Biofilms: implications in bioremediation

Biofilms: implications inbioremediationRajbir Singh*, Debarati Paul* and Rakesh K. Jain

Institute of Microbial Technology, Sector 39-A, Chandigarh-160036, India

Review TRENDS in Microbiology Vol.14 No.9

Biofilms are assemblages of single or multiplepopulations that are attached to abiotic or bioticsurfaces through extracellular polymeric substances.Gene expression in biofilm cells differs from planktonicstage expression and these differentially expressedgenes regulate biofilm formation and development.Biofilm systems are especially suitable for the treatmentof recalcitrant compounds because of their highmicrobial biomass and ability to immobilize compounds.Bioremediation is also facilitated by enhanced genetransfer among biofilm organisms and by the increasedbioavailability of pollutants for degradation as a resultof bacterial chemotaxis. Strategies for improvingbioremediation efficiency include genetic engineeringto improve strains and chemotactic ability, the useof mixed population biofilms and optimization ofphysico–chemical conditions. Here, we review theformation and regulation of biofilms, the importanceof gene transfer and discuss applications of biofilm-mediated bioremediation processes.

Biofilm communities and bioremediationBiofilms

Since the inception of microbiology, microorganisms haveprimarily been characterized as planktonic, freelysuspended cells and have been described on the basis oftheir morphological and physiological properties andgrowth characteristics in nutritionally rich culture media.However, in most natural environments, microbes arecommonly found in close association with surfaces andinterfaces in the form of multicellular aggregates gluedtogether with the slime they secrete [1,2].

Biofilms are clusters of microbial cells that are attachedto a surface. They occur in nearly every moist environmentwhere sufficient nutrient flow is available and surfaceattachment can be achieved. A biofilm can be formed bya single bacterial species, although they can also consist ofmany species of bacteria, fungi, algae and protozoa.Approximately 97% of the biofilm matrix is either water,which is bound to the capsules of microbial cells, or solvent,the physical properties of which (such as viscosity) aredetermined by the solutes dissolved in it [3]. The diffusionprocesses that occur within the biofilm matrix aredependent on the water-binding capacity and mobility ofthe biofilm. Besides water and microbial cells, the biofilmmatrix is a complex of secreted polymers, absorbed

Corresponding author: Jain, R.K. ([email protected]).* Both authors contributed equally.Available online 18 July 2006

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nutrients and metabolites, cell lysis products and evenparticulate material and detritus from the immediatesurrounding environment [4]. Therefore, in additionto peptidoglycan, lipids, phospholipids and other cell com-ponents, all major classes of macromolecules (i.e. proteins,polysaccharides, DNA and RNA) can be observed within abiofilm environment [3]. The anionic property of biofilmsis conferred by the presence of uronic acids (such asD-glucuronic, D-galacturonic and mannuronic acids)or ketal-linked pyruvates [4]. The phenomenon of masstransport in biofilms is influenced by biofilm structure,which in turn depends upon the local availability ofsubstrates. Solute transport in biofilms is driven byconvective transport within pores and water channelsand also by diffusion in the denser aggregates [5]. Thus,the matrix displays a high degree of microheterogeneitybecause of the numerous microenvironments that co-existwithin it.

Bioremediation

The swift growth of chemical industries over the past fewdecades has resulted in contamination of the environmentdue to toxic waste effluents [6]. The persistence of chemicalpollutants and consequent environmental problemshas brought the possibility of long-term environmentaldisasters into the public conscience [7]. Therefore, variousstrategies are being developed and further research iscurrently underway to develop means of sustainingthe environment. Bioremediation is an emerging in situtechnology for the clean-up of environmental pollutantsusing microorganisms. The biological processes fortreating toxic effluents are better than chemical andphysical methods in terms of their efficiency and economy[6] and the potential of biofilm communities for bioreme-diation processes has recently been realized.

Biofilm-mediated bioremediation presents a proficientand safer alternative to bioremediation with planktonicmicroorganisms because cells in a biofilm have a betterchance of adaptation and survival (especially during per-iods of stress) as they are protected within the matrix [8].Owing to the close, mutually beneficial physical and phy-siological interactions among organisms in biofilms, theusage of xenobiotics is accelerated and, consequently, bio-films are used in industrial plants to help in immobiliza-tion and degradation of pollutants. Akinson [9] reportedthe use of biofilms for water and wastewater treatments inthe early 1980s. However, it is only during the pastfew decades that biofilm reactors have become a focus ofinterest for researchers in the field of bioremediation.

ed. doi:10.1016/j.tim.2006.07.001

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Table 1. Techniques commonly used for analysis of biofilm communities

Method of analysis Examples and modifications Use(s) Refs

Direct observation Epifluorescent microscopy, confocal scanning laser

microscopy

Morphological observation, enumeration [1]

Molecular methods In situ hybridization, comparative sequence analysis Community analysis, taxonomy [1,11]

Fluorescent labeling Fluorescence in situ hybridization in combination

with microautoradiography and microsensors

Characterization of biofilm communities [10,12]

Detection of gene expression Reporter protein assay, in vivo expression

technology (IVET), recombination based IVET (RIVET)

Investigation of gene activity [10,13]

PCR Direct in situ PCR Characterization of genetic and phylogenetic

properties

[14]

390 Review TRENDS in Microbiology Vol.14 No.9

Numerous studies have determined the composition ofcommunities present in biofilms in various environments[1,10–14] and a growing number of microscopic and mole-cular methods have facilitated the analysis of spatialorganization and phylogenetic properties of microbes ina biofilm community (Table 1). This Review article dis-cusses the formation and regulation of biofilms, the impor-tance of gene transfer and applications of biofilm-mediatedbioremediation processes.

Genetic and environmental regulation of biofilmformationBiofilm formation is a complex multifactorial processwhereby microorganisms of single and/or multiple speciesgrow on a surface and produce extracellular polymers,which results in alterations in the organism phenotypewith respect to growth rate and gene transcription. Cellsurface properties, specifically the presence of fimbriae,flagella and surface-associated polysaccharides or pro-teins, are important for surface attachment and mightprovide a competitive advantage for one organism whereamixed community is involved [15,16]. Figure 1 depicts thesteps involved in formation of a functional biofilm, startingwith the initial attachment and establishment followed bymaturation and, finally, detachment of cells. Various envir-onmental factors regulate bacterial biofilm formation suchas variations in pH, availability of nutrients and oxygenand concentration of bacterial metabolites, which causebiofilms to differ from each other so that a biofilm formed ina stream differs from one formed on biological tissue. Thisfact was also substantiated by Allegrucci et al. [17], whoobserved that environmental factors affect de novo proteinproduction within biofilms in Streptococcus pneumoniae;simultaneously, an increase in cell number and biomasswas also observed. Quorum sensing (the ability of bacteriato communicate and coordinate behavior through signalingmolecules) also regulates changes in mature biofilms bycontrolling the formation of channels and pillar-like struc-tures that ensure efficient nutrient delivery to the cells.This architecture helps in the adequate dispersal of nutri-ents when there is increased competition for food at highcell density within biofilms [16,18].

To gain an insight into the genes involved in biofilmformation, DNA microarrays representing >99% of theannotated Bacillus subtilis open reading frames were usedto follow temporal changes in gene expression thatoccurred during the transition from the planktonic tobiofilm state. Approximately 6% of the genes involved inmotility, phage-related functions, metabolism and

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transcription of B. subtilis were differentially expressed[19]. The transcription factor RpoS affected biofilm forma-tion during different phases in Pseudomonas aeruginosaand Escherichia coli as indicated by microarray studies[16]. Whiteley et al. [20] also demonstrated that certainsubpopulations in a biofilm exhibit a different pattern ofgene expression than the planktonic cells or other meta-bolically active cells. Lazazzera [21] reported the repres-sion of flagellar genes and hyperexpression of genesresponsible for adhesion and formation of ribosomal pro-teins at various stages of biofilm formation. In E. colibiofilms, ribosomal genes were expressed at an increasedlevel when compared with planktonic cells in stationaryphase but not in exponential phase [22]. Therefore, variousmicroorganisms show differences in their gene expressionpatterns during biofilm formation.

Quorum sensing could also be important for the aggre-gation of bacteria on solid surfaces [18], thus influencingbiofilm development in several species. In Staphylococcusaureus, the cyclic-peptide-dependent accessory gene reg-ulator (agr) of its quorum-sensing system represses severalsurface adhesins thatmediate contact with the hostmatrixand, hence, facilitate its attachment to various surfaces[18,23]. Vibrio cholerae (an enteric pathogen) uses quorumsensing to regulate the production of secreted exopolysac-charides (EPS) encoded by the vps operon, which mediatesintercellular aggregation and adherence to surfaces [23].Acyl-homoserine lactone (HSL)-based quorum sensinginfluences biofilm maturation in Serratia liquefaciens [24]and it also controls cellular aggregation in Rhodobactersphaeroides [25]. Mutations in the acyl-HSL synthase ofthe R. sphaeroides quorum-sensing system, called cer (forcommunity escape response), resulted in hyperaggregationof cells in liquid culture [25]. Using confocal scanning lasermicroscopy, Stanley et al. [19] observed that glucose inhib-ited biofilm formation through the catabolite control protein(CcpA) inB. subtilis. Studies on differential gene expressionin bacterial biofilms are still in their infancy and scientistsare in search of a ‘universal’ biofilmgeneexpressionpattern,the regulation of which would lead to the formation and/ordestruction of biofilms.

The flexible nature of bacterial gene expression permitsthe survival of bacteria in diverse environments withrapidly changing conditions and it is this adaptability thathas enabled bacteria to thrive far and wide [26]. Aggrega-tion of bacteria into complex microbial communities alsoconfers some special advantages that are not enjoyed bydiscrete bacteria; for example, organisms within a biofilmcan withstand shear forces, nutrient deprivation, pH

Figure 1. Diagrammatic representation of cyclic steps involved in the formation of an active biofilm. Cells initially attach by physico–chemical interactions or extracellular

matrix protein secretion to form a cell monolayer, in which cells express pili and have twitching motility and/or the ability to undergo chemotaxis. Cells proliferate in the

monolayer and other microbes attach to form an active biofilm, the development and distortion of which is influenced by environmental factors such as hydrodynamic and

mechanical stress. Cells in the mature biofilm are motile and undergo chemotaxis, which leads to spreading of biomass and an increased rate of horizontal gene transfer. As

cells die, active bioconversion and/or biodegradation leads to solute transfer to or from the bulk liquid, which results in eventual biofilm detachment. The processes of

formation and detachment of cells are repeated in a cycle, thereby enabling further development of similar biofilms, which can subsequently attain new dimensions as a

result of environmental influences. The approximate time period for which each of the phases persist is shown on the left.

Review TRENDS in Microbiology Vol.14 No.9 391

changes and antibiotics in a more pronounced mannerbecause of the mechanical strength provided by the EPS[26,27]. This renders biofilms more advantageous for bior-emediation purposes when compared with free cells.


Gene transfer within biofilmsTransformation, transduction and conjugation in bacterialbiofilms result in gene transfer between biofilm organisms[28–30]. Many oral streptococcal species are naturally


competent and take up free DNA that occurs in the mixedoral biofilm environment of humans and animals [29].There are increased opportunities in biofilms for the trans-fer of mobile genetic elements (e.g. plasmids) that mightencode beneficial traits, such as resistance to antibioticsand/or heavy metals and the ability to degrade pollutants[29,30]. Some conjugative transposons such as Tn916-liketransposons have a broad host range and have beenintroduced into >50 different species and 24 genera ofbacteria [29,31]. Bacteriophage-mediated transfer oftetracycline resistance has been demonstrated in S. aureusbiofilms [29]. Horizontal gene transfer in bacteria underboth natural and engineered environments occurs throughtransposons and the transformation of naked DNA orplasmids, which leads to transfer of genetic traits.

Plasmid-mediated gene transfer in biofilms

Genes that encode the degradation of xenobioticcompounds are often located on plasmids. Therefore, thehorizontal exchange of catabolic genes among bacteria inmetabolic pathways could help in the construction of novelcatabolic pathways and strategies for bioremediation.Gene dissemination has been used as a tool to enhancebioremediation in biofilms in several cases and furtherresearch is underway. Goris et al. [32] studied the transferof plasmid pC1 of Delftia acidovorans tagged with amini-Tn5 transposon encoding the gene for oxidativedeamination of 3-chloroaniline. The labelled plasmidwas transferred to Pseudomonas putida and the effectwas later monitored on activated sludge bacteria. Manytransconjugants displayed mineralization of 3-chloroani-line. These observations substantiate that gene transferoccurs with a high frequency in biofilms and this propertycan be explored further to optimize bioreactor efficiency incases where degradation is limited by the low biomass andpaucity of degradative genes. Ghigo [28] postulated thatconjugative plasmids might enhance biofilm formationbecause plasmids repressed for horizontal gene transferretained the ability to form biofilms even if the biofilmpopulation included plasmid-free recipient cells.

Gene transfer by transformation

Natural competence is a pervasive phenomenon amongbacteria. Hence, it could be expected that transformation ofDNA would occur efficiently in dense bacterial populationssuch as biofilms that are regulated by quorum sensing.Streptococcus mutans cells grown in a biofilm were trans-formed at a frequency up to 4 � 10�3 per cell and, in mostcases, at rates of ten to 600-fold higher than planktonic S.mutans cells [33]. In many streptococci, includingS. mutans, a quorum-sensing system mediated by a com-petence-stimulating peptide (CSP) pheromone regulatesgenetic competence and biofilm formation [34]. This phe-nomenon is encoded by the comC gene and the centralcomponent of this system is ComX, which functions as analternative sigma factor to activate competence genesinvolved in DNA uptake and processing. Aspiras et al.[34] demonstrated a positive correlation between comXpromoter (pcomX) activity, natural transformation andcompetence development in biofilms using a transcrip-tional fusion of pcomX with lacZ. Whitchurch et al. [35]


presented evidence that extracellular DNA could mediateadhesion of cells to one another or to other surfaces in theinitial phase of biofilm formation. com genes involved inregulating competence might also be involved in biofilmformation where optimal conditions for DNA transfer pre-vail [33,34]. However, such processes that take place in thecell microenvironment need to be better understood toimprove the mineralization process for bioremediation.Impending developments in the field include furthergenetic improvement of strains and the adaptation ofexisting methodologies to large-scale and in situ deconta-mination processes.

Transposon-mediated gene transfer

Conjugative transposons are probably the most promiscu-ous of all mobile elements. These elements integrate intothe host cell genome and do not need to provide their ownreplication machinery or rely upon interactions with thehost cell for stable replication and maintenance. Springaelet al. [36] studied community shifts in the 3-chlorobenzo-ate-degrading biofilm reactor using P. putida BN210 thatcarried the self-transferable clc-element (encoding degra-dation of 3-chlorobenzoate) on the chromosome. Theyobserved a loss of strain BN210 from the reactors togetherwith the appearance of several novel 3-chlorobenzoate-mineralizing bacteria and the presence of a copy of a clc-element in many isolates, which suggested transposon-mediated acquisition of degradative genes. Roberts et al.[31] also investigated horizontal gene transfer of a con-jugative transposon Tn5397 (tetracycline resistant) in amixed species oral biofilm and found another tetracycline-resistant Streptococcus species in the community, whichdemonstrated the presence of Tn5397 in the genome. Theirinvestigation reconfirmed the transfer of genes in biofilmcommunities among related or unrelated strains.

Gene transfer is autocatalytically promoted in biofilmsowing to their dense population and packed structure. Thisenables an increased local gene and plasmid transfer rate.Furthermore, DNA release and transformation processesseem to be involved in the biofilm-mediated life cycle,which presents new possibilities for bioenhancement stra-tegies and for developing strains with versatile metabolicabilities [33]. Compared with bioaugmentation (the exo-genous application of degrading organisms) for in situclean up, the indirect introduction of catabolic capabilityby such gene transfer mechanisms is beneficial becausethis involves the adaptive success of the existing popula-tions. Licht et al. [37] postulated that effective mixing inshaken and/or stirred cell suspensions could result incomplete and effective transfer of genes into cells. Variousstudies on 2,4-dichlorophenoxyacetic acid degradationhave also authenticated that gene transfer enhances bio-degradation [38,39]. Therefore, this phenomenon mighthave the potential to speed up the physiological and cata-bolic performance of complex communities by the forma-tion of new degradation pathways.

Applications of biofilms in bioremediationSuccessful application of a bioremediation process reliesupon an understanding of interactions among microorgan-isms, organic contaminants and soil or aquifer materials.

Table 2. Bioremediation of hydrocarbons using biofilms in bioreactorsa

Pollutant Reactor or experimental

conditions

Organisms or culture Overall efficiency of

degradation

Refs

Chlorophenols

2-Chlorophenol Silicone tube membrane

bioreactor

Anaerobic sludge from a swine

wastewater treatment plant

>90% [67,68]

4-Chlorophenol Granular activated-carbon

biofilm reactor

Bacterial consortium from

rhizosphere of Phragmites australis

70–100% [69,70]

2,4-Dichlorophenol Rotating perforated tube biofilm

reactor

Pseudomonas putida �100% [49]

2,4,6-Trichlorophenol Fluidized bed biofilm reactor Pseudomonas sp., Rhodococcus sp. �100% [50]

2,3,4,6-Tetrachlorophenol Fluidized bed biofilm reactor Pseudomonas sp., Rhodococcus sp. �100% [50]

Pentachlorophenol Fluidized bed biofilm reactor Pseudomonas sp., Rhodococcus sp. �100% [50]

Pyrene, phenanthrene Biofilm grown directly on liquid

medium

Polaromonas sp., Sphingomonas sp.,

Alcaligenes sp., Caulobacter and

Variovorax sp.

�50% (pyrene); �98%

(phenanthrene)

[71]

o-Cresol, naphthalene, phenol,

1,2,3-trimethylbenzene

Biofilm grown in NAPLs Pseudomonas fluorescens Not determined [72]

n-Alkanes Rotating biological contactors Prototheca zopfii �65% [73]

Carbon tetrachloride Continuous flow fixed biofilm

reactor

Providencia stuartii, Pseudomonas

cepacia

�100% [74]

Toluene Hollow-fibre membrane biofilter

reactor; continuously fed

biodrum reactor

Secondary sludge from wastewater

treatment plant; various aerobic and

anaerobic bacteria

�84%; 65% [75,76]

Azo dyes

Acid Orange 10, 14 Laboratory-scale rotating drum

biofilm reactor

Methylosinus trichosporium �60% [77]

Everzol Turquoise Blue G Laboratory-scale activated

sludge unit

Coriolus versicolor �82% [78]

Herbicides

MCPP; 2,4-D Granular activated-carbon

biofilm reactor

Mixed culture of herbicide-degrading

bacteria

MCPP (partial); 2,4-D

(complete)

[79]

aAbbreviations: 2,4-D, 2,4-Dichlorophenoxyacetic acid; MCPP, 2-(2-methyl-4-chlorophenoxy) propionic acid.

Table 3. Bioremediation of heavy metals using biofilms inbioreactors

Reactor or

experimental

conditions

Methods of

remediation

Heavy

metals

remediated

Refs

Anaerobic–anoxic–

oxic (A2O) biofilm

process

Biosorption Zn, Cd, Ni [80]

Biofilm formed on

moving bed sand

filter

Biosorption,

bioprecipitation

Cu, Zn, Ni, Co [81]

Rotary biofilm

reactor for algae

immobilization

Immobilization Co [82]

Biofilm developed

over granular

activated carbon

Adsorption Cd, Cu, Zn,

Ni

[83,84]

Bacteria-

immobilized

composite

membrane reactor

Bioprecipitation Cd, Zn, Cu,

Pb, Y, Co,

Ni, Pd, Ge

[85]


Physiological properties of the microorganisms such asbiosurfactant production and chemotaxis enhance bioa-vailability and, hence, degradation of hydrophobic com-pounds [6,7]. Microorganisms that secrete polymers andform biofilms on the surface of hydrocarbons are especiallywell suited for the treatment of recalcitrant or slow-degrading compounds because of their high microbial bio-mass and ability to immobilize compounds by biosorption(passive sequestration by interactions with biological mat-ter), bioaccumulation (increased accumulation of microbesunder influence) and biomineralization (formation of inso-luble precipitates by interactions with microbial metabolicproducts) [40]. Biofilms support a high biomass densitythat facilitates the mineralization processes by maintain-ing optimal conditions of pH, localized solute concentra-tions and redox potential in the vicinity of the cells. This isachieved by the unique architecture of the biofilm andcontrolled circulation of fluids within it [4,5]. Biofilm-basedreactors are commonly used for treating large volumes ofdilute aqueous solutions such as industrial and municipalwastewaters. The main biofilm reactors are categorizedaccording to the methods they use, such as the upflowsludge blanket (USB), biofilm fluidized bed (BFB),expanded granular sludge blanket (EGSB), biofilm airliftsuspension (BAS) and internal circulation (IC) methods. InUSB, BFB and EGSB reactors the particles are keptfluidized by an upward liquid flow. In BAS reactors, asuspension is obtained by pumping air into the system,whereas in IC reactors, the gas produced in the systemdrives the circulation and mixing of the liquid and solids inan airlift-like reactor [41]. Table 2 lists some bioreactors


that are currently being used for bioremediation of varioushydrocarbon contaminants and Table 3 lists those used inbioremediation of heavy metals.

Role of chemotaxis in biodegradation and biofilm

formation

Chemotaxis is the movement of organisms in response to achemical nutrient or chemical gradient. It helps bacteria tofind optimum conditions for growth and survival and is anintegral feature of biodegradation [6,7]. Under conditionsof limited carbon and energy sources, it is possible that


chemotaxis is selected as an advantageous behaviour inbacteria, along with xenobiotic degradation capabilitiesafter exposure to such compounds. Although bacterialdegradation of pollutants has proved efficient for bioreme-diation of contaminated sites in many cases, the applica-tion of bacteria that exhibit chemotaxis towards pollutantshas received less attention.

The first step in bioremediation is the bioavailability ofthe compound to the bacterial cells. Bioavailability oforganic contaminants has been identified as a major lim-itation to the efficient bioremediation of contaminated sites[6,7,42] and can be improved by exploiting chemotacticbacteria. Cells displaying chemotaxis can sense chemicalssuch as those adsorbed to soil particles in a particular nicheand swim towards them; hence, the mass-transfer limita-tions that impede the bioremediation process can be over-come. Once the cells are brought into close contact with asurface, the mechanism of biofilm formation and surfac-tant production commences, which leads to enhanced bioa-vailability and biodegradation. When the targetcontaminants are dissolved in an aqueous medium, therate of biodegradation is improved compared with thosehydrophobic pollutants that remain adsorbed in the non-aqueous phase liquid (NAPL) associated with contami-nated soils [42,43]. Bacteria access these target compoundseither by dissolution of the target compounds in the aqu-eous phase or by direct adhesion to the NAPL–water inter-face, a process that is facilitated by biofilm formation.

Chemotaxis has an important role in biofilm formationin several microorganisms [15,44]: it guides bacteria toswim toward nutrients (hydrophobic pollutants) that areadsorbed to a surface, which is followed by surface attach-ment using the bacterial flagellum. Flagella are requiredfor attachment to abiotic surfaces and facilitate the initia-tion of biofilm formation [15,42]. In addition, chemotaxisand/or motility might be required for bacteria within adeveloping biofilm to move along the surface to grow andspread [42]. McCarter et al. [45] observed that the swarm-ing motility of Vibrio parahaemolyticus is induced whenthere is any constraint in the movement of polar flagella.Watnick and Kolter [46] demonstrated that the flagellummediates spreading of a biofilm along abiotic surfaces, andthat the EPS is involved in the formation of the 3D biofilmarchitecture in V. cholerae. Lee et al. [47] reported thedecreased ability of a mutant to form biofilms in Vibriovulnificus. In this mutant, the flgE gene, which encodes acomponent of the flagellum, was knocked out to study theinvolvement of flagella in biofilm formation. Therefore,flagella seem to be important for biofilm formation invarious Vibrio species. This is supported by the work ofStanley et al. [48], who studied the attachment of P.aeruginosa to stainless steel surfaces. After cells weremechanically sheared in a blender to remove their flagella,the authors observed that the rate of cell attachmentdecreased by at least 90% when flagella were removed,thereby further highlighting the need for motility in bio-film formation.

Role of biofilms in remediation of hydrocarbons

Chlorinated aromatic compounds are recalcitrant chemi-cals that are present in several chemical industry effluents


and can migrate quickly through soils. They are classifiedas one of the most widespread contaminants of soil andgroundwater and are carcinogenic even at extremely lowconcentrations [49,50]. To remove 2,4-dichlorophenol(DCP) from synthetic wastewater, Kargey and Ekker[49] used a rotating perforated tube biofilm reactor thatcontained a mixed microbial biomass of activated sludgeculture supplemented with DCP-degrading P. putida;nearly 100% of the DCP was degraded. Similarly, bacteriaspecialized in adhesion to polyaromatic hydrocarbons(PAHs) facilitate PAH degradation [51,52]. This has beenshown for diclofop-methyl, methyl 2-[4-(2,4-dichlorophe-noxy)phenoxy] pyruvate, a two-ring chlorinated herbicide(diclofop-methyl), which accumulated in biofilms byadsorption to microbial exopolymers. The biofilm commu-nity metabolized the accumulated diclofop-methyl duringstarvation [53].

Nitroaromatic compounds are another group of xeno-biotics that have found multiple applications in the synth-esis of foams, pharmaceuticals, pesticides and explosives.The presence of a nitro group makes these compoundsresistant to biodegradation and microbial conversion oftenleads to the production of harmful metabolites [54,55].Lendenmann and Spain [55] used a mixed culture thatdegraded an isomeric dinitrotoluene (DNT) mixture in afluidized-bed biofilm reactor; the reactor was fed withaqueous solutions containing 2,4-DNT (40 mg L�1) and2,6-DNT (10 mg L�1). Degradation efficiencies higher than98% for 2,4-DNT and 94% for 2,6-DNT were achieved at allloading rates. Degradation of 4,6-dinitro-ortho-cresol (oneof the oldest synthetic pesticides) was reported in batchcultures and in fixed-bed column reactors [54].

Improving strains by engineering metabolic pathwaysand enzymes involved in degradation or by increasing thecopy number of degradative genes could further enhancebiofilm-mediated bioremediation. Strains with biodegra-dation and chemotactic capabilities would also be benefi-cial for biofilm formation; for example, a chemotactic strainengineered to contain catabolic genes would be highlyproficient at biodegradation. Perumbakkam et al. [56]introduced atrazine-degrading genes to biofilms of Acine-tobacter sp. BD413, thereby developing a biofilm-mediatedprocess to degrade atrazine. Developing specific mixedcommunities can also facilitate bioremediation [56]. Otherreports suggest that co-adhesion and synergistic interac-tion with biofilm-forming species might be an alternativestrategy for the persistence and propagation of otherstrains [57,58]. Therefore, a combination of geneticengineering of microorganisms and optimization ofphysico–chemical parameters and substrate concentrationin bioreactors is of pronounced importance for developingbioremediation strategies [59].

Role of biofilms in remediation of heavy metals

Another promising application of biofilms is in heavymetaland radionuclide remediation [40,60]. The distribution anddiversity of microbes that inhabit contaminated sites andof the genes that encode for phenotypes responsible formetal–microbe interactions are crucial elements in metaland radionuclide bioremediation. Heavy metal bioreme-diation can be achieved by immobilization, concentration


and partitioning to an environmental compartment,thereby minimizing the anticipated hazards [40,60]. In astudy byWhite and Gadd [61], sulphate-reducing bacterialbiofilms grown in continuous culture and exposed to amedium that contained 20–200 mM copper were found toaccumulate it in the form of copper sulfide. A simultaneousincrease in the EPS content of the biofilm was alsoobserved, which suggested the role of EPS and biofilmsin the entrapment of metal precipitates. Macaskie et al.[62] observed that polycrystalline NaUO2PO4 accumulatedin and around the cell wall of Citrobacter sp. N14 byadsorption to lipopolysaccharide and, hence, aided in itsbioprecipitation of the uranium salt. In another study ofmetal precipitation, Labrenz et al. [63] observed the for-mation of sphalerite (ZnS) by members of the aerotolerantDesulfobacteriaceae in a natural biofilm. In this process, Znwas concentrated and metal sulfides were then precipi-tated by sulfate-reducing bacteria in the second phase of acombined sulfur oxidation–reduction biotreatment techni-que. Costley and Wallis [64] reported �84% removal ofCd2+, Cu2+ and Zn2+ in synthetic wastewater using arotating biological contactor with alternating sorptionand desorption cycles.

Biofilms can also affect the fate of other compounds intheir vicinity as a consequence of their physiologicalresponse during the absorption of water and inorganicor organic solutes [4], which might also be beneficial forremediation purposes. Valls and Lorenzo [65] have dis-cussed a variety of instances in which a cellular trait(already present in some strains) can be combined orimproved through genetic engineering. Molecularapproaches enable the construction of improved strainswith specific metal-binding properties through the expres-sion of metal-chelating proteins and peptides, the improve-ment of metal precipitation processes and the introductionof metal transformation activities in robust environmentalstrains.

Other applications

Biofilms can also be applied to various other aspects ofbioremediation. NAPLs pose great challenges to the reme-diation of contaminated soil and sediments because resi-dual NAPLs that are trapped in pores lead to small-scaleheterogeneity in contaminant distribution and hindertheir transfer to the surrounding aqueous phase [43].Law and Aitken [43] suggested that chemotaxis providedan increased cell density at or near an interface fromwhicha chemical attractant desorbs or dissipates, thereby lead-ing to increased degradation rates of the attractant. Bio-films also have a role in the removal of acid in acid-minedrains, which is one of the challenging long-term environ-mental problems attributed to various mining activities.The oxidation of sulphide particles in tailings, waste-rockpiles and open-pit walls is limited by the transport ofoxygen. Bacterial biofilms significantly improve rates ofremediation under such conditions by improving the avail-ability of oxygen [66].

Concluding remarksMost bacteria in natural ecosystems exist in complexassemblages that comprise one or more species. Biofilms


are an example of such an assemblage and are made up ofhighly structured matrix-enclosed communities separatedby a network of open water channels. This architecture isan optimal environment for cell–cell interactions includingthe intercellular exchange of genetic material, communi-cation signals and metabolites that enable diffusion ofnecessary nutrients to the biofilm community. Study ofbiofilm communities and gene transfer within biofilmswould facilitate the development of better techniques forthe bioremediation of contaminated sites andwastewaters.Exploration of the properties of soil biofilms for bioaug-mentation of contaminated sites and development of engi-neered biofilm processes with enhanced biodegradationkinetics has the potential to improve in situbioremediation.

AcknowledgementsWe are thankful to Anuradha Ghosh and Archana Chauhan for theirhelp. The authors are supported, in part, by the Council of Scientific andIndustrial Research (CSIR), India, and the Department of Biotechnology(DBT), India. This is IMTECH communication no. 37/2005.

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doi:10.1111/j.1747-6593.2005.00010.x

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81 Diels, L. et al. (2003) Heavy metal removal by sand filters inoculatedwith metal sorbing and precipitating bacteria. Hydrometallurgy 71,235–241

82 Travieso, L. et al. (2002) BIOALGA reactor: preliminary studies forheavy metals removal. Biochem. Eng. J. 12, 87–91

83 Scott, J.A. and Karanjkar, A.M. (1998) Immobilized biofilms ongranular activated carbon for removal and accumulation of heavymetals from contaminated streams. Water Sci. Technol. 38, 197–204

84 Scott, J.A. et al. (1995) Biofilms covered granular activated carbon fordecontamination of streams containing heavy metals and organicchemicals. Minerals. Eng. 8, 221–230

85 Diels, L. et al. (1995) The use of bacteria immobilized in tubularmembrane reactors for heavy metal recovery and degradation ofchlorinated aromatics. J. Memb. Sci. 100, 249–258

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