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Phenol Biodegradation by Halophili 2016 International Biodeterioration Bio
Phenol Biodegradation by Halophili 2016 International Biodeterioration Bio
Phenol Biodegradation by Halophili 2016 International Biodeterioration Bio
a r t i c l e i n f o a b s t r a c t
Article history: Phenol is a toxic aromatic compound produced as a by-product of industrial activities. Biological
Received 13 July 2015 treatment of highly saline wastewaters containing phenol can be performed through halophilic micro-
Received in revised form organisms. In this study, the ability of halophilic archaeal isolates to degrade phenol was investigated.
16 November 2015
Among 103 tested isolates, the strain designated A235 was identied as having the highest phenol
Accepted 18 November 2015
Available online 1 December 2015
degradation capacity on solid and liquid media containing 20% (w/v) NaCl and phenol as the sole carbon
and energy source. The strain was adapted sequentially to increasing phenol concentrations. The removal
of phenol via cross-toluene adaptation was increased by 14% in the medium. The growth kinetics of strain
Keywords:
Halophilic archaea
A235 during growth on phenol was found to t the Monod model. The values of mmax and Ks were
Phenol biodegradation calculated to be 0.015 h1 and 71.4 g l1, respectively. For an initial phenol concentration of 100 ppm, the
Adaptation biodegradation by A235 was found to be optimal at pH 7.5, 37 C and 200 rpm when the culture con-
Catechol 2,3-dioxygenase tained 20% (w/v) NaCl, 0.025% yeast extract and the inoculum size was set at 10%. A preliminary enzyme
screening indicated that the degradation of phenol was achieved through a meta-cleavage pathway
involving a catechol 2,3-dioxygenase. Catechol 2,3-dioxygenase displayed its highest catalytic activity at
42 C, 2 M KCl, and pH 8. To the best of our knowledge, this is the rst report showing the ability an
extremely halophilic archaeon to metabolize phenol at higher salt concentrations.
2015 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ibiod.2015.11.016
0964-8305/ 2015 Elsevier Ltd. All rights reserved.
E. Acikgoz, B. Ozcan / International Biodeterioration & Biodegradation 107 (2016) 140e146 141
been suggested to overcome the phenol toxicity (Masque et al., was calculated by plotting the colony forming units (CFUs) of the
1987; Lob and Tar, 2000). Among these approaches, adaptation to culture vs. optical density measured at 600 nm.
higher phenol concentrations is the most effective to overcome Phenol concentration was determined spectrophotometrically
substrate inhibition (Masque et al., 1987). In addition, pre-adaptation at 500 nm by monitoring the production of 4-aminoantipyrine
to phenol may have signicant effects on the degradation patterns (Yang and Humphrey, 1975). This method is based on rapid
such as shorter degradation time or higher degradation rates condensation with 4-aminoantipyrine followed by oxidation with
(Kwon and Yeom, 2009). The mechanism of microbial adaptation to alkaline potassium ferricyanide. All samples were analysed three
phenol degradation was explained on the basis intracellular times to calculate an average value. The specic growth rates for
enzyme induction (Yeom et al., 1997). Microbial degradation of various initial phenol concentrations were calculated from the
phenol under aerobic conditions usually proceeds via an initial growth kinetic slope.
hydroxylation to generate catechol which is cleaved through the
ortho or meta ssion pathways (Bonfa et al., 2013). The ortho 2.3. Adaptation to phenol and toluene
cleavage catalysed by the catechol 1,2-dioxygenase (EC 1.13.11.1)
leads to the formation of cis,cis-muconic acid and the meta-cleavage Adaptation of isolate A235 to phenol was achieved by gradually
catalyzed by the catechol 2,3-dioxygenase (EC 1.11.13.2) leads to the increasing the phenol concentration of the medium, from 50 to
formation of 2-hydroxymuconic semialdehyde (Gurujeyalakshmi 250 ppm and simultaneously decreasing the yeast extract con-
and Oriel, 1989). centration from 0.075 to 0%. Similarly, toluene adaptation was
In this work, we have investigated the ability of the halophilic achieved by increasing the concentration of toluene. The initial
archaeon strain A235 to use phenol a sole carbon source at high salt toluene concentration was set at 10 ppm and it was changed
concentrations and we have obtained preliminary evidence that in stepwise to 20, 30, 40 and 50 ppm. The adapted cells were then
this strain, phenol degradation proceeds through the meta-cleavage tested for their phenol degradation capacities.
pathway. We also determined its growth kinetics during the
degradation of phenol in batch culture. The effects of phenol and 2.4. Effects of growth conditions on phenol degradation
toluene adaptation as well as the effects of various growth condi-
tions on phenol degradation were also investigated. The optimal cultural conditions for phenol biodegradation by
strain A235 were determined by using different concentrations of
2. Materials and methods phenol (50, 75, 100, 150 and 200 mg l1), NaCl (10, 15, 20 and 25 M)
and yeast extract (0.3, 0.1, 0.005, 0.025%), and different inoculum
2.1. Screening for phenol-degrading archaeal strains sizes (2.5, 5,10, 15% v/v), pH (6, 7, 7.5, 8 and 8.5), temperatures (25,
30, 37, 45 and 50 C) and agitation rates (120, 140, 170 and
A total of 103 halophilic Archaea isolated from different parts of 200 rpm). For each variable, optical density and phenol concen-
Turkey were routinely cultured in SehgaleGibbons (SG) medium as trations were monitored during the cultivation.
explained before (Ozcan et al., 2006). The agar plates contained 2%
(w/v) agar. The liquid cultures were incubated at 120 rpm and at 2.5. Rothera test for the detection of ortho- and meta-cleavage
37 C. products
The halophilic archaeal isolates were screened for their ability to
degrade phenol at 37 C on a solid or liquid mineral salt medium The ring cleavage pattern of catechol by isolate A235 was
containing phenol (PMS). The PMS medium was modied from determined by the Rothera's test (Ottow and Zolg, 1974). The test
Halohandbook online protocol (Dyall-Smith, 2009). The medium was performed as follows: the cell pellet was suspended in 2 ml
contained (g l1): NaCl 200; MgSO4.7H2O 25; MgCl2.6H2O 10; 0.02 M Tris buffer (pH 8), then 0.5 ml of toluene and 0.2 ml of 4 mM
CaCl2.2H2O 1.25; KCl 5; K2HPO4 8.7; KH2PO4 6.8; trisma baze 6; catechol were added. Following vigorous shaking of the tube, the
NH4Cl 0.2; phenol and trace element 2 ml l1 containing ((g/ development of a yellow colour within 5 min was considered as an
100 ml) MnCl2.4H2O 0.03; ZnSO4.7H2O 0.1; FeCl3 0.25; CuSO4.5H2O indication of meta-cleavage. In the absence of yellow colour,
0.01; Na2MoO4.2H2O 0.03; H3BO4 0.3; NH4NO3 0.2). The pH of the ammonium sulphate (1 g) was added and the mixture was further
medium was adjusted to 7.35. Preliminary screening was carried incubated at 30 C for 1h. The addition of freshly prepared sodium
out by inoculating archaeal cell suspensions on PMS-agar plates nitroprusside and 2.0 ml of concentrated ammonium hydroxide
containing 50 ppm (0.5 mM) phenol as sole carbon source. Cells develops a strong violet colour when the degradation proceeds
capable of degrading phenol were successively transferred to an through the ortho-clevage pathway.
identical medium containing increasing concentrations of phenol
ranging from 100 to 200 ppm. The highest phenol tolerant strains 2.6. Enzyme assays
were inoculated in liquid PSM containing 100, 150, 200 and
250 ppm (1.1, 1.6, 2.1 and 2.7 mM, respectively) phenol for quan- 2.6.1. Preparation of cell extracts
titative screening. The cultures were incubated at 120 rpm. The Cells grown to exponential phase under optimal degradation
most efcient phenol degrading strain (strain A235) was selected conditions were harvested by centrifugation at 15,000 rpm for
by monitoring the optical density (OD600) of the cultures after 15 15 min at 4 C and washed twice with 50 mM TriseHCl buffer (pH
days of incubation. Cells employed for phenol degradation were 7.35). The pellet was suspended in the same buffer containing KCl
obtained from the late exponential phase cultures (OD600~3) by and the cells were disrupted by sonication (Bandelin Sonopuls GM
centrifugation at 5000 rpm for 30 min at 4 C and washing twice 200) on ice for a total time of 6 min (30 s on/30 s off). Cell debris
with 50 mM TriseHCl buffer (pH 7.35). All cultures were incubated was removed by centrifugation at 15,000 rpm for 30 min at 4 C and
at 37 C. the supernatant was used to assay the enzymes activities.
2.2. Analysis of archaeal growth and phenol degradation 2.6.2. Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase
activities
Archaeal growth was carried out with various initial phenol Catechol 1,2-dioxygenase (EC 1.14.13.1) and catechol 2,3-
concentrations ranging from 50 to 200 mg l1. Cell concentration dioxygenase (EC 1.13.11.2) activities were measured
142 E. Acikgoz, B. Ozcan / International Biodeterioration & Biodegradation 107 (2016) 140e146
spectrophotometrically. All assays were performed at room tem- as sole carbon and energy sources (Emerson et al., 1994; Fairley
perature and one unit of activity was dened as the amount of et al., 2002; Cuadros-Orellanaa et al., 2012), we are not aware of
enzyme transforming 1 mmol of substrate in 1 min under the assay any previous study showing the ability of members of this group to
conditions. Catechol 1,2-dioxygenase was assayed by measuring degrade phenol.
the absorbance at 260 nm, corresponding to the formation of cis,cis-
muconic acid (Hegeman, 1966). The reaction mixture contained 3.2. Adaptation to phenol and toluene
0.1 ml of crude extract, 50 mM TriseHCl buffer (pH 7.5) and 3.3 mM
2-mercaptoethanol. Following the addition of catechol, the activity During the acclimatization process, the microorganisms develop
was monitored for 5 min. The activity of catechol 2,3-dioxygenase strategies to overcome the substrate inhibition problems (Lob and
was measured as previously described (Feist and Hegeman, 1969), Tar, 2000). Although, phenol has signicant inhibitory effects on
from the increase of absorbance at 375 nm, corresponding to the growth of microorganism at higher concentrations (Kumar and
production of 2-hydroxymuconic semialdehyde, the meta-cleavage Kumar, 2005), the isolate A235 was gradually adapted simulta-
product of catechol. The reaction mixture was composed of crude neously to increased concentrations of phenol and lower concen-
extract (0.2 ml), 50 mM TriseHCl buffer (pH 7.5) and catechol tration of yeast extract in the growth medium. As shown in Fig. 1,
(0.2 ml). The reaction was incubated for 5 min. phenol biodegradation by the adapted cells was two times higher
than for the non-adapted ones. In a similar study, Kwon and Yeom
2.6.3. The effects of salt concentrations, temperatures and pH on (2009) indicated that pre-adaptation enhanced the phenol degra-
catechol 2,3-dioxygenase activity dation rate of Pseudomonas uorescence KNU417 by almost two
The effect of KCl concentrations (1.5e3.5 M) on the enzyme times. In the current study, in addition to phenol adaptation we also
activity was determined at 25 C in 50 mM Tris buffer (pH 7.5). The develop toluene-adapted cells. The toluene-adapted cultures
effects of temperatures and pH were determined at the optimal KCl degraded phenol signicantly better than the phenol-adapted cells
concentration by carrying out reactions at various temperatures (Fig. 1). A previous report also showed that benzene- or toluene-
(from 20 to 52 C) and pH (from 5 to 8.5). adapted cultures degraded phenol more efciently than the
In the present study, all tests were performed in triplicate. phenol-adapted ones (Yeom et al., 1997).
3.1. Screening of phenol degrading strains The proles of growth and of phenol degradation by isolate
A235 at different initial phenol concentrations (50e200 mg l1) are
In this study, 103 archaeal strains were screened on solid PMS showed in Fig. 2. At the initial phenol concentration of 50 mg l1, no
containing between 50 and 200 ppm phenol as sole carbon source. lag phase was observed. However, when phenol was used as a sole
Among the 103 isolates, 8 were able to tolerate 200 ppm phenol. substrate at initial concentrations of 75, 100, 125, 150 and
These eight isolates were grown in liquid PMS containing 50 ppm 200 mg l1, lag phases of respectively 48 h, 72 h, 98 h, 144 h and
phenol. After 20 days incubation, the tested isolates were able to 240 h were observed (Fig. 2A). The observation that the lag phase
degrade between 5 and 42% phenol. Three isolates (A235, A405 and increases when phenol concentration is increased has also been
A138) were selected as they displayed phenol degradation above reported previously (Kumar and Kumar, 2005; Agarry et al., 2008).
20%. These isolates were inoculated in liquid PMS containing 100, Our data clearly show that, for all tested concentrations, the
150 and 200 ppm phenol. Isolate A235 was nally retained for isolate A235 degraded between 20 and 80% of phenol within 15
further work, as it was the most effective phenol-degrading strain days. The rate of degradation was reduced when the initial phenol
(reaching 32% degradation at 150 ppm phenol). There was no concentration was increased, probably due to a substrate inhibition
growth in the medium containing 200 ppm phenol. effects (Fig. 2B). Agarry et al. (2008) reported a similar observation
In our previous studies, it was demonstrated that 95 of the 103 for a Pseudomonas strain grown on phenol. Peyton et al. (2002) also
isolates contained the phytanil diether moieties which is a typical observed that the specic growth rates decreased as the initial
feature of Archaea (Ozcan et al., 2006). In addition, 45 of them were phenol concentration increased in high salt solutions. These results
subjected to 16S rRNA gene sequencing analyses (Yildiz et al., 2012; suggest that phenol is an inhibitory substrate for halophilic
Ozcan et al., 2012, 2007) which revealed that they were members of microorganisms.
Halobacteriaceae family. We have not determined the precise The growth kinetics of isolate A235 at phenol concentrations
taxonomic position of strain A235 which was the best phenol between 50 and 200 mg l1 has been determined using the Monod
degrading strain. However, according to our previous ndings model. The Monod parameters values were respectively 0.015 h1
(Ozcan et al., 2006), this strain was found to bear several features for the mmax and 71.4 mg l1 for the Ks. The observed specic
common to archaea and on the basis of its protein prole, it clus- growth rates showed an increasing tendency up to 200 mg l1.
tered with the archaea isolate designated A283. It was clearly
shown that the isolate A283 belongs to the genus Haloarcula from
120
the domain Archaea on the basis of its 16S rDNA gene sequence and
Phenol concentration (mg l )
Bastos et al., 2000; Munoz et al., 2001; Alva and Peyton, 2003; 40 No adaptation
Maskow and Kleinsteuber, 2004; Bonfa et al., 2013). Hong-Xia 20
(2011) reported that the bacterial strain SM5 which was isolated
0
from a marine environment degraded more than 93% phenol at
0 100 200 300 400
5e25 g l1 NaCl. Although there are a few studies showing the Time (h)
ability of halophilic archaea to use aromatic substrates (e.g. p-
hydroxybenzoic acid, benzoic acid, cinnamate, phenylpropanoate) Fig. 1. Biodegradation of phenol by toluene and phenol adapted A235 strain.
E. Acikgoz, B. Ozcan / International Biodeterioration & Biodegradation 107 (2016) 140e146 143
1.6 0.8
1.4
1.2 0.6
1
0.4
0.8
0.6
0.2
0.4
0.2
0
0 0 50 100 150 200 250 300 350 400
0 100 200 300 400
Time (h)
Time (h) A
A
120
200 80
60
150
40
20
100
0
50 0 50 100 150 200 250 300 350 400
Time (h)
0 B
0 100 200 300 400
Time (h)
B Fig. 3. Effects of inoculum size on (A) growth and (B) phenol degradation capacity of
A235 strain at 100 mg/l phenol (lled diamond, 2.5%; open square, 5%; lled triangle,
Fig. 2. Cell growth (A) and phenol degradation (B) capacity of A235 isolate at various 10%; asterisk, 15%).
initial concentrations of phenol. Concentrations (mg/l) were represented as follows:
(lled diamond) 50, (open square) 75, (lled triangle) 100, (open triangle) 125; (lled
square) 150, (open circle) 200. containing toxic compounds, like phenol. In another study (Beshay
et al., 2002), the rate of phenol biodegradation was signicantly
affected by the inoculum size; by increasing it, the lag phase
Studies on the growth kinetics of archaea are limited and the cur- decreased and the rate of phenol biodegradation was enhanced.
rent study is the rst one reporting phenol degradation by a It has been shown that several neutral or alkaliphilic microor-
halophilic archaeal strain. Christen et al. (2011) reported that Sul- ganisms such as Arhodomonas aquaeolei, Modicisalibacter tuni-
folobus solfataricus 98/2, a thermoacidophilic archaeon, acclima- siensis, Penicillium chrysogenum, Candida tropicalis and Alcaligenes
tized to phenol on a mixture of glucose and phenol showed a faecalis have the ability of degrading phenols at various NaCl con-
specic growth rate value of 0.034 h1 at phenol concentrations centrations (ranging from 0.25 to 15%) (Hinteregger and
lower than 365 mg l1. Furthermore, the Ks value was 77.7 mg l1 Streichsbier, 1997; Bastos et al., 2000; Alva and Peyton, 2003;
for the cells of S. solfataricus 98/2 acclimatized on 51e745 mg l1 of Bonfa et al., 2013; Haddadi and Shavandi, 2013). Optimal biodeg-
phenol. While most of the Ks values found for non-halophilic mi- radation of phenol by a moderately halophilic bacterial consortium
croorganisms ranged from 1 to 110 mg l1 (Onysko et al., 2000; was performed in the presence of 5% (w/v) NaCl (Veenagayathri
Kumar and Kumar, 2005), we found Ks as 71.4 mg l1 in the cur- and Vasudevan, 2011). However, at higher NaCl concentrations,
rent study. longer lag periods were observed and longer times were required
for complete phenol degradation which required respectively 50 h
3.4. Optimization of phenol biodegradation and 100 h a 9% and 14% (w/v) NaCl (Hinteregger and Streichsbier,
1997).
The effects of the inoculum size on the rate of phenol degra- In the current study, we investigated the effects of various NaCl
dation and biomass production were examined for cultures grown concentrations on the growth of isolate A235 and on its ability to
in the presence of 100 mg l1 phenol. The phenol biodegradation degrade phenol. We found that it could degrade phenol at high
rate as well as the biomass increased as the inoculum size increased NaCl concentrations. The highest degradation of phenol (over 80%)
whereas, the lag phase dropped (Fig. 3A). Haddadi and Shavandi and shortest lag phase were obtained in cultures containing 20%
(2013) also found a similar relationship between the inoculum (w/v) NaCl (Fig. 4). However, the degradation capacity of A235
size and the rate of phenol biodegradation and biomass production decreased to 40% when NaCl concentration was increased to 25%
for a Halomonas sp. In their study, the optimal inoculum size was (w/v). At this high concentration, the growth rate was also
found to be 10% corresponding to approximately 80% of phenol decreased; suggesting extreme salinity might become a natural
degradation. In our study, the phenol degradation was increased by barrier to hydrocarbon metabolism as suggested previously by
about 30% at optimal inoculum size (Fig. 3B). A similar result has Ward and Brock (1978).
been reported for the strain SM5 grown on phenol where the At the end of the 15th day of incubation, the highest phenol
optimal inoculum size was 12.5% (Hong-Xia, 2011). Santos and degradation, associated with the highest growth of the isolate, was
Linardi (2004) noticed that the starting inoculum was a very obtained at neutral pH (7.5). It is well documented that the majority
important parameter when cultivation was carried out in media of halophilic archaea grow at neutral pH (Bowers and Wiegel,
144 E. Acikgoz, B. Ozcan / International Biodeterioration & Biodegradation 107 (2016) 140e146
0.9 0.9
0.7
0.7
0.5
0.5
0.3
0.3 0 100 200 300 400
0 100 200 300 400
Time (h) Time (h)
A
A
120
120
Phenol concentration (mg l )
-1
100
Data show that isolate A235 possesses the meta pathway, because reported that the intracellular enzyme from Haloferax volcanii was
the development of a yellow product was observed which indicates more active at higher concentrations of KCl when compared with
the presence of 2-hydroxymuconic semialdehyde, the product of NaCl. It is also established that the [Na] is usually lower inside the
catechol 2,3-dioxygenase (Li and Humphrey, 1989). To conrm the cells than outside, while the [Cl] remains approximately equal to
results, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase the external concentration, and [K] is higher inside than outside
activities were assayed in cell-free extracts. Absence of absor- the cells (Grant, 2004).
bance at 260 nm indicated the absence of the ortho-cleavage The maximal catechol 2,3-dioxygenase activity occurred at 42 C
pathway. However, absorbance at 375 nm, which is due to formation and at 2 M KCl. (Fig. 6B). Catechol 2,3-dioxygenase activity isolated
of 2-hydroxymuconic semialdehyde, conrmed the meta-cleavage from non-halophilic organisms was reported for Pseudomonas sp.
pathway. Therefore, it is likely that strain A235 metabolizes phenol ZJF08, Planococcus sp. strain S5, having activity at 30e70 C (Zhou
through the meta-cleavage pathway. et al., 2007; Hupert-Kocurek et al., 2012).
The inuence of pH on the catechol 2,3-dioxygenase activity of
the isolate A235 was determined at 42 C in the culture containing
3.6. Effects of salt concentrations, temperatures and pH on catechol
2 M KCl using catechol as a substrate (Fig. 6C). The optimal pH was
2,3-dioxygenase activity
determined to be pH 8.0. The optimal pH for the catechol 2,3-
dioxygenase from Planococcus sp. S5 was also found to be pH 8.0
The effects of salt concentration on the catechol 2,3-
(Hupert-Kocurek et al., 2012). The optimal pH for catechol 2,3-
dioxygenase activity of the strain A235 was determined for the
dioxygenase for the thermo-acidophilic archaeon S. solfataricus
salt concentrations ranging from 1.5 to 3.5 M KCl and NaCl. The
strain 98/2 was found to be lower (pH 7.0e7.5) (Murakami et al.,
activity of the enzyme was more strongly stimulated by KCl than by
1998).
NaCl. The maximal activity was obtained at 2 M KCl (Fig. 6A). KCl
plays an important role in the cell by maintaining the enzymes in
an active state (Oren and Mana, 2002). Ortega et al. (2011) also Acknowledgements
6
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