Screening of xylose utilizing and high lipid producing yeast strains as a potential candidate for industrial application

BACKGROUND: Sustainable production of oil for food, feed, fuels and other lipid-based chemicals is essential to meet the demand of the increasing human population. Consequently, novel and sustainable resources such as lignocellulosic hydrolysates and processes involving these must be explored. In this paper we screened for naturally-occurring xylose utilizing oleaginous yeasts as cell factories for lipid production, since pentose sugar catabolism plays a major role in efficient utilization of lignocellulosic feedstocks. Glycerol utilization, which is also beneficial in yeast-based oil production as glycerol is a common by-product of biodiesel production, was investigated as well. Natural yeast isolates were studied for lipid accumulation on a variety of substrates, and the highest lipid accumulating strains were further investigated in shake flask cultivations and fermenter studies on xylose and hydrolysate. RESULTS: By collecting leaves from exotic plants in greenhouses and selective cultivation on xylose, a high frequency of oleaginous yeasts was obtained (> 40%). Different cultivation conditions lead to differences in fatty acid contents and compositions, resulting in a set of strains that can be used to select candidate production strains for different purposes. In this study, the most prominent strains were identified as Pseudozyma hubeiensis BOT-O and Rhodosporidium toruloides BOT-A2. The fatty acid levels per cell dry weight after cultivation in a nitrogen limited medium with either glucose, xylose or glycerol as carbon source, respectively, were 46.8, 43.2 and 38.9% for P. hubeiensis BOT-O, and 40.4, 27.3 and 42.1% for BOT-A2. Furthermore, BOT-A2 accumulated 45.1% fatty acids per cell dry weight in a natural plant hydrolysate, and P. hubeiensis BOT-O showed simultaneous glucose and xylose consumption with similar growth rates on both carbon sources. The fatty acid analysis demonstrated both long chain and poly-unsaturated fatty acids, depending on strain and medium. CONCLUSIONS: We found various natural yeast isolates with high lipid production capabilities and the ability to grow not only on glucose, but also xylose, glycerol and natural plant hydrolysate. R. toruloides BOT-A2 and P. hubeiensis BOT-O specifically showed great potential as production strains with high levels of storage lipids and comparable growth to that on glucose on various other substrates, especially compared to currently used lipid production strains. In BOT-O, glucose repression was not detected, making it particularly desirable for utilization of plant waste hydrolysates. Furthermore, the isolated strains were shown to produce oils with fatty acid profiles similar to that of various plant oils, making them interesting for future applications in fuel, food or feed production

On yeasts from traditional fermented foods - Characterization, phytate degradation, strain improvement and applications

Plant materials naturally contain minerals of iron, zinc and calcium. However, plants also contain a compound called phytic acid, which can chelate the minerals and form insoluble complexes. Minerals from plant foods are unavailable for intestinal uptake when they are bound in phytate complexes. Cereal-based diets low in meat, can thereby lead to micronutrient deficiencies of e.g. iron and zinc.Yeasts were isolated from food fermentations, and studied with the aim to find starter strains able to degrade phytate in cereal-based matrices. The overall phytate degrading ability of different strains was studied, followed by analysis of release of the phytase (phytate degrading) enzyme to the surrounding media by the two strains Pichia kudriavzevii TY13wt and TY1322, and of factors affecting the phytase release. Yeasts isolated from the traditionally fermented goat milk from the Yaghnob valley, Tajikistan, were genetically identified by ITS1-4 sequencing and restriction fragment length polymorphism. Phenotypic characterization was done by studying e.g. growth at various pH and temperatures, on different carbon sources, in presence of ox bile, ethanol or lactic acid, under osmotic and oxidative stress. Selected strains were used as starters for fermentation of some plant-based substrates.A prominent phytate degradation was observed from the strain P. kudriavzevii TY13wt, previously isolated from Tanzanian Togwa. Through mutagenesis by ultraviolet light exposure of strain TY13wt, mutant strain TY1322 was acquired, having improved phytate degrading ability. Strain TY1322 showed about eight times higher phytate degradation compared to wild type strain TY13wt under certain conditions. The phytase synthesis from strain TY1322 was not repressed by high phosphate levels (26mM), as opposed to the wild type strain. Both TY13wt and TY1322 were able to release phytase to the surrounding media, which was induced by yeast extract medium. Strains TY13wt and TY1322 could grow at pH 2, at 46\ub0C, in presence of 2% ox bile or 6% ethanol, and under osmotic stress. The phytase produced showed two pH optima (3.5 and 5.5) and one temperature optimum (55\ub0C). Yeast isolation from fermented goat milk of the Yaghnob valley resulted in 52 isolates belonging to Kluyveromyces marxianus (29), Pichia fermentans (12), Saccharomyces cerevisiae (10) and Kazachstania unispora (1). Characterization of the strains revealed i) two genetically different groups among the K. marxianus strains, ii) Yaghnob S. cerevisiae strains forming a separate cluster in a phylogenetic tree constructed of 350 previous S. cerevisiae isolates of various origins, iii) phenotypically intriguing traits of several strains, e.g. S. cerevisiae strains able to grow at elevated temperatures, 37\ub0C (all ten strains), 40\ub0C (9), 42\ub0C (2), and 46\ub0C (1). The phenotypic intra-species variations found among some of the Yaghnob strains may potentially indicate isolation of some new species. Finally, application of selected yeasts and lactic acid bacteria for soy milk and soy flour fermentations showed strong phytate degradation in fermentations containing strain P. kudriavzevii TY1322, and the phytate degrading effect of TY1322 was improved in co-cultures with Yaghnob strains K. marxianus AL2 or BL8.Traditional food fermentations constitute a valuable source of microbial strains. This work demonstrates the usefulness of phytase-active strains for phytate degradation in plant-based substrates, and new strain isolation revealed several strains able to grow under stress conditions that may occur during fermentation, or inside the gastrointestinal tract. This work can contribute to future strain selection for food and feed processes

On yeasts from traditional fermented foods - Characterization, phytate degradation, strain improvement and applications

Plant materials naturally contain minerals of iron, zinc and calcium. However, plants also contain a compound called phytic acid, which can chelate the minerals and form insoluble complexes. Minerals from plant foods are unavailable for intestinal uptake when they are bound in phytate complexes. Cereal-based diets low in meat, can thereby lead to micronutrient deficiencies of e.g. iron and zinc.Yeasts were isolated from food fermentations, and studied with the aim to find starter strains able to degrade phytate in cereal-based matrices. The overall phytate degrading ability of different strains was studied, followed by analysis of release of the phytase (phytate degrading) enzyme to the surrounding media by the two strains Pichia kudriavzevii TY13wt and TY1322, and of factors affecting the phytase release. Yeasts isolated from the traditionally fermented goat milk from the Yaghnob valley, Tajikistan, were genetically identified by ITS1-4 sequencing and restriction fragment length polymorphism. Phenotypic characterization was done by studying e.g. growth at various pH and temperatures, on different carbon sources, in presence of ox bile, ethanol or lactic acid, under osmotic and oxidative stress. Selected strains were used as starters for fermentation of some plant-based substrates.A prominent phytate degradation was observed from the strain P. kudriavzevii TY13wt, previously isolated from Tanzanian Togwa. Through mutagenesis by ultraviolet light exposure of strain TY13wt, mutant strain TY1322 was acquired, having improved phytate degrading ability. Strain TY1322 showed about eight times higher phytate degradation compared to wild type strain TY13wt under certain conditions. The phytase synthesis from strain TY1322 was not repressed by high phosphate levels (26mM), as opposed to the wild type strain. Both TY13wt and TY1322 were able to release phytase to the surrounding media, which was induced by yeast extract medium. Strains TY13wt and TY1322 could grow at pH 2, at 46\ub0C, in presence of 2% ox bile or 6% ethanol, and under osmotic stress. The phytase produced showed two pH optima (3.5 and 5.5) and one temperature optimum (55\ub0C). Yeast isolation from fermented goat milk of the Yaghnob valley resulted in 52 isolates belonging to Kluyveromyces marxianus (29), Pichia fermentans (12), Saccharomyces cerevisiae (10) and Kazachstania unispora (1). Characterization of the strains revealed i) two genetically different groups among the K. marxianus strains, ii) Yaghnob S. cerevisiae strains forming a separate cluster in a phylogenetic tree constructed of 350 previous S. cerevisiae isolates of various origins, iii) phenotypically intriguing traits of several strains, e.g. S. cerevisiae strains able to grow at elevated temperatures, 37\ub0C (all ten strains), 40\ub0C (9), 42\ub0C (2), and 46\ub0C (1). The phenotypic intra-species variations found among some of the Yaghnob strains may potentially indicate isolation of some new species. Finally, application of selected yeasts and lactic acid bacteria for soy milk and soy flour fermentations showed strong phytate degradation in fermentations containing strain P. kudriavzevii TY1322, and the phytate degrading effect of TY1322 was improved in co-cultures with Yaghnob strains K. marxianus AL2 or BL8.Traditional food fermentations constitute a valuable source of microbial strains. This work demonstrates the usefulness of phytase-active strains for phytate degradation in plant-based substrates, and new strain isolation revealed several strains able to grow under stress conditions that may occur during fermentation, or inside the gastrointestinal tract. This work can contribute to future strain selection for food and feed processes

Biofortification of fermented foods with selected yeasts for raised folate content and mineral availability

The goal of this project is to achieve natural biofortification of iron, zinc and folate in foods via selected yeasts. Deficiencies of iron, zinc and folate are prevalent in many parts of the world, especially where cereal based food comprises a large part of the food intake, but also in the western world where meat-free diets, such as vegetarian or vegan diets, has become more popular. Cereals contain several minerals such as iron, zinc and calcium, but it also contains the anti-nutrient phytate, which limits the bioavailability and gastrointestinal uptake of those minerals (1). Folate and mineral deficiencies are related to a range of health issues from mild to lethal (2). Deficiencies can sometimes be prevented by synthetic fortification, but this is not always a feasible solution due to e.g. availability or economy. There are also studies showing an increased risk of overdosing from synthetic supplements (3), which can lead to increased risk of developing some types of cancer (4). Fortification with synthetic folate may also mask deficiency of vitamin B12, something that can result in neurological damages (5).For mentioned reasons, it is of great significance to find ways to naturally fortify foods with folic acid, and to increase the bioavailability of the naturally present minerals in cereal based foods to limit the development of mineral deficiencies. This project focuses on natural biofortification using yeast strains that i) can degrade phytate and ii) synthetize natural folate. Yeast synthesized folates are believed to not mask vitamin B12 deficiencies (5). Phytate degrading yeasts allow natural release of minerals already present in the food matrix, hence less need for synthetic fortification and thereby less risk of overdosing

Bioethanol production using algal biomass produced on waste material as substrate

For growth and ethanol production, microorganisms need sugars, which commonly are provided from agricultural and forestry products containing starch or cellulose. There are several drawbacks with those substrates for fuel ethanol production, such as interference with food production when using agricultural crops, or high formation of inhibitory compounds from pretreatment of lignin-containing forestry products. Algae biomass could be an alternative sustainable raw material since cultivations can be set up in places where food crops cannot grow thus not interfering with food production. Most microalgae cells do not contain lignin and therefore pretreatment will result in less formation of inhibitors. Microalgal cultivations can contribute to environmental benefits by capturing carbon dioxide from flue gas and utilize nutrients such as nitrogen and phosphorus from municipal waste water, resulting in purification of such waste streams together with production of energy-rich biomass. The feed of municipal waste water results in a continuous addition of algal species and microorganisms other than the initial alga of the inoculum which gives a mixed population in the cultivation system. This, together with seasonal variations, may cause changes in the population dynamics; hence different times of harvesting may result in different microbial composition.This project investigates the potential of using microalgal biomass produced on waste streams as substrate in ethanol production. The algae used were produced in the development plant in Ume\ue5 run by SLU, using flue gas of the heat and power plant (Ume\ue5 Energi) as carbon source, municipal waste water from the waste water treatment plant (Umeva) as nutrient source and sunlight as energy source. Algal biomass from 8 time points of a cultivation season were used for i) determination of macromolecular composition in biomass, ii) investigation of different biomass pretreatment processes to yield fermentable sugars, iii) analysis of fermentability and ethanol production in algal hydrolysates and vi) investigation of changes in species composition during the cultivation season. Both enzymatic and acid hydrolysis were investigated as pretreatment process and so far the best results have been obtained with acid hydrolysis. Additionally, small scale fermentations in acid pretreated algal hydrolysates were performed to study fermentability and ethanol production. Variations in species composition of the community at different sampling times will be investigated by terminal restriction fragment length polymorphism (T-RFLP) based on species variations in the 18S rDNA gene

Biofortification of fermented foods with selected yeasts for raised folate content and mineral availability

The goal of this project is to achieve natural biofortification of iron, zinc and folate in foods via selected yeasts. Deficiencies of iron, zinc and folate are prevalent in many parts of the world, especially where cereal based food comprises a large part of the food intake, but also in the western world where meat-free diets, such as vegetarian or vegan diets, has become more popular. Cereals contain several minerals such as iron, zinc and calcium, but it also contains the anti-nutrient phytate, which limits the bioavailability and gastrointestinal uptake of those minerals (1). Folate and mineral deficiencies are related to a range of health issues from mild to lethal (2). Deficiencies can sometimes be prevented by synthetic fortification, but this is not always a feasible solution due to e.g. availability or economy. There are also studies showing an increased risk of overdosing from synthetic supplements (3), which can lead to increased risk of developing some types of cancer (4). Fortification with synthetic folate may also mask deficiency of vitamin B12, something that can result in neurological damages (5).For mentioned reasons, it is of great significance to find ways to naturally fortify foods with folic acid, and to increase the bioavailability of the naturally present minerals in cereal based foods to limit the development of mineral deficiencies. This project focuses on natural biofortification using yeast strains that i) can degrade phytate and ii) synthetize natural folate. Yeast synthesized folates are believed to not mask vitamin B12 deficiencies (5). Phytate degrading yeasts allow natural release of minerals already present in the food matrix, hence less need for synthetic fortification and thereby less risk of overdosing

Assessing phytase activity–methods, definitions and pitfalls

Phytases are nutritionally important for increased bioavailability of dietary minerals and phosphate for monogastric animals including humans. Release of minerals and phosphate is accomplished by the enzymatic stepwise degradation of phytate (inositol hexaphosphate, IP₆). Activity determinations of phytase is often based on analysis of total released phosphate (P_i), but phytase activity in its purest form represents released product per time from IP₆ only. Microbial and plant preparations often also contain mixtures of phosphatases and organic phosphate compounds; hence some released phosphate in enzymatic assays may originate from non-phytase phosphatases degrading non-phytate molecules. Moreover, even purified enzyme extracts assessed via P_i release may result in errors, since commercial IP₆ commonly contains contamination of lower inositol phosphates, and further, the products of phytase IP₆ hydrolysis are also substrates for the phytase. These facts motivate a quantitative comparative study. We compared enzyme activity determination in phytase assay samples at four different time points, based on analyzing the substrate IP₆ versus the product P_i using different selected methods. The calculated activities varied substantially. For example, at 15 min into enzymatic assay, variations from 152 mU/ml (by IP₆ analysis on HPIC) to 275-586 mU/ml (by P_i analysis using several methods) was detected. Our work emphasizes the importance of defining the type of activity assessed, showing that phytase activity based on released P_i may yield false positive results and/or overestimations. We propose to differentiate between <em>phytase activity</em>, being the activity by which IP₆ is degraded, and <em>total inositol phosphatase activity</em>, corresponding to total released phosphate during the enzymatic reaction

Assessing phytase activity–methods, definitions and pitfalls.

Phytases are nutritionally important for increased bioavailability of dietary minerals and phosphate for monogastric animals including humans. Release of minerals and phosphate is accomplished by the enzymatic stepwise degradation of phytate (inositol hexaphosphate, IP6). Activity determinations of phytase is often based on analysis of total released phosphate (Pi), but phytase activity in its purest form represents released product per time from IP6 only. Microbial and plant preparations often also contain mixtures of phosphatases and organic phosphate compounds; hence some released phosphate in enzymatic assays may originate from non-phytase phosphatases degrading non-phytate molecules. Moreover, even purified enzyme extracts assessed via Pi release may result in errors, since commercial IP6 commonly contains contamination of lower inositol phosphates, and further, the products of phytase IP6 hydrolysis are also substrates for the phytase. These facts motivate a quantitative comparative study. We compared enzyme activity determination in phytase assay samples at four different time points, based on analyzing the substrate IP6 versus the product Pi using different selected methods. The calculated activities varied substantially. For example, at 15 min into enzymatic assay, variations from 152 mU/ml (by IP6 analysis on HPIC) to 275-586 mU/ml (by Pi analysis using several methods) was detected. Our work emphasizes the importance of defining the type of activity assessed, showing that phytase activity based on released Pi may yield false positive results and/or overestimations. We propose to differentiate between phytase activity, being the activity by which IP6 is degraded, and total inositol phosphatase activity, corresponding to total released phosphate during the enzymatic reaction

Strain improvement of Pichia kudriavzevii TY13 for raised phytase production and reduced phosphate repression

In this work, we present the development and characterization of a strain of Pichia kudriavzevii (TY1322), with highly improved phytate-degrading capacity. The mutant strain TY1322 shows a biomass-specific phytate degradation of 1.26 mmol g−1 h−1 after 8 h of cultivation in a high-phosphate medium, which is about 8 times higher compared with the wild-type strain. Strain TY1322 was able to grow at low pH (pH 2), at high temperature (46\ub0C) and in the presence of ox bile (2% w/v), indicating this strain\u27s ability to survive passage through the gastrointestinal tract. The purified phytase showed two pH optima, at pH 3.5 and 5.5, and one temperature optimum at 55\ub0C. The lower pH optimum of 3.5 matches the reported pH of the pig stomach, meaning that TY1322 and/or its phytase is highly suitable for use in feed production. Furthermore, P. kudriavzevii TY1322 tolerates ethanol up to 6% (v/v) and shows high osmotic stress tolerance. Owing to the phenotypic characteristics and non-genetically modified organisms nature of TY1322, this strain show great potential for future uses in (i) cereal fermentations for increased mineral bioavailability, and (ii) feed production to increase the phosphate bioavailability for monogastric animals to reduce the need for artificial phosphate fortification

Secretion of non-cell-bound phytase by the yeast Pichia kudriavzevii TY13

AimsMineral deficiencies cause several health problems in the world, especially for populations consuming cereal-based diets rich in the anti-nutrient phytate. Our aim was to characterize the phytate-degrading capacity of the yeast Pichia kudriavzevii TY13 and its secretion of phytase. Methods and ResultsThe phytase activity in cell-free supernatants from cultures with 100% intact cells was 35-190mUml(-1) depending on the media. The K-m was 028mmoll(-1) and the specific phytase activity 032Umg(-1) total protein. The phytase activity and secretion of extracellular non-cell-bound phytase was affected by the medium phosphate concentrations. Further, addition of yeast extract had a clearly inducing effect, resulting in over 60% of the cultures total phytase activity as non-cell-bound. ConclusionsOur study reveals that it is possible to achieve high extracellular phytase activity from the yeast P.kudriavzevii TY13 by proper composition of the growth medium. Significance and Impact of the StudyTY13 could be a promising future starter culture for fermented foods with improved mineral bioavailability. Using strains that secrete phytase to the food matrix may significantly improve the phytate degradation by facilitating the enzyme-to-substrate interaction. The secreted non-cell-bound phytase activities by TY13 could further be advantageous in industrial production of phytase