Assessing the potential of wild yeasts for bioethanol production

Bioethanol fermentations expose yeasts to a new, complex and challenging fermentation medium with specific inhibitors and sugar mixtures depending on the type of carbon source. It is, therefore, suggested that the natural diversity of yeasts should be further exploited in order to find yeasts with good ethanol yield in stressed fermentation media. In this study, we screened more than 50 yeast isolates of which we selected five isolates with promising features. The species Candida bombi, Wickerhamomyces anomalus and Torulaspora delbrueckii showed better osmo- and hydroxymethylfurfural tolerance than Saccharomyces cerevisiae. However, S. cerevisiae isolates had the highest ethanol yield in fermentation experiments mimicking high gravity fermentations (25\ua0% glucose) and artificial lignocellulose hydrolysates (with a myriad of inhibitors). Interestingly, among two tested S. cerevisiaestrains, a wild strain isolated from an oak tree performed better than Ethanol Red, a S. cerevisiae strain which is currently commonly used in industrial bioethanol fermentations. Additionally, a W. anomalus strain isolated from sugar beet thick juice was found to have a comparable ethanol yield, but needed longer fermentation time. Other non-Saccharomycesyeasts yielded lower ethanol amounts

Phenotypic evaluation of natural and industrial Saccharomyces yeasts for different traits desirable in industrial bioethanol production

Saccharomyces cerevisiae is the organism of choice for many food and beverage fermentations because it thrives in high-sugar and high-ethanol conditions. However, the conditions encountered in bioethanol fermentation pose specific challenges, including extremely high sugar and ethanol concentrations, high temperature, and the presence of specific toxic compounds. It is generally considered that exploring the natural biodiversity of Saccharomyces strains may be an interesting route to find superior bioethanol strains and may also improve our understanding of the challenges faced by yeast cells during bioethanol fermentation. In this study, we phenotypically evaluated a large collection of diverse Saccharomyces strains on six selective traits relevant for bioethanol production with increasing stress intensity. Our results demonstrate a remarkably large phenotypic diversity among different Saccharomyces species and among S. cerevisiae strains from different origins. Currently applied bioethanol strains showed a high tolerance to many of these relevant traits, but several other natural and industrial S. cerevisiae strains outcompeted the bioethanol strains for specific traits. These multitolerant strains performed well in fermentation experiments mimicking industrial bioethanol production. Together, our results illustrate the potential of phenotyping the natural biodiversity of yeasts to find superior industrial strains that may be used in bioethanol production or can be used as a basis for further strain improvement through genetic engineering, experimental evolution, or breeding. Additionally, our study provides a basis for new insights into the relationships between tolerance to different stressors

Effects of microbial activity and diversity on the adaptation of nitrifying and denitrifying communities to zinc stress in soil

Dit onderzoek handelt over de toxische effecten van de spoormetalen zink (Zn) en koper (Cu) op de functie en de structuur van microbiële gemeenschappen in de bodem. Het onderwerp is het proces van herstel van de microbiële functie na een initieel toxische situatie, dit is de adaptatie van de microbiële gemeenschap aan de verontreiniging. Adaptatie kan veroorzaakt worden door phenotypische of genotypische veranderingen of veranderingen in de gemeenschapsstructuur. In het bijzonder wordt de adaptatie van de bodemnitrificatie en -denitrificatie bestudeerd na zinkvervuiling van de bodem. De aanleiding van dit onderzoek was de waarneming dat adaptatie langer duurt in het geval van de nitrificatie (&gt; 1 jaar) dan voor de denitrificatie (< 3 maanden) na zinkvervuiling. Vermoedelijk is de adaptatie van denitrificatie sneller door de hogere groeisnelheid en microbiële diversiteit van de denitrificerende gemeenschap vergeleken met die van de nitrificerende gemeenschap. Bij een hogere groeisnelheid kunnen tolerante populaties sneller dominant worden of kunnen genotypische veranderingen sneller optreden. Een grotere diversiteit verhoogt de kans op de aanwezigheid van tolerante soorten. Er werden experimenten opgezet om te testen of de adaptatie wordt versneld met (i) toenemende groeisnelheid of (ii) toenemende microbiële diversiteit.De eerste hypothese werd getest door de adaptatie aan Zn te meten onder verschillende condities van microbiële activiteit. De hypothese was dat toenemende activiteit de groeisnelheid bevordert. Bodems werden vervuild met Zn (0-4000 mg Zn kg-1) in een factoriële proefopzet met substraatdosissen die de activiteit stimuleren. Voor nitrificatie werd gebruik gemaakt van NH4+-N, voor denitrificatie van gemalen hooi. De bodems werden in open lucht geïncubeerd in potten. De Zn-concentraties in het poriewater van de bodem waarbij de nitrificatie was gehalveerd stegen met meer dan een factor 10 met stijgende NH4+-N-dosis vergeleken met de controles die geen NH4+-N kregen toegediend. Dit toont aan dat stijgende activiteit de adaptatie aan Zn versnelt. Tezelfdertijd veranderde de microbiële gemeenschapsstructuur waardoor een Zn-tolerante gemeenschap ontstond. Deze toegenomen Zn-tolerantie werd toegeschreven aan de AOB-gemeenschap, aangezien de AOB/AOA-ratio steeg van 0.4 in de controlegemeenschap tot 1.4 in de meest tolerante gemeenschap (AOB: ammonium oxiderende bacteriën; AOA: ammonium oxiderende archaea). Bovendien veranderde het AOB amoA DGGE-profiel tijdens Zn-adaptatie, terwijl het AOA amoA DGGE-profiel onveranderd bleef. In de series waaraan hooi was toegediend, herstelde de denitrificatie binnen de 6 maanden tot minstens 80% van de denitrificatie in de controlebehandeling (geen Zn). Grote inhibitie was nog steeds waarneembaar in de serie waaraan geen hooi werd toegevoegd. Deze snellere adaptatie in de series met hooi werd gedeeltelijk, maar niet volledig, toegeschreven aan effecten op de Zn-biobeschikbaarheid als gevolg van de behandelingen. Het herstel ging samen met het herstel van de nosZ genaantallen (van 10.000.000 kopijen g-1 bodem in de behandelingen met een afgenomen activiteit tot 500.000.000 kopijen g-1 bodem in de andere behandelingen). In tegenstelling tot de nitrificerende gemeenschap werden er voor de denitrificerende gemeenschapsstructuur slechts kleine veranderingen waargenomen als gevolg van Zn-vervuiling die echter wel van belang zouden kunnen zijn in adaptatie. Deze experimenten bevestigden de tragere adaptatie van de nitrificerende gemeenschap aan Zn vergeleken met de denitrificerende gemeenschap. Bovendien was de adaptatie versneld bij hogere microbiële activiteit en verbonden met veranderingen in de gemeenschapsstructuur of -grootte. De tweede hypothese over de rol van microbiële in adaptatie werd enkel getest voor de nitrificatie. De diversiteit werd gevarieerd op 2 manieren. Ten eerste werden 8 bodems met een verschillende diversiteit geselecteerd. Ten tweede werd de AOB-diversiteit experimenteel gevarieerd door steriele bodem te inoculeren met bodems en bodemmengsels (5 wt%). Zo werden 13 behandelingen verkregen met gelijke abiotische bodemeigenschappen, maar een variërende AOB-diversiteit. De diversiteit werd gekwantificeerd met behulp van verschillende diversiteitsindices gebaseerd op de DGGE-profielen. Adaptatie was niet statistisch gecorreleerd met de gekozen diversitietsindices. Indien de diversiteit werd gevarieerd volgens de eerste manier was het moeilijk om de resultaten te interpreteren omwille van abiotische factoren die ook een rol speelden. De verschillen in Zn-adaptatie tussen de 13 behandelingen van experiment 2 waren gerelateerd aan de initiële nitrificatieactiviteit van de geïnoculeerdebehandelingen. De microbiële activiteit is dus de voornaamste factor die adaptatie beïnvloedt.De hierboven beschreven experimenten toonden aan dat de nitrificerende gemeenschapsstructuur duidelijk verandert bij de ontwikkeling van Zn-tolerantie. In het laatste hoofdstuk werd de vraag gesteld of de Zn-tolerante gemeenschappen een gelijkaardige structuur verkrijgen onafhankelijk van de originele structuur. Eerder werd metaalcotolerantie (bv. Zn en Cu) aangetoond in metaalverontreinigde bodems. De vraag is of deze co-tolerantie geassocieerd is met de ontwikkeling van gelijkaardige populaties tussenverschillende bodems vervuild met Zn of Cu. De gemeenschapsstructuur werd bepaald met behulp van DGGE en populaties werden geïdentificeerd met behulp van sequenering. Acht bodemseries, elk met een Zn- of Cu-vervuilingsgradient, werden geselecteerd, waaronder 4 series met vergelijkbare Zn- of Cu-vervuilingen. Zink- of Cu-vervuiling verhoogde de metaaltolerantie in vervuilde bodems van elke serie. Cotolerantie voor Zn in Cu-vervuilde bodems werd aangetoond in 2 bodems en cotolerantie voor Cu werd enkel aangetoond in 1 artificieel met Zn vervuilde bodem. Cotolerantie bleek niet altijd geassocieerd met de ontwikkeling van identieke dominante fylotypes in of tussen de bodems. Detolerante gemeenschappen van verschillende bodems die gelijkaardig vervuild waren met Zn of Cu convergeerden niet naar een gemeenschappelijke structuur. Veranderingen van de gemeenschapsstructuur als gevolg van spoormetaalvervuiling werden bepaald door de initiële gemeenschapssamenstelling en -structuur en tijdens blootstelling vorm gegeven door het type metaal en de dosis. We besloten dat metaaltolerantie cotolerantie voor andere metalen kan induceren, maar dit bleek niet noodzakelijk geassocieerd met de selectie van identieke fylotypes.Samengevat toont deze studie aan dat verschillen in de microbiële activiteit de adaptatie aan Zn verklaarden tussen verschillende bodems, bodembehandelingen en microbiële groepen. De invloed van de microbiële diversiteit blijft onduidelijk, mogelijk omdat de microbiële bodemgemeenschap intrinsiek al een relatief grote diversiteit heeft vergeleken met andere ecosystemen. De tolerante microbiële bodemgemeenschap van verschillende bodems convergeert niet naar een gelijke structuur en is afhankelijk van de originele structuur en samenstelling of het type en de dosis van het metaal (Znof Cu).Dankwoord................I Samenvatting.............III Summary..................VII List of abbreviations....XI Table of Contents .......XIII Chapter 1 Adaptation of nitrification and denitrification in metal contaminated soils: environmental context, definitions, rationales and research objectives (adapted from Soil Biol Biochem 42: 766-772)............................................ 1 Chapter 2 Stimulated activity of the soil nitrifying community accelerates community adaptation to zinc stress (adapted from Soil Biol Biochem 42: 1581-1587)...................................... 17 Chapter 3 Dynamics of the nitrous oxide reducing community during adaptation to zinc stress in soil ........................................... 41 Chapter 4 Effects of the microbial diversity on the adaptation of nitrification in Zn contaminated soils ......................................... 61 Chapter 5 Co-tolerance to zinc and copper of the soil nitrifying community and its relationship with the community structure ...............................................91 Chapter 6 General conclusions and future outlook ...............................................111 Appendix.......................................119 Reference list ................................121 List of publications...........................133nrpages: 151status: publishe

Copper toxicity in soils amended with copper containing fungicides

The long-term application of copper containing pesticides can cause a significant accumulation of Cu in soils. In order to assess the potential toxic effects of these increased Cu concentrations, a comprehensive comparison of Cu toxicity in a set of soils with high Cu concentrations due to the application of Cu pesticides and corresponding reference soils was performed. Soils with a long history of application of Cu containing fungicides were sampled at 11 vineyards across Europe including an uncontaminated control soil outside each vineyard. Standard ecotoxicity tests for plant yield, root length, nitrification and invertebrates (Enchythraeus albidus) reproduction were performed on each soil of both the vineyard gradient and the corresponding control soil spiked with CuCl2. For all vineyard gradients, the increased Cu concentration (maximum 349 - 689 mg Cu/kg) did not decrease the response of any test compared to the corresponding control soils. Spiking these control soils with CuCl2 however resulted in clear dose-response curves and toxicity data for these freshly spiked control soils are in the range of toxicity data for these endpoints reported in the European Voluntary Risk Assessment Report (VRAR) on Cu and its compounds. Soil limits (Predicted No Effect Concentration, PNEC) calculated according to this VRAR are a factor 2.2 to 5.7 below the Cu concentrations observed in the sampled vineyard soils and therefore seem to be over-protective for soils contaminated with Cu due to the long term use of Cu containing pesticides. It is assumed that long-term accumulation of Cu in soils due to the annual application of small amounts of Cu, as is the case for Cu plant protection products, may result in stronger attenuation of Cu availability with time and hence a lower toxicity of Cu in soils compared to the default assumptions in the VRAR on the effect of ageing on toxicity (as reflected in the so-called leaching-ageing factor). Therefore, a larger scenario-specific leaching/ageing factor is proposed for the effects assessment of Cu in soils affected by long-term application of Cu containing pesticides.status: accepte

Co-tolerance to zinc and copper of the soil nitrifying community and its relationship with the community structure

Previous studies showed that microbial communities develop trace metal tolerance after long term exposure to increased trace metal concentrations in soil. Co-tolerance is the development of tolerance to other trace metals than those to which the microbial community had been exposed to due to, e.g., similar physiological tolerance mechanisms. We determined co-tolerance of nitrifying communities to Zn and Cu in different soils and assessed the nitrifying community structure using AOB amoA DGGE. We selected soils from five locations which were contaminated with either Cu or Zn or with both. Increased Zn and Cu tolerance was observed in response to corresponding soil contamination, however, co-tolerance was only detected in few cases. Similarly contaminating soil with either Zn or Cu selected for identical phylotypes in one soil, however, co-tolerance was not observed in the Zn contaminated treatment. In contrast, similar Zn and Cu contamination in a second soil both resulted in the development of a co-tolerant nitrifying community that was composed of different dominant phylotypes. The Zn tolerant nitrifying community structures from different soils that inherently have a different native community did not converge to a similar community structure.posterstatus: publishe

Recovery of soil ammonia oxidation after long-term zinc exposure is not related to the richness of the bacterial nitrifying community

A soil sterilization-reinoculation approach was used to manipulate soil microbial diversity and to assess the effect of the diversity of the ammonia-oxidizing bacteria (AOB) on the recovery of the nitrifying community to metal stress (zinc). Gamma-irradiated soil was inoculated with 13 different combinations of up to 22 different soils collected worldwide to create varying degrees of AOB diversity. Two months after inoculation, AOB amoA DGGE based diversity (weighted richness) varied more than 10-fold among the 13 treatments, the largest value observed where the number of inocula had been largest. Subsequently, the 13 treatments were either or not amended with ZnCl2. Initially, Zn amendment completely inhibited nitrification. After 6 months of Zn exposure, recovery of the potential nitrification activity in the Zn amended soils ranged from 100 % of the potential nitrification activity in the corresponding non-amended soils. This recovery was neither related to DGGE-based indices of AOB diversity nor to the AOB abundance assessed 2 months after inoculation (p > 0.05). However, recovery was significantly related (r = 0.75) to the potential nitrification rate before Zn amendment and only weakly to the number of soil inocula used in the treatments (r = 0.46). The lack of clear effects of AOB diversity on recovery may be related to an inherently sufficient diversity and functional redundancy of AOB communities in soil. Our data indicate that potential microbial activity can be a significant factor in recovery.status: publishe

Effect of the microbial diversity on the adaptation of the soil nitrifying community to zinc

Background and aims After long term exposure the nitrification is not affected by increased trace metal concentrations in contrast to short term exposure. It is shown that this is related to the development of a tolerant community. In addition, we showed that the Zn adaptation rate (6-12 months) increases if the growth rate of the nitrifying community in soil is stimulated. Here, we assess the role of microbial diversity on the adaptation rate and extent. Methods Gamma-irradiated sterilized soil was inoculated (5 wt%) with 13 different soil samples with different amoA DGGE profiles. The soil inocula were, for 5 treatments, selected mixtures of 3-20 soil samples or, for 8 treatments, single soil samples, all non-metal contaminated soils. Soils were amended with 150 mg NH4+ kg-1 to stimulate the growth during the initial incubation period at 25 °C. When the nitrification was restored, soils were either or not spiked with ZnCl2 (1500 mg Zn kg-1) and leached 1 week later. Potential nitrification rate and the Zn tolerance were determined 4 months after spiking. The ammonium oxidizing community was determined by PCR-DGGE of the bacterial amoA gene. Results Two months after inoculation, the nitrification in the inoculated soils had recovered and no detectable nitrification was measured in the sterile control soil. The initial Zn tolerances (Zn EC50 values) prior to exposure to Zn were within a factor 6 among all treatments, illustrating that the initial tolerance is relatively similar among soils compared to differences previously found in tolerant communities. The DGGE analysis revealed a different amoA DGGE profile for the 13 treatments. Currently, soils have received 150 mg NH4+ /kg to stimulate growth and are incubated at 25 °C. Conclusions The experimental set-up was successful to produce one soil with 13 different treatments of soil microbial diversity but similar initial Zn tolerance. The effect of this diversity on adaptation to Zn will be reported at the conference.status: accepte

The Potential Consequences of the Hungarian Red Mud Disaster for Soil

In October 2010 a dam of a waste reservoir of the Hungarian Aluminium Cooperation broke resulting in a red mud (pH=12) spill across the Torna river flooding the cities of Devecser and Kolontar in Hungary. Approximately 800 ha of land have been contaminated with red mud. Red mud was characterized and its toxicity for plants was measured to evaluate the soil contamination risks. Increasing red mud doses were mixed into the soil up to a 16.5% dry weight fraction resulting in a maximal soil pH increase of 1.7 units. Plant toxicity and trace metal availability were determined and a reference experiment was set up with NaOH dose soil to reach the same pH. Trace element concentrations in plant shoots (Cu, Cr, Ni, B and Fe) increased significantly with increasing red mud dose, but were well below phytotoxicity thresholds. Nevertheless, plant growth was significantly decreased at the two highest doses due to increased Na uptake. Plant growth was also impaired in leached soil mixtures and in NaOH amended soils due to Na stress. It is concluded that the salinity of the red mud, not trace metals, is the main ecological concern regarding the Hungarian red mud disaster.status: publishe

Copper toxicity in soils under established vineyards in Europe: a survey

Copper (Cu) containing fungicides have been used for more than one century in Europe on agricultural soils, such as vineyard soils. Total Cu concentrations in such soils can exceed toxicological limits that are commonly derived using artificially spiked soils. This study surveyed Cu toxicity in vineyard soils with reference to soils spiked with CuCl2. Soil was collected in six established European vineyards. At each site, samples representing a Cu concentration gradient were collected. A control (uncontaminated) soil sampled nearby the vineyard was spiked with CuCl2. Toxicity was tested using standard ecotoxicity tests: two plant assays (Lycopersicon esculentum Miller (tomato) and Hordeum vulgare L. (barley) growth), one microbial assay (nitrification) and one invertebrate assay (Enchytraeus albidus reproduction). Maximal total Cu concentrations in the vineyard sites ranged 435-690 mg Cu kg-1, well above the local background (23-105 mg Cu kg-1). Toxicity in spiked soils (50% inhibition) was observed at added soil Cu concentrations from 190 to 1039 mg Cu kg-1 (mean 540 mg Cu kg-1) depending on the assay and the site. In contrast, significant adverse effects were only found for three bioassays in vineyard samples of one site and for two bioassays in another site. Biological responses in these cases were more importantly explained by other soil properties than soil Cu. Overall, no Cu toxicity to plants, microbial processes and invertebrates was observed in vineyard soil samples at Cu concentrations well above European Union limits protecting the soil ecosystem.status: publishe

Substrate Addition Enhances the Adaptation Rate of Nitrifying and Denitrifying Communities in Zinc Contaminated Soils

Previous studies showed that nitrifying and denitrifying soil microbial communities are able to adapt to increased Zn concentrations. A striking difference in adaptation rate was observed between denitrification (within 3 months after contamination) and nitrification (not within 6 months). It is hypothesized that this difference is related to the growth rate of the microbial communities. Experiments were conducted to determine the adaptation rate of both processes in artificially zinc contaminated soils as affected by growth stimulating substrate additions (NH4+ and organic matter). Without substrate additions, no adaptation of nitrification was observed in 12 months whereas denitrification activity restored during that period. Substrate addition enhanced the adaptation rate of both processes within 6 months of incubation.status: publishe