Biogeochemical activity of microbial biofilms in the water column overlying uranium mine tailings.

AIMS: To describe microbial diversity, biofilm composition and biogeochemical potential within biofilms in the water overlying uranium tailings characterized by high pH, high metal concentration and low permeability. METHODS AND RESULTS: To estimate microbial diversity in biofilms formed in water columns overlying uranium mine tailings, culture-dependent and culture-independent methods were employed. High-throughput sequencing revealed the presence of 11 phyla; however, the majority of the sequences were affiliated with four major lineages (Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) as confirmed by culture-based methods. Dominant phylotypes were closely related to methylotrophs (Methylobacterium) and bacterial groups able to utilize complex hydrocarbons (Aquabacterium and Dechloromonas). Microbial diversity in biofilms from the 13 m depth was significantly different that in biofilms from 1 to 41 m (P < 0·05). Phylotypes closely related to iron-reducing bacteria were identified at each depth; whereas sulphate-, thio-sulphate-, sulphite- and sulphur-reducing bacteria, at low abundance, were only detected at lower depths. Confocal scanning laser microscopy (CSLM) was used to investigate polymer quantity and composition of the biofilm components, and principal component analysis of the CLSM data revealed that the relative abundance of α-L-fucose and N-acetyl-glucosamine/lipopolysaccharide residues separated tailings-water interface biofilms from those from other depths. Reduced (ferrous) iron was detected within all the biofilm samples examined by scanning X-ray transmission microscopy. CONCLUSIONS: Microbial communities within the water column covering a highly alkaline uranium tailings body form biofilms with microenvironments where iron reduction takes place. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the biogeochemical potential of microbial biofilm communities in the water column covering an alkaline uranium tailings body; specifically, the nature of the bacterial groups detected (Aquabacterium, Dechloromonas) and the presence of reduced iron suggest that complex hydrocarbons are available for bacterial growth and geochemical change, such as iron reduction, can occur even though the system bulk phase is predominantly oxic.

Microbial communities in low permeability, high pH uranium mine tailings: characterization and potential effects.

AIMS: To describe the diversity and metabolic potential of microbial communities in uranium mine tailings characterized by high pH, high metal concentration and low permeability. METHODS AND RESULTS: To assess microbial diversity and their potential to influence the geochemistry of uranium mine tailings using aerobic and anaerobic culture-based methods, in conjunction with next generation sequencing and clone library sequencing targeting two universal bacterial markers (the 16S rRNA and cpn60 genes). Growth assays revealed that 69% of the 59 distinct culturable isolates evaluated were multiple-metal resistant, with 15% exhibiting dual-metal hypertolerance. There was a moderately positive correlation coefficient (R = 0·43, P < 0·05) between multiple-metal resistance of the isolates and their enzyme expression profile. Of the isolates tested, 17 reduced amorphous iron, 22 reduced molybdate and seven oxidized arsenite. Based on next generation sequencing, tailings depth was shown to influence bacterial community composition, with the difference in the microbial diversity of the upper (0-20 m) and middle (20-40 m) tailings zones being highly significant (P < 0·01) from the lower zone (40-60 m) and the difference in diversity of the upper and middle tailings zone being significant (P < 0·05). Phylotypes closely related to well-known sulfate-reducing and iron-reducing bacteria were identified with low abundance, yet relatively high diversity. CONCLUSIONS: The presence of a population of metabolically-diverse, metal-resistant micro-organisms within the tailings environment, along with their demonstrated capacity for transforming metal elements, suggests that these organisms have the potential to influence the long-term geochemistry of the tailings. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first investigation of the diversity and functional potential of micro-organisms present in low permeability, high pH uranium mine tailings.

Occurrence of contaminants of emerging concern in the Eerste River, South Africa: Towards the optimisation of an urban water profiling approach for public- and ecological health risk characterisation.

The study evaluated the presence and fate of various contaminants of emerging concern (CECs) from a South African wastewater treatment works (WWTW) and surface waters located around an urban setting. A total of 45 CECs were quantified from nine sampling locations over an 11-month period. Daily loads (g/day) of the target analytes in the WWTW showed persistence of some CECs, along with population-normalised daily loads (mg/day/1000inh) of pharmaceuticals and drugs of abuse (DOA) that were estimated for the first time in the study area. Multiple chemical markers were recorded in river water located upstream of the WWTW discharge throughout the study period, suggesting a high degree of diffuse pollution from urban communities in the study area that are not connected to sewage networks or where sanitation services are limited. The potential of using defined surface water locations to perform community-wide substance use profiling for non-sewered communities was also explored. Environmental risk characterisation for the WWTW effluent and surface waters throughout the study period provided multiple risk quotients (RQ) for the target list of CECs spanning over various sentinel trophic levels. High risk profiles (RQ > 1.0) with a frequency of exceedance (FoE) larger than 75 % were recorded for several CECs in both WWTW effluent and surface water locations that suggest potential long-term ecological health risk impacts of pollution hotspot areas in the river catchment situated around the urban area. We present challenges in surface water quality within the study area that is relatable, or may even present more challenging, in other low- or middle-income country (LMICs) settings. The study also highlighted some challenges and limitations associated with the much-needed application of wastewater-based epidemiology (WBE) intervention in non-sewered communities that can inform on public health and communal substance use profiles of the entire urban setting.

Wastewater-based epidemiology in hazard forecasting and early-warning systems for global health risks.

With the advent of the SARS-CoV-2 pandemic, Wastewater-Based Epidemiology (WBE) has been applied to track community infection in cities worldwide and has proven succesful as an early warning system for identification of hotspots and changingprevalence of infections (both symptomatic and asymptomatic) at a city or sub-city level. Wastewater is only one of environmental compartments that requires consideration. In this manuscript, we have critically evaluated the knowledge-base and preparedness for building early warning systems in a rapidly urbanising world, with particular attention to Africa, which experiences rapid population growth and urbanisation. We have proposed a Digital Urban Environment Fingerprinting Platform (DUEF) - a new approach in hazard forecasting and early-warning systems for global health risks and an extension to the existing concept of smart cities. The urban environment (especially wastewater) contains a complex mixture of substances including toxic chemicals, infectious biological agents and human excretion products. DUEF assumes that these specific endo- and exogenous residues, anonymously pooled by communities' wastewater, are indicative of community-wide exposure and the resulting effects. DUEF postulates that the measurement of the substances continuously and anonymously pooled by the receiving environment (sewage, surface water, soils and air), can provide near real-time dynamic information about the quantity and type of physical, biological or chemical stressors to which the surveyed systems are exposed, and can create a risk profile on the potential effects of these exposures. Successful development and utilisation of a DUEF globally requires a tiered approach including: Stage I: network building, capacity building, stakeholder engagement as well as a conceptual model, followed by Stage II: DUEF development, Stage III: implementation, and Stage IV: management and utilization. We have identified four key pillars required for the establishment of a DUEF framework: (1) Environmental fingerprints, (2) Socioeconomic fingerprints, (3) Statistics and modelling and (4) Information systems. This manuscript critically evaluates the current knowledge base within each pillar and provides recommendations for further developments with an aim of laying grounds for successful development of global DUEF platforms.

Biofilm form and function: carbon availability affects biofilm architecture, metabolic activity and planktonic cell yield.

AIMS: To investigate carbon transformation by biofilms and changes in biofilm architecture, metabolic activity and planktonic cell yield in response to fluctuating carbon availability. METHODS AND RESULTS: Pseudomonas sp. biofilms were cultured under alternating carbon-replete and carbon-limited conditions. A shift to medium without added carbon led to a 90% decrease in biofilm respiration rate and a 40% reduction in planktonic cell yield within 1 h. Attached cell division and progeny release were shown to contribute to planktonic cell numbers during carbon limitation. Development of a significantly enlarged biofilm surface area during carbon limitation facilitated a rapid increase in whole-biofilm metabolic activity, cell yield and biomass upon the re-introduction of carbon after 8 days of limitation. The cumulative number of planktonic cells (>10(10) CFU) released from the biofilm during the cultivation period contained only 1·0% of the total carbon available to the biofilm, with 6·5% of the carbon retained in the biofilm and 54% mineralized to CO(2) . CONCLUSIONS: Biofilm-derived planktonic cell yield is a proliferation mechanism. The rapid response of biofilms to environmental perturbations facilitates the optimal utilization of resources to promote both proliferation and survival. Biofilms function as efficient catalysts for environmental carbon transformation and mineralization. SIGNIFICANCE AND IMPACT OF THE STUDY: A greater understanding of the relationship between biofilm form and function can inform strategies intended to control and/or promote biofilm formation.

Qualitative and quantitative detection of SARS-CoV-2 RNA in untreated wastewater in Western Cape Province, South Africa.

Recent studies have shown that the detection of SARS-CoV-2 genetic material in wastewater may provide the basis for a surveillance system to track the environmental dissemination of this virus in communities. An effective wastewater-based epidemiology (WBE) system may prove critical in South Africa (SA), where health systems infrastructure, testing capacity, personal protective equipment and human resource capacity are constrained. In this proof-of-concept study, we investigated the potential of SARS-CoV-2 RNA surveillance in untreated wastewater as the basis for a system to monitor COVID-19 prevalence in the population, an early warning system for increased transmission, and a monitoring system to assess the effectiveness of interventions. The laboratory confirmed the presence (qualitative analysis) and determined the RNA copy number of SARS-CoV-2 viral RNA by reverse transcription polymerase chain reaction (quantitative) analysis from 24-hour composite samples collected on 18 June 2020 from five wastewater treatment plants in Western Cape Province, SA. The study has shown that a WBE system for monitoring the status and trends of COVID-19 mass infection in SA is viable, and its development and implementation may facilitate the rapid identification of hotspots for evidence-informed interventions.

Microbial response to environmental gradients in a ceramic-based diffusion system.

A solid, porous matrix was used to establish steady-state concentration profiles upon which microbial responses to concentration gradients of nutrients or antimicrobial agents could be quantified. This technique relies on the development of spatially defined concentration gradients across a ceramic plate resulting from the diffusion of solutes through the porous ceramic matrix. A two-dimensional, finite-element numerical transport model was used to predict the establishment of concentration profiles, after which concentration profiles of conservative tracers were quantified fluorometrically and chemically at the solid-liquid interface to verify the simulated profiles. Microbial growth responses to nutrient, hypochloride, and antimicrobial concentration gradients were then quantified using epifluorescent or scanning confocal laser microscopy. The observed microbial response verified the establishment and maintenance of stable concentration gradients along the solid-liquid interface. These results indicate the ceramic diffusion system has potential for the isolation of heterogeneous microbial communities as well as for testing the efficacy of antimicrobial agents. In addition, the durability of the solid matrix allowed long-term investigations, making this approach preferable to conventional gel-stabilized systems that are impeded by erosion as well as expansion or shrinkage of the gel.

Wastewater-based epidemiology and enantiomeric profiling for drugs of abuse in South African wastewaters.

The current study is aimed to introduce a wastewater-based epidemiology (WBE) approach for the first time on the African continent where substance abuse data is limited. The study included the quantification of several drugs of abuse (DOA) in raw wastewater samples. Quantification of urinary metabolites as drug target residues (DTR), as well as enantiomeric profiling of chiral DOA was performed to distinguish between consumption and direct disposal into sewage. Monitoring campaigns were undertaken at two South African wastewater treatment works (WWTWs) located within two provinces of the country. The presence of non-racemic 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine, as well as the metabolite of cocaine, benzoylecgonine (BEG), confirmed their consumption within the areas investigated. Enantiomeric profiling further pointed to the abuse of methamphetamine as the primary DOA with use estimates calculated between 181.9 and 1184.8mg·day-1·1000inhabitants-1. Population-normalised mass loads for MDMA and cocaine confirmed their status as secondary DOA within the study sites. Use estimates for the new psychoactive substance (NPS) mephedrone were performed for one WWTW. The minor metabolite of heroin, O-6-monoacetylmorphine (O-6-MAM), was also detected at one WWTW and served as a qualitative indicator for heroin abuse within the area. These findings provide a novel comparison of the WBE approach in a developing-country with other global studies, with the aim to strengthen this approach as a tool to inform drug prevention strategies in countries where substance abuse data is limited due to financial constraints and lack of government structures to facilitate conventional monitoring.

Distribution and Biogeochemical Importance of Bacterial Populations in a Thick Clay-Rich Aquitard System.

To investigate the distribution of microbial biomass, populations and activities within a clay-rich, low hydraulic conductivity (10-11 to 10-12 m s-1) aquitard complex, cores were aseptically obtained from a series of overlying clayey deposits; a fractured till, unfractured till (20-30 ka BP), a disturbed interfacial zone (20-30 ka BP), and a Cretaceous clay aquitard (71-72 Ma BP). The results of confocal microscopy studies, culture methods, molecular approaches, and extractive fatty acid analyses all indicated low bacterial numbers that were non-homogeneously distributed within the sediments. Various primers for catabolic genes were used to amplify extracted DNA. Results indicated the presence of eubacterial 23S rDNA, and the narH gene for nitrate reductase and ribulose-1,5-biphosphate carboxylase (RuBP carboxylase). Although there was no evidence of limitation by electron acceptors or donors, sulfate-reducing bacteria were not detected below the fractured till zone, using PCR, enrichment, or culture techniques. Denaturing gradient gel electrophoresis (DGGE) analyses indicated differences in community composition and abundance between the various geologic units. Results of FAME analyses of sediments yielded detectable extractable fatty acids throughout the aquitard complex. Bacterial activities were demonstrated by measuring mineralization of (14C) glucose. Porewater chemistry and stable isotope data were in keeping with an environment in which extremely slow growing, low populations of bacteria exert little impact. The observations also support the contention that in low permeability sediments bacteria may survive for geologic time periods.

Efficacy enhancement of trisodium phosphate against spoilage and pathogenic bacteria in model biofilms and on adipose tissue.

A two-step approach for enhancing the efficacy of trisodium phosphate (TSP) was evaluated using meat spoilage and pathogenic bacteria in flow cell biofilms and adipose tissue model systems. The process was based on the plasmolysis of attached bacteria (biofilms) with a hyperosmotic solution (1.5 M NaCl) and the subsequent deplasmolysis of cells with a low-osmotic-strength solution containing different concentrations of TSP (0.1, 0.25, 0.5, 0.625, and 1.0 % [wt/vol]). Escherichia coli, Salmonella Enteritidis, Pseudomonas sp., Listeria monocytogenes, and Brochothrix thermosphacta strains were cultivated for 24 h as pure culture biofilms in glass flow cells with complex media and were then treated with either 0.1, 0.25, 0.5, 0.625, and 1.0% TSP, or the same TSP concentrations delivered in conjunction with plasmolysis-deplasmolysis (PDP). Confocal scanning laser microscopy, a commercial fluorescent viability probe, and image analysis were then used to quantify the relative abundances of living and dead cells remaining after the different treatment regimes. With the exception of L. monocytogenes (which was resistant to TSP concentrations of up to 5%), the PDP process increased the sensitivity of the test strains to TSP. However, when similar experiments were conducted with pork adipose tissue, it became evident that higher TSP concentrations were necessary to produce significant decreases in the number of viable cells and that the PDP process generally failed to enhance TSP efficacy. An exception was L. monocytogenes, which exhibited an increase in sensitivity to TSP when inoculated tissue was pretreated with 1.5 M NaCl. It is thought that factors contributing to the failure of the PDP process to enhance the activity of TSP in meat systems involves the mode of TSP antimicrobial activity, alkaline pH stress, and the chemically complex, buffered nature of meats. It remains to be determined whether the PDP process is suitable for use with other food grade antimicrobial agents or can be used in nonfood biofilm control applications.

Microbial distribution and diversity in saturated, high pH, uranium mine tailings, Saskatchewan, Canada.

Microbiological analyses were conducted on core samples collected along a vertical profile (0-66 m below surface) from the tailings management facility (TMF) at the Rabbit Lake uranium mine in northern Saskatchewan, Canada. Bacterial numbers in the core materials were similar to surrounding soils and surface waters, regardless of the seemingly unfavorable pH (mean=9.9) and temperature (approximately 0 degrees C) in the TMF. The greatest number of viable cells (105 CFU/g) was detected at the interface between the tailings and overlying standing water, below which cell counts decreased rapidly with depth. Whole-community metabolic profiles for samples from the different depths grouped into 3 clusters; however, these groups could not be positively correlated with sampling depth, temperature, redox potential, pH, or ore-mill feed. Flow-cell studies demonstrated microbial communities in the tailings surface water could develop biofilms and maintain cell activity at both pH 10 and 7, and altering the pH between these 2 values had little effect on biofilm viability. These results demonstrate the resilience and adaptive nature of naturally occurring microbial communities and signify a potential role of microbial activity in the long-term geochemical evolution of the TMF.

Adhesion of Lactobacillus plantarum 423 and Lactobacillus salivarius 241 to the intestinal tract of piglets, as recorded with fluorescent in situ hybridization (FISH), and production of plantaricin 423 by cells colonized to the ileum.

AIMS: To determine which intestinal section of pre and postweaned piglets are colonized by Lactobacillus plantarum 423 and Lactobacillus salivarius 241, and follow production of plantaricin 423 in a gastro-intestinal model. METHODS AND RESULTS: Lactobacillus plantarum 423 and Lact. salivarius 241, single or in combination, were administered to 1-, 14- and 28-day-old (postweaned) piglets. According to results obtained by fluorescent in situ hybridization (FISH), Lact. plantarum 423 adhered strongly to the ileum and posterior colon and Lact. salivarius 241 to the duodenum in preweaned piglets. High numbers of strain 241 were recorded in the duodenum and posterior colon of postweaned piglets, whereas strain 423 remained localized to the ileum. Lowering in Enterococcus faecalis cell numbers were recorded when preweaned piglets were challenged with strain 241. Plantaricin 423 was produced for 96 h in the ileum section of a gastro-intestinal model. CONCLUSIONS: Lactobacillus plantarum 423 and Lact. salivarius 241 adhere to different sections of the intestinal tract, depending on the piglet's age. Ent. faecalis were inhibited in vivo, probably by plantaricin 423. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescent in situ hybridization proved valuable in the detection of probiotic bacteria adhered to the intestine. This is the first report of bacteriocin production in a model simulating the porcine gastro-intestinal tract.

Microbial exopolymers link predator and prey in a model yeast biofilm system.

Protistan grazing on biofilms is potentially an important conduit enabling energy flow between microbial trophic levels. Contrary to the widely held assumption that protistan feeding primarily involves ingestion of biofilm cells, with negative consequences for the biofilm, this study demonstrated preferential grazing on the noncellular biofilm matrix by a ciliate, with selective ingestion of yeast and bacterial cells of planktonic origin over attached and biofilm-derived planktonic cells. Introducing a ciliate to two biofilm-forming Cryptococcus species, as well as two bacterial species in a model biofilm system, fluorescent probes were applied to determine ingestion of cellular and noncellular biofilm fractions. Fluoromicroscopy, as well as photometric quantification, confirmed that protistan grazing enhanced yeast biofilm metabolism, and an increase in biofilm biomass and viability. We propose that the extracellular polymeric matrix of biofilms may act as an interface regulating interaction between predator and prey, while serving as source of nutrients and energy for protists.

Xylose utilization and short-chain fatty acid production by selected components of the intestinal microflora of a rodent pollinator (Aethomys namaquensis).

Namaqua rock mice (Aethomys namaquensis) consume nectar xylose when visiting Protea flowers. Whole-animal metabolism studies suggest that the gastrointestinal microflora plays an important role in xylose metabolism in A. namaquensis. We collected caecal contents under anaerobic conditions, cultured caecal microflora both aerobically and anaerobically, and assessed caecal microbial xylose utilization using a (14)C-xylose incubation assay. All four mice sampled hosted culturable caecal micro-organisms that tested positive for xylose utilization. These were classified by 16S rRNA based taxonomy as: Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Shigella boydii, Arthrobacter sp. and members of the fungal genera Aspergillus and Penicillium. Cultures of these isolates were then analyzed by gas chromatography to determine the types and quantities of short-chain fatty acids produced by xylose fermentation. These results are discussed in the context of other studies of gut microflora in vertebrates.

Response of the microbial community to copper oxychloride in acidic sandy loam soil.

AIMS: Determining the response of different microbial parameters to copper oxychloride in acidic sandy loam soil samples using cultivation-dependent and direct microscopic techniques. METHODS AND RESULTS: Culturable microbial populations were monitored for 245 days in a series of soil microcosms spiked with different copper oxychloride concentrations. Microbial populations responded differently to additional Cu. Protistan numbers and soil metabolic potential decreased. Experiments with more soil samples revealed that metabolic potential was not significantly affected by < or =100 mg kg(-1) additional Cu. However, a negative impact on protista was noted in soil containing only 15 mg kg(-1) EDTA-extractable Cu. The negative impact on protistan numbers was less severe in soils with a higher phosphorous and zinc content. CONCLUSIONS: Bacterial populations responded differently, and protista were most sensitive to elevated Cu levels. Protistan numbers in soil from uncultivated land were higher and seemed to be more sensitive to additional Cu than the numbers of these organisms in soil originating from cultivated land. SIGNIFICANCE AND IMPACT OF THE STUDY: Protistan sensitivity to small increases in Cu levels demonstrates the vulnerability of the soil ecosystem to Cu perturbations, especially when the importance of protista as link in the flow of energy between trophic levels is considered.

Determination of diffusion coefficients in biofilms by confocal laser microscopy.

Microbial exopolymer may hinder the diffusion of nutrients, antibiotics, and other materials to the cell surface. Studies of diffusion in biofilms have been limited to indirect measurements. This study demonstrated the use of fluorescein and size-fractionated fluor-conjugated dextrans in conjunction with scanning confocal laser microscopy to directly monitor and determine diffusion coefficients within biofilms. The monitoring approaches were simple and, when combined with computerized image collection, allowed assembly of a data set suitable for calculation of one-dimensional diffusion coefficients for biofilm regions. With these techniques, it was shown that regional variability in the mobility of the dextrans occurred within mixed-species biofilms. Some regions exhibited rapid diffusion of all test molecules, while adjacent regions were only penetrated by the lower-molecular-weight compounds. The effective diffusion coefficients (D(e)) determined in a mixed-species biofilm were a function of the molecular radius of the probe (i.e., fluorescein, D(e) = 7.7 x 10 cm s; 4,000 molecular weight, D(e) = 3.1 x 10 cm s; and 2,000,000 molecular weight, D(e) = 0.7 x 10 cm s). These results demonstrated that diffusion in the biofilm was hindered relative to diffusion in the bulk solution. The study indicated that in situ monitoring by scanning laser microscopy is a useful approach for determining the mobility of fluorescently labeled molecules in biofilms, allowing image acquisition, appropriate scales of study, both xy and xz monitoring, and calculation of D(e) values.

The role of interactions, sessile growth and nutrient amendments on the degradative efficiency of a microbial consortium.

A degradative microbial consortium consisting of at least nine bacterial and one algal species was isolated from soil with diclofop methyl as the sole carbon source. In continuous flow culture, the presence of the algae increased diclofop methyl degradation and removal by 36%. Batch culture experiments with 14C-labeled diclofop methyl confirmed algal involvement in the mineralization of diclofop methyl as there was no significant difference in the amount of 14CO2 evolved by the bacterial consortium with and without the algal activity when the consortium was cultivated in the dark to inhibit algal growth, while 11% more 14CO2 was produced in the light by the algal-bacterial consortium. Pure cultures isolated from the bacterial consortium could not individually mineralize diclofop methyl as the sole carbon source. However, when supplied with an additional carbon source, two strains could mineralize diclofop methyl. Addition of either the complex growth medium, or a cell-free filtrate from the algal-bacterial consortium to batch systems containing 14C-labeled diclofop methyl resulted in a significant increase in the production of 14CO2 by the bacterial consortium, suggesting co-metabolism of diclofop methyl in the presence of a labile carbon source. Removal of diclofop methyl by the bacterial consortium was increased by 36% when a larger surface to volume ratio was provided by glass beads that allowed extensive biofilm formation. The requirement for exogenous carbon sources and the inability of isolated pure cultures to degrade diclofop methyl indicated that interspecies interactions are necessary for degradation. The positive effect of sessile growth suggested that spatial organization of cells may also be important for degradation.

Adaptation of bacterial communities to environmental transitions from labile to refractory substrates.

The aim of this work was to assess the adaptation of bacterial communities to environmental transitions from labile to refractory substrates. This involved testing the hypothesis that bacteria self-organize and propagate not only as individual cellular systems, but also as functional sets of interacting organisms. A biofilm community was cultivated in a flow-cell irrigated with tryptic soy broth and subjected to a cyclic series of environmental transitions, from labile to refractory substrates, followed by a period of starvation (30 days). The appearance and disappearance of specific colony morphotypes when the emigrants were plated onto tryptic soy agar was used to monitor the restructuring of the community. Confocal laser microscopy of flow cells showed that these transitions decreased the biofilm thickness and coverage. Substrate shifts also changed the architecture of the biofilm communities. Repeated inoculation of flow-cell communities with a composite inoculum increased the number and diversity of emigrants. Their biofilms were thicker and covered a wider area than those of communities that had been inoculated only at the beginning of the experiment. With repeated inoculation, the time required for the community to restructure and stabilize decreased during most transitions. This suggested that organismal recombination acted as a mechanism of adaptation, enhancing the growth of microbial communities exposed to environmental stresses. Changes in the profiles of emigrants during the adaptation of biofilm communities to environmental transitions showed the appearance and disappearance of discrete sets of organisms. This suggested that the biofilm communities responded to environmental stresses as sets of interacting organisms. Enhanced growth of biofilm communities due to repeated environmental cycling suggested that the functionality of cellular positioning accrued from one cycle to the next and was thus heritable, although it was not necessarily genetically encoded.

Impact of nutrient composition on a degradative biofilm community.

A microbial community was cultivated in flow cells with 2,4,6-trichlorobenzoic acid (2,4,6-TCB) as sole carbon and energy source and was examined with scanning confocal laser microscopy and fluorescent molecular probes. The biofilm community which developed under these conditions exhibited a characteristic architecture, including a basal cell layer and conspicuous mounds of bacterial cells and polymer (approximately 20 to 30 (mu)m high and 25 to 40 (mu)m in diameter) occurring at 20- to 200-(mu)m intervals. When biofilms grown on 2,4,6-TCB were shifted to a labile, nonchlorinated carbon source (Trypticase soy broth), the biofilms underwent an architectural change which included the loss of mound structures and the formation of a more homogeneous biofilm. Neutrally charged fluorescent dextrans, which upon hydration become cationic, were observed to bind to mounds, as well as to the basal cell layer, in 14-day biofilms. In contrast, polyanionic dextrans bound only to the basal cell layer, indicating that this material incorporated sites with both positive and negative charge. The results from this study indicate that nutrient composition has a significant impact on both the architecture and the physicochemistry of degradative biofilm communities.