Investigation of biofilm growth and attrition in a three‐phase airlift bioreactor using 35SO42− as a radiolabelled tracer

The growth and biomass loss pattern for immobilized cells growing on diatomaceous earth particles in a three‐phase airlift bioreactor (TPALB) is studied using ³⁵S as a radiolabelled tracer. A monoculture of Beneckea natriegens was grown immobilized on support particles in a 3 L TPALB. Sterile conditions were maintained during the experiments, and n‐propanol was used as the sole carbon source. After the system reached steady state, the unlabelled sulfate (SO₄^2?) in the feed tank was substituted by a radioactive grade (³⁵SO₄^2?), and the assimilation of ³⁵S in the immobilized and in the suspended cells was monitored. The results indicated that the growth rate of immobilized biomass was not uniform throughout the biofilm. More specifically, it was concluded that cells growing closer to the external biofilm layer exhibit a higher growth rate, and that biomass loss from the biofilm was through attrition at the outer biofilm surface rather than by sloughing off of whole sections of biofilm. Copyright © 2006 Society of Chemical Industry     

Simulation of microbial mass and its variation in biofilm systems using STELLA

Biofilm systems have been extensively used for treating different types of wastewater. Difficulty in determination of microbial mass in fixed‐film reactors has been always the greatest problem in evaluating effects of loading rates on the microbial population in such reactors. For this reason, the effect of operating parameters such as organic loadings on the available microbial mass in the system and solids retention time (SRT) have not been discussed in detail. In this study an innovative methodology was developed to simulate the quantity of microbial mass in an aerated submerged fixed‐film reactor (ASFFR) reactor. After determination of kinetic parameters, a dynamic model was developed using STELLA, popular dynamic modeling software, to simulate the microbial mass in the reactor at run time. The pilot plant study was performed with two different surface media and at different loading rates from 2.37 to 19.56 g m^?2 d^?1. Furthermore, the effect of different organic loadings on the accumulation of microbial mass and SRT have been studied and the relevant mathematical relationships were presented. This method makes the evaluation of biofilm system simple and practical without taking samples to quantify microbial mass in reactors. Copyright © 2006 Society of Chemical Industry     

Continuous bioremediation of phenol‐polluted air in an external loop airlift bioreactor with a packed bed

An external loop airlift bioreactor with a small amount (99% porosity) of stainless steel mesh packing inserted in the riser section was used for bioremediation of a phenol‐polluted air stream. The packing enhanced volatile organic chemical and oxygen mass transfer rates and provided a large surface area for cell immobilization. Using a pure strain of Pseudomonas putida, fed‐batch and continuous runs at three different dilution rates were completed with phenol in the polluted air as the only source of growth substrate. 100% phenol removal was achieved at phenol loading rates up to 33 120 mg h^?1 m^?3 using only one‐third of the column, superior to any previously reported biodegradation rates of phenol‐polluted air with 100% efficiency. A mathematical model has been developed and is shown to accurately predict the transient and steady‐state data. Copyright © 2006 Society of Chemical Industry     

Surface phenomena and hydrodynamic effects on the deposition of pseudomonas fluorescens

Biofilm adhesion to metals (copper, aluminium and brass) was studied at two different velocities and pH values of 7 and 9. Both bacteria and metals showed negative surface charges at those values of pH, which tends to slow down adhesion. Film densities increased with the fluid velocity and were also affected by the pH and by the growth rate of the bacteria. Long duration tests based on heat transfer measurements were run at five different fluid velocities and at pH = 7, showing in general an asymptotic behaviour and a control of deposition by adhesion and growth phenomena.     

丁香酚对解淀粉芽孢杆菌气液界面生物膜的抑制作用

为研究丁香酚对解淀粉芽孢杆菌DY1a生物膜的抑制作用及成膜早期界面黏附聚集能力的影响,分析了不同质量浓度的丁香酚对生物膜形成、生物膜表面微观结构、膜内活菌数及生物膜基质多糖和蛋白质含量的影响,并评价其对成熟生物膜的清除能力,通过细菌运动能力实验、细胞表面疏水性、细胞表面Zeta电位及细胞自聚集能力,综合分析丁香酚对生物膜形成早期界面黏附聚集能力的影响。结果表明:丁香酚对解淀粉芽孢杆菌的最低生物膜抑制质量浓度(MBIC)为1.500mg/mL,MBIC的丁香酚对成熟生物膜清除率为28.85%。添加1/2 MBIC、1/4 MBIC的丁香酚培养基气液界面形成的生物膜较为单薄,表面较为光滑平整,生物膜内活菌数显著降低。丁香酚对腐败菌泳动能力抑制率为22.16%~100.00%,对丛集能力的抑制率为43.86%~97.50%,使细胞表面疏水性、细胞表面负Zeta电位和自聚集率均降低。此外,丁香酚显著抑制了腐败菌胞外多糖和蛋白质合成。丁香酚对芽孢杆菌生物膜的抑制作用主要是通过抑制细菌运动能力,降低细胞表面的疏水性、自聚集性,从而干扰早期菌体在成膜界面上的黏附能力,并通过抑制胞外聚合物组分的合成分泌延迟生物膜的形成和成熟。丁香酚可作为抑制腐败解淀粉芽孢杆菌气液界面生物膜形成的潜在抗生物膜剂。     

群体感应信号分子AI-2调控乳酸菌生物膜形成机制的研究进展

乳酸菌生物膜具有黏附性、抗逆性以及抗菌活性等多种物理特性和生理功能,被广泛应用于改善食品质构以及食品的生物保鲜中。乳酸菌生物膜的形成需经历附着、形成、成熟、老化脱落和重新附着5个阶段,其形成受到群体感应系统的调控。群体感应系统(quorum sensing system,QS)是细菌中广泛存在的一种基因表达调控系统,该系统通过信号分子靶向调控相关基因的表达,从而调控细菌的生理功能。LuxS/AI-2型QS是调控乳酸菌益生特性的主要群体感应系统。明悉乳酸菌的LuxS/AI-2型QS及其调控生物膜形成的机制,对于乳酸菌在食品工业中的进一步应用至关重要。重点介绍了乳酸菌生物膜的形成过程,以及LuxS/AI-2型QS调控乳酸菌生物膜形成的5个调控元件,即信号分子AI-2(autoinducer-2)、关键调控基因(luxS、tuf、fba、gap)、关键调控蛋白(LuxS、LacI、AraC、PadR以及Rbs家族蛋白)、信号分子AI-2的可能受体蛋白(LuxP、LsrB、RbsB和含有dCACHE结构域的受体蛋白)以及关键代谢路径的最新研究进展;总结了信号分子AI-2调控乳酸菌生物膜形成的可能分子机制,以及信号分子AI-2受体蛋白的筛选策略。希望为深入了解LuxS/AI-2型QS调控乳酸菌生物膜的形成提供理论指导。     

基于网络药理学-分子对接的藤椒精油抗解淀粉芽孢杆菌生物被膜机理

为探讨藤椒精油抗解淀粉芽孢杆菌生物被膜的关键活性分子及其胞内作用机理,采用PharmMapper数据库进行靶点预测,结合基因组注释信息对筛选后的靶点进行清洗过滤,预测筛选藤椒精油关键活性组分及抗生物被膜的作用靶点,并应用Cytoscape软件构建藤椒精油活性分子-潜在靶点互作网络,通过String数据库对潜在靶点进行GO与KEGG通路富集分析。应用STITCH数据库获得潜在靶点互作关系,Cytoscape软件进行网络拓扑分析,筛选关键核心靶点。采用CB-DOCK2在线分子对接服务器进行藤椒精油活性分子与核心靶点的分子对接。最后通过离体培养结合结晶紫染色法、细菌运动能力实验验证藤椒精油关键活性分子的抗生物被膜活性。结果表明,通过网络药理学方法共筛选获得了21种藤椒精油活性组分,133个潜在作用靶点,8个核心靶点。GO富集分析共获得17个GO二级条目,包括生物学过程条目6个、分子功能条目8个和细胞组分条目3个。KEGG通路富集分析共获得8条信号通路,涉及次级代谢产物的生物合成、氨基酸的生物合成等通路。分子对接结果表明,藤椒精油关键活性组分中桉叶醇、金合欢醇乙酸酯、反式法尼醇可通过氢键和疏水相互作用等分子间作用力与核心靶点OdhA、CarB、PurF紧密结合并形成稳定构象。离体生物被膜培养验证实验结果表明,0.32~1.28μL/mL的金合欢醇乙酸酯、反式法尼醇对解淀粉芽孢杆菌均有显著的抗生物被膜活性。研究结果旨在为藤椒精油抗细菌生物被膜的胞内分子机理提供新的研究思路。     

Escherichia coli O157:H7 Biofilm Formation on Romaine Lettuce and Spinach Leaf Surfaces Reduces Efficacy of Irradiation and Sodium Hypochlorite Washes

Abstract: Escherichia coli O157:H7 contamination of leafy green vegetables is an ongoing concern for consumers. Biofilm-associated pathogens are relatively resistant to chemical treatments, but little is known about their response to irradiation. Leaves of Romaine lettuce and baby spinach were dip inoculated with E. coli O157:H7 and stored at 4 °C for various times (0, 24, 48, 72 h) to allow biofilms to form. After each time, leaves were treated with either a 3-min wash with a sodium hypochlorite solution (0, 300, or 600 ppm) or increasing doses of irradiation (0, 0.25, 0.5, 0.75, or 1 kGy). Viable bacteria were recovered and enumerated. Chlorine washes were generally only moderately effective, and resulted in maximal reductions of 1.3 log CFU/g for baby spinach and 1.8 log CFU/g for Romaine. Increasing time in storage prior to chemical treatment had no effect on spinach, and had an inconsistent effect on 600 ppm applied to Romaine. Allowing time for formation of biofilm-like aggregations reduced the efficacy of irradiation. D₁₀ values (the dose required for a 1 log reduction) significantly increased with increasing storage time, up to 48 h postinoculation. From 0 h of storage, D₁₀ increased from 0.19 kGy to a maximum of 0.40 to 0.43 kGy for Romaine and 0.52 to 0.54 kGy for spinach. SEM showed developing biofilms on both types of leaves during storage. Bacterial colonization of the stomata was extensive on spinach, but not on Romaine. These results indicate that the protection of bacteria on the leaf surface by biofilm formation and stomatal colonization can reduce the antimicrobial efficacy of irradiation on leafy green vegetables. Practical Application: Before incorporating irradiation into the overall GMP/GHP chain, a packer or processor of leafy green vegetables must determine at what stage of processing and shipping the irradiation should take place. As a penetrating process, irradiation is best applied as a postpackaging intervention. Time in refrigerated storage between packaging and processing may alter the antimicrobial efficacy of irradiation. Irradiation on a commercial scale should include efforts to minimize the time delay between final packaging and irradiation of leafy vegetables.     

Microscopic observation of multispecies biofilm of various structures on whey concentration membranes

The objective of this study was to evaluate biofilm formation on polyamide reverse osmosis (RO) whey concentration membranes. Biofilms were observed with scanning electron and fluorescence microscopy. For scanning electron microscopy, pieces of 6-, 12-, and 14-mo-old membranes were allowed to air dry at room temperature (22°C) for 24 h followed by sputter coating with a 5-nm layer of gold and microscopic observations. Scanning electron microscopy images revealed that the hydrophilic layer, used to prevent membrane plugging, was not evenly distributed on the surface. Although this hydrophilic layer seemed to prevent the attachment of proteins, it supported biofilm formation. Three different structures of multispecies biofilm were observed on the retentate side of the membrane: 1) a mono layer, 2) a 3-dimensional structure of a dense matrix of extracellular polymeric substances where different types of bacterial cells were embedded, and 3) cell aggregates. In some of the biofilms, a smooth layer (shell) covered cell aggregates. In the 6-mo-old membranes, part of the shell layer was broken off. Biofilms as observed on the RO membrane were described as having a hill-and-valley type of structure, with hills showing a mushroom-like appearance and valleys comprising dense matrices of extracellular polymers with embedded bacterial cells. Fluorescence microscopy showed live cells on the surface of the biofilm. It is concluded that both cells in the deep layers of biofilm and surface cells may resist cleaning and sanitation. The extent of biofilm formation and the presence of live cells on RO membranes after regular clean in place cycles indicate the need for a more effective cleaning regimen customized for dairy separation systems.     

Nitritation performance and biofilm development of co- and counter-diffusion biofilm reactors: Modeling and experimental comparison

A comparative study was conducted on the start-up performance and biofilm development in two different biofilm reactors with aim of obtaining partial nitritation. The reactors were both operated under oxygen limited conditions, but differed in geometry. While substrates (O₂, NH₃) co-diffused in one geometry, they counter-diffused in the other. Mathematical simulations of these two geometries were implemented in two 1-D multispecies biofilm models using the AQUASIM software. Sensitivity analysis results showed that the oxygen mass transfer coefficient (K_i) and maximum specific growth rate of ammonia-oxidizing (AOB) and nitrite-oxidizing bacteria (NOB) were the determinant parameters in nitrogen conversion simulations. The modeling simulations demonstrated that K_i had stronger effects on nitrogen conversion at lower (0-10 m d⁻¹) than at the higher values (>10 m d⁻¹). The experimental results showed that the counter-diffusion biofilms developed faster and attained a larger maximum biofilm thickness than the co-diffusion biofilms. Under oxygen limited condition (DO < 0.1 mg L⁻¹) and high pH (8.0-8.3), nitrite accumulation was triggered more significantly in co-diffusion than counter-diffusion biofilms by increasing the applied ammonia loading from 0.21 to 0.78 g NH₄⁺-N L⁻¹ d⁻¹. The co- and counter-diffusion biofilms displayed very different spatial structures and population distributions after 120 days of operation. AOB were dominant throughout the biofilm depth in co-diffusion biofilms, while the counter-diffusion biofilms presented a stratified structure with an abundance of AOB and NOB at the base and putative heterotrophs at the surface of the biofilm, respectively.