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Applied Microbiology and Biotechnology (v.55, #6)
Polyester and polycarbonate synthesis by in vitro enzyme catalysis by R. Gross; B. Kalra; A Kumar (pp. 655-660).
Enzyme technology has significantly expanded in scope and impact over the past 10 years to include organic transformations in non-traditional environments. This is in part due to an increased understanding and capability of using enzyme catalysis in a wide variety of organic solvents, at interfaces, and at high temperatures and pressures. This review focuses on a relatively new but rapidly expanding research activity where in vitro enzyme catalysis is used for the synthesis of non-natural polyesters and polycarbonates. The inclination to use of enzymes for polymer synthesis has been fueled by a desire to carry out these reactions in the absence of heavy metals, at lower temperatures, and with increased selectivity. Aspects of this work that include enzyme-catalyzed step-growth condensation reactions, chain-growth ring-opening polymerizations, and corresponding transesterification of macromolecular substrates are discussed.
The use of transgenic plants in the bioremediation of soils contaminated with trace elements by U. Krämer; A. Chardonnens (pp. 661-672).
The use of plants to clean-up soils contaminated with trace elements could provide a cheap and sustainable technology for bioremediation. Field trials suggested that the rate of contaminant removal using conventional plants and growth conditions is insufficient. The introduction of novel traits into high biomass plants in a transgenic approach is a promising strategy for the development of effective phytoremediation technologies. This has been exemplified by generating plants able to convert organic and ionic forms of mercury into the less toxic, volatile, elemental mercury, a trait that occurs naturally only in some bacteria and not at all in plants. The engineering of a phytoremediator plant requires the optimization of a number of processes, including trace element mobilization in the soil, uptake into the root, detoxification and allocation within the plant. A number of transgenic plants have been generated in an attempt to modify the tolerance, uptake or homeostasis of trace elements. The phenotypes of these plants provide important insights for the improvement of engineering strategies. A better understanding, both of micronutrient acquisition and homeostasis, and of the genetic, biochemical and physiological basis of metal hyperaccumulation in plants, will be of key importance for the success of phytoremediation.
Bacterial cellulose production under oxygen-enriched air at different fructose concentrations in a 50-liter, internal-loop airlift reactor by Y. Chao; Y. Sugano; M. Shoda (pp. 673-679).
Bacterial cellulose (BC) production by Acetobacter xylinum subsp. sucrofermentans BPR2001 was carried out in a 50-l internal-loop airlift reactor in air at an initial fructose concentration of 40 g/l. The BC production rate was 0.059 g/l per h. When oxygen-enriched air was supplied instead of air, the BC production rate increased to 0.093 g/l per h, and the BC yield was enhanced from 11% in air to 18%. When the initial fructose concentrations were varied from 30 to 70 g/l, the highest BC yield (35%) the highest production rate (0.22 g/l·per h), and the highest concentration of BC produced (10.4 g/l) were observed at 60–70 g/l fructose. From the carbon mass balance calculated at the final stage of cultivation, it was observed that enhanced BC production was reflected as a decrease in both CO2 evolution and the concentration of other unknown substances, suggesting the efficient utilization of energy for BC synthesis despite O2 limitation.
Efficient pyruvate production by a multi-vitamin auxotroph of Torulopsis glabrata: key role and optimization of vitamin levels by Y. Li; J. Chen; S.-Y. Lun; X.-S. Rui (pp. 680-685).
A multi-vitamin auxotroph, Torulopsis glabrata strain WSH-IP303, which can use ammonium chloride as a sole nitrogen source for pyruvate production, was selected. To optimize pyruvate yield and productivity, a simple but useful, orthogonal design method, was used to investigate the relationship between thiamine, nicotinic acid, pyridoxine, biotin, and riboflavin. Thiamine was confirmed to be the most important factor affecting pyruvate production. When the concentration of thiamine was 0.01 mg/l or 0.015 mg/l, glucose consumption was improved by increasing the nicotinic acid concentration. When the concentrations of nicotinic acid, thiamine, pyridoxine, biotin, and riboflavin were 8.0, 0.015, 0.4, 0.04, and 0.1 mg/l, respectively, pyruvate concentration and yield reached 52 g/l and 0.52 g/g, respectively, in a 48-h flask culture. By employing a combination of the optimum vitamin concentrations, a batch culture was conducted in a 2.5-l fermentor with an initial glucose concentration of 112 g/l; and the pyruvate concentration reached 69 g/l after 56 h (yielding 0.62 g/g).
Glycerol production by a novel osmotolerant yeast Candida glycerinogenes by J. Zhuge; H.-Y. Fang; Z.-X. Wang; D.-Z. Chen; H.-R. Jin; H.-L. Gu (pp. 686-692).
Candida glycerinogenes, an osmotolerant yeast isolated from a natural sample in an environment of high osmotic pressure, had a modest sugar-tolerance and an extremely high glycerol productivity. The optimum conditions for glycerol formation by C. glycerinogenes were a temperature of 29–33 °C and a pH of 4–6. The optimum medium for glycerol production consisted of 230–250 g glucose/l, 2 g urea/l and 5 ml corn steep liquor/l (55–65 mg phosphates/l); the pH was not adjusted. The highest yield of glycerol was 64.5% (w/w) based on consumed glucose from 240 g glucose/l, and the highest concentration of glycerol was 137 g/l from 260 g glucose/l. These results were obtained by using a 30-l agitated fermentor under optimal fermentation conditions. In ten batch-fermentations carried out in a 50,000-l airlift fermentor, an average yield of glycerol of 50.67% (w/w) and an average glycerol concentration of 121.9 g/l were obtained from an average 240.6 g glucose/l.
Effects of stress factors, bioregulators, and synthetic precursors on indole alkaloid production in compact callus clusters cultures of Catharanthus roseus by J. Zhao; Q. Hu; Y.-Q. Guo; W.-H. Zhu (pp. 693-698).
Compact callus cluster (CCC) cultures established from Catharanthus roseus consist of cohesive callus aggregates displaying certain levels of cellular or tissue differentiation. CCC cultures synthesize about two-fold more indole alkaloids than normal dispersed-cell cultures. Our studies here show that additions of KCl, mannitol, and a variety of synthetic precursors and bioregulators to the CCC cultures markedly improved indole alkaloid production and release of these alkaloids into the medium. Treatment with 250 mM mannitol and 4 g/l KCl yielded 42.3 mg l–1 and 33.6 mg l–1of ajmalicine, respectively; these amounts were about four-fold higher than the control. Succinic acid, tryptamine, and tryptophan feedings also significantly increased ajmalicine (41.5 mg l–1, 36.9 mg l–1 ,and 31.8 mg l–1, respectively) and catharanthine (21.1 mg l–1, 17.2 mg l–1, and 18 mg l–1, respectively) production by the CCC cultures, while geraniol feeding inhibited biomass and alkaloid accumulation. We also found that tetramethyl ammonium bromide could significantly improve ajmalicine production (49.3 mg l–1) and catharanthine production (18.3 mg l–1) in C. roseus CCC cultures. The mechanisms responsible for these treatment effects are discussed herein.
Enzymatic modification of kraft lignin through oxidative coupling with water-soluble phenols by M. Lund; A. Ragauskas (pp. 699-703).
The aromatic polymer lignin can be modified through promotion of oxidative coupling between phenolic groups on lignin and various phenols. The reaction is initiated by an oxidation of both components, e.g., by using the oxidoreductases laccase or peroxidase. Coupling between phenolic monomers and lignin has previously been studied by the use of radio-labeled phenols. In this study, incorporation of water-soluble phenols into kraft lignin, using laccase as catalyst, was investigated. Several phenols with carboxylic or sulfonic acid groups were used as markers for the incorporation. The modified lignin was isolated and the amount of phenol incorporated was characterized by means of titration, quantitative 1H-NMR, and quantitative 31P-NMR after modification with 2-chloro-4,4,5,5-tetramethyl-1,2,3-dioxaphospholane. Only a few of the phenols studied were found to be incorporated into lignin. When the phenol guaiacol sulfonate was incorporated into kraft lignin, the lignin became water-soluble at pH 2.4 and a low ionic strength due to the introduction of sulfonic acid groups. The content of sulfonic acid groups in the product was 0.5–0.6 mmol/g lignin. A lower amount of 4-hydroxyphenylacetic acid was incorporated under similar conditions.
Production of manganese peroxidase by pellet culture of the lignin-degrading basidiomycete, Pleurotus ostreatus by H-C. Ha; Y. Honda; T. Watanabe; M. Kuwahara (pp. 704-711).
Pleurotus ostreatus No. 42 produced the ligninolytic enzymes, manganese peroxidase (MnP) and laccase, in agitation culture in glucose/peptone/wheat-bran medium. Formation of mycelial pellets 1–2 mm in diameter was essential for the production of MnP; and the concentration of dissolved oxygen in the culture medium greatly influenced the production of MnP, a concentration over 5 ppm being necessary for MnP production. The maximal activity of MnP was obtained on days 7–9 of culture, after the consumption of nutrient glucose. Introduction of oxygen from the start of the cultivation caused large pellet formation, which resulted in a low MnP activity level. P. ostreatus No. 42 produced two MnP isozymes in agitation culture. The major isozyme, F-2, was 36.4 kDa and had a pI of 3.95. The MnP characteristics, K m values, dependence on Mn2+ and optimum pH showed the similarity between this isozyme and MnP 3, which was produced under different culture conditions. Analysis of the N-terminal amino acid sequence indicated the close similarity of F-2 to MnP 3.
Characterization of a Saccharomyces cerevisiae mutant with oversecretion phenotype by B.-D. Wang; D.-C. Chen; T.-T. Kuo (pp. 712-720).
An oversecreting mutant of Saccharomyces cerevisiae was obtained from about 400 meiotic segregants derived from thediploid cells made by crossing the HBsAg-induced mutant NI-C with the wild-type strain Sey6211. When transformed with a plasmid containing mouse α-amylase cDNA, the mutant (NI-C-D4) exhibited an increased capacity (up to 13-fold) for the secretion of mouse α-amylase, higher than the parental strains and other standard wild-type strains. It was also shown that α-amylase secreted by the oversecreting mutant had a higher activity and contained more of the non-glycosylated form than the glycosylated form. This isolated oversecreting, low-glycosylation mutant may prove to be a potential S. cerevisiae host for the production of foreign proteins. Further genetic analysis suggested that the mutation responsible for the mutant's oversecretion was partially dominant and that both the oversecretion and low-glycosylation phenotypes were governed by a single chromosome mutation. These pleiotrophic phenotypes may be attributed to a defect in the synthesis of an ER-resident chaperone.
Expression of benzene dioxygenase from Pseudomonas putida ML2 in cis-1,2-cyclohexanediol-degrading pseudomonads by R. Swift; S. Carter; D. Widdowson; J. Mason; D. Leak (pp. 721-726).
Benzene dioxygenase (BDO; EC 1.14.12.3) from Pseudomonas putida ML2 dihydroxylates benzene to produce cis-1,2-dihydroxy-cyclohexa-3,5-diene. As well as oxidising benzene and toluene, cell-free extracts of Escherichia coli JM109 expressing recombinant BDO oxidised cyclohexene, 1-methylcyclohexene and 3-methylcyclohexene. In an attempt to construct a novel metabolic pathway for the degradation of cyclohexene (via an initial BDO-mediated dihydroxylation of cyclohexene), cis-1,2-cyclohexanediol-degrading bacteria were isolated by enrichment culture. The bedC1C2BA genes encoding BDO (under the control of the tac promoter) were sub-cloned into pLAFR5, successfully conjugated into seven of the Gram-negative cis-1,2-cyclohexanediol-degrading isolates and stably maintained and expressed in three of them. However, despite their ability to grow on cis-1,2-cyclohexanediol as sole carbon source, express an active BDO and oxidise cyclohexene, none of the three strains was able to grow on cyclohexene as sole carbon source. Analysis revealed that BDO oxidised cyclohexene to a mixture of two products, a monohydroxylated (2-cyclohexen-1-ol) product and a dihydroxylated (cis-1,2-cyclohexanediol) product; and failure to grow on cyclohexene was attributed to the toxicity of metabolic intermediates accumulating from the 2-cyclohexen-1-ol metabolism.
Xanthomonas campestris pv. campestris secretes the endoglucanases ENGXCA and ENGXCB: construction of an endoglucanase-deficient mutant for industrial xanthan production by K. Schröter; E. Flaschel; A. Pühler; A. Becker (pp. 727-733).
Xanthomonas campestris pv. campestris secretes at least two cellulose-degrading endoglucanases. One of these endoglucanases is encoded by the engXCA gene of X. c. pv. campestris 8400 that was previously characterized by Gough et al. [Gene (1990) 89: 53–59]. An additional endoglucanase encoded by the engXCB gene was identified in X. c. pv. campestris 8400 and FC2. The engXCB gene product that was grouped into the endoglucanase family E contains a putative N-terminal signal peptide, suggesting a secretion by the type II secretion system. The ENGXCB protein contributed approximately 8% to the cellulase activity in xanthan preparations. Deletion of engXCA and engXCB resulted in a fivefold reduction of the cellulose-degrading activity in xanthan preparations. The cellulase activity determined in xanthan preparations of the engXCA-engXCB mutant was only slightly higher than the activity found in preparations that were subjected to heat treatment. Mutations in engXCA and engXCB did not affect the growth rate and xanthan production of X. c. pv. campestris FC2 under several cultivation conditions. The engXCA-engXCB deletion mutant is markerless, which makes this mutant a valuable strain for xanthan production and approaches aimed at inactivating further genes encoding extracellular enzymes.
Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha by H. Kang; W.-K. Hong; J.-H. Sohn; E.-S. Choi; S. Rhee (pp. 734-741).
The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized. In contrast to the actin genes of other ascomycetous yeasts, which have only one large intron, the H. polymorpha ACT gene was found to be split by two introns. The H. polymorpha ACT introns were correctly processed in the heterologous host Saccharomyces cerevisiae despite appreciable differences in the splice site sequences. The promoter region of H. polymorpha ACT displayed two CCAAT motifs and two TATA-like sequences in a configuration similar to that observed in the S. cerevisiae actin promoter. A set of deleted H. polymorpha ACT promoters was exploited to direct expression of the bacterial hygromycin B resistance (hph) gene as a dominant selectable marker in the transformation of H. polymorpha. The resistance level of H. polymorpha transformants to the antibiotic was shown to be dependent on the integration copy number of the hph cassette. The selectivity of the hygromycin B resistance marker for transformants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT motif, from the H. polymorpha promoter. The dosage-dependent selection system developed in this study should be useful for genetic manipulation of H. polymorpha as an industrial strain to produce recombinant proteins.
RAPD discrimination of Agaricus bisporus mushroom cultivars by A. Moore; M. Challen; P. Warner; T. Elliott (pp. 742-749).
Cultivars of the white button mushroom Agaricus bisporus are difficult to differentiate, which has made strain protection problematic for this crop species. We have used RAPDs to discriminate between 26 strains of A. bisporus, 24 of which were commercial cultivars, and to characterise the genetic relatedness of these strains. Using 20 primers, 211 RAPD markers were identified and used in hierarchical cluster, patristic distance and parsimony analyses. All strains could be differentiated using the aggregated primer data. Although no one primer could differentiate all 26 strains, several individual primers yielded unique fingerprints for a variety of strains. The greatest differences (up to 28% variation) were observed in comparisons with or between two wild collections of A. bisporus. Quondam cultivars, commercial brown and off-white varieties proved more variable than the widely grown 'hybrid' types. Of the 15 hybrid varieties analysed, only one differed substantially (20% or more variable). The patristic and parsimony analyses both demonstrated the gross similarity of the hybrids, many of which appear to be essentially derived varieties from two original hybrid cultivars. RAPD analyses can assist mushroom strain identification and could play a role in the protection of novel cultivars.
Cloning, nucleotide sequence and expression of a hydantoinase and carbamoylase gene from Arthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from Arthrobacter aurescens DSM 3747 by Anja Wiese; Burkhard Wilms; Christoph Syldatk; Ralf Mattes; Josef Altenbuchner (pp. 750-757).
The genes encoding hydantoinases (hyuH1) and carbamoylases (hyuC1) from Arthrobacter aurescens DSM 3745 and Arthrobacter aurescens DSM 3747 (hyuH2, hyuC2) were cloned in Escherichia coli and the nucleotide sequences determined. The hydantoinase genes comprised 1,377 base pairs and the carbamoylase genes 1,239 base pairs each. Both hydantoinases, as well as both carbamoylases, showed a high degree of nucleotide and amino acid sequence identity (96–98%). The hyuH and hyuC genes were expressed in E. coli under the control of the rhamnose promoter and the different specific activities obtained in E. coli crude extracts were compared to those produced by the original hosts. For purification the hyuH2 gene was expressed as a maltose-binding protein (MalE) and as an intein–chitin binding domain (CBD) fusion in E. coli. The expression of malE-hyuH2 resulted in the production of more soluble and active protein. With respect to temperature stability, optimal pH and optimal temperature, substrate and stereospecificity, the purified fusion enzyme exhibited properties similar to those of the wild-type enzyme.
Biosynthesis of methionine from homocysteine, cystathionine and homoserine plus cysteine by mixed rumen microorganisms in vitro by M. Or-Rashid; R. Onodera; S. Wadud (pp. 758-764).
This study quantitatively investigated the biosynthesis of methionine (Met) and the production of related compounds from homocysteine (Hcys), cystathionine (Cysta), and homoserine (Hser) plus cysteine (Cys) by rumen bacteria (B) or protozoa (P) alone and by a mixture of these bacteria and protozoa (BP). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39 °C for 12 h. Hcys, Cysta, and Hser plus Cys were catabolized by all rumen microbial fractions to different extents. B, P, and BP converted Hcys to Met with 2-aminobutyric acid (2AB) and methionine sulfoxide . The Met-producing ability of B (83.2 µmol g–1 microbial nitrogen; MN) from Hcys was about 3.6 times higher than that of P in a 6-h incubation period. The ability of BP, during the same incubation period, was about 30.0% higher than that of B. Hcys, Met, and 2AB were formed when Cysta was incubated with B, P, or BP. Rumen microbial fermentation of Hser plus Cys led to the formation of Cysta, Met (through Hcys), and 2AB. Thus the results indicated that a trans-sulfurylation pathway for Met synthesis was operating in the rumen bacteria and protozoa. The results mentioned above have been demonstrated for the first time in B, P, and BP in the present study.
Attenuation of monochromatic and polychromatic lights in Chlorella vulgaris suspensions by Y.-S. Yun; J. Park (pp. 765-770).
A quantitative description of light attenuation in microalgal suspensions is a prerequisite for kinetic modeling of microalgal photosynthesis and/or growth activity depending upon the light distribution inside photobioreactors. In this study, the light attenuation coefficients in Chlorella vulgaris suspensions were theoretically calculated from light absorption spectra and spectral irradiances of various light sources. By using this method, errors occurring in the direct measurement of the attenuation coefficients can be avoided. The obtained light attenuation coefficients were used for evaluating light attenuation models such as the Beer-Lambert, Cornet, and hyperbolic models. Furthermore, advantages and disadvantages of these models are discussed with respect to prediction of performance, mechanistic background, and usefulness for further application to calculation of the light distribution inside photobioreactors.
Absence of aflatoxin biosynthesis in koji mold (Aspergillus sojae) by Kenichiro Matsushima; Kumiko Yashiro; Yoshiki Hanya; Keietsu Abe; Kimiko Yabe; Takashi Hamasaki (pp. 771-776).
Ten strains isolated from industrial soy sauce producing koji mold were identified as Aspergillus sojae and distinguished from Aspergillus parasiticus morphologically and physiologically. There was no detectable aflatoxin in any culture extracts of A. sojae strains. Strain 477 was chosen as a representative strain of industrial A. sojae for further molecular analysis. All enzymatic activities associated with the aflatoxin biosynthesis were not detected or negligible in strain 477 compared with that of the A. parasiticus strain. Southern analysis suggested that the genomic DNA of strain 477 contained aflatoxin biosynthetic pathway genes. In contrast, all industrial strains lacked detectable transcripts of aflR, the main regulatory gene for aflatoxin biosynthesis, under the aflatoxin-inducing condition. Our data suggest that defects in aflR expression cause the lack of expression of aflatoxin-related genes which results in the absence of aflatoxin biosynthesis in A. sojae strains.
Flocculation and coflocculation of bacteria by yeasts by X. Peng; J. Sun; D. Iserentant; C. Michiels; H. Verachtert (pp. 777-781).
Biotransformations in natural environments frequently involve interactions between microorganisms. Although there are many reports on the interactions between bacteria, interactions between yeasts and bacteria have not been extensively studied. Previously we reported on the flocculation and coflocculation of Pediococcus damnosus by Saccharomyces cerevisiae. Now we report that several other yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, and Schizosaccharomyces pombe, induce flocculation with several industrially or medically relevant bacteria, including Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Candida utilis was one of the best flocculation inducers. The results are discussed with respect to interactions between yeasts and bacteria and their applications in industry and medicine.
Exopolysaccharides of Xanthomonas pathovar strains that infect rice and wheat crops by R. Sunish Kumar; N. Sakthivel (pp. 782-786).
In order to understand the mode of action of the taxonomically related pathogens Xanthomonas campestris pv. translucens, Xanthomonas oryzae pv. oryzae, and Xanthomonas oryzae pv. oryzicola, which attack wheat and rice crops, we examined the compositional differences of their exopolysaccharides (EPSs). Maximum production of polysaccharide in shake cultures of these pathogens was observed between 24 and 72 h. X. campestris pv. translucens, the leaf streak pathogen of wheat, produced a higher amount of polysaccharide (46.97 µg/ml) at 72 h compared to X. oryzae pv. oryzae (42.02 µg/ml), the bacterial blight pathogen of rice, and X. oryzae pv. oryzicola (41.91 µg/ml), the bacterial leaf streak pathogen of rice. Infrared (FTIR) spectra suggested that the polysaccharides of all three Xanthomonas pathovar strains have an –OH group with intermolecular hydrogen bonding, a C–H group of methyl alkanes, an aldehyde (RCHO) group, a C=C or C=O group, and a C-O group. FTIR spectra also revealed the presence of an acid anhydride group in X. oryzae pv. oryzae, a secondary aromatic or aliphatic amine group in X. campestris pv. translucens, and a primary aromatic or aliphatic amine group in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Nuclear magnetic resonance (NMR) spectra revealed the presence of unsubstituted sugars, an acetyl amine of hexose or pentose, and a β-anomeric carbon of hexose or pentose in the polysaccharides of all bacteria. NMR spectra also identified the α-anomeric carbon of hexose or pentose in all strains, and a branching at the fourth carbon of the sugar only in X. campestris pv. translucens; the presence of an uronic acid molecule (acid anhydride group) in X. oryzae pv. oryzae; and a deoxy sugar, rhamnose, in X. oryzae pv. oryzicola.
Microbial sulfate reduction with acetate: process performance and composition of the bacterial communities in the reactor at different salinity levels by W. Muthumbi; N. Boon; R. Boterdaele; I. De Vreese; E. Top; W. Verstraete (pp. 787-793).
Microbial sulfate reduction with acetate as carbon source and electron donor was investigated at salinity levels between 0.53 and 1.48%. The experiment was carried out in a 2.3-l upflow anaerobic sludge blanket reactor inoculated with granular methanogenic sludge. A pH of 8.3, a temperature of 32±1 °C and a chemical oxygen demand (COD)/SO4 2–-S ratio of 2 were maintained in the reactor throughout the experiment. Sulfate reduction and the composition of the dominant bacterial communities in the reactor were monitored. The results showed that a maximal conversion rate for SO4 2–-S of 14 g l–1 day–1 and a conversion efficiency of more than 90% were obtained at a salinity level of 1.26–1.39%. A further increase in the salinity level led to reactor instability. Denaturant gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from total bacterial DNA extracted from the inoculum and reactor sludge showed that salinity level had an impact on the composition of the bacterial communities in the reactor. However, no clear relationship was found between reactor performance and the composition of the dominant bacterial communities in the reactor.
Isolation and characterization of psychrotrophic bacteria from oil-reservoir water and oil sands by T. Kato; M. Haruki; T. Imanaka; M. Morikawa; S. Kanaya (pp. 794-800).
Four psychrotrophic strains, which grew at 4 °C but not at 37 °C, were isolated from Japanese oil-reservoir water (strains SIB1, SIC1, SIS1) and Canadian oil sands (strain CAB1). Strains SIB1, SIS1, and CAB1 had a maximum growth rate at 20 °C and grew to the highest cell densities at the cultivation temperature of 0–4 °C. Strain SIS1 was capable of growing even at –5 °C. The growth profile of strain SIC1 was rather similar to that of a mesophilic bacterium. Strains SIB1, SIC1, and SIS1 were identified as members of the genus Shewanella, and strain CAB1 was a member of the genus Arthrobacter. All these strains exhibited weak degradation ability against catechol, a hydroxylated aromatic hydrocarbon, and tributyrin. These strains are expected to be of potential use in the in situ bioremediation technology of hazardous hydrocarbons and esters under low-temperature conditions.
Design of PCR primers and a gene probe for extensive detection of poly(3-hydroxybutyrate) (PHB)-degrading bacteria possessing fibronectin type III linker type-PHB depolymerases by K. Sei; M. Nakao; K. Mori; M. Ike; T. Kohno; M. Fujita (pp. 801-806).
For rapid and sensitive detection of poly(3-hydroxybutyrate) (PHB)-degrading bacteria, a PCR primer set (PHB primers) and a gene probe (PHB probe) were designed, based on the homologous regions of six fibronectin type III linker domain-encoding sequences laid on a variety of PHB depolymerase genes listed in the GenBank. PCR using PHB primers amplified DNA fragments with the expected sizes from all the tested bacterial strains used for primer design; and all of the amplified fragments gave positive signals by Southern hybridization with the PHB probe. No amplified fragments were observed from negative controls. To evaluate the availability of the PHB primers and PHB probe, they were applied to 57 wild-type, PHB-degrading bacteria newly isolated from a variety of environments. The PHB primers amplified DNA fragments with expected sizes from 50 of the 57 wild-type strains, while the PHB probe showed positive signals against the amplified fragments from 47 strains. These results suggest that the primer and probe system established in this study can detect a considerable proportion of the potential PHB-degrading bacteria and can be applied to evaluate PHB-degradation potential in a natural environment, in combination with direct DNA extraction methods.