Biodegradation of Phenol Review

Critical Reviews in Environmental Science and Technology, 42:16311690, 2012
Copyright Taylor & Francis Group, LLC

ISSN: 1064-3389 print / 1547-6537 online
DOI: 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 an
integrated approach that may be adopted to get synergistically en-
hanced removal rates and to treat the contaminated efuents in
an ecologically favorable process.
KEY WORDS: biodegradation, bioreactors, immobilization, kinet-
ics, phenol, wastewater
1. INTRODUCTION
Chemical and petroleum industries generate a wide variety of highly toxic
organic pollutants, which have led to cumulative hazardous effects on the
Address correspondence to Muftah H. El-Naas, Department of Chemical and Petroleum
Engineering, UAE University, PO Box 17555, Al-Ain, United Arab Emirates. E-mail: muftah@
uaeu.ac.ae
1631
1632 T. Al-Khalid and M. H. El-Naas
environment. The efuents of these industries often contain aromatic organic
compounds 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 hazardous
to human health.
13
Many aromatic compounds show carcinogenic, terato-
genic, or mutagenic properties.
4
Nonbiodegradable organic compounds must
be 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 great
concern worldwide. Phenols are well known for their high toxicity for hu-
man life, aquatic life, and others.
57
They are considered to be among the
most hazardous contaminants, and they are certainly the most difcult to
remove.
8
Phenol is a pollutant that is usually found in many industrial efuents
such as wastewaters from coal processing plants, oil reneries, pulp and
paper 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 it
quickly penetrates the skin and may cause severe irritation to the eyes, the
mucous 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 phenol
is reported to be lethal to humans.
11
The concentration of phenols in wastewater may vary from 10 to
300 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 at
concentrations of 525 mg/l. This imparts medicinal taste and objectionable
odor to drinking water even at a much lower concentration of 2 g/l.
9,13
Due 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 and
their potential toxicity has led the U.S. Environmental Protection Agency to
dene them as priority pollutants
16
and has set a water purication standard
of phenol concentration less than 1 g/l in surface waters.
14,17
The European
Council 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 the
European Union is presently undergoing considerable change. Adopted mea-
sures emphasize that surface water deterioration must be prevented, bodies
of water protected, and pollution from discharges of hazardous substances
reduced 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
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 safe
disposal to the environment. Efcient treatment methods are available for
the removal of phenol such as activated carbon adsorption, ion exchange,
liquidliquid extraction, and chemical oxidation; however, they often suffer
from serious drawbacks such as high cost. In addition, most of these tech-
niques do not degrade phenol, but rather move it to another phase, which
results in the formation of hazardous byproducts (secondary pollution). On
the other hand, biodegradation is considered a more environmental friendly
and cost-effective alternative. Biological treatment of phenols has therefore
been 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 a
compound 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 no
byproducts. However, the practical application of this technology is rather
limited because of its poor adjustability to uctuation in the phenolic load.
23
The inability of conventional biological treatments to effectively remove
many toxic pollutants indicates that novel biological treatment systems are
needed, and the use of pure and mixed cultures of organisms is considered
a favorable and most promising approach.
7
Although there has been a considerable amount of research carried
on 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 mediating
biodegradation of phenols and the mechanism involved. In this article we
offer a comprehensive review of the literature work on the aerobic biodegra-
dation of phenols during the past decade, with emphasis on evaluating the
present state of the area and exploring future research possibilities. The
review 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; recent
advances 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 Properties
Phenol 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
FIGURE 1. Chemical structure of common phenolic compounds.
synthetic organic compounds. The chemical structures of phenol and some
commonly studied phenolic compounds are given in Figure 1.
As an organic substance, phenol is soluble in most organic solvents
and it is slightly soluble in water at room temperature, but entirely water
soluble above 68
C.
24
Phenols are distributed either as natural or articial
monoaromatic 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
Because of the aromatic structure of phenol, it is resistant to natural
biodegradation and phenolic compounds have been reported to have high
stability 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 ring
cleavage using the enzyme present in the microorganism.
9
2.2 Microorganisms in Phenol Biodegradation
In general, toxic and hazardous xenobiotics, which have a structure that is
different from naturally occurring compounds, are more difcult to degrade.
In recent years, however, an array of microorganisms has been identied
that 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
Although
aerobic and anaerobic microorganisms are able to degrade phenol, con-
ventionally aerobic processes are preferred.
3032
Aerobic microorganisms are
more efcient for degrading toxic compounds because they grow faster
and usually transform organic compounds to inorganic compounds (CO
2
,
H
2
O).
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 in
the utilization of anaerobic bacteria for the degradation of phenols. Several
studies have been reported in the literature in this regard.
3643
Since most
biological treatments studies have used aerobic biomasses, in this review we
focus on aerobic biodegradation.
A large number of microorganisms including bacteria, fungi, and al-
gae
30
are capable of degrading phenol. The biodegradation of phenol and
its derivatives by bacteria has been extensively studied and a large number
of phenol-degrading bacteria have been isolated and characterized at the
physiological 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 different
biotechnological 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 that
its cells could use diverse protective mechanisms for survival in various ex-
treme environments. These studies could help in synthesizing new bacterial
strains with enhanced degradation capability and improved tolerance to toxic
pollutants.
52
Fungi share a signicant part in the recycling of aromatic compounds in
the biosphere and several studies have shown that diverse fungi are capable
of 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
fungi in polluted environments are yeasts. Some yeasts such as Candida
tropicalis, Fusarium occiferium, and Trichosporon cutaneum are capable
of 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 development
of technologies for purication of contaminated soils and waters.
50
Rubilar
et al.
54
analyzed the degradation of chlorophenols by white rot fungi, which
are a group of organisms very suitable for the removal of chlorinated pheno-
lic compounds. They are robust organisms that are tolerant to the presence
of high concentrations of various pollutants, even with a low bioavailability
and this ability is mainly due to their very powerful extracellular oxidative
enzymatic system.
The bioremoval of phenols by bacteria and fungi has been extensively
studied, but only recently there has been more interest in investigating the
capabilities of some algae for phenol biodegradation. The biodegradation
of phenol by microalgae occurs only under aerobic conditions. While some
algae have low tolerance to the acute toxicity of phenols, both cyanobacteria
and eukaryotic microalgae (e.g., Chlorella sp., Scenedesmus sp., Selenastrum
capricornutum, 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 Biodegradation
Metabolic 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 ultimate
source of energy.
24
The biodegradation process requires the presence of
molecular oxygen to initiate enzymatic attack on the aromatic rings. A typical
pathway for metabolizing phenol is to hydroxylate the ring, by the enzyme
phenol 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 phenol
degradation.
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 is
cleaved by the enzyme catechol 2,3-dioxygenase (C23O). Thus, the ring is
opened and then degraded.
1,24,56
The ring cleavage can occur in two different
orientations and this difference in cleavage site is used to classify catechol
dioxygenases in two groups; the intradiol (such as C12O) and extradiol (such
as C23O) cleaving enzymes.
57
The genes of ring-cleavage dioxygenases may
serve as good targets for monitoring the biodegrading biomass, thus pro-
viding a rapid method for monitoring the microbial community during the
treatment process.
58
Discussion of biodegradation pathways and mechanisms
can 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 assays
proved 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 was
shown that between the two enzymes of the o- and m-pathways, the phenol
hydroxylase, which hydroxylates phenol to catechol, was the key enzyme
for phenol biodegradation. The hydroxylation reaction in phenol metabolism
was the key determinant of biodegradation velocity. The assay of enzyme
activity proved that phenol was assimilated by o-ssion of catechol. On the
other hand, Khleifat
16
reported that most bacteria use the m-pathway of cat-
echol degradation. The author evaluated the degradation of phenol by the
bacterium Ewingella americana and detected the activity of C23O, indicat-
ing that the catechol ring ssion occurs through the m-pathway, not through
the o-pathway. Similar results were shown for strains of Bacillus cereus, a
bacterium with high potential for phenol degradation and high tolerance.
62
Stoilova et al.
6
reported that little is known about phenol metabolism
by fungi. In most previous studies, phenol was metabolized by the -
ketoadipate pathway, through o-ssion of catechol.
6
The properties of the
involved key enzyme(s) play a key role in determining the efciency of a
certain catabolic pathway. Vallini et al.
63
investigated the metabolism path-
ways of alkyphenols by a yeast strain, classied as Candida aquaetextoris. A
novel metabolic route for the microbial degradation of 4-(1-nonyl) phenol,
at least in certain yeasts, was proposed.
2.4 Factors Affecting Biodegradation of Phenols
Biodegradation is a multifaceted process in which many biotic and abiotic
factors 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, and
physical properties of contaminants.
1,8,24,59
Each of these factors should be
optimized 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
biodegradation by microbial cells has generally been known to be inhib-
ited by phenol itself, especially at higher concentrations. Although phenol is
biodegradable both aerobically and anaerobically, it can inhibit the growth
of microorganisms at elevated concentrations, even for those species that can
use it as a substrate.
1,11,64
Adjei and Ohta
65
reported that phenol was com-
pletely inhibitory to cyanide utilization by the bacteria Burkholderia cepacia
strain 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 its
growth. Consequently, laboratory studies on phenol degradation are usually
carried out at or near neutral pH values (pH = 7.0). Each organism has a
certain 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 phenol
at 50
C.
24
El-Naas et al.
8
believed that sudden exposure to temperatures
higher than 35
C may have detrimental effect on the bacterial enzymes that

are usually responsible for the benzene ring cleavage, which is the key step
in the biodegradation process. On the other hand, exposure to temperatures
much lower than 30
C was expected to slow down the bacterial activity and

enhance the inhibitory effect of phenol on the bacteria, especially for high
phenol concentrations. Most of the studies on phenol biodegradation had
been carried out in the temperature range 2535
C (Table 1).
Some other factors that can affect the biodegradation of phenols are
chemical structure and compound toxicity. The chemical structural effect is
reected by the number of substituents, type of substituents, position of sub-
stituents and degree of branching. The greater the number of substituents
in the structure, the more toxic and less degradable it becomes. For exam-
ple, substituted phenols such as mono, di-, tri-, and pentachlorophenol are
less degradable than unsubstituted phenol. In addition, o- and p-substituted
phenols are more degradable than m-substituted phenols.
24
Toxicity prevents or slows metabolic reactions. This depends on the
exposed 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 or
mixed cultures. It is likely that an application of the mixed culture permits
faster 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 the
biodegradation rate of phenol can be improved by immobilizing the cells and
entrapping 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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6
f
o
r
C
o
=
9
4
9
7
1641
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-gauze
hybrid 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 immobilization
method is not toxic to the cells and is inert and practical.
14
There are various
technologies for bacteria immobilization, including bead entrapment, carrier
binding, adsorption techniques, encapsulation, cell coating, and lm attach-
ment.
69
Immobilization of bacterial biomass for the degradation of phenol
is an important and effective technique that is usually employed to serve
several purposes, including protection of the bacteria from high phenol con-
centrations as well as ease of separation and reutilization of the biomass. It
has been reported that the use of free bacterial cells for wastewater treatment
in activated sludge processes creates problems such as solid waste disposal,
while immobilized microorganisms are capable of effective treatment with
little sludge formation.
2,21
The merit of immobilization is due to the high
surface area available for biolm formation, which results in high biomass
concentration of 3040 g VSS/l, compared with 1.52.5 g VSS/l for activated
sludge systems.
70
In addition, the systems with immobilized cultures are
more stable to shock loadings than the suspended cultures with free cells
53
and immobilization can provide high degradation capacity compared with
free cells.
71
Kim et al.
72
showed that calcium alginate immobilization of mi-
crobial cells effectively increased the tolerance of P. putida MK1 to phenol
and improved the degradation of pyridine in a binary mixture of the two
compounds.
The performance of a biodegradation system may be affected by the
presence of metabolic inhibitors or competing substrates.
12,67
There are con-
straints imposed by the occurrence of the environmental contaminants in
mixtures as the degradation of one component can be inhibited by other
compounds in the mixture, and because different compounds within the
mixture may require different treatment conditions.
50
The ability of microbial communities to degrade pollutants is affected
by 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
)
2
SO
4
enhances the ability to degrade phenols. Gladyshev et al.
73
reported that biodegradation of phenolic compounds is known to increase
at higher concentrations of inorganic nutrients, while it is inversely affected
by higher concentrations of organic nutrients. The process of cometabolism
is an important example of the inuence of substrate interaction during the
biodegradation of pollutants.
74
Cometabolism is dened as the degradation
of a compound only in the presence of another organic material that serves
as the primary growth substrate. This phenomenon has been attributed to
the production of broad-specicity enzymes, where the primary substrate
and the other compound compete for the same enzyme.
26
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 researchers
in free and immobilized forms, batch and continuous modes, and pure and
mixed cultures, using different types of bioreactors.
8
During the biodegradation of phenol, the contaminant concentration
decreases with time while the biomass is generated. Figure 2 is a typical
illustration of how the concentrations change with time. The gure also
clearly shows the effect of lag times at the beginning of the process, especially
when the biomass cells are not preadapted to the contaminant medium. The
lag 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 phenol
to the microorganism at high initial phenol concentrations. The biodegrada-
tion rate, which is a measure of the biomass activity, starts to decrease if the
initial phenol concentration is above the maximum concentration that can
be 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 (C
o
= 1100 mg/l). According to the study, higher efciencies are
obtained 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.
FIGURE 3. Biodegradation rate of phenol as a function of initial phenol concentration.
concentration of phenols, type of reactor, type of microorganisms, and the
removal efciency that could be achieved. In general, these studies have
addressed important research topics, and hence some of their main ndings
are discussed in the following sections.
3.1 Immobilization
In view of its major advantages, the immobilization of biomasses has been
receiving considerable attention. Using immobilized cell is a well-known
approach for incorporating bacterial biomass into an engineering process.
74
Continuous degradation of phenol at an inuent concentration of 100 g/l
with immobilized P. putida was investigated by Mordocco et al.,
67
who
pointed out the signicance of this low range of concentrations in light of the
potential 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 free
cells, the superiority of the immobilized cell system was more pronounced.
A bead diameter between 1 and 2 mm was found to be optimal for phenol
degradation at low levels.
Physical entrapment of organisms inside a polymeric matrix has been
extensively used for whole-cell immobilization.
14,74
The effectiveness of this
method has also been investigated by El-Naas et al.
8
in a study to assess
the biodegradation of phenol by P. putida immobilized in PVA gel matrix
at 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
phenol uptake per mass of PVA, which has a signicant economical consid-
eration in designing an industrial process for wastewater treatment. At high
phenol concentration, the PVA particle size was found to have negligible
effect on the biodegradation rate. However, for low concentrations, particle
size seemed to show a small effect, which conrmed the importance of mass
transfercontrolled mechanisms.
Subsequent studies in the batch and continuous modes in a spouted
bed bioreactor (SBBR),
22,51
which is characterized by intense mixing, proved
that mass transfer limitations have considerable effect on the biodegradation
rate and the dynamics of the system is mainly controlled by the internal mass
transfer (at the biomass interface).
Wang et al.
21
used PVA carrier for immobilization and reported that the
immobilized cells could tolerate higher phenol levels and wider changes
in temperature and pH. Immobilization increased the thermal stability of
the microbial cells under the protection of PVA carrier. The immobilized
cells possessed better storage stability and could be reused for at least 50
cycles, which demonstrated that PVA carrier cubes had good exibility and
retained a high mechanical strength. More examples for the biodegradation
of phenols with immobilized microorganisms are presented in Table 1.
3.2 Bacteria for High Phenol Concentrations
Kumar et al.
9
carried out a kinetic study utilizing P. putida MTCC 1194 in
the initial concentrations of 1000 and 500 mg/l of phenol and catechol,
respectively. Even the well-acclimatized cultures showed lag phase due to
the high concentration of phenol. The higher the initial concentration, the
longer was the lag phase (inhibitory effect). No bacterial growth could be
detected at phenol concentrations of 1200 mg/l, even after 20 days. For
catechol, the growth of the bacterial strain was inhibited when exposed to
600 mg/l.
According to Wang et al.,
21
little information on bacteria with a high
phenol tolerance and high metabolizing activity is available. Therefore, there
still 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.
55,7682
It is a novel wastewater treatment technology that can quickly decontaminate
highly contaminated wastewater. Aerobic granules yield a very high biomass
concentration (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
Laser scanning microscopy test revealed that live cells are principally dis-
tributed throughout the surface layer of the granule, with an extracellular
polymeric substance layer covering them to protect the cells from phenol
toxicity.
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-strength
phenol wastewater. The acclimated granules degraded high concentration
phenol, of up to 5000 mg/l, at an acceptable rate, even after acid or alkaline
pretreatment.
3.3 Bacteria From Contaminated Sites
Shourian et al.
7
reported that many pollution problems resulting from re-
leasing aromatic chemicals occur in rivers, lakes, groundwaters, and process
efuents 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 and
unique candidate for rapid and efcient phenol removal, particularly for
the shorter lag time at high phenol concentrations (up to 1000 mg/l) com-
pared with those occurring in other Pseudomonas species. Other researchers
have utilized microbial biomasses isolated from phenol-contaminated soil,
2
wastewater plants,
16,25
efuents of coke processing units,
44
and municipal
gas 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 contains
phenol as well as other aromatic compounds.
20,85
Alva and Peyton
85
reported
the 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 saline
environments (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
eutropha is one of these microorganisms; Ralstonia is a newly designated
genus 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 being
reported. R. pickettii strain LD1 can metabolize monochlorophenols, which
represent an important challenge as they may be particularly formed during
the 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 using
pure species of microorganism are available, reports on using mixed cultures
of microorganism are scant.
88
Liu et al.
2
evaluated a mixture of Acinetobac-
ter sp. XA05 and Sphingomonas sp. FG03 strains for phenol degradation
and reported better degradation efciency by the mixed cells than by the
pure cultures. The authors attributed that to the fact that the strains of XA05
and FG03 were isolated from different environment conditions, and they
have different enzyme systems and different ways to degrade phenol. When
mixed, 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 chemicals
by microbes using pure cultures can produce toxic intermediates and that
this problem may be overcome by the use of mixed cultures, which have
a wider spectrum of metabolic properties. On the other hand, the same au-
thors reported a higher efciency when selecting a pure culture of P. putida
DSM 548 for phenol degradation. This discrepancy may be attributed to the
difference in the phenol concentration range between the two studies, being
high for the study of Liu et al.
2
and low for the study by Monteiro et al.
75
It
is believed that the synergism effect of different strains is enhanced at high
concentrations of phenol.
3.6 Research on Mixed Substrates and Industrial Efuents
Biodegradation 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 transformation
of a nongrowth substrate by cells growing on a growth substrate.
12,13
In this
regard, the results from a study on phenol biodegradation in a binary mixture
of phenol and sodium salicylate by free P. putida CCRC 14365 in a batch
mode proved that the cells adapted with comparatively hard-to-degrade sub-
strate (sodium salicylate, in this case) facilitated the overall biodegradation
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 simulating
industrial 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 ATCC
17484 immobilized in calcium-alginate gel beads was reported by Gonzalez
et al.
89
In a batch operation, phenol concentration of up to 1000 mg/l was
degraded. Compared with a degradation capacity of 2000 mg/l from previ-
ous work by the authors with model solution,
90
the negative effect of the
nonphenolic compounds in the industrial wastewater was indicated. Also
Agarry et al.
91
treated renery wastewater in their study, which investigated
the phenol-biodegrading potential of two indigenous Pseudomonas species.
3.7 Fungi and Algae in Phenol Biodegradation
The 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, and
others. Inhibition was observed for all substrates above an inuent concen-
tration of 2000 mg/l. Upon repeated reuse of the cells, the cell adaptation to
phenol 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 activated
sludge. The mutant strain CM 2 possessed the strong capacity to degrade
phenolic compounds, especially phenol and 4-chlorophenol (4-CP). In this
dual substrate biodegradation system, the mode of interaction was revealed
in such a way that low-concentration phenol could enhance biodegradation
of 4-CP, while very little 4-CP could greatly inhibit phenol biodegradation.
The fungal strain Aspergillus awamori NRRL 3112 was reported to have
high 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 special
interest, which is the interaction of substrates in substrate mixtures. The
main nding was that for all combinations containing dichlorophenol, the
diauxy 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 both
substrates were metabolized simultaneously (Figure 5). The diauxic growth
characteristics were also investigated by other researchers.
96
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 pathways
for catabolism of phenol in fungi are carried out similar to bacteria, where
phenol 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.
of the two enzymes, C
12
O and C
23
O, were reported in terms of enzymatic
activities at different pH and temperature. Mineral salts added in culture have
an inhibition on both enzymes. The activity range of C
12
O was from pH 3 to
pH 8.8 and from 30 to 50
C, being optimal at pH 6.8 and 40
C. The activity of
C
23
O was slightly more sensitive to pH, being higher in the range of 7.410.6
and 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. Lika
and Papadakis
30
developed a mechanistic model for the aerobic degradation
of 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 for
inhibitory effects at high phenol concentrations and to include mixtures of
phenolic compounds as well as mixed cultures. A study by Semple
97
showed
that Ochromonas danica, a eukaryotic alga, is capable of growing on phenol
and 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 Modeling
In biodegradation reactions, kinetics studies give a measure of how effec-
tively the microbial system is functioning.
24
Knowledge of such kinetics will
help 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 microbial
growth 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 and
2, respectively:
dX
dt
= X k
d
X =
net
X or
d lnX
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
(hr
1
); 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),
TABLE 2. Biodegradation models (kinetics and mass transfer)
Name of model Equation
Example
references
Monod =

max
S
K
s
+ S
[A] 8, 9, 88, 99
Haldane (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
0
e
t
[I]
=

max
S
0
K
s
+ S
0
+
_
S
2
0
K
i
_ [J]
Monod: sum kinetics
Binary mixture, no
interaction
=

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

max,2
S
2
K
s,2
+ S
2
[K] 109
Monod: sum kinetics
Binary mixture,
purely competitive
interaction
(inhibition)
=

max,1
S
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,
noncompetitive
inhibition
=

max,1
S
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,
uncompetitive
enzyme inhibition
=

max,1
S
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)
TABLE 2. Biodegradation models (kinetics and mass transfer) (Continued)
Name of model Equation
Example
references
SKIP
b
Binary mixture,
unspecied type of
interaction
=

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

max,2
S
2
K
s,2
+ S
2
+ I
1,2
S
1
[O] 19, 109
SKIP
Three-compound
mixture,
unspecied type of
interaction
=

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

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

max,3
S
3
K
s,3
+ S
3
+ I
1,3
S
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
1
S
+
1
V
max
[R] 57
J D-factor J
D
=
k
L
G
N
2/3
Sc
= KN
(1n)
Re
[S] 14, 86
Ficks Law
d
d
2
C
dr
2
+
2
r
dc
dr
=

p
D
e
v [T] 3, 116
Thiele Modulus
e
= r
0
_
k
D
e
[U] 3, 100, 116
a
R is the specic consumption rate of substrate (mg/mg.hr), k
s
is the saturation constant for substrate
consumption (mg/l); f(i) represents the functional relationship of the effect of metabolic intermediates on
phenol degradation; and K
p
is a proportionality constant.
b
I
ij
is the interaction parameter that indicates
the degree to which substrate i affects the degradation of substrate j.
c
For 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.
d
C 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 diffusion
coefcient of phenol within the bead, respectively. is the actual degradation rate (mg/g.hr).
e
r
0
is
the radius of the particle, k
is the rate constant = k

p
where k is rst-order degradation rate constant
(cm
3
/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 dependence
on substrate concentration. The rst considers phenol as noninhibitory com-
pound and, therefore, neglects the inhibitory effect, whereas the second
takes into consideration the inhibitory effect of phenol.
8
These two models
can be used to predict the variations of the biodegradation rate with initial
phenol concentrations, utilizing the relation in Equation 2 and assuming that
Y is constant over the concentration range. This assumption is valid if the
phenol concentration is much higher than K
s
(i.e., S >>K
s
).
8
However, the
Haldane equation has been used extensively to describe phenol microbial
degradation in pure and mixed cultures. Although the model is based on
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 is
used because of its wide applicability and mathematical simplicity for repre-
senting cell growth kinetics of inhibitory substrate, as it has less parameters
and is easily used for representing continuous biological reactors.
9,12
The Haldane (or sometimes referred to as Haldane-Andrews) equation
represents a modication of the original Monod equation to account for
inhibitory effects when a substrate (or substrate biotransformation interme-
diate) is toxic to the degrading population. The effects of this self-inhibition
are incorporated into the expression with an inhibition term, S/K
i
in the
denominator. A larger K
i
value indicates that the culture is less sensitive
to substrate inhibition. It should be noted that when K
i
is very large the
Haldane 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+
_
S
2
K
i
_, (4)
which leads to the linearized Haldanes equation (Table 2, Equation C).
9,102
Monods 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 the
linearized-Haldanes model were used to provide initial guess values of the
Haldane model parameters. A typical growth curve showed a decline in cell
population after the complete consumption of substrate (endogenous or de-
cay coefcient), which was explained by drop in oxygen and pH values
toward 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 the
growth of a microbial consortium at different concentrations of substrate. The
culture followed substrate inhibition kinetics that could be tted to Haldane
and Han-Levenspiel models. Between the two models, the Han-Levenspiel
model (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
that may be formed during degradation, whereas Haldanes model is based
on 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. putida
CCRC 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 Onysko
et al.
103
for phenol biodegradation by P. putida Q
5.
; little difference was
found 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 model
and 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 proles
only when different sets of model parameters were used (Table 3).
11
Differ-
ent Haldane kinetic parameter values were obtained even when the same
microbial 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. The
inadequacy of the Haldane equation has been attributed to the inhibition of
metabolic intermediates of phenol degradation.
6,11,24,105
These discrepancies
may 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 intermediates
has been conrmed by Bajaj et al.
70
The phenol degradation prole indi-
cated that the presence of acetate that represents an intermediate of phenol
degradation retarded the phenol degradation. The highest phenol removal
rate observed in batch assay was 1.51 g/l.day in the presence of acetate
and it increased to 3.54 g/l.day after acetate depletion.
70
Monteiro et al.
75
explained the variation of kinetic parameters by the different inoculums
history. 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 constant
and not limiting.
23,61,75
A lag phase had also been observed experimentally.
The length of the lag phase before the exponential growth phase increased
linearly with phenol concentration between 1 and 100 mg/l.
75
Wang and Loh
105
found that the Haldane equation was not sufcient for
modeling phenol degradation, especially for high initial phenol concentra-
tions, although it could correlate specic growth rates with initial substrate
concentrations very well. Nuhoglu and Yalcin
11
raised an argument that the
degree 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
critical substrate concentration S
where d/dS = 0) is relative to

max
, and
thus, 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
, the
Haldane 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 average
constant, which is not always true, especially at extremes of specic growth
rate. Consequently, a new phenol degradation model was proposed by Wang
and Loh
105
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 concentrations
of 25800 mg/l. Nuhoglu and Yalcin
11
conrmed that the model could rectify
the failure of the classical Haldane model, especially at high initial phenol
concentrations (Table 3). Another proposed model that accounts for the de-
terminant role of metabolic intermediates is the two-step model.
10,106
Phenol
biodegradation was assumed to take place in two steps by two microbial
populations constituting the whole biomass. In the rst step, phenol is de-
graded by a fraction of the total biomass, which grows and produces one
or several metabolic intermediates. In the second step, the intermediate is
mineralized by another microbial population. Because phenol and the inter-
mediates are considered inhibitory substrates, the specic growth rates of the
two 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 on
the composition of the medium affecting the degradation efciency. Shourian
et al.
7
found that phenol-containing M9 minimal medium supplemented with
mannitol and casein showed the highest degradation rate among all carbon
and nitrogen nutrients tested. The highest inhibitory effects were attained by
addition of sucrose and glycerol. In another study by Stehlickova et al.,
101
higher biodegradation rate and more intensive growth were observed dur-
ing the cultivation in presence of potassium humate (humic substance) in
comparison with the cultivation without its addition. The results suggested
that effect of the humate (0.005% [w/v]) on phenol biodegradation consisted
of the interaction between the bacterial cell and the humic substance, which
may serve as a protective layer on the cell surface (Cupriavidus metallidu-
rans). Another reason for increased biodegradation efciency could be a
decrease of phenol toxicity to the bacterial strain by reversible bond be-
tween phenol and humate. At 400 mg/l, the specic growth rate was 0.0503
hr
1
without humate compared with 0.1741 hr
1
with humate.
The Monod equation (Michaelis-Menten) has also been used to describe
the kinetics of oxygenation. The catechol dioxygenase activity was followed
by monitoring the production of the main cleavage products (Table 2, Equa-
tion R).
57
T
A
B
L
E
3
.
S
u
m
m
a
r
y
o
f
g
r
o
w
t
h
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)
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(
m
g
/
l
)
m
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(
h
r
1
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s
(
m
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i
(
m
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)
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1656
TEMPERATURE SIGNIFICANCE IN KINETICS
Li et al.
107
reported that there has been little focus on microorganisms that
can function at low temperature, with scarce information in this regard.
Therefore, it is necessary to nd a kind of organism that can adapt to the
signicant daily and seasonal uctuations in environmental temperatures,
with focus on cold-adapted microorganisms in environments where they are
needed. Conventionally, these organisms can be divided into psychrophilic
and psychrotrophic organisms. Psychrophiles are organisms that have mini-
mum, optimum and maximum growth temperatures of 0, 15, and 20
C,
respectively. The corresponding temperatures for psychrotrophs are 05,
>15, and >20
C. It is evident that the most interesting organisms are often

the psychrotrophs, as they are also active at temperatures above 20
C. The
bacterial strain P. putida LY1 as a psychrotrophic microorganism was shown
to grow on phenol as the sole carbon and energy source and survive well in
a 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 design
parameters, such as the critical dilution rate, which corresponds to the limit
above which biomass washout occurs and, hence, failure of the biological
treatment 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 dependence
of cell growth and phenol biodegradation kinetics of the psychrotrophic bac-
terium P. putida Q5 in both batch and continuous cultures in the range of
1025
C. The Haldane equation was found to be most suitable to model the

growth kinetics P. putida Q5 on phenol. There are essentially two types of
temperature models presently in use for
max
. The Arrhenius model, given
as
max
= Ae
H
RT
(5)
includes the temperature characteristic (or apparent activation energy) H
(kJ/mol), assumed to be constant, A is the frequency factor (s

1
), and the
empirical square-root model (b
1
is the proportionality constant

C
1
h
0.5
):
max
= b
1
(T T
0
) (6)
As the investigators expected from the well-known effect of temperature
upon 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,
indicating that the degree of inhibition decreased at higher temperatures.
The Haldane parameters
max
and K
i
were best modeled by a square-root
dependency on temperature. However, the Arrhenius model provided a bet-
ter prediction of the temperature dependence of K
s
. S a 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
1
T
2
)
, the constant was determined from the experimental results.
MIXED SUBSTRATE KINETICS
Although microbial growth on substrate mixtures is commonly encountered
in biological treatment processes, mathematical modeling of mixed substrate
kinetics has been limited.
109
Reardon et al.
109
investigated the kinetics of
P. putida F1 growing on benzene, toluene, phenol, and their mixtures, and
compared mathematical models to describe the results. The three aromatics
are each able to act as carbon and energy sources for this strain. Biodegra-
dation rates were measured in batch cultivations following a protocol that
excluded mass transfer limitations for volatile substrates. Because these three
compounds are homologous substrates that are catabolized by the same
enzymes in P. putida F1, their mixture can serve as a good model sys-
tem for mixture biodegradation studies. Toluene signicantly inhibited the
biodegradation rate of both of the other substrates, and benzene slowed
the consumption of phenol (but not of toluene). Phenol had little effect
on the biodegradation of either toluene or benzene. It was observed that
growth continued after phenol concentrations fell to zero, which indicated
the formation (and consumption) of one or more intermediates. Models were
tested that incorporated both substrate and intermediate inhibition (compet-
itive and noncompetitive; Equations K, L, M, and N). Of the models tested, a
sum kinetics with interaction parameters (SKIP) model and unspecied type
of interaction best described the paired substrate results. This model, with pa-
rameters determined from one- and two-substrate experiments, provided an
excellent prediction of the biodegradation kinetics for the three-component
mixture (Table 2, Equations O and P).
Martn et al.
19
also used the SKIP model to describe the biodegradation
kinetics of P. putida CECT 324 growth on formic acid, vanillin, phenol, and
oxalic acid mixtures. It was found that phenol and oxalic acid inhibit P.
putida growth, and formic acid consumption strongly affects the biodegra-
dation of oxalic acid.
19
According to Jiang et al.,
95
the Haldane equation was selected for as-
sessing the dynamic behavior of the CTM 2 grown on phenol, but was
not suitable to describe the 4-CP biodegradation, which imposed a stronger
inhibition than phenol on the cells. Thus, the kinetic behaviors were de-
scribed using the kinetic model proposed in their lab. An inhibition constant
for cell growth (K
i
1
) was appended in Haldanes equation (Table 2, Equa-
tion Q). The specic degradation rates of phenol and 4-CP in dual-substrate
biodegradation were described by Equations 7 and 8, respectively:
S1
= A
1
X1
+ B
1
(7)
S2
= A
2
X2
+ B
2
(8)
The total cell specic growth rate is given by
X
=
X1
+
X2
(9)
Bhatt et al.
26
reported that metabolic rates in general are several orders of
magnitude higher than cometabolic rates and gave an example that
max
/K
s
values for phenol degradation were three orders of magnitude higher than
the values for trichloroethene in several phenol-oxidizing bacteria.
26
Although microbial transformations via cometabolism have been widely
observed, the few available kinetic models of cometabolism have not ad-
equately addressed the case of inhibition from the growth and nongrowth
substrates.
110
A study by Hao et al.
110
investigated the degradation kinetics
of self-inhibitory growth (phenol) and nongrowth (4-CP) substrates, present
individually and in combination. The 4-CP transformation by the Acineto-
bacter species growing on phenol was modeled using a competitive kinetic
model of cometabolism, which included growth and nongrowth substrate
inhibition and cross-inhibition terms. The model successfully predicted the
synchronous disappearance of the phenol and 4-CP mixture.
RESPONSE SURFACE METHODOLOGY
Response surface methodology (RSM) is the most widely used statistical tech-
nique for bioprocess optimization.
111
RSM is a graphical statistical approach
and an empirical modeling technique used to evaluate the relationship be-
tween a set of controllable experimental factors and observed results.
112
Basically this optimization process involves three major steps, which are per-
forming the statistically designed experiments, estimating the coefcients in a
mathematical model, and predicting the response and checking the adequacy
of the model. Response surface methodology requires a prior knowledge of
the process to achieve a statistical model,
74,112
in the following form:
Y = f (x
1
, x
2
, x
3
, x
4
. . . x
k
) (10)
For statistical calculations the variable x
i
is coded X
i
according to X
i
=
(x
i
x
0
)/x.
The mathematical relationship of the response of these variables can
be approximated by the following quadratic (second-degree) polynomial
equation:
Y = b
0
+b
i
X
i
+b
ii
X
2
i
+b
i j
X
i
X
j
(11)
where Y is the predicted response, b
0
is the offset term, b
i
is the linear effect,
b
ii
is the square effect, and b
ij
is the interaction effect. The low, middle, and
high levels of each variable (equally spaced) are designated 1, 0, and +1,
respectively.
Annadurai et al.
74
employed RSM to optimize the medium composition
for the degradation of phenol by P. putida (ATCC 31800). The mathematical
expression of the relationship of phenol degradation with variables such as
glucose, yeast extract, ammonium sulfate, and sodium chloride was found
and a mathematical model was then developed to show the effect of each
medium composition and their interactions on the biodegradation of phenol.
By this model, the response for the above variables can be predicted at any
time.
Annadurai et al.
111
also employed RSM to examine phenol degrada-
tion using mixed liquors of P. putida and activated sludge. The effects of
pH, temperature, carbon, and nitrogen sources were discussed. Experimen-
tal design should allow selecting optimal conditions in such biodegradation
processes. In another study,
71
an attempt was made to study the degrada-
tion of phenol under various initial phenol concentrations, initial pH val-
ues, and temperatures using immobilized cells and then modeling the data
using RSM, which develops a relationship between maximum percentage
of phenol degraded and fundamental variables, namely initial phenol con-
centrations, initial pH values, temperatures, and diameter of immobilized
P. pictorumalginate beads.
Bhattacharya and Banerjee
112
and Bhattacharya et al.
113
employed RSM
for the study of enzyme-mediated biodegradation of 2, 4-dichlorophenol.
The method has also been used by others.
114,115
4.2 Mass Transfer
As kinetic models for phenol degradation in biolms are complex and dif-
cult to develop, it is assumed that kinetic models for suspended cell cultures
apply equally well to biolm cultures, when the internal diffusion resistances
are not accounted for in the inherent growth kinetics.
70,100
Several authors
have developed microkinetic models that attempt to cover all mass transfer
and bioconversion processes in the bioreactors.
Although the immobilization of biomass by entrapment has several ad-
vantages, its major drawback is nutrient or product diffusion limitation be-
cause of the resistance of protective structure. Diffusion limitation is a general
problem that often decreases the efciency of degradation.
17,86
The biomass
is not easily accessible to pollutant because the majority of sites will lie
within the bead. Thus, a good support material for biomass immobilization
should be rigid, chemically inert, and inexpensive. It should bind the cells
rmly and should have high loading capacity and a loose structure for less
diffusion limitations.
86
For any phenol biodegradation process in a moving bed reactor that
involves immobilized biomass, there are three basic processes occurring in
the bioreactor: (a) transport of oxygen from the gas phase into the bulk
liquid; (b) transport of phenol, oxygen, and other nutrients from the bulk
liquid phase to the surface of the biolm; and (c) simultaneous diffusion and
reaction of phenol, oxygen, and other nutrients within the biolm. The last
step (c) is not dependent on ow conditions or turbulence in the reactor, as
it is a molecular phenomenon.
100
As for the rst process (a), it is directly related to the presence of dis-
solved oxygen. Melo et al.
32
reported in their study that phenol biodegrada-
tion can be affected by insufcient oxygen due to oxygen transfer limitations.
The rate of mass transfer of oxygen would increase ve times if oxygen were
used instead of air. Alternatively, increasing stirring speed proportionally in-
creased the mass transfer coefcient of oxygen and also resulted in improved
phenol degradation.
In process (b), transfer of the substrate into biolm is assumed to occur
in two steps: (a) transfer of the substrate from bulk liquid to the surface
of bioparticle and (b) diffusion through the microorganism (biolm) layer.
Many studies have focused on this process as the rate of transport of phenol
from the bulk phase to the surface of the biolm would directly inuence the
biochemical reaction taking place in biolm. Thus, knowledge of external
mass transfer coefcients for the transfer of phenol from the bulk phase
to the surface of the biolm is essential in the design and modeling of
bioreactors. Vinod and Reddy
100
developed a diffusion-reaction model for
phenol biodegradation by P. putida immobilized on solid particles in a
uidized bed bioreactor, assuming that cell growth and limiting substrate
utilization kinetics obtained from suspended cell culture can be applied
equally well to the immobilized microorganism. Other assumptions included
a rst-order rate expression (this is correct at low phenol concentrations
where Monod equation applies, K
s
> S), plug ow, no axial dispersion,
negligible bioadsorption of the limiting substrate onto support particles, and
steady state. The external mass transfer coefcient was found to increase with
increasing feed concentration, air ow rate, and feed ow rate. A correlation
with Reynolds number revealed that the mass transfer coefcient increased
with increase in Reynolds number, which is an expected trend as turbulence
at higher ow rates could have contributed to this increase in mass transfer
coefcient. A dimensionless correlation was developed for the mass transfer
coefcient in terms of Sherwood, Reynolds, and Schmidt number.
100
Holding the same modeling assumptions employed by Vinod and
Reddy,
100
Tepe and Dursun
86
investigated the combined effects of exter-
nal mass transfer and biodegradation rates of phenol by calcium-alginate
immobilized R. eutropha in a packed bed reactor. The mass transfer rate (r
m
,
mg g
1
hr
1
) of phenol from the uid bulk to the surface of the immobilized
beads was considered to be proportional to the area of mass transfer (a, cm
2
g
1
) and the concentration difference between the bulk (S
b
) and the external
surface of the immobilized beads (S
i
):
r
m
= k
L
a(S
b
S
i
) (12)
where k
L
is the external mass transfer coefcient (cm hr
1
). It is common
practice to correlate the mass transfer coefcient with uid properties in term
of J
D
-factor (Table 2, Equation S), where is the uid density (gml
1
); K is
constant; G is the mass ux (g cm
2
hr
1
); and N
Sc
and N
Re
are the Schmidt
and Reynolds numbers, respectively. In the literature, the value of n varies
from 0.1 to 1.0 in these correlations. The biodegradation rate can be given
as the following:
r = kS
i
(13)
where k is the intrinsic rst-order biodegradation rate constant (ml g
1
hr
1
).
Based on continuity equation, at steady state the rate of mass transfer equals
the rate of biodegradation rate.
The intrinsic rst-order biodegradation rate constants and the external
mass transfer coefcients were calculated and then the combined effects of
these rates on the observed rst-order biodegradation rate constants were
also investigated. In the view of obtained results, with the estimated values
of K = 1.34 and n = 0.65, the mass transfer correlation J
D
= 1.34N
0.35
Re ac-
curately predicted the experimental data for the biodegradation of phenol in
the packed bed reactor. A similar study had been carried out earlier by Aksu
and B ulb ul
14
using calcium-alginate gel-immobilized P. putida in a packed
bed column reactor. Employing the previous theoretical approach with the
same previous assumptions, the mass transfer correlation J
D
= 1.625Re
0.507
accurately predicted the experimental data. Comparing the obtained mass
transfer rate and intrinsic biodegradation rate constants gives an idea
about the magnitudes of kinetic parameters and the controlling step of
biodegradation, which helps in making realistic engineering estimates. It was
reported that the effect of external mass transfer limitations on observed reac-
tion rate was signicant and should not be ignored in any engineering anal-
ysis.
14,86
The same topic has also been addressed by Agarwal and Ghoshal.
5
On the other hand, when a microorganism is attached to a porous
carrier matrix the internal mass-transfer limitations considerably affect the
intrinsic kinetics. It is important, therefore, to develop comprehensive models
that take into consideration the internal diffusion effects as the substrates
and product diffuse into and out of the immobilized particles.
116
Banerjee
et al.
117
proposed a mathematical model that accounted for internal and
external mass transfer limitations. A double-layer reaction-diffusion model
was developed by Xiaoqiang et al.
118
Assuming an isothermal porous structure that is spherical in shape with
uniform distribution of microorganisms in the particle and rst-order reaction
kinetics, then the diffusion rate is equal to the reaction rate at steady state
and Ficks law applies (Table 2, Equation T). The effectiveness factor, , and
Thiele modulus, , are usually utilized to measure the extent to which the
reaction rate is lowered by resistance to mass transfer. The rst is dened
as the ratio of the actual reaction rate to that without considering diffusion
limitations. The Thiele modulus (Table 2, Equation U) is related to the ef-
fectiveness factor and serves as a measure of the ratio of the reaction rate to
diffusion rate in the biolm (intraparticle diffusion).
3,100
For small values of
( < 0.5) approaches 1 and the internal mass transfer has less effect on
the rate and the degradation reaction step controls the overall rate. These
conditions mean that the rate at the center of the bead is the same as the rate
at the surface, and all of the volume of bead is fully effective. On the con-
trary, for large values of ( > 5; << 1), the internal diffusion has a large
effect on the rate and it would be the limiting step.
17,69,116
In essence, this
indicates that diffusion into the particle is relatively slow, so that biodegra-
dation occurs before the substrate has diffused far into the particle and only
the surface near the outer periphery of the particle is effective.
17,116
In such
cases, the effective diffusion coefcient is determined by the molecular dif-
fusivity and the pore structure of the immobilized particles.
116
In the work
of Chung et al.
3
and Chen et al.,
69
was close to 5, in the range 3.04.3,
indicating the combined effect of intraparticle diffusion and chemical reac-
tion in the system. In both works, it was shown that the growth kinetics of
the immobilized culture was slower than that of free cells due to the intra-
particle diffusion limitation. Moreover, Chung et al.
3
detected the presence
of the intermediate catechol only when an immobilized culture was used.
Consequently, it was proposed that the occurrence of diffusion resistance in
the immobilized system could be employed as a useful tool for the detection
of intermediates. Using small particles, a high degree of turbulence around
the particles and high substrate concentrations was recommended by Dur-
sun and Tepe
116
to overcome diffusion resistances. The particle size should
be as small as possible, but within the constraints of particle integrity and
resistance to compression.
Mordocco et al.
67
reported that increasing particle size had signicant
effects on mass and gas transfer in the gel matrix. The authors believe that
microenvironments can develop within beads, and these may alter intraparti-
cle growth, metabolism, and product formation. El-Naas et al.
51
also proved
that the mass transfer and hence the accessibility of the biomass to phenol
was enhanced by decreasing the PVA particle size, as biodegradation of
phenol was carried out in a spouted bed bioreactor that is characterized by
intense mixing, and thus the external mass transfer resistance was ignored
and only the intraparticle diffusion resistance was assumed.
22,51
5. ADVANCES: REACTOR TYPES
Biological reactors involve a variety of geometries and hydraulic systems.
Batch or semibatch reactors are often used for laboratory studies, anaero-
bic digestion, and manufacture of pharmaceuticals. Flow reactors are often
employed for aerobic treatment of municipal and industrial wastes.
119
Batch
operation is usually associated with very low initial substrate concentra-
tion, which affects the process productivity. On the other hand, continuous
reactors require low dilution rates to avoid instability or low conversion.
Processes involving immobilized cells often encounter problems of nutrient
and oxygen transfer.
35
In ow reactors, the two extremes in mixing are represented by well-
stirred (stirred tank reactor) and plug ow reactors. Intermediate degrees of
mixing are often described by well-stirred reactors in series or by plug ow
reactors with axial dispersion. More complex mixing models can be devised
but their use is constrained by limitations in knowledge of the system. The
overall model of the reactor is obtained by combining the equations for
the hydraulic regime and the kinetics of the reactions.
119
An advantage of
a well-mixed reactor is the uniformity of oxygen consumption rates by the
microorganisms. With a plug ow reactor, the oxygen demand is usually
greatest at the inlet region where the substrate concentration is high. Thus,
the design of aerators for a plug ow reactor is constrained by the ability
to provide more oxygen transfer in the inlet region. A plug ow reactor
generally produces a higher conversion of substrate in a given volume than
a well-mixed reactor. If no biomass enters a plug ow reactor, no reaction
can occur and the reactor washes out. On the other hand, the inuent to a
well-stirred reactor is mixed with vessel uid containing biomass so that the
reaction can be sustained even in the absence of biomass in the feed stream.
At the end, the two types of reactors are idealized models that are difcult
to achieve in large-scale biological reactors. The advantage of a stirred tank
reactor is that operating it and adjusting its retention time is simple.
31
Use
of a stirred tank reactor had been documented in the batch mode
19,109
and
continuous mode.
90,103
The immobilization process affects the bioreactor performance. The
main advantage of immobilization is the retention of active biomass on
support particles, giving shorter hydraulic retention times and no biomass
washout.
70
Another advantage is the exibility of reactor design and the im-
proved thermal and operational stability. Because the biomass can be reused,
a large volume of wastewater can be continuously treated using a dened
quantity of immobilized cells.
14
A summary of the main advantages and disadvantages of the different
types of bioreactors is presented in Table 4. Brief descriptions of major
bioreactors are given in subsequent sections.
T
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5.1 Sequencing Batch Reactor
Silva et al.
120
employed this reactor to biological ammoniacal nitrogen re-
moval and it proved to be useful for phenol biodegradation. Reductions
around 95% and 99% were achieved for both pollutants, respectively. The
reactor was operated on a ll-and-draw basis, with a cycle of 360 min con-
sisting of 260 min in aerobic condition and the last 100 min in an anoxic
one. An SBRs cycle involves ve operational phases described as ll, react,
settle, decant, and idle/waste sludge. The basic advantage of SBR design is
its inherent exibility of cyclic phasing, providing different operating modes.
5.2 Fixed-Film Reactor (Biolm Reactor)
It consists of a solid surface of an attached layer of biomass. Substrate and
oxygen diffuse into the layer and react to form products and additional
biomass. The most common types of xed-lm reactors are the trickling
lter and the rotating biological contactor.
119
Advanced xed-lm bioreac-
tors can be constructed in a variety of congurations that include rotating
biological contactors, uidized beds, packed beds, rotating ber discs, and
microporous membranes. It is likely that xed-lm bioreactors with rotating
disks or packed beds produce the greatest efciency and stability, especially
when a high degree of degradation is desired. Irrespective of the congura-
tion, all these systems exhibit characteristics that make them advantageous
over more conventional suspended growth reactors such as activated sludge
systems, and the low performance xed-lm systems such as trickling lters.
Jou and Huang
121
designed a new xed-lm bioreactor system to
improve on the efciency of the traditional techniques of bioremedia-
tion. A xed-lm bioreactor system was constructed with highly porous
polyurethane foam to incubate microorganisms at concentrations up to
8000 mg/l. A unique feature of this system is the tremendous surface area
that allows for attachment and growth of microorganisms. The xed-lm
system has four principal advantages over other presently available systems:
(a) It is simpler to operate, (b) It handles shock loads (i.e., the sudden in-
creases in inuent contaminant concentration) with greater efciency, (c) it
forms less solid sludge wastes, and (d) it is more energy efcient, as it re-
quires less power to operate. Hydraulic ow is gravity driven in a bottom-up
fashion, concurrent with airow. This mode of operation could minimize air
stripping of volatiles. As evident from pilot-scale tests, the superior properties
of this biological treatment technology are emphasized. More than 85% COD
removal and near 100% degradation of phenol from industrial oil renery
wastewater was consistently achieved with 8-hr hydraulic retention time. The
system was extremely stable under the pilot test conditions. Compared with
the traditional methods of wastewater treatment, this study demonstrated that
the xed-lm bioreactor technology exhibited higher removal rate, greater
stability of the system, and less sludge formation under real-world condi-
tions, which led ultimately to a greater overall efciency of the bioreactor. In
summary, the pilot system decreased sludge formation and land area usage
by two-thirds compared with more traditional methods. Bajaj et al.
70
studied
the aerobic degradation of high phenol-containing synthetic wastewater (up
to 5.17 g/l) by an immobilized mixed consortium in a continuous xed bed
reactor. Lin et al.
38
developed in their study the kinetic biolm model for
phenol biodegradation with chromium (VI) reduction and veried the model
by conducting experiments in a laboratory-scale xed-biolm reactor. The
biolm reactor was capable of achieving high simultaneous removal of phe-
nol and Cr(VI) at feed concentrations of 500 mg phenol/l and 5 mg Cr(VI)/l,
respectively.
5.3 Packed Bed Column Reactor
Phenol biodegradation was investigated in this reactor in continuous mode
using Ca-alginate-immobilized P. putida.
14
It was proved that phenol removal
in the packed column system is strongly affected by solute diffusion into the
pores of Ca-alginate gels.
14
In another study, the immobilized bacteria was R.
eutropha,
86
while Tziotzios et al.
18
investigated the ability of bacterial popu-
lations originating from olive pulp to degrade phenol in a pilot-scale packed
bed reactor with gravel as support material. Gerrard et al.
53
proposed a sim-
ple, plug-ow model to represent continuous biodegradation in a packed
bed reactor.
5.4 Trickling Filter
It is a bed packed with rocks or plastic structures. Wastewater is distributed
over the top of the packing and allowed to trickle through the bed. S a and
Boaventura
108
used a trickling bed biolm reactor packed with PORAVER
(Dennert PORAVER GmbH, Germany) particles to evaluate phenol and total
organic carbon removal efciencies by P. putida DSM 548. The mean hy-
draulic residence time in the reactor was determined by the tracer injection
technique. The average residence time, t
r
, in the trickling bed reactor is a
function of the hydraulic loading rate, L, and the column depth, D. For the
experimental conditions used in this study, t
r
= 0.034 D/(L
0.85
).
5.5 Rotating Biological Contactor
Trickling lters (and biological towers) are examples of devices that rely
on microorganisms that grow on the surface of the rocks, plastic, or other
media. A variation on this attached growth idea is exemplied by the rotat-
ing biological contractor.
119
A rotating biological contactor consists of many
plastic discs attached to a central drive shaft. The discs are parallel to each
other with intermediate spacing to permit movement of uid between them.
The discs are partially submerged in the liquid so that they are exposed
alternately to substrate in the wastewater and oxygen in the air during rota-
tion.
32,119
These devices are easier to operate under varying load conditions
than trickling lters.
29
5.6 Fluidized Bed Bioreactors
Biodegradation of phenol in uidized bed bioreactors (FBRs) has been re-
ported because of their superior performance and some inherent advantages.
A sparger is provided at the bottom of the reactor through which air can be
sparged into the reactor. In this type of reactor, the transport of substrate,
oxygen, and other nutrients takes place from bulk solution to the surface of
the biolm. Therefore, knowledge of phenol mass transfer coefcient, from
bulk solution to the surface of the bioparticle, is essential for design and
operation of FBRs.
100
In an FBR, high volumetric degradation rates can be achieved by high
specic biolm surfaces. The prevailing turbulence may release part of the
biomass that covers the solid particles, thus maintaining a suitable mass
transfer rate through the microbial lm. However, a limitation is imposed on
excessive detachment and washout of the microbial biolm adhered to solid
particles as it could diminish the volumetric reaction rate. Biomass detach-
ment could be reduced using porous particles as carriers, such as granular
activated carbon and a nonturbulent regime in the FBR. After comparison
of these two processes it seems that better phenol degradation efciency
is obtained with a FBR rather than with a stirred tank.
102
Gonzalez et al.
90
found that best results for biodegradation of phenol with P. putida were
obtained when a continuous uidized bed bioreactor with immobilized cells
was operated. In another study,
122
the continuous FBR loaded with bioparti-
cles of C. tropicalis immobilized onto granular activated carbon was capable
of efciently removing phenol at volumetric loading rates as high as 60 mg
phenol/l.hr when fed phenol was the sole carbon source. When operated
with a feed consisting of a mixture of phenol and 4-CP, the FBR was able
to remove more than 98% of 4-CP in the range of volumetric loading rates
of 4.1 mg 4-CP/hr.l and 55 mg phenol/l.hr with no apparent deterioration
of bioreactor performance.
122
The bioreactor was operated in the biphasic
(gas-liquid) and triphasic (gas-liquid-solid) modes, the solid phase being the
granular activated carbon.
5.7 Spouted Bed Bioreactor
A specially designed bioreactor characterized by a systematic intense mixing
due the cyclic motion of particles within the bed, which is generated by a
single air jet injected through an orice in the bottom of the reactor. The SBBR
is known to provide better mixing than conventional airlift bioreactors.
22
It has many advantages over the conventional bubble column and other
ow bioreactors, including better mixing and contact between substrate and
cells, and faster oxygen transfer rate, which lead to higher rates of phenol
removal.
51
5.8 Internal Loop Airlift Bioreactor
Recent studies have focused on employing and exploring new types of
bioreactors with high performance for practical utilization in the treatment
of wastewater. It is reported that the biodegradation of phenol was faster
in airlift bioreactor than in bubble column.
123
Airlift reactors are known for
mechanical simplicity, good mixing behavior, low shear rate, high capac-
ity, the absence of mechanical agitators, and low cost, which make them a
versatile type of bioreactor. The advantages of airlift reactors and uidized
beds have been utilized by combining the two into one single bioreactor.
One such reactor comprised two concentric tubes with air sparged in the
inner tube: the internal loop airlift reactor.
124
An internal loop airlift biore-
actor was preferred to be used in phenol removal to conventional type of
reactors, mainly due to its various advantages such as better mixing, intimate
contact between phases, faster oxygen transfer rate, higher operational ex-
ibility, shorter reaction time, and greater processing capability.
123
Some of
the inherent problems of most internal airlift reactors are gas entrainment in
the downcomer, and some cellular damage caused by the adherence of the
light particles to the bubbles and subsequent disengagement and bursting
of the bubbles. More importantly, ow cycling that exists in internal loop
reactors results in an inherently unstable phenomenon induced by the liq-
uid circulation in these reactors.
124
Quan et al.
125
developed an internal loop
airlift bioreactor packed with honeycomb-like ceramic as the carrier to immo-
bilize Achromobacter sp. for simultaneous phenol and 2,4-dichlorophenol
biodegradation under fed-batch and continuous operations. A study by Sar-
avanan et al.
126
demonstrated that the performance of this reactor in treating
industrial wastewater containing phenolic compounds was enhanced by us-
ing mixed culture predominantly Pseudomonas sp. Signicantly, the scale-up
of the process was shown to be highly effective.
A mathematical model for simulating the dynamic behaviors of batch
phenol biodegradation processes in internal loop airlift bioreactors with gas
recirculation using free cells of the yeast C. tropicalis was established by
coupling the uid dynamic model and the mass balance model.
123
Proved
to be valid, the proposed model was recommended to provide valuable
directions for reactor design, optimization, and scale-up in full-scale applica-
tions, which would signicantly reduce the cost of unnecessary experimental
work.
5.9 Pulsed Plate (Reciprocating Plate) Column
The use of packed bed reactors or trickle bed reactors with immobilized
cells in biotechnological processes presents a series of problems, whereas
uidized bed bioreactors involve other problems (Table 4).
127
The applica-
tion of a perturbation in the form of pulsation into the xed bed reactor can
minimize these problems, due to the generation of the slow movements of
the bed, which favors the distribution of the substrate as well as the elimina-
tion of dead zones. Pulsed plate column is a column of pulsating plates, in
which the pulsation is generated by the rising-descending motion of the pile
of perforated plates. It has numerous advantages over the more conventional
stirred tank or bubble column type of gas-liquid contactor. A study by Shetty
et al.
127
used a pulsed plate bioreactor for the biodegradation of phenol with
Nocardia hydrocarbonoxydans cells immobilized on glass beads. Axial con-
centration prole measurements revealed that the pulsed plate bioreactor
shows continuous stirred tank behavior. About 100% degradation of phenol
in wastewater (500 mg/l) was achieved and the percentage degradation was
found to increase with the increase in frequency and amplitude of pulsation.
It was also found that the rate of degradation is more affected by external
mass transfer limitation than internal diffusional resistance of the biolm.
Performance studies proved the efciency of the pulsed plate bioreactor
with immobilized cells in wastewater treatment.
5.10 Microbial Fuel Cell
In the microbial fuel cell (MFC), electrons released from the substrate oxi-
dation in the anode are transferred via the external circuit to the cathode,
where the electrons are eventually consumed by the terminal electron ac-
ceptors.
64
The terminal electron acceptors can be easily replaced (e.g., using
oxygen in ambient air as the electron acceptor). MFC combines the benet of
power generation with the biodegradation of organic contaminants such as
phenol. Luo et al.
64
constructed a packing-type MFC using granular graphite
as the packing material in anode and cathode chambers. Both electrodes
were made of carbon cloth. This type of MFC was associated with improved
efciency in electricity generation as it provides larger surface areas for en-
hanced bacterial growth. The maximal power generation was obtained when
a phenol-glucose mixture was used. This might be justied by a stimulated
growth of microbes and the generation of more electrons in unit time from
the synchronous degradation of phenol and glucose in the cosubstrate.
5.11 Rotating Rope Bioreactor
A rotating rope bioreactor is a novel bioreactor specially developed for the
treatment of pollutants that cannot be treated effectively in conventional
stirred tank bioreactors or by activated sludge process due to their high
volatility and high water solubility (e.g., pyridine).
128
In general, biolter,
suspended growth, and packed bed bioreactors have been used for the bi-
ological treatment of such compounds. However, these bioreactors suffer
from some drawbacks, which include poor oxygen transfer due to limita-
tions imposed by the volatile nature of compounds on the use of forced
aeration and intense mixing. To overcome these disadvantages associated
with conventional systems, recent research efforts have addressed the de-
velopment of novel bioreactors, which are able to effectively degrade the
volatile compounds at higher concentration and loading rates (and also at
shorter hydraulic retention time). Mudliar et al.
128
described the features of
a novel xed-lm rotating rope bioreactor for the treatment of wastewater-
containing compounds with characteristics of high volatility along with high
water solubility and low microbial yields.
5.12 Microporous Membrane Bioreactor, Hollow Fiber
Membrane Bioreactor
Sometimes, efuents containing organic pollutants exhibit extremes of pH
and/or high salt concentrations, one or both of which may prevent microbial
growth or at least make it difcult to sustain.
129
Also, at high concentrations,
it is necessary to protect the cells from being damaged, by constructing a
barrier between the toxic high phenol concentration and the cells.
130
In such
cases, the use of cells immobilized on polymeric membranes, particularly in
the form of hollow bers, can be employed in membrane bioreactors for
biotechnology applications. The membrane provides physical separation of
the wastewater from biological medium where degradation takes place. The
microporous hollow bers can act as barriers, which regulate the passage of
one or more species through the pores. The microporous membrane biore-
actor was reported to have better degradation efciency than an extractive
membrane bioreactor (Table 4).
66
Juang and Wu
129
conducted experiments in microporous polypropylene
hollow bers for the degradation of 500 mg/l of phenol by P. putida BCRC
14365 in the solutions containing 01.78 M NaCl. Chung et al.
66
also used the
same module bioreactor, operated in a dynamic mode, for the degradation of
high-strength phenol (up to 2800 mg/l) by P. putida under different operating
conditions. A thicker biolm formed on the outer surface of the bers played
a positive and signicant role on improved degradation of high-strength
phenol solutions. It was recommended that high-strength phenol solutions
could be rst degraded in hollow ber module to a level below 600 mg/l,
and then treated by freely suspended cultures with larger degradation rates.
A membrane bioreactor (hollow ber module) was also used by Marrot
et al.
102
for the biodegradation of high phenol concentration by activated
sludge. Li and Loh
130
investigated phenol degradation at high concentrations
under continuous operation in an immobilized-cell hollow ber membrane
bioreactor by P. putida. Continuous biological operations often suffer from
problems such as oxygen limitation, low degradation rate, and biofouling
from uncontrolled biomass growth. The results demonstrated that long-term,
continuous operation of the bioreactor can be sustained without signicant
biolm fouling on the membranes.
5.13 Two-Phase Partitioning Bioreactor
The two-phase partitioning bioreactor concept is based on the use of a
water-immiscible and biocompatible organic solvent that is allowed to oat
on the surface of a cell-containing aqueous phase.
4,131
By introducing a
water-immiscible solvent it is possible to maintain a low concentration of
the toxic/inhibitory compound in the aqueous phase, while the overall con-
centration remains high.
132
By carefully choosing the organic solvent, the
substrate partitions into the aqueous phase at a concentration that is not sub-
stantially inhibitory to the cells, on the basis of their metabolic demand and
the thermodynamic equilibrium of the system. This system has the advantage
of being self-regulatory, whereas the xenobiotic substrate is delivered to the
aqueous phase according to the consumption rate of the cells, and the prob-
lem of substrate inhibition associated with high concentrations in a batch
system is thus effectively eliminated. Thus, it presents a treatment option
for contaminants that are difcult to remediate by conventional biological
methods. A dynamic model for continuous operation of the two-phase par-
titioning bioreactor was developed.
131
However, liquid second phases may have limitations that hinder two-
phase partitioning bioreactor performance such as bioavailability (Table 4).
These limitations could be overcome by using a polymer phase as the ab-
sorption/desorption component of the two-phase partitioning bioreactor.
However, the polymer using absorption/desorption component has a low
capacity and rate of absorption and some polymers are very expensive.
4
In
order to overcome these drawbacks, Zhao et al.
4
proposed in their study
the use of organic modied montmorillonite (OMMT) as delivery agents for
phenol to microorganisms in a two-phase partitioning bioreactor. OMMT has
been reported to be effective as a sorbent of phenol, which can then be com-
pletely released into water and removed by microorganism. To resolve the
problem of silt formation, the powder of OMMT was wrapped within polysul-
fone to form OMMTpolysulfone capsules. Abu Hamed et al.
132
investigated
the biodegradation of benzene, toluene, and phenol in a two-phase sys-
tem, individually and in mixtures. The three aromatics could be degraded at
higher initial concentrations and at shorter times relative to the single-phase
system.
5.14 Triphasic System
A modication of the two-phase system is the triphasic system (solvent ex-
traction + biphasic biodegradation). In a two-phase bioreactor, the solvent
can dissolve high levels of xenobiotic organics, which then partition to the
aqueous phase at low levels. A local nonequilibrium is created as the biomass
experiences only low levels of the toxic organics, although large amounts
are added to the bioreactor. A triphasic process in a continuous mode
was therefore proposed to remove toxic organics from saline and acidic
solutions.
133
In this process, the organics were extracted (partitioned) from
the saline solution to an organic solvent in one vessel and back-partitioned
to the aqueous cell medium in another vessel. The organic solvent could be
recycled as long as the volatility and water solubility is sufciently low.
Jia et al.
134136
developed a three-dimensional transient model to simu-
late the local hydrodynamics of a gas-liquid-solid three-phase reactor using
the computational uid dynamics method.
There are other types of reactors listed in Table 4. Some of the reactors
in Table 4 are novel ones. However, most of these reactors have difculty in
long-term operation and scale-up, which limit their practical application in
any industrial process.
51
6. OUTLOOKS OF FUTURE DEVELOPMENT AND RESEARCH
Many years of industrial activities have led to the emergence of new pol-
lutants that are harmful to the environment and increasing concentrations
of toxic substances, challenging the capacity of the microbial communities
to deal with them.
59
Because these industrial activities will continue to be
accompanied by the production of hazardous wastes, it is essential to de-
velop efcient strategies for waste management.
1
There is a growing interest
in this area of environmental biotechnology, as the degradation of aromatic
organic pollutants by microorganisms has a unique environmental and eco-
logical impact. Moreover, rigorous pollution control and strict legislation in
many countries have resulted in an intensive search for new and more ef-
cient waste treatment technologies. As priority pollutants, phenols have
been proved to be efciently removed by biological treatment as an en-
vironmentally friendly and cost-effective alternative. However, some of the
critical challenges lie ahead of researchers toward the full understanding
of the biodegradation process and some of the existing issues need to be
addressed and resolved.
During the past decade, a variety of microorganisms have been isolated
and characterized for their ability to degrade different phenols. However,
there still exits the need to isolate new phenol-degrading microorganisms.
Continuous investigations are going on for testing the degrading abilities of
new microorganisms for a variety of phenolic compounds, under laboratory
and eld conditions. Special consideration should be given to obtaining mi-
crobes, which are capable of degrading incompatible compounds. This is
because biological treatment of wastewaters and contaminated soils always
involve degradation of some incompatible mixtures rather than single com-
pounds. As an example, methyl- and chlorinated phenolic compounds are
known to be incompatible substrates and often exist together.
24
In view of
the wide diversity and remarkable specicity of microorganisms, obtaining
such microbes with the ability to degrade incompatible substrate mixtures
is of special importance. Moreover, extreme environmental conditions (low
or high pH, toxicity of other pollutants) necessitate that organisms need
to be selected based not only on their ability to degrade phenolic com-
pounds, but also their tolerance to the additional stresses and their ability to
compete effectively with indigenously microbial populations within the en-
vironment in which they will be operating.
65
Screening of organisms, which
degrade phenols, or produce enzymes that degrade phenols, may prove to
be an environmentally protable line of future work in the 21st century. A
screening program for such organisms and enzymes is required.
28
It is note-
worthy mentioning that fungal and algal groups represent new frontiers for
searching potent phenol-degrading microorganisms, as limited research has
been utilizing microbial strains from both groups compared with bacterial
strains.
148
Environmentally derived bacterial isolates often degrade only a narrow
range of organic matter. Thus, special consideration should be given to co-
operative processes involving a consortium of strains with complementary
capacities. The catabolic cooperation resulting from the natural coexistence
of bacteria and fungi in soil suggests that fungal-bacterial interactions may
be of importance for phenols degradation in the natural environment. A
universal and efcient system can be designed by simultaneous applica-
tion of all the microorganisms to get synergistically enhanced rates.
60,149
In this context, many yet unknown species contribute to biodegradation,
particularly in view of the large microbial diversity, for which it has been
estimated that <1% of the natural bacterial population can be cultured in
the laboratory.
59
This would preferably direct the research to enhancement
of indigenous microorganisms, which copes with the worldwide concern
of conservation of wild species in local environment. Hence, indigenous
rather than foreign microbes should be rst considered for in situ or ex situ
biotreatment.
96
In recent years, many reports have been published about genetically
modied microorganisms and their potential in bioremediation. Employing
genetic engineering tools can produce novel strains with desirable prop-
erties for bioremediation applications. Further study is required in genetic
modication of microorganisms to meet the required recombinant strain.
33
The idea of recombinant strains, which can degrade different compounds
under extreme conditions, must be subject for further development. How-
ever, there is much controversy over whether to use natural or genetically
engineered microorganisms in biodegradation. Government agencies are
mostly resisting the release of genetically engineered microorganisms into
the environment due to the potential risks associated with unforeseen eco-
logical impact. Thus, it is inevitable to do meticulous investigations of the
regulation of physiological parameters and their effects on the biodegrada-
tion of organic pollutants.
7,16
In essence, biodegradation is a natural complex phenomenon. Nature-
like experiments are difcult to simulate in laboratory due to the great num-
ber of parameters occurring during the biodegradation process.
27
Most of the
priority pollutants are biodegradable under well-controlled conditions in the
laboratory. It has been noticed that researchers have primarily approached
their biodegradation studies by simulating the industrial waste efuents in
shake asks with synthetic model phenol solutions (Table 1). However, it
is generally difcult to apply the specically adapted microorganisms in a
traditional biological treatment plant because many substrates are actually
present in a eld of industrial application.
129
The observed rates of biodegra-
dation in the site are often much lower than would be anticipated based
solely on the results of laboratory tests with similar microbial populations
and similar water/sediment mixtures. This may be attributed to issues as-
sociated with up-scaling from a simple batch system to a more complex
subsurface context. In a general sense, the problem arises from both differ-
ences in biological activity and decreased availability of the contaminants to
microbes (i.e., bioavailability). Consequently, determining the bioavailabil-
ity of substratesand not only the intrinsic biodegradation potentialplays
an important role in estimating the feasibility of biodegradation or biore-
mediation at a site.
150
Thus, further investigations are required in order to
employ the microorganisms for in situ elimination of toxic contaminants.
7,150
In this regard, large-scale pilot studies and eld-scale applications have a
signicant role. A particular challenge in this regard is to address the effects
of cocontamination on bioavailability as they need to be more thoroughly
incorporated into numerical models and site remediation plans.
There is still limited information about the enzymes involved, particu-
larly the real scenario in which biodegradation takes place. New technolo-
gies such as stable isotope probing will help in identifying intermediates and
the bacterial populations that carry each step. Also, molecular ngerprint-
ing techniques based on DNA arrays are allowing a better characterization
of the environment, and precise proling of the local bacterial population.
Recently, proteomic technologies that are novel methods dealing with the
high-throughput analysis of gene products directly at the protein level has
been shown as a powerful tool to provide straightforward information on
enzymes and bacterial physiology in biodegradation processes.
52,59
Bioin-
formatics tools will also help in the identication of more proteins in the
future. The diversity of the data presents a tremendous challenge from the
data management and data mining points of view.
Based on the fact that biodegradation involves a series of enzyme-
catalyzed reactions, it has been pointed out that the enzymes, instead of the
microorganisms, should be studied for phenol degradation. This is because
they are more tolerant to wider pH and temperature ranges and many of
the adverse factors involved in microbial utilization will be either reduced or
eliminated when enzymes are used.
24,54,112,113,151
As mentioned previously, the pure cultures of Pseudomonas, and par-
ticularly P. putida, have been the most commonly used in the studies on
phenol biodegradation because of their high removal efciency. However,
Ryan et al.
87
reported in their review study on R. pickettii that though
P. putida and P. uorescens are of the best characterized for phenol biodegra-
dation, they have drawbacks that may limit their use. P. uorescens has been
demonstrated to cause n rot in sh and P. putida has also been shown to
cause disease in sh. The use of these bacteria as biodegraders is, therefore,
not advisable as environmental release could lead to environmental dam-
age, such as disease in plants and depletion of sh stocks, and the potential
cause of disease in humans. The use of these organisms could also raise
public concern. In light of this illustration, the preference of P. putida by
many researchers has to be validated, and its environmental impact should
be assessed in future work. This may also include other candidate microbial
strains, which have been proved to be potent biodegraders of phenol.
Within the scope of development of new techniques, the development
of the MFC using recalcitrant contaminants as fuels is still in its infancy and
warrants further research.
64,143
If the device can be successfully developed,
it should be a novel cost-effective method in enhancing biodegradation of
recalcitrant contaminants such as phenol in practical applications, while the
concomitant electricity generation can be an additional advantage. Develop-
ment efforts should also focus on other novel types of bioreactors targeting
practical utilization and efcient long-term performance. It is essential to
develop and design efcient reactors that would reduce mass transfer limi-
tations and enhance the degradation rate such as the SBBR. Novel processes
are required as well.
Additionally, the shortage of unpolluted freshwater sources and the high
costs associated with water desalination have led many countries to develop
more stringent legislations for determining water quality. Furthermore, rigor-
ous and reliable analytical methods for fast determination of pollutants are
increasingly required. As several phenols can be present in aquatic environ-
ments, it is more suitable to monitor total phenols instead of the quantica-
tion of individual species, thus saving time and costs.
152
In summary, it is forecasted that the combination of high-throughput
technologies, applied in novel and imaginative ways, together with proper
data management, could in the future allow researchers to address even more
interesting and challenging aspects of the complex process of biodegrada-
tion. The synergy of all these data could provide alternative tools to deal
with one of the major problems in the modern world: chemical pollution.
7. SUMMARY
Within the frame of the sustainable development, there is a growing aware-
ness of the environmental impacts imposed by the continuing generation
of large volume of organic material bearing wastes from various industries,
which resulted in contamination of soils and water. As priority pollutants,
phenolic compounds must be eliminated to preserve the environmental qual-
ity. Among various methods available, biological treatment is environmen-
tally friendly, cost-effective, and most promising, and has therefore gained
an increasing attention in pollution prevention.
Biodegradation of phenols can be mediated by bacteria, fungi, and
algae. It involves the breakdown of organic compounds through biotrans-
formation into less complex metabolites, and through mineralization into
inorganic minerals, H
2
O, CO
2
(aerobic), or CH
4
(anaerobic). In essence, mi-
crobial metabolism is a process of energy conversion and it is governed
by enzymatic mechanisms, where reaction intermediates play a vital role.
Biodegradation is a multifaceted process that incorporates several important
factors and not only the intrinsic biodegradation potential in estimating the
feasibility of biodegradation or bioremediation at a site. These factors may
include bioavailability, temperature, pH, oxygen availability, substrate con-
centration, and biomass abundance as well as the physical properties of the
contaminants. Thus, to devise a bioremediation system, all these factors are
to be counted for, with particular consideration of the substrate concentration
because it inhibits the growth of the organism at higher concentrations.
Both bacteria and fungi have been extensively studied for their ability to
degrade phenolic compounds, but the work in the algal group is scant. Most
of research work has been focused on the bacterial species of Pseudomonas
genus, and specically P. putida, because of their proved high phenol re-
moval efciencies. The present status of research work has addressed a va-
riety of issues including research on mixed cultures of microorganisms and
studies with high initial phenol concentrations. Additionally, a lot of research
work has been directed to study the kinetics of the phenols biodegradation,
and several models have been proposed. The Monod and the Haldane mod-
els are the most commonly used. The mass transfer modes and regimes play
a signicant role in determining the efciency of the biodegradation process.
Several research studies investigated the effects of external and internal mass
transfer limitations.
The continued development and application of biotechnologies for
phenol biodegradation imposes a variety of challenges and opens new
frontiers of prospective research and exploration. Protable lines of future
work should be directed to the isolation and identication of new microor-
ganisms and testing their phenol-degrading capacities, both under laboratory
and eld condition with special consideration of large-scale pilot studies
and eld-scale applications. In addition, the development of microbial pro-
cesses that are efcient in the extreme environmental conditions, such as low
pH and contaminant toxicity, is required to ensure a competitive technol-
ogy. Exploring and developing new types of bioreactors presents a research
challenge as well. Further study is required in genetic modication of mi-
croorganisms. Moreover, the implementation of advanced technologies such
as proteomics and bioinformatics should be investigated to provide more
knowledge regarding the enzymatic mechanisms and intermediates involved
in metabolic activities during biodegradation. In this regard, research may be
also focused on examining the possibility of using the enzymes rather than
the microorganisms in biological treatment. To sum it up, the synergistic
performance of various microorganisms and various technologies is to be
considered a research topic of high priority.
ACKNOWLEDGMENTS
The authors would like to acknowledge the nancial support provided by
the United Arab Emirates University as part of the PhD Scholarship Program.
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C may have detrimental effect on the bacterial enzymes that

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