Effects of alkalinity and co-substrate on the performance of an upflow anaerobic sludge blanket...

Bioresource Technology 96 (2005) 633–643

Effects of alkalinity and co-substrate on the performanceof an upflow anaerobic sludge blanket (UASB) reactor

through decolorization of Congo Red azo dye

Mustafa I+ık, Delia Teresa Sponza *

Environmental Engineering Department, Engineering Faculty, Dokuz Eylul University, Buca Kaynaklar Campus, 35160, Izmir, Turkey

Received 23 July 2003; received in revised form 26 May 2004; accepted 1 June 2004

Available online 7 August 2004

Abstract

The effect of substrate (glucose) concentrations and alkalinitiy (NaHCO3) on the decolorization of a synthetic wastewater

containing Congo Red (CR) azo dye was performed in an upflow anaerobic sludge blanket (UASB). Color removal efficiencies

approaching 100% were obtained at glucose-COD concentrations varying between 0 and 3000 mg/l. The methane production

rate and total aromatic amine (TAA) removal efficiencies were found to be 120 ml per day and 43%, respectively, while the

color was completely removed during glucose-COD free operation of the UASB reactor. The complete decolorization of CR

dye under co-substrate free operation could be attributed to TAA metabolism which may provide the electrons required for

the cleavage of azo bond in CR dye exist in the UASB reactor. No significant differences in pH levels (6.6–7.4), methane pro-

duction rates (2000–2700 ml/day) and COD removal efficiencies (82–90%) were obtained for NAHCO3 concentrations ranging

between 550 and 3000 mg/l. However, decolorization efficiency remained at 100% with decreasing NaHCO3 concentrations as

low as 250 mg/l in the feed. An alkalinity/COD ratio of 0.163 in the feed was suggested for simultaneous optimum COD and

color removal.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Azo dye; UASB; Glucose-COD; Alkalinity; Decolorization; Aromatic amines; Color

1. Introduction

Azo colorants are substances that have a coloring ef-

fect of one or more azo groups (N‚N double bonds) in

their chemical structure. They can be used in synthetic

and natural textile fibers, plastics, leather, paper, min-

eral oils, waxes, and even (with selected types) foodstuffs

0960-8524/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2004.06.004

* Corresponding author. Tel.: +90 232 4531008/1119; fax: +90 232

4531153.

E-mail addresses: [email protected] (M. I+ık), delya.

[email protected] (D.T. Sponza).

and cosmetics (Geisberger, 1997). Azo dyes are the class

of dyes most widely used in textile finishing, having aworld market share of 60–70%. They have become of

concern in wastewater treatment because of their color,

bio-recalcitrance, and potential toxicity to animals and

humans (Yoo et al., 2000).

The azo dyes are not readily degradable under natu-

ral conditions and are typically not removed from

wastewater by conventional wastewater-treatment sys-

tems. Several chemical and physical decolorizationmethods such as coagulation/flocculation and precipita-

tion, oxidation, adsorption, electrolysis, and membrane

extraction are available (Cooper, 1993). These methods

are costly. In recent years there has been a tendency to

mailto:[email protected]

mailto:[email protected]

634 M. Is�ık, D.T. Sponza / Bioresource Technology 96 (2005) 633–643

use biological treatment systems to treat wastewaters

containing azo dyes. The recalcitrant nature of azo dyes

together with their toxicity to microorganisms makes

aerobic treatment difficult (Knackmuss, 1996). Under

anaerobic conditions, azo dyes are readily cleaved via

a four electron reduction through the linkage of azobonds generating aromatic intermediates (Wuhrmann

et al., 1980; Weber and Wolfe, 1987). Various azo dyes

were shown to be decolorized by anaerobic sludge, sedi-

ments and pure cultures in batch reactors (Rajaguru

et al., 2000; I+ık and Sponza, 2003). Currently, in the

biological treatment of textile wastewater, studies have

been focused on anaerobic biotechnology since anaero-

bic treatment can achieve color removal. Furthermorethe anaerobic treatment has classical advantages which

includes low sludge formation, no aeration requirement

and methane gas formation when compared to aerobic

treatment systems (Delee et al., 1998). Generally during

the anaerobic process a reduction in COD of up to 60–

70% can also be achieved (Zaoyan et al., 1992). Hence it

seems that an anaerobic processes hold promises for the

effective treatment of textile wastewaters on the basis ofCOD and color. Upflow anaerobic sludge blanket

(UASB) reactors are the most commonly used high rate

anaerobic systems. They are generally used for waste-

waters that have a low suspended solid concentration

and can be used for the treatment of dye wastes (An

et al., 1996; Razo-Flores et al., 1997; Delee et al.,

1998; O�Neill et al., 2000a).

Several studies have reported that suitable co-sub-strate and alkalinity in a bioreactor are necessary for

color removal. The electrons required for decoloriza-

tion are provided by an electron donating carbon

source such as glucose, VFA or starch (Banat et al.,

1996; Brown and Laboureur, 1983; Chinwetkitvanich

et al., 2000; Bras et al., 2001). Gingell and Walker

(1971) and Carliell et al. (1995) reported that decolori-

zation take places under anaerobic conditions if co-subsrate is present to donate electrons. Co-substrates

were used in these studies for effective decolorization

of azo dyes. Bicarbonate alkalinity was used to provide

favorable conditions for conversion of substrate to

methane and enough electrons from electron transport

chain for concurrently decolorizing of azo dyes (Carliell

et al., 1996). However, studies performed by Razo-

Flores et al. (1997) and Beydilli et al. (1998) showedthat decolorization of dyes did not depend on COD

since the dye itself can be used as carbon source by

the anaerobic microorganisms. It is generally assumed

that the aromatic amines generated from the linkage

of azo bound compounds are not further degraded

under anaerobic conditions (Brown and Hamburger,

1987; Haug et al., 1991). However, several studies dem-

onstrated that the aromatic amines could potentially befully or partially biodegradable in anaerobic environ-

ments (Razo-Flores et al., 1997; O�Connor and Young,

1993; Battersby and Wilson, 1989; Kalyuzhnyi et al.,

2000).

One of the major problems is the inadequate influent

alkalinity/influent COD ratio in the operation of the

UASB system treating textile industry wastewaters con-

taining azo dye. This may cause the minimum pH in thebed to fall below about 6.2–6.6, which can lead to failure

of the system. Carbon dioxide produced via micro-

organisms often exceeds the weak acids in aqueous

anaerobic systems. Therefore a sufficient bicarbonate

alkalinity must be present to neutralize it and is, there-

fore, of prime importance. If the acid concentrations

(H2CO3 and total volatile fatty acid––TVFA) exceed

the available alkalinity, the reactor will sour, severelyinhibiting microbial activity, especially the methanogens

(Speece, 1996). Souza et al. (1992) and Moosbruger et al.

(1993) found that an alkalinity/COD ratio of 0.5 in the

effluent decreased the pH to 6.6 which is considered as

the lower limit recommended for the anaerobic digestion

processes. With carbohydrate wastes the alkalinity

requirement is 1.2–1.6 g alkalinity as CaCO3/g influent

COD which is sufficient to maintain the pH above 6.6(Speece, 1996).

Limited studies have been performed investigating the

effect of co-substrate and alkalinity on the treatment effi-

ciencies using continuous plug flow systems. The effect of

different alkalinity and substrate concentrations on the

treatment efficiencies using continuous anaerobic reac-

tors treating azo dyes has not been extensively reported

in previous studies. For instance, Chinwetkitvanich etal. (2000) reported that the supplementation of tapioca

starch as a co-substrate apparently gave a better color re-

moval performance. However, excessively high concen-

trations of tapioca did not enhance the process

capability in terms of color removal efficiencies in an

UASB system. O�Neill et al. (2000b) suggested that if

the color removal efficiency decreases, carbonhydrate

should be added to UASB reactor operating atsludge loading rates varying between 0.11 and 0.15 kg

COD/kg TVSday. However, azodisalicylate azo dye (75

mg/l) could be continuously used as the sole carbon and

energy source with a removal efficiency of 89% in an

UASB reactor at a HRT of 26 (Razo-Flores et al., 1997).

The alkalinity required to buffer a wastewater con-

taining azo dyes and the supplementation of external

carbon source adversely affect the economical feasibil-ity of the anaerobic treatment. The cost for alkalinity

and sometimes for additional co-substrate supplemen-

tation to wastewater can easily exceed the value of

methane produced in UASB reactors. This study was

designed to investigate the effects of glucose-COD

and NaHCO3 alkalinity concentrations on the reactor

performances (COD, color, TAA removal efficiencies,

VFA and methane gas productions) in a lab-scale up-flow sludge blanket (UASB) reactor treating Congo

Red azo dye.

M. Is�ık, D.T. Sponza / Bioresource Technology 96 (2005) 633–643 635

2. Methods

2.1. Experimental design and operating conditions

In the first step of the study (Runs 1–5) the glucose-

COD was reduced from 3000 to 0 mg/l in order to deter-mine the effect of substrate on the color, COD removals

and methane gas productions. The operating conditions

during the operation in UASB reactor are summarized

in Table 1. The NaHCO3 concentrations were adjusted

by taking into consideration the alkalinity required for

each COD level under anaerobic conditions. Although

it was aimed to study at constant HRTs, they varied be-

tween 15.7 and 19 h due to some difficulties in adjustingthe dosage pump. The flow rate taking into considera-

tions are the mean values.

In the second step of the study (Runs 6–11) the COD

concentrations were kept constant at 2000 mg/l and the

effect of decreasing NaHCO3 concentrations (from

3000 to 250 mg/l) on the COD, color removal efficiencies,

TVFA accumulation and methane gas productions were

investigated. Although it was aimed to study at constantflowrates the HRTs varied between 15.54 and 21.83 h due

to the weekly variations in dosage pump (see Table 1).

The UASB reactor used in this study was previously

used for removal of synthetic wastewaters containing

Reactive Black 5 and Congo Red azo dyes (Sponza

and I+ık, 2002; I+ık and Sponza, 2002). After that, the

reactor was operated under dye-free conditions at an or-

ganic loading rate of 4.54 kg COD/m3day, with an HRTof 16 h in the beginning of these studies (Run 1). This

volumetric organic loading rate used in this study is near

the optimum organic loading rate range (2–4 kg COD/

m3day) proposed for UASB reactors treating textile

wastewater (Manu and Chaudhari, 2003). The COD re-

moval efficiency, gas production rate, methane percent-

age, pH and TVFA concentrations in effluent of UASB

reactor were 88%, 4280 ml/day, 80%, 7.3 and 78 mg

Table 1

Operation parameters and loading conditions of UASB reactor

Stage Period

(days)

Glucose-COD

(mg/l)

NaHCO3

(mg/l)

HRT

(h)

Effect of glucose-COD on UASB reactor performance

Run 1 0–16 3000 5000 15.7

Run 2 17–23 500 667 19

Run 3 24–30 250 333 18

Run 4 31–37 100 333 18.3

Run 5 38–50 0 333 18.9

Effect of alkalinity on UASB reactor performance

Run 6 51–56 2000 3000 20.4

Run 7 57–63 2000 3000 15.54

Run 8 64–70 2000 1500 15.54

Run 9 71–77 2000 750 18.19

Run 10 78–91 2000 550 21.83

Run 11 92–103 2000 250 17.6

CH3COOH/l, respectively, before starting the aforemen-

tioned runs, indicating the steady-state conditions for

effective continuous operation of UASB reactor. For

each run, steady-state conditions were assumed to be

achieved if the effluent COD concentrations and gas

productions values did not differ by more than ±5%for at least four consecutive days.

2.2. Experimental lab-scale reactors and seed

The anaerobic UASB reactor used for Congo red

decolorization was 6 cm in diameter, 100 cm in length

and had an effective volume of 2.5 l. The schematic con-

figuration of the sequential UASB reactor is illustratedin Fig. 1. Partially granulated anaerobic sludge was used

as seed in the UASB reactor and was taken from a meth-

anogenic reactor treating industrial effluent from the

Pakmaya Yeast Baker Factory in Izmir. The UASB

reactor was operated at 37 �C using an electronic heater

in the medium part of the system.

2.3. Azo dyes and composition of synthetic wastewater

Congo Red (Direct Red 28) which is a banned azo

dye including the carcinogenic aromatic amines (benzi-

dine) was used in dissolved form. The color index num-

ber, the maximum wavelength of absorbance, and the

COD value of 100 mg/l of CR dye solution are 22120,

497 nm, and 74.6 mg/l, respectively. The structure of this

dye is depicted in Fig. 2. Vanderbilt mineral medium(Speece, 1996) was used as feed.

2.4. Analytical procedures

Total suspended solid (TSS) in granulated sludge were

measured by filtration technique using membrane filters

with pore sized 0.45 lm (APHA, 1992). The COD in

influent and effluent samples were detected by using a

Org. load

(kg COD/m3day)

Dye load rate

(g dye/ m3h)

Upflow vel.

(m/h)

4.54 0 0.064

0.845 5.3 0.053

0.437 5.6 0.056

0.286 5.5 0.055

0.195 5.4 0.054

2.3 0 0.049

3.27 6.43 0.064

3.27 6.43 0.064

2.86 5.62 0.056

2.42 4.76 0.048

2.99 5.88 0.056

6 cm

10 c

m5

cm15

cm

15 c

m20

cm

20 c

m15

cm

100

cmTotal Gas Measurement2% (w/v) H SO 10% (w/v) NaCl solution2 43% NaOH (w/v) solution

Methane Measurement

Gas

Out

let

Time ControlledPerilstaltic Pump

UASB FeedingTank+4 C24 L

Sam

lplin

g Po

rts

Heater

EffluentTank

o

Fig. 1. The schematic configuration of the UASB reactor.

Fig. 2. The structure of Congo Red azo dye.


closed reflux colorimetric method following standard

methods (APHA, 1992). Total volatile fatty acid (TVFA)

and bicarbonate alkalinity (B.Alk.) concentrations in the

effluent of UASB reactor were measured using the titri-

metric method described by Anderson and Yang

(1992). pH was determined immediately after sampling

to avoid any change due to the CO2 evolution using a

pH meter (WWT pH 330). Gas production was meas-ured with a liquid displacement method. Total gas was

measured by passing it through a liquid containing 2%

(v/v) H2SO4 and 10% (w/v) NaCl (Beydilli et al., 1998).

Methane gas was detected using a liquid solution con-

taining 3% NaOH (w/v) (Razo-Flores et al., 1997).

Color in the influent and effluent samples of the

UASB reactor was measured by an Optima Photomech

301-D UV–VIS spectrophotometer (Nova, UK) at max-imum wavelength. The samples were centrifuged at 7000

rpm for 10 min and the absorbance values of superna-

tants were recorded for color measurements. Abiotic

tests were performed with autoclaved anaerobic par-

tially granulated sludge and a mineral medium in batch

serum bottles at 37 �C to determine the biological dye

degradation.

The total aromatic amines were determined colori-

metrically at 440 nm after reacting with 4-dimethylami-nobenzaldehyde–HCl according to the method

described by Oren et al. (1991). Total aromatic amines

(TAA) released from anaerobic, and chemical reduction

were quantified using benzidine as a standard at absorb-

ance maxima of 440 nm. The chemical reduction of azo

dyes was carried out with sodium dithionite. The condi-

tions of the reduction process are as follows: 0.06 g of dye

sample heated in boiling temperature with 1 M NaOHfor 1 h. After 30 min 0.6 g sodium dithionite was added.

A 100 mg/l sample of dye was reduced according to

method mentioned by Pielesz (1999). The aromatic

amine recoveries were calculated from the TAA values

of effluents to expected TAA in influent samples.

2.5. Statistical analysis

The values given in figures are mean ± standard devi-

ation (SD) and n is the numbers of samples. Error bars

represent the SD.


Regression analysis between y and x variables was

performed using the EXCELL in Microsoft Windows�(HP, USA). The linear correlation was assessed with r2.

The r2 value is the correlation coefficient and reflects sta-

tistical significance between dependent and independent

variables.

3. Results and discussion

3.1. Effect of glucose-COD on reactor performance

The effect of glucose-COD on the color and the COD

removal efficiencies are shown in Fig. 3. Color was re-moved very effectively (approximately 100%), at all glu-

cose-COD concentrations varying between 100 and 500

mg/l. This shows that the electrons required for the

reductive cleavage of azo dyes was not depend to glu-

cose-COD through decolorization under anaerobic con-

ditions. A slight linear relationship between color

removal and COD concentrations was observed for

the r2 values of 0.42 but it was not statistically significant(P = 0.34 and F = 1.47). The electrons providing the re-

duced environment appear to be sufficient for the cleav-

age of the azo bond at COD concentrations as low as

100 mg/l for complete decolorization. Both COD levels

were able to perform more than 98–99% color removal

in CR dye. In Run 5, the co-substrate was excluded in

the influent whether CR azo dye could be degraded as

the sole carbon and energy source by the anaerobicmicroorganisms in the UASB reactor. As shown in

Fig. 3, almost complete (99%) decolorization was

achieved even at substrate-free operation. This shows

that the dye and the degradation products could be used

as a carbon source by methanogens as reported by

Manu and Chaudhari (2002). The abiotic decolorization

studies with nonviable granulated sludge indicated that

no significant color removals (E = 1–2%) were observedin co-substrate containing and co-substrate free samples.

0

10

20

30

40

50

60

70

80

90

100

500 250 100 0Glucose-COD concentration in feed (mg/l)

CO

D a

nd c

olor

rem

. eff

. (%

)

0

2

4

6

8

10

12

TA

A c

onc.

(m

g be

nzid

ine/

l)

COD rem. eff. Color rem. eff. TAA conc.

Fig. 3. Effect of glucose-COD on color, COD removals, and TAA

concentrations in UASB reactor (mean ± SD, n = 3).

This indicates that the dye decolorization occurs only

with biodegradation.

The COD removal efficiencies decreased from 78 to

68% as the glucose-COD concentrations decreased from

500 to 100 mg/l. This could be attributed to a low co-

substrate concentration which was a limiting factor sincethe methanogens have very low growth rates, and their

metabolism is usually considered rate limiting in the

anaerobic processes (Bras et al., 2001; Beydilli et al.,

1998; Lun et al., 1995). When the co-substrate was not

a limiting factor (3000 mg/l of glucose-COD), high

COD removal efficiencies (88%) were obtained (I+ıkand Sponza, 2002). In Run 5 without co-substrate oper-

ation low COD removal efficiencies (28%) could bemainly attributed to the inert COD originated from

the CR dye together with the anaerobic extracellular

products as reported by Germirli et al. (1991). Regres-

sion analysis indicated that the linear relationship be-

tween glucose-COD and COD removal efficiency was

not statistically significant (r2 = 0.69, P = 0.15 and

F = 5.14).

As shown in Fig. 4, the methane production rates de-creased from 445 to 125 ml/day while the glucose COD

in the feed was reduced to 100 from 500 mg/l. The meth-

ane production was recorded as 73 ml/day at substrate-

free operation. The COD dependence of methane gas

production was statistically significant (r2 = 0.99,

P = 0.004 and F = 236).

The concentrations of TAA varied between 8.2 and 9

mg benzidine/l in the effluent samples at glucose-CODconcentrations of 100 and 500 mg/l, respectively, in the

feed. No significant differences in TAA concentrations

(7.6 mg benzidine/l) were obtained through decoloriza-

tion of CR dye while the UASB reactor was fed without

co-substrate. Regression analysis indicated that there

was no linear relationship between TAA and COD con-

centrations (r2 = 0.03, P = 0.82 and F = 0.03). TAA and

COD removal efficiencies were observed to be 43% and28%, respectively, in substrate-free operation (see Fig.

4). The CR dye was degraded to aromatic amines

0

20

40

60

80

100

500 250 100 0Glucose-COD concentrations (mg/l)

TA

A r

emov

al e

ffic

ienc

y (%

)

0

100

200

300

400

500

600

Met

hane

pro

duct

ion

rate

(m

l/day

)

TAA Methane production rate

Fig. 4. Effect of glucose-COD on the TAA removal efficiencies and

methane production rates in UASB reactor (mean ± SD, n = 3).


through the cleavage of dye chromophore. The color re-

moval was provided by the electrons produced from the

TAAs, which can be used as carbon and energy sources

through anaerobic decolorization. These findings agree

with the studies performed by Razo-Flores et al.

(1997). They indicated that the reducing environmentwas provided by the electrons released from the aro-

matic amines through the cleavage of azo dye azodisal-

icylate (ADS). This dye was used as carbon and energy

sources by the methanogens in the UASB reactor feed-

ing with glucose-free wastewater. As aforementioned,

the aromatic amines provides electrons supporting the

reduction of the dye resulting in complete decolorization

even during substrate-free operation. The TAA removalefficiencies were 40% and 38% at COD concentrations

500 and 100 mg/l, respectively, indicating the partial

degradation of benzidine under anaerobic conditions

(see Fig. 4). Supporting these findings, Kalyuzhnyi

et al. (2000) and Razo-Flores et al. (1997) also reported

that the aromatic amines could be further mineralized

under anaerobic conditions in an UASB reactor. O�Con-

nor and Young (1993) found that some aromatic amines(2-aminophenol, 4-aminophenol) can be mineralized by

methanogenic consortia. However, Brown and Labour-

eur (1983), Haug et al. (1991) and Bras et al. (2001)

showed that aromatic amines are not degraded anaero-

bically in co-substrate containing and co-substrate-free

reactors.

3.2. Differences in absorbance spectra in influent and

effluent samples at various co-substrate levels

The absorbance spectra at varying wavelengths in the

samples taken from the UASB reactor effluent in the dif-

ferent runs and in the feed containing 100 mg/l of CR

dye with mineral medium is illustrated in Fig. 5. These

spectra exhibited marked alterations as exemplified in

the aforementioned figure. These changes may be ex-plained by structural modifications of the CR dye mol-

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

300 400 500 600

Wavelengt

Abs

orba

nce

of e

fflu

ents

3000 mg/l Glucose-COD and dye-free

glucose-COD free and 100 mg/l CR

Fig. 5. Visible absorbance spectra of CR in influent and efflue

ecule due to azo bond reduction under anaerobic

conditions. The absorbance peak in the influent (feed)

samples of the UASB reactor was obtained at a wave-

length of 497 nm while the absorbency peak was at a

wavelength of 340 nm in the effluents of UASB reactor

through Runs 2 and 5. In other words, the absorbancepeak at 497 nm completely disappeared after the feed

decolorized in the UASB reactor. This suggests that a

transformation of CR dye to dye-intermediates had

taken place under anaerobic conditions. The absorbance

levels in the effluent samples of the UASB reactor were

higher (0.37) compared to that of dye-free samples

(0.26). This shows that the aromatic amines produced

through the cleavage of the azo bond were not ulti-mately degraded. The partial degradation of TAAs in

the anaerobic UASB reactor resulted in higher absorb-

encies in the effluent samples containing dye compared

to dye free samples as reported by Rajaguru et al.

(2000). This suggests a biological mineralization of

amines. Since the the aromatic amine removal efficien-

cies varied between 25% and 42% (see Fig. 4) it can be

conluded that most of the aromatic amines released weremetabolized in UASB reactor. Razo-Flores et al. (1997),

Rajaguru et al. (2000), Kalyuzhnyi et al. (2000) and I+ıkand Sponza (2002) demonstrated the degradability of

azo dye metabolites under anaerobic conditions.

3.3. Effect of NaHCO3 alkalinity on reactor performance

Traditionally, the total alkalinity in an anaerobic di-gester includes all the bicarbonate alkalinity and

approximately 80% of the volatile fatty acids. Because

only bicarbonate alkalinity is usable for neutralizing vol-

atile acids, total alkalinity does not always represent the

available buffering capacity in a digester (Anderson and

Yang, 1992). A stable anaerobic treatment system re-

quires a balance among all organisms. The maintenance

of this balance is normally indicated by a low VFA con-centration and a stable pH. When the anaerobic system

700 800 900

h (nm)

0.00.30.50.81.01.31.51.82.02.32.52.8

Abs

orba

nce

of f

eed

500 mg/l glucose-COD and 100 mg/l CR

Feed

nts of reactor at different glucose-COD concentrations.

0

500

1000

1500

2000

2500

3000

3000 1500 750 550 250

NaHCO3 concentration (mg/l)

Met

hane

pro

duct

ion

rate

(m

l/day

)

0

2

4

6

8

10

12

14

16

18

20

TV

FA

/B.A

lk *

10 a

nd p

H

Methane production VFA/B.Alk*10 pH

Fig. 7. Effect of decreasing concentrations of NaHCO3 alkalinity on

TVFA/Alk ratio, methane productions and pH levels (mean ± SD,

n = 3).


is in balance, the methanogens could be inactivated by

unfavorable environmental conditions, e.g., pH decrease

due to insufficient alkalinity, accumulation of VFA and

toxicity of intermetabolites (Kuai et al., 1998).

The effect of bicarbonate alkalinity on the COD, col-

or removal efficiencies and VFA productions in theUASB system is illustrated in Fig. 6. Color was effec-

tively removed (greater than 99%) at all NaHCO3 con-

centrations. No significant differences in COD removal

efficiencies (E = 84–88%) were obtained while the alka-

linity decreased to 550 from 3000 mg/l. The NaHCO3

was not a significant parameter influencing the COD re-

moval. Regression analysis indicated that the linear rela-

tionship was not statistically significant (r2 = 0.14, P =0.52 and F = 0.51). However, the COD removals de-

creased to 68% while the alkalinity decreased to 250

mg/l in the feed since the bicarbonate alkalinity could

not buffer the pH in the UASB reactor. Similarly, the

VFA concentrations increased from 81 to 403 mg/l as

the NaHCO3 concentrations in feed decreased to 250

mg/l from 3000 mg/l. No statistically significant linear

relationship was observed between NaHCO3 and VFAconcentrations (r2 = 0.26, P = 0.37 and F = 1.07).

If a UASB reactor is stable, the TVFA/B.Alk ratio of

reactor effluent is lower than 0.4 (Behling et al., 1997).

The TVFA/B.Alk. ratios were between 0.03 and 0.28

as the NaHCO3 concentrations varied between 550

and 300 mg/l in the feed (see Fig. 7). However, this ratio

increased to 0.68 when the NaHCO3 concentrations de-

creased to 250 mg/l in the feed, indicating the instabilityof the UASB reactor. The regression analysis indicated

that the linear relationship between NaHCO3 concentra-

tion and the TVFA/B.Alk. ratio was not statistically sig-

nificant (r2 = 0.32, P = 0.31 and F = 1.43). The pH

ranges decreased from 7.3 to 6.2 as the bicarbonate alka-

linity decreased from 3000 to 250 mg/l (see Fig. 7). The

regression analysis indicated that the linear relationship

between pH and B.Alk. was statistically significant(r2 = 0.74, P = 0.06 and F = 8.66). The well being of a

0102030405060708090

100

3000 1500 750 550 250


CO

D a

nd c

olor

rem

oval

eff

. (%

)

0

100

200

300

400

500

TV

FA

(m

g C

H3C

OO

H)

COD removal eff. Color removal eff. TVFA


COD, color removal efficiencies, and VFA concentrations in UASB

reactor (mean ± SD, n = 3).

UASB reactor can often be judged by the effluent pH.

The pH in a normally functioning reactor is close to

neutral and controlled by a system that consist of bicar-

bonate and volatile fatty acids. Although acidogenic

phase was not preferred as dominant phase, the color

could be removed together with acidogenic and metha-

nogenic consortia in the buffered anaerobic reactors

(Behling et al., 1997; Chinwetkitvanich et al., 2000).TAAs released through the cleavage of CR dye and

corresponding removal efficiencies and recoveries are

shown in Fig. 8. The TAA concentrations increased to

10.1 from 5.83 mg/l as the bicarbonate concentration

in influent was increased from 250 to 3000 mg/l. CR

breakdown products such as benzidine were greatly re-

moved and the corresponding aromatic amine was

recovered by less than 44%. This indicates that the aro-matic amines released from azo dye cleavage was being

partly metabolized.

As shown in Fig. 8, no significant difference in meth-

ane production rates (2000–2400 mg/l) was observed as

the NaHCO3 concentrations decreased from 3000 to

0

2

4

6

8

10

12

3000 1500 750 550 250


TA

A c

once

ntra

tion

(

mg

benz

idin

e/l)

0

20

40

60

80

100

TA

A r

ecov

erie

s (%

)

TAA concentration TAA recoveries


the TAA productions and recoveries (mean ± SD, n = 3).

Table 2

Variations of alkalinity in influent and effluent samples in UASB reactor (mean values, n = 3)

NaHCO3 mg/l in feed As mgCaCO3/l equivalent Effluent bicarbonate alkalinity (mg CaCO3/l) Effluent total alkalinity (mg CaCO3/l)

3000 dye-free 1786 1857 1870

3000 1786 2387 2406

1500 893 1510 1556

750 446 1127 1133

550 327 682 720

250 149 378 595


550 mg/l in the feed. The regression analysis indicated

that the linear relationship between methane production

and NaHCO3 concentrations was not statistically signif-

icant (r2 = 0.24, P = 0.46 and F = 2.66). Since 550 mg/l

of NaHCO3 concentration (as 327 mg CaCO3/l) pro-

vided the optimum buffering capacity to effectively con-

vert glucose-COD to methane and VFA, it could be

suggested as the optimum bicarbonate alkalinity con-centration in influent to maintain the pH above 6.6. This

value corresponds to an Alk./COD ratio of 0.163 for

optimum operation, which is lower than the ratios pro-

posed by Gonzalez et al. (1998) (0.4), Souza et al. (1992)

and Moosbruger et al. (1993) (0.5), and Speece (1996),

(1.2). The total and bicarbonate alkalinity levels in the

influent and effluent samples of the UASB reactor are

summarized in Table 2. Increases in alkalinity concen-tration was observed in the effluent of the UASB reactor

compared to influent samples. For example when 1500

mg/l NaHCO3 (93 mg CaCO3/l) was added to the feed

the bicarbonate and the total alkalinity concentrations

were 1510 and 1556 mg CaCO3/l, respectively. This

shows that the alkalinity was regenerated in the upper

layer (effluent) of UASB reactor. The VFA will be con-

sumed more rapidly than they can be produced by themethanogens and subsequently the VFA were converted

to methane. The VFA alkalinity will be regenerated to

bicarbonate alkalinity. Furthermore, since the NH4-N

concentration (NH4Cl = 400 mg/l) in the feed is high,

00.1

0.2

0.30.40.50.6

0.70.80.9

300 400 500 600

Wavelength

Abs

orba

nce

of e

fflu

ents

3000 mg/l NaHCO3-CR free1500 mg/l NaHCO3-100 mg/l CR250 mg/l NaHCO3-100 mg/l CR

Fig. 9. Visible absorbance spectra of CR in influent and effl

the generated ammonium bicarbonate alkalinity con-

tributes to total alkalinity in effluent samples. However,

since the reserve alkalinity is the bicarbonate alkalinity

that maintains the pH above 6.2–6.6 in the UASB reac-

tor, it can be assumed that this alkalinity was provided

in our system since the lowest pH level recorded was 6.3.

3.4. Differences in absorbance spectra in influent and

effluent samples at various bicarbonate alkalinity levels

The change of UV–visible spectra of the influent

(feed) and effluent samples containing different bicarbo-

nate alkalinity levels in the UASB reactor are displayed

in Fig. 9. The feed spectrum has a peak in the visible re-

gion at 560 nm, which accounts for the color and a peak

at 390 nm. In the anaerobic treated samples containingdifferent bicarbonate alkalinity levels and in the dye-free

samples, there is a peak in the visible region at spectra

380 nm. The absorbance peak at 560 nm completely dis-

appeared after anaerobic decolorization. This indicates

that CR dye was degraded and exhibited maxima

absorbance peak at 380 nm. Similarly the absorbance

spectrum in the dye-free samples was obtained at a

wavelength of 380–390 nm. Although different bicarbo-nate alkalinity and dye containing UASB reactor efflu-

ent samples exhibited similar maxima spectra the

observed absorbance levels was found to be quite differ-

ent. For example the absorbance levels increased to 0.88

700 800 900

(nm)

0.00.30.50.81.01.31.51.82.02.32.52.8

Abs

orba

nce

of f

eed

3000 mg/l NaHCO3-100 mg/l CR550 mg/l NaHCO3-100 mg/l CRFeed, 100 mg/l CR

uents of reactor at different NaHCO3 concentrations.


from 0.4 as the bicarbonate alkalinity levels increased to

1500 mg/l from 250 mg/l in the effluent of UASB reactor

with the exception of 3000 mg/l of bicarbonate alkalinity

containing effluents (A = 0.65). This findings demon-

strated that the alkalinity could change the dominated

acidogen and methanogen bacteria resulting in modifi-cations in chemical structure of the intermetabolite

products released through cleavage of CR. The modifi-

cations in the bacterial genus and species from methano-

genic and acidogenic bacteria probably affect their

metabolic pathway through degradation of CR dye

resulting in different metabolic products.

4. General discussion

The findings of this study showed that the decoloriza-

tion of CR dye in an UASB reactor did not correlate

with COD concentrations, COD removal or bicarbonate

alkalinity. It seems that the reducing environment pre-

vailing in the UASB reactor by the electrons releasing

from the CR dye and its inter-metabolite products,mainly from the aromatic amines provides the color

removal.

Although some studies showed that the addition of

an external carbon source to the anaerobic reactor im-

proved the color removal which helps ascertain a reduc-

ing environment and possible increase in the

concentration of enzyme cofactors (Carliell et al.,

1995; Gingell and Walker, 1971; Banat et al., 1996;Brown and Laboureur, 1983; Chinwetkitvanich et al.,

2000), other studies have indicated that the dyes can

be used as sole carbon source (Chang et al., 2001; Chung

and Stevens, 1993; Razo-Flores et al., 1997; Kalyuzhnyi

et al., 2000; Blumel, 1998; Coughlin et al., 1997) which

are in agreement with findings in this study.

Several researchers have shown that the aromatic

amines could be decolorized under anaerobic conditions(Razo-Flores et al., 1996, 1997; Battersby and Wilson,

1989; O�Connor and Young, 1993). The results of this

study showed that partial mineralization of benzidine re-

leased from the cleavage of CR azo dye provides elec-

trons to support continued reduction of CR dye,

supporting the published references given above. How-

ever, these results conflict with the studies performed

by Haug et al. (1991), Brown and Hamburger (1987),and Field et al. (1995) indicating the aromatic amines

could not be degraded further in anaerobic reactors.

In a UASB system treating textile wastewaters con-

taining azo dye, alkalinity is lost through NH4-N con-

version to organic nitrogen, the buffering of H2CO3

acidity and the VFA generation by the granules. The

VFA that was generated increased the acidity allowing

the partial phase separation of acidogenesis from meth-anogenesis in the lower active zone in UASB reactor. In

the upper active zone of the granular bed the VFA con-

verted to methane, regenerating the alkalinity and

increasing the pH.

The textile industry in Turkey is one of the most

important industrial sectors both in terms of its contri-

bution to economy and environmental emissions. There-

fore, besides cost-effective anaerobic treatment process,additional external carbon sources and alkalinity

requirements should be kept in mind for economical

costs. If alkalinity is required to buffer the textile indus-

try wastewaters containing azo dyes, this could ad-

versely affect the economy of the anaerobic treatment

(Speece, 1996). However, this study showed that 100

mg/l of CR azo dye could be used as substrate and a

bicarbonate alkalinity as low as 500 mg/l is enough forsimultaneous decolorization (E = 100%) and COD re-

moval (E = 56).

5. Conclusions

Congo Red could be completely decolorized under

glucose-COD concentrations as low as 100 mg/l. In thecase of co-substrate-free operation, greater than 99%

color removal was achieved. In the COD-free operation

the CR dye was used as a carbon and energy source

by the anaerobic microorganisms. Meanwhile, the azo

bond was cleaved initially, aromatic amines was pro-

duced second and then the color removal could be main-

tained by TAAs which are used as carbon end energy

source. Fifty-eight percent COD, 100% color, 39%TAA removal efficiencies and 380 ml/day methane pro-

duction rates were obtained in 100 mg/l COD concentra-

tion as external co-substrate while 25% COD, 99%

color, 40% TAA removal efficiencies and 320 ml/l meth-

ane production rates were monitored in co-substrate free

operation.

The results of this study showed that the alkalinity

requirement is 0.16 HCO3Alk/g influent COD providing84% COD and 99% color removals at a pH 6.5 through

decolorization of 100 mg/l CR dye with 2000 mg/l glu-

cose-COD in the UASB reactor. Moreover, the methane

production rate was 2000 ml/day and the VFA was neu-

tralized resulting in a VFA concentration of 250 mg/l in

the effluent. Although no significant differences in pH

and methane production rates were observed up to a

NaHCO3 concentration of 550 mg/l in the influent ofUASB reactor, the color was effectively (greater than

99%) removed at all NaHCO3 concentrations.

Acknowledgments

The Turkish Scientific and Technical Research Coun-

cil (TUBITAK) and Turkish Government PlanningInstitution (DPT) and Dokuz Eylul University Research

Foundation funded this project. The authors would like


to thank them for the financial support to project with

grant numbers 199 Y 110 and 0 908 20 0001.

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