Degradation Profile of Phenol in Sequential Batch...

Degradation Profile of Phenol in Sequential

Batch Reactor

Farah Khan, Mohammad Zain Khan and Suhail Sabir* Environmental Research Laboratory, Department of Chemistry,

Aligarh Muslim University, Aligarh-202002, India

E-mail: [email protected]

Tel/Fax: 0915712703515

Abstract

Compact well settling granules were cultivated for degrading 3.9 kg m-3

d-1

of phenols. Formed granules were studied qualitatively using SEM and it was found

that a large number of bacterial types including bacterial rod, cocci, diatoms are

dispersed in the extracellular polymeric substance (EPS). Degradation studies show

that initially the removal rate of phenol was fast but later on it becomes slow. Low

concentration of 50 mg l-1

, 100 mg l-1

, 200 mg l-1

phenol comes down below detection

limit in about 170 min of the SBR cycle but high concentration of 400 mg l-1

, 650 mg

l-1

took around 240 min for complete removal. This study demonstrates the utilization

of aerobic granulation for treating high concentration of phenol and other xenobiotics.

Keywords: Granules, Degradation, Sequential batch reactor, Aerobic sludge.

Introduction

Various industries such as oil refineries, petrochemical plants, coke

conversion, pharmaceuticals and resin industries produces many toxic substances as

their effluent, phenol being one of it. Phenol is also most frequently found pollutant in

rivers and land fill runoff water (Prasad and Ellis, 1978). Phenol concentration of up

to10 000 mg l-1

has been reported in much industrial waste water (Fedorak and

Hrudey, 1988).

Phenol is toxic at relatively low concentrations and is listed as priority

pollutants by the United States Environment Protection Agency (Ghisalba, 1983). The

toxicity of phenol often results in the reduction of wastewater biotreatment even at

relatively low concentration (Hinteregger et al., 1992).

A wide range of adverse effects have been reported following well

documented human exposure to phenol by dermal, oral or intravenous routes. Phenols

are also toxic to some aquatic species at low concentration as low as 1 mg l-1

(Brown

Environment & We

An International

Journal of Science

& Technology

Available online at www.ewijst.org

ISSN: 0975-7112 (Print)

ISSN: 0975-7120 (Online)

Environ. We Int. J. Sci. Tech. 5 (2010) 123-135

Khan et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 123-135

124

et al., 1967) and far lower concentration caused taste and odor problem in drinking

water (Rittmann and McCarty, 2001). Hence the removing phenol is significant.

For the removal of aromatic compounds from the ground water various

physical, chemical and biological methods are used (Strier et al., 1980; Atlas et al.,

1981; Kobayashi et al., 1982; National Academy of Sciences 1983; Gills et al., 1986).

Biological methods have advantage over physical and chemical methods. In chemical

and physical methods the intermediate formed are even more toxic than the original

compounds (Strier et al., 1980 and Gils et al., 1986). In the case of phenol the use of

non biological methods like solvent extraction, mineralization etc. suffers from high

cost and formation of hazardous by-products (Loh et al., 2000). Because of lower cost

and possibility of complete mineralization and complete oxidation to carbon di oxide

and water, biological methods are most commonly used. Phenolic wastewater is

treated in activated sludge process. This system is sensitive to high phenol loading

rates and to fluctuations in phenol loadings (Kibret et al., 2000 and Watanabe et al.,

1999). These difficulties arise because of substrate inhibition, whereby growth (and

consequently phenol degradation) is inhibited because of phenol toxicity.

The inhibition difficulties associated with high strength phenolic wastewater

can be overcome by strategies such as bioagumentation (Watanabe et al., 2002) and

the other one is cell immobilization (Keweloh et al., 1989). Aerobic granulation

technology (a cell immobilization technique) has been developed for treating a wide

variety of wastewaters (Peng et al., 1999; Moy et al., 2002; Lin et al., 2003; Yang et

al., 2003). This technology is also been extensively investigated (Morgenroth et al.,

1997; Beun et al., 1999; Tay et al., 2001; Liu and Tay 2004). Most studies have either

focused on granule cultivation through various substrates or treating efficiency of

various granule processes (Arrojo et al., 2004; Kim et al., 2004; McSwain et al., 2004;

Wang et al., 2004; Hu et al., 2005).

Aerobic granulation represents a cell immobilization technology that has

attracted recent research attention (Tay et al., 2001, 2002). Without the inert support

for biofilm attachment aerobic granules that are microbial aggregate are cultivated in

SBR. The compact microbial structure can confer on aerobic granules the good

settleability and the high biodegradation capacity for toxic and recalcitrant

compounds (Jiang et al., 2002; Tay et al., 2005a; Yi et al., 2006). Flocculated

activated sludge fed with phenol as sole carbon source aerobic granules has been

successfully cultivated (Jiang et al., 2002). With the kinetic data it is indicated that

phenol degrading aerobic granule have potential to treat wastewater with high phenol

loading and also possess highly active compact structure and good settleability.

The main object of this work is to study the aerobic degradation profile of

phenol through an aerobic granulation technology by a sequential batch reactor

(SBR).

Materials and Methods

Reactor set up and operation

A column type sequential aerobic sludge blanket reactor (5 cm diameter and

150 cm high) made of transparent perfex glass with total volume of 1.5 l (working


125

volume 1.4 l) was used. The reactor was housed in a thermostat at about 30±20C. The

reactor was fed with phenol as a sole carbon source and was left open for the growth

of natural mixed population. At the bottom of column fine bubble aerator was fitted

for supplying air at superficial air velocity of 2.67 cm s-1

. Reactor was operated

sequentially in 4 hrs cycle (2 min of influent filling, 205-230 min of aeration, 2-30

min of settling and 5 min of effluent withdrawal). To avoid excess loss of sludge, the

reactor was initially operated at high settling time of 30 min. Later the settling time

was reduced to 10 min and finally to 2 min. At the initial stage, feeding time was kept

high which was decreased gradually as the operation proceeds. The reactor set-up

included two sampling port, arranged along the height of column type reactor, one at a

distance of 70 cm and, other at 30 cm from the bottom of the reactor. The effluent was

withdrawn through 70 cm port at a volumetric exchange ratio of 50% giving a

hydraulic retention time (HRT) of 8 hrs. The HRT of 8 hrs was kept constant during

the analysis. Port at 30 cm height was used for the collecting samples for

MLSS/MLVSS determinstion and periodical sludge wasting. The operation proceeded

with abiotic losses of phenol which were negligible under identical operation

condition.

Seed sludge and wastewater

The source of aerobic digested sludge with mixed liquor suspended solid

(MLSS) and mixed liquor volatile suspended solid (MLVSS) content of 3.5 gm l-1

and

0.43 gm l-1

respectively was taken from secondary clarifier of Star Paper Mills

Saharanpur, India. For the period of first 15 days, it was conditioned in an aeration

tank where the content was fed with phenol as an only source of carbon along with

nutrient (Jiang et al., 2002) and micronutrient (Moy et al., 2002). The acclimatization

towards phenol was done in a high precision water bath at a temperature of 30±20C.

After the period of acclimatization, the aeration tank was fed in sequential batch

reactor and started with 50 mg l-1

phenol in mineral salt medium which was gradually

increased to 650 mg l-1

in a stepwise process.

Wastewater feed

A stock solution of phenol was prepared having concentration of 10 gm l-1

(10000 mg l-1

). The desired concentrations of phenol for experiments were obtained

by proportionate dilution with double distilled water and fed into SBR along with

nutrients solution) with following composition (Moy et al., 2002) Table 1. Stock

solution of micronutrients was prepared at 1 000 times concentration (Table 1). For

optimal growth 1 ml of this solution was added to the feed solution. SBR was

operated at a constant temperature of 30±20C and the pH was maintained at around

7.5-7.7 throughout the study.

Microbial granulation depends upon factors such as pH and temperature. A

slight alkaline pH (around 7.5) is also necessary for proper aerobic granulation and

granulation cease to occur at pH> 8.5 as reported by (Hailei et al., 2006). Yang et al.,

(2008) reported that a slight low pH favors fungi dominating granules where as a

slight alkaline pH favors bacterial granules. Temperature change also affects the

reactor performance upto a certain degree. Song et al., (2009) reported that 300C is

optimum temperature for matured granule cultivation.


126

Table 1 Nutrients composition as given by Moy et al., 2002.

Nutrients Amount required (gm l-1

)

Macro Nutrients

KH2PO4 1.35

K2HPO4 1.65

MgSO4.7H2O 0.13

NH4Cl 0.20

Micro Nutrients

H3BO3 0.05

ZnCl2 0.05

CuCl2 0.03

MnSO4.H2O 0.05

Mo7O24.4H2O 0.05

AlCl3 0.05

CoCl2.6H2O 0.05

NiCl 0.05

Analytical Methods

Measurement of pH, suspended solids, mixed liquor suspended solids

(MLSS), mixed liquor volatile suspended solids (MLVSS), chemical oxygen demand

(COD) were conducted in accordance with the Standard Methods (APHA, 1998). The

phenol concentrations were determined spectrophotometrically using the absorbance

values at 500 nm λmax with a UV/VIS spectrophotometer (GENESYS 20,

Thermospectronic) according to APHA 1995.

Optical density (O.D.600) was calculated using UV/VIS spectrophotometer at

600 nm as specified by Ziagova et al., 2007. O.D. 600 gives density of the microbes

present in the SBR. All the experiments were performed in duplicate.

Morphology and surface structure of granules were observed qualitatively

with a scanning electron microscope (STEREO SCAN 360). Granules were prepared

for SEM image by washing with a phosphate buffer and fixing with 2%

glutaraldehyde overnight at 40C. Fixed granules were washed with 0.10 M sodium

cacodylate buffer, dehydrated by successive passages through 25, 50, 75, 80, 90, 95

and 100% ethanol and dried with a CO2 Critical Point Dryer.

Result and Discussion

Phenols are most common compounds, distributed in waste discharge of pulp

and paper industries. The microorganisms are already acclimatized for phenol and its

derivatives through natural selection. In our experiment conditioning period was

reduced due to already acclimatized sludge. This is the reason for choosing the

aerobic sludge from Star Paper Mills Saharanpur, India. However, for getting more

specific microbes, the sludge was initially acclimatized for about 15 days to allow

biomass to adopt phenol. The SBR cycle was performed 45 days.


127

Microbial density in SBR system (O.D.600)

Optical density (O.D. 600) gives the density of microbes present in the SBR.

Figure 2 shows the O.D.600 of the acclimatized sludge is around 0.63 which increases

after inoculating in to SBR. This acclimatized sludge shows two sharp decreases on

day 13 and day 23 due to reduction settling time from 30 to 10 min and from 10 to 2

min. From first day to day 12, when the settling time was 30 min, O.D.600 increases

from 0.63 to 0.82, but on day 13 when the settling time was reduced to 10 min the

O.D.600 of SBR decreases to 0.69 and then increases gradually up to day 22 with same

settling time. There is again a decrease in the in the value of O.D.600 to 0.98 on day 23

due to reduction in settling time to 2 min and then the value of O.D.600 increases for

rest of the cycle till day 38 and then stabilized at 1.65.

Figure 1 Sequencing batch reactor (SBR) design principle.

Shape and Size of Microbial Granule

In SBR stable granule were obtained. Stable granules were compact and strong

but not much spherical. However, high degradation efficiency of granules is due to

their compact structure (Khan et al., 2009) and not effected by their shape. It has also

been proved earlier that granules have a high resistant to toxic xenobiotic due to their

compact structure (Glancer et al., 1994; Jiang et al., 2002, Tay et al., 2005b, Bergsma-

vlami et al., 2005) The diameter of formed granules is about 1-2 mm. Figure 3 shows

close packed structures with evenly distributed channels which facilitates movement

of waste water inside the granules.


128

0 5 10 15 20 25 30 35 40 45

0.6

0.8

1.0

1.2

1.4

1.6

1.8

SB

R O

.D. 6

00

Days

Figure 2 O.D.600 of SBR during degradation of Phenol.

Effluent and Influent concentration

Figure 4 shows concentration graph of effluent and influent with respect to

number of days. According to the graph, initially the concentration of effluent is high

27.56 mg l-1

due to the absence of compact granules although the concentration of

influent added was quite low 52 mg l-1

. The concentration of effluent did not

decreases remarkably in the first few days of the SBR cycle but decreases gradually

for the rest of the cycle inspite of being increase in the influent concentration from 52

to 656 mg l-1

.

Effluent and Influent COD (mg l-1

)

Figure 5 shows COD graph of effluent and influent Vs days. This graph

shows that initially, the COD of effluent was high 215 mg l-1

even though the COD of

influent was 516 mg l-1

, because the lack of stability in aerobic granules. After few

days of operation, compact granules were formed and the COD of influent was very

much high but the effluent COD was low. At the end of cycle, influent and effluent

COD were 7418 and 263.31 mg l-1

respectively.

Phenol Removal Profile

The degradation profile shows that initially the degradation of phenol was

high later on degradation rate decreases till the end of cycle (Figure 6). This is due to

the fact that initially microorganism has large amount phenol available, which is food

for them, so they degrade it faster in the beginning. During the first 60 min of 240 min

cycle (with 50% volumetric exchange giving 8 hrs HRT) sufficient amount of phenol

was removed but in next 180 min its concentration reduces to as low as 2 mg l-1

.

Figure 4 also shows that concentration of 50, 100 mg l-1

of phenol decreases to below

detection limit in just 150 min but high concentration of phenol (200, 400, 650 mg l-1

)

took around 220-230 min for complete removal. Just after addition of influent, a sharp


129

decrease in phenol concentration was observed (as shown in Figure 6 first 20 min),

this is due to dilution.

Figure 3. SEM image of mature granule at 15000 magnifications.

0 10 20 30 40 50

0

100

200

300

400

500

600

700

Co

nce

ntr

ati

on

(m

g l

-1)

Days

Influent

Effluent

Figure 4 Concentration of Effluent and Influent VS time.

COD Removal Profile

Similarly the removal rate of COD was high in the beginning but later on it

goes on decreasing. Just after addition of influent in the SBR there is sharp decrease

in COD which is attributed to dilution but this value of COD further increases to give

COD concentration below 50 mg l-1

by microbial action (Figure 7).


130

0 10 20 30 40 50

0

1000

2000

3000

4000

5000

6000

7000

8000

CO

D (

mg

l-1

)

Days

Influent

Effluent

Figure 5 COD of Effluent and Influent Vs Time.

COD Removal Efficiency with Phenol Concentration

Figure 8 illustrates that COD removal efficiency increases with phenol

concentration and attains maxima (94.54%) at 600 mg l-1

. This is because at high

concentration of phenol, influent COD was itself very high hence high removal

efficiencies were achieved by using the following formula

% COD removal = [(Influent COD - Effluent COD) / Influent COD] * 100

However, at very high concentration of phenol, removal efficiency decreases

owing to microbial inhibition caused by high substrate concentration.

Biomass Concentration (MLSS and MLVSS)

The initial sludge has MLSS concentration of 3.5 gm l-1

and MLVSS of 0.43

gm l-1

. After conditioning period, MLSS increases 5.2 gm l-1

and MLVSS to 0.82 gm

l-1

. Then the sludge was inoculated in to SBR for the cultivation of aerobic granules.

Aerobic granules developed along with amorphous flocs were first observed after 10

days of inoculation. However the biomass concentration of MLSS and MLVSS

reduces to 3.5 gm l-1

and 0.94 gm l-1

due to reduced settling time from 30 min to 10

min. The biomass with small settling velocity had been washed out of the reactor.

Small granules grew rapidly in subsequent week, while more floc like sludge washed

out from reactor gradually. The MLSS and MLVSS concentrations increases till 5.6

gm l-1

and 2.1 gm l-1

and again shows a sharp decrease, due to decrease in settling

time from 10 to 2 min on day 23. The granules become dominant form of biomass as

evident from gradual increase in biomass concentration beyond day 24. MLSS and

MLVSS concentration finally shows an upward trend and stabilizes at 7.3 gm l-1

and

4.1 gm l-1

(Figure 9).


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0 50 100 150 200 250

0

100

200

300

400

500

600

700

50 mg l-1

100 mg l-1

200 mg l-1

400 mg l-1

650 mg l-1

Time (min)

Ph

eno

l R

emo

va

l (m

g l

-1)

Figure 6 Concentration profile of phenol during single cycle.

0 50 100 150 200 250

0

1000

2000

3000

4000

5000

CO

D (

mg

l-1

)

Time (min)

50 mg l-1

100 mg l-1

200 mg l-1

400 mg l-1

650 mg l-1

Figure 7 COD removal profile of phenol during single cycle.


132

0 100 200 300 400 500 600 700

78

80

82

84

86

88

90

92

94

96

Per

cen

t C

OD

rem

ov

al

Phenol Concentration (mg l-1

)

Figure 8 COD removal efficiency VS phenol concentration.

0 5 10 15 20 25 30 35 40 45

0

1

2

3

4

5

6

7

8

Bio

ma

ss C

on

cen

tra

tio

n (

gm

l-1

)

Days

MLSS

MLVSS

Figure 9 Variation of MLSS and MLVSS concentration of Phenol during its

degradation

Conclusion

This study illustrate that compact aerobic granule cultivated in SBR can

tolerate fluctuation in COD load of 1000 mg l-1

and higher COD load of 7500 mg l-1

.

Optical density (O.D. 600) of SBR system was stabilized at 1.65 which shows


133

maximum retention of biomass. The concentration and COD of influent and effluent

shows that after the formation of compact granule phenol degradation was more

efficient. The COD removal efficiency was above 94%. Degradation profile illustrates

that during first hour of cycle removal rate of phenol and COD were very fast but later

on becomes slow. MLSS and MLVSS stabilized at 7.3 gm l-1

and 4.1 gm l-1

. The

process appears very useful for treating waste water containing phenols and other

toxic xenobiotics. This technology is advantageous as the same biomass can be used

over a long period. This technology can withstand high organic load as well as

fluctuation in organic load so it can be exploited for treating high strength municipal

and industrial waste both.

Acknowledgments

Authors wish to thank Prof. (Mrs.) A. Lal, Chairperson, Department of

Chemistry for providing necessary research facilities. F.K. and M.Z.K. thank UGC for

providing fellowship.

Authors' contributions: Farah Khan performed experiment, wrote the manuscript and peformed

calculations; and Mohammad Zain Khan performed a portion of experiment and helped in the

calculations and Dr. Suhail Sabir (Associate Professor), contributed in experiment design and final

editing of the manuscript;

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