Intrinsic fermentation kinetics of lactose in acidogenic biofilms

The intrinsic fermentation kinetics of lactose in acidogenic biofilms were investigated in situ in a continuous flow fermentor at 35 degrees C and pH 4.6. The external and internal mass transfer resistances to lactose molecules from bulk solution to inside the biofilms were experimentally minimized or eliminated in a thin biofilm and recycled medium. In a chemically defined culture medium, the immobilized acidogens converted lactose mainly to acetate and butyrate; the minor products included ethanol. propionate, lactate, and hydrogen. The utilization rate of lactose, as a function of lactose concentration in the fermentor, can be described by a Michaelis-Menten equation, as can the formation rates of acetate, butyrate, and ethanol. The production rates of propionate and lactate had a liner relationship with lactose concentration under the experimental conditions. The low pH (4.6) of culture medium could depress the formation of propionate, and intermediate which is most difficulty digested by acetogenic bacteria located in the second fermentor in a two-phase process. Production rate of acetate quickly reached a constant, and additional utilization of lactose produced more butyrate and other minor products. (c) 1993 John Wiley & Sons, Inc.     

Effects of calcium on development of anaerobic acidogenic biofilms

Anerobic biofilms with dominantly acidogenic bacteria were grown in fixed-bed recycle reactors. The influence of calcium concentration in the culture medium on biofilm mass accumulation, immobilized calcium concentration, and biofilm-specific activity was investigated. The results indicate that the biofilm mass accumulation was increased by the presence of calcium in the growth medium when calcium concentration was not higher than 120mg/L. Calcium accumulated in the biofilms increased in proportion to the calcium level in the feed. The biofilms for an increased input calcium concentration showed a trend of decrease in specific activity. The biofilms with a thickeness of less than 0.5 mm had the highest specific activity. The optimum calcium concentration for substrate consumption by the biofilms was 100 to 120 mg/L. The biofilms transferred from higher calcium medium to lower calcium medium were more susceptible to sloughing from their support surfaces, which indicates calcium's role in the stability of the biofilm structure. (c) 1995 John Wiley & Sons, Inc.     

Diffusion profiles in L. lactis biofilms under different conditions

Despite being an important topic in biofilm research, we still know little about diffusion in biofilms. Emerging biofilms of Lactococcus lactis growing in custom‐made flow‐cells were monitored and diffusion constants across the height of the biofilms recorded. The biofilms showed different diffusional behavior with regard to flow rate and pH variations, despite growing to similar thickness. At a higher flow rate, the biofilm exhibits slower diffusion compared to the reference cultivation at lower flow rate. By increasing pH, the biofilm exhibited fast growth and little difference in diffusion compared to the reference cultivation. Furthermore, the diffusion inside of the biofilms differed depending on the position in the flow‐cell. The present study reveals new insights in how external factors can affect structure and density of biofilms. The method can be reliably used for L. lactis biofilms with a thickness up to 120 μm.     

Diffusion of lactose in kappa-carrageenan/locust bean gum gel beads with or without entrapped growing lactic acid bacteria

Effective diffusion coefficients (De) of lactose in kappa-carrageenan (2.75% wt/wt)/locust bean gum (0.25% wt/wt) (LBG) gel beads (1.5-2.0-mm diameter)with or without entrapped lactic acid bacteria (LAB) were determined at 40 degrees C. The effects of lactose concentration, bacteria strain (Streptococcus salivarius subsp. thermophilus and Lactobacillus casei subsp. casei) and cell content at various steps of the fermentation process (after immobilization, pre-incubation of the beads and successive fermentations) were measured on De as a first step for process modelling. Results were obtained from transiend concentration changes n well-stirred lactose solutions in which the beads were suspended. A mathematical model of unsteady-state diffusion in a sphere was used, and De was obtained from the best fit of the experimental data. Diffusivity of lactose in cell-tree beads was significantly lower than in pure water mainly because of the obstruction effect of the polymer chains and the hydration region. Furthermore, effective diffusivity and equilibrium partition factor were independent of lactose concentration in the range from 12.5 to 50 g/L. No significant difference was found for De (effective diffusivity) and Kp (partition) coefficients between beads entrapping S. thermophilus (approximately 5 x 10(9) CFU/mL) and cell-free beads. On the other hand higher cell counts obtained with L. casei (close to 1.8 x 10(11) CFU/mL) increased mass transfer resistance resulting in lower effective diffusivities and Kp. Finally, the effects of the type of bacteria and their distribution in the beads on the diffusivity were also discussed.     

Reaction kinetics in biofilms

A novel in situ microtechnique allows evaluating parameters of diffusion-controlled reactions in biofilms. A microprobe, 15 mum in diameter, was used to simultaneously measure the dissolved oxygen concentration and the optical density at different depths in a submerged biofilm. Based on the results, the biofilm diffusion coefficient for dissolved oxygen, D(f) the dissolved oxygen flux through the biofilm surface, J(02), and the half velocity coefficient, K(s), have been calculated.     

Hyperbaric biofilms on engineering surfaces formed in the deep sea

Biofouling is a major problem for long-term deployment of sensors in the marine environment. This study showed that significant biofilm formation occurred on a variety of artificial materials (glass, copper, Delrin^? and poly-methyl methacrylate [PMMA]) deployed for 10?days at a depth of 4700?m in the Cayman Trough. Biofilm surface coverage was used as an indicator of biomass. The lowest biofilm coverage was on copper and PMMA. Molecular analyses indicated that bacteria dominated the biofilms found on copper, Delrin^? and PMMA with 75, 55 and 73% coverage, respectively. Archea (66%) were dominant on the glass surface simulating interior sensor conditions, whereas Eukarya comprised the highest percentage of microflora (75%) on the glass simulating the exterior of sensors. Analysis of Denaturing Gradient Gel Electrophoresis profiles indicated that copper and Delrin^? shared the same community diversity, which was not the case for glass and PMMA, or between PMMA and copper/Delrin^?. Sequence alignment matches belonged exclusively to uncultivable microorganisms, most of which were not further classified. One extracted sequence found on glass was associated with Cowellia sp., while another extracted from the PMMA surface was associated with a bacterium in the Alterominidaceae, both γ-proteobacteria. The results demonstrate the necessity of understanding biofilm formation in the deep sea and the potential need for mitigation strategies for any kind of long-term deployment of remote sensors in the marine environment.     

Cryosectioning of biofilms for microscopic examination

A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.     

Interspecies bacterial interactions in biofilms

Interactions among bacterial populations can have a profound influence on the structure and physiology of microbial communities. Interspecies microbial interactions begin to influence a biofilm during the initial stages of formation, bacterial attachment and surface colonization, and continue to influence the structure and physiology of the biofilm as it develops. Although the majority of research on bacterial interactions has utilized planktonic communities, the characteristics of biofilm growth (cell positions that are relatively stable and local areas of hindered diffusion) suggest that interspecies interactions may be more significant in biofilms.     

Liquid flow in heterogeneous biofilms

Liquid flow was studied in aerobic biofilms, consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. Fluorescein microinjection was used as a qualitative technique to determine the presence of flow in cell clusters and voids. Flow velocity profiles were determined by tracking fluorescent latex spheres using confocal microscopy. Liquid was flowing through the voids and was stagnant in the cell clusters. Consequently, in voids both diffusion and convection may contribute to mass transfer, whereas in cell clusters diffusion is the dominant factor. The flow velocity in the biofilm depended on the average flow velocity of the bulk liquid. The velocity profiles in biofilms were linear and the velocity was zero at the substratum surface. The velocity gradients within biofilms were 50% of that near walls without biofilm coverage. The influence of the biofilm roughness on the flow velocity profiles was similar to that caused by rigid roughness elements. (c) 1994 John Wiley & Sons, Inc.     

生物制造高附加值乳糖衍生物的研究进展

乳糖来自于乳清,是奶酪行业的低附加值副产物.由于亚洲人普遍存在乳糖不耐受性,因此乳糖缺乏有效的经济增值途径.随着我国乳制品行业的不断发展,利用乳糖制备高附加值产品具有重要的经济价值和产业升级意义.本文就近年来发展的利用生物技术转化乳糖为稀少糖或益生寡糖的方法进行了综述,以期对该领域研究提供借鉴.     

Osteomyelitis and the role of biofilms in chronic infection

Understanding the mechanisms implicated in the initial attachment, development, and maturation of a biofilm phenotype are of tremendous importance for their effect on the medical, industrial, and public health arenas. This review explores the current understanding of the nature of biofilms and the impact that molecular interactions between the bacteria themselves, as well as between bacteria and the host, may have on biofilm development and phenotype using the nonmotile Gram-positive coccus, Staphylococcus aureus, as an example.     

Measurement of local mass transfer coefficient in biofilms

Local mass transfer rates for an electrochemically formed microsink in an aerobic biofilm was measured by a mobile microelectrode using limiting current technique. Mass transfer coefficients varied both horizontally and vertically in the biofilm. The results implied the existence of an irregular biofilm structure consisting of microbial cell clusters surrounded by tortuous water channels. An unexpected increase of the local mass transfer coefficient just above the biofilm surface suggested the existence, of local flow instability in this region. As expected, the influence of bulk flow velocity on the local mass transfer rate decreased with increasing depth into the biofilm. Mass transfer coefficients fluctuated significantly inside microbial cell clusters, suggesting the existence of internal channels through which liquid could flow. A new conceptual model of biofilm microbial cluster structure is proposed to account for such biofilm microstructure irregularities. (c) 1995 John Wiley & Sons, Inc.     

Diffusion coefficients of metabolites in active biofilms

Diffusion coefficients of actual metabolites in completely active biofilms can be determined by applying a new concept that is based on a constant local activity in the entire biofilm. In that case, a concentration step will be transmitted unattenuated. Subsequently, the diffusion coefficient can be calculated from the response monitored with a microelectrode positioned in the biofilm without quantitative knowledge of the local microbial kinetics. The conditions required for such a constant microbial biofilm activity were formulated in terms of the Thiele modulus and the substrate concentration in the bulk liquid. This proposed method was successfully applied to determine diffusion coefficients of oxygen and glucose in agar gels containing various fractions of active immobilized microorganisms. The values obtained were compared to experimental results from well-defined inert systems. The transient response of oxygen was far more affected by the presence of the immobilized cells than glucose. This can be explained by partition of the diffusing solute between the microbial cells and the aqueous phase.     

The sociobiology of biofilms

Biofilms are densely packed communities of microbial cells that grow on surfaces and surround themselves with secreted polymers. Many bacterial species form biofilms, and their study has revealed them to be complex and diverse. The structural and physiological complexity of biofilms has led to the idea that they are coordinated and cooperative groups, analogous to multicellular organisms. We evaluate this idea by addressing the findings of microbiologists from the perspective of sociobiology, including theories of collective behavior (self-organization) and social evolution. This yields two main conclusions. First, the appearance of organization in biofilms can emerge without active coordination. That is, biofilm properties such as phenotypic differentiation, species stratification and channel formation do not necessarily require that cells communicate with one another using specialized signaling molecules. Second, while local cooperation among bacteria may often occur, the evolution of cooperation among all cells is unlikely for most biofilms. Strong conflict can arise among multiple species and strains in a biofilm, and spontaneous mutation can generate conflict even within biofilms initiated by genetically identical cells. Biofilms will typically result from a balance between competition and cooperation, and we argue that understanding this balance is central to building a complete and predictive model of biofilm formation.     

乳糖诱导木聚糖酶基因在大肠杆菌中的可溶性表达

以构建好的大肠杆菌工程菌BL21(DE3)/xylanase为研究对象,研究了以IPTG和乳糖作为诱导剂时重组蛋白的表达规律。在摇瓶发酵条件下研究了诱导剂浓度、诱导时机、诱导培养时间和诱导培养温度对目标蛋白表达的影响。实验结果表明,乳糖作为诱导剂时,重组菌产酶活力33.9 U/mg略高于IPTG作为诱导剂时重组菌产酶活力28.10 U/mg,这为乳糖作为诱导剂应用于重组大肠杆菌生产木聚糖酶提供了参考依据。     

Antibiotic resistance of biofilms

Microbial biofilms are notably recalcitrant towards treatment with antibiotics, biocides or disinfectants that would adequately control the same organisms growing in planktonic mode. Much of this resistance has been attributed to an organisation of the biofilm cells within exopolymer matrices. Whilst such exopolymers are unlikely to hinder the diffusion and access of antimicrobial agents to the underlying cells, they will chemically quench reactive biocides such as chlorine and peroxygens, and bind highly charged antibiotics, such as tobramycin and gentamycin, thereby providing some protection to the more deep lying cells. Extracellular enzymes, bound within the glycocalyx and able to degrade the treatment agents, will further reduce the access of susceptible compounds. Diffusion limitation however, is unlikely to be the sole moderator of the resistance properties of microbial biofilms. In addition, gradients of oxygen and nutrients established across the biofilm community will cause growth rates to be much reduced at points remoted from the accessible nutrient. Slow growth rates, and the associated induction of stringent responses further contribute towards this resistance. Finally, there have been recent demonstrations that attachment of microorganisms to surfaces promotes the expression of genes that are not normally expressed in planktonic culture. Whether or not the expression of such genes alters the phenotype in a manner which alters the response of the cells to antimicrobial agents remains to be demonstrated.     

Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB

The force-deflection and removal characteristics of bacterial biofilm were measured by two different techniques before and after chemical, or enzymatic, treatment. The first technique involved time lapse imaging of a biofilm grown in a capillary flow cell and subjected to a brief shear stress challenge imparted through increased fluid flow. Biofilm removal was determined by calculating the reduction in biofilm area from quantitative analysis of transmission images. The second technique was based on micro-indentation using an atomic force microscope. In both cases, biofilms formed by Staphylococcus epidermidis were exposed to buffer (untreated control), urea, chlorhexidine, iron chloride, or DispersinB. In control experiments, the biofilm exhibited force-deflection responses that were similar before and after the same treatment. The biofilm structure was stable during the post-treatment shear challenge (1% loss). Biofilms treated with chlorhexidine became less deformable after treatment and no increase in biomass removal was seen during the post-treatment shear challenge (2% loss). In contrast, biofilms treated with urea or DispersinB became more deformable and exhibited significant biofilm loss during the post-treatment flow challenge (71% and 40%, respectively). During the treatment soak phase, biofilms exposed to urea swelled. Biofilms exposed to iron chloride showed little difference from the control other than slight contraction during the treatment soak. These observations suggest the following interpretations: (1) chemical or enzymatic treatments, including those that are not frankly antimicrobial, can alter the cohesion of bacterial biofilm; (2) biocidal treatments (e.g., chlorhexidine) do not necessarily weaken the biofilm; and (3) biofilm removal following treatment with agents that make the biofilm more deformable (e.g., urea, DispersinB) depend on interaction between the moving fluid and the biofilm structure. Measurements such as those reported here open the door to development of new technologies for controlling detrimental biofilms by targeting biofilm cohesion rather than killing microorganisms.     

Structural changes in S. epidermidis biofilms after transmission between stainless steel surfaces

Transmission is a main route for bacterial contamination, involving bacterial detachment from a donor and adhesion to receiver surfaces. This work aimed to compare transmission of an extracellular polymeric substance (EPS) producing and a non-EPS producing Staphylococcus epidermidis strain from biofilms on stainless steel. After transmission, donor surfaces remained fully covered with biofilm, indicating transmission through cohesive failure in the biofilm. Counter to the numbers of biofilm bacteria, the donor and receiver biofilm thicknesses did not add up to the pre-transmission donor biofilm thickness, suggesting more compact biofilms after transmission, especially for non-EPS producing staphylococci. Accordingly, staphylococcal density per unit biofilm volume had increased from 0.20 to 0.52 μm^–3 for transmission of the non-EPS producing strain under high contact pressure. The EPS producing strain had similar densities before and after transmission (0.17 μm^–3). This suggests three phases in biofilm transmission: (1) compression, (2) separation and (3) relaxation of biofilm structure to its pre-transmission density in EPS-rich biofilms.