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Applied Microbiology and Biotechnology (v.60, #6)

Microbial silica deposition in geothermal hot waters by F. Inagaki; Y. Motomura; S. Ogata (pp. 605-611).

A combined use of molecular ecological techniques and geochemical surveys revealed that thermophilic or hyperthermophilic microorganisms living in geothermal environments are likely to be implicated in the formation of biogenic siliceous deposits. Electron microscopic observations indicated that numerous microorganism-like fabrics were preserved in naturally occurring siliceous deposits such as siliceous sinter, geyserite, and silica scale, which suggests microbial contribution to silica precipitation. Molecular phylogenetic analyses suggested that extreme thermophilic bacteria within the genera Thermus and Hydrogenobacter are predominant components among the indigenous microbial community in siliceous deposits formed in pipes and equipment of Japanese geothermal power plants. These bacteria seem to actively contribute to the rapid formation of huge siliceous deposits. Additionally, in vitro examination suggested that Thermus cells induced the precipitation of supersaturated amorphous silica during the exponential growth phase, concomitant with the production of a specific cell envelope protein. Dissolved silica in geothermal hot water may be a significant component in the maintenance of position and survival of microorganisms in limited niches.

Diatom cultivation and biotechnologically relevant products. Part I: Cultivation at various scales by Thierry Lebeau; Jean-Michel Robert (pp. 612-623).

Biotechnological applications of diatoms are still in development. Further development at the industrial scale will depend on optimisation of the culture process with the aim of reducing costs. Because of the photoautotrophic status of the majority of diatoms, microalgal cultures suffer from the limitation of light diffusion, which requires the development of suitable photobioreactors. Thus, genetically engineered microalgae that may be cultivated in heterotrophic conditions present a new opportunity. Other limiting factors, such as nutrients (phosphate, nitrogen, silicon), pH, temperature, bioturbation and many more must be taken into account. Most of the time, metabolic stress conditions lead to an overproduction of the products of interest, with a decrease in biomass production as a consequence. Outdoor cultures in open ponds are usually devoted to aquaculture for the feeding of shrimps and bivalve molluscs (commercial production), while closed axenic indoor/outdoor photobioreactors are used for biotechnological compounds of homogeneous composition (still at the laboratory scale). In addition to the optimum culture conditions that have to be taken into account for photobioreactor design, the localisation of produced metabolites (intra- or extracellular) may also be taken into account when choosing the design. Microalgal cell immobilisation may be a suitable technique for application to benthic diatoms, which are usually sensitive to bioturbation and/or metabolites which may be overexpressed.

Diatom cultivation and biotechnologically relevant products. Part II: Current and putative products by T. Lebeau; J.-M. Robert (pp. 624-632).

While diatoms are widely present in terms of diversity and abundance in nature, few species are currently used for biotechnologically applications. Most studies have focussed on intracellularly synthesised eicosapentaenoic acid (EPA), a polyunsaturated fatty acid (PUFA) used for pharmaceutical applications. Applications for other intracellular molecules, such as total lipids for biodiesel, amino acids for cosmetic, antibiotics and antiproliferative agents, are at the early stage of development. In addition, the active principle component must be identified amongst the many compounds of biotechnological interest. Biomass from diatom culture may be applied to: (1) aquaculture diets, due to the lipid- and amino-acid-rich cell contents of these microorganisms, and (2) the treatment of water contaminated by phosphorus and nitrogen in aquaculture effluent, or heavy metal (bioremediation). The most original application of microalgal biomass, and specifically diatoms, is the use of silicon derived from frustules in nanotechnology. The competitiveness of biotechnologically relevant products from diatoms will depend on their cost of production. Apart from EPA, which is less expensive when obtained from Phaeodactylum tricornutum than from cod liver, comparative economic studies of other diatom-derived products as well as optimisation of culture conditions are needed. Extraction of intracellular metabolites should be also optimised to reduce production costs, as has already been shown for EPA. Using cell immobilisation techniques, benthic diatoms can be cultivated more efficiently allowing new, biotechnologically relevant products to be investigated.

Candicidin biosynthesis in Streptomyces griseus by J. Gil; A. Campelo-Diez (pp. 633-642).

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase (pabAB) from S. griseus IMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.

New developments in oxidative fermentation by O. Adachi; D. Moonmangmee; H. Toyama; M. Yamada; E. Shinagawa; K. Matsushita (pp. 643-653).

Oxidative fermentations have been well established for a long time, especially in vinegar and in L-sorbose production. Recently, information on the enzyme systems involved in these oxidative fermentations has accumulated and new developments are possible based on these findings. We have recently isolated several thermotolerant acetic acid bacteria, which also seem to be useful for new developments in oxidative fermentation. Two different types of membrane-bound enzymes, quinoproteins and flavoproteins, are involved in oxidative fermentation, and sometimes work with the same substrate but produce different oxidation products. Recently, there have been new developments in two different oxidative fermentations, D-gluconate and D-sorbitol oxidations. Flavoproteins, D-gluconate dehydrogenase, and D-sorbitol dehydrogenase were isolated almost 2 decades ago, while the enzyme involved in the same oxidation reaction for D-gluconate and D-sorbitol has been recently isolated and shown to be a quinoprotein. Thus, these flavoproteins and a quinoprotein have been re-assessed for the oxidation reaction. Flavoprotein D-gluconate dehydrogenase and D-sorbitol dehydrogenase were shown to produce 2-keto-D-gluconate and D-fructose, respectively, whereas the quinoprotein was shown to produce 5-keto-D-gluconate and L-sorbose from D-gluconate and D-sorbitol, respectively. In addition to the quinoproteins described above, a new quinoprotein for quinate oxidation has been recently isolated from Gluconobacter strains. The quinate dehydrogenase is also a membrane-bound quinoprotein that produces 3-dehydroquinate. This enzyme can be useful for the production of shikimate, which is a convenient salvage synthesis system for many antibiotics, herbicides, and aromatic amino acids synthesis. In order to reduce energy costs of oxidative fermentation in industry, several thermotolerant acetic acid bacteria that can grow up to 40°C have been isolated. Of such isolated strains, some thermotolerant Acetobacter species were found to be useful for vinegar fermentation at a high temperature such 38–40°C, where mesophilic strains showed no growth. They oxidized higher concentrations of ethanol up to 9% without any appreciable lag time, while alcohol oxidation with mesophilic strains was delayed or became almost impossible under such conditions. Several useful Gluconobacter species of thermotolerant acetic acid bacteria are also found, especially L-erythrulose-producing strains and cyclic alcohol-oxidizing strains. Gluconobacter frateurii CHM 43 is able to rapidly oxidize meso-erythritol at 37°C leading to the accumulation of L-erythrulose, which may replace dihydroxyacetone in cosmetics. G. frateurii CHM 9 is able to oxidize cyclic alcohols to their corresponding cyclic ketones or aliphatic ketones, which are known to be useful for preparing many different physiologically active compounds such as oxidized steroids or oxidized bicyclic ketones. The enzymes involved in these meso-erythritol and cyclic alcohol oxidations have been purified and shown to be a similar type of membrane-bound quinoproteins, consisting of a high molecular weight single peptide. This is completely different from another quinoprotein, alcohol dehydrogenase of acetic acid bacteria, which consists of three subunits including hemoproteins.

Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli by A. Shokri; A. Sandén; G. Larsson (pp. 654-664).

This paper is a review of strategies to introduce protein into the liquid medium of Escherichia coli K-12 industrial production cells. The cell design strategies are generally based on one of two general mechanisms. The first strategy involves a two-stage translocation using active transporters in the cytoplasmic membrane followed by passive transport through the outer membrane. Passive transport is achieved through either external or internal destabilization of the E. coli structural components. The latter can be achieved by transplantation of destabilizing components (lysis proteins) that work by permeabilization of the outer membrane from the interior of the cell, or by using cells carrying mutations of structural components. Passive transport can also be achieved by a chemical, mechanical, or enzymatic permeabilization directed from outside the cell. The second strategy is realized through transplantation of proteins capable of active transport over one or both of the membranes. This involves the transplantation of secretion mechanisms into the K-12 cell from pathogenic E. coli as well as from other species. The process design strategies are dependent on environmental conditions and must take into account changes in physical parameters, medium design, and influx of limiting carbon source in fed-batch cultivation.

Molecular biology and application of plant peroxidase genes by K. Yoshida; P. Kaothien; T. Matsui; A. Kawaoka; A. Shinmyo (pp. 665-670).

Peroxidases are a family of isozymes found in all plants; they are heme-containing monomeric glycoproteins that utilize either H₂O₂ or O₂ to oxidize a wide variety of molecules. These important enzymes are used in enzyme immunoassays, diagnostic assays and industrial enzymatic reactions. Peroxidase genes and their promoters can be used for molecular breeding of useful plants. Transgenic techniques have also been used to investigate the physiological and molecular functions of peroxidase genes in plants. Here, we review transgenic studies of peroxidase genes, including the functional analyses of the enzymes and their promoters. Regarding application of peroxidase genes, it has been reported that overexpression of the tomato TPX2 gene or the sweet potato swpa1 gene conferred increased salt-tolerance or oxidative-stress tolerance, respectively. The growth stimulation effect in transgenic tobacco and hybrid aspen upon overexpression of horseradish peroxidase gene is also discussed.

A novel circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrica) sponge for the bioconversion of raw cassava starch to ethanol by N. Roble; J. Ogbonna; H. Tanaka (pp. 671-678).

A circulating loop bioreactor (CLB) with cells immobilized in loofa sponge was constructed for simultaneous aerobic and anaerobic processes. The CLB consists of an aerated riser and a non-aerated downcomer column connected at the top and bottom by cylindrical pipes. Ethanol production from raw cassava starch was investigated in the CLB. Aspergillus awamori IAM 2389 and Saccharomyces cerevisiae IR2 immobilized on loofa sponge were placed, respectively, in the aerated riser column and non-aerated downcomer column. Both α-amylase and glucoamylase activities increased as the aeration rate was increased. Ethanol yield and productivity increased with an increase in the aeration rate up to 0.5 vvm, but decreased at higher aeration rates. The CLB was operated at an aeration rate of 0.5 vvm for more than 600 h, resulting in an average ethanol productivity and yield from raw cassava starch of 0.5 g-ethanol l^–1 h^–1 and 0.45 g ethanol/g starch, respectively. In order to increase ethanol productivity, it was necessary to increase the dissolved oxygen (DO) concentration in the riser column and decrease the DO concentration in the downcomer column. However, increasing the aeration rate resulted in increases in the DO concentration in both the riser and the downcomer columns. At high aeration rate, there was no significant difference in the DO concentration in the riser and downcomer columns. The aeration rate was therefore uncoupled from the liquid circulation by attaching a time-controlled valve in the upper connecting pipe. By optimizing the time and frequency of valve opening, and operation at high aeration rate, it was possible to maintain a very high DO concentration in the riser column and a low DO concentration in the downcomer column. Under these conditions, ethanol productivity increased by more than 100%, to 1.17 g l^–1 h^–1.

Effect of nutrient limitation on product formation during continuous fermentation of xylose with Thermoanaerobacter ethanolicus JW200 Fe(7) by H. Hild; D. Stuckey; D. Leak (pp. 679-686).

Thermoanaerobacter ethanolicus JW200 Fe(7) was grown in continuous culture, using xylose as the primary carbon source, with progressively lower concentrations of supplementary yeast extract. This enabled the comparison of metabolic flux to fermentation end-products under carbon-limited and carbon-sufficient (yeast extract-limited) conditions and the determination of process data under fully mass-balanced conditions. Under carbon-limitation, the specific ethanol-formation rate was described by q p=40.34 µ+3.74, the specific rate of substrate utilisation for maintenance was 0.31±0.02 g g^–1 h^–1 and the maximum cell yield on xylose, corrected for maintenance requirements, was 0.15±0.04 g g^–1. Based on the product profiles, these corresponded to a maintenance coefficient of m _ATP=4.1±0.5 mmol g^–1 h^–1 and a maximum cell yield of $$ Y_{x/{ m{ATP}}}^{{ m{max}}} $$ = 14.7±0.8 g mol^–1. Limitation by a component in yeast extract resulted in incomplete xylose utilisation, increased catabolic flux rates (primarily resulting in increased lactate production, due to limitations in the flux through the phosphoroclastic reaction), a reduction in cell yield $$ Y_{x{ m{/ATP}}}^{{ m{max}}} $$ = 10.0±1.0 g mol^–1 and an increase in maintenance energy requirements of m _ATP=7.95±0.7 mmol g^–1. The latter was also reflected in a shift from ethanol to acetate production at lower growth rates. An analysis of ethanol and acetate tolerance indicated that any high-intensity process employing this strain would require a bioreactor design which incorporated continuous ethanol stripping.

Size-effect on the physical characteristics of the aerobic granule in a SBR by S. Toh; J. Tay; B. Moy; V. Ivanov; S. Tay (pp. 687-695).

Owing to a fast growth rate, aerobic granules display a wide range of sizes, approximately 0.3–5.0 mm in diameter. As the diameter increases, the aerobic granule undergoes serial morphological and physical changes that could cause problems to the reactor operation, a phenomenon which, however, has not been fully studied hitherto. In this study, aerobic granules from a sequencing batch reactor were mechanically separated into various size-categories in order to investigate their physical properties, including sludge volumetric index (SVI), settling velocity (sv), specific surface hydrophobicity, granule strength, total solids, percentage volatile solids and other structural properties. Also, the live and dead biomass distribution was examined under a confocal laser scanning microscope after treatment with nucleic acid viability stains. Regardless of size, the biomass (both live and dead) was densest in the outer layer of the granule, which was about 600±50 µm thick. The live cells appeared only in the peripheral zone, while dead biomass spread into the inner zone. The biomass distribution pattern justified the changing physical properties of the granules as they grew bigger. As size increased, the sv, granule total density and biomass density increased but not in parallel with the size increment, while the granule strength, specific surface hydrophobicity and SVI decreased. Nonetheless, beyond a threshold size (4.0 mm diameter), the granules presented peculiar values in those properties, deviating from the initial trends. This was due to both inner and outer structural changes. The physical properties associated significantly with the size factor, for which the correlation coefficients were above 0.67. In view of biological viability and physical properties, the operational size-range suggested for optimal performance and economically effective aerobic SBR granular sludge is a diameter of 1.0–3.0 mm.

Inducive effect of cresoquinone on microbiological transformation of L-tyrosine to 3,4 dihydroxy phenyl L-alanine by Aspergillus oryzae NG-11^P1 by I. Haq; S. Ali; M. Qadeer; J. Iqbal (pp. 696-699).

The present work describes the inducive effect of cresoquinone on microbiological transformation of L-tyrosine to 3,4 dihydroxy phenyl L-alanine (L-DOPA) by Aspergillus oryzae NG-11^P1. Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinases, β-carboxylases and tyrosine hydroxylases are intracellular enzymes. The maximum conversion of L-tyrosine to L-DOPA (0.428 mg/ml) was achieved after 60 min of biochemical reaction. To enhance the production of L-DOPA, cresoquinone was added to the reaction mixture. Best L-DOPA biosynthesis results were observed when the concentration of cresoquinone was 3.5×10^–6 M (1.686 mg/ml L-DOPA produced with 1.525 mg/ml consumption of L-tyrosine). Cresoquinone not only increased enzyme activity but also enhanced cell membrane permeability to facilitate secretion of enzymes into the reaction broth. Comparison of kinetic parameters revealed the ability of the mutant to yield L-DOPA {Y _p/x [i.e., mg L-DOPA formed (mg cells formed)^–1] =7.360±0.04}. When the culture grown on various cresoquinone levels was monitored for Q _p, Q _s and q _p [Q _p: mg L-DOPA produced ml^–1 h^–1; Q _s: mg substrate consumed ml^–1 h^–1; q _p: mg L-DOPA formed (mg cells)^–1 h^–1], there was significant enhancement (P<0.025) of these variables.

Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2 by Y. Xiao; X. Tu; J. Wang; M. Zhang; Q. Cheng; W. Zeng; Y. Shi (pp. 700-707).

A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58,522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11–12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50°C, respectively. The half-life of the enzyme at 75°C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K _m values of the enzyme toward substrates 2,2′-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 µM, respectively, and the corresponding V _max values are 670, 66.8, and 79 µM min^–1 mg^–1, respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN₃ or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 µmol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 µmol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.

Laccase-catalysed synthesis of coupling products of phenolic substrates in different reactors by R. Pilz; E. Hammer; F. Schauer; U. Kragl (pp. 708-712).

Substrate oxidation of aromatic substances by the enzyme laccase followed by a heteromolecular coupling with a co-substrate is a promising possibility for the synthesis of new compounds. To find a suitable reactor for the effective production of new compounds, the laccase-catalysed coupling of 3-(3,4-dihydroxyphenyl)propionic acid with 4-aminobenzoic acid was investigated as a model system. Based on the kinetic parameters, a mathematical model was used to predict the reaction yield and oxygen demand in a discontinuously stirred tank reactor and a continuously operated stirred tank reactor. Membrane processes were used for bubble-free aeration of the system and to recover the soluble enzyme.

Surrogate biochemistry: use of Escherichia coli to identify plant cDNAs that impact metabolic engineering of carotenoid accumulation by C. Gallagher; M. Cervantes-Cervantes; E. Wurtzel (pp. 713-719).

Carotenoids synthesized in plants but not animals are essential for human nutrition. Therefore, ongoing efforts to metabolically engineer plants for improved carotenoid content benefit from the identification of genes that affect carotenoid accumulation, possibly highlighting potential challenges when pyramiding traits represented by multiple biosynthetic pathways. We employed a heterologous bacterial system to screen for maize cDNAs encoding products that alter carotenoid accumulation either positively or negatively. Genes encoding carotenoid biosynthetic enzymes from the bacterium Erwinia uredovora were introduced into Escherichia coli cells that were subsequently transfected with a maize endosperm cDNA expression library; and these doubly transformed cells were then screened for altered carotenoid accumulation. DNA sequencing and characterization of one cDNA class conferring increased carotenoid content led to the identification of maize cDNAs encoding isopentenyl diphosphate isomerase. A cDNA that caused a reduced carotenoid content in E. coli was also identified. Based on DNA sequence analysis, DNA hybridization, and further functional testing, this latter cDNA was found to encode the small subunit of ADP-glucose pyrophosphorylase, a rate-controlling enzyme in starch biosynthesis that has been of interest for enhancing plant starch content.

Indigo production by Escherichia coli carrying the phenol hydroxylase gene from Acinetobacter sp. strain ST-550 in a water–organic solvent two-phase system by N. Doukyu; K. Toyoda; R. Aono (pp. 720-725).

Acinetobacter sp. strain ST-550 produces indigo from indole in the presence of a large volume of diphenylmethane and a high level of indole. Particular proteins increased remarkably in strain ST-550 grown in the two-phase culture system for indigo production. One of the proteins showed a N-terminal amino acid sequence that was identical to that of the largest subunit of phenol hydroxylase (MopN) from A. calcoaceticus NCBI8250. The indigo-producing activity was strongly induced when ST-550 was grown with phenol as a sole carbon source. Genes coding for the multicomponent phenol hydroxylase were cloned, based on the homology with mopKLMOP from A. calcoaceticus NCBI8250. Escherichia coli carrying the genes produced indigo from indole. E. coli JA300 and its cyclohexane-resistant mutant, OST3410, carrying the hydroxylase genes and the NADH regeneration system were grown in the two-phase culture system for indigo production. The OST3410 recombinant produced 52 µg indigo ml^–1 of medium in the presence of diphenylmethane. This productivity was 4.3-fold higher than that of the JA300 recombinant.

Comparison of modular and non-modular xylanases as carrier proteins for the efficient secretion of heterologous proteins from Penicillium funiculosum by M. J. C. Alcocer; C. S. M. Furniss; P. A. Kroon; M. Campbell; D. B. Archer (pp. 726-732).

Genes encoding three enzymes with xylanase activity from the filamentous fungus Penicillium funiculosum are described. Two of the encoded xylanases are predicted to be modular in structure with catalytic and substrate-binding domains separated by a serine and threonine-rich linker region; the other had none of these properties and was non-modular. In order to develop P. funiculosum as a host for the secreted production of heterologous proteins, each of the xylanases was assessed for use as a carrier protein in a fusion strategy. We show that one of the modular xylanases (encoded by xynA) was an effective carrier protein but the other (encoded by xynB) and the non-modular xylanase (encoded by xynC) were not effective as secretion carriers. We show that the β-glucuronidase (GUS) protein from Escherichia coli is secreted by P. funiculosum when expressed as an XYNA fusion but that the secreted GUS protein, cleaved in vivo from XYNA, is glycosylated and enzymatically inactive.

Alginate production by an Azotobacter vinelandii mutant unable to produce alginate lyase by M. Trujillo-Roldán; S. Moreno; D. Segura; E. Galindo; G. Espín (pp. 733-737).

Alginate is an industrially relevant linear copolymer composed of β-1,4-linked D-mannuronic acid and its C-5 epimer L-guluronic acid. The rheological and gel-forming properties of alginates depend on the molecular weight and the relative content of the two monomers. Alginate produced by Azotobacter vinelandii was shown to be degraded towards the end of the culture, an undesirable situation in terms of potential alginate applications. A gene (algL) encoding the alginate lyase activity AlgL is present within the alginate biosynthetic gene cluster of A. vinelandii. We constructed strain SML2, an A. vinelandii strain carrying a non-polar mutation within algL. No alginate lyase activity was detected in SML2. Under 3% dissolved oxygen tension, higher values of maximum mean molecular weight alginate were obtained (1,240 kDa) with strain SML2, compared to those from the parental strain ATCC 9046 (680 kDa). These data indicate that AlgL activity causes the drop in the molecular weight of alginate produced by A. vinelandii.

GltS, the sodium-coupled L-glutamate uptake system of Corynebacterium glutamicum: identification of the corresponding gene and impact on L-glutamate production by C. Trötschel; S. Kandirali; P. Diaz-Achirica; A. Meinhardt; S. Morbach; R. Krämer; A. Burkovski (pp. 738-742).

A screening procedure was established to identify Corynebacterium glutamicum transposon mutants with an altered L-glutamate excretion behaviour. By this microtiter plate-based approach seven non- or less excreting C. glutamicum strains and two hyper-excreters were found. The subsequently carried out molecular analysis of a hyper-producing clone led to the identification of the gltS gene, which codes for the sodium-coupled secondary L-glutamate uptake system in C. glutamicum. Characterization of a gltS deletion strain revealed that this transporter has a weak but significant impact on L-glutamate production induced by biotin limitation in the wild type. Obviously, GltS leads to the re-uptake of excreted L-glutamate causing a futile cycle. In accord with this hypothesis, the overexpression of gltS decreased L-glutamate production.

Achievement of rapid osmotic dehydration at specific temperatures could maintain high Saccharomyces cerevisiae viability by C. Laroche; P. Gervais (pp. 743-747).

Various methods have been tried to prevent cell mortality during dehydration, but the reasons why microorganisms die when submitted to dehydration and rehydration are not well understood. The aim of this study was to further investigate the reasons for yeast mortality during dehydration. Osmotic dehydration and rehydration of Saccharomyces cerevisiae W303-1A were performed at different temperatures. Two different approaches were used: isothermic treatments (dehydration and rehydration at the same temperature), and cyclic treatments (dehydration at an experimental temperature and rehydration at 25°C), with significant differences in viability found between the different treatments. Dehydration at lower and higher temperatures gave higher viability results. These experiments allowed us to propose a hypothesis that relates mortality to a high water flow through an unstable membrane during phase transition.

The isolation and use of iron-oxidizing, moderately thermophilic acidophiles from the Collie coal mine for the generation of ferric iron leaching solution by P. H.-M. Kinnunen; W. J. Robertson; J. J. Plumb; J. A. E. Gibson; P. D. Nichols; P. D. Franzmann; J. A. Puhakka (pp. 748-753).

Moderately thermophilic, iron-oxidizing acidophiles were enriched from coal collected from an open-cut mine in Collie, Western Australia. Iron-oxidizers were enriched in fluidized-bed reactors (FBR) at 60 °C and 70 °C; and iron-oxidation rates were determined. Ferrous iron oxidation by the microbiota in the original coal material was inhibited above 63 ˚C. In addition to four iron-oxidizers, closely related to Sulfobacillus spp that had been earlier isolated from the 60 °C FBR, one heterotroph closely related to Alicyclobacillus spp was isolated. The Alicyclobacillus sp. isolated from the Collie coal mine tolerated a lower pH than known Alicyclobacillus spp and therefore may represent a new species. The optimum temperature for growth of the iron-oxidizing strains was approximately 50 °C and their maximum temperatures were approximately 60 °C. The FBR was adjusted to operate at 50 °C and was inoculated with all of the isolated iron-oxidizing strains. At 60 °C, an iron-oxidation rate of 0.5 g Fe²⁺ l^–1 h^–1 was obtained. At 50 °C, the iron-oxidation rate was only 0.3 g Fe²⁺ l^–1 h^–1. These rates compare favourably with the iron-oxidation rate of Acidianus brierleyi in shake-flasks, but are considerably lower than mesophilic iron-oxidation rates.

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Applied Microbiology and Biotechnology (v.60, #6)

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Applied Microbiology and Biotechnology (v.60, #6)