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e> Pergamon Wat. ScL Tech. Vol.34, No. 5-6. pp. 495-500.1996.CopyrightC 1996IAWQ. PublishedbyElsevierScienceLtd .

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AZO-DYE BIODEGRADATION UNDERANOXIC CONDITIONS

U. Zissi and G. Lyberatos

Department ofChemical Engineering, University 0/Patras andInstinae ofChemicalEngineering and High Temperature Chemical Processes. P. O. Box 1414,GR·26500 Patras, Greece

ABSTRACf

Biological oxidation of azo-dyes is important for wastewater treatment. Azo-dyes are synthetic organiccolorants that exhibit great structural variety. A large majority of these dyes are released into theenvironment. The textileindustry and dyestuffmanufacturing industry are two majorsourcesof releasedazo­dyes. In the present study, we focus on the anoxic degradation of a disperse azo-dye, p-ammoazobenzene(pAAB). a simple azo-dye, by a pure culture of Bacillussubtilis, growing on a synthetic medium. Bacillussubtilis is a bacterium capable of using nitrate andlor nitrite as terminal electron acceptor under anoxicconditions. This bacterium lacks the capability (or fermentation. The degradation of p-aminoazobenzene byBacillus subtilis was examined through batch experiments in order to elucidate the mechanism of dyedegradation. The results provedthat Bacillussubtilisco-metabolizes p-aminoazobenzene under denitrifyingconditions, in the presenceof glucose as carbon source, producing aniline and p-pbenylenediamine as thenitrogen-nitrogen double bondis broken. Copyright iC> 1996lAWQ. Published by ElsevierScienceLtd.

KEYWORDS

Azo-dyes, Bacillus subtilis; degradation; denitrifying conditions; p-aminoazobenzene.

INTRODucnON

The removal of color from textile industry and dyestuff manufacturing industry wastewaters represents amajor environmental concern. Organic dyes cause both organic pollution and a higher coloration of theeffluents. Dye compounds are difficult to treat because of their ability to resist light and oxidizing agents(McKay et 01., 1988). They are not readily degradable and are typically not removed from water byconventional wastewater treatment systems (Anliker, 1979; Pagga and Brown, 1986).

Azo-dyes are synthetic organic colorants that exhibit great structural diversity (Anliker, 1979). They arecharacterized by the presence of nitrogen to nitrogen double bonds (-N=N·) . The color of azo-dyes is due tothe azo-bond, the associated auxochromes and a system of conjugated double bonds (aromatichydrocarbons). A large majority of these dyes are released into the environment. Release may occur asparticulate dispersions or as true solutions. In the treatment of textile wastewater. a considerable amount ofdyes and their metabolites are either sorbed or trapped in bioflocs and end up in sludges. Negligible totalbiodegradation (mineralization) of azo-dyes by aerated sludge has been document (Ogawa et 01.• 1974)

495

496 U.ZISSIandG.LYBERATOS

Biodegradationof azo-dyes can occur in both aerobic and anaerobic environments. In both cases, the initialstep in the biodegradation is the reductive cleavage of the azo-bond. Under anaerobic conditions no furtherdegradation has been observed (Meyer, 1981), whereas permeability through the cell wall has been foundoften to be the rate-limitingstep in the reduction process (Mechnsneret al., 1982).Under aerobic conditionsthe initial step of azo-bond cleavage is typically followedby hydroxylationand ring opening of the aromaticintermediates. Kulla et al. (1983), however, investigating the aerobic degradation of two sulfonated azo­dyes, did not observe complete mineralization. Zimmermanet al. (1982) demonstratedthat the azoreductaseunder aerobic conditions is highly specific and this specificity plays an important role in determining whichazo-dyes are susceptibleto bacterial attack.

In the present study, we focus on the anoxic degradationof a disperse azo-dye, p-aminoazobenzene (pAAB),a simple azo-dye, by a pure culture of Bacillussubtilis, growing on a synthetic medium. Such a well definedsystem secures reproducibilityof the results and allows a clear accounting of all experimentalobservations.Bacillus subtilis is a bacterium capable of using nitrate andlor nitrite as terminal electron acceptor underanoxic conditions. This bacterium lacks the capability for fermentation.

METHODS

Mjcroor&anjsm

Bacillus subtilis was purchased from the American Type Culture Collection (ATCC 6051). The organismwas maintainedon nutrient agar slants at 4·C.

Culture medjum and &rowth conditiQns

The nutrient agar contained: Bacto-beef extract (3g/l), Bacto-peptone (5g/l), Bacto-agar (l5g/l). The mineralsalts medium used consisted of IgIl NH4CI, IgIl NaCl, 0.2 gil MgS04,7H20, 0.0264 gil CaCI2, IS gilKH2P0 4, 50 gil KHP04. The medium was also supplementedwith I dropll trace elements ( 0.5%w/v MnCl2CUS04 , FeCI), Na2Mo04,2H20), 0.090 gil yeast extract and 0.090 gil peptone. Glucose (0.920 gil) wasadded as sole carbon and energy source and ammonium chloride as nitrogen source, Psaminoazobenzenewas added in the medium at a concentration of -10 mg/l, and nitrate at a concentrationof 210 mgll. All theexperiments were carried out under sterile conditions. The pH was adjusted to 7.1. Three loopfuls of cellsmaintained on agar were precultured on a rotary shaker at 25·C in 11 Erlenmeyer flasks containing 500 ml ofthe above medium.

Experimental system

The precultured cells (150 ml) were inoculated in a F-2000 Multigen Bench Fermentor with 1500 mlworking volume. Air was excluded from the culture system by purging the fermenter with high purity argon.Temperature was controlled by a Solid State Temperature Controller with a fast response ThermowellThermistor Sensor. Agitation was provided by means of a heavy duty drive motor magneticallycoupled to amagnet assembly located inside the vessel. The fermentor is illustrated in Fig. I.

Analytical methods

Bacterial growth was determined by measuring the absorbance at 660 om in a Milton Roy Spectronic 601Spectrophotometer. A calibration curve relating cell concentration and absorbance was constructed.Glucosewas determined using a reagent kit (Bioquant 10709-Merck).The concentration of p-aminoazobenzene was measured spectrophotometrically, at the dye's absorptionmaximum of 370 nm.

Aniline and p-phenylenediamine, the products of the azo-bond cleavage, were determined by high pressureliquid chromatography on a model 9010 Solvent Delivery System (Varian) and a model 9050 UV-VISDetector (Varian). The column used was a Nucleosil CI8 (reversed phase 25 em, 4.6 mm). The elution was

Azo-dye biodegradation under anoxic conditions 497

isocratic and the mobile phase consisted of 60% methanol and 40% water (HPLC grade). At a flow rate ofImllmin the retention time was 4.1 min for aniline and 3.2 min for p-phenylenediamine. The UV-Visdetector was set at 238 nm and 254 nm for aniline and p-phenylenediamine respectively. Nitrate and nitriteconcentrations were determined by ion chromatography on a model DX300 using an AS II column and aCDM-3 conductivity detector. The elution was gradient and the mobile phase consisted of NaOH:Hp at aflow rate of 2.0 mJlmin. The chromatogram was developed within II min and the retention time for nitrateand nitrite was 5.3 and 3.4 min respectively.

Figure I. Experimental apparatus .

RESULTS AND DISCUSSION

In this study. the anoxic degradation of p-aminoazobenzene was investigated under denitrifying and carbonlimited conditions. Denitrification is considered to be an anoxic process. occuring when nitrate or nitrite isused instead of oxygen as the terminal electron acceptor and requires an organic substrate for energy(electron donor) and cell synthesis. A batch experiment was carried out under glucose (carbon) limitedconditions. Thus the concentration of nitrate-nitrogen was used in excess (210 mgll) in order to avoid growthlimitation by nitrate. Glucose (920 rng/l) was used as the sole carbon and energy source. The concentrationsof nitrate. nitrite, biomass and glucose were measured during the growth of Bacillus subti/is under anoxicconditions (Fig . 2). No growth took place after the exhaustion of glucose. Nitrate was consumed by thebacterium. accumulating an appreciable quantity of nitrile as an intermediate product The initialconcentration of pAAB was 10.23 mgll. Aniline and p-phenylenediamine were obtained as the products ofreduction cleavage of the azo-bond.

According to the stoichiometry of the reaction, reduction of one mole of pAAB Should yield one moleaniline and one mole p-phenylenediarnine. The experimental results of Fig. 3 are in complete agreementwith this expected stoichiometry.

498 U. ZlSSIandG. LYBERATOS

• glucose • biomass • •I>. N03-N 0 NOZ-N ••I>.

I>. I>. I>.I>.

•I>. I>.I>. I>.

• • I>. •• ! I>.

• •I>. I>.

• • I>.

• 1>.1>.

•• •. • .•• •

• 0000

• • 000 00 0 0 •0

00 0 0 0

•·

200 300

,... ,...t:: 2S0~CD

ISo-!El......

= =.2 200'~..~ i8 u

loog ISO ~u u<n :z:; ',.

10000 z

SO ~

SO

1600

1400,...~ 1200El......l:l.2 1000

El:l 800~o~ 600<noU:I 4006

200

oo 5 10

Time

IS 20o

25o

Figure 2. Experimental profilesof biomass, glucose.nitrate-nitrogen and nitrite-nitrogenconcentrations underanoxic conditionsin the presence of pAAB <c;.AABo"10.23mgll.C.1oc.=92Omgll. C'I03-Na'"210 mgll,pH:7 .1}.

12

10 • • • • • • • • • • • ••

8 pUB • •~ • • •a • aniline.....

p-pheuJleuodiaminog D

.= Ci•::sl:l8 .-l:l

82 D

i D DD Ii ~ Ii ••g ~ g Ii • •

0 g g g

0 s 10 15 20 25

Timo (h)

Figure3. Conl:Cntration profile. of p-aminoazobenzene, anilineand p-phenylenediamine with time(CpAABc?10.23mgll,CsJucr=92Omgll, CN03-Na'"210 mgll, pH=7.1).

Azo-dye biodegradation underanoxicconditions

Consequently the reaction for the anoxic degradation of p-aminoazobenzene by Bacillussubtilis is:

499

<>-N=~_}NH' -..... <>-NH2 +

p-aminoazobenzene aniline p-phenylenediamine

The maximum specific utilization rate of p-aminoazobenzene is 1.1 mg pAABtg cellsh, Another experimentwas carried out under the same conditions with~ut p~. The effect of pAAB on cell growth is clearlyshown in Fig. 4. We observed a.15% decrease 10 specific growth rate. whereas the growth yield was notaffected.

0 without pAAB0 •

with 10.23 mgtl pAAB •• 000 o.

•0

0 •0

0 •0 •0 •

0

•0 •

c • •c••

•i i

I

200

e-bb ISO!c:Io':l

i 100

8

I 50iii

oo 5 10 15

Time (h)

20 25

Figure4. Influence of p-aminoazobenzene on bacterial growth rate.

These results confirm that the azo-dye is partially metabolized under anoxic conditions producing aromaticamines as products of reductive azo-bond cleavage. Lack of oxygen hinders the mineralization and leads toaccumulation of aromatic amines (Meyer. 1981). Oxygen is often an inhibitor of azo-reduction. Degradationof pAAB by Bacillus subtilis was also observed under aerobic conditions and with the same metabolicproducts (Zissi et al.• 1996) although at a faster rate (1.8 mg pAABtg cells·h). This bacterium cannot furtherutilize these amines in subsequent enzymatic reactions. The ability of Bacillus subtilis to degrade pAABunder both aerobic and anoxic conditions may be attributed to an oxygen insensitive azo-reductase, found tobe present in the soluble fraction of biomass (Horitzu, 1977).

CONCLUSIONS

• Experiments were performed in a b~tch react~r containing a pure cUl~re of Bacillus subtilis and p-aminoazobenzene as a eometabolite, p-aminoazobenzene was partially degraded during anoxicgrowth of Bacillussubtilis.

• The results presented in this study demonstrate that aniline and p-phenylenediamine are thestoichiometric products of p-aminoazobenzene indicating that aza-linkage is reduced yielding thecorrespoding amines.

sao U. ZISSI and G. LYBERATOS

• The presence ofpAAB in the culture medium causes a 15% decrease in the specific growth rate.

• p-aminoazobenzene degradation rates are lower under anoxic conditions than under aerobicconditions.

REFERENCES

Anliker. R. (1979). Ecotoxicology of dyestuffs-A Joint effort by Industry. 3, S9-74.Horitzu. H.• Takeda. M.• ldaka, E.• Tomoyeda, M. and Ogawa, T. (1971). Degradation of p-aminoazobenzene by Bacillus subtilis,

Eur.J. AppLMicrobiol. 4,217-224.Kulla, H. G. (1981). Aerobic bacterial degradation of azo-dyes. In Microbial Degradation of Xenobiotics and Recalcitrant

Compounds, Leisinger. T.•Cook, A. M.• Nuesch, 1. and Hutter. R. (eds), pp. 387·399. Academic Press London.Kulla, H.• Klausener, F.• Meyer. U.• Ludeke, B. and Leisinger. T. (1983). Interference of aromatic sulfo groups in microbial

degradation of the azo-dyes Orange I and Orange n. Arch.Microbiol. 135, 1·7.McKa,y G., Geundi, M. EL. and Nassar. M. M. (1988). External mass transport processes during the adsorption of dyes onto

bagasse pith. Wat. Res.22(12). IS27-IS33.Mechsner, K. and Wuhrmann. K. (1982). Cell permeability as a rate limiting factor in the microbial reduction of sulfonated azo­

dyes. Eur.J. AppL Microb. Biotech.1S, 123·126.Meyer. U. (1981). Biodegradation of synthetic organic colorants. in Microbial Degradation of Kenobiotics and Recalcitrant

Compounds, Leisinger. T.•Cook. A. M., Nuesch, J. and Hutter. R. (eds), pp. 371·38S. Academic Press. London.Ogawa, T.. ldaka, E.• Hirabayashi, and Takase. (1974). Biological treatments of acid azo-dyes by microbial populations in

activated sludges. J. Soc. FiberSci. Technol. Jpn. 30, TlS3-IS9Pagga, U. and Brown. D. (1986). The degradation of dyestuffs. n. Behavior of dyestuffs in aerobic biodegradation tests.

Chemosphere 15,479-491.Zissi. U.• Lyberatos, G. and Pavlou, S. (1996). "Biodegradation of p-aminoazobenzene by Bacillus subtili« under aerobic

conditions". WatRes. (submitted).Zimmermann. T.• Kulla, H. and Leisinger. T. (1982). Properties of purified Orange n azoreductase the enzyme initiating azo-dye

degradation by Pseudomonas KF46. Eur.J. Biochem. 129, 197·203.