13 research outputs found
Screening For Yeast Phytase Leads to the Identification of a New Cell-Bound and Secreted Activity in Cyberlindnera jadinii CJ2
Get PDFPhytic acid is an anti-nutritional compound able to chelate proteins and ions. For this reason, the food industry is looking for a convenient method which allows its degradation. Phytases are a class of enzymes that catalyze the degradation of phytic acid and are used as additives in feed-related industrial processes. Due to their industrial importance, our goal was to identify new activities that exhibit best performances in terms of tolerance to high temperature and acidic pH. As a result of an initial screening on 21 yeast species, we focused our attention on phytases found in Cyberlindnera jadinii, Kluyveromyces marxianus, and Torulaspora delbrueckeii. In particular, C. jadinii showed the highest secreted and cell-bound activity, with optimum of temperature and pH at 50°C and 4.5, respectively. These characteristics suggest that this enzyme could be successfully used for feed as well as for food-related industrial applications
APPLICATION OF NON-CONVENTIONAL YEASTS IN BIOPROCESSES
Get PDFSustainability is one of the most pressing challenge of our century, this term is becoming a main keyword of political agendas and more in general of mass media. To increase the “greenness of bioprocesses”, academia and industry, especially in the biotechnological and chemical fields, are focusing their studies with the scope to shift from traditional organic synthesis to new processes with reduced ecological foot-print. A good way to increase sustainability could be set up bioprocesses exploiting microorganisms. Nowadays, companies are searching new organisms that, differently from the well characterized Saccharomyces cerevisiae, show to be more resistant to the harsh conditions commonly occurring in industrial fermentations (high salt concentration, temperature and pressure). Due to their peculiar features, non-conventional yeasts (NCYs) seem to be a promising solution. On the other hand, the disadvantage to use these new organisms is related to the few studies and literature data available, especially compared to S. cervisiae. To fill this gap researchers have started to characterize these new species.
My PhD work had dual aim:
• First to identify good candidates, with specific physiological properties, that could be exploited in bioprocesses.
• Second to characterize new promising enzymatic activities useful for industrial applications.
In the first studies, I focused my attention on marine yeasts. I chose yeasts isolated from this environment, because their use gives the possibility to perform a seawater-based bioprocess saving large amount of fresh waters, reducing both cost and environmental impact. From our laboratory yeasts collection, I selected, for their halotolerance, two different Debaryomyces hansenii strains. Hence mechanisms involved in osmotic stress response have been investigated employing flow cytometry. I showed that hyper-osmotic stress elicits membrane depolarization and decreases membrane permeability to cationic compounds. This phenomenon reduces ions permeability and can negatively affect the uptake of charged substrate during bioprocesses. My research proceeded with the set up of new fermentation protocols in seawater-based media composed by a mixture of hexose and pentose sugar and cheap nitrogen sources. In these conditions we obtained high biomass yield (0.627) in 40 h of bioprocess.
In the second part of my PhD project, I studied NCYs as sources of enzymes. With this aim I identified a nitrilase of marine strain of Meyerozyma guilliermondii, that displayed high activities on aromatic substrate, but also on arylaliphatic and aliphatic ones. These activities were maintained also in presence of high salts concentration. In particular M. guilliermondii nitrilase was able to perform complete dynamic resolution of mandelonitrile in seawaters within in 8 h.
In the last part of my PhD, I identified a novel extracellular and cell-bound phytase activity in Cyberlindnera jadinii. This enzyme is suitable as feed additive, indeed activities at pH 4.5 and 37°C (animals gastric pH and temperature) were 26.25 mU/mgd.w. and 58.36 mU/mgd.w., detected as extracellular and cell-bound respectively. Phytase activities had their optimum at 50°C, reaching 37.2 mU/mgd.w. (extracellular) and 146 mU/mgd.w. (cell-bound).
Data reported in my PhD work suggest that could be interest to proceed with further characterization on NCYs. New “green” bioprocesses characterized by high productivity could be a key for reach sustainability reducing the ecological impact of industrial production
Utilization of nitrate abolishes the "Custers effect" in Dekkera bruxellensis and determines a different pattern of fermentation products
Get PDFNitrate is one of the most abundant nitrogen sources in nature. Several yeast species have been shown to be able to assimilate nitrate and nitrite, but the metabolic pathway has been studied in very few of them. Dekkera bruxellensis can use nitrate as sole nitrogen source and this metabolic characteristic can render D. bruxellensis able to overcome S. cerevisiae populations in industrial bioethanol fermentations. In order to better characterize how nitrate utilization affects carbon metabolism and the yields of the fermentation products, we investigated this trait in defined media under well-controlled aerobic and anaerobic conditions. Our experiments showed that in D. bruxellensis, utilization of nitrate determines a different pattern of fermentation products. Acetic acid, instead of ethanol, became in fact the main product of glucose metabolism under aerobic conditions. We have also demonstrated that under anaerobic conditions, nitrate assimilation abolishes the "Custers effect", in this way improving its fermentative metabolism. This can offer a new strategy, besides aeration, to sustain growth and ethanol production for the employment of this yeast in industrial processes
Bioprocesses with reduced ecological footprint by marine debaryomyces hansenii strain for potential applications in circular economy
Get PDFThe possibility to perform bioprocesses with reduced ecological footprint to produce nat-ural compounds and catalyzers of industrial interest is pushing the research for salt tolerant microorganisms able to grow on seawater‐based media and able to use a wide range of nutrients coming from waste. In this study we focused our attention on a Debaryomyces hansenii marine strain (Mo40). We optimized cultivation in a bioreactor at low pH on seawater‐based media containing a mixture of sugars (glucose and xylose) and urea. Under these conditions the strain exhibited high growth rate and biomass yield. In addition, we characterized potential applications of this yeast biomass in food/feed industry. We show that Mo40 can produce a biomass containing 45% proteins and 20% lipids. This strain is also able to degrade phytic acid by a cell‐bound phytase activity. These features represent an appealing starting point for obtaining D. hansenii biomass in a cheap and environmen-tally friendly way, and for potential use as an additive or to replace unsustainable ingredients in the feed or food industries, as this species is included in the QPS EFSA list (Quality Presumption as Safe—European Food Safety Authority)
Physiological performance of Kazachstania unispora in sourdough environments
Get PDFIn this work we explored the potential of several strains of Kazachstania unispora to be used as non-conventional yeasts in sourdough fermentation. Properties such as carbohydrate source utilization, tolerance to different environmental factors and the performance in fermentation were evaluated. The K. unispora strains are characterized by rather restricted substrate utilization: only glucose and fructose supported the growth of the strains. However, the growth in presence of fructose was higher compared to a Saccharomyces cerevisiae commercial strain. Moreover, the inability to ferment maltose can be considered a positive characteristic in sourdoughs, where the yeasts can form a nutritional mutualism with maltose-positive Lactic Acid Bacteria. Tolerance assays showed that K. unispora strains are adapted to a sourdough environment: they were able to grow in conditions of high osmolarity, high acidity and in presence of organic acids, ethanol and salt. Finally, the performance in fermentation was comparable with the S. cerevisiae commercial strain. Moreover, the growth was more efficient, which is an advantage in obtaining the biomass in an industrial scale. Our data show that K. unispora strains have positive properties that should be explored further in bakery sector. Graphic abstract: [Figure not available: see fulltext.
Engineering cytoplasmic acetyl-CoA synthesis decouples lipid production from nitrogen starvation in the oleaginous yeast Rhodosporidium azoricum
Get PDFBackground: Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. Results: In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. Conclusion: Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates
Characterization of lipid accumulation and lipidome analysis in the oleaginous yeasts Rhodosporidium azoricum and Trichosporon oleaginosus
No full textThe influence of cultural conditions on lipid production was investigated in two species, Trichosporon oleaginosus and Rhodosporidium azoricum. We showed that nitrogen limitation is not the main factor triggering the mechanism of lipid accumulation in T. oleaginosus. Moreover, a scarce availability of oxygen negatively affected lipid synthesis to a lesser extent in T. oleaginosus than in R. azoricum. This highlights how the importance of controlling fermentation parameters is strictly linked to the yeast species employed. We showed that these parameters affect the activity of important enzymes, influencing the metabolic fluxes into different pathways, in particular pentose phosphate pathway and cytoplasmic pyruvate bypass. Furthermore, T. oleaginosus exhibited wider substrate flexibility, faster growth and higher lipid accumulation in fed-batch cultivation. Microbial oils obtained from both yeasts proved a valuable feedstock, alternative to vegetable oils, for advanced diesel biofuel production
Galactose utilization sheds new light on sugar metabolism in the sequenced strain Dekkera bruxellensis CBS 2499.
Get PDFDekkera bruxellensis and Saccharomyces cerevisiae are considered two phylogenetically distant relatives, but they share several industrial relevant traits such as the ability to produce ethanol under aerobic conditions (Crabtree effect), high tolerance towards ethanol and acids, and ability to grow without oxygen. Beside a huge adaptability, D. bruxellensis exhibits a broader spectrum in utilization of carbon and nitrogen sources in comparison to S. cerevisiae. With the aim to better characterize its carbon source metabolism and regulation, the usage of galactose and the role that glucose plays on sugar metabolism were investigated in D. bruxellensis CBS 2499. The results indicate that in this yeast galactose is a non-fermentable carbon source, in contrast to S. cerevisiae that can ferment it. In particular, its metabolism is affected by the nitrogen source. Interestingly, D. bruxellensis CBS 2499 exhibits the 'short-term Crabtree effect', and the expression of genes involved in galactose utilization and in respiratory metabolism is repressed by glucose, similarly to what occurs in S. cerevisiae
Effects of oxygen availability on acetic acid tolerance and intracellular pH in Dekkera bruxellensis
Get PDFThe yeast Dekkera bruxellensis, associated with wine and beer production, has recently received attention, because its high ethanol and acid tolerance enables it to compete with Saccharomyces cerevisiae in distilleries that produce fuel ethanol. We investigated how different cultivation conditions affect the acetic acid tolerance of D. bruxellensis. We analyzed the ability of two strains (CBS 98 and CBS 4482) exhibiting different degrees of tolerance to grow in the presence of acetic acid under aerobic and oxygen-limited conditions. We found that the concomitant presence of acetic acid and oxygen had a negative effect on D. bruxellensis growth. In contrast, incubation under oxygen-limited conditions resulted in reproducible growth kinetics that exhibited a shorter adaptive phase and higher growth rates than those with cultivation under aerobic conditions. This positive effect was more pronounced in CBS 98, the more-sensitive strain. Cultivation of CBS 98 cells under oxygen-limited conditions improved their ability to restore their intracellular pH upon acetic acid exposure and to reduce the oxidative damage to intracellular macromolecules caused by the presence of acetic acid. This study reveals an important role of oxidative stress in acetic acid tolerance in D. bruxellensis, indicating that reduced oxygen availability can protect against the damage caused by the presence of acetic acid. This aspect is important for optimizing industrial processes performed in the presence of acetic acid
Marine microorganisms for biocatalysis : selective hydrolysis of nitriles with a salt-resistant strain of meyerozyma guilliermondii
Get PDFA screening among marine yeasts was carried out for nitrile hydrolyzing activity. Meyerozyma guilliermondii LM2 (UBOCC-A-214008) was able to efficiently grow on benzonitrile and cyclohexanecarbonitrile (CECN) as sole nitrogen sources. A two-step one-pot method for obtaining cells of M. guilliermondii LM2 (UBOCC-A-214008) endowed with high nitrilase activity was established; the resulting whole cells converted different nitriles with high molar conversions and showed interesting enantioselectivity toward racemic substrates. Nitrilase from M. guilliermondii LM2 (UBOCC-A-214008) displayed high activity on aromatic substrates, but also arylaliphatic and aliphatic substrates were accepted. Salt-resistant M. guilliermondii LM2 (UBOCC-A-214008) was used in media with different salinity, being highly active up to 1.5 M NaCl concentration. Finally, hydrolysis of nitriles was efficiently performed using a bioprocess (yeast growth and biotransformation with resting cells) entirely carried out in seawater
