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Applied Microbiology and Biotechnology (v.54, #2)
Fruiting body production in basidiomycetes by U. Kües; Y. Liu (pp. 141-152).
Mushroom cultivation presents an economically important biotechnological industry that has markedly expanded all over the world in the past few decades. Mushrooms serve as delicacies for human consumption and as nutriceuticals, as “food that also cures”. Mushrooms, the fruiting bodies of basidiomycetous fungi, contain substances of various kinds that are highly valued as medicines, flavourings and perfumes. Nevertheless, the biological potential of mushrooms is probably far from exploited. A major problem up to now is that only a few species can be induced to fruit in culture. Our current knowledge on the biological processes of fruiting body initiation and development is limited and arises mostly from studies of selected model organisms that are accessible to molecular genetics. A better understanding of the developmental processes underlying fruiting in these model organisms is expected to help mushroom cultivation of other basidiomycetes in the future.
Ice crystallization by Pseudomonas syringae by N. Cochet; P. Widehem (pp. 153-161).
Several bacterial species can serve as biological ice nuclei. The best characterized of these is Pseudomonas syringae, a widely distributed bacterial epiphyte of plants. These biological ice nuclei find various applications in different fields, but an optimized production method was required in order to obtain the highly active cells which may be exploited as ice nucleators. The results presented here show that P. syringae cells reduce supercooling of liquid or solid media and enhance ice crystal formation at sub-zero temperatures, thus leading to a remarkable control of the crystallization phenomenon and a potential for energy savings. Our discussion focuses on recent and future applications of these ice nucleators in freezing operations, spray-ice technology and biotechnological processes.
Utilisation of saccharides in extruded domestic organic waste by Clostridium acetobutylicum ATCC 824 for production of acetone, butanol and ethanol by A. M. López-Contreras; P. A. M. Claassen; H. Mooibroek; W. M. De Vos (pp. 162-167).
Domestic organic waste (DOW) collected in The Netherlands was analysed and used as substrate for acetone, butanol and ethanol (ABE) production. Two different samples of DOW, referred to as fresh DOW and dried DOW, were treated by extrusion in order to expand the polymer fibres present and to obtain a homogeneous mixture. The extruded material was analysed with respect to solvent and hot water extractives, uronic acids, lignin, sugars and ash. The total sugar content in the polymeric fractions of the materials varied from 27.7% to 39.3% (w/w), in which glucose represented the 18.4 and 25.1% of the materials, for fresh and dried DOW, respectively. The extruded fresh DOW was used as substrate for the ABE fermentation by the solventogenic strain Clostridium acetobutylicum ATCC 824. This strain was grown on a suspension of 10% (w/v) DOW in demineralised water without further nutrient supplement. This strain produced 4 g ABE/100 g extruded DOW. When C. acetobutylicum ATCC 824 was grown on a suspension of 10% (w/v) DOW hydrolysed by a combination of commercial cellulases and β-glucosidases, the yield of solvents increased to 7.5 g ABE/100 g extruded DOW. The utilisation of sugar polymers in both hydrolysed and non-hydrolysed DOW was determined, showing that only a small proportion of the polymers had been consumed by the bacteria. These results indicate that growth and ABE production on DOW is mainly supported by soluble saccharides in the medium.
The use of silica gel prepared by sol-gel method and polyurethane foam as microbial carriers in the continuous degradation of phenol by T. Brányik; G. Kuncová; J. Páca (pp. 168-172).
A mixed microbial culture was immobilized by entrapment into silica gel (SG) and entrapment/ adsorption on polyurethane foam (PU) and ceramic foam. The phenol degradation performance of the SG biocatalyst was studied in a packed-bed reactor (PBR), packed-bed reactor with ceramic foam (PBRC) and fluidized-bed reactor (FBR). In continuous experiments the maximum degradation rate of phenol (q s max) decreased in the order: PBRC (598 mg l−1 h−1) > PBR (PU, 471 mg l−1 h−1) > PBR (SG, 394 mg l−1 h−1) > FBR (PU, 161 mg l−1 h−1) > FBR (SG, 91 mg l−1 h−1). The long-term use of the SG biocatalyst in continuous phenol degradation resulted in the formation of a 100–200 μm thick layer with a high cell density on the surface of the gel particles. The abrasion of the surface layer in the FBR contributed to the poor degradation performance of this reactor configuration. Coating the ceramic foam with a layer of cells immobilized in colloidal SiO2 enhanced the phenol degradation efficiency during the first 3 days of the PBRC operation, in comparison with untreated ceramic packing.
Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans by A. Kademi; N. Aït-Abdelkader; L. Fakhreddine; J. Baratti (pp. 173-179).
The thermostable esterase from the moderate thermophile Bacillus circulans was purified to homogeneity using a four-step procedure. Esterase activity was associated with a protein of molecular mass 95 kDa, composed of three identical subunits of 30 kDa. The esterase activity was thermostable with a maximum activity at 55 °C using initial rate assay. The half-inactivation temperature was 71 °C after a 1-h treatment, which compared favorably to that of other enzymes. Activity at temperatures of 30–37 °C was high (about half of maximum), making this new enzyme very attractive for applications in this moderate temperature range. The esterase also showed high activity at a rather alkaline pH (higher than 10). The specificity pattern showed a marked specificity for mid-chain-length fatty acids (3–8 carbon atoms), which classified the enzyme as a carboxylesterase.
A genetically modified solvent-tolerant bacterium for optimized production of a toxic fine chemical by J. Wery; D. I. Mendes da Silva; J. A. M. de Bont (pp. 180-185).
The aim of the study was to investigate whether toxic fine chemical production can be improved using the solvent-tolerant Pseudomonas putida S12 in a two-liquid-phase system consisting of aqueous media and a water-immiscible octanol phase with production of 3-methylcatechol from toluene as the model conversion. For this purpose the genes involved in this conversion, todC1C2BAD from P. putida F1, were introduced into P. putida S12 with high stable expression. Production of 3-methylcatechol was monitored in batch incubations with different media using a single medium and a two-liquid medium–octanol system. The maximum concentration of 3-methylcatechol increased two-fold using the two-liquid medium–octanol system, irrespective of the selected medium.
Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme by M. Liebergesell; S. Rahalkar; A. Steinbüchel (pp. 186-194).
The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (M r 40,950) and PhaC (M r 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed.
Coexpression of the Bacillus pumilusβ-xylosidase (xynB) gene with the Trichoderma reeseiβ-xylanase 2 (xyn2) gene in the yeast Saccharomyces cerevisiae by D. C. La Grange; M. Claeyssens; I. S. Pretorius; W. H. Van Zyl (pp. 195-200).
The xynB gene encoding the Bacillus pumilusβ-xylosidase was expressed separately and jointly with the Trichoderma reeseiβ-xylanase (xyn2) gene in the yeast Saccharomyces cerevisiae. Both genes were placed under the transcriptional control of the glucose-derepressible alcohol dehydrogenase 2 promoter (ADH2 P ) and terminator (ADH2 T ) sequences. The xynB gene was fused in frame to the yeast mating factor α1 secretion sequence (MFα1 S ) to effect secretion in S. cerevisiae. The fusion protein was designated Xlo1. Xlo1 produced in S. cerevisiae exhibited low affinity for xylobiose, but eventually hydrolyzed xylobiose and xylotriose to the monomeric constituent, d-xylose. Coproduction of Xyn2 and Xlo1 by S. cerevisiae led to a 25% increase in the amount of reducing sugars released from birchwood xylan compared to S. cerevisiae producing only the Xyn2 β-xylanase. However, no d-xylose was produced from birchwood xylan, presumably due to very low Xlo1 β-xylosidase activity and its low affinity for xylobiose.
Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation by T. Colin; A. Bories; G. Moulin (pp. 201-205).
1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied. The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than the glycerol fermentation. μ max was inversely proportional to the initial concentration of 1,3-propanediol (0–65 g l−1). For glycerol at 20 g l−1, the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l−1. However, for an initial 1,3-propanediol concentration of 50 g l−1 and glycerol at 70 g l−1, the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l−1(1.1 M), with complete consumption of the glycerol. Therefore, during the fermentation, the strain tolerated a 1,3-propanediol concentration higher than the initial inhibitory concentration (65 g l−1). The addition of 1,2-propanediol or 2,3-butanediol (50 g l−1) in the presence of glycerol (50–100 g l−1), showed that 2-diols reduced the μ max in a similar way to 1,3-propanediol. The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol mixtures did not indicate any differences between these compounds. The hypothesis of diol inhibition was discussed. Taking into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for optimising production were considered.
Changes in the physiological and agricultural characteristics of peat-based Bradyrhizobium japonicum inoculants after long-term storage by C. Revellin; G. Meunier; J. J. Giraud; G. Sommer; P. Wadoux; G. Catroux (pp. 206-211).
Commercial soybean inoculants processed with sterilised peat and stored at 20 °C for 1–8 years were used as experimental materials to assess the changes in the physiological activity of Bradyrhizobium japonicum after storage. Viable counts decreased and physiological characteristics of the bacterium changed during storage, with an increase in the time taken for colony appearance on a medium without yeast extract, an increase in the lag time for nodule appearance on soybean grown in glass tubes and a decrease in survival on seeds. All the inoculants produced a significant increase in grain yield in a field experiment. The percentage of efficient cells in the field (relative to the plate counts) decreased as the length of storage increased. These results suggest that the physiological activity of B. japonicum cells changes after storage. Practical implications for inoculant quality control are discussed.
Metabolic characterization of high- and low-yielding strains of Penicillium chrysogenum by B. Christensen; J. Thykær; J. Nielsen (pp. 212-217).
A recently developed method for analyzing metabolic networks using 13C-labels was employed for investigating the metabolism of a high- and a low-yielding strain of Penicillium chrysogenum. Under penicillin-producing conditions, the flux through the pentose phosphate (PP) pathway in the high- and the low-yielding strains was estimated to 70 and 66, respectively. When the high-yielding strain was cultivated in a medium without the penicillin side chain precursor, phenoxyacetic acid, the PP pathway flux was estimated as 71. Thus, in all three experiments, the flux through the PP pathway was almost constant with an average value of 69 ± 3, and the method therefore allows for a very reproducible estimation of the PP pathway flux. Phenoxyacetic acid was found to be a source of cytosolic acetyl-CoA and thereby a source of precursors for the biosynthesis of 2-aminoadipic acid, which is a central amino acid in penicillin biosynthesis. However, the labeling patterns also indicated the presence of an unrecognized pathway to cytosolic acetyl-CoA.
Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126 by H. M. Alvarez; R. Kalscheuer; A. Steinbüchel (pp. 218-223).
The time course of the accumulation of triacylglycerols (TAGs) in Rhodococcus opacus PD630 or of TAGs plus polyhydroxyalkanoates (PHA) in Rhodococcus ruber NCIMB 40126 with gluconate or glucose as carbon source, respectively, was studied. In addition, we examined the mobilization of these storage compounds in the absence of a carbon source. R. opacus accumulated TAGs only after the exhaustion of ammonium in the medium, and, with a fixed concentration of the carbon source, the amounts of TAGs in the cells increased with decreasing concentrations of ammonium in the medium. When these cells were incubated in the absence of an additional carbon source, about 90% of these TAGs were mobilized and used as endogenous carbon source, particularly if ammonium was available. R. ruber accumulated a copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate already during the early exponential growth phase, whereas TAGs were synthesized and accumulated mainly during the late exponential and stationary growth phases. In the stationary growth phase, synthesis of TAGs continued, whereas PHA was partially mobilized. In the absence of an additional carbon source but in the presence of ammonium, mobilization of TAGs started first and was then paralleled by the mobilization of PHA, resulting in an approximately 90% and 80% decrease of these storage compounds, respectively. During the accumulation phase, interesting shifts in the composition of the two storage compounds occurred, indicating that the substrates of the PHA synthase and the TAG synthesizing enzymes were provided to varying extents, depending on whether the cells were in the early or late exponential or in the stationary growth phase.
Kinetic analysis of oil biosynthesis by an arachidonic acid-producing fungus, Mortierella alpina 1S-4 by M. Certik; S. Shimizu (pp. 224-230).
Kinetic analysis of arachidonic acid (AA)-oil biosynthesis by Mortierella alpina 1S-4 growing under lipid-accumulating (LN medium) and non-lipid-accumulating (HN medium) conditions was investigated and compared with industrial AA fermentation. Various kinetic parameters of these cultivation processes demonstrate a characteristic pattern of the lipogenesis in this fungus, where growth phase, phase of oil accumulation and phase of AA synthesis are distinct from each other. The fungus utilizing LN medium synthesized 32.3 g fatty acid 100 g−1 glucose on the 4th day of cultivation and reached the maximum daily fatty acid accumulation (expressed as differential specific rate q D(FA/B)) of 9.5%. Our results also indicate that a q D(FA/B) value of about 2.5% might be critical for lipid overproduction in M. alpina. AA was rapidly incorporated into triacylglycerols (90% of total AA) at the later cultivation phase and overall AA yield was directly related to the total yield of fatty acid.
Artificial biofilm model – a useful tool for biofilm research by M. Strathmann; T. Griebe; H.-C. Flemming (pp. 231-237).
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.
Isolation of Penicillium chrysogenum PEX1 and PEX6 encoding AAA proteins involved in peroxisome biogenesis by J. A. K. W. Kiel; R. E. Hilbrands; R. A. L. Bovenberg; M. Veenhuis (pp. 238-242).
In Penicillium chrysogenum, key enzymes involved in the production of penicillin reside in peroxisomes. As a first step to understand the role of these organelles in penicillin biosynthesis, we set out to isolate the genes involved in peroxisome biogenesis. Here we report the cloning and characterization of P. chrysogenum PEX1 and PEX6, which encode proteins of the AAA family of ATPases. The second AAA module, which is essential for the function of Pex1p and Pex6p in peroxisome biogenesis, is highly conserved in both PcPex1p and PcPex6p. PcPEX1 and PcPEX6 contain three and two introns, respectively.
Bacterial response to acetate challenge: a comparison of tolerance among species by D. R. Lasko; N. Zamboni; U. Sauer (pp. 243-247).
Although acetate formation and tolerance are important criteria for various aspects of biotechnological process development, available studies on acetate tolerance in different species are disparate. We evaluate the response of eight bacterial strains, including two variants of Escherichia coli, two variants of Staphylococcus capitis, and one each of Acetobacter aceti, Gluconobacter suboxydans, Lactobacillus acetotolerans, and L. bulgaricus, to acetate challenges under identical conditions. Our findings were: (1) wild-type organisms of species that are considered tolerant of acetate perform only slightly better than E. coli in unadapted shaker cultures; (2) the ability to tolerate acetate is strongly dependent on the carbon source, and is, especially for E. coli, much greater on glycerol than on glucose; (3) respiration is not as important to acetate tolerance in E. coli and S. capitis as has been reported for the acetic acid bacteria; (4) S. capitis was the least affected by acetate under all conditions and grew at up to 44 g/l acetate without any preconditioning; and (5) qualitative high-throughput screening of growth characteristics can be achieved with relatively inexpensive multiwell plate readers.
Toluene vapour removal in a laboratory-scale biofilter by M. Zilli; A. Del Borghi; A. Converti (pp. 248-254).
A bench-scale biofilter with a 0.5-m high filter bed, inoculated with a toluene-degrading strain of Acinetobacter sp. NCIMB 9689, was used to study toluene removal from a synthetic waste air stream. Different sets of continuous tests were conducted at influent toluene concentrations ranging over 0.1–4.0 g m−3 and at superficial gas velocities ranging over 17.8–255 m h−1. The maximum volumetric toluene removal rate for the biofilter (242 g m−3 h−1) was obtained at a superficial gas velocity of 127.5 m h−1 (corresponding to a residence time of 28 s) and a toluene inlet concentration of 4.0 g m−3. Under these operating conditions, toluene removal efficiency was only 0.238, which suggested that effective operation required higher residence times. Removal efficiencies higher than 0.9 were achieved at organic loads less than 113.7 g m−3 h−1. A macro-kinetic study, performed using concentration profiles along the bioreactor, revealed this process was limited by diffusion at organic loads less than 100 g m−3 h−1 and by biological reaction beyond this threshold.
Estimating the availability of polycyclic aromatic hydrocarbons for bioremediation of creosote contaminated soils by G. D. Breedveld; D. A. Karlsen (pp. 255-261).
Bioremediation of soil contaminated by organic compounds can remove the contaminants to a large extent, but residual contamination levels may remain which are not or only slowly biodegraded. Residual levels often exceed existing clean-up guidelines and thereby limit the use of bioremediation in site clean-up. A method for estimating the expected residual levels would be a useful tool in the assessment of the feasability of bioremediation. In this study, three soil types from a creosote-contaminated field site, which had been subjected to 6 months of bioremediation in laboratory column studies, were used to characterize the residual contamination levels and assess their availability for biodegradation. The soils covered a wide range of organic carbon levels and particle size distributions. Results from the biodegradation studies were compared with desorption rate measurements and selective extractability using butanol. Residual levels of polycyclic aromatic hydrocarbons after bioremediation were found to be strongly dependent on soil type. The presence of both soil organic matter and asphaltic compounds in the soil was found to be associated with higher residual levels. Good agreement was found between the biodegradable fraction and the rapidly desorbable fraction in two of the three soils studied. Butanol extraction was found to be a useful method for roughly estimating the biodegradable fraction in the soil samples. The results indicate that both desorption and selective extraction measurements could aid the assessment of the feasability for bioremediation and identifying acceptable end-points.
Factors influencing the biosorption of gadolinium by micro-organisms and its mobilisation from sand by Y. Andrès; G. Thouand; M. Boualam; M. Mergeay (pp. 262-267).
The present work was devoted to the study of the biosorption capacities of various microbial species (Bacillus subtilis, Pseudomonas aeruginosa, Ralstonia metallidurans CH34 previously Alcaligenes eutrophus CH34, Mycobacterium smegmatis, Saccharomyces cerevisiae) for ions of the lanthanide gadolinium (Gd3+). The uptake by sand of this element was also measured. Saturation curves and Scatchard models were established for all biosorbants used in this work. The results enabled us to determine the binding affinities and the maximum capacities for biosorption of Gd3+, which ranged from 350 μmol g−1 for B. subtilis to 5.1 μmol g−1 for S. cerevisiae. This study demonstrated the usefulness of optimisation of experimental conditions in biosorption investigations. Experimental results showed that biosorption could be influenced by the growth stage and by the composition of the growth medium of microbial cells. Finally, particular attention was given to the transfer of gadolinium ions from a loaded sand to a bacterial suspension.
Biodegradation of azo dyes in a sequential anaerobic–aerobic system by P. Rajaguru; K. Kalaiselvi; M. Palanivel; V. Subburam (pp. 268-273).
A sequential anaerobic–aerobic treatment process based on mixed culture of bacteria isolated from textile dye effluent-contaminated soil was used to degrade sulfonated azo dyes Orange G (OG), Amido black 10B (AB), Direct red 4BS (DR) and Congo red (CR). Under anaerobic conditions in a fixed-bed column using glucose as co-substrate, the azo dyes were reduced and amines were released by the bacterial biomass. The amines were completely mineralized in a subsequent aerobic treatment using the same isolates. The maximum degradation rate observed in the treatment system for OG was 60.9 mg/l per day (16.99 mg/g glucose utilized), for AB 571.3 mg/l per day (14.46 mg/g glucose utilized), for DR 112.5 mg/l per day (32.02 mg/g glucose utilized) and for CR 134.9 mg/l per day (38.9 mg/g glucose utilized).