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International Biodeterioration & Biodegradation 92 (2014) 6e11

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International Biodeterioration & Biodegradation

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Atrazine biodegradation by a monoculture of Raoultella planticolaisolated from a herbicides wastewater treatment facility

Nissim Swissa a,b, Yeshayahu Nitzan b, Yakov Langzamb, Rivka Cahan a,*

aDepartment of Chemical Engineering, Ariel University, Ariel 40700, Israelb The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

a r t i c l e i n f o

Article history:Received 7 November 2013Received in revised form1 April 2014Accepted 2 April 2014Available online

Keywords:AtrazineDegradationRaoultella planticolaToxic solventsSludgeBiofilm

* Corresponding author. Tel.: þ972 3 9066606; faxE-mail address: rivkac@ariel.ac.il (R. Cahan).

http://dx.doi.org/10.1016/j.ibiod.2014.04.0030964-8305/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

This research describes indigenous Raoultella planticola bacterial cells which were isolated from thewastewater treatment plant of a herbicide factory. The optimum conditions for degrading atrazine wereat pH ¼ 7 and 28 �C, with a degradation rate of 10 mg L�1 h�1. Biodegradation was observed at tem-peratures of 45 and 4 �C and partial degradation was also observed at extreme pH values (3 and 10). Thedegradation rates to reach 50% depletion of atrazine were 9.42, 7.42 and 5.42 mg L�1 h�1 in the presenceof acetonitrile, phenol or toluene, respectively. Successful inoculation of R. planticola into the originalsludge from the herbicide factory led to atrazine degradation within 3 h, instead of 3 days without theinoculation. R. planticola developed a massive biofilm when exposed to atrazine. The results indicate thatthe isolated R. planticola strain can be added to the arsenal of atrazine-degrading bacterial cells that havethe ability to degrade this substance under unfavorable conditions, such as those existing in the sludge ofherbicide factories. In addition, the isolated strain showed an ability to form a biofilm, which can beutilized for improving the wastewater treatment.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) is a member of the s-triazine group of herbicides andhas been used extensively inmany parts of theworld for controllinga variety of weeds for the past 50 years (Tomlin, 1994). Atrazine’stoxicity for susceptible plants is due to its binding to the quinone-binding protein in photosystem II, thus inhibiting photosyntheticelectron transport (Chan and Chu, 2005). It is considered to be arelatively persistent chemical, with a half-life ranging from a fewweeks to several months and with very little mineralization (Seileret al., 1992). Atrazine is mobile in soil and due to its moderate watersolubility (33 mg L�1 at 20 �C), has frequently been detected ingroundwater at concentrations of up to 3 mg L�1 (Schiavon, 1988;Kruger et al., 1993).

Biodegradation is one of the key processes for decreasing atra-zine in soil, groundwater and wastewater. Previous research onatrazine biodegradation focused on pure cultures of atrazine-degrading bacterial cells (Mandelbaum et al., 1995; Rousseauxet al., 2003; Aislabie et al., 2005). There are several reports of

: þ972 3 9066323.

bacterial isolates from atrazine-contaminated sites which are ableto degrade or mineralize atrazine, including Pseudomonas, Rhizo-bium and Agrobacterium strains (Siripattanakul et al., 2009). Pseu-domonas sp. ADP, isolated from soil contaminated with atrazine, isthe best-characterized bacterial strain capable of degrading atra-zine and was shown to completely mineralize its s-triazine ring(Mandelbaum et al., 1995). However, there are bacterial strainswhich have only part of the genes encoding for enzymes in theatrazine degradation pathway. For example Arthrobacter aurescensstrain TC1 metabolizes atrazine to cyanuric acid via TrzN, AtzB, andAtzC, leading to cyanuric acid accumulation (Sajjaphan et al., 2004).There are researches that describe atrazine degradation by mixedcultures in which each strain performs part of the catabolic path-ways which enable the degradation process (Siripattanakul et al.,2009; Yang et al., 2010). Atrazine-degrading bacteria generallyinitiate the degradation through dechlorination or dealkylationfollowed by deamination, finally leading to cyanuric acid that isthen completely mineralized to CO2 and NH3 (de Souza et al., 1996).

Atrazine is very useful for the control of undesirable weeds.However, it has been reported to be toxic for amphibians andhumans. It has been shown to be a potential disruptor of normalsexual development in male frogs and may therefore pose seriousecological risks (Hayes et al., 2002; Rhine et al., 2003; Murphy et al.,2006). Atrazine may also alter some aspects of the immune

N. Swissa et al. / International Biodeterioration & Biodegradation 92 (2014) 6e11 7

response (Christin et al., 2004). Since 2003, the United States’ EPA(Environmental Protection Agency) has beenworking to determinea level of concern that is based on studies which mimic conditionsin natural communities and ecosystems, where effects on aquaticplants have been measured at constant atrazine concentrations.This analysis allows the agency to better understand and evaluatethe various concentrations and durations of exposures monitoredin natural systems. The EPA estimates that the aquatic ecosystem’slevel of concern is approximately 10 ppb for atrazine over a 60-dayperiod. However, the level of concern for humans in a long-termexposure is 3 ppb (EPA, 2013).

It is therefore very important to find indigenous atrazine-degrading bacterial cells and to use them for biodegradation pro-cesses. Wastewaters from the manufacture of pesticides thatcontain s-triazine, such as atrazine, simazine, propazine and cyan-azine, are especially problematic due to high concentrations of re-sidual chlorinated s-triazine compounds and other manufacturingbyproducts (Shapir et al., 1998). The indigenous bacterial cells in thewastewater of herbicide factories commonly collapse, due to unfa-vorable conditions such as organic solvents and extreme pH values(Kauffmann and Mandelbaum, 1998).

The purpose of this study was to isolate atrazine-degradingbacteria from the wastewater treatment facility of a herbicide fac-tory and to evaluate their effectiveness for degradation of theatrazine in the factory’s effluents. This research describes theisolation of indigenous Raoultella planticola bacterial cells fromthe wastewater facility of Maktheshim Agan Industries, Israel.

2. Materials and methods

2.1. Medium for bacterial growth

Minimal salt medium (MM): MM pH 7, contained (per liter ofdeionized water) 1.6 g K2HPO4; 0.4 g KH2PO4; 0.2 g MgSO4$7H2O;0.1 g NaCl; 0.02 g CaCl2; 10 mL of a sodium citrate stock solution(100 g L�1) to a final concentration of 1 g L�1; 1 mL of a traceelement solution; 1 mL of a vitamin stock solution and 1 mL of aFeSO4

. 6H2O stock solution (5 g L�1). The trace element solutioncontained 2 g L�1 boric acid; 1.8 g L�1 MnSO4

. H2O; 0.2 g L�1 ZnSO4;0.1 g L�1 CuSO4; 0.25 g L�1 Na2MoO4. The vitamin stock solutioncontained 100 mg L�1 of thiamin-HCl and 40 mg L�1 of biotin. TheFeSO4

. 6H2O stock solution, vitamin stock solutions, and sodiumcitrate were filter sterilized, kept at 4 �C and added to the mediumafter autoclaving.

Minimal salt medium with atrazine (MMA): MMA pH 7, whichwas used for bacterial growth, was supplied with 30 ppm atrazine(from a 10 mg mL�1 stock solution, in methanol), unless otherwiseindicated. The opaque solid medium used for bacterial selectioncontained the same mineral salts and carbon source as the liquidmedium, but was supplied with 1000 ppm of atrazine and 2% Nobelagar.

2.2. Isolation of microorganisms from sludge

The bacterial strainwas isolated by selective enrichment. Sludge(25 mL) from the aerobic treatment facility at Maktheshim AganIndustries, Israel, was incubated in a 250 mL Erlenmeyer flask un-der aerobic conditions at 28 �C with addition of atrazine (97% pu-rity, was a gift fromMaktheshim Agan Industries, Ashdod, Israel) asthe sole nitrogen source. The atrazine portions were added by astepwise increase in the concentrations from 7 to 30 ppm over thecourse of 14 days. A same portion of atrazine was added to MM andserved as an abiotic control for natural degradation. Every 24e48 h,the sludge (1 mL) was centrifuged at 16,000g for 10 min and theatrazine concentration was analyzed in both the supernatant and

the sediment using HPLC. The atrazine from the sediment was firstextractedwith 300 mLmethanol and then analyzed. After 4 cycles ofatrazine degradation, the filtrated sludge (using Whatman paper)was diluted 1:10 in MMA and incubated at 28 �C for 48 h. Theaddition of atrazine was repeated 6 times over the course of 3weeks. This was followed by spreading a sample of the culture onLB agar plates which were incubated at 28 �C for 24 h. Threedifferent colonies were detected and identified by 16S rRNA genesequence analysis (Hy-labs Ltd., Israel).

2.3. Bacterial identification

2.3.1. 16S rRNA gene sequence analysisDNA was extracted from a loopful of an isolated colony using

the AccuPrep Genomic DNA Extraction Kit (Bioneer, Korea). PCRamplification of the first 800 bp of the 16S rRNA gene was carriedout using a Hy-BID PCR kit (cat no. 5032, Hy-labs Ltd., Israel) withthe following cycle parameters: initial denaturation at 94 �C for5 min, followed by 35 cycles of 94 �C for 30 s, 55 �C for 30 s, and72 �C for 30 s, and a final incubation at 72 �C for 10 min.Amplification products were purified and sequenced using theBigDye Cycle Sequencing Kit (Applied Biosystems, Inc.) on the ABIPRISM 3730 Genetic Analyzer. The obtained sequences werealigned and compared with archived NCBI sequences for geneidentification.

2.3.2. Biochemical testsA Rapid 20E kit, Ilex medical LTD, Israel was used for

biochemical tests.

2.4. Analysis of atrazine using HPLC

Atrazine was detected and identified using reverse-phase high-performance liquid chromatography (HPLC) on a Jasco systemwithsolvent delivery module model PU-1580 and intelligent UV/VSdetector model UV-1575 (Jasco Corporation, Tokyo, Japan). Thechromatography column was C-18 Supelco 250 � 4.6 mm. Thesolvents used were acetonitrile and methanol with a mobile phaseflow rate of 1 mL min�1. Gradient elutionwas carried out accordingto the following program: 12 min from 50 to 80% acetonitrile. Theworking wavelength for quantitative analysis was 220 nm, theatrazine retention time was 5.6 min. The area of the atrazine peakwas calculated and calibration curves were prepared in order todetermine the concentration of each peak.

2.5. Biodegradation experiments

In the biodegradation experiments with monoculture ofR. planticola, the bacterial cells were grown in MMA pH 7 or in amicrocosm consisting of herbicide factory sludge, with aeration in arotary shaker at 150 rpm and incubated at 28 �C, unless otherwiseindicated. When atrazine biodegradation was carried out as afunction of different pH values, MMA was adjusted to pH 3, 5, 7, 9and 10 using HCl or NaOH. The initial OD590 nm value of the culturewas indicated for each experiment. Atrazine concentration wasmeasured using HPLC analysis. The biodegradation of atrazine inthe presence of toxic organic solvents (phenol, toluene and aceto-nitrile at concentrations of 400, 200 and 400 mg L�1, respectively)was examined in MMA at 28 �C, pH 7 with aeration.

2.6. Scanning electron microscopy (SEM)

Samples of plankton R. planticola bacterial cells and the biofilmwere washed and fixed with 2% glutaraldehyde for 2 h, followed by1% osmium tetroxide. The cells were then dehydrated by incubation

Fig. 1. Dependence of atrazine degradation on initial OD value of R. planticola mono-culture. 0.1 OD (-); 0.3 OD (C); 0.5 OD (:) and an abiotic control (>).

N. Swissa et al. / International Biodeterioration & Biodegradation 92 (2014) 6e118

in increasing concentrations of ethanol. The specimens were gold-coated using an LKB device. Scanning was performed with a JEOL840 scanning electron microscope at an accelerating voltage of20 kV.

2.7. Confocal scanning laser microscopy (CSLM)

Examination of R. planticola biofilm viability on polymers suchas PET (polyethylene terephtalate) fibers was performed usingCSLM analysis. R. planticola bacterial cells were grown in MMA inthe presence of PET fibers, after 72 h the substratum was washedwith phosphate buffered saline, fluorescently stained using a Live/Dead Kit L7007 (Molecular Probes, Israel) and examined usingCSLM (Zeiss LSM 700).

2.8. Statistics

Each experiment was performed at least in triplicate. All pri-mary data are presented as means � standard deviations of themean.

3. Results and discussion

3.1. Isolation of microorganisms from sludge

A sludge sample was taken from the wastewater treatment fa-cility and incubated with a stepwise addition of atrazine concen-trations ranging from 7 to 30 ppm. A sample from this microcosmwas centrifuged every 24e48 h, and the atrazine concentration inthe supernatant as well as in the sediment was monitored by HPLC.The degradation of the first cycle of atrazine addition (7 ppm)lasted 3 days, whereas the second portion (15 ppm) lasted 4 days.During these two cycles, atrazine was detected only in the super-natant. After the third and fourth atrazine additions (25 and30 ppm, respectively), atrazine was detected in the supernatant aswell as in the sediment. Atrazine degradation during the third andfourth cycles lasted 4 days. The degradation rate in the supernatantof the first cycle was 0.1 mg L�1 h�1 and at the forth cycle itincreased to 0.31 mg L�1 h�1. No degradation was observed in thecontrols, where the same atrazine portions as in the experimentwere added to MM (data not shown).

After four cycles of atrazine degradation, the sludge was filteredand the supernatant enriched with atrazine-degrading bacterialcells was inoculated into MMA. The bacterial cells from theenriched culture were isolated on LB agar and the three differentcolonies, designated NS-1, NS-2 and NS-3, were sent for identifi-cation. 16S rRNA gene analysis indicated that strain NS-1 showed ahigh similarity of 98% with Pseudomonas nitroreducens, strain NS-2exhibited 99% similarity to Raoultella ornithinolytica or R. planticolaand strain NS-3 exhibited 99% similarity with Stenotrophomonasacidaminiphila. Each of the strains was inoculated separately onMMA agar and incubated at 28 �C in order to determine which ofthe strains has atrazine degradation ability. After 4 days, only strainNS-2 developed colonies, and a clear zone appeared around thecolonies a week later. On the basis of 16SRNA gene analysis, strainNS-2 was found to either belong to the species R. ornithinolyticaor R. planticola. However, biochemical assays were more consistentwith the features that characterize R. planticola thanR. ornithinolytica. The biochemical tests using RAPID 20 E kit: 2nitrophenyl-ßD-galactopyranoside, L-lysine, urea, tri-sodium cit-rate, 4-nitrophenylalannine, sodium malonate, esculin ferric cit-rate, L-arabinose, D-xylose, D-adonitol, L-rhamnose, D-cellobiose,D-melibiose, D-saccharose, D-trehalose, D-glucose, sodium pyruvateand oxidase were positive. L-ornithine and L-tryptophane werenegative.

3.2. Dependance of atrazine degradation on initial bacterial cellconcentrations

The atrazine (30 ppm) degradation rates for different initial ODvalue of R. planticola cells suspension is shown in Fig 1. Bacterialcells at different OD values were added to MMA and incubated at28 �C. The results showed that R. planticola bacterial cells with 0.5OD led to complete atrazine degradation within 2 h, whereas in aculture with 0.1 OD the atrazine was reduced by only 57% within6 h. The degradation rate at R. planticola initial concentration of 0.3OD was 10 mg L�1 h�1. No atrazine degradation occurred in thecontrol sample in which atrazine was added to a sterile MM.

3.3. Dependance of atrazine degradation on differentenvironmental conditions

Unfavorable conditions in wastewater treatment facilities ofherbicide factories, such as extreme temperatures and pH values aswell as solvents, can kill or harm the inoculum and suppress itsdegradation capability (Kauffmann and Mandelbaum, 1998).

Examination of the ability of the isolated R. planticola to degradeatrazine was carried out as a function of different environmentaltemperatures, pH values and solvents. The typical temperature inthe wastewater treatment facility of Maktheshim Agan Industriesranges between 20 and 35 �C and the pH value of the wastewater isabout 5e8.5. However, the biodegradation of atrazine was exam-ined under extreme temperatures (4e45 �C) and extreme pHconditions (3e10) with the aim of considering its use in otherwastewater facilities. The solvents, phenol, toluene and acetonitrileare commonly found in the herbicide industry’s sewage.

A monoculture of R. planticola (0.3 OD) was inoculated intoMMA supplied with atrazine (35 ppm) at 4 �C, 28 �C, 45 �C (Fig 2).Atrazine degradation at 45 �C and 28 �C occurred within 1 h and3 h, respectively, while at 4 �C the atrazine concentration wasreduced to 10 ppm within 6 h. Total reduction of atrazine at 4 �Coccurred after 24 h. Degradation of atrazinewas not observed in thecontrol at all temperatures.

A monoculture of R. planticola (0.3 OD) was inoculated intoMMA and incubated at 45 �C. When the atrazine concentrationdiminished to 0e3 ppm, another portion of atrazine was supplied.This procces was repeated 3 times (Fig 3). The ability of R. planticola

Fig. 2. Atrazine degradation by R. planticola (0.3 OD) at different tempratures : 4 �C(-); 28 �C (:); 45 �C (C); and an abiotic control (>).

Fig. 4. Atrazine degradation by the monoculture of R. planticola in presence oftoluene e 100 ppm (:); phenol e 400 ppm (C); acetonitrile e 400 ppm (A); 0.1%sodium citrate (B) and without the addition of carbon source (-).

N. Swissa et al. / International Biodeterioration & Biodegradation 92 (2014) 6e11 9

bacterial cells to degrade atrazine within 1 h was maintained fortwo cycles. After addition of the third atrazine portion, degradationlasted 1.5 h, while no degradation ocurred after the fourth additionof atrazine. No colonies from the fourth cycle were observed whena sample of the culture was pour-plated on LB agar. However, whenthe same experiment was carried out at 28 �C, atrazine degradationwas observed for 10 cycles, each lasting 3 � 0.5 h. It is important toindicate that the 10th cycle also degraded atrazine within 3 � 0.5,and we may therefore assume that more cycles could have beenperformed.

Atrazine degradation using R. planticola was carried out as afunction of different pH values in MMA adjusted to pH 3, 5, 7, 9 and10 (data not shown). Optimum atrazine biodegradation occurred atpH 7, where the degradation process lasted only 3 h. At pH 5 and 9,atrazine degradation lasted 4 and 5 h, respectively, whereas atextreme pH values of 3 and 10, atrazine degradation after 5 h wasonly about 40%.

Atrazine degradation was examined in the presence of phenol,toluene and acetonitrile, common solvents in effluents of herbicidefactories. Atrazine degradation was examined with a culture of 0.3OD at 28 �C and pH 7 (Fig 4). When sodium citrate was added, thedegradation rate of 50% from the initial atrazine concentration was11.42 mg L�1 h�1 and without addition the degradation rate was10.52 mg L�1 h�1. Atrazine degradation continued even in thepresence of acetonitrile, phenol and toluene. However, the degra-dation rate slowed to 9.42, 7.42 and 5.42 mg L�1 h�1, respectively.

3.4. Atrazine degradation in sludge enriched with R. planticolabacterial cells

A culture of R. planticola was inoculated into sludge microcosmto a final OD of 0.3 followed by the addition of atrazine (30 ppm)

Fig. 3. Atrazine degradation by R. planticola at 45 �C.

(Fig. 5). The atrazine concentration in the sludge supernatant aswell as in the sediment was monitored by HPLC. Complete atrazinedegradation occurred in the supernatant as well as in the sedimentwithin 3 h. However, degradation of atrazine occurred after 4 daysin the sludge without the addition of R. planticola bacterial cells,which served as a control.

The absence or the insufficient amount of naturally occurringatrazine-mineralizing bacteria as well as unfavorable conditions forbiodegradation appear to be the major reasons for the persistenceof this herbicide after wastewater treatment. The limitations ofmicrobial cultures’ acclimation for wastewater treatment of atra-zine are due to unfavorable conditions, such as extreme pH, tem-perature, salinity, toxins, predators, and high concentrations ofaromatic organic compounds (Kauffmann and Mandelbaum, 1998).Some researchers have succeeded in adapting Pseudomonas sp.strain ADP to the biodegradation of atrazine in highly salinewastewater (Shapir et al., 1998).

3.5. Biofilm development in the presence of atrazine

During repeated atrazine degradation cycles, a massive biofilmdeveloped on the surface of the Erlenmeyer flask within 72 h, andaggregates of several millimeters of bacterial cells were observed.The biofilm did not develop within this period when this bacterialstrain was grown in MM supplied with ammonium sulfate insteadof atrazine as the sole nitrogen source. The biofilm which wasdeveloped in the presence of atrazine (Fig. 6b and c) and theplankton bacterial cells which were grown in the presence ofammonium sulfate (Fig. 6a) were fixed with glutaraldehyde andanalyzed using SEM. The biofilm which was formed after 3 days on

Fig. 5. Atrazine degradation in a sludge microcosm. Supernatant (C) and sediment(:) of sludge enriched with R. planticola. Supernatant (B) and sediment (D) of sludgewhich was not enriched with R. planticola.

Fig. 6. SEM micrographs of R. planticola. A, Magnification � 10,000 of plankton bacterial cells grown in the presence of ammonium sulfate; B and C, the biofilmwhich was formed inthe presence of atrazine after 72 h, magnification � 10,000 and 80,000, respectively; D, biofilm (72 h) on PET fibers stained with a live/dead kit and examined using CSLM(magnification � 600).

N. Swissa et al. / International Biodeterioration & Biodegradation 92 (2014) 6e1110

polymers such as PET fibers was stained with a live/dead kit andexamined using CSLM. Most of the cells were stained with thegreen color which indicates that the cells in the biofilm are a live(Fig. 6d). It was recently reported that a biofilm of Pseudomonasstutzeri T102 has high ability to degrade naphthalene and survive inpetroleum-contaminated soils compared to the planktonic cells(Shimida et al., 2012).

4. Conclusions

In this study, the isolated R. planticola showed high atrazinedegradation ability at pH 7 and 28 �C. However, partial atrazinedegradation occurred even under extreme pH values such as 3 and10. At extreme temperatures, such as 45 �C, atrazine degradationwas even faster than at 28 �C, but lasted only 3 cycles, and slowdegradation was observed even at 4 �C. Atrazine degradation was

also observed in the presence of toxic organic solvents. Inoculationof R. planticola into the original sludge from a herbicide factorywastewater treatment facility showed good acclimation for atra-zine degradation which lasted the same period as in the MMAexperiments. Thus, enrichment of the indigenous bacterial cells inwastewater treatment sludge with the isolated R. planticola maylead to improve atrazine degradation and may reduce its risks tothe environment. The phenomenon of the isolated R. planticolabacterial cells to grow on substratum as biofilm may enable thedevelopment of a facility composed of particles covered with thebiofilm for maintaining atrazine bioremediation for a long period.

Acknowledgments

This research was supported in part by the Samaria and JordanRift Valley Regional R&D Center, the Research Authority of the Ariel

N. Swissa et al. / International Biodeterioration & Biodegradation 92 (2014) 6e11 11

University, Israel and the Rappaport Foundation for MedicalMicrobiology, Bar-Ilan University, Israel (to Y.N.).

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