A non-invasive fluorescent staining procedure allows Confocal Laser Scanning Microscopy based imaging of Mycobacterium in multispecies biofilms colonizing and degrading polycyclic aromatic hydrocarbons

To study the micro scale interactions of Mycobacterium with bacteria belonging to other genera by means of Confocal Laser Scanning Microscopy (CLSM), a procedure was developed to non-invasively and fluorescently stain Mycobacterium without compromising the signal produced by commonly used fluorescent reporter genes. The procedure makes use of the commercial non-specific nucleic acid stain Syto62 and was optimized to efficiently stain Mycobacterium cells in suspensions and biofilms. The staining procedure was found non-invasive towards overall cell viability, biofilm architecture and fluorescence signals emitted by other organisms expressing the fluorescent reporter genes gfp and dsRed. The procedure was successfully applied to visualize the comportment of the PAH-degrading Mycobacterium sp. VM552 in triple species biofilms containing, in addition to strain VM552, the GFP labeled PAH-degrading Sphingomonas sp. LH128-GFP and DsRed-labeled Pseudomonas putida OUS82(RF), and colonizing a glass substrate coated with phenanthrene crystals in flow chambers. CLSM imaging and subsequent appropriate image processing of the biofilms show that the comportment of strain Mycobacterium sp. VM552 was largely affected by the presence of the other organisms. The data support the value of the staining procedure to study ecological questions about micro scale behavior and niche occupation of Mycobacterium in multi-species systems.     

A non-invasive fluorescent staining procedure allows Confocal Laser Scanning Microscopy based imaging of Mycobacterium in multispecies biofilms colonizing and degrading polycyclic aromatic hydrocarbons

To study the micro scale interactions of Mycobacterium with bacteria belonging to other genera by means of Confocal Laser Scanning Microscopy (CLSM), a procedure was developed to non-invasively and fluorescently stain Mycobacterium without compromising the signal produced by commonly used fluorescent reporter genes. The procedure makes use of the commercial non-specific nucleic acid stain Syto62 and was optimized to efficiently stain Mycobacterium cells in suspensions and biofilms. The staining procedure was found non-invasive towards overall cell viability, biofilm architecture and fluorescence signals emitted by other organisms expressing the fluorescent reporter genes gfp and dsRed. The procedure was successfully applied to visualize the comportment of the PAH-degrading Mycobacterium sp. VM552 in triple species biofilms containing, in addition to strain VM552, the GFP labeled PAH-degrading Sphingomonas sp. LH128-GFP and DsRed-labeled Pseudomonas putida OUS82(RF), and colonizing a glass substrate coated with phenanthrene crystals in flow chambers. CLSM imaging and subsequent appropriate image processing of the biofilms show that the comportment of strain Mycobacterium sp. VM552 was largely affected by the presence of the other organisms. The data support the value of the staining procedure to study ecological questions about micro scale behavior and niche occupation of Mycobacterium in multi-species systems.     

Microbial degradation of pentachlorophenol

Pentachlorophenol (PCP) was the most prevalent wood preservative for many years worldwide. Its widespread use had led to contamination of various environments. Traditional methods of PCP clean-up include storage in land-fill sites, incineration and abiotic degradation processes such as photodecomposition. Some aerobic and anaerobic microorganisms can degrade PCP under a variety of conditions. Axenic bacterial cultures, Flavobacterium sp., Rhodococcus sp., Arthrobacter sp., Pseudomonas sp., Sphingomonas sp., and Mycobacterium sp., and fungal cultures, Phanerochaete sp. and Trametes sp. exhibit varying rates and extent of PCP degradation. This paper provides some general information on properties of PCP and reviews the influence of nutrient amendment, temperature and pH on PCP degradation by various aerobic and anaerobic microorganisms. Where information is available, proposed degradation pathways, intermediates and enzymes are reviewed.     

Degradation and metabolism of synthetic plastics and associated products by Pseudomonas sp.: capabilities and challenges

Synthetic plastics, which are widely present in materials of everyday use, are ubiquitous and slowly‐degrading polymers in environmental wastes. Of special interest are the capabilities of microorganisms to accelerate their degradation. Members of the metabolically diverse genus Pseudomonas are of particular interest due to their capabilities to degrade and metabolize synthetic plastics. Pseudomonas species isolated from environmental matrices have been identified to degrade polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyurethane, polyethylene terephthalate, polyethylene succinate, polyethylene glycol and polyvinyl alcohol at varying degrees of efficiency. Here, we present a review of the current knowledge on the factors that control the ability of Pseudomonas sp. to process these different plastic polymers and their by‐products. These factors include cell surface attachment within biofilms, catalytic enzymes involved in oxidation or hydrolysis of the plastic polymer, metabolic pathways responsible for uptake and assimilation of plastic fragments and chemical factors that are advantageous or inhibitory to the biodegradation process. We also highlight future research directions required in order to harness fully the capabilities of Pseudomonas sp. in bioremediation strategies towards eliminating plastic wastes.     

Phylogeny of Sphingomonas species that degrade pentachlorophenol

Four pentachlorophenol (PCP)-degrading bacteria isolated from geographically diverse areas have been examined in detail as regards their physiology and phylogeny. According to traditional biochemical methods, these strains had been classified as members of the genera Arthrobacter, Flavobacterium, Pseudomonas, and Sphingomonas. The PCP degradation pathway has been studied extensively in Sphingomonas (Flavobacterium) sp strain ATCC 39723 and the first three degradation steps catalyzed by a PCP-4-monooxygenase (PcpB) and a reductive dehalogenase (PcpC) that functions twice are well established. A fourth step appears to involve ring-fission of the aromatic nucleus (PcpA). Molecular analyses revealed that the PCP degradation pathway in these four strains was rather conserved, leading to a phylogenetic analysis using 16S rDNA. The results revealed a much closer phylogenetic relationship between these organisms than traditional classification indicated, placing them into the more recently established genus Sphingomonas where they may even represent a single species. With 16S rDNA analysis, many bacterial isolates involved in degradation of xenobiotic compounds that were previously classified into diverse genera have been reclassified into the genus Sphingomonas. Received 14 April 1999/ Accepted in revised form 20 July 1999     

Diversity in surface colonization behavior in marine bacteria

Using laminar flow chambers and time-lapse video imaging, colonization of surfaces by four marine bacteria revealed a diverse range of morphological characteristics and cell-cell interactions. The strain SW5 formed a compact, multilayered single- and double-cell biofilm on hydrophobic surfaces but developed long multicellular chains on hydrophilic surfaces. The morphologically similar SW8 showed unusual proximal vertical packing of cells on both substrata.Vibrio sp strain S14 exhibited cyclical colonization-detachment events on both substrata.Pseudomonas sp strain S9 initially displayed reversible and then irreversible adhesion apparently triggered by a cell density phenomenon that led to the development of regular microcolonies on both substrata with individual cells translocating between the colonies. The length of time bacteria were exposed to and their density at a surface influenced behavioral traits, with diverse and distinctive species-specific behavioral events.     

Exopolysaccharide production and attachment strength of bacteria and diatoms on substrates with different surface tensions

Attachment strength and exopolysaccharide (EPS) production of Pseudomonas sp. (bacteria) and the diatom Amphora coffaeformis were studied on six different substrata with surface tensions between 19 and 64.5 mN m^–1. Test panels of the materials were exposed to bacterial cultures between 3 and 120 hours, and to diatom cultures between 48 and 72 hours. Exopolysaccharide production by surface-associated cells was measured using the phenol sulfuric acid method. Attachment studies were run by exposing test panels to laminar flow pressure using a radial flow chamber. Highest EPS production by bacteria and diatoms was recorded on substrata with surface tensions above 30 mN m^–1. Lowest EPS production occurred on substrata between 20 and 25 mN m^–1. Highest EPS production and strongest adhesion was found on polycarbonate (33.5 mN m^–1). Both test organisms improved their attachment strength with exposure time on most materials. However, amounts of produced EPS and improvement of attachment indicated that mechanisms other than polysaccharide production are more important on substrata with low surface tensions (<25 mN m^–1). Simply producing more polysaccharides is not sufficient to overcome weak attachment on materials with low surface tensions. For example, adhesion of Pseudomonas sp. and A. coffaeformis on polytetrafluorethylene/perfluor-copolymer (PFA; 22 mN m^–1). and glass (64.5 mN m^–1. was equally strong although EPS production was much higher on glass than on PFA. This is somewhat surprising for A. coffaeformis because polysaccharide production has been considered the most important attachment mechanism of A. coffaeformis.     

The role of microorgansisms in maintaining the chitin balance in the Barents Sea

In this study, we investigated chitin hydrolysis by the bacteria inhabiting the ground of the Barents Sea. Four microbial cultures isolated from the ground were described as the genera of Rhodococcus sp., Bacillus sp., Pseudomonas sp., and Acinetobacter sp. Protein complexes with endochitinase and exochitinase activities were purified from the culture liquid. These microorganisms can participate in chitin degradation in sea water. The average molecular weight of the protein fraction with the chitinolytic activity constituted 92–135 kDa. The ratio of the endo-/exochitinase activities of the enzymatic systems was increased in the order Pseudomonas sp. < Bacillus sp. < Acinetobacter sp. < Rhodococcus sp.     

Biofilm-forming bacteria with varying tolerance to peracetic acid from a paper machine

Biofilms cause runnability problems in paper machines and are therefore controlled with biocides. Peracetic acid is usually effective in preventing bulky biofilms. This study investigated the microbiological status of a paper machine where low concentrations (≤15 ppm active ingredient) of peracetic acid had been used for several years. The paper machine contained a low amount of biofilms. Biofilm-forming bacteria from this environment were isolated and characterized by 16S rRNA gene sequencing, whole-cell fatty acid analysis, biochemical tests, and DNA fingerprinting. Seventy-five percent of the isolates were identified as members of the subclades Sphingomonas trueperi and S. aquatilis, and the others as species of the genera Burkholderia (B. cepacia complex), Methylobacterium, and Rhizobium. Although the isolation media were suitable for the common paper machine biofoulers Deinococcus, Meiothermus, and Pseudoxanthomonas, none of these were found, indicating that peracetic acid had prevented their growth. Spontaneous, irreversible loss of the ability to form biofilm was observed during subculturing of certain isolates of the subclade S. trueperi. The Sphingomonas isolates formed monoculture biofilms that tolerated peracetic acid at concentrations (10 ppm active ingredient) used for antifouling in paper machines. High pH and low conductivity of the process waters favored the peracetic acid tolerance of Sphingomonas sp. biofilms. This appears to be the first report on sphingomonads as biofilm formers in warm water using industries.     

The diversity of extradiol dioxygenase (edo) genes in cresol degrading rhodococci from a creosote-contaminated site that express a wide range of degradative abilities

Analysis of the bacterial population of soil surface samples from a creosote-contaminated site showed that up to 50% of the culturable micro-organisms detected were able to utilise a mixture of cresols. From fifty different microbial isolates fourteen that could utilise more than one cresol isomer were selected and identified by 16S rRNA analysis. Eight isolates were Rhodococcus strains and six were Pseudomonas strains. In general, the Rhodococcus strains exhibited a broader growth substrate range than the Pseudomonas strains. The distribution of various extradiol dioxygenase (edo) genes, previously associated with aromatic compound degradation in rhodococci, was determined for the Rhodococcus strains by PCR detection and Southern-blot hybridization. One strain, Rhodococcus sp. I1 exhibited the broadest growth substrate range and possessed five different edogenes. Gene disruption experiments indicated that two genes (edoC and edoD) were associated with isopropylbenzene and naphthalene catabolism respectively. The other Rhodococcus strains also possessed some of the edo genes and one (edoB) was present in all of the Rhodococcus strains analysed. None of the rhodococcal edo genes analysed were present in the Pseudomonas strains isolated from the site. It was concluded that individual strains of Rhodococcus possess a wide degradative ability and may be very important in the degradation of complex mixtures of substrates found in creosote.     

Persistence and degrading activity of free and immobilised allochthonous bacteria during bioremediation of hydrocarbon-contaminated soils

Rhodococcus sp. and Pseudomonas sp. bioremediation experiments were carried out using free and immobilized cells on natural carrier material (corncob powder) in order to evaluate the feasibility of its use in the bioremediation of hydrocarbon-contaminated soils. Terminal restriction fragment length polymorphism analysis was performed on the 16S rRNA gene as molecular fingerprinting method in order to assess the persistence of inoculated strains in the soil over time. Immobilized Pseudomonas cells degraded hydrocarbons more efficiently in the short term compared to the free ones. Immobilization seemed also to increase cell growth and stability in the soil. Free and immobilized Rhodococcus cells showed comparable degradation percentages, probably due to the peculiarity of Rhodococcus cells to aggregate into irregular clusters in the presence of hydrocarbons as sole carbon source. It is likely that the cells were not properly adsorbed on the porous matrix as a result of the small size of its pores. When Rhodococcus and Pseudomonas cells were co-immobilized on the matrix, a competition established between the two strains, that probably ended in the exclusion of Pseudomonas cells from the pores. The organic matrix might act as protective agent, but it also possibly limited cell density. Nevertheless, when the cells were properly adsorbed on the porous matrix, the immobilization became a suitable bioremediation strategy.     

Application of Biofilm-Forming Bacteria on the Enhancement of Organophosphorus Fungicide Degradation

This study aimed to develop technology enhancing the biodegradation efficacy against organophosphorus fungicide with biofilm-forming bacteria in situ. Using the crystal violet staining method, two bacterial strains having biofilm formation capability were isolated and identified as Pseudomonas sp. C7 and Bacillus sp. E5. Compared with the culture of tolclofos-methyl degrader Sphingomonas sp. 224, biofilm formation was improved by co-inoculation with biofilm-forming bacterium Bacillus sp. E5. Evaluated in liquid culture conditions, this two-species mixed consortium was observed to degrade tolclofos-methyl more effectively than Sphingomonas sp. 224 alone, with an approximately 90% degradation efficiency within 48 h of dosing. The improved effectiveness of the consortium biofilm was reflected using soil in situ with an approximately 7% increased degradation ratio over Sphingomonas sp. 224 alone. This is the first report demonstrating improved bioremediation degradation efficacy against tolclofos-methyl exhibited by a consortium biofilm. This work presents a possible effective bioremediation strategy using a specific biofilm composition against pollutants containing organophosphorus compounds in situ.     

Bioremediation of hydrocarbon-contaminated polar soils

Bioremediation is increasingly viewed as an appropriate remediation technology for hydrocarbon-contaminated polar soils. As for all soils, the successful application of bioremediation depends on appropriate biodegradative microbes and environmental conditions in situ. Laboratory studies have confirmed that hydrocarbon-degrading bacteria typically assigned to the genera Rhodococcus, Sphingomonas or Pseudomonas are present in contaminated polar soils. However, as indicated by the persistence of spilled hydrocarbons, environmental conditions in situ are suboptimal for biodegradation in polar soils. Therefore, it is likely that ex situ bioremediation will be the method of choice for ameliorating and controlling the factors limiting microbial activity, i.e. low and fluctuating soil temperatures, low levels of nutrients, and possible alkalinity and low moisture. Care must be taken when adding nutrients to the coarse-textured, low-moisture soils prevalent in continental Antarctica and the high Arctic because excess levels can inhibit hydrocarbon biodegradation by decreasing soil water potentials. Bioremediation experiments conducted on site in the Arctic indicate that land farming and biopiles may be useful approaches for bioremediation of polar soils.     

Interaction of Sphingomonas and Pseudomonas strains in the degradation of chlorinated dibenzofurans

We have studied the concerted degradation of two monochlorodibenzofurans by a bacterial consortium, consisting of the chlorodibenzofurans-cometabolizing and chlorosalicylates-excreting strain Sphingomonas sp RW16, and Pseudomonas sp RW10, which mineralized the released chlorosalicylates. Neither of the organisms was able to grow with chlorodibenzofurans alone. Degradation of 2-chloro- and 3-chlorodibenzofuran proceeded to the end products 5-chloro- and 4-chlorosalicylate, respectively, when the initial dioxygenase of Sphingomonas sp RW 16 attacked the unchlorinated aromatic ring of the heterocyclic dibenzofuran molecule. 2-Hydroxypenta-2,4-dienoate, formed upon meta-cleavage of the intermediary chlorotrihydroxybiphenyls, served as a growth substrate for the sphingomonad. Presumably, most of the chlorosalicylates were excreted and degraded further by Pseudomonas sp RW10. Mineralization of both chlorosalicylates proceeded through a converging pathway, via 4-chlorocatechol, and protoanemonin. Chlorosalicylates were mineralized by the pseudomonad only when their concentration in the culture medium was below 1.5 mM. In the case of initial dioxygenation taking place on the chlorinated aromatic ring, salicylate and chlorinated hydroxypentadienoates should be formed. The metabolic fate of putative chlorohydroxypentadienoates is not clear; ie, they may be channeled into unproductive catabolism and, thus, represent the critical point in the breakdown of the carbon of these two chlorodibenzofurans by Sphingomonas sp RW16. Received 01 May 1999/ Accepted in revised form 26 July 1999     

Diversity and phylogeny of bacteria on Zimbabwe tobacco leaves estimated by 16S rRNA sequence analysis

Microorganisms play important roles in the tobacco aging process. However, microbial communities on flue-cured tobacco leaves (FCTL) remain largely unknown. In this study, the total microbial genomic DNA of unaged and aging FCTL from Zimbabwe were isolated using a culture-independent method, and the bacterial communities were investigated through analyzing two 16S rRNA gene libraries. Eighty-four and 65 operational taxonomic units were obtained from the libraries of the unaged and aging FCTL, respectively. The following genera were represented more than 4% in both libraries (aging and unaged library): Sphingomonas (4.84%, 4.18%), Stenotrophomonas (4.84%, 5.23%), Erwinia (5.81%, 4.88%), Pantoea (19.35%, 18.47%), and Pseudomonas (21.29%, 24.04%). The dominant species varied between the two libraries. Specifically, several dominant species in unaged FCTL including Pseudomonas fulva, Pseudomonas sp. (AM909658), Klebsiella sp. (HM584796), and Pantoea sp. (AY501386) were not identified in aging FCTL, while several dominant species in aging FCTL such as Pantoea sp. (GU566350), Pseudomonas sp. (EF157292), and Buttiauxella izardii were not found in unaged FCTL. The phylogenetic analysis showed that bacteria from unaged and aging FCTL were divided into two clades, and two unique subclades were identified in aging FCTL. Our results revealed for the first time the bacterial diversities on Zimbabwe tobacco, and provided a basis for clarifying the roles of bacteria in aging process of FCTL.     

Auxin production by bacteria associated with orchid roots

Bacteria associated with the roots of greenhouse tropical orchids were shown to produce indole-3-acetic acid (IAA) and to excrete it into the culture liquid. The presence and activity of IAA were demonstrated colorimetrically, by thin-layer chromatography, and by biotests. The associated bacteria varied in their ability to excrete indole compounds (1–28 µg/ml nutrient broth). Addition of tryptophan to the growth medium enhanced phytohormone production. Upon addition of 200 µg/ml tryptophan, the bacteria isolated from Dendrobium moschatum roots (Sphingomonas sp. 18, Microbacterium sp. 23, Mycobacterium sp. 1, Bacillus sp. 3, and Rhizobium sp. 5) produced 50.2, 53.1, 92.9, 37.6, and 60.4 µg IAA/ml, respectively, while the bacteria isolated from Acampe papillosa roots (Sphingomonas sp. 42, Rhodococcus sp. 37, Cellulomonas sp. 23, Pseudomonas sp. 24, and Micrococcus luteus) produced 69.4, 49.6, 53.9, 31.0, and 39.2 µg IAA/ml. Auxin production depended on cultivation conditions and on the growth phase of the bacterial cultures. Treatment of kidney bean cuttings with bacterial culture liquid promoted formation of a root brush with a location height 7.4- to 13.4-fold greater than the one in the control samples. The ability of IAA-producing associated bacteria to act as stimulants of the host plant root development is discussed.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 55–62.Original Russian Text Copyright © 2005 by Tsavkelova, Cherdyntseva, Netrusov.     

Simultaneous anaerobic and aerobic degradation of the sulfonated azo dye Mordant Yellow 3 by immobilized cells from a naphthalenesulfonate-degrading mixed culture

The naphthalenesulfonate-oxidizing bacterium Sphingomonas sp. BN6 was immobilized in calcium alginate. These beads were incubated under aerobic conditions in a medium with the sulfonated azo dye, Mordant Yellow 3 (MY3), and glucose. The immobilized cells converted MY3, but only a marginal turnover of the dye was found under these conditions with freely suspended cells of Sphingomonas sp. BN6. Under anaerobic conditions, suspended cells of Sphingomonas sp. BN6 reductively cleaved the azo bond of MY3 to 6-aminonaphthalene-2-sulfonate (6A2NS) and 5-aminosalicylate. The turnover of MY3 by the immobilized cells under aerobic conditions resulted in the formation of more than equimolar amounts of 5-aminosalicylate, but almost no (6A2NS) was detected. Cells of Sphingomonas sp. BN6 aerobically oxidize 6A2NS to 5-aminosalicylate. It was therefore concluded that the cells in the anaerobic center of the alginate beads reduced MY3 to 6A2NS and 5-aminosalicylate and that 6A2NS was oxidized to 5-aminosalicylate by those cells that were immobilized in the outer aerobic zones of the alginate beads. The presence of oxygen gradients within the alginate beads was verified by using oxygen micro-electrodes. A coimmobilisate of Sphingomonas sp. BN6 with a 5-aminosalicylate degrading bacterium completely degraded MY3. The immobilized cells also converted the sulfonated azo dyes Amaranth and Acid Red␣1. Received: 6 May 1996 / Received revision: 6 August 1996 / Accepted: 12 August 1996     

Drip irrigation biofouling with treated wastewater: bacterial selection revealed by high-throughput sequencing

Clogging of drippers due to the development of biofilms weakens the advantages and impedes the implementation of drip irrigation technology. The objective of this study was to characterise the bacterial community of biofilms that develop in a drip irrigation system supplied with treated wastewater. High-throughput sequencing of 16S rRNA gene amplicons indicated that the bacterial community composition differed between drippers and pipes, mainly due to changes in the abundance of the genus Aquabacterium. Cyanobacteria were found to be involved in the biological fouling of drippers. Moreover, bacterial genera including opportunistic pathogenic bacteria such as Legionella and Pseudomonas were more abundant in dripper and pipe biofilms than in the incoming water. Some genera such as Pseudomonas were mostly recovered from drippers, while others (ie Bacillus, Brevundimonas) mainly occurred in pipes. Variations in the hydraulic conditions and properties of the materials likely explain the shift in bacterial communities observed between pipes and drippers.     

Effects of organochlorines on microbial diversity and community structure in Phragmites australis rhizosphere

This study investigated the impacts of an organochlorine (OC, γ-hexachlorocyclohexane and chlorobenzenes) mixture on microbial communities associated to Phragmites australis rhizosphere. Seventy-eight distinct colony morphotypes were isolated, cultivated and analysed by 16S rDNA sequence analysis. Toxicity tests confirmed sensitivity (e.g. Hevizibacter, Acidovorax) or tolerance (e.g. Bacillus, Aeromonas, Pseudomonas, Sphingomonas) of isolates. Rhizosphere analysis by pyrosequencing showed the microbial adaptation induced by OC exposure. Among the most abundant molecular operational taxonomic units, 80 % appeared to be tolerant (55 % opportunist, 25 % unaffected) and 20 % sensitive. P. australis rhizosphere exposed to OCs was dominated by phylotypes related to α-, β- and γ-Proteobacteria. Specific genera were identified which were previously described as chlorinated organic pollutant degraders: Sphingomonas sp., Pseudomonas sp., Devosia sp. and Sphingobium sp. P. australis could be suitable plants to maintain their rhizosphere active microbial population which can tolerate OCs and potentially improve the OC remediation process in part by biodegradation.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution