Experimental and Mathematical Methodology on the Optimization of

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Experimental and Mathematical Methodology on the Optimization of Bacterial Consortium for the Simultaneous Degradation of Three Nitrogen Heterocyclic Compounds Cui Zhao,† Donghui Wen,*,† Yin Zhang,‡ Jing Zhang,† and Xiaoyan Tang† †

College of Environmental Sciences and Engineering, The Key Laboratory of Water and Sediment Sciences (Ministry of Education), Peking University, Beijing, 100871, People’s Republic of China ‡ Department of Environmental Engineering, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, 200235, People’s Republic of China S Supporting Information *

ABSTRACT: This study aims to establish a systematic method to optimize the bacterial consortium for the simultaneous biodegradation of multixenobiotics in wastewater. Three nitrogen heterocyclic compounds (NHCs), pyridine, quinoline, and carbazole, were chosen as the target compounds with each about 200 mg/L. Different consortia originated from six bacteria for degrading pyridine (Paracoccus sp. BW001 and Shinella zoogloeoides BC026), quinoline (Pseudomonas sp. BW003 and BW004), and carbazole (Pseudomonas sp. BC039 and BC046) were tested for the capacity of NHCs simultaneous degradation. Mathematical methods including dummy-variable-laden kinetic modeling, cubic spline regression and interpolation, and dimensionality reduction were employed to evaluate the complex impacts of cocontaminants and coexisting bacteria on the simultaneous biodegradation, and the most efficient consortium was determined. The influences of cocontaminants on the bacterial degradation activity were far greater than the interactions among the mixed bacteria. Integrating the experimental results and mathematical analysis, consortium M19 (BC026, BW004, BC039, and BC046 with dose rate of 1:1:0.5:0.5) was the best one, which degraded over 95% of pyridine, quinoline, and carbazole simultaneously in 15.4 h. The research methodology in this study could be applied to the optimization of a bacterial consortium which might be used in the bioaugmentation and bioremediation of multixenobiotics removal.

1. INTRODUCTION Bacteria play an important role in natural substance circulation in the ecosystem. For the artificial contaminants discharged from human society, the biological treatment that depends mainly on bacterial metabolism is also regarded as a feasible and inexpensive method of biodegradation.1 Furthermore, bioaugmentation is more effective for wastewater treatment2,3 and in situ remediation4 when a certain kind of xenobiotic is present. In more prevalent case of multixenobiotics’ pollution, assembling of a bacterial consortium is usually required for effective bioaugmentation because most of the specific degrading bacteria equip catabolic pathway for only one kind of xenobiotic.5 Therefore, an understanding of the interactions among toxic compounds and bacterial flora is essential for the design of a bioaugmentation system. A number of studies6,7 interpreted the complex interactions among cocontaminants © 2012 American Chemical Society

and coexisting bacteria from diverse perspective. To sum up, the influences of cocontaminants on the bacteria’s degradation activity appear in three modes: enhancement,8 inhibition,6 and cometabolism;7 the interactions among coexisting bacteria include positive synergy,9 negative competition,10 and nonsignificant effect. Based on this knowledge, a proper combination and dosage of mixed bacteria could be proposed for the operation of a bioaugmentation system. Although the overall degradation efficiency of a bacterial consortium was always the focus, little attention was paid to the methodology of the consortium optimization. In some previous studies on contaminated soil bioremediation8 and bioaugmented wasteReceived: Revised: Accepted: Published: 6205

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Table 1. Key Characteristics of the NHC-Degrading Strains target compound

RT95 (h)b

optimal pH

optimal T (°C)

Paracoccus sp. BW001 (CGMCC 2225)

pyridine

9.12

5.2−7.5

23.4−32.6

+c

+c

Shinella zoogloeoides BC026 (CGMCC 2224)

pyridine

8.89

6.5−7.9

23.9−34.3

+c

+c

Pseudomonas sp. BW003 (CGMCC 3008) Pseudomonas sp. BW004 (CGMCC 4221) Pseudomonas sp. BC039 (CGMCC 4222) Pseudomonas sp. BC046 (CGMCC 4223)

quinoline

5.45

6.5−7.5

26.2−36.1

+c

−d

16S-EU192073 nirS-EU192074 nosZ-EU192075 16S-EU346730 nosZ-EU346731 16S-EU371554

quinoline

5.53

6.4−7.5

26.8−34.7

unknown

unknown

16S-HQ710831

carbazole

19.20

7.4−8.2

29.5−34.1

+c

+c

16S-HQ105008

14

carbazole

18.49

6.7−7.3

29.9−34.3

+c

+c

16S-HQ105014

14

strain (CGMCC no.)a

nitrificational potential

denitrificational potential

GenBank accession no. of gene sequence

ref 12

27 13

a

A culture of each strain has been deposited in the China General Microorganism Culture Center (CGMCC) with an accession number. bRT95 is the required time for degrading 95% of a NHC by a degrading bacterium. cPossesses the potential. dDoes not possess the potential.

Table 2. Constitutions of 27 Bacterial Consortia and Their RT95 and Confidence Interval (CI) for the Simultaneous Degradation of NHCs consortium code

strain (OD602)

M1 M2 M3 M4 M5 M6 M7 M8 M9

BC026 (0.1), BW003 (0.1), BC039 (0.1) BC026 (0.1), BW003 (0.1), BC046 (0.1) BC026 (0.1), BW004 (0.1), BC039 (0.1) BC026 (0.1), BW004 (0.1), BC046 (0.1) BW001 (0.1), BW003 (0.1), BC039 (0.1) BW001 (0.1), BW003 (0.1), BC046 (0.1) BW001 (0.1), BW004 (0.1), BC039 (0.1) BW001 (0.1), BW004 (0.1), BC046 (0.1) BC026 (0.05), BW001 (0.05), BW003 (0.05), BW004 (0.05), BC039 (0.05), BC046 (0.05) BC026 (0.05), BW001 (0.05), BW003 (0.1), BC039 (0.1) BC026 (0.05), BW001 (0.05), BW003 (0.1), BC046 (0.1) BC026 (0.05), BW001 (0.05), BW004 (0.1), BC039 (0.1) BC026 (0.05), BW001 (0.05), BW004 (0.1), BC046 (0.1) BC026 (0.1), BW003 (0.05), BW004 (0.05), BC039 (0.1) BC026 (0.1), BW003 (0.05), BW004 (0.05), BC046 (0.1) BW001 (0.1), BW003 (0.05), BW004 (0.05), BC039 (0.1) BW001 (0.1), BW003 (0.05), BW004 (0.05), BC046 (0.1) BC026 (0.1), BW003 (0.1), BC039 (0.05), BC046 (0.05) BC026 (0.1), BW004 (0.1), BC039 (0.05), BC046 (0.05) BW001 (0.1), BW003 (0.1), BC039 (0.05), BC046 (0.05) BW001 (0.1), BW004 (0.1), BC039 (0.05), BC046 (0.05) BC026 (0.05), BW001 (0.05), BW003 (0.05), BW004 (0.05), BC039 (0.1) BC026 (0.05), BW001 (0.05), BW003 (0.05), BW004 (0.05), BC046 (0.1) BC026 (0.05), BW001 (0.05), BW003 (0.1), BC039 (0.05), BC046 (0.05) BC026 (0.05), BW001 (0.05), BW004 (0.1), BC039 (0.05), BC046 (0.05) BC026 (0.1), BW003 (0.05), BW004 (0.05), BC039 (0.05), BC046 (0.05) BW001 (0.1), BW003 (0.05), BW004 (0.05), BC039 (0.05), BC046 (0.05)

M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27

RT95 for pyridine (h)

CI for pyridine (h, h)

RT95 for quinoline (h)

CI for quinoline (h, h)

RT95 for carbazole (h)

16.74 15.18 15.70 15.80 20.14 20.08 20.16 20.38 16.36

(16.6, 16.92) (15.08, 15.24) (15.64, 15.78) (15.72, 15.88) (20.08, 20.2) (20, 20.16) (20.08, 20.22) (20.3, 20.44) (16.3, 16.42)

7.08 6.80 5.50 5.52 5.76 7.50 3.90 4.92 3.88

(7.04, 7.12) (6.76, 6.84) (5.48, 5.52) (5.5, 5.54) (5.76, 5.78) (7.46, 7.52) (3.88, 3.92) (4.9, 4.94) (3.86, 3.88)

21.00 20.76 23.24 22.84 18.74 17.12 19.82 18.40 20.40

(20.88, (20.24, (23.02, (22.64, (18.64, (17.04, (19.72, (18.18, (20.14,

21.14) 24) 23.42) 23.04) 18.84) 17.2) 19.94) 18.56) 21.04)

18.82 14.54 14.56 15.36 14.50 14.50 20.70 20.66 14.48 14.48 20.28 20.50 15.40

(18.76, 18.88) (14.5, 14.56) (14.52, 14.58) (15.28, 15.42) (14.46, 14.54) (14.46, 14.54) (20.66, 20.74) (20.62, 20.7) (14.44, 14.52) (14.44, 14.52) (20.22, 20.34) (20.46, 20.54) (15.34, 15.46)

3.90 3.90 3.90 6.98 6.32 5.80 4.06 3.94 7.16 4.12 3.94 5.82 4.30

(3.88, 3.92) (3.88, 3.92) (3.88, 3.92) (6.96, 7.02) (6.28, 6.34) (5.78, 5.82) (4.04, 4.08) (3.92, 3.96) (7.1, 7.2) (4.1, 4.14) (3.92, 3.94) (5.8, 5.84) (4.3, 4.32)

14.56 17.88 19.12 15.50 17.00 15.64 15.58 15.32 17.06 15.36 15.04 14.62 17.40

(14.54, (17.64, (18.98, (15.44, (16.92, (15.54, (15.46, (15.12, (16.98, (15.22, (14.92, (14.58, (17.12,

14.58) 18.18) 19.26) 15.58) 17.08) 15.76) 15.74) 15.46) 17.14) 15.46) 15.18) 14.66) 17.7)

15.86

(15.8, 15.94)

4.04

(4.02, 4.06)

14.62

(14.52, 14.76)

15.28

(15.2, 15.32)

3.92

(3.9, 3.94)

16.72

(16.66, 16.78)

17.64

(17.62, 17.68)

5.40

(5.38, 5.44)

19.36

(19.32, 19.42)

17.32

(17.26, 17.38)

5.52

(5.5, 5.52)

20.22

(20.16, 20.26)

20.82

(20.8, 20.84)

3.88

(3.86, 3.9)

18.44

(18.36, 18.5)

water treatment,2,11 several bacterial strains2,8 or bacteria plus other microorganisms, such as fungus,11 were simply mixed and delivered into the contaminated sites or treatment reactors. In recent years, a well-known statistical method, response surface methodology (RSM), has been applied to optimize the bacterial consortium for decoloring dyes.11,12 However, since each

CI for carbazole (h, h)

variable is independent in the black-box of RSM, little is known about the interactions among the target compounds and bacterial strains. In this study, we established a systematic method to optimize the bacterial consortium for multixenobiotics’ biodegradation based on the understanding of the interactions among 6206

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Three groups of biodegradation experiments were conducted. First, to investigate the influences of cocontaminants on a bacterium, the degradation courses of three NHCs by single bacterium were compared. Second, to detect the interactions among coexisting strains, the degradation courses of each NHC by 3 bacteria (consortia M1−M8), each of which could degrade one NHC, were compared. Finally, to screen the most efficient consortium for the three NHCs simultaneous degradation, all 27 consortia were compared by their degradation capacity for each NHC, as indicated by the index, RT95. The RT95 is the required time for degrading 95% of a NHC by a bacterial consortium. In all experiments, flasks were sealed with sealfilm, shaken at 30 °C, 180 rpm, and sampled periodically. All incubations were not interfered by light. A portion of each sample was fully vortexed, centrifuged, and filtered through a 0.2-μm membrane for pyridine and quinoline analysis. The rest of the sample was extracted with isometric ethyl acetate for carbazole analysis. All experiments were performed in triplicate, and the average values were used as the experimental results. 2.5. NHCs Analytical Methods. The concentrations of pyridine, quinoline, and carbazole were analyzed by a highperformance liquid chromatography (HPLC) system (Shimadzu LC10ADVP, SPD10AVP UV−vis detector; Rheodyne 7725i manual injector; Diamonsil C18 reverse-phase column, 250 × 4.6 mm, 5 μm). Methanol and water solution (4:1 for pyridine and quinoline detection; 9:1 for carbazole detection) was used as the mobile phase in isocratic mode at the flow rate of 1.0 mL/min. Pyridine and carbazole were detected at 254 nm, and quinoline was detected at 275 nm wavelength. 2.6. Mathematical Methods. 2.6.1. Dummy Variable Regression Using Kinetic Models. In data analysis for the second group experiment, the kinetic modeling and dummy variable regression22 were applied to describe the degradation course and assess the impacts of the coexisting bacteria on the degradation capacity of a bacterium. The zero- and first-order kinetic models with dummy variable were tried in experimental data fitting as follows:

compounds and bacteria. Three nitrogen heterocyclic compounds (NHCs) were chosen as the target compounds, i.e. pyridine, quinoline, and carbazole, which are known to be toxic and mutagenic, and are often discharged simultaneously from coking, oil refining, and pharmaceutical industries. 13,14 Recently, the biodegradation of pyridine,15 quinoline,16 and carbazole17 by individual isolates have been investigated, and the degradation pathways and mechanisms have been proposed.13,18,19 However, there is no report on the multiNHCs’ influences on the degradation activity of a NHCdegrading bacterium or on the complex interactions among the NHC-degraders. As a result, the optimization of bacterial consortium for NHCs simultaneous degradation is still unexplored. Our study is an attempt to reveal the complexity by combining biodegradation experimental data with a series of mathematical analyses.

2. MATERIALS AND METHODS 2.1. Chemicals. Standard samples of pyridine, quinoline, and carbazole were purchased from Chemservice Inc., USA, AccuStandard Inc., USA, and Sigma-Aldrich Inc., USA, respectively. Dimethyl sulfoxide (DMSO) was obtained from Amresco, USA. Tryptone and yeast extract were obtained from Oxoid Ltd., UK. Solvents for HPLC analysis were chromatographic grade. All other chemicals used were of analytical grade. 2.2. Bacterial Strains. Degrading bacterial strains that utilize one NHC as their sole carbon, nitrogen, and energy source, were isolated by plate streaking method from the activated sludge of the coking wastewater treatment plants of Capital Iron and Steel Group, Beijing, and Wuhan Iron and Steel Group, Hubei Province, China. They were named in BC and BW serials according to their respective origins. Six highly efficient NHC-degrading strains were selected as the candidates for bacterial consortia, including two pyridine-degraders, two quinoline-degraders, and two carbazole-degraders. Table 1 summarizes key characteristics of the strains. A phylogenetic tree of the 16S rDNA of the strains, compared to the GenBank database, is shown in Figure S1. 2.3. Media. Luria−Bertani (LB) medium20 was used for bacteria enrichment and maintenance. Mineral salt medium (MSM),21 which does not contain C and N sources, was used as the basic ingredient in the degradation experiments. Pyridine and quinoline solutions were filtered through 0.2μm membranes. Carbazole was dissolved in DMSO (30 g/L). Three compounds were added separately or jointly into the MSM as the carbon, nitrogen, and energy sources in the biodegradation experiments. All media were sterilized at 121 °C for 20 min before use. 2.4. Biodegradation Experiments. A series of 250-mL Erlenmeyer flasks was used in the experiments. Each flask carried 100 mL of MSM with about 200 mg/L of one target NHC or three NHCs and an inoculum of a corresponding degrading strain or a bacterial consortium. From our previous studies, all six bacteria can degrade their target NHC but hardly degrade the other two NHCs in the “single bacterium−single substrate” system. Therefore, in order to obtain the simultaneous degradation of pyridine, quinoline, and carbazole, a total of 27 bacterial consortia were generated from the 6 candidates (Table 2). The inoculum concentration was controlled by the optical density at 602 nm (OD602, measured by Shimadzu UV2401, Japan). The dosage of each strain was 0.1 if one NHC-degrader was added, or 0.05 if two NHCdegraders were added for degrading this NHC.

CA = CA0 − (k1 + Dk 2) × t

(Model 1)

CA = CA 0e−(k1+ Dk 2) × t

(Model 2)

where CA0 is the initial concentration of a NHC, mg/L; CA is the NHC concentration at time t, mg/L; t is the contact time, h; k1 and k2 are the model coefficients, mg/L·h in Model 1 and L/h in Model 2. For both models, the kinetic rate constants (K) are (k1 + Dk2). D is the dummy variable, acting as a ‘‘switch’’ that turns various impacts on and off in a single regression equation,23 D = 1 if a bacterial consortium was used (other bacteria presence), and D = 0 if single NHC degrader was used (other bacteria absence). k2 gives weight to the dummy variable. If k2 is a positive value, it indicates a positive synergy among the coexisting bacteria; if k2 is a negative value, it indicates a negative competition among the bacteria. Furthermore, if k2 shows no significance (i.e., sig >0.05), it means there is no significant interaction among the coexisting bacteria. The mathematical analysis was performed using SPSS (V. 16 for Windows, SPSS Inc.). 2.6.2. Cubic Spline Regression and Spline Interpolation. In data analysis for the third group experiment, the cubic spline 6207

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Figure 1. Biodegradation of the three nitrogen heterocyclic compounds (NHCs) by (A) BW001, (B) BC026, (C) BW003, (D) BW004, (E) BC039, or (F) BC046. Co-contaminants degradation of (●) pyridine, in the (MSM + 3 NHCs); (▼) quinoline, in the (MSM + 3 NHCs); and (■) carbazole, in the (MSM + 3 NHCs). Single contaminant degradation of (○) pyridine, in the (MSM + pyridine); (∇) quinoline, in the (MSM + quinoline); and (□) carbazole, in the (MSM + carbazole). n−1

R=

regression was applied to fit to each NHC degradation course by different bacterial consortium. The equation is 24−27

∑ ri(t ) × [ai(t − ti)3 + bi(t − ti)2 + ci(t − ti) + di] i=1

6208

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Here R is the NHC residue at time t, %; t is the contact time, h. The function precision, i.e. the step of t, Δt (ti+1 − ti), was set at 0.02 h. The cubic spline interpolation was applied for curve fitting of RT95 in a 3-dimensional space, which corresponds to the bacterial dose rate. The equation is

degradation activity of BW001 was mainly due to carbazole. The presence of pyridine might promote the pyridine-degrader to acquire an ability to degrade quinoline, but not carbazole. Second, regarding another pyridine-degrader, BC026 (Figure 1B), the presence of quinoline and carbazole also prolonged the complete degradation of pyridine, as 8 and 18 h were needed in the single contaminant system and the cocontaminants system, respectively. Co-metabolism of pyridine and quinoline was carried by BC026, which did not need a period of acclimatization for quinoline and degraded all quinoline within 18 h, too. Similar to BW001, BC026 could not degrade carbazole throughout the experiment, indicating that carbazole was the main inhibitor on the pyridine degradation activity of BW026. Third, regarding the quinoline-degraders, BW003 (Figure 1C) and BW004 (Figure 1D), the presence of pyridine and carbazole did not influence the quinoline degradation by BW003 and enhanced the quinoline degradation by BW004. In the first 4 h, BW004 degraded quinoline at rates of 19.7 mg/L·h and 49.0 mg/L·h in the single contaminant system (MSM + quinoline) and the cocontaminants system (MSM + 3 NHCs), respectively. Both strains could utilize a small amount of pyridine, but could hardly utilize carbazole throughout the experiment, indicating that the presence of quinoline could not assist the quinoline-degraders to acquire a full ability to degrade other NHCs, especially carbazole. Finally, regarding the carbazole-degraders, BC039 (Figure 1E) and BC046 (Figure 1F), the presence of pyridine and quinoline had a little inhibition on the carbazole degradation activity of both strains. By BC039, respectively 97.8% and 71.8% of carbazole in the single contaminant system (MSM + carbazole) and the cocontaminants system (MSM + 3 NHCs) were degraded at 15 h; by BC046, respectively 93.3% and 89.0% of carbazole in the single contaminant system and the cocontaminants system were degraded at 15 h. Acclimated to the cocontaminants system, both carbazole-degraders gradually degraded nearly 50% of quinoline and a small amount of pyridine throughout the experiment. 3.2. Interactions among Coexisting Bacteria. By different bacterial consortium (M1−M8, all combinations of three degraders for the three NHCs) or single NHC-degrading bacterium, the degradation courses of the NHC were compared, as shown in Figure 2. The degradation of pyridine, quinoline, and carbazole by either pure strain or mixed strains showed similar trends. To distinguish the interactions among the mixed bacteria, all removal rates of the NHCs in the degradation courses were calculated, and the dummy-variable-laden kinetic models were applied for data fitting. By trailing the data trends, Model 1 was determined as the proper model for pyridine and quinoline degradation (R2 ≥ 0.940), and Model 2 was determined for carbazole degradation (R2 ≥ 0.968). The coefficients and statistical parameters of all models were obtained and listed in Table S2. First, concerning the impacts of coexisting bacteria on the pyridine degradation, all the significances (Sig) of dummy variable coefficient (k2) were greater than 0.05, indicating that the bacterial interactions were not significant. Neglecting the small impacts, pyridine removal rate constant was steady at 22.90 mg/L·h, as reflected by the mean value of the regression coefficient k1. The values of k2 were positive in consortia M1− M4, indicating a little positive synergy among the bacteria, which contributed slightly to the pyridine removal; but k2

n−1

RT95 =

∑ ri(X ) × ⎡⎣ai(X − Xi)3 + bi(X − Xi)2 i=1

+ ci(X − Xi) + di⎤⎦

Here RT95 is the required time for degrading 95% of a NHC by a bacterial consortium with random dose rate; X is a vector value of a point in the 3-dimension space, where the spatial location of the point determines the bacterial dose rate. The function precision, i.e. the step of X, ΔX (Xi+1 − Xi), was set at 0.02. The analysis of spline regression and interpolation were carried out with Matlab (V. 2010R for Windows, Mathwork Inc.). 2.6.3. Dimensionality Reduction. The degradation efficiencies of three NHCs were affected by the strains and their dosages in the consortium. Regarding the constitutions of a consortium, as many as 6 strains could be chosen as 6 variables, representing 6 coordinates. The 6-dimension system was demoted using dimensionality reduction, a key technique to extract the meaningful structures and merge the correlationships in the multivariate data and a visualization tool to present multivariate data in human accessible form.28−30 In the simultaneous degradation experiments, the dosage of each strain, 0, 0.05, or 0.1 (value of OD602), was converted to the dose rate, 0 (zero), 0.5 (half), or 1 (full). The total dose rate of one kind of NHC-degrader(s) in any consortium was identically equal to 1. Therefore, the linear dimensionality reduction can be carried out by matrix (6 × 6) multiple at the condition that the matrix determinant does not equal 0. After the multiplication, the 6 strains were grouped into 3 new variables as pyridine (P), quinoline (Q), and carbazole (C) degraders, which formed a 3-dimension space. Moreover, the dimensionality reduction technique follows a lower bounding lemma, i.e., the distance between any two transformed objects is never greater than that in the original space.31 The values of the 3 variables were in the interval of [1, 3] on the P, Q, and C axes. The detailed information of the dimensionality reduction for the 6 strains is shown in Table S1.

3. RESULTS 3.1. Influences of Cocontaminants on the Bacteria. By single pure bacterium, the degradation courses of the single contaminant and the cocontaminants were compared, as shown in Figure 1 First, regarding the pyridine-degrader, BW001 (Figure 1A), the presence of quinoline and carbazole inhibited the pyridine degradation remarkably. Pyridine was degraded respectively by 99.7% in the single contaminant system (MSM + pyridine) and 65.5% in the cocontaminants system (MSM + 3 NHCs) at 15 h. Quinoline could not be utilized by BW001 until 18 h. After acclimatization to the cocontaminants system for 18 h, in addition to pyridine degrading at a steady rate, the strain degraded quinoline almost completely in 3 h with a high rate of 54.5 mg/L·h. Carbazole could not be degraded throughout the experiment, indicating that the inhibition on the pyridine 6209

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slight benefit to BC026, but slight harm to BW001, i.e. BW001 was a little more sensitive in a mixed bacterial system. Second, concerning the impacts of coexisting bacteria on the quinoline degradation, a same finding was that all the significances of k2 were greater than 0.05, indicating no significant bacterial interaction. Neglecting the small impacts, the quinoline removal rate constant was steady at 28.78 mg/ L·h, as reflected by the mean value of k1. The values of k2 were positive in M1 and M4, and negative in the other consortia. There was no noticeable difference between BW003 and BW004 as the quinoline-degrader in a consortium. Finally, concerning the impacts of coexisting bacteria on the carbazole degradation, although a first-order kinetic model was used, the same finding was that all the significances of k2 were greater than 0.05, indicating no significant bacterial interaction. Neglecting the small impacts, carbazole was removed at a rate constant of 0.159/h, as reflected by the mean value of k1. The values of k2 were all positive in M1−M8, indicating that the coexistence of other NHC-degraders impacted similar (though slightly) on the carbazole-degrader, BC039 or BC046. 3.3. Most Efficient Bacterial Consortium. By different bacterial consortium (M1−M27, all combinations of 3−6 degraders for the three NHCs), the degradation courses of pyridine, quinoline, and carbazole in the cocontaminants system (MSM + 3 NHCs) were compared, as shown in Figure 3. The percentages of residual NHCs along with the contact time were calculated, all data were fitted by the cubic spline regression, and in total 81 spline regression curves were obtained in the pattern of “single NHC residue by one bacterial consortium” (figures not shown). All RT95 could be acquired at 5% of residual NHCs. All values of RT95 and the confidence interval (CI) are shown in Table 2. After the dimensionality reduction for the six strains, the 27 consortia were dotted in the 3-dimension coordinate system, according to their bacterial constitutions (Figure 4A). Based on the RT95 of the 27 consortia, the functions of RT95 for degrading the three NHCs by any bacterial dosages (X) were obtained using cubic spline interpolation. The numerical magnitudes of RT95 were diversified and displayed in color in the 3-dimensional space (Figure 4B, 4C, and 4D). The mixed bacteria needed 13−22 h, 3.5−7.5 h, and 14−23 h to degrade 95% of pyridine, quinoline, and carbazole, respectively. The minimum RT95 values were distinguished, as 13.80 h for pyridine at (1.50, 1.00, 1.44), 3.35 h for quinoline at (2.50, 2.56, 1.94), and 14.43 h for carbazole at (2.12, 1.68, 1.00) in the (P, Q, C) coordinates. The complex constitutions of the consortia for each NHC’s fastest degradation are listed in Table S3. However, the most efficient consortium was defined as the one which had the minimum RT95 for the simultaneous degradation of the three NHCs, a compromised and practical constitution had to be proposed. Comparing the RT95 values in Table 2, the consortium M19, containing BC026, BW004, BC039, and BC046 with dose rate of 1:1:0.5:0.5, was the most efficient one, which degraded over 95% of pyridine, quinoline, and carbazole simultaneously in 15.4 h; three other consortia, M13, M15, and M23, were more efficient ones, which degraded over 95% of the NHCs simultaneously in 15.5 h, 15.6 h, and 15.9 h, respectively.

Figure 2. Biodegradation of (A) pyridine, (B) quinoline, and (C) carbazole by different bacterial consortium (M1−M8) or pure strain.

values were negative in consortia M5−M8, indicating a little negative competition among the bacteria. The only difference between M1−M4 and M5−M8 was that they had different pyridine-degraders, as BC026 in M1−M4 and BW001 in M5− M8. Therefore, the coexistence of other NHC-degraders gave

4. DISCUSSION NHCs usually appear simultaneously in many industrial wastewaters.13,14,32 Bacterial consortium is often used in the 6210

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bioaugmented treatment of the wastewater. In our study, six degrading bacteria were selected as the candidates for an efficient consortium for the simultaneous degradation of pyridine, quinoline, and carbazole. The strains require similar environmental conditions, i.e. pH 5.2−8.2 and temperature 23−36 °C (Table 1); and possessed complementary degradation capacity, which enables the degradation of multiple NHCs. Using a single degrading strain to deal with the mixture of 3 NHCs, pyridine was most difficult to be degraded, and carbazole inhibited its degradation greatly (Figure 1A and 1B); quinoline was easier to be degraded, and the other 2 NCHs did not inhibit its degradation (Table 1, Figure 1C and 1D); carbazole was the most difficult to be degraded (Table 1), and pyridine and quinoline inhibited its degradation to some extent (Figure 1E and 1F). Using a bacterial consortium to deal with the mixture of 3 NHCs, the maximum RT95 for quinoline was even lower than the minimum RT95 for either pyridine or carbazole (Figure 4B, 4C, and 4D). Differing from our previous studies,15,17,33 a new phenomenon was discovered in this study that the pyridine-degraders, BW001 and BC026, could completely degrade quinoline (Figure 1A and 1B); and the carbazole-degraders, BC039 and BC046, could partially degrade quinoline (Figure 1E and 1F) in the cocontaminants system. All the strains do not code the quinoline-degrading genes, qorMSL and oxoOR,16 in their genomes and plasmids, therefore, we postulated that they evolved other necessary metabolic pathways after prolonged exposure to quinoline in our laboratory. However, quinolinedegraders, BW003 and BW004, could not acquire the degradation capacity for pyridine or carbazole (Figure 1C and 1D) in the cocontaminants system. The interactions among the coexisting bacteria were not significant (Figure 2 and Table S2). Therefore, it could be predicted that the NHC-degraders would keep their independence in any bacterial consortium. Nevertheless, a consortium had the advantage that the cocontaminants’ inhibition could be alleviated or eliminated by their degraders. Another new phenomenon was noticed in the pyridine degradation using different bacterial consortia, that all pyridine concentrations increased in the first 6 h and decreased thereafter (Figure 3A). Such phenomenon did not occur in the degradation of quinoline or carbazole, nor in the degradation of pyridine in the single contaminant system (Figure 2A). Considering the NHCs’ chemical structures, possible degradation pathways, and the degradation rates in the experiments, we reasoned a conjecture, which has never been reported, that a small part of quinoline was transformed into pyridine or pyridine-derivatives. Figure 1 (except 1B) strengthens this proposal since pyridine concentration raised appreciably while quinoline concentration reduced in the cocontaminants system. We then made a further conjecture that pyridine or its derivatives might be the intermediates in the new metabolic pathways of quinoline degradation by BW001, BC026, BC039, or BC046. In determining the most efficient consortium for the simultaneous biodegradation of the three NHCs, the experimental results (Figure 3) could not give a definite answer; therefore, the cubic spline regression and interpolation were chosen and employed to predict the trend,34 i.e. the correlation between bacterial dose rate and RT95. Because quinoline could be degraded much faster than the other two NHCs by all consortia, the issue in the study was simplified as the simultaneous biodegradation of pyridine and carbazole.

Figure 3. Biodegradation of (A) pyridine, (B) quinoline, and (C) carbazole by bacterial consortium M1 (−•−), M2 (−○−), M3 (−▼−), M4 (−△−), M5 −■−), M6 (−□−), M7 (−⧫−), M8 (−◊−), M9 (−▲−), M10 (−▽−), M11 (−●−), M12 (− ◯−), M13 (···•···), M14 (···○···), M15 (···▼···), M16 (···△···), M17 (···■···), M18 (···□···), M19 (···⧫···), M20 (···◊···), M21 (···▲···), M22 (···▽···), M23 (···●···), M24 (···◯···), M25 (--•--), M26 (--○--), or M27 (--▼--).

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Figure 4. Cubic interpolation analysis of the function RT95. (A) 27 bacterial consortia dotted in the 3-dimension space; and the slice vision of RT95 for the degradation of (B) pyridine, (C) quinoline, and (D) carbazole in the (MSM + 3 NHCs).

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However, the distribution of RT95 shown by mathematical modeling (Table S3) and 3-dimension output (Figure 4) indicated that the efficient degradation of pyridine and carbazole occurred in different spaces, i.e. occurred with different bacterial dose rates. Due to the complex mechanisms of NHCs simultaneous biodegradation by a consortium, the correlation between bacterial dose rates and overall degradation efficiency remains unclear. The rank of the three most efficient consortia that degraded the NHCs simultaneously within 15.6 h is M19 > M13 > M15 (Table 2). Three key features of the constitutions of the consortia are (1) four strains were needed; (2) BC026, BW004, and BC046 were always included; (3) half dosage of BC026, BW004, and BC046 was replaced by BW001, BW003, or BC039, respectively. The consortium M4 (basic combination of BC026, BW004, and BC046) degraded over 95% of the NHCs in 22.8 h, therefore, the replaced fourth strain played an important role in harmonizing the NHCs simultaneous degradation. The research methodology developed in our study could be applied to the determination of an optimal bacterial consortium, which would be used in the bioaugmentation technology for multixenobiotics removal.

ASSOCIATED CONTENT

S Supporting Information *

Phylogenetic tree of the six degrading strains, and the same genera of corresponding bacteria (Figure S1), dimensionality reduction for the NHCs simultaneous degradation by 6 bacterial strains (Table S1), coefficients of kinetic models for the NHCs degradation by dummy variable regression (Table S2), and bacterial constitutions of the most efficient consortium for the NHCs degradation using cubic spline interpolation (Table S3). This information is available free of charge via the Internet at http://pubs.acs.org/

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 10 62751923; fax: +86 10 62751923; e-mail: [email protected]. Notes

The authors declare no competing financial interest. 6212

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ACKNOWLEDGMENTS This study was accomplished under an“863” Follow-up Exploration Project (2009AA06Z309) granted by the Chinese Ministry of Science and Technology.

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