ORIGINAL PAPER

Biodegradation of pyridine raffinate by two bacterial co-culturesof Bacillus cereus (DQ435020) and Alcaligenes faecalis(DQ435021)

Ram Chandra • Sangeeta Yadav •

Ram Naresh Bharagava

Received: 22 July 2009 / Accepted: 19 October 2009 / Published online: 2 November 2009

� Springer Science+Business Media B.V. 2009

Abstract This study deals with the optimization of bac-

terial degradation of pyridine raffinate by previously iso-

lated two aerobic bacteria ITRCEM1 (Bacillus cereus) and

ITRCEM2 (Alcaligens faecalis) with accession number

DQ4335020 and DQ435021, respectively. The degradation

of pyridine raffinate was studied by axenic and mixed

bacterial consortium at different nutritional and environ-

mental conditions after the removal of formaldehyde from

pyridine raffinate (FPPR). Results revealed that the opti-

mum degradation of pyridine raffinate was observed by

mixed bacterial culture in presence of glucose (1% w/v)

and peptone (0.2% w/v) at 20% FPPR, pH 7.0, temperature

30�C and 120 rpm at 168 h incubation period . The HPLC

analysis of degraded pyridine raffinate samples has indi-

cated the complete removal of a, b and c picoline. Further,

the GC–MS analysis of FPPR pyridine raffinate has shown

the presence of pyrazine acetonitrile (6.74), 1,3-dioxepin

(8.68), 2-pyridine carboxaldehyde (11.26), propiolactone

(12.06), 2-butanol (13.10), benzenesulfonic acid (16.22)

and 1,4-dimethyl pyperadine while phenol (17.64) and 3,4-

dimethyl benzaldehyde as metabolic products of FPPR.

Keywords Alcaligenes faecalis � Bacillus cereus �Degradation � GC–MS analysis � Pyridine raffinate

Introduction

Pyridine raffinate is residual reaction mixture after

extraction of pyridine, discharge as pollutants from pyri-

dine manufacturing industries. This contains large quanti-

ties of pyridine, formaldehyde, phenolics, and picolines

with high alkalinity (pH 12.0) and water solubility

(Chandra and Singh 2005). Pyridine is naturally produced

from coal also and widely used as industrial solvent and

raw materials in pharmaceutical, dyes, pesticides, herbi-

cides manufacturer and agrochemical industries. Pyridine

and its derivatives (a, b and c-picoline) have low octanal

water partition coefficient (Kow) (Verschueren 1983) and

high water solubility (Sax and Lewis 1987) due to which

all these act as major environmental pollutants.

The toxicity of pyridine, picoline, formaldehyde and

phenolics has been well documented (Mohammad et al.

1983; Sim et al. 1986; Chandra and Singh 2005). The pH

dependent toxicity of pyridine raffinate for common

duckweed (Lemna minor) has been reported in aquatic

ecosystem due to the interconversion of its constituent

(Chandra and Singh 2005). United States Environmental

Protection Agency (USEPA) has listed pyridine as one of

the major organic pollutants (Richards and Shieth 1986).

Moreover, pyridine is reported to be toxic for several

bacterial species at the concentration of 340 mg l-1 (Ver-

schueren 1977) and due to its toxic nature and nauseous

odor, discharge of pyridine raffinate causes irreversible

damage to human health and environmental quality.

The biological degradation of pollutants is considered as

an environment friendly, feasible technique requiring low

cost and minimum maintenance. It has been reported that

pyridine could be used as the sole source of carbon and

nitrogen during the degradation process by soil microor-

ganisms (Houghton and Cain 1972; Shukla and Kaul 1975;

R. Chandra (&) � S. Yadav � R. N. Bharagava

Environmental Microbiology Section, Indian Institute of

Toxicology Research (CSIR), Post Office Box No. 80,

M. G. Marg, Lucknow, Uttar Pradesh 226 001, India

e-mail: ramchandra_env@indiatimes.com;

rc_microitrc@yahoo.co.in

123

World J Microbiol Biotechnol (2010) 26:685–692

DOI 10.1007/s11274-009-0223-z

Watson and Cain 1975; Korosteleva et al. 1981). It

revealed that microorganisms oxidize pyridine for energy

generation and release nitrogen atom from pyridine ring as

ammonium ions (Sim and Sommers 1985). In addition,

Shiu and Cheng (1997) have reported the anaerobic bio-

transformation of pyridine in estuarine sediments and

metabolism of pyridine is initiated either by ring reduction

or ring hydroxylation (Holenberg and Stadtman 1969;

Watson and Cain 1975). Most of the aerobic biodegrada-

tion involves the general hydroxylation steps followed by

dioxygenolytic cleavage of the heteroaromatic ring prior to

cleavage (Fetzner 1998).

However, the inhibitory effect of phenol for pyridine

degradation is reported (Kim et al. 2006). But, the bacterial

degradation of pyridine raffinate is not reported so far due

to the high content of formaldehyde, phenol, pyridine and

picoline in mixed conditions. The microbial degradation of

pyridine raffinate in aerobic conditions could be a better

approach for its safe disposal into the environment.

Recently, we isolated and identified two aerobic bacterial

strains Bacillus cereus (DQ 435020) and Alcaligenes fae-

calis (DQ435021), high capability for pyridine degradation

in presence of picoline, phenol and formaldehyde in mixed

condition (Chandra et al. 2009). Hence, the objectives of

this study were to optimize degradation of pyridine raffi-

nate by these strains and to characterize its metabolic

products by HPLC and GC–MS–MS analysis for safe

disposal.

Materials and methods

Collection of pyridine raffinate sample

The pyridine raffinate was collected aseptically in plastic

containers (Capacity 25 l) from M/S Jubilant Organosys

Ltd, Gajraula (UP), India. The freshly collected pyridine

raffinate samples were transparent pale with pungent smell

of formaldehyde, phenol, picoline, and pyridine.

Physico-chemical analysis of pyridine raffinate

The freshly collected pyridine raffinate was highly alkaline

in nature due to presence of complex residual mixture of

phenolic, formaldehyde and pyridine. To investigate the

status of different constituents of pyridine raffinate at dif-

ferent pH, the physico-chemical parameters of pyridine

raffinate were analyzed at pH 4.0, 5.0, 6.0, 7.0, 9.0 and 12.0

as per standard methods for water and wastewater analysis

(APHA 2005). The different pH of raffinate was main-

tained by addition of 5.66 N HCl. The biological oxygen

demand was done by 5 days test, chemical oxygen demand

by open reflux method, total nitrogen (Micro kjeldahl),

phenol (chloroform extraction method), sulfate (Gravi-

metric method), color (visual color comparison method),

total solid, total suspended solid, total dissolved solids as

per methods specified by APHA (2005). Whereas, ammo-

nium, sodium, potassium, nitrate, chloride was done with

ion meter by their respective electrode (Ion meter, Orion

960). The pyridine and formaldehyde contents were

determined by colorimetric method (Nash 1953) as well as

by HPLC analysis (Waters 515 model, Equipped with UV-

Vis detector, 2487, Milford, USA). Heavy metals (Fe, Cr,

Zn, Cu, Cd, Ni and Pb) were analyzed by Inductively

Coupled Plasma spectrophotometer (ICP, model-8440,

Plasma Lab, Australia).

Formaldehyde removal from pyridine raffinate

The formaldehyde is reported to have inhibitory effect on

microbial growth. Hence the formaldehyde was removed at

optimized conditions from pyridine raffinate before bacte-

rial treatment (data under patent filing). This formaldehyde

pretreated pyridine raffinate was designated as FPPR.

FPPR at pH 7 was favorable for bacterial growth hence this

sample was optimized for bacterial degradation of pyridine

raffinate and its constitute.

Optimization of bacterial growth at different FPPR

concentration, temperature and shaking rate

The minimal medium (100 ml) containing glucose 1.0%

and peptone 0.2% in 250 ml flasks was sterilized by

autoclaving at 121�C for 15 min. After cooling at room

temperature, different concentrations 15, 20, and 25% (v/v)

of FPPR was added aseptically after sterilization by Mil-

lipore membrane filtration with 0.22 lm pores to this

minimal medium. The flasks were inoculated with 1% (v/v)

of previously isolated bacteria ITRCEM1 (B. cereus; DQ

435020) and ITRCEM2 (A. faecalis; DQ 435021) in axenic

and mixed condition, incubated at different temperature

(27, 32, 37 and 42�C) and rpm (100, 120 and 150) in

temperature controlled shaking incubator (New Brunswick

Innova 4230, USA). During the bacterial degradation of

FPPR, the bacterial growth was monitored spectrophoto-

metrically (GBC Cintra-40, Australia) at 620 nm. The

reduction in formaldehyde, picoline, and pyridine content

during the bacterial degradation was measured at regular

interval (24 h). The ammonium and nitrate ions content

were also measured from culture media at every 24 h by

using Ion meter (Orion 960).

Analysis of bacterial degraded FPPR

The biodegradation of pyridine raffinate was measured in

terms of loss of pyridine (mineralization) from aqueous

686 World J Microbiol Biotechnol (2010) 26:685–692

123

phase suspension samples at periodic intervals. The bacte-

rial biomass was separated by centrifugation at 5000 rpm

for 5 min at 4�C and the pyridine content present in aqueous

phase was determined colorimetrically at 450 nm. An ali-

quot of a suspension containing 0.5 ml of NaOH (0.2% w/

v), 5 ml of distilled water, 5 ml of cyanogen bromide (10%

w/v) and 2.0 ml of sulphanilic acid (10% w/v) in 10% (v/v)

NH4OH, which was adjusted to pH 4.5 with 0.1 N HCl. The

color intensity of this suspension was measured at 450 nm

using a blank reagent (Mohan et al. 2003).

The pyridine degradation was also confirmed by HPLC

(waters 515 HPLC pump equipped with UV-Vis-2487,

detector) analysis. The culture supernatant obtained after

centrifugation at 5,000 rpm for 5 min at 4�C was passed

through anhydrous sodium sulfate (Na2SO4) to remove the

excess of water from samples and 20 ll of this concentrated

sample was injected into HPLC and the wavelength was set

at 254 nm to monitor the degradation of pyridine raffinate.

The column used in this study was Lichrospher-100 RP-18

(size 250 9 4 mm); Merck with particle size 5 lm and the

mobile phase consisted of acetonitrile and water (70:30, v/

v) with flow rate of 1 ml min-1. The pyridine and picoline

(a, b and c) standards were run at the same conditions

(Chandra et al. 2009). The concentration of pyridine and

picoline was calculated by using the following formula:

ðAreaof sample

� Concentration of standardÞ=Area of standard:

Metabolite characterization by GC–MS analysis

For the GC–MS analysis, the culture supernatant obtained

after centrifugation at 5000 rpm for 5 min at 4�C was

extracted thrice with ethyl acetate at pH 7.0 to get the

residual pyridine. The upper organic layer was taken and

dried over anhydrous sodium sulfate prior to GC–MS

analysis. An aliquot of 2 ll was injected into the injector

port of the GC–MS, which was equipped with a PE auto

system XL gas chromatograph interfaced with Turbo mass

selective selector. The analytical column connected to the

system was a PE-624 capillary column (30 m 9 0.25 mm

IDX 1.4 lm film thickness) internal diameter 0.18 lm film

thickness. Helium gas was used as carrier gas with flow

rate 1 ml min-1. The column temperature was set to 50�C

(5 min); 50–250�C (10�C min-1 hold time; 5 min). The

injector temperature was maintained at 250�C and the

transfer line and ion source temperature was maintained at

200 and 250�C, respectively. A solvent delay of 3 min was

selected. In the full scan mode, electron ionization mass

spectra in the range of 30–500 (m/z) were recorded at

electron energy of 70 eV. The identification of different

pyridine intermediate and residual compounds in media

was done by comparing their mass spectra with that of the

mass spectra available in NIST library provided with the

instrument and also by comparing the retention time with

those of authentic compounds reported in literature.

Results

Physico-chemical characteristics of pyridine raffinate at

different pH

The physico-chemical analysis of control and bacteria

treated pyridine raffinate samples at different pH (4.0, 5.0,

6.0, 7.0, 9.0, and 12.0) has indicated the presence of sig-

nificant amount of formaldehyde, pyridine, picoline (a, band c) and phenolics (Tables 1, 2). The change in pH from

basic to acid lead to a decrease in total nitrogen, nitrate,

chemical oxygen demand (COD), biological oxygen

demand (BOD) and total phenol content compared to ori-

ginal pyridine raffinate at pH 12. Some heavy metals were

also detected in pyridine raffinate where iron (Fe) was in

higher concentration followed by Zn, Cu and Cr (Table 1).

This might be generated from the metallic reactor vessel

during the downstream harvesting of pyridine. On the other

hand, potassium, sodium, sulfate, total solids (TS), total

dissolve solids (TDS), total suspended solids (TSS) and

color showed sharp increase as pH decreases. Physico-

chemical analysis revealed that increase in BOD, COD

with increase of pH, due to pH dependent increase solu-

bility of organic compounds. The harmful effects of pyri-

dine raffinate at higher pH (12) might be due to high BOD,

COD and phenolics. Similar observation has been also

noted earlier (Chandra and Singh 2005).

Bacterial degradation of FPPR at different

environmental conditions

The bacterial degradation of pyridine raffinate at different

concentration, pH, temperature and rpm has shown the

consistent growth upto 168 h and further incubation sup-

ported neither the bacterial growth nor pyridine degrada-

tion. Interestingly, it was observed that the growth of

individual strain B. cereus and A. faecalis in FPPR was

slow compared to mixed culture. It revealed that mixed

culture was more effective for pyridine raffinate degrada-

tion compared to individual strain of B. cereus and

A. faecalis. The initial low bacterial growth rate upto 48 h

in medium may be attributed to the substrate inhibition, i.e.

pyridine, picoline and phenol (Kim et al. 2006; Bai et al.

2009) and subsequently, the acclimatization has enhanced

the bacterial growth by metabolizing the constituents

present in pyridine raffinate.

In addition, during the pyridine degradation by axenic

culture, picoline and phenol present in FPPR act as

World J Microbiol Biotechnol (2010) 26:685–692 687

123

potential inhibitor for bacterial growth as well as for pyr-

idine degradation. But, when the mixed culture of Bacillus

and Alcaligenes was added to FPPR, the pyridine degra-

dation by this mixed culture was higher than by axenic

culture. This indicated that the potential inhibitory effect of

picoline and phenol was alleviated by this mixed bacterial

culture in FPPR. This might be due to the utilization of

metabolic product of one bacterial strain by another (Bai

et al. 2009). Consequently, the maximum biomass pro-

duction was noted in mixed bacterial culture (Fig. 1a). The

optimum growth of bacterial culture was observed at 20%

FPPR (Fig. 1b) at pH 7, temperature 37�C and 120 rpm in

mixed condition (Table 3). This showed complete degra-

dation of pyridine and picoline (a, b and c) from FPPR at

pH 7.0 after 168 h incubation period (Fig. 2a). Therefore,

it was not detectable during analysis (Table 2). While

bacterial culture showed prolonged lag phase in 25% FPPR

at same growth condition. This indicated substrate inhibi-

tion of bacterial culture at higher concentration of FPPR

(Fig. 1b). Simultaneously, it was also observed that the

nitrate content decreases with increase in ammonium

content in medium during the course of bacterial growth

and pyridine raffinate degradation (Fig. 2b). These findings

corroborated with previous observations (Rhee et al. 1997;

Ronen et al. 1998).

HPLC analysis

The HPLC analysis of 20% FPPR has shown the retention

time (RT) of pyridine, a, b and c picoline at 3.34, 4.11,

5.01 and 5.77 min, respectively, when compared with their

respective standard chromatogram. Further, analysis in

bacterial treated 20% FPPR showed disappearance of all

peaks. This indicated complete degradation of pyridine and

picoline (a, b and c) from FPPR after 168 h.

GC–MS analysis of residual pyridine from degraded

FPPR

The degradation of FPPR and generation of metabolic

products was confirmed by GC–MS analysis after 168 h

bacterial treatment (Table 4). The total ion chromatogram

Table 1 Physico-chemical characteristics of pyridine raffinate at different pH

Parameters Pyridine raffinate at different pH

4.0 5.0 6.0 7.0 9.0 12.0

Total nitrogen 41410 ± 953 53480 ± 1069 66080 ± 848 81480 ± 940 80030 ± 860 84280 ± 1069

Nitrate 286 ± 10.01 312 ± 8.73 370 ± 18.50 369 ± 7.75 425 ± 9.12 502 ± 8.69

Ammonium 378 ± 8.16 352 ± 6.61 298 ± 12.51 302 ± 9.02 350 ± 7.13 367 ± 6.90

Potassium 1870 ± 50.49 1960 ± 73.30 1731 ± 48.21 1645 ± 38.19 980 ± 39.20 86 ± 2.60

Sodium 100 ± 3.86 280 ± 4.90 233 ± 4.89 210 ± 3.99 50 ± 1.05 30 ± 0.99

Chloride 177 ± 4.34 1135 ± 54.48 1178 ± 47.82 1110 ± 41.63 976 ± 18.06 1350 ± 28.35

Total phenol 388 ± 10.51 400 ± 9.88 432 ± 7.32 488 ± 5.55 505 ± 8.70 598 ± 16.19

Sulfate 22814 ± 342 10182 ± 109 NA 9261 ± 89.53 7889 ± 81.53 2630 ± 72.90

TS 280970 ± 1072 319841 ± 1516 292830 ± 1617 184190 ± 2131 154650 ± 1992 21640 ± 557

TDS 216120 ± 2890 205120 ± 4332 199876 ± 3881 179210 ± 3769 137880 ± 2426 14024 ± 307

TSS 64850 ± 1861 114721 ± 1031 92960 ± 1976 4980 ± 76.69 16770 ± 637 7616 ± 282

COD 356000 ± 4984 384000 ± 4100 398000 ± 6145 428000 ± 5312 468000 ± 1012 524000 ± 14115

BOD 178000 ± 5212 199000 ± 6632 205000 ± 4169 214000 ± 3813 220000 ± 4221 222000 ± 3630

Color* 13500 ± 370 13500 ± 360 13500 ± 340 12600 ± 162 10500 ± 210 6000 ± 180

Formaldehyde 400 ± 12.65 400 ± 11.42 410 ± 13.85 441 ± 12.84 440 ± 16.52 450 ± 18.85

Pyridine 4615 ± 128 4615 ± 137 4612 ± 140 4617 ± 125 4734 ± 112 4828 ± 138

Picoline a 4200 ± 165 4290 ± 136 4270 ± 105 4270 ± 133 4280 ± 111 4325 ± 121

Picoline b 4100 ± 182 4170 ± 180 4180 ± 134 4188 ± 108 4185 ± 102 4200 ± 86

Picoline c 3000 ± 110 2900 ± 46 2900 ± 66 2800 ± 68 2900 ± 88 3100 ± 102

Fe 6.80 ± 0.006 6.63 ± 0.007 6.48 ± 0.005 6.25 ± 0.005 6.07 ± 0.010 6.55 ± 0.010

Cr 0.08 ± 0.000 0.10 ± 0.001 0.11 ± 0.001 0.12 ± 0.001 0.15 ± 0.001 0.20 ± 0.001

Zn 0.12 ± 0.000 0.15 ± 0.000 0.12 ± 0.001 0.09 ± 0.000 0.08 ± 0.000 0.11 ± 0.001

Cu 0.11 ± 0.001 0.11 ± 0.000 0.11 ± 0.000 0.07 ± 0.001 0.09 ± 0.001 0.12 ± 0.000

Cd, Ni and Pb ND ND ND ND ND ND

All values are mean (n = 3) ± SD in mg l-1 except *color (Co–Pt unit)

ND not detectable

688 World J Microbiol Biotechnol (2010) 26:685–692

123

(TIC) (Fig. 3a, b) corresponding to the metabolic products

has shown the presence of pyrazine acetonitrile (6.74), 1,3-

dioxepin (8.68), 2-pyridine carboxaldehyde (11.26), pro-

piolactone (12.06), 2-butanol (13.10), benzenesulfonic acid

(16.22) and 1,4-dimethyl pyperadine (17.64) as new met-

abolic products present in pyridine raffinate while all these

compounds disappeared in bacterial degraded FPPR

(Table 3). The traces of only phenol (16.90) and

Table 2 Physico-chemical characteristics and pyridine and picoline contents in pyridine raffinate after formaldehyde removal and bacterial

degradation

Parameters (mg l-1) Pyridine raffinate

(pH 7.0)

FPPR (pH 7.0) 20% FPPR

(pH 7.0)

Bacterial treated

FPPR (pH 7.0)

Compliance with

CPCB and EPA

Total nitrogen 81480 ± 940 29400 ± 299 5880 ± 109 110 ± 4.28 Yes

Nitrate 369 ± 7.75 198 ± 2.03 120 ± 0.86 0.32 ± 0.01 Yes

Ammonium 302 ± 9.02 234 ± 8.93 220 ± 1.93 0.41 ± 0.10 Yes

Potassium 1645 ± 38.19 11.56 ± 1.53 3.50 ± 0.53 0.35 ± 0.10 Yes

Sodium 210 ± 3.99 7.68 ± 0.12 1.40 ± 0.08 0.10 ± 0.01 Yes

Chloride 1110 ± 41.63 74.85 ± 0.43 15.90 ± 0.43 0.80 ± 0.17 Yes

Total phenol 488 ± 5.55 377 ± 12.13 76.40 ± 1.25 0.68 ± 0.03 Yes

Sulfate 9261 ± 89.53 8561 ± 162 1712 ± 62.31 1.65 ± 0.05 Yes

TS 184190 ± 2131 13070 ± 107 2614 ± 37.45 375 ± 32.54 Yes

TDS 179210 ± 3769 12021 ± 280 2404 ± 80.12 210 ± 10.69 Yes

TSS 4980 ± 76.69 1049 ± 31.83 210 ± 8.33 149 ± 4.80 Yes

COD 428000 ± 5312 142000 ± 1697 28400 ± 697 240 ± 8.53 Yes

BOD 214000 ± 3813 70000 ± 1400 1400 ± 32.85 70 ± 3.25 Yes

Color* 12600 ± 162 9000 ± 168 1800 ± 68.35 80 ± 4.57 Yes

Formaldehyde 441 ± 12.84 ND ND ND Yes

Pyridine 4617 ± 125 4600 ± 55.55 920 ± 11.24 ND Yes

Picoline a 4270 ± 133 4200 ± 65.80 840 ± 25.80 ND Yes

Picoline b 4188 ± 108 4100 ± 80.00 820 ± 18.00 ND Yes

Picoline c 2800 ± 68 2735 ± 86.35 560 ± 6.35 ND Yes

Heavy metals

Fe 6.25 ± 0.005 6.10 ± 0.10 5.10 ± 0.01 0.10 ± 0.01 Yes

Cr 0.12 ± 0.001 0.12 ± 0.01 0.11 ± 0.01 0.02 ± 0.01 Yes

Zn 0.09 ± 0.000 0.09 ± 0.001 0.07 ± 0.001 ND Yes

Cu 0.07 ± 0.001 0.07 ± 0.001 0.05 ± 0.001 ND Yes

Cd, Ni and Pb ND ND ND ND Yes

All values are mean (n = 3) ± SD in mg l-1 except *color (Co–Pt)

ND not detectable, FPPR formaldehyde pretreated pyridine raffinate

(a)

0

20

40

60

80

100

120

140

0 24 48 72 96 120 144 168

Incubation Time (h)0 24 48 72 96 120 144 168

Incubation Time (h)

Bio

mas

s (m

g l-1

)

ITRC EM-1

ITRC EM-2

Mixed

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Opt

ical

Den

sity

(62

0nm

)

20% FPPR

15% FPPR

25% FPPR

(b)Fig. 1 Bacterial growth during

the pyridine raffinate

degradation (a) and effect of

different concentration of FPPR

on growth of mixed bacterial

culture (b). FPPR formaldehyde

pretreated pyridine raffinate

World J Microbiol Biotechnol (2010) 26:685–692 689

123

3,4-dimethyl benzaldehyde (22.56) were detected as new

metabolites constituently two rudimentary peaks were only

visible. This study revealed that the developed bacterial

co-culture was capable to degrade pyridine raffinate uti-

lizing its constituent as sole source of carbon, nitrogen and

energy. The GC–MS analysis showed the disappearance of

peaks in bacterial treated FPPR, which might be due to the

biodegradation of compounds resulting in the removal of

various organic constituents. Phenol and 3,4-dimethyl

benzaldehyde were detected as persistent organic com-

pounds in bacterial treated FPPR (Table 4).

Conclusion

In this study, the bacterial co-culture was found more

effective compared to axenic bacterial culture of Bacillus

cereus (DQ435020) and Alcaligenes faecalis (DQ435021)

for the degradation of pyridine raffinate. The maximum

degradation of pyridine raffinate by bacterial co-culture was

observed in presence of glucose (1%) and peptone (0.2%) at

20% formaldehyde pre-treated pyridine raffinate (FPPR).

The HPLC analysis of degraded pyridine raffinate sample

has shown the complete removal of a, b and c picoline.

Further, the GC–MS analysis of degraded pyridine raffinate

sample has indicated the presence of phenol and 3,4-dime-

thyl benzaldehyde as persistent metabolites produced during

the bacterial degradation of FPPR and other compounds

Table 3 Optimized FPPR concentration and environmental condi-

tions for FPPR degradation by mixed bacterial culture

Sr. No. Parameters Optimized

1. FPPR concentration 20%

2. pH 7

3. Temperature 37�C

4. Shaking rate 120 rpm

FPPR formaldehyde pretreated pyridine raffinate

0

100

200

300

400

500

600

700

800

900

1000

0 24 48 72 96 120 144 168

Incubation Time (h)0 24 48 72 96 120 144 168

Incubation Time (h)

Pyri

dine

& P

icol

ine

(mg

l-1)

Pyridine (C) Alpha Picoline (C)Beta Picoline (C) Gamma Picoline (C)Pyridine (D) Alpha Picoline (D)Beta Picoline (D) Gamma Picoline (D)

0

50

100

150

200

250

300

350

m

g l-1

Ammonium

Nitrate

(a)

(b) Fig. 2 Pyridine, picoline (a, band c) degradation (a) and

release of ammonium and

nitrate ions (b) in bacterial

treated 20% FPPR. FPPRformaldehyde pretreated

pyridine raffinate, C control, Ddegraded FPPR

Table 4 Compounds identified

in control and bacterial treated

FPPR

a Confirmed by match of

retention time (RT) with known

standards

Identified compounds a RT (min) Fig 3 (a) Fig 3 (b)

Pyrazine acetonitrile 6.74 + -

1,3-dioxepin 8.68 + -

2-pyridine carboxaldehyde 11.26 + -

Propiolactone 12.06 + -

2-butanol 13.10 + -

Benzenesulfonic acid 16.22 + -

1,4-dimethyl pyperadine 17.64 + -

Phenol 16.90 - +

3,4-dimethyl benzaldehyde 22.56 - +Metabolites

Compounds in pyridine raffinate

690 World J Microbiol Biotechnol (2010) 26:685–692

123

present in raffinate were degraded. Hence, it is concluded

that the developed bacterial co-culture was capable to

degrade pyridine raffinate utilizing its constituents as sole

source of carbon, nitrogen and energy.

Acknowledgments We are grateful to Department of Biotechnol-

ogy (DBT), New Delhi and Council for Scientific Industrial Research

under SIP08, New Delhi for their financial assistance.

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(b)

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Time

0

100

%

RB2-22-161106 Scan EI+ TIC

1.93e411.26

6.74

6.26

8.68

8.42

10.62

13.10

12.06

17.64

16.22

14.74

13.7614.96

17.34

24.3621.40

20.10

18.50

22.20

(a)

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Time

0

100

%

std Pyb17-131106 Scan EI+ TIC

1.06e7

16.90

22.56

0

100

%

25 50 75 1000

100

%

94

6539

38 40

66

748495105

94

66393827

6567

93 95

R:924 Nist 1

0

100

%

20 40 60 80 100 120 1400

100

%

133

10577323940 5063 103 106119

134

135

133

105

773927 51 63 9198

106115

135

R:916 Nist 6182: BENZALD

(I) (II)

Fig. 3 Total ion chromatogram

of control (a) and degraded

FPPR (b) by mixed bacterial

culture. FPPR: formaldehyde

pretreated pyridine raffinate; Iand II: Nist library matched

chromatogram

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