The reduction of bacterial growth and biological fouling by organic-carbon and nutrient removal has enjoyed widespread acceptance in industrial and process engineering. Earlier studies investigating the impact of organic carbon perturbations on biofilm development often associated a reduction in the availability of a growth-related substrate to biofilms that were thinner and contained less biomass than those growing under nutritionally sufficient conditions (16
). Likewise, our data obtained from microtiter plate experiments showed that the A600
s in carbon-impoverished BF biofilms were significantly lower than those in SE biofilms. Because of this well-established relationship, the quantification of the organic-carbon content and its various important constituents, like AOC, has been used extensively in biofilm monitoring. However, the general usefulness of AOC is limited by the complexity and low speed involved in its determination. Furthermore, the use of pure-culture inocula of Pseudomonas fluorescens
P17 and Spirillum
sp. strain NOX is innately controversial, and an alternative using a natural microbial consortium has been suggested (14
The microtiter plate assay described here avoids the drawbacks associated with these bioassays. The biofilm formation potential of a water sample can be assessed directly in less than 2 h by using indigenous microorganisms, which form biofilms on the walls and at the bottom of each well. A high R2
value between the A600
and biovolume measurements further suggests that this assay provides representative biomass estimates, because the biovolume was independently determined using intact biofilm samples collected by polyacrylamide gel embedding (10
In the process of biofilm development, surface colonization is known to proceed by motility-assisted locomotion (15
) or clonal growth (42
). In the latter case, distinct patterns of colonization behavior, including spreading, shedding, and packing maneuvers, have been described (23
). Microcolonies produced by spreading tend to be poorly defined, with cell-to-cell separation reaching as much as 5 to 20 μm. Shedding does not produce microcolonies, because daughter cells move away from the immediate vicinity to colonize a new location. Surface colonization by these two maneuvers thus produces an unstructured assortment of single cells distributed over the surface—a pattern resembling the random arrangement of individual cells in the monolayers of BF biofilms. In contrast, microcolonies produced by packing tend to be dendritic and compact. This characteristic was observed in the SE_2 biofilm, where compact microcolonies (Fig. ) were produced by some SE members, as well as in laboratory-cultured P. fluorescens
) and naturally occurring pond water biofilms (7
). However, the appearance of different morphotypes when microcolonies developed into larger aggregates suggests that recruitment of secondary microorganisms is also significant in the development of SE biofilms.
Biofilm structural development and the resultant architecture have often been associated with substrate availability. Mathematical models (37
) relate a decrease in nutrient availability to an increase in biofilm porosity, and this was repeatedly shown in mixed-community biofilms (34
). Likewise, the BF_12 biofilm in run 2, consisting of a cell monolayer with low surface coverage and a high S
ratio (Fig. ), suggests that an open architecture with a probable optimized cell-to-cell separation was maintained in the BF biofilm. This adaptation maximizes the cells' surface area exposed to the nutrient flow and reduces resource competition in a carbon-restricted environment. However, the relationship between substrate availability and biofilm architecture is not always predictable. In run 1, the porosity of BF_12 biofilms appeared to be lower than that of the corresponding SE_12 biofilms. This discrepancy has also been noted in P. aureofaciens
) cultivated under different carbon concentrations. The apparent inconsistency in these results suggests that the biofilm architecture may have been influenced by microenvironmental conditions, such as the localized concentration gradient of substrates in the hydrodynamic boundary layer. Further research using microelectrode probes to examine localized microenvironments (34
) is anticipated to provide valuable data for the elucidation of this relationship.
In addition to the effect on biofilm development, environmental perturbations in the availability of organic-carbon sources can also lead to the selection of distinct biofilm communities (22
). Using T-RFLP, a temporal change in the community structure or bacterial succession was observed in both SE and BF biofilm communities. This phenomenon has been well researched in human dental biofilms, where biofilm formation is initiated predominantly by a defined group of pioneer colonizers consisting of actinomycetes and streptococci (28
). Similarly, the emergence of pioneer colonizers was observed here, and they appeared to be common to both SE- and BF-cultivated biofilms. Selection for pioneer colonizers is probably not affected by the organic-carbon content but by the ability to adhere to the substratum, which can be dependent on several physicochemical parameters (6
). The ability to produce exopolysaccharides can also enhance cellular adhesion, and this physiological trait is common among many members of the Sphingomonadaceae
). Other cellular appendages, such as the prostheca on Caulobacter
cells, are also known to mediate bacterial attachment. Together with the putative identification of the 111-bp T-RF in the SE_2 and BF_2 biofilms, Sphingomonadaceae
- and Caulobacter
-related organisms thus appear to be potential primary colonizers in these biofilms.
Following initial colonization, a subsequent loss of species richness suggests that competitive interactions exist between biofilm organisms in which some pioneer colonizers are outcompeted. As the ecological conditions in the SE and BF habitats were not the same, organisms with different physiological characteristics were selected in the two biofilms. For example, the 100-bp T-RF in the BF biofilms was associated with phylotypes related to Aquabacterium
. This genus is typically found in oligotrophic environments, like drinking water systems (18
), where bacterial activity can be severely restricted by low contents of organic carbon (25
) and, possibly, phosphorus (32
). The proliferation of Aquabacterium
phylotypes in BF biofilms suggests that biofiltration exerts a selection pressure that favors the growth of organisms physiologically adapted for survival under low-nutrient conditions. This was further supported by the exclusive occurrence of phylotypes BF74, BF89, and BF115, closely related to Legionella
, a genus that is often found in oligotrophic freshwater and drinking water biofilms (4
To adapt to low-nutrient conditions, some organisms selected for in the BF habitat also appeared to be metabolically versatile. Phylotypes (BF160 and BF161) associated with the 453-bp T-RF were related to Azospira oryzae
, which is known to fix nitrogen (39
). These phylotypes were present only in the BF biofilms, suggesting that nitrogen fixation can be an important mode of cellular growth in localized microenvironments where microaerobic conditions prevailed in the biofilm. The ability to fix dinitrogen as a source of cell nitrogen freed these phylotypes from dependence on fixed forms of nitrogen (like ammonia) and thus conferred an ecological advantage for their survival in a nutrient-limited environment.
Despite the different ecological pressures in the SE and BF niches, certain groups of bacteria continued to thrive in both biofilm communities. This was exemplified by the increase in abundance of the 111-bp T-RF, possibly representing either the Sphingomonadaceae
spp. in Alphaproteobacteria
. Alphaproteobacterial populations were also selectively enriched at a higher rate in SE- than in BF-cultivated biofilms. This suggests that although the growth of these alphaproteobacterial populations was affected by the removal of organic carbon by biofiltration, they could still compete favorably with other biofilm bacteria, presumably due to their ability to catabolize a large variety of organic substrates (5
). For example, Caulobacter
spp., which are known to survive under low-nutrient conditions (38
), were found to be dominant in the BF_10 biofilm but not in the SE_10 biofilm.
Although the effect of the organic-carbon concentration on biofilm formation has been well established, this study suggests that the correlation may not be a simple one, due to the selection of biofilm organisms that are adapted for survival under low-substrate conditions. These organisms, including those with low nutritional requirements (e.g., Aquabacterium, Caulobacter, and Legionella) and others that are metabolically versatile (Azospira and sphingomonads), tend to form open biofilm structures that maximize the influx of nutrients into the biofilm. These adaptive strategies imply that carbon limitation may not be an effective barrier to biofilm growth. For this reason, long-term biofouling control may not be achievable using carbon limitation strategies alone, and it would thus be prudent to assess the biofilm formation potential of a water sample by using a combination of organic-carbon-based measurements and direct biofilm quantification. As described above, the microtiter plate assay is a powerful tool for direct biomass determination in terms of speed, simplicity, and representation. In the same way, the molecular analyses of temporal biofilm communities can uncover useful information for the formulation of specific countermeasures. For example, biofouling caused by SE effluents may be more appropriately dispersed by a mixture of polysaccharidases than by conventional oxidizing biocides, as Sphingomonadaceae-related organisms are likely to form exopolysaccharide-ensconced biofilms. When incorporated as a side stream device, the techniques described here are expected to provide insight into biofilms developed on inaccessible locations (such as on the interior surfaces of reverse-osmosis membranes) and to assist in the timely mitigation of biofouling.