Domestic hot-water boilers harbour active thermophilic bacterial communities distinctly different from those in the cold-water supply.

Running cold and hot water in buildings is a widely established commodity. However, interests regarding hygiene and microbiological aspects had so far been focussed on cold water. Little attention has been given to the microbiology of domestic hot-water installations (DHWIs), except for aspects of pathogenic Legionella. World-wide, regulations consider hot (or warm) water as 'heated drinking water' that must comply (cold) drinking water (DW) standards. However, the few reports that exist indicate presence and growth of microbial flora in DHWIs, even when supplied with water with disinfectant residual. Using flow cytometric (FCM) total cell counting (TCC), FCM-fingerprinting, and 16S rRNA-gene-based metagenomic analysis, the characteristics and composition of bacterial communities in cold drinking water (DW) and hot water from associated boilers (operating at 50 - 60 °C) was studied in 14 selected inhouse DW installations located in Switzerland and Austria. A sampling strategy was applied that ensured access to the bulk water phase of both, supplied cold DW and produced hot boiler water. Generally, 1.3- to 8-fold enhanced TCCs were recorded in hot water compared to those in the supplied cold DW. FCM-fingerprints of cold and corresponding hot water from individual buildings indicated different composition of cold- and hot-water microbial floras. Also, hot waters from each of the boilers sampled had its own individual FCM-fingerprint. 16S rRNA-gene-based metagenomic analysis confirmed the marked differences in composition of microbiomes. E.g., in three neighbouring houses supplied from the same public network pipe each hot-water boiler contained its own thermophilic bacterial flora. Generally, bacterial diversity in cold DW was broad, that in hot water was restricted, with mostly thermophilic strains from the families Hydrogenophilaceae, Nitrosomonadaceae and Thermaceae dominating. Batch growth assays, consisting of cold DW heated up to 50 - 60 °C and inoculated with hot water, resulted in immediate cell growth with doubling times between 5 and 10 h. When cold DW was used as an inoculum no significant growth was observed. Even boilers supplied with UVC-treated cold DW contained an actively growing microbial flora, suggesting such hot-water systems as autonomously operating, thermophilic bioreactors. The generation of assimilable organic carbon from dissolved organic carbon due to heating appears to be the driver for growth of thermophilic microbial communities. Our report suggests that a man-made microbial ecosystem, very close to us all and of potential hygienic importance, may have been overlooked so far. Despite consumers having been exposed to microbial hot-water flora for a long time, with no major pathogens so far been associated specifically with hot-water usage (except for Legionella), the role of harmless thermophiles and their interaction with potential human pathogens able to grow at elevated temperatures in DHWIs remains to be investigated.

Scale-up of phosphate remobilization from sewage sludge in a microbial fuel cell.

Phosphate remobilization from digested sewage sludge containing iron phosphate was scaled-up in a microbial fuel cell (MFC). A 3litre triple chambered MFC was constructed. This reactor was operated as a microbial fuel cell and later as a microbial electrolysis cell to accelerate cathodic phosphate remobilization. Applying an additional voltage and exceeding native MFC power accelerated chemical base formation and the related phosphate remobilization rate. The electrolysis approach was extended using a platinum-RVC cathode. The pH rose to 12.6 and phosphate was recovered by 67% in 26h. This was significantly faster than using microbial fuel cell conditions. Shrinking core modelling particle fluid kinetics showed that the reaction resistance has to move inside the sewage sludge particle for considerable rate enhancement. Remobilized phosphate was subsequently precipitated as struvite and inductively coupled plasma mass spectrometry indicated low levels of cadmium, lead, and other metals as required by law for recycling fertilizers.

BioMig--A Method to Evaluate the Potential Release of Compounds from and the Formation of Biofilms on Polymeric Materials in Contact with Drinking Water.

In contact with water, polymeric materials (plastics) release compounds that can support suspended microbial growth and/or biofilm formation. The different methods presently used in the European Union to test plastics take 7-16 weeks to obtain a result. In industry, this delays material and product development as well as quality testing. Therefore, we developed a method package (BioMig) that allows testing of plastic materials with high reproducibility in 2 weeks for their potential biofilm (or biomass) formation and release of carbonaceous migration products when in contact with water. BioMig consists of (i) an extended migration potential test (seven times for 24 h at 60 °C), based on the European norm EN 12873-1 and the German UBA (Umweltbundesamt) guideline, and (ii) a biomass formation potential (BFP) test (14 days at 30 °C), which is a modified version of the Dutch biofilm production potential test. In the migration potential test, the amount of carbon released into water by the specimen is quantified by monitoring total and assimilable organic carbon over time; furthermore, the modular design of the test also allows one to assess additional parameters such as pathogen growth potential on the migration water or toxic effects on microbial growth. Flow cytometry (FCM)-based total cell counting (TCC) is used to quantify microbial growth in suspension and on surfaces after removal with mild sonication without affecting cell integrity. The BFP test allows one to determine both the planktonic (pBFP) and the sessile (sBFP) cell fractions. The sBFP consists of surface-attached cells after removal (>90% efficiency). Results for four standard test materials (PE-Xa, PE-Xc, EPDM 2%, and EPDM 20%), plus positive (PVC-P) and negative (glass) controls are presented. FCM-based TCC demonstrates that the release of growth-supporting carbon and proliferation of surface-attached cells stops increasing and stabilizes after 14 days of incubation; this allows for faster assessment of growth-supporting properties of plastics with BioMig compared to established tests.

Effect of Global Regulators RpoS and Cyclic-AMP/CRP on the Catabolome and Transcriptome of Escherichia coli K12 during Carbon- and Energy-Limited Growth.

For heterotrophic microbes, limited availability of carbon and energy sources is one of the major nutritional factors restricting the rate of growth in most ecosystems. Physiological adaptation to this hunger state requires metabolic versatility which usually involves expression of a wide range of different catabolic pathways and of high-affinity carbon transporters; together, this allows for simultaneous utilization of mixtures of carbonaceous compounds at low concentrations. In Escherichia coli the stationary phase sigma factor RpoS and the signal molecule cAMP are the major players in the regulation of transcription under such conditions; however, their interaction is still not fully understood. Therefore, during growth of E. coli in carbon-limited chemostat culture at different dilution rates, the transcriptomes, expression of periplasmic proteins and catabolomes of strains lacking one of these global regulators, either rpoS or adenylate cyclase (cya), were compared to those of the wild-type strain. The inability to synthesize cAMP exerted a strong negative influence on the expression of alternative carbon source uptake and degradation systems. In contrast, absence of RpoS increased the transcription of genes belonging to high-affinity uptake systems and central metabolism, presumably due to reduced competition of σ(D) with σ(S). Phenotypical analysis confirmed this observation: The ability to respire alternative carbon substrates and to express periplasmic high-affinity binding proteins was eliminated in cya and crp mutants, while these properties were not affected in the rpoS mutant. As expected, transcription of numerous stress defence genes was negatively affected by the rpoS knock-out mutation. Interestingly, several genes of the RpoS stress response regulon were also down-regulated in the cAMP-negative strain indicating a coordinated global regulation. The results demonstrate that cAMP is crucial for catabolic flexibility during slow, carbon-limited growth, whereas RpoS is primarily involved in the regulation of stress response systems necessary for the survival of this bacterium under hunger conditions.

Microbial growth and physiology: a call for better craftsmanship.

Virtually every microbiological experiment starts with the cultivation of microbes. Consequently, as originally pointed out by Monod (1949), handling microbial cultures is a fundamental methodology of microbiology and mastering different cultivation techniques should be part of every microbiologist's craftsmanship. This is particularly important for research in microbial physiology, as the composition and behavior of microbes is strongly dependent on their growth environment. It has been pointed out repeatedly by eminent microbiologists that we should give more attention to the media and culturing conditions. However, this is obviously not adhered to with sufficient rigor as mistakes in basic cultivation principles are frequently found in the published research literature. The most frequent mistakes are the use of inappropriate growth media and little or no control of the specific growth rate, and some examples will be discussed here in detail. Therefore, this is a call for better microbiological craftsmanship when cultivating microbial cultures for physiological experiments. This call is not only addressed to researchers but it is probably even more important for the teaching of our discipline.

Characterization of the Escherichia coli σ(S) core regulon by Chromatin Immunoprecipitation-sequencing (ChIP-seq) analysis.

In bacteria, selective promoter recognition by RNA polymerase is achieved by its association with σ factors, accessory subunits able to direct RNA polymerase "core enzyme" (E) to different promoter sequences. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), we searched for promoters bound by the σ(S)-associated RNA polymerase form (Eσ(S)) during transition from exponential to stationary phase. We identified 63 binding sites for Eσ(S) overlapping known or putative promoters, often located upstream of genes (encoding either ORFs or non-coding RNAs) showing at least some degree of dependence on the σ(S)-encoding rpoS gene. Eσ(S) binding did not always correlate with an increase in transcription level, suggesting that, at some σ(S)-dependent promoters, Eσ(S) might remain poised in a pre-initiation state upon binding. A large fraction of Eσ(S)-binding sites corresponded to promoters recognized by RNA polymerase associated with σ(70) or other σ factors, suggesting a considerable overlap in promoter recognition between different forms of RNA polymerase. In particular, Eσ(S) appears to contribute significantly to transcription of genes encoding proteins involved in LPS biosynthesis and in cell surface composition. Finally, our results highlight a direct role of Eσ(S) in the regulation of non coding RNAs, such as OmrA/B, RyeA/B and SibC.

Using coagulation to restrict microbial re-growth in tap water by phosphate limitation in water treatment.

Extensive microbial re-growth in a drinking water distribution system can deteriorate water quality. The limiting factor for microbial re-growth in a tap water produced by a conventional drinking water treatment plant in China was identified by determining the microbial re-growth potential (MRP) by adding different nutrients to stimulate growth of a natural microbial consortium as inoculum and flow-cytometric enumeration. No obvious change of MRP was found in tap water after addition of carbon, whereas, a 1- to 2-fold increase of MRP was observed after addition of phosphate (P). This clearly demonstrated that microbial re-growth in this tap water was limited by P. Most of the re-grown microbial flora (>85%) consisted of high nucleic acid content cells. A subsequent investigation of the MRP in the actual water treatment plant demonstrated that coagulation was the crucial step for decreasing MRP and producing P-limited water. Therefore, a comparison concerning the control of MRP by three different coagulants was conducted. It showed that all the three coagulants efficiently reduced the MRP and shifted the limitation regime from C to P, but the required dose was different. The study shows that it is feasible to restrict microbial re-growth by P limitation using coagulation in water treatment.

Improved productivity of poly (4-hydroxybutyrate) (P4HB) in recombinant Escherichia coli using glycerol as the growth substrate with fed-batch culture.

BACKGROUND: The most successful polyhydroxyalkanoate (PHA) in medical applications is poly(4-hydroxybutyrate) (P4HB), which is due to its biodegradability, biocompatibility and mechanical properties. One of the major obstacles for wider applications of P4HB is the cost of production and purification. It is highly desired to obtain P4HB in large scale at a competitive cost. RESULTS: In this work, we studied the possibility to increase P4HB productivity by using high cell density culture. To do so, we investigated for the first time some of the most relevant factors influencing P4HB biosynthesis in recombinant Escherichia coli. We observed that P4HB biosynthesis correlated more with limitations of amino acids and less with nitrogen depletion, contrary to the synthesis of many other types of PHAs. Furthermore, it was found that using glycerol as the primary carbon source, addition of acetic acid at the beginning of a batch culture stimulated P4HB accumulation in E. coli. Fed-batch high cell density cultures were performed to reach high P4HB productivity using glycerol as the sole carbon source for cell growth and 4HB as the precursor for P4HB synthesis. A P4HB yield of 15 g L-1 was obtained using an exponential feeding mode, leading to a productivity of 0.207 g L-1 h-1, which is the highest productivity for P4HB reported so far. CONCLUSIONS: We demonstrated that the NZ-amines (amino acids source) in excess abolished P4HB accumulation, suggesting that limitation in certain amino acid pools promotes P4HB synthesis. Furthermore, the enhanced P4HB yield could be achieved by both the effective growth of E. coli JM109 (pKSSE5.3) on glycerol and the stimulated P4HB synthesis via exogenous addition of acetic acid. We have developed fermentation strategies for P4HB production by using glycerol, leading to a productivity of 0.207 g L-1 h-1 P4HB. This high P4HB productivity will decrease the total production cost, allowing further development of P4HB applications.

Abundance and composition of indigenous bacterial communities in a multi-step biofiltration-based drinking water treatment plant.

Indigenous bacterial communities are essential for biofiltration processes in drinking water treatment systems. In this study, we examined the microbial community composition and abundance of three different biofilter types (rapid sand, granular activated carbon, and slow sand filters) and their respective effluents in a full-scale, multi-step treatment plant (Zürich, CH). Detailed analysis of organic carbon degradation underpinned biodegradation as the primary function of the biofilter biomass. The biomass was present in concentrations ranging between 2-5 × 10(15) cells/m(3) in all filters but was phylogenetically, enzymatically and metabolically diverse. Based on 16S rRNA gene-based 454 pyrosequencing analysis for microbial community composition, similar microbial taxa (predominantly Proteobacteria, Planctomycetes, Acidobacteria, Bacteriodetes, Nitrospira and Chloroflexi) were present in all biofilters and in their respective effluents, but the ratio of microbial taxa was different in each filter type. This change was also reflected in the cluster analysis, which revealed a change of 50-60% in microbial community composition between the different filter types. This study documents the direct influence of the filter biomass on the microbial community composition of the final drinking water, particularly when the water is distributed without post-disinfection. The results provide new insights on the complexity of indigenous bacteria colonizing drinking water systems, especially in different biofilters of a multi-step treatment plant.

sigmaS, a major player in the response to environmental stresses in Escherichia coli: role, regulation and mechanisms of promoter recognition.

Bacterial cells often face hostile environmental conditions, to which they adapt by activation of stress responses. In Escherichia coli, environmental stresses resulting in significant reduction in growth rate stimulate the expression of the rpoS gene, encoding the alternative σ factor σ(S). The σ(S) protein associates with RNA polymerase, and through transcription of genes belonging to the rpoS regulon allows the activation of a 'general stress response', which protects the bacterial cell from harmful environmental conditions. Each step of this process is finely tuned in order to cater to the needs of the bacterial cell: in particular, selective promoter recognition by σ(S) is achieved through small deviations from a common consensus DNA sequence for both σ(S) and the housekeeping σ(70). Recognition of specific DNA elements by σ(S) is integrated with the effects of environmental signals and the interaction with regulatory proteins, in what represents a fascinating example of multifactorial regulation of gene expression. In this report, we discuss the function of the rpoS gene in the general stress response, and review the current knowledge on regulation of rpoS expression and on promoter recognition by σ(S).

Kinetics and yields of pesticide biodegradation at low substrate concentrations and under conditions restricting assimilable organic carbon.

The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter(-1). We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.

Poly(4-hydroxybutyrate) (P4HB) production in recombinant Escherichia coli: P4HB synthesis is uncoupled with cell growth.

BACKGROUND: Poly(4-hydroxybutyrate) (P4HB), belonging to the family of bacterial polyhydroxyalkanoates (PHAs), is a strong, flexible and absorbable material which has a large variety of medical applications like tissue engineering and drug delivery. For efficient production of P4HB recombinant Escherichia coli has been employed. It was previously found that the P4HB synthesis is co-related with the cell growth. In this study, we aimed to investigate the physiology of P4HB synthesis, and to reduce the total production cost by using cheap and widely available xylose as the growth substrate and sodium 4-hydroxybutyrate (Na-4HB) as the precursor for P4HB synthesis. RESULTS: Six different E. coli strains which are able to utilize xylose as carbon source were compared for their ability to accumulate P4HB. E. coli JM109 was found to be the best strain regarding the specific growth rate and the P4HB content. The effect of growth conditions such as temperature and physiological stage of Na-4HB addition on P4HB synthesis was also studied in E. coli JM109 recombinant in batch culture. Under the tested conditions, a cellular P4HB content in the range of 58 to 70% (w w(-1)) and P4HB concentrations in the range of 2.76 to 4.33 g L(-1) were obtained with a conversion yield (Y(P4HB/Na-4HB)) of 92% w w(-1) in single stage batch cultures. Interestingly, three phases were identified during P4HB production: the "growth phase", in which the cells grew exponentially, the "accumulation phase", in which the exponential cell growth stopped while P4HB was accumulated exponentially, and the "stagnation phase", in which the P4HB accumulation stopped and the total biomass remained constant. CONCLUSIONS: P4HB synthesis was found to be separated from the cell growth, i.e. P4HB synthesis mainly took place after the end of the exponential cell growth. High conversion rate and P4HB contents from xylose and precursor were achieved here by simple batch culture, which was only possible previously through fed-batch high cell density cultures with glucose.

A microbiology-based multi-parametric approach towards assessing biological stability in drinking water distribution networks.

Biological stability of drinking water implies that the concentration of bacterial cells and composition of the microbial community should not change during distribution. In this study, we used a multi-parametric approach that encompasses different aspects of microbial water quality including microbial growth potential, microbial abundance, and microbial community composition, to monitor biological stability in drinking water of the non-chlorinated distribution system of Zürich. Drinking water was collected directly after treatment from the reservoir and in the network at several locations with varied average hydraulic retention times (6-52 h) over a period of four months, with a single repetition two years later. Total cell concentrations (TCC) measured with flow cytometry remained remarkably stable at 9.5 (± 0.6) × 10(4) cells/ml from water in the reservoir throughout most of the distribution network, and during the whole time period. Conventional microbial methods like heterotrophic plate counts, the concentration of adenosine tri-phosphate, total organic carbon and assimilable organic carbon remained also constant. Samples taken two years apart showed more than 80% similarity for the microbial communities analysed with denaturing gradient gel electrophoresis and 454 pyrosequencing. Only the two sampling locations with the longest water retention times were the exceptions and, so far for unknown reasons, recorded a slight but significantly higher TCC (1.3 (± 0.1) × 10(5) cells/ml) compared to the other locations. This small change in microbial abundance detected by flow cytometry was also clearly observed in a shift in the microbial community profiles to a higher abundance of members from the Comamonadaceae (60% vs. 2% at other locations). Conventional microbial detection methods were not able to detect changes as observed with flow cytometric cell counts and microbial community analysis. Our findings demonstrate that the multi-parametric approach used provides a powerful and sensitive tool to assess and evaluate biological stability and microbial processes in drinking water distribution systems.

Genome Sequence of a Freshwater Low-Nucleic-Acid-Content Bacterium, Betaproteobacterium Strain CB.

Betaproteobacterium strain CB is a typical minute freshwater bacterium, representing the small-cell bacteria that are numerically dominant in most freshwater environments. The genome of betaproteobacterium CB consists of a circular 2,045,720-bp chromosome, and the information we report will provide insights into the mechanisms underlying its survival and ecological function.

A defined, glucose-limited mineral medium for the cultivation of Listeria spp.

Members of the genus Listeria are fastidious bacteria with respect to their nutritional requirements, and several minimal media described in the literature fail to support growth of all Listeria spp. Furthermore, strict limitation by a single nutrient, e.g., the carbon source, has not been demonstrated for any of the published minimal media. This is an important prerequisite for defined studies of growth and physiology, including "omics." Based on a theoretical analysis of previously published mineral media for Listeria, an improved, well-balanced growth medium was designed. It supports the growth, not only of all tested Listeria monocytogenes strains, but of all other Listeria species, with the exception of L. ivanovii. The growth performance of L. monocytogenes strain Scott A was tested in the newly designed medium; glucose served as the only carbon and energy source for growth, whereas neither the supplied amino acids nor the buffering and complexing components (MOPS [morpholinepropanesulfonic acid] and EDTA) supported growth. Omission of amino acids, trace elements, or vitamins, alone or in combination, resulted in considerably reduced biomass yields. Furthermore, we monitored the specific growth rates of various Listeria strains cultivated in the designed mineral medium and compared them to growth in complex medium (brain heart infusion broth [BHI]). The novel mineral medium was optimized for the commonly used strain L. monocytogenes Scott A to achieve optimum cell yields and maximum specific growth rates. This mineral medium is the first published synthetic medium for Listeria that has been shown to be strictly carbon (glucose) limited.

Cryptosporidium spp. in drinking water. Samples from rural sites in Switzerland.

In most rural areas and small communities in Switzerland the drinking water is supplied to the consumers after a minimum or even no treatment at all. However, it is just in these areas where drinking water from sources of agricultural activities can be contaminated by liquid manure and faeces of pasturing animals. The Swiss drinking water regulations are limited to the monitoring of E. coli, Enterococcus spp. and total plate counts only. Hence, resistant pathogens, as for example Cryptosporidium spp., remain unnoticed. During a drinking water survey, which lasted from June 2003 to December 2004, water samples were collected from 3 selected rural sites in Switzerland. The drinking water was investigated for Cryptosporidium spp., E. coli, Enterococcus spp., Clostridium perfringens and other parameters. In all samples oocysts of Cryptosporidium spp. were detected at elevated concentrations of up to 0.18 oocysts/l. Between 28% and 75% of the oocysts were found to be vital by the excystation method. Sampled oocysts collected from the three sites were subjected to genotyping and in one case the isolate was found to belong to the genotype of C. parvum. No evidence for increased incidents of diarrhoea in the past years was noted by local authorities.

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS-FCM method). The method requires 120 min and can discriminate 'viable' and 'membrane-damaged' cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also 'viable but not culturable' (VNBC) cells are detected by our method, this result was expected. The IMS-FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1-12. This and the fact that no Lp-containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS-FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems.

Competition of Escherichia coli O157 with a drinking water bacterial community at low nutrient concentrations.

In contrast to studies on (long-term) survival of enteric pathogens in the environment, investigations on the principles of their growth and competition with autochthonous aquatic bacteria are rare and unexplored. Hence, improved basic knowledge is crucial for an adequate risk assessment and for understanding (and avoiding) the spreading of waterborne diseases. Therefore, the pathogen Escherichia coli O157 was grown in competition with a drinking water bacterial community on natural assimilable organic carbon (AOC) originating from diluted wastewater, in both batch and continuous culture. Growth was monitored by flow cytometry enabling enumeration of total cell concentration as well as specific E. coli O157 detection using fluorescently-labelled antibodies. An enhanced competitive fitness of E. coli O157 with higher AOC concentrations, higher temperatures and increased dilution rates (continuous culture) was observed. A classical "opportunist" versus "gleaner" relationship, where E. coli O157 is the "opportunist", specialised for growth at high nutrient concentrations (µ(max): 0.87 h(-1) and K(s): 489 µg consumed DOC L(-1)), and the bacterial community is the "gleaner" adapted to nutrient-poor environments (µ(max): 0.33 h(-1) and K(s): 7.4 µg consumed DOC L(-1)) was found. The obtained competition results can be explained by the growth properties of the two competitors determined in pure cultures and it was possible to model many of the observed dynamics based on Monod kinetics. The study provides new insights into the principles governing competition of an enteric pathogen with autochthonous aquatic bacteria.

Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440.

BACKGROUND: Pseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain. RESULTS: The genes encoding xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h(-1) and a final cell dry weight (CDW) of 2.5 g L(-1) with a maximal yield of 0.5 g CDW g(-1) xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w(-1) of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions. CONCLUSIONS: Sequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.