A simple method for quantifying biomass cell and polymer distribution in biofilms

Biofilms are ubiquitous and play an essential role in both environmental processes and hospital infections. Standard methods are not capable of quantifying biomass concentration in dilute suspensions. Furthermore, standard techniques cannot differentiate biomass composition. In this study, a user-friendly technique was developed for measuring biomass cell and polymer content in detached biofilms using a standard coulter counter. The method was demonstrated for an environmentally relevant strain of Pseudomonas aeruginosa (Schroeter) Migula grown in a bioreactor and also for a medically relevant strain of P. aeruginosa (PAO1) grown on standard growth pegs. Results were compared and validated by standard assays, including EPA method 1684 for measuring biomass, microscopic direct counts, and a crystal violet staining assay. The minimum detection limit for the coulter counter method (0.07 mg-biomass L^− 1) was significantly lower than the EPA method 1684 (1.9 ± 0.4 mg/L) and the crystal violet assay (1.1 ± 0.2 mg L^− 1). However, the coulter counter method is limited to dilute biomass samples (below 204 ± 16 mg L^− 1) due to clogging of the aperture tube. While biomass measurements are useful, the major advantage of the coulter counter method is the ability to directly determine EPS, cell, and aggregate fractions after mild chemical treatment. The rapid technique (4–5 min per sample) was used to measure biomass fractions in dispersed P. aeruginosa (Schroeter) and PAO1 biofilms. This technique will be critical for understanding biofilm formation/dispersal.     

Detachment forces and their influence on the structure and metabolic behaviour of biofilms

The biofilm reactor has been frequently applied in wastewater treatment. The formation, structure and metabolic activity of the biofilms are closely associated with the detachment forces in the reactors. This paper reviews the essential role of detachment forces in the biofilm process. A more compact, stable and denser biofilm can be formed at a relatively higher detachment force. The detachment force has significant influence on the structure, mass transfer, production of exopolysaccharides, metabolic and genetic properties of the biofilm. In an engineering sense, the detachment forces can be manipulated, as a control parameter, to produce a more stable and compact biofilm for use in wastewater treatment. The molecular and genetic mechanisms responsible for the detachment force-associated phenomena are not yet fully understood.     

Exposure of biofilms to slow flow fields: the convective contribution to growth and disinfection

A previously introduced degenerate diffusion-reaction model of biofilm growth and disinfection is extended to account for convective transport of oxygen and disinfectants in an aqueous environment. To achieve this in a computationally efficient manner we employ a thin-film approximation to the (Navier)-Stokes equations that can be solved analytically. In numerical experiments, we investigate how the convective transport of nutrients and disinfectants due to bulk flow hydrodynamics affects the balance between growth and disinfection processes. It is found that the development of biofilms can be significantly affected by the flow field even at extremely low Reynolds numbers. While it is natural to expect that increased bulk flow velocities imply increased mass transfer of both, nutrients and disinfectants, and hence an acceleration of both, growth and decay of biomass, it is found, furthermore, that in many instances the actual flow conditions, determine the success or failure of disinfection, i.e. persistence or extinction of a biofilm community.     

A new mathematical model for combining growth and energy intake in animals: The case of the growing pig

A simultaneous model for analysis of net energy intake and growth curves is presented, viewing the animal's responses as a two dimensional outcome. The model is derived from four assumptions: (1) the intake is a quadratic function of metabolic weight; (2) the rate of body energy accretion represents the difference between intake and maintenance; (3) the relationship between body weight and body energy is allometric and (4) animal intrinsic variability affects the outcomes so the intake and growth trajectories are realizations of a stochastic process. Data on cumulated net energy intake and body weight measurements registered from weaning to maturity were available for 13 pigs. The model was fitted separately to 13 datasets. Furthermore, slaughter data obtained from 170 littermates was available for validation of the model. The parameters of the model were estimated by maximum likelihood within a stochastic state space model framework where a transform-both-sides approach was adopted to obtain constant variance. A suitable autocorrelation structure was generated by the stochastic process formulation. The pigs’ capacity for intake and growth were quantified by eight parameters: body weight at maximum rate of intake (149-281 kg); maximum rate of intake (25.7-35.7 MJ/day); metabolic body size exponent (fixed: 0.75); the daily maintenance requirement per kg metabolic body size (0.232-0.303 MJ/(day×kg^0.75)); reciprocal scaled energy density ; a dimensional exponent, θ₆ (0.730-0.867); coefficient for animal intrinsic variability in intake (0.120-0.248 MJ^0.5) and coefficient for animal intrinsic variability in growth (0.029-0.065 kg^0.5). Model parameter values for maintenance requirements and body energy gains were in good agreement with those obtained from slaughter data. In conclusion, the model provides biologically relevant parameter values, which cannot be derived by traditional analysis of growth and energy intake data.     

Interaction between biofilm development, structure and detachment in rotating annular reactors

In biotechnology, composition of biofilms and suspended bioaggregates can be crucial for system performance or product quality. Consequently, understanding biofilm dynamics is important for any process optimisation. The aim of this study was to investigate biofilm development and detachment under different hydrodynamic conditions and varying glucose load. Confocal laser scanning microscopy proved to be a fast method providing information about structure, distribution and volume ratio of bacteria and extra cellular polymers (EPS) within biofilms and detached biomass. As a result, it could be shown that biofilm structure, in terms of density and EPS volume, was largely influenced by hydrodynamic conditions. Furthermore, it was demonstrated that the EPS:bacteria ratio and distribution was largely influenced by substrate load. Finally, the characteristics in biofilm structure and development were reflected in the composition and quantity of the detached biomass.     

A microplate spectrofluorometric assay for bacterial biofilms

A spectrofluorometric assay was developed for quantification of bacterial biofilms grown on a microtiter plate. The method involved staining biofilms formed by gram-negative and gram-positive bacteria with wheat germ agglutinin-Alexa Fluor 488 conjugate, which selectively binds to N-acetylglucosamine residues in biofilms. The fluorescence of stained biofilms was measured with a fluorescent plate reader. This method was compared with a widely used microplate colorimetric assay involving crystal violet staining of biofilms formed by both gram-negative and gram-positive bacteria. A strong linear association existed between the two methods (r ²=0.99/0.94). Being more sensitive and specific as compared to colorimetric method, the spectrofluorometric assay provides a better alternative for quantification and characterization of bacterial biofilms.     

Porosity and tortuosity relations as revealed by a mathematical model of biofilm structure

A biofilm is assumed to be submerged in a fluid with given viscosity and low Reynolds number. The interaction between fluid and bacteria is modeled through streamlines. We use finite-difference and boundary element numerical schemes to predict streamlines within the biofilm. The results show that this approach can provide information about prior distribution and geometry of the biofilm structure. Theoretical values of porosity and tortuosity are computed and compared with published data.     

A general description of detachment for multidimensional modelling of biofilms

A general method for describing biomass detachment in multidimensional biofilm modelling is introduced. Biomass losses from processes acting on the entire surface of the biofilm, such as erosion, are modelled using a continuous detachment speed function F(det). Discrete detachment events, i.e. sloughing, are implicitly derived from simulations. The method is flexible to allow F(det) to take several forms, including expressions dependent on any state variables such as the local biofilm density. This methodology for biomass detachment was integrated with multidimensional (2D and 3D) particle-based multispecies biofilm models by using a novel application of the level set method. Application of the method is illustrated by trends in the dynamics of biofilms structure and activity derived from simulations performed on a simple model considering uniform biomass (case study I) and a model discriminating biomass composition in heterotrophic active mass, extracellular polymeric substances (EPS) and inert mass (case study II). Results from case study I demonstrate the effect of applied detachment forces as a fundamental factor influencing steady-state biofilm activity and structure. Trends from experimental observations reported in literature were correctly described. For example, simulation results indicated that biomass sloughing is reduced when erosion forces are increased. Case study II illustrates the application of the detachment methodology to systems with non-uniform biomass composition. Simulations carried out at different bulk concentrations of substrate show changes in biofilm structure (in terms of shape, density and spatial distribution of biomass components) and activity (in terms of oxygen and substrate consumption) as a consequence of either oxygen-limited or substrate-limited growth.     

A simple in vitro model for growth control of bacterial biofilms

The perfused biofilm fermenter was found to be unsuitable for the long-term culture and growth rate control of Staphylococcus aureus and Pseudomonas aeruginosab biofilms. In a simplified approach, biofilms of these organisms were grown within Sorbarod filter plugs which were perfused with culture medium. Pseudo-steady states were established which were stable over several days at which the growth rate of the biofilm was reproducible, measurable and significantly slower than in broth culture. Environmental scanning electron microscopy of dissected Sorbarods demonstrated an association of cells with the surfaces of individual cellulose fibres, and growth characteristic of biofilms. Relatively high cell numbers recovered from the Sorbarod model facilitated biochemical investigations of biofilm populations and cells released spontaneously from them.
SDS-PAGE demonstrated significant differences between the protein profiles of biofilm and eluted populations, which include, in Staph. aureus , the repression of a 48 kDa protein and increased expression of a 21 kDa protein relative to planktonic controls cultured at equivalent growth rates. The paper demonstrates the suitability of the approach for the culture of biofilm samples which are suitable for biochemical analysis.     

A simple in vitro model for growth control of bacterial biofilms

The perfused biofilm fermenter was found to be unsuitable for the long-term culture and growth rate control of Staphylococcus aureus and Pseudomonas aeruginosa biofilms. In a simplified approach, biofilms of these organisms were grown within Sorbarod filter plugs which were perfused with culture medium. Pseudo-steady states were established which were stable over several days at which the growth rate of the biofilm was reproducible, measurable and significantly slower than in broth culture. Environmental scanning electron microscopy of dissected Sorbarods demonstrated an association of cells with the surfaces of individual cellulose fibres, and growth characteristic of biofilms. Relatively high cell numbers recovered from the Sorbarod model facilitated biochemical investigations of biofilm populations and cells released spontaneously from them. SDS-PAGE demonstrated significant differences between the protein profiles of biofilm and eluted populations, which include, in Staph. aureus, the repression of a 48 kDa protein and increased expression of a 21 kDa protein relative to planktonic controls cultured at equivalent growth rates. The paper demonstrates the suitability of the approach for the culture of biofilm samples which are suitable for biochemical analysis.     

Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics

Abstract A simple in vitro system was developed as a model structure of biofilms and to evaluate their susceptibility to antibiotics. Viable Escherichia coli cells were entrapped in agar gel layers and incubated for 2 days in a minimal salt medium supplemented with glucose. After subsequent culture for 3 weeks under metal ion depletion, the biomass distribution inside the gel layer was highly heterogeneous. The cell concentration reached 10¹¹ cfu/g gel in the outer regions of the agar structure whereas the inner gel areas were less colonized (10⁹ cfu/g gel). Immobilized cells displayed enhanced resistance to latamoxef as compared with free microorganisms. Moreover, a 3-week-old immobilized-cell membrane was less susceptible to the antibiotic than a younger (2 days old) one. The exposure for 11 h to 64 μg/cm³ latamoxef killed about 90% of the bacteria entrapped in the older agar layer, whereas the number of killed cells was 100-fold higher in the younger structure. Effective diffusivity measurements showed that the diffusion of latamoxef in the biofilm-like agar structures was moderately restricted as compared to that in water, and independent of the immobilized-cell content.     

不同温度对中国对虾生长及能量收支的影响

研究了18～34℃6个不同恒温下中国对虾(Fenneropenaeus chinensis)的生长、饵料转化率及能量收支。结果表明,中国对虾的体重及能量特定生长率分别变动在1．22％～3．27％和1．33％～1．45％之间,在18～31℃温度范围内随温度升高而升高,34℃下则显著下降．对虾的摄食量及对饵料的消化率总体上随温度升高而升高,但在34℃下则有所降低;饵料重量转化率和能量转化率分别在28．99％～53．09％和15．70％～7．24％之间,总体上随温度升高而有所下降．根据拟合的多项式方程推算得到的中国对虾的最佳生长温度为29．7℃,生长能和呼吸能的变化主导着中国对虾的能量收支模式,随温度升高生长能占摄食能的比例逐渐降低,而呼吸能比例则逐渐升高。本研究表明,对虾在适宜温度下获得的较高生长率主要归因于较高的摄食量和食物消化率。     

A suggested standardised method for testing photocatalytic inactivation of aeroterrestrial algal growth on TiO2-coated glass

Aeroterrestrial green algae form conspicuous biofilms on man-made surfaces. The self-cleaning properties of photocatalytic coatings prevent their growth and can probably replace biocides. The aim of this study was to develop a laboratory method to investigate the efficiency of photocatalytic materials against algal growth. Two algal isolates (“Chlorella” luteoviridis, SAG 2196, and Coccomyxa sp., SAG 2040) functioned well as model organisms because they grew on almost all test specimens at 100% humidity and low UVA radiation. With these species, we examined algal growth prevention using photocatalytic glass. No effects on algal growth were detected, although the coated surfaces were photocatalytically active and degraded methylene blue. Because their cells are protected well against photocatalytically generated hydroxyl radicals, aeroterrestrial algae survive various stress factors. Nevertheless, the newly developed experimental design may be useful for assessing the biological function of other photocatalytic materials or stress factors.     

Amino acid metabolism and the energetics of growth

The nonessential amino acids are involved in a large number of functions that are not directly associated with protein synthesis. Recent studies using a combination of transorgan balance and stable isotopic tracers have demonstrated that a substantial portion of the extra‐splanchnic flux of glutamate, glutamine, glycine and cysteine derives from tissue synthesis. A key amino acid in this respect is glutamic acid. Little glutamic acid of dietary origin escapes metabolism in the small intestinal mucosa. Furthermore, because glutamic acid is the only amino acid that can be synthesized by mammals by reductive amination of a ketoacid, it is the ultimate nitrogen donor for the synthesis of other nonessential amino acids. Because the synthesis of glutamic acid and its product glutamine involve the expenditure of adenosine triphosphate (ATP), it seems possible that nonessential amino acid synthesis might have a significant bearing on the energetics of protein synthesis and, hence, of protein deposition. This paper discusses the topic of the energy cost of protein deposition, considers the metabolic physiology of amino acid oxidation and nonessential amino acid synthesis, and attempts to combine the information to speculate on the overall impact of amino acid metabolism on the energy exchanges of animals.     

Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms

Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21°C. Biofilm formation was maximal ca. 20–50 h after inoculation of the liquid medium and, as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spent culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C₆) exogenous homoserines can trigger such responses in P. fluorescens, its own signal substance is likely to possess a longer (>C₈) fatty acyl chain.     

A cellular growth model for root tips

Growth of the root tip is modeled using a one-dimensional string of cells. Each cell is characterized by three distinct phases, division, elongation-only or maturity. In this model two hypothetical phytohormones, one produced at the root tip and the other at the shoot, determine the behavior of the cell, and therefore the growth of the entire tip. While the division rate is taken to be a step function of the string coordinate, the growth rate of each cell is assumed to be piecewise linear and composed of linear functions of cell length. Thereafter, suitable operators for the calculation of the velocity and relative growth rate distributions are given. The results of the model are finally compared to measurements of Arabidopsis thaliana, Nicotiana tabacum and Pisum sativum roots.