Isolation and characterization of a Klebsiella oxytoca strain for simultaneous azo-dye anaerobic reduction and bio-hydrogen production
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- Yu, L., Li, W., Lam, M.H. et al. Appl Microbiol Biotechnol (2012) 95: 255. doi:10.1007/s00253-011-3688-2
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A facultative anaerobic bacteria strain GS-4-08, isolated from an anaerobic sequence batch reactor for synthetic dye wastewater treatment, was investigated for azo-dye decolorization. This bacterium was identified as a member of Klebsiella oxytoca based on Gram staining, morphology characterization and 16S rRNA gene analysis. It exhibited a good capacity of simultaneous decolorization and hydrogen production in the presence of electron donor. The hydrogen production was less affected even at a high Methyl Orange (MO) concentration of 0.5 mM, indicating a superior tolerability of this strain to MO. This efficient bio-hydrogen production from electron donor can not only avoid bacterial inhibition due to accumulation of volatile fatty acids during MO decolorization, but also can recover considerable energy from dye wastewater.
KeywordsAnaerobicBio-hydrogenDecolorizationElectron donorKlebisiella oxytocaMethyl Orange (MO)
Intensive research has been recently conducted in treatment of azo-dye wastewaters, which are known to be toxic, carcinogenic and causing serious environmental pollution (O’Nell et al. 2000; Méndez-Paz et al. 2005). Biological method has been recognized as an inexpensive, environmental friendly and sustainable way for dye wastewater treatment (Rajaguru et al. 2000; Yu et al. 2011). However, azo dyes are mostly xenobiotic compounds that contain one or more azo groups. Decolorization, i.e., breakage of the azo bond, is the first step of azo dye degradation (van der Zee and Villaverde 2005). Anaerobic–aerobic treatment has long been a common practice for azo dye biodegradation. In the anaerobic process, however, methanogenesis can be seriously inhibited by azo dye or its reduced products, resulting in VFA accumulation and low biogas production. And the increased VFA concentration would, in turn, further inhibit the biogas production (Isık and Sponza 2004; Méndez-Paz et al. 2005). As a result, almost no valuable energy products can be recovered from dye-containing wastewaters, leading to a vast waste of potentially precious resources.
This problem might be addressed if some powerful bacterium is available that can simultaneously reduce the dyes and convert VFA into valuable products (ethanol or hydrogen gas). However, no such single strains have been reported so far. Fortunately, a bacterium capable of simultaneous dye reduction and hydrogen production, strain GS-4-08, was discovered and successfully isolated by us. In this study, we characterize this strain and evaluate its performances in azo dye decolorization and bio-hydrogen production. To the best of our knowledge, this is the first report on simultaneous anaerobic reduction of azo dyes and bio-hydrogen production by a single strain.
Materials and methods
Chemicals and cultivation media
The azo dyes, Acid Red 88 (AR88), Reactive Black 5 (RB5), Disperse Orange 3 (DO3), Direct Red 81 (DR81), were purchased from Sigma-Aldrich without further purification. N’,N-dimethyl-p-phenylenediamine (DPD) was procured from Aladdin. Other chemicals were of analytical grade and were procured from Sinopharm Chemical Reagent (China).
The Luria–Bertani (LB) medium was used for aerobic cultivation. The basal medium was used as the screening medium, which contained (g l−1 of tap water): glucose (1.0), K2HPO4 (1.11), KH2PO4 (2.09), NaHCO3 (0.5), NH4Cl (0.28), MgSO4·7H2O (0.1), yeast extract (0.1). Azo-dye decolorization and sucrose fermentation tests were performed using a simulated dye wastewater containing (g l−1 of tap water): sucrose (6.84), Methyl Orange (MO, 0.033), K2HPO4 (1.0), KH2PO4 (0.5), (NH4)2SO4 (2.0), MgSO4·7H2O (0.1), 1 ml of a trace element solution containing (mg l−1) FeSO4·7H2O (5), ZnSO4·7H2O (0.011), MnCl2·4H2O (0.1), CuSO4·5H2O (0.392), Co(NO3)2·6H2O (0.248), NaB4O7·10H2O (0.177), NiCl2·6H2O (0.025) (O’Nell et al. 2000).
An azo-dye tolerant strain, GS-4-08, was isolated from an anaerobic sequencing batch reactor for treatment of dye wastewater (Yu et al. 2011). Firstly, a 2-ml mixture of anaerobic sludge was inoculated into 100 ml screening medium with 0.1 g l−1 MO, and incubated at 30 °C and pH 7.0 under static conditions. After the color of medium disappeared, a new round of screening operation was performed with an inoculation ratio of 3% (v/v). The screening process was repeated five times, and finally the concentration of target bacteria was 1,000-fold diluted. A sloop of 50 μl aliquots of the dilution was plated on LB agar containing 0.1 mM MO. After purification by single colony isolation, stain GS-4-08 was identified and preserved at −70 °C in LB with 30% glycerol.
16S rRNA sequencing and phylogenetic analysis
To identify the strain, the bacteria culture was subjected to 16S rRNA sequencing and phylogenetic analysis. DNA from 50 ml of samples was isolated and purified using a spin column bacteria genomic DNA reagent kit (Sangon Biotech Co., Ltd, China). Then 16S rRNA gene was amplified by using the polymerase chain reaction (PCR) method with two universal primers, 1492R and 27F. The PCR amplification products were used for the determination of 16S rRNA gene sequence (Sangon Biotech CO., Ltd., China). The sequences amplified from this strain were compared with those in the GenBank nucleotide database by using BLAST program packages. A phylogenetic tree was constructed from distance matrix via neighbor-joining method by using the program of MEGA 4.1.
Decolorization and hydrogen production tests
The strain was first cultivated under aerobic conditions at 30 °C overnight in LB medium and then harvested by centrifugation (8,000 × g, 25 min). The pellet was washed twice in 10 ml Na–K phosphate buffer (pH = 7.0) and then re-suspended in 20 ml fresh Na–K phosphate buffer. Cell suspension was transferred into 250-ml serum bottles containing simulated dye wastewater (100 ml) with an initial cell mass of approximately 0.3 ± 0.01 g l−1 (cell dry weight [CDW]). After being purged with N2 gas to ensure the anaerobic condition, the serum bottles were sealed with butyl rubber stoppers and incubated statically under anaerobic conditions at 30 °C and pH 7.0. The sucrose (20 mM) and MO (0.1 mM) were used as electron donor and acceptor, respectively, unless otherwise stated. Then, a series of tests on dye decolorization and hydrogen production were performed.
Liquid samples were extracted from serum bottles at regular time interval for analysis of the residual dyes and reduced products. The dye concentrations in supernatant were determined by a UV/visible spectrophotometer (UV-2550; Shimadzu, Japan). The concentration of aromatic amines was analyzed by an HPLC (Waters 2695; Waters Inc., USA) according to Yu et al. (2011). Reduced sugars were determined using the anthrone method (Koehler et al. 1952), while the CDW was measured according to the Standard methods (APHA 1998). VFA concentration was detected by a GC-FID (GC-6890N; Agilent Inc., USA) equipped with a fused-silica capillary column (DB-FFAP) as reported previously (Mu et al. 2007). Gas production was periodically measured by releasing the pressure in bottles using a glass syringe according to the method described by Owen et al. (1979). The hydrogen was measured using a gas-tight syringe (1.0 ml injection volume), analyzed by a GC-FID (GC-9790; FuLi Inc., China) coupling with a 5-Å molecular sieve column with argon as the carrier gas.
The bacteria samples were collect at the end of the exponential growth phase, washed by 50 mM phosphate buffer solution (PBS) for three times, and then fixed using 2.5% glutaraldehyde overnight. After decanting the fix solution, the samples were rinsed with PBS for three times again. The washed samples were dehydrated by ethanol solution, then vacuum-dried and coated by Au prior to SEM (S-4700; Hitachi, Japan) observation.
Morphological and phylogenetic analysis of strain GS-4-08
Decolorization characteristics of K. oxytoca GS-4-08
The decolorization performances under varied biomass concentration were also investigated in this study. As shown in Fig. 4b, the specific decolorization rates (SDRs) in all the batches increased significantly in the initial 14 h, and then declined gradually till the end of cultivation at 48 h. The overall decolorization efficiencies increased with the initial biomass concentration. Low decolorization efficiencies of 35.53 ± 1.61 and 54.63 ± 1.03% were obtained, respectively, at low CDW batches (0.5 and 1.5 g l−1). The appropriate initial CDW for decolorization of 0.1 mM MO was 3.0 g l−1, exceeding which no obvious enhancement of SDR was observed. A similar result was obtained by Cosmarium sp. in biodegradation of Malachite Green in a previous study (Daneshvar et al. 2007).
To determine the maximum tolerable concentration of MO for K. oxytoca GS-4-08, the decolorization performances under different initial MO concentrations (0.1–0.5 mM) were investigated. At an initial MO concentration of 0.1 mM, the decolorization efficiency was about 86.93 ± 1.83% after 24 h cultivation and reached 96.86 ± 1.48% after 30 h. Further increase the dye concentration lowered the decolorization efficiency, but the dyes could be almost entirely decolorized when extending the incubation time. As shown in Fig. 4c, at initial dye concentration of 0.2 mM the decolorization efficiencies reached 96.53 ± 1.25% after 48 h, while 96 h was needed to achieve 92.96 ± 0.88% decolorization at an initial dye concentration of 0.3 mM. When the initial dye concentration was further increased to 0.5 mM, a rapid decolorization was achieved in the initial 48 h. But, after that it slowed down, and finally reached a decolorization efficiency of about 75%. This result implies that K. oxytoca G-04-08 can tolerate a MO concentration as high as 0.5 mM.
Bio-hydrogen production from electron donors by K. oxytoca GS-4-08 in MO decolorization
Effect of initial dye concentration on the sucrose degradation by K. oxytoca GS-4-08
The isolated strain K. oxytoca GS-4-08 exhibited considerable anaerobic decolorization ability to a broad range of azo dyes. The highest SDR for MR was 131.15 mg g cell−1 h−1 under a high initial concentration of 1 mM. This high performance might be due to fact that MR could easily penetrate though the cell membranes and be degraded efficiently (Chen et al. 2010), while those dyes with highly polar sulfonate groups cannot penetrate through the cell membrane and thus have lower SDRs (Table 1). Notably, no decolorization occurred under shaking aerobic condition, indicating an anaerobic culturing nature of this bacterium toward azo dyes. Electron donor is necessary in dye anaerobic decolorization, and the type of applied electron donor can directly affect the decolorization performance. Sucrose was identified as the favorite electron donor for MO decolorization, with a highest SDR of 4.04 ± 0.09 mg g cell−1 h−1 achieved at a sucrose concentration of 20 mM. Higher sucrose concentration caused no further increase in SDR, possibly because that although the bacteria preferentially utilize sucrose as carbon source for anaerobic growth, a too high sucrose concentration may result in the “latent period” (the initial 6 h cultivation as shown in Fig. 4a) and lower the decolorization efficiency (Wang et al. 2009).
Comparison between the decolorization ability of K. oxytoca GS-4-08 and those reported in the literature
Klebsiella pneumoniae RS-13
Wong and Yuen (1996)
Klebsiella oxytoca GS-4-08
Kocuria rosea MTCC 1532
Parshetti et al. (2010)
Ceriporia lacerate P2
Xu et al. (2008)
Klebsiella oxytoca GS-4-08
Enterobacter sp. hEC3
Wang et al. (2009)
Klebsiella oxytoca GS-4-08
It has been reported that high concentration of azo dye during anaerobic degradation would inhibit the methanogenesis, which may induce the accumulation of VFAs and further inhibit biogas production (Isık and Sponza 2004). However, no obvious VFA accumulation was observed in this study even at high MO concentration of up to 0.5 mM (Table 2). Only low concentration of acetate (0.17–0.19 g l−1) was detected in all batch runs, suggesting that the produced VFAs have been effectively utilized for hydrogen and ethanol production. The hydrogen yields remained almost unchanged (ranging slightly from 8.04 to 9.33 mmol g−1 sucrose) independent of the initial MO concentration (Table 2). These results suggest that this strain possesses a remarkable high hydrogen production capability as well as azo dye adaptive ability.
Comparison between the hydrogen production yields and rates obtained in this study to those reported in the literature
Klebsiella oxytoca GS-4-08
Klebsiella oxytoca HP1
Long et al. (2005)
Engineered Escherichia coli strain
Mathews et al. (2010)
Klebsiella pneumoniae DSM 2026
Liu and Fang (2007)
Klebsiella sp. HE1
Wu et al. (2008)
The authors wish to thank the NSFC-JST joint project (21021140001), the National 863 Program of China (2008BADC4B18), the JSNSF (BK2010256), the Chinese Universities Scientific Fund and the Suzhou Environmental Protection Technology project for the partial support of this study.