Bull Environ Contam Toxicol (2015) 94:358–364 DOI 10.1007/s00128-014-1366-7

Isolation of a Naphthalene-Degrading Strain from Activated Sludge and Bioaugmentation with it in a MBR Treating Coal Gasification Wastewater Peng Xu • Wencheng Ma • Hongjun Han Shengyong Jia • Baolin Hou

•

Received: 10 July 2014 / Accepted: 16 August 2014 / Published online: 2 September 2014 Ó Springer Science+Business Media New York 2014

Abstract A highly effective naphthalene-degrading bacterial strain was isolated from acclimated activated sludge from a coal gasification wastewater plant, and identified as a Streptomyces sp., designated as strain QWE-35. The optimal pH and temperature for naphthalene degradation were 7.0 and 35°C. The presence of additional glucose and methanol significantly increased the degradation efficiency of naphthalene. The strain showed tolerance to the toxicity of naphthalene at a concentration as great as 200 mg/L. The Andrews mode could be fitted to the degradation kinetics data well over a wide range of initial naphthalene concentrations (10–200 mg/L), with kinetic values qmax = 0.84 h-1, Ks = 40.39 mg/L, and Ki = 193.76 mg/ L. Metabolic intermediates were identified by gas chromatography and mass spectrometry, allowing a new degradation pathway for naphthalene to be proposed for the first time. Strain QWE-35 was added into a membrane bioreactor (MBR) to enhance the treatment of real coal gasification wastewater. The results showed that the removal of chemical oxygen demand and total nitrogen were similar between bioaugmented and non-bioaugmented MBRs, however, significant removal of naphthalene was obtained in the bioaugmented reactor. The findings suggest a potential bioremediation role of Streptomyces sp. QWE35 in the removal of naphthalene from wastewaters. Keywords Andrews mode  Bioaugment  Metabolites  Naphthalene  Streptomyces sp.

P. Xu  W. Ma  H. Han (&)  S. Jia  B. Hou State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China e-mail: [email protected]

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Naphthalene is a hazardous compound present in many waste streams from various industrial processes, such as coal conversion, petroleum refining, and synthesis of organic chemicals (Chang et al. 2009; Horng et al. 2009). It is generally a toxicant to plants as well as microorganisms. Especially in China, fast expanding and developing chemical industries are an increasing source of environmental pollution (Gu et al. 2011). As a result of growing awareness over pollution caused by naphthalene release, efforts are being made to minimize their adverse effect. Many treatment techniques such as activated carbon adsorption, chemical oxidation, and biodegradation have been developed to remove naphthalene from contaminated environment (Filonov et al. 2010; Gu et al. 2013). Of these options, physicochemical methods have proven to be costly and have the inherent drawbacks of causing secondary pollution (Maillacheruvu and Pathan 2009). However, biodegradation appears to be environmentally friendly, and has turned out to be a favorable alternative. Previous studies showed that a number of bacterial strains are able to utilize naphthalene and its derivatives under aerobic and anaerobic conditions, e.g., Pseudomonas sp., Desulfo indolicum, Burkholderia pickettii, Bacillus fusiformis and white rot fungus (Lin et al. 2010; Mollea et al. 2005, O’Loughlin et al. 1996). Majority of these strains showed poor degrading capability, particularly under high naphthalene concentrations ([200 mg/L) (Farjadfard et al. 2012). Search for new strains is still of great scientific and industrial significance. In this report, a highly effective naphthalene-degrading bacterial strain, designated QWE-35, was isolated from activated sludge from a coal gasification wastewater plant and identified as Streptomyces sp. The optimal conditions for degrading naphthalene with strain QWE-35 were examined. Understanding the biodegradation mechanisms

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of naphthalene by strain QWE-35 also required an analysis of the biodegradation kinetics, along with the identification of intermediate products using gas chromatography and mass spectrometry (GC–MS). Finally, the bioaugmentation of strain QWE-35 into activated sludge for naphthalene degradation has also been investigated.

Materials and Methods Naphthalene was of chromatographic grade, and obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other chemicals used were of analytical grade, and purchased from the local suppliers. Two kinds of media were used to assess naphthalene biodegradation. The Luria–Bertani (LB) medium was used for bacterial growth and maintenance. Mineral salt medium (MSM), described by Lin et al. (2010) was used in the biodegradation experiments. Naphthalene solution filtered with 0.2 lm membrane was added into the MSM as the sole carbon and energy source. Solid medium contained 1.9 % (w/v) agar. All media were sterilized at 121°C for 15 min before use. A naphthalene-degrading bacterium was isolated from activated sludge from a coal gasification wastewater plant by enrichment culture in MSM containing 50 mg/L naphthalene at first. 200-mL activated sludge solution was centrifuged at 5,0009g for 10 min, the centrifuged deposition was then washed twice with 0.01 M sodium phosphate buffer (KH2PO4 72.3 mg/L and K2HPO4 104.5 mg/ L, pH 7.0). One gram of the centrifuged deposition was transferred into the MSM contain 50 mg/L naphthalene and incubated at 30°C and 180 rpm. After 1 week of incubation, 5 mL of the culture was transferred to 95 mL fresh MSM in a new 250-mL flask with 50 mg/L naphthalene. This operation was repeated until the degradation of naphthalene came to a stable level. And then the culture was diluted and spread onto agar (1.9 %) plates with MSM and naphthalene. The agar plates were incubated at 35°C for 4 days. A pure culture of the isolate was started from a single isolated colony. The isolate was stored in 15 % glycerol in the refrigerator at -10°C. The isolated strain was identified by colony color and morphological characteristics. Gram staining, capsule staining, spore stain, methyl red (M.R), nitrate reduction, acetyl methyl carbinol (V.P) were tested as described previously (Filonov et al. 2006). Further identification was undertaken using the 16S rDNA sequencing. Genomic DNA was isolated using standard procedures (Lee et al. 2003). 16S rDNA was amplified with the primers 27F (50 AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -TACCTTGTTACGACTT-30 ). The fragment of rDNA was amplified using a Gene Amp PCR System (PE, USA). PCR

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had been carried out with 1 cycle at 94°C for 2 min, followed by 30 cycles at 94°C for 30 s, 50°C for 30 s and 72°C for 2 min. This amplification process ended with 1 cycle at 72°C for 15 min. After the gene had been amplified, the products were separated by agarose (0.8 %) gel electrophoresis (Jajuee et al. 2007). DNA sequencing was performed using ABI 3700 (Applied Biosystems, USA) following the manufacturer’s instructions. Finally, using the BLAST program, 16S rDNA homology searches against the NCBI database were carried out in order to find the 16S rDNA database for similar sequences. The main environmental factors that could determine biodegrading suitability were examined to understand their influence on the biodegradation of naphthalene. The experiments were performed in a series of 250 mL Erlenmeyer flasks with 100 mL MSM. The bacterial cells growing in LB medium were harvested by centrifuging at 5,0009g for 5 min. Then the cells were washed twice with 0.01 M sodium phosphate buffer. The bacterial deposition was resuspended and diluted with sodium phosphate buffer to an optical density of 1.0–1.2 at 602 nm (OD602). Each flask was inoculated with 2 mL of bacterial suspension to make the final cell density 1.0 9 108–1.0 9 109 cfu/mL. Various conditions are briefly described: incubation temperatures of 20, 25, 30, 35 and 40°C; medium pH of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0.; initial naphthalene concentrations of 10, 30, 50, 100, 150, 200 mg/L; and other available carbon sources, such as methanol (50 mg/L), glucose (50 mg/L), biphenyl (50 mg/L) and aniline (50 mg/L). All experiments were carried out in duplicate. To study the degradation kinetics of naphthalene, the experimental data obtained from the batch mode degradation experiments were fitted with Andrews kinetic mode. The Andrews mode is as follows (Zheng et al. 2009): q¼

qmax S0 Ks þ S0 þ S20 =Ki

ð1Þ

where, q is the degradation rate, (1/h); qmax is the maximum degradation rate, (1/h); Ki is the inhibition constant, (mg/L); Ks is the half-rate constant, (mg/L); and S0 is the substrate concentration, (mg/L). The values of the kinetic parameters for Andrews mode were obtained from the nonlinear regression analysis using Orgin 8.6 SR1 (OriginLab Corporation, USA). In order to trace the metabolites during the degradation of naphthalene, the experiments were carried out in a series of 250-mL Erlenmeyer flasks containing 100 mL of the MSM, supplemented with naphthalene at 100 mg/L and inoculated with 2 mL of bacteria suspension. This suspension was prepared by harvesting exponentially growing QWE-35 by centrifugation and washed twice with 0.01 M sodium phosphate buffer before inoculation. Negative controls were prepared similarly except for the bacteria

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inoculum. All flasks were sealed with sealfilm and incubated at 35°C, 180 rpm. To determine the metabolic intermediates, the samples were taken at regular intervals and filtered through 0.2 lm membrane. Naphthalene metabolites were identified using gas chromatographymass spectrometry. Experiments were conducted to evaluate the potential of bio-augmentation with QWE-35 to improve the degradation of recalcitrant naphthalene during wastewater treatment in two membrane bioreactors (MBRs). The effective volume of each reactor is 8.0 L. The submerged hollow fibre polythene (PE) membranes (nominal pore size: 0.4 lm; membrane surface area: 0.08 m2) installed in MBRs were purchased from Mitsubishi Rayon Co. Ltd. Japan. Zhao et al. (2009) discovered that the MBR (same membrane) was effective with reduction by more than 75 % of COD and 25 % of TN when treating coke wastewater collected from the Beijing Steel Company, China. In the present study, air of 0.12 m3/h was aerated through perforated pipe underneath the membrane module in order to scour the membrane surface and maintain the dissolved oxygen (DO) between 3 and 5 mg/L throughout the experiment. Membrane filtration was carried out with a filtration/pause ratio of 8 min:2 min. The seed sludge was obtained from the full-scale aerobic process in the same plant and was added in each reactor. The volatile suspended solids in the reactors were around 1,500 mg/L. The feed water to the MBRs was the real coal gasification wastewater inlet to the treatment plant in the China Coal Longhua Harbin Coal Chemical Industry Co. Ltd. The COD, total nitrogen (TN) and naphthalene of real wastewater were 1,080.7–1,210.9, 178–204 and 34.8–40.6 mg/L, respectively. The MBR systems were run for 30 days at a hydraulic retention time of 24 h. On day 10, 200 mg/L of QWE-35 inoculum was added once in MBR #1 as the bioaugmented reactor, while MBR #2, as the control reactor, was added 200 mg/L of seed sludge. The influent and effluents from both reactors were sampled every day for analysis of the concentrations of COD and naphthalene. COD, TN and volatile suspended solids were measured according to Standard Methods (Andersen et al. 2008). The concentration of naphthalene was determined with a gas chromatography (Agilent 6,890) equipped with flame ionization detector and 30.0 m 9 0.32 mm 9 0.25 lm capillary column (HP-5). Samples of 10 mL were taken periodically from the flasks, and filtered through 0.20 lm membrane. The injector and detector temperatures were kept at 260 and 280°C, respectively. The column temperature was increased from 120 to 170°C at the rate of 10°C/ min, and kept for 5 min. Nitrogen was used as the carrier gas. The detection limit for this method was 0.1 mg/L. Naphthalene metabolites were extracted with three equal volumes of ethyl acetate and identified using gas

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chromatography–mass spectrometry (GC–MS, Thermo, USA) with EI mode (70 eV). The detailed procedures were described in the previous paper (Seo et al. 2007). Identification was obtained by probability-based matching with mass spectra in NIST library as well as by matching with the mass spectra and retention time of the standard reference compounds used (Kang et al. 2003).

Results and Discussion Strain QWE-35, which was able to grow on naphthalene as the sole carbon and energy source, was isolated from the acclimated activated sludge of a coal gasification wastewater plant. Colonies of QWE-35 appeared entire, wet, convex, and opaque with smooth surface during growth on agar plates for 4–5 days. Cells of QWE-35 were motile and spore-forming, 2–3 lm in length and 0.8–0.9 lm in width. It was negative in tests such as Gram staining, catalase, gelatin liquefaction, M. R and V. P. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain QWE35 belonged to Streptomyces sp. It was closely related to Streptomyces sp. FKY (MZ071831) and Streptomyces sp. SZA (DQ101053), with 99 % sequence identity. In combination with the physiological-biochemical tests and gene sequence analysis, the isolated strain QWE-35 was tentatively identified as Streptomyces sp., and named Streptomyces sp. QWE-35. Figure 1a shows that the biodegradation efficiencies of naphthalene by strain QWE-35 ranged from 53.7 % to 87.2 % as the incubation temperatures increased from 20 to 45°C and the maximum biodegradation efficiency was achieved at 35°C. One possible explanation is that the increased solubility of naphthalene at higher temperatures will cause a noticeable improvement in the bioavailability of naphthalene molecules. However, further increase in temperature will lead to the decline in oxygen solubility. Figure 1b shows that when the initial pH of the media increased from 4.0 to 9.0, the maximum biodegradation efficiency was achieved at pH 7.0. Biodegradation efficiency significantly declined when the pH was \6.0 or [8.0. These results are consistent with most studies, where microorganisms favoured growth at pH levels ranging from 6.0 to 8.0. As charges distributed on the surface of bacteria would changed with different pH, which might alter the interaction between naphthalene and the microorganisms and resulted in the variation of removal efficiency. The effect of other available carbon sources (glucose, methanol, biphenyl, aniline) on the biodegradation of naphthalene was shown in Fig. 1c. Among the four additional carbon sources, the presence of methanol and glucose significantly increased ([5 %) the biodegradation efficiency of

Biodegradation efficiency (%)

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361

(a)

100

(b)

100

90

90

80

80

70

70

60

60

50

50 15

20

25

30

35

40

45

50

4

5

6

Biodegradation efficiency (%)

Temperature (°C)

7

8

9

pH 100

100

(c)

95

(d)

90 80 70

90

60 85 50 40

80 Gluc

Meth

Biph

Anil

Control

available carbon sources

0

30

60

90

120

150

180

210

Initial concentration (mg/L)

Fig. 1 The effects of a Temperature; b pH; c Other available carbon sources; d Initial concentration on the naphthalene degradation. Control, without other carbon source

naphthalene, while the increase caused by the presence of biphenyl and aniline was very limited (\5 %). The explanation is that glucose and methanol are primary substrates, which may aid in reducing the toxicity and growth inhibition of toxic compounds on cells, thereby increasing the transformation rate of these compounds (Lee et al. 2003). The effect of initial concentration of naphthalene on its biodegradation was shown in Fig. 1d. When the initial concentrations of naphthalene were 10, 30, 50, 100, 150 and 200 mg/L, biodegradation efficiencies were 98.4 %, 96.7 %, 90.2 %, 70.3 %, 60.9 % and 47.8 % respectively after incubation for 4 days. The removed amount of naphthalene with strain QWE-35 however increased as the initial concentration of naphthalene rose, which is due to the initial concentration that provides an important driving force to overcome all mass transfer resistances of the naphthalene between the aqueous and solid phases. This suggests that strain QWE-35 could survive and degrade a high concentration of naphthalene at 200 mg/L. Naphthalene degradation profiles for its various initial concentrations were shown in Fig. 2a. It can be seen that an increase in concentration prolonged the degradation time. This could be attributed to the recalcitrance and the toxicity

of naphthalene. The Andrews kinetics mode was used here to express the kinetics of naphthalene degradation. The value of R2 was 0.9481, which demonstrated that the experimental data was well correlated by Andrews equation (Fig. 2b). The values of kinetic constants for naphthalene degradation obtained in this work were qmax = 0.84 h-1, Ks = 40.39 mg/L, and Ki = 193.76 mg/ L. The maximum specific degradation rate occurred at low substrate concentration. On further increasing the initial substrate concentration, significantly lower values of the degradation rates were achieved. In this paper, the QWE35 strain could use naphthalene as a sole carbon and energy source and could tolerate the toxicity of the naphthalene at a concentration of up to 200 mg/L. The Ks and qmax values in this study were comparatively larger than that previously reported. The findings suggest a potential bioremediation role of Streptomyces sp. QWE in the removal of naphthalene from wastewaters. Based on the NIST library, five intermediates were identified by GC–MS during naphthalene degradation. They were identified as 1,4-dihydroxynaphthalene, 1,4naphthaquinone, trans-2-carboxybenzalpyruvic acid, benzoic acid and adipic acid (Table 1). With standard chemicals analyzed under the same condition, those compounds

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10 mg/L 30 mg/L 50 mg/L 100 mg/L 200 mg/L

(a)

200

Concentration (mg/L)

Fig. 2 a Naphthalene degradation profiles for its various initial concentrations; b Experimental and predicted q due to Andrews mode

150

100

50

0

0

15

30

45

60

75

90

105

Time (h)

experimental Fitted curve

(b)

0.9

q (1/h)

0.6

0.3

0.0

0

20

40

60

80

100

120

140

160

180

200

220

Initial concentrations (mg/L)

Table 1 Gas chromatography retention times and prominent fragment ions of metabolites Metabolites

Retention time (min)

Prominent fragment ion (m/z)

7.2

172(M?), 148 (M?-24), 130 (M?-42),

1,4-naphthaquinone

8.6

118 (M?-54) 158(M?), 130 (M?-28), 116 (M?-42)

Trans-2carboxybenzalpyruvic acid

8.9

186(M?), 154 (M?-32), 128 (M?-58)

Benzoic acid

9.4

150 (M?), 105 (M?-45), 77 (M?-73)

Adipic acid

13.1

1,4dihydroxynaphthalene

104 (M?), 58 (M?-46)

were confirmed. These metabolites were not detected in sterile controls. Based on the identified intermediates, a new pathway for the degradation of naphthalene is proposed and is shown in Fig. 3. In the proposed pathway, the degradation of naphthalene is initiated by hydroxylation at position C2 and C4 to form 1,4-dihydroxynaphthalene which is then oxidized to 1,4-naphthaquinone. The for-

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mation of trans-2-carboxybenzalpyruvic acid and benzoic acid may result from the further hydrogenation at the C=O bond and its subsequent cleavage. Benzoic acid can be further metabolised into adipic acid and finally CO2 and H2O. The major pathway, for catabolism of naphthalene, has suggested that the initial conversion steps were carried out similar to that of Alcaligenes faecalis (Shah et al. 2012). The bioaugmentation of strain QWE-35 into activated sludge for naphthalene degradation was investigatived. During the continuous treatment, the effluent COD, TN and naphthalene from both reactors is shown in Fig. 4. Comparing the data after day 10, the average removal efficiencies of COD and TN were 80.6 % and 31.5 % by the bioaugmented MBR #1; and 75.7 % and 28.2 % by the control MBR #2, respectively. Regarding naphthalene removal, Fig. 4c shows that the effluent naphthalene concentration from MBR #1 was significantly lower than that from MBR #2. After day 10, the average influent naphthalene concentration was 38.2 mg/L, and the effluent values were 9.2 and 22.8 mg/L for MBR #1 and #2, respectively. Adding QWE-35 into the MBR appeared to contribute to the improvement of naphthalene removal.

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Fig. 3 Proposed pathway for the degradation of naphthalene by stain QWE-35

Fig. 4 Treatment efficiency for a COD; b TN and c naphthalene by the MBRs

Influent Effluent of MBR #1(bioaugmented since day 10)

COD (mg/L)

Effluent of MBR #2 (control) 1200 1000 800 300 200

TN (mg/L)

100 0

5

10

15

20

25

30

0

5

10

15

20

25

30

0

5

10

15

20

25

30

200 160

Naphthalene (mg/L)

120

40 30 20 10

Time (day)

Conclusions A novel strain that is capable of degrading naphthalene at high concentrations was isolated from acclimated activated sludge. This strain was identified by 16S rDNA sequencing and physiological-biochemical tests, and designated Streptomyces sp. QWE-35. Batch degradation tests indicated that the strain could tolerate the toxicity of the naphthalene at a concentration of up to 200 mg/L and possesses potential application in the treatment of the wastes containing the naphthalene. The experimental data were fitted

to the Andrews mode using nonlinear regression analysis. On the basis of these findings and GC–MS analysis, it is possible to propose a metabolic pathway of QWE-35 for naphthalene degradation. Furthermore, the bio-augmentation with QWE-35 showed a significant improvement in the naphthalene degradation efficiency of the activated sludge in a membrane bioreactor. Acknowledgments This work was supported by Harbin Institute of Technology (2013DX10) and the Sino-Dutch Research Program (zhmhgfs2011-001).

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