Artificial biofilm model – a useful tool for biofilm research

For biofilm studies, artificial models can be very helpful in studying processes in hydrogels of defined composition and structure. Two different types of artificial biofilm models were developed. Homogeneous agarose beads (50–500 μm diameter) and porous beads (260 μm mean diameter) containing pores with diameters from 10 to 80 μm (28 μm on average) allowed the embedding of cells, particles and typical biofilm matrix components such as proteins and polysaccharides. The characterisation of the matrix structures and of the distribution of microorganisms was performed by confocal laser scanning microscopy. The physiological condition of the embedded bacteria was examined by redox activity (CTC-assay) and membrane integrity (Molecular Probes LIVE/DEAD-Kit). Approximately 35% of the immobilised cells (Pseudomonas aeruginosa SG81) were damaged due to the elevated temperature required for the embedding process. It was shown that the surviving cells were able to multiply when provided with nutrients. In the case of homogeneous agarose beads, cell growth only occurred near the bead surface, while substrate limitation prevented growth of more deeply embedded cells. In the porous hydrogel, cell division was observed across the entire matrix due to better mass transport. It could be shown that embedding in the artificial gel matrix provided protection of immobilized cells against toxic substances such as sodium hypochlorite (0.5 mg/l, 30 min) in comparison to suspended cells, as observed in other immobilized systems. Thus, the model is suited to simulate important biofilm matrix properties. Received: 21 December 1999 / Received revision: 7 March 2000 / Accepted: 10 March 2000     

Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination

The marine diatom Haslea ostrearia was immobilized in a tubular agar gel layer introduced into a photobioreactor of original design with internal illumination for the continuous synthesis of marennin, a blue-green pigment of biotechnological interest. Marennin was produced for a long-term period (27–43 days) and the volumetric productivity was maximum (18.7 mg day⁻¹ l⁻¹ gel) at the highest dilution rate (0.25 day⁻¹) and lowest agar layer thickness (3 mm). Heterogeneous cell distribution in the agar layer revealed diffusional limitation of light and nutrients. However, the 3 mm gel thickness led to a more homogeneous cell distribution during incubation and to an increase of the whole biomass in the agar gel layer. Received: 22 October 1999 / Received revision: 14 February 2000 / Accepted: 18 February 2000     

Immobilisation of whole bacterial cells for anaerobic biotransformations

Anaerobically grown cells of Escherichia coli were immobilised within a range of entrapment matrices and packed into a column under standard conditions, and the ability of the immobilised cells to reduce nitrite (0.5 mM) was measured at a range of flow rates using sodium formate (20 mM) as the electron donor for nitrite reduction. A flow-rate/activity plot was constructed for each flow-through reactor and RA_1/2 values (residence time corresponding to 50 % nitrite removal) calculated for each reactor type. Cells immobilised in flat and hollow-fibre membranes were the most effective (RA_1/2 = 0.35 h and 0.47 h respectively), with cells entrapped by dialysis membrane (1.53 h), alginate beads (1.93 h), Hypol foam (2.31 h) and polyacrylamide gel (50 % nitrite not removed at maximum residence time tested: 4.9 h) performing progressively less effectively. Cells grown as a biofilm on a range of support materials were also tested in comparable packed-bed reactors. Cell loss from these supports was extensive and contributed to poor performance of the reactors despite high initial biomass loadings (RA_1/2 values using raschig rings, coke and activated-carbon supports: 1.6 h, 2.3 h and 1.0 h respectively). Biofilms grown on Pharmacia microcarrier supports and used in packed and also fluidised beds were more stable and the performance of these reactors was superior to that of biofilm reactors using other supports, and comparable to that of the membrane reactors (RA_1/2 values for Cytoline 2, Cytopore 2 and Cytodex 3: 0.76 h, 0.56 h, 0.68 h respectively). Received: 12 August 1996 / Received revision: 14 November 1996 / Accepted: 15 November 1996     

Monitoring nitric oxide from immobilised denitrifying bacteria, Pseudomonas stutzeri, by the use of chemiluminescence

A chemiluminescence detector was used to measure the production of nitric oxide, NO, from the denitrifying bacteria Pseudomonas stutzeri. NO is an intermediate when P. stutzeri converts nitrate into nitrogen gas. The reaction between NO and ozone is selective and sensitive in generating chemiluminescence. Calibrations were made down to 1 nM, with a signal-to-noise ratio of 3. Bacteria were immobilised in alginate beads. Denitrification experiments were made in an anaerobic non-growth medium by adding nitrate to a certain concentration in the reactor. The bacteria were exposed to nitrate in the concentration range 1 pM–5 mM. The lowest concentration to give a measurable NO response was 100 nM. Received: 16 October 1997 / Received revision: 20 January 1998 / Accepted: 24 January 1998     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000     

Mean residence time of molecules diffusing in a cell bounded by a semi-permeable membrane: Monte Carlo simulations and an expression relating membrane transition probability to permeability

 The rapid exchange of water across erythrocyte membranes is readily measured using an NMR method that entails doping a suspension of cells with a moderately high concentration of Mn²⁺ and measuring the rate of transverse relaxation of the nuclear magnetisation. Analysis of the data yields an estimate of the rate constant for membrane transport, from which the membrane permeability can be determined. It is assumed in the analysis that the efflux rate of the water is solely a function of the rate of membrane permeation and that the time it takes for intracellular water molecules to diffuse to the membrane is relatively insignificant. The limits of this assumption were explored by using random-walk simulations of diffusion in cells modelled as parallel planes, spheres, and biconcave discs. The rate of membrane transport was specified in terms of a transition probability but it was not initially clear what the relationship should be between this parameter and the diffusional membrane permeability P _d. This relationship was derived and used to show that the mean residence time for a water molecule is determined by P _d when the diffusion coefficient is above a certain threshold value; it is determined by the distance to the membrane below that value. Received: 7 January 2000 / Revised version: 4 April 2000 / Accepted: 4 April 2000     

1H NMR studies: dynamics of water in gelatin

 Proton magnetic resonance was used to characterize the dynamics of water in gelatin. Both sol and gel states were investigated. Transverse relaxation rates (R ₂) were dependent on the proton frequency measurement. (R ₂) measured with the Carr-Purcell-Meiboom-Gill pulse sequence was dependent on pulse spacing. These observations were interpreted in terms of chemical exchanges between water protons and those of the macromolecules in the sol state, whereas in the gel state the contribution of diffusion through microheterogeneities in the sample seems to provide an additional transverse relaxation mechanism. Received: 10 May 1999 / Revised version: 13 December 1999 / Accepted: 25 January 2000     

Periodical cicadas

The evolution of periodicity and synchronicity of magical cicadas is studied by means of mathematical models. Received: 28 January 1999 / Revised version: 4 November 1999 / Published online: 3 April 2000     

Continuous production of enantiopure 1,2-epoxyhexane by yeast epoxide hydrolase in a two-phase membrane bioreactor

A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (>98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l⁻¹ h⁻¹. The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane. Received: 14 February 2000 / Received revision: 15 June 2000 / Accepted: 18 June 2000     

Growth of Photorhabdus luminescens in batch and glucose fed-batch culture

Photorhabdus luminescens, a bacterial symbiont of entomopathogenic biocontrol nematodes, was grown in batch and glucose fed-batch culture. The cell density, bioluminescence, production of antibiotic substances, number of cells with inclusion bodies, glucose concentration and oxygen uptake rate were recorded. The addition of 12.4 g l⁻¹ glucose prolonged the growth, and the yield almost doubled, from 6.85 g l⁻¹ to 12.45 g l⁻¹ dry mass. The production of antibiotic substances increased by 140%. Bioluminescence was higher in the batch culture. A shift of P. luminescens to phase II variants was not detected. Received: 21 January 2000 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Molecular studies on isopenicillin N synthases

The isopenicillin N synthases isolated thus far are related to oxidases from other microorganisms and plants. These enzymes maintain a non-heme monoferrous-dependent catalytic centre comprising a HisXAsp(53–57)XHis motif and a crucial substrate-binding pocket with an ArgXSer motif for their functionality. The elucidation of these motifs was dependent on information collated from studies on structural chemistry, structural biology, site-directed engineered mutations and biochemical experiments. It is envisaged that these enzymes can potentially be improved through molecular breeding and protein engineering. Received: 15 December 1999 / Received revision: 26 January 2000 / Accepted: 27 January 2000     

Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli

 The expression of a recombinant pectate lyase from Bacteroides thetaiotaomicron strain 217 was studied in Escherichia coli strain HB101(pBT4). First, two sets of complete 2⁴ factorial designs were used to evaluate the influences of casamino acids, glucose, magnesium, calcium, tetracycline, ampicillin, tryptophan and MOPS buffer on pectate lyase production in a basal medium. While casamino acids, glucose and magnesium were found to be the prevalent factors, the presence of tetracycline, ampicillin and MOPS buffer were necessary for the reproducibility of the process, probably by increasing the plasmid stability. Secondly, application of the Doehlert design, a response-surface methodology, allowed a good prediction of pectate lyase production according to the variation in glucose and magnesium concentrations. This optimization strategy allowed the production of biomass and recombinant pectate lyase respectively to be increased from 0.2 g l^-1 to 1.9 g l^-1 (dry weight) and from 10 units ml^-1 to 210 units ml^-1 within 24 h at 30°C in shake flasks. Received: 26 July 1995/Received revision: 22 January 1996/Accepted: 29 January 1996     

Immobilisation of Thiobacillus ferrooxidans cells on nickel alloy fibre for ferrous sulfate oxidation

The immobilisation of the iron-oxidising bacteria Thiobacillus ferrooxidans on nickel alloy fibre as support is described. This matrix showed promise for application in iron oxidation under strongly acidic conditions. The influence on the colonisation process of T. ferrooxidans exerted by the initial pH of the medium and by temperature has also been studied. Results showed that immobilisation of T. ferrooxidans cells was affected by changes of temperature between 30 °C and 40 °C and in pH from 1.4 to 2.0. Received: 25 January 2000 / Received version: 20 April 2000 / Accepted: 1 May 2000     

Solid matrix characterization of immobilized Pseudomonas putida MTCC 1194 used for phenol degradation

Characterization studies of calcium alginate beads with encapsulated Pseudomonas putida MTCC 1194, used for the biodegradation of phenol, were carried out to investigate the reactivity, reusability and structural strength of the solid matrix. Various techniques were employed to improve the structural stability of the immobilized solid necessary for its use in commercial reactors like packed bed flow reactor, fluidized bed and CSTR systems. Experiments were performed to establish the optimum conditions for durability, strength and steady biochemical reactivity. During a batch run of 40 h a gradual decline in the rate of phenol degradation was observed with the immobilized system. The calcium alginate beads with high structural strength yielded decreased activity. Treatment with a hardening agent like glutaraldehyde for different concentrations and treatment times led to variations in structural stability, reusability and the extent of phenol degradation. Scanning electron microscope studies of the immobilized solid indicated the internal distribution pattern of the cells encapsulated in a calcium alginate bead. Received: 13 November 1998 / Received revision: 27 January 1999 / Accepted: 31 January 1999     

Competition between n-alkane-assimilating yeasts and bacteria during colonization of sandy soil microcosms

An n-alkane-assimilating strain of Candida tropicalis was selected in sandy soil inoculated with microorganisms from contaminated sites. Competition experiments with n-alkane utilizers from different strain collections confirmed that yeasts overgrow bacteria in sandy soil. Acidification of the soil is one of the colonization factors useful for the yeasts. It can be counteracted by addition of bentonite, a clay mineral with high ion exchange capacity, but not, however, by kaolin. Strains of different yeast species showed different levels of competitiveness. Strains of Arxula adeninivorans, Candida maltosa, and Yarrowia lipolytica overgrew strains of C. tropicalis, C. shehatae or Pichia stipitis. Two strains of C. maltosa and Y. lipolytica coexisted during several serial transfers under microcosm conditions. Received: 20 October 1999 / Received revision: 26 January 2000 / Accepted: 27 January 2000     

Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures

The entomopathogenic nematode–bacterium complex Heterorhabditis megidis–Photorhabdus luminescens was cultured in 10-l internal loop bioreactors with marine impellers at aeration rates of 0.3 vvm and 0.7 vvm. Process parameters like impeller velocity and oxygen saturation were controlled at equal set points. The bacterial density was assessed at 24 h. Nematode dauer juveniles (DJ) were then inoculated and the development to adults after 8 days and final DJ yields after 16 days were recorded. The bacterial population density and the nematode inoculum development was variable and was not influenced by the aeration rate. A significant effect on the yield was recorded at the highest aeration rate. This result was confirmed by a direct comparison in two 5-l internal loop glass bioreactors at 0.3 vvm and 1.0 vvm, which were inoculated with nematode and bacterium pre-cultures from the same flask culture. Possible reasons for the positive correlation between aeration rate and DJ yield are discussed. Received: 27 September 1999 / Received revision: 21 January 2000 / Accepted: 23 January 2000     

Denitrification with methane as electron donor in oxygen-limited bioreactors

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10⁷ cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10⁷ cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10⁴ cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. “In vivo”¹³C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification. Received: 19 October 1999 / Received revision: 11 January 2000 / Accepted: 14 January 2000     

Fracture mechanics of the cell wall of Chara corallina

Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but there appears to be no published study of their failure properties. The mechanical strength of single large internode cell walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with age whereas their strength was little affected. The strength of unnotched walls was estimated as 47 ± 13 MPa, comparable to that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The strength was notch-sensitive and the critical stress intensity factor K _1c was estimated to be 0.63 ± 0.19 MNm^−3/2, comparable to published values for grasses. Received: 4 April 2000 / Accepted: 21 July 2000     

Effect of cations, pH and sulfate content on the viscosity and emulsifying activity of the Halomonas eurihalina exopolysaccharide

The effects of monovalent and divalent cations on the rheological behavior of Halomonas eurihalina exopolysaccharide (EPS) were studied. Sodium, potassium, magnesium and calcium were added and the relative abilities to increase viscosity were as follows: KCl > NaCl > MgCl₂ > CaCl₂. The highest viscosity value was measured in acidic 10⁻⁴ M KCl, in which a gel formed. A loss of sulfate content seemed to correlate with the increase of viscosity. H. eurihalina produced EPS in all growth media. Addition of hydrophobic substrates to culture media produced changes in chemical composition and emulsifying activity of the EPS. Xylene was the most effectively emulsified substance and the EPS produced on tetradecane and on corn oil the most active emulsifier. Received 25 July 1997/ Accepted in revised form 30 January 1998