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Applied Microbiology and Biotechnology (v.52, #5)
Nutrient requirements of lactococci in defined growth media by E. W. J. van Niel; B. Hahn-Hägerdal (pp. 617-627).
Many attempts have been made for the last six decades to design defined media for species of the lactococcus group. The general outcome of the studies suggests that this group is heterogeneous with respect to specific requirements for nutrients. Lactococcal species are limited in various metabolic pathways. Early attempts to trace the required nutrients were not always successful because of the poor quality of analysis and the presence of impurities in the medium components.
Production, recovery and purification of bacteriocins from lactic acid bacteria by E. Parente; A. Ricciardi (pp. 628-638).
Bacteriocins produced by lactic acid bacteria are a heterogeneous group of peptide inhibitors which include lantibiotics (class I, e.g. nisin), small heat-stable peptides (class II, e.g. pediocin AcH/PA1) and large heat-labile proteins (class III, e.g. helveticin J). Many bacteriocins belonging to the first two groups can be successfully used to inhibit undesirable microorganisms in foods, but only nisin is produced industrially and is licensed for use as a food preservative in a partially purified form. This review focuses on the production and purification of class I and class II bacteriocins from lactic acid bacteria. Bacteriocin production is growth associated but the yield of bacteriocin per unit biomass is affected by several factors, including the producing strain, media (carbohydrate and nitrogen sources, cations, etc.) and fermentation conditions (pH, temperature, agitation, aeration and dilution rate in continuous fermentations). Continuous fermentation processes with cell recycle or immobilized cells can result in a dramatic improvement in productivity over batch fermentations. Several simple recovery processes, based on adsorbing bacteriocin on resins or silica compounds, have been developed and can be used to build integrated production processes.
Role of microorganisms in corrosion inhibition of metals in aquatic habitats by J. S. Potekhina; N. G. Sherisheva; L. P. Povetkina; A. P. Pospelov; T. A. Rakitina; F. Warnecke; G. Gottschalk (pp. 639-646).
Bacterial activity at metal surfaces may result in corrosion induction or corrosion inhibition. An important effect of chemoorganotrophic bacteria under aerobic conditions is the removal of oxygen from the metal surface. This is most effectively done by biofilm-forming microorganisms and causes corrosion inhibition. Anaerobes promote corrosion if they consume molecular hydrogen, for example sulfate-reducing bacteria and certain Fe(III)-reducing organisms such as Shewanella putrefaciens. Chemoorganotrophic Fe(III) reducers are of special interest because they remove corrosion products and hence destroy ecological niches for sulfate-reducing bacteria beneath the deposits. Also, these chemoorganotrophic bacteria may cause passivation of the metal surface through the consumption of dissolved oxygen, lowering the open circuit potential and the creation of a protective layer of atomic hydrogen on the metal surface.
A kinetic model incorporating energy spilling for substrate removal in substrate-sufficient batch culture of activated sludge by Y. Liu; G. H. Chen; J. L. Rols (pp. 647-651).
Batch assays are currently used to study the kinetic behavior of microbial growth. However, it has been shown that the outcome of batch experiments is greatly influenced by the initial ratio of substrate concentration (S o) to biomass concentration (X o). Substrate-sufficient batch culture is known to have mechanisms of spilling energy that lead to significant nongrowth-associated substrate consumption, and the Monod equation is no longer appropriate. By incorporating substrate consumption associated with energy spilling into the balance of the substrate oxidation reaction, a kinetic model for the observed specific substrate consumption rate was developed for substrate-sufficient batch culture of activated sludge, and was further verified by experimental data. It was demonstrated that the specific substrate consumption rate increased with the increase of the S o/X o ratio, and the majority of substrate was consumed through energy spilling at high S o/X o ratios. It appears that the S o/X o ratio is a key parameter in regulating metabolic pathways of microorganisms.
Immobilization of pancreatic islet cells with preserved secretory potential by W. J. Malaisse; E. Olivares; A. Belcourt; K. Nilsson (pp. 652-653).
Dispersed pancreatic islet cells from rats were cultured overnight in the presence of macroporous gelatin microcarriers. The cells attached to the microcarriers were then incubated for 90 min in the absence or presence of 15.0 mM d-glucose and/or 1.25 mM theophylline. The release of insulin during incubation was about three times higher in the simultaneous presence of these two secretagogues than in their absence. This procedure could thus be used for the immobilization of pancreatic islet cells with preserved secretory potential.
A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles by J. E. Gavagan; R. DiCosimo; A. Eisenberg; S. K. Fager; P. W. Folsom; E. C. Hann; K. J. Schneider; R. D. Fallon (pp. 654-659).
A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C3–C6 dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746.
Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I by V. Monchois; M. Vignon; R. R. B. Russell (pp. 660-665).
Related streptococcal and Leuconostoc mesenteroides glucansucrases are enzymes of medical and biotechnological interest. Molecular modelling has suggested that the catalytic domain contains a circularly permuted version of the (β/α)8 barrel structure found in the amylase superfamily, and site-directed mutagenesis has identified critical amino acids in this region. In this study, sequential N-terminal truncations of Streptococcus downei GTF-I showed that key amino acids are also present in the first one-third of the core domain. Mutations were introduced at Trp-344, Glu-349 and His-355, residues that are conserved in all glucansucrases and lie within a region which is a target for inhibitory antibodies. W344L, E349L and H355V substitutions were assayed for their effect on mutan synthesis and also on oligosaccharide synthesis with various acceptors. It appeared that Trp-344 and His-355 are involved in the action mechanism of GTF-I; His-355 may also play a role in a binding subsite necessary for oligosaccharide and glucan elongation.
Cloning and functional expression of the d-β-hydroxybutyrate dehydrogenase gene of Rhodobacter sp. DSMZ 12077 by K. Krüger; G. Lang; T. Weidner; A. M. Engel (pp. 666-669).
Nucleotide sequence and biochemical analysis of d-β-hydroxybutyrate dehydrogenase (EC 1.1.1.30), isolated from Rhodobacter sp., indicate functional oligomers composed of subunits of 257 amino acids with a calculated M r of 26,800 and a pI of 5.90. Compared to mammalian short-chain alcohol dehydrogenases, the bacterial enzyme lacks a C-terminal lipid anchor domain and was found to be highly active upon expression in Escherichia coli even without lipid supplement. The recombinant enzyme could be highly enriched using a single chromatography step and was shown to be stable over a broad range of pH and temperature.
Effect of dilution rate, cellobiose and ammonium availabilities on Clostridium cellulolyticum sporulation by S. Payot; E. Guedon; M. Desvaux; E. Gelhaye; E. Petitdemange (pp. 670-674).
The nutritional and physiological factors affecting sporulation of Clostridium cellulolyticum were studied using steady-state continuous cultures grown in both complex and synthetic media. Under cellobiose limitation, the probability that cells will sporulate appears to be directly related to the growth rate. In complex medium, the highest percentage of sporulation was 20% at a dilution rate of 0.015 h−1 whereas in synthetic medium it was 10% at 0.035 h−1. In both media, when the dilution rate was either higher or lower the percentage of sporulation decreased by between 2% and 4%. At low dilution rates, endospore formation was repressed under cellobiose-sufficient concentrations, suggesting catabolite repression by cellobiose. Furthermore, the concentration of ammonium was important in determining the percentage of sporulation, as ammonium limitation induced extensive sporulation at low growth rates even in an excess of cellobiose. The sporulation process is not triggered when cells are cellobiose-exhausted both in complex and synthetic media. These data suggest that, in C. cellulolyticum, an exogenous supply of carbon is required throughout the sporulation process. In the experimental conditions used in this work, no relationship between glycogen accumulation or glycogen mobilization and endospore formation was detected in C. cellulolyticum.
Production of halostable β-mannanase and β-mannosidase by strain NN, a new extremely halotolerant bacterium by M. Wainø; K. Ingvorsen (pp. 675-680).
An extremely halotolerant mannan-degrading bacterium (strain NN) was isolated from the Great Salt Lake, Utah, USA. Strain NN grew at salinities from 0 to 20% NaCl with optimal growth at 0% NaCl. When grown on 0.2% (w/v) locust bean gum as the carbon source at 10% NaCl, both β-mannanase and β-mannosidase activities were produced. β-Mannosidase activity was shown to be cell-associated, while at least 23% of the total β-mannanase activity was extracellular. The optimum temperature and pH for β-mannanase activity were 70 °C and 7.6, and for β-mannosidase 25 °C and 7.0. The β-mannanase system retained full activity after 24 h of incubation at 60 °C and 10% NaCl. β-Mannanase activity was maximal at 1% NaCl and β-mannosidase activity at 0.5% NaCl. Despite these low salinity optima, 50% and 100% respectively of the initial β-mannanase and β-mannosidase activities remained after 48 h of incubation at 20% NaCl, indicating a high degree of halostability. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed the presence of at least eight different mannan-degrading proteins in the cell-free culture supernatant of cultures grown on locust bean gum.
Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 α-galactosidase by K. M. J. Van Laere; R. Hartemink; G. Beldman; S. Pitson; C. Dijkema; H. A. Schols; A. G. J. Voragen (pp. 681-688).
Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an α-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083T. It was found to act with retention of configuration (α→α), releasing α-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As α-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of α-galactosides using the transgalactosylation activity of Bi. adolescentisα-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 °C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp and tetrasaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate.
Production of ligninolytic enzymes and synthetic lignin mineralization by the bird's nest fungus Cyathus stercoreus by A. Sethuraman; D. E. Akin; K.-E. L. Eriksson (pp. 689-697).
Production of ligninolytic enzymes and degradation of 14C-ring labeled synthetic lignin by the white-rot fungus Cyathus stercoreus ATCC 36910 were determined under a variety of conditions. The highest mineralization rate for 14C dehydrogenative polymerizates (DHP; 38% 14CO2 after 30 days) occurred with 1 mM ammonium tartrate as nitrogen source and 1% glucose as additional carbon source, but levels of extracellular laccase and manganese peroxidase (MnP) were low. In contrast, 10 mM ammonium tartrate with 1% glucose gave low mineralization rates (10% 14CO2 after 30 days) but higher levels of laccase and manganese peroxidase. Lignin peroxidase was not produced by C. stercoreus under any of the studied conditions. Mn(II) at 11 ppm gave a higher rate of 14C DHP mineralization than 0.3 or 40 ppm, but the highest manganese peroxidase level was obtained with Mn(II) at 40 ppm. Cultivation in aerated static flasks gave rise to higher levels of both laccase and manganese peroxidase compared to the levels in shake cultures. 3,4-Dimethoxycinnamic acid at 500 μM concentration was the most effective inducer of laccase of those tested. The purified laccase was a monomeric glycoprotein having an apparent molecular mass of 70 kDa, as determined by calibrated gel filtration chromatography. The pH optimum and isoelectric point of the purified laccase were 4.8 and 3.5, respectively. The N-terminal amino acid sequence of C. stercoreus laccase showed close homology to the N-terminal sequences determined from other basidiomycete laccases. Information on C. stercoreus, whose habitat and physiological requirements for lignin degradation differ from many other white-rot fungi, expands the possibilities for industrial application of biological systems for lignin degradation and removal in biopulping and biobleaching processes.
Screening and characterization of bioflocculant produced by isolated Klebsiella sp. by W. Dermlim; P. Prasertsan; H. Doelle (pp. 698-703).
Sixteen strains of polymer-producing bacteria were isolated from the activated sludge samples taken from two seafood processing plants in Southern Thailand. Their culture broths possessed the ability to flocculate kaolin suspension in the presence of 1% CaCl2. Based on the flocculating activity, the strain S11 was selected and identified to be a Klebsiella sp. using the partial 16S rRNA sequencing method. The growth of the isolated Klebsiella sp. was maximal (1.026 g l−1 dry cell mass) after 1 day cultivation while the highest polymer yield (0.973 g l−1) was achieved after 5 days cultivation. The flocculating activity of the culture broth, however, was highest after 2 days cultivation. The polymer was identified to be an acidic polysaccharide containing neutral sugar and uronic acid as its major and minor components, respectively. Results on the properties of the partially purified polysaccharide from Klebsiella sp. S11 revealed that it consisted of galactose, glucose and mannose in an approximate ratio of 5:2:1. It was soluble in acidic or basic solutions but not in organic solvents. Its molecular mass was greater than 2 × 106 Da. Infrared spectra showed the presence of hydroxyl, carboxyl and methoxyl groups in its molecules. Differential scanning calorimetry of the polysaccharide indicated the crystalline melting point (T m) at 314 °C. The optimum dosage of polysaccharide to give the highest flocculating activity was 15 mg l−1 in the presence of 1% CaCl2.
Isolation and characterization of a 2-methylphenanthrene utilizing bacterium: identification of ring cleavage metabolites by J. Sabaté; M. Grifoll; M. Viñas; A. M. Solanas (pp. 704-712).
A bacterial strain capable of utilizing 2-methylphenanthrene (2-MP) as its sole source of carbon and energy for growth was isolated from creosote contaminated soil. The isolate was identified as a strain of Sphingomonas sp. and was designated strain JS5. Utilization of 2-MP by strain JS5 was demonstrated by an increase in bacterial biomass concomitant with a decrease of 2-MP in liquid mineral medium with this compound as sole source of carbon and energy. Growth yield indicated a 23% assimilation of 2-MP carbon. Washed-cell suspensions of strain JS5 incubated with 2-MP accumulated a major metabolite identified as 1-hydroxy-6-methyl-2-naphtoic acid, according to its UV, mass and NMR spectra, and a minor compound with HPLC R t and UV spectrum indistinguishable from 5-methylsalicylate. The identification of those metabolites, and the demonstration of 2,3-catechol dioxygenase activity in 2-MP induced cells show that the biodegradation of 2-MP by strain JS5 is initiated via dioxygenation and meta-cleavage of the non-methylated aromatic ring, and then proceeds by reactions similar to those reported for phenanthrene. Incubation of the strain with a MP-containing mixture from a pyrolytic fuel oil demonstrates that strain JS5 also acts on other methylated phenanthrenes.
Characterization of a biosurfactant, mannosylerythritol lipid produced from Candida sp. SY16 by H.-S. Kim; B.-D. Yoon; D.-H. Choung; H.-M. Oh; T. Katsuragi; Y. Tani (pp. 713-721).
One yeast strain, SY16, was selected as a potential producer of a biosurfactant, and identified as a Candida species. A biosurfactant produced from Candida sp. SY16 was purified and confirmed to be a glycolipid. This glycolipid-type biosurfactant lowered the surface tension of water to 29 dyne/cm at critical micelle concentration of 10 mg/l (1.5 × 10−5 M), and the minimum interfacial tension was 0.1 dyne/cm against kerosene. Thin-layer and high-pressure liquid chromatography studies demonstrated that the glycolipid contained mannosylerythritol as a hydrophilic moiety. The hydrophilic sugar moiety of the biosurfactant was determined to be β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by nuclear magnetic resonance (NMR) and fast atom bombardment mass–spectroscopy analyses. The hydrophobic moiety, fatty acids, of the biosurfactant was determined to be hexanoic, dodecanoic, tetradecanoic, and tetradecenoic acid by gas chromatography–mass spectroscopy. The structure of the native biosurfactant was determined to be 6-O-acetyl-2,3- di-O-alkanoyl-β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by NMR analyses. We newly determined that an acetyl group was linked to the C-6 position of the d-mannose unit in the hydrophilic sugar moiety.
4-Chlorophenol degradation by a bacterial consortium: development of a granular activated carbon biofilm reactor by M. Caldeira; S. C. Heald; M. F. Carvalho; I. Vasconcelos; A. T. Bull; P. M. L. Castro (pp. 722-729).
A bacterial consortium that can degrade chloro- and nitrophenols has been isolated from the rhizosphere of Phragmitis communis. Degradation of 4-chlorophenol (4-CP) by a consortium attached to granular activated carbon (GAC) in a biofilm reactor was evaluated during both open and closed modes of operation. During the operation of the biofilm reactor, 4-CP was not detected in the column effluent, being either adsorbed to the GAC or biodegraded by the consortium. When 4-CP at 100 mg l−1 was fed to the column in open mode operation (20 mg g−1 GAC total supply), up to 27% was immediately available for biodegradation, the rest being adsorbed to the GAC. Biodegradation continued after the system was returned to closed mode operation, indicating that GAC bound 4-CP became available to the consortium. Biofilm batch cultures supplied with 10–216 mg 4-CP g−1 GAC suggested that a residual fraction of GAC-bound 4-CP was biologically unavailable. The consortium was able to metabolise 4-CP after perturbations by the addition of chromium (Cr VI) at 1–5 mg l−1 and nitrate at concentrations up to 400 mg l−1. The development of the biofilm structure was analysed by scanning electron microscopy and confocal laser scanning microscopy (CLSM) techniques. CLSM revealed a heterogeneous structure with a network of channels throughout the biofilm, partially occupied by microbial exopolymer structures.
Kinetics and characteristics of 70 °C, VFA-grown, UASB granular sludge by R. Lepistö; J. Rintala (pp. 730-736).
We studied in batch reactors the kinetics and characterization of 70 °C, volatile fatty acids (VFAs)-grown, upflow anaerobic sludge blanket granular sludge with 55 and 35 °C sludge as reference. The half-saturation constant (K s), the inhibition constant (K i), the maximum specific methane production rate (μCH4max), and the inhibition response coefficient (n) of the 70 °C sludge were 6.15 mM, 48.2 mM, 0.132 h−1, and 2.48, respectively, while no inhibition occurred at 55 and 35 °C, where the K s was 3.67 and 3.82 mM, respectively. At 70 °C, the highest initial specific methanogenic activity (ISMA, 0.311 gCH4-COD per gram volatile solids per day) on VFAs was about 12–15% lower than that on acetate and three to four times less than the ISMA for the 55 and 35 °C sludge. In the acetate conversion study, residual acetate (79 mg l−1) at 70 °C was three to five times higher than that at 55 and 35 °C. Further, the methane produced as percentage of the acetate consumed at 70 °C (89%) was lower than that at 55 (95%) and 35 °C (97%). At 70 °C, 10% of the ISMA remained after 15 days of starvation as compared to 26% (55 °C) and 92% (35 °C) after 30 days of starvation. Thus, the kinetics of the 70 °C granular sludge seem to differ from those at 55 and 35 °C.