Mixed Population Biofilms Research Articles

Significance of biofilm structure on transport of inert particulates into biofilms

Evolutional changes in interior structures of mixed population biofilms grown on domestic wastewater were quantitatively analyzed using a cryosectioning technique and an image analysis. Meanwhile, transport of particulates into the biofilms was also experimentally investigated using fluorescent microbeads as tracers to relate the biofilm structure and particulate transport into the biofilm. Microscopic observation of the cryomicrotomy biofilm sections indicated the biofilms were very porous and consisted of interwinded filamentous biomass acting as a framework of the biofilm. A honeycomb structure was often found, which would make the biofilm more resistant to water flow. There were micropores with the diameter of about 10 µm microcolony aggregates attached to filamentous biomass and macropores with the diameter of 20-200 µm in the biomass matrix. These pores did not clog during two months of cultivation. Areal porosity was about 30% in the bottom biofilm and more than 90 % in the surface. Significant difference in transport efficiency was not observed for various sizes of microbeads due to the presence of macropores. Therefore, even 10 µm tracer beads could quickly traverse throughout a biofilm 640 µm thick via water channels or macropores and then penetrated into the micropores. Convective transport from the bulk to the bottom biofilm, rather than molecular diffusion, was responsible for this rapid transport. Based on experimental results, it can be concluded that the biofilm structure seems to be well designed to maximize the transport efficiency of substrates and products and the strength of biofilm structure.

Significance of the spatial distribution of microbial species in mixed‐population biofilms

Dynamic changes in spatial microbial distribution in mixed‐population biofilms resulting from interspecies competition between heterotrophs and nitrifiers were experimentally investigated using a microslicer technique. Biofilms cultured in partially submerged rotating biological contactors (RBC) with synthetic wastewater were used as test materials. The results showed that variation in the carbon loading rate (CLR) and the composition of the feed substrate resulted in rapid and substantial changes in the spatial distribution of nitrifiers in mixed population biofilms within a week, which significantly influenced the NH4‐N removal rate. Heterotrophs were more successful than nitrifiers in acquiring dissolved oxygen and space and dominated throughout the biofilms, especially in the surface biofilm. Nitrifiers were therefore diluted in the surface biofilm and mainly found in the inner biofilm. The fraction differences of NH4‐oxidizers and NO2‐oxidizers over the biofilm depth were about 2 orders and 3–4 orders of magnitude at CLR=0.8 and 1.6 g‐C m‐2 d‐1 respectively, indicating a steep spatial gradient of nitrifiers. Although total areal density of NH4‐oxidizers in the biofilms were in the same order, the NH4‐N flux decreased by about 9% and 24% after one month at CLR=0.8 and 1.6 g‐C m‐2 d‐1 respectively. This decrease of NH4‐N flux was attributed to the extent of the stratified spatial distribution of NH4‐oxidizers. Heterotrophs lie near the surface and act as a diffusion barrier, resulting in an increase in internal dissolved oxygen and NH4‐N diffusion resistance for NH4‐oxidizers. These results suggest that simplifying or neglecting the spatial distribution of microbial species can lead to substantial errors in biofilm kinetic parameters determined from measured substrate removal rates. For implication in reactor design, the extent of the spatial distribution of microbial species in a biofilm is especially important for interpretation of nitrification efficiency in the presence of organic matter.

Uptake and release of inert fluorescence particles by mixed population biofilms

Inert fluorescent microparticles were used as tracers to investigate the dynamics of spatial distribution of particulate components in mixed population biofilms. The tracer bead spatial distributions in the biofilm were experimentally measured by sectioning the biofilms with a microslicer. The experimental results were compared with model simulations using the biofilm model (BIOSIM) to evaluate the assumption that advective transport (displacement) of particulates balances with cell growth in the model. The tracer beads could traverse throughout a biofilm 360 microm thick within less than 23 minutes, which cannot be explained solely by their attachment to the surface followed by molecular diffusion. Advective transport of the tracer beads via "voids and pores" could be responsible for such rapid bead penetration. Observation by confocal scanning laser microscopy (CSLM) clearly showed that the biofilm consisted of a thick loose surface layer, varying in thickness, and a semicontiguous base layer separated by water channels. About 80% of attached tracer beads remained in the biofilm for over 20 days. The trapped tracer beads were gradually transferred from the depth of the biofilm to the surface. The observed bead release rate was much slower than the model predictions. This is probably because the cell density increased predominantly near the substratum, resulting in an unbalance of advective transport of the tracer beads and cell growth. The pores, voids, and cell-free spaces in the biofilm were first filled with growing biomass, thereafter, displacement of the beads took place once the cell density reached certain levels. The model assumptions of the temporal and spatial constant cell density and the continuum concept (flat biomass) are clearly oversimplified and should be revised. It was concluded that the dynamics of the inert microbeads in the biofilm was strongly influenced by not only microbial growth, but also by the biofilm structure and growth pattern. Therefore, one dimensional modeling is not adequate for the accurate description of the transport of particulates in a biofilm. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 459-469, 1997.

Colonization and disinfection of biofilms hosting coliform-colonized carbon fines

The documented release of carbon fines from granular activated carbon filters is a concern for drinking water utilities, since these particles may carry coliform and even pathogenic bacteria through the disinfection barrier. Such a breakthrough could have an impact on distribution system biofilms. Using total cell counts, specific monoclonal antibody staining, and computerized image analysis, we monitored the colonization of introduced Klebsiella pneumoniae associated with carbon fines in mixed-population biofilms. The particles transported the coliforms to the biofilms and allowed successful colonization. Chlorine (0.5 mg/liter) was then applied as a disinfectant. Most K. pneumoniae along with the carbon fines left the biofilm under these conditions. The impact of chlorine was greater on the coliform bacteria and carbon fines than on the general fixed bacterial population. However, 10% of the introduced coliforms and 20% of the fines remained in the biofilm. The possibility that this represents a mechanism for bacteria of public health concern to be involved in regrowth events is discussed.

Effect of growth conditions and substratum composition on the persistence of coliforms in mixed-population biofilms.

Laboratory reactors operated under oligotrophic conditions were used to evaluate the importance of initial growth rate and substratum composition on the long-term persistence of coliforms in mixed-population biofilms. The inoculum growth rate had a dramatic effect on the ability of coliforms to remain on surfaces. The most slowly grown coliforms (mu = 0.05/h) survived at the highest cell concentration. Antibody staining revealed that Klebsiella pneumoniae existed primarily as discrete microcolonies on the surface. Both coliforms and heterotrophic plate count bacteria were supported in larger numbers on a reactive substratum, mild steel, than on polycarbonate.

Evaluation of physiological staining, cryoembedding and autofluorescence quenching techniques on fouling biofilms

Physiological staining, cryoembedding, cryosectioning and autoftuorescence quenching techniques were evaluated for their applicability to undefined mixed population biofilms collected from environmental or engineered systems. Four different biofilms from two cooling towers, a paper mill machine and the effluent ditch of a wastewater treatment plant were tested. The redox dye 5‐cyano‐2,3‐ditolyl tetrazolium chloride (CTC) was used in combination with the DNA stain 4’,6‐diamino‐2‐phenylindole (DAPI) to distinguish respiring and nonrespiring cells. Positive CTC staining, as evidenced by the development of pink or red color, was successful in all samples examined except for paper mill biofilm. The structural integrity of frozen sections deteriorated when biofilms contained rigid or fibrous material. Autofluorescence generally impaired the ability to distinguish specific staining from natural background fluorescence. Two physical and three chemical methods were tested to quench autofluorescence. Quenching with...

Relationship between population dynamics of nitrifiers in biofilms and reactor performance at various C:N ratios

The relationship between time dependent population dynamics of nitrifiers and heterotrophs in undefined mixed-population biofilms and their nitrification efficiency was experimentally investigated at various C:N ratios of feed solutions. Five types of biofilms were cultured in partially submerged rotating biological contactors (RBC's) at different C:N ratios and were used as test materials. The results indicated that initial microbial composition in the biofilms and substrate composition (e.g. C:N ratio) strongly influenced the later population dynamics and the nitrification efficiency. Higher influent C:N ratio retarded accumulation of nitrifying bacteria, especially NO 2-oxidizers, resulting in a considerably long start-up period for complete and stable nitrification due to competition for dissolved oxygen and space in the biofilm. Furthermore, a start-up inoculum was very important to keep start-up time of nitrification to a minimum. Time-dependent population dynamics in the biofilms reflected well the bulk water quality and microbial community structure in the bulk liquid. These results suggest that the structure of microbial community in the biofilm can be predicted from monitoring the water quality and microbiology of the bulk liquid. Physiologically inactive cells in the biofilm were determined by an INT dehydrogenease assay. These cells gradually accumulated up to about 30% of the total bacterial population within the biofilms. The results of this study will provide a rational basis for developing and controlling desired biofilm population dynamics to maximize nitrification efficiency of wastewater biofilms

Spatial microbial distributions of nitrifiers and heterotrophs in mixed-population biofilms

Spatial microbial distributions of nitrifiers and heterotrophs in undefined mixed-population biofilms were experimentally investigated using a microslicer technique and correlated with nitrification efficiency of the biofilm system. The general stratification of different bacterial groups in the biofilm was simulated using a one-dimensional (1-D) mathematical biofilm accumulation model (BAM) and compared with the experimental results. Biofilms were cultured at three C : N ratios of feed solutions in a partially submerged rotating biological contactor (RBC). It was shown that the biofilms were vertically stratified (from biofilm surface to substratum). At C : N = 0, heterotrophs and nitrifiers coexisted in the outermost biofilm and heterotrophs dominated in the innermost biofilm. At C : N = 1.5, heterotrophs outcompeted nitrifiers for dissolved oxygen and space; thus, heterotrophs dominated in the outermost biofilm and nitrifiers were present only in the deeper biofilm. Nitrifiers and heterotrophs coexisted in the innermost biofilm. An increase in the influent C : N ratio resulted in stronger stratification of microbial species, as well as inhibition of nitrification. In batch experiments, NH(4)--N utilization rate (R(NH(4)--N)) was almost the same at each substrate C : N ratio even though NH(4) oxidizers were predominantly present in the deeper biofilm. The biofilm performance could not be sufficiently explained by the obtained microbial spatial distribution, suggesting that one-dimensional description of microbial distribution was not good enough and three-dimensional measurements of microbial spatial distribution is necessary. Total bacterial densities increased by a factor of 3-17 with biofilm depth. The metabolically active cell fraction decreased from 35 +/- 13% in the outermost biofilm to 15 +/- 4% in the innermost biofilm, presumably due to substrate limitation. The model predicted more pronounced stratification of nitrifiers and heterotrophs than the observed results. This discrepancy could be attributed to the real biofilms that were structurally heterogeneous (e.g., water channels), which could not be described by the one-dimensional model. The results of this study clearly indicate the limitation of 1-D biofilm models to describe the extent of stratification of nitrifiers and heterotrophs and suggest a 3-D model is necessary.

Dynamic changes in spatial microbial distribution in mixed-population biofilms: experimental results and model simulation

Dynamic changes in spatial microbial distribution in mixed-population biofilms were experimentally determined using a microslicer technique and simulated by a biofilm accumulation model (BAM). Experimental results were compared with the model simulation. The biofilms cultured in partially submerged rotating biological contactors (RBC) with synthetic wastewater were used as test materials. Experimental results showed that an increase of substrate loading rate (i.e., organic carbon and NH4-N) resulted in the microbial stratification in the biofilms. Heterotrophs defeated nitrifiers and dominated in the outer biofilm, whereas nitrifiers were diluted out in the outer biofilm and forced into the inner biofilm. At higher organic loading rates, a stronger stratified microbial spatial distribution was observed, which imposed a severe internal oxygen diffusion limitation on nitrifiers and resulted in the deterioration of nitrification efficiency. Model simulations described a general trend of the stratified biofilm structure. However, the actual stratification was stronger than the simulated results. For implication in the reactor design, when the specific carbon loading rate exceeds a certain limit, nitrification will be deteriorated or require a long start-up period due to the interspecies competition resulting in oxygen diffusion limitation. The extend of microbial stratification in the biofilm is especially important for determination of feasibility of nitrification in the presence of organic matters.

Dynamic changes in spatial microbial distribution in mixed-population biofilms: Experimental results and model simulation

Dynamic changes in spatial microbial distribution in mixed-population biofilms were experimentally determined using a microslicer technique and simulated by a biofilm accumulation model (BAM). Experimental results were compared with the model simulation. The biofilms cultured in partially submerged rotating biological contactors (RBC) with synthetic wastewater were used as test materials. Experimental results showed that an increase of substrate loading rate (i.e, organic carbon and NH4-H) resulted in the microbial stratification in the biofilms. Heterotrophs defeated nitriflers and dominated in the outer biofilm, whereas nitrifiers were diluted out in the outer biofilm and forced into the inner biofilm. At higher organic loading rates, a stronger stratified microbial spatial distribution was observed, which imposed a severe internal oxygen difuitsion limitation on nitrifiers and resulted in the deterioration of nitrification efficiency. Model simulations described a general trend of the stratified biofilm structure. However, the actual stratification was stronger than the simulated results For implication in the reactor design, when the specific carbon loading rate exceeds a certain limit, nitrification will be deteriorated or require a long start-up period due to the interspecies competition resulting in oxygen diflijsion limitation The extend of microbial stratification in the biofilm is especially important for determination of feasibility of nitrification in the presence of organic matters

A statistical analysis of the effect of substrate utilization and shear stress on the kinetics of biofilm detachment

One of the least understood processes affecting biofilm accumulation is detachment. Detachment is the removal of cells and cell products from an established biofilm and subsequent entrainment in the bulk liquid. The goal of this research was to determine the effects of shear stress and substrate loading rate on the rate of biofilm detachment. Monopopulation Pseudomonas aeruginosa and undefined mixed population biofilms were grown on glucose in a RotoTorque biofilm reactor. Three levels of shear stress and substrate loading rate were used to determine their effects on the rate of detachment. Suspended cell concentrations were monitored to determine detachment rates, while other variables were measured to determine their influence on the detachment rate. Results indicate that detachment rate is directly related to biofilm growth rate and that factors which limit growth rate will also limit detachment rate. No significant influence of shear on detachment rate was observed.A new kinetic expression that incorporates substrate utilization rate, yield, and biofilm thickness was compared to published detachment expressions and gives a better correlation of data obtained both in this research and from previous research projects, for both mono- and mixed-population biofilms.

NMR and Microelectrode Studies of Hydrodynamics and Kinetics in Biofilms

AbstractProfiles of flow velocity and dissolved oxygen concentration were studied by NMR and microelectrodes near mixed population biofilms growing on polycarbonate substratum. The profile of dissolved oxygen concentration within the diffusion boundary layer and profiles of flow velocity within the hydrodynamic boundary layer can be described by exponential equations.