study has reported
the use of a living fungus, Coriolus versicolor, for Cr(VI)
reduction to date, which used ceramics to immobilize the
fungus (Sanghi and Srivastava 2010).
The objectives of this research were to (i) isolate a
novel Cr(VI)-reducing fungus and study its Cr(VI)
removal efficiency, (ii) select appropriate immobilization
supporting matrices for the Cr(VI)-reducing fungus used
for Cr(VI) bioremediation, (iii) evaluate the Cr(VI)
reducing abilities of free and immobilized Cr(VI)-reducing
fungus and (iv) study the reusability of fungus-embedded
beads for repeated Cr(VI) reduction.
Materials and methods
PVA (mol. wt 75 000–80 000, 87–90 mol% of hydrolyzation) and SA (high viscosity) were obtained from Sinopharm Group Chemical Reagent Co., Ltd (Shanghai,
China). Potassium chromate (K2CrO4) was used as a
source of hexavalent chromium [Cr(VI)]. A sterilized
Cr(VI) stock solution was added to sterile medium to
obtain the desired concentrations of Cr(VI). All other
chemicals were of analytical grade. Tests of Cr(VI) reduction by the free and immobilized fungus were performed
in 250-ml shake flasks. All experiments were performed at
least three times, and unless otherwise mentioned, the
data shown are from one representative experiment performed with triplicate cultures. The data are expressed as
the mean with the standard deviation (n = 3).
Isolation and identification of Cr(VI) resistant and
reducing fungi
A soil sample was collected from the subsurface soil
(0–15 cm) in a Tieshan iron mine (11454¢43¢¢E,
3013¢10¢¢N), Daye county, Hubei province, China.
Cr(VI)-resistant fungi were isolated from the sample by
adding 10 g of soil to 100 ml of a 0Æ85% sterile NaCl
solution, and the sample was then shaken at room
temperature for 30 min. The mixed solution was serially
diluted tenfold and poured onto modified Martin broth
(MMB) plates (composed of 5 g of tryptone, 20 g of glucose, 2 g of yeast extract, 1 g of KH2PO4, 0Æ5 g of MgSO4
and 15 g of agar in 1 l distilled water, pH 6Æ6) supplemented with 42 mg l)1 of K2CrO4. The plates were then
incubated at 28C for 1 week. The colonies were restreaked several times to obtain pure isolates. The Cr(VI)
resistance level of the fungus was determined by inoculating 1% of the original spore suspension (OD660 = 1Æ0)
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Journal of Applied Microbiology 112, 651–659 ª 2012 The Society for Applied Microbiology
H. Liu et al.
into MMB medium with different concentrations of
K2CrO4. The growth of each treatment was observed after
incubation at 28C for 7 days. The minimal inhibitory
concentration (MIC) of Cr(VI) that completely inhibited
the growth of strain LG04 was determined (Sarangi and
Krishnan 2008).
The fungus was identified by its morphological characteristics and 18S rRNA gene and internal transcribed
spacer (ITS) sequences. The 18S rRNA gene was amplified
by PCR using universal primers NS1 (5¢-GTAGTCATATGCTTGTCTC-3¢) and NS8 (5¢-TCCGCAGGTTCACCTACGGA-3¢) (Simon et al. 1992). For the amplification
of the ITS sequence, universal primers ITS1 (5¢-TCCGTAGGTGAACCTGCGG-3¢) and ITS4 (5¢-TCCTCCGCTTATTGATATGC-3¢) were used (Sert et al. 2007). The
18S rRNA gene and ITS sequences were analysed using
the BLASTn searching tools (http://www.ncbi.nlm.nih.
gov/blast). The morphologies of the colony and spores were
observed after incubation on MMB plates for 3–5 days at
28C. The fungi were examined for sporangia, sporangiospores and mycelia under an optical microscope and a
scanning electron microscope (SEM; JSM-6390, JEOL,
Tokyo, Japan) with an accelerating voltage of 20 000 V.
Cr(VI) reduction by free Rhizopus sp. LG04 fungus
The fungus was grown on MMB plates at 28C for 5 days
and then fully mixed with sterile deionized (sdd) water.
The spores in suspension were collected by a sterile muslin cloth filter and adjusted to 1Æ0 at OD660 (Lele et al.
1996). In total, 1 ml of the spore suspension was added
into 100 ml of MMB medium containing 42 mg l)1 of
Cr(VI), which was then incubated at 28C on a rotary
shaker rotating at 150 rev min)1. Controls without fungal
inoculation were also incubated under the same conditions to monitor abiotic Cr(VI) reduction. Samples were
taken at regular intervals. The concentration of the residual
soluble Cr(VI) in the supernatant was determined using
the 1,5-diphenylcarbazide (DPC) reagent at an absorbance
value of 540 nm using a UV spectrophotometer (DU800,
Beckman, Brea, CA, USA; APHA 1995) (Urone and
Anders 1950). The total Cr concentration was also determined with the DPC by a permanganate oxidation method
(Saltzman 1952). The elemental analysis of the surface and
interior structure of the fungus with and without exposure
to 42 mg l)1 Cr(VI) was performed using SEM coupled
with an energy dispersive X-ray spectroscope (EDX) and
TEM (Transmission electron microscope).
Chromate reduction using immobilized Rhizopus
strain LG04 spores for Cr(VI) reduction experiments.
Additionally, the effects of using the MMB media with
different nutrition ratios (100, 75, 50 and 25%) on
Cr(VI) reduction were investigated. The PVA–SA biobeads were prepared as follows: (i) different concentrations
(1–4%) of PVA and SA (1–4%) were mixed in 100 ml of
sdd water and heated three times (with a 12-h interval
between each heating) to 80C for 1 h; (ii) when the
solution was cooled to 40C, 5 g of the fresh spore suspension (OD660 = 1Æ0) was added, and the solution was
mixed aseptically; and (iii) the biobeads (3–4 mm in
diameter) were produced by adding the mixture dropwise
into the immobilization phase containing 4% (w ⁄ v)
CaCl2 and 2% (w ⁄ v) boric acid with a sterile 50-ml disposable plastic syringe and 21-G needle and then immersing the beads for 12 h at 4C.
Reusability of the LG04 biobeads for repeated Cr(VI)
reduction
The immobilized biobeads were washed three times with
sdd water and incubated in 50% MMB broth at 28C for
3 days rotating at 150 rev min)1 to activate spore germination. Then, 10 g of PVA–SA biobeads was added aseptically into 100 ml of 50% MMB broth containing
42 mg l)1 Cr(VI) and incubated as describe above. Samples were taken every 12 h, and the Cr(VI) and total Cr
concentrations were determined.
The analysis of the continual Cr(VI) reduction efficiencies of the immobilized biobeads was performed using
two methods. The first method involved repeated additions of Cr(VI). In total, 100 ml of the 50% MMB broth
was supplemented with 42 mg l)1 of Cr(VI). When the
Cr(VI) reduction was nearly finished, Cr(VI) was added
into the culture to a final concentration of 42 mg l)1 (for
a total of four cycles). In the other method, when one
reduction cycle was nearly complete, the immobilized
LG04 biobeads were washed three times with sdd water
and added into another 100 ml of the 50% MMB broth
containing 42 mg l)1 Cr(VI). The Cr(VI) concentration
in the cell-free samples was also determined over the
course of Cr(VI) reduction. In addition, the pH values of
the solution during the continual Cr(VI) reduction by the
LG04 biobeads were determined. The morphology of the
immobilized LG04 biobeads was examined by microscopy.
Results
Immobilization of Rhizopus sp. LG04 spores
Isolation and identification of a Cr(VI)-reducing fungus
Different concentrations of a synthetic PVA matrix and
the natural material SA were combined to immobilize
The LG04 fungus strain was isolated from the soil from
an iron mine in Daye county and showed high resistance
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Chromate reduction using immobilized Rhizopus
H. Liu et al.
(a)
(b)
(g)
(c)
(d)
(h)
(f)
(i)
to Cr(VI) with a MIC of 10 mmol l)1. After cultivating
the fungus on MMB plates at 28C for 3 days, white mycelia covered the entire surface of the plates (Fig. 1a),
which turned black in 5 days because of the production
of black spores (Fig. 1b). The diameters of the sporangia
and hyphae were 20–30 and 3–8 lm, respectively
(Fig. 1c). The hyphae contained no septa (Fig. 1d). The
sporangia (Fig. 1e) were round, and the sporangiospores
(Fig. 1f) were oval-shaped.
A phylogenetic analysis based on the 18S rRNA gene
and ITS sequences indicated that strain LG04 was closely
related to Rhizopus microsporus UPSC 1758 (GenBank:
AF548092, 18S rRNA gene sequence) and R. microsporus
US-A2 (GenBank: HQ404248, ITS sequence), with 99Æ9%
similarity to both. The 18S rRNA gene and ITS sequences
were submitted to the NCBI GenBank (http://
www.ncbi.nlm.nih.gov/), and their accession numbers are
HQ876464 and HQ876465, respectively. On the basis of
the morphological characteristics and the analysis of the
18S rRNA gene and ITS sequences, this strain was identified as a Rhizopus member and designated as Rhizopus sp.
LG04.
Cr(VI) reduction using free LG04 fungus
After cultivation of strain LG04 for 3 days with and without 42 mg l)1 of Cr(VI), the morphology of the fungi
changed from dispersed spores to a loosely fluffy clump
resulted from aggregation of the spores and mycelia. The
mycelia were observed under a SEM coupled with an
EDX unit. The surface of the mycelia became smooth
654
after culture with Cr(VI) (Fig. 1h) compared with the
control (Fig. 1g). Cr was detected on the surface of the
LG04 fungus by EDX analysis (Fig. 1i), which may be due
to a small amount of physical adsorption or covalent
bonding of Cr. But there was no detection of Cr inside of
the LG04 cell (data not shown). The TEM images showed
no obvious change in the interior structures of the LG04
fungus after Cr(VI) reduction (data not shown).
Figure 2 shows the time course of the Cr(VI) reduction
by free LG04 fungus. In the first 2 days, the rate of
Cr(VI) reduction was slow. After 4Æ5 days, almost all of
the Cr(VI) was reduced. The MMB medium supplemented with 42 mg l)1 of Cr(VI) without fungus inoculation showed no obvious Cr(VI) reduction. The total Cr
50
Residual Cr(VI) (mg l–1)
(e)
Figure 1 Morphological characters of the
fungus LG04 determined by SEM + EDX and
light microscope: fungus LG04 incubated on
modified Martin broth (MMB) agar plate for
3 days (a) and 5 days (b); light micrographs of
sporangium (c), mycelia (d); SEM photographs
of LG04 sporangium (e) and sporangiospore
(f); SEM photographs of LG04 mycelia in
MMB medium without Cr(VI) (g) and with
42 mg l)1 Cr(VI) (h), incubated for 3 days; the
EDX spectrum of strain LG04 cultured in
MMB medium containing 42 mg l)1 Cr(VI) for
3 days (i). EDX, energy dispersive X-ray spectroscope; SEM, scanning electron microscope.
40
30
20
10
0
0
0·5
1
1·5
2
2·5 3
Time (d)
3·5
4
4·5
5
Figure 2 Cr(VI) reduction curves by free fungus LG04. The residual
Cr(VI) concentration with fungus LG04 incubated ( ) and an abiotic
control without fungus inoculation ( ), (¤) the total Cr concentrations. Error bars represent standard deviation of triplicate tests.
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H. Liu et al.
concentrations did not change significantly and were
decreased only by 7Æ1 ± 0Æ35% (Fig. 2). The Cr(VI)
reduction products were still soluble in the medium, suggesting they were most likely the soluble organo-Cr(III)
complex (Puzon et al. 2005).
The repeated reduction of Cr(VI) using immobilized
LG04 biobeads
When different concentrations of PVA and SA were used
in the immobilization experiments, the combination of
3% PVA plus 3% SA formed the most stable bead in
which to embed LG04 spores. Furthermore, efficient
Cr(VI) reductions were obtained with the 3% PVA plus
3% SA embedded LG04 biobeads cultured in 100, 75 and
50% MMB media containing 42 mg l)1 Cr(VI). However,
there was serious leakage of biomass from the LG04
biobeads in the 100 and 75% MMB media. In the 25%
MMB medium, the reduction efficiency was low, and the
reduction rate decreased sharply as cycles increased (data
not shown). Because there was no obvious difference in
Cr(VI) reduction rate when the LG04 biobeads were incubated in 100, 75 and 50% MMB media, and because the
leakage was effectively controlled in the 50% medium, the
continual Cr(VI) reduction by immobilized LG04 biobeads was performed in the 50% MMB medium. In the first
Cr(VI) reduction test, the Cr(VI) was completely reduced
by the LG04 biobeads in 5 days. The abiotic control and
the beads without fungi embedded in them showed no
obvious Cr(VI) reduction (Fig. 3a).
The reusability of the immobilized LG04 biobeads for
further Cr(VI) reduction was tested by two methods as
described above in the Materials and methods. The first
method was performed to test the ability of the entrapped
biobeads to repeatedly reduce Cr(VI) by adding Cr(VI) to
a final concentration of 42 g l)1 after the previous Cr(VI)
was metabolized in a total of four cycles. As shown in
Fig. 3b, the first round of 42 mg l)1 of Cr(VI) was almost
fully reduced within 5 days with an average reduction rate
of 0Æ70 mg l)1 h)1. When the culture was added with
42 mg l)1 of Cr(VI) again, 98Æ2% of the Cr(VI) was
reduced over another 2 days (0Æ86 mg l)1 h)1). After the
third addition of 42 mg l)1 Cr(VI), the concentration of
Cr(VI) was reduced to 6Æ05 mg l)1 over another 2Æ5 days
(0Æ60 mg l)1 h)1). During the fourth cycle, the Cr(VI)
reduction efficiency decreased, and approximately 72Æ5%
of the Cr(VI) was reduced in 8Æ5 days. Overall, the results
showed that the immobilized biobeads were able to
reduce <4 cycles of repeatedly amended Cr(VI) without
the addition of any extra nutrients in the period of
18 days.
In the second method, repeated operations for the
reduction of Cr(VI) in media with a concentration of
Chromate reduction using immobilized Rhizopus
42 mg l)1 of Cr(VI) using immobilized LG04 biobeads
were carried out. Before each operation, the biobeads
were washed and transferred into fresh yellow coloured
50% MMB medium containing 42 mg l)1 of Cr(VI). After
each cycle, the yellow colour of the solution was disappeared, which most probably due to the reduction of
K2CrO4, while the colour of the biobeads changed from
white to light yellow and to yellow along with the
increase in treatment cycles. The LG04 biobeads could be
reused while completely reducing the Cr(VI) for more
than 30 cycles, up to a total of 82 days of operation.
Interestingly, with the increase in the cycles, enhanced
reduction rates were observed. The complete reduction
time stabilized at 2Æ5 days at approximately the fourth
cycle (Fig. 3c). The pH value of the supernatant decreased
from 6Æ60 ± 0Æ10 to 3Æ85 ± 0Æ15 (n = 4) at the start and
the end of 19th, 22nd, 27th and 30th cycles. Other cycles
showed similar trends. The LG04 biobeads, after being
reused for 30 cycles were bigger (Fig. 3d II, about two
times in diameter) than the biobeads before the treatment
(Fig. 3d I). One explanation for this is that the LG04 fungal spores germinated and grew in the beads and a small
amount of Cr was adsorbed. In addition, the LG04 biobeads became harder with the increased use, which favoured
the stability of the biobeads and limited the fast growth
of the internal mycelia.
The interior and exterior structures of the immobilized
LG04 biobeads were observed. Several mycelia were
observed inside the LG04 biobeads when they were incubated in the 50% MMB medium for 2 days (Fig. 4a), and
the mycelia filled the beads by 3 days (Fig. 4b). After the
Cr(VI) reduction, there were some changes both in
the exterior and in interior of the beads, with or without
the embedded LG04 spores. The surface of the beads
without the immobilized LG04 fungus was wrinkled and
creviced (Fig. 4c,e), but the surface of LG04 biobeads was
smooth and integrated with mycelia (Fig. 4d,f). The
interior of the LG04 biobeads was full of mycelia
(Fig. 4h), but there was no analogous structure in the
beads without the LG04 spores embedded (Fig. 4g).
Discussion
So far, most studies have used immobilized bacteria or
dead fungi to remediate Cr(VI) (Table 1). It was previously reported that the immobilized dead fungal biomass
of Rhizopus cohnii could be reused for five cycles for the
adsorption of Cr(VI) (Li et al. 2008). The immobilized
dead fungus Rhizopus nigricans could be reused for more
than 25 cycles (Bai and Abraham 2003). However, the
number of functional groups on the cell surface of dead
fungi is limited. Most of them may have lost the adsorptive ability during the desorption process, and the adsorp-
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Journal of Applied Microbiology 112, 651–659 ª 2012 The Society for Applied Microbiology
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Chromate reduction using immobilized Rhizopus
H. Liu et al.
Residual Cr(VI) (mg l–1)
Residual Cr(VI) (mg l–1)
(a)
(d)
50
40
II
30
1 cm
20
10
0
I
0
(b)
50
45
40
35
30
25
20
15
10
5
0
0
0·5 1 1·5 2 2·5 3 3·5 4
Time (d)
1
2
3
4
5
6
4·5 5
7
8
9 10
Time (d)
11
12
13
14
15 16
17
18
Residual Cr(VI) (mg l–1)
(c)
60
50
40
30
20
10
0
0 2
4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82
Time (d)
Figure 3 Cr(VI) reduction by immobilized LG04 biobeads. Error bars represent standard deviation of triplicate tests. (a) The Cr(VI) reduction using
(m) the immobilized biobeads, (¤) an abiotic control and ( ) the 3% polyvinyl alcohol and 3% sodium alginate beads without LG04 spores
embedded; (b) method 1: the LG04 biobeads could be reused four times continually with repeated adding of 42 mg l)1 Cr(VI) into 50% modified
Martin broth (MMB) medium without adding extra nutrient; (c) method 2: the LG04 biobeads could be reused for more than 30 times, before
each cycle, LG04 biobeads were washed with sterile deionized water and transferred into a fresh 50% MMB medium containing 42 mg l)1 Cr(VI);
(d) comparison of LG04 biobeads before (I, 3–4 mm in diameter) and after (II, 4–6 mm in diameter) the treatment of 42 mg l)1 Cr(VI) for 30
cycles, which was mixed together. Bar, 1 cm.
tion efficiency decreased with the increased frequency of
long-term use (Li et al. 2008). Thus, these methods were
less efficient for complete removal of Cr(VI). Because
there is no restriction on saturation and desorption of
functional groups, applying immobilized living fungi for
Cr(VI) bioreduction should be capable of repeated
removing Cr(VI).
In this study, the free Rhizopus sp. LG04 fungus could
completely reduce 42 mg l)1 of Cr(VI) within 4Æ5 days.
After Cr(VI) reduction, the surface of the LG04 fungus
656
became smooth, which is similar to the morphology
reported for a Cr(VI)-reducing bacterial strain, Lysinibacillus fusiformis ZC1 (He et al. 2011), possibly because of
the interaction between the functional groups on the fungal surface and Cr(VI) and ⁄ or Cr(VI)-reducing products.
The EDX spectrum showed the presence of Cr, which
might be due to physical adsorption and ⁄ or covalent
bonding. The TEM images revealed no differentiation of
the interior structure of the LG04 fungus, which indicated
that there was almost no Cr accumulation inside the cells.
ª 2012 The Authors
Journal of Applied Microbiology 112, 651–659 ª 2012 The Society for Applied Microbiology
H. Liu et al.
Chromate reduction using immobilized Rhizopus
(a)
(c)
(e)
(g)
10 µm
X33
(b)
500 µm
X1,000
(d)
10 µm
(f)
X1,000
10 µm
X1,000
10 µm
(h)
10 µm
X33
500 µm
X1,000
10 µm
Figure 4 The internal and external morphologies of LG04 biobeads immobilized with 3% polyvinyl alcohol (PVA) and 3% sodium alginate (SA). a
and b represent light micrographs of biobead incubated for 2 and 3 days, respectively. Others are the scanning electron microscope photographs
of exterior (c and e) and internal (g) morphologies of the 3% PVA and 3% SA beads without LG04 spores, and exterior (d and f) and internal (h)
morphologies of biobeads incubated in 50% modified Martin broth medium containing 42 mg l)1 Cr(VI) for two cycles. Bars: 10 lm except for c
and d (500 lm).
Table 1 Cr(VI) reduction or sorption rates for different immobilized bacteria and fungi
Micro-organism
Bacteria
Streptomyces griseus
Intrasporangium sp. Q5-1
Desulfovibrio vulgaris
Desulfovibrio desulfuricans
Fungi
Rhizopus spore
Coriolus versicolor
Rhizopus cohnii
R. cohnii
Rhizopus nigricans
Lentinus sajor-caju
Rhizopus arrhizus
Immobilization matrix
PVA-alginate
PVA–SA
Agar
Polyacrylamide gel
PVA-alginate
Ceramics
Polyurethane
Alginate
Polysulfone
Carboxymethylcellulose
Alginate
Cr(VI) removal efficiency
(mg l)1 h)1)
0Æ26*
1Æ50*
1Æ09*
1Æ02*
0Æ70*
1Æ07*
5Æ70
46Æ65
11Æ20
1Æ18
7Æ94
Cr(VI) removal
cycle
References
4
ND
ND
ND
Poopal and Laxman (2008)
Yang et al. (2009)
Humphries et al. (2005)
Tucker et al. (1998)
>30
1 year
5
5
25
5
ND
This study
Sanghi and Srivastava (2010)
Li et al. (2008)
Li et al. (2008)
Bai and Abraham (2003)
Arica and Bayramoglu (2005)
Prakasham et al. (1999)
ND, no data available; PVA, polyvinyl alcohol; SA, sodium alginate.
*The removal of Cr(VI) by bioreduction.
The immobilized micro-organisms listed here are living unless otherwise mentioned.
The removal of Cr(VI) by biosorption.
Therefore, we concluded that the detoxification of Cr(VI)
by the LG04 fungus was conducted predominately via
bioreduction coupled with a small amount of biosorption
on the fungus surface.
The immobilized LG04 biobeads remained stable and
showed a satisfactory Cr(VI) reduction efficiency and
could be reused for Cr(VI) reduction for more than 30
cycles when the LG04 biobeads were washed and then
transferred into a fresh Cr(VI)-containing solution. After
the first three cycles, each complete Cr(VI) reduction period was stabled at approximately 2Æ5 days, which was faster than using the free fungus (4Æ5 days). This result may
be due to spore germination and biomass increase, which
results in an increase in metabolic reducing substances
and ⁄ or electron donors. The pH value of the solution
decreased after each cycle, which could be due to the
metabolic reduction products generated by the fungus.
Another explanation is that the accumulation of organic
acid salt, such as the organo-Cr(III) complex, changed
the pH. In addition, the acidic environment might be
favourable to the protonation of functional groups on the
surface of the fungus, allowing them to capture the
Cr(VI) anion and promote the Cr(VI) reduction (Sanghi
and Srivastava 2010). The sporangiospores were shown to
germinate and grow inside the PVA–SA beads through
light microscopy and SEM, which was the basis for the
ª 2012 The Authors
Journal of Applied Microbiology 112, 651–659 ª 2012 The Society for Applied Microbiology
657
Chromate reduction using immobilized Rhizopus
H. Liu et al.
continuous bioreduction of Cr(VI). Owing to the restriction of the PVA–SA net, the growth of the internal mycelia was slow and the biobeads were not broken after the
82 days.
To immobilize the fungus for Cr (VI) reduction, different materials were considered to embed the spores. Most
natural matrices have the disadvantages of being abrasive
and biodegradable (Poopal and Laxman 2008). Coupling
natural matrices with synthetic materials can conquer
these drawbacks and have complementary advantages
with an appropriate combination. PVA is a promising
synthetic polymer and is suitable for the entrapment of
fungus spores because it is stable and nontoxic to microorganisms. Our results showed the LG04 spores embedded in the PVA–SA beads exhibited a significantly
repeated Cr(VI) reduction activity. However, such combination could not embed other fungus strains successfully
(data not shown). The optimal support matrix for immobilization appears to be quite different between individual
bacteria and fungi (Table 1).
The 50% MMB medium showed the best choice for
the repeated Cr(VI) reduction. However, this medium
contained tryptone and yeast extract that were still
costly. According to our experience using both free and
immobilized bacterial cells, complete media were much
more efficient for Cr(VI) reduction than minimal media
(Yang et al. 2009; He et al. 2011; and unpublished data).
In our future studies, cost-efficient media will be applied
to cut down the expenses for fungal Cr(VI) reduction,
such as the modified Lee’s minimal medium (LMM; Lee
et al. 1975) or the potato dextrose broth (Ahluwalia and
Goyal 2010) media.
In summary, this study reports the successful use of
immobilized living fungus for the repeated reduction of
Cr(VI). The LG04 biobeads have many advantages over
free cells and immobilized dead cells in that the LG04
biobeads are more stable and durable, are easier to reuse
and leak less biomass in a continuous system. Given these
extraordinary properties, the immobilized LG04 biobeads
have great potential for use in a repeated bioprocess for
the remediation of Cr(VI)-contaminated wastewater.
Acknowledgements
This work was supported by a Major International Joint
Research Project from the National Natural Science Foundation of China (31010103903).
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