Article

Morphological and ecological similarities: Wood-boring beetles associated with novel xylose-fermenting yeasts, Spathaspora passalidarum gen. sp. nov. and Candida jeffriesii sp. nov

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Abstract

Ascomycete yeasts that both ferment and assimilate xylose were reported previously as associates of insects living in woody substrates. Most notable have been reports of Pichia stipitis-like yeasts that are widely associated with the wood-boring beetle, Odontotaenius disjunctus (Coleoptera: Passalidae), in the eastern United States. Our continuing investigation of insect gut yeasts has lead to the discovery of two new xylose-fermenting yeasts that phylogenetic analysis places as sister taxa. The beetle hosts, O. disjunctus and Phrenapates bennetti (Coleoptera: Tenebrionidae), are similar in habitat and appearance, and the presence of similar gut yeasts is an additional common feature between them. Here we describe the new yeast genus Spathaspora, the type species S. passalidarum, and its sister taxon Candida jeffriesii and discuss their natural history, including a comparison with Pichia stipitis, another member of a guild of xylose-fermenting yeasts with similar metabolic traits. In addition a morphologically distinct yeast ascospore type is described for Spathaspora.

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... S. passalidarum is a fungi of the phylum Ascomycota, first found on the guts of woodboring beetles in the state of Louisiana (USA) by Nguyen et al. (2006). Due to earlier findings and literature reports on xylose-fermenter yeast associations with woodboring beetles, tests have been run to prove the hypothesis that new yeasts with the ability to ferment pentoses would be found in association with those insects. ...
... Since isolation of novel xylose-fermenting yeasts S. passalidarum (Nguyen et al. 2006), most researches have been focusing on understanding and enhancing production of ethanol from different raw materials, mostly from lignocellulosic biomasses, due to the capacity of this microorganism to ferment a wide range of sugars present on lignocellulosic materials (Nguyen et al. 2006) and the commercial interest of ethanol as a more sustainable biofuel (Garcia-Ochoa and Gomez 2009). Many approaches have been adopted throughout the years to improve ethanol production. ...
... Since isolation of novel xylose-fermenting yeasts S. passalidarum (Nguyen et al. 2006), most researches have been focusing on understanding and enhancing production of ethanol from different raw materials, mostly from lignocellulosic biomasses, due to the capacity of this microorganism to ferment a wide range of sugars present on lignocellulosic materials (Nguyen et al. 2006) and the commercial interest of ethanol as a more sustainable biofuel (Garcia-Ochoa and Gomez 2009). Many approaches have been adopted throughout the years to improve ethanol production. ...
Chapter
Lignocellulosic biomass comprehends the most abundant and renewable material in the world, being its efficient fractionation crucial to develop economically viable biorefineries. Chemical, physical, physical-chemical, biological, or enzymatic conversion can be used as strategies to produce important bioproducts as carbohydrates, bioactive compounds, and lignin derivatives. Carbohydrates as xylose and glucose can be used for food, chemical blocks, materials, and biofuels production by microorganisms like the yeast Spathaspora passalidarum for the production of xylitol, ethanol, acetoin, and 2,3-butanediol. Besides that, the lignocellulosic biomass is an important substrate for the production of several enzymes such as glycohydrolases (cellulases and hemicellulases) and oxidoreductases (laccase, peroxidases, and polysaccharide monooxygenases). Hemicellulases are necessary enzymes to achieve the required degree of polymerization of xylooligosaccharides, a new class of prebiotics extracted from the hemicelluloses fraction. Chemicals derived from lignin have found applications in various industries including nanoparticles, composites, antioxidants, polymer, among others. The focus of this chapter is to review the state of the art with regard of the characterization and valorization of lignocellulosic feedstock, as well as the process involving in the biomass fractionation, bioproducts recovery, and production.
... Second, more and more nonconventional yeast species naturally capable of metabolizing xylose were exploited (Radecka et al. 2015), but most of them cannot consume xylose until glucose depletion and form byproducts, resulting in low ethanol yields. Interestingly, Spathaspora passalidarum, a wood-boring beetle-associated yeast, was discovered to be capable of simultaneously assimilates glucose and xylose with a high ethanol yield (Nguyen et al. 2006;Long et al. 2012), indicating its great potential for application in industrial bioethanol production from lignocellulosic biomass. Third, fermentation performance and ethanol tolerance of S. cerevisiae as well as xylose utilization capacity of non-conventional yeast, such as Pichia stipitis (Scheffersomyces stipitis), were combined by using intergeneric genome shuffling, endowing the hybrids with desirable traits for bioethanol production (Zhang and Geng 2012). ...
... All the yeast stains used in this study are listed in Table S1, Supporting Information. S. passalidarum NRRL Y-27907 obtained from the culture collection of the Agricultural Research Service (ARS) is a natural xylose-fermenting yeast (Nguyen et al. 2006). Saccharomyces cerevisiae ScY01 is an evolved thermotolerant strain derived from an industrial diploid strain ScY in our lab (Shui et al. 2015), which can grow and ferment well at 40 • C. Yeast cells were propagated in YP medium (per liter, 10 g yeast extract, 20 g peptone, pH 5.5) containing 20 g L −1 glucose at 30 • C. Pure yeast cultures were stored at −80 • C with addition of 20% glycerol (v/v). ...
... Aiming to obtain strains that can efficiently co-ferment glucose and xylose to ethanol at high temperature, our strategy was to take both the advantages of a non-conventional yeast strain with a superior xylose fermentation capacity and a widely used industrial S. cerevisiae strain in bioethanol production with a desirable high-temperature glucose fermentation performance by using intergeneric genome shuffling, followed by adaptive evolution. To be specific, the non-conventional yeast S. passalidarum is a newly identified species capable of producing ethanol more efficiently from xylose (Nguyen et al. 2006;Hou 2012;Long et al. 2012;Su, Willis and Jeffries 2014). Notably, its xylose metabolism related genes were also recently explored to enhance xylose fermentation of engineered S. cerevisiae strains (Wohlbach et al. 2011;Cadete et al. 2016). ...
Article
Enhanced capability of co-fermenting glucose and xylose at high temperature is highly desirable for yeast application in second-generation bioethanol production. Here, we obtained hybrid strains with improved glucose-xylose co-fermentation properties at high temperature by combining genome shuffling and adaptive evolution. Genome resequencing of these strains suggested predominantly inherited genetic information from one parental strain Spathaspora passalidarum SP rather than the other parental strain Saccharomyces cerevisiae ScY01, possibly due to that the CUG codon system of S. passalidarum might have systematically eliminated most of the functional proteins from S. cerevisiae through misfolding. Compared to SP, one-copy loss of a 146-kb fragment was found in the hybrid strain and regained after being evolved for a while, whereas one-copy loss of an 11-kb fragment was only found after being evolved for a longer time. Besides, the genes affected by nonsynonymous variants were also identified, especially the mutation S540F in the endoplasmic reticulum chaperon Kar2. Structural prediction indicated that S540F might change the substrate binding activity of Kar2, and thus play a role in preventing protein aggregation in yeast at high temperature. Our results illustrated genomic alterations during this process and revealed some genomic factors that might be involved to determine yeast thermotolerance.
... S. passalidarum was isolated from woodboring beetles' guts in 2006 and can naturally consume and ferment xylose [16]. The genera names come from the Greek words spathe and spora, which means broadsword and seed, respectively. ...
... The genera names come from the Greek words spathe and spora, which means broadsword and seed, respectively. S. passalidarum reproduces asexually by budding; its ascospores are elongated; its vegetative cells are mostly globose and tapering at the ends with a membrane running along the long axis of the spore [16]. It has been demonstrated its capability to co-ferment glucose, xylose, and cellobiose with similar consumption rates with increased ethanol production using xylose as a sole carbon source [15,17]. ...
... In the past years, many species screened in samples from forest [32], wood-feeding termite [33], and woody-boring beetles [16] were reported as xylose consumers. That includes E s c h e r i c h i a c o l i, y e as t a n d fi l a m e nt ou s f u n gi , Caldicellulosiruptor, Clostridia, Proteobacteria, and the domain Archaea [30]. ...
Article
Full-text available
Bioethanol from lignocellulosic biomass (namely second-generation bioethanol) has been considered to be a great opportunity to increase production of biofuels from renewable sources without expanding the world planted area. Spathaspora passalidarum has shown great potential to convert sugars derived from lignocellulosic materials, which comprises hexoses and pentoses, into bioethanol. Besides its unusual ability to produce ethanol naturally from pentoses, this microorganism can also be used to obtain other products of interest. This review summarizes and discusses fermentative and kinetic parameters from the state of the art of ethanol production by S. passalidarum in synthetic medium and hemicellulosic hydrolysates. Additionally, S. passalidarum performance for xylose consumption and ethanol production are discussed in comparison with other wild yeasts and genetically modified Saccharomyces cerevisiae. In addition to ethanol, the production of other chemical blocks such as xylitol, glycerol, acetoin, and 2,3-butanediol are also discussed, elucidating the potential of S. passalidarum for application in biorefineries. The potential of S. passalidarum for studies of mutagenesis, evolutionary, and genetic modifications is also reviewed in this work.
... Recently, genome sequencing of several yeasts has been reported, including for Spathaspora passalidarum (Wohlbach et al. 2011), Scheffersomyces stipitis (Jeffries et al. 2007), Candida tenuis (Wohlbach et al. 2011) and M. guilliermondii (Butler et al. 2009). The genus Spathaspora has been described recently (Nguyen et al. 2006), and only a few species have been identified of which only a few genomes are available (Lobo et al. 2014;Lopes et al. 2017). Moreover, among these, only S. passalidarum presents a high quality genome annotation (Wohlbach et al. 2011). ...
... A phylogenetic tree based on concatenated alignment of ITS and D1/D2 sequences showed the placement of the new isolates into the independent clades Spathaspora and Meyerozyma (Fig. 1). The Spathaspora genus was previously described by Nguyen et al. (2006) and contains D-xylose-fermenting yeasts generally associated with rotting wood substrates and insects (Nguyen et al. 2006;Cadete et al., 2009Cadete et al., , 2012Cadete et al., , 2013Cadete et al., , 2016aCadete and Rosa 2017). The Meyerozyma genus was proposed by Kurtzman and Suzuki (2010) to accommodate yeasts such as M. guilliermondii and M. caribbica, species that are widely dispersed in natural environments (Vaughan- Martini et al. 2005;Kurtzman and Suzuki 2010;Romi et al. 2014;Corte et al. 2015). ...
... A phylogenetic tree based on concatenated alignment of ITS and D1/D2 sequences showed the placement of the new isolates into the independent clades Spathaspora and Meyerozyma (Fig. 1). The Spathaspora genus was previously described by Nguyen et al. (2006) and contains D-xylose-fermenting yeasts generally associated with rotting wood substrates and insects (Nguyen et al. 2006;Cadete et al., 2009Cadete et al., , 2012Cadete et al., , 2013Cadete et al., , 2016aCadete and Rosa 2017). The Meyerozyma genus was proposed by Kurtzman and Suzuki (2010) to accommodate yeasts such as M. guilliermondii and M. caribbica, species that are widely dispersed in natural environments (Vaughan- Martini et al. 2005;Kurtzman and Suzuki 2010;Romi et al. 2014;Corte et al. 2015). ...
Article
Xylitol is a five-carbon polyol of economic interest that can be produced by microbial xylose reduction from renewable resources. The current study sought to investigate the potential of two yeast strains, isolated from Brazilian Cerrado biome, in the production of xylitol as well as the genomic characteristics that may impact this process. Xylose conversion capacity by the new isolates Spathaspora sp. JA1 and Meyerozyma caribbica JA9 was evaluated and compared with control strains on xylose and sugarcane biomass hydrolysate. Among the evaluated strains, Spathaspora sp. JA1 was the strongest xylitol producer, reaching product yield and productivity as high as 0.74 g/g and 0.20 g/(L.h) on xylose, and 0.58 g/g and 0.44 g/(L.h) on non-detoxified hydrolysate. Genome sequences of Spathaspora sp. JA1 and M. caribbica JA9 were obtained and annotated. Comparative genomic analysis revealed that the predicted xylose metabolic pathway is conserved among the xylitol-producing yeasts Spathaspora sp. JA1, M. caribbica JA9 and Meyerozyma guilliermondii, but not in Spathaspora passalidarum, an efficient ethanol-producing yeast. Xylitol-producing yeasts showed strictly NADPH-dependent xylose reductase and NAD+-dependent xylitol-dehydrogenase activities. This imbalance of cofactors favors the high xylitol yield shown by Spathaspora sp. JA1, which is similar to the most efficient xylitol producers described so far.
... For instance, Scheffersomyces stipitis and Spathaspora passalidarum are capable of fermenting xylose to ethanol with high yields and productivity (Veras et al. 2017), whereas Yarrowia lipolytica showed a respiratory metabolism, and it is capable of synthesizing and accumulate high levels of intracellular lipids (Beopoulos et al. 2009). In addition to the low number of xylose-consuming yeast strains reported among almost 1000 yeast taxa, there is still few information about physiology and genetics of many species (Nguyen et al. 2006;Kurtzman 2011b). ...
... The genus Spathaspora was described by Nguyen et al. (2006) to accommodate the teleomorphic species Spathaspora passalidarum, which form specific allantoid asci, with a single, elongated, and curve-ended ascospore, very distinct from any other known yeast. The first species was isolated from the gut of the wood-boring beetle Odontotaenius disjunctus (Coleoptera: Passalidae), collected in Louisiana (USA). ...
... The vegetative cells are predominantly globose, formed by budding, and septate hyphae and pseudohyphae are present. In sexual reproduction, an allantoid ascus is formed without conjugation and contains the single ascospore surrounded by a persistent membrane (Nguyen et al. 2006;Kurtzman 2011b). Supported by phylogenetic analyses of D1/D2 LSU, the genus also included other related taxa as the anamorphic species Candida jeffriesii and Candida materiae (Nguyen et al. 2006;Barbosa et al. 2009). ...
Chapter
Demands for clean and sustainable processes and products that are environmentally friendly are challenging biotechnologists to develop new strategies to produce fuels and chemicals. As the petroleum demands rise together with the concern of climatic and environmental changes, there is an increasing interest for renewable energy. Sugars present in the lignocellulosic biomass can be used as raw material in biotechnological processes employing yeasts as catalysts. Several known yeasts such as Saccharomyces cerevisiae assimilate glucose but lack the efficiency to consume xylose. Due to industrial interest, there has been an increasing effort to discover and construct new xylose-assimilating yeast strains. In this sense, due to the diversity and metabolic potential, several non-conventional yeasts species were isolated, identified, and physiologically and genetically characterized in the last years. The current review sought to summarize the main characteristics as well as the biotechnological applications of non-conventional yeasts for xylose utilization. First, it will present and discuss the data about non-conventional yeasts that naturally and efficiently assimilate xylose as Scheffersomyces, Meyerozyma, Candida, Spathaspora, and Kluyveromyces. Then the yeasts Komagataella, Yarrowia, and Ogataea that do not assimilate xylose or poorly assimilate xylose justifying genetic manipulation to increase xylose utilization will also be presented. In each case, basic information about yeast taxonomy, morphology, and physiology will be presented, and the clearest biotechnological application will be introduced.
... Spathaspora passalidarum is a wood-boring beetle-associated yeast [15]. It can ferment xylose under both aerobic and anaerobic conditions with a higher efficiency than that of S. stipitis [16]. ...
... A type strain of S. passalidarum, ATCC MYA-4345 (CBS 10155 = NRRL Y27907), has been isolated from the gut of Odontotaenius disjunctus (wood-boring beetle) in the eastern part of the United States [15]. Other strains of S. passalidarum have been isolated from rotting wood collected, wood-boring beetles and log galleries sampled in Brazilian Amazonian Forest [24,25] and rotten wood in central China [26]. ...
... In this study, S. passalidarum CMUWF1-2 was found to have different characteristics from those of S. passalidarum ATCC MYA-4345 that were previously reported [15]. ATCC MYA-4345 is unable to utilize L-Ara for growth, while CMUWF1-2 was shown to utilize L-Ara even at high temperatures. ...
Article
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Background Efficient bioconversion of lignocellulosic biomass to bioethanol is one of key challenges in the situation of increasing bioethanol demand. The ethanologenic microbes for such conversion are required to possess abilities of utilization of various sugars including xylose and arabinose in lignocellulosic biomass. As required additional characteristics, there are a weak or no glucose repression that allows cells to simultaneously utilize various sugars together with glucose and thermotolerance for fermentation at high temperatures, which has several advantages including reduction of cooling cost. Spathaspora passalidarum ATCC MYA-4345, a type strains, isolated previously have mainly of these abilities or characteristics but its thermotolerance is not so strong and its glucose repression on xylose utilization is revealed. ResultsNewly isolated S. passalidarum CMUWF1–2 was found to have a high ability to produce ethanol from various sugars included in lignocellulosic biomass at high temperatures. The strain achieved ethanol yields of 0.43 g, 0.40 g and 0.20 g ethanol/g xylose at 30 °C, 37 °C and 40 °C, respectively. Interestingly, no significant glucose repression was observed in experiments with mixed sugars, being consistent with the strong resistance to 2-deoxyglucose, and antimycin A showed no effect on its growth in xylose medium. Moreover, the strain was tolerant to glucose and ethanol at concentrations up to 35.0% (w/v) and 8.0% (v/v), respectively. ConclusionsS. passalidarum CMUWF1–2 was shown to achieve efficient production of ethanol from various sugars and a high ethanol yield from xylose with little accumulation of xylitol. The strain also exhibited stress-resistance including thermotolerance and no detectable glucose repression as beneficial characteristics. Therefore, S. passalidarum CMUWF1–2 has remarkable potential for conversion of lignocellulosic biomass to bioethanol.
... Nguyen, S.O. Suh & M. Blackw (2006) (Saccharomycetales, Debaryomycetaceae) was introduced, based on a single species, Spathaspora passalidarum, which was isolated from a passalid beetle in Louisiana, USA (Nguyen et al. 2006). This species produces asci containing elongate ascospores with curved ends, a unique trait of this genus (Nguyen et al. 2006;Nguyen et al. 2011). ...
... Suh & M. Blackw (2006) (Saccharomycetales, Debaryomycetaceae) was introduced, based on a single species, Spathaspora passalidarum, which was isolated from a passalid beetle in Louisiana, USA (Nguyen et al. 2006). This species produces asci containing elongate ascospores with curved ends, a unique trait of this genus (Nguyen et al. 2006;Nguyen et al. 2011). Subsequently, Spathaspora arborariae, Sp. boniae, Sp. brasiliensis, Sp. girioi, Sp. gorwiae, Sp. hagerdaliae, Sp. piracicabensis, Sp. roraimanensis, Sp. suhii and Sp. ...
Article
Full-text available
Spathaspora is an important genus of d-xylose-fermenting yeasts that are poorly studied in China. During recent yeast collections in Yunnan Province in China, 13 isolates of Spathaspora were obtained from rotting wood and all represent undescribed taxa. Based on morphological and phylogenetic analyses (ITS and nuc 28S), five new species are proposed: Spathaspora elongata , Sp. mengyangensis , Sp. jiuxiensis , Sp. parajiuxiensis and Sp. rosae . Our results indicate a high species diversity of Spathaspora waiting to be discovered in rotting wood from tropical and subtropical southwest China. In addition, the two Candida species, C. jeffriesii and C. materiae , which are members of the Spathaspora clade based on phylogeny, are transferred to Spathaspora as new combinations.
... The strain used was Spathaspora passalidarum NRRL Y-27907 [36]. The microorganism was stored in YPDX medium (1% yeast extract; 2% peptone; 1% dextrose; and 1% xylose, in w/v) with glycerol (1:1 v/v) at − 80 °C. ...
... This behavior was observed only for this condition due to the greater number of samples collected, which does not discard the possibility that the same behavior may have occurred in fermentations with free cells. Long et al. [36] and Hou [22] reported the same behavior for sugar consumption, who used free cells of S. passalidarum at the initial concentration of 1.23 g/L. However, the effect of catabolite repression, characterized by the sequential consumption of glucose and xylose, is reported in the literature for this microorganism [23,34,40,41]. ...
Article
Full-text available
The immobilization of S. passalidarum in calcium alginate beads for second-generation ethanol production (2G ethanol) was evaluated in a medium that simulated a hemicellulosic hydrolysate of sugarcane bagasse pretreated with diluted sulfuric acid in terms of sugar composition. Three sets of sequential batch fermentations (SBF) were carried out with free cells or immobilized cells in high (HSC) and moderate (MSC) initial sugar concentration (120 and 70 g/L, respectively). SBF were characterized by five consecutive batches performed in shaker, at 30 °C and 110 rpm. Better results were observed for the SBF with immobilized cells in MSC medium when compared to HSC (Y’P/S of 0.27 ± 0.02 and 0.19 ± 0.03 g/g, respectively), in the second batch cycle. The value for YP/S in MSC was similar to the obtained with free cells (0.30 ± 0.02 to 0.33 ± 0.02 g/g). However, QP was lower for MSC with immobilized cells, reaching 0.81 ± 0.04 g/L.h in the second batch, while for free cells the QP varied from 1.06 ± 0.02 to 1.16 ± 0.22 g/L.h. A technique for determining the concentration of immobilized cells in the alginate beads was applied, which made it possible to determine the specific rates for the SBF performed. According to the results obtained, it was possible to demonstrate that S. passalidarum can be immobilized in calcium alginate and reused through SBF, with performance similar to free cells, which can be a good strategy for fermentation of hemicellulosic hydrolysates.
... S p at h as p o r a pa s s a l i d a r u m , S . a r b o r a r i a e , and Scheffersomyces stipitis have shown potential for the production of ethanol from xylose [18][19][20][21][22], whereas species such as Candida tropicalis, Meyerozyma caribbica, Spathaspora sp. JA1, and Wickerhamomyces anomalus have shown potential for the production of xylitol [16,[23][24][25][26][27]. ...
... JA1, and Meyerozyma caribbica JA9 [27], and 4 with fermentative metabolism-S. arborariae Y-48658 [22], Wickerhamomyces anomalus 740 [25], and S. passalidarum NRRL Y-27907 [21], Scheffersomyces stipitis NRRL Y-7124-these last two strains kindly provided by the ARS-NRRL culture collection (Peoria, USA). ...
Article
Lignocellulosic hydrolysates will also contain compounds that inhibit microbial metabolism, such as organic acids, furaldehydes, and phenolic compounds. Understanding the response of yeasts toward such inhibitors is important to the development of different bioprocesses. In this work, the growth capacity of 7 industrial Saccharomyces cerevisiae and 7 non-Saccharomyces yeasts was compared in the presence of 3 different concentrations of furaldehydes (furfural and 5-hydroxymetil-furfural), organic acids (acetic and formic acids), and phenolic compounds (vanillin, syringaldehyde, ferulic, and coumaric acids). Then, Candida tropicalis JA2, Meyerozyma caribbica JA9, Wickerhamomyces anomalus 740, S. cerevisiae JP1, B1.1, and G06 were selected for fermentation in presence of acetic acid, HMF, and vanillin because they proved to be most tolerant to the tested compounds, while Spathaspora sp. JA1 because its xylose consumption rate. The results obtained showed a dose-dependent response of the yeasts toward the eight different inhibitors. Among the compared yeasts, S. cerevisiae strains presented higher tolerance than non-Saccharomyces, 3 of them with the highest tolerance among all. Regarding the non-Saccharomyces yeasts, C. tropicalis JA2 and W. anomalus 740 appeared as the most tolerant, whereas Spathaspora strains appeared very sensitive to the different compounds.
... In this context, Spathaspora passalidarum, a recently isolated yeast reported to be capable of fermenting pentose sugars [8], is a potential platform for industrial applications aiming to produce second-generation ethanol from lignocellulosic biomass. In addition to having the capacity to ferment xylose and arabinose, S. passalidarum can also consume xylose and glucose simultaneously in aerobic processes [8,9]. ...
... In this context, Spathaspora passalidarum, a recently isolated yeast reported to be capable of fermenting pentose sugars [8], is a potential platform for industrial applications aiming to produce second-generation ethanol from lignocellulosic biomass. In addition to having the capacity to ferment xylose and arabinose, S. passalidarum can also consume xylose and glucose simultaneously in aerobic processes [8,9]. In addition, a study by Cadete et al. (2016) [10] indicated that S. passalidarum encodes a xylose reductase (XR), encoded by the XYL1.2 gene, that has a higher affinity for the coenzyme NADH than for NADPH, generating a smaller redox cofactor imbalance in the xylose fermentation pathway compared to other native xylose-consuming yeasts. ...
Article
This study aimed to elucidate the impact of temperature on the first-generation fermentation process (Melle-Boinot) applied to second-generation ethanol production by the native xylose-fermenting yeast Spathaspora passalidarum. The Melle-Boinot process consists of fed-batch fermentations with cell recycling and was performed in this study under three conditions: fixed temperature of 30 °C, temperature decay of 1 °C per cycle and fixed temperature of 27 °C. The physiological adaptation of S. passalidarum was observed throughout the recycles under all conditions regardless of the temperature applied, as confirmed by the improvement in the kinetic and fermentation parameters. Regarding the fixed 30 °C condition, the best condition of this study for industrial application, the ethanol yield, ethanol volumetric productivity and ethanol titer increased 31.77, 33.58 and 33.55%, respectively, from the first to last fermentation cycle. The Melle-Boinot fermentation process was shown to be a worthy strategy for second-generation ethanol production by native yeasts without the need for genetic modifications.
... They are usually endosymbioticly associated with wood-boring-beetles that occupy rotting wood. Spathaspora passalidarum (Figure 3), the first identified species of genus Spathaspora, was isolated from the gut of passalid beetle Odontotaenius disjunctus [9,[21][22][23][24]. Notably, S. arborariae, S. gorwiae, S. hagerdaliae, and S. passalidarum ferment xylose to produce bioethanol, whereas the rest within the Spathaspora clade are thought to be xylitol producers [9]. ...
... Notably, S. arborariae, S. gorwiae, S. hagerdaliae, and S. passalidarum ferment xylose to produce bioethanol, whereas the rest within the Spathaspora clade are thought to be xylitol producers [9]. S. passalidarum was firstly described in 2006 by Nguyen et al. [21]. The authors speculated that Spathaspora mainly exists in the beetle's biosphere rather than the beetle's gut microbiota, and it may be only by coincidence that O. disjunctus beetles ingested decaying wood contaminated by yeasts. ...
Article
Full-text available
Currently, the fermentation technology for recycling agriculture waste for generation of alternative renewable biofuels is getting more and more attention because of the environmental merits of biofuels for decreasing the rapid rise of greenhouse gas effects compared to petrochemical, keeping in mind the increase of petrol cost and the exhaustion of limited petroleum resources. One of widely used biofuels is bioethanol, and the use of yeasts for commercial fermentation of cellulosic and hemicellulosic agricultural biomasses is one of the growing biotechnological trends for bioethanol production. Effective fermentation and assimilation of xylose, the major pentose sugar element of plant cell walls and the second most abundant carbohydrate, is a bottleneck step towards a robust biofuel production from agricultural waste materials. Hence, several attempts were implemented to engineer the conventional Saccharomyces cerevisiae yeast to transport and ferment xylose because naturally it does not use xylose, using genetic materials of Pichia stipitis, the pioneer native xylose fermenting yeast. Recently, the nonconventional yeast Spathaspora passalidarum appeared as a founder member of a new small group of yeasts that, like Pichia stipitis, can utilize and ferment xylose. Therefore, the understanding of the molecular mechanisms regulating the xylose assimilation in such pentose fermenting yeasts will enable us to eliminate the obstacles in the biofuels pipeline, and to develop industrial strains by means of genetic engineering to increase the availability of renewable biofuel products from agricultural biomass. In this review, we will highlight the recent advances in the field of native xylose metabolizing yeasts, with special emphasis on S. passalidarum for improving bioethanol production.
... The discovery of D-xylose-fermenting yeasts began in the 1980s [42], and since this decade, we can list some species of nonconventional yeasts studied for ethanol production: Pachysolen tannophilus [43], Kluyveromyces cellobiovorus [44], Scheffersomyces (Candida) shehatae [45], and Scheffersomyces (Pichia) stipitis [45][46][47]. Most recently, Spathaspora arborariae [48,49], Sp. passalidarum [50][51][52][53], and Sp. piracicabensis are other species described as able to perform this activity [54]. ...
... D-xylose fermenting yeasts have been isolated for example from the gut of wood-boring insects such as passalid beetles (S. stipitis, S. shehatae, Sp. passalidarum, C. jeffriesii, and Sp. allomyrina) [51,65,66], rotting wood (Sp. arborariae and Sp. ...
Chapter
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Yeasts not belonging to species of the Saccharomyces genus, called nonconventional yeasts, have gained prominence recently in the biotechnological scenario. For many years, they have been generally characterized as undesirable contaminants in fermentative processes. However, several studies pointed them as useful for many biotechnological applications. This chapter will cover some of these applications, highlighting the most widely employed nonconventional yeasts. The use of non- Saccharomyces strains in (I) xylose fermentation for the production of ethanol and xylitol, (II) brewing industry, (III) improvement of coffee and cocoa fermentation, and (IV) plant growth promotion will be presented.
... Investigation of new asexual and sexual states may contribute to further studies on their distribution, sexual compatibility and evolutionary processes affecting their diversity. Several of the yeasts detected can ferment xylose, the second most abundant monosaccharide in woody plant matter (Kurtzman, 1990;Nguyen et al., 2006;Wohlbach et al., 2011). Rhodotorula species can utilize monoterpenes (as carbon and energy sources) present in coniferous wood (Cook, 1992;Nguyen Thanh et al., 2004;Pohl et al., 2011). ...
Article
Abundance of fungi and richness of fungal communities were studied in coarse wood debris of Scots pine in stands that were unmanaged or managed (by sanitary cutting, commercial thinning and timber harvesting) in an 85-year-old Scots pine forest in western Poland, in June 2014. Samples consisted of 20 × 20 × 20 cm pieces of logs, fallen branches and stumps, in the 1st, 2nd and 3rd decay classes. Fungal communities were analysed using high throughput Illumina MiSeq sequencing of fungal rDNA internal transcribed spacer. From a total of 80 076 OTUs from 34 samples, 58 436 (72.98%) were of culturable fungi and 21 640 (27.02%) were of non-culturable fungi and other organisms. Fungi from Glomeromycota, Zygomycota, Ascomycota and Basidiomycota were detected. In the unmanaged stand the frequency of Glomeromycota was 0–0.2%, of Zygomycota 0.01–0.29%, of Ascomycota 30.47–79.08%, of Basidiomycota 1.56–16.45%, and of non-culturable fungi 7.28–65.51%. In the managed stand the frequency of Glomeromycota was 0–0.12%, of Zygomycota 0.04–1.48%, of Ascomycota 52.25–68.33%, of Basidiomycota 8.01–18.05%, and of non-culturable fungi 10.54–26.09%. Fungal communities in coarse dead wood were shown to be huge complexes which include many previously undifferentiated taxa that often occupy specific ecological niches. Samples were colonized by at least 260 taxa of fungi. Fungi were most abundant and fungal communities most species-rich in the managed stand and in the initial stages of wood decay. Known wood-decay species were most abundant in the less decayed wood. Management practice influenced density and diversity of fungal communities in decayed wood. Illumina technology proved to be more effective in detection of fungi than analysis based on fungal morphology in culture or Sanger DNA sequencing.
... Scheffersomyces stipitis (formerly known as Pichia stipitis) belongs to a group of yeasts that naturally ferment xylose while accumulating low amounts of by-products such as xylitol [97]. Other known representatives of the group of native xylose-fermenting yeastsare, Candida shehatae (teleomorph form is known as Scheffersomyces shehatae), Pachysolen tannophilus, Spathaspora passalidarum, and Ogataea polymorpha [38,128,160,162]. As a rule, most natural xylose-fermenting yeasts inhabit the guts of passalid beetles that degrade white-rotted hardwood [125,130] with the exception of O. polymorpha strains that have been isolated from other natural habitats [46]. ...
Article
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This review summarizes progress in the construction of efficient yeast ethanol producers from glucose/sucrose and lignocellulose. Saccharomyces cerevisiae is the major industrial producer of first-generation ethanol. The different approaches to increase ethanol yield and productivity from glucose in S. cerevisiae are described. Construction of the producers of second-generation ethanol is described for S. cerevisiae, one of the best natural xylose fermenters, Scheffersomyces stipitis and the most thermotolerant yeast known Ogataea polymorpha. Each of these organisms has some advantages and drawbacks. S. cerevisiae is the primary industrial ethanol producer and is the most ethanol tolerant natural yeast known and, however, cannot metabolize xylose. S. stipitis can effectively ferment both glucose and xylose and, however, has low ethanol tolerance and requires oxygen for growth. O. polymorpha grows and ferments at high temperatures and, however, produces very low amounts of ethanol from xylose. Review describes how the mentioned drawbacks could be overcome.
... These landscapes allow diverse microbial populations to establish, suggesting that such anatomical properties and separation of these compartments may be beneficial to the processes of energy extraction from woody biomass 15 . Isolation and analysis of O. disjunctus' fungal microbiota, particularly in the posterior hindgut region (PHG), has identified a large diversity of yeasts with xylose fermentation capabilities, suggestive of their role in host metabolism [19][20][21][22][23][24] . Previous research has demonstrated that passalid beetle digestive tracts harbour diverse bacterial and archaeal populations, with steep exterior-to-interior oxygen gradients allowing close coexistence of aerobic and anaerobic microorganisms 10 . ...
Article
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Beneficial microbial associations enhance the fitness of most living organisms, and wood-feeding insects offer some of the most striking examples of this. Odontotaenius disjunctus is a wood-feeding beetle that possesses a digestive tract with four main compartments, each of which contains well-differentiated microbial populations, suggesting that anatomical properties and separation of these compartments may enhance energy extraction from woody biomass. Here, using integrated chemical analyses, we demonstrate that lignocellulose deconstruction and fermentation occur sequentially across compartments, and that selection for microbial groups and their metabolic pathways is facilitated by gut anatomical features. Metaproteogenomics showed that higher oxygen concentration in the midgut drives lignocellulose depolymerization, while a thicker gut wall in the anterior hindgut reduces oxygen diffusion and favours hydrogen accumulation, facilitating fermentation, homoacetogenesis and nitrogen fixation. We demonstrate that depolymerization continues in the posterior hindgut, and that the beetle excretes an energy- and nutrient-rich product on which its offspring subsist and develop. Our results show that the establishment of beneficial microbial partners within a host requires both the acquisition of the microorganisms and the formation of specific habitats within the host to promote key microbial metabolic functions. Together, gut anatomical properties and microbial functional assembly enable lignocellulose deconstruction and colony subsistence on an extremely nutrient-poor diet. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.
... For heterologous xylose pathway expression in the eukaryotic host system, Sc. stipitis genes encoding xylose pathway have been more widely used than any other eukaryotic xylose pathway [6][7][8][9]. Spathaspora passalidarum is a relatively recently identified beetle-associated yeast [26] and currently the most promising native xylose-fermenting yeast in terms of growth and ethanol fermentation on xylose [27,28]. Sp. passalidarum has NADH-preferred XR that allows Sp. passalidarum to more efficiently consume xylose and produce ethanol under both aerobic and anaerobic culture conditions as compared to Sc. stipitis and other native xylose-fermenting yeasts [27]. ...
... Candida is a highly diversified and polyphyletic genus that has been reformulated (Lachance et al. 2011). This genus appears to be ubiquitous in insects and has not been associated with a specific host since it has previously been detected in association with DT from a wide range of insects such as Odontotaenius disjunctus (Coleoptera: Passalidae), Phrenapates bennetti (Coleoptera: Tenebrionidae) (Nguyen et al. 2006), among others. ...
Article
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The interaction between insects, both larval and adult, and yeasts associated with their digestive tract (DT), has been of interest in recent years, since it can be beneficial for both partners. Studies focusing on this habitat have contributed to the expansion of knowledge about diversity, biogeography and functional characterization of yeasts, especially in ecosystems still poorly exploited, such as the Brazilian Cerrado. We investigated the interaction between larvae of Phylloicus spp. (Trichoptera: Calamoceratidae), which is an aquatic insect, and the yeasts isolated from its DT. The larvae were collected from first-order Cerrado streams of two States (Mato Grosso – MT and Pará – PA) in Brazil. Yeasts were cultivated and identified based on sequence analysis of the D1/D2 domains of the large subunit of rRNA genes. A total of 20 yeast species, belonging to six genera of Ascomycota and five Basidiomycota, is harbored in the DT of the larvae. The most frequent genera were Candida, Papiliotrema, Rhodotorula (19.3% each) and Issatchenkia (15.8%). Candida parapsilosis and Rhodotorula mucilaginosa were only yeast species isolated from the DT of larvae in both locations. The most species-rich community was that associated with DT of Phylloicus spp. in MT samples (H′ = 1.48) as compared to PA samples (H′ = 0.67). All species were accidental (frequency < 25%), which is indicative of a loose association of these yeasts with their host. This is the first report of the association of yeasts with the DT of the shredders group of aquatic insects.
... While yeast species were historically identified by metabolic differences, recent studies have shown that many of these classic characters are subject to rampant homoplasy, convergence, and parallelism (Hittinger et al. 2004;Hall and Dietrich 2007;Wenger et al. 2010;Slot and Rokas 2010;Lin and Li 2011;Wolfe et al. 2015). Despite the considerable progress in classifying yeasts using multi-locus DNA sequence data, critical gaps remain (Kurtzman and Robnett 1998, 2007, 2013Nguyen et al. 2006;Kurtzman et al. 2008Kurtzman et al. , 2011Kurtzman and Suzuki 2010); many genera are paraphyletic or polyphyletic, while circumscriptions at or above the family level are often poorly supported (Hittinger et al. 2015). ...
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Understanding the phylogenetic relationships among the yeasts of the subphylum Saccharomycotina is a prerequisite for understanding the evolution of their metabolisms and ecological lifestyles. In the last two decades, the use of rDNA and multi-locus data sets has greatly advanced our understanding of the yeast phylogeny, but many deep relationships remain unsupported. In contrast, phylogenomic analyses have involved relatively few taxa and lineages that were often selected with limited considerations for covering the breadth of yeast biodiversity. Here we used genome sequence data from 86 publicly available yeast genomes representing 9 of the 11 major lineages and 10 non-yeast fungal outgroups to generate a 1,233-gene, 96-taxon data matrix. Species phylogenies reconstructed using two different methods (concatenation and coalescence) and two data matrices (amino acids or the first two codon positions) yielded identical and highly supported relationships between the 9 major lineages. Aside from the lineage comprised by the family Pichiaceae, all other lineages were monophyletic. Most interrelationships among yeast species were robust across the two methods and data matrices. However, 8 of the 93 internodes conflicted between analyses or data sets, including the placements of: the clade defined by species that have reassigned the CUG codon to encode serine, instead of leucine; the clade defined by a whole genome duplication; and of Ascoidea rubescens. These phylogenomic analyses provide a robust roadmap for future comparative work across the yeast subphylum in the disciplines of taxonomy, molecular genetics, evolutionary biology, ecology, and biotechnology. To further this end, we have also provided a BLAST server to query the 86 Saccharomycotina genomes, which can be found at http://y1000plus.org/blast .
... [10] On the other hand, most native xylose-utilizing yeasts use the oxidoreductase pathway composed of xylose reductase (XR) and xylitol dehydrogenase (XDH). [11] Other naturally existing xylose catabolic routes are the Dahms and Weimberg pathways, also called xylose oxidative pathways (XOP). [12] Compared to XOP, the XI and XR-XDH pathways are more widely studied. ...
Article
Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns over limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources toward engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Here, recent advances in metabolic engineering of yeast is summarized to address bottlenecks on xylose assimilation and to enable simultaneous co‐utilization of xylose and other substrates in lignocellulosic hydrolysates. Distinct characteristics of xylose metabolism that can be harnessed to produce advanced biofuels and chemicals are also highlighted. Although many challenges remain, recent research investments have facilitated the efficient fermentation of xylose and simultaneous co‐consumption of xylose and glucose. In particular, understanding xylose‐induced metabolic rewiring in engineered yeast has encouraged the use of xylose as a carbon source for producing various non‐ethanol bioproducts. To boost the lignocellulosic biomass‐based bioeconomy, much attention is expected to promote xylose‐utilizing efficiency via reprogramming cellular regulatory networks, to attain robust co‐fermentation of xylose and other cellulosic carbon sources under industrial conditions, and to exploit the advantageous traits of yeast xylose metabolism for producing diverse fuels and chemicals.
... Xylose-utilizing microorganisms in nature use two different catabolic pathways to convert xylose into xylulose, which can be channeled into the glycolytic pathway through innate xylulokinase (XK) and the pentose phosphate pathway (PPP) (Fig. 1). Most native xylose-utilizing yeasts, including representative yeasts Scheffersomyces stipitis, Candida shehatae, and Spathaspora passalidarum, convert xylose to xylulose through the oxidoreductase pathway composed of xylose reductase (XR) and xylulose dehydrogenase (XDH) (Bruinenberg et al., 1984;Nguyen et al., 2006). Native XRs exhibit dual cofactor preference for NADPH and NADH with few exceptions such as NADH-preferred S. passalidarum Xyl1.2 (Hou, 2012), but XDH uses only NAD + (Fig. 1). ...
Article
Numerous metabolic engineering strategies have allowed yeasts to efficiently assimilate xylose, the second most abundant sugar component of lignocellulosic biomass. During the investigation of xylose utilization by yeasts, a global rewiring of metabolic networks upon xylose cultivation has been captured, as opposed to a pattern of glucose repression. A clear understanding of the xylose-induced metabolic reprogramming in yeast would shed light on the optimization of yeast-based bioprocesses to produce biofuels and chemicals using xylose. In this review, we delved into the characteristics of yeast xylose metabolism, and potential benefits of using xylose as a carbon source to produce various biochemicals with examples. Transcriptomic and metabolomic patterns of xylose-grown yeast cells were distinct from those on glucose—a conventional sugar of industrial biotechnology—and the gap might lead to opportunities to produce biochemicals efficiently. Indeed, limited glycolytic metabolic fluxes during xylose utilization could result in enhanced production of metabolites whose biosynthetic pathways compete for precursors with ethanol fermentation. Also, alleviation of glucose repression on cytosolic acetyl coenzyme A (acetyl-CoA) synthesis, and respiratory energy metabolism during xylose utilization enhanced production of acetyl-CoA derivatives. Consideration of singular properties of xylose metabolism, such as redox cofactor imbalance between xylose reductase and xylitol dehydrogenase, is necessary to maximize these positive xylose effects. This review argues the importance and benefits of xylose utilization as not only a way of expanding a substrate range, but also an effective environmental perturbation for the efficient production of advanced biofuels and chemicals in yeasts.
... Nos anos 2000, novas linhagens naturalmente capazes de fermentar pentoses foram descritas, tais como Spathaspora passalidarum (NGUYEN et al., 2006;HOU, 2012) e Spathaspora arboriae (CADETE et al., 2009;CUNHA-PEREIRA et al., 2011) (RYABOVA et al., 2003;DMYTRUK et al., 2008;GALAFASSI et al., 2011;NITIYON et al., 2011;RODRUSSAMEE et al., 2011;VERHO et al., 2011;CADETE et al., 2013;DOGARIS et al., 2013;LORLIAM et al., 2013;KALHORINIA et al., 2014;SUKPIPAT et al., 2017;MATOS et al., 2017;VALINHAS et al., 2018). Nesta mesma década, Barbosa (2017) A D-xilose (C 5 H 10 O 5 ) é uma das principais unidades monossacarídicas que compoem as hemiceluloses e a mais abundante pentose encontrada nos vegetais (JUGWANTH et al., 2020;LACHKE, 2002;MAURYA et al., 2015). ...
... As we homogenized beetles completely without separating the guts from the body, beetle-associated fungal communities likely comprised species that were on the body surface, inside mycangia and in the gut system. Guts of insects, including beetles, host diverse communities of yeast-like fungi (Nguyen et al., 2006;Suh et al., 2005), many of which help to digest wood and fungal material consumed by insect larvae (Douglas, 2009). The beetle gut might thus have contributed to the diversity of beetleassociated fungi in our study, particularly of endosymbiotic mutualists such as Candida spp. ...
Article
Spore characteristics of wood-inhabiting fungi suggest that wind is their predominant dispersal vector. However, since they are restricted to ephemeral habitats, colonizing new patches should benefit from dispersal by animals with similar habitat preferences because the directed, resource-searching movement of animals increases the likelihood of reaching suitable habitats. Here we determine which fungal guilds are carried by wood-inhabiting beetles and what influences beetle-associated fungal communities. High-throughput sequencing identified >1800 fungal taxa from beetle communities that emerged from 64 experimental logs. Beetle-associated fungi included mutualistic, decomposing, pathogenic and mycorrhizal fungi; decomposers were the most diverse. Partial-procrustes analysis revealed that the total beetle-associated community and mutualists were correlated (p 0.05) with beetle community composition and decomposers were marginally correlated (p 0.10) with beetle community composition. All three groups were marginally correlated with the total fungal communities that inhabit the dead wood. Our results show that beetles carry a broad range of wood-inhabiting fungi and beetle-associated fungal communities are determined by environmental factors and the vectoring beetle community and to some degree by the fungal source community. This suggests that wood-inhabiting beetles contribute to fungal dispersal, including directed dispersal, which could affect fungal community assembly and ecosystem processes like wood decomposition.
... Nos anos 2000, novas linhagens naturalmente capazes de fermentar pentoses foram descritas, tais como Spathaspora passalidarum (NGUYEN et al., 2006;HOU, 2012) e Spathaspora arboriae (CADETE et al., 2009;CUNHA-PEREIRA et al., 2011) (RYABOVA et al., 2003;DMYTRUK et al., 2008;GALAFASSI et al., 2011;NITIYON et al., 2011;RODRUSSAMEE et al., 2011;VERHO et al., 2011;CADETE et al., 2013;DOGARIS et al., 2013;LORLIAM et al., 2013;KALHORINIA et al., 2014;SUKPIPAT et al., 2017;MATOS et al., 2017;VALINHAS et al., 2018). Nesta mesma década, Barbosa (2017) A D-xilose (C 5 H 10 O 5 ) é uma das principais unidades monossacarídicas que compoem as hemiceluloses e a mais abundante pentose encontrada nos vegetais (JUGWANTH et al., 2020;LACHKE, 2002;MAURYA et al., 2015). ...
Chapter
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Micro-organismos fermentadores de pentose formam um grupo heterogêneo de espécies microbianas com aplicação potencial em indústrias de base biotecnológica. Para a indústria do etanol de 2ª geração esses micro-organismos representam a possibilidade de aumentar entre 20% e 50% a produtividade do etanol a partir da mesma tonelagem de biomassa lignocelulósica processada. A disseminação do conhecimento sobre a evolução das pesquisas com micro-organismos fermentadores de pentoses coopera para a construção e consolidação da cadeia de produção de bioetanol lignocelulósico. Nesta revisão da literatura científica, detacam-se a cronologia dos estudos sobre micro-organismos fermentadores de pentose desde os anos 50; seu metabolismo peculiar; as estratégias empregadas para contornar a presença de inibidores nos hidrolisados lignocelulósicos; e a importância de fatores físico, químicos e nutricionais que condicionam a eficiência do processo fermentativo.
... Therefore, it seems reasonable to continue the search for yeasts with a higher capacity for ethanol conversion from xylose, such as those from the genera Spathaspora and Scheffersomyces (Cadete et al. 2012;Chandel et al. 2014). Spathaspora passalidarum has been used in the fermentation of xylose to ethanol, and shows a great potential in the production of lignocellulosic ethanol (Nguyen et al. 2006;Hou and Yau 2011;Cadete et al. 2012;Long et al. 2012). ...
Article
In this study, we evaluated the potential of yeasts isolated from Amazon to produce second-generation ethanol from sugarcane bagasse delignified with alkaline hydrogen peroxide and hydrolysed with commercial enzyme preparation. The best efficiency savings in glucose and release of xylose were determined by considering the solids and enzyme loads. Furthermore, we selected Spathaspora passalidarum UFMG-CM-Y473 strain with the best fermentative parameters. Fermentations used bagasse hydrolysate without any nutritional supplementation, a significant difference from previous studies, which is closer to industrial conditions. Ethanol yield of 0.32 g/g and ethanol productivity of 0.34 g/L h were achieved after the consumption of 78% of the sugar. This hydrolysis/fermentation technology package could represent the input of an additional 3180 L of ethanol per hectare in areas of average sugarcane productivity such as 60 ton/ha. Thus, we concluded that Sp. passalidarum UFMG-CM-Y473 has a clear potential for the production of second-generation ethanol from delignified and enzyme-hydrolysed bagasse.
... However, efficient ethanol production from lignocellulosic biomass requires a diverse microbial flora capable of metabolizing various carbon sources such as pentoses and hexoses (Sànchez Nogué and Karhumaa 2015). To address this issue, native yeasts such as Spathaspora passalidarum and Scheffersomyces stipitis have been studied for their ability to efficiently assimilate carbon from sources such as xylose, arabinose, glucose, and cellobiose present in lignocellulosic hydrolysates (Nguyen et al. 2006;Cadete et al. 2012). ...
Article
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Understanding the mechanisms involved in tolerance to inhibitors is the first step in developing robust yeasts for industrial second-generation ethanol (E2G) production. Here, we used ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) and MetaboAnalyst 4.0 for analysis of MS data to examine the changes in the metabolic profile of the yeast Spathaspora passalidarum during early fermentation of hemicellulosic hydrolysates containing high or low levels of inhibitors (referred to as control hydrolysate or CH and strategy hydrolysate or SH, respectively). During fermentation of SH, the maximum ethanol production was 16 g L⁻¹ with a yield of 0.28 g g⁻¹ and productivity of 0.22 g L⁻¹ h⁻¹, whereas maximum ethanol production in CH fermentation was 1.74 g L⁻¹ with a yield of 0.11 g g⁻¹ and productivity of 0.01 g L⁻¹ h⁻¹. The high level of inhibitors in CH induced complex physiological and biochemical responses related to stress tolerance in S. passalidarum. This yeast converted compounds with aldehyde groups (hydroxymethylfurfural, furfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) into less toxic compounds, and inhibitors were found to reduce cell viability and ethanol production. Intracellularly, high levels of inhibitors altered the energy homeostasis and redox balance, resulting in lower levels of ATP and NADPH, while that of glycolytic, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways were the most affected, being the catabolism of glucogenic amino acids, the main cellular response to inhibitor-induced stress. This metabolomic investigation reveals interesting targets for metabolic engineering of ethanologenic yeast strains tolerant against multiple inhibitors for E2G production. Key points • Inhibitors in the hydrolysates affected the yeast’s redox balance and energy status. • Inhibitors altered the glycolytic, pentose phosphate, TCA cycle and amino acid pathways. • S. passalidarum converted aldehyde groups into less toxic compounds.
... Actinomycetes with antimicrobial properties are widespread in O. disjunctus galleries Passalid beetles of the species O. disjunctus (formerly known as Passalus cornutus, and commonly referred to as 'bessbugs', Figure 1A) are widely distributed across eastern North America, where they are important decomposers of rotting timber (Ceja-Navarro et al., 2014;Ceja-Navarro et al., 2019;Gray, 1946;Pearse et al., 1936). This role has prompted interest in the O. disjunctus gut microbiota as a potential source of lignocellulose-processing microbes for biofuel efforts (Ceja-Navarro et al., 2014;Ceja-Navarro et al., 2019;Nguyen et al., 2006;Suh et al., 2003;Suh et al., 2005;Urbina et al., 2013). O. disjunctus is subsocial, with mating pairs establishing galleries within decaying logs where they rear their larvae (Schuster and Schuster, 1985;Wicknick and Miskelly, 2009). ...
Article
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Some insects form symbioses in which actinomycetes provide defense against pathogens by making antimicrobials. The range of chemical strategies employed across these associations, and how these strategies relate to insect lifestyle, remains underexplored. We assessed subsocial passalid beetles of the species Odontotaenius disjunctus, and their frass (fecal material), which is an important food resource within their galleries, as a model insect/actinomycete system. Through chemical and phylogenetic analyses, we found that O. disjunctus frass collected across eastern North America harbored multiple lineages of Streptomyces and diverse antimicrobials. Metabolites detected in frass displayed synergistic and antagonistic inhibition of a fungal entomopathogen, Metarhizium anisopliae, and multiple streptomycete isolates inhibited this pathogen when co-cultivated directly in frass. These findings support a model in which the lifestyle of O. disjunctus accommodates multiple Streptomyces lineages in their frass, resulting in a rich repertoire of antimicrobials that likely insulates their galleries against pathogenic invasion.
... These strategies mostly target xylose fermentation as these species have been documented as efficient xylose fermenters (Nguyen, Suh, Marshall, & Blackwell, 2006). Despite their xylose fermentation abilities, some challenges are still inherent in the production of bioethanol using these species. ...
Article
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Bioethanol production from monomeric sugar is performed by several yeasts. But there are several limitations associated with yeast strains such as their low tolerance to ethanol, toxic inhibitors, and high sugar concentration. Genetic and metabolic engineering of potential yeast strains can overcome the above limitations. The present article summarized current genetic and metabolic engineering approaches for the development of yeast strain for efficient ethanol production. The review systematically examined bioethanol generations based on substrate utilization, criteria for strain selections, strategies for strain improvements including randomized mutagenesis, genetic engineering, metabolic engineering, genome editing, whole genome (re)sequencing, promoter engineering, quantitative trait locus analysis, protein engineering, and evolutionary engineering. Different fermentation technologies employed in hydrolysate fermentation including low gravity (LG), high gravity (HG), and very high gravity (VHG) as well as challenges of yeast strains development and its future prospect have been critically evaluated in this article. Significant engineering efforts are imminent for yeast-based second-generation biofuel to leave a demonstration phase through strain improvement and become economically competitive with fossil fuel. Practical Applications This is a comprehensive review of yeast strain development for bioethanol production. The readers should be able to acquire some basic knowledge on: • The accompanied substrates for bioethanol generations as well as the technologies and challenges behind them. • The criteria to consider in selecting yeast strain for bioengineering development. • Different strategies and their reported applications employed in yeast strain development including randomized mutagenesis, genetic engineering, metabolic engineering, genome editing, whole genome (re)sequencing, promoter engineering, quantitative trait locus (QTL) analysis, protein engineering, and evolutionary engineering. • Challenges and merits of different fermentation technologies employed in hydrolysate fermentation including LG, HG, and VHG. • Possible challenges to encounter in developing yeast strain for bioethanol production. • Desirable traits to consider in the selection and development of yeast strains for bioethanol production.
... In the context of 1G and 2G process integration, C6-rich stream could be fermented in already established first-generation ethanol mills, decreasing heat demand and capital costs. Among the naturally xylose-consuming yeasts is Spathaspora passalidarum, which was isolated from the intestine of wood-boring beetles [16]. In the past decade, the potential application of this yeast for C5 fermentation has been explored in several studies [17][18][19][20][21], proving that this yeast can be used for C5-rich stream fermentation. ...
Article
In this work, a strategy to promote ethanol fermentation through the complete utilization of fermentable sugars released from sugarcane bagasse was proposed. Initially, the sugarcane bagasse was subjected to a diluted sulfuric acid pretreatment, leading to 89.5% of hemicellulose solubilization, in which 82% was recovered as monomeric sugars (xylose and arabinose) in the liquid stream. The cellulignin obtained was subjected to enzymatic hydrolysis, resulting in 51.2 and 95.08% of cellulose and hemicellulose yields, respectively. The C5-rich hemicellulosic hydrolysate was fermented to ethanol by the pentose-fermenting yeast Spathaspora passalidarum NRRL Y-27907, achieving an ethanol yield of 86.11%. The C6-rich enzymatic hydrolysate, on the other hand, was fermented by Saccharomyces cerevisiae CAT-1 through the application of a cell-recycle batch fermentation system. This strategy improved the overall fermentation performance throughout the cycles, with a substantial increase in the ethanol productivity of 361% from the first to the last batch. Based on a mass balance with input of 1000 kg of sugarcane bagasse and taking into account a complete pentose and hexose fermentation, 191.96 kg of ethanol could be produced from this integrated platform, which corresponded to 243.3 L of ethanol.
... In general, Malassezia is mainly colonized in the skin (50). However, the availability of lipid nutrition within the GI tract may facilitate the localization and survival of Malassezia in the gut, and its nutrition can be accessed from host diet or intestinal fungal synthesis (51,52). It is speculated that Malassezia might immigrate to the GI tract along with the diet; however, solid evidence is lacking. ...
Article
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Accumulating evidence indicates that patients with inflammatory bowel disease (IBD) have a significantly higher risk of developing different cancers, while the exact mechanism involved is not yet fully understood. Malassezia is a lipid-dependent opportunistic yeast, which colonizes on mammalian skin and internal organs. Also, dysbiosis in fungal communities accompanied by high level of Malassezia are fairly common in inflammatory diseases such as IBD and various cancers. In cancer patients, higher levels of Malassezia are associated with worse prognosis. Once it is ablated in tumor-bearing mice, their prognostic conditions will be improved. Moreover, Malassezia manifests multiple proinflammatory biological properties, such as destruction of epithelial barrier, enrichment of inflammatory factors, and degradation of extracellular matrix (ECM), all of which have been reported to contribute to tumor initiation and malignant progression. Based on these facts, we hypothesize that high levels of Malassezia together with mycobiome dysbiosis in patients with IBD, would aggravate the microecological imbalance, worsen the inflammatory response, and further promote tumorigenesis and deterioration. Herein, we will discuss the detrimental properties of Malassezia and explore the key role of this fungus in the correlation between IBD and cancer, in order to take early surveillance and intervention to minimize the cancer risk in individuals with IBD.
... Yeast-insect interactions are widespread and encompass chemical signaling Becher et al., 2018;Witzgall et al., 2012), environmental detoxification, and diet supplementation (Bellutti et al., 2018;Soto-Robles et al., 2019;Spitaler et al., 2020;Witzgall et al., 2012). Some insects are known to harbor different yeast communities (Lachance et al., 2001;Stefanini, 2018;Suh et al., 2005), and new yeast species have been described in association with several insect orders (Nguyen et al., 2006(Nguyen et al., , 2007Rosa et al., 1999;. Yeasts can be vectored by insects (Brysch-Herzberg, 2004;da Costa Neto & de Morais, 2020;Ganter, 1988;Lachance et al., 2001), usually attracting the insect host by chemical volatiles (Ljunggren et al., 2019;Scheidler et al., 2015). ...
Article
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Insects interact with a wide variety of yeasts, often providing a suitable substrate for their growth. Some yeast–insect interactions are tractable models for understanding the relationships between the symbionts. Attine ants are prominent insects in the Neotropics and have performed an ancient fungiculture of mutualistic basidiomycete fungi for more than 55–65 million years. Yeasts gain access to this sophisticated mutualism, prompting diversity, ecological, and biotechnological studies in this environment. We review half a century research in this field, surveying for recurrent yeast taxa and their putative ecological roles in this environment. We found that previous studies mainly covered the yeast diversity from a small fraction of attine ants, being Saccharomycetales, Tremellales, and Trichosporonales as the most frequent yeast or yeast-like orders found. Apiotrichum, Aureobasidium, Candida, Cutaneotrichosporon, Debaryomyces, Meyerozyma, Papiliotrema, Rhodotorula, Trichomonascus, and Trichosporon are the most frequent recovered genera. On the other hand, studies of yeasts’ ecological roles on attine ant–fungus mutualism only tapped the tip of the iceberg. Previous established hypotheses in the literature cover the production of lignocellulosic enzymes, chemical detoxification, and fungus garden protection. Some of these roles have parallels in biotechnological processes. In conclusion, the attine ant environment has a hidden potential for studying yeast biodiversity, ecology, and biotechnology, which has been particularly unexplored considering the vast diversity of fungus-growing ants. Take Away • Yeasts are symbionts in attine ant colonies and often found in high abundance. • Substrates foraged by ants are the primary source of yeasts. • Attine ant gardens are interesting sources of yeasts for biotechnology.
... Spathaspora passalidarum is a wild-type yeast strain with relevance in biofuel research, based on its natural ability to ferment xylose into ethanol (Nguyen et al., 2006). Its recently sequenced genome has revealed insights into the metabolic pathways for pentose assimilation, and work has been done on the optimization of these pathways in S. passalidarum itself or on the construction of systems for heterologous expression of S. passalidarum genes (Lopes et al., 2016). ...
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The transition to sustainable development with the replacement of gasoline and petroleum-based products with biofuels and green chemicals is a common goal for current concepts of the bioeconomy. In this sense, biorefinery integration appears as a promising route to replace chemical technologies. Moreover, there is the possibility of using existing first-generation (1G) bioethanol plants facilities as the host for the development of new processes using the huge amount of lignocellulosic biomasses available in the world. In order to maximize profitability, all sugars released after biomass pre-treatment and hydrolyses must be converted into target products. Regarding the pentose fraction, sugar conversion by microorganisms is not optimally performed, compared to glucose, to give rise to the production of bioproducts in a sustainable and competitive manner. Thus, this review focuses on the recent progresses and emerging strategies aiming towards pentose utilization, efficient assimilation and conversion into industrially relevant bioproducts.
... While the expression of SsXUT1 in DLG-K1 allowed ethanol production from xylose or glucose/xylose mixtures, SsHXT2.6 led to the production of both ethanol and xylitol, and SsQUP2 generated mainly xylitol during xylose consumption. Since the genome of other new xylose-fermenting yeast species (e.g., Sp. passalidarum [37,38] or Sp. arborariae [39,40]) have genes encoding putative transporters with high amino acid sequence similarity (78-80%) to SsXUT1, we decided to clone and characterize these putative permeases in the DLG-K1 platform strain. ...
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In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78–80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3′-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.
... Due to its nutritional composition, biofunctionality, and capacity to act as a probiotic, there has been an increasing interest in its application as a supplement in the food protein industry, in the formulation of animal feed and as food additive (flavor enhancer). It is important to mention that there is no reported evidence of pathogenicity of S. passalidarum [25], which is a desirable characteristic when it comes to the feed industry. In previous studies [10,11], researchers performed fermentations with S. passalidarum from xylose, glucose, and a mixture of the two sugars (50:50), focusing on ethanol production. ...
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Spathaspora passalidarum is a naturally pentose-fermenting yeast with the potential to be applied for biotransformation of sugars from lignocellulosic biomasses. Despite being mostly investigated for ethanol production from sugarcane bagasse sugars (mainly xylose and glucose), this microorganism is also capable of producing high xylitol concentrations as a by-product of the ethanol fermentation. The integration of ethanol and xylitol production can improve the economic viability of the process due to the lower sale price of ethanol and the higher added-value of xylitol, a sucrose-substitute sugar with healthier properties. Considering the metabolic pathways interaction from glucose and xylose, it is essential to understand the effect of different glucose and xylose concentrations in the production of ethanol and xylitol by S. passalidarum. In this way, a simultaneous production of both products for the development of an integrated platform for food and chemical and biofuel industries can be accessed. Yeast biomass can also be recovered and applied as a protein source. Therefore, in this study, the fermentative performance of S. passalidarum was investigated in batch fermentations with different xylose and glucose concentrations, using synthetic substrate. The ATP levels and the enzymatic activities of xylose reductase (XR), xylitol dehydrogenase (XDH), and alcohol dehydrogenase (ADH) were also determined for each condition. The results indicated that low amounts of glucose (35%) were necessary to promote a higher xylitol production (10.58 ± 0.29 g/L) without losses on ethanol yield (78.99 ± 4.41%), being the most interesting condition for simultaneous formation of both products.
... As mentioned before, S. passalidarum was tested at different initial glucose and xylose concentrations and proportions in a concept of 2G fermentation, since this yeast is not able to ferment sucrose and only grows in this carbohydrate. 49 S. passalidarum fermentation under the lowest glucose ratio (20% glucose and 80% xylose under 60 g RS ·L −1 and 90 g RS ·L −1 , Figure 1b,d) has shown a different profile of ...
... Since the genome of other new xylose-fermenting yeast species (e.g. Spathaspora passalidarum [37,38] or Sp. arborariae [39,40]) have genes encoding transporters with high sequence similarity (78-80%) to SsXUT1, we decided to clone and characterize these putative permeases with our hxt-null DLG-K1 strain. ...
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: In our previous work we had developed an hxt-null Saccharomyces cerevisiae strain displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities that proved to be useful as a chassis strain to study new xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Spathaspora passalidarum and Spathaspora arborariae have in their genomes genes with high sequence similarity (78-80%) to SsXUT1. To characterize these putative transporter genes (SpXUT1 and SaXUT1, respectively) they were expressed in the same chassis strain as SsXUT1. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested, although the strains expressing the SsXUT1 and SpXUT1 permeases, after growth on maltose, showed the presence of 14C-glucose and 14C-xylose transport activity. An important feature of these permeases is that SsXUT1 lacks lysine residues in its N-terminal domain with high-confidence ubiquitinylation potential, and has only one at the C-terminal domain, while the SpXUT1 transporter had several of such residues at its C-terminal domain. When the SpXUT1 gene was cloned in a truncated version lacking such lysine residues, the permease allowed grow on glucose or xylose, and even promoted xylose fermentation by the hxt-null strain. In another approach, we deleted two arrestins known to be involved in sugar transporter ubiquitinylation and endocytosis (ROD1 and ROG3), but only the rog3Δ strain allowed modest growth on these sugars. Taken together, these results suggest that to allow efficient sugar transporter expression in S. cerevisiae the lysines involved in transporter endocytosis should be removed from the sequence of the permease.
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Investigations on the fungal community associated with the digestive tract (DT) of insects have provided insights into the diversity of associated microorganisms and their potential roles in the interaction with their hosts. However, most studies have focused on terrestrial insects, with few studies focusing on aquatic insects in Neotropical regions. We studied fungal taxa associated with the DT of larval stages of the aquatic shredders Phylloicus amazonas, P. elektoros and P. fenestratus in the Brazilian Amazon Forest. Filamentous fungi were isolated, purified and screened for cellulolytic activity. A total of 33 fungal taxa was identified through the combination of classical and molecular taxonomy. The genus Penicillium was the most frequent in DT of Phylloicus spp. (18.75%). The occurrence of fungal taxa among hosts was quite variable, with more than half of the associated fungi being exclusive of each host species. A significant portion of the fungal community associated with each host presented cellulolytic activity (± 50%). It was concluded that the fungal community associated with Phylloicus spp. larvae consist mainly of fungal taxa from food items, which come from riparian vegetation (whose plant species are variable) or are indigenous of the aquatic ecosystems, which is the habitat of these larvae.
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We investigated the performance of fluidized and packed-bed bioreactors using cells of Spathaspora passalidarum UFMG-CM-469 immobilized on LentiKats® in the fermentation of lignocellulosic biomass hydrolysates. Acid (AH-CYE) and enzymatic (EH-CYE) hydrolyses of a 1:1 mixture of oat and soybean hulls, minimally supplemented with crude yeast extract, were used as substrates. Continuous cultures at dilution rate (D) of 0.05 h−1 were run in the fluidized and packed-bed bioreactors, using both AH-CYE and EH-CYE, showing ethanol yields varying from 0.30 to 0.45 g•g−1 and productivities from 0.39 and 0.99 g•(L•h)−1. Continuous cultures in the fluidized bioreactor, which proved to be better than the packed bed, operating at higher dilution rates of 0.1 h−1 and 0.2 h−1 increased ethanol productivity, reaching 1.67 g•(L•h)−1 when using AH-CYE and 2.65 g•(L•h)−1 with EH-CYE. These results show the possibility of exploring the fermentation of lignocellulosic biomass hydrolysates in continuous cultures of immobilized S. passalidarum UFMG-CM-469 to produce second-generation ethanol under a concept of biorefinery.
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The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44 g/g) and productivity (1.02 g/L.h) were obtained using Sp. passalidarum grown in 100 g/L xylose at 32 °C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells.
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Efficient bioconversion of lignocellulosic biomass is one of the key challenges for the production of bioethanol and chemicals. Therefore, the present work focuses on finding a robust microorganism able to convert all sugars in lignocellulosic hydrolysates efficiently. The fermentation performance showed that Kluyveromyces marxianus CICC 1727-5 could produce ethanol from glucose with productivity 4.2 g/L/h and higher ethanol yields (0.44 g/g) under 40 °C, outdistance the productivity 0.258 g/L/h of S. passalidarum ATCC MYA-4345. The xylose utilization of S. passalidarum ATCC MYA-4345 was faster than K. marxianus CICC 1727-5 with the ethanol yield 0.31 g/g at 30 °C. However, K. marxianus CICC 1727-5 could produce xylitol from xylose with the yield 0.58 g/g at 40 °C. Meanwhile, the two yeasts both had the ability to use arabinose naturally, but K. marxianus CICC 1727-5 could consume arabinose completely and quickly. Furthermore, the two yeasts both could ferment glucose and xylose simultaneously, but K. marxianus CICC 1727-5 showed much better performance in the cofermentation. The peak ethanol concentration of K. marxianus CICC 1727-5 and S. passalidarum ATCC MYA-4345 was 42.6 and 31.9 g/L, respectively. In the saccharification and cofermentation (SSCF) process using non-detoxificated corncob, K. marxianus CICC 1727-5 showed better performance. K. marxianus CICC 1727-5 was more tolerant in the presence of formic acid, acetic acid, and mix inhibitors and even was capable to grow in the medium with the acetic acid concentration up to 15 g/L. K. marxianus CICC 1727-5 is a promising candidate strain for further metabolic engineering to develop robust industrial strains for the lignocellulosic ethanol.
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The horned passalus (Odontotaenius disjunctus) is one of the most extensively studied saproxylic beetles in the eastern United States. For several decades this species has been the subject of investigations into the behaviors associated with subsociality as well as physiological responses to stress, and, most recently, the composition of its gut microbiome has been closely examined. However, no published study to date has characterized this beetle’s broad-scale population genetic structure. Here, we conducted intensive geographic sampling throughout the southern Appalachian Mountains and surrounding areas and then assessed mitochondrial DNA (mtDNA) sequence variation among individuals. Unexpectedly, we discovered two divergent, yet broadly sympatric, mtDNA clades. Indeed, the magnitude of divergence between- vs. within-clades ranged from 5.9 to 7.5×, depending on the dataset under consideration, and members of the two lineages were often syntopic (i.e., found in the same rotting log). Given the potential implications for past and future studies on behavior, physiology, and the gut microbiome, we developed a simple cost-efficient molecular assay (i.e., polymerase chain reaction restriction fragment length polymorphism; PCR-RFLP) to rapidly determine mtDNA clade membership of O. disjunctus individuals. We suggest that the evolutionary processes that gave rise to the emergence and persistence of divergent sympatric lineages reported here warrant investigation, as this type of spatial-genetic pattern appears to be rare among southern Appalachian forest invertebrates.
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First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G-E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2, XKS1 genes and a mutant ([4-59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G-E2G production process.
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In recent years, many novel xylose-fermenting yeasts belonging to the new genus Spathaspora have been isolated from the gut of wood-feeding insects and/or wood-decaying substrates. We have cloned and expressed, in Saccharomyces cerevisiae, a Spathaspora arborariae xylose reductase gene (SaXYL1) that accepts both NADH and NADPH as co-substrates, as well as a Spathaspora passalidarum NADPH-dependent xylose reductase (SpXYL1.1 gene) and the SpXYL2.2 gene encoding for a NAD+-dependent xylitol dehydrogenase. These enzymes were co-expressed in a S. cerevisiae strain over-expressing the native XKS1 gene encoding xylulokinase, as well as being deleted in the alkaline phosphatase encoded by the PHO13 gene. The S. cerevisiae strains expressing the Spathaspora enzymes consumed xylose, and xylitol was the major fermentation product. Higher specific growth rates, xylose consumption and xylitol volumetric productivities were obtained by the co-expression of the SaXYL1 and SpXYL2.2 genes, when compared with the co-expression of the NADPH-dependent SpXYL1.1 xylose reductase. During glucose-xylose co-fermentation by the strain with co-expression of the SaXYL1 and SpXYL2.2 genes, both ethanol and xylitol were produced efficiently. Our results open up the possibility of using the advantageous Saccharomyces yeasts for xylitol production, a commodity with wide commercial applications in pharmaceuticals, nutraceuticals, food and beverage industries.
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During the pretreatment and hydrolysis of lignocellulosic biomass to obtain a hydrolysate rich in fermentable sugars, furaldehydes (furfural and hydroxymethylfurfural), phenolic compounds, and organic acids are formed and released. These compounds inhibit yeast metabolism, reducing fermentation yields and productivity. This study initially confirmed the ability of Spathaspora passalidarum to ferment xylose and demonstrated its sensibility to the inhibitors present in the hemicellulosic sugarcane bagasse hydrolysate. Then, an adaptive laboratory evolution, with progressive increments of hydrolysate concentration , was employed to select a strain more resistant to hydrolysate inhibitors. Afterward, a central composite design was performed to maximize ethanol production using hydroly-sate as substrate. At optimized conditions (initial cell concentration of 30 g/L), S. passalidarum was able to produce 19.4 g/L of ethanol with productivity, yield, and xylose consumption rate of 0.8 g/L.h and 0.4 g/g, respectively, in a sugarcane bagasse hemicellulosic hydrolysate. A kinetic model was developed to describe the inhibition of fermentation by substrate and product. The values obtained for substrate saturation and inhibition constant were K s = 120.4 g/L and K i = 1293.4 g/L. Ethanol concentration that stops cell growth was 30.1 g/L. There was an agreement between simulated and experimental results, with a residual standard deviation lower than 6%.
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We investigated the fermentation of a mixture of oat and soybean hulls (1:1) subjected to acid (AH) or enzymatic (EH) hydrolyses, with both showing high osmotic pressures (> 1200 Osm kg−1) for the production of ethanol. Yeasts of genera Spathaspora, Scheffersomyces, Sugiymaella, and Candida, most of them biodiverse Brazilian isolates and previously untested in bioprocesses, were cultivated in these hydrolysates. Spathaspora passalidarum UFMG-CM-469 showed the best ethanol production kinetics in suspended cells cultures in acid hydrolysate, under microaerobic and anaerobic conditions. This strain was immobilized in LentiKats® (polyvinyl alcohol) and cultured in AH and EH. Supplementation of hydrolysates with crude yeast extract and peptone was also performed. The highest ethanol production was obtained using hydrolysates supplemented with crude yeast extract (AH-CYE and EH-CYE) showing yields of 0.40 and 0.44 g g−1, and productivities of 0.39 and 0.29 g (L h)−1, respectively. The reuse of the immobilized cells was tested in sequential fermentations of AH-CYE, EH-CYE, and a mixture of acid and enzymatic hydrolysates (AEH-CYE) operated under batch fluidized bed, with ethanol yields ranging from 0.31 to 0.40 g g−1 and productivities from 0.14 to 0.23 g (L h)−1. These results warrant further research using Spathaspora yeasts for second-generation ethanol production.
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We describe two related species of Metschnikowia isolated from Conotelus spp. (Coleoptera: Nitidulidae) of Costa Rican Convolvulacae. Metschnikowia similis sp. nov. is a sister species to Metschnikowia dekortorum (Lachance & Bowles 2002). These two species are distinguishable only by the ability to grow in the presence of 5 % NaCl, the sterility of hybrid asci, and rDNA sequencing. Metschnikowia colocasiae sp. nov. is a sister species to Metschnikowia arizonensis and can be differentiated from other species by a combination of the utilization of D-gluconate and L-lysine, growth at 34°C, and by the lack of ascospore formation in hybrids. The two new species occur in nature as haploid mating types and form acicular ascospores that reach 50 to 100 μm in length.
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Two new haplontic heterothallic species of Metschnikowia were isolated from floricolous insects and flowers. Metschnikowia lochheadii was recovered from insects found in various flowers on the Hawaiian Islands of Kauai and Maui, and from Conotelus sp. (Coleoptera: Nitidulidae) in northwestern Guanacaste Province, Costa Rica. The morphology, physiology, and sexual cycle are typical of the large-spored Metschnikowia species, and the partial ribosomal DNA large subunit (D1D2) sequences suggest that the new species is most closely related to Candida ipomoeae. Metschnikowia lochheadii is nearly indistinguishable from its ascogenous relatives and conjugates freely with Metschnikowia continentalis, forming sterile asci. It also exhibits asymmetric mating with Metschnikowia hawaiiensis. Metschnikowia drosophilae was found in morning glory (Ipomoea sp.) flowers and associated Drosophila bromeliae on Grand Cayman Island. Its nutritional profile is atypical of the genus, being the only species that does not utilize sucrose or maltose as carbon sources, and one of the few that does not utilize melezitose. D1D2 sequences show that Metschnikowia drosophilae is a sister species to Candida torresii, to which it bears considerable similarity in nutritional profile. The type cultures are: Metschnikowia lochheadii, strains UWO(PS)00-133.2 = CBS 8807 (h+, holotype) UWO(PS)99-661.1 = CBS 8808 (h-, isotype); and Metschnikowia drosophilae, strains UWO(PS)83-1135.3 = CBS 8809 (h+, holotype) and UWO(PS)83-1143.1 = CBS 8810 (h-, isotype).
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Over the past century the recognition of the presence of endosymbionts in a variety of arthropods has become well established (Buchner, 1965). Intense interest in the rickettsial endosymbionts, widespread among insects (van Meer italic., 1999), led to the discovery that they may induce sterility of the host, and increased rates of speciation have been attributed to their presence (Shoemaker italic., 1999). Bacteria also have long been known for their nutritional contributions to insects, but more recently indigenous gut bacteria have been recognized for their ability to prevent colonization of non-indigenous microbes. In fact the insect gut is considered a ‘hot spot’ of bacterial gene exchange and bacterial adaptation (Dillon & Dillon, 2004). Thus, important attributes that affect speciation, habitat utilization, and survival are provided by prokaryotic symbionts. By contrast, although there were a number of early reports of fungal endosymbionts of insects, fewer of them were substantiated after the original reports (Buchner, 1965). More recent work, however, indicates that insect–yeast interactions abound in nature, although the exact nature of many of the interactions is less well understood (Suh & Blackwell, 2005; Vega & Dowd, 2005). Yeasts and yeast-like fungi from the guts of a small group of planthoppers (Homoptera) and beetles in three families (Coleoptera: Anobiidae, Cerambycidae and Scolytidae) have been studied most extensively. These fungi have a yeast growth form with single cells and asexual reproduction by budding, the hallmark of the ‘yeast habit’, although many yeasts actually have filamentous growth as well. © Cambridge University Press 2007 and Cambridge University Press, 2009.
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This chapter focuses on yeast ecology in general, and its possible future developments. Yeasts do not occur randomly throughout the biosphere. They form communities of species. Each community may be defined by its habitat, which is the actual place where an assemblage of yeasts lives, and by the niches of its component species. The niche consists of the attributes that make yeast capable of sharing a habitat with other members of the community. Thus, the niche is the sum of all physical, chemical, or biotic factors required for successful existence. The members of a community may have different niches that happen to overlap in a given habitat. By their very nature, yeasts are generally limited in the range of habitats they can occupy. In addition to various mineral nutrients that serve as building blocks for most of their cell constituents, yeasts require significant amounts of an organic source of carbon and energy of relatively small molecular weight. The high surface/volume ratio of yeasts favors rapid nutrient absorption. Their unicellular nature often makes them better suited for deep liquid substrates or moist and uneven surfaces, unlike molds, which form hyphae that can penetrate and liquefy semisolid substrates or spread over smooth, inert surfaces. Yeasts usually grow over a broad range of pH values, allowing them in particular to colonize materials that have already been the site of fermentative activities by bacteria. Yeasts also fulfill an important role in the food chain. Insects not only feed on substrates known to serve as yeast habitats, but also exhibit several adaptations to yeast utilization as a feedstock.
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— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
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'BLAST 2 SEQUENCES', a new BLAST-based tool for aligning two protein or nucleotide sequences, is described. While the standard BLAST program is widely used to search for homologous sequences in nucleotide and protein databases, one often needs to compare only two sequences that are already known to be homologous, coming from related species or, e.g. different isolates of the same virus. In such cases searching the entire database would be unnecessarily time-consuming. 'BLAST 2 SEQUENCES' utilizes the BLAST algorithm for pairwise DNA-DNA or protein-protein sequence comparison. A World Wide Web version of the program can be used interactively at the NCBI WWW site (http://www.ncbi.nlm.nih.gov/gorf/bl2.html). The resulting alignments are presented in both graphical and text form. The variants of the program for PC (Windows), Mac and several UNIX-based platforms can be downloaded from the NCBI FTP site (ftp://ncbi.nlm.nih.gov).
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Publisher Summary This chapter focuses on the methods used for the isolation, maintenance, and identification of yeasts. Yeasts have been recovered from widely differing aquatic and terrestrial sources, as well as from the atmosphere. Many types of yeast occur widely, whereas some appear to be confined to restricted habitats. Yeasts seldom occur in the absence of either molds or bacteria. Consequently, selective techniques are often used for recovery of yeasts, employing media which permit the yeast to grow while suppressing molds and bacteria. The composition of such media is determined by the fact that yeasts are, as a rule, capable of developing at pH levels and water activities, which reduce or inhibit the growth of bacteria. Antibiotics may also be used to suppress bacteria. When yeasts are present in low numbers, their isolation may require enrichment using media and conditions which favor the growth of yeasts over other microorganisms. Yeast cultures are best maintained on a medium which contains glucose as the only source of carbon as this reduces the risk of changes in growth and fermentative patterns due to the selection of mutants. Many basidiomycetous yeasts do not survive well during prolonged storage on a glucose-peptone medium, although they grow well on it. Potato-dextrose agar is used when cultures of such yeasts are to be kept for a long time. The majority of yeasts may be stored at temperatures between 4 and 12° C and subcultured at intervals of 6 to 8 months. Yeasts such as Arxiozyma and Malassezia, may have to be subcultured every month.
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Xylose utilization is essential for the efficient conversion of lignocellulose to ethanol. The objective of this review is to trace the development of xylose-fermenting yeast strains from their discovery in 1980. following initial reports, screens of known yeast identified five species of interest: Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis, Candida shehatae strains can be deivided into three varieties. Pachysolen tannophilus and Pichia stipilis have been studied most extensively and have the best-understood genetic systems. Improved mutants of P. tannophilis have been obtained by selecting for an inability to oxidize ethanol (eth) and for rapid growth on xylitol and nitrate,. Improved P. stipitis mutants have been obtained by selecting for flocculation decreased utilization of glucose,a nd growth on noninductive carbon sources. Bacterial xylose isomerase has been cloned and expressed in S. cerevisiae and Schizosaccharomyces pombe, but he heterologous enzyme is inactive. Xylose reductase and xylitol dehydrogenase have been cloned from P. stipitis and expressed in Saccharomyces cerevisiae, giveing rise to transformant S. cerevisiae that grow on oxylose but that ferment it poorly. A transformation and expression system based on the URA3 marker has recently been developed for P. stipilitis so that contemporary genetic methods may be brough to bear on this organism.
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Approximately 500 species of ascomycetous yeasts, including members of Candida and other anamorphic genera, were analyzed for extent of divergence in the variable D1/D2 domain of large subunit (26S) ribosomal DNA. Divergence in this domain is generally sufficient to resolve individual species, resulting in the prediction that 55 currently recognized taxa are synonyms of earlier described species. Phylogenetic relationships among the ascomycetous yeasts were analyzed from D1/D2 sequence divergence. For comparison, the phylogeny of selected members of the Saccharomyces clade was determined from 18S rDNA sequences. Species relationships were highly concordant between the D1/D2 and 18S trees when branches were statistically well supported.
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'BLAST 2 Sequences', a new BLAST-based tool for aligning two protein or nucleotide sequences, is described. While the standard BLAST program is widely used to search for homologous sequences in nucleotide and protein databases, one often needs to compare only two sequences that are already known to be homologous, coming from related species or, e.g. different isolates of the same virus. In such cases searching the entire database would be unnecessarily time-consuming. 'BLAST 2 Sequences' utilizes the BLAST algorithm for pairwise DNA-DNA or protein-protein sequence comparison. A World Wide Web version of the program can be used interactively at the NCBI WWW site (http://www.ncbi.nlm.nih.gov/gorf/bl2.++ +html). The resulting alignments are presented in both graphical and text form. The variants of the program for PC (Windows), Mac and several UNIX-based platforms can be downloaded from the NCBI FTP site (ftp://ncbi.nlm.nih.gov).
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A new haplontic heterothallic species of Metschnikowia and two related asexual yeast species were discovered in morning glory flowers and associated insects. Metschnikowia santaceciliae came from Conotelus (Coleoptera: Nitidulidae) and other insect species associated with flowers of Ipomoea indica (purple morph) in Costa Rica. Candida hawaiiana and Candida kipukae were found in I. indica (syn. I. acuminata) and its insects in Hawai'i, and the former was also isolated in a specimen of Conotelus collected on Merremia tuberosa (Convolvulaceae) in Costa Rica. The three species have nearly identical physiological profiles, typical of the genus Metschnikowia. The sequences of the D1/D2 domains of their large subunit ribosomal DNA confirm that the species belong to the Metschnikowia clade, even though they share a very low degree of inter-relatedness. M. santaceciliae is a sister species to Metschnikowia continentalis. C. kipukae is a basal member of the large-spored Metschnikowia subclade, and C