Bioprocess Biosyst Eng DOI 10.1007/s00449-013-1089-x
ORIGINAL PAPER
Kinetics study of pyridine biodegradation by a novel bacterial strain, Rhizobium sp. NJUST18 Jinyou Shen • Xin Zhang • Dan Chen • Xiaodong Liu • Libin Zhang • Xiuyun Sun • Jiansheng Li • Huiping Bi • Lianjun Wang
Received: 10 September 2013 / Accepted: 1 November 2013 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Biodegradation of pyridine by a novel bacterial strain, Rhizobium sp. NJUST18, was studied in batch experiments over a wide concentration range (from 100 to 1,000 mg l-1). Pyridine inhibited both growth of Rhizobium sp. NJUST18 and biodegradation of pyridine. The Haldane model could be fitted to the growth kinetics data well with the kinetic constants l* = 0.1473 h-1, Ks = 793.97 mg l-1, Ki = 268.60 mg l-1 and Sm = 461.80 mg l-1. The true lmax, calculated from l*, was found to be 0.0332 h-1. Yield coefficient YX/S depended on Si and reached a maximum of 0.51 g g-1 at Si of 600 mg l-1. Vmax was calculated by fitting the pyridine consumption data with the Gompertz model. Vmax increased with initial pyridine concentration up to 14.809 mg l-1 h-1. The qS values, calculated from Vmax , were fitted with the Haldane equation, yielding qSmax = 0.1212 g g-1 h-1 and q* = 0.3874 g g-1 h-1 at Sm0 = 507.83 mg l-1, Ks0 = 558.03 mg l-1, and Ki0 = 462.15 mg l-1. Inhibition constants for growth and degradation rate value were in the same range. Compared with other pyridine degraders, lmax and Sm obtained for Rhizobium sp. NJUST18 were relatively high. High Ki and Ki0 values and extremely high Ks and Ks0 values indicated that NJUST18 was able to grow on pyridine within a wide
J. Shen X. Zhang D. Chen X. Liu L. Zhang X. Sun J. Li H. Bi L. Wang (&) Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province, China e-mail: [email protected] J. Shen e-mail: [email protected]
concentration range, concentrations.
especially
at
relatively
high
Keywords Pyridine Biodegradation Kinetics Haldane model Gompertz model Rhizobium List of symbols K Fitting parameter of the Gompertz model (h-1) Ki Inhibition coefficient for Haldane’s growth kinetics (mg l-1) Ks Half saturation coefficient for Haldane’s growth kinetics (mg l-1) Ki0 Inhibition coefficient for Haldane’s degradation kinetics (mg l-1) Ks0 Half saturation coefficient for Haldane’s degradation kinetics (mg l-1) qS Specific degradation rate (g g-1 h-1) qS * Apparent maximum specific degradation rate (g g-1 h-1) qSmax True maximum specific degradation rate (g g-1 h-1) Ss Pyridine concentration (mg l-1) Sc Pyridine consumed (mg l-1) Si Initial pyridine concentration (mg l-1) Sm Pyridine concentration at which l = lmax (mg l-1) 0 Sm Pyridine concentration at which qS = qSmax (mg l-1) t Time of incubation (h) topt Time of maximum pyridine degradation rate (h) Vmax Maximum volumetric rate of pyridine degradation (mg l-1 h-1) X Concentration of biomass (mg l-1) Xopt Concentration of biomass at topt (mg l-1) YX/S Yield coefficient [g biomass (g substrate)-1]
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Bioprocess Biosyst Eng
Greek a, b l l* lmax
symbol Fitting parameter of the Gompertz model (mg l-1) Specific growth rate (h-1) Apparent maximum specific growth rate (h-1) True maximum specific growth rate (h-1)
isolate that had the nature of strength degrading pyridine. The present study aimed to determine the growth kinetics parameters of Rhizobium sp. NJUST18 and pyridine biodegradation kinetics parameters. Both the inhibition tendency of pyridine toward biomass and pyridine degradation performance of Rhizobium sp. NJUST18 were evaluated based on these kinetics parameters obtained in this study.
Introduction Materials and methods As a representative of nitrogen heterocyclic compounds (NHCs), pyridine is widely used in manufacturing of dye, herbicides, pesticides and pharmaceuticals, causing serious environmental problems because of its toxic, teratogenic and carcinogenic effects [1]. For this reason, its presence in wastewater is severely regulated and there is interest in removing it from contaminated sites. Many treatment technologies have been developed for pyridine removal from contaminated environment. However, due to its high solubility and structural stability, physico-chemical methods, such as oxidation, adsorption and incineration, have been proven to be cost and energy intensive [2]. Biological treatment is often thought to be environmental friendly and cost effective, becoming an attractive means for decontamination of the environment and industrial wastewater [3]. However, due to its heterocyclic structure, pyridine is recalcitrant to biodegradation and persistent in nature. Up to date, several new and efficient microbial species have been isolated for pyridine biodegradation. These microorganisms include genera Arthrobacter [4], Bacillus [5], Lysinibacillus [6], Nocardiodes [7], Paracoccus [8–10], Pseudomonas [2, 11], Rhodococcus [1], Shinella [3], Shewanella [5] and Streptomyces [12]. However, due to the limited number of collections of such bacteria, knowledge about pyridine biodegradation is yet limited. Consequently, for pyridine biodegradation, isolation of new bacterial strains indigenous to sites contaminated by pyridine is still an important issue. What is more, for the evaluation of the persistence of pollutants such as pyridine, knowledge on the biodegradation kinetics is of importance [13, 14]. Biodegradation kinetics is helpful for understanding both the inhibition tendency of the pollutants toward biomass and degradation performance of biomass. Microbial growth on pyridine has been successfully described by substrate-inhibition models, such as Haldane’s equation, which has provided satisfactory correlations with experimental data. However, to the best of our knowledge, kinetics on pyridine biodegradation has been seldom investigated in previous studies. A novel bacterial strain was previously isolated from a site contaminated by pyridine and was named Rhizobium sp. NJUST18. Rhizobium sp. NJUST18 reported here seemed to be the first documented rhizobial bacterium
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Microorganism and culture media Pure culture of Rhizobium sp. NJUST18, which was isolated in this laboratory previously, was used throughout our work. Rhizobium sp. NJUST18 was identified based on morphological, physiological and biochemical tests and genospecies. Store culture was maintained by periodical transfer onto mineral salt agar plates supplemented with 1,000 mg l-1 pyridine and stored at 30 °C for further study. For long-term maintenance, pyridine degrading strain was cultivated aerobically in liquid mineral salt medium (MSM) containing 1,000 mg l-1 pyridine and stored in 20 % glycerol at -80 °C in an ultralow temperature freezer. The MSM used in this study contained Na2HPO412H2O (1.529 g l-1), KH2PO4 (0.372 g l-1), MgSO47H2O (0.1 g l-1), CaCl2 (0.05 g l-1) and SL-4 (10 ml l-1). In MSM, Na2HPO412H2O and KH2PO4 served as the phosphate buffer (7 mM, pH 7.0). A certain amount of pyridine was added into MSM as the sole carbon and nitrogen sources. The composition of SL-4 was described previously [15]. Experimental procedures Luria–Bertani (LB) medium was inoculated with a pure culture of NJUST18 and incubated on a rotary shaker at 180 rpm and 30 °C. 1,000 mg l-1 pyridine was supplemented into LB medium for acclimation of biomass. About 48 h later, the biomass grew into the logarithmic phase and was then harvested by centrifugation at 6,0009g for 5 min. The deposition was resuspended and washed three times using 100 ml MSM. At last, the bacterial deposit was resuspended by vortexing and diluted with MSM to optical density (OD600) of about 1.5. The bacterial suspension was immediately employed as the inoculum during the following biodegradation experiment with inoculum size of 5 %. To investigate the effect of pyridine concentration on pyridine degradation and biomass growth, inoculum of NJUST18 strain described as above was inoculated into the liquid MSM (initial pH 7.0) containing 100, 200, 300, 400, 600 and 1,000 mg l-1 pyridine, respectively. The cultures
Bioprocess Biosyst Eng
pffiffiffiffiffiffiffiffiffiffi Ks Ki
were incubated on a rotary shaker at 180 rpm and 30 °C (both NJUST18 growth and pyridine degradation were optimal at this condition when using pyridine as the sole carbon and nitrogen sources). A series of 150 ml Erlenmeyer flasks was used as batch reactors. Each flask contained 50 ml liquid MSM supplemented with a certain amount of pyridine. The variation of pyridine concentration and OD600 during the incubation process was monitored at suitable time intervals throughout the study. Each result was reported as an average of three independent experiments, with maximum deviations from the average (error bars) indicated.
Sm ¼
Analytical methods
For the calculation of specific degradation rate (qS), the pyridine consumption data (Sc) were modelized with Gompertz equation [19]:
Pyridine in the samples was quantified by HPLC through authentic standard. The mobile phase was a mixture of 70 % methanol and 30 % water pumped at a flow rate of 1.00 ml min-1. Biomass produced during the biodegradation process was monitored by OD600. Due to the strong self-flocculation/aggregation ability of NJUST18, before determination of OD600, it is necessary to resuspend/deflocculate the growth media by vortexing for 10 min. Cell dry weight was determined gravimetrically by drying the harvest cells in an oven at 105 °C for 24 h after centrifugation and washing with sterilized ddH2O. A linear equation was found between the value of OD600 and the corresponding cell dry weight. The OD value was then converted to dry cell mass using the calibration curve obtained by plotting the dry weight of biomass per liter against OD of the suspension.
Kinetics models development
Microbial growth on pyridine has been successfully described by substrate-inhibition models, especially the Haldane equation (Eq. 1) [2, 5, 6, 16]: l¼
l Si Ks þ Si þ S2i =Ki
Replacing Sm in Eqs. 1, 4 could be obtained: lmax ¼
ð1Þ
The specific growth rate (l) at each initial pyridine concentration (Si) could be determined according to the following equation during the exponential growth phase [17]: X ln ¼ lt ð2Þ X0 According to Christen et al. [18], lmax occurred when dl/dS = 0 at:
l qffiffiffiffiffiffiffiffiffiffi 1 þ 2 Ks=Ki
ð4Þ
The cell mass yield coefficient YX/S was estimated by linearizing the cell mass density increase with pyridine consumption [17], as given below: YX=S ¼
Xm X 0 Ss S0
ð5Þ
Biodegradation kinetics model
Sc ¼ aexpðbexpðktÞÞ
ð6Þ
Through the modeling of Sc data with the incubation time t, fitting parameters such as a, b and k were obtained. Maximum volumetric degradation rate (Vmax) at each initial pyridine concentration (Si) was then calculated as follows: Vmax ¼ 0:368 ak
ð7Þ
The corresponding time (topt) for each initial pyridine concentration (Si) was given as follows: topt ¼
Lnb k
ð8Þ
At a given Si, the corresponding Xopt for each topt could be directly read from the biomass growth curve. The specific degradation rate (qS) at each initial pyridine concentration (Si) was then calculated according to Eq. 9: qS ¼
Growth kinetics model
ð3Þ
Vmax Xopt
ð9Þ
The Haldane model was then applied to model qS vs Si, with the fitting parameters qS*, Ks0 and Ki0 calculated. Finally, Sm0 and qSmax were calculated based on Eqs. 3 and 4 applied to qS*, Ks0 and Ki0 . In this study, the parameters of the Haldane model were obtained by a nonlinear least squares technique using MATLAB 7.0, based on Windows XP.
Results and discussion Characterization of strain NJUST18 Strain NJUST18, which was able to grow on pyridine as the sole carbon and nitrogen source, was isolated from the
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Bioprocess Biosyst Eng Fig. 1 Biodegradation of pyridine (a) and growth of NJUST18 strain (b) at different initial pyridine concentrations
soil from a site contaminated by pyridine in a chemical factory. Mineralization of pyridine in the presence of strain NJUST18 was observed in our previous study. Basic morphological, physiological and biochemical characteristics of this strain were investigated for the identification of this strain. Colonies of NJUST18 appeared circular and white with smooth surface during growth on MSM plates for 96 h. As indicated by transmission electronic microscope (TEM) of NJUST18, cells of NJUST18 were motile and rod shaped, 2–3 lm in length and 0.8–0.9 lm in width. Strain NJUST18 was a rod-shaped bacterium with flagellum, exhibiting strong self-flocculation/aggregation ability. It was negative in tests such as Gram staining, catalase, oxidase, urease and starch hydrolysis, casein hydrolysis and gelatin hydrolysis. For further identification of NJUST18, the partial 16S rRNA sequence was determined. The 16S rRNA sequence (comprising 1,381 nucleotides) was deposited in the GenBank database under accession no. JN106368. The phylogenetic analysis was carried out based on this 16S rRNA sequence. The strain was closely related to Rhizobium sp. R-24658 (GenBank accession no. AM084043.1) and Rhizobiales bacterium D11-28.1 (GenBank accession no. AM403228.1), with 99 % sequence identity. In combination with the morphological, physiological and biochemical tests and genospecies, the isolated strain NJUST18 was tentatively identified as Rhizobium sp. and named Rhizobium sp. NJUST18.
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Effect of initial pyridine concentration on strain growth and pyridine degradation To evaluate the effect of initial pyridine concentration on microbial growth and pyridine degradation, Rhizobium sp.NJUST18 was cultivated in mineral salts media at pyridine concentrations between 100 and 1,000 mg l-1. As Fig. 1a shows, in the mineral salts media with an initial pH of 7.0, NJUST18 strain was capable of completely degrading pyridine at a concentration as high as 1,000 mg l-1. At initial pyridine concentrations of 100, 200, 300, 400, 600 and 1,000 mg l-1, complete degradation was achieved within 36, 46, 51, 62.5, 75 and 102 h, respectively. Correspondingly, the biomass increased from around 50 mg l-1 to 89.54 ± 3.56, 105.67 ± 5.40, 145.90 ± 3.81, 253.92 ± 4.59, 345.88 ± 15.42 and 491.67 ± 17.56 mg l-1, respectively, at the time of complete exhaustion of pyridine (Fig. 1b). The lag phase was observed even at relatively low concentrations, demonstrating positive correlation between pyridine biodegradation and cell biomass growth. In addition, with the increase of initial pyridine concentration, the lag phase was extended, although well-acclimatized inoculum was used during the experiments. A similar phenomenon was observed by others in the biodegradation systems for pyridine removal [3, 5, 8], which could be attributed to the recalcitrance and the toxicity of pyridine.
Bioprocess Biosyst Eng
Growth kinetics To estimate the growth kinetic parameters of Rhizobium sp.NJUST18 in the presence of pyridine, the specific growth rate (l) for each initial pyridine concentration (Si) was determined during the exponential growth phase. In these experiments, initial pyridine concentrations varied in the range of 100–1,000 mg l-1. After the lag phase, linear plots of biomass vs time were obtained at all initial pyridine concentrations (Fig. 1b). The slope of the natural logarithm of the biomass vs time was calculated for each Si during the exponential phase according to Eq. 2 and was identified as specific growth rate (l). The experimental specific growth rates (l) were plotted against initial pyridine concentrations (Si). Figure 2 shows a typical trend in which specific growth rates first increased with the increase in initial pyridine concentrations up to a certain concentration level, and then decreased with increase in the initial pyridine concentrations. The value of maximum experimental specific growth rate was found to be equal to 0.0401 h-1, which was achieved at an initial pyridine concentration of 400 mg l-1. The decline trend of specific growth rates beyond the initial pyridine concentration of 400 mg l-1 (Fig. 2) confirmed that pyridine was an inhibitory type of substrate and the inhibition effect of pyridine became predominant above 400 mg l-1. Haldane’s growth kinetics model (Eq. 1), which has mathematical simplicity [5, 20, 21], was used in this study for growth kinetics parameter estimation in the presence of pyridine, which was an inhibitory substrate. In addition to the experimental specific growth rates of NJUST18, the predicted specific growth rates due to Haldane’s model are also included in Fig. 2. The values of growth kinetic parameters obtained through nonlinear least squares
Fig. 2 Experimental and predicted specific growth rates at different initial pyridine concentrations
method using MATLAB are listed in Table 1. Growth kinetics constants reported in other literature [2, 5, 6, 16] for pyridine biodegradation are also included in Table 1. In all of the reported pyridine biodegradation systems, a confusion was observed between the apparent maximum growth rate (l*) and the true maximum growth rate (lmax) [2, 5, 6, 16]. In the literature, the values reported for lmax were overestimated with respect to the true lmax. Thus, Sm and lmax both in this study and in the literature were all calculated with Eqs. 3 and 4, respectively, and are also listed in Table 1. Sm of 461.80 mg l-1 and lmax of 0.0332 h-1 matched with those observed in Fig. 2. As Sm was the pyridine concentration corresponding to lmax, it could be regarded as the value below which biomass growth was limited by the substrate, while above which cell growth was increasingly subject to substrate inhibition. Compared with the calculated Sm from data given in the literature [7, 8], Sm obtained for Rhizobium sp.NJUST18 in this study was the highest (Table 1), which was an advantage of Rhizobium sp.NJUST18 for the treatment of pyridine-containing wastewater. In general, lmax value reported in this work was also larger than that estimated in the literature [7, 8]. The large lmax value indicated that pyridine could be degraded by Rhizobium sp.NJUST18 more rapidly than other microorganisms [17]. However, the lmax value of Rhizobium sp. NJUST18 was lower compared with microorganisms degrading other inhibitory substrates, such as phenol, which typically ranged between 0.047 and 0.385 h-1 [18]. The low lmax values observed in the pyridine biodegradation system was probably due to the high toxic and inhibitory effects of pyridine toward microorganisms. For possible application, this drawback in terms of low lmax values for Rhizobium sp. NJUST18 could be counterbalanced by the relatively high Sm, compared with those reported in the phenol degrading systems, which typically ranged between 23.3 and 219.7 mg l-1 [18]. The inhibition constant (Ki) indicated both the inhibition tendency and the toxicity degree of the substrate toward microorganisms [17]. This value was particularly important for subsequent wastewater treatment application since it defined a concentration threshold which should not be exceeded. The Ki value found in this study (268.60 mg l-1) was in the middle range of values reported in the literature (Table 1). The Ks value was influential on the growth kinetics of biomass in the low concentration region [5, 18]. A low value of Ks indicated high affinity of biomass toward substrate. However, the Ks value (793.97 mg l-1) in this study was extremely high, compared with that in the literature [2, 7, 8]. The unique kinetic characteristic of Rhizobium sp. NJUST18 with a high Ki value and an extremely high Ks value indicated that, in comparison to other cultures generally grown on pyridine, Rhizobium sp.
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Bioprocess Biosyst Eng Table 1 Comparison of Haldane’s growth kinetics parameters for various microorganisms grown on pyridine lmax (h-1)
l* (h-1)
Ks (mg/L)
Ki (mg/L)
Sm (mg/L)
T (oC)
System
Cultures
Reference
0.0224
0.0424
122.74
616.37
275.05
30
Batch reactor
Shewanella putrefaciens
[5]
0.0226
0.0481
134.28
418.69
237.11
30
Batch reactor
Bacillus sphaericus
[5]
–
0.0083
37.29
–
–
30
Batch reactor
Pseudomonas pseudoalcaligenes-KPN
[2]
–
0.0045
5.37
–
–
–
Completely mixed activated sludge process
Pseudomonas pseudoalcaligenes-KPN
[2]
0.0190
0.0925
60.28
35
Batch reactor
Lysinibacillus cresolivorans
[6]
–
0.0212
–
–
–
30
Batch reactor
Unknown culture
[16]
0.0332
0.1473
793.97
268.60
461.80
30
Batch reactor
Rhizobium sp. NJUST18
This work
16.17
31.22
NJUST18 was able to grow on pyridine within a wide range of concentrations, especially at relatively high concentrations. A similar phenomenon of high Ks value was also observed in the phenol biodegradation system by Rhizobium sp. CCNWTB 701 [20]. The results indicated the low affinity of Rhizobium sp. toward substrates such as pyridine and phenol.
Table 2 Vmax, qS and YX/S for pyridine degradation by Rhizobium sp.NJUST18 at different initial pyridine concentrations (Si) Si (mg/L)
100
200
300
400
600
1,000
Vmax (mg/L/h)
4.228
6.134
9.397
9.786
12.932
14.809
qS (g/g/h)
0.0669
0.0776
0.1172
0.1206
0.1188
0.1035
YX/S (g/g)
0.35
0.30
0.33
0.50
0.51
0.46
Biodegradation kinetics To calculate the specific degradation rate (qS) for each initial pyridine concentration (Si), the Gompertz model (Eq. 6) was used for modeling the pyridine consumption data, with the parameters a, b and k determined (R2 ranged from 0.9871 to 0.9998). Vmax and topt were then calculated based on these fitting parameters according to Eqs. 7 and 8. Then, Xopt for each Si was graphically determined according to topt at each Si (Fig. 1b). Finally, the specific degradation rate (qS) for each Si was calculated according to Eq. 9. As indicated in Table 2, the calculated maximum volumetric degradation rate (Vmax) increased with the increase of initial pyridine concentration (Si). However, similar with specific growth rates (l) vs initial pyridine concentrations (Si), the pattern of specific degradation rate (qS) vs initial pyridine concentration (Si) also displayed a substrateinhibition behavior (Table 2). As reported in other works, for the inhibitory substrates, such as phenol, the relationship between qS and Si could be adequately described by the Haldane’s equation as well [18]. Both experimental and predicted specific degradation rates (qS) of pyridine due to Haldane’s model are shown in Fig. 3. The fitting of parameters yields the following values: qSmax = 0.1212 g g-1 h-1 and q* = 0.3874 g g-1 h-1 at Sm0 = 507.83 mg l-1, Ks0 = 558.03 mg l-1 and Ki0 = 462.15 mg l-1. Inhibition constants for both biomass growth and pyridine degradation were in the same range, indicating that pyridine affected both the biomass growth and
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Fig. 3 Experimental and predicted specific degradation rates at different initial pyridine concentrations
pyridine degradation ability of Rhizobium sp. NJUST18 likewise. The magnitudes of kinetic parameters for pyridine biodegradation, such as qSmax, Sm0 , Ks0 and Ki0 , which indicated pyridine biodegradation performance by microorganisms, were still lacking in the present literature with regard to pure cultures. In a pyridine biodegradation system based on aerobic granules, the values of maximum specific degradation rate (qSmax) and Sm0 were found to be 0.073 g g-1 h-1 and 250.00 mg l-1, respectively [22],
Bioprocess Biosyst Eng Table 3 Degradation kinetics parameters for pyridine qSmax (g/g/h)
qS* (g/g/h)
0.0730
–
0.1212
0.3874
Ks0 (mg/L)
Ki0 (mg/L)
Sm0 (mg/L)
Vmax (mg/L/h)
Cultures
Reference
–
–
250.00
63.700
Aerobic granules
[22]
558.03
462.15
507.83
4.228–14.809
Rhizobium sp. NJUST18
This work
which were much lower than that obtained in this study (qSmax value of 0.1212 g g-1 h-1 and Sm0 of 507.83 mg l-1) (Table 3). However, the Vmax reported in the literature was much higher than those in this study, probably due to the huge biomass in the SBR reactor [22]. In addition, the qSmax value in this study was in the lower range of values reported for biodegradation of phenol, which ranged from 0.102 to 0.940 g g-1 h-1 [18]. As for lmax, even though Rhizobium sp.NJUST18 displayed low qSmax, its great capacities in terms of wide pyridine concentration range and high tolerance toward pyridine toxicity made it adequate for pyridine removal applications. Yield factor For each Si, the observed yield coefficient (YX/S) was determined by plotting the biomass produced against the pyridine consumed according to Eq. 5. As initial pyridine concentration varied from 100 to 1,000 mg l-1, the yield coefficient varied between 0.30 and 0.51 g g-1 (Table 2). The yield coefficient obtained by linear regression was confident enough since the R2 of the regression lay between 0.910 and 0.994. The yield coefficient in this study seemed to be low compared with that reported for other inhibitory substrates such as phenol [18]. However, low yield coefficients were also observed in other biodegradation systems treating highly recalcitrant and toxic compounds, such as 2,4,6-trinitrophenol [17]. Besides, the profile of cell mass yield as a function of initial pyridine concentration was similar to that of specific growth rate and specific degradation rate (Table 2; Figs. 2, 3). The yield maximized at pyridine concentration of 400 and 600 mg l-1 where l was also maximum. Beyond 600 mg l-1, considerable decrease in the values of mass yield coefficient was observed with increase in pyridine concentration up to 1,000 mg l-1. Similar results of decreasing YX/S with increasing pyridine concentration in the inhibitory region were also reported in the literature [3, 8]. This phenomenon could be reasoned based on the fact that the percentage of the total substrate carbon converted to energy for cell growth and maintenance increased as the specific growth rate decreased, when the inhibition effect of pyridine became predominant above the initial pyridine concentration of 600 mg l-1. More energy was required to overcome the effect of substrate inhibition, while the percentage of total substrate carbon assimilated into biomass
decreased in the inhibitory concentration range. In addition, the production and accumulation of intermediates might also be responsible for the decreased cell mass yield at high pyridine concentrations, as indicated by high residual TOC concentrations at high initial pyridine concentrations (data not shown). Thus substrate inhibition was known to reduce not only the specific growth rate, but also cell growth yield.
Conclusions The paper described the kinetics of pyridine biodegradation by a novel bacterial strain, Rhizobium sp. NJUST18. The following conclusions were derived: (1)
(2)
(3)
Pyridine inhibitory growth could be adequately described by the Haldane’s model. The kinetic constants of the Haldane’s equation were lmax = 0.0332 h-1 at Sm = 461.80 mg l-1, Ks = 793.97 mg l-1 and Ki = 268.60 mg l-1. The cell mass yield coefficient decreased with increasing substrate concentration in the inhibitory region. The Gompertz model was successfully used to model pyridine consumption profiles. Specific degradation rate was also subject to substrate inhibition and the relationship between qS and Si was adequately described by the Haldane’s equation (qSmax = 0.1212 g g-1 h-1 at Sm0 = 507.83 mg l-1, Ks0 = 558.03 mg l-1 and Ki0 = 462.15 mg l-1). Inhibition constants for growth and degradation rate value were in the same range. Compared with other pyridine degraders, lmax and Sm obtained for Rhizobium sp.NJUST18 was the highest, which was an advantage for further treatment of pyridine-containing wastewater. High Ki and Ki0 values and extremely high Ks and Ks0 values indicated that Rhizobium sp.NJUST18 was able to grow on pyridine within a wide concentration range, especially at relatively high concentrations.
Acknowldgments This research is financed by Major Project of Water Pollution Control and Management Technology of P. R. China (No. 2012ZX07101-003-001), National Natural Science Foundation of China (No. 51208258, 51378261 and 51348007), Natural Science Foundation of Jiangsu Province (No. BK2011717), Research Fund for the Doctoral Program of Higher Education of China
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Bioprocess Biosyst Eng (20123219120009), Environmental Protection Scientific Research Project of Jiangsu Province (201103), Fundamental Research Funds for the Central Universities (No.30920130122007) and Zijing Intelligent Program (No. 2013-ZJ-02-19).
12.
13.
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using Pseudomonas sp. and isolation of plasmid responsible for degradation. Waste Manag 23:167–171 Li J, Cai W, Cai J (2009) The characteristics and mechanisms of pyridine biodegradation by Streptomyces sp. J Hazard Mater 165:950–954 Bai J, Wen JP, Li HM, Jiang Y (2007) Kinetic modeling of growth and biodegradation of phenol and m-cresol using Alcaligenes faecalis. Process Biochem 42:510–517 Loh KC, Yu YG (2000) Kinetics of carbazole degradation by Pseudomonas putida in presence of sodium salicylate. Water Res 34:4131–4138 Shen J, Zhang J, Zuo Y, Wang L, Sun X, Li J, Han W, He R (2009) Biodegradation of 2,4,6-trinitrophenol by Rhodococcus sp. isolated from a picric acid-contaminated soil. J Hazard Mater 163:1199–1206 Lodha B, Bhadane R, Patel B, Killedar D (2008) Biodegradation of pyridine by an isolated bacterial consortium/strain and bioaugmentation of strain into activated sludge to enhance pyridine biodegradation. Biodegradation 19:717–723 Shen J, He R, Zhang J, Zuo Y, Li Y, Sun X, Li J, Wang L, Han W (2009) Biodegradation kinetics of picric acid by Rhodococcus sp.NJUST16 in batch reactors. J Hazard Mater 167:193–198 Christen P, Vega A, Casalot L, Simon G, Auria R (2012) Kinetics of aerobic phenol biodegradation by the acidophilic and hyperthermophilic archaeon Sulfolobus solfataricus 98/2. Biochem Eng J 62:56–61 Acun˜a ME, P_erez F, Auria R, Revah S (1999) Microbiological and kinetic aspects of a biofilter for the removal of toluene from waste gases. Biotechnol Bioeng 63:175–184 Wei G, Yu J, Zhu Y, Chen W, Wang L (2008) Characterization of phenol degradation by Rhizobium sp. CCNWTB 701 isolated from Astragalus chrysopteru in mining tailing region. J Hazard Mater 151:111–117 Wang L, Li Y, Yu P, Xie Z, Luo Y, Lin Y (2010) Biodegradation of phenol at high concentration by a novel fungal strain Paecilomyces variotii JH6. J Hazard Mater 183:366–371 Adav SS, Lee D-J, Ren NQ (2007) Biodegradation of pyridine using aerobic granules in the presence of phenol. Water Res 41:2903–2910
ORIGINAL PAPER
Kinetics study of pyridine biodegradation by a novel bacterial strain, Rhizobium sp. NJUST18 Jinyou Shen • Xin Zhang • Dan Chen • Xiaodong Liu • Libin Zhang • Xiuyun Sun • Jiansheng Li • Huiping Bi • Lianjun Wang
Received: 10 September 2013 / Accepted: 1 November 2013 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Biodegradation of pyridine by a novel bacterial strain, Rhizobium sp. NJUST18, was studied in batch experiments over a wide concentration range (from 100 to 1,000 mg l-1). Pyridine inhibited both growth of Rhizobium sp. NJUST18 and biodegradation of pyridine. The Haldane model could be fitted to the growth kinetics data well with the kinetic constants l* = 0.1473 h-1, Ks = 793.97 mg l-1, Ki = 268.60 mg l-1 and Sm = 461.80 mg l-1. The true lmax, calculated from l*, was found to be 0.0332 h-1. Yield coefficient YX/S depended on Si and reached a maximum of 0.51 g g-1 at Si of 600 mg l-1. Vmax was calculated by fitting the pyridine consumption data with the Gompertz model. Vmax increased with initial pyridine concentration up to 14.809 mg l-1 h-1. The qS values, calculated from Vmax , were fitted with the Haldane equation, yielding qSmax = 0.1212 g g-1 h-1 and q* = 0.3874 g g-1 h-1 at Sm0 = 507.83 mg l-1, Ks0 = 558.03 mg l-1, and Ki0 = 462.15 mg l-1. Inhibition constants for growth and degradation rate value were in the same range. Compared with other pyridine degraders, lmax and Sm obtained for Rhizobium sp. NJUST18 were relatively high. High Ki and Ki0 values and extremely high Ks and Ks0 values indicated that NJUST18 was able to grow on pyridine within a wide
J. Shen X. Zhang D. Chen X. Liu L. Zhang X. Sun J. Li H. Bi L. Wang (&) Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province, China e-mail: [email protected] J. Shen e-mail: [email protected]
concentration range, concentrations.
especially
at
relatively
high
Keywords Pyridine Biodegradation Kinetics Haldane model Gompertz model Rhizobium List of symbols K Fitting parameter of the Gompertz model (h-1) Ki Inhibition coefficient for Haldane’s growth kinetics (mg l-1) Ks Half saturation coefficient for Haldane’s growth kinetics (mg l-1) Ki0 Inhibition coefficient for Haldane’s degradation kinetics (mg l-1) Ks0 Half saturation coefficient for Haldane’s degradation kinetics (mg l-1) qS Specific degradation rate (g g-1 h-1) qS * Apparent maximum specific degradation rate (g g-1 h-1) qSmax True maximum specific degradation rate (g g-1 h-1) Ss Pyridine concentration (mg l-1) Sc Pyridine consumed (mg l-1) Si Initial pyridine concentration (mg l-1) Sm Pyridine concentration at which l = lmax (mg l-1) 0 Sm Pyridine concentration at which qS = qSmax (mg l-1) t Time of incubation (h) topt Time of maximum pyridine degradation rate (h) Vmax Maximum volumetric rate of pyridine degradation (mg l-1 h-1) X Concentration of biomass (mg l-1) Xopt Concentration of biomass at topt (mg l-1) YX/S Yield coefficient [g biomass (g substrate)-1]
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Bioprocess Biosyst Eng
Greek a, b l l* lmax
symbol Fitting parameter of the Gompertz model (mg l-1) Specific growth rate (h-1) Apparent maximum specific growth rate (h-1) True maximum specific growth rate (h-1)
isolate that had the nature of strength degrading pyridine. The present study aimed to determine the growth kinetics parameters of Rhizobium sp. NJUST18 and pyridine biodegradation kinetics parameters. Both the inhibition tendency of pyridine toward biomass and pyridine degradation performance of Rhizobium sp. NJUST18 were evaluated based on these kinetics parameters obtained in this study.
Introduction Materials and methods As a representative of nitrogen heterocyclic compounds (NHCs), pyridine is widely used in manufacturing of dye, herbicides, pesticides and pharmaceuticals, causing serious environmental problems because of its toxic, teratogenic and carcinogenic effects [1]. For this reason, its presence in wastewater is severely regulated and there is interest in removing it from contaminated sites. Many treatment technologies have been developed for pyridine removal from contaminated environment. However, due to its high solubility and structural stability, physico-chemical methods, such as oxidation, adsorption and incineration, have been proven to be cost and energy intensive [2]. Biological treatment is often thought to be environmental friendly and cost effective, becoming an attractive means for decontamination of the environment and industrial wastewater [3]. However, due to its heterocyclic structure, pyridine is recalcitrant to biodegradation and persistent in nature. Up to date, several new and efficient microbial species have been isolated for pyridine biodegradation. These microorganisms include genera Arthrobacter [4], Bacillus [5], Lysinibacillus [6], Nocardiodes [7], Paracoccus [8–10], Pseudomonas [2, 11], Rhodococcus [1], Shinella [3], Shewanella [5] and Streptomyces [12]. However, due to the limited number of collections of such bacteria, knowledge about pyridine biodegradation is yet limited. Consequently, for pyridine biodegradation, isolation of new bacterial strains indigenous to sites contaminated by pyridine is still an important issue. What is more, for the evaluation of the persistence of pollutants such as pyridine, knowledge on the biodegradation kinetics is of importance [13, 14]. Biodegradation kinetics is helpful for understanding both the inhibition tendency of the pollutants toward biomass and degradation performance of biomass. Microbial growth on pyridine has been successfully described by substrate-inhibition models, such as Haldane’s equation, which has provided satisfactory correlations with experimental data. However, to the best of our knowledge, kinetics on pyridine biodegradation has been seldom investigated in previous studies. A novel bacterial strain was previously isolated from a site contaminated by pyridine and was named Rhizobium sp. NJUST18. Rhizobium sp. NJUST18 reported here seemed to be the first documented rhizobial bacterium
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Microorganism and culture media Pure culture of Rhizobium sp. NJUST18, which was isolated in this laboratory previously, was used throughout our work. Rhizobium sp. NJUST18 was identified based on morphological, physiological and biochemical tests and genospecies. Store culture was maintained by periodical transfer onto mineral salt agar plates supplemented with 1,000 mg l-1 pyridine and stored at 30 °C for further study. For long-term maintenance, pyridine degrading strain was cultivated aerobically in liquid mineral salt medium (MSM) containing 1,000 mg l-1 pyridine and stored in 20 % glycerol at -80 °C in an ultralow temperature freezer. The MSM used in this study contained Na2HPO412H2O (1.529 g l-1), KH2PO4 (0.372 g l-1), MgSO47H2O (0.1 g l-1), CaCl2 (0.05 g l-1) and SL-4 (10 ml l-1). In MSM, Na2HPO412H2O and KH2PO4 served as the phosphate buffer (7 mM, pH 7.0). A certain amount of pyridine was added into MSM as the sole carbon and nitrogen sources. The composition of SL-4 was described previously [15]. Experimental procedures Luria–Bertani (LB) medium was inoculated with a pure culture of NJUST18 and incubated on a rotary shaker at 180 rpm and 30 °C. 1,000 mg l-1 pyridine was supplemented into LB medium for acclimation of biomass. About 48 h later, the biomass grew into the logarithmic phase and was then harvested by centrifugation at 6,0009g for 5 min. The deposition was resuspended and washed three times using 100 ml MSM. At last, the bacterial deposit was resuspended by vortexing and diluted with MSM to optical density (OD600) of about 1.5. The bacterial suspension was immediately employed as the inoculum during the following biodegradation experiment with inoculum size of 5 %. To investigate the effect of pyridine concentration on pyridine degradation and biomass growth, inoculum of NJUST18 strain described as above was inoculated into the liquid MSM (initial pH 7.0) containing 100, 200, 300, 400, 600 and 1,000 mg l-1 pyridine, respectively. The cultures
Bioprocess Biosyst Eng
pffiffiffiffiffiffiffiffiffiffi Ks Ki
were incubated on a rotary shaker at 180 rpm and 30 °C (both NJUST18 growth and pyridine degradation were optimal at this condition when using pyridine as the sole carbon and nitrogen sources). A series of 150 ml Erlenmeyer flasks was used as batch reactors. Each flask contained 50 ml liquid MSM supplemented with a certain amount of pyridine. The variation of pyridine concentration and OD600 during the incubation process was monitored at suitable time intervals throughout the study. Each result was reported as an average of three independent experiments, with maximum deviations from the average (error bars) indicated.
Sm ¼
Analytical methods
For the calculation of specific degradation rate (qS), the pyridine consumption data (Sc) were modelized with Gompertz equation [19]:
Pyridine in the samples was quantified by HPLC through authentic standard. The mobile phase was a mixture of 70 % methanol and 30 % water pumped at a flow rate of 1.00 ml min-1. Biomass produced during the biodegradation process was monitored by OD600. Due to the strong self-flocculation/aggregation ability of NJUST18, before determination of OD600, it is necessary to resuspend/deflocculate the growth media by vortexing for 10 min. Cell dry weight was determined gravimetrically by drying the harvest cells in an oven at 105 °C for 24 h after centrifugation and washing with sterilized ddH2O. A linear equation was found between the value of OD600 and the corresponding cell dry weight. The OD value was then converted to dry cell mass using the calibration curve obtained by plotting the dry weight of biomass per liter against OD of the suspension.
Kinetics models development
Microbial growth on pyridine has been successfully described by substrate-inhibition models, especially the Haldane equation (Eq. 1) [2, 5, 6, 16]: l¼
l Si Ks þ Si þ S2i =Ki
Replacing Sm in Eqs. 1, 4 could be obtained: lmax ¼
ð1Þ
The specific growth rate (l) at each initial pyridine concentration (Si) could be determined according to the following equation during the exponential growth phase [17]: X ln ¼ lt ð2Þ X0 According to Christen et al. [18], lmax occurred when dl/dS = 0 at:
l qffiffiffiffiffiffiffiffiffiffi 1 þ 2 Ks=Ki
ð4Þ
The cell mass yield coefficient YX/S was estimated by linearizing the cell mass density increase with pyridine consumption [17], as given below: YX=S ¼
Xm X 0 Ss S0
ð5Þ
Biodegradation kinetics model
Sc ¼ aexpðbexpðktÞÞ
ð6Þ
Through the modeling of Sc data with the incubation time t, fitting parameters such as a, b and k were obtained. Maximum volumetric degradation rate (Vmax) at each initial pyridine concentration (Si) was then calculated as follows: Vmax ¼ 0:368 ak
ð7Þ
The corresponding time (topt) for each initial pyridine concentration (Si) was given as follows: topt ¼
Lnb k
ð8Þ
At a given Si, the corresponding Xopt for each topt could be directly read from the biomass growth curve. The specific degradation rate (qS) at each initial pyridine concentration (Si) was then calculated according to Eq. 9: qS ¼
Growth kinetics model
ð3Þ
Vmax Xopt
ð9Þ
The Haldane model was then applied to model qS vs Si, with the fitting parameters qS*, Ks0 and Ki0 calculated. Finally, Sm0 and qSmax were calculated based on Eqs. 3 and 4 applied to qS*, Ks0 and Ki0 . In this study, the parameters of the Haldane model were obtained by a nonlinear least squares technique using MATLAB 7.0, based on Windows XP.
Results and discussion Characterization of strain NJUST18 Strain NJUST18, which was able to grow on pyridine as the sole carbon and nitrogen source, was isolated from the
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Bioprocess Biosyst Eng Fig. 1 Biodegradation of pyridine (a) and growth of NJUST18 strain (b) at different initial pyridine concentrations
soil from a site contaminated by pyridine in a chemical factory. Mineralization of pyridine in the presence of strain NJUST18 was observed in our previous study. Basic morphological, physiological and biochemical characteristics of this strain were investigated for the identification of this strain. Colonies of NJUST18 appeared circular and white with smooth surface during growth on MSM plates for 96 h. As indicated by transmission electronic microscope (TEM) of NJUST18, cells of NJUST18 were motile and rod shaped, 2–3 lm in length and 0.8–0.9 lm in width. Strain NJUST18 was a rod-shaped bacterium with flagellum, exhibiting strong self-flocculation/aggregation ability. It was negative in tests such as Gram staining, catalase, oxidase, urease and starch hydrolysis, casein hydrolysis and gelatin hydrolysis. For further identification of NJUST18, the partial 16S rRNA sequence was determined. The 16S rRNA sequence (comprising 1,381 nucleotides) was deposited in the GenBank database under accession no. JN106368. The phylogenetic analysis was carried out based on this 16S rRNA sequence. The strain was closely related to Rhizobium sp. R-24658 (GenBank accession no. AM084043.1) and Rhizobiales bacterium D11-28.1 (GenBank accession no. AM403228.1), with 99 % sequence identity. In combination with the morphological, physiological and biochemical tests and genospecies, the isolated strain NJUST18 was tentatively identified as Rhizobium sp. and named Rhizobium sp. NJUST18.
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Effect of initial pyridine concentration on strain growth and pyridine degradation To evaluate the effect of initial pyridine concentration on microbial growth and pyridine degradation, Rhizobium sp.NJUST18 was cultivated in mineral salts media at pyridine concentrations between 100 and 1,000 mg l-1. As Fig. 1a shows, in the mineral salts media with an initial pH of 7.0, NJUST18 strain was capable of completely degrading pyridine at a concentration as high as 1,000 mg l-1. At initial pyridine concentrations of 100, 200, 300, 400, 600 and 1,000 mg l-1, complete degradation was achieved within 36, 46, 51, 62.5, 75 and 102 h, respectively. Correspondingly, the biomass increased from around 50 mg l-1 to 89.54 ± 3.56, 105.67 ± 5.40, 145.90 ± 3.81, 253.92 ± 4.59, 345.88 ± 15.42 and 491.67 ± 17.56 mg l-1, respectively, at the time of complete exhaustion of pyridine (Fig. 1b). The lag phase was observed even at relatively low concentrations, demonstrating positive correlation between pyridine biodegradation and cell biomass growth. In addition, with the increase of initial pyridine concentration, the lag phase was extended, although well-acclimatized inoculum was used during the experiments. A similar phenomenon was observed by others in the biodegradation systems for pyridine removal [3, 5, 8], which could be attributed to the recalcitrance and the toxicity of pyridine.
Bioprocess Biosyst Eng
Growth kinetics To estimate the growth kinetic parameters of Rhizobium sp.NJUST18 in the presence of pyridine, the specific growth rate (l) for each initial pyridine concentration (Si) was determined during the exponential growth phase. In these experiments, initial pyridine concentrations varied in the range of 100–1,000 mg l-1. After the lag phase, linear plots of biomass vs time were obtained at all initial pyridine concentrations (Fig. 1b). The slope of the natural logarithm of the biomass vs time was calculated for each Si during the exponential phase according to Eq. 2 and was identified as specific growth rate (l). The experimental specific growth rates (l) were plotted against initial pyridine concentrations (Si). Figure 2 shows a typical trend in which specific growth rates first increased with the increase in initial pyridine concentrations up to a certain concentration level, and then decreased with increase in the initial pyridine concentrations. The value of maximum experimental specific growth rate was found to be equal to 0.0401 h-1, which was achieved at an initial pyridine concentration of 400 mg l-1. The decline trend of specific growth rates beyond the initial pyridine concentration of 400 mg l-1 (Fig. 2) confirmed that pyridine was an inhibitory type of substrate and the inhibition effect of pyridine became predominant above 400 mg l-1. Haldane’s growth kinetics model (Eq. 1), which has mathematical simplicity [5, 20, 21], was used in this study for growth kinetics parameter estimation in the presence of pyridine, which was an inhibitory substrate. In addition to the experimental specific growth rates of NJUST18, the predicted specific growth rates due to Haldane’s model are also included in Fig. 2. The values of growth kinetic parameters obtained through nonlinear least squares
Fig. 2 Experimental and predicted specific growth rates at different initial pyridine concentrations
method using MATLAB are listed in Table 1. Growth kinetics constants reported in other literature [2, 5, 6, 16] for pyridine biodegradation are also included in Table 1. In all of the reported pyridine biodegradation systems, a confusion was observed between the apparent maximum growth rate (l*) and the true maximum growth rate (lmax) [2, 5, 6, 16]. In the literature, the values reported for lmax were overestimated with respect to the true lmax. Thus, Sm and lmax both in this study and in the literature were all calculated with Eqs. 3 and 4, respectively, and are also listed in Table 1. Sm of 461.80 mg l-1 and lmax of 0.0332 h-1 matched with those observed in Fig. 2. As Sm was the pyridine concentration corresponding to lmax, it could be regarded as the value below which biomass growth was limited by the substrate, while above which cell growth was increasingly subject to substrate inhibition. Compared with the calculated Sm from data given in the literature [7, 8], Sm obtained for Rhizobium sp.NJUST18 in this study was the highest (Table 1), which was an advantage of Rhizobium sp.NJUST18 for the treatment of pyridine-containing wastewater. In general, lmax value reported in this work was also larger than that estimated in the literature [7, 8]. The large lmax value indicated that pyridine could be degraded by Rhizobium sp.NJUST18 more rapidly than other microorganisms [17]. However, the lmax value of Rhizobium sp. NJUST18 was lower compared with microorganisms degrading other inhibitory substrates, such as phenol, which typically ranged between 0.047 and 0.385 h-1 [18]. The low lmax values observed in the pyridine biodegradation system was probably due to the high toxic and inhibitory effects of pyridine toward microorganisms. For possible application, this drawback in terms of low lmax values for Rhizobium sp. NJUST18 could be counterbalanced by the relatively high Sm, compared with those reported in the phenol degrading systems, which typically ranged between 23.3 and 219.7 mg l-1 [18]. The inhibition constant (Ki) indicated both the inhibition tendency and the toxicity degree of the substrate toward microorganisms [17]. This value was particularly important for subsequent wastewater treatment application since it defined a concentration threshold which should not be exceeded. The Ki value found in this study (268.60 mg l-1) was in the middle range of values reported in the literature (Table 1). The Ks value was influential on the growth kinetics of biomass in the low concentration region [5, 18]. A low value of Ks indicated high affinity of biomass toward substrate. However, the Ks value (793.97 mg l-1) in this study was extremely high, compared with that in the literature [2, 7, 8]. The unique kinetic characteristic of Rhizobium sp. NJUST18 with a high Ki value and an extremely high Ks value indicated that, in comparison to other cultures generally grown on pyridine, Rhizobium sp.
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Bioprocess Biosyst Eng Table 1 Comparison of Haldane’s growth kinetics parameters for various microorganisms grown on pyridine lmax (h-1)
l* (h-1)
Ks (mg/L)
Ki (mg/L)
Sm (mg/L)
T (oC)
System
Cultures
Reference
0.0224
0.0424
122.74
616.37
275.05
30
Batch reactor
Shewanella putrefaciens
[5]
0.0226
0.0481
134.28
418.69
237.11
30
Batch reactor
Bacillus sphaericus
[5]
–
0.0083
37.29
–
–
30
Batch reactor
Pseudomonas pseudoalcaligenes-KPN
[2]
–
0.0045
5.37
–
–
–
Completely mixed activated sludge process
Pseudomonas pseudoalcaligenes-KPN
[2]
0.0190
0.0925
60.28
35
Batch reactor
Lysinibacillus cresolivorans
[6]
–
0.0212
–
–
–
30
Batch reactor
Unknown culture
[16]
0.0332
0.1473
793.97
268.60
461.80
30
Batch reactor
Rhizobium sp. NJUST18
This work
16.17
31.22
NJUST18 was able to grow on pyridine within a wide range of concentrations, especially at relatively high concentrations. A similar phenomenon of high Ks value was also observed in the phenol biodegradation system by Rhizobium sp. CCNWTB 701 [20]. The results indicated the low affinity of Rhizobium sp. toward substrates such as pyridine and phenol.
Table 2 Vmax, qS and YX/S for pyridine degradation by Rhizobium sp.NJUST18 at different initial pyridine concentrations (Si) Si (mg/L)
100
200
300
400
600
1,000
Vmax (mg/L/h)
4.228
6.134
9.397
9.786
12.932
14.809
qS (g/g/h)
0.0669
0.0776
0.1172
0.1206
0.1188
0.1035
YX/S (g/g)
0.35
0.30
0.33
0.50
0.51
0.46
Biodegradation kinetics To calculate the specific degradation rate (qS) for each initial pyridine concentration (Si), the Gompertz model (Eq. 6) was used for modeling the pyridine consumption data, with the parameters a, b and k determined (R2 ranged from 0.9871 to 0.9998). Vmax and topt were then calculated based on these fitting parameters according to Eqs. 7 and 8. Then, Xopt for each Si was graphically determined according to topt at each Si (Fig. 1b). Finally, the specific degradation rate (qS) for each Si was calculated according to Eq. 9. As indicated in Table 2, the calculated maximum volumetric degradation rate (Vmax) increased with the increase of initial pyridine concentration (Si). However, similar with specific growth rates (l) vs initial pyridine concentrations (Si), the pattern of specific degradation rate (qS) vs initial pyridine concentration (Si) also displayed a substrateinhibition behavior (Table 2). As reported in other works, for the inhibitory substrates, such as phenol, the relationship between qS and Si could be adequately described by the Haldane’s equation as well [18]. Both experimental and predicted specific degradation rates (qS) of pyridine due to Haldane’s model are shown in Fig. 3. The fitting of parameters yields the following values: qSmax = 0.1212 g g-1 h-1 and q* = 0.3874 g g-1 h-1 at Sm0 = 507.83 mg l-1, Ks0 = 558.03 mg l-1 and Ki0 = 462.15 mg l-1. Inhibition constants for both biomass growth and pyridine degradation were in the same range, indicating that pyridine affected both the biomass growth and
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Fig. 3 Experimental and predicted specific degradation rates at different initial pyridine concentrations
pyridine degradation ability of Rhizobium sp. NJUST18 likewise. The magnitudes of kinetic parameters for pyridine biodegradation, such as qSmax, Sm0 , Ks0 and Ki0 , which indicated pyridine biodegradation performance by microorganisms, were still lacking in the present literature with regard to pure cultures. In a pyridine biodegradation system based on aerobic granules, the values of maximum specific degradation rate (qSmax) and Sm0 were found to be 0.073 g g-1 h-1 and 250.00 mg l-1, respectively [22],
Bioprocess Biosyst Eng Table 3 Degradation kinetics parameters for pyridine qSmax (g/g/h)
qS* (g/g/h)
0.0730
–
0.1212
0.3874
Ks0 (mg/L)
Ki0 (mg/L)
Sm0 (mg/L)
Vmax (mg/L/h)
Cultures
Reference
–
–
250.00
63.700
Aerobic granules
[22]
558.03
462.15
507.83
4.228–14.809
Rhizobium sp. NJUST18
This work
which were much lower than that obtained in this study (qSmax value of 0.1212 g g-1 h-1 and Sm0 of 507.83 mg l-1) (Table 3). However, the Vmax reported in the literature was much higher than those in this study, probably due to the huge biomass in the SBR reactor [22]. In addition, the qSmax value in this study was in the lower range of values reported for biodegradation of phenol, which ranged from 0.102 to 0.940 g g-1 h-1 [18]. As for lmax, even though Rhizobium sp.NJUST18 displayed low qSmax, its great capacities in terms of wide pyridine concentration range and high tolerance toward pyridine toxicity made it adequate for pyridine removal applications. Yield factor For each Si, the observed yield coefficient (YX/S) was determined by plotting the biomass produced against the pyridine consumed according to Eq. 5. As initial pyridine concentration varied from 100 to 1,000 mg l-1, the yield coefficient varied between 0.30 and 0.51 g g-1 (Table 2). The yield coefficient obtained by linear regression was confident enough since the R2 of the regression lay between 0.910 and 0.994. The yield coefficient in this study seemed to be low compared with that reported for other inhibitory substrates such as phenol [18]. However, low yield coefficients were also observed in other biodegradation systems treating highly recalcitrant and toxic compounds, such as 2,4,6-trinitrophenol [17]. Besides, the profile of cell mass yield as a function of initial pyridine concentration was similar to that of specific growth rate and specific degradation rate (Table 2; Figs. 2, 3). The yield maximized at pyridine concentration of 400 and 600 mg l-1 where l was also maximum. Beyond 600 mg l-1, considerable decrease in the values of mass yield coefficient was observed with increase in pyridine concentration up to 1,000 mg l-1. Similar results of decreasing YX/S with increasing pyridine concentration in the inhibitory region were also reported in the literature [3, 8]. This phenomenon could be reasoned based on the fact that the percentage of the total substrate carbon converted to energy for cell growth and maintenance increased as the specific growth rate decreased, when the inhibition effect of pyridine became predominant above the initial pyridine concentration of 600 mg l-1. More energy was required to overcome the effect of substrate inhibition, while the percentage of total substrate carbon assimilated into biomass
decreased in the inhibitory concentration range. In addition, the production and accumulation of intermediates might also be responsible for the decreased cell mass yield at high pyridine concentrations, as indicated by high residual TOC concentrations at high initial pyridine concentrations (data not shown). Thus substrate inhibition was known to reduce not only the specific growth rate, but also cell growth yield.
Conclusions The paper described the kinetics of pyridine biodegradation by a novel bacterial strain, Rhizobium sp. NJUST18. The following conclusions were derived: (1)
(2)
(3)
Pyridine inhibitory growth could be adequately described by the Haldane’s model. The kinetic constants of the Haldane’s equation were lmax = 0.0332 h-1 at Sm = 461.80 mg l-1, Ks = 793.97 mg l-1 and Ki = 268.60 mg l-1. The cell mass yield coefficient decreased with increasing substrate concentration in the inhibitory region. The Gompertz model was successfully used to model pyridine consumption profiles. Specific degradation rate was also subject to substrate inhibition and the relationship between qS and Si was adequately described by the Haldane’s equation (qSmax = 0.1212 g g-1 h-1 at Sm0 = 507.83 mg l-1, Ks0 = 558.03 mg l-1 and Ki0 = 462.15 mg l-1). Inhibition constants for growth and degradation rate value were in the same range. Compared with other pyridine degraders, lmax and Sm obtained for Rhizobium sp.NJUST18 was the highest, which was an advantage for further treatment of pyridine-containing wastewater. High Ki and Ki0 values and extremely high Ks and Ks0 values indicated that Rhizobium sp.NJUST18 was able to grow on pyridine within a wide concentration range, especially at relatively high concentrations.
Acknowldgments This research is financed by Major Project of Water Pollution Control and Management Technology of P. R. China (No. 2012ZX07101-003-001), National Natural Science Foundation of China (No. 51208258, 51378261 and 51348007), Natural Science Foundation of Jiangsu Province (No. BK2011717), Research Fund for the Doctoral Program of Higher Education of China
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Bioprocess Biosyst Eng (20123219120009), Environmental Protection Scientific Research Project of Jiangsu Province (201103), Fundamental Research Funds for the Central Universities (No.30920130122007) and Zijing Intelligent Program (No. 2013-ZJ-02-19).
12.
13.
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