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Yeast immobilization in beer fermentation has recently regained attention, due to the expansion of the craft beer market and the diversification of styles and flavors. The aim of this study was to evaluate the physiological differences between immobilized and free yeast cells with a focus on flavor-active compounds formation. Three strains of Saccharomyces spp. (SY025, SY067, SY001) were evaluated in both free and immobilized (using a cellulose-based support, referred as ImoYeast) forms during static batch fermentations of 12 °P malt extract. Immobilized cells showed higher glycerol (SY025, 40%; SY067, 53%; SY001, 19%) and biomass (SY025, 67%; SY067, 78%; SY001, 56%) yields than free cells. Conversely, free cells presented higher ethanol yield (SY025, 9%; SY067, 9%; SY001, 13%). Flavor-active compounds production exhibited significant alterations between immobilized and free cells systems, for all strains tested. Finally, a central composite design with varying initial biomass (X0) and substrate (S0) concentrations was conducted using strain SY025, which can be helpful to modulate the formation of one or more flavor-active compounds. In conclusion, yeast immobilization in the evaluated support resulted in flavor alterations that can be exploited to produce different beer styles.

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Androsta-4-ene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD), and 9α-hydroxy-4-androstene-3,17-dione (9-OHAD), which belong to C-19 steroids, are critical steroid-based drug intermediates. The biotransformation of phytosterols into C-19 steroids by Mycolicibacterium cell factories is the core step in the synthesis of steroid-based drugs. The production performance of engineered mycolicibacterial strains has been effectively enhanced by sterol core metabolic modification. In recent years, research on the non-core metabolic pathway of steroids (NCMS) in mycolicibacterial strains has made significant progress. This review discusses the molecular mechanisms and metabolic modifications of NCMS for accelerating sterol uptake, regulating coenzyme I balance, promoting propionyl-CoA metabolism, reducing reactive oxygen species, and regulating energy metabolism. In addition, the recent applications of biotechnology in steroid intermediate production are summarized and compared, and the future development trend of NCMS research is discussed. This review provides powerful theoretical support for metabolic regulation in the biotransformation of phytosterols.

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AIMS: While gas-fermenting acetogens have been engineered to secrete non-native metabolites such as butyrate, acetate remains the most thermodynamically favourable product. An alternative to metabolic engineering is to exploit native capabilities for CO-to-acetate conversion by coculturing an acetogen with a second bacterium that provides efficient acetate-butyrate conversion. METHODS AND RESULTS: We used dynamic metabolic modelling to computationally evaluate the CO-to-butyrate conversion capabilities of candidate coculture systems by exploiting the diversity of human gut bacteria for anaerobic synthesis of butyrate from acetate and ethanol. A preliminary screening procedure based on flux balance analysis was developed to identify 48 gut bacteria which satisfied minimal growth rate and acetate-to-butyrate conversion requirements when cultured on minimal medium containing acetate and a simple sugar not consumed by the paired acetogen. A total of 170 acetogen/gut bacterium/sugar combinations were dynamically simulated for continuous growth using a 70/30 CO/CO2 feed gas mixture and minimal medium computationally determined for each combination. CONCLUSIONS: While coculture systems involving the acetogens Eubacterium limosum or Blautia producta yielded low butyrate productivities and CO-to-ethanol conversion had minimal impact on system performance, dynamic simulations predicted a large number of promising coculture designs with Clostridium ljungdahlii or C. autoethanogenum as the CO-to-acetate converter. Pairings with the gut bacterium Clostridium hylemonae or Roseburia hominis were particularly promising due to their ability to generate high butyrate productivities over a range of dilution rates with a variety of sugars. The higher specific acetate secretion rate of C. ljungdahlii proved more beneficial than the elevated growth rate of C. autoethanogenum for coculture butyrate productivity. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study demonstrated that metabolic modelling could provide useful insights into coculture design that can guide future experimental studies. More specifically, our predictions generated several favourable designs, which could serve as the first coculture systems realized experimentally.

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AIMS: To evaluate the production of biosurfactants by fungi isolated from the Amazonian species Piper hispidum (Piperaceae), and to determine the physico-chemical properties of the crude biosurfactant obtained from the most promising fungi. METHODS AND RESULTS: A total of 117 endophytic fungi were isolated, and 50 were used to verify the production of biosurfactants. Of these, eight presented positive results in the drop collapse test, and emulsification index ranging from 20 to 78%. The most promising fungi, Ph III 23L and Ph II 22S (identified as Aspergillus niger and Glomerella cingulata, respectively) were recultivated for extraction and analysis of the biosurfactant's physico-chemical characteristics. The cultivation broth that presented the greatest decrease in surface tension (36%) was that of the A. niger, which reduced it from 68·0 to 44·0 mN m-1 . The lowest critical micellar concentration value was found for the same endophyte (14·93 mg ml-1 ). CONCLUSIONS: Endophytes of P. hispidum proved to be interesting producers of biosurfactants and presented promising physico-chemical characteristics for applications in diverse industrial sectors. SIGNIFICANCE AND IMPACT OF THE STUDY: Piper hispidum endophytic fungi can be used as a new source of biosurfactants, as these molecules present a significant market due to their wide industrial applications.

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The health enhancer yeast Saccharomyces cerevisiae (SC) is widely used in diets for different animals. Two main types of SC-based products are commercially available, one containing live yeasts and one containing SC fermentation by-products, which are supposedly not dependent on live yeasts for their physiological effects in vivo. Culture-based techniques were applied to study yeasts in two types of commercial products: a product containing live SC (LSC) and a SC fermentation product (SCFP). Three temperatures (25, 30 and 39°C) and two pH levels (4 and 7) were tested. The product with LSC contained an average of 1·21 × 109 colony-forming units (CFUs) of yeasts per g contents (min: 1 × 108 , max: 3 × 109 ). In contrast, the SCFP contained an average of 4·67 × 103 (min: 3 × 102 , max: 1·9 × 104 ) CFUs per g contents (c. 1 million times less than the concentration of yeasts in the product with LSC). Both temperature and pH level affected the number of CFUs but this effect differed between the two products. Biochemical tests identified the two yeasts as SC, which differed in their ability to ferment maltose (negative in the SCFP). This report encourages more research on commercial microbial strains for animal nutrition that can lead to a better understanding of their mode of action in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: Probiotics (or direct fed microbials) are increasingly popular in Animal Nutrition. Different products containing live micro-organisms or microbial-derived products are commercially available to enhance health and boost commercial traits. The characteristics of these products dictate their physiological effects and determine their potential to increase profitability from livestock. For the first time, this report presents data about the numbers and phenotype of the health enhancer Saccharomyces cerevisiae in two widely available commercial products in Animal Nutrition. These findings may be useful for scientists and producers around the globe and have the potential to open up novel venues for research.

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Selenium-enriched yeast (selenium yeast) are one of the most popular sources of selenium supplementation used in the agriculture and human nutritional supplements industries. To enhance the production efficiency of selenium yeast, we sought to develop a method to identify, and ultimately select for, strains of yeast with enhanced selenium accumulation capabilities. Selenite resistance of four genetically diverse strains of Saccharomyces cerevisiae was assayed in various conditions, including varying carbon sources, nitrogen sources, and phosphate amounts, and they were correlated with selenium accumulation in a commercially relevant selenium-containing growth medium. Glycerol- and selenite-containing media was used to select for six yeast isolates with enhanced selenite resistance. One isolate was found to accumulate 10-fold greater selenium (0.13 to 1.4 mg Se g-1 yeast) than its parental strain. Glycerol- and selenium-containing medium can be used to select for strains of yeast with enhanced selenium accumulation capability. The methods identified can lead to isolation of industrial yeast strains with enhanced selenium accumulation capabilities that can result in greater cost efficiency of selenium yeast production. Additionally, the selection method does not involve the construction of transgenic yeast, and thus produces yeasts suitable for use in human food and nutrient supplements.

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AIMS: This study aimed to characterize yeasts isolated from the environment of artisanal cachaça production and brewer's spent grain-bearing in mind their further application in bioprocesses. METHODS AND RESULTS: Cell morphology, growth and fermentative parameters, and karyotyping were employed for the selection and grouping of yeast strains. The results showed that from 134 yeast strains studied, 14·2% exhibited cells with snowflake morphology, which is not appropriate for bioethanol production. The fermentation in sugarcane syrup was carried out with 71 Saccharomyces cerevisiae, 19 Torulaspora delbrueckii, eight Wickerhamomyces anomalus, six Candida parapsilosis, five Pichia mashurica, three Candida intermedia, two Clavispora lusitaniae and one Candida aaseri. Among the most important ethanol-producing strains, T. delbrueckii LMQA BSG 7 and S. cerevisiae LMQA SNR 65 presented biomass yield, ethanol yield and productivity similar or higher than PE-2 and CAT-1 (bioethanol industrial strains). CONCLUSIONS: This study showed a high potential for industrial application of the strains LMQA SNR 65 (S. cerevisiae) and LMQA BSG 7 (T. delbrueckii). It was found that the use of the chromosomal profile is not adequate to qualify yeasts concerning their technological performance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reported yeasts isolated from uncommon sources that present significant characteristics for potential application in bioprocesses.

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In this work the fermentation performances of seven vineyard strains, together with the industrial strain EC1118, have been investigated at three differing yeast assimilable nitrogen (YAN) concentrations (300 mg N l-1 , 150 mg N l-1 and 70 mg N l-1 ) in synthetic musts. The results indicated that the response to different nitrogen levels is strain dependent. Most of the strains showed a dramatic decrease of the fermentation at 70 mg N l-1 but no significant differences in CO2 production were found when fermentations at 300 mg N l-1 and 150 mg N l-1 were compared. Only one among the vineyard strains showed a decrease of the fermentation when 150 mg N l-1 were present in the must. These results contribute to shed light on strain nitrogen requirements and offer new perspectives to manage the fermentation process during winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected vineyard Saccharomyces cerevisiae strains can improve the quality and the complexity of local wines. Wine quality is also influenced by nitrogen availability that modulates yeast fermentation activity. In this work, yeast nitrogen assimilation was evaluated to clarify the nitrogen requirements of vineyard strains. Most of the strains needed high nitrogen levels to express the best fermentation performances. The results obtained indicate the critical nitrogen levels. When the nitrogen concentration was above the critical level, the fermentation process increased, but if the level of nitrogen was further increased no effect on the fermentation was found.

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AIMS: Identify novel bacterial taxa that could increase the availability of branched-chain amino acids and the amount of distinctive volatiles during skim milk fermentation. METHODS AND RESULTS: We recovered 344 bacterial isolates from stool samples of healthy and breastfed infants. Five were selected based on their ability to produce branched-chain amino acids. Three strains were identified as Escherichia coli, one as Klebsiella pneumoniae and other as Klebsiella variicola by molecular and biochemical methods. HPLC and solid-phase microextraction with GC-MS were used for the determination of free amino acids and volatile compounds respectively. The consortium formed by K. variicola and four Lactobacillus species showed the highest production of Leu and Ile in skim milk fermentation. In addition, the production of volatile compounds, such as acetoin, ethanol, 2-nonanone, and acetic, hexanoic and octanoic acids, increased in comparison to commercial yogurt, Emmental and Gouda cheese. Also, distinctive volatiles, such as 2,3-butanediol, 4-methyl-2- hexanone and octanol, were identified. CONCLUSION: The use of K. variicola in combination with probiotic Lactobacillus species enhances the availability of Leu and Ile and the amount of distinctive volatiles during skim milk fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The identified consortium increases the functional potential of fermented dairy products.

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In this study we determined the influence of different sugar concentration in media, time of rehydration and type of strain on relative expression level of GPD1 and SIP18 genes of active dry cider-making yeast strains, followed by the assessment of the impact of rehydration on the fermentation process. High expression of SIP18 at the beginning of rehydration was shown to be due to high transcription of the gene during the drying process. High sugar concentrations of media initiated transcription of the GPD1 gene and triggered the cellular glycerol biosynthesis pathway in examined strains. Rehydration time and type of strain showed to have no statistically significant impact on the course of the fermentation; RT qPCR results depended mainly on the time of rehydration and sugar concentration of the medium. This is the first attempt to confront rehydration time and molecular mechanisms acting upon rehydration with the course of the fermentation process.

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Anaerobic digestion of organic residues offers economic benefits via biogas production, still methane (CH4 ) yield relies on the development of a robust microbial consortia for adequate substrate degradation, among other factors. In this study, we monitor biogas production and changes in the microbial community composition in two semi-continuous stirred tank reactors during the setting process under mesophilic conditions (35°C) using a 16S rDNA high-throughput sequencing method. Reactors were initially inoculated with anaerobic granular sludge from a brewery wastewater treatment plant, and gradually fed organic urban residues (4·0 kg VS m-3  day-1 ) . The inocula and biomass samples showed changes related to adaptations of the community to urban organic wastes including a higher relative proportion of Clostridiales, with Ruminococcus spp. and Syntrophomonas spp. as recurrent species. Candidatus Cloacamonas spp. (Spirochaetes) also increased from ~2·2% in the inoculum to >10% in the reactor biomass. The new community consolidated the cellulose degradation and the propionate and amino acids fermentation processes. Acetoclastic methanogens were more abundant in the reactor, where Methanosaeta spp. was found as a key player. This study demonstrates a successful use of brewery treatment plant granular sludge to obtain a robust consortium for methane production from urban organic solid waste in Mexico. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the selection of relevant bacteria and archaea in anaerobic digesters inoculated with anaerobic granular sludge from a brewery wastewater treatment plant. Generally, these sludge granules are used to inoculate reactors digesting organic urban wastes. Though, it is still not clearly understood how micro-organisms respond to substrate variations during the reactor start-up process. After feeding two reactors with organic urban residues, it was found that a broader potential for cellulose degradation was developed including Bacteroidetes, Firmicutes and Spirochaetes. These results clarify the bacterial processes behind new reactors establishment for treating organic wastes in urban areas.

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AIMS: The tyraminogenic potential of the strains Enterococcus faecalis EF37 and ATCC 29212 was investigated in a synthetic medium containing defined amounts of tyrosine and phenylalanine at different temperatures. METHODS AND RESULTS: Enterococci growth and the production of biogenic amines (BA) were evaluated in relation to their pre-growth in medium containing tyrosine. Significant differences between the two strains were evidenced at metabolic level. Both the pre-adapted strains grew faster in all the tested conditions, independently of the presence of the precursor. Temperatures of 30 and 40°C positively affected the growth parameters. The tyrosine decarboxylase (tyrDC) activity of the strain EF37 was positively affected by pre-adaptation, while ATCC 29212 showed a faster and higher tyramine accumulation with not-adapted cells. The expression analysis of the gene tyrDC confirmed the influence of the growth conditions on gene transcription. CONCLUSIONS: The small differences found between the two strains in the maximum transcript level reached rapidly after the inoculum and the different behaviour in the tyramine accumulation suggested the possible involvement of complex regulation mechanisms on the tyrDC or on the membrane transport systems, which could affect the different BA accumulation trend. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives deeper insight into the metabolic regulation of tyrDC activity of enterococci.

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Wine yeast deals with many stress conditions during its biotechnological use. Biomass production and its dehydration produce major oxidative stress, while hyperosmotic shock, ethanol toxicity and starvation are relevant during grape juice fermentation. Most stress response mechanisms described in laboratory strains of Saccharomyces cerevisiae are useful for understanding the molecular machinery devoted to deal with harsh conditions during industrial wine yeast uses. However, the particularities of these strains themselves, and the media and conditions employed, need to be specifically looked at when studying protection mechanisms.

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AIMS: To assess the influence of non-Saccharomyces yeasts on the pyranoanthocyanins and polymeric pigments formation after the addition of (+)-catechin and procyanidin B2 to fresh red grape must. METHODS AND RESULTS: The fermentation of red grape musts was done with non-Saccharomyces yeasts either alone or in sequential fermentations with the Saccharomyces cerevisiae species. The characterization of both pyranoanthocyanin and polymeric pigments has been carried out with liquid chromatography coupled to mass spectroscopy (HPLC-DAD-ESI/MS). Red wines were also characterized by infrared spectroscopy (FTIR), gas chromatography (GC-FID) and spectrophotometry (UV-Vis). It has been observed that fermentation with the species Schizosaccharomyces pombe led to higher concentrations of pigments of all types: anthocyanins, polymeric pigments and pyranoanthocyanins, particularly vitisin A. CONCLUSIONS: The use of non-Saccharomyces yeasts improve the formation of stable pigments in red wines thanks to the differences in the microbial metabolism from among the yeasts studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Colour stability as one of the main organoleptic properties in red wines, may be improved by the controlled use of selected non-Saccharomyces yeasts during red must fermentation.

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AIMS: In order to improve the quality and to create a biological basis for obtainment of the protected denomination of origin (PDO), indigenous yeast were isolated and characterized for use in Salinas city (the Brazilian region of quality cachaça production). MATERIAL AND METHODS: Seven thousand and two hundred yeast colonies from 15 Salinas city distilleries were screened based on their fermentative behaviour and the physicochemical composition of cachaça. Molecular polymorphic analyses were performed to characterize these isolates. RESULTS: Two Saccharomyces cerevisiae strains (nos. 678 and 680) showed appropriate characteristics to use in the cachaça production: low levels of acetaldehyde and methanol, and high ethyl lactate/ethyl acetate ratio respectively. They also presented polymorphic characteristics more closely related between themselves even when compared to other strains from Salinas. CONCLUSIONS: The application of selected yeast to cachaça production can contribute for the improvement of the quality product as well as be used as a natural marker for PDO. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the use of selected yeast strains could contribute to obtain a cachaça similar to those produced traditionally, while getting wide acceptation in the market, yet presenting more homogeneous organoleptic characteristics, and thus contributing to the PDO implementation.

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AIMS: In order to improve the availability of geranyl diphosphate (GPP) in the mevalonate pathway for enhancing (S)-linalool production in Saccharomyces cerevisiae. METHODS AND RESULTS: A (S)-linalool synthase (LIS): AaLS1 from Actinidia arguta was coexpressed with FPPS with different peptide linkers to redirect the flux from geranyl diphosphate (GPP) to (S)-linalool production in S. cerevisiae. The strain with the best peptide linker ((GGGGS)3 ), produced 101·55 ± 2·97 µg l(-1) (S)-linalool, a 69·7% increase compared to those with two independent LIS and FPPS expressed. In a 3-l fermenter, the (S)-linalool titre was further improved to 240·64 ± 5·31 µg l(-1) . CONCLUSIONS: The results demonstrate that the fusion proteins catalysing consecutive steps in a metabolic pathway significantly improved the (S)-linalool production with GPP as precursor. SIGNIFICANCE AND IMPACT OF THE STUDY: The fusion protein strategy co-expressing AaLS1 and FPPS, assembled with a long peptide linker made S. cerevisiae produced the highest reported (S)-Linalool titre to date.

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The main goal of our study was to evaluate the effect of the individual administration of five lyophilized lactic acid bacteria strains (Lactobacillus fermentum 428ST, Lactobacillus rhamnosus E4.2, Lactobacillus plantarum FCA3, Lactobacillus sp. 34.1, Weissella paramesenteroides FT1a) against the in vitro simulated microbiota of the human colon using the GIS1 system. The influence on the metabolic activity was also assessed by quantitative determination of proteins and polysaccharides at each segment of human colon. The obtained results indicated that the lactic acid bacteria L. rhamnosus E4.2 and W. paramesenteroides FTa1 had better efficiency in synthesising exopolysaccharides and also a better probiotic potential and therefore could be recommended for use in probiotics products or food industry.

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UNLABELLED: Coffee is among the most preferred nonalcoholic drinks, and its consumption is distributed globally. During the coffee fruiting process, however, a large amount of waste is generated in the form of pulp, mucilage, husks, and water waste. The pulp and mucilage have the chemical composition to support the growth of micro-organisms and the production of value-added product. The aim was testify pulp coffee can be considered as carbon and inductor source for ß-glucosidase by Bacillus subtilis CCMA 0087. The response surface methodology (RSM) based on a central composite rotatable design (CCRD) was employed for this optimization. The methodology used in the optimization process was validated by testing the best conditions obtained and comparing them with the values predicted by the model. The highest ß-glucosidase production (22·59 UI ml(-1) ) was reached in 24 h of culturing at coffee pulp concentration of 36·8 g l(-1) , temperature of 36·6°C, and pH of 3·64. SIGNIFICANCE AND IMPACT OF THE STUDY: Countries whose economy is based on agricultural activities generate a great deal of liquid and solid waste. Thus, it is important to develop new alternatives for using this waste rather than disposing it in the environment. The production of enzymes, and particularly cellulase, is one such alternative. In this study, we proposed to produce ß-glucosidase production from pulp coffee extract using a Bacillus subtilis strain.

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Deinococcus spp are among the most radiation-resistant micro-organisms that have been discovered. They show remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation and oxidizing agents. Traditionally, Escherichia coli and Saccharomyces cerevisiae have been the two platforms of choice for engineering micro-organisms for biotechnological applications, because they are well understood and easy to work with. However, in recent years, researchers have begun using Deinococcus spp in biotechnologies and bioremediation due to their specific ability to grow and express novel engineered functions. More recently, the sequencing of several Deinococcus spp and comparative genomic analysis have provided new insight into the potential of this genus. Features such as the accumulation of genes encoding cell cleaning systems that eliminate organic and inorganic cell toxic components are widespread among Deinococcus spp. Other features such as the ability to degrade and metabolize sugars and polymeric sugars make Deinococcus spp. an attractive alternative for use in industrial biotechnology.

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Phenyllactic acid (PLA) is an antimicrobial compound naturally synthesized in various fermented foods and its D-form of PLA is known to be more active than the L-isomer. In this study, Leuconostoc mesenteroides ATCC 8293 cells, elaborating D-lactate dehydrogenase (D-ldh) were used to produce D-PLA from phenylpyruvic acid (PPA). When cultured in the presence of PPA (≤50 mmol l(-1)), growing cells produced a maximum yield of 35 mmol l(-1) of D-PLA, and the yields were between 75·2 and 83·3%. Higher conversion yields were obtained at pH 6·0-7·0 when growing cells were used, while the optimum pH range was broader for resting cells. The time required for the complete conversion of PPA into PLA could be shortened to 3 h using resting cells. D-ldh, an enzyme encoded by the LEUM_1756 gene of Leuc. mesenteroides ATCC 8293, was found to be responsible for the conversion of PPA into PLA. The Km and kcat values of the enzyme for PPA were found to be 15·4 mmol l(-1) and 5645 s(-1), respectively. The conditions required for the efficient production of D-PLA were optimized for both growing and resting cells of Leuc. mesenteroides, with special emphasis on achieving high stereoselectivity and conversion yield. Significance and impact of the study: This is the first study on the production of D-phenyllactic acid, which is a natural antimicrobial compound, from phenylpyruvate using Leuconostoc mesenteroides cells. The strain, ATCC 8293, that was used in the study, possesses high stereoselectivity and delivers a high yield. Therefore, it might be a promising candidate for use in large-scale production facilities and in fermented foods.

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