900

Journal of Scientific & Industrial Research J SCI IND RES VOL 68 OCTOBER 2009
Vol. 68, October 2009, pp. 900-903

Biodegradation kinetics of benzoic and anthranilic acids by Micrococcus sp.

Karuppan Muthukumar*, Chakravarthy Bharath, Velan Pugalenthi and Manickam Velan
Department of Chemical Engineering, Anna University, Chennai 600 025, India

Received 17 July 2007; revised 07 July 2009; accepted 15 July 2009

This study presents biodegradation of benzoic acid (BA) and anthranilic acid (AA) by Micrococcus sp. under
aerobic conditions. Initial concentrations of BA and AA were varied (500-2500 mg/l) and almost complete biodegradation of
BA and AA was observed within 30 h. Andrews kinetic model for single substrate was fitted to obtain maximum specific
growth rates, half saturation and substrate inhibition constants. Cell growth with degrading BA (µm,BA = 0.645 h-1) was faster
than with degrading AA (µm,AA = 0.63 h-1). Under biodegradation of BA and AA, first order rate constant values decreased with
increase in initial concentration.

Keywords: Andrews model, Anthranilic acid, Benzoic acid, Biodegradation, Micrococcus sp.

Introduction
Biological degradation of toxic organic compounds
offer advantages than other methods that are cost
intensive and end up with secondary pollutants1,2. Waste-
water from some industries consists of benzoic acid (BA)
and its derivatives. Benzoate is an intermediate during
biodegradation of aromatics and recently has been
reported as an intermediate of anaerobic biodegrada-
tion of benzene 3,4. Biodegradation of benzoate by
Pseudomonas putida and its degradation pathway has
been reported5. Nelson et al6 reported biodegradation
of benzoate by Burkholderia cepacia G4. Biodegra-
dation of benzoate using P. cepacia occurred through
ortho and meta pathways, induced simultaneously3.
Genetically modified microorganism also employed for
degradation of chloro and methyl substituted BAs and
compounds were metabolized by ortho cleavage route7.
Degradation of BA in a three phase fluidized bed reac-
tor has been reported8. Chlorobenzoates were degraded
effectively by Burkholderia sp. and Bradyrhizobium
sp9. Ajithkumar & Kunhi10 reported that P. aeruginosa
3mT degrade high concentrations of 3-chlorobenzoate
and 4-chlorobenzoate and degraded 4-CBA (> 99%)
through formation of respective chlorocatechol, via a
modified ortho-pathway. Miguez et al 11 reported
mechanism of uptake of BA and 2,4-dichlorobenzoic
*Author for correspondence
E-mail: m_kumar77@yahoo.co.in; muthukumar@annauniv.edu
acid by Alcaligenes denitrificans BRI 3010, BRI 6011
and Pseudomonas sp. strain B13 and these organisms
were capable of degrading various isomers of chlorinated
benzoic acids. David et al12 reported that Enterobacter
aerogenes, Raoultella ornithinolytica,
Stenotrophomonas maltophila, Xanthomonas
campestris and Stenotrophomonas acidaminophila
degrade BA (up to 9 mM) under anoxic conditions with
nitrate as electron acceptor.
Many of nitro- and amino aromatics, produced in large
amounts by chemical industries, are toxic to
environment. Anthranilic acid (AA) is reported as an
intermediate during biodegradation of o-nitrobenzoate and
carbazole13,14. Mineralization of AA as a sole carbon and
energy source using methanogenic granular sludge was
studied and treatment was carried out in batch and in an
anaerobic expanded granular sludge bed reactor at low
concentrations under anaerobic conditions15. AA has been
completely oxidized to CO2 and NH 4+ by anaerobic
degradation with two strains of denitrifying bacteria of
Pseudomonas 16. Sludge isolated from wastewater
treatment plant has been successfully used for biodegra-
dation of 2, 3 and 4-iodobenzoic acids17. Biodegradation
of halogenated BAs has also been reported18-20.
This study presents feasibility of BA and AA
biodegradation at higher initial concentrations and studies
growth kinetics using Andrew’s model.
 MUTHUKUMAR et al: BIODEGRADATION OF BENZOIC AND ANTHRANILIC ACIDS BY MICROCOCCUS SP. 901

5000
Materials and Methods
Culture Media Preparation a) 5 0 0 m g /l
21 1 0 0 0 m g /l
Mineral salt medium (MSM) (pH 7) contained: 4000
1 5 0 0 m g /l
NH4NO3, 1; (NH4)2SO4, 0.5; NaCl, 0.5; MgSO4, 0.5; 2 0 0 0 m g /l

COD, mg/l
2 5 0 0 m g /l
K2HPO4, 1.5; KH2PO4, 0.5; FeSO4, 0.01; and CaCl2, 3000

0.01 g/l.
2000

Microorganism Growth Conditions

Microorganism utilizing BA and AA as a sole source 1000

of carbon was isolated from petroleum refinery

environment by enrichment culture in MSM containing 0
0 5 10 15 20 25 30
200 mg/l of BA and AA. Enrichments were incubated
t, h )
for 5 days at 30°C on rotary shaker operated at 180 4000
rpm, and portions of enrichments were then transferred
500 mg/l
to fresh medium. After five 58 h interval transfers, b)
1000 mg/l
culture was streaked on plates containing solidified agar 3000
containing MSM. A pure culture of isolate was 1500 mg/l
maintained in refrigerator at 4°C. Isolated bacterium, 2000 mg/l

COD, mg/l
identified as Micrococcus sp., degraded BA and AA 2500 mg/l
2000
effectively.
Degradation of Benzoic Acid (BA) and Anthranilic Acid (AA)
1000
Free cell suspension (3 ml) was inoculated into an
Erlenmeyer flask (250 ml) containing sterile MSM
(97 ml) and agitated and aerated in a rotary shaker at
0
30°C and 180 rpm. Initial BA and AA concentrations 0 5 10 15 20 25 30
(200-2500 mg/l) were used in batch experiments carried
t, h
out at pH 7.
Fig. 1— Biodegradation at different initial concentrations by
Micrococcus sp. of: a) BA; and b) AA
Cell Density Measurements and COD Determination
Bacteria concentrations were determined by optical
density measurement at 610 nm using a UV-VIS digital required 29 h. Micrococcus sp. was found more efficient
spectrophotometer and correlated to biomass in degrading BA than AA.
concentration. BA and AA concentrations were Kinetic Studies
assessed in terms of chemical oxygen demand (COD), First order kinetics generally has following form:
which was determined by APHA method. lnC = -Kt + A. …(1)
where C, initial concentration of BA or AA in terms of
Results and Discussion
Biodegradation of Benzoic Acid (BA) and Anthranilic Acid (AA) COD (mg/l); K, first order kinetic constant, h-1; t, time in
With increase in incubation time, COD decreased s; and A, constant. Half-life of first order reaction of BA
(Fig. 1a) and complete destruction of BA (500 mg/l) was and AA by Micrococcus sp. can be obtained as
achieved within 10 h, whereas 24 h required for
ln 2
destruction of BA at 2500 mg/l. Micrococcus sp. utilized t1 / 2 = …(2)
K
BA as a sole source of carbon with faster degradation
rate. As initial concentration increases, time required for Experimental data were fitted with Eqs (1) and (2)
complete mineralization also increases due to increase and kinetic equations obtained for BA and AA
in organic load. biodegradation (Table 1) gave a better fit for first order
Initial COD increased with an increase in initial model. Increase in initial concentrations of BA and AA
concentration of AA and decreased with an increase in significantly decreased first-order rate coefficient (K),
incubation time (Fig. 1b). Complete destruction of AA indicating dependence of biodegradation rate on initial
(500 mg/l) was in 12.5 h, whereas AA (2500 mg/l) concentration of substrate employed.
902 J SCI IND RES VOL 68 OCTOBER 2009

Growth Kinetics 0.7

Among substrate inhibition models, Andrews kinetic a)
0.6
model is most widely used22,23 . Gouder et al24 reported
that Andrews model also acts as a good representation 0.5
of experimental data. Andrews model25 describing
substrate inhibition kinetics of a single substrate is given 0.4

µ, h-1
as 0.3 Mod el d ata
Ex per ime ntal d ata
1 dX µmS 0.2
µ= = …(3)
X dt K s + S + S 2 /K I 0.1

0
where S is substrate concentration (mg/l); t, time (s); ¼ 0 50 0 10 00 15 00 20 00 25 00 30 00
specific growth rate (h-1); X, biomass concentration (g); BA concentration, mg dm
) -3

µm, maximum specific growth rate (h-1); Ks, half saturation

constant (mg/l) and KI is substrate inhibition constant 0.7
b)
(mg/l). Constants were evaluated by using nonlinear least
0.6
squares technique. Biomass concentrations were
measured with time for different substrate concentrations
0.5
(200-2500 mg/l) of BA and AA. Specific growth rate,
µ, for various initial concentrations of BA and AA was
µ, h-1
0.4
determined using semi logarithmic plots of biomass versus
time. Using specific growth rates obtained, Andrews 0.3
kinetic model for single substrate was fitted and relevant
plots drawn (Fig .2). Model equations and their regression 0.2
M o d e l d a ta
coefficients are given as E xp e ri m e n ta l d a ta
0.1
0 . 65 S 2
For BA µ = ( R = 0 . 99 )
11 + S + S 2 /12500 0
0 500 1000 1500 2000 2500 3000

AA concentration,) mg l -1
0 .63 S
For AA µ = ( R 2 = 0 .98 )
5 + S + S 2 /4000 Fig. 2 — Specific growth rate of Micrococcus sp. on: a) BA;
and b) AA

Table 1 — Kinetic equations for biodegradation of BA and AA by Micrococcus Sp

 MUTHUKUMAR et al: BIODEGRADATION OF BENZOIC AND ANTHRANILIC ACIDS BY MICROCOCCUS SP.
Maximum ¼ values obtained for BA (0.65 h-1) and
AA (0.63 h-1) were higher than those reported26 for µ max
(0.27 h -1 ) of Pseudomonas sp. during aerobic
biodegradation of phenol at 30°C. Aerobic shake flask
biodegradation of phenol24 by microorganism gave µ max
at 0.251 h-1 . KS values (11 mg/l for BA and 5 mg/l for
AA) indicate that microorganism has ability to grow at
lower concentration of substrates. KI values for BA
(12500 mg/l) and AA (4000 mg/l) illustrate that inhibition
is significant only at higher concentrations.
Conclusions
BA and AA can be degraded effectively by
Micrococcus sp. Increase in initial concentration
increased time required for complete degradation. Also,
biodegradation kinetics described by first-order reaction
model indicated that rate of degradation depends on initial
concentration of substrate. Microorganism growth
kinetics was described by Andrews kinetics model and
predicted microorganism growth well on BA and AA.
Also, BA was found better substrate for Micrococcus
sp. than AA.
References
1 Singleton I, Microbial metabolism of xenobiotics: fundamental
and applied research, J Chem Technol Biotechnol, 59 (1994) 9-
23.
2 Grady C P L, Biodegradation: its measurement and microbio-
logical basis, Biotechnol Bioengg, 27 (1985) 660-674.
3 Hamzah R Y & Al-Baharna B S, Catechol ring cleavage in
Pseudomonas cepacia: the simultaneous induction of ortho and
meta pathways, Appl Microbiol Biotechnol, 41 (1994) 250-256.
4 Caldwel M.E & Suflita J M, Detection of phenol and benzoate
as intermediates of anaerobic benzene biodegradation under dif-
ferent terminal electron-accepting conditions, Environ Sci
Technol, 34 (2000) 1216-1220.
5 Loh K C & Chua S S, Ortho pathway of benzoate degradation in
Pseudomonas putida: induction of meta pathway at high sub-
strate concentrations, Enzym Microb Technol, 30 (2002) 620-
626.
6 Nelson M J K, Montgomery S O, Mahaffey W R & Pritchard P
H, Biodegradation of trichloroethylene and involvement of an
aromatic biodegradative pathway, Appl Environ Microbial, 53
(1987) 949-954.
7 Muller R H, Deckwer W D & Hecht.V, Degradation of chloro
and methyl substituted benzoic acids by a genetically modified
microorganism, Biotech Bioeng, 51 (1996) 528-537.
8 Hecht V, Langer O & Deckwer W D, Degradation of phenol and
benzoic acid in a three phase fluidized bed reactor, Biotechnol
Bioeng, 70 (2000) 391-399.
9 Chatterjee D K, Kellogg S T, Hamada S, & Chakrabarty A M,
Plasmid specifying total degradation of 3-chlorobenzoate by a
modified ortho pathway, J Bacteriol, 146 (1981) 639-46.
903
10 Ajithkumar P V & Kunhi A A, Pathways for 3-chloro- and 4-
chlorobenzoate degradation in Pseudomonas aeruginosa 3mT,
Biodegrad, 11 (2000) 247-261.
11 Miguez C B, Greer C W, Ingram J M & MacLeod R A, Uptake
of Benzoic Acid and chloro-substituted benzoic acids by Alcali-
genes denitrificans BRI 3010 and BRI 6011, Appl Environ
Microbiol., 61 (1995) 4152-4159.
12 David K N, Muniru K T & Hamadi I B, Benzoic acid-degrading
bacteria from the intestinal tract of Macrotermes michaelseni
Sjostedt, J Basic Microbiol, 47 (2007) 87-92.
13 Chauhan A & Jain R K, Degradation of o-nitrobenzoate via
anthranilic acid (o-aminobenzoate) by Arthrobacter
protophormiae: a plasmid-encoded new pathway, Biochem
Biophys Res Commun, 267 (2000) 236-241.
14 Kasuga K, Nojri H, Hisakazu Y & Omori J, Genes of enzymes
involved in the biodegradation of carbazole, dibenzofuran, fluo-
rene and dibenzo-p-dioxin by bacteria, Wat Sci Technol, 36 (1997)
9-16.
15 Flores E R, Smulders P, Boldu F P, Lettinga G & Field J A,
Treatment of anthranilic acid in an anaerobic expanded granular
sludge bed reactor at low concentrations, Wat Sci Technol, 40
(1999) 187-194.
16 Braun k & Gibson D T, Anaerobic degradation of 2-
aminobenzoate (Anthranilic acid) by denitrifying bacteria, Appl
Env Microbiol, 48 (1984) 102-107.
17 Santos L M F, New A & Kingswood M, Biodegradation of 2, 3-
and 4-iodobenzoic acids, J Chem Technol Biotechnol, 74 (1999)
815-820.
18 Higson F & Focht D, Degradation of 2-bromobenzoic acid by a
strain of Pseudomonas aeruginosa, Appl Environ Microbiol, 56
(1990) 1615-1619.
19 Van den Tweel Will J J, Kok J B & de Bont J A M, Reductive
dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and
hydrolytic dehalogenation of 4-chloro-, 4-bromo-, and 4-
iodobenzoate by Alcaligenes denitrificans NTB-1, Appl Environ
Microbiol, 53 (1987) 810-815.
20 Marks T S, Smith A R W & Quirk A V, Degradation of 4-
chlorobenzoic acids by Arthrobacter sp, Appl Environ Microbiol,
48 (1984) 1020-1025.
21 Ehrhart H M & Rehm H J, Semicontinuous and continuous
degradation of phenol by Pseudomonas putida p8 absorbed on
activated carbon, Appl Microbiol Biotechnol, 30 (1989) 312-
317.
22 Sokol W, Oxidation of an inhibitory substrate by washed cells
(oxidation of phenol by Pseudomonas putida), Biotechnol
Bioengg, 30 (1986) 921.
23 Tang W T & Fan L S, Steady state phenol degradation in a draft
tube gas- liquid -solid fluidized bed reactor, AICHE J, 33 (1987)
239-245.
24 Goudar C T, Ganji S H, Pujar B G & Strevett K A, Substrate
inhibition kinetics of phenol biodegradation. Wat Environ Res,
72 (2000) 50-55.
25 Andrews J F, A mathematical model for the continuous culture
of microorganisms utilizing inhibitory substrates, Biotechnol
Bioeng, 10 (1977) 707-723.
26 Polymenekou P N & Stephenar E G, Effect of temperature and
addition of carbon sources on phenol degradation by an indig-
enous soil pseudomonas, Biodegrad, 16 (2005) 403-413.