Studies on formation,control and application of biofilm formed by food related microorganisms

Biofilms are sessile microbial aggregates on the interfaces, and they were usually considered as microbial contamination sources in medical care and various industries. We studied the control and application of biofilms formed by food-related microorganisms, and mechanism of the biofilm formation was also investigated. We studied the biofilm formation in mixed cultures using various combinations of two strains of food-related microorganisms. There were various microorganisms that showed decreased or increased biofilm formation in the mixed culture in comparison with that in a single culture. Biofilm formed by lactic acid bacteria and yeast isolated from traditional fermented food, Fukuyama pot vinegar, exhibited unique feature in that structure and formation mechanism, and expected to be used as an immobilized microorganism in fermentation production. Here our studies on the control and application of biofilms and the mechanisms of its formation were described.     

Modeling and simulation of competition between two microorganisms for a single inhibitory substrate in a biofilm reactor

A simple biofilm model was developed to simulate the competition between two microorganisms for a common inhibitory substrate. The following assumptions were made for the simulations: (1) the biofilm has a uniform thickness and is composed of 5 segments, (2) growth of two microorganisms A and B which utilize the common substrate is expressed by the Haldane kinetics with a spatial limitation term and is independent of the other microorganism in the biofilm reactor, and (3) diffusion of the substrate, movement of the microorganisms, and continuous loss of the biomass by shearing are expressed by Fick's Law-type equations. The qualitative behavior of the biofilm reactor is characterized by five regions, I-V, depending on the operation conditions, the substrate concentration in feed, and the dilution rate. In region I, both microorganisms are washed out of the biofilm reactor. In region II, microorganism B is washed out, and in region III, microorganism A is washed out of the biofilm. In region IV, both microorganisms coexist with one another. In region V, both microorganisms coexist with a sustained oscillatory behavior. Convergence to regions I-V depends on the initial conditions. In regions II-V, washout of either or both microorganisms is also observed with initial conditions too far away.     

In vitro modeling of dental water line contamination and decontamination

The contamination of dental unit water lines (DUWL) is an emerging concern in dentistry. The aim of this study was to use an in vitro DUWL to model microbial contamination and evaluate the decontamination efficacy of tetraacetylethylenediamine (TAED) solutions. A DUWL biofilm model used to simulate clinical conditions was used to generate a range of biofilms in DUWL. Three distinct biofilms were generated: (1) biofilm from water, (2) biofilm from a mix of water + contaminating human commensal bacteria, (3) biofilm from water with contaminating oral bacteria added after biofilm formed. The contaminating oral species used were Streptococcus oralis, Enterococcus faecalis and Staphylococcus aureus. Decontamination by simple water flushing or flushing with TAED was evaluated (2, 5 and 10 min intervals). The DUWL tubes were split and samples were plated onto a range of media, incubated and bacteria enumerated. Water flushing did not reduce the number of microorganisms detected. Bacteria were not detected from any of the TAED sampling points for any of the biofilm types tested. Interestingly, if contamination was introduced to new DUWL along with the waterborne species a biofilm was formed containing only the waterborne species. If however, an existing biofilm was present before the introduction of "contaminating" bacteria then these could be detected in the biofilm. This implies that if the DUWL are new or satisfactorily cleaned on a regular basis then the associated cross-contamination aspects are reduced. In conclusion, TAED provides effective control for DUWL biofilms.     

The Pseudomonas quinolone signal (PQS), and its precursor HHQ, modulate interspecies and interkingdom behaviour

The Pseudomonas quinolone signal (PQS), and its precursor 2-heptyl-4-quinolone (HHQ), play a key role in coordinating virulence in the important cystic fibrosis pathogen Pseudomonas aeruginosa. The discovery of HHQ analogues in Burkholderia and other microorganisms led us to investigate the possibility that these compounds can influence interspecies behaviour. We found that surface-associated phenotypes were repressed in Gram-positive and Gram-negative bacteria as well as in pathogenic yeast in response to PQS and HHQ. Motility was repressed in a broad range of bacteria, while biofilm formation in Bacillus subtilis and Candida albicans was repressed in the presence of HHQ, though initial adhesion was unaffected. Furthermore, HHQ exhibited potent bacteriostatic activity against several Gram-negative bacteria, including pathogenic Vibrio vulnificus. Structure-function analysis using synthetic analogues provided an insight into the molecular properties that underpin the ability of these compounds to influence microbial behaviour, revealing the alkyl chain to be fundamental. Defining the influence of these molecules on microbial-eukaryotic-host interactions will facilitate future therapeutic strategies which seek to combat microorganisms that are recalcitrant to conventional antimicrobial agents.     

Heterogeneity in the efficacy of dental chemical disinfectants on water-derived biofilms in vitro

Abstract

Conditions in dental unit waterlines are favourable for biofilm growth and contamination of dental unit water. The aim of this study was to assess the effect of several chemical disinfectants on bacteria in a biofilm model. Water-derived biofilms were grown in a static biofilm model (Amsterdam Active Attachment model), using two growth media. Biofilms were challenged with Alpron/Bilpron, Anoxyl, Citrisil, Dentosept, Green & Clean, ICX and Oxygenal in shock dose and maintenance doses. The concentration and the composition of the chemical disinfectants influenced the number of culturable bacteria in the biofilms. The application of a single shock dose followed by a low dose of the same chemical disinfectants resulted in the greatest suppression of viable bacteria in the biofilms. Exposure to Citrisil and ICX consistently resulted in failure to control the biofilms, while Alpron/Bilpron had a substantial and relevant effect on the number of bacteria in the biofilms.     

Resistance of the oil-oxidizing microorganism <Emphasis Type="Italic">Dietzia</Emphasis> sp. to hyperosmotic shock in reconstituted biofilms

A number of halotolerant and halophilic bacterial strains were isolated from the Romashkinskoe oil field (Tatarstan) stratal waters having a salinity of up to 100 g/l. The isolation of pure cultures involved biofilm reconstitution on M9 medium with paraffins. The associations obtained were dispersed and reinoculated onto solid media that contained either peptone and yeast extract (PY medium) or paraffins. It was shown that such associations included both oil-oxidizing bacteria and accompanying chemoheterotrophic bacteria incapable of oil oxidation. The pure cultures that were isolated were used for creating binary biofilms. In these biofilms, interactions between halophilic and nonhalophilic bacteria under hypo-and hyperosmotic shocks were investigated. We conducted a detailed study of a biofilm obtained from an oil-oxidizing halotolerant species (with an upper growth limit of 10–12% NaCl) identified as Dietzia sp. and an extremely halophilic gram-negative bacterium (growing within the 5–20% NaCl concentration range) of the genus Chromohalobacter that did not oxidize paraffins. If these microorganisms were grown in a mixed suspension (planktonic) culture that was not supplemented with an additional amount of NaCl, no viable cells of the halophilic microorganism were detected after reinoculation. In contrast, only halophilic cells were detected at a NaCl concentration of 15%. Thus, no mutual protective influence of the microorganisms manifested itself in suspension culture, either under hypoor under hyperosmotic shock. Neither could halophile cells be detected after reinoculating a biofilm obtained on a peptone medium without the addition of NaCl. However, biofilms produced at a NaCl concentration of 15% contained approximately equal numbers of cells of the halophilic and halotolerant organisms. Thus, the halophile in biofilms sustaining a hyperosmotic shock exerts a protective influence on the halotolerant microorganism. Preliminary data suggest that this effect is due to release by the halophile of osmoprotective substances (ectoine and glutamate), which are taken up by the halotolerant species. Such substances are diluted by a large medium volume in suspension cultures, whereas, in biofilms, their diffusion into the medium is apparently hampered by their interaction with the intercellular polymer matrix.     

Impact of flow velocity on the dynamic behaviour of biofilm bacteria

The impact of flow velocity (FV) on the growth dynamics of biofilms and bulk water heterotrophic plate count (HPC) bacteria in drinking water distribution systems was quantified and modeled by combining a logistic growth model with mass balance equations. The dynamic variations in the specific growth and release rates of biofilm bacteria were also quantified. The experimental results showed that the maximum biofilm biomass did not change when flow velocity was increased from 20 to 40 cm s(-1), but was significantly affected when flow velocity was further increased to 60 cm s(-1). Although the concentration of biofilm bacteria was substantially reduced by the higher shear stress, the concentration of bacteria in the bulk fluid was slightly increased. From this it is estimated that the specific growth rate and specific release rate of biofilm bacteria had doubled. The specific release (detachment) rate was dependent on the specific growth rate of the biofilm bacteria.     

Multiple substrate growth kinetics of Leptothrix discophora SP-6

The growth parameters of Leptothrix discophora SP-6 were quantified on the basis of the steady-state concentrations and utilization rates of pyruvate, dissolved oxygen, and concentration of microorganisms in a chemostat operated at 25 degrees C, pH 7.2, and an agitation rate of 350 rpm. The results showed that the microbial growth was limited by both pyruvate and dissolved oxygen. A combined growth kinetics model using Monod growth kinetics for pyruvate and Tessier growth kinetics for oxygen showed the best correlation with the experimental data when analyzed using an interactive multiple substrate model. The growth kinetics parameters and the respective confidence limits, estimated using the Monte Carlo simulation, were mu(max) = 0.576 +/- 0.021 h(-1), K(sMp) = 38.81 +/- 4.24 mg L(-1), K(sTo) = 0.39 +/- 0.04 mg L(-1), Y(X/p) = 0.150 (mg microorganism mg(-1) pyruvate), Y(X/o) = 1.24 (mg microorganism mg(-1) oxygen), the maintenance factors for pyruvate and oxygen were m(p) = 0.129 (mg pyruvate consumed mg(-1) microorganism h(-1)) and m(o) = 0.076 (mg oxygen consumed mg(-1) microorganism h(-1)), respectively.     

Population dynamics and in situ kinetics of nitrifying bacteria in autotrophic nitrifying biofilms as determined by real-time quantitative PCR

Population dynamics of ammonia-oxidizing bacteria (AOB) and uncultured Nitrospira-like nitrite-oxidizing bacteria (NOB) dominated in autotrophic nitrifying biofilms were determined by using real-time quantitative polymerase chain reaction (RTQ-PCR) and fluorescence in situ hybridization (FISH). Although two quantitative techniques gave the comparable results, the RTQ-PCR assay was easier and faster than the FISH technique for quantification of both nitrifying bacteria in dense microcolony-forming nitrifying biofilms. Using this RTQ-PCR assay, we could successfully determine the maximum specific growth rate (mu = 0.021/h) of uncultured Nitrospira-like NOB in the suspended enrichment culture. The population dynamics of nitrifying bacteria in the biofilm revealed that once they formed the biofilm, the both nitrifying bacteria grew slower than in planktonic cultures. We also calculated the spatial distributions of average specific growth rates of both nitrifying bacteria in the biofilm based on the concentration profiles of NH4+, NO2-, and O2, which were determined by microelectrodes, and the double-Monod model. This simple model estimation could explain the stratified spatial distribution of AOB and Nitrospira-like NOB in the biofilm. The combination of culture-independent molecular techniques and microelectrode measurements is a very powerful approach to analyze the in situ kinetics and ecophysiology of nitrifying bacteria including uncultured Nitrospira-like NOB in complex biofilm communities.     

常见变质水性工业产品中的微生物种类和耐药性分析

为了科学治理常见变质水性工业产品中的腐败微生物,对其进行分离、鉴定和分类,同时以卡松、布罗波尔、甲基异噻唑啉酮和苯并异噻唑啉酮四种杀菌防腐剂对6种标准细菌菌株的最小抑菌浓度(MIC)作为耐药性标准,对腐败细菌进行耐药性评估分析.结果显示,日化用品变质样中革兰氏阴性细菌约占80.00％(克雷伯氏菌属、假单胞菌属、伯克霍尔...     

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination.     

Determination of the kinetic parameters of the phenol-degrading thermophile Bacillus themoleovorans sp. A2

Phenolic compounds are pollutants in many wastewaters, e.g. from crude oil refineries, coal gasification plants or olive oil mills. Phenol removal is a key process for the biodegradation of pollutants at high temperatures because even low concentrations of phenol can inhibit microorganisms severely. Bacillus thermoleovorans sp. A2, a recently isolated thermophilic strain (temperature optimum 65 degrees C), was investigated for its capacity to degrade phenol. The experiments revealed that growth rates were about four times higher than those of mesophilic microorganisms such as Pseudomonas putida. Very high specific growth rates of 2.8 h(-1) were measured at phenol concentrations of 15 mg/l, while at phenol concentrations of 100-500 mg/l growth rates were still in the range of 1 h(-1). The growth kinetics of the thermophilic Bacillus thermoleovorans sp. A2 on phenol as sole carbon and energy source can be described using a three-parameter model developed in enzyme kinetics. The yield coefficient Yx/s of 0.8-1 g cell dry weight/g phenol was considerably higher than cell yields of mesophilic bacteria (Yx/s 0.40-0.52 g cell dry weight/g phenol). The highest growth rate was found at pH 6. Bacillus thermoleovorans sp. A2 was found to be insensitive to hydrodynamic shear stress in stirred bioreactor experiments (despite possible membrane damage caused by phenol) and flourished at an ionic strength of the medium of 0.25(-1) mol/l (equivalent to about 15-60 g NaCl/l). These exceptional properties make Bacillus thermoleovorans sp. A2 an excellent candidate for technical applications.     

Dynamics of Biofilm Formation and the Interaction between Candida albicans and Methicillin-Susceptible (MSSA) and -Resistant Staphylococcus aureus (MRSA)

Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL^-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.     

Effects of short-time drying on biofilm-associated bacteria

Microorganisms tend to form biofilms consisting of cells embedded in a highly hydrated extracellular polymeric matrix. The biofilm protects its inhabitants from antimicrobial agents, pH alterations, and confers protection against drying. It is known that biofilm-associated bacteria can survive for a while in the absence of water. When rehydrated, metabolic processes are quickly restored and microorganisms resume life. The aim of this study is to evaluate the survival of heterotrophic bacteria, sulphate-reducing bacteria and amoeba against short-time drying. Biofilms were allowed to grow for 30 and 60 days on stainless steel (316, 2B) coupons in annular biofilm reactor, which was fed with drinking water network under constant, non-turbulent shear stress and temperature. The results presented in this study indicate a role for biofilm layer in protecting biofilm-associated microorganisms from drying. The current study has provided that short-time (24 h) absence of water could not affect biofilm-associated heterotrophic microorganisms significantly, in terms of cell viability.     

Doubly entrapped baker's yeast survives during the long-term stereoselective reduction of ethyl 3-oxobutanoate in an organic solvent

To attain long-term bioreaction in organic solvents with living microorganisms, we tried to protect the microorganisms from the toxicity of the solvent by immobilization. In this study, baker's yeast, which is not tolerant to organic solvents such as isooctane, was selected as a model microorganism and the immobilized living yeast cells were examined for activity in the steroselective reduction of ethyl 3-oxobutanoate to ethyl (S)-3-hydroxybutanoate in isooctane; an activity that correlated well with the viability of the yeast cells. It was found that double entrapment, that is, further entrapment of calcium-alginate-gel-entrapped cells with a urethane prepolymer, made it possible for the yeast to remain viable in isooctane, although other conventional immobilization methods, such as single entrapment using polysaccharide or synthetic resin prepolymers, were insufficient for its protection. Furthermore, doubly entrapped living yeast cells could carry out the stereoselective reduction in isooctane repeatedly for a long period (more than 1200 h) with occasional cultivation. Thus, double entrapment enabled a microorganism sensitive to organic solvents to survive over long-term bioreaction in an organic solvent. Received: 29 August 1997 / Received last revision: 24 December 1997 / Accepted: 13 January 1998     

Quantification of airborne microorganisms and investigation of their interactions with non-living particles

A fluorescence method was developed for the direct counting of airborne microorganisms and for the examination of their interactions with other aerosol particles. The method is based on a combination of the aerosol sampling technique using a cascade impactor and the selective dyeing of the trapped microorganisms with ethidium bromide. The method enables both the total microorganism concentrations and their counts in clusters to be evaluated. The new method and the cultivation method, enabling the colony-forming units (CFU) to be determined, were compared. Based on the results obtained by the fluorescence technique, a procedure is suggested for the conversion of CFU data to bacteria and yeast concentrations.     

Impact of flow velocity on the dynamic behaviour of biofilm bacteria

Abstract

The impact of flow velocity (FV) on the growth dynamics of biofilms and bulk water heterotrophic plate count (HPC) bacteria in drinking water distribution systems was quantified and modeled by combining a logistic growth model with mass balance equations. The dynamic variations in the specific growth and release rates of biofilm bacteria were also quantified. The experimental results showed that the maximum biofilm biomass did not change when flow velocity was increased from 20 to 40 cm s^?1, but was significantly affected when flow velocity was further increased to 60 cm s^?1. Although the concentration of biofilm bacteria was substantially reduced by the higher shear stress, the concentration of bacteria in the bulk fluid was slightly increased. From this it is estimated that the specific growth rate and specific release rate of biofilm bacteria had doubled. The specific release (detachment) rate was dependent on the specific growth rate of the biofilm bacteria.     

Growth yield, maintenance requirements, and lipid formation in the oleaginous yeast Apiotrichum curvatum

For guiding and improving the efficiency of the production of lipid, complete insight into the flow of carbon and energy during growth and product formation is necessary. Therefore, data have been collected to determine various important growth parameters for the oleaginous yeast Apiotrichum curvatum. Chemostat experiments at specific growth rates, ranging from 0.05 to 0.15 h(-1) and recycling experiments with 100% biomass retention, with growth rates decreasing from 0.10 to 0.004 h(-1), demonstrated that maintenance requirements of A. curvatum are very low, compared to maintenance requirements described for other yeasts as Saccharomyces cerevisiae and Candida parapsilosis.It also appeared that growth and lipid production are proportional to substrate consumption when specific growth rates are higher than approximately 0.02 h(-1), but that lipid production stops at growth rates below this value. The practical consequences of these data are that fed batch cultures, which are often applied in fermentation industry, can only be useful with lipid producing yeasts when the growth rate in the process is carefully monitored to ensure specific growth rates higher than 0.02 h(-1). Dilution of the culture, partial recycling and/or a continuously increasing nutrient feed are solutions for the expected problems at low growth rates.     

Identification of a microorganism that links its growth to the reductive dechlorination of 2,3,5,6-chlorobiphenyl

Anaerobic bacteria reductively dechlorinate polychlorinated biphenyls (PCBs) in aquatic sediments, but these microorganisms remain uncultured and, until now, unidentified. Through denaturing gradient gel electrophoresis (DGGE) of 16S rDNA from a highly enriched ortho -PCB dechlorinating culture, the growth of a single microorganism was shown to be dependent upon the presence and dechlorination of 2,3,5,6-tetrachlorobiphenyl. This is the first identification of a microorganism that catalyses the reductive dechlorination of a PCB. The organism, bacterium o -17, has high sequence similarity with the green non-sulphur bacteria and with a group that includes Dehalococcoides ethenogenes . Bacterium o -17 required acetate for dechlorination and growth. H₂:CO₂ (80:20 at 101 kPa) did not support dechlorination or growth of the dechlorinator. Archaeal 16S rDNA was not detected in actively dechlorinating bromoethanesulphonate-treated non-methanogenic cultures, which indicated that methanogenic Archaea were not required for dechlorination. The consistent association with dechlorinating activity combined with high similarity to other known dechlorinating microorganisms indicates that bacterium o -17 catalyses the reductive ortho -dechlorination of 2,3,5,6-tetrachlorobiphenyl.     

The effect of bacteria and fungi on chemical weathering and chemical denudation fluxes in pine growth experiments

Vascular plants and associated microbial communities affect the nutrient resources of terrestrial ecosystems by impacting chemical weathering that transfers elements from primary minerals to other ecosystem pools, and chemical denudation that transports weathered elements out of the system in solution. We performed a year-long replicated flow-through column growth experiment to isolate the effects of vascular plants, ectomycorrhiza-forming fungi and associated bacteria on chemical weathering and chemical denudation. The study focused on Ca²⁺, K⁺ and Mg²⁺, for which the sole sources were biotite and anorthite mixed into silica sand. Concentrations of the cations were measured in input and output solutions, and three times during the year in plant biomass and on exchangeable cation sites of the growth medium. Weathering and denudation fluxes were estimated by mass balance, and mineral surface changes, biofilm and microbial attachments to surfaces were investigated with scanning electron microscopy. Both bacteria and fungi increased weathering fluxes compared to abiotic controls. Without a host plant denudation rates were as large as weathering rates i.e. the weathering to denudation ratio was about one. Based on whole year fluxes, ectomycorrhizal seedlings produced the greatest weathering to denudation ratios (1.5). Non-ectomycorrhizal seedlings also showed a high ratio of 1.3. Both ectomycorrhizal hyphal networks and root hairs of non-ectomycorrhizal trees, embedded in biofilm (microorganisms surrounded by extracellular polymers), transferred nutrients to the host while drainage losses were minimized. These results suggest that biofilms localize both weathering and plant nutrient uptake, isolating the root-hypha-mineral interface from bulk soil solution.