Marko Kolari

Mechanisms of biofilm formation in paper machine by Bacillus species: the role of Deinococcus geothermalis

Mechanisms for the undesired persistence of Bacillus species in paper machine slimes were investigated. Biofilm formation was measured for industrial Bacillus isolates under paper machine wet-end-simulating conditions (white water, pH 7, agitated at 45°C for 1–2 days). None of the 40 tested strains of seven Bacillus species formed biofilm on polished stainless steel or on polystyrene surfaces as a monoculture. Under the same conditions, Deinococcus geothermalis E50051 covered all test surfaces as a patchy thick biofilm. The paper machine bacilli, however, formed mixed biofilms with D. geothermalis E50051 as revealed by confocal microscopy. Biofilm interactions between the bacilli and the deinococci varied from synergism to antagonism. Synergism in biofilm formation of D. geothermalis E50051 was strongest with Bacillus coagulans D50192, and with the type strains of B. coagulans, B. amyloliquefaciens or B. pumilus. Two B. licheniformis, one B. amyloliquefaciens, one B. pumilus and four B. cereus strains antagonized biofilm production by D. geothermalis. B. licheniformis D50141 and the type strain of B. licheniformis were the strongest antagonists. These bacteria inhibited deinococcal growth by emitting heat-stable, methanol-soluble metabolite(s). We conclude that the persistence of Bacillus species in paper machine slimes relates to their ability to conquer biofilms formed by primary colonizers, such as D. geothermalis. Journal of Industrial Microbiology & Biotechnology (2001) 27, 343–351.

Firm but Slippery Attachment of Deinococcus geothermalis

Bacterial biofilms impair the operation of many industrial processes. Deinococcus geothermalis is efficient primary biofilm former in paper machine water, functioning as an adhesion platform for secondary biofilm bacteria. It produces thick biofilms on various abiotic surfaces, but the mechanism of attachment is not known. High-resolution field-emission scanning electron microscopy and atomic force microscopy (AFM) showed peritrichous adhesion threads mediating the attachment of D. geothermalis E50051 to stainless steel and glass surfaces and cell-to-cell attachment, irrespective of the growth medium. Extensive slime matrix was absent from the D. geothermalis E50051 biofilms. AFM of the attached cells revealed regions on the cell surface with different topography, viscoelasticity, and adhesiveness, possibly representing different surface layers that were patchily exposed. We used oscillating probe techniques to keep the tip-biofilm interactions as small as possible. In spite of this, AFM imaging of living D. geothermalis E50051 biofilms in water resulted in repositioning but not in detachment of the surface-attached cells. The irreversibly attached cells did not detach when pushed with a glass capillary but escaped the mechanical force by sliding along the surface. Air drying eliminated the flexibility of attachment, but it resumed after reimmersion in water. Biofilms were evaluated for their strength of attachment. D. geothermalis E50051 persisted 1 h of washing with 0.2% NaOH or 0.5% sodium dodecyl sulfate, in contrast to biofilms of Burkholderia cepacia F28L1 or the well-characterized biofilm former Staphylococcus epidermidis O-47. Deinococcus radiodurans strain DSM 20539(T) also formed tenacious biofilms. This paper shows that D. geothermalis has firm but laterally slippery attachment not reported before for a nonmotile species.

Detection and quantitation of colored deposit-forming Meiothermus spp. in paper industry processes and end products

Colored biofilms cause problems in paper industry. In this work we used real-time PCR to detect and to quantitate members of the genus Meiothermus from the process samples and end products from 24 machines manufacturing pulp, paper and board in four countries. The results obtained from 200 samples showed the importance of members of the genus Meiothermus as ubiquitous biofoulers in paper machines. This genus was the dominant biofouler in some mills. From ≤104 to 1011 copies of Meiothermus 16S rRNA genes were found per gram of process deposit (wet weight). Meiothermus spp. were found in paper and board products with colored defects and connection between deposit-forming microbes and end-product spots was shown. 16S rRNA gene sequences of 29 biofilm producing bacterial isolates from different mills were determined. Based on sequence data, 25 of the isolates were assigned to the genus Meiothermus, with Meiothermus silvanus and M. ruber as the most frequent species.

Architecture of Deinococcus geothermalis biofilms on glass and steel: a lectin study

Deinococcus geothermalis is resistant to chemical and physical stressors and forms tenuous biofilms in paper industry. The architecture of its biofilms growing on glass and on stainless acid proof steel was studied with confocal laser scanning microscopy and fluorescent lectins and nanobeads as in situ probes. Hydrophobic nanobeads adhered to the biofilms but did not penetrate to biofilm interior. In contrast, the biofilms were readily permeable towards many different lectins. A skeletal network of glycoconjugates, reactive with Dolichos biflorus and Maclura pomifera lectins, was prominent in the space inside the biofilm colony core but absent on the exterior. Cells in the core space of the biofilm were interconnected by a network of adhesion structures, reactive with Amaranthus caudatus lectin but with none of the 65 other tested lectins. The glycoconjugates connecting the individual cells to steel reacted with Phaseolus vulgaris lectin whereas those connecting to glass mainly reacted with A. caudatus lectin. Envelopes of all cells in the D. geothermalis biofilm reacted with several other lectins, with many different specificities. We conclude that numerous different glycoconjugates are involved in the adhesion and biofilm formation of D. geothermalis, possibly contributing its unique survival capacity when exposed to dehydration, biocidal chemicals and other extreme conditions.

Community structure of biofilms on ennobled stainless steel in Baltic Sea water

ca 400 mV), the biofilm on the steel surface was characterized using confocal laser scanning microscopy (CLSM) in combination with functional and phylogenetic stains. The biofilm consisted of microbial cell clusters covering 10–20% of the surface. The clusters were loaf-formed, with a basal diameter of 20–150 μm, 5–20 per mm−2, each holding >104 cells in a density of 1–5 × 107 cells mm−3. The typical cluster contained mainly small Gram-negative bacteria (binding the EUB338 probe when hybridized in situ on the steel surface), and often carried one to three spherical colonies, either homogeneously composed of large Gram-negative cocci or more often small bacterial rods in high density, 108–109 cells mm−3. The clusters in live biofilms contained no pores, and clusters over 25 μm in diameter had a core nonpenetrable to fluorescent nucleic acid stains and ConA lectin stain. Fluorescently-tagged ConA stained cells at a depth of <5 μm, indicating the presence of cells with α-d-mannosyl and α-d-glucosyl residues on surfaces. ethidium bromide (log Kow −0.38) penetrated deeper (17 μm in 15 min, corresponding to >10 cells in a stack) into the cluster than did the less polar dyes SYTO 16 (log Kow 1.48) and acridine orange (log Kow 1.24), which stained five cells in a stack. Fluorescent hydrophobic and hydrophilic latex beads (diameter 0.02, 0.1 or 1.0 μm) coated patchwise the cluster surface facing the water, but penetrated only to depths of ⩽2 μm indicating a permeability barrier. About 1/3 of the stainable cells hybridized in situ with Alf1b, while fewer than 1/7 hybridized to GAM42, probes targeted towards α- and γ-Proteobacteria, respectively. Our results represent a microscopic description of an ennobling biofilm, where the ennoblement could follow the sequence of redox events as suggested by the model of Dickinson and Lewandowski (1996) for the structure of corrosive biofilms on a steel surface.

Quantitative contributions of bacteria and of Deinococcus geothermalis to deposits and slimes in paper industry

Deinococcus geothermalis has frequently been isolated from pink colored deposits of paper industry processes. Laboratory studies have shown that D. geothermalis is capable of forming on nonliving surfaces patchy biofilms that are resistant to adverse agents such as extreme pH, desiccation, solubilising detergents and biocides. This study was done to quantitatively assess the role of D. geothermalis as a biofouler in paper industry. Colored deposits were collected from 24 European and North American paper and board machines and the densities of the bacterial 16S rRNA genes and those of the red slime producers D. geothermalis and Meiothermus spp. were measured by QPCR (quantitative real time PCR). D. geothermalis was found at nine machines, usually from splash area deposits, but its contribution was minor, 0.001–1%, to the total bacterial burden of 8.3 to log 10.5 log units per gram wet-weight of the deposits. When D. geothermalis was found in a measurable quantity, Meiothermus spp. also was found, often in bulk quantity (7–100% of the total bacteria). The data are in line with the properties of D. geothermalis known from laboratory biofilm studies, indicating this species is a pioneer coloniser of machine surfaces and may help other bacteria to adhere and grown into biofilms, rather than competing with them.

The effect of filters on aseptic pipetting lifetime of mechanical and electronic pipettors and carryover during pipetting

The aseptic pipetting lifetime of air displacement pipettors during serial pipetting was investigated. When a Micrococcus luteusT culture was pipetted using no filter, less than 100 pipettings were required to contaminate the interiors of both mechanical and electronic pipettors with more than 103 cfu (n = 1890 pipettings). When a filter was placed in the pipettor tip cone, the sterility of the pipettor barrel was maintained for more than 500 pipettings (n = 2620) or 50–250 pipettings (n = 2520), depending on the filter type used. When a radioactive liquid or plasmid DNA was pipetted using no filter, contamination of the pipettor barrel occurred within less than 100 pipettings. A filter placed in the pipettor tip cone protected the interior of the pipettors against radioactive contamination for more than 250 pipettings and against DNA contamination for more than 500 pipettings. A pipettor barrel contaminated with M. luteusT caused carryover in 13% of the pipetting series. When a SCF filter was placed in the tip cone, no carryover was observed within 2620 pipettings. A tip cone filter replaced at intervals of 50–250 (polyethylene filter) or 500 (SCF filter) pipettings will protect the pipettor barrel from contamination and the samples from carryover.