JOURNAL OF SCIENCE & TECHNOLOGY • No. 87 - 2012
A STUDY ON BIODEGRADATION KINETICS OF 1,4-DIOXANE (1,4-D) BY MIXED CONSORTIUM OF BACTERIA IN BATCH REACTOR
NGHlfeN CUU DONG HQC QUA TRINH PHAN HUY 1,4-DIOXANE (1,4-D) B A N G N H 6 M VI K H U A N P H A N LAP TRONG H$ T H I £ T BI L £ N MEN G I A N DOAN
Nguyen Thi Thu Ha ' ^ Do Khac Uan \ Ick T. Yeom * ' Sungkyunkwan University. Korea ' Energy and Environment Consultancy JSCo, Vietnam ' Hanoi University of Science and Technology. Vietnam
Received March 1, 2012; accepted April 25.2012 ABSTRACT
1.4-Dioxang (1.4-D) has been classified as a human carcinogen. Microbial degradation of 1,4-D would be an attractive bioremediatlon approach. In this study, Ihe kinetics of 1,4-D btodegradathn was determined in a t>atch reactor by a mixed consortium of bacteria isolated from Industrial sludge contaminated with 1,4-D. As a result, the kinetic coefficients of Monod equation were found tobeqm = 0.0051 mg 1,4-D/mg VSS.hr. K, = 7.47 mg/L. ka = 0.0023 mg/mg.hr, and Y = 0.41 mg VSS/mg 1.4-0.
In addition, the 1.4-D degradation rate and specific degradation rale were increased with the increase in 1,4-D concentrations, ranging from 5 to 2000 mg/L. showing that the enriChed culture has high affinity to 1,4-D biodegradation.
T6M TAT
1.4-Dioxane (1.4-D) Id mdt trong nhQng hgp chdt gdy ung thir cho con ngu&i. Phdn hdy hgp chdt ndy bdng vi sinh v$t Id mdt cdch tidp cdn rdt dugc chd trgng. Nghidn c&u ndy tidn hdnh xdc iTinh cdc thdng sd ddng hoc cua qud trinh phdn bdy sinh hqc 1,4-D trong thidt bj Idn men gidn doan sir dung h$ vi khudn phdn l$p tu- bun thdi dd bi d nhidm 1.4-D. Kdt qud thu dugc <^1 v&i cdc h$ sd ddng hqc cua phuang trinh Monod idn lugt Id q„ = 0 0051 mg 1.4-Q/mg VSS.hr. K, = 7.47 mg/L. k^ = 0 0023 mg/mg.hr. vd Y = 0.41 mg VSS/mg 1.4-D. Ngodi ra. kdt qud nghidn cOu cho thdy tdc dd phdn huy 1.4-D vd tdc dd phdn gidi ridng phdn ddu tdng khi ndng dd 1.4-D ban ddu tdng tir 5 ddn 2000 mg/L. Nhu vdy. mdi tru&ng cd hdm lugng co chdt cdng cao cdng c6 Igi cho qud trinh phdn huy 1.4-D.
..INTRODUCTION [7]; Flavobacterium sp. [8]. There were, 1,4-dioxane (1,4-D) is a cyclic ether however, only few studies investigated on 1.4- which was widely used as a stabilizer in ^ biodegradation m mixed cultures. For chlorinated solvems in various industries such example, Zenker et al. have studied the 1,4-D as paints, lacquere, cosmetics, textile. biodegradation on consortium isolated ^m deodorants, fumigants and detergents [I]. Due aquifer sediment contaminated with 1,4-D [9].
to its carcinogen properties and recalcitrance in '" ° ' ' ' " study, the removal rate of I.4-dioxane natural water, 1.4-D has been classified as a and the dominant species were evaluated in the priority pollutant [2]. 1.4-D degradation has activated sludge from polyester manufactures shown a significant challenge and attracted ['0]- '" addition, the biodegradation potential great attention in both academic and of 1,4-D in different natural bacteria sources engineering fields [3]. has also been examined [11]. So far. however, D.>^.>„.i.- I ,1 r» u- J- .• 1. L ^^^"^ ^i3s not been any study on the kinetics of Recently, 1,4-D bioremediation has been . . . , . . . _ . . ^ . . . ^
^,„ J- J - jff. , r • u- 1 biodegradation by mixed cu lure of ,4-D.
studied in different processes of microbial
metabolism, but mostly in pure cultures [4]. A In this study, therefore, the kinetics of number of single strains were isolated and 1-4-D biodegradation was first examined in a characterized for 1,4-D biodegradation such as batch reactor by a mixed consortium of bacteria Cordyceps sinensis fungus [3]; Pseiidonocardia Isolated from industrial sludge contaminated sp. [5]; Graphium sp. [6]; Mycobacterium sp. ^^i'h L4-D.
JOURNAL OF SCIENCE & TECHNOLOGY * No. 87 - 2012 2. MATERIAL AND METHODS
Chemicals. All chemicals and solvents used for experiments were the highest purity available. 1,4-D (99.9%) was obtained fi-om Sigma-Aldrich (USA). The chemicals used for 1,4-D extraction such as Methylene Chloride, Sodium Chloride, Sodium Sulfate Anhydrous were obtained from Fisher Scientific Co.
(Korea) and Kanto Chemical Co. (Japan). The basal salts medium (BSM) used in this study was prepared in one litter of distilled water containing a given concentration of 1.4-D as sole carbon source and 3.24g K2HPO4: 1.000 g NaHjPOa, HjO; 2.000 g NH4CI; 0.200 g MgS04.7HiO; 0.012 g FeS04.7HjO; 0.003 g ZnSO^; 0.003 g MnSO* and 0.001 g C0CI2 [4],
1.4-D biodegradation kinetics. The experiment of batch tests was conducted in duplicated 500 mL bottles containing 200 mL of medium culture amended with 200 mg/L of 1,4-D. The duplicate of abiotic controls prepared with same 1,4-D concentration in test sample but without inoculation of mixed culture were to control the volatile portion of 1.4-D.
While biotie controls inoculated with enriched culture and without adding carbon source were to determine the decay rate of mixed consortium. All tests were incubated aerobically at DO of about 3 mg/L in a stirrer at room temperature.
idation of differeni 1.4-D concentrations. The experiments were conducted in a matrix of 100 mL flasks containing 50 mL of sample with different 1,4- D concentrations. The enriched culture was distributed equally in 16 separate flasks containing 50 mL of BSM, while duplicated seven fiasks amended with different 1,4-D concentrations as sole carbon source, ranging from 5, 50, 200, 500, 1000, 1500, and 2000 mg/L. Another 2 flasks were not served 1,4-D used for biotie control. Two abiotic control flasks containing 200 mg/L and 1000 mg/L of 1,4-D were not inoculated with consortium. All tests were prepared in duplicated, incubated at room temperature in a shaker at 560 rpm. The 1,4-D disappearance and cell growth were monitored daily.
Analysis methods The 1,4-D concentration was determined using liquid-
liquid extraction combined with gas chromatography/mass spectrometry (GC/MS) [12], The soluble constituents of the samples used for analyses were filtered through the filter papers with the mesh size of 0.45 nm (GD/X PVDF, Whatman. UK). Volatile suspended solids (VSS) and chemical oxygen demand (COD) were determined in accordance with Standard Methods [13].
3. RESULTS AND DISSCUTION 3.1 Enrichment of mixed consortium of bacteria degrading 1,4-D
The activated sludge taken from a wastewater treatment system at Yongsal Industrial Zone and then was enriched in the presence of increasing concentration of 1,4-D from 20 to 100 mg/L. The cell growth and biodegradation of 1,4-D were observed simultaneously and then transferred to fi-esh BSM medium with higher concentration of 1,4- D. As a result, after 2 months of enrichment, the mixed culture was able to degrade 1,4-D as sole carbon source. Then, mineralization of 1,4-D by the enriched culture was tested in sealed 500 mL bottle. In the test samples, culture (VSS = 340 mg/L) was grown in BSM containing IOO mg/L of 1,4-D, while in biotie control cuhure was inoculated (VSS = 340 mg/L) into BSM without 1,4-D, in abiotic control BSM was amended with IOO mg/L of 1,4-D without seeding. The cells growth measured as the increase in VSS to further investigate the role of t,4-D in cell associated.
After 60 hr, 1,4-D was degraded to 1.2 mg/L, the biodegradation time was referred to preliminary study and fixed the end point al 60 hr.
3.2 Estimation of 1,4-D biodegradation kinetics
The balch lesls were conducted in a series of 500 mL bottles containing 200 mL medium. Initial concentration of 1,4-D and biomass were 200 mg/L and 522 mg/L, respectively. The tests were done in duplicated with biotie and abiotic control. The batch reactor model was applied to detennine the kinetic parameters of 1,4-D biodegradation.
in batch reactors, while the microorganisms are consuming 1,4-D, no
JOURNAL OF SCIENCE & TECHNOLOGY * No. 87 - 2012 substrate was added or removed from batch
reactor. The rate of 1,4-D utilization can be assumed based on the following Monod equation [14]:
dt ' q.S
Ks+S
[x^+ns'-s)] (I)
where: S" and S: concentration of substrate (1,4-D) at initial and at any time t, respectively (mg/L); X,': initial biomass concentration (mg/L); qn,: maximum specific degradation rate (mg 1,4-D/mg VSS.hr); Kji half velocity constant (mg/L).
Fig 1. shows the time-course variations of 1,4-D and biomass concentrations during incubation. The duplicated abiotit controls were to determine the loss of 1,4-D as volatilization.
This volatile portion was calculated as 4.35%
based on the initial and the end point concentration of 1,4-D. The cell yield (Y) can be estimated by linearizing cell mass increase with 1,4-D consumption, as following equation [14):
_dXJdl__Cell^
' lis I lit ~1,4.Z)„,, (2) In this study, the celt yield was estimated bas^ on the observation of increased biomass per utilized substrate in test sample. The yield coefficient was 0.41 mg VSS/mg 1,4-D which was about 0.23 mg VSS/mg COD (I mg 1,4-D is equal to 1.81 mg COD). The obtained result was relatively low compared to the cell yield obtained for normal substrate (0.5-0.7 mg VSS/mg COD [15].
The endogenous decay is the cells oxidize themselves when Ihe substrate completely consumed to provide energy demanded for maintenance the activities of cells such as motility, repair, resynlhesis. transport [14]. A typical curve showed a decline in cell population when the complete consumption of substrate, this also can be seen in the biotie control lest which was not provided carbon source for inoculated biomass. The biotie control used to determine the decay rate of mixed culture in this study. The rate of endogenous decay has been modeled by equation [15]:
^*r=C-r
I, l l ^
(3)
The experiment was conducted for initial biomass concentration of 522 mg/L. Fig. 1 also shows the decrease of biomass concentration (for biotie control) and the increase of biomass concentration (for test sample) atler 70 hr in the 3 point-curve.
Fig. I. Biodegradation of 1.4-D at 200 mg/L at initial concentration
Fig. 2. Reduction of 1.4-D during biodegradation
The negative slope gave the decay rate coefficient (kj) thai was found in this study to be about 0.023 day'. The kinetic coefficients of 1,4-D biodegradation obtained in this study by application of the experimental results from Fig. 2 and estimated cell yield to Monod equation of substrate utilization (eq, 2), were listed in Table 1,
JOURNAL OF SCIENCE & TECHNOLOGY * No. 87 - 2012 The specific degradation rate of 1,4-D in
this study was found to be much lower than those reported in previous study on pure culture (q„= 0.1 mg I,4-D/h.mg protein of Pseudonocardia benzenivorans B5 [16], This was expected that the mixed culture maybe only include some kinds of bacteria which could degrade 1,4-D. It should be noted that the K, (7.47 mg/L) obtained in this study was relatively low. This could be implied that the enriched culture has high affinity to 1,4-D during biodegradation.
Table L Kinetic coefficients of 1.4-D biodegradation
Kinetic parameters K,(mgl,4-D/L) q.„(mg 1,4-D/mg VSS.hr) kj(mg VSS/mg VSS.day) Y(mgVSS/mgl,4-D) IVuCday)'
Value 7.47 0.0051
0.023 0.41 0.053 3.3 EfTect of substrate concentration on 1,4- D biodegradation
The effect of substrate concentration on mixed consortium was studied at various initial 1,4-D concentration ranging from 5, 50, 200, 500, 1000, 1500, and 2000 mg/L. All tests were inoculated with the same biomass concentration (VSS = 655 mg/L).
"r
Fig 3. Degradation of 1.4-D at various initial concentrations
It can be seen from Fig. 3 that the complete degradation of 1,4-D was obtained at all initial concentrations. Il can be concluded that the 1,4-D is not a self-inhibition during
biodegradation by the enriched culture. This can be reconfirmed in increase of specific degradation rates.
Other kinetic parameter (q^u and Ks) were delermined by using Ihe Monod equation at various initial concentration of 1,4-D. As shown in Fig 4, the degradation rate increased with the increase of initial 1,4-D concentrations.
It meant that the 1,4-D was Ihe unlimited substrate for enriched culture.
Fig. 4. Degradation rate al various initial 1.4- D concentration
4. CONCLUSIONS
The biodegradation of 1,4-D, one of the probable human carcinogens, was studied in batch reactor. After 2 months of enrichment, a mixed consortium of bacteria was isolated from industrial sludge contaminated with 1,4-D. The batch tests were conducted with initial concentration of 1,4-D and biomass of 200 mg/L and 522 mg/L, respectively to estimate the kinetic parameters. The kinetic coeOieients of Monod equation were found to be qn, - 0.0051 mg 1,4-D/mg VSS.hr, K, = 7.47 mg/L, the decay rate kj = 0.0023 mg/mg.hr and the cell yield Y = 0.41 mg VSS/mg 1,4-D. A wide range of initial 1.4-D concentrations from 5 ppm lo 2000 ppm was also studied in the batch reactors, the degradation rale and specific degradation rale were increased with the increase in initial concentration within the studied range. The results were indicated that 1,4-D was unlimited substrate for enriched culture.
.lOl KNALOF.SCIKMlidk 1ECHNOLOGY4NO.87-20I2
R E F E R E N C E S
Chiang D.S.. Glover W.E.. Pclerman J.. Harrigan J.. Dihuiseppi B. and Woodward D.S., Evaluation of natural attenuation at ii 1.4-dioxane contaminated site. Wiley Inter Sci.
Remcdialion. 19: 19-37(2009)
USEPA. Treatment technologies for 1,4-dioxanc fundamentals and field application. US Environmental Protection Agency EPA-542-R.06-009. Washington. DC. USA (2006).
Nakamiya K.. Hashimoto S.. Ito IL, Edmonds J.S. and Morita M.. Degradation of 1.4-Dioxane and cyclic ethers by an isolated Fungus. Appl. Environ. Microbiol.. 7 1 : 1254-1258 (2005).
Vainberg S.. McClay K.. Masuda II.. Root 1).. Condce C . Zylstra G.J. and Steffan R.J., Biodegradation of ether pollutants by Pseudonocardia sp. strain l - \ V 4 7 8 . Appl. Environ.
Microbiol., 72:5218-5224 (2006).
Parales R.E.. I d a m u s J.E.. White N. and May H.D.. Degradation of I.4-Dioxane by an Actinomycetc in pure culture. A p p l . Environ. Microbiol.. 60' 4,S27-4530{I994).
Mahendra S.. Pelzold C.J.. Baidoo E.E., Kcasling J.D. and Alvarez C.L.. Identification of the inlermediales of in vivo ovidation of 1. 4-dioxanc by monooxygenase-coniaining bacteria.
Environ. Sci. Teehnol.. 4 1 : 7330-7.Ki6 (2007).
Kim Y.M.. Jeon J.R.. Murugesan K. Kim E.J.. Chang Y S . Biodegradation of I. 4-dioxane and transformation of related cyclic compounds by a newly isolated Mycobacterium sp. PH-06.
Biodegradation, 20: 511-519 (2009).
Bozhi S.. Kenton K.. Juliana A.R., Biodegradation of J.4-dioxane by a Flavobaclerium.
Biodegradation, 22: 651 -659 (2011).
Zenker M.J. Borden R.C. and Bariaz M.A. Vlincrulizalion of t,4-Dioxane in the presence of a structural analog. Biodegradation. 11: 239-246 (2000),
, Han J.S. So M.H. and Kim C.G.. Optimization of Biological wastewater treatment condition for 1.4-Dioxane decomposition in polyester manufacturing processes. Water Sci, Teehnol., 59: 995- 1002(2009).
, Kazunari S.. Takashi K.. Daisuke I.. Saloshi S . Masanori F., Michihiko I., Evaluation of the biodegradation potential of 1.4-dioxane in river, soil and activated sludge samples.
Biodegradation, 21: 585-?'J 1 (2010).
, Katharine Nunn (2004) Standard operation procedures for the measurement of 1.4-dioxane in aqueous samples by CiC MS-SIM. FPA CLP Method OLM03.1 (1994).
, APHA, Standard methods for the examination of water and wasiewaier. 2 l s t Edition. American Public Health Association. Wasliiiiykni. DC. L S A ( 2 0 0 5 ) .
Bruce E.R. and Pcrr> I. C . linvironmenlal biolcchnologj': Principles and applications, McGraw ilill.Nc\\ York. LSA (2005).
. Vlelcalfand Eddy, \\'.istc\vater cnmriccniii;, ireatmenl. disposal and reuse. 3rd edition, McGraw Hill.N'cu York. I S A (2003).
. Vlahcndia s , Alvarez C.I . Kinetics of !.4-dioxanc biodegradation by Monooxygenase- cxpressing bacteria. Environ. SLI Tcchnol.. 40: 5435-5442 (2006),
Do Khac Uan - Tel: (-84) 1202.136.818 - Liiuiil: u.mdk-incMiniuil.hul.edu.vn Instilutc For Knvironnicnlal SLK-IKC .uui Technology
Hanoi 1 iii\cisit\' I't Science and '1 echnolog\
No 1 Dai Co \ Id Sir., i la \ n i , Vicl \ ; i m "
