Plastic debris in lakes and reservoirs.

Plastic debris is thought to be widespread in freshwater ecosystems globally1. However, a lack of comprehensive and comparable data makes rigorous assessment of its distribution challenging2,3. Here we present a standardized cross-national survey that assesses the abundance and type of plastic debris (>250 µm) in freshwater ecosystems. We sample surface waters of 38 lakes and reservoirs, distributed across gradients of geographical position and limnological attributes, with the aim to identify factors associated with an increased observation of plastics. We find plastic debris in all studied lakes and reservoirs, suggesting that these ecosystems play a key role in the plastic-pollution cycle. Our results indicate that two types of lakes are particularly vulnerable to plastic contamination: lakes and reservoirs in densely populated and urbanized areas and large lakes and reservoirs with elevated deposition areas, long water-retention times and high levels of anthropogenic influence. Plastic concentrations vary widely among lakes; in the most polluted, concentrations reach or even exceed those reported in the subtropical oceanic gyres, marine areas collecting large amounts of debris4. Our findings highlight the importance of including lakes and reservoirs when addressing plastic pollution, in the context of pollution management and for the continued provision of lake ecosystem services.

Engineering cytoplasmic acetyl-CoA synthesis decouples lipid production from nitrogen starvation in the oleaginous yeast Rhodosporidium azoricum.

BACKGROUND: Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. RESULTS: In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. CONCLUSION: Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates.

Cloning the putative gene of vinyl phenol reductase of Dekkera bruxellensis in Saccharomyces cerevisiae.

Vinylphenol reductase of Dekkera bruxellensis, the characteristic enzyme liable for "Brett" sensory modification of wine, has been recently recognized to belong to the short chain dehydrogenases/reductases family. Indeed, a preliminary biochemical characterisation has conferred to the purified protein a dual significance acting as superoxide dismutase and as a NADH-dependent reductase. The present study aimed for providing a certain identification of the enzyme by cloning the VPR gene in S. cerevisiae, a species not producing ethyl phenols. Transformed clones of S. cerevisiae resulted capable of expressing a biologically active form of the heterologous protein, proving its role in the conversion of 4-vinyl guaiacol to 4-ethyl guaiacol. A VPR specific protein activity of 9 ± 0.6 mU/mg was found in crude extracts of S. cerevisiae recombinant strain. This result was confirmed in activity trials carried out with the protein purified from transformant cells of S. cerevisiae by a his-tag purification approach; in particular, VPR-enriched fractions showed a specific activity of 1.83 ± 0.03 U/mg at pH 6.0. Furthermore, in agreement with literature, the purified protein behaves like a SOD, with a calculated specific activity of approximatively 3.41 U/mg. The comparative genetic analysis of the partial VPR gene sequences from 17 different D. bruxellesis strains suggested that the observed polymorphism (2.3%) and the allelic heterozygosity state of the gene do not justify the well described strain-dependent character in producing volatile phenols of this species. Actually, no correlation exists between genotype membership of the analysed strains and their capability to release off-flavours. This work adds valuable knowledge to the study of D. bruxellensis wine spoilage and prepare the ground for interesting future industrial applications.

Galactose utilization sheds new light on sugar metabolism in the sequenced strain Dekkera bruxellensis CBS 2499.

Dekkera bruxellensis and Saccharomyces cerevisiae are considered two phylogenetically distant relatives, but they share several industrial relevant traits such as the ability to produce ethanol under aerobic conditions (Crabtree effect), high tolerance towards ethanol and acids, and ability to grow without oxygen. Beside a huge adaptability, D. bruxellensis exhibits a broader spectrum in utilization of carbon and nitrogen sources in comparison to S. cerevisiae. With the aim to better characterize its carbon source metabolism and regulation, the usage of galactose and the role that glucose plays on sugar metabolism were investigated in D. bruxellensis CBS 2499. The results indicate that in this yeast galactose is a non-fermentable carbon source, in contrast to S. cerevisiae that can ferment it. In particular, its metabolism is affected by the nitrogen source. Interestingly, D. bruxellensis CBS 2499 exhibits the 'short-term Crabtree effect', and the expression of genes involved in galactose utilization and in respiratory metabolism is repressed by glucose, similarly to what occurs in S. cerevisiae.

Cold exposure affects carbohydrates and lipid metabolism, and induces Hog1p phosphorylation in Dekkera bruxellensis strain CBS 2499.

Dekkera bruxellensis is a yeast known to affect the quality of wine and beer. This species, due to its high ethanol and acid tolerance, has been reported also to compete with Saccharomyces cerevisiae in distilleries producing fuel ethanol. In order to understand how this species responds when exposed to low temperatures, some mechanisms like synthesis and accumulation of intracellular metabolites, changes in lipid composition and activation of the HOG-MAPK pathway were investigated in the genome sequenced strain CBS 2499. We show that cold stress caused intracellular accumulation of glycogen, but did not induce accumulation of trehalose and glycerol. The cellular fatty acid composition changed after the temperature downshift, and a significant increase of palmitoleic acid was observed. RT-PCR analysis revealed that OLE1 encoding for Δ9-fatty acid desaturase was up-regulated, whereas TPS1 and INO1 didn't show changes in their expression. In D. bruxellensis Hog1p was activated by phosphorylation, as described in S. cerevisiae, highlighting a conserved role of the HOG-MAP kinase signaling pathway in cold stress response.

Candida milleri species reveals intraspecific genetic and metabolic polymorphisms.

Candida milleri, together with Candida humilis, is the most representative yeast species found in type I sourdough ecosystems. In this work, comparison of the ITS region and the D1/D2 domain of 26S rDNA gene partial sequences, karyotyping, mtDNA-RFLP analysis, Intron Splice Site dispersion (ISS-PCR) and (GTG)5 microsatellite analyses, assimilation test of different carbohydrates, and metabolome assessment by FT-IR analysis, were investigated in seventeen strains isolated from four different companies as well as in type strains CBS6897(T) and CBS5658(T). Most isolates were ascribed to C. milleri, even if a strong relatedness was confirmed with C. humilis as well, particularly for three strains. Genetic characterization showed a high degree of intraspecific polymorphism since 12 different genotypes were discriminated. The number of chromosomes varied from 9 to 13 and their size ranged from less than 0.3 to over 2 Mbp. Phenotypic traits let to recognize 9 different profiles of carbon sources assimilation. FT-IR spectra from yeast cells cultivated in different media and collected at different growth phases revealed a diversity of behaviour among strains in accordance with the results of PCR-based fingerprinting. A clear evidence of the polymorphic status of C. milleri species is provided thus representing an important feature for the development of technological applications in bakery industries.

Microplastics removal in wastewater treatment plants: A review of the different approaches to limit their release in the environment.

In last 10 years, the interest about the presence of microplastics (MPs) in the environment has strongly grown. Wastewaters function as a carrier for MPs contamination from source to the aquatic environment, so the knowledge of the fate of this emerging contaminant in wastewater treatment plants (WWTPs) is a priority. This work aims to review the presence of MPs in the influent wastewater (WW) and the effectiveness of the treatments of conventional WWTPs. Moreover, the negative impacts of MPs on the management of the processes have been also discussed. The work also focuses on the possible approaches to tackle MPs contamination enhancing the effectiveness of the WWTPs. Based on literature results, despite WWTPs are not designed for MPs removal from WW, they can effectively remove the MPs (up to 99 % in some references). Nevertheless, they normally act as "hotspots" of MPs contamination considering the remaining concentration of MPs in WWTPs' effluents can be several orders of magnitude higher than receiving waters. Moreover, MPs removed from WW are concentrated in sewage sludge (potentially >65 % of MPs entering the WWTP) posing a concern in case of the potential reuse as a soil improver. This work aims to present a paradigm shift intending WWTPs as key barriers for environmental protection. Approaches for increasing effectiveness against MPs have been discussed in order to define the optimal point(s) of the WWTP in which these technologies should be located. The need of a future legislation about MPs in water and sludge is discussed.

Traditional and biodegradable plastics host distinct and potentially more hazardous microbes when compared to both natural materials and planktonic community.

Microplastic particles are persistent micropollutants that provide a substrate for the growth of bacterial biofilms, posing a threat to the environment. This study explores the changes in commercially available food containers made of conventional (polypropylene PP, polyethylene terephthalate PET), innovative biodegradable (Mater-Bi) and natural (wood and cellulose) materials, when introduced in the surface waters of Lake Maggiore for 43 days. Spectral changes revealed by FT-IR spectroscopy in PET and Mater-Bi, and changes in thermal properties of all human-made material tested indicated a degradation process occurred during environmental exposure. Despite similar bacterial richness, biofilms on PET, PP, and Mater-Bi differed from natural material biofilms and the planktonic community. Human-made material communities showed a higher proportion of potential pathogens, with PET and PP also exhibiting increased abundances of antibiotic resistance genes. Overall, these findings stress the need for dedicated strategies to curb the spread of human-made polymers in freshwaters, including innovative materials that, due to their biodegradable properties, might be perceived less hazardous for the environment.

Monthly variability of floating plastic contamination in Lake Maggiore (Northern Italy).

The monitoring of plastics in freshwater ecosystems has witnessed a significant increase in recent years, driven by the awareness that approximately 80 % of marine plastic litter originates from terrestrial sources transported to the seas through lakes and rivers. Consequently, it is imperative to develop monitoring plans that offer a comprehensive understanding of plastic contamination in these aquatic environments, given their seasonal variations in hydrochemical characteristics and anthropogenic sources. Historically, most global lake monitoring campaigns have been limited to one-time or, at most, seasonal sampling. In this context, the primary objective of the present study was to assess the quantitative and qualitative monthly variations of floating plastics in Lake Maggiore, a large European lake with high ecological and economic significance. Twelve transverse transects were conducted from January to December 2022 using a Manta-net with a 100 µm mesh. Characterization of each plastic particle was performed using a µ-Fourier Transform Infrared Spectroscope (µFT-IR). The results revealed relatively low levels of contamination in Lake Maggiore when compared with other lakes worldwide exclusively from a secondary origin. However, a considerable heterogeneity was observed, both quantitatively and qualitatively. Notably, we identified a 13-fold difference between the minimum (0.02 plastics/m3 in September) and maximum (0.29 plastics/m3 in December) concentrations of plastics, accompanied by significant variations in polymer composition. Our monitoring underscored the necessity of also considering the temporal variation as a potential factor influencing plastic contamination in a lake. Moreover, frequent sampling emerged as a crucial requirement to accurately gauge the extent of plastic pollution, yielding robust and valuable data essential for effective environmental management.

Freshwater plastisphere: a review on biodiversity, risks, and biodegradation potential with implications for the aquatic ecosystem health.

The plastisphere, a unique microbial biofilm community colonizing plastic debris and microplastics (MPs) in aquatic environments, has attracted increasing attention owing to its ecological and public health implications. This review consolidates current state of knowledge on freshwater plastisphere, focussing on its biodiversity, community assembly, and interactions with environmental factors. Current biomolecular approaches revealed a variety of prokaryotic and eukaryotic taxa associated with plastic surfaces. Despite their ecological importance, the presence of potentially pathogenic bacteria and mobile genetic elements (i.e., antibiotic resistance genes) raises concerns for ecosystem and human health. However, the extent of these risks and their implications remain unclear. Advanced sequencing technologies are promising for elucidating the functions of plastisphere, particularly in plastic biodegradation processes. Overall, this review emphasizes the need for comprehensive studies to understand plastisphere dynamics in freshwater and to support effective management strategies to mitigate the impact of plastic pollution on freshwater resources.

Utilization of nitrate abolishes the "Custers effect" in Dekkera bruxellensis and determines a different pattern of fermentation products.

Nitrate is one of the most abundant nitrogen sources in nature. Several yeast species have been shown to be able to assimilate nitrate and nitrite, but the metabolic pathway has been studied in very few of them. Dekkera bruxellensis can use nitrate as sole nitrogen source and this metabolic characteristic can render D. bruxellensis able to overcome S. cerevisiae populations in industrial bioethanol fermentations. In order to better characterize how nitrate utilization affects carbon metabolism and the yields of the fermentation products, we investigated this trait in defined media under well-controlled aerobic and anaerobic conditions. Our experiments showed that in D. bruxellensis, utilization of nitrate determines a different pattern of fermentation products. Acetic acid, instead of ethanol, became in fact the main product of glucose metabolism under aerobic conditions. We have also demonstrated that under anaerobic conditions, nitrate assimilation abolishes the "Custers effect", in this way improving its fermentative metabolism. This can offer a new strategy, besides aeration, to sustain growth and ethanol production for the employment of this yeast in industrial processes.

Osmotic stress response in the wine yeast Dekkera bruxellensis.

Dekkera bruxellensis is mainly associated with lambic beer fermentation and wine production and may contribute in a positive or negative manner to the flavor development. This yeast is able to produce phenolic compounds, such as 4-ethylguaiacol and 4-ethylphenol which could spoil the wine, depending on their concentration. In this work we have investigated how this yeast responds when exposed to conditions causing osmotic stress, as high sorbitol or salt concentrations. We observed that osmotic stress determined the production and accumulation of intracellular glycerol, and the expression of NADH-dependent glycerol-3-phosphate dehydrogenase (GPD) activity was elevated. The involvement of the HOG MAPK pathway in response to this stress condition was also investigated. We show that in D. bruxellensis Hog1 protein is activated by phosphorylation under hyperosmotic conditions, highlighting the conserved role of HOG MAP kinase signaling pathway in the osmotic stress response. Gene Accession numbers in GenBank: DbHOG1: JX65361, DbSTL1: JX965362.

Increased similarity of aquatic bacterial communities of different origin after antibiotic disturbance.

Stochastic or deterministic processes control the bacterial community assembly in waters and their understanding is a fundamental question to correctly manage aquatic environments exposed to the release of antibiotics from anthropogenic sources. It has been suggested that microdiversity (i.e. the rare biosphere) convers freshwater communities with stability, meaning that previously rare taxa bloom when the community is disturbed. Since there might be a seed bank of similar, but not abundant, bacterial taxa in different waters, we tested whether a disturbance by an antibiotic cocktail would increase similarity in bacterial communities from different freshwater systems (a wastewater effluent and two lakes). In a continuous culture set-up in chemostats, we show that disturbance with antibiotics causes communities from different environments to become more similar. Once the antibiotic pressure is released the communities tend to become more dissimilar again. This shows that there is a similar shift in community composition even in waters from very different origins when they are disturbed by antibiotics, even at low concentrations. Antibiotics impact the bacterial communities at the cell and the community level, independently by the original degree of anthropogenic stress they are adapted to, altering the original phenotypes, genotypes, and the relations between bacteria.

Size-age population structure of an endangered and anthropogenically introgressed northern Adriatic population of marble trout (Salmo marmoratus Cuv.): insights for its conservation and sustainable exploitation.

Salmonid species are main actors in the Italian socio-ecological landscape of inland fisheries. We present novel data on the size-age structure of one of the remnant Italian populations of the critically endangered marble trout Salmo marmoratus, which co-occurs with other stocked non-native salmonids in a large glacial river of the Lake Maggiore basin (Northern Italy-Southern Switzerland). Like other Italian native trout populations, the Toce River marble trout population is affected by anthropogenic introgression with the non-native brown trout S. trutta. Our sample includes 579 individuals, mainly collected in the Toce River main channel. We estimated the length-weight relationship, described the population size-age structure, estimated the age-specific growth trajectories, and fit an exponential mortality model. A subset of the sample was also used to measure numerical and biomass density. The estimated asymptotic maximum length is ~105 cm total length (TL). Mean length at first maturity is ~55 cm TL, and mean length at maximum yield per recruit is ~68 cm TL. Approximately 45-70% of the population are estimated to die annually, along with a fishing annual mortality of ~37%, with an exploitation ratio of ~0.5. The frequency distribution of length classes in a sample collected by angling shows that ~80% of the individuals that could be retained according to the current recreational fishing regulations likely never reproduced, and large fish disproportionally contributing to recruitment are fished and retained. We identify possible overfishing risks posed by present regulations, and propose updated harvest-slot length limits to mitigate such risks. More detailed and long-term datasets on this system are needed to more specifically inform the fishery management and monitor the effects of any change in the management strategy on the size-age structure of the marble trout population of the Toce River.

Class 1 integron and related antimicrobial resistance gene dynamics along a complex freshwater system affected by different anthropogenic pressures.

The risk for human health posed by polluted aquatic environments, and especially those carrying antibiotic resistance genes (ARGs) of clinical interest, is still debated. This is because of our limited knowledge of the dynamics of antimicrobial resistance in the environment, the selection mechanisms underlying the spread of ARGs, and the ecological factors potentially favoring their return to humans. The Class 1 integron is one of the most effective platforms for the dissemination of ARGs. In this study we investigated a freshwater system consisting of a lake-river-lake continuum, determining the abundance of class 1 integrons and their associated ARGs by a modulated metagenomic approach. Bacterial abundance and community composition were used to identify the potential carriers of class 1 integrons and their associated ARGs over a period of six months. Class 1 integrons and their ARG cargoes were significantly more abundant in riverine sampling sites receiving treated wastewater. Further, class 1 integrons carried ARGs ranked at the highest risk for human health (e.g., catB genes), in particular, genes encoding resistance to aminoglycosides. Genera of potential pathogens, such as Pseudomonas and Escherichia-Shigella, were correlated with class 1 integrons. The lake-river-lake system demonstrated a clear relationship between the integrase gene of class 1 integrons (intI1) and anthropogenic impact, but also a strong environmental filtering that favored the elimination of intI1 once the human derived stressors were reduced. Overall, the results of this study underline the role class 1 integrons as proxy of anthropogenic pollution and suggest this genetic platform as an important driver of aminoglycoside resistance genes, including high risk ARGs, of potential concern for human health.

Microplastics pollution in the terrestrial environments: Poorly known diffuse sources and implications for plants.

Research on microplastics (MPs) in the terrestrial environment is currently at a still embryonal stage. The current knowledge concerning poorly known diffuse sources of MPs pollution in terrestrial ecosystems have been considered in this work. In addition, a particular focus on the presence, mechanism of absorption and effects of MPs in plants has also been provided. Research concerning microplastics in urban areas and their intake by Tyre and Road Wear Particulates (TWRP) demonstrated a high contribution of this plastic debris to microplastic pollution, although quantification of these inputs is challenging to assess because studies are still very few. Around 50% of particles are expected to remain in the roadside soil, while the rest is transported away by the runoff with high concentrations of TRWP with a size ranging between 0.02 and 0.1 mm. Natural and anthropic environments like temporary ponds, stormwater retention ponds and small waterbodies were considered sensitive connecting ecosystems rich in biodiversity between terrestrial and aquatic environments. Even if studies are not yet exhaustive and just eight studies were currently published concerning these ecosystems, considerable values of MPs were already observed both in the sediment and water phase of ponds. Although still poorly explored, agricultural environments were already demonstrated to hide a significant number of microplastics linked mainly to agricultural activities and practices (e.g. mulch, sewage and compost fertilisation). However, the microplastics transportation processes into the soil are still understudied, and a few works are available. Microplastics and primarily nanoplastics presence was also observed in common edible plants (fruit and vegetables) with alarming Estimated Daily Intakes ranging from 2.96 × 1004 to 4.62 × 1005 (p kg-1 day-1) for adults depending on species. In addition, adverse effects on plants growth, photosynthetic activity, antioxidant system and nutritional values of several common fruits and vegetables were also demonstrated by several studies.

Microplastic retention in small and medium municipal wastewater treatment plants and the role of the disinfection.

Wastewater treatment plants (WWTPs) efficiently retain microplastic particles (MPs) generated within urban areas. Among the wastewater treatment steps, disinfection has not been characterized for its potential MPs retention activity, although it has been reported that processes used to abate the bacterial load could also affect MPs concentration. For this reason, we evaluated the MPs concentration across the overall wastewater treatment process and before and after the disinfection step in four small/medium WWTPs located in the north of Italy. Most of the MPs found in the samples were fibers or fragments, smaller than 500 µm, mainly composed of polyethylene, polypropylene, or polyethylene terephthalate. The retention efficiency at the outlets was higher than 94% in all the plants analyzed. More interestingly, the disinfection treatments adopted by the different WWTPs reduced MPs concentration from a minimum of 9.1% (UV treatment) to a maximum of 67.6% (chlorination), promoting a further increase of the overall retention efficiency of the WWTPs from 0.4 to 0.7%. Quantitatively, the disinfection contributes to the MPs reduction in the outlets by retaining 0.5-6.7 million MPs per day, in WWTPs that discharge 2.7-12 million MPs per day. The results of the present work underline the importance of a careful choice of the steps that constitute the wastewater treatment, including disinfection, in order to minimize MPs discharge into the natural ecosystems.

Candida albicans--a pre-whole genome duplication yeast--is predominantly aerobic and a poor ethanol producer.

Yeast species belonging to the lineage that underwent the whole genome duplication (WGD), and including Saccharomyces cerevisiae, can grow under anaerobiosis and accumulate ethanol in the presence of glucose and oxygen. The pre-WGD yeasts, which branched from the S. cerevisiae lineage just before the WGD event, including Kluyveromyces lactis, are more dependent on oxygen and do not accumulate large amounts of ethanol in the presence of excess oxygen. Yeasts that belong to the so-called 'lower branches' of the yeast phylogenetic tree and diverged from S. cerevisiae more than 200 million years ago have so far not been thoroughly investigated for their physiology and carbon metabolism. Here, we have studied several isolates of Candida albicans and Debaryomyces hansenii for their dependence on oxygen. Candida albicans grew very poorly at an oxygen concentration <1 p.p.m. and D. hansenii could not grow at all. In aerobic batch cultivations, C. albicans exhibited a predominantly aerobic metabolism, accumulating only small amounts of ethanol (0.01-0.09 g g(-1) glucose). Apparently, C. albicans and several other pre-WGD yeasts still exhibit the original traits of the yeast progenitor: poor accumulation of ethanol under aerobic conditions and strong dependence on the presence of oxygen.

Generation of an evolved Saccharomyces cerevisiae strain with a high freeze tolerance and an improved ability to grow on glycerol.

Glycerol is a residue generated during biodiesel production and represents around 10% of the total product output. Biodiesel production is currently having a significant impact on glycerol price, leading to an increased interest in the use of glycerol as a cheap substrate for fermentation processes. We have analysed the growth kinetics of two wild-type strains of Saccharomyces cerevisiae grown on synthetic media containing glycerol as the sole carbon and energy source. Both strains were initially unable to grow when cultivated under these conditions, and an unusually long lag phase was necessary prior to the appearance of slow-growing cells. Following the application of an "evolutionary engineering" approach, we obtained S. cerevisiae strains with an improved ability to grow on glycerol. We report here the isolation of an evolved strain that exhibits a reduction of the lag phase, a threefold increase of the specific growth rate and a higher glycerol consumption rate compared to wild-type strains. The evolved strain has retained its fermentative activity, producing ethanol at the same rate and yield as the wild type. Interestingly, the yeast biomass obtained by cultivating the evolved strain on synthetic glycerol-based media also showed a high viability after prolonged storage at -20°C. The strategy adopted in our study could be easily applied to obtain S. cerevisiae strains with new industrially relevant traits, such as an improved ability to use cheap substrates and high resistance to freeze and thaw procedures.

Dekkera/Brettanomyces yeasts for ethanol production from renewable sources under oxygen-limited and low-pH conditions.

Industrial fermentation of lignocellulosic hydrolysates to ethanol requires microorganisms able to utilise a broad range of carbon sources and generate ethanol at high yield and productivity. D. bruxellensis has recently been reported to contaminate commercial ethanol processes, where it competes with Saccharomyces cerevisiae [4, 26]. In this work Brettanomyces/Dekkera yeasts were studied to explore their potential to produce ethanol from renewable sources under conditions suitable for industrial processes, such as oxygen-limited and low-pH conditions. Over 50 strains were analysed for their ability to utilise a variety of carbon sources, and some strains grew on cellobiose and pentoses. Two strains of D. bruxellensis were able to produce ethanol at high yield (0.44 g g(-1) glucose), comparable to those reported for S. cerevisiae. B. naardenensis was shown to be able to produce ethanol from xylose. To obtain ethanol from synthetic lignocellulosic hydrolysates we developed a two-step fermentation strategy: the first step under aerobic conditions for fast production of biomass from mixtures of hexoses and pentoses, followed by a second step under oxygen limitation to promote ethanol production. Under these conditions we obtained biomass and ethanol production on synthetic lignocellulosic hydrolysates, with ethanol yields ranging from 0.2 to 0.3 g g(-1) sugar. Hexoses, xylose and arabinose were consumed at the end of the process, resulting in 13 g l(-1) of ethanol, even in the presence of furfural. Our studies showed that Brettanomyces/Dekkera yeasts have clear potential for further development for industrial processes aimed at production of ethanol from renewable sources.