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. ª 2012 The Authors Journal of Applied Microbiology 112, 651–659 ª 2012 The Society for Applied Microbiology 651 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) ª 2012 The Authors 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 ª 2012 The Authors Journal of Applied Microbiology 112, 651–659 ª 2012 The Society for Applied Microbiology 653 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. ª 2012 The Authors Journal of Applied Microbiology 112, 651–659 ª 2012 The Society for Applied Microbiology 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- ª 2012 The Authors Journal of Applied Microbiology 112, 651–659 ª 2012 The Society for Applied Microbiology 655 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. 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