Type 2 quorum sensing monitoring, inhibition and biofilm formation in marine microrganisms.

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 µM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 µM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that QSI compounds must be selected in the specific system in which they are to function, and they cannot easily be transferred from one QS system to another.

Modeling of the Van Der Waals Forces during the Adhesion of Capsule-Shaped Bacteria to Flat Surfaces.

A novel model is developed to evaluate the van der Waals (vdW) interactions between a capsule shaped bacterium (P. putida) and flat minerals plates in different approach profiles: Vertically and horizontally. A comparison of the approaches to the well-developed spherical particle to mineral surface (semi-infinite wall and spherical) approach has been made in this investigation. The van der Waals (vdW) interaction potentials for a capsule-shaped bacterium are found using Hamaker's microscopic approach of sphere to plate and cylinder to plate either vertically or horizontally to the flat surface. The numerical results show that a horizontal orientated capsule shaped bacterium to mineral surface interaction was more attractive compared to a capsule shaped bacterium approaching vertically. The orientation of the bacterial approaching a surface as well as the type and topology of the mineral influence the adhesion of a bacteria to that surface. Furthermore, the density difference among each type of bacteria shape (capsule, cylinder, and sphere) require different amounts of energy to adhere to hematite and quartz surfaces.

Isolate-specific effects of patulin, penicillic Acid and EDTA on biofilm formation and growth of dental unit water line biofilm isolates.

Dental unit water line (DUWL) contamination by opportunistic pathogens has its significance in nosocomial infection of patients, health care workers, and life-threatening infections to immunocompromized persons. Recently, the quorum sensing (QS) system of DUWL isolates has been found to affect their biofilm-forming ability, making it an attractive target for antimicrobial therapy. In this study, the effect of two quorum-sensing inhibitory compounds (patulin; PAT, penicillic acid; PA) and EDTA on planktonic growth, AI-2 signalling and in vitro biofilm formation of Pseudomonas aeruginosa, Achromobacter xylosoxidans and Achromobacter sp. was monitored. Vibrio harveyi BB170 bioassay and crystal violet staining methods were used to detect the AI-2 monitoring and biofilm formation in DUWL isolates, respectively. The V. harveyi BB170 bioassay failed to induce bioluminescence in A. xylosoxidans and Achromobacter sp., while P. aeruginosa showed AI-2 like activity suggesting the need of some pretreatments prior to bioassay. All strains were found to form biofilms within 72 h of incubation. The QSIs/EDTA combination have isolate-specific effects on biofilm formation and in some cases it stimulated biofilm formation as often as it was inhibited. However, detailed information about the anti-biofilm effect of these compounds is still lacking.

Investigating the effect of patulin, penicillic acid and EDTA on biofilm formation of isolates from dental unit water lines.

This study investigated the effect of patulin and penicillic acid, two known quorum-sensing inhibitors, and the common biocide ethylenediaminetetraacetic acid (EDTA) on the biofilm formation and auto-inducer (AI)-2 production of three isolates from dental unit water lines, Klebsiella sp., Bacillus subtilis and Bacillus cereus. Penicillic acid on its own had no effect on the biofilm formation of all isolates, whereas in combination with EDTA, it enhanced biofilm formation significantly in Klebsiella sp. and B. cereus. EDTA at concentrations greater than 10 microM promoted biofilm formation in B. cereus and B. subtilis. Patulin was found to promote biofilm formation in B. cereus up to 25 microM. A significant increase in biofilm formation was observed in B. cereus and B. subtilis at concentrations greater than 10 microM of patulin when combined with EDTA. The Vibrio harveyi BB170 AI-2 bioassay showed a positive response for Klebsiella sp. AI-2 production with a maximum fold induction at the late exponential growth phase. Addition of glucose prolonged the AI-2 production phase considerably. No significant effect of patulin, penicillic acid alone as well as in combination with EDTA was observed on AI-2 production by Klebsiella sp. The findings have important implications for the design of biofilm prevention and eradication strategies.

Investigating and modelling the development of septic sewage in filled sewers under static conditions: a lab-scale feasibility study.

This paper describes a lab-scale study of the physical and bio-chemical processes associated with the development of septic conditions in sewer pipes filled with static sewage. The study has concentrated on the uptake of oxygen (OUR) and the subsequent changes in chemical oxygen demand (COD), sulphate, sulphide and nitrate concentration and the formation of volatile fatty acids (VFA). OUR of raw sewage ranged from 2 to 13 mg L(-1) h(-1). Apparent nitrate uptake and sulphide generation rates in static sewage varied between 0.2-0.7 mgNO(3) L(-1) h(-1) and 0.02-0.05 mgH(2)S-S L(-1) h(-1), respectively. A logistic function was used to simulate the sulphide generation process in static sewage. It was found that total COD (COD(total)) influenced the apparent sulphide generation rate while nitrate concentrations greater than 4 mg L(-1) controlled the onset of sulphide production in experiments without added sediment phase. Introducing a sediment phase appeared to accelerate hydrolysis and fermentation processes as evidenced by 5-14 times greater dissolved COD generation rates in the bulk water phase.

Anaerobic treatment of olive mill wastewater and piggery effluents fermented with Candida tropicalis.

Olive mill wastewater (OMW) contains high concentrations of phenolic compounds that are inhibitory to many microorganisms making it difficult to treat biologically prior to discharge in waterways. The total mono-cyclic phenol reduction in OMW in this study was carried out by aerobic pre-treatment using the yeast Candida tropicalis in a 18 L batch reactor at 30 degrees C for 12 days followed by anaerobic co-digestion. A COD removal of 62% and a reduction in the total mono-cyclic phenol content by 51% of the mixture was achieved in the aerobic pre-treatment. Pig slurry was added as co-substrate to supplement the low nitrogen levels in the olive mill wastewater. Subsequent anaerobic treatment was carried out in a 20L fixed-bed reactor at 37 degrees C and HRT between 11 and 45 days. After a long start-up period, the OLR was increased from 1.25 to 5 kg COD m(-3)day(-1) during the last 30 days, resulting in subsequent increase in overall COD removal and biogas production, up to maximum values of 85% and 29 L(biogas)L(reactor)(-1)day(-1), respectively. Methane content of the biogas produced from the anaerobic digestion ranged between 65% and 74%.

Characterization of the cell surface and cell wall chemistry of drinking water bacteria by combining XPS, FTIR spectroscopy, modeling, and potentiometric titrations.

Aquabacterium commune, a predominant member of European drinking water biofilms, was chosen as a model bacterium to study the role of functional groups on the cell surface that control the changes in the chemical cell surface properties in aqueous electrolyte solutions at different pH values. Cell surface properties of A. commune were examined by potentiometric titrations, modeling, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. By combining FTIR data at different pH values and potentiometric titration data with thermodynamic model optimization, the presence, concentration, and changes of organic functional groups on the cell surface (e.g., carboxyl, phosphoryl, and amine groups) were inferred. The pH of zero proton charge, pH(zpc) = 3.7, found from titrations of A. commune at different electrolyte concentrations and resulting from equilibrium speciation calculations suggests that the net surface charge is negative at drinking water pH in the absence of other charge determining ions. In situ FTIR was used to describe and monitor chemical interactions between bacteria and liquid solutions at different pH in real time. XPS analysis was performed to quantify the elemental surface composition, to assess the local chemical environment of carbon and oxygen at the cell wall, and to calculate the overall concentrations of polysaccharides, peptides, and hydrocarbon compounds of the cell surface. Thermodynamic parameters for proton adsorption are compared with parameters for other gram-negative bacteria. This work shows how the combination of potentiometric titrations, modeling, XPS, and FTIR spectroscopy allows a more comprehensive characterization of bacterial cell surfaces and cell wall reactivity as the initial step to understand the fundamental mechanisms involved in bacterial adhesion to solid surfaces and transport in aqueous systems.

The polymer physics and chemistry of microbial cell attachment and adhesion.

The attachment of microbial cells to solid substrata is a primary ecological strategy for the survival of species and the development of specific activity and function within communities. An hypothesis arising from a biological sciences perspective may be stated as follows: The attachment of microbes to interfaces is controlled by the macromolecular structure of the cell wall and the functional genes that are induced for its biological synthesis. Following logically from this is the view that diverse attached cell behaviour is mediated by the physical and chemical interactions of these macromolecules in the interfacial region and with other cells. This aspect can be reduced to its simplest form by treating physico-chemical interactions as colloidal forces acting between an isolated cell and a solid or pseudo solid substratum. These forces can be analysed by established methods rooted in DLVO (Derjaguin, Landau, Verwey and Overbeek) theory. Such a methodology provides little insight into what governs changes in the behaviour of the cell wall attached to surfaces, or indeed other cells. Nor does it shed any light on the expulsion of macromolecules that modify the interface such as formation of slime layers. These physical and chemical problems must be treated at the more fundamental level of the structure and behaviour of the individual components of the cell wall, for example biosurfactants and extracellular polysaccharides. This allows us to restate the above hypothesis in physical sciences terms: Cell attachment and related cell growth behaviour is mediated by macromolecular physics and chemistry in the interfacial environment. Ecological success depends on the genetic potential to favourably influence the interface through adaptation of the macromolecular structure, We present research that merges these two perspectives. This is achieved by quantifying attached cell growth for genetically diverse model organisms, building chemical models that capture the variations in interfacial structure and quantifying the resulting physical interactions. Experimental observations combine aqueous chemistry techniques with surface spectroscopy in order to elucidate the cell wall structure. Atomic force microscopy methods quantify the physical interactions between the solid substrata and key components of the cell wall such as macromolecular biosurfactants. Our current approach focuses on considering individually mycolic acids or longer chain polymers harvested from cells, as well as characterised whole cells. This approach allows us to use a multifactorial approach to address the relative impact of the individual components of the cell wall in contact with model surfaces. We then combine these components to increase complexity step-wise, while comparing with the behaviour of entire cells. Eventually, such an approach should allow us to estimate and understand the primary factors governing microbial cell adhesion. Although the work addresses the cell-mineral interface at a fundamental level, the research is driven by a range of technology needs. The initial rationale was improved prediction of contaminant degradation in natural environments (soils, sediments, aquifers) for environmental cleanup. However, this area of research addresses a wide range of biotechnology areas including improved understanding of pathogen survival (e.g., in surgical environments), better process intensification in biomanufacturing (biofilm technologies) and new product development.

Interpretation of standard leaching test BS EN 12457-2: is your sample hazardous or inert?

A slag sample from a lead refiner has been obtained and given to two analytical laboratories to determine the release of trace elements from the sample according to BS EN 12457-2. Samples analysed by one laboratory passed waste acceptance criteria, leading it to be classified as an inert material; samples of the same material analysed by the other laboratory failed waste acceptance criteria and were classified as hazardous. It was found that the sample preparation procedure is the critical step in the leaching analysis and that the effects of particle size on leachability should be taken into account when using this standard. The purpose of this paper is to open a debate on designing a better defined standard leaching test and making current waste acceptance criteria more flexible.