Bioresource Technology 100 (2009) 1746–1753

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Bioresource Technology

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Bioaugmentation of microbial communities in laboratory and pilotscale sequencing batch biofilm reactors using the TOL plasmid

S. Venkata Mohan a,b,*, Christina Falkentoft a,c, Y. Venkata Nancharaiah a,d, Belinda S. McSwain Sturm a,e,Pierre Wattiau a,f, Peter A. Wilderer a,g, Stefan Wuertz a,h, Martina Hausner a,i

a Institute of Water Quality Control and Waste Management, Technical University of Munich, Am Coulombwall, D-85748 Garching, Germanyb Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Hyderabad 500 007, Indiac European Patent Office, Erhardtstrasse 27, 80331 Munich, Germanyd Water and Steam Chemistry Laboratory, BARC Facilities, Kalpakkam 603102, Indiae Department of Civil, Environmental and Architectural Engineering, The University of Kansas, Learned Hall, 1530 W 15th Street, Lawrence, KS, USAf Veterinary and Agrochemical Research Institute, Department of Bacteriology, Groeselenberg 99, B-1180 Brussels, Belgiumg Institute for Advanced Studies on Sustainability, Amalienstr. 75, 80799 Munich, Germanyh Department of Civil and Environmental Engineering, University of California, One Shields Avenue, Davis, CA 95616, USAi Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, Ontario, Canada M5B 2K3

a r t i c l e i n f o

Article history:Received 28 June 2008Received in revised form 18 September2008Accepted 22 September 2008Available online 17 November 2008

Keywords:BioaugmentationGene transferConjugationSequencing batch biofilm reactorTOL plasmid

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.09.048

* Corresponding author. Address: BioengineeringIndian Institute of Chemical Technology, Hyderabad27160123x2664.

E-mail address: vmohan_s@yahoo.com (S. Venkata

a b s t r a c t

The aim of this study was to investigate the effectiveness of bioaugmentation and transfer of plasmidpWWO (TOL plasmid) to mixed microbial populations in pilot and laboratory scale sequencing batch bio-film reactors (SBBRs) treating synthetic wastewater containing benzyl alcohol (BA) as a model xenobiotic.The plasmid donor was a Pseudomonas putida strain chromosomally tagged with the gene for the red fluo-rescent protein carrying a green fluorescent protein labeled TOL plasmid, which confers degradationcapacity for several compounds including toluene and BA. In the pilot scale SBBR donor cells were disap-peared 84 h after inoculation while transconjugants were not detected at all. In contrast, both donor andtransconjugant cells were detected in the laboratory scale reactor where the ratio of transconjugants todonors fluctuated between 1.9 � 10�1 and 8.9 � 10�1 during an experimental period of 32 days. BA deg-radation rate was enhanced after donor inoculation from 0.98 mg BA/min prior to inoculation to1.9 mg BA/min on the seventeenth day of operation. Survival of a bioaugmented strain, conjugative plas-mid transfer and enhanced BA degradation was demonstrated in the laboratory scale SBBR but not in thepilot scale SBBR.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Bioaugmentation is the application of indigenous or allochtho-nous wild type or genetically modified organisms to polluted haz-ardous waste sites or bioreactors in order to accelerate the removalof undesired compounds (Van Limbergen et al., 1998). Bioaugmen-tation has been used to improve the start-up of a reactor (Wildereret al., 1991), to enhance reactor performance (Stephenson andStephenson, 1992; Jianlong et al., 2002), to protect the existingmicrobial community against adverse effects (Hajji et al., 2000;Quan et al., 2003; Bathe, 2004; Venkata Mohan et al., 2005a,2006), to accelerate the onset of degradation (Bathe et al., 2005;Hu et al., 2008; Venkata Mohan et al., 2007; Park et al., 2008) orto compensate for organic or hydraulic overloading (Chong et al.,

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and Environmental Centre,500 007, India. Tel.: +91 40

Mohan).

1997). Bioaugmentation has been also used as a treatment strategyfor contaminated soils (Brandon, 1997; Halden et al., 1999; Newbyet al., 2000; Roane et al., 2001; Rojas-Avelizapa et al., 2003; Topet al., 1999, 2002; Zhang et al., 2000; Sarma et al., 2006; VenkataMohan et al., 2006, 2008, 2009; Shailaja et al., 2007; Rodrigoet al., 2008; Jezequel and Lebeau, 2008). Bioaugmentation withgenetically modified organisms carrying plasmid-encoded cata-bolic genes has the potential to enhance the breakdown of xenobi-otic compounds by increasing the degradation potential of anindigenous microbial population via horizontal gene transfer. Hor-izontal gene transfer is considered to be an important mechanismfor microorganisms to rapidly adapt to changing environments.Conjugation, the exchange of genetic elements via cell-to-cell con-tact is thought to be significant for bacteria residing in biofilms andother bioaggregates which represent high cell density environ-ments (Hausner and Wuertz, 1999;Wuertz et al., 2004). Novel cat-abolic traits can be acquired as a result of horizontal gene transferand can be directly inherited by future generations (Amabile-Cuevas and Chicurel, 1996).

S. Venkata Mohan et al. / Bioresource Technology 100 (2009) 1746–1753 1747

In open engineered systems such as wastewater treatmentplants, cell densities and growth rates are much higher and the im-pact of horizontal gene transfer may be enhanced by rapid divisionof transconjugants passing their newly acquired genetic informa-tion to the next generation (Hausner and Wuertz, 1999; Wuertzet al., 2002). Conjugation is likely to play a major role in spreadinggenetic information and can be exploited in bioaugmentation. Cat-abolic plasmid transfer has been previously studies in model aero-bic suspended growth microcosms (Molin and Tolker-Nielsen,2003; Bathe, 2004; Bathe et al., 2005; Boon et al., 2000; McClureet al., 1991). Sequencing batch reactor (SBR) technology has beendeveloped on the basic scientific assumption that periodic adapta-tion of microorganisms to defined process conditions is effectivelyachieved in a batch fed system in which xenobiotic exposure timeand the frequency of exposure can be set independently of any in-flow conditions (Wilderer et al., 2001). Thus, SBR technology caninfluence the distribution and physiological state of microorgan-isms, which are selected to grow in the reactor. Bioaugmenta-tion-mediated enhanced degradation of xenobiotics in asequencing mode activated sludge system has been shown previ-ously (Bathe, 2004; Bathe et al., 2005). However, bioaugmentedspecies often fail to compete with the indigenous population andmay also cause process inhibition (Bouchez et al., 2000a, b). Simi-larly, possible instability of the catabolic plasmid and varied sur-vival success of the introduced strain in the system do notensure effective degradation (Lewis et al., 1986; McClure et al.,1991). Therefore, it is imperative to monitor the survival of theintroduced strain and plasmid transfer (emergence of transconju-gants) along with the performance of the reactor. For this purpose,cultivation independent in situ techniques using fluorescent mar-ker genes along with microscopy are useful as reviewed by Soren-sen et al. (2005). One such culture independent approach for in situtracing of the inoculated strain or evaluating the extent of thespread of catabolic plasmid is to use fluorescent protein genes suchas those for the green fluorescent protein (GFP) or red fluorescentprotein (DsRed) as markers (Boon et al., 2000; Nancharaiah et al.,2003; Bathe, 2004; Bathe et al., 2005).

The aim of this study was to evaluate process enhancementusing bioaugmentation and horizontal gene transfer of the TOLplasmid, which confers degradation capacity for several com-pounds including toluene and benzyl alcohol (BA) (Harayamaet al., 1986), to mixed microbial biofilm communities in biofilmscultivated in laboratory and pilot scale sequencing batch biofilmreactors (SBBRs) treating synthetic wastewater containing benzylalcohol (BA) as a model xenobiotic compound. Conjugal transferof the plasmid and the fate of donor cells were investigatedin situ with confocal laser scanning microscopy (CLSM) and GFPand DsRed as genetic markers. Studies were performed separatelyin two independent SBBRs (pilot and laboratory scale) at low andhigher volume levels.

2. Methods

2.1. Donor strain and plasmid

The donor organism was a modified Pseudomonas putida strainKT2442 (Christensen et al., 1998). The strain was chromosomallylabeled with the DsRed gene and carried a gfp5 tagged TOL plas-mid, as described in detail by Nancharaiah et al. (2003). The donorstrain shows both green and red fluorescence, whereas transconju-gant cells which received the TOL plasmid fluoresced only green.The differential fluorescence emission was the basis for micro-scopic tracking and differentiation of introduced donors and trans-conjugants that had acquired the gfp-tagged plasmid uponconjugation.

2.2. Sequencing batch biofilm reactors (SBBRs)

2.2.1. Pilot scale SBBRPilot scale glass SBBR with 38 l of total volume and 21 l of work-

ing volume was used in the experiments. Clay beads (Biolith claybeads, Phillip Muller, Germany) with a diameter of 4.5–8 mmand porosity of approximately 0.33 were used as carrier materialfor biofilm formation. The reactor was operated in plug flow modewithout back washing. A sludge retention time (SRT) of 14 dayswas maintained throughout the study by daily removal of a fixedamount of carrier beads and replacement with fresh beads. Thewater exchange ratio was 50%. A data acquisition and control sys-tem with a computer program written in the graphical program-ming language Lab View 5.1 (National Instruments, Germany)was used to control cycle sequence and duration, air supply, sub-strate addition, effluent withdrawal and recirculation. The reactorwas operated in a sequencing batch mode with a total cycle peri-od/hydraulic retention time (HRT) of 8.2 h (cycle configuration: fillphase – 15.5 min, aeration phase – 480 min, draw phase – 4.5 min).

2.2.2. Laboratory scale SBBRA glass laboratory scale SBBR with a volume of 1.6 l and an

exchangeable water volume of 1 l was used. Glass beads with adiameter of 7–10 mm (average diameter �8.5 mm) and porosityof approximately 0.34 were used as carrier material for biofilm for-mation. The reactor was operated in a completely mixed configura-tion with an SRT of 10 days by daily removal of 160 ml of glassbeads. The reactor was operated with an 8 h cycle (HRT) comprisedof 16 min filling phase, 450 min aeration with recirculation phaseand 14 min drawing phase with 100% water exchange. Two controlreactors were operated separately under the same operating condi-tions without bioaugmentation and bioaugmentation with BA.Therefore, control reactor received BA but not the donor strainand was used to evaluate the effect of bioaugmentation on BA deg-radation and COD removal, whereas control reactor received nei-ther BA nor the donor strain and was used to assess the effect ofBA on COD removal.

2.3. Inoculum and operation of reactors

To develop biofilm in the pilot scale SBBR, an inoculum col-lected from a biofilm reactor operating in the laboratory treatingmunicipal wastewater was used. The laboratory scale SBBRs wereinoculated with wastewater collected from the aeration tank outlet(TSS = 1630 mg/l; MLVSS = 1108 mg/l) of a municipal wastewaterplant (WWTP Grueneck, 23 Munich, Germany). Biomass was har-vested by centrifugation, washed twice with PBS [in g/l, NaCl, 8,KCl, 0.2, Na2HPO4, 1.44, NaH2PO4, 0.2, pH 7.0], and stored as a glyc-erol culture at �80 �C until further use. Before inoculation, thestored culture was washed twice in PBS, resuspended in syntheticwastewater, incubated overnight at 30 �C, harvested by centrifuga-tion and resuspended in the synthetic wastewater before beingadded to the laboratory scale SBBR.

After inoculation, all reactors were fed with synthetic wastewa-ter without benzyl alcohol (BA) and operated until stable operatingconditions with respect to carbon removal were achieved. The com-position of the synthetic wastewater was as follows (in mg l�1): so-dium acetate, 61.7; citric acid, 66.7; D-(+)-glucose, 46.7; sodiumgluconate, 61.7; BA, 108.2; yeast extract, 1.0; NH4Cl, 26.7; NaH2-

PO4 � 2H2O, 75.5; MgSO4 � 2H2O, 90; CaCl2, 14; NaHCO3, 275.4;FeCl3 � 6H2O, 0.45; H3BO3, 0.045; CuSO4 � 5H2O, 0.009; KI, 0.054;MnCl2 � 4H2O, 0.036; Na2MoO4 � 2H2O, 0.018; ZnSO4 � 7H2O, 0.036;CoCl2 � 6H2O, 0.045; and disodium EDTA, 3.0. The organic substrateand nutrient stock solutions were prepared separately prior toexperimentation and stored in refrigerator and subsequently usedby further diluting with tap water. The substrate oxidation in stocks

1748 S. Venkata Mohan et al. / Bioresource Technology 100 (2009) 1746–1753

were monitored periodically and variation was found to be negligi-ble and hence, the nitrification. After obtaining stable performancewith respect to carbon removal by biofilm community, the reactorswere fed with benzyl alcohol (pilot scale reactor – 0.5 mM BA[0.162 Kg BA/m3 day accounting for COD of 135 mg COD/l] andsmall scale reactor – 1.0 mM BA [0.316 Kg BA/m3 day accountingfor COD of 271 mg COD/l]) was introduced into feed as co-sub-strate/selective pressure separately by maintaining same organicloading rate (adjusting the concentration of D-glucose). Organicloading rate of 2.19 Kg/m3 day was maintained constant for theboth the reactors throughout the experiment.

Pre-programmed timers were used to control cycle sequenceand duration, air supply, substrate addition and withdrawal ofthe effluent and recirculation. Aqueous phase dissolved oxygen(DO) was maintained in the range of 6.0–7.0 mg/l during operation.Since genetically modified microorganisms were used, all reactorswere mounted inside a specially constructed safety laminar flowhood. All the experiments were performed at room temperature(20 ± 2 �C).

2.4. Bioaugmentation

After achieving stable COD removal, the reactors were inoculatedwith an overnight culture of the donor P. putida strain TUM-PP12(laboratory scale SBBR – 3 � 104 cells/ml; pilot scale SBBR – 1.67 �104 cells/ml) with a ratio of 1:10 with feed volume. Prior to inocula-tion an overnight culture was harvested, washed and subsequentlysuspended in synthetic wastewater. The reactors were operated un-der the operating conditions specified earlier. The pilot scale SBBRwas bioaugmented 122 days after addition of BA. The laboratoryscale SBBR was bioaugmented on day 28, 14 days after BA addition.

2.5. Effluent and biofilm sampling

Effluent samples were collected during the entire test sequenceat pre-defined time intervals from the top of the reactor and werefiltered through a 0.22 lm filter (Millipore) and stored at �20 �Cprior to analysis. Samples were analysed for COD and BA (APHA,1998). COD analysis was performed by a close refluxing method(Dr. Lange GmbH and Co., Düsseldorf). BA analysis was performedby means of gas chromatography (GC; HP Series II, Avondale, PA)with a capillary column (30 m length, 0.53 mm internal diameter,1 lm – film) by injecting 2 ll of sample with helium as carriergas. All analytical values are reported as an average of three inde-pendent measurements.

Biofilm samples were removed from glass/clay beads by agita-tion (�5 min) in autoclaved ultra pure water (Millipore) and vor-texed at high speed for 15 min. The biofilm suspension wasremoved from the supernatant after centrifugation, washed twicewith PBS and stored in 2 ml of PBS at �20 �C.

2.6. Microscopic monitoring of donors and transconjugants

Confocal laser scanning microscopy (CLSM) was performed withan inverted LSM 410 microscope (Carl Zeiss, Jena, Germany) to visu-alize cells expressing green and red fluorescent proteins. A 63�water immersion lens with a numerical aperture of 1.4 was usedfor image generation. The 488 nm laser line and a 515–545 nmband-pass emission filter, and 543 nm laser line and a 570 nmlong-pass emission filter were employed to detect GFP and DsRed,respectively. Approximately 15–20 images (200–300 cells) wereobtained for each sample and analysed with Adobe Photoshop 5.5(Adobe Systems, Inc., San Jose, CA). To monitor the abundance ofdonors, DAPI (4,6-diamidino-phenylindole; 2–2.5 lg/ml) stainingwas used. Five microliter of PFA fixed biofilm sample was appliedto each well on a microscopic slide, air-dried and stained with

30 ll of DAPI (1 lg/ml) for 30 min. Slides were washed with auto-claved double distilled water, air-dried and mounted in an antifad-ing agent (Citifluor Ltd., Leicester, UK) for microscopic analysis. Afluorescent microscope (Leica, Wetzlar, Germany) with specific fil-ter sets was used to estimate total cells (DAPI stained cells), donors(green and red fluorescing cells) and transconjugants (green fluo-rescence). A minimum of ten fields were counted.

3. Results

3.1. Reactor performance before addition of donor organism

3.1.1. Pilot scale SBBRCOD removal was used to evaluate the SBBR performance and

thus, the establishment of a native mixed culture biofilm (Fig. 1).The mixed population biofilm showed consistent performance interms of COD removal after 40 days of the reactor start-up andthe removal rate subsequently remained steady. More than 90%COD removal was evidenced within the first 60 min of the cycleoperation. After obtaining stable operation, BA (0.16 Kg 18 BA/m3 day) was added along with the feed as co-substrate. Com-plete degradation of BA was observed within 26 min of the cycleoperation. The degradation pattern of BA observed for all subse-quent days was consistent as shown in Fig. 2. The pilot scalereactor was operated for a period of eight months continuously.

3.1.2. Laboratory scale SBBRThe performance of the reactor on the basis of COD removal was

assessed during the cycle operation as depicted in Fig. 3. The mixedpopulation SBBR achieved stable operation with respect to COD re-moval within 10 days of the reactor start-up. COD removal wasquite rapid and complete removal was noticed within 80 min ofthe cycle operation. After obtaining stable operation, the reactorwas fed with BA as co-substrate (0.316 Kg BA/m3 day) and the per-formance of the reactor in terms of BA degradation was monitored(Fig. 4). In comparison to the pilot scale SBBR, a higher BA concen-tration was supplied to the laboratory scale reactor to ensure thepresence of sufficient selective pressure throughout the cycle peri-od. Addition of higher concentrations of BA along with feed ex-tended the time required for complete COD removal (Fig. 3).Complete removal was observed after 450 min of the cycle opera-tion suggesting that BA was less readily degraded in the reactorthan the other carbon sources in the feed. It is evident that withoutselective pressure (in form of BA), the biofilm reactor showed highand rapid COD removal rate (Fig. 3).

3.2. Reactor performance after bioaugmentation (addition of donororganism)

3.2.1. Pilot scale SBBRThe performance of the pilot scale reactor was found to be un-

changed by the addition of the donor strain (Fig. 1). After the addi-tion of the donor strain, the reactor was operated continuously fora period of 45 days under the same operating conditions. CODreduction was observed during the first 60 min of the cycle opera-tion. This is comparable to the performance prior to donor inocula-tion. Subsequent days of the reactor operation exhibited identicalCOD removal rates. Degradation profiles of BA also did not showvisible variation in degradation patterns after donor strain additionas the pattern is almost similar to the pattern of BA degradationprofile before donor strain addition (Fig. 2).

3.2.2. Laboratory scale SBBRFollowing the addition of the donor strain, considerable

enhancement in the performance of the laboratory scale SBBR

0

100

200

300

400

500

600

700

800

0 15 10 20 30 40 50 60 120 240 360 450Time (minutes)

CO

D (m

g/l)

Without BAWith BA (11 day)With BA (14 day)12 hours after inoculation18 days after donor inoculation22 days after donor inoculation43 days after donor incoulation

Fig. 1. COD removal during one 8.2 h cycle of the pilot scale SBBR in the presence and absence of benzyl alcohol (BA) and with and without added plasmid donor strain.

S. Venkata Mohan et al. / Bioresource Technology 100 (2009) 1746–1753 1749

was observed as reflected by enhanced COD removal and BA deg-radation rates. Prior to donor inoculation, complete degradationof substrate was observed after more than 420 min of the cycleoperation (Fig. 3). However, after inoculation, the carbon degrada-tion rate was enhanced and complete COD removal was observedwithin the first 360 min of the cycle operation. BA degradation ratealso enhanced after donor inoculation from 0.98 mg BA/min priorto inoculation to 1.9 mg BA/min on the seventeenth day ofoperation (Fig. 4). These rates were calculated based on the BAconcentration decline in the corresponding profiles in Fig. 3. Dur-ing the enhanced biodegradation period, microscopic observationsrevealed that donors persisted and transconjugants appeared(Table 1).

0 5 10 15 20 20

5

10

15

20

25

Benz

yl A

lcoh

ol (m

g/l)

Time (min

Fig. 2. Benzyl alcohol (BA) removal during one 8.2 h cycle of the pil

3.3. Monitoring of gene transfer

3.3.1. Pilot scale SBBRBiofilm samples from the pilot scale SBBR reactor were analysed

for the presence of donors and transconjugants on the basis ofgreen and/or red fluorescence using fluorescence microscopy.Microscopic observation of biofilm samples revealed the presenceof donor cells up to 84 h after the donor addition (data not shown).The number of donors present in the biofilm decreased over time.After 84 h of donor addition no further donor cells in biofilm couldbe detected. Transconjugants were not detected in the investigatedbiofilm samples.

5 30 60 90 120utes)

Before Inoculation 12 h after donor inoculation 18 h after donor inoculation

22nd

day after donor inoculation

25th

day after donor inoculation

35th

day after donor inoculation

ot scale SBBR before and after addition of plasmid donor strain.

0

200

400

600

800

0 16 30 60 90 120 240 360 420Time (minutes)

COD

(mg/

l)

Control without BA (10 day)

Control with BA (20 day)Control with BA (29 days)

12 hours after donor inoculation9 days after donor inoculation

24 days after donor inoculation

Fig. 3. COD removal during one 8 h cycle of the laboratory scale SBBR in the presence and absence of benzyl alcohol (BA) and with and without added plasmid donor strain.Control SBBR did not receive donor cells.

0 20 40 60 80 300 4000

50

100

150

200

250

300

Ben

zyl a

lcoh

ol (m

g/l)

Time (minutes)

Control with BA before inoculation (20th

day)

Control with BA before inoculation (28th

day) 12 h after inoculation

9th day after inoculation

12th

day after inoculation

Fig. 4. Benzyl alcohol (BA) removal profiles in laboratory scale SBBR. Control SBBR received benzyl alcohol but was not bioaugmented with plasmid donor strain.

1750 S. Venkata Mohan et al. / Bioresource Technology 100 (2009) 1746–1753

3.3.2. Laboratory scale SBBRFrom the microscopic analysis, it is evident that donor as well

as transconjugant cells were present in the biofilm of the labora-tory scale SBBR throughout the 32 days of operation (Table 1;Nancharaiah et al., 2003). The percentage of donor cells with re-spect to total cells varied during the reactor operation (Table 1).Twelve hours after inoculation, donor strain cells were 2.9% ofthe total cells. The donor strain percentage increased to 4.5%in samples obtained at 96 h of operation and remained relativelystable at 2.5% until day 9 (216 h) after inoculation. The percent-age of donor cells fluctuated between 0.5% and 1.7% from day 11(264 h) to day 32 (768 h) of reactor operation. It can be con-

cluded that the donor strain persisted after inoculation in thelaboratory scale SBBR. This observation is in contrast with therapid disappearance of inoculated donor cells in the pilot scaleSBBR within 84 h after donor inoculation.

In the laboratory scale SBBR, the frequency of conjugativeplasmid transfer (expressed as the ratio of transconjugant (T)cells to donor (D) cells) was 2.9 � 10�1 T/D at 12 h after inocu-lation and subsequently increased to 6.1 � 10�1 T/D after 24 hfollowing inoculation (Table 1). The T/D frequency increased to8.9 � 10�1 T/D on day 24 (576 h) and day 22 8.3 � 10�1 T/D atthe end of the experiment (day 32, 768 h). The ratio of transcon-jugant (T) cells to recipient (R) cells also varied during reactor

Table 1Ratio of transconjugants to donar or recipient cells monitored in the biofilm of thelaboratory scale SBBR

Time after donorinoculation (h)

Donorspresent (%)

Ratio oftranscongugantsto donors (T/D)

Ratio oftranscongugantsto recipients

12 5.91 2.9 � 10�1 8.4 � 10�3

24 (day 1) 3.00 6.1 � 10�1 1.8 � 10�2

48 (day 2) 2.97 1.9 � 10�1 5.6 � 10�3

96 (day 4) 4.52 4.6 � 10�1 2.1 � 10�2

120 (day 5) 2.69 4.6 � 10�1 1.2 � 10�2

264 (day 11) 0.50 6.7 � 10�1 3.4 � 10�3

336 (day 14) 0.49 5.9 � 10�1 2.9 � 10�3

576 (day 24) 1.48 8.9 � 10�1 1.3 � 10�2

768 (day 32) 1.72 8.3 � 10�1 1.43 � 10�2

Results shown are averages of analysis of 15–20 images (T/D) and at least tenmicroscopic fields (T/R).

S. Venkata Mohan et al. / Bioresource Technology 100 (2009) 1746–1753 1751

operation. After 12 h of inoculation, the frequency was found tobe 8.4 � 10�3 T/R. It then varied between 10�3 and 10�2 and in-creased to 1.43 � 10�2 T/R at the end of the experiment. How-ever, these figures may also reflect vertical gene transfer, i.e.the division and growth of transconjugants and thus, may notrepresent the true conjugation frequency.

4. Discussion

Bioaugmentation via gene transfer in wastewater engineering islikely to be successful if there is an initial survival of the inoculateddonor strain and subsequent spreading of the genetic informationto the native biofilm population in conjunction with enhanced sys-tem performance (substrate removal) and without the inhibition ofother metabolic processes. Survival and establishment of the donorstrain carrying degradative genes represents ‘‘classical” bioaug-mentation. The first prerequisite for successful transfer of geneticinformation is the survival of the inoculated donor strain and itsretention in the system long enough to transmit degradative genesto indigenous microorganisms. In the present study, survival andmaintenance of the donor strain in addition to the emergence oftransconjugants was observed throughout the reactor operationin the laboratory scale SBBR. We were able to visualize individualP. putida TUM-PP12 donor cells in biofilm samples due to theirbright dual fluorescence (Nancharaiah et al., 2003) using both fluo-rescence and confocal microscopy. This allowed us to follow thefate of this strain after introduction into the complex wastewatermicrobial community. Donor cells tend to adhere to the existingbiofilm within few hours upon bioaugmentation. Donor cells werealso detected inside eukaryotic organisms (data not shown), sup-porting earlier studies which indicated that grazing is a major bio-logical process affecting survival of bacteria introduced intoactivated sludge (Leung et al., 2000). The survival and persistenceof the donor strain suggests that the growth rate of the organismmay be higher than washout and grazing activity which may reducethe initial cell density of the inoculated species (Goldstein et al.,1985; Watanabe et al., 1998). In the laboratory scale reactor,microscopy demonstrated the emergence of transconjugants after12 h of donor addition. Donor and transconjugant cells persistedin the biofilm of laboratory SBBR up to the termination of the exper-iments (32 days). The appearance of transconjugants in the labora-tory scale SBBR indicated successful transfer of the TOL plasmid tothe native biofilm population. After addition of the donor strain, BAdegradation rate in the laboratory scale SBBR was enhanced.

Contrary to the laboratory scale SBBR, the donor strain in the pi-lot scale SBBR could be detected only up to 84 h after inoculation.In addition, transconjugants were not observed in the biofilm dur-ing the entire experimental period. The performance of the reactorwith respect to carbon removal and BA degradation was unaffected

by bioaugmentation. The unchanged performance of the pilot scaleSBBR, failure of the donor strain to persist and the lack of emer-gence of transconjugants may be attributed to an inadequate selec-tion pressure (complete removal of BA was observed within thefirst 60 min of cycle operation and the remaining 87.5% or168 min of cycle period was operated without any carbon source).Since the initial inoculated donor cell densities were comparable inboth reactors (1.67 � 104 cells/ml and 3 � 104 cells/ml in the pilotand laboratory scale SBBRs, respectively), low inoculum densitymost probably did not play a role. Stable enhanced performanceoccurred in the augmented laboratory SBBR until the terminationof the experiment (37 days), suggesting that bioaugmentationwas effective and not ephemeral. Ramadan et al. (1990) showedthat a Pseudomonas cepacia, at an inoculated density of4.3 � 103 cells/ml could multiply and degrade p-nitrophenol,whereas the bioaugmented cells disappeared after 13 h of additionwhen inoculated at a density of 2–3 � 102 cells/ml. In anotherstudy, the inoculation of donor showed disturbance of the ecosys-tem’s equilibrium resulting in an overgrowth of bacterial predators(protozoa) leading to a perturbation of the nitrification process(Bouchez et al., 2000a). Initial BA concentration, SRT and operationmode were different for lab scale and pilot scale reactors. Hence,the successful bioaugmentation in the lab scale reactor could alsobe a result of the different operating conditions. This assumptioncould be validated further by comparing two reactors with differ-ent volumes operated under identical conditions. Additionally,maintaining of selective pressure until the termination of the cycleperiod in the pilot scale reactor may have had a positive influenceon the bioaugmentation success.

Only a few successful cases of bioaugmentation using indige-nous and genetically modified microorganisms in wastewaterengineering have been reported (Boon et al., 2000; McClure et al.,1991; Top et al., 2002; Bathe, 2004; Sarma et al., 2006; VenkataMohan et al., 2005a, 2006). In one study, even though a stable pop-ulation of the introduced strain in sludge environment was estab-lished, no enhanced degradation was observed (McClure et al.,1991). In other studies, bioaugmentation failures could neitherbe attributed to the instability of the catabolic plasmid nor to theviability of the introduced strain (Bouchez et al., 2000a, b; Lewiset al., 1986; McClure et al., 1991). To understand whether theintroduction and dissemination of catabolic genes in a reactor isa feasible bioaugmentation strategy, more knowledge is neededon plasmid exchange rates, and their retention and disseminationin complex microbial communities.

The introduction of catabolic plasmids to SBBR’s seems to be apromising bioaugmentation strategy. SBR operation is a periodicdiscontinuous process with variable individual components at eachprocess step, thus, subjecting microorganisms 1 s to altering feastand famine conditions, resulting in high uptake of substrate undernutritional shift conditions (Chiesa et al., 1985; Wilderer et al.,2001; Irvine and Moe, 2001; Venkata Mohan et al., 2005a, b,2006). The accumulation regeneration theory states that if microor-ganisms were able to store more substrate during the imposed tran-sient phase, then the subsequent reuse of stored compounds forgrowth during substrate limiting conditions would give them acompetitive advantage (Grau et al., 1982). The survival of inocu-lated microorganisms is affected by the physiological status of cellssince cells starved for a short period of time seem to survive longer(Watanabe et al., 2000). Venkata Mohan et al. (2005a, 2006) re-ported successful bioaugmentation in an SBBR reactor operated un-der anaerobic conditions to treat high sulfate containing chemicalwastewater. In this study, alternating feast and famine conditionsdue to periodic discontinuous operation may have facilitated thelong survival of the inoculated strain in the small scale reactor.Inadequate selection pressure might have effected the survivaland retention of the donor strain in the pilot scale reactor. If the

1752 S. Venkata Mohan et al. / Bioresource Technology 100 (2009) 1746–1753

donor strain persisted initially, the periodic discontinuous opera-tion coupled biofilm configuration might be able to sustain the sur-vival of gene and subsequent transconjugants. Research conductedon conjugation in wastewater biofilm systems (Ehlers and Bouwer,1999; Ashelford et al., 2001; Bathe et al., 2005) showed that thesesystems are favourable for conjugation due to high cell densityand thus, greater potential for cell-to-cell contact. The dense popu-lation structure of biofilms may increase plasmid transfer by conju-gation resulting in efficient bioaugmentation.

5. Conclusion

In our study, the addition of a P. putida catabolic plasmid donorstrain led to successful bioaugmentation as demonstrated by donorsurvival and emergence of transconjugants in a laboratory scalebiofilm reactor operated in a sequencing batch mode. The survivaland retention of the bioaugmented donor strain and transfer of theTOL plasmid concurred with enhanced benzyl alcohol degradation.The survival of the donor strain and the subsequent emergence andpersistence of transconjugants might be also facilitated by suffi-cient selective pressure.

Acknowledgements

S.V.M. wishes to express his deep sense of gratitude to the Alex-ander von Humboldt (AvH) Foundation for providing fellowshipand Y.V.N. is grateful in acknowledging the financial support fromGerman Aerospace Center (DLR). This work was supported in partby the Bavarian Ministry for Land Development and EnvironmentalIssues.

The authors would like to dedicate this publication to LarissaHendrickx, a fellow microbiologist and a dear friend. Larissa passedaway suddenly in Honolulu, Hawaii, on March 3rd 2008, just oneday after her 35th birthday, while attending the 7th InternationalSymposium on Advanced Environmental Monitoring. Larissa wasa researcher at the Belgian Nuclear Research Centre since 2002,where she worked primarily on the development of a closed artifi-cial microbial ecosystem. Larissa was well known to the scientistswho authored this paper. She worked on her Ph.D. thesis at theInstitute of Water Quality Control and Waste Management at theTechnical University of Munich under the direction of Dr. StephanWuertz from 1998 to 2001. Her Ph.D. thesis and subsequent publi-cations generated significant insights into the extent of natural ge-netic transformation in biofilms. Her studies provided a first in situlook at the uptake of foreign DNA by biofilm-forming naturallycompetent cells. During this time, Larissa was a valued colleagueand a dearly loved friend to all authors of this publication. She isgreatly missed. Her quick scientific intuition, sharp mind, sense ofhumor, friendliness and immense personal warmth will stay inour memories forever.

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