A slightly pink-coloured strain, strain DFL-11(T), was isolated from single cells of the marine dinoflagellate Alexandrium lusitanicum and was found to contain the genes encoding two proteins of the photosynthetic reaction centre, pufL and pufM. 16S rRNA gene sequence analysis revealed that the novel strain belonged to the alpha-2 subgroup of the Proteobacteria and was most closely related to Stappia aggregata (97.7 % similarity), Stappia alba (98.0 %) and Stappia marina (98.0 %). Dark-grown cells of strain DFL-11(T) contained small amounts of bacteriochlorophyll a (bchl a) and a carotenoid. Cells of strain DFL-11(T) were rods, 0.5-0.7 x 0.9-3.0 microm in size and motile by means of a single, subpolarly inserted flagellum. The novel strain was strictly aerobic and utilized a wide range of organic carbon sources, including fatty acids, tricarboxylic acid cycle intermediates and sugars. Biotin and thiamine were required as growth factors. Growth was obtained at sea salt concentrations of between 1 and 10 % (w/v), at a pH between 6 and 9.2 and at a temperature of up to 33 degrees C (optimum, 26 degrees C). Nitrate was not reduced and indole was not produced from tryptophan. Strain DFL11(T) was resistant to potassium tellurite and transformed it to elemental tellurium. The major respiratory lipoquinone was ubiquinone 10 (Q10). The polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, an unidentified aminolipid and the glycolipid sulphoquinovosyldiacylglyceride. The fatty acids comprised 16 : 1 omega7c, 16 : 0, 18 : 1 omega7c, 18 : 0, 11-methyl 18 : 1 omega6t, 11-methyl 20 : 1 omega6t, 20 : 1 omega7c, 22 : 0, 22 : 1 and the hydroxy fatty acids 3-OH 14 : 0, 3-OH 16 : 0 (ester-linked), 3-OH 18 : 0, 3-OH 20 : 1 and 3-OH 20 : 0, all of which are amide-linked. The DNA G+C value was 56 mol%. Comparative analysis of alpha-2 subgroup 16S rRNA gene sequences showed that the type species of the genus Stappia, Stappia stellulata, is only distantly related to S. aggregata (95.3 % sequence similarity). Based on the combination of the 16S rRNA gene sequence data, a detailed chemotaxonomic study and the biochemical and physiological properties of members of the genera Stappia, Pannonibacter and Roseibium, it is proposed that S. aggregata, S. alba, S. marina are transferred to a new genus, Labrenzia gen. nov., as Labrenzia aggregata comb. nov., Labrenzia alba comb. nov. and Labrenzia marina comb. nov. The type species of the new genus is Labrenzia alexandrii sp. nov., with strain DFL-11(T) (=DSM 17067(T)=NCIMB 14079(T)) as the type strain. The pufLM genes of the photosynthesis reaction centre were shown to be present in some, but not all, species of the new genus Labrenzia and they were identified for the first time in S. stellulata. In accordance with the new data collected in this study, emended descriptions are provided for the genera Pannonibacter, Roseibium and Stappia.

Within a collection of marine strains that were shown to contain the photosynthesis reaction-centre genes pufL and pufM, a novel group of alphaproteobacteria was found and was characterized phenotypically. The 16S rRNA gene sequence data suggested that the strains belonged to the order Rhizobiales and were closest (98.5% sequence similarity) to the recently described species Hoeflea marina. The cells contained bacteriochlorophyll a and a carotenoid, presumably spheroidenone, in small to medium amounts. Cells of the novel strains were small rods and were motile by means of single polarly inserted flagella. Good growth occurred in complex media with 0.5-7.0% sea salts, at 25-33 degrees C (optimum, 31 degrees C) and at pH values in the range 6-9. With the exception of acetate and malate, organic carbon sources tested supported poor growth or no growth at all. Growth factors were required; these were provided by small amounts of yeast extract, but not by standard vitamin solutions. Growth occurred under aerobic to microaerobic conditions, but not under anaerobic conditions, either in the dark or light. Nitrate was not reduced. Photosynthetic pigments were formed at low to medium salt concentrations, but not at the salt concentration of sea water (3.5%). On the basis of smaller cell size, different substrate utilization profile and photosynthetic pigment content, the novel strains can be classified as representatives of a second species of Hoeflea, for which the name Hoeflea phototrophica sp. nov. is proposed. The type strain of Hoeflea phototrophica sp. nov. is DFL-43(T) (= DSM 17068(T) = NCIMB 14078(T)).

The Roseobacter lineage is a phylogenetically coherent, physiologically heterogeneous group of alpha-Proteobacteria comprising up to 25% of marine microbial communities, especially in coastal and polar oceans, and it is the only lineage in which cultivated bacteria are closely related to environmental clones. Currently 41 subclusters are described, covering all major marine ecological niches (seawater, algal blooms, microbial mats, sediments, sea ice, marine invertebrates). Members of the Roseobacter lineage play an important role for the global carbon and sulfur cycle and the climate, since they have the trait of aerobic anoxygenic photosynthesis, oxidize the greenhouse gas carbon monoxide, and produce the climate-relevant gas dimethylsulfide through the degradation of algal osmolytes. Production of bioactive metabolites and quorum-sensing-regulated control of gene expression mediate their success in complex communities. Studies of representative isolates in culture, whole-genome sequencing, e.g., of Silicibacter pomeroyi, and the analysis of marine metagenome libraries have started to reveal the environmental biology of this important marine group.

The influence of light and starvation on bacteriochlorophyll a (bchl a) and biomass formation of some aerobic anoxygenic photosynthetic bacteria was investigated in chemostat culture. Three species recently isolated from dinollagellatecultures were compared, Dinoroseobacter shibae, which contained relatively high amounts of bchl a, and Stappia sp. DFL-11 and Hoeflea phototrophica, which both have very low amounts of photosynthetic pigments. Simulating day/night alternation D. shibae was subjected to 7 light:dark cycles = 8 h: 16 It and 3 light:dark cycles = 16 h:8 h. Using a dilution rate of 0.1 h(-1) and succinate as a substrate (8.5 mmol/l) the concentration of bchl a decreased during the light period due to inhibition by light and recovered always to about the previous level during the dark period. Biomass increased during the light period showing that additional energy was generated in the light.Nutrient deficiency in the absence or presence of light was studied by exposing continuous cultures growing in the dark under the conditions described above to 8 h of illumination, starvation, i.e. interruption of medium supply, or both. The cultures of D. shibae exhibited a bchl a base level of 2.5 nmol/mg protein, which decreased reversibly in the light and increased significantly during starvation in the dark to reach a level of 4 nmol/mg protein 16 h after medium supply was resumed, indicating a slow regulatory response towards periodic starvation in D. shibae. Under simultaneous illumination and starvation conditions, these two effects apparently cancelled one another out, resulting in unchanged levels of bchl a. By contrast, the cultures of Stappia sp. and H. phototropica were characterized by bchl a contents of almost zero in the dark, little change under illumination or starvation, but a very marked increase after simultaneous illumination and starvation, reaching 0.16 and 0.24 nmol bchl a/mg protein, respectively, 16 h after medium supply was resumed. These data suggest the presence of a regulatory mechanism integrating light stimuli and starvation related metabolic signals. The different physiological behaviour of the two groups is discussed in terms of the significance of aerobic anoxygenic photosynthesis in their natural environment. (c) 2006 Elsevier Ltd. All rights reserved.

Among a group of marine isolates that were found to be positive for genes of the bacterial photosynthetic reaction centre, a strain was selected for characterization that was phylogenetically close to the genus Roseovarius. The strain, designated DFL-24T, originated from a culture of Alexandrium ostenfeldii (dinoflagellate) and contained small amounts of bacteriochlorophyll a, corresponding to about 1 % of the amount found in intensely pigmented aerobic phototrophs such as Roseobacter litoralis. Cells were rods of 0.5-0.7x1.3-3.0 microm often with uneven ends, suggesting a budding mode of division. True motility was not observed. Electron micrographs of ultrathin sections revealed a Gram-negative cell-wall structure. Cultures did not grow without addition of sea salts and tolerated up to 10 % (w/v) sea-water salts. Acetate, butyrate, tricarboxylic acid cycle intermediates, glutamate and glycerol were used as growth substrates, but not glucose or fructose. Biotin, thiamine and nicotinic acid were required as growth factors. The DNA G+C content was 63 mol%. 16S rRNA gene sequence analysis placed the strain within the Roseobacter lineage of the 'Alphaproteobacteria'. Its closest phylogenetic neighbour was Roseovarius tolerans showing 96.4 % 16S rRNA gene sequence similarity. Based also on its physiological and biochemical characteristics, the strain is considered to represent a novel species of the genus Roseovarius, Roseovarius mucosus sp. nov. Strain DFL-24T (=DSM 17069T=NCIMB 14077T) is the type strain. The 16S rRNA gene sequence of DFL-24T was found to have a similarity of 99.7 % with an unidentified strain, 253-13 (=DSM 17070), which was likewise characterized and shown to constitute another strain of the species.

A Gram-negative, spore-forming anaerobe, KS(T), was isolated from an enrichment culture that was set up for anaerobic degradation of the aliphatic polyester poly(propylene adipate). The strain had the cellular organization of Sporomusa, vibrio-shaped cells and terminal round spores, and fermented sugars and sugar alcohols to propionic and acetic acid. Based on the morphological and physiological features as well as on a 16S rRNA gene similarity of 98 %, it was grouped with Propionispora vibrioides. A relatively low DNA-DNA hybridization value with the type strain of this species (47 %), and differences in substrate utilization and spore morphology, suggested that the strain should be classified in a separate species, Propionispora hippei sp. nov., with KS(T) as the type strain (=DSM 15287(T)=ATCC BAA-665(T)).

Six Clostridium strains which ferment glycerol to 1,3-propanediol were tested for their taxonomic and phylogenetic relatedness. All but one were known as C butyricum. By physiological tests, 16S rDNA sequences and fatty acid composition two groups were distinguished. The first comprised the strains VPI 3266, DSM 2478 and DSM 523 (C. "kainantoi") and was consistent with the type strain of C. butyricum in almost all characters. The second group comprising the strains DSM 5430, DSM 5431 and E5 was related to C. beijerinckii. The 16S rDNAs of these strains were almost identical with that of the type strain of C. beijerinckii, DSM 791. The DNA-DNA hybridization value of DSM 5431 and ES with C. beijerinckii DSM 791 was markedly but not decisively lower (67 and 72%, respectively). However, there were significant physiological differences to C. beijerinckii which suggested to describe the strains as a separate species, Clostridium diolis with strain SH1 (= DSM 5431) as the type strain. The new species is distinguished from C. beijerinckii, which requires complex nutrients, by its ability to grow in glucose mineral medium with biotin as the only growth factor and by differences in substrate utilization. "C. kainantoi" Takeda and Matsui was recognized as a later synonym of C. butyricum.

The need for a sustainable resource supply, the rapid advances in plant biotechnology and microbial genetics and the strategic shift of major chemical companies into the area of life sciences are some of the driving forces for renewed interest in producing bulk chemicals from renewable resources by biological processes. The microbial production of 1,3-propanediol as briefly reviewed in this article and compared with the competing chemical processes demonstrates the promise and constraints of bioprocesses for bulk chemicals. The new concept of biorefinery and biocommodity engineering and future research needs in this area are also outlined.

The amino acid analogue selenomethionine (SeMet) is shown to be efficiently incorporated into recombinant proteins expressed in Escherichia coli grown in a simple minimal medium without the addition of synthetic amino acids. Furthermore, satisfactory SeMet incorporation is obtained with a methionine-prototrophic strain transformed with commonly used vector systems. As examples, purified tryparedoxin 1 from Crithidia fasciculata, alkylhydroperoxide reductase (AhpC) from Mycobacterium marinum and the 16-kDa antigen from M. tuberculosis are shown to be efficiently labelled with SeMet, using the culture conditions and the host/vector systems described here. Enzymatic analysis reveals no differences between native and SeMet-labelled tryparedoxin 1 enzyme. Both proteins yield crystals under similar conditions. The culture conditions and host vector systems described greatly facilitate selenium-labelling of proteins for 3-D structure determination.

The fermentation of glycerol by Clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3-propanediol, butyric and acetic acids and ethanol. More than 60 g/l glycerol was utilized, and up to 17 g/l butanol was produced. Fed-batch cultures did not offer an advantage. When molecular nitrogen was used as a nitrogen source, the fermentation time was prolonged by a factor of 1.5. Fermentations at constant pH values between 4.5 and 7.5 did not reveal significant differences in product formation except for an increase in the ethanol content starting at pH 6.5. Chemostat cultures also yielded predominantly n-butanol, but in some fermentations, the 1,3-propanediol fraction was relatively high. The pH auxostat cultures, which were operated at a glycerol excess, contained 1,3-propanediol as the main product. As a whole, the fermentations were characterized by a certain variability in product formation under seemingly equal or slightly varied conditions. It appears that the regulation of the numerous fermentation pathways occurring in this organism is not very strict.

Anaerobic enrichment cultures, with erythritol as substrate, resulted in the isolation of a strain with properties not yet found in an existing genus in this combination. The strain, FKBS1, was strictly anaerobic, stained gram-negative and formed spores. Cells were small motile vibrios with flagella inserted at the concave side of the cell. Spores were located terminally and caused only slight swelling of the cells if compared to related spore-forming genera. FKBS1 fermented fructose, mannitol, sorbitol, xylitol and erythritol to propionic acid, acetic acid, CO2 and small amounts of H2 to balance the difference in the oxidation-reduction value between substrate and cell mass. The 16S rDNA sequence revealed relationship to the Sporomusa-Pectinatus-Selenomonas group. However, the phylogenetic distance to any of its members was too great to allow it to be placed in one of the existing genera. Morphologically the strain resembled Sporomusa, which, however, performs an acetogenic type of fermentation. The propionic-acid-forming genera of the group are either not spore-formers or, in the case of Dendrosporobacter quercicolus (syn. Clostridium quercicolum), morphologically different. It is therefore proposed to classify strain FKBS1 as a new genus and species, Propionispora vibrioides.

The butanol and acetone-producing strain DSM 2152, invalidly described as ‘ Clostridium saccharoperbutylacetonicum ’ is compared with the type strain C. acetobutylicum , DSM 792, with respect to solvent and acid formation at varying pH values and growth rates. Batch cultures, product-limited chemostat and pH-auxostat cultures were used for characterization. Under all conditions strain DSM 2152 produced much lower amounts of butyric and acetic acids than the type strain. The pH optimum for solvent formation was higher, ie 5.5 instead of 4.5. Solvent formation occurred at higher dilution rates, but below 0.1 h −1 a lower solvent concentration was obtained, indicating that acid production was too low to provide a sufficient amount for acetone formation. The results are discussed in the light of recent publications on the taxonomy of butanol-acetone producing clostridia using 16S rRNA sequence analysis and other nucleic acid data. The presently suggested ‘phylogenetic’ classification of the collective species, C. acetobutylicum , is also reflected in the fermentation characteristics.

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO2, H2, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH2, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a “green” polyester is a more cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism.

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this pH range. 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed.

The continuous fermentation of 1,3-propanediol from glycerol by Clostridium butyricum was subjected to cell recycling by filtration using hollow-fibre modules made from polysulphone. The performance of the culture system was checked at a retention ratio (dilution rate/bleed rate) of 5, dilution rates between 0.2 h−1 and 1.0 h−1 and glycerol input concentrations of 32 g l−1 and 56 g l−1. The near-to-optimum propanediol concentration of 26.5 g l−1 (for 56 g l−1 glycerol) was maintained up to a dilution rate of 0.5 h−1 and then decreased while the propanediol productivity was highest at 0.7 h−1. The productivity could be increased by a factor of four in comparison to the continuous culture without cell recycling. By application of the model of Zeng and Deckwer [(1995) Biotechnol Prog 11: 71–79] for cultures under substrate excess, it was shown that the limitations resulted exclusively from product inhibition and detrimental influences from the cell recycling system, such as shear stress, were not involved.

Glycerol fermentation and product formation of two product-tolerant mutants of Clostridium butyricum DSM 5431 were investigated in continuous culture at increasing glycerol feed concentrations. Under conditions of glycerol excess (above 55 g l(-1) at D = 0.15 h(-1)), the mutants maintained a constant level of glycerol consumption and product formation, whereas the parent strain exhibited a substantial decrease in substrate conversion, 1,3-propanediol and butyrate formation, and an increase in acetate formation. The activities of the glycerol dehydrogenase, the glycerol dehydratase and the 1,3-propanediol dehydrogenase showed only slight changes with glycerol concentrations in the mutants, but dropped markedly at high concentrations in the wild type. Intracellular concentrations of NADH, NAD(+) and acetyl-CoA remained at a relatively constant level in the mutants, but increased sharply with the wild type strain. The NADH content was always higher than the NAD(+) content in the mutants as well as in the wild type.

The effect of methyl viologen addition, and iron and phosphate limitation on product distribution during glycerol fermentation of Clostridium butyricum DSM 5431 was investigated in continuous culture. Special attention was paid to the gaseous products H2 and CO2, which were measured on-line. In all three cases, an increased yield of 1,3-propanediol linked to a decreased hydrogen release was observed, indicating that a higher proportion of electrons was channelled from reduced ferredoxin towards NADH2 production. The specific substrate consumption rates and the specific production rates revealed that this increase in propanediol yield was not obtained at the expense of glycolysis products but by an increased substrate conversion (overflow metabolism). The acetate/ butyrate ratio during glycerol fermentation was essentially influenced by the availability of iron. It was substantially increased when the culture turned from iron excess to iron-limited conditions. Therefore iron limitation proved to be a suitable means to achieve high 1,3-propanediol yields and to reduce butyrate formation.