Biodegradation of Phenol Review

8/11/2019 Biodegradation of Phenol Review

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Critical Reviews in Environmental Science and Technology, 42:16311690, 2012Copyright Taylor & Francis Group, LLCISSN: 1064-3389 print / 1547-6537 onlineDOI: 10.1080/10643389.2011.569872

Aerobic Biodegradation of Phenols: A Comprehensive Review

TAGHREED AL-KHALID and MUFTAH H. EL-NAAS Department of Chemical and Petroleum Engineering, UAE University, Al-Ain,

United Arab Emirates

Phenol and its derivatives are hazardous pollutants that are highly toxic even at low concentrations. The management of wastewater containing high concentrations of phenols represents major eco-nomical and environmental challenges to most industries. Biotech-nology has been very effective in dealing with major environmental challenges through utilizing different types of bacteria and bio-catalysts to develop innovative processes for the biodegradation,biotreatment, and biosorption of various contaminants and wide range of hazardous materials. Biological treatment has proved to be the most promising and most economical approach for the removal of many organic water pollutants such as phenol. Numerous studies have been published in the literature dealing with the biodegrada-tion of phenols utilizing different types of biomasses and different types of reactors. The authors offer a comprehensive review of the present research on the biodegradation of phenols and presents trends for future research and development, with emphasis on anintegrated approach that may be adopted to get synergistically en-hanced removal rates and to treat the contaminated efuents inan ecologically favorable process.

KEY WORDS: biodegradation, bioreactors, immobilization, kinet-ics, phenol, wastewater

1. INTRODUCTION

Chemical and petroleum industries generate a wide variety of highly toxicorganic pollutants, which have led to cumulative hazardous effects on the

Address correspondence to Muftah H. El-Naas, Department of Chemical and PetroleumEngineering, UAE University, PO Box 17555, Al-Ain, United Arab Emirates. E-mail: [email protected]

1631


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1632 T. Al-Khalid and M. H. El-Naas

environment. The efuents of these industries often contain aromatic organiccompounds that are rather resistant to natural degradation and therefore per-sist in the environment. This makes them capable of long-range transporta-tion and bioaccumulation in human and animal tissue. Organic pollutants

represent a potential group of chemicals that can be seriously hazardousto human health. 13 Many aromatic compounds show carcinogenic, terato-genic, or mutagenic properties. 4 Nonbiodegradable organic compounds mustbe pretreated into biodegradable or less toxic compounds.

Contamination of soil, surface water, and underground water by aro-matic organic pollutants such as phenol and its derivatives has caused greatconcern worldwide. Phenols are well known for their high toxicity for hu-man life, aquatic life, and others. 57 They are considered to be among themost hazardous contaminants, and they are certainly the most difcult toremove. 8

Phenol is a pollutant that is usually found in many industrial efuentssuch as wastewaters from coal processing plants, oil reneries, pulp andpaper manufacturing plants, resins and coke manufacturing, steel industries,pharmaceutical industries, plastic and varnish industries, textile units, pesti-cide plants, tannery, and smelting and related metallurgical operations. 9,10

Phenol may be fatal by ingestion, inhalation, or skin absorption, as itquickly penetrates the skin and may cause severe irritation to the eyes, themucous membranes, and the respiratory tract. 8 Oral exposure to phenol may cause severe damage to the liver and kidney and ingestion of 1 g of phenolis reported to be lethal to humans. 11

The concentration of phenols in wastewater may vary from 10 to300 mg/l, but this can rise to 4.5 g/l in highly polluted wastewaters. Moreover,it is possible that toxic polychlorinated phenols are formed when phenol-bearing water is chlorinated for disinfection. 1214 In addition to being poten-tial carcinogens, phenol and its derivatives are either toxic or lethal to sh atconcentrations of 525 mg/l. This imparts medicinal taste and objectionableodor to drinking water even at a much lower concentration of 2 g/l. 9,13Due to these adverse health effects of phenols, the World Health Organi-zation has set a guideline of 1 g/l to regulate the phenol concentration in

drinking waters.15

The high-volume use of phenols in the United States andtheir potential toxicity has led the U.S. Environmental Protection Agency todene them as priority pollutants 16 and has set a water purication standardof phenol concentration less than 1 g/l in surface waters. 14,17 The EuropeanCouncil Directive has set a limit of 0.5 g/l to regulate the phenol concen-tration in drinking waters. 18 It is worth mentioning that water policy in theEuropean Union is presently undergoing considerable change. Adopted mea-sures emphasize that surface water deterioration must be prevented, bodiesof water protected, and pollution from discharges of hazardous substancesreduced by 2015. 19 The legislations in the UAE limit the total phenols in

industrial water discharged to the marine environment to 0.1 mg/l.20


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Aerobic Biodegradation of Phenols 1633

Therefore, to save the soils and aqueous ecosystems, it has been manda-tory worldwide for industries to treat their wastewater efuents before safedisposal to the environment. Efcient treatment methods are available forthe removal of phenol such as activated carbon adsorption, ion exchange,

liquidliquid extraction, and chemical oxidation; however, they often sufferfrom serious drawbacks such as high cost. In addition, most of these tech-niques do not degrade phenol, but rather move it to another phase, whichresults in the formation of hazardous byproducts (secondary pollution). Onthe other hand, biodegradation is considered a more environmental friendly and cost-effective alternative. Biological treatment of phenols has thereforebeen an increasingly important process in pollution control. 2,21,22 Moreover,compared with physicochemical methods, the biodegradation method of phenol removal is universally preferred, because of the possibility of com-plete mineralization of phenol, 2,8 which results in complete conversion of acompound to its inorganic mineral constituents. 1

Efuents containing phenols are traditionally treated in continuous ac-tivated sludge processes with a relatively lower processing cost and nobyproducts. However, the practical application of this technology is ratherlimited because of its poor adjustability to uctuation in the phenolic load. 23The inability of conventional biological treatments to effectively removemany toxic pollutants indicates that novel biological treatment systems areneeded, and the use of pure and mixed cultures of organisms is considereda favorable and most promising approach. 7

Although there has been a considerable amount of research carriedon the biological treatment of wastewater containing phenolic compounds,there is a lack of comprehensive reviews of the published literature. Agarry et al.24 reviewed briey the mechanisms and kinetics of microbial degrada-tion of phenols, while Nair et al. 1 focused on the microorganisms mediatingbiodegradation of phenols and the mechanism involved. In this article weoffer a comprehensive review of the literature work on the aerobic biodegra-dation of phenols during the past decade, with emphasis on evaluating thepresent state of the area and exploring future research possibilities. Thereview addresses the following topics: theoretical aspects that cover basic

information on phenol structure and properties, microorganisms and mech-anisms of microbial pathways, and factors affecting biodegradation; recentadvances in the use of biological treatment focusing on kinetics, modeling,and reactor types; future scope and directions; and a concluding summary.

2. THEORETICAL ASPECTS

2.1 Phenol Structure and PropertiesPhenol is an aromatic hydrocarbon containing a hydroxyl group (OH) at-

tached to the benzene ring; it is a basic structural unit for a variety of


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FIGURE 1. Chemical structure of common phenolic compounds.

synthetic organic compounds. The chemical structures of phenol and somecommonly studied phenolic compounds are given in Figure 1.

As an organic substance, phenol is soluble in most organic solventsand it is slightly soluble in water at room temperature, but entirely watersoluble above 68 C.24 Phenols are distributed either as natural or articialmonoaromatic compounds in various environmental sites as major pollutants.Natural sources of phenol include forest and rangeland res and natural

decay of lignocellulosic material.1,21,24


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Because of the aromatic structure of phenol, it is resistant to naturalbiodegradation and phenolic compounds have been reported to have highstability due to the difculty of cleaving the benzene ring. However, sev-eral microorganisms can tolerate phenol and use it as a carbon and energy

source.25

The biological degradation is accomplished through benzene ringcleavage using the enzyme present in the microorganism. 9

2.2 Microorganisms in Phenol BiodegradationIn general, toxic and hazardous xenobiotics, which have a structure that isdifferent from naturally occurring compounds, are more difcult to degrade.In recent years, however, an array of microorganisms has been identiedthat use xenobiotics for their survival. 26

Depending on the microbial abilities to grow in specic conditions, or-ganic material can be degraded aerobically or anaerobically. 2729 Althoughaerobic and anaerobic microorganisms are able to degrade phenol, conventionally aerobic processes are preferred. 3032 Aerobic microorganisms aremore efcient for degrading toxic compounds because they grow fasterand usually transform organic compounds to inorganic compounds (CO 2,H2O).31,33,34 Aerobic processes are also preferred due to the low costs as-sociated with this option. 35 For these reasons, there is a limited interest inthe utilization of anaerobic bacteria for the degradation of phenols. Severalstudies have been reported in the literature in this regard. 3643 Since mostbiological treatments studies have used aerobic biomasses, in this review wefocus on aerobic biodegradation.

A large number of microorganisms including bacteria, fungi, and al-gae 30 are capable of degrading phenol. The biodegradation of phenol andits derivatives by bacteria has been extensively studied and a large numberof phenol-degrading bacteria have been isolated and characterized at thephysiological and genetic levels. 21,4449 Pure and mixed cultures of the Pseu-domonas genus are the most commonly utilized biomass for the biodegrada-tion of phenols 50 and they are believed to have good potential for differentbiotechnological applications. Specically, Pseudomonas putida has been

commonly used for the biodegradation of phenol due to its high removal ef-ciency. 17,51 Responses of P. putida to chemical stresses have indicated thatits cells could use diverse protective mechanisms for survival in various ex-treme environments. These studies could help in synthesizing new bacterialstrains with enhanced degradation capability and improved tolerance to toxicpollutants. 52

Fungi share a signicant part in the recycling of aromatic compounds inthe biosphere and several studies have shown that diverse fungi are capableof phenols mineralization. They are capable of consuming a wide variety of carbon sources by enzymatic mechanisms, thus providing possibilities for

metabolizing phenols and other aromatic derivates.50

The most abundant


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fungi in polluted environments are yeasts. Some yeasts such as Candidatropicalis , Fusarium occiferium , and Trichosporon cutaneum are capableof utilizing phenol as the major carbon and energy source. 24,53 Moreover,there are studies attesting the ability of strains from Penicillium , Aspergillus ,

Graphium , and Phanerochaete genera to disintegrate aromatic compounds.This makes them an interesting subject for studies aimed at the developmentof technologies for purication of contaminated soils and waters. 50 Rubilaret al.54 analyzed the degradation of chlorophenols by white rot fungi, whichare a group of organisms very suitable for the removal of chlorinated pheno-lic compounds. They are robust organisms that are tolerant to the presenceof high concentrations of various pollutants, even with a low bioavailability and this ability is mainly due to their very powerful extracellular oxidativeenzymatic system.

The bioremoval of phenols by bacteria and fungi has been extensively studied, but only recently there has been more interest in investigating thecapabilities of some algae for phenol biodegradation. The biodegradationof phenol by microalgae occurs only under aerobic conditions. While somealgae have low tolerance to the acute toxicity of phenols, both cyanobacteriaand eukaryotic microalgae (e.g., Chlorella sp., Scenedesmu s sp., Selenastrumcapricornutum , Tetraselmis marina , Ochromonas danica , Lyngbya gracilis , Nostoc punctiforme , Oscillatoria animalis , Phormidium foveolamm ) are ca-pable of biotransforming phenolic compounds. 30

2.3 Mechanism of Phenol BiodegradationMetabolic processes are governed by the action of enzymes, which are spe-cic for each type of reaction; thus microbial metabolism is a process of energy conversion sustained by specic reactions, providing the ultimatesource of energy. 24 The biodegradation process requires the presence of molecular oxygen to initiate enzymatic attack on the aromatic rings. A typicalpathway for metabolizing phenol is to hydroxylate the ring, by the enzymephenol hydroxylase, form catechol, and then open the ring through ortho -

(also termed -ketoadipate pathway) or meta- oxidation.14,30,32,55

Phenol hy-droxylase represents the rst enzyme in the metabolic pathway of phenoldegradation.

Both o- and m -pathways are distinguishable by measuring their charac-teristic enzyme activities. In the o-pathway, the aromatic ring is cleaved by the enzyme catechol 1,2-dioxygenase (C12O). In the m -pathway, the ring iscleaved by the enzyme catechol 2,3-dioxygenase (C23O). Thus, the ring isopened and then degraded. 1,24,56 The ring cleavage can occur in two differentorientations and this difference in cleavage site is used to classify catecholdioxygenases in two groups; the intradiol (such as C12O) and extradiol (such

as C23O) cleaving enzymes.57

The genes of ring-cleavage dioxygenases may


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serve as good targets for monitoring the biodegrading biomass, thus providing a rapid method for monitoring the microbial community during thetreatment process. 58 Discussion of biodegradation pathways and mechanismscan be found in the literature. 1,24,5961

Wang et al.21

estimated the enzymatic activities for phenol biodegra-dation by the bacterial strain Acinetobacter sp. PD12. The enzyme assaysproved that the crude extract from strain PD12 only showed C12O activity, which indicated that the strain PD12 metabolized phenol in the o-pathway. According to Jiang et al., 23 the efciency of a certain catabolic pathway of-ten depends on the properties of the involved key enzyme. In their study of phenol biodegradation by the bacterial strain Alcaligenes faecalis , it wasshown that between the two enzymes of the o- and m-pathways, the phenolhydroxylase, which hydroxylates phenol to catechol, was the key enzymefor phenol biodegradation. The hydroxylation reaction in phenol metabolism was the key determinant of biodegradation velocity. The assay of enzymeactivity proved that phenol was assimilated by o-ssion of catechol. On theother hand, Khleifat 16 reported that most bacteria use the m -pathway of cat-echol degradation. The author evaluated the degradation of phenol by thebacterium Ewingella americana and detected the activity of C23O, indicat-ing that the catechol ring ssion occurs through the m-pathway, not throughthe o-pathway. Similar results were shown for strains of Bacillus cereus , abacterium with high potential for phenol degradation and high tolerance. 62

Stoilova et al.6 reported that little is known about phenol metabolismby fungi. In most previous studies, phenol was metabolized by the -ketoadipate pathway, through o-ssion of catechol. 6 The properties of theinvolved key enzyme(s) play a key role in determining the efciency of acertain catabolic pathway. Vallini et al. 63 investigated the metabolism pathways of alkyphenols by a yeast strain, classied as Candida aquaetextoris . Anovel metabolic route for the microbial degradation of 4-(1-nonyl) phenol,at least in certain yeasts, was proposed.

2.4 Factors Affecting Biodegradation of PhenolsBiodegradation is a multifaceted process in which many biotic and abioticfactors are involved. 59 There are many factors that can affect the degrada-tion ability or metabolism of microorganisms by either preventing or stim-ulating growth of the organisms. These factors may include temperature,pH, oxygen content and availability (aeration and agitation), bioavailability (availability of the contaminants to microbes), substrate concentration, andphysical properties of contaminants. 1,8,24,59 Each of these factors should beoptimized for the selected organism to achieve the maximum degradation of the organic compound of choice. The optimization of the substrate concen-

tration in phenol biodegradation is particularly important because phenol


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biodegradation by microbial cells has generally been known to be inhib-ited by phenol itself, especially at higher concentrations. Although phenol isbiodegradable both aerobically and anaerobically, it can inhibit the growthof microorganisms at elevated concentrations, even for those species that can

use it as a substrate.1,11,64

Adjei and Ohta65

reported that phenol was com-pletely inhibitory to cyanide utilization by the bacteria Burkholderia cepaciastrain C-3.

Extreme pH values of the medium (less than 3 or greater than 9) as well as sudden changes in pH in which the microbe is present can inhibit itsgrowth. Consequently, laboratory studies on phenol degradation are usually carried out at or near neutral pH values (pH = 7.0). Each organism has acertain temperature range for growth. A strain of P. putida has successfully been used to degrade phenol at low temperature of 10 C, while a bacterium Bacillus stearothermophiles has also been used to effectively degrade phenolat 50C.24 El-Naas et al.8 believed that sudden exposure to temperatureshigher than 35 C may have detrimental effect on the bacterial enzymes thatare usually responsible for the benzene ring cleavage, which is the key stepin the biodegradation process. On the other hand, exposure to temperaturesmuch lower than 30 C was expected to slow down the bacterial activity andenhance the inhibitory effect of phenol on the bacteria, especially for highphenol concentrations. Most of the studies on phenol biodegradation hadbeen carried out in the temperature range 2535 C (Table 1).

Some other factors that can affect the biodegradation of phenols arechemical structure and compound toxicity. The chemical structural effect isreected by the number of substituents, type of substituents, position of sub-stituents and degree of branching. The greater the number of substituentsin the structure, the more toxic and less degradable it becomes. For exam-ple, substituted phenols such as mono, di-, tri-, and pentachlorophenol areless degradable than unsubstituted phenol. In addition, o- and p-substitutedphenols are more degradable than m-substituted phenols. 24

Toxicity prevents or slows metabolic reactions. This depends on theexposed microorganisms and the concentrations of specic toxicants. Toxi-city is induced by a blockage that may result from gross physical disruption

of the microbial cell structure or hindrance in its enzymatic activity.24

Bac-terial abundance is another factor in determining the overall efciency of biodegradation. The degradation of phenol can be performed by pure ormixed cultures. It is likely that an application of the mixed culture permitsfaster phenol degradation than a pure culture. 53

The microbial processes for phenol degradation employ suspended(free) living microbial cells or immobilized cells. It has been shown that thebiodegradation rate of phenol can be improved by immobilizing the cells andentrapping them on solid support particles such as alginate, polyacrylamide,chitosan (a natural nontoxic biopolymer), diatomaceous earth, activated car-

bon, sintered glass, polyvinyl alcohol (PVA), and polymeric membrane to


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T

A B L E 1

. E x a m p l e s o f b i o d e g r a d a t i o n s t u d i e s a n d p r o c e s s c o n d i t i o n s r e p o r t e d i n t h e l i t e r a t u r e

C o n c e n t r a t i o n

T e m p e r a t u r e

R e m o v a l

D e g r a d a t i o n

C o n t a m i n a n t

M i c r o o r g a n i s m

T y p e o f r e a c t o r

( m g / l )

p H

( C )

e f c i e n c y ( % )

t i m e ( h r )

R e f e r e n c e

S y n t h e t i c p h e n o l

F r e e P . p u t i d a

D S M 5 4 8

B a t c h ( s h a k e

a s k s )

1 1 0 0

6 . 8

2 6 0 . 5

1 0 0 ( f o r C o =

2 3 . 4

m g / l )

1 4

7 5


I m m o b i l i z e d

P . p u t i d a A T C C

1 1 1 7 2

C o n t i n u o u s

( b u b b l e c o l u m n

b i o r e a c t o r )

1 0 0

5 . 5 6 . 0

2 5 3 0

9 7 . 5

6 7



P . p u t i d a

B a t c h ( b u b b l e

c o l u m n


5 1 5 0

7 . 0

3 0

1 0 0

0 . 3 5

8



P . p u t i d a

B a t c h ( s p o u t e d

b e d b i o r e a c t o r )

4 0 1 9 0

7 . 0

3 0

7 4 1

0 0

0 . 8 1

. 8

2 2



P . p u t i d a

C o n t i n u o u s

( s p o u t e d b e d


1 0 1 5 0

7 . 0

3 0

8 8 9

7

0 . 6 7 2

. 5 5

5 1



M T C C 1 1 9 4


a s k s )

P h e n o l : 1

0 0 0

7 . 1

2 9 . 9

0 . 3

1 0 0

P h e n o l : 1

6 2

9

S y n t h e t i c c a t e c h o l

C a t e c h o l : 5 0 0

C a t e c h o l : 9 4


F r e e a n d

i m m o b i l i z e d

A c i n e t o b a c t e r s p .

s t r a i n P D 1 2


a s k s )

1 0 0 1 1 0 0

7 . 2

( o p t i m u m )

3 0

( o p t i m u m )

F r e e c e l l s : 9

9 . 6

( f o r C o =

5 0 0 m g / l )

9

2 1


M i x t u r e o f

A c i n e t o b a c t e r s p .

X A 0 5 a n d

S p h i n g o m o n a s s p .

F G 0 3 ( 1 : 1 ) ,

f r e e

a n d i m m o b i l i z e d


a s k s )

2 0 0 1 0 0 0

7 . 2

( o p t i m u m )

3 0

( o p t i m u m )

> 9 5

( f o r C o =

8 0 0 m g / l )

3 5

2


F r e e P s e u d o m o n a s

s p .

S A 0 1


a s k s )

3 0 0 1 0 0 0

7 . 0

3 0

1 0 0

2 0 8 5

7


F r e e E w i n g e l l a

a m e r i c a n a


a s k s )

0 1 0 0 0

7 . 5

3 7

1 0 0 ( f o r C o =

3 0 0 m g / l )

2 4 ( s t a r v e d

c e l l s )

1 6


F r e e A l c a l i g e n e s

f a e c a l i s


a s k s )

0 1 8 0 0

7 . 2

3 0

1 0 0 ( f o r C o =

1 6 0 0 m g / l )

7 6

2 3


F r e e O c h r o b a c t r u m

s p .


a s k s )

5 0 4 0 0

8 . 0 ( o p t i m u m )

3 0

4 5 . 2

( f o r C o =

6 2 m g / l , u p o n

a d d i t i o n o f

m o l a s s e s )

9 6

2 5

( C o n t i n u e d o n n e x t p a g e )

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T

A B L E 1

. E x a m p l e s o f b i o d e g r a d a t i o n s t u d i e s a n d p r o c e s s c o n d i t i o n s r e p o r t e d i n t h e l i t e r a t u r e ( C o n t i n u e d )


T e m p e r a t u r e

R e m o v a l

D e g r a d a t i o n

C o n t a m i n a n t

M i c r o o r g a n i s m

T y p e o f r e a c t o r

( m g / l )

p H

( C )

e f c i e n c y ( % )

t i m e ( h r )

R e f e r e n c e



R a l s t o n i a

e u t r o p h a


a s k s ) a n d

c o n t i n u o u s

p a c k e d b e d

2 5 5

0 0

7 . 0

3 0

B a t c h : 6 8 ( f o r C o =

1 0 0 m g / l )

8 6


F r e e i n d i g e n o u s

m i x e d c o n s o r t i u m


a s k s )

1 0 0 8 0 0

7 . 0

2 7

1 0 0

1 0 6 9

8 8


F r e e s u s p e n d e d

m i x t u r e o f P .

a e r u g i n o s a a n d

P . u o r e s c n c e

B a t c h b i o r e a c t o r

1 0 0 5 0 0

3 0

1 0 0

2 4 9 6

9 8


F r e e m i x e d b a c t e r i a l

c o n s o r t i u m


a s k s )

2 3 . 5

6 5 8

7 . 2

2 5 2

9 0 1

0 0

1 1 0

9 9


F r e e m i x e d c u l t u r e

f r o m a n a c t i v a t e d

s l u d g e

F u l l y m i x e d b a t c h

r e a c t o r

2 5 1 4

5 0

2 5 1

9 7 1

0 0

1 7 3

1 1


F r e e m i x e d a e r o b i c

a c t i v a t e d s l u d g e

a n d a n a e r o b i c

s l u d g e

C o n t i n u o u s

4 0 0 , 1 0 0 0

7 . 0

3 0

> 9 5

W i t h i n

6 0

6 4

S y n t h e t i c b i n a r y

m i x t u r e o f p h e n o l

a n d s o d i u m

s a l i c y l a t e


C C R C

1 4 3 6 5

B a t c h

1 . 0 6 m M

a n d

3 . 1 8 m M

( t o t a l )

7 . 0

3 0

1 0 0

1 6 4 3

1 2

P h e n o l a n d

n o n p h e n o l i c

c o m p o u n d s i n r a w

w a s t e w a t e r

I m m o b i l i z e d P .

p u t i d a A T C C

1 7 4 8 4

- B a t c h ( s h a k e

a s k s )

- C o n t i n u o u s

u i d i z e d b e d

2 0 0 1 0 0 0

2 5 0 2 5 0 0

6 . 6

6 . 6

3 0 3 0

1 0 0

> 9 8

B a t c h : 2 6 0 f o r

C o =

1 0 0 0

8 9


- F r e e P . p u t i d a

A T C C 1 7 4 8 4

- I m m o b i l i z e d

P .

p u t i d a A T C C

1 7 4 8 4

C o n t i n u o u s s t i r r e d

t a n k b i o r e a c t o r

C o n t i n u o u s

u i d i z e d b e d

b i o r e a c t o r

1 0 0 0

1 0 0 0

6 . 6

6 . 6

3 0 3 0

> 9 5

> 9 8

9 0


I m m o b i l i z e d P .

p u t i d a N C I M 2 1 7 6

C o n t i n u o u s

u i d i z e d b e d

b i o r e a c t o r

5 0 2

5 0

7 . 0

3 0

1 0 0

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R e s t i n g C . t r o p i c a l i s

N C I M 3 5 5 6


a s k s )

2 0 0 0 1

0 0 0 0

N A

2 8

1 0 0

2 4

9 3


R e c y c l e d r e s t i n g C .

t r o p i c a l i s N

C I M

3 5 5 6


a s k s )

2 0 0 0

N A

2 8

7 0 a f t e r 5 c y c l e s

5 0 a f t e r 5 c y c l e s

2 4

9 4

M o d e l b i n a r y m i x t u r e s

o f p h e n o l , c

a t e c h o l ,

2 , 4 - d i c h l o r p h e n o l

a n d 2 , 6 -

d i m e t h o x y p h e n o l

A s p e r g i l l u s a w a m o r i

N R R L 3 1 1 2


a s k s )

T o t a l 1 0 0 0 ( 1 : 1

)

N A

3 0

2 5 1

0 0 ( f o r p h e n o l )

1 6 8

5 0

S y n t h e t i c p h e n o l +

h u m a t e p o t a s s i u m

F r e e C u p r i a v i d u s

m e t a l l i d u r a n s

- B a t c h ( s h a k e

a s k s )

- C o n t i n u o u s

( b i o l m r e a c t o r

w i t h c e r a m i c

c l a y l l i n g )

4 0 0 1

4 0 0

4 0 0 1

4 0 0

6 . 6

5 . 5 7 0 . 7 1

2 3 . 5 1 . 4

1 0 0 f o r C o =

4 0 0

> 9 9 f o r C o =

4 0 0 8 0 0

4 5

1 0 1

- S y n t h e t i c p h e n o l

- C a t e c h o l ( a s a

m e t a b o l i c

i n t e r m e d i a t e )

F r e e H a l o m o n a s

c a m p i s a l i s


a s k s )

1 3 0

8 1 1

3 0

1 0 0

5 0 @ p H =

9 . 5 , 1 0 0 g / l

N a C l

8 5


A e r o b i c

g r a n u l a r s l u d g e


a s k s )

5 0 0 5

0 0 0

7

3 0

1 0 0

1 8 5

4 0

8 3

S y n t h e t i c m i x t u r e o f

p h e n o l a n d 4 - C P

C T M 2 ( m u t a n t

s t r a i n o f w i l d C .

t r o p i c a l i s )


a s k s )

- 4 - C

P : 4 0 0 i n t h e

p r e s e n c e o f

0 8 0 0 p h e n o l

- P h e n o l : 2

5 0 0 i n

t h e p r e s e n c e

o f 0 3 0 4 - C P

3 0

1 0 0 ( f o r a m i x t u r e o f

3 0 0 p h e n o l &

4 0 0 m g / l 4 - C

P )

5 0 . 5

9 5

- P h e n o l

- p - c r e s o l

e u k a r y o t i c a l g a e :

O c h r o m o n a s

d a n i c

0 3

8 0 0 4 3 2

2 5

1 0 0

4 8 f o r C o =

9 4

9 6 f o r C o =

9 4

9 7

1641


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obtain the maximum degradation capability. 2,7,8,17,21,66,67 Jianglong et al. 68 re-ported the development of a novel immobilization carrier, that is, PVA-gauzehybrid carrier. It was found that biodegradation rate of quinoline by the mi-croorganisms immobilized on PVA-gauze hybrid carrier was faster than that

by the microorganisms immobilized in PVA gel beads. The immobilizationmethod is not toxic to the cells and is inert and practical. 14 There are varioustechnologies for bacteria immobilization, including bead entrapment, carrierbinding, adsorption techniques, encapsulation, cell coating, and lm attach-ment. 69 Immobilization of bacterial biomass for the degradation of phenolis an important and effective technique that is usually employed to serveseveral purposes, including protection of the bacteria from high phenol con-centrations as well as ease of separation and reutilization of the biomass. Ithas been reported that the use of free bacterial cells for wastewater treatmentin activated sludge processes creates problems such as solid waste disposal, while immobilized microorganisms are capable of effective treatment withlittle sludge formation. 2,21 The merit of immobilization is due to the highsurface area available for biolm formation, which results in high biomassconcentration of 3040 g VSS/l, compared with 1.52.5 g VSS/l for activatedsludge systems. 70 In addition, the systems with immobilized cultures aremore stable to shock loadings than the suspended cultures with free cells 53and immobilization can provide high degradation capacity compared withfree cells.71 Kim et al.72 showed that calcium alginate immobilization of mi-crobial cells effectively increased the tolerance of P. putida MK1 to phenoland improved the degradation of pyridine in a binary mixture of the twocompounds.

The performance of a biodegradation system may be affected by thepresence of metabolic inhibitors or competing substrates. 12,67 There are con-straints imposed by the occurrence of the environmental contaminants inmixtures as the degradation of one component can be inhibited by othercompounds in the mixture, and because different compounds within themixture may require different treatment conditions. 50

The ability of microbial communities to degrade pollutants is affectedby the presence of naturally occurring carbon sources. In general, adaptation

to variations in the concentration of nutrients such as glucose, yeast extract,and (NH 4)2SO4 enhances the ability to degrade phenols. Gladyshev et al. 73reported that biodegradation of phenolic compounds is known to increaseat higher concentrations of inorganic nutrients, while it is inversely affectedby higher concentrations of organic nutrients. The process of cometabolismis an important example of the inuence of substrate interaction during thebiodegradation of pollutants. 74 Cometabolism is dened as the degradationof a compound only in the presence of another organic material that servesas the primary growth substrate. This phenomenon has been attributed tothe production of broad-specicity enzymes, where the primary substrate

and the other compound compete for the same enzyme.26


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3. ADVANCES IN BIODEGRADATION OF PHENOLS

The degradation of phenol by the bacterial Pseudomonas genus, and specif-ically, the strain P. putida is very well documented. Because of its reported

high removal efciency, P. putida has been studied by many researchersin free and immobilized forms, batch and continuous modes, and pure andmixed cultures, using different types of bioreactors. 8

During the biodegradation of phenol, the contaminant concentrationdecreases with time while the biomass is generated. Figure 2 is a typicalillustration of how the concentrations change with time. The gure alsoclearly shows the effect of lag times at the beginning of the process, especially when the biomass cells are not preadapted to the contaminant medium. Thelag time increases with increased initial concentration of phenol.

Figure 3 shows another phenomenon usually encountered in experi-mentation on phenol biodegradation, which is the inhibitory effect of phenolto the microorganism at high initial phenol concentrations. The biodegrada-tion rate, which is a measure of the biomass activity, starts to decrease if theinitial phenol concentration is above the maximum concentration that canbe tolerated by the microorganism.

Monteiro et al. 75 studied the phenol-inhibitory growth kinetics of P. putida DSM 548 in batch experiments for low initial phenol concen-trations (Co = 1100 mg/l). According to the study, higher efciencies areobtained when selecting a pure culture for phenol degradation.

Table 1 presents a summary of recent studies on biodegradation of various phenolic compounds. It includes the details of pH, temperature,

FIGURE 2. Phenol and biomass concentration as a function of time.


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FIGURE 3. Biodegradation rate of phenol as a function of initial phenol concentration.

concentration of phenols, type of reactor, type of microorganisms, and theremoval efciency that could be achieved. In general, these studies haveaddressed important research topics, and hence some of their main ndingsare discussed in the following sections.

3.1 ImmobilizationIn view of its major advantages, the immobilization of biomasses has beenreceiving considerable attention. Using immobilized cell is a well-knownapproach for incorporating bacterial biomass into an engineering process. 74Continuous degradation of phenol at an inuent concentration of 100 g/l with immobilized P. putida was investigated by Mordocco et al., 67 whopointed out the signicance of this low range of concentrations in light of thepotential toxicity of phenol at concentrations as low as 5 mg/l. Comparing the

performance of the immobilized cells in calcium alginate beads to that of freecells, the superiority of the immobilized cell system was more pronounced. A bead diameter between 1 and 2 mm was found to be optimal for phenoldegradation at low levels.

Physical entrapment of organisms inside a polymeric matrix has beenextensively used for whole-cell immobilization. 14,74 The effectiveness of thismethod has also been investigated by El-Naas et al. 8 in a study to assessthe biodegradation of phenol by P. putida immobilized in PVA gel matrixat different conditions. They reported that microbial acclimatization (adap-tation) to phenol is necessary as phenol is inhibitory to bacteria growth at

concentrations above 75 mg/l. The study emphasized the importance of the


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phenol uptake per mass of PVA, which has a signicant economical consid-eration in designing an industrial process for wastewater treatment. At highphenol concentration, the PVA particle size was found to have negligibleeffect on the biodegradation rate. However, for low concentrations, particle

size seemed to show a small effect, which conrmed the importance of masstransfercontrolled mechanisms.Subsequent studies in the batch and continuous modes in a spouted

bed bioreactor (SBBR), 22,51 which is characterized by intense mixing, provedthat mass transfer limitations have considerable effect on the biodegradationrate and the dynamics of the system is mainly controlled by the internal masstransfer (at the biomass interface).

Wang et al. 21 used PVA carrier for immobilization and reported that theimmobilized cells could tolerate higher phenol levels and wider changesin temperature and pH. Immobilization increased the thermal stability of the microbial cells under the protection of PVA carrier. The immobilizedcells possessed better storage stability and could be reused for at least 50cycles, which demonstrated that PVA carrier cubes had good exibility andretained a high mechanical strength. More examples for the biodegradationof phenols with immobilized microorganisms are presented in Table 1.

3.2 Bacteria for High Phenol ConcentrationsKumar et al.9 carried out a kinetic study utilizing P. putida MTCC 1194 inthe initial concentrations of 1000 and 500 mg/l of phenol and catechol,respectively. Even the well-acclimatized cultures showed lag phase due tothe high concentration of phenol. The higher the initial concentration, thelonger was the lag phase (inhibitory effect). No bacterial growth could bedetected at phenol concentrations of 1200 mg/l, even after 20 days. Forcatechol, the growth of the bacterial strain was inhibited when exposed to600 mg/l.

According to Wang et al., 21 little information on bacteria with a highphenol tolerance and high metabolizing activity is available. Therefore, therestill exits the need to isolate new phenol-degrading bacteria that can grow

at elevated concentration of phenol. A new phenol-degrading bacterium Acinetobacter sp. strain PD12 was isolated from activated sludge.Research attention in recent years has focused on developing aerobic

granules in sequencing batch reactors. The aerobic granule process, a self-immobilization of microorganisms, has been extensively investigated. 55,7682It is a novel wastewater treatment technology that can quickly decontaminatehighly contaminated wastewater. Aerobic granules yield a very high biomassconcentration (up to 15000 mg/l) and have an ability to degrade high-strength wastewater (up to 15 kg chemical oxygen demand [COD]/m 3/day). 76 Aero-bic granules have a dense and strong microbial structure, good settling abil-

ity, high biomass retention, and tolerance of highly toxic substrates.76,79,80


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Laser scanning microscopy test revealed that live cells are principally dis-tributed throughout the surface layer of the granule, with an extracellularpolymeric substance layer covering them to protect the cells from phenoltoxicity. 76,79 Adav et al.82 reported that a short settling time is preferred in

aerobic granulation processes and seemed to enhance the granulation of aerobic granules. Ho et al. 83 used aerobic granules to degrade high-strengthphenol wastewater. The acclimated granules degraded high concentrationphenol, of up to 5000 mg/l, at an acceptable rate, even after acid or alkalinepretreatment.

3.3 Bacteria From Contaminated SitesShourian et al. 7 reported that many pollution problems resulting from re-leasing aromatic chemicals occur in rivers, lakes, groundwaters, and processefuents from the industry. Accordingly, environmental bacterial strains iso-lated from contaminated sites are expected to play a vital role in the biore-mediation of contaminated areas. In their study, Pseudomonas sp. SA01 was isolated from pharmaceutical wastewaters. The bacterial strain was ex-amined for phenol biodegradation and was suggested as an excellent andunique candidate for rapid and efcient phenol removal, particularly forthe shorter lag time at high phenol concentrations (up to 1000 mg/l) com-pared with those occurring in other Pseudomonas species. Other researchershave utilized microbial biomasses isolated from phenol-contaminated soil, 2 wastewater plants, 16,25 efuents of coke processing units, 44 and municipalgas works. 23 Recently Corynebacterium glutamicum , an industrial soil mi-croorganism, was proved to be very effective for the bioremediation of phenol-contaminated soil. 84 The authors suggested that a suitable dose of C. glutamicum treatment was sufcient for the large scale remediation of phenol-contaminated soil.

An industrial efuent of great environmental concern is petroleum ren-ery spent caustic, which is an alkaline and saline waste stream that containsphenol as well as other aromatic compounds. 20,85 Alva and Peyton 85 reportedthe rst study of phenol and catechol biodegradation under combined saline

and alkaline conditions by the haloalkaliphilic bacterium Halomonas camp-isalis . Haloalkaliphiles are bacteria that thrive in saline and alkaline envi-ronments such as soda lakes. The haloalkaliphilic bacterium H. campisalis degrades phenol and catechol in alkaline (pH values of 811) and salineenvironments (0150 g/l NaCl).

3.4 New Strains of Bacteria Although, the Pseudomonas genus have been widely used to treat phenols,considerable attention has been recently directed towards new and more ef-

cient microorganisms for this purpose. Tepe et al.86

reported that Ralstonia


17/60


eutropha is one of these microorganisms; Ralstonia is a newly designatedgenus that includes former members of Burkholderia species. 87 R. pickettii has been reported to have the ability to use a variety of aromatic com-pounds as energy and carbon sources. It has several advantages over other

candidate strains being studied such as P. putida in that it is only weakly pathogenic with no phytopathogenic or animal pathogenic incidents beingreported. R. pickettii strain LD1 can metabolize monochlorophenols, whichrepresent an important challenge as they may be particularly formed duringthe chlorination of wastewaters. 87

3.5 Utilization of Mixed Cultures A mixed community of microbes may be needed for the complete miner-alization of phenols. Although many reports on phenol degradation usingpure species of microorganism are available, reports on using mixed culturesof microorganism are scant. 88 Liu et al.2 evaluated a mixture of Acinetobac-ter sp. XA05 and Sphingomonas sp. FG03 strains for phenol degradationand reported better degradation efciency by the mixed cells than by thepure cultures. The authors attributed that to the fact that the strains of XA05and FG03 were isolated from different environment conditions, and they have different enzyme systems and different ways to degrade phenol. Whenmixed, they may act synergistically to overcome the inhibition of substrate.This synergistic effect seems to be consistent with that reported by Mon-teiro et al., 75 who suggested that the biodegradation of organic chemicalsby microbes using pure cultures can produce toxic intermediates and thatthis problem may be overcome by the use of mixed cultures, which havea wider spectrum of metabolic properties. On the other hand, the same au-thors reported a higher efciency when selecting a pure culture of P. putidaDSM 548 for phenol degradation. This discrepancy may be attributed to thedifference in the phenol concentration range between the two studies, beinghigh for the study of Liu et al. 2 and low for the study by Monteiro et al. 75 Itis believed that the synergism effect of different strains is enhanced at highconcentrations of phenol.

3.6 Research on Mixed Substrates and Industrial EfuentsBiodegradation of a compound in a mixture could be strongly impacted by other components of the mixture. This is related to the special class of biolog-ical transformation called cometabolism, which refers to the transformationof a nongrowth substrate by cells growing on a growth substrate. 12,13 In thisregard, the results from a study on phenol biodegradation in a binary mixtureof phenol and sodium salicylate by free P. putida CCRC 14365 in a batchmode proved that the cells adapted with comparatively hard-to-degrade sub-

strate (sodium salicylate, in this case) facilitated the overall biodegradation


18/60


efciency of mixed homologous carbon and energy substrates. 13 Moreover,most of the works that appeared in the literature concerning the biodegrada-tion of phenol dealt with model solutions (lab-prepared solutions simulatingindustrial efuents). 89 Only a limited amount of work has been reported

on the treatment of industrial wastewater. The biodegradation of high phe-nol concentrations from industrial wastewaters by cells of P. putida ATCC17484 immobilized in calcium-alginate gel beads was reported by Gonzalezet al.89 In a batch operation, phenol concentration of up to 1000 mg/l wasdegraded. Compared with a degradation capacity of 2000 mg/l from previ-ous work by the authors with model solution, 90 the negative effect of thenonphenolic compounds in the industrial wastewater was indicated. Also Agarry et al.91 treated renery wastewater in their study, which investigatedthe phenol-biodegrading potential of two indigenous Pseudomonas species.

3.7 Fungi and Algae in Phenol BiodegradationThe degradation of phenols has also been observed in fungal strains.C. tropicalis was demonstrated to use phenol as the sole carbon and energy source. 92 Varma and Gaikwad 93 identied a high phenol-degrading yeast C.tropicalis NCIM 3556, which was further investigated for its potential to de-grade other derivatives of phenol such as o-cresol, m-cresol, -naphthol, andothers. Inhibition was observed for all substrates above an inuent concen-tration of 2000 mg/l. Upon repeated reuse of the cells, the cell adaptation tophenol increased with each cycle. 94

Jiang et al.95 obtained a mutant strain CTM 2 by the He-Ne laser irradia-tion on wild-type C. tropicalis , which was isolated from acclimated activatedsludge. The mutant strain CM 2 possessed the strong capacity to degradephenolic compounds, especially phenol and 4-chlorophenol (4-CP). In thisdual substrate biodegradation system, the mode of interaction was revealedin such a way that low-concentration phenol could enhance biodegradationof 4-CP, while very little 4-CP could greatly inhibit phenol biodegradation.

The fungal strain Aspergillus awamori NRRL 3112 was reported to havehigh potential for degrading high concentrations (up to 3.0 g/l) of phenol,

catechol, 2,4-dichlorophenol, and 2,6-dimethoxyphenol.6

. A later study by Stoilova et al.50 investigated the ability of this fungus to degrade binary mix-tures of the above phenolic compounds. It also focused on a topic of specialinterest, which is the interaction of substrates in substrate mixtures. Themain nding was that for all combinations containing dichlorophenol, thediauxy phenomenon was observed; it took equal time (4 days) for the nearly complete degradation of the compound. The assimilation of the second phe-nolic derivative was suppressed until dichlorophenol had been completely degraded (Figure 4). In other mixtures, no diauxy was observed and bothsubstrates were metabolized simultaneously (Figure 5). The diauxic growth

characteristics were also investigated by other researchers.96


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FIGURE 4. Diauxy in a binary phenolic mixture.

In a study involving the biodegradation of phenol by the strain Fusar-ium sp. HJ01,57 it has been revealed that the major enzymatic pathwaysfor catabolism of phenol in fungi are carried out similar to bacteria, wherephenol is transformed into the intermediate catechol. The dihydroxylated in-

termediates proceed into the o- or m-cleavage pathway. The characteristics

FIGURE 5. Simultaneous substrate utilization in a binary phenolic mixture.


20/60


of the two enzymes, C 12O and C23O, were reported in terms of enzymaticactivities at different pH and temperature. Mineral salts added in culture havean inhibition on both enzymes. The activity range of C 12O was from pH 3 topH 8.8 and from 30 to 50 C, being optimal at pH 6.8 and 40 C. The activity of

C23O was slightly more sensitive to pH, being higher in the range of 7.410.6and was more stable at higher temperatures from 30 to 75 C. Although algae have been shown to have the ability to degrade phenols,

there has been limited work devoted to their use in recent literature. Likaand Papadakis 30 developed a mechanistic model for the aerobic degradationof phenolic compounds by photosynthetic algae, under photoautotrophic,heterotrophic, or mixotrophic conditions. The model can be applied to a wide variety of aromatic compounds and can be extended to account forinhibitory effects at high phenol concentrations and to include mixtures of phenolic compounds as well as mixed cultures. A study by Semple 97 showedthat Ochromonas danica , a eukaryotic alga, is capable of growing on phenoland its methylated derivative p-cresol as the sole carbon sources. However,the biodegradation rate of phenol was almost twice that of p-cresol.

4. ADVANCES: KINETICS, MODELING, AND MASS TRANSFER

4.1 Kinetics and ModelingIn biodegradation reactions, kinetics studies give a measure of how effec-

tively the microbial system is functioning.24

Knowledge of such kinetics willhelp improve the process control and phenol removal efciency. 75,91 Model-ing any biodegradation process involves relating the specic growth rate of the biomass to the consumption rate of the substrate (contaminant). 8 A vari-ety of kinetic models have been used to describe the dynamics of microbialgrowth on phenol (Table 2).

Based on material balance, the rate of biomass growth, and the rate of substrate utilization (both in mg/l.hr) can be represented by Equations 1 and2, respectively:

dX

dt = X k d X = net X or d ln X

dt = net (1)dS

dt = X Y

, (2)

where Y is the cell mass yield (g/g) = dX/dS ; X is the biomass concentration(mg/l); S is the substrate concentration (mg/l); k d is the decay coefcient(hr1); and is the specic growth rate (hr 1).9,12 Two of the most widely used models for the biodegradation of phenol are the Monod model (Table

2, Equation A) and the Haldane (Andrews) model (Table 2, Equation B),


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TABLE 2. Biodegradation models (kinetics and mass transfer)

Name of model EquationExample

references

Monod = max S

K s +S [A] 8, 9, 88, 99Haldane (Andrews) =

max S

K s +S +S 2 K i

[B] 9, 11, 12, 31,75, 99

Linearized Haldane 1

=1

max +S

K i max[C] 9, 102

Han-Levenspiel = max 1

S S m

n

K s +S 1 S S m

m [D] 88, 99

Yano

=

max S

S + K s +S 2 K i 1 +

S K

, K is a constant [E] 13, 62, 103, 104

Edwards = max exp S

K i exp S

K s [F] 13, 62, 103, 104

Wang-Loh a dS

Xdt = R max S

k s +S + f (i ) [G] 105

f (i ) =(S 0 S )

2

K p[H]

X = X 0e t [I]

= max S 0

K s +S 0 +S 20

K i

[J]

Monod: sum kineticsBinary mixture, nointeraction

= max ,1S 1

K s ,1 + S 1 + max ,2S 2

K s ,2 + S 2[K] 109

Monod: sum kineticsBinary mixture,purely competitiveinteraction(inhibition)

= max ,1S 1

K s ,1 + S 1 + K s ,1 K s ,2

S 2

+ max ,2 S 2

K s ,2 + S 2 + K s ,2 K s ,1

S 1

[L] 109

Binary mixture,noncompetitiveinhibition

=

max ,1S 1

( K s ,1 + S 1) 1 + S 2 K s ,2+

max ,2 S 2

( K s ,2 + S 2) 1 + S 1 K s ,1

[M] 109

Binary mixture,uncompetitiveenzyme inhibition

= max ,1S 1

K s ,1 + S 1 1 + S 2 K s ,2

+ max ,2 S 2

K s ,2 + S 2 1 + S 1 K s ,1

[N] 109

(Continued on next page)


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TABLE 2. Biodegradation models (kinetics and mass transfer) (Continued)

Name of model EquationExample

references

SKIPb

Binary mixture,unspecied type of interaction

= max ,1S 1

K s ,1 + S 1 + I 2,1S 2+

max ,2S 2 K s ,2 + S 2 + I 1,2S 1

[O] 19, 109

SKIPThree-compoundmixture,unspecied type of interaction

= max ,1S 1

K s ,1 + S 1 + I 2,1S 2 + I 3,1 S 3+

max ,2S 2 K s ,2 + S 2 + I 1,2S 1 + I 3,2 S 3

+ max ,3S 3

K s ,3 + S 3 + I 1,3S 1 + I 2,3 S 2

[P] 19, 109

Proposed by Jiang

et al.

= max S

K s +S + S 2

K i + S 3

K i

[Q] 95

Michaelis-Menten c v =V max S k m + S

or 1

v =k m

V max

1S +

1V max

[R] 57

J D-factor J D =k LG

N 2/ 3Sc = KN (1n)Re [S] 14, 86

Ficks Law d d 2C

dr 2 +2r

dcdr =

p D e

v [T] 3, 116

Thiele Modulus e =r 0 k D e [U] 3, 100, 116a R is the specic consumption rate of substrate (mg/mg.hr), k s is the saturation constant for substrateconsumption (mg/l); f(i) represents the functional relationship of the effect of metabolic intermediates onphenol degradation; and K p is a proportionality constant. b I ij is the interaction parameter that indicatesthe degree to which substrate i affects the degradation of substrate j . cFor Catechol dioxygenase activity,v is the initial velocity of the reaction (mg/s), K m is the Michaelis constant, and V m is the maximum velocity. dC is the phenol concentration within the immobilized particles (mg/l), r is the radial position within the bead, and p and D e are the density of dried microorganism (g /cm 3) and effective diffusioncoefcient of phenol within the bead, respectively. is the actual degradation rate (mg/g.hr). er 0 isthe radius of the particle, k is the rate constant = k p where k is rst-order degradation rate constant(cm3/g.hr).

where K s (mg/l) is the half-saturation coefcient (it shows the microorgan-

ism afnity to the substrate) and K i is the substrate inhibition constant (mg/l).These two models are used to describe the specic growth rate dependenceon substrate concentration. The rst considers phenol as noninhibitory com-pound and, therefore, neglects the inhibitory effect, whereas the secondtakes into consideration the inhibitory effect of phenol. 8 These two modelscan be used to predict the variations of the biodegradation rate with initialphenol concentrations, utilizing the relation in Equation 2 and assuming thatY is constant over the concentration range. This assumption is valid if thephenol concentration is much higher than K s (i.e., S >> K s).8 However, theHaldane equation has been used extensively to describe phenol microbial

degradation in pure and mixed cultures. Although the model is based on


23/60


the specic growth rate, it may also be related to the specic substrate con-sumption rate. The value is determined based on the exponential phase of growth curve. 12 In their work, Jiang et al. 23 related phenol degradation pro-cess directly to the growth rate of A. faecalis , assuming a constant biomass

yield Y :

S = A X + B (3)

where X and S are the specic rate of cell growth and substrate consump-tion, respectively, and A and B are kinetic constants. The Haldane model isused because of its wide applicability and mathematical simplicity for repre-senting cell growth kinetics of inhibitory substrate, as it has less parametersand is easily used for representing continuous biological reactors. 9,12

The Haldane (or sometimes referred to as Haldane-Andrews) equationrepresents a modication of the original Monod equation to account forinhibitory effects when a substrate (or substrate biotransformation interme-diate) is toxic to the degrading population. The effects of this self-inhibitionare incorporated into the expression with an inhibition term, S/K i in thedenominator. A larger K i value indicates that the culture is less sensitiveto substrate inhibition. It should be noted that when K i is very large theHaldane equation simplies to the Monod equation. 31

At higher substrate concentrations, S >> K s, the Haldane equation re-duces to the following:

= max S

S+S2 K i

, (4)

which leads to the linearized Haldanes equation (Table 2, Equation C). 9,102Monods model and the linearized Haldanes model are two-parameter mod-els, while Haldanes growth kinetics model has three parameters. In a study by Kumar et al.,9 the estimated parameters in Monods model and thelinearized-Haldanes model were used to provide initial guess values of the

Haldane model parameters. A typical growth curve showed a decline in cellpopulation after the complete consumption of substrate (endogenous or de-cay coefcient), which was explained by drop in oxygen and pH valuestoward the end of the substrate consumption curve. 6

Saravanan et al. 88 used an extended Monod-type model originally pro-posed by Han-Levenspiel, which was shown to be efcient in explaining thegrowth of a microbial consortium at different concentrations of substrate. Theculture followed substrate inhibition kinetics that could be tted to Haldaneand Han-Levenspiel models. Between the two models, the Han-Levenspielmodel (Table 2, Equation D) was found to give a better t. According to

Bajaj et al.,99

the Han-Levenspiel model is based on the effect of a product


24/60


that may be formed during degradation, whereas Haldanes model is basedon the effect of a substrate on a culture growth.

Other substrate-inhibition kinetic models include Yano model and Ed- wards model (Table 2, Equations E and F, respectively). Juang and Tsai 13

tested these two models for the biodegradation of phenol by P. putidaCCRC 14365 and compared their predictions to that of the Haldane model.The predictions by each model overlapped and the obtained kinetic pa-rameters were comparable. The same behavior was conrmed by Onyskoet al.103 for phenol biodegradation by P. putida Q5.; little difference wasfound among the predictions by the different models at phenol levels be-low 300 mg/l. Banerjee and Ghoshal 104 showed that the growth kinetics of B. cereus strains isolated from oil sites were best tted by the Yano modeland the Edwards model.

It has been reported in the literature that for a wide range of phenol con-centrations the Haldane equation could simulate phenol degradation prolesonly when different sets of model parameters were used (Table 3). 11 Differ-ent Haldane kinetic parameter values were obtained even when the samemicrobial strain was studied. It is evident that there is a considerable lack of consistency in the Haldane parameters for specied combination of an or-ganism and a substrate and there are different reasons for this variability. Theinadequacy of the Haldane equation has been attributed to the inhibition of metabolic intermediates of phenol degradation. 6,11,24,105 These discrepanciesmay also be attributed to different microorganisms and media that were used,different initial substrate concentrations, and the mode of growth cultivation(batch vs. continuous cultivation) that were used. The role of intermediateshas been conrmed by Bajaj et al. 70 The phenol degradation prole indi-cated that the presence of acetate that represents an intermediate of phenoldegradation retarded the phenol degradation. The highest phenol removalrate observed in batch assay was 1.51 g/l.day in the presence of acetateand it increased to 3.54 g/l.day after acetate depletion. 70 Monteiro et al. 75explained the variation of kinetic parameters by the different inoculumshistory. Moreover, in most of the studies listed in Table 3, the growth rate was assumed to be only limited by phenol concentration. The inuence of

oxygen was not considered. It was assumed that the aeration provided by shaking the asks is sufcient to keep the oxygen concentration constantand not limiting. 23,61,75 A lag phase had also been observed experimentally.The length of the lag phase before the exponential growth phase increasedlinearly with phenol concentration between 1 and 100 mg/l. 75

Wang and Loh 105 found that the Haldane equation was not sufcient formodeling phenol degradation, especially for high initial phenol concentra-tions, although it could correlate specic growth rates with initial substrateconcentrations very well. Nuhoglu and Yalcin 11 raised an argument that thedegree of inhibition is determined by K s/ K i ratio, and not just by K i alone.

The larger the value of K s/ K i, the smaller ( value corresponding to


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critical substrate concentration S where d /dS = 0) is relative to max , andthus, the greater the degree of inhibition. Wang and Loh 105 argued that par-ticularly for S 0 K s K i , which is the critical substrate concentration S , theHaldane model will erroneously model substrate consumption, especially when the substrate has been consumed to a signicant extent. Furthermore,it has been customary to assume that the yield coefcient Y is an averageconstant, which is not always true, especially at extremes of specic growthrate. Consequently, a new phenol degradation model was proposed by Wangand Loh105 by incorporating the inhibition effects of metabolic intermediates(Table 2, Equations G, H, I, and J) and accounting for a variable cell mass yield. According to the authors, the new model successfully simulated phe-nol degradation proles in the entire range of initial phenol concentrationsof 25800 mg/l. Nuhoglu and Yalcin 11 conrmed that the model could rectify the failure of the classical Haldane model, especially at high initial phenolconcentrations (Table 3). Another proposed model that accounts for the de-terminant role of metabolic intermediates is the two-step model. 10,106 Phenolbiodegradation was assumed to take place in two steps by two microbialpopulations constituting the whole biomass. In the rst step, phenol is de-graded by a fraction of the total biomass, which grows and produces oneor several metabolic intermediates. In the second step, the intermediate ismineralized by another microbial population. Because phenol and the inter-mediates are considered inhibitory substrates, the specic growth rates of thetwo steps, 1 and 2, are modeled according to a Haldane-type equation.The model involves a single set of kinetic parameters.

The rate of degradation was also proved to be strongly dependent onthe composition of the medium affecting the degradation efciency. Shourianet al.7 found that phenol-containing M9 minimal medium supplemented withmannitol and casein showed the highest degradation rate among all carbonand nitrogen nutrients tested. The highest inhibitory effects were attained by addition of sucrose and glycerol. In another study by Stehlickova et al., 101higher biodegradation rate and more intensive growth were observed dur-ing the cultivation in presence of potassium humate (humic substance) incomparison with the cultivation without its addition. The results suggested

that effect of the humate (0.005% [w/v]) on phenol biodegradation consistedof the interaction between the bacterial cell and the humic substance, whichmay serve as a protective layer on the cell surface ( Cupriavidus metallidu-rans ). Another reason for increased biodegradation efciency could be adecrease of phenol toxicity to the bacterial strain by reversible bond be-tween phenol and humate. At 400 mg/l, the specic growth rate was 0.0503hr1 without humate compared with 0.1741 hr 1 with humate.

The Monod equation (Michaelis-Menten) has also been used to describethe kinetics of oxygenation. The catechol dioxygenase activity was followedby monitoring the production of the main cleavage products (Table 2, Equa-

tion R).57


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T

A B L E 3

. S u m m a r y o f g r o w t h k i n e t i c s f r o m l i t e r a t u r e ( b a s i c a l l y H a l d a n e e q u a t i o n )

M

i c r o o r g a n i s m


r a n g e

( m g / l )

m a x

( h r

1 )

K s ( m g / l )

K i ( m g / l )

T e m p e r a t u r e ( C )

p H

R e f e r e n c e

P . p

u t i d a D S M 5 4 8

0 1

0 0

0 . 4 3 6

6 . 1 9

5 4 . 1

2 6 0 . 5

6 . 8

7 5

E w i n g e l l a A m e r i c a n a

0 1 0 0 0

0 . 2 9

0 . 3 2 a

5 . 1 5 6

1 0 3 3

. 7 2

3 7

7 . 5

1 6

I n d i g e n o u s m i x e d

c o n s o r t i u m

0 8

0 0

0 . 3 0 8 5

0 . 3 7 b

4 4 . 9

2

1 4 4 . 6 8 b

5 2 5 . 0 0

2 7

7 . 0

8 8

P . p

u t i d a M T C C 1 1 9 4

P h e n o l : 0 1 0 0 0

C a t e c h o l : 0 5 0 0

0 . 3 0 5

0 . 2 1 6 b

0 . 1 7 5 c

0 . 3 2 6

0 . 1 4 3 b

0 . 2 2 9 c

3 6 . 3

3

2 0 . 5

9 b

2 9 . 8

9 9 . 6 6 b

1 2 9 . 7 9

2 5 8 . 7 2 c

1 5 6 . 7 8 c

2 9 . 9

0 . 3

7 . 1

9

P . p

u t i d a C C R C 1 4 3 6 5

5 0 3

0 0

0 . 2 4 5

1 2 . 1

1 1 8 5

. 7

3 0

7 . 0

1 2

A l c a l i g e n e s f a e c a l i s

0 1 6 0 0

0 . 1 5

2 . 2 2

2 4 5 . 3 7

3 0

7 . 2

2 3

M

i x e d b a c t e r i a l

c o n s o r t i u m

2 3 . 5 6 5 8

0 . 3 0 9 5

7 4 . 6

6 4 8 . 1

2 5 2

7 . 2

9 9

M

i x e d c u l t u r e

( a c t i v a t e d s l u d g e )

5 0 0 2

5 0 0

0 . 4 3 8

2 9 . 5

7 2 . 4

A m b i e n t

6 . 5

1 0 2

M



5 0 1

0 0 0

0 . 1 1 9

1 1 . 1

3

2 5 0 . 8 8

2 0

U n c o n t r o l l e d

3 1

M



2 5 1

4 5 0

0 . 1 4 3

0 . 1 4 5 e

8 7 . 4

5

8 8 . 5

e

1 0 7 . 0 6

1 4 5 e

2 5 1

1 1

P . p

u t i d a A T C C 4 9 4 5 1

2 5 8

0 0

0 . 9

6 . 9 3

2 8 4 . 3

3 0

1 0 5

A c i n e t o b a c t e r

3 5 0

0 . 8 3

1 . 5

1 8 8

3 0

1 1 0

P . p

u t i d a

0 5

4

0 . 1 1 0 . 0 1

3 2 2 . 4

3 0

1 0 9

- A c t i v a t e d s l u d g e

- A e r o b i c g r a n u l a r

s l u d g e

0 3 0 0 0

0 5 0 0 0

8 7 . 5

d

4 0 4 d

1 9 8

6 5 0

3 5 7

1 1 4 0

3 0

7

8 3

a F o r s t a r v e d c e l l s

. b M o n o d . c

L i n e a r i z e d H a l d a n e . d

m g ( g

1 V S S ) h r

1 . e P r o p o s e d m o d e l b y W a n g a n d L o h . 1 0 5

1656


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TEMPERATURE SIGNIFICANCE IN KINETICSLi et al.107 reported that there has been little focus on microorganisms thatcan function at low temperature, with scarce information in this regard.Therefore, it is necessary to nd a kind of organism that can adapt to thesignicant daily and seasonal uctuations in environmental temperatures, with focus on cold-adapted microorganisms in environments where they areneeded. Conventionally, these organisms can be divided into psychrophilicand psychrotrophic organisms. Psychrophiles are organisms that have mini-mum, optimum and maximum growth temperatures of 0, 15, and 20C,respectively. The corresponding temperatures for psychrotrophs are 05,> 15, and > 20C. It is evident that the most interesting organisms are oftenthe psychrotrophs, as they are also active at temperatures above 20 C. Thebacterial strain P. putida LY1 as a psychrotrophic microorganism was shownto grow on phenol as the sole carbon and energy source and survive well ina wide range of temperatures. The intrinsic kinetics in the range 0800 mg/l was described well by the Haldane model. 107

The temperature-dependent performance of biological processes may be strongly inuenced by their content of psychrotrophic bacteria. It is re-ported that the effect of even 5 C change in temperature can be consider-able. 31 As a result, temperature signicantly affects critical process and designparameters, such as the critical dilution rate, which corresponds to the limitabove which biomass washout occurs and, hence, failure of the biologicaltreatment process. 103 According to Onysko et al., 103 there is a lack of suf-

cient information about the change of microbial kinetics as a function of temperature. The authors attempted to model the temperature dependenceof cell growth and phenol biodegradation kinetics of the psychrotrophic bac-terium P. putida Q5 in both batch and continuous cultures in the range of 1025C. The Haldane equation was found to be most suitable to model thegrowth kinetics P. putida Q5 on phenol. There are essentially two types of temperature models presently in use for max . The Arrhenius model, givenas

max

= Ae

H

R T (5)

includes the temperature characteristic (or apparent activation energy) H (kJ/mol), assumed to be constant, A is the frequency factor (s 1), and theempirical square-root model ( b 1 is the proportionality constant C1h0.5):

max =b 1(T T 0) (6) As the investigators expected from the well-known effect of temperatureupon chemical and biochemical reactions, max in the Haldane equation in-creased with increasing temperature, as did the half-saturation constant, K s.

The inhibition constant, K i, also increased as the temperature was increased,


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indicating that the degree of inhibition decreased at higher temperatures.The Haldane parameters max and K i were best modeled by a square-rootdependency on temperature. However, the Arrhenius model provided a bet-ter prediction of the temperature dependence of K s. Sa and Boaventura 108

reported that it is documented in literature that the removal efciency, , varies with temperature, T , in accordance with the expression ( 1 / 2 ) = (T 1T 2 ), the constant was determined from the experimental results.MIXED SUBSTRATE KINETICS Although microbial growth on substrate mixtures is commonly encounteredin biological treatment processes, mathematical modeling of mixed substratekinetics has been limited. 109 Reardon et al. 109 investigated the kinetics of P. putida F1 growing on benzene, toluene, phenol, and their mixtures, andcompared mathematical models to describe the results. The three aromaticsare each able to act as carbon and energy sources for this strain. Biodegra-dation rates were measured in batch cultivations following a protocol thatexcluded mass transfer limitations for volatile substrates. Because these threecompounds are homologous substrates that are catabolized by the sameenzymes in P. putida F1, their mixture can serve as a good model sys-tem for mixture biodegradation studies. Toluene signicantly inhibited thebiodegradation rate of both of the other substrates, and benzene slowedthe consumption of phenol (but not of toluene). Phenol had little effecton the biodegradation of either toluene or benzene. It was observed thatgrowth continued after phenol concentrations fell to zero, which indicatedthe formation (and consumption) of one or more intermediates. Models weretested that incorporated both substrate and intermediate inhibition (compet-itive and noncompetitive; Equations K, L, M, and N). Of the models tested, asum kinetics with interaction parameters (SKIP) model and unspecied typeof interaction

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Biodegradation of Phenol Review