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Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.

For many industrial applications in which the yeast Saccharomyces cerevisiae is used, e.g. beer, wine and alcohol production, appropriate flocculation behaviour is certainly one of the most important characteristics of a good production strain. Yeast flocculation is a very complex process that depends on the expression of specific flocculation genes such as FLO1, FLO5, FLO8 and FLO11. The transcriptional activity of the flocculation genes is influenced by the nutritional status of the yeast cells as well as other stress factors. Flocculation is also controlled by factors that affect cell wall composition or morphology. This implies that, during industrial fermentation processes, flocculation is affected by numerous parameters such as nutrient conditions, dissolved oxygen, pH, fermentation temperature, and yeast handling and storage conditions. Theoretically, rational use of these parameters offers the possibility of gaining control over the flocculation process. However, flocculation is a very strain-specific phenomenon, making it difficult to predict specific responses. In addition, certain genes involved in flocculation are extremely variable, causing frequent changes in the flocculation profile of some strains. Therefore, both a profound knowledge of flocculation theory as well as close monitoring and characterisation of the production strain are essential in order to gain maximal control over flocculation. In this review, the various parameters that influence flocculation in real-scale brewing are critically discussed. However, many of the conclusions will also be useful in various other industrial processes where control over yeast flocculation is desirable.

Ever since the introduction of controlled fermentation processes, alcoholic fermentations and Saccharomyces cerevisiae starter cultures proved to be a match made in heaven. The ability of S. cerevisiae to produce and withstand high ethanol concentrations, its pleasant flavour profile and the absence of health-threatening toxin production are only a few of the features that make it the ideal alcoholic fermentation organism. However, in certain conditions or for certain specific fermentation processes, the physiological boundaries of this species limit its applicability. Therefore, there is currently a strong interest in non-Saccharomyces (or non-conventional) yeasts with peculiar features able to replace or accompany S. cerevisiae in specific industrial fermentations. Brettanomyces (teleomorph: Dekkera), with Brettanomyces bruxellensis as the most commonly encountered representative, is such a yeast. Whilst currently mainly considered a spoilage organism responsible for off-flavour production in wine, cider or dairy products, an increasing number of authors report that in some cases, these yeasts can add beneficial (or at least interesting) aromas that increase the flavour complexity of fermented beverages, such as specialty beers. Moreover, its intriguing physiology, with its exceptional stress tolerance and peculiar carbon- and nitrogen metabolism, holds great potential for the production of bioethanol in continuous fermentors. This review summarizes the most notable metabolic features of Brettanomyces, briefly highlights recent insights in its genetic and genomic characteristics and discusses its applications in industrial fermentation processes, such as the production of beer, wine and bioethanol.

The aging and consequent changes in flavor molecules of a top-fermented beer were studied. Different aging conditions were imposed on freshly bottled beer. After 6 months of aging, the concentration changes were recorded for acetate esters, ethyl esters, carbonyls, Maillard compounds, dioxolanes, and furanic ethers. For some flavor compounds, the changes with time of storage were monitored at different temperatures, either with CO(2) or with air in the headspace of the bottles. For some molecules a relationship was determined between concentration changes and sensory evaluation results. A decrease in volatile esters was responsible for a reduced fruity flavor during aging. On the contrary, various carbonyl compounds, some ethyl esters, Maillard compounds, dioxolanes, and furanic ethers showed a marked increase, due to oxidative and nonoxidative reactions. A very high increase was found for furfural, 2-furanmethanol, and especially the furanic ether, 2-furfuryl ethyl ether (FEE). For FEE a flavor threshold in beer of 6 mug/L was determined. In the aged top-fermented beer, FEE concentrations multiple times the flavor threshold were observed. This was associated with the appearance of a typical solvent-like flavor. As the FEE concentration increased with time at an almost constant rate, with or without air in the headspace, FEE (and probably other furanic ethers) is proposed as a good candidate to evaluate the thermal stress imposed on beer.

Lambic and Gueuze are special Belgian beers obtained by spontaneous fermentation. Micro organisms involved in this fermentation were counted and differentiated using several selective growth media. Micro-organisms were also isolated from samples of Lambic of different age and originating from different casks and brews and identified by classical tests. The following general pattern of microbial development was observed. After 3 to 7 days the fermentation started with the development of wort Enterobacteriaceae and strains of Kloeckera apicu/ata. These organisms were overgrown after 3 to 4 weeks by strains of Saccharomyces cerevisiae and S. bayanus. These were responsible for the main fermentation, lasting for 3 to 4 months. This was followed by a strong bacterial activity. This period was dominated by the growth of strains of Pediococcus cerevisiae. These reached their maximal numbers during the summer months and were responsible for a five fold increase in lactic acid concentration. In some casks they caused ropiness. After the main fermentation period Lambic is very sensitive to spoilage by acetic acid bacteria of the genus Acetomonas. The presence of air may be the determining factor for their development. After 8 months a new increase in yeast cells was noted. These belonged now mainly to the genus Brettanomyces bruxellensis and Br. lambicus. They caused a further slow decrease in residual extract and the appearance of special flavours. Oxidative yeasts of the genera Candida, Cryptococcus, Torulopsis and Pichia were also detected and may be responsible for the formation of a flim on the beer surface after the main fermentation.

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Various techniques are used to adjust the flavors of foods and beverages to new market demands. Although synthetic flavoring chemicals are still widely used, flavors produced by biological methods (bioflavors) are now more and more requested by consumers, increasingly concerned with health and environmental problems caused by synthetic chemicals. Bioflavors can be extracted from plants or produced with plant cell cultures, microorganisms or isolated enzymes. This Mini-Review paper gives an overview of different systems for the microbial production of natural flavors, either de novo, or starting with selected flavor precursor molecules. Emphasis is put on the bioflavoring of beer and the possibilities offered by beer refermentation processes. The use of flavor precursors in combination with non-conventional or genetically modified yeasts for the production of new products is discussed.