Huddling together to survive: Population density as a survival strategy of non-spore forming bacteria under nutrient starvation and desiccation at solid-air interfaces.

Acclimation and flexible response mechanisms are survival adaptations allowing prokaryotic cells to colonize diverse habitats and maintain viability in nature. Lack of water significantly impacts cellular response, which can be partially compensated for through community interactions and accessing survival means beyond the cell's boundaries. In the present study, higher numbers of cultivable Gram-positive Arthrobacter sp. and Gram-negative Pseudomonas stutzeri cells were found on surfaces when high population density was used after prolonged periods of desiccation and nutrient starvation. Total cell counts during desiccation periods decreased slower than culturable cell counts independently from initial population density. The presence of homogenate, prepared by filtering homogenized cultures through a 0.2 µm filter, extended culturability of Arthrobacter sp. cells, while intact heat-killed cells extended the culturability of Arthrobacter sp. and P. stutzeri. Our results suggest very slow cell membrane breakdown for desiccated bacterial cells at solid-air interfaces over extended time spans, which may serve as reservoirs of nutrients, and may potentially provide trace amounts of water for surviving cells. Higher initial population density and recycling of resources from "zombie"-like cells, may support growth in a similar fashion as access to cell lysates or the contents of heat-killed cells analogous to dead-phase cultures where some cells experience cryptic growth.

Evaluating CO2 emissions from continuous flow and batch growth systems under autotrophic mode: Implications for GHG accounting of biological nutrient removal.

The oxidation of ammonia by autotrophic bacteria is a central part of the nitrogen cycle and a fundamental aspect of biological nutrient removal (BNR) during wastewater treatment. Autotrophic ammonia oxidation produces protons and results in net-CO2 production due to the neutralizing effect of bicarbonate alkalinity. Attention must be paid to the propensity for this produced CO2 to be transferred to the atmosphere where it can act as a greenhouse gas (GHG). In the context of BNR systems, bicarbonate-derived CO2 emissions should be considered distinct from the biogenic CO2 that arises from cellular respiration, though this distinction is not made in current GHG accounting practices. The aim of this study was to evaluate the performance of two experimental systems operated under autotrophic mode and buffered with bicarbonate, to investigate the relationship between ammonia removal and gaseous CO2 emissions. The first system consisted of continuously aerated lab-scale batch reactors, which were effective in demonstrating the important link between ammonia oxidizer activity, pH, and gaseous CO2 production. Depletion of the buffer system always led to a rapid decline in system pH and cessation of CO2 emissions when the pH fell below 7.0. The second system was a tubular continuous-flow biofilm reactor which permitted comparison of ammonia removal and CO2 emission rates. A linear relationship between ammonia removal and CO2 emissions was demonstrated and the quantified CO2 production was relatively close to that which was predicted based on the stoichiometry of nitrification, with this CO2 being detected in the gas phase. It was apparent that this system offered minimal resistance to the mass transfer of CO2 from the liquid to gas, which is an important factor that determines how much of the bicarbonate-derived CO2 may contribute to greenhouse gas emissions in engineered systems such as those used for BNR.

Interaction between CO2-consuming autotrophy and CO2-producing heterotrophy in non-axenic phototrophic biofilms.

As the effects of climate change become increasingly evident, the need for effective CO2 management is clear. Microalgae are well-suited for CO2 sequestration, given their ability to rapidly uptake and fix CO2. They also readily assimilate inorganic nutrients and produce a biomass with inherent commercial value, leading to a paradigm in which CO2-sequestration, enhanced wastewater treatment, and biomass generation could be effectively combined. Natural non-axenic phototrophic cultures comprising both autotrophic and heterotrophic fractions are particularly attractive in this endeavour, given their increased robustness and innate O2-CO2 exchange. In this study, the interplay between CO2-consuming autotrophy and CO2-producing heterotrophy in a non-axenic phototrophic biofilm was examined. When the biofilm was cultivated under autotrophic conditions (i.e. no organic carbon), it grew autotrophically and exhibited CO2 uptake. After amending its growth medium with organic carbon (0.25 g/L glucose and 0.28 g/L sodium acetate), the biofilm rapidly toggled from net-autotrophic to net-heterotrophic growth, reaching a CO2 production rate of 60 µmol/h after 31 hours. When the organic carbon sources were provided at a lower concentration (0.125 g/L glucose and 0.14 g/L sodium acetate), the biofilm exhibited distinct, longitudinally discrete regions of heterotrophic and autotrophic metabolism in the proximal and distal halves of the biofilm respectively, within 4 hours of carbon amendment. Interestingly, this upstream and downstream partitioning of heterotrophic and autotrophic metabolism appeared to be reversible, as the position of these regions began to flip once the direction of medium flow (and hence nutrient availability) was reversed. The insight generated here can inform new and important research questions and contribute to efforts aimed at scaling and industrializing algal growth systems, where the ability to understand, predict, and optimize biofilm growth and activity is critical.

A Novel System for Real-Time, In Situ Monitoring of CO2 Sequestration in Photoautotrophic Biofilms.

Climate change brought about by anthropogenic CO2 emissions has created a critical need for effective CO2 management solutions. Microalgae are well suited to contribute to efforts aimed at addressing this challenge, given their ability to rapidly sequester CO2 coupled with the commercial value of their biomass. Recently, microalgal biofilms have garnered significant attention over the more conventional suspended algal growth systems, since they allow for easier and cheaper biomass harvesting, among other key benefits. However, the path to cost-effectiveness and scaling up is hindered by a need for new tools and methodologies which can help evaluate, and in turn optimize, algal biofilm growth. Presented here is a novel system which facilitates the real-time in situ monitoring of algal biofilm CO2 sequestration. Utilizing a CO2-permeable membrane and a tube-within-a-tube design, the CO2 sequestration monitoring system (CSMS) was able to reliably detect slight changes in algal biofilm CO2 uptake brought about by light-dark cycling, light intensity shifts, and varying amounts of phototrophic biomass. This work presents an approach to advance our understanding of carbon flux in algal biofilms, and a base for potentially useful innovations to optimize, and eventually realize, algae biofilm-based CO2 sequestration.

Species Interaction and Selective Carbon Addition During Antibiotic Exposure Enhances Bacterial Survival.

Biofilms are multifaceted and robust microbiological systems that enable microorganisms to withstand a multitude of environmental stresses and expand their habitat range. We have shown previously that nutritional status alters antibiotic susceptibility in a mixed-species biofilm. To further elucidate the effects of nutrient addition on inter-species dynamics and whole-biofilm susceptibility to high-dose streptomycin exposures, a CO2 Evolution Measurement System was used to monitor the metabolic activity of early steady state pure-culture and mixed-species biofilms containing Pseudomonas aeruginosa and Stenotrophomonas maltophilia, with and without added carbon. Carbon supplementation was needed for biofilm recovery from high-dose streptomycin exposures when P. aeruginosa was either the dominant community member in a mixed-species biofilm (containing predominantly P. aeruginosa and S. maltophilia) or as a pure culture. By contrast, S. maltophilia biofilms could recover from high-dose streptomycin exposures without the need for carbon addition during antibiotic exposure. Metagenomic analysis revealed that even when inocula were dominated by Pseudomonas, the relative abundance of Stenotrophomonas increased upon biofilm development to ultimately become the dominant species post-streptomycin exposure. The combined metabolic and metagenomic results demonstrated the relevance of inter-species influence on survival and that nutritional status has a strong influence on the survival of P. aeruginosa dominated biofilms.

Microbes at Surface-Air Interfaces: The Metabolic Harnessing of Relative Humidity, Surface Hygroscopicity, and Oligotrophy for Resilience.

The human environment is predominantly not aqueous, and microbes are ubiquitous at the surface-air interfaces with which we interact. Yet microbial studies at surface-air interfaces are largely survival-oriented, whilst microbial metabolism has overwhelmingly been investigated from the perspective of liquid saturation. This study explored microbial survival and metabolism under desiccation, particularly the influence of relative humidity (RH), surface hygroscopicity, and nutrient availability on the interchange between these two phenomena. The combination of a hygroscopic matrix (i.e., clay or 4,000 MW polyethylene glycol) and high RH resulted in persistent measurable microbial metabolism during desiccation. In contrast, no microbial metabolism was detected at (a) hygroscopic interfaces at low RH, and (b) less hygroscopic interfaces (i.e., sand and plastic/glass) at high or low RH. Cell survival was conversely inhibited at high RH and promoted at low RH, irrespective of surface hygroscopicity. Based on this demonstration of metabolic persistence and survival inhibition at high RH, it was proposed that biofilm metabolic rates might inversely influence whole-biofilm resilience, with 'resilience' defined in this study as a biofilm's capacity to recover from desiccation. The concept of whole-biofilm resilience being promoted by oligotrophy was supported in desiccation-tolerant Arthrobacter spp. biofilms, but not in desiccation-sensitive Pseudomonas aeruginosa biofilms. The ability of microbes to interact with surfaces to harness water vapor during desiccation was demonstrated, and potentially to harness oligotrophy (the most ubiquitous natural condition facing microbes) for adaptation to desiccation.

USMB-induced synergistic enhancement of aminoglycoside antibiotics in biofilms.

This study evaluated the effect of combining antibiotics with ultrasound and microbubbles (USMB) toward the eradication of biofilms. Pseudomonas aeruginosa PAO1 biofilms were treated with the antibiotics gentamicin sulfate or streptomycin sulfate, or a combination of USMB with the respective antibiotics. Biofilm structure was quantified using confocal laser scanning microscopy with COMSTAT analysis, while activity was measured as whole-biofilm CO2 production in a continuous-flow biofilm model. The combined antibiotic-USMB treatment significantly impacted biofilm biomass, thickness and surface roughness compared to antibiotics alone (p<0.05). USMB exposure caused the formation of craters (5-20µm in diameter) in the biofilms, and when combined with gentamicin, activity was significantly lower, compared to gentamicin, USMB or untreated controls, respectively. Interestingly, the CO2 production rate following combined streptomycin-USMB treatment was higher than after streptomycin alone, but significantly lower than USMB alone and untreated control. These results show strong evidence of a synergistic effect between antibiotics and USMB, although the varied response to different antibiotics emphasize the need to optimize the USMB exposure conditions to maximize this synergism and ultimately transfer this technology into clinical or industrial practice.

Measuring microbial metabolism in atypical environments: Bentonite in used nuclear fuel storage.

Genomics enjoys overwhelming popularity in the study of microbial ecology. However, extreme or atypical environments often limit the use of such well-established tools and consequently demand a novel approach. The bentonite clay matrix proposed for use in Deep Geological Repositories for the long-term storage of used nuclear fuel is one such challenging microbial habitat. Simple, accessible tools were developed for the study of microbial ecology and metabolic processes that occur within this habitat, since the understanding of the microbiota-niche interaction is fundamental to describing microbial impacts on engineered systems such as compacted bentonite barriers. Even when genomic tools are useful for the study of community composition, techniques to describe such microbial impacts and niche interactions should complement these. Tools optimised for assessing localised microbial activity within bentonite included: (a) the qualitative use of the resazurin-resorufin indicator system for redox localisation, (b) the use of a CaCl2 buffer for the localisation of pH, and (c) fluorometry for the localisation of precipitated sulphide. The use of the Carbon Dioxide Evolution Monitoring System was also validated for measuring microbial activity in desiccated and saturated bentonite. Finally, the buffering of highly-basic bentonite at neutral pH improved the success of isolation of microbial populations, but not DNA, from the bentonite matrix. Thus, accessible techniques were optimised for exploring microbial metabolism in the atypical environments of clay matrices and desiccated conditions. These tools have application to the applied field of used nuclear fuel management, as well as for examining the fundamental biogeochemical cycles active in sedimentary and deep geological environments.

Effect of carbon on whole-biofilm metabolic response to high doses of streptomycin.

Biofilms typically exist as complex communities comprising multiple species with the ability to adapt to a variety of harsh conditions. In clinical settings, antibiotic treatments based on planktonic susceptibility tests are often ineffective against biofilm infections. Using a CO2 evolution measurement system we delineated the real-time metabolic response in continuous flow biofilms to streptomycin doses much greater than their planktonic susceptibilities. Stable biofilms from a multispecies culture (containing mainly Pseudomonas aeruginosa and Stenotrophomonas maltophilia), Gram-negative environmental isolates, and biofilms formed by pure culture P. aeruginosa strains PAO1 and PAO1 ΔMexXY (minimum planktonic inhibitory concentrations between 1.5 and 3.5 mg/l), were exposed in separate experiments to 4000 mg/l streptomycin for 4 h after which growth medium resumed. In complex medium, early steady state multispecies biofilms were susceptible to streptomycin exposure, inferred by a cessation of CO2 production. However, multispecies biofilms survived high dose exposures when there was extra carbon in the antibiotic medium, or when they were grown in defined citrate medium. The environmental isolates and PAO1 biofilms showed similar metabolic profiles in response to streptomycin; ceasing CO2 production after initial exposure, with CO2 levels dropping toward baseline levels prior to recovery back to steady state levels, while subsequent antibiotic exposure elicited increased CO2 output. Monitoring biofilm metabolic response in real-time allowed exploration of conditions resulting in vulnerability after antibiotic exposure compared to the resistance displayed following subsequent exposures.

Live-streaming: Time-lapse video evidence of novel streamer formation mechanism and varying viscosity.

Time-lapse videos of growing biofilms were analyzed using a background subtraction method, which removed camouflaging effects from the heterogeneous field of view to reveal evidence of streamer formation from optically dense biofilm segments. In addition, quantitative measurements of biofilm velocity and optical density, combined with mathematical modeling, demonstrated that streamer formation occurred from mature, high-viscosity biofilms. We propose a streamer formation mechanism by sudden partial detachment, as opposed to continuous elongation as observed in other microfluidic studies. Additionally, streamer formation occurred in straight microchannels, as opposed to serpentine or pseudo-porous channels, as previously reported.

Pronounced effect of the nature of the inoculum on biofilm development in flow systems.

Biofilm formation renders sessile microbial populations growing in continuous-flow systems less susceptible to variation in dilution rate than planktonic cells, where dilution rates exceeding an organism's maximum growth rate (micro(max)) results in planktonic cell washout. In biofilm-dominated systems, the biofilm's overall micro(max) may therefore be more relevant than the organism's micro(max), where the biofilm micro(max) is considered as a net process dependent on the adsorption rate, growth rate, and removal rate of cells within the biofilm. Together with lag (acclimation) time, the biofilm's overall micro(max) is important wherever biofilm growth is a dominant form, from clinical settings, where the aim is to prevent transition from lag to exponential growth, to industrial bioreactors, where the aim is to shorten the lag and rapidly reach maximum activity. The purpose of this study was to measure CO(2) production as an indicator of biofilm activity to determine the effect of nutrient type and concentration and of the origin of the inoculum on the length of the lag phase, biofilm micro(max), and steady-state metabolic activity of Pseudomonas aeruginosa PA01 (containing gfp), Pseudomonas fluorescens CT07 (containing gfp), and a mixed community. As expected, for different microorganisms the lengths of the lag phase in biofilm development and the biofilm micro(max) values differ, whereas different nutrient concentrations result in differences in the lengths of lag phase and steady-state values but not in biofilm micro(max) rates. The data further showed that inocula from different phenotypic origins give rise to lag time of different lengths and that this influence persists for a number of generations after inoculation.

Metabolic differentiation in biofilms as indicated by carbon dioxide production rates.

The measurement of carbon dioxide production rates as an indication of metabolic activity was applied to study biofilm development and response of Pseudomonas sp. biofilms to an environmental disturbance in the form of a moving air-liquid interface (i.e., shear). A differential response in biofilm cohesiveness was observed after bubble perturbation, and the biofilm layers were operationally defined as either shear-susceptible or non-shear-susceptible. Confocal laser scanning microscopy and image analysis showed a significant reduction in biofilm thickness and biomass after the removal of the shear-susceptible biofilm layer, as well as notable changes in the roughness coefficient and surface-to-biovolume ratio. These changes were accompanied by a 72% reduction of whole-biofilm CO2 production; however, the non-shear-susceptible region of the biofilm responded rapidly after the removal of the overlying cells and extracellular polymeric substances (EPS) along with the associated changes in nutrient and O2 flux, with CO2 production rates returning to preperturbation levels within 24 h. The adaptable nature and the ability of bacteria to respond to environmental conditions were further demonstrated by the outer shear-susceptible region of the biofilm; the average CO2 production rate of cells from this region increased within 0.25 h from 9.45 +/- 5.40 fmol of CO2 x cell(-1) x h(-1) to 22.6 +/- 7.58 fmol of CO2 x cell(-1) x h(-1) when cells were removed from the biofilm and maintained in suspension without an additional nutrient supply. These results also demonstrate the need for sufficient monitoring of biofilm recovery at the solid substratum if mechanical methods are used for biofouling control.

CO2 production as an indicator of biofilm metabolism.

Biofilms are important in aquatic nutrient cycling and microbial proliferation. In these structures, nutrients like carbon are channeled into the production of extracellular polymeric substances or cell division; both are vital for microbial survival and propagation. The aim of this study was to assess carbon channeling into cellular or noncellular fractions in biofilms. Growing in tubular reactors, biofilms of our model strain Pseudomonas sp. strain CT07 produced cells to the planktonic phase from the early stages of biofilm development, reaching pseudo steady state with a consistent yield of approximately 10(7) cells.cm(-2).h(-1) within 72 h. Total direct counts and image analysis showed that most of the converted carbon occurred in the noncellular fraction, with the released and sessile cells accounting for <10% and <2% of inflowing carbon, respectively. A CO(2) evolution measurement system (CEMS) that monitored CO(2) in the gas phase was developed to perform a complete carbon balance across the biofilm. The measurement system was able to determine whole-biofilm CO(2) production rates in real time and showed that gaseous CO(2) production accounted for 25% of inflowing carbon. In addition, the CEMS made it possible to measure biofilm response to changing environmental conditions; changes in temperature or inflowing carbon concentration were followed by a rapid response in biofilm metabolism and the establishment of new steady-state conditions.

Studies of the extracellular glycocalyx of the anaerobic cellulolytic bacterium Ruminococcus albus 7.

Anaerobic cellulolytic bacteria are thought to adhere to cellulose via several mechanisms, including production of a glycocalyx containing extracellular polymeric substances (EPS). As the compositions and structures of these glycocalyces have not been elucidated, variable-pressure scanning electron microscopy (VP-SEM) and chemical analysis were used to characterize the glycocalyx of the ruminal bacterium Ruminococcus albus strain 7. VP-SEM revealed that growth of this strain was accompanied by the formation of thin cellular extensions that allowed the bacterium to adhere to cellulose, followed by formation of a ramifying network that interconnected individual cells to one another and to the unraveling cellulose microfibrils. Extraction of 48-h-old whole-culture pellets (bacterial cells plus glycocalyx [G] plus residual cellulose [C]) with 0.1 N NaOH released carbohydrate and protein in a ratio of 1:5. Boiling of the cellulose fermentation residue in a neutral detergent solution removed almost all of the adherent cells and protein while retaining a residual network of adhering noncellular material. Trifluoroacetic acid hydrolysis of this residue (G plus C) released primarily glucose, along with substantial amounts of xylose and mannose, but only traces of galactose, the most abundant sugar in most characterized bacterial exopolysaccharides. Linkage analysis and characterization by nuclear magnetic resonance suggested that most of the glucosyl units were not present as partially degraded cellulose. Calculations suggested that the energy demand for synthesis of the nonprotein fraction of EPS by this organism represents only a small fraction (<4%) of the anabolic ATP expenditure of the bacterium.