APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2003, p. 1721–1727 Vol. 69, No. 30099-2240/03/$08.00�0 DOI: 10.1128/AEM.69.3.1721–1727.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Natural Genetic Transformation in Monoculture Acinetobacter sp.Strain BD413 Biofilms

Larissa Hendrickx,1† Martina Hausner,2 and Stefan Wuertz1*Department of Civil and Environmental Engineering, University of California, Davis, Davis, California 95616,1 and

Institute of Water Quality Control and Waste Management, Technical University of Munich,85748 Garching, Germany2

Received 5 March 2002/Accepted 22 August 2002

Horizontal gene transfer by natural genetic transformation in Acinetobacter sp. strain BD413 was investi-gated by using gfp carried by the autonomously replicating plasmid pGAR1 in a model monoculture biofilm.Biofilm age, DNA concentration, and biofilm mode of growth were evaluated to determine their effects onnatural genetic transformation. The highest transfer frequencies were obtained in young and actively growingbiofilms when high DNA concentrations were used and when the biofilm developed during continuous exposureto fresh medium without the presence of a significant amount of cells in the suspended fraction. Biofilms werehighly amenable to natural transformation. They did not need to advance to an optimal growth phase whichensured the presence of optimally competent biofilm cells. An exposure time of only 15 min was adequate fortransformation, and the addition of minute amounts of DNA (2.4 fg of pGAR1 per h) was enough to obtaindetectable transfer frequencies. The transformability of biofilms lacking competent cells due to growth in thepresence of cells in the bulk phase could be reestablished by starving the noncompetent biofilm prior to DNAexposure. Overall, the evidence suggests that biofilms offer no barrier against effective natural genetic trans-formation of Acinetobacter sp. strain BD413.

A great variety of synthetic chemicals have been releasedinto the environment, thereby causing serious pollution. Bio-logical treatment processes offer a way to reduce the amountsof xenobiotic compounds in the environment. Bioremediation(treatment of waste with microorganisms) (49) and phytore-mediation (treatment of polluted soil with plants) (17) areregarded as cost-efficient technologies for the clean-up of pol-luted waters or soils. Many wastewater treatment plants rely onbiofilms. One of the advantages of biofilm reactors is theircompactness. The small footprint areas of biofilm reactors aredue to two factors, high volumetric biomass concentrations(29) and high retention times (6). The extended retention timeof bacteria growing near the substratum is the reason why agreater variety of microorganisms can develop compared tothe variety in planktonic cultures (8). Another reason is theformation of microniches due to concentration gradients ofnutrients and electron acceptors. Chemical pollutants can ad-sorb or be degraded further, contributing to the zonation on amicroscale. Hence, extended retention times of solids (6), spe-cies diversity, higher local nutrient concentrations (14), andhigh retention times for recalcitrant compounds due to adsorp-tion in the extracellular polymeric matrix (48) in biofilm reac-tors facilitate remediation of synthetic pollutants. Further-more, biofilms allow interspecies interactions by signaling andnutrient cycling (20) and offer resistance to toxicant (30), phys-ical (13), or environmental stresses (44).

However, for a system to be able to degrade xenobiotic

compounds, the biocatalysts that are needed for degradation ofa specific compound need to be present in the system (22). Ifreactors lack the desired biocatalysts, they need to be bioaug-mented to obtain efficient degradation. By addition of exoge-nous optimally constructed bacteria (11, 43) or functionallyadapted bacterial consortia (18) and communities (47), enrich-ment of the total gene pool can be obtained. Likewise, a re-actor can be augmented by in situ gene transfer (42). It issurprising that in spite of several successes, few studies haveevaluated bioaugmentation by horizontal gene transfer in ac-tivated sludge wastewater treatment reactors (33, 34, 50) oractivated sludge microcosms (16, 36, 41). When plasmids car-rying catabolic genes were integrated into indigenous bacterialorganisms, increased and more rapid degradation of the targetcompound was observed (33, 34, 36). Likewise, there is littleinformation available regarding bioaugmentation of biofilmreactors by in situ gene transfer by bacterial conjugation (1, 4,12, 15, 53). Conjugation inside a biofilm matrix offers a greatadvantage in terms of both gene transfer frequency (1, 15, 19)and subsequent transconjugant stability (15). Still, the effect ofin situ natural genetic transformation in order to obtain bioen-hancement in activated sludge- or biofilm-based biological pro-cess engineering systems has not been studied.

To investigate the feasibility of bioaugmentation by genetictransformation, in situ natural genetic transformation was in-vestigated with a model system consisting of biofilm-culturedAcinetobacter sp. strain BD413 (24) with the autonomouslyreplicating gfp-carrying plasmid pGAR1 (19) as the transform-ing DNA. Acinetobacter species are ubiquitous, strictly aerobic,nonmotile organisms that can be isolated from soil, water, orwastewater (23). Acinetobacter strains can degrade recalcitrantaromatic and alicyclic compounds, as well as some aromaticamino acids, mineral oils, and synthetic polymers (3, 5, 7, 40).In addition, Acinetobacter strains produce biosurfactants, like

* Corresponding author. Mailing address: Department of Civil andEnvironmental Engineering, University of California, Davis, OneShields Avenue, Davis, CA 95616. Phone: (530) 754-6407. Fax: (530)752-7872. E-mail: swuertz@ucdavis.edu.

† Present adress: Laboratory for Microbiology, Radioactive Wasteand Cleanup, Belgian Nuclear Research Center, Mol, Belgium.

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emulsan (25, 45) and alasan (2), that enhance the bioavailabil-ity of poorly soluble compounds. Furthermore, Acinetobactersp. strain BD413 (24) is amenable to gene manipulation byconjugation, transformation, and transduction (23), and thisproperty makes the strain particularly interesting as a tool forbiologically enhancing the catabolic properties of hazardouswaste treatment facilities.

In this study the following conditions were investigated todetermine their effects on natural genetic transformation in amodel biofilm: biofilm age, free DNA concentration, andgrowth of the biofilm in the presence or absence of cells in thebulk fluid.

MATERIALS AND METHODS

Bacteria, plasmids, and media. Acinetobacter sp. strain BD413 (24) was usedas the model strain for evaluation of natural genetic transformation in monocul-ture biofilms. pGAR1 was isolated from Escherichia coli strain GM16 (19).pGAR1 is pRK415 (26), a Mob� Tra� Tetr Inc P1 plasmid, carrying the wild-type gfp (green fluorescent protein [GFP]) gene (Clontech, Palo Alto, Calif.)under regulation of a Plac promoter. Induction of the promoter by addition ofisopropyl-�-D-thiogalactopyranoside was not necessary. The fluorescence inten-sity of single BD413(pGAR1) cells could be clearly distinguished from thefluorescence intensity of unlabeled single BD413 cells by epifluorescence andconfocal laser scanning microscopy (CLSM). Rich Luria-Bertani medium (38)and minimal medium M9 (0.2% gluconate) (46) were used during transforma-tion experiments. Standard microbiological techniques (spectrophotometricDNA concentration measurement, bacterial cultivation techniques, DNA extrac-tion and purification) were performed as described by Sambrook et al. (46).

Transformation in biofilms. Transformation of biofilms was performed by themethods described by Wuertz et al. (54). Biofilms of Acinetobacter sp. strainBD413 were grown in a flow cell containing four separate flow channels (4 by 4by 40 mm) for 3 days in rich Luria-Bertani medium by using a flow rate of 2.4ml/h after DNA was added with minimal medium M9 (0.2% gluconate). Trans-formation involved incubation of DNA during 1 h of continuous flow withDNA-containing medium, unless indicated otherwise. After overnight incubationin minimal medium M9 (0.2% gluconate) without DNA at a flow rate of 2.4 ml/h,which allowed expression of the received gene, biofilms were prepared for mi-croscopic monitoring.

Microscopic monitoring, image acquisition, and data processing. Biofilm cellsin Acinetobacter sp. strain BD413 monoculture biofilms undergoing transforma-tion were visualized with the general nucleic acid stain Syto 60 (MolecularProbes, Eugene, Oreg.) and were detected with an LSM 410 CLSM (Zeiss, Jena,Germany). The 633-nm laser line and a 665-nm long-pass emission filter wereused to detect cells stained with Syto 60. The 488-nm laser line and a 515-nmlong-pass emission filter were used for detection of cells expressing gfp (Clon-tech). Automated image acquisition and data processing were performed by themethods described by Wuertz et al. (54).

Colocalization experiments. Pure-culture BD413(pGAR1) biofilms grown for46 h in selective minimal medium (minimal medium M9 containing 0.2% glu-conate and 20 �g of tetracycline per ml) were stained with Syto 60 to testcolocalization of the two signals. Approximately 18.5% of the signals in theBD413(pGAR1) biofilm were colocalized GFP and Syto 60 signals, 22.6% weresingle Syto 60 signals, and 58.9% were single GFP signals. Therefore, in trans-formation experiments the total cell volume was obtained by adding the Syto 60signals and the GFP signals and subtracting the overlapping signals. GFP signalswere considered transformants. Potential underestimation of the 22.6% GFPsignals was not considered during calculation of the results because it was notpossible to check whether cells still contained the introduced plasmid. When noDNA was added to the medium, no signals were detectable with CLSM settingsfor detection of GFP signals in two separate tests. In an additional test, signalswere obtained by using the 515-nm long-pass emission filter, but no overlap wasdetectable with the Syto 60 settings. These false-positive signals might have beendue to autofluorescent impurities in the inlet medium and could be clearlydistinguished from fluorescence-expressing cells on the basis of form and size.Hence, to avoid overestimation of GFP due to autofluorescent impurities, im-ages had to be checked manually to discard possible false-positive signals on thebasis of form and size.

Mathematical parameters. The mathematical equations describing volumesand transformation frequency are given below.

Calculation of the volume of transformants and the volume of recipients wasbased on the following equation:

VX,e � �i�0

e�1

�z�i�1� � zi� � Xi (1)

where VX,e is total volume of transformants or recipients (in cubic micrometers),Xi is the area covered by cells of interest at position i (in square micrometers), zi

is the distance from the substratum at position i (in micrometers), i is thescanning position in the z direction starting at the biofilm substratum, and e is thelast scanned position in a biofilm in the z direction.

The equation was adapted as follows with the trapezoidal rule to obtain a morecorrect approximation of the numerical integral as described by Kuehn et al.(28):

VX � VX,e � 1/2 � �z1 � z0� � X0 � �i�1

e�2

�z�i�1� � zi� � Xi � 1/2 � �ze � z�e�1�� � Xe (2)

The transformation frequency (TF) was the fraction of transformant volume pertotal cell volume: TF � VT,e/VR,e, where VT,e is the volume of transformantsobtained with equation 2 (in cubic micrometers) and VR,e is the volume ofrecipients obtained with equation 2 (in cubic micrometers).

Below, transfer frequencies are expressed as the volume of transgenic cells pervolume of recipient cells, unless indicated otherwise.

To obtain a reproducible estimate of the normalized distance from the sub-stratum, we limited the total cell volume and biofilm thickness to contain 98% ofthe scanned biomass (VR,e) starting from the substratum towards the biofilm-bulkfluid interface within the biofilm volume investigated. The normalized distancefrom the substratum was calculated by dividing the distance from the biofilmattachment surface by the biofilm thickness: di � zi/zk for VR,k � VR,e � 0.98,where VR,k is the volume of recipients calculated with equation 2 limited to 98%of the total scanned biomass (in cubic micrometers), di is the normalized distancefrom the substratum at position i (in micrometers), zi is the distance from thesubstratum at position i (in micrometers), zk is the biofilm thickness or distancefrom the substratum at position k (in micrometers), and k is the position in abiofilm where VR,k reached 98% of VR,e.

Reproducibility of in situ natural genetic transformation in monocultureAcinetobacter sp. strain BD413 biofilms. Biofilms should be observed noninva-sively and with confidence that reliable and reproducible results will be obtained.Direct in situ detection of large areas is, therefore, desirable. A study to deter-mine statistically representative areas of Pseudomonas fluorescens biofilmsshowed that a minimal area of 1 � 105 �m2 should be scanned to obtainreproducible results in biofilm investigations (27). In the present study a minimalarea of 2.4 � 105 �m2 was scanned to monitor a biofilm volume of at least 1.2 �107 �m3. The standard transformation frequencies with 0.2 �g of pGAR1/ml,derived from four separate experiments monitoring a minimum volume of 1.2 �107 �m3, ranged from logarithmic values of �3.5 to �3.1, with a mean value of�3.3 and a standard deviation of 0.15. In contrast to the standard method inwhich planktonic cells are used (38), the in situ method in which CLSM was usedprovided reliable and reproducible results.

RESULTS

Effect of biofilm age on natural genetic transformation. Totest the transformability of biofilm cells at different growthstages, experiments were conducted with 0.2 �g of pGAR1DNA/ml (Table 1). The responses of cells to DNA exposurewere measured with two different exposure times. Flow cell

TABLE 1. Transformation frequencies obtained for biofilms ofdifferent ages exposed to 0.2 �g of plasmid pGAR1

DNA/ml for different periods of timea

Time of exposureto DNA (min)

Transformation frequency

1-day-old biofilm 3-day-old biofilm

15 1.9 � 10�2 7.7 � 10�4

45 1.3 � 10�2 7.4 � 10�4

a Data were obtained by quantitative microscopy.

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experiments were performed with 1- and 3-day-old biofilms.The thickness of Acinetobacter sp. strain BD413 biofilms grownin rich medium reached a maximum after 2 days for fourseparately investigated biofilms, and after this the biofilmthickness remained at a steady state due to lysis, detachment,and subsequent growth, indicating a mature biofilm stage (re-sults not shown). A 1-day-old biofilm could therefore be con-sidered an actively growing biofilm, and a 3-day-old biofilmcould be considered a mature biofilm. Although cells in youngand growing biofilms are more readily transformed, the biofilmheterogeneity in 3-day-old biofilms still ensured the presenceof a significant fraction of competent cells that were able totake up DNA (Table 1; Fig. 1). Furthermore, cells respondedwithin 15 min to the addition of exogenous DNA.

Effect of concentration of added free DNA. To investigatethe influence of DNA concentration on transformation fre-quency and transformant location in a mature biofilm of thehighly competent organism Acinetobacter sp. strain BD413,natural genetic transformation was investigated in biofilms thatwere exposed for 1 h to a specific pGAR1 DNA load.

The DNA concentration in the inlet medium ranged from 1

� 10�9 to 1.5 �g of pGAR1/ml. The transformation frequencyincreased as a function of DNA concentration within the rangeof DNA concentrations investigated (Fig. 2).

The observed relationship between DNA concentration andtransformation frequency corresponds to the results of previ-ous studies involving batch transformation experiments (38).However, a saturation point was not reached in this study.Further examination with DNA concentrations of 100 �g/ml ormore would be needed to see if a saturation point is reached orif transformation keeps increasing until 100% of the cells aretransformed. Such high concentrations were not investigated inthis study as they are unlikely to occur in nature or are not verypractical for bioaugmentation of bioreactors.

With low concentrations of pGAR1 in the feed, transfor-mants were formed at the biofilm attachment surface. Expo-sure to increasing amounts of pGAR1 resulted in gradualaccumulation of transformants at the bottom of the biofilm(Fig. 3), where the biofilm density was the greatest, and not inthe middle or upper part of the biofilm. If it were true that thefraction of competent cells is homogeneously distributed insidea biofilm, most transformants would be found in the layers with

FIG. 1. Microscopic images showing pGAR1 transformants in 3-day-old (A) and 1-day-old (B) Acinetobacter sp. strain BD413 biofilms. Theblack and white images show GFP signals (panels I) or Syto 60-stained cells (panels II). Superimposed single optical images (panels III) show gfptransformants (yellow, green) against a background of recipient Acinetobacter sp. strain BD413 biofilm cells (red). The edges of each imagecorrespond to a length of 90 �m.

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the highest biofilm density and the transformation frequencywould be equally high throughout the biofilm. However, thiswas observed only 5 of 10 times. Hence, other factors may havecontributed to the distribution of frequencies detected.

Effect of biofilm ontogenesis on natural genetic transforma-tion. Biofilms can grow either in a batch mode (on the surfaceof a shaken container containing a bacterial suspension) or ina continuous mode (by constant feeding of a biofilm grown ona surface with fresh medium in a flow channel). A biofilmgrown in a shaken container emerges with suspended cellspresent in the bulk fluid, while a biofilm in a flow channelgrows in an environment continuously rinsed with suspendedcells in the medium. All previous experiments were performedwith biofilms grown in continuously fed flow cells. How woulda biofilm that had developed in the presence of stationary-

phase bacterial cells in the bulk phase respond to the additionof free DNA? To answer this question, flow cells were contin-uously fed with medium containing stationary-phase Acineto-bacter sp. strain BD413 cells to mimic biofilm ontogenesis inthe presence of a batch-grown suspended culture. After this,the biofilms were rinsed with a 0.01 M MgSO4 to remove anysuspended cells from the flow channel. Cell-free minimal me-dium containing 0.1 �g of pGAR1 DNA/ml was added to theflow cells for 1 h. The preparations were incubated overnight incell-free minimal medium, and after this the biofilms werestained, washed, microscopically investigated, and quantified.No transformation was detected in the biofilms, which hadgrown in the presence of cells in the bulk fluid.

Next, we investigated the effect of prestarvation on transfor-mation of biofilm cells grown in the presence of cells in thebulk phase. Suspended cells were removed as described aboveby rinsing with 0.01 M MgSO4. The biofilm was then starved byfeeding the flow cell either with a continuous supply of minimalmedium M9 without a carbon source or with a salt solution(0.01 M MgSO4) for 24 h. Subsequent transformation wasdone with identical experimental transformation steps, as de-scribed above. Transformation was observed at low but mea-surable frequencies (9.1 � 10�5 and 1.9 � 10�5 for biofilmsstarved in the presence of minimal medium M9 and in thepresence of the salt solution, respectively).

When biofilms that were grown with cells present in the inletmedium were rinsed with 0.01 M MgSO4 and subsequently fedwith cell-free rich medium for 24 h before transformation,again no transformation was observed in the biofilms.

DISCUSSION

Standard transformation experiments in which Acinetobactersp. strain BD413 is used require cells to be transformed in astate of competency (38). Acinetobacter sp. strain BD413reaches competence at the early log phase. The cells remaincompetent during the log phase, and competence drops toalmost zero when the stationary phase is reached (31). Instandard batch culture experiments (31) or soil microcosms(35), additional transformation events were not detected aftera prolonged period that was more than 12 h long. Similar toexponentially growing suspended cultures, young and growingbiofilms allowed a high frequency of transformation events. Inaddition, the biofilms still contained significant fractions ofcompetent cells for at least 3 days after inoculation. The bio-film mode of growth could therefore be compared to the con-tinuously exponentially growing steady-state batch or turbi-dostat cultures of Palmen et al. (37). These authors studiedAcinetobacter sp. strain BD413 cultured in a continuous modein order to prolong the period of competency. After 3 days theywere still able to observe a detectable transformation fre-quency, but it was less than the initial transformation frequen-cies (37); this was impossible in transformation experimentsperformed with Acinetobacter sp. strain BD413 cultured in abatch mode (31, 35).

The DNA concentration in the inlet was positively corre-lated with increasing transformation frequency. Natural ge-netic transformation occurred readily at high frequencies inmonoculture Acinetobacter sp. strain BD413 biofilms. When1.2 �g of pGAR1/ml was used, transformation frequencies as

FIG. 2. Transformation in monoculture strain BD413 biofilms withpGAR1 DNA at various concentrations in the inlet medium expressedas a semilog plot. The insert shows the same data points on a log-logscale.

FIG. 3. Distribution profiles for the volume of gfp transformants asa function of the normalized distance from the substratum when or-ganisms were exposed to 1 � 10�9 �g of DNA/ml (F), 1 � 10�7 �g ofDNA/ml (�), 1 � 10�4 �g of DNA/ml (}), 1 � 10�1 �g of DNA/ml(‚), and 1.5 �g of DNA/ml (�).

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high as 2.4 � 10�3 were observed. Furthermore, it was possibleto obtain detectable transformation frequencies with minuteamounts (as little as 1 fg of pGAR1/ml) in biofilms. The lowesttested DNA concentration used for transformation of Acineto-bacter sp. strain BD413 reported previously was approximately1 ng of DNA/ml (38). Transfer frequencies even higher thanthose reported here may be obtainable if a feed with a DNAconcentration greater than 10 �g of pGAR1/ml is added to abiofilm. For bioaugmentation via gene transfer, however, it isnot necessary to have maximum transformation frequencies.Even if transfer frequencies are low, transformants may un-dergo cell division, and the transgenic strain could thereforestill establish itself in a reactor system. In such a case, low butsignificant transfer frequencies should be enough to obtainsuccessful bioaugmentation.

It was interesting that an increased DNA concentration inthe influent resulted in accumulation of transformants in thebiofilm substratum. However, it seems odd that most transfor-mation events take place at the biofilm base. This observationimplies that free DNA first has to diffuse through the biofilmbefore competent cells take up and subsequently replicate theintegrated plasmid. It is possible that immobilization of cellswas responsible for the occurrence of most transformationevents at the bottom of the biofilm. The location of transfor-mation events seemed to be simply a matter of chance, and theprobability increased with increasing cell density at a certaindistance from the substratum. In the case of monoculture Acin-etobacter sp. strain BD413 biofilms the cell density was greatestnear the biofilm attachment surface. Therefore, the chance fortransformation was greater near the biofilm substratum. Thissuggests that the chances for transformation to occur are high-est in a tightly packed biofilm (a biofilm with a low porosityvalue). However, it is not correct that porous biofilms are illsuited for transformation. Young biofilms, for example, arevery porous (Fig. 1), and they allowed transformation at in-creased rates due to enhanced competency levels during expo-nential growth. Porosity plays an important role only in maturebiofilms. Therefore, it is necessary to investigate the trueimpact of biofilm density and porosity on natural genetictransformation. If density played an important part in naturalgenetic transformation, anthropogenic manipulation of thefactors that decrease biofilm porosity could lead to enhancedoccurrence of transformation events. van Loosdrecht et al. (51)included flow rate, nutrient loading rate, and growth rate of thebiofilm cells as parameters that influence biofilm thickness andporosity. In addition, cell-to-cell signaling molecules influencebiofilm structure (10) and may indirectly influence transforma-tion events.

Another important parameter in this study was the mode ofgrowth in which the biofilm itself emerged. In many biofilminvestigations scientists use batch-grown biofilms in 96-wellmicrotiter plates (9, 39). In contrast to batch-grown biofilms,biofilms grown in flow chambers are in continuous contact withfresh medium that does not contain suspended cells. Oneneeds to consider that biofilms grown in different modes couldpossess different qualities. In the present study, differences inthe transformability of Acinetobacter sp. strain BD413 biofilmcells grown in the presence and in the absence of cells in thebulk fluid were observed. The presence of high numbers ofcells in the surrounding medium inhibited transformation in

biofilms (Hendrickx, results not shown). Likewise, when bio-films developed in the presence of high numbers of cells in thebulk fluid but were rinsed to eliminate cells present in thesurrounding medium before they were exposed to naked DNA,transformation was strongly inhibited. Also, differences in themorphotypes of the bacterial cells were observed. Acineto-bacter sp. displays two different morphotypes, the bacillar mor-photype and the coccoid morphotype (21). In biofilms fed withcell-free medium, both the bacillar and coccoid forms weredetected. In contrast, when biofilms were allowed to emerge inthe presence of medium containing suspended bacterial cells,the cells exhibited almost exclusively the bacillar morphotype.

As determined by microscopy, batch-cultured stationary-phase Acinetobacter sp. strain BD413 cells also displayed thebacillar morphotype when the organism was grown in richmedium. Hence, it is possible that the lack of transformabilityin biofilms grown in the presence of cells in the bulk fluid is dueto increased attachment of bacterial cells that have entered thestationary phase (and hence have become noncompetent) inthe surrounding medium.

Biofilms grown in biofilm reactors may encounter manypassing free-floating bacteria. This would discourage bioaug-mentation by in situ transformation if the problem of inhibitedgene transfer due to the presence of suspended cells duringbiofilm ontogenesis were unsolvable. Rinsing a biofilm to re-move most cells present in the drifting fraction, followed by astarvation period, could reinduce in situ transformation whenthe cells are exposed to a nutrient-containing substrate withplasmid DNA. James and coworkers (21) discovered that uponstarvation, bacillar cells revert to the coccoid form by reductiondivision, resulting in conservation of biomass but increased cellnumber. In this study the CLSM investigation revealed that thebiofilms treated with starvation-inducing medium containedmany cells with the coccoid morphotype, as observed whenbiofilms were grown in the absence of cells in the bulk fluid.Thus, all signs suggest that coccoid cells are the competentcells and bacillar cells are not competent for DNA uptake.Still, when cell shape was checked as a function of distancefrom the substratum, it was observed that cells at the biofilmsubstratum had the bacillar shape, while the prominent mor-photype of cells at the biofilm-medium interface was coccoid(Hendrickx, data not shown). However, most transformantswere formed at the biofilm substratum, where most bacillus-shaped cells resided. Further experiments are therefore need-ed to establish if other parameters related to the amount orlocation of bacillus-shaped or coccoid cells can be correlatedwith transformation frequency.

Nevertheless, the observed differences in transformation fre-quency in biofilms grown in the presence and in the absence ofplanktonic cells show that care should be taken in designingbiofilm experiments. In addition to the effects of standardenvironmental parameters (temperature, pH, nutrient content,substratum, moisture content, biotic and abiotic stresses, flowrate, etc.), the results might differ considerably depending onwhether the biofilms are grown under batch or continuous-flowconditions.

It should be noted that the experiments which we performedwere not designed to prove the general feasibility of in situbiofilm cell transformation with any highly competent soil bac-terium in naturally occurring biofilms. Our results do indicate

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that transformation has potential as a tool for bioaugmentationof biofilm reactors. It is interesting that transformation withAcinetobacter sp. strain BD413 resulted in transformationfrequencies in nonsterile groundwater and wet soil micro-cosms that were as high as those obtained under sterile con-ditions (32). Likewise, transformation of Acinetobacter sp.strain BD413 biofilm cells embedded in river epilithon was notinhibited by the presence of indigenous organisms (52). It canbe speculated that the presence of an ambient community inwastewater treatment systems should have few negative effectson transformation of Acinetobacter sp. strain BD413 cells. Fu-ture research should elucidate the efficiency of transformingcells which reside in natural biofilms inside bioreactors andother systems.

ACKNOWLEDGMENTS

This work was supported by the Research Center for FundamentalStudies of Aerobic Biological Wastewater Treatment, Munich, Ger-many (grant SFB411), and by the University of California, Davis.

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