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Yeasts belonging to the genus of Hanseniaspora are widespread in nature and are generally part of the microbiome of ripe fruits, such as apples, pears and grapes. Hanseniaspora spp. form 50-75% of the microorganisms found at the onset of grape must fermentations, H. uvarum often being the most abundant. H. uvarum is also a common isolate from other fruits, such as oranges, pineapples, mangoes, apples and pears. Since H. uvarum, known for producing high amounts of acetic acid and ethyl acetate, dominates the onset of spontaneous grape must fermentations, it greatly influences the aroma profile of wines. Besides its important role in fermentations, more biotechnological research has been reported in recent years, highlighting the universal value of H. uvarum beyond the beverage industry.
In this thesis, I characterized the aroma-production profile of seven different Hanseniaspora spp. in simultaneous co-fermentations with Saccharomyces cerevisiae. Significant differences could be detected between fermentations using Hanseniaspora spp. belonging to the fast-evolving lineage (FEL) and the slow-evolving lineage (SEL). Fermentations with yeasts of FEL showed increased amounts of ethyl acetate, whereas yeasts belonging to SEL revealed no meaningful difference compared to the pure S. cerevisiae fermentation. Furthermore, co-fermentations resulted in lactic acid depletion (H. osmophila), extremely high isoamyl acetate levels (H. guilliermondii), reduction in glycerol (H. occidentalis), increased terpenols (H. opuntiae) as well as esterified terpenes (H. guilliermondii and H. uvarum). This is the first study to investigate the use of H. nectarophila and H. meyerii in relation to wine, of which fermentations using H. nectarophila revealed high levels of acetate esters.
Nitrogen sources in form of amino acids have a major impact on the resulting aroma-production profile of yeasts as they serve as backbones for several volatile organic compounds. I explored amino acid uptake preferences of Hanseniaspora spp. of both lineages (H. uvarum = FEL; H. osmophila = SEL), S. cerevisiae (all glucophilic), as well as the fructophilic yeasts Starmerella bacillaris and Zygosaccharomyces rouxii in synthetic must, spiked with single amino acids. Among yeasts investigated, H. uvarum showed the most rapid amino acid uptake. Additional research on yeast fermentations with synthetic must supplemented with an amino acid mix resulted in amino acid-dependent synthesis of specific aroma compounds by the respective yeast. This study reveals similarities in both preferences in amino acid uptake and nitrogen source depending aroma compound synthesis of H. osmophila and S. cerevisiae.
I investigated the transcriptomic profile of H. uvarum and S. cerevisiae to gain insight into the different regulation mechanisms involved in the synthesis of aroma compounds. Furthermore, I explored the impact of different levels of nitrogen (nitrogen deficiency/ diammonium hydrogen phosphate (DAHP) supplemented must) within the grape must on the gene expression profile and therefore on the resulting aroma profile. Even though the different nitrogen regimes did not significantly affect transcriptional levels, a major impact on both fermentation kinetics and the aroma profile could be monitored. H. uvarum unravelled possessing additional genes including alcohol acetyltransferase activity leading to a rapid esterification of higher alcohols to acetate esters. In addition, the lack of genes involved in the synthesis of higher alcohols and ethyl esters, as well as an up-regulation of genes responsible for the synthesis of acetyl-coA and acetate esters favour the high acetate ester production.
In this study, I developed the first genetic modification tool for H. uvarum by constructing a synthetic marker gene encoding the hygromycin marker under the control of a H. uvarum promoter, flanked by ~1 000 bp homology region to the target locus, enabling the knock-out of one copy of the alcohol acetyltransferase 1 (ATF1) gene. By increasing the concentration of the antibiotic during selection, I could identify a knock-out of both ATF1 alleles in the diploid yeast, generating a homozygotic atf1 mutant. Fermentations resulted in a significant reduction of the acetate ester synthesis, proving the function of ATF1 as an important gene involved in the ester synthesis in H. uvarum.
Genetic modification of H. uvarum was optimized by establishing a rapid PCR-based gene targeting approach for one-step gene replacement. Generation of two novel synthetic antibiotic resistance genes (pFA-hygXL and pFA-clnXL) provided resistance against hygromycin and nourseothricin, respectively. Short flanking-homology regions of 56-80 bp added to these selection markers via PCR enabled gene targeting and the deletion of both the H. uvarum LEU2 and LYS2 genes via two rounds of consecutive transformations, generating a leucine auxotrophic leu2/leu2 mutant and a lysine auxotrophic lys2/lys2 mutant, respectively. The genetic modification approach of H. uvarum using PCR-based gene targeting was further validated by targeted complementation of the auxotrophic leu2/leu2 strain.
A substantial area of the terrestrial surface is used for agricultural production. In Europe, many farmland species and ecosystems have evolved with traditional agricultural practices and rely on extensive cultivation for their survival. Today, agrobiodiversity is threatened by the abandonment of traditional agricultural land use.
The cultivation of wine is one of the oldest forms of agriculture. On steep slopes, viticulture has shaped landscapes, cultural traditions, and unique ecosystems in many regions. In Germany, steep slope viticulture has been declining dramatically in recent decades due to insufficient profitability. The abandonment of these traditional cultural landscapes threatens the cultural heritage and the flora and fauna adapted to viticultural management. One solution to halt the decline of steep slope viticulture could be a change of cultivation system from vertically oriented rows to modern terraced vineyards. Here, the rows run parallel to the hillside, facilitating cultivation, and thus reducing production costs. At the same time, a large portion of terraced vineyard consists of uncultivated embankments, which can provide valuable habitats within the agricultural system. However, little is known about the effects of a cultivation system change and vineyard abandonment on biodiversity. In addition, information on how to sustainably revegetate terrace embankments is missing.
Within the framework of this dissertation, the overall aim was to evaluate the potential of terraced vineyards to promote biodiversity and investigate measures for sustainable revegetation of terrace embankments. In addition, the effects of distinct vineyard management (vertically oriented vs. terraced) and vineyard abandonment on biodiversity were assessed. The main part of this thesis is based on a field study comparing three vineyard types (vertically oriented vineyard, terraced vineyard, abandoned vineyard) that were arranged in 15 triplets along the Upper Middle Rhine Valley (UMRV) in Germany. The effects of vineyard types, local vineyard parameters, and landscape structure on biodiversity were evaluated using four species groups. Studied groups represented plants and arthropod species (grasshoppers, wild bees, spiders). In a second field study, three newly constructed terrace embankments were revegetated using different seeding techniques and seed mixtures. In addition, measures to support plant establishment were evaluated. Plant establishment was assessed for three consecutive years in permanent vegetation plots.
The first study on the three vineyard types revealed that there was no uniform positive effect of one vineyard type on all investigated groups. Instead, the responses were group and taxon-specific and driven by management-related disturbance intensities, local vegetation structures, soil and landscape structure. In total, the heterogeneous landscape and small-structured vineyard agroecosystem of the UMRV supported high species diversity, hosted high numbers of rare and threatened species, and diverse species communities across vineyard types. Plant species richness was enhanced by vineyard management and particularly benefited from terraced vineyards and rather nutrient-poor and extensively managed embankments. In comparison, plant communities of fallows were completely different with lower plant diversity. Grasshoppers were supported by vineyard management, and bare ground in vineyard inter-rows supported heat- and open-adapted species. Taller vegetation stands on terrace embankments enhanced grasshopper species richness while woody structures and vineyard abandonment reduced grasshopper diversity and abundance at the local and landscape scale. Wild bee species richness and abundance were strongly determined by their nesting needs. For ground-nesting bees, the suitability of soils for nesting was important, irrespective of vineyard management types whereas above-ground nesting bees were enhanced by woody structures of fallows at the local and landscape scale. Moreover, fallows further provided valuable floral and non-floral resources for both ground- and above-ground nesting bees. For spiders, there was no difference in species richness and abundance between vineyard types but many xerophilic species adapted to open habitat structures prevailed in terraced vineyards. Vineyard fallows supported highly distinct spider communities, including some late-successional species. In conclusion, the vineyard cultural landscape of the UMRV with both actively managed and abandoned vineyards led to a high diversity of plant and arthropod communities. Hence, for its preservation, a spatially heterogeneous landscape comprising both (semi) natural and viticultural-management elements is important.
Results from the second study showed that terraced vineyards and especially terrace embankments can contribute to promoting biodiversity, especially if they are revegetated with regional seed mixtures. Seeds of locally adapted populations and hydro-seeding provided higher vegetation cover which is needed to prevent severe erosion whilst non-locally adapted standard seed mixtures and manual seeding had a higher failure risk and were more prone to severe precipitation events. Taken together, vineyard terracing appears to be a win-win solution for both winegrowers and biodiversity and thus helps to maintain viticulture on steep slopes.