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Applied Microbiology and Biotechnology (v.62, #5-6)
Novel bioreduction system for the production of chiral alcohols by M. Kataoka; K. Kita; M. Wada; Y. Yasohara; J. Hasegawa; S. Shimizu (pp. 437-445).
Chiral alcohols are useful intermediates for many pharmaceuticals and chemicals. Enzymatic asymmetric reduction of prochiral carbonyl compounds is a promising method for producing chiral alcohols. There have been many attempts to construct bioreduction systems for the industrial production of chiral alcohols. This review focuses on the establishment of a novel bioreduction system using an Escherichia coli transformant co-expressing genes for carbonyl reductase and cofactor-regeneration enzyme. This bioreduction system could be useful as an all-purpose catalyst for asymmetric reduction reactions.
Diversifying microbial natural products for drug discovery by V. Knight; J.-J. Sanglier; D. DiTullio; S. Braccili; P. Bonner; J. Waters; D. Hughes; L. Zhang (pp. 446-458).
Historically, nature has provided the source for the majority of the drugs in use today. More than 20,000 microbial secondary metabolites have been described, but only a small percentage of these have been carried forward as natural product drugs. Natural products are in tough competition with large chemical libraries and with combinatorial chemistries. Hence, each step of a natural product program has to be more efficient than ever, starting from the collection of environmental samples and the selection of strains, to metabolic expression, genetic exploitation, sample preparation and chemical dereplication. This review will focus on approaches for diversifying microbial natural product strains and extract libraries, while decreasing genetic and chemical redundancy.
Methionine biosynthesis and its regulation in Corynebacterium glutamicum: parallel pathways of transsulfuration and direct sulfhydrylation by H.-S. Lee; B.-J. Hwang (pp. 459-467).
There are two alternative pathways leading to methionine synthesis in microorganisms: The transsulfuration pathway involves cystathionine as the intermediate and utilizes cysteine as the sulfur source, but the direct sulfhydrylation pathway bypasses cystathionine and uses inorganic sulfur instead. While most microorganisms synthesize methionine via either one of these pathways, Corynebacterium glutamicum utilizes both pathways, which appear to be fully functional. In C. glutamicum, each pathway is catalyzed by independent enzymes and is tightly regulated by methionine. Although the physiological significance of parallel pathways remains to be elucidated, their presence suggests metabolic flexibility and efficient adaptation of the organism to its environment.
Biotechnological production and applications of pullulan by T. D. Leathers (pp. 468-473).
Pullulan is a unique biopolymer with many useful traits and hundreds of patented applications. However, despite the fact that pullulan has been in commercial production for more than 25 years, few of these potential uses have been widely adopted. In large part this may be due to the relatively high price of pullulan. Nevertheless, the last few years have seen a resurgence in interest in pullulan, particularly for higher-value health and pharmaceutical applications.
Biorefinery by H. Ohara (pp. 474-477).
The biorefinery produces fuels, solvents, plastics and food for human beings. In some countries, these biorefinery products are made from waste biomass. The main processes in the biorefinery involve ethanol fermentation and lactic acid fermentation. For the biorefinery, many hybrid technologies were developed from different fields, such as bioengineering, polymer chemistry, food science and agriculture.
Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles by H. Furukawa; R. Shimojyo; N. Ohnishi; H. Fukuda; A. Kondo (pp. 478-483).
Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30°C, above which the flocculated particles—which absorbed a large amount of avidin due to their large surface area—were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.
Production of tobacco aroma from lutein. Specific role of the microorganisms involved in the process by G. Maldonado-Robledo; E. Rodriguez-Bustamante; A. Sanchez-Contreras; R. Rodriguez-Sanoja; S. Sanchez (pp. 484-488).
A mixed culture formed by Bacillus sp. and Geotrichum sp. produced tobacco aroma compounds from the carotenoid lutein through the formation of the intermediate β-ionone. Both microorganisms can grow independently in a medium supplemented with lutein, but only Geotrichum produces β-ionone. This intermediate was incorporated by the bacilli, converted to aroma and this product excreted to the culture medium. Bacillus sp. did not utilize β-ionone for growth but modified it. We conclude that, in the bioconversion of lutein to products with tobacco aroma, Geotrichum sp. is involved in carotenoid oxidation to produce β-ionone and Bacillus sp. is responsible for the norisoprenoid reduction to produce 7,8-dihydro-β-ionone and 7,8-dihydro-β-ionol.
Co-fermentation of glucose and citrate by Lactococcus lactis diacetylactis: quantification of the relative metabolic rates by isotopic analysis at natural abundance by S. Goupry; E. Gentil; S. Akoka; R. J. Robins (pp. 489-497).
The simultaneous catabolism of citrate and glucose by growing Lactococcus lactis subsp. lactis biovar. diacetylactis to obtain energy was followed quantitatively, using a non-enrichment isotopic technique. Both citrate and glucose are precursors of pyruvate, which may either be reduced to lactate, the principle product that accumulates, or be converted to diacetyl and acetoin. Under suitable conditions, both routes regenerate NAD+. Until recently, however, the quantitative relationships between the two substrates and these three products were poorly defined. It was recently shown, by exploiting differences in natural abundance 13C/12C ratios in the two substrates, that there is no metabolic separation of the catabolism of these two carbon sources. In this study, it is shown that the relative consumption rates change throughout the growth phase, citrate being preferentially metabolised at the onset of a culture of energy-depleted cells, with a subsequent evolution towards a metabolism dominated by glucose consumption. Additionally, it is shown that the relative consumption rates are influenced by environmental factors, notably initial pH and temperature.
Control of Lactobacillus contaminants in continuous fuel ethanol fermentations by constant or pulsed addition of penicillin G by D. P. Bayrock; K. C. Thomas; W. M. Ingledew (pp. 498-502).
The addition of penicillin G to combat microbial contamination in continuous fuel alcohol fermentations was performed using both continuous and pulsed addition regimes. In continuous fermentations where both Saccharomyces cerevisiae and Lactobacillus paracasei were present, the mode of addition of penicillin G determined final numbers of viable L. paracasei. When the same overall average concentration of penicillin G was added in both pulsed and continuous modes, the initial viable number of L. paracasei (8.0×109 cfu ml−1) decreased to a greater degree (1.02×105 cfu ml−1 L. paracasei) when penicillin G was pulsed at 6 h frequencies at an overall average concentration of 2,475 U/l than when penicillin G was added continuously at 2,475 U/l (2.77×105 cfu ml−1 L. paracasei). Pulsed additions over longer frequencies at 2,475 U/l were not as effective in reducing viable bacteria. Viable yeasts increased during both treatment conditions by more than 2-fold. The two addition regimes also eliminated the 40% decrease in ethanol concentration caused by the intentional bacterial infection. Although there was 3 times more bacterial death with 6 h pulsed additions compared to continuous additions of penicillin G at 2,475 U/l, there was, by that point, no practical difference in either final ethanol concentration or relative ethanol recovery.
Composites of bacterial cellulose and paper made with a rotating disk bioreactor by R. Mormino; H. Bungay (pp. 503-506).
Suspended solids in the nutrient medium for Acetobacter xylinium in a rotating disk bioreactor become incorporated into the gelatinous mat of bacterial cellulose as it forms. Embedding fibers of ordinary cellulose creates composites with enhanced strength and the toughness of bacterial cellulose. Purified cellulose and elongated fibers from paper are incorporated differently than are spherical particles such as silica gel. About 90% of the final cellulose can come from scrap paper, and dried composite sheets were much stronger than plain bacterial cellulose per unit area.
Isolation and characterization of a new strain of Achromobacter sp. with β-lactam antibiotic acylase activity by K. Plháčková; S. Bečka; F. Škrob; P. Kyslík (pp. 507-516).
A bacterial strain producing a β-lactam antibiotic acylase, able to hydrolyze ampicillin to 6-aminopenicillanic acid more efficiently than penicillin G, was isolated from soil and characterized. The isolate was identified as Achromobacter sp. using the phenotypic characteristics, composition of cellular fatty acids and 16S rRNA gene sequence. The enzyme synthesis was fully induced by phenylacetic acid (PAA) at a concentration of 2 g l−1. PAA at concentrations up to 12 g l−1 had no negative effect on the specific activity of acylase and biomass production, but slowed down the specific growth rate. Benzoic or 4-hydroxyphenylacetic acids can also induce synthesis of the enzyme. The inducers were metabolized in all cases. Acylase activity in cell-free extracts was determined with various substrates; ampicillin, cephalexin and amoxicillin were hydrolyzed 1.5- and 2-times faster than penicillin G. A high stability of acylase activity was observed over a wide range of pH (5.0–8.5) and at temperatures above 55°C.
Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells by T. Matsui; H. Nakayama; K. Yoshida; A. Shinmyo (pp. 517-522).
Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.
Sequential cloned gene integration in the yeast Kluyveromyces lactis by Y.-C. M. Wang; L. L. Chuang; F. W. F. Lee; N. A. Da Silva (pp. 523-527).
Two integrating vectors developed for use in Saccharomyces cerevisiae were successfully employed for cloned gene integration in the yeast Kluyveromyces lactis. A δ-integrating vector carrying the dominant selection marker neo allowed tandem integrations of a CUP1 p -lacZ cassette into one or two chromosomal sites. A δ/UB-integrating vector, which contains a reusable selection cassette, enabled multiple rounds of integration and the sequential insertion of stable, dispersed copies of CUP1 p -lacZ. Subsequent gene expression was closely correlated with integrated copy number illustrating the promise of this method for metabolic engineering in K. lactis. While both vectors contain an S. cerevisiae δ target sequence, the presence of δ-like elements in K. lactis has not been confirmed. Given the degree of illegitimate recombination in this yeast species, the insertions likely occurred at random locations in the chromosomes.
The constitutive AHSB4 promoter—a novel component of the Arxula adeninivorans-based expression platform by T. Wartmann; C. Bellebna; E. Böer; O. Bartelsen; G. Gellissen; G. Kunze (pp. 528-535).
An Arxula adeninivorans-AHSB4 gene, encoding histone H4, was isolated and characterized. The gene includes a coding sequence of 363 bp disrupted by a 51-bp intron, similar to the situation in other fungal H4 genes. The identity of the gene was confirmed by the high degree of homology of the derived amino acid sequence with that of other H4 histones. The gene is strongly and constitutively expressed, maintaining this expression profile under salt-stress conditions. The AHSB4 promoter was tested for suitability in heterologous gene expression using genes encoding the intracellular green fluorescent protein and the secreted human serum albumin (HSA) for assessment. Plasmids incorporating respective expression cassettes were used to transform the host strain A. adeninivorans LS3, which forms budding cells at 30 °C, and strain 135, which forms mycelia under these conditions. Transformants of both types were found to harbor a single copy of the heterologous DNA. Strong constitutive expression was observed during culture in salt-containing and salt-free media, as expected from the expression profile of AHSB4. In 200-ml shake-flask cultures, maximal HSA levels of 20 mg l−1 culture medium were achieved. This productivity could be increased to 50 mg l−1 in strains harboring two copies of the expression cassette. The AHSB4 promoter thus provides an attractive component for constitutive heterologous gene expression under salt-free and salt-stress conditions.
Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans by D. K. Y. Solaiman; R. D. Ashby; T. A. Foglia (pp. 536-543).
The phaZ gene of Pseudomonas resinovorans codes for a poly(hydroxyalkanoates) (PHA) depolymerase. Two phaZ mutants of Pseudomonas resinovorans NRRL B-2649, FOAC001 and FOAC002, were constructed by an in vitro transposition procedure followed by chromosomal integration via homologous recombination. A detailed mapping of the transposon insertion sites and an analysis of the resultant sequences showed that putative fusion polypeptides PhaZFOAC001 (239 amino-acid residues) and PhaZFOAC002 (85 amino-acid residues) could result from the mutant phaZ genes of FOAC001 and FOAC002, respectively. In vivo PHA degradation data indicated that PhaZFOAC001 might still retain a partial PHA depolymerization activity, while PhaZFOAC002 is completely devoid of this function. The cell yields and PHA contents of B-2649, FOAC001, and FOAC002 were similar when the cells were grown either under a limiting nitrogen-source (low-N) condition for up to 5 days or in excess N-source (high-N) for 3 days. A dramatic decrease in PHA content was observed in the PhaZ-active B-2649 and FOAC001 cells during prolonged cell growth (5 days) in high-N medium or in response to a shift-up in nitrogen-source. The repeat-unit compositions of the PHAs from FOAC001 and FOAC002 contained slightly lower amounts of β-hydroxyoctanoate and higher β-hydroxytetradecenoate than that of the wild-type B-2649 when grown under a high-N condition. While the molecular masses of the PHAs from FOAC001 and FOAC002 did not vary under any conditions used in this study, those of the wild-type B-2649 were markedly increased in cells either grown for 5 days under a high-N condition or subjected to a nitrogen-source shift-up. These phaZ mutants thus provide a valuable system to study the influence of PHA depolymerase on the accumulation and properties of medium-chain-length PHA.
Diversification of exogenous genes in vivo in Neurospora by D. E. A. Catcheside; J. P. Rasmussen; P. J. Yeadon; F. J. Bowring; E. B. Cambareri; E. Kato; J. Gabe; W. D. Stuart (pp. 544-549).
We have adapted the meiotic recombination hotspot cog of Neurospora crassa for shuffling exogenous DNA, providing a means of generating novel genes in situ from sequences introduced into chromosomes. Genes to be diversified are inserted between the his-3 locus and cog. Diversification crosses are heterozygous both for alleles of the exogenous DNA and for auxotrophic alleles of his-3. Progeny selected for ability to grow without histidine supplementation are enriched for exchange events within the exogenous DNA. Exchange events initiated by cog can propagate past DNA sequences mismatched for more than 370 bp and complete exchanges in patches of matched sequence as short as 24 bp, parameters that make the system suited for use in the directed evolution of genes for protein engineering. Here we demonstrate the system by shuffling human immunoglobulin kappa chain genes and also endoglucanase genes derived from different species of fungi.
Enrichment and properties of an anaerobic mixed culture that reductively deiodinates 5-amino-2,4,6-triiodoisophthalic acid, an X-ray contrast agent precursor by D. Lecouturier; A. Rochex; J.-M. Lebeault (pp. 550-556).
5-Amino-2,4,6-triiodoisophthalic acid (ATIA), both a precursor and a degradative intermediate of triiodinated contrast media, was anaerobically converted by sludge from a wastewater treatment plant. ATIA conversion took place only when an electron donor such as ethanol was added. A stable mixed culture was established by transfer to a defined synthetic mineral medium with ATIA and ethanol. It could be maintained for 1 year when the sulfate concentration was kept below 30 µM. Transient appearance of 5-amino-2,4-diiodoisophthalic acid, iodide release (2.7 mol iodide/mol ATIA) and accumulation of 5-aminoisophthalic acid indicated that ATIA was reductively dehalogenated. The enriched mixed culture also dehalogenated ATIA derivatives but deiodination remained incomplete. ATIA was the sole terminal electron acceptor used by the mixed culture during deiodination. The ratio of electrons transferred to ATIA, 0.83, was consistent with a respiratory metabolism. Formate, acetate, lactate, butyrate and hydrogen were also used as electron donors. Deiodination was inhibited by a headspace of air or by addition of nitrate, sulfite or thiosulfate. The reaction was 2.6 times slower with sulfate than without.
Utilisation of structurally diverse organophosphonates by Streptomycetes by A. Obojska; B. Lejczak (pp. 557-563).
A group of streptomycete strains was found able to utilise a wide range of structurally diverse phosphonates as a sole phosphorus source. No relation could be observed between ability to synthesise compounds containing a direct carbon-to-phosphorus (C–P) bond and biodegradative potential towards phosphonates in the strains studied. Streptomyces morookaensis DSM 40565 could degrade 2-amino-4-phosphonobutyrate as a sole nitrogen and phosphorus source in a stereoselective-like manner. This result suggests the existence of a new metabolic pathway for C–P bond breakage.
Characterization of the metabolic shift of Saccharomyces bayanus var. uvarum by continuous aerobic culture by A. Serra; P. Strehaiano; P. Taillandier (pp. 564-568).
Saccharomyces bayanus, being of interest for wine-making, is not as well known as S. cerevisiae and, due to many changes in the yeast classification, accurate data concerning its metabolic activity are difficult to find. In order to produce this yeast as an active dry yeast to be used as a starter in wine-making, its sensitivity to glucose was determined as the objective of our work. Using the pulse technique in continuous culture, it was found that growth in a synthetic medium was not limited by vitamins or mineral salts. We determined the critical dilution rate of a continuous culture and performed an aerobic continuous culture, measuring the respiratory quotient on-line in order to observe the metabolic shift from respiratory to fermentative metabolism. The S. bayanus var. uvarum strain studied was Crabtree-positive (glucose-sensitive) but had a weaker respiratory capacity than S. cerevisiae since the dilution rate of the metabolic shift was only 0.15 h−1. These new data provide essential information for the biomass production of this yeast strain for wine-making.
In vitro reduction of hexavalent chromium by a cell-free extract of Bacillus sp. ES 29 stimulated by Cu2+ by F. A. O. Camargo; B. C. Okeke; F. M. Bento; W. T. Frankenberger (pp. 569-573).
Chromium-resistant bacteria (CRB) isolated from soils can be used to reduce toxic Cr(VI) from contaminated environments. This study assessed in vitro reduction of hexavalent Cr using a cell-free extract (CFE) of CRB isolated from soil contaminated with dichromate. One isolate, ES 29, that substantially reduced Cr(VI) was identified as a Bacillus species by 16S rRNA gene-sequence homology. The isolate reduced Cr(VI) under aerobic conditions, using NADH as an electron donor and produced a soluble Cr(VI)-reducing enzyme stimulated by copper (Cu2+). The CFE of the bacterial isolate reduced 50% of Cr(VI) in 6 h. The Cr(VI)-reduction activity of the CFE had a K m of 7.09 μM and a V max of 0.171 μmol min−1 mg−1 protein. Mercury inhibited the enzyme, but not competitively, with a V max of 0.143 μmol min−1 mg−1 protein, a K m of 7.07 μM and a K i of 1.58 μM. This study characterizes the enzymatic reduction of Cr(VI) by Bacillus sp. ES 29 which can be used for the bioremediation of chromate.
Metabolic activities in Azospirillum lipoferum grown in the presence of NH4 + by V. Tsagou; I. Kefalogianni; K. Sini; G. Aggelis (pp. 574-578).
The utilization of some agro-industrial wastes as soil conditioners to provide free-living nitrogen-fixing bacterial populations (e.g. Azospirillum spp.) with carbon and energy sources, may be an interesting perspective for agriculture. However, the presence of ammonium nitrogen in cultivated soils and/or various wastes could inhibit the growth of the nitrogen-fixing populations. The present investigation shows that growth of Azospirillum lipoferum was restricted at a dissolved oxygen (DO) concentration equal to 135 μM, when the initial NH4Cl concentration increased from 0.5 to 0.9 g/l. The activities of both citrate synthase (CS) and isocitrate dehydrogenase were significantly decreased in the presence of 0.9 g/l NH4Cl (e.g., 40% and 66%, respectively, in cells incubated for 95 h), while ammonium assimilation occurred via the glutamate dehydrogenase reaction. Furthermore, growth limitation occurred even in the presence of 0.5 g/l NH4Cl, when the DO concentration decreased from 135 to 30 μM. The activities of both CS and succinate dehydrogenase were dramatically decreased in cells grown at the lower DO concentration (e.g., 90% and 93% respectively, in a 95 h incubation), while ammonium assimilation was limited due to the low activities of both glutamate dehydrogenase and glutamate synthase. It is concluded that the threshold of ammonium concentration at which growth of A. lipoferum is limited, depends on the DO concentration in the medium.
Metabolism of benzyl alcohol via catechol ortho-pathway in methylnaphthalene-degrading Pseudomonas putida CSV86 by A. Basu; S. S. Dixit; P. S. Phale (pp. 579-585).
Pseudomonas putida CSV86 metabolizes 1- and 2-methylnaphthalene through distinct catabolic and detoxification pathways. In spite of the similarity in the steps involved in the methylnaphthalene detoxification and the toluene side-chain hydroxylation pathways, the strain failed to utilize toluene or xylenes. However, it could grow on benzyl alcohol, 2- and 4-hydroxybenzyl alcohol. Metabolic studies suggest that the benzyl alcohol metabolism proceeds via the benzaldehyde, benzoate, and catechol ortho-cleavage pathway, in contrast to the well established catechol meta-cleavage pathway. Carbon source-dependent enzyme activity studies suggest that the degradation of aromatic alcohol involves two regulons. Aromatic alcohol induces the upper regulon, which codes for aromatic alcohol- and aromatic aldehyde-dehydrogenase and converts alcohol into acid. The aromatic acid so generated induces the specific lower regulon and is metabolized via either the ortho- or the meta-cleavage pathway. CSV86 cells transform 1- and 2-methylnaphthalene to 1- and 2-hydroxymethyl naphthalene, which are further converted to the respective naphthoic acids due to the basal level expression and broad substrate specificity of the upper regulon enzymes.
Effect of dissolved oxygen concentration on sludge settleability by A. M. P. Martins; J. J. Heijnen; M. C. M. van Loosdrecht (pp. 586-593).
This laboratory study presents a detailed evaluation of the effects of dissolved oxygen concentration and accumulation of storage polymers on sludge settleability in activated sludge systems with an aerobic selector. The oxygen and substrate availability regime were simulated in laboratory sequencing batch reactor systems. The experiments showed that low dissolved oxygen concentration (≤1.1 mg O2 l−1) had a strong negative effect on sludge settleability, leading to the proliferation of filamentous bacteria (Thiothrix spp., Type 021N and Type 1851). This negative effect was stronger at high chemical oxygen demand loading rate. This indicates that a compartmentalised (plug flow) aerobic contact tank, designed at short hydraulic residence time to guarantee a strong substrate gradient, with low dissolved oxygen concentration, might be worse for sludge settleability than an "overdesigned" completely mixed contact tank. Contrary to the general hypothesis, the maximum specific acetate uptake rate, poly-β-hydroxybutyrate production rate, and resistance to short starvation periods are similar in both poor- and well-settling sludge. The results of this study support our previous hypothesis on the importance of substrate gradients for the development of filamentous structures in biological flocs, from soluble organic substrate gradients to dissolved oxygen gradients in sludge flocs.
Bacterial diversity in an industrial wastewater bioreactor by M. Bramucci; H. Kane; M. Chen; V. Nagarajan (pp. 594-600).
Industrial wastewater bioreactors are potentially important sources of novel biocatalysts. However, the microbial populations in these bioreactors are not well characterized. The microbial community in an industrial wastewater bioreactor was surveyed by extracting DNA from a sample of activated sludge, followed by PCR amplification and sequencing of cloned 16S rRNA genes. A total of 407 cloned 16S rRNA gene sequences were compared with 88 bacterial isolates cultured from the same sample of sludge using a variety of standard media. Most of the bacteria detected by the PCR-based approach were β-subdivision Proteobacteria, whereas most of the cultured bacteria were γ-subdivision Proteobacteria. Only a few types of bacteria were detected by both approaches. These observations indicate that multiple techniques are necessary to characterize the microbial diversity in any complex ecosystem.
Monitoring of white-rot fungus during bioremediation of polychlorinated dioxin-contaminated fly ash by H. Suhara; C. Daikoku; H. Takata; S. Suzuki; Y. Matsufuji; K. Sakai; R. Kondo (pp. 601-607).
Bioremediation is a low-cost treatment alternative for the cleanup of polychlorinated-dioxin-contaminated soils and fly ash when pollution spread is wide-ranging. An interesting fungus, Ceriporia sp. MZ-340, with a high ability to degrade dioxin, was isolated from white rotten wood of a broadleaf tree from Kyushu Island in Japan. We have attempted to use the fungus for bioremediation of polychlorinated-dioxin-contaminated soil on site. However, we have to consider that this trial has the potential problem of introducing a biohazard to a natural ecosystem if this organism is naturalized. We have therefore developed a monitoring system for the introduced fungus as a part of the examination and evaluation of bioremediation in our laboratory. We have also developed a PCR-based assay to reliably detect the fungus at the bioremediation site. DNA isolated from the site was amplified by PCR using a specific primer derived from internal transcribed spacer region (ITS: ITS1, 5.8S rDNA and ITS2) sequences of Ceriporia sp. MZ-340. We successfully monitored Ceriporia sp. MZ-340 down to 100 fg/µl DNA and down to 2 mg/g mycelium. We also successfully monitored the fungus specifically at the bioremediation site. The polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran content was observed to decrease in response to treatment with the fungus. The species-specific PCR technique developed in the present work is useful in evaluating the possibility of on-site bioremediation using the fungus Ceriporia sp. MZ-340.