The natural habitats of prokaryotes are remarkably diverse (
188,
268). Prokaryotes can inhabit any environment that is suitable for higher life forms, as well as a variety of inhospitable settings that the majority of higher life forms would find extremely objectionable (
152). Their ability to persist throughout the biosphere is due, in part, to their unequaled metabolic versatility and phenotypic plasticity. One key element of their adaptability is their ability to position themselves in a niche where they can propagate. Numerous positioning mechanisms have been discovered in bacteria. The most common mechanism is flagellar motility and different methods of surface translocation, including twitching, gliding, darting, and sliding (
102). However, there are other mechanisms utilized by bacteria to position themselves in response to their environment. Some species are able to affect their position by synthesizing cellulose, thereby forming a fibrous pellicle that places cells near the air-water interface. In addition, cellulose synthesis aids in attachment to surfaces such as plant cells (
216). Other bacteria, such as the purple sulfur bacterium
Amoebobacter purpureus, modulate their density in order to position themselves. These photosynthetic bacteria position themselves at different levels in the water column in response to light intensity by producing gas vesicles for bouyancy or synthesizing carbohydrates or forming aggregates in order to sink (
187). In addition, some species have magnetosomes (intracellular structures consisting of a crystal of a magnetic mineral surrounded by a membrane) that cause the cells to passively align with the Earth's geomagnetic field, thereby restricting lateral excursions (
11,
227). One of the most important positioning mechanisms is aggregation or attachment. Aggregation enhances cell-cell interaction as well as the sedimentation rate of cells. Through attachment, the bacteria not only position themselves on a surface; they can form communities and obtain the additional benefit of the phenotypic versatility of their neighbors. Since a surface-attached lifestyle is ubiquitous, it is likely that this type of sessile community-based existence is a critical characteristic for persistence of the bacteria. Organisms can exist in an environment independently, but in many cases they proliferate more effectively by interacting and forming communities (
23). Some of the concepts discussed in the following sections are illustrated in Fig.
1.
Bacterial communities in nature play a key role in the production and degradation of organic matter, the degradation of many environmental pollutants, and the cycling of nitrogen, sulfur, and many metals. Most of these natural processes require the concerted effort of bacteria with different metabolic capabilities, and it is likely that bacteria residing within biofilm communities carry out many of these complex processes. Studies in bioreactors and enrichment cultures have shown that biofilms are involved in the processing of sewage (see below), in the treatment of groundwater contaminated with petroleum products (
155), and in nitrification (
58). Biofilms also form in many extreme environments, such as in acid mine drainage (at a pH of 0), where they contribute to the cycling of sulfur (
67). Cyanobacterial mat biofilms have been intensively studied in thermal springs (
204,
261), and recently, researchers have started to investigate biofilms in the “desert-like” lake ice cover in Antarctica (
190). Complex structured communities in these extreme environments have been found to conduct a variety of biological processes, such as photosynthesis, nitrogen fixation, and fermentation.
Another type of biofilm community that is being investigated is the bacterial assemblages associated with suspended particles of organic and inorganic material in the marine environment. Researchers have shown that these macroscopic particles, often referred to as marine snow, are enriched in microbial biomass, nutrients, and trace metals and are involved in biogeochemical transformation of particulate organic carbon in the pelagic environment (
28,
189). Although the importance of microbial communities associated with these macroscopic particles has not been thoroughly investigated, methanogenesis (
121), nitrogen fixation (
191), and sulfide production (
228) have been detected in these particles, indicating microbial activity. Moreover, microbial production of methane or sulfide as well as nitrogen fixation only occurs under anoxic conditions; therefore, the data indicate that anaerobic metabolism is being performed in an otherwise oxygenated environment. Also, these aggregates have been examined with oxygen microelectrodes, and steep redox gradients were found in these biofilms, providing additional evidence of anaerobic metabolism (
191). In a study by Rath et al., the phylogenetic diversity of the bacterial community associated with marine snow was assessed by amplifying and classifying small-subunit ribosomal DNA (rDNA) fragments from nucleic acids extracted from samples of marine snow collected in the northern Adriatic Sea (
208). These experiments showed that bacterial colonization of marine snow can result in diverse and complex assemblages, with specific phyla being associated with the particles. Also, the nature of the associated phylogenetic groups was found to be similar to that of other assemblages found in marine sediments and terrestrial soils.
Biofilm Structure
The application of confocal scanning laser microscopes (CSLM) to biofilm research radically altered our perception of biofilm structure and function (
140). Before the use of CSLM, electron microscopy was the method of choice to examine microbial biofilms under high resolution. Unfortunately, sample preparation for electron microscopy results in dehydrated samples. Consequently, this approach provided a deceivingly simplistic view of biofilms, since the biofilm collapsed when water was removed. On the other hand, CSLM, which allows the visualization of fully hydrated samples, has revealed the elaborate three-dimensional structure of biofilms (
47,
56,
57). CSLM has been used very effectively to monitor biofilm development in flow cells. Flow cells are small continuous-flow systems with a viewing port that allows direct observation of the biofilm without disrupting the community. These systems are often once-flow, meaning that fresh medium enters the system, passes through the cell, and is collected as waste—the medium is not recycled through the flow cell. A number of descriptions of flow cell and related techniques have been reported (
64a).
Interestingly, biofilms formed from single species in vitro and those produced in nature by mixed species consortia exhibit similar overall structural features (
47,
52,
264). Most biofilms have been found to exhibit some level of heterogeneity in that patches of cell aggregates, not monolayers, are interspersed throughout an exopolysaccharide matrix that varies in density, creating open areas where water channels are formed. An example of a mature single-species biofilm of
Vibrio cholerae is shown in Fig.
2.
The microcolonies that constitute the biofilm can be composed of single-species populations or multimember communities of bacteria, depending on the environmental parameters under which they are formed. Numerous conditions, such as surface and interface properties, nutrient availability, the composition of the microbial community, and hydrodynamics, can affect biofilm structure (
240). For example, under high shear stresses, such as on the surface of teeth during chewing, the biofilm (dental plaque) is typically stratified and compacted (
15,
274). Biofilms have also been examined under various hydrodynamic conditions such as laminar and turbulent flows, and it was shown that biofilm structures are altered in response to flow conditions (
241). Biofilms grown under laminar flow were found to be patchy and consisted of rough round cell aggregates separated by interstitial voids. Biofilms grown in the turbulent flow cells were also patchy, but elongated “streamers” that oscillated in the bulk fluid were observed. Moreover, by observing biofilm development under continuous flow, this group was able to evaluate the effect of perturbations on established biofilms. They showed that the biofilm was polymorphic and structurally adapted to changes in nutrient availability.
In biofilms formed in upflow anaerobic sludge bed reactors (continuous-flow systems comprising multiple microbial species, where the flow occurs from bottom to top of the vessel), aggregates consisting of complex bacterial communities (referred to as flocs or sludge granules) predominate (
151). This is primarily due to the fact that the degradation of organic materials to methane and carbon dioxide is a community-level process that is driven by the close contact of multiple guilds interacting in a food web (
159,
224). In addition, through aggregation, the bacteria are advantageously positioned in these reactors. Since there is no surface area for attachment in this type of reactor except for the walls, the formation of granular sludge is a mechanism by which the biofilm communities settle to the bottom of the reactor, which prevents their being washed out of the system. Furthermore, through granular sedimentation, the biomass is more readily exposed to the continuous supply of nutrients being pumped into the bottom of the reactor. Hence, biofilm structure is affected by both the microbial biology and environmental parameters. Structural organization is clearly a hallmark of biofilm communities that differentiates this mode of growth from conventional suspension cultures.
The interstitial voids or channels are also an integral part of the biofilm structure. Using particle-tracking techniques, researchers have been able to demonstrate water flow through these channels (
242). Therefore, the channels are, in essence, the lifeline of the system, since they provide a means of circulating nutrients as well as exchanging metabolic products with the bulk fluid layer (
45). For instance, in situ measurements of dissolved oxygen using microelectrodes revealed that oxygen is available in the biofilm as far down as the substrata, indicating that the channels are transporting the oxygenated bulk fluid throughout the biofilm to the surface (
143). Also, in situ measurements of toluene degradation in a multispecies biofilm indicated that toluene was available to cells deep within the biofilm, indicating transport through channels (
168). Presumably the channels are a vital part of the biofilm structure and function, and therefore there are likely to be mechanisms for the formation as well as the maintenance of these structures. This is clearly a key area for future investigations.
Structure and Function Studies
The identification and quantification of members of particular microbial communities, as well as a clear understanding of the functional relationship between members, are required before we can fully appreciate and possibly manage the complex processes that these communities perform. Recent technological advances have aided in attaining this goal. The remarkable breakthrough in rRNA-based phylogenetic analysis (
276) has provided a means of developing tools with which to investigate microbial communities. The development of fluorescently labeled rRNA-targeted oligonucleotides, a variety of microsensors, real-time image analysis, and confocal microscopy has provided researchers with noninvasive means to monitor populations in situ (
5,
24,
25,
260). In addition, one of the key advances in the study of microbial communities has been the development of various tools for cultivating communities, such as chemostats, continuous-flow slide cultures, microstats, and colonization tracks (
22). These techniques have been used to identify and quantify specific populations within a variety of complex microbial mixtures.
As discussed above, the anaerobic degradation of complex organic material to methane and carbon dioxide is a community-level process carried out by multiple microbial populations interacting in a food web (
159). This process is one of the most complex interactions between bacterial populations known to exist. Although anaerobic food chains have been studied extensively, our understanding of community-level processes in anaerobic food webs is still limited. Due to the important role of microbes in wastewater treatment, an extensive amount of research and method development has been performed in order to increase our understanding of the processes involved in the degradation of organic materials. Here we will describe some of the research in this area in order to illustrate one of the primary goals of biofilm research, that is, connecting structure and function.
It has been discovered that surface-attached biofilms as well as sludge granules readily form in anaerobic reactors (
151,
281). Moreover, the development of these biofilm communities results in more efficient processing of contaminants in wastewater. rRNA probes have been used to identify and quantify phylogenetically defined populations of organisms in sludge granules (
120,
206,
207,
256,
257). In a recent study by Raskin et al. (
207), changes in the composition of two metabolically competitive populations (methanogens and sulfate-reducing bacteria [SRB]) were examined in a biofilm reactor in response to the availability of sulfate. Both of these metabolic types catalyze final stages in the anaerobic mineralization of organic matter, and both depend on other microorganisms (fermentative bacteria) to convert complex organic matter to simpler compounds, such as hydrogen and acetate, which in turn can serve as their substrates. Hence, the methanogens and SRB compete for the same substrates. The generally accepted paradigm of SRB and methanogens in their natural habitats is that of mutual exclusion. Typically, environments that are rich in sulfate select for SRB, and environments that are sulfate depleted select for methanogens. However, it is becoming clear that the interactions of these two groups are more complex than previously envisioned. The coexistence of methanogens and SRB has been observed when sulfate is available (
112,
178), and large populations of SRB have been found in sulfate-depleted environments (
247).
In order to determine the composition of the SRB and methanogen community under different conditions, a portion of the attached biofilm was removed from the reactor at a specific time point. Experiments were carried out in biofilm reactors, and the populations of bacteria were monitored by quantitation of specific 16S rRNA organisms compared to total 16S rRNA (
5). The nucleic acids were extracted from the samples and probed for specific populations (e.g., universal,
Archaea, Bacteria, various methanogens, and various SRB). In addition, various metabolic activities, including sulfide and methane production, were assayed. Using this approach, it was found that in the absence of sulfate, certain types of SRB were still present at high levels in the reactors. The authors state that this ability to persist without sulfate may be explained by the ability of certain SRB to function as fermenters or as proton-reducing acetogens, and the potential for SRB to have these metabolic capabilities has been reported previously (
91,
245). Upon addition of sulfate to the reactor, the levels of sulfate reduction were found to increase with a concomitant increase in the SRB population. Also, methane production and the methanogenic population decreased immediately following the addition of sulfate. However, the opposite did not occur. When the sulfidogenic reactor was reversed to sulfate-free medium, it took a long time (50 days) for any significant amount of methane to be produced, indicating that under these condition the SRB population can more readily reestablish itself in the environment than the methanogenic bacteria. These experiments illustrate how an rRNA-based approach can be combined with functional assays in order to monitor population dynamics in conjunction with metabolic changes in a biofilm community.
Other researchers have used identification of cells hybridized in situ with fluorescent rRNA-targeted probes to study the diversity and spatial distribution of populations within the community. In a recent study by Amann and colleagues, the microdiversity in a municipal activated-sludge sample was assessed using fluorescent rRNA-targeted probes (
4). The primary reason for this study was that high microdiversity (i.e., clusters of closely related yet distinct 16S rRNA sequences with similarities of between 95 and 99%) is commonly reported in complex environmental samples. It has become a concern that this technique may be providing misleading results. Cultivation-independent comparative rRNA analysis relies on PCR amplification of rRNA from nucleic acids extracted from environmental samples (
5), and therefore there are several factors at each step of the process that can give rise to artificial sequence diversity, or lack there of, in rRNA gene libraries (
5,
196,
203,
210). By using in situ probes and CSLM, these researchers investigated the potential for high microdiversity in a natural microbial community without the selective bias of cultivation, extraction, or amplification. Evidence for high diversity was shown, indicating that high diversity within a relatively narrow phylogenetic group (in this case beta-1
Proteobacteria) is present in this environment.
Studies combining fluorescent
in situ hybridization (FISH) with microelectrode analysis for determining pH, oxygen, or sulfide profiles have been performed in order to evaluate the distribution of different populations in relationship to chemical profiles (
92a,
93,
205,
225,
226). In a study by Harmsen et al. (
92a), FISH was used to localize organisms belonging to the bacterial domain (two syntrophic propionate-oxidizing bacteria) and various types of methanogens in sludge granules (Fig.
3). It was shown that the outer layers of the granules were populated with a variety of bacterial colonies most likely involved in hydrolyzing complex organic matter, while the interior of the granule contained methanogenic microcolonies. Moreover, the syntrophic strains, which require low hydrogen partial pressures in order to oxidize propionate, were found to be tightly associated with the methanogens in microcolonies. Consequently, these experiments provided convincing evidence of a layered microbial architecture in sludge granules where the bacteria on the surface of the granule hydrolyze complex organic materials, thereby providing the anaerobic bacteria in the interior of the biofilm with an energy source.
In a recent comprehensive study by Schramn et al., multiple methods were used to investigate the occurrence of anaerobic processes, such as denitrification and sulfate reduction, in well-aerated activated-sludge samples (
226). In this set of experiments, microsensors were used to measure oxygen, nitrite, nitrate, and sulfide concentrations, and
15NO
3− and
35SO
42−were used to measure denitrification (
177) and sulfate reduction (
76), respectively. In addition, the three-dimensional structure of the flocs was examined with CSLM, and the SRB population was monitored by FISH and by PCR with primers specific for the dissimilatory sulfate reductase gene (
258). Also, a newly designed flow system including microelectrodes (
199) was used in the experiments. It was discovered that anoxic microniches and denitrification can occur in well-aerated activated sludge, but this potential appeared to be the exception rather than the rule. In four of the six samples examined, no anoxic zones developed during aeration of the granules, indicating that the respiratory capacity of the microbial community is simply not sufficient to create an anoxic environment when they are well aerated. In addition, sulfate reduction was not detected in any of the flocs, but SRB were found to be present, although in very small numbers. These findings are significant because the development of anaerobic niches in aerated sludge granules is detrimental to the degradation of contaminants. Anoxic habitats can support the persistence of SRB, resulting in the production of hydrogen sulfide and subsequent problems in the treatment process.
In addition to the techniques mentioned above, by using hybridization with fluorescent probes or by staining cells with acridine orange (AO), researchers are able to evaluate growth rate by determining cellular ribosome (rRNA) content. The direct correlation between ribosome content and growth rate is based on early observations in microbial physiology (
223). Therefore, by using FISH in combination with digital microscopy, researchers have been able to quantify the cellular content of rRNA and thereby estimate the growth rate of cells in a biofilm. Using this technique, it was discovered that cells (the SRB PT2) in a young biofilm (initial colonization in the bioreactor) had a doubling time of 33 h, and cells in long-established (presumably steady-state) biofilms had doubling times of at least 70 h (
200). Interestingly, a subset of cells observed in the mature biofilm were significantly more fluorescent (corresponding to a doubling time of 33 h) than any of the other surrounding cells. These data indicate that populations of cells within the biofilm may have different growth rates, which may reflect the heterogeneity of microniches within a biofilm in that some cells may be in a better position to obtain nutrients. One limitation of the rRNA-based quantitation technique is that a standard curve is required in order to quantify ribosome content, and therefore cells must be isolated before their examination. By using AO staining to determine the RNA-DNA ratio, the need for isolation is eliminated. When AO complexes with nucleic acids, it will emit red fluorescence when it is attached to single-stranded templates and green fluorescence if the nucleic acids are double stranded (
211). Therefore, AO can be used to differentially stain RNA and DNA in cells. Moreover, the amount of light from AO-stained cells can be quantified by using image analysis software (
167). The measurements included determination of cell volume, frequency of dividing cells, and simultaneous quantitative measurement of RNA and DNA by AO staining. Using this combinatorial method, it was shown that
Pseudomonas putida cells residing in a biofilm exhibited a constant growth rate that was independent of the dilution rate of the chemostat and, hence, independent of nutrient availability. These data indicate that other factors (e.g., oxygen availability or physical constraints) may be limiting the growth of bacteria in the biofilm.
Researchers have also combined FISH with specific enzyme activity probes (e.g., phosphatase activity) so as to assign functions to certain phylogenetic groups (
125). In this study it was discovered that strains that cluster with the cytophaga-flavobacteria group are involved in the release of inorganic phosphate during wastewater treatment. Previously it had been reported that this group of microorganisms was not involved in the removal of phosphate (
14), but the combined use of FISH with the phosphatase localization probe method clearly illustrated the colocalization of phosphatase activity and the cytophaga-flavobacteria probe. Moreover, a significant amount (35 to 45%) of the total phosphatase activity was detected associated with the cytophaga and flavobacteria, indicating that this group not only has activity but is responsible for a significant portion of the total phosphatase activity in the sludge. In addition, the authors point out that the synthesis of other precipitating, fluorogenic substrates for various enzymatic activities should be possible, and therefore this approach should prove useful in addressing a variety of biological questions.
Plant-Associated Biofilms
Soils constitute a heterogeneous environment with numerous fluctuating parameters that can affect microbial growth and survival (
193). Like many natural environments, soil is nutrient poor (
272). Soil organic matter varies in concentration from 0.8 to 2.0%, with the bulk of the carbon in recalcitrant forms, such as humic acids. Therefore, bacteria indigenous to soil must constantly contend with nutrient deprivation (
252). The rhizosphere (the root surface and the region immediately surrounding a root, typically ∼2 mm) constitutes an ecological niche in soil where nutrients are more readily available, and certain bacteria have developed mechanisms to take advantage of this niche. Rhizodeposition (the release of organic material from the roots as they grow through the soil) enhances microbial growth and drives the structuring of the microbial communities in the rhizosphere (
27). Rhizodeposition consists of a variety of compounds, including (i) exudates, such as amino acids, simple sugars, and organic acids that are passively released from the roots; (ii) actively secreted compounds such as carbohydrates and enzymes; (iii) mucilage (sloughed-off cells and cell lysates); and (iv) gases, such as carbon dioxide and ethylene (
267). This deposition accounts for a significant amount of the plant's photosynthate, estimated to be ∼20% of the carbon allocated to the root system. Thus, numerous bacteria are attracted to the rhizosphere and compete in order to colonize this oasis in soil (
266). Moreover, the interactions between the plant and the surrounding microorganisms select for the establishment of only certain microbial populations (rhizobacteria). Therefore, structured microbial communities attached to the roots and the surrounding soil particles could be viewed as a biofilm community. This suggests that a highly evolved association may exist between the nutritionally rich photosynthesizing plants and the nutrient-deprived bacteria residing in soil. An example of a biofilm on a plant root is shown in Fig.
4. There are many indications of biofilm communities in the rhizosphere. First of all, it is evident that bacteria attach to roots, and various mechanisms have been described for attachment that involve a variety of cell components, such as outer membrane proteins, wall polysaccharides (capsules), lipopolysaccharide (LPS), and cell surface agglutinin (
164). Second, exopolysaccharide (EPS) is produced by bacteria in the rhizosphere (
7). This not only provides many advantages to bacterial cells (as described below), it also enhances soil aggregation, which in turn improves water stability, which is critical to the survival of the plant. Hence, there is a strong selective advantage for the production of EPS in the rhizosphere. Third, microcolonies have been observed in the root system (
231) along with an increase in the frequency of conjugation between certain bacteria (
Pseudomonas species) in the areas immediately adjacent to the roots, indicating cell-cell contact (
251).
Another part of the plant root system where microbial biofilms are formed is on the surface of symbiotic fungi associated with roots. Arbuscular mycorrhizal fungi form an association with plants in which the fungi colonize the cells of roots obtaining carbon from the plant, and in turn, the fungi develop a network of external hyphae which absorb and transfer phosphates and other minerals from the soil to the root (
94). It has been estimated that as much as 80% of the extant species of plants form this type of symbiosis (
92), and it follows that bacteria have evolved to take advantage of this common ecological niche. Bacteria described as good root colonizers, i.e.,
Pseudomonas fluorescens (
31), have been shown to form biofilms on mycorrhizal fungi (reference
194 and references therein). Although the significance of microbial attachment to the fungi is not known, it is likely that this is a positioning mechanism that allows the bacteria to more readily obtain nutrients and propagate. Another area of active biofilm research in the plant world is the study of biofilms on leaves of plants (known as the phyllosphere) (
169,
170). Early studies have shown that phyllosphere biofilms consist of diverse morphotypes of bacteria embedded in an exopolymer present on a variety of leaf surfaces. However, the nature of the microbial community and the role they play in this unique environment have not yet been determined.